Autism Draft 1 (This is a preliminary draft which currently contains excessive text Apologies)

[The inserted documents at the end relate to Wiggins-water-structure etc. (which, perhaps especially induced by glycosylation including by anonic polysacharides, putativley are the ultimate determinants of pathology and therefore seem likely to be of general relevance to autism]

NEW THINKING ON AUTISM

(Non-medical-scientific) Discussion of Possible Pollutants Involved in the Aetiology of Autism) suggests
a new hypothesis of autistic spectrum disorders.

A Literature mining survey links autism spectrum disorders (ASDs) to organohalogen plus heavy metal xenobiotic intoxication which leads to disturbances in

glycation especially that involving heparan sulfate (HS) and related nitric oxide signaling
(including during perinatal development) D. Grant, Ph.D., *a New Deer, Scotland, UK, AB53 6SX
Footnotes: after references

Index
1.Summary
1.1 Autism is a major global public health problem + 1.2 Persistent xenobitoic intoxications {Effect of persistent xenobiotic intoxication by halogenated organic (especially aromatic) substances plus antimony, lead, mercury, aluminium arsenic and cadmium may be a cause of autism} 1.3 Heparan sulfate (HS) dependent tissue protection 1.3.1 Further Notes on the Role of Heparan Sulfate (HS) and Other Polysaccharide Biochemistry in Autism and Other Diseases 1.3.1.1 Therapeutic use of Guar Gum, Fulvic Acid Etc. 1.3.2 The possible critical need for retention in vivo of a high effectiveness of H/HS as metal ion binders 1.4 Accumulation of antimony(Sb) by HS(PG) may perturb (nervous system) development 1.4.1 Accumulation of lead, mercury, aluminum, arsenic, antimony beryllium and cadmium by HS

2. Polychlorinated benzenes (PCBs) and related substances

{Pesticides may disturb HS biochemistry to produce an altered (including innate) immune response} 2.1 Possible Critical Role of Hexachlorobenzene (HCB) + Other Xenobiotics in Autism, Hypertension, Obesity and Type 2 Diabetes 2.2 Formation of PCBs (polychlorobiphenyls), OCS (octachlorstyrene) and HCB (hexachlorobenzene) during combustion and incineration

[OCS is a marker of accidental chlorinated aromatic synthesis] 2.2.1 Contribution of Aryl Hydrocarbon Receptor induced inflammation from exposure to dioxins 2.2.2 HCB as a Hormonal Disrupter 2.2.3 HCB and gap junctions 2.2.4 HCB Toxicology Further Reading 2.2.5 HCB and the Aubrey-Van Wazer Equilibrium Inorganic Scrambling Catalysts in the Aubrey Van Wazer Equilibrium 2.2.6 Claude Rimington a pioneer of HCB intoxication studies 2.2.7 Evidence from Specific Environmental Locations & Prelevance of Autism [PCBs HCB produced by Road Traffic (Old or defective) Incinerators & Metallurgical Plants] Unfiltered Dust PM2.5 PM1

3. A new hypothesis of autistic spectrum disorders (ASDs):

Autism may be caused by Pesticides and other environmental inputs which produce (via a disturbance of HS biochemistry) an altered immune response (Possible Anthropogenically Introduced Pollutants Involved in the Aetiology of Autism)

4.

Epidemiological Studies

The worldwide prevalence of ASD may be much higher than previously thought. A large twins study suggests an environmental cause of autism. Most autism is produced by environmental factors (which dominate over genetic factors).
But which highly toxic environmental input over the last thirty years might most closely match the dramatic increase in autism? [cf. Footnote b. ]

4.1 Reported Studies of Korean children 4.1.1 Large twins study

5.
Tendency for the current universal human intoxication by xenobiotics to produce a (relative apparent mild for most persons but more severe for genetically susceptible individuals) chronically enhanced inflammatory state which is enhance by Dietary Antioxidant Depletion
[Heparanome-defect hypothesis of autism and Heavy Metal intoxication].

6. 7.
` 8.

The Possible Synergistic Interaction of Xenobiotics with the Immune System Intoxication of the Global Human and Animal Populations by POPs Sb intoxication & alpha lipoic acid

Some Previously Suggested Hypotheses of Autism Dietary deficiency of selenium and zinc 8.1 Causative Inducing Role of Vaccination 9. Diminished magnesium (Mg) and calcium (Ca) 9.1 Calcium homeostasis and ASD 10. Epigenetics and Autism 11. Chronic retroviral infection and ASD 12. Hypothesized role of electromagnetic radiation in the aetiology of ASD 13. Role of environmental input of antimony (Sb) Newer uses of Sb 13.1 Multiple toxic metal intoxication hypothesis including from lead cadmium mercury nickel arsenic cadmium aluminum beryllium and antimony

14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

13.2 Mercury (Hg), Lead (Pb), Cadmium (Cd) Are ASDs phenomenologically related to cancer Calcium Oxalate Stone Formation

[HS inhibition of oxalate stone formation]

Oxidized lipids excess glucose homocysteine omega-3 fatty acids retinoic acid ascorbate. [Effect on HSPG integrity] Cholesterol Lipid Rafts Cytokines Glucosamine HS(PG) status and fear response Secretin Possible future use of (sulfated) polysaccharide-based chelation Chlorinated Phenols Phthalate Synergistic metalloid organohalogen interaction Automobile Brake Friction Surface Dust Friedel Krafts reactions Tyrosine nitration Aubrey-Van Wazer reactions ME/CFS Myalgic Encephalomyelitis Chronic Fatigue Syndrome 29-1 (Mark Purdey) Barium intoxication and multiple sclerosis
(Cf. Cu/Zn Mn3+ electron phonon PrPC coupling) IAG Other Reported Biochemical Markers of Autism Proton NMR of urine; MRI

Nitration of tryptophan Aluminium Calcium carbonate crystallization Possible relatedness of ASD with Alzheimers disease Sulfate depletion in soils Dark Chocolate (therapy?) Water activity Inorganic Si Gallstones Gut Defects

40.

Appendix
Notes A1 A2 Why are anthropogenic substances not easily detoxified Possible Unique Role of Mercury (Hg) to HCB etc. Environmental Possible Unique Role of Antimony (Sb) amongst toxic elements associated Are AD and ASD produced by similar toxins? G. Bell et al. U.K. Scottish Autistic Subject hair element survey
Communicated Archival Sb Hair-Element Data

Augmentation A3 with Autism A4 A5 (Severity) A6 A6-1 A7 A8 A9 A10 A10-1 A10-1-1 A10-2 Advanced Glycation End Products Lead (Pb) Intoxication and Altered Glycation Purine Metabolism Glutathione-S-Transferase More on Sb Intoxication; Re Glycation Alteration of Heparan Sulfate by Systemic Oxidant Overload Is HS too complex for present day? Sb may be more strongly bound than Al to HS Unzipping of HS by toxic metal plus excess nitrite

Some Other Reported Hair Element Alteration Studies Relating to Autism

A11 Sb binding greatly diminishes the ability of Sb-polluted alginates to bind other toxic heavy metals A12 The metallome 41. References
Selected References References (General arranged alphabetically according to first named author) Further Sb Intoxication References (Lipoic Acid) Further HCB-Re lated References [Within References see also marked sections: Zinc & Autism Further Notes on Antimony & Autism Toxic Metals & Heparan Biochemistry Tyrosine nitration How Heavy Metals (which have been Associated with Autism) Induce Cytokines Latitudinal Variation of the Prevalence of Multiple Sclerosis and Perhaps also Autism Serotonin & Autism Oxytoxin & Autism]

Footnotes Footnote a - footnote *-*The author

More on ME/CFS
Footnotes a-d a Genetics. chromosome and telemere damage vs. environmental insult

Concluding statement

Research suggstions from literature relating to possible therapeutic intervention in autism. The bioavailabilty of mercury may be altered by the anthropogenic alteration of soil organic matter and its augmentation in natural waters
Discarded Text

BIOLOGICAL WATER STRUCTURE [Inserted Documents 1-16 (at end of original document)] --------------------------------------------------------------------------------------------------------------------------------------------------

Doc 1 Wiggins Two State Water Theory Doc 2 Reconcliling Wiggins, Cairns-Smith and Kornberg Theories of the beginning of Life Doc 3 Liquid water as a Van Wazer stochastic sytem Doc 4 Hydration water structures of sparingly soluble Ca salts Doc 5 Abbreviated Reconciling Wiggins, Cairns-Smith and Kornberg Theories of the Beginning of Life Doc 6 How polyphosphate could be part of a more general definition of life Doc. 7 The generation of specific species of silica sol by seeeding Doc 8 Inorganic element association with heparin Doc 9 Water and Life Doc 10 Water & Biology Doc 11The Structure of Water and Hydrates- Its Importance to Biology Doc 12 The Chemistry of Hydrogen Bonding Doc 13 Water Life and Prebiotic Evolution Doc 14 Notes + Classical Papers on Biologically-Relevant Hydration Doc 15 HEPARAN SULPHATES AS BIOLOGICAL EFFECTORS THROUGH THE MODULATION OF WATER
STRUCTURE

Doc 16 Biological Nucleation

The first (preliminary Autism Draft) document [below] is derived from ideas gleaned from peer-reviewed academic studies and grey internet posted documentation, a selection of which sources are collected at the end of this document. I also have included unpublished research results (archival data) kindly communicated to me in response to a written request. These are presented together with personal databases of possible relevance to the subject of this paper. These data were partly assembled at the University of Aberdeen.

============================================

. 1.1 Autism is a Major Global Health Problem While recent reports now indicate that an improvement of instrumentation might permit autism to be diagnosed on the sole basis of urine nuclear magnetic resonance (NMR) pattern recognition or analogous magnetic resonance imaging (MRI) evaluation, the idea has emerged that ASDs are a group of possibly aetiological-related disorders with some relatedness to other more genetically defined diseases all of which are to at least, to a significant extent, engendered by xenobiotic intoxication. Of especial relevance for this scenario is the formation of dioxin and related toxins during sub-optimal industrial (re-)processing in newly industrialized countries (cf. Weber et al.); input of volatile toxins into the atmosphere apparently commonly occurs during such activities and the international redistribution of these highly toxic substances via input into the atmosphere can, it seems, potentially account for the current apparent global intoxication of all animal species by these anthropogenic inputs. Such a mechanism may be at least in part why, throughout the world, a number of mystery illnesses including autism have shown recent increases in prevalence. 1.2 Persistent xenobiotic intoxication by halogenated organic (especially aromatic) substances plus antimony, lead, mercury, aluminium, arsenic, and cadmium may be the especial environmental cocktail which gives rise to autism in the foetus and developing child; this idea seems to be in general agreement with the outcome of a recent large twins study (cf Section 4.1.1) which has indicated that autism is largely an environmentally-induced disease.

Summary

1.3

Heparan sulfate (HS) - dependent tissue development and protection

If numerous mystery diseases including autism which are now major global public health problems

are held to be at least in part be induced by the current environmental presence of anthropogenically-introduced toxic chemical substance(s), then a possible underlying cause of these pathologies might be the disturbance of the HS (PG) [a primitive tissue regulation and non-specific immune regulator system which has signalling connectedness with most or all animal biochemical systems and for which the primary and post-synthetic biosynthesis and re-synthesis pathways are apparently ultra-sensitive to the presence of exogenous substances]. In depth studies of the inorganic biochemistry of heparan sulfate (HS) and related polysaccharides show that these substacnes are primary system managers therefore are likely to be directly implicated in the aetiologies of numerous diseases (cf. the list of publications listed at the end of this document [cf. also Grant (2000) and Long (2003)]. Heparan sulfate and its post-synthetic scission products which are heparin-like (HSmimicking) polyanions can simultaneously bind the entire range of 60+ inorganic elements which occur in seawater and which also occur (in approximatly correlated amounts) in human blood serum (cf. Haraguchi). (Such binding of inorgnic ions apparently occurs analogously but more efficiently than the similar previouslyestablished behaviour of marine alginates). Heparan sulfates exist as sidechains in protoglycans (HSPGs). While the core proteins contribute to the biochemical activites of HSPGs the side chains most often play the dominant biochemical roles (cf. Bernfield). Sidechains and core proteins may, however, act cooperatively for the binding of inorognic ions. The HSPG system is centrally involved in development (e.g. brain development requires correct HS microstructures, cf. Tornberg et al.) and wound healing. The activity of HSPGs in development etc. apparently also critically depends on the presence of appropriate inorganic cofators (cf. Rudd et al.). This means that intoxication by anthropogenically introduced heavy metals etc. is likely to (potentially greatly) alter HSPG activity. This HSPG activity is also subject to an apparent servo-control feedback control by nitric oxide and its metabolites. This also involves ascorbate and Cu (and perhaps also Zn) (cf. Ding et al.). [this can probably explain Linus Paulings findings regarding the benefit of ascorbate for combatting degenerative diseases]. This means that HSPG is probably sensitive to the systemic and local redox status and to the the presence of anthropogenic pro-oxidant pollutants (e.g.HCB and dioxins in fatty tissues) which tend to create a chronic pro-oxidant state and so (potentailly greatly) alter HSPG activity and thereby promote a range of diseases. The HSPG system is, of course, part of a wider glycome (N- and O-glycosylation). [Arthritic diseases are now thought to arise at least in part because of a pathological alterationof the glycosylation e.g. of immunoglobins (cf. IgG) [cf. Raju]]. There is also current interest in the effect of altered glycosylation in autistic spectrum diseases (cf. Pivac, {cf. selected refs.}, who reported than an altered (N-) glycoslyation was associated with ADHD [attention deficit hyperactive disorder]). It is now suggested that O-glycosylation (and especialy of HSPG) alteration(s) including during development) are responsible for ASDs. This hypothesis is supported by studies of selective changes in neurodevelopmental proteins (which are known from other informatio to be subject to HSPG control) cf. e.g. the mouse model of ASD study reported by Stephenson DT. et al. loc cit.

1.3.1 Further Notes on the Role of Heparan Sulfate (HS) and Other Polysaccharide Biochemistry in Autism and Other Diseases

Sugar biochemistry could be a useful focus for unraveling the complexities of the rest of biochemistry and pathology. Obviously the role of the monosaccharide glucose as a carbon energy source (the need for animals to retain glucose homeostasis) is a key factor for health. It should be noted that alteration in blood glucose levels are known to also signal for an alteration in primary HS biosynthesis. Elevated blood glucose essentially damages the ability of HS (cf. e.g. Moreno) to provide for multiple tissue protective functions. A similar potential therapeutic system feeding into HS microstructure may also conceivable be designed to offer therapeutic benefit for autism (perhaps especially since autism and obesity seem in some way to be linked etiologically as indicated by the epidemiological correlations which have been observed between these conditions in large population surveys).
Previous studies (Paka et al.) using apolipoprotein E-/-mice had indicated a heparan sulfate mediated effect of pro-atheromic lipid dietary components in generating inflammatory responses relevant to the etiology of atherosclerosis. The oxidized lipids caused a diminution of tissue protective heparan sulfate biosynthesis. The occurrence of different amounts of endogenous heparin in the blood stream (in an amount negatively correlated with LDL cholesterol, cf. H. Engelberg publications {THIS ENDOGENOUS HEPARIN MAY ALSO BE SUBJECT TO REDOX STATUS AND NITRIC OXIDE DEPLETION, cf. D. Grant internet documents, cf. dg2 iNOS is promoted by cytokines TNF, IFN IL-1 and IL-2 but suppressed by TGF, IL4, IL8 IL10 and IL13 ) may be part of the reason why there is a marked variation between individuals in their response to the alleviation of the symptoms of CFS by heparin. Another problem is the possible variation of the potency of heparin for this purpose between brands. Part of this variation could be the presence of aluminum in some brands heparin as indicated by the studies of Bohrer et al. loc. cit.

1.3.1.1 Therapeutic Use of Guar Gum, Fulvic Acid Etc.

Guar gum (is a plant-derived polysaccharide) The dietary use of guar gum (often referred to as [soluble] dietary fiber) provides an illustration of sugar biochemical approach to provide a possible therapy for obesity (Krotkiewski). The ability of plant derived poly- and oligo-saccharides (such as guar gum, but also such chemical systems as, carrageenans and even soil-derived fulvic acids; semi-synthetic xylan sulfates, heparin analogues derived from birch wood and marine algae) to enter mammalian biochemical signaling seems ultimately seems to derive from the very ancient evolutionary use of sugars in the wide-ranging regulation of biological activities and protect them from pathogens. It can be argued logically that sugars provided tissue protection and other functions during the earliest evolutionary stages in the development of life forms. [This continued bio-friendly aspect of sugars seems to be why there have been found to be a large range of potential therapeutic uses of sufated xylans, e.g. include apart from blood anticoagulation also interstitial cystitis and inhibition of prion diseases such as variant Creutzfeldt Jakob disease]. Part of a wider potential use of sugars and their derivatives as well tolerated therapeutic agents is the finding that guar gum is an effective modulator of blood glucose and obesity. The mechanism of this effect may however be achieved via interaction of between the guar oligosaccharides and the heparan sulfate (sulphate) (HS) present as side chains in proteoglycans (e.g. syndecans, glypicans, agrin, type XVIII collagen) is a major cell surface and extracellular matrix component HS signaling system. The effect is either direct or secondary to the reduction achieved by guar on blood glucose level elevation of which negatively alters the tissue protective functions of HS.

1.3.2 The critical need for a high effectiveness of H/ HS as metal ion binders. The multi-inorganic element content of heparin, a model structure for the most highly sulfated regions of heparan sulfate, is found to consist of a pure metallomic matrix type of chemical stoichiometry. Although the function of this state-ofmatter has not been fully explored, preliminary indications suggest that increased

anti-oxidant protection (e.g. heightened non-enzymic superoxide dismutase activity) might be afforded by the multi-inorganic element loaded heparin-like polyanions (relative to e.g. a single alkali metal counterion form normally used pharmaceutically) which, it should be noted, are the most anionic bio- molecules known and are likely to sequester the full range of inorganic ions which occur in the seawater-like extracellular fluids of animals including humans. [A metal-related HS action of possible relevance to the aetiology of ASDs: Progesterone (cf. Grant ECG) may promote ASD via. Mg (Zn) down regulation of HSPG. [Progesterone may have a (hexachlorobenzene) HCB-like role]. Cf. Zn and Mg lowering effects of maternal progesterone; (cf. birth pill lowers Mg, this lowers HSPG and Zn affects NO recycling from endogenous HSPG NO stores cf. Mani et al., loc. cit.)].

1.4 Accumulation of antimony (Sb) by HS(PG) may perturb (nervous sytem) development This action of Sb may promote ASDs
Antimony was indicted by (unpublished) preliminary results (obtained at Aberdeen University) to be more strongly bound to H/HS than were any other of the 37 (cf. Haraguchi) commonly encountered blood-serum-inorganic-elements; this circumstance suggests that the relatively high and increasing current environmental abundance of antimony might now be sufficient in gentically susceptible individuals to perturb human HS (PG) signaling (and this may be of especial relevance to how an (often subtle) alteration may arise in the development of the foetal and infant brain which putatively causes childhood obesity and other mystery illnesses in adults) and if this hypothesis can be corroborated, it is evident that the topic of the environmental pollution by antimony and its abatement, must be of prime relevance for attaining any fuller understanding of the etiology of autism and related illnesses and for designing measures to combat these illnesses. This hypothesis accords with the dramatically increased use of compounds of antimony (e.g. as components of friction surfaces used in automobiles, as flame retardants in plastics and textiles and in production polymeric substances used as food and drink containers where, especially during microwave cooking, which seems to produce an enhanced leaching of antimony into foodstuffs). 1.4.1 Accumulation of lead (Pb), mercury (Hg), aluminium (Al), arsenic (As)), antimony (Sb) and cadmium (Cd) by HS. Studies of the effectiveness of a sulfonate ion-exchanger to remove multi-elements from heparin (H) (a surrogate form HS) suggested (Grant) that the H/HS system is capable of sequestering the full range of metal ions which occur in seawater and in blood serum (cf. Haraguchi). A corollary to this finding is that while ultratrace loading of H/HS may be benign, the over-loading of H/HS may trigger pathological responses (in those growth factor pathways in e.g. neurological assembly processes which seem to require both HS and multi-valent metal ion cofactors, (cf. Rudd et al. Kan et al.; Purdey)).

2. Polychlorinated benzenes (PCBs) dioxins and related substances

Possible Critical Role of Halogenated Xenobiotics in Autism:
{Pesticides may disturb HS biochemistry to produce an altered (including innate) immune response}

Cf. refs., (first named authors) Canzoniero, Dingeman (Rose), {Dunstan} Guariglia, Kalkbrenner, Kim, Kuriyama, Larsson, Miodovink, Morei, Mazzariol, Park, Pereira, Randi, Roberts, Smink, Weber, Windham, Zhang and appended Further HCB-Related References.

2.1 Possible Critical Role of Hexachlorobenzene (dioxins + dioxin-likeHCB) + Other Xenobiotics in Autism, Hypertension, Obesity and Type 2 Diabetes
Pesticide Residues, Polychlorinated Biphenyls, Hexachlorobenzene + Heavy Metals (Mercury, Cadmium, Lead, etc.) and Metalloids (Antimony etc.) Cf. refs (first named authors) Adams, (Cardinas), Choi, Cloy, (Collins), De Burbure, Dodds, Dufault, El-Bay, Elsheshtawy, Geir, Grant [ECG], Haldimann, Hall, Hallmayer, Hongve, (Johnson), (Kaji), (Kan), Kim, Landigran, Marlowe, Marth, Mariea, Palmer, Priya, Roberts, Roze, Rossignol, Seelig, Shotyk, Shepherd, Stejskal, (Templeton), Utschig, Vali, Windham, Yashuda, (Yin). Autism, Hypertension, Obesity and Type-2 Diabetes have been indicated at least by some researchers (cf. Haldimann) to enhance the possibility of direct dietary ingestion of antimony, and arsenic-like inorganic elements. Cf. also the possible role of pesticide plus heavy metal intoxication in the aetiology of amyotrophic lateral sclerosis (ALS) (Johnson et al.). 2.2 Formation of PCBs (polychlorinated biphenyls) , OCS (octachlorostyrene) and HCB and brominated aromatic xenobiotics during combustion and incineration (N.b. OCS is a marker of accidental chlorinated aromatic molecule synthesis) Literature search tends to confirm a possible key role of pentachlorophenol and its metabolic precursor hexachlorobenzene (HCB). The unique pathway by which these molecules are formed in relatively large amounts (accidentally during combustion conducted with polluted air intake) is why these substances are highlighted. 2.2.1 Contribution of Aryl Hydrocarbon Receptor Induced Inflammation from Exposure to
Dioxins

Cf. D Wu et al. (Activation of aryl hydrocarbon receptor induces vascular inflammation and promotes atherosclerosis in apolipoprotein E-/-mice) Arteriocler Thromb Vasc Biol. 2011 31 1260-7 [The results suggest that CXCR2 mediate the atherogenic activity of environmental pollutants such as dioxins and contributes to development of inflammatory response by activating the AhR signaling pathway

{The CXC subfamily of chemokines and their receptors are believed to be involved in inflammation, wound healing , growth regulation angiogenesis and tumorigenesis; CXCR2 is reported to bind IL-8 GRO-, NAP-2 and GPC-2}]. 2.2.2 HCB as a Hormonal Disrupter
Cf. SM Lelli et al. (Hexachlorobenzene as hormonal disrupter-studies about glucocortcoids. Their hepatic receptors, adrenal synthesis and plasma levels) Biochem Pharmacol. 2007 73 (6) 873-9 HCB was found to diminish Wistar rat plasma corticosterone and hepatic glucocortiosteroid receptor numbers (especially after porphyria has become well established). This seemed to be a major part of the mechanism of how HCB can diminish phosphoenolpyruvate-carboxylkinase (PEPCK) and related gluconeogenesis activity.

HCB causes a distinct glucocorticoid and glucocorticoid receptor depletion in Wistar rats. This affects gluconeogenesis. HCB has been reported (Li) to act synergistically with TCDD dioxin to induce thyroid dysfunction. 2.2.3 HEXACHLOROBENZENE & GAP JUNCTIONS HCB (a gap junction disrupter and liver and kidney dysfunction inducer; and also a immune suppressor, neurotoxin, cardiotoxin, and calcium metabolism and reproductive system disturber) which has separately been epidemiologically associated with the foetal development alterations which are believed to cause childhood obesity (Smink). That autism might also be (partly) caused by HCB intoxication and perhaps also HCB-related induced HS dysfunction(s) is confirmed from epidemiological evidence which has linked the prevalence of autism and unexplained childhood obesity.

2.2.4 HCB Toxicology: Further Reading

Cf., e.g.JAGM van Raaij Reduction of thyroxane levels in the circulation and in the brain of hexachlorobenzene-exposed rats Thesis TNO Nutrition and Food Research Institute Rikswijk The Netherlands Cf. also the references relating to HCB toxicity listed after the alphabetically-listed-byfirst-author references
2.2.5 HEXACHLOROBENZENE and the Aubrey Van Wazer Equilibrium [Scrambling] It is now suggested that the global intoxication of animals and humans by the highly lipid soluble (aqueous phase sparing soluble aromatic persistent organic pollutants (exemplified by hexachlorobenzene [HCB] the formation of which from a range of chlorinated precursors is driven by the Aubrey-Van Wazer equilibrium) suggests a mechanism by which HCB by entering the key lipid rafts could perturb HS tissue development. This might account at least in part for why low levels of HCB intoxication can effectively disrupt growth factor signalling needed for correct nervous system assembly and might explain why childhood obesity which is apparently correlated with maternal HS intoxication (cf. Smink).

Inorganic Scrambling Catalysts

Possible Unique Role of Mercury (Hg) to HCB etc. Environmental Augmentation Possible Unique Role of Antimony (Sb) to HCB etc. Environmental Augmentation

The formation of scrambled aromatic halogenated substances is, however, subject to both negative and positive catalysis by inorganic substances and attempts have been made to diminish their amounts by this method
2.2.6 .CLAUDE RIMINGTON a pioneer of HCB intoxication studies cf. footnote d

PCBs and HCB were originally used industrially and produced for this purpose by deliberate chemical processing, but these substance (together with HCB and OCS) but can also arise accidentally during common combustion of fossil fuel usages (e.g. in exhaust gases from power plants automobile and truck engines). Since OCS has never been deliberately employed in industry, the environmental presence of this substance is a valid marker for the presence of all of the other accidental synthesis generated xenobitics produced during (chlorinated) waste incineration or fossil fuel combustion and indicates that the current continuation of the presence of such substances in food is not entirely due to the release of such substances from the soil reservoirs or left-over pollutants from the pre-ban historical industrial pollution soil reservoirs but at least some of the HCB etc. is formed recently during chlorocarbon pyrolysis via scrambling of other chlorinated hydrocarbons (or nonchlorinated hydrocarbons plus chloride anions).
There has been indicated from a general reading of the literature that a critical role may be played by hexachlorobenzene (HCB) and related halogenated aromatic and intoxication by those halogenated aliphatic substances which can rearrange (especially under the influence of heavy metal catalysis) to produce HCB which further becomes transformed in vivo into pentachorophenol. This substance uniquely of several suspected inducers of ASD has been specifically associated with several indices of learning disablement in children (Roze). HCB self-assembles during the pyrolysis/incineration of large tonnage waste materials. This arises because at the temperatures occurring in common combustion systems the basic chemistry is outwith the usual (near room temperature) mechanistic control processes (which had been defined by classical organic chemists). Instead thermodynamic equilibrium processes (where numerous mechanisms may give rise to the same end products but the thermodynamic parameters are what determines the outcome) arise. The first approximation model of the organic-chlorine scrambling system is the formation of atom interchanges between HCB, CCl4 and C2Cl6 (a more accurate model includes C2Cl2 and C2Cl4 and in the presence of O2 also, by a related process leads to the production of dioxins and perchlorodibenzenes (PCBs). It is also commonly found under mild pyrolysis or common incineration conditions that random halogen interchange occurs (e.g. Cl can be randomly replaced by Br) (cf. Aubrey & Van Wazer) The pro-ASD xenobiotics arise during common combustion of fossil fuel etc. in which ubiquitous thermal rearrangement reactions produce the same toxic cocktail from a range of starting molecules containing some carbon and chlorine atoms or ions. It should be noted that major international effort had previously attempted (but as yet not succeeded) in preventing HCB from entering the environment (although some success have been attained in banning the deliberate manufacture of other members of the dirty dozen pesticides which has been proscribed under the Stockholm Convention). Currently the highly human health damaging cocktail arising directly or indirectly (by the secondary effect of ash elements which create a second wave of HCB etc. derived from fossil fuel combustion) from waste disposal becomes transported intercontinentally in such a manner to intoxicate the entire animal kingdom environment. Fossil fuel combustion also creates toxic metal aerosols (e.g. containing Sb and Hg [it seems possible that when coal deposites were laid down cycads and other less evolved palnt species were then more abundant than today and the uptake and tolerance of Hg by such plants can acount for its occurence in elevated amounts in coal etc. and also perhaps also pose a neurological health risk to presentday cycad eaters]). These elements when present in air intakes can increase the rate of formation of HCB and dioxins. Such anthropogenic HCB, transported over large distances in the atmosphere and concentrated up the food chain is known to act as a potent endocrine disturbing substance which at the current tissue loading can create the kind of subclinical chronic inflammation which is thought to be associated with autism. HCB is also known to disturb foetal development (and this could explain why the amount of HCB in cord blood can be correlated directly with the observed degree of obesity in offspring (Smink) ; the HCB load in adipose tissue has furthermore also putatively been associated with obesity in adults). It should be noted that an epidemiological overlaps have been indicated between autism and unexplained obesity and also between obesity and hypertension. A PCB intoxicated environment was used to test the hypertension-PCB linkage (cf. Goncharov). Organochlorine production facilities always produce HCB (as a byproduct of PCB manufacture) which is suspected to give rise to large scale soil contaminations. The presence of a range of persistent inorganic and organic pollutants in the environment and their enrichment up the food chain has apparently allowed the global human population to become subject to a general intoxication which has induced a general, most often mild, but sometimes relatively severe, pro-inflammatory state (perhaps this can be regarded as having an equivalent effect to that of a vaccine adjuvant).

Such enhanced inflammatory states putatively enable the sudden onset of chronic fatigue syndrome (CFS) or autistic spectrum diseases to be induced upon additional pathogen infection or, perhaps more frequently than in former times, by the excessive use of pharmaceutical agents in medical interventions.

Cf. also Footnote 1a-2 2.2.7 Although the entire atmosphere has a low (but biologically relevant) level of halogenated plus heavy metal intoxication Specific Locations which may have enhanced antioxidant depletion in affected individuals include industrial sites, old industrial sites and sites close to highways (cf. Volk) or adjacent to pesticide treated crops (cf. Roberts). Unfiltered Dust PM2.5 PM1 A major source is uncontrolled incineration (vinyl input) has been suggested to be the ultrafine unfiltered dust arising from otherwise highly controlled municipal incinerations.

3. A new hypothesis of autistic spectrum disorders (ASDs): HCB and related Pesticides (via the disturbance of heparan sulfate (HS) biochemistry) produce an altered (including innate) immune response
The Putative Heavy Metals + Persistent Organic Pollutants (POPs) Roles in the Aetiology of Autism (A maternal and infant intoxication by synergistic heavy metals- halogenated organic xenobiotic interaction hypothesis)

A new hypothesis of autism and related conditions: these arise in genetically susceptible individuals as a consequence of persistent xenobiotic intoxication by molecules produced during the combustion of fossil fuels which cause an impairment of heparan sulfate (HS)-dependent tissue protection, developmental and immunological signaling

Hypotheses are derived below relating to how an environmental presence of xenobiotics e.g. several persistent organic pollutants (POP) chlorinated aromatic substances (cf. Kim) and their metabolites may contribute to the aetiology of the autism spectrum disorders (ASDs) which may primarily arise from neurodevelopmental defects causing impairment in verbal and non verbal communication skills. These hypothesis were derived from a critical in depth review (mining) of the literature conducted from the perspective of how environmental pollutants might alter HS signalling involved in nervous system development so as to promote ASDs and other developmental illnesses which, since these diseases seemed not to be consistently associated with well defined generally agreed-on markers (e.g. as seen in blood urine and also in the commonly applied hair element analyses), it was apparent that ASDs

comprise a highly heterogeneous group of disorders which can only be identified on the basis of a list of physician-assessed observations.

The majority of ASDs seem likely to be the result of environmentally determined foetal and continued childhood intoxication by xenobiotics (e.g. the persistent organic pollutants (POPs) and heavy metals which are the eventual outcomes of scientific researches by industrial chemists especially those who have researched the fundamental chemistry of chlorinated organic compounds); this suggests that the databases of such industrial chemists might offer clues as to how these disorders arise and putatively also might offer how improved therapeutic interventive strategies might be developed. Of prime importance for the elucidation of the aetiology of ASDs seem to be the pervasive occurrence of halogenated aromatic xenobiotics which now apparently intoxicate the entire human population (and putatively induce obesity as well as hypertension in adults following an initial foetal intoxication in pregnant women). The metabolic imbalance consequent on common xenobiotic intoxication which evidently also causes a dyshomeostasis of the essential major and minor inorganic ions (perhaps due to kidney and liver function disruption and also the back-up more primitive tissue homeostasis systems which although insufficiently studied, seem to depend on the anionic polysaccharides which occur principally in the glycocalyxes of extracellular matrix and cell surfaces). The overall xenobiotic intoxication process seems to produce an inability to detoxify the non-essential inorganic elements; this leads to the accumulation of lead (Pb), mercury (Hg), aluminium (Al), arsenic (As), antimony (Sb) and cadmium (Cd) [with the storage of these elements in the glycocalyx and skeleton] and which shows up in hair element analyses. It is known from cell culture work that HS biosynthesis is highly sensitive to negative perturbation by heavy metal presence (cf Fujiwara). A direct effect of PCBs and related substances does not seem to have been studied but evidence that PCB intoxication is mediated via nitric oxide (cf. Lorella) and the known ability of nitric oxide to cleave HS in a heavy metal catalyzed manner could suggest that co-intoxication by PCBs and heavy metals will negatively perturb HS signalling so as to impair neurodevelopment.. A sub-group of ASD is now also suggested to arise more specifically from a synergistic intoxication interaction between hexachlorobenzene (HCB) and the metalloid element Sb. Other sub-ASDs types may partly arise from direct Hg intoxication (e.g. derived from amalgam and intoxicated fish consumption as well as controversially from the continued presence of Hg in some vaccines).

4. Epidemiological Studies
4.1 Studies of Korean children seem to have indicated a possible chlorinated aromatic and phenol-related substance origin of autism. The worldwide prevalence of ASD may be much higher than previously thought apparently affecting 1 in 38 children of age 7-12 (as suggested by a recent study (Kim) of South Korean children).

4.1.1
Large Twins Study Implicates A Major Environmentally-Related Cause of Autism

A recent large epidemiological study of twins has indicated that autism spectrum disorders (ASDs) could most typically be the outcome of the presence of (currently stated-to-be-unknown) environmental factors*b and these, to a much larger extent than was previously widely believed, are now the target of researches aimed at fully uncovering the cause(s) of ASDs. These conditions are no longer believed as formerly was the case, a consequence of specific genetic deficiencies. Perhaps as little as 5% of current ASDs could be caused by primary DNA defects.

5.
The putative anthropogenically mildly enhanced inflammatory state

throughout the entire human population

is putatively due to the universal animal intoxication by multiple xenobiotics which leads to ANTIOXIDANT DEPLETION Redox Status and Heparan Sulfate Dyshomeostasis Antioxidant System Dysfunction in Autism apparently includes dysfunction of
Heparan sulfate /Li

Se, S, Mg -3 -6 Vit C Vit D and Vit B12 Vit B6 Depletion of key vitamins and minerals which deplete antioxidant detoxification and associated enzyme systems including glutathione (cf. e.g., Bernhoft) and related substances will lead to oxidative stress. Such deficiencies plus intoxication by polychlorinated aromatic POPs including HCB tends to increase the redox status of the organism [causing oxidant damage to a range of biological molecules and systems and a related nitration of key amino acids tyrosine and tryptophan and inappropriate nitrosative degradation of heparan sulfates (HS)]; the system manager HS sulphated polysaccharide information-encoded system (which is centrally involved in development and wound healing cf. Perrimon; Bernfield) also displays a general antioxidant protection function (cf. Grant et al (1)) which is subject to diminution under the influence of toxic heavy metals (Grant et al. (2) and xenobiotics; HS, which was originally discovered because of dramatic anticoagulant activity also has a wider function in the regulation of hemostasis, lipid metabolism, wound healing (including. the regulation of angiogenesis) and inhibition of a wide range of types of pathogenic organisms and toxic substances, which especially can damage the kidney and the liver. HS protects against urolithiasis by an apparent servo feedback control mechanism which attempts to create specific anti-Ca oxalate seed inhibitors. The urinary presence of elevated urate, it should be noted, seems to be a characteristic feature of autism 1a-2.

6. Possible Synergistic Interactions of Xenobiotics with the Immune System

Chlorinated and brominated xenobiotics could have possible roles in autism Possible key role of principal HCB metabolite pentachlorophenol (PCP) possible synergism with heavy metal intoxication The level of intoxication by pentachlorophenol (the major metabolite of HCB) in mothers was found in a n=62 academic environmental study which to be associated with a worsening severity in offspring at age 5-6, of coordination, sensory integration, attention deficit and coordinated visionary integration functions (cf. Rose et al. listed under Dingemans) [this study also established that the different polybrominated diphenyl ethers similarly crossed the placenta to change these and related functions compared with matched control subjects but the effect of this class of xenobiotic could be both negative and positive for individual congeners; overall the foetal intoxication level of these substances seemed to correlate with later (5-6 year old) child behaviour disorders. One study (Martin) had compared the breast milk organohalogen xenobiotic toxic levels (of brominated aromatic DDE, HCHs, PCBs HCB and PBDEs) for a Lancaster North East of England (n=27 sample) which was a ca. 2 fold less toxic substance loaded than a that in the London South East of England (n=27 sample). The prevalence of autism is known from other reports to be several fold higher in South East England than it is in rural Wales (and perhaps tends to confirm the local pollution relationship). Grey literature internet reports also mention that the prevalence of autism (as well as infant mortality) is greater in the vicinity of (traditional but semi-updated) municipal incinerators (being suggested e.g. to be greater downwind of Edmonton, London and also in between two incinerators in Birmingham, England). Studies in California Texas and elsewhere in the USA (which have been published in peer-reviewed journals) have similarly linked increased environmental pollution with the prevalence of autism. (An hypothesis that Hg intoxication is of especial relevance to autism and could perhaps partially explain why rural Amish populations have much lower rates of autism, seems however to have slanted the discussed against the possible role of other more abundant xenobiotics which co-occur together with Hg, e.g. in fine particulate matter produced by the combustion of fossil fuel, in this population) The presence of PCB congeners in gannet colonies studied in East and west Scotland seems to have decreased historically and then increased again for some of the congeners but not for others; this suggests an incinerator origin of the newer PCB inputs into the environment. A similar new input mechanism (perhaps from road traffic plus incinerators might also be the reason why the antimony (Sb) loading of peat in Scotland has shown evidence of a historical decrease due to the cessation of coal mining and fossil fuel use but the Sb in the peat is being now being augmented from a new source). 7. Sb intoxication & alpha lipoic acid
A Putative Central Mechanism of Autism Induction Blockage of -Lipoic Acid Dependent Redox Contorl (and also Heparan Sulfate and Nitric Oxide Signaling) by Antimony The diminustion in a-lipoic acid dependent (antioxidant) functions arising from the formation of strong Sb-alipoic acid complexes (together with those of other toxic metal e.g. Pb, Cd and Hg. Sb rather

than Hg can be argued to be prime pro-autism toxic inorganic element trigger since Sb may block lipoic acid functions more effectively than by Hg or Pb complexation. (N.b.the normal activities of this small two divalent sulphur containing carboxylated molecule include a range of tissue protective antioxidant related functions and also protective chelation of heavy metals). That such high-affinity complexes arise between a-lipoic acid and the Sb present in intoxicated human tissues was suggested from a grey literature report that employing -lipoic acid chelation for removal of Hg fromn an autistic child initially produced only an excretion of Sb-(putatively as an lipoic acid complex); only after the Sb had been removed could the Hg be removed by use of this chelating agent. [Cf . Dr. Amy Holmes web.bbbautism.com.dan_amy_holmes.htm downloaded October 4 2011]. The increase in intoxcation by Sb from dietary rather than via the traditionaly route of (coal-related) air-borne Sb intoxication (arising from the recent decadal greatly extended industrial applications of Sb, including those in food containers) might therefore act to promote autistic spectrum disorders by perturbing the -lipoic acid tissue homeostasis via the ability of Sb to deactivate -lipoic acid by the formation of highly stable and putatively inactive complexes. [Evidence that Sb rather than Hg might be the most relevant inorganic environmental toxin to further investigate for attaining insight into the etiology of autism, including that invovling functions of lipoic acid, is that Hg has been in use from about 1835 in large amounts in amalgams and therefore will also occur in human tissues (perhaps being a factor in causing MS which first became evident at the same time as the major Hg intoxciation of humans from the amalgam route ca 1835 cf. Shepherd, loc. cit.). However while there has been a gradual diminution of the amalgam use over the same recent decades which have seen a dramatic increase in the prevalence of autism (and which seems to parallel the recent increased environmental pollution which have followed from the new industrialization uses of Sb). The likely effect of Sb intoxication is that it disturbs both the direct -lipoic acid tissue protection as well as the more important indirect lipoic acid effects which include the -lipoic acid dependent glutathione biosynthesis process and also the ability of -lipoic acid to conserve the ascorbate redox system tissue protetion mechanisms. Ascorbate sufficiency seems especially to be of importance to allow for proper HS biosynthesis and signaling. The anti-cancer effect of ascorbate (studied by Linus Pauling) which was fomerly thought to mainly due the promotion of the crosslinking of collagen (and hence diminishing metastasis) by asorbate is putativley more likely to arise from an up-regulation of HS sulfation and eliminating inappropriate nitrite HS-degradation].[
{-lipoic acid has also been indicated to act as an direct antidote to Sb (tartar emetic) intoxication} -lipoic acid also diminishes DNA strnd brakage (cf. e.g. Jia et al. Mol Cell Biochem. 2009 323 (1-2) 131-8)

Autism, like a number of other diseases involving oxidative stress are, it should be noted, characterized by the occurrence of pathological tyrosine nitration, an indicator of reactive nitrogen tissue intoxication which could be a consequence of Sb diminution of -lipoic acid activity. [-lipoic acid is also believed to control the entry into the cell of cysteine (from cystine) needed for glutathione [GSH] biosynthesis, regulation of the activity of the -glutamyl cysteine synthase [GGS]). Dihydrolipoic acid was reported to regenerate greater amounts of ascorbate at a much faster thae than equivalent amounts of GSH (cf., Guo O et al.. Ascorbate-dependent recycling of the Vitamin E homologus Trolox by dihydrolipoic acid or GSH; Free Rad Biol Med 2000 29 (3-4) 368-74). Cf., Packer L., ibid., 1995 19 277-30) a-lipoic acid acts as a biological antioxidant membranes by interacting with ascorbate and GSH. Lipoic acid increases the de novo synthetis of cellular GSH by improving cystine utilization (Han D et al. Biofactors1997 6 (3) 321-8). This process enables glutamylcysteine synthase needed for GSH biosynthesis]. The chelationof Cu by dihydrolipoic acid has also been idnicated to prevent high low density lipoprotein peroxidation a process belived to promote atherosclerosis (Lodge JK et al., Free Rad Biol Med. 1998 25 (3) 287-97). This process is also abrogated by sulfated polysacharides also putativley by the binding of the redox metal to the polysaccharide ligand

-----------------------------------------------------------------------------------------------------------------------------------------------------------------------

8.

Some Previously Suggested Hypotheses of Autism

Dietary Deficiency of Selenium and Zinc

[cf., possible lack of those essential elements which are needed to combat Hg intoxication}
Cf. Infantile zinc deficiency has been associated with ASD (Yasuda) Possible Zn dietary supplementation therapy for ASD?

8.1 Causative Inducing Role of Vaccination

Mercury (Hg) A major discussion of the possible role of Hg intoxication had centered on the use of Thimerosal (ethyl Hg) as a preservative (formerly or currently) present in some vaccines. This hypothesis has been dismissed by what seems to be the vast majority researchers. However the issue is still a subject of scientific debate and no real consensus seems to have been arrived at. Although there is apparently strong evidence from numerous peer reviewed scientific papers which seem convincingly to disprove the hypothesis that autism is a consequence of intoxication by ethyl Hg, it should be noted that the toxicity of ethyl Hg is analogous to ethyl Pb for which the cessation of the addition of ethyl Pb to gasoline has become well accepted but there seems to be some resistance to a similar complete rejection of ethyl Hg in vaccines and the prohibition of the possible formation of methyl Hg generated by bacterial action from the presence of very large amounts of Hg present in dental amalgams. Hence the possible role of Hg intoxication in ASD should not be ruled out. It is apparent that Hg serves no known role in animal physiology and animal studies have confirmed that ASD-like symptoms may arise from Hg intoxication (e.g. from accidental poisoning); Hg intoxication could especially arise from the ingestion of Hg intoxicated fish, from the Hg in dental amalgams transformed by bacteria into methyl Hg and similarly from the Hg present in consumer products (e.g. the fluorescent low energy light bulbs and from batteries) or the direct olefactory brain entry route of Hg containing nanoparticulates transported intercontinentally in the atmosphere from nonfiltered particulates emitted from unregulated incineration of waste products in backyards etc. in underdeveloped countries. Some (allbeit small but apparently scientifically rigorous) epidemiological studies of ASD hair elements have indicated a characteristic increase in the Hg content of this tissue which further indicates the likely presence of Hg in the blood of ASD subjects. The presence of Hg in blood will cause it to react strongly with S-H residues containing (cysteine) in proteins as well as the anionic surfaces of glycosaminoglycans polysaccharides. (Cf. heparin (a common model of HS) is known to selectively sequester ultratrace amounts of non-physiological elements which may occur dissolved or dispersed in the bloodstream (Grant cf. Haraguchi); such elements will putatively

become enriched at the ubiquitous anionic blood vessel wall heparin-like segments in HS. It should be noted that Hg (and other toxic metal ions e.g. Al3+, Pb2+ and Sb(III); (V) and F-) can disturb major enzymic systems which are regulated by HS including those implicated in the etiology of ASDs. Studies relevant how kidney damage may arise from Hg intoxication indicated that Hg2+ can negatively perturb HSPG biosynthesis. The other toxic metal ions and F- which have also been suggested might be relevant to the etiology of ASD also negatively perturb HSPG biosynthesis. Hg can also putatively disturb how HS becomes involved as a key player in the innate immune response. 9. Diminished magnesium (Mg) and diminished calcium (Ca) and elevated aluminum (Al) Studies of hair elements in ASD subjects suggest a decrease in Mg, Ca and increase in Al. Linking the physiological alkaline earth element deficiency with an elevation of Al in ASD could indicate some etiological overlap between ASD and Alzheimers disease (AD) where a possible Al intoxication effect could be correlated with a Ca and Mg defect (cf. Kobayashi) [WF Long (who jointly with FB Williamson headed the University of Aberdeen polysaccharide research laboratory) had proposed in 1979 and backed this hypothesis up by arranging a major research effort in the following decades, that that cell surface HS glycosylation system had evolved principally to regulate calcium (Ca) ion activities (especially where these are involved in the control of cell proliferation]
9.1 Calcium Homoestasis and ASD The hypothsis that ASDs arise in part due to Ca signaling and Ca dyshomeostasis defects could indicate an underlying HS defect in ASD. Cf., e.g. HS binds Ca in a physiologically relevant manner, and can affect numerous Ca related activities e.g. cf., HS/ heparin can bind Ca and slow spermatozoa decondensation and HS can inhibit pathological calcification;

HS defect is also associated with atherosclerosis. 10. Epigenetics & Autism (This Conceptually also involves HS) A conventional epigenetic process which could tag ASD to DNA also suggest another kind of linkage between HS and ASD. Global methylation DNA tagging reveals a candidate gene for ASD subjects with sever language defects (cf. Nguyen). This gene (RORA) product may be invovled in nitric oxide biosynthesis and hence Cu-related HSPG biochemistry. The ability of DNA methylation defect to potentially be repaired by methylation inhibitors.may point the way to future targeted epigentic therapy for autism.
The etiology of ASDs may also arise from HS-related defects in Ras/Raf/ERK1/2 signaling (Yang) which can be putatively be corrected for by exogenous heparin or heparinoid administration (cf. Medeiros). This method also could promise a new type of therapy for ASD. The hypothesis that ASDs arises primarily from a number of defects in HS microstructure however requires an improved method of sequencing of HS so that this idea can putatively be fully connected and allow for monitoring of attempted therapeutic correction of HS defects. This approach might in the future offer a rigorous scientific basis for an HS-centered therapeutic strategy to combat ASD (and other diseases such as rheumatoid arthritis and some forms of cancer).

11 Chronic Retroviral Infection and ASD The hypothesis that ASDs may arise in part due to latent retroviral infection can be associated with HS deficiency. HS provides part of the natural anti-viral defense mechanism. Heparin and heparin mimetics are potent multi- pathway inhibitors of HIV-1 and a range of other viruses.

12. Role of Electromagnetic Radiation in the Etiology of ASD:

Cf. Mobile phone use has risen in tandem with increase in the prevalence of ASD.

13. Role of Environmental Input of Antimony (Sb)

--------------------------------------------------------------------------------------------

Cf. New uses of Sb have been suggested to have increased in tandem with an increase in the prevalence of ASD. However it has also been noted (e.g. Mutter J et al., Neurodedcrinol Lett 2005 26 (5) 439-46) that Hg increase has also mirrored ASDA markedly disordered metabolism of all metals has, however, been indicated to occur in autistic subjects (1-1).

cf. Footnote *b

13.1 Autistic subjects may suffer from multiple toxic metal intoxications including from lead cadmium mercury nickel arsenic lead cadmium aluminum beryllium and antimony Autistic subjects been indicated by a range of preliminary academic researches and numerous anectdotal reports to commonly suffer from multiple toxic metal intoxications which show up as elevated hair contents (e.g. of lead, aluminium and antimony and perhaps other toxic metal ions). The results however seem to indicate a considerable variation in the relative mounts of the specific elements which are augmented. (Autistic subjects, furthermore, also seem to suffer from altered levels of essential metal ions calcium and magnesium, zinc and copper and the possibly also the putatively essential element lithium and, of especial interest for groups of parents is the putative role of mercury intoxication, e.g. that arising from maternal dental amalgam and vaccination antibiotic Thimerosal).

Lead, cadmium mercury and nickel intoxication as indicated by cell culture experiments likely to account for metal induced nephropathy of renal mesangial cells (Templeton), and also the primary in vivo biosynthesis of HS proteglycans (HSPGs) in other organs and this phenomenon together and the discovery that HS is a multiinorganic element binder could further suggests that various mystery development disorders which have been suggested to arise from the contamination of the environment by these and various other kinds of pollutants do so by inducing errors in HSPG signaling (this alteration diminishes the ability of the kidney to modulate the levels of metal ions and also alters the ability of the kidney to detoxify xenobiotics).

The apparent strong binding of aluminium, antimony, lead, arsenic and mercury to HS (Grant) seems to occur by a selective sequestration process which encourages the preferential collection of these elements from biological bathing fluids and allows the selective enrichment of these otherwise ultratrace inorganic elements at HS surfaces, a phenomenon which is likely to lead to major growth factor and other HS-dependent tissue management function perturbations. This HS binding may be part of the bodys defensive mechanism against the deleterious effect of heavy metal ions. The HS-toxic metal system is a putative transporter of toxic metals into the bone. The ability of bone to hold onto these toxic metals could be related to how bisphosphonate therapy (commonly employed for osteoporosis) but which also increases the average lifespan of patients reviewing this therapy by and average of five years (cf. Center). Arsenic (As) in groundwater has also been regarded as a possible a risk factor for autism. As tends to occur together with antimony (Sb) and furthermore the toxicology of Sb and arsenic are thought to be similar. While the highly toxic nature of As is publicly well known, this understanding does not extend to Sb, so this element has been permitted to become very widely used in new technologies and thereby enter into the environment in such a manner as to likely adversely affect human health. This suggests that Sb intoxication studies from the perspective of autism research is more pertinent than e.g. mercury intoxication. The effect of multiple toxic elements bound to HS surfaces may however reinforce each other so that perhaps the toxic profile is of more fundamental relevance than the attempt to conduct toxicological studies based on studies of single toxic inorganic elements. Causative role in autism of intoxication by heavy metals and the metalloids arsenic (As) and antimony (Sb)
[Possible Link to HS vide supra)

13.2 Mercury, Lead, Antimony, Cadmium Intoxication Hypothesis of Autism Role of new environmental inputs of mercury and antimony
14 The Hypothesis that ASD is Phenomenonologically Related to Cancer

A major component of the etiology of cancer is thought to be the creation of multiple aberrant mutations of DNA sequences e.g.as a result of the effect of specific chemical substances (Hg etc. carcinogens, which include those xenobiotics which also have been implicated in the etiology of ASD). A conceptually related hypothesis (which is not well known by the general public) is that cancer arises form mutations in HS anionic patterns analogous to how cancer is thought to arise from some mutations in DNA. (Such HS damage can logically be demonstrated in vitro to arise from the effect of specific toxic chemical substances e.g. on the primary HS biosynthetic machinery or in the post-synthetic HS modification processes; these defects can also be promoted by dietary insufficiencies which promote redox status disturbances leading to oxidative and nitrosative stress) A variety of defects in HS signaling could promote the growth of tumor cells but their ability to secrete the mammalian heparanase (which correlates with the disease severity) allows

the degradation of extracelluar matrices and thus spread of tumor cells to distant sites. Such metastasis processes are widely acknowledged to be the ultimate reason for cancer mortality. It seems possible that analogous processes are also of critical relevance to the etiology of ASDs. A further cancer research interest is angiogenesis, the process of the stimulation of the growth of new blood vessels by which tumors acquire a blood system nutrient supply. This process includes an important input from vascular endothelial growth factor (VEGF); the inhibition of this activity is thought to be useful method of providing anti-cancer therapy. VEGF is believed to submit to HS control. A conceptually equivalent process by which HS error putatively similarly contributes to the etiology of ASD might similarly involve the enzyme which cuts HS chains, heparanase which is thought (by its presence at the nucleus, cf. Chen et al. loc. cit.) to inhibit the expression of VEGF, the increased presence of which is believed to be an etiological feature of ASD. [Observations of decreased VEGF in blood serum could indicated that this molecule contributes to the symptoms of the most severe type of ASD]

--------------------------------------------------------------------------------

15. Calcium Oxalate Stone Formation The ability of HS to prevent calcium oxalate crystal formation is now suggested to have also become compromised by a further systemic defective in HS
Cf., Shaw, William Internet search term cf file entitiled OXALATES CONTROL IS A MAJOR NEW FACTOR IN AUTISM THERAPY, accessed September 9 2012.

Heparan sulfate (HS)/oxalate Defective HS can arise from an altered redox biochemical balance which feeds into nitric oxide signaling which can be perturbed by oxalate
The increase in autism seems to have been accompanied by an increase in (an often associated) childhood urinary stone formation which includes that associated with an increased amount of oxalate in the urine (cf. Shaw) While this is usually thought to be associated e.g. with infestations of the gut and other organs by Candida species (perhaps induced by the environmental presence of ultratrace amounts of antibiotics which has caused the gut to change its microbial population, an action which seems to

be aided by the presence of trace amounts of lead, antimony, mercury and steroids in foods especially that from more polluted parts of the sea). The increased oxalateinduced formation of highly pro-inflammatory Ca oxalate crystals (in kidney as well as in other organs) is believed to be prevented, under normal healthy kidney functioning conditions, by an increased synthesis of specifically microstructured HS polysaccharides which can effectively block the seeding process needed to allow the growth of the Ca oxalate crystal stones beyond the less harmful nidus stage (cf. Borges). The inflammatory effect of Ca oxalate will only occur under normal dietary conditions, it might be argued, if this key HS anti-oxalate crystal formation inhibitory system becomes defective. This could especially arise e.g. from a deficiency of glucosamine N-sulfonate microstructure in the HS (cf. Grant-1). Such structures seem to be subject to non-enzymic redox metal or H+ catalyzed de-sulfonation. This might occur if metal ion homeostasis becomes seriously disturbed. Perhaps the formation of Ca oxalate stones in autistic subjects arises at least in part from defects in the HS feedback assembly process designed to halt the formation of life-threatening pathological crystallizations.
16. OXIDIZED LIPIDS EXCESS GLUCOSE HOMOCYSTEINE OMEGA-3 FATTY ACIDS RETNIOC ACID ASCORBATE

The above argument supports the idea that the primary cause of autism might be a defective HS proteoglycan assembly and postsynthetic scission disease process similar to the alteration of HS biosynthesis perturbation indicated to occur from cell culture experimentation models of how the presence of heavy metals can alter the microstructure of HS. It should be noted that while toxic heavy metals seem to damage HS (an analogous effect arises from the presence in cell cultures of oxidized lipids, excess glucose and homocysteine but the reverse is true of a range of beneficial dietary components, e.g. w-3 fatty acids, retinoic acid, ascorbate which in agreement with the HS-microstructure driven nature of the etiology of autism, also have been indicated when administered as dietary supplementation to be of benefit to autistic subjects.
17. CHOLESTEROL LIPID RAFTS

A further aspect of HS signalling which could impinge on the aetiology of autism is the key role of cholesterol in forming the lipid rafts which are required for e.g. for fibroblast growth factor 2 HSPG-dependent signaling [cf. Cho et al.]) and also perhaps part of how FGF 4 interaction with glucosamine N-sulphonate motifs in HS on which is especially dependent embryonic stem

cell differentiation depends could point to future therapeutic intervention strategies for various developmental diseases (cf. Lanner) which may include autism. 18. Cytokines Further evidence for the involvement of HS biochemistry in autism is the circumstance that the specific cytokine immune signaling defects which are believed to be at least partly controlled by HS (and need correct HS-microstructures to accomplish this) are also those which have been indicated to be specifically altered in autistic subjects (cf. Goines and related listed refs) 19. GLUCOSAMINE
As well as redox normality (and sufficiency of ascorbate), the non-redox metal ion Mg is probably essentially required for the proper biosynthesis of HS; also required is the presence of adequate inorganic sulfur (which can be made available in vivo from the amino acid cysteine). The apparently highly approved-by -the-public (but perhaps less so by some health care providers) for the alleviation of the symptoms of some forms of arthritis (especially osteoarthritis), the success of the over-the-counter provision of glucosamine sulfate as a dietary supplement most likely derives its numerous prohealth benefits from the circumstance that HS is an alternating copolymer composed of uronic acids (iduronic acid and glucuronic acid) and glucosamine sulfate.
This structure in HS evidently evolved from a bacterial polyanionic cell surface evolutionary precursor which lacks the glucosamine units (cf. the bacterial polysaccharide-like polysaccharide heparanosome is the starting substance which present day animals use to start off their HS biosynthetic process in the Golgi apparatus). Glucosamine confers additional reactivity absent from the earlier models of cell surface polyanionic polysaccharides (e.g. for metal ion uptake) since it can be rapidly cleaved by nitrite under in response to pathogen insult. Under chronic conditions which mimic this kind of insult, however, tissue protection and other function of HS become so seriously diminished as to create complex down-line pathologies. {This seems to be the case with cardiovascular dysfunction and rheumatic disease but it is now suggested that autism may also be another related disease which is caused by altered nitrosative cycling of HS). The glucosamines in HS are mostly N-sulfonated which stabilizes them against nitrosative scission, but when this sulfonate is removed (a process catalyzed by the presence iron and copper ions and perhaps other non-physiological redox elements {perhaps antimony?} or rather non-physiological low pH values) then the kind of chronic nitrosative stress which characterises autistic spectrum diseases is predicted to unzip HS thereby abolishing its tissue protective properties. The first stage of this unzipping the partial removal of N-SO3- side groups from glucosamine sulfate HS also sufficiently diminishes the ability of HS to perform its normal functions. An example of this is the ability to prevent plaque formation modelled in vitro by CaCO3 or Ca oxalate crystallisation. HS is very effective at preventing this but de-N-SO3-d HS is either inactive for or it perhaps also could augment plaque formation This sort of behaviour can impair blood capillary function in the brain and in the gut.

20. Heparan status and fear response Secretin

Further Evidence for HS Biochemical Alteration in Autism An effect found in the rat of HS-oligosaccharides on brain functions might also benefit human autism spectrum disorder patients. HS-derived oligosaccharides have been reported (Lorens et al.) to abolish the brain amygdala-associated fear response (in rats). That this is relevant to a fuller understanding of autism is indicated because a similar response to hostile faces (in 12 autistic humans compared with control subject studied by MRI) apparently identified changes in the secretin - active brain region which is implicated in autism (Yurgelin-Todd). (N.b. secretin [a 27 amino acid peptide] therapy is known to benefit some autistic subjects).
SECRETIN

Autism might then, it might be conjectured, also be potentially corrected for (as regards the secretin mechanism pathway) by use of an appropriate oligo-HS therapy either by direct administration or by some dietary supplementation which indirectly produces the required oligo-HS in vivo at the secretin-sensitive brain region. Such oligo-HSs can, it should be noted, arise naturally either by scission by hydrated electrons, by nitrite or by heparanase. There is also a possible metal ion effect mediated via metalloproteinase activity in the generation of such oligo-HS. It should be further noted however that a conceptually similar oligo-HS mimetic (pentosan polysulfate SP54) when directly infused into the v-Creutzfeld Jacob disease [v-CJD] (mad cow disease attributed to misfolded prion pathogens) in animal models (cf. Dringer, results which later confirmed in human patients) seems also to be capable of reversing the neurological damage caused by misfolded prions and to extend the life in human victims of v-CJD (cf. Highfield). 21. FUTURE POLYSACCHARIDE CHELATION The role of heavy metals in the promotion of autism as outlined in the preceding paragraphs might similary eventually be treatable by a polysaccharide-based metal chelation system

(analogous to use of EDTA). The use of an EDTA-like HS-based chelation therapy is conceptually possible. 22.CHLORINATED PHENOLS
Autism has also been tentatively linked directly with the likely environmental presence of chlorinated aromatic pesticides (cf. e.g., Roberts et al.) which can metabolize in vivo to similar range of chlorinated phenols to those produced by OCs (e.g. HCB). Studies of the amount of HCB in maternal blood by Smink, confirmed several earlier hints that had linked the amount of HCB in the maternal blood to childhood obesity. HCB intoxication has also been associated with adult obesity ( Moreno) and a variety of ongoing health defect problems have continued even 40 years after a large scale accidental human population intoxication incident (Jarrels). 23.Phthalate It should also, however, be noted that not-halogenated, endocrine system disrupting environmental pollutants have also been specifically linked with the prevalence of specific diseases (cf. Bornehag et al. reported that contamination of the domestic environment by benzyl phthalate can be epidemiologically associated with rhinitis and eczema and di(2-ethylhexyl) phthalate with asthma). 24.Synergystic metalloid organohalogen interactions 24.SYNERGYSTIC METALLOID ORGANOHALOGEN INTERACTIONS Additionally, synergistic interactions between antimony and organochlorines also widely distributed in the environment and in food might also be of major relevance to a fuller understanding of how individual mystery illnesses might arise. Of interest here is how e.g. chlorinated phenols might become more toxic in the presence of heavy metals and toxic metalloids. The metals which occur together with silver in ores (lead and antimony) are therefore likely to be present in ultra-trace amounts in the environment but which are no doubt without health risk unless they become subject to bacterial methylation. This scenario also affect the possible release of toxic metals into human tissues from dental amalgams (however the bio-markers for such elements (urine, blood and hair) are considered not to be reliable indicators of tissue loadings (cf. WHO). The perturbation (e.g. by fungal gut infestation) of the metal ion detoxification system in autism may be an important etiological situation in this and related diseases (cf. e.g. Lane) which includes important input from glutathione (GSH) and metallothioneine which normally function together with such ligands as ceruloplasmin (Cu binding) and ferritin and transferrin (Fe binding). Heavy metal and xenobiotic (e.g. hexachlorobenzene [HCB}intoxication has also been associated with altered glutamyl-cysteinyl-glycine (GGT) (a key regulator of GSH) homostasis, [It is of interest in this context that GGT levels are associated both with heavy metal intoxication and HCB intoxication]. The actual working surfaces which are most susceptible to toxic metal perturbation of tissue homeostasis might, however, be argued to be the HS information system which is now becoming to be recognized to be dependent on correct metal ion availability [e.g. for Ca2+, Zn2+ Cu(II)] to enable correct biochemical function. This indicates that knowledge of the multi-inorganic element content of this polyanionic polysaccharide is

the most relevant index of inorganic (including heavy metal) intoxication. This area of research, however, has scarcely begun. It might nevertheless be surmised from the frequency of reports on the internet of toxic inorganic element presence in the hair of autistic subjects that there may be a specific HS-linked Sb intoxication induction of autism (with etiological overlap of types of autism caused by developmental and later intoxication by toxic inorganic elements). It might further be suggested that the strong binding of toxic elements to HS the major HS polyanion which regulates embryo development causes an alteration of the information transmission ability of this system. This accords with the preliminary observation (made by the author) that those ultratrace inorganic elements which occur in biological fluids (and e.g. in sea water) can become highly concentrated at HS surfaces. An indication of the ability of HS polyanion ligands to bind heavy metals and related elements is obtained by the study of the natural multi-element contents of these and also other natural polyanions. This seems to suggest that Sb interacts especially strongly with such polyanions and therefore if present at all in the natural environment will tend to become bio-concentrated at the surfaces of such key system management ligands.
This means that if there is an anthropogenic augmentation in the maternal and fetal blood of these toxic elements (e.g, due to their widespread use in brake pads, flame retardants or catalyst residues present in microwave ovens food containers) then the effect of this intoxication acting synergistically with HCB and related xenobiotics can be predicted to produce an alteration of the development of the fetal tissues including the brain).

A similar interaction between the biochemistry of toxic metals, HS and HCB seems to be involved in the etiology of unexplained obesity (Grant 2009). The most relevant perchlorocarbon inducer of unexplained obesity, may be hexachlorobenzene (HCB) a-1 a substance which has previously been indicated to participate in the etiology of childhood obesity is now hypothesized also to be a key cofactor of the induction of autism by heavy metals (principally Sb). [HCB is, it should be noted, is well established to be common man-made pollutant in urban air (cf. e.g. Zhang) (and also now more generally distributed widely in the planetary atmosphere and ecosystem; HCB is also an indicator of the co-presence of smaller amounts of the more toxic dioxins)]. 25.BRAKE FRICTION SURFACES That Sb (the sulfide is now introduced into the environment as the principal automotive brake pad ingredient) should be included in the autism inducing environmental pollutant cocktail is now suggested by the report (Volk) that increased autism can be epidemiologically associated with maternal residence near a high volume traffic route. [It should be noted that HCB and smaller amounts of more toxic related dioxin and chlorinated biphenyl molecules can also be synthesized de novo during combustion in automobile engines and during the incineration of chlorinated organic molecules which may be present in cigarette tobacco. Also the incineration of chlorinated organic molecules in domestic, municipal or industrial waste. The presence of catalytic amounts of Sb and other inorganic elements (e.g. present in fly ash) however enables HCB and associated dioxins to be formed more readily under the above circumstances. It is suggested that carbon particulate adsorbed HCB plus Sb may be involved in the mechanism of transfer into the victim of the strong pro-autistic stimulus adjacent to freeways].

Sb may also be introduced from sue of Sb2O3 as a fire retardant and the Sb catalyst residues which can be removed on heating the plastics containing them (e.g. during their use as trays for microwave cooking (cf. Haldimann). 26.FRIEDEL KRAFTS Being able to function as a cofactor in Ziegler Natta (where perchlorocarbon additives can cause redox recycling of metal polymer growth sites cf. Grant et al 1979 ) or Friedel Krafts catalyst when coordinated to an aromatic ligand may be a useful indicator of which of the inorganic elements are possible autism spectrum diseases promoters via synergistic actions with endocrine disruption substances which inter alia, can as suggested by animal experiments to likely subtly affect fetal and child development, especially that of the brain. 27.TYROSINE NITRATION While the possible neurobiological basis of autism is commonly stated to remain poorly understood, perhaps the positive correlation with an increased tyrosine nitration, decreased serum adiponectin (also putatively linked to the presence of the pollutant bisophenol A) decreased serum glutathione (related to a pro-oxidant redox status) and (at least in some subtypes of autistic subjects) an increased urinary content of (probably tryptophan gut bacteria or fungal derived) indoylacryloglycine (Shattock and Whitely), and a spectrum of porphyins (porphyria) following perturbation of heme biosynthesis (e.g. by heavy metals [this perturbation has also been associated with Sb intoxication (cf .Vali). This adds weight to the hypothesis that Sb could act in concert with HCB perhaps the most pertinent environmentally present endocrine disrupting intoxicant which now occurs in all humans worldwide and which is thought to dose-dependently alter brain development in such a manner as to promote childhood obesity {which has further been correlated with autism and a range of other disorders. It should be noted that the disturbance of heme biosynthesis is a hallmark of autism but to a lesser extent in Aspergers syndrome (cf. Nataf) a milder form of autism (Geir et al.) While these authors suggested that their results were consistent with the known ability of Hg intoxication to produce the observed disruption of heme biosynthesis which causes porphyria it is now indicated that the similar effects of HCB + Sb (and other toxic metal) disturbance provides an equally or more credible explanations of the numerous indications that heavy metal intoxication is an ultimate cause of autism.

28.AUBREY VAN WAZER REACTION

All perchlorocarbons (e.g. tetrachloroethylene used as a dry cleaning solvent (e.g. Grant 1974)) and mixtures of hydrocarbons with elemental chlorine (and numerous other substance as carbon sources cf. e.g., when heated in an oven in sealed tubes are converted into HCB (plus carbon tetrachloride hexachloroethane and HCB). The chlorinated organic molecule rearrangement process is catalyzed by a range of inorganic substances (most notably by salts of Fe and Cu, (Hg was relatively inactive); {since Fe and Sb are used industrially for the related catalysis of the industrial manufacture of chlorobenzene and related substances it seems likely that Sb will also

efficiently catalyze HCB formation during incineration of organic wastes). This kind of catalyzed molecular rearrangement process evidently occurs ubiquitously during backyard trash burning or in municipal or medical incinerators, crematoria and heavy industry (especially metallurgical processing) sites. These facilities all produce HCB (together with PCBs and dioxin congeners). A large additional source of HCB is internal combustion engines in which the ubiquity under high traffic conditions of Sb loaded dust [containing Sb2O3 as well as (CH3)3Sb-ciontaining particles which apparently show consistent Sb/Cu ratios (this ratio is apparently determined mainly from the use of Cu plus Sb2S3 in friction brake pads) near freeways associated with children showing a greater prevalence of autism (cf. Volk et al.). Road traffic is also associated with particles containing e.g. Ba, Ca, Ce, Cu, Fe, La, Mo, Mn, Pb as well as Sb (cf. Hueglin) + Fe, Ni, Cr) containing particles which also typically contain inorganic elements. The amounts of the above listed inorganic elements present in the head hair of a cohort of (normal) schoolboys has been correlated with the amounts of these elements present animal mast cell heparin (a heparan sulfate-like substance) (cf. Grant 2000) points to how elevation of the amounts of some of these elements might perturb the developmental processes in the brains and other organs of human fetuses (e.g. by perturbing fibroblast growth factor receptor (FGFR) assembly, a HS-dependent process which is putatively of relevance to the role of Ba intoxication in the etiology of multiple sclerosis (Purdey)}. FGFR activities may be implicated, it should be noted, in the etiology of autism via dysfunctions of the klotho system (John).

29. ME/CFS
Myalgic Encephalomyelitis Chronic Fatigue Syndrome 29-1 (cf. Purdey Cu/Zn Mn3+ electron phonon PrPCcoupling ) Mark Purdeys ideas may be relevant to how altered environmental insults (including organophosphates Mn overoad and Cu deficiency coupled with infrasound) has possibly contriubted to the generation a range of diseases e.g.\prion idseases, and chronic fatigue syndrome (Purdey) The putative role of H2S in ME/CFS (cf. Lemle) might suggest that SbH3 or other compounds containing Sb-H might also have relevance to the etiology of autism (where the presence of H2 in exhaled breath seems to be a possible diagnostic marker).

The symptoms of the autisitc spectrum disorder ME/CFS can be alleviated by the administration of heparin (the end-member of the HS family of glycosaminoglycans). It should be noted that heparin may bind Sb with an exceptionally high affinity (authors personal research notes from Marischal College Aberdeen). It should be noted that the administration of heparin (perhaps partially because it can act like an EDTA chelator) benefited at least in a significant proportion (e.g. about a quarter as indicated by Wright (personal communication) of ME/CFS patients. The ability of heparin to dramatically improve the symptoms of ME/CFS is an idea which

was reported and publicized by S. Berg who believed that a cohort of ME/CFS patients could be classified seaprately because they were suffering from blood hypercoagulation (i.e. a (Hughes?) syndrome). The Dundee ME/CFS group (Vance Spence et al. with Prof Belch [Dundee University]), who had established that that separate etiologies could be identified for ME/CFS-type illnesses on the basis of acetyl choline stimulation of peripheral vascular function , however disagreed with the Berg hypercoagulability hypothesis of the origin of ME/CFS since the Dundee group apparently had found no evidence of hypercoagulability in their studied ME/CFS GFS and OPP subgroup subjects.. Perhaps the ability of heparin to bind and de-activate numerous types of potentially toxic particles substance (e.g. HCB) and toxic inorganic ions (e.g. the ability of heparin to bind and deactivate such ions (e.g. Al3+ and unliganded Cu2+), rather than the anticoagulant properties might however have been the origin of the anti-ME/CFS effect of heparin. 29.1 Purdey

8. Autoimmune Hypothesis of Autism

30. IAG
The Discovery by the Sunderland Group (Shattock) of the Increased Urinary Excretion of Indoyl-3acryloylglycine in Autistic Patients. Could this be Related to The Discovery by Claude Rimmington of the Induction of Porphyria by HCB? (Cf., copropoprphryin levels have been reported to be elevated in children with autistic disorders [R Nataf et al. (Porphyrinuria in childhood autistic disorders: Implication for environmental toxicology) Toxicol Appl Pharmacol 2006 214 (2) 99-108, cf , also DA Geier and MR Geier Neurotoxicity Res 10 (1) 57-63) It should be noted that the unique indoyl-3-acryloylglycine marker of autism (cf. G Bull et al. Med Sci Monit 2003 9 (10) cr422-5) could also be a possible indicator of role of some central role played HCB intoxication in the etiology of autism While the biochemical origin of this indoyl derivative as a consistent urinary marker of autism is, however according to the published literature on this subject, currently considered to be obscure, the consistent formation of such a marker seems most likely to arise following a disturbance (e.g. by chlorinated environmental pollutants) of enzymes involved in indoyl derivative biosynthesis or metabolism, most probably of trypophan (analogously to how chlorinated aromatic substances induce porpyria by disrupting the heme biosynthesis pathway, HCB being used to induce porphyria by this mechanism in experimental animals d). Indoyl-3-acryloylglycine may possibly arise, it can now be rationally suggested as an effect of disturbance of the proteolysis of neuropeptides containing terminal tryptophan-glycine (e.g. the cephalomyotropins, cf .,GM Holman et al., Compar Biochem Physiol Pt C Compar Physiol 1986 84 (2) 271-76). The biochemical relatedness of the essential amino acid (needed to be present in diet) tyrptophan to serotonin (5-hydoxytryptophan) and melatonin seem pertinent. Cf. many fibromyalgia patients are thought to have low serotonin levels.

Tryptophan (e.g. from dark chocolate) might, if tryptophan biochemistry is defective in autism and other autistic spectrum disorders be useful dietary supplements for the alleviation of the symptoms of these diseases.

31. Nitration of tyrosine and tryptophan

Whereas the nitration of tyrosine as a disease marker is well established (Cf Ishipourous) the nitration of tryptophan may also have relevance to pathologies [cf. H Kawasaki et al.]. (Nitration of tryptophan in riobsomal proteins is a novel post-translational modification of differentiated and nave PC12 cells) Nitric Oxide 2100 e-Pub ahead of print. Another role of tryptophan and tyrosine could be as (a part of an essential) anti-nitrant protection mechanism. The key to understanding autistic spectrum disorders could come dwon most simply to this. An providing relief from the symptoms might most simply be viewed as by achieving the upregulation of this protection including by the pharmaceutical use of suitable anti-nitrant agentsg.

The nitration of tyrosine is a hallmark of numerous diseases the etiologies of which have been at least to some extent thought to arise via auto-immune processes involving the over-stimulation of the cellular immune response and it is of interest that a study of tyrosine nitration in the hair and nails has been correlated with the degree of severity of autism in children (Lakshimi). HCB intoxication is also known to alter tryptophan biochemistry and HCB seems to decrease the production of serotonin in the small gut (Llamban et al., cf. Shattock). This may be of relevance to the HCB led etiology of autism.

32. ALUMINUM
Putative Role of Al3+ and Aluminum Hydroxide (and related substances e.g. Alum vaccine advujant) in the induction of Autisitic Spectrum Disorders

Al3+ ions have been reported to potently inhibit the activity of superoxide dismutase (Shainken-Kestenbaum). Aluminum intoxication will therefore be expected to be proinflammatory in nature. The ingestion my macrophages of foreign substance such as the aluminum hydroxide used as a vaccine adjuvant has been found to cause macrophage myofascitis which generate inflammation via switching the Th1 to Th2 response (n.b. this switch in the cellular immune system response is a characteristic of autistic spectrum disorders such as ME/CFS) [Cf. RH Gherardi et al. Brain 2001 124 1821-31 (Macrophage myofascitus lesions arises long term perspective of vaccine-derived aluminium hydroxide in muscle) {cf also P Chrin and J Authier (Macrophage myofascites) Orphanet Encyclopedia. Aug 2001; cf .also RL Blaylock (Vaccines, neurodevelopment and autism spectrum disorders) web.whale/vaccine/blalock1.html}.

Aluminum hydroxide strongly binds to heparin and (putatively) also to heparan sulfate thus abrogating signaling mediated by these polyanions (D.Grant et al University of Aberdeen research results). 33 CALCIUM CARBONATE CRYSTALLIZATION A similar binding may pacify exogenous particulates including aluminum oxide hydroxide in a similar manner to the inhibition of seeded crystallization of CaCO3

which is achievable by the surface adsorption of heparin/heparan sulfate onto active crystal growth sites thus completely stopping this particle formation process (cf. D. Grant et al., Biochem J 1997 cf. also Med Hypoth. 1992 38 49-55) This property is lost following d-N sulfonation of these polyanions (this type of structural modification can, it is believed, arise as a result of acidosis or the binding of copper or iron ions to these polymers {this suggests a possible mechanism by which iron overload could abrogate the ability of heparin-like polymers to protect against inflammation triggered by proinflammatory particulates). [There seems to be a possible similar mechanism of the promotion of plaque formation in Alzheimers disease, cf. Brunisma et al; the suggestion of a role of copper or iron overload in the promotion of Alzheimers disease (e.g. by copper in tap water) may conceivably be related to the ability of excess free copper to produce de-N sulfonated heparan sulfate which lacks the ability of the fully N-sulfonated polysaccharide to prevent plaque formation]. It should be noted that HS biosynthesis seems to be altered (perhaps in a designed servo feedback manner) in an attempt by the kidney to prevent the crystallization of Ca oxalate stones (cf., FT Borges et al., Kidney Int. 2005 68 1630-42). The presence of excess oxalate seems to occur at least in some kinds of autism. A role of perturbation of heparan sulfate signaling and tissue protection related to the engulfment of pro-inflammatory iron containing particulates in macrophages was advance by F.B. Williamson (a former academic researcher and sufferer from a CFSlike illness who in 2002-3 conducted a series of discussions with the author and Vance Spence on this topic). Related phenomena of particulate ingestion with altered cytokine signaling is thought to be the basis of silicosis and it should be noted also that a traditional mechanism of atherosclerosis postulated the ingestion of oxidized LDL into macrophages to form foam cells.

34 Possible relatedness of ASD and Alzheimers disease (AD)

It can be deduced from published studies that ASDs resembles Alzheimers disease and ASD diseases may occur via a HS biochemical defect (involving aparent xenobiotic induced a redox overload which for Alzheimers disease is perhaps an Al/Si defect related etiology but ASD is a halogenated xenobiotoic plus heavy metal xenobiotic related aetiology). The role of the extracellular matrix in regulating tissue construction and repair includes a critical role played by the complex heparan sulfate system manager which putatively operates under a system of nitric oxide (copper/ascorbate) linked servo control singnaling which also putatively engages in cross talk with the metallome so as to create a complex system of protein activity regulation which includes growth factor regulation and which is ultimately determined by the system of fuzzy information processing afforded by the polyanionic HS side chains of HS proteglycans (the cell membrane associated syndecans and glypicans (phosphatidyl inositol membrane linked) and extracellular agrin, perlecan, collagen XVIII etc. This system and processes enabled by it can be considered to be what to a major extent defines epigenetics. This is how extra-genetic information systems control the interaction of the organism with the environment and which normally allows for evolution to occur in order to accommodate to environmental changes and insults but where the latter stimulae overwhelm this system this leads to major pathologies. An example of this is now

suggested to include the autistic spectrum disorders. These seem putatively to be classifiable according to the elevation of serum copper which shows up as elevated hair copper which apparently is elevated in direct proportion to autism severity in a manner almost exactly reminiscent of rhematoid and other arthritic conditions (Priva). It is likely that similar considerations also apply to schizophrenia and cancer but this of course is a highly controversial idea. The published information on this idea as regards ASD is much less than is the possible linkage between HS biochemistry and amyloid-beta (a peptide formed by proteolysis from the beta-amyloid precursor protein) which is thought to be a critical etiological factor in Alzheimers disease. The levels of the Alzheimer beta amyloid protein has also been found to be correlated with the severity of autism but this disease was indicated (Sokol) to be associated with an increased alpha-secretase pathway (anabolic) the opposite of what is seen in Alzheimers disease. Autism is putatively associated with an increased secretion of amyloid precursor
protein-alpha. Autism has also been associated with an elevation of nitrosative stress. Amyloid precursor protein has been reported to modulate the Cu/Zn NO catalyzed degradation of HS (Cappai).

The amyloid (putatively protective) system and it interaction and perhaps control by HS PGs which seems to be involved in an as-yet-not-fully-understood manner in the etiology of Alzheimers disease could also define autism since some autistic children have a high level of secreted -amyloid precursor protein and since this seems to be correlated with the severity the autistic condition (Ray) putatively could provide a valuable diagnostic marker. Amyloid precursor protein APP processing and amyloid-beta production has been reported (Hoe) to be regulated by Reelin (a large glycoprotein the putative possible HS binding region which does not seem to bind to heparin but which has been indicated to control brain layer buildup during development and defects in this may give rise to autism). The nitric oxide determined scission of HS may be how Reelin generates nitric oxide which established HS signaling profiles during neurological development and function. Similar Reelinrelated activity defects has been indicated to be related both to schizopherenia as well as autism.

Perhaps these are related to HS sequence metal ion (especaily copper/zinc) related defects in these and other neurological diseases; (cf. Cheng et al.) The HS-side chains of syndecan-1 HSPG has been associated with the formation of amyloid plaques (Watanabe). (This is now thought more likely to be a protective mechanism than a toxic effect as was originally believed). An alteration of HS sulfation may be required to trigger this protection system and to prevent the formation of amyloid plaques. (N.b. these were originally thought to be toxic but are now thought by some investigators to be part of a wide-ranging protective mechanism (e.g. to inter alia protect synapses against damage mediated by soluble amyloid-beta oligomers the formation of which is now believed to cause Alzheimers disease). The HS system (putatively also again in conjunction with copper/zinc) is also believed to control the fate and pathology of prions and protect against misfolded prion instigated disease (which is also insoluble fibril related). An HS-oligomer and heparin mimetic pentosan polysulfate (PPS a post extraction sulfated beech wood xylan) has been demonstrated to provide therapeutic benefit against the neurological degradation processes thought to occur in prion diseases, a phenomenon discovered in other mammals, but also found to be applicable for treatment of the nv-Creutzfeldt Jacob disease (mad cow) disease in humans. HS-related therapeutic agents putativley also have been indicated to possibly have similar benefits for Alzheimers disease therapy. This could suggest a possible future extension of this paradigm also to ASD. In the context of the possible modus operandi of PPS in the amelioration of the symptoms of neurological dysfunctions, it has been reported that PPS is highly effective (likely it acts as an HS-oligmer surrogate) as an activator switch mechanism control operator for ADAM12 (a mechanism which may also apply to other members of the ADAM family; n.b. these metalloproteinases are thought to play key sheddase roles in the regulation of amyloid protein precursor protein which upon secretase cleavage generate the amyloid peptide fragments which have been implicated in the etiologies of Alzheimers and autism spectrum diseases).

35
Possible Effect of Plant Inorganic Nutrient Depletion of Soils The switchover from coal to oil and natural gas for electrical power generation has diminished the supply of inorganic sulphate to the soil which could diminish the necessary inorganic sulphate is required for HS sulfation in the Golgi apparatus. The putatively beneficial effect on the food chain might have been due to inorganic byproducts of fossil fuel combustion which can act as plant nutrients and which have been altered by the change from coal to clean coal and oil which has altered the supply of such nutrients to the food chain. The reduction in the delivery of sulfur to the soil may be the prime difference between the traditional use of coal as an energy source and the modern systems of energy provision.
Cf Latitudinal Variation of the Prevalence of Multiple Sclerosis and Perhapsalso Autism This seems to be explicable in terms of deficiency of in vivo transport of inorganic sulfate (which is depenent on Vit D which is further dependent on sunlight UV). This could also be affected by the sulfur cycle alteration following the decrease in acid rain (attributable to suphuric acid produced by the changes introduced in the methods of using coal as a prime energy source). This alteration might also have, counter-intuitively, been at least part of the cause of the increased presence of mercury in the tissues of marine animals (cf. 35.1).

35.1 The reduction in delivery of sulfur (and sulfide production in natural waters) can also augment the human intoxication by methyl mercury
Cf. Graham et al., and Hongve et al.

36. Dark Chocolate

G. Kennedy et al. (Easter eggs are good for you. Dark chocolate inhibits collagenstimulated platelet aggregation) reduction in platelet adhesion as well as to diminish the NOS biosynthesis of NO. This file also reported that another research group C Keen, U. Cal., Davies) had also found evidence that dark chocolate could also suppress ADPstimulated platelet adhesion This could suggest that ingestion of dark chocolate might also be a useful method of combating those autistic spectrum disorders which have been associated with vascular wall disturbance, hypercoagulation and/or inappropriate excessive secretion into the blood circulation of NO (e.g. as a consequence of chronic over-expression of iNOS due to presence of xenobiotics). And also mentioned in the Edition42:04 file was a study which had indicated that in a rabbits on a cholesterol-rich diet further intoxicated with copper from tap water developed brain lesions reminiscent of Alzheimers disease. Could dark chocolate also be of benefit for the alleviation of the symptoms of this disease?

37 Water activity

The HS signaling system depends on specific extracellular ultraanionic aminosulfate and sulfate half ester anionic group sequences which can act as antennae for the Haraguchi distribution of seawater multi-elements in extracellular spaces. This may have major relevance for the attainment and control of water activity (ultimately the microstructure of liquid water which can be fine -tuned by the presence of inorganic ions). There is an increasing awareness that the presence of inorganic cofactors is an essential part of the HS signaling process (which seems in contrast starkly to that used by DNA which is an exact one to involve a more fuzzy logic system). This could be why different toxic metal cocktails can apparently produce similar overall intoxication effects on the HS system. Although inorganic Si as a dietary component is a poorly researched subject the circumstance that inorganic Si is ubiquitously associated with HS is likely to be part of the reason why Si is an essential nutrient. [Deficiency of dietary Si could increase the toxicity of aluminium (Birchall) and might be a risk factor for autism]. Another often overlooked critical heath problem is the formation in tissues of sharp crystal surfaces. The importance of the need for animal organisms to prevent the crystallization in tissues of sparingly soluble phases including calcium carbonate, phosphate and oxalate is a very basic one for biological viability. In circumstances where crystals (e.g. of calcium oxalate) are allowed to form in animal tissues, a chronic pro-inflammatory oxidative damage inevitably follows (this arising from the aberrant stimulation of the immune system). 38 Gallstones The apparent change in the chemical composition of gallstones over the past decades (cf. e.g., Schafmayer) has reflected the change in the environmental air and food pollution and the changed use of antibiotics and steroidal hormones etc. which have increased in the environment, a circumstance which has, over the recent decades, paralleled the dramatic increase in the

prevalence of obesity as well as autistic spectrum disorders, asthma, type-2 diabetes and other mystery illnesses).
[HS biochemistry also impacts on the biochemistry of gall bladder secretions and defects in this could contribute to the symptoms of autism (and how these relate to cholesterol dyshomeostasis). Heparin (a HS family member) appeared to aid the dissolution of gallstones by bile acids (cf. Furnival et al.). A HS dependent system The aging-process-related-protein Klotho appears to have a central role in bile acid homeo stasis (Moschetta)].
39. Gut Defect A defect in the gut has been suggested to be an essential part of the etiology of autism. The very wide range of HS- dependent physiological and pathological situations include the integrity of the gut wall. Since the HS related pharmaceutical heparin has been found to improve leaky gut behavior is seems conceivable that an appropriate HS oligosaccharide therapy might promise the future development of some effective HS-based gut leak healing strategy which might partly combat the symptoms of autism.

40 APPENDIX
A1 Why are anthropogenic substances not easily detoxified? WHY ARE ANTHROPOGENIC SUBSTANCES ARE NOT EASILY DETOXIFIED It is evident that HCB and the other pollutant OCs would have been absent at current concentrations in the environments where the multi-cellular animals first evolved and therefore against which animal species probably now still lack any evolved appropriately specific detoxification mechanisms. This seems to allow numerous overtly anthropogenic substances even at very low concentrations to potentially cause multiple major pathological effects. These evidently include the developmental disturbances in the fetus and ongoing metabolic and nervous system disturbance in adults which seem to be the origins of autism and autistic spectrum disorders.
[The alternative view is that the increase in the prevalence of these disorders is entirely due to an increased interest in conducting extensive diagnostic tests seems not to agree with the majority of expert opinion (cf. Grant {ECG}]

A2
Possible Unique Role of Antimony to HCB etc Environmental Augmentation

The augmentation of HCB and other scrambled products is partly an outcome of inorganic input into trash since chlorocarbon scrambling is subject to positive catalysis by iron, copper and putatively also antimony chlorides. The latter input includes the Sb from fire retardants in plastics and PET bottles. e.g.
Antimony +[Heavy Metals]+ (Hydroxylated] Chlorocarbons Oxidative stress depletion of nitrosative heparan system tisssue protection

Sb present as surface adsorbate on ultrafine particulates residues form flame retardant and plastic catalyst resides which is recycled in urban dust is likely also to augment HCB etc formation especially in the road traffic environment Sb dust which may reenter the combustion process is produced from Sb(+Cu) containing friction surfaces (brakes and clutches). Should this hypothesis be confirmed by further research it is good news for future generations as this would allow a focussed chemistry science coupled with improved technology for waste disposal for the scientific prevention of future ASD occurrences. The alternative hypotheses that ASDs are primarily genetic predispositions cannot easily be combated at the present time because the necessary stem cell therapy which is currently not sufficiently advanced to enable this.

A3

Possible Relevance of Hg Possible relevance of Sb

Possible Unique Relevance of Antimony (Sb) Amongst Toxic Elements Associated with Autism The toxic inorganic elements which have been indicated to be present in the bloodstream of autistic subjects as indicated by in hair sample analysis include: lead (Pb) cadmium (Cd), tin (Sn) aluminum (Al), mercury (Hg) and antimony (Sb).

Of these, Sb is now suggested to have an especial relevance because Sb differs from the other elements listed in having found a wide range of new industrial uses over the recent decades including in food containers (as catalysts residues) and as fire retardants in textiles which might allow rapid human intoxication pathways. This increased potential rate of contamination of the environment by Sb seems also to have, uniquely of the potential pro-autism inorganic toxins, closely tracked the increase in the rate of prevalence of autism. At the same time studies of seabirds have suggested that chlorinated biphenyls and related substances have again increased in the environment (in spite of an international ban on their manufacture); these substances commonly occur together with hexachlorobenzene and octachlorostyrene OCS (n.b. OCS which has never been used for any defined industrial purpose but it is known to be produced during the incineration of common industrial waste products as a result of operation of thermodynamic driving forces which

govern this process. A recent report has noted that OCS diminishes glutathione in liver cells. Sb(III) has also been indicated to diminish glutathione GSH protection. Sb, perhaps by acting synergistically with OCS and similar xenobiotics, may especially be able to diminish GSH give rise to ASDs in genetically susceptible subjects.
The circumstances surrounding current increasing use of Sb in commerce should make this element of a priori interest to researchers seeking the most likely potential etiological factors which are responsible for a range of human disease. Such Sb usages have apparently increased (uniquely) in tandem with the increasing reported prevalence autism and other endocrine system disruption diseases which are thought to be a consequence of chronic inflammation (e.g. induced by endocrine disrupting substances acting to diminish the effectiveness of the GSH (and also glutathione S-transferase, catalyse and superoxide dismutase tissue protection systems).

A4

Are AD and ASD produced by similar toxins?

This above ASD-related list of altered inorganic elements has also been reported to permit, by the establishment of their blood levels, a highly effective method of diagnosis of AD as indicated by
Bocca et al. who reported that the amounts of 26 metals in whole blood and serum of 20M and 40F AD patients compared wit 44 control subjects suggested that altered balances in the accepted neurotoxic elements (Al, Cd, Hg and Mn) in combination with a disturbance from normal levels in Ca, Cu, Fe, Mg and Zn is a characteristic feature of AD and that this disease likely arose as a consequence of redox control dysfunction; the analysis of seurm Al,Ca,.Co and Si and related oxidant stress levels was able to correctly diagnose 94% of AD using these serum analytical markers alone). Cf. also Brunisma et al. loc. cit. and appended comments.

The possible role of antimony (Sb) intoxication in AD does not seem to have been widely addressed. One ICP-MS study (Gerhardsson) reported the concentrations of: Mg, Ca, V, Mn, Fe, Co, Ni, Cu, Zn, Se, Rb, Sr, Mo, Cd, Sn, Sb, Cs, Hg and Pb in the plama and CSF of 173 AD and 87 AD plus minor vascular components subjects, showed up statistically highly significantly increased Mn and Hg in plasma of the AD and AD vasc. cohorts. (In the CSF there was a statistically significant reduction in V, Mn, Rb, Sb Cs and Pb in AD and AD+ vasc. vs. controls. (Mn has been indicated to be a relevant promoter of prion diseases and Hg to be a suspected pormoter of ASD). That Mn is decreased in CSF and increased in plasma might indicate that the brain content of this element is of interest to the aetiology of AD (and perhaps the finding of an AD-related diminution of CSF of Mn, Sb and Pb could implicate a possible role of Sb uptake in the brain in the aetiology of AD). An increase in Sn in AD brains was reported by Corrigan loc. cit. Sn is also increased in ASD hair (cf. Hall loc. cit.). (An inferred relationship can be made between Sb- gluconic acid and Alzheimers disease (AD), the most direct link is the IL1 B gene).

A pro-autism stimulus may arise, it is now suggested from the direct presence of Sb in human tissues as inferred from some but not all of the reported hair element analysis of autistic subjects. In all of these cases the absolute amount of Sb in hair is very small.

A5
Some Reported Hair Element Alteration Studies Relating to Autism (Severity)
Priva & Geertha (India); Majewska (Poland) & G. BELL et al (Scotland UK).

That Sb is a cofactor for autism (cf. Hall report on SCOTTISH AUTISTIC SUBJECT HAIR ELEMENT STUDIES researches conducted by G. Bell et al., at Stirling University) promoting illness (and a possible HCB intoxication cofactor) is consistent with the circumstance of the explaining the supposed Silicon Valley electronic industry autistic clusters, the mode of occurrence of Sb its presence in solder, in semiconductor components being present in automobiles, the plastic covers of computers etc., in the polyhalogenated flame retardant treatments of childrens clothes, domestic furniture and (together with Cu and Ba etc.) in automotive brake friction pads as well as likely being leached into fast food from microwave oven plastic trays and also in drink containers perhaps most importantly those which contain additional ligands for Sb. [The presence of Sb in PET might explain why the use of bottled water mirrors the prevalence of autism as reported on the internet; this correlation is must be noted however is more likely to have arisen because of the entry of Sb oxides and as methylSb compounds into the environment from the eventual incineration of the PET containers; the amount present in most fresh produced bottles is, however, conceivably relatively safe]. A further link between Sb (cf. Richardson) + HCB intoxication and sudden infant death syndrome (SIDS) might be indicated (cf. e.g. the attempts to backtrack use of PVC (a source of residual HCB plus dixoins leftover form the manufacturing process.
Communicated Archival Sb Hair-Element Data [The following Communicated archival material Information w.r.t. antimony in autistic subject hairis abstracted from a personal communication dated 30 August 2011 ex
Professor G. Bell of Stirling University, Scotland, UK FK9 4LA]

Antimony (Sb) in child hair samples hair of 24 [patients} diagnosed with autism (and 5 with Aspergers syndrome {data not listed} compared with {an apparently randomized interpolated} normal children hair n= 8 which act as controls which from information provided in other text data are assumed to be the values labeled by me as } The *normal range 0.000-0.030 (presumably this was what was found for Scottish controls); Fig. 12 of the original draft paper indicated that the maximum normal range of Sb in hair was taken as 0.030 ppm (stated in the caption); the average values of Sb ppm for autism was 0.149 (n=24) {for Aspergers ?} and for controls (n=8) 0.064 ppm {The data which were present separated in graphic form suggest that the hair elements for Aspergers child hair samples are very similar to those of the n=8 controls. I.e. hair element analysis sharply distinguished autism from Aspergers syndrome. Autism is associated with blood intoxication by a range of heavy metals. This intoxication does not occur with Aspergers syndrome}. Here are the hair Sb (ppm) data for autistic and normal controls:
Patient No. Age ppm Sb 47 55 1 14 2 18 54 3 4 5 0.065

16 <0.025 7 5 9 18 13 11 8 0.092 0.137 0.093 0.126 0.069 <0.025 0.159

50 49 37 8 60 7 64 46 63 19 29 38 26 39 41 59 25 35 30 33 40 28 36

9 3

0.04 0.118 0.049 0.051 0.352 0.229 0.047 0.057 0.071 0.109 0.139 0.035 0.095 0.455 0.339 0.457 <0.025 0.264 0.085 0.08 0.025 0.064

8 4 8 3

8 4 3 5 8

6 6 7 5 8

9 3 4 8 9 8

these seem [authors provisional attempt to identify on the baiss of the verbal and 5 0.102 figurative discription of the tabulated received data from the given archival data printouts kindly porvided to me] to be the controls ; other data deems to from autistic subjects Half the controls and all of the autistic subjects showed Sb above the recommended level of 0.030ppm It is evident that a larger study is required to confirm the suggested elevation of Sb in the hair of aASD subjects. cf Bass DA et al., Altern Med Rev. 2001 6 (5) 472-81 Table 2 Accuracy of Sb determ. Chinese hair CRM GBW Report 1, Sb 0.064; Report 2 0.069; Report 3 0.046 %RSD 20 Table 4 Results of Split Samples from Two Laboratories Sb, Lab #1 0.095 Lab #2 0.041 cf also Gebel T et al. Int Arch Occup Environ Health found that subjects (n=89) who had supposedly been occupationally exposed to Sb had hair averaging 0.026 ppm vs. 0.045 ppm for controls (n=47) [Bowen had earlier reported that Sb in soil ranges from 0.2-10 ppm; such Sb is believed to be augmented by coal mining activities). A US Public Health compilation from Sept. 1992 cites Muramatsu and Oar 1988 who reported an average hair Sb level of 0.12 ppm and Tagaku et al. 1986 0.096 ppm The US EPA document (by Hollowman JW and Hammons AS) gives hair Sb as 0.00-0.31 ppm (n=39) 0.06 ppm (n=10) Ali. loc. cit., gives 0.068ppm for normal hair Sb NIEW No 5 hair sample. Numerous values of reported hair Sb, which are are higher than the normal range <0.030 assumed by Prof. Bell (often cited by commercial hair elements analysts casts doubt on any direct correlation between hair Sb and autism. However the correlation between hair elements and a standard (Aberdeen University former polysaccharide laboratory used) heparin multi-inorganic element sample confirmed that the value 0.03ppm for normal hair Sb is an appropriate baseline value from which to judge if a hair analysis indicates the possibility of the existence of Sb intoxication in the subject. Goull JP et al. (Forensic Sci Int. 2005 153 39-44) list the multi-inorganic elements present in blood plasma urine and hair as determined by IPC-MS ; the reference value range for hair was 0.003-0.13 (av. 0.008) ppm from 45 healthy subjects. It should be noted that Tl is listed here as 0.0001-0.0004 and Hg as 0.31-1.66 pp/

The above tabulation of Prof Bells data could suggest that the majority of the patients exhibit a typical (former) coal mining district -related elevated Sb level of ca 0.06ppm while a minority (6/32 patients) show a much higher level. Perhaps Sb intoxication is related to social dysfunction which have in the past also been thought to be more prevalent in coalmining districts. Similar higher levels of Sb have been reported to be associated with autism by parents on the internet. These higher levels are also associated with larger than normal ppm hair element values for Al, and Pb and sometimes also as indicated by internet postings, Be, Hg, Cd and Tl. [Prof. Bells autistic subjects also demonstrated an elevation of Pb and Al in their hair. The Pb levels elevation in autism was statistically relevant (this was not the case for Sb). All of these elements are characterized as being preferentially bound by HS (Grant D et al. data).
Pb, Sn, Sb, Al and 29 other inorganic element contents in the hair (collected from the 2 cm of hair growing close to the scalp at the nape of the neck) of Scottish child patients (aged 3-16) with ASD disorders autism n=24 and Aspergers syndrome n=5 and 8 controls with no diagnosis) were by Bell et al. using ICP-MS (by employing a commercial diagnostic laboratory Great Smokies Diagnositc Lab NC USA). An apparent Pb elevation in autism was (apparently indicated to be) correlated with body tissue deposition (in bone aorta liver and kidney). The Sb content (ppm) of the autistic child hair samples, while not statistically significant (p=0.066) was indicated to be almost 5x greater in the autism group than the controls where the assumed for the normal range (Sb content 0.0000.030 ppm); while all children with autism exhibited elevated Sb, 50% of the control group and 40% for the Aspergers group also exhibited this toxic element elevation. Children with autism showed significantly higher hair Sn compared with controls or by comparison with children with Aspergers syndrome. The calcium content of hair was significantly lower than in the autism group (but not in the Aspergers group) however 38% of the children in the control group also exhibited depressed Ca levels. The Na and K hair contents were also higher in the autism group compared with controls which may be indicative of an imbalance in electrolyte and fluid control perhaps related to kidney dysfunction. In general the differences in elemental composition observed in the study groups suggest that children with autism have a problem with accumulation of certain toxic elements.which may have direct effects on neural and immune function with impinge on the etiology of the condition. These increased concentrations of toxic elements may arise from an inability to excrete these toxins due to dysfunctional detoxification mechanisms, and while they may not be directly causative in the development of autism, may exacerbate some of the symptoms found in children with autism. Heparan sulfate (HS) is likely to contribute both directly and indirectly to blood detoxification mechanisms including by the ability of this ultra anionic substance to bind cations. HS is also sensitive to dietary nutrient status and putatively changes in response to inorganic element Vitamin C and fatty acid composition of the diet. This may be an important reason why imbalances in minerals, ascorbate and -3 and -6 fatty acids contribute to pathologies. Further Comment on the Above Research Results by D. Grant. The association of Sb intoxication which was suggested by Professor Bells researches (putatively also by SH-binding plus additional metal redox afforded disturbance not achievable by Pb intoxication) may, it is suggested, may be more pertinent than Pb intoxication to the fuller understanding of the etiology of these diseases. While backup tissue culture experimentation results of how Sb might alter HS(PG) biosynthesis has not yet been conducted, several studies have shown that Pb intoxication can decrease HSPG core protein biosynthesis (and this may account for the possible role of Pb intoxication in the promotion of elevation of blood pressure in Pb intoxicated individuals (cf. Kaji et al.). The suggestion that the metal ion intoxication in with autism might be associated with kidney dysfunction (my italicized section above), accords with the finding that toxic metals can diminish HS function in the kidney (cf. Templeton); this heavy metal effect plus an organohalogen intoxication scenario (e.g. that of water-chloroform or food fatty tissue HCB intoxication) indicates a useful hypothesis of a primary event in the etiology of autism. A personal unsolicited communication from C. Rimington to the author (in 1986) had indicated the possibility that chlorinated organic toxins might seriously diminish HS functions in the liver (indicating the similar possibility in the kidney especially when there is a dual insult from heavy metal intoxication).

N.b. the discussion of a possible role of Sb in the aetiology of autism currently appears only in the grey literature (Hall loc. cit. : Sb was found to be elevated by ca. five fold relative to controls in the hair of a small cohort of autistic subjects. (These data, however fell below statistical significance as Sb intoxication occurs in the general population especially in former mining areas). Tin (Sn), Pb) and Al were also apparently elevated in the austitic hair (but not in Aspergers (n=5) hair). The data obtained for an autism Pb- intoxication association has been noted previously. This agrees with the statistically significant increase in Pb noted in the Hall report of in the hair elements of

autistic subjects. It should be also noted, however that while Pb together with Sb has been known for a long time to occur in the soil and dust (especially in former mining areas) and also to occur together with Cd in cigarette smoke, the recent decadal rate of combined Sb input into the environment putatively matches the rate of prevalence increase in autism more closely than does the rate of input of the other heavy metals into the environment. Of especial interest is the new use of Sb in the brominated aromatic substances fire retardants present in childrens bedding, clothing, in carpets and car seats and also the additional new use of Sb catalyst (which leads to particulate matter residues which ultimately enter the environment) in the widely used (sometimes recycled|) polyethylene terephthalate food and drink containers (from which Sb might additionally leach out e.g. during microwave cooking), add to increasing use of Sb compounds in automotive [brake and clutch] friction surfaces (which can form Sb containing dust in urban areas and along highways) to the continuing use of Sb in lead acid batteries and in other alloys, in enamels including those used in cookwares, in solders and semiconductors form which volatile antimony hydrides may also arise. The perhaps especially highly toxic methylated Sb (hydride) compounds which evidently also occur in small amounts in urban dust and perhaps also in some damp (mouldcontaining) house air seems to be microbial transformation produces of the more abundant inorganic Sb present in the environment. In some of the automotive urban dusts, Sb occurs together with Cu which is also used as a component of the friction surfaces, which together with Pb, Hg, Cd and Al could all act as part of the autism- promoting urban and transport artery toxic environmental cocktail. [It should be noted that Al intoxication especially that in drinking water and perhaps also urban automotive Sb intoxication to some extent also may be countered by inorganic silicon (Si) in drinking waters (cf. Birchall) so a deficiency of dietary Si might stimulate Al and perhaps other harmful toxic inorganic metal uptake. In this way the tendency for decreased Si in modern diets putatively creates a pro-autism stimulus (cf. use of steel rather than stone seed milling may be to some extent a contributory factor to the promotion of autism).
(It should be noted that substances which are chemically similar to the kinds of chlorinated aromatic substances which have been banned from deliberate manufacture because of their known highly adverse effect on human and animal health can be produced in large amounts accidentally, e.g. during incineration of polyvinylchloride); Sb is a known catalyst of this resynthesis process; hence the combination of an increased amount of Sb in municipal waste and the processing this waste by pyrolysis and incineration will putative tend to increase the amount of halogenated furans, dioxins and dioxin-like endocrine disrupting substances in the environment which further could promote autism and other diseases which are thought to be associated with increased oxidative stress which is known to be promoted by these xenobiotics. The presence of Sb in urban dusts which also contain halogenated organic substances will similarly catalyze the formation of furans, dioxins and dioxin-like substances in internal combustion engines).

A6

(Defective) Glycation and Advanced Glycation End Products (AGE) - possible

link to autism.
Glycation can aid the B-cell directed immune response and the use of polysaccharideadducts to create a much more effective vaccine seems to have become a comon practice and indeed has been hypothesized (Richardson, loc. cit.) per se to have led to the observed increase in autism over the last decades. While the exact biochemistry of ASDs and attention-deficit hyperactivity disorder ADHD have not yet been established ASD and ADSH could (at least partly) arise as a consequence of general other-thanvaccine glycation defects. While N-glycation defects were not detected with the most typical form of autism spectrum disorders (ASDs))) these defects were, however, evidently present in{ADHD) subjects..
The existence of proteins is normally assumed to be a prime biological circumstatnce. However proteins may have amongst their primary functions the provision of a platform for the attachment of sugars. This idea has been promoted by Dweck (Oxford) In turn these sugars could control the supramolecular structure of water. The latter structuring may ultimately be what creates all biological activity (cf. PM Wiggins).

A somewhat unorthodox group of scientists also believe that glycation (i.e. the attachment of sugars and complex saccharide oligomers and polymers) is the prime central essential requirement for all multicellular life forms. Proteins can be viewed in this context as merely the providers of a suitable platform for the attachment of the actual working surfaces which can be argued to consist of the glycome: its major components are systems of N- and O- glycation. Glycation has e.g. been discovered to determine antigen recognition and this is thought be a major factor in how rheumatoid arthritis (RA) may arise from an alteration of N-glycation. Defective glycosylation is probably also involved in other pathological conditions including, it is now suggested, autism where a mechanistically separate type of protein alteration (derived from nonenzymic (spoilage or browing reactions) may occur between amino groups and sugars (which is also classified as advanced glycation end product AGE a phenomenon related to the kind of oxidative stress which occurs in diabetes and a range of other illnesses). AGE-affected products are thought to be taken up and processed by a dedicated AGE receptor system [RAGE (in which a further O-glycation system, that of heparan sulfate (HS) proteglycans HSPGs is also apparently involved]. Since autism is thought to be included in the list of oxidative stress determined disorders for which AGE/RAGE biochemistry is believed to be highly relevant this implicates HS biochemistry are being relevant to the fuller understanding of autism. A growing understanding of HSPG biochemistry now indicates that vital processes which are influenced by this polysaccharide system can be improved by the exogenous application of HS related saccharides or their mimetics. This research may in the future provide a therapeutic benefit to such diseases as ASD. A6-1 Lead (Pb) Intoxciation and Altered Glycation {Cf. also., numerous epidemiological studies have associated ADHS with intoxication he xenobiotic substsnce lead (Pb) intoxciation which is known to alter O-glycation by information encode anionic patterned heparan sulfate (HS) also called the heparanome). Pb is also known to alter HSPG provided tissue protection via the ability of Pb to diminish the HSPG core protein biosynthesis.

Cadmium (Cd) intoxication is known to diminish HS sulfation. This sulfation an be however augmented by ascorbate).

A7
Purine Metabolism & Sb Purine metabolism alteration, which seems to have been observed in some autistc subjects, is believed to be perturbed by the inhibition of phosphofructokinase. Antimony Sb (Sb(III) is a highly effective inhibitor of this enzyme (which also the rate limiting enzyme of erythrocyte glycolysis/ Sb. Arsenic (As) also inhbits this enzyme but Sb is apparently much more effective than is As inhibition.

A8
Glutathione S-Transferase & Sb Sb(III) is also an-ultra effective inhibitor of glutathione-S-transferase (GST) therefore potentially greatly depletes antioxidant protection. Sb intoxicated subjects can be predicted to lack critical glutathione dependent antioxidant and heavy metal detoxification protection . Sb intoxicatied individuals are expected to become more hypersensitive to intoxication by other heavy metals e.g. Pb2+

A9
More on Sb Intoxication (Re Glycation) The ubiquitous Sb environmental presence and similar rate of rise in the input of Sb in a domestic rather than in an industrial setting might however suggest that autism may most often arise indirectly by the Sb catalysis of the augmentation of the rate formation of dioxin and related substances during fossil fuel combustion (engines etc) which use polluted air intakes. Nevertheless, the direct in vivo effect of Sb as an inducer of autism should be addressed since Sb(III) is known to be an specially highly effective example of the heavy metal depleter of glutathione antioxidant protection and the generation of advanced glycation end products (AGE). Since Sb intoxication, has putatively also been separately indicated from hair element analyses to be a risk factor for autism its role in inhibiting GST could be key part of its modus operandi. Sb(III) also inhibits phosphofructosekinase and diminishes erythrocyte glycolysis and could conceivably also impair fructosamine-3-phophokinase activity (an enzyme is thought to play a critical role in protecting cells from AGE-related intracellular protein and nucleic acid damage which arises from oxidative damage n.b. it has been observed experimentally the AGE-RAGE (receptor for AGE) axis seems to be defective in autism (Boso)} suggesting that Sb intoxication could act via several pathways (reduce GSH, augment AGE and inhibit anti-AGE protection and also slow energy metabolic processes).

An unknown but probably ultra-low level of Sb may negatively affect many of all autism subjects. and intensify the effect of Pb intoxication of autism subjects (giving rise to oxidative tissue damage). Another possible mechanism by which Sb could contribute to increase ex-vivo perchlorocarbon toxin formation is the ability of Sb to act as a catalyst for the formation of OCS and related end products of pyrolysis (arising during fossil fuel combustion in automobiles and trucks as well as in power plants and municipal incinerators). The appended literature search individually discussed by sub-notes to references arranged at the end of this document by alphabetical order according to the first named author now tentatively identifies those halogenated organic persistent organic pollutants which can enter the food chain so as to give rise to chronic inflammation in the global human population as being a possible primary inducer of ASDs. It is likely that dioxins, furans, hexachlorobenzene (HCB) and polyhalogenated biphenyls and similar substance which are synthesized during combustion in such a manner as to allow their uncontrolled entry into the environment can pass through air filters. The fossil fuel combustion process which commonly use polluted air input and which contain therein small amounts of heavy metal catalyst-doped nanoparticulates are likely to augment the toxic human and animal load. It should be noted that Sb residues occur in high tonnage food packaging materials which seems to allow the leaching of this element into food but also causes air pollution as a consequence of incineration allows the forming nanoparticle Sb doped catalysts when these plastic containers textiles and plastic mouldings with Sb-based fire retardants are incinerated (especially by) backyard trash disposal or (from low cost medical waste disposal systems which may exist both in the developed and developing counties). Sb aerosols act as catalyst system for the subsequent generation of dioxinlike substances in internal and other fossil fuel combustion chambers and the exhausts from which may not be effectively scrubbed by the currently-used catalytic converter systems. Furthermore the incinerator generated Sb doped nanoparticles, which can be transported intercontinentally augment other Sb-+Cu containing nanoparticles present in ambient air from the local use of Sb in friction surfaces of automotive brakes and clutches. It is now suggested that this mechanism of Sb recycling and dioxin synthesis is relevant to the promotion of autistic spectrum disorders (ASDs) in genetically susceptible individuals. These illnesses form part of a larger effect (unexplained obesity, asthma Alzheimers disease (AD) and other mystery illnesses) which arise from the global human and animal population following xenobiotic intoxication by halogenated aromatic organic pollutants. Bell (cf. Hall) found that lead Pb was the most statistically relevant elevation of toxic metal associated with autism (but not with Aspergers syndrome) subjects. Al and Sn were also elevated (also not with Aspergers). The large amount of Al present in the autism subjects might suggest that this is of especial relevance as this element has also been associated with AD (putatively in a Sidietary-status related manner). The overlap of specific toxic metals which have been associated with ASD also extends to cadmium (Cd) and mercury (Hg).

The Bell (cf. Hall data) suggest that ASD (but again not Aspergers) is associated with increased hair tissue Ca. A large number of reports also suggest that ASD is associated with diminished Mg, Zn and Cu.

A10
Alteration of Heparan Sulfate by Systemic Oxidant Overload
The Heparanome Defect Hypothesis of Autism It is now hypothesized that the most typical form of autism, and indeed possibly all forms of related diseases which include ADHD also partly arise as a consequence of defects in HSsugar-O-sulfate and glucosamine-N-sulfate based information processing (including the redox sensitive nitric oxide metabolite scission oligosaccharide generation second messenger process) which in vitro cell culture studies have indicated to be sensitive to the types of heavy metal intoxication cocktails which seem to be associated both with typical ASD and also with ADHD. The alteration of HS by the pro-oxidant effect of systemic oxidant overload seems also to provide a credible hypothesis of the correlations which have been found to exist between autism and maternal hypertension, obesity and cardiovascular disease. Since the heparanome seems especially designed to strongly interact with various external factors as part of its function to protect cellular environment against toxic insults it therefore seems to be a credible hypothesis that the aetiology of ASDs is determined to a large extent by HS biochemistry. ASDs could arise in genetically susceptible infants following the disturbance of HS functions including in utero nervous system developmental (e.g. perhaps even specifically by the widely distributed xenobiotic hexachlorobenzene (HCB) the cord blood content of which has been correlated with later childhood obesity). Since HCB (together with other chemically related dioxins and dioxin-like substances) now intoxicate the entire global human and animal populations the subtle alteration of the nervous system caused by this intoxication might lead either to obesity or autism (or a range of other mystery illnesses) according to the specific individual genetic susceptibilities. A further role of heavy metal xenobiotic intoxication is also suspected, however. Controversially, the symptoms of autism are suddenly triggered by metal ion intoxication [perhaps most especially by aluminium (Al)] which might augment the general background, wide environmental pollution by small amounts of lead (Pb), mercury (Hg), cadmium (Cd), nickel (Ni) and fluoride (F-) which will, as indicated by in vitro studies, especially in subjects who additionally have an insufficiency of magnesium (Mg) and sulfate, cause an alteration of the HS microstructure which putatively leads to major heparanome dysfunction. (The above listed specific ions have either been shown in cell culture experiments to directly perturb the assembly of HSPGs in the Golgi apparatus or to create oxidative/nitrosative stress (where tyrosine nitration occurs as is the case with ASDs) a process which can negatively alter primary HS biosynthesis or cause HS postsynthetic structure change. The otherwise carefully controlled HS scission by reactive nitrogen generated second messenger oligomers seems especially at risk from the effects of anthropogenic xenobiotic insult. Al and the metalloids antimony (Sb) and arsenic (As) (all of which commonly but perhaps more sporadically co-occur with autism) are well known to be capable of creating in vivo oxidative/nitrosative stresses.

In summary: the various xenobiotics which have been indicated to promote autism also potentially perturb the entire molecular structure of HS in such a manner as to disturb systemic cellular immune and growth factor signaling in development and wound healing. Further evidence in favour of a HS-led hypothesis of autism is that a high penetration mutation in genes encoding various synaptic cell adhesion molecules has also been associated with ASDs. These molecules require HS and other related anionic polysaccharides (Thorne) for their correct functioning. That HS could play a major part in major electrolyte balance could seem to be a totally unacceptable extremely unorthodox hypothesis. This can be perturbed by heavy metals. There is, however, considerable experimental confirmation in the peer-reviewed literature in support of this kind of hypothesis that (other-than-protein ion channel) sugar systems contribute to the creation of primitive electrolyte buffer systems which back up the usual protein based systems. The polysaccharide systems are retained by modern animal organisms (cf., e.g. the specific amounts of HS and related sulfated polysaccharides were discovered to be required to protect the integrity of tissues in fifteen aquatic invertebrate; the amounts of these polysaccharides varied according to the salinities of their environments and this was precisely defined exactly by mathematical relationships relating the amount of HS required exponentially with the salinities of the various aquatic habitats converted by the study. HS polysaccharides are, however, more commonly associated with their first-discovered function, namely: hemostais (where a key event is a heparin pentasaccharide-HS anthrombin binding also involving Ca). Later studies uncovered a wide range of other biochemical processes including neurological development, lipid metabolism and more generally the control of growth factor activities in foetal assembly and wound healing]. Defects in HS including its ability to regulate electrolytes such as Ca2+ and how these can be perturbed by Pb2+, Al3+ and putative also by e.g. Sb(III) could, it is now suggested, be of major relevance to why HS biochemistry has been implicated in neurodegenerative diseases [cf. Cappari et al.] and analogously in ASD which is believed to be, at least in part a neurological dysfunction condition arising from disturbances in perinatal developmental programmes.

A10- 1
Altered Metal Ions Can Alter HS Activity BUT IS HS MICROSTRUCTURE TOO COMPLEX FOR PRESENT DAY? [The suggestion now being entertained in this essay that defective HS is the origin of autism can be logically countered by the valid criticism that HS is so closely interwoven into all of animal biochemistry and physiology that any attempt to pinpoint any single HS defect must be fruitless since HS is now implicated in multiple mechanisms (as is also the case for any disease process apparently as complex as autism) requiring all organic diseases to be at least partially dependent on HS dysfunctions].

A10-1-1 Sb may be more strongly bound than Al to HS

It had been indicated from preliminary observations (1b) that antimony may be more strongly bound to HS than is aluminum, lead and arsenic which in turn are more strongly bound than such normal physiological elements as calcium, copper and zinc (which are known to be naturally required cofactors for HSPG signaling (1c). [The use of seaweed which contain HS-like polysaccharides to remove heavy metals from polluted water has been reported (Bakir) to be subject to a Sb(III) antagonistic effect; the presence of Sb(III) in natural water can apparently greatly dimishish the effectiveness of
the algal polysaccharides to sequester a the kind of toxic metal which have been putatively been associated with autism spectrum disorders].

A10-2
Unzipping of HS by toxic metal plus excess nitrite
[Cf. Copper and Zinc Dependence of Heparan Sulfate Signalling (cf. Mani)}

Road traffic and incinerators are known to generate the above-listed and other xenobiotics (these incude dioxins, polychlorinated biphenyls and brominated analogues) which putatively act together with Al, Sb, As, Cu, Ba, Be, Bi, Cd. Pb, Hg, Sn Ti, Tl, Zr and Ce containing particulates to increase a potential pro-inflammatory stimulus and with the Al introduced from numerous domestic industrial and medical application (cf. the wide use of Al (hydr)oxide in vaccine adjuvants) to activate the inappropriate nuclear transcription of cytokines which produce nitric oxide (NO) [a molecule used normally as as a major response of the cellular immune system to generate NO]; (xenobiotic heavy metals seem to induce abnormal formation of NF kappaB and IL-6 etc. which further leads to inappropriately augmented synthesis of NO}. NO on conversion to nitrite quickly unzips HS. Originally this was not thought to apply to physiological conditions but this classical assumption seems to be incorrect (cf. Vilar). The HSPG polysaccharide-based animal system manager (the heparnome) had traditionally been most well known for its ability to direct the activities of numerous proteins (the first of which to be discovered being those of the blood hemostasis system; later the use the HS-related system heparin as a blood anticoagulant provided evidence that HS also efficiently promoted the metabolism of fatty acids. Heparin is the most anionic of any biopolymer and binds a wide range of counterions at modest but physiologically relevant
association constant values. The binding mechanism is more complex than being a purely electrostatic phenomenon since anions can also bind strongly to heparin. Numerous studies of metal ion and anion binding to heparin have indicated that heparin/HS likely serves e.g., at cell surfaces and in extracellular matrix as a proton conductor as well as a multi-inorganic element collector transport

is able to provide a protective shield against entry into the cell of toxic ions including those listed above.
and storage vehicle for the physiological elements and

Of these, Sb, which occurs widely naturally in small amounts in the environment, and has no known physiological role, appears as indicated by preliminary observations to be more strongly bonded to heparin/HS than any other inorganic element. Therefore any exposure to Sb might especially create adverse effects on HSPG behaviour. This suggests a possible mechanism by which Sb intoxication might, through its negative perturbation of HSPG (e.g. growth factor) signalling causes autism..
--------------------------------------------------------------------------------------------Low levels of Hg, Sb, and Cd intoxication may negatively affect HS proteoglycans (HSPG) Hg intoxication is known to potentially inhibit HSPG biosynthesis (Templeton). Cd intoxication is also known to potentially inhibit HS sulfation (Cardenas) Published cell culture experiments have demonstrated that Hg Cd and Pb can negatively perturb heparan sulfate proteoglycan biosynthesis. [This effect of Pb on HSPG (via the inhibition of the biosynthesis of the core protein) has been postulated to be a major cause of Pb-induced high blood pressure (Fujiwara)]. Preliminary studies of the binding of Al and Sb etc. to heparin conducted at Aberdeen University show the this metal ion combination may causes an altered pH homeostasis elicited by heparin like molecules. A possible role of Ba intoxication of heparan action may also be part of the etiology of multiple sclerosis. (Purdey studied this element in an attempt to correlate its natural abundance in the soil etc. with epidemiological data for multiple sclerosis (MS) e.g. in N.E. Scotland) and published a hypothesis of the etiology of MS on the basis; Ba might e.g. perturb HS dependent growth factor signalling in wound healing of nerve coatings so causing MS. It seems worthwhile to also re-examining the possible deleterious effect of the co-binding of Ba, Sb and Hg as a possible aetiological factor in heparan sulfate dysfunction-determined autism and other diseases. A hypothesis is now advanced that low level Sb intoxication can negatively affects HSPG biochemistry disproportionately via an unusual high affinity of Sb for HSPG (this may include a high affinity for the sulfated polysaccharide side chains as well as for the conserved HSPG core protein cysteines which serve as nitric oxide reservoirs used in controlled signaling oligosaccharide generation (cf. Mani et al.). This idea arose from a re-evaluation of old laboratory data which suggested that Sb co-occurs in the sulfated polysaccharide heparin together with a wide range of other inorganic elements including toxic elements, and is perhaps uniquely difficult to remove from such multiinorganic heparin (a pharmaceutical industrial product commonly used as a model system for HS). This heparin demonstrated a correlation between these multi-elements in heparin and in standard hair samples.. [Other indications from published data indicate that a perhaps similar strong binding of Sb occurs to marine algal sulfated polysaccharides.

A-11
Sb binding has been reported (Bakir) to diminish alginate toxic metal sequestration Sb binding has been reported to greatly diminish the ability of Sb-polluted alginates to bind to other toxic heavy metals. A similar perturbation by Sb of cross-talk between the heparanome and the metallome could therefore give rise to developmental disorders of which autism is now generally considered to be a prime example.

The entire list of the toxic inorganic elements which have been previously suggested to negatively affect human health (cf., e.g. Stejskal) (while putatively binding to heparin less strongly than Sb) seem nevertheless to bind more strongly to heparin and putatively also to HSPG than do the normal physiological elements.

A-12
The metallome The metallome (a branch of science revived in 2004 by H Haraguchi) originated by RJP Williams) which relates to the system of multi-inorganic elements which exist in geology but is also found and putatively is of fundamental importance to biology. The metallome concept suggests a control system analogous to the proteome, genome, etc. which is highly relevant to animal physiology. Seawater, human and animal blood serum and bone are all Haraguchi metallomic matrices (and show fairly well-defined intra-matrix compositional correlations) as are human bone, nails and scalp hair (which is commonly used in alternative medical and putative diagnostic marker of autistic subjects). All of types of highly anionic polysaccharides which occur at cell surfaces and extracellular matrices are also likely to be prime metallomic matrices. This idea arose from the observations that a standard heparin (an end member of the major HS system of signaling polysaccharides [the heparanome] is a very well defined metallomic matrix (cf Grant, 2000). The inorganic element profile of this standard heparin (gifted some thirty years ago to a range of academic institutions from a former UK pharmaceutical facility at Liverpool) was found to correlate with most of the elements in human hair which tends to confirm the relevance of the study of hair elements for the diagnosis of metal ion intoxication. There was some doubt about what is the correct baseline for Sb. (cf. the wide range of values of reported baseline for Sb in human subjects; how much Sb is indicative of serious systemic intoxication?). The (metallomic) correlation which seems to exist between the multi-inorganic content of heparin and a cohort of normal schoolboys hair provides supporting evidence that the value 0.030ppm commonly assumed by commercial hair analysis laboratories (including that used by Prof. J Bell) is about the correct upper limit. [It should be noted that literature reports of hair elements especially form coal mining and metallurgical procession regions are much greater than this; This may be why there are higher potential numbers of autism subjects from such regions]. HS biochemistry can also explain gut dysfunction in autism. Pre and post natural disruption of the gut surface is believed to be formed via HSPG signaling. A known link between Sb intoxication and biochemisty connects the adrenomodullin (ADM) gene with autism and also with nitric oxide biochemistry (Zoroglu) which is part of HSPG biochemistry (e.g., cf. Mani).

-------------------------------------------------------------------

Footnotes * The author

The author (1) has personal research involvement in studies which has established of
the central role of the formation of hexachlorobenzene during pyrolysis of organochlorine substances. This substance is usually discussed in the context of human an animal health concerns in terms of the previous use of this (now banned) substance as an insecticide (it also had specific industrial and military uses). While current input into the environment from residues leftover from former deliberate utilization sources (indeed persons intoxicated during the large HCB intoxication incident in Turkey remain affected by the intoxication) may still have an affect on current intoxication of the environment the circumstance that this substance is positioned in a prime fundamental endpoint of reorganization chemistry needs to be addressed which points to this circumstances has greatly outweighing its former agrochemical employment. The reorganization chemistry dictates the obligatory formation of HCB as a principal product during deliberate or accidental pyrolysis or incineration of numerous starting materials most prominently polyvinylchloride. The toxicology of HCB may have prime relevance for the evaluation of epidemiological and environmental studies aimed at understanding the prevalence of mystery illnesses which might have been induced by environmental pollution. (1- 3) Following training as a chemist at the University of Glasgow Scotland, UK, B.Sc.,1959, Ph.D., 1962, (Thesis A study of phosphites) the author was engaged as a Postdoctoral Fellow in a program (hosted by Monsanto research laboratories in St Louis, Missouri, USA) where JR Van Wazer had achieved international fame in phosphorus chemistry (by the use of NMR he and colleagues had discovered the relevance of structural reorganization concepts to the elucidation of chemical nature of a number of inorganic phosphorus based polymer systems and following on from this an international research team had been assembled to study the relevance of the new theories of chemical stability of inorganic polymers and related substances proposed by Van Wazer; it seemed that this work could be the basis of the establishment of fundamental theories of general chemical stability which should apply to all types families of monomer polymer systems throughout the Periodic Table. While this new structural reorganization theory was most relevant to inorganic chemistry, in the realm of organic chemistry was also found to be especially relevant to the thermal behavior of organochlorine compounds. The fundamental basis of organochlorine thermal stability and outcome of incinerations from the point of view of structural reorganization chemistry is the central role played in the reorganization chemistry by the formation of HCB The fundamental basis of organochlorine thermal stability and outcome of incineration from the point of view of structural reorganization chemistry is the central role played in the reorganization chemistry by the formation of HCB, (carbon tetrachloride and hexachloroethane) [This was firmly established by NE Aubrey and JR Van Wazer J Amer Chem Soc. 1964b86 4380-3 and later further studied by the author (reporting from I.C.I. Ltd., UK in J Appl Chem Biotechnol. 1974, 24, 4958). The thermal rearrangement during pyrolysis or incineration of all substances containing carbon and chlorine atoms (i.e. all known carbon chlorine compounds and their mixtures) was found to be determined not kinetically as had hitherto been believed, but the production of HCB (etc.) in amounts determined purely be of a mathematically determined chemical process similar to that which also determined the outcome of structural reorganization of other systems such as bisphosphonates (cf. D.

Grant Eur Polym J. 1979, 15 1161-5 which also mimicked the reorganization behavior of phosphate, sulfate, borate or silicate esters or polyphosphates (as well as the behavior of polyborates, polysilicates as well as e.g. polysulfides and polyselenides cf. J. Amer Chem. Soc. 1964, 86 3012-7, (the pyrolysis chemistry of these families of chemical substances is determined by the operation of the similar types of putative thermodynamically-driven reversible processes. (A list of the early references to this subject are given in D. Grant, J Inorg Nucl Chem. 1967, 29 69-81). The thermal rearrangement of organochlorines to HCB is a central outcome of the incineration of municipal wastes (cf. G.C. Choudry and O. Hutzinger, Current Topics in Environmental and Toxicological Chemistry Vol. 4 Gordon and Breach Science Publishers, 1983). Further Notes The HS assembly process is known to be sensitive to Ca, Mg as well as the toxic inorganic elements Pb Hg and Cd. HS biosythesis is also sensitive to redox ststus and pH. Also to excess fluoride (F-). Aluminium (Al) putatively can disturb cell surface pH by binding to HS. This conceivably could occur in conjunction with F. Chlorinated aromatic substances can cause systemic disturbance in tissue redox status (and nitric oxide biochemistry). This kind of thinking points to the halogenated aromatic xenobiotics and heavy metals which alters nitrosative HS recycling and de-novo biosynthsis which in turn alter Ca and other metal ion homostasis as being a prime trigger of ASD.

More on ME/CFS
Myalgic Encephalphalitis/Chronic Fatigue Syndrome (ME/CFS) also called (although it has also been indicated that these diseases can be separately diagnosed using an effect of topical application of acetylcholine which augments peripheral blood flow and the recovery of this to normal is especially inhibited in CFS patients in comparison with controls (Cf. F Khan et al. with Vance Spence, Clin Physiol Funct Imaging. 2003 23 (5) 282) or patients with related diseases of Gulf War Syndrome and farmers affected with a common organo-phosphorus insecticide intoxication allowing this procedure to distinguish differences in these pathological conditions which evidently include variations in the deficiencies in vascular biochemistry associated with these different medical conditions. There are numerous internet documents discussion ME/CFS. A useful 2004 literature survey by Charles Weber (Chronic Fatigue Syndrome (ME, CFS or CFIDS) and Fibromyalgia-some possible helps) notes that the most consistent laboratory abnormality in patients with CFS is an extremely low erythrocyte sedimentation rate (ESR) which approaches zero. Also an increased secretion of citrate in the urine which might be responsible for an increased excretion (and resultant depletion in blood serum) of Mg. CFS has also been associated with viral infections (associated with 2-5 synthase enzymic conversion of ATP to 2-5 oligoadenylate and activation of the RNase L enzyme which degrades viral and single strand RNA) and environmental toxins (which in the presence of heat shock protein activity also induces the preceding RNAse L system {K de Meilier presented a discourse on this topic and its relevance to the etiology and therapy for ME/CFS at a Workshop on this topic held in the University of

Dundee in 2003 attended by the authorb} ). Such toxins may include aluminum (which is apparently elevated in CFS patients, cf. Van Rensberg) and aspartame; a related phenomenon is chemical hypersensitivity augmentation of nitric oxide (Pall, 2001); there seems to be some relief from such effects by the administration of methylsulfonylmethane (MSF). [This conceivably could be related to sulfur compound (e.g. on glutathione isomerase or glycosaminolgycan especially heparan sulfate) status] Of possible relevance to CFS and autism is the presence in the urine of specific markers which include heme biosynthesis alteration products and altered tryptophan containing derivatives. Tyrptophan metabolism via serotonin is altered in rats with HCB induced porphyria (Lamban et al).

Footnote a-1 Footnote a-2

Footnote a-2 Oxalate OXALATE [It is of interest that reported that autism can be characterized by a highly significant increased urinary presence of oxalate, this could arise it is now pointed out from the altered gut bacterial population which is thought alto to define the autistic condition; depletion of the bacterial species which consume oxalate in anaerobic metabolism (a-21), and normally perform the function of limiting the presence of oxalate and their diminution may augment the occurrence of kidney damage in autistic subjects. Xenobiotics and oxalate crystal damage is associated with the presence of markers of such damage in the urine (e.g. gamma glutamyl transferase GGT.). The amount of urinary GGT has also been correlated with obesity, HCB and xenobiotic intoxication. The damaging effect of Ca urate crystals acting in concert with nitrosative deaminative cleavage will, it is suggested promote HS damage and further damage the antioxidant protection etc. afforded by this system manager. HS related deficiency may be especially relevant to the impairment in the vascular system causing poor oxygen transport to the brain which is thought to be part of the etiology of autism (cf. the reported alleviation of the symptoms of autism by countering cerebral hypofusion attenuation by using HBOT as discussed by Rossignol]. Footnote a-2-1
Footnote a-2-1 Cf. Sidhu H et al. J Amer Soc Nephrol. 1999 Supl. 14 S334-340 (Oxalobacter formigen imputed in maintaining oxalate homeostasis) Pieraer E et al. J Biol Chem. 2010 285 (52) 40515 (Identification and characterization of oxalate oxidoreductase, a novel thiram pyrophosphate defined 2oxo-acid oxido reductase that enables anaerobic growth on oxalate

Footnote b Footnote b Notes from this Workshop prepared by the author (related file Note C

).

Note C Anti-inflammatory effects of heparin ANTI-INFLAMMATORY EFFECTS OF HEPARIN The i.v. injection of heparin has been indicated (in internet documents including blogs accessed via Google search term heparin chronic fatigue syndrome ) to a useful tool for eliminating the effect of environmental airway inflammation and related chemical substance hypersensitivity. Perhaps heparin can also inhibit the pro-oxidant effects of macrophage myofacities. A beneficial use of injection of heparin has been indicated (e.g. in these blogs as well as more generally in the scientific literature) to (temporarily) reverse the symptoms of chronic fatigue in some individuals but not in others.
Footnote e Footnote e

Hormonal Effect of Heparin Relating to Putative Cancer Therapy Hormonal Effect of Heparin Relating to Putative Cancer Therapy.
An anti- angiogenesis effect of heparin activity is putatively mediated by actions mediated by (an unconventional) steroid hormone effect (J Folkman et al). Footnote g Footnote g g The destructive effect of reactive nitrogen moieties is analogous to that of the more well-known reactive oxygen species. The existence of a semi-conserved system of cysteine- tyrosine throughout biota seems to be analogous to the synergistic antioxidant effects of hindered phenols and bivalent redox metal ligands which is a basic principle of industrial man-made polymer life-extension technology. The human polymer age boost seems to mimic animal biochemistry systems (cf. D. Grant et al., Med Hypoth. 1997 ). However in the later case an additional insult from reactive nitrogen species may also be countered by the synergism of bivalent sulfur and aromatic hydroxyl containing tissue protective agents.

Research Suggestions FROM LITERATURE Relating to Possible Therapeutic Intervention in Autism

Purging of HCB Use of oily fish diet. Perhaps the EyeQ indicated from academic research reported in Mail Online by James Chapman (web dailymail.co.uk/health/article-74554/Hew-fish-diet-helps-autistic-children) {the supposed scientific basis of this benefit was however different: it being suggested to improve cognition via neural cell membrane function improvement). Fish diet removal of HCB and other halogenated substances Fish diet removal of HCB and related substances from adipose tissue stores Inhibition of oxalate augmentation Inhibition of oxalate augmentation by probiotic containing Oxalobacter formingens. Drugs which putative boost HS protection Bisphosphonate therapy Pentosan polysulfate therapy _ Hyperbaric therapy

Dark Chocolate Therapy

-----------------------------------------------------------------------------

41. References

41.1 Selected References

[1] [The review of literature which is given in the following first named author list of alphabetically arranged references and annotations, could to shed light on the aetiology of ASDs study which cause ASDs in genetically susceptible subjects. A theme which emerged early in this survey was that Sb was a key ingredient in the pro-ASD toxic cocktail. Sb is known to be a highly toxic inorganic element which has no known physiological role in animals and furthermore, uniquely amongst the list heavy metals this metalloid stands out from other possible triggers because in recent years Sb has been increasingly been introduced into the global environment at a rate which uniquely tracks the contemporaneous increase in the prevalence of ASDs and this alone could support the hypothesis that this element somehow acts as the principle stimulus trigger of ASDs. On the other hand the rate of introduction of Al, Pb and Hg, other contenders for the role of the principal environmental xenobiotic stimulus for ASDs have not varied in a manner which is supportive of such a role. Although there are sporadic reports of Sb in academic studies, the possible role of Sb intoxication in the aetiology of ASDs seems to be discussed disproportionately in the grey literature internet blogs of informed concerned parents. A small Scottish academic institute study of autistic children also seems to have identified Sb uniquely as being augmented in hair samples. This report which had been relegated to the grey literaure is now discussed in detail below since it could support the hypothesis that Sb is also a key pro-autism factor by its direct in vivo pro-inflammatory behaviour. Larger similar epidemiological studies to Sb intoxication in ASDs is now suggested to be warranted. Hair elements analysis (as an indicator of the presence of toxic blood elements) in ASDs has previously generally been conduced in order to refine discredited grey hypotheses relating to mercury (Hg) (e.g. from food, dental amalgams and most controversially from the Thimerosal in vaccines). Other candidate toxic metals such as lead (Pb), cadmium (Cd), aluminium (Al) etc. intoxications may, however, perhaps induce ASDs. Toxic element antimony (Sb) intoxication could be more likely that the above-mentioned toxic metals to induce ASD since the amounts introduced into the environment over that last three decades has paralleled with the increased reported prevalence of ASD. Discussion of any role of this element in the aetiology of ASD has, however been restricted to the grey literature. It should be noted that a possible problem exists with the use of hair analysis for estimating the body load of Sb is that this element can become very strongly bound to biological molecules, making the amount of Sb in the hair unrepresentative of the true body burden, which could be greater.. [Also, possible errors which may be introduced into the results obtained from exogenous impurities and the possible existence of errors arising from procedural differences between laboratories which especially could affect the low levels of trace toxic trace elements which are of interest, evidence for the heavy metal hypothesis of autism is now quite well-documented (but most often, again, in the grey literature) (1a). There seems to be an indication nevertheless from the totality of the internet-accessible reports (especially those which do not restrict their inorganic element analyses to the

amounts of the toxic elements) that ASDs are typified first of all by with major disturbances in the major essential elements, especially of Ca. Putatively, major diminution of Ca may define the typical form of ASD and an increase in Ca may define the Aspergers syndrome type of ASD. There is also a general tendency, for magnesium (Mg) to be deficient in all ASDs subjects and this is reflected in hair analyses. There are typically augmented toxic trace elements in hair from typical ASD subject (but perhaps not from Aspergers subjects)

Selected References
(1) Pivac N. et al. (Human plasma glycome in attention-deficit hyperactivity disorder and autism spectrum disorders) Mol Cell Proteomics 2011 10 M110.004200 ---------------------------------------------------------------------------------------------------------(1-1) Studies using Spisula and in a mouse animal models of autism of the effect of chloroform, bromoform and tetrachloroethylene (three commonly detected halogenated byproduct contaminants present [at very low concentrations] in some domestic water supplies) has indicated that these substances when acting tri-synergistically, but not singly or in pairs, can alter fetal nervous system development. This seems to point to a similar critical multi-system synergistic effect between toxins as being the origin of autism via a disturbance of nervous system developmental processes in human fetuses. The animal model halogenated substance autism-inducing effect seemed to be mediated via disturbance of cAMP-dependent protein kinase activity (Kreiling). This finding could further implicate cell surface and extracellular matrix (ECM) polysaccharide (heparan) dependent fibroblast growth factor signaling which is known to play a central role in embryo developmental regulation and for which the key ECM polysaccharide which is subject to alteration in primary or secondary structural alteration by disturbance of tissue redox state (and other effects of toxic metals and xenobiotics). [A similar intoxication by (municipal) water chlorinated byproducts during pregnancy which has been associated (Dodds) with the prevalence of stillbirth reinforces the notion that halogenated organic substances can induce nervous system defects causing human autism in human (animal studies (Kreiling) had indicated that a mixtures of three halogenated organic substances (chloroform, bromoform and tetrachloroethylene which can sometimes occur in municipal water supplies) are able to cause albeit at much higher concentration than occur in tap water, a disruption of the nervous system development of the fetus; a conceptually similar mechanism seems also to underpin the association between the halogenated organic substance intoxciation which has been associated with the prevalence of unexplained obesity in children (cf. Smink) as well as in adults (1.1) Walsh WJ Usman A Tarpey J Americal Psychiatric Association Annual Meeting May 2001 New Orleans (Disordered metal metabolism in a large autism population)

[499/503 patients showed evidence of altered blood metal ion values especially evident for elevation of the Cu/Zn ratio which was 1,78 for the ASD subjects but was 1.15 for healthy controls] {The central role of Cu and Zn in nitric oxide dependent HS recycling (cf. Mani et al.; Cheng et al.) putatively extends the alteration in the ratio of these elements as likely causing a major shift in the HS oligomer tissue protection and wound healing process (including that of the kidney and liver)}. (1a) When HS becomes structurally defective it is predicted to fail as a protective sequester of toxic inorganic ions (and transporter of such ions into the bone reservoir) this will putatively also diminish the protection afforded by HS for protection against pathological crystal formation. The heparanome is an information-encoded polysaccharide [heparan sulfate, {HS}which is now known to play a prime role in animal development. This signaling system is redox status sensitive and metal ion dependent which seems to allow for a designed response to the environment but when HS encounters anthropogenicically produced substances which were ( present at much lower than the current concentrations in the pre-human environments in which HS systems evolved (at the same time as the first multicellular animals), the HS-dependent tissue protective mechanisms can be overwhelmed. This allows pathological conditions to result from halogenated organic substance acting synergistically with toxic (e.g. heavy metal) intoxication ASD may further arise following pathogen endotoxin induced alterations to HS biosynthesis either directly or via the modulation action on HS biosynthesis of excessive nitric oxide formation (which also gives rise to a characteristic nitration of tyrosine, a phenomenon which accompanies found a wide range of diseases (cf. Ischiropoulos) including those which are thought to arise by anthropogenic (xenobiotic) organic substances synergism with overloaded redox ion copper, which can be enhanced by the presence of ultratrace amounts of aluminium, antimony, barium, lead (and perhaps also mercury) ions (2). 1-a Grant D
Grant D 1 Grant D et al., Biochem J. 1989 259 41-5 (Inhibition by glycosaminoglycans of CaCO3 (calcite) crystallization) [The anti-calcification effect of heparin and HS was similar (and also similar to that of ethane hydroxy, 1-1- diphosphonate, a commercial calcification inhibitor used e.g. to protect oilfield boreholes. {In a primate model of atherosclerosis such oilfield anti-calcification agents were found to de-plaque arteries suggesting that the use of bisphosphonates as anti-cancer agents might partly be due to their abilities to diminish pro-inflammatory pro-cancer effects of pathological sparingly soluble calcium salt including urate crystallization; a similar therapeutic effect has been indicated to be of value in protecting bioprosthetic heart valves (cf. Science 228 190). This might indicate the possible use of bisphosphonate a reportedly very well tolerated drug system in the therapeutic intervention in the autistic disease process That bisphosphonate therapy is of wider possible benefit than the current use for the treatment of osteoporosis (cf. the old work on how osteoporosis inhbits arterial calcification) was indicated by an epidemiological survey by J Center and J Eisman of Garvan Institute, Sydney Australia who found that

patients who had received long term bisphosphonate therapy inexplicably lived five years longer than controls. A possible explanation was the role of bones as repositories of toxic metals. Cf . web.eva-news.com/health/osteoporosis/osteoporosis-bisphosphonates-can-add-five-years-to-yourlife-scientists-announce-surprising-discovery/2822412; cf. J Clin Endocrinol Metab. 2011 96 1006-14. Another facet of bisphosphonate is that it is a metal ion chelator (which somewhat resembles EDTA in having high association constants but differs from EDTA in also behaving as a potent crystallization inhibitor of a similar potency per weight as HS (cf. Grant-1) ; perhaps the chelation and anti-crystal (anti-inflammatory) efficiency of bisphosphonate drugs merits their trial as anti-ASD agents.

Preliminary indications (made by Grant D. in the former polysaccharide research laboratory located at the former Marischal College campus of the University of Aberdeen) from the observed ability of cation exchange resin multi-inorganic element removal effectiveness indicates that antimony is more resistant to removal and therefore more strongly bound than other elements present. This metalloid has generally been considered to be of a similar high toxicity to animals and humans as arsenic (while arsenic is now thought to be biologically essential to humans in small amounts, at relatively low levels it becomes highly toxic a well known historical discussed phenomenon, which is thought to be facilitated by the binding and alteration of the activity of S-H groups in numerous proteins (cf. Yan et al.).
Confirmation of the ability of heparin (analogously to other natural anionic polysaccharides) to simultaneously bind all elements which occur in seawater can be deduced from a consideration of mass spectroscopic multi-inorganic element profiles (published on the internet of nitric acid leachates from heparinized blood sample containers; the use of polyethylene terephthalate produced using an antimony based catalyst seems however to increase the relative amounts of antimony in some of these profiles). Evidence that the use of hair element analysis for the indication of the occurrence of toxic metal overload in animal organisms might be questionable is countered by a 1985 University of Aberdeen obtained result of the inorganic element profile of heparin (a mast cell product and putatively an indicator of the multi-element content of animal blood serum and commonly used model of HS heparan sulfate a major cell surface polyanion receptor) has been found to be correlated (a linear log-log correlation which also mimics a similar correlation which can be demonstrated with the inorganic elements in seawater) with that of human hair most especially that of boys (cf. D. Grant, {Marischal College, University of Aberdeen studies discussed on the internet} In this plot, however, Zn was the only inorganic element studied which was markedly displaced from the exponential correlation; the amount of Zn in hair seemed to be much greater than that predicted from the correlation). Grant D Research Interests Continued from Aberdeen University Researches Cf. listed references by Prof W.F. Long
[Natural heparin-like /heparan sulfate (H/HS) polyanions which occur at the cell surface and extracellular matrices of animals apparently are multi-nutrient but also toxic inorganic ion chelators analogously to bur seeming to have overtly lesser but still physiologically relevant binding constants for individual counterions than does EDTA (and therefore any side effects cause by too much chelation of essential elements which might occur with EDTA chelation therapy is avoided). Animals have however evolved more effective polyanionic polysaccharides for binding metal than those employed by bacteria or algae (which include the well known algal polysaccharides (e.g. alginates and carrageenans) which have previously been observed to behave as full metallome ligands which putatively act in vivo primarily as essential nutrient gatherers from the seawater bathing fluid. This type of function early on in animal evolution may also have been that of H/HS (that this activity may remain of physiological relevance is suggested by the report that a range of 15

present-day aquatic invertebrates contained of cell surface HS and related ligands in strictly mathematically determined amounts (an exponential function of the salinities of their various habitats).

Grant D Chemistry Preprint Archive Volume 2000, Issue 10, October 2000, Pages 94-103 (Multi-ion content of heparin) [The relevance of the submitted prepint arose out of recent seminar discussions of redox metals in heparin/heparan sulfate which might be involved in catalysis of nitric oxide metabolite nitrosylation and deaminative cleavage processes of relevance to degenerative diseases] CPS: biochem/0010002 [download achieved on 10 October 2000[ from web.preprint.chemweb.com/biochem/001002) [The most strongly bound inorganic elements to this heparin (surrogate for heparan sulfate) sample determined by comparison of the SSMS mass spectrometric analysis using a cation exchange resin Tl+ ion replacement procedure cf. Grant D et al., Biochem J. 1987 244, 143-9 were (the ratio before and after Tl replacement being shown in parenthesis) Sb (1.2) ,Ce (2.0), Pb (4.), Nd (5.), La (7.), Ag (8.), Zn (11.), As (15.), Zr, (16.6), Ni (17.) cf. however, Ca ca. 1000Cu (>146), Si (ca. 100), K (ca. 50 [a misprint in the above ref value of 140 form K in Tl heparinate should read 40] {a general principle could be that major physiological ions are relatively weakly bound to heparin/HS by comparison with non-physiological elements for which heparin or HS binding may provide for a physiological safety net filter {this may include As which seems to be essential but is highly toxic at superoptimium levels). This filtration activity could be come depleted as a result of the type of chronic nitrosative stress which is thought to cause extensive tyrosine nitration. The putative role of Cu toxicity in autism and other diseases could result from the ability of Cu to catalyze de-N-sulfonation of heparin perhaps in a chlorinated organic cofactor mode. [Chlorinated organic cofactors (e.g., carbon tetrachloride or butyl perchlorocrotonate) activities are used industrially for obtaining high yields in Ziegler Natta-type polyolefin preparation (cf. Grant D. et al. Eur J Polymer. 1974 ) by a redox recycling mechanism which is reminiscent of the Cu-dependent redox recycling mechanism of reaease of nitric oxide from cysteine stores in the nitrosative oligosaccharide signaling mechanism (cf. Ding et al., J.Biol Chem 2002, loc. cit ). This may, it is now suggested, become defective due to cofactor overload in autism]. Evidence for inorganic cofactor overload in autism The putative ability of chelating agents used by alternative medical practitioners in attempt to remove toxic metals from autistic subjects seems to approximately correlate to the above order of binding to sulfated polysaccharide (model of ECM and cell surface glycocalyx) which is therefore confirmed as a component of the reservoir of toxic metals in humans. A large number of hair analyses are available on the internet but there is no at first sight easy correlation between hair element suggested intoxication and the existence of autism in the subject. Nevertheless there seem to be general fuzzy logical conclusions to be made. One possible highly commonly but not ubiquitously encountered element in autistic subjects is Sb. Cf. also (More on Mercury Intoxciation cf. e.g., (Amalgam Illness ) web noamalgam.com [It can be logically suggested (cf. Cutler AH) on the basis of published researches that Hg intoxication may be highly relevant as causes of numerous human illnesses; these may include attention deficit hyperactivity disorder, fibromyalgia, chronic fatigue, anorexia, recurrent depression, Alzheimers disease, Addisons disease, Parkinsons disease, psychoses, rheumatoid arthritis, schizophrenia, scabies sleep disorders etc. etc.] Cf. also Shaw W et al. (Biological Treatment for Autism and PDD). Cf. Hall loc. cit 911 Mystery illnesses and heavy metal intoxication dust which was produced by terrorist fire demolition of the World Trade Center. The apparent mystery illnesses may have arisen not solely from the irritating slow cancerogenic effect of crysotile asbestos dust but also and perhaps principally by the organochlorine (especially dioxinPCB heavy metal) HS tissue protection disruption mechanism N.b. Sb was reported to occur in dangerous amounts in 911 dust (together with large amounts of asbestos Hg, Ba and Pb of the most toxic inorganic (which also included Zn, Co, Cu and Mo)

components in a garment held sample collected by Yehuda Kaplaoun analysis arranged by the New York Post (cf. web. davidworbyproductions.com/news/nypost-040906.pdf) Possible public Sb intoxication is also indicated by data presented in the official (internet published) documents e.g. web.tera.org/peer/WTC/COPC%20-%Benchmark%20Report%20with%20appendices.pdf and web.cdc.gov/niosh/docs/2011-197/pdfs/2011-197.pdf

1-a-1 Rudd et al, papers on heparan sulfate (see internet, Glycobiol Carbohydr Res 2009) ; (also Ed. Yates, University of Liverpool, personal communication)

Cf. an earlier review by Coombe DR, Kett WC (HS protein interactions: therapeutic potential through structure-function insights Cell Mol Life Sci. 2005 62 410-24) which briefly mentioned the dependence of HSPG signaling on metal ion cofactors. Previous indications that a range of M3+ cations (of Fe, Al, La, Ce, Er, Sm, Yb, In, Ru, and Eu) as well as Fe2+ had a strong affinity for HS (deduced by their exchangeability with 45Ca2+) were indicated by Kojima S et al. (Elevated uptake of 67Ga and increased HS content in liver-damaged rats) Eur J Nucl Med 1983 8 52-9) It is perhaps somewhat surprising that anions (e.g. SO42-) also have a strong affinity for HS. This must be assumed not be accomplished by an electrostatic mechanism which evidently facilitates the binding of Sb (as well as As, P and B) to HS. 1-b Cohen D et al. J Autism Dev Disord. 2005 35 (1) 103-16 (Specficicgenetic diseases and autism: clinical contribution towards their identification) [The following genetic diseases are associated with autism symptoms, viz. Fragile X, tuberous sclerosis, Angelman syndrome, Duplication of 15q11-9B. Down syndrome, San Filippo syndrome, MECP2 related disorders, phenylketouria, Smith Magenis syndrome, 22q13 deletion, adenylsuccinate lyase deficiency, Cohen syndrome, Smith-Lerndi-Opitz syndrome (caused by cholesterol deficiency)]; cf. also Schiff M et al., PloS one 2011 6 (7) e21932 (Should metabolic disease be systematically screened in non-syndrome autistic spectrum disorders?) [Ca. 10-20% of cases of ASD can, it is now believed be attributed to known genetic defects. This means that 80-90% of ASD might similarly arise from HS defects (i.e. alterations in specific anionic pattern sugar sulfate codon specific information held in the HS tissue regulator which may, e.g. arise as is also the case with DNA from high energy radiation production of hydrated electrons, or insult by non-physiological molecules which are now becoming universally deposited in human and animal tissues) It should be noted that Schiff et al. indicated that metabolic screening is unlikely to contribute to the causative diagnosis of nonsyndromic ASD]. (2) {Cf. it has been suspected that the etiology of autism partly depends on the perturbing effect on zinc and copper balance of lead and other toxic metal and metalloid

intoxication. This is in accord with the ability of toxic inorganic elements to interact negatively with heparan sulfate dependent systems involved in tissue protection and in tissue development, wound healing, antioxidant defense, anti-pathogen defense, haemostasis and lipid metabolism etc}.
__________________________________________________________________________________ _______________________________________________________

14.2 References (General), cont. Articles designated in the text by the first named author, arranged alphabetically below
Adams JB. et al. Nutritional and metabolic status of children with autism .. [n=55] Nutr Metab (Lond). 2011 8 (1) 34 Cf Analysis of toxic metals and essential minerals in the hair of Arizona children with autism .Biol Trace Elem Res 2009; cf also the ten fold increase in bottled water consumption coincides nearly exactly with an apparent tenfold increase in autism over the same time period [web .greatplainslaboratory.com/home/eng/lithium.asp] this may be a consieqnce of ultralow Sb present as a PET catalyst residue; cf internet reports of (preliminary results from?) hair analysis apparently conducted by G Bell (Scotland) which indicated that elevated Sb seemed to be associated with autism) Adjarov D et al., Toxicology, 1982, 23, 73-7 Aubrey NE and Van Wazer JR J Amer Chem Soc 1964 86 4380-4383 Cf. also Grant D. J Appl Chem Biotechnol. 1974 24 49-58 Lenoir D et al., Chemosphere 2001 43 107-114; Taylor PH Lenoir D Sci Tot Environ 2001 269 1-24 and Choudhry GG Hutzinger Curr Top Environ Toxicl Chem 1983 Vol. 4 (Gordon and Breach Science Publishers); Barber J (Eurochlor representing the chlor-alkali industry) web.eurochlor.org/upload/documents/document187.pdf and refs cited therein.

Cf. e.g., Oberg T & Bergstrom JGT (HCB as an indicator of dioxin production from combustion) Chemosphere 1985 14 1091-6) Angyal SB. Chem Soc Rev .1980 9 (4) 415-28 (Haworth Memorial Lecture. Sugar-cation complexes-structure and applications) [This review which is restricted to polyols (which are much weaker ligands than the polyanionic polysaccharide starts which are not discussed) starts with the observation that since under physiological conditions, sugars occur in solutions which also contain salts, it is relevant to query whether any association exits between these two different types of compoundsover the years to 1980 sufficient evidence had accumulated to indicate that at least some sugars form complexes with some cations. (When this subject was reviewed in 1966 172 references could be cited but it was not clear which sugars from complexes with which cations? What are the structure of the complexes? What are the configurational or conformation features required for complex formation?). By 1980 it had been established that certain optimal arrangements of COH in neutral sugars allow for the weak-moderate strength (but physiologically relevant) binding of physiological cations ( e.g. for epi-inositol the Ca2+ complex has a stability constant of 3M-1).

The high abundance of sugars in biological media indicates that the low-medium affinity of multi-valent cations for polyols will provide for a natural primitive homostasis system form such cations].
Bailley-Hamilton P Altern Comp Med 8(2) 185-92; Cf. Sect 2) [Evidence that POPs give rise to obesity might also suggest a further link to autism] Bakir A et al. Clean Soil Air Water 2009 37 (9) 712-9 DOI: 10.1002/clen.200900164 (Competetive sorption of antimony with zinc nickel and aluminum in a seaweed-based fixed bed sorption column) [The putative polyanionic polysaccharides of Ascophyllum nodosum used sulphonate, carboxylate, alcholic and ether ligand sites for the binding of the studied element removal from water where the presence of Sb(III) was found to considerably inhibit the otherwise efficint ability of the seaweed (probably via polysaccharide polyanions) to bind Ni(II), Zn(II) and Al(III) efficiently. This antagonistic effect of Sb(III) was however absent with the polyanions of Polysiphonia lanosa (which had a greater array of weak acid binding groups in addition to sulphonate and carboxyl ). It might be conjectured that the behaviour of A. nodosum more closely resembles HS than does P. lanosa. This might suggests that Sb binding to HS could perturb the normal binding of physiological elements to this animal equivalent of the marine algal polysaccharides.

Bernfield M. et al. Ann Rev Biochem 1999, 68 729-77 Cf. also Perrimon N and Bernfield M. Nature 2000, 404 725-8 and Bishop JR. et al. ibid., 2007 446 1030-7 Bernhoft R Buttar R. Autism: a multi system oxidative and inflammatory disease Townsend Letter 2008 April 86-90 [Other grey literature surveys relating to autism which are probably the most useful include Jonathan Tommey Report web.theautismline.com/autismfilearticles/autismfile10.pdf and Willis S. Langford A Comprehensive Guide to Managing Autism [KlickStart.doc April 8,2002] Bhaki JS et al. Microchim Acta 62 (5) 909-13 (Sensitive colorimeric method of microdetection of flavenoids) [Sb(III) forms a colored complex with flavenoids; this allows the detection of quercetin at 0.4-6g/ml] {Cf. Oral quercetin was used as an antidote to ME/CFS by a biochemist (Vance Spence University of Dundee, personal communication) who personally suffered from this illness; this substance available in a pure form was found by him to be the most useful therapeutic agent which he had tested for the alleviation of his symptoms. Could the ability of this antioxidant to act as a chelator of heavy metals and metalloids such as Sb by relevant to this finding?} Birchall JD. (The role of silicon in biology) Chem Brit. 1990 26 (2) 141-4 [Hypothesis that adequate inorganic Si nutrition is required to counter Al intoxication; cf. rats on low Si diets accumulated Al in their brains] Cf. also ibid., 1983 19(1) 18-19 (Al and Alzheimers) ibid., 1994 30 (6) 470 [Birchall wrote of the precipitate rejection of the aluminium hypothesis in consequence of a far from definitive experiment has led to a reduction in suport for research on the role of this element. The failure to detect aluminum in Alzheimer brains was stated by Birchall to have been due to an inappropriate choice of insufficiently sensitive method (scanning proton microscopy) which was incapble of the task in hand This what now seems to be a major error of scintific judgement and also seems to have been responsible for the long delay in acceptance at least by some of the scientific community, that the original hypothesis that aluminum intoxication contributes to Alzheimers disease (cf. e.g. the Kawahara et al., loc. cit., which strongly argues that the aluminium hypothesis is essentially correct. Birchall had noted that low levels of Al which do not show up by scanning proton microscopy (e.g. 1010 M) are known to effect tubulin aggregation; at 10-6M Al can have aprofound effect on InsP3-evoked Ca2+ signaling. In suport of the possible role of Al intoxciation in AD is the correlation between the prevalance of AD and the Al content of water (which however might be controlled by the amount of Si in water which determined the bio-availabilty of Al cf. Edwardson JA .Lancet. 1993 342, 211 cf. ibid. 1994 343 3). Another indicator of the likely role of Al in promoting neurodegenerative disease was the ability of Al to alter the processing of tau protein also seen in brains of renal dialysis patients who accumulate Al.]. {The ability of HS to bind Al3+ may, however, be part of the defense against Al uptake (e.g. the HS in the BBB performing this function). A corollary is that excessive nitrosative stress which can depolymerize HS might diminish this. Si is also commonly associated with HS and an adequate amount of Si might be requried for HS barriers to perform an effective Al3+ dequestration function; the ability of Si to protect tissues could however be dependent on the absence of a chronic elevation in redox balance}. It should be noted tht Al was elevated in the hair of n=32 autism subjects (av. value 10.7ppm vs. controls 6.3 ppm) but not in n=8 Aspergers subjects (4,3ppm) (cf. Hall). Although an inverse relationship was found between municipal water Al and Si, a correlation between these data and the prevalence of AD could not be established Forbes WF McLachlan DR. J Epidem Community Health. 1996 50 401-3 suggested that a lack of correlation between Al and AD indicated by e.g. Taylor et al. ibid., 1995 49 323-4 compared with

positive correlation found in Ontario where the Al loading was greater could be accounted for by this 9ncreased Al inotxication potential in Ontario which was also, however, apparently affected by other factors putatively the presence of F-. Jacqmin-Gadda H et al Epidemiology 1996 7 281-5 d found an association between cognitive impairment and Al depended o the pH of the available drinking water and the concentration of Si therein related to how high Al levels, when Si levels are low, could have a deleterious effect above a threshold Al concentration level of 3.5g/L. [Later workers (Bocca et al, cf. Brunisma) demonstrated that AD could be diagnosed on the basis of blood serum measurements of Al, Ca, Co and Si (and related overproduction of oxidant species)] {This could suggest that the etiology of AD is HS-driven Al/Si related process which might also explain why HS-related therapy has potential therapeutic value for AD (cf e.g. , Eur Pat. 0293974. There is concern about the presence of Al in some HS-related pharmaceutical agents cf. Bohrer et al. loc cit. {further refs relating to HS and AD: Ferroro ME et al. Biochem Soc Trans. 1989 362; Lorens SA et al. Semin Thromb Hemacrit. 1992 17 (Suppl 2) 104; Kilsilevsky R et al. Nature Medicine. 1995 1; Taylor GA et al. J Epidemiol Commun Health. 1995 49 (3) 323-45; Snow AD et al. Am J Path. 1988 133 (3) 456-63, cf. ibid., 1990 137 (5) 1253-70; 1994 144 (2) 337-47; ibid. 1999 155 (6) 2115-25 (HSPG iin AD is agrin; Multhaup Get al. Biochemie 1994 76 (3-4) 304}. Cf also Cheng et a. Bishop JR et al. Nature. 2007 446 1030-7 (Heparan sufate proteoglycans fine-tune mammalian physiology) [The anionic patterns present in the polysaccharide side chains of HS proteoglycans provide a postcode-like information transmission system which had been named the heparanome by Turnbull J et al. Trends Cell Biol. 2001 11 (2) 75-82 (The heparanome seems to provide a self-regulating system capable of wide-ranging high level system management behaviour which can affect the cellular immune response, cardiovascular function, wound healing as well as energy belance. Cf. also e.g. Bernfield M et al. Ann Rev Biochem. 1999 68 729-77; cf. Nature 2000 404 725-81 and Guimond S et al. Biochem Soc Trans 2001 29 (20) 177-81. There are hints that many of the proteins which currently use HS receptors have evolved specifically to make use of the pre-existing library of such structure cf. Esko JD and Lindahl U. J Clin Ivest. 2001 1008 169-73 Cf. Feyzi E et al., J Biol Chem. 1998 273 13395-8 implicated heparan microstructural change central to the mechanism of aging (in animals including humans). EPIGENETICS Cf. Nature. 2011 477 cf. p534 Epigenetics explores how genetically associated non-DNA entities whether cells or the whole organism, display different characteristics and how these are inherited. The old standard view that DNA is always the ultimate central manager of the cell needs to be reassessed. This has downgraded the role of the gene. In an analogy which pictures the cell as a theatrical production in which the gene was traditionally regarded as the director now required that the gene is seen to be an important actor but only one of the entire cast of actors. Currently epigenetics is focused on DNA methylation and histone acetylation. It should be noted however that HS occurs at the nucleus in mammalian cells and could act as a major system manager (i.e. is a major player in epigenetics). Cf., e.g. oocyte HS it is believed to orchestrate sperm decondensation (protaine replacement by histones) (cf. Romanoto et al. loc. cit). Nuclear HS (and the diminution of HS by heparanase) is thought to enable a system of HS control of gene expression (cf. Chen L et al. loc. cit.) The common reflections on epigentics taken from popular reviews of the subject however generally do not mention HS. Since this complex information processor e.g. is able to selective bind and remove chromatin from DNA). HS is a prime candidate for researchers aiming to find new therapeutics for such diseases as ASD, Alzheimers and some or most types of cancer. Discussions of epigenetics should be read after also reading e.g., Bishop JR et al. loc. cit which seem to imply that HS is now the prime candidate for the role of director of (adherent) animal cells. Cf. also Prydz K Dalen KT loc. cit. Bishop NJ et al., New Engl J Med. 1997 336 1557-62 (Aluminum neurotoxicity in pre-term infants receiving intravenous-feeding solutions) [In randomly assigned n=90 vs. control infants selected from 227 preterm infants with gestational ages less than 34 weeks and birth weights of less than 1850g who required intravenous feeding, showed that

the use of an Al-depleted nutrient improved mental development relative to a standard (Alcontaining( feed. Mental Developemnt Index of 102+17 vs. 92+20 appeared to be atributatable to Al intoxication, i.e,. a prolonged intravenous feeding with solutions containing Al impairs neurologic development {Cf. comparison (by Bocca B et al., Ann Ist Super Sanita. 2005 41 (2) 197-203) of the amounts of 26 metals in whole blood and serum of 20M + 40F Alzheimer patients compared with 44 control subjects indicated that metal ion dyshomeostasis is a characteristic feature of this disease which is associated with elevations in Al (as well as Cd, Hg and Mn) (n.b. all of these metals have been indicated to be neurotoxins) in combination with abnormal amounts of Ca, Cu, Fe, Mg and Zn; this metal ion imbalance apparently depleted antioxidant capacity; previous studies had established that there is a defined role for Al in the AD-like illnesses which had been found in early kidney dialysis treatment patients (cf. A. Sanz-Medel. Analusis. 1998 26 (6) M76-80). {N.b. Al has been found to be elevated in the hair of ASD but not Aspergers subjects (Hall, cf.Bell loc. cit)} Bode L et al. Am J Physiol Gastointest Liver Physiol. 2005 288 (5) G1015-23 HS depletion amplifies TNF induced leakage in an in vitro model of protein-losing enteropathy [This depletion of HS.may explain how leaky gut arises in autistic subjects; i.e. it arises via the increased TNF which has e.g. been reported to arise followoing stinulation of the gut by cows milk] Bohrer D et al. (Concern about the presence of alumnium in pharmaceutical agents) RBAC (Brasil). 2004 36 99-103 [Cf., ALS (internet documentweb.alsglobal.se/hem2005/pdf/blood_collection-tubes_eng.pdf) relating to multi-inorganic element ICP-MS leachates from heparinized blood collection vessels indicated that heparin tends to be enriched in Ag, Al, Ca, Fe, Pb and U]. Cf. ~WRL005 23 Sept 2010 www.scribd/doc/37999700/WRL005 ~WRL251 Revised 10 April 2010; ~WRL 3217 contiud. [Al3+ + 3F- AlF3OH may perturb G protein signaling n ASD] Borges FT et al. Kidney Int. 2005 68 94) 1630-41 (Crystallization and glycosaminnoglycans in tubular epithelial cells : calcium oxalate and excess ions effect) [Kidney tubule cells seem to increase the synthesis of HS glycosaminoglycans to protect them from the toxic insult of calcium oxalate crystals and oxalate ions] Cf, e.g. Matsuo M. Kurume Med J. 2008 55 (1-2) 1-28 and H Sidhu et al. Eur Urol. 1989 16 (1) 45-7 {The ability of HS also prevent CaCO3 crystallization but de-N sulfonated HS is ineffective cf. Grant D et al. Biochem J. 1989 259 41-5; The ability of selectively modified heparins as modulators of CaCO3 crystallization seems to exact match their ability to facilitate the decondensation of spermatozoa as reported by Romanoto et al. loc cit.} Bornehag CG et al. Environ Health Prespect. 2004 112 (14) 14 October (The association between asthma and allergic symptoms in children and phthalates in house dust: a nested case-control study) Bocca B et al. Ann Ist Super Sanita. 2005 41 (2) 197-203 Boso M et al., (Alterations of circulating endogenously secreting RAGE and S100A9 levels indicating dysfunction of AGE-RAGE axis in autism) Neurosci Lett. 2006 410 (3) 163-73

[The etiology of autism has been indicated to involve a subtle dysfunction of the AGERAGE S100A9; (Probably this is the S100A8/S100A9)

Cf., Advanced glycation end-products AGE (carboxylated proteins) are derived from chronic oxidative process non-enzymic involving Schiff base and Amajori rearrangements; RAGE is an endogenous signal transducing receptor for AGE]
{N.b., S100A9 and S100A8 bind with high affinity to heparin and HS}. Blocking of S100A8/S100A9 is a putative novel therapy for atherosclerosis (a disease which like autism is believed to arise from endemic oxidative stress). Substances which achieve this blockage might also be of value for the alleviation of the synmptoms of autism. Such blocking is likely be afforded by HS mimetics e.g. semisynthetic algal sulfated polysaccharides or SP54 (a birch wood HS mimetic) which has been in use for many years as an anticoagulant and also as a nhibitor of cystitis. It should be noted that SP54 has been found to be effective in the inhibition of neurodegeneration associated with CJD (cf. Highfield loc.cit.). Brasky TM et al. Cancer Cause Control. Jun 25 2011 PMID21706174 [Glucosamine dietary supplementation (but not chondroitin sulfate) was found to inhibit lung adenocarcinoma as indicated by a n=76, 904 survey by questionaire; this finding may indicate that HS boost consequent on this supplementation and the resultant tissue protection afforded is responsible for the apparent anti-cancer effect of glucosamine] Braun JM et al. (Exposure to environmental toxicants and ADHD in U.S. children) Environ Health Perspect. 2006 114 (12) 1904-9 [Blood Pb (as well as prenatal but not postnatal exposure to tobacco smoke) was predictive of ADHS; data for n=4794 subject children were obtained from NHANES 1999-2002 ). A number of other reported reviewed by Bellinger DC. (PMICD PMC3155319) confirms this conclusion regarding the link between Pb intoxication and ADHD] {Pb intoxciation is known to alter HSPG biosynthesis, e.g. by reducing synthesis of the required core protein, cf. Fujiwara et al. loc. cit.} Brunisma IB et al., (Sulfation of heparan sulfate associated with amyloid-beta plaques in patients with Alzheimers disease) Acta Neuropathol. 2010 119 (2) 211-20; PMID 19636575 [Altered sulfation of HS was associated with amyloid-beta plaques in patients with Alzheimers disease; these authors indicated that the formation of fibrils was promoted by sufation-this is an opposite effect to that by which increased sulfation diminishes inorganic Ca-related plaque formation Cf. also Timmer NM et al., Neurosci Res. 2010 66 (4) 380-9 found that heparin and HS were much more potent than other GAGs for the reduction of Abeta induced cellular toxicity and that the effectiveness of this reduction in toxicity was an important role in the etiology of AD and related disorders. The removal of sulfate groups abolished this ability. This reduction in toxicity may, however, have been achieved by a process by which these GAGs caused Abeta to aggregate. {De-N sulfonation of HS might, however, it is now suggested, be a key biochemical process in the etiology of Alzheimers disease} {It is likely that the catalytic effect of Fe and other acidifying counterions which cause selective deNsulfonation of heparin [observed in in vitro experiments conducted at Aberdeen University] will abolish the protective effect of heparin and HS in AD brains and that this is a possible part of the etiology of AD and related diseases} Cf also Bocca B et al., Cf. also Bue et al. loc. cit .who reported that binding of HS PG to Alzheimers amyloid is mediated in part by the N-terminal part of A4 peptide

Bue L et al.
(Binding of vascular heparan sulfate protoeglycan to Alzheimers amyloid precursor protein is mediated in part by the N-terminal region of A4 peptide) Brain Res. 1993 627 (2) 198-202 Cf. also Watanabe et al. loc. cit. who reported that glypican-1 probably facilitates amyloid deposition in Alzheimers disease brains.

Tornberg J. et al. PNAS. 2011 108 (28) 11524-9; cf. Blow HE et al., Neuron 2004 41 (5) 723-6; Van Vactor D. et al., Curr Opin Neurobiol 2006 16 (1) 40-51and Mendez-de-Aguiar CBN. et al. Cell Mol Neurobiol. 2008 28 (6) 795-80 Canadian Gov Report (Canadian Update on PCBs Furans and HCB) Canadas Update.on.Emissions_Inventory_findings Canadas Update on Dioxins/Furans and HCB Emissions Inventory CEC North American Strategy for early Zero Compliance in Dioxins Furans and HCB December 10th 2009 Mexico City Mexico (Downloaded from internet 3 August 2011) [Whereas of the top 10 sources of dioxin/furans {an also putatively HCB} emissions from MSW incineration and iron and steel manufacture has declined between 1990-2007 and that from backyard trash burning has remained relatively constant, that from HD diesel engines has increased] Canzoniero LMT et al. (Involvement of nitric oxide/protein kinase G pathway in polychlorinated biphenyl-induced cell death in SH-SY 5Y neuroblastoma cells) J Neurosci Res. 2006 84 692-7 [PCB intoxication dose dependently caused a Ca dependent putative neuroNO synthase related damage to cells] Dioxin and PCB may diminish cytokine production in such a manner to reduce airway inflammation (the opposite of what has been anticipated) as indicated by Devos S et al., Eur Cytokine Network. 2004 15 (20 145-51] Cappai R. et al., J Biol Chem. 2005 280 (14) 13913-20 (The amyloid precursor protein (APP) of Alzhheimer disease and its paralog, APLP2, modulate the Cu/Zn nitric oxide catalyzed degradation of glycpican-1HS in vivo) [The research reported in this paper which is possibly relevant to an improved understanding of the etiology of Alzheimers disease may also be of value for allowing a greater insight into the etiology of ASDs. This paper shows that a functional relationship exists between the HS and Cu ion binding activities of APP/APLP2 and that this controls how hormone-like HS fragments may arise from the HS side chains of glypican-1; this mechanism is putatively part of an ultra-epigenetic system of HS-gene control system. It is now suggested to be ultra-sensitive to perturbation by xenobitoics and this allows very low tissue loading of ultratoxic substances to promote an altered central and peripheral nervous system development and function. This is clearly possibly relevant to ASD; {A further aspect of nitrosative signaling in ASD and AD is that nitration of tyrosine occurs which points to a defective regulation of nitric oxide signaling. The disturbance of the cellular immune system in ASD which disturbs the Th1-Th2 balance as indicated by the cytokine profiles) in ASD and chronic fatigue syndrome suggests that both conditions evidently could ultimately arise from a similar proinflammatory synergistic interaction between chlorinated-aromatic plus heavy metal xenobiotic intoxication cf. also Cardenas et al. vide infra for other (also putatively ASD) related heavy metal HS signaling perturbation effects. Cardenas A et al. Toxicology. 1992, 76, 219-231 [Cd2+ diminishes HS sulfation] Cf. Templeton DM Proc Trace Element Health Disease IUPAC Symp 1990 p. 209-219 [Hg, Cd, Mn, Ni and Pb perturb HS biosynthesis] Cf. also Furukawa et al. loc cit. Inorganic Si may also be required for correct HS biosynthesis (cf. McCarty loc. cit.) Corrigan FM et al. (Multiple element analysis of the frontal cortex, temporal cortex and basal ganglion in schizophrenia) Trace Elem Med. 1990 7(1) 1-7; Chem Abs. 114 21843q There was an apparent average schizophrenic vs. non-schizophrenic brains (frontal cerebral cortex) decrease in In 0.00610.0012 vs. 0.00740.007 ppm and also a decrease in Ce 2.330.47 vs. 2.71 0.39 ppm [There, perhaps surprisingly, is a possible tissue protective role of Ce(IV) oxides nanopoarticles as free-radical scavengers in the brain cf. Tsai Y-Y et al. Nanomed 2007 2 (3) 325-32; n.b. such particle have evidenced neuroprotection in adult rat spinal cord neurons cf. Das M et al. Biomaterials. 2007 28

(10) 1918-25; CeO2 coordinatiom could be HSPG as indicated by the difficulty of ion exchange removal of Ce from heparin (Aberdeen Unversity research results which indicated that Ce becomes uniquely storngly bound to heparin and by inference to HS by comparison with most other inorganic elements with the exception of Sb and possibly Cd] Corrigan et al. in Eur. Pat. Applicn. 0298644 (1989) suggested that dietary supplementation by Ce (e.g. as salts of n-5 and n-3 essential fatty acids which were also significantly reduced in schizophrenic brains) might be a therapy for schizophrenia. Cf. also: internet document (Grant D.) web/scribd.com//The-Possible-Relevance-of-theNeuroprotective-Properties-of-Ce. Which discussed how Ce attached to HS might be of value as an an antioxidant. Center JR et al. J Clin Endocrinol Metab. 2011 96 1006-14 doi.10.1210/jc20102739 [This paper reported the unexpected finding from epidemiological evidence that bisphosphonate therapy (for osteoporosis) inexplicably increases the recipient life span by five years] {Cf. the discussion of these results by Grzbek E., February 2 2010 (web.eta-news.com/health/osteoporosis/osteoprosis-bisphosphonate-can-add-five-years-to-your-lifescientists-announcesurprsing-discovery/2822412) Bisphosphonates can add five years to your like-scientists announce surprising discovery made when 2000 people were treated with bisphosphonates for 3 years. When compared with other sub-groups taking other forms of treatment, such as Vitamin D (with or without calcium) or hormone therapy, the longer life-associated with bisphosphonate treatment was marked and clear. The findings applied equally to male and female. In a group of women with osteoporotic fracture over the age of 75, the expected mortality rate over a period of five years of 0% dropped to 10% and for a group of younger women patients with an expected mortality rate of 20-25% this add decreased to zero. The mechanism of the life extension was speculated to be something to do with the fact that bone acts as a repository for toxic heavy metals such as lead and cadmium. The effect of bisphosphonate suggested here is that it has the ability to prevent the re-dissolution of bone which stops the release of bone-held toxins. Since the same toxic metals are also putatively involved as causative agents in the etiology of autism this might promise some similar beneficial bisphospohnate therapy might also be possible for autism. The lack of an effective bone transport and reservoir function as a key etiological event in autism might be remedied by some designed phosphonate-related therapy. Greater success of chelation of toxic metals in autistic subjects might be achievable if an agent which also positively affects bone biochemistry. EDTA is likely to lack this additional ability. The use of silicate dietary supplementation for racehorses to improve their bone structure might also conceivably be why silicate dietary supplementation might also be of benefit for the treatment of autism. The mechanism by which heavy metals are secured in bone may involve their transport via HS . It was observed that intact HS and bisphosphonate exhibited similar abilities to inhbit the formation of CaCO3 (calcite) a putative model system for hydroxylapatite crystallization a model of bone formation. (HS also is able to modify hydroxylapatite crystallization and in so doing is now thought to be a major control agent for bone [calcium hydroxylapatite] formation (cf. Reid DG et al., Chem Mater 208, 20, 3579-86 cf., ibid., 2010 6109-16) who found that sulfated polysaccharides, rather then the previously believed protein systems, were the major control agents for this process; cf. also Grant D et al Med Hypoth 1992 38 49-55 whee on p 52 the Van Wazer The Chemistry of Phosphorus textbook notes that apatites often approximate to a unit cell of stoichiometry M10X2(PO4)6 where M=Ca, Pb, Na, K, Sr, Mn, Zn, Cd, Mg, Fe, Al carbonate and the rare earths especially Ce and PO4 may be replaces by AsO4, VO4, SO4 and SiO4) . [N.b. Sb(III) (and Mn2+) doped calcium halophosphors (obtained from calcium chlorapatites are structurally related to the apatites) are widely used (in fluorescent mercury vapor lamps) the Sb being a likely P substituion atom, [cf. Hoekstra AK. Doctoral thesis, 1967, web.alrxandria.tue.nl/repository/books/23899.pdf]. The ability of Sb to alter the physiochemical characteristics of biologically produced apatite like minerals is worthy of further study. E.g. has this any relevance to the etiologies of e.g. autism; or adverse physiological outcomes of RF radiation etc? Hydroxylapatite column separation is effective for fractionation of nucleic acids and also glycosaminoglycans. For heparin a peptization process also occurs in which colloidal hydroxylapatie particles occur attached to the heparin which demonstrates a superposition of phosphate and sulfate infrared anionic group vibrations the latter being shifted to lower energy.

(related studies of the binding of a range of metal ions to heparin showed that no similar alteration of sulphate vibrations similar to those found for hydroxylapatite particle interaction, occurred (the heparin carboxylates were however now altered systematically). A large diminution of the ability to modulated CaCO3 calcification processes was observed following the partial structural modification of native HS (cf., Grant D et al. Biochem J 1987 259 41-55; cf. also Med Hypoth, 1992, 38 49-55). The most dramatic effect was seem when the N-SO3- groups were removed from HS when the ability of HS to inhibit the formation of CaCO3 was abolished

{N.b. HS is also a key system manager is known to engage in multi-inorganic ion dependent biochemical mechanisms which putatively include the prevention of damaging effects of toxic metal ions; HS is putatively also a prime metallomic matrix which is highly implicated in the embryonic and postnatal developmental biology which becomes perturbed during the autism-inducing intoxication processes}.
Chen AY et al. (Prevalence of obesity among children with chronic conditions) Obesity (Silver Spring). 2010 18 (1) 210-3 BMI indicated obesity prevalence was found to increase from 12.2% in normal (age 10-17 n=467007) children to 23.4, 19.7, 19.3, 18.9, 18.4 % for autism, asthma, learning disability, attention deficit disorder, and hearing/vision impairment respectively, i.e. autism has a more pronounced tendency compared to these other chronic conditions to be correlated with obesity. Cf. also Curtin C et al., BMC Pediat. 2010 10 11 (cf. ibid. 2005 5 48) Who found that (for a studied n=86272 age 3-17 population database) the prevalence of obesity in children with autism was 30.4% compared to 23.6% in children without autism. {this baseline prevalence seems however to be very similar to that found for normal subjects in the above Chen et al. study but this can probably be attributed to a difference in their treatment of the data}. Further indication of the validity of the autism-obesity linkup was provided by the work of Mills et al. [Clin Endocrinol (Oxf). 2007 67 (2) 230-7)] who also implicated a role for insulin (like)growth factor IGF activities in the observed correlation between autism and obesity. (cf. also Van Cleave J et al. JAMA 2010 30 (1) 70-6; Xiong N et al. Res Dev Disabil. 2009 30 (1) 70-6; Whiteley P et al. Pediatr Int. 2004 46 (5) 531-7). It should be noted that HCB intoxication has been associated with perturbation of IGF signaling during HCB co-carcinogen action. (cf. Randi et al. loc.cit.)

Chen L et al. (Heparanase regulates levels of syndecan-1 in the nucleus) PloS ONE 2009 4 (3) e4947 PMC 2654539 [Although HS function at the nucleus is poorly understood there is emerging evidence that it may act to repress gene expression; this includes VEGF which is thought to be incorrectly regulated in ASDs]. The presence of HS at the nuclear membrane and at the nucleus and also the ability of HS fragments to travel from the cell membrane to the nucleus allows information held in HS sequences (the heparanome) to putatively direct gene expression and indicates that HS may be the master system overlord of DNA and hence the prior excessive interest in DNA defect as what leads to cancer can be suggested to be a partly erroneous assumption. In order to obtain effective therapeutic intervention in cancer and putatively also in ASD it us suggested that first of all it will be necessary to achieve a greater understanding of the HS high level manager system]
Cheng F. et al. (Suppression of amyloid beta A11 antibody immunoreactivity by vitamin C: possible role of heparan sulfate oligosaccharides derived from glypican-1 by ascorbate induced nitric oxide (NO) catalyzed degradation) J Biol Chem. 2011 286 (31) 27559-72

Chevrier J. et al., Amer J Epidemiol. 2008 168 (3) 298-310 Chez MG et al. (Elevation of tumor necrosis factor alpha TNF in autism) Ped Neurol. 2007 36 361-5 Cf. Young loc. cit. Choi AL Grandjean P (Methylmercury exposure and health effects in humans) Environ Chem 2008 5 112-20 [Methylmercury is a worldwide contaminant found in seafood and freshwater fish; this is the dominant source of human exposure to methylmercury which promotes negative effects on brain function and probably also on heart function but in the past awareness of these negative effects has tended to be masked by the opposite, beneficial effects of -3 fatty acids also present in methylmercury contaminated seafoods. There is evidently now a growing awareness of the negative impact of methylmercury intoxication of the global human population and a world health need to curb Hg input into the environment and to improve the biomonitoring of Hg intoxication] {These findings indicate that while Hg possibly is a partial causative agent for ASD it seems to be a likely aetiological factor in a very large number of diseases (cf. Cutler loc. cit a grey review which lists a very wide possible list of diseases for which literature evidence is available for multiple pathological outcomes of Hg intoxication; the possible contribution of Hg intoxication to multiple sclerosis is hinted at since the first description of this highly identifiably disease apparently coincided historically with the commencement of the widespread use of amalgam fillings cf. Shepherd loc. cit.)} Chu CL et al. Biochem J. 2004 379 331-41 (Heparan sulphate proteoglycans modulate fibroblast growth factor 2 binding through a lipid raft mechanism) [The key function of cholesterol in the lipid rafts which control HSPG signaling studied by Chu et al. could also be relevant to the putative role of cholesterol dyshomeostasis in the aetiology of ASD (in which levels of cholesterol have been suggested to be abnormally low). A genetic defect in cholesterol biosynthesis is associated with a type of autism (Smith-Lemli-Opitz Syndrome, SLOS) of known etiology (it being corrected by administration of cholesterol. Cholesterol is also implicated somewhat analogously in a neurological defect disease producing more severe symptoms in children which have a partial similarity to autism). This (Niemann-Pick disease an extremely rare genetic disease arising from the buildup of glycosphingolipids especially in the CNS) which has a HS-nitric oxide biochemistry aspect, (cf. Leteux et al. loc. cit.), may possibly to be subject to beneficial sulfated cylcodextrin therapy. Cholesterol excess is also well known to the public as a problematic agent which instigates cardiovascular dysfunction by promoting plaque formation at vascular surfaces. This process, as noted by Chu et al is ultimately controlled by HSPG biochemistry. The presence of endogenous heparin which varies between individual has been traditionally associated with a natural protection against the occurrence of vascular plaque formation via the effect of heparin on HS signaling. The general intoxication of animals including human with the lipid soluble (aqueous phase very sparingly soluble) OC hexachlorobenzene and related highly chlorinated aromatic persistent organic pollutants (e.g. PCBs) suggests a mechanism by which such OC toxins might impinge on HSPG and lipid raft modulation of HSPG dependent growth factor activities: by the alteration of the physical chemical nature of the lipid rafts and thereby altering HS signaling]. Cloy JM et al. (Retention of As and Sb in ombrotrophic peat bogs: records of As, Sb and Pb deposition at four Scottish sites) Environ Sci Technol. 2009 43 (6) 1756-62 {Because of new sources of atmospherically transported Sb (and As) post 1980, [e.g. from use as a flame retardant and PET bottle component incineration] caused the amounts of peat-bound As and Sb to dimished 2-3 fold) to be diminished post 1980 (when use in Scotland of leaded gasoline gradually diminished) much less so than the decrease in Pb were considerably less Pb 4-7 fold. Sb, As and Pb are strongly (irreversibly) chelated by soil humic matter (a putative surrogate for HS} Cf. Ceriotti G, Amarasiriwardena D. Microchem J. 2009 91 (1) 85-93

[Brake pad derived Sb was correlated with traffic volume. Binding of Sb to soil humic acid (where Sb(III) is oxidised to Sb(V))] {This behaviour of polyanion Sb sequestration may mimic the binding of Sb to HS in humans and animals} Collins, Vicky (The Herald United Kingdom July 22, 2002) reported in web. rawfoodinfo.com/articles.art_autismtoxoins.html (downloaded September 19 2011) A scientist in Scotland revealed new research which could indicate a linkage between autism and MMR by showing that autistic children have abnormally high levels of toxins in their body. The study .. also raises the possibility that autism may not be geneticand instead be a physical and therefore potentially treatable condition. Lead, aluminium and antimony (similar to arsenic but more toxic) are found to be present in children suffering from autism at a significantly higher level than other children. All three toxins weaken the immune system and when present in high levels they could affect the response to the MMR jab.; the immune system cold be too weak to react appropriately to the triple toxin triggering the onset of autism Cone M cf. web.scientificamerican.com/article.cfm?id=link-between-autism-and-vinyl; cf also Wikipedia Phthalates Cook EH et al. (Autism or atypcial autism in maternally but not paternally derived proximal 15q duplication) Amer J Hum Genet. 1997 60 928-34) [Chromosomal alteration in autism] Cookson Clive (Financial Times (London ) (article (photocopied 22Aug 1990) ex personal Aberdeen U polysacch. lab archive)) The sweet taste of a sugar chain. Clive Cookson tells why scientists believe that carbohydrate molecules may help fight diseases Sugars are rapidly assuming an importance in biotechnology that was undreamt of five years ago. Then scientists believed that the two significant types of bio-molecules were proteins, which carry out the main biological functions and DNA, which holds the genetic code for making proteins. Biotechnologists ignored the branching chins of sugar molecules which bedeck most proteins in animals and plants. But recent research shows that the sugars are not an irrelevant adornment- they actually control the functioning of the proteins. ....One possibility is that there is an underlying sugar code waiting to be unraveled similar to the DNA-based genetic code which determines the sequence of amino acids in proteins. But this glyco-code if it exists, must be far more complex than the genetic code. The state of scientific knowledge about sugars today- and the growing excitement about themis reminiscent of DNA and proteins in the 1940s before Crick and Watson got together in Cambridge to crack the genetic code. Who is going to decipher the sugar code? {It should be noted (as e.g. suggested by research conducted at Aberdeen U.. that there may be several glyome codes. The most dominant for developmental disorders is putatively the heparan sulfate system acting in conjunction with the metallome}. Corrigan FM et al. (Tin and fatty acids in dementia) Prost Leuk Essent Fatty A. 1991 43 (4) 229-38 [Serum Al, Sn, and V were found to be elevated in Alzheimers disease (AD) brains compared to multi-infarct dementia (MID) brains ; Sn was highly correlated with phopholipid fatty acids (positively with 16:0 and 18:1 n-9 and negatively with essential fatty acids C20 n-3 and n-6 ( -3 and 6) ) suggesting that the pathological role of Sn in fatty acid metabolism could also be relevant to the etiology of AD] Sn has also been indicated to be significantly elevated (by a factor of ca. 4, similar to the degree of augmentaion of Pb and Sb) Al was also elevated in the hair of Alzheimer subjects, but to a lesser degree in the hair of autistic, but not at all in Aspergers subjects (as indicated in communicated draft archival data by from Prof Bell Stirling University [cf. also Hall loc. cit.]) Croonenberghs J et al. (Activation of the inflammatory response system in autism) Neuropsychobiology. 2002 45 (1) 1-6

[An increase in the production of some proinflammatory cytokines was indicated to have a potential role in the etiology of ASDs] Cf. also Malik et al. loc. cit. who later reported that the NF-B pathway is unlikely to be responsible for the apparent inflammatory situation associated with ASD. Whilst there was a large elevation of whole blood IFNg and IL1 receptor antagonist (IL01RA) and some elevation of IL-6 and TNFa there was no significant differences in serum concentrations of IL-6, IL-2R and IL-1RA between ASD and normal subjects] Cyhlarova E et al., Eur Neuropsychopharmacol. 2007 17 (2) 116-21 (Membrane fatty acids, reading and spelling in dyslexia and non-dyslexic adults) De Burbure C et al., (Renal and neurological effects of Cd, Pb, Hg and As in children, evidence of early effects and multiple interactions at environmental exposure levels) Environ Health Perspect. 2006 114 (4) 584-590 [This large n>800 mulit-European, former non-ferrous industrial pollution child populations indicated that good statistical evidence of significant kidney and neurological damage arose from the still evident ultra-low exposures to the listed heavy metals which while greater in the studies also applied more generally to a much wider cohort of children. The effect of Cd, Pb and Hg seems to be explicable by the known ability of these metals to alter the microstructure of HSPG e.g. that which is required for effective glomerular filtration; the results also seemed to imply that heavy metal intoxication causes altered HSPG dependent nitric oxide signaling]) Cf. Braun JM et al. loc. cit. De Lange FP et al. Brain. 2008 131 (8) 2172-8 (Increase in prefrontal cortical volume following cognitive behavioural therapy in patients with chronic fatigue syndrome) Dietrich CP et al. Arch Biochim Biophys. 1996 325 (1) 129 [Endotoxin (added to cell culture) causes decreased sulfation of HS] The major achievements made in advancing the understanding of HS PG and heparin by CP Dietrich et al were reviewed in 2006 in Insights into Carbohydrate Structure and Biological Function; Ed. Hugo Verle (Transworld Research Network, Trivandrum, Kerala India_ web.umc/br/noticias/755/artigo.pdf) viz., cf., Sampaio LO et al. (Heparins and heparan sulfates. Structure, Distribution and protein interactions); Bouas RI et al. (Heparin and heparin derivative and their effect on hemostasis) and. Nascimento FD et al. (Heparan sulfate proteoglycan as regulator of protein function). Ding K et al., (Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosothiols) J. Biol Chem. 2002 277 33353-60 Cf. also Mani K et al. loc.cit. and Belting M et al. (Proteoglycan involvement in polymine uptake) Biochem J. 1999 338 317-23 and Vilar RE et al., loc. cit. Cf. Lahiev JP (Fe(II) selectively degrades heparin) BioMetals. 1996 9 10; Chem Abs. 124 109969t Cf. Lahiri et al. (Depolymerization of heparin by complexes ferrous ions) Arch Biochem Biophys. 1992 293 54-68 Dingemans MM et al.

(Neurotoxicity of brominated flame retardants; (in)direct effect of parent and polybrominated diphenyl ethers on the (developing) nervous system Environ Health Perspect. 2011 119 (7) 900-7. Cf. Rose E et al. Prenatal exposure to organohalogens, inclkuding brominated flame retardants, influences motor , cognitive and behavioral performance at school age Ibid., 2009, 117 (120 1953-8 [This study showed both positive and negative outcomes in the child at age5-6 of fetal intoxication by the studied substance intoxication in mothers and later childhood (at age 5-6) motor behavioural and cognitive outcome. The principal HCB metabolite pentachlorophenol, also a major environmental pollutant which showed up in all mothers, uniquely amongst the substances identified as being transferred across the placenta so as to potentially cause altered neurodevelopment, apparently dose dependently produced what seems to be ASD-like symptoms (viz. impairment of coordination, sensory integration, attention and visomotor integration). The authors found that the brominated flame retardants caused both possible and negative outcomes in the severity of the studies symptoms (producing worse manipulative skills and attention but improved coordination visual perception and behavior.) This n=62 study is however especially important as it established for the first time that maternal polybrominated aromatic flame retardants intoxication (measured at the 35th week of pregnancy) allows these substances together with other xenobiotics (heavy metals?) to cross the placenta to potentially to alter the brain development of the fetus so as to account for (putatively ASD-like) motor, intellectual and behavioural deficiencies in children at age 5-6, the study also included in the study the apparent effect of the larger similar transfer of organochlorine compounds DDT and derived DDE, major (OH) PCBs and (pentachlorophenol, the major metabolite of HCB {also the major thermodynamic process determined end product of fossil fuel combustion/incineration of organic wastes, commonly also formed together with with PCBs}[it is less likely that the use of pentachorophenol as a wood preservative mentioned by Rose et al. is the principal reason why this substance showed up in in their studies]. Diringer H, Ehlers B (Chemoprophylaxis of scrapie in mice) J Gen Virol. 1991 72 451-68 Dodds L et al., Epidemiol (Cambridge Mass) 2004 15 (2) 179-86 (Trihalomethanes in public water supplies and risk of stillbirth) Cf. also Kreiling JA et al., loc. cit. Dronca M Craium EC et al. (Antioxidant protection in autism) Ann West Univ Timisoara Ser Chemistry. 2007 16 (2) 169-74 [Erythrocyte SOD was reduced in atypical autism but not in autism] Dufault R et al. (Mercury exposure, nutritional deficiencies and metabolic disruption may affect learning in children) Behavioral and Brain Functions. 2009 5 44 doi: 10.1186/1744-9081-5-44 PMID19860886 [Zn deficiency, implicated in brain dysfunction might promote Hg intoxication both of which seem to be implicated in the etiology of ASDs] {Both dietary Zn deficiency and Hg intoxication from environmental sources are likely to impair HSPG functions; cf. Ohkawara et al.{listed under Fujiwara et al., loc.cit,) reported that Zn reversed the deleterious Pb of Cd induced diminution of HS sulfation} Cf. (A macroepigenetic approach to identify factors responsible for the autism epidemic in the United States) Clin Epigenetics 2012 4 (1) 6 PMID 22490277 [Zn deficiency + intoxication by e.g. Hg and organoP pesticides identified as possible cause of autism {gene varn. of paroxonase-1 was assoc. with autism in US but not in Italy}] Dunstan RH et al. Med J Australia. 1995 163 (6) 294-9; ibid., 1996 164 (4) 251

{HCB intoxication of humans is associated with ME/CFS]

Dweck RA [Cf. In 1990 Clive Cookson (the scientific editor of the Finincial Times) noted in this newpaper that the work of

Dwek (Oxford Glycobiology Unit) and others had caused a re-think of the role of glycation which docorates animal proteins in such a manner to require that glycation should be the prime structural agents by which biotechnolgy needed to address in order to create new effective therapeutic agents]. Ecker C et al. J Neurosci. Doi: 10.1523/jneurosci.5413-09.2010 (Describing the brain in autism in five dimensions MRT-assisted diagnosis of ASD using a multiparameter classification approach) J Neurosci. 2010 11 30 (32) 1612-23 A complex method of evaluating subtle differences in MRI scans could effectively diagnose ASD (and it seems clearly distinguish ASD from ADHD). This confirms that ASD is likely to be, at least in part, a brain function disorder Cf. New Scientist issue no. 2772 [Sofware that identifies the anatomical signature of autism] Cf.. also Boddaert N et al. PloS ONE 4(2) e4415 2009 which was also successful in identifying brain abnormalities 67/77 in non-syndromic autistim subjects. El-Bay F et al. (Hair mercury measurements in Egyptian autistic children) Egypt J Hum Genet. 2011 11 135-41 [A highly significant increase relative to controls was observed in n=32 autistic subjects Hg 0.790.51vs.0.120.086 ppm p<0.001] El-Mowafy AN et al. (Eicosapentaenoic acid ablates valproate induced liver oxidative stress) Biochem Biophys Acta. 2011 1811 (7-8) 460-7 Emanuele E et al. (Serum levels of VEGF and its receptors in patients with severe autism) Clin Biochem. 2010 43 (3) 317-9 [In a n=22 ASD (vs. n=28 control) study, serum VEGF levels were lower and soluble VEGF receptor 1 were found to be higher in the ASD patients.] Engelberg H (Endogenous heparin deficincy in atherosclerosis) Clin Appl Thromb /Hemostais. 1996 2 (2) 83-93 (Heparin is a potential anti-cancer drug) Cancer. 1999 85 (23) 257-72 [Endogenous circulating heparin, which varies between individuals, negatively correlates with LDL cholesterol and protects against a range of degenerative diseases] {Conceivably the endogenous heparin status could protect against adverse outcomes of multiple chemical sensitivity e.g. that which seems to be an etiological facet of ASD]; cf. heparin administration has been indicated to quickly counteract multiple chemical sensitivity cf. (grey literature web.forums.phoenixrising.me/archive/index.php/t-7646.html accesses Aug 9 2011 Cf. also Feizi E et al. loc. cit. and Murata K Yokoyama Y. Atherosclerosis (Shannon Irl.) 78 (1) 69-79 Chem Abs. 111 1513431 [Age-dependent alteration in abundance and microstructure of HS at arterial walls]. Exogenous heparin or heparin mimetics can putatively exert a pro-health effect by altering endogenous HSPG biosynthesis (so as to increase the amount of highly sulfated regions) Cf. Medeiros et al. loc. cit. [Exogenous HS fragment mimetics putatively show beneficial effects in neurological defect diseases] Fatemi SH et al. (Reduction in anti-apoptotic protein bcl-2 in autistic cerebellum) Neuroreport. 2001 12 (5) 929-33

[HS biochemistry is implicated in bcl-2 activity regulation; (n.b., bcl-2 is a potent anti-apoptotic regulatory protein which occurs at abnormally low amounts in schizophrenic brains). HS controls multiple protein activities involved in the regulation of neurodevelopment is relevant to a fuller understanding of the etiology of autism; The putative etiology of ASD proposed is that xenobiotics promote ASD by an alteration HS microstructure. This can in principle might be subject to repair processes boosted by dietary components (e.g. ascorbate, retinoic acid, -3 polyunsaturated fatty acids}which have demonstrated up-regulation of HS sulfation) {The marker of a specific HS epitope (10E4) putatively associated with a N-unsubstituted GlcN and /or a specific arrangement of GlcN-SO3- and GlcN-Ac sequenced HS microstructure is of interest for a fuller understanding of (e.g.) breast phyllode tumour cell proliferation. This HS marker was inversely correlated by 10E4 immunohistochemically with the intensity of bcl-2 (and positively associated with p53) expression as reported by Koo C-Y et al. (Immunohistochemical expression of heparan sulfate correlated with stromal cell proliferation in breast phyllodes tumors) Modern Pathology. 2006 19 1344-50} Cf. Reelin Nitric Oxide and HS Reelin in Schizophrenia Alzheimers Disease and Autims Reelin is believed to control the layering of the brain during development Fatemi SH et al (Regulation of Reelin and bcl-2 proteins in autistic cerebellum) J Autism Dev Disord. 2001 31 (6) 529-35 [More on Reelin HS and Autism. It should be noted that Wei et al. loc. cit. have reported that the focal adhesion kinase(FAK)-Src complex is activated by upstream Reelin and integrin 1 and other processes involving mitogen activated protein kinases were indicated to be defective in ASD which likely leads to altered brain cell migration and adhesion behaviour e.g. during synaptogeneis. (The authors had actually studied B lymphocytes as models for this; these cells were defective as regards HSPG mediated adhesion and migration in ASD subjects compared with controls, the ASD B cells were also defective providers of IgG). Feige JJ et al. (Differential effect of heparin, fibronectin and laminin. The phosphorization of basic fibroblast growth factor by protein kinase C and the catalytic subunit of protein kinase A) J Biol Chem. 1989 109 (6) 3105-14 [Heparin and other glycosaminoglycans inhibit the ability of protein kinase C (PKC) to phosphorylate bFGF; in contrast heparin can directly increase the phosphorylation of bFGF by PKA. {PKC is phospholipid and Ca dependent; cf. protein kinase A (PKA) is cAMP-dependent).

Feize E et al., J. Biol Chem. 1998 273 13395-98 [HS is the only biopolymer which alters systematically with age and this effect of HS at vascular surfaces can account for the age-related dependence of atherosclerosis;] Cf. also Engelberg loc. cit. Fordham PJ et al. Food Additives and Contaminants. 1995 12 (5) (Element residues in food contact plastics and their migration into food simulants, measured by inductively-coupled plasma mass spectrometry) [The results showed the leaching of Sb and to a lesser degree Co, from PET containers] {The use of PET containers for blood collection seemed to allow leaching of Sb and Co which attached to heparin or EDTA used as anticoagulants. [This seems to confirm that EDTA is likley to act as an effective in vivo chelator of Sb (and other toxic)

elements]. Comparison between the blood serum multi-inorganic elements and the multi-element contents of heparin indicated by mass spectrometric results obtained at Aberdeen University and those published on the internet by ALS showed that when the results for Sb and Co were removed from the list of elements the correlation coefficient R2 value of the log-log plot correlations was increased]
Frzard F et al. Molecules 2009 14 2317-36 (Pentavalent antimonials: new perspectives for old drugs) [Cf. strong complexes might be predicted to form between Sb(V) and anionic polysaccharides such as HS similarly to the high stability complexes with adenine ribonucleoside putative via binding to C-OH groups; it should be noted that it is thought that stibogluconate is a potent inhibitor of protein tyrosine phosphatases which lead to an increase in cytokine responses ] {Heparin antimonates could conceivably be of interest as anti-lieshmaniasis therapy} {Antimoniotungstate was cited [DATA] as having similar anti-HIV activity to polyacrylates, polyvinylsulfate and heparin and related polysaccharides which indicate that the arrays of anionic oxyacid side-chains in HS and related signaling systems could mimic natural inorganic oxy-acid polymer systems which in turn mimic crystalline mineral structures; this could further indicate a plausible role of such purely inorganic structures in the events which predated the evolution of cellular life as it now exists}

Funita N et al. (Speciation analysis of antimony in size classified airborne particulate matter by HPLCICPMS) web.1.uni-jena.de/Antimony2011/T18%-%20Furutat.pdf Cf. Funita N et al. J Environ Monit. 2005 7 1155-61; Zheng J et al. J Anal Atom Spectrom.16 812-8 and Iijima A et al., ibid., 2010 25 356-63
[Using citric acid to extract Sb from particulates showed that presence of Sb(III) probably as SbO43-, Sb(V) e.g. SbO43- and trimethylantimony hydroxide [(CH3)3SbOH]+ with more Sb(V) being present in the smaller particles and the amount of methyl-Sb being of the least amount Furnival CM et al., Austr. NZ J Surg 1977 47 (6_ 828-31 (In vitro studies of gallstone dissolution: the effect of additive heparin in bile salt solutions) Fujiwara Y Kaji T. Toxicology. 1999 133 (2-3) 159-69 (Lead inhibits the core protein synthesis of a large heparan sulfate proteoglycan perlecan by proliferating vascular endothelial cells in culture) Cf. ibid., 1999, 133 (2-3) 147-57 (Possible mechanism for lead inhibition of vascular endothelial cell proliferation: a lower response to basic fibroblast growth factor through inhibition of heparan sulfate synthesis) {A similar mechanism was proposed to account for the putative effect of barium intoxication in the etiology of multiple sclerosis by Purdey M loc. cit. It is now further suggested that Sb (plus the heavy metals (by acting synergistically with HCB) may similarly deplete HS signaling and that this process could be a key event in the aetiology of autism. Cadmium also depletes HSPG in this case by the reduction of the degree sulfation of heparan sulfate (cf. Cardenas et al. loc.cit.) Cf. also Ohkawa S et al., J Toxicol Environ Health. 1996 47 (2) 183-93 Showed that Zn could protect HS from the effect of Cd which diminished sulfation. {this could suggest that the adverse effects of toxic metal intoxication e.g. in ASD might be inhibited by Zn which further suggests the importance of Zn status for the protection against heavy metal toxicity]. In cultivated vascular endothelial cells Pb chloride was reported (by Kaji T et al. Toxicology 1997 118 (1) 1-10) to markedly diminish HS sulfation especially of a subclass of low molecular weight HSPG (but this metal only slightly diminished the sulfation of chondroitin and dermatan sulfate). This reduced the binding of FGF-2 (Fujiwra Y Kaji T Toxicology 1999 133 (2-3) 147-157. On the other hand ascorbate increases the degree of sulfation of HS (Kao J et al., Exp Mol Pathol. 1990 53 1-10, Edward M Oliver RF. Biochem Soc Trans. 1983 11 313; cf. also Grant D scribd.com/doc/26994439/publication-2-web for a wide-ranging discussion of this and related topics).

This suggests that certain toxic metal ions (e.g. Pb2+ + Cd2+) under the conditions of ascorbate deficiency induced redox dyshomeostasis can cause serious damage to HS and HSPG integrity and anionic density. This may promote pathologically altered growth factor signaling and altered brain assembly. Cf. Qiao D et al., J Biol Chem. 2003 278 (18) 16045-53 (Heparan sulfate proteoglycans as regulators of fibroblast growth factor-2 signaling in brain endothelial cells. Specific role for glypican-1 in glioma angiogenesis.) [Previously it had been indicated (Hondermarck H et al. Dev Brain Res. 1992 68 (2) 247-53) that HS plays an important function in the control of the biological activity of FGF-2 during brain development. This may be why heavy metals which have been associated autism alters brain development in such a manner as to induce autism (perhaps both in the fetus and also under other perinatal wound healingrelated conditions)]. Geir DA et al. (A prospective study of prenatal mercury exposure from maternal dental amalgams and autism severity) Acta Nerobiol Exp. 2009 69 1-9 PMID19593333 [Severe autism in offspring was associated with mothers with > 6 amalgams as indicated in the n=100 study] {The increased fetal blood Hg (together with other commonly present toxins e.g. HCB) will distrub HSPG signaling so as putatively to cause the brain developmental anatomical changes observed in ASD subjects} N.b. a later larger study Herz-Picciotto I et al. (Blood mercury concentration in CHARGE study children with and without autism) Environ Health Perpect 2010 118 (1) 161-6, which measured blood Hg found that ASD subjects (n=332) and normal controls (n=166) demonstrated a very similar adjusted blood Hg concentration viz. 0.26 (ASD) vs. 0.24 g/L(controls). [This study seems to conclusively indicate that intoxication by Hg is not a principal cause of autism] {A possible criticism of the above study however is that the blood Hg intoxication of the studied population seemed overall to arise from the consumption of fish; the anti inflammatory and HSPG normalization effect of fish oils might conceivably have overall counteracted the negative pro-ASD effect of Hg intoxication in this population group. This might indicate that a non-fish eating population might have been more appropriate study group with which to unravel the possible role of Hg intoxication per se in ASD; it should be noted however that Herz-Picciotto derived from the statistical analysis of their data that separate from any pathway mediated by variation in fish consumption blood Hg was 12% lower in children with ASD compared with controls}. Wajewska MD et al. (Age-dependent lower or higher levels of hair mercury in autistic children than in healthy controls) Acta Neurobiol Exp (Warsaw) 2010 70 (2) 196-208 PMID 20628443 [A n=91 vs 75 controls study of autistic children indicated that autistic children differ from normal children in their ability to metabolize Hg which shows up as a significant age-related difference in hair Hg contents between autistic subjects and age-matched controls (viz. younger autistic children had less Hg while older autistic children had higher Hg levels; the authors also reported that autistic children characteristically suffered a greater tendency towards an adverse reaction to vaccination] Cf. also (Biomarkers of environmental toxicity are susceptible to autism) J Neurological Sci doi:10.1016/j.jns2008.08.02/ Accesses from internet with search term fetal isocoproporphyrin autism; article in press [While the results for urinary porphyrins pentacarboxyporphyrin, precoproporphyrin and the ratio pentacarboxyporphyrin/UP, preproporphyrin/UP, coproporphyrin I & II/UP and precoproporphyrin + pentacarboxyporphyrin/UP (where UP is uroporphrins I & II) indicated a marked augmentation between mild (n=14) and severe (n=14) autism subject was thought to have arisen as a consequence of Hg intoxication an alternative explanation of the results might be that the effect was due to a multiple intoxication phenomenon which included major input from HCB + Sb intoxication] Cf. also Nataf R et al loc. cit. and Woods JS et al. loc.cit. Cf. Geier MD Geir D (internet document assessed 19 July 2011); cf. Grant ECG loc. cit. (The avalanche of new mercury autism studies) July 25 2010 High Pb and Hg intoxication levels have been associated with autism

[58 empirical reports had linked autism (spectrum disorders) with heavy metal intoxication, 43 studies were suggestive of this but were less certain and 13 studies found no such linkage; there were some indication that the severity of autism might be directly associated with Cu intoxication which showed up as higher Cu in hair and nails] Cf. Elsheshtawy E et al. Study of some biomarkers in hair of children with autism) Middle East Current Psychiatry. 2011 18 (1) 6-10. [Pb and Cu were higher and Hg and Zn were lower in autistic subjects however CARS score was significantly correlated with both Hg and Cu and IQ with Pb intoxication]. Cf., Fido A Al-Saad (Toxic trace elements in the hair of children with autism) Autism 2005 9 (3) 290 [High Pb Hg and U in hair but no significant differences in other elements]. Adams JB et al. (Analysis of toxic metals and essential minerals in the hair of Arizona children with autism and mental retardation) Biol Trace Elem Res. 2006 110 (3) 193-209 [Of 39 elements in affected children, their mothers and controls I, Li and K levels were lower in autistic subjects {low Li was also found in the mothers}. It was suggested that a further investigation of the I and Li physiological level status in autism was warranted]. Zinc & Autism Cf. also Hair Zn is commonly low in diabetes and in association with ADD/ADHD and autism (Cf. internet reported observations arising from commercial provision of hair element analytical data) Hair Al is commonly elevated in children /adults low Zn and behavioral /learning disorders such as ADD, ADHD and autism. It should be noted that the use of hair element determination as an indicator of intoxication by toxic elements is a subject of disagreement (e.g. as indicated by internet postings). The routine use of preanalysis hair washing has been e.g. suggested to be a major potential source of errors as it may remove of some elements of interest (e.g. Cu). A recent Roy Soc Chem. review (loc. cit.) confirmed that hair analysis was a valid procedure. Obviously, however, on the other hand the contamination of the hair from dust and of toxic elements externally applied cosmetic hair treatments will also cause potential erroneous elevation of such elements if these are assumed to have arisen from the blood system of the organism. In contrast to the common assumption that appears to have been made by most commercial reporters of hair element analysis that the amounts of toxic inorganic elements in hair (e.g. determined by ICPMS) is a direct indicator of systemic intoxication, it should be noted that in the paper from M.I.T. by LinWen Hu et al. (loc. cit. vide infra) it is assumed that the hair is a terminal depot for heavy metal detoxification. These authors proposed that element determined ratio of Hg/Zn was a useful as a diagnostic marker of autism it being assumed that the presence of these elements in hair has arisen because of an attempt by the organism to achieve detoxification of excess normal or overtly toxic elements by transferring them into the hair. This mechanism seems to be the origin, when comparing same Zn hair contents in both populations, an apparent decrease in the relative amount of Hg in autistic subjects relative to that present in the control subjects. Cf., Yorbik O et al. (Zinc status in autistic children) J Trace Elem Exper Med. 2004 17 (2) 101-7 [Zn was found to be low in plasma and erythrocytes of the autistic Turkish subjects studied]. Cf. Lin-Wen Hu et al., (Neutron activation analysis of hair samples for the identification of autism) Internet document web.sarnel.org.lib/MIT%20-%20Hghair%20autism%20vs%20control.pdf accessed 19 July 2011. [Focusing on regressive autism (late-onset) cases where the child originally healthy, developed autism suddenly under circumstances where the suspected primary cause was metal poisoning it was found that a plot of Hg vs. Zn hair contents compared with controls showed lower Hg in the hair of autistic children; this phenomenon could be used to diagnose this subtype of autism in which the hair Hg level plotted as a function of the Zn level was depleted relative to controls. {The authors interpreted their results in terms of them supporting a hypothesis that autistic subjects might have lower metals in their hair due to this organ being a terminus of a part of the toxic metal excretion mechanism where hair deposition is used to remove toxins from the body and furthermore that regressive autism arises from a malfunction of this mechanism perhaps, however, the dominant effect is that low Zn dominates the hair element results in autistic subjects]. There are however problems relating the amount of Zn in hair to that in other organs. Zn is higher than its blood level would otherwise have suggested (cf. Grant 1-a, loc.cit.)

Further Notes on Antimony and Autism

Could Sb be synergistic with HCB for the augmentation of its toxicity in human tissues? (Is there a further Sb-Al synergy at HS surfaces?) (similarly to how TCDD dioxin can, as has been reported (Li), to greatly augment HCB intoxication; or the possible synergistic action of copper (Grant D, hypothesis) in the observed synergism of chloroform, bromoform, tetrachloroethylene perturbation of fetal development in a pro-autistic manner, cf. Kreiling et al., loc cit.). Toxic metal [e.g. Sb from fossil fuel and brake pads] intoxication (e.g. from ambient urban dust or from medical intervention) might putatively act synergistically to promote overt dioxin toxin effect exacerbation (e.g. for inducing liver damage).

It is now proposed that HCB acts synergistically with heavy metals to induce autistic spectrum diseases in humans. The incineration of organic waste materials especially under uncontrolled or poorly controlled conditions allows the co-introduction into the environment of particulates containing both heavy metals such as Cu, Pb, Cr, Ni, Ba and Sb together with HCB as well as other dioxin (-like) endocrine disrupting substances or substances. This mixture may also act, it is further proposed to increase the toxicity of certain human dietary additives, agricultural chemicals, pharmaceutical agents and cause them to further increase the prevalence and etiological complexity of the various kinds of autistic spectrum disorders. In this regard, possible candidate proautism agents include insecticides and related substances and their in vivo interaction with vaccine ingredients (organomercury antibacterial agent thimerosal and adjuvants such as aluminum hydroxide or other metal e.g., Al oxides which may also have been used for this purpose cf. also Hall (loc. cit.) noted from academic information sources that Sb intoxication from fire retardants could sensitize susceptible infants to adverse MMM outcome) especially in synergy with the effects of particulates which include those ingested via airways from ambient air [there has been reported to be a very general correlation between this kind of pollution and mortality] and by medical intervention via e.g. intra-vascular or intra-muscular injection. The latter has been reported (cf. internet blogs) to have produced severe muscular fatigue in some individuals so treated. The airborne particulates may also contain immune system disrupting heavy metals and polycyclic hydrocarbons from use of additives to fossil fuel in internal combustion engines.
Cf. Google search term nails hair Sb autism accessed documents which suggested that: excessive exposure to Sb may deplete glutathione pools. The presence of Sb negatively affects liver function. Deposition of Sb(V) in bone, kidney, and endocrine organs have been associated with fatigue, hypotension, angina and immune system dysfunction. [{Unexplained cot deaths which had been postulated (Cook Report BBC TV documentary) to be caused by the presence of Sb flame retardant (especially in PVC covered bedding) and childrens clothes; cf. Expert group to Investigate Cot Death Theories-Final Report May 1998, internet document}. (Sb intoxication has been associated with ECG abnormalities and an increased risk of sudden death (cf. Gunnar Nordbey Handbook on the toxicity of metals). The volatile gas SbH3 (stilbene), it should be noted is produced (in small amounts) by the nascent hydrogen formed during the charging of lead-acid car batteries. Methyl substituted stilbenes are believed to arise from mold.. This could be why it is thought that autism putatively increases in homes where molds are more prevalent. Also the apparent tendency for autism to be greater in such countries as Scotland which are damp. The increase in recent years in this country seems to have been greater than elsewhere suggesting perhaps that the greater pollution from use of automobiles the greater use of Sb-containing PET water and food containers as well as a great increase in the use of PVC windows etc. as well as for interior floor coverings may be at least partly to blame for this phenomenon.

Gerdhardsson L et al. (Metal concentrations in plasma and cerbrospinal fluid in patients with Alzheimers disease) Dement Geriatr Cogn Disord. 2008 25 (6) 508-15 PMID 18463412 Goines PE et al., (Increased midgestational IFN, IL-4 and IL-5 in women bearing a child with autism: A Case-control study) Mol Autism. 2011 2 13 [ASD and DD risk in offspring (n=84 and 49 respectively compared with n=159 controls) was assessed in terms of maternal cytokine and chemokine level variation. Increased IFNg, IL-4 and IL-5 was associated with ASD while increased IL-2, IL-4 and IL-6 was associated with DD. Further work was indicated to be required to probe this] Cf. Ashwood P et al. (Elevated plasma cytokines in autism spectrum disorders provide evidence of immune dysfunction and are associted with imparied behavioral outcome) Brain Behav Immun. 2011. 25 (1) 40-5 [Increased IL-1, IL-6, IL-8 and IL-12p40 levels in ASD subjects correlated with disturbances in behavior was indicated from a n=94 ASD, n=39 DD vs. n=87 control study] Cf. also Suzuki K et al. (Plasma cytokine profiles in subjects with high-functioning autism spectrum disorders) PloS ONE 2011 6 (5) e20470 [The levels of IL-1, IL-1RA, IL-5, IL-8, IL12(p70) and GRO were significantly higher in a n=28 cohort of high functioning ASD subjects compared to n=28 controls] {The above reports agree that ASD is associated with IL-1, IL-8 and IL-12 variants; of especial interest may be that elevation of these cytokines is associated with increased expression of nitric oxide synthases. [n.b., HS binding to GRO which is upreguated by TNF, enhances monocyte adhesion to target sites (cf. Weber et al. Eur J Immunol. 1999 29 (2) 700-12; cf Massena S et al., Blood, 2010 116 (11) 1924-31) HS controls the chemotaxis chemokine afforded gradient which directs circulating leukocytes to the required site by a process which seems equivalent to how cells are controlled by HS during embryogenesis, the perturbation of which is what provides the primary tissue defects responsible for the ASD phenotypes]. The role of nitric oxide induced tissue damage (e.g. those of the kidney being of putative especial relevance to the etiology of ASD) includes aberrant HS scission also putatively enhanced by unliganded (not protected, catalytically active) redox metal ions} {Of possible further relevance to the above scenario is that (unliganded non-redox active) Zn2+ has been observed to strongly enhance the potential of IL-1 to act as a IFN production amplifier and therefore also is a immunoregulator; this Zn2+ acts in vivo to dramatically alter cytokine biology (cf.. Poleganov MA et al., J Interferon Cytokine Res 2007 27 (12) 997-1001); the observed major dyshomeostasis of Zn2+ in ASD could therefore be relevant how altered nitric oxide signaling contriibutes to oxidative stress arises in these diseases. The level of Zn2+ is reported to be lower than the normal level in ASD subjects, this is likely to cause defective Zn signaling including the Znafforded IFN cytokine regulation system; cf. also, Zn2+ is also known to participate in nitric-oxidedependent-HS scission and HS-hormone-like molecule generation (cf. Cappai et al. loc. cit.); Zn2+ seems to act in concert with Cu(I)-Cu(II) ascorbate dependent redox recycling for nitric oxide HS-core protein SH reservoir regulation for this function; it should be noted that in ASD Cu levels are elevated (this Cu elevation seems to similarly correlate to oxidative and nitrosative stress in both ASD and rheumatoid arthritis; cf. this elevation of Cu is traditionally thought to be part of the acute phase response; the ASD-related increase in Cu, decrease in Zn (the decrease in Mg which was more closely related perhaps to the reduction in Se seem to be interlinked at least in the small cohort of ASD subjects e.g. as reported on by Priya and Geetha loc. cit which also evidenced an increase in (hair analysis values of) Hg (and also a larger increase in Pb) which seemed to correspond to the decrease in Se and for which perhaps the most significance for HS biosynthesis integrity is Mg > Pb > Hg; possible addition roles of Al3+ and F- intoxication negatively affecting HS tissue protection (via altered biosynthesis) is also suspected but has not yet been specifically reported on}

-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Grant D. see 1-a (vide supra)

Grant Ellen CG (Kingston-upon-Thames) BMJ. Feb 2011 342 d854 (Increases in the neurodevelopmental disorders of autism, environmental toxins, toxic metals and oxidative stress) [This review which highlights the possible role in the etiology of autism of environmental intoxication mainly by Cd, but also of Ni, Hg, and Pb and insufficiency of Zn, Mg, Cr, Se, Mn, Mo and/or B vitamins disagrees with the findings of I McClure ibid., 2011 342 doi: 10.1136/bmj/d852 which suggests that the autism epidemic is not real and has arisen by an increases diagnostic efficiency]. {It should be noted that Cd intoxication causes alteration of HS microstructure, a decreased sulfation: cf. Cardenas et al. loc. cit.). Cf also The Autism Epidemic and The Pill www.ecomed.org.uk/upload/2011.09/7grant.pdf http//bm/com/cgi.eletred/329/7466/588=b#7417.13sep2004 http//www.bmf.com/content/329/7466/588.3/reply
Toxic Metals & Heparan Biochemistry {It should be noted that the specific toxic inorganic elements listed by Grant ECG (loc. cit.) as being augmented in autistic subject are also those which have been indicated to negatively affect HS biosynthesis and the essential inorganic elements which were highlighted by this author as being insufficient in autistic subjects are also precisely those elements which have been identified {cf. Grant D Publication-2-Web) as being essentially required for the proper HS biosynthesis. This could indicate that autism is principally a HSPG-led pathology} Cf., Grant D et al., Biochem Soc Trans. 1996 (Heparin is an essential antioxidant) Chemweb 2000 (Multi-inorganic element content of heparin) Cf also Publication-2 web (scribd) 2009 and other internet documents [~WRL005 23 Sept 2010 web.scribd.com/doc/37999700/WRL0005; ~WRL2521 Revised 10 Apr 2010 web.scribd.com/doc/38671527/WRL2521-Revised and ~WRL3217 comtiued modif 20 Apr 2005 web.scribd.com/doc/342055543/WRL3217-contiued-modif web.scribd (Heparin and putatively heparan sulfate are Haraguchi-type metallomic matrices). The multi-inorganic elements in heparin correlated with those in human hair most especially boys. The presence of additional Sb putatively from PET containers shows up in multi-inorganic profiles of blood collection vessels reported on the internet web.alsglobal.se/hem2005/pdf/blood_collection_tubes_eng.pdf the data listed include EDTA a s a phase boundary enhancing agent which is rich in lanthanides etc. The multi-inorganic element hypothesis of heparin and HS is substantiated by this document which indicates that heparin leached from blood container surfaces by dilute nitric acid contains the anticipated full seawater-like multi-inorganic element presence thus confirming the University of Aberdeen research results which had previously found that heparin including in ion-exchanged heparin demonstrated very well defined of seawater-like multi-inorganic element profiles. HS sulfate signaling are implicated in hemostasis and related growth factor signaling including by endothelial growth factor VEGF. It has been proposed that regulation of VEGF activity is of relevance to the etiology of autism. Cf. Emanuele E et al. (Serum levels of vascular VEGF and its receptors in patients with severe autism) Clin Biochem. 2010 43 (3) 317-19 [Imbalance between VEGF and its receptors may be involved in the etiology of autism]

{It should be noted that VEGF activity is involved in angiogenesis; it has been suggested (Rossignol) that HBOT helps autistic subjects may stimulate angiogenesis of small blood vessels in the brain thereby improving the supply of oxygen}] Could (possibly defective VEGF or analogous) blood capillary effects be involved in gut disorders which have been consistently found in all autism subjects?

N.b. VEGF is secreted by mast cells, the degranulation of which is believed to be dysregulated in autism. It is thought likely that the effect of polychorinated aromatic hydrocarbon intoxication may also be mediated via the heparin/heparan sulfate nitric oxide redox signaling and tissue protective system which is believed also to include an intrinsic anti-oxidative protection function as well as being a co-factor for antioxidant enzyme activity (including for the binding of extracellular superoxide dismutase [SOD]). Polychlorinated aromatic hydrocarbon intoxication is known to affect glucose homeostasis. This may negatively affect HS (perhaps, analogously to how guar can correct for elevated blood glucose, the blood glucose is the actual chemical entity which affects denovo HS biosynthesis or the presence of the chlorinated toxic molecules may directly alter the HS assembly process [although the actual experiments relating to chlorinated aromatic intoxicants which are required in order to confirm or elaborate these ideas have no yet been conducted, a general principle of HS biochemistry is emerging which suggests that those chemical substances which are known to be toxic (e.g. Cd, Pb, Hg {perhaps also As and Sb} as well as F or excess blood glucose) negatively alter HS biosynthesis but those chemical substances which are known to be beneficial to health (e.g. sufficient inorganic sulfate, Mg and Mn, {perhaps also Si} as well as ascorbate, retinoic acid and those dietary lipids which are known to be of benefit to health) positively affect HS biosynthesis. HS biosynthesis has also been reported to change in order to combat the toxic effects of Ca oxalate stones. {A similar mechanism may allow HS to inhibit the pathological effects of the formation of damaging plaques both including those of inorganic and organic chemical natures [cf. Grant D et al. Med Hypotheses. 1992 38 36-48]}. Dietary augmentation by glucosamine (or glucosamine sulfate) improves health at least partly via the augmentation of HS biosynthesis. (N.b. the use of isotopically 14C labeled glucosamine in cell cultures is a standard procedure for metabolic labeling of HS in cell culture experiments). In this context the correlation between the anti-lung cancer effect of such dietary supplementation which was found in a large epidemiological study likely derives from the improved tissue protection achieved by this mechanism.
Lithium (Li) in heparan sulfate signaling. Li alters the conformation of HS (Grant cf. Long 2003). This conceivably could be of relevance to alteration in development biology mechanisms which are centered on HS biochemistry. Very small amounts of Li appear to be required for optimal mental health and deficiency of Li also seems to be associated with autism. Cf. Adams et al. loc. cit.). N.b. commercial sulphonate proton conductors depend on counterion modulated sulphur oxyacid hydration clusters which alter in size with alkali metals e.g. Na and Li. The ability Li to alter HS sulphate half ester hydration seems inevitably to alter its proton conductivity in an analogous manner which suggests that HS biochemistry might encompass systems which mimic

man-made technology (or vice versa) but this phenomenon has as yet not been investigated in the biological context nor has the biological system been investigated in technological context. Other Electronic Industry Sb-Related Notes LiSbO3 is a component of lead-free piezoelectric ceramics. Doping of apatities by Sb is the basis of a common type of phosphor. Goncharov A et al. (Blood pressure in relation to concentrations of PCB congeners and chlorinated pesticides) Environ Health Perspect. 2011 119 (3) 319-25 [Serum concentrations of PCBs (especially those with multiple ortho chlorines) were strongly associated with both sytosolic and diastolic blood pressure. An example of a highly PCB intoxicated population was available adjacent to a former industrial plant which had produced these compounds prior to 1971]. A milder intoxication scenario of the general global human population by PCBs and chlorinated aromatic substances putatively arises from the scrambling of organic halogenated substances during pyrolysis or incineration (also during common fossil fuel combustion processes. The Link Betwee Xenobiotics: Blood Pressure Oxytocin & Autism (Pb, Cd Halogented Organic Substances (PCBs and pentachlorophenol) Perinetal Pb intoxication is known to cause substantial elevation of later adult blood pressure (Cf. Prozialeck WC et al., Toxicol Sci 2008 102 (2) 207-8) Intoxication by PCBs the metabolite of HCB pentachlorophenol, other halogenated organohalogen compounds the formerly used insecticide DDT derived 4-4 DDE and bromodiphenyl ethers intoxication is strongly associated with motor cognitive and behavioural dysfunctions (cf. Roze et al., loc. cit) (i.e. developmental disorders on the autism spectrum) via alteration of endocrine systems. Graham AM et al. Environ Sci Technol 2012 46 2715-2723 (Dissolved organic matter enhances microbial mercury methylation ) The amount and (and putatively also the type) of dissolved organic matter present in natural waters can enhance the uptake of mercury and promote the formation of methyl mercury. In absence of dissolved organic matter normally abundant in municipal water sulfide bound Hg(II), HgS particles undergo self-assemble into sufficiently large particles which prevents their attachment to cell surface receptors at microorganism cell surfaces (as suggested by studies with Desulfovibrio desulfurico) but dissolved organic matter can inhibit the HgS aggregation process so as to increase the bioavailability of Hg via allowing uptake of small Hg S particles {The amount of CO2 in the ocean can similarly be affected by the amount and type of dissolved organic matter (fulvate is an ultra-effective inhibitor of the aggregation of CaCO3 and hence the supersaturation of CO32- in the ocean with respect to precipitation of CaCO3, a major mechanism by which atmospheric CO2 is controlled) is potentially controlled by dissolved organic matter which in turn can be augmented by anthropogenically augmented humic matter run-off from the land and from municipal effluent discharge etc. This may be a major (possibly the dominant route by which humans affect the climate). The paper by Graham et al. which seems to have followed from earlier studies (Hsu H and Sedlak DL Environ Sci Technol 2003 37 (12) 2743-2749 cf. ibid. 2005 39 (11) 4035-4041) which had indicated that Hg(II) sulfide forms a strong complex with S2- in natural water (the association constant for Hg(II) binding with sulfide (or perhaps polysulfide) having a very high association constant value of >1030). Cf. Hongve D et al. (Decline of acid rain enhances mercury concentration in fish)

Guariglia SR et al. Neurotoxicology. 2011 Jun 29 E pub ahead of print PMID 21740927 (Chlorination byproducts induce gender specific autistic-like behavior in CD-1 mice) Cf. also Kreiling JA et al. loc. cit.
Haldimann M et al.

(Exposure to antimony from polyethylene terephthalate (PET) trays used in ready-to-eat meals) Food Addit Contam. 2007 24 (8) 860-8 [Transfer from the PET of 8-38g/kg Sb occurred on microwave heating] {This suggests a possible major source of Sb intoxication of humans and that this method of food preparation should be considered a potential risk factor in the induction of autism e.g. in utero] {It should be noted that other authors have however concluded that the microwave oven food trays studied by them transferred an insufficient amount of Sb to be considered harmful to human health. {A further point of interest in the possible health hazards of use of microwave food preparation was that scientific studies had caused the former USSR to ban this procedure in 1976 but this ban was later lifted to conform with free market norms; (cf. web.powerwatch.org.uk/rf/microwave.asp)}. [It might be suggested that the possible roles of microwave cooking as an environmental inducer of autism should be re-investigated].
Since increased antimony usage for multiple purposes which could allow this element to enter the food chain in larger amounts has apparently mirrored the recent increase in autism this could indicate that the toxicology of this element might have an especial central relevance to the aetiology of autism.

Hall, Terry A. Flame Retardant News October 2002 {This internet-available document S27_Research-AutismFRN02 (downloaded 3 Aug 2011) discusses results of an academic researcher who apparently chose to report his the (presumably preliminary) work verbally at scientific conferences but also to inform the public in a newspaper (e.g. as cited by Dr D van Steenis web. p-a-in.co.uk/docs2.htm ref 279 as The Herald, Scotland; Bell G., 22 July 2002 {Raised lead, antimony and aluminium in Scottish autistic children) [cf. Collins, loc. cit]. TA Hall indicated in the Flame Retardant News draft document (found on the internet) that findings presented at Stirling University by Professor G. Bell based on hair element analysis of 24 children (compared with 8 normal controls) showed the presence of 5x the normal amount of Sb, (also higher Pb and to a lesser extent higher Al). Hall noted that Sb2O3 is likely to be present mostly from its use in flame retardants, and that this Sb might act synergistically in combination with brominated substance and Zn borate based fire retardants as an induction system for autism, perhaps by the ability of Sb intoxication to weaken the immune system and the childs response to the MMM vaccine. This is a key paper. The findings deserve further research.
This source is not covered in PubMed which however does list other work reported by G. Bell relating to -3 and -6 lipids in erythrocytes of autistic subjects and the role of dietary supplementation by suitable lipids to increase the reading ability of autistic (as well as normal) subjects

[The peer reviewed publications of JG Bells autism spectrum disorder related researches however are focussed on lipid-related alterations in autistic subjects; cf.

Bell JG. et al. Br J Nutr. 2010 103 (8) 1160-9 which indicated that collectively autism does not qualify as an underlying phospholipid defect disease although erythrocyte lipid studies indicate that an intake of essential, highly unsaturated fatty acids may describe a cohort of autistic subjects]}.
Similar findings were reported (by Dr Amy Holmes et al. Austin Treatment Center) in an internet document (web. flw.com.arsenicc.html) accessed on 22 August 2011] where it was stated that in the hair of 300 autistic children very high antimony content was almost universal; the geographical region was supposed to be arsenic rich geologically. Highlighted example of a hair analysis (normal values in parenthesis) was: Al 11 (<8), Sb 1.506 (<0.066), As 0.16 (<0.08), Be <0.01 (<0.02), Bi 0.48 (<0.13), Cd 0.077 (<0.15), Pb 1.12 (<1), Hg 0.43 (<0.4) Ti 1.3 (<1) {i.e. there is an elevation (by a factor of ca.23) of Sb together with some elevation of Bi and As in this subject). One hair analysis result reported in detail on an internet site (Hair Test Results-Mercury Babies) over a six year period is perhaps useful. This was a hair test results over a six year period of an autistic child posted on the internet freewebs.com/mercury babies accessed on 24 August 2011, which indicated a much higher than normal value of Sb, As, Pb, Ag and Sn (but less evidently Hg). The kinetic curves of the removal of Sb, As, Pb, Ag and Hg deduced from the over-six-years chelation monitoring history by hair analysis, suggested that continuation of EDTA chelation intervention caused a marked initial augmentation of these five chelated-out elements (Zn, K and Na also showed some initial increase but this was absent for Al, Bi, Sr,, Ba, Sn, Ni; the amount of Zr extracted seemed, however, to continue to increase through the six years treatment).

Ce, La and Nd values in urine were not reported in this blog. It should be noted that the amount of Ce in the brain has been tentatively associated with anti-schizophrenic antioxidant protection (indications from Corrigan loc. cit. Lochgilphead, Argyll and Bute). [Nb., Ce may provide (as indicated by tests carried out in nervous tissues) a natural antioxidant redox recycling protection mechanism; this is in agreement with the observations that Ce contents have been suggested to be diminished in the brains of schizophrenic subjects). The curves for Sb, Pb, As, Ag are uniquely different form the others and these element seem also to be uniquely strongly held to HS as indicated above}. Ce was found, like Sb to be strongly bound to heparin. (Grant, D. unpublished results obtained at Aberdeen University)}. [Ce may act as a neuroprotectant antioxidant cocneivable when bound to HS, cf. also Corrigan et al. loc. cit.] The amounts of Sb was elevated by x5, of Pb was elevated (x3) in 92% of the subjects with autism (and also Sn but Ca was diminished). This was indicated from :-

Hallmayer J et al. Arch Gen Psychiatry. Jul 4, 2011 (doi:1001/archgenpsychiatry.2011.76) Cf. New York Times (New Study Implicates Environmental Factors in Autism) web.nytimes.com/211/07/05/health research/ Haraguchi H. J Anal At Spectrom. 2004 19 5-14 [The concept of metallomics, originated by RJP Williams for ca. 20 physiological inorganic elements was reviewed in this article.. Haraguchi extended the concept to include all seawater elements which occur also in blood serum; these also occur in heparin cf. Grant D 2009, and putatively also in HS; the elements which have been suggested to cause autism bind to H/HS which may act to inhibit their toxic activites]. Herrmann G et al. Nitric oxide and Reelin both modulate neuronal plasticity in developing and mature synaptic networks. Hypothesis: Reelin and nitric oxide signaling pathways influence each other. J Chem Neuroanat. 2008 36 (3-4) 160-0 [Evidence was presented of reciprocal signaling between Reelin/NSsGC and ApoEr2/nNOS expression in olfactory bulb] {The olfactory system provides a mechanism by which atmospheric polution by e.g. Sb and PCB or HCB loaded particulates can directly enter the brain}. Highfield R Telegraph/.co.uk 10/11/2007 Dramatic results of CJD treatment [Pentosan polysulfate {xylan sulfate, SP54} therapy for vCJD] This was a rather less informative than might have been desired press release concerning the use of a HS mimetic for the alleviation of the symptoms of vCJD; further details of this therapeutic method were discussed on the internet by Graham Steel of the CJD Alliance; Cf. also Grant D. (16 July 2010) Ms for G.S.IV (web.scribd.com/doc/34358542/Ms-for-G-S-IV); cf. also web.scribd.com/doc/37999700/WRL005. {This example of the ability of HS oligo mimetics to prove therapeutic benefit in a neurological disease in humans could suggest both the central role of HS biochemistry in neurological dysfunction diseases in general} Ho HH et al., Focus Autism Other Dev Disord. 1997 12 (3) 187-92; Cf. a further indication of the overlap of the aetiologies of autism and obesity is suggested by the report (Fujitat-Shimizu A. et al., Prog Neuro-Psychopharmacol Biol Psy. 2010 34 (3) 455-8) which indicated that the adipokine adiponectin is decreased in the serum of adiponectin in subjects with autism compared with controls.

[The notion that both autism and obesity are triggered by a similar environmental pollutant mechanism agrees with a report that the severity of autism was positively

correlation with obesity in a study group of 54 Canadian school children (HH Ho et al). Much larger epidemiological studies seem to confirm this hypothesis (cf. Chen)]
Hong SR et al. Nutr Res Pract. 2009 3 (3) 212-9 A [n=80 (F)] Korean study showed a direct asociation of the hair element contnets of Na, K, Cr and Cd with body mass index (BMI) Hongve D et al. Decline of acid rain enhances mercury concentrations in fish Environ Sci Technol 2012 46 (5) 2490-2491 [The authors postulated that acid rain might have protected against Hg uptake into fish and humans. The Hg concentrations in lake water ecosystems has increased leading to increasing Hg intoxication of fish because of the declining sulfate concentration in rain which caused a decline in sulfide (and polysuylfide ?) concentration of lake water This sulfide strongly binds HgII) Thus, as a consequence of international protocols which have led to a reduction of sulfur emissions in Europe (Geneva Convention on Long-range Transboudary Air Pollution, implemented in 1983) the anthropogenic process by which environmental Hg becomes bio-available (has counter-intuitively) has become greatly enhanced so that the amount of methyl Hg in fish to have greatly increased. This may have arisen via a process which also depends on the type and activity of natural water dissolved organic matter which has possibly also shown a parallel increase (cf. Graham et al. loc. cit.)] Hsieh C-J et al., BMC Res Notes. 2011 4 291 (The Taiwan birth panel study: a prospective cohort study for environmentally related child health) [Includes data on heavy metals Pb, As, Hg, Cd, Ba,Sb, Ba, Ce, Pt, Th, Mn, Zn, Cu, Si, Co, Mo, Ga and U as well as perfuloroalkyls and non-persistent chlorpyrifos insecticide] Huegline C et al. (Chemical characterization of PM2.5 , PM10 and coarse particles at urban near-city and rural sites in Switzerland) Atmos Environ. 2005 39 (4) 637-51 [The abundances in particles of Ba, Ca, Ce, Fe, La, Mo, Mn, Pb, Sb, Rh gradualy decreased from the kerbside to the background but the amounts of Al As Cd K V in the particulate matter were less dependent on road traffic] Hu W-L Regoeczi E. Biochem Cell Biol. 1992 70 (7) 535-8 (Hepatic heparan sulphate proteoglycan and the recylcling of transferrin) [This study shows the importance of glycosylation. The HSPG proteoglycan ligand which can bind to and help to transport the key iron protein transferin when deglycosylated to the he core protein did not bind to transferrin. The ability of HSPG to bind to transferrin depended on a synergy between the core protein and the anionic polysaccharide side chains. The two lobes release Fe in a complementary, opposite mechanism when bound to the true# receptor compared with HSPG receptor at acid pH. This study also indicated that HSPG from the liver can mimic some of the known functions of more traditional transferrin receptors and may provide form a parallel system of tranfer of tranferrin and associated ions through hepatic cells] Cf. Geir DA et al. (A prospective study of oxidative stress biomarkers in autistic disease) Electonic J Appl Psychol: Innovations in Autism 2009 5 (1) 2-10 [This paper which reports that GSH defect correlates with severity of autism as indicated by a n=28 subject study, also develops the hypothesis (James 2006) that ASDs arise from oxidative stress which putative arises from a combination of increased oxidative insult e.g. form Hg intoxication and inability of the battery of antioxidative systems which oppose this, part of which is briefly noted to be transferrin [which as found by Hu et al., acts in conjunction with HS} {An essential antioxidant protection function is also associated with HS and related molecules; this intrinsic antioxidative effect is combined with a helper-antioxidant effects of HS e.g. that where HS binds to SOD and other antioxidant and related enzymes. This indicates that defective HS antioxidant and co-antioxidant and nitric oxide control functions which are putatively the ultimate basis of antioxidative stresses which promote a range of disorders cf. also

Long 2003 [Grant et al. 1996 etc; Ross et al. 1996 etc.] which can now also be hypothesized to include ASDs} {It should be noted that the diagnostic test for all types of congenital disorders of glycosylation is analysis of sera transferrin glyoforms (cf. Sparks SE Krasnewich DM (Cognitive disorders of glycosylation overview) web.ncbi.nlm.gov/books/NBK1332/ )] Ischiropoulos H. Arch Biochem Biophys. 1998 356 1-11 Tyrosine nitration is associated with a range of diseases [This marker of nitrosative stress which can dysregulate HS signaling is now also known to be associated with ASDs] James SJ et al., (Metabolic endophenotype and related genotypes are associated with oxidative stress in children with autism Am J Med Genet B Neurophych Genet. 2006 (4) 947-56

Jarrels J et al., Reprod Toxicol. 1998 12 (4) 469-76; cf. ibid., 2002 16 (1) 65-70 Jaya P Kurup PA. J Biosci. 1986 10 187-93 [Mg2+ is requried for HSPG biosynthesis] John GB. Amer J Kidney Diseases. 2011 58 (1) 127-34 (Role of klotho in aging, phosphate metabolism and CKD) [Klotho is a HS-biochemistry linked -glucuronidase (which is thought to control aging, phosphate metabolism and be disturbed in chronic kidney dysfunction (CKD)]
{N.b. the etiology of autism may involve klotho activity} Johnson FO. et al., The role of environmental mercury, lead and pesticide exposure in development of amyotrophic lateral sclerosis Neurotoxicology 2009 30 (5) 761-5 Jyonouchi H et al., (Impact of innate immunity in a subset of children with autism spectrum disorders: a case control study) J Neuroinflamm. 2008 21 5 52 PMCID 2625336 [A subset of ASD who were especially subject to frequent (viral etc.) infections (which caused the severity of ASD symptoms to increase) showed evidence of altered innate immune, altered Toll-like receptor (TLR) responses. (The innate immune responses were assessed by measuring the production of pro-inflammatory and counter regulatory cytokines by peripheral blood mononuclear cells in response to agonists of TLR with or without pretreatment with lipopolysaccharide (LPS); a n=19 subgroup demonstrated increased PBMC production of IL-23 with a TLR4 agonist without LPS pretreatment] {It should be noted that TLR4 is a HS controlled system cf., Platt et al., loc. cit. Defects in HS (e.g. following binding or other consequences of xenobiotics) causing an inappropriate initiation of the innate immune system (in autoimmunity) are indicated to be relevant to a fuller understanding of at least a subset of ASD subjects} Kaji et al. Pb intoxication leads to HSPG diminution: cf. Fujiwara loc. cit.

Kalkbrenner AE et al. (Perinatal exposure to hazardous air pollutants and autistic spectrum disorders at age 8) Epidemiol. 2010 21 (5) 631-41 [This study conducted in N. Carolina and W Virginia where outdoor pollutants are lower than was the case for a previous California study, confirmed previous indications that methylene chloride, CH2Cl2, is

a likely inducer of ASD as also are quinoline and styrene were possible causative agents that merit further study as triggers of ASD. {Methylene chloride might be a pre-ASD stimulus because it can induce chlorination of other molecules in vivo when present in conjunction with redox metal catalysts. It is likely to share this property with other chlorinated molecules e.g. CCl4; cf. a preferred co-catalyst acting in this way in an industrial process is butylperchlorocrotonate which can re-oxidize vanadium catalyst centres in a variation of a Ziegler Natta olefin polymerization procedure (cf. Duck EW et al. Eur Polym J. 1974 10 (6) 481-8} {the Kalkbrenner et al. paper also seems to have highlighted 1,3 dichloropropene, propylene dichloride, tetrachloroethylene, butadience xylenes and mercury compounds as possibly relevant inducers of ASDs}. The olfactory bulb route allows air-borne pollutants to enter the brain and directly alter neurological systems. From a selected list of 25 air pollutant positive associations with ASD prevalence were found for the chlorinated solvent group, the metal group as well as the above named specific chemical substances] Kan M et al. (Divalent cations and heparin/HS cooperate to control assembly and activity of fibroblast growth factor receptor complex) J Biol Chem. 1996 271 26143-8 {This paper was the stimulus of a hypothesis [Purdey loc. cit.] of how Ba2+ intoxication might be of etiological relevance in multiple sclerosis but could also suggest how other toxic Mm+ (e.g. Hg2+, Pb2+, Cd2+ and Al3+) might similarly perturb FGFR assembly processes required for neurological development Kane PC Kane E Autism: Bioxidation (Peroxisomal disturbances occur in ASD involving very long chain fatty acids (VLCFAs) nervone (C24:19), lignocene (C24:0) [ these are associated with erythrocyte membrane elevation of docoasapentaenoic (C28:53), docosahexanoic (C22:6s) [These fatty acids are believed to alter the assembly of HS in the Golgi apparatus; Of relevance to this is the activities of 5desaturase - insulin dioxin, HCB induced thyroid immune system dysfunctions] Kao H-T et al., PloS ONE 2010 5 (2) e 372 (The correlation between rates of cancer and autism: an exploratory ecological investigation) [Correlations were found between autism rates and the incidence of in situ breast cancer in F {but no correlations were found between autism and other types of cancer) This database research had been prompted because of the known occurrence of high rates of genetic aberration in both cancer and autism]. {HCB and other chlorinated aromatic molecule (e.g. polychlorinated biphenyls [PCBs]) POP intoxication has, it should also be noted, elsewhere similarly been associated with an increased prevalence of breast cancer in females and therefore, together with the other suggestion that obesity might be correlated with autism, can putatively confirm the hypothetical link between global HCB intoxication with an increased prevalence of autism}. Kawahara M Kato-Negishi M. (Link beteen aluminum and the pathogenesis of Alzheimers disease: The integration of the aluminum and amyloid cascade hypothesis) Int J Alzheimers Dis. 2011 2100 276396 PMID 21423554 [The previous doubts about the aetiological link between Al3+ intoxication and Alzzheimers disease have now been overcome following recent detailed researches and multiple epidemiological studies. There are numerous (e.g. ca. 200) potential disease-promoting actions (e.g. on enzymic activity) which have been associated with the presence of Al3+ in human tissues including altered expression of oxidative stress marker genes SOD1 and glutathione reductase.] {It is also likely that Al3+ intoxication contributes to the aetiology of ASD (cf. e.g. Al was elevated in the hair of ASD subjects, but not in Aspergers subjects cf. Hall for Bell); Although the increase in Al in this study fell below the statistical relevance level the average value of Al was 10.7 ppm (n=24 cf controls n=8, 6.3 ppm and Aspergers n=5, 4.3 ppm. Grey literature posted on the internet also reports that Al may be frequently augmented in ASD hair.

Of relevance to the putatively etiological role of Al in ASD is that such Al is ca. an order of magnitude greater than the amount of Pb or Sn in ASD hair and two orders of magnitude greater than the amount of Sb} Kempuraj D et al., (Mercury induces inflammation mediator release from mast cells) J Neuroinflamm 2010 7 20 [HgCl2 administration led to VEGF, IL6 release from mast cells] Cf. Theoharides TC et al. (Mast cell activation and autism) Biochim Bophys Acta. 2010 PMID 21193031 [Mast cell activation may contribute to the fourfold increase in ASD in preterm infants] Cf. also Angelidou et al., loc. cit. Kennedy G University of Dundee press release of 17 April 2003 posted on the internet; also reported at the XIX Congress of the International Society for Thrombosis and Haemostasis (held at Birmingham UK) on a medical news-sheet internet site labeled Edition 42:04-18 August 2003) found (from a study of 30 subjects and matched controls) that dark chocolate could inhibit blood clotting so as to minimize the risk of strokes. The effect was attributed to the ability of flavenoid polyphenols in cocoa to act via the COX-1 cyclooxygenase system which can inhibit thromboxane-A2 induced platelet adhesion. [Tryptophan in chcolate might also benefit autistic subjects]

Kerzenil JR. Curr Treat Options Gastroenterol. 2000 3 95-8 (Heparin: an emerging, counterintuitive therapy for inflammation) [Heparin appears to offer an useful therapy for IBD by a mechanism which likely extends beyond the ability of heparin to offer anticoagulant protection {e.g. by the augmentation of anti-inflammatory protection}{these findings, thought to be of relevance to the therapeutic intervention in Crohns disease may conceivably also be of relevance to the therapeutic intervention in autism related bowel dysfunction (e.g. cf., in focal-enhanced gastritis in regressive autism)}]. {It should be noted, however, that children with autism have gastrointestinal disease no more frequently than children without autism cf., Black C et al. Br Med J. 2002 324, 419-21)} {It should be noted that the endogenous heparin which is thought to provide protection against atherosclerosis and perhaps also other degenerative diseases (Engelberg, loc.cit.) and also against pathological organisms perhaps including HIV and aberrantly folded prions, is believed to derive from endothelial HS. This is a possible example of how defects in HS could be the ultimate cause of a range of diseases, putatively now suggested also to include ASDs} Kilpinen H. Genetic Mechanisms Underlying Autism Spectrum Disorders web/helda.helsinki.fi/bitstream/handle/10138/23650/geneticm.pdf?sequence=1 Cf. Association of (Disruption I Schizophrenia-1) DISC1 in autism and Aspergers syndrome Mol Physiol. 2008 13 187-96; cf. the genes which mediate synaptic signaling. E.g. neurexin which also are implicated in HSPG mechanism of memory are also indicated to be altered in ASD It should be noted that free radical insult seems to be a key factor in the etiology of s schizophrenia cf. e.g. Reddy RD Yao JK Prot Leuk Ess FA. 1996 55 (1-2) 33-43 Corrigan et al. loc cit. found that the cerium (Ce) content of schizophrenic brains was less than that of controls. Ce (like Sb and Cd) seem to be exceptionally strongly bound to some sites in HS, and perhaps this is part of how it acts as an antioxidant (cf the Ce(III)-Ce(IV) one electron system.

This finding is one of the very rare instance of evidence for possible tissue protective roles of Ce but CeO2 nanoparticles have also been reported to confer nervous system antioxidative protection. Kim Y-S et al., Amer J Psychiatry. 2011 168 (9) 904-12 PMID 21558103 (Prevalence of autism spectrum disorders in a total population sample) [This article noted that experts disagree about the cause of autism and the significance of the recent apparent increases in the prevalence of these diseases. This study suggested that previous screening methods did not identify the majority of autistic subjects] Cf. The New York Times (Claudia Wallis May 9th 2011) (Study in Korea puts autismss prevalence at 2.6%, surprising experts) (Cf. PMID 20023608 puts autisms prevalence at 0.9% for 8 year old children in 11 ADDD sites in the USA. That was in 2006.) {N.b. it is likely that the prevalence of ASD is increasing. It is probably incorrect to assume that the rate of increase is that indicated from the 0.9%(2006) to 2.6%(2011); the correlation with unexplained obesity and overweight (where the percentage of affected individuals is greater by an order of magnitude than those affected with ASD might suggest that the unraveling of the cause of this increase is of major public health concern. If this is confirmed to be a byproduct of industrial activities it is essential that these byproducts be properly identified and brought under control. It is evident that this might become the most pressing task with which the scientific community in the future may urgently be required to become engaged. Kim SM et al. (Exposure to environmental toxins in mothers of children with autism spectrum disorder) Psychiatry Investig. 2010 7 (2) 122-7 [This study aimed to test the hypothesis that South Korean mothers of autistc children were less aware of the possible environmental intoxicants which putatively give rise to autism {this especially applies to PBDE, PCDD, PCB and BPA which are possible oxidative stimulant originators of these disorders]. Cf. Kim et al., the preceding ref. where the prevalence of autism putatively arising from the environmental presence of PCBs etc. was indicated to be ca. 2.6% of the child population] Kojima S et al. Eur J Nucl Med. 1983 8 62-9 Kocyigit A et al. (Antimonial therapy induces circulating proinflammatory cytokines in patients with cutaneous leishmaniasis) Infect Immun. 2002 70 (12) 6589-91 [The authors showed that the administartion of therpay using the Sb drug Glucantime was asssocited with a tenfold increase in IL-6 and a twenty fold increased IL-8 putatively following macrophage and possibly other cell type activation]. {and therefore tends to support the hypothesis that Sb intoxication will cause nitrosative stress to HSPG via IL-6 and other cytokine induced nitric oxide production}. It should be noted that autism is associated with increased IL-6 in the cerebellum as indicated by Wei et al (cf. Young et al).

It should also be noted that some other heavy metals (especially those which have been indicated to be elevated in the hair of autistic subjects which seems to exclude Cr) have also been indicated to cause an increase in IL-6 (CdCl2 at very low levels putatively is associated with air pollution, was found by Marth et al. to cause an increase of 3-9 fold in IL-6 in peripheral blood mononuclear cells) Kreiling JA et al. Environ Tox Pharm 2005 19 9-19 (A mixture of environmental contaminants increases PKA-RII expression in Spisula embryos)
[cf. also (Early embrryonic exposure to PCBs disrupts heat shock protein 70 cognate expression in Zebrafish embryos J Tox Environ Health. (A) 2007 70 (12) 1005-8]

Cf. also Guariglia et al. loc cit. Cf. Dodds L et al. J Autism Dev Disord 2010 (e pub ahead of print)[PubMed 20922473] (The role of prenatal, obstetric and neonatal factors in the development of autism) and Epidemiol (Cambridge Mass). 2004 15 (2) 179-86 (Trihalomethanes in public water supplies and risk of stillbirth) cf. also the power point presentation available (27 August 2011) on the internet web.obs-gyn-atlantic.ca/Assets/Presentation2009/Dodds,%20Linda%20-%20Risk %20Factors%20for%20Autism.pdf notes, inter alia, that while there is an monozygotic twins indicate that there is also a very strong environmental factor role in the etiology of autism obvious genetic predisposition to autism studies of; cf., in 1994 Miller et al. had reported that children who had been affected by maternal medically induced intoxication by thalidomide (a substance later discovered to disturb human fetal development) and who shown abnormalities at the external parts of their ears (but without malformed limbs) were at much higher risk of autism. It is evident from hair element analyses that autism is highly associated with prooxidant anthropogenic metal intoxication (cf. Pb, Sb, Sn elevated by a factor of 4 and Al also elevated in a n=-28 vs n=8 control study of Scottish autistic children) which together with dietary insufficiency of antioxidant ascorbate and e.g. -3 fatty acids likely creates a systemic pro-oxidant condition in such children. This likely augments existing pre and perinatal induced neurological damage and associated genetic susceptibilities to neurological dysfunction leading to the symptoms of autism. Such finding tend to confirm the central role of anthropogenic environmental intoxication and food related associated human intoxication in autism. This kind of intoxication also seems to promote criminal behavior. Cf. it is of interest that violent offenders showed a pronounced oxidative stress signature in their tissue lipids/fatty acids compared with normally-behaved control subjects (cf. Corrigan T et al. J Forensic Psychiatry. 1994 5 (1) 83-92 this included decreases in -3. Studies of patients with dementias by the same group had previously identified heavy metal intoxication in these diseases and also similar lipid abnormalities to those evidenced in the violent offenders. The role of lipid alteration in brain function seems to be the basis of altered behviour in criminality autism as well as in such diseases and schizophrenia and Alzheimers disease. Mast cell activation has been correlated with ASD. Mast cells contain heparin etc. This can arise from experimental stimulationby HgCl2 (cf. Kempuraj et al. loc. cit.)

Perhaps also by other anthropogenic metal salts. Could the protective effect of heparin of msst cells become overwhelmed? It is e.g. negatively affected by uptake of Hg bound to this heparin. Or perhaps most especially by Sb(III) which seems to deactivated analogous sulfated polysaccharides from seaweed. Krotkiewski M Br J Nutr. 1984 52 97-1-5 (Effect of guar gum on body-weight, hunger ratings and metabolism) Cf. Butt MS et al. Crit Rev Food Sci Neutr 207 47 (4) 389-90 (Guar gum is a miracle therapy for hypercholesterol, hyper-glycemia and obesity) {Guar gum might conceivably offer a therapeutic window for the treatment of autism)
Kuin L Haussmann R (Pennche novel pitocin as an early ADHD biomarker of neurodevelopmental risk?) J Attn Disord. 2011 15 (5) 423-31 Kuriyama SN et al. Environ Health Perspect. 2005 113 (2) 149-54 (Developmental exposure to low-dose PBDE-99: effects on male fertility and neurobehaviour in rat offspring) [In utero exposure to a single low dose, 60-300 g.kg body wt. of 2,2,4,4,5 penta bromo diphenyl ether, a component of commercial fire retardants, caused permanent effects on the male rat reproductive system and also behavioural abnormalities]. Cf. Hertz-Picciotto J et al., Environ Health .2011 10 1; cf. Environ Int.2010 Oct 14 E Epidemiol. 2008 19 (6) S75 [The possible role of polybrominated diphenyl ethers PBDEs as a risk factor for autism include the epidemiological indications that PBDEs are related in complex ways to cognitive disorders; It has been indicated that PBDE intoxication causes mice to become hyperactive] Kalantzi OI et al. (Different levels of polybrominated diphenyl ethers (PBDEs) and chlorinated compounds in breast milk from two U.K. regions) Environ Health Perspect. 2004 112 (10) 1085-91 [Sample extracts with the lowest contaminant levels n=7 were from Lancaster and those containing the highest n=7 contaminant levels were from London two of the sample extracts closely aligned together. The relative abundance of contaminants was DDE >PCB 153,138>180>HCB> PBDEs all of which occurred in higher average amounts in milk from London than in that from Lancaster]. Kiven O et al. (Expression of cyclooxygenase 2 and pro-inflammatory cytokines induced by 2,2',4,4',5,5'hexachlorobiphenyl (PCB-153) in human mast cells) Biol Pharm Bull. 2002 25 (9) 1165-8 [PCB-153 a ubiquitously present environmental POP was observed to highly enhance the human mast cell expression of cyclooxygenase-2 and IL 6. This pro-inflammatory outcome which was depended on NFB pathway dependent and could be inhibited by pyrrolidine dithiocarbamate. This PCB apparent had little influence on the expression of TNF- and IL1- by mast cells. It should be noted that mast cells and also intoxication by PCBs and proinflammatory cytokine augmentaion have been implicated in the etiology of autism; Also, autism has been associated with polymorphisms of PTGS2 the gene which encodes cyclooxegenase 2 (Yoo et al. loc. cit.) Kobayashi S et al., (Hair Al in normal aged and senile dementia of Alzheimer type) Prog Clin Biol Res. 1989 317 1095-109 [While the less than statistical significance of the increase in hair Al which was observed in AD vs aged controls seemed to rule out the use of hair Al as a diagnostic for AD, the overall effect in the AD plus non-AD aged population who showed that decreased hair Ca and Mg was associated with an increase of Al uptake by the brain and that this correlated with decreased blood flow determined by Xe133 inhalation observed in this comparison of AD and age matched control subjects]

{It should be noted that a similar apparent less than statistical significant elevation of Al accompanied by a significant decrease in Ca (and likely also Mg) was also apparent in a hair element study of ASD subjects vs. controls cf. Hall loc. cit.). Other workers Naylor GJ et al., (Tissue aluminium concentration stability over time, relationship to age, and dietary intake) Biol Psychiatry. 1990 15 27 (8) 884-90 failed, however, to link AD to Al intoxication. This failure might conceivably be to do with how Si is critically involved in Al turnover and as part of the mechanism by which Al intoxication is prevented. This further also conceivably could center on how both Al and Si are bound to polysaccharides. Cf also the putative tissue protective role of Si seems to be supported by findings reported by a comparison of tissue values of Al between demented and non-demented patients Roberts NB et al. (Increased absorption of Al from a normal dietary intake in dementia) J Inorg Biochem. 1998 69 (3) 171-6, however, showed up a strong disease-related correlation and also that Si was excreted with the elevated Al in such patients putatively as a key part of the mechanism by which this was accomplished. This puts Si as a biochemical factor which is worth considering e.g. as a anti-Al protective agent (cf. Birchall loc. cit.) It should be noted that HS binds both Si and Al and is invovled kidney function including glomerular filtration. The binding of Si to HS seems to enhance HS activity. The association of HS-like polysaccharides and Si has been suggested to have originated from the very start of life on Earth (cf. Iler RK. The Chemistry of Silica Wiley 1979 and also Grant D. et al. Med Hyoth. 1992 38 (1) 46-8).

Lakshmi P Geeha A (A biochemical study on the level of protein and their percentage of nitration in the hair and nails of autistic children) Clin Chem Acta. 2011 412 (11-12) 1036-42. Lelli SM et al., (Hexachlorobenzene as a hormonal disrupter...)
Biochem Pharmacol. 2007 73 (6) 873-9. Leteux C et al. (with T Feyzi) J Biol Chem. 2001 276 12539-45 Cf. also Mani K et al. Glycobiology.2004 14 599-603 and J Biol Chem. 2007 282 21934-) Landrigan PJ. Curr Opin Pediatr. 2010 22 (2) 219-25 (What causes autism? Exploring the environmental case) [This paper draws attention to the likelihood that the biological basis of autism is the alteration of brain development, especially the early part of this by exposure to environmental toxins such as Pb, EtOH and Me-Hg, but there is no credible explanation in terms of vaccinations; it was suggested that useful insight might be achieved by probing the toxicology of thalidomide, and the effects of valproic acid, maternal rubella virus infection {cf. also Megson loc.cit.} and pesticides especially chloropyrofos] Lane KS (internet document 2010 accessed 21 August 2011) web.slideworld.org/slideshow.aspx/Acetaminophen-and-Gliotoxin-Etiology-of-Autism-by-ppt2846786; discusses metallothionein dysfunction in the etiology of autism and suggests, inter alia, that gliotoxin (from gut fungal infections) is present in autistic subjects where it interacts via sulfide rearrangement with glutathione GSH and reduced glutathione GSSG so as to deplete the antioxidant and heavy metal (including Hg) detoxification and nitric oxide homeostasis role of GSH and metallothioneine. {It should be noted that disruption of nitric oxide as well as Zn and Cu homeostasis in autism which this author indicates to putatively play a central role in the etiology of this disease are the same factors which are involved centrally in HS- oligosaccharide generation via deaminative cleavage (which is also thought to involve ascorbate for the re-cycling of Cun+ a phenomenon which might indicate how HSoligomer mimetics (e.g. SP54) are able to inhibit such neurological disruption diseases as nvCJD as

well as putatively in HIV-AIDS and Alzheimers disease. Hence a possible future SP54 mimetic therapy for autism?} Cf. Tatum AH [Upstate Medical University Syracuse, New York (Internet avial. Doc.)] (Pentosan polysulfate PPS (Elmiron) inhibition of neuronal antibody reactivity) Reported an in vitro study of the effect of PPS on the binding of human neural antiodies including those from patents with Guillain-Barr (GB) syndrome, paraprotein neuropathies, systemic lupus erythematosis and diabetes mellitus serum; samples were compared with those from normal person which contained low IgG anti-axonal antibodies possibly to neurofilaments. It was found that antibodies to axonal antigens in lupus were readily inhibited by PPS but the effectiveness of PPS for blocking GB and diabetic anti-neural antibodies was more variable with IgM generally more resistant to PPS inhibition than IgG. However many of normal sera containing IgG axonal antibodies were inhibited by PPS. That the above studies could also be extended to some kinds of autism and language disabilities was indicated by Dalton P et al. Ann Neurol. 2003 53 (4) 533-7 who administered neuronal antibodies from one each of autism and language disorder subjects to an altered mouse model brain development that was apparent as a result of the administration of autism neuronal antibodies. This idea needs to be confirmed by larger scale animal studies and additional human studies but it could suggest that the symptoms of autism and related disorders might be inhibited by the administration of PPS. This idea is supported by the report that the administration of heparin upgrades HSPG biosynthesis via focal adhesion proteins, Ras/Raf/ERK /MEK MAP and Ca2+ - nitric oxide signaling systems which are also putatively also altered in ASD subjects (cf. Medieros et al. loc. cit. A very large epidemiological study (Keil A et al., Epidemiology. 2010 21 (6) 805-8 confirmed that a general (if overall weak) correlation was apparent between 19 autoimmune diseases and autism in offspring suffering from these disease. The odds ratio was 1.6 for maternal and 1.4 for paternal. Several maternal autoimmune diseases were especially associated with autism in offspring as was rheumatic fever in both parents. This could suggests that HS turnover by Cu/Zn nitrite could be relevant to the etiology of autism and therpeutic intervention using this concept is then conceivably possible. The drastic method of PPS injection directly into the brain which afforded therapeutic benefit for CJD is not a viable method of administration. A simple trial of oral PPS is warranted with autistic subjects. Perhaps a cohort requiring Elmiron therapy for interstitial cystitis can be found and monitored for improved ASD symptoms. Octachlorstyrene produced in municipal incinerators etc. (e.g. via. HCB + dichloroacetylene) and now occurring widely in the environment and potentially could boost autism by greatly diminish the amount of GSH in liver cells (cf. Park loc.cit.) {It should be noted that HCB + OCS {most likely from incinerators OCS has never been used for any designed industrial purpose an is therefore a marker for this source}occurs inter alia together with other POPs in human milk and this can be correlated with developmental disorder crytochiridism as reported by Damgaard IN et al.. Environ Health Perspect 2006 114 (7) 1173-8. It is possible that the diminution of GSH protection which occurs in the presence of OCS etc. will be augmented (perhaps synergystically) by the additional presence of Sb(III) (cf. Poon loc. cit.) Larsson M et al. Neuro Toxicology. 2009 30 (5) 822-31 (Association between indoor environmental factors and parental-reported autistic spectrum disorders in children 6-8 years of age) [Cf. also M Cone, comment on this article in Scientific American Scientists find baffling link between autism and vinyl flooring [This study identified the ca. doubled risk of autism due to the presence of PVC floor coverings especially in the parents bedroom; a similar rate of increased risk of autism also arose from maternal smoking {these floor covering s have been suggested to emit ultra small amounts but perhaps relevantto-autism amounts of HCB mimetics]. {Both smoking and PVC are also thought to release true dioxins into the environment; It should be noted that dioxins are likely to be formed together with HCB and other chlorinated substances) as well {in the case of PVC) with Bisphenol A release. A major effect of these toxins is on thyroid and adrenal function. HCB, a dioxin-like substance is now suggested may be the dominant pro-autism substance (able to act synergistically with dioxins and perhaps Bisphenol A) present in the identified pro-autism home environments.

[The Wikipedia entry on Phthalate accessed on 21 August 2011 noted that B Weiss a co-author of the Larsson et al. paper had noted that these results which were intriguing and baffling at the same time had turned up by accident. The importance of the Larsson et al. study (which seems to have sought to identify environmental causes of childhood asthma) is that it is one of the few studies which provides evidence for the genuine environmental triggers of autism viz. somehow being related to PVC usage. A prior (controversial) PVC-usage hypothesis of unexplained cot death had included consideration of the possible effect of Sb intoxication (arising from the widespread use of Sb2O3 as a fire retardant). PVC had also been indicated (less controversially) as a likely cause of childhood asthma which like autism has show a dramatic increase over the last several decades. {Cf. Bornehag C-G et al. Environ Health Perspect. 2005 113 (1) b1393-1397 found that butyl benzyl phthalate [BBzP] was significantly associated with physician-diagnosed rhinitis and eczema whereas di-(2-ethylhexyl) phthalate [DEHP] was associated with physician-diagnosed asthma. The dust concentrations of BbBzP a\nd DEHP displayed quite different associations with different symptoms indicating a genuine biologic response and not due to just lifestyle or demographic factors. Cf. Dickinson J (of the Wolfson Institute of Preventive Medicine) {personal communication, 10 January 2005 in reply to my suggestion that HCB intoxication might be an important part of the etiology of ME/CFS} replied by sending a document CJ FNPVC 24th December 2004 entitled Could polyvinyl chloride account for the increase in asthma in the developed world? Cf. also a publication in book form by this author, 21 Medical Mysteries The Book Guild Ltd. Sussex England, 2000 Chapter 2, p5 Asthma: why is it increasing identified increasing exposure to PVC and also in Chapter 12 Chronic Fatigue Syndrome so called ME which suggested that this disease arose from damage to the reticular activating system (RAS) of the upper brain stem and to its cortical projections}. Lee DH et al., Free Radical Res. 2009 43 (6) 533-7 Lemie MD Hypothesis : Chronic fatigue syndrome, mitochondrial hypo-function and hydrogen sulfide Medical Hypotheses 2008 article (accessed at: web. aboutmecfs.org.violet.arvixe.com/Rsrch/H2S/H2S.pdf) [This paper notes that previous studies had indicated that H2S administration to mice had induced a hibernation-like state which putatively resembled ME/CFS and which was ascribed to a diminution of mitochondrial function] [ME/CFS is an ASD related disease] Li Sin (see supplementary refs) Li SMA et al. Toxicol Envviron Chem 1989 22 (104) 215-27 [HCB acted synergisticallly with 2,3,5,7 dioxin to induce thyroid atropy] Liu BJ. Huan Jing Ke Xue. 2009 30 (3) 907-12 (PMID 19432349) [Sb, As and Hg were indicated to be augmented in hair in Sb mining areas and this intoxication was beleived to potentially negatively affect health] {The very high levels of Sb>As>Hg reported in these studies, viz. 15.9 (0.53) 4.2 (0.28) and 1.79 (0.34) for mining area and control non-mining area, suggests that an effective mechanism of elimination of these toxins into the hair is used by humans as a physiological detoxification route} Long WF. University of Aberdeen (2003) internet listed publications of Marischal College polysaccharide group web.abdn.ac.uk/~bch118/publications2003march.doc [This summary of publications from the Aberdeen polysaccharide group includes studies of metal ion HS interactions] Long WF Williamson FB (Glycosaminoglycans Ca ions and the control of cell proliferation) IRCS J Med Sci. 1979 7 429-34 Cf. Boyd J et al. (Physico-chemical study of heparin. Evidence for a Ca-induced co-operative conformational transition)

J Mol Biol. 1980 175-90 Cf. also Takeuchi Y et al. (Extracellular Ca regulates distribution and transport of heparan sulfate proteoglycans in a rat parathyroid cell line) J Biol Chem. 1990 265 13661-8 Cf. also K Burger et al. (The effect of cations on the Ca ion coordination of heparin) Inorg Chim Acta. 1984 92 173-6 Cf. also Liang JM et al. (An essential role for the 2-sulfamino group in the interaction of Ca ions with heparin) Carbohydr Res. 1982 106 101-9 Cf. also Gargus JJ et al. (Ann NY Acad Sci 2009 1157 133-56) indicated that Ca signaling abnormalities are of fundamental importance to the etiologies of autism and a range of other diseases. Cf. Ji L et al. Life Sci. 2009 85 (23-26) 788-93; PMID 19863947 who reported that Na+/K+ATPase and Ca2+/Mg2+ ATPase are altered in some brain regions of autistic subjects Lorens SA et al. (A heparin derived oligosaccharide normalizes the fear response of old Brown Norway rats) Behav Brain Res. 2003 147 (1-2) 65-72 [Abnormal fear response is thought to be a characteristic of some ASD subjects} Llamban EBC et al., (Tryptophan metabolism via serotonin in rats with HCB experimental porphyrina) Biochem Pharmacol. 2003 66 (1) PMID 1281836 [This report is relevant to the other indication (e.g. Yap et al. loc. cit.) that disturbance of tyrptophannicotinamide pathway metabolism is a critical part of the etiology of autism] Lozac N B-S (Central Role of Voltage Gated Calcium Channels and Intercellular Calcium Homeostasis in Autism) web.autismcalciumchannelopathy.com/index.html [A freestanding literature assessment of the etiology of ASD highlights calcium homeostasis, immune dysfunction and possible role of viral infections] {This document which discusses calcium ion dyshomeostasis determined dysfunctions which may be relevant to the aetiology of ASDS but inter alia also draws attention to similarities between induced neurological dysfunctions in both HIV/AIDS and in ASDs} Luc le E Wrenchal JDE (Regulation of local immune responses by HS-bound IL-2) 12th International Congress of Histochemistry and Cytochemistry, La Jolla, 2004 Abstr. S82 Ma Q et al. (Heparin oligosaccharides as potential therapeutic agents in senile dementia) Curr Pharm Design. 2007 13 1607-16 Cf. Kisilevsky R et al. (Heparan sulfate as a therapeutic target in amyloidogeneis: prospects and possible complications) Amyloid. 2007 14 (10 21-32 and Fransson L- et al. (Novel aspects of glypican glycobiology) Cell Mol Life Sci. 2004 61 1-9 Cf. also Mani et al. loc. cit Lortaat-Jacob H. (Interferon and heparan sulphate) Biochem Soc Trans. 2006 34 (3) 461-4 [Cf. also Medeiros et al. loc. cit] Mani K et al., (Endogenous interanal degradation of heparan sulphate during recycling of glypican-1 in vascular endothelial cells)

Glycobiology. 2000 10 577-86 Cf. Ding K et al., (Copper-dependent autocleavage of glypican-1 heparan sulfate by nitric oxide derived from intrinsic nitrosthiols J. Biol Chem. 2002, 277 33353-60 (Invovlement of glycosylphosphatidylinositol-linked ceruloplasmin in the copper/zinc nitric oxidedependent degradation of glypican- heparan sulfate in rat C6 glioma cells) ibid., 2004 279 (13) 1298-23 Marrack P et al. (Towards an understanding of the adjuvant activity of aluminium) Nature Reviews Immunity 2009 9 287-93 Marlowe M et al. J Orthomol Pychiatry. 13 (2) 117-22 (Decreased magnesium in the hair of autistic children) [The most notable difference between ASD (n=28) subjects and (n=23) controls which is very evident from the results tabulated in this paper is the dramatic approximate factor of ca.4.decrease s in both Ca and Mg relative to normal value contents of hair which is accompanied by a smaller elevation in both Na and K hair values. On the other hand the various toxic elements Pb, As, Hg, Cd.Al, Ni, Be, etc. were found to be essentially the same in both the ASD and the control group hair samples. Perhaps the large relative increases in Hg etc. which appears in the grey literature (but also in peer-reviewed academic studies cf. Marlowe M et al. J Orthomol Med. 1 (1) 43-9 and also in e.g. El-bay et al. loc. cit.) but may apply to perhaps non-typical cohorts of ASD subjects, could divert attention away from the need research to find the actual major ASD trigger. This could be the potent organohalogen xenobiotic mixture which, it should be noted, is thought to produce the kind of adrenal overload which has been supposed to lead to the type of cortisol disturbance which is thought to lead to the kind of linked decreased Mg and Ca - increased Na and K which has been reported in some of the hair element autism studies.. While the confirmation of this hypothesis requires further research, the animal studies of Valkusz Z et al., Physiol Behav. 2011 103 (5) 421-31 PMID 21419145 who confirmed that HCB intoxication rates below those currently thought to be safe produces adrenal dysfunction perhaps likely to lead to the above-mentioned chronic adrenal overload in humans leading to the major electrolytes in intoxicated subjects. It should be noted that HCB and related organohalogen xenobiotics become greatly enriched in maternal as well as in cows milk. Oily fish derived dietary food additives (but without the anthropogenic Hg which is unfortunately also found in some fish extracts) have been suggested by animal models to eventually rid the host of halogenatied xenobiotics. This procedure may be the most appropriate treatment by which to seek to ameliorate the symptoms of ASDs. But Aspergers syndrome seems to have a quite separate etiology as indicated by the major electrolyte hair contents (this disorder is then likely to require a different form of therapy). as indicated by the results of Bell et al. reported in the grey literature under Hall (loc cit.; and also kindly partly communicated to the author draft manuscript archive material from by Prof. Bell) which showed that that Ca hair values were greatly depleted in the hair of autistic subjects but not in Aspergers subjects where the Ca hair values were augmented. Bell et al. however also showed that some toxic elemetns e.g. Sb were elevated in the hair of autistic subjects. Marth E et al. (The effect of heavy metals on the immune system at low concentrations) Int J Occup Environ Health. 2001 14 (4) 375-86 [CdCl2 at 5mol/L caused elevation of mRNA for IL-6 by 3-9, for IL-1a, Il-ib and IL-8 by 5-6 and TNFa by 1.6 fold in peripheral blood mononuclear cells isolated from heparinzed venous blood by Ficoll gradient centrifugation. This work was indicated to be an attempt to find the cause of the (urban) pollutant related increase in allergic reactions]. {It suggests that low-level heavy metal intoxication can e.g. augment IL-6 also found later to be especially elevated in autistic subjects (Wei, loc cit, cf. Young) and to putatively alter synaptic activity via its alteration of adhesion molecules (these include HSPG)}.. Malik M et al. (NF-B signaling in the brain of autistic subjects)

Mediators Inflamm. 2011, 2011: 785265 PMID 22046080 [While IKK kinase which phosphorylates the inhibiting subunit of IB was found to be significantly increased in the cerebellum of ASD subjects, overall the expression of NF-B(p65) and the phosphorlation /activation of NF-B(p65) at Ser 536 was found not to be significantly changed in the cerebellum and cortex of both ASD subjects and in BTBR mice (a model of ASD) indicating that the NF-B signaling pathway is not dysregulated in the brain of ASD subjects and is unlikely to be invovled in the augmentation of inflammation observed in ASD.} Mariea TJ Carlo GL (Wireless radiation and the etiology and treatment of autism: clinical observations and mechanisms) J Austl Coll Nutr Envir Med. 2007 26 3-7 [The authors state that This study presents the first clinical data to to link wireless technology-related EMR in the environment to Autism This report supports the hypothesis that electromagnetic radiation (EMR) may be at least a part of the environmental disturbances which lead to autism {and perhaps other mystery illnesses including asthma, chronic fatigue and fibromyalgia? }and further indicates that an EMR-free environment is useful for achieving a successful (dietary factor based chelation?) energetic nutrition for Autism therapeutic removal of (the putative the key major disease promoting) toxic inorganic elements. The study apparently involved a retrospective analysis of data from n=21 ASD subjects. The paper however includes detailed tabulated data for hair and feces inorganic elements which indicates that successful removal was achieved by use of non-conventional (but not clearly indicated exactly what) chelation therapy coupled with an EMR-free environment. The metals and metalloids the removal of which was of great benefit to ASD subjects were Al, As, Sb, Hg, Pb and to a lesser extent Be and U. Of possible major relevance was the observation that Sb and Hg were indicated to be inter-related in the clearance profile and this seemed to hint that intoxication by elements might somehow be involved in how anthropogenic augmentation of electromagnetic induction might act as a primary cause autism including by preventing ASD subjects from being able to eliminate toxic elements from their bodies perhaps by creating some kind of physical membrane-associated or cytoskeleton-related barrier]. {It should be noted that all cell surfaces throughout biota are associated with inorganic polyphosphate (cf. Brown et al. PNAS ) which may be intercalated with poly--butyrate and Ca2+ . On the adherent cells of animal these polyanions are accompanied by abundant anionic polysachcairdes (HS). This membrane associated polyanion creates a glycocalyx which can putatively create a conductive and EMR-responsive soft doped apatite which e.g. can modulate the formation of ferromagnetic particle formation thought to facilitate interactions between cells and organism with the Earths electromagnetic field (e.g. that which allows bird migration). {This paper could indicate a role for HS-metal ion adduct or other polyanion adduct role in EMR interactions. Also this paper can be read as being supportive of the idea that autism is a HS-led pathology which (because of this) can be beneficially modified by modified dietary intervention strategies. It should be noted that a n=18 vs. n=15 control subject open non randomized controlled study of a vegan diet vs. a non-vegan diet was reported (Kaartinen K et al., Scand J Rheumatol. 2000 29 (5) 30813; PMID 11093597) to greatly benefit fibromyalgia patients who additionally showed a reduction in their BMI and serum cholesterol levels. Another study indicated likely benefits of vegan or vegetarian diets (as well as fasting) on rheumatoid arthritis (Kjeldsen-Kragh et al. PMID 1681264) but a later review of several similar randomized or controlled clinical trials Hagen et. al., PMID 19160281) failed to confirm such possible benefit of vegan and vegetarian diets exist for rheumatoid arthritis patients; the current database seems however, not to allow a proper scientific evaluation of the subject. The idea that gut microflora e.g. consequent on a probiotic diet also seems not to find easy support from attempts to find alteration in such flora following Mediterranean and vegan dietary intervention in rheumatoid arthritis also lacked experimental verification as indicated by Michalsen et al. PMID 16372904. A vegan diet can, however, be rationally argued to promote increased HS sulfation and microstructural integrity (e.g. indicated by reported cell culture studies of the effect on HS biosynthesis of ascorbate, retinoic acid and fatty acids); this idea emerged from an attempted comprehensive literature survey on heparin/HS conducted by the author initially started as part of a formal academic research program at Aberdeen University (from1981 fulltime to1992 thereafter part time and informally after 1996 but also concerned (also informally) with ME/CFS literature search (in conjunction with a former Aberdeen University academic staff member and others) which started in 2002)

Mazzelli MB et al. Arch Toxicol 78 (10 25-33 (HCB impairs glucose metabolism in a rat model of porphria cutana tarda) McCarty MF. Macarty M (Reported antiatherosclerotic activity of silicon may reflect increased endothelial synthesis of heparan sulfate proteoglycans) Med Hypoth. 1997 49 (2) 175-6 [This is hypothesis only and requires back up researches which have not yet been done. The circumstances why that Si is an essential nutrient for humans may be to do with its role in HS biochemistry. Si always co-occurs with HS and other anionic polysaccharides. SiO2 nanoparticles (which self assemble from Si(OH)4) also demonstrate a surprising similarity to biological cells. The seem to be capable of reproduction of form without having genes. In this respect they resemble normal inorganic seeded crystallization. CaCO3 reproduces true to form without needing the help of DNA or RNA to accomplish this (cf. Lima-de-Faria Evolution without Selection, Elsevier 1988)) and may have been associated with sugar-like molecules since life began (cf. Grant D et al cf. also RK Iler). Life may have started off with a protective SiO2 coat. Studies aimed to find uses of SiO2 nanoparticles as pharmaceutical agents could open up novel dietary supplement based therapies. Some op these may benefit ASD subjects. Currently available oral inorganic SiO2 related preparations are claimed to protect gut walls]. [Inorganic Si e.g. as colloidal SiO2 or silicic acid may be an essential nutrient for human and other animals because it assists with HS biosynthesis] {The possible traditionally held view that the prime role of inorganic Si nutrition in providing a buffer against intoxication by Al3+ may also be a related function. Si may assist in the crosslinking of HS to allow the engulfment of not only Al3+ but also of other toxic elements; (cf. the Si(OH)4 crosslinking of B(OH)3 in plant rhamnoglactan II; (cf. also Nader HB et al. Cell Physiol. 1989, 140, 305-10) and hence lack of dietary Si may increase the tendency for disease promotion arising from a poor HS-(Pb etc.) toxic metal uptake protection activity. SiO2 colloids could have been involved in early evolution of animals (cf. Grant D et al. Med Hypoth. 1992 38 36-8; this may also be related to the ability of SiO2-heparin to separate both cations and anions on the same chromatography column (Takeuchi et al. Analysus. 1998 29) Could a poor Si dietary provision therefore be a possible key factor in the promotion of autism?}. It should be noted that colloidal SiO2 [Silicol] has been apparently been successfully used as a gut barrier improver. Similar preparations have been indicated from internet blogs to have possible benefit in autism. This SiO2 iis likely to crosslink the HS and collagen. {HS is known to become degraded under chronic intestinal inflammation conditions cf. Murch et al. loc. cit.}

Medeiros VP et al. (with Nader HB) (Heparin-integrin interaction in endothelial cells: downstream signaling and heparan sulfate expression) J Cell Physiol. 2011 Sep 6 DOI: 10.1001/jcp 23018 PMID 21898414 [Pharmaceutical heparin putatively acts like an endogenous HS servo control signaling fragment messenger to signal for increased biosynthesis of anticoagulantly-active HS polysaccharide chains (normally endothelial HS lack such antithrombin binding pentasaccharide microstructures). This was found to be achieved by the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca2+/NO pathways {heparin enduced ERK1/2 phosphorylation and inhibition of Ras and MEK decreased heparin sependent HSPG synthesis; heparin also induced intracellular Ca2+ release, phospholipase C1 and calcium calmoulin kinase II activation as well as increased NO production] {It should be noted that upregulation of Ras/Raf/ERK1/2 signaling and ERK5 activities may be critically involved in the aetiology of ASDS cf . Yang et al loc. cit.} Heparin-like and other microstructured fragments of HS are generated by nitrosative scission (which originally was believed not to apply to physiological pH conditions by this hypotheses was shown to be incorrect by Vilar et al. loc.cit. who found that nitrite the major metabolite of nitric oxide deaminatively cleaved heparin and HS at pH 7.2 in the presence of a phosphate buffer but not an imidazole buffer. The phosphate type of buffer is known to contain sufficient transition metal redox catlysts to initiate the removal of SO3- groups from GlcN residues in HS the necessary first step in the

deaminative cleavage mechanism. This may be why dysregulation of metal ion homeostasis which produced an equivalent method of allowing trace amounts of transition metals etc. which can initiate the HS deaminative cleavage reaction is associated with rheumatic illnesses and also Alzheimer s disease (which are putatively associated with defective HS signaling). Another mechanism by which the dysregulation of HS biochemistry is thought to promote diseases by the inappropriate production of heparanase. This is a major part of the mechanism by which tumor metastasis is enhanced. This is the key event in cancer-related mortality. Heparanase has been indicated (Chen et al. loc. cit.) to regulate the levels of HS syndecan-1 in the nucleus where the presence of HS is thought to repress inappropriate transcriptional activity [this is part of an epigenetic mechanism afforded by HS]. Heparanase produced by tumor cells by decreasing the Amount of repressor HS at the nucleus is thought to increase the expression of MMP-9, VEGF tissue factor and perhaps other effectors that promote cancer aggressiveness. Megson MN (Is autism a G-alpha protein defict reversible with natural Vitamin A?) Med Hypoth. 2000 54 (6) 979-83 PMID 10867750 [This hypothesis draws on autism subset and inheritable genetic defect in eyesight associated with Gprotein pertussis toxin related second messenger signaling during development. It suggests tht the symptoms of autism may in part be reversed by retinoids] {It should be noted that G-apha i-3 regulate the secretion of HSPG from the Golgi apparatus (studied with LK-PKI cells by Stow et al. loc. cit.) and this process is potentially affected by pertussis toxin; other bacterial bacterial endotoxins have also been reported to diminish HSPG biosynthesis. Bacterial infections or additional toxins added to some vaccines might therefore be able to diminish HSPGafforded tissue protection and this is a possible origin of autism The possible adverse effect of multiple vaccination on infant health seems to be stongly supported by a comparison of infant mortality rates (IMR) between nations. The number of vaccinations which varies between nations has been shown Miller NZ Goldman GS Hum Expt Toxicol. 2011 30 (9) 1420-8.to correlate with the IMR data for the individual nations. This paper also draws attention to the prevalence of sudden infant cot death and vaccination against diptheria-pertussis-tetatanus toxins. {It is of interest in this context that a controversial UK grey research related hypothesis of sudden infant cot death and methyl-antimony intoxication was apparently disproved by later supposedly indepth investigations, cf. Emsley J. Chem Brit. 1999 35 54} The possible role of the measles virus in MMM vaccines as being a causative agent for induction of ASD in genetically susceptible (and/or) xenobiotical intoxicated subjects has similarly been firmly discredited, [or has it ? cf. Reich ES (Fresh debate about MMR fraud. Nature. 2011 479 157-8]} If it can be shown that large enough cohort of Amish or other subpopulation groups of children who are not submitted to mass vaccination and as a consequence of this have a much lower prevalence of ASD (and that this is not because they are genetically predisposed to be resistant to ASD) {a topic accessible on the internet using the search term Olmsted) then the hypothesis that the induction of ASD must be at least in part a consequence of modern methods of vaccination of children would re-emerge and hopefully stimulate new research initiatives to establish why the prevalence of ASDs may be increasing at an alarming rate (from 1/10000 in 1970 to 1/150 in 2007 to 1/40 in 2011? (Or is this only in some regions of Korea?) Might a common global atmospheric pollutant (e.g. by nanoparticles containing antimony and chlorinated aromatic xenobiotics pose a new major a public health risk. Could this (primarily by affecting the mother) have caused an increased infant susceptibility to all types of vaccines? Cf. also Walrad CS (The Autistic Spectrum Regressive Autism Understanding What Regressive Autism Us and Effective Strategies for Stress Reduction with The SCIO) web. qxconference.com/upload/speakers-files/the_autistic_spectrum_notes.pdf [This document discusses ASD from the perspective of homeopathy but is of wider interest e.g. in the context that ASDs are Ca dyshomeostasis and immune system related disorders]. [Cf. also Lozac loc. cit ]. Melke J et al. (Abnormal melatonin synthesis in autism spectrum disorders) Mol Psychiatry. 2008 13 90-8

[Low melatonin levels cuased by a primary deficiency of the ASTM (acetylserotonin methyltransferase) gene encoding the last stage of melatonin synthesis is a risk factor for ASD as indicated by a n=250 vs. 255 controls study)] {ASTM is a non specific N-methyl transferase. Are other methylation processes defective in autistic spectrum disorders? E.g those relating to methylation of As and Sb?} Miller NZ Goldman GS (Infant mortality rates {IMR}regressed against number of vaccine doses routinely given: Is there a biochemical or synergistic toxicity?) Hum Exptl Toxicol. 2011 30 (9) 1420-8 [This paper suggests that an increased {IMR} arises from vaccine use; a related hypothesis (Richardson, loc cit.) suggests that use of more effective polysaccharide modified vaccines might be responsible for the increase in autism in recent decades]. {The grey literature also suggests that IMRs and autism also increase downwind of incinerators} Miodovnik A Wolff MS Encyclopedia of Environmental Health p648-58 doi:10.1016/B978-0-444-52272-600142-2 Discusses HCB etc. It is hypothesized that certain chemical exposure. account for increases in neurodevelopmental abnormalities observed over recent years i.e. Autism (HCB and related intoxication). Molloy CA et al. (Elevated cytokine levels in children with autism spectrum disorders) J Neuroimmunol. 2006 172 (1-2) 198-205 This n=20 vs. control study of children with ASD had increased activation of both Th2 and Th1 arms of the adaptive immune response with a Th2 predominating and without the compensatory increase in the regulating cytokine IL-10 The Comparative Toxigenomics Database citing Molloy et al. inferred that autism was also associated with IL-5 and 10 and IFN { accessed information from internet search term Snitrosoglutathione autistic disorder search}. {This was indicated from measured peripheral blood mononuclear cell contents of IL-2, -4, -5, -10 and 13 and IFN }. [Earlier work by Croonenberghs et al. loc cit. had indicated that autism might be characterized by an activation of the monocytic and an increase in IFN via the activation of Th1 arm of the inflammatory response system. [It should be noted cf. Lortat-Jacob loc. cit., that the immunomodulator IFN is dependent on its binding to a specific type of (a possibly fuzzy logic) sugar sequence in HS. This could mean that the ability of an encoded sequence in HS to modulate IFN (cf. also Sader R et al. J Biol Chem.1998 273 (18) 10919), as well as those other cytokines which have been indicated to bing to specific microstructures in HS chains which include IL-8 (cf. Spillman DS et al., J Biol Chem 1998 273 (25) 15487-93 and IL-2 (cf. Najjam S et al. Glycobiology. 1998 8 (5) 509 are altered in autistic subjects); it should be noted that these cytokines could also be centrally relevant to the etiology of atustic spectrum disorders. N.b., cf., IFN binds to and is thereby controlled by HS (IFN is primarily produced by NK cells, cytotoxic CD8+ T cells and the Th1 subset of T helper cells; HS is believed to control the blood clearance and subsequent tissue targeting of local accumulation of IFN and also to limit the extent of IFN degradation. HS binding thus enhances the activity of this cytokine (on the other hand unliganded IFN is rapidly inactivated); HS ensures a local action of IFN ; the IFN -binding domain of HS has been indicated to consist of an extended internal domain that is precominatnly N-acetylated and glucuronic acid rich (NA domain) flanked by two small glucosamine N-SO3- NS domains. It should be further noted that HS biosynthesis is apparently highly sensitive to the presence of anthopogenically-introduced toxins (such as Cd2+, Pb2+, and Hg2+) which are known to disrupt HS proteoglycan biosynthesis. HCB and PCBs etc. are also likely to do likewise perhaps indirectly via altering redox status especially in the liver and the effect of eicoapenaenic acid ablation of liver oxidative stress (cf. El-Mowafy et al. loc cit.) is relevant to how this fatty acid can provide therapeutic benefit for ASD subjects.

{It is believed that while other IFN are primarily anti-viral, IFN mainly promotes the activity of the components of the cell-mediated immune system such as macrophages, NK cells and cytotoxic T-cells and also induces the production of MHC-1 and MHC-II molecules, stimulates the differentiation of T4 lymphocytes into Th1 cells and inhibits the proliferation of Th2 cells. It helps regulate B-cell differentiation and stimulates the production of IgG subclasses that activate the complement pathway; IFN is thus involved in both innate and adaptive immunity and is believed to modulate the activity of virtually every component of the immune system as well as regulate the biosynthesis of the matrix components involved in tissue remodeling, cell adhesion, differentiation and proliferation. Aberrant expression of IFN (as indicated in transgenic mice studies) can cause pathological inflammation induce severe tissue destruction.} How Heavy Metals (which have been Associated with Autism) Induce Cytokines It should be noted that Sb had been indicated to greatly augment IL-6 and IL-8 (cf. Kocyigiy loc. citt.) A similar effect has also been indicated for Cd (cf. Math et al.) and As (cf. e.g. Das S et al., Toxicol Appl Pharm 2005 204 91) 18-29. Hg has been indicated (Kempuraj et al. loc cit. ) to induce inflammatory mediator (VEGF and IL-6) release from mast cells (which also have heparin-containing granules). Nb. also mast cell activation has been correlated with autism (cf. Theoharides et al. loc. cit.). The abilities of Ag and Al but not Au containing manoparticles to stimulate macrophage NFkappa-B IL-6 etc. (Nishanth RP et al. Nanotoxiol PMID 21417802) could indicate that Ag and Ag but not Au nanoparticlulates might also be potential pro-autism agents {cf also Braydich-Stolle LK et al PMID 20553840 who also showed that Al nanoporticles agumented IL-6 etc.} Moreno, Juan Pedro Arrebola. Doctoral Dissertation, University of Granada, 22 June 2007 [Internet accessed 2009](This dissertation showed data and plots of inter-relationships of body mass index (BMI) HCB as well as DDE intoxication)]. Cf .Smink A. loc.cit. The HCB (+ PCB + related polyhalogenated organic POPs) induce fetal nervous system related obesity is suggested to be of more general relevance including for the occurrence of ASD in individuals who have with a gnetic predisposition for that disease, following their intoxication with low level HCB and related POPs. Mostafa GA Al-ayadhi L (Increased serum levels of anti-ganglioside M1 auto-antibodies in autistic children: relation to disease severity) J Neuroinflamm 2011 8 39 [This marker of autoimmune inflammation (ex the silylated glycosphingolipids which are thought to be involved together with HS in the mechanism of memory transmission of membranes) indicated that a subtype of ASD showed a disease severity correlated presence of antibodies to this marker. Part of the background to this investigation was the prior discovery of ileocolonic lymphonodular hyperplasia or enterocolitis (related to the gut-brain connection) in autism] Mori C et al., (Effects of organochlorines, individually and in mixtures, on B cell proliferation in marine mammals and mice) J Toxical Environ Health. 2008 71 (4) 266-75 [The commonly used mouse model failed to predict the immunotoxicity due to organochlorines (OCs) and the toxic equivalency (TEQ) approach failed to predict immunotoxicity in marine mammals. OCs exhibited both synergistic and antagonistic interactions. Lymphoproliferative responses were modulated by OCs in most species in such a manner as to tend to increase susceptibility to infections]. Cf. also e.g., Levin M et al. ibid, 2007 70 (1) 73-83 [The use of the mouse model for OC intoxication does not predict the effect of OC in marine mammals or humans; the use of toxic equivalency TEC values similarly was non-predictive of OC toxicity effects on the immune system. Complex mutual synergistic and suppressive effects occurred between OC toxins as indicated e.g. by this study of the respiratory burst]

{The human intoxication by OCs and the occurrence of synergy between e.g. PCBS and dioxins will putatively tend to make current infants less tolerant to infections and putatively also to vaccines than was the case prior to the general human population intoxication by these OCs}. Cf.also Mazzariol S et al. PloS ONE. 2011 6 (5) e19417 [This study of the stranding of whales showed high OC tissue contents and also heavy metals especially Hg including methyl Hg and likely immune system impairment with a putative neurological effect (perhaps involving synergy between Hg and DDT plus PCBs intoxication mechanism) may have contributed to the stranding (where whales could not return to the open sea evidently becoming disoriented and starved (which was indicated to have caused release of xenobiotics especially PCBs and DDT related residues but with smaller amounts of HCB present evidently released from adipose tissue stores)] {This intoxication scenario may provide a model for how chlorinated OC plus heavy metal intoxication (putatively acting synergistically) induces ASD in humans}. Moschetta A Kliewer SA. J Clin Invest. 2005 115 (8) 2075-7 (Central role of Klotho in bile acid homeostasis) [Cf, HS is beleved to control Klotho via FGF-4] {Cf. Amigo L et al. Gastroenterol 2000 118 (4) 772-0 (Impaired bliary cholesterol secretion and decreased gallstone formation in apolipoprotein E deficient mice fed a high cholesterol diet} Motamedi-Shad N et al., (Kinetic analysis of amyloid formation in the presence of heparan sulfate) J Biol Chem. 2009 43 29921-34 [In this study of the denaturation of human muscle acylphosphatase with a standard HS which was purchased from Sigma showed that the HS initiated a fast aggregation and a slower HS mediated fibril formation. This was suggested to be a useful model for the disease causing fibrils formed from serum amyloid A, transerythrin, cystatin, amyloid-beta peptides, islet amyloid polypeptide and prions as well as alpha synuclein, fragments of gelsolin, beta2 microglobulin and tau protein; all of these proteins produce fibrils seemingly always co-occurring in association with HS]. {A possible defect in the logical design and thinking relating to the above and other related study is that HS in vivo (or pharmaceutical heparin which occurs in different salt forms and in different degrees of single counterion enrichment) are not a single molecular species (e.g. antithrombin binding blood anticoagulantly active and non-antithombin-binding-blood- anticoagulantly inactive forms of HS types exist in vivo ; HS fragments also exist in vivo; such different forms of HS might be expected to show different pro- and anti-fibril forming abilities; the presence of absence of GlcNsulfate may be of critical importance to the ability of HS to promote fibril formation (cf. Brunisima loc. cit.); this ability is likely to be subject to change following the binding of various counterions etc. to HS; e.g. the acidic HS surface hydration environments produced by the binding of Al3+ might be expected to differ from physiologically counterion-substituted HS in their abilities to promote the formation of pathological fibrils a process which is known to be speeded up under more acidic conditions; Al may occur in some heparin samples and its removal from such heparin by cation exchange prior to use for hemodyalisis was recommended by Bohrer et al. (cf. loc. cit.); heparin has been shown to promote fibril formation from 2 microglobulin; these types of amyloid fibrils are thought to promote a pathology related to long-term dialysis (cf. Relini A et al. J Biol Chem. 2008 283 4912-20. A possible tissue protective effect of heparin/HS may, however be induced under normal physiological conditions since these sulfated polyanions may encourage the formation of non-toxic fibrils instead of their precursors the apparently cytotoxic pre-fibril aggregates which putatively could be the actual pathological agents which promote tissue damage in amylodoses. Whether heparin and related molecules act as tissue protection or as tissue damaging agents is a matter of current debate (cf. Vilasi S et al.(Heparin induces harmless fibril formation in amyloidogenic W7W14F apomyoglobin and amyloid aggregation in wild type protein in vitro) PloS ONE 2011 6 (7) e22076 Murch SH et al. The Lancet. 1993 341 711-714 (Disruption of sulphated glycosaminoglycans in intestinal inflammation) [The authors, from a study of the substantial loss of GAGs from lamina propria and subepithelial basal lamina etc. in Crohns disease and ulcerative colitis, suggested that inflammatory disruption of vascular and connective tissue GAGs may be an important pathogenetic mechanism contributing to the leakage

of protein and fluid, thrombosis and tissue remodelling seen in these inflammatory bowel diseases. The importace of the highly charged HS (together with dermatan sulphate and sialic acid) to the permeability of the vascular endothelium. These findings seem pertinent to autism which also seems to be associated with intestinal surface dysfunction]. Cf. also Pender SLF et al. (Proteolytic degradation of intestinal mucosal extracellular matrix after lamina propria T cell activation ) Gut. 1996 39 284-90 Nader HB et al. Comp Biochem Physiol. 1983 76 433-6 [An exact mathematical relationship exists between the bathing habitat salt conectrations (studied in fifteen species of marine Crustacea, Pelecypoda and Gastropoda invertebrates) and average total sulfated glycosaminoglycans including HS] Naish J. Mail online 21 February 2012 (article-2103940) [Some children with autism may simply grow out of it (Renitha Tutuin (cf. her book My Child is Autistic) had apparently found that training to play a musical instrument had produced a major therapeutic benefit for her autistic child (perhaps by affecting the left-right brain connectivity?) The article noted that, while some academics ({e.g. A Zimmerman who had suggested that of 1,356 parents of children who had been diagnosed as autistic, 453 had later reported that the child no longer had the condition and D Fein had indicated that 10-20 % of autistic children might improve sufficiently so that the diagnosis needs to changed to Aspergers syndrome a less severe form of autism} seemed to support the idea that autism might become less severe; perhaps intensive care can diminish in the severity of autism or autistic subjects simply learn to cope better} other academics and members of the public who commented on this article, did not agreeing with the general idea that autism can be cured, but indicated that autism must always remain a lifelong condition] Nataf R et al. (Porphyrinuria in childhood autistic disorder. Implication for environmental toxicity Toxical Appl Pharmacol 2006 214 99-108 [This study (n=106) showed that coproporphyrin levels were elevated in autism relative to normal subjects (but were unchanged for Aspergers syndrome). Of especial relevance was that the atypical precoproporphyrin, thought to be a special indicator of heavy metal intoxication, was also elevated in autistic subjects. Of possible therapeutic relevance for intervention in autism was the finding that the treatment of a subgroup of autistic patients with the heavy metal chelator dimercaptosuccinic acid (DMSA), a substance with a somewhat similar molecular structure to lipoic acid, produced a significant decrease in urinary porphyrin excretion] Cf. also Geier DA et al. loc. cit. Nguyen AT et al., (Global methylation profiling of lymphoblastoid cell lines reveals epigenetic contribution to autism spectrum disorders and a novel autism candidate gene RORA where protein product is reduced in autistic brain) FASEB J. 2010; DOI:1096/fj. 10-154484 {A putative nitric oxide biosynthetic function of this gene product agrees with it being assoicated with atherosclerosis, breast cancer and macular degeneration) Okada K et al. J Health Sci. 2010 56 (1) 1-13 (Biological function of protein disulfide isomerase : target of phenolic endocrine-disruping chemicals) [The putative role of PCB in autism e.g. via the alteration of brain development (as established in animal studies) may be mediated inter alia by phenolic PCB metabolites which like bisphenol A {BPA}, which bind to and alter the biological activity of protein disulfide isomerase PDI] Osius N et al. (Expsure to PCBs and levels of thryoid hormones in children) Environ Health Prespect 1999 107 843-9 [A +ve correlation was found between PCG 118 and TSH; a ve correlation was found between PCBs 138,153,180,183, 187 and thyroid F T(3) but no association was found between PCB intoxication and F T(4) nor was any correlation found between the presence of Pb or Hg and thyroid factors but Cd blood values were found to be correlated with TSH increase and decrease of F T(4)

Padhyre U (Excess dietary iron is the root cause for increase of child autism and allergies) Med Hypoth. 2003 61 (2) 220-2

This seems to have been confirmed by Walsh W (2004) (but only reported in the internet, grey lit.?) who in a n=3000 study found that a third of these had very high serum feritin Fe. Two thirds of ASDs had normal serum ferritin Fe. Obrenovich ME et al. (Biol Trace Elem Res. 2011 PMID 21755304) found deposition of As, Hg, Cu and Fe in hair in amount consistent with a redox metal induce oxidative stress aetiology of ASD. Cf. Hall, loc. cit. Sb, Pb and Sn are also elevated in ASD hair. Cf., however no significant association between (haemochromatosis HFE gene) p.H63D nor p.C282Y polymorphism and autism was found in a n=25 subject study (Gebril OH Mequid MA. Dis Markers. 2011 31 (5) 289-94). Also Fe deficiency occurs more commonly than with normal subject with ASD (cf.. Latif A et al. Autism 2001 6 (1)1-3-114 and confirmed by Herqner S et al. Eur J Pediatr. 2011 PMID 21643649) in a n=116 study where 24% of subjects showed (serum feritin indicated) Fe deficiency and 15.5% had anaemia. This is consistent with the report that Fe supplementation seemed to improve sleep disorders in n =33 ASD subject study (Dosman CF et al., Pediatr Nutrn. 2007 36 (3) 152-8). Low Fe in ASD may arise from low serum ferritin. It is also possible that an altered glycosylation of ferritin occurs in ASD and this affects how the Fe is processed. The transferrin receptor is also glycosylated. Including apparently by HSPG as indicated controversially by the Fransson group in the 1980s (cf.. JBC 1986 261 (26) 12079-88). This HSPG related system might e.g., become dysfunctional following heavy metal loading. The above reports, however, seem to indicate that Fe dyshomeostasis on its own is unlikely to be the principal cause of ASD. But perhaps Fe overload and resultant oxidant stress perhaps that not correlated with the usual serum markers is relevant to a major subset of ASD.
Pro-oxidant processes which can also arise from halogen atom transfer processes catalyzed by Fe and heavy metal (especially the redox active ones) points to the possible relevance of synergistic interactions between (Fe +) Cu and Sb with organohalogens in the now suggested to be the specific pro-oxidant pro-autism HS disrupting cocktail}.

Palmer RF et al., (Environmental mercury release, special education needs and autism disorders: an ecological study of Texas) Health & Place 2006 12 (2) 203-9 Ibid., 2006 12 (4) 751-2 Cf. Lewandowski TA ibid., 2006 12 (4) 749-50 [These reports discussed the possible link between mercury emissions from coal burning power plants and the prevalence of autism and the need for special educational provision] {The formation of carbonaceous particulate matter which can adsorb the heavy metals and dioxin-like substances such as HCB is now suggested to be of much greater potential relevance to the aetiology of autism than is the amount of Hg present in ultrafine and other particulate dusts emitted by power plants and urban activities including traffic. A range of toxic metals in addition to Hg including Pb, Cd and Sb occur in coal and the chloride therein allows the formation of HCB, dioxins and other chlorinated aromatics

which can enter the atmosphere from the combustion of coal and other fossil fuels (e.g. in electrical power plants, incinerators and heavy industrial metallurgical operations) but the precise amounts vary with the type of coal etc., the effectiveness of smoke stack clean up techniques employed etc. The focus on Hg seems to have arisen from the hypothesis (now discredited) that ethyl-Hg containing vaccine use is the sole principal cause of (sudden onset) autism. A large epidemiological study (Hertz-Picciotto loc cit.) established that the amount of Hg in blood could not be correlated with autism. Smaller studies (e.g. in Egypt and Poland) have however found that autism is associated with an elevation of hair Hg. While the Hg in some vaccines and in atmospheric dust and food may be of only secondary relevance to the aetiology of most cases of autism, other aspects of vaccine composition may, however be more relevant. It has been indicated that further research is required to establish why the infant mortality rate seems to be correlated (between nations) with the intensity of childhood vaccination (Miller loc cit.). It has also been suggested that increased use of bacterial polysaccharide conjugated vaccines which leads to an augmentation of their effectiveness might relevant to the aetiology of (sudden-onset) autism (Richmond loc. cit.) Park EJ Park K (Induction of oxidative stress in human Chang liver cells by octachlorostyrene OCS, the persistent and bioaccumulative toxicant) Toxicol In Vitro. 2008 22 (2) 367-75 [Although OCS has never been commercially used its has become widely distributed in the environment. {This must indicate an incinerator type of input route} This substance is assumed to have a similar toxicology to HCB. The study assessed the possible impact of OCS on human health by observing how OCB promotes the formation of reactive oxygen species (ROS) and GSH depletion which occurs in the cytosol of liver cells. The ROS decrease observed was related to the decrease in the level of GSH] {Decrease in GSH and a reduced ability to detoxify heavy metals is a characteristic feature of ASD. The likely presence of small amounts of HCB + OCB in the global human population could mean that humans are now more likely to suffer diseases such as ASD which can be promoted by lack of adequate GSH protection}.
Pawalowska-Goral K et al. Fluoride. 1998 31 193-202 [Excess F- perturbs HS biosynthesis] Fujiwara Y Kaji T J Health Sci 2002, 48, 460-6; Toxicology 1999 133 159-69 [Pb perturbs HS biosynthesis] Cf . also the requirement of sufficient essential nutrient Mg2+ (Jaya et al., loc. cit.) and ascorbate (Edwards et al., loc. cit.) for HS biosynthesis Essential nutrient Si may also be required for effective HS assembly (cf. McCarty loc. cit.) [Perhaps this is because this Si-HS is involved in protecting against Al3+ {and perhaps also counters As and Sb} intoxication] Pea J et al. Nature. 2011 472 437-42 (Shank3 mutant mice display autistic-like behaviors) [Shank3 is a postsynaptic protein whose genetic disruption is putatively responsible for 22q13 deletion syndrome (Phelan-Macdermid syndrome) and other non syndromic ASDs] {Shak3 may be involved in clustering and organization of receptors at the synapse by a process which may resemble how HSPG modulates the clustering of acetylcholine receptor clusters} Cf. also ibid., 478 9 for comments on an article by Geschwind D et al., Cell. 2010 147 235-46 (Autism in a mouse) Deletion of gene Cntnap 2 which in humans is associated with epilepsy; treatment with

the anti-psychotic drug risperidine prevents epileptic repetitive behaviour but not social interaction related behaviour. {Shank occurs together with HSPG (syndecan2) AMPAR CASK synbindin etc. in dentritic spines [cf. Nature Rev 2001 880] (which are thought to serve as storage sites for synaptic strength and to contribute to cognition and memory and putatively if defective also to autism}. Pereira MG et al. (Long term trends in mercury and PCB congener concentrations in gannet (Morus bassanus) eggs in Britain) Environ Pollut. 2009 157 155-63 Available at web.nora.nerc.ac.uk/3705 {Some PCB congeners have shown a long term decline from 1970-90 (probably related to cessation of manufacture) but other PCB congeners (putatively formed during incineration/combustion) showed a long term increase in East Scotland (North Sea) offshore island Bass Rock which has ca. a 10 fold greater level of pollution than has the West Scotland (Atlantic Ocean) Aisla Craig (Clyde Coast) (the more polluted area was downwind of land); PCBs in gannet eggs both sites were dominated by PCB congeners 153,138, 180, 118, 170 and 105 of which from1989-2004 PCB 170 increased, 180 increased slightly 153 changed little118 decreased slightly but 138 and 128 decreased considerably; it is evident that 2/3 of the most abundant PCBs showed some increase between 1989 and 2002} It should be nnoted that PCB-153 (2,2',4,4',5,5'-hexachlorobiphenyl) has been observed to augment the expression of cyclooxygenase-2 (COX-2) and IL6 in human mass cells (Kiven et al. loc. cit.) Perrimon N et al. Nature. 2000 404 725-8 Cf. Bernfield M et al. Annu Rev Biochem. 1999 68 729-77 Cf . also Pyrda and Dalen loc cit. Cf. Bishop JR.loc cit. Cf. Ding et al. loc cit. Phillips CA Gladding T and Maloney S Chem Brit. 1994 30 (8) 646-656 (Clouds with a quicksilver lining) [This article discusses mercury toxicity and especially suggests, on the bases on the preliminary epidemiological studies of the authors) the likely serious health hazard posed by mercury vapor arising from crematoria and its deposition in the surrounding soil etc.] Platt JL Rosenberg HF (Toll-like receptors, endogenous ligands, and constitutive control ..) J Leukocyte Biol. 2007 82 286-7 Cf. Johnson GB et al., (Receptor-mediated monitoring of tissue well-being via deletion of soluble heparan sulafate by Tolllike receptor 4 (TLR-4)) [The type of innate immune system present in plants where polyanions are recognized following tissue injury; this is reminiscent of how bacterial polysaccharide (lipopopolysaccharide, LPS) initiates an immune reponse in humans; probing this uncovered the existence of a similar system in animals; HS fragments however, play a key role in the Toll-like receptor system innate immunity of animals] [This phenomenon is relevant to a fuller understanding of the etiology of ASDs cf. Jyonouchi et al. loc. cit.]. {HS fragments which might initiate the innate and leads to activation of the adaptive system could arise from action of heparanase or nitrosative scission as well as non-enzymic HS degradation, e.g. putatively following de-N sulphonation by the action of Al3+ induced local H+ release}. {The stimulus for the discovery of the role played by HS in the innate immune system in humans stemmed from an interest of the above researchers in the mechanism by which organ transplants were rejected by an immunological mechanism. It should be noted that early work in this field had indicated that algal sulfated polysaccharides which putatively mimicked HS had shown some promise as inhibitors of the rejection process} Plante I et al., Carcinogenesis 23 (7) 1243-9

(Decreased gap junctional intercellular communication in HCB induced gender specific hepatic tumor formation in the rat) {HSPG biochemistry is also implicated in gap junctional integrity, cf., Spray DC et al., J Cell Biol. 1987 105 541-51} Poon R Chu I (Effects of trivalent Sb on human erythrocyte glutathione-S-transferases) J Biochem Mol Toxicol. 2000 14 (3) 169-75 [The relative effectivness of heavy metal inhibitors of GST was indicated to be Sb(III)>Hg2+>Cu2+>Cd2+>Cr3+>Fe2+] Cf. ibid. 1998 (12) 4227-33 Prandota J (Autistic spectrum disorders may be due to cerebral toxoplasmosis associated with chronic neruoinflammation causing persistent hypercytokinemia that resulted in an increased lipid peroxidation, oxidative stress, and depressed metabolism of endogenous and exogenous substances) Res Autism Spect Disord. 2010 4 119-55 Priya L P Geetha A (Level of trace elements (copper, zinc, magnesium and selenium) and toxic elements (lead and mercury) in the hair and nails of children with autism) Biol Trace Elem Res. 2011 142 (2) 148-58 [ASD subjects were divided into three groups (n=15) according to the degree of severity of autism. The most severe group showed alteration in all of the measured inorganic elements in hair in which the amount of Cu appeared to be augmented according to the severity of the autism. (thus the mildest group showed no elevation, the medium affected group showed elevation of Cu by ca x2 and the most affected group showed an elevation of Cu by ca 3 fold.), Cf. Youssef AAR et al., (Serum copper: a marker of disease activity in rheumatoid arthritis) J Clin Pathol. 1983 36 14-17 [The classification of autism by Priya and Geetha on the basis of hair copper (which will reflect serum copper) and the similar classification of the severity of rheumatoid arthritis (RA) in n=60 patients by Youssef et al. shows that ASD and RA are almost identically copper status dependent. The arthritic conditions are acknowledged to be extracellular matrix dyshomeostasis diseases arising from chronic autoimmune inflammation. {Could the alleviation of the symptoms of osteoarthritis by glucosamine also be of possible relevance to future therapeutic intervention in ASDs?} Prydz K Dalen KT (Synthesis and sorting of proteoglycans) J Cell Sci. 2000 113 193-205 Purdey M Principal ideas relating to mammalian prion diseases and other environmentally induced disease were generated from farming during the period of bovine spongiform encephalopathies epidemic in the UK Cf. web. markpurdey.com/mark_purdey.htm (accessed 9 July 2012) TSE pathologies could have been initiated once Mn replaces Cu in PrPC. The role of H/HS in the modulation of PrPC biochemistry suggests a further role of alteration of H/HS in these diseases (Grant D e-mail and letter correspondence with Mark Purdey) Cf. also Purdey M. Med Hypoth. 2004 62 746-54 (Chronic barium intoxication disrupts sulphated protoeglycan synthesis: a hypothesis for the origins of multiple sclerosis) (Ba intoxication in multiple sclerosis {MS}) {N.b., this hypothesis that perturbation of HSPG signaling by Ba2+ is involved in the etiology of MS was suggested following discussions with the present author and others} [This study of the presence of excessive amount of Ba in soil/water and plant samples from the N.E. Scotland locations which had been identified (by DI Shepherd (loc. cit.) MD Thesis 1975 University of Aberdeen [Shepherd suggested, as a result of his exhaustive historical researches into this research topic, that the widespread use of mercury in dentistry around 1835 had coincided with the first recorded

description of MS (furthermore this disease present such unique symptoms which inevitably would have been noted by physicians prior to this if it had been prevalent); i.e. Hg/Pb + (Sb?) intoxication is indicated as a likely inducing factor in this disease and the highly efficient provision of medical and dentistry services in Scotland may furthermore have been a factor in inadvertently causing the prevalence of multiple sclerosis in the UK, to be amongst the highest in the world. Purdey discussed the etiology in terms of the possible (additional and perhaps dominant) role of Ba intoxication in MS via the perturbation of the bivalent metal ion dependent fibroblast growth factor (and especially the related receptor) signaling pathway. Other hints that HS signaling might be central to the etiologiy of multiple HS as well as e.g. autism is the association of diminished inorganic sulfate ion presence in blood and other disturbances of the sulfur cycle in these conditions. Latitudinal Variation of the Prevalence of Multiple Sclerosis and Perhaps also Autism The dependence of sulphate transporter activity on 1,25 diOH-vitamin D and iodothyonine [T(3)} (Dawson PA Markovich P Pflugers Arch-Eur J Physiol. 2002 444 (3) 353-50; PMID 12111243) suggests a mechanism by which the augmentation of sulfate transport and optimization of sulfate homeostasis could be mediated by sunlight intensity which might further explain a well-defined latitudinal variation in the prevalence of MS which is especially evident in Australia (and more generally globally) as well as the putative role of metal ion intoxication in multiple sclerosis and autism due to the role of sulfation in HS signaling (this is critically dependent on the sulfation pattern of this anionic polysaccharide system).and this could be negatively affected by the general disturbance in these diseases of sulfur and sulfur-dependent molecule processing which seems to be a characteristic feature of the etiologies of ASDs. Cf. Markovich P. PMID 21298488, the physiological roles of the renal sulfate transporters NaS1 (Slc13a1) and Sat1(Slc26a1) encode respectively renal anion transporters that mediate proximal tubular sulfate reabsorption and therefore regulate blood sulfate levels. Targeted disruption leads respectively to hyper or hyposulfatemia and liver damage. Loss of Sat1 leads additionally to hyperoxaluria with calcium oxalate urolithiasis. Oxalate Crystal Formation in ASD [That the deletion of Sat1 causes hyposulfatemia and calcium oxalate crystallization likely arises because of defective sulfation of the sulfated polysaccharide HS which is required to prevent calcium oxalate crytallization. This seems to be a related phenomenon to how hyposufatemia in autism is associated with calcium oxalate crystal fromation in that disease]. Other mark Purdey Hypotheses which maight be of relevance to the etiology of autism: The Metal Microcrystal Nucleator Hypothesis The ability of e.g. Sr, Ba and Ag containing microcrystalline seeds (arising e.g. from incineration) may enter the brain via the nasal olefactory tract and e.g. initiate protein misfolding. This mechanism was thought of possible relevance to the etiology of transmissible spongiform encephalopathies, TSEs. An earlier (2000, cf. Med Hypoth 2000 54 (2) 278-306) Purdey Mn3+ (Cu deficiency) hypothesis of TSEs, has provisionally been confirmed by other workers (e.g. G. Multhaup who confirmed that Mn3+ could stimulate prion protein misfolding). Raju TS. et al. Glycobiology. 2000 10 (5) 477-486 cf. e.g. Patel TP. et al., Biochem J. 1992 285 839-845 Randi AS et al. (HCB is a tumor co-carcinogen and induces alterations in insulin growth factor pathway in the rat mammary gland) Toxicol Sci. 2006 89 (1) 83-92 [This study showed that HCB is a co-carcinogen for N-nitroso N-methyl urea induced mammary tumors in the rat] Cf. Pontillo CA et al. (Activation of cSrc/HER 1/STAT5b and HER1/ERK1/2 signaling pathways and cell migration by HCB in MDA MB-231 human breast cancer cell line) ibid. 2011 120 (2) 284-96; [HCB is an inducer of cell proliferation and c-SRC kinase activity in MCF-7 breast cancer cells] Rao G et al.

(Reactive oxygen species mediate high glucose-induced hyparanase-I production and heparan sulphate proteglycan degradation in human and rat endothelial cells) Diabetologia. 2011 54 (6) 1527-38 [The content of HS at vascular walls is reduced under hyperglycemic conditions which also promotes reactive oxygen free radial damage and this mechanism may contribute to the etiology of atherosclerosis]. {The release of HS fragments could also induce the innate immune response, cf. Platt et al. loc. cit., which is believed to be an etiological feature of a sub-set of ASD subjeccts} Ray B et al. (Increased secreted amyloid precursor protein-alpha (sAPP) in severe autism: proposal of a specific anabolic pathway and potential biomarker) PloS ONE.. 2011 6 (5) e20405 [HS protoglycans bind amyloid proteins and putatively are involved in their regulation cf. Bue L et al. and Bruinisma et al., loc.cit. ] {While this potential critical role of HS biochemistry has been noted for Alzheimers disease the similar relevance of HS biochemistry to the etiology of autism does not seem to have been noted {except however from the link which is apparent between the dependence of the innate immune system dependence on HS via TLR-4, cf. Jeffrey loc. cit.} the possible critical role of GlcN sulfation is indicated in the etiology of autism. A chronic halogenated xenobiotic augmentation of nitric oxide metabolite associated with elevated unliganded redox metal ion GLNsulfate de-sulfation, is now suggested to contribute to the etiology of ASDs} Richardson & SIDS (Cf. e.g. Expert Group Investigation on Cot Death Theories-Final Report 1998. (UK)} [Downloaded from the internet Aug 2011} [This document summarizes (unsuccessful) attempts to confirm a link between use of PVC mattresses which had been fire-protected by Sb (+As) compounds and the prevalence of SIDS; it covers a wide range of follow-up studies. Perhaps a fault in this examination of the possible toxicological and epidemiological evidence and which might have been the most useful, a comparison between Japanese populations which did not use the PVC infant mattress and the target population, which was briefly alluded to, was not probed in depth] [It should be noted that female workers exposed occupationally to Sb showed an increased incidence of spontaneous late abortions compared to controls (cf. ref. citation in S Schonwald (author of book) Medical Toxicology, accessed on internet] Roberts EM et al. (Maternal residence near agricultural pesticide applications and autism spectrum disorders arisen in children in the California Central Valley) Environ Health Perspect. 2007 115 (10) 1482-9 [It was suggested in a n= 249 subject study relating distance from source and level of pesticide usage could indicate that that organochlorine pesticides (e.g. dicofol and endosulfan) had induced autism spectrum disorders e.g. via the perturbation of fetal development in the affected population]. Maternal residence near sites where chlorinated aromatic or perchlorocarbon group or related structured insecticides had been employed was dose dependently correlated with the prevalence of autism in offspring; similar but less precise indications have been made for other endocrine system disrupter toxins especially the organophosphates and as well as toxic heavy metals (which include metalloids) such as Pb, Sb, Cd, Hg, and As which may also be a potential risk factors for autism (cf., e.g. Hall et al. loc. cit.). (Cf. also Volk et al. loc cit. which could suggest that maternal intoxciaton by road traffic toxins might relevant to the etiology of ASD) Romanoto M et al., (Heparan sulphate a putative decondensing agent for human spermatozoa in vivo) Hum Reprod. 2003 18 1861-73 [While as chromatin decondensating agents heparin and HS were equally active, completely de-O / deN sulfated heparins were completely inactive. Partially de-sulfated heparins showed a heirarchy of different intermediate degrees of activity. Of especial importance was the presence of N-sulfonation (N-acetylation, however could serve almost equally as well).

The hierarchy of dependence of the ability to decondense chromatin from DNA seemed surprisingly close to the hierarchy of the abilities of chemically modified heparins to inhibit the seeded crystallization of CaCO3 (cf. Grant et al. also cf. Borges et al. loc. cit.]. It should be noted that CaCO3 seeded crystallization is of central interest to hypotheses relating to a non-nucleic acid definition of life (cf. Lima de Faria Evolution without Selection . Different HS structural features seem also to mediate innate immunity via Toll-like receptors and xenobiotic assisted alteration in such structures could be relevant to the aetiology of (as subset ) of ASD conditions. Cf. (The presence of heparan sulfate in the mammalian oocyte provide a clue to human sperm decondensation in vivo) Ibid., 2008 23 (5) 1145-50 [HS provides the necessary protamine sink which in the presence of glutathione allows sperm DNA to transfer to histone binding in the oocyte. The HS from other mammals was found to be equally effective; perhaps later studies of HS microstructure might reveal differences between distant animal species in the details of how sperm chromatin becomes decondensed by HS during fertilization since the unpacking of sperm DNA is thought to control zygote gene activation during fertilization; this might suggest a key role for HS in future epigenetics theories] Rossignol D. - (1) (The use of urinary porphyrins analysis in autism) Medical Veritas 2007 4 91) 1276 It is reported that the urinary porphyrin test is apparently more effective for the diagnosis of autism than chelation challenge in detecting body burdens of toxic metal (e.g. Hg, Pb, and As) which are thought to responsible for the perturbation of heme biosynthesis and impairment of heme oxygen transport in autism. This may contribute to diminished oxygen transport to the brain. This might be alleviated it was proposed (Med Hypoth. 2007 68 (6) 1206-7) by the use of hyperbaric oxygen therapy (HBOT). Cf. also Nataf R et al. (Porphyinuria in childhood autistic disorders implication for environmental toxicity) Toxicol Appl Pharmacol. 2006 214 (2) 99-108 Cf. Urinary Porphyrin Profiles Metramix internet doc Ed RS Lal et al. Ch 8 in Toxicology and Detoxification. The possible role of HCB intoxication in perturbation of the tyrptophan nicotinic acid metabolic pathway seems to be indicated by the report of Llamban et al. loc. cit. [A more recent study of urine analysis for the diagnosis of ASD {Yap et al. loc cit.) described how pattern recognition of proton NMR spectra can be a highly effective procedure for the diagnosis of ASD; these results also indicate disturbance of the tryptophan nicotinic acid pathway in ASD] Rossignol D. - (2) Discussion of the possible stratagem for autism therapy via stimulating capillary growth Cf. angiogenesis is probably one way that HBOT helps autism (web.vsan.org/rok-ag/hbot.HBOTcap.growth.pdf) [Cf. also Emanuele loc. cit.] [The putative deficiency of the supply of oxygen to the brain as a key art of the aetiology of autism may, it is further indicated putatively could be offset by anticoagulant therapy e.g. using low molecular weigh heparin or pentosan polysulfate (PPS); the ability of brown chocolate to inhibit platelet determined blood coagulation [cf. Kennedy] could also point to a possible benefit of therapeutic use of this foodstuff] Royal Society of Chemistry (UK) Aluminium in Food and in the Environment. Special Publication No 73, Ed Massey RC and Taylor D, 1988 [This document provides evidence for the neurotoxicity of Al] Cf. also Kempson IM Lombi E. Chem Soc Rev. 2011 40 (7) 3915-40 (Hair analysis as a biomonitor for toxicology, disease and health status) {the validity of inorganic element hair analysis for epidemiology and etiology studies was upheld by this review). Cf. also Smolders R et al. Environ Health 2009 8 8

A study conducted at the University of Aberdeen showed that Al3+ binds to heparin and HS (sufficiently strongly to displace Ca2+) but perhaps most significantly greatly reduces the pH value of Al-heparinate by comparison with Na or Ca-heparinates. The presence of Al3+ ions created a pH 4.3 buffer in the vicinity of the heparin (and putatively also heparan sulfate) molecules; ( in sharp contrast to the binding to heparin of this non-physiological toxic countercation, the binding of a range of physiological counterions to heparin created a pH-stat buffer of pH 7.2 which is much closer to physiological pH value). While the ability of HS to bind Al3+ may contribute to a natural barrier function (including of the BBB) the generation of H+ adjacent to heparin HS following the total replacement of the major counterions by excess Al3+ will also promote de-N sulphonation. This alteration in HS seems also to be associated with Alzheimers disease (Brunisma PMID 19636575). This de-N sulphonation is a major microstructural alteration. De-N-sulfonated heparin, e.g. completely fails to inhibit calcification (plaque formation) a phenomenon which N-sulfonated heparin accomplishes highly efficiently (cf., Grant D et al. Biochem J. 1989 259 41-5). Aberrant, defective HS mis-inhibition of (clacified) plaque (crystal) formation may be relevant to a fuller understanding of the aetiologies of various degenerative diseases (cf. D. Grant et al Med Hypoth 1992, 38, 49-55). HS microstructure integrity is likely also to be required for correct neurological HS-related functions putatively subject to perturbation as a consequence of intoxication by heavy metals in autistic subjects. Another study conducted at the University of Aberdeen established that heparin and therefore also putatively the heparin (more sulfated) regions of HS are Haraguchi (loc. cit.) type multi-inorganic element matrices. In this regard they resemble marine alginates which previously had been indicated to exist in this form in nature. The inorganic profiles of heparin are correlated with blood serum and also loosely with the multielements contents of the sea (cf. Haraguchi loc. cit.) and also with the multi-element contents of human scalp hair. This reinforces the validity of the use of scalp hair analysis for the evaluation of inorganic ion dyshomeostasis or intoxication and teneds to validate the alternative medical use of hair analysis of autistic subjects (n.b. this is a child health monitoring procedure which is also enforced by the Kempson Lombi (UK) and Smolders et al. (EU) reviews). The latter however noted that the concentration of trace elements can vary 5 or 10 fold due to specific exposure situation, such as living in the vicinity of coal and metal mining and smelting operations... When using hair and finger/toe nails to quantify exposure to environmental contaminants, caution is needed in the interpretation of exposure data. Many contaminants have been proved to reach har and finger/toenails by two major routes: endogenous (xenobiotics reach the hair matrix through blood) and exogenous (atmospheric deposition) hence it may be difficult to distinguish between contaminants taken up and those related to external contamination. For methylmercury which is generally taken up through food or dental amalgam, hair is by far the preferred matrix for biomonitoring and large scale biomonitoring campaigns have clearly linked methylmercury with neurodevelopmental deficiencies (Choi loc. cit.) Roze E. et al., Environ Health Perspect. 2009 117 1953-8

Rudd TR et al. (Influence of substitution pattern and cation binding on conformation and activity of heparin derivatives) Glycobiology. 2007 17 (9) 983-93 PMID 17580314 Cf. Guimond SE et al., Biochemistry 2009 48 (22) 4772-9 and Hughes AJ. et al. (A zinc complex of HS destablizes lysoyme and alters its conformation) Biochem Biophys Res Commun. 2012; PMID 22884801
Salvemini et al. Drug News Perspect.1998 11 (4) 204 Sanderson J, Chem World 2006 3,2 8 (HEALTH Siome plastic bottles continuously leach antimony Toxic risk in bottled water?) [Bill Shattock et al.

at the University of Heidelberg indicated that a possible health risk arises from the continuous leaching of small amoutns of Sb from the PET containers used for bottled water] Seelig MS. Amer J Cardiol. 1989 63 4G-21G (Cardiovascular consequences of magnesium deficiency and loss: pathogenesis prevalence and manifestations. Magnesium and chloride ion in refractory potassium repletion) [Dietary Mg deficiency is highly prevalent e.g. in the USA; this especially affects the young] Cf. Keenoy BM et al. J Amer Coll Nutr. 2000 19 (3) 374-82 (Magnesium status and parameters of the oxidant-antioxidant balance in patients with chronic fatigue: effects of supplementation with magnesia]. There are numerous grey literature reports of Mg deficiency in ASD. Shainkin-Kestenbaum R et al. Clin Sci.1989, 77 (5) 463-6 [Al intoxication will impede superoxide dismutase (SOD) action] Cf. also Nephron. 1990 55 (3) 251-3

More on Al Intoxication
Chainy GB et al. (Res Commun Mol Pathol Pharmacol. 1996 94 (2) 217-20), however, found that aluminum intoxication of rats caused microsomal lipid peroxidation not via SOD but by inhibiting catalyase. Moumen R et al. (J Trace Elem Med Biol. 2001 15 (2-3) 89-93), however, found that in rats aluminum intoxication increased xanthine oxidase but decreased glutathione peroxidase concentration. These effects might have caused an increased oxygen free radical production which stimulated an increase in SOD. Gmez M et al. (Free Radic Biol Med. 2005 38 104-110) found that aluminium administration caused oxidative stress in studies of a range of antioxidant enzymes expressed in rat hippocampi but this could be countered by the administration of melatonin which offered a possible therapeutic interventive strategy to counter such oxidative stress. Cf. also Garcia T et al. (Hippocampus. 2010 20 (1) 218-25) where this idea was confirmed in a mouse model of Alzheimer disease. It should be noted that HS microstructure (cf. Kuberan B et al. J Biol Chem. 2004 279 5053-6) also changes in the pineal gland as a consequence of light exposure. This effect is thought to be independent of melatonin. Al hydroxide is the principal adjuvant used in vaccine preparation (cf. e.g. Ch 4-2 in Metallotherapeutic drugs .. by M Gielin and ERT Tiekin). The modus operandi of this use of aluminum is not established but may be related to the surface adsorptive properties of antigens onto colloidal aluminum hydroxide which could further implicate glycan-aluminium hydroxide nanoparticulate interaction. Cf. the binding of Al3+ to HS and heparin is abnormally strong and also creates local acidosis. This phenomenon may be of relevance to the elucidation of why vaccination has been associated in the grey literature with the initiation of autism. It should be noted that interaction of antigen sugar structures (glycosylation) is what to a large extent underpins the molecular interactions which control immune system modulation by vaccination. Most of the studies in this field have, however, been concerned with N-glycosylation. (HS is invovled in Oglyocsylation which also impacts on immune surveillance). Examples of N-glycosylation studies of immunoglobulins are: (cf. Arnold JN et al. (The impact of glycosylation of athe biological function and structure of human immunogloblulins) Annu Rev Immunol 2007 25 21-50 (Species-specific variation in the glycosylation of IgG) Glycobiol. 2000, 10 (*5) 477-86) (cf. eg. web.bioch.ox.ac.uk/glycob/archive/index.html); cf. Pyi M et al. High throughput isolation and glycosylation anlysis of IgG..)Mol Cell Proteomics. 2100 10 MIII010090) N Pivac et al. ibid., MIIIO.0004200 found no change in the plasma glycome(i.e. N-glycosylation) in (n=86) ASD but a significant alteration was found in 99 children with ADSD (an increased antennary fucosylation of biantennary glycans) comapred with 340 controls.. Melatonin (N-acetyl-5-methoxytryptamine) and melanin maybe anti-nitrant as well as anti-oxdiant (also anti-Al?),

Shattock P and Whitely P. (The Role of Tryptophan in Autism and Related Diseases) [The Nutrition Pract, Summer 2006 {accessed from internet 2011}] This review of autism focussed on the putative role of serotinin (5-Hydroxyl tryptamine) which while mainly in the gastrointestinal tract also affects the CNS and a diminution of serotonin action is possibly a key part of the etiology of autism and related conditions. {Cf. serotinin is converted into melatonin (N-acetyl-5-methoxytryptamine) (cf. prev ref.). Both require tryptophan, the amino acids which is essentially required from diet. Melatonin and melanin may be an anti-nitrant as well as an anti-oxidant. Melatonin supplementation has bene indicated {cf., PMID 21615492} to ameliorate oxidative stress and inflammatory signaling induced by streuous excercise in adult human males}. Tryptophan per se may also be of benefit as an anti-psychiatric drug which can be administered as a purified form or as a normal food component. Also tryptophan, in conjunction with cysteine and tyrosine juxtapostiions in proteins, is putatively a necessary part of the tissue protective anti-oxidant and anti-nitrant defense system}. Cf. also Rossignol loc.cit. related notes: {HCB and putatively also the HCB-mimetics PCBs etc., appear to block the major -lipoic acid related electron transfer NAD invovled in energy metabolism; Sb (As and Hg) might impair the action of a-lipoic acid further. This may be why the tyrptophan-nicotinic acid pathway is perturbed in autism

{Yap et al., loc. cit., using advanced pattern recognition for urine NMR spectra seems to have confirmed the disturbance of the tryptophan nicotinic acid pathway in autism}.
{Other possible therapuetic agents which might be considered for the relief of autism symptoms. Luteolin and thiosalicylicate which are indicated to couter inflammation via the inhibition of VEGF release from mast cells (cf. PMID 21244751) and Quercetin which has been indicated (cf. PMID 21562239) to confer (antioxidative?) tissue protection by suppressing amphiregulin/EGFR signaling}. Shaw W. E.g. internet document Oxalates Control Is A Major New Factor in Autism Therapy (The Great Plains Laboratory) The amount of oxalate in urine of autistic children (n=100) was found to be consistently much higher than in normal controls (n=16) This was hypothesized to have arisen from fungal overgrowth. The principal damaging effect of oxalate was believed to be the formation of sparingly soluble Ca oxalate crystals which both physically damaged tissues and also promoted inflammation. {However the ability of HS to prevent Ca oxalate stone formation could point to a primary defect in HS anti-crystal seed protection as being a possible contributory factor to hyperoxaluria; HS is also primarily subject to adverse dietary factor perturbation. HS was indicated from Sb-heparin removal studies to be especially sensitive to the effects of Sb intoxication}.

Shepherd DI. Multiple Sclerosis in North East Scotland Univeristy of Aberdeen Doctor of Medicine dissertation (1976) Vol I, used as the basis of an epeidmeiolgical study of MS later conducted by Purdey M loc.cit. (with the assistence of the author). Shepherd identified those areas in Aberdeenshire with an especially high number of cases and included districts with amongst the highest rates of this disease in the world . This disease was indicated to have some environmental-factor origin. [The hotspots of multiple sclerosis identified by Shepherd were later found by Purdey to have high concentrations of barium which seems to have derived from the geological presence of a baryte rich zone which follows the northern border of the Central Rift Valley. This seems to have led to a high soil, water and plant tissue barium contents which putatively caused a human population intoxication by this element which may lead to further pathological susceptibilities]. It should be noted that Shepherd also suggested that the first historical description of multiple sclerosis in the UK etc. had coincided

with the start of wide use of dental amalgam around 1835. The use of silver mercury etc. intoxication caused by the widespread use of dental amalgam might have at least partly been a trigger of MS.
[It should be noted that the metals used for dental amalgam commonly contain small amounts of other putatively toxic elements [even in some cases antimony] (e.g. which might under pathogen modification could perhaps lead to volatile methyl-mercury or methyl-(other metal) formation. Even the presence of ultra-low amounts of certain elements (cf. Marth loc. cit.) can induce inflammatory cytokine production]. Shepherd noted that MS had been indicated in an (n=150) epidemiological study to be promoted by childbirth, trauma and vaccination. Shepherd also noted the apparently inexplicable finding (EJ Field) of the persitently raised antibody titre to measles in MS patients and also a report by Lumsden in 1970 of histological evidence for the optic nerve and visual pathway invovlement in MS. [It should be noted that a similar internationally high prevalence of autism also affects the Scottish population]. Also there seems to be some similararity between autism and MS regarding the associations with measles antibodies and visual pathway invovlment. Shotyk W. web. medicine.org/toxin-leaches-inotbottled-water-in-pet-containers/ Levels of Sb rise the longer the Sb stays in the bottle. Cf. Keresztes S et al., Sci Tot Environ 2009 407 (16) 4731-5 and the review by Sax L. Environ Health Perspect. 2010 118 45-8 which draws attention to the ned for more research into the possible public health hazard posed by the leaching of endocrine disrupting amounts of Sb from PET bottles; perhaps this is enhanced by the presence of acidic food components. Cf. also Fordham PJ loc. cit. {Another possible effect of Sb (plus phthalate) intoxication arising form commonly used plastic food containers might be considered: the alteration of gut microflora e.g. as found in autistic subjects.} Intolerance to dietary factors seems to affect at least a sub-group of ASD subjects which could suggest some overlap between ASD and Celiac disease (which results from intolerance to dietary gluten (gliadin) causing inflammation of and altered architeture of the small intestine which becomes completely resored following a gluten-free diet [cf. Gianfrani et al. J Immunol. 2006 177 4178-86]; the inflammatory response to gliadin is believed to arise from NFB induced T-cell cytokine (including I:L2 and IFNg). This causes an increase in itric oxide synthesis which can cause inappropriate HSPG degradation. It should be noted that aberrant NFkB expression has been demonstrated in ASD (Young et al loc cit)]. {Phthalate contamination of distilled water used some 25 years ago in an Aberdeen U. lab. was traced to a specific PVC plasticizer (using comparison of published with observed ir spectra) which might have been used in the municipal supply system at that time. The small amouts there present evidently had slowly accumulated at the colder parts of the automatic glass distillation apparatus. This produced very large alteration in biochemical parameters related to blood homostatis; this contamination might , in principle have affected blood flow in capillaries at gut and brain in humans. Further work is required to check this out}. Other information obtained from Aberdeen University studies which are of possible relevance to how the etiology of ASD might be a consequence of environmentally present toxins including Sb intoxication was the likelihood that HS binds strongly to Sb (this was indicated from Sb-heparin removal studies {Moffat, CF Aberdeen U. related multi-inorganic element content of heparin} {Sb (together with As, Be, Cd, Cr, Hg, Ni and Tl (all of which metals can become selectively enriched at natural polyanion surfaces including those at human cell surfaces and ECM, cf . mass spectrometric studies of heparin reported by Moffat, Grant and later confirmed by ALS loc. cit) are also those which have been especially indictated (Levine SA Reinhardt JH. J Orthomol Psychiatry. 1983 12 (3) 166-83) to be associated with chemical hypersensitity which incidentally (on internet blogs) which has further been indicated to be commonly countered by exogenous heparin administration. Shearer TR et al. (Minerals in hair and nutrient uptake of autistic children) J Autism and Dev Disord. 12 (1) 25-33

[The hair element contents of Ca, Mg, Zn, Cu and Pb did not differ significantly between n=12 ASD subjects and controls. However the amount of Cd was diminished by 62% in the ASD group hair samples]. {This early small cohort study could suggest that the elevation in ASD hair of toxic heavy metals which was reported in later studies might not be of critical relevance to the origin of the ASD disease process but may be a seconday outcome of this}. A sub-group of ASD subjects may not show any characteristic alteration in hair elements. This might apply more to the Aspergers type of ASD than the more typical type of ASD howeve cf. the findings of Bell (cf. Hall, loc. cit.). It should be noted that subchronic Cd intoxication could negatively affect nitric oxide determined vascualar relaxation (cf. Skoczynska loc. cit.). Shute N. Scientific American. October 2010 81-5 (Desperate for an autism cure. Diagnoses have soared, but valid treatments are few. Parents have turned instead to dubious, and often risky, alernative therapies) Serotonin & Autism It was noted by Shute (preceding ref.) that administration the neurotransmitter serotonin (mainly prpduced in the gut which is of diminished effectivness for this purpose in autism) and which might accout for the formation of IAG (cf. Shattock] a putative marker of ASD) has been indicated to be very effective in reducing the repetetive hand motions of obsessive-compulsive disorders but it seems that serotonin therapy is ineffective for the alleviation of the repetive motions typcial of autism. Oxytocin & Autism Among the new candidates for autism therapy is oxytoxicn (a medication that enhances REM sleep) which in one (n=13) French study was reported, as noted by Shute, to improve face recognition by Aspergers syndrome subjects.

Oxytocin (and its receptor, a rhodopsin G-protein) may also offer wider therapeutic benefit for ASD therapy and include a normalization of nitric oxide dependent pathways including those centred on HSPG biochemistry. It should be noted that the fear (brain) response which can (as reported for experiemntal animals) be reversed by HS-oligomer administration. The use of exogenous oxytoxin to induce rapid childbirth might, however be a causative factor in autism according to Stillerman (loc. cit.) since this is a possible cause of the trauma and brain injury which could at least partly lead to the kind of brain damage which is believed can also trigger autism in the neonate
Skoczynska A Martynowicz H. (The impact of subchronic cadmium poisoning on the vascular effect of nitric oxide in rats) H. Hum Exp Toxicol. 2005 24 (7) 353-61 [Cd intoxciaton decreases the serum NO concentration and the sensitivity of vascular resistance to inhibition of e-NO synthase and the effect on this of acetylcholine (ACh)] {It should be noted that the effects of Cd intoxication has been linked to the etiology of ASD; of possible relevance to this perhaps the finding of University of Dundee researchers (Kennedy loc. cit.) who studied sensitivity of vascular ACh response quantified using a doppler laser techniqe to classify Chronic Fatigue Syndrome (CFS)-like dysfunctions (especily in order to distinguish CFS/ME from Gulf War Syndrome and Organophosphate intoxiication induced chronic fatigue-like syndromes). Discussions between this group and FB Williamson (Aberdeen University) had explored the role of HS biochemistry/NO biochemistry in vascular tone and the known effects of Cd, Pb and Zn and other prooxidant xenobiotics on this. The outcome of these deliberations {which are partly responsible for the present discourse}are putativley also relevant to ASD research interests Blood pressure may be linked to both heavy metal as well as PCB intoxciaton (cf. Goncharov et al. Enviorn Health Perspect. 2011 119 (3) 319-25.

The intoxication by PCBs which induce obesity and type-2 diabetes seem to link with the cause of ASD. A possible mechanism of such a linkage is via HSPG tissue reguation. High blood glucose e.g. decreases HS integrity via HS microstructural perturbation.. Smink A et al. Acta Paedriat. 2008 97 (10) 1465-9 PMID 18665907 [This study estblished the direct correlation between maternal intoxication by hexachlorobenzene (HCB) and obesity in the child; Other larger studies [cf. Moreno, loc.cit.] (have also implicated HCB and related intoxications by PCBs in the etilogy of unexpalined obesity in adults] {The perurbation of developmental biochemical signaling relating to nervous system assembly might be resonsible for this relationship and also for the putative role of HCB in the induction of autism}. Srensen HP et al. (Heparan sulfate regulates ADAM12 through a molecular switch mechanism J Biol Chem. 2008 283 (46) 31920-32) {The exgenous HS regulating actiivy can be mimicked by the therpuetic application of xylan sulfates PPS e.g. SP54 or algal sulfated polysaccharides produced at Aberdeen Univerisity. PPS has been used traditionally for the relief of interstitial cystitis and as a blood anticoagulant} Stejskal J Stejskal VDM. Neuroendocrinol Lett.1999 20 351-64 (The role of metals in autoimmunity and the link to neuroendocrinology) Stephenson DT et al. Histopathological characterization of the BTBR mouse model of autistic like behaviour reveals selective change in neurodevelopmenal proteins and adult hippocammpal neurogenesis Mol Autism. 2011 2 7 [3/4 of the markers identified are glycosaminoglycan-linked viz. NG2 (a chondroitin sulfate PG), PSANCAM and NeuroD (both of which are linked to HSPG biochemistry)] {This paper provides supportive evidence for an HSPG-related etiologicy of ASDs} N.b. HSPG in dendritic spines has been traditionally propsed to be invovled in cognitio and memory. Dysfunction of dendritic spines has also been hypothesized to be part of the etiology of ASDs. (cf Wikipedia internet) Pb2+ selectively affects synthesis of versican chondroitin sulfate which seems, as indicated above, to be centrally implicated in the biochemicsty of neurodevelopmental PSA-NCAM markers associated with autism.(cf. Fujiwara Y et al. J Health Sci. 2003 49 (6) 534-40 who also discussed how Pb2+ inhibits wound healing by a related mechanism, it seems that Pb and Cd specfically inhibit the condroitn and dermatan core protein synthesis without affecting the sulftated side chain synthesis whereas other of these syntheses can be inhibited during HSPG synthesis in the presence of these heavy metals). Stillerman E. (The truth about Pitocin) Massage Today. 2006 06 (03) [Pitocin, oxytocin extracted from animal tissues, has been used for a long time to induce labor but according to this author this might alter the normal childbirth process so as to induce autism producing head injuries in the infant] Stow JL. (Heterotrimeric G protein, G-apha i-3 in Golgi membraneregulates the secretion of HSPG in LLC-Pki epithelial cells) J Cell Biol. 1991 114 (6) 1113-24 [The G-alpha i-3 protein dependent HSPG secretion system may become perturbed in ASD as a result of childhood vaccine based protection therapy as postulated by Megson (loc. cit.) who suggested that sutism is a G-protein signaling defect (especially that afforded by G-alpha i-3) disease]. {The related alteration of HSPG biosynthesis following dietary lipid augmentation or alteration in palmitoylation may conceivably be a related phenomenon invovling G-protein activity modification. If theMegson hypothesis that autism is a G-protein defect condition is confirmed this obviously potentially implicates the role of G-protein signaling directed HSPG biochemistry in the etiology of autism. A related effect of the Golgi disrupter brefeldin allows HS recycling to be interrupted and studied in the HS oligo generation mechanism studied by Ding et al., loc.cit.}

Templeton DM (Metal-proteoglycan interactions in the regulation of renal mesangial cells: implications for metal induced nephropathy) Proc Trace Element Health Disease. 1991, pp209-218; Cambridge UK Royal Society of Chemistry, Aito A. Ed., Proc Jo Nord Trace Elem Soc/Union Pure Appl Cehm Int Symp 1990 cf. Chem Abs. 111 129 40712 [This cell culture study indicates how heavy metal intoxication might induce HS microstructural change] {This might trigger autism and/or other diseases in which altered kidney function is implicated} Teoh MLT et al. Cancer Res. 2009 69 6355-63 [Linkage exists between redox status (oxidant and nitrant overload) and HS acitivity] {E.g. relevant to HCB/heavy metal induced redox status disturbance} Theoharides TC Zhang B. (Neuro-inflammation, blood brain barrier, siezures and autism) J Neuroinflamm. 2011 8 168 [Brain mast cells activated by allergic environmental and /or stress triggers could lead to focal disruption of the BBB and neuro-inflammation thus conributing to the development of seizures] {The role of HS in the BBB suggests that this is a target for such disrutpion} Thompson NW et al. (Early thimerosal exposure and neuro psychological outcome at 7 and 10 yr) New Eng J Med. 2007 357 (13) 1281-92 [No negative outcome of this kind of Hg exposure was observed] Utschig LM et al. (Mercury-199 NMR of the metal receptor site in MerR and its protein DNA complex) Science 1995 268 380-385 [MerR metalloregulatory protein which acts as a selective and strong Hg(II) bindier and sensitive metal ion (putatively three coordinate ligand to Hg) sensor activates expression of the stress responsive mercury detoxification (mer) genes; it reponds to nanomolar Hg(II) concentrations and stimulates transcritiption at this level by binding Hg(II) specifically at high effinity {in the absence of Hg(II) MerR binds tightly to DNA and represses transcription}. Metal binding to the proteinDNA complex stimulates transcriptionm by altering the conformation of the DNA Hg(II) was also found to substute readily into a variety of Cu and Zn metalloproteins {Putatively Hg intoxication as a promotion stimulus for at least some of the symptoms of autism could be conditional upon defects of autistic subjects of MerR gene activity effictivness; this suggests that bacterially proudced MerR proteins or related Mer proteins which bring very strongly toHg(II) and might be useful for the therapeutic (partial) alleviation of the symptoms of autism}. Van Birgelen APJM. Environ Health Perspect., 1998 106 (11) 683-8; cf., Shen H et al. Hum. Reprod., 2008, 23 (1) 201-10 Vali F et al. (Effect of coproporphyrin test of drugs used in the treatment of parasitic disease Brit J Industr Med. 1965 22 260 [The use of Sb tartarate (tartar emetic) as a treatment for bilharzial infection caused an elevated urinary excretion of coproporphyrin {which might have been confused with the effect of Pb intoxication}] {Cf. A highly publicized case of the urinary porphyria and mental derangement the UK monarch King George III which likely resulted from the therapeutic use of very large dose Sb tartarate as indicated by moderns investigations of his medical notes which indicted he had been administered James mixture ; an attempt to obtain a multi-element investigation of a hair sample seemed however indicated the presence of very large amounts of arsenic indicating that the King has been affected by As intoxication but other information from a Diary of a Mad Kings Mistress (web.authorsden.com/visit.viewArticle.asp?id=26650) seems to indicate that the hair studied was actually froom a wig Van Wazer JR Cf. e.g. Amer Sci. 1962 50 450

Cf . Lockhart JC. Chem Rev. 1965 65 131-151 Vilar RE et al., Biochem J. 1997 255 183-91 {This study showed that nitrite derived from the oxidation of nitric oxide can depolymerize HS under physiooical conditions. The authors were unable to fully explain why a phosphate buffer enabled this reaction but an imidazole buffer failed to act similarly. It is possible that the usual contamination of phosphate buffers with iron ions which can act as HS de-N-sulfonation catlayst is the expplanation. This work is considered to have key relevance to how dyshomeostasis of metal ions and augmentation of toxic metal ions which have been associated with ASDs can diminishe tissue protection afforded by HSPG and that this diminution is the ultimate origin of ASD defects}. Vilas GI et al. (Effect of a-lipoic acid on HCB porphyria) Biochem Mol Biol Int. 1999 47 (5) 815-23 Volk HE et al. (Residential proximity to freeways and autism in the CHARGE study) Environ Health Perspect. 2011 119 (6) 873-77 Waring R et al. (Sulphur metabolism in autism) J Nutrn Environ Med. 2000 10 25-32 [Evidence for rhodanese dysfunction/inhibition in autism which impairs the detoxification of cyanide and thiosulfate thiocyanide] {Mitochondrial environment? Possible effect of HCB-DNA binding?} Watt NT The role of zinc in Alzheimers disease (Pdf internet) Watanabe N et al. (Glypican-1 as an A-beta binding HSPG in the human brain: its localization in DIG {detergent insoluble glycosphingolipid-enriched) domains {where Abeta is produced} and possible roles in the pathogenesis of Alzheimers disease FASEB J. 2004 18 (9) 1013-5 Cf. also Brunisma et al. loc. cit. Weber R et al. Environ Sci Pollut Res Internat 2008 15 (5) 363-393 PMID 18697132 [Persitent Organic Pollutants {especially the dioxins and related chemical toxins generated as a byproduct of the chlor-alkali industry and more recently during recycling of plastic, especially as conducted in third world or currently fast-industrializing countries}. The authors draw attention to the problem of enforcing international health standards (and the prinicple that the polluter pays) regarding dioxin and related POP input into the atmosphere and the global pollution thereby caused] Willenbourg DO et al. J Immunol 140 (10) 3401-15 [Report that HS mimetics inhibit allergic encephalomyelitis in the rat (a model of MS)] Wilson SC et al. (The chemistry and behviour of antimony in the soil environment with comparison to arsenic) Environ Pullut. 2010 158 1169-81 [Sb binds to organic matter]. {This suggested Sb binding to the humic polyanions may mimick Sb binding to key animal extracellular anionic polysaccharides such as HS which are invovled in tissue development} Windham B

Mercury exposure levels from amalgam dental fillings: documentation of mechanism by which mercury causes over 30 chronic health conditions; resultsof replacemnt of amalgam fillings and occupational effects on dental staff Discussion of reported rapid recent increase in Hg levels in humans; While mercury was detected in the blood of 2% of women aged 18-49 in the 1999-2000 NHANES survey , that level rose to 30% of women by 2005-2006; surveys in all studies using hair tests have found dangerous levels of mercury in an average of 22% of the population, with over 30% in some states like Florida and New York. Cf. Autism and Schizophrenia subgroup related to blockage by toxic exposure of enaymes processing gluten and casein web.flcv.c0m/autismgc.html Windham GC et al. (Autism spectrum disorders in relation to distribution of hazardous air polutants in the San Francisco Bay area) Environ Health Perspect 2006 114 (9)1438-44 [A n=284 study of ASD subjects suggested and association between ASD and intoxciation by urban air pollutants especially heavy metals and metalloid (As, Cd, Hg, and Ni ) and chlorinated organic solvents (especially trichloroethylene and tetrachloroethylene) where the adjusted odds ratio were elevated 50% in the top quartile by heavy metals and chlorinated solvents but not for aromatic solvents] {A later epidemiological study [Goldman S M Ann Neurobiol .2011 (14) DOI: 10.1002/ana22629] reported that intoxication by trichloroethylene is a risk factor for Parkinsons disease but could require a long incubation period (10-40 years) which concnevably might indicate that a slow in vivo transformation occurs into chlorinated aromatic molecules which are the actual disease producing factors}. Wei H et al., J Neuroinflammation 2011 19 8 52 demonstrated that IL-6 was 86% (p<0.01) elevated in the brains of 6 autistic subjects (compared to 6 controls) and that this was associated with imparied cellular adhesion and migration of granule cells also stimulated related excitory synapses while not affecting inhibiting synapses or dendritic spines. {This cellular and adhesion migration is putatively associated with HSPG and hence the nitrosative degradation of HS following on the inappropriate elevation of IL-6 could be of central relevance to the etiology of autism. Cf .also this author et al., Am J Pathol 2011 179(1) 66-74 where it was reported that abnormal lymphocyte adhesion occurs in ASD.. B-lymphoblast adhesion (a model sytem) demonstrated such abnormal adhesion. Reduced neural cell migration and associated alteration of dendritdic morphology, axonal branching and synapse formation could cause autism. This adhesion was dependent on integrin b1 and focal adhesion kinase and Src expression were decreased as was IGg protein expression in ASD. Cf. also Naik US et al., PloS ONE. 2011 6(5): e19488 reported elevated NF-B expression in autistic subjects (n=67) compared with controls 3.14/1.40/ Cf. also Chez MG et al. Ped Neurol. 2007 36 361-5 reported that THFalpha (a potent inducer of NFB) was elevated in the CFS of 10 autistic subjects (by a factor of 53.7) [It should be noted that Malik et al. loc. cit. also recentlly reported that NFkB signaling pathway was not dysregulated in the brains of ASD subjects and therefore NFB was unlikely to explain why ASD was associated with inflammation]. Woods JE et al. (Cf .Medscape Today web medscape.com.viewarticle.730552_4 (Urinary pophyrin excretion in neurotypical and autistic children) It can be suggested that the uro- and precoproporphyrins are compatible between normal and autistic children of the same age [n.b., however it should be noted that the global intoxication by e.g. HCB + heavy metals means that urinary porphyrins will putatively now have increased for all humans by comparison to the pre-industrial period] Cf. also Correia et al. 2006; (cf. Olivein et al. Ponselle et al.) (Krishnamurthy et al.) Deficient mitochondrial porphyrin uptake mediated by prophryin transporter

Abcb6 [Mitochondrial dysfunction may account for differences in urinary porphyrin levels between autistic and normal children] {Mitochondrial DNA has been indicated on internet blogs to bind halogenated pesticide molecules and heavy metals including Sb} Yamaguchi Yu (Proteoglycans in the developing nervous system) Semin Cell Dev Biol. 2001 12 (2) 99-106 HSPG syndecan-2 is involved in brain dendritic spine development (these structures are thought to be involved in memory and cognition) Yan H et al. Anal Chem. 2009 81 (10) 414-52 (Identification of arsenic-binding proteins in human cells by affinity chromatography and mass spectrometry) [The mechanism of As intoxication which can induce cancer of the bladder, lung, skin and kidney is unknown but is thought to involve cysteine S-H coordination. Analysis of subcellular fractions of A549 human lung carcinoma cells identified 50 proteins in the nuclear fraction and 24 proteins in the membrane/organelle fraction that could bind arsenic] Yang K et al. (Upregulation of Ras/Raf/ERK1/2 signaling and ERK5 in the brain of autistic subjects Genes Brain Behav. 2011 10 (8) 834-43 PMID 21848643 [Gene defects in Ras/Raf/ extracelular signal-regulating kinase ERK were previously assoicted with the prevalence of ASD (this signaling systtem is also believed to be invovled in neural progression, learning and memory. This system was found to be altered in a mouse model of ASD (BTBR). The upregulation of the above signaling in the frontal cortex of ASD subjects was indicated to occur in ASD.] {Since the Ras/Raf/ERK1/2 system is putativelly also under HSPG control this could add weight to the hypothesis that ASD is an HS defect driven disease} Yao Y et al. (Altered vascular phenotype in autism) Arch Neurol 2006 63 1161-4 [Autism, studied with n=26 vs 12 controls showed a direct biochemical signature of platelet and vascular endothelial activation and was characterized by increased oxidative stress; cf. Abramsson et al. loc cit. studies which showed that platelet and vascular development which is imparied by HSPG defective in HS N-SO3- microstructure anionic binding site {morphogen} dysarrangement]. {These putativley xenobiotic-induced HS code defects may be the ultimate cause of barrier structural .dysfunctions which is likely to explain gut and brain barrier dysfunctions in autism} Yap IKS et al. (Urinary metabolic phenotyping differentiates children with autism from their unaffected siblings and age-matched controls) J Proteome Res. 2010 9 2996-3004 [A pattern recognition comparison of urine NMR gives for the first time a simple diagnostic test for autism; a primary defect in the tryptophan nicotinic acid metabolic pathway in autistic subjects seems to be indicated by these results which also show up a large gut flora differentiation and also seem to confirm the general overriding redox dyfunctional nature of the etiology of autism] Yashuda H et al. (Infantile zinc deficiency: association with autism spectrum disorders) Sci Rep 2011 1 129 PMID 22355646 [This paper indicates that Zn dietary supplementaion may offer therapeutic benfit for the treat ment of autism] Yin A et al. (Methylmercury-induced alterations in astrocyte functions are attenuated by ebselen) Neurotoxicity. 2011 32 (3) 291-9

[Phosphroylation of ERK and the dissipation of the astrocyte mmtrochondrial membrane potential are beleived to be invovled in MeHg toxicity but it has been shown for the first time that ebselen (a Se containing oxidant) can stabilize the mitochondrial membrane potential against this effect of MeHg intoxication. This observation could indicate why Se-deficiency and Hg intoxication have been associated with autism (e.g. cf Priya L ). Perhaps ebselen therapy might be useful for autism]. Zn is believed to be required for effective Zn detoxicfication. This may be why infantile Zn defiiciency has been associated with ASD (Yashuda H et al.) Ebselen has also been reported (Smith SME et al, Chem & Bio 2012 19 (6) 752-63 PMID 22726689) to inhibit the (inappropriate) formation of reactive oxygen species starting from NADPH Yoo HJ et al. (Association between PTGS2 polymorphism and ASD in Korean trios Neurosci Res 2008 62 (1) 66-9 [This gene encodes cyclooxygenase-2 which is also known to be promoted by one of the most abundant environmental exenobiotics, PCB-153 also putativley associated with ASD induction cf. refs discussed under Pereira et al. loc. cit. Young AMH et al. (Aberrant NF-kappa B expression in autsim spectrum conditions: a mechanism for neuroinflammation) Front Psychiatry. 2011 2 article 27; doi:10.3389/fpsyt.2011.00027 [A similar pro-inflammatory mechanism was indicated to apply to Alzheimers disease, Parkinsons disease and MS; this could further suggest that the inappropriate HS de-N-sulphonation (cf. Brunisma) and cnsequent nitrostive degaradation of HS at these sites plays an etiological role in these diseases and putativley also in autism]. Yurgelin-Todd D (Secretin is active in a brain region activity which is implicated in autism) Press release 01.11.2002 web.test.innovation-report.com/html/reports/medicien-health/report-14140.html McLean Hospital and Repliglen announce results of brain imaging by secretin Zhang H and Forman HJ. Am J Respir Cell Mol Biol. 2009 41 (5) 509-75 Cf. Jean JC et al., Am J Physiol Lung Cell Mol Physiol. 2002 283 L766 Zhang L et al. (Concentration and gas particle partitioning of HCB in the ambient air before and after the Beijing Olympic Games) Bull Environ Contam Toxicol. 2010 85 (1) 1-4 Cf. also the Canadian Gvt. Document outlining the relative importance of various sources which include a major of HCB and dioxin/furan pollution from vehicles. Also presence of HCB (+HCH, DDT, PCB and PAA) in Hugli river sediments adjacent to Calcutta (Guzzella L et al. Envir Int. 2007 33 (5) 685-93) but various specific chlorinated aromatic substances manufactured for pesticide use (dieldrin etc.) were absent.

Further Sb Intoxication References

An internet survey accessed the textbook on Medical Toxicology by Seth Schonwald The antidotes to Sb poisoning noted were: dimercapto chelating agents. Female workers affected by occupational exposure to Sb (comparrted to control subjects) showed an increased incidence of spontaneous late abortions. Such occupational exposure was also associated with an increase of lung cancer with a latency period of 20yr. Keritner M et al., Int Arch Occup Environ Health 1995 67 (2) 119-23 [Sb2O3 and SbH3 show equivalent pulmonary absorption and renal elimination characteristics] Cf. Lauwerys R studies which showed that the Sb excreted in bile is partly reabsorbed in the intestine. The methylation of Sb seemed to be less than for As.

The log-log correlation between the airborne occupational concentration of Sb/creatine and in the urine of workers post-shift was highly significant. In a textbook (by Gummar Nordberg [available on the internet]), Handbook on the toxicity of metals results from lung Sb of 113 workers measured by X-ray spectroscopy was found (by McCallum et al, 1970) to increase to 12 mg/cm2 after 45 years (in the surveyed population the lung Sb loading increased approx. linearly with the period of employment). (Severe) Sb intoxication has been associated with ECG abnormalities and risk of sudden death (Sullivan & Krieger 1992) [for 124 workers exposed to Sb2S3 at air contents of 0.6-5.5mg/m2 6 died suddenly]. In animal studies of Sb intoxication changes in the T wave of the ECG pattern were noted Sudden death, evidently related to this has also been recorded for patients treated with Sb-based medication for parasitic diseases (cf. Halawani, 1963, Rugemaleto 1980). Moinsour SE and Reese SE. Exp Paratiol. 196516 148-57, proposed that use of tartar emetic (Sb tartarate) is an augmenting factor in the development of schistosomal myopathy. A similar Sb-based intoxication circumstance seems to pertain to the neurological illness of King George III (United Kingdom) who received James powder therapy (this apparently is a form of tartar emetic) [discussed separately above]. Sb intoxication has been associated with increased liver-damage associated enzymes GOT and GTP (Woodruff 1969) Kim et al. (1999) reported alteration of cytokine and immunoglobin levels [lower IgG1 and IgE) in Sbexposed workers SbH3 was similarly toxic to AsH3. Both substances are employed in semi-conductor manufacture. Sb also can produce SbH3 during soldering The emission of SnH3 (together with AsH3) during the use of lead acid batteries is of relevance to their safe use in non-nuclear powered submarines and more generally as a phenomenon of urban pollution by high volume road traffic. Gallichio et al (2000) reported that SbH3 induced haemolysis of erythrocytes . Other Sb Intoxciation Mechanisms Evidence that DNA binds Sb(III) (with a 1:1 stoichiometry) Ka= 1.4x106 L mol-1 under physiological conditions was reported by Li Y et al. (J Anal At Spectrom 2011 26 94-9). [Sb(V), however, apparently did not bind to DNA under the conditions studied bby these authors} The observation of the formaiton of a well defined DNA- Sb adduct is in keeping with the observation (made in previous sections) that Sb also binds strongly to HS. Sb - DNA adducts also show up (together with other apparently common instances of Cd (pr from tobacco smoke) Ni, Pb, Hg, and Mn and also (common?) chlorinated aromatic pesticide (related to HCB ingestion?) in DNA from peripheral blood leucocyte test method (using genomic DNA) as indicated by McLaren-Howard J. (J Nutr Env Med. 2002 12 19-31). Internet blogs also suggest simlar Sb-DNA findings could be common in autistic spectrum disorders. (Using mitochondrial DNA?). More controlled studies are needed. Comparison of Sb (III) and Sb(V) uses in medicine for respectively anti-helminith and anti-protozoal (Leishmania) with Pt(II) as an anticancer agent in humans is of interest to determine how drug resistence resistance is developed in these species. The common human cancer therpy drugs ciisplatin (and related drugs) suffer from poor effectivness caused by this. Elevation of GSH and metallothionene (used for other toxic metals) seems not is not to be employed, instead a P-glycoprotein like tranport system seems to be responsible. Sb and Pt intoxications seem to arise in general by a chemical mechanism which resemble each other and is different from that which applies to other toxic metals. It is apparent that both Pt as well as Sb may similarly share a stronger binding to HS (as well as to nucleic acids) than is the case for other metal ions. (The Pt(II)-heparin binding was found to be enhanced relative to that of Al(III) or Zn2+-heparin cf. Grant D et al., Biochem Soc Trans 1996 24 194S; the existecne of strong Sb-heparin binding (stronger than that of 38 other elements was inferred from the ease of removal of multielements from heparin using a styrene sulfonate anioni exchange column discssed in earlier sections).

Further HCB-Related References

from 19_REFERENCES.htm
Debets FM et al. Int J Biochem 1986 12 1019-25 (Metabolism as a prerequisite for the porphyinogenic action of polyhalogenated aromatics with special reference to HCB and polybrominated biphenyls (firemaster BP-6); Chem Biol Internat 37 77-94 (Effects of dietary antioxidants on the biotransformation and porphyrinogenic action of HCB in two strains of rats) Toxiclogy 15 181-95 (Effects of pentachlorophenol De Matters F (with Rimington C) et al) Nature 1961 191 363-6 (Nervous and biochemical disturbances following HCB intoxication) Den Besten C et al. Toxical Appl Pharmacol 119 181-94 (The role of oxidative metabolism in HCB-induced porphyria and thyroid hormone homeostasis. A comparison with pentachlorobenzene in a 13-week feeding study) Dobson S et al. IARC Sci Publ 1986 77 203-9 (Evaluation of environmentalimpact of HCB) Elder GH et al Morris CR et al. HCB: Proceeding of and International Symposium ibid., 1986 77 441-8 (Immunochemical studies of the uroporphyriogen decarboxylase effect caused by HCB. In Enriquez De Salamanca RE Med Hypoth 1990 33 (1) 69-7 (Is HCB human overload related to porphyria cutanea tarda? A speculative hypothesis) Feldman ES et al. Hepatology 1989 9 686-92 (Hepatic mitochondrial oxidative metabolism and lipid peroxidation in experimental HCB induced porphyria with dietary carbonyl iron overload) Fernandez-Tome M et al Kidney Blood Pressure Research 200 23 20-6 (Heme metabolism and lipid peroxidation in rat kidneys in HCB induced porphyria. A comparmentalized study of biochemical pathogens) Finley B et al. Organohalogen Compounds. 1998 38 325-29 (Does the available toxicological evidence warrant identification of HCB as a dioxin-like chemical?) Goerz G. IARC Sci Publ 1986 77 513-5 (Effects of chloroquine and HCB on the HCB-induced porphyria in rats) Goopalaswamy UV et al. ibid. 1986 77 267-76 (Biotransformation and toxicity of lindane and its metabolite HCB in mammals) Greeb JA et al Biochem Soc Trans 1989 17 1016-7 (Sexual dimorphism of cytochrome P-450 induction by HCB in rats) Grimalt JO et al. Int J Cancer.1994 56 200-3 Risk excess of soft-tissue sarcoma and thyroid cancer in a community exposed to airborne organochlorinated compound mixtures with a high HCB content) Gustapin DL et al. Toxicol Sci.2000 53 245-52 (Comparative hepatocarcinogenicity of HCB, pentachlorobenzene, 1,2,4,5 tetrachlorobenzene and 1,4 dichlorobenzene application of a medium-term liver faces bioassay and molecule and cellular indicator) Hahn ME et al. Biochem J. 1988 254 245-52 (The role of the Ah locus in HCB-induced porphyria) Arch Biochem Biophys 1989 270 344-55 (Interaction of HCB with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo) Hansteing WG et al. Biochim Biophys Acta 1981 678 293-99 (Effects of HCB and iron loading on rat liver mitochondria) Ingebrigtsen K. IARC Sci Publ. 1986 77 277085 (Comparative studies on the distribution of 14C-HCB by whole-body autroradiography) Ivanov E et al. ibid .1986 77 611-8 (Changes in some intestinal enzyme activities in experimental HCB-induced porphyria and modifying effects of diet) Jannsson B et al Chemosphere 1978 7 (3) 257-68 (Sulfur-containing derivatives of HCBN metabolites in the rat) Jarrell J et al Pure Appl Chem 2000 72 (6) 1015-21 (A review of human and sub-human primate toxicity of HCB Kenmnedy SW et al Biochem Pharmacol 1990 40 1381-8 (Dose-respnse relationships in HCB-induced porphyria)

Khanna RN et al IARC Sci Publ 1986 77 319-21 (Distribution, excretion and in-vivo metabolism of 14C-HCB and the influence of iron overload in C57BL/10 mice) Kim JJ et al. Arch Dermatol 1999 135 459060 (HCB and porphyria cutanea tarda) Kleiman de Pisareo DL et al Biochem Pharmacol 1990 39 817-25 (Thyroid function and thyroxine metabolism in HCB-induced porphyria) cf J Endocrinol Invest 12 767-72; ibid., 18 271-6 Kodiba RT . IARC Sci Publ 1986 77 371-8 (Evalutaion of global literature for difinition of dose-response relationship for HCB toxicity Kon G et al ibid 1986 77 261-6 (Excretion of metabolites of HCB in the rat and in man) Krishnan K et al Toxical Lett 1992 61 167-74 (Modulation of HCB-induced hepatic porphria by methyl isobutyl ketone in the rat) Kurper-Goodman T et al IARC Sci Publ 1986 77 343-6 (Subchronic toxicity of HCB in the rat, clinical biochemical, morphological and morphometric findings) Lackmann GM et al. Pediatr Res 2000 47 (5) 598-601 (Parental smoking and neonatal serum levels of polychlorinated biphenyls and HCB) Lambrecht RW et al. (Effects of etheylenediaminetetraacetic acid on HCB-induced changes in rats) IARC Sci publ 1986 77 505 7 Lane DA et al Environ Sci Technol 1992 26 (1) 126-33 (Gas- and particle-phase concentration of alpha-hexachlorocyclohexane, gammahexachlorocyclohexane, and HCB in Ontario air) Lecavalier PR et al. J Environ Sci Health 1994 B29 (5) 951-61 (Combined effects of mercury and HCB in the rat) Li Sin et al., Toxicol Environ Chem 1989 22 215-227 (Hexachlorobenzene biochemical effects and synergistic toxic interactions with 2,3,7,8 tetrachlorodibenzo-p-dioxin) Lilienthal H et al Arch Toxicol 1996 70 174-181 (Impairment of schedule-controlled behavior by pre and postnatal exposure to HCB in rats) Linko P et al J Biochem Toxicol 1986 1 95-107 (Induction of cytochrome P450 isozymes by HCB in rats and aromatic hydrocarbon (Ah) responsive mice) Loaiza-Perez AI et al Endocrinol 1994 140 (9) 4142-51 (HCB, a dioxin-type compound increases malic enzyme gene transcription through a mechanism involving the thyroid hormone response element) Loose LD et al J Reticuloendothelial Sci 1977 22 (3) 213-267 (Polychlorinated biophenyls and HCB induced humoral immuno-suppression) Luo K et al Biomed Chromatogr 1995 9 (3) 113-22 (Isolation and characterization of new porphyrin metabolites in human prophyuria cutanea tarda and in rats treated with HCB by HPTLC, HPLC and liquid secondary ion mass spectrometry) Mansour M et al IARC Sci Publ 1986 77 53-9 (Assessment of the persistence of HCB in the ecosphere) Mansim A et al Biochem Biophys Res Commun 1988 151 320 326 (The effect of iron overload on the mitochondrial porphyrin level in the HCB induced experimental porphyrias) Mathews DR et al World J Microbiol Biotech 2000 16 415-21 (Biodegradation of HCB by basidiomycetes in soil contaminated with industrial residues)

Mathews HB IARC Sci Pub 1986 77 253-60 (Factors determining HCB distribution and persistence in higher animals) Mendoza CE et al Toxical Appl Pharmacol 1976 35 447-53 (Effects of HCB on malathion LD50 and on cholinesterease and carboxylesterase activities in organs of the sucking albino rat) [HCB increased by a factor of 2+ the LD50 value for malathion in 17-18 day old pups; it also increased the content of liver esterases catalyzing the hydrolysis of indoylphenyl acetate without any change in the liver cholinesterase activity] Michielsen CPPC et al (HCB induces immunomodulation and skin and lung lesions. A compariosn between Brown Norway, Lewis and Wistar rats) Toxicol Appl Pharmacol 1997 144 12-26 Cf (HCB-induced esoinophilic and granulomatous lung inflammation is associated with in vivo airways hyperrespnsibeness in the brown Norway rat Toxicol Appl Pharmacol 2001 172 11-20 Mill T et al (The environmental fate of HCB) IARC Sci Publ 1986 77 61-6 Miskiewisz AG et al (Organochlorine pesticides and HCB in tissues of fish and invertebrates caught near a sewage outflow) Environ Polut 1994 84 269-77 Mollenhauer HH et al (Ultrastructural changes in liver of the rat fed HCB) Am J Vet Res 1975 36 1777-81 Morley A et al (HCB pesticides and porphyria) Med J Aust 1973 1 565 Mybchrest E et al (Studies on the mechanism of uroporphyrinogen decarboxylase inhibition in HCB induced porphyria in the female rat) Toxicl Appl Pharmacol 1997 145 23-33 Nakashima Y et al (Masking of guar gum-induced acceleration of HCB excretion by its rapid excretion through lactation in adult female rats) J Agric Food Chem 1998 46 (6) 2241-7 Nikolaeva et al (Rat liver plasma membrane damage in HCB intoxication and its potentiation by ethanol) Toxicol Lett 1986 32 269-73 Noren K et al (Distribution of PCB congeners, DDE, HCB and methylsulfonyl metabolites of PCB and DDE among various fractions of human blood plasma) Arch Environ Contam Toxicoll 1999 37 408-414 Oberg T et al (HCB as an indicator of dioxin production from combustion) Chemosphere 1985 14 1081-6 Ockner RK et al (Acquired porphyria in man and rat due to HCB intoxication) Nature 1061 Feb. 11 499

Peters HA et al (Neurotoxicity of HCB-induced porphyria turcica) IARC Sci Publ 1986 77 575-9; (Pophyria turcica. HCB-induced porphyria. Neurological manifestations and therapeutic trials of ethylenediaminetetraaceticacid in the acute syndrome ibid., 1986 77 581-3; cf., (HCB induced pophyria. Effect of chelation on the disease, porphyrin and metal metabolism) Am J Med Sci 1966 251 314-22 (Epidemiology of HCB-induced porphyria I Turkish clinical and laboratory follow-up after 25 years) Arch Neurol 1982 39 744-9 Pohl HR et al (Public health assessment of HCB) Chemosphere 2001 43 903-908 U.S. Department of Health &Human Services http://www/atsdr.cdc.gov/toxrpofiles/tp90.pdf (HEXACHLOROBENZENE TOXICOLOGICAL PROFILE)

Footnotes
Footnote a
GENETICS CHROMOSOME AND TELOMERE DAMAGE vs. ENVIRONMENTAL INSULT Although the complete knowledge of the etiologies of autism and related diseases have not yet been achieved, autism seems to be associated with a high level of chromosome and telomere damage a-1-1 a phenomenon which it also shares with cancer [a circumstance which prompted a database search (Kao) which established that a direct significant statistical correlation exists between autism and female breast (but not other kinds of) cancer which is known also to associated with the environmental presence of chlorinated aromatic POPs including polychlorinated biphenyls (PCBs) dioxins and HCB]. The mixture indicated in the preceding paragraph is a useful starting point for seeking the origin of the findings that genetic factors are not of dominant importance to the etiology of autistic spectrum disorders (indicated on the basis of very large population studies, which have now overturned (Hallmayer) former belief s(on the basis of small twins studies) that genetic factors were the dominant causal influences in autism. Autism is now apparently attributed by mainstream medical experts to intoxication of the general human population by the environmental presence of POPs, but the precise composition of the inducing POP mixture is currently commonly stated to be unknown. Since autism is a wide spectrum of related diseases and not a single category of disorders it is likely that different mixtures of toxins including halogenated substances may give rise to a range of different overlapping categories diseases which arise from a variety of specific in utero environments which produce (genetically variable) subtle developmental alterations in the assembly of the nervous system consequent on endocrine disruption by the presence of halogenated substances (including HCB, its mimetics and metabolites such as pentachorophenol) acerbated by traces of Sb, Pb Cd, Sn, Al, Be and Hg compounds present in the maternal blood, enhanced further by the effects of dietary inadequacy of major non-toxic elements sulfur as sulfate (n.b this is

needed via phosphoadenosylphosphosulfate [PAPS] to sulfation of heparan sulfate the key glycosaminoglycan modulator of developmental biochemistry including that of the brain), inorganic silicic acid, Na, K, Mg, Mn, Mo, etc. (and perhaps also some of the rare earths) as well as the small amounts of lithium salts and, in addition possible, major effects of copper and iron overload induced oxidative and nitrosative stress (especially that which leads to the nitration of tyrosine). A similar scenario is evidently responsible for unexplained obesity which like autism is now a global epidemic. Of the persistent organic pollutants (POPs) which have been indicated to induce obesity the most outstanding (cf. Bailley-Hamilton) evidence centered on HCB intoxication. The degree of obesity in the child at age 6 could be directly correlated (Smink) with the level of maternal intoxication by HCB. Intoxication by HCB and DDE (a commonly found decopostion product of DDT) have also been lineraly correlated with adult obesity.

Footnote b By asking the question which highly toxic environmental input over the last thirty years might most closely match the dramatic increase in autism? suggests that (1) Mercury (Hg) especially methyl-Hg in fish; perhaps autism and other human health problems have arisen as a unpredicted side effect of the clean air legislation which has conter-intuitively led to an augmentiaon of methyl-Hg in fish. Buring of fossil fuel allows sulfur to become avialable (so as to lock up Hg(II)) in natural waters. The binding of sulfide and polysulfide to Hg(II) is believed to lead to aggregation into particles with poor bioavailaiblity. But this process can be prevented and Hg uptake boosted by the presence of sufficient dissolved organic matter of the kind which has been increased over recent decades owing to greater human food production and soil degradation. Then, the dual effect of increased (fulvate) water organic matter and the reduction of (sulfate->) sulfide concenention has caused athe bioavialitiblty of Hg to greatly increase. Cf. Graham et al and Hongve et al., loc. cit. The increase in natural water fulvic (dissolved organaic matter) also has putatively increased the degree of supersaturation of CaCO3 in natural waters incuding the ocean and thereby has (putatively) affected the amount of CO2 in the atmosphere (This CO2 level is known to accurately have correlated with the global human population over the last several decasdes and putatively since the start ofhuman agriculture). The mechanism of increased autism may have in part been via anthropogenic Hg augmentation via a global change in agricurlture and (perhpas more locally) those industrial atmospheric emissions which are benifiical as regards anti-Hgintoxiication sulfur provision). (2) Antimony (Sb), its compounds, alloys and mixtures might also have an especial relevance to autism.

Of possible especail relevance is the leaching of Sb (into water) from polyethylene terephthalte (PET) bottles (cf. Sanderson). Perhaps especially the relatively new uses of Sb*b in brake pads and as plastic (toxic phenol releasing) food container polymerization catalysts and the use of Sb (together with polybrominated aromatic substances) as a fire retardants. The additional presence of Sb from these newer uses will augment the toxic burden of Hg and Pb (the traditional coal derived toxic metals) which also include Al, As, Ba, Be, Bi and Cd which have been indicated to occur in elevanted amounts in the hair of autistic spectrum disorder subjects. That Sb intoxication (arising now most often from road traffic) might have an especial relevance for a fuller understanding of the etiology of the current epidemic of autism was suggested by epidemiological findings (Volk) that maternal residences adjacent to high volume freeways are associated with an increased prevalence of autism. An earlier academic study (Bell cf. Hall etc.) of 24 Scottish autistic, 5 Aspergers and 8 control Scottish had suggested that autistic children (but not Aspergers children) have unusually elevated levels of Sb in their hair (averaging five times the assumed 0.03ppm normal hair Sb content baseline) whereas only half of the the control children showed (a smaller) increase in their hair Sb. (This type of study, which had suggested that while autistic children also showed increased Pb (which was more statistically significant than the results for Sb) as well as elevated Al, Sn, Na and K (but decreased Ca) levels, can now be reassesed in the light of the more recent findings that increased road traffic density increases the prevalance of autism, to reinforce the possibility that Sb intoxication might have an especial relevance for the etiology of autism. This clearly warrants larger follow-up studies.

Footnote a-1-1
Footnote a-1-1 Damage to DNA promoted by HCB and related substances

Oxidative damage to DNA is promoted by HCB and related substances

Combustion and incineration produces HCB plus dioxins (cf. e.g. the presence of small amounts of a range of environmentally stable chlorinated organic substances in the air intake of internal combustion vehicles will allow the thermal rearrangement in the combustion chamber and exhaust of chlorinated aliphatic molecules (e.g. those used as solvents) converting them to perhalogenated aromatic hexachlorobenzene HCB (plus a range of dioxins and PCBs). [A similar process is likely to apply to flulorinated and brominated substances]. which become adsorbed onto small carbon particles (e.g. those especially produced by diesel engines) formed by incomplete combustion which occur in urban pollution particulates. This may be why autism is associated with telomere defect effects Sentikumar PK et al. Toxical Lett. 2011 204 (1) 64-76 (Airborne PCBs reduce telomerase and shorten telomere length)

[The authors studied Chicago Airborne Mixture containing PCB 28 and 52 which cuased telemoere dmage so as to tend to shorten life and promote cancer Cf Williams JE Telomeres the Secret of Life; a Google search of telomeres HCB indicated that HCB is one of amny compounds which can accelerate the shortening of telomeres a Google search of teleomeres autism indicated a putative linkage between telomere damage and autism (e.g. the work of Miranda KK Nair).

Footnote a-1
HCB HCB
The endocrine perturbing agent 1 a dioxin-like substance which was formerly used as a pesticide but now mainly injected into the environment from the incineration of polyvinyl chlorine perhaps mainly in Asia and Africa but eventually transported in the atmosphere thousands of miles to become concentrated up the food chain.. HCB is known to have become widely distributed in the global environment and apparently continues to increase at least in some locations. HCB now to occurs in all humans. The known toxicology of this substance mainly gleaned form animal experimentation suggest that HCB (or the structurally related chlorinated aromatic moiety containing molecules) are the most likely prime developmental biology disrupting substances which might trigger autism especially by acting synergistically with other toxins (such as heavy metals). This hypothesis suggests that an ultra-trace-ultra-toxic cocktail which differs somewhat between locations and has been responsible for the historical prevalence and etiology of the autistic spectrum and other conditions which have been characterized by an anthropogenically altered developmental biology. A further extension of this hypothesis suggests that a key system which becomes perturbed is the heparan sulfate system which is indicated to be sensitive to environmental conditions (e.g. the ability of redox metals and ascorbate to orchestrate the deaminative cleavage process leading to signaling oligosaccharides). The status of HCB as a possible principal player in some common physiological prooxidant scenario is reinforced by the circumstance that HCB is an unavoidable byproduct of the industrial scale manufacture and disposal of all organo-chlorine containing organic matter. [cf the Van Wazer mathematical model theory underlying the descriptive chemistry of the rearrangement processes which occur during the decomposition of families of chemical substances during incineration and related chemical processes]. HCB is e.g. formed during the incineration of all chlorinated organic substances as well as non-chlorinated organic compounds when admixed with inorganic chloride. HCB is believed to self assembly due to the existence fundamental (quasi-thermodynamic) driving forces which dictates the outcome of thermal rearrangements (cf. Aubrey). An automatic self-assembly process dictates that HCB will always be formed during incineration of common domestic waste (which also gives rise to smaller amounts of, chlorinated biphenyls dioxins and furans). The formation of HCB is to a major extent, ultimately, however, the eventual consequence of the manufacture use and incineration

of polyvinylchloride (PVC). It has also been indicated (cf. Greenpeace internet file) that small amounts of dioxins can also be released from manufactured products containing polyvinylchloride parts without the need for incineration. The low-level intoxication of the human population during the use and disposal of commonly used industrial products which are commonly assumed per se to be of low toxicity but as a consequence of degradation during hydrolysis, aging, accidental overheating or incineration of their chemical components can also emit toxic trace substances (e.g. Sb which is also contained in urban dust from vehicle brake friction surfaces or catalyst residues leached from polyethyleneterepthalate (PET) food and drink containers and Ti, Cr and V from polyethylene and polypropylene as well as Al from urban dust and vaccine adjuvant use, organo-Hg anti-bacterial use and Cu from domestic water supplies ) may conceivably act syergistically with HCB and aromatic plasticizers (such as bisphenol-A) to induce the kind of chronic oxidative stress which is putatively a characteristic feature of autism, obesity and various other mystery illnesses. It should be noted that a low degree of exposure of HCB, at a level which had been previously thought to be without adverse effect on human health, has been reported to perturb thyroid function during pregnancy (Chevrier). By 1998, HCB had become the most abundant dioxin-like substances present in human milk (cf. e.g.. van Birgelen). The restriction of the deliberate manufacture and use of HCB following the Stockholm Convention (2004) should in principle have diminished the amount of HCB in milk but the inevitable continuation of input of HCB into the environment is likely to continue to allow HCB to contaminate milk. HCB, it should be noted, is a dioxin-like substance. A further aspect of HCB toxicology is that HCB seems to increase the toxicity of TCDD dioxin. While much less potent than true dioxins its mechanism of formation and the enhancement of its effectivness as a toxin by synergistic interactions with other toxins suggests that HCB is the principal inducer or at an important least part of the toxic cocktail which engenders autistic spectrum disorders.
Continbution of Aryl Hydrocarbon Receptor Induced Inflammation from Exposure to Dioxins

Cf web.biograph.be/concept/graph/C0019432/C0019134 Concept Interactions: genes expressed shown [ HEXACHLOROBENZENE ]

IFN gamma IL15 (Malignant neoplasm of breast) Polysacchairdes Behcet Sundrome Suramin Oligosaccharides (Liver Cirrhosis) Glycosaminoglycans Vascularitis FGF5 FGFR4 FGF10 FGFR2 FGFR1 [ HEPARIN ] ------------ ------------- --------------- -----------Cf. also web.biograph.be/concept/show/C0919455 BioGraph Relations between Autistic Disorder and Heparin

1L10

[Autistic Disorder]

5 methyl tetrahydrofolate Methionine Cysteine Vitamin B12 pioglitazone Galantamine Glutathione disufide Quinolinic acid POMC MTHFR Liver Cirrhosis Alzheimers disease FGF10 Sinus Thrombosis Intracranial ABCC1 FGF1 Heparin, low molecular weight

[Heparin]
--------------

------------

-------------

------------ ------------

Cf. web.biograph.be/concept/graph/C0004352/C0003380 [Autistic Disorder] (Haloperidol) (Mirtazapine) (Fluoxitine) (Sertraine) STATH IL4 PITX1 C4B ADM SCD LSS structural constituent of tooth enamel TUFT1 (Leishmaniasis)

[Antimony] ------------ ------------- ----------- ----------- ------------- ------------- --------

Footnote d
d

The oxidative damage outcome of HCB intoxication was first studies by Claude Rimington, a pioneer of the study of the biochemical mechanism of induction of porphyria. A personal communication form this author had indicated that he was also aware that chlorinated substances could also cause a major alteration of the mucopolysacharides in the liver. This could indicate that HCB and related substances at least in part cause autistic spectrum disorder by further causing a major biochemical disturbance of the /nitric oxide heparin/heparan sulfate system. This may be part of the reason why a cohort of ME/CFS patients from heparin therapy since this pharmaceutical agent is apparently able to up-regulate tissue protective heparan sulfate biosynthesis as apart of its repertoire of behavioral response to stress. Cf. HCB induced porphyria produces altered porphyrins (cf GH Elder Biochem J 1972 126 (4) 877-91)

Concluding Statement

The production of toxic molecule cocktails during incineration or pyrolysis of organic waste products arises from scrambling of starting substances of low toxicity in the presence of antimony (e.g. the antimony-containing brominated aromatic flame retardants) encourages multiple of backbone and side chain atom redistribution to greatly increase the toxic nature of the potential emissions relative to the relative benign nature of the starting feedstock. This is how HCB and other OCs enter the environment. The chronic low level intoxication of the human global population by HCB and related chlorinated aromatic xenobiotic POPs now means that the human population worldwide has become contaminated by a wide range of man-made chemicals (xenobiotics). Halogenated toxic substances which have been introduced by human actions into the environment can become augmented up the food chain in such a manner as to cause either obesity or autism or both in genetically susceptible individuals. The historical rate of increase in such pollution has mirrored the increase in autistic spectrum

disorders the prevalence of which have increased dramatically globally over the last thirty years. It seems self-evident that human and animal intoxication by POP substances (e.g. the endocrine disrupting pesticides which are known to damage the liver and other organs) and which have become globally distributed in the atmosphere and deposited in the soil in such a manner so as to facilitate their entry into the food chain, are likely be relevant to a fuller understanding of the etiologies of a range of poorly understood diseases. This suggested that autistic spectrum disorders are directly caused by intoxication by the environmental presence of such POPs. While HCB, together with eleven other substances are subject to prohibition from deliberate manufacture and use under the Stockholm Convention because of their established high toxicity to humans and animals, HCB in marked distinction from the other eleven banned substances, nevertheless continues to be injected into the environment because it is formed because of fundamental chemical principles which governs the ubiquitous formation of this substance(together with PCBs and dioxins and their related brominated and putatively also fluorinated halogenated aromatic molecules) as a consequence of the occurrence of the Aubrey Van Wazer equilibration (apparently thermodynamically determined process by which organic molecules aalso containing halogen atoms or even mixtures of non-halogenated molecules with halogen ions, rearrange under pyrolysis conditions [especailly in the presence of antimony catlaysts]). The extent of global industrialization (the ultimate source of HCB etc.) has increased dramatically over the last thirty and the resultant increasing extent of environmental pollution by xenobiotics can therefore putatively provide a rationale for the in tandem induction of obesity, autism and other autistic spectrum disorders. The sudden onset of autism (regressive autism) which conceivably could arise as a unforeseen consequence of medial intervention and may e.g. involve a sudden influx into the organism of toxic substances might also be in part caused by the pre-existence of other toxic substances in the affected subjects especially as this affects mast cell activation. The toxicogenomics of a range of toxic elements and xenobiotics is therefore most likely relevant to a fuller understanding of the induction of a wide range of range of diseases which include unexplained obesity (cf. Bailley-Hamilton) and autism (cf. Bernhoft). Similar toxic cocktails are putatively also involved in the etiologies of the unexplained epidemics of type 2 diabetes, obesity and breast cancer in women). HCB is likely to be a critical component of the POP mixture which is responsible for autism since this commonly encountered xenobiotic is now so widely distributed to be apparently present in virtually all humans worldwide. This is because HCB is an end product in the scrambling system of carbon and chlorine atoms. HCB ubiquitously will be formed by the structural reorganization of all chlorinated organic molecule precursors. Copper and iron additionally catalyze this process. To summarize and extend the above argument: the tendency for the now ubiquitous intoxication of all humans by ultratrace amounts of chlorinated organic substance cocktails produced by industrial process to promote an increased human body mass index (BMI) also appears to be related to an increase the risk of developmental disruption which gives rise to birth defects including autism in the offspring of obese mothers. It should be noted that the body burden of environmentally present (albeit other) chlorinated organic substances (chlorinated insecticides including hexachlorobenzene

[HCB] and polychlorinated biphenyls) have also been directly associated with obesity both in adults and in offspring. Organohalogen intoxication by (e.g., chloroform, bromoform, tetrachloroethylene,
hexachlorbenzene, polychlorinated biphenyls hexachlorobenzene DDT and DDE and dioxins) may exacerbate aluminum and other toxic metal ion dysregulation of HSPG function so as to cause autistic spectrum disorders in susceptible individuals.

Such low level multi-substance intoxication is likely to alter HS signaling in the fetus so as to perturb development of nervous tissue. The same synergistic interactions between the three chlorination products which seem to induce autism-like conditions in experimental animals (which have been detected in very small amounts in domestic water supplies in areas where autism rates seem to be high than average) may be part of the spectrum of xenobiotic insult which is the ultimate origin of human autism. The possible additional role of toxic metals expecially in older un-modernized houses, lead pipes some of these will aslo contain the metalloid antimony which is now also being introduced into the environment from the increasing use of antimony as compounds as fire retardants, components of brake pads and as catalysts for the preparation of PET food containers. A toxic metal risk arises from excessive copper dissolution into some kinds of municpal water supplies (especially near stray electrical currents which cause pinhole formation in water pipes as they pass over electrical cables)..

A literature search made in the context of possible roles of HS biochemistry (which is believed to be involved inter alia hemostasis, lipid metabolism, anti-pathogen protection, wound healing and a related growth factor regulation process in fetal nervous system development) revealed that perturbation of this signalling system might be of prime relevance to the fuller understanding of how autism and related illnesses could arise following intoxication by endocrine system damaging environmental pollutants. This review especially suggested that pro-autism intoxication process might critically involve synergistic heavy metal organohalogen interactions which disturb the metal ion (and putatively) nitric oxide dependent heparan sulfate (HS) foetal and continued postnatal nervous system development procesess (and also are involved in wound healing and anti-pathogen protection). Does EDTA act like heparan sulfate as a heavy metal remover? A commonly used alternative medicine therapeutic treatment for autistic spectrum disorders is to remove heavy metal toxins by intravenous administration of EDTA (a man-made very high

affinity multi-metal ion affinity chelator). While this procedure been indicated to have had some success it may also have undesirable side effects relating to depletion of non-toxic, essential metal ions. It is now pointed out that EDTA may serve as a substitute for the apparent glycome-dependent including the anionic polysaccharide-based metal ion multi-functional tissue regulator. Heparan sulfate [HS] is a low-medium affinity inorganic ion chelating system which is now believed to be the master fuzzy information-encoded manager of animal physiology and as such defects in HS can be predicted impinge on essentially all pathologies. HS putatively first of all provides for an apparent primitive system of electrolyte balance (which is retained in humans and other highly evovled species together with more evolved systems dependent on e.g. kidney function). HS ligands occur ubiquitously in the extracellular matrices and at adherent cell membranes of all animals. The HS ligand system putatively contributes to multi-inorganic ion homeostasis (including a provision for removing toxic metal ions) but apparently includes a facility for provision for the simultaneous multi-inorganic element ions binding to the surfaces of all glycosaminoglcyan (including HS-based) proteoglycans. In this often high affinity binding process which to target the non-physiological ions and colloidal particles which occur widely but in ultratrace amounts in natural aqueous environments (e.g. the sea). A related function is the control of calcification (e.g. in bone formation) and for the prevention (by acting as a seed particle inhibitor) of unwanted crystal and fibril deposition (plaque formation) in both the blood and urinary systems. HS also is well known to control hemostasis, lipid metabolism and pathogen infection and its key roles in developmental biology (by allowing for the correct orchestration of growth factor and growth factor receptor provision) has also been subjected to major research efforts. (These show e.g. that HS directs the activity of numerous protein activities e.g. during fetal development where HS actions are known to affect the development of the nervous system).

The status of correct microstructured HS at blood vessel walls is essentially required for correct tissue protection functioning; an efficient functioning of the liver and of the kidney are also dependent of HS actions (e.g., HS assists in glomerular filtration), The HS-dependent lipoprotein lipase activities (including in the liver) of HS are also critical for energy provision. While some of the normal functions of HS have been found to essentially require metal ion cofactors (Ca2+, Mg2+, Mn2+,Cu2+and Zn2+) the presence of the toxic metal ions (Cd2+ , Pb2+ and Hg2+ and excess F-) have been found to negatively perturb HS microstructure formed during primary biosynthesis e.g. Cd2+ disrupts HS sulfation while Pb2+ disrupts core protein production). A system of HS nitrosative scission (dependent on Cu, Zn and ascorbate) is also believed to provide for a inter and intracellular signalling which is sensitive to redox status. The ability of dioxin and dioxin-like toxins to alter systemic redox status is likely to perturb this nitric oxide determined HS recycling system and so affect the ability of HS to provide tissue protection and wound healing related activities. HSPG perhaps because it can create phase change engulfment (a phenomenon which may also be related to poorly researched critical for animal nutrition presence of inorganic silicate which always co-occurs with HS and related polysaccharides) be normally constituted to provide a polysaccharide-based (backup?) toxic metal purification system. This complex microstructure system seems to vary systematically as a function of age perhaps causing an effective programmed diminution of the ability of the organism to protect the cell surface environment; indeed the HS system is the only biomolecule which is known to behave in this way (e.g. as reported by Feyzi et al.). Understanding HS is therefore thought to be a key scientific endeavour in the quest to more fully understand the ageing process. HS in old age is also known to become in essence more like old HS under certain pathological systems. These can be thought of as accelerating ageing. An example is in

diabetes under which conditions vascular HS also is indicated to become structurally altered. From the above argument it seems likely that the patholgy autism is likely to involve changes produced in HS microstructures as a result of toxic substance ingestion coupled with dietary deficiencies of essential inorganic elements, vitamins and lipids. Since administration of pharmaceutical agents which mimic HS seems to be able to re-boot some of the HS protective systems a possible future therapeutic use of HS-mimetic-related therapy to illnesses additional to those involving blood hemostasis and urinary system dysfunctions is anticipated. The organohalogen heavy metal containing toxic dust induced illnesses caused by the 9/11 terrorist attack may also be further examples the seem kind of intoxication induced alteration in HS. A survey of the literature shows that heavy metal environmental pollutants can diminish membrane and extracellular matrix metallome-heparanome dependent tissue functions (e.g. growth factor and cytokine control mechanisms and tissue protection which including the ability of HS to protectively sequester toxic metals) may be defective in autistic subjects. I.e. autism is an HS defect driven disease.
This activity of HS may also be dependent on adequate dietary provision of magnesium and sulfate which promotes HS biosynthesis as well as inorganic silicon (a poorly understood essential dietary requirement) to allow for sufficient inorganic silicate attachment to HS (which is putatively involved in promoting a phase change engulfment high-affinity metal ion binding process which is distinct from the type of electrostatic binding usually assumed to occur between metal ions and HS).
A relevant task for estblisheing how autism spectrum disorders may arise seems to be to question how HS might be invovled in the etioligoy of these diseases. It should be noted that resembles DNA in the circumstance that both of these biopolymers (which are linear information endcoding systems) might how, their structures might become pathologically altered (e.g. by the effect of intoxication of humans by the persistent environmental pollutants) and how this alteration might produce the multiple follow-on biological consequences which are a charactersitic feature of autistc spectrum disorders.

While DNA is correctly regarded as the king of bio-molecules, the more complex but less precise HS information processing (which is asembled using DNA encocoded enaymes but also subject to post-synthetic structure alteration by non-enzymic directed mechanisms ) is a useful focus system (of especial relevance for the post-human gneome project downgrading of the centrality of DNA to pathology) is still a relatively unknown system by comparison, but it is increasingly becoming evident that this information holding and processing system is of prime interest to unraveling the etiologies of mystery illnesses since the loss of the correct fuzzy logic HS microstructures can potentially cause multiple downstream biological consequences.

The outcome of the (e.g. synergistic multi-ion heavy metal intoxication and /or chronic low level endocrine disrupting chemical) intoxication) of the HS-mediated animal fetal development process and postnatal wound healing is now proposed to re-define the (previously identified as polygenic) predispositions of autism a , autistic spectrum disorders (viz. chemical substance sensitivity, attention deficit disorder, hyperactivity; dyslexia) and the putatively circumstantially linked diseases: childhood asthma, rhinitis, eczema, and in later life arthritis, infertility, chronic fatigue syndrome, fibromyalgia, type 2 diabetes as having some etiolgical tie-up with atherosclerosis, Alzheimers disease, Parkinsons disease, multiple sclerosis, some forms of cancer [especially e.g. breast cancer in women] and also the current global epidemic of unexplained obesity.. While DNA mutations seem to explain only ca. 10-20% cases of autism spectrum diseases the remainder as suggested by literature surrvey suggests that HS sequence alteration and consequent distruption of HS signaling causes the majority of cases of autism. (this is the ususal and not the DNA genetic defect in enzymes which are requried for HS catabolism which is reponsible for the range of diseases labelled as mucopolysahharidoses). mercury other toxic metal and metalloid toxins This is a system of anionic patterns sulfated alternate copolymer glucosamine-uronic acid anionic polysaccharides (heparan sulfate]) seems likely to suffer damage from which can act synergistically with the genetic DNAas well as analogous defects . Autism May Arise From A SynergisticHeavy Metal/Organohalogen And
Nitric Oxide Metabolite Disruption of Heparan Sulfate Signaling

The above scenario could also be a key event in non-genetic autism spectrum disease etiology. The key autism-causing intoxication(s) is however also suggested to require a synergistic augmentaion of the HEAVY METAL intoxication effect by the presence of HALOGENATED ORGANIC MOLECULES which by synergistic interaction can disurb the HS system of tissue protection provides anti-pathogen, anti-free radical and CRITICAL anti-crystal insult protection. This possibility could explain why a range of different toxic metal ions (including mercury, lead, aluminium, arsenic, and barium as well as inappropriate homeostasis of copper and zinc) seems to have been implicated in autism (e.g. as indicated be a review of reported autistic subject hair element studies). However

The idea is further supported by the preliminary indication that antimony may become uniquely strongly bound to HS. Further research is warranted to probe this. [What about studies of genetically susceptible subjects endocrine systems, pre-and post-natal growth factor and cytokine regulation by heparan sulfate]. It should be noted that while the ubiquitous presence in the environment of anthropogenically-introduced toxic substances, especially the endocrine system disrupting organochlorine [OC] molecules and heavy metals has long been thought to be a possible primary cause of a range of pathological conditions, the primary mechanisms involved in these aetiologies have, however, remained obscure. This intoxication is now suggested to induce defects in HS signaling.
The heavy metal intoxications which have been (mostly anecdotally) associated with autism (viz. lead, mercury, cadmium etc.) are also those (as indicated from cell culture studies) which cause major diminutions/perturbations of HS biosynthesis.

The various dietary factors (including w-3 fatty acids etc.) and ascorbate which have been found to beneift autistic subjects are also those which have been indicated to increase the efficincy of HS biosyntheiss (cf. Grant).

A major difference between the DNA and HS information encoded systems is that DNA is rigorously protected against structural alteration while HS (which is present as both an extracellular and intracellular system) is intrinsically much less well protected and may indeed be required to become systematicaly structurally altered in response natural to various natural alterations in environmental conditions. There could be a mechanism whereby HS becomes altered by chronic environmental changes which require follow on DNA alteration (i.e. the putative invovlement of HS in the mechanism of animal species). This environmental response might however be incapable of appropriately interacting with the numerous (many thousands) of new challenges imposed very rapidly in the geological timescale upon all biota (from the presence of the kinds of persitent organic polluatants poorly bio-degradable herbicides, laundry detergents and residues from steroid hormones and antibiotics and levels of heavy metals which currently contaminate the environment as a result of recent human industrial activities). Since these chemicals were either completely abesent or present at much less than their present day amounts at the time of the evolution of HS (concident with the first evolution of multi-celluar animal organisms) the system by which HS has been carfully conserved throughout animal evolution has now become partially overwhelmed. Animal biochemistry has not been given sufficient time to evolve the appropriate response to the current evironmental pollution cocktails in order to safeguard the coninuation of the previous complexity of biological species.
While genetic defects in DNA sequences might explain ca. 10-20% of cases of autism (1b), by far the majority of autistic spectrum disorders, it seems, must arise as a consequence of some (environmental) toxin(s) of unknown nature(s).

including hexachlorobenzene).

synergistic interaction between organic and inorganic pollutants which perturbation of

It might be anticipated that the most pertinent of the putatively autism-inducing toxic inorganic elements is

(e.g., lead, aluminium, antimony, mercury and arsenic)

and numerous kinds of toxic inorganic elements (including lead aluminium arsenic and mercury) together with those agrochemicals which resist biodegradation (e.g. phosphonates). This scenario serves to create a multiplechemical substance cocktail.

(e.g. heavy metals and organocarbons).

unexplained dependent on in which are potentially subject to seem most likely to be (partly but largely) distrubance by xenobitoic intoxication but which (controversially) only become evident only following a later immune system overload
identify

the ultimate origin of autism and related disorders might produced

these developmental defects

Reports of researches on autistic spectrum disorders (ASD of researches on autistic spectrum disorders (ASD) The

Acting together with redox elements as disturbers of heparan sulfate signaling these chemical toxins may be the ultimate cause of autism. That lead (Pb) (Sb), tin Sn and perhaps aluminum (Al) might have a key role in the etiology of autism (but not in Aspergers syndrome)

They can be formed during incineration of e.g. domestic trash from non-toxic precursors by the occurrence of atomic scrambling processes first identified some fifty years ago. and organohalogen intoxication. Polybrominated and polychlorinated substances which resist biodegradation in the enviornment and are known to be highly toxic and may cause atuism via an additional synergstic interaction with antimony. (perhaps this includes the inorganic and organic flame retardant compositions) a synergistic interactions between the toxic inorganic elements and

stimulated nitsoative stress which disturbs

heavy metal chelation and normal metal cofactor dependent signaling (e.g. that dependent on Cu and Zn (cf Mani et al.).
or by vaccination overload and an asociated system. which has already become compromised by the effects of th
It seems that the proper discussion of the possible role of such intoxication in the etiology of autism has been greatly hindered by the relegation of this controversial subject to the realm of grey scientific publication.

dependent functions by toxic heavy metal plus halogenated aromatic environmental pollutant synergytic intoxication iby fire retadrants
by Prof. J. Bell which leads to an inappropriate systemic in vivo formation of large amounts those nitric oxide metabolites which regulate HSPG signaling. conducted in 2001 has only as yet in the grey literature by Scottish (which can occur both in vivo and ex vivo

perinatal brain development and broad spectrum protection afforded by by halogenated aromatic persistent organic pollutants and heavy metals.
(a nitric oxide-glucosamine-dependent information system invovled in the etioogy of neurological diseases (and that this process is aalso exacerbrated by infections). A possibly relevant distrubance of HSPG stucture is by inappropriate depolymerization which is a redox status dependent phenomenon and is thought to occur during chronic autoimmune disturbances (e.g.in the etiology of arthrtic diseases which have together with stroke, multiple sclerosis, Alzheimers disease, amyotrophic lateral sclerosis, Huntingtons and Parkinsons diseases been asociated with deleterious actions of nitric oxide metabolites (cf. Salvemini et al.). Both redox acitve heavy metal intoxication and the presence in tissues of halogenated aromatic organic substances in tissue are potential sources of redox status disturbance which are likely to cause increase the amounts of deleterious nitric oxide metabolites. This mechansm may be a key part of the process by which an alteration of development biochemistry can lead to obesity and/or to It has recently been suggested (Kilpinen)

The glycome is now thought in the post-human gneome era to be of major importance in how multiclelular animal organisms function. autism etiology in order to suggest possible novel therapies for this disease is impeded because of the lack of the same kind of in depth knowledge of olysacchairdes biochemistry compared with other major polymer systems which impact on health viz. proteins and DNA. The HSPG signaling system may be uniquely perturbable by anthropgenically intoduced bio-unfriendly environmental factors. Xenobiotic perturbation of HS is now suggested to induce ASD in geneticaly susceptible subjects. and and the circumstance that the major O-glycome system seems to be designed to closely interface the envioronment suggests that HS is the most relevant part of the Oglycation system to seek to related toASDs It is likely that the toxic metal cocktail which has been associated with infants with autism and putativley also the fetus will to induce subtle changes in HS microstructures so as to alter the brain development the toxic cocktain of toxic metals present in autistic subjects or their mothers is likely so as to promote autism. This idea is consistent with the circumstance that HSPG dependent signaling is believed to infludence. Autism and autistic spectrum disorders (ASDs) however might arise from defects in Oglycation, especilally the highly conserved systme of animal informational processing enabled by the heparnome which is mediated by HS proteoglycan [HSPG] O-glycation signaling.

by heparan sulfate functions

but historically the absence of template for the assembly of this complex managerial system has historically hindered the full understanding of its functions but it is now understood that functioning again this process is likely to be perturbed by the presence of ultratrace amount of the listed toxic elements in the tissues of ASD subjects

the perturbation of O-glycation Disorders May arise from

Are such factors also those which could (as reported in the peer-reviewed litereature) perturb HS signaling (and thereby perhaps also perturb the major essential inroganic elements)? This could to be the case.

including that which is dependent on nitric oxide.

Zinc (Zn) seems to be over-represented in hair.

in general it seems that studies of inorganic element in hair samples have often been regarded as suspect owing to the . chronic heavy metal cocktail which conceivably by acting together with other environmentally universally distributed xenbiotics such as organic pesticides and fire retardants can lgocially be proposed, a priori to be possible inducers of the The highly toxic element antimony (Sb) which has increasingly become intorduced into the environment and food chain over the same time period as the dramatic inrease in ASD could be at least partly responsible for ASD. This possibility arose following an extensive literature survey which New which cause ASDs in genertically susceptible subjects. but confirmed that ASDs are also highly dependent on some (unknown) environmental factor(s than had hitherto been thought likely. including of the use of to identify possible xenobiotic inorganic elements which might be part of the environmental cause of ASDs suggests that antimony (Sb) might be especailly relevant. It should be noted that Sb(III) has been reported to uniquely inhibit the formation of glutathione, a key antioxidant defense molecule and also to inhibit a rnage of other enzymic activities.

between antimony, heavy metals and halogenated organic persistent organic pollutants
Polychlorinated biphenyls (PCBs), octachlorostyrene (OCS), hesachlorobenzene (HCB Polychlorinated biphenyls (PCBs), octachlorostyrene (OCS), hexachlorobenzene(HCB) It is now thought to be useful to dicuss the possible xenobiotic chemical environmental stimulae which migh be associated with the etiology of autism. One or more of the anthopogenically introduced environmentally introduced toxic chemical substances which are now ubiquitously present in human tissues could be a major part of the environmental cause of autism. Other autism inducers which putativley enhance the toxic effect of HCB are dioxins and polychlorinated biphenyls (PCBs) as well as octachlorostyrene (OCS) and chlorinated solvents including carbon tetrachoride, trichloroethane and tetrachoroethylene and the metabolites of the chlorinated aromatics. of the contribution made by environmental factors to the etiology . Etiology of Autism & Other Neurological Diseases

also in a sub-class of autism subjects act to increase heavy metal and metalloid uptake and related intoxication.

Discarded Text Subject headings

Alteration of HS may explain autism, maternal hypertension, obesity and cardiovascular disease correlation [Mercury from amalgams and/or via crematoria via fish etc. Role of vaccination ? {Vaccination Hg Vaccinations (general)}

Role of dental amalgam Hg; Power plant coal derived (Hg + Sb etc .) ] Mercury from fish Epigenetics & autism (this conceptually also involves HS) Schizophrenia link Acquired gene defect link Reported Possible Anti-ASD Benefit Research Suggestions Relating to Possible Therapeutic Intervention in Autism Fish diet removal of HCB and other halogenated POPs Hyperbaric therapy blood oxygenation Drugs which putatively boost HS protection [Dark chocolate Diminishes the viscosity of blood could improve oxygen supply to the brain. Autoimmune Hypothesis of Autism Glycation deficit hypothesis of ADDH Myalgic encephalomyelitis/ Chronic fatigue syndrome (ME/CFS) link to autism Possible Synergistic Interactions of Xenobiotoics with the Immune System Role of aluminium plus toxic halogenated substances? Could Sb intoxication be synergistic with HCB intoxication? The metallome crosstalk with the heparanomeSome Role of new environmental inputs of mercury and (Cheng et al ascorbate nitric oxide and heparan sulfate scenario) Oxidized lipids, excess glucose, homocysteine, omega-3 faty acids, retinoic acid and ascorbate cause Alteration of Heparan Sulfate

Dietary deficiency of selenium and zinc Low levels of Hg, Sb and Cd diminish HS activity
Calcium Carbonate Crystallization Aluminium)

-----------------------------------------------------------------------------------------------------------------------------------------

The bio-avaialabiity of mercury may be altered by anthropogenic alteration of soil organic matter and its augmentation in natural waters. A similar mechanism has been suggested to apply to the alteration of the rate of precipitationof CaCO3 from the sea The ability of fulvate (dissolved organic matter from agricutural soil) to greatly diminish the rate of precipitation of CaCO3 from the sea may diminish the ability of the sea to act as a CO2 sink. This suggests a hypothesis of a major role of agricultural degradation of soil humus in the mechanism of CO2-led climate change Cf. the following documents

(cf. David Grant, Chemical World (Letters), June 2011 which was an edited version of the folowing documents))

Joseph Needham, CO2 and Global Warming Cf., Emissions from India & China since AD 2000 A short news article which appeared in Chemistry in Britain in 1995 (1) stated that Joseph Needham, the leading Western expert on the history of science and technology in China, who died recently, may have saved the world from global warming. Needham once told a colleague that Mao Tse-tung had asked his advice about whether he should allow the Chinese people to have cars or make them stay with bicycles. Needham was a Cambridge don, and cycled everywhere. He told Mao this. Fine said Mao then well stick with bicycles. Given the size of the Chinese population, this decision may have delayed global warming by decades. China has now become this major car ownership industrialized superpower but the kind of marked increase in atmospheric CO2 and associated global warming by to follow from the abandonment of the use of bicycles by China which had predicted in this 1995 article has not happened. The temporal increase of CO2 present in the atmosphere however shows a direct linear relationship to the change in the size of the human global population (i.e. it must be presumed to be anthropogenic). The same relationship, however seems both to described the pre and the post AD 2000 data. The amount of CO2 in the atmosphere does not appear from this method of comparison to have been directly affected by the large increase in the annual rate of fossil fuel usage by Indian and China. The same CO2 human population relationship which has apparently dictated the amount of anthropogenic CO2 present in the atmosphere from the start of human society continues to be valid. This is apparently quite independent of fossil fuel use, a circumstance which points to some other mechanism e.g., a food production agricultural runoff alteration of the ability of the sea to hold CO2 being those which are principally responsible for the present anthropogenic elevation of atmospheric CO2. (1) Anon, On your bike, Chem. Brit., 1995, 31 (6) 450 (2) suggested by the Mauna Loa measurements
Why has the atmospheric CO2 content apparently increased as a linear function of the global human population? Research Note by D. Grant, MRSC, New Deer, Aberdeenshire, AB53 6SX.

The amount of CO2 in the atmosphere has shown an approximately linear dependence on the size of the global human population (cf. Fig. 1). While this appears to be the most exact for the post-1959

data (1), (for which the R2 value for the least squares fit can be shown to be >0.99) a plot of the most recent data (similar to that given in ref. (1)) together with a representative amount of the earlier data (which has generally been much less accurately determined) produces a reasonably good fit to a single linear correlation curve (shown in Fig. 1 curve a) which putatively describes all of the sufficiently accurately determined atmospheric CO2 content data from over the last several millenia. It seems that an increasing dominance of the human species over the planetary ecosystem over this period of time could have allowed the size of the global human population to commensurately perturb a sufficiently large portion of the biosphere in an approximately linear human-populationdependent manner so as to translate into the observed linear dependence of the amount of CO2 in the atmosphere on the human population number. Several possible major sources of anthropogenic CO2 which could sum to the observed net linearly-transmitted human influence could be of interest. These are likely to include terrestrial and marine sources and sinks for CO2 as well as, conceivably, human inputs into cloud forming mechanisms.
These may include some processes which are very subtle.

Nevertheless the soil and the sea are perhaps the largest most obvious sources and sinks which should be considered in order to elucidate the nature of such anthropogenic perturbations. While the most well-known uniquely anthropogenic, potentially climate altering influence is that caused by the highly unnatural CO2 (8GtC/yr) (emitted together with particulates and toxic gases) from fossil fuel combustion, it is possible that the combined amount of the more natural anthropogenic emissions of CO2 from agriculture (including that emitted from agricultural soils (ca.13GtC/yr (2)) and those emitted from the respiration of agricultural animals (ca.3GtC/yr (1))) may be considerably larger than the amounts emitted from fossil fuel usage. This suggests that, at least prior to the industrial revolution, retention of anthropogenic CO2 in the atmosphere in amounts determined by

the human population size has been directly and/or indirectly associated with agricultural activities, and that this has caused, and may be continuing to cause, the amount of CO2 in the atmosphere to continue to depend on the size of the global human population. A possible, hitherto not widely discussed agricultural mechanism which might even have been, and perhaps remains, a major source of (natural mechanism input) anthropogenic CO2, is now tentatively suggested. While the human population increases which have required a more intensive food production could incidentally have caused a greater direct emission of CO2 from altered soils (2) (especially in recent decades as the effects of augmented atmospheric greenhouse gases have caused the earth to warmed up and increase the use made by soil dwelling bacteria of the long lived soil organic matter as a carbon source), a concomitant process which is believed to be associated with a gradual-over-centuries loss of humus from agricultural soils which can lead to an increased rate of water-soluble organic matter (fulvate) runoff from fields into the sea, could have stimulated the emission of additional CO2 into the atmosphere from the sea as a result of the incipient blockage of the inorganic cycle of the sea via an augmentation of the amount of humified organic matter present therein. {N.b., the presence of this kind of surface active (mineral surface binding) organic matter in the sea is known to abolish the lysocline (3) and also to increase the degree of supersaturation of CaCO3 phases (4) in surface waters}. This putatively has enabled the process of slow anthropogenic humus degradation to increase the amount of CO2 which is available in surface waters for exchange with the CO2 in the atmosphere (5). In confirmation of the credibility of this hypothesis, a standard fulvic acid obtained by the traditional humic matter fractionation procedure from a typical Scottish agricultural soil, was confirmed by studies conducted under in vitro conditions, to be uniquely effective as an inhibitor of CaCO3 precipitation (6). Such fulvic acid was found to be a very effective inhibitor of CaCO3 seed crystals upon which the surface adsorption of fulvic acid

evidently occurs with high efficiency. A Freundlich isotherm plot comparison of the rates of inhibited seeded crystallization suggested that such fulvic acid was about up to 2 orders of magnitude more efficient on a weight basis as a calcification inhibitor than e.g., ethane,1-hydroxy,1,1,bisphosphonate (one of the most successful commercial products used for this purpose e.g. in oil-well boreholes). This confirms the plausibility of the hypothesis that a fulvate runoff mechanism could be responsible for at least a part of the anthropogenic augmentation of atmospheric CO2 content. Further research into this possible mechanism of anthropogenic augmentation of atmospheric CO2 is now warranted.
References

K. Onozaki, Population is a Critical Factor for Global Carbon Dioxide Increase, J. Health Sci., 2009, 55, 125; cf. Y.T. Prairie and C.M. Duarte, Biogeosci. Discuss., 2006, 3, 1781. (2) B. Bond-Lamberty and A. Thomson, Nature, 2010, 464, 579; Biogeosci. Discuss., 2010, 7, 1321 (3) P.J. Wangersky, Limnol. Oceanogr., 1969, 14, 929 (4) P. Zuddas et al., Chem. Geol., 2003, 201, 91; A.R. Hoch et al., Geochim. Cosmochim. Acta, 2000, 64, 61; W.P. Inskeep and P.R. Bloom, Soil Sci. Soc. Amer. J. 1986, 50, 1167.
(1)
[That fulvate-like polyanions are perhaps the most potent natural substances known which can prevent CaCO3 precipitation, and could be key part of the putative global CO2 homeostasis mechanism provided by the sea, was also discussed by D. Grant in web.scribd.com/doc/37544394/Anthropogenic-Augmentation-of-Marine-Fulvate-ampClimate-Change; web/ scribd.com/doc/44604504/Climate-Change; web.scribd.com/doc/41898831/New-Thinkingon-Climate-Change-Abbrev.-III]

(5) Y. Kitano (Calcification and atmospheric CO2) in Biomineralzation and Biological Metal Accumulation, Ed. P. Westbroek and E.W. de Jong, D. Reidel Publishing, 1983, p.89. Cf., K.E Chave & E. Suess, Limnol. Oceanogr., 1970, 15, 633; Y. Kitano and D.W. Hood, Geochim. Cosmochim. Acta, 1965, 29, 29; J.R. Morse, Rev. Mineralogy, 1983, 11, Carbonates in Mineralogy and Chemistry, Ed. R.J. Reeder, Mineralogy Soc. Amer., p 227-284; A. Pentecost, Thalassas, 2004, 20, 45. (6) Studies conducted at Aberdeen University using procedures described in D. Grant, Biochem. J., 1989, 259, 41
Fig. 1 & Additional Notes
The amounts of CO2 expired from domestic animals can exceed the amounts of CO2 produced from fossil fuel combustion in the less developed countries(1). The surge in

fossil fuel usage in India and China has not greatly changed the population vs. CO2 curve shown in Fig. 1 which has tended to continue to follow the earlier linear dependence on human population. This, however, may be partly accounted for by a changeover to fossil fuel burning from wood burning (which, however could not to have been the cause of the early anthropogenic CO2 input which caused an apparent 20ppm augmentation of atmospheric CO2 cf. W.F. Ruddiman, Rev. Geophys., 2007, 45, RG 4001, which is believed to have arisen from an alteration of the sea by prehistoric agricultural human activity).

While curve b of Fig.1 shows an overall highly non-linear dependence on human population values of fossil fuel usage, the addition of a hypothetical amount of carbon from plant material fuels will tend to produce a more linear variation.
The amount of fossil fuel usage is unlikely, however, to be the principal contributor to curve a in Fig. 1 since this, currently at 8GtC/yr. is much less the amount of CO2 recycled annually in the carbon cycle which includes the of CO2 emitted from the soil which alone amounts to 98+12GtC/yr. (of which 13GtC/yr. is agricultural and therefore anthropogenic).

While R. Revelle and H.E. Suess, Tellus, 1957, 9,18, on the basis of a study of the stable isotopes of carbon, found that the residence time of CO2 in the atmosphere was ca.10 years, later studies (e.g. by S. Solomon et al., PNAS, USA, 2009, 106, 1704) suggested that the CO2 emitted from fossil fuel anthropogenic sources remains in the atmosphere for many hundreds if not for thousands of years. The amount of CO2 remaining in the atmosphere deduced from the latter hypothesis agrees with the
currently observed amount. I.e. a large proportion of the CO2 emitted from fossil fuel use after 1800AD seems to have remained in the atmosphere and this amount alone can entirely account for the observed historical data for increase in atmospheric CO2. Indeed a highly linear correlation curve can be demonstrated in support of this idea

(cf. G.W. Harding How Much of Atmospheric Carbon Dioxide is Anthropogenic web.strom.clemson.edu/becker/prtm320/commons/carbon3.html.)
However, for the discussion of the ultimate origin of anthropogenic climate change, perhaps the most fundamentally relevant consideration is the existence of the linear atmospheric CO2 content dependence on the global human population shown in Fig. 1. [The origin of this phenomenon, also discussed in ref.1 and in e.g. web.columbia.edu/itc/sipa/esp/math_review/MathProb2.pdf http://faculty.washington.edu/blewis/papers/co2/co2b.html and in web.scribd.com/46952647/Fulvate-II].

Inserted Could curve b (the fossil fuel usage which is inevitably a source of toxic inroganic element e/g/ Hg and Sb air pollutants) be related to the global prevalence of autism?

Fig. 1 was drawn using the data sources cited in ref.(1).

Inserted Document

Wiggins Two State Water Theory Its Relevance to Biology and non (or quaasi-) biological self-assembling [inorganic] systems.
List of papers received from P.M. Wiggins (Auckland, New Zealand) Papers 1 and 2 discussed above together with Water Digest Sept Oct 2006 pp 78-82 (Some Watery Science) [This discusses the origin of life]. Cellular and Molecular Biology (Ed. K.-L. Halbhuber, Jena, Germany) 2001 47 (995) 735-744 (High and Low Density Intracellular Water)/

Microbiological Reviews Dec. 1990 p. 432-449 [Vol. 54 No. 4] (Role of Water in Some Biological Processes)
[This paper starts off by noting that in 1956 Troschin (a Russian scientist) had proposed that the cytoplasm contains water in an altered structural state; this idea which was further developed in 1962 by Ling (which strongly disagreed with the traditional view of biological cells as membranous bags of aqueous solution). This theory apparently was well accepted in Russia but not in the English-speaking

world (where confusion existed e.g. about the interpretation of NMR data and by the polywater controversy so that ideas relating to water structure in general were thought to be fraudulent). The traditional Gibbs-Donnan membrane concept previously applied to polyelectrolyte gels required to be re-evaluated following the work of Wiggins and Van Ryan (who used density bottles to obtain evidence for the diminution of the density of water in such gels which had been treated with polyethylene glycol (PEG) which was too big to enter the pores of the gel which imbibed pure water from the external solution so that water decreased in density to 0.96g.cm-3; the intra-gel water density decreased with increasing PEG concentration in the external solution; charged gels imbibed water until the mixing is optimal and then ions and water equilibrate [a diminution of water density was also found by Garriogos et al. for myosin associated cleft water]). The previously-believed putatively apparently quite erroneous concepts regarding such gels had arisen from a misunderstanding of the physical and chemical nature of water especially how water microstructure varies in respect to solute ion effects. Wiggins indicated that the low density water (LDW) in gels tends to have straight ice-like hydrogenbonds whereas water in the denser [high density water (HDW)] tends to form weaker, bent, hydrogenbonds. A continuous spectrum of water structures between LDW and HDW could be envisaged to exist. For understanding the basic water-structure-related driving force of biology it is necessary to consider the effect of ion hydration effects on the production of such water structuring. The observed distribution of (inorganic) ions between two aqueous phases of different densities in gels which was studied by Wiggins et al. suggests that small highly hydrated cations tended to accumulate in the HDW while larger [e.g. singly charged cations such as K+ and Na+] preferred the LDW. The stability of gelinduced water structure per se evidently created the characteristic type of biologically observed inorganic ion selectivity. The Hofmeister (lyotropic) series ranks ions for this behaviour. I.e. water structure was confirmed by its direct association with the Hofmeister series effect to be the principal driving force for protein structure stabilization or de-stabilization (denaturation). [D.G. insert A similar tie-up between water structure and nucleic acid and polysaccharide structuring is also evident, i.e. the absolute nature of biological tissue is entirely water-structure-driven]. While primitive water structure pumping mechanisms contribute to ion pumping in biological membranes, where ATP-driven cation pumps are also well-established to function, the mechanism by which ATP action is coupled to the active transport of ions also showed an underlying tie-up with a highly hydrated gel ionic selectivity. Wiggins proposed that the inward diffusion of e.g. Ca2+ and actions of Mg2+ are at least partly waterstructuredetermined. The hydrophobic effect, which had been previously mis-understood but could now be better explained by a water-structure related dependency of the cooperativity of hydrogen-bonding between water molecules. Wiggins and Van Ryan pointed out that the water molecules adjacent to hydrophobic surfaces, because they make fewer hydrogen-bonds, are in a state of higher energy (this causing individual water molecules to move apart (to decrease their chemical potential to a greater extent) than those water molecules which are further away from such a surface. This kind of hydrophobic surface water structure discriminating effect is the exact opposite to that produced on water-structure by hydrophilic surfaces (e.g. those containing ionically charged surfaces). The new Wiggins gel-water-structure theory was thought to permit a better understanding of the modulation of cellular volume and replace the former theory that this was enabled by a Donnan-Gibbs semipermeable membrane system and replace the previously held notion that there must some kind of osmotically inactive water phase inside biological cells, an idea that seems to have been engendered by the use of an incorrect model of the role of water-structure in biological cells. Structured water adjacent to actin filaments were believed by Wiggins to contribute to cytoplasmal structure and muscle activity. Structured water power was also how all enzymes can achieve hydrolysis of proteins and other biopolymers as well as to promote the reverse processes (i.e. the catalysis of the formation of biopolymers by a process of removal of water molecules from the monomer components).

A related water energy transduction mechanism was also proposed to enable those enzymic actions which produce ATP from ADP + Pi. This paper concluded by noting (my underlining) that: Soluble, sulfated, carboxylated, or phosphorylated polymers are ubiquitous, often in association with gels. A soluble sulfated mucus, for example, is associated with a solid mucus gel with considerable hydrophobic components. Exploration of the properties of water associated with these various biopolymers should be revealing. It is safe to predict that it will also be confusing and frustrating, because when two extreme forms of water coexist at a single polymer surface, or in a single binding cavity, direct elucidation of their individual properties is fraught with difficulties. This experimental block is one reason why controversy has surrounded the properties of water in gels and cells for so long and why a significant biological force has remained hidden. [D.G. Comment: A water-structure dependent mechanisms of action are seen to especially underpin how the extracellular matrix mucus sulphated polysaccharides [especially heparan sulfate proteoglycans in animals] perform multiple actions as information processors and high level system managers [e.g., for the control numerous cellular tissue protection processes, embryo assembly and wound healing] . These can be seen, due to the work of Wiggins et al. (and others), ultimately to be due to the ability of such counterion binding water structure building to be elicited by complex microstructured anionic sulphated polysaccharide (and also phosphorylated biopolymers etc.) in order (putatively) to provide a smart servo-feedback system which potently modulates specific deterministic chaotic water structure formation in a highly controlled [cofactor and environmentdependent] manner; Cf. also Grant D. et al. Biochem J. 1991 277 569-71; NIR News 1991 9-13 Biochem Soc Trans. 1990 18 1283-4; ibid., 1988 17 1029-30; ibid.,1985, 13 389; 1984 12 302; 1983 11 96; Communication from D. scribd 2011]. Physica A 314 (2002) 485-491 (Water in complex environments such as living systems) Physica A 238 (1997) (1-4) 113-128 (Hydrophobic hydration, hydrophobic forces and protein folding) [Discusses LDW which has stronger hydrogen-bonds and lower intrinsic entropy than normal water and which has been shown to from outside the double layers which occur with polyelectrolytes. The paper also discussed the effects of the co-operativity of water-water hydrogen-bonding and the heat capacity on dissolution in water of hydrocarbons and denaturation of proteins, proteins as polyelectroytes, temperature effects on protein stability and LDW effects as a determinant of the Hofmeister series as well as LDW effects in lipid bilayers, etc. Hydrophobic hydration which (is not, as it was previously assumed) due to oil/water incompatibility but is actually due to oil/LDW incompatibility]. LANGMUIR 1995 11 1984-1986 (Microosmosis, a Chaotic Phenomenon of Water and Solutes in Gels) JOURNAL OF RAMAN SPECTROSCOPY Vol 26 3-8 (1995) (Micro-Raman Spectroscopic Study of Organ Cultures Cornea) Diana C.W. Siew and Gillian M. Clover Ralph P. Cooney Phillipa M. Wiggins Prog. Polym. Sci. Vol. 20 1121-1163, 1995 (HIGH AND LOW DENSDITY WATER IN GELS) CELL BIOCHEMISTRY AND FUNCTION VOL 13: 165-172 (1995) (Micro-Osmosis in Gels, Cells and Enzymes) Cell Biology International 1996, Vol. 20, No. 6 429-435

Schematic for Origin of Life [and Origin of Water-Structure Pathology] Based on Wiggins Water Theory & Silica Sol Pore Theory

HIGH AND LOW DENSITY WATER AND RESTING, ACTIVE AND TRANSFORMED CELLS -----------------------------------------------------Other papers in File Draft of a discussion of protein folding and disease -----------------------Water and the biology of prions and plaques Graham K. Steel & Philippa M. Wiggins http://hdl.handle.net/10101/npre.2008.138.1.1(2007) ----------------- ------------------Life depends upon two kinds of water Web Isbu.ac.uk/water/monograph200904pw/pdf ----------------------------PLoS ONE 2008 3(1)e1406

--------------------------------------------------

Bird Box File

DG Grant D. Literature Collection A highly selective collection of papers. Graham Steel (& Philippa Wiggins) discussions about prion diseases.

Index Introduction Polysaccharides act in concert with water structure Ion Partition ATP Fibril & Plaque Formation Prions Lipids, HDL & LDL
Footnotes (Wiggins) HDW/LDW & Sulphated Polysaccharides Heparin/Heparan sulphate (H/HS) H/HS inorganic counterion binding & water structure Deficit in H/HS system manager function in disease Chemical structure of H/HS includes Haraguchi multi-elements H/HS and Ca 2+ ion activity regulation The basis of biology is liquid water Extrathermodynamic Relationships Industrial Process SiO 2 Particle Replication Prion stress and Ca 2+ Regulation (Purdey) Prion disease Mn 3+ hypoth. (HS) Ba 2+ hypoth. of MS) Prion-Cu(II) (Supattopone) phospholipase TSE hypoth Wiggins Theory of Water further notes Silica gel and prebiotic evolution Haraguchi metallomics further notes Hg intoxication & HS Nephropathy

Introduction
Heparin/Heparan sulphate (H/HS) Related Topics
(Including Silica (SiO2)-Based Life Forms)

The glycosaminoglycan (GAG) polysaccharide proteoglycan (PG) systems include, as a highly specialized and information storage and processing system, the H/HS sulphated polysaccharide (occurring in vivo as sidechains of proteoglycan cores, H in mast cells and HS at cell surfaces) which are systems of ultrahighly anionic structure enabled to bind strongly to water molecules so as to modulate adjacent water (soft-ice and liquid) structure formation in a co-ion binding and sorting manner.
H/HS also binds to silicate and, in common with other natural polyuronides normally occurs naturally in combination with inorganic silicon (most likely in the form of silicic acid aggregates).

HS is also known to be a wide-ranging anti-disease and anti-pathogen system.

Hypothesis 1: All biopolymers including polysaccharides such as HS act in concert with water structuring energy flow modulation via Wiggins high density/low density HDW/LDW Glycosaminoglycans include HS (as well as chondroitin sulphates and hyaluronan) have been indicated to interact ( in conjunction with the presence of other biopolymers) with (Wiggins) HDW/LDW (high density/low density forms of microstructured liquid water).

Hypothesis 2: HS putatively acts as a therapeutic agent in concert with density-variable water structuring. The Wiggins PM (extrathermodynamic) high density water /low density water (HDW/LDW ) (water structure/activity-basis-of life) theory (cf., PloS ONE 2008 (1) e1406) is considered to provide a credible explanation of those water structure and physical property anomalies which may be the ultimate causes of a range of diseases. {Cf., the health-related concept of water structure which has also been discussed by e.g. Martin Chaplin in an internet literature collection}.
Those biological reactions which are deemed to depend on transmission of ATP energy are actually accomplished, according to Professor Philippa M. Wiggins, entirely by the existence of two-state microsomotic energy facilitating water structure. With the two state water theory there is no need to invoke the classical energy transduction theory which can be credibly be re-evaluated in terms of HDW/LDW which shows that biological energy is not provided directly by the free energy of the hydrolysis of ATP but, instead, indirectly by the alteration of water structure produced as a consequence of enzyme phosphorylation which changes the HDW/LDW ratio. Such modulation of HDW/LDW can also explain how enzyme activities can be re-activated for continued use.

According to Wiggins: The activation of small cations and reversal of hydrolysis (e.g. of ATP) are inevitable consequences of the presence of LDW

Behaviour (e.g. of an enzyme) as a concentrated strong acid is also an inevitable consequence of the presence of HDW Crystallization of bone becomes possible if Ca2+ and PO4 3- are first concentrated in HDW and then precipitated when they convert HDW to LDW The Wiggins Theory considered the effectiveness of individual solute molecules or ions for the induction either of LDW or HDW in the bulk water in their neighborhood. E.g. Na+ was shown to partition preferentially into HSW and induce LDW but K+ partitions preferentially into LDW and induces HDW.
Enzymes start in an inactive state, are converted to an active state which performs the characteristic reactions, but must then revert to the inactive state in order to be able to repeat the cycle. If the active state contained LDW, then K+ would demolish it and restore the inactive state. This mechanism by which small solutes demolish their own preferred environment has proved of general application.

Wiggins Two State Water Theory & How protein denaturation, fibril and plaque formation occur (in vivo). Hypothesis 3:

In general (all?) plaque or insoluble fibril-determined pathologies (at least to some extent?) can be considered to arise as a consequence of dysfunction of the physiological regulation of water structure [i.e. the HDW/LDW system].
The Wiggins theory of water structure is suggested to be of major relevance to a fuller understanding of degenerative diseases in general and the etiology of both Alzheimers disease (AD) and prion diseases transmissible spongiform encephalopathies (TSEs), in particular.

(Cf., the Wiggins Theory (cf. also footnotes: A pertains e.g., to the Supattopone group researches [loc. cit. footnote b-2]) on lipoprotein dependent mechanism of prion misfolding, the formation of other misfolded proteins and how insoluble Ca2+ salt plaques can be induced to form).
Heat shock proteins [water structure modulators]) and other chaperones such as heparan sulphate putatively function by altering and regulating HDW/LDW
Cf. the ability of chaperones to alter the rate of the process of PrPSc seeded conversion of PrP to the protease resistant form PrP-res was reported by DeBurman SK et al. PNAS 1997 94 25 13938-43: the heat shock chaperone proteins GroEF (Hsp10) and Hsp104 promoted, while the chemical chaperones, sucrose, trehalose and dimethylsulfoxide (DMSO) inhibited prion misfolding; the presence of N-linked sugars and GPI anchor in normal PrP also putatively act (albeit modestly) as PrP stabilizing chaperones. These studies provide further evidence of the key involvement of HDW/LDW water structure regulation in the mechanism of the prion misfolding.

Anionic surfaces may have a key role to play in the induction of HDW/LDW but this includes the ability of subclasses of such surfaces to promote the formation of pathological plaques. A sub-class of such surfaces can inhibit the formation of such pathological plaques. A role for pro- and anti-anionic surface classes for assisting correcting or causing incorrect protein folding can be included in this mechanism. The HDW/LDW theory seems likely to be centrally implicated in this, however.
The more energetic HDW form of water can, per se, putatively, according to Wiggins, cause non-enzymic hydrolysis (of e.g. sulphate half-ester groups from glycosaminoglycans). This could remove N-SO3- (creating de-N-sulfonated H/HS which, in sharp contrast to the NSO3- form of HS) is inactive as an anti-plaque agent.
{Cf. Grant D. et al. Inhibition by glycosaminoglycans of CaCO3(calcite) crystallization Biochem J 259 41-5, which suggested that fully N-sulphonated H(HS) may be essential both for the prevention of pathological Ca salt precipitation and also (putatively) for the prevention of the formation of amyloid plaques in AD; cf. also Grant D. et al. Degenerative and inflammatory diseases may result from defects in anti-mineralization mechanism afforded by glycosaminoglycans Med Hypoth. 1992, 38 49-55 . While native H/HS is a highly effective scale inhibitor, de-N-sulphonated H actually promotes the formation of insoluble Ca salts. The ability of free H+ and unliganded active-Fe, active-Cu and other redox-active ions to non-enzymically cleave N-SO3- could be why the unregulated in vivo presence of such ions poses a severe health risk. (The mechanism of HS damage involving de-N sulphonation of HS is considered to pose a similar level of risk as to the abilities of excessive amounts of unbound redox ions to promote (Fenton reaction) oxygen free-radical chain reaction damage to proteins and DNA).

The above scenario also indicates, when considered in the context of HDW/LDW water structure that HS-N-SO3- may induce a tissue-protective HDW/LDW ratio which might be abolished if such HS-N-SO3- become depleted.
[The above ideas seem to accord with a 2010 report by Bruinsma B. et al. (Acta Neuropathol 119 (2) 211-20] which indicated that a lack of NSO3- in HS might be a critical pro-disease modification HS which is associated with the Alzheimers disease (AD) toxic fibril formation; it should be noted that insoluble proteinaceous plaque forming diseases including AD seem invariably also to be associated with metal ion, especially Fe ion, dyshomeostasis].

Good & Bad Lipids, Cholesterol & Wiggins HDW/LDW

The effect of correctly-sequenced GAG microstructures, especially of HS (PGs), is further suggested to be centrally relevant to the etiology of cardiovascular health and the etiology of atherosclerosis. The correct role of high density HDL and low density lipoprotein LDL may be to act as a stimulus for the modulation of correct HS microsequence production during primary HS biosynthesis as well as via an ability of an appropriate HS microstructure to optimally regulate lipid metabolism. The general association of hydrophobicity with lipid groups suggests that the concept of good and bad cholesterol is a surrogate or subsidiary concept to the ultimate pathological stimulus which is a deviation of pathological water structure from the correct cell surface Wiggins-HDL/LDW normal physiological equilibrium level. This would be in keeping with the Wiggins hypothesis that (all) life ultimately is an outcome of the modulation by biological molecules by the occurrence of HDL/LDL in liquid water ; (the corollary to this hypothesis is that all aspects of the health of animals, in the most general sense, must ultimately depend on the maintenance of critical-for-life HDW/ LDW balances. Deviation from the correct balance creates circumstances which promote atherosclerosis, AD and TSEs, it can be argued. The etiologies of such diseases can then be seen to be influenced by inappropriate lipids (including bad cholesterol in the wrong place and time) because such lipids induce inappropriate HDL/LDL ratios. A prime function of extracellular polysaccharides including the HS (PG) system may be to regulate the HDL/LDL ratio in particular in vivo locations.

Following on from this idea suggests an alternative rationalization of the influence of inappropriate lipids on cardiovascular diseases. It should be noted that tweaking the HS system (which is possible by the exogenous administration of HS fragments etc.) has been indicated by numerous investigators over many decades of research to produce beneficial therapeutic outcomes on the cardiovascular system. (In the 1960s Engelberg H. (vide infra) discovered that the amount of endogenous heparin in blood (probably derived ex vascular surface HS) correlated positively with LDL [the good cholesterol lipoprotein which has become a commonly believed pro-health dietary component]).

{Cf. it should be noted that the original good cholesterol HDL (cardiovascular health) hypothesis which is still generally believed could be subject to serious error since no correlation was recently found between expected heart attack risk in a genetically enhanced HDL sub-population which had been thought to be more at risk; furthermore there was a lack of vascular therapeutic benefit shown by (cholesteryl ester transfer protein (CETP) inhibitor) LDL cholesterol lowering drugs (Cf. The Royal Society of Chemistry Chem. World 2012 09 7 article by Houghton S., entitled: HDL drug class struggling after flop Cholesterol drug failure threatens HDL hypothesis). It should be also noted that while the statin type of drugs are believed to have a similar efficacy as standard heparin for the prevention of, and for the alleviation of the symptoms of atherosclerosis (cf. a 2008 animal model study reported by Vijayabaskai P. et al., Afr. J. Biochem. Res. 2(5) 120-7 which reinforced this old idea (which came to light decades ago (cf. Engelberg H. Pharmacol. Res. 1988 36 91-110) that use of pharmaceutical heparin as a blood anticoagulant can also modulate and improve beneficial lipid turnover and that individuals who have more endogenous heparin (which likely is produced by vascular wall HSPG) in their bloodstream exhibit a correspondingly greater resistance to the occurrence of atherosclerosis suggesting that a correct functioning of the H/HS system is a more accurate measure of cardiovascular health than is HDL/LDL and that use of heparin-based therapy (or dietary therapy linked to augmentation of endogenous heparin status) is likely to be the most effective therapeutic method now available to improve human cardiovascular health without the possible adverse effects of some which have apparently been associated with some of the current exogenous drug treatments. It can therefore be rationally suggested that future research should aim to optimize this H/HS cardiovascular protection system. This could be argued to ultimately actually be a HS-directed Wiggins LDW/HDW balance strategy. It should also be noted that a major part of this HS-directed LDW/HDW regulation seems to modulated by nitric oxide (via the effects of nitric oxide metabolites which can deaminatively cleave HS at pre-primed de-N-SO3- groups; this is a system which can be perturbed by redox dyshomeostasis which in turn can be induced by xenobiotic intoxication). A recent paper of relevance to this idea is Cheng F. et al. J. Biol. Chem. 2011 286 (11) 27559-72.

Footnotes

A Wiggins HDW/LDW & HS-associated water structure Although it is perhaps unfortunate that a surrogate sulphated polysaccharide (dextran sulphate) instead of the more appropriate glycosaminoglycan system and especially HS was used by Wiggins in her quest to establish why water is the single most important substance in living organisms, selection of this semisynthetic (sulphated bacterial polysaccharide, while it weakens the arguments derived from the experimental

researches somewhat, clearly does not invalidate the Wiggins theory of HDW/LDW/HDW as it applies to animal biochemistry. The discussion on p 10 of PloS ONE 2008 (1) e1406, draws attention to why dextran suphate etc. were chosendextran sulphate, glass beads, silica gel and anion and cation exchangers these polyelectrolyte systems are included under a single heading because their interactions with water and ions are most relevant to biology [my underlining].
The reason why dextran sulphate was chosen (Grant D e-mail discussion with Wiggins PM) (and seemingly also the other chosen polyelectrolytes) was that these non-animal polymer species were considered to be entirely suitable laboratory equivalents of HS and other GAGs this use of dextran sulphate is OK as a starting-off model of primitive polysaccharides but further work might usefully consider the uniqueness of HS relative to other sulphated polysaccharides in terms of its ability to engender HDW/LDW/HDW.

[N.b., dextran sulphate derives its anionic character largely from sugar-O-SO3- groups; it should be noted such anionic polysaccharides also ubiquitously contain small amounts of inorganic Si (as well as a wide range of other inorganic elements [additional to the polysaccharide covalently bound O, H, S and N]; Si is the most abundant of these non-core-structure inorganic elements; which occurs in a currently unknown chemical form; this seems most likely to be: hydrogen-bonded SiO2(H2O)n sol nanoparticulates). Aberdeen polysaccharide workers (cf., Grant D et al. Med. Hypoth. 1992 cf. p. 48) had earlier suggested that elucidation of the chemical mechanism by which silicate minerals exert present-day biological (including pathological) effects may provide clues about how prebiotic evolution might have been achieved by silicate minerals (by a related mechanism centred on (amorphous but with e.g. discriminated chaotic order) to that proposed by Cairns-Smith A.G. (Genetic Takeover and the Mineral Origins of Life published by Cambridge University Press in 1982 which had proposed that early life had depended on the putative gene-like activities afforded by the well-known type of ordering which occurs in crystalline clay minerals). A-1

H/HS, Inorganic Counterion Binding & Water Structure Studies at Aberdeen U.

A broad-scientific-interest Aberdeen University polysaccharide group (which had been headed by Long WF and Williamson FB) had focused on in-depth researches into the inorganic biochemistry of H/HS. (Cf. web. abdn.acuk/bch~118/publications2003march.pdf : this gives a list of the majority of the published literature of the group which reveals that a recurrent theme of this research had been the inter-relationship between inorganic ion - H/HS interactions and hydration effects (cf. also e.g. the papers by Grant D. et al. listed in the above 2003 literature list included communications entitled Altered water structure in mixtures of heparin and metal ions Biochem. Soc. Trans. 1984 12 310, A role for glycosaminoglycans in cellular adhesion of relevance to the cancer state , ibid., 1985 13 389; Polystyrene surfaces may require structured water for effective cell adhesion ibid. 1988 16 1029-30, The dependence on counter-cation of the degree of hydration of heparin ibid., 1990 18 (6) 1213 and Near and fundamental region IR spectroscopy of heparin-metal cation complexes suggests involvement of water molecules in the complexation, NIR News 1991 9-13).

Deficit of a putative high level H/HS centred system manager function may be involved centrally in the aetiologies of numerous human diseases
allowimg exogenous application (as well as possibly the stimulation of endogenous provision) of H/HS could, it might be indicated, elicit numerous possible beneficial therapeutic activities.

The detailed chemical microstructure of sulphated (sulfated) polysaccharide-based multicellular animal information system H/HS comprising heparin/heparan sulfate proteoglycans (syndecan, glypican, agrin, perlecan, betaglycan, type XVIII collagen etc.) can become defective under pathological conditions as regards an ability to perform designated tasks. It can be argued that a central ultimate cause of many diseases is because of a major defect has arisen in the affected subjects H/HS (water-

structure-inorganic ion regulating) tissue protection system. Exogenous administration of H/HS molecules or substances (this includes H/HS fragments) which can boost improved correct de novo HS biosynthesis are credibly achievable therapeutic intervention strategies for a wide range of degenerative diseases. Such a, perhaps surprising, conclusion must inevitably, it is suggested, be reached from any attempted comprehensive literature review of the now extensive H/HS biochemical literature. The Chemical Structure of H/HS

Although the chemical structure of the core polysaccharide itself a complex as yet not fully solved problem it has become apparent that (-COO-, -OSO3- and -N-SO3-) anionic patterning of the polysaccharide which creates a bar-code like information system can also be modified post-synthetically including by non-enzymic de-N- sulphonation which primes the molecule for rapid nitrosative scission (by nitric oxide metabolites) [a reaction which is subject to Cu and Zn and putatively other redox ion catalytic effects cf. Cheng F. et al., loc. cit vide supra]; this complexity of modus operandi is compounded by the ability of anionic polysaccharides to simultaneously sequester a range of 60+ inorganic element counter-cations and anions. This inorganic moiety sequestration process is believed to alter the biological (signalling) activities of the various H/HS-PGs information bits. A correct water structure, which in turn is dependent on a correct inorganic cofactor arrangement at the H/HS working surfaces, is likely therefore to be required to attain such an optimum H/HS function and this, furthermore, may be what becomes deficient if too high a concentration of toxic inorganic ions are permitted to replace the correct required inorganic water structure inducing cofactors. The effect of H/HS (inorganic-cofactor) microstructure is transmitted, it is tentatively proposed into discretely different HDW/LDW water structure microstructures which were proposed (cf. Wiggins 2008, loc. cit.) to be the ultimate modus operandi machine of biological enzymic activities. H/HS and Ca2+ ion activity regulation The ability of Ca2+ to bind to an alter the conformation of H/HS may especially allow Ca2+ to selectively control H/HS (conformation associated water structure) activity and also to conversely allow H/HS water structure to control Ca2+ activity (cf. Long WF & Williamson FB 1979 Glycosaminoglycans, calcium ions and the control of cell proliferation IRCS J Med Sci 7 429-34) The more general regulation of other-than-Ca2+-metal-ion activities may be a possible very primitive role of H/HS. Prion proteins, which seem to be intimately involved in HS biochemistry may also participate in the above H/HS mechanism of metal ion regulation of water structure a .
Multiple Metal ion H/HS interaction.

Cf. the series of studies of the binding of individual metal ions to H/HS etc. reported by Long WF et al. (cf. web.abdn.ac.uk/~bch118/publications2003march.doc); Cf. e.g.,
Grant D Long WF and Williamson FB. Infrared spectroscopy of heparin-cation complexes Biochem. J. 1987 244 143-149 [This paper reported that the various inorganic counterions salts of heparin (H) show large cationspecific alterations of the infrared vibrational bands associated especially with carboxylate and hydration. Such water-structure dependent spectroscopic alteration is enhanced in the overtonecombination NIR band regions.

A standard pharmaceutical industry sodium heparin which had been kindly supplied to various UK academic institutions in the 1980s; when this was subjected to mass spectroscopic multi-inorganic element analysis was found (unexpectedly) to contain

large amounts of a wide range of other elements; these could however be reduced in amount by the use of a cation exchange columna1 [N.b., such ion-exchanged heparin while enriched in a selected counterion remained however, intrinsically a similarly multi-inorganic-element matrix classifiable-state-of-matter, just as the unpurified form of heparin had been. Both purified and native heparin showed log-log (linearly) approximate correlations with the multi-inorganic element profiles which had been identified by Haraguchi c as ubiquitously occurring in animal cells and blood serum and also occurs in (modern) seawater (evidently because the ancestors of such species evolved in the sea and continue to require the unique 60+ element inorganic element profile composition (which induces, it can be further deduced following the Wiggins HDW/LDW theory, the specific HDW/LDW/HDW water microstructure similar to that present in that marine multi-ion solution). That H (and by inference also HS) must be expected always to occur in association with a seawater-like distribution of 60+ inorganic multi-elements seems to have been confirmed in 2006 (in an internet document published by ALS {a major internationally validated provider of such analytical data}which independently reported (all-element) mass spectroscopic data obtained from elution of heparins from the surfaces of heparinized blood collection vessels. ---------------------------------------------------------------------------------------------A-2 The basis of biology seems to depend to a greater extent than on any other chemical substance on water and its supramolecular structure Water is generally acknowledged to be the sine qua non of and for life. It is now suggested in line with numerous prior similar statements like it not circumstantial that water is by far the most abundant chemical surface in viable biological cells. Biology is not at its most basic level the outcome of the presence of DNA or any other biopolymer but is to do with how the various chemical substances which occur in living organisms interact with water {Many scientists have hit on this idea, cf. e.g. Luck W.A.P. in Top Curr Chem 1975 5 115-80 and Bernal J.D. (Cf. Symp Soc Exptl Biol 1965 19 17-32 [Chem. Abs. 65 9242f]) Philippa M. Wiggins Prog Poly Sci. 1995 20 1121-63 cf., PloS ONE 2008 e1406. A-3 Extrathermodynamic relationships in water Silica Sols in water & Wiggins HDW/LDW An important driving force of how liquid water may facilitate biology is to do with its reluctance to obey reversible thermodynamic rules such as the second law of thermodynamics (cf. the phenomenon of Brownian motion which seem to be the basis of the validation of the Evans Fluctuation Theorem [the exponential probability of occurrence of reverse time entropy is an exponent of entropy/time {a process which leads to entropy enthalpy compensation in rate processes}and indication for how reverse entropy flow can be especially facilitated by aqueous solutions]. The sacrilegious idea that genes per se cannot be the basis of the origin and nature of life was also discussed by (n.b., a renowned geneticist) Lima-de-Faria A. in Evolution without Selection a book published by Elsevier in 1988, which drew attention to the

ability of various types of CaCO3 crystal modifications to engage in true-to-form seeded inorganic-particle reproduction. This reproduction is achieved, it should be noted, in water. Genes played no part in this type of abiotic reproduction process which, like the similar reproduction of specific types of SiO2 sol particles (vide infra) seems to mimic biological reproduction (of species which are designed by their unique forms).
There have also, it should be noted, been suggestions that abiotically formed amino acid derived pseudo biopolymers e.g. protein-like thermal condensates, might have induced life since they were demonstrated to undergo a form of abiotic cell scission when dispersed in water (cf. Fox S. loc cit.) A-2-1 Industrially Observed SiO2 Sol Replication

Earlier, in 1965-6 during industrial researches, I had observed what seemed to be a abiotic SiO2 cellular reproduction process which was however more like a true noncrystal cellular reproduction phenomenon which had been achieved by a purely inorganic, formally amorphous (i.e. to conventional X-ray diffraction evidenced crystallinity) species of silica sol. This finding arose from research which was aimed at reproducing a specific type of silica sol formed by de-polymerizing pre-formed silica gel in an autoclave; structural disruption and re-assembly occurred under these condition had been known to the art to create sols by a seeded growth process. This could apparently be accomplished most conveniently by using previous batch heel seeds. I found that this seeding could be accomplished by using seeds produced by a particle assembly from silicic acid process. It was possible to make sols resembling those produced by the depolymerization of gel by use of seeds of the former type in an apparatus of the latter type. My thoroughly researched study (set forth in a 1966 draft manuscript) which had discussed my silica sol assembly process results floated the idea that the seeded growth of silica sols resembled biological cellular reproduction, but his idea turned out to be too highly controversial at that time (which was before the Fox proteinoid work had become generally known). So much so that it did not get out of the lab. and into conventional publication. It seemed in 1966 that life must always depend on nucleic acids and anything else could not be entertained. (The laboratory experiments were later however, I believe used highly successfully commercially, e.g. for the reproducible production of a silicon wafer (chip) abrasive agent). The censorship of my preliminary inorganic cell reproduction data is unfortunate as these observations (if confirmed) could allow fundamental insight to be attained into how water-binding systems contribute to the phenomenon of life and conversely how the mechanisms of life could critically depend on supramolecular water structures. (N.b. silica gels can be produced having very high water contents; this also applies to biologically produced polysaccharides and living cells in general). The idea that the modern type of DNA gene-based biology could have evolved from an original silicon-based entirely inorganic gene system was later also suggested by Cairns-Smith AG. (cf. Genetic takeover and the Origin of Life Cambridge University Press, 1982) [however in my correspondence with Cairns-Smith AG. relating to the kind of primitive inorganic biology suggested by silica sol cellular particle chemical researches, it should be noted that I failed to convince this scientist of the possible relevance of silica sols researches as a basis for unraveling the origin of terrestrial lifee). Diagram of Putative Pre-Biotic Silicic Acid Aggregate (Silica Sols with Pores) Generator of HDW/LDW Energy (Pro-life) Potential

Appendix e
Water Structure and Protein Folding

Hypothesis: The Ultimate Dysfunctions Leading to Many Pathologies including Prion Protein Misfolding Diseases are Caused by Perturbations of Water Structure.
Cf. Luck WAP. (cf. Top. Curr. Chem. 1975 5 115-80) established that the Hofmeister lyotropic series correlates with altered liquid water vibrational spectroscopic energy levels.

Cf. also studies of e.g. Dr A. et al. e.g. (Salts, interfacial water and protein conformation) Biotechnol. Biotech. Equ. 2008 22 (1) 629-63; cf. J. Phys. Chem. 2007 111 (19) 5344-50
The paradigm shifting HDW/LDW theory of how ATP energy provision arises suggests a possible scenario for the pre-biotic origin of life With the Wiggins two state water (HDW/LDW Theory) there is no need to invoke energy transduction in regard to the explanation of the mechanism of operation of ATP- (or [inorganic polyphosphate (afforded energy provision e.g.) for enzymatic pumping of Na+ from a low concentration inside the cell to a higher concentration outside the cell. This process, according to Wiggins, depends entirely on the inevitable alteration of HDW/LDW caused by phosphorylation of the sodium pump which produces an energy-transduction-affording change in the water structure near the phosphorylated site. Since reverse running of the pump causes ATP to be re-synthesized from ADP, this further suggests that water structure environment change effects can allow both the uphill transport of Na+ (as well as the de-novo synthesis of ATP from ADP + Pi). These processes are postulated by Wiggins to be achieved in modern organisms via an alteration of the water structure inside the active cleft of the enzyme which causes this water (following phosphorylation of the sodium pump enzyme) to have stronger interwater-molecule linkages. This creates HDW which further induces the formation of an adjacent

accumulation of LDW. The existence of (hydrated) phosphorylated (or sulphated) surfaces in proteins and polysaccharides when dispersed in liquid water thus causes the formation of a HDW/LDW potential energy system which can act as an energy source and ultimately (according to Wiggins) this is what defines life at its most fundamental, basic, level. The ability of LDW (e.g. at SiO2 surfaces) to dramatically specifically select for L over D amino acids can, it seems, can fully explain the chirality of biology (e.g. as it pertained to the origin of life on Earth) (cf., Wiggins P., Water Digest. 2008 Sept-Oct 78-82). LDW is believed to have a similar structure to Ice 1h

Cf. The Chemical Structure of Liquid Water. [Philippa Wiggins Theory (cf. DG, the present authors e-mails with Prof Wiggins Auckland New Zealand discussion paper drafting assisted by Graham Steel cf. also (Water and the Biology of Prions) Wiggins, Philippa and Steel, Graham, Nature Proceedings http://hdl.handle.net/10101/npre.2007.1381.12007 Cf. Wiggin P. (Life Depends upon Two Kinds of Water) PloS ONE. 2008 3(1): e1406 The traditional high-energy phosphate (squiggle P-O-P) energy transduction hypothesis becomes redundant if the Wiggins water chemistry concept is regarded as the key to how biology is energized. A prior form of this idea had been promoted by Bernal, Ling, Luck and others. The Wiggins Theory extends water structuring effects to include the bulk water. {An extrathermodynamic effect which may especially be facilitated by water structuring may also have fundamental relevance for the operation of water-related biological energy systems (and which is outwith the ATP linkage system) is the phenomenon of entropy-enthalpy compensation in rate processes whereby putatively energy derived from reverse entropy flow (as allowed by the Evans Fluctuation Theorem) can be harnessed by biology}. [Although Bernal had postulated that long-range alteration of water structure was caused by solutes and surface, the alternative idea that only short-range water structure alteration took place at biological surfaces became to be widely believed by most biochemists who also firmly held the view that water layers at protein surfaces were only at most two water molecules thick and therefore did not affect the bulk water which was therefore considered to be unaffected so that intracellular water was considered to behave simply as a relatively inert supporting medium for the supposed key-to life nucleic acid-based biopolymers; this view of water structure was challenged especially by Bernal, Luck, Franks and Ling and evidenced directly by Wiggins P.M et al., as well as by e.g. Pashley K.M. et al. (Science 1985 229 1088) who established experimentally that biological systems probably had available to them a hierarchy of attractive forces that can operate between hydrophobic moieties and that these forces depended on the dimensions and geometry of these surfaces and were much stronger, longer ranged and more variable than classical colloid science had previously indicated]. A simple demonstration of alteration by LDW of hydrophobic glass bead surfaces was illustrated on p.10-11 of Wiggins P.M. PloS ONE 2008 1406, where glass bead flotation was produced by the addition of K+, Cs+, Ca2+ or Mg2+ ions but conversely, precipitation occurred with Li+, Na+ or H+ ions. Such counterion salt effects were attributed to the presence of LDW/HDW, especially via a LDW augmentation at surfaces which caused SiO2 bead aggregation (including by the prevention of air trapped between the beads from escaping). This bead behavior phenomenon occurred only with H2O (or D2O) and not when the bead dispersant was hexane or DMSO). [The 44-60m diameter dimethyldichlorosilane hydrophobized glass beads (of negative charge due to Si-O-) had the ability to discriminate between the two classes of

physiological counterions; this was suggested to arise via the discrimination effects elicited by LDW present on the silica bead surfaces and their associated small air bubbles]. An internet-posted document:
Cf. web.Isbu.ac.uk/water/monograph200904pw.pdf (downloaded from the internet during 2007) contained additional social comments about myths which apparently had been removed from the 2008 PloS ONE edition of this paper. This discussed the ATP myth . Wiggins wrote: Energy transduction: the great biochemical myth Michael King.. in his Penguin History of New Zealand described the Great New Zealand Myth. This was published in 1909 and thereafter in the School Journal, which was read by all primary school children in New Zealand. It described in detail how the great Polynesian navigator, Kupe, discovered New Zealand in 950AD. Generations of New Zealanders have believed that this was authentically derived from Mauri oral tradition. In fact it was pure invention by a non-Maori. A similar status is enjoyed by the biochemical concept of energy transduction which appeared in early text books and is believed by generations of biochemistry to be in the great tradition of Willard Gibbs and other classical thermodynamicists.
The final Wiggins PloS ONE 2008 e1406 article omitted the chat about the New Zealand Myth continued as in the original interent posting, viz. Energy transduction says that the free energy of hydrolysis of ATP or of dissipation of a cation gradient can be harnessed by enzymes to do work. In fact the free energy of any spontaneous reaction is always dissipated as heat. Reactions are coupled, in a thermodynamic sense, only when the product of one reaction is a reactant of the second. So the energy required to transport cations against gradients or to make the filaments slide or to synthesize ATP or peptides or polynucleotides or to perform many of the reactions idiosyncratic of life, must have another source. That hydrolysis of ATP is an essential part of the overall reactions of cation pumps and motor molecules is not in question, but the mechanism is. Since water is a reactant in these reactions and is also the solvating medium in which they take place, its involvement is inevitable. Phosphorylation of the enzyme by ATP must change the local environment in which the reaction takes place, so that its free energy change becomes negative. That environment is water close to the enzyme surface.

Hydrolysis of peptides and polynucleotides. Proteases and Dnases hydrolyze peptide bonds and oligonucleotide bonds, respectively, at neutral pH and 38C or ambient temperature. To break these bonds in vitro requires boiling in 6M HCl. Somehow these enzymes-in-solution have acquired the properties of hot concentrated strong acids. Again it is impossible to ignore a contribution of water, which is present and is required for the development of acid properties. Formation of bone. The mineral component of bone is Ca3(PO4)2. In vivo this forms at pH 7.4 and 38C from the norrmal concentrations of calcium and phosphate (approximately 2mM) in blood. Similar ceramics require a temperature of 1200C, presumably, to dry the product. The concentration of the species PO4 3- in the blood is probably .of the order of 10-6M, making it far too dilute to precipitate out at all. Indeed , we known that the solubility product is not exceeded in blood because bone forms only in conjunction with specialized cells (the osteoblasts). Crystallization of Ca3(PO4)2 is a process which depends entirely upon the solvent, which is water, and the electrostatic attraction between Ca2+ and PO4 3-. Only changes in the solvent properties of water could allow crystallization of bone at pH 7.4 and 38C. Sodium and potassium. Many enzymes discriminate with exquisite precision between sodium and potassium. Carboxyl groups show a slight preference for K+ over Na+ (2:1) but nothing approaching the 30:1 of the Na,K-ATPase and the many other enzymes which absolutely require either K+ (e.g. pyruvate kinase) or Na+ (e.g. Na+-dependent active transporters). A scrutiny of the aqueous solution chemistry of the two cattions shows that their only significant difference lies in their effects upon the water in which they are dissolved. Na+ slightly increases water structure and K+ slightly decreases water structure

Footnotes a1
Prions
Prion stress and Ca2+ regulation. Does this invovle HS? N.b. both PrPC and glypican HS-PG are glycosylphosphatidylinositol-anchored cell surface proteins

Beraldo F.H. et al. reported (in J. Biol. Chem. 2010 285 (47) 36542-50) that the mechanism of prion transmission of stress signals involved an augmentation of intracellular Ca2+ levels which were increased following the interaction of PrPC with stress inducible protein-1 (SIP-1).

N.b. It has separately been suggested that the biochemistry of PrPC (e.g. its metabolic fate) is intimately linked with that of HS-PGs. Ca2+ may be the primary signal which is regulated by a prion-HS switch but Cu2+, Zn2+ and other metal ion signals and also nitric oxide scission of HS may similarly be regulated and perhaps be subject to the effect of perturbation by non-physiological toxic metal ions (e.g. Ba2+, Hg2+ and Cd2+) as well as by Pb2+ and Al3+). [Cf. Sulkowski E. (FEBS 307 2 129-30) suggested that binding of transition metals e.g. Co2+, Ni2+, Cu2+ and Zn2+ (presumably in an abnormal toxic concentration etc.) to the apparent transition metal binding histidine and tryptophan residues in PrPC, caused the spontaneous formation of infectious PrPSc. If such ions occurred in vivo in inappropriately high concentration (e.g. at HS surfaces adjacent to PrPC) then, it follows from the preceding arguments set out above that the ultimate cause of prion misfolding must be an altered water HDW/LDW structure. It should be also be noted that Kawhara M. et al. (Metallomics. 2011 3 7 726-34) in keeping with a role of prions as metal homeostasis and water structuring agents, found that in cultured rat hippocampal neurons, Zn2+, Cu2+ ions and carnosine ( alanyl histidine) could attenuate the sheet and fibril formation and neurotoxicity of prion fragment PrP106-120. It might be further suggested that this attenuation was further dependent on the association of HS with the prions and the resultant modification of the adjacent HDW/LDW/HDW.

a
Multi-inorganic element polyanions
Polyanions likely become multi-inorganic matrices as a consequence of the multi-inorganic nature (as discussed later by Haraguchi, loc cit.) of their biological bathing fluids. There seemed to have been some difficulty in correctly reporting the multi-inorganic element chemical nature of heparin since this idea jarred with conventional notions that some biochemists strongly believed about this topic as well as the supposedly high status of a standard purification method via cation ion exchange resin to remove the contaminating multi-elements in heparin. This lack of clarity as to the chemical nature of heparin creates a possible source of quality control error and could account for at least some of the problems reported in the literature over the years in reproducing the biological effects of heparin (e.g. as has emerged from angiogenesis research)}. Cf. Grant D. (Uploaded 10 October 2000 at 10:47 GMT) Multi-Ion Content of Heparin CPS biochem/0010002 This paper reported the 38 element content of sodium heparin in standard and sodium cation heparin percolated through an thallium (Tl) ion exchange column to give Tl- enriched heparin. The exchange of Na and non-Na elements with Tl allows comparison of the relative binding strengths of various inorganic elements with heparin. The presence of redox active elements in heparin (and putatively also heparan sulphate (HS) seem most likely to control the rate of nitrosative scission). This was the subject of seminars at the Univeristy of Glasgow in 1988/9 which are briefly reported in this CPS paper. Calcium Heparin Preparation Using Phase Change Mechanism

Whereas prior art had used reversible cation exchange processes in which the exchange of countercations being reversible intrinsically always gave rise to the kind mixed salt

form (e.g. similar to all element blood serum or seawater cf. Haraguchi loc. cit.); if a phase separation method were employed, however, this could achieve an improved pharmaceutical grade singly counterion type of heparin preparation. Cf. Process for the preparation of heparin salts: (UK Patent Application GB2 176 200 A Jun 1986 Kerey G. et al. [Budapest Hungary]). A high yield of calcium or other heparinate could only be prepared by the use of an irreversible phase separation process especially that achieved by forming the insoluble quaternary ammonium heparinates as a primary product and then converting these into the desired salt forms.
Calcium heparinate (e.g. containing less than 1000ppm Na) which had been prepared in this manner, was considered to be a more therapeutically effective heparin than the salt form which contains sodium as the principal countercation (and which had been the almost exclusively prior used form of heparin for blood anticoagulation but which had also been associated with the occurrence of hemorrhaging; this undesirable side-effect had been found (by Kakkar VV) to be less apparent when calcium heparin had been employed as a blood anticoagulant.

b
The protein-only theory of TSE, although commonly accepted as being an approximation to reality has also been indicated to be incomplete. Other cofactors seem to contribute to the etiologies of TSEs. Cf., The role of ionic strength and transition metals in PrPSC conversion-inducting activity (and protease resisance.

b-1
The Transmissible Spongiform Encephalopathies (TSE)-related work of Mark Purdey Mark Purdey had drawn attention to metal ion dyshomeostasis and intoxication as having a possible role in the aetiologies of TSEs and also multiple sclerosis (MS). Purdey originally proposed an organophophosphate hypotheis of prion misfolding and obtained an apparent experimental confirmation of this hypothesis by use of cell culture experimentation. (Purdey later proposed a metal microcrystal nucleator hypothesis of prion misfolding; cf. the internet document entitled Draft Abstract of Chapter for Trends in Prion Research By Mark Purdey (downloaded from internet 19 August 2005 23:39) who wrote This chapter charters the flaws in the conventional consensus on the aetiology of (TSEs) which decrees that the protein-only misfolded prion represents the primary causal agent. Purdey continued with an alternative hypothesis: PrP-ferritin fibril crystals initiate TSEs by a process also involving Sr, Ba and Ag present in soils and foodchains. Purdey had earlier proposed (Medical Hypotheses. 2004 62 746-754) that multiple sclerosis (MS) arose from a disruption of heparan sulfate (HS) signalling following Ba intoxication (Grant D. correspondence and telephone conversations with Mark Purdey). Cf. also Purdey M. Medical Hypotheses. 2000 54 (2) 278-306, p 280 where in Fig.1, a Mn intoxication scenario involving a multifactorial aetiological template mechanism pathogenesis of sporadic TSEs was hypothesized; this involved the formation of Mn3+ and binding to cellular prion protein as a key event. This Mn3+ is suggested to initiate chain reactions of auto oxidation involving multi site radical attack on PrP and other CNS membrane/cytoskeletal proteins. Cu deficiency was believed to represent a key factor for allowing the formation of Mn3+ .

b-1-1
Old Internet-Posted Hypotheses from Mark and Nigel Purdey Cf.,

Mark Purdeys Organophosphate and BSE Page. htm Cf. The NRJ Purdey Environmental Home Page A Nov 2002 download dealt with hypotheses relating to a range of enviornmentallyinduced pathologies [cf. organophosphates, BSE, CJD, manganese, copper, scrapie, prions, chronic fatigue syndrome, pesticides, industrial pollutants soils and diet). Cf. The manganese loaded copper depleted bovine brain fails to neutralise incoming shockbursts of low frequency infrasound: the origins of BSE? Purdey M in Cattle Practice 2002 10 (4), suggested that TSEs will only emerge in those infrasound-rich environments which are simultaneously predisposed to specific environmental factors that induce a high Mn/low Cu /low Zn ratio in brains of local mammalian populations. It was proposed that PrP Cu component performed a role in the conduction and distribution of electromagnetic energy transduced from incoming ultraviolet, acoustic, geomagentic etc. sources. TSE pathology was initiated once Mn replaced Cu whereupon the piezoelectric Mn atoms absorbed and blockaded that energy flow instead of conducting it. The epidemic of BSE in the UK (was suggested to have) resulted from the combined simultaneous exposure of the bovine to three environmental factors: Cu chelating insecticides, Mn inclusion in milk replacer/mineral blocks, and intense infrasonic shock waves from trubojet aircraft. Compulsory, exclusive high dose formulation of systemic phosomet warblecides penetrated the CNS and deprived PrP of its Cu component, enabling the excesses of Mn to substitute at the vacant Cu domain on PrPC resulting in the formation of a non-pathogenic, protease resistant trivalent Mn3+ PrP isoform. The final stage of pathogenesis was considered to come into play once a low frequency wave of infrasonic shock metamorphosed the atomic structure of the Mn3+ component of the prion thereby priming the sleeping prion into its fully fledged, pathogenic TSE isoform - where the paramagnetic status of the Mn3+ atom become transformed into a stable ferrimagnetic lattice work, due to the strong electron-phonon coupling, specific to the trivalent Mn species. I.e. prion infectivity was a misnomer and was more correctly (should be) defined as the magnetic/ reactive free radical generating capacity of the Mn3+ component of the prion. Cf. also Bounias M, Purdey M Sci Tot Environ 2002 297 (103) 1-19 and The Ecologist Nov 2002 (High dose exposure to phosmet ..) Med Hypth 1998 (Ecosystems supporting clusters) ibid., 2000 (Does an ultraviolet photooxidation of the manganese-loaded-copper-depleted prion protein in the retina initiate the pathogenesis of TSE? ) ibid., 2001

b-2 Metal Ions in Prion Diseases

Copper may have at least two roles in HS modulation of prion activity by(1) acting as a co-catalysts for oligomer HS formation (via nitrosative deaminative cleavage {n.b. there seems to be evidence of reduced nitric oxide signalling in prion diseases}) and (2) by stabilizing prion HS-complex formation. Cf. the latter mechanism was evidenced by R. Gonzlez-Iglesias, MA Pajares C Ocal JC Espinosa B Oesch M Gasset

Prion protein interaction with glycosaminoglycan occurs with the formation of oligomeric complexes staiblized by Cu(II) bridges

J. Mol. Biol. 2002 319 527-540 The authors start off by noting that prion diseases are fatal neurodegenerative disorders characterized by an aberrant metabolism of the cellular prion proteins (PrPC) resulting in the generation of an ensemble of alternative conformers displaying lethal self-propagating properties. The process by which PrPC converts into scrapie PrP (PrPSc) has been ascribed to a conformational change in which a helix-rich structure rearranges into a high -sheet form. This transition apparently promotes a change in the association state and precludes the protein clearance in vivo. Until recently, prion accumulation was thought to be an irreversible process. However blockage of the precursor PrPC supply revealed that prions are dynamic assembles exhibiting a prolonged but real turnover. These findings have changed the perception of the degree of participation of PrPC in prion diseases, being now considered both the prion precursor and the determinant of its perpetuation.
The authors suggest that GAGs are likely physiological ligands for prion proteins via the octarepeat region of the prion; this interaction seems to be boosted by the formation of oligomers crosslinked by Cu(II). N.b. The His residues in the prion octarepeats are potential Cu(II) ligands. The other cations which can substitute for Cu(II) probably include Zn(II) [but not Ni(II)]. The authors were aware that GAGs are also likely controllers of misfolded prion clearance. Defect of such a GAG-determined mechanism was therefore at least in part the likely basis of prion diseases and explained why administration of suitable GAG molecules (e.g. pentosan polysulphate SP53 which is a mimic of HS oligomer, e.g. as produced by Cu-dependent NO scission) can putatively reverse the progression of prion diseases. [It should be noted that glycosylphosphatidyinositol-linked caeruloplasmin was reported) (Mani K et al. J. Biol. Chem. 2004 279 (13) 1298-23) to be involved in the copper/zinc nitric oxide- dependent degradation of glypican-1 heparan sulfate (studied in rat C6 glioma cells)]. {This process may produce hormone-like HS oligosaccharide messengers}.

Supattopone S. et al. (using an ultra-efficient in vitro PMCA [PCA for DNA-like] replication
method demonstrated that Cu(II) and Zn(II) (etc.) and polyanions (e.g. GAGs and nucleic acids) could greatly enhance the rate of conversion of PrPC to PrPSc. Poyanions were later, however found not to be essential for this process (Piro JR and Supattapone S. Prion 2011 5(2) 49-51. Purified PrPC apparently contains a stoichiometric amount of co-purified lipid. This may be a critical cofactor needed to generate replication and disease. e.g. possibly implicating abnormal hydrophobicity which in turn seems to point to a Wiggins HDW/LDW/HDW driven pathology. The traditional (Prusiner) protein-only (prion-only) hypothesis of the nature of the infective agent seems actually to be incorrect. Later studies by Supattapone et al. (Delaut N.R. et al. PNAS. 2012 109 (22) 8546-51) indicated that endogenous brain phosphatidylethanolamine (PE) was the likely natural brain agent which facilitated the conversion of PrPC to PrPSc in vivo. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrPSc molecules. Such cofactor molecules can regulate the defining features of mammalian prions viz. PrPSc conformation, infectivity and strain propagation. Thus such cofactor molecules are integral components of infectious prions. [Cf. PMID 22711839]. While the exact role of polyanions including HS in the mechanism of prion disease is still unclear HS etc. may act as scaffolds to bring PrC and PrSc together to allow templating to occur as well as by acting as catalyst for the refolding process. This ability of HS to regulate phopholipase activity may be highly relevant, also however.

The above results suggests that the ability of HS polyanions (or mimetics such as pentosan polysulfate SP54) to modulate phospholipase activity could be a critical anti-TSE function of HS and mimetics and be relevant to a fuller understanding of why pentosan polysulfate is apparently a successful therapeutic agent for human TSEs. (Cf. Roger Highfield Dramatic results of CJD treatment Telegraph .co.uk 12:01am GMT 10/11/2007; {this CJD patient was sadly reported to have died suddenly and unexpectedly in 2011}). [Other phospholipids and related substances were unable to achieve the conversion in the absence of RNA]. (Previously it had been shown that this misfolded conversion could be accomplished in vitro by RNA and lipid molecules but later studies indicated that nucleic acids were not essential for this conversion and there must have existed an alternative propagation cofactor in brain tissue).

The Wiggins Theory of Liquid Water is Highly Relevant to Prion Misfolding

This asserts that high and low density forms of water molecule hydrogen-bonded aggregates [i.e. HDW and LDW] occur and these generate specific water microstructure variations. This affects the overall density (and can be directly measured) as well as vibrational spectrocospy determined H-O bands etc. Water activity is held to depend on the HDW/LDW ratio.
The HDW and LDW microdomains which are believed to exist are considered to be in thermodynamic equilibrium which can be perturbed by the presence of solute molecules.

The Wiggins theory proposes that a hydrophobic molecule such as the cellular prion protein PrPC can (if released from cells) accumulate into HDW where it will induce the formation of LDW. Protein folding depends on water structure determined by HDW/LDW. The misfolding of prion proteins can therefore putatively arise as a consequence of the alteration of HDW/LDW.
Whereas in bulk solution such an accumulation has a thermodynamic cost which is offset in principle by the process of correct folding, this folding may however occur in an inappropriate manner, and this process is putatively enabled by the accumulation of HDW. This relates normal-pathological conditions to a switchover from LDW to HDW.

Silica (Gel) derived life relevant to Wiggins Theory silica pore generation of HDW/LDW
The final form of the Philippa M. Wiggins HDW/LDW theory (PloS ONE 2008 paper, should, it is suggested, be read in conjunction an earlier Wiggins paper which reported on silica gel; the key to the final outcome, the LDW/HDW theory seems to have been generated by Wiggins studies of both inorganic and organic high water content gels. It should be noted that both inorganic and organic gels can contain large amounts (>98%w/w) of water. Inorganic gels were perhaps thought inappropriate to include in the final PloS ONE 2008 discussion which were restricted entirely to organic gels. Such gels are familiar to the target academic biochemist reader of this paper, and for them only organic gels are credible mimic of biological systems and for whom inorganic gel studies can be considered to have been censored by the commercial use of such gels in industrial processes. Purely inorganic gels are however of interest as conceivably offering an useful hypothesis of the ultimate origin of life. This is of especial interest to the future search for extraterrestrial forms of (early or primitive) life. It is now pointed out that the groundbreaking investigations of such gels reported in the Wiggins paper (Biophys. J. 1973 13 (4) 385-98) are highly pertinent to pre-biotic research; this

study illustrates the unusual behaviour of water (e.g. in regard to its ability to select between inorganic ions) in silica gel. This paper was apparently left out of the 1995 (Prog. Polym. Sci. 20 1121-63) and later discussions on LDW/HDW. It might have been assumed that purely organic gels are considered to be more relevant to how water drives biology (which is assumed to be a phenomenon which is restricted to nucleic acid driven cells composed on organic molecule including polymer and constituted operating systems). This is I think inappropriate since inorganic silicon putatively in a form resembling hydrated silica gel is known to be an essential component of the nutrition for human and other animal as well as other species. (Silica gel can be formed with an ultra high water content likely because of the tetrahedral linking similarities between liquid water and silica). The organic polymer gel water discussion in High and low density water in gels Prog. Polym. Sci. 1995 20 1121-63 describes the experimental development of the theory HDW/LDW and acknowledges the work of G.W. Robinson et al. as being the origin of the idea. Other parts of this paper to be underlined include e.g. cf. on p. 11130m A gel in contact within a solution looses water when a soluble polymer too large to penetrate its interstices is added .Wiggins and van Ryn found that the density of water in Biogel P-100 beads decreased down to 0.96 g ml-1 with increasing osmotic stress imposed by polyethylene glycol 20M, Table I on p. 1131 links entropies of hydration and the degree of selectivity of gels for these ions, and notes that: the solvent properties of LDW are at least partially entropy-driven. The development of full LDW (phase formation) was also sometime irreproducible and very slow (needing days) and also showed (day-scale) oscillations in the deduced internal volume changes (putatively due to LDW/HDW ratio oscillations). The LDW/HDW balance seems to involve phase change and (deterministic chaos?) ; (furthermore it seems that extrathermodynamic rules apply here, e.g. those which establish entropy-enthalpy compensation and which further (putatively) allow breach of thermodynamic restriction on entropy flow vs. time which promote order to increase with time as is a critical hallmark of biology. Also of note is the phenomenon by which formally negatively and positively charged surfaces behave similarly to each other and the water association effect which allows highly negatively charged sulphate anion coated surfaces to seem to bind avidly to negatively charged counterions a circumstance which counters the previously held (Manning) electrostatic origin of this binding.. c

Haraguchi Metallomics
The groundbreaking paper: Metallomics as integrated biometal science [Haraguchi H. in J Anal At Spectrom. 2004 19 5-14] discussed inter alia the similarity between seawater and (human) blood serum as regards both essential and non-essential inorganic element contents; this review paper suggests a new science of metallomics (which broadens the concept of metallomics, which had originally been introduced by R.J.P. Williams to include all of the inorganic elements which occur in seawater (n.b. this includes most of those in the Periodic Table); a corollary to this finding is that the supposedly non-physiological elements actually play some (as yet undiscovered) role in animal and other higher species physiologies most probably, it may be assumed (from consideration of the implications of the Wiggins [loc. cit. theory] as a consequence of the modulation of HDW/LDW by the presence of ions of such multi-inorganic-elements ion which co-occur in all natural fluids [i.e. intracellular, extracellular and habitat aqueous solutions]. Some of the elements (perhaps especially Hg and Cd and to some extent also Al and Pb) may be almost entirely pathological in their effect on HDW/LDW. The (Aberdeen University) experimentally-observed occurrence of H/HS (ubiquitous surface components of adherent animal cells) as existing (putatively naturally) in (Haraguchi-type) multiinorganic element matrices is likely therefore to be relevant to a more complete definition of metallomics and the mechanism of metal ion influence on physiological water structure; (hitherto [Williams-] metallomics had largely been dedicated to the study of those metal ions which are required for protein function and this theme rather than the wider field of how metal ions and biological anions regulate water structure was the previous mainstream inorganic biochemical intellectual focus). Each metal or other inorganic ion at the surface of H/HS will, it is now predicted, elicit a particular HDW/LDW supramolecular structure and energy profile. This creates life. This also pertains to pathological disturbances of vital processes.

A possible primitive additional function of HS (PG) and GAG PGs is to act as a filter to sequester engulf and protect against toxic inorganic ions (and their ability to adversely affect water structure). This may be coupled to the recycling of HS and GAGs and the removal of toxic ions.
Mercury Intoxication & H/HS

Compared with MerR (the Hg(II) binding regulator) HS is probably a much lower affinity but less specific metal binding ligand but perhaps this is more flexible and can achieve high binding strength by phase change. Putative (Potentially) Toxic Counterion Impurities in (Some) Heparin (Preparations) Cf. Boher D. et al. [RBAC. 2004 36 (2) 99-103; Aspectos importantes na determinao do nvel de alumnio no sangue de pacientes em tratamento regular de hemodilise{Aluminium level in serum anlysis and its importance in hemodialyis treatment}] reported that some commercial heparins may contain up to 22ppm Al which can easily be removed using a cation exchange resin percolation e.g. before being employed as anticoagulant for hemodyalysis).
Metal-Proteoglycan Interactions in the Regulation of Renal Mesangial Cells: Impliciations for Metal Induced Nephropathy

Cf. the work of Templeton D.M. Reported in Proceedings Trace Element Health Disease 1991 pp. 209-218 Cambridge UK Royal Society of Chemistry
Aito A Ed. Proc J NORD TRACE ELEM SOC/UNION PURE APPL. CHEM. INT. SYMP. 1990 Source Chemical Abstracts. 111 12940712 The author hypothesized that proteoglycans (PGs)(an important class of growth regulators in some if not all adherent animal cells) were subject to charge reduction as a consequence of metal ion binding. This may especially affect the function of the renal glomerulus. The presence of (toxic) divalent metals in the kidney included an alteration of biosynthesis, secretion and anti-mitogenic properties of PGs. The NaCl gradient separation of glomerular PGs showed diminution of both HS-PG and dermatan sulphate -PG by addition of divalent ions of the following metals (Ni<Hg<Cu<Cd) (lesser effects were however produced by divalent ions of Mn, Co, Ni and Zn) [the latter PG was present in greater amounts in absence of e.g. Cd but in lesser amounts in the presence of Cd {this effect may be caused by diminution of core protein synthesis and (perhaps also partly or predominantly as suggested by reports from other workers) by alteration of polysaccharide biosynthesis]. HS is believed to control cell proliferation and determine mesangial cell quiescence. Regulation of glomerular filtration mesangial cells was evidently subject to a major effect of the diminution of dermatan sulphate PG contents by Cu, Hg and Cd with a corresponding enhancement of HS-PG. Additionally there was an especial effect of Ni which inhibited the insertion of HS-PG into the glomerular basement membrane

Silica sol replication

A cellular reproduction process which had apparently occurred during the seeded growth of silica sols in a silicic acid medium offered an explanation of light scattering results observed during experiments made to model conventional industrial nanotechnological growth of amorphous silica sols on a heel from the previous batch; it was well-established by plant technicians that a heel from the previous batch was indeed an essential component of the process but this had been supposed by chemical engineers involved to be an essential part of the quality control only because it critically

improved the heat transfer processes which were needed to achieve a correct silicic acid aggregation into the desired type of sol particle. I reinterpreted the need for this heel on the basis of a light scattering (plus other physiochemical evaluation) based re-evaluations (using a laboratory scale model of the plant and a modification of this) the formation of particular kinds of SiO2 sols could depend on the occurrence of a specific microstructure generating seeding mechanism similar to that which operates when crystals are grown from seeds; the growing sol particles seemed critically also to undergo some kind of simultaneous scission or deaggregation (which might have generated new seeds) which seemed to evoke some similarity between the process of the creation of specific types (species) of sol particles; this process which somehow mimicked biological reproduction (cf. Grant D et al. Medical Hypoth. 1992 38 46-48 which briefly mentioned this idea if somewhat obliquely). Later correspondence about this idea with concerned parties and others was apparently met with (mild) dis-approval but the new idea about sol replication seemed to be of especially most positively accepted by scientists at the polysaccharide lab in Aberdeen University during the 1980s. The origin-of-life-from-SiO2-sols-idea was also the subject of an answered 2007 letter from Grant D. to (the Nobel laureate) Arthur Kornberg {I had mentioned in my letter that I had worked in J.R. Van Wazers lab. in St Louis (cf. e.g. Grant D. et al. J. Polym. Sci. A1 1967 5 57-75); this industrial scientist had previously become well known as the author of Phosphorus and its Compounds a groundbreaking treatise on phosphorus chemistry}. Kornberg (to a greater extent than Cairns-Smith seemed to show some positive interest in the SiO2 sol idea of the origin of life in Earth perhaps; Kornberg, it should be noted, had later in his career become interested in the idea that poly-inorganic phosphate, which is known to currently exist at the surfaces of numerous cells throughout biota, had played a critically central role in pre-biotic evolution (cf. e.g., Brown M.R.W. & Kornberg A. PNAS. 2004 101 (46) 16085-7); my (brief) communication with Arthur Kornberg generated an additional idea to that earlier proposed (in Grant D. Medical Hypothesis article (vide supra)): selfreplicating cellular particles of (silica (SiO2) sol water pore cum (poly) phosphate (in the pores) might more a realistically provide a possibly more relevant model for the kind of early pre-biotic cellular reproduction which eventually might have evolved into the current ATP-based system which Wiggins further showed is likely centrally dependent on the energy transduction mechanism afforded by her LDW/HDW/LDWdependent phosphorylation; this abiotic phosphorylation could have affected of a range of pre-biotic organic substances (which, may have, early-on, included carboxylated polymethylene humic-like units (cf. Grant D. Nature. 1977 270 709-10) and the range of small organic molecules which occur in space and their chemical transformation products enabled by the energetic LDW/HDW/LDW within SiO2 sol pores; such water induced chemical transformation allows energy for the self-replicating pre-biomorphs to move around and evolve by competition for nutrients etc. and eventually to get more complex with time [but only at much later time might similar activities plus evolutionary pressure generate the evolution of sugars , polysaccharides, proteins, their matching RNA and finally DNA].)
It should also be noted that in 1973 Phillipa M. Wiggins (who later published the final-in series paper Life depends on two kinds of water in PloS ONE 2008 e1406) had, early-on in her researches, also studied the chemical nature of the water structure in (the completely inorganic) pores present in silica gel, finding that such pore water structures were qualitatively similar to those present in most living cell membranes (e.g. in the pores therein are the same for both living and non-living pore-bearing systems); both show a critical-for-biology behaviour of having an increased solvent power for water-structure-breaking ions (cf. K+ is water structurebreaking {as also are N+ based ions) and a decreased solvent power for water-structure-making

ions (especially Na+, Ca2+, Mg2+ and H+). It seems that pore-water-structure-effects in (the completely inorganic) silica gel pores could allow for a similar-to-biological-cell-type ionic selectivity to be accomplished; (This SiO2-inorganic-ion-selectivity was found by Wiggins to be broken by urea, this confirming that it likely arose entirely from an altered water structure). This inorganic ion selectivity provides a mechanism for the distribution of Na+ and K+ between extra and intracellular spaces in both conventional biological cells as well as in those pre-biotic cell like particles which putatively could mimic biological cells in several ways (e.g., it is now suggested in the proteinoid spheres {Fox S., New Scientist. 1969 450-2} and the (selfreplicating?) hydrated pore-containing SiO2 sols {Grant D. et al. Med. Hypoth. 1992 46). True biology and quasi biology (which might have been the precursor of the former) seem to be determined primarily by the extra and intracellular difference in the chemical forms of water. Life-like properties must presumably have evolved first of all in some kind of water-borne prebiotic cells; [Fox proposed that such abiotic cells comprising poly - D- +L- amino acids could have formed at siliceous hot rock surfaces; he produced racaemic poly-amino acids in this manner and found that they gave electron micrographs which resembled bacteria and which also could bud and split i.e. reproduce, seemingly in a similar manner to that which could also have occurred with self replicating SiO2 sols, vide supra}. This idea seems to somehow accord with how energy could arise for the process of cell breakup etc. at a critical silica sol size as suggested in a 1973 paper by Wiggins P.M. Ionic partition between surface and bulk water in silica gel (Biophys. J. 14(4) 385-98) [this seems to have anticipated development of the later Wiggins theory of the low density water/high density water (LDW/HDW) balance suggested by the cellulase acetate (cf. Wiggins and van Ryan 1986 {cf. Wiggins P.M., Cell Mol. Biol. 2001 47 (5) 735-44}) and polysaccharide gel studies by this mechanism as being due to the critical chemical structure of liquid water which allow biologically-relevant pores to engage in ion selection via pore accumulation of LDW which also is the energy generator of the ATP + enzyme ion pumps via the LDW-HDW gradient which these facilitate]. LDW is chemical-energy-endowed (according to Wiggins) to transform ADP plus Pi into ATP. This mechanism (according to Wiggins) is how ATP becomes regenerated enzymatically in vivo. This phenomenon which could have arisen naturally first of all in inorganic colloids (e.g. consisting of SiO2 particles) is, of course, retained in modern organisms and is one of the hallmarks of biological cells. It should be noted that inorganic Si(O2)(H2O)n shows up in biological systems attached to all polyuronides. Purely inorganic polyphosphate (in complex with Ca2+ and probably also polyhydroxy butyrate) also shows up on all (or most) biological cells. A possible primary function of these inorganic surface modifiers could be to modify the HDW/LDW equilibrium. Schematic Summary of Pre-Biotic Evolutionary Process Hypothesis Key Role of LDW in Pores within Silica (Sols)

Notes on Reprints of papersa on High Density Water/Low Density Water (HDW/LDW), kindly provided by Philippa M Wiggins (Genesis Research and Development Corporation Limited Auckland, formerly of Department of Medicine University of Auckland)
Received by David Grant Turriff UK on February 10 2007 in response to request made in a letter of January 24 2007

sent together with a paper originating from Aberdeen University {re: possible roles of silica sols in prebiotic evolution and modern pathology which might indicate that water structure elicited by such sol particles is relevant to these topics)}.

Cf. also the previous interest of the author in the subject of how water structure might be modified by polyanions acting in concert with metal ions cf., Grant D. et al., Biochem. Soc. Trans. 11, 96 where it was noted that in near infrared spectra of

heparinates The first overtone region 6300-7400cm-1 , of the H-O stretchcan be interpreted in terms of two broad classes of water structure 1) Philippa M. Wiggins High and low density water and resting, active and transformed cells Cell Biology International. 1996 Vol. 20, No. 6, 429-435 {This paper suggests that resting and active cells differ in their (metastable, short- lived individual water molecule environment; two broad classes of water structure designated (high density water) HDW and (low density water) LDW and the HDW/LDW balance may be suggested to play a central role in normal physiology but become greatly altered in cellular transformation and death (n.b. this water structure, its formation at charged polymer surfaces, and its alterations (as well as its kinetic and thermodynamic properties and abilities to selectively discriminate between inorganic ions and small molecules) are putatively the principal driving forces which caused biology to come into existence and also, at the most basic level to perform essential

Wiggins hypothesized that LDW predominates in the resting cell and is converted to HDW in the active cell.
(It seems that THE SWITCHING RATE BETWEEN HIGH AND LOW DENSITY WATER IS CRITICALLY IMPORTANT. Putatively this switching process in water structure hydrogen-bonding an integral part of cellular function but this ability is lost when cells are transformed either to a state of rigor or to a hyperactive state (i.e. the process which creates neoplasia) The mechanism of REGULATION OF HDW/LDW is then putatively OF CENTRAL RELEVANCE TO CELLULAR TRANSFORMATION). functions, and is therefore also of central relevance to pathology).
[This paper reviewed the Wiggins high density water/low density water (HDW/LDW) water structure theory which postulates that HDW/LDW gradients occur in the intraand extracellular matrices of proteins and other biopolymers.
Fig 1 illustrated a protein in solution together with its both positive and negative counter ions which move through the solution with the protein creating an osmolaility gradient and an associated water density gradient Figs. 2 and 3 in this paper indicated that

the charged (hydrophilic) surfaces attract HDW {which forms a thin layer} which then, further out from these surfaces, induces the formation of LDW which tends to form a still thicker (growing?) layer; The mobility of the dissolved polyelectrolyte causes it to shed the LDW and HDW from its surface so limiting the surface growth of these phases. This shedding is less when the polyelectrolyte forms a gel. (so that e.g. weakly charged polyamide gels ended up containing mainly LDW at their external and abundant internal {pore} surfaces).
around a diffusing polyelectrolyte containing

Hydrophobic surfaces, on the other hand, attracted (only or mostly) LDW. The above two phase chemical nature of liquid water encourages the self-assembly of solute or dispersed hydrophobic moieties (or the induced protein folding) to occur therein in which the LDW formed at both hydrophobic and hydrophilic surfaces becomes the dominant {potential energy} force.
Dextran sulphate dissolved in water (which because it can attain the same water content as a cell or extracellular matrix can be considered to be a simple in vitro model of the in vivo microdomains occurring, e.g., in cytoplasm) was shown to undergo the above type of gel phase separation via the HDW/LDW microosmotic force which can be generated by the addition of KCl (but not NaCl) above a critical electrolyte concentration. The dextran sulphate molecules aggregated into a gel phase and expelled water

(including some high activity water) but the later addition of NaCl was shown to reverse this by selectively partitioning into the low-activity HDW. It should be noted that Na+ is a cell activity stimulating ion (Ca2+ also elicits it biological signalling by acting via water structure change) achieved ultimately by altering the HDW/LDW ratio. Rigor arises when ATP is exhausted and Ca2+ cannot be transported back to the sarcoplasmic reticulum causing a state of extreme and irreversible rigidity, this is especially the result of the expulsion of large amounts of water from cellular bio-gels. N.b. LDW is high enthalpy {high activity} water (which is of lower density and is more strongly hydrogen-bonded than normal water). HDW is low enthalpy {low activity} water (which is of higher density and is less strongly hydrogen-bonded than normal water). HDW is also highly fluid. HDW is extremely reactive. The accumulation of hydrophobic carcinogens was suggested to transform cells into an irreversibly active state which contains more water and more Na+ than occurs in normal cells. Movement of proteins etc. causes structured water shedding. This creates a dynamic system. The Gibbs free energy TS term enables self assembling protein microstructuring to occur via thermodynamic forces generated via water micro osmotic forces which arise from stress created by water density decrease in the zones of high water activity. [N.b. not discussed by Wiggins in her papers: Reactions conducted in water dispersant also show changes in reaction rate constant enthalpy terms which are often exactly compensated for by changes in entropy; this phenomenon can be argued to allow for selective breaches of the second law of thermodynamics which can permit entropy to increase with time and therefore allows biological evolution to occur. This phenomenon which, could depend on the existence of HDW/LDW in liquid water, tends to add weight to the Wiggins Theory of (the origin and continued critical relevance to the functioning) of biology].

The Wiggins Theory of Liquid Water Structure is truly revolutionary in that it requires that the classical theory of osmosis (formulated assuming a single state liquid phase in water) be dramatically modified to accommodate HDW/LDW especially how the conversion of some LDW domains into HDW and some HDW domains into LDW, creating microdomain areas. Increase in the density and chemical potential of water gives rise to micro-omosis which is the ultimate basis of enzymic action in muscle function (vide infra). Cf. 2) Philippa M. Wiggins Enzyme reactions and two-state water Journal of Biological Physics and Chemistry. 2002 2 25-37 Robinson G.W. et al., (as well as Stanley H.E. et al. and Chaplin M.F.{amongst others, carried forward a hypothesis which had been first discussed by Rntgen W.K. (Ann Phys Chem N.F. 1892 45 91-7}) which had proposed a model for liquid water involving two phases (later considered to be a rapidly exchanging equilibrium between microdomains). These ideas were adapted by Wiggins who proposed a high and low density liquid water (HDW and LDW) theory. LDW seemed to resemble ice 1h with an open structure while HDW seemed to be a collapsed version of LDW with bent, weaker hydrogen-bonds. Wiggins et al. also, importantly, experimentally demonstrated that HDW has a density of ~ 1.1.8g/ml; furthermore LDW has a lower entropy than HDW, which had lower enthalpy than LDW. The Wiggins HDW/LDW theory extends the ideas of Ling, G.N. who e.g. in A Physical Theory of the Living State: the Association Induction Hypothesis [New York Blaisdell Publishing Co., 1962] had proposed that polarized structured water multilayers are important determinants of cellular homeostasis and of George, P. et al. who, in An enquiry into the importance of solvation effects in phosphate ester and anhydride reactions (Biochim. Biophys. Acta. 1970 1-15) argued that the actions of ATP are achieved via the formation of phosphorylated surfaces which attain different water structures. Wiggins extended the prior theories of the role of water structure in biology, noting that Biochemistry is the original nanotechnology: all reactions take place within a nm or so of a protein surface in contact with water.

Altered water chemistry at surfaces allows for the biologically-observed ion selectivities. The creation of osmotic pressure gradients associated with the induction either LDW or HDW is the fundamental inorganic chemical process which evidently permits biology to arise. The creation of osmotic pressure gradients and the essential for life HDW/LDW systems can apparently be achieved by a wide range of inducers, including silica gels or sols [SiO2(H2O)n] (such gels and sols can exhibit a wide variety of forms and can attain very high water contents evidently enabled because of the isostructural resemblances between networks of silicate tetrahedra and hydrogen-bonded networks of water molecules). {Although the induction of HDW/LDW by purely inorganic highly hydrated systems were not mentioned in Wiggins in paper 2 (which is restricted to explaining how protein-based enzymes function via the existence of an associated water-structure) the circumstance that a similar-to-biology HDW/LDW system can arise in a purely inorganic system because of the ability of such a system to imbibe and modify a very large associated water contents seems surely of critical relevance to how life on Earth or elsewhere might have arisen by a process which initially does not require the presence of organic compounds}. [It should be noted that Wiggins et al. had previously studied the purely inorganic (silica gel ) chemical induction of HDW/LDW and reported the results in a major peer-reviewed journal (Biophys J. 1974 385). I had alluded to this groundbreaking work in my letter of 24 January 2007 showed at the beginning of this document] This earlier, 1974, research by Wiggins is now suggested to be especially relevant to the idea that Life depends on two kinds of water (cf. the review PLoS ONE. 2008 3(1) e1406 paper) since life in its most general form cannot then be restricted only to those systems which contain specific organic substances such as nucleic acids or indeed carbon-based compounds. Such ideas were also discussed e.g. by Cairns-Smith A.G. and Luck W.A.P. The idea that a primitive form of life can be based entirely on high water content soft gel SiO2(H2O)n systems which can induce discriminated water structure (potential energy systems) seems highly credible and can explain why natural and artificially produced silica sols seem to behave in some respects like biological systems (cf, Grant D. Med. Hypoth. 1992 38 46-8). [The research results in this field are, however, not widely known]. In paper 2) Wiggins suggested that the necessary-for-biology water-structuring could arise by five mechanisms, viz.: l) the surface effects which induce HDW directly at the surface and LDW further out; 2) the effect of counterions in a zone adjacent to the biological surfaces which create another osmotic pressure gradient similar to 1) but which is independent of the chemistry of the surface moieties (other than their electrical charge) and the chemistry of the counterion; 3) induction of HDW in the double layer by the efect of the fixed charges on biopolymers which are strong chaotropes; 4) by the the water structure modifying chaotropic effect of the principal intracellular countercation, K+ (which is pumped into the cell by the Na,K-ATPase pump, {which also pumps Na+ out of the cell}); 5) by a change in the thickness of the double layer (effected by added solutes {which produce otherthan-counterion-effects, by altering the dielectric constant which decrease the excess concentrations of counterions and therefore the osmotic pressure gradients}). How Ion Channels Work via Water Structure Change Putatively, micro-osmosis is the ultimate mechanism of ion channel action. K+ and anion accumulate in pores containing LDW; this converts the LDW into HDW this is then becomes an open channel for kosmotropic ions. If there is a filter region at the entry to the pore, which is specific for Na+, Na+ then diffuses spontaneously into the cell (followed by an anion through an anion channel to maintain electroneutrality); the K+ and the anion which opened the channel for Na+ then diffuses out having destroyed the LDW which attracted it in in the first place. If the filter region were specific for K+ this channel might also serve as a K+ channel. In experiments with mouse embryos, it was found that solutions devoid of chaotropic anions and cations induced a state of dormancy in the cells by inhibiting metabolism (e.g. without K+ the Na,KATP ase was inhibited and univalent anion Na+ and Ca2+ channels remained closed. Raffinose, which was too large to enter ion channels, also tended to keep them closed).

How ATP Provides Biological Energy via Water-Structure Change The conventional view of the mechanism of biological energy that it is achieved by a change in enzyme shape (conformation) following its phosphorylation by ATP was re-assessed by Wiggins in terms of changed hydration following surface phosphorylation. Philippa Wiggins found experimental evidence for a role for water structure in ATP action. I.e. this (quite different from the traditional model) idea provides a putative modus operandi for ATP now suggests that all biological work is actually accomplished via the change in protein hydration caused by phosphorylation of the enzyme surfaces. The negatively charge phosphorylated surfaces induce low density water [LDW] which further induces the formation of high density water [HDW]. This phenomenon can be evidenced at all kinds of charged surfaces (including non-biological ones, e.g. hydrophobic glass beads [and, as described in her earlier Biophys. J. 1974 385, silica gel structured water paper, in the pores which occur in siica gel]).

[Mg2+ was believed to act in conjunction with ATP in the following water-structuredependent mechanism.].
When MgATP phosphorylates proteins on a hydrophilic residue, the total number of negative charges in the cell does not change, but a charge is transferred from the MgATP complex to a protein. In the MgATP complex, Mg2+ neutralizes two negative charges, and Mg2+ and ATP move through the solution together. When however, the charge is on the surface of a protein, it moves with the protein, i.e. the Mg2+ is relatively immobilized at the surface. Mg2+ remains as counter-cation because of its very high affinity for HDW and because of its double charge. Locally, therefore the combination of osmotic pressure gradients leads to a large zone of LDW. The reversal of ATP hydrolysis ([K+ , LDW] + Pi + ADP ATP) was demonstrated to occur in dense films of cellulose acetate Rapid hydrolysis of biopolymers (e.g. proteins and DNA) can also be accomplished by HDW alone which occurs closely adjacent to vely charged surfaces with divalent (e.g. Ca2+) counterions. (Cf. many proteases are Ca2+ activated). HDW seems to exist, e.g. in the first hydration cell of lysozyme (cf. Merzel F. et al., PNAS, 2002 99 5378-83).

The ATP-dependent change in water structure could e.g. explain how hydration difference allowed the separation of Na+ from K+; (cf. the effectiveness of LDW for cation partition into was found by Wiggins et al. to vary as: K+<Rb+Cs+<NH4+ and similarly for the anions Cl-<HCO3-<H2PO4-<NO3<HSO4-<N(CH4)4+; cations were selectively partitioned into HDW in the order: Na+<Li+<H+<Ca2+<Mg2+ as were the anions F-<SO42- = HPO42- as were also those non-electrolytes which contain a predominantly hydrophobic group. The five (above listed) modes of producing HDW/LDW can both oppose or reinforce each other. This can explain why, the Na+ kosmotrope can paradoxically induce less LDW than does chaotropic K+ Experiments conducted on the aggregation of silanized glass beads demonstrated the effects of some of the permutations of the five modes of HDW/LDW formation when salts were added as solutes.
A solute which prefers LDW induces HDW (this is achieved by the equalization of the solute concentration throughout the solution by the re-adjustment the relative amounts of LDW and HDW present) was illustrated in Fig. 2 of this paper which explained, e.g., how chaotropic solutes can decrease the water structure by increasing its entropy and kosmotropic solutes can increase the water structure and also decrease its entropy.

[Biological Surfaces are Smart Generators of HDW/LDW] Biological proteins, polynucleotides and polysaccharides (n.b. these are very water soluble) characteristically contain some chaotropic plus some kosmotropic segments and others which are chaotropic so that when such molecules are solutes in water, the overall cost of abolishing the osmotic pressure gradient and the overall preference for HDW or LDW, are both slight.
Solutes which are highly effective kosmotropes (e.g. trimethylamine oxide and betaine) partition into HDW in the double layer and induce some LDW and decrease the dielectric constant thereby thinning the double layer and increasing the osmotic pressure gradient.

The fraction of liquid water which occurs in HDW microstructure, generally increased with increasing temperature and pressure and also increased with increasing water purity (i.e. the presence of decreased amounts of those contaminants which induce LDW). Paradoxically, increasing the temperature and pressure and the purity of liquid water all promote the formation of LDW at surfaces (as a consequence of the increased pressure gradient which induces more LDW when the water equilibrium is otherwise shifted strongly toward HDW). An interesting feature of a characteristic partition-and-destroy mechanism of water structure is that, at surfaces, solute and water cannot always be in equilibrium together. Accumulation of a chaotropic molecules puts water out of equilibrium and displacement of the water equilibrium puts the chaotrope out of equilibrium. This can cause the system to oscillate (this

process was demonstrated in vitro to be manifested over periods of days; the slowness of the process was considered have arise because of the stiffness of the {e.g. microporous polyamide beads} polymer surfaces which were used inter alia by Wiggins to create oscillations in water-structure dependent effects). HDW/LDW is relevant to the myosin ATPase action in mechanical energy production in muscle. ATP when enzymically hydrolyzed, releases H2PO4 2- which is a powerful chaotrope, which together with K+, remains in the LDW associated with the myosin headgroup. Wiggins proposed that muscles generate mechanical energy by a mechanism which ultimately depends on inorganic ion induced microosmosis via HDW/LDW plus Mg2+ which generate a pressure gradient in water in a form which can easily become converted into mechanical energy. More generally, the existence of HDW/LDW water explains how proteins fold in a countercation dependent manner. It should also be noted that the complex (e.g. pore) surface-dependent interactions which create HDW/LDW gradients can be easily observed to occur in purely abiotic (including entirely inorganic systems such as hydrated silica) which are seen to be relevant models for biological (ion selective) protein cavities. Such systems conceivably could be modified through a statistically generated increased sol particle generation rate (equivalent to biological evolutionary pressure) which allows the passage of time to the generate a system of mechanical energy in a purely inorganic silica sol system which had adsorbed inorganic phosphate and by the chemical action of LDW in pores had generated polyphosphate then used the energy obtained from the hydrolysis of polyphosphate to create HD/LDW gradient afforded motion.
The HDW/LDW Origins of Chirality.

Wiggins showed that water-structure enabled the separation of D- from L- glucose and D- from L- lysine. This experimental demonstration indicated that the kind of amino acid and sugar chirality which is now a hallmark of biology is likely to have arisen during pre-biology as a consequence of the highly selective solvent properties of LDW and HDW.
The LDW Afforded Condensation Reactions Achieved by Dehydration of Biopolymer Monomers.

(The possibility of a non-enzymic synthesis of peptides by means of channelled water structure energy was exemplified by the formation of poly L-lysine from and DL lysine. (the condensation reaction was more effective with the L-lysine) plus glycine at 38oC; this condensation process was demonstrated to occur in small-pored silica gel where the SiOH and SiO- surface groups (which are both chaotropes) stabilize LDW which apparently caused the observed preferential L-amino acids polymer formation. (HDW , when present, conversely, caused the polypeptide to hydrolyze).

The above scenario is evidence for the ability of SiO2 - induced water structure to create biopolymers in a pre-biotic evolutionary scenario.

{Entropy Enthalpy Compensation

It should be noted that Wiggins did not, however, discuss another possible driving force in prebiology which is accelerated in water, namely the possible role for both pre- and modern biology of the enthalpy-entropy compensation (extrathermodynamic) rule [a phenomenon which seems to be greatly enhanced in those processes which can occur in liquid water and which might also be explained by the HDW/LDW theory]; this compensation process allows structure build-up over time, the putative basis of evolution of complex form } .

Continue INSERTED DOCUMENTS

DOCUMENT 1 Water & Biology Philippa M Wiggins (cf., water putatively forms high and low density forms HDW/LDW cf. the paper Life depends on two kinds of water PloS ONE 2008 3(1): e1406]) had earlier reported (in 1973 in a paper entitled Ionic partition between surface and bulk water in a silica gel. A biological model, Biophys. J., April; 13(4)

385-398 PMID 4348836) what seems to be directly related research findings: that water in (pores present in) silica gel selects K+ from Na+ and demonstrates a range of other biologically-relevant inorganic ion selectivities; this seems to indicate that the ability of polyelectrolytic surfaces such as would occur in the associated pores present in silicate gels can create a markedly altered form of water. (Wiggins and her coworker(s) later papers (cf. that with Rene T van Ryan [1] showed that (putatively the same general type of altered) water also occurs in the small rather hydrophobic pores which exist in dense films of cellulose acetate; this water was reported to show an increased H2O-H2O inter-molecule hydrogen-bond strength and a markedly altered solvent properties and these effects could be further amplified by those solute molecules which could not enter the cellulose acetate pores because of their sizes. The chemical activity of the water-in-the-pore becomes markedly higher than the chemical activity of the water in the external solution. The 1990 paper [1] entitled Changes in ionic selectivity with changes in density of water in gels and cells discussed the chemically anomalous nature of water in pores present in hydrated organic polymer gels (but which is now suggested can be surely also be considered possibly to be equivalent to the kind of anomalous water also found by her in the 1973-studied (silicate-based glass) capillaries). The 1990 Wiggins group groundbreaking paper showed that the water in the pores of commonly used biologically selective gels is highly anomalous chemically: it contains a stretched form of water structure having a markedly lower density and a markedly higher viscosity (and also a markedly altered infrared absorption energies compared with normal water). The Wiggins LDW is clearly definable as anomalous water which is formed in pores or capillaries. It may also be identical to the Deryaugin polywater which had apparently, however, eventually been shown not to exist. The Wiggins work therefore suggests that the polywater controversy is not resolved. The totality of reports from the Wiggins research group indicates the likelihood that water structure alteration in the presence of solutes, surfaces (especially those in pores) etc. can provide a highly credible mechanism to explain why liquid water has a central role in biology and further allows new insight to be gleaned into how the existence of liquid water could have facilitated the establishment of biology in the first place. The existence of ordinary liquid water is, however, it should be noted, apparently quite insufficient on its own for the establishment of life according to this hypothesis. What seems to be highly essential, however, is the existence of a suitable pore system which is capable of generating the stretched anomalous form(s) of water which can, it seems, eventually allow for the natural self assembly from non-biologically starting materials over presumably geologically extended time scales, of biology. It is now pointed out that the circumstance that the pores formed in silica gel apparently are similarly endowed with chemical structure which enabled them to generate the essential pro-biological anomalous water (as also are the pores in cellulose acetate or other biologically formed gel-forming substances), also indicates that biology could have emerged from a silicate pore plus liquid water basis alone. Life could therefore have emerged from a purely inorganic chemical source. A key part of why this relevance should be emphasized, however, and which is not generally known to the scientific community is that silicate in the form of colloidal size

e.g. 1000 sol particles can demonstrate per se a range of quite close-to-biological cell like behaviour in the laboratory. This includes abilities of such hydrated sol particles (which are cellular forms) to demonstrate a large variation in species types and an apparent possible ability of at least some of such types to generate daughter particles capable of quasi-biological cellular reproduction. Silica sols can seed the formation of similar sol particles and also become senescent and age. This phenomena apparently involves an altered hydration (and putatively an altered water structure). These biological mimic phenomena perhaps arise because of the ability of pores and surface comprised of amorphous silica to generate the unique-to-life altered form of water (n.b., discovered by Wiggins to occur in silica gel) to putatively generate what later became life under pre-biotic situations. A purely inorganic glass capillary system can create the type of anomalous water which, according to later publications by the Wiggins group is what putatively enables life to evolve in the first place and what remains the most central system which confers the most primitive form energy transduction and ion and other solute selection upon biological cells. [1] Wiggins P van Ryn R (1990) Biophys. J. 56 585-96. [2] Deryagins polywater idea (now thought to have been mistakenly identified) could actually to be of a similar chemical nature to that of the Wiggins-van Ryan viscous, stretched (also highly anomalous) low-density [0.96g/ml] water. It furthermore evident that Deryagin silicate glass capillary polywater could have been formed in the polyanionic silicate surfaces present in the silica gel studied by Wiggins in 1973. (Cf. , common silica-glass, artificially-manufactured capillaries may have chemically similar water modifying polyanionic surfaces as the pores which occur in silica gel (which are obviously equivalent to capillaries). Silica gel pore water has (putatively) the anomalous physical properties e.g. higher viscosity and altered infrared absorptions seems to be similar to the anomalous water generated in organic gel pores. It should be noted that the claim that anomalous water per se can be formed in glass capillaries was retracted by Deryaguin: the (previously) supposed anomalous properties of glass capillary water was then assumed to have been mistaken and to have been due entirely to the presence of impurities (e.g. silicates) in the supposed polywater. It is surely, if this assertion turns out to be correct therefore now justified to resurrect the polywater idea but perhaps in an altered format and not the polywater idea as prematurely put forward in the 1960s and 70s which, was, it seems, incorrect. Mainstream science however has tended to shun the topic of anomalous capillary water per se in pores in clay etc. A possible adverse risk to reputation might arise from any discussion of a phenomenon which has a low credibility status. This may have required that direct reference to silica capillary generation of anomalous water is wisely to be avoided while the organic gel-generation of anomalous-in-pore water remains an acceptable scientific topic for peer reviewed scientific paper submission review. It is now suggested that the Wiggins theory of high and low density water might actually be entirely equivalent to a silica capillary polywater-related theory. Discussion of any possible chemical similarity of silica gel polywater to organic gel polywater may have been deliberately avoided (cf. , however, Wiggins in her 2008 PloS ONE paper however, detailed (including photographs of) the abilities of (putatively) the

altered surface hydration produced differential flotation and phase separation behviour of silanized glass beads when bathed with aqueous solutions with different (inorganic) ions present. [The hydrophobized glass spheres of a range of sizes were further discussed in Wiggins P.M., J. Biol. Phys. Chem., 2002 2 25-37]. The silica gel pore mechanism the biologically relevant altered water generation is, however, it is now suggested, highly relevant to the topic of the 2008 PloS ONE 3910: e1406 Life Depends upon Two Kinds of Water paper, which has developed a revolutionary groundbreaking how water is essential to life hypothesis.

Water Studies INSERTED Liquid water might be considered to be a Van Wazer type of stochastic system analogous to polysilicate esters (cf. Grant D. J Inorg. Nucl Chem 1967 29 69-81) in which an essentially infinite number of types of unstable hydrogen-bonded aggregates can arise by random scrambling of the hydrogen-bonds. This type stochasitc reorganization process also creates the glassy state found in common sodium silicate glass. Silica gels when formed in the presence of individually shaped organic molecules achieves high selctivity for the absorption from solution of the solute moleucles which were present during gelation apparently because it contians pores of the specifically matched morphology. Silicic acid (which can commonly arise in natural waters and may be especially enriched in volcanic hot springs) self-assembes into (putativley self-seeding-of-pore-structure) cells of colloidal sol aggregates and also into glassy silica gels; these stochasitc pore-containing systems can also form adducts the hydrogen-bonded aggregates wich are present in liquid water from which specific structures can apparently be sequestered within the randomly sized and distributed pores. Such gels might conceivably thereby select specifically shaped (albeit fragile) water hydrogen-bonded aggregates analogously to how the silica gels can act as a specific adsorbing/templating system for organic molecucles. This mechanism of selective absorption seems to possibly explain why water in silica gel pores can become enriched in the Wiggins pro-life low density form of liquid water (LDW). (LDW can also, however, become selectively enriched in water contaning pores present in polyamides and polysaccharides; [ the polysaccharides may have been the first biopolymers to become evolved perhaps because they could perform the biologicallyessential LDW enrichment process without the aid of silicates {it may however also be of interest in this context, however, that many or perhaps indeed all polysaccharides still occur naturally in a close association with some form of inorganic silicate}]. Such LDW was shown by Wiggins to demonstrate a wide range of primitive quasi-enzymic biological activities (being able e.g. to selective absorb and (dehydrate so as to) link together various hydroxylated and aminated organic molecules (and thereby to synthesize peptides and polysaccharides, as well as being able to correctly selectively absorb the correct L and D enantiomers from mixtures to create true biopolymers, to provide a transduction mechanism for high energy P-O-P bond energy untilization and to select K+ from Na+ in mixed salt solutions (as well as accompalish a range of similar ionic and molecular discriminations). The self-assembling hydrated silica gels and related sols were likely to have been highly favoruble environments in which the

evolution of early life on earth could have been accomplished. The co-adsorption within silica gel and sol pores of the range of organic molecules which naturally occur in space, are present in some comets and meteorites and could arise following electrical discharge activation of the small organic molecules plus water, ammonia, carbon dioxide, hydrogen sulphide, phosphine and methane which may have been present in the earths atmosphere, could have provided the starting point mixture to allow the LDW to create the first biopolymers which might have added to the complexity of the host cellular silicate-based systems. The inclusion of the easily-decomposed organic molecules within gel pores also provided a natural protection mechanism against ionizing radiation. At a point of build up of sufficient complexity we then have a primitive form of life. The reproduction of such cells was apparently possible without the organic matter being present but the inclusion of organic polymers seems to have allowed greater flexibility, this allowing a carbon-based life to eventually to become master of the original host silicate-based cellular systems.

Inserted Document
DOCUMENT 2

Reconciling Wiggins, Cairns-Smith and Kornberg Theories of the Beginning of Life

by including ideas suggested by Wang et al. , Lima-de-Faria , Bernal, Ling, Luck, Orgel and

others

Pore surface polyphosphate, polysilicate plus Evans-anti-entropy conditions could have been necessary to create the (helical?) pre-life supramolecular water structures which could have enabled the first life to emerge on earth. The same structures may currently hinder the ageing process and be of relevance to modern biology and pathology. The modification of water structure in pores seems to be an example of how small mechanisms over short periods of time can behave counter to the laws of classical thermodynamics. DG August 2012 Contents: Introduction
Kornberg Polyinorganic Phosphate Cairns-Smith Clay Minerals Wiggins High and Low Density Water Pro-Life Anti-Entropy

Restatement and More

Were RNA & DNA Present in the First Living Organisms? Fox Protenoids Chance & Self-Assembly Probabilities LDW. Role in Biology of Reverse Time/ Anti-Entropy

Chaplin Buckyballs Reusch et al. Helices Silica Gels & Sols

Conclusions Selected References Additional Notes

Nucleic Acids & The Putative Role of Negative Entropy in Biology Anti-Disease Anti-Ageing Water Structure? Is The Reusch et al. Double Helix Piezoelectric Preo-Life LDW Force Transducer ?

Introduction: Arthur Kornberg Polyinorganic Phosphate A. Kornberg et al. have proposed that high molecular weight polyinorganic phosphate, which is an ubiquitous component of virtually all biological cell surfaces, could have played a key role in the early evolution and survival of life, but the mechanisms by which such a purely inorganic system might have accomplished these tasks are unknown. These mechanisms may, it is now suggested, include the known ability of inorganic (poly)Ca-polyphosphate to form helices, to modulate the structure of water, to inhibit crystal formation in aqueous solutions, and putatively also to enhance antientropy generation (a currently poorly understood process which may conceivably depend specifically on the helical structure adopted by, and/or the piezoelectric properties which are inherent in various polyanionic systems).
The (Kornberg) poly-inorganic phosphate could have arisen, it is now pointed out, in the first instance by Wiggins low density water (LDW) enzymic activity (vide infra) generation; [LDW -high-energy-water concnetrated in some types of silicate pores can apparently produce high energy phosphate bonds; e.g. monomeric phosphate anions + LDW likely can roduces the type of poly-inorganic phosphate discussed by Kornberg. A similar LDW pore water system might then also have generated poly-phosphodiester ribose molecules by a similar mechanism during the earliest period of the development of the earliest ancestors of biological cells, (Cf. Orgel et al. has shown (by used a man-made carbodiimide chemical dehydration system) that calcium phosphate or hydroxylapatite can act as a selection template for a solid phase assisted process of synthesis of polypeptides and polynucleotides; the dehydrating activity of LDW could have enabled a similar process to occur)].

Cairns-Smith Clay Minerals A.G. Cairns-Smith proposed that ionic charge-arrays on clay mineral surfaces could have provided some kind of pre-nucleic-acid-like-information system which was used by early life forms. Water is essential for clay layer pores to expand. It is generally acknowledged also that liquid water, e.g., by some consequence of its unique properties, is somehow essential to life. The possibility that Wiggins LDW [a form of energy rich polywater (vide infra)] might form in clay pores adds possible weight to the Cairns Smith early-life hypothesis. Wiggins Polywater : High and Low Density Water HDW & LDW Formed in Capillaries

P.M. Wiggins proposed that the reason why all life depends on the unique properties of liquid water is because of the occurrence of low and high density forms of liquid water. Altered hydrogen-bonded water molecule aggregate structures can apparently exist, e.g., adjacent to polyanionic surfaces. This phenomenon is especially pronounced adjacent to those polyanions which occur at the surfaces of various capillary pores. A similar water structuring effect is apparently also associated with numerous types of solutes. Hydrated carbohydrates or silicate gels possessing polyelectrolyte pore surfaces seem uniquely able to generate high concentrations of a low density form of water [lowdensity-water (LDW)] which (according to Wiggins) demonstrates a dramatically wide range of specific pro-life activities and therefore could have played a key role in the appearance of early life (cf. Wiggins, 2006). LDL can, on its own, also create biological chirality by selectively absorbing D over L sugars and L over D alpha amino acids and produce high energy P-O-P bonds. (LDW can form such bonds from inorganic phosphate). LDW can also apparently convert amino acids into polypeptides. The Wiggins LDW apparently, then, has a very broad-ranging enzymic activity indeed.
This water structuring and its associated intrinsic enzymic activity seems also likely to occur in and around Ca polyinorganic phosphate helices, nucleic acid helices as well as inside the pores generated by the aggregation of various sparingly soluble Ca salts. The formation of pro-life LDL in pores present in aggregated CaCO3 and hydroxylapatite could account respectively for the reported ability of CaCO3 to select L over D amino acids and for hydroxylapatite to act as a template for nucleic acid oligomerization (cf. Orgel et al.).

The pro-life liquid water structuring effects can, it seems especially easily arise at silicate surfaces and this especially applies to the capillary pores which exit inside silica gels and sols and probably also inside clay interlayers. Since the necessary pro-life process of the alteration of liquid water were shown experimentally by Wiggins in 1974 to occur on the surfaces in the types of pores which occur inside man-made silica gels, LDW will probably also form in the clay interparticle spaces which have been proposed by Cairns-Smith to have been involved in early life. Possible Pro-Life Anti-Entropy Water Structuring Effects The self assembly processes needed to generate life may critically have required additional processes which are separate from those considered above and which may include some kind of anti-entropy (perhaps most conveniently termed a negative entropy). The generation of this kind of (extrathermodynamic) order may be part of a critical-for-life force. The first appearance of life and its continuation, as well as the evolution of more complex living organisms, may, it is now therefore also proposed, have critically depended on those specific shaped systems which can act as nano-machines for the cogeneration of pro-life water structures under those conditions which favour the production of Evans anti-entropy. Such systems may even include the well known helical structures present in biological systems. These include the fiber forming inorganic polyphosphates, collagen, anionic polysaccharides and nucleic acids as well as cell like colloidal size silica sols and clay particles. The Evans Fluctuation Theorem (cf. Wang et al., 2002) proposes that the second law of thermodynamics is not the previously supposed universal law but can be broken; entropy production is actually a size dependent process so that entropy can naturally be required to decrease over short times in small sized (machine-like) systems such as mitochondria.

The Evans negative entropy production process may be a fundamental pro-life force.
The Evans Fluctuation Theorem which proposes that the probability that entropy will naturally decrease (i.e. that anti-entropy will increase) relative to the probability that it will naturally increase depends on the exponent of entropy.time. This permits small scale engines to work in reverse for short time intervals. This seems to be equivalent to a reversal of time (anti-time or reversal of time) in such machines. Experimental systems which seem to demonstrate these extrathermodynamic properties apparently include micron sized beads [suspended in water] as reported by Wang et al. (2002).

Summary of this section

The proposed pro-life time reversal process may, it is further proposed, be enabled by certain specific (hydrated) helix systems and helical molecular machine systems which allows them to create order in other molecular systems and also to greatly extend the lifetimes of such systems. This ability to create order and increased thermal stability will, it is suggested apply to aqueous solutions and dispersions of polysaccharides, nucleic acids and also to poly-Ca-polyphosphate. The modus operandi of such biologically- essential liquid water supramolecular water- structure generators may be to enhance the lifetimes of unstable water molecule in pore systems present in gels, helix aggregates or geometrically regular cluster structures e.g. the (Chaplin) buckyball system so as to generate virtual anti-entropy channels which can force the Evans fluctuation virtual time reversal processes. This can explain the mystery force which seems to be critically associated with biology and which generates a systematic opposition of the effects of the second law of thermodynamics. Restatement and More. LDW/HDW water Liquid water was needed to create the first life and remains the most essential as well as being the most abundant chemical substance present in all living cells. The first living cells must also have contained a system to separate out the most pro-life water structure(s). This seems to include the LDW which was identified by Philippa M Wiggins. Wiggins identified separate high and low density forms of water in polyetheylene glycol and in the pores present in various polysaccharide gels.
{It should be noted that in her early work, an entirely inorganic silica gel pore system was also studied by Wiggins and this also seems to have created a similar LDW HDW separation process which was similarly later also attained in her organic gel research systems}.

HDW and LDW LDW was found to have the ability to selectively absorb D-sugars from D+L sugar mixtures (and also select L-amino acids from D+L amino acid mixtures). This circumstance further indicates that the critical water (re)structuring role of water in pores could have enabled the early biotic selection and enrichment processes which led to the currently observed chirality of biology and also eventually allowed the evolution of highly complex systems by random selection processes. Water structure in pores, it can now be suggested, was the critical organizing factor which could have eventually generated nucleic acid containing cells. Water and Entropy Liquid water putatively possesses some additional essential-for-life ability to tune into Maxwell-Demon-like mechanisms in order to enable the essential properties of living organisms to counter the normal temporal increase in disorder which is required by the second law of thermodynamics. It should be noted that it is commonly accepted that the second law of thermodynamics undergoes local breaches during Brownian motion (and this phenomenon seems to have

been the origin of Wangs 2002 demonstration of Evans fluctuation theorem which quantifies how reverse entropy arises). This, however, it is now further suggested, actually reflects on some hitherto-notconsidered extrathermodynamic dimension in virtual space-time processes (cf. Grant 2012). Life putatively can access these extradimensional reverse time event pathways via virtual enthalpy-entropy compensation channels (which can e.g. also be demonstrated in non-biological seeded nucleation of inorganic form situations).
Living cells seem to reproduce themselves true to form both via cortical (e.g. via cell surface glycation information codes) as well as by nucleic acid coded protein control mechanisms both of which are further potentially enabled by water structure manipulation and both of which again can separately be adapted to allow a (sometimes-fast) evolution of more complex structures to occur. Biology may thus [controversially] actually be achieved by some direct and indirect uses of reverse [Evans] time which enables the evolution of more complex systems to respond to possible future events. These improved more-evolved systems could actually be created to meet imaginary but perceived future needs. The evolution of species is then actually equivalent to a virtual temporal tunnelling processes which may be achieved [again controversially] both by the straightforward Darwinian mechanisms of engagement of organisms in competitive activities as well as by the use of reverse time and extradimensional space to separately encourage the evolution of heightened system complexities.

Were RNA & DNA Present in the First Living Organisms? The nucleic acid based systems of RNA or DNA are, of course, commonly believed to be what defines life. The reason why liquid water, as outline above, is believed to support all forms of life is the same reason why the most important prime-for-life-essential initial component of life from the very beginning of biology on earth was also liquid water. Life, it seems, further critically still continues to critically depend on the existence of a system for the maintenance (at least?) two kinds of water structure. These are (according to Wiggins) low density water, LDW and high density water, HDW. A more general definition of life can now be attempted according to the ideas mentioned in the above discussions and my follow-on notes and references. According to this alternative hypothesis: the new [Wiggins] definition of life is centred on LDW, Such a definition of life needs, perhaps, to be further modified by an inclusion of an Evans reverse entropy pro-life force: The unique chemical and physical properties of liquid water directed by this force might be considered to be central to what ultimately is actually essential for life.
This leaves out DNA or RNA as the primary initiators of life since it can be logically deduced that DNA or RNA or indeed proteins may simply be additional facets of the currently observed terrestrial life forms (even although they are believed to have been retained by earths biological systems for some perhaps three plus thousand million years). So, for a few hundred million years before the advent of nucleic acids other related older systems (e.g. the clay mineral surfaces as suggested by Cairns-Smith) simpler water pore systems were in place and these systems eventually by virtue of their actual high complexity gave rise to the currently used genetic code etc.

[The circumstance that RNA contains ribose (which is acknowledged to be a likely evolutionary precursor of deoxyribose) suggests that protein sequence information was encoded by a series of progressively more complex more highly evolved sugar phosphates; hence this system needed the pre-existence of simpler sugars (perhaps starting from poly-formose) and therefore obviously the highly complex systems of DNA or RNA encoded information must have evolved far on into the evolutionary process. Hence this idea that life is always DNA-essential for evolution can be suggested to be irrational. The evolution of an improved version of RNA needed the existence of deoxyribose which also needed pre-existing active water processing (quasi or actual enzyme) systems. The prior existence of sugars seems, however also to be a prime critical logic block: a necessary deduced requirement to allow RNA and its precursors to evolve in the first place. Hence the system of simpler sugars and polymers based on some much simpler system (with enzymic activities?) must have existed (in quasi-biological cells) before nucleic acids or their evolutionary precursors could evolve. This may mean that biological cells containing sugars and polysaccharides first. These substances could have excited without proteins or nucleic acids since their presence is a pre-evolution requirement for the creation of the protein - nucleic acid evolutionary process which eventually became necessary to conserve the by-now-highly-complex special information coder which allows for the numerous types of proteins to be accurately reproduced and to be passed on down the generations].

Fox Proteinoids

Perhaps, however, early polyamino acids (such as the Fox proteinoids) evolved in parallel with polysaccharides and nucleic acids. The first polyamino acids (and other quasi biopolymers) were however formed most likely, not as suggested by Fox at hot lava surfaces, but in Wiggins LDW-rich pores at near ambient temperatures. The pores could, at least at first, have been those in inorganic gels, most likely silica gel or sols which are known to be formed in volcanic springs. The retention of fragile biologically relevant polymers within such pores would have aided their survival in the early earth which is believed to have been bombarded by high energy cosmic radiation. Chance & Self Assembly: Relative Probabilities
First Biopolymers were Polyphosphates Carbohydrates + PHBs Polymethylene lipids ??

The laws of chance demand that the simpler the biopolymer then the more likely was the probability that such a biopolymer was produced by random events. Such a stoichiometrically simple system as Si(OH)4 is known to spontaneously polymerize in liquid water at ambient temperature to create random Si-O-Si linked structures which can further form hydrogen bonds between SiO-H groups and hydrogen bonded-bonded water. Very high water content silica gels and sols can arise spontaneously by this method. This can easily be demonstrated in the laboratory. These sols have individual speciation of form with intricate pore structure. These forms can engage in primitive reproduction following growth achieved by additional Si(OH)4 provision (cf. Grant 1992). This is quasi life at it simplest. It is analogous to e.g. CaCO3 crystal replication. A seeded process which generates daughter morphologies. This allows for a spontaneous generation of the water-restructuring pro-life Wiggins LDW generating systems which can give rise to organic and organometallic polymers; (cf., e.g. by the condensation reaction processes allowed within silica sol pores or calcite containing LDW which dehydrates small organic molecules containing C-OH (as well as group B metal OH) bonds. This allows spontaneous random polymer assembly). Consider also the circumstance that the stoichiometry of sugars, the carbohydrates [CH2O]n is the simplest of any biopolymer. Such a stoichiometrically-simple chemical system is intrinsically more likely to form (e.g. in the silica or calcite pores) by chance events than are systems consisting of alpha amino acids which in turn are more likely to form by self-assembly than are the nucleic acids. Carbohydrates are also very water-like; they resemble water aggregates; they actually may directly mimic the LDW-HDW-LDW-HDW system. Pore Water first. Then evolution (via the basic for life [Evans] reversal of second law entropy vs. time) gave rise to polysaccharide water-look-a-likes which could then perhaps improve on the original pore water generating mechanisms.
For the above purposes the poly-inorganic phosphates, O-{P(O)[O-]}n-O- , can be noted to be of especial simple stoichiometry, as also are polyhydoxybutyrates [PHB] [O-CH(CH3)C(O)-]n which are, however, more complex than the very stoichiometrically simple polymethylene (CH2)n- part of soil humic matter {(CH2)n>32[CO]{C(O)O-)}(cf. Grant 1977) [which perhaps was also present in pre-biotic soils (having arrived in comets etc.) which, following its inclusion in the inorganic (silica sol) pores could have eventually promoted the use of biological lipids which are currently present in cell walls and other membranes putatively perhpas to improve the efficiency of other-polyanion-induced pore formation systems there present which can induce the appropriate water structure].

The stoichiometry of water, H2O: this seems ultra-simple but Wiggins and others have shown that this apparent simplicity hides an extreme complexity of (unstable) hydrogen-bonded supramolecular complexity. This complexity of course selfassembles. The silica sols with pores also self assemble. LDW Reverse Time & Anti-Entropy: Its Possible Key Role in Biology The need for a non nucleic acid (re) definition of life must (re)consideration of how life processes tend to occur in opposition to the second law of thermodynamics. The fundamental physical laws are time symmetric. Life, a system of extreme complexity based on an apparent self-assembly mechanisms outlined above obviously continues to generate new systems of even greater complexity over geological time. Life therefore tends to occur in opposition to the intuitive effects of the positive direction of time. Some mechanism (perhaps involving water structuring) may allow access of biological systems to a negative time processing of matter. Time running backwards as it were must, it seems, be implicit in the ultimate nature of the phenomenon of life. On the other hand the conventional thermodynamic laws seem to demand that over thousands of millions of years that a severe system decay must arise; this would be expected to completely destroy all pre-existing biological complexities. The use of the now well-known smart system of nucleic acid unwinding, reforming and repair of codes and cellular reproduction must surely ultimately be quite insufficient to both preserve and to continue to increase the system complexity of biota. If this contention were true, then life must combat information decay by its ability to use alternative strategies. Perhaps these include the harnessing of reverse entropy vs. time. This idea is now a realistic possibility following the apparent demonstration by Wang et al. of virtual reversal of time with particles the size of a biological cell or organelle (or biomolecule). This pro-life harnessing process may also, it is further suggested, is perhaps accomplished via an entropy-enthalpy compensation system. This seems to be a very common if generally overlooked extrathermodynamic process [which shows up throughout not only biology but also chemistry and physics (cf. Grant 2012)] and which seems at least as common as are those processes which are controlled by classical thermodynamic equilibria]; the compensation process may operate via an imaginary space-time overlap phenomenon (cf. Grant 2012); it should be noted that entropyenthalpy compensation has commonly often been associated with reactions (including gel microstructure structure seeding) which are accomplished in aqueous solutions. Following the Wang et al. 2002 report that aqueous solutions contain some intrinsic potential allowance for a the reverse-time running mode (for small machines), it might be indicated that the Wiggins LDW pro-life nano machines could not only define life but also provide the water-aggregate nano-machines which are enabled to operate in reverse time. Perhaps the phenomenon of the reversal of time which might be achievable in water aggregates via LDW action can also be achieved by the hydration associated with

DNA and RNA as well as by the hydrated polyanions which are known to act as Wiggins LDW water structure generators. The (pseudo-)equilibrium between LDW and HDW water microstructures in pores could be driven by the (extrathermodynamic) entropy-enthalpy compensation process which affects all associated chemical and physical reaction rate processes. This specifically could enable a useful-for-life countering of the second law of thermodynamics via the the generation of reverse entropy (=virtual reverse time) and perhaps also vacuum energy utilization (cf. Grant 2012). This might be a phenomenon which can be achieved by a wide range of biopolymers.
It is worth again re-stating at this point the amazing properties of LDW: this active form of water is able to remove the elements of water from simple molecules to produce e.g. P-O-P bonds and also putatively to synthesis polysaccharides from a mixture of simple sugars polypeptides from amino acids etc. and to efficiently act as an inorganic ion selector (e.g. allowing K+ to become concentrated inside cells and leaving Na+ outside). as well also to potentially self-assemble sugar-phosphodiesters Again re-stating: These diverse abilities of LDW are probably why liquid water is the single most essential chemical component of biological cells and why a reproducible mechanism for the separation of water phases was necessary for the evolution of the first kinds of pre-biotic cells in the early earth. The role of ATP and nucleic acids also seem, from the view point of the analysis of current biological systems, to be a critical component of all living cells. These may have self assembled under the guidance of LDW. The Wiggins theory of water can also explain the basic mechanisms of e.g. muscular action, osmotic shock and rigor. While the nucleic acid systems including ATP, RNA and DNA seems to be (modern) life essentials, any straightforward logical deduction also could soon suggest that these systems are unlikely to have been in place in the first living cells. Over the first few hundred million years of early biology replicating cells may have contained the much simpler polyinorganic phosphate and perhaps also the polybetahydroxybutyrate + calcium (+ other 50 Hofmeister water structure regulating seawater elements) (as do ATP equivalent systems and perhaps also other simple memory systems for handing on cellular uniqueness to new generations of early cells). Incidentally the 50+ seawater counterion system seems to be continued by the heparan sulphate information encoded cell surface growth factor regulators (present at the cell surfaces of adherent animal cells) in a further layer of complexity to how water structure is tweaked for biological advantage by the more highly evolved biological species).

Reusch et al: Hypothesis of the co-occurrence of calcium ions, poybetahydroxybutyrate and polyinorganic phosphate helices The lesser known aliphatic polyesters which apparently occur at virtually all current cell surfaces and sub-cellular organelles throughout biota together with the long-chain poly-inorganic phosphate [which has been discussed by Arthur Kornberg et al. in the context of the putative role of these polymers in the (early) evolution and survival of species] may perhaps have been assembled by random actions but have been retained throughout evolution in order to continue to improve the specific efficiency of generation of LDW. This possible ability to separate the separate phases of water by such helices may have been inherited from pre-biotic times and used as a LDW generator.
The putative major potential helical structure water structure generator associated with inorganic polyphosphate is polybetahydroxybutyrates, also present in a further association with calcium ions in the form of a double or twin helical structure (as indicated by Reusch et al.). [Such inter-twined double helices cannot e.g. be relicts of pre- nucleic acid information-encoding systems, as they do not seem to for allow for individual polymer component variety]. Are the poly-Ca -inorganic-poly phosphate-polybetahydroxybutyrate systems a primitive essential-to -life water tune-into extrathermodynamic reverse entropy system? And do they continue to provide this kind of service to modern organisms?

Cell surface and nucleus helical structures may perhaps offer an especially effective water structure generator and LDW HDW separator and/or Evans reverse entropy facilitator, e.g. via rapid hydrogen bond fluctuation vacuum energy overlap (cf. Grant 2012). Further research to test this hypothesis is warranted.
[The continued presence of the high molecular weight poly-inorganic phosphates were originally thought to be present in modern species simply as molecular fossils but are now thought to continue to play back-up roles as protection systems, to act as Ca2+

channels and also as reserve high energy phosphate energy sources but also will provide pores for tissue protection and water retention].

Silica Gels & Sols Possible Key Role Players in Pre-Life & the First Evolution of Separate Biological Species Various kinds of naturally formed self-assemblies of particles (silicic acid aggregates) can apparently also exemplify the critical-for life water structure separation in the exact form which was discovered by Wiggins. These include silicic acid aggregates with pores containing poly-inorganic phosphate composite systems may have, in the early earth, facilitated the separation out of the two chemically distinct (necessary-for-life) forms of water which have been identified by Wiggins (cf. PloS ONE 2008 3(1) e1406. Pore water in hydrated silica gel (as reported by Wiggins in 1973) apparently can create the pro-life LDW (which can catalyze the formation of P-O-P high energy phosphate bonds) and acting together with silica gel (which can sequester [as well as be highly protective of] all of the relatively fragile organic molecules which can be been formed from electrical discharges or deposited from comets etc.) can potentially perform a host of different classes of primitive biosynthesis. It can be assumed that the high water content pore containing gels which could have sequestered the various pre-biotic organic substances could also engage in various random assembly building test runs of the various possible building blocks of life because of the presence therein of LDW. The families of natural silica sols (which however are currently poorly understood except that these are silicic acid aggregates) can per se can engage in like-like particle seeded reproduction by the quasi-biological seeding process (which is analogous to that demonstrated by CaCO3 particles {cf., Lima-de-Faria} (i.e. by a process which mimics biological cellular reproduction without DNA or RNA being present); this cellular selfseeding of form could have been a critical-for-life event and be the primary origin of the creation of individual biological species. These started off in the first place following the generation of a range of different species of CaCO3 particle morphologies or different pore distributions in species of silica sols and related gels. {Although the silica sol particle systems were formally thought to be entirely amorphous [at least to the traditional use of X-ray diffraction to identify well-developed crystallinity] these could contain low-order crystallinity or even some other type of order (perhaps a discriminated chaotic order which like pure crystalline order which augments the efficiency of the process of seeding of daughter particles cf., Grant et al., 1992}. While modern biologically- and industrially-produced polymers can create the necessary pores for life required water transient phase separations, the ability of pores in purely inorganic systems to produced the necessary-for-life pores which create stretched LDW. This state of matter was identified by Wiggins et al. and proposed by this research group to have created the first life.
A silicate-based biotic process is obviously of possible special relevance for the understanding of pre-biotic evolution since the polysaccharide- or polyamide- based organic gels (which formed the main focus of Wiggins et al.s researches reported in 1995) surely could not have existed (at least for these types polysaccharides and protein-like gels in sufficient quantities) before the appearance of the first truly biological cells. This circumstance does not apply to clays, silica gels and sols which can arise in a quasi-biological cellular form entirely by abiotic processes.

The reason why inorganic silicon is an essential element for animal nutrition is not known, but once again this could be something to do with the critical-for-life pore forming abilities of silicate-based polymers (e.g. zeolites); these systems putatively induce negative entropy (and [favorable-to-life-extension] water structuring). Perhaps inter alia the binding of natural silica sol particles to the extracellular matrix polyuronide (anionic) polysaccharides (e.g. heparan sulphates, chondroitin sulphates and hyaluronan) also provides for an especially useful water-holding pore healthpromoting probiotic system. The primitive anti-increase-in-disorder force exerted by capillary pore water per se, however, may be what underpins all life and well as what determines good health. The pore water force, first of all perhaps, from a natural philosophy point of view seems to allow for a more exact definition of life. Life then can be suggested to be defined thus: The eventual outcome of a general anti-entropy extrathermodynamic phenomenon enabled by the effects of the operation of the Fluctuation Theorem in liquid water present in pores bounded by anionic surfaces. The Lima-de-Faria Evolution Without Selection Hypothesis Limade-Faria proposed in 1988 that the mechanism by which the growth of crystals from seeds occurs is also what underpins biology but the basic mechanism by which this inorganic seeding is accomplished lies entirely outwith biology; it is thought that this process depends is regulated by water structure change but the details are not fully understood. The anti-entropy water structuring force discussed above may also be relevant to solution of a fuller understanding of what enables abiotic seeding (n.b. this seems to be purely a physical chemical process). Inorganic seeding allows an exact replication of complex form (e.g. of formally amorphous chaotic structural forms such as occur in silica sols as well as in well-developed crystalline solids. This process can e.g. enable sparingly soluble (in-water) Ca salt crystals, e.g., CaCO3 (which can exist in hundreds of different forms) to be capable of self-seeding [putatively by a templated process which includes a sloughing off of parts of parent particles which allow for the production of daughter particles of the same distinct morphological forms].
The ultimate mechanism behind the existence of current biological species could therefore be that simple, a repetition of inorganic crystal morphology replication, but underpinning such processes surely lies the Evans negative entropy-water structure mechanisms discussed above.

According to Lima-de-Faria the most basic pro-biotic process may simply be the seeding of sparingly soluble Ca salts, this resembles the reproduction process which is used by organisms to create skeletal units and which arises in its simplest form without the assistance of genes and which clearly indicates that reproduction of form per se cannot be any unique property of DNA or RNA but, instead must be a consequence of the fundamental property of inorganic or organic particles to engage in the process of the seeding of similar daughter particles; this is reproduction in its most fundamental form , the assembly of unique form but which, it should be noted can be accomplished most typically in liquid water. The use of water by biology may therefore hinge on this circumstance; how this unique hydrogen-bonded liquid chemical system encourages the fundamental reproduction of form is the basis of biology.

(Cf. e.g. purely inorganic seeds e.g. of CaCO3 when added to aqueous solutions containing Ca2+ and CO32- ions can produce selective growth on some of the surfaces of pre-existing CaCO3 particles or on the surfaces of CaCO3 seeds shed from these particles which reproduce the specific morphological forms of the parent particles; these processes are observed in aqueous solutions to which the addition of polyanions e.g. polyphosphates can greatly alter the shapes of the particles formed; such polyanions behave as secondary morphogens The biological polyanions share this ability together with the purely abiotic polyanions. The biological polyanions which demonstrate intrinsic morphogen activity for inorganic Ca salt crystallization (which include DNA and RNA as well as the similarly structurally complex anionic polysaccharides present in the glycocalyx) seem to transfer different (hierarchy) microstructures into different inorganic salt particle morphologies via their abilities to selectively alter the kinetics of crystallization; studies of the kinetics of this seeding process shows up an entropy-enthalpy compensation in the kinetic rate parameters and this process is tied up with water structure manipulation. It may further be logically suggested that the extrathermodynamic compensation process inherent in water structure manipulation may have some bearing on the ability of morphogens to create order by the pro-life process of opposition to the second law of thermodynamics. The ultimate cause of the extrathermodynamic entropy-enthalpy compensation in the seeding of structure is likely to be coupled to negative entropy assisted water restructuring at phase boundary surfaces, most especially those present in water holding pores.
Conclusions

The several organic gel pore systems which were studied by PM Wiggins (cf., e.g. her 2008 PLoS ONE paper) were shown to efficiently separate low from high density water [LDW, HDW]. During early pre-biology this important mechanism could have enabled pore water to select D over L sugars (and the corresponding reverse process for alpha amino acids). While this bio-chiral selectivity seems also to occur following absorption of amino acids on calcite, the ability of low density pore water per se to create biochemical chirality may have been the more important primary selection mechanism. Thus the chirality of biology likely derives from a fundamental chiral selection process afforded by pore water microstructure. The necessary pores can occur in purely inorganic substrates, e.g., in pores present in silica gels and some sols or in the interlayers which hold clay water. The different LDW and HDW water phases identified by Wiggins normally co-exist in rapid equilibrium with each other. The hydrogen bond rearrangements in these LDW/HDW systems may create a mechanism of enthalpy-entropy compensation which enables pore water to behave contrary to the classical laws of thermodynamics especially by the negation of the usual second law increase in entropy with time (which is the possible driving force for biological evolutionary trend towards a greater system complexity). The ability of aqueous solutions or dispersions of purely inorganic polyanions to negate entropy may be why the Ca polyphosphate helix (plus its associated water system) has been carefully retained at the surfaces of modern biological cells throughout their evolution.

Such inorganic polymer systems may conceivably generate anti-entropy and have done so throughout geological time (in agreement with the Kornberg hypothesis that polyinorganic phosphate was needed for the generation and safeguarding of life). The ability of numerous types of polyanions to create apparently similar disturbances in the microstructure of liquid water, however, seems to require future research into the critical nature of the apparent anti-entropy force which can be exemplified by the Brownian motion phenomenon. The present note re-states the Wiggins (polyanions create two phase water systems which are essential to life) hypotheses with the additional proviso that Evans reverse time is also a critical requirement which is needed for biological systems to succeed. Polybetahydroxybutyrate which seems to co-occur throughout biota together with intercalated Ca inorganic polyphosphate polymer helices may also have an associated (perhaps especially pro-life?) type of water-structure which might further enhance the anti-second law effect which is suggested to be associated with the simpler inorganic polyphosphate hydration. In the early earth phosphate copolymers may have generated sugars from formose trapped in pores. This process may eventually have engendered the formation of the precursors of nucleic acids; Life then, it seems, for its start-up (and for its continuation) critically depended on the negative entropy force achieved via water variable density separation by the antientropy polyanion and/or pore systems which can separate bent hydrogen-bonded from straight hydrogen-bonded water structures, viz. HDW from LDW. Selected References Acevedo OL Orgel LE Nature 1986 321 790-2 Brown MDW Kornberg A PNAS 2004 101 16085-7 Chaplin MF (Water structure references, internet) cf. Biophys Chem 1999 83 211-21 Cairns-Smith AG Genetic Takeover and the Mineral Origins of Life Cambridge Univ. Press 1982 Fox S In the beginning life assembled itself New Scientist 1969 41 450-2 (cf. Nature 1964 201 336-337 Grant D Nature 1977 270 709-710; et al., Med Hypoth. 1992 38 46-8; (2012) web. scribd.com/doc/87757336/D; ibid./ 922082877/DG-Note-27-3 and 96573303/A hypothesis1a37 Lima-de-Faria A. Evolution without Selection Elsevier 1988
The seeding of inorganic crystal morphology buildup is, a process which according to Lima-de-Faria is not to be yet fully understood but could reveal the ultimate nature of biology.

(Cf. on page 80 of the above book, this author writes How can these minerals or such a simple chemical as water already behave
in an essentially similar way to that of a living organism without having genes that maintain their constancy of pattern and that create their variation? The reason is simple. The genes have little to do with these two basic phenomena. As a geneticist I should feel outraged by such a statement but this is sad news for those who try to solve all biological problems by resorting solely to genes. Water has no genes, calcite has no genes, yet they already possess mechanisms that at present are considered fundamental gene attributes

Ling GN Biophys J 1973 13 807-16 Reusch RN et al., Proc Soc Expt Med Biol 1989 191 377-81
High molecular weight inorganic [Ca2+] polyphosphate forms a double helix with polybethydroxybutryic acid, a polymer system which apparently shows up throughout biota at cell surfaces.

Wiggins PM Biophys J 1974 385-98; 1984 13 385-98; For high and low density water in polysaccharides and polyamide gels see Prog Poly Sci 1995 29 1121-63. For (high and low density water in resting, active and transformed cells) see Cell Biol Int 1996 20 429-35. For enzyme reactions and two state water see J Biol Phys Chem 2002 2 25-37. For the Wiggins Origin of Life Theory see: Water Digest SeptOct 2006 pp 78-82) and for Water and the biology of prions and plaques (co-author: Steel GK) see the internet file: precedings.nature.com/documents/1381.version/1; (2007). For a general review of the Wiggins Theory of Water etc. see: PLoS ONE 2008 3 e1406
[Professor Philippa A Wiggins and her co-workers (working in Auckland University, New Zealand) have identified two kinds of supramolecular structures which occur in liquid water and also discussed a general theory of water relating to how water is essential for biology. The overall topic might be entitled Low Density Water (LDW) and its role in biology. [An important property of LDW (n.b. this equates in part to pore water e.g. in inorganic as well as conventional biologically produced gels) can not only select D-sugars and L- amino acids from D+ L mixtures but can also select K+ from Na+ and also perform all the functions of all other ion pumps. LDW can also engender the formation of P-O-P bonds starting from inorganic phosphate P-OH. LDW was also observed to allow the formation of peptide linkages and create polypeptides from amino acids, so it must be presumed that LDW likely created the first proteins and therefore the first enzymes. Presumably also LDW can simultaneously create P-O-C bonds, including those in ribose phosphate and the polyribose phosphate backbone of RNA type nucleic acids or their precursors. Could this have been coordinated with protein structure also using the ability of sparingly soluble Ca salts (created e.g. via modulation of LDL/HDW) to selective bind nucleic acids and other bipolymers. This process by which LDW in pores can dehydrate hydroxyl and amide group containing molecules allows for the self-assembly of proteins consisting of L-amino acids only. Obviously if LDW has been correctly defined and characterized by Wiggins, the state of matter named LDW was of likely critical importance to how life started. The Pre Life stage of biology may have arisen randomly in a purely inorganic-system which contained self-assembling pores and/or surface systems which demonstrated an enhanced ability to make LDW. This process may have taken only over a few million years. This inorganic water structure-based mechanism seems ultimately also to be how modern living organisms continue to be able to access energy from ATP, the basic biological energy process which was shown to require Wiggins LDW/HDW hydration to transfer the energy to e.g. alter protein conformations etc.(cf. Wiggins, 2002)].
Surprisingly, perhaps because of the original negative scientific reputation of capillary water research arising from the (1960s) capillary polywater fiasco, Wiggins seems not to directly discuss her silica gel polywater researches in her 2008 paper.

Wang GM et al. Phys. Rev. Lett., 2002 89 (5) 050601; cf. New Scientist 19 July 2002 ; cf. web.newscientist.com/article/dn2572-second-law-of-thermodynamics broken.html; Scientific American July 24 2002; Nature web,nature.com/nsi/020727/020727-2.html; web. chemweb.com/alchem/articles/1027071098597.html BBC NEWS (Whitehouse D)web.news.bbc.co.uk/2/hi/sci/tech/2135779.stm
This result has profound consequences for any chemical or physical process that occurs over short times and in small regions In systems away from equilibrium over a finite time t the ratio between the irreversible entropy production probability takes on a value A and the probability that it takes the opposite value A will be exponential in At. The virtual time reversal processes which apparently applies to relatively large sized particles, e.g. micron-sized plastic beads towed through water, suggests that the reverse-entropy process will always affect the operation of mitochondria and smaller biologically relevant chemical reaction mechanisms.

Nucleic Acids & The Putative Role of Negative Entropy in Biology The now universally biologically-employed nucleic acid genetic code (and the acknowledged likely prior existence of RNA which predated DNA) which by their

templated reproduction systems obviously tend directly to inhibit the decay of the twodimensional crystal information held therein, seems, however, be required by the second law of classical thermodynamics to be subject of a process of relatively rapid decay. Or at least to decay sufficiently rapidly so as to cause even the slight errors which are known to devastate the homeostasis systems present in all complex organisms. This decay will, if left unhindered, clearly defeats life. This pathological decay is usually thought to arise because of reactive oxygen or nitrogen based free radicals etc. and systems of antioxidants and antinitrants are evidently in place to meet such threats. This decay does not, however, happen as easily as might have been expected even in the absence of sufficient antioxidants. Why? The conventionally understood mechanism of double helix unwinding + templating ability of repair mechanisms plus antioxidant, heat shock system etc. afforded protection may be assisted somehow by the nano-machines which can make use of the Evans anti-entropy force which could be inherent in the existence within the nucleus of hydrated nucleic acid helices and the similar existence of inorganic polyphosphates and polysaccharides at membrane surfaces. These polymeric system may per se confer virtual time reversalin-small-engine processes which act to additionally protect DNA so as to allowed the cherished protein sequences to have been retained over geological time in opposition to the second law of thermodynamics which demands that there must arise an increase of entropy over this time sufficient to completely delete all such information many times over. The Wiggins LDW may also be part of such reverse entropy protection and (putatively) exists per se in (a short-lived flickering cluster) helical or equivalent forms having intrinsic anti-entropy properties.
Anti-Disease Helical Anti-Ageing Water Structures?

The (Evans) negative entropy vs. time process or force negates the natural second law decrease in order over time and could help to create the kind of increase in order over time which typifies biology. In absence of this force disorder must overwhelm any evolution of living organisms. The force will tend to apply more weakly {although perhaps still be of great significance for sub-system components} (as required by the Fluctuation Theorem) to the much larger complex multicelluar organisms generated by the association of cells. The older view of thermodynamic laws would have demanded a total erasure of ancient nucleic acid genetic information. Part erasure is DNA damage, where this occurs in the part of DNA which is transcribed into proteins is believed to be associated with e.g. neoplasia and ageing. Where it occurs in what was formerly thought to be junk genes similar damage is likely also eventually to arise. The process of cellular (e.g. oncogenic) transformation is, furthermore, apparently accompanied by a major change in the water structure of the affected cell (Wiggins 1996). Alteration in water structure may then be regarded as an indication of heath. This essentially is what magnetic resonance imaging (MRI) picks up.: the abnormal water structure associated with pathology. The ability of biological species to retain intact genetic information over long periods of time can now be understood in terms of the (not yet widely acknowledged) Evans (1992) Fluctuation Theorem which, it is now proposed, can suggests the mechanism by

which the anti-entropy opposition to the second law of thermodynamics may be achieved. This mechanism evidently depends on water differentiation into different structures within pores to avert normal entropy vs. time decomposition processes in such environments Some very simple ancient pore-forming systems are now suggested to be retained by modern organisms in order to inhibit the various types of life-threatening irreversible organic and organometallic molecule decompositions. An example of such a system could be the poly-Ca-phosphate which is especially associated with mitochondria. Such diseases as chronic fatigue and autism may be associated with mitochondrial dysfunction. Is The Reusch et al. Double Helix a Pro-Life LDW Piezoelectric Force Transducer ? Inorganic polyphosphates have been observed [e.g. by Reusch et al.] to occur ubiquitously at membranes throughout biota in conjunction with Ca2+ and polybetahydroxybutyrate conjoined in a (putative) twinned helical structure. This helix-generating polyanion composite systems and their associated water structures could have been a further prime pre-life factor during the pre-nucleic acid stage of early biology. It seems potentially to be part of the mechanism by which pro-life water structures might have been produced. While not known if this applies to the surfaces of modern cells, when studied separately, these polymers have demonstrated intrinsic piezoelectric properties. This perhaps bizarre coincidence, which may be worth of further probing is this intertwined double helix composite system. Perhaps an ability of such systems to generate electrical or proton currents from applied mechanical forces and vice versa could have given a pro-biotic evolutionary boost to the ability of polyinorganic phosphates to modulate the known abilities of such polymers to control the water structure and control the induced seeding of Ca2+ plus CO32- , HPO42-and so allow for an enhanced modulation of this process in the presence of additional adsorbed organic molecules so as to produce a controlled aggregation and particle formation mechanism which may have aided the creation of polypeptides, sugars, glycans, etc. in response to external mechanical forces.

INSERTED DOCUMENT 3 Liquid water might be considered to be a Van Wazer type of stochastic system analogous to polysilicate esters (cf. Grant D. Inorg. Nucl Chem 1967 29 69-81) in which an essentially infinite number of types of unstable hydrogen-bonded aggregates can arise by random scrambling of the hydrogen-bonds. This type stochastic reorganization process also creates the glassy state found in common sodium silicate glass. Silica gels when formed in the presence of individually shaped organic molecules achieves high selectivity for the absorption from solution of the solute molecules which were present during gelation apparently because it contians pores of the specifically matched morphology. Silicic acid (which can commonly arise in natural waters and may be especially enriched in volcanic hot springs) self-assemblies

into (putatively self-seeding-of-pore-structure) cells of colloidal sol aggregates and also into glassy silica gels; these stochastic pore-containing systems can also form adducts the hydrogen-bonded aggregates which are present in liquid water from which specific structures can apparently be sequestered within the randomly sized and distributed pores. Such gels might conceivably thereby select specifically shaped (albeit fragile) water hydrogen-bonded aggregates analogously to how the silica gels can act as a specific adsorbing/templating system for organic molecucles. This mechanism of selective absorption seems to possibly explain why water in silica gel pores can become enriched in the Wiggins pro-life low density form of liquid water (LDW). (LDW can also, however, become selectively enriched in water containing pores present in polyamides and polysaccharides; [ the polysaccharides may have been the first biopolymers to become evolved perhaps because they could perform the biologicallyessential LDW enrichment process without the aid of silicates {it may however also be of interest in this context, however, that many or perhaps indeed all polysaccharides still occur naturally in a close association with some form of inorganic silicate}]. Such LDW was shown by Wiggins to demonstrate a wide range of primitive quasi-enzymic biological activities (being able e.g. to selective absorb and (dehydrate so as to) link together various hydroxylated and amine-ated organic molecules (and thereby to synthesize peptides and polysaccharides, as well as being able to correctly selectively absorb the correct L and D enantiomers from mixtures to create true biopolymers, to provide a transduction mechanism for high energy P-O-P bond energy utilization and to select K+ from Na+ in mixed salt solutions (as well as accomplish a range of similar ionic and molecular discriminations). The self-assembling hydrated silica gels and related sols were likely to have been highly favourable environments in which the evolution of early life on earth could have been accomplished. The co-adsorption within silica gel and sol pores of the range of organic molecules which naturally occur in space, are present in some comets and meteorites and could arise following electrical discharge activation of the small organic molecules plus water, ammonia, carbon dioxide, hydrogen sulphide, phosphine and methane which may have been present in the earths atmosphere, could have provided the starting point mixture to allow the LDW to create the first biopolymers which might have added to the complexity of the host cellular silicate-based systems. The inclusion of the easily-decomposed organic molecules within gel pores also provided a natural protection mechanism against ionizing radiation. At a point of build up of sufficient complexity we then have a primitive form of life. The reproduction of such cells was apparently possible without the organic matter being present but the inclusion of organic polymers seems to have allowed greater flexibility, this allowing a carbon-based life to eventually to become master of the original host silicate-based cellular systems.

INSERTED
DOCUMENT 4 Hydration water structures of sparingly soluble Ca salts: possible role in pre-biological evolution biological energy provision and inorganic ion homeostasis and related induction of water structure. DG 28/8/12

The Hofmister series of ions (which was originally suggested as an index of protein denaturation) is believed to constitute a ranking of individual solutes and especially ions which can additively achieve an alteration in the water structures which arise from the inter water molecule hydrogen- bonding system. This phenomenon has been apparently harnessed by biology. And may have led to the evolution of biological species in the first place (by putatively allowing for the regulation of the Wiggins HDW/LDW liquid water microstructure system a central mechanism which has recently been proposed to be the most fundamental process underpinning life). The formation of high energy water in the pores of gels (such as silica gel and putatively also in the pores in aggregated sparingly soluble Ca salt particles e.g. containing hydroxide, silicate, carbonate, sulphate, phosphate and fluoride) can putatively provide model systems which create a biological-mimicking system of separation of Wiggins HDW from LDW and allows the high activity of LDW to perform biological energy transfer including protein folding utilization for the performance of work. A related basic phenomenon needed by biology is the control of sparingly soluble Ca salt formation and pH by polyanions. Related further to most basic-for-biology need for the control of high and low density water by polyanions. The above need may have developed as a consequence of a general principle illustrated as follows. During the (spontaneous) precipitation of calcium by the carbonate anion are formed a series of phases with have progressively decreased enthalpies and free energies: related further is phase hydration and putatively to the creation of a system of high and low density water by the carbonate anion and the aggregated carbonated anions in initiallyformed hydrated aggregates. These may act as seeds for the build up of (semi-) amorphous particles a process which is (essentially reproduction of form similar in principle perhaps to biological reproduction) illustrated especially by the first-formed amorphous calcium carbonate hydrates which can give rise (by energy loss) to (progressively less hydrated) anhydrous amorphous calcium carbonate which eventually leads to lower energy forms of crystalline calcium carbonates ({progressively} vaterite, aragonite and calcite). Cf. Radha AV et al. PNAS. 2010 107 164-43. Of especial relevance is the circumstance that the initial (formally amorphous structured) formed phases (which are formed during spontaneous calcium carbonate (and putatively other oxy-anion precipitations and post-precipitation structural modifications) are (highly?) hydrated. {Cf., more generally initially formed phases calcium silicate, polysilicates, phosphate, polyphosphates, phosphite, (possible roles of phosphonate and glass forming polyphosphonates) sulphate, [sulphide, however, is {colloidal}iron-related and free electronic control detemined rather than hydrogen bond protonic control determined], borate also hydrated? And also (semiamorphous?}. Cf. the use of present-day use (by all) organisms of high molecular polyphosphate (at membranes) and by some (only?) organisms of amorphous (polysilcicates). And the co-presence of silicate, phosphate, carbonate and adsorbed H3O+ etc. at the surfaces of heparin (a model for heparan sulphate which in turn is a model for the surface polysaccharide system which regulates protein activity and wide range overall all-system regulation and homeostasis in animals).

The hydrogen-bonded potential energy in sparingly soluble amorphous, semiamorphous and regulated crystalline Ca salt hydrates is likely to have a fundamental biological role and relevance (both for how biology arose in the first place and how the use of Ca by biology continues in modern biological systems and be further related to how liquid water in the high and low density form provide a basic energy transduction mechanism for biology (Wiggins) a process in which the regulation of the formation of sparingly Ca salts may be considered to be of central relevance. Modern natural waters contain soluble and colloidal humic matter (humic and fulvic acids) which are potent inhibitors of the seeded crystallization of CaCO3 (calcite) as well as other sparingly soluble phases. This inhibition appears to be a major possible mechanism of seawater homeostasis of inorganic ions including carbonate. Also putatively is involved in atmospheric chemistry (via CO2 homeostasis). The present-day humic polymers may conceivably have had pre-biotic equivalents e.g. formed form formose of polybuyrate. Perhaps then the carbonate anion is of especial primary significance as natural organic (carbohydrate-based) biopolymers with carboxylate carbonate anion-mimicking) It is of especial interest that the surfaces of adherent biological cells commonly contain anionic polysaccharides (perhaps the most primitive of these contains carboxylate carbonate anion-like anionic groups which can be considered to act with respect to the control of the precipitation of calcium carbonate. For multi-cellular animals (commonly considered to have evolved in seawater and which continue to re-create this specific water structure system, cf. the ionic content of human blood serum) the anionic polysaccharides occurring at membrane surfaces and in extracelluar space include the glycosaminoglycans which contain sequences ( information-encoded) in the form of various (metal ion associated} O-sulphate and glucosamine 2Nsulphonate (these are strongly ionized and the less strongly ionized carboxylate uronic acid groups which occur in an evidently more highly evolved from in multicellular animals as iduronate (evidently an improved form of glucuronate which is its biological precursor evolutionary which is a process repeated during biosynthesis These are polyanions showing specific metal ion dependent hydration as well as metal ion sequestering and inorganic (including calcium) crystal formation and inhibitory and structural modulation properties.

INSERTED DOCUMENT 5 Reconciling Kornberg and Wiggins theories of the beginning of life. How polyphosphate and polysilicate could be part of a more general definiton of life DG August 2012 The now universally biologically-employed nucleic acid genetic code may actually reinforce an earlier system of how life more-or-less always attempts to opposes the second law of thermodynamics. This anti-increase-in-disorder force seems to underpin all life.

This force can, however, be evidenced non-biologically. It can be seen in an ability of (abiotic) seeded systems to reproduce complex forms, a phenomenon which seems to exist in physical chemistry outwith biology and which e.g. can enable sparingly soluble Ca salts crystals, e.g., of CaCO3 (which exists in hundreds of different forms) to be capable of self-seeding [putatively by a templating process which includes sloughing off of parts of parent particles allowing the production of further particles with the same distinct morphological forms]. The ultimate mechanism behind the existence of biological species could therefore be purely inorganic. [According to Lima-de-Faria this is the seeding of crystallization, a repoduction processes which arise without the assistance of genes which indicates that life is not any unique property of DNA or RNA but, instead is a consequence of the fundamental property of particles to engage in the seeding of unique form (which is however a process which is not yet fully understood) which can be accomplished by the presence most commonly in liquid water (e.g. purely inorganic seeds e.g of CaCO3 when added to aqueous solutions containing Ca2+ and CO32- ions produce growth on the srufaces of pre-existing CaCO3 particles or on the surfaces of shed CaCO3 seeds where the latter reproduce the morphological forms of the former)]. [A similar seeding of phosphate copolymers with sugars (perhaps byproducts of Ca polyphosphates) may conceivably have, in early pre-biotic situations, engendered the formation of the precursors of nucleic acids]. Arthur Kornberg has argued that {high molecular weight} poly-inorganic phosphate (originally thought to be present in modern species as a molecular fossils but which) occurs ubiquitously at membranes throughout biota (albeit in conjunction with Ca2+ and polybetahydrxybutyrate at least in some and perhaps in all instances [cf. Reusch]) could have been the prime factor which enabled the appearance of early life. {A bizarre perhaps coincidence which may be worth of further probing: both the polybetahydroxybutyrate and the Ca polyphosphate with which it is believed to form an intertwined helix system at the surfaces of modern cells, have peizeoelectric properties. Perhaps this ability to generate electrical current from force and vice versa could have allowed a pro-biotic evolutionary boost given to the ability of polyinorganic phosphate to interact according to circumstances so as to control the seeding of Ca2+ plus CO32- , HPO42-and allow a modulation of this process with adsorbed organic molecules so as to produce controlled aggregated quasi cellular particle formations}. Life also, it seems, for its start-up have also critically depended on the presence of systems for separation of HDW from LDW (vide infra) Some naturally formed self-assemblies of particles can do this. These may include the obovementioned polyinorganic phosphate composite systems. These which could in the early earth perhaps have especaily allowed the separation out of the two chemically distinct (necessary-for-life) forms of water which were identified by Wiggins. This can happen in pores. This suggests that the Kornberg polyinorganic phosphate could have arisen in the first instance by the pore water mechanism together with an ability of the polyphosphate associated pore water to create the biological precursors of first phosphodiester ribose molecules. While modern biologically and industrialy produced polymers can produce the necessary pores for life required water transient phase separation, the ability of pores in purely inorganic systems which occur abundantly in natural waters, could, it is now

suggested also have produced the same necessary-for-life kind of stretched LDW water which was identified by Wigins et al. and proposed by this research group to have created the first life. This was obvioiusly of especialy relevance to pre-biotic evolution since the polysaccharide or protein based organic gels (which formed the main focus of Wiggins et al.s later researches) could surely not have existed (at least in sufficient quantities) before the appearance of the first biological cells. This circumstances does not apply to clays, silica gels and sols which can arise entirely by abiotic processes. Pore water in hydrated silica gel can (as reported by Wiggins in 1973) seems to be the same type of LDW which can catalyse the formation of P-O-P high energy phosphate bonds and together with the ability of silica gel can sequester and link together (as well as be highly protective of) the relatively fragile organic molecules which may have been formed from electrical discharges or deposited from comets etc.; these gels may have collected the various pre-biotic organic substances and engaged in random assembly of the builidng blocks of life. A futher ability of silica sols{which however is currently pooly understood}is that these silicic acid aggregates can per se engage in self assembly by a seeding process which is analogous to that demonstrated by CaCO3 particles {vide supra} (i.e. by a process which similary mimicks biological cellular reproduction without DNA orRNA); this cellular self-seding of form could have been a further critical for life event and be the primary origin of how pre-biotic evolution started off in the first place following the generated a range of different species silica sols and gels which could reproduce their own unique forms by the universally applicable inorganic self seeding morphogenic phenomena. {The silica sol particle systems were formally thought to be entirely amorphous [at least to the traditional use of X-ray diffraction to identify well-developed crystallinity] but could contain low-order crystallity or even some other type of order (perhaps a discriminated chaotic type of order which like pure crystaline order is also capable of the seeding of daughter particles)}. Restatement and more. The first living cells must, it is to be assumed, have contained water plus a system to separate out HDW and LDW; (cf. Philippa M Wiggins: LDW/HDW water in pores {which included polysaccharides but also included entirly inorganic} silica gel pores} has the ability to selectively absorb D-sugars from D+L sugar mixtures (and also select L-amino acids from D+L amino acid mixtures) further indicated how this critical role of water in pores was a probable cause of the pre-biotic or early biotic selection and enrichment processes which led first of all to the currently observed chirality of biology and also allowed the evolution of highly complex systems which eventually created by random selection processes which gave rise to nucleic acid containing cells). This shows up of course why water is the essential and principal chemical substance present in all living cells. Water plus the additional essential system to tune into the unique physiochemical properties of water in order to enable the essential properties of living organisms which are perhaps first of all the ability to act counter to the second law of thermodynamics (i.e. to move in non-random Brownian motion manner [cf Yangs demonstration of Evans fluctuation theorem] which creates or reflects an extrathermodynamic (separate from non-living systems) dimension in space-time [life attmpts to counter the second

law {life putativley accesses reverse time via enthalpy-entroppy compensation which shows up e.g. in seeded nucleation of form which seems to enable life also to reproduce true to form in manner which [controversally] uses reverse [Evans] time to evolve more complex systems which generates separate species this is [controvesally} in additon to the engagement in competitive activities which encouraged which increases the evolution of system complexity}]. The nucleic acid based systems of RNA or DNA is, of course, commonly believed to be what defines life. A more general definition can, however, can be attempted according to the above discussion. The [Wiggins] definition of life: the unique chemical and physical properties of liquid water are seen to be what is ultimately essential according to this alternative hypothesis. This leaves out DNA or RNA since it can be logically deduced that DNA or RNA or indeed proteins could simply be additional facets of currently observed terrestrial life forms (even although they are believed to have been retained by earths biological systems for e.g. four thousand million years). Liquid water, as outline above, is believed to support life and was likely to be the prime for-life essential component from the beginning of biology on earth. Life, it seems, further critically depends on the existense of (at least?) two kinds of water structure. Low density water LDW and high density water HDW. Low density water LDW was identified by Philippa A Wiggins in Auckland New Zealand. LDW (pore water e.g. in inorganic as well as conventional biologically produced gels) can engender the formation of P-O-P bonds starting from inorganic phosphate P-OH. This process was of likely critical imprtance ot how life started Life putatively arose in a purely inorganic-system which self assembled suitable pores and/or surface systems which had an enhanced ability to make LDW. This seems ultimalty also to be and how living organisms continue to be able to access energy from ATP a basic process which seems to require hydration to tranfer the energy. The Wiggins theory of life suggests that the most bsasic definition of life is that it depends on the selection between two types of water which occur in rapid equilibrium in liquid water from which it is, however, possible by use of gel forming systems to separate out these two chemically distinct forms, one being more active than the other (e.g. the active form of water is able to remove the elements of water from simple molecules to produce e.g. P-O-P bonds {and also putatively to synthesis polysaccharides from simple sugars sugar-phosphodiesters etc. and polypeptides from amino acids and also to act as an inorganic ion selector e.g. allowing K+ to become concentrated inside cells a nd leaving Na+ outside}. These abilities are probably why liquid water is the single most essential chemical component of biological cells and the separation of water phases was necessary for the evolution of the first kinds of pre-biotic cells. The role of ATP and nucleic acids also seem from the view point of the analysis of current biological systems to be a critical component of all living cells. A lesser known additional occurrence at the surface of all current biological cells however is polyinorganic phosphate. [as has been discussed by Arthur Kornberg]

This seems strangely to be associated with (perhaps also ubiquitously) with polybetahydroxy butyrate again perhaps in association with calcium ions]. This biopolymer system (and the nucleic acids together with other biopolymers in general) could conceivably retain an ablity inerited from pre-biotic precursors to separate out the separate pahses of water. While the nucleic acid systems including ATP, RNA and DNA seems to be (modern) life essentials but logical deduction suggests that these systems were unlikely to have been in place in the first living cells which may have contained the much simpler polyinorganic phosphate and perhaps also to polybetahydroxbutyrate and calcium (as ATP equivalent systems and perhaps also memory systems for handing on cellular uniqueness to new generations of early cells).. Could the polyinorganic phosphate-polybetahydroxybutyrate have in the first instance of pre-biology the necessary water tune-into system. (Equivalent to the pore systems studied by PM Wiggins which distinguish high and low density water [HDW LDW] An important mechanism enabled by water is the selection of D over L sugars (and the corresponding process of amino acids). While this has also been found to be a propoerty of calcite the similar ability of liquid water in pores may have been the more important. Thus the chirality of biology likely derives from a fundamental selection process afforded by pore water. The pores however, can occur in a purely inorganic substrate e.g. in silica gel or in clay interlayers. The prior existence of RNA predating DNA. The circumstance that RNA contains ribose ( which is acknowledged to be a likely evolutionary proecursor of deoxy ribose) is more complex, thus more highly evolved sugar; hence this system needed the pre-existence of simpler (formose) sugars and thererfore it evolved later. Hence this idea that life is DNA-essential for evolution is irrational. The evolution of an improved version of RNA needed the existence of deoxyribose which needed pre-existing active water processing (quasi or actual enzyme) systems The prior existence of sugars seems, however also a prime critical logic block: a necessary deduced requirement to allow RNA to evovle in the first place. Hence the system of simpler sugars and polymers based on these (with enzymic acitivites?) must have existed (in quasi-biological cells) before nucleic acids could evolve. This may mean that biological cells containing sugars and polysaccharides could have exited without proteins or nucleic acids since their presence is a pre evolution requirement for the creation of the protein - nucleic acid evolutionary process which eventually became necessary to conserve the by now highlyt complex special information allowing the numerous types of proteins to be acurated reproduced and be passed on down the generatios. Perhaps early polyamino acids (such as the Fox proteinoids) evolved in parallel with polysaccharides and nucleic acids.

The laws of chance suggest that the simpler the biopolymer than the more likely would its probability of it being produced by random events. Consider the circumstance that the stoichiometry of the sugars is the simplest of any biopolymer (cf. the carbohydrate [CH2O]n system is more likely to form than are alpha amino acids which in turn are more likely to form than are nucleic acids. Especailly stoichionmetrically simple are the polyinoganic phosphates O-{P(O)[O-]}O- ; PHB is [O-CH(CH3)C(O)-]n being less simple that the polymethylene containing (CH2)n-[CO]{C(O)O-) soil humic matter system constituent (Grant) which perhaps was also present in pre-biotic soils. Stoichiometry can of course be misleading. H2O seems very simple but Wiggins and others have shown that this apparent simplicity hides an extreme complxity of hydrogen-bonded supramoleuclar complexity.

The definition of life must somehow invovle consideration of how life opposes the second law of thermodynamics. Life opposes the positve time direction. Negative time processing seem implicit in the phenomenon of life Thus the thermodynamic laws which would have caused decay of information are effectively aborted over thousands of millions of years by the process of cellular reproduction and especially but not exclusively by the use of the genetic code. Life seems [controversally] to use reverse time accessed by entropy-enthapy compensation [which may be part of how complex structures are formed following their seeding].

INSERTED
DOCUMENT 6 How polyphosphate could be part of a more general definiton of life The definition of life must somehow invovle consideration of how life opposes the second law of thermodynamics. Life opposes the positve time direction . Negative time processing seem implict in the phenomenon of life Thus the thermodynamic laws which would have caused decay of information is aborted over thousands of milliond of years by the process of cellular reproduction and especilaly by the use of the genetic code. The nucleic acid code may actualy reinforce the ability of seeded systems to reprodce complex forms which sems to exist with the system of sparingly soluble Ca salts, e.g. CaCO3 which exists in hundrreds of different forms which are capable of self seeding ditinct forms without the assistence of genes The ultimate mechanism of the existence of biolgical species seems [according to Lima-de-Faria] not to be due to DNA or RNA but to be about how seeding of form can be accomplished by CaCO3 seeds added to aqeous solutions containing Ca2+ and CO32- ion.

Ca2+ phosphates in similar seeding were also likely, however to have been invovled in early pre-biotic situaitons which could have engendered nucleic acids. A Kornberg has argues that poly-inorganic phosphate which occurs ubiqiteously (albeit in conjunction with Ca2+ and polybethydrxybutyrate at least in some instaces) throughout biota was a prime factor for the appearanc of early life. PA Wiggins had shown that pore water (e.g. in inorganic as well as conventional biologicaly produced gels) can create P-O-P bonds starting from inorganic phosphate. This suggests that the Kornberg polyinorganic phosphate arose in the first instance by this mechanism. That silica gel can apparently create the P-O-P high energy phsopahte bonds suggests that silicic acid genrated silcia gels acted in this way inearly pre-biotic seas. Thus nucleic acid based sytem is commonly believed to be what defines life. A more general definiton can also be attmepted however which leaves out DNA or RNA since it can be logicaly deduced that DNA or RNA or indeed proteins are additional facets of currently observed terrestrial life forms (although they are believed to have been retained for fourthousand millon years). Water is, however likely to be the prime essential compnent. Water is believed to support life. The Wiggins theory of life indicates is that it depends on the selecotion of two types of water which occur in rapid equiobrium in liquid water from which it is, however, posible by use of gel forming systems to separate out these two chemicaly distinct forms one being more active than the other (e.g. it is able to remove water from simple molecules e..g to produce P-O-P bonds (and also puatively to synthesis popysaccharides from simple sugars sugar-phosphodiesters etc. and polypeptides from amino acids and also to act as an inorangic ion selector e.g. allowing K+ to become concentrated inside cells a nd leaving Na+ outside). These abilities are probably why liquid water is the single most essential chemical component of biological cells and was necessary for the evolution of the first kinds of pre-biotic cells. The role of ATP and nucleic acids also seem from the view point of the analysis of current biological systems also to be a critical component of all living cells. A lesser known additional occurrence at the surface of all current biological cells however is polyinorganic phosphate [as has been discussed by Arthur Kornberg]. This seems strangely to be associated with (perhaps also ubiquitously) with polybetahydroxy butyrate again perhaps in association with calcium ions]. While the nucleic acid sytems incuding ATP, RNA and DNA seems to be (modern) life essentials but logical deduction suggests that these sytems were unlikely to have been in place in the first living cells which may have contained the much simpler polyinorganic phsphate and perhaps also to polybetahydroxbutyrate and calcium (as ATP equivalent systems and perhaps also memory systems for handing on cellular uniqueness to new generations of early cells).. Could the polyinorganic phosphate-polybetahydroxybutyrate have in the first instance of pre-biology the necessary water tune-into system. [Equivalent ot the pore systems studied by PM Wiggins [PM Wiggins showed that water in pores {which included polysaccharides but also incding inorganic substance e.g. silica gel} pores} had the ability to to selecively absorb

D-sugars from D+L sugars (and also L-amino acids from D+L amino acids further indicating how this critical role of water in pores was a probable cause of the pre-biotic or early biotic selection and enrichment proceses which led first of all to the currently obsrved chriality of biolgogy and also allowed the evolution of highly complex systems which eventually created by random selection processes which gave rise to nucleic acid containing cells)] The first living cells must, it is assuimed have contained water plus a ssytem ot separate out HDW and LDW. This shows up of course as the principal chemical substance present in all living cells. Water plus a system to tune into the unique physiochemical properteis of water in order to enable the essential properties of living organisms which are perhaps first of all an ability to act counter to the second law of thermodynamics (i.e. to move in non-random manner which creates or reflects an extrathermodynamic (separate from non-living systems) dimension in space-time; also to reproduce true to form; also to evovle more complex systems which generates separate species which enagae in competive activities which encouraged the evolution of increses system complexity. An imporant mechanism afforded by water is the selection of D over L sugars (and the corresponsing proces of amino acids). Thus the chirality of biology likely derives fromthis fundamental selection process afforded by water. This however is water in proes. The pores however, can be inorganic e.g, silica gel. or clay. The prior existence of RNA predating DNA. The circumstance that RNA contains ribose which is a likely evolutionary proecursor of eoxy ribose is more complex, thus more highly evovled sugar; hence this evovled later. Hence this idea that life is DNA-essential for evoution is irrational. The evolution of an improved version oif RNA using the deoxyribose The prior existence of the sugars seems, however alaso a critical logical a necessary deduced requirement to allow RNA to evovle in the first place. Hence the system of sugars must have existed (in biological cells) before nucleic acids could evolve. This may mean that biological cells containing sugarrs and poysaccharides could have exited without proteins or nucleic acids since their presence is a pre evolution requirement before the nucleic acids could evolve. Perhaps however some early polyamijno acids eveolved in paralled with poysaccharidees and nucleic acids became necessary to conserve the special information allowing the proteins to be reproduced as required and the informationpassed on down the generations. The laws of chance suggest that the the simpler the biopolmer the more likely would its probability of its being produced by random events. Consider the circumstance that the stoichiometry of the sugars is the simplest of any biopolymer (cf. the carbohydrate CH2O system is more likely to form than are alpha aminoacids which in turn are more likelky to form than are nucleic acids.

INSERTED
DOCUMENT 7 The generation of specific species of silica sol by seeding from inorganic alkaline earth salts, suggests that these salts were the original genes Hyde et al (2004) showed that barium carbonate (witherite Check) could precipitate a highly structures witherite/ silica acid microstructured aggregate which mimicked the microscopic appearance of supposed very ancient (32.8 billon year) terrestial microfossils and apparently also Martian fossil form a meteorite. Hyde et al (loc cit) also mentioned that calcium (carbonate) could also precipitate (similar?) aggregates. Silica sols have been proposed (Grant 2004, cf Grant et al 1992) to be the originators of biology. This was suggested to have been accomplished by their competitive reproduction and subsequent Darwinian evolutions (initially by competition between species of sol for silicic acid nutrient). The greater propensity of tetracoordinate silicate units for forming a wide range of structured nanoparticulates seems the most rational and reasonable explanation for silica sols to be the precursors of living organisms as they provide self asembling system capable of generating the greatest degree of complexity via competetive evolutionary pressures. Different silica sols (species) are probably produced in industrial plants as an accidental consequence of differences in trace substances present during the polymerisation of silicic acid. These may include inorganic or organic substances from ion exchange columns. Inorganic carbonate (carbonic acid) will arise form dissolution of atmospheric CO2 in the alkaline solutions used to cook the silica sols to final form. (Could trace inorganic calcium (or even other alkaline earths or other salts) be present in plant water or in some technical sodium hydroxide solutions?). A similar mechanism in prebiotic environmental niches is hypothesised to have enabled the formation of a range of silica sol species. The presence of trace alkaline earth carbonates during the process of silicic acid polymerisation in the formation of silica sols could have generated (seeded) the formation of specific sub-crystalline aggregate structures which were capable of replication (e.g. by cellular scission or by otherwise generating seed particles). The most likely generator of microstructures by seeding seems most likely to have been calcium carbonates (perhaps basic calcium carbonates) or phosphates (perhaps basic phosphates or even phosphites or similar basic strontium (or barium phosphates or phosphites). (The possibility of phosphites arises if it is considered that the early earth had a reducing atmosphere; phosphorous acid may be produced from phosphine which has been detected on Jupiter (Check)) The presence of (basic) calcium phosphate/carbonate (apatites) as seeds might have been the ultimate ancestor of the use of phosphate in biology as nucleotides (e.g. ATP and nucleic acids).

The simpler salts suggested above may, however, have been the primitive type of genes associated with primitive quasi-biological systems, as suggested by the present hypothesis. The widespread use of calcium ions as second messengers in biological systems (and calcium intracellular concentration oscillations also having some fundamental function) suggests that salts of the inorganic carbonate/phosphate etc salts (but perturbed by the presence of a range of foreign elements?) rather than other alkaline earth salts were the proposed ancestral genes. A major interaction between (extracellular) anionic polysaccharides and calcium ions which include the modulation of the crystallisation and creation of specific microstructures of calcified aggregates seems to be a linked topic (cf Long & Williamson 1979; Grant et al 1987, 1992). The incorporation of gallium into the structure of silica sols is a possible mechanism for creating altered microstructure. Possible gallium oxide at the surface of gallium metal which has been reported to create unusual amorphous silica wires (JACS 124 1812) [metallic gold etc., however, seeds the production of single crystalline silica wires]. A possible role of gallium in biological sislioca including its putative role in early evolution is worth considring. References JACS 124 1812 Grant et al Biochem J 1987; Med Hypoth 1992 38-40 et seq Hyde et al (with Carnerup Christy and Larson et al Scince 2003 302 1194-7 Cf New Scientist 2003 article (22 Nov 14-15) Cf also Brasier M et al Phil Trans Roy Soc B 2006 361 887-9092

INSERTED
DOCUMENT 8 Draft of a communication by D Granta Inorganic element association may contribute to heparin/heparan sulphate functions in vivo. (Footnote a) a Results from researches conducted when this author was a Research Fellow at Aberdeen University]
(With thanks to RJP Williams [University of Oxford] for discussions and suggestions relating to the reporting of this topic)

This communication suggests that the simultaneous association of a wide range of inorganic elements to glycosaminoglycans could be physiologically relevant and furthermore that determination of the multi-element content of these polysaccharides could serve (in an analogous manner to the use of hair) as a diagnostic marker of inorganic ion dyshomeostasis or intoxication in animals including humans. Recent studies have revealed possibly important nitric oxide/metal ion oligosaccharide signalling systems which might be relevant to both the biochemsitry of plants b and

animals where primed heparan sulphate chains generate oligosaccharides in a redoxCu2+/Cu+ (and poorly understood Zn2+ ion) dependent process of oligosaccharide generation. This inorganic redox system may have a feedback potential linked to the heparanase dependent enzymic scission process. The growting list of other reported metal ion dependent heparin/heparan sulphate functions include Ca2+ binds to heparin at a physiologically relevant affinity a consistent with the requirement of Ca2+ ions for annexin V binding to heparin (which may be a model for how heparan sulphate promotes apoptosis) as well as apparently to correctly allow heparan sulphate assembly of basic fibroblast growth factor receptors. Heparan sulphhte metabolism also appears to be dependent on Mg and Mn, Sr may affect glycosaminoglycan biosynthesis in the context of bone mineralization control and Pb and Cd have been reported to adversely affect heparan sulphate biosythnesis Footnote b Recent findings also suggest that some analogous reactive nitrogen dependent nonenzymic scission may also contribute to oligosaccharide (saccharins) signalling in plants which, originally thought be a hormone like action, is now thought to be dependent on a Ca2+ second messenger modulation mechanism.

INSERTED
DOCUMENT 9 Apple II document DG 8/12 Jan 20 1997 Re-processed 8/8/12

Water and Life

(dg8/2)

We propose that life arises by interaction between systems capable of generating large numbers of structures (the numbers approaching infinity) for periods of time tending towards infinity. A suitable buffered environment, however, including agents to prevent unwanted crystallization would have been critically required top have been long-term maintained. Thus life will surely arise by the random assembly of components, being especially the consequence of interaction between polymer systems with an inherent randomness of sorting such as polyanionic silicate glasses. Silicates and phosphate stochastic structural rearrangement systems have been quantified and water may also be reasonably described as a random hydrogen-bonded cluster distribution. For sodium silicates the ratio of capping-off Na+ to Si determines the possible building blocks which sort themselves out if allowed to do so without the interruption of nucleation of crystal formation, into a large number of individual kinds of molecules ; if actually completely random this number would be infinity. Water has hydrogen-bonded aggregates which demonstrate unusual electrical properties including potential for conduction and switching. The, highly flexible, rapidly rearranging

supramolecular structure of water is apparently modifiable by adjacent surfaces. Thus an array of water molecules shows commercially efficient proton conduction. Water can be thought of as a potential source of electronic components capable of being randomly selected onto a randomly arrangeable silicate circuit board. Random assembly requires the prevention of nucleation of crystalline phases. Modern living organisms do this very efficiently and so it would have been in the beginning polyphosphates are actually especially good at this. Concepts which are not those to which mainstream science is currently devoted, where apparent facts do not seem to fit neatly into established theory, should perhaps be perused for clues about the fundamental role of water in life processes. Perhaps the most important possible property of water is where some kind of templated nucleic-acid like information might be preserved somehow in a non-biological water-rich colloidal system, albeit at a much lower level of detail and accuracy than DNA. It has been frequently assumed that DNA was necessary for life to start; (thus, Orgel modelled prebiotic oligomerization of nucleotides at hydroxylapatite surfaces). But perhaps reproduction of prebiotic cells could have occurred without DNA. Cairns-Smith has suggested silicate minerals provided DNA functions; DNA is then seen to be a highly evolved improved mechanism of information storage. Other hypotheses of key prebiotic chemistry centre on pyrites (Popper, 1982) ; clearly extant oxidation-reduction mechanisms will reflect early available environments, but this seems an evolved rather than a key prebiotic cellular process. Another difficult area of science is those systems seeming to breach the second law of thermodynamics; this could conceivably allow systems of increased complexity to occur spontaneously. There are known to be, but by quite unknown cause, a compensation effects between entropy and enthalpy terms in the rate and similar processes for certain types of chemical reaction and other processes. Where this occurs there also is a characteristic temperature where, e.g., a series of compensated chemical reactions proceed at the same rate, including those between molecules dissolved in water; in fact the occurrence of compensation in aqueous solution is sometimes said to be the most easily explicable compensation situation since the properties of water as so quite unusual, it being the most anomalous liquid known. We suggest that life, at least for multicellular organisms, shows the existence of a characteristic temperature making us look for some compensated effects of key reactions especially involving water molecules. The usual homoisothermic temperature for us and many of our fellow creatures is of course 37C which may favour reactions between water clusters leading to release of hydrogen-bonding energy to biological substrates. Compensation behaviour in a system may produce the Maxwell-Demons-like breach of the second law of thermodynamics otherwise requiring entropy to increase and enthalpy to decrease with real time, this defines the direction of time. Arrangements of molecules would otherwise get more random with positive time flow. The occurrence of evolution, the formation of single cells from non-biological precursors, and the development of multicellular organisms, seem prima facie cases of breaches in the second law of thermodynamics. The division of a cell to give daughter offspring of course also is non-thermodynamic. It is likely however, that this type of event can occur without the usual molecules oof life, i.e. it is observable in colloidal suspensions. Even seeding of inorganic crystal growth may be of this nature. Water

containing colloidal particles of e.g., 1000, with high water to surface contacts, so that water molecules are perturbed by biological-like surfaces, are hypothesised to be capable of acting against the laws of thermodynamics. Colloidal silica systems of this size and with internal pore structure seem to show these properties in laboratory experiments; suitable curvature/contact effects may enable water arrays to act as proton conduction switches. The usually very rapid but modulatable switching of hydrogen bonds in flickering clusters of water molecules is known to occur. It may be argued that although multicellular organisms usually utilise temperatures around 30-40C, most commonly around 37C, many organisms like different temperatures, e.g. fish in polar regions, where an antifreeze protein is employed; although multicellular organisms do not inhabit environments with T>50C specialized single cell organisms can occur above normal water boiling point, with the proviso that conditions must support the presence of liquid water. This applies to both eukaryotes and prokaryotes. The presence of liquid water is thus fundamental to life. Organisms are around 8085% water by weight. The chemical and physical properties of water make life tick; the presence of biologically evolved additives let these organisms adapt to other temperatures than 37C; first evolved cells seem most likely to have used what is most commonly used today to utilise those properties of liquid water which are most manifested at 37C and are likely to be something to do with lucrative energy storage and transfer potential, e.g., use by essential vital processes of the arrays of hydrogenbonded aggregates with fluxional activity. The properties of water molecules adopted for vital purposes include arrays of water molecules attached to certain hydrophilic groups such as SO3- and probably analogous silicate and phosphate systems which exhibit high proton conductivity. Bulk water, of course has unusual electrical conductivity properties; energy differences between hydrogen-bonded aggregate forms of water present under any given condition are likely to be quite small and capable of changing rapidly and not easily separable into discrete structures; but they are also likely to interact differently with electromagnetic fields; study of such interactions and effects is warranted and is expected to give greater insight into the subtle multiple polymeric forms present in water and their weak modes of interaction. The structure of liquid water is currently far from being perfectly understood. A reasonable model however (based on known facts), can be proposed. This is hydrogen-bonded aggregates of water molecules undergoing rapid structural rearrangement to give stochastic distribution of structures at any given time (with average bond lifetime shorter than one thousandth of a second, although the exchange rates are likely to be slowed up or otherwise modulated by the presence of other molecules such as biopolymers or polysilicic acid). Thus, water is likely to contain real but short-lived randomized structures akin to a random silica garden (ref required) including wall to wall structures, cages, chains, oligomers and monomer. Silica and phosphate glasses also contain such stochastic structures but in these cases the structures are produced by exchange of parts between different environments of Si-O-Si or P-O-P bonds and are easily described by NMR; in the case of water the structures are considered to be produced by exchange of parts between different environments of OH O bonds and are not easily detected by NMR. Some of these structures still are capable of transporting protons and other entities rapidly and both generating electromagnetic fields and interacting with external electromagnetic radiation. They are also capable of alignment and attachment to various kinds of dissolved molecules,

polymers and solid phases with which they form boundaries ; thereby causing mutual modification of the chemical and physical properties of such molecules and the associated water systems. Highly watery co-gels can form e.g. with silicic acid owing to the high surface areas of such systems the properties of the water aggregates adjacent to the surfaces will be considerably altered. Water is the most non-characteristic liquid known being anomalous in virtually all properties when compared to other liquids (Bernal 1968). The rapid hydrogen-bond rearrangements occurring in water suggest a seething constant motion of reforming colloid-size aggregate assemblies. The random proton motion is akin to aromaticity but of course quite differently delocalized in space but capable of being modulated quite differently by adjacent surfaces. This effect might be the cause of the unusual catalytic properties of water . Water also is modified by small entities like small ions and perhaps active oxygen radicals the effect of such large scale structural modification in this way by hydrogen ions giving rise to the widely employed , biologically important , concept of pH. Salts also modify water structure: physiological saline is important. The Hofmeister series ranks in the order of their effects on water structure as detectable by their orders of protein denaturation. Polyelectrolytes are likely to have especially strong local effects. Important biopolymers like DNA, RNA and glycosaminoglycans are polyelectroytes. Heparin is especially highly charged. The law of mass action, the usual effect of molecules in solution, an underlying concept in chemistry, does not seem to apply at least for certain well-defined conditions, in the presence of such polyelectrolytes. Water has high level of infrared absorbance and has a complex combination and overtone spectrum which is sensitive to differences in supramolecular structure. Difficulties in rationalising infrared spectra of water in different environments, although the spectra are easily and very reproducibly obtained, has inhibited mainstream science from studying it more deeply. Previous attempts to do so have burned fingers as in the case of the polywater fiasco. However NIR spectra of water-rich milieu are routinely used for commercial of quality of biological samples albeit by purely empirical procedures. The NIR spectrum of liquid water obtained in a pressurised vessel , cuvette, does not show any discrepancy at the usual boiling point; discontinuities however occur, e.g. , in the 1.5 micron region of overtone vibrations, at the freezing point (but supercooling is possible) and near the critical temperature of 380C (Luck 1964) . A structural transition in the liquid is evident over the temperature region 20-40C centred around 37C. This is most likely due to a partial melting or rearrangement of the hydrogen-bonding arrays present over this temperature interval. The NIR spectral data certainly support the notion that water contains large aggregates of water molecules; the detectable number of non-hydrogen bonded HO groups from ca 50[?] to ca20 over the temperature region of 0-100C. At the usual boiling point liquid water is still largely a polymer (Luck 1964).

The hydrogen bonded aggregate size is calculable as around 50 [?] at 37C but changes with temperature or the addition of ions. Small changes in temperature or ions can exert quite large effects on the average structure. Urry (1996) has suggested that biological systems induce small changes in water temperatures to access hydrogen-bonding energy from the water, particularly by randomizing pentagonal water polymer structures stabilized as hydrophobic icebergs at protein surfaces. As mentioned above a possible model fro the structures present in liquid water can be constructed using random sorting of polymer parts by analogy with silicate or phosphate glass stochastic reorganization behviour. The structural parameter which describes the stoichiometry with silicate esters is the RO/Si ratio. The equivalent parameter for water is temperature or ionic strength; all other stochastic systems require the stoichiometry to define the composition, only water uses a variable which is much more flexible and convenient for biological use. This allows water polymers to re-adopt [to] many shapes and sizes very rapidly when required e.g. complete in 10-7 [?] sec. Effectively without changing appreciably the polymer stoichiometry. Water thus has a potential complex switching capability conceivably capable of logic in an abstract definition of this term. Incidentally, water-rich polyamide gels with voltage-dependent shrinkage have development potential as switching devices in conventional electronic circuits as described in a prototype form by [Tanaka et al] Science [218 467]. So a drop of water seems to have the built-in capacity for acting as a computer. One might predict there to be a critical size of water droplets, in a hydrophobic medium, to act as switches. This might be the origin of ion pumps in biomembranes and much else. How brains got started. These effects are worthy of [further] experimentation. The presence of surfaces has the property of stabilising or destabilising various types of water aggregates perhaps the best known of which is hydrophobic hydration where fused pentagonal hydrogen bonded arrays occur as in the by X-ray crystallography determined structure of crambin (ref. required). Direct measurement of the presence of water aggregates distant from a planar hydrophobic surface showed that water structured by hydrophobic hydration reaches much further out from the hydrophobic surface than does the structure from hydrophilic surfaces, it being one ot two orders of magnitude greater in terms of water structuring power than the weak interactions usually encountered between molecules (Van der Walls forces) structured water (Pashley et al 1985). Flow plus hydrophobic hydration can cause the surfaces to be abraded: cavitation. Degranulation of eosinophils (Beinveniste 1988) Small amounts of impurities such as silicates, buffer molecules or heparin [could have assisted the phenomena reported]. And be the basis of homoeopathy.

Not that this diminishes homeopaths or anything else; just shows that water structure is dependent on boundary surface[s]. If glass rather than polycarbonate is required, surely a simple experiment. But as far as I know not reported, this will give the vital clue. Biology is about water with controlled boundary conditions. This includes buffer molecules etc.; this seems more likely than imprinted immunological information including electronically digitalized information, capable of activat[ing] water alone, but only further experimentation will solve this. A similar situation also seemed to be the cause of apparently anomalous water properties in the poly water saga, again scientific reputations became somewhat bruised as a result of that scandal where the boundary conditions were ignored and results assumed to arise from pure water. The author has found that the NMR but more easily seen with near infrared spectroscopy [NIR] spectrum of water is dependent on the shape of the cuvette. Water in capillaries gave markedly different spectra from planar films. Part of the difference may be due to multiple reflections in the former case but long range water structuring effects of curves surfaces might be part of the difference. The main difference is the relative [NIR] intensities of fundamental and overtone bands in the two situations. The phenomenon is very easy to show experimentally and the results are very reproducible. A previous report that the NMR spectrum of water differs between microsample holders of different shape; [this] might however be more readily explained as due to different diamagnetic susceptibilites in these two cases. Biomolecules are hydrated as a rule; the hydrophobic and hydrophilic water effects control protein shapes. Phosphorylation of proteins e.g. controls their activity, by altering their water interaction. Cancer might be said to arise by abnormal activation. It is clearly to neat to say such a thing so boldly but simple principles often underlie complex effects. Cancer cells have abnormal water structure detectable by NMR or NIR. Biomolecules are likely to use hydrogen bonded water energy (e.g. as a central pool for muscle activity, cf. Urry 1996). The Hofmeister series relates aqeuous solution to salts etc. in the order of their protein denaturant ability. Urry et al discussed the concept of protein denaturation as a one concept for understanding bio-utilization of proteins especially the hydrophobicity hydration aggregates. Water is a powerful catalyst. Many reactions seem not to proceed in its absence. Often the catalytic effects of water are overlooked since only small amounts may be involved. How then is water and life linked? It is not even easy to give an unbiased definition of life.

We may use systems using DNA or RNA as a definition, but perhaps other information storage and retrieval could work just as well, e.g., on another planet. Even water may have in-built information storage and computational potential without the aid of biopolymers. A good pre-hypothesis for life and water is that water droplets can act as switches permitting primitive logic devices to form. Perhaps we could note that life has arisen as far as science is concerned as a consequence of evolutionary processes occurring over long periods of time, perhaps sufficiently long that the laws of thermodynamics where disorder usually occurs spontaneously normally encountered under laboratory conditions, no longer applies. And the presence of water structures perhaps in combination with silicate and phosphate operate Maxwell demon-like doors to increase system complexity with positive time rather than the otherwise thermodynamically driven increase in disorder. Clearly terrestrial life is water linked, e.g. [can be] 85% by weight and evolved in water environment and employs the properties of water in ways we are dimly or not aware of at present. It seems most likely that a mixture of prebiotic chemicals (amino acids and polyphosphate) existed in an aqueous environment at around usual body temperature, but with access to hot spots and flow zones will gradually evolve into living cells. Proteinoids, random polymers of primitive amino acid produced in the laboratory [can be shown] to have primitive cellular and reproductive powers of highly hydrated colloids. The maintenance of say a temperature of around 30C for four hundred million years will, it might be suggested, make the[se] water based system[s] increase in complexity to give a true biological cell. References Popper ICR (1990) Nature 344 38777 [Urry Chem Brit 1996] Pharmacuetical sodium heparin may contain significant amounts of Ca, Cu and Zn. It is now suggested that this is arises naturally from an in vivo source and not from contamination. These and a range of other elements (discussed below) could have become associated with heparin/heparan sulphate in vivo (and when injected in to the bloodstream it is likely that the normal blood serum concentration of calcium will lead to the formation of Ca2+ etc. substituted heparin in vivo. also contains the range of other inorganic ions in blood serum could likewise be predicted to become associated with heparin in vivo where the heparin/heparan sulphate ion exchanger might behave similarly to related polyuronide anionic ion exchangers such as alginate in marine algae. The relatedness between the inorganic elements in seawater and blood serum has long been noted but the concept was recently extended to include the non-physiological ultratrtace elements in a new of the concept of metallomics c Footnote c from the original 20 elements proposed by Williams to comprise this system to a wider range of elements.

Synthetic ion exchangers are known to become enriched in the less abundant solute ions. This concept evidently also applies to the natural ion exchange system in the cell wall of marine algae since alginates demonstrate an approximate inverse relationship is apparent between the degree of enrichment and the concentration in seawater. By analogy a similar relationship may also apply in vivo to the analogous animal polysaccharide ion exchangers which will include heparin, and the heparin-like segments of heparan sulphate proteoglycans (e.g. syndecans and glipicans) which occurring in an extracellular environment will be bathed in the (seawater-like) extracellular multilement containing fluids. This idea is consistent with several reports have noted the presence of a range of inorganic elements in commercial heparin preparations and with the findings that a purified form of heparin obtained (similar to a process used industrially to reduce the toxic and heavy metal content normally associated with native heparin) retained residual amounts of the starting range of inorganic in the same relative amounts to those present in blood serum and other biological (seawater-related) matrices. An experimental laboratoary sample of thallium heparin (believed to represent the trace contents present in heparins used pharmaceutically) actually contained similar levels of trace elements to the quantities present in kelp biomass (where the principal metal binding ligand is the algal poyuronide, alginate (n.b. the commercial plant and animal nutritional value of kelp is thought to depend largely on the full range of inorganic mineral nutrient elements from a matrix containing this level of trace elements). Heparin posses an uniquely high multiple inorganic ion binding facility consistent with this being the most anionic ion-exchange biopolymer known. Heparin seems to possess an additional facility for promoting of metal ion aggregation at the heparin surface which may apply to a range of the common redox active metals (Cu, Fe, Ni, Co, Ti, Ni, V, Mn, Cr). Fe and Cu can apparently become sequestered in sufficiently strongly complexed form** to suggest that this binding has an antioxidant role (similarly to what has similarly been observed when Fe ions are sequestered to hylauronan and chitin). Heparin and by inference HS in vivo therefore contains variable amounts of redox metals Cu, Mn, Cr, and V in a pathological related manner. Excessive NO is associated with degenerative diseases processes (during which typical pathological tyrosine nitration has been found to be a useful marker) which suggested a redox-status dependence hypothesis of HS action. It was proposed that the e.g. anti-degenerative disease action of ascorbate could be accounted for by a role of this vitamin in retaining a correct balanceof HS/NO redox metal ion signalling. Age-dependent downgrading of necessary exact control of such redox metal ion HS binding could be a contributory factor to the aetiology of such degenerative disease processes (e.g. serum ferritin increases in absence of disease, but serum ferritin levels were strongly correlated with caridovascular associated mortality in men, vascular HS seems to show a strong age-dependent change in microstructure which has also been associated with such cardiovascular dysfunctions The use of a heparin probe to quantify and probe the possible roles of metal ion dyshomesasis in degenerative disease process could be suggested (by e.g. the mass spectroscocpic assay of multi-elements in heparan sulphates or heparin derived from body fluids or tissue samples, e.g. the use of ultrapure heparin as a biologically relevant ligand by which to obtain a biologically relevant (cf. heparan sulphates ubiquitously present in extracellular matrixes and cell walls probably bind such inorganic elements in vivo and are thereby subject to activity perturbation) element enriched sample from blood fraction or other body fluid assay for metal ion intoxication etc., in a similar

manner to use of tissues such as hair or nails by mass spectroscopic multi-element evaluation). Footnote a A number of heparin dependent serpin actions are known to be modulated by Ca2+ (however the mechanism is thought to be an indirect one) but a E.g. pectin fragments which are known to regulate host pathogen defence (including apoptosis), growth, morphogenesis and ageing (senescence). b Long & Williamson (the Aberdeen polysaccharide group) proposed that in 1979 that calcium ion second messenger actions were especially influenced iduronate containing heparan sulphates (HS) and by implication by oligosaccharides derived from them. This idea was probed experimentally until recently; an internet listing in 2003 includes studies of Ca2+ interactions with HS and related systems *) e.g. studies of roles of M2+ in HS dependent protein activation or deactivation processes as well the interactions between Ca2+ containing surfaces and other inorganic ions and surfaces with HS and other anionic polysaccharides. A number of hypotheses arose from these studies suggestive of putative roles of polysaccharides in cellular activities as well as more basic mechanisms as modulators of hydration, antioxidant, anti-nitrant actions and anti-pathological crystal control and general physiological liganding of metal ions (of relevance to reports from other laboratories which showed that growth factor, immune system and apoptosis functions of HS can show absolute requirements for divalent metal cations).. One of the (originally thought trivial) findings of this research group was that heparin* simultaneously bound, amounts greater than 1ppm, some 40 inorganic elements present in seawater and biological fluids, but that these could be readily exchanged (e.g., for a desired principal countercation) by use of a sulphonated ion exchange of heparin Other related studies, some of which have not been published, were conducted on other glycosaminglycans (chondroitins, dermatans, keratans, hyaluronan, cellulose derivatives, plant wall xylans, algal wall xylans, their sulphated forms, alginates, carrageenans and humic materials etc.). ** Also heparin acts as a ferroxidase for Fe(II) and generated FeO.OH alagaenite, identified by X-ray diffraction. *A mast cell (porcine mucosa being a traditional tissue of origin) cocktail of highly heparan sulphate higher sulphated domain oligo- and polysacharides) but readily available from its longstanding medical employment as a blood anticoagulant which affords a convenient protein-free laboratory model of the highly sulphated domains of HS). *** Many years before the important second messenger role of nitric oxide (nitrite) had been identified, the highly specific reaction of HS was originally used as a convenient laboratory method of identifying HS in mixtures of other polysaccharides including the other glycosaminoglycans. NO metabolites and transition metal catalysis are now believed to enable release of NO from cysteine clusters. This signalling system may be further modulated by polyamines which evidently signals for the biosynthesis of pre-primed HS scission sites.

This is especially relevant to how oligosaccharides are generated via redox metal ions *** since the in vivo nitric oxide metabolite subject to catalysis by which create physiologically active oligosaccharides (these are formed in vivo from polysaccharides both via enzymic scission but uniquely in the case of the HS system, by a non-enzymic, nitric oxide metabolite mode of formation via de-aminate cleavage of primed sites (where blocking N-sulphonates are converted to NH2 groups)

INSERTED
DOCUMENT 10 Water & Biology Philippa M Wiggins [cf., water putatively forms high and low density forms HDW/LDW cf the 2007 paper Life depends on two kinds of water PloS ONE reported earlier in 1973 that water in (pores present in) silica gel selects K+ from Na+ and demonstrates a range of other biologically relevant inorganic ion selectivity; this seems to indicate that the ability of polyelectolytic surfaces such as would occur in such silicate surfaces in spores can create a markedly altered form of water (Wiggins later, with Rene T van Ryan [1] 1986 that (putatively the same general type of altered) water also occurs in small rather hydrophobic pores of dense films of cellulose acetate; this water showed increased hydrogen-bond strength and altered solvent properties and these effects could be amplified by solutes which could not enter the cellulase acetate pores because of their sizes; the chemical activity of the water-in-the-pore became higher than the chemical activity of the water in the external solution; the 1990 paper [1] Changes in ionic selectivity with changes in sensitivity of water in gels and cells continued this discussion and noted that water in pores of gel (which can be considered to be equivalent to (silicate-based glass) capillary water cf. [2] This [1] groundbreaking paper showed that water in the pores of commonly used biologically selective gels contains stretched water with lower density and higher viscosity (and also altered infrared absorption energies compared with normal water). Other reports from this research group indicated the likelihood that as a result of the effect on water structure of presence of solutes, surfaces (especially those in pores) provided a credible mechanism to explain the centrality of liquid water in biology and allowed new insight to be attained into how liquid water facilitated the establishment of biology in the first place. It is now pointed out that the circumstance that the pores formed in silica gel apparently behaved similarly to the pores in cellulose acetate or other biologically formed gel forming substances has a profound relevance for how biology could have emerged from a silicate plus water basis. A key part of this relevance however is that silicate in the form of sol particles can demonstrate quite close to biological cell like behaviour in the laboratory. This includes a cellular form with abilities to show large species variation in types and the apparent possible ability of such types to generate daughter particles capable of quasi biological reproduction. Silica sols also become senescent and age.

These phenomena could perhaps arise because of the ability of pores and surface comprised of amorphous silica to generate the unique-to-life altered form of water. Thus a purely inorganic glass capillary system creates the anomalous water which, according to later publications by the Wiggins group is what enabled life to evolve and what remains the most central system which confers energy transduction and selection [2] Deryagin polywater seems strangely also to be possibly of a similar nature to Wiggins-Van Ryan visous, stretched (also anomalous) low-density [0.96g/ml] water. It furthermore as later reported by Deryagin also was initially suggested to be formed in silica gel (i.e. common silica glass) capillaries. This water had anomalous physical properties e.g. higher viscosity and altered infrared absorptions. Later this claim that anomalous water per se was formed in glass capillaries was retracted by Deryaguin and the supposed anomalous properties were then supposed to have arisen entirely from the presence of impurities (e.g. silicates) in the supposed polywater. The Wiggins theory of high and low density water is it seems apparently entirely equivalent to polywater. It is surely now justified to resurrect the polywater idea but in an altered format. The complete rubbishing of the polywater idea was perhaps correct at the time but no longer is warranted. This supposed fraud seems to have engendered reluctance in the scientific community to engage in the needed further probing of the polywater fiasco. Wiggins seems to have (deliberately but why?) omitted the silica gel pore mechanism of generating the biologically relevant altered water in her final 2007 PloS ONE hypothesis.

INSERTED Document 11
The Structure of Water and Hydrates- Its Importance to Biology
D Grant, WF Long & FB Williamson Department of Molecular & Cell Biology Marischal College University of Aberdeen Aberdeen AB9 1AS Scotland, UK (written in 1991, transferred to Microsoft word 18/4/08) Water is, by far, the most abundant molecular component of cells and, it may be argued, its a prerequisite for life is a likely requirement for prebiotic evolution (Stillinger 1980). Water structure and water activity and their alteration by physiologically active substances may therefore have a fundamental importance to biological function; a corollary to this is that those agents which have a special influence upon water structure may also be active physiologically. Thus physiological metal ions, nucleotides and biopolymers such as glycosaminoglycans , proteins and polyamines inter alia are expected to have important effects on water structure.

Anti-inflammatory drugs were suggested (Warner, 1973) to possess key functional groups positioned in such a way as to interlock with the crystal structure of ice as is the case with hydrocortisone, aspirin, indomethacin , flufenisal and naproxen; water molecules in cell membranes were also believed to be similarly ordered. The discontinuity in water associated molecules detectable by near i.r. spectroscopy at near 37oC (Luck, 1966) may suggest that this is common homoisothermal temperature is used by many species allowing advantage to be taken of a rapid change in water structure in this temperature region perhaps finding use for this in anti-microbial defence. Biopolymers, including nucleic acids, proteins and glycosaminglycans are highly hydrated ; such coordinated water molecules may be important for stabilising conformations and for the modulation of the reactivity of these molecules. In the cases of the glycosaminoglycans, the anionic groups display different water binding tendencies, that of the -SO3- group being especially high (cf. Zundel & Murr, 1969; Atkins, 1974). Hydrating water molecules may be highly mobile, rapid reorientation typically being about an order of magnitude slower than the corresponding process in bulk water (Piculell, 1985). Organic solvent water mixtures contain water monomer dimer and timer or tetramer and in some cases other related adducts where water could bind to phosphoryl groups, and in high polymer equilibria ; organic solvents with a single base site were believed to stabilise a cyclic water trimer (Johnson et al., 1967). The unusual behaviour of water towards non-polar solutes and non-polar side groups attached to biopolymers has long been recognised (Stillinger, 1980) and termed the hydrophobic bond. Typical non-polar solutes inducing hydrophobic bonding are the noble gases and hydrocarbons; none can hydrogen-bond to water and all are sparingly soluble in water but dissolved in water they cause water structure making between the water molecules by reorganising the random hydrogen-bonding network of liquid water; computer modelling shows that insertion of inert space filling entities in liquid water causes the hydrogen bond network to rearrange to form a local clatharate-like imperfect convex cage; another aspect of the hydrophobic interaction is the orientation preference of water molecules next to a non-polar solute; the water molecules tend to straddle the inert solute, pointing two or three tetrahedral directions tangential to the surface of the occupied space and forming water cluster polyhedra like those present in supercooled water (Stillinger, 1980). Pairs of non-polar solutes in water experience an entropy driven net attraction for one another (the hydrophobic bond) which may determine the native conformation of biopolymers. For water at around room temperature and below, it has been demonstrated by computer simulation (Geiger et al., 1979; Stillinger, 1980) that the liquid lies above the critical percolation threshold for hydrogen-bonding; i.e. any macroscopic sample of the liquid will inevitably will have un-interrupted hydrogen-bond paths running in all directions, spanning the entire volume of the sample, being analogous to a gel point. These network pathways, which are random , but with a local preference for tetrahedral geometry, but containing a large proportion of strained and broken bonds, provide natural routes for rapid transport of H+ and OH- (a proton hole) ions by a directed sequence of exchange hops; the strained hydrogen bonds appear to be more reactive and to be important kinetically (Stillinger, 1980). Studies of supercooled water by Angell et al. (1980)) suggests that an anomaly occurs at 45oC which is somewhat below the experimental limit of supercooling attainable easily but suggests the working of a structural order-disorder phenomenon in the hydrogen-bonded network of supercooled water; in the supercooled water the

properties are evidently caused by the concentration and spatial distribution of the relatively unstrained and hence bulky hydrogen-bond polyhedra embedded within and linked to the random network, possibly including an octameric unit similar to that which occurs in hexagonal ice. These polyhedra can share edges without the introduction of mutual strain, consequently they are able to link up with one another more readily than a strained and an unstrained polyhedron can, so that the ideal unstrained structures find it advantageous to clump together. At temperatures close to 0oC the infrequent unstrained polyhedra form a dilute gas dispersed throughout the predominantly strained and defective network, but as the temperature declines, the polyhedra become more and more frequent since they incorporate stronger hydrogenbonds, the cluster sizes evidently diverge as the T approaches 45oC. Study of the OH-stretching region of the Raman spectrum of aqueous solutions provides a sensitive method of detecting of structure breaking and structure making effects of solutions when compared to pure water. The 3650cm-1 region of the spectrum of liquid water is due to non-hydrogen-bonded OH oscillators and the region near 3250cm-1 to OH oscillators vibrating in phase and involved in a fully hydrogenbonded tetrahedral assembly of five H2O molecules. Loss of intensity in this region is the case for aqueous sugar solutions relative to pure water, indicating that the concentration of OH oscillators involved in such an in-plane motion, decreases. ClO4which does not form H-bonds, has an opposite effect to sugars on the water structure (Walrafen & Fischer, 1986); ClO4- efficiently breaks up water structure as confirmed by the increase in the intensity of the absorption due to dangling OH oscillations at 3570cm-1. Luck (1966) studied fundamental, combination and overtone vibrations of water molecules in clusters and estimated the proportion of free OH groups as a function of temperature up to the critical point. The results suggested that the extent of the hydrogen-bond system in the neighbourhood of the melting point amounted to several hundred H2O molecules. The 1 of H2O in monomer , dimer, trimer, tetramer and polymer was suggested to be 3725, 3700, 3545, 3510, 3398 and 3355cm-1 resp. The angular dependence of the shift caused by hydrogen-bonding in the H2O, H-O fundamental vibration frequency was reported. Free OH, in unbonded H2O absorbs at 8748cm-1, free OH in H2O molecules with one free OH group, OH group in a hydrogen-bond in a cyclic double bridge between two H2O molecules having bond angle of 109o. Water bridges may interlink basic amino acids resides with heparin (Grant et al., 1991) and have relevance for glycosaminoglycan-protein binding, particularly in a pericellular environment. Water has a number of unusual properties with anomalies in practically every single one of its physical properties; e.g. it increases in volume on freezing, it has a density maximum at 4oC, an isothermal compressibility minimum at 46oC, has a high dielectric constant, anomalously high melting, boiling and critical temperatures, shows increasing liquid fluidity with increasing pressure, the heat of melting is only 13% of the sublimation energy of the solid and has a high mobility for transport of H+ and OH- ions (Stillinger, 1980) and has a highly anomalous surface tension, attributable to a highly preferential internal orientation of the hydrogen bonds (Bernal, 1965). It might have been expected on comparison with H2S that water would have been a gas at room

temperature since H2S, on account of its greater molecular weight, might have been expected to have a more coherent structure. A consideration of complex dimensionality of the crystalline state as discussed by Bernal suggests that while liquid water possess a lower degree of order than allowed for in traditional crystallography, the structure of water may, however, be conceived as having crystallinity in the sense that liquid water is a state of ordered matter demonstrating a gradation between the three-dimensional crystalline state and the liquid state which under ideally limiting conditions would exhibit a no dimensional order (exemplified by liquid mercury); biological membranes also possess a type of crystallinity less than three dimensional in this case they contain two-dimensional crystal-like order. Water molecule aggregates, evidently up to several 100 in size , might also be considered to be a branch of colloidal science (Bernal, 1965) cf., also Stillingers (1980) discussion of supercooled water. The sort of order apparent in amorphous silicates as, e.g., suggested by 29Si nmr spectra of sodium silicate and possibly capable of nucleating similar ordered amorphous particles (Grant et al. 1992) may be in some way analogous to the type of ordered structures present in liquid water. When units have low symmetry, whether intrinsically or by random movement, or made random by relatively rapid structure alterations, it is impossibly to obtain regular three-dimension arrangements over time scales required for diffraction measurements of crystallinity (Bernal, 1965); the possibility of nucleation of loworder crystal aggregate structures in liquid water, should be considered; particular nucleating agents might have the property of nucleating slightly different distribution of water aggregates, at least on a short enough time scale which retain physiological activities; convincing experimental methods for the detection of such possibilities have evidently yet to be developed. Possibly nmr evidence for water aggregates similar to those in amorphous silicates (detectable by high resolution 29Si nmr) comes form the coalesced proton average nmr spectra of water molecules in hydrocarbon mixtures which showed different chemical shifts and peak widths for water aggregates composed of in differently shaped micelles containing different quantities of stabilising oil and surfactant molecules (Shah & Hamlin, 1971). Long-range, thermodynamically metastable, but with long term stability quasiperiodic order, is exhibited by quasicrystals with crystallographically forbidden symmetries (cf. Fujiwara & Ogawa, 1990); new theories of crystallinity of which the five-fold rotational symmetry may be but one facet, may have some relevance for random assemblage of low order aggregates of crystallite-like water structures. Bienveniste suggested the existence of ghost putative water memory structures in very dilute aqueous solutions of physiologically active agents by using immunological methods (the procedure employed in these experiments has, however. been vigorously criticized). The numerous crystalline polymorphs of solid water, at least nine in number, include some that form at elevated pressure (Stillinger, 1980) seem to lend weight to possibilities that liquid water could contain perhaps under different stimuli, a variety of different low order metastable crystalline structures which might nevertheless be capable of different physiological interactions.

The properties of water depend on how far the hydrogen is form the oxygen, i.e., the acidity of the medium. At low pH the hydrogen moves out from the oxygen as far as 1.05; at high pHs it moves to 0.85, in neutral water it is at 0.96 (Bernal, 1965). The results of quantum mechanical studies lend support to a notion, that of Frank and Wen (1957) and Frank (1958), that hydrogen bonding in water is cooperative (Stillinger, 1980); the hydrogen bonds mutually reinforce each other, encouraging chains of hydrogen-bonds to form. A calculated hydrogen-bond pair occurrence potential as a function of pair potential at different temperatures indicates an invariant point at 4kcal/mol, suggesting that a thermally activation bond breakage occurs which transfers pairs across this invariant point as the temperature rises (Stillinger, 1980). Water possesses unusual proton transfer properties which are of importance to its chemistry. The symmetrical hydrogen-bonded H5O2+ and H5O2+(H2O)4 were studied by ab initio calculations by Muniz et al. (1985), the results being that the energy minimum of the former cation in which the proton is located midway between the two hydrogen atoms, is broken down by solvation into two minima corresponding to structures with un-symmetric hydrogen-bonds. When considering an adiabatic transfer model, solvation parameters were suggested to take part in the reaction coordinate of the proton-transfer model, the inversion of solvation distances produces spontaneous proton transfer, the proton adjusting its position to the changes in solvation. Given that there is an unsymmetrical contracted hydration shell around the H3O+ unit, and an expanded one around the H2O part of the cation, the symmetry of the energetic profile was suggested to be destroyed if solvent relaxation was not allowed, this being supposed to prevent proton tunnelling from taking place; in order for this to occur, the symmetry of the energy profile had to be re-established by means of solvent movement to make the hydration shells around the two water molecules between which the proton is transferred, identical. Ling (1973) has stressed the role of structured water in cellular phenomena, e.g. on the permeability properties of natural membranes which are extremely permeable to water molecules in disagreement with a hydrocarbon solubility theory of permeability. A possible model for the water permeability was considered to be cellulose acetate film, where gaps between the cellulose acetate fibres are filled with water channels in which the water occurs as a polarised multilayer structure (cf. Anon. 1973). Such structural water channels in polysaccharide gels and effects of cations on them, may be probed by X-ray diffraction analysis of oriented hydrated fibres (Arnott, 1989). Similar studies carried out with glycosaminoglycans (Atkins, 1973) showed that the conformation of the polysaccharide and the degree of hydration may be dependent on the cations present. The Hofmeister series ranks the potency of aqueous solutions of ions for the denaturation of proteins . The molecular origin of the series is not known but it is believed to be related to the water structuring ability of the ions. Geometric molecular structure of water & related The detailed molecular structure of water is as outline above is essentially unknown; one model is that flickering clusters (Frank & Wen, 1957, 1962) of individual

molecular aggregates are held together by hydrogen-bonds continually rearranging, the precise arrangements and rate of interchange can be conceived as being alterable by solutes. The presence of aggregates of a quartz-like structure was proposed for liquid water by Bernal & Fowler (1933) based on the known crystalline ice structures, and 46 molecule aggregate of poly-pentagonal dodecahedral structures by Pauling (1959); these structures were based on atomic radial distribution curves determined by X-ray diffraction, there is a problem of accommodating various fixed structures of aggregates to the density, dispersion of dielectric constant and the known structure of ice, which has several forms). Leyendekkers (1985) explained the anomalies in the heat capacities and acoustic characteristics of water in terms of unbonded and singly bonded water molecules attached to aggregates, the transitions of these special water molecule environments probably were the critical factor underlying the anomalous properties of water and an activation mechanism of an equilibrium involving their rearrangements governing gas phase transition of the second kind . The experimental values of unbonded water molecules used in these calculations (for H2O) at different temperature and pressure was reported by Luck (1980) from the near i.r. (NIR) spectrum of water. Luck (1967) reported the i.r. spectrum of H2O in the temperature region from 50oC to 400oC showing that H2O , as well as CH3OH and C2H5OH , possessed less non-H-bonded OH groups than most theories of liquids claimed. An exact interpretation of the i.r. spectra was not possible, but the model of the structure of liquid water which was adopted had flickering clusters possessing fissure planes consisting of free OH groups dividing groups with closed H-bonds in icelike structures. The smaller proton mobility in liquid water in comparison with that in ice had to conform with the presence of large clusters. The mobiliety of the cluster surfaces was believed to determine mobility in the liquid state. Long range order The origin of this order was far form clear and its spatial extent is a matter for controversy (Franks, 1979). The degree of orientation of water on extended surfaces varies considerably. Near the surface, up to 10-20 distant, water is believed to be bound in the form of ice. Beyond that, up to 100 or so, the water is less tightly bonded and is able to accommodate ions, though with restricted possibilities of movement. This is about the limit to which the structure of water itself is liable to be affected by hydrophilic surfaces. However beyond that there is an influence exerted thought water for quite great distances, measured up to 4000 but probably further (cf. a colloidal sol, long range force, cf. coacervate phenomenon (Bernal, 1965)). Bernal hypothesised that in other than very primitive cells, there was an almost complete lack of spherical organelles; water in contact with the rough side of the endoplasmic reticulum and the cristae of the mitochondria was more viscous being in a partly gel-like state than water on the smooth side named by him the endolymp) which was seen to communicate with the extracellular water, even when the membrane is continuous, by a process of forming and separating of small globules of liquid (micropinocytosis) . No part of the cell except that in the interior of vesicles was at a distance of more than a few hundred from some other part of the membrane and consequently a force should always act between them producing interacted water molecules.

The rate-determing processes in crystallization from aqueous solutions of Ca salts such as CaCO3, are likely to involve water molecules in the transition states of the rate determining reactions, since these ions are hydrated in solution but become dehydrated in the insoluble salt crystalline forms. CaCO3 exists in many forms, the nucleation of which demonstrated the non-genetic inheritance of form as was pointed out by Lima de Faria (1986); this may provide a clue about the fundamental processes inherent in both crystal and cells about which, e.g. for the molecular mechanism of crystallization and nucleation, little is known, but which if understood, might shed further light on the still less understood biological non-genetic determined processes. Of these, aspects of water structuring may be involved in the control of crystallization from aqueous solution. Modulated r.f./microwave fields modify calcium binding at cell surfaces and modulate a host of calcium dependent intracellular enzyme mechanisms which regulate the flow of intracellular messages (protein kinases), cell metabolites and cell growth; the use of imposed fields might reveal the essential importance of biological organization at the atomic rather than the molecular level and in physical rather than chemical terms; with coherent states between adjacent molecular electric charges and enormous cooperativity in energy release by very weak triggers as the physical essence of living matter (Adey, 1988). Water structures are also implicated in the mechanism of cellular adherence to substrates (Curtis et al., 1986) as indicated by a dependence of adherence of BHK cells on growth surface hydroxyl group spacing. Nucleation of ice crystal formation is more directly involved with ice nucleation bacterial Erwina anana, which was increased with increasing supercooling and required the presence of a water glass interface; the nucleation was inhibited by sugar (Watanabe et al., 1987). Silica is formed of joined tetrahedra in a similar fashion to joined hydrogen-bonded water tetrahedra; this appears to allow silica gels and water structures to be mutually accommodating. Similar considerations apply to sugars and polysaccharides cf., the water structuring properties of glycosaminolycan-rich extracellular space and to such materials as polyacrylamide gels; phase transitions occur in these latter materials, considered (Tanaka et al., 1982) to be as a consequence of change in the balance between three classes of forces which contribute to the osmotic pressure in the gel viz., 1) rubber elasticity, 2) polymer-polymer affinity and 3) the hydrogen ion pressure (Tanaka et al, 1982); drastic changes in the state of a gel can be brought about by small changes in the external conditions (e.g. temperature, pH, ionic conditions or solvent). Gels share mathematics with other critical processes (chaotic processes-the possibility that at least some biological control mechanisms may straddle potential chaotic systems of such water-rich gel systems might be worth considering, e.g. the effect of small changes in ionic (e.g. Ca2+) concentrations); in the gels fluctuations create local variations in the density of the polymer networks so that small aggregations of polymer are constantly forming and disintegrating ; a divergence of scattered light occurs at the critical point where the polymer network becomes infinitely compressible; this can be considered in terms of phase transitions and critical phenomena.

The Rees (1968) hypothesis of polysaccharide gels gives an equivalent model of a similar phenomenon where the points involving the cooperative association of long regions of m polymer chains occur, so that sol-gel transformation may involve a conformational change (Bryce et al., 1974). Various gel-like mixtures (electrorheological fluids ER) which may contain e.g. silica, water and polyelectrolytes (however although systems without water have been described particularly good ER systems contain water) which exhibit applied voltage dependent gel formation have been formulated by empirical methods (Webb, 1990). ER fluids are able to convert rapidly (millisecond) and repeatedly between a fluid and a solid when an electric field is applied or removed (of interest for mechanism (such as clutches etc. in robotic systems). It seems possible that alteration of gelation by modulation of hydrogen bonding arrangements in H2O polyhedra or related hydrogen-bonded clusters is the basis of the mechanisms of these fluids; lack of understanding of the molecular origin of the phenomeneon has severely limited the development of ER fluids for engineering use. Aspects of such hydrated gel structures are relevant to an understanding of biological tissue both multi-cellular extracellular and cytosolic. The arrays of microtubules which are arranged in parallel arrays in axon, cilia, mitotic spindle and other cellular formations participate in many cellular processes, including directed division, excitability etc., and show a dependence on the electric events occurring at the level of the cell surface or cytoplasmic organelles, naturally occurring fields in the range 20500mV/cm have been implicated (Jaffe & Nucitelli, 1977). Microtubules were found to align in parallel arrays and may be involved in the mechanism of the action of electromagnetic fields on some biomolecular processes (Vassilev et al. 1982). I.r. spectroscopy, especially near i.r. spectroscopy, gives insight into water structure. Whilst the band positions observed are known to correspond to combination and overtone bands, the causes of relative intensities in the near i.r. are less understood. There is a lack of any direct correlation between hydrogen-bond strength and water structure, apparent from consideration of i.r. spectra of H2O and D2O (Bonner, 1970). Expected effects of hydrogen bonding, however, suggesting more complex than hitherto appreciated aspects of water structures come for the much greater than expected observed difference between the n.m.r. signals of 18O(-H2O) compared with 16 O-(-H2O) when hydrogen bonded to organic molecules (Pinchas & Meshulam, 1970). The enthalpies and entropies of transfer of non-electrolytes and individual ions from H2O to D2O show linear correlation (compensation effect). These results were interpreted as indicating that there seems to be a wide variety of perturbations of liquid water which evoke the same type of response in water Even in relatively strong aqueous solutions there is evidence for structural arrangements characteristic of the bulk structure of water which are unaffected by the presence of the solute, even in rather high concentrations of electrolytes Drost-Hansen, 1971). The factors giving rise to entropy-enthalpy change correlations as well as the detailed structure of water have not been established, but it is evident that such knowledge might aid a fuller understanding of biological processes. Plasma membrane lipid head groups are hydrated and this is believed to be involved in their phase transitional behaviour. This can be studied in a water-tetradecane solvent

system (Rand et al., 1990) where inverse hexagonal phases containing cylindrical water cores were detected. It was necessary to take into account two modes of interaction between lipid polar groups and molecules, one mode of interaction being of a normal solvation solvent affinity type, due to hydrogen bonding of the solute with water, and the other mode of interaction resulting from the geometrical properties of lipid bilayers when large amounts of water molecules are taken up by them (non-local solvation) which arises when a spontaneous curvature of the lipid monolayers would be at a free-energy minimum in a geometry which would create voids or low-density regions in the liquid crystalline structure, voids that are filled with compatible water. Without enough water the hydrophilic cores of the hexagonal II phase shrinks; without enough alkane, the inverse hexagonal phase will often simply cease to exist; curvature energy is important in these situations. Crystal deposition diseases of the joints are age-dependent and may be related to paralleled age-dependent changes in the joint glycosaminoglycans; highly hydrated joint proteoglycans secreted by chondrocytes occur adjacent to a calcified (hydroxyapatite) zone interlocking with bone, the glycosaminoglycans are predominantly chondritin-6-sulphate and keratan sulphate arranged like a bottle brush on a protein core (cf. Dieppe & Calvert, 1983). Sulphated polysaccharides may be capable of producing both hydrophobic and hydrophilic forces at interfaces. Sequences of hydrophilic and hydrophobic regions may occur in heparan sulphate (more hydrophobic NAc-rich regions occur in blocks). Studies of adsorbed double chained quaternary ammonium salt on mica indicated that hydrophobic forces are strong and long-ranged. Hydrophobic processes are thought to play a key role in biological self-assembly. Long-range forces reflect interactions due to surface-induced water structure and are 10 to 100 times stronger than the van der Waals forces that would operate in the absence of any surface-induced order in water. The situation is in direct contrast with that observed for hydrophilic bilayers where the forces have been indicated to be short range (Pashley et al., 1985). Electric fields, e.g., generated by Ca2+ influence the proton potentials of C-OH-OPHOP bonds (Scihberg & Zundel, 1976) as well as by their degree of hydration (Carmona & Garcia-Ramos, 1985) and possibly water structures. Bulk dimagnetic susceptibility differences may occur between water spheres, cylinders and lamelae producing differences in the n.m.r. spectra well as electrical and birefringence properties (Shah & Hamlin, 1971). References Adey T (1988) Nature 333, 401 Amost LA (1982) In Electron Microscopy of Proteins (Ed. JR Haris, Vol III pp 207250, Academic New York Angell CA in Water, a comprehensive treatise Ed F Franks, Plenum, New York, 1980, cf. Stillinger (1980)

Anon (1973) New Scientist 20 September 670, cf. also ibid., 59, 378 Arnott S (1973) Roy Soc Chem Carbohydrate Groups Spring Meeing Cranfield p. 2931 Atkins EDT Isaac DH Nieduszynski IA Phelps CF Perlin AS 263-71 (1973) Polymer 15

Bernal JD (1959) in Hydrogen bonding, Proc Symp Ljubliana 1959 Ed Hadzi D & Thompson HW Pergamon Press London, New York Paris & Los Angeles p 7-22 Bernal JD Symp Soc Exptl Biol 1965 19 17-32 Bernal JD & Fowler RH (1933) J Chem Phys 1, 515-48 Bonner OD (1970) J Amer Chem Soc 92 4197-9 Cf Bonner OD & Curry JD (1970) Infrared Physics 10 91-4 Carmona P & Garcia-Ramos JV (1985) J Chem Soc Faraday Trans 81 925-35 Bryce TA McKinnon AA Morris ER Rees DA Thom D (1974) Faraday Discuss Chem Soc 57 221-9 Curtis ASG Forrester JV Clark P (1986) J Cell Sci 86 9-24 Dieppe PA & Calvert P (1983) Crystals & Joint Disease Chapman & Hall London Drost-Hansen W (1971) Cf. Chemistry of the Cell Surface Academic New York; Structure and properties of water at biological intrfaces 1970; Cell-associated Water Academic 1979; Biologically allowable thermal pollution limits EPA 660/3-74-003, May 1974 Frank HS (1958) Proc Roy Soc Lond Ser A 247 481Frank HS Wen WY (1957) Discuss Faraday Soc 24 133 Franks F (Ed) Water, a comprehensive treatise Vol 6 p16 (1979) Fujiwara T & Ogawa T (Eds.) (1990) Quasicrystals, Proceedings of the 12th Taniguchi Symposium, Shima, Mie Prefecture, Jappa, 14-19 November 1989, Springer-Verlag, Berlin, Heidelberg New York Paris Tokyo Hong Kong Barcelona Geiger A Stillinger FH Rahman A (1979) J Chem Phys 70, 4185-92 Grant D Long WF Williamson FB (1991) Biochem J 277 569-71 Cf ibid 275 193-7; ibid 244 143 Biochem Soc Trans13 389 ibid 18 1283-4; Jaffe LF Nuccitelli R (1977) Ann Rev Biophys Bioeng 6 445-476 Johnson JR Christian SD Affsprung HE J Chem Soc 1980 1924-1928

Leyendekkers JV (1985) Trans Faraday Soc 81 519-536 Lima de Faria Evolution without selection Elsevier 1988 Ling GN (1973) Biophysical J 13 807 Luck WAP (1968) Ber Bunsenges 69 626-637 Luck WAP (1980) Angew Chem Int Ed Engl 19 28 Muniz MA Bertran J Andres JL Duran M Lledos A (1985) J Chem Soc Farady Trans 1, 81, 1547-1554 Pashley RM McGuiggan PM Ninham BW Evans PI (1985) Science 229 1088-9 Pauling L (1959) in Hydrogen Bonding, Proc Symp Ljubliana 1957, Ed D Hadzi & HW Thompson, Pergamon Press London New York Paris & Los Angeles p 1-6 Picuelell L 1985 Water 17-O and 2-H spin relaxation in aqueous colloidal systems, Lund Pinchas S & Meshulam E (1970) Chem Commun 1147 Rand RP Fuller NL Gruner SM Parsegian VA (1990) Biochemistry 29 76-87 Rees DA Chem Ind 1968 Cf Biochem J 1972 126 257-73 Sachi G (1964) Bull Chem Soc Japan 37 1685 Shah DO Hamlin RM (1971) Science 171 483 Schiberg D & Zundel G (1976) Chem Phys Lett 38 335 Stillinger FH (1980) Science 209 451-457 Tanaka T Izumi N Shao-Tang S Shizue U-N (1982) Science 218 467-9 cf., (1981) Scientific American 244 124-38 Vassilev PM Dronzine RT Vassileva MP Georgiev GA (1982) Biosci Reports 2 10251029 Watanabe M & Arai S (1987) Agric Biol Chem 51 557 [Cf ibid 50 169-75 and Wanatabe et al. Mol Microbiol 4 1871-9] Walrafen GE Fischer MR (1985) in Methods in Enzymology 127 Academic Press 102-105 Warner D (1973) Reported in Chem Week, June p73 Webb N (1990) Chem in Brit 26 338-340

Zundel S Murr A (1969) Journal de Chimie Physique 66 246-249

INSERTED
DOCUMENT 12 The Chemistry of Hydrogen Bonding Hydrogen-Oxygen

Hydrogen-Nitrogen Hydrogen-Boron Hydrogen-Phosphorus {Hydrogen-Silicon Hydrogen-Sulphur} O----H---O Hydrogen Bonding - Anomalous Water Water in Pores has higher viscosity and higher chemical potential and selects K+ from + Na+ Necessary pre-condiiton for life. Quantum mechanical tunnelling & Entrapment? Biological relevance seems possible. B----H----B Could boron hydrides create a boron-based life analogous to water-based life? P-H Bonds The existence of reducing conditons in the early earth has suggested that the llower oxyacids of P were then present [Cf Faraday Soc Discuss 2004]. The importance of phosphate (oxidation state five) to biology cannot be overstated. [ATP, DNA, RNA, phosphorylation of proteins to activate them]. What contribtion, however, could lower oxidation state P make to biology especially to early biology? Phosphorous acid P(3) vs. Phosphoric acid P(5); P(3) Does not easily form polymers linked by P-O-P bond; Forms much weaker metal ion comples pyrophosphite vs pyrophosphate.

Highly efficient P-H migration to form phosphonates which also form fmailies of polyphosphonate [E.g. acetic anhydride plus phosphorous acid poly(hydroxy ethane 1,1, diphosphonate based families of P-O-P and P-O-C linked polymers initially formed as a glass]. (While phosphonates where originally thought not to be of much relevance to modern bioogy but this belief has now been overturned).

INSERTED
DOCUMENT 13 Re-typed 11 Aug 2012 from old computer system printout Water Molecules Cation/Anion/Gas/Radical Sulphated Carbohydrate Interaction D Grant 15/12/97 The function of extracellular polysaccharides such as heparan sulphate is not fully understood. One of their primitive biological functions may have been to sequester physiological ions such as cations, amines and other small entities and molecules. Binding of physiological ions such as calcium and iron may be especially important. Binding of toxic moieties might, however, also protect cells (e.g. from the effects of reactive oxygen species). Numerous metal ions were found to be present in heparin (Glaxo 008) e.g. spark souce mass spectrometry quantified important amounts of the physiological cations. Iron did not manifest an expected paramagnetic effect on nmr spectra pointing to some unusual binding mechanism for this ion e.g. invovling sequestration via phase change. Literature data on any reported multiple cation forms of natural polysacharides might be usefully collected (e.g. alginate from brown algae occurred as a mixed na-Mg-Ca-Sr salt (Mo, F Brobak TJ & Siddqui IR (1985) Carbohydrate Research 145 13). These authors also discussed electrical charge distribution in such complexes where delocalisation of charge over various sites was evident (this phenomenon may be of general occurrence); Ca2+ was surprisingly not specifically bound to carboxylate but rather to hydroxyl groups, their presence in a favoured conformation for cation binding seemed to be the more important parameter (as previously reviewd by Angyal SJ 1981, Chem Soc Rev, 415-428 cf Cook WJ & Bugg CE 1975 Biochemica et Biophysica Acta 389, 428-435). It is further evident from published X-ray crystallographic structures of sugar cation complexes that water molecules participate, the sugar hydroxyls replacing the ususal cation hydration sphere. The degree and strength of hydration of heparin was found to be strongly dependent on the counterication (Grant D Long WF & Williamson FB 1990, Biochem Soc Trans 18 (6) 1283-1284. Comparison of the degree of hydration of sodium counterion suphated polysaccharides also

suggested a strong dependent of hydration on the degree of sulphation There being a tendencey towards six water molecules bound to sulphate half ester groups. The proton conductivity properties of hydrated sulphonate groups have found commercial application Nafion, Dupont). These substances also exhibit cation dependent hydration dependence and the proton conductivity is dependent on the arrays of water molecules held at the sulphonate groups (cf Chemistry of Solid State Materials Proton Conduction Solids, Membranes and Gels Material and Devices Ed P Columban, Cambridge University Press, 1992) The above proton conduction properties of related polymer systems suggests that similar properties might also occur in e.g. cell membrane of nuclear envelope heparan sulphates and is worthy of experimental investigation. Such conduction would, it seem likely, be modulated by degree of hydration which can be tuned by cation binding e.g. cations, polyamines and nucleotides. Other related situations Carbon dioxide binding to carbohydrates under alkaline conditions was Evidenced by Frahn JL (1968) Aust J Chem 21 811-814. The mode of binding seemed to be C-O- Na+ - C-O-CO2 Na+. Heparin pH titrations adding salt solutions-release of H+. Indications that heparin buffers solutions against pH change. Heparin Binds H+ and releases it upon metal ion coordination. Heparin appeared to bind colloidal hydroxyapaptite. Evidence from ir spectroscopy. [Later handwritten addition: 1999 What about CO Cf. NO and CO both may be invovled in regulation of blood pressure]. -Water Life and Prebiotic Evolution DOCUMENT 14

INSERTED FILE
DOCUMENT 15 Biologically-Relevant Hydration Notes plus some Classical Papers
Wiggins Phillipa [M.] Life Depends upon Two Kinds of Water PloS ONE 3(1): e1406 [2008]

Enzyme reactions and two-state water J Biol Chem 25-37

[2002]

Ionic Partition between Surfaces and Bulk Water in a Silica Gel A Biological Model Biophys J. 13(4) 385-98 [1973] {Cf Schmidt RJ Water solution to amino acid question Chem World 2007 Jan 32 [discussion of Wiggins paper Water in complex environments such as living systems Physica A 2002 314 485} Tanaka T et al Collapse of Gels in an Electric Field Science 218 4679 [1982]

Water Molecules Cation/Anion/Gas/Radical Sulphated Carbohydrate Interaction Notes Water and the origin of life Liquid water is usually assumed to be a pre-requisite for life and living organisms in their active forms are largely water. Water is their main nutrient. Specialized organisms are known for habitats with temperature above 100C provided the pressure is sufficient to maintain liquid water. The evolution of current biological systems may have been the consequence of the interaction of several stochastic systems held together in the long-term stable buffer. We propose these systems to be water, phosphate and silicate glass, polyhydroxyl ethers and proteinoids. Water is possibly a stochastic hydrogen bonded mixture containing arrays with electrical and electronic potential which can be stabilized at surfaces; phosphate and silicate glasses are random assemblages of linked units; sugar-like polyethers are also random polymers; proteinoids are produce by the random joining of amino acids

Cf. Columban P & Novak A {Book} Nagel JF et al (Hydrogen Bonded Chain Mechanism of Proton Conduction and Proton Pumping) J Membrane Biol. 74 1-14 Hadzi F (Proton Transfer in Biological Mechanisms) J Mol Struct. 177 1-21 Glew DN Mak HD Rath (Aqueous non-electrolyte solutions. Water stabilization by non-electrolytes). Chem Commun. 1968 264 These 1H n.m.r. chemical shifts provide a striking conformation for the strengthening of water-water H-bonding through watershell formation around weakly ion interaction PD Cratin BK Robertson Nuclear magnetic resonance , dielectric , near infrared and cryoscopic studies of solubilized and emulsified water. The system cyclohexane carbon tetrachloride-Duomeen T Dioleate J Phys Chem 69 (4) 1087 (1965) Jorge Cerbn Nuclear magnetic resonance of water in microorganisms Biochim. Biophys. Acta 88 (1964) 444-447 {my underlining} A portion of the study was directed toward ascertaining the cause of the broadening of the water signal, specifically, if related to any special kind of water structure. Different water systems were designed: free water, water with ions, water in emulsions, water in organic gels, water in inorganic gels, interstitial water in hydrophobic latex particles, water in crystals and various combinations of these systems. The NMR spectra of water in the different systems studied show that the water spectrum in microorganisms appears similar to those in inorganic gels that have an oriented structure of water with mobility limited by the adsorption forces. The little mobility of cellular water is suggested by its broad signal, which implies the existence of physiochemical properties different from free water. This fining could help to understand the parallelism between the physical properties of the bound water in hydrophilic colloid systems and biological systems as well as the differences between diffusion processes through artificial versus natural membranes [This study was reported to have been conducted with Nocardia asteroides strain N-51 using a Varian A-60 {60MHz continuous wave NMR spectrometer n.b., the use of continuous wave can reveal the amount of bond water directly; later use of Fourier transform etc. is often in conjunction with computed subtraction etc. of the water resonances which can loose water structure information]. Jean V Leyendekkers Aqueous Solutions Part 3.- Thermal and Acoustic Characteristics of Water PAPER 4/879 [Trans Faraday Soc (?) 1984 519 18 pages clipped

The anomalous heat capacity has been resolved into two components. Overall the results show that the I-bonded and O-bonded equilibrium is probably the critical factor underlying the anomalous properties of water and that the activation mechanism for this equilibrium could be a quantal one . That is, a time delay in the redistribution of energy between vibrational and rotational (or pure rotational) degree of freedom could govern the hydrogen-bond equilibium. The dispersions hydrogen-bond energy, H , identified previously, is of the right order to represent the quantum energy involved. It was found that anomalous increases in density fluctuations (with which energy fluctuations are linked) are almost as pronounced at constant volume as at constant pressure, the conclusion being that geometrical factors are the critical determinants of the cooperative behaviour of water.

Sachio Goto Studies of the Hydration and the Structure of Water and Their Roles in Protein Structure. I. (The Effective Volumes of 1-1- Electrolytes in Aqueous Solutions and the Electrostriction of Water) Bull Chem Soc Japan 1964 64 (11) 1685-1689 The volumes of inorganic salts in aqueous solutions are apparently smaller than the volumes in their respective crystals, due to the contraction of the solvent by electrostatic attraction between the ion sand water dipoles. However the volume of lithium iodide in solution is greater then that of its crystals. The order of the molal electrostriction of univalent cation was found to be as follows Na+>K+>Rb+>Cs+>Li+. Therefore the behavior of the lithium ion in an aqueous solution seems to be unusual [the results supported the idea that there is a clathrate around the lithium ion (cf. clathrate around inert gases). Hydration water seems to exist partly as a clathrate around the Li ion, the density of which is low also a clathrate around the Rb and Cs ions, as well as a clathrates round the Br- and I- and around tetraalkylammonium ions also. II, Toshizo Izemura And Sachio Goto (The Hydration of Electroytes by Ultasonic Inteferometry and Its Temperature Dependence) Ibid. 1690-1693 This paper gives further evidence for the existence of clathrates as hydration shells around inorganic ions I aqueous solution The numbers of hydration of various electrolytes at 20oC (when their compressibilities are assumed to be zero) decrease in the following order Sr2+>Ba2+>Ni2+>Co2+>Mn2+>Mg2+>Ca2+>Cd2+>Na+>Rb+>K+>Li+>Cs+. This finding contradicts the results obtained from the dielectric constants, entropy, viscosity and sonar. The disagreement may be due to differences between the definition of hydration. III Saucgui Goto and Toshizo Isemura (Proton Magnetic resonance Shifts of Water in Aqueous Solutions of Electrolytes and Their Relation to the Structure of Water) Ibid. 1693-1697 Fig. 1 showed uncorrected relative shifts of water protons indicating concentration dependent increase in in the order NaSCN>KI>KBr>NaBr> Acetone> RbCl, NaCl>CsBr, urea, LiBr> HCCOONa decr. with LiCl>CH3COONa, {CH3)4NCl The direction of shift agrees with the effect of breaking of hydrogen bonds by ions; the number of hydrogen bonds tends to decrease with ion size increase Li+ requries a different mechanism from other ions studied. Be2+ and Mg2+ (and also but differently? Li) cause not only the contact of water molecules with the ions but also exert and ordering effect upon the outer molecules by means of hydrogen bonding. These polarizing ions are expected to denature proteins by this mechanism. The phenomenon of protein denaturation by urea is however more complicated. Fig 2 showed the results of Fig 1 corrected (using the methyl shifts of acetone effect on water for comparison) for bulk diamagnetic susceptibilities [the results were in general simialar but with the relative effects of individual salts on changed somewhat] Fig 3 reported the chemical shift, , of water vs. the ionic radium of ions showing linear curves for monovalent cations and monovalent anions , the chemical shifts. IV Sacgui Goto and Toshizo Isemura The Hydration of Amino Acids and Oligopepties Ibid. 1697-1701 Another point of interest is that the hydration of the hydrophobic side chains of amino acids depends greatly on the temperature although the hydration of strong electrolytes and the ionic sites of amino acids depends only slightly on the temperature.. the number of hydration water at 20oC and at 40oC increases in the following order: glycine, alanine, valine and leucine. This pheonomenon is to be regarded as additional evidence for the hypotheses that the icebergs melt with an increase in the temperature or that the structure of ahe chlathrates is broken down at higher temperatures The hydration of the side chain of methionine is similar to that of the aliphatic hydrocarbon chain in valine. The phenyl radial is also hydrated The hydration of cyclic compounds is very dependent on the temperature. The peptide bond is hydrated The hydration of the peptide bond is probably like that of hydrophilic groups.. The hydration of amino acids can be classified roughly into the following three types : HYDRATION OF (1) IONIC SITES (2) HYDROPHPILIC SITES AND (3) HYDROPHOBIC SITES, The hydration of the hydrophobic sites differ in number and temperature dependence.

Jonathan W Clark Peter G Hall Alan J Pidduck Christopher J Wright (Molecular Diffusion in Monolayer Films of Water Adsorbed on a Silica Surface) Ibid. 1985 81 2067-2082 Quasieleastic neutron scattering showed the presence of two phases of sorbed water molecules with different dynamics. One component was immobile while those other demonstrated a two-dimensional diffusion. Jack H Kolaian and Philip F Low (Calorimetric Determination of Unfrozen Water in Montmorillonite Pastes) Soil Sci 1963 95 376-384. Water adjacent to clay mineral surfaces has many unusual properties. One of these is its resistance to freezing at sub-zero temperatures. Unfrozen water was measured calorimetrically in Li, Na and K-montmorillonite. The experimental observations reported here support the idea that the lack of freezing of clay-adsorbed water can be attributed to an alteration of the structure and properteis of this water induced by forces inherent in the mineral surface S Pinchas E Meshulam (The Nuclear Magnetic Resonance Spectrum of H218O in Dioxan-CDCl3 Solutions) Chem Commun 1970 1147 The chemical shift of a dilute H218O solution is ca. 0.3ppm lower than that of H216O. Previous work had indicated that nmr signals of the acetonitrile protons hydrogen-bonded to the oxygen atoms of H218O molecules appeared 12Hz higher than in the corresponding H216O case. Although this difference falls in line with a large number of similar anomalous phenomena peculiar to 18O compounds this appreciable difference in the chemical shift of a proton as a result of exchanging its hydrogen bonding oxygern 16 for and oxygen 18 atom cannot as yet be explained on the basis of existing theories. It might suggest that nuclear particles participate in chemical (hydrogen) bonds and hence contribute to the molecular structure of liquid water. Tsutomu Arakawa Serge N Timasheff (Preferential Interactions of Protein with Solvent Components in Aqueous Amino Acid Solutions) Arch Biochem Biophys 1983 24 (10) 169-177 High precision densimetery studies suggested that bovine serum abumin and lysozyme were preferentially hydrated in all of the amino acids examined (glycine, - and -alanine) (additionally, the effects of betaine was studied; these effects differentiated betaine form those of the amino acid additives). It was indicated that addition of the studies amino acids resulted in an unfavorable free energy change . In the case of the three studied amino acids which are known to have a positive surface tension increment, their perturbation of the surface free energy of water is consistent with their preferential exclusion from the protein surface. In the case of betaine which does not increase the surface tension of water, preferential exclusion form the protein surface must have affected the chemical structure of this cosolvent which is considerably more hydrophilic than that of the three studied amino acids. The activity of many enzymes were not affected by the presence of betaine at high concentration which allows betaine to act as a regulator of osmotic pressure (previously studies in the cytoplasm of halophyte and halotolerant bacteria adapted to low water potential). The action mechanisms of betaine and glycine for non-polar substances should be different including the different interactions with denatured proteins Previous work had established that the ability of certain solvent additives to stabilize the structure of proteins at high concentration was related to a preferential (protein) hydration in the aqueous solution containing the additives (e.g. 2-methyl-2,4-pentanediol, sugars, salts and glycerol all seemed to stabilize proteins by this mechanism. In the case of sugars and some salts the preferential interaction paralleled the ability of these additives to increase the surface tension of water. Glycreol, however, lowers the surface tension of water but also induces the preferential hydration of proteins while urea which increases the surface tension of water is bound preferentially to protein peptide bonds through hydrogen bonding. Most organic substances lower the surface tension of water but diand mono-saccharides are an exception to this rule.

WF Long FB Williamson Marischal College Aberdeen University)( HEPARAN SULPHATES AS BIOLOGICAL EFFECTORS THROUGH THE MODULATION OF WATER STRUCTURE) Evidence from studies of cell surface heparan structure and function conducted at Marischal College [Near infra-red spectroscopy of aqueous heparin solutions; Influence of metal ions on water-heparin interactions; Importance of water in protein-heparan interactions; Altered NMR chemical shifts of heparin nuclei; NMR relaxation studies and Heparin/heparan interaction with artificial surfaces suggests that

heparans are modulators of water structure

Anon., New Scientist 15 June 1978. Fish antifreeze nips ice crystals in the bud. The antifreeze action is caused by the glycoproteins specific ability to inhibit the growth of ice crystals by binding to small regions of ice-like order in water. Franks and Morris (Biochim. Biophys Acta 540 346) showed that such an ability is likely to depend on the presence of sugar groups on the glycoprotein which have a very specific spatial arrangement and orientation with respect to each other(It was hypothesized that) the sugar groups can arrange themselves flat along the protein surface so as to present a very regular hydrophilic array to the surrounding water. Anomalous Water References, Collection of
Leland C Allen (An Annotated Bibliography for Anomalous Water) Journal of Colloid and Interface Science 1971Vol 36 (4) August

Arakawa T et al., (Why preferential hydration does not always stabilize the native structure of globular proteins) Biochemistry 1990 29 (7) 1824-31 Masamichi Tsuboi (On the Melting Temperature of Nucleic Acid in Solution) Bull Chem Soc Japan 1964 37 (10) 1514-1522
Heating aqueous solutions of double strand DNA commonly causes the coil to unwind and engage in strand separation to give random coils. This transition occurs over a narrow temperature region. This is considered to be melting of a one-dimensional crystal. This varies according to the base composition, salt concentration, the pH and the presence of some solutes. The melting temperature is greatly influenced by the ionic strength of the aqueous solution. Spermine also causes an increase in the melting temperature of DNA. The hydration structure present in spermine phosphate hexahydrate where there is a sheet which consists of HOPO32- ions and water molecules formed by the OHH hydrogen bonds seems to be similar as regards the disposition of the phosphate groups to that which occurs in spermine-DNA.

The nucleic acid melting process demonstrates a linear entropy enthalpy relationship.
Cf., Kolata G , (Z-DNA) Science 214 1108 Cf., Conner BN et al. (The molecular structure of d(CpCpGpC1) a fragment of righthanded double helical A-DNA) Nature 295 294
Hydration of A DNA is different from that in B DNA . In a 35% methylpentanediol (the solvent used to crystallize DNA) crystals of B-DNA show all the N and O groups on the edges of base pairs in the major grove are hydrated by a monolayer of solvent molecules but the bulk water with the major groove appears disordered. Similarly except for these examples where a water molecule is trapped in a clathrate-like manner between a phosphate group and a thymine methyl, hydration of B-DNA along the phosphate backbone is also un-localized. The most characteristic feature, however, of B-DNA hydration is a zigzag line of (H2O)n in the minor groove. Possibly the breaking of this (H2O)n line is the first step in B-DNA A-DNA. This spline of (H2O)n is absent in A-DNA. B-DNA is favored where H2O activity is high. This activity can be altered by the addition of EtOH.

INSERTED
DOCUMENT 16

Biological Nucleation. D Grant Modified to 3/12/00

CONTENTS 1. Nucleation of Non-random Structure Assembly. Relevance of Non-Crystalline Order in Highly Metastable Polymeric Systems to Conditions for Life. 2. Nucleation of Heparin/heparan Sulphate (Polyionic) Binding. 3. Nucleation of Prion Infectivity. 4. Nucleation may Promote Autommune Diseases 5. Are Aluminosilicate Cores of Alzheimer's Disease Plaques Nulceation Factors? 6. Is Iduronate Flexibility Coupled to Nucleation Activity? 7. Hydration, Water & (Poly)ol Structuring as Factors in Nucleation Events. 8. Non-genetic Inheritance and Heparin/heparan Sulphate Coded Information. 9. Nucleation Functions in Silicate Structures. 10. Control of Calcification. 11. Reversible Polymerization Processes in Stochastic Systems. 11a Critical Temperature and Entropy and Enthalpy Compensation Systems 12. PRECONDITIONS FOR BIOLOGY & CENTRALITY OF nucleation out of STOCHASTIC SYSTEMS TO INITIATION OF BIOLOGY. 14.Notes from "Evolution without Selection" by Lima de Faria (1988). 15. Effect of Electromagnetic Fields on Nucleation. 16. Polysaccharides Involved in (Biological) Nucleation of Nanocomposites. 17 Amorphous Ca/Mg Pyrophosphates and Nucleation. 18.Transition Metal Centres in Enzymes and Ziegler Natta Catalysts 19. References 1. Nucleation of Non-random Structure Assembly. Relevance to Non-crystalline Order in Highly Metastatic Polymer Systems and Conditions for Life. We seek here to define life in terms of the modulated/amplified vibrational complexity and structured/micro systems present in water and poly(O-H)and (N-H) molecular cluster systems, it being postulated that there are surface field effects therein of proton fluxes related at a fundamental level to the existence of the biological phenomenon. We assume that life requires the presence of liquid water because of the unique physical chemistry of water. The hypotheses arrived at, suggesting the important influence of nucleation of a highly ordered but not-crystalline, not-solid colloidal structures in molecular composite structures required for the attainment of biological complexity, an additional facet of which, also central to phenomenon of the existence of such naturally occurring systems is a possible involvement of thermodynamic principles governing highly metastable states where, paradoxically, complexity may tend to increase with time if inhibitors of the decay of such states are present, such factors are seen to influence the effect of water structure builders (ions and polymers formed by water elimination condensation

reactions to give polymers with side chains with water structure and proton conductance modulation); The following observations are thought to be significant 1) the likely importance of nucleation of water-rich inorganic dispersions (silica sols) previously thought of as amorphous colloidal systems which seemed to possess properties (shape, size, reproduction and ageing) reminiscent of biological cells; 2) water is well established to have anomalous properties derived apparently from unstable flickering clusters of hydrogen-bonded aggregates with a potential for rapid proton translation (causing an infrared absorption continuum in the fundamental bond stretching region); an infrared overtone vibration spectroscopic well-defined phase-liketransition occurs in liquid water at 36-40C; this apparent phase change will depend on nucleation phenomena but may be perturbed by noise in bands ascribable to nucleation effects on water aggregates; 3) that living cells and organisms are mainly water suggests that the water serves a function more central to biology than that of an inert supporting medium; the likely central requirement of the special properties present in liquid water for the operation of biological cells is illustrated by the non-trivial critical requirement for the presence of sufficient polyhydroxyl water substitute sugars to permitting suspension of life allowing a complete long-term dehydration in certain organisms with almost immediate return to life upon replacement of the water. 4) a likely involvement of nucleation in heparin/heparan sulphate biochemistry. Manning predicted that the ionic charge alone not the chemical nature of the ion controls such polyanion co-ion binding in disagreement with experimental results of heparin ion binding which showed potential nucleation dependent phase separation behaviour; this is also the predicted situation for heparin/heparan sulphate involvement in growth factor signalling (e.g. during embryogenesis). 5) possible involvement of hydration of polysaccharides in proton conduction. Such effects are also intimately tied up with supramoleuclar structures involving water molecules which occur in counterion dependent clusters at sulphonate/sulphate groups in heparin/heparan sulphate suggesting a facility of gating of fast proton conduction along the polysaccharide chains (which are chemically similar to Nafion, a sulphonate based water cluster proton conductor); this phenomenon may be connected with unusually hydrogen bonding arrangements involving highly polarizable (very short, symmetrical) hydrogen bonds giving rise to extreme continuous absorption regions in the infrared; structures associated with proton conduction effects (cf Colomban 1992) seem to be the ones which support such hydrogen and are the ones favoured by biopolymers ( viz -C(=O)-O- groups e.g. in acid salts (sodium hydrogen acetate (Hadi), some phosphorus oxy acid acid salts (Grant & Payne 1966) and sulphonated polymers (Zundel)).

5) the tendency of biological systems to generate increased complexity with time, which seems to violate the general application of the second law of thermodynamics although this has been suggested to not be applicable to open as opposed to closed systems, may alternatively be related to less well understood thermodynamics of nucleation which often favours less stable forms. Experimental data including nucleation kinetics shows inexplicable correlation between entropy and enthalpy factors . The occurrence of such apparent compensation between entropy and enthalpy (and the implied blurring of the direction of time) which tends to violate the laws of thermodynamics as applied to stable systems i.e. the second law of thermodynamics, is perhaps inherent in all biological state metastable systems. The rate of all chemical variants in such systems undergo a particular type of reaction at constant rate at the characteristic temperature. Nucleation of crystallization is a well established phenomenon but owing historically to the success of crystal structure determination methods "less interest was focussed on correlating crystal structure with morphology.... the use of dopants for inducing a particular crystal morphology is a widespread technological tool, even though the mechanism of the effect is scarcely understood" (Addadi et al 1985). Biological tissue is customarily supersaturated with respect to formation calcium containing crystals, inhibition of nucleation of such crystallization being required to prevent such insults as arterial calcification and ice formation (and degenerative diseases may be augmented by fault in such systems (Grant et al 1992)). However crystallite shapes and composite microstructures are utilized biologically, being responsible for the many forms of substances such as calcite or skeletal apatite which are readily conceptualized as arising by (controlled) nucleation processes. Molecular structure aggregations of forms analogous to crystals, but possessing, not lower, but different, degrees of order than crystals are now postulated to be fundamental to all biological operational systems. Water and related liquids seems to be capably of forming loose hydrogenbonded aggregates the surfaces of which may possess tuneable energy levels useful for biological systems. The tendency of water rich gels to form ionic units showing continuous apparently unresolved quantization of vibration may include arrays of symmetrical hydrogen bonds possessing unusual delocalization of proton movement. Biological systems might even be considered to be uniquely centred on the nature of such metastable poly-O-H water-rich aggregate structures, such a may be responsible for the anomalous effects attributable to the polymeric nature of water and -OH rich systems, and their ability to engage in complex phase-like transitions (additional to overt solid-liquid transition), sensitivity to nucleation control; soft composite biology may even be ultimately definable by proton energy fluxes. Nucleated structuring is considered relevant to such

complex soft tissue morphology as well as to the formation of hard composites in shells, teeth, bones and calcium-rich granules. Hysteresis in metastable phase transitions in polysaccharide systems (e.g. of bacterial, algal as well as mammalian origin) demonstrate influence by nucleation factors; previously such phenomena have tended to be regarded as trivial or a nuisance; such effects are likely to be common factors in in water rich gel/sol systems including those responsible for the realm of living organisms. Nucleation of the formation of highly hydrated solids often containing large numbers of water molecules such as methane hydrates (where nucleated formation of insoluble water clathrates "snow" may block pipes; cf also carburettor iceing); water is a good solvent and dispersant and the effects of the usual presence of often trace amounts of multiple inorganic and organic components and their effects (possibility of providing seed for nucleation or inhibitor factors) upon water aggregation or de-aggregation in their vicinity, is likely to be highly relevant to many chemical and biochemical situations. The denaturation of proteins involves often obvious irreversible insoluble phase formation (which is likely subject to nucleation and the effects of promoters and inhibitors of such nucleation e.g. polysaccharides especially glycosaminoglycans, heparin especially can inhibit thermal denaturations). The Hofmeister series ranks salts for their protein denaturing abilities; the origin of this effect has been somewhat obscure but nevertheless it has been considered an important pointer to likely fundamental biological vectors; it has been established that the Hofmeister effect also applies to the observed order of alteration of the supramolecular structure of water in salt solutions (detected by the near infrared spectroscopic absorptions of water molecule aggregates, cf Luck 1987, Marburg symposium); salt effects further resemble the effect of temperature upon water aggregation. The temperature dependence of a near infrared band at ca 1.18 microns in lamellar films of liquid water showed an apparent discontinuity consistent with a phase change-like alteration in less-than-crystalline-order aggregate structure in the biologically important temperature region of 36.5-38C seemingly affecting principally the sharp first overtone O-H stretching band and apparently less the fundamental O-H bond vibration region (e.g that of HOD as reported by Ford & Falk 1968 Fig 2 although the bending mode reported in this Fig shows the ca37C transition but in a somewhat irreproducible manner); on the other hand an apparent structural transition in water between 20 and 30C shows up preferentially in the 0.75 micron, 13000cm-1 band and is apparently absent from the 0.95micron, 10526cm-1 band. (cf Luck 1965 cf Part II Figure 6, in the 1.15 micron band at ca 8475cm-1; this band is assigned to "pendant" H-O groups in water clusters of various sizes should be compared with Figure 1 in Ford & Falk 1968 for HOD melting). The apparent irreproducibility of the 2OC and the 37C transitions in liquid water detected by IR spectroscopy could suggest the involvement of nucleation phenomena in the formation or decomposition of these evidently metastable states.

The 8475cm-1 band is attributed to the first overtone (2x fundamental vibration frequency combination with the water bending frequency, so it should be simultaneously detectable in other spectral regions unless the structure change is distorted by other factors unusual nucleation effects (in different laboratories ?) variable signal to noise ratios, variable extinction coefficients in the different parts of the spectrum (this phenomenon has does not seem to have attracted previous attention except for the mention by Luck (1965) (the melting of ice at 0C is of course well known to be dramatically affected by nucleation inducers and inhibitors). Although overtone regions may be dependent on the quite different extinction coefficients reflecting impure absorptions involving scattering for different vibrational overtones of water aggregates absorptions) but further study might offer insight into nucleation effects in such water structures. It is suggested that an enhancement of overtone occurs via a systematic, specifically nucleated (could there be direct auto-nucleation caused by the presence of adjacent vibrations at the overtone frequency?), causing abrupt change in hydrogen bond rearrangement rates and induced electron and proton as well as molecular fluxes e.g. resembling flow patterns. Similar temperature dependent shifts in O-H are found in the spectrum of methanol indicating that the the -O-H group rather than the water molecule and its tendency to form hydrogen bond; Abundant alcoholic O-H groups compounds in sugars and polysaccharides are expected to show similar transitions. The formation of charged surfaces between phases is associated with effects of electromagnetic radiation and magnetic fields on crystallization; heating effects of vibrationally absorbed radiation may facilitate the formation of such surfaces in liquid water, perhaps acting as nucleation factors for structure formation (cf Evans 1984). Amplification of low energy transitions between unstable structures (e.g. at cell walls) may be a core phenomenon of biological systems (cf. Adey 1988). Do most homoisothermic biological systems "ride out" the 37C water structure phase change via their nucleation abilities to modulate and control water aggregate supramolecular structure by linkage of osmolality-temperature with polymer conformation? Urry has discussed the transition around 37C in terms of rearrangement of pentagonally arrayed water molecules of hydrophobic hydration around proteins. Nucleated formation-rearrangement of pentagonal arrays in water alone may be responsible for the 37C phase like transition in the 1.15 micron band. Such nucleation may be cuvette surface, impurity, and heating effect induced flow pattern dependent. Comparing repeat spectra of water in the overtone region (Grant et al 1982) using a dispersive (non FT spectrometer) showed that signal to noise ratios were inconstant for some but not all overtone absorptions, being especially noisy in spectral regions ascribable to water aggregate absorptions (e.g. a shoulder at 6950cm-1 on the sharper 7050cm-1 first overtone absorption of water in capillaries) at which are evidently more sensitive to chaotic changes in the average structure present at any given time. Unstable water aggregates responsible for the 6950cm-1 absorption could conceivably be

formed by sloughing off surface templates and are capable of persisting for highly variable periods of time (in some (deterministic?) chaotic manner?) Such signal to noise effects could lead to sharper bands showing up particular aggregate vibrations but less well detectable by other bands. The biological effects of such water aggregates, perhaps augmented by heparin, may have contributed to the apparently irrational experimental results, subject to unusual editorial disclaimers and post publication evaluation, of basophile degranulation triggered apparently by water alone, obtained in a sequence of critical mixing protocols (Glick 1988, Davenas et al 1988) starting from IgE dispersions . Similar ultra-dilution phenomena were observed, it was reported, for vortexed ethanol and propanol experiments but not for dimethylsulphoxide indicating that the property may arise from O-H group containing solvents rather than being unique to water further strengthening a possible explanation including heparin polysaccharide activities (Glick, 1988). In the infrared studies of water and some other -O-H containing liquids reported by Luck, a similarity in the effect of temperature on the NIR-detected water aggregation and alcoholic solvent aggregation was observed. The irreproducibility of metastable phase transitions (in which phase boundaries will have associated electrical fields) in liquid water may be due to microheterogenity produced by persistent vortexed flow ("whirlpools") with surface energy levels sensitive to protein binding site signals; the vortex state may therefore encourage encourage the nucleation of biologically active metastable water molecules aggregates. Biology requires both high order of space segmentation and high accuracy of phase specification, since biological function is facilitated by precise semipermeable boundaries at cell and organelle walls, but also more subtle boundaries at hydrated biomolecular, especially at highly polyanionic nucleic acids (nucleation of nucleic acid template activity being an established requirement) proteoglycan, glycosaminoglycans surfaces as well as two dimensional phospholipid bilayer liquid crystals. In general, then, the nucleation of the formation of complex chemical phases characterized by less than solid crystalline order, can be rationally postulated to be of likely fundamental importance to biology, characterized as this is by an extreme non-randomness and assemblages of highly specified molecular structures; the hypothesis is here advanced that these structures are dependent on their assembly and correct functioning by the action of correctly sequenced heirarchical nucleation processes. These are proposed to permit spatial and temporal amplification of signals relating to morphogenesis. Such polysaccharides as heparan sulphate are proposed to be central in such a function by enhancing and controlling nucleation. Such nucleation activities, especially in relation to water chemistry as modulated by biological surfaces, may even be the true essence of the nature of biological system - that which makes them most different from abiological systems. Life is seen in this model to need access to a sort of biological less-thanstochastic or deterministic chaos in which rearrangement of hydrogen-bonded

water or -O-H aggregates undergoing spontaneous flickering but governed by rules of engagement - these are highly centred on the control of nucleation activities. Thus such 'biochemical potential water structures inherent in water itself (and having close association with properties of other poly O-H containing molecules) may be the leading potential driving force for biological events centred on nucleation events. The toxic nature of deuterium oxide for higher organisms is perhaps an indicator of the high specificity such water structure related effects as is the critical importance of the presence of liquid water as a pre-condition for life. What then is life? Can we define life in its most basic form in terms of stable cellular activities centred on metastable amplifiable states of (potential, vibrational including proton conduction band field energetics) of water aggregate structures? The sort of thing detectable in near infrared spectra of water? Such "flickering cluster frequencies" of loose aggregates of water molecules at lipid surfaces are likely influenced by precise boundary conditions in cells produced by polysaccharide and protein polymers with arrays of hydrophobic and hydrophilic and related capillary effects. Faculty for alteration of water aggregate structure and the vibrational energetics associated with these by boundary conditions, salt and polyol concentration and temperature is now postulated to be critical to biological activity and define life. Such highly evolved substances as glycosaminoglycans offer polyionic modulation of such effects by smart surface alteration coupled with a high level of ion binding and translocation potential determined and controlled by and offering enhancement of nucleation.

In summary, growth dependent on nucleation is more generally required for the self-assembly of any structures more complex than random. Anionic polysaccharides long understood to serve as biological nucleation control agents in calcification processes in bivalves and mammals also more recently have been found to modulate the activities of growth factors and adhesion molecules in embryogenesis, wound healing and probably in cancer. Such polysaccharide activity will also be dependent on cooperatively formed biologically structured water. The key to understanding how such activity is controlled might be found in consideration of their nucleation activities. Heparin/heparan sulphate modulation of the crystallization of calcite was found to be centred on the de-N-sulphonate groups in heparin/heparan sulphate (removal of these groups greatly diminishes this activity (cf. Grant et al 1989). (Damage to such N-sulphonate sugar arrays by nitrous acid or other active nitrogen metabolites of the nitric oxide system perhaps catalysed by unprotected iron or other redox metals may also be a more general mechanism of pathology than has hitherto been appreciated (Grant 2000)). In heparan sulphate these hydrophilic N-sulphonate groups generally occur in arrays separated by hydrophobic N-acetyl rich domains which, it can be postulated, may achieve different significance in such arraying of the Nsulphonate groups by suitable conformational switching, most likely being influenced by, and influencing, salt and temperature-associated modulation of

water macromolecular structures around polyol (polysaccharide) created microdomains in water. Glycosaminoglycans seem to resemble mineral surfaces such as silicates or apatites and it is evident how formation of polyanionic interlinks involving ionic interactions could resemble crystal formation and how biological systems could have first been formed by interactions of mineral surfaces with lipids and primitive organic polymers (formed by condensation polymerization involving elimination of water molecules). It became evident that (highly sulphated) polysaccharides containing iduronate sugars have been present throughout animal evolution (present in both vertebrates and invertebrate (Nader et al 1988)) where they evidently serve fundamental functions. In plants, less anionic polysaccharides (Albertsheim) may serve a similar functions. Evidently more highly charged groups may be associated with greater motility needed in animal tissue. Boundary conditions in the amorphous states (which may contain various short range rather than by long range degrees of order) of polyphosphates and silica may additionally provide models of biological systems since they demonstrate the ability to undergo polyanion determined manipulations to form precise morphological shapes and may participate in colloidal chemical behaviour mimicking biological behaviour. 2. Nucleation of heparin/heparan sulphate (polyionic) ionic binding. The functions of heparan sulphate proteoglycans influencing nucleation of crystallization of calcite etc but perhaps have more dramatic involvement in soft tissue morphogenesis; (the actions of Hedgehog (involved in tissue patterning during both vertebrate and invertebrate development) FGFand Wnt depend on them (Perrimon and Bernfied 2000 cf Lin et al 2000); . This modern work follows from earlier ideas implicating cortical control of cell division (cf Caplan et al 1983). Heparan sulphates seem to provide structurally specific binding sites ("smart glue" - an recent example is the requirement of heparan sulphate PGs and integrin for adhesion of human skin fibroblasts to adhesion mediator Cyr 61 (Chen et al 2000)) with regulatory function for protein processing in control of cellular activity including during embryonic development. The likelihood that such binding behaviour of heparan sulphate during these processes may be triggered by nucleation factors has not so far been addressed. Such binding is believed to be non-covalent and be similar to salt formation so that the model studies of ion binding to heparin are thought to be valid for the more general evaluation of heparin/heparan sulphate protein binding. Experimental results (Grant et al 1992c,d,e) of heparin salt ion binding, using a number of different techniques, showed that this did not obey the usual rules governing single phase behaviour but was more in keeping with (nucleated) phase change behaviour, it demonstrating hysteresis effects. Other aspects of heparin co-ion binding such as the lack of general paramagnetic NMR broadening following from high paramagnetic iron loading,

independence of acetate side chain resonances from factors affecting more central resonances, and the effect of nucleation factors in inducing supramolecular structuring of algal polysaccharides and similar substances are also consistent with this idea. It is important that hysteresis and nucleation control be confirmed for these important interactions since such salt linking (highly tied up with surface hydration effects) is likely to occur both with inorganic ion binding and protein binding to anionic biopolymers and be central to the mechanisms of their biochemical activities. 3. Nucleation of Prion Infectivity. Is this the key to its Aetiology? A topical example of possible critical pathological nucleation, involving noncrystalline order, could be the basis of prion pathology, supposed (in he Prusiner hypothesis) to be the consequence of a mismatched secondary structure; the normal protein contains the same primary structure but altered conformation and evidently altered function. The pathological activity evidently derives from a powerful nucleating activity of the pathogen for changing the normal into the aberrant form of the protein; (however, reversal of such pathologically conformated of scrapie prions by highly sulphated polysaccharides (e.g. heparin and xylan polysulphate) is, apparently possible (Diringer & Ehlers 1991; Gabizon et al 1993)) and may provide an general antidote to prion diseases); hydration effects are expected to contribute to nucleating of aberrant structures in prion diseases. 4. Nucleation may promote autoimmune diseases. The importance of nucleation of aberrant biopolymer structures by various pathological organisms could be a factor in the aetiology of autoimmune diseases (e.g. by the triggering of the immune responses by components via pathogen cell walls). 5. Are aluminosilicate cores in Alzheimer's disease plaques nucleation factors? Inorganic nucleation of amyloid plaque formation by alumoinosilicates is implied in the findings of Edwarson, Candy et al in Alzheimer brain tissue as well as in the effects of toxic solids such as cancer inducing silica fibres, crystalline silica (in silicosis) and high iron forms of asbestos (in asbestosis). There are some indications that heparin/heparan sulphates can also provide antidotes in such situations. 6. Is iduronate flexibility coupled to a nucleating activity? The unusually highly flexibility of iduronates (e.g. Sanderson et al 1985) in heparin/heparan sulphate suggests that iduonate evolved from glucuronate to give more efficient highly flexible matching templates for nucleation of protein conformation or organelle morphology (their main disaccharide :- iduronate2-deoxy 2 amino D-glucosamine N-suphonate residues are known to facilites correct conformational folding of proteins for specific functions as are specialized sulphated sugar sequences within the heparin/heparan sulphate chains, the most well known of which is the AT(III) binding site which promotes antithrombin activity via a conformational switch in AT(III), this being the main basis of its anticoagulant activity).

7. Hydration and Water Structuring as Factors in Nucleation Events. The nucleation of ice structure and the occurrence of highly evolved antifreeze proteins, in the cysteine-threonine arrays in insect antifreeze the surface of the antifreeze protein is considered very specific to fit ice crystal nuclei. The ice nucleation gene product AGYGSTLT has 122 imperfect repeats where up to 68 octapeptides could be deleted without abolishing ice nucleation; deletions however caused a change in the shape of the temperature-frequency profile rather than a uniform reduction in freezing at all temperatures (Green & Warren 1985). Water chemistry, is influenced to an unusual degree by surfaces (discussed by Bernal, 1965) where extremely long range effects are to anticipated. Water in glass capillaries give NIR spectra which show relative enhancement, compared to conventionally studied lamellar liquid films, of the resolved absorptions in the overtone region (although the effect is likely to include differential scattering and effects of micro temperature gradients and flow); an apparent broadening of the bands may indicate the presence of enhanced amounts of aggregated water (deducible from correlations between band positions and the degree of polymerization of water in aggregates as discussed by Luck (also NIR results of Grant et al 1984, unpublished). Such infrared absorption effects provide evidence that highly reproducible long-range surface influences can modulate biologically relevant energetics; similar highly reproducible spectra, dominately by water absorptions are utilized in commercial financial evaluation of agricultural samples using empirical comparative methods, a situation which does not, however, win merit from academic spectroscopists more interested in correlating more definable aspects of infrared absorption. A somewhat similar situation exists in medical use of proton NMR tissue imaging where water proton relaxation contributions dominate. Many biological situations mimic such nearness of water to surfaces and will therefore be influenced critically by such proximity, the ultimate situations being such properties as proton conduction in water "wires" evidently present in cell membrane protein channels this phenomenon continues to be dealt with by theoretical calculations of increasing sophistication. Similar proton translocating "wires" are likely to occur on the surfaces of glycosaminoglycans (which have similar counterion dependent water clusters associated with sulphonate (cf. Nafion, Dupont) and sulphate half-ester group. The presence of the correct adhesion friendly water environments is correlatable with NIR vibrations due to water aggregates and their presence at variously treated polystyrene surfaces accords with the ability of such surfaces to support cellular adhesion and growth (Grant et al 1988). Urry (1996) postulated that energy conversion in living organisms may result from "changing not the temperature but the temperature interval over which proteins fold and reassemble within and surrounding cells;..on lowering the temperature from just above to just below body temperature (37C) the

hydrophobic residues cluster away from the hydrophilic residues and fold back on themselves, separating out from their water cell environment and allowing the protein chains to associate... below the critical temperature region the protein hydrophobic groups are surrounded by pentagonal arrangements of water molecules"; the rearrangement of these was seen as being an energy source ca37C and will surely be a source/sink tapped into by living organisms from the phase-change-like alteration in NIR detected water structure at 37C. Hydrophobic forces between uncharged hydrophobic surfaces make "available to biological systems a hierarchy of attractive forces that operate between hydrophobic molecules; these forces depend upon the dimensions and the geometry of the surfaces and are much stronger, longer ranged and more variable than classical colloid science previously indicated" (Pahsley KM et al 1985). Phospholipid bilayers biophysics is intimately dependent on associated water e.g. in the phase change between sol and gel but water mimicking polyols (especially trehalose + zinc ions) are able to stabilization of biological membranes by inhibition of the nucleation of phase change) and is used by certain organisms to survive dehydration (Crowe et al 1987); this a stunning philosophical demonstration, rather than a trivial outcome, of the fact that living is to do with systems centred on water chemistry; suitable water substitute sugar (especially trehalose) protects in suspended animation those complex organisms which can become completely dehydrated but revived simply by adding water. Such metastable states derived from water aggregates and hydrated biopolymers are postulated to be kernel of the biological phenomenon. We discuss the possible chaotic nature of biological order as in its most primitive form- water structure at near (usual) biological temperature- where an apparent metastable phase transition occurs involving water aggregates of evidently differing structure, likely dependent on differently arranged hydrogen bonded aggregates. Difficulties in experimentally observing this transition are suggested to arise from the unstable nature of the transition and its great sensitivity to nucleation effects which facilitates stored energy release from such batteries of hydrogen-bonding rearrangements. Another possible feature of such metstable situations is that the laws of thermodynamics may be differently applicable, the facility for spontaneously increase in order with time, i.e. decreased entropy with time in apparent violation of the second law of thermodynamics may be allowed. Similar metastable states of matter present in certain reaction rate determining intermediates may be at the heart of otherwise disallowed compensation effects between energy and entropy changes. The metastable water transitions favoured by biological water is suggested to be such an example of this type of metastable matter. 8. Non-genetic inheritance and heparin/heparan sulphate (nucleationrelated?) information coding. Heparin/heparan sulphate are coded information carriers (possessing specific 'conserved' sugar/hydration sequences analogous to the genetic DNA and

RNA information storage systems). Although these sulphated polysaccharide-based information carriers are ultimately derived from gene products, they may also possess, a carried-over, somewhat independent influence (perhaps the label "non-genetic inheritance" is not too far-fetched); their critical roles in developmental biology (cf. Perrimon & Bernfield 2000) which evidently include sequenced control of ionic binding of various proteins such as growth factors to their receptors during embryology nucleation activities are now suggested to be of critical significance. (Such highly ionic systems will also alter water structure by the Hofmeister effect and such hydration effects may also be part of the mechanism transmission of such information as nucleation site structure). Heparin/heparan sulphate and analogous polymers, especially in the pericellular environment of relevance to cellular recognition and adhesion, can be thought of as providing a sort of information/processor function involved in 'upgrading' and 'processing' information derived from genes i.e. allowing protein primary structures adaptation by inducing correct supramolecular structure folding and provision of templates for component assembly and conformational repair which will include correct hydration structure effects. We hypothesize therefore that the mechanisms of polysaccharide biological function could be centred on the need for nucleation of the formation of specific biological phases and further that hydration structures are of central relevance to such processes. 9. Nucleation functions in silicate structures. Biological polysaccharides (including sulphated polysaccharides) also apparently nucleate inorganic structures in the control of biological silicification (Perry et al 1987) and calcification. This linkage may have relevance for consideration of mechanisms of early prebiotic events in the development of biological cells in general. Silica is an abundant component of the surface of the earth's crust, capable of dissolution to monomeric units in water in which spontaneous self assembly of more complex structure can start from the soluble, highly hydrated silicic acid monomer unit giving rise to "structured" colloidal silica sols (Grant et al 1992). Such sols may have provided templates for the formation of nascent biological systems in a similar fashion to the present-day glycosaminoglycans. Such silicate polymers assembled into cell-like units seem also to allow the enrichment of non-random structures in a similar manner to the proteinoid microspheres described by Fox et al, evidently being subject to some form of nucleation of structure. Silica gels have apparently been described which, if formed in the presence organic isomers reproduce such shapes in the solidified gel allowing a molecular sieve type of isomer separation (discussion with Dr Chilton, Monsanto, personal communication old pre-1964 patent literature). Silicate structures formed by the scratching of the walls of a soda glass test tube can nucleate specific crystallization from supersaturated solutions in the absence of a seed of the required crystal.

Silicate glass capillaries produce aqueous solutions with apparently anomalous properties ascribed to an unusual form of water ("polywater"), a claim which was later refuted, but high water content silica gels (a well known characteristic of aqueous silica gel/sol chemistry) may contributed to the effects observed. Silicate 'contaminants' in heparin (present in the ultradilution experiments of Benveniste et al.) where biological activity apparently survived dilution beyond the Avogadro number, could have allowed antigen imprinting (immunologically nucleating templates) at the surfaces of hydrated polyanions perhaps enhanced in quantity and activity by vortex mixing (including mechanical release of solids from vessel walls). Biological silicas are believed also to be assembled by a self-assembly mechanism and are therefore of particular interest for studies of the influence of factors which are able to nucleate such unique forms adopted by these assemblages especially a they might give clues about the origin of biological evolution. The underlying chemistry of silicates encountered in mono-phase silicate ester systems is characterized by an essential random ligand exchange process leading to facile structural reorganization; in the case of aqueous dispersion they may encourage increased formation of non-random assemblage into discrete structures influenced by nucleating forces involving specific hydration effects. Related structuring effects may occur during the apparent creation by polysaccharides of specific form in biogenic silicas which although still retaining their pure chemistry property of undergoing self-assembly within the modern biological cell, are evidently encouraged by adjacency of specific polysaccharide surfaces to adopt specific forms (Perry et al 1987) illustrated by the formation of botanical "amorphous" silicas occurring in many speciesdependent morphologies which are useful taxonomicaly (cf. Bunney 1985). Industrial amorphous silicas such silicate areogels and silica sols display a complex taxonomy of "species" characteristics (valid for such preparations as as Ludox (Dupont) Syton (Monsanto and Nalcoag (Nalcoa) (e.g. as revealed by light scattering Zimm plots etc.) (the differences arise from empirically arrived at preparation procedures which incidentally induce desirable properties including biological properties; these situations are surely not trivial and deserve more focussed scientific attention). Nucleation effects involving glycosaminoglycans may mimic an older system based on minerals such as silicates; this is suggested to be a useful model for the formation of the unique non-crystalline but highly ordered chemical structures present in biological systems. Heparin/heparan sulphate and nucleic acids and collagens evidently retain an ability to strongly bind silicates, and pathological effects of silicates on phospholipid membranes may reflect early evolutionary interdependence of these systems (Grant et al 1992a).

That the binding of heparin/heparan sulphated polysaccharides to other ions or molecules is a general phenomenon best described the formation of separate phases potentially subject to the influence or control by nucleation events was suggested by Grant et al 1992 who observed an independence of counterion binding isotherms on the polyanion concentration showing that the law of mass action as applicable for a single phase solution binding behaviour was not obeyed. Phase separation seemed a more appropriate description of the binding behaviour than a previous hypothesis due to Manning (which postulated a characteristic calculable critical ionic density responsible for inner and outer core co-ion binding depending on ion charge alone as discussed by Grant et al 1992). Similar consideration may be an alternative conceptualization of codonanticodon recognition where Poisson-Boltzman equation that incorporates the screening effects of the electrolyte, the exclusion of ions by the molecule, the molecular shape and the different polrizabilities of the solvent and tRNA shows up a binding hole caused by invagination of potential contours is not found in simple Coulombic calculations (Sharp et al 1990). 10. Control of nucleation of calcification. The inhibition or promotion by glycosaminoglycans and modified glycosmainoglycans of nucleated crystallization of calcite (Grant et al 1989) shows how polyanions can control of nucleation of crystal growth (soil humic polymers are also potent modulators of calcite crystallization, (Grant, unpublished studies, revealed effects of soil polyanioncs which exert a powerful influence on such crystallization and are of interest from the point of view of discussion of global carbon dioxide balance influenced by possible marine supersaturation enhancement by such effects). In studies of BaSO4 crystallization (Grant et al 1992, unpublished) (easier to perform than calcite crystallization, but a similar in general effect of polysaccharides) a series of alginates of defined microstructure were found to control the crystallization rates in a systematic polysaccharide-structuredependent manner (such influence on the rate of crystallization could, conversely, in principle, have been used to identify such polysaccharide microstructures). Nucleation events involving other anions than polysaccharides are also likely to be involved in skeletal calcification where amorphous Ca/Mg pyrophosphate granules are believed to perform this role (Taylor 1990); heparin/heparan sulphate may also, apparently, retain the ability to bind such polyphosphates. Nucleation is also invovled in the initiation of DNA replication ("priming" by oligo-RNA being required). Nucleation of calcite formation in the oceans is an important factor for atmospheric carbon dioxide via the influence of nucleation factors in controlling the carbon dioxide composition of the sea where the presence of natural inhibitors (which may include land soil polyanioncs such as humic and fulvic acids perhaps augmented by agricultural mismanagement) of the nucleation of insoluble calcium carbonate crystallization increase the degree of supersaturation present.

11. Reversible Polymerization Processes & Stochastic Reaction Products. Van Wazer et al (cf bibliography given by Grant 1967, cf also Van Wazer 1962) investigated polymers and related systems containing B sub-group metal such as phosphorus in polyphosphates and phosphate esters, silicon in siloxanes and polysilicate esters, polysulphates, and polyethers which are characterized at easily accessible temperatures and catalytic conditions by reversible ligand exchange leading to change of function of polymer/monomer groups, most easily observable in the liquid phase, giving rise to reproducible mixtures of small ring, oligomer and polymer molecules formed in stoichiometrically, not mechanistically, determined amounts by operation of reversible (quasi)equilibrium processes (an analogous situation also exists for the pyrolysis of some C-C based polymer systems but charring in C-H rich systems but not C-Cl rich systems (Grant 1974) masks an experimental clarity of the process). The stable form of matter in the liquid phase under such apparent stable equilibrium conditions consists of a part interchange between multitudes of different molecular structures. But the operation of nucleation can throw the system out of stable (pseudo)equilibrium by the seeding out by nucleation growth process favouring particular forms. It has been observed that nucleation often lead to a less thermodynamically favoured form being produced thus may favour the formation of metstable states. Biological form is obviously very highly ordered indeed, definitely not being manifestly crystalline, but definitely not a random structure not an amorphous state of matter. Such high structural order must needs be achieved by operation of complex sequential hierarchical nucleations of structure Solution of equations describing the simultaneous reversible (pseudo)equilibria gives the total amounts of structure building units which are conceptually randomly sorted (except by ring/chain balance restriction) into all possible molecular combinations; experiments were undertaken with considerable success to analyse such mixtures and compare the results of calculated and experimental product distributions (this "blue-sky" research at Monsanto Co by JR Van Wazer et al in the 1950s and 1960s was likely justified by much of the commercially rewarding phosphorus chemistry being influenced by such processes). Silicate esters and related polymers (also of industrial importance) were also found to be describable by similar stochastic models. A typical early mathematical model (Grant 1967) of such a system of (pseudo)equilibria between functional groups in polysilicate esters (which are capable of changing function based on 1H-NMR) is: end groups + end groups = ortho(monomer) groups + middle groups in chains K=0.46(0.375) middle groups + middle groups = end groups + 3-way branch groups K=0.44(0.444) 3-way branch groups + 3-way branch groups = middle groups + 4-way branch groups

K=0.28(0.375) the K values for the above experimental equilibria are compared to the random distribution values shown in parenthesis. This approach has been verified, but not updated, by 29Si NMR results. In the case of sodium silicates or molten silicates a similar, essential random distribution of structures (excepting the control exerted by ring/chain equilibria) was confirmed by later 29-Si NMR results confirmed the preliminary findings of the essentially "random" + tendency-to form-mediumsized-rings nature of sodium silicate and silicate esters). The presence of large numbers of individual molecular structures (tending towards infinity) in such systems slows crystallization and encourages glass formation but in the presence of sufficiently strong nucleating agents allows specific molecular structures may be crystallized out. In general such random scrambling allows the simultaneous existence of large number of complex molecular structures from which individual synthesis of seemingly highly complex improbable molecular structures may be achieved by correct seeding of the crystallization of such a structure from such a reorganizing mixture (e.g. as from a silicate melt where constituents include catalysts for the reorganization mixture and the viscosity and temperature are favourable) following nucleation of such a phase and its separation out by growth onto the seeds and continued rearrangement of the remaining melt to permitting high yields of the specified molecular structure. Something like nucleation may also occur in the lower-than-crystalline degree of order present in some "amorphous" silicate formations also seems possible and such structures as well as the better understood highly crystalline silicates could have provides seeds for assemblage of proteinoid polymers discovered by Fox et al thereby inducing in them non-random assemblages. 11a Critical Temperature and the Entropy Enthalpy Compensation Systems. The existence of compensation between allowable entropy and enthalpy changes show up in e.g. collections of rate data which show a linear relationship between temperature dependent and temperature independent terms. The phenomenon is wider than this but has not received the discussion it merits since it uncomfortably suggests there is some serious flaw in our understanding of the basic laws of thermodynamics. The occurrence of compensated energy enthalpy phenomena is believed to be relevant both to water chemistry and to biology similarly interconnected by largely unknown or unacknowledged processes. Compensated systems exhibit a critical temperature where rates become constant for variation of usual rate determining parameters. One idea is that degrees of freedom can be mobilized from usually unavailable energy levels to give a unexpected boost to energy flow. 12. Biological systems may initially have started through (nucleation) perturbation of stochastic polymer systems.

All known present-day biological polymers, in general, at biologically relevant physiological temperature and catalytic conditions, seem, in principle, capable of reversible stochastic polymerization/reorganization processes. A strange corollary to this situation may be that systems containing nonbiological polymers which are capable of such reactions also can demonstrate biological-like properties. This suggests a central requirement of biology may be that such reversible stochastic activity is a fundemental requirement for initiating that state, perhaps particularly in its early stages of evolution. The place that biological nucleation plays in this scenario is that it enables such systems to generate "required" complex molecular structures in high yield. This principle may also be applicable to such biological systems as mobile lipids in cell membranes etc. Currently evolved species, however, mask the requirement for such an effect by elaborate strategies to stabilize individual labile polymer systems. In prebiotic evolutionary conditions, however, the centrality of stochastic potential requirements would have been more apparent. 13. Nucleation and "Biological Polymerization" of Silica Sols. Poly -O-H - rich silicic acid and its aggregates and partially dehydrated, densified silica sol derivatives exemplify chemistries in which the aggregation capacity of -O-H groups play important roles. This includes effects of interactions with biological molecules but also collidfal behaviour which, like that of water aggregates, seems to offer insight into the fundamental importance of the aggregation behaviour of -O-H groups to biology in general especially to its inception. Silica sols differ from polymeric silicate esters in that the system is aqueous and contains more complex boundary constraints which have not yet been fully established but it is likely that the fundamental basis of such chemistry is similar in principle to that of silicate esters. An interesting feature of these and similar systems is that they strangely resemble biological cells (some of such features are listed by Iler 1979) including an apparent ability to undergo some type of apparent replication resembling biological reproduction (Grant 1965). (Although silica sols were originally classified as being amorphous to X-rays as originally studied, and are therefore "essentially amorphous states of matter" by this usual definition, it seems likely that a non-crystalline degree of order exist in certain of these preparations (so that preparations obtained by various modifications of the preparation reactions involving aggregation and densification of silicic acid monomer hydrates leads to sols of different physico-chemical characteristics and even different abilities to react with biological cells such as differing abilities to haemolyse erythrocytes, furthermore such cells from different animal species have remarkably different susceptibilities in this regard; biological interaction with silica surfaces can be highly toxic (as is the case for quartz cf induction of silicosis in mine dusts) or carcinogenic (fine fibrous silica contaminant of flour O'Neil et al 1980) certain forms of asbestos have been correlated for carcinogenicity with their iron contents tentatively suggesting that free radical initiation of DNA damage is the cause of their toxicity, but a pathological nucleation effect might also be worthy of consideration here as in other pathological effects of mineral surfaces (DATA REQD WHAT ABOUT EXFAS etc?)).

The degree of order in silica sols can be reproduced by seeded nucleation in an analogous manner to nucleation of the formation of crystals. Evidence for this sort of phenomenon was termed by the author "biological polymerization" but this did not gain credibility (Grant 1965). Silica sols are (or are derived from) aqueous dispersions of colloidal particles (of various 'nanocomposite' controlled sizes ranging from ca 50-1000 Angstroms consisting of hydrated, densified silicic acid, produced industrially, e.g., by slow addition of low molecular weight silicic acid (obtained by passage of sodium silicate through a cation exchange resin in the hydrogen form) to a dispersion seeded with pre-assembled silica sols; another process used depolymerization (peptization of silica gel in the presence of a heel from the previous batch which evidently acts as a seed) to produce silica sols, which have different specification (average particle size and distribution and surface characteristics) from those produced by the polymerization route. In an attempt to produce particles of the latter type by the former process, using light scattering analysis (Zimm plots) to chart the process, it was indicated that a reversibility of the polymerization process occurred in such a way to engender the notion that such seeded sol formation was analogous to biological reproduction ("biological polymerization") as became apparent from an analysis of the light scattering results. It seemed as though the silica sol particles were behaving like reproducing microorganisms in that by adding low molecular weight silcic acid monomer units onto seeds of the required type of silica particles, the uniquely morphologically specified colloidal particles in the seeded solution increased in size then underwent some kind of particle beakup or cellular division which tended to maintain the integrity of the system. The presence of nucleation seeds was also apparently useful in the depolymerization route for achievement of the desired type of silica sol dispersion. Whilst the build-up of like-structured silicate aggregates on a seed particle and its depolymerization seems logical, the requirement for a seed particle to facilitate correct depolymerization is less clear. The chemical basis of this process may be to do with the mechanism of the reversibility of the Si-O-Si bond forming reactions in the undensified silicic acid aggregates. Seeded polymerization of "amorphous" sols was evidently similar to seeded crystallization of a specific phase. Silica sols find numerous applications owing to their high surface/mass area one of which is to provide a substrate for thin layer chromatogrphy. (It may also be of some interest that pores in such sol particles might have biologically relevant "capillary electrophoresis" properties which are dependent on the ionization of silica surface SiOH groups). Another related biologically relevant colloidal chemistry situation seems to exist with Ca/Zn pyrophosphate granules believed to act as nucleation catalysts in skeletal calcification in vertebrates and also as metal ion sequesters in some invertebrates; these granules were originally believed to be amorphous but low angle high energy X-ray scattering revealed a low but detectable degree of order (REF REQUIRED).

The principles governing symmetry, phase rule, crystallization potential/degree of supersaturation etc. extrapolated to the low symmetry situations in colloidal systems are relevant to biological development. Further experimental studies of these systems are warranted. Silicate structures can be isomorphous with ice structures and the polymeric nature of water may fit in well with that of silicates, e.g., allowing gels of very high water contents to be achieved and the possibility of existence of a glassy state. A similar situation also arises with other polymeric substances rich in OH groups such as polysaccharides. It should not be forgotten that the chemistry of water may be the number one fundamental factor in the chemistry of biology since living organisms are mostly water (and the polymers used are derived by the elimination of water between monomer units). Perhaps even water manifests a similar colloidal microstructural basis to that of silica sols reported in the previous paragraph, with albeit lower stability associated with reversible formation of links between structural building units (hydrogen-bonded aggregates but of specific microstructure capable of be altered by seeding processes (in plots of temperature dependence of aggregate structure reported by Luck et al a discontinuity occurs at near 37C in the manner of a phase change, which may have relevance for adoption of this temperature by many homoi-isothermic species) (such considerations might be the basis of homeopaty or the (much criticized) reported (Benveniste) effects of ultradilute solution on degranulation of eosinophils. Another outrageously disreputable fate was accorded the evidently contaminated highly hydrated silica sol/silicic acid preparations erronously accorded the "polywater" label and the largely hostile reaction (cf Nature) to the book by Sheldrake (1981) who suggested that "morphogenic fields" which emanated from nucleation sites(?) might influence developmental biology and memory. The morphogenic fields of Sheldrake are much wider than the nucleation of ordered structure effects in adjacent solutions, and are extrapolated into and explanation of the paranormal but in some cases substitute for the effects of nucleation factors and self assembly interactions during crystallization. Sheldrake's ideas greatly extend the notion of the importance of nucleation of structures outside usual scientific contexts and emphasizes the lack of fundamental knowledge governing such processes in an embryological context; similar discussion but from a less controversial viewpoint, and perhaps affording a more suitable starting point for further experimentation, is presented in the monograph by Lima de Faria in 1988. 14. Notes from "Evolution without Selection" by Lima de Faria (1988) This monograph highlights parallels between biological development and crystal growth giving prior references (e.g. to the work of Bernal). This author drew attention to the complexity of ice crystal forms, their patterns having possible biological significance. Although a geneticist, Lima de Faria emphasized that: (1) the mechanism of evolution is not known;

(2) self assembly of biological molecules and cell-like assemblages which is well established to occur is a visibile consequence of autoevolution; (3) this resembles crystallization, which is far from being understood; (4) the same mineral can produce completely different forms, e.g. calcite (CaCO3) can occur in about 600 forms and over 2000 combinations; (5) minerals and other pure chemicals have no genes, yet they already display two basic features which had been postulated by biologists to be unique to genes, viz. a constancy of pattern and ability to change it by forming a very large number of forms. "How can these minerals or such a simple chemical as water already behave in an essential similar way to that of a living organism without having genes that maintain their constancy of pattern and that create their variation?", it was asked. The reason was simple. "Genes have little to do with these two basic phenomena which are intrinsic to crystallization" (including nucleation of form). ------------------------------------------------------------------------------------------------------------------(Further notes from this book) The similarity between reproduction and crystallization was already emphasized by Bernal (1965) who pointed out that "the essential principle of reproduction is implicit in crystallization itself".. ..."The explanation of biological development needs to await the explanation of crystal growth.....The fact that there is an intermediate state between the crystalline and the amorphous which exhibits properties found in living organisms", this led the author to think that "the evolution from minerals to living organisms had used four different routes: the solid crystalline, the liquid crystalline, the quasicrystalline and the amorphous".... In Chapter 10 it is noted that "there are two processes of mineral evolution which can now be seen as obligatory bridges in the transfer of isofunctional information form minerals to biological structures. The first is the role of mineral surfaces in the polymerization of macromolecules. The second is the importance of crystallization in reproduction. Mineral surfaces are considered to increase the concentration of organic reactants in water medium." The compounds would adsorb to clay surfaces. This adsorption would lead to the formation of stereospecific compounds in an ensuing polymerization. Aminoacetonitrile adsorbed to kaolin, led to the synthesis of glycine polypeptides (Akabour 1965). The origin of asymmetric synthesis in primordial organisms could be attributed to "the presence of disymmetrical mineral crystals which would have had favoured asymmetric catalysis (Beerstecher 1964)". .... "Reproduction is the biological process by which a structure is copied, ie, in which atoms and molecules assemble along identical ones. The copying of cells involves a number of organelles and several chemical processes, yet its essential characteristics are already present in the minerals. The copying of DNA from DNA is assisted by proteins but the assembly of the phosphates and the bases in the DNA chains occurs initially without their intervention (Kornberg 1980). It is a phenomenon as purely physical as the assembly of similar atoms along the layers of a crystal." .. "The growth of crystals and the growth of DNA molecules both demand an initial seeding..

DNA replication is preceded by the formation of an RNA primer, i.e. , a small piece of RNA that is formed first and later discarded and which allows the DNA growth to start (Dahlberg 1977, Kornberg 1980)"..... "Hence DNA is a prisoner of the process of crystal formation that occurs at the underlying level of evolution. It could not liberate itself from using a similar seeding process." Chapter 14 includes discussion of the work of Fox et al who originally noted that "in the beginning life assembled itself" cf: "The proteinoid microspheres come into existence with remarkable ease, arising in astronomical numbers and showing already many unexpected properties. The non-randomness of the reactions became evident from the experiments of Melium and Sheng (1975) in tyrosine-containing tripeptides. Fox(1974) found that lysine-rich proteinoids catalyse each of the two principal syntheses of biomacromolecules: the synthesis of peptides and the synthesis of nucleotides.....and provide the basis for the emergence of the genetic code within cells." ----------------------------------------------------------------------------------------------------------------15. Effect of electromagnetic fields on nucleation. Weak electromagnetic fields have been demonstrated to exhibit crystallization nucleation activities by unknown mechanism, the phenomenon being found for aromatic systems suggesting molecular electron currents may be perturbable in such a way as to induce specific nucleation electron currents. Ice crystallization is influenced by non-uniform electric fields and a magnetic field appears to be transmitted by an unknown mechanism across the surface of liquid water (Evans 1985). These effects are likely to be of importance to the phydical chemistry involved in colloidal behaviour and especially to biological interfaces. An effect of magnetic fields on prevention of calcification in boiler scale water (Anon 1990) might be due to a similar effect of such fields in modulating the activity of (e.g.) transition metal probably iron containing) nucleation centres. This effect has been confirmed by independent research (Cranfield) but the existence of the effect of magnetic fields remains controversial. Mixed valency iron complexes such as the Fe(II)-Fe(III) O Fe(III) with the {Fe3O}6+ core (triangular) obtained by auto-oxidation of ferrous acetate or more complex related species, show electron delocalization dependent on associated ligands (cf Lippard et al 1991), of interest to magnetism studies as they demonstrate unusual electrochemical properties, may conceivably have a role to play in biological nucleation . Some transition metal complexes with sugars and polysaccharides including those formed with polyanions like heparin/heparan sulphate may be relevant to understanding of as yet pooly understood biological nucleation. Since carboxylate bridging ligands are common features of the Fe3O core systems the formation of complexes containing heparin/heparan sulphate with such cores may also be biologically relevant and are worth studying.

16. Polysaccharides involved in nucleation of (biological) iron nanocomposites? A role for polysaccharides in the biological modulation of iron particle formation is suggested by the study of Cornell (1985) who demonstrated the effect of sugars on the alkaline transformation of ferrihydrite into goethite and haematitie. The likelihood that sulphated polysaccharides such as glycosaminolgycans may be involved in similar activity (including in mammalian physiology) is suggested by the effect of sulphonated nanocomposite magnetitie particle formation (Cahn 1992). The mechanism of the biological formation of (similar) nanocomposites in e.g. bacterial cell walls is unknown (cf Day 1996). 17. Amorphous Ca/Mg pyrophosphate granules and nucleation. Amorphous Ca/Mg pyrophosphate granules are found in membrane vessicles of numerous species, in vertebrates they are thought to be involved in the nucleation of skeletal calcification and may be subject ot doping by zinc ions; they have the property of sequestering metal ions in the order Zn>Mn>Fe>Co (Taylor et al 1990). Pyrophosphate granules were used by Grant (1977) to remove paramagnetic ions from soil extracts thereby allowing NMR spectroscopic evaluation of samples. The possible complexation of such ligands to heparin and related molecules suggests a mechanism for removal of potentially damaging reactive oxygen generating active iron from blood of interest to iron overload treatments. The incorporation of phosphorus in heparin in relatively large amounts and the peptization of hydroxylapatite by heparin (Grant, unpublished) might suggest that pyrophosphate anions might also be bound to heparin thereby providing high affinity metal binding (and skeletal calcification nucleation sites?). Spark source mass spectrometry showed the presence of large amounts of phosphorus bound to unpurified heparin (Grant 2000). 18. Transition Metal Centres in Enzymes and Ziegler Natta Catalysts. Complex magnetic-related behaviour includes such redistribution reactions in clusters. Such studies are also of relevance to understanding the mechanism of catalytic action of such complexes. (Redistribution reactions are of wider interest than to magnetism effects; such effects are used for Ziegler Natta and related catalysts). Enzymes also often rely on the unique reactivity and ligand modulation effects of metal-centred complexes, such activity being central to biology and may include specific nucleating effects. The polymerization of olefins at transition metal centres resembles the operation of a nanometric knitting machine, the addtion rate onto e.g. V-C bonds being near that of bond vibration rate (Grant et al 1973).

The binding, and modulation of ligand exchange and activity of central metals by such ligands as nitric oxide to (similarly active) transition metal enzyme centres is believed to be of physiological and pathological significance. 19. References. Addadi L Berkovitch-Yelin Z Werissbuch I van Mil J Shimon LJW Lahav M Leiseerowitz L (1985) Growth and dissolution of organic crystals with "taylor -made" inhibitorsimplication in stereochemistry and materials science Angew Chem Int Ed Engl 24 466-484 Anon (1990) Scaling down the water problem Chem in Brit, March, 209 Bernal JD (1965) The structure of water and its biological implications Symp Soc Exptl Biol 19 17-32 CA 65 9242f Cahn RW (1992) Transparent ferromagnetic nanocomposites Nature 359 591 (Fe2+ coordinated at polymeric sulphonate groups is hydrolyzed and heated in the presence of hyhdrogen peroxide to form nonocomposites of small particle sized gamma Fe2O3, much more strongly magnetic than previous products such as FeBO3 and FeB); cf article by P Day Chemistry in Brit July 1996 p31 re Fe particles in bacteria and pigeons clearly indicates optimized morphology of magnetic particles and controlled particle formation) Caplan AI Fiszman MY Eppenberger HM (1983) Molecular and cell isoforms during development Science 221 921-927 Chen N et al (2000) Adhesion of human fibroblasts to Cyr 61 is mediated through integrin alpha 6 beta 1 and cell surface heparan sulphate proteoglycan J Biol Chem 275(32) 24953-61 Ca 133 264513d Colomban E (Ed Proton Conductors, Cambridge Univ Press 1992) Cornell RM (1985) Effect of simple sugars on the alkaline transformation of ferrihydrite into goethite and hematite Clays and Clay Minerals 33 (3) 219-227 Crowe JH Crowe LM Carpenter JF Aurell Wistrom C (1987) Stabilization of dry phospholipid bilayers and proteins by sugars

Biochem J 242 1-10 Day P (1996) Chemistry in Britain July 31 de Terra N (1974) Cortical control of cell division Science 184 530-537 Davenas E Beauvais F Amara J Oberbaum M Robinzon B Miadonna A Tedeschi A Pomeranz B Fortner P Belon P Sainte-Luady J Poitevin B Benveniste J (1988) Human basophil degranulation triggered by very dilute antiserum against IgE Nature 333 816-818 Diringer H Ehlers B (1991) Chemopropylaxis of scrapie in mice J General Virology 72 457-460 (Although these authors do not accept the Prusnier hypothesis of the nature of prion diseases but prefer to consider them slow viral diseases but their findings of the effect of heparin analogues on these diseases is possible fundamental importance in unravelling the nature of these diseases) Evans GJ (1985) Influence of external fields on nucleation and crystal growth J Chem Soc Faraday Trans (1) 81 673-687 Ford TA Falk M (1968) Hydrogen bonding in water and ice Canad J Chem 46 3379-3386 Gabizon R et al (1993) J Cell Physiol 157(2) 319 CA 120 28472a Ganz E (1936) Uber das absorptionspectrum vom wasserigen Losungen zwishen 0.70 micron bis 0.90 micron Z physikal Chem B 33 163-178 Glick JL (1988) Only the smile is left Nature 334 376 Grant D (1965) (Grant, industrial lab {Ruabon Wales}, 1965, internal report a requested permission for publication of a manuscript reporting experimental results (denied, perhaps justifiably from the viewpoint of a commercial laboratory in a competitive field, owing to the critical nature of the experimental data and the nature of the hypothesis derived from it which might have tended to bring

ridicule and disrepute on the laboratory in question* as has been the historical fate of other related hypotheses; attempting to learn of what had happened to this report after I moved to another company I sent a review of prebiotic evolution of proteinoid microspheres and their bacterial like "reproduction" by Fox, in which proteinoid microspheres had been reported (including critical electron micrograph evidence) to undergo similar biological-like particle breakup); the written reply led me to think publication of my weird results was not even to be thought of; a similar request to allow publication of a study of redistribution reactions in polymeric alkyl silicates (Grant 1967) had, however, been granted. I am making a fuss about this mater since these results may have provided clear experimental evidence for the existence of similar autodirected assembly processes for apparently amorphous polymers similar to what occurs with crystalline assemblages involving well-defined principles of retention of symmetry etc. but of especial importance is the occurrence of nucleation seeding of particular phases akin to the assembly of components of an organism such as a virus or the build up of body parts during embryogenesis. (That silicate colloidal systems even in biological tissue undergo self assembly is usually stated). (cf also review by Grant et al 1992a) *Caution about such too rapid publication (especially by industrial chemists) was certainly justified by a later over rapid publication of a monograph based on highly erroneous assignments of chemical shifts caused by unsuspected dissolution by the starting ingredients of the glass NMR tubes used for 19-F NMR studies; a stunne industrial lab. management seemed then to abandon such in-house academic research (thereby unjustifiably tarnishing all prior work arising from this group). The basis of scientific reporting is surely to allow other workers to repeat and confirm or refute experimental any results; inevitable mistakes can surely be tolerated if they arose in an honestly-admitted fashion (mind sets of industrial labs often encourage over-optimistic self confidence due to profit-oriented pressure). Grant D (1967) Redistribution reactions in polymeric alkyl silicates J Inorg Nucl Chem 29 69-81 Grant D (1974) The pyrolysis of chlorocarbons J Appl Chem Biotechnol 24 49-58 Grant D (1977) Chemical structure of humic stbstances Nature 270 (5639) 709-710 Grant D Long WF Williamson FB (1988) Polystyrene surfaces may require structured water for effective cell adhesion Biochem Soc Trans 16 1029-1030

Grant D Long WF Williamson FB (1989) Inhibition by glycosainoglycans of CaCO3 (calcite) crystallization Biochemical Journal 259 41-45 Grant D Long WF Williamson FB (1992a) A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern pathological afflictions Medical Hypotheses 38 46-48 Grant D Long WF Williamson FB (1992b) Degenerative and inflammatory diseases may result from defects in antimineralization mechanism afforded by glycosaminoglycans ibid 38 49-55 Grant D Long WF WIlliamson FB (1992c) A study of Ca - heparin complex -formation by polarimetry Biochem J 282 601-604
2+

Grant D Long WF Moffat CF Williamson FB (1992d) Cu - heparin interaction studied by polarimetry ibid 283 243-246
2+

Grant D Long WF Moffat CF Williamson FB (1992e) Zn2+-heparin interaction studied by potentiometric titration ibid 287 849-853 Grant D (2000) (Home web page dealing with heparan sulphate reaction with nitrous acid in the etiology of degenerative diseases) web.ukonline.co.uk/dgrant Hadi D {Hydrogen bonding cf internet} Iler RK (1979) The Chemistry of Silica: Solubility, Polymerization, Colloidal and Surface Properties and Biochemistry. Wiley-Interscience, New York, 1979 Kornberg A {Publications listed on internet} Lima-de-Faria A (1988) Evolution without selection: form and function by autoevolution Elsevier Science Publishing B.V (Biomedical Division) Amsterdam Lin X et al (2000) Disruption of gastrulation and heparan sulphate biosynthesis in EXT-1 defficient mice Dev Biol 224(2) 299-31 CA133 279284d Luck WAP (1965)

Zur Assoziation des Wassers II. Salzeffekte auf die Ultrarotbanden des Wassers Ber Bunsengeselschaft 69 (1) 69III. Die Temperaturabhangigkeit der Wasserbanden bis zum kritischen Punk ibid 69 (7) 626-637 cf also Ganz E (1936) Z Phys Chem B 33 12 cf Fig 13 and nteractions of Water in Ionic and Nonionic Hydrates Hubertus Kleeberg (Ed) Springer Verlag Berlin Proceedings of a Symposium in Honor of the 65th Birthday of WAP Luck Marburg 2-3 April 1987 Nader HB Ferreira TMPC Toma L Chavante SF Dietrich CP Casu B Torri G (1988) Maintenance of heparan sulfate throughout evolution: Chemical and enzymic degradation, and 13C-spectral evidence Carbohydr Res 184 292-300 O'Neil CH Hodges GM Riddle PN Jordan PW Newman RH Flood RJ Toulson EC (1980) A fine fibrous silica contaminant of flour in the high oesophageal cancer area in North-East Iran Int J Cancer 26 617-628 Pashley KM McGuiggan PM Hinham BW Evans DF (1985) Attractive forces between uncharged hydrophilic surfaces. Direct measurement in aqueous solutions Science 229 1088 Perry CC Williams RJP Fry S (1987) Cell wall biosynthesis during silicification of grass hairs J Plant Physiol 126 437-448 Poganiuch P Liu S Papaefthymiou GC Lippard SJ (1991) J Amer Chem Soc 113 4645-4651 Sanderson PN Huckerby TN Nieduszynski IA (1985) Glycoconjugate J 2 109-120 Sharp KA Honig B Harvey SC (1990) Electrical potential of transfer RNAs: Codon-anticodon recognition Biochemistry 29, 340 Sheldrake R (1981) A new science of life Blond & Briggs Ltd London

Taylor MG Greaves GN Simkiss K (1990) Biotransformation of intracellular minerals by zinc ions in vivo and in vitro Eur J Biochem 192 783-789 Urry D (1996) Chemistry in Britain 32 (10) (cf von Stackelberg M Muller HR (1951) Naturwiss 38 456) Van Wazer JR (1962) Structure re-organization through ligand interchange American Scientist 50 450-472 Zundel G. List of publications [added later: see internet Wichtige wissenschaftiche Publikationen van Georg Zundel