D. Grant (Turriff) draft articles : Page 1. Document 1.

Notes on the Inorganic Biochemistry of Heparin/Heparan Sulphate

Page 115 Document 2
Recent Heparan Sulphate Research May Explain Why a Vegan Diet is Good For You
Page 127 Doc. 3 The emerging paradigm of heparan sulphate related therapy(Doc 3a is an earlier edition of Doc.1);
Page 137 Doc. 4 (This article argues for a role of heparan sulphate biochemistry in animal evolution); Page 142 Doc.

5
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DOC. 1. Notes on the Inorganic Biochemistry of Heparin/Heparan Sulphate

An Incompletely Edited ‘Scrapbook’ by David Grant, B.Sc., M.Sc., Ph.D. (Turriff, U.K.)*
Preliminary Draft of a Discussion Document (Drafted for Internet Posting, 2008;

5/2/09-still needs more editing)

Research Continuation of Ideas Generated From University of Aberdeen Marishal College Research*
Contents:
(page 8) Summary

2(page9) Introduction
Heparin (H) can apparently sequester the full range of inorganic ions which
occur in seawater/biological fluids by a mechanism which tends to favour the uptake of
the least abundant elements present

2.1 (page 10) A suggested inorganic ion nutrient gathering and other functions of extracellular
polysaccharides

2.2 Available information on the association of multi-inorganic-elements with natural

polyanionic substances
Possible quality control problems with use of H for academic researches and
for future therapeutic applications

2.2-1 Countercation-specific (polarization-power-related) interation of metal ions

with –C-(O)O- groups in H/Heparan Sulphate (HS)

2.3 The Haraguchi Hypothesis Extended to Polysaccharides

Metallomics & polysaccharides

2.4(page 12) The Heparanome Metallome

A putative H/HS inorganic environment interface system

2.4-1(page13) Summary of the origin of the heparanome-metallome hypothesis

2.4-1-1(page 14)Examples of literature reports of metal ion driven H/HS signaling

2.4-1-2 Comparison of ‘metallomic’multi-inorganic-element arrays in anionic polysaccharides

with those in other matrices

2.4-2 Further evidence that multivalent inorganic element metal ions binding to H and HS
proteoglycans in vivo
2.4-3 H/HS is an evolutionary designed flexible metal ion binding system

Polysaccharides, especially the polyuronates and highly anionic glycosaminoglycans, seem to be especially
designed to act, in conjunction with other anionic systems, as multi-element metallomic ligands.

2.5 (page 16) Evidence for ‘all-element’- decorated H

2.5-1 SSMS-determined inorganic elements associated with an experimental Na H

(more accurately Na/Ca = ca.6, H) (believed to represent the inorganic components
present in mast cell derived pharmaceutical H before final ‘heavy metal clean-up’)

2.5-2 SSMS determined inorganic elements associated with an experimental Tl H

2.6 Summary of apparent effect on metallomic profiles of Heparin leached from blood
 collection containers
ICP-MS studies of H

2.6.1(page 18) Re-evaluation of SSMS Data for NaH and comparison with ICP-MS data

2.7 (page 18) Toxic Inorganic Elements in the Natural Anionic Polysaccharides

2.7-1 Toxic inorganic elements in H

2.7-2 Multi-inorganic-elements in common laboratory reagents

2.8 Other suggested metallomic-related activities of animal polysaccharides: modulation of

water supramolecular structure, calcification and deactiviation of Fenton reaction
catalysts

2.8-1(page 19) Other cell surface polyanion (putative metallomic) systems:

polyphosphate, poly-β − hydroxybutyrate & teichoic acids

2.8-2 Possible inorganic phosphate-containing high afffinity metal ion binding sites in H

2.9 Metal ion assisted polysaccharide-protein binding could be relevant to the current
discussions of how different HS polymers selectively binding by variants of FGF in vivo

2.9-1 Servo-feedback signaling via the heparanome-metallome could have facitlitated

animal evolution

2.9-2(page20) Inorganic cofactors for nitrosative structural alteration of H/HS

2.9-3 The selective degradation of Heparin/HS by action of redox metal ions

2.9-4 How different HS polymers could selectively bind variant of FGF in vivo: hints
from lipoprotein binding studies

2.10(page 21) Comparison of Different Multi-Inorganic-Element Matrices

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3 (page21) Ca2+ signaling via the heparanome –metallome

The suggested primary functions of the heparanome-metallome;

The hypothesis that Ca2+ and other inorganic ions modulate HS activites
[The Long-Williamson1 hypothesis : HS controls Ca2+activity
The control of activities of Ca2+ (e.g. at pericellular environments) were suggested to be the
primary
function of HS. This general idea can now, however, be modified to suggest that Ca2+ and other
inorganic ions might modulate HS activities by modulating biopoymer water activities which
affect
H/HS-protein interactions]

3.1(page 22) In plants Ca2+ and other inorganic ions are believed to modulate polyanonic polysaccharide
activities

3.2 Evidence from knockout mice that HS N-SO3- groups potentiate HS-determined Ca2+
activities required for skeletal muscle function
(Ndst-1 -/- mice are reported to show reduced Ca2+ kinetics in myotubes)

3.3(page 22) Heparin/HS & control of calcification

 3.3-1(page23)N-SO3- is involved in Ca2+ binding by Heparin/HS

3.4 Proposed roles of inorganic borate, silicate, arsenic (oxides) and phosphate
attached to H/HS

3.4-1 Specific nucleation activities (e.g. afforded by natural ‘SiO2’ nanoparticles) may be
required for supramolecular structure formation in polysaccharides

3.4.-2 Polyoxymetalates and biologically relevent Heparin/HS catalytic activity

Possible role of anionic polysaccharides in the assembly of polyoxymetalates-
polysaccharides with primitive enzymic activity

3.4-3(page 24) Heparin/HS & Metalloproteinases (MMPs): a putative further example of metal ion (and
water structure?) determined activity –related effects

3.4-3-1 Modulation of metalloproteinase activity by arsenic suggests a possible therapeutic

application of As2O3- H/HS as an anti-cancer agent

3.4-4(page 24) Heparin/HS & Sulfatase activites; postsynthetic editing of HS sequences; role of
metallomics?
6-O-Endosulphatase (Sulf) action in ‘smart’ HS microstructure modulation

3.4-5 Heparin/HS & kallikrein: could exemplify how HS determines water structure/activity
HS signaling (thyroid hormone dependent) in skeletal growth & mineralization –[inorganic
biochemistry related systems]

3.4-6 The use use of barium acetate buffer for the electrophoretic separation via selective
binding of Ba2+ to Heparin/HS

3.5 Mineralization – (inorganic biochemistry related systems)

3.5-1 Thyroid hormone dependent skeletal growth etc.

Plaque formation & Heparin/HS

The inhibition of various forms of proteinaceous and inorganic plaques by the
most highly sulphated fractions of Heparin/HS and related substances (such as PPS)
could be why such preparations show great promise for the therapeutic intervention
in those diseases which are believed to be promoted by the formation of
such plaques ** [Cf. also Section 6.3].

3.5-2-1(page26) Phospholipid Heparin/HS interaction with metal ions

3.5-2-2 Polysaccharide metal ion dependent aggregation

3.5-2-3 (page26) Nucleation events (subject to potential inhibition by Heparin/Heparan S)

may determine the progression of amyloidoses

3.5-2-4 Polysaccharide inorganic ion dependent gelation and melting

3.5-3(page 27) Binding of anionic polysaccharides to crystal surfaces

3.5-3-1 Relevance of control of inorganic crystal morphology to evolution of precursors of life

3.5.4(page29) How information in anionic polysaccharides can be ‘read-off’ by crystal structures

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4 WATER & LIFE

–ROLE OF POLYSACCHARIDES
4.1 Water chemistry & compensations between allowable entropy and energy changes
Phenomena Related to Water Structure

4.2(page 29) How life depends on the chemistry of water

4.2-1 Possible key role of Wiggins-Water -Structure

Possible key role of H/HS surface Wiggins-water-structure

4.3(page30) Role of water structure in polysaccharide biochemisty

Possible role of nucleation of associated water
& hydrate phase/conformation change in the metallome-heparanome

4.4 General water structuring effects of polysaccharides

4.5(page 31) Bacterial mucilages

Mucilage structure adhesion forces

4.6 Evidence for a role of water adheison forces in the anticoagulation mechanism of H and in the
binding of poly-L-lysisne and poly-L-arginine to H
Evidence for biologicaly relevant polysaccharide-determined water structure adhesion forces
The binding of poly-L-arginine and poly-l-lysine to an optimally hydrated (Na-counterion containing)
Heparin occurs initially via a Heparin-bound water structure adhesion mechanism. This could be observed
when a hydrophobic environment was used for near infrared spectroscopic detection of the changes in the overtone
water bands asociated with Heparin surface bound water present in mixtures of Heparin and polyamine

Adhesiveness of metal ion dependent water associated with H/HS elicits protein binding
similar to how metal ion dependent hydration affects polysaccharide lectin binding etc
Evidence for water adhesion forces in H-protein interactions
Adhesiveness of metal ion dependent water associated with H/HS may control glycocalyx
assembly and calcification

4.7 Other evidence for biologically relevant polysaccharide determined water structure adhesion
forces

4.8 Manning (electrostatic) binding of counterions to polyanions

4.9(page 33) Water activity model of metal ion dependent H//HS signaling
(Metal ions may participate in H/HS signaling in part via modulation of water activities
at H/HS surfaces). Do HS actions ultimately depend on the modulation of water structure
to achieve selective protein binding and directed refolding?

4.10(page 34) HS as a proton pump

Proton Conduction
H/HS probably occur naturally as mixed salt
forms in which complex hydration patterns
are associated with the sulphate half-ester and N-
sulphonate groups which are putativley involved
in H+ and Ca2+ transport and energy transduction
Water Channel (HS)Related Proton Conduction
Arrays of hydrated sulphate half ester groups attached to polysaccharides could be involved in energy (e.g. proton) transduction by a similar mechanism to
that by which sulphonated man-made ionomers (in which water clusters are attached to sulphur oxy-anions) are believed to confer conductivity (James et
al. 1) and which further seem to be subject to stabilization by the incorporation of similar amounts of inorganic silicate nanoparticles to those which occur
naturally with polyuronides (Schwarz1). There may also be roles for in such systems for semiconductor modulator functions of various rare earth ions

4.11(page 35) HS as a mimetic of humic polymer buffers in natural waters

High molecular weight polyanionic (including organic polymer) multi-inorganic-ion
sequestering and buffering systems putatively enable bio-friendly water structures to be formed
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5. (page 35) Nitric Oxide Biochemistry & H /HS Signaling

5.1 The selective degradation of H/HS by actions of redox metal ions

5.2 Metal ions & water structure dependent nitrite cleavage of H/HS
Possible key roles of metal ions in the physiological nitrite cleavage of H/HS

Early studies of metal ion effects in nitrosative processes

5.3-1 Identifying HS by deaminative cleavage with nitrous acid
5.3-2(page 36) Tyrosine nitration in degenerative diseases
5.3-3 Inorganic ion catalysis of nitrosative reactions associated with stomach cancer

5.4 (page 38) Internet postings: “Ascorbate & Nitric Oxide in Redox Control of Heparan Sulphate”
Internet posting: “Ascorbate & Cancer”

5.5 Additional Hypothesis : Hydrogen Sulphide & Nitric Oxide are Invovled in
Redox Control of HS

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6 (page 38) Relevance of H/HS biochemistry to pathology

6.1 Putative pathological inorganic element perturbation of H/HS signaling

6.1-1(page 39) H/HS biochemistry & degenerative diseases

6.1-2 Possible perturbation of H/HS signaling by Ba2+ in multiple sclerosis

6.1-3 Possible perturbation of H/HS signaling by Al3+, Zn2+ (and Cu2+/+) in Alzheimer’s disease
6.1.4(page 41) H-mimetic pentosan polysufate: anti-misfolded prion & anti- HIV activities

6.1-5(page 42) General anti-degenerative disease potency of H/HS

6.2 The defective nitrosative cleavage which has been indicated to occur in Niemann-Pick
Disease could also be relevant to the etiologies of cancer and other degenerative diseases
Possible role of dyshomeostasis of nitric oxide and cholesterol in Alzheimer’s disease

6.3(page42) Cancer & HS Biochemistry. Inhibition of Carcinogenesis by H & H-Like Molecules

Overall HS biochemistry, including the of specific signaling sequences of sulphate half ester anionic groups
seems to be influenced by redox status (which include ascorbate, oxygen pressure, homocysteine) and the
correct inorganic ions perhaps involved in generation of water structure (but a range of toxic inorganic ions
seem to negatively perturb HS biosynthesis).

6.3-1(page 45) Inorganic tumour suppressors and tumour promoters in H

6.4(page 47) Does HS provide for a servo feedback system which directly interfaces the environment and
which allows dietary manipulation to produce beneficially altered HS microstructure?

6.5 Pacification of redox active iron ions by their incorporation into polysaccharide aggregates

6.6 Alternative and ethnic medical use of metallomic matrices.

Could some of the non-anticoagulant suggested uses of H and claimed benefits of Ayurvedic
Shilagit etc. be dependent on the co-occurrence of similar ultratrace elements in these
preparations?
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7(page 49) Interference from inorganic constituents in H during blood analysis
Interference from multiple inorganic constituents associated with H used as an anticoagulant
during determination of inorganic elements in blood
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8 Variation between manufacturers of the degree of final purification of H

9 Could HS signaling generate a systemic biological clock

which is speeded up during degenerative disease processes
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10 (page 51) Examples of Literature Reports of
Metal Ion Driven H/HS Signaling
10.1 Table I Involvement of metal ions in H/HS interactions thought to be involved in blood
anticoagulation, arterial calcification, breast cancer, basic fibroblast factor
dimerization, annexin-V binding, endostatin (collagen –XVIII), collagen -V and
prion interactions, as well as the activities of a range of other proteins and in the
nitrosative scission of H/HS

10.2(page 54) Table II Reports of (apparently direct) modulation of HS microstructure and/or HS PG

synthesis in response to the presence of inorganic ions etc.

10.3(page 56) Table III Some examples of reported effects of organic molecules on modulation of HS
microstructure and/or alteration of HS PG synthesis

10.4 (page60) Table IV Inorganic elemental contents (mg/kg) of kelp, H & human scalp hair
Comparison of ‘metallomic’ multi-inorganic-element arrays
in anionic polysaccharides with those in other matrices
Examples of ‘metallomic’ arrays associated with H, kelp & human scalp hair

10.5 Further background to the studies of metal ion binding and the multi-element content of H

10.6(page62) Table IVa Comparison of inorganic elements in H and chitosan

10.7 Further Notes

10.8(page 64) Log-Log plots Figs. 1-5

Some interrelationships between multi-inorganic element contents of H-related
matrices, seawater, human hair
Inorganic element contents compared with H

11 (page 68) References

11.1 General reviews of H/HS

11.1- 2 Reference 1
References arranged alphabetically according to first-named author

11.2(page 93) Further references (and notes) referred to by number

and also arranged according to subject matter

11.2-1 The metallome-heparanome

(References to metal ions in H/HS signaling)
11.2-2
(page 93)Ref. 1A-1 Published peer-reviewed articles which include mention that inorganic ions might
participate in H/HS signaling
Ref. 1A-2 How metal ions are currently known to assist in H/HS nitrosative signaling
Ref. 1A-3 Degenerative diseases: putative roles of H/HS signaling
References 1A3-1 H/HS & Cancer
(Gene expression seems to be linked to H/HS)
1A3-1-1 Histones
1A3-1-1-1 Inhibition of Histone Acetyltransfersases (HAT) by GAGs
1A3-1-1-2 Tumour cell surface HS is a ‘cryptic system’ which can act both
as a promoter or inhibitor of tumor growth
1A3-1-1-3 System of trans repression by H

1A3-1-1-4 The Sulf system & cancer

1A3 1-1-4-1 Sulf loss influences N-, 2-O, and 6-O sulphation of multiple HS
proteoglycans and modulates FGF signaling

1A3-1-1-5 Glypican & Cancer

1A3-1-1-5-1 Glypican 1 HS PG & Cancer
Pro-tumor effect of glypican-1 HS PG asociated with human
gliomas
1A3-1-1-6 Glypican 3 HS PG & Cancer
Antibody against the 30kD fragment of (Glypican-3 HS) PG with
anti-tumour therapeutic potential
Glypican-3 HS PG is a potential lung tumor suppressor

1A3-1-1-6-1 Glypican 4 Akt

1A3-1-1-6-2 Glypican 5 HSPG & Cancer

1A3-1-1-6-3 Glypican 6 HSPG & Cancer

1A3-1-1-7 Heparanase & Cancer

1A3-1-1-7-1 Selectin, H & Cancer metastasis

1A3-1-1-7-2 Heparanase-Akt activity and tumorigenesis

1A3-1-1-8 Angiogenesis, HS & Cancer

1A-1-1-1-8-1 Angiopoietin HS & Cancer
1A-3-1-1-8-2 Endostatin HS PG Zn2+ & Angiogenesis
Induced Nitric Oxide Synthase Expression & Cancer
Nitric oxide can apparently directly activate MMP-9
Putative modulation of angiogenesis by H/HS
1A3-1-1-8-3 Classical research by J Folkman et al
1A3-1-1-8-4 Further studies related to VEGF affecting angiogenesis
Pericytes and H
VEGF antibodies are showing promise
as anti-cancer therapeutic agents
1A3-1-1-9 Dietary polyunsaturated acids & Cancer
1A3-1-1-10 Glucosamine therapy for degenerative diseases?

1A3-1-2 Earlier reports of relevance to H/HS & Cancer

Engelberg (historical) review

1A3-1-2-1 Other (historical) reviews

1A3-1-3 Tumour suppressors & H/HS

Exotosin I (EXT1) and exotosin 2 (EXT2)
(are glycotransferases required for biosynthesis of HS)

BRCA-1

1A3-1-3-1 HS can be both pro- and anti-cancer,

the difference may be related to HS microstructure
 1A3-1-3-2 Recent reports of potential H-related anti-cancer therapeutics

1A3-1-3-3 Low molecular weight H and oligosaccharides: anti-cancer

effects

1A3-1-3-3-1 H derivative for oral administration with claimed potent anti-

cancer activity and low toxicity

(page 102) 1A3-1-3-4 Pentosan polysulfate (PPS) anti-cancer effect

1A3-1-4 Anti-cancer metal and metallioid ions (which could be sequestered
from biological fluids by H

1A3-1-5 Altered HS in transformed cells and cancer tissues

1A3-1-5-1 Putative differences in H iduronate conformation in transformed
cells

1A3-1-6 Water structure & cancer

1A-4 H and Atherosclerosis

1A-5 H and Dementia

H-derived oligosaccharides for inhibiton of senile dementia
H/HS claimed to normalize age-related fear response

(page 104) 1A-6 Further Notes on

H and Antioxidant Protection
(Smoke inhalation injury)
Antioxidant & antinitrant activity (related to medulation of superoxide
dismutase by H/HS) and sequestration and deactivation of redox active
iron, copper & silver ions, etc. by H/HS
11.3 (page105) Further references Refs. 1a1-14; 1b-1h
11.3-1 Refs. 6a, 6a-1, 6b-,. 6b-2, 6b-2-1, 6b2-2, 6b2-3; 7-9, 9b-3, 9b-3-1, 9c; 10-
13, 13a; 14-17
12 Concluding Remarks
12-1 (page 111) Suggestions for Further Research
APPENDIX
Author’s Curriciulum Vitae (page 142)
Acknowlegements (page 144)
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1. Summary

Heparin (H) is believed to be most anionic biopolymer, and has traditionally been used by
biochemists to provide a readily available laboratory model of heparan sulphate (HS).
Evidence is now discussed which suggests that the biochemical reactivities of
H and the H-like segments of HS may be influenced by the presence in the hydrated supramolecular
structure of these polysaccharides of small amount of the wide range of inorganic ions which occur in
biological fluids and natural waters.

Low abundance inorganic solutes are known to become selectively sequestered by the hydrated anionic
polysaccharides which occur in the cell walls marine algae and by the polysaccharide-like soil and
natural water humic/fulvic polymers. A similar multi-inorganic element sequestration process is now
suggested also to occur with the H-like animal anionic polysaccharides abundantly present in HS at
adherent animal cells and extracellular matrices.
H/HS seem to preferentially sequester the less abundant ions present in biological fluids.

The HS system of polysaccharides which as is now becoming apparent, may serve as a ‘super-
hub’ control system which influences a wide range of biological acitivies. HS may, however, have
been selected during early stages of animal evolution to serve as a multi-inorganic element
nutrient gatherer but later evolved into a general system of inorganic ion regulation of cellular
activites by HS proteoglycans.

Glycocalyx polysaccharide-reservoir-based inorganic ion (including oligomeric inorganic ion)

provision is suggested to allow the heparanome-metallome system to achieve and control a system of
delicately balanced HS-(metal ion-[H2O]n) in H-like supramolecular structure which is now proposed
to influence HS reactivity as regards protein binding and nitrosative generation of oligosaccharides.
An anthopogenic or ortherwise induce inorganic ion dyshomeostasis perturbation of HS biochemical
signaling could be of relevance to several pathologies.
The control of activities of Ca2+ (e.g. at pericellular enviornments) had formerly been
suggested (the Long-Williamson hypothesis) to be a primary function of HS.
This general idea can, it is suggested, be usefully modified to suggest that Ca2+ and
other inorganic ions can modulate HS activities by affecting biopoymer water activities
which affect H/HS-protein interactions

Future biological metallomics research might usefully consider how polysaccharides collect and
deploy counterions and other inorganic moieties present in their natural bathing solutions and
seek to establish if the ternary association of specific metal ions with HS + proteins is a critical
part of the HS signaling mechanism.

Keywords:
Heparin, heparan sulphate, the heparanome, metallomics, degenerative diseases, cancer, multiple sclerosis,
Alzheimer’s disease, prion misfolding diseases, water structure, hydration, biological clock, inorganic ion
contamination of heparin, inorganic elements in blood serum, nitric oxide, nitrite, glycocalyx.

Abbreviations:
ACE: agiotensin converting enzyme; ChS: chondroitin sulphate; CSE: cystathionine gamma-lyase; DeS: dermatan
sulphate; EHDP: ethane hydroxyl, 1,1-diphosphonate; ECM: extracellular matrix; FGF: fibroblast growth factor; H:
heparin, HS: heparan sulphate; GAG: glycosaminoglycan; HDL: high density lipoprotein; HIF: hypoxia inducible factor;
i.v.: intravenous; iNOS: induced nitric oxide synthase; ICP-MS: inductively coupled plasma mass spectroscopy; I: ionic
strength; LDL: low density lipoprotein; LPL: lipoprotein lipase; LPS: lipopolysaccharide; MMP: metalloproteinase;
Ndst: N-deacetylase/N-sulfotransferase; PAPS: 3/phosphoadenosine 5/phosphosulfate; PET: polyethylene terephthalate;
SLE: systemic lupus erythromatosis; SRCD synchrotron radiation circular dichroism, SSMS: spark source mass
spectrometry; TIMP: tissue inhibitor of metalloproteinase; uPA: urokinase plasminogen activator; VEGF: vascular
endothelial growth factor.
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Foreword : This document is an attempt to produce a general overview of heparin (H)/heparan sulphate (HS) biochemistry from the
perspective of novel academic polysaccharide laboratory researches formerly conducted by the author as a member of a team led by W.F.
Long & F.B. Williamson which had led to numerous publications in peer-reviewed journals [cf. Long1, 2003 ]); each of these papers dealt
with sub-facets of the results obtained, but no overview of these findings has been attempted before now. Viewed from the perspective of
an industrial quality control standard chemist (e.g. where rigorous purification and the reagents employed as monomers, catalysts and
solvents etc. are well known to be essential prerequisites for the preparation of polyolefins) it seem now to be apparent that medically
employed reagents and polymers (such as glycosaminoglycans) should be subjected to a more thorough evaluation of standard quality
control methods in regard to the possible variation in co-purification with these polysaccharides of a wide range of inorganic ions. This
phenomenon was formerly regarded as of little fundamental scientific interest being regarded as a relatively trivial random contamination
phenomenon which fails to merit the elvel of relevance and scientific value which is required to allow any discussion in mainstream peer-
reviewed journals. Some recent reports (cf. Boher et al.1) have, however, discussed (albeit in a non-English language peer reviewed
journal) the possible risks to patients undergoing kidney dialysis of the presence of potentially toxic elements present in the presently
considered type of blood anticoagulant H, which as the most anionic of biologically encountered polymer systems is therefore also the
most likely of commonly employed pharmaceutical agents to show an especially enhanced ability for becoming highly enriched in those
ultratrace (e.g. counter-cation) metal elements which are now known to exist in biological fluids. This ability may, however be a normal
designated property of H and H-like polysacchrides (including HS) and may be of fundamental importance to the in vivo modus operandi
of HS PG for the regulation of protein binding by these polysaccharides and also be of relevance following, e.g., anthropogenic,
pathological or normal age associated perturbation of blood inorganic ions contents, to a range of pathologies.
Inorganic input to the heparanome is suggested to pertain inter alia to the thyroid hormone dependence of inorganic sulphate
transport, the biosynthetic thyroid hormone inhibitory roles in HS structure modification and the requirements for inorganic
cofactors for the nitrosative scission of HS chains (which generate HS oligosaccharides used as signal messengers) and for
metalloproteinase (MMP) control of HS shedding. HS and other polysaccharides putatively also contribute to animal plasma
membrane hydration and water activity regulation.

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2. Introduction
Heparin (H) (n.b. a protein and nucleic acid free pharmaceutical industrially prepared polysaccharide mixture)
is obtained from its mast cell proteglycans containing highly H-substituted serine glycine rich core protein); H
contains abundant salt-forming anionic (sulphate half ester, sugar uronic (carboxylate) side groups which confer
upon this polysaccharide system an almost inorganic outer surface.

It should be noted that H is a highly heterogeneous system (cf. Nader et al.1, 1981) which does not correspond to what is
classically definable as a chemical substance, namely a specific, single formula/structure chemical molecule. H
should perhaps more accurately be described as a library of many different chemical molecules, which are structurally
related only in the restricted sense of the non-mathematical fuzzy logic used by chemists to label related systems of
families of chemical compounds.
The administion of H to humans (e.g. as an aerosol) has been reported to show many health
benefits, including the inhibition of asthma, lung damage in hypoxia, bleomycin induced lung
fibrosis and the inhibition of lung cancer (cf., Gabr et al. 1 a study of rat lung proteomics following
H instillation).
For these uses, as well as the traditional use of H as a blood anticoagulant, a quality control
problem might be anticipated from a consideration of the available data on the variations in
associated inorganic elements arising from different procedures used to remove heavy metals and
other unwanted inorganic elements from H.
While an approximate similarity is apparent between different Hs (e.g. all seem to approximate
to the seawater-metallomic-fingerprint) differences are also evident between the residual multi-
inorganic element contents of some commercial pharmaceutical H preparations for which
analytical data have been made public.

A recent study (Rudd et al.,1) which included use of highly sensitive method of
synchrotron radiation circular dichroism, has confirmed previous putatively
made suggestions (e.g. by the former Aberdeen polysaccharide group, cf.,
Long, 1,2003 ) which had proposed that HS controls Ca2+activity. It is now evident
that the association of Na+, K+, Mg2+, Ca2+, Mn2+, Cu2+ and Fe3+ counterions with
H and modified Hs could create structurally distinct uronic acid conformations.
This means that the metallomic profile of counterions must be included in
considerations of any proposed H/HS signaling system and different counterion
forms of H/HS are predicted to have
quite different biological activites {e.g. Al3+, Be2+ or Ba2+ intoxication might
pathologically perturb growth factor signaling by H/HS , (cf., Purdey, 1, 2004)}.
The range of inorganic ions (in addition to Na+ , K+, Mg2+ and Ca2+ ) present biological fluids (and also present in H Tables I and
IV) will putatively participate in H/HS –([H2O]n)-inorganic ion-protein adduct formation as well as
perhaps participating in self-assembly of formally highly charged GAG chains.

The binding of a range of metal counterions to pharmaceutical H (cf., e.g. Grant, 1997, Long 2003) showed
that this polysaccharide system behaved similarly to phosphorus oxyanion systems which had been
previously investigated by the author* (both inorganic oxyacid systems show similar systematic shifts in observed
infrared absoptions which correlated with chemical reactivity for different salt forms). Studies of H using
polarography, equilibrium dialysis, potentiometric titration, osmolytic titration, NMR spectroscopy and infrared
spectroscopy 6b-2, 6b2-1 indicated that the counterion binding processes failed to obey the law of mass action as required
for ideal solution state dissolved ion interactions; instead, the effective concentration or activity of H remained
essentially constant over a wide range of apparent dissolved H concentrations; counterion binding isotherms further
demonstrated characteristic discontinuities in the linear isothermal binding curve at characteristic metal
ion/polysaccharide ratios.

2.1 A suggested inorganic ion nutrient gathering and other functions of extracellular polysaccharides
[N.b. blood serum and other biological fluids contain some 60+ inorganic elements (cf. Haraguchi1)].

A similar range and distribution of inorganic elements pertains both to marine alginates and the animal polysaccharide H, a
circumstance which suggests that the highly anionic polysaccharides in animals behave similarly to those in marine bacteria
and algae which act as smart ligands for the simultaneous sequestration of the full range of cations and anions which occur
in the natural bathing fluids (cf. Figs.1-6, p. 83-87).

Inorganic ion nutrient gathering, it is suggested, could have been the original primary functions of extracellular anionic
polysaccharide-rich mucilages.
It is further suggested that during the early evolution of animals in the sea, the glycosaminoglycans (including the H-
like polysaccharide system of HS (analogously to carrageenans and alginates in marine algae) provided osmolyte
buffer and crystallization control agent functions. The latter encouraged
the formation of defined (non-thermodynamic) inorganic salt solution compositions which can be argued to
have been critically needed for the maintenance of biological functions.
This can explain why the amount of HS and other GAGs present in the tissues of aquatic invertebrates seems to be strictly
related mathematically to the salinity of their habitats (Nader et al., 1 (1983) ; 5). Extracellular polyanionic polysaccharides can
also provide protection against oxidising radiation (cf., the general ability of H-like molecules to act as anti-free radical
agents cf., Grant et al.1, 1987, 1994, 1996. Ross et al.1, Mackintosh et al.1, Long et al.1, 1994. The extracellular polyanionic
polysaccharides also can inhibit predation and serve as an effective sink for toxic organic molecules and deactify potentially
damaging inorganic particles which can create promote free-radical induced oxidative and nitrative damage as well as
unconventional damage via the inappropriate nucleation of phase changes (such as may be required for the formation of
toxic proteinaceous fibrils and both calcified and non-calcified plaque formation).
A critical influence of polyanionic substances, which is often overlooked by biochemists, is that these substances
(exemplified by humic polymers and anionic polysaccharides) can strongly inhibit biologically relevant nucleation
dependent phase changes including the preciptiation of insoluble phases from aqueous solutions which also allows such
polyanionic substances to regulate water activity.
It seems likely that primitive osmolyte balance activities and water structuring activites ultimately can be held
responsible for the numerous properties of H-like polysaccharides (e.g. those present in the glycocalyx (cf., Rubio-
Gayosso et al.1) which originally was neded to establishe the existence of the first animal organisms in the sea but
remain of continued relevance to the ion balance physiological control mechanisms in use in modern highly evolved
animal organisms.

2.2 Available information on the association of multi-inorganic-elements with H.

Possible quality control problem with use of H for academic researches and proposed therapeutic
applications
The data currently available suggest that H metallomic profiles approximately conform to the common biological
serum/seawater type of distribution and hence are unlikely to have mainly arisen from in vitro sources of post extraction
contamination.

A preliminary test of the relevance of metallomic thinking to GAG biochemistry is to ask whether the wide range of
inorganic elements found to be present in a range of pharmaceutical H samples studied by mass spectroscopic
techniques (e.g. Moffat1 and an internet report3b-10 as well as sporadic reports in older literature) conforms to the
pattern of blood serum like multi-element distribution in H, which might indicate that the multi-inorganic elements,
which seem always to be present in many commercial polysaccharides, will also occur in vivo, or, alternatively they
simply represent an artifactual contamination, e.g. arising from industrial processing, or from dust particles.

It was originally uncertain whether the characteristic metallomic inorganic element profiles which had been detected
in early mass spectrometric evaluations of heparin (H) (cf., Grant1, 2000) applied to to all H samples or simply
represented some accidental multi-inorganic-element contamination.
Results 3b-10 which have recently been made public by commercial laboratories engaged in blood inorganic element
analysis (cf. Table IV and Figs. 6) now tend to confirm the older indications
(cf. Bowen3b-1 (cf. also 3b2-8-1) ) that similar multi-inorganic element arrays to those previously found by the Aberdeen
polysaccharide group always seem to ‘decorate’ H.

[Multi-inorganic-element analytical data are currently available for alginates e.g., Wassermann1
(and later studies, cf. Table I) which have some resemblances to the analytical data available for H: e.g., leached from
blood collection vessels3b-10, obtained from an industrial Na H sample and a derived single counterion enriched salt
form (Moffat1 cf., Grant et al., 1, 1987, Grant 1, internet 2000; and data from numerous reports (refs. 3b-1, 3b-2, 3b-3, 3b-4,
3b-5, 3b-6, 3b-7, 3b-8, and 3b-8-1) which suggest a variable presence of individual elements in H].
Irrespective of how the multi-inorganic elements had been accumulated by H, the possible occurrence of variable
amounts of inorganic elements in different batches and brands of H poses a serious quality control problem for the
use of H as a convenient laboratory model for HS as well as the proposed used of H for the treatment of human
diseases for which preliminary reports (reviewed below) suggest is an achievable goal. In the event that the multi-
element contents of H and HS are confirmed by further studies to be a general phenomenon, a careful reappraisal
may be required of some previous H/HS biochemical researches.

It will be necessary to draw up appropriate quality control protocols for future researches in this field.

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

It be noted that H, perhaps because this is the most anionic biopolymer, H seems have an unusual ability to
simultanesously sequester (apart from the wide range of dissolved inorganic ions and molecules) various colloidal
sized particles which can occur in biological fluids; apparently H acts thereby in such a manner so as to inhibit the
potential seeding by such surfaces of the formation of pathological plaques
(vide infra, cf. also Grant et al. 1,1992). H shares this property of the prevention of thermodynamically required
precipitations (e.g. of sparingly soluble Ca salts) with other polyanionic substances (e.g. polyphosphate and fulvic
acids) by the ability to stabilize aqueous solution supersaturation.

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

2.2.-1 Countercation-Specfic [Polarization-Power-Related] Interaction of Metal Ions with

–C(O)O- groups in H/HS

The carboxylate groups the principal disaccharide repeats in H showed infrared absoptions which altered

according to the counterion polarization power. Similar results were reported by Panov & Ovsepyan1,

Grant et al. 1 1987b) and Rudd et al.1, confirming that counter cations can directly modify the electronic

environments (and putatively the electrostatic activities) of Heparin–C(O)-O- groups and thereby contribute

to the overall mechanism of how inorganic cations modify the attractive potencies of H/HS.

The principal efffect of counterions on H–SO3- groups appeared to be the alteration of the water clusters

associated with these groups.

2.3 The Haraguchi Hypothesis Extended to Polysaccharides

Haraguchi1 noted that most of the elements in the periodic table normally occur in animal cells and discussed the
existence of a similar range and distribution of multi inorganic elements in both blood serum and seawater.
It is now pointed out that this seawater-like multi-inorganic-element phenomenon is most strikingly illustrated
by the highly sulphated anionic animal polysaccharides.
Haraguchi seemed to imply that the scientific field (to be termed “metallomics”) should be concerned inter alia with the roles of the entire
seawater range of inorganic ions. He proposed that “all elements” occur in biological fluids and therefore might be relevant to biochemistry, but
he did not specifically discuss any role for polysaccharides in the preferential uptake of such ultra trace elements which could perhaps be required
to modulation of water structures present at the surfaces of key information-encoded H and HS anionic polysaccharides which seem to naturally
become enriched in such elements. This idea is my extrapolation of his ideas. The only biopolymers for which metallomics were relevant,
according to Haraguchi, were proteins. However, it is now suggested that the inorganic element reservoirs seemingly present in anionic
polysaccharides may participate in evolved signaling mechanisms and therefore be invovled directly in the transmission of information encoded
within the anionic patterns of the ultra anionic animal polysaccharides H and HS so as to enable these polysaccharides to control protein
activities.

The Work of Rudd et al. Confirms the Major Relevance of the (Haraguchi) Inorganic Element Phenomenon to
How Heparin & Heparan Sulphate Function In Vivo
Rudd et al.1, in a recent groundbreaking study, reported the effect of a range of metal ions on the CD and NMR
behaviour of H and modified Hs and also the drastic effect of K+ and Cu2+ substitution of H upon FGF2/FGFR1c
action; the presence of different metal counterions creates a distinct chemcial signal which indicates that it is
necessary to include such metal ions as part of the information encoding mechanism provided by the H/HS protein
control system.
An important additional part of this control, it is now suggested, could be provided by the wide range of ultratrace element which
occur in biological fluids but which previously were not thought to be of relevance to biochemistry, but which ,nevertheless, seem to
become (sometimes) greatly enriched at H/HS surfaces where the could provide an inorganic ion encoded signaling system addtional
to that offered by Fe, Cu, Ca, Mg, Zn, K and Na ions implied by Rudd et al., to enable the formation of specific H/HS sugar
conformations and electronic phase coupling systems which could behave as discrete biological signals.

2.4 The Heparanome-Metallome

It is suggested that by combining together two recently proposed hypotheses (Haraguch1 and Turnbull et al.a which
discussed the “heparanome” a and the metallome [putatively analogues of the proteome and the geneome]) gives a useful,
but at present tentative, intellectual framework with which to address the roles of inorganic cofactors (and also to address
the role of water structures) in H/HS signaling.

According to the general principles of physical chemistry, it would be expected that the same range of inorganic ions to
those which occur in H, should also occur in HS especially in the H-like segments of HS. {This notion is also in apparent
agreement with a number of research publications6}. [for ref. 6 go to page 126]

The possession by anionic polysaccharides of an intricate information endoced bar-code or fuzzy logic microstructure which
selectively determines protein folding and signaling is the basis of the heparomea,
a mechanism which is thought to enable the HS polysaccharide-based to engage in systemic control of multicellular
discriminated behaviour in animals (this is a ‘super-hub’, around which numerous biochemical activities can rationaly be
proposed to be controlled [perhaps even the most important hub and information system which determines the whole of
animal biochemistry] [it might alterntive by viewed as an auxiliary system which enable the genome to perform many more
functions than would have otherwise been possible {cf., the much lower than expected number of genes which have been
found to occur in humans}. The presence of a correct HS reservoir of inorganic ions may then be supposed to be required
for optimum human health. The actions of the heparanome could also be relevant to a fuller understanding of the roles of
bacterial, algal and plant anionic polysaccharides. The reactivities of all such biological anionic information systems could
however, actually be potentiated via inorganic cofactor effects which in turn could ultimately be potentiated by water
structuring effects (although the actual nature of liquid water has not been established perhaps the hypothesis proposed by
Wiggins1 provides, at the present time, the most suitable working model). [Viz. the surface multi-ion compositions create
exact water activites and ionic strengths which thereby allow their associated polysaccharides to determine the specific
folding patterns of associated proteins which rather than possessing intrinsic abilities to fold correctly (as was the traditional
view) are now believed to be critically dependent for such folding on the protein surface water activities (cf. Wilse-
Robinson et al.1, Wiggins1)].
a
J. Turnbull et al. in TRENDS in Cell Biol. 2001, 11 (2) 75-82 suggested the use of the term ‘the heparanome’ for the new concept
of the dynamic expression of HSs possessing differing sugar sequences produced by particular cells or tissues from a precursor in
which glucuronic acid-N-acetylglucosamine units are subjected to structural alteration in the Golgi apparatus by sulphation (by
2-O, 6-O and 3-O- sulfotransferases) and epimerization (C5 epimerase) and postsynthetic de-N sulphonation and scission by nitric
oxide metabolites and also by the action of 6-O-endosulphatases. These processes also allow for environmental - HS
microstructure crosstalk for modulation of HS functions, which are according to L. Kjellin & U. Lindahl (Annu. Rev. Biochem.,
1991, 60, 443-465) in a reveiew of the function of proteoglycans of which HS are prominent members: as being present in
multicellular animals for the provision of mechanical support, negative charge, regulation of cell migration and aggregation, the
development and stabilization of synaptic structures, endothelial regeneration, the stabilization of basement membranes, the
modulation of collagen fibrilogenesis including the transparency of the cornea, the regulation of cell growth, urinary trypsin
inhibition, provision of a filtration barrier, various roles in morphogeneis, the provision of links to cytoskeleton and ECM, the
mediation of adhesion and morphogensis, the assembly of the matrix phosphatidyl–inositol linkage, the provision of reservoirs for
fibroblast growth factors, allow for the regulation of blood coagulation, the mediation of transferrin functions, and the uptake of
antigen presentation.
The initial step in biosynthesis of H/HS is known to be the formation of the protein linkage region GlcA-Gal-Gal-Xyl-Ser by
transfer of xylose from the UDP-xylose to specific Ser residues in the core protein. In their extensive 1999 review of HS systems
(the most common members being now known to be glycoylphosphoinositide-linked glypicans and transmembrane syndecans)
Bernfield et al. ibid., 68, 729-77, noted that the linkage region, which is the same in HS and chondroitin sulphate ChS, evidently
has been highly conserved “from flies to humans” and that HS provides the most abundant receptor system at adherent animal
cell surfaces for tissue morphogenesis and wound repair as well as for host defense and energy metabolism processes which are
achieved via interactions including those with the HS molecules present in the extracellular matrix.

Perhaps the most fully understood epitope in H/HS is the pentsaccharide antithrombin-binding site which occurs most characteristically in
mast cell H and in some HSs. Pharmaceutical H, which has been used for many years as a blood anticoagulant, is now available in a
variety of salt and molecular weight forms which includes the single epitope, an artificially produced pentasaccharide molecule as well as
unfractionated H (‘Na heparin’) from porcine mucosa (but also from bovine lung) which derives a major part of its anticoagulant activity
from the antithrombin epitope content.

A putative H/HS inorganic ion environment interface system

It is suggested in vivo discrimination process by which information held within H/HS sequences is transmitted into
biochemical activities requires inorganic cofactors (putatively multi-inorganic elements) suggesting the use of a fuzzy logic, ‘analogue’ recognition
system of a polymer rather than oligomer-associated metallomic array rather than an exact ‘digital’ type of specific microstructure electrostatic
recognition similar to the hydrogen-bonding etc. used by the DNA double helix.

It is proposed that the the multiple inorganic elements which occur in H (and putatively also in HS) and which are
also part of an essential glycocalyx reservoir system permits the release of inorganic cofactors in amounts needed to
achieve correct biological signaling by the heparanome; this may includes a system of fast track provision to achieve
a required inorganic cofactor cocktail to permit a selective H/HS protein binding. Such inorganic cofactors seem to
be needed to assist in muscle action in the lung (cf. Jenniskens et al. 1) (and likely also elsewhere including for HS
functions performed in the glycocalyx of the heart) and (possibly a range of ) redox metal ions which is needed
together with Zn2+ are required for nitrosative scission of HS chains (cf., e.g. Ding et al. 1) and for the modulation of
the actions of enzymes invovled in the assembly of H/HS and HS PG disassembly activities of MMPs (e.g. for
shedding of HS PGs from plasma membranes).

2.4-1 Summary of the Origin of the Heparanome-Metallome Hypothesis

Virtually all elements in the periodic table can be detected in biological samples by commonly used mass spectroscopic
multi-inorganic-element analysis techniques.
This circumstance prompted Haraguchi1 to update the earlier concept of biochemical ‘metallomics’ which was restricted to
the 20 or so highly essential inorganic elements utilized by biochemical processes previously proposed by R.J.P. Williams.
The Haraguchi ‘new biochemical metallomics’ is now, it seems, to be concerned with all stable elements in the periodic
table.
Individually additive contributions of each of these ions present in biological fluids will enable the fine tuning of the
structure of liquid water (cf. Luck1). Since “Life Depends on Two Kinds of Water” (Wiggins1,2008) it follows this chemical
system is the most important determinant of all biochemical processes and the most central way metallomics could be of
relevance to biology is by the ability of metallomic matrixes to fine tune such water structures.

The other primcipal biochemical system for which the new metallomics also seems to be of especial relevance is the anionic
polysaccharides which apparently also naturally exist as multi-inorganic-element metallomic matices. This phenomenon is
found to the greatest extent with pharmaceutical H, a traditional industrial product which is derived from bovine and
porcine mast cell H protoeglycans which has found wide use in medicine as a blood anticoagulant. H is thought to be the
most anionic of any biopolymer which confers on this polysaccharide system an ultra-high electrostatic charge which
should promote the (chemical nature independent) electrostatic counterion collection process (a mechanism which is at least
partly responsible for the uptake of counterions) to enable the simultaneous binding to H of the multitude of types small
amounts of metal ions which exist in multi-element containing natural waters including biological fluids. Other H-like
medications include marine algal ** and plant-derived xylan fractions (e.g. pentosan polysulfate (PPS)) which are produced
by an additional sulphation carried out post-extraction from tissue. These preparations are also suggested to function as
metallomic matrices and to require use of multi-inorganic-element mass spectrometric techniques for quality control
purposes.

All H samples examined to date (and discussed in more detail below) seem to have approximately (log-log) inter-related
multi-inorganic element compositions which apparently define them as blood serum/seawater type Haraguchi-new-
metallomic matrice systems (cf. Section 10.8 (page 83 et seq.).

These metallomic profiles inevitably include small amount of a range of toxic elements the removal of which has been
suggested Bohrer et al., 1 to be a critical clinical requirement for certain uses of H.

[Different degrees of ‘purification’ of H achieved by different manufacturers can lead to different degrees of enrichments by
the principal conterion (e.g. Ca2+, Na+, Li+ or NH4+) containing different amounts of residual ‘toxic element’ contents.
Such differences in H activites should be addressed by researchers before attempting to use H as a model of HS.
This urgently requires further research].

2.4-1-1 Examples of Literature Reports of Metal Ion Driven H/HS Signaling

Cf. Tables I-II (page 66 lists selection of available information on the requirement of inorganic cofactors for H/HS signaling.

The modus operandi of the heparanome is believed to depend on selective interactions between proteins and HS
proteoglycans which seem to be less determined by the HS proteglycan core-proteins [cf., Iozzo1, Powell et al.] than
the microstructure of the (H)-like segments of the HS polysaccharide side chains, but which also involves the
inorganic ions putatively associated with such H-like segments. (HS core proteins will, however, augment the ionic
strength (I) at sites of interaction, and thereby also influence the rate of formation of ternary complexes between
metal ions, glycosaminoglycans (GAG) and target proteins and may also serve to promote the formation of
oligomeric metal ions with enhanced abilities to bind HS side chains).

Some examples of the growing literature database on the interdependence of H/HS biochemistry and
inorganic factors are collected in Table I which includes a list of reports which have noted that metal ions
are required to facilitate H/HS protein binding; in Table II lists reports which have suggested that inorganic ions or
particles can also directly modify HS microstructures (evidently by influencing the
primary assembly or by postsynthetic structural modification).

An essential requirement for inorganic cofactors has been reported to allow the binding of H/HS to endostatin (Ricard-Blum
et al.1), of H/HS to annexin-V (Capila et al.,1) and for the H/HS promoted basic fibroblast growth factor receptor assembly
process (Kan et al. 1).
The Aberdeen polysaccharide group redox hypothesis of H/HS signaling (cf. Grant 1, 2000) putatively also implicates the
activities of redox metal ions in the etiologies of degenerative diseases.
Such putative roles of the inorganic-element matrix associated with H/HS include the generation of oligomers by the action
of nitric oxide metabolites are now known (cf. Ding et al. 1) to be dependent on the presence of Cu and Zn cofactors.
General chemical considerations also suggests that oxygen-metal ion containing (polyoxometallates) and related
sulphur bridged metal ions might also be considered as possible cofactors for protein HS interation and regulators of
H/HS actitities since a ruthenium red polycation [(NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5] 6+ has ben reported (Utsumi &
Ota1) to bind to cell surface HS and cause cellular agglutination via a HS intercellular crosslinking mechanism. [N.b.
small amounts of Ru were detected by ICP-MS in H]. Further research is warranted into the mechanism by which
ruthenium red inhibits immunological responses (Dwyer et al., 1) since HS activities and their modulation by Ru
(which can also bind to nitric oxide) could conceivably be implicated in such activities.

Ba2+ is known to selectively bind (and precipitate) H (cf., Nader et al. 1, 1981). A perturbation of HS signalling
following intoxication by Ba2+ might perturbs FGF-II (b-FGF) assisted myelin sheath renewal and thereby
contribute to nervous systems pathologies (this was suggested by Purdey1 to explain an apparent correlation between
the presence excess Ba2+ in soil and plant samples and the geographical prevalence of multiple sclerosis (e.g. in North
East Scotland)).

Other possible candidate metal ion cofactors for aberrant HS signalling include the the rare earth metals (which
have found general use as animal growth promoters**) as well as ‘Zr4+’ , Ga3+ and Tl+ which seem to be prominent
components of industrial H before its final ‘clean up’. Be2+ and Al3+ (e.g. the polycations formed of Al3+ which binds
more strongly to H when in an aggregated form**, might also promote of HS cell surface polysaccharide activation,
deactivation or crosslinking in vivo).

(Table III (page 72) lists some analogous effects of organic molecules etc., which influence HS synthesis).

2.4-1-2 Comparison of ‘Metallomic’ Multi-Inorganic-Element Arrays in Anionic Polysaccharides

with Those in Other Matrices (Cf., Table IV)

Spark source mass spectrometry (SSMS) and inductively coupled plasma mass spectrometry (ICP-MS)

multi-element analytical data for several H samples from different manufacturers suggest that H always

contains an enhanced seaweed-like inorganic element content (recent ICP-MS reports of blood sampling

tube H-associated ‘contaminants’ suggested3b-10 the co-occurrence with H of the full seawater range

of some 80 elements 4). Such single ion enriched Na and Li H (e.g. obtained by use of ion -exchange resin

column technology for the preparation of medical-grade H) as well as ‘native’ H, appears to be greatly

enriched in those elements which occur in the least amounts in natural waters, a situation which is

reminiscent of the cell wall polysaccharides of plants1a-10 where a similar range of inorganic ions as are

believed to contribute to cell wall control over Ca2+ second messenger actions e.g., via the borate crosslink

polysaccharide oligosaccharide generator1a-10.

The occurrence in marine algal tissues of major cell wall polysaccharides in multi-counterion salt forms

rather than, as was originally thought, in a mainly free acid form, was established by A. Wasserman3

and confirmed by W.A.P. Black & R.L. Mitchell3a and later workers. It is now thought that marine

algal polysaccharides contain most or all of the inorganic elements (60+ in number) which occur in

seawater3b from which solution the anionic polysaccharide ligand is apparently able to simultaneously

sequester the full range of inorganic counterions and particles there present, by a mechanism which

apparently enables these polymeric ligand systems to become selectively enriched in those elements which

are the least abundant in this bathing solution.

Haraguchi4 suggests that an updating of the metallomic concept required that many elements

which had hitherto been classified as being ‘non-physiological’ must now be included as being potentially

physiologically relevant since the amounts of these elements in human blood serum are approximately

correlated with the amounts present in seawater (as suggested by a log-log correlation between these

metallomic matrices4). Possible evidence for the relevance of the former ‘non-physiological’ elements in

blood serum to animal biochemistry is that these elements can become selectively enriched in H (cf. Table

IV) suggesting a possible modulatory role of these elements for for H/HS** activities incuding for the

formation of ternary polysaccharide-metal ion-protein complexes (e.g. similar to those identified by

Cornwell & Kruger1).

Table IV (page 77) also compares the metallomic profiles of alginate, H and human hair, a tissue commonly

employed for the presence of toxic metals in humans (cf., Fig. 2 (page 83))

Table IVa lists results reported for metal ion impurities present in chitosan (their presence in this case

was attributed to their uptake from a final washing with tap water13a but further studies to more fully

establish the nature and origian of the multi-inorganic elements in chitosan seems to be warranted).

Polysaccharide-rich biomasses of land plants, brown and red marine algae, as well as mollusc shells, bone, mast cell H
and H-like segments of HS may constitute a related system of natural (‘salinity induced’) metallomic matrices (cf., Fig. 3
(page 83) which seems also to be related to the inorganic element contents of human hair15 (cf., Fig.2) (which is currently
the most well-researched multi-inorganic element containing matrix) and chitosan13a
cf. Table IVa).
Human scalp hair (e.g., 15 ; except for Zn, which is augmented in hair) is comparable (cf. ratio A/C Table IV) with the data
shown for ‘Na’ and Tl H. This correlation is best for the least perturbed samples, from schoolboys 15 (Fig.2)

The binding of a range of metal counterions to H seemed more akin to a (first stages of a) counterion
induced phase-separation process (but where final aggregation into solid particles was strongly inhibited)
which nevertheless yielded nanoscale quasi liquid phases of similar structures to the non-crystalline
polysaccharide glasses obtainable by casting H and similar polysaccharides onto plane surfaces (a method
which is commonly employed in industrial laboratories to study thin films of polymers which was adapted
by the author for the evaluation of glycosaminoglycans by attenuated total reflectance and related
infrared spectroscopic methods (cf. Grant et al.1, 1987).

H when added an anticoagulant for the analysis of metal ions in whole blood has been traditionally associated with
interference by a range of metal ions (cf. Bowen 3b-1 and other 3b refs.) which seems to accord with the present hypothesis of a
ubiquitous H- associated metallomics scenario.

H in mast cells was shown, by histological methods, also to bind {Ru2+ 3}6+ [present as ammonia linked metals in ruthernium
red]. A range of similar publications also suggest that multivalent metal ions bind to sulphated polysaccharides when used
in histological stains or for the spectroscopic evaluation of H 6.
Ruthenium red has also been reported to bind and crosslink HS-like molecules at cell surfaces during agglutination of
ascites hepatoma cells by this substance (Utsumo & Oda1).

The use of Ga3+ in medical scintigraphic visulization of tissues has similarly been associated with the binding of this metal
ion to HS. Related in vitro and in vivo binding studies showed that Ga3+, and a wide range of other multivalent metal ions,
bonded strongly to H and HS proteoglycans (Kojima et al., 1, 6).
The reported anti-tumour actions of Ga3+ might conceivably arise by a related mechanism.
H and putatively also H-like segments of HS have intrinsically or specifically been ‘designed’ by evolution to
incorporation an additional design features relative to their evolutionary precursors which allows for a more
flexible system of metal ion binding (cf. Whitfield et al. 1) [whereby the intrinsically low inherent chelation-
binding power for metal ions of older polysaccharides is boosted by use of an iduronate plus some glucuronate
rather than exclusively glucuronate (as in more primitive extracellular polysacchrides) and also by using both O-
sulphate and glucosamine N-sulphonate anionic patterns which seems to enablethe selective but flexible easily
alterable mode of binding of the ultratrace elements which occur in both blood serum etc. in order to corrrectly
facilitate water structure and the resultant binding of specific HS segments within HS chains to their
designated protein binding sites].
Biologically-centred metallomic studies should now perhaps be re-focussed on how the inorganic biochemistry
animal anionic polysaccharides function in vivo, including e.g. how they confer tissue protecton. It is indicated that
there could be a major role for multi-inorganic elements in the promotion or the inhibition of the formation of
plaques in arterial and other tissues including those of the central and peripherial nervous systems.

[A long list can be drawn up of academic laboratory reports (cf. Tables I-III (pages 66-79) which seem to
confirm that inorganic cofactors are of potential major importance to H/HS signaling; these findings can be
tentatively extrapolated to suggest that hitherto not-hitherto-thought-biologically-relevant ultratrace elements
become enriched in H/HS where they serve essential functions such as those responsible for increase animal
growth rates and affect thyroid functions etc.** when such elements are added to animal feeds].

Perhaps a major system of heparanome-metallome-crosstalk underpins much of animal physiology.

2.4-3 H/HS is an evolutionary designed flexible metal ion binding system

Natural polyanionic systems (e.g. the complex anionic polysaccharides systems of heparin, pectin, alginates as well as the anionic humic materials in
soils and natural waters) can apparently simultaneously sequester the full range of inorganic ions which occur in the natural bathing solutions by
mechanisms which tend to favour the preferential uptake of the least abundant elements present

2.5 Evidence for ‘all-element’ decorated H

Rabenstein1 has noted that sodium H when injected into the blood stream will bind the Ca2+ ions there present, to convert
into the calcium salt form. It is now suggested that while this is probably a good initial approximation of the in vivo
behaviour of H, a more accurate description seems to be the formation of a Haraguchi metallomic ‘all-element’ decorated H
similar to that which was first identified by Moffat1 and later discussed on the internet by Grant1 (2000) and confirmed by later
reports of ICP-MS analysis to multiple inorganic elements present in of several H samples used in blood collection tubes
(cf. Table IV).

2.5-1 SSMS results for Na H [this H was donated by a (former) major manufacturer at Runcorn, U.K] (more accurately
this is a Na/Ca = ca. 6 H)
Ca (30,000), Si (5900), Cl (5600), K (2000), Mg (1300), Fe (1100), F (890), Cu (730), P (440), Ti (390),
Ta (280), Ni (170), Ba (140), Br(130), Co(80), Zn(80), Sr(65), Cr(30), B(25), Ga(20), Pb(16), As(15), I(10), Cs(9), Tl(8),
La(7), Ce(7), Mo(7), W(5), Sn(5), Nd(5), Zr(5), Ag(4), Rb(3), V(3), Y(3) , Sb(2) and Cd(1).
This Na H can now be seen to be an especially well-defined example of the type of biological metallomic matrix first
identified by Wassermann1 and later (less specifically) discussed by Haraguchi1 where the inorganic elements present
in biological matrices approximately correlate with those in seawater and human blood serum.

These results were obtained in 1983 by C. M. Moffat1 a graduate student working with W.F. Long and F.B. Williamson (the then
directors of a Marischal College (Aberdeen University, Scotland, U.K.) polysaccharide research laboratory) who together with J.
R. Bacon, a specialist in multi-inorganic element analysis at the Macaulay Institute (also in Aberdeen) conducted a spark source
mass spectrometric (SSMS) multi inorganic element analysis (by a method described e.g. in Filip et al.,1 ) of a carefully selected
sample of a typical porcine sodium H (which seems also have been distributed to several academic research laboratories)
Moffat extensively dialysed this H against deionised water and following lyophilisation of the non-diffusible material submitted it
to SSMS analysis without being pre-ashed.
[This H had, however, apparently not been ‘cleaned-up’ by the manufacturer, e.g. by using the standard final ion exchange single
counterion enrichment process; this was possibly a deliberate omission since this H had been specifically allocated for
fundamental academic inorganic biochemical studies (and had obviously not been intended for medical use, e.g., as a blood
anticoagulant, since it greatly exceeded the Pharmacopeial recommendations for heavy metal contents, but otherwise showed the
normal 13C and 1H NMR spectra of porcine H and behaved in the expected manner as a blood coagulant)].

Although the inorganic elements present in this H probably altered only slightly its anticoagulant activities it was suggested
by comparison with other H samples containing less associated inorganic elements studies of the binding Zn2+ to this and
other H samples (cf. Woodhead et al., 1 and results obtained by other postgraduate student working with F.B. Williamson &
W.F. Long) that the presence of different amounts of multielements attached to different H samples might have affected its
ability to bind further metal ions and also could have augmented their antioxidant and ferrioxidease activites**. The
presence of a number of redox metal ions in H and related substances also suggested augmented redox-metal assisted
nitrosative reaction processing by such H-like moleucles (cf. Grant, 1, 2000 internet).
This idea has not been properly tested by specifically designed experiments since it only emerged as a possible origin of
discrepancies of apparent differences in H samples some years after the original laboratory studies had been completed.

In his Ph.D. thesis, Moffat1 had partly reported the SSMS results given in Section 2.5-1 of this document where it is indicated that the major
counterion is Na (at ca. 96,000ppm). [Although Moffat1 listed SSMS data for for Ti, Zn, B, V, Co, Ni, Y, Zr and Sb in Table 17 (data for Tl H) in his
thesis, he did not list the full results for NaH (which were partly reported in Table 15 of his thesis); this was explained as being due to the possible
interferences possibly assoicted with the missed out data which also affected, to a lesser extent those SSMS results which he had chosen to report
viz. those for F, As, Br Mo, and Ta. These SSMS data are, however, now shown to be essentially correct since they are comparable to values
reported by others using ICP-MS instrumentation (cf., Table IV, vide infra) [results reported later on the Internet for other pharmaceutical,
medical grade H samples]. All of commercial H samples examined thusfar seem to contain similar amounts of the same multiple inorganic
elements to that derived from the Na H reported in Section 2.5-1 following a single percolation through a cation exchange resin (to give the (Tl+)
salt form.

2.5-2

SSMS Determined Inorganic Elements Associated with an Experimental Tl Heparin

When the above multi-inorganic Na H (2-1-1) was treated with a standard cation exchange resin procedure the
resultant single counter-ion enriched H showed an SSMS spectrum indicating that an effective replacement by the
selected single counter-ion had been achieved (cf. Grant 1 (1987)); this procedure produced a form of H similar to the
types of (‘single counterion’) H commonly used in medicine as also used as laboratory model for HSc. The results
are listed in Table IV (page 69). The inorganic elements present in Na(Ca) nad Tl H are compared in Fig. 1
(page76).

This ‘single counterion’ thallium Hd was prepared by a standard cation exchange process achieved with a
sulphonated polystyrene resin (Rohm & Hass Amberlite IR 120) column substituted with Tl+ (Moffat1). The SSMS
analyses of the starting Na-H and the resultant Tl-H samples indicated that a single passage through this type
standard cation ion exchange column achieved replacement by Tl+ of more then 99% of
the principal ‘contaminant’ inorganic (mainly cat)ions which had been present in this (evidently highly
‘contaminated’) starting commercial ‘sodium’ H (cf. Grant 1(1987)). (A similar effectiveness of use of ion exchange
resin columns for the removal of the Al which is present in some commercial H samples has recently been confirmed
by Bohrer et al., 1).

2.6 ICP-MS Studies of H3b-10 Dilute Nitric Acid Leachates from Evacuated Plastic Blood Sample Tubes.
Summary of apparent effect on metallomic profiles of H leached from blood collection containers compared to whole
H

H being a mixture of many individual molecular structures of a related nature which confer different microstructure held
information related to the ability to selectively bind counterions and other inorganic ions (as well as proteins). This means
that leaching of the H mixture of molecules from polyethylene terephthalate (PET) blood vessel containers (a procedure
used to obtain ICP-MS results which have been posted on the internet3b-10) would be expected to fractionate the H in terms
of the mass spectroscopic results when compared with mass spectroscopic results obtained by taking, without fractionation,
an entire H mixture as received from the manufacturer.
A preliminary evaluation of the currently available information on the metallomic profiles of H samples collected in Table
IV suggests some fractionation by leaching of H from blood collection tubes by dilute nitric acid applies to the rare earths
which seem more abundant in entire H compared to leached H.
While the data corresponding to whole H samples used for the preparation of whole blood containers used for clinical
analysis is not available to the author an indication of the possible differences between leached H and intact H can be made
from a comparison of the available older whole SSMS H and
more recent ICP-MS H studies3b-10.

[Evidently this information was published to provide backup information on how contaminating inorganic ions are leached
from containers and the anticoagulants (including H) used in the makeup of such containers which are believed to be
fabricated from PET {which is expected to adsorb H}]
The report gives detailed tabulations of ICP-MS results for multiple inorganic elements, which allows the multi-element
contents of several commercial samples to be extracted from the reported data; the tabulated data also provided system
blanks from non-heparinized tubes. The data confirm earlier indications (Moffat, loc. cit.) that commercial H is a highly
substituted multi-inorganic matrix.

ICP-MS data for H can be extracted from data which appeared on the Internet in 2005 to indicate that
a selective removal of more soluble H fractions occurred during the leaching procedures used to obtain
multi-inorganic-element (H-related) for study (cf. discussion of results in footnotes of Table IV). {It is
known that H adsorbs at blood collection vessel surfaces, this effect being greater with glass than plastic
cf., Tunbridge et al. 1 but this difference may be less for the most up-to-date PET plastic used for blood
sampling. Further studies are obviously required to check out this.

2.6.1 Re-Evaluation of the Moffat et al. SSMS Data for Na H and Comparison with ICP-MS Data
A re-evaluation of the old Aberdeen (Moffat et al.) SSMS data (by Grant 1 (2000)) indicted that the ion
exchange ion replacement procedure which had been used to prepare the Tl –H (and conducted in
association with the author) had been more complex than required for a simple counterion replacement
process. Some of the ultratrace elements present (e.g. cerium) is now suggested to be bound much more strongly to
the H than would have been anticipated by a simple electrostatic counterion attachment process (cf. Grant internet, 2000a).
The ion exchange process was re-assessed creating skewed but essentially retained the distinct original multi-
inorganic element matrix related to that of the starting Na-H, albeit this was greatly diluted with a greatly enhanced
presence of Tl+ couterions.

These data, when compared with those from non-mass-spectroscopic inorganic analytical reports 3b-1 to 3b-8
and (somewhat more accurate ICP-MS) mass spectrometric multi-element analyses data 3b-10 later reported
for further H samples confirmed the notion that H and H-like polysaccharides always occur in vivo as
multi-element organometallic composite mixtures, which, even after extensive purification, retain a
skewed remnant polyinorganic profile related to the original ‘native’ H metallomic profile (which
approximately correlates with the metallomic profiles of seawater, human blood serum and many other
biological metallomic matrixese and hence suggests the possibly co-existence of an abundant metallomic
array with the (e.g. juxtaposed) polysaccharide chains of H/HS and other proteoglycans in vivo.

2.7 Toxic Inorganic Elements in Natural Anionic Polysaccharides

A range of ‘toxic’ elements occurs in algal polysaccharides, chitin and chitosan as well as in fulvic acid
soil and fossilized soil derived humic polymer. The relative amounts of such elements seem to be similar to
those present in H which suggests that they arise from a similar process of preferential sequestration by
naturally occurring polyanionic polysacchrarides and analogous polycarboxylic acid substituted organic humic polymers**.
2.7-1 Toxic elements in H
Currently available evidence suggests that H is an optimum metallomic matrix (hence H is expected also naturally to
contain {unless subjected to ion exchange purification} exceptionally large amounts of ‘toxic’ inorganic elements, (e.g.
many commercial H samples were found by Bohrer et al.1,2004 to contain unacceptable amounts of Al (which was efficiently
removed by percolation through an ion exchange column); As was also found by Bohrer et al., 1,2005 to occur in
commononly used clinical chemicals including H) [N.b., when bound to H (e.g. when used medically as a blood
anticoagulant), these elements may, howver, normally be inert in vivo. A similar inertness of toxic metals in the H-like
segments of HS proteoglycans can be predicted. Inappropriate nitrosative scission of HS, however, may occur under
pathological conditions (cf., e.g., Grant 1, 2000, internet dg2)] which could over-ride the toxic metal pacification mechanism and
result in tissue damage arising from release of such toxic heavy metals within the patient.

2.7-2 Multi-inorganic-elements in common laboratory reagents

It should be noted that multi-inorganic-elements are also found by modern instrumentation to occur ubiquiteously in various
common laboratory reagents (e.g. in simple salts and man-made compounds such as ethylendiaminetetraacetic acid (EDTA)
which is used (analogously to H) as an anticoagulant in blood collection tubes sometimes used together with inorganic gels
(e.g. those which are silicate-based blood clot stabilizing agents) (cf., 3b-10). The metallomic profiles for EDTA and the clot
stabilization gels seemed at first glance to have a H-like multi-inorganic-element nature, but a closer examination suggests
that the inorganic profiles of the man-made chemicals are distinctively different and less ‘contaminated’ that are those of
biological ligands; the multielements in the man-made chemicals could also perhaps ultimately be derived from those
present in rock salt NaCl (derived ultimately from seawater) but is generally found to be less by one to two orders of
magnitude on a molar basis than those which are associated with H. (3b-10).

2.8 Other suggested metallomic-related activities of animal polysaccharides: modulation of water

supramolecular structure, calcification and deactiviation of Fenton reaction catalysts
Animal polysaccharides are known to modulate normal and pathological calcification and provide antioxidant and
antinitrant protection by binding and deactifying toxic metals including inappropriately unliganded redox metals
such as Fe3+, Fe2+ and Cu2+. In animals an altered HS biosynthesis seems to occur in direct response to changes in
Ca2+, Mg2+ and Mn2+ as well as NO and H2S and putatively other inorganic factors such as silicate nanoparticles and
rare earths elements (the presence of optimal amounts of which may be required for optimal vascular health and to
achieve promotion of growth and wound healing). This can putatively be achieved by increasing the sequestration
power (e.g. for an insufficient presence of essential inorganic elements or for a need to pacify toxic inorganic
elements) by an alteration of the Golgi apparatus biosynthesis of HS chains (cf. the reported alteration of HS
biosynthesis in reponse to the presence of enhanced amounts of those inorganic elements (such as Pb, Cd and Hg and
perhaps also Ba and Al and Be) which are detrimental to health, as noted in Table II.

2.8-1 Other cell surface polyanionic (potential metallomic) systems:

polyphosphate, poly-β − hydroxybutyrate & teichoic acid
Various metallomes H/HS, polyphosphate16and polyβ hydroxybutyrate 16a may determine, perhaps in a cooperative
manner, the metal ion binding and associated water activities at cell such surfaces and intracellular organelles.

Putative metallomic matrices are constituted by anionic phosphate groups which include apatite minerals and hydroxyapatite composites
of bones and teeth and the high molecular weight polyphosphates which have been reported Kornberg 16 to occur at cell surfaces
throughtout biota; the association of P with heparin (cf. Table IV) may indicate the occurrence of phosphate-bound metals here also;
polyβ hydroxybutrate which is also apparently present (in a similar to anionic polysaccharides) at the cell surfaces of a wide range of
species.

Cf. also associated with Gram positive bacterial peptidoglycans are highly anionic teichoic acids and teichuronic
acids (e.g., Bhavsar et al.1 ) which constitute metal ion binding (and water activity regulating) activities.

Related phenomenon will also, it is suggested, pertain to the geneome**.

2.8-2 Possible inorganic phosphate –containing high affinity metal io binding sites in H

There is a possible role of inorganic phosphate/polyphosphate associated with H for the creation of sparse high affinity metal ion binding
sites (these show up in the electrometric titration of H e.g. with Cu2+ (cf. Grushka & Cohen 1).

Studies of the binding of Cu2+ to H reported in the literature (Grushka & Cohen1) suggested that H might possess small amounts of high
affinity metal ion binding sites. Later studies suggested that especially in multi-inorganic H suggested the presence of such high affinity
Cu2+ binding sites (these might e.g. have been at inorganic P associated with H) were further implicated in the ability of H to act as an
antioxidant and perhaps also as a ferrioxidase. Further work is required to more fully establish this possibility.**
Use of putatively ion exchange cleaned up “Celsus” H for modern spectroscopic evaluation of the major mode of Cu2+ binding clearly
identified a tetracoordinated Cu2+ ion bound to carboxylic acid group, the ring oxygen of iduronate-2-O-sulphate, the glycosidic oxygen
between this reside and the adjacent (towards the reducing end) glucosamine and the 6-O-sulphate group (Rudd et al., 1).

2.9 Metal ion assisted H/HS protein binding

A re-evaluation of the author’s published (mostly peer-reviewed) studies of how metal ions bind to H (cf. Grant et al.,
1, 1984-96
; cf. also the comprehensive list given by Long1, 2003) is now attempted in the context of an evaluation of the
above heparanome-metallome hypothesis. A selection of relevant literature reports are collected in Tables I-III
(pages 62-73). A key additional paper which further lends support to the notion that inorganic cofactors are
essential for H/HS signaling, is the careful investigation of metal ion binding to modified Hs using modern CD and
NMR methods reported by Rudd et al., 1.

2.9-1 Servo Feedback Signaling via the Heparanome-Metallome could have Facilitated Animal
Evolution (Cf. also Section 6.3)
A system of post-synthetic editing and servo feedback control modulation is suggested by the data collected in Tables
I-III (vide infra pages 62-73) to be afforded by the metallome. This is suggested to be achieved by the requirements
for metal ions for HS-synthetic and HS-degradating enzymes and the facilitation by metal ions of HS binding to
target protein and also the catalysis of nonenzymic pathways of degradation of HS (e.g. by nitric oxide metabolites).
These pathways seems to point to a system of similar or greater complexity to that achieved by the previously
suggested servo feedback signaling processes which are believed to be afforded by H-degrading (Zn-dependent)
endosufatases [cf., Morimoto-Tomita et al.1].
The ability of the heparanome-metallome to interact with the geneome could suggest a mechanism by which alteration of
genes might be facilitated via the servo controlled system facilitated by HS in order to counterbalance sustained
environmental stresses. This provides a new working hypothesis for how animal evolution could occur**.

A system of biofeedback involving altered biosynthetic and postsynthetic alteration of HS microstructure, which
evidently can occur in response to various environmental inputs, is suggested by the data currently available

Such data includes the effects of inorganic cofactors have been reported to engage in direct metal ion dependent HS
segment-protein interactions cf., e.g., Kan et al. 1 cf., also Rudd et al.1, and similar reports listed in Table I and Table
II is a collection of reports which suggest that the presence of Na+, Ca2+, Mn2+, Zn2+ and Cu2+ ions can influence HS
synthesis and alter HS activity and Table III lists reports which suggests that various small organic molecules, which
might influence redox status, e.g., ascorbate, can signal for the upreguation of altered, e.g., more highly sulphated,
HS molecules.

2.9-2 Inorganic Cofactors for Nitrosative Structural Alteration of H/HS

Inorganic cofactors which can contribute to the nitrosative structural alterations of H/HS (e.g., as described by
Cappai et al.1, cf. Ding et al.1) include Cu1+/2+ and Zn2+ ions.

The range of inorganic cofactors which occur in pharmaceutical H and which could also occur in HS PG
in vivo include ions viz., Ag, Au, Ce and other rare earths, Fe, Hg, Mn, Mo, Ni, Os, Pd Pt, Rh, Ru, Sn,Tl, Ti, U, and Zn (cf.
Table IV and also Grant 1, 2000 internet dg2)
which should be tested as possible catalysts for augmenting nitric oxide metabolite reactivities.

2.9-3 The Selective Degradation of H/HS by Actions of Redox Metal Ions

This includes the older findings of Z. Liu and A.S. Perlin who showed 1c that Cu2+ caused selective free-radical degradation
of H and a related study by R.N. Rej et al 1d, which found that trace amounts (ca. 1/185 mol ratio with respect to H)
together reactive oxygen containing free-radicals generated from added H2O2, plus ascorbate, caused a marked reduction in
the anti-FactorXa activity of H without causing any detectable alteration in the NMR spectrum of the polysaccharide (i.e.
the proportion of sites affected was too small to
show up in NMR spectra).
The recent study by complementary SRCD, NMR, FTIR and EPR of the site-specific complexation of small amounts of Cu
and larger amounts by H reported by Rudd et al.1 extends the understanding of the possible role of Cu as a modulator of H
activity. The initial binding phase (15-20 Cu(II)/H molecule)
involved specific coordination to four binding sites: the carboxylic acid groups and the ring oxygen of Id2OS, the
glycosidic O behind this resiude and the adjacent glucosamine and the 6O-S group. A later binding phase showed little
structural specificity. The porcine mucosal H used for this study was obtained
from Celsus, USA [who in an internet document cite the Grant et al.1, 1987a work where a somewhat
overoptimistic assessment had unwittingly been given of the single counterion nature of standard medical grade H)

Nagasawa et al.1 reported that oxidative damage to H caused by actions of redox-active (e.g. iron) ions
were of a highly selective nature (e.g. causing preferential destruction of D-glucuoronate rather than L-iduronate anionic
groups).

2.9-4 How different HS polymers could selectively bind variants of FGF in vivo: hints from lipoprotein binding
studies
The divalent cations which facilitate FGF receptor assembly (Kan et al., 1 ,cf . Rudd et al.1)) by HS
seems to be a related phenomenon to how multivalent metal ions form ternary complexes with
polysaccharides and lipoproteins (the basis of laboratory blood lipoprotein evaluation methods, e.g., using
Mn2+ and Ca2+ ion plus sulphated polysaccharides (preferably H) a process which was discovered during
early researches into lipoprotein fractionation to allow the efficient precipitation of lipoproteins containing
phospholipids and thereby to further provide a quick assay for blood serum LDL/HDL (Cham 1, cf. also e.g.
Seidel et al. 1, Cornwell & Kruger1).

It is now tentatively proposed that similar metal ion modulated polysaccharide protein interaction could also be
relevant to the current discussions of the mechanisms by which different HS polymers selectively bind variants of
FGF in vivo (Kreuger et al. 1 cf., Powell et al.1).

Early researches had established that exogenous H released lipoprotein lipase (LPL) from (putatively HS at)
vascular surfaces (for which action the N-SO3- groups in H/HS were of relevance to the interaction between these
polysaccharide and the enzyme). {Later workers (Sivaram et al.1) established that endothelial cells synthesize a H-
releasable high affinity LPL-binding protein hrp(H-releasable protein}.

Sulphated polysaccharides including H were found to be useful reagents for the fractional precipitation determination of
cholesterol linked lipoproteins in human blood. For this purpose the formation of ternary complexes between the sulphated
proteins, multi-valent metal ions and the proteins was fount to be useful.

The required inorganic cofactors to enable correct selection of HS – protein interactions are putatively
provided by an in-built reservoir of inorganic cofactors which seems normally exist in juxtaposition to
H/HS chains. This is proposed to be analogous to the retention in costal cartilage by chondrotin-4 sulphate
of ca. 1Ca2+ /tetrasaccharide (cf., the purified chonroitin-4 sulphate chains were much less able to bind
Ca2+ than when attached to the core proteins as noted by Woodward & Davidson1) (this is now suggested to
be due to an enhanced water structure in the in the vicinity of the cartilage proteoglycans; such bound
water seems to augment the ability of polyanions to bind counterions).

2.10 Comparison of Different Multi-Inorganic-Element Matrices

The process by which the amounts of bound inorganic elements can be changed by pitting one ion exhange system agaisnt
another as in the percolation of a dispersion/solution of H through a man-made polystyrene sulphonate resin column in a
single counterion enriched form seems to be mimicked by natural ion exchange systems such as those formed of skeletal
calcifications and marine algal tissues which, together with the muti-element contents of Hs produced by different
manufacturers show relatedness between the inorganic element contents (e.g. in log-log plots as suggested by Fig 3
(page81)**.
Ca2+ is the most abundant multi-valent ion present in such a series of materials and is also is most easily exchanged (viz. the
exchange of Tl+ for Ca2+ occurred in H with an efficiency of 103 while Ce(III/IV) was exchanged by Tl+ (the least efficient
exchange) occurred at an efficiency of 0.5 (cf. Table IV)). The ‘residual’ Ca in each of the samples defines the ‘purity’ or
the degree of ion exchanged purification. This gives a simple, but apparently also a scientifically robust, method for
classifying various commercial Hs which have been purified to different extents starting apparently from a similar multi-
element containing crude tissue extract giving rise, following extensive purification (during which the least strongly held
countercations such as Mn and Mg are lost) to the sort of Na H listed in Table IV (arising from natural buffer systems
supporting multi-element solutions like biological fluids or uncontaminated seawater).
It is found that a single log-log relationship applies to the amounts of alkaline earths present in a range of Hs (reported by
Harrison & Sutton3b-2), the H samples reported by Moffat1 as the contents of the alkaline earths in calcified materials and
marine algae** (these data are plotted in Fig. 3 (page 81)). (It should be noted that the same method of classifying index
does not seem to apply to algal polysaccharides affected by highly polluted waters but it does apply to such substances
obtained from organisms grown in natural seawaters).

The results presented for what seems to be a unrefined Na H (Table IV) suggest that an apparent systematic enrichment of
trace and ultratrace multi- inorganic elements from some seawater(-like) animal tissue bathing fluid or perhaps brine used
during manufacture (which requires further research to confirm the relative importance of each route) similar to the
enrichment of seawater elements by marine algal polysaccharides.

3. Ca2+ signaling by the heparanome metallome

Metal ion dependent H/HS signaling – the special role of Ca2+ ions
The Long/Williamson hypothesis had suggested that
HS evolved to potentiate Ca2+ signaling
The results of studies relating to the nature of the conformation changes elicited by the
interaction of Ca2+ with H (Boyd et al.1, 1980) and the effect of Ca2+ on the biochemical
interactions and reactivity of H and related polysaccharides with proteins
(cf., list of publications given by Long, et al., 1, 2003) together with a comprehensive review
of the peer-reviewed literature had suggested some thirty years ago that the primarly
function of HS in multicellular animals might be to potentiate Ca2+ signaling activity
(Long & Williamson 1, 1979).
A NMR study of Ca2+ binding to H (Boyd et al. 1) showed this to be accompanied by a conformation change in H which suggested how the biological
activities of H and HS could be altered by Ca2+ binding. This ideas was in keeping with earlier X-ray crystallographic studies (Gould et al.1) which had
suggested that Ca2+ (and Na+) ions, by their unique abilities to form coordinate links between water molecules, uronate groups, and the sugar oxygens of
cell wall polysaccharides, could form functionally active organometallic supramolecular structures, e.g., of the “egg box” type, (cf., Grant et al.1 and Seale
et al.. 1). Similar HS polysaccharide-based organometallic supramolecular structures held together by the ability of polysacharides to modulate the binding
of metal ions to arrays of water molecules could also explain why HS proteoglycans were able to self -associate under appropriate conditions (cf., L.A.
Fransson & B. Havsamark1) and be present in basement membranes as double-tracked structures (Inoue et al. 1).

The presence of Ca2+ in biological fluids seems to allows HS to bind proteins in a Ca2+-dependent manner. Scott1 has pointed out that the self-association
of GAG chains, like DNA is dependent on a balance between attractive forces (e.g. hydophobic [this is essentially the asociated water structure] and
hydrogen bonding) and electrostatic repulsive forces, so that whereas hyaluronan and chondroitin 6-sulphate can self-associate, this is less easy for
chondroitin 4 sulphate and the more highly sulphated GAGs).

The neturalization of the anionic charges on such GAGs which can allow them to self-assemble,can however be accomplished by binding of multivalent
metal ions perhaps the most evident of which are Fe3+, Al3+ ** and also La3+, Ce3+ and Ce4+ (which are also redox active especially in acidic aqueous media,
(cf. Grant & Payne1 , Grant 1,1964).

While Ca2+ ions seem, as suggested by some recent studies (e.g. those listed in Table I) to more likely to
potentiate HS activities rather than vice versa, the Long-Williamson hypothesis agrees with the recent report
that instillation of H into rat lungs was associated with inceased expression of proteins involved in Ca2+
signaling (including annexin V, VI and S100A6).
[It should also be noted that espression of proteins involved in cytoskeletal control and for
immune function actions were also increased following the instillation of H].
The effect in mice of HS N sulphate knockout seems also to have caused a large alteration in
the potentiation by HS of Ca2+ kinetics required for lung skeletal muscular activity after birth
(Gabr et al.,1) discussed in 2.1-1 below).
3.1
In plants, anionic pectin derived oligosaccharides are thought to modulate Ca2+-dependent signaling
processes , analogously to how in 1979 1 Long & Williamson had suggested that the activity of Ca2+ ions in animal
1a-8

biochemistry could, in principle be subject to the regulation of its activity by the actions of HS.

3.2 Evidence from knockout mice (Jenniskens et al.1) suggests that HS N-SO3- groups potentiate HS-determined Ca2+
activities required for skeletal muscle function

Although the lung defect in newborn Nsdt-1 -/- mice which prevented them from breathing at birth might also have been
due to a lack of an effective lung surfactant/ hydration action, which can also be affected by a lack of HS N-sulphation in
lung surface HS PGs) (cf. sections 2.1-1) Jenniskens et al.1 found a disturbance of Ca2+ kinetics caused by the genetic
deletion of N-deacetylase/Nsulfotransferase-1 (Ndst-1 -/-) which reduces the amount of HS N-sulphation and therefore also
affects HS related Ca2+ binding capacity which can be suggested to be the origicn of the observed inhibition of Ca2+ kinetic
processes required for lung skeletal muscle myotube action (which these authors also studied separately in Ndst-1 -/-
cultured myotubes).

3.3 H/HS and Control of Calcification

H was found to peptize hydroxyapatite (used as a column packing); the colloidal H-mineral associated

particles obtained showed infrared spectra with superposed vibrations from both the mineral and the

polysaccharide components but which showed a notable displacement of the S=Oas vibrations of H which

indicated some degree of covalent interaction**.

The mineral, apatite is (like H) a multi-inorganic ion substituted calcium phosphate (cf., Van Wazer 1, 1964)

also present, in a sub-crystalline form in bone, the formation of which is now known to depend on the

presence at growth sites of sulphated polysaccharide morphogenic agents (cf. Wise et al.1) which could

suggest that the secondary inorganic ionic constituents associated with such H-like polysaccharides could

enable a less than total crystalline ordered but optimized nanostructured solid matrix structure to be formed

during nascent bone formation. It seems possible that the transfer of such secondary ions from HS could

facilitate this. A similar polysaccharide-dependent process seems also to permit functionally optimized

marine bivalve exoskeletal nanostructures to be formed (cf. 17).

The presence of phosphate moieties attached to H/HS is expected to augment the binding strength for

redox active metal ions thereby conferring on such H an added antioxidant and pro-ferrioxidase potency.
It should be noted that H- like polysaccharides can also bind to mineral surface in a similar manner to
oligomers of phosphorus oxy-acids, evidently reflecting an analogous inorganic binding property for both
phosphorus and sulphur oxy-anion based systems (cf., Grant et al., 1,1989). By comparing diphosphate,
triphosphate and a commonly used anti-scale phosphonate (ethane,1-hydroxyl,1,1-diphosphonate (EHDP)
(cf., Grant 1,1979) with a range of anionic sulphate half-ester substituted polysaccharides (which included
sulphated cellulose, carrageenan, H and HS ) it was established that these polyanion systems act
as efficient inorganic morphogens by modulating templates for crystal growth so as to be
able to stongly modulate or inhibit crystallization (cf., Grant et al., 1979 for the modulation of CaCO3
crystallization) perhaps analogous in some respects to how the key roles played enable ATP/ADP via Mg2+
complex formation are enabled by water structure induction (cf. Wiggins1).

3.3-1 N-SO3- is involved in Ca2+ binding by H/HS

The neonatal mortality of Ndst-1 -/- mice can then at least partly be attributed to a lack of an effective lung muscular action
produced by the inability of Ca2+ ions to interact correctly with N sulphate groups in HS in myotubes.
This mechanism aggrees with in vitro studies which had indicated that in the absence in H of H - N-sulphate groups, the
binding of Ca2+ to H is greatly diminished (cf. Liang et al.1, Ayotte & Perlin1).
A similar requirement for H-N-sulphate groups seem also to underpin the ability of HS to act as a potent
inhibitor of calcification, a phenomenon of relevance to the etiology of atherosclerosis (cf. Grant et al., 1 1992).

3.4 Roles of Inorganic Borate, Silicate and Phosphate Attached to H/HS

It is suggested that the co-occurrence of multiple metal ions with H (and perhaps also with HS) further
enables the binding of this highly anionic polysaccharide to other anionic entities such as inorganic
sulphate, borate, silicate and phosphate, a phenomonon which may be of considerable relevance to
heparanome signaling and therefore animal physiology and biochemistry since H-like molecules are
believed to be present as key morphogens of tissue assembly and bone formation (cf. Wise et al.1) a process
which also depends on the presence of inorganic Si (SiO2?) associated with HS**; a related phenomenon
is the inhibition of urinary stone formation and
arterial calcification (cf. Grant et al.,1,1992).

It has been suggested that the silicon-rich precipitates (which show elemental analysis and vibrational spectra suggestive of
a multi-element containing amorphous silicate-glycosaminoglycan complex) which are formed in the urinary system of
male rats is of critical relevance for the induction of cancer which is also associated with high dietary saccharin intake
(Cohen et al. 1). This seems analogous to the abilities of some forms of silicate minerals to induce cancer and indeed the
association of cancererous tissues with other solid phases (cf. Grant et al.,1, 1992a,b) as well as the induction of cellular
proliferation by certain forms of calcium phosphates.

HS putatively could perform polyelectrolyte functions similarly to how H, like polyphosphates acts a peptizing
agents for the stabilization of colloidal-sized mineral particles, including sparingly soluble carbonates and
phosphates. [In biological fluids these particles would also be expected also to contain phospholipids].

Natural polyuronic acid rich (metal-ion binding) mucilages from marine microorganisms are apparently being
evaluted as potential industrial surfactant and emulsifying agents
(Guierrez et al. 1) suggesting that similar polysaccharides in animal tissues may also perform such functions in
vivo.

3.4-2 Polyoxymetalates and biologically relevant catalytic activity

There are possible roles of anionic polysaccharides in the assembly of polyoxymetalate-polysaccharides having primitive
enzymic activities (cf., Rajeswari et al. 1).
The ability of H to bind both cations and anions of a range of inorganic salts also reported in a Patent
specification (Fo-We1) which noted that H bound “hydroxides of certain metals or with certain inorganic
salts to form water-soluble compositions”.

Such ability of H to bind to inorganic salts is in broad agreement with the author’s experience.

The ability of H to act as a superoxide dismutatse and ferroxidase could possibly be dependent on the presence of sufficient
amounts of multi-inorganic elements attached to the H**.

This could suggest that during the early evolution of animals primitive enzymic activities could have been achieved by the
formation of metallomic-polysaccharide assemblages**.

3.4-3 H/HS & Metalloproteinases

(These are dependent on Zn2+-related activity).

The activity of metalloproteinases (MMPs) is implicated in H/HS PG biochemistry including as a method of controlled release of soluble
HS PGs.

The proteolytic activity of MMPs depends on the Zn2+-containing active site.

The activity of MMPs could also be subject to modulation by the presence of additional metal ions. MMP-2 and MMP-9 activities can
apparently be inhibited by the presence of additional Zn2+ (and also, but less effectively by Cu, Sn and Hg ions) (de Souzo et al.1, Santos
et al.1).

Nitric oxide also apparently can directly influence the reactivity of MMPs (McCarthy 1) (putatively via interaction with the Zn-
site) and elsewhere so as to either augment or inhibit MMP activity. Reactive thiols (e.g. in glutathione) also seem to have
inhibitory activity for MMPs putatively elicited via interactions with the active Zn2+ containing site (Pei et al. 1).

3.4-3-1 Modulation of Metalloproteinase activity by arsenic suggests a possible therapeutic

application of As2O3- H/HS as an anti-cancer agent)
Arsenic (in the form of As2O3) has been reported (cf., Park et al. 1, 2005 ) to modulate MMP activity and suppress tumor invasiveness by this
mechanism.
This suggested that dietary insufficiency of As might be a contributory factor to carcinogenesis.

Arsenic is consistently found to co-purify with polysaccharides (including H) which seem to sequester this element present natural water
and biological bathing fluids.
A possible role of As modulation of MMP determined ECM degradation suggests that this element may play a normal role in the
biochemistry of the heparanome.

3.4-4 6-O-Endosulfatase (Sulf) Action in ‘Smart’ HS Microstructure Modulation

(Cf. Ai et al.1 Remodelling of HS sulphation by extracellular enodosulfatases
[Qsulf: quail sulf; Hsulf human Sulf]
Sulfs have unique 6-0-endosulfatase activity, different from lysosomal 6-O-exosulfatase. Sulfs
differ from lysosomal sulfatases by their endolytic mode of action during post biosynthetic
modification of HS chains.
Qsulf-1 preferentially release 6-O-sulphate groups from IdoA2S-GlcNS6S units, showing lesser
activity for
GlcA-GlcNS6S units and no activity for IdoA-GlcNS6S sequences or 6OSGlcNAc or GlcNS residues
within the alternating NS/NS domains. In mouse embryos Sulf-1 is expressed dynamically in
somatic muscle and motor neuron progenitors. Sulf 2 has overlapping localized pattern of
expression in neuronal and mesodermal tissues.
Sulf 1 and Sulf 2 are also expresssed in a diversity of adult tissues.
Qsulf-1 shares a conserved cysteine with other Sulfs involved in postranslational modification to
form a formylglycine derivative required for catlyatic acitivation of all sulfatases (if this cysteine is
mutated the sulfatase acitivity is inactivated). [Sulfs are positive regulators of Wnt signaling. (It
was noted that considring Wnt proteins are posttranslationally lipid-modified, it is intriguing that
HS PGs control Wnt signaling)].
QSulf-1 expresion had been shown to modify the 6-O-sulphation state of HS of expessing cells but
not neighbouring cells as assayed by loss of cell surace 10E4 epitope espression. However it has
been reported that Hsulf 1 and 2 in part are excreted into the media i.e. they likely modify HS on
neighbouring cells. Hsulf-1 also acts on Heparin.
The inhibitory effect of Qsulf-1 on FGF signaling is dependent on catalytic sulfatase activity as
over-expression of catalytically inactive Qsulf-1 (with conserved cysteine mutated to alanine) is
eneffective for FGF signaling. {Cell surface HS that has been 6-O-desulfated by Qsulf-1 will still
bind FGF2 through 2-sulphate groups but cannot bind the receptor which requires both 2-O and 6-
O sulphate groups}.
HSulf-1 is differentially expressed in different human tumours suggesting that Sulfs have diverse
roles in tumorogenesis.
Hsulf-1 can act as a tumour suppressor.
Expression of HSulf-1 in cancer cell lines seems to down-regulate the FGF-dependent activation of
extracellular signal regulating kinase (ERK). Although ca.50% of pancreatic carcinomas have
increased HSulf-1 mRNA, a majority of ovarian hepatocellular head and neck squamous
carcinomas showed down-regulation of Hsulf-1 mRNA expression (and exhibited enhanced
proliferation and mobility in response to FGF and HGF; cell lines from these kinds of cancers show
resistance to staurosporin and cisplatin induced apoptosis).
Sulf-1 structurally modified heparin inhibits FGF-induced angiogenesis (in vitro) suggesting such
heparin has anti-cancer activity.
G.K. Dhoot et al. Science, 2001, 293, 1663-1666 who drew attention to the how developmental biology was dependent upon not –direct-
genetically-controlled extracellular post synthetic alteration of HS microstructures by actions of 6-O-endosulphatase).

Sulfatase (Sulf) activity putatively involves EDITING of HS information and associated cellular feedback loops
(Extracellular Postsynthetic Sulphatase Activities can systematically alter HS microstructures)
Sulfatese activies including 6-O- endosufatase action are putatively involved in cellular servo-feedback
control mechanisms for regulation of cellular signaling via postsynthetic editing of the sulphation patterns feeding back to the
altered expression of HS biosynthetic enzymes.
Such altered sulphation of HS chains will cause altered Ca2+ binding other inorganic binding potential and altered water
structure which is highly dependent on the presence in H of O-SO3- groups.

Sulfatase 2 (Sulf-2) up-regulates glypican 3 thereby promoting FGF and Akt activity and acts as a tumour promoter but sulfatase 1 (Sulf-
1) also an endosulfatase functions as a tumour suppressor (cf. Lai et al.1)

3.4-5
H/HS & kallikrein: a putative example of HS-determined water structure/activity-related effects

That the activity of kallikreins (K) are known to be affected by anionic Hofmeister effects (Angelo et al.1 ) implicates, in the ultimate
modus operandi of Ks, a central role for water structure elicited by the presence of ions incuding those assoicated with GAG surfaces.
(H/HS and other GAGs are knwn to modulate kallikrein activities (cf., Gozzo et al,1)).
(Human kallikrein-6 (hK6) is reported to be highly expressed in the central nervous system and be implicated in the etiology of
Alzheimer’s disease and in other demyleinating disease processes; kallikrein activites are also believed to be centally implicated in the
etiology of cancer [cf. e.g., Schmitt et al. 1]).

3.4-6 The use of barium acetate buffer for the electrophetic separation via
selective binding of Ba2+.
[H was reported to contain e.g. 120 different types of individual H molecules (Nader et al. 1,1981)].
It seems to have been assumed that individual molecular heterogeneity is less for HS but perhaps this notion
needs to be re-evaluated.
[It might tentatively be suggested that the existence of heterogeneity could be as part of some evolved fuzzy
logic systemic signaling and computation process employed by animal biology; further research is required to
test this hypothesis].

3.5 H/HS & Mineralization (Inorganic Biochemistry-Related Systems)

3.5-1 HS Signaling (Thyroid Hormone Dependent) in Skeletal Growth & Mineralization –[Inorganic Biochemistry
Related Systems]
Signaling by HS and HS-like molecules (mast cell H) is believed to be involved in the control of the growth and
mineralization of bone (cf., Wise et al.1).

Thyroid hormone has been indicated to negatively regulate HS biosyntheis at the growth plate and is apparently involved in
a feedback loop which includes PTHrP, Ihh and FGF signaling (Bassett et al., 1)

3.5-2 Plaque formation & H/HS

H/HS exhibits a general ability to inhibit the formation of pathological plaques composed of sparingly soluble mineral-rich
phases as well as those formed from sparingly soluble (fibril) proteins. This phenomenon could be, at least in part, why
such H-like drugs as pentosan polysulfate (PPS) are uniquely useful therapeutic agents for the inhibition of prion diseases
and putatively also for other amyloidoses including Alzheimer’s disease
(cf., ref. 1A-5).

3.5-2-1 Phospholipids and H/HS seem especially to interlink by way of metal ion crosslinks
(E.g. –P(O)2-O-Ca2+-O-S(O)2-N-H [cf., Burnstein et al. 1]). This was the traditional model for lipid binding to HS-like
molecules at blood vessels which had suggested a key role for glucosamine N-SO3- groups in this process and further
indicated that defects in HS (if such groups were less abundant) could suggest a mechanism of atherosclerosis. A
related in vivo role of GlcNSO3- containing polysaccharides for the inhibition of pathological calcification (n.b. de-N-
sulphonated H can promote calcification**) could also suggest a role for de N-sulphonation of H (which apparently
can be achieved nonenzynically by iron ions**) in the occurrence of this disease in an age-determined manner (cf.
Feyzi et al.1 proposed a related mechanism of age-dependent alteration of arterial surface HS which augments the
binding of pro-artheroma amines to the form of HS which is augmented with increased age); an increase in serum
ferritin with age (Jarrett et al., 1) suggests an alterantive mechanism by which unliganded iron might remove anti-
calcification HS N-SO3- groups in this disease. (Iron ion including oligomeric iron multi-chanrged ion seem to
promote the formation of various kinds of plaques and ultimately roles of H/HS as tissue protectors to hinge on an
ability to inhibit such actions of poly-iron moieties**).

There is some support for this idea in the findings that arterial calification can be inhibited or even removed by the
exogenous application in an experimental primate model of bisphosphonate anti-calcification agents (Levy et al.,1)
(which have shown in in vitro experiments (Grant et al., 1,1989) equal potency to H/HS for this activity).

3.5-2-2 Polysaccharide Metal Ion Dependent Aggregation

In plants, anionic pectin derived oligosaccharides are thought to modulate Ca2+-dependent signaling processes 1a-8,
analogously to how in 1979 1 Long & Williamson (working at Aberdeen University, U.K.) had suggested that the activity of
Ca2+ ions in animal biochemistry could, in principle be subject to the regulation of its activity by the actions of HS. A NMR
study of Ca2+ binding to H (Boyd et al. 1) showed this to be accompanied by a conformation change in H which suggested
how the biological activities of H and HS could be altered by Ca2+ binding. This ideas was in keeping with earlier X-ray
crystallographic studies (Gould et al.1) which had suggested that Ca2+ (and Na+) ions, by their unique abilities to form
coordinate links between water molecules, uronate groups, and the sugar oxygens of cell wall polysaccharides, could form
functionally active organometallic supramolecular structures, e.g., of the “egg
box” type, (cf., Grant et al.1 and Seale et al.. 1). Similar HS polysaccharide-based organmetallic supramolecular structures
held together by the ability of polysacharides to modulate the binding of metal ions to arrays of water molecules could also
explain why HS proteoglycans were able to self -associate under appropriate conditions (cf., L.A. Fransson & B.
Havsamark1) and be present in basement membranes as double-tracked structures (Inoue et al. 1). The presence of Ca2+ in
biological fluids seems to allows HS to bind proteins in a Ca2+-dependent manner. Scott1 has pointed out that the self-
association of GAG chains, like DNA is dependent on a balance between attractive forces (e.g. hydophobic [this is
essentially the asociated water structure] and hydrogen bonding) and electrostatic repulsive forces, so that whereas
hyaluronan and chondroitin 6-sulphate can self-associate, this is less easy for chondroitin 4 sulphate and the more highly
sulphated GAGs). The neutralization of the anionic charges on such GAGs which can allow them to self-assemble,can
howeverbe acocmplished by binding of multivalent metal ions perhaps the most evident of which are Fe3+, Al3+ ** and also
La3+, Ce3+ and Ce4+ (which are also redox active especially in acidic aqueous media, (cf. Grant & Payne1 , Grant 1,1964).

3.5-2-3 Specific nucleation activities (e.g. afforded by natural ‘SiO2’ nanoparticles) may be
required for supramolecular structure formation in polysaccharides and be relevant to how
inorganic elements can contribute to H/HS signaling
The high Si content in both human hair and H and the well-known association of Si with
glycosaminoglycans3b-6 is in accord with the likely importance of this element for mammalian biochemistry.
Although Si is known to be an essential animal nutrient and be critically involved with glycosaminoglycans, especially with
H/HS, the mechanisms of binding and physiological action are unknown.
Si could be the key element linking geological and biological “metallomics”, suggesting the need for
a wider “geoglyco-metallome” conceptualization by which the inorganic “metallome” influences the heparanome, operating
in a manner receptive to inorganic matrices via cooperative interactions involving ultra-anionic polysaccharide side chains
for which an absolute requirement for specific metal ions e.g. Ca, Zn and Cu have already been established for several such
processes. An analogous role for Si might be anticipated.

The association of metal ions and inorganic moieties with anionic polysaccharides seemed more to resemble a phase change
process than, e.g., an electrostatic controlled counterion condensation process (the Aberdeen polysaccharide laboratory
studies of metal ion binding to H reported by Grant et al. 6b-2-2 suggest this hypothesis). Such a phase change would be
subject, according to the general principles governing the formation of phases in physical chemistry, will occur via a
nucleation-seed-sensitive mechanism (e.g, facilitated by suitable inorganic or organometallic nanoparticles) .

The actual chemical nature of the putative nucleation seed particles in use by biological systems are currently unknown. A
possible silica particle role in this function, however, seems credible. Si-containing particles (putatively amorphous SiO2
sols similar to those discussed by Grant et al. 6b-2-2) seem always to be a characteristic component of anionic polysaccharides
(Schwarz6b-1). Their presence in such an association to fulfill a nucleation of phase change role might account at least in part
for the fact that Si is an essential nutrient for animals and other species.

3.5-2-4 Polysaccharide Inorganic-Ion-Dependent Gelation & Melting

Preliminary skeches copied from original diagrams (kept by author in archival documents retained from Marischal College)




Cations studied K+, Cs+, Na+ and NH4+

3.5-3
Binding of Anionic Polysaccharides to Crystal & Other Inorganic Surfaces

A major additional potential signaling and information processing ability of HS is suggested to be afforded by
the specific interactions between HS epitopes and specific kinds of inorganic surfaces. Binding of HS to
colloidal inorganic particles (e.g. to crystal seeds which might otherwise cause pathological crystallization) can
apparently occur in a functionally relevant way; this seems to have been established for the specific crystal
surfaces of calcium oxalate appear to induce (evidently by some form of biofeedback) an alteration in the kidney
biosynthesis of HS so as to augment the specific anti-Ca oxalate crystallization abilities8 of HS microstructures
which are subsequently produced. The results presented in ref. 8 also suggest (although less clearly) that
hydroxyapatite crystals are also similarly capable of prompting for a similar specific alteration of HS
microstructures capable of inhibiting by surface adsorption and deactivation of potentially damaging seed
crystals of this composition.

Similar specific interactions between crystal surfaces and alginates were studied by the Aberdeen polysaccharide
group; a range of protein-free alginates polysaccharides of different defined microstructures appeared to be
adsorbed onto inorganic seed crystals and caused an inhibition of crystallisation; the relative degree of inhibition
showed a mathematical relationship between the crystal growth rate constants (which could be accurately
determined) and the detailed “information encoded” known microstructures of these polysaccharides9.
Essentially this is an in vitro demonstration of how information in polysaccharides with complex microstructure
can be directly “read-off” by an inorganic microstructure.
Calcium salt crystallization rates were indicated from similar studies of the kinetics of crystal growth in their
presence to be similarly dependent on H/HS microstructure (n.b. the similar binding of HS fragments to
sparingly soluble calcium salt seeds is thought to be relevant to the mechanism of control of both urinary and
cardiovascular calcification9b and also to the age-associated human cardiovascular dysfunction in which
(programmed?) changes in blood vessel wall HS microstructures are believed to contribute to1a-5. By an
extension of this hypothesis, both normal ageing and degenerative diseases in animals could have an ultimate
link to age-related changes in HS sugar sequences which may include an age-dependent alteration in the
suggested inorganic cofactor effects).

3.5-3-1
Relevance of Control of Inorganic Crystal Morphology to Evolution of Precursors of Life

Lima de Faria has suggested10 that the study at the most fundamental level of the seeded crystallization

of specific complex crystalline forms of CaCO3 can gives insight into how biology could have arisen

without the help of DNA-based genes10 since the numerous forms of CaCO3 are seemingly capable of

reproduction in the manner often assumed to be restricted to biological cells and assumed to be a unique

property of nucleic acid-determined genetics. This further suggests how the occurrence of a biological-

cell-like ‘reproduction’ of ‘species’ of particles similar to specific kinds of CaCO3 particles discussed by

Lima de Faria, could have been capable of Darwinian evolution into more complex true biological

forms**. The ability of polysaccharides to bind strongly to such CaCO3 surfaces and thereby modulate

their abilities to replicate, could have had pre-biotic relevance since it seems more likely that

polysaccharide-like molecules than other present day types of biopolymers could have spontaneously arisen

(e.g. by the oligomerisation and rearrangement of formaldehyde polymers) (cf. Grant1 1992a).

A corollary to this idea is that information held in specific (e.g. anionic) sugar sequences in H/HS is

capable of being modulated by inorganic surfaces or membranes to which the inorganic elements which also

occur (albeit mostly in very small amounts) in biological fluids and tissues as well as in seawater4.

(These will include single ions, ion pairs and aggregates which are potential crystal growth nuclei (n.b.

physiological solutions are supersaturated as regards the formation of solid phases of sparingly soluble Ca
salts e.g. CaCO3 (calcite), apatite etc. Apatites characteristically are, similarly to H, non-stoicheiometric

salts which contain a range of isomorphously substituted inorganic ions (cf. Van Wazer 1, 1964).

Colloidal complexes of hydroxyapatite - H can also be prepared in the laboratory1h ** . Such entities

putatively have biological significance as have colloidal SiO2 particles (cf. Grant 1, 1992a)

Purely inorganic systems including silicic acids (cf. Grant et al.,1, 1992b) can engage in self-assembly of

complex forms which, superficially at least resemble living organisms (cf. Colfen & Mann1)

(the form of seeding by such sub crystalline particles also resembles biological reproduction (cf., Hyde et

al.,1).

3.5-4 How information in anionic polysaccharides can be ‘read-off’ by crystal structures.

A method of discrimination between differently microstructured alginates and a general method for the
elucidation of the structural heterogeneity of alginates (as well as, in principle, H and HS) is enabled by the
measurement of BaSO4 seeded crystallization rates in the presence of a subject polysacchride {the second order
crystal growth rate constants are found to vary according to the ability of different microstructures with in
anionic polysaccharides to selective bind to the crystal growth sites (it being found that the second order rate
constants of seeded growth ot BaSO4 crystals vary as a direct function of the microstructure of a series of
alginates determined by other methods8a)}.
These experiments show, in a general fashion, how information held in sequences of anionic polysaccharides can
be be ‘read-off’ by crystal surfaces and, in principle, ‘translated’ into morphogenesis.

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

4 Water & Life

4.1 Water Chemistry & Compensations Between Allowable Entropy and Energy Changes
Life and its evolution from non-living system could depend on a critical supporting driving force enabled by
commonly found (cf. Leffler1) (but not of known cause) compensation between allowable entropy and energy changes
which is characteristic of “water chemistry”.

Compensation of entropy and enthalpy in water chemistry which can be suggested to arise from an engendered perturbation
of the laws of thermodynamics under certain at present obscure physical and chemical conditions which encourage more
complex systems to form spontaneously over the course of time and which may have contributed to the self-assembly of
complex (inorganic) quasi biological assemblies (e.g., of the kind suggested by Cairns-Smith, cf. Grant 1, 1992a)) which later
evolved into living organisms.

The nucleation of an order-disorder transition in κ carrageenan occurs at rates repoted byAusten et al, cf., Williamson1
which were reported by apparently restricted by the compensation effect suggesting a role of water structure regulation in
this nucleation process.
4.2 HOW LIFE DEPENDS ON THE CHEMISTRY OF WATER
A characteristic feature of all biology is its acknowledged essential dependence on the presence of liquid water which
is, by far, the most abundant molecular component of cells, and which, it can also logically be argued, is a central
pre-requisite for setting up the physical and chemical conditions required
for the prebiotic events which preceded life. This form of liquid water seems likely not, however, to have been the
pure chemical substance, but rather some form ‘muddy water ’, a multi-inorganic ion and trace organic molecule
(including humic polymer) containing aqueous solution somewhat similar in complexity to present day natural
waters most closely exemplified by seawater; this idea is supported buy recent observations which suggest that
aliphatic poly carboxylated polymers approximately similar in stoicheometry to humic polymers apprently are
present in comets [cf. report by Sanford et al., cf. Wickramasingh et al. 1] ) which can be suggested to engenders the
formation of microdomains of hydrogen-bonded water clusters within coacervates allowing functional separation of
the different density and reactivity water domains needed, according to Wiggins1, to initiate life.
4.2-1 Possible Key Role of Wiggins-Water-Structure
Wiggins1 has (on the basis of her groundbraking polysaccharide and silica gel surface studies) proposed the existence
in liquid water of two types of water structure with low and high density (with long and short hydrogen bonds
similar to those in related structural forms of ice). She has argued that this is the ultimate driving force of the
evolution of early living organisms is the water-in (e.g. silica) pore system which allows the separation of L- over D-
amino acids, the separation of Na+ / Ca2+ from K+ / Mg2+, for providing energy sinks for condensed phosphate P(O)-
O-P(O) bond and P(O)-O- interconversions and later on determining how proteins fold (Wiggins1 , cf. also , Luck1,
Stillinger1, Bernal1, and Chaplin1).
A critical aspect of associated water structure related phase change processes seems also to underlie the observed phenomena which
require separation of two forms of water in liquid water which are induces by surfaces and solutes and seem to allow a system of
hysteresis in distribution of thermodynamically determined amounts of the two types of water which a different phenomenon from a
conventional multi-phase system of liquid water. The Wiggins1 system of two high and low density hydrogen bonded phases is
subject to the modulation of the detailed interchange between the two phases following the additional presence of solutes of which
the sugars, polysaccharides and inorganic ions as well, perhaps, as micro gas bubbles thought by others to create long range water
structure, seem to have an especially strong influence. It seems possible that the generation of bio-friendly Wiggins supramolecular
structures in liquid water is the ultimate pre-requisite for life but a further essential factor which could have led to the creation of a
stable sytem of seawater multi-inorganic element aqaueous system is porposed to have engendered by prebiotic polymers resembling
the (formose reaction product) anionic humate polymers which evidently formed first of all from primordeal ingredients (e.g. similar
to those which Wickramasinghe1 has suggested to occur in intergalactic space and recently found in meteorites).

4.2-1-1 Inorganic ions and particles associated with H/HS surfaces may influence the Wiggins low density/high density
water microstructure balance.

Biochemistry which could ultimately depend on the behavior of liquid water determined by multi-hydrogen bonded

liquid water aggregate systems (e.g. Wiggins1) which is further subject to modulation of its structure by the present of

solute molecules of which inorganic ions are known to play dominant roles. The ultra ionic nature of H/HS therefore

could be of especial relevance to uniquely alter water structure towards that required for the evolution of the precursors

of mutlicellular animal organisms. The close association of both inorganic ions and clusters of water molecules with

the surfaces of highly anionic polymeric systems (Grant et al.1(1990)) will determine the activity of H/HS surface water

systems.
4.3 Role of water structure in polysaccharide biochemistry
Possible role of nucleation of associated water & hydrate phase/conformation change in the metallome-heparanome

If it turns out that DNA, RNA and HS actions are ultimately dependent on the physics of the not-completely-
understood phase change processes (which can display hysteresis and the formation and transformation thereof are
subject to inhibition and promotion effects elicited by chemical and physical systems which are capable their
nucleation) then the provision of nucleation potencies by inorganic complexes of polysaccharide seems especially
relevant to such discussions which include the discussions published in a monograph by Lima-de Faria10 which
denotes the relevance of seeded nucleation phase change processes to the ultimate definition of life. Nucleation
events are known to determine the formation of a wide range of possible forms of insoluble CaCO3 phases, which
can, it seems, be assembled by actions which closely mimic biological reproduction, a scenario which seems to
downgrade the presumed status of DNA as an essential ingredient for life. Lima-de-Faria10 (n.b., a renowned
nucleic acid biochemist) suggested that the presence of DNA per-se is not anything to do with the ultimate nature
of life which perhaps arose by similar processes to those which determine inorganic crystal morphogenesis. Indeed
there seems to be some kind of similararity between patterns adopted by various living organisms and mineral
aggregates. Both inorganic and multicelllar biological systems can surprisingly generate the same kind of complex
shapes. A fuller understanding of this, he argues, would allow for a more rigorous definition of the nature of
biology. A non-biological inorganic system (e.g. based on the silicon biochemistry of clay minerals, cf. Cairns
Smith1, or semi-amorphous silica sols cf., Grant et al., 1992a) can rationally be proposed to have further evolved
into carbon and DNA based life forms (Experimental evidence of this process has been obtained by the author
whilst studying the mechanism of seeded growth of commercial silica sols) and briefly referred to in the Medial
Hypothesis article). This system, it may now further be suggested benefits from the detailed kind of
supramolecular stuctures generated in multi-inorganic ion solutions similar to seawater.
This, it is now further suggested, is somehow interlinked with why water structuring is
intimately intertwined with the chemistry of polyol molecules which include, inter alia,
polysilicates, humic polymers and polysaccharides and their adducts which seem to be able to
form a highly stable multi-inorganic element composition, stable-over-geological-time
homeostatic buffer system for seawater-like solutions.

It should be noted that H/HS and other anionic polysaccharides have the ability to directly translate the information
held in their linear anionic sulphate half ester, N-sulphonate and uronic acid patterns plus bound inorganic cofactors
into inorganic morphologies via the the perturbation of seeded/ nucleated processes (cf. Grant et al., 1989,1992, 8a ). A
similar interaction between polysaccharide-like substances and inorganic self-assembling nanostructures (e.g. silica
sols) could have been associated with the early evolution of biota (cf., Iler1); polysaccharides are now known to
currently perform similar functions in multicelluar animals and other classes of biological systems, and putatively
have done so from the time of the first organisms. Such functions may, however, have been performed, especially
in the more primitive organisms, in tandem with primary nutrient gathering and water structure forming functions.

4.4 GENERAL WATER STRUCTURING EFFECTS OF POLYSACCHARIDES

Mucilage structure adhesion forces

Extracellular Slimes & Role of Water Structure in Biochemistry
The general control of water activities, osmoregulation, nutritent gathering, attachment to surfaces and protection against
pathogenic organisms and toxins (including toxic metals) as well as cellular recognition seem to be major functions of
glycans abundantly present in prokaryotic extracellular slimes and capsules.
[which are currently of interest, e.g. for their multi-metal ion binding and surfactant properties, cf., Gutierrez et al. 1].

4.5 Bacterial Mucilages

Common soil bacterial species18 can produce large amounts of highly hydrated polysaccharide-rich extracellular matrices and are used for
functions similar to those which have also been proposed for the animal extracellular HS proteoglycans viz. the potentiation of
morphogenesis, differentiation, adhesion and the facilitiatation of cellular movement. Bacterial polysaccharide-rich slimes can also, it is
believed, bind and protect the cells from heavy metals and protect them against a range of bacterial and viral infections, against the
phagocytosis of toxins and against dehydration. This suggests that polysaccharides throughout biota including HS proteoglycans in
animals, provide similar primitive functions. (cf. 18) from which the more complex functions which are apparently orchestrated by the
heparanome-metallome, have evolved.
Possible Evidence that (Extracellular) HS Polysaccharides Support Biological Water Activity
For aquatic invertebrates, the exact mathematical relationship which has been demonstrated (Nader1, 1983; 5) between tissue (GAGs)
including HS and the salinity of habitats of invertebrates suggests that HS proteoglycans directly exert osmoregulation / water activity at
those animal cell surfaces which are exposed to the habitat salinities.

Do Normal HS Actions Ultimately Depend on the Modulation of

Water Structures to Achieve Selective Protein Binding and Directed
Refolding?

Previously believed to be an intrisic property of proteins (determined by hydrophobic/hydrophilic nature of amino acids) protein folding
has been suggested cf. Wiggins1, Wilse Robinson1 Chaplin1) more likely to be controlled by the supramolecular structure of water layers
ajacent to protein surfaces. This also subject modulation by Hofmeister effects (e.g. elicited by inorganic ions {cf. Luck1} such as the
metallomic arrays).
It seem likely that this water structure is modulated by glycosylation of proteins as well (in a servo-controllable manner) by the non-
convalent association of proteins with polysaccharides including GAGs and for multicellular animals in a high evolved manner by the
heparnome.

4.6 Evidence for role of water adhesion forces in the anticoagulation mechanism of H
and in the binding of poly-L-lysine and poly-L-arginine to H

Pedersen & Jorgensen1 found that rate constants of the rate determining reactions responsible the antithrombin binding to H
varied between LiCl, NaCl and KCl solutions used as reaction media. These rate constants were also directly proportional
to the vibrational energies (reported by Grant et
al. 1(1984)) for the water molecules present in these salt forms of H (where they are believed to be preferentially bound to H-
SO3-. This suggests that the different vibrational energy properteis of such –SO3- bound water molecules infuence the rates
of interaction of H with antithrombin.

The NMR chemical shifts of C atoms adjacent to the glucosamine 3-OSO3- (hypersulphated) groups in the
centre of the active antithrombin-binding H pentasaccharide sequences were also found by to be selectively
perturbed by these and other inorganic counterions to a much greater extent than was apparent for the non-
antithrombin binding disaccharide repeat units in H which lacked such 3-OSO3- groups Grant et al.1(1986;1987).
This points to the existence of an NMR-detectable influence of metal ions on the activity of anionic groups in H.
Sulphate half-ester linked water molecules attached to the anionic groups rather than the naked H-SO3- groups per se could
determine at least the initial stages of protein binding and general biochemical activities of H/HS. This notion was
confirmed by experiments in which H binding to poly L-arginine and poly L-lysine was studied in an hydrophobic medium
(Grant et al. (1991)) where a marked displacement of the infrared overtone bands of such water molecules was observed.

The sulphate half ester linked water aggregates present at H surfaces can apparently act as effective ‘smart’

glue-like agents to promote H binding to poly-L-lysine and poly-L-arginine.

The ability of exogenously appied H, HS or Des to bind to endothelial glycocalyx (and alter is ability to bind
leukcocytes) (cf., Constantinesu et al., 1) may similarly be dependent on the adhesivenss of metal ion associated
sulphated polysaccharide hydration layers.
4.7 Other evidence for biologically relevant polysaccharide determined water structure adhesion forces

The adhesion of animal cells to polystyrene culture dish surfaces seemed to be dependent on a
presence of the correct type of surface-water structure generated by arrays of surface C-OH
groups or, alternatively by adsorbed H-like molecule adducts which shows up as discriminated
overtone water structures at suitably chemically modfied polystyrene surfaces
(Grant et al.,1(1988a)).

The binding of O- and N-linked glycans to lectins is also apparently dependent on the presence of a correct kind of
specificmbiopolymer surface-associated water structure; this is apparent from the X-ray determined crystal
structures of polysaccharide lectin adducts the formation of which requires the presence of polyvalent metal ions in
order to generate adhesive ‘cements’ surfaces consiting of water molecule chains formed of water-water bonds;
Ca2+ ions also show up as a constituent of this kind of highly hydrated protein lectin polysaccharide supramolecular
aggregated water cement system as revealed by X-ray diffraction structure determination (Bourne et al.1). A
similiar kind of ternary polysaccharide-lectin-Ca2+ aggregation process is also believed to form with lipoproteins
 present in human blood which undergo formation of ternary complexes containing H, metal ions and lipoproteins.
The differing solubilities of such protein-M2+-H complexes forms the basis of rapid methods of estimation of blood
HDL cholesterol (cf., Cornwell & Kruger1, Cham1, Burnstein et al., Warnick et al.1, Vijayagopal et al. 1) and the
formation of a similar ternary complex has been adapted to provide a rapid assay method for HS in tissue digests
(Wu & Cohen1).
The process by which Ca2+ and Mn2+ ions cause aggregation of DNA seems to occur by an analogous process to
this. [ Cf. also the discussion of polysaccharide metal ion water interactions, below}
4.8 Manning (Electrostatic) Binding of Counterions to Polyanions
Manning (Electrostatic) interactions between H/HS and counterions etc. may be augmented by water
structure adhesion crosslinks

Older rationalisations of the binding of counterions to polyelectrolyte had proposed a purely electrostatic binding
mechanism. However Grant et al. 6b-2-1 in a series of studies of the binding of counterions to H, showed that various
counterions of the same charge which were predicted by electrostic theory to bind to H with identical binding
isotherms, failed to do so in the manner predicted by the electrostatic theory. Further indications that the binding of
inorganic ions to H is not an entirely electrostatically-determined process is that simple anions bind strongly to a
formally highly anionic H, a phenomenon which should not occur according to the electrostatic binding theory.
These anionis include SO42- (Helbert & Mariani1) and probably also HPO42- ** . Such amphoteric behaviour seems to
underlie the mechanism by which an H-SiO2-based inorganic ion chromatographic separation column can efficiently
separate both anions and cations on the same column (as reported by Takeuchi et al.1).
The binding of counterions to polyanions (including H, DNA, RNA and humate) is characterized by a step-wise polyelectrolye
adsorption behaviour which was traditionally explained by electrostatic binding models (e.g. that of Manning,) in which all equally
charged couterions were supposed to bind to polyanions such as H, DNA or humate with equal efficiency. In-depth experimental
investigations of polyelectrolyte counterion binding processes, however, suggested that that some quite different, but more correct
description of the binding processed requires additional interactions than purely electrostatic ones to be taken into account. The
overall binding process may be more akin to a phase change process (cf. Grant et al. 6b-2-1) (i.e. this suggests that such binding will be
subject to modulation or initiation by nucleation phenomena). Similar such other than electrostatic mechanisms are likely also apply
to the binding of H/HS with organic counterions (viz. HS target proteins) as well as to inorganic counterions and also to determine
how counterions can potentiate DNA aggregation and melting (cf. Sitco et al.1) (and the melting of high water content
polysaccharide gels, cf Section

Straighforward electrostatic counterion attractions (the Manning theory) while clearly partly responsible for counterion and protein
binding do not fully explain the physical chemical counterion binding activities of the sulphated polysaccharides. The principal
interaction process evidently involve additional interactions which are needed to achieve correct discriminated crosslinks to proteins,
including those afforded by the binding factilitated by associated water structures; such water structures may in turn be elicited by the
occurrence of an array of (correct) inorganic ions which are associted with the anionic –CO(O)O- and SO32- sites in H. Water structures
are engendered by the inorganic chemical environment in the neighborhood of the polysaccharide chains. The specific arrays of inorganic
ions putatively induce specific water structuring effects analogously to how Hofmeister activities influence protein folding** a
phenomenon which has been shown to be dependent induced water structure effects (cf. Luck1). The correct water structures are also
needed at the surfaces of plastic cell culture dishes (cf. Grant et al. 1, 1988). The reason why the presence of a seawater range of inorganic
elements need to be attached to H is then apparent: this is needed to induce a correctly finely tuned water structure which resembles the
water structure in which the first animal organisms evovled. This could originally been achieved via the buffering actions of earlier
bacterial mucilages which in turn were preceded by an organic humic (highly carboxylic acid substituted) polymer solution (which in
present-day waters as well as at the time of evolution of the first animal cells, is derived mainly from bacterial and algal mucilages) which
were putativley needed to coexist with the primitive abiotic precursors of animal cells in order to create the exact bio-friendly fine
structure of water aggregates. The chemical composition of cometary organic matter seems to support the hypothesis that input to the
early Earth from similar sources could have created a pool of abiotic humic matter. The precursors of animal cells, hover, have evidently
evolved a more effective water structure builder namely the sulphated polysaccharides especially the HS PGs.

4.9 Water Activity Model of Metal Ion Dependent H/HS Signaling

Metal Ions May Participate in H/HS Signaling via Modulation of
Water Structure Effects of H & Putatively HS.
Evidence for Adhesiveness of metal-ion-dependent-water associated with H/HS which elicts protein binding similarly to how metal ion
dependent hydration affects polysaccharide lectin binding etc.

A fundamental prerequisite for biology may be the pre-existence of a specific multi-inorganic-ion induced water structure, as
discussed above.
Luck1 established that the overtone infrared absorptions of aqueous electrolyte solutions vary in accord with the Hofmeister
series. This indicates that the Hofmsister effect is due to the alteration of supramolecular arrangements in hydrogen bonded water
molecule aggregates.

Since animal GAGs evidently evolved from ancestral bacterial capsular polysaccharide similar to those present in
Escherichia coli (K5 {N-acetyl H/HS(-precursor) heparanosan} and K4 {ChS-like} polysaccharide), it can be presumed
that similar primitive bacterial polysaccharide functions are potentially still available to eukaryotes (cf., this accords with
the traditionally held view is that animal GAGs (including H/HS and ChS) are generally involved in osmoregulation and
hydration including via the ability of charged carboxylate and sulphate side groups on GAG polysaccharide chains to
become neutralizaed by cations enabling them to attract and retain large quantities of water molecules by osmotic forces
which further allows for the expansion of tissues and their equilibration against tensile forces provided, e.g., by the presence
of verterbral discs, which create hydraulic spaces of fixed volume and alterable dimension (cf., Osmo1)).

Furher evidence that GAGs including HS contribute to the regulation of water activity is that the integrity of human lung
hydration is believed to be highly dependent on such hydration (cf. Souza-Fernandes et al.1).

The degree of hydration and the ease of dehydration of different metal ion substituted H samples depend strongly on the
chemical nature of the metal counterions
(cf., Grant et al. 1,1987)

Confusion currently exists as to the how H/HS microstructural information is transmitted to enable the

modulation of the numerous protein activities by, e.g., specific H/HS sequences.

Implicit in the Long & Williamson1 hypothesis is the notion that inorganic cofactors might

determine much of H/HS signaling. This hypotheis, however, has not been universally accepted.

The groundbreaking work using modern CD and NMR methods (Rudd et al.1, Yates et al.1) now tends to

confirm this hypotheses that H/HS supramolecular stucture and hence signaling must include an input from

the inorganic elements which become intimately associated with these signaling polysaccharides.

Consideration of the roles of inorganic cofactors and their associated water structures might enable a fuller
understanding of the reasons why microstructures present in intact H/HS macromolecules which apparently
can highly selectively bind specific proteins in vivo, fail to selectively bind their identified isolated
oligosaccharide segments in vitro as was reported by Kreuger et al.1 in studies of in vitro binding of in vivo
identified HS epitopes for FGF growth factor variants.
Fragments of HS (Kreuger et al. 1) were studied in a highly purified form in vitro. These had been selected from in vivo information which had been predicted
such epitopes on their own to be capable of selective binding of FGF4, FGF7 and FGF8b. A possible explanation of these findings is that in vivo HS
discrimination involves additional hydrogen bonding or Van der Walls effects which the low molecular weight epitope lack. The failure of these studies of
purified oligosaccharide epitopes could, however, also suggest that multi-inorganic ions co-spheres might have provided some essential inorganic cofactor
for a discriminated HS epitope binding suggesting that e.g. a quaternary HS-FGF-FGFR-divalent metal ion complex might have been required to allow the
in vivo discrimination. Kreuger et al. seem to suggest that apart from the antithombin dedicated pentasaccharide binding site of H/HS, other dedicated
H/HS protein binding sites may not exist. Amongst the evidence against this hypothesis is the large body of data which indicates that discriminated
signaling by intact HS molecules requires specific HS microstructures. Examples include
IFN-γ binding to HS (Sarrazin et al.1) where binding requires a large HS sequence which encompasses an internal HS N-acetyl domain flanked at both
side by two HS N-SO3- domains (which are thought to control the blood clearance, subsequent tissue targeting and local accumulation of this cytokine).
The antibody identifiable inflammatory N and 6-O-sulpahted HS domains which have been found to be increased in glomerular endothelial cells in lupus
nephritis (cf. Rops et al.,1) seem to be another
example of specific HS microstructure engagement in signaling.

It seems likely that the additional factors (e.g., inorganic ions) which were absolutely required for
the highly discriminated in vivo selectivity between epitopes are removed during attempts to clean up fractions in the
laboratory using enzymic digestion and subsequent fractionation etc.

Evidence for the existence of such inorganic cofactor requirements for FGF signaling was reported
by Kan et al.1 who established that divalent cations were absolutely required for HS-assisted b-FGF

receptor assembly; Ricard-Blum et al. 1 similary established that Zn2+ cofactors (in this case confirmed

by these workers to be removed by over-purification) are also needed for HS endostatin (collagen XVIII)

binding (strong binding between these molecules is believed to occur in vivo but the highly purified protein

and normally purified HS failed to bind but could be induced to do so by the addition of Zn2+).

Zn2+ ions have been consistently found to bind selectively H (cf. Parrrish & Fair1) (cf. also Table IV and

Grant et al., 1992d)). Ricard-Blum et al.1 (2005) also reported that Ca2+ ions (apparently naturally associated

with HS in vivo were essentially required cofactors for collagen(V) binding to HS proteoglycans, an activity

for which Zn2+ ions could not in this case substitute for Ca2+.

H is known mainly for its use in medicine as a blood anticoagulant via the dedicated antithrombin binding

pentasaccharide epitope as well as via the heparin cofactor II mechanism. Both of these mechanisms

require (indirect) actions of Ca2+ ions (cf. details discussed in the refs. etc. listed in Table I).

Eckert & Ragg1 have additionaly reported that Zn2+ apparently induces a conformation change in heparin

cofactor II that favours interaction with H (Cu2+ and Ni2+ were also found to have a similar, lesser effect but

Ca2+ in this assay apparently was without effect).

Zn2+ counterions associated with H may seem to especially alter the H-associated water structure (cf.

Grant et al. (1984, 1987a, 1992d, etc.)).

Presumably currently used standard laboratory procedures for the separation and purification of

biopolymers will sometimes reduce the amounts of Zn ions etc. necessary to allow HS epitopes to bind to

their specific protein sites.

4.10 HS as a proton pump

Similarity Between Cation-Dependence of Hydration of H/HS and Commercial Ionomers
Proton conduction by [-SO3- Mn+(H2O)m] metal ion hydration dependent arrays
The commercial ionomer Nafion® which exhibits proton conduction, is also believed to depend
upon interactions between water clusters associated with –SO3- groups for this activity** (e.g.,
James et al., 1). The degree of hydration of these sulphonate groups depend upon the counterion
in a similar manner to H being maximum at 6 H2O/SO3- for Na+ and minimum at ca.0 H2O/SO3- for
(CH3)4N+ (Grant et al., 1, 1990).
The addition of colloidal SiO2 to the commercial ionomer (in similar amounts to the occurrence of
Si putatively as SiO2 nanoparticles in H/HS) has been reported to improve its function (Adiemian
et al.,1).
(cf. section 9-1, vide infra).
Iler1 has suggested that SiO2 has been associated with polyuronides since life began perhaps
because of an ability to improve the biological activities of such polysaccharides.

H/HS putatively occur naturally as mixed salt forms which change their degree of hydration according to the chemical nature of
the associated counterions in a similar manner to man-made industrial ion membranes which depend for their activities on
counterion dependent hydration of functional sulphonate ionic groups.

HS proteoglycans (e.g. glypicans, syndecans, perlecan, agrin and collagen XVIII it can be rationally suggested,
must exist in vivo in mixed salt forms similar to H).
The functions of the mixed salt forms, like man-made ionomers based on arrays of –SO3- groups will depend
critically on the inorganic counterions which induce a functionally important associated water cluster
systems**. It had been previously reported (Nader et al.5, 1(1983)) that the amounts of HS and other sulphated
polysaccharides present in fifteen species of aquatic animal organisms increased with the habitat salinity.
This was thought to be a requirement to inhibit high ionic strength induced tissue dis-aggregation, but it is now
suggested that these organisms require the correct (multi-inorganic element) inorganic seawater constituents,
including similar amounts of Na+ + arrays of amaller amounts of other counterions associated with
polysaccharide–SO3- hydrate structures to form a specific membrane HS water cluster structure. Apart from
the principal ions needed to form the polysaccharide - SO3- Na+ (H2O)6 array system, both natural as well as
man-made ionomer functions may require an additional fine tuning (like the doping of a semiconductor) by some
of the ultratrace inorganic components which occur seawater (cf., doped SiO2 nanoparticles, cf Adiemian et al.1
might be supposed to fine tune the acquired water structure in man-made water cluster ionomer systems.

A corollary to the above hypothesis is that the dietary absence of the correct amounts of SiO2 nanoparticles and
ultratrace elements (listed by Haraguchi1 as occurring in blood serum etc.) may give rise to ion channel
pathologies, e.g., those associated with chronic fatigue (Chronic fatigue syndrome is of unknown etiology but
numerous hyotheses have been advanced relating to this disease including the dyshomeostasis of Cu and Zn
ions**).

H/HS probably occur naturally as mixed salt forms with complex hydration patterns which are associated with
clusters of the sulphate half ester and N-sulphonate groups and which are putatively involved in Ca2+transport
and energy transduction. This action could, it is now suggested be dependent on hydration effects which can be
physiologically modulated or pthologically perturbed by the changed hydration which is dependent on the
chemical nature of individual counterion.s

Evidence for involvement of HS in energy transduction for lung muscle function (detected by electrically
unduced Ca2+ spikes) is greatly diminished in Nsdt-1 -/- mice (Jenniskens et al. 1).

4.11 HS as a Mimetic of Humic Polymer Buffers in Natural Waters**

Although present-day anionic humic polymers (which are the largest class of organic molecules on Earth) are obviously
derived from the decomposition of the organic molecules which exist in present-day biota, a polymeric system of a similar
chemical nature might have existed on earth before biology started off. Such a polymer system could conceivably, e.g.,
have arisen from the rearrangement of condensed formaldehyde (cf., Wickramasinghe1) and an inorganic core. This might
have been largely composed of polymers of silicic acid (Grant et al., 1992a; 6b-2-3). This could have provided a stable system of
biofriendly hydration which was highly conducive to the evolution of life.

The binding and redistribution of the inorganic elements by natural polyuronic acid ion exchange systems is suggested to be
an important determinant of the observed distribution of inorganic elements in seawater (which contains ion exchanger
organic matter as ca. 2µ g /ml polyuronate-like humic acid containing humic–C(O)O- anionic groups which are analogous
to such groups in animal and plant anionic polysaccharides; nb., this quantity of dispersed humic organic matter is of a
similar magnitude to the amount of dissolved H/HS in human blood (cf. Engleberg1).
Such a structured humate system in seawater is suggested to be an important part of a system of natural anionic inorganic
ion homeostasis which is evident in both seawater and seawater-like biological fluids (present and presumably acitve in
primitive as well as in highly evolved animal organisms). Such buffer action is achieved by the ability of such polyanonic
molecules to act as highly efficient inhibitors of crystallization enabling them to promote the sort of long-term
supersaturated solutions which exist both in seawater and biological fluids.
This is clearly shown by the results of studies of the comparative rates of seeded crystallization of e.g. CaCO3 and BaSO4 in
the presence of such anionic materials (i.e., humic and fulvic acids from marine and land humified organic matter as well as
the more accurately molecular structure defined polyanionic polysaccharides)**.
H, like humate and fulvate polymers seems to have the ability both to simultaneously bind a wide range of inorganic ions
and also to modulate the rates of crystallization of salts containing these ions so as to perform a buffering action to create a
stable multi-ion solution.
-----------------------------------------------------------------------------------------

5 Nitric oxide biochemistry & H/HS signaling

Metal Ions & Water Structure Dependent Nitrite Cleavage of H/HS

Vilar et al reported1e (an unexpected effect) the ability of nitrite to deaminatively cleave H under physiologically relevant
conditions (cleavage occurred when the reaction was performed in the presence of phosphate buffer but not when an
imidazole buffer was used; the critical factor here could be that the former buffer ubiquitously contains trace amounts of
redox metal ions). Hitherto it had been thought essential that highly acidic non-physiological conditions were required to
achieve nitrosative scission of H/HS, which was further known to require the presence of unsubstituted glucosamine –NH2
groups in the H/HS chains, such groups had been thought not to exist naturally in H/HS under in vivo conditions. Later
work, however, found that these assumptions were incorrect 1f. It has now become fairly well established that a major HS
signaling mechanism depends on the controlled insertion into HS of unsubstituted glucosamine-NH2 groups which thereby
prims the HS chains for nitrosative cleavage under slightly acidic conditions in response to biochemical stimulae; these are
apparently coupled to servo feedback coupled microstructural alteration of HSf.
----------------------------------------------------------------------------------------------------------------------------------------------
f
HS-based dynamic servo-feedback control process seem also to be dependent on the regulation of HS microstructure by HS-6-O-
endosulfatases. (cf. Dai et al. 1 and Section 3.4-4)
-----------------------------------------------------------------------------------------------------------------------------------
H+ ions and/or redox-metal-ion-dependent non-enzymic transformations apparently can also directly prime for nitrosative
cleavage by promoting the (heparan)-N-SO3àNH2 reaction 1g,h.

The transition metal ions associated with H can be predicted, on the basis of well established inorganic chemistry to
potentially facilitate further complexation at such sites and might, e.g. account for the sequestration of nitric oxide by H (cf.
Upchurch, but further research is required to probe this idea ); a difficult-to-interpret ESR spectrum of H [F.B. Williamson
et al., Aberdeen unpublished results discussed with the author] could have arisen because of electronic interactions between
the H-bound bound transition metal ions).

Redox metal ions (as well as Ca2+ and Zn2+) present in the metallomic profile of H/HS could, it is now proposed, be of
especialy relevance to a fuller understanding of the mechanism of nitrosative scission of HS which is now believed to major
animal protein control and signaling system which generates ‘hormone’-like HS oligomer messenger molecules which
engage in such tissue protective and other functions. This type of nitrosative cleavage which has been researched by the L-A
Fransson et al. group (cf. Mani et al.; Ding et al.1) and has been found to be relevant to the etiology of Niemann-Pick
Disease [vide infra, section 6] and can explain (cf., Lofgren et al., 1) the effective inhibition of prion diseases by H-like
sulphated xylans (especially by PPS SP54, cf. Steel1) could also be relevant to the etiologies of cancer and other
degenerative diseases (e.g., ostosarthritis [an alternative mechanism {cf., McCarty1} by which dietary glucosamine was
thought to directly augment HS biosynthesis (and perhaps increase the dgree of sulphation) and which might have acounted
the beneficial effects of dietary supplementation by glucosamine on this disease process was not confirmed in recent studies
[Qu1]). Such diseases can alterntively be suggested to be vulnerable to pathological perturbation of more subtle effects on
HS signaling by toxic metal ions which are also subject to glucosamine input (cf. Grant 1 (2000, internet)).

5.3 Some Early Indications of Metal Ion Catalysis of Biologically-Relevant Nitrosative Processes

Identifying HS by deaminative cleavage with nitrous acid

The traditional, highly effective, method of identifying HS in a mixture of polysaccharides is by reaction

with nitrous acid. This is highly specific for H and HS and can reveal the presence of HS in proteoglycan
mixtures. This reaction was, however, originally thought to be an entirely non-physiological laboratory

curiosity.

5.3-2 Tyrosine nitration in degenerative diseases

Nitrite/nitrous acid promotion of stomach cancer is attributed to a pro-cancer mutagenic action of nitrite etc.

on DNA.

Damaging effects of excessive nitric oxide generation and associated nitrite formation may also be a key event in the
etiology of Alzheimer’s disease and other neurodysfunctions (cf., Vural et al., 1) and account for the putative beneficial
effects of “anti-nitrant” reagents; such effects may also contribute to the activities of “Memantine” a novel type of
antidepressant (cf. Almeida et al.1) Alzheimer’s disease and putatively also anti-multiple sclerosis drug. Vural et al. 1
reported that a range of othe neurodysfunctional conditions were also associated with diminished plasma levels of Mn and
arginase and elevated nitrite.

H/HS modification by nitrite/nitrous acid seems to allow a specifically targeted signaling process during

nitric oxide signaling (n.b., nitrite is a primary metabolite of nitric oxide).

Such a highly specific interaction with HS biochemistry should perhaps have alerted classical researchers

concerned with researching the mechanism of nitrite promotion of stomach cancer to the likelihood that the

pathological actions of nitrite (nitrous acid in the stoach) were related to this specific reaction, but this only

became a credible hypothesis following the later, quite unexpected, and for a time disbelieved, discovery

that nitric oxide is actually an in vivo messenger molecule employed widely throughout in animal

physiology.

Apart from the long-known ability of nitrous acid to rapidly deaminatively depolymerize H and HS, nitric

oxide is also apparently deactivated by H-like molecules by an aparently separate process

(Upchurch et al., 1).

It was further discovered was that if nitric oxide formation and metabolism became unbalanced, appreciable

pathological in vivo protein tyrosine nitration always ensues and such nitration became known as a

hallmark of many, or indeed perhaps of all, degenerative diseases 1a-8.

5.3-3 Inorganic ion catalysis of nitrosative reactions associated with stomach cancer
Prior to the discovery of the normal physiological roles of nitric oxide, B. C. Challis et al.1 had

established that various inorganic catalysts (e.g. copper, iron, silver iodide and thiocyanate ions,
the latter being augmented in the serum of cigarette users) catalyze nitrosative reactions.

A corollary to this scenario can now be made that anthropogenic inputs from such inorganic redox ions

might inappropriately perturb nitric oxide-dependent HS signaling processes and therby contibute to the

promotion of cancer and other degenerative diseases which were known to somehow be inhibited by

Vitamin C (ascorbic acid). A highly efficient reversal of inappropriate nitrite formation is known to be

achievable by the reaction of nitrite with ascorbate which efficiently reconverts it back to nitric oxide.

Recently a role of ascorbate in nitrosative HS signaling has also been identified, where ascorbate acts to

reduce Cu2+ to Cu+ a reaction held to be essential for recycling of nitric oxide held at HS-core-protein

cysteine stores1a-9 , a critical part of the mechanism of generation of HS oligomers putatively involved in

‘hormone’ –like activites.

5.3-4 Inhibition of soluble guanylate cyclase by H and carrageenans; role of Mn2+

5.3-4 Inhibiton of soluble cuanylate cyclase by H and carrageenans: role of Mn2+
M.A. Liebel & A..A. White, Biochem. Biophys. Res Commun,. 1982, 104 (3) 957; Chem. Abs., 138749q
reported that soluble guanylate cyclase was inhibited by H in a Mn2+ dependent manner.
This points to a linkage of nitric oxide sythesis in a H, Mn2+ interlinked pathway;
sulphated algal polysaccharides also demonstrated an H-like modulatory activity for this process in which the order of
effectiveness for carrageenans was λ >ι > κ .

5.4 “Ascorbate & Nitric Oxide in Redox Control of Heparan Sulphate”

THE HEPARAN REDOX HYPOTHESIS

This hypothesis was proposed in an internet document

[http:..web/ukonline.co.uk/dgrant/dg4 which should be read in conjunction with the
file http:../ukonline.co.uk/dgrant/dg8]
which suggested on the basis of literature and Aberdeen University laboratory studies that a
linkage occurs between ascorbate, heparan sulphate, lipid and nitric oxide biochemistries.

“Ascorbate & Cancer”

The possible anti-cancer activity of ascorbate can be at least partly explained by

direct and indirect effects on HS management.
[This was proposed in an interent document
[http:../ukonline,co..uk/dgrant/dg4 which should be read in conjunction with the file http:../ukonline.co.uk/dgrant/dg8].

An update of this hypothesis including discussion of the action of hypoxia induced factor HIF-1α
(cf., Li et al.1) in relation to ascorbate and HS recycling has been given by Fransson & Mani1.
Cf., also Frie et al.1

5.5 Hypothesis: Hydrogen Sulphide & Nitric Oxide are Involved in Redox Control of HS
The heparan redox hypothesis suggests roles for small redox molecules (H2S and CO) in the NO dependent redox signaling
alters HS microstructure in response to environmental input.
H2S was found to inhibit nitric oxide production (Oh et al.1) and nuclear factor kappa B via heme oxygenase-1 expression in
RAW264.7 macrophages stimulated with polysaccharide.
The role of hydrogen sulphide gas, a potent reducing agent and ligand for metal ions (endogenously synthesized from L-
cysteine by cystathionine-gamma lyase (CSE)) as a vasodilator and a modulator of nitric oxide synthesis suggests that
hydrogen sulphide and polysulfides are invovled (in conjunction with nitric oxide) in the direct and indirect systemic
heparan sulphate structure management system in animal biochemistry.

H2S production is believed to be supported by cytosolic glutathione levels, reduced thiols in or on the red blood cell
membranes and H2S generated by allyl sustituted polysulfides. Dietary garlic augmentation of polysulfides is believed to be
the basis of the correlation between the consumption of garlic and the inhibition of cardiovascular diseases (Benavides et al.
1
).

In a purely inorganic chemical context organic polysulfides demostrate redox-dependent stochastic structrual reorganization (cf. Grant & Van Wazer,1, 1964)
which is subject to catlalysis by free radical generators (e.g., as used in commerical rubber vulcanization; aspects of this intrinsic physical chemistry
behaviour seems to been incorporated into biochemistry as suggested by roles of organic polysuphides in the generation of H2S (to thereby further inhibit
iNOS activity (cf., Oh et al.1) and also as a redox related formation of disulphide linkages between proteins including HS core proteins, which are invovled
in a metal-ion-dependent manner in the regulation of nitric oxide scisssion of HS side chains (cf. Ding et al. 1))

6. Relevance of H/HS Biochemstry to Pathology

A comprehensive literature research in the H/HS and natural polyanion fields, conducted over many years in tandem with
laboratory experimentation suggested that HS, a system which normally is without deleterious effects in the animal
organism (except for the genetic defect conditions which lead to mucopolysaccharideoses and the aberrant conditions
leading to tumour growth which is subject to promotion as well as to inhibition by HS PGs1A3-1) is also a master system for
control of multi-activities (which is now suggested to include a postsynthetic editing via metal ion dependent nitrosative
scission servo-feedback controls) circumstances which can be suggested to allow a novel dietary-based general system of
rational healthcare to counter most (or indeed, perhaps, all types of) pathogenic insults and degenerative diseases. Such
broadly functional tissue protective functions provided by H-like molecules include antioxidant functions, achieved
apparently, in part, by the formation of clatharate like H-redox metal complexes.

Prior research (cf. Tables I-III) suggest that presence of overtly toxic ions (e.g. Pb2+ and Cd2+) and damaging organic
factors (e.g. excess glucose and saturated fatty acids) negatively perturb the HS structure, conversely, pro-health
dietary factors (e.g. ascorbate and ω -3 fatty acids) positively enhance the effectiveness of HS structures involved in
tissue protection and anti-pathogen roles.
HS microstructure in the pineal gland has also been reported to be altered by light, this linking HS biochemistry to circadian
rhythm which is linked to HS 3O sulfation and melatonin production (a situation which is of possible relevance to anti-
cancer potency of H/HS and HS3ST-2 (3O-sulfation is also putatively inorganic counterion binding related).
A designed modification of HS may allow for a rational generally applicable dietary-based therapeutic intervention
system based on the liklihood that the HS system is a ‘hub’ system impacting to numerous biochemical pathways
which further suggests that intervention in HS biochemistry can be adapted to combat diseases inclkuding cancer.
This notion accords with reports that H/HS based therapies seem to promise potential successful interventions in a
wide variety of diseases which also include prion diseases, viral and bacterial infections, atherosclerosis and
osteoarthritis.
The range of diseases for which H/HS therapy seems able to influence is so very great (full utilization of H/HS
biochemistry could cause a medical revolution where a single family of optimally effective drugs is able to combat all types
of illness) a situation which engenders extreme reticence amongst scientific reviewers (e.g., Lindahl1; Garg et al. 1) who are
understadably shy of openly discussing this possibility.

6.1 Putative pathological inorganic element perturbation of H/HS

signaling
The present document suggests that H/HS provides an essential inorganic reservoir system of inorganic cofactors for correct in
vivo H/HS activity is that such activities may be subject to pathological disturbance of inorganic components, e.g., by
anthropogenic environmental inputs and even by electromagnetic radiation.

The etiologies of some forms of cancer and other degenerative diseases1a could centrally involve perturbation of polysaccharide protein
cross-linking (especially that facilitated by Ca2+) involving an altered modulation of the activities of growth factors involved in wound
healing which are putatively perturbed in all degenerative disease processes.

Epidemiolgical evidence that serum ferritin (“iron overload”) was highly significantly correlated with cardiovascular dysfunction
determined mortality in Eastern Finnish men (Salonen et al. 1) coud suggest that, e.g., inappropriate redox active iron catalysed de-N-
sulphonation of HS which might occur under these conditions could render the resultant vascular surface HS polysaccharides defective
for protein activity regulation (this includes mediation of immune function) and for the inhibition of pathological calcification (Grant et
al., 1(1992)). Such de-N-sulphonation might also be involved in the etiology of arthritic diseases which are a charcterized by dyshomeostasis
of nitric oxide (cf. Stichenoth & Frolich1).

Prions both bind copper ions and HS (the biochemistry of which seems central to their function) which could suggest that some
anthropogenic alteration of metal ion concentration (e.g. under condition of copper depletion, the presence of excess manganese could
promote the Mn3+** ion induced misfolding of prions cf. Purdey; Treiber et al.1). This scenario might be involved in the etiologies of
prion diseases for which H-mimetics apparently exhibit an highly effective therapeutic potential (cf., Grant1 (Discussions with M. Purdey)).

6.1-1 H/HS Biochemsitry & Degenerative Diseases

6.1-1-1
Nucleation events (subject to inhibiton by H/HS) may determine the progression of amyloidoses**

The correct H/HS microstructure has been indicated by in vitro studies to be needed to faciltate the effective inhibition of pathological
seeding; e.g., whereas H was highly effective for CaCO3 seed particle inhibition, de-N sulphonated H, could enhance somwhat rather
than inhibit the activity of seed particles (cf., Grant et al. 1 , 1989, 1992).

Such a dependence on correct microstructure of plaque seeding inhibition by H-like molecules could suggest that a diesase or an age-
related alteration in the microstructures of anti-seed H/HS polysaccharides, so as to diminish their surface adsoption and deactivating
effectiveness, might be the ultimate origin of amyloidoses.
A corollary to this is that therapeutic intervention in amyloidoses should be achievable by administration of correctly-microstructured
anti-seed agents such as parmaceutical H or H-mimetics (such a possiblilty seems to be supported by in vivo studies**).
The putative age-or pathological related diminution of the anti-plaque effectiveness of HS by diminusion of its N-SO3- content might be
subject to redox metal ion catlaysis (as has been suggested by in vitro observations). The presence of inappropriate amounts of redox
metal ions which are capable of such catalysis might be promoted by the increasing tendency for serum ferritin to increase with age (cf.
Jarrett et al., 1). The apparent association of excess manganese under conditions of depletion of inorganic elements invovled in
antioxidant protection or the presence of elevated amounts of silver (occurring together with strontium and barium in a suggested
piezolelectric particle system) which has also been suggested to promote scrapie-like illnesses2 may do so at least in part via promotion of
the putative redox metal ion catlalysis of desulphonation of HS.

6.1-2
Possible Example of Anthropogenic Metal Ion (e.g. Ba2+) Perturbation of HS Biochemistry in the
Etiology of Multiple Sclerosis
[Cf. The epidemiological studies of Mark Purdey2 **]

The established roles of intoxication by Al in dialysis encelipathy and by Cu in Wilson’s disease were

discussed in a review of metal ion intoxication (Andersen1) and its treatment by chelation therapy (e.g. with

dimercaptopropane sulphonic acid DMPS) the need for improved methods of removing aged deposits of

e.g. Al, Cd, Fe, Hg and Cu especially using orally administered chelating agents was identified. The use of

polysaccharide-based chelators does not seem to have been considered for general medical use

although suggestions that e.g. sulphated anionic polysaccharides are useful as antidotes to accidental

intoxication by radioactive elements (such as occurred during the Chernobyl incident).

Such polysaccharides including H/HS proteoglycans (which have both polysaccharide side chain
anionic –SO3- and core protein S-H metal chelating groups [cf. also present in DMPS]) may perform such a

detoxification role in vivo, further suggeting that defects in such protection might may be invovled in the

mechanism of metal ion intoxication such as the intoxication by Al which has (controversially) been

associated with Alzheimer’s disease (cf. Andersen1) and the Ba intoxication with multiple sclerosis2 .

It has recently been noted (Praline1) that the symptoms of acute Hg intoxication closely resemble those of

amyotrophic lateral sclerosis. It has been pointed out by D.I. Shepherd that such a highly distinctive

disease as multiple sclerosis would much earlier have come to the attention of medical science than its

first observation which was also coincident with the use of mercury amalgams in dentistry (suggesting a role

for Hg intoxication in the etiology of this disease.

Evidence for an anthropogenic metal ion perturbation of H/HS signaling in the etiology of multiple

sclerosis could also be suggested by geographical/geological studies of the prevalence of this disease;

although the prevalence of the disease tends on a global scale to increase with increasing latitude the highest

prevalence in England was found to be in the most southerly county (Cornwall) suggesting that e.g. the

minerals derived from older rocks there present which leached into water supplies and thereby into biota or

regional variation in genetic factors also promoted the disease.

The latitudinal factors implicate the quality of sunlight UV exposure and vitamin D and calcium

dyshomeostais. A possible connection between Vitamin D isoforms generated by UV B and the inorganic

sulphate transporter (Dawson & Markovich1) suggestes that the availability of inorganic sulphate which is

essential for HS assembly might be compromised under conditions of Vitamin D isoform and sunlight

quality inadequacy. This might mbe further compromised under modern conditions of inorganic sulpahte

depletion of soil (previously supplied from the combustion of coal). Ba2+ intoxication, perhaps in

combination with such deficiencies, possibly augmented by tissue disruptive effects of infrasonic shock

waves associated with (military) aviation and industrial mining and quarrying activities were proposed to

affect the folding of metal ion prion adducts2 [cf., it seems likely that the normal role of prions could be in

provision and transport of metal ions perhaps in concert with HS] might also promote multiple sclerosis

since prion biochemistry seem centrally linked to that of HS.

Multiple sclerosis migh also be dependent upon defective FGF growth factor signaling (a Ca2+-dependent

HS directed growth factors/growth factor receptor assembly processe putatively subject to Ba2+ and Sr2+

perturbation) thought to be essential for myelin sheath renewal2.

The primary current use of interferon gamma (IFN-γ ) therapy is apparently for therapeutic alleviation of the

symptoms of multiple sclerosis. Such a use of this anti-inflammatory cytokine may, however, apparently

could depend on its correct interactions with HS (via interactions of IFN-γ with specific sites believed to

consist of N-acetylated glucosamine rich domain and two highly sulphated sequences, cf. Sarrazin et al.1)

It might further be worth considering the putative roles of inorganic factors in such binding (both in the

normal function and therapeutic intervention situations); the anti-inflammatory effects of cytokines such as

IFN-γ elicited by HS interactions could require additional roles of water structure/inorganic cofactors which

in turn could also be subject to possible anthropogenic pathological input.

The electrophoresis of H in a Ba2+ containing buffer solution while providing an effective method of
demonstrating the complexity of the mixture also suggests how in vivo signaling or its disruption could
depend on interactions of H with alkaline earth cations. This may be relevant for the mechanism of
fibroblast growth factor (FGF) signaling thought normally to depend on a quaternary complex of HS-
FGF -(Ca or Mg)-fibroblast growth factor receptor (FGFR)9b-3 and how replacement of Ca or Mg with
other metal ions might account for disruption FGF-FGFR signaling in demyelinating degenerative
diseases which have been suggested might have been triggered by the chronic exposure to
inappropriate amounts of Cu, Zn, Pb, Al, Ba or Mn ions {Cf., also Rudd et al.1}.

6.1-3 Possible Perurbation of H/HS Signaling by Al3+ , Zn2+ (Cu2+/+) in Alzheimer’s Disease

Al3+ (-HS related?) induction of dementias (cf. Snow1). The amyloid precursor protein of Alzheimer’s
disease has also been reported to modulate the Cu/Zn nitric oxide deaminative cleavage of HS (Cappai
et al., 1) indicating an possible role for altered redox balance/metal ion related HS tissue protective
surveillance systems in this disease which may be why HS oligomer mimetics (e.g., PI 88) have been
found to offer therapueutic interventive benefit in this disease (as well as for the inhibition of pro-tumor
angiogenesis and heparanase activities)**.

6.1-4 Anti-misfolded prion and anti-HIV activites of the H-mimetic pentosanpolysulfate (PPS):

The H-mimetic Pentosanpolysufate (PPS): putatively protects agaisnt misfolded

prions and is also a potent HIV antiviral agent.
H-mimetic drugs such as pentosan polysulphate (PPS) provide an effective therapeutic
intervention strategy for prion diseases; this was first reported by Diringer & Ehlers1 and confirmed
by later workers; H/HS and PPS are also highly effective against intractable viruses such as HIV-1 and
II (cf., e.g., De Clercq, TiPs., 1990, 11, 198-205; similar results suggesting that such polysaccharides
were exceptionally active antivirals, and can be argued to be the best way of obtaining more effective
anti-AIDS therapies (Aberdeen polysaccharide research of a andti-HIV-1, algal PPS-like
polysaccharide**).
H-like molecules also inhibit hepatitis C virus entry into mammalian cells (Basu et al.1)

-----------------------------------------------------------------------------------------------------------------------
6.1-5 General anti-degenerative disease potency of H/HS
It can rationally be suggested that the systemic dysfunction of inorganic-ion-determined
supramolecular structures in H/HS glycocalyxes is relevant to the provision of improved methods of
therapeutic intervention in a wide range of degenerative diseases incuding cancer, atherosclerosis and
dementias (including Alzheimer’s disease) for which H-centred therapeutic intervention seems to offer
benefit 1a (Engelberg reviews). A problem with such H-based therapy is that the quality control of H is at
present not adequate, a
situation which this article seeks to address.

6.2
Defective Nitrosative Cleavage which has been Indicated to Occur in Niemann-
Pick Disease Could also be Relevant to the Etiologies of Cancer and other
Degenerative Diseases

Defective nitric oxide-dependent deaminative cleavage of HS (and pathological nitration) has been

reported to be associated with Niemann-Pick disease (K. Mani et al. 1a-13-2 )(a condition where an HS-

dependent recycling of cholesterol is defective but this becomes less severe in presence of adequate

ascorbate which is believed to increase defective nitric oxide availability, e.g. via the redox metal ion

catalytic process for nitric oxide cycling).

Alterations in cholesterol biochemistry (e.g., Cibickova et al.) and nitric oxide homoestasis seem also to be associated with Alzhiemer’s as well as a range
of other diseases (cf., e.g., Vular et al., 1) implicating altered nitrosative modulation of HS as possible key processes in the etiology of such diseases.

6.3 Cancer & H/HS Biochemistry.

Inhibition of Carcinogenesis by H & H-Like Molecules

{References to this section are listed in ref. A1.3-1 from which the following synopsis was suggested}.

Cancer is a degenerative disease process about which much disagreement still exists but

traditionally was believed to arise from a critical genetic damage load which eventually causes

uncontrollable cellular transformation (and inhibition of cellular differentiation associated with cellular

immortalization) followed by growth, clonal extension, pro-tumour angiogeneis and tumour metastasis.

A newer model of cancer proposes that this can be initiated by a cancer stem cell system (cf., Lobo et al.1).

The failure of tissue protection actions of key tumour suppressor proteins such as p53 is also thought by

some to be the basis of cancer. The role of the heparanome for the modulation of animal wound healing

and in developmental biology are clearly also likely to be relevant to a fuller understanding of the etiology

of cancer which seems to arise from aberrant cellular differentiation, proliferative and behaviourly activites
following a perturbation of the complex heparanome biofeedback signaling systems.

Much of the supposed genes needed to account for why humans and other most animal species can be

argued to be more complex organisms than bacteria or even C. elegans has been tentatively accorded to the

existence in animals of a more complex encoded HS extracellular polysaccharide control system for

cellular assembly and regulation of e.g. peripheral and central nervous system organization (and associated

cognition) and the elaboration of complex tissues with highly evolved organ functions).

The mechanisms by which HS PG can provide tisuue protection and more generally affect biochemical

signaling involve actions of both the core proteins and the HS side chains. Antibodies against HS core

proteins can confer anti-tumour benefit. Numerous researches also suggest that the presence of the correct

degree of sulphation and perhaps also the presence (e.g. bound to HS in a multi-inorganic-element

cosphere) of the correct amounts and types of inorganic cofactors (cf. those potential inorganic ionic

cofactors suggested in Table I) participate in the actions of HS which are relevant to carcinogenesis.

A wealth of information now suggestes that tumour growth can be both promoted and inhibited by H-like molecules. This
provides a rational focus for designing novel therapeutic interventive strategies for cancer but the incompleteness of the current
knowledge on H/HS microstructure and especially the role of inorganic cofactors in creating such microstructure is a problem
here. Ga3+ ions occur in at least some H [cf. Table IV]. Pt also seems to occur in at least some Hs, suggesting potential anti-cancer
actions known to be afforded by such inorganic ions).

The HS master systems of multicellular animal biology.

[HS has a greater intrinsic complexity and initial flexibilibty than DNA and can be rationally suggested to
extend the information storage and processing capabiities of the sparse human geneome (which the human
genome project has shown to have at least an order of magnitude less genes than had previously
been deemed necessary for humans) suggesting that some other anionic system (e.g. HS) upgrades the
equivalent gene number effectiveness in multicellular animal. HS seems to be the most likely candidate for
the suggested extra anionic system since HS chains are thought to be possess and almost DNA barcode or
postcode-like information system which requires at least some minimum correctness of sugar sequence
integrity to function properly, and where the sequences are incorrect, in principle this could promote
diseases, including perhaps some forms of cancer similar to how genetic disturbances, either inherited
tendencies or mutated genes can undoubtedly be key events in carcinogenesis, the notion of an analogous
disturbance of the heparanome (e.g. invovling non-enzymic postsynthetic redox metal catalysed nitric oxide
metabolite-determined structural alteration) should perhaps also be considered as a possible contributory
factor in carcinogenesis and other degenerative diseases. This idea in principle is an extension of the
concept that genetic defects can lead to defective HS synthesis or metabolism which contributes to
mucopolysaccharidoses].

HS while being obviously highly dependent on gene products is also affected by independent-of-the-

geneome systems of postsynthetic alteration (remodelling) of HS e.g. by actions of 6-O-sulphatases (e.g.

Sulf-1) which seem to postsynthetically alter the HS in expressing cells as well as in neighbouring cells;

while it is currently undertain if metal ions influence Sulf actions it seems likely that such actions are relvant

to cancer as is the major postsynthetic alteration system of HS by reaction with nitric oxide metabolite is

known also to require metal ions and ascorbate and other essential dietary factors to achieve critical

signaling activities which could also potentially also involve other inorganic cofactors (e.g. silicates and
other inorganic crystals), some of which seem able to further directly or indirectly modulate enzymic

(including heparanase and sulfatases) additional to the overtly non-enzymic activites of nitric oxide

metabolites for the deaminative cleavage of HS.

Anti-cancer effects of the genetic ablation of inducible nitric oxide synthase (iNOS)
Kisley et al 1 reported that the genetic ablation of iNOS decreased mouse lung tumorigenesis.
Arguing from the possible role of excess nitric oxide generation in the etiology of rheumatoid arthritis and the role in the
etiology of this disease of excessive active iron and other redox metal catalysed deaminative cleavage of HS, it was
hypothesized that the drug enabled reduction of iNOS which seemed to offer benefit to rheumatoid arthritic patients might
also confer similar anti-cancer benefits (cf., Grant 2000 internet dg2, dg4). This scenario seems to have been confirmed despite
contrary reports which indicate that nitric oxide has a general anti-cancer effect. It is now suggested that the difference
might be due to redox metal status. Where redox metal can augment the nitrosative degradation then attenuation of iNOS is
anti-cancer but otherwise not. It should be noted that, in general the authors of articles dealing with the beneficial
therapeutic effects of the attenuation of iNOs did not consider the likelihood that the effects observed related to the role
which HS biochemistry is here accorded in the etiology of cancer.

Traditional therapeutic intervention strategies against cancer rely on the use of highly toxic chemical

agents e.g. platinium complexes which crosslink DNA and induce apoptosis, alkylating agents (e.g.,

mustard gas derivatives) and targeted toxic radiation, the new HS PG-related strategiesd of therapeutically

inhibiting carcinogenesis are likely to intrinsically be associated with less adverse side effects.

d
Suggested HS PG anti-tumour actions accord with the known manner in which HS PGs play a
central role in the control of intracellular communication, adhesion and apoptosis, processes which
become highly disturbed in carcinogenesis.
H and H-mimetics like PPS are potential HS-like therapeutic agents of low toxicity agent with which to
influence HS PG biochemistry in order to attempt to counter tumour growth.
H is currently believed to inhibit cancer via promoting enhanced cellular (e.g. H enhances mixed leucocyte reaction/cytotoxic antitumour responses) and
non specific immune reponse, by inhibiting cellular proliferation (e.g. that promoted by H-binding growth factors and cellular proliferation initiated
phorbol ester (TPA), serum via attenuation of levels of c-fos and c-myc RNA; H-like molecules (evidently mimicking endocytosed HS fragments
produced either via heparanase or nitroastive cleavage) seem to perform a trans regulator role in gene expression (cf., 2(hydoxynapthyl)-β -D-
xylopyronoside priming of HS and nuclear targeting of the products attenuatates tumour growth. The ability of H to modulate histone gene interactions and
inhibit histone acetyltransferase may be a phenomenon which can be harnassed for therapeutic benefit).
Heparanase has both enzymic polysaccharide chain scission actions on HS as well as separate signaling activities including Akt mediated activation of
endothelial nitric oxide synthase eNOS (a modulator of angiogenesis). H can both promote and inhibit angiogenesis (by influencing the activities of H-
binding growth factors) and also direcly inhibit heparanase actions which promote cancer cell metastasis (the virulence of cancer cells can apparently be
directly correlated with their heparanase activites).
The promotion of metastasis by selectin is also cancelled by H.
The collagen XVIII HS PG protein fragment endostatin suppresses angiogenesis.
The pro-angiogenesis factor Ang3 binds to HS but this inhibited by H.

The attenuation of Sulf-2 endosulfatase is known to up-regulate glypican HS PG in a manner which promotes tumour
growth; the promotion of Sulf-1 tumour suppressor functions [cf. Lai et al.1].
Although ca. 50% of pancreatic carcinomas show increased HSulf1 RNA, the majority of ovarian, hepatocellular and head
and neck squamous carcinomas show downregulation of HSulf1 [cf. Garg et al. Antibodies to HSPG have been demonstrated to
attenuate cancer; e.g. use of that against glypican-3 (which also influences Akt/mitogen activated protein kinase activites which are
relevant to tumourigenesis} [Cf. HS PG glypican-3 is overexpressed in colorectal carcinoma, hepatocellular carcinoma, ovarian cancer
and extragonodal germ line cancers; glypican-1 is considered to be a hallmark of human gliomas and the downregulation of which can
decrease tumour growth. [Antibodies to glypican -3, -5 and 6- show promising anti-tumour activities with specificities
against mesotheliomas, pancreatic cancer, ovarian cancer, liver cancer and sarcomageneis.
VEGF (an H-binding growth factor) which promotes tumour augmentation by aniogiogenesis) seems also to offer by its inhibition, a
range of effective anti-cancer therapeutic agents].
-------------------------------------------------------------------------------------------------------------------------------

Correction of erroneous signaling by H-like HS fragments seems also to allow both pharmaceutical H and

exogenously applied HS fragments or semisynthetic xylan sulphates etc., to act as anticancer agents (e.g.

pharmaceutical H or mimetics thereof and enzymically modulated H (e.g., QSulf-1-attenuated-H) inhibits

FGF induced angiogenesis / tumour growth (cf., Garg et al.1)).

It is also believed that the HS system is subject to servo control change of HS-microstructure-dependent

action by the presence of molecules like ascorbate, dietary supplementation or the direct intravenous

administration of this substance is evidently highly capable of controlling the several disease processes

including cancerogenesis. How this is achieved could include by the process of by feeding back into a

servocontrol system to ‘correct’ errors or ‘edit’ pro-cancer HS microstructures** Input into this feed-back

mechanism can evidently change HS PG biosynthesis from a pro- to an anti-cancer mode.

6.3-1 Inorganic Tumour Suppressors and Tumour Promoters in H

As discussed in the preceding section, a range of degenerative diseases including cancer seem to be

attenuated by the administration of H A1.3-1 (cf., Engelberg1), modified H or H mimetics but some of this

attenuation may, however, be mediated via the inorganic elements attached to H which can include the

tumour suppressor inorganics Ga, Pt and As (cf., Park et al. 1).

Alterantively some of the inorganic elements known to occur in H (e.g., Cd, Pb and Al) could

also conceivably promote cancer. To serve as an anti-cancer agent such elements should therefore

be removed from H.

A pathological perturbation of the ability of the heparanome to interact with extracellular environments

could also suggest how cancer could might be induced via HS microstructures modified by the actions of

tumour promoter inorganic surfaces such as asbestos fibres calcium phosphate particles, glass fibres or (cf.

Grant et al., 1 1992b). Similar pro- cancer glycosaminoglycan-containing silicate deposits seem to be formed

in the urinary systems of saccarin-fed male rats (Cohen1)).

Tl (an inorganic ion of moderate abundance in the environment and considered to be highly toxic) appears
suprisingly reproducibly in H (cf. Table IV). The amount present in blood serum seems to be less well
defined. It is however possible that the occurrence of Tl attached to H and perhaps also HS in vivo may
have physiological relevance e.g. I regrd to the ability of H and related polysacchrides to confer anti
degenertive diseases properties.

6.3-2 Induction of cancer by inorganic arrays could arise from the disturbance of extracelluar
heparanome control systems
N.b. H/HS contain mineral-like sulphate anionic group arrays; these perhaps following their inappropriate

interaction with exogenous mineral surfaces, per se can induce altered primary biosynthesis of HS

microstructure, cf. Borges et al.,1 in an apparent attempt to limit the potential negative impact of such

crystal structures on tissues.

The occurrence of tyrosine nitration is a characteristic feature of all degenerative diseases including cancer

(e.g., Masri et al. 1). This includes tyrosine nitration produced by nitric oxide metabolites. Tyrosine

nitration may occur by a redox metal e.g. catalysed process invovling nitric oxide metabolites (additional to

peroxynitrite which has traditionally been believed to be the principal active form of reactive nitrogen

responsible for tyrosine nitration) which are known also to react and deaminatively cleave H/HS**.

A dependence of nitric oxide metabolite HS-related tissue damage on insufficiency of

ascorbate could then, can rationally be suggested be centrally relevant to the etiology of cancer which can

be argued to be promoted by diminished tissue protection engenedered by a redox-related HS scenarioc (an

idea supported by the observation that ascorbate in cell culture experimental observations greatly increased

the rate of synthesis and degree sulphation of fibroblast HS (evidently via the modulation of the primary

biosynthesis1a-7 and that ascorbate efficiently reverses the reaction by which tissue damaging nitrite is

formed by oxidation of nitric oxide).

Excess nitrite present in cured meat (where iron nitroso compounds also appear to be present) had been traditionally
associated with the promotion of stomach cancer which was thought to occur by inappropriate nitrosation of key biological
signaling molecules which were nitrosated by nitrous acid plus redox metal ions and thiocyantate (an anion which is
apprently augmented in a direct dose dependent fashion with cigarette smoking). The deaminative cleavage of H/HS by
nitrous acid seemed also to be subject to catalysis by inorganic redox ions including iron which had been indicated to create
pro-cancer nitrosations
Ascorbate has known anti-cancer effects which at least in part can be attributed a diminution of exessive nitrosative
cleavage of HS.
c
The possible benefits of ascorbate therapy for the treatment of cancer and viral infections was discussed by Linus Pauling.
An update of this work was recently reported by Frei et al.1 who discussed the recent findings of Chen et al that i.v. ascorbate administration inhibits cancer
much more effectively than than the dietary supplementation method studied by Pauling.
Such effects can now be rationally ascribed in part to an augmentation of HS tissue protection afforded by ascorbate therapy (cf. D Grant, internet
discussions posted in 2000 at web.ukonline.co.uk/dgrant/dg4/ [similar addresses with dg2/ /dg5/ and dg8/].
A preferred rationalization of the anti-cancer action of ascorbate is via tumour inhibition following the formation of hydrogen peroxide. A separate
mechanism invovling HS has been recnetly discussed by Fransson & Mani1 et al.

A similar list can be suggested for effect of diet on nitric oxide biochemistry but the details of how this fully impinges on HS biochemistry are less clear
apart from the utilization of nitrosative scission for generation of HS oligomers used as inter- and intracellular signaling molecules.

Tumour cell surface HS PG can both inhibit and promote tumour growth via actions of H-binding growth

factors, including the inhibition of angiogenesis for the generation of tumour blood vessel nutrient supply,

and the inhibition of heparanase dependent metastasis, scenarios which have suggested the design of a range
of anti-cancer H-like therapeutic agents.

Each cell type seems to be controllable by specifically microstructured HS activities including control of

proliferation and differentiation, cell-cell communication, signaling to the nucleus and growth factors some

of which activities may also critically depend on the presence of specific inorganic ions including sulphate

[cf. also the list of metal ions suggested by Tables II &III] as well as redox status (dertermined by such

input as ascorbate, nitric oxide, hydrogen sulphide and homocysteine). This suggests a future possibility for

designed alteration of HS sulphation patterns so as to optimize the tissue protection functions of H/HS.

Nitric oxide directly activates MMP9 which releases pro-angiogeneis VEGF (a H/HS binding growth factor which requires
the presence for such binding of 2OS anionic groups in the H/HS hexuronic acids).

Nitric oxide overexpression seems to be a promoter of cancer. This effect may be due to an altered nitrosative processing of
HS under pro-cancer conditions.

ZnO (e.g. as used in dental prostheses) inhibits MMP2 and MMP9.

Syndecan 2 suppresses MMP2 activation (and thereby suppresses metastasis).
PDGF promotes angiogeneis by promoting VEGF/pericyte release of pro-angiogenic mediators.

Integrins are surface adhesion receptors putatively subject to modulation by H/HS + multivalent ions involved in breast
cancer via P13/Akt pathways

Sulf 1 and 2 (HS 6O-endosulfatases) regulate the activities of growth factors (e.g. FGF and Wnt);
Sulf 1 is a potent inhibitor of myoloma tumour growth and also a liver tumour suppressor; Sulf 2 can also
act as an oncogene promoter.
A transmembrane HS related TGF-β receptor has been reported to counter prostate and breast cancer
Expression of p30 in rat lung was boosted by H. p53 is potentiated by S100/Ca2+/HS [S100A6 expression is boosted by H.
The tumour suppressor p53, defective in many cancers, negatively regulates S100A6].

A list and brief discussion of references which implicate H/HS biochemistry in the etiology of cancer is

given below (Ref. 1A3-1).

------------------------------------------------------------------------------------------------------------------------
6.4 Does HS provide for a servo feedback system which directly interfaces the
environment and which allows dietary manipulation to produce beneficially altered
HS microstructure ?

While ascorbate, retinoic acid, and ω -3 polyunsaturated fatty acids (e.g. in aggregated form) [cf., Table
III], apparently induce beneficial HS structural alterations, excess blood glucose, oxidized lipids (and
other inappropriate lipids?), virus infections etc. 1a-7, and toxic ions (Pb2+, Cd2+ and Hg(II)) as well as
very high concentrations of F- (cf. Table II) can apparently negatively perturb HS microstructure and
associated activity. (It is conceivable that trace amounts of inorganic elements which hitherto have
been considered to be toxic might also be required to optimize the H/HS metallomic matrix chemical
composition, perhaps by inducing a fine-tuned water structure, cf. section 4). HS microstructure, it
seems, can also be induced to change in direct response to the presence of pathological organisms
(perhaps sensing their surface ionic patterns) and specific inorganic surface/crystal structures (e.g.
calcium oxalate crystal surfaces which can apparently signal for the synthesis of a matching HS
microstructure to optimally inhibit the growth of such crystals; cf. F.T. Borges et al.,8 ; these studies
also could suggest a possible similar scenario with hydoxyapatite). There seems to be overall
sensitivity of HS microstructure flexibility to attempt to combat or enhance a wide variety of
extracellular and environmental signals.

The attainment of correct inorganic element supramolecular structuring is also likely to be highly dependent

on the existence of correct H/HS microstructures as well as the presence of the correct distribution of

inorganic elements in the bathing fluids sources of the inorganic ions and particles which attach to H and

HS. Pathological influences will include alteration of H/HS microstructure during biosynthesis or

postsynthetic structural alterations so as to diminish the structures needed to accommodate an optimum

metallomic matrix and also the possible inclusion into this matrix of inappropriate amounts of

anthropologically augmented toxic inorganic elements from the bathing fluids.

It should be noted that H/HS polysaccharides seem to preferentially sequester those ions which occur in

such fluids in ultra trace amounts thereby permitting such elements to potentially amplify their putative

H/HS-related biochemical signaling inputs including the morphological modification (e.g., by Si) of

inorganic matrices such as nascent bone which are believed to controlled by adjacent HS-related

polysaccharide surfaces (cf., Wise et al.1).

The efficient binding by H of, e.g., Ca2+ ions or CaCO3 crystallization seeds requires GlcNSO3 groups and

depletion of such groups in H/HS greatly diminishes such binding of Ca2+ (Grant et al., 1). Redox

metal ions may both stimulate pathological calcification and Fenton reactive oxygen radical

generation. The metallomic nature of H/HS could input into normal and pathological calcification,

proteinaceous plaque formation and the ability of H/HS to provide antioxidant protection gates.

6.5 Pacification of redox active iron ions by their incorporation into polysaccharide aggregates.

Redox active Cu and Fe ions deactivated for potential free radical generation by their transformation into

sparingly soluble metal ion aggregates containing H/HS proteoglycans may act analogously to the

apparent antioxidant actions factilitated by similar aggregates formed by iron ions with other anionic

polysacchrides (Sipos et al.1 , Merce et al.1) and ternary complexes containing proteins such as amyloid
fibrils 1a-3.

Correctly structured H/HS including the optimal metallomic environment and water structure is likely also

to be required to accomplish these tissue protective actions.

H Fe and Cu-containing nanoparticles showed lack of paramagnetic ion penetration into the bulk solution phase (as suggested by
NMR)**.The major physiological counterions seem to have high mobility within the nanoparticle environment demonstrating fast
exchange on the NMR time scale but ESR spectra of Fe and Cu parmagnetic ions were difficult to interpret and indicated possible
multiple electronic interactions between the bound paramagnetic ions and H.** [cf. later reported ESR studies of Cu-H by Rudd et al., 1
where the interaction of Cu with H (especially when studied in D2O) was found to be somewhat different].

H/HS biochemistry has also been implicated in the mechanism of antioxidant protection additional to the

more commonly acknowledged antioxidant role of HS in potentiating the activities of enzymic superoxide

dismutases and selenoprotein antioxidative systems1a-6 the efficiency of which could also depend on the

presence of the correct inorganic elements cofactors needed both for active centre generation in the protein

and also to bind these proteins to the correct H/HS microstructures needed to sequester the proposed

optimal inorganic element matrix components.

An alternative explanation of the blood vessel wall change in HS could be suggested to a servo-feedback
response to counter the age-related diminution of fibrinolytic activity, increase of serum ferritin
(Jarrett et al.l ) or tendency for formation of hydroxyapatite crystallites at blood vessel surfaces which could conceivably
signal for altered HS microstructure generation to combat these potential threats (this process is suggested by the results
published by F.T. Borges et al. 8).

The hitherto unacknowledged roles of perturbation (e.g. by their lower or higher than optimal concentration

or by the inappropriate presence) of inorganic cofactors may explain why H/HS molecules, their fractions

or inorganic adduct polymers, oligomers and their mimetics have been reported to both inhibit

(Engelberg1,1999) as well as to promote (cf., Ouida et al.,1 and refs. 1A3 et seq.) tumour growth and similarly

both inhibit or promote a range of other degenerative diseases as augment, diminish or promote the

activities of pathological organisms and the possibly related puzzle of why HS-related molecules can

apparently both promote as well as inhibit prion-dysfunction diseases.

6.6 Alternative and ethnic medical use of metallomic matrices**.

Could some of the non-anticoagulant suggested uses of H and claimed benfits
of Ayrvedic Shilagit etc. be dependent on the co-occurrence of similar
ultratrace elements in these preparations?

The alternative medical preparations Ayurvedic ‘Shilajit’ and some North American
(perhaps originally derived from Native folklore) seems to be classifiable as metallomic
matrices preparations (apparently derived from of multi-inorganic-element containing
geological fulvates). The seaweed preparation ‘Kelp’ is also a multi-inorganic-element
anionic polysaccharide rich metallomic matrix.
It has been claimed (cf. internet documents**) that the presence of trace and ultratrace
inorganic elements additional to those which are currently accepted as being required for
human nutrition in these preparations, has nutritional or therapeutic value, and it has
further been argued that modern intensive industrial agricultural practices has
diminished such trace and ultratrace elements in normal supermarket marketed foods.
Both the geological fulvates and Kelp apparently resemble H in the possession of such a
trace/ulttratrace repertoire of associated inorganic elements.
It might be suggested that at least some of the non-anticoagulant therapeutic benefit
which has been identified for H preparations (which have also been reported to vary
between manufacturers for their provision of such activities), and the apparent health
benefit of ‘Shilajit’ and ‘Kelp’ is due to the similar multi-element nature of these
preparations which could act by providing a source of the full range of trace and
ultratrace elements which are required for optimum human health.

7 Interference from Inorganic Constituents in H during Blood Analysis

(Interference from the multiple inorganic constituents associated with H used as an
anticoagulant during the dertermination of inorganic elements in blood)

Multi elements occur at the greatest abundance in the traditional form of unfractionated Na H but they also

occur, albeit in reduce amounts, in “Lithium H” (cf. Table IV).

Such (claimed) single counterion forms of H are now suggested to be more correctly be described as

modified multi-element matrices.

This circumstance confirms older observations3b-1 that, if commercial H is employed as an anticoagulant for

blood inorganic element determinations, account needs to be taken of the potential interference for the

accurate determination of the inorganic elements in blood samples; the ‘inorganic contaminants’ present

in H clearly especially affect the accurate determination of those elements which occur in ultratrace

amounts in biological samples.

A recent study 3b-10 of the leaching of inorganic elements from a range of PET blood collection tubes

confirmed the traditionally held view that the presence of H (and to a lesser extent with other additives

used) in such tubes can cause large blank values for the assessment of wide range of inorganic elements

present in human blood. For the attempted accurate determination of any specific trace or ultratrace

element in blood, the attention must be paid to a careful selection of the most appropriate type of blood

collection tube for that element in order to minimise the blank correction required.

8. Variation between manufacturers of the degree of final purification of H

It is suggested that possible variation in ‘purification’ procedures between commercial H manufacture

operations can give rise to large differences in the multi-inorganic element contents of H which might

account for the previously reported but hitherto inexplicable irreproducibility in the biological
activities of commercial H samples from different manufacturers 3b-10, 7.

Variation in the Al content of H from commercial manufacturers has been noted (Bohrer et al.1)

The presence of some elements likely comes from containers, e.g. Sb and Co from catalysts used in PET

manufacture (which seems to affect the ICP-MS results of leached H listed in Table IV (Fig. 6) and Ti from

HDPE which might account for the higher than expected value of this element in the SSMS (Aberdeen

results) listed in Table IV (cf. Fordham et al.,1).

Unacceptable variation between commercial H preparations for the clinical laboratory analysis of plasma

HDL cholesterol using the H plus divalent metal ion method can apparently be avoided by use of the

semisynthetic H-mimetic dextran sulphate (or suphated α cycoldextran) ([Warnick 1982, 2001], Cham1).

The amounts of alkaline earths in seven H samples varied in a manner suggestive of the notion that all

of these Hs (from four manufacturers) had started off with similar (higher) alkaline earth content

native forms present in the mast cells and the difference in the final Hs arose as a result of different

degreees of ion exchange resin ‘clean-up’ (cf. Fig. 3 (page 80)) .

For fundamental biochemical researches, it seems evident that the actual in vivo, putatively multi-inorganic

forms of H and HS should always be employed, but this hypothesis requires to be more fully established by

further work.

9. Could Systemic HS Signaling Generate a Biological Clock which is Speeded

up During Degenerative Disease Processes?

The ‘hub system’ of HS protoglycans (in concert with Na+, K+, Ca2+, Mg2+ other metal ions and nitric oxide) may
empower temporal control systems which include those directing cellular assembly during embryogenesis as
well as the controlled alterations of HS microstructures which influence the course of the normal ageing
processes in individual tissues and possibly also within in the entire animal organism. Results tabulated by
Murata & Yokoyama1 suggested that a linear age-dependent diminution occurs of the relative amounts HS
occurs at arterial surfaces; E. Feyzi et al.1a-5 detected a linear age-related alteration of the microstructure of HS
(such a change in HS was considerd to be the only example of an uniquely age-dependent biopolymer system
structural variation which accurately depended on the age of the animal organism and which is ultimately
responsible for the age-related increase in atheroma at vascular surfaces; cf. ‘correctly’ microstructured HS (but
not, e.g., HS depleted of NSO3- groups, cf. Grant et al., 1(1992) ) which are known to strongly inhibit the formation
of sparingly soluble Ca salt deposits could further suggest that the age-determined alteration of HS
microstructure provides the basis of the biological clock which ultimately determines lifespan. A short time
span HS-determined timing mechanism may also influence the cell cycle. Cf. HS has been reported by B.
Roussel et al. 1, to occur in the mitotic spindle and an HS-microstructural alteration has been associated with the
absorption of light (cf. circadian rhythm) by the pineal gland1a-6 and vascular surface HS is thought to act as a
flow meter at endothelial surfaces (e.g., Siegel et al.1 who proposed that this function was enabled by a Ca2+/Na+
dependent alteration in HS conformation). Such an alteration, if it occurs in a cyclical manner, could be the
mechanism by which the apparent time regulated clock-like actions of HS are achieved.

The dynamic regulation of HS microstructure and generation of small oligomers of HS by nitrosative scission
may also occur in concert with a similar but apparently separate mechanism of post-synthetic alteration of HS
microstructure achieved by 6-O sulfatase (Sulf) actions.
Both time regulated HS processes could occur in concert with inorganic cofactors (especially Ca2+) with input
from the water structures elicited by entire metallome to create temporal and spatial patterns of signals involved
in the ageing processes but which seem to be capable of being accelerated during degenerative disease processes.

HS biochemistry can be suggested from published studies also to be involved in cognition.

While a discussion of these papers is outwith the scope of this article it is worth making the point that the
regulation of temporal activities could perhaps be the most primitive form of cognition.

10 Tables

10. 1 Table I

Examples of putative metal-ion driven H/HS signaling

A Putative H/HS Inorganic Ion Environmental Interface

Some examples of reported of metal-ion-dependent HS signaling by, e.g.,
involvement of metal ions in H/HS interactions with proteins are thought to be involved in blood anticoagulation, arterial
calcification, breast cancer, basic fibroblast factor dimerization, endostatin (collagen XVIII), collagen V and prion
interaction, the actitivities of a range of proteins and nitrosative scission of H/HS

HS potentiation of Ca2+ signaling

(Hypothesis that HS is centrally involved in inorganic biochemistry)
W.F.Long & F.B.Williamson, IRCS J. Med. Sci., 1979, 7, 429-434

Strong evidence was recently obtained that HS is involved in Ca2+ kinetics in muscle action; electrically induced Ca2+ spikes had significantly lower
amplitude in skeletal muscle physiology; this comes from studies of skeletal muscle tissue derived from NDST-1 -/- mice and NDST-1 -/- cultured
myocytes [but such effects were not observed with NDST-2 -/- of heterozygous myotubes]).
A defect in lung muscle action was suggested to account for the release of Ca2+ induced by inositol 1,2,5-trisphosphate (from rat liver microsomes) which
was inhibited by H (P. Cullen et al., FEBS Lett., 1988, 228 (1) 57-59).

Ca2+
Blood Anticoagulant Action of H (Antithrombin mechanism)
Indirect cofactor of H mediated blood homeostasis

W.F.Long & F.B. Williamson, Biochem. Biophys. Res. Commun., 1982, 104, 363-368
Potentiation by Ca2+ of the antithrombin (III) inhibition of thrombin

Cf., R.N.Pike et al., FEBS J., 2005, 272, 4842-4851

Ca2+ is needed to overcome the negative effect of Gla domains of Factor Xa on the templating action
mediated by H;
{The antithrombin-binding pentasaccharide sequence has been firmly established to be the
principal mechanism by which H influences blood coagulation (cf., e.g., Bourin & Lindahl1)}

Blood Anticoagulant Action of H (Heparin cofactor II mechanism)

Ca2+ Potentiates
Heparin cofactor II binding to H

F. Zhang et al., Clin. Appl. Thromb. Haemost., 2004, 10, 245-257

Zn2+ R. Eckert & H. Ragg (loc. cit.) found that Zn2+ had a much greater cofactor effect
than did Ca2+ for the promotion of this interaction

Ca2+ & Arterial Calcification

The binding of LDL cholesterol to GAGs at arterial walls is believed to be a possible trigger of
atherosclerosis.
Whereas the precipitation of ternary complexes of GAG-Ca2+-LDL is apparently a delicately balanced
process for HS (being highly dependent on pH, the presence of Ca2+ and I) it is much less so for chondrotin
sulphate-dermatan sulphate which more readily form insoluble ternary complexes (cf., P. Vijayagopal et al.,
1981, 1983, loc.cit.). It seems that a sufficiently high ratio of HS/chonroitin sulphate-dermatan sulphate at
arterial walls is required to inhibit calcification. It may further be suggested that the value of I at HS
surfaces might also critically depend on the array of ultratrace multivalent metal ions other than Ca2+ which
occur in blood serum (Haraguchi, loc. cit.) and potentially could become highly enriched at HS surfaces (cf.
Table IV).

Ca2+ & Breast Cancer

Ca2+ enhanced HS containing PGs activities which may modulate tumour cell growth
B. Vandewalle et al., J. Cancer Res. Clin. Oncol. 1994, 120 (7) 389; Chem. Abs. 121, 105487j
Ca2+ activated S100 proteins (which bind to HS (Robinson et al. 1) are upregulated in a p53 dependent manner (this tumour suppressor and hence S100 (cf.,
S100A6, Orre1) and Ca2+ is believed to be closely linked with the etiology of (breast) cancer).

Bivalent cations
(e.g. Ca2+, Mg2+, Mn2+) are required for HS modulation of
Basic fibroblast factor receptor dimerization;
M. Kan et al., J. Biol. Chem., 1996, 271, 26143-26148; G. Siegel, et al., Cardiovasc. Res. 2003, 58, 696-705.
(This apparently also requires the antithrombin binding (H pentasaccharide epitope) in HS
W.L. McKeehan et al. ibid., 1999, 31, 21511-21514 which is especially sensitive to the effect of counterions as revealed by comparison of
the shift induced by counterions in the NMR signals between the antithrombin binding site and regular disaccharide repeat units in H. (Reported by D.
Grant et al., Cell Biol. Intern. Rep. 1987, 11, 220)
{Cf. also T.R. Rudd et al., Glycobiology, 2007, 17 (9) 9983-993}

Ca2+ Collagen V HS binding

S. Ricard-Blum et al., J Biol. Chem. 2006, 281 (35) 25195-25204
(Zn2+ was found to be inactive as a cofactor of this binding;
it was believed that the initial binding of Ca2+ was to HS not to the collagen)

Similar HS protein interactions, discussed by Kan et al. loc. cit. include

Bivalent cations are
required for correct HS interaction with
β 2 and β 3 integrins,
Platelet/endothelial cell adhesion molecule-1 (PECAM)-1)
Cadhedrin and
L-selectin;
Cf., L-selectin in nonlymphoid endothelial cells;
K.E. Norgard-Sumnich et al., Science. 1993; 261: 480-483.
N.b. Integrins are highly significant surface adhesion receptors (and are involved in many types of signaling with HS PGs are also
involved in) contain alpha subunits containing multiple Ca2+ binding loops and beta chains which contain a second type of metal ion
binding site. Integrins seem to form ternary complexes with ligands and metal ions in which the metal ions can modify reactivity both
positively and negatively. Some such adducts are currently known to involve HS PGs and it is suggested that this involvement of the
heparanome and the metallome may be more general than is currently established.

Ca2+ is also required for HS mediated

Annexin V assembly on cell surfaces
I. Capilla et al. Structure (Camb.) 2001; 9: 67-64, cf., A Lewit-Bentley et al., Eur. J. Biochem., 1992; 210: 73-77

Bivalent cations
Zn2+ etc. are required for
Endostatin binding to H and HS
S. Ricard-Blum et al. J. Biol. Chem. 2004: 275: 33688-33696);
2+
Ca is required for
Serum amyloid P binding to HS
H Hamakai, J. Biol. Chem. 1987; 262 ; 1456-1460);
2+
Ca enhances
Serum amyloid P aggregation inhibition by H and HS
E.H Nielson et al., APIMS. 1994; 102: 420)

Ca2+is required for H modulation of the

Acrosome reaction
and also the required
Ca2+ flux for Capacitation
A. Valencia-Sanchez, et al., Mol. Androl., 1995, 7 (1,2) 57, Chem Abs. 122, 2821787

Ca2+ transport for ATPase activity inhibited by H in sarcoplasmic reticulum of skeletal muscle, the plasma membrane of red cells and dense tubular
sytsem of blood platelets but not the Na+/K+ ATPase of the mitochondrial F1 ATPase in the reversal of the Ca2+ pump
H DeMers et al., J. Biol. Chem. 1994, 269 (20) 14529

Divalent cations are required for H binding to

Fibronectin
M. Hayashi & K.M. Yamada, J. Biol. Chem. 1982, 257, 5263-5267

Ca2+ is required for extracellular

Fibrillin-1 binding to HS proteoglycans
K. Tidemann et al., J. Biol. Chem. 2001; 276: 36035-36042

Extracellular [Ca2+ ]
regulates the distribution and transport of HS proteoglycans
and fragments in a rat parathyroid cell line
Y. Takeuchi et al. J. Biol. Chem., 1990, 265 (23) 13661-13668

Ca2+ is required for aggregation of

Syndecan-1 transfected cells, mediated by HS,
M.J. Stanley et al., J. Biol. Chem. 1995; 270: 5077-5083

Zn2+ and Ca2+ affect the neutralizing ability of

Histidine rich glycoprotein by H
Y. Kazama & T. Koide, Thrombosis Haemostasis, 1992; 67: 50;
Cf. also A.L. Jones et al., J. Biol. Chem., 2004, 279, (29) 30114-30122)

Zn2+ promotes the association of HS with

biotyinylated H-Kininogen
T. Renne, J. Biol. Chem., 2000; 275: 33688-33696)

Zn2+ (Evidence for a Zn modulated H superfamily)

Amyloid β protein precursor
A.J. Bush et al., J. Biol. Chem. 1994, 269 (43), 26618
Zn2+ (to a lesser extent Cu2+ and Ni2+) H binding to heparin cofactor II
R. Eckert & H. Ragg, FEBS Lett., 2003, 541 (3) 121-125

Cu(II) forms bridges in oligomeric complexes in

Prion protein - HS and GAG complexes
R. Gonzales-Iglesias et al., J. Mol Biol., 2002: 319: 527-540)

Cu(II) as Cu(II) tripeptiede (GHK Augments GAG expression (as well as

collagen,metalloporoteinase, elastin and protease expression)
Simeon et al. J. Invest. Dermatol., 2000, 115 (6) 962-968

Cu ions promote the inhibition of angiogenesis by H

[H can act as a Cu ion carrier (in a similar manner to ceruloplasmin and glycyl-L-histdyl-L-lysine)]
K. Raju et al., J. Natl. Cancer Inst., 1982, 69, 1183-1188

Copper-Dependent
Nitrosative Scission of HS
K. Ding et al., J Biol. Chem., 2002, 277, 33353-33360

Nitrosative scission of HS is a Cu2+/+, Zn2+ dependent mechanism;

Nitric Oxide can be stored e.g. at conserved cysteines in the HS core protein, which together with
Cu2+/+ and Zn2+ can potentiate
Nitrosative scission of H/HS at GlcNH2 sites
Cf. , e.g., F. Cheng et al. J. Neurochem., 2006, 98, 1445-1457
A mechanism by which various environmental inputs (both chemical and physical) can positively or
negatively impart into HS activity include direct effects of diet on nitric oxide biochemistry and metal
ion-dependent nitrosative postsynthetic alteration of HS. This could ultimately account for how diet
controls health. Cf., 1a-7 which lists how some known dietary factors have been reported to effect change
in HS structured.

Mn2+ potentiates the inhibition of

Soluble Guanylate Cyclase by H (and other sulphated polyanions)
 M.A. Liebel et al., Biochem. Biophys. Res. Commun., 1982, 104, 957-964
[Cf. the invovlement of the L-arginine-nitric oxide, cyclic guanosine phosphate pathway in the antidepressant actions of
“memantine” (Almeida et al. 1 ) and related studies which implicate roles for elevated nitrite (and associated low plasma
Mn and diminished arginase activity) in a range of neurodegenerative diseases cf. Vular et al.1].
Arginase (which hydolyses arginine to urea) activity is dependent on the presence of Mn2+-Mn2+ . (Kango et al.,
Nature, 1996, 383, 554-557) also it is believed, further loosely associated Mn2+ completes this active site, the later is
apparently subject to blocking by
B(OH)4 - [N. Carvajal et al., J. Inorg. Biochem., 1999, 77 (3-4) 163-1667

Fe2+ (and Cu2+) ions apparently catalyse HS-GlcNSO3- à HS-GlcNH2 i.e. prime HS for nitrosative cleavage
D. Grant et al. (unpublished observations, Aberdeen University)

Na2SO4 hK3 kininogen is strongly activated by this salt and also by H

hK6 kininogen is also affected by Hofmeister anion effects

(Cf., P.F. Angelo et al., J. Biol. Chem. 2005 MS 10096200)

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

Cu2+, Zn2+
Cf. Fe3+-related labelled Biotinylated
H – Binding to Lactoferrin
was augmented by 100µ MCu2+ but inhibited by >500µ M Cu2+ or >40µ M Zn2+
S. Zou et al. Comp. Biochem. Physiol.,1992, 103B (4), 889-895
Cf., in vivo diffusion of lactoferrin in brain extracellular space is regulated by interaction with HS
(R.G. Thorne, et al. Proc.Nat. Acad. Sci, USA, 2008)

Cf., HS proteoglycan and the hepatic recycling of transferrin

W.L. Hu & J.Y-P Sheng, Biochem. Cell. Biol. 1992, 70, 535-538

Possible Clue as to why Si occurs in H/HS-to Act as a multi-elelment conveyor and sorter
SiO2-based chromatographic packing medium-H allows both cations and anions can be
separated on a single heparinized chromatography column
T. Takeuchi et al. Analusis. 1998, 26, 61-64

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

10.2 Table II

Reports of (apparently direct) modulation of HS microstructure and/or HS PG synthesis in response

to the presence of inorganic ions etc.

Ca2+
Direct influence on HS biosynthesis:
Extracellular [Ca2+ ] directly signals for increased HS synthesis in thyroid cells
Y. Takeuchi et al., J. Biol. Chem., 1990, 265, 13661-13668

(Ca2+ related effect of high affinity metal ion chelator?)

Ethane hydroxydiphosphonate (anion) increased HS/Chondroitin sulphate ratio
(also associated with sn liver tumour cell metastatic potential)
J. Timar et al., Int. J. Cancer, 1995, 62 (6) 755-761
Inhibition of HS biosynthesis by Ca2+ ionophore A23187
in cultured vascular endothelial cells implication of
intracellular calcium assimilation in lead inhibition of endothelial PG synthesis
[Y. Fujiwara & T. Kajo, J. Health Sci., 2002, 48 (5) 460-466]

Mn2+ and Ca2+

a GlcNAc transferase I can use both Mn2+ and Ca2+ but a-GlcNAc transferase II can use only Mn2+
TA Fritz et al., J Biol. Chem., 1994, 269, 28809-28814;
[Cf., the X-ray structural data which included the location of the catalytic Mn2+ ions needed for glucuronyltransferase activity for HS and ChS assembly
which were reported by L.C. Pedersen et al., J. Biol. Chem. 275 (44) 34588]

Mn2+ increases HS synthesis

A.Z. Kalea et al., Biometals, 2006,19, 535-416

Mg2+ is required for HS synthesis

P. Jaya & P.A. Kurup, J. Biosci., 1986, 10, 487-493;

Pb2+ (related to [Ca2+]) diminishes HS synthesis

Y. Fujiwara & T. Kaji, J. Health Sci., 2002, 48, 460-466; Toxicology, 1999, 133, 159-169

Cd2+ diminishes HS synthesis

A. Cardenas et al., Toxicology, 1992, 76, 219-231

Interaction between
Cd2+ - Zn2+
affects production and sulphation of GAGs in cultured bovine endothelial cells
S Ohkawara, T.Kaji et al., J. Toxicol. Environ. Health, 1996, 47 (2) 183-193
(Zn 20µ M and Cd 2µ M prevented Cd-induced decrease in 35S uptake but not Cd-induced increase in 3H glucose uptake;
Zn may protect cells from Cd-induced inhibition of DNA and protein synthesis)
Cf., R.C. Chambers et al., Am. J. Respir. Cell. Mol. Biol., 1998, 19, 498-506
(Cd inhibits proteoglycan and procollagen production in cultured lung fibroblasts; greatest inhibition of proteoglycan synthesis occurred with the
major matrix-associated proteoglycans versican, decorin and the large HS proteoglycans; a lesser diminution occurred with other HS proteoglycans)

Hg(II),
Cd2+,
Mn2+,
Ni(II),
Pb2+ diminished HS synthesis
D.M. Templeton, Proc. Trace Element Health Disease, IUPAC Symp., 1990, p. 209-219;

Na+ depletion increased HS synthesis in spontaneously hypertenisve rats (but not normal rats)
G.N. Jysthirmayi et al., Res. Commun. Mol. Pathol. Pharmacol., 1995, 90 (1) 115; Chem. Abs. 123, 311506j; cf.,
High
[NaCl ] alters HS synthesis in spontaneously hypertensive rats (but effect suggested to be lactate and NAD/NADH dependent)
M. Cechowska-Pasko et al., Exp. Toxicol. Pathol., 2000, 52 (2) 123; Chem Abs., 133, 264382k

Extracellular
[NaCl] HS synthesis has apparently become adjusted during evolution to balance this
(Salinity of seawater and aquatic habitat i.e. mainly an increase in [NaCl] correlated with increased HS and GAG contents
of tissues for 15 species of aquatic invertebrates)
Cf., H.B. Nader et al., Comp. Biochem. Physiol., 1983, 76, 433-436

NH4Cl blocks lysosmal catabolism of HS

M.J. Bienkovski & H.E. Conrad, J. Biol. Chem., 1986, 261, 13575-13580

High
[F- ] depletes HS synthesis
K. Pawalowska-Goral et al., Fluoride, 1998; 31: 193-202

SO42- (sulphate tranporter requires thyroid factor and Vit. D isoform (1,25 dihydroxy D3) {suggesting related input from UV B
exposure})
P.A. Dawson & D. Markovich, Pflugers Arch.-Eur. J. Physiol,. 2002, 444, 353-359
Depletion of inorganic sulphate, however, in cell culture experiments was found not to diminish HS sulphation**
(cf., Iozzo 1, 1989; lack of sulphation of HS in colon carcinoma; reduced sulphation of HS is generally associated with
cancer)

[SO42-] produced an altered PG synthesis by cartilage

K Iti et al., J Biol Chem., 1982, 259 (2) 917-925

Cf., L.J. Hoffer et al., Am. J. Physiol., Regul. Integr. Comp. Physiol., 2005; doi: 10.1152ajphysiol.00325.2005
The extracellular fluid was found to be the predominant source of free inorganic sulphate for biosynthetic reactions in the body.
[Humans are believed to have the smallest extracellular inorganic pool of all mammals; this is depleted by the consumption of a
low protein diet and the ingestion of drugs which are metabolized by sulphation]. [For a review of cytosolic sulphation see
M.N.H. Coughtree, Pharmacogenomics J., 2002, 2(5) 297-305]
Cf., low
[PAPS] produces an altered HS microstructure
(P. Carlsson et al., J. Biol. Chem., 2008, 283 (29) 20008-20014)

Possible cross effect of HS sulphate depletion by drugs which reduce extracellular inorganic sulphate concentration; these include
acetaminophen cf., L.J. Hoffer et al., Am. J. Physiol. Regul. Integr. Comp. Physiol. (loc. cit.)

Calcium oxalate
crystal (surfaces) upregulated the formation of HS optimized for calcium oxalate crystallization inhibition
F.T. Borges et al., Kidney Int., 2005, 68, 1630-1642
[The most clear-cut nresults were obtained with calcium oxalate crystals, similar but less statistically significant results were
 shown by these authors for (hydroxyl)apatite]
Si (probably in the form of’ ‘SiO2’; suggested input into HS biosynthesis; possible (bone) hydroxypatite-related effect)
M.F.McCarty, Med. Hypoth., 1997, 49, 177-179

Sr2+ may augment and modulate GAG biosynthesis (possibly for HS structure optimized to hydoxyapatite {bone} surfaces?)
Y. Henrotin et al., J. Bone Miner. Res., 2001, 16, 299-308

Cf., E.R. Wise et al., Chem. Mater., 2007, 19, 5055-5057

It was confirmed by using advanced surface NMR procedures that bone calcification requires the presence of sulphated
polysaccharides at the active surface; the organic mineral interface during bone formation is mainly polysaccharide,
putatively HS.

Li+ (Addition of LiCl to cell culture inhibits the synthesis of phosphatidylinositol {required for glypican HS anchor} and
negatively
affects nuclear signaling by recyclied HS)
Cf., Involvement of phosphatidylinositol and insulin in the coordinate regulation of HS metabolism and hepatocyte growth
M. Ishihara et al., J. Biol. Chem., 1987, 262 (10) 4708-4716

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

Depolymerization of H by Actions of Metal Ions

Fe
B. Lahiri et al., Arch. Biochem Biophys., 1992, 293, 54-65

Cu
E. Liu & A.S. Perlin, Carbohydr. Res., 1994, 255, 183-191
------------------------------------------------------------------------------------------------------------------------------------------------------
HS metal ion binding- flow transduction mechanism

Ca/Na has been suggested to act by altering HS conformation to participate in a flow sensor mechanism:
S Siegel et al., Colloids & Surfaces A Physiol. & Energy Aspects 1998, 138, - 351
Cf. also, e.g., J.A. Florian et al., Proc. 2003 Summer Bioengineering Conference June 25-29, Sonesta Beach Resort
Biscayne, Florida (HS proteoglycan is a mechanosensor on endothelial cells).
Read at http://www.tulane.edu/~sbc2003/pdfdocs/0683.PDF

Mechanical stress has also been reported to regulate syndecan-4 HS expression in vascular smooth muscle cells
L. Li & E.L. Chaikof, Arterioscler. Thromb., 2002, 22, 61-68, and

Lamellar flow was found to induce cell polarity which leads to the rearrangement of proteoglycan metabolism in endothelial cells
(J. Grimm, et al., Thromb. Haemost., 1988, 60 (3) 437-441

Mechanical strain was also found to regulate the chicken Glypican-4 HS PG gene expressionin the avian eggshell gland
I. Lavelus et al., Am. J. Physiol. Regul. Integr. Comp. Physiol., 52, 12853-12867

Unsaturated HS disaccharides produced bacterial heparinase can inhibit of the

Na+/Ca2+ exchanger and the value of
cytosolic Ca2+
(K Shinko et al., J. Biol. Chem., 2002, 277, (50) 48227-48233)

Effect of reactive oxygen species (ROS) on the biosynthesis and structure of proteoglycans
(A. Panasyuk, et al., Free Rad. Biol. Med., 1994, 16 (2) 157-187)
Low concentration of ROS stimulated while high concentrations of ROS inhibted the incorporation of 35-S and 3-H-glucosamine into proteoglycans

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

HS & Cancer

A general principle seems to be that HS is greatly altered in cancer. Cellular tranformation is often associated with defective HS biosynthesis including a
reduction in the degree of sulphation of HS (cf., Iozzo 1, 1989, where in human colon carcinoma were found fully elongated glucuronic acid unsulphated
heparan proteoglycan, supporting the accepted model of HS biosynthesis which is believed to start from N-acetyl heparanosan; this work also showed
that depletion of inorganic sulphate could not account for this defect as sulphate for HS biosynthesis can be alternative supplied from methionine and
cysteines).

There is a growing literature on the role of HS in the etiology of cancer.

An attempt is made to review these in refs 1A3-1 (vide infra).
-------------------------------------------------------------------------------------------------------------------
10.3 Table III

Some Reported Examples of Effects of Inorganic & Organic Molecules on Alteration of

HS Microstructure and/or HS PG Synthesis

Putatively ‘beneficial-health’ effects on HS biosynthesis are produced by:

Ascorbic acid (stimulates GAG (especially HS) synthesis in cultured fibroblasts [J. Kao et al., Exp. Mol. Pathol. 1990, 53 (1) 1-10 (this confirms the
earlier reports of M. Edward and R.F. Oliver, Biochem. Soc. Trans., 1983, 11, 383; ibid., 12, 304) that this nutrient increased the degree of sulphation of
HS)
[This effect of ascorbate on modulation of HS microstructure suggests a mechanism by which dietary supplementary ascorbate counters viral
infections and cancer, cf., 1Grant (2000) Internet, {e.g. “Ascorbate & Cancer”}]
Recent studies (discussed by Frei et al.1) have suggested that i.v. ascorbate can achieve anti-cancer activity via the intermediate formation of
hydrogen peroxide]
-----------------------------------------------------------------------------------------------------------------------------------------------

Retinoic acid and cAMP up-regulation of 3-O-sulfotransferase-1 led to a dramatic augmentation of the anticoagulant form of HS in F9 embryonal
carcinoma cells [L. Zhang et al., (with R.D. Rosenberg) J. Biol. Chem., 2003, 273, 27998-28003];
Cf., Retionoids increased HS synthesis in human skin fibroblasts
[M. Edward, Br. J. Dermatol., 1995, 133, 223-230]
-------------------------------------------------------------------------------

Omega-3 polyunsaturated fatty acids regulate syndecan-1 (HS proteoglycan) expression in human breast cancer cells
[H. Sun, et al., Cancer Res., 2005, 65, 4442-4447]
--------------------------------------------------------------------------------------------------------------------------------------------------
Apo-lipoprotein E (apoE) containing HDL stimulates endothelial production of HS rich in H-like domains
[L. Paka et al, J. Biol. Chem., 1999, 274 (6) 4816-4823].
Perlecan HS proteoglycan mediates the antiproliferative effect of apoE
[L. Paka et al., ibid., 1999, 274, 36403-36408].
This HS, but not chondroitin or dermatan sulphate synthesis, was boosted by apoE which explains its beneficial anti-atherosclerotic effect on blood
vessels; cf., the paper by
A.E. Mullick et al., Arterioscler. Thromb. Vasc. Biol., 2007, 27 (2) 339-345
which suggests that apo-lipoprotein E3 and nitric oxide-dependent modulation of endothelial cell inflammatory responses are involved in anti-
atherosclerotic effects (which will involve HS cf., HS oligomers as intracellular signals with anti-mineral deposition potency can be generated by
nitrosative scission)
[LDL (on the other hand) was found by
R.S. Chana et al., Nephrol. Dial. Transplant, 2000, 15 (2) 167-22
to stimulate mesangial cell proteoglycan and hyaluronan synthesis]

Cf.also oxidized LDL induce aortic smooth muscle cell PG synthesis with enhanced native LDL binding properties
[M.L. Chang et al., J. Biol Chem., 2000, 275, 4766-4773]

[O2]

‘Diminished’ O2
Hypoxia Inducible Factor (HIF) related activities signal to GAG synthesis
J. Li, et al., J. Cell Sci., 2002, 115, 1951-1959
Hypoxic conditions led to increased HS synthesis with increased degree of sulphation
HS core protein expression also increased
Cells cultured under hypoxic conditions showed an increase in the ratio of cell surface HS to ChS and well
as an increase in the number of low affinity (HS-assoicated ) binding sites for FGF-2 which resulted in enhanced
responsiveness of hypoxic compared to normoxic endothelial cells to FGF-2 stimulation
 increased HS synthesis in large vein and cadiac microvascular endothelial cells
[J. Li, et al., J. Cell Sci., 2002, 115, 1951-1959]

‘Augmented’ O2 hyperbaric O2
(e.g. produced in a pressure chamber) also changed PG synthesis.
Hyperparic oxygen was reported to induce an increase in glycosaminglycan synthesis in wound fibroblasts
G.P. Roberts & K.Y. Harding, Br. J. Dermatol., 1994, 131 (5) 630-633.

-----------------------------------------------------------------------------------------------------------------
Polyamines
Inhibition of the endogenous synthesis of polyamines modulates glypican-1 HS structure
[K. Ding et al., J. Biol Chem., 2001, 276 (6) 3885-3889]
-----------------------------------------------------------------------
+Catechin increased HS synthesis
[W. Sinn et al., Biochem. J., 1981, 200 (1) 31

------------------------------------------------------------------------------------------------------------------------------------------------------------
Heparin (H)
H induced synthesis of more highly charged HS by vascular smooth muscle cells
[R. M. Mason & S.P. Williams, Biochem. Soc. Trans., 1990, 18 (5) 96].
Following intratracheal instillation of H the protoeomic profile of whole rat lung suggested the general reduction in cytosolic protein expression there were
significant increases of groups of proteins associated with calcium signaling (annexin V, annexin VI and calcyclin, cytosolic organization, immune
response and tumour suppression p30/hyaluronic binding protein-1 DJ-1/PARK-7 and HABP-1,
[A.A. Gabr et al., Respir. Res., 2007, 8 (1) 36].
Treatment of MRL lpr mice with low molecular weight H prevented changes in expression of ‘inflammatory’ N-, and 6-O-SO3- specific antibody-detected
HS domains in the glomerular basement membrane and on glomerular endothelial cells
(A.L. Rops et al., Nephrol. Dial. Transpl. 2007, doi: 10.1093/adt/gfm194)
H stimulates the synthesis and modulates the sulphation pattern of HS proteoglycans from endothelial cells)
[H.B. Nader, V. Buonasisi, P. Colburn, C.P. Dietrich, Cell Physiol., 1989, 140, 305-310]
Binding of anticoagulant H (plus a necessary compound Y cofactor) to endothelial cells was found to stimulates the biosynthesis of antithrombotically
active HS proteoglycan,
[M.A.S. Pinal et al., Braz. J. Med. Biol. Res,. 1994, 27p, 2191-2195
(Cf., Antithrombotic agents which stimulate the biosynthesis of anticoagulant HS having an increased degree of sulphation (achieved with a minimum
(pentasulphated) tetrasaccharide polysaccharide chain length.
Pharmaceutical low molecular weight H
Hs CY216, CY222 (from Sanofi/Choay) , OP 622 and OP 386 (from Opocrin) and
oligosaccharides produced from H by heparanase II digestion as well as
unmodified H) [M.A.S. Pinhal et al. Thromb.. Res., 1994, 74 (2) 143]
----------------------------------------------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------------------------------------------------
Glucosamine
Has been suggested to promote endothelial production of HS proteglycans and thereby to retard atherosclerosis
[M.F. McCarty, Med. Hypoth. ,1997, 48, 245; Chem. Abs. 127 12883u]
{This hypothesis of a direct boost of HS in osteoarthritis (for which glucosamine dietary supplementation has become a mainstream medically employed
therapeutic intreventive strategy) was not confirmed in well-designed human and animal study [C.Qu, (read at web uku.fi/vaitokset/2007/isbn978-951-27-
0756-0.pdf). However these authors might have come to a different conclusion if
a role for glucosamine as an HS-oligomer mimetic in nitrosative signalling feedback had been more usefully explored}
[Glucosamine does not seem to boost ChS synthesis in articular cartilage (C. Qu et al., loc. cit.)]
------------------------------------------------------------------------------------------------------------------------------------------------------------
Butyrate (sodium) altered HS biosynthesis (with increased sulphation) and also inhibited normal breast epithelial cell and cancer cell proliferation (e.g.
via a FGF-2 related mechanism)
V. Lambrech, et al., Exp. Cell Res., 1998, 245 (2) 239-244
_____________________________________________________________________________________________________
TGFβ (and Matrigel) stimulated basal lamina HS protoglycan production in basal lamina of mouse uterine epithelium
K. Nagasawa et al., Carbohydr. Res., 1992, 236, 165-189;
Also induced increased sulphation of HS in normal breast epithelial cells (but not breast cancer cells studied)
V. Lambrecht et al., Exp. Cell Res.., 1998, 245 (2) 239-244
---------------------------------------------------------------------------------------------------------
Thyroid hormones were reported (Y. Tshiba et al. DATA ) to caused an increase of protoglycan synthesis in fibroblasts in culture.
(Further work is required to establish the precise effect of such hormones on HS microstructure)
_________________________________________________________________________________________________________
Phorbol Ester PMA induces shift from chondroitin to HS correlated with fibronectin adhesion of MDS human leukaemia cells
J Timar et al., Anticancer Res., 1994, 14 (3A) 1227-1231
----------------------------------------------------------------------------------------------------------
Glucocorticoid regulation of proteoglycan synthesis in mesangial cells
M. Kuroda et al., Kidney Int., 2002 62 (3) 780-789
Pathogensis of diabetic vascular disease: evidence for reduced HS production
[T. Jensen, Diabetes, 1997, 46, Suppl. 2, S98-100]

Amyloid fibres were reported to stimulate GAG biosynthesis

M.J. Palmoski & K.D. Brandt, Biochem. J., 1975, 148 (1) 145-147
[Amyloid fibres have also been found to increase hyaluronan biosynthesis leading to decreased HS, vide infra]
---------------------------------------------------------------
Melanoma-cell –derived-factors were reported to stimulate glycosaminoglycan synthesis by fibroblasts
cultured as monlayers and within collagen lattices
M. Edward et al., J. Cell. Sci., 1986, 85, 217-229

ACE inhibition was reported to preserve HS protoglycans in the glomerular basement membranes of rats with
established adriamycin nephropathy
[B.S. Kasinath et al., J. Cell. Physiol., 1998, 167 (1) 131-136]
-----------------------------------------------------------------------------------------------------
Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated
Gene transfer
[M. Belting & P. Peterson, J Biol. Chem., 274, 19376]
------------------------------------------------------------------------------------------------
------------------------------------------------------------------------------------

Putentially both beneficial and/or deleterious effects are produced by:

Electromagnetic Radiation : ‘Light’

HS microstructure is subject to alteration by light
(linked to circadian rhythm/pineal gland)
B. Kuberan, R.D. Rosenberg, et al., J. Biol Chem., 2004, 279, 5053-5054
(Light induced 3-O-sulfotransferase expression alters pineal HS fine structure- a surprising link to circadian
rhythm)

2 deoxy β D xylose, D glucose and

Ethane,1-hydoxyl ,1-,1-bisphosphonate)(EHDP) (a high affinity metal ion e.g. Ca2+ ion chelator)
and
5 Hexyl 2 deoxy uridine (believed to be an especially effective reagent)
Was reported to alter HS synthesis (and to produce an increased HS sulphation)
EHDP was found to be an especially effective inhibitor of chondroitin sulphate synthesis
(but eleicited an increase the HS/chondroitin sulphate ratio) (this semed also to be associated with an increased liver metastasis
potential )
[J. Timar et al., Int. J. Cancer, 1995, 62 (6) 755]

Cortisol (hydrocortisone)(1.4.10-7M)
In a study of GAG synthesis in relation to in vitro ageing of (W1-38) diploid human fibroblasts, cortisol increased the proliferation rate and the saturation
density of cells but was associated with some overall decrease of GAG biosynthesis (similarly for hyaluronan, dermatan sulphate chnondrotin sulphate and
heparan sulphate) ; Phase II (senescent) cells showed an elevated sensitivity to the inhibitory effect (now while other GAGs than hyaluronan were
inhibited the latter was augmented).

VEGF165
This growth factor was reported to unduce synthesis of perlecan substituted with shorter HS chains but with unaltered sulphation via VEGF receptor 2 in
cultured human brain microvascular endothelial cells
[T. Kaji et al., Biochim. Biophys. Acta, 2006, 1760 (9) 1465-1474
---------------------------------------------------------------------------------------------------------

Putatively adverse or negative effects on HS biosynthesis are produced by:

Conventional (laboratory GAG biosynthesis inhibitor reagents e.g.)
Low [Sulphate]
Altered the synthesis of HS proteoglycans
[B. Tyree et al., Arch. Biochem. Biophys., 1986, 250 (1) 202-210]

Inadequate
[PAPS] The concentration of PAPS (3’phosphoadenosine 5’phosphosulfate) was found to alter the microstructure of HS during
primary synthesis (via modulation by PAPS of N-deacetylase, sulfotranferase action)
[In absence of PAPS, NDST creates random (and sparse) sulphation of HS.
This would seem to potentially be disease promoting]
In presence of higher PAPS concentrations NDST enabled systematic (non-random) progressive sulphation. This suggests
that defective PAPS provision could pathologically impact to altered HS biosynthesism (e.g. via defective PAPS
synthetases (PAPSS1 or PAPS2) genetics or expression). This might feedback to altered HS microstructure by altering
PAPS concentration as might deficiency of inorganic sulphate transport (which also seems to depend on thyroid factor and
VitD isoform activity elicited by UV light cf. Dawson et al.1
(P. Carlsson et al., J Biol Chem., 2008, 283 (29) 20008-20014)

β -D-xylosides
Modulate the synthesis of HS;
The effect depends on the aglycone
[T.A. Fritz et al., J. Biol.Chem., 1994, 269 (1) 3007]
Chlorate
Inhibits HS synthesis [e.g., H.E. Conrad, Methods Mol. Biol.,2001, 171, 325-328]

Chloroquine blocked catabolism of HS in lysosomes

[M. Ishihara et al., J. Biol. Chem., 1986, 261, 13575-13580]

Monesin
diminished the degree of sulphation of HS produced in cell culture
[L.O. Sampaio et al., J. Cell. Biochem., 1992, 50 (1) 147; Chem. Abs. 117, 188863v;

Thrombin
Reduced large HS protoglycan production in cultured vascular endothelial cell layers through inhibition of core protein
synthesis
[N. Fuji et al., Thromb. Res. 1997, 88, (3) 299-307]
Cf., Induction of synthesis of a large HS PG perlecan by thrombin in cultured human coronary smooth muscle cells
(this, it was suggested, occurred in advanced atherosclerosis)
[C. Yamamoto et al., Biochem. Biophys. Acta, 2005, 1722 (1) 92-102]

Thyroid Hormone negatively regulates HS protoglycan expression in skeletal growth plate

T3 negaively regulates the expression of core protein Gpc6, the HS polymerase EXT-1 and the HS modification enzyme HS6ST2
[J.H.D. Bassett et al., Endocrinology, 2006, 147, 295-305]
(Also affects inorganic sulphate transport cf., Dawson & Marocvich1)

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

Insulin
Altered the synthesis of proteoglycans in cultured human arterial smooth muscle cells
[V.B. Thogersen et al., Eur. J. Endocrin. 1996, 134 (3) 326-339]
and may activate the cell surface phospholipase C which cleaves (glypican) HS proteoglycan form its phosphatidyl anchor in
the hepatocyte plasma membrane
Cf. also
Insulin ( plus Myo-inositol / Phosphatidylinositol) actions were implicated in the
Li+ dependent inhibition of phosphatidylinositol anchor synthesis.

Both the synthesis and internalization of HS (including to the nucleus where an involvement in cell division and contact inhibition override was evident)
were dependent on the metabolism of myo-inositol by hepatocytes (involvement in the coordinate regulation of proto-HS metabolism and hepatocyte
growth and signaling to the nucleus by recycled HS)
[M. Ishihara et al., J. Biol. Chem. 1987, 262, (10) 4708-4716].

Elevated [Glucose] Alteration of HS in Diabetes & Related Conditions

High [Glucose] changed basement membrane HS proteoglycan perlecan gene expression in glomerular
epithelial cells [B.S. Kasomath et al., J. Cell Physiol., 1996, 167, (1) 131-136].
Adverse modification of HS due to
high glucose was also reported in mouse glomerular epithelial cells [S. Morano et al.,
Diabetes/Metab. Res. & Rev. 1999, 15 (1) 13-20] and mesangial and epithelial cells [S. Morano et al., Diabetes Metab.
Res. Rev. 1999, 15 (1) 13-20] as well as altered the production and degree of N- sulphation of HS in
cultured adipocyte cells [N. Parthasarathy et al., Mol. Cell. Biochem., 2000, 3, (1-2) 1-9]
and promote hyaluronan biosynthesis and diminish sulphated GAG biosysnthesis (this
was attributed to the induction of the formation of close packed cells (cf. contact inhibiton due to nuclear targeting by HS
(discussed by Ishihara et al., loc. cit.) [cf., T.P. Anastassiades et al., Arthritis Rheum. 1979, 22 (8) 871-876].
TGFβ 1 induced by
High [Glucose] regulated mesangial production of HS proteoglycans
[V. Kolm et al., Am. J. Physiol. 1996, 167 (1) 131-136]
also altered basement membrane HS proteoglycan perlecan gene expression in glomerular cells and promoted
Glomerular nephritis which is also associated with the masking of anioionic HS sites by Ig deposition
and associated albuminuria. (This process has also been described also in
Lupus nephritis where decreased HS expression in
glomerular basement membranes also showed increased presence of ‘inflammatory’ N- and O-SO3- domains identified by
antibodies (EWD3D10, AO4B08 and EW4G2) on glomerular endothelial cells in experiemental animals and SLE
patients). These actions were believed to be facilitated by the inflammatory cytokines (e.g.
TNF-α and IL-1β ) [A.L. Rops et al., Nephrol. Dial. Transpl., 2007, doi:10.1093/ndt/gfm194].

apoB-48 lipoprotein in diabetic mice was also

associated with a decreased HS proteoglycan production [T. Ebara et al., J. Clin. Invest., 2000, 105 (12) 1807-1818

Glucose uptake affected proteoglycan synthesis was the dominant activity in both rheumatoid and non rheumatoid
 fibroblasts in culture which
[Cf., S. Yung & T.M. Chang, Perit. Dial. Int., 2007, 27 (4) 375-390;
and F.H. Wapstra et al., Exptl. Nephrol., 2001, 9 (1) 21-27]

---------------------------------------------------------------------------------------------------------------------------
Hydrogen peroxide
is a mediator of the transcriptional regulation of HS biosynthesis,
(Cf.., recent reports have suggested that the anti-cancer activity of i.v. ascorbate is actually due to the killing of cancer cells
by hydrogen peroxide {Frie et al.1})
and
Catalyase overexpression induces an increase in anti-HS antibody 10-E4 epitope expression
{Related to presence of unsulphated glucosamines which are primed for rapid nitrosative scission}
[F. Nakayama et al., Cell Mol. Biol. Lett., 2008, 13 (3) 475-492]

Oxidised LDL (Ox-LDL)

regulates the synthesis of monkey aortic smooth muscle cell proteoglycans (including HS) that have enhanced native LDL
binding properties
[M.Y. Chang et al., J. Biol. Chem., 275 (7) 4766-4773

Circulating HS and H “attach to the venule walls and attenuate Ox-LDL induced leukocyte immobilization” (and associated
glycocalyx damage thought to be relevant to the etiologiy of atherosclerosis)
[A.A. Constantinescu, et al., Arterioscl, Thromb. Vac., Biol., 2003, 23, 1541-1547]

Homocysteine
diminished anticoagulant HS expression in cultured porcine aortic endothelial cells
[M. Nishinaga, et al., J. Clin. Invest., 1993, 92, 1381-1386]

Nonesterified fatty acids (NEFA)

reduced the degree of sulphation of HS (this apparently cuase an increased permeability to albumin); in smooth muscle cells
NEFA-albumin increased expression of the genes for core proteins of HS mediated by
peroxisome proliferator-activated receptor gamma; the matrix produced by cells treated with NEFA-albumin had a high
affinity for LDLs
[G. Camejo et al., Curr. Atheroscler. Rep., 1999, 1 (2) 142-149]

Cholesterol & Low Density Lipoproteins in blood plasma

Endogenous H appears to be negatively correlated with blood cholesterol
[H. Engelberg, e.g., Clin Appl Thomb./Hemostas. 1996, 2(2) 83-93
Cf., J.C. Klock, US Pat. 6291439]

Lysolecithin
may adversely alter subendothelial HS proteoglycan production
[P. Sivaram et al., J. Biol. Chem., 1995, 270, (30) 29760-29765];

Human-rIL1-b and TNFα

suppressed the formation of H-like compounds on cultured porcine aortic endothelial cells
[M. Kobayashi et al., J. Cell. Physiol., 1990, 144, 383-390]

Infection with
Herpes simplex virus type I in vitro
inhibited proteoglycan synthesis in human endothelial cells,
[R. Kanner et al., Am. J. Respir. Cell. Mol. Biol,. 1990, 2, 423-431]

Colchicine and vinbalstine (Antimicrotubular agents)

inhibited glycosaminoglycan synthesis by embryonic chick tibia and chondrocytes in culture
[H.W. Jansen & P. Bornstein, Biochim. Biophys. Acta, 1974, 362, 150-159];

Fibrin D-dimer
impaired the accumulation and anticoagulant properties of HS by rabbit coronary endothelial cells
[N.Y. Yevdokimova et al., Acta Biochim. Polon., 2003, 50 (1) 280]

Amyloid fibrils
stimulated the biosynthesis of GAGs in cell cultures in which hyaluronan was increased
but HS was decreased
[M.J. Palmoski & K.D. Brandt, Biochem,. 1975, 148 (1) 145-147]
[Cf., L.Li & E.L. Chaikof, Arterioscler. Thromb. Vasc. Biol., 2002, 22 51-68;

Endotoxin
caused a loss in HS of regions rich in sulphate half-ester groups at non-reducing end of GAG chain
[P. Colburn et al. (with C.P. Dietrich) Arch. Biochem. Biophys., 1996, 325 (1) 128

Endosulfatases
Sulfatase 2 (Sulf 2) up-regulated glypican HS PG in a manner which promoted tumour growth.
------------------------------------------------------------------------------------------------------------

10.4 Table IV
Examples of ‘metallomic’ arrays associated with H, kelp & human scalp hair
[n.b. H is a highly purified, protein free, animal mast cell-derived polysaccharide mixture]

Inorganic Elemental Contents (mg/kg) of Kelp, H & Human Scalp Hair

Baltic Seaweed3a-4 Commercial Kelp3a-1 Na+ Heparin Li Heparin Tl+ Heparin,3b-9 Human Scalp Hair15
3b-9 3b-10
Element (A:before ion exchange) (E after ion exchange)
(a) (s) (b) (c)
(D after ion exchange)

(commercially produced) (academic lab. product)

A D E1 E2 B C --------A/C--------
Si ? 1642 5900 116 126 1170 100 510(s) 11.6
Mg 4300-6000 2130 1300{<620}{<1001}{<260} 10 28.0(a) 59 3.6 28
Mn 410-420 1235 [1-65](d) 0.4 (0) (0) (0) 6 (s)
Ca 1200-5400 19040 30000 171 (0) 125 30 450 (a) 67 11
Sr 2180-2210 749 65 2 0.04 1 1.4 1.2(a) 54
Ba 372-381 12.8 140 1 0.3 14 1 0.16-1.6(b) ca.90+ 9
Na 460-510 principal 143 336 90
K 2000-7000 12800 2000 23 42 22 40 20.4(s) 98
Rb 0.05 3 0.1 <0.02 0.1 0.2 0.051(s) 59
0.15 (s) 20
Cs 0.047-1.08 trace 9 0.01 <0.01 0.01 0.1
Tl 2.93 8 0.2 0.1 0.001 principal
Cr 1.8 trace 30 0.5 <0.5 0.2 1 0.99(a) 30.3
Cu <10-20 6.3 730 16 7 10 <5 22.1(a) 33 70
Fe 280-320 896 1100 5 {<4} 24 10 19.0(a) 58 24
Ni 9.2-13.2 35 <170 8 1.3 0.6 10 1.49(a) ca.100 ca.125
Co 12.2 <80 {<0.01}<80 0.1 2 0.67(a) ca.100
Sn 0.06 5 {<0.31} 0.17 0.5 0.3 0.24 21
Mo <0.027 15.9 7 0.01 {<0.02} 0.2 <1 0.43(a) 16 96
Al 120-140 193 3-35f n.d. 14 16 16 n.d.
Ti 0.12 <390 0.2 0.0001 2 4 0.79(a) <493 <126
As 327 <15{<78} 0.08 0.3 1 0.274(s) <55 <167
Zr 10.7 0.01 5 0.05 0.002 0.02 0.3 0.21(s) 24
Ce trace 7 0.02 {<0.014}{trace} 3.5 0.093(s) 75
Ag 0.04 4 0.002 0.003 0.01 0.5 0.034(s) 118
Nd 5 0.01 0.003 0.04 1 0.039(s) 128
W 0.33 5 {<0.02} 0.01 0.07 <1 0.037(s) 135
La 0.19 7 0.005 0.003 0.003 1 0.045(s) 156
Zn 250-310 35.2 <80 16 34 49 7 150(a) <0.5
185(s) <0.43
Pb 0.14 16 0.8 0.2 0.4 4 7.1(a) 2.2
Ga trace 20 0.005 0.003 0.003 1 3.4(s) 5.9
Sb 0.07 1.4 2 3 3 0.003 1.7
Cd (1) 0.3 0.12 0.1 1.5

P 2110 440 61 77 135 ca.30 195(a) 2.3

169(s) 2.6
V 0.6-0.9 5.3 3 {<0.3}{<0.2} 0.7 <1 1.22(a) 2.5
B 194 <25 10 16 93 1 1.41(s) <17.7
I 130-160 624 10 1.3 {<0.08} 2 <1 2.9(s) 3.4
Cl 36800 5600 n.d. n.d. n.d. 1000 <580(s) >10
F 326.5 890 n.d. n.d n.d. 4 - -
Br 140-160 trace 130{<256} 34 3 0.9 4.46(s) 29

Notes:
 Data for Na H (which had been recommended as being suitable as a standard reference material to be distributed amongst a number of academic
institutions by a major U.K. manufacturer, which was further purified by extensive dialysis) before ion exchange and Tl H after ion exchange are taken
from ref. 3b-9. (This counterion was thought to be of especial interest because it enabled 205Tl NMR studies).
Data for commercial Na (D) and Li H (E1 and E2) (all of these Hs had evidently been ion exchange ‘purified’) are taken from ref. 3b-10, being for
E1 column t11 and for E2 the average of columns t2 and t3 (evidently similar Hs). The t10 (Na H) and t11 t2-t3 (Li H) given in ref 3b-10 have
been normalized to µ g inorgnic element/gH to allow direct comparison with the data from SSMS. (Additional data for further two H samples in
general agreement with the multi-inorganic element nature of H are given in ref 3b-10, columns t1 and t4). For a number of elements the values
reported are listed above as “?” (blanks) from leaching of ‘empty’ tubes were greater than when H was present in similar tubes [specially designed
experiments are needed to find the true blank values; the blank values Mg (160µ g/L) and Br (34µ g/L) are excessive and suggest these elements
were enriched in the plastic tubes and leached therefrom; the maximum value of Mg leached from H is howver be listed, thus{ }; the blanks for
Co (15ng/L) and Ce (2.2 ng/L) were also larger than probable true blanks.
An analysis of the tabulated data given in ref 3b10 suggests that the five Li H samples for which multi-elemnt data was listed tended towards similarity (when the data are normalized for inorganic sulphur which can be assumed to indicate the concentration of H present

and the amounts of individual elements in e.g., t3 were compared with the average amounts of elements in t1-5 . \the data suggest that the amounts of Ag, Al and perhaps also Ni and Zn in H tend towards a single exact H-inorganic element stoicheometry also

approximated for Cr, Co and Cu (1.4 times greater); amounts of Ti, Tl, Fe and As were 2-2.5 times greater in t3 suggesting that H has an especially high complexation activity for these latter elements.

Table IV compares H with alginate-based biomasses3a and human hair (data from the internet). The inorganic elements of a Baltic seaweed3a-4 seem to
include a large anthropomorphic component while the commercial kelp inorganic element values suggest an oceanic seawater origin of these elements the
distribution of which also shows greater similarity than does the Baltic data to the native Na H data of Table I which further suggests that the inorganic
components of the H samples listed in Table I are derived from a natural source. [These tabulated results suggest that Rb, Cu, Ni and Sn bind more strongly
to the combined NSO3- –OSO3-, C(O)O- anionic system of heparin than to single C(O)O- anionic groups system of kelp alginate, the most likely ligand in
that matrix].

G.E. Harrison & A. Sutton3b-2 had also reported , many years ago, the Ca, Sr and Ba contents of five commercial H samples {These data are plotted in Fig.
3}; these data appear also to be approximately related to the contents of these elements in a range of other (polysaccharide containing) matrices reported
by H.J.M. Bowen14 who in 1966 had listed the then known reported information on the multi-element contents of H (µ g/g) as: Mn 3.6, Ca 30-2900, Sr 5-
92, Ba 2.5-12, Cu 0.4 and Zn 28 3b-1.
Further Notes:
(a) ref. 15; (s) ref. 15a; (b) other internet listings; (c) ref. 15b ; (d) ref. 15d.
(e ) This (highly toxic counterion form) had been selected for in vitro 205Tl NMR (which showed rapid along-chain site interchange and perturbation of the
205resonances; the standard sulphonated polystyrene resin exchange cation exchange column method used for covertign the NaH to TlH was confirmed to
be highly effective (e.g. the residual Na content of the TlH was 90ppm). The SSMS results are semi quantitative; as well as the elements shown in Table
IV vanadium was also present but no figure is listed since there were complication with the spectral analysis for this element; the aluminium content also
cannot be listed because aluminium wa used to prepare the electrode for SSMS analysis; the 13C spectrum of the TlH was generally similar to that of the
NaH studies except for perturbation of the ring C resonances around 70 ppm and the carbonyl carbons and the anomeric carbons of Id2S and GlcNS).
Tl seems to occur in natural H in similar amounts to that found in kelp but when present in such matrices may not act as a high risk human intoxicant3b-9
but further specific studies are required to confirm this hypothesis [Cf. C.F. Moffat, 1987, loc. cit.].
(f) The amounts of Al in a range of commercial Hs have been listed by Bohrer et al.1
10.5 Further Background to the Studies of Metal Ion Binding and the Multi-Element Content of H
Commercial H, readily available as a highly purified, protein-free polysaccharide mixture, avidly sequesters both cations and anions in a relatively
indiscriminate manner and therefore potentially can host a full repertoire of inorganic ions6, 6a (both from ex vivo contamination13a and from uptake from the
wide range of small amounts of inorganic elements which occur in biological fluids4). Although it is possible that the toxic ions in H are bound in some
less toxic form, their occurrence in H used for dialysis and parenteral nutrition solutions is a topic of current concern3b-7,8. Although L.B. Jaques in his
excellent 1978 review6a had emphasised the unique physiological relevance of the sequestration of both positively and negatively charged inorganic ions by
H, at the present time there seems to be lack of awareness or interest in the wider relevance of this phenomenon. An analyses of a carefully selected
typical porcine sodium H by spark source mass spectrometry (SSMS) (C.F. Moffat “Synthesis, characterisation and applications of chemically modified
heparins”, Ph.D. Thesis University of Aberdeen, 1987) had revealed the presence of some 38 inorganic elements in amounts equal to or greater than1ppm.
An even larger range of inorganic elements could be detected in purified H studied by ICP-MS (in the context of commercial H anticoagulant employment
in PET blood collection tubes); the ICP-MS results for inorganic elements obtained from the extraction of blood collection vessels containing
anticoagulants with 0.01M HNO3, however, agrees in general with the SSMS results previously reported for native and ion exchange purified Hs
(confirming the presence of the 38 elements previously detected together with Al as well as the presence of smaller amounts ((Au)),((Be)),(Bi),((Dy)),(Gd),
Ge, Hf, Ho, (Ir), Li, Lu, Nb, (Os), Pd, Pr, Pt, Re, Rh, Ru, Sc, Se, (Sm), Ta, (Tb), Te, Th, Tm, U, Y and Yb (N.b. these remain in the pharmaceutical type of
single counterion enriched, purified forms of H).
The ICP-MS results also confirm a consistent occurrence of Tl in H.
Data in parenthesis and double parenthesis are where the occurrence of the element in question in H is progressively less well established than those
without parenthesis3b-10.
The SSMS results were for all elements which were present in H in amounts >1ppm whereas the ICP-MS results were obtained by leaching H from blood
collection vessels. The leaching procedure used may have skewed the distribution of elements somewhat (e.g. previously it had been suggested that Ce
was difficult to remove from H by ion exchange- the amount of Ce in the leachate studied by ICP-MS seems somewhat lower than would have been
expected from its high natural abundance suggesting a tendency for Ce containing H fractions to be less easily leached from PET surfaces.

Comparison of the data for multi-inorganic elements leached from blood collection tubes containing H or K2EDTA anticoagulants suggest
that on the basis of anionic equivalents, the purified, medical grades of anticoagulant H which had been used in these containers,
contained greater amounts of associated inorganic elements than did anticoagulant K2EDTA, especially for Pb, Fe, Al, Na, Ca, Mg, Ag,
Cd, Cu, I, Si, Sm and V which appeared to occur in the medical grade H at some 100 times greater molecular abundance, Au, Bi, Ga, Zr,
Yb, Y, Tm, Th, Sb, Ru, Re, Pd, Os, Gd, Zn, Rh, Ir, Ho, Hg, Ge, Er, Dy, Cr, Ce, As, W, Tl, Tb, Ta, Sr, Pr, Ni, Cs, Co, Be and Ba at 10-50
greater molecular abundance than in K2EDTA, Ti, Pt, Lu, and Hf in 5 fold greater amounts, whereas La, Mn, Eu, Mo, B, P, Sc and Mn
were present at similar abundance in both K2EDTA and purified H. Much larger amounts of Rb, La and Li were indicated to occur in the
K2EDTA than in the medical grade H 3b-10.
Pb 0.14 16 0.2-0.8 4 0.59
10.6 Table IVa
Comparison of Inorganic Elements in H and Chitosan
Comparison of the elemental content (mg/kg)
of chitosan from crab shells data from ref. 13 (obtained by using a final wash with demineralised water which avoids the uptake of metal ions from tap water, a source of
suspected but not documented contamination), data for unrefined and experimental Tl heparin are from ref1, Grant 2000a) and for commercial heparins are estimated from data
given in ref. 3b-10
Element Pure Kelp Heparin Tl Heparin Chitosan
unrefined commercial experimental
Fe 898 1100 4-24 10 ‘high’
Cu 6.3 730 7-16 5 1.03
Ni 35 170 0.6-8 10 2.03
Cr trace 30 0.2-0.5 1 0.36
V 5.3 3 <0.2-0.7 <1 0.12
Mn high <1 (0.4) <1 0.09
Pb 0.14 16 0.2-0.8 4 0.59
Ag 0.04 4 0.002-0.01 0.5 0.02
Cd trace <1 0.1-0.3 1.5 0.22
Hg 1.9 <1 0.003 <1 0.025

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

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10.7 Further Notes
The controlled uptake and release of inorganic ions from anionic polysacchrides (H-like polysaccharides in
animals) could, it is proposed, be part of a wider servo-control-feedback system used by organisms to exert
systemic controls and perhaps also to evolve new species.

The presence of multiple elements is a common phenomenon in naturally occurring salts as well as industrially
produced chemical reagents (including K2EDTA and gels employed to improve blood clot phase separation 3b-10).
Naturally occurring polyanion systems include polysaccharides, proteins (such as keratin responsible for the
multi-elements in hair and nail), the apatite mineral of bone (as well as naturally occurring apatites {cf. Van
Wazer 1, 1964}, polyphosphates (which occur ubiquitously at cell surfaces throughout biota) and soil and natural
water humic salts (which include a fraction termed fulvate). It is entirely in keeping with the general occurrence
of multi-element in salts found in nature (unless these have been subjected to chemical purification by
crystallization and recrystallization processes) that the ultra salt-like highly sulphated polysaccharides H and HS
which are the extreme forms of such biologically utilized salt systems exist as multiple inorganic ion arrays and
that inorganic ions could influence or control the heparanome. The HS inorganic array may, however be subject
to pathological or anthropogenic skewing. In general the metallome seems to influence the supramolecular
structure of polysaccharides and thereby affects their biological activities.
Some Log-Log Interrelationships Between Multi-Inorganic Element Contents

The inorganic content of the ultra anionic animal polysaccharide H (a purified protein-and nucleic

acid free pharmaceutical industry preparation) especially before its final rigorous purification processes

which is designed to remove the supposed inappropriate amounts of toxic elements present, also

demonstrated an especially enhanced multi-inorganic element fingerprint, i.e. H is so constituted

chemically to enable it to simultaneously bind enhanced amounts of the metallomic profile (cf. Table IV).

The logarithmic inter-relationship which shows up between the multi-inorganic element contents of human

blood serum and seawater (Haraguchi1) and also describes how the inorganic contents of H is correlate with

other natural matrices biological metallomicb profile formats.

Human hair shows interdependence of its multi-inorganic element contents and other Haraguchi1 seawater

like matrices, e.g., human hair inorganic element contents are approximatly log-log correlated with those

of H (cf. Fig. 3).

The alkaline earth contents of two H samples ( a (Tl) H sample obtained by ion exchange replacement of a commercial
Na(Ca)H sample) determined by Moffat in 1984 are in Fig. 3 compared with the reported alkaline earth contents of five
commercial H samples which had been earlier reported by Harrison & Sutton and similar data which had been tabulated for
a range of marine algae, coral and mollusc shells (e.g. by W.A.P Black & R.L. Mitchell and also by H.J.M. Bowen) which
suggests that the differences between various seawater (blood serum)-related matrices submitted to differeing degrees of ion
exchange purificiation in different manufacturing plants form a regular series of curves (when plotted as logarithims of
inorganic element contents) suggesting a common kind of starting native H which contains higher levels of multi alkaline
earths (and other inorganic cations) than any commercial H (e.g. perhaps natural H has a high (mollusc-like?) alkalkine
earth content).

Approximately linear log – log interrelationships ** can be also demonstrated between

the multi-inorganic element contents of natural waters including seawater, the bound inorganic elements

present in kelp and geological fulvates (attributable respectively chiefly to the sequestration to the abundant

carboxylate (uronic acid) anionic groups present in alginates and in humic polymers (cf., Grant1).

Comparison of the multi-element compositions of the earth’s crust with seawater and H however, showed

no obvious correlation.

b
The ubiquitous presence of trace and ultratrace elements found by mass spectrometric analysis in biological matrices has led H. Haraguchi1 to
propose that many elements, which formerly were classified as being non-physiological, should now be included within a revised definition of the
“metlallome” and “metallomics” (a branch of science originated by R.J.P. Williams to the study of some 20 physiological metal ions concerning,
inter alia, the connectivity between geological and biological matrices). Mast cell derived highly purified anionic polysaccharide H (used as a
blood anticoagulant) which had been purified to different extents during manufacture are good examples of metallomic matrices. This included a
highly multi-element substituted ‘sodium’ H as well as purified forms of H, e.g., ‘lithium H’ (the ‘toxic’ element contents now conform with
Pharmacopoeia requirements) which suggested that the retained multi-inorganic element contents in all H mostly derived from their original in
vivo sequestration from extracellular multi-element containing bathing fluids. This sequestration process was apparently more complex than a
simple counterion condensation since both cations and anions as well as colloidal inorganic particles were apparently simultaneously sequestered.

Inorganic Elements: Seawater Compared with H

That the full 60+ inorganic ion compositions of animal (especially extracellular) biological fluids mimic

that of seawater4 agrees with with the hypothesis that multicellular animals first evolved in a

marine environment. This may also by why the total amounts of HS and other glycosaminoglycans

occurring in the tissues of in fifteen species of aquatic including marine invertebrates obey a fairly

exact exponential mathematical relationship with the degree of salinity of the habitats5. This finding could

add support to the notion that the sequestration of inorganic elements from natural growth media might

have been the primary reason for the evolution of HS during the early stages of the evolution of

multicellular animals (HS appeared at the same time as this1a-1,2) in the sea, a phenomenon which could still

be of relevance to the full understanding of the modus operandi of these polysaccharides in present day

organisms).

The deproteinated H samples (Table IV) show a (skewed but) characteristic seawater/extracellular

biological fluid like multi-inorganic element ‘fingerprint’ similar to that of seawater bathed marine

polysaccharide matrices, indicating that the structurally closely related HS will also simultaneously

sequester the full range of inorganic ions and particles which can naturally occur in their normal bathing

solutions; the protein cores of HS PG will bind additional metal ions.

Numerous reports6a confirm that a variety of metal ions, including highly charged multivalent ultratrace

elements, bind at often modest but physiologically relevant (and rather similar) affinities to HS under in

vitro and in vivo conditions (such investigations include those relating to scintigraphic visualisation of

tumours and a arange of multivalent metal ions used for the histochemical staining of tissues6a).

Anions are known also to bind to H. These include SO42- which commonly contains ca. 12% S in this

form 6a-1 . It can be suggested that the binding of such negatively charged anions as suphate and phosphate

to H will depend on the presence of metal ions at the H surface which could act to counter the negative

charge of H which would rationally be suggested to repel rather than bind other anions.

-------------------------------------------------------------------------------------
10.8 Log-Log Plots (Figs. 1-6)
Fig.1
Inorganic elements in Na(Ca) Heparin vs Tl Heparin preprared from it (values in ppm)

Fig. 2 Inorganic Elements in Human Hair Compared to Na(Ca) Heparin
Plot of inorganic element contents ppm, (log10 scale)

Fig. 3 Alkaline Earths in Heparin, Mollusc Shells, Kelp and Marine Algae
Data for H from Moffat (loc. cit.) and from Harrison & Sutton3b-2 from other matrices from Bowen**

Fig. 4 Comparison of Inorganic Elements in Heparin (Na) Compared with This Arising from a (Hypothetical)
Sequestration from Seawater via Selective Uptake of Least Abundant Elements Present

Fig. 5 Log Ratio of Elements in Heparin/Seawater vs. Log Concentration of Elements in Seawater

Fig. 6
Comparison of Na Heparins x-axis Na Hep [believed to have been subjected to ion exchange resin clean up] leached from a
commercial blood collection tube
[Believed to be fabricated from polyethylene terephthalate (PET) which is expected to contain Sb and Co used during
manufacture as catalyst and accelerator

y-axis Na Hep SSMS (Aberdeen)[believed sampled prior to ion exchange resin clean-up]
Inorganic element contents, log10µ g/g

Fig.6


11

References

Proteoglycans containing HS (e.g. the syndecans with transmembrane core proteins and the glypicans
with phosphidylinositol membrane anchors at cell surfaces and perlican and agrin at basement membranes) contain
highly anionic, variously sulphated linear uronic acid-glucosamine-disaccharide-based polysaccharide side chains
which engage a wide variety of biochemical functions. Supramolecularly structured H/HS proteoglycan-inorganic-
 glycocalyxes can be predicted to be formed at all adherent animal cell surfaces and extracellular matrices.
Such organometallic matrices are suggested to potentiate H/HS participatation inter alia, in
blood anticoagulation, antioxidant protection, protection against pathogen insult, energy metabolism
including the modulation of lipoprotein lipase activities as well as complex cellular activities such as
embryogenesis, wound healing and immune surveillance.

11.1 General Reviews of H/H:

Garg, H. R.J. Linhard & C.A. Hales (Eds) et al., “Chemistry & Biology of Heparin & Heparan Sulfate” 2005 Elsevier
Bernfield, M. et al., Ann. Rev. Biochem., 1999, 68, 729-777 cf., also J. Turnbull et al., Trends Cell Biol., 2001, 11, 75-82

A library of epitopes similar to those which are believed to allow heparnome heparan sulphates (HS) to discriminatly bind and thereby control
protein activites occurs in pharmaceutical H and allows this (protein and nucleic acid free polysaccharide mixture) to serve as a convenient
laboratory model of the heparanome in which specific polysaccharide microstructures can evidently select between target proteins by use of a
range of different anionic patterned epitopes, the synthesis of which, e.g., during development and wound healing, is apparently strictly
spatiotemporally regulated;
cf., P.W. Park et al., J. Biol. Chem., 2000, 275, 29923-29926; M. Lyon and J.T. Gallagher, Matrix Biol., 1998, 17, 485-493;
K. Mishra-Gorul et al with JJ Castellot Jr., Trends Glycosci. Glycobiol., 1998, 10 (52) 193-200
N. Perrimon & M Bernfield Nature. 2000, 404, 725-728
J.D. Esko & U. Lindahl have listed (in a J. Chem Invest. Supplement) “A Suggested reading list” entitled “Molecular
diversity of heparan sulfate” (available at http://www.jci.org/content/full/108/2/169/DC1)
Cf. also Editorial in The New England Journal of Medicine, 2001, 344, 673-675;
B. Mullow, An. Acad. Bras. Cienc. 2005 ,77 (4) 651-664,
H. Habuchi, Glycoconjugate J., 2004, 21, 47-52; A.K. Powell et al., Glycobiology, 14, 17R-30R
S. Yung & T. M. Chan, Perit. Dial. Int., 2007, 27 (4) 375-390

M. Yanagishita & V.C. Hascall (Cell surface HS protoglycans; Minireview) J. Biol. Chem., 1992, 267, 9451-9454 was cited as a key introductory paper
(which intimated a key activity of HS is that cell surface GAGs turn over within one eighth to one third of a cell cycle and hence as noted by L.M.
McDowell (ibid., 2006, 282 (11) 6924-6930) the quantity and the fine structure of GAGs (especially of HSs) may alter in response to the presence of a
variety of stimuli.
At the cell surface 4 syndecans, 6 glypicans and CD44 (the hyaluronan receptor) can be substituted by HS side chains
which also occur in ECM
perlecan and aggrecan.

----------------------------------------------------------------------------------------------------------------------------------
11.1-1 Reference(s) 1
(Alphabetically arranged according to the first named author)

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Adiemian, K.T. et al., J. Electrochem Soc., 2002, 149, A256-261

(Silicon oxide Nafion® composite for proton-excahnge membrane fuel cell operation at 80-140oC)

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(Eds.) H.G. Garg, R.J. Linhardt & C.A. Hales, Elsevier, 2005

Albertini, R., et al., Int. J. Mol. Med., 2000, 6, 129-136

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Almeida, R.C. et al., Behav. Brain Res., 2006, 168 (2) 318-322
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{Prior to the evolution of diatoms some 500 millon years ago the oceans probably contained much more dissolved Si(OH)4 and related chemical species.
Cf., Grenne, Tor, Slack, J.F., Geology, 2003, 31, 319-322 (Paleozoic and Mesozoic silica-rich seawater: evidence from hematic chert (jasper) deposits)
The amounts of inorganic elements asocited with ‘unrefined’ H probably corresponds to the seawater levels some 1000 million years ago where Si and Fe
were probably higher than in modern oceans}

Andersen, O., Chem. Rev., 1999, 99 (9) 2683-2710

(Principles and recent developments in chelation treatment of metal intoxication)
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Angyal, S.J. Chem. Soc. Rev., 1980, 9 (4) 415-428

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Ayotte, I & A.S. Perlin, Carbohydr. Res., 1986, 145, 267-277

(Nmr spectroscopy observations related to the function of sulphate groups in H. Calcium binding vs. biological activity)

Bando, Y & K. Aki, Biochem. Biophys. Res. Commun., 1990, 168 (2) 389-395
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Basu, A. et al., J. Virol., 2007, 81 (8) 3933-3941

(Sulfated homologues of H inhibit hepatitis C virus entry into mammalian cells)
(Cf. however the minireview by Hahn T. & C.M. Rue, J. Biol. Chem., 2008, 283, 3689-3693 which, it can be suggeted, tends to
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Bassett, J.H.D., et al., Endocrinology, 2006, 147 (1) 295-305

(Thyroid hormone regulates HS protoglycan expression in the growth plate)
[T3 regulates HS protoglycan core protein production and HS synthesis and modulating enzyme expression
Gpc6, EXT-1 and HS6ST; the regulation of HS PG expresson provides an important additional link between FGF and Indian hedgehog (Ihh) signaling and
T3]

Beckman, J.S. et al., Proc. Natl. Acad. Sci., USA, 1990, 87, 1620-1624
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[Human red blood cells were found to convert garlic derived organic polysufides into H2S;
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(The homeostais of H2S is also supported by glutathione levels).
Allyl polysufides undergo nucleophilic substituion at the α C of the allyl group thereby forming RSnH, a key intermediate in the formation of H2S.
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Table II (p,102) of this article reports the aluminium contents of some commercial H : viz. a selection of H samples were shown to contain Al (amounts
reported in µ g/L were 732, 783 (ca. 35ppm), 72, 436, 137, 168 and 190) which suggests that purchased H solutions normally contain greater amount of Al
than do commercial solutions of EDTA (Na), sodium tungstate and sodium sulphate which contained respectively 3.37, 26 and 27 mg/L (26.6).

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[As in H etc.]

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[Important lectin-(water)2)-sugar linkaged identified close to bivalent metal (Ca2+ )(Mg2+ ions seem also to assist lectin assembly) site and other water
molecules forming a water-linked binding channel; similar water structure cross-linking cementation
seems also to allow binding of structurally quite different ligands by a process of molecular mimicry which is also dependent on ligand hydration (cf. Goel,
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Brown, D.R. et al., Nature, 1997, 390, 684-687

(The cellular prion protein binds copper in vivo)
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Cf., Brown, D.R., Trend. Neurosci., 2001, 24 (2) 69-127
Cf. also C. Treiber, A. Simons & A. Multhaup, Biochemistry, 2006, 45, 6674-6680
(Mn ions and less severely Cu ions can re-fold normal prions to the pathological misfolded form;
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[Cf., also N.G. Rainov et al. (with K. Doh-ura) Expert Opin. Biol. Therapy, 2007, 7 (5) 714]
[Perez, M., et al., Biochem. J. 1998, 335, 243-246 (Sulphated glycosaminoglycans prevent the neurotoxicity of a
human prion protein fragment)]
A putative role of HS-nitric oxide signaling in TSEs (Depletion of both nitric oxide and copper invovled?)
The putative copper-centred prion scenario further sugges a central role of nitric oxide biochemistry TSEs: cf.,
scrapie infection of neuroblastoma cells precludes nitric oxide production when the cells are challenged with
lipopolysaccharides (this was reported by H. Lindegren et al,. J. Neurosci. Res., 2003, 71, 291-299); diminution of nitric oxide
(as noted by K. Mani et al., Glycobiology, 2004, 14, 599-607) will affect the extent of S-nitrosylation of HS glypican and the
subsequent generation of HS fragments used as hormone-like signaling messenger molecules (these may also be involved in
cognition processes as well as tissue protection and ‘wound helaing’); to probe such ideas further studies are needed to establish
the variation between healthy and diseased (misfolded prion affected) brain tissues of the nitrosative cleavage markers (anhydro
mannose-containing degradative products produced by nitric oxide metabolite cleavage of HS)
(K. Mani et al. further note in this paper that the HS-specific epitope 10E4 is nitric oxide sensitive but partly inaccessible in
glypican-1 HS).
N.b., it had also been established by these authors that glypican-1 core protein nitric oxide recycling for nitrosative cleavage is
dependent on the Cu2+,óCu+ system and Cu deficiency has been demonstrated by others to occur in brain tissues of humans
afflicted with neurodegenerative diseases).
How the infectious prion theory has been modified by the finding of later workers to include effects of metal ion dyshomeostasis in TSEs. Cf. the work of
Supattapone S. et al. [Supattopone S., et al., J. Biol. Chem., 2004, 279, (39) 40788-40794 {Ionic strength and transition metals control PrPSc proteoase
resistance and conversion-inducing activity}] and other studies [Delaut, N.R. et al., Proc. Nat. Acad. Sci,. USA, 2007, 104, (23) 9741-9746 (Formation of
native prions from minimal components in vitro (Saa P. et al., J. Biol. Chem., 2006, 281, 35245-35252); this work employed sonication technology in a
cyclic amplification loop (PCMA ‘cyclic amplification’) which was critically dependent on the presence of a polyanion cofactor (this could be polyA,
polyT, DNA or HS) which was needed to achieve the greatly enhanced yield of PrPSc; related studies further suggested that the active infective agent (an
aggregated form of PrPSc) generated by PCMA was held together by metal ions**. Prior to the PCMA work the prion-only hypothesis of TSEs had lacked
a suitable mechanism to explain how the very low primary misfolded infectious particle (originally defined as a slow virus) titre could realistically be
demonstrated to achieve the observed pathology in vivo].
Cf ., also ref. 2 (vide infra)
Sulphated glycosaminoglycans also promote the assembly of microtubule-associated protein tau into Alzheimer-like filaments
(cf. M. Goedert et al., loc. cit.)

Burger, K. et al., Inorg. Chim. Acta, 1984, 92, 173-176

(The effect of cations on the calcium ion coordination of H)

Burnstein, M. et al., J. Lipid Res., 1970, 11, 583; Cf., Olivecrona, T. et al., Biochem. Biophys. Res. Commun., 1971, 43, 524-529 (where it is suggested that
ionic binding links milk lipoproteinlipase to H)

Busch, S.J., et al., J. Cell Biol. 1992, 116, 31-42

(Trans-repression activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription)
[Results are part of growing awareness that H-responsive pathways, involving direct modulation of gene expression by H, allow a selection of processes
which dominate at certain time in development or can analogously promote or inhibit tumor formation and growth under pathological situtions.
Pentosan polysulfate (PPS) seems to have H-like actions but ChS does not]
(Cf., Zaslau, S., et al., Amer. J. Surg., 2006, 192, (5) 640-643
(Pentosan polysulfate (Elmiron): in vitro effects on prostate cancer cells regarding cell growth and vascular endothelial growth factor (VEGF) production)
[The reduction in cell growth and VEGF indicate that PPS may at as an antiangiogenic agent with possible application for the treatment of prostate cancer]

Bush, A.J. et al., J. Biol. Chem. 1994, 269 (43) 26618-26621

(Amyloid beta protein precursor and its mammalian homologs. Evidence for zinc modulated H superfamily)

Bruce, J.S., M.W. McLean, F. B. Wiliamson & W. F. Long, Eur. J. Biochem., 1985, 152, 75-82
(Flavobacterium heparinum 6-O-sulphatase for N-substituted glucosamine 6-O-sulphate)
[Enhancements of activity of 12% and 30% were effected by Mg2+ and Ca2+ ions, respectively;
inorganic phosphate and sulphate inhibited the enzymic effectivness by 48% and 50%, respectively]
Brule, S., et al., Glycobiology, 2006, 16 (6) 488-501
(The shedding of syndecan-4 and syndecan-1 from HeLa cells and human primary macrophages is accelerated by SDF-1/CXL12 and mediated by the
matix metalloproteinase-9)

Cairns-Smith, A.G. “Genetic Takeover and the Mineral Origins of Life”, Cambridge University Press, Cambridge, 1982
[A theory which suggests that crystalline silicates could have been the precursors of DNA]

Capila, I., et al., Structure (Camb.) 2001, 9, 57-64

(Ca2+ ions crosslink H to annexin-V suggesting that a similar crosslinking is needed for apoptosis)

Cardenas A., et al Toxicology, 1992, 76, 219-231

(Incorporation of [35S]sulfate inro glomerular membranes of rats chronically exposed to cadmium and its relation with urinary glycosiminoglycans and
proteinuria)

Carlsson, P., et al., J. Biol. Chem., 2008, 283 (29) 20008-20014

(H/HS biosynthesis: progressive formation of N-sulfated domains)
[PAPS concentration regulates the microstructure of HS]

Cappai, R., et al., J. Biol. Chem., 2005, 280, 13913-13920

(The amyloid precursor protein (APP) of Alzheimer Disease and its paralog APLP2 modulate the Cu/Zn-nitric oxide-catalysed degradation of glypican-1
HS in vivo)

Challis, B.C., et al. See Ref, 1a-14

Cham, B.E., Clin. Chem., 1976, 22 (11) 1812-1816

[Phospholipid is required for the precipitation of apoVLDL and apoLDL by H-CaCl2 used for serum lipoprotein characterization]
Cf., formerly H-MnCl2 was the preferred method for the separation of LDL and HDL-chloresterol e.g., Bachorik, P.S., et al., J. Lipid Res., 1982, 23 (8)
1326-1242; Chem. Abs., 98, 69580e, and Lippi, U., et al., Lab. (Milan) 1980, 7(6) 573-577; Chem. Abs., 95, 76460g;
(However, Nishida, T. & U. Cogan, J. Biol. Chem., 1970, 245 (18) 4689-4697, in a study of Mg2+ -dextran sulphate assisted LPL complex
formation/precipitation, identified a complex interplay of the modulation by Mg2+ interactions involving both positive and negative charges on the
lipoprotein and the conformation of the anionic polysaccharide to be implicated in lipoprotein fractionations; the role of phosphate groups in phospholipids
were less critical for this Mg2+-assisted precipitation (than was the case fro Mn2+ assisited precipitation)
Cf., also Kim, Y.C. & T. Nishida, J. Biol. Chem., 1979, 254 (19) 9621-9626
(Nature of the interaction of dextran sulphate with high density and low density lipoproteins in the presence of Ca2+)
UPDATE of the use of H- and H-like polyanions for LDL precipitation
Warnick, R. G., et al., Clin. Chem. 2001, 47, 1579-1596 {discussed how variation of Hs between manufacturers is a problem for clinical laboratory
use of H as a polyanion for the separation of lipoproteins; an improvement was achieved by use of the semisythetic H analogue dextran sulphate}
(cf., Warnick, R.G. et al., ibid., 1982, 28 (6) 1379-1388). The H-mimetic, sulphated α cyclodextran + Mg2+ was employed for LDL/HDH fractionation
for automated clinical plasma lipid HDL cholesterol determination}.
(Cf., also Cornwell & Kruger, loc. cit. and Constantinesu et al., loc cit.)

Chang, M.F. et al,, J. Biol. Chem., 2000, 275, (7) 4766-4773

(Oxidized low density lipoproteins regulate synthesis of monkey aortic smooth muscle cell proteoglycans that have enhanced narive low density
lipoprotein binding properties)

Chaplin M., 2008, Water structure and science, Internet document

http://www/Isbu.ac.uk/water/index2.html

Chmiela, M. et al., APMIS, 1995, 103, 469-474

(The role of HS-binding activity of Helicobacter pylori bacteria in their adehsion to murine macrophages)

Cibickova, L. et al., Physiol. Res., 2007, 56, 765-776

(Differential effects of statins and alendronate on cholinesterases in serum and brains of rats)
[This study discusses the putative beneficial role of cholesterol lowering drugs in alleviating the
progression of Alzheimer’s disease and includes bisphosphonate.
Cf., Carrugia, A., et al., Boll. Soc. Ital. Biol. Spec., 1974, 50, (17) 1416-1422
(Diphosphonate (disodium etidronate) lowers cholesterol and total lipid levels in the blood of humans)

Coelho T.C. et al., Reactive & Functional Polymers, 2007, 67 (5) 468-475
(Effect of H coating on epichlorohydrin cross-linked chitosan)
[The H coating increased (ca.2x) the adsorption capacity for Cu2+ and changed the adsorption behaviour from Langmuir to a more Fruendlich-like
isotherm. Electron paramagnetic resonance showed the formation of tetrahedrally distorted square planar signals from H-bound Cu]

Cohen, S.M., et al., Carcinogensis, 1991, 12, 1551-1555

(A proposed role for silicates in the proligerative effects of saccharin in the male rat urethelium)
Cf. ibid., 2000, 21, 783-792

Colfin, H, & S. Mann., Angew. Chem. Int. Ed., 2003, 42, 2350-2365
(Higher-order organization by mesoscale self-assembly and transformation of hybrid nanostructures)

Comner, W.D., Interplay Genet Phys. Processes Dev. Biol. Form Workshop (1994, Pub. 1995) p.121. Ed. D. Beysens et al., World Scientific, Singapore;
Chem. Abs., 124, 79513e
(Evaluation of how the development of extracellular matrix is correlated with the sulphation of matrix macromolecules to form highly variable ion binding
templates)

Constantinesu, A.A., et al., Atheroscler. Thromb. Vasc. Biol., 2003, 23, 1541-1547
(Endothelial cell glycocalyx modulates immoblilzation of leukocytes at the endothelial surface)
[Circulating HS and H attach to the venuele wall and attenuate Ox-LDL induced leukocyte immobilization]

Corrigan, F.M., et al., BioMetals, 2005, 6 (3) 149-152

(Hippocampal tin, aluminium and zinc in Alzheimer’s disease)

Cornwell, D. G. & F.A. Kruger, J. Lipid Res., 1961, 2 (2) 110-134

(Molecular complexes in the isolation and characterization of plasma lipoproteins)
[That chelating agents dissociate these complexes is evidence for the role in their formation of the presence of multivalent metal ions; it is believed that
related ternary adducts, also dependent on the presence of multivalent metal ions are formed between proteins and other polyanions such as nucleic acids,
phosphotungtic acids and phosphorylated proteins ; such the metal ions allow interactions to occur between the negatively charged polysaccharides and
‘amphions’ (protein at lower pH than the isoelectric point where the protein is no longer cationic); other examples are egg lecithin –La3+-arabinate and
signaling –Zn2+ -chondrotin sulphate. Multivalent metal ions, ionic strength and pH were believed to generally contribute to the specificity of
polysaccharide-protein complex formation]
This work is suggested to be of especial relevance to a fuller understanding of how the related HS protoglycans (the basis of the heparnome)
function in vivo
The information holding, transmission and processing abilities of HS proteoglycans (including those enabled by the selective interaction between
HS segments with proteins) are further suggested to be dependent on the nature of the anions (especially phosphate and putatively
polyphosphates) associated with the sulphated polysaccharides (as was suggested by research into the fractionation by sulphated polysaccharides
of blood lipoproteins which needed also to containing phospholipids [cf., Cram1]. It seems possible that this could be a key to the full elucidation
of how the fine-tuning required to accomplish the multiple interactions required to discriminate between different proteins is achieved.
(Cf., Cham, loc. cit.)
{The formation of ternary complexes between Ca2+, polyphosphate polyanions and milk proteins seems also to be relevant to processed cheese
manufacture , cf., Mizuno, R & Lucey J.A., J. Dairy Sci., 2005, 88, 3070-3078}

Costello, A.G.R., T Glonek & J.R. Van Wazer, Inorg. Chem., 1976, 15, 972-974
(Phosphorus-31 chemical shift variations with counteraction and ionic strength for the various ethyl phosphates)
Cf., Menses, P & T. Glonek, J. Lipid Res., 1985, 29, 679-689
Cf., also Brow, R.K. et al., J. Amer. Ceram. Soc., 2005, 74 (6) 1287-1290
[Data reported here suggest that counterions modify the P-O
ionic character (also hydration especially where P-OH groups are also present**)
A similar linearity of the curves relating chemical shift to added salt were found with H**
[Cf., the discussion given in the following paper; the relative 31P ∆ δ shift effects for inorganic PO43- for the following counterions are:
(CH3)4N+ -0.61, K+ -0.62, Ca2+ -0.65, Li+ -0.71, Cs+ -0.77, NH4+ -0.82, Na+ -1.21, Mg2+ -1.65, Rb+ -1.66,
+
Shift from (CH3)4N 0.00 0.01 0.04 0.10 0.16 0.21 0.40 1.04 1.05
It is evident that here the effect of K+ ,which is similar to Ca2+, (is considerably less than) the effect of Na+ which is similar to Mg2+
Cf., Costello, A.J.R., T. Glonek & T.C. Myers, Carbohydr. Res., 1976, 46, 159-171
(31P nuclear magnetic resonance –pH titrations of myo-inositol hexaphosphate)
{The form of phytate (e.g. its degree of hydration and conformation) in aqueous solution, in analogy with that in the solid state, seems to vary with the
counteractions and I where the lack of complexation between the phosphate half ester anionic groups and tetrabutylammonium is the only type of cation to
approximate to the ideal state (i.e. no influence of the counteraction on the electronic structure of the anionic group).
The effect of increasing salt concentration and I with metal salts causes shifts the 31P resonances (a linear shift in the δ values with increased added salt
concentration where the effects are due to the cations in the salts and not the anions).
Na+ moves the 31P resonances in a manner consitent with short-range interactions between Na+ , H2O molecules and/or the phosphate anionic groups under
observation according to the relationship ∆ δ = 180∆ χ o-147 ∆ η -A∆ θ
π
( J.H. Letcher & J.R. Van Wazer, J. Chem. Phys., 1966, 44, 815-829; K. Moedritzer, Inorg. Chem., 1967, 6, 936-939)
where ∆ χ o is the change in the effective electronegativity of the PO4 oxygen atoms caused by solvent effects,
∆η is the concomitant change in the P δ −orbital due to change in the π character of the P-O bonds and ∆ θ is change in the C-O-P bond angle
π π
caused by solvent effects (which can usually be neglected because of the low value of A). This suggests that a shift in
δ 31P to more positive values upon addition of salt corresponds to more σ electron withdrawal by the oxygen atoms form the P or less
p −> d donation by the O atoms to the P atom, or both. The results suggested depletion of signaling water around the phosphate ester groups is
π π
produced increased I and also for the poly-phosphate group substituted phytate, more electronic interactions between the adjacent anionic phohphate half
ester groups occur with higher I.

Cross, A.J. et al., PLoS Med. 2007, 4 (12) : e325doi:10.1371/journal. pmed.0040325

(A prospective study of red and processed meat intake in relation to cancer risk)
[Strong evidence was obtained linking red meat ingestion and colorectal and lung cancer. A tenth of colorectal and lung cancers could be avoided by
reduced ingestion of red meat according to this survey of an initial cohort of 567,169 persons reduced with exclusions (which included pre-existing cancer)
to 4,94,036. The ingestion of red meat (which included bacon) was also linked to increased incidence of esophagal and liver cancer but was negatively
correlated with endometrial cancer]
Cf. also Taylor, E.F. et al., Br. J. Cancer , 2007 96 (7) 1139-1146
(Meat consumption and risk of breast cancer in the UK Woman’s Cohort Study)
[Women both pre-and post menopausal, who consumed the most meat had the highest risk of breask cancer. Processed meat seemed to post the greatest
risk]
[Cf. In a series of extensive discussions held in 1999 between the author and Prof. K.E. McColl (Glasgow University) relating to the HS nitrosative
dysfunction model of cancer, it was mentioned that upper stomach cancer is believed to be more prevalent in men and post-menopausal women than in pre-
menopausal women; this tends to support the notion that iron catalysis of inappropriate nitrosative cleavage of HS could be involved in the etiology of this
type of cancer]

Cullen, P.J., et al., FEBS Lett., 1988, 228, (1) 57-59

(H inhibits the inositol 1,4,5,-trisphosphate [Ins(1,4,5)P3] –induced Ca2+ release from rat liver microsomes)
[It is believed that H competes with Ins(1,4,5)P3 for its specific binding site so that H at micromolar concentrations can inhibit the Ca2+ release]
{Cf. also Hill, T.D., et al., Biochem. Biophys. Res. Commun., 1987, 149 (3) 897-901}
(H inhibits inositol trisphosphate-induced release from permeabilized rat liver cells)

Dai, Y., et al., J. Biol. Chem., 2005, 280 (48) 40066-40073

Dais, P., et al., Carbohydr. Res.,1987, 168, 163-179

(A relationship between 13C-chemical shift displacements and counterion condensation theory in the binding of calcium ion by H)
[cf., Grant et al.**., found similar results to those reported in this study indicating that a simple application of electrostatic attraction hypothesis of H-
counterion binding failed to explain the observed shifts in NMR parameters; different H2O/D2O ratios in the solvents used for NMR in such studies
showed a systematic correlated shift in iduronate C3, C2 and C1 with glucosamine C2, C3 and C6 indicaing water structure/ion effects in altering the
conformation of the polysaccharide chains; a similar hydration
driven effect could also account for how the CaH NMR spectrum became largely shifted to a NaH spectrum when CaH was ‘dissolved’ in physiological
0.15M NaCl].

David, T.A., et al., Water Res., 2003, 37, 4311-4333

(A review of the biochemistry of heavy metal biosorption by brown algae)
Cf., also Appl. Biochem. Biotechnol., 2003, 110, 75-90 and Envion. Sci. Technol., 2003, 37, 261-2667)

Dawson, P.A. & D. Marcovich, Pflugers Arch.-Eur. J. Physiol., 2002, 444, 353-350

De Clercq, E., J. Antimicrob. Chemother. (Suppl A) (1989) 23-46

(Potential drugs for the treatment of AIDS)
Cf. TiPS, 1990, 11, 198-205
(Targets and strategies for the antiviral chemotherapy of AIDS)

Dietrich C.P., et al., Biochem. Biophys. Res. Commun., 1983, 111, 865-871
(Structural difference of HS according to the tissue and species of origin)

Dietrich C.P., Braz. J. Med. Biol. Res., 1984, 12, 5-15

(A model for cell-cell recognition and control of cell growth mediated by sulphated glycosaminoglycans)

Ding, K., et al., J. Biol. Chem., 2002, 277, 33353-33360

(Copper-dependent autocleavalge of glypican-1 HS by nitric oxide mderived from intrinsic nitrosothiols)
(Nitrosative signalling catalyzed by metal ions) cf.,
K. Mani, et al., Glycobiology, 2006, 16 (8) 711-718 and refs. cited
K. Mani, et al.. J. Biol. Chem., 2007, 282, 21934-21940
R. Cappai, et al., J. Biol. Chem. 2005, 289; 13913-13920;
K. Mani, et al., ibid., 2004, 279, 12918-12923;
K. Mani, et al., J. Biol. Chem., 2003, 278, 38956-38965
K. Mani, et al., Glycobiology, 2000, 10 (6) 577-586
Cf. M. Belting, et al., J. Biol. Chem., 2003, 278 (47) 47181-47189
F. Cheng, et al., ibid., 2002, 277, (46) 44431- 44439
Cf. D.Grant, Chemistry Preprint Archive 2000, Oct. 94-104

Dong, M. et al., J. Clin. Invest., 2007, 117, 206-217

(The type III TGF-beta receptor suppresses breast cancer progression)
[This receptor is a HSPG (betaglycan)]

Drzeniek, Z. et al., Blood, 1999, 93 (9) 2884-2897

(HS proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells)

Dwyer, D.S., et al., Int. J. Immunopharmacol. 1995, 17 (11) 931-940

Eckert, R. & H. Ragg, FEBS Lett., 2003, 541 (3) 121-125

(Zinc ions promote the interation between H and heparin cofactor II)

Engelberg, H., Clin. Appl. Thromb./Hemostasis, 1996, 2 (2) 83-93

(Endogenous H activity deficiency and atherosclerosis)
[This activity was provided both by H and HS (fragments) which occurred in blood plasma at 1-2.4 mg/L
and with which serum cholesterol was inversely related. The lower endogenous circulating H activity was indicated to be an important determinant of the
lipid abnormalities which seemed to be involved in the pathogensis of atherosclerosis]

Engelberg, H., Cancer, 1999, 85 (23) 257-272

[H is a potential anti-cancer drug]
Fan, T.-Y & S.R. Tannenbaum, J. Agr. Food Chem., 1973, 21 (2) 237-240
(Factors influencing the rate of formation of nitrosomorpholine form morpholine and nitrite: acceleration by thiocyanate and other anions)

Feyzi, E. et al., J. Biol. Chem., 1998, 273, 13395-13398

[HS varies with age, being the only biopolymer to do so in such a precise manner]

Filip, Z, J.J. Alberts, M.V. Cheshire , B.A. Goodman & J. R. Bacon, Sci. Tot. Environ., 1988, 71, 157-172

Fordham, P.J. et al., Food Additives & Contamination, 1995, 12 (5)

(Element residues in food contact plastics and their migration into food simulants, measured by inductively-coupled plasma mass spectrometry)
[Polyethylene terephthalate manufacture uses Sb catalyst as well as a Co accelerator which would be expected to leach out during dilute nitric acid
treatment]

Fo-We (Forschungs und Verweltungs Anstalt) Brit. Pat. Appl., 1962 890,622
(Therapeutically active product)
[ “….Heparin has the property of combining with hydroxides of certain metals or with certain inorganic salts to form water-soluble composition of which
the cations of the hydroxides used and the cations and anions of the metallic salts are very stongly bound to the polysaccharides….The invention consist in
a method of producing a therapeuticlay active complex of heparin with a halide or sulphate of sodium, potassium, cobalt, a halide of manganese or copper
or a hydroxide or carbonate of cobalt manganese or copper.. These therapeutic properties are especially interesting in the combination of heparin-iodine-
metal…..Moreover, in atherosclerosis , iodides act synergistically on heparin, when they promote lipid catabolism, especially that of cholestereol….To
prepare the heparin-iodide product … the reaction proceeds faster at 100oC Example XII…A solid product, soluble in waer, is obtained which is a
heparin-MnI2 complex”]

Fransson, L.-A., & B. Havsmark, Carbohydr. Res., 1982, 105, 215-225

Fransson, L.-A., ibid., 1982, 110, 127-133; Eur. J. Biochem., 1981, 120, 251-255

Fransson, L-A., Proc. Natl. Acad. Sci., USA., 1984, 81 (18) 6557-6661
(Binding of transferrin to the core protein of fibroblast protoheparan sulphate)
[Later workers, Schmidtchen, A. et al., Biochem. J., 1990, 265 (1) 289-300 could not confirm this binding
perhaps due to differences in the inorganic ion cofactors which might also be required to facilitate such binding]

Fransson, L.-A., & K. Mani, Trends in Molecular Medicine, 2007, 13 (4) 143-149
(Novel aspects of vitamin C: how important is glypican-1 recycling)
[Possible benefit of high intake of Vitamin C in health, which previously was obscure, is via hypoxic-inducting factor, nitric oxide synthase and HS PG
glypican-1 which is deglycanated in a Vitamin C and copper dependent reactions.
{These, it might be further postulated, exert effects on cell growth gene transcription and host resistance to infection by the uptake of polyanions and the
clearance of misfolded proteins, the latter being why HS H-like fragments combat bacterial and viral infections}.
Cf., Grant, D., 2000 (internet, loc. cit.) “Ascorbate and nitric oxide in redox control of heparan sulphate”
Cf. also the following ref.:

Frei, B., et al., Proc. Natl. Acad. Sci., USA, 2008, 105 (48) E96
(Cf., Frei B & S. Lawson, ibid., 2008, 105 (32) 11037-11038
Chen, Q. et al., ibid., 2008, 105 (32) 11105-11109
Cameron E. et al., ibid., 1976, 73, 3685-3689; 75, 4538-4542
[Ascorbate may inhibit cancer via intermediate formation of hydrogen peroxide; much higher, more effective anti-cancer, concentrations of ascorbate are
achievable by i.v. administration than by dietary supplementation]
[Ascorbate is also known to boost HS biosynthesis (leading to synthesis of HS of increased degree of sulphation, cf. Table III)
and also to diminish the availability of HS-degrading nitrite]

Fujiwara, T. & J. Kaji, Toxicology, 1999, 133 (2,3) 159-169

(Lead inhibits the core protein synthesis of a large HS protogelycan perlecan by prokliferating vascular endothelial cells in culture)

Fukuchi K. et al., Front Biosci, 1998, 3, d327-337

[Links between HS and Alzheimer’s disease]

Fritzsch, J., et al., Thromb. Hemost., 2008, 100 (6) 1166-1175

(Melanoma cell adhesion can be blocked by H in vitro suggesting VLA-4 as a novel target for antimetastatic approaches to cancer therapy)

Gabizone, R. et al., J Cell. Physiol,. 1993, 157, (23) 317; Chem. Abs., 123, 249546j
(H-like molecules bind differentially to prion proteins and change their metabolic fate)

Gabr, A.A. et al., Respir. Res., 2007, 8 (1) 36 doi: 10.1186/1465-9921-8-36

(Alteration in cytoskeletal and immune function-related proteome profiles in whole rat lung following intratracheal instillation of Heparin)
[Aerosol administration of H into the rat lung induced increases at 0.24h in the following proteins (ranging respectively from 30.05 fold to 1.99 fold):
Parkinson disease protein, annexin V, rat tumour suppressor p30, heat shock protein k71, annexin VI, septin 2, capping G protein, actin related protein,
protoeosome related subunit PA 28 IFNγ inducible protein, moesin, Rho GDP dissociation inhibitor, calcyclin, carboylesterase 3, phosphatidyl inositol
transfer protein PITPN, heat shock protein 70-90 and proteosome subunit macrophain alpha PSMA-1]

Gallagher, J.T. et al., Biochem. J., 1986, 236, 313-325

(Structure and function of HS protoglycans)
Cf., Int. J. Biochem., 1992, 24 (4) 553; Glycobiol., 1992, 2 (6) 523
Garg, H.G., R.J. Linhardt, & C.A. Hales (Eds.) (Numerous authors) “Chemistry and Biology of Heparin and Heparan Sulfate”, 2005, Elsevier

Ghael, D., et al., Biochem. J., 1997, 324, 473-497

(Nitric oxide degradation of H and HS)

Gilat, D., et al., J. Exp. Med., 1995, 181 (5) 1929; Chem. Abs., 128, 39743j
(Molecular behaviour adapts to context: heparanase functions as an extracellular matrix-degrading enzyme or as a T-cell adhesion molecule, depending
on the local pH)

Goedert M. et al., Nature, 1996, 383, 550-553

(Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans)

Gould, S.H. et al. (with D.A Rees) J. Chem. Soc. Perkin Trans. II, 1975, 237-242
[Cf., Jarvis, M.C. & D.C. Apperley, Carbohydr. Res. 1995, 275, 131-145 who used solid-state NMR to probe the transformation of the (21) “egg box”
conformation of calcium pectate during drying to the 31 conformation; this conformation change was confirmed to be inhibited by the presence of
monovalent cations]

Gozzo, A.J. et al., Braz. J. Med. Biol. Res., 2003, 36 (8) 1055-1059
(Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors)
[H, HS, DeS, Ch4S and Ch6S reduced thectalytic efficiency of kallikrein on the hydrolysis of plasminogen and increased the enymic efficiency of Factor
XII. On the other hand H, HS and DeS increased kallikrein inhibition of AT while Ch4S and Ch6S reduced it]

Grant, D., Foreword

Scientific hypothesis building can benefit from on easy access of information from highly compartmentalized systems of scientific knowledge; absence of
such data can deny biomedical researchers key information needed to enable progression of fundamental ideas.
The author had been permitted to participate in a wide range of research activites (projects ranging from pure inorganic chemistry to biomedical research,
applied inorganic chemistry, organophosphorus chemistry, the preparation of inorganic polymers, the preparation of organic polymers by Ziegler-Natta
catalysts (in a company also involved with soil stabilization using organic polymers), the study of the fundamental chemical structure of soil organic matter
(involving how humic matter bound to inorganic ions and also how humic polymers could inhibit crystallization of sparingly soluble salts) and other
aspects of soil chemistry and the basic inorganic chemistry of animal polysaccharides which are believed to be involved in much of animal physiology.

Connections between different research interests of the author

A simple illustration that the reactivity of inorganic anions can be strongly dependent upon the chemical nature of the metal counterions present was
obtained by the author during comparison of the infrared spectra, thermogravimetric analytical data and metal ion complexing abilities of phosphorus oxy-
anions containing covalent P-H bonds which showed an unexpected dependence of their chemical properties upon the chemical nature of the
counteractions associated with them. [E.g. in P-H oxyanions, the P-H covalent bond vibrational frequencies varied with the metal counterions present; this
indicated that the non-covalent association of counterions could modulate the electronic reactivity of the phosphorus anion system and thereby affect its
reactivity, e.g. the abilities to undergo P-H bond rearrangements and P-O-P bond forming reactions (cf., Grant 1962)] perhaps analogously to how well-
known polyphosphate (oxidation state five) activies are modulated by adjacent Mg2+ ions which determine the key biological adenosine triphosphate (ATP)
adenosine diphosphate (ADP) interconversion functions (n.b. these P-based polyanions occur as Mg complexes under physiological conditions).
{The actual active centres for energy transduction by ATP have been further argued to consist of water molecule aggregates of different energy
state achieved by way of the ADP –(Mg2+) interconversion with ATP (cf. Wiggins1}.
Heparin can, fairily directly, it seems, also contribute to the phosphorus oxy-anion dependent Ca2+ inositol 1,4,5-trisphosphate activity, cf., Cullen1). The
interaction of counterions may also contribute to the electronic energy levels of non-oxygenated sulphur containing ions (cf., Grant & Van Wazer1, 1964 ) and
such bonds in protein sulphur tyrosine clusters (Grant et al., 1,1992) similar to the suggested computed effect of counterianons on Te42+ outer π bond related
electronic energy levels (Rothman et al., 1)}

Grant, David. (Turriff) [Examples of the Range of Research Topics Undertaken is Illustrated by the Following Publications.]

Grant, D., & D.S. Payne, Anal. Chim. Acta, 1961, 25, 422-428
Cf. Grant, D., J. Inorg. Nucl. Chem., 1964, 26, 337-346
(Thermal instability of cerium(IV) sulphuric acid solutions)
[Catalysis of generation of free radicals from Ce(IV) + H2O by Cu2+ and Ag+; and inhibition of this reaction by Hg(II)]

Grant, D. (1962) Ph.D. Thesis, University of Glasgow “A Study of Phosphites”

Cf., Grant, D., J. Inorg. Nucl. Chem., 1964, 26, 2103-2111; ibid., 1964, 26, 337

Grant D., & J.R. Van Wazer (1964), J. Amer. Chem. Soc., 86, 3012-3017
(Exchange of parts between molecules at equilibrium. V. Alkyl-terminated chain polysulfides and polyselenides)
Cf., Van Wazer., J.R., D. Grant & C.H. Dungan, ibid., 1965, 87, 3333-3339
(Exchange of parts between molecules at equilibrium. VIII. Dimethylpolysulfates)
Grant, D. & J.R. Van Wazer, J. Organometallic Chem., 4, 229-236

Grant, D., et al., (1966) J. Polym. Sci, Pt. 1, Polymer Chem., 5 (1) 57-75
(α , ω -Disubtitued polymethylpolyphosphonates and polyphenylpolyphosphonates from condensation polymerizaton)

Grant, D. (1967) J. Inorg. Nucl. Chem., 29, 69-81

(Redistribution reactions in polymeric alkyl silicates)

Grant, D. & J.O. Wright (1969) Brit. Pat. Specs. 1,134,118 and 1,143,019 (Chem. Abs. 70, P50947e and P98389b respectively) to
Monsanto Chemicals Ltd.
 [These Patents described the preparation of colloidal SiO2 dispersions. This was the background to studies of the
“seeded” polymerization process of SiO2 on a heel of preformed particles which suggested a mechanism of
how primitive life like cells could have self assembled and reproduced and since variation types of such sols exists,
their competition for silicic acid could have started a Darwinian evolutionary process which could have led to
improvement in competitive abilities through their incorporation of substances composed of elements other than Si, O
and H which could further have evolved.]

Grant, D. (1974) J. Polym. Sci. Polymer Lett. Edit., 13 (1) 1-9

(The pertinence of the scrambling behaviour of ligands on transition-metal centres to Ziegler-Natta catalyst activites)

Grant, D. (1974a) J. Appl. Chem. Biotechnol., 24, 49-58

(The pyrolysis of chlorocarbons)
[An attempt was made to probe the relevance of Aubrey & Van Wazer’s discovery that all compounds containing
carbon and chlorine atoms (when heated in a closed vessel) all underwent a similar process of of structural
reorganization to produce CCl4, C2Cl6 and C6Cl6 (bz); it was of interest to determine if this type of pyolysis was also
followed by other known chlorocarbons and mixtures of chlorocarbons and elementary chlorine
(especially C6Cl6bz + Cl2) which had not been tested by Aubrey & Van Wazer. The occurrence of the proposed general
structural reorganization was confirmed in general but a more exact reoganization product description required the
inclusion within the reaction scheme of the formation of lesser amounts of elementary chlorine, chlorine radicals,
tetrachloroethylene and dichoroacetylene together with the main pyrolysis products which had been previously
identified by Van Wazer & Aubrey; it was also discovered that the reactions were not fully reversible hence not really
controlled by thermodynamics as had been proposed by Aubrey & Van Wazer. Catalysis of the pyrolysis by a range of
metal ions especially Cu and Fe and SiO2 surfaces was also noted to be important. The outcome of these studies
suggests that while the general existence of structural reorganization behaviour (viz. non-kinetic-mechanism-controlled
stochastic behaviour) which is apparent for families of compounds containing elements from throughout the period
table is confimed, the traditional thermodynamic rationalization of the phenomena is suggested not to be correct. An
alternative system of correlating chemical reactivity based on the rules which seem to govern enthalpy and entropy
compensated reactions (governed by some additional “thermodynamic process” which seems additional to the
traditional system of thermodynamics) is proposed to be a more useful concept with which to approach chemical
reactivity under pyrolysis conditions**)

(Cf., Duck, E.W. et al., Grant, D. et al., Eur. Polym. J. 1974, 10, 77-83)
Studies of Ziegler-type catalysts for the polymerization of ethylene and propylene. I.
[e.g. Grignard reagent + TiCl4 + an active support (SiO2 aerogel [Monsanto Santocel FRC]) produced
an especially active catalyst and improved polymers e.g. ethylene propylene rubber]
Part II. The polymerisation of ethylene with VOCl3-Et3Al2Cl3- (Chlorinated Activator) catalyst
Grant, D. et al., Ibid., 10, 481-488
Part IIII. Studies of Ziegler-type catalysts for the polymerisation of ethylene and propylene-III, the polymerisation of propylene with TiCl4-MgCl2 based
catalysts
Ibid., 15, 625-626
(This series of papers arose from studies of novel second generation Ziegler Natta catalysts based on Ti, and V were carried out at the International
Synthetic Rubber Co., Hythe, Southampton, with Prof. E.W. Duck (a U.K. national who obtained his Ph.D. in the immediate aftermath of world war two
Germany under K. Ziegler (awarded the Nobel prize for the discovery of TiCl3-catalyst enabled linear high density polyethylene) [It is very necessary to
pay close attention to the purity of substances for the preparation of such hyperactive catalysts in order to obtain meaningful, reproducible (very high) rates
of reaction of olefin polymerization; these catalysts are somewhat analogous in structure and reactivity to biological metalloprotein catalysts. This
concern with purity had suggested that similar considerations might also to apply to the roles of the inorganic cofactors which seem to occur in heparin
upon the reactivity of heparin and related polysaccharides]

Grant, D. (1977) Nature, 279, 709-710

(The chemical structure of humic substances)
[This study highlighted the normal presence of long (polyethylene-like) polymethylene chains in natural humic acids;
this was clearly an important reason for their long-term stability in the soil since man-made long chain polyethylene
also is known to be highly resistant to biodegradation in the soil]
[Related research by the author was in assistance into inorganic humates included work on Zr(IV) oxyhydroxide
sequestration by humic polymers, Cf., Kibblewhite, M.G. & J. Tinsley, J. Soil Sci., 1979, 30, 303-314]

A more comprehensive collection and discussion of NMR spectra of humic materials was presented by D. Grant at an
Interntional Symposium on Soil Organic Matter held at Brauschweig in1977
Cf., Grant, D. (1977) International Peat Society, Symposium, Braunschweig, F.R.G., Abstract No. 11
(Molecular composition of soil organic matter components using proton and 13C-NMR-techniques)
{This study gave well resolved 13C NMR spectra of carboxyl group containing carbohydrate-like humic matter. Most
of the supposed lignin-like derivatives previously presumed to occur widely in humic matter seem to have been
extraction artefacts}

Grant, D. (1979) Eur. Polym. J., 15, 1161-1165

(The rearrangement polymerization of phosphorous acid with acetic anhydride)
[This study had been conducted in Glasgow University around 1961 where it was confirmed that phosphorous acid
reacted with acetic anhydride to give bisphosphonates (this study started from an attempt to produce polyphosphorous
acid analogously to how polyphosphoric acids can be obtained by gentle heating of orthophosphoric acidwith acetic
anhydride { polyphosphoric P(V) acid reorganizes in a random manner to give structures retaining the phosphoric acid
unit whereas polyphosphorous P(III) acid reorganizes in a non-random manner to products not directly related to the
 starting substance). I used a reaction flask attached to a high vaccum system; following evacuation a series of clear
glasses were obtained for a range of starting ratios of the starting materials [these glasses seem to have
remained stable for decades (being kept in their original reaction flasks and not exposed to the atmosphere] the
glasses were submitted in to a range of traditional and the newly available (in 1961) analytical techniques of NMR.
[The reaction of phosphorous acid with acetic anhydride was confimed to produce ethane, hydroxyl, 1-1 diphosphonic
acid [EHDP] condenstates in almost quantitative yield].
[Later it was found that EHDP showed an almost identical anti-CaCO3 crystallizataion ability to Heparin and
Heparan Sulphate, cf. Grant et al., 1989) suggesting a similar strong affinity of EHDP for inorganic counterions
when present at crystal surfaces]

Grant D., Duck E.W. (1981) Paper presented by D. Grant at: The G.M. Burnett Symposium held in the Department of Chemistry,
University of Aberdeen, 3rd July 1981. (Soluble VOCl3 –Chlorinated Activator Ziegler-Natta Catalysts) [The very
efficient ability of perchlorocarbons to promote pro-oxidant states is utilized commercially to recycle ‘spent’ V-based
olefin polymerization catalysts which become deactivated by chemical reduction. The efficiency of perchlorocarobons for
the studied reaction was discussed in terms of the instability of perchlorocarbon moieties (the basis of the Aubrey Van
Wazer (Monsanto Co., St Louis research) reoganization of perchlorocarbons, cf. Grant, D. (1974a) and structural
reorganization behaviour of V compounds (viz., as illustrated by the systems VOCl3-CH3Si(OCH3)3 and VOCl3-
(CH3)3SiOSi(CH3)3 {data from research conducted by D. Grant also at Monsanto, St Louis and previously briefly
presented by J.R. Van Wazer at symposia [the International IUPAC Symposium on Transition Metal Complexes held in
Vienna in 1965 and a Symposium on Inorganic Polymers held at the Northern Polytechnic in London in1967]

Grant, D., et al., Biochem. Soc. Trans., 1983, 11, 96

(Analysis by infrared spectroscopy of the association of water and metal ions with H)

Grant, D., W.F. Long & F.B. Williamson (1984) Biochem. Soc. Trans., 12, 302
(Altered water structure in mixtures of H and metal ions)

Grant, D., et al., (1984a) Eur. J. Cell Biol., 36, 13

(The binding of copper to H. Physico-chemical studies suggest interaction of pharmacological significance)

Grant, D. et al. (1984b) Ibid., 36,14

(Extracellular sulphated galactans from proliferating and non-proliferating tissues of Porphyra umbicialis)

Grant, D., et al. (1984c) Ibid., 36, 14

(Differences in the properties of HS from BHK and PyY cells)
(Infrared and proton nuclear magnetic resonance spectra of carbohydrates from BHK and PyY cells)

Grant, D., et al. (1985) Biochem. Soc. Trans., 13, 389

(A role for glycosaminoglycans in cellular adhesion of relevance to the cancer state)

Grant, D. et al. (1987) Proc. XIIIth Meeting of the Federation of European Biochemical Societies, Ljubljana, 28 June-3 July 1987
(Possible conformational states of heparins and heparans)

Grant, et al., (1987) Proc. IXth International Symposium on Glycoconjugates, Lille, 6-11 July, 1987
(Heparin and heparan interaction with cations studied by wide-range infrared spectroscopy)

Grant, D et al. (1987a) Biochem. J., 1987, 244, 143-149 (and

Cf., Grant, D. et al., Biochem. Soc. Trans., 1990, 18 (6) 1283-1284)
(Infrared spectroscopy of Heparin-cation complexes);
Cf., also Grant, D. et al., Biochem. J., 1989, 261, 1935-1938
(Infrared spectroscopy of chemically modified Heparins)
Cf., also Grant, D. et al., Biochem. Soc. Trans., 1989, 17, 498-499
(Carboxylate symmetric infrared absorbances of chemically modified Heparins)
Cf., also Grant, D et al., ibid., 1989, 17, 502-503
(N-acetyl amide-II and methyl C-H beinding infrared absorbances of chemically modified Heparins)
Cf., also Grant, D. et al., ibid.,1991, 275, 193-197
(Infrared spectroscopy of Heparins suggest that the region 750-950 cm-1 is sensitive to changes in iduronate residue ring
conformation)
Different counterion salt forms of H gave infrared spectra showing shifts in the vibrational energies of the carboxylate, and
oxy-anion associated hydration structures which were analogous to the shifts produced in vibrational bands in salts of
phosphorous acid where the observed vibrational energies of the anions were found to strongly depend on the chemical
nature of the metal counter cations present (Grant et al. (1984)(1987a)).
Similar findings were reported by both Grant et al., (1987a) and Panov & Ovsepyan, loc. cit.
The reported effect (Costello et al.,1) [who were later colleagues of J.R. Van Wazer] of inorganic counterions on the 31P NMR spectra of phosphate
and phosphate half-ester groups in inositol phosphates, is analogous (but not identical) to the effects of counterions on inorganic phosphate,
phosphite and sulphate half-ester and uronic acid groups in anionic polysaccharides (e.g. the sulphur based oxyanions in glycosaminoglycans
including H) are predicted to similarly for their modus operandi, on the interplay between metal ions, oxyanion electronic activity and their
associated hydration states (e.g. this being applicable to the signaling by H-like molecules which are known to control numerous cellular activities
including embryo development, wound healing and putatively also degenerative diseases including carcinogenesis).

Grant, D. (1986, 1987b), W.F. Long & F.B. Williamson, Thrombosis Research, 1986;
Cf., Supplement VI, 92; Cell Biol. Int. Reports, 1987, 11, 220
 (Cation interaction with H at antithrombin-binding sites differ from that occurring elsewhere in the polymer. It was
noted that especially larger shifts in NMR absorptions due to metal ion interaction were observed close to the
hypersulphated region which occurs in the centre of this sequence;

Grant., D. et al., (1987c-1) Med. Hypoth., 1987, 23, 67-71

(Pericellular heparan may contribute to the protection of cells from free radcials)

Grant, D. et al., (1987c) Med. Hypoth., 1987, 24, 141-146

(A model of two conformational forms of H/HS suggested by infrared spectroscopy)

Grant D et al., (1987e) Cell Biol. Int. Rept., 11, 221

(Cation interaction with H and heparans is not explicable in terms of simple electrostatic effects)

Grant, D. et al., (1988a) Biochem. Soc. Trans., 16, 977-978

(Heparans as pericellular antioxidants)

Grant, D. et al., (1988) Biochem. Soc. Trans., 16 (6) 1030-1031

(Effect of H on dismutation of superoxide ions)

Grant, D., et al., (1988a) Biochem. Soc. Trans., 16, 1029-1030

(Polystyrene surfaces may require structured water for effective cell adhesion)
[The ability of polystyrene surfaces to support eukaryotic cell adhesion and proliferation was enhanced by chemical
treatments which caused hydroxylation and allowed the formation of putatively ‘correct’ water structures to be
engendered by the modified surfaces. Evidence for this was obtained from the study of the near infrared spectra of
hydroxyl and water absorption bands of modified and unmodified cell culture Petrie dish surfaces]

Grant D., et al., (1989) Biochem. J., 259, 41-45

(Inhibition of glycosaminoglycans of CaCO3 (calcite) crystallization)
(H and HS showed similar anti-calcification effectiveness, similar to that of EHDP
[Cf., also Levy et al., loc. cit.] but this effectiveness was less than that fulvic fractons of humic polymers {which were
also very much more effective than lignin derivaties, [the results for humic and fulvic acids etc. had not, however, been
not included in the above article but in a related manuscript. The methodologies used here were the same as that in the
above Biochem. J. article but the subject matter was of little obviously interest to biochemistry, the main objective of the
project during which the experiments were performed but the resutls can belatedly be published elsewhere, it being
pertinent to discussions of how global atmospheric and marine carbon dioxide balance is achieved; similar
findings have by now also been published by others**].
De-N-sulphated H showed negligible anti-calcification effectiveness (and could demonstrate promotion of calcification
suggesting that if damge to HS occurred, e.g. by de-N sulphonation due to acidosis, or by catalytic actions of redox metals
such as Fen+, the normal strong inhibition of arterial calcification which apparently is centred on the presence of correct
endothelial ‘intact’ HS microstructures, would be greatly diminished) [cf. also, Liang et al., loc. cit.]
Cf., also the Hyman Engelberg reviews relating endogenous H to cholesterol and atherosclerosis (H inhibits
atherosclerosis) and also Klock, J.C., US Pat. 6291438 (2001-09011)
(Methods for diagnosing atherosclerosis by measuring endogenous H and methods for treating atherosclerosis using H)

Grant, D., et al., Proc. British Society for Cell Biology and British Society for Developmental Biology, St. Andrews, 3-6 April, 1989 (Biophysical clues to
glycosaminoglycan function)

Grant D., et al., (1989b) Med. Hypoth., 28, 245-253

(A comparison of the antioxidant requirements of proteins with synthetic polymers suggests an antioxidant function for
clusters of aromatic and bivalent sulphur-containing amino acid residues)

Grant, D., et al. (1990) Biochem. J., 1989, 259, 41-45

(The dependence on counter-ion of the degree of hydration of H)
Cf., Grant, D., et al., Biochem. Soc. Trans., 1984, 12, 302
[Estimated values of the ratio H2O/H disaccharide were, for the following salt forms of H: Na+ : 16, Mg2+ : 12, Li+:10,
Zn2+ and K+: 9, Cu2+ , Ca2+ and Tl+ (but here TlOH formation may also occur): 4 and (CH3)4N+: 0]

Grant, D., et al. (1990) Proc. 34th Harden Conf. of the Biochemical Society (Free radicals: cell growth, disease and repair
mechanisms (ed. Burden, R.H. & Rice Evans, C.A.), 1990, p. 52
(Structural features of glycosaminoglycans and proteins relevant to free radical biochemistry)

Grant, D., et al., (1991) Biochem. J., 277, 569-571

(H-polypeptide interaction. Near infrared spectroscopy in an anhydrous dispersant allows the involvement of polymer-
associated water to be assessed)
[Infrared overtone study using an hydrophobic medium of the glue –like action of water molecules at H surfaces for
poly-L lysine and poly-L arginine binding]

Grant, D., et al. (1991) Biochem. Soc. Trans., 1991, 19, 392S
(A relationship between cation-induced changes in H optical rotation and H-cation association constants)
[The binding of Ca2+ to H and modified Hs is associated with a discontinuity in the binding isotherm which
suggested the following empirical relationship: the value of this transition occurs at values of [Ca2+]/[polymer disaccharide] for H like molecules according
to 0.20(carboxylate) + 0.18(N-sulphonate)+0.08(Nacetyl)+0.06(O-sulphate)]
Grant, D., et al. (1991a) Biochem. Soc. Trans., 1991, 19, 393S
For a series of counteractions, the maximal change (increase or decrease in the optical rotation of H, seen when a high concentration of cation is added to
the polyanion, is directly related to an independently derived association constant form the interaction of that cation with the polylanion. A linear to non-
linear transition occurred in the binding isotherms for Ca2+ to H and modified Hs at the value of [Ca2+]/[polymer disaccharide] = 0.20 x carboxylate
group/disaccharide + 0.18 x N-sulphonate groups/disaccharide + 0.08 x N-acetyl group/disaccharide + 0.06 x O-sulphate group/disaccharide which
suggests a greater importance of the carboxylate and N-sulphonate groups and a lesser importance of the N-acetyl and O-sulphate groups for the H
interaction with Ca2+ indicated by the linear portion of the isotherm and disagrees with the electrostatic hypothesis of the origin of this phenomenon.

Grant, D., et al, (1992) Med. Hypoth., 1992, 38, 49-55

(Degenerative and inflammatory diseases may result in effects of anti-mineralization mechanisms afforded by glycosaminoglycans)

(In this review of anti-mineralization actions of glycosaminoglycans it was suggested that defects in this protection might contribute to degenerative
diseases and also (in an immediately preding article Grant, D., et al.,1992) that the early evolution of life might have depended on polyanions such a
polyphosphates and the precursors of glycosaminoglycans acting in cooperative interactions with metal ions in minerals related to apatite; it should be
noted that the interaction of Ca2+ with H and HS was found to be greatly diminished following de-N-sulphation (cf. Laing T.N. et al., Carbohydr. Res.
1982, 106, 101-109, Ayotte L & A.S. Perlin, ibid., 1986, 145, 267-277 Grant, D., et al., (1991a) and Grant D., et al., (1992b) Biochem. J. 282, 601-604
(Ca2+ H complex formation studied by polarimetry). (Such loss of NSO3- groups could arise by actions of unliganded redox metals**)
Cf., also Grant D., et al. (1988b) Biochem. Soc. Trans. (1988c), 1028-1029 (NMR data suggested that under 0.15M physiological NaCl concentration
conditions, Na+ and Ca2+ bound to H at similar affinities in an approximately stochastical fashion but with some compensation effect arising from a
cooperativity between adjacent binding sites for increased Ca2+ binding)

Grant, D., et al., (1992a) Med. Hypoth., 38, 46-48 (A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to
modern pathological afflictions).

Grant, D et al., (1992b) Biochem. J., 1992, 282, 601-604

(A study of Ca2+ heparin complex-formation by polarimetry)
[One of the studies of the author and colleagues of the interaction of H with metal cations which suggested that counterion bound to H by a phase change
mechanism]

Grant D., et al. (1992c) Biochem. J., 1992, 285, 477-480

(A potentiometric titration study of the interaction of H with metal cations)

Grant, D., et al. (1992d) ibid., 1992, 287, 849-853

(Zn2+ H interaction studies by potentiometric titration)
Cf., Wooodhead N. E., et al., ibid., 1983, 237, 281-284 (Binding of zinc to H. Analysis by equilibrium dialysis suggests the occurrence of two entropy
driven processes);
Cf., Grant, D., et al., (1986) IRCS, J. Med. Sci. (Library Compendium, 1986, 14, 805-806 (A study by equilibrium dialysis suggests the occurrence of two
types of H Ca2+ interactions)
(Unpublished similar researches were conducted into Mg2+, Mn2+ and Al3+ H binding indicated that therelative strength of complexation suggested by pH
changes effected under standard conditions by binding of metal ions to Li H was
Mg2+ 1.0, Mn2+1.14, Fe2+ 1.34, Ca2+,1.75, Cu2+ 3.0, Fe3+ 3.24, and Al3+ 4.54 (this cation differed greatly from the other metal ion by its creation a pH 4.5
buffer system following saturated binding to H whereas the other cations studied created a near physiological pH buffer system under these conditions)

Grant, D. et al. (1992e) Biochem. J.,1992, 283, 243-246

(Cu2+ H interaction studied by polarimetry)
Cf., Biochem. Soc. Trans., 1992, 20; 214S
(13C-nmr spectroscopy of Cu2+-H suggests phase separation of the complex from Cu2+ in aqueous solution)
Cf., also Park J.W. & Mukherjee, loc. cit.

Grant, D., et al. (1992f) Biochem. J., 1992, 285, 477-480

(A potentiometric titration study of the interaction of H with metal cations)
This paper described potentiometric tritrations of H carried out at ionic strengths (I values achieved by a range of different inorganic salts) ranging from
0.0057 to 0.0336 suggested that Ca2+, Cu2+, Li+ and Na+ bind to H in a manner that depends on the identity of the cation not its charge as predicted by the
Manning hypothesis. A somewhat more electrostatic type of binding was found for Mg2+ which binds weakly and electrosically below I=0.050; K+ bound
to H weakly at I= 0.0057. Cu2+ bound to H essentially overtly irreversibly, i.e., being potentially subject to nucleation effects.

Grant, D et al. (1992g) Biochem. Soc. Trans. (1992) 20, 4S

(Multiple-specular-reflectance i.r. spectroscopy of glycosaminoglycan cetylpyridinium complexes)

Grant D et al., (1992h) “Making Light Work, Advances in Near Infrared Spectrscopy”, 1991, (pub.1992; Ed. Murray I, & Cowe I.A.) VCA Weinheim,
Germany) p. 633; Chem. Abs., 118, 164498z
[Near and fundamental region infrared spectroscopy of HS isolated from the surface of normal and transformed fibroblasts]

Grant, D. et al. (1994) Agents & Actions, 1994, C214-C215

(Heparins as essential antioxidants)

Grant, D. et al. (1996) Biochem. Soc. Trans., 1996, 24, 194S

(The antioxidant activity of Heparin)
[Cf. also Grant, D., et al. (1988) and 1988a) loc. cit.)
Some indications that ‘single chemical substance’, ‘single salt forms’ of H, having been depleted of their natural multi-inorganic-element components, are inappropriate models for biochemical researches into H/HS signaling were obtained during studies

of antioxidative and related properties were apparently considerably less than that of native H (with a large mutli-inorganic element content), but further research is required to fully establish this.

Grant, D. (2000b)
Discussion Documents (Internet)
Nitrosative H/HS Signaling
Nitrosative H/HS signaling catalyzed by metal ions putatively constitutes a major inorganic ion assisted H/HS information processing and control system
in animals which seems to have been highly conserved throughout animal evolution (cf. 1a-1,2) and which could have enabled environmental inputs to
influence the course of such evolution. This system might have arisen from an earlier system where HS evolved to mimic the natural humic (poly
carboxylic acid) polyanionic buffer systems of natural waters. This notion is in keeping with the report that concentration of dispersed or soluble HS in
blood (cf., W. Manley et al., 1) is similar to that of the poly-carboxylated poly-counterion salt containing natural (humic) polymers in natural waters
including the sea where they putatively contribute to inorganic ion homeostasis (including by the potent inhibition of crystallization of sparingly soluble
salts such as CaCO3**).

Grant, D. (2000a)
Multi-Ion Content of Heparin
Chemistry Preprint Server CPS biochem/0010002 Internet available
[The multi-inorganic elements present in H are variously bound; comparison of the amounts present before and after ion exchange column percolation
gives an indication of the different binding strengths of individual elements; the range of elements associated with H suggests that H and similar molecules
collect nutrient elements and serve as storage site for their supply]

Cf. also refs. 3b1 – 1b-10 (vide infra)

H (Moffat1, Grant et al., 1 (1987) cf., Jaques1 and alginate have long been known to bind a cosphere of inorganic ions and particles e.g. acquired in vivo from
multi-inorganic-ion bathing solutions. It is suggested that analytical data which indicates that H can simultaneously sequester the full range of inorganic
ions which occur in seawater and blood serum has biological relevance. Such putative, uniquely wide-spectrum-based inorganic ligand provision
behaviour, apart from giving a focus for the study of biological metallomics (the suggested new branch of science concerned with the elucidation of
interrelationships between geology and biological evolution, cf. Haraguchi1) is now suggested also to be relevant to the fuller understanding of how the HS
family of polysaccharides function in vivo.

Internet
Grant, D. (2000)
Ascorbate & nitric oxide in redox control of HS structure
dgrant@ukonline.co.uk/dgrant/

files ( through Yahoo)

from
http://web.ukonline.co.uk/dgrant/dg1/
http://web.ukonline.co.uk/dgrant/dg2/
http://web.ukonline.co.uk/dgrant/dg3/
http://web.ukonline.co.uk/dgrant/dg4
http://web.ukonline,co,uk/dgrant/dg5/
http://web.ukonline.co.uk/dgrant/dg8/
[the latter file is a list of the references cited in the previous files]

Grant, D.
Discussions with F.B. Williamson (Aberdeen). Included HS hypothesis of animal evolution
Metal ion dependent H/HS signaling;
Inorganic (“Metallomic”) Cofactors Can Modulate Heparin & Heparan Sulphate Activities
Putative H/HS-based inorganic ion – environment interface system suggests this.
HS by its sensitivity vial nitrosative processes to environmental changes can also signal systemically for nitrosative selective genetic mutation. These
events can link to create systemic response to altered food availability and other environmental changes.
(Cf. also A Varki, Cell, 2006, 126, 841-845 (Nothing in glycobiology makes sense except in the light of evolution) has suggested that polysaccharides in
general might be centrally involved in evolutionary pathways)
That multiple metal ion and other small (inorganic) ion cofactor binding seems to have become optimized with the most anionic of
polysaccharides (and biopolymers) H, and by inference also with the H-like segments of HS, suggests that such polysaccharides might depend
upon their bound inorganic ions to enable high rank managerial systemic control functions in animals

Grant, D.
Discussions with Mark Purdey (Taunton)
Cf. M. Purdey, Med. Hypoth., 2004, 62, 746-754;
and on Roles of H/HS in Prion Dysfunction Diseases
(continued with Graham Steel)
HS has been indicated to determine the metabolic fate of prions which also seem to bind and can be misfolded by bound metal ions (for which Mnn+ ions
cf. Treiber et al.1, seem to be of especial relevance for the possible augmentation of prion misfolding under the conditions of Cu depletion and Mn
intoxication. Cf. also the putative antioxidant roles of Cu ions in prion biochemistry discussed by D.R. Brown et al. 1). The H/HS-mimetic pentosan
polysulfate (PPS) which can apparently prevent or inhibit misfolded prion diseases may do so, at least in part, by influencing the putative redox metal ion-
HS-prion-nitric oxide signaling system, the efficiency of which could influence how prions are normally prevented from misfolding in vivo.
----------------------------------------------------------------------------------------------------------------------------------------
Discussion on Mark Purdey’s HS wound healing perturbation hypothesis of the etiology of multiple sclerosis indicated that the efficiency of a sulphate
transporter which was implicated in HS biosynthesis depended on a vitamin D isoform availability (with an associated input from UV B exposure)
suggesting an explanation for the latitudinal dependence of the prevalence of multiple sclerosis and especially the more exact latitudinal variation of the
prevalence which is especially marked in Australia and the USA;
cf., also the internet discussion at
http://www.medicine.ox.ac.uk/bandolier/booth/neural/MSgeog.html
which includes the results reported by
I.A.F. van der Mei et al., Neuroepidemiol. 2001, 20, 168-17
-----------------------------------------------------------------------------------------------------------------------------------------------------------
Discussions with Graham Steel (Formerly of the CJD Alliance)
PPS has been found to offer therapeutic benefit for the treatment of
nvCJD** [A large number of research documents relating to the long term inhibition of neurological dysfunction associated with prion diseases by the
direct infusion of a low dose of PPS into the brain]

Grant, G.T. et al., (with D.A. Rees), FEBS Lett., 1973, 32 (1) 195-198

Grenne, T.S. et al., Geology, 2003, 31, 319-322

(Paleozoic and Mesozoic silica-rich seawater: evidence from hematic chert (jasper) deposits)
Cf., also Anbar & Knoll, loc. cit.

Grushka, E. & A.S. Cohen, Anal. Lett., 1982, 15(B16) 1277-1288

The binding of Cu(II) and Zn(II) to H
(This study showed the presence in ca 1/20 disaccharide units in H of a higher association binding site for Cu(II);
Cf., Stivala & Liberti et al. in Arch. Biochem. Biophys., 1967, 122 (1) 40-54, had previously suggested that the ability of Cu2+ to bind H could be used to
predict the anticoagulant activities of H samples)
Cf., Rudd, T.R. et al., Carbohydr. Res., 2008, 343, 2184-2193

Gutierrez, T., et al., Appl Environ. Microbiol., 2008, 74 (15) 4867-4876

(Emulsifying and metal ion binding activity of a glycoprotein exopolymer produced by Pseudoalteromonas sp. Strain TG12v)
[Although the authors failed to establish the nature of the exopolymer discussed in this report largely because conly aobut a third could be
hydrolysed, it was believed to be a polyuronic acid]

Halliwell, B., Acta Neurol. Scand. 1989, 126, 23-33

(Oxidants and the central nervous system: some fundamental questions. Is oxidant damage relevant to Parkinson’s sidesdaes, Alzhiemer’s disease,
traumatic injury or stroke?)

Haraguchi, H., J. Anal. At. Spectrom., 2004, 19, 5-14

(Metallomics as integrated biometal science)
Although Haraguchi included “heparin” in a list of substances for which metallomic was relevant, this was because of the presence of the large
amounts of S, present as sulphur oxyacid esters in this polysaccharide.
This author seemed to suggest that biochemical metallomic research was only of value for the study of metallo-protein formation but a more informed
judgement might have indicated that metallomic anionic polysaccharide research was also relevant.

Haraguchi here seems to suggest that the similarities apparent in log-log plots of the inorganic element contents of geological matrices which
include natural waters (cf. 2.3) and biological fluids (a phenomenon which may extend to all elements in the periodic table) is of fundamental
scientific significance and requires its own branch of science: ‘metallomics’. Harguchi further proposed that previously believed limit of some 20
inorganic elements which had formerly been thought to be essential for animal biology should now be extended to include most of the remaining
elements in the periodic table since these elements are also commonly detected by modern analytic methods to be (ubiquiteously) present in
biological matrices which seem to be examples of “all-element phenomena”.

[A typical porcine unfractionated sodium H which was found to contain numerous inorganic elements (cf. Grant et al., 1 (1987)) with relative
abundances corresponds with the types of inorganic element distributions reported by Haraguchi (vide infra) to occur in human blood serum and
seawater].

Haraguchi, H., internet file, downloaded on 5/9/06 from

http://www.apchem.nagoya-u.ac.jp/06-III-3/index-e.html
“All materials on the earth, incuding man as well as plants and animals, contain all the chemical elements on the periodic table. We propose this hypotheis
as Extended all present theory of the elements”

Hedlund, B.E. & P.E. Hallaway, Biochem. Soc. Trans., 1993, 21, 340-343
(High-dose systemic iron chelation attenuates reperfusion injury)

Helbert, J.R. & M.A. Marini, Biochem. Biophys. Acta, 1964, 83, 122-134

Hu, W.L. & J.Y.-P. Sheng, Biochem. Cell. Biol., 1992, 70, 535-538
(Hepatic HS proteoglycan and the recycling of transferrin)

Hochstein, P & A.S. Atallah, Mutation Res., 1986, 202, 363-375

(The nature of oxidants and antioxidant systems in the inhition of mutation and cancer)

Holmborn K. et al., with L. Kjellen, J. Biol. Chem., 2004, 279 (41) 42355-42358
(HS synthesized by mouse embryo stem cells)
[This work suggests that a rethink is required in the Lindahl mechanism of HS synthesis. HS synthesized by mouse embryo stem cells deficient in NDST1
and NDST2 completely lacked N sulphation but still contained 6-O-sulphate groups demonstrating that N sulphation was not required as the intermediate.
The 6-O-sulphated HS lalcking N-sulphate also have N-unsubstituted groups in contradiction to the conventional mechanism by which HS is thought to be
assembled (such C-NH2 groups are primed for nitrosative cleavage]

Hyde, S.T., et al., Physica A, 2004, 339, 24-33

(Self-assembly of carbonate-silica colloids between living and non-living form)
[Cf. also Carnerup, A.M. et al., “Silica-carbonate biomorphs and implications for identification of microfossils, in J. Seckbach et al., “Life In The
Universe: From The Miller Experiment To The Search For Life On Other Worlds (Cellular Origins, Life in Extreme Habitats and Astrobiology”) Kluwer
Academic Publications, 2004]
[Cf., Grant, D., et al., Med. Hypoth., 1992, 38, 46-48
(A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern pathological afflications)]
Cf. also Iler, R.K. loc. cit. and.
Cairns-Smith, A.G. (mineral origins of life etc.) loc. cit.
See also the following ref.

Iler R.K. “The Chemistry of Silica”, Wiley

The Chemistry of Silica: “Solubility Polymerization, Colloidal and Surface Properties and Biochemistry”
Wiley –Interscience, New York, 1979
Cf. p. 762 in this textbook where it is noted that “Chemically the silica is bound in tissues with glycosaminoglycans and polyuronides {as reported by K.
Schwarz, Proc. Nat. Acad. Sci., USA, 70 (5) 1608 et seq. About 800 ppm SiO2 was bound to purifed hyaluronic acid, chondroitin 4-sulfate and heparan
sulphate (HS). This corresponds to one SiO2 per 200 repeating units of polymer. {cf., Moffat, loc. cit., cf. Table IV, reported that a relatively unrefined H
contained 5900ppm Si suggesting the possibility that the Si content associated with H-like GAGs in tissue may be somewhat higher perhaps approaching
one SiO2 per 10 or less repeating units of polymer in some cases}. Silica is also reported bound in pectin and alginic acid. It is present attached to these
polysaccharides in a for, not dialyzable or reactive with molybdic acid and stable against autoclaving with 8M urea…..
Some association of polysaccharides with silica has probably existed since life began”

The possible existence of biological polysaccharide linked-SiO2 systems had been suggested by the groundbreaking studies of K. Schwarz in 1973; the
amount of non-dialyzable Si in H found by Moffat was 5900ppm Si (equivalent to possible (extrapolated value of 2% dry wt. of hydrated silica gel or sol
particles). The highest prior amount of Si associated with an anionic polysachride was 200ppm in pectin (reportd by Schwarz). Previously reported values
for H were 300-800ppm (this agrees with the results obtained by Moffat for an ion exchanged “single counteion” form of H favoured for use in medicine).
Lambert, J.B. et al., e.g., J. Amer. Chem. Soc., 2004, 126 (31) 9611-9625 more recently reported the formation of soluble 2/1 sugar/basic silicic acid
adducts; these were formed with certain furanose (but not pyranose) sugars capable of forming five membered diolato rings.
[If the SiO2 particles in interstellar dust are like the SiO2 particles of commerce (and perhaps also those in some modern organism for which there is much
less knowledge) this provides an alternative mechanism by which non-DNA held “genetic” information could be transmitted over large distances. The
particles of (highly hydrated?) polyoxymethylene on SiO2 the presence of which has been suggested (by N.C. Wickramansingh, Nature, 1974, 252, 462-
463) to account for spectroscopic properties for intersellar dust, could conceivably be transfomed e.g. as a result of UV radiation and high energy particles,
into something like “humic acid” the ubiquitous soil polyanonic polymer system which is loosely analogous to major extracellulr polyanionic systems of
animals and marine algae GAGs including HS and alginic acid, repectively. Such humic acid-like water structuring multiple inorganic ions buffering
systems, present in natural waters attached to SiO2 particles could have aided some natural selection for fitness by a quasi Darwinian evolutionary process
which depends on natural selection of chemical systems most fitted for purpose to enable the eventual development of carbon and DNA based organisms
on Earth or elsewhere**]

The existence of biologically relevant silica sol-HS glycocalyx structures could be be relevant to a wide range of suggested uses of amorphous silica for
the treametnt of osteoporosis, for the inhibtion of dental caries, skin acne, psoriasis, stomach mucosal dysfunction (dyspepsia treatments using direct silica
sol application and use of silica in conjunction with an anionic algal polysaccharide) and perhaps also cancer (by an aluminium zeolite absorbent method).
McCarty (loc. cit.) has suggested that Si e.g. as SiO2 can upregualte the synthesis of tissue protective forms of HS **.

Ikegami, A., J. Polymer Sci., A, 1964, 2, 907-924

(Hydration and ion binding of polyelectrolytes)
[Measurement of refractivity changes caused by polyanion counterion association is ascribed to an alteration of the two layered structure of water around
poyanions. Two regions of hydration around the polyanion were a first layer of intrinsic hydration region containing water molecules near the individual
charged groups and a second layer containing water molecules rearranged by the cooperative action of two or more charged groups on the polyanion. Ba2+
and La3+ destroyed both water layers in polyacrylic acids being bound to the carboxylic acid groups. Na+, was, however, bound by the second layer of
water moleucles]

Inoue, S., et al., J. Histochem. Cytochem., 1989, 37, (5) 597-602

Iozzo, R.V., J. Clin. Invest., 2001, 109 (2) 165-167

Cf., Iozzo, R.V., J. Biol. Chem., 1989, 264 (5) 2690-2699

(Unsulfated HS chains in HS proteoglycans of human colon carcinoma)

Ishihara, M. et al., J. Biol. Chem., 1987, 262 (10) 4708-4716

Iwada, H. et al., Proc. 7th Urolithiasis Symp., 1994, 185

(HS proteoglycans efficiently inhibit the crysallization of Ca oxalate in urine)

Jaques, L.B. ref. 6a (vide infra)

Jaques, L.B., Biochem. J., 1943, 37,189-195

(The reaction of H with proteins and complex bases)
[The solution equilibrium salt formation hypothesis of H-protein interaction previously proposed by Fisher & Astrup (1935) [Biochem. Z. 278, 326] was
probed in this study the data given however can be now be reinterpreted in terms of the formation of
phase-separated non-equilbrium complex formaton; results for the displacement of bound dyestuff from H show differences in the effects of salt (additions
of e.g. trichloroacetic acid ZnSO4, CaCl2 and NaCl which indicate the occurrence of competive processes of displacement of soluble ionic adducts and the
formation of phase separated nanoparticles with the inorganic salt additions demonstrated by the overt selective precipitation of lipoproteins by inorganic
salts which later became used for the determination of LDL/HDL in blood serum [the formation of trichloracetic acid H adducts can be demonstrated by
infrared spectroscopy**]
{An important feature of small ion H binding isothermal binding curves is that these do not obey the law of mass action for changes in the H
concentration, cf. Grant, D. et al. studies of counteraction binding by H, listed above}

James, P.L., et al., J. Materials Sci., 2000, 35, 5111-51119

(Hydraton of Nafion® (studied by AFM and X-ray scattering)
Cf., Adiemian et al., loc. cit.

Jarrett, D. J. R. et al., J. Clin. Exp. Gerontol., 1989, 11 (3-4) 145-154

[How the availability of iron ions capable of catalysing fee radical oxidative and nitrative damge, increases in blood serum with age
in absence of disease; other age related alterations which might affect this are the systematic alteration of endothelial surface HS microstructure, cf., Feyzi,
E., et al., loc. cit., arterial HS (Murata & Yokayama , loc. cit., and an diminution in the effectiveness of glucosamine biosynthesis with increasing age**]

Jenniskens, G.J., et al., J. Cell Sci., 2003, 116 (11) 2187-2193

(Effects of Ndst deficiency on Ca2+ kinetics in myotubes from Ndst-1 and Ndst-2 deficient mice indicating a novel role for HS in skeletal muscle
physiology)
[It was somewhat surprising, in view of the numerous biochemical systems which are thought to be dependent on HS PG in animals, that Ndst-1 -/- mice
survived until birth. This was attributed to abilities of other Ndst enzymes (especially Ndst-2 which is responsible for H synthesis in mast cells) to
substitute for Ndst-1 activity]

Jones, L.S., et al., Mol. Cell. Proteomics, 2004, 3, 746-769

Polyanions and the proteome.
Emerging evidence for the ubiquitous nature of polyanionc/protein interactions perhaps traceable to early (pre-biotic) pre-cellullar interactions.
This review also discusses use of GAG-mimetics as therapeutic agents, e.g., for Alzheimer’s disease

Kao, J., et al., Exp. Mol. Pathol., 1990, 53 (1) 1-10

(Ascorbic acid stimulates production of GAGs in cultured fibroblasts)
[Ascorbate induced HS augmentation is accompanied by increased degree of sulphation]

Khoury, J. & D. Langleben, Am. J. Physiol. Lung Cell Mol. Physiol., 2000, 279, L252-261
(H-like molecules inhibit pulmonary vascular pericyte proliferation in vitro)

Kisilevsky, R., et al., Amyloid, 2007, 14 (1) 21-32

(HS as a therapeutic target in amyloidogeneis: prospects and possible complications)

Kisley, L.R. et al., Cancer Res., 2002, 62 (23) 6850-6856

(Genetic ablation of inducible nitric oxide synthase (iNOS) decreases mouse lung tumorigenesis)[This mouse cancer is a model for human lung cancer]
Cf. Takahashi, T. et al., Carcinogenesis, 2008, 29 (8) 1608-1613
(Increased expression of iNOS in N-nitrosobis(2-oxopropyl)amino-induced hamster pancreatic carcinogenesis and prevention of cancer development by
ONO-1714)[Elevated iNOS is also observed in human pancreatic cancer suggesting that this hamster model of the human disease and the anti-cancer
action of iNOS inhibitors applies also to human pancreatic cancer]
Cf., Calvise, D.F. et al., Carcinogenesis, 2008, 29 (8) 1639-1647
(Aberrant iNOS signaling is under genetic control in rodent liver cancer and potentially prognostic for the human disease)
[Progressive induction of iNOS correlates with aggressiveness of lesions; the mechanism favoured was via cross talk of iNOS with NF-κ B and
HaRAS/ERK cascades]
Cf., Chen, J., et al., Carcinogenesis, 2005, 26 (6) 1035-1043
(Differential requirement of signal pathways for benzo[a]pyrene (B[c]P) induced iNOS in rat esophageal epithelial cells)[This suggested that the
carcinogenic action of B[c]P in human cancers also was a result of increased iNOS activity and that iNOS inhibitors are appropriate anti-cancer therapeutic
agents]
Cf., Crowell, J.A. et al., Molecular Cancer Therapeutics, 2003, 2, 815-823
(Is iNOS a target for chemoprevention?)
[In this review article it is noted that iNOS expression is increased in colon, breast, prostate, bladder, skin, oral cavity, head and neck, esopahgus, lung and
gastric cancer but in some circumstances, for unknown reasons, increased iNOS expression can, however, also counter tumor growth but this is less
common than the opposite effect]

Kojima S. et al., e.g. in Eur. J. Nucl. Med., 1983, 8, 52-50; (Elevated uptake of 67Ga and increased HS content in liver-damaged rats)
Cf.., ibid.; 1984, 9, 51-56
[These authors showed that cations which have a high affinity for HS in vitro and in vivo include Al3+, La3+, Ce3+, Er3+, Sm3+, Yb3+, In3+ , Ru3+, Eu3+ as well
as Fe3+ and Fe2+]

Kouretas, P.C. et al., J. Mol. Cell. Cardiol., 1998, 30 (12) 2669; Chem. Abs., 130, 246661j
(Non-anticoagulant H increases endothelial nitric oxide synthase activity)

Kraemer, P.M., J Cell Physiol., 1968, 71, 109-120

(Production of H related glycosaminoglycans by an established mammalian cell line)
[This was the first report of the occurrence of free cell cytosol HS, not linked to protein cores; the author had also noted that N-linked –SO3- groups were
extremely labile in comparison with the O- linked –SO3- groups; this phenomenon assumed greater importance following the discovery that such lability is
of possible critical relevance to how inappropriate HS microstructural alterations could occur in degenerative disease processes since the hydrolysis of N-
linked SO3- produces glucosamine-NH2 groups which are primed for rapid reaction with nitric oxide metabolites such as nitrite which is characteristically
found to be associated with degenerative disease conditions which are also characterized by the nitration of tyrosine groups in proteins,
cf., Ischiropoulos H, ref. 1a-8, vide infra]

Kreuger, J., P. Jemeth, E. Sanders-Lindberg, L.Eliahu, D. Ron, C. Salmivirta and U. Lindahl, Biochem. J., 2005; 389, 145-150;
Cf., Kreuger, J., et al., J. Cell. Biol., 2006, 34 (3) 461-469
{Difficulty with a “DNA-analogy signaling system model” for HS}.
The smart information-encoded system manager modus operandi where dedicated patterns of anionic sugar
sequences is supposed to act as specific sites for protein binding is questioned here by Kreuger
et al., who propose that the antithrombin pentasaccharide sequence is only true type of unique H/HS
epitope.
This idea is contradicted by e.g., the more recent report by Vanpouille et al. 1 that Cyclophilin A binds to H/HS via a specific type of microstructure
consisting of rare sites containing 3-O-sulphated, N-unsubstituted glucosamine groups;
similarly it had been earlier indicated that differently structured H/HS fragments differentially inhibit the binding of lipoproteinase to α 2-macroglobulin
(cf., Larnkjaer et al. 1).
It is now suggested that this observed lack of sensitivity found by Kreuger et al. could have arisen from a diminution of the
essential inorganic cofactors which had been present in intact H/HS polymers during the oligomer preparation and fractionation procedures. This idea is
consistent with the findings (e.g. Grant, D., et al.1 and Rovo, J. & E. Morava1) that fragments of H generally bind metal ions less effectively that do intact
polymers and that lower molecular weight; lower molecular weight dextran sulphates are also less effective than higher molecular weight dextran
sulphates as selective complexing agents for precipitating Mn2+ -low density lipoprotein during a traditional assay of serum lipoproteins (cf. also Warnick,
G.R. et al., Clin. Chem., 1982, 28 (6) 1379-1388 and
Rudd, T.R., et al., loc. cit.).

Lahiri B. et al., Arch. Biochem. Biophys., 1999, 293, 54-65

(Depolymerization of H by complexed ferrous ions)

Lai, J-P. et al., Hepatology, 2008, 47 (4) 1211-1222

(Sulfatase 2 up-regulates glypican 3, promotes FGF signaling and decreases survival in hepatocellular carcinoma)
[Sulfatse 2 expression was negatively correlated with recurrence of hepatocellular carcinoma after surgery]

Laihev, J.P., Biometals, 1996, 9 (1) 10; Chem. Abs., 124, 109969t
(Fe(II) selectively degrades H)

Larnkjaer, A. et al., Biochem J. , 1995, 307 (1) 205-214; Chem. Abs. 122, 285358v
(Structure of H fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to α 2-macroglobulin-receptor/low density
lipoprotein receptor –related protein by H fragments)

Lauri, S.E. et al., J. Neurosci., 1999, 19 (4) 1226-1235

(Regulatory role oand molecular interactions of a cell-surface HS PG (N-syndecan) in hippocampal long-term potentiation)

Leffler, J.E., J Org. Chem., 1955, 20, 1202-1231

(The enthalpy-entropy relationship and its implications for organic chemistry)
[This gives a comprehensive compilation of compensated organic reactions where at the ‘isokinetic’ temperture all reactions in a given series (such as
dehydrochlorination reactions) occur at the same rate].

Levy R.J., et al., Science, 1985, 228, 190-192

(Inhibition of calcification of bioprosthetic heart valves by local controlled-release diphosphonate [bisphosphonate EHDP]
Cf., Kramsch, D.M., et al. ibid.,1981, 213, 1511-1512
(Atherosclerosis: prevention by agents [bisphosphonates including EHDP] not affecting levels of blood lipids)
{Cf., Grant, D., et al., (1992) and (1989) and Liang, J.N., et al., Carbohydr. Res., 1982, 106, 101-109
(An essential role for the 2-sulfamino group in the interaction of calcium ion with H)

Li, J. et al., J. Cell Sci., 2002, 115, 1951-1959

(Increased responsiveness of hyoxic endothelial cells to FGF2 is mediated by HIF-1α -dependent regulation of enzymes involved inm synthesis of HS
FGF2 binding sites)
[Hypoxia generally augmented HS sites required for FGF2 binding this apparent increase in HS was accompanied by a decrease in ChS; (however
hypoxia diminished syndecan-1,-2 and -4 expression but did nor affect perlecan expression; reduced core protein expression was thus an opposite effect to
the apparent increase in HS which was attributed to increased expression of HS2ST]

Liang, J.N. et al., Carbohydr. Res., 1982, 106, 101-109

(An essential tole for the 2-sulfamino group in the interations of calcium ion with H)

Lidholt, K. & U. Lindahl, Biochem. J., 1992, 287 (2) 21-29

(Biosynthesis of H and heparinoids from E. Coli capsular polysaccharides)
Cf., Klaus K., et al., Brit. UK Pat. Appl., GB 2,254,083; Chem. Abs., 118, 55633u
Ideas arising from experimentation coupled with an extensive literature review suggested that
HS evolved from its evolutionary precursor (similar to bacterial heparanosan (cf. Bhattacharya1 this starting material is retained for Golgi apparatus
generation of animal HS polysaccharides).
Isomerization of most of the precursor glucuronate to iduronate achieves a more flexible system of Ca2+ buffering which could permit HS to more
effectively act in a feedback control loop together with Ca2+ activity.

Liebel, M.A. & A.A., White, Biochem. Biophys. Res. Commun., 1982, 104 (3) 957; Chem. Abs., 96, 138749q
(Inhibition of soluble guanylate cyclase by Mn2+ and sulphated polysaccharides, H and sulphated algal polysaccharide carrageenan , order of effectivness
λ >ι >κ )
[These data could suggest that this and other redox metal ions may be associted with HS as a control mechanism for the modulation of nitric oxide
biochemistry (and other H-like moleucles might participate in similar modulation).
Cf. also Vural et al., (loc. cit.) and other related studies which indicated that augmention of nitric oxide in a range of degenerative and inflmmatory
diseases may occur via inhibition of arginase (an enzyme which employs a Mn2+ stabilized Mn2+-Mn2+ active site) or dysfunction of natural mechanism of
endogenous nitric oxide synthase inhibiton, e.g., by asymmetric dimethyl arginine]

Lindahl, U., Thromb. Haemostasis, 2007, 98, 109-115

(HS-protein interations. A concept for drug design)

Liu, D et al., J. Biol. Chem. 1999, 274, 4089-4095 (Roles of Metal Ions in Bacterial Heparanase)
(The Ca binding site of bacterial heparanase I is essential for its activity)
Cf., also Mihailescu, G, K.J. Nitelea, Rom. Pat. RO 76, 508; Chem. Abs., 99, 93718x.
(The addition of zinc acetate was indicated to prevent loss of H due to heparinase activity during
extraction of H from animal tissues)
Shaya, D et al., J Biol. Chem., 2006, 261, (22) 15525-15535 reported that a Zn2+ ion is bound within the central domain and
plays an essential structural role in the stabilization of a loop forming wall of the substrate-binding site (from studies of
heparinase II from Pedobacter heparinus (Flavobacterium) and its complex with HS-related disaccharide

Lobo, N.A., et al., Annu. Rev. Cell Dev. Biol., 2007, 23, 675-699
(The biology of cancer stem cells); Cf., Dalerba, P. et al., Annu. Rev. Med., 2007, 58, 267-284 (Cancer stem cells: models and concepts)

Lofgren, K., et al., Eur. J. Neurosci., 2008, 28 (5) 964-972

(Involvement of glypican-1 autoprocessing in scrapie infection)
[Confocal immunofluoresce microscopy was used to follow the intial processes involving intracellular nitrosative cleavage of HS sidechains [to give PPS
like fragments] (which are dependent on ascorbate and Cu) in glypican-1 are indicated to be involved in the clearance of misfolded prion PrPSc responsible
scrapie-like diseases; normally glypican-1 and Cu-prions are attached by their glycosylphophatidyl anchors to lipids rafts, from which Cu is belived to
stimulate internalization to the endosomes; this study
suggests how PPS prevents and reverses prion diseases].

Long, W.F., & F.B. Williamson, IRCS J. Med. Sci. (Library Compendium) 1979, 7, 429-434;
Cf., Med. Hypoth. 1983, 11, 285-308
(Glycosaminoglycans, calcium ions and the control of cell proliferation)
[The patterns of GAGs extracellular space and pericellular environments seemed to influence cell movement, growth and development. The GAGs
associated with tumours and cultured transformed cells were notably different from those of normal tissue and cells.
Of the GAGs HS seems to have an especial relevance. The less sulphated GAGS tend to promote cellular growth while the more sulphated GAGs inhibit
such growth
Calcium ions are involved, e.g. in muscular action, coupling of secretion and excitation at nerve endings, the funntion of exocrine andendocrine glands and
the mediation of a number of hormones, a common feature of which is that stimulus arriving at the cell surface, is transformed into an intracellular signal
responsible for generating biological activity. It was suggested that this signaling was facilitated by the binding of calcium ions to sulphated
polysaccharides so as to change their conformations. Calcium ions show an especially marked effect on cellular proliferation of transformed cells which
proliferate in media with low concentrations of calcium ions, suggesting that alteration of the mechanism by which calcium ions are controlled (putatively
involving glycosaminoglycans in the pericelluar mediaum) is part of a general mechanism of neoplastic transformation.
It was suggested tht during the G1 phase of the cell cycle there is an increased transformation of intracellular calcium (and other) ions into the cytosol
involving a coopertive release from GAG-calcium complexes enabled by conformation changes of GAG polysaccharides. Neoplastic transformation
involved removal of GAG-enabled restrictions in calcium provision. It was further suggested that the outlined mechanism for control of animal cells by
GAGs via calcium, also applied throughout biota and suggested that a primary function for all extracellualr polysacchrides invovles binding and release of
metal ions.

Long, W.F. (1994) Proc. of a satellite meeting “Cellular Antioxidants” of the 7th Biennial Meeting of the International Society for Free Radical Research,
Queenstown, New Zealand
(Sulphated polysaccharides protect erythrocytes against free radical-induced damage)

Long, W.F. (2003) http://www.abdn.ac.uk/~bch118/publications2003march.doc

(Research & Scholarship. Publications of Professor W.F. Long and former coworkers in the Aberdeen Polysaccharide Group)

Luck, W., Ber. Bunsen. Phys. Chem., 1965, 69 (1) 69; ibid., 1962, 66, 766, 1965, 69, 626, 826
(These reports include results of studies by near infrared spectroscopy of salt effects on the association of water)
(This and related work established that the origins of the Hofmeister effect were probably the effects of
Hofmeister agents on the supermolecular structure of liquid water)

Cf. Kleeberg E. Ed. (Proc. Symp. (2-3 Apr. 1987 Marburg in Honor the 65th
birthday of Werner A.P. Luck, 1987, Marburg FRG)
“Interaction of Water in Ionic and Nonoionic Hydrates”. Springer Verlag, Berlin, 1987;
Luck, W.A.P., Topics Curr. Chem., 1976, 5, 115-180
(Is life mainly inorganic chemistry?)
and
Kleeberg, H. & W.A.P. Luck, Naturwiss., 1977, 64, 223
(Near-infrared spectroscopy of H2O sorbed in bovine nasal cartilage)
[Water is not uniformly hydrogen-bonded at various H2O concentrations in cartilage]
Lyon, M.E., Clin. Biochem., 1995, 28 (1) 79-84
(Specific H properties interfere with simultaneous measurement of ionized Mg and ionized Ca)
[Time-dependent bias was observed in ionized Mg and Ca concentratons determined with use of Zn H but not with Li H or electrolyte-balanced H]

Lyon, M. & J.T. Gallagher, PCT Int. Appl 1994, WO 9421689; Chem. Abs. 122, 47510u
(HS oligosaccharides having hepatocytic growth factor (HGF) binding affinity)
[HGF binds to HS oligosaccharides with 12-14 monosaccharide residues with a relatively high proportion of 6-O-sulphate hexosamines; claimed to have
therapeutic benefit]

Lyon, M. & J.T. Gallagher, Matrix Biol, 1998, 17 (7) 485-493

(Biospecfic sequences and domains of HS and the regulation of cell growth and adhesion)

Mackintosh, G et al., Proc. 7th Biennial Meeting, Int. Soc. Free Radical Research, Sydney, 6-10 Nov. 1994
(H inhibits hydroxyl radical production – an ESR spectroscopic study)

Marczynski, B., Med. Hypoth., 1988, 26 (4) 239-249

(Carcinogenesis as the result of the deficiency of some trace elements)
[This hypothesis suggested that a deficiency of Ga(III), Ge (IV) Si (silica) As(V) and Se(IV) in DNA of eukaryotic cells could be involved with a
semiconductor function which influences the control of genetic expression]
[N.b. these elements are also found by ICP-MS to occur consistently in different H samples (prior SSMS data for H had showed the especial abundance
Ga, Si and As in H) which could suggest an alternative, analogous hypothesis of the role of deficiencies of such elements attached to HS in the promotion
of carcinogeneis]

Masri, F.A. et al., Amer. J. Respir. Crit. Care Med., 2005, 172, 597-605
(Protein nitration is elevated in lung cancer)
Cf. also tyrosine nitration is elevated in
breast cancer (Samoszuk, M., et al., Breast Cancer Res. Treat., 2002, 74 (3) 271-278),
pancreatic cancer (Vickers, S.M. et al., Arch. Surg., 1999, 134, 245-251),
human glioma (Fiore, G., et al., Neurosci Lett., 2006, 394, (1) 57-62),
B cell non-Hodgkin’s lymphoma and
multiple myeloma (Mendes, R.V. et al., Histopathol., 2001, 39 (2) 172-178

McCarthy, S.M, et al., Biochemistry, 2008, 47 (21) 5832-5840

(Nitric oxide regulation of MMP-9 activation and its relationship to modification of the cysteine moieties )
[Data suggest that nitric oxide is capable of directly activating proMMP and that S-nitrosylation of MMP-9 by nitric oxide donors is unrelated to their
abilities to regulate MMP-9 activity]

McCarty, M.F. Med. Hypoth., 1997, 48, 245

(Glucosamine may retard atherosclerosis by promoting endothelial production of HS PG)

Merce, A.L. et al., J. Inorg. Biochem. 2002, 89, 212-218

(Complexes of Fe(III) with hyaluronan)
[Cf. also Sipos, P. et al.,loc. cit.]

Meschino, J.P., Dynamic Chiropracter, 2002, 20, 2 (interenet file)

(The research status of glucosamine sulfate)
[It seems that the availability of enzymes required for fructose-6 phosphate dependent synthesis of glucosamine declines with age]

Moffat, Colin. F., Ph.D. Thesis, University of Aberdeen, 1987:

“Synthesis, characterization and applications of chemically modified heparins”;
the multielement data for ‘Na’H are given in this thesis on p.187-189 and for Tl H on p.195-196.
The procine sodium H which was subjected to inorganic analysis had been selected and donated (apparently to several academic research laboratories) by
Dr. W.E. Lewis, a scientist involved at that time in the commercial manufacture of H at a (former) major (Runcorn, UK) pharmaceutical industrial facility.

Morimoto-Tomita, M. et al., J. Biol. Chem., 2002, 277, (51) 49175-49185

(Cloning and characterization of two extracellular H-degrading endosulfatases in mice and humans)
[Mammalian Sulfs are extracellular endosulfatases with strong potential for modulating the interations of HS proteoglycans in the extracellular
environment]
Cf. also Lamanna, W.C. et al., ibid., 2008, 283 (41) 27724-27735
(Sulf loss influences N, 2-O and 6-O-sulfation of multiple HS proteoglycans and modulates fibroblast growth factor signaling.
[Findings indicate a dynamic influence of Sulf deficiency on the HS biosynthesis machinery. Sulfs are involved in a potential cellular feed-back
mechanism, in which they edit the suphation of multiple HS proteoglycans and modulate the expresion of HS biosynthetic enzymes thereby potentially
regulating cellular signaling]

Morissete, C., et al., Potential application of glycosaminoglycan-related compounds in Alzheimer’s disease

Pharmacological Mechanisms in Alzheimer’s Disease
Springer, New York, 2008, p. 255-272

Multhaup, G., et al., Biochemie, 1994, 76 (3-4) 304

Murata K & Y. Tokayama, Atherosclerosis, 1989, 78, 69-79; Chem. Abs., 111, 151343q
(Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissue)
[The aterial GAG data tabulated by these authors show that the HS content of affected arteries diminished linearly with age]

Nader, H.B., et al., Int. J. Biol. Macromol., 1981, 3 (6) 356-360

(Heterogeneity of H: characterization of one hundred components with different anticoagulation activities by a combination of electrophoretic and affinity
chromatography methods)
[Ba acetate electrophoresis method employs selective binding of Ba2+ to different molecules probably caused by the presence therein of different
microstructures]

Nader, H.B., et al. (1983) see ref. 5 (vide infra)

Nader, H.B., et al., Carbohydr. Res., 1988, 184, 292-300

(Maintenance of HS throughtout evolution: chemical and enzymic degradation, and 13-C-n.m.r. spectral evidence)

Nagasawa, K, et al., Carbohydr. Res. 1992, 236, 165-180

Oh, G.S. et al., Free Radic. Biol. Med., 2006, 41 (1) 106-119
(Hydrogen sulfide inhibits nitric oxide production and nuclear factor kappa β via heme oxygenase 1 expression in RAW 264.7 macrophages stimulated
with lipopolysaccharide (LPS))
[This accords with the report that nitric oxide production in LPS-stimulated macrophages expressing the H2S generating CSE the mRNA of which was
significantly reduced by the additon of L-cysteine.
Overexpresison of CSE stimulates smooth mucscle cell apoptosis due to increased endogenous production of H2S]
Cf. Benavides G.A. et al., loc. cit.

Ogamo, A., et al., Carbohydr. Res., 1989, 193, 165-172

(Reactivity toward chemical sulfation of hydroxyl groups of H)
[An improved method for the chemical desulfation of H employed the pyridinium salt plus 3:6:1 (v/v) 1,4-dioxane-dimethylsulfoxide-methanol with the
intermittent addition of pyridinium chloride (this achieved 100% N and 6-O desulfation of 2-amino-2-deoxy-D-glucose and 91.5 % 2-O desufation of L-
idosiduronic acid unit; after re-N-sulfation the desulfated H was converted to a tributylammonium salt and sulphated with pyridine SO3 in N,N
dimethylformamide to achieve O sulfation (the re-O- sulfation showed the order of reactiviey of the hydroxyl groups to be HO-6 in GlN>>HO-2 in IdoA>
other HO (viz. HO-3 in GlN, HO-3 in IdoA and HO-2 in GlcA.). DeN-sulfated N-acetylated H showed a tendency to molecular association; free amino
groups of de-N sulphated H can act to stabilize the residual O-sulfate groups by internal salt formation]
{The conditions used to allow chemical modification of H correspond to those where the water structure which normally stabilizes the sulfation of H is
greatly diminished in the presence of quaternary N counteractions}

Orre L. “Proteomic analysis of DNA damage induced stress signaling with focus on p53”
Thesis, published by the Karolinska Inst. Stockholm, 2008
Includes a list of relevant publications and submitted papers by Orre, L.M., et al.

Osmo
Anionic polysaccharides in osmoregulation. Provisional Reference List
Ca2+ ions play an important part in the stabilization of soil by polysaccharides.
{Cf. ,e.g.,Yormah, T.B.R. & P.O. Egbenda UNESCO AJST, 2005, 6 (1) 90-96 and the references cited in this article}

This ability to Ca2+ to improve the binding of polyanions like polyvinyl alcohol and anionic polysaccharides to soil particles can
be rationally proposed to be a related phenomenon to how Ca2+ and other inorganic cofactors act as anionic polysaccharide and
anionic polyphosphate protein ionic linking agents in animal biochemistry.

{Cf., Role of extracellular bacterial polysaccharides as soil stabilizers

Soil organic matter is believed to be stabilized by (decomposition products structurally related to extracellular bacterial
polysaccharides was suggested by pioneeeing work by Haworth (Birmingham University, UK)
Cf., Haworth, W.N., F.W. Pinard & M. Stacey, Nature, 1946, 158, 836-837 (Function of bacterial polysacchrides in soil)
[cf., also J. H. Quastel, Annu. Rev. Plant Physiol., 1954, 5, 75-92]).

Negatively charged groups such as uronic acid and sulphated sugars determine the adhesive capacity of extracellular
polysaccharides for the stabilization of interstitial sediments attached to diatoms
{Staats, N., et al., Eur. J. Phycol., 1999, 34 (2) 161-169
(Isolation and characterization of extracellular polysaccharides from the epipelic diatoms Cylindrotheca closterium and Navicula
salinarum)}.

Cf., also, Prieto, A. et al., Mycological Res., 2008, 112 (3) 381-388
(A polysaccharide from Lichina Pygmaea and L. Confines supports the recognition of Lichinomycetes)

Cf. also Web document - bacterial polysaccharides of biotechnological interest

The recent web document gives a useful discussion of the current technological interest in extracellular bacterial polysaccharides
web. fedoa.unina.it/2574/01/Carannante_Science_Chimiche.pdf
[Bacterial polysaccharides include lipopolysacharies, lipooligosaccharides, capsular polysaccharides and intracellular
polysaccharides; in pathogenic bacterial capsular extracellular polysaccharides and O-antigenic lipopolysacchrides are involved in
the immune response, representing the first line of defense against bacteriophages.
Extracelllular polysaccharides (e.g. as slimes) are thought to inhibit desiccation, phagocytosis and phage activity as well as
the actions of antibiotics and toxic compounds including toxic metal ions and osmotic stress, they are believed to be involved in
bacterial adhsion to solid sufaces, the formation of biofilms and in cellular recognition.
This document cites Hascall, V.C. & G.K. Hascall, “Cell Biology of Extracellular Matrix” Plenum, 1981;
 Lidholt, K., Biochem. Soc. Trans., 1997, 25, 866-870 and Wim J. van Blitlerswijk et al., Compl. & Alt. Med., 2003, 3, 2.
For information relevant to extracellular polysaccharide prevention of desiccation, cf. Robertson E. & M. Firestone, Appl. Environ.
Microbiol., 1992, 58, 1284-1291; for biofilms, cf., Costerton, J.W., et al., Annu. Rev. Microbiol., 1987, 41, 435-464

Ouida, M.-O., et al., Biochem. Biophys. Res. Commun., 2007, DATA

(Heparanase I generated HS polyanonic sequences by scission at highly sulphated regions which promoted tumour growth while heparnase III (??)
generated HS sequences which inhibited tumour growth via scission at less sulphated regions.
The modus operandi was believed to be relevant to HS microstructure-dependent modulation of basic fibroblast growth factor activity)

Paka, L. et al., J Biol. Chem., 1999, 274 (8) 4816-4823

(Apolipoprotein E-containing high denisty lipoprotein stimulates endothelial production of HS rich in biologically active H-like domains. A potent
mechanism for the anti-atherosclerotic action of vascular apolipoprotein E)

Panov V.P. & A.M. Ovsepyan, Visokomol. Soed., 1984, 26, (9) 1963-1970
(Study of H salts by spectroscopic methods)
[This well performed study reports how counterions affect the infrared and NMR spectra of H; the asymmetric CO2- stretching frequency in H dissolved in
D2O varied with cation in proportion to the polarization power of the counter cation; Grant, D et al. (im an independent study which was conducted before
the publication of the Panov & Ovespyan article) found a similar relationship between the polarization power for the symmetric vCO2- vibration of H metal
ion complexes studied as cast films by multiple specular reflectance infrared spectroscopy, a method which allowed the alteration of the infrared
absorptions of water molecules interlinking H and metal ions to be observed; this phenomenon seemed to be an important aspect of such films. The
author had employed a variant of the multiple specular reflectance method from previous experience of its ability to provide highly resolved spectra gained
at the International Synthetic Rubber Co.]

Parrish, R.F. & W.R. Fair, Biochem. J., 1981, 193, (2) 407-410
(Selective binding of zinc ions to H rather than to other glycosaminoglycans)
Cf., Grant, D., et al., ibid., 1992, 287 (3) 849-853 and Woodhead N.E. et al., ibid., 237 (1) 231-4

Park, J.W. & C. Mukherjee, Bull. Korean Chem. Soc., 1980, 1, (3) 83-87
(Spectroscopic studies on Cu2+ and Ca2+ binding with glycosaminoglycans)
[Cu2+ bound to H, by a partly chelation mechanism which also showed logK to vary with I (logK= 3.98-2.98I 0.5) in accord with electrostatic predictions;
Ca2+ bound to H less strongly than did Cu2+ for which ESR suggested 4-fold coordination].
Cf. Rudd, T.R. et al., Carbohydr. Res., 2008, 343, 2184-2193

Park, M.J., et al., J. Cell. Biochem., 2005, 95(5) 955-969

(Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear kappa β and reactive oxygen species)

Pauling L., Science 160, 265

Cf. Cameron, E., & L. Pauling, Proc Natl. Acad. Sci. USA, 1985, 75, 4538-4542; Cf., ibid., 79, 3685-3689
(Supplemental ascorbate in the supportive treatment of cancer: prolongation of survivial times in erminal human cancer)

Park S.H. & A.E. Aust, Cancer Res., 1998, 58, 1144-1148
(Participation of iron and nitric oxide in the mutagenicity of asbwstos in hgprt-gpt+ Chinese hamster V79 cells

Pei, P., et al., Free Radic. Biol. Med., 2006, 41 (8) 1315-1224
(Reduced nonprotein thiols inhibit activation and function of MMP-9)

Perez, M et al, Biochem. J., 1998, 335, 369-374

(Sulphated glycosaminoglycans prevent the neurotoxicity of a human prion protein fragment)

Petersen, L.C. & M. Jorgensen, Biochem. J., 1983, 211, 91-97

Kinetic rate constants for the H enhanced reaction between thrombin and antithrombin obtained in LiCl, NaCl
or KCl electrolytes at 90mM and 300mM, suggested that the rate determining process for the principal H-mediated anticoagulation mechanism was highly
sensitive to the chemical nature and/or the ionic strength (I) of these electrolyte solutions; it seemed possible that that the different alkali metal cations
(e.g. by their different modes of binding to H and possibly related water activity changes) could markedly influence transition state reactivities.

[The apparent first order rate constants of thrombin proteolysis (in the presence of a Tris.Mops buffer plus these alklai metal salt electrolytes) were for
90mM LiCl, NaCl, and KCl, 0.013, 0.024 and 0.029 s-1 and for 300mM LiCl, NaCl and KCl 0.033, 0.012 and 0.003 s-1 respectively, i.e. different
countercations present in the electrolyte solutions had an opposite influence on the reaction rates for I values of 90mM compared with 300mM.
A similar opposite effect of this range of I values was confirmed to affect how the counterion binding to H from LiCl, NaCl and KCl solutions releases H+
(e.g., from clusters of water molecules associated with H).

In a series of experiments (Grant et al., 1992f, Biochem. J., 285, 477-480) designed to test the effect of ionic strength (I) (which including the above range
of I) upon counterion binding to H, it was confirmed that binding of H to Li+, Na+ and K+ varied with both the value of I and the chemical nature of the
cation (it would have been predicted to have been the same for all these cations of unit formal charge if purely electrostatic Manning binding had
occurred). [Binding was also less discriminated between counterions at low, non-physiological pH values than at higher pH values]. For K+, a lower
degree of binding to H was noted at I = 0.0057 than at higher I values (this e.g., being putatively relevant to the binding of K by H under extracellur
physiological conditions (but rapid release of K+ from H could occur under lower ionic strength conditions) cf., such differential binding of K by H might
be used to tranport K+ and also to selectively bind K+ or Na+). At I = 0.336 Mg2+, Ca2+ as well as Na+ and K+ were bound to H in a much less
discriminatory manner than was the case at the lower I values studied where the binding of Mg2+ became weaker than the binding of Na+ which however
remained more strongly bound than did Li+ which at I = 0.0057 became bound to H more weakly than did (CH3)4N+ (but both of these counterions were
very weakly bound under these conditions).
Such effects of I (and pH) on the discriminated cation binding to H might be explicable by a dependence on an alteration of water structures present with
the different countercation binding.
Such a possibility also concurrs with the role of nucleation in binding of counterions to H; this process seems to be an essentially irreversible, ‘not-a-
simple-thermodynamic-drive’ process which however can approximate to a reversible process; it seems further to deviate from reversibilty in a counterion
sensitive manner.
The rate constants reported by Petersen & Jorgensen in the above communication can also be provisionally related to the way H binds water molecules in a
counterion differentiated manner. The H-O overtone vibrational energies found with solid samples (Grant et al., (1983) reported these values for LiH
6400cm-1, for NaH 6520cm-1 and for KH 6550cm-1) showed a direct proportionality to the rate constants reported by Petersen & Jorgensen for the
90mM, I value electrolytes and an inversely proportionality for the 300 mM, I value electrolytes suggesting that the thrombin-antithrombin reaction rate is
inhibited by greater water structuring at the H salt surfaces acting in combination with the blocking of antithrombin binding by the counterion-water-
polysaccharide complexes.
(Cf. also the effect of LiCl, NaCl and KCl on pH values of H solutions reported by Grant et al., (1992f) suggest the relative strength of binding of
counterions is reversed from K>Na>Li at I =ca. 90mM to Li>Na>K at I=ca. 300mM).

Pinhal, M.A.S. et al., Thromb. Res. 1994, 74 (2) 143

(Antithrombotic agents stimulate the synthesis and modify the sulpathion pattern of a HS PG from endothelial cells)

Powell, A.K. et al., Glycobiology, 2004, 14, (4) 17R-30R

(Review. Interactions of H/HS with proteins: Appraisal of structural factors and experimental approaches)
[Althought the evidence that proteins bind with a variety of affinities to particlar sequences within H and HS chains is fairly strong, the mechanism by
which H/HS regulates the activty of proteins throught such binding is still unknown]

Praline J., et al., Chem. Neurol. Neurosurg., 2007, 109 (10) 880-883

Purdey, M., Med Hypoth., 2004, 54, 278-306

[See also related discussions and literature search by D. Grant]

Qu, C., Doctoral Dissertation, Kuopiou University, Finland

“Articular Cartilage Proteoglycan Biosynthesis and Sulfation”
Kuopio University Publications D., Medical Sciences 419. 2007. 78p
[The hypothesis that the modus operandi of therapeutic action of dietary supplementation by glucosamine
in osteoarthritis is by an alteration of the primary biosynthesis of GAGs was not supported by this study]
{Cf., Valvason, C., et al., loc .cit, who suggest that the modus operandi of gluocosamine could be to reduce oxidative stress
including a reduction in iNOS leading to diminution of (inappropriate) nitrosative degradation of HS;
cf. also Grant D., interent file
http://web.ukonline.co.uk/dgrant/dg2/ }
{Cf., also Kelly, G.S., Intern. Med. Rev., 1998, 3 (1) 27-39
(The role of glucosamine sulphate and chondroitin sufates in the treatment of degenerative joint disease);
Russell, A.L., Med. Hypoth. 1998, 61 (4) 347-349
[Glucosamine “after a latent period, is rapidly developing a position in the treatment of osteoarthritis as well as a potential
therapeutic palce in wound healing and gastrointestinal disorders”. The usefulness of glucosamine sulphate for the treatment of of
arthritis and gastrointestinal disorders had been long known but the mechanism was a puzzle].
Cf., Reginster, J.Y., et al., The Lancet, 2001, 357, 251-256
(Long-term effects oof glucosamine sulphate on osteoarthritis progression: a randomised, placebo-controlled clinical trial)
[The long-term combined structure-modifying and symptom-modifying effects of glucosamine sulphate suggest that it could be a
disease modifying agent in osteoarthritis]

Rabenstein D.L. et al., Carbohydr. Res., 1995, 278. 239-256

Multinuclear magnetic resonance studies of the interaction of inorganic cations with H

Rajeswari J., et al., Bull. Cat. Soc. India, 2005, 4, 109-126

(Comparison of electrochemical behaviour of polyanions with that of biological species)

Reisenstein, P., Med. Oncol. Tumor Pharmacother., 1991, 8 (4) 229-233

(Iron free radicals and cancer)

Reusch R.N. et al., Proc. Soc. Expt. Biol. Med., 1989, 191, 377-381
(Poly-β -hydroxybutyrate/calcium polyphosphate complexes in eukaryotic membranes)

Ricard-Blum, S. et al. J. Biol. Chem. 2004, 279, 2927-2936

(The binding of endostatin to H requires the presence of Zn2+ ions)
Cf. ibid., 2006, 281 (35) 25195-25209
(The binding of collagen V to H/HS requires Ca2+ ions {Zn2+ ions apparently cannot substitute for Ca2+ here})

Ringvali, M. et al., J. Biol. Chem., 2000, 275, 25926-25930

(Defective HS biosynthesis and neonatal lethality in mice lacking N-deacetylase/N-sulfotrandferase-1)

Robinson, M.J. et al., J. Biol. Chem., 2002, 277 (5) 3658-3665

(The S100 family heterodimer MRP-8/14 binds with high affinity to H and HS GAGs on endothelial cells)

Rops, A.L. et al., Nephrol. Dial. Transpl., 2007, doi:10.1093/ndt/gfm194

(Expression of glomerular HS domains in murine and human lupus nephritis)
[Specific cytokine induced augmentation of “inflammatory N and 6-0 sulphated HS domains”]
Rosenberg, R.D. & A.I. De Agnostini, Adv. Exp. Med. Biol., 313, (Heparin Related Polysaccharides) 307-16

Ross, M.A., et al., Biochem. Soc. Trans., 1992, 20, 6S

(H reduces Fe(II)-catalyzed peroxidation of linolenic acid
Cf., ibid., 1992, 20, 216S; ibid., 1992, 20, 364S

Rothman, M.J., Bartell L.S., Ewig, C.S. & Van Wazer, J.R., J. Comput. Chem., 1980, 1, (1) 64-68

Roussel, B., et al., Clin. Exp. Immunol., 1990, 82 (2) 294-299; Medline 91055393
(A human monoclonal IgM autoantibody acitivity against HS and the mitotic spindle)
[HS could be an essential part of all membrane structures where its could modulate their formation and activities]

Rovo, J. & E. Morava, Kiserl. Orvostud., 1981, 33 (5) 528; Chem. Abs. 96, 24695p

Roy, R. et al., Material Research Inovation online 9-4:1433-075X p577-608

(The structure of liquid water: novel insights from material research of potential relevance to homopathy)

Rubio-Gayosso, I., et al., Am. J. Physio. Heart Circ. Physiol., 2006, 290 (6) 112247-112256
(Reactive oxygen species mediate modification of glycocalyx during ischemia reperfusion injury)
[N.b. the glycocalyx is a poorly understood GAG-containing covering of the luninal surface of endothelial cells which affects vascular rheology and
permeability; intravenous administration of H could inhibit the effects ischemia repefusion injury to the the glycocalyx]

Rudd, T.R. et al., Glycobiology, 2007, 17 (9) 983-993

(Influence of substitution pattern and cation binding in conformation and activity in H derivatives)
[Eight systematically modified H derivaties were studied by synchrotron radiation circular dichroism (SRCD) which is sensitive to uronic acid
conformation which was altered by (sulphation) substituion patterns. SRCD spectra of these derivatives following conversion to different countercation
(Na+, K+, Mg2+, Ca2+, Mn2+, Cu2+ and Fe3+ ) forms also revealed that almost all substition/cation combinations resulted in unique spectra showing each to be
structurally distinct. These findings were confirmed by NMR (including by NOE and by T2 measurement. Conversion to the K+ and Cu2+ forms seemed
also to drastically modify biological activity (e.g., from inactive to active in a cell-based assay of FGF21/FGFR1c signaling), this effect not being
reproduced by the free cations. These results confirm previous indications of this behaviour of different salt forms of H evident from literature reports
(also suggesting the existence of a common ‘quality control’ problem caused by different trace (residual) countercations present on H or differences caused
in vivo by differences of contact times, pH etc. with mutil-inorganic element containing biological fluids which will tend transfer the most highly charged
and also least abundant of the dissolved inorganic ions present in extracellular matrices to H). The traditional view that signaling by H/HS depends on the
poylysaccharide anionic sequence is apparently incorrect and the metallome-heparanome concept is shown to be the more-likely-to-be-correct hypothesis].
Cf., also Rudd, T.R. et al., Glycobiology, 2009, 19 (1) 52-57
(Glycosaminoglycan origin and structure revealed by multivariant analysis of NMR and CD spectra)

Rudd, T.R., et al., Carbohydrate Res., 2008, 343, 2184-2193

(Site-specific interations of copper(II) ions with heparin revealed with complementary (SRCD, NMR, FTIR and EPR) spectroscopic techniques)
[The high affinity Cu(II) binding site corresponded with a tetrahedral Cu(II) on the 4-L-IdoA2S-α -4D-GlcNS(orNAc)6S-α -1- predominant H repeat
unit]

Salonen, J.T et al., Circulation, 1992, 86 (3) 803-811

(High stored iron levels are associated with excess risk of myocardial infarction in eastern Finnish men)
{This could suggest that release of iron ions could negatively impact by uptake and possible alteration of HS PG blood vessel walls}

Santos, M.C.L.G et al., J. Oral Rehab., 2004, 31 (7) 660-664

(Inhibition of human pulpal gelatinases (MMP-2 and MMP-9) by zinc oxide cements)
[Dysfunction of MMPs is believed to cause tooth decay and promote gum inflammatory diseases;
This process may be a possible example of how metal ions can modulate HS PG-growth factor activities]

Sarrazin, S et al. J Biol. Chem., 2005, 280 (45) 37558-37564

(HS mimicry. A synthetic glycoconjugate, that recognizes the H binding domain of interferon-γ inhibits the cytokine activity)

Schmitt, M. et al., Haematologica Reports, 2005, 1 (9) 28-33

Schwarz K.A., Proc Natl. Acad. Sci., USA, 1973, 70, 1608-1612
(Bound form of silicon in glycosaminoglycans and polyuronides)
Cf. McCarty M.F. Med Hypoth., 1997, 49, 177-179
(Anti-atherosclerotic activity of silicon may reflect increased endothelial synthesis of HS proteoglycans)

Scott, J.E., J. Physiol., 2003, 553 (2) 335-343

(Elasticity in extracellular matric ‘shape modules’ of tendon, cartilage, etc. A sliding proteoglycan-filament model)
[Key role of anionic GAGs include stabilization of water structure (so as to act like a shock absorber fluid)]

Seale, R., E.R. Morris & D.A. Rees, Carbohydr. Res., 1982, 110, 101-112

Shepherd, David Ian, M.D. Thesis “Multiple Sclerosis in North-East Scotland” Volumes I & II, University of Aberdeen, 1976
(An academic epidemiological study of the incidence of multiple sclerosis in North Scotland, a region with amongst the highest international prevalences
of this disease)

Shinko, K., et al. (with C.P. Dietrich and H.B. Nader (H/HS Disaccharides obtained by bacterial heparinase could control cytosolic Ca2+ by inhibition of
Na+/Ca2+exchanger) (H and HS disaccharides bind to the exchanger inhibitor peptide region of Na+/Ca2+ exchanger and reduce the cytosolic calcium of
smooth muscle cell lines (oligosaccharides need C4-C5 unsaturation and
1->4 glycosidic linkage for activity) J. Biol. Chem., 2002, 277, (50) 48227-48233

Siebler, T. et al., Bone, 2002, 30, 259-266

[Mast cell H is possibly involved in modulation of bone formation]

Siedel, D., et al., J. Clin. Invest., 1969, 48, 1211-1223; ibid., 1970, 49, 2396-2407
Cf., Neubeck, W., et al., Clin. Chem., 1977, 23 (7), 1296-1306

Siegel, S. et. al.. Colloids & Surfaces A: Physiol. & Energy Aspects, 1998, 138, -351
(HS may act as a biosensor and act as a flow sensor for flow control via biofeedback at blood vessel walls via HS conformation determined by bound
Na/Ca)

Sipos, P., et al., J. Inorg. Biochem., 2003, 95, 55-63

(Formation of spheroidal iron(III) oxyhydroxide nanoparticles sterically stabilized by chitosan in aqueous solution)
[A similar phenomenon seems to occur with H **]

Sitko, J.C., et al., Biophys. J., 2003, 84 (1) 419-431

Sequence –dependent DNA condensation and the electrostatic zipper )
[DNA condensation is a theroetical puzzle since the Possion-Boltzman theory of electrostatic replulsion between polyanions prohibits such adhesion]
Bloomfield lab. studies suggested that the MnCl2 condensation of DNA requires non-electrostatic theory for its explanation.
Cf also., Polyanichko, A.M. et al., Nucl Acids Res., 2004, 32 (6) 989-996 suggested on the basis of circular dichroism studies, etc. that the effect of Mn2+ on
DNA interaction was mediated by water (structure); Cf. also Duguid, J.G. et al., Biophys. J. 1995, 69, 2623-2641

Sivaram, P., et al., J. Biol. Chem., 1992, 267, 16517-16522

(Identification of an H-releasable lipoprotein lipase binding protein from endothelial cells)
{Cf., Bengtspm G et al., Biochem J., 1980, 189, 625-633
(Interaction of LPL with native and modified H-like polysaccharides)}

Snow, A.D., et al., Am. J.Pathol., 1988, 133,(3) 456-463

(The presence of HS proteoglycans in neurite plaques and congophilic angiopathy in Alzheimer’s disease)
Cf., also ibid., 1990, 137 (5) 1253-1270; 1994, 144 (2) 337-347 and M.M. Verbeek et al., ibid., 1999, 155 (6) 2115-2125 (which identified agrin as the
principal HS proteoglycan in Alzheimer’s disease plaques)
Zn dyshomeostasis seems to be involved in Alzhiemer’s disease
Zn2+ amyloid protein precursor was indicated by G. Multhaup et al., (Biochemie, 1994, 76 (3-4) 304) to be mediated by HS proteoglycans in Alzheimer’s
disease.
Al3+& HS (are putatively mutually strongly interactive sytems) D. Grant**
Cf., M.E. Ferrero et al., Biochem. Soc. Trans. 1989, 360
(Ateroid, a HS-containing mixture used in Italy for the treatment of dyslipemia (ascribiable to the lipidemic actions of non-anticogualnt H present in
Ateroid) (cf., Seethansthan, P., et al., Mol. Cell. Biochem., 1975, 8 (3) 177-183 found that the major GAGs in Ateroid were H and HS with some additional
DeS) was a promising therapeutically effective inhibitor of Alzheimer’s disease).
[Cf., also:
Cf. also Lorens, S.A. et al,. Seminar Thromb. Hemost. ,1991, 17 (Suppl. 2) 164
{Cf. also: HS mimetics as therapeutic agents for Alzheimer’s disease: Kisilevsky, R. et al., Nature Medicine, 1995, 1, 143-148}
Other authors (e.g., Taylor, G.A., et al., J. Epidemiol. Community Health, 1995, 49 (3) 323-324;
have suggested that Al3+ intoxication (perhaps related to Si ingestion) might induce Alzheimer’s disease plaque formation
Preliminary studies by D. Grant et al., have suggested that Al3+ uniquely binds and significantly reduced the pH buffering capacity of H/HS (Al-H in
solution seemed to produce pH4.6 whereas a similar amount of NaH produced pH7.3]
[The pH buffereing of CO2 containing solutions by physiological counterion substituted Hs seem to create a near-physiol. pH value which may be a
‘primitve’ function of Na and other physiological counterion Hs; this property may be part of the mechanism by which H affects cellular proliferation, cf.,
H inhibits intracellular alkalinization essential to proliferation of pericytes thought to involve augmentation by H of p21 (cyclin-dependent kinase
inhibitor) expression (cf., Khoury & Langleben, loc. cit.)}

Sobel, M. et al., J. Vasac. Surg., 2001, 33 (3) 587-594

(H modulates integrin function in human platelets)
[This seems to be the mechanism by which H (plus multivalent metal ions) causes activation and aggregation of platelets]

Souza-Fernandez A.B. et al., Critical Care, 2006, 10, 237 doi:10.1186/cc5069

(Bench-to-bedside review: The role of glycosaminoglycans in respiratory disease)
[HS was noted to be the most structurally complex of the lung GAGs (which also incude hyaluronan and chondrotin sulphate) and such GAGs seemed to
influence lung hydration homeostasis, tissue integrity and immune response. For lung function, the integrity of the HS system is now believed to be of
especial importance (The lung is also a rich source of mast cell H)]

Steel, Graham, Cf., e.g. McDougall, L., Sunday Herald, Oct 3 2004, article discussing first survivor of vCJD
[Cf. also internet search results for “Graham Steel PPS vCJD”]

Stichtenoth, D.O. & J.C.Frolich, Br. J. Rheum. 1998, 37, 246-257

(Nitric oxide and inflammatory joint diseases)
(It would be of interest to compare (the effect of metal ions) in the reactivity towards nitric oxide of normally and abnormally synthesisied HS: this idea
was discussed by the author in a presentation made to a Glasgow University seminar arranged by Prof. KE. McCall in 1999, and was also the subject of a
posting on the internet by the author (Grant, D., 2000,.. dg2)
Stillinger, F.H., Science, 1980, 209, 451-457
(Water revisited)
Cf., Kraemer D. et al., Proc Nat. Acad. Sci., USA, 2008, 105 (2) 437-442
(Temperature dependence of the two dimensional infrared spectrum of liquid H2O)

Sun, H. et al., Cancer Res., 2005, 65, 4442-4447

(Omega-3 polyunsaturated fatty acids regulate syndecan-1 (HS proteoglycan) expression in human breast cancer cells)

Sun, Y., Free Radicals Biol. Med., 1990, 8, 583-599

(Free radicals, antioxidant enzymes, and cacinognesis)

Takeuchi, T., et al., Analusis, 1998, 26, 61-64

(Ion exchange chromatography using anion exchanger modified with H)
[A Heparinized-(modified-SiO2) chromatographic column can be used, most unusually, is reported to achieve a good separation of both cations and anions
from a single elution over this form of heparanized silica; this tends to confirm other observations (e.g. Helbert, J.R & M.A. Marinin 6a-1, that (formally
highly anionic) H adsorbs (also highly anionic) SO42- ions) and H can form a complex containing metal salts (Fo-We, 1) and that H binds cations as well as
anions such as SO42-, PO43- and acetate
(Jaques. L.B. 6a);
This phenomenon may point to a role for SiO2 in H/HS in vivo (n.b. Si, putatively as SiO2, seems always occur chemically bound to polyronides, cf. Iler,
R.K. 1)

Takeuchi, Y., et al., J. Biol. Chem., 1990, 265 (23) 13661-13668

(Extracelllular ccalcium regulates distribution and transport of HS PGs in a rat parathyroid cell line)

Tan L. et al, Talanta, 2007, 71 (2) 827-832

(Electrochemical determination of H using methylene blue probe)
[Methylene blue bound to H with an association constant of 7.2x105
Ba2+ could be competively kinetically controlled pitted against methylene blue for binding to H]

Treiber, C. (with G. Multhaup) Biochemistry, 2006, 45, 6674

Tunbridge J.N. et al., Amer. J. Hosp. Pharm. 1981, 38, 1001-1004

(Stability of diluted H sodium stored in plastic sytinges)

Turley, R.S. et al., Cancer Res., 2007, 67, 1090-1096

(The type III transforming growth factor beta receptor as a novel tumor suppressor gene in prostate cancer)
[This receptor is a HSPG (betaglycan)]

Upchurch, G.R. et al., Cardiovasc. Pharmacol. Ther. 2001, 6 (2) 163-173

(H reacts with and inactivates nitric oxide)

Utsuimi, K & T. Oda, J.Cell Sci., 1973, 13, 901-911

(Ruthenium-red-induced cell agglutination and surface glycoprotein and mucopolysaccharide)
[Cell surface HS was identified at the likely target for (Ru2+ammoniate 3)6+]

Vanpouille, C. (with M. Lyon and D.G. Fernig) et al., J. Biol. Chem., 2007, 282 (33) 24416-24429
(Rare HS motifs facilitate selective binding of target proteins)

Valvason, C et al., Rheumatorlogy, 2008, 47 (1) 31-35

(Influence of glucosamine sulphate on oxidative stress in human osteoarthritic chondrocytes: effect of HO-1, p22Phox and iNOS)
[This papers suggests that dietary glucosamine and related compounds may act against osteoarthritis including by reversal of IL1β stimulated iNOS
expression thereby inhibiting potentially damaging excessive nitrosative activities]

Van Wazer, J.R.,, J. Dent. Res., 1964, 43 (6) 1052-1064

(The chemistry of phosphates)
[This paper deals with the general relvenance of the inorganic chemistry of phosphorus(V) oxyacids and their Ca2+ salts to biochemistry]
[The precipitates formed between chain polyphosphates and proteins can be considered to be in the class of insoluble phosphate salts.
It is likely that these also incorporate bivalent metal ions as crosslinking agents. Fig. 11 of this paper shows how in the presence of various amounts of egg
white the formation of a complex containing protein Ca2+ and polyphosphate polyanion alters the of the sequestering power. This is a complex function of
the chain length of the polyphosphates and the amount of protein present (the sequestering power is a function of the complexing ability of Ca2+ by the long
chain phosphates)] [cf. also a common use of polyphosphate additived in milk which form ternary Ca2+ protein adducts]{polyphosphate + saline + water
can be used to increase the weight of meat and meat products (e.g. as used in food processing and even apparently in adulteration where the inclusion of
polyphosphate keeps the added water in a stable form associated with the tissue}
Cf. also
Van Wazer, J. R., “Phosphorus and its compounds”, Vol 1., 1958 Intescience New York
Cf. also Amer. Sci., 1962, 50, 450

Van Wazer, JR & C.F. Callis, Chem Rev. 1958, 58, 1011 cf. p. 1018
(In this paper chain polyphosphates were noted to be less effective at holding iron in solution than at holding calcium in
solution, however chain polyphosphates were extremetly good peptizing agnets for holding iron in colloidal suspension which
from a practical point of view is equivalent to a strong complexing action.
 [A similar situation may pertain to the removal of paramagnetic ions from biological fluids by complexation of such metal ions to
H/HS proteoglycans** as the removal of paramagnetic ions (believed to be chiefly iron and manganese) from humic matter which
thereby allowed the NMR evauations of the organic structures present (briefly mentioned by Grant, D. (1977) loc. cit.]

Cf., also Nooner, D.W. & Oro J., J. Mol Evol. 1974, 3(1) 79-88
(Direct synthesis of peptides. Polycondensation of α amino acids by polymetaphosphate esters)
[This reaction may have enabled the formation of protein-like materials prior to the evolution of life]

Vijayagopal P. et al., Biochim. Biophys. Acta, 1983, 758, 70-83

(Hemostatic properties and serum lipoprotein binding of a HS proteoglycan from bovine aorta)
[This investigation of the formation of soluble an insoluble arterial wall proteolgycan-CaCl2-LDL cholesterol complexes showed that the propensity of HS
proteoglycan for rendering serum LDL insoluble was very finely balanced by the conditions of pH and I
and [Ca2+] whereas the propensity for the formation of ternary complex containing arterial chonroitin-dermatan sulphate was much greater and less finely
balanced. I.e., HS tends to inhbit processes which might promote arterial atherosclerosis and calcification while chondroitin-dermatan sulphate tends, in
absence of HS to promote this. The ratio of HS to chondroitin sulphate-dermatatan sulphate is then seen to be critical for the prevention of inosoluble
phase formation which can promote atherosclerosis at arterial surfaces
in agreement with the findings of Murata & Tokayama1 that this ratio is related to the presence of arterial plaque and cerebral atherosclerosis in human
subjects (the HS as percent of proteoglycans also diminished in direct propotion to the age of subjects)
The formation of such insoluble complexes was found by Vijayagopal et al. to occur only at or below pH 6.0 and I = 0.18, where in the presence of 30 mM
Ca2+ the amount of LDL converted into insoluble complex by two 100 µ g/ml arterial wall HS preprations was 4.5 and 12.8% whereas arterial wall
chondroitin sulphate-dermatan sulphate at 40µ g/ml and physiological pH converted 98% of added LDL into an insoluble complex in the presence of this
concentration of Ca2+. The formation of soluble compexes of LDL and HS was studied at physiological pH and I in the presence of added Ca2+ (2.7 µ M
Ca2+) where for two arterial HS preparations 15.4-18.9 % of proteoglycan was recovered in the top fractions after ultracentrifugation. This value was 4.5-
6.2% in the absence of added Ca2+
A similar difference between HS and chondroitin-dermatan sulphate was found by Grant et al. (1989) for the inhibition of calcification using an in vitro
seeded CaCO3 crystal growth model. These studies suggested that some dermatan and keratan sulphates could promote calcification but (unmodified) HS
was a potent inhibitor of this process. {However, HS molecules which had been modified by de-N sulphonation (a process which putatively might arise
under pathological situations at arterial walls) instead of being inhibitors of calcification, could become promoters of this process**).
Vijayagopal P et al., J. Biol. Chem., 1981, 256, 8234-8241
[The importance of the core protein for the formation of ternary complexes GAG-Ca2+-LDL cholesterol]

{Cf., also Vijayagopal P. et al., Biochim. Biophys. Acta, 1985, 837 (3) 251-261 and Biochem. J., 1996, 315 (3) 95-1000}

Vilar, R.E. et al., Biochem. J., 1997, 324, 473-497

(Nitric oxide degradation of H and HS at physiologically pH)
[Cf., Ghael, S. et al., Biochem. Mol Biol. Int., 1997, 183; Chem. Abs., 128, 39743j

Vural, H., et al., Biol. Trace Elem. Res., 2008 PMID 19099206
(The role of arginine-nitric oxide pathway in patients with Alzheimer’s disease)
Silverman, R.B., Acc. Chem. Res., 2009., Jan20 (Design of selective nitric oxide synthase inhibition for the prevention and treatment of neurodegenerative
diseases).
Cf., Dorheim, M.A. et al., Biochem. Biophys. Res. Commun., 1994, 205 (1) 659-665
(Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer’s disease)
Alkam, T. et al., J. Pharmacol. Exp. Ther., 2008, 327 (1) 137-147 (The extensive nitration of neurofilament light chain in the hippocampus is associated
with the cognitve impairment induced by amyloid beta in mice)
{Related studies: Sarban, S., et al., Biol. Trace Elem. Res., 2007, 115 (2) 97-106 (Relationship between synovial fluid and plasma manganese, arginase,
and nitric oxide in patients with rheumatoid arthritis); Yanik, M., et al., Eur. Arch. Psychiatry Clin. Neurosci., 2004, 254 (1) 43-47 (The role or arginine-
nitric oxide pathway in the pathogenesis of bioplar affective disorder);
Cf., also Neuropsychology, 2003, 47, 61-65 (Is the arginine-nitric oxide pathway involved in the pathogeneiss of schizophrenia?);
Baylis, C., Am. J. Physiol. Renal Physiol., 2008, 294 (1) F1-9 (Nitric oxide deficiency in chronic kidney diseases); Selly M.Y., et al., Neurobiol. Aging,
2003, 24 (7) 903-4 (Increased concentration of nitric oxide and symmetric dmethylarginine and decreased concentration of nitric oxide in the plasma of
patients with Alzheimer’s disease); Toutouzas, K et al., Horm. Metab. Res., 2008, 40 (9), 655-659 (Asymmetric dimethylarginine and endogenous nitric
oxide synthase inhibition as an important cause of vascular insulin resistance); Masuda, H., et al., Int. J. Urol., 2008, 15 (2) 128-134 (Significance of nitric
oxide and its modulation mechanisms by micturition disorders and erectile dysfunction). [A modulation of Mn-dependent nitric oxide synthase and
arginase activity might be worth considering under the conditions suggested by M. Purdey relating to epidemiologically suggested interrelationship
between Mn intoxication and neurodegeneration].

Warnick, G. Russell., & J.J. Albers, J. Lipid Res., 1978, 19, 65-76
(A comprehensive evaluation of the H-manganese procedure for estimation of high density lipoprotein cholesterol)
[The specific precipitation of apolipoprotein B (apoB) containing lipoproteins by sulphated polysaccharides especially by
H-Mn(II) which is a convenent reagent for blood; many studies of such ‘ternary complex’ formation between proteins, (sulphated) polysaccharides and
multivalent metal ions had been reported in ealier researches (reviewed by Cornwell & Kruger, loc. cit.)]
(Cf. also Warnick, G Russell., et al., Clin Chem., 1982, 28 (6) 1379-1399 and ibid., 2001, 47, 1579-1596; ibid., 2008, 54 (5) 923-924

Wassermann, A., Ann. Bot., 1949, NS, 13 (49) 79-88

(Cation adsoption by brown algae: the mode of occurrence of alginic acid
[Alginates occur naturally as multi-element salts rather than as free alginic aicd; under continuous flow conditions only cations were adsorbed but in static
systems anion adsoption could also be detected].
Numerous later investigations have confirmed that alginates adsorb a rnage of counterions from seawater.
Recent examples studies of exopolysacchrides which are similary thought to act as collectors of nutrent ions from seawater are
Nicols, C.M., et al., Appl. Envir. Microbiol, 2005, 71, 3519-3523
(Effect of incubation temperature on growth and production of exopolysaccharides by an Antartic Sea Ice Bacteria grown in batch culture);
Gutierrez T. et al., ibid., 2008, 74 (15) 4867-4876
(Emulsifying and metal ion binding activity of a glycoprotein)

Watanabe, A. et al., J Neurooncol., 2006, 77 (1) 25-32

(Expresion of syndecans, HS PG in malignant gliomas; participation of NF-κ B in upregulation of syndecan-1 expression)
[Normal tissue showed the expression of syndecans -2, -3 and -4 but syndecan-1 protein was localized at the cell surface of all malignant glioma cell lines
by flow cytometry]

Chitosan has also been proposed as a ligand for the collection from seawater of naturally occurring zinc, cadmium, lead and copper ions (e.g., Muzzarelli,
R.A.A., Talanta, 1971, 18, 853-858)

Whitfield, D.M. & B. Sarkar, Biopolymers, 1992, 32, 597-619

(Heavy metal binding to H disaccharides. II. First evidence for zinc chelation)
Cf., Whitfield, D.M. & B. Sarkar, J. Inorg. Biochem., 1991, 41, 157-170
(Metal binding to H monosaccharides: D-glucosamine-6-sulphate, D glucuronic acid, and L-iduronic acid)
[Only weak interations with the sulphate group were found. The 4C1 ring conformation of a sodium methyl β -D-glucopyranosiduronate was not perturbed
by binding of its carbohydrate and little evidence was found for chelation which was in marked contrast to the behviour of the sodium methyl-α -L-
idopyranosiduronate which was affected by the addition of Zn2+>Pb2+>Cd2+>Ca2+>>K+ ions where cation binding was observed to shift the equilibrium
mixture of 2So and 1C4 conformations towards the latter, an effect which seemed to have an electrostatic contribution although each cation had a different
effect, {Zn2+ was considered to bind to the carboxylate and the ring oxygen}). These results were in keeping with the hypothesis that the control of the
flexibility of the iduronate ring conformation by metal ions was involved in physiological control systems)]

Wickramasinghe, N.C., Nature, 1974, 252, 462-463

(Formaldehyde polymers in interstellar space)
{The existence of formalin plus H2O and SiO2 in interstellar space could suggest the origins of life on Earth derived from organic matter transported by
comets etc.}
Later studies tend to confirm this hypothesis, especially the direct collection of cometry organic matter by a spacecraft.
Cf., Sandford, S.A. et al., Science, 2006, 314 (5806) 1720-1724
(Organics captured from comet Wild 2 by the Stardust spacecraft)
[Elevated organic matter –CH2-/CH3 ratios in the comet is quantitatively consistent with what is expected from radiation processing into polymeric organic
matter of chemically-simpler species such as HCO and H2CO (e.g. adsorbed on astrophysic ices (plus SiO2); the apparent presence of aromatic groups in
the organic matter were found to be artefacts, the organic matter is essentially aliphatic]
{Such organic matter seems to be structurally similar to the most abundant organic polymer on the Earth’s surface namely soil humic material, the structure
of which was identified and discussed by Grant, D 1 (1977) ; a further property of this poly carboxylic acid polymers is that it can act as a natural water buffer
systes capable creating the current oceanic multi-inorganic ion homostais system which further suggested that this multi-inorganic ion solution creates a
(putative) pro-life water structure and hence is a (putative) driver of both a possible inorganic initiation which in principle could kick start the evolution of
modern carbon-based cells and organisms}.

Wiggins, P. (M.) Life depends on two kinds of water PloS One 2008 Jan 9 3(1) e1406;
Cf. also
http://www.Isbu.ac.uk/water/monograph200904pw.pdf
Physica A, 2002, 414, 458

{To this is may be added that: thhe fundamental prerequisite for biology of the presence of liquid water may further be dependent on the pre-existence of
high molecular weight polyanionic (including polysaccharides and their mimetic) multi-inorganic-ion sequestering and buffering systems to create specific
bio-friendly water structures}
{The Wiggins hypothesis is now suggested to apply also to the H/HS anionic surface environment
Where counterion binding determines water structure in the vicinity of the polyanion. And this issuggested to be the ultimate
basis of the heparanome}

Although it is generally acknowledged that the presence of liquid water is somehow associated with an
ability to generate complex supramolecular structures which are necessary for life (e.g. as recently
discussed by Wiggins (cf. above) how life first started on Earth is unknown, but fundmentally important
roles for the unique structure of liquid water (Chaplin, loc.cit., Luck, loc. cit.) may have been part of the necessary background
to allow the self-assembly and evolution of more complex forms in the first (possibly inorganic) precursors of
living organisms.

Cf., Wiggins, P.M., Prog. Polym. Sci., 1995, 20, 1121-1161

(High and low-density water in gels)
Cf., Robinson, G.W. & C.B. Cho, Biophys. J., 1999, 77, 3311-3318
(Role of hydration water in protein unfolding)

Cf., Wiggins, P.M. & T.T. van Ryn,, Biophys. J., 1990, 58, 585-596
(Changes in ionic slectivity with changes in density of water in gels and cells)
[It was proposed that a continuous spectrum of water structures can exist in two compartment systems present in
gel polymer solution slurries in which water in the pores of the gel was decreased in density to 0.96 as increasing osmotic
stress was applied. When the gel water had a higher activity than external water it was K+ selective, when it had a lower
activity than external water it was Na+ selective]
Williamson, F.B., Ph.D. Thesis, University of Edinburgh, 1970 DATA (also personal communication)
(Addition of Hofmeister salts causes a dose-dependent alteration of the melting point of 1% agarose gels (a sketch of these results is shown in Section 3.5-
2-4, page 35) ; also studied was the hysteresis of melting and the melting temperature of agar furcellaran, κ carrageenan and ι carageenan as a function
of the % gel in H2O, the melting temperature at a given concentration was observed to decrease in the order listed (of these polysaccharides agar has the
greatest gel forming capacity; the melting temperature of 1% agarose gels (prepared from the K salt) were determined as a function of the concentration of
anions (of F-, Cl-, Br-, I- and SCN- studied, the latter two anions produces the greatest effect showing a linear decrease in melting point with increased
concentration) and cations (K+, Cs+, Na+ and NH4+)). The
effects of the presence of inorganic ions was in accord with the generation of greater or less amount of ‘water structure’ according to the Hofmeister series.
Cf. also Austen et al., Biopolymers, 1988, 27, 139-155; included a study of the rate of nucleation (determined by stopped flow polarimetry) of a disorder-
order transition in κ carrageenan occurring at a range of temperatures determined by the presence in the aqueous solution of a range of anions in a
tetramethylammonium cation environment; a compensation between allowable entropy enthalpy changes were evident in the reported rate constants (the
authors however did not note or discuss this which could indicate that the nucleation process was dependent on the occurrence e.g. of an activated water
structure, e.g., of the kind proposed by Wiggins1 which had nucleation activity)

Williamson, F.B., Biochem. J., 1972, 129 (2) 28P

(Double-helical conformation of sulphated polysaccharides and their potential biological function)
[The acidic sulphate half ester groups confer ion exchange properties to the gels. Studies of ligand-induced conformational transitions and selective
cation binding may be involved in the maintenance of intercellular ionic environments. Calcium binding studies suggested that certain carrageenans
controlled the calcification of seaweeds]

Williamson, F.B., et al., Biochem. Soc. Trans., 1992, 20 (4) 360S

(The effect of H on the UV absoption properties of Fe(II) and Fe(III)

Wise, E.R. et al., Chem. Mater., 2007, 19, 5055-5057

[Bone formation: key structure forming role of surface HS-like molecules was suggested by use of novel NMR techniques]

Woodhead, N.E., W.F. Long & F.B. Williamson, Biochem J., 1986, 237, 281-284
(Binding of zinc ions to H)
[This paper reported a low and high affionity process for the binding of Zn2+ to H (both were entropy driven) This study used Na H derived from the same
high multi-element (putatively ‘native’) Na H studied by Moffat (loc. cit., cf., Table IV and section 2.2 (vide supra) which was part-way converted to a
single counterion form.
A later study by Grant et al. (1992d) (vide supra), used a different starting H which showed a similar but distinguishable binding behaviour of Zn2+ to H
from that reported by Wooodhead et al. This difference can putatively be attributed to the effect of the presence, in the Woodhead et al., studies, of greater
amounts of multi-elements attached to H which putatively affects the binding of further metal ions. Further work is, however, required to confirm this
hypothesis].
Cf. also Parrish, R.F. & W.R. Fair 1

Woodward, C. & E.H. Davidson, Proc. Natl. Acad. Sci. USA, 1968, 60, 201-203
(Structure-function relationships of protein-polysaccharide complexes: specific ion binding properties)
[The Ca2+ binding ability of chondroitin-4 sulphate when attached to the core protein present in porcine costal cartilage
is much greater than for the isolated polysaccharide. This may suggest that in vivo the juxtaposition of the sulphated polysaccharide
etc. aids the uptake of counterions (in vivo this reaches 1Ca2+/tetrasaccharide unit).
[The de-calcification process for extraction of chondroitin-4 sulphate involved dialysis vs. EDTA with the EDTA being removed by further dialysis vs.
H2O]

Wu, V.-Y. & M.P. Cohen, Anal. Biochem., 1984, 139, 218-223
(A competitive binding assay for measurement of HS in tissue digests)

Yang, Y-C et al., Blood, 1995, 86 (7) 2526-2533; Chem. Abs., 123, 246454a
H influences cytokine expression via control of mRNA

Zou, S. et al., Comp. Biochem. Physiol., 1992, 103B(4), 889-895

(H-binding properties of lactoferrin and lyzozyme)
[Binding of labelled biotinylated H to lactoferrin and lyzozyme in presence of Na+, Ca2+, Cu2+, Zn2+, Fe(II) and Fe(III) showed a complex buffer, pH and
especially cation-dependent behaviour. H binding was unaffected by 10µ M Zn2+ or Cu2+ but was inhibited above 500 µ M Cu2+ or 40 µ M Zn2+]

11.2

Further References (with Notes) Referred to by Number. Arranged According to Subject Matter

11.2-1 The Metallome-Heparanome (References : Metal Ions in H/HS Signaling)

The inorganic components of H and putatively HS may also normally find use in animal tissues to link specific
parts of H/HS to proteins as part of a cooperative inorganic ion/H/HS manner, is suggestive of a “metallomic-
heparanome” nature of the inorganic biochemistry of polysaccharides. A possible function of small colloidal
particles containing e.g. Ca, Sr, Ba, P, Si, Fe, V etc. in H/HS could be to provide links similar to the borate
crosslinks in plant polysaccharides (in a similar manner to how hyaluronan2a and chitosan act as a ligand for
Fe-rich particles2b).
Currently there is fairly strong evidence that single Ca, Mg, Sr, Mn, Zn and Cu ions have been reported to be required to allow a number
of specific protein HS microstructures to interlink (cf. Table I). Exogenously administered H (which it is now pointed out also includes
multi-inorganic elements) is also reported to induce an increased secretion of H-like HS fragments from the endothelium11, the
mechanism of which may at least perhaps partly be due to (a now established) Cu (redox-ascorbate)/Zn dependent nitric oxide/ HS
oligosaccharide generation12 process as well as the effects of such inorganic ions on the Golgi apparatus exemplified by the effect of the
presence of Ca2+ (perhaps Sr2+), Mg2+ and Mn2+ in cell culture experiments which boost HS biosynthesis (toxic elements, which can also
occur in H (e.g., Pb2+ ,Cd2+ , Hg(II) and F- (cf. Table II) were found in similar experiments to inhibit this).
(Overpurification of polysaccharides used for in vitro studies could have removed the necessary inorganic cofactor to
achieve HS-protein interaction)
(An idea which is suggested by the published as well as unpublished concepts suggested from Aberdeen University
polysaccharide laboratory studies).

Key Findings of Inorganic Ion Assisted HS Signaling

An HS-dependent basic FGF receptor dimerization activity has been reported (Kan et al. 1) to require the
absolute presence of divalent cations. Redox metal ions, especially Cu2+/Cu+ are apparently absolutely required (Ding et al.
1
) for nitric oxide-dependent H/HS oligomer signaling/messenger molecule generation.

Other likely inorganic ion inputs into HS biochemistry include: core protein proteolytic shedding actions of Zn-dependent
metalloproteinases 1A-3-6-1 Selectins, H and metastasis (induced by cellular stress, mechanical, heat shock and hyperosmolality1)
induction or seeding of H/HS or H/HS-protein supramolecular structure or aggregate formation; effects of inorganic
cofactors in DNA expression, m-RNA production or activity and the modulation of the intracellular Golgi apparatus
assembly process and putatively also the roles of metal ions for the modulation of enzymic post-synthetic HS processing by
bacterial heparanase (Lui et al. 1), and other GAG structure processing enzyme activites1).

11.2-2
Ref. 1A-1
Published Peer-Reviewed Articles which Include Mention that Inorganic Ions Might Participate in H/HS Signaling
Perhaps the most pertinent reference is the recent study and discussion by CD and NMR (Rudd et al.1)
A review article which covers (inter alia) inorganic ions in HS signaling
D.R. Coombe & W.C. Kett. Cellular & Molecular Life Sciences, 2005, 62 (4) 410-4249d cf.,
Sasabi et al., EMBO J. web//emboj.oupjournals.org/cgi/ content/full/18/22/6240
and the Discussion Section of a paper reporting the structural requirements for H/HS binding to type V collagen
by S. Ricard-Blum et al., J. Biol. Chem., 2006, 281 (35) 25195-25204
Putative Example of an Absolute Requirement for Bivalent Metal Ions for HS Signaling
M. Kan et al., J. Biol. Chem., 1996, 271, 26143-26148

Ref. 1A-2 Metal Ions Are Currently Known To Assist in H/HS Nitrosative Signaling
Nitrosative cleavage of H/HS is believed to constitute a major signaling pathway involving oligosaccharide
messengers the formation of which depends on redox cycling by Cu2+ / + apparently assisted by Zn2+ ions.

Cf., F. Cheng et al J. Neurochem. 2006, 98, 1445-1457; R. Capai et al., J. Biol. Chem. 2005, 280, 13913-13920

Examples of perhaps more general, type of non-redox-metal ion-determined-H/HS-signaling

include inter alia, the absolute requirements for divalent cations (e.g. Ca2+) for the facilitation of basic
fibroblast growth factor receptor assembly (cf., Kan et al. 1) the inhibition of inositol trisphosphate Ca2+
ion release by H (cf., Hill et al.1a4) and the binding of specifically unsaturated sugar H dimer to
membrane Na+/Ca2+ ion exchanger allows bacterial heparanase to inhibit this activity (Shinjo et al., ).

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

Pharmaceutical H is a multi-chemical molecular species system

Although pharmaceutical H is essentially protein-and nucleic acid free, it is not a single stoicheiometric molecule, but a library of at least
120 chemically distinct molecular species (cf., Nader et al. 1, 1981) to which a further level of complexity should be added, namely, that this
library supports a metallomic multielement, organometallic array containing some 60+ inorganic elements as normal constituents,
selectively highly enriched in elements which occur only in ultratrace amounts in normal biological fluids.
This presence in pharmaceutical H of multiple inorganic elements (a phenomenon also found with natural humic polyanions derived from
soil and marine alginates and also suggested by indirect evidence to apply to H present in mast cells and HS proteoglycans present at
adherent animal cell surfaces and extracellular matrices) suggests that hydration of the polyanion surfaces is greatly modified by the
presence of bound multiple inorganic ions which allow for a Hofmeister-effects driven, organometallic-array-assisted-HS-system of water
structure transmission system to enable information encoded in H microstructures (analgous to “bar-codes” or “postcodes”) to be
transmitted into altered protein folding. This could ultimately explain how the H/HS management system is achieved via a system
somwhat analgous but more complex that that of DNA and RNA in that primary information is encoded linearly in all of these system as
sequences of codons provided principally by variation in the detailed microstructure produced by sulphation substitution of the sugar
backbones in which the iduronate rings show enhanced flexibility for metal ion selection (e.g. for Zn2+) coupled with a complex system of
indpendent enyzmic and non-enymic scission and feedback signaling to achieved altered synthesis of different suphation patterns. A
phase change mechanism involving hydration changes seems to be the origin of the discontinuities found in H-counterion binding
isotherms; the existence of phase separation suggests some role for nucleating agents; such agents could also underpin the mechanism by
which H-like molecules act as morphogens for inorganic crystal (e.g. CaCO3) formation as well as for HS-determined cellular
organization during development and wound healing (cf. FGF-II receptor binding (dependent on HS) seems to absolutely require the
presence of multivalent cations, suggesting a continuation of a H-determined inorganic morphogenic process where the inorganic effects
have been retained from some more primitive cellular, perhaps inorganic, replicative system [cf. Cairns Smith1, Grant et al., 1 1992a). A
further primitive type of role in energy transduction achieved by counterion dependent hydration of polysaccharides is suggested by the
occurrence in H-like polysaccharides of an apparently identical counterion dependent hydration behaviour to that which enables the
proton conduction of commerical sulphonate salt-based industrial ionomers (where proton conduction is thought to arise from polyanion
site water cluster overlap, a phenomenon which is also subject to modulation by the chemical nature of the counterions present via their
alteration of the hydration structure associated with the man-made [intrinsically H-like] sulphonated polyanions commonly employed
[James 1]).

References 1A-3
Degenerative Diseases:
Putative Roles of H/HS Signaling

Refs. 1A3-1

H/HS & Cancer

There is a newly identified (in this article)

quality control problem in regard to differences in inorganic cofactors present which
could affect the reactivity of H/HS which might require carful reassessment of prior researches in this field.
A lack of awareness of variation in the inorganic elements which may be present in Hs includes
some (e.g. Ga3+ iond and Pt complexes) which are known to inhibit tumour growth and, others such as Ge, Si and Se
containing molecules which have been suggested to offer (putatively anti- cancer) pro-health effects. Such trace and
ultratrace elements are apparently present in different amounts in different brands of H.
(cf .Table IV and footnotes thereto) and also probably between in different molecular weight forms of H, are suggested
to have contributed to the confusion regarding the anti-tumour effectiveness of H]

HS signaling seems to depend on the presence of correct HS anionic (including sulphation) patterns.
Alteration of such HS signaling pattern then, like a mutation of a gene, should in prinicple of inducing
cancer like cellular behaviour disturbance in the absence of any genetic aberration.

The presence of H-like molecules at the cell nucleus (which can be augmented by exogenously applied H and putatively by
PPS) are believed to regulate gene expression of immediate entry or competence class genes activated during development,
a process which if it becomes dysfunctional is believed to lead to cancer. This putatively puts the regulatory role of H or its
dysfunction as a central mechanism of tumor devoloment. Cf., e.g., S.J. Busch et al,. J. Cell Biol., 1992, 116, 31-42 and refs.
cited therein.

H was found to block the phorbol ester (TPA)-induced progression of nontransfomed cell through GO/G1 phase or G1 to S
phase of the cell cycle; H-like molecules seem to be involved in regulation of cellular proliferation via decreased RNA and
transcriptional regulation of cell growth where nuclear H plays a trans regulatory role to enhance recovery of endogenous
AP-1 specific DNA binding acitvities, altereing patterns of inducible gene expression; previous studies had indicated that H
modulated cellular proliferation induced by TPA or serum via attenuation of levels of c-fos and c-myc RNA.

1A3-1-1
Gene expression modulation by histones seems to be linked to H/HS signaling
which may also be subject to H/HS regulation of histone inhibition via modulation of histone acetyltransferase activity.
Cf.:
1A3-1-1-1
Inhibition of Histone Acetyltransferases (HAT) by GAGs
H and HS were found to be the most active inhibitors of HAT activiation
(J.A. Buczek-Thomas et al., J. Cell Biochem., 2008, 105 (1) 108-20)

1A3-1-1-2 Tumour cell surface HS is a ‘cryptic system’

Tumour cell surface HS is a ‘cryptic system’ which can act both as a promoter or inhibitor of tumor growth and metastasis
(e.g,. depending of the microsquence signals which are presented in a dynamically balanced system between distinct
polysaccharide populations).
Study of the microstructures present in the presented arrays of HS at cell surfaces is now thought to be of critical
importance, in the post-genome age, to enable a fuller understanding of how the genotype translates into the phenotype
which should enable the more rational design of effective anti-cancer polysaccharide-based therapy (cf., Liu et al., Proc.
Nat., Acad. Sci., USA, 2002, 99 (2) 568-573

1A3-1-1-3 System of transcription repression by nuclear H-like molecules (cf. S.F. Busch, J. Cell Biol., 1992, 116 (1) 31-42) (and by H-
like semisynthetic polysaccharides like PPS and putatively also by other polyanions like Suramin) enable
potential therapeutic intervention in numerous diseases,
including cancer, which are obviously associated with inappropriate cellular proliferation.

1A3-1-1-4
Sulf loss influences N-, 2O-and 6O- sulphation which is associated with tumour growth 1A3-1-1-4
H/HS & Sulfatase activities (Cf. ref. 1A.3-1-1-4)
Environmental initiated stimulae apparantly change HS Microstructure via Sulf actions.
HS 6-O-endosulphatases (Sulfs) seem especailly to be ESSENTIAL FOR MAMMLIAN DEVELOPMENT AND SURVIVAL,being putatively
involved in a cellular SERVO-FEED-BACK mechanisms which include the regulation of cellular signaling, and apparently the
EDITING of the sulphation patterns of numerous HS proteoglycans, and modulation of the expression of various HS biosynthetic
enzymes.

The Sulf SYSTEM & CANCER HS 6-O Sulfatase activities and tumor growth
Cf. also section 3.4-4
Sulf-1 and Sulf-2, HS 6-O-endosulphatases, are known to regulate the activity of multiple growth factors including FGF
and Wnt.
Sulf-1 and Sulf-2 are also potent inhibitors of myeloma tumour growth in vivo
(Y. Dai et al., J. Biol. Chem., 2005, 280 (48) 40066-40073
Cf. X. Yue et al., J. Biol. Chem., 2008, 283 (29) 20397-20407)

1A3-1-1-4-1
Sulf loss influences N-, 2-O, and 6-O- sulphation of multiple HS proteoglycans and modulates FGF signaling;
W.C. Lamanna et al., ibid. 2008, 283 (41) 27724-27735 discussed how
Sulf-1 functions as a liver tumor suppressor but
Sulf-2 acts as an oncogenic promoter by upregulating glypican-3 HS PG and promotion of FGF signaling and Akt/mitogen-
activated protein kinase pathway which decreases survival of hepatocellular carcinoma patients
(J.-P Lai, , et al., Hepatology, 2008, 47 (4) 1211-1211

1A3-1-1-5 Glypican & Cancer

1A3-1-1-5-1 Glypican 1 HS PG & Cancer

Glypican 1 HS PG derived both from cancer and host cells modulates the angiogenic and metastatic potential of human and
mouse cancer cells via interactions with H-binding growth factors. Downregulation of glypican-1 HS PG using an
antisense approach resulted in prolongation of doubling times and decreased anchorage independent growth; athymic mice
that lacked glypican 1 HS PG exhibited decreased tumour angiogenesis and metastasis following tumour implantation and
fewer pumonary metastases following intravenous injection and hepatic endothelial cells isolated from such mice exhibited
attenuated response to VEGF (T. Aikawa et al., J. Clin. Invest., 2008, 118 (1) 89-99).

Pro-tumor effect of glypican 1 HS PG is associated with human gliomas

Whereas normal brain tissues expressed glypicans -2,-3 amd 4 mRNA but not glypican-1 mRNA; glioma cell lines
expressed glypican-1 (together with NF-κ B) (A. Watanabe et al., J Neurooncol., 2006, 77 (1) 25-32.
The pro-tumor effect of overexpressed glypican 1 HS PG, associated with human gliomas was believed to enhance FGF-2
activity
(G. Su, et al., Am. J. Pathol., 2006, 168 (6) 2014-2026).
[Cf., Nitrosative scission of glypican-1 HS side chains is promoted by Zn2+ ions or Cu(II) ions (e.g. those which can be
provided by prions or amyloid β (K. Mani et al.1, 2003)]

Glypican 1 HS PG is overexpressed in human breast cancer cells enabling it to modulate the mitogenic effects of multiple
H-binding growth factors
(K. Matsuda et al., Cancer Res. 2001, 61, 5562-5569)

Low expression of glypican 1 HS PG causes cell cycle arrest at mitosis or the G2 phase of the cell cycle and inhibits the
anaphase –promoting complex/cyclosome (APC/C) mediated degradation of mitotic cyclin and securin while a high level of
glypican 1 induces metaphase arrestand centrosome overproduction
(D. Qiao et al., Mol. Biol. Cell, 2008, 19 (7) 2789- 2801)

Glypican 1 HS PG regulates growth factor activities in pancreatic carcinoma cells and is overexpressed in human pancreatic
cancer
(J. Kleiff et al., J. Clin. Invest., 1998, 102 (9) 1662-1678)

1A3-1-1-6

Glypican 3 HS PG & Cancer

Glypican 3 HS PG is upregulated by action of HS-sidechain degrading endo sulfatase 2 (Sulf 2) leading to a promotion of
FGF signaling and phophorylation of extracellular signal regulating kinase and Akt and decreased survival of heparocelllar
carcinoma patients. Forced expression of Sulf 2 promoted hepatocellular carcinoma; on the other hand
Sulf 1 HS-sidechain endo sulfatase functions as a tumour suppressor
(J.-P. Lai et al., Hepatology, 2008, 47 (4) 1211-1222)

Overexpression of glypican 3 HS PG may have an important role in the genesis and development of colorectal cancer and
be useful for diagnostic purposes.
Cf., expression of glypican 3 in colorectal cancer (D.H. Yuan, Zhonghua Xue Za Zhi, 2008, 88 (22) 1540-1542 PMID
18956635)

Glypican 3 HS PG seems also to be an effctive serum and histochemical marker for hepatocellular carcinoma (O. Capurro,
et al., Gastroenterology, 2003, 125 (1) 89-97) and for testicular germ cell tumours
(D.L. Zynger et al., Am. J. Clin. Pathol,. 2008, 130 (2) 224-230) {it being highly expressed as suggested by histology in
63.6 % of tissue samples of hepaocellular carcinomas and also highly expressed in tissues from squamous cell carcinoma of
the lung, testicular nonseminomatosis germ cell tumours and liposarcomas (D. Baumhoer et al., ibid., 2008, 129 (6) 89-
908)}.
Although glypican 3 was scarcely expressed in normal thyroid glands the expression of glypican 3 was apparently
dramatically enhanced in thyroid-related cancers
(K. Yamamaka et al., Oncology, 2007, 73 (5-6) 389-394)

Cf ., also
Expression of glypican 3 HS PG in ovarian and extragonodal germ cell tumours
(D. L. Zynger et al., Am. J. Clin. Pathol., 2008, 130 (2) 224-230)
[33/33 yolk sac tumours expressed glypican 3 HS PG].
1A3-1-1-6-1
Anti- glypican 3 antibody as a potential anti-tumour agent
(T. Ishiquori et al., Cancer Res., 2008, 68 (23) 9832-9838)
Antibodies against the 30kD terminal fragment of
glypican 3 HS PG caused antibody dependent cellular toxicity aginst human hepatocellular carcinoma cells, with
therapeutic potential (Nakano, K. et al., Biochem. Biophys. Res. Commun., 2009, 378 (2) 279-284
Glypican-3 HS PG can be targeted to allow this PG to become a potential lung tumor suppressor
H. Kim et al., Am. J. Respir. Cell. Mol. Biol., 2003, 29, 694-728

Glypican 4 HS PG is a bactericidal/permability increasing protein (BPI) induces ERK1/2 and

Akt activity
(P. Geraldes, et al., Inves. Opthalmol. Vis. Sci., 2007, 48, 5750-5755
(Glypican 4, a membrane binding protein for bactericidal/permeability –increasing protein signaling pathway in retinal
pigment epithelial cells)

Glypican 5 HS PG & Cancer

A major role of glypican 5 HS PG has been suggested in sarcomagenesis where glypican 5 HS PG is believed to be an
attractive target for therapeutic intervention
(Role for amplification and expression of glypican 5 HS PG in rhabdomyosarcoma,
D. Williamson et al., Cancer Res., 2007, 67 (1) 57-65)

Glypican 6 HS PG & Cancer

Roles of glypican 6 HS PG have been suggested in mesotheliomas, ovarian cancer and pancreatic cancer
J. Filmus, Glycobiology, 2001, 11 (3) 19R-23R

1A3-1-1-7 Heparanase & Cancer

Heparanase activity has classically been regarded as having a principal role in tumor metastasis and indicators of tumour
virulence (cf ., E.A McKenzie , Br. J. Pharmaco.l, 2007, 157 (1) 1-14)
Tumour metastasis inhibition by H Tumor metastasis was found by several groups to be promoted by tumour cell heparanase which can be inhibited e.g.
by H or by H-like agents.
Cf., H derivative for oral administration with claimed potent anti-cancer activity and low toxicity
D.Y. Lee et al., Clin. Cancer Res., 2008, 14 (9) 2841-2849 (Antimetastatic effect of an orally active H derivative ‘LHD’) This agent inhibited
experimentally induced metastasis which could suggest that this is a promising new type of first line anticancer drug which can be used for preventing
metastsis and recurrence and furthermore demonstrates potentially low side effects. The drug can be orally absorbed.

Heparanase apparently modulates expression and shedding of syndecan-1 HS PG and its expression by bone marrow environment is a
bad prognosis factor in multiple melanoma patients
(K. Mahtouk. et al., Blood, 2007, 109 (11) 4914-4923)

Cf., S. Munesue et al., J Biol. Chem., 2007, 282 (38) 28164-28174 (A novel function of
syndecan-2 HS PG, suppression of matrix metalloproteinase-2 (MMP-2) activation which causes suppression of metastasis.

HS degradation by heparanase is believed to promote cancer especially in tumours which home to or grow within bones
(cf., R.D. Sanderson et al., Matrix Biol., 2004, 23 (6) 341-352)
Dowregulation of the expression of heparanase inhibits the invasiveness, angiogenesis and metastasis of human hepatocellular carcinoma (Y. Zhang et
al., Biochem. Biophys. Res. Commun., 2007, 385 (1) 124-129); FGF-2 binding, signaling and angiogenesis believed to be modulated by heparanase in
metastatic melanoma cells (J. Reiland et al., Neoplasia, 2006, 8 (7) 596-606)

Roles of heparanase involving the p38 pathway have been suggested in the etiology of cancer
heparanase functioning both as as an enzyme and as a signaling molecule
Cf., J. Shajat et al., Neoplasia, 2007, 9 (1) 909-916
(Heparanase levels are elevated in the plasma of pediatric cancer patients and correlated with reponses to anticancer
treatment)
{Heparanase levels in cancer patients was studied by ELISA being found to be elevated by a factor of 4 664+ 143/163+8
pg/ml, in cancer patients and 429+82 following anticancer therapy; the heparanase level was an indication of the advanced
nature of the disease with poor prognosis}.

Low afinity (low abundance) as well as high affinity (high abundance HS PG) receptors mediate cellular uptake of
heparanase
(O. Ben Zaken et al., Int. J. Biochem. Cell Biol., 2008 40(3) 530-542)
[There is evdence for a hierarchy of receptors binding to which are enabled by heparanase concentration]
1A3-1-1-7-2
Heparanase – Akt kinase activity & tumorigenesis
Akt kinase activity is believed to play a central role in tumorigenesis (cf., J.R. Testa & A. Bellacosa , Proc. Natl. Acad. Sci., USA, 2001,
98 (20) 10983-10985)
Akt kinase mediates the activation of endothelial nitric oxide synthase (eNOS), an important modulator of angiogenisis and vascular
tone
Heparanase is also thought to induce Akt phosphorylation via a lipid raft receptor mechanism (O. Ben-Zaken., et al., Biochem. Biophys.
Res. Commun., 2007, 361 (4) 829-834
and heparanase has been reported to induce endothelial cell migration via protein kinase B/Akt activation
(S. Gingis-Velitski et al., J Biol Chem., 2004, 279, 23536-235441)

1A3-1-1-7-1
Selectins, H & metastasis
J.L. Stevenson et al., Clin. Cancer Res., 2006, 11 (19) 7003-7011 (Differential metastasis inhibition by clinically relevant levels of
HS correlation with selectin inhibition, not antithrombotic activities)
Cf., Thrombosis Res., 2007, 120, (Suppl. 2.) S107-111 (H attenuates metastasis mainly due to inhibiton of P and L selectin but
non-anticoagulant Hs can have additive effects)
L. Borsig, Physiology, 2004, 19 (1) 16-21 (Selectins facilitate carcinoma metastasis and H can prevent them)

1A3-1-1-8
Angiogenesis, HS & Cancer
Angiogenesis inhibitors (including HS-related) are also in general anti cancer. (Cf. internet discussion D. Grant (2000)1
1A3-1-1-8-1
Angiopoietin, HS & Cancer
The Angiopoietin (Ang) family of factors are believed to have important roles in angiogeneis.
Ang-3 was reported to be tethered on cell surface HS protoglycan (perlecan) to enabled contol of its activity by modulation of its cell
surface concentration
(Cf., Xu, Y. et al., J. Biol. Chem., 2004, 279 (39) 41179-41188

1A3-1-1-8-2
Endostatin HS PG, Zn2+ & Angiogenesis
Endostatin (a proteolytic fragment of collagen XVIII, a HS PG) is a potent inhibitor of angiogenesis and tumour growth
(cf., Brideau et al., Cancer Res. 2007, 67, 11528-11535). Endostatin is believed to bind to H/HS by a process which requires Zn2+ (cf.
Ricard-Blum et al., loc. cit.)

Inducible Nitric Oxide Synthase (iNOS) & Cancer

It was suggested (Grant, D. 2000 internet dg4 ) that iNOS inhibitors might be expected to demonstrate useful anti-cancer activities
[this was predicted on the basis of the putative relevance of a HS-(redox metal)-aberrant nitrosative-cleavage-related-
mechanisms suggested during K.E.L. McColl et al., (Glasgow University, seminars held in 1999 in the context of nitrite
induced stomach cancer and role of nitric oxide and its therapeutic diminution in arthritic diseases). Numerous reports now
confirm from studies in animal models that iNOS inhibitors show promise as potential wide-range human anti-cancer agents
(cf., L.R. Kisley et al.1 and appended list of articles) but without reference to any possible role of HS signaling in this effect
(the preferred mechanism was via NF-κ B related signaling (which as is discussed elsewhere cf. Watanabe et al.1 is
associated with altered HS PG expression in cancer).

Recent large population epidemiological finndings (Cross et al., 1) confirm previous indications that red meat ingestion is
associated with several important human cancers, and tends to add support to the nitrosative/heparanome dyshomeostasis
hypothesis of cancer.

Nitric oxide can directly activate MMP-9

(via reacting with the active Zn2+ site)
Cf., S.N. McCarthy et al., Biochemistry, 2008, 47 (21) 5832-5840
(Nitric oxide regulation of MMP-9 activation and its relationship to modifications of the cysteine switch)
[Nitric oxide seem to be capable of directly activation proMMP-9 via Zn2+ active site]

Putative Modulation of angiogenesis by H/HS

include interactive roles of
nitric oxide and Zn2+ on MMP-9 induced H/HS shedding

1A3-1-1-8-3
Classical researches conducted by J. Folkman et al., 7
Suggested that (a new class of) steroid inhibits tumour angiogenesis in the presence of H (but possibly requires the additional presence of copper or zinc
ions)
Cf., R. Crum et al., Science , 1985, 230, 1375-1379; cf., J. Folkman et al., ibid., 1983, 221, 7190 ;
[Some commercial H samples were, however, without effect; it had previously been reported (Raju et al., J. Natl. Cancer Inst., 1982, 69, 1183-1188) that
copper was required to be present in H in order for inhibiton of angiogenesis to occur; cf. also 1A3-3-3-8-3 re. zincH had been known to stimulate
angiogenesis (but Folkman7 showed that) H + cortisone can inhibit angiogenesis)
Cf., W.D. Thompson et al., Microcirculation, 1987, 2, 807-808
(Compound 48/80, a mast cell activator, stimulates angiogenesis)
Folkman et al. had reported in 1983 that steroid treatment plus H inhibited (putatively pro-cancer) angiogenesis
(but some of the pharmaceutical H samples tested were inactive for unknown reasons but perhaps due to insufficincy of Zn2+ ions which (as oxide) can
inhibit the angiogenesis promoter MMP-9 which is believed to release VEGF to induce pro-cancer angiogenesis) Cf., M.C.L.G. Santos et al., J. Oral
Rehabilitation, 2004, 31 (7) 660-664
(Zn2+ oxide inhibits of MMP-2 and MMP-9 activity)]

Cf., also S.A. Mousa et al., (with R.J. Linhardt) J. Cardiovasc. Pharmaccol., 2006, 48 (2) 6-
Synthetic oligosaccharides (H-mimetics) were found to stimulate and stabilizes angiogenesis: structure function relationships and potential mechanism
involving α ν β 3 and MAPK signalling.
Unfractionated H and low molecular weight H are known to stimulate angiogenesis. Similarly, pro-angiogeneis effects of H-mimetics are observed.
Sulphated oligosaccharides promote FGF-2 and VEGF mediated angiogenesis as well as stabilizing capillary networks formed.

1A3-1-1-8-4
Further studies related to how VEGF affects angiogenesis
VEGF apparently exibits both a positive and negative effect on angiogenesis
(cf., J.J. Greenberg et al., Nature, 2008, 456, 809-813)
A specific antagonistic relationship is believed to exist between platelet-derived growth factor (PDGF) and VEGF actions during neovascularisation.
PDGF was indicated to induce neovascularisationby by promoting VEGF/pericyte release of pro-angiogenic mediators in which VEGF
playes a negative regulator function via pericyte coverage of vascular sprouts for vessel stabilization coordinated by VEGF and PDGF.

VEGF165 regulates perlecan HS PG synthesis in brain microvasculature via VEGFR2 (rVEGF causing increased x1.4 and 1.6 HSPG
formation in cell layer and medium in cultured human brain microvascular endothelial cells)

Pericytes & H
The smooth muscle like cells invovled with (protection of) small blood vessel walls .
H/HS inhibit pulmorary vascular pericyte proliferation (cf., H induces p21 a potent inhibitor of cyclin dependent kinases; H maintains
pericytes in the Go/G1 growth phase)
(J Khoung & J. Langleben, Am. J. Physiol. Lung Cell Mol. Physiol., 2000, 279, L252-261)

[Both VEGF and PDGF activities are subject to modulation by H/HS epitopes]

H regulates VEGF165 dependent mitogenic activity, tube formation and its receptor phosphorylation of human endothelial cells
S. Ashikari-Hada et al., J. Biol. Chem., 2005, 286 (36) 31508-31515
The presence of 2-O-SO3- groups of hexuronic acids as well as 6-O-SO3- groups of GlcNSO3- in H/HS are, however, necessary for
VEGF165 activity.
H enhancement of VEGF included an effect mediated by induced phopshorylation of VEGFreceptor2.

Integrin alpha 9 beta 1 directly binds to VEGF-A and contriubtes to VEGF A-induced angiogenesis.
N.E. Vlahakis et al., J.Biol. Chem. 2007, 282 (10) 10787-10796
VEGF release by MMP-9 mediates HS cleavage and induces colorectal cancer angiogenesis
(L.J. Hawinkels et al., Eur. J. Cancer, 2008, 44 (13) 1904-1913)

VEGF antibodies are showing promise as anti-cancer therapeutic agents

E.g., N Ferrara et al., Nature Rev. Drug. Disc., 2004, 3, 391-400
[Cf. Avastin®, bevacizumab]

1A3-1-1-9
Dietary polyunsaturated acids & cancer
Modulation of HS PG related to apoptosis of breast,
cancer cells
Cf ., H. Sun et al., Cancer Res., 2005, 65, 4442-4447
Omega-3 polyunsaturated fatty acids appear to regulate syndecan-1 HS proteoglycan expression in human breast cancer
cells. Treatment of MCF breast cancer cells (but not normal cells) with n-3 PUFA, (but not n-6 enhanced LDL),
increased syndecan HS proteoglycan mRNA by a mechanism involving peroxisome proliferators-activated receptor
gamma (PPARγ ); (PPARγ -mediated up-regulation of syndecan-1 by n-3 fatty acids promotes apoptosis of human
breast cancer cells, Ibid., 2008, 68 (8) 2912-291
Cf ., I.J. Edwards et al., J Biol. Chem., 2008, 283 (26) 18441-18447
In vivo and in vitro regulation of syndecan-1 in prostate cells by n-3 polyunsaturated fatty acids

Cf . also C.D. Allred et al., J. Nutr., 2008, 138 (2) 250-256

PPARγ as a molecular target of eicosapentaenoic acid in human colon cancer (HT-29) cells

1A3-1-1-10
Glucosamine therapy for degenerative diseases?
Perlecan HS PG many be highly sensitive to dietary glucosamine input
(Glucosamine is currently employed for the treatment of osteoarthritis but only a small increase in cellular
 glucosamine can be achievable by dietary supplementation which seems unlikely to does affect the sythesis of the
principal aticular cartilage GAG (aggrecan ChS PG) [Qu, loc. cit.]).
It may, however, be that HS is ultrasensitive to a variety of environmental and dietary factors.
allowing interventive articular cartilage HS (perlecan) modulation to be achieved by levels of such factors which do not
affect ChS synthesis.

The HS in articular cartilage (G. Stocker et al., Fresnius J. Anal. Chem. 1992, 343 (1) 128 has been reported to regulate
FGF-II activity cf. T. Vincent & J Saklarvali, Biochem. Soc. Trans., 2006, 34 (3) 456.
This supports the notion that HS is the system manager of cartilage and that dietary supplementaion by glucosamine may
achieve beneficial alteration of HS synthesis under conditions where no alteration of the principal GAG (ChS) is achieved
in agreement with the findings of Qu (loc. cit.) who reported that dietary supplementation by glucosamine or related
substances did not affect the synthesis of the prinicpal aggrecan chondroitin sulphate componnent of articular cartilage
(and hence casting doubt on the ability of dietary glucosamine to affect cartilage in vivo and hence be a genuine
therapeutic agent, cf., however, vide infra)

Possible Antioxidant Role of Glucosamine

Modulation of Nitric Oxide Synthase activity suggests that glucosamine tissue protection may occur
via the HS oligomer/nitric oxide signaling system
Cf., C. Valvason et al., Rheumatology, 2008, 47 (1) 31-35
(Influence of glucosamine sulphate on oxidative stress in human osteoarthritic chondrocytes: effect of HO-1, p22Phox and
iNOS)
[The modulation of gene expression of selected oxidant induced factors by glucosmine sulphate in response to oxidative
stress]

[Could oral glucosamine boost of HS synthesis by why glucosamine shows anti-tumour activity?
Glucosamine may counter putative defective HS pro-cancer scenarios]
(Cf., J.H. Quastel & A. Cantaro, Nature, 1953, 171, 252-254 (Inhibition of tumour growth by D-glucosamine) identified
glucosamine as having anti-tumour potency in mice; at that time a plausible mechanism of action was not avialable;
Pauling later identified a potent anti-tumour action (and antiviral action) of ascorbate dietry supplementation in cancer
patients; similarly progress with these findding seem to have been hampered for general application because of the lack of
a plausible mechanism).

The author has suggested that the principle mechanism by which ascorbate affects cancer is by the effectiveness of
ascorbate for boosting the synthesis of HS (and increasing the degree of sulphation of HS) and for countering aberrant HS
nitrostive cleavage (Grant, D (2000), Internet posting)
M.F. McCarty (Med. Hypoth. 51 (1) 11-15) suggested that dietary supplementation by glucosamine, known to
demonstrate anti-inflammatory activity in rodents and anecdotally of benefit to inflammatory bowel disease patients might
generally promote endothelial HS barrier function which incidentally is known also to, e.g., inhibit tumour metastasis).
This author also proposed that glucosamine retards atherogeneosis by promoting endothelial production of HS
(Ibid., 1997, 48 (3) 245-251). Problems with acceptance by physicians of glucosamine as a first line treatement for
osteoarthritis were discussed by this author (Ibid., 42, (5) 323-327). An alternative hypothesis that boost of hyalronan
synthesis was the basis of the anti-osteoartritic effects of glucosamine was also proposed by this author (Ibid., 50 (6) 507-
510.

1A3-1-2 Some Earlier Reports of Relevance of H/HS Biochemistry to Cancer

V.P. Chiarugi & S. Vannucchi, J. Theor. Biol., 1976, 61, 459-475 (Surface HS as a control element in eukaryotic cells:
a working model)

Cf. Exp. Cell Biol., 1976, 44, 251-9 (Cell coat GAGs in cellular transformation and differentiation)

Cf. C.P. Dietrich, Braz. J. Med. Biol. Res., 1984, 17, 5-15 9 (A model for cell-cell recognition and control of cell
growth by sulphated glycosaminoglycans)

Cf., S. Nagasawa, J. Osaka Dent. Univ., 1993, 27 121-33 [Some GAGs promote and othes inhibit cellular growth;
growth regulation seems to require the complete GAG integrity]

N Volpi et al., Exp. Cell Res., 1994, 215, 119-130 [H and derivatives inhibit cell proliferation and increase cell
differentiation suggested by studies of U937 leukemia cells regualation of which seemed to depend on charge density
and H-NSO3- groups]

J.D. Esko et al., Science, 1988, 241, 1092-1096

(Tumor formation is dependent on proteoglycan biosynthesis;
HS involvement in immune system regulation was highlighted)

M. Ishihara et al., J Biol. Chem., 1987, 262 (10) 4708-4716

(Myo-inositol (phosphate) amount in extracellular medium affects the amount of recycled HS in nucleus which
apparently affects rate of cellular proliferation and ability of cells to demonstrate contact inhibition of obvious
relevance to cellular transformation and cancer)
 Review Articles: H/HS & Cancer

(The Engelberg Review)

H. Engelberg, Cancer (New York) 1999, 85 (23), 257-272.

Other reviews on the relevance of H biochemistry as applied to the etiology of cancer include:

R. Castelli et al., Vascular Med. 2004, 9, 205-213 (The Hs and cancer: review of clinical trials and biolgocial properties
suggests that there is sufficient experimental data to suggest that H may interfere with various aspects of cancer:
proliferation, angiogenesis and metastasis to suggest that large scale clinical trials should be conducted).

L.R. Zacharski L.R. & A.Y.Y. Lee, Expert Opin. Investigated Drugs, 2008, 17 (7) 1029-1037
(Heparin as an anticancer therapeutic)

J.-P. Li et al., Anticancer Agents in Medicinal Chemistry, 2008, 8 (1) 64-76; J. Jiminez-Barberi et al., ibid., 52-63;
T.M. Wrodnigg , ibid., 77-85;
Cf., S.M. Smorenburg & C.J.F. Van Norden Pharmacol. Rev. 2001, 53 (1) 93-105 (The complex effect of H on cancer
progression and metastasis in experimental studies).
[This study suggests that low molecular weight H might have therapeutically verifiable anti-cancer therapeutic
value but the results available from unfractionated H are less certain].

Cf., also K. Ono et al., Br. J. Cancer, 2002, 86, 1803-1812,

R.J. Linhardt (H-induced cancer cell death) Chemistry & Biology, 2004, 11, 420-422
9 Cf. Vandewalle et al., (Ca2+ regulation of HS proteoglycans in breast cancer cells)
J. Cancer Res. Clin. Oncol., 1994, 120 (7) 389; Chem Abs.,121, 105487j
Cf., R. Dziarski (Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by H)
J. Immunol., 143 (1) 356-365 and P.A. Steck et al., Cancer Res., 1989, 49, 2096-2103
(Altered expression and distribution of HS PGs in human gliomas)

K. Mani et al. Glycobiology, 2004, 14, (5) 387-394

(Tumour attenuation by 2(hydroxynapthyl)-β − D-xylopyronoside requires priming of HS and nuclear targeting of the
products; cf., ibid., 2006, 16 (12) 1251-61 (Constitutive and vitamin C induced nitric oxide catalysed release of HS from
recycling glypican-1 in late endosomes).

[Tumour cells expressed about three times more HS and hyaluronan in their growth media and on their cell surfaces than did the normal cells thus
indicating that the cellular transformation had been accompanied by an augmented biosynthesis of HS., however a lower production of HS of decreased
degree of sulphation is the usual effect of oncogenic cellular transformation {cf. H. Rahmoune, J.E. Turnbull, J.T. Gallagher et al., Biochem. Soc. Trans.
1996, 24 (3) 355S}].

1A3-1-3
Tumour suppressors & H/HS
Tumour suppressors Exotosin1 (EXT1) and Exotosin 2 (EXT2)
are glycotransferases required for biosynthesis of HS
Cf., T. Lind. et al., J. Biol. Chem., 1998, 273 (41) 26265-26268)
EXT1 alters the expression of cell surface HS
Cf., C. McCormick et al., Nature genetics 1998, 19, 158-161
Thyroid hormone negatively modulates EXT1 expression
(cf., J.H.D. Bassett et al., Endochrinology, 2006, 147, 285-305)

Claimed as a novel
tumour suppressor: Betaglycan
(a transmembrane HS (+ChS) PG (HS-related TGF-β receptor) has been reported to counter prostate [R.S. Turley et al., Cancer Res. 2007, 67, 1090-
1098] and breast cancer [M. Dong,., et al., J. Clin Invest. 2007, 67, 1090-10].

Tumor suppressor gene BRCA-1

is believed to have a caretaker role in controlling genome integrity and inactivation of which is associated with a high incidence of familial breast and
ovarian cancer, may be subject to modulation by H/HS + multivalent metal ions.
A study the effect of ErbB-2 activation by heregulin (HRG) on BRCA-1 function suggested that the extracellular matrix (ECM) affected the expression
and HRG dependent phosphorylation of BRCA-1 (T. Miralem & H.K. Avraham, Mol. Cell Biol. 2003, 23 (2) 579-59.
(The ECM enhances HRG-dependent BRCA-1 phosphorylation and suppresses BRCA-1 breast cancer susceptibility gene expression product through its
C terminus) viz. the ECM affected the expression and the phosphorylation of BCRA-1.
 It was previously known that in ovarian and breast cancer cells α 6β 4 integrin co-immunoprecipitated with ErbB-2 and
ligation of this integrin increased ERbB-2 phosphorylation and invasion through the activated P13K/Akt pathway.
{The pro-tumour effect of dysfunction of this tumor suppressor seems to involve dysfunction of basement
membrane/ECM integrins suggesting that HS PG ultimately can modulate the activity of the BRCA-1 system}
{Nb., Integrins are highly significant surface adhesion receptors putatively subject to modulation by H/HS +
multivalent metal ions which contain alpha subunits containing multiple Ca2+ binding loops and beta chains
which also contain a second type of metal ion binding sites}

p21 (a potent inhibitor of cylcin-dependent kinases) is induced by H (cf. Khoury & Langeleven1)
Rat p30 (tumor suppressor)
Expression of p30 in rat lungs was boosted by aerosol instillation of H
(Cf. the proteomic study of the proteins boosted by H, confirms the general upregulation by H of various singling systems cf., A.A. Gabr
et al. 1)

p53 tumour suppressor activity is putatively potentiated by S100/ Ca2+/HS activities

A putative mechanism can be proposed linking the HS PG regulation system with tumour suppression by p53 via actions of multiple EF-hand Ca2+ binding
proteins (S100 series proteins which are small acidic Ca2+ binding proteins believed to bind to receptors following the formation of hydophobic patches
created by Ca2+ binding).
Cf., S100A8 and S100A14 were observed to bind with high affinity to H and HS at endothelial surfaces
(M.J. Robinson et al., J Biol. Chem. 2002, 277,(5) 3658-3665)

S100A4 oligomers were suggested to regulated of several matrix-degrading enzymes and modulation of the transcriptional activation function of tumor
suppressor p53 in rheumatoid arthritis synovial fibroblasts
(L. Oslekskovic et al., Ann. Rheum Dis. 2008, 67, 1499-1504).

S100A6 activity(Calcyclin) {is implicated in carcinogenesis}; this protein is overexpressed in cancer cells
(Cf. Lukas Orre (Karolinska Inst. Stockholm) Thesis, 2008
(“Proteomic Analaysis of DNA Damage Induced Stress Signaling with Focus on p53” [posted on the internet])
This study reported that p53 was involved in the negative regulation of S100A6 activity
(p53 seems to be involved in negative regulation of homologous recombination following DNA damage, e.g., induced by ionizing radiation).
S100A6 expression is boosted (by a factor of 2.71) by instillation of H (from an aerosol) in rat lungs as established by proteomic strudies (A.A. Gabr et al.
Respir. Res., 2007, 8 (1) 36 [Other calcium signaling proteins also boosted were annexin V (x27) and annexin VI (x6.2)
S100A6 activity

1A3-1-3-1
HS can both be pro-cancer and anti-cancer, the different effects may be related to HS microstructure
In a liver tumour metastasis model attenuation of HS by GAG biosynthesis inhibitors produced an anti-cancer effects relating to surface GAGs especially
pro-metastasis HS variants; anti-HS agents were therefore suggested to potentially useful for anti-metastasis therapy. J. Timar et al., Int. J. Cancer, 1995,
62 (6) 755-761;
On the other hand EXT-2 and EXT- 2 putative anti-tumour agents are enzymes required for HS biosynthesis.

1A3-1-3-2
Other Recent Reports of Potential H-Related Anti-Cancer Therapeutics

The sulphated oligosaccharide P1-88 is a promising low molecular weight H-mimetic anti-cancer agent (which inhibits heparanase activity as well as
growth factor activities in angiogenesis promoted tumour growth)
(cf. E.A. McKenzie Br. J. Pharmacol., 2007, 151, 1-14).
[PI-88 also inhibits the cell-cell spread of Herpex simplex virus (K. Nyberg et al., Antiviral Res., 2006, 63 (1) 15-24)]

Cf., also:
J. Rak & J.I. Weitz, Arterioscler. Thromb. Vasc. Biol. 2003, 23 1954-1955 (H and angiogenesis: size matters), and

C.Y Pumphrey et al. Cancer Res. 2002, 62 (13) 3722-3728 (Neoglycans, carbodiimide-modified GAGs: a new class of anticancer
agents that inhibit cell proliferation and induce apoptosis)

1A3-1-3-3

Low molecular weight H and oligosaccharides: anticancer effects

 J. Hassan et al. (with J.T. Gallagher et al.) Clin. Cancer Res., 2005, 11, 8172-8179 (described H octasacharides represent novel
antiangiogenesis compounds that can be given without the anticoagulant effects of low molecular weight Hs)
C.P.W. Klerk, J. Clin. Oncol., 2005, 23 (10) 2130-2135 (The effect of low molecular weight H on survival in patients with
advanced malignancies)

1A3-1-3-4
Pentosan Polysulfate (PPS): anti-cancer effects
This is an H-like semi-synthetic sulphated xylan from beechwood
Zaslau, S. et al,. Amer. J. Surg., 2006, 192 (5) 640-643
(Pentosan pollysulfate (Elmiron®): In vitro effects on prostate cancer cells regarding cell growth and vascular endothelial
growth fator production).
[The reduction in cell growth and VEGF levels indicate that PPS may act as an antiangiogenic agent and may have
application in the treatment of prostate cancer].
PPS is currently used for the treatment of interstitial cystitis and nvCreutzfeld Jacob disease (cf. Graham Steel1).

1A3-1-4
Anti-cancer metal and metalloid ions
(which could be sequestered from biological fluids by H)
The presence of Ga (probably as Ga3+) and Sn (in unknown form) in some H samples (cf. Table IV) could confer associated
anti-cancer activities.
[Cf., gallium nitrate and other salts show potential anti-tumour activity, cf.,
e.g., C.R. Chitamba et al., J. Pharmacol. Exp. Ther. ,2007, 322 (3) 1228-1236];
This possibility suggests further targeted research.
The antiproliferative activities of some organo-Sn compounds (e.g., C. Pettinan et al., J Inorg. Biochem. 2006, 100 (1) 58-
69) which suggest the possibility of useful Sn-based anti-tumour therapeutic agents which might include H-based organo Sn
complexes (cf., Sn seems to be sequested by suggesting that this element occurs in vivo in association with both H and HS).
The presence of As (putatively both as As(III) and As(V) oxides in H may depend, as seems to be the case with humic matter
(Cf. A. Redman, internet) on the binding of As oxides to Fe and Al oxyhydroxides. Since As is also known to have anti-
cancer activity (e.g. J. Lu et al., Proc. Natl. Acad. Sci., USA, 2007, 104 (30) 12288-12283 including by the action of As2O3
which is believed to inhibit tumour cell invasion by modulating the MMP/TIMPs and uPA related systems of ECM
degradation (M.J. Park et al., J. Cell. Biochem., 2005, 95 (5) 955-969 , the presence of As in H might affect
the ability of H to inhibit cancer (further work is required to probe this idea).
Pt may also naturally occur in ultratrace amounts in H (as suggested by ICP-MS)
Pt ions are also known to bind strongly to H (Cf. D. Grant et al. Biochem Soc. Trans., 1996, 24, 204S);
such Pt-H adducts should be evaluated for anti-tumour activity.

B. Marczynski (loc cit.1) suggested that carcinogenesis migh be promoted by deficiency of Ga, Ge, Si, As and Se
elements which seem consitently to co-occur with pharmaceutical H and therefore might also be naturally required for H/HS
function including the control of cellular activities such as apoptosis and proliferation involved in angiogenesis and
carcinogenesis [This author however suggested a related trace-element deficiciency-related DNA semiconductor hypothesis]

The claimed ant-cancer effects of such folk remedies as kelp, Shilagit and other geological fulvate-derivated preparations
might also derive from a similar provision of trace elements such as those listed above. Small amounte of Ge occur natually
in soil and biological matrices including H (as suggested by ICP-MS). Although not thought to be an essential nutrient Ge-
based dietary supplements (including those from yeast) have also been proposed for use as anti-tumor therapeutic agents (cf.
also B.J. Kaplan et al., J. Altern. Complemnt. Med. 2004, 10 (2) 337-34 and website germaniumsesquioxide. org

1A3-1-5
Altered HS in transformed cells and cancerous tissue.
The degree of sulphation of HS is believed to influence cellular proliferation and adhesion
(suggesting a role of reduced sulphation in how transformed cells loose contact inhibition and easily become detached from
culture vessel surfaces. The general principle seems to be that HS varies with cell type and the alteration of HS between
normal to transformed cells is no exception to this rule. A further tendence seems to be a lower molecular weight HS with
lesser degree of sulphation is often (but not always associated with cellular transformation).
Recent studies (Fritzsch et al. 1) have suggested that the potent antimetastatic clinical benefit of H in cancer patients arises
from blockage by H of selectin-dependent VLA-4/VCAM-1 actions.

H/HS (PGs) can however have both anti and pro cancer effects. The presence of certain HS PGs in tumours which are
 absent or less abundant in normal tissues allows for HSPG-directed. Several Glypican HSPGs (vide supra) have been
indicated to be upregulated in cancer cells allowing a therapeutic approach invoving anti HSPG antibody treatment has
potential to inhibit tumour growth. It might be privisonally suggested that the core proteins rather than the polysaccharide
side chains are the main determinants of such pro-cancer effects of HS PGs.

Lambrecht V. et al., Exp. Cell Res., 1998, 245 (2) 239-244 found evidence that changes in HS PGs are the key processes to
be considered in the mechanism of breast epithelial cell growth regulation.

Cf., also, N.C. Woohead et al., IRCS Med. Sci. (Library Compendium) 1984, 14, 427-428
(HS from fibroblasts exhibiting a temperature-dependent transformed growth trait)
Biochem. Soc. Trans., 1987, 9, 555-556
(The HS from normal and virus-transformed hamster fibroblasts);
H.H.K Watson. et al., Biochem. Soc. Trans., 1980, 8, 134-136
(Sulphated gllycosaminoglycans of exponentially growing and non-proliferating BHK-21/C13 cells)
D. Grant et al., Eur. J. Cell Biol., 1985, 36, 14
(Infrared and proton nuclear magnetic resonance spectra of carbohydrates from BHK and PyY cells)
D. Grant et al., ibid., 1985, 36, 14
(Differences in the properties of Heparan (SPG) from BHK and PyY cells)

Cf., also a similar degree of sulphation variation to that which seems to determine H/HS activity to meet required designed extracellular
and in vivo temporal and tissue postcode requirements may also regulate the activity of algal polysaccharides as suggested by the report:
D. Grant et al, Eur. J. Cell Biol. 1985, 36, 14
Possible altered sulphation of Porphyra umbicalis in accord with proliferative potency of tissues (analogy with proliferative status of animal cells)

1A3-1-5-1
Putative Differences in HS Iduronate Conformation in Transformed Cells
This was suggested by D. Grant et al., Biochem. Soc. Trans. 1990, 18, 1282 from studies of near and fundamental region IR
spectroscopy of HSs isolated from the surface of normal and transformed fibroblasts.
[Cf., also Proc. Int. Cong. Near Infrared Spectroscopy, Aberdeen, 1991 (Publ. 1992); Ed I. Murrya & I.A. Cowe, VCA
Weinhiem. Germany; Chem Abs., 118, 164498a]

1A3-1-6
Water Structure & Cancer
It was suggested (Grant et al.. 1985) that the alteration of HS microstructure induces in cancerous tissues, a water structure
less conducive to cellular adherence.
Cf., also IR spectroscopy of HSs isolated from the surfaces of normally and cvirally transformed fibroblasts reported by D.
Grant et al., Proc Int. Conf. Aberdeen, Near Infrared Spectrscopy 1991 pub 1992, Ed. I. Murrray & I.A. Cowe, VCA
Weinheim, Germany, Chem. Abs. 118, 164498z ; NIR News, 1992, 19-21; cf., also ibid., p 22-24, NIR spectroscopy shows
that animal cell adhesion to non-biological solid surfaces may required surface water structuring.

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

Ref 1A-4
(H & Atherosclerosis)
Cf. K. Murata & Y. Yokoyama Atherosclerosis
J.R. Klock US Pat. 6291439 (2001;

H. Engelberg, Clin. Appl. Thromb. /Hemostas., 1996, 2 (2) 83-93

(Endogenous H-active deficiencies and atherosclerosis); cf.,

H. Engelberg, Pharmacol. Rev. 1984, 35, (2) 91-104

Cf. H. Engelberg, Circulation, 1961, 23, 573-577 (Cf. role of natural serum H)

Cf. D. Grant, W.F. Long & F.B. Williamson (Degenerative and inflammatory diseases may result from defects in anti- mineralization
mechanisms afforded by glycosaminoglycans) Med. Hypoth. 1992, 38, 49-55, Cf. Biochem J.,1989, 259, 41-45

Ref 1A-5
(H & Dementia)
H. Engleberg, Dement. Geriat. Cogn. Disord., 2004, 18, 3-4

Cf. J.D. Snow et al. (cf. also notes listed under Snow, J.D. et al.,1 and Morrisete C. et al.,1

Cf., Morissete et al.,1

‘Ateroid’
 Non-anticoagulant H is reported to be the principal component of ‘Arteroid’
which has been extensively studied including by medium-sized clinical trials in
which significant benefit was reported
Cf., L. Conti et al., Prog. Neuropsychopharmacol. Biol. Psychiatry, 1984, 13 (6)
977-981
(Glycosaminoglycan polysufate (Ateroid) in old-age dementias: effects upon
depression symptomatology in geriatric patients) Cf., T.A. Ban et al., ibid., 1992,
15 (3) 135-138 and ibid., 1992, 16, 661-678; Semin. Thromb. Hemost. 1991, 17,
Suppl. 2, 161-163; L.C. Morley et al., Neuropsychobiology, 1988, 19 (3) 135-
138.

M. Passeri & D. Cucinotta, Mod. Probl. Pharmacopsychiatry, 1989, 23, 85-94

(Ateroid in the clinical treatment of multi-infarct dementias)

M. Rose et al., Brain Res., 2004, 1015 (1-2) 96-102

(Glycosaminoglycan C3 protects against AF64A induced chloinotoxidity in a
dose dependent and time dependent manner)

U. Cornelli & T.A. Ban, US Pat., 3,007,787

(Administration of Ateroid was found to benefit Alzheimer disease Patients).

VEGF 165 regultes perlecan HS PG synthesis in brain microvasculature via VEGFR2 (rVEGF causing increased x1.4 and
1.6 HSPG formation in cell layer and medium in cultured human brain microvascular endothelial cells)
T Kaji et al., Biochim. Biophys. Acta, 2006, 1760 (9) 1465-1474

H-derived oligosaccharides for inhibition of senile dementia

Useful for the treatment of senile dementia were claimed to have been obtained via γ radiation depolymerization of H to
give MW 2400D oligomers.
Effect on τ − 2 protein suggested the product was useful for the treatment of Alzheimer’s disease
U. Cornelli et al., US Pat. 6979680
(Such low molecular weight H oligomers are believed to pass through the blood brain barrier)

Novel HS analogues: inhibition of β -secretase cleavage of amyloid precursor protein

S.J. Patey, E.A. Yates & J.E.Turnbull, Biochem. Soc. Trans., 2005, 33 (5) 1116-1117

H/HS claimed to normalize

Age-Related FEAR RESPONSE
Old brown rats exhibited impaired spatial learning and have an exaggerated response to stress. These effects, attributed to age-related
brain damage were found to be reversed by the administration of H-derived oligosaccharides.
(S.A. Lorens et al., Behavioural Brain Res,. 2003, 147 (1-2) 65-72)

Ref. 1A-6 Further Notes

H & Antioxidant Protection

Antioxidant & Antinitrant Activity (Related to Modulation of Superoxide Dismutase by H/HS) and
Sequestration and Deactivation of Redox Active Iron, Copper & Silver Ions, Etc. by H/HS)

Severe Smoke Inhalation Injury

O. Tasaki et al., Crit. Care Medicine, 2002, 30 (3) 637-643 reported on the effects in sheep of inhalation of nebulized Heparin compared
with nebulized saline on smoke inhalation injury; although treatment with nebulized H alone did not reveal any statistical efffect, a
beneficial effect of nebulized H was achieved when sheep where also systemicically treated with lisofylline (1-(5-R-hydroxyhexyl)3,7-
dimethylxanthine)

The following articles discuss related topics:

H/HS-Superoxide Dismutase
T. Adachi & S.L. Marklund, J. Biol. Chem., 1989, 264 (15) 8557
J. Biochem. (Tokyo) 1995, 117 (3) 586-590
Biol. Pharm. Bull., 1998, 21 (10) 1090; Chem. Abs. 130, 64588h

G.E. Arteel et al., Biol Chem. 381, (3) 265-268, Chem. Abs., 133, 85803f
(Selenium containing antioxidant which probably binds to endothelial wall HS)

-------------------------------------------------------------------------------------------------------------------
Cf. also,
D. Grant et al., Biochem. Soc. Trans., 1996, 24, 194s (Hs as essential antioxidants)
Ibid., 1988, 16, 1030-1031 (Effect of H on dismutation of superoxide anion)
Ibid., 1992, 20, 361s (Complexation of Fe2+ based ions by H)

D. Grant et al., Med. Hypoth., 1987, 23, 67-71 (Review article: Pericellular heparans may contribute to the protection of cells from free radicals)
D. Grant et al. had found the absence of NMR detected paramagnetic effects expected for high levels of Fe and Cu in some H samples; this was
rationalised as being due to occurrence of these elements within the H supramolecular structure in a separate non-solution phase attached to H.
Cf., G. Mackintosh, Ph.D. Thesis “Heparin-Iron Interaction and its Possible Relevance to Antioxidant Activity” University of Aberdeen, 1995
[This study involved in vitro and in vivo investigations into possible inflammatory actions of Fe(II) ions which are putatively inhibited by binding of such
ions to H/HS or to H-mimetics such as pentosan polysulphate]
Cf. also M.A. Ross, M.Sc. Thesis, “Heparin as an Antioxidant”, University of Aberdeen, 1992
M.A. Ross et al. Biochem J. 1992, 287, 849-853
Cf.,
R. Albertini et al., Int. J. Mol. Med. 2000, 6, 129-136; FEBS Lett., 1995, 377, 240-242

F.B. Williamson et al. (unpublished, personal communication) showed that Fe(II) H underwent auto-oxidation to generate
Fe(III) (O.OH) akagaenite crystals detectable by their characteristic X-ray diffraction pattern.
Incompleted studies had suggested that the multi-inorgnaic element content of H was associated with a ferroxidase catalytic property.
Other related unpublished studies have indicated that iron ions can promote pathological calcification and fibrin aggregate formation which is upon
ingestion by macrophages and resultant inappropriate cytokine induction, promotes degenerative disease processes .

Related solid phase, antioxidant function indicated by removal of potentially damaging redox iron ions by other anionic polysaccharides have been
reported by Merce et al.2a and Sipos et al. 2b :
P. Sipos, O. Berkesi, E. Tombacz, T.G. St Pierre & J. Webb, J. Inorg. Biochem., 203; 95: 55-63;
A.L. Merce, L.C. Marques Carrera, L.K. Santos Romanholi & M.A. Lobo Reico, J. Inorg. Biochem., 2002; 89: 212-218.

Cf., also D.J.R. Jarrett et al. J. Clin. Exp. Gerontol., 1989, 11, (3-4) 145-154
Ageing as a cause of raised serum ferritin in the absence of disease.

Cf. also, J.T. Salonen et al. (Circulation, 1992, 86, (3) 803-811) found an epidemiological correlation between mortality in Eastern Finnish men with
appropriately corrected serum ferritin values. This study supported a possible role of redox active iron ions in degenerative cardiovascular dysfunction.
This can be further suggested to include a redox iron related alteration of HS signaling.

Cf. also
T. Aoyagi et al., Biol. Pharm. Bull. 1994, 17 (2) 340, Chem. Abs., 120, 294925b
Reported an age-dependent decrease in fibrinolytic enzyme activity in serum of healthy subjects.

Internet discussions (e.g. by Meschino1) suggest that the biosynthesis of glucosamine becomes less efficient with increasing age in humans.

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

11.3
Further References
(1a 1-14; 1b-1h)

1a -1 H.B. Nader, T.M.P.C. Ferreira, L. Toma, S.F. Chavanto, C.P. Dietrich, B. Casu and G. Torri,
Carbohydr Res. 1988, 184, 292-300
(Maintenance of HS thoughout evolution)

1a -2 H.B. Nader, H.K. Takahasi, J.A. Guimaraes and C.P. Dietrich, Int. J. Biol. Macromols. 1981; 356-360
1a-3 D. Grant, W.F. Long and F.B. Williamson, Med. Hypoth., 1987, 23, 67-73; cf. ibid.29, 245-253

1a-4 D. Grant, W.F. Long and F.B. Williamson Biochem J. 1989; 259; 41-45; cf. also Med Hypoth. 38, 49-55

T.D. Hill et al., Biochem Biophys. Res. Commun., 1987, 149 (3) 897-901

1a-5 E. Feyzi, T. Saldeen, E. Larsson, U. Lindahl & M. Salmivirta, J Biol. Chem., 1998, 273, 13395-13398

1a-6 B. Kuberan et al. (with R.D. Rosenberg) J. Biol. Chem. 2004, 279, 5053-5054 (Light induced 3-O-sulfotransferase expression alters pineal HS fine
structure-a surprising link to circadian rhythm) (i.e. HS microstructure is subject to alteration by light).

1a-7 H/HS, alginates and sulphated marine anionic polysaccharides, e.g., carrageenans and soluble humic acids (fulvic acids)
act as potent modulators of the crystallization of the sparingly soluble salts: CaCO3 and BaSO4, and hence influence the
amounts of Ca2+, Ba2+, CO3- and SO4- present in solution.

The naturally occurring amounts of HS in blood serum and fulvic acid in seawater required to achieve such buffering activity (ca. 2 mg/L) corresponds
with the amount [1.6mg/L] of HS in blood reported by W.C. Manley et al., Clin. Chim. Acta, 1984, 137, (3) 315

1a-8 H. Ischiropoulos, Arch. Biochem. Biophys., 198, 356, 1-11

Cf. H. Tohgi et al,. Ann. Neurol, 1999, 46, 129-131
(Remarkable increase in cerebrospinal fluid 3-nitrotyrosine in patients with sporadic amylotrophic lateral sclerosis)

1a-9 Cf., K. Mani, F. Cheng, B. Havsmark, M. Jonsson, M Belting & L.-A. Fransson, J. Biol. Chem., 2003, 278, 38956-38965

1a-10 F. Baluska, J. Samaj, P. Wojraszek, D. Volkman and D Menzel, Plant Physiol. 2003: 133: 482-491
E . Melotto & J.M. Labovitch, R Bras Fisiol Bras Veg. 1994; 6: 75-82
M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Ann Rev Biochem. 1984; 6: 75-82
cf., T. Matsunaga & T Ishii, Anal. Sci. 2004.; 20: 1389-1393;
(cf., borate crosslinks in plant rhamnogalacturonan II)
[In plants pectin-like oligosaccharides were originally supposed to engage in hormone-like activities but are now thought to
influence Ca2+ second messenger actions]

The system of plant anionic polysaccharide, pectin generated oligosaccharide dependent Ca2+ second messenger action could serve as a provisional
model for HS signaling in animals which is known, at least in part, to require Ca2+ and Zn2+ as well as Cu+/Cu2+ (for nitric oxide/(ascorbate) dependent
oligosaccharide generation).

1a-11 D. Lagunov & G. Warren, Arch. Biochem. Biophys., 1962, 99, 396-400; J.E.Shivley & H.E. Conrad, Biochemistry, 1970, 8
(1) 33-43

1a-12 Cf. C. Leteux et al., J. Biol Chem., 276 (16) 12539-12545

[Scrapie antigen indicated co- occurrence with HS with GlcNH2, unsulphated motif: this is the site which is targeted by
nitric oxide metabolites to create HS oligosaccharides]

1a-13 H. Diringer & B. Ehlers, J. Gen Virol. 1991, 72, 457

[Initial communication dealing with the inhibition of scrapie by sulphated polysaccharides]

1a-13-1 Communication to the author from Graham Steel, Glasgow (2007) (including information communicated in a Professor Ian
Bone Report)
The H-mimetic PPS has a stimulatory effect of proteoglycan synthesis and a potent suppressor of angiogeneis and tumour
growth.
In a pivotal study by Doh-ura (2004) PPS was confirmed to suppress CJD attributable to misfolded PrP. The drug was
directly infused into the brain using Tg7 mice expressing hamster than endogenous mouse PrP). Later work on some 20+
human patients worldwide corroborated the effect in humans.

1a-13-2 PPS-like molecules produced by normal stress-related deaminated cleavage of HS may act as cofactors for clearance of
misfolded prions from cells.
Cf. K. Mani et al., J. Biol. Chem., 2007, 282, (30) 21934-21944
(In Niemann-Pick fibroblasts where nitrosative degradative processing of HS is defective carboxylated proteins were
abundant when glypican-1 was silenced in normal fibroblasts the level of such proteins increased, suggesting that the
deaminative cleavage of HS is involved in the clearance of such abnormal including misfolded proteins)

1a-14 B.C. Challis et al., IARC Sci. Publ., 1978, 19 (Environ Aspects N-Nitroso Compd.) 127-142; Chem. Abs. 89, 196583x
Cf. E. Boyland & S.A. Walker, Nature, 1974, 248, 601-602;
T.Y. Fan & S.R. Tannenbaum, J. Agr. Food Chem. 1973, 21(2) 237-240;
B.C. Challis & J.R. Outram JCS Chem. Commun. 1978, (16) 707-708 (article highlighting silver catalysis of nitrosative
processes)

1b Cf., Grant, D., W.F. Long and F.B. Williamson, Biochem J. 1987, 244, 143-149
(The infrared spectra of different H salts show individual counterion input into H structure as detected by vibrational
spectroscopy in solution and solid phases which are different from a purely electrostatic effects)
Cf. Panov, V.P. & A.M. Ovespan (refs. 1)
1c Z. Liu & A.S. Perlin, Carbohydr. Res., 1994, 255, 183-191

1d R.N. Rej et al. ibid., 1990, 207, 143-152

1e R.E. Vilar et al., Biochem. J. 1997, 324, 473-497

1f J. Van den Born et al. J. Biol Chem., 270 (52) 31303

1g F. Cheng et al., J. Neurochem., 2006, 98, 1445-1457; K. Mani et al., J. Biol. Chem., 2003, 278, 38956-38965
and refs. cited

1h D. Grant et al. unpublished observations, Marischal College, University of Aberdeen

2 M. Purdey, Med. Hypothes., 2004, 62 (5) 746-754

(Chronic barium intoxication disrupts sulphated proteoglycan synthesis: a hypothesis for the origins of multiple sclerosis);
Cf., Ibid., 2000, 54, 278-306
(Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical-generating divalent cation manganese and deficiencies
of antioxidant co-factors Cu, Se, Fe, Zn. Does a foreign cation substitution at prion protein’s Cu domain initiate TSE?)
Ibid., 2001, 56 (1) 29-45
(Does an ultraviolet photooxidation of the manganese-loaded/copper depleted prion protein in the retina initiate the pathogenesis of TSE?);
Cf., Ibid., 2003, 60 (6) 797-820
(Does an infrasonic acoustic shock wave resonance of the manganese 3+ loaded/copper depleted prion protein initiate the pathogenesis of
TSE?);
Ibid., 2004, 63, 793-809
(Elevated levels of ferromagnetic metals in food chains supporting the Guam cluster of neurodegeneration: Do metal nucleated crystal
contaminants evoke magnetic fileds that initiate the progressive pathogenesis of neurodegeneration)
Ibid., 2004, 63 (5) 793-809
(Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: Do Ag/Ba/Sr
piezoelectric crystals represent the transimissible pathogenic agent in TSEs);
cf., http://www.purdeyenvironment.com/floodstudy.htm (Fresh research report of a trace metal-CWD study at Saskatoon University
is being exploited to discredit a misrepresented version of Purdey’s theory)
Ibid., 2005, 65, 448-477
(Metal microcrystal pollutants: the heat resistant, transmissible nucleating agents that initiate the pathogensis of TSEs?)
Cf., also, ibid., 2006, 66 (1) 197-199
Cf., also M. Purdey, Draft Abstract for Chapter in “Trends in Prion Research” [communicated to the author by Graham Steel, formerly CJD
Alliance press officer etcbased in Glasgow] PrP-ferritin-‘fibril’ crystals were suggested to be nucleators of solid phases which were proposed to
be
the true pathological entities responsible for prion diseases.
Cf. also article by Mark Purdey in ACRES Aug.-Oct 2003 vol 33, entitled “Educating Rida. An eco-detective journey”
Cf. also M. Bounias & M. Purdey, Sci. Tot. Environ., 2002, 297 (1-3) 1-19
(Transmissible spongiform encephalopathies: a family of etiologically complex diseases—a review)

Cf. also Brown, D.R., ref. 1

2a A.L. Merce et al., J. Inorg. Biochem. 2002, 89, 212-218

2b P. Sipos et al.,ibid., 2003, 5,55-63 (Spheroidal iron(III) oxyhydroxide nanopartilces sterically stabilised by chitosan in
aqueous solution)

3 A. Wassermann, Ann. Bot. 1949, NS 13 (49) 79-88;

Cf. W.A.P. Black & R.L. Mitchell. J. Mar. Biol. Assoc. U.K ., 1952, 30 (3) 575-584
Cf. brief mention by F. Mo et al., Carbohydr. Res., 1985, 145, 13-24 (cf. also refs. cited herein ).

3a-1 E.g., The presence of inorganic elements in alginate have been reported by
Ascophylum nodosum polysacchride-based ion exchanger kelp elements are tabulated by several internet sites, e.g.
http://www/alginure.co.uk/ascophylum-nodosum.html
3a-2 for Ecklonia maxima at http://www.gairesearch.co.za/kelp.html.

3a-3 Cf., also T.A. David, et al., Appl. Biochem. Biotechnol.,2003, 110, 75-909 Su

3a-4 K. Truus, et al., Proc. Eston. Acad. Sci., Chem., 2001, 50, 95-103 for apparently anthropogenically polluted seaweed
from the Baltic). Data from this paper are listed in Table I which also lists similar multi-element analysis (which was
apparently provided by the Norwegian Seaweed Research Institute) of less polluted polysaccharide from the open ocean.

3b-1 The presence of inorganic elements in H of relevance to the contamination of blood samples have been discussed by
H.J.M. Bowen in Trace Elements in Biochemistry, Academic Press, London, 1966 where on p.63 it is noted that since
H often contains appreciable amounts of Ba, Ca, Cu , Mn, Sr and Zn this can causes serious
interference problems for the estimation of inorganic elements in blood. This seemed especially to apply to the
determination of Mn since the apparently results for the elemeent can be entirely due to the Mn present in the H added
as an anticoagulant whole blood samples);
Cf. also Katz, S.A., Amer. Biotechnol. Lab. 1984, 2, 24-30

3b-2 (for Ca, Sr, Ba in H) G.E. Harrison & A. Sutton, Nature, 1963, (4869), 809
 Cf. M.E. Lyon, Clin. Biochem. 1995; 28: 79

3b-3 The presence of paramagnetic metals in natural H can also affect NMR resonances
cf. G. Gatti et al., Macromoleucles, 1979, 12 (5) 1001-1007
Cf. also the presence of the metal ion chelator EDTA in commercial H
(B. Casu et al., European Patent, 1987, 0245813) suggests its industrial employment for removal of
metals thought to be undesirable for commercial medical applications.

3b-4 (for V in H ) G. Heineman & W. Vogt, Biol. Trace Elem. Res. 2000, 75, 227-234

3b-5 (for Cr, Mn in H) N.W. Alcock Serum versus plasma for trace metal analysis, Elem. Metab. Man, Anim. Proc. Int.
Symp. 4th, 1981 (Pub 1982). Eds. J.M. Gawthorme, J.M.M.M.C. Howell, C.L. White, p. 6578-680; Springer, Berlin;
Chem. Abs., 96, 213646

3b-6 (for Si in H/HS) K.A. Schwarz., Proc. Natl. Acad. Sci. USA, 1973; 70: 1608-16124
Cf. R.K. Iler, The Chemistry of Silica ,Wiley, New York, 1979
and D. Grant, W.F. Long and F.B. Williamson, Med. Hypoth., 1992, 38, 49-55

3b-7 (for As in H) D. Bohrer et al., J Parenteral Enteral Nutrition, 2005, 29 (1), 1-7;
(Unacceptably high levels of Al and As still occur in some commercial Hs
alginate, carageenan etc., and calcified skeletal matrices perhaps forming crosslinks analogous to borate in plant
rhamnogalacturonan II (cf. ref. 1a-10)

3b-8 (for Al in H) D. Bohrer et al., RBA, 2004, 36(2) 99-103

3b-8-1 Be2+ ions interact strongly with H

J.G. Hall, Immunology, 1988, 64, 345-351

3b-9 The presence of 38 seawater multielements in H was confirmed in Na and Tl H of the elements listed at a-f (except Al
added during the spark source mass spectrosocpic (SSMS) analysis of Mg, K, Rb, Cs, Tl, Fe, Ni, Co, Sn, Mo,Ti, Zr,Ce,
Ag, Nd, W, La, Pb, Ga, Sb, Cd, B and I in Na H and Mg, K, Rb, Cs, Cr, Fe, Ni, Co, Sn, Mo, Ti, Zr,Ce, Ag, Nd, W, La,
Pb,Ga, Sb, Cd, P, B, I, Cl, F and Br in Tl H (D. Grant, Chemistry Preprint Archive, 2000 Oct., 94-104; available at
http://preprint.chemweb.com/biochem/0010002; cf. also a preliminary summary of these data was given by D., Grant,
et al., Biochem J.,1987, 244: 143-149) and more completely for Na and Tl Hs in
C.F. Moffat, Ph.D. Thesis University of Aberdeen, 1987, briefly discussed the 205Tl NMR spectrum of Tl H (on p.144)
and the possible lack of toxicity of 8 ppm Tl in H is also discussed on p. 189.
The relevance of larger amounts of possibly toxic or therapeutic elements (such as Ga which has demonstrated anti-
tumour activities) are worth pursuing.

The original prime functions of the heparanome-metallome could have been for nutrient gathering and cellular protection

It is suggested that nutrient inorganic ion binding to anionic polysaccharides (such as those which constitute the heparnome) both enabled the early
evolution of multicellular animal organisms in the sea and continue to enable the multiple functions which are now known to be dependent on the
heparanome in present day animal species
(cf. Section 11-1).

3b-10 (ICP-MS evaluation of dilute nitric acid leachates from

Heparinzed blood collection tubes)
web.alsglobal.se/hem2005/pdf/blood_collection_tubes_eng.pdf (2005) tabulated multi-element data obtained by ICP-
MS for types of H commonly present as additives in blood assay tubes which were extracted with 0.01M HNO3 . This
study also gave an assessment of the possible inorganic element inputs from empty tubes and needles employed for
blood sampling which can serve as blanks for correction of the H analyses for background contamination; the data also
reported ICP-MS data of other substances commonly added to blood collection tubes (EDTA and a phase boundary
enhancing agent which is rich Si, Al and in lanthanides etc). The data for extracts from six types of heparinized blood
collection tubes (believed to be polyethylene terephthalate and of the evacuated type) confirm the seawater multielement
pattern of inorganic cofactors present in H previously identified the author.
Such data can be extracted from the tabulated information (which included inorganic S -derived from H –SO3- groups
present in amounts corresponding to the principal counterion for the employed single counterion-enriched forms of
H.

4. H. Haraguchi, J. Anal. At. Spectrom., 2004, 19, 5-14

5 H.B. Nader, M.G.L. Medeiros, J.F. Paiva, V.M.P. Paiva, S.M.B. Jeronimo, T.M.P.C. Ferreira & C.P. Dietrich
Comp. Biochem. Physiol., 1983, 76, 433-436
This study established that an exact mathematical relationship exists between the bathing habitat salt concentrations for fifteen
species of Crustacea, Pelecypoda and Gastropoda. The average total sulphated glycosaminoglycans (GAGs) present in these
organisms increased with increased habitat salinity from 125 to 5265 µ g/g dry weight. This suggests that differing salinity
induces evolution/biosynthesis of the exactly balancing amounts of GAG- SO3- groups required to titrate against Na+ (the
results of ref.1h suggest that the sum of the –SO3-(H2O)n groups present i.e. the total GAG-(O)-SO3- + GAG- (N)-SO3- rather
than the GAG-C(O)O- contents correlated with how individual GAG types were ‘induced’ by different habitats with a range of
different natural saline, principally NaCl habitats (i.e. seawater and brackish seawater etc.)
[This explanation seems more likely than that offered by Nader et al., in this paper, who had suggested that the presence of
 greater amounts of GAGs was to prevent tissue disintegration under the higher ionic strength salinity conditions]

6 The work of S. Kodima et al., (e.g. Eur. J. Nucl. Med., 1983, 8, 52-59, cf., Y. Hama et al., ibid., 1984, 9, 51-56 who by in vivo and in vitro multivalent
ion binding studies e.g. of labelled 45Ca to HS (relating to the scintigraphic observation of tumours via 67Ga binding which were indicated to occur at
HS sites) support the notion that HS will occur in vivo as multi-inorganic- element-substituted systems.

Mast cells in vivo histochemicaly stain with ruthenium red by binding to H in the mast cell granules
(E. Phil, Histochem. Cell. Biol., 1970, 22, 302-315)

Cf., also: the formation of the adduct 99Tc-H enabled the bio-distribution of H to be achieved in the rat
N. Vigneron et al., J. Biophys. Med. Nucl. 1984, 8 (2-3) 192;
Cf., M. Decousus et al., ibid., 1984, 8 (2-3) 180; cf.,
99m
Tc-H binding studies by Mosberk et al., Nuclearmedizin (Stuttgart), 1981, 18, 343; Chem. Abs., 96, 30896w; the use of
111
InCl3-H for visualisation of thrombosed arteries, Chem Abs., 97, P168866w;
cf. related studies of 169Yb and 161Yb H binding, Chem. Abs., 98, 194186g;
cf. also 167Tm has been reported to have strong affinity for tumour tissues especially to HS and other glycosaminoglycans by
A. Ando et al., Eur. J. Nucl. Med. Mol. Imaging, 1983, 8 (10), 440-446

Eu(III)-H complex formation enables H to be spectrophotometrically assayed as reported by

M. Rizk, Spectroscop. Lett., 1995, 28 (8) 1235 (Eu3+ -H formation enhanced the emission of a forbidden Eu3+ band at
616nm allowing ratio of complexed/uncomplexed Eu3+ to be determined; Log10Kassoc. for Eu3+ -H was reported as 5.079;
cf., the log10Kassoc.value for La3+ -H was found by Rabenstein et al. to be 4.5 (cf. ref. 6b-2-1)

11-3-1
Refs 6, 6a, 6a-1, 6b-1, 6b-2. 6b2-2, 6b2-3; 7-9. 9b-3. 9b3-1. 9c, 10-13, 13a; 14-17

6a

Reviews of the binding of inorganic elements include:

L.B. Jaques, Science. 1978; 206: 528-533;
and in Ions in Polymers (Based on a symposium by the Division of Polymer Chemistry at the 176th Meeting of the American
Chemical Society, Miami Beach Fla., Sept 11-15, 1978) Amer. Chem. Soc. Adv. Chem. Ser. 187 (Pub. 1980) p. 350-360
These perceptive articles remain highly relevant.

J.R. Dunstone, Biochem. J., 1962, 85, 336-351 and

J.E. Scott, Ch. 12, p 171-187 in
The Chemical Physiology of Mucopolysaccharides, Ed., G. Quintarelli, Churchill, London, 1968

The formation of adducts with a range of inorganic salts (e.g. iodides) with H was discussed in a Patent
(Fo-We Forschung und Verweltungs-Anstalt [Therapeutically active product ] Brit. Pat. 890,622, 1959)

6a-1

The Binding of Inorganic Sulphate to H

J.R. Helbert & M.A. Marini, Biochem. Biophys. Acta, 1964, 83, 122-134
Studied the binding of inorganic sulphate anions to H

K. Simon Naturwiss. Rundschau 1982, 35 (11) 452-453

discussed the occurrence of ca. 12% of total S in H in the form bound inorganic SO42-

6b-1
K.A. Schwarz, Proc. Natl. Acad. Sci., USA, 1973, 70, 1608-1612
(Bound form of silicon in glycosaminoglycans and polyuronides)

6b-2
A list of published studies from the former Aberdeen polysaccharide group (W.F. Long, F.B. Williamson et al.) which includes
reports of studies by a variety of physical chemistry methods of metal ion and polyamine binding to H and related roles
of hydration and phase separation, was posted on the internet by W.F. Long (and accessed at the time by the author at)
http://www.abdn.ac.uk/~bch118/publications 2003march.doc

6b-2-1
Articles which deal with the binding of metal ions by H include:
D. Grant et al., Biochem. J., 1991, 275, 193-197; ibid., 1992, 287, 849-853; ibid., 1992, 282, 601-604; ibid., 1992, 283, 243-
246; ibid., 1992, 285, 477-480; Biochem. Soc. Trans., 1991, 18, 1281-1282; ibid., 18, 1282-1283; ibid., 1992, 20, 361S;
ibid., 1991,; ibid., 1991, 19, 390S; ibid, 1996, 24, 203S; ibid., 1996, 24, 204S;
D.L. Rabenstein et al., Carbohydr. Res., 1995, 278, 239-256
L. Herwats, et al., Nov. J. Chem. 1977, 1, 173-176

6b-2-2
D. Grant et al., Med Hypoth. 1992, 38, 46-48 (A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern
pathological afflictions).
Silicate anions in aqueous solution above a threshold concentration (ca. 50 ppm) undergo self-association which is likely to be enhanced at H/HS surfaces
in keeping with the strong association of polyuronide with inorganic silicon in biological tissues where they are believed to contribute to the mechanism of
nucleation of bone formation. There is also some evidence that insufficiency of dietary Si is associated with augmented oxidative damage to tissue
(soluble silicate in drinking water has been negatively correlated with the incidence of degenerative diseases such as atherosclerosis (cf., M. F. McCarty,
Med Hypoth.,1997, 49, 177-179 ).

Both HS and redox metal dyshomeostasis may, however, be to be implicated in this effect of Si where a putative role of silicate enhanced redox metal ion
sequestration and pacification can be suggested.

A variety of types of SiO2 sols and gels have been form many years available from the chemical industry; during research into the self assembly of such
materials it became evident to the author that a form of nucleation behaviour which mimicked biological activity was apparent. A hypothesis of silicic acid
generated seeding/Darwinian evolution is rationally derivable from these observations. This type of structural seeding could continue to be of importance
in present day organisms especially in how HS supramolecular inorganic composite material structures are nucleated.

6b-2-3
Some Aberdeen Polysaccharide Group Studies (cf. also ref. 6b-2-1)
These had confirmed the results of previous workers (as well as data from industrial use of sulphonated organic polymer membranes) that H contains a
similar system of interacting -SO3- - associated water cluster structures. The details of which vary between individual metal counterions, but the greatest
degree of hydration, 6H2O/-SO3-, is found for Na+ counterions and the least for quaternary N+ counterions (D. Grant et al., Biochem. Soc. Trans. 1990, 18,
1293-1294; ibid., 1983, 11, 96; ibid., 1984; 12: 302. The H-associated water clusters showed a systematic change in their near infrared water cluster
absorptions upon poly-L-lysine or poly-L-arginine binding. (Glue-like surface clusters of H-metal ion associated of water structure (cf. 9d ) become more
obvious in a hydrophobic medium. (D. Grant et al., Biochem J., 1991, 277, 569-572). Such types of interactions are reminiscent of the reversible
adsorptions thought to involve both the protein core and polysaccharide side chains of cartilage proteoglycans with chromatographic media9c which have
been described as “non-specific”).
New research is required to search for ion conducting properties of GAG hydration superstructures which can be predicted to exist since this is how man-
made organic polymer-SO3-(H2O)n membrane proton conductors are employed in commercial use, their mechanism of action is believed to depend on the
properties of such water clusters (cf., e.g., James et al., J. Materials Sci., 2000, 35: 5111-5119); these man-made sulphonated polymers also find use in
commercial fuel cell membranes which show improved thermal stability when the membrane contains additional SiO2 particles (Adjemian et al., J.
Electrochem. Soc., 2002, 149, A256-261) suggesting a related role for the presence of Si (e.g., as SiO2 particles) in GAGs (Schwarz, 1973, loc. cit., cf.
Grant et al. Med Hypoth. 1992; 38: 36-48). Further hints of how interaction between SiO2 and GAGs, especially, H/HS, may be physiologically relevant
comes from the report by Takeuchi et al . (Analusis, 1998, 28, 61-64) that a heparinised SiO2 chromatographic medium separated both cations and anions
on the same column.
Si(OH)4 perhaps forms crosslinks analogous to B(OH)3 in plant rhamnogalacturonan II (cf. ref. 1a-10)

Anionic Polysaccharides Resemble Humic Polymers as Multi-Element Matrices

In vitro studies of H indicates an ability, which it seems to share with other anionic polysacharides such as the alginate-rich biomass of kelp, to simultaneously sequester a wide range non-
physiological (and therefore potentially harmful) ions from the full range of trace and ultratrace ions etc. which occur in natural waters and biological fluids including blood serum. This
property is also shared with soil humic acids. The perturbation of NMR resonance due to the presence of abundant Mn and Fe in humic acids for a long time prevented the true chemical nature of
these polymers to be determined; a commonly held view was that they were aromatic compounds related to lignin; D. Grant, Nature, 1977, 270, 709-710 suggested a procedure by which the
paramagnetic ions could be removed by placing sparingly soluble detergent formulated pyrophosphate granules in solutions of humic substances. This caused a slow removal of the paramgnetic
ions into the solid chelator and a gradual narrowing of the solution phase NMR peaks. In the case of H containing 1100ppm, Fe and 730 ppm Cu there seemed to be no detectable paramagnetc
broadening. H evidently can coordinate these metals sufficiently strongly ,and more than humic polymers, so to prevent NMR resonance paramagnetic perturbation under the conditions studied.
This is indirect evidence that H can behave as an antioxidant and antinitrant (by blocking of the promotion of reactive free radicals which are known to be formed by Cu and Fe ions when
present in an available solution phase forms ).

Review articles in preparation by D. Grant suggest that a range of cytokines, dietary and physical factors greatly influence HS biosynthesis. This could be part of the reason why
favourable diets can elicit anti-cancer effects (cf. ref. 1A-3 (vide supra) which briefly reviews the anti-cancer effects of H/HS and tend also to suggest that such activities can
depend on H/HS ‘integrity’).

7 J. Folkman, Science, 1983, 221, 719-725, cf. p.722-3

Cf. also J. Folkman & M. Klagsburn, Science, 1987, 235, 442-447
(steroid induced H-related anti-angiogenesis effects differed between Hs from different manufacturers; these authors were
unable to idenfiy what these differences were. Later similar work by J. Folkman et al. in Nature, 1989, 339, 58-61 appears to
have been conducted to avoid use of H]
Cf.,
W.F. Long & F.B. Williamson, Med. Hypoth., 1984, 13, 385-394
(Heparan structure and the modulation of angiogenesis)

J.-M. Herbert & J.-P. Maffand, J. Cell. Physiol., 1989, 138, 424-432
(HS oligosaccharides generated non-enzymically from H from different manufacturers differed markedly (but why was
unknown) in vascular endothelial and smooth muscle cells antiproliferative potencies)
 More recent studies (cf., ref. 1A-3, vide supra) have however confirmed the original concept historically promoted by
J. Folkman which indicated that H and H- like compounds are powerful anti-angiogenesis (anti-cancer) agents (Hs are now
known to have additional anti-cancer activities, including anti-proliferation and anti metastasis activities).

8 F.T. Borges et al., Kidney Int. 2005, 68, 1630-1642

Cf. G. Embery et al. Scand. J. Dent. Res. 1979, 87, 318-324

(Interaction of acid glycosaminoglycans with hydroxyapatite; the order of binding was H>HS>dermatan sulphate/chondrotin
sulphate>hyaluronan. Ca2+ (and saliva) a pretreatments led to an increase in hyaluronan and chondrotin 4 and 6 sulphate
adsorption, but a slight decrease in H adsorption; the presence of F- led to decreased adsorption of hyaluronan and chondrotin
sulphate).

Cf., J.L. Arias et al. J Mater Chem. 2004, 14 (14) 2154-2160

(Sulphated polymers inbiological mineralzation: a plausible source for bio-inspired engineering)

8a
A simple illustration of how different microstructures in complex polysaccharides which (analogously to HS display complex information encoding
sequences by which they can control complex chemical activities) is the alginate-inhibited seeded crystallization of BaSO4 which occurs by consecutive
second order rate processes which show systematic variation in their rate constants which are apparently mathematically dependent on the alginate
microstructure (which is conventially described in terms of guluronate and mannuronate blockiness)**.

Cf., D. Grant, M.I. Tait, W.F. Long & F.B. Williamson “Microstructure-dependent modulation of crystallization by alginates”, a communication presented
in the form of a poster by M.I. Tait at the International Seaweed Symposium, Vancouver, British Columbia, Canada in 1989. This reported that the rate
constants of BaSO4 crystal growth on seed crystals, obtained in a similar manner to that described by Grant et al., Biochem J. 1989; 259: 41-45 for a range
of alginates (detailed by J. Boyd, “Enzymic approach to the structure and function of alginic acid”, Ph.D. Thesis, University College of North Wales,
1975, cf., J. Boyd & J.R. Turvey, Carbohydr. Res. 1978, 66, 187-194 and A.J. Currie in “The structure and biosynthesis of alginic acid”, Ph.D. Thesis
University College of North Wales, 1983) were dependent on the microstructure of M (mannuronate) and G (guluronate) environments rather than degree
of polymerisation. Individual alginates: (1) [“SS/DJ”, mainly a random structure with some preponderance of polyguluronate units] (2) [“F387” , an
industrial product obtained from Ascophyllum nodosum also studied by Grasdalen et al. (cf. Carbohydr. Res. 1981; 89;: 179-11) which contained
proportions of M =0.58, G =0.42 of which were in diads with MM = 0.40, GG = 0.24, MG and GM = 0.18, triad MMM = 0.08, MMG = 0.08, GMM =
0.08, GMG = 0.10, GGG = 0.18, GGM = 0.06, MGG = 0.06 and MGM = 0.12] (3) [polymannuronic acid from fruiting bodies of Ascophyllum nodosum];
(4) [poly M blocks]; (5) [ poly alternating blocks] and (6) [poly G blocks] (4)-(6 ) were obtained from commercial samples by enzymic degradation
followed by fractionation according to the methods of Haug et al. (Acta. Chem. Scand. 1967, 21; 691-704). Second order rate constants were for (1)-(6)
respectively 0, 55, 55, 54, 62 and 81 for the primary and 59.6, 18.8, 15.2, 14.6. 6.0 and 36.6 for the (principal) secondary reaction processes. The relative
potency of the alginates was further correlated with infrared spectrocopic variation in the 630-1190cm-1 region which was indicative of the microstructural
differences.

9b-3 D. Grant W.F. Long & F.B. Williamson, Biochem J., 1991, 277, 569-571

9b-3-1 M. Kan, F. Wang, M. Kan, B. To, J.L. Gabriel & W.L. McKeehan, J Biol Chem., 1996, 271, 26143-26148
(Although the interaction of H with FGF was not affected by divalent cations, the presence of such cations especially Ca2+ or
Mg2+ 9b-3-1 is reported to be absolutely required for high affinity (Kd= 10nM) binding of H to FGFR).

9c L.J.J. Hronowski & T.P., Anastassiades, Biochem. Biophys. Res. Commun. 1990; 167; 81-88;
Anal. Biochem. 1990, 191 (1) 50-57
(Proteoglycans show a broad specficity adhesive property binding to chromatogrpahic media such as silica gel, glass surfaces,
cellulose, sepahrose and various polyoleiifing used as test tubes but the interactions were also found to be highly dependent on
the ionic compositions of the buffers used and the presence of chaotropic agents)

10 A. Lima de Faria “Evolution without selection: form and function by autoevolution” Elsevier Science Publishers B.V.
(Biomedical Division) Amsterdam, & New York 1988
That biological reproduction in its simplest form is mimicked by inorganic substances such as CaCO3 crystals which reproduce
as true-to-form “species” by a seeding process (which also can describe the behaviour of formally amorphous aggregates of
silicic acid (silica sols), cf. D. Grant et al. 1992a)

11 M.A.S Pinal et al., Braz J. Med Biol Res. 1994, 27p, 2191-2195
H.B. Nader, V. Buonassisi, P. Colburn & C.P. Dietrich, Cell. Physiol. 1989; 140: 305-310

12 R. Cappai, F. Cheng, G.D. Ciccotosto, B.E. Needham, C.L. Masters, G.Multhaup. L.-A. Fransson and K. Mani, J Biol.
Chem. 2005; 280: 13913; cf., K Mani et al., ibid., 2004; 279, 12918-12923; ibid., 2003, 278: 38956-38965;

13 D. Grant suggested in 2000 a similar mechanism of redox metal ion and ascorbate nitric oxide metabolite dependent HS
oligosacchride signalling in preliminary draft discussion articles entitled “Ascorbate and Nitric Oxide in Redox Control of
Heparan Sulphate” and “Ascorbate & Cancer” originally posted on web.ukonline.co.uk/dgrant/dg4/- etc are now available
only as cached files from Yahoo (but not now Google) using the search terms /dgrant/dg4- and /dgrant/dg5- (with the
References posted at /dgrant/dg8/-) cf., also /dgrant/dg2- and /dgrant/dg5-)

13a R.A.A. Muzzarelli, Polymer Sci. Technol. 1983; 23: 359-374

(which describes the preparation of H-like derivatives of chitosan which are evidetly highly ‘contaminated’ with iron etc., cf.,
Table IVa
14 H.J.M. Bowen, J. Mar. Bio. Assoc. 1956; 35: 451-458

15 a V.N Senofonte and S Caroli, J. Trace Elem. Med. Biol., 2000, 14, 6-13

b I. Rodushkin and M.D. Axelsson, Sci. Total Environ., 2000, 250, 83-100;

c I. Rodushkin and M.D. Axelsson, ibid., 2000, 262, 21-36;

[cf. also http://www.analytica.se/hem2001/human/research.asp; cf., D.A. Bass et al., Altern. Med. Rev., 2001, 6, 472-481 and
A.R. Bleise et al. Analytical Qualtity Control Services, (Hair Elements Reference Sheet), International Atomic Energy Agency,
A-1400,Vienna, Austria RAA

16 A. Kornberg (with M.R.W. Brown) Proc. Natl. Acad. Sci. USA, 2004, 46, 16085-16087
(Inorganic polyphosphate in the origin and survival of species)

16a R. N. Reusch et al., Proc. Soc. Expt. Biol. Med., 1989, 191, 377-381
Cf., S. Das S et al. (with R.N. Reusch), Proc Natl. Acad. Sci. USA, 1997, 94 (17) 9075-9079
(Proof of a non proteinacous calcium seletion channel in Escerichia coli by total synthesis from (R)-3-hydroxybutanoic acid and
inorganic polyphosphate)

17 J.M. Somers, M.L. Tait, W.F Long & F.B, Williamson, Hydrobiologica, 1990; 204/205: 491-497

18 M.L. Merrous M.L. et al. Chemosphere, 2003, 52, 113-120

(Lanthanum fixation by Myxococcus xanthus: cellular location and extracellular polysaccharides observed)
[A study of La3+ binding to extracellular polymers secreted by Myxococcus xanathus]

12 Concluding Remarks

If the ability to bind and utilize inorganic cofactors associated with H is also replicated with HS, this could suggest a
new paradigm of metallome-heparanome crosstalk for the modus operandi of this control system.

Polysaccharides apart from consisting of complex information-containing sequences of differently sulphated

polysaccharides (the heparanome) also co-exist with an array of inorganic ions and particles including alkali metal
and alkaline earth counterions present in biological fluids as well as trace and ultratrace elements also present in
such fluids.
Unfractionated H is apparently the most optimized known example of any biological metallomic matrix system
which, if this is confirmed, could suggest that the evolution of H-like structures in HS present in all
animal species and mast cell H present in many animal species must have been, at least in part, driven by
the potential advantages conferred by the presence (especially in extracellular glycocalyx environments) of
such potential inorganic ion reservoir matrices containing such elements in largely available forms.

The complex patterns of sulphated anionic sugar HS proteoglycans, perhaps existing partly as flexible metallo-polysaccharide complexes
can logically be proposed to be of sufficient functional complexity to fulfill the sought after role of a non-genetic biochemical information
handing and storage system suggested to be required for the post-human genome era. The information-encoded H/HS system is that it is
known to be involved in a systemic signaling and could explain why the human geneome is much smaller than expected.

It is suggested that the modulation of the activity of HS sequences by metal ions could act as an amplification loop to extend the
information processing abilities of otherwise inadequate, limited size genomes may allow the heparanome to contribute to the
development and perhaps also the evolution of complex animal organisms.

HS- associated multi-inorganic ion arrays are now suggested to promote the formation of functionally relevant HS supramolecular
structures, which, further subject to the efficiency of probably required nucleation agents, and perturbation by pathological and
anthropogenic factors, are suggested to be highly relevant to the etiologies of those degenerative diseases in which HS biochemistry has
been implicated.
Sequence patterns in HS arise via servo feedback controlled alterations of the sequential utilization of the enzymic
modifications during their Gogli apparatus assembly as well as via related postsynthetic modifications which also
include an important non-enzymic (environment-linked) putatively metallomic input.
H and HS are now known able to bind and modulate the activities of such a wide range of proteins, many of which are
known to require inorganic cofactor. This circumstance can be putatively be extrapolated to suggest that H/HS is
involved, in addition to embryogenesis and ageing, also in virtually all physiological and pathological processes which
further could suggest that the complex inorganic multi-element metallomic biochemistry of H/HS could contribute to the
mechanism of evolutionary change of animal genes, probably by servo control feedback processes enabled by HS
signals which are known to inteconnect extracelullar space with the geneome
12.1 Suggested Further Research

Further research seems to be warranted in the field of metallomics as applied

to sulphated polysaccharides.
It is required to test the hypothesis that transmission of information-encoded in the HS glycosaminoglycans in the
form of specific anionic sulphate half ester patterns may be dependent, at least in part, on the co-occurrence in these
highly anionic polysaccharides of Ca, Mg, Mn, Cu and Zn as well as a range of other inorganic elements including
those (e.g. Si, Al, As, Pb, Sr and the rare earth elements) which have been tentatively implicated to be required in
animal biochemistry, and dyshomeostasis of which may promote degenerative diseases by perturbing their putative
inorganic cofactor heparan sulphate dependent biochemical signaling functions.

It is required to test the hypothesis that small amounts of phosphate or polyphosphates attached to H/HS act to
increase the antioxidant and/or the ferrioxidase activity of H/HS (e.g. by facilitiating high affinity binding of
unliganded redox metal ions such as Cu2+ which otherwise could initiate Fenton reaction oxidative degradations).

Further research is required to establish the optimal inorganic ion array to allow correct in vitro H
modelling of HS in vivo behaviour and also determine how different types of final purification procedures
adopted by different manufacturers alters the inorganic array associated with H.

[The single counterion diluted forms of H exhibit skewed inorganic ion distributions relative to the less purified
form a distribution which evidently can vary between different manufacturers, depending on the details of the
purification procedures used which is a source of potential often ignored variation in the activities of H used to
provide a convenient molecular structural model library system for biochemical investigations of HS
activities].

Purified oligosaccharide epitopes: do such preparations lack the natural multi-inorganic ions normally associated with the intact
polysaccharide molecules and which, can be rationally suggested to be essential cofactors which allow the necessary precise
discrimination between HS microstructures needed to accomplish developmental regulation?

The Mark Purdey Hypotheses of TSEs: Update

The origianl hypothesis that organophosphates were cofactors of TSE infective agent generation was later replaced by one based on
mangangese overload (under conditions of copper deficiency). Later still hypotheses that infrasonic shock waves could promote TSEs
and evidence for possible imput from the presence of anthopogenic nuclides were presented.
The accepted hypothesis is that TSEs depends both on prion misfolding with some of metal ion dyhomesotasis in agreeement with how
scrapie infective particles can now be genrated by a sonication-based cyclic amplification protocols where in the presence of a necessary
polyanion cofactors, aggregated misfolded prions are created which are capable of transmitting the disease. Since HS is the most likely
polyanion invovled in this process in vivo, the rate determining phase change process (not an electrostatic ‘Manning’ process) by which
H/HS binds metal ions (and putatively also some or indeed possibly many proteins) could therefore also be criticlly important to the
scrapie factor infection process. Such phase changes will be subject to general nucleation of seeded initiation. This could allow high
energy inputs from various sources to promote the seeding of the pathological agents. Such ionizing energy from nuclides (cf. H studies
by ICP-MS contains a full range of inorganic elements which include e.g., uranium ) will anable the concentration of ionizing radiation
and energetic particle emitting centres consequent on biological fluid ultra ultra trace element adsorption enrichment at HS-like surfaces
which have been identified by the author (discussed above). It is suggeted that such energy hotspots assist with prion
misfolding/aggregation.

Such putative sequestration of active nuclides at the H/HS surfaces also could provide the basis of a practical technolgical method for the
removal of radioactive materials from contaminated environments.

*A former industrial inorganic chemist with later appointments in academic soil science and polysaccharide
laboratories

Heparan sulphate biochemistry is suggested to be a super-hub system permeating animal physiology/biochemistry and this might depend on the nucleation
of phase changes involving inorganic moietites
The origins of this somwhat unorthodox hypothesis seems to need some explanation.
While all kinds of scientific unerstanding is part of a now gigantic unitary system of knowledge, much of it uncorrelated due to the hindrance to
scientific progress arising from the rigid compartmentalization of science which seriously limits appropriate application of many facets of scientific
knowledge. Information which is buried in subcompartments can sometimes become evident only following employment jumping in changes made
fairly randomaly in response to both a search for improved prospects or in reponse to economic downturn closures. Normally scientists are
encouraged to stay in a single stream career path. This hinders access to most of known scientific data. A sideways movement between jobs I think
has enabled me to gain novel insights into the biochemistry of polysacchardes.
At the risk of being boring I feel the need to mention some details of this including key names which will be unknown to biochemists.
* Curriculum Vitae
The author (from Turriff, Scotland, UK, AB53 6SX) was formerly a Senior Research Chemist at Interntional Synthetic Rubber, Hythe, U.K. (this followed previous appointments with Monsanto Co. (Central Research, St Louis, U.S.A.), Monsanto Chemicals Ltd.

(Nickell Laboratories, Ruabon, North Wales, U.K.), Imperial Chemical Industries Ltd. (Head Office Division, Central Instruments Research Laboratory, Bozedown House, Oxon., England, U.K.) a Research Student (from 1975-1976) and a Research Fellow in the

Department of Soil Science, King’s College University of Aberdeen, Scotland, UK (from 1976-81) and (from 1981-92) a Research Fellow at Marischal College University of Aberdeen, and thereafter for a time an Honorary Research Fellow following which I have

engaged in private freelance research mainly relating to previous research interests. At the invitation of Dr F.B. Williamson, a retired senior lecturer who formerly was a co-manager of the former Marischal College, Aberdeen University polysaccharide laboratory, I have

conducted literature research on Chronic Fatigue Syndrome. At the same time I had attempted to continue previously started literature researches in the field of H/HS biochemistry (a project commenced in 1981); a general consensus seems to be emerging here that this

polysaccharide system is a ‘super-hub’, high ranking management system in animal biology.

Forty plus years ago in St Louis I was priviledged to help with fundamental chemical reseach then being conducted at a pure science group headed by John R. Van Wazer at Monsanto Company which sought to extend ideas John had gleaned from hig academic approach

to industrial phosphorus chemistry, by which it was hoped to explain the behaviour, especially the thermal stability of other-than-phosphorus systems of compounds (especially of polymers). This extension of research interests included sulphur oxyacids systems of

which heparin/heparan sulpahte is a major biochemical representative. Similar principles (controlling molecular reactivity, also first identified by the van Wazer group) involved studies of a system of exchange of atoms between small molecules and polymers consiting

of such units as monomers. Similar principles applies to such systems of inorganic polymers thoughout the perioidc table. The theories developed by this group are now somewhat out of fashion but I think are still highly relevant to a fuller understading of both pure

chemistry and biochemical sciences (especially as to how libraries of related systems of polymers (e.g., proteins , polysacharides and nucleic acids as well as polysislicates etc.) of differing strurcture can be formed by self-assembly processes). J. R. Van Wazer was of

the opinion that in inorganic chemistry organic reaction mechanisms are red herrings as regards usefulness for the understanding of polymers. Another current private research initiative stems from the N.E. Aubrey & J.R.Van Wazer (J. Amer. Chem. Soc. 86, 4380)

discovered that all perchlorocarbons try to reorganize into carbon tetrachloride, hexachloroethane and hexachlorobenzene. The chlorine containing analgoue of polytetrafluoroethylene easily falls apart in this manner but such instability is absent with the

perfluorocarbons. A similar scrambling behaviour of perchlorocarbons seems to apply to all compounds containing carbon and chlorine. I revisited this scenario in greater detail at ICI, Ltd. cf., Grant, D., J. Appl. Chem. Biotechnol, 1974, 24, 49-58; this confirmed the

validity of the Aubrey-Van Wazer concept. A further concept which I became aware of, also when working with ICI, was that all dehydrochlorination reactions of organic molecules always obey strict compensation rules in which the temperature dependent and

temperature independent parts of dehydrochlorination rate constants are strictly correlated. The ultimate scientific origin of this phenomenon is unknown. It seems to apply to reactions which are heterogeneous as well as those which are thought to occur homogeneously

in the gas phase. Similar compensation phenomenon had however been the subject of numerous literature reports going back many years and it was the subject of a review in 1955 by J.E. Leffler but current science seems not to be interested in this topic. The

compensation effect also applies to the heats and entropies of vaporisation from solutions and pure unassociated liquids, to Eotvos surface tension factors, to the thermionic emission of electrons from solid surfaces and to the electrical conductivity of solids, including

amino acids and proteins, to the heamolysis of erythrocytes and to the denaturation of proteins. According to Lumry & Biltonen (1969) “it is probable that the enthalpy and entropy change during conformational changes in proteins is the single most important physical-

chemical characterization of protein function”. [The renowned reaction kinetics scientist C. Hinshelwood was well aware of the mystery but unable to offer any convincing explanationof it]. Much of chemistry and physics is, I think, affected or even determined by this

correlation. Especially it applies to reactions conducted in aqueous solution. I think that the tendency of biological systems to create order out of non-order is a related phenomenon. There is a major additional restriction required to fully establish thermodynamic/kinetic

theories relating to life processes. This phenomenon is also relevant to how pyrolysis of compounds containing carbon and chloride (or chloride) can give rise to dioxin-like hexachlorobenzene. This could be part of the mechanism of promotion of Myalgic

Encepalomyolitis/Chronic Fatigue Syndrome ME/CFS which might arise via hexachlorobenzene intoxication (this substance was formerly used as an insecticide including for threatment of scabies) which promotes the pro-inflammatory state and nitric oxide

dyhomeostasis. It is of some interest in this regard that heparin therapy has been reported to offer dramatic therapeutic benefit to, at least a significant proportion of ME/ CFS afflicted persons. There seems to be some similarity here with how perchlorocarbons are highly

effective and find industrial use for promoting higher oxidation states of transition metals used as olefin polymerization catalyst sites (I had discussed this in a Eur. Polym. J. 10, 41 article and also in a Symposium Presentation at the Chemistry Department at Aberdeen

Univeristy in 1981)

I have written and contributed to a wide range of (peer-reviewed) published scientific studies [cf. Grant, D. et al. 1] (e.g. on the structural reorganization of organo tin monomers, organic phosphonate polymers,
polysulphates, polyvanadates and organic bivalent sulpur compounds. The latter is relevant to redox balance of thiol and disulphide group equilibria in biochemisty. Unpublished data indicates that disulphides can

self cluster (e.g. into octamers) in a manner which shows up in the NMR spectra. This may have biochemical relevance as is the previously mysterious nature of organic silicate esters and silicic acid polymers.
Related systems of silica-based inorganic colloids were also found to ‘self assemble’ by a seeded biological-cell-like replication process in which a “heel” of the previous batch needed to be retained to act as a

template seed to obtain the desired types of final product particles. I had left a preliminary manuscript describing this work at Monsanto Chemicals Ltd. (Ruabon, Wrexham, Wales, U.K.) to be continued by others
but commecially sensitive issues seemed to have intervened with getting this idea known, nevertheless I contined to think about my preliminary results on these self-replicating inorganic colloids when I became a
mature student at Aberdeen University (Scotland, U.K.) where discussions for a time centred on how silicate interactions with anionic polysaccharides could be critically involved in human health and might also
have been involved in the generation of life on earth in the first place (cf., Med. Hypoth., 28, 245-534). My later post-career Aberdeen biochemical studies also encouraged me to think about how ageing might

occur in living organisms. Such thinking benefited from knowledge gained during organic polymer ageing studies (which I had earlier conducted at (former) International Sythetic Rubber, Hythe, Southampton,
Hants., U.K., laboratories where I had been for a time a group leader, correlating research results (our results on hyperactive Ti- and V- based organometallic catalysts were reported in Eur. Polym. J .
[later when I found that these and other redox active elements also can occur in association with heparin, this encouragaed me to think how the redox properties of these and other elements (which also occur in such
biological fluids as blood serum) when (putatively) normally present in sulphated polysaccharide complexes might be of biochemical relevance; I had studied the redox properties of Ce at Glasgow and again this

prior interest allowed me to question the role of rare earths as putative heparan sulphate cofactors]. Ageing in animals might be controlled by a related similar chemical structure dependent antioxidant mechanisms
so those which determine the life expectancy of articles fabricated from isotactic-polyproplyene stored in the dark (in air) which appeared to depend entirely on the presence of a preferred type of synergistic

combination of a hindered phenol plus a bivalent sulphur iron chelating ligand, life-enhancing, system. (The idea that a similar system of antioxidant protection was present in proteins arose from a comparison of
the amino acid sequences of a very large number of proteins which suggested a tendency of natural selection to incorporate cysteine tyrosine clusters after the occurrence of atmospheric oxygen; it seemed that the

non-biological antioxidant consisting of hindered phenols plus a disulphide iron chelator which is similar to the tyrosine-cysteine cluster antioxidant system had been discovered by laboratory workers seeking to
inhibit oxidative degradation of industrial polymers had accidently mimicked a type of antioxidant system which nature had previously identified. The Med. Hypoth. Vol. 28, 248, article which outlined these
findings, however, I now think needs to be updted to consider the tyrosine-cysteine clusters provision of anti-nitrant activity (and how this might be enhanced by heparan sulphate proteoglycans which can also be
argued to generally promote antioxidant protection systems including via iron and other redox metal chelation scenarios as well as by providing binding sites for extracellular superoxide dismutatase). Earlier,

during my first career postgraduate research studies (at the Univeristy of Glasgow, funded by Albright & Wilson Manufacturing Ltd., who directly benefited from my pilot study interpretation of of the reaction
products of phosphorous acid with acetic anhydride which clearly did not to form acetyl phosphite as they had believed, but proceeded to a quantitative formation of condensed acetylated poly ethane,1- hydroxyl

1,1-diphosphonate glasses) I redscovered a method of sythesis of bisphosphonates which had originally been reported in 1897 by H. von Baeyer & K.A. Hoffman (Ber., 30, 1973) (but this groundbreaking research
had strangely been forgotted a half century later by Engish speaking world). At Glasgow I had also carried out studies of numerous salts (including acid salts) of phosphorous acid and pyrophosphorous acid using

acid base titration, ceriometric oxidation, chromatographic, spectroscopic, thermogravimetric and polarographic methods. It became evident that the reactivity of anionic structures containing H-P(O)(X)O- [X=
-O-,OH or O-] groups depended critically on the nature of the (mostly metal) countercations. Poly [–O- [H-P(O)]-O-] was found to easily undergo a non-random structural reorganization to produce molecules
n
lacking P-H groups. Water binding and the chelation of metal ions was involved somehow in this. About a quarter of a century during my mature student days Frank Williamson suggested that I study the overtone
infrared spectra of water and how this hydration affected polysaccharide activities and the activities of cells with such polysaccharides at or near their surfaces (these studies eventualy extended to cell culture
metabolic labelling, gel fractionation etc., i.e. routine biochemical research methods but addtiional studies also were carried out on the extraction of xylans from seaweed, the sulphation and assessment for
spectroscopic, metal ion binding hydration and anti HIV activies of such xylans which have heparin like qualitites and are highly active as anti-scale agents, and are also highly effective anti-virals). Sulphated

xylans are now known also to include anti-scrapie, (anti-prion) actions which allows for medical intervention in nvCJD a subject which I contributed to a little by engaging in long discussions and literature
exchanges with Granham Steel (formerly of the CJD alliance).
I had formerly experimentally probed the effect of humic acids, fulvic acids and sulphated polysaccharides as modulators of crystallization (e.g., of CaCO [calcite]). This is also the subject of current thinking and correspondence relating to how
3
anthropogenic alteration of the oceanic and atmospheric buffereing of CO might be controlled by agriculture and deforestation via altered humic-fulvate input into the sea. I think this is relevant to how humic polymers affect ocanic carbonate and also
2
how animal polysaccharides inhibit numerous types of pathological plaque formations. I am endebted to the late Joe Tinsley and the late John Parsons (who had been consecutively Heads of the Department of Soil Science at Aberdeen University) for

encouraging me to investigate the chemical nature of humic polymers by using methods I had learned about during my researches in industrial laboratories. I found that humic materials could be made to give highly resolved NMR spectra only if the

paramagnetic ions were removed therefrom (this was achieved in a manner uniquely suited to humic polymer evalutation by the selection of an industrial polyphosphate composite metal ion chelator which had been designed as detergent builder

employment of which enabled distinct resonances to be teased out of dispersed humic matter clearly showing 13C and 1H NMR resonance attributable to polymethylene (known to me to be highly soil=environment-stable from my work with man-made

polymers at ISR) as well as various mainly aliphatic poly(C(O)O-) anionic group substituted aggregates. I think this was the first demonstration of the chemical nature of humates the most abundant type of organic polymer (really a composite system) on

Earth [it was found that they were not highly aromatic as had previously been supposed]. Although this work led to a prleminary article in Nature in 1977 most of my collection of studies of humic polymers still has not been properly published. [These

data had been present at an International Symposium in Braunschweig in 1978, but there seems to have been lack of funds to allow the Proceeding of this Symposium to become more widely distributed)]. I hope to re-submit these data for publication on

the internet together with an update of literature on the subject which seems in general agreement with the Aberdeen work although some workers continue to use older lignin-based models of humic polymers.

I later applied the crystal growth techniques used by G.M. Nancollas (in who’s laboratory I had briefly worked in 1959 where a highly accurate method had been developed to enable the reproducible determination of seeded crystallization rate constants).

Using these methods I conducted studies into the inhibition of the seeded crystallization (of e.g. CaCO [calcite]) in the presence of condensed phosphates, ethane, hydroxyl, 1,1, diphosphonate, various glycosaminoglycans including heparin, chemically
3
modified heparin and heparan sulphate, soil humates, fulvates and lignin-derived compositions. Heparin resembles inorganic salts of phosphorus in the sense that the activity of these key polysaccharides seem to be greatly influenced by attached

inorganic cofactors; similarities were apparent between to phosphite-like chelation- metal-ion-hydration-dependent reactivities. Counterions seem to be important for the reactivity of key heparin/heparan sulphate biopolymers.

Acknowedgements
I thank Professor W.F. Long and Dr F.B. Williamson (former managers of the Marischal College, University of Aberdeen former polysaccharide laboratory) for their support over many years,

Professor K.E.L. McColl and Ms J,. Grant, Univeristy of Glasgow for discussions, Professor R.J.P. Williams of Oxford University for conducting a long correspondence offering advice and criticism of the subject matter of this communication, Professor P.M. Wiggins

(Auckland, New Zealand) for discussions relating to her theories of water structure, Graham Steel (formerly of the CJD alliance Glasgow) and the late Mark Purdey (Taunton) for many discussions, scienfitic communications and encouragement.

** Review of these data is outwith the scope of this communication [but further communications are in preparation].

{Apologies for remaining spelling mistakes and lack of conciseness and also for two sets of references from merged
documents (there are two main lists, those arranged alphabetically and those referred to by a number}.

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More on WATER & LIFE – ROLE OF POLYSACCHARIDES

Is the pre-existence of polyanionic (including organic polymer) multi-inorganic-ion sequestering and buffering systems
which promote bio-friendly environments including bio-friendly water structures, a fundamental pre-requesite for life?
Bio-friendly Wiggins supramolecular structures in liquid
water, the putative ultimate pre-requisties for life, could
have been engendered by prebiotic polymers resembling
anionic polysaccharides
APPENDIX
Phenomena Related to Water Structure
A hypothesis** of how water chemistry contributed to the self assembly of complex quasi biological assemblages capable of eventual
evolution into DNA-based life forms.

The complex nature of hydrogen-bonded networks in liquid water is also perhaps the most common type of the example of a
chemical system of families of labile polymers in which occur reversible chemical structure interconversions are achieved by
compensated rate processes which are both restricted (by at present unknown mechanisms) by the existence of mathematically
precise permitted inter-relationships between entropic and enthalpic changes which tends to promote the formation of both
scrambled libraries of related structures but paradoxically also can eventually evolve, it is now suggested, into highly-ordered
self- replicating non-crystalline systems** similar to colloidal silica dispersions (cf., Grant et al., 1, 1992a). Such chemical systems
also previously had been found to engaging in “structural reorganization by ligand interchange” scrambling processes which are
fairly common throughout chemistry (cf., Van Wazer1 , Grant,1 1966, 1967, 1974 ). This phenomenon which also occurs in liquid water
and ligand exchange and polymer rearrangement dominated systems, is now suggested to be achieved via general entropy-
enthalpy compensation processes which, governed by the allowable outcomes of compensated chemical reaction rate processes,
encourage, near the isokinetic temperature, the creation of increasingly complex systems, which over long periods of time (in
apparent violation of the second law of thermodynamics) can attain the degree of high non-crystalline order characteristic of
living organisms.

The existence in present-day natural waters of humic polymers (these are polyanionic organic molecule assemblages with abundant
carboxylate-rich side groups as well as associated amorphous silicate) which seems to have provided** to be an important part of an
ancient planetary surface homeostasis buffering system whereby the multi-inorganic ion composition of seawater is achieved over
geological times. While present day humic polymers are clearly mainly the residues of living organisms, some primitive humic polymer
system similarly constituted and capable of buffering seawater could, it can rationally be suggested, could have arisen prebiotically, e.g.,
from the formaldehyde polymer (hydrated) SiO2 particle system which apparently exists in interstellar space (cf., e.g., Wickramasinghe1).
It is now further suggested that cell-surface anionic polysaccharides, intially those possessing only carboxylate anionic groups similar to
alginate and hyaluronate, specifically evolved to replicate the buffering and water activity regulation
functions afforded by the humic-silicate polymer systems but following evolution of multicellular animals the adherent cell surface
polysaccharides associated with these organisms which still retain Si (amorphous silicate components similar to those preseent in
humates) dramatically evolved with increased complexity which seems initially to have increased the metal ion chelating activity by use
of sulphate half ester, N-sulphonate and iduroronate polysaccharide side-chains which achieve increased flexibility for inorganic ion
binding and structure processing via nitric oxide metal ion dependent mechanims. These more flexible metal ion ligands enable animal
cellular, protein and genetic processing which depend, it is now suggested, on central roles for the HS-inorganic ion and associated
(water) interactions.

At their most fundamental level of biological function, it is suggested that the anionic polysaccharides act as smart buffer
systems for the creation of optimized multi-inorganic element aqueous solutions of compositions which, e.g., can fullfill the
specialized compartamentalized physiological ionic strength and water activity (water-structure) requirements of
multicellular animals. This role of polysaccharides can be further elaborated in terms of the role of polysaccharides for the
separation in pores of the low density and high density forms of liquid water, a phenomenon which according to the theory
of P.M. Wiggins1, allowed, in the early Earth where biology first arose, the separation of D- and L-forms of amino acids, the
separation of Na+ and Ca2+ from K+ and Mg2+ and provided an essential component of the transduction mechanism of the
biological energy systems used throughout biology, namely the adensosine diphosphate adenosine triphosphate
equilibration.
This ability of polysaccharides to act as primitive ion pumps and ion exhange membranes seems to be retained by animal
organisms where the function of the kidneys includes an anionic barrier function for HS proteoglycans.
Evidence of retention of metal ion transport functions of HS is that an independent HS proteoglycan Fe pump may co-exist
with transferrin in liver cells (cf., Hu et al.1) and the perturbation of essential Ca2+ kinetics required for muscle function in
Nsdt-1 -/- knockout mice with defective N-SO3- depleted Ca2+ binding abilities of HS PGs (Jenniskens et al.1)

The inorganic sequestering and buffering abilities of natural polyanions can be rationally suggested to have been a key
factor by which the unique properties of liquid water can be harnessed in a manner to explain why this liquid is centrally
associated with the existence of life (cf. Chaplin1).

Multicellular animals which are believed to have initially evolved in the sea will have sequestered their required nutrient
inorganic ions from the surrounding seawater via initial binding to extracellular polysaccharides especially to those
glycosaminoglycans containing enhanced anionic domains- the H/HS types of polysaccharides which contain a system of
N-SO3- and iduronate carboxylate anionic groups (cf. Whitfield & Sarkar1) which seem especially to have been selected by
evolution to more effectively bind inorganic moieties and thereby more efficiently generate humate-like biofriendly
hydration pericellular environments in multicellular animals e.g. to re-create the ‘all element’ inorganic ion buffer systems
in natural waters.

The specific hydration patterns of ionic polymers induced by inorganic co-ions can be regarded as being an intrinsic part of
their molecular structures**.

The multiple C(O)O- contaning soil organic matter humic polymers polymers seem to provide a multi-inorganic ion and
inorganic particle buffer system therein which which seems to have kept the inorganic ion composition of the sea
approximately constant over geological time a phenomenon which can also be suggested to be part of the reason why the
unique properties of liquid water (e.g. cf., Wiggins1) is a fundamental pre-requisites for the phenomenon of life. The
inorganic chemistry of humic polymers seems to been mimicked by possible precursors of the evolution of multicellular
animals which were the evolutionary precursors of highly anionic polysaccharides such as the glycosaminoglycans. For
animals the sulphated polysaccharide system of HS PGs seems to be of especially important in this role. HS first seems
first to have evolved coincident with the early evolution of multicellular animals in the sea some 109 years ago.
The ubiquitous existence of small amounts of polyanionic humic matter in multi-inorganic element containing natural
waters may be the key factor which provides the multi-inorganic ion buffering system of natural waters. This then could
have been a critical chemical system which was needed to have been in place first of all before biology could exist. The
retention of water and nutrients in soil is an acknowledged role of present day humic substances.
It is suggested that organisms evolved similar polyanionic systems (e.g. the uronic acids in the extracellular polysaccharide
systems containing multiple –C(O)O- anionic groups) in order to achieve multi-element buffering capacity and putatively
achieve control of the fine-tuned water structures associated with specific composition of multi-inorganic element aqueous
solutions which can be argued to determine intra and extracellular activities. Those polysaccharides which form gels (e.g.
agarose) which contain e.g. 98% aqueous salt, also show a well-defined Hofmeister-dependent variation of ‘melting’
temperature which indicates that water structure formation is assisited by such polysaccharides in a manner which is also
dependent on the chemical nature of the inorganic solutes also present. The adaptation in multicellular animals by the
evolution of in-house polyanionics is acted out again during Golgi apparatus synthesis of HS starting with heparanosan a
mimic of bacterial capsular polysaccharides is the starting point for assembly of HS PGs. Modern retention of this system
as a multi-inorganic buffer is evidenced by the possession of aquatic invertebrates of a glycosaminoglycan-based (including
HS PG) buffering capacity which is found to be directly related with mathematical precision to the salinity of aquatic
habitats (Nader et al. 1, 1983); such a inorganic ion buffereing system seems (from the presence of such molecules on all
adherent animal cells) apparently also to contribute to the inorganic homeostais in animals which also use internal blood
systems with more hghly evolved protein hormonal controlled kidney functions where kidney anionic barriers are still
required to fulfil the HS filtration functionality).

Document 2

The following paper was submitted to a vegan publication in 2001 but may not
have been considered suitable for inclusion because at that time the suggested
roles of heparan sulphate made in this article were less well known outside
polysaccharide scientific circles than is now the case.

Recent Heparan Sulphate Research Results May Help Explain Why a Vegan Diet is Good for You

David Grant
Ashbank, AB53 6SX, U.K.

Summary.
Dietary factors can affect health by influencing heparan sulphate polysaccharide
systems on which important tissue protection activities are centred. A vegan
diet, which tends to be close to that under which our species evolved may
promote health principally by avoiding iron-overload which is associated with
inappropriate heparan sulphate degradation by nitric oxide metabolites and the
formation of lipid oxidation products which negatively perturb heparan sulphate
biosynthesis.
Introduction.
Of the three major classes of polymer systems used by our cells, the nucleic
acids and proteins are better understood than are the polysaccharides. But a
picture is gradually emerging in which key sulphated glucosamine-containing
polysaccharides
especially the heparan sulphate proteoglycans (HSPGs) - are found to be linked
to many aspects of health and disease. These polymers are members of the
glycosaminoglycan (GAG) proteoglycan families which include chondroitin and
keratan sulphates as well as hyaluronan. A critical dependence of human health
on heparan sulphate biosynthesis supports the notion that evolutionary
pressure, at the very eary stages of the evolution of multicellular organisms in
our ancestral lineage, led to the selection of these polymers to be the basis of
an auxiliary information storage system. If we are allowed to make a computer
hardware analogy in which the memory chips are the DNA-containing genes,
then the keyboard and processor are HSPG-based systems providing signaling
channels, ion stores and water based osmolality buffers between extracellular
and intracellular space. The computer analogy is not an exact one since
biological systems seem to use precisely microsequenced polymers for both
software and hardware functions. (Although there is poorly understood non-
genetically inheritance with which GAGs may even be involved, GAGs are
highly dependent on gene products for their biosynthesis). Protein primary
sequence structures are determined by genetic information, but their correct
folding (e.g. as required for growth factor modulation, antioxidant anchoring and
haemostasis activities) is often determined by binding to HSPGs which may,
however, be susceptible to inappropriate oxidative damage under some
pathological and adverse dietary situations.

Some HSPG Biochemical Systems.

Haemostasis was the first tissue-protective HSPG role to be discovered. The
inhibition of blood coagulation by heparin, the most sulphated of the heparan
sulphate-like polysaccharides (discovered ca. 1913 by McLean & Howell)
involves conformational alteration of antithrombin(lll) (AT(lll)) by a highly specific
heparin pentasaccharide sequence (as intensively researched by Rosenberg,
Lindahl, Choay and others).

Dietrich and coworkers predicted a cellular recognition role for HSPGs which
seems to be inherent in the complexity of their shape and anionic charge array
patterns enhanced by the highly flexible iduoronic acid sugar ring (formed by
epimerization
of the more primitive glucuronic acid CS present in ancestral bacterial
polysaccharides) and they recently demonstrated that administration of
anticoagulant heparin (including the now popular low molecular weight forms)
upregulated AT(lll)-binding sequences in blood vessel cell wall HSPGs and
increased their antithrombotic properties.
Nitric oxide under some circumstances does likewise, in keeping with recent
indications of biofeedback between nitric oxide and heparan sulphate tissue
protective systems.

HSPG-controlled systems are also employed by organisms for the regulation of

cell-cell interactions including cells of the immune system and in regulation of
growth factor signalling, most researched for basic fibroblast growth factor (b-
FGF). EXT1 and EXTZ anti-tumour factors were recently found to probably be
heparan sulphate biosynthesis enzymes (this came as a surprise to many
biochemists). HSPGs are centrally involved in lipid processing and can limit
transfer of DNA cationic lipid complexes into and within cells. Although the
involvement of heparin/HSPG with lipid biochemistry was discovered many
decades ago from the routine pharmaceutical use of heparin for inhibition of
blood coagulation, a mechanism is currently emerging that suggests that the
induction of atherosclerosis may be countered by ‘good’ lipids typical of
vegetable-rich diets (and some fish oils) by promotion of the biosynthesis of
beneficially microstructured HSPG for the inhibition of pathological calcification.
HSPG involvement in tissue regeneration in wound healing is part of their global
cellular control function which is also highly relevant to embryogenesis (recently
reviewed by Perrimon & Bernfield) especially for nervous system development.
So it comes less of a surprise to learn that dysfunction of HSPG biochemistry
may be implicated in neural degenerative diseases such as BSE and CJD.
Heparin/heparan sulphate and analogous substances seem to directly inhibit
prion diseases as well as stopping injurious crystal (stone) formation in blood
and urinary vessels (including inhibition of calcium oxalate crystallization, a
possible metabolite problem potentially associated with long-term high dose
Vitamin C (ascorbate) dietary supplementation). Many viruses (including type-O
bovine foot and mouth virus) bind to cell wall HSPGs which, together with
analogous substances, are broad spectrum antivirals (inhibiting enveloped
viruses such as HIV-1, HIV-2, cytomegalovirus, herpes simplex virus (including
mutants resistant to acyclovir) togaviruses and rhabdoviruses).
HSPG biochemistry involvement together with bacterial adhesion has been
proposed for Helicobacter pylon induction of stomach ulceration and cancer.
HSPGs are also apparently involved in the biochemistry of endotoxins,
polyamines, homocysteine, prostaglandins and non-steroidal antinflammatory
agents and HSPG biosynthesis is adversely affected by toxic heavy metals,
excess serum glucose and oxidative stress including that associated with iron
overload.
Heparin anticoagulant therapy apparently demonstrates anti-cancer side effects
(reviewed by Engelberg in 1998) often associated with restriction of tumour
blood vessel growth (angiogenesis).

HSPGs and Ageing.

Binding to, and supramolecular structure induction of collagens by GAGs,
especially by HSPGs, provides very important structural integrity and water-
holding properties. A natural depletion, linear with age, in the content of HSPGs
(combined with the effect of an apparent natural increase in serum ferritin, the
iron storage protein, with age) may even be a major biochemical clock
responsible for the ageing process. (Research into atherosclerosis showed a
linear depeletion of HSPGs with increasing age to occur in total GAGs at human
artery surfaces; perhaps this indicates the ultimate secret of beauty and what
makes skin look young, possibly allowing future dramatic improvement in the
cosmetics trade when such HSPGinfluenced mechanisms of ageing are more
fully understood).
An inverse correlation with age, reported by Aoyagi et al in 1994, shows up in
the blood cogulation and fibrinolysis activities of heparin/HSPG-dependent
urokinase, plasmin and thrombin. Similar effects observed in Alzheimer’s
disease patients suggested that a derangement of such HSPG-linked processes
was accelerated in such degenerative diseases possibly by the effects of trace
redox metal ion overload and pathogen perturbations (e.g. situations leading to
chronic inflammation) capable of tipping the balances involving THSPG
signalling mechanisms.

Our general health, as well as the way we look, can be seen then to depend on
the structural integrity of the GAG-rich rubbery polymers which hold our cells
together in tissue, cartilage and bone structures, and provide binding sites for
protective enzymes and growth factors employed in wound healing and
haematopoiesis.

HSPGs in Antioxidant Defense.

The above reasoning is the basis of antioxidant theories of ageing and of ill-
health. Degradative damage to the extracellular matrices which, like corrosion of
metals or similar degradation of plastics, drastically impairs their performance.
An HSPGbased antioxidant theory of health can be made on this basis. Nutrient
elements used by antioxidant enzyme defense systems include selenium
required for glutathione peroxidase, manganese and iron for intracellular-, and
copper and zinc for extracellular-superoxide dismutases (SO Ds). A newly
discovered selenium-containing antioxidant is probably another endothelial cell
surface, HSPG-bound enzyme.
Since heparan sulphate is a linearly codified information polymer (in this way
analagous to DNA or RNA) even slight damage at key sites (especially N-
sulphonates) may significantly diminish their physiological activities and impair
health. The rate at which such defective HSPG is removed and replaced by de
novo HSPG biosynthesis obviously require to equate in order to maintain HSPG
tissue protective potency. Such processes can, however, be affected by dietary
factors.

Bench Observations Modelling Oxidant & Nitrant Tissue Damage.

Antioxidant protection can be illustrated by a comparison of the effect of
antioxidants on test mouldings containing trace metal (especially iron-binding)
sulphur compounds plus phenolic antioxidant mixtures developed by industrial
chemists in ignorance of the surprisingly similar natural biological antioxidant
systems which have only more recently been discovered by biochemists. After
storage for two years in a light-free drawer an antioxidant containing test piece
retained physical and mechanical properties, but an unprotected test piece had,
dramatically, totally disintegrated into dust.

Similar damage to HSPG polymer systems can easily be observed in vitro

(which is perhaps why, in vivo, these key molecules provide the main blood
vessel wall binding sites for extracellular SOD and other antioxidant enzymes).

HSPGs Need ANTI-NITRANT PROTECTIONS

Nitrous acid is a highly specific and efficient heparin/heparan sulphate
nitrosylating-depolymerization agent. Originally the antioxidant concept of
avoiding tissue damage in ageing and disease was thought to be centred
exclusively on the importance of pro-oxidant (e.g. hydroxyl radical)
damage, but the unusually corrosive nature of nitric oxide metabolites
needs an extention of this notion to include “anti-nitrant” protection.
Nitric oxide (and therefore its metabolites including nitrous acid) can be
critically increased by being induced by cellular (including auto) immune
responses elicited against pathogical stimulae.
Whilst aimed at killing pathogenic organisms such activity may seriously
diminish host HSPG if the rate of replacement of HSPG is less than the rate of
its destruction, as may easily be the case under vitamin and lipid dietary
insufficiency conditions.

Despite the fact that nitrous acid in vitro is well known to rapidly cleave heparan
sulphate polymers into two-sugar unit fragments (c) and this historically well
known reaction continues to be find routine employment in biochemical
laboratories for sequencing of HSPG/GAG chain sugar microstructures and also
provides a faithful test for the presence of heparan sulphate polymers in GAG
mixtures, no chemist in his wildest dreams in olden times would ever have
predicted that such vigorous nitrous acid deaminative cleavage could be of
major in vivo relevance. How wrong we were. Originally it was thought that
nitrogen oxides were only implicated in health as pollutants from car exhausts
and the like, but it has now become firmly established that nitric oxide is used
very extensively throughout mammalian physiology.

Physiological Use of pH for Stabilization of Heparan Sulphate.

Amongst the important reasons for the requirement of maintenance of strict pH
control in human physiology is evidently the prevention of nitric oxide metabolite
damage to HSPGs; “naked” (redox active) metal ions (not easily formed in
healthy subjects but may occur in chronic sepsis or under adherent
macrophages) may override this. Nevertheless the pH 7.4 hydrogen ion buffer
stasis system is seen to be an efficient anti-nitrant defense strategy.

Activated Iron and Copper Ion Catalysis of Deaminative cleavage of

HSPGs.
Vilar et al and Ghael et al, reported in 1997 that deaminative depolymerization of
heparan sulphate could occur at physiological pH when the pH buffer used was
phosphate based. Such a buffer is often (perhaps even always) contaminated by
trace redox metals. The deaminative reaction does not occur in an imidazole
buffer.
Special “nakedness” induced in metal ion phosphate adducts then seems a likely
culprit for causing serious tissue damage but further research is needed to
clarify this.

Iron and copper ions were found by in vitro infrared studies (not yet published)
to remove sulphonate from glucosamine N-sulphonate. This is thought to be the
first stage of deaminative cleavage. It remains to be estblished if other known
redox active ions (especially nickel, manganese, vanadium and titanium) can
behave similarly.
Challis and colleagues in the1970s had found that iron and copper salts were
potent catalysts of nitrosytation of primary amines in studies of carcinogenic
nitrosamine formed by reaction of nitrogen oxides with dietary amines; these
results provide published confirmation of the likely importance of redox metals in
deaminative damage to HSPG systems.
Such metal ion and nitric oxide biochemical effects could also be the basis of the
pro-cancer threats posed by copper-rich betel nuts, and even explain the
carcinogenic effects of asbestos fibres (which evidently involves nitric oxide and
iron-activated-by-silicate-surface effects) as well as the effects of silicate plant
hairs present in the contaminated bread which had apparently been associated
with a previous incidence of increased of oesophageal cancer in Iran.

Redox Status, Nitric Oxide Metabolites & HSPGs.

Nitrous acid can be directly reductively countered in tissue by ascorbate and
reduced glutathione.
Ascorbate will be likely an important factor for control of redox potential but this
substance has a kinetically favourable special efficiency for reduction of nitrous
acid
to nitric oxide (of especial importance to HSPG integrity).
Alpha tocopherol (vitamin E), glutathione peroxidase, catalase, vitamins A and
K, beta carotene and urate are probably further cofactors for such protection.

The effect of naked iron ions and other redox metal ions, may, however, more
specifically negate the effects of ascorbate protection and even lead to induction
of oxidant damage via ascorbate redox cycling aided by complex formation
between ascorbate and redox metal ions.

It had long been suspected that nitrite in meat, especially in hams containing
nitrite-iron adducts formed during the curing process, was a causative factor for
the promotion of stomach cancer, against which a protective role of dietary
ascorbate had been evident (ascorbate is abundantly secreted in saliva if dietary
intake is sufficient) by protecting against the damaging effects of nitrous acid
against HSPGs. Cigarette smoking is also strangely associated with a pro-
cancer nitrous acid dependent nitrosamine formation catalysed by salival
thiocyanate secretion (smokers show serum thiocyanate levels directly
proportional to the number of cigarettes smoked) and thiocyanate iron adducts
may particpate in such nitrosylation reactions (isothiocyanates which show anti-
tumour activity may also particpate in tumour redox enzyme iron centre
complexing in this case leading to an inhibition of tumour growth).
Experiments are required to test if thiocyanate catalyses nitrous acid cleavage
of HSPGs in the presence of redox metal ions.
A greater incidence in men than in pre-menopausal but not post-menopausal
women of upper stomach cancer in which ntrous acid and HSPG biochemistry is
implicated is indicative of a pro-cancer role of iron overload.

Relevance of Nitric Oxide Metabolite Damage to HSPG in the Aetiology of Degenerative

Diseases.
Much research effort has been devoted to establishing the totality of the biological roles of nitric oxide
which, on its own is an efficient antioxidant and a tissue protective agent. The
current interest in nitric oxide stemmed from the realization that nitric oxide is
responsible for blood vessel dilation (a previously identified endothelial-derived
relaxing factor turned out to be this simple two atom molecule; such an
unexpected finding was initially greeted with disbelief); nitric oxide has
subsequently been found to have a host of other physiological applications
(including its use as a second messenger in brain biochemistry). It is believed to
be held under homostasis by binding to haem iron. There are inducible and
constitutive forms of nitric oxide synthsase (NOS), Larginine being the starting
point for its biosynthesis. Nitric oxide, calcium and soluble guanylate cyclase
biochemistries are interlinked.
Nitric oxide was dubbed the “molecule of the year” by the journal Science in
1992.

Relatively few workers have yet, however, fully considered the implicthions of potentially very
damaging HSPG depolyrnerization processes caused by nitric oxide metabolites inevitably associated
with the wide physiological utilization in an oxygen environment of the various nitric oxide
mechanisms1. It is likely that in the course of time the significance of the importance of this reaction in
the aetiology of degenerative diseases will be more generally recognized. It is currently acknowledged
however (e.g. by Salvemini et al 1998) that nitric oxidebiochemistry is strongly
implicated in the aetiology of stroke, multiple sclerosis, Alzheimer’s disease,
amylotrophic lateral sclerosis, Huntington’s and Parkinson’s diseases; it is,
however, generally believed that redox metal ion disfunction is involved in the
aetiology of at least some of these diseases.

The presence of nitric oxide metabolites have been found to be effective

markers for the occurrence of arthritis in patients in which nitrate in urine greatly
exceed ingested nitrate. by amounts which correlate with the severity of the
arthritic diseases (reviewed in 1998 by Stichentoch and Frolich who advocated
the therapeutic use of nitric oxide synthase inhibitors; initial trials of these agents
produced encouraging results suggesting that further investigations into their
effects in other degenerative diseases might be warranted).
Successful intervention in osteoartritis is apparently achieved by glucosamine
therapy which may owe its effectiveness to an increased produced in the rate of
heparan sulphate biosynthesis as postulated by McCarty in 1997.
Dietary glucosamine supplementation coupled with iron chelation therapy as a
couter-measure to degenerative diseases, may also warrant investigation.

Another Damaging Nitric Oxide Metabolite is Peroxynitrous Acid.

Tissue damage arising from peroxnitrous acid formed from superoxide anions
and nitric oxide is prevented by the elimination of superoxide radical anions by
superoxide dismutase. Clusters of tyrosine and cysteine in a number of at-risk
proteins previously discussed by the present author in 1992, may perform a
similar anti-peroxynitrous acid function since the formation of tyrosine nitrate is
an accepted indicator of the presence of peroxynitrite in tissue. It used to be
thought that hydroxyl radicals induced by trace rogue iron ions from superoxide
radical anions were the principal perpetrators of ill-health. Peroxynitrite formed
from the addition of superoxide to nitric oxide is now believed (following the work
of Beckman et al, 1990) to be responsible for much of the DNA and lipid
damage formerly attributable to hydroxyl radicals.
The possibility of such peroxynitrous acid damage to DNA has received greater
literature attention for consideration of possible mechanisms of cancer induction
than have the effects of nitrous acid damage to HSPG which could, however, have greater
potential therapeutic relevance.

Effect of Lead Ions on HSPG Biosynthesis.

Toxic, or less physiologically utilized, metal ions such as lead and cadmium
have also been found to alter heparan sulphate proteoglycan biosynthesis. Lead
intoxication from air and water sources by negatively affecting heparan sulphate
biochemistry may even account for hypertension (Fujiwara et al 1999). The
effect of perturbation of lead, cadmium and perhaps mercury may be
contributory factors acting together with dietary insufficiency of ascorbate in
causing HSPG system disfunction.

Toxic Metals Released from Chitosan by Nitrous add Cleavage.

Rapid deaminative nitrous acid cleavage can occur with other glucosamine
based polysaccharide systems including chitosan (obtained from deacetylation
of exoskeletal chitin) used as a food additive, from which deaminative cleavage
will promote the release of bound trace metals including cadmium and mercury
originating from marine pollution sources. Although such toxic metals are less
dangerous to health when bound to the highly polymeric chitosan, there is a
worry about employment of such materials as food additives under conditions
where nitrite and nitrous acid might be formed (e.g. in the stomach).
Cadmium released from chitosan can diminish glomerular HSPG sulphation and
associated anionic density related filtration efficiency.
Disadvanges of Non-Vegan Diets by Impairment of HSPG Protective Mechanisms.
Non-vegan diets may on occasion be associated with various kinds of
patholgical organisms (and residual chemicals from vetinary interventions) and
even prions, which are, of course, often potentially dangerous to human health
because of the relatedness of our species to the animal species being used as
food. Immune responses elicited against pathological organisms potentially
dramatically increase nitrous acid formation with resultant heparan sulphate
depolymerization and thereby accelerate tissue damage and ageing.
An especially pertinent danger of pro-oxidant stress arises from chronic iron
overload associated with excess consumption of red meat. This is believed to
permit de-N-sulphonation of glucosamine-N-S03- groups priming the heparan
polymer chain for subsequent deaminative cleavage.

The vegan diet, of course, goes beyond the vegetarian diet by avoiding the
additional hazard posed by inappropriate fats and growth factors present in milk
and dairy products of possible involvement in breast and prostate cancer
promotion (as discussed by Professor Jane Plant in her recent book).

The effects of cholesterol LDLIHDL (high density lipoprotein/low

density lipoprotein) on health are now found to be explicable by the
effects of such factors on heparan sulphate biochemsitry e.g. the anti-
atherosclerotic effects of HDL- (containing apolipoprotein E) probably
stems from up-regulation of AT(lll)-binding heparan sulphate
biosynthesis at arterial walls (Paka et al 1999) and saturated lipids
present in meat, if consumed to excess, tend to inappropriately
diminish biosynthesis of heparan sulphate relative to other GAGs. A
further negative lipid-related factor has been identified to be an effect
of oxidised lipids which can similarly potentially negate anti-tissue
protection via heparan sulphate mechanisms.

Non-vegan Western diet by deviating markedly (including from the

required balance of omega three lipids) from that in which our
ancerstor’s polysaccharide biochemistry evolved to deal with,
potentially impairs heparan sulphate biochemistry for optimum tissue
protective efficiency.
Pauling: Vitamin c and cancer.
Linus Pauling, a brilliant scientist, incredibly gained two Nobel prizes.
Future generations may, however, come to more greatly praise his dietary supplementation
discoveries than his chemical or his political accomplishments.
His studies on cancer prevention seem to have been poorly accepted in his lifetime and continue to be
disbelieved since a plausible biochemical mechanism for them was lacking at that time (a suitably
believable mechanism could involve HSPG and nitric oxide biochemistry).
Pauling’s epidemiological evidence of benefit achieved by ascorbate
supplementation against the common cold and cancer appears to be sound.
A conference arranged in 1991 in response to the controversy surrounding
Pauling’s claims somewhat restored public credibility in the main thust of his
contentions. The cancer studies had been conducted in Scotland using previous
clinical records in place of the paralleled double blind placebo experimental
protocol which would perhaps have been more convincing. All patients receiving
the vitamin C supplements apparently benefited. Attempts to repeat these
studies in the USA were unsuccessful but Pauling believed he had an
explanation for this - in the repeat experiments, the US patients died following a
too-sudden withdrawal of their ascorbate supplementation as discussed by
Pauling in his letters to Chemistry in Britain in 1985.
A previous explanation for the anti-cancer benefit of ascorbate had centred on
its known effect in hydoxylation of collagen which tends to inhibit cancer cells
metastasizing. The additional likely effect on heparan sulphate microstructure
and total environment is now seen to be a more significantly wide benefit-
providing mechanism.
Pauling’s results suggest that a vegan diet will tend to counter the common cold
and inhibit cancer.

Ascorbate-induced increase in heparan sulphate activity and protection of heparan activity by

neutralizing the anti-heparan sulphate activity of nitric oxide
metabolites may well be the main reason for Pauling’s findings. Increased
heparan sulphate sulphation and total biosynthetic formation in fibroblast cell
culture experiments resulted from ascorbate additions to the culture medium
(Edward & Oliver 1983, Kao et al 1992). The signifcance of this finding is greater
than it might at first seem as it is likely to occur with various other cell types as it
has been well known for some time that ascorbate was involved in GAG
biosynthesis. Further research into this important research finding and its
relevance to human health is warranted.

Pro-cancer effects of ascorbate have, however, also been reported. Ascorbate

can be shown to increase free-radical production in vitro in presence of redox
metals like iron. Iron toxicity effects might be capable of reducing or reversing
the tissue protective effects of ascorbate. Such iron toxicity has been associated
with cardiovascular disfunction but there may be a dietary link with overtly non-
vegan diets.

Salonen et al : Iron Overload and Cardiovascualr Disease.

A major beakthrough linking iron overloadwith ‘heart attack’, the major killer in
Western society was communicated in 1992 by Salonen and his team from
Finland. The origin of this iron toxicity effect is now postulated to include HSPG
damage at blood vessel walls caused by the effect of nakect iron ions which can
prime heparan sulphate N-sulphonate groups for subsequent deaminative
cleavage as well as promote peroxynitrite and hydroxy radical generation via
Fenton reactions catalysed by such iron ions.
Iron overload, then, which can arise from a red-meat rich diet can, together with
the effects of nitric oxide metabolites (which can arise from dietary arginine via
nitric oxide synthase (induced by the immune response to pathogens or
normally cellularly produced) as well as from nitrite in meat or from that
produced by molybdenum-containing enzymes of mouth bacteria, is capable of
depleting these critical substances by snipping off key N-sulphonate sites or
even totally unzipping the polymers which are involved in blood vessel wall
sensing and antioxidative protection by providing binding sites for superoxide
dismutase and the selenium-containing antioxidant molecules.
Such effects of iron overload are probably countered by a vegan diet.

The Suggested Biochemical Basis of the Benefit of a Vegan Diet.

The results of chemical and biochemical research summarized here may
provide part of the key to understanding why wide-ranging health benfits ensue
from certain ascorbate-rich and lipid-friendly vegetarian and vegan diets
(esspecially if free of natural and man-made toxins).
Ascorbate directly reduces nitric oxide metabolites back to tissue-protective
nitric oxide, but perhaps of equal or greater importance is that this vitamin is
capable of augmenting appropriate heparan sulphate biosynthesis and may be a
cofactor in promoting its tissue protective role.
A similar effect has been found with certain vegetable fats) (polyunsaturates especially). A
boost of heparan sulphate via ascorbate or lipids is the likely outcome of a vegan diet which
has an important additional factor of limiting the effects of ~haemoglobin iron.
It is now dramaticlly apparent that many hitherto poorly-understood
degenerative diseases in which the extracellular matrix is degraded
such as bone disease, apoptosis, tissue damage from chronic
inflammation involving release of growth factors and cytokines could
be potentially linked to inappropriate redox-catalysed degradation of
heparan sulphate by nitric oxide metabolites; these situations are also
highly relevant to the aetiology of cancer as pointed out by Vilar et al
in 1997.
Future 1-leparan Sulphate linked Therapy?
HSPG replacement therapy (e.g. by sulphated polysaccharides
derived from artificially sulphated xylans from birch trees or seaweed
or pharmaceutical compounds like suramin and analogues) may, in
principle, be possible. The current use of dietary glucosamine
supplements, popular in alternative medicine, may be to do with
boosting HSPG protection. Heparin/heparan sulphate therapy seems
to show benfit to Alzheimer’s and other degenerative disease
patients. But a more satisfactory procedure would clearly be to seek
non-invasive protection and correction of aberrant heparan sulphate
systems through a precise monitoring of dietary control of heparan
sulphate status and cofactor disfunction via urinary or tissue sampling
and microstructure analysis. Future medical procedures might even
allow routine monitoring and correction by such methods. This would
need to include a correction of iron (and perhaps other redoxmetals
such as manganese which is required for heparan sulphate
biosynthesis enzymes).

Acknowlegements
I am indebted to help received from former colleagues at the
University of
Aberdeen (especially to Frank Williamson and Bill Long) and I am
grateful to Prof KEL McColl of the Dept of Medicine and Therapeutics
at the University of Glasgow for discussions of his work.

Selected References & Further Reading

Adachi T Marklund SL (1989)

Interaction between human extracellular superoxide dismutase C and
sulphated polysaccharides
J Biol Chem 264 (15) 8537
Adachi T Yamada H Futenma A Kato K Hirano K (1995) Heparin-
induced release of extracellular dismutase form V to plasma
J Biochem (Tokyo) 117 (3) 586-90; cf also Adachi et al (1998) Biol
Pharm Bull 21
(10) 1090 CA 130 64588h
which reported decreased extracellular-superoxide dismutase in
patients with coronary artery atherosclerosis

This list of references is truncated since a similar list is given

with document 1 (pages
Update draft for document 2




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DOCUMENT 3




DOCUMENT 3A




















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Document 4

Outline draft of a paper

Anionic Polysaccharides (Including Heparin/Heparan Sulphates) May Function as Multi-Inorganic Ions Ligand
Systems

Communication from David Grant, PhD (formerly of the Dept of Soil Science and the Polysaccharide Research Laboratory,
Marischal College, University of Aberdeen)
(written 1/12/06 following helpful correspondence with Professor RJP Williams,University of Oxford)

Summary
An important aspect of heparin/heparan sulphate (HS) biochemistry, of relevance to the development

of theories of how this ubiquitous animal protein management system operates in vivo, is suggested to

be the ability of heparin-like polyanionic systems to switch between electrostatic and non-electrostatic

mechanisms of counterion binding which enables ligands to be bound fairly weakly and thereby engage

in rapid site interchange, and allow a rapid transference to higher affinity protein binding sites, or

to be bound with greater strength and specificity so as to act as a detoxifier, an antioxidant or create a

stable reservoir of (e.g. essential nutrient) metal ions and other inorganic elements. The latter function

surprisingly resembles that of polyanionic humic polymers in the soil, which similarity it is now further

suggested to be biologically relevant to the overall role of polysaccharides in biology as well as to the

possible involvement of such polymers in the early stages of biological evolution.

Introduction

The voluminous literature reports on heparin/heparan sulphate (HS) are combined with previously

incompletely reported data from a discontinued academic laboratory (dedicated over some thirty

years to studies of the binding of polysaccharides to metal ions) to support the notion that HS provides

for animal biochemistry a unique ultra-wide-ranging control system which is designed to act a

relay system between the environment and intracellular compartments and especially interacts with the

binding and processing of second messenger Ca2+ by which HS modulate the activities of many

proteins including growth factors involved in wound healing and assembly of the embryo.

Another important second messenger is nitric oxide which cleaves HS at unsubstituted C-NH2 groups

of glucosamine residues in HS. This reaction is also subject to metal ion activation.

Metal ions could be the absolute cofactors for all HS actions. This has already been established in a

number of cases.

The known scope of apparent HS functions now encompasses such a wide range of animal

biochemical systems that HS must now be considered to be the prime candidate for construction of

post-neo-Darwinism hypotheses of the mechanisms by which animals control gene expression during

differentiation, development and evolution. These could have an especial input from

HS oligosaccharides generated non-enzymically by nitric oxide metabolite reactions (which may exert
a hormonal like control somewhat analogous to actions of oligosaccharides in plants).

The inorganic ion sequestration abilities of the HS system are now suggested to also confer antioxidant

protection, and may also be an essential part of the wide-ranging molecular chaperone activities

relating to protein folding including the inhibition of pathological prion formation but much remains to

be learnt about how inorganic components might influence such events.

The differential binding of metal ions by HS and HS-oligosaccharides is therefore a suitable platform

with which to explore a number of such outstanding biological puzzles as well as individual situations

e.g. the bFGF binding HS epitopes which seem in vitro not to discriminate between

isolated oligosaccharide binding sites selected on evidence that they perform such selection in vivo

under multi-metal ion biolgical fluid bathed whole HS-molecule milieu.

An unsuspected critical influence of metal ions on this process might be the reason for this apparent

paradox.

This is a particular example of the problem of what constitutes the correct physiological buffer for

laboratory studies.

The Role of Water Aggregates on Biological Functions

(How Important are the Ultradilute Aqueous Solutions of Non-Physiolgical Metal ions in Blood Serum & Biological
Fluids?)

The general roles of metal and inorganic ions and their ligands is part of the wider subject of the

supramolecular structuring of water. It is always noted that water is some 70% of viable cells. This

water when studied by vibrational spectroscopic methods, reveals a strong interdependence on water-

aggregate absorptions on temperature (cf is apparently especially sensitive at the temperature range

employed by homoisothermic animals), pH and the presence of inorganic ions which, per se, may have

a major influence the folding of proteins in physiological solutions as a consequence of altered solute

microstructure. Such supramolecular water structures are the apparent origin of the Hofmeister series

of salts etc. for protein denaturing (involving irreversible folding, as well as reversible folding which is

the ultimate origin of how HS and other chaperone systems modulate protein activity and eventually

multicellular assembly. Metal ions are obviously bound so tightly with high affinity to such

evolutionarily conserved proteins as calmodulin but requires a much weaker but adjustable highly

low/high affinity flexible binding mechanism evidently afforded by polysaccharides by which to

modulate this folding.

Why biological and inorganic morphologies seem to have some common controlling factors.

It should be noted that biological polysaccharides are highly efficient modulators of inorganic (e.g.

CaCO3) crystal growth and assembly. The reason could be to do with how nucleation of crystallization

can be modified to control morphology. Growth of crystals from an aqueous solution has been found

to have much in common with the self assembly of cells into organs and organisms. This process may

be initiated in both instances by control of nucleation events which create template structures.

The interaction of inorganic ions with polysaccharides and nucleic acids and the hormonal effects of

HS oligosaccharides as selective modulators of chromatin binding to DNA.

Aspects of such interactions can give new insight into the current debate on whether

evolution occurs via A) random DNA mutations (neo-Darwinism) or B) non-random DNA alteration.

DNA, at least in eukaryotes, is apparently so constituted so as to vigorously resist change and to do so

requires the cooperation of other major influences which are known to include inorganic ions and may

also involve the effects of inorganic ion binding to the non-nucleic acid polyanionic system (which

include polyphosphate, polyβ hydroxy-butryate and glycosaminoglycans GAGs) the principal candidate

for which at present for this action is the most complex GAG, the HS, a system which, as noted above may

be strongly influenced by purely inorganic processes involving metal ions (principally, Na, K, Ca and Mg,

but also Cu and Zn) and nitric oxide and its metabolites.

Heparin can be used as a convenient model for HS

Heparin acts as a multi-element ligand in such a way that metal ions can be safely predicted to

probably greatly influence HS biochemistry.

Unrefined heparin turned out to contain a seawater-like array of inorganic ions. This was also the case for a sample of
heparin refined by a conventional single counterion enrichment process. Other polysaccharides seem to have similar arrays.
[Confirmatory reports from other laboratories of this seemingly fundamental property of anionic polysaccharides are at
present relatively few and such reports cover at present only a small fraction of the seawater-range of elements present; the
confirmation of a likely similar binding of multielements by heparin and HS comes from in vitro and in vivo competitive Ca
binding studies in the context of scintigraphic analysis of pathological lesions by Ga-HS].

The mechanism of homeostasis of seawater elements in blood serum is a fundamental biological puzzle.

Evidently the precursors of modern multicellular animals evolved first of all in the sea and for

unknown reasons apparently needed to strongly conserve the full elemental composition of that sea
within their biological fluids. In the early stages of animal evolution it can be proposed that in addition

to the protein-based metal ion channels and hormonal-controlled systems now in place in modern

animals, a more primitive ancestral animal metal ion and pH buffering mechanism was based on the

actions of the cell surface anionic GAGs including HS system (and could explain why modern aquatic

organisms seem to preserve the need to synthesise different amounts of these polysaccharides

according to the salinities of their habitats). This type of phenomenon can be further demonstrated by a

relatedness between such elemental composition and the amounts present in biological matrices.

Examples are human hair, alginate-rich seaweed biomass and unrefined heparin.

The existence of such an inorganic array in heparin provides an indication that similar constitution of

HS when equilibrated with extracellular fluids.

The Mechanism by which Heparin Acts as a Multi-Element Inorganic Ion Binding Matrix

The polyelectrolytes heparin, DNA and humic acids have been suggested to behave as purely

electrostatic counterion collectors by a mechanism proposed by Manning which does not discriminate

between equally charged ions, i.e., the Irving Williams series will not be obeyed.

Heparin seems, at least under some circumstances also to have the ability to bind counteions by a

“regular” Irving Williams mechanism in keeping with the wide validity of this rule throughout

chemistry, biology and geology.

The full multi-element binding characteristic were found to display a curious reverse-order binding

behaviour in which those elements present in biological and natural water environments are bound with

greater affinity than those which occur most abundantly. The latter tend to obey Manning, the former

non-Manning behaviour. This may be due to the designed heterogeneity of heparin structure with

minor sites having being evolved to accomplish covalent interactions with ultra-trace metal ions while

major sites tend to bind mainly by electrostatic ionic force linkage. But even here departures from a

pure electrostatic binding behaviour are evident. Although the Manning hypothesis is a good

approximation to metal binding to heparin, and also for comparison of polyanionic substances with

different anionic densities, each metal ion essentially behaves somewhat differently from that

predicted by this theory.

It is instructive to deal with the alkali metals, alkaline earths , transition metals and lanthanides etc.

separately. In each case the more abundant elements are present in approximate amounts predicted by the

Manning theory (i.e. there is no selectivity except by chemical charge) but the least abundant elements in

amounts in accord with selective covalent binding selection according to the Irving Williams series.

The presence of apparent non-physiological elements and ions in heparin, blood serum and seawater is

of some interest as is how this apparent stable buffered system came into being in the first place and

how it is currently maintained.

A major contributory role of polyanionic substance (including humic matter in natural waters and
anionic polysaccharides in biological fluids) is now suggested. Whilst this idea is not new, the relevance of the uniquely
high efficiency of humic fractions as inhibitors of CaCO3 has not been widely discussed, and incompletely reported data
which is now supported by similar data now available for humic fractions from a range of environments strongly support the
humic quasi-polysaccharide hypothesis of the homeostsis of supersaturated multielement solutions in natural waters.

There are hints of a common chemical mechanism underlying both the geological and biological

systems whereby supersaturated Ca2+ aqueous solutions are stabilized against the thermodynamically

required solubility product precipitation process by the inhibition of e.g. CaCO3 precipitation.

ANIONIC POLYSACHARIDES AND HUMIC MATTER (WHERE ANIONIC SITES ARE

GENERATED BY ARRAYS OF CARBOXLATE GROUPS) appear to share such a common

mechanism as demonstrated by laboratory experiments. This further could suggest a common pre-

biotic evolution of both systems (and also an important influence of ‘humic matter’ as a key ingredient

of inorganic and pre-biotic organic morphology (i.e. the self assembly of complex forms which in some

cases also possessed the ability to disintegrate similarly to the spontaneous behaviour of some kinds of

inorganic crystals (e.g. sodium thiosulphate hexahydrate) or semi-amorphous colloids so that

reproduction of form was possible) in the pre-biotic stage of the evolution of biota.

The importance of this sort of organic polymer (but not yet non-protein-based) system as a barrier to

ionizing radiation and as a buffer for inorganic ions may have been important for early biological

evolution. It is evident that as nucleic acids are composed of sugars and inorganic phosphate that the

polysaccharides must have been in place certainly prior to the evolution of nucleic acids and probably

also of proteins. The inorganic components of the early seas probably also included amorphous

inorganic colloids e.g. silicic acids which would have preserved polysaccharide systems and may

account for why such materials remain as ubiquitous component of modern polysaccharides.

What is also apparent from laboratory experiments is that the polysaccharide polyanionic buffer
systems may still exist as contributors to biological fluid homeostasis e.g. at vascular walls (the

inhibition of pathological calcification) and urinary system (the inhibition of Ca oxalate formation).

Suggestion for Further Work

Studies of HS epitopes should consider whether single metal counterion Na+ system commonly used, is an appropriate
model for a biological fluid system which produces an ultratrace enrichment of the multi-elements present in biological
fluids. Such enriched multielemnt forms of HS epitopes may be the correct forms for such studies

Animal evolution is suggested to be a process which could principally depend on interactions between the

environment and HS further relayed to the nucleus. This could provide the intellectual framework of a new

theory of animal evolution which circumvents major difficulties inherent in the neo-Darwinism concept of a

slow random DNA mutation process.

Current thinking tends to exclude polysaccharides from debated mechanisms of possible biological

evolutionary processes which might involve metal ions.

This discussion document draws attention to how polysaccharide-like substances which simply from

chemical logic (i.e. the ease of their abiotic formation by the formose reactions which could have

created a primitve humic polymer, evolutionary related in the abiotic earth to similar environmentally

stable multi-metal ion binding polymeric (CH2)n>32(H-C-OH)m (C(OO-)(SiO2 etc)) structures which

are the core polymer units in present-day humic substances which later might have been evolved within a

primitive organism into the first biopolymers- polysaccharides by polymerization processes aided by their

hydrogen bonded binding to inorganic crystals, a process which is also responsible for the creation of multi-

element omeostasis in natural waters (the biochemical actions of such substances reported in a now

voluminous literature) must be prime candidates (rather than primitive nucleic acids or proteins) for the

original biopolymers. Such a central role which may have been played by polysaccharides in biology

has been obscured by the enormous advances made in nucleic acid and protein fields which has

inevitably ascribed functions to these other biopolymer systems which are actually, it is now suggested,

determined by polysaccharides.

______________________________________________________________________________________
DOCUMENT 5 (Cf. Document 1 which is a later edition of this)

Preliminary Draft of a Discussion Document by David Grant Ph.D. (Turriff, U.K.)

(Continuation of University of Aberdeen Research* On Roles of Inorganic Cofactors in H/HS Signaling)
Inorganic (“Metallomic”) Cofactors Could Determine Heparin & Heparan Sulphate Activities
The animal anionic polysaccharide heparin can simultaneously bind the range of inorganic elements which occur in
seawater.
Transmission of information-encoded in heparan sulphates in the form of specific anionic sulphate half ester
patterns present in substituted glycuronic-glucosamine disaccharide repeat sequences may be dependent, at least in
part, on the co-occurrence in these highly anionic polysaccharides of Ca, Mg, Mn, Cu and Zn as well as a range of
other inorganic elements including those (e.g. Si, Al, As, Pb, Sr and the rare earth elements) which have been
tentatively implicated to have normal roles in animal biochemistry, and dyshomeostasis of which may promote
degenerative diseases by perturbing their putative inorganic cofactor heparan sulphate dependent biochemical
signaling functions.
Contents
1 Summary
2 Introduction
3 Review of studies of relevance to a fuller understanding of how metal ions could affect H/HS signaling
3-1 The Long-Williamson hypothesis that a primary function of HS is for control of Ca2+ activity
(This can now be updated to suggest the reverse hypothesis that Ca2+ and other inorganic ions modulate HS activities)
3-1-1 The multi inorganic elements associated with H
3-1-2 Re-evaluation of old SSMS data for H

3-2 Possible key roles of metal ions in the physiological nitrite cleavage of H/HS
3-2-1 Early studies of metal ion effects in nitrosative processes

3-3 Water activity model of metal ion dependent H/HS signaling

(Metal ions may participate in H/HS signaling in part via modulation of water activities at H/HS surfaces) (Evidence for water
adhesion forces in the anticoagulation mechanism of H)

3-3-1 Inorganic ions and particles associated with H/HS surfaces may regulate the Wiggins low density/high density water
microstructure balance
3-3-2 Electrostatic (Manning) interactions between H/HS and counterions etc. may be augmented by water structure adhesion forces
3-3-3 Evidence for water adhesion forces in the anticoagulation mechanism of H and in the binding of poly-L-lysine and poly-L
arginine to H

3-3-4 Countercation-specific binding to –C-(O)O- groups in H

4. A putative H/HS inorganic ion environment interface

4.1 Table I Involvement of metal ions in H/HS interactions thought to be involved in blood anticoagulation, breast cancer,
basic fibroblast factor dimerization, annexinV binding, endostatin (collagen XVIII), collagen V and prion
interactions, as well as the activities of a range of other proteins and the nitrosative scission of H/HS
4.2 Table II Reports of (apparently direct) modulation of HS microstructure in response to the presence of inorganic ions etc.

4.3 Table III Some examples of reported effects of organic molecules on HS

5. Comparison of ‘metallomic’ multi-inorganic-element arrays in anionic polysaccharides with those in other matrices
5-1 Examples of ‘metallomic’ arrays associated with H, kelp & human scalp hair
5-1-1 Table IV Inorganic elemental contents (mg/kg) of kelp, H & human scalp hair
5-1-2 Table IVa Comparison of inorganic elements in H and chitosan
5-2 Log-log interrelationships between multi-inorganic element contents
5-2-1 Inorganic elements: seawater compared with H

6. Relevance H/HS biochemistry to disease

6.1 Possible pathological perturbation of inorganic signaling by H/HS by metal ions
6.1-1 H/HS biochemistry & degenerative diseases
6.1-2 Possible perturbation of H/HS signaling by Ba2+ in multiple sclerosis
6.1-3 Possible perturbation of H/HS signaling by Al3+ , Zn2+ (and possibly Cu2+/+) in Alzheimer’s disease
6.1.4 H-mimetic Pentosanpolysufate: putative anti-misfolded prion and potent HIV antiviral activities
6.2 The defective nitrosative cleavage which has been indicated to occur in Niemann-Pick Disease could also be relevant to the
etiologies of cancer and other degenerative diseases

6.3 Does HS provide for a servo feedback system which directly interfaces the environment?

6.4 Alternative and ethnic medical use of metallomic matrices.

Can some of the non-anticoagulant suggested uses of H and claimed benefits of Ayurvedic Shilagit etc. be dependent on the co-
occurrence of similar ultratrace elements in these preparations?

6.4 Interference from inorganic constituents in H during blood analysis

Interference from multiple inorganic constituents associated with H used as an anticoagulant during determination of inorganic
elements in blood

7-1 Variation between manufacturers of the degree of final purification of H

8. Binding of anionic polysaccharides to crystal surfaces

8-2 Specific nucleation activities (e.g. afforded by natural ‘SiO2’ nanoparticles) may be required for supramolecular structure
formation in polysaccharides and be relevant to how inorganic elements contribute to H/HS signaling

8-3 Nucleation events (subject to potential inhibition by H/HS) may determine the progression of
amyloidoses

9. Could HS signaling generate a systemic biological clock which is speeded up during degenerative disease processes

9-1 HS as a proton pump

10. H/HS as a mimetic of humic polymer buffers in natural waters

11 References

11-1 References
References arranged according to first-named author

‘Ref. 1’Alphabetically arranged according to the first named author

11.2 Further references (and notes) referred to by number

and also arranged according to subject matter
and
11.2-1 The metallome-heparanome (References: metal ions in H/HS signalling)

11.2-2 Ref 1a-1

Recently published discsussions of how inorganic ions might participate in H/HS signalling

11-2-2 6-O-Endosulphatase processing of HS

Further references (1a1-14; 1b-1h)

Refs 6a,6a-1, 6b-,. 6b-2,6b-2-1, 6b2-2, 6b2-3; 7-9, 9b-3, 9b-3-1, 9c; 10-13, 13a; 14-17

1a Antioxidant & antinitrant activity (related to modulation of superoxide dismutase by H/HS) and
sequestration and deactivationofredox active iron, coper and silver ions, etc., by H/HS

1a-1 Published discussions in peer-reviewed articles of how inorganic ions might participate in H/HS signaling
1a-2 How metal ions are currently known to assist H/HS signaling
1a-3 Degenerative diseases: putative roles of inorgnic elements in H/HS signaling
1a-4 H and atherosclerosis
1a-5 H and dementia
1a-6 Antioxidant & antinitrant activity (related to medulation of superoxide dismutase by H/HS) and
sequestration and deactivation of redox active iroj, copper & silver ions, etc. by H/HS

Refs 6a,6a-1, 6b-,. 6b-2,6b-2-1, 6b2-2, 6b2-3; 7-9, 9b-3, 9b-3-1, 9c; 10-13, 13a; 14-17
12 Concluding Remarks
12-1 Suggestions for Further Research
1. Summary
Of a range of natural polyanionic substances for which multi-inorganic-element analytical data are

available, heparin, perhaps because this is the most anionic biopolymer, has been found to be uniquely

capable of sequestering multi-inorganic elements and therefore seems to offer an optimal model system for

the study of biological metallomics (a branch of science which is concerned inter alia with

interrelationships between geology and biology). The putative existence in heparin of a characteristic

metallomic matrix was suggested by the spark source mass spectrometric (SSMS) analyses of heparin

samples conducted during multidisciplinary researches into of the inorganic biochemistry of heparin (at

Marischal College, Aberdeen, U.K.) during the1980s. At that time it was uncertain whether these results

were generally relevant to all heparin samples or represented simply an accidental contamination e.g.

during industrial processing. Later results confirmed the notion that multi-inorganic elements always

‘decorate’ heparin.

If the ability to bind potential inorganic cofactors found with heparin is also replicated with heparan

sulphate this then could suggest a new paradigm of metallome-heparanome crosstalk. Metal ion modulation

of the activity of the clusters of water molecules associated with the sulphate half ester groups of heparin

and heparan sulphate can rationally be suggested to play a critical role in the biochemistry of these

polysaccharides.

Keywords: Heparin, heparan sulphate, the heparanome, metallomics, degenerative diseases, cancer, multiple sclerosis,
Alzheimer’s disease, prion misfolding diseases, water structure, biological clock, inorganic ion contamination of heparin for
determination of inorganic elements in blood serum
2. Introduction

Heparin (H) has traditionally been used as a convenient laboratory model for the study of the animal

“heparanome”a , which is believed to regulate numerous protein activities by a signalling process which

depends mainly on polysaccharide-side chain microstructures, rather than some general proteolgycan core-

protein-based (cf., Iozzo1) recognition system, which select between target proteins by a range of

differently anionic patterned epitopes, the synthesis of which, e.g., during development and wound healing,

is apparently strictly regulated both temporally and positionally; the library of such epitopes which occur

in H allows this protein and nucleic acid free pharmaceutical polysaccharide mixture to serve as a

convenient laboratory model of the heparanome.

An additional, highly characteristic physical chemical feature of H, however is the apparently unique ability

of this ultra ionic polysaccharide system to acquire a cosphere containing a wide retinue of inorganic

ions and particles (cf., e.g. Jaques1 ; Long1(2003)). Haraguchi1 has recently suggested that the science

of metallomics could be highly pertinent to biochemistry, which may especially pertain to how H and

related polysaccharides depend for their biological functionalities upon the presence of correctly formulated

Haraguchi-metallomic cospheres.
a
J. Turnbull et al. TRENDS in Cell Biol. 2001, 11 (2) 75-82, suggested the use of the term ‘the heparanome’ for the new concept of the dynamic expression
of HSs with differing sugar sequences produced by particular cells or tissues from a precursor in which glucuronic acid-N-acetylglucosamine units are
subjected to structural alteration in the Golgi apparatus by sulphation (by 2-O, 6-O and 3-O- sulfotransferases) and epimerization (C5 epimerase) and
postsynthetic de-N sulphonation and scission by nitric oxide metabolites and also by the action of 6-O-endosulphatases. These processes also allow for
environmental - HS microstructure crosstalk. L. Kjellin & U. Lindahl (Annu. Rev. Biochem. , 1991, 60, 443-465 in a review of the structure and function
of proteoglycans of which HS are prominent members, listed these as: provision of mechanical support, negative charge, regulation of cell migration and
aggregation, roles in the development and stabilization of synaptic structures, endothelial regeneration, stabilization of basement membranes, modulation
of collagen fibrilogenesis including the transparency of the cornea, regulation of cell growth, urinary trypsin inhibition, provision of a filtration barrier, role
in morphogeneis, link to cytoskeleton and ECM, mediation of adhesion and morphogensis, assembly of matrix phosphatidyl–inositol linkage, reservoir for
basic fibroblast growth factor, regulation of blood coagulation, mediation of transferrin functions, and uptake of antigen presentation. The initial step in
biosynthesis is the formation of the protein linkage region GlcA-Gal-Gal-Xyl-Ser by transfer of xylose from the UDP-xylose to specific Ser residues in the
core protein. The same linkage region occurs with HS and chondroitin sulphate had been highly conserved “from flies to humans”. Bernfield et al. ibid.,
1999, 68, 729-77 also provided an extensive review updating similar studies of HS systems (the most common members being now known to be
glycoylphosphoinositide-linked glypicans and transmembrane syndecans) which provide the most abundant receptor system at adherent animal cell
surfaces for tissue morphogenesis and wound repair as well as for host defense and energy metabolism) via interactions with the extracellular matrix, e.g.
to control growth factors.
Perhaps the most fully understood epitope in H/HS is the pentsaccharide antithrombin-binding site which occurs most characteristically in mast cell H and
in some HSs. Pharmaceutical H, which has been used for many years as a blood anticoagulant, is now available in a variety of salt and molecular weight
forms which includes the single epitope, an artificially produced pentasaccharide molecule as well as unfractionated H (‘Na heparin’) from porcine mucosa
(but also from bovine lung) which derives a major part of its anticoagulant activity from the antithrombin epitope content.Polysaccharides could also
contribute to the essential protective processes of animal plasma membrane hydration and water activity regulation and also modulate normal and
pathological calcification and by binding unliganded redox metals such as Fe3+ which will confer antioxidant and antinitrant protection). Altered HS
biosynthesis seems to occur in response to changes in Ca2+Mg2+ and Mn2+ as well as ascorbate, glucose and a wide range of other dietary factors
concentrations (those which promote health tend to boost the biosynthesis of HS of increased its degree of sulphation whilst those which are detrimental
to health seem to diminish this).

H/HS Putatively Occur Naturally as Mixed Salt Forms with Complex Hydration Patterns Associated with Sulphate Half-Ester Groups
HS proteoglycans (e.g. glypicans, syndecans, agrin and collagen XVIII) must it can be rationally suggested, exist in vivo in mixed salt
forms similar to H. [Evidence for this hypothesis is further developed later in this communication].
The functions of the mixed salt forms, like man-made ionomers based on arrays of –SO3- groups will depend critically on the inorganic
counterions which induce a functionally important associated water cluster systems**. It had been previously reported (Nader et al.
 that the amounts of HS and other sulphated polysaccharides present in fifteen species of aquatic animal organisms increased with
1(1983))

the habitat salinity. This was thought to be a requirement to inhibit high ionic strength induced tissue dis-aggregation, but it is now
suggested that these organisms may require the correct inorganic seawater constituents including similar amounts of Na+ and
polysaccharide–SO3- to form the correct membrane HS water cluster system. Apart from the principal ions needed to form the
polysaccharide - SO3- Na+ (H2O)6 array system both natural as well as man-made ionomer functions may require a form of doping by
seawater ultratrace inorganic components (e.g. doped SiO2 nanoparticles).

In 1984, C. M. Moffat1 a graduate student working with W.F. Long and F.B. Williamson (directors of the

then Marischal College (Aberdeen University) polysaccharide research laboratory) and with Macaulay

Institute (Aberdeen) scientists including J. Bacon, a specialist in multi-inorganic element analytical

techniques, obtained a spark source mass spectrometric (SSMS) analysis of carefully selected sample of a

typical porcine sodium H (which had been extensively dialysed against deionised water by Moffat but

apparently had not originally been ‘cleaned-up’ by the manufacturer e.g. using the standard final ion

exchange purification process; this might have been a deliberate omission since this H was specifically

allocated for fundamental biochemical studies (and not designated for medical use). This H

was found to contain a type of multi-inorganic-element profile similar to those which were later discussed

(and termed metallomic matrices) by Haraguchi who emphasised the occurrence of a similar range of

major, trace and ultratrace inorganic elements in oceanic seawater and human blood serum.

3 Review of studies of relevance to a fuller understanding of how metal ions could affect H/HS
signaling

3-1
The Long-Williamson hypothesis suggested that a primary function of HS is for control
Ca2+activity
This can now be updated to suggest the reverse hypothesis that Ca2+ and other inorganic ions
modulate HS activities

In plants, anionic pectin derived oligosaccharides are thought to modulate Ca2+-dependent signaling

processes 1a-8, in a manner similar to how Ca2+ activity in animal biochemistry was earlier suggested by

W.F. Long & F.B. Williamson1 to be regulated by HS. It was found that the saturated Ca2+ binding to H

was accompanied by a NMR detectable conformation change suggesting that the biological activity of H

could be highly dependent on the presence of Ca2+ (Boyd et al. 1). Earlier X-ray crystallographic studies

(Gould et al.1) had also suggested that Ca2+ ions (and also Na+ ions), by forming coordinate links between

water molecules, uronate groups, and sugar oxygens in cell wall polysaccharides could generate complex

supramolecular structures, e.g. of the “egg box” type, (cf., Grant et al.1 and Seale et al.. 1) ; a related form

of supramolecular structure generation by metal ions and water molecules might also explain why HS

proteoglycans can self -associate under appropriate conditions (cf., L.A. Fransson & B. Havsamark1) and
perhaps also explain why proteins in Ca2+ containing biological fluids often bind selectively to HS epitopes

in a Ca2+-dependent manner (cf. Table I).

Hints that other inorganic elements than Ca2+ might participate (e.g. in pathologically induced alterations) in

H/HS activities were suggested by the reports of Z. Liu and A.S. Perlin who showed 1c that Cu2+ caused

selective free-radical degradation of H and, in the related study by R.N. Rej et al 1d, which found that trace

amounts (ca. 1/185 mol ratio with respect to H) together reactive oxygen containing free-radicals generated

from H2O2, plus ascorbate, caused a marked reduction in the anti-FactorXa activity of H without causing

any detectable alteration in the NMR spectrum of the polysaccharide.

3-1-1 The SSMS Determined Multi-Inorganic Elements Associated with the Na H (Moffat1) Selected for Fundamental
Biochemistry Research
Listed in the order of their abundance (in ppm) : Na (96,000), Ca (30,000), Si (5900), Cl (5600), K (2000), Mg (1300), Fe (1100), F
(890), Cu (730), P (440), Ti(390), Ta (280), Ni (170), Ba (140), Br(130), Co(80), Zn(80), Sr(65), Cr(30), B(25), Ga(20), Pb(16),
As(15), I(10), Cs(9), Tl(8), La(7), Ce(7), Mo(7), W(5), Sn(5), Nd(5), Zr(5), Ag(4), Rb(3), V(3), Y(3) , Sb(2) and Cd(1).

When the above multi-inorganic H was treated with a standard cation exchange resin procedure the

resultant single counter-ion enriched H showed by SSMS that an effective replacement of these elements by

the selected single counter-ion had occurred (cf. Grant 1 (1987)); this procedure produces a form of H similar

to the types of H commonly used in medicine as well as laboratory model for HSc.

This ‘sodium’ H had been donated to this and to other academic laboratories by a (former) major

manufacturer (from factory based in Runcorn, U.K. )) had been used to prepare a ‘single

counterion’ thallium Hd by cation exchange achieved with a sulphonated polystyrene resin (Rohm & Hass

Amberlite IR 120) column substituted with Tl+ (Moffat1). The SSMS analyses of the starting Na-H and the

resultant Tl-H samples indicated that a single passage through this type standard cation ion exchange

column achieved replacement by Tl+ of more then 99% of the principal ‘contaminant’ inorganic (mainly

cat)ions which had been present in this (evidently highly ‘contaminated’) starting commercial ‘sodium’ H

(cf. Grant 1(1987)). (A similar effectiveness of use of ion exchange resin columns for the removal of the Al

which is present in some commercial H samples has recently been confirmed by Bohrer et al., 1).

3-1-2 Re-Evaluation of the Moffat et al. SSMS Data for Na H

A re-evaluation of the old Aberdeen (Moffat et al) SSMS data (by Grant 1 (2000)) indicted that the ion

exchange ion replacement procedure had skewed but essentially retained a distinct original multi-inorganic

element matrix of the starting Na-H, in the Tl -H, albeit this was greatly diluted with a greatly enhanced

presence of Tl+ couterions.

These data, when compared with those from earlier inorganic analytical reports and (somewhat more

accurate ICP-MS) mass spectrometric multi-element analyses data later reported for further H samples

confirmed the notion that H and H-like polysaccharides always occur in vivo as multi-element

organometallic composite mixtures, which, even after extensive purification, retain a skewed

remnant polyinorganic profile related to the original ‘native’ H metallomic profile (which approximately

correlates with the metallomic profiles of seawater, human blood serum and many other biological

metallomic matrixese and hence suggests the co-existence of an abundant metallomic array with H/HS
polysaccharides in vivo.

c
Some indications that such ‘single chemical substance’, ‘single salt forms’ of H, having been depleted of their natural multi-inorganic-element
components, are inappropriate models for biochemical researches into H/HS signaling were obtained during studies of antioxidative and related
properties (e.g. the activity of pure seemed much less than native H for ferrioxidase activity, but further research is required to fully establish
this).

d
This counterion was thought to be of especial interest because it enabled 205Tl NMR studies.

e. Other putative metallomic matrices are constituted by anionic phosphate groups which include apatite minerals and hydroxyapatite composites of bones
and teeth and the high molecular weight polyphosphates which have been reported Kornberg (ref. 16) to occur at cell surfaces throughtout biota; the
association of P with heparin may indicate the occurrence of phosphate-bound metals here also; polyβ hydroxybutrate which is also apparently present (in
a similar to anionic polysaccharides) at a the cell surfaces of a wide range of species.

3-2 Possible Roles of Metal Ions in the Nitrite Cleavage of H/HS

Vilar et al reported1e the unexpected ability of nitrite to deaminatively cleave H under physiologically

relevant conditions (cleavage occrred when the reaction was performed in the presence of

phosphate buffer but not when an imidazole buffer was used; the critical factor here could be

that the former buffer ubiquitously contains trace amounts of redox metal ions). Hitherto it had been

thought essential that highly acidic non-physiological conditions were essentially required to achieve

nitrosative scission of H/HS, which was known further to require the presence of unsubstituted

glucosamine –NH2 groups in the H/HS chains, which were also further believed not to exist naturally in

H/HS under in vivo conditions. Later work found that these assumptions were incorrect 1f . It has now

become fairly well established that a major HS signaling process occurs by the controlled insertion into HS

of unsubstituted glucosamine groups primed for nitrosative cleavage under slightly acidic conditions in

response to biochemical stimulae; these are apparently coupled to servo feedback coupled microstructural

alteration of HSf. Such H+ and/or redox-metal-ion-dependent non-enzymic transformations apparently

also directly primes for nitrosative cleavage by promoting the (heparan)-N-SO3àNH2reaction 1g,h .

Redox metal ions (as well as Ca2+ and Zn2+) present in the metallomic profile of H/HS could, it is now

proposed, facilitate nitrosative scission of HS which is now believed to major animal protein control and

signaling system which generates ‘hormone’-like HS oligomer messenger molecules which engage in

such tissue protective and other functions. This type of nitrosative cleavage which has been researched by

the L-A Fransson et al. group (cf. Mani et al.; Ding et al.1) and has been found to be relevant to the
etiology of Nieman-Pick Disease [vide infra, section 6] could also be relevant to the etiologies of cancer

and other degenerative diseases a mechanism which is, however, vulnerable to pathological perturbation by

toxic metal ions (cf. Grant 1 (2000, internet))..

f HS-based dynamic servo-feedback control process seem also to be dependent on the regulation of HS microstructure by HS-6-O-endosulfatases. (cf. Dai
et al. 1)

3-2-1 Early Studies of Metal Ion Effects in Nitrosative Processes (Role of Metal Ions)

A traditional, highly effective, method of identifying HS in a mixture of polysaccharides is by reaction

with nitrous acid which is highly specific for H and HS, being widely used in the laboratory to show their

presence in proteoglycan mixtures. This was originally thought to be an entirely non-physiological

action. Nitrite/nitrous acid was also known to promote stomach cancer, an effect which had been

attributed to a pro-cancer mutagenic action on DNA. That nitrite/nitrous acid was such a highly specific

target reagent for a major HS animal cell surface receptor should perhaps have alerted researchers

to the possibility that perturbation of HS biochemistry might have accounted for such pathological

actions of nitrous acid, but this only became a credible hypothesis following the later, unexpected,

discovery that nitric oxide is actually a major in vivo messenger molecule employed in animal physiology.

N.b., nitrite is a primary metabolite of nitric oxide. It was further discovered was that if nitric oxide

formation and metabolism became unbalanced, appreciable pathological in vivo protein tyrosine nitration

always ensues and such nitration is apparently also a hallmark of many, or indeed perhaps of all,

degenerative diseases 1a-8.

Prior to the discovery of the normal physiological roles of nitric oxide, B. C. Challis et al.1had

established that various inorganic catalysts (e.g. copper, iron, silver iodide and thiocyanate ions,

the latter being augmented in the serum of cigarette users) catalyze nitrosative reactions. A corollary to

this scenario is that anthropogenic inputs from such inorganic redox ions might inappropriately

perturb nitric oxide-dependent HS signaling processes.

A reversal of inappropriate nitrite formation is however achievable by the reaction of nitrite with

ascorbate which efficiently reconverts it back to nitric oxide. Another role for ascorbate in nitrosative HS

signaling has also been identified, this being for the reduction of Cu2+ to Cu+ needed to enable the

recycling of nitric oxide held at HS-core-protein cysteine stores1a-9.

M.A. Liebel & A..A. White, Biochem. Biophys. Res Commun. 1982, 104 (3) 957; Chem. Abs., 138749q
reportd that soluble guanylate cyclase was inhibited by H in a Mn2+ dependent manner.
This points to a linkage of nitric oxide sythesis in a H, Mn2+ interlinked pathway;
sulphated algal polysaccharides also demonstrated an H-like modulatory activity for this process in which the order of effectiveness for carrageenans was
λ >ι > κ .

3-3 Water Activity Model of Metal Ion Dependent H/HS Signaling

Metal Ions May Participate in H/HS Signaling via Modulation of Water Activities at H/HS Surfaces

Confusion currently exists as to the how H/HS microstructural information is transmitted to enable the

modulation of the numerous protein activities by H/HS.

The original proposition (implicit in the Long & Williamson1 hypothesis) that inorganic cofactors

determine much of H/HS signaling has not been universally accepted.

It is worthwhile to probe the circumstances under which microstructures present in intact H/HS

macromolecules apparently can highly selectively bind specific proteins in vivo, but either fail to bind, or do

so less selectively under highly purified in vitro conditions e.g. those described for how HS epitopes are

shared between and do not select between FGF growth factor variants (chosen on the basis of a supposed

found in the elegant in vitro modeling attempts reported by Kreuger et al.1;

Fragments of HS (Kreuger et al. 1) were studied in a highly purified form in vitro. These had been selected from in vivo information which had been predicted
such epitopes on their own to be capable of selective binding of FGF4, FGF7 and FGF8b. A possible explanation of these findings is that in vivo HS
discrimination involves additional hydrogen bonding or Van der Walls effects which the low molecular weight epitope lack. The failure of these studies of
purified oligosaccharide epitopes could, however, also suggest that multi-inorganic ions co-spheres might have provided some essential inorganic cofactor
for a discriminated HS epitope binding suggesting that e.g. a quaternary HS-FGF-FGFR-divalent metal ion complex might have been required to allow the
in vivo discrimination. Kreuger et al seem to suggest that apart from the antithombin dedicated pentasaccharide binding site of H/HS other suggested
similarly dedicated protein bidning H/HS sites do not exist. Amongst the evidence against this hypothesis is the large body of data which suggest that
discriminated singnaling depends on intact HS macromolecularly supported epitopes. An example of this is that for IFN-γ binding to HS (which is
thought to control the blood clearance, subsequent tissue targeting and local accumulation of this cytokine) binds not to isolated HS N-SO3- domains in HS
but requires a larger sequence that encompasses an internal HS N-acetyl domain flanked at both side by two HS N-SO3- domains (Sarrazin et al.1).

Key additional factors (e.g., inorganic ions) may also be needed for highly discriminated selectivity

between epitopes were possibly removed during enzymic digestion, fractionation etc. ; this is the expected

behaviour of the metallomic multi-inorganic-ion-arrays initially present in the intact HS molecules.

(Evidence that such inorganic cofactors may be required for FGF signaling is that divalent cations were

found by Kan et al. 1 to be absolutely required for HS-assisted b-FGF receptor assembly).

Further evidence for the requirement for essential inorganic cofactors to allow correct HS signaling is that

over-purification seems capable of removing such factors. Cf., the report by Ricard-Blum et al. 1 who

discussed how the discovery that Zn2+ ions were absolutely required to achieve HS

endostatin (collagen XVIII) binding (strong binding between these molecules is believed to occur in vivo)

had only became evident when highly purified protein failed to bind to normally purified HS and a

search for the possible binding cofactors removed during purification of the protein revealed that this was
apparently Zn2+ since binding could be restored by the addition of this inorganic ion.

Zn2+ is a component of the metallomic matrix of H (cf. Table IV and Grant et al., 1992d)) and probably co-

exists with HS in vivo. Zn2+ seems to selectively bind to H/HS (Parrish)

Zn2+ counterions associated with H seem also to characteristically alter the H-assoicated water structure (cf.

Grant et al. (1984, 1987a, 1992d, etc.)).

Presumably currently used standard laboratory procedures for the separation and purification of

biopolymers can reduce the amounts of Zn etc. necessary to allow HS epitopes to bind to selected protein

sites.

H is known mainly for its use in medicine as a blood anticoagulant achieves this action via the dedicated

antithrombin binding pentasaccharide epitope as well as via the heparin cofactor II mechanism. Both of

these mechanisms seem also to require indirect actions of Ca2+ ions (cf. refs. etc. listed in Table I).

Ricard-Blum et al.1 (2005) also reported that Ca2+ ions (apparently naturally associated with HS in vivo

were essentially required cofactors for collagen(V) bindning to HS proteoglycans, an activity for which

Zn2+ ions could not substitute.

3-3-1 Inorganic ions and particles associated with H/HS surfaces may influence the Wiggins low density/high
density water microstructure balance

Biochemistry which could ultimately depend on the behavior of liquid water determined by multi-hydrogen bonded

liquid water aggregate systems (e.g. Wiggins1) which is further subject to modulation of its structure by the present of

solute molecules of which inorganic ions are known to play dominant roles. The ultra ionic nature of H therefore could

enable this polysaccharides to uniquely alter water structure. The close association of both inorganic ions and clusters

of water molecules with the surfaces of highly anionic polymeric systems (Grant et al.1(1990)) could suggest that such

inorganic ions could determine the activity of such surface water systems.

3-3-1-1 Similarity Between Cation-Dependence of Hydration of H/HS and Commercial

Ionomers

The commercial ionomer Naficon® which exhibits proton conduction, is believed to depend upon
interactions between water clusters associated with –SO3- groups for this activity**. The degree of
hydration of these sulphonte groups depend upon the counterion in a similar manner to H being
maximum at 6 H2O/SO3- for Na+ and minimum at ca.0 H2O/SO3- for (CH3)4N+.
The addition of colloidal SiO2 to the commercial ionomer (in similar amounts to the occurrence of
Si putatively as SiO2 nanoparticles in H/HS) has been reported to improve its function.
(cf section 9-1, vide infra).
3-3-2 Electrostatic (Manning) interactions between H/HS and counterions etc. may be augmented by water
structure adhesion forces
Straighforward electrostatic counterion attractions (the Manning theory) while clearly partly responsible for counterion and protein
binding does not fully explain the physical chemical activities of sulphated polysaccharides. The principal interaction process evidently
employs additional interactions, including those afforded by the associated water structures; these are evidently also needed to achieve
correct discriminated crosslinks to proteins; such water structures may in turn be elicited by the occurrence of inorganic ions at anionic
SO32- groups of H. These water structures seem to be under direct control of the inorganic chemical environment in the neighborhood of
the polysaccharide chains putatively inducing water structuring analogous to how Hofmeister activities influence protein folding in turn
putatively also dependent on the induced water structure effect. A possible reason for the presence of a seawater range of inorganic
elements attached to H is then apparent: this complex multi-inorganic ion and organic humic (highly carboxylic acid substituted) polymer
solution is needed to create the correct bio-friendly fine structure of water aggregates. (Luck1 has established that the overtone infrared
absorptions of aqueous electrolyte solutions vary in accord with the Hofmeister series. This indicates that. The Hofmsister effect is due to
the alteration of supramolecular arrangements in hydrogen bonded water molecule aggregates.

3-3-2-1 Other evidence for biologically relevant polysaccharide determined water structure adhesion forces

The adhesion of animal cells to polystyrene surfaces seemed to be dependent on the correct water structure which shows up e.g. as
discriminated overtone infrared absorptions

3-3-3 Evidence for water adhesion forces in the anticoagulation mechanism of H

and in the binding of poly-L-lysine and poly-L-arginine to H

Pedersen & Jorgensen1 found rate constants for the antithrombin action of H to vary markedly between

LiCl, NaCl and KCl solutions used as reaction media. The rate constants reported by these authors were

also noted by the present author to be directly proportional to the vibrational energies (reported by Grant et

al. 1(1984)) for the water molecules preferentially bound to –OSO3- groups in a metal ion dependent

manner, associated with these salt forms of H.

The NMR chemical shifts of C atoms adjacent to the glucosamine 3-OSO3- (hypersulphated) groups in the

centre of the active antithrombin-binding H pentasaccharide sequences were also found by to be selectively

perturbed by these and other inorganic counterions to a much greater extent than was apparent for the non-

antithrombin binding disaccharide repeat units in H which lacked such 3-OSO3- groups

Grant et al. 1 (1986;1987) .

Sulphate half-ester linked water molecules attached to the anionic groups rather than the naked SO3- groups

per se might be responsible for protein binding and general biochemical activities of these sulphated

polysaccharides, a notion that was consistent with the displacement of the infrared overtone

bands of such water molecules during poly L-arginine and poly L-lysine binding when studied in an

hydrophobic medium (Grant et al. (1991)). The sulphate half ester linked water aggregates present at H

surfaces can apparently act as effective ‘smart’ glue-like agents to promote H binding to poly-L-lysine and
poly-L-arginine.

3-3-4 Countercation-Specfic Binding to –C(O)O- groups in H

The principal disaccharide repeats in H also showed, however, a general metal ion-dependence of infrared

vibration of the uronic acid, carboxylate groups (Panov & Ovsepyan1 Grant et al. 1 (1987b)) confirming that
counter cations in addition to changing the water structures around –OSO3- groups, can also directly modify

the electronic environments (and putatively the electrostatic activities) of –C(O)-O- groups and thereby

contribute to the overall mechanism of how inorganic cations modify of the attractive potencies of H/HS.

Various metallomes include those associated with H/HS, polyphosphate and polyβ hydroxybutrate) could determine
in a cooperative manner the water activities at cell such surfaces and intracellular organelles. Related phenomenon
will also, it is suggested, pertain to the geneome.

4. Examples of Literature Reports of Metal Ion Driven H/HS Signaling

A general redox hypothesis of H/HS signaling which is dependent on the activities of redox metal ions in the context of the
etiologies of degenerative diseases was proposed by Grant 1(2000).
Putative roles of the inorganic-element matrix associated with H/HS include the generation of oligomers by the action of
nitric oxide metabolites which are now known to be dependent on the presence of Cu and Zn cofactors (cf. Ding et al. 1);
there is an additional mechanism of essential requirement for inorganic cofactors to allow the binding of H/HS to endostatin
(Ricard-Blum et al.1) , of H/HS to annexinV (Capila et al.,1) and for the H/HS promoted basic fibroblast growth factor
receptor assembly process (Kan et al. 1). A selection similar of available information on the requirement of inorganic
cofactors for H/HS signaling is listed in Tables I-III.

Some examples of the growing literature database on the interdependence of H/HS biochemistry and

inorganic factors are collected in Table I which includes a list of reports which have noted that metal ions

are quite often needed to facilitate H/HS protein binding, and the reports listed in Table II which have

suggested that inorganic ions or particles can also directly modify HS microstructures by influencing the

primary assembly or postsynthetic structural modification of HS.

(Table III lists some analogous effects of organic molecules etc., which influence HS synthesis).

4. 1 Tables
Examples of putative metal-ion driven H/HS signaling

4.1.1 Table I A Putative H/HS Inorganic Ion Environmental Interface

Some examples of reported of metal-ion-dependent HS signaling by, e.g.,
involvement of metal ions in H/HS interactions with proteins are thought to be involved in blood anticoagulation, breast
cancer, basic fibroblast factor dimerization, endostatin (collagen XVIII), collagen V and prion interaction, the actitivities of a
range of proteins and nitrosative scission of H/HS

HS potentiation of Ca2+ signaling

(The original hypothesis that HS was centrally involved in inorganic biochemistry)
W.F.Long & F.B.Williamson, IRCS J. Med. Sci. 1979, 7, 429-434

Ca2+ Blood Anticoagulant Action of H (Antithrombin mechanism)

Indirect cofactor of H mediated blood homeostasis
Ca2+ is needed to overcome the negative effect of Gla domains of Factor Xa on the templating action mediated by H

R.N.Pike et al., FEBS J. 2005, 272, 4842-4851

The antithrombin-binding pentasaccharide sequence, which has been firmly established to be the
principal mechanism by which H influences blood coagulation (cf., e.g., Bourin & Lindahl1).

Ca2+ Blood Anticoagulant Action of H (Heparin cofactor II mechanism)

Potentiates
Heparin cofactor II binding to H
F Zhang et al., Clin. Appl. Thromb. Haemost. 2004, 10, 245-257
Ca2+ & Breast Cancer
Ca2+ enhanced HS containing PGs activities which may modulate tumour cell growth
B. Vandewalle et al., J. Cancer Res. Clin. Oncol. 1994, 120 (7) 389; Chem. Abs. 121, 105487j

Bivalent cations
(e.g. Ca2+, Mg2+, Mn2+) are required for HS modulation of
Basic fibroblast factor receptor dimerization;
M. Kan et al., J. Biol. Chem., 1996, 271, 26143-26148; G. Siegel, et al., Cardiovasc. Res. 2003, 58, 696-705.
(This apparently also requires the antithrombin binding (H pentasaccharide epitope) in HS
W.L. McKeehan et al. ibid., 1999, 31, 21511-21514 which is especially sensitive to the effect of counterions as revealed by comparison of
the shift induced by counterions in the NMR signals between the antithrombin binding site and regular disaccharide repeat units in H. (Reported by D.
Grant et al., Cell Biol. Intern. Rep. 1987, 11, 220)

Ca2+ Collagen V HS binding

S. Ricard-Blum et al., J Biol. Chem. 2006, 281 (35) 25195-25204
(Zn2+ was found to be inactive as a cofactor of this binding;
it was believed that the initial binding of Ca2+ was to HS not to the collagen)

Similar HS protein interactions, discussed by Kan et al. loc. cit. include

Bivalent cations are
required for correct HS interaction with
β 2 and β 3 integrins,
Platelet/endothelial cell adhesion molecule-1 (PECAM)-1)
Cadhedrin and
L-selectin;
Cf., L-selectin in nonlymphoid endothelial cells;
K.E. Norgard-Sumnich et al., Science. 1993; 261: 480-483.

Ca2+ is also required for HS mediated

Annexin V assembly on cell surfaces
I. Capilla et al. Structure (Camb.) 2001; 9: 67-64, cf., A Lewit-Bentley et al. Eur J Biochem 1992; 210: 73-77

Bivalent cations
Zn2+ etc. are required for
Endostatin binding to H and HS
S. Ricard-Blum et al. J Biol Chem. 2004: 275: 33688-33696);
Ca2+ is required for
Serum amyloid P binding to HS
H Hamakai, J Biol Chem. 1987; 262 ; 1456-1460);
Ca2+ enhances
Serum amyloid P aggregation inhibition by H and HS
E.H Nielson et al. APIMS. 1994; 102: 420)

Ca2+is required for H modulation of the

Acrosome reaction
and also the required
2+
Ca flux for Capacitation
A. Valencia-Sanchez, et al., Mol. Androl., 1995, 7 (1,2) 57, Chem Abs. 122, 2821787

Ca2+ transport for ATPase activity inhibited by H in sarcoplasmic reticulum of skeletal muscle, the plasma membrane of red cells and dense tubular
sytsem of blood platelets but not the Na+/K+ ATPase of the mitochondrial F1 ATPase in the reversal of the Ca2+ pump
H DeMers et al., J Biol. Chem. 1994, 269 (20) 14529

Divalent cations are required for H binding to

Fibronectin
M. Hayashi & K.M. Yamada, J Biol Chem. 1982; 257: 5263-5267)

Ca2+ is required for extracellular

Fibrillin-1 binding to HS proteoglycans
K. Tidemann et al., J Biol Chem. 2001; 276: 36035-36042)

Extracellular [Ca2+ ]
regulates the distribution and transport of HS proteoglycans
and fragments in a rat parathyroid cell line
Y. Takeuchi et al. J. Biol. Chem. 1990, 265 (23) 13661-13668

Ca2+ is required for aggregation of

Syndecan-1 transfected cells, mediated by HS,
M.J. Stanley et al., J. Biol. Chem. 1995; 270: 5077-5083

Zn2+ and Ca2+ affect the neutralizing ability of

 Histidine rich glycoprotein by H
Y. Kazama & T. Koide, Thrombosis Haemostasis, 1992; 67: 50;
Cf. also A.L. Jones et al. J Biol Chem., 2004, 279, (29) 30114-30122)

Zn2+ promotes the association of HS with

biotyinylated H-Kininogen
T. Renne, J. Biol. Chem. 2000; 275: 33688-33696)

Zn2+ (Evidence for a Zn modulated H superfamily)

Amyloid β protein precursor
A.J. Bush et al., J. Biol. Chem. 1994, 269 (43), 26618

Cu(II) forms bridges in oligomeric complexes in

Prion protein - HS and GAG complexes
R. Gonzales-Iglesias et al., J Mol Biol., 2002: 319: 527-540)

Cu(II) as Cu(II) tripeptiede (GHK Augments GAG expression (as well as

collagen,metalloporoteinase, elastin and protease expression)
Simeon et al. J. Invest. Dermatol., 2000, 115 (6) 962-968

Nitrosative Scission
Nitrosative scission of HS is aCu2+/+, Zn2+ dependent mechanism;
Nitric Oxide can be stored e.g. at conserved cysteines in the HS core protein, which ntogether with
Cu2+/+ and Zn2+ can potentiate
Nitrosative scission of H/HS at GlcNH2 sites
Cf. , e.g., F. Cheng et al. J. Neurochem., 2006, 98, 1445-1457
A mechanism by which various environmental inputs (both chemical and physical) can positively or
negatively impart into HS activity include direct effects of diet on nitric oxide biochemistry and metal
ion-dependent nitrosative postsynthetic alteration of HS. This could ultimately account for how diet
controls health. Cf., 1a-7 which lists how some known dietary factors have been reported to effect change
in HS structured.

Mn2+ potentiates the inhibition of

Soluble Guanylate Cyclase by HS (and other sulphated polyanions)
M.A. Liebel et al., Biophys. Res. Commun., 1982, 104, 957-964

Fe2+ (and Cu2+) ions apparently catalyse HS-GlcNSO3- à HS-GlcNH2 i.e. prime HS for nitrosative cleavage
D. Grant et al. (unpublished observations, Aberdeen University)

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

Cu2+, Zn2+
Cf. Fe3+-related labelled Biotinylated
H – Binding to Lactoferrin
was augmented by 100µ MCu2+ but inhibited by >500µ M Cu2+ or >40µ M Zn2+
S. Zou et al. Comp. Biochem Physiol. ,1992, 103B (4), 889-895
Cf., in vivo diffusion of lactoferrin in brain extracellular space is regulated by interaction with HS
(R.G. Thorne et al PNAS USA 2008)

Possible Clue as to why Si occurs in H/HS-to Act as a multi-elelment conveyor and sorter
SiO2-based chromatographic packing medium-H allows both cations and anions can be
separated on a single heparinized chromatography column
T. Takeuchi et al. Analusis. 1998, 26, 61-64

O2 Hypoxia HS synthesis and degree of sulphation is increased

HS core protein expression also increased
Hypoxia Inducible Factor (HIF) related activities
J. Li et al., J Cell Sci. 2002, 115, 1951-1959
Hyperbaric O2 apparently can also induce an increase in glycosaminglycan synthesis in wound fibroblasts
G.P. Roberts & K.Y. Harding, Br. J. Dermatol. 1994, 131 (5) 630-633.

4. 2 Table II

Reports of (apparently direct) modulation of HS microstructure in response to the

presence of inorganic ions etc.
Direct influence on HS biosynthesis:
Extracellular [Ca2+ ] directly signals for increased HS synthesis in thyroid cells
Y. Takeuchi et al., J Biol Chem,1990, 265, 13661-13668

(Ca2+ related effect of high affinity metal ion chelator?)

Ethane hydroxydiphosphonate (anion) increased HS/Chondroitin sulphate ratio
(also associated with sn liver tumour cell metastatic potential)
J. Timar et al., Int. J. Cancer, 1995, 62 (6) 755-761
Inhibition of HS biosynthesis by Ca2+ ionophore A23187
in cultured vascular endothelial cells implication of
intracellular calcium assimilation in lead inhibition of endothelial PG synthesis
[Y. Fujiwara & T. Kajo J. Health Sci., 2002, 48 (5) 460-466]

Mn2+ and Ca2+

a GlcNAc transferase I can use both Mn2+ and Ca2+ but a-GlcNAc transferase II can use only Mn2+
TA Fritz et al., J Biol Chem. 1994, 269, 28809-28814;
[Cf., X-ray structural data which included the location of the catalytic Mn2+ ions needed for glucuronyltransferase activity for HS and ChS assembly were
reported by L.C. Pedersen et al., J. Biol. Chem. 275 (44) 34588]
Mn2+ increases HS synthesis
A.Z. Kalea et al., Biometals. 2006; 19: 535-416

Mg2+ is required for HS synthesis

P. Jaya P.A. Kurup, J Biosci. 1986; 10: 487-493;

Pb2+ (related to [Ca2+]) diminishes HS synthesis

Y. Fujiwara T. Kaji, J Health Sci. 2002; 48: 460-466; Toxicology, 1999; 133: 159-169

Cd2+ diminishes HS synthesis

A. Cardenas et al., Toxicology, 1992; 76: 219-231
Interaction between
Cd2+ and Zn2+ effects on production and sulphation of GAGs in cultured bovine endothelial cells
S Ohkawara, T.Kaji et al., J. Toxicol Environ. Health, 1996, 47 (2) 183-193
(Zn 20µ M and Cd 2µ M prevented Cd-induced decrease in 35S uptake but not Cd-induced increase in 3H glucose uptake;
Zn may protect cells from Cd-induced inhibition of DNA and protein synthesis)
Cf., R.C. Chambers et al., Am. J. Respir. Cell. Mol. Biol. 1998, 19, 498-506
(Cd inhibits proteoglycan and procollagen production in cultured lung fibroblasts; greatest inhibition of proteoglycan synthesis occurred with the
major matrix-associated proteoglycans versican, decorin and the large HS proteoglycans we well as a less diminution of other HS proteoglycans)

Hg(II), Cd2+, Mn2+, Ni(II), Pb2+ diminished HS synthesis

D.M. Templeton Proc. Trace Element Health Disease IUPAC Symp .1990 p 209-219;

Na+ depletion increased HS synthesis in spontaneously hypertenisve rats (but not normal rats)
G.N. Jysthirmayi et al., Res. Commun. Mol. Pathol. Pharmacol., 1995, 90 (1) 115; Chem. Abs. 123, 311506j; cf.,
High [NaCl ] alters HS synthesis in spontaneously hypertensive rats (but effect suggested to be lactate and NAD/NADH dependent)
M. Cechowska-Pasko et al., Exp. Toxicol Pathol., 2000, 52 (2) 123; Chem Abs., 133,, 264382k

Extracellular [NaCl]: HS synthesis had apparently become adjusted during evolution to balance this
(Salinity of seawater and aquatic habitat i.e. mainly an increase in [NaCl] correlated with increased HS and GAG contents of
tissues for 15 species of aquatic invertebrates)
Cf., Nader et al., Comp. Biochem. Physiol. 1983, 76, 433-436
High
F- depleted HS synthesis
K. Pawalowska-Goral et al., Fluoride. 1998; 31: 193-202
SO42- (tranporter requires thyroid factor and Vit. D isoform (1,25 dihydroxy D3) {suggesting related input from UV B exposure})
P.A. Dawson & D. Markovich, Pflugers Arch.-Eur. J. Physiol,. 2002, 444, 353-359
Low
[SO42-] produced an altered PG synthesis by cartilage
K Iti et al., J Biol Chem., 1982, 259 (2) 917-925

Calcium oxalate
crystal (surfaces) the presence of which upregulated the formation of HS optimized for calcium oxalate crystallization inhibition
F.T. Borges et al., Kidney Int. 2005, 68, 1630-1642

Si (probably in the form of’ ‘SiO2’; suggested input into HS biosynthesis; possible (bone) hydroxypatite-related effect)
M.F.McCarty Med. Hypoth. 1997, 49, 177-179

Sr2+ may augment and modulate GAG biosynthesis (possibly for HS structure optimized to hydoxyapatite {bone} surfaces?)
Y. Henrotin et al., J Bone Miner Res. 2001, 16, 299-308
 Cf., E.R. Wise et al., Chem. Mater. 2007, 19, 5055-5057
Confirmed using surface NMR study that bone calcification requres surface sulphated polysaccharides; the organic
mineral interface during bone formation is mainly polysaccharide, putatively HS.
--------------------------------------------------------------------------------------------------------------------------

Depolymerization of H by Actions of Metal Ions

Fe
B Lahiri et al., Arch. Biochem Bio
phys. 1992, 293, 54-65

Cu
E. Liu & A.S. Perlin, Carbohydr. Res. 1994, 255, 183-191
------------------------------------------------------------------------------------------------------------------------------------------------------
HS metal ion binding flow transduction

Ca/Na has been suggested to act by altering HS conformation to participate in a flow sensor mechanism:
S Siegel et al., Colloids & Surfaces A Physiol. & Energy Aspects 1998, 138, - 351
Cf. also, e.g., J.A. Florian et al., Proc 2003 Summer Bioengineering Conference June 25-29, Sonesta Beach Resort Biscayne,
Florida (HS proteoglycan is a mechanosensor on endothelial cells). Read at
http://www.tulane.edu/~sbc2003/pdfdocs/0683.PDF

Unsaturated HS disaccharides produced bacterial heparinase can inhibit of the

Na+/Ca2+ exchanger and the value of cytosolic Ca2+
K Shinko et al., J. Biol. Chem. 2002, 277, (50) 48227-48233

-------------------------------------------------------------------------------------------------------------------
Mechanical stress has also been reported to regulate syndecan-4 HS expression in vascular smooth muscle cells
L. Li & E.L. Chaikof, Arterioscler. Thromb. 2002, 22, 61-68
biosynthesis

4.1.3 Table III

Some Examples of Reported Effects of Organic Molecules on HS Biosynthesis

Putatively beneficial effects on HS biosynthesis are produced by:

Ascorbic acid (stimulates GAG (especially HS) synthesis in cultured fibroblasts [J Kao et al., Exp. Mol. Pathol. 1990, 53 (1) 1-10 (confirming the earlier
reports of M. Edward and R.F. Oliver, Biochem. Soc. Trans. 1983, 11, 383; ibid., 12, 304) that this nutrient increased the degree of sulphation of HS.
Retinoic acid and cAMP up-regulation of 3-O-sulfotransferase-1 led to a dramatic augmentation of the anticoagulant form of HS in F9 embryonal
carcinoma cells [L. Zhang et al., (with R.D. Rosenberg) J. Biol. Chem., 2003, 273, 27998-28003];
Cf., Retionoids increased HS synthesis in human skin fibroblasts
[M. Edward, Br. J Dermatol., 1995, 133, 223-230]

Polyamines.
Inhibition of the endogenous synthesis also modulates
glypican-1 HS structure
[K. Ding et al., J. Biol Chem., 2001, 276 (6) 3885-3889]

+Catechin also increased HS synthesis [W.Sinn et al., Biochem. J., 1981, 200 (1) 31

H stimulates the synthesis and modulates the sulphationpatern of HS proteoglycans form endothelial cells)
[H.B. Nader, V. Buonasisi, P. Colburn, C.P. Dietrich, Cell Physiol. 1989; 140: 305-310]
Binding of anticoagulant H (plus a necessary compound Y cofactor) to endothelial cells was found to stimulates the biosynthesis of antithrombotically
active HS proteoglycan,
[M.A.S. Pinal et al., Braz. J. Med. Biol. Res. 1994, 27p, 2191-2195
(Cf., Antithrombotic agents which stimulate the biosynthesis of anticoagulant HS having an increased degree of sulphation (achieved with a minimum
(pentasulphated) tetrasaccharide polysaccharide chain length,
pharmaceutical low molecular weight
Hs CY216, CY222 (from Sanofi/Choay) , OP 622 and OP 386 (from Opocrin) and
oligosaccharides produced from H by heparianase II digestion as well as
unmodified H) [M.A.S. Pinhal et al. Thromb.. Res. 1994, 74 (2) 143]

Apolipoprotein E containing HDLP stimulates endothelial production of HS rich in H-like domains [L. Paka et al, J. Biol. Chem. 1999, 274 (6) 4816-
4823].
Cf., LDLP (on the other hand) stimulates mesangial cell proteoglycan and hyaluronan synthesis
R.S. Chana et al., Nephrol. Dial. Transplant, 2000, 15 (2) 167-22

Glucosamine
may promote endothelial production of HS proteglycans thereby retarding atherosclerosis
[M.F. McCarty, Med. Hypoth. 1997, 48, 245; Chem. Abs. 127 12883u]
Thyroid hormones were reported (Y. Tshiba et al. DATA ) to caused an increase of protoglycan synthesis in fibroblasts in culture.
(Further work is required to establish the precise effect of such hormones on HS microstructure)

Glucoccorticoid regulation of proteoglycan synthesis in mesangial cells

M. Kuroda et al., Kidney Int., 2002 62 (3) 780-789
Pathogensis of diabetic vascular disease: evidence for reduced HS production
[T. Jensen, Diabetes, 1997, 46, Suppl. 2, S98-100]

-----------------------------------------------------------------------------------------------------
Intracellular accumulation of secreted proteoglycans inhibits cationic lipid-mediated
gene transfer
[M. Belting & P Peterson, J Biol. Chem. 274, 19376]

Putatively adverse effects on HS biosynthesis are produced by:

Conventional (laboratory GAG biosynthesis inhibitor reagents e.g., Xylosides

Modulation of the biosynthesis of HS by β -D-xylosides depends on the aglycone
T.A. Fritz et al., J. Biol.Chem., 1994, 269 (1) 3007
Cf.,
β D xylose, 2 deoxy D glucose and ethane,1-hydoxy,1-,1-bisphosphonate
Cf also 5 hexyl 2 deoxy uridine (an especially effective reagent which increased HS sulphation
All induced marked (reagent specific) changes in GAG including HS synthesis
(The high affinity metal ion chelator)
Ethane, 1-hydroxy,-,1-bisphosphonate was an especially effective inhibitor of chondroitin sulphate ChS (incr. HS/ChS ratio and this was thought to be
associated with increased liver metastasis potential )
[J. Timar et al., Int. J Cancer., 1995, 62 (6) 755]

High [Glucose]
negatively affects HS production by mesangial and epithelial cells
S. Morano et al., Diabetes Metab. Res. Rev. 1999, 15 (1) 13-20Glucose uptake was a dominant activity in both rheumatoid and non rheumatoid fibroblasts
in culture;
N.F. van Det et al., DATA; Kasinath et al Kolm et al
promoted hyaluronan biosynthesis and diminished suilphated GLG biosysnthesis attributed to a
formation of close packed cells
[T.P. Anastassiades et al., Arthritis Rheum. 1979, 22 (8) 871-876]

Hydrogen peroxide
Nishingae et al

Nonesterified fatty acids (NEFA) reduced the degree of sulphation of HS (this apparently cuase an increased permeability to albumin); in smooth
muscle cells NEFA-albumin increased expression of the genes for core proteins of HS mediated by peroxisome proliferator-activated receptor gamma; the
matrix produced by cells treated with NEFA-albumin had a highly
affinity for LDLs
[G. Camejo et al., Curr. Atheroscler. Rep. 1999, 1 (2) 142-149]

Homocysteine also diminished anticoagulant HS expression in cultured porcine aortic endothelial cells [M. Nishinage et al., J. Clin Invest. 1993, 92,
1381-1386] and high Glucose adversely modifies HS biosynthesis by mouse glomerular epithelial cells [S. Morano et al., Diabetes/Metab. Res. & Rev.
1999, 15 (1) 13-20] ;
Cf. also,
Lysolecithin may also adversely alter subendothelial HS proteoglycan production
[P. Sivaram et al., J. Biol Chem. 1995, 270,(30) 29760-29765];
Human-rIL1-b and TNFα suppressed the formation of H-like compounds on cultured porcine aortic endothelial cells [M. Kobayashi et al., J. Cell.
Physiol., 1990, 144, 383-390]
Infection with
Herpes simplex virus type I in vitro also inhibited proteoglycan synthesis in human endothelial cells,
[R. Kanner et al., Am. J. Respir. Cell. Mol. Biol. 1990, 2, 423-431] and
Endotoxin causes loss in HS of region rich in sulphate half ester groups at non-reducing end of GAG chain
P. Colburn et al. (with C.P. Dietrich) Arch. Biochem. Biophys., 1996, 325 (1) 128;

Antimicrotubular agents
colchicine and vinbalstine inhibited glycosminglycan synthesis by embryonic chick tibia and chondrocytes in culture [H.W. Jansen & P Bornstein,
Biochim. Biophys. Acta 1974, 362, 150-159];
Amyloid fibrils apparently stimulated the biosynthesis of glycosaminoglycans in cell cultures in which hyaluronan was increase but HS was decreased
[M.J. Palmoski & K.D. Brandt, Biochem. 1975, 148 (1) 145-147]
[L.Li & E.L. Chaikof, Arterioscler. Thromb. Vasc. Biol., 2002, 22 51-68;

Monesin diminished the degree of sulphation of HS produced in cell culture [L.O. Sampaio et al., J. Cell. Biochem.,1992, 50 (1) 147; Chem. Abs. 117,
188863v;
(Albumin bound)
Cf. Oxidized LDL regulates the synthesis of monkey aortic smooth muscle cell proteoglycans (including HS) that have enhanced native LDL binding
properties [M.Y. Chang et al., J. Biol. Chem. 275 (7) 4766-4773
-------------------------------------------------------------------------------------

5. Comparison of ‘Metallomic’ Multi-Inorganic-Element Arrays in Anionic

Polysaccharides with Those in Other Matrices

Spark source mass spectrometry (SSMS) and inductively coupled plasma mass spectrometry (ICP-MS)
multi-element analytical data for several H samples from different manufacturers suggest that H always

contains a seaweed-like inorganic element content (recent ICP-MS reports of blood sampling tube H-

associated ‘contaminants’ suggested3b-10 the co-occurrence with H of the full seawater range

of some 80 elements 4). Such single ion enriched Na and Li H (e.g. by use of ion exchange resin column

technology as used in medical grade H preparation) as well as native H appears to be greatly enriched in

those elements which occur in the least amounts in natural waters, a situation which is reminiscent of the

cell wall polysaccharides of plants1a-10 where a similar range of inorganic ions as are believed to

contribute to cell wall control over Ca2+ second messenger actions via the borate crosslink polysaccharide

oligosaccharide generator1a-10.

The occurrence in marine algal tissues of major cell wall polysaccharides in multi-counterion salt forms

rather than, as originally thought, free acid forms, was established by A. Wasserman3 and confirmed by

later workers including W.A.P. Black & R.L. Mitchell3a. It is now thought that marine algal

polysaccharides contain most or all of the inorganic elements (60+ in number) which occur in seawater3b

from which anionic polysaccharide ligands apparently are able to simultaneously bind a wide range of

inorganic counterions and particles by a mechanism which apparently enables these polymeric ligand

systems to become selectively enriched in those elements which are least abundant in their natural bathing

solutions.

H. Haraguchi4 suggested that for updating of the metallomic concept, many elements which had hitherto

been classified as being ‘non-physiological’ must be included, furthermore it has now become evident

that such elements occur both in seawater and human blood serum (these solutions demonstrate a

degree of inter-relatedness in their inorganic element metallomic compositions4).

The non-physiological elements also show up in H cf. Table III, indicating that a possible physiological

role of such element might be to modulate the biochemical activities of H/HS.

Table III compares the metallomic profiles of alginate, H and human hair, a common

used tissue employed for detection of metal ion status of humans and for providing evidence of metal ion

intoxication. Comparison of the multi-element compositions of the earth’s crust with seawater and H
show much less correlation (data not shown). Table IIIa lists results reported for metal ion impurities

present in chitosan which have been attributed to uptake from a final washing with tap water13a.
Polysaccharide-rich biomasses of land plants, brown and red marine algae, as well as mollusc shells, bone, mast cell H
and H-like segments of HS may constitute a related system of natural (‘salinity induced’) metallomic matrices which seems
also to be related to the inorganic element contents of human hair15 (which is currently the most well researched multi-
inorganic element containing matrix) and chitosan13a ( cf. Table IIIa).
Human scalp hair (e.g., 15 ; except for Zn, which is augmented in hair) is compared with the data shown in Table I for ‘Na’
and Tl H. This correlation is best for the least perturbed samples, from schoolboys 15.

5. 1 Examples of ‘metallomic’ arrays associated with H, kelp & human scalp hair

5-1-1 Table IV Inorganic Elemental Contents (mg/kg) of Kelp, H & Human Scalp Hair
Baltic Seaweed3a-4 Commercial Kelp3a-1 Na+ Heparin Li Heparin Tl+ Heparine,3b-9 Human Scalp Hair15
3b-9 3b-10
Element (A:before ion exchange) (E after ion exchange)
(a) (s) (b) (c)
(D after ion exchange)

(commercially produced) (academic lab product)

A D E1 E2 B C A/C
Si 1642 5900 116 126 1170 100 510(s) 11.6
Mg 4300-6000 2130 1300 ? (30) 260 10 28.0(a) 59 3.6 28
Mn 410-420 1235 [1-65](d) ? (0) ? (0) 6 (s)
Ca 1200-5400 19040 30000 171 <50 125 30 450 (a) 67 11
Sr 2180-2210 749 65 2 0.04 1 1.4 1.2(a) 54
Ba 372-381 12.8 140 1 0.3 14 1 0.16-1.6(b) ca.90+ 9
Na 460-510 principal 143 336 90
K 2000-7000 12800 2000 23 42 22 40 20.4(s) 98
Rb 0.05 3 0.1 <0.002 0.1 0.2 0.051(s) 59
0.15 (s) 20
Cs 0.047-1.08 trace 9 0.01 <0.002 0.1
Tl 2.93 8 0.2 0.0008 principal
Cr 1.8 trace 30 trace 1 0.99(a) 30.3
Cu <10-20 6.3 730 16 7 10 <5 22.1(a) 33 70
Fe 280-320 896 1100 5 <4 24 10 19.0(a) 58 24
Ni 9.2-13.2 35 <170 trace 1.3 0.6 10 1.49(a) ca.100 ca.125
Co 12.2 <80 trace 2 0.67(a) ca.100
Sn 0.06 5 trace 0.3 0.24 21
Mo <0.027 15.9 7 trace <1 0.43(a) 16 96
Al 120-140 193 3-35f ? 14 16 ?
Ti 0.12 <390 0.2 (0) 2 4 0.79(a) <493 <126
As 327 <15 ? 0.08 0.3 1 0.274(s) <55 <167
Zr 10.7 0.01 5 ? 0.3 0.21(s) 24
Ce trace 7 ? (0) 3.5 0.093(s) 75
Ag 0.04 4 0.002 0.003 0.01 0.5 0.034(s) 118
Nd 5 0.04 0.008 0.1 1 0.039(s) 128
W 0.33 5 0.03 <1 0.037(s) 135
La 0.19 7 0.005 0.004 0.003 1 0.045(s) 156
Zn 250-310 35.2 <80 16 33 49 7 150(a) <0.5
185(s) <0.43
Pb 0.14 16 0.8 0.2 0.4 4 7.1(a) 2.2
Ga trace 20 0.005 0.003 0.003 1 3.4(s) 5.9
Sb 0.07 1.4 2 3 ? 0.003 1.7
Cd (1) 0.3 0.12 0.1 1.5

P 2110 440 61 77 435 ca.30 195(a) 2.3

169(s) 2.6
V 0.6-0.9 5.3 3 ? 0.7 <1 1.22(a) 2.5
B 194 <25 16 1 1.41(s) <17.7
I 130-160 624 10 1.3 (0) 2 <1 2.9(s) 3.4
Cl 36800 5600 1000 <580(s) >10
F 326.5 890 4 - -
Br 140-160 trace 130 30 3 0.9 4.46(s) 29
Notes:
Data for Na H (which had been recommended as being suitable as a standard reference material to be distributed amongst a
number of academic institutions by a major manufacturer, which was further purified by extensive dialysis) before ion exchange and Tl
He after ion exchange are taken from ref. 3b-9.
Data for Na and Li H after ion exchange are taken from ref. 3b-10.

Further Background to the Studies of Metal Ion Binding and the Multi-Element Content of H
Commercial H, readily available as a highly purified, protein-free polysaccharide mixture, avidly sequesters both cations and anions in a relatively
indiscriminate manner and therefore potentially can host a full repertoire of inorganic ions6, 6a (both from ex vivo contamination13a and from uptake from the
wide range of small amounts of inorganic elements which occur in biological fluids4). Although it is possible that the toxic ions in H are bound in some
less toxic form, their occurrence in H used for dialysis and parentereal nutrition solutions is a topic of current concern3b-7,8. Although LB Jaques in his
excellent 1978 review6a had emphasised the unique physiological relevance of the sequestration of both positively and negatively charged inorganic ions by
H, at the present time there seems to be lack of awareness or interest in the wider relevance of this phenomenon. An analyses of a carefully selected
typical porcine sodium H by spark source mass spectrometry (SSMS) revealed the presence of some 38 inorganic elements in amounts equal to or greater
than 1ppm. A larger range of inorganic elements remained in purified H studied by ICP-MS (in the context of commercial H anticoagulant employment in
blood collection tubes); the ICP-MS results agree surprisingly closely with those previously reported for both native and ion exchange purified H
previously studied by SSMS. C.F. Moffat Synthesis, characterisation and applications of chemically modified heparins, Ph.D. Thesis University of
Aberdeen, 1987.
The ICP-MS results show the occurrence in H of ultratrace amounts of ((Au))((Be))(Bi)((Dy))(Gd)Ge Hf Ho (Ir) Li Lu Nb (Os) Pd Pr Pt Re Rh Ru Sc Se
(Sm) Ta (Tb) Te Th Tm U Y and Yb (including in single counterion enriched, purified forms of H_
Data in parenthesis and double parenthesis are progressively less well established than those without parenthesis3b-10.

Comparison of the data for multi-inorganic elements present when H or K2EDTA were used as the anticoagulant in blood collection tubes suggested that on
the basis of anonic equivalents purified H contains a higher inorganic element profile than K2EDTA, especially for Pb, Fe, Al Na Ca Mg Ag Cd Cu I Si Sm
and V which appeared to occur in purified H in amounts some 100 times greater whereas the amounts of La Mn Eu Mo B Ti Pt Lu and Hf were present in
similar amount in both K2EDTA and purified H. Much larger amounts of Rb and Cs occurred in the K2EDTA than in the purified H 3b-10.

Table I shows the multi-element contents of a range of H and of alginate-based biomasses3a. The inorganic elements of a Baltic seaweed3a-4 seem to include
a large anthropomorphic component while the commercial kelp inorganic element values suggest an oceanic seawater origin of these elements the
distribution of which also shows greater similarity than does the Baltic data to the native Na H data of Table I which further suggests that the inorganic
components of the H samples listed in Table I are derived from a natural source.
[The results shown in Table III also suggest, however that Rb, Cu, Ni and Sn bind more strongly to the combined
NSO3- –OSO3- ,C(O)O- anionic system of heparin than to single C(O)O- anionic groups system of kelp alginate, the most likely ligand in that matrix].

G.E. Harrison & A. Sutton3b-2 had also, many years ago, reported the Ca, Sr and Ba contents of five commercial H samples; these data appear also to be
approximately related to the contents of these elements in a range of other (polysaccharide containing) matrices reported by HJM Bowen14. This author, in
1966 had listed the then known reported information on the multi-element contents of H (µ γ ./g) as:
Mn 3.6, Ca 30-2900, Sr 5-92 Ba 2.5-12, Cu 0.4 and Zn 28 3b-1.

Further Notes:
(a) ref. 15; (s) ref. 15a; (b) other internet listings; (c) ref. 15b ; (d) ref. 15d.
e
This (highly toxic counterion form) had been selected for in vitro 205Tl NMR (which showed rapid along-chain site interchange and perturbation of the
205resonances; the standard sulphonated polystyrene resin exchange cation exchange column method used for covertign the NaH to TlH was confirmed to
be highly effective (e.g. the residual Na content of the TlH was 90ppm). The SSMS results are semi quantitative; as well as the elements shown in Table
IV vanadium was also present but no figure is listed since there were complication with the spectral analysis for this element; the aluminium content also
cannot be listed because aluminium wa used to prepare the electrode for SSMS analysis; the 13C spectrum of the TlH was generally similar to that of the
NaH studies except for perturbation of the ring C resonances around 70 ppm and the carbonyl carbons and the anomeric carbons of Id2S and GlcNS).
Tl seems to occur in natural H in similar amounts to that found in kelp but when present in such matrices may not act as a high risk human intoxicant3b-9
but further specific studies are required to confirm this hypothesis [Cf C.F. Moffat 1987, loc cit].
(f) The amounts of Al in a range of commercial H were listed by Bohrer et al. (loc. cit.)

5-1-2 Table IVa Comparison of Inorganic Elements in H and Chitosan

Comparison of the elemental content (mg/kg) data from ref. 13
of chitosan from crab shells (obtained by using a final wash with demineralised water

which avoids the uptake of metal ions from tap water, a source of suspected but not documented contamination)
Element Pure Kelp Na heparin Tl Heparin Chitosan

Fe 898 1100 10 ‘high’

Ni 35 170 10 2.03
Cu 6.3 730 5 1.03
Cr trace 30 1 0.36

V 5.3 3 <1 0.12

Mn high <1 <1 0.09

Pb 0.14 16 4 0.59
Ag 0.04 4 0.5 0.02

Hg 1.9 <1 <1 0.025

Cd trace <1 1.5 0.22
The controlled uptake and release of inorganic ions from H-like polysaccharides could, it is proposed, be part of a wider servo-control feedback system.
The presence of multiple elements in naturally occurring salts as well as chemical reagents (including K2EDTA and gels employed for blood clot phase
separation 3b-10) is a common phenomenon.
(Such naturally occurring salts include apart from polysaccharides, proteins such as those responsible for the multi-elements in hair and nails, the apatite
mineral of bone (as well as naturally occurring apatites), polyphosphates (which occur ubiquitously at cell surfaces throughout biota) and soil and natural
water humic salts (which include a fraction termed fulvate). It is entirely in keeping with the general occurrence of multi-element in salts found in nature
(unless these have been subjected to chemical purification by crystallization and recrystallization processes) that the ultra salt-like highly sulphated
polysaccharides H and HS which are the extreme forms of such biologically utilized salt systems exist as multiple inorganic ion arrays and that inorganic
ions can influence or control the heparanome. The HS inorganic array may, however be subject to pathological or anthropogenic skewing. In general the
metallome can influence the supramolecular structure of polysaccharides and thereby its biological activities.

5-2 Log-Log Interrelationships Between Multi-Inorganic Element Contents

Approximately linear log – log interrelationships can be demonstrated between the multi-inorganic element

contents of natural waters including seawater, the bound inorganic elements present in kelp and

geological fulvates (attributable respectively chiefly to the sequestration by the abundant carboxylate

anionic groups present in alginates and in humic polymers (cf., Grant1).

A similar approximately logarithmic inter-relationship also shows up between the multi-inorganic element

contents of human blood serum and seawater (Haraguchi1). These matrices show typical metallomicb

profiles. The inorganic content of the ultra anionic animal polysaccharide H, studied in the form of a

purified protein-and nucleic acid free pharmaceutical industry preparation, especially before its final

rigorous purification processes which is designed to remove the supposed inappropriate amounts of toxic

elements present also demonstrated a similar but more enhanced multi-inorganic element fingerprint, i.e. it

H is so constituted to acquire an enhanced metallomicb profile (cf. Table IV).

b
The ubiquitous presence of trace and ultratrace elements found by mass spectrometric analysis in biological matrices has led H. Haraguchi1 to
propose that many elements, which formerly were classified as being non-physiological, should now be included within a revised definition of the
“metlallome” and “metallomics” (a branch of science originated by R.J.P. Williams to the study of some 20 physiological metal ions concerning,
inter alia, the connectivity between geological and biological matrices). Mast cell derived highly purified anionic polysaccharide H (used as a
blood anticoagulant) which had been purified to different extents during manufacture are good examples of metallomic matrices. This included a
highly multi-element substituted ‘sodium’ H as well as purified forms of H, e.g., ‘lithium H’ (the ‘toxic’ element contents now conform with
Pharmacopoeia requirements) which suggested that the retained multi-inorganic element contents in all H mostly derived from their original in
vivo sequestration from extracellular multi-element containing bathing fluids. This sequestration process was apparently more complex than a
simple counterion condensation since both cations and anions as well as colloidal inorganic particles were apparently simultaneously sequestered.

5-2-1 Inorganic Elements: Seawater Compared with H

In agreement with the hypothesis that multicellular animals first evolved in a marine environments, the

full 60+ inorganic ion compositions of animal (especially extracellular) biological fluids can be shown to

mimic that of seawater4

This phenomenon may by why the total amounts of HS and other glycosaminoglycans occurring in the

tissues of in fifteen species of aquatic including marine invertebrates demonstrated an fairly exact

exponential mathematical relationship with the degree of salinity of their habitats5. This finding could add

support to the notion that the sequestration of inorganic elements from natural growth media might have
been the primary reason for the evolution of HS during the early stages of the evolution of multicellular

animals (HS appeared at the same time as this1a-1,2) in the sea, a phenomenon which could still be of

relevance to the full understanding of the modus operandi of these polysaccharides in present day

organisms).

A further confirmation of this notion is that H, [n.b. this is a highly purified, protein free, animal mast

cell- derived polysaccharide] apparently always clearly demonstrates, (e.g. when studied by mass

spectrometric techniques cf. Table II) a (skewed but) characteristic seawater/extracellular biological fluid

like multi-inorganic element ‘fingerprint’ similar to that of seawater bathed marine polysaccharide

matrices, suggesting that H and the structurally closely related HS (a likely difference, however, is that the

protein cores of HS can also bind additional metal ions) simultaneously sequester the full range of

inorganic ions and particles which can naturally occur in their normal bathing solutions.

Numerous reports6a confirm that a variety of metal ions, including highly charged multivalent ultratrace

elements, bind at often modest but physiologically relevant (and rather similar) affinities to HS under in

vitro and in vivo conditions (such investigations include those relating to scintigraphic visualisation of
tumours and a arange of multivalent metal ions used for the histochemical staining of tissues6a).

Anions are also known to bind to H. These include SO42- which commonly contains ca. 12% S in this
form 6a-1.

6. Relevance of H/HS Biochemstry to Disease

6.1 Possible Pathological perturbation of inorganic signaling by H/HS by metal

A corollary to the above discussion that H/HS provides an essential inorganic reservoir system of

inorganic cofactors for correct in vivo H/HS activity is that such activities may be subject to pathological

disturbance of inorganic components, e.g., by anthropogenic environmental inputs.

The etiologies of some forms of cancer and other degenerative diseases1a could centrally involve

perturbation of polysaccharide protein cross-linking (especially that facilitated by Ca2+) involving an

altered modulation of the activities of growth factors involved in wound healing which are putatively

perturbed in all degenerative disease processes. Epidemiolgical evidence that serum ferritin (“iron

overload”) was highly significantly correlated with cardiovascular dysfunction determined mortality in

Eastern Finnish men (Salonen et al. 1) which might suggest that, e.g., inappropriate redox active iron

catalysed de-N-sulphonation of HS which might occur under these conditions could render the resultant
vascular surface HS polysaccharides defective for protein regulation and for the inhibition of pathological

calcification (Grant et al., 1(1992)). Such d-N-sulphonation might also be involved in the etiology of arthritic

diseases which are a charcterized by dyshomeostasis of nitric oxide (cf. Stichenoth & Frolich1)

Prions both bind copper ions and HS (the biochemistry of which seems central to their function) which

could suggest that some anthropogenic alteration of metal ion concentration (e.g. under condition of copper

depletion, the presence of excess manganese could promote the Mn3+** ion induced misfolding of prions

(cf. Treiber et al.1). This scenario might be involved in the etiologies of prion diseases for which H-

mimetics apparently exhibit an highly effective therapeutic potential (cf., Grant1 (Discussions with M Purdey).

6.1-1 H/HS Biochemsitry & Degenerative Diseases

Possible Example of Anthropogenic Metal Ion Perturbation of HS Biochemistry in the Etiology of
Multiple Sclerosis
The primary current use of interferon gamma (IFN-γ ) therapy is apparently for therapeutic alleviation of the

symptoms of multiple sclerosis. Such a use of this anti-inflammatory cytokine may, however, apparently

could depend on its correct interactions with HS (via interactions of IFN-γ with specific sites believed to

consist of N-acetylated glucosamine rich domain and two highly sulphated sequences, cf. Sarrazin et al.1)

It might further be worth considering the putative roles of inorganic factors in such binding (both in the

normal function and therapeutic intervention situations); the anti-inflammatory effects of cytokines such as

IFN-γ elicited by HS interactions could require additional roles of water structure/inorganic cofactors which

in turn could also be subject to possible anthropogenic pathological input.

Evidence for anthropogenic metal ion perturbation of H/HS signaling in the etiology of multiple sclerosis

is suggested by epidemiological and geographical/geological studies of the prevalence of this disease. In a

discussion of the incidence of multiple sclerosis in North Scotland (a region with amongst the highest

international prevalences of this disease) D.I. Shepherd1 noted that this highly distinctive disease

had first came to the attention of medicine coinidentllay with a greater use in dentistry of

mercury amalgams; although the prevalence of the disease tends on a global scale to increase with

increasing latitude the highest prevalence in England was found to be in the most southerly county

(Cornwall) suggesting that e.g. the minerals derived from older rocks there present which leached into water

supplies and thereby into biota or regional variation in genetic factors also promoted the disease

The latitudinal factors implicate the quality of sunlight UV exposure and vitamin D and calcium

dyshomeostais. A possible connection between Vitamin D isoforms generated by UV B and the inorganic
sulphate transporter (Dixon & Markovich1) suggestes that the availability of inorganic sulphate which is

essential for HS assembly might be compromised under conditions of Vitamin D isoform and sunlight

quality inadequacy. This might mbe further compromised under modern conditions of inorganic sulpahte

depletion of soil (previously supplied from the combustion of coal). Ba2+ intoxication perhaps in

combination with such deficiencies possibly augmented by tissue disruptive effects of infrasonic shock

waves associated with (military) aviation and industrial mining and quarrying activities were proposed to

affect the folding of metal ion prion adducts2 might also promote multiple sclerosis since prion biochemistry

seem centrally linked to that of HS. Multiple sclerosis migh also be dependent upon defective FGF growth

factor signaling (a Ca2+-dependent HS directed growth factors/growth factor receptor assembly

processe putatively subject to Ba2+ and Sr2+ perturbation) thought to be essential for myelin sheath

renewal2.
The electrophoresis of H in a Ba2+ containing buffer solution while providing an effective method of demonstrating the complexity of
the mixture also suggests how in vivo signaling or its disruption could depend on interactions of H with alkaline earth cations. This may be relevant for the
mechanism of fibroblast growth factor (FGF) signaling thought normally to depend on a quaternary complex of HS-FGF -(Ca or Mg)-fibroblast growth
factor receptor (FGFR)9b-3 and how replacement of Ca or Mg with other metal ions might account for disruption FGF-FGFR signaling in demyelinating
degenerative diseases which have been suggested might have been triggered by the chronic exposure to inappropriate amounts of Cu, Zn, Pb, Al, Ba or Mn
ions.

6.1-2 Possible Perurbation of H/HS Signaling by Al3+ , Zn2+ (Cu2+/+) in Alzheimer’s Disease

Al3+ (-HS related?) induction of dementias (cf. Snow1). The amyloid precursor protein of Alzheimer’s

disease has also been reported to modulate the Cu/Zn nitric oxide deaminative cleavage of HS

(Cappai et al., 1) indicating an possible role for altered redox balance/metal ion related HS tissue protective

surveillance systems in this disease which may be why HS oligomer mimetics (e.g. PI 88) have been found

to offer therapueutic interventive benefit in this disease (as well as for the inhibition of pro-tumor

angiogenesis and heparanase activities)**.

6.1-3 H-mimetic Pentosanpolysufate (PPS): putatively protects agaisnt misfolded prions and also is a potent
HIV antiviral agent
H-mimetic drugs such as pentosan polysulphate (PPS) provide an effective therapeutic
intervention strategy for prion diseases; this was first reported by Diringer & Ehlers1 and confirmed by later workers; H/HS
and PPS are also highly effective against intractable viruses such as HIV-1 and II (cf., e.g. E. De Clercq TiPs. 1990, 11, 198-
205; similar results were obtained by Aberdeen polysaccharide researchers for an algal PPS-like polysaccharide).

-----------------------------------------------------------------------------------------------------------------------
6.1-4
It can rationally be suggested that the systemic dysfunction of inorganic-ion-determined supramolecular

structures in H/HS glycocalyxes could also be relevant to the etiology of a wide range of degenerative

diseases incuding cancer, atherosclerosis and dementia for which H therapy has also been suggested to be of
benefit 1a (Engelberg reviews).

6.4 The Defective Nitrosative Cleavage which has been Indicated to Occur in Niemann-
Pick Disease Could also be Relevant to the Etiologies of Cancer and other
Degenerative Diseases

Defective nitric oxide-dependent deaminative cleavage of HS (and pathological nitration) has been

reported to be associated with Niemann-Pick disease (K. Mani et al. 1a-13-2 )(a condition where an HS-

dependent recycling of cholesterol is defective but this becomes less severe in presence of adequate

ascorbate which is believed to increase defective nitric oxide availability, e.g. via the redox metal ion

catalytic process for nitric oxide cycling).

A related dependence of aberrant nitric oxide metabolite HS-related tissue damage on insufficiency of

ascorbate is probably also centrally relevant to the etiology of cancer in a redox-related HS scenarioc (an

idea supported by the observation that ascorbate in cell culture experimental observations to greatly

increases the degree sulphation of fibroblast HS (evidently via the modulation of the primary

biosynthesis1a-7).
------------------------------------------------------------------------------------------------------------------------

6.3 Does HS provide for a servo feedback system which directly interfaces
the environment ?

While ascorbatec, retinoic acid, and other beneficial fatty acid types (e.g. in aggregated form),

apparently induce beneficial HS structural alterations, excess blood glucose, oxidized lipids (and other

inappropriate lipids?), virus infections etc. 1a-7, and toxic ions (Pb2+, Cd2+ and Hg(II)) as well as very high

concentrations of F- (cf. Table II) can apparently negatively perturb HS microstructure and associated

activity. (Many elements which hitherto have been considered to be toxic might also conceivable normally

be required to optimize the H/HS metallomic matrix chemical composition and perhaps induced water

structure, cf. section 3.3). HS microstructure, it seems, can also be induced to change in direct

response to the presence of pathological organisms (perhaps sensing their surface ionic patterns) and

specific inorganic surface/crystal structures (e.g. calcium oxalate crystal surfaces which can apparently

generate the correct HS microstructures to optimally inhibit the growth of such crystals; cf. F.T. Borges et

al.,8 ; these studies also could suggest a possible similar scenario with hydoxyapatite). There seems to be

overall sensitivity of HS microstructure flexibiluity to combat or enhance a wide variety of extracellular and
environmental signals.
A similar list can be suggested for effect of diet on nitric oxide biochemistry but the details of how this fully impinges on HS biochemistry are less clear
apart from the utilization of nitrosative scission for generation of HS oligomers used as inter- and intracellular signaling molecules.

The attainment of correct inorganic element supramolecular structuring is also likely to be highly dependent

on the existence of correct H/HS microstructures as well as the presence of the correct distribution of

inorganic elements in the bathing fluids sources of the inorganic ions and particles which attach to H and

HS. Pathological influences will include alteration of H/HS microstructure during biosynthesis or

postsynthetic structural alterations so as to diminish the structures needed to accommodate an optimum

metallomic matrix and also the possible inclusion into this matrix of inappropriate amounts of

anthropologically augmented toxic inorganic elements from the bathing fluids.

It should be noted that H/HS polysaccharides seem to preferentially sequester those ions which occur in

such fluids in ultra traceamounts thereby permitting such elements to potentially amplify their putative

H/HS-related biochemical signaling inputs including the morphological modification (e.g. by Si) of

inorganic matrices such as nascent bone which are believed to controlled by adjacent HS-related

polysaccharide surfaces (cf. Wise et al.1).

The efficient binding by H of e.g. Ca2+ ions or CaCO3 crystallization seeds requires GlcNSO3 groups and

depletion of such groups in H/HS greatly diminishes such binding of Ca2+ (Grant et al., 1). Redox

metal ions may both stimulate pathological calcification and Fenton reactive oxygen radical

generation. The metallomic nature of H/HS could input into normal and pathological calcification,

proteinaceous plaque formation and the ability of H/HS to provide antioxidant protection.

Pacification of iron ions by their incorporation into polysaccharide aggregates

Redox active Cu and Fe ions deactivated for potential free radical generation by their transformation into

sparingly soluble metal ion aggregates containing H/HS proteoglycans may act analogously to the

apparent antioxidant actions factilitated by similar aggregates formed by iron ions with other anionic

polysacchrides (Sipos et al.1 , Merce et al1) and ternary complexes containing proteins such as amyloid

fibrils 1a-3.

Correctly structured H/HS including the optimal metallomic environment and water structure is likely also

to be required to accomplish these tissue protective actions.

H/HS biochemistry has also been implicated in the mechanism of antioxidant protection additional to the
more commonly acknowledged antioxidant role of HS in potentiating the activities of enzymic superoxide

dismutases and selenoprotein antioxidative systems1a the efficiency of which could also depend on the

presence of the correct inorganic elements cofactors needed both for active centre generation in the protein

and also to bind these proteins to the correct H/HS microstructures needed to sequester the proposed

optimal inorganic element matrix components.

An alternative explanation of the blood vessel wall change in HS could be suggested to a servo-feedback
response to counter the age-related diminution of fibrinolytic activity, increase of serum ferritin (Jarrett et al.l )or
tendency for formation of hydroxyapatite crystallites at blood vessel surfaces which could conceivably signal for altered HS microstructure generation to
combat these potential threats (this process is suggested by the results published by F.T. Borges et al. 8).

The hitherto unacknowledged roles of perturbation (e.g. by their lower or higher than optimal concentration

or by the inappropriate presence) of inorganic cofactors may explain why H/HS molecules, their fractions

or inorganic adduct polymers, oligomers and their mimetics have been reported to both inhibit (H.

Engelberg1) as well as to promote (cf., M.O. Ouida et al., 1) tumour growth and similarly both inhibit or

promote a range of other degenerative diseases as augment or diminish or promote the activities of

pathological organisms and the possibly related puzzle of why HS-related molecules can both apparently

promote as well as inhibit prion-dysfunction diseases.

The possible benefits of ascorbate therapy for the treatment of cancer and viral infections was discussed by Linus Pauling.
Such effects can now be rationally ascribed to the augmentation of HS protection afforded by ascorbate therapy (cf. D Grant, internet discussions posted in
2000 at web.ukonline.co.uk/dgrant/dg4/ [similar addresses with dg2/ /dg5/ and dg8/].

6.4 Alternative and ethnic medical use of metallomic matrices**.

Can some of the non-anticoagulant suggested uses of H and claimed benfits of
Ayrvedic Shilagit etc. be dependent on the co-occurrence of similar ultratrace
elements in these preparations?

The alternative medical preparations Ayurvedic ‘Shilajit’ and some North American
(perhaps originally derived from Native folklore) seems to be classifiable as metallomic
matrices preparations (apparently derived from of multi-inorganic-element containing
geological fulvates). The seaweed preparation ‘Kelp’ is also a multi-inorganic-element
anionic polysaccharide rich metallomic matrix.
It is claimed that the trace and ultratrace inorganic elements additional to those which
are currently accepted as being required for human nutrittion in these preparations have
nutritional or therapeutic value, it being further argued that intensive industrial
agricultural practices has diminished such trace and ultratrace elements in normal foods.
Both the geological fulvates and Kelp resemble H in the apparent possession of such a
trace/ulttratrace repertoire of associated inorganic elements.
It might be suggested that at least some of the non-anticoagulant therapeutic benefit
which have been identified for H preparations (which have also been reported to vary
between manufacturers for such activities) is of a similar origin in providing a supply of
trace and ultratrace elements which could be required for optimum human health.

7. Interference from Inorganic Constituents in H during Blood Analysis

(Interference from the multiple inorganic constituents associated with H used as an
anticoagulant during the dertermination of inorganic elements in blood)

Multi elements occur at the greatest abundance in the traditional form of unfractionated Na H but they also

occur, albeit in reduce amounts, in “Lithium H” (cf. Table IV).

Such (claimed) single counterion forms of H apparently should more correctly be described as modified

multi-element matrices.

This circumstance confirms older observations3a that, if commercial H is employed as an anticoagulant for

blood inorganic element determinations, account needs to be taken of the potential interference for the

accurate determination of the inorganic elements in blood samples; the ‘inorganic contaminants’ present

in H clearly especially affect the accurate determination of those elements which occur in ultratrace

amounts in biological samples.

7-1 Variation between manufacturers of the degree of final purification of H

It is suggested that possible variation in ‘purification’ procedures between commercial operations can

also gives rise to the previously reported but hitherto inexplicable irreproducibility in the biological

activities of commercial H samples from different manufacturers 3b-10, 7.

For fundamental biochemical researches the in vivo forms of H and HS seem optimally to be required;

these are most likely the natural multi-element forms. This hypothesis requires to be more fully

established by further work.

8. Binding of Anionic Polysaccharides to Crystal & Other Inorganic Surfaces

A major additional potential role as part of this signaling and information processing of HS is that the

binding to colloidal inorganic particles (e.g. to crystal seeds which might otherwise cause pathological

crystallization) can apparently occur in a functionally relevant way; this seems to have been established for

the specific crystal surfaces of calcium oxalate appear to induce (evidently by some form of biofeedback) an
alteration in the kidney biosynthesis of HS so as to augment the specific anti-Ca oxalate crystallization

abilities8 of HS microstructures which are subsequently produced. The results presented in ref. 8 also

suggest (although less clearly) that hydroxyapatite crystals are also similarly capable of prompting for a

similar specific alteration of HS microstructures capable of inhibiting by surface adsorption and

deactivation of potentially damaging seed crystals of this composition.

Similar specific interactions between crystal surfaces and alginates were studied by the Aberdeen polysaccharide group; a
range of protein-free alginates polysaccharides of different defined microstructures appeared to be adsorbed onto inorganic
seed crystals and caused an inhibition of crystallisation; the relative degree of inhibition showed a mathematical
relationship between the crystal growth rate constants (which could be accurately determined) and the detailed “information
encoded” known microstructures of these polysachcarides9.
Essentially this is an in vitro demonstration of how information in polysaccharides with complex microstructure can be
directly “read-off” by an inorganic microstructure.
Calcium salt crystallization rates were indicated from similar studies of the kinetics of crystal growth in their presence to be
similarly dependent on H/HS microstructure (n.b. the similar binding of HS fragments to sparingly soluble calcium salt
seeds is thought to be relevant to the mechanism of control of both urinary and cardiovascular calcification9b and also to the
age-associated human cardiovascular dysfunction in which (programmed?) changes in blood vessel wall HS microstructures
are believed to contribute to1a-5. By an extension of this hypothesis, both normal ageing and degenerative diseases in
animals could have an ultimate link to age-related changes in HS sugar sequences which may include an age-dependent
alteration in the suggested inorganic cofactor effects).

Lima de Faria has suggested10 that the study of the seeded crystallization of specific complex

crystalline forms of CaCO3 at the most fundamental level gives insight into how biology could have arisen

without DNA-based genes10; the numerous forms of CaCO3 seem to be capable of reproduction in a

manner often assumed to be restricted to biological cells and normally associated with nucleic acid

determined genetics. This further suggests how reproduction of ‘species’ of pre-biotic particles (which

presumably could have been capable of Darwinian evolution into more complex true biological forms)

similar to specific kinds of CaCO3 particles could occur. The ability of polysaccharides to bind strongly to

such surfaces and modulate such processes could have had pre-biotic relevance since it is more likely that

polysaccharide-like molecules would have been able to spontaneously form (e.g. by the oligomerisation and

rearrangement of formaldehyde polymers) (cf. Grant1 1992).

A corollary to this idea is that information held in specific (e.g. anionic) sugar sequences in H/HS could

potentially be modulated by inorganic surfaces or membranes to which the inorganic elements which also

occur (albeit mostly in very small amounts) in biological fluids and tissues as well as in seawater4.

(These will include single ions, ion pairs and aggregates which are potential crystal growth nuclei (n.b.

physiological solutions are supersaturated as regards the formation of solid phases of sparingly soluble Ca

salts e.g. CaCO3 (calcite), apatite etc. Apatites characteristically are, like H, non-stoicheiometric salts
which contain a range of isomorphously substituted inorganic ions.

Colloidal complexes of hydroxyapatite - H can be prepared in the laboratory1h ** Such entities putatively

have biological significance.

8-2 Specific nucleation activities (e.g. afforded by natural ‘SiO2’ nanoparticles) may be
required for supramolecular structure formation in polysaccharides and be relevant to
how inorganic elements contribute to H/HS signaling
The high Si content in both human hair and H and the well-known association of Si with glycosaminoglycans3b-6 is in accord
with the likely importance of this element for mammalian biochemistry.
Although Si is known to be an essential animal nutrient and be critically involved with glycosaminoglycans, especially with
H/HS, the mechanisms of binding and physiological action are unknown.
Si could be the key element linking geological and biological “metallomics”, suggesting the need for
a wider “geoglyco-metallome” conceptualisation by which the inorganic “metallome” influences the heparanome, operating
in a manner receptive to inorganic matrices via cooperative interactions involving ultra-anionic polysaccharide side chains
for which an absolute requirement for specific metal ions e.g. Ca, Zn and Cu have already been established for several such
processes. An analogous role for Si might be anticipated

The association of metal ions and inorganic moieties with anionic polysaccharides seemed more to

resemble a phase change process than e.g. an electrostatic controlled counterion condensation process

(the Aberdeen polysaccharide laboratory studies of metal ion binding to H reported by Grant et al. 6b-2-2

suggests this hypothesis). Such a phase change would be subject, according to the general principles

governing the formation of phases in physical chemistry, will occur via a nucleation-seed-sensitive

mechanism (e.g, facilitated by suitable inorganic or organometallic nanoparticles) .

The actual chemical nature of the putative nucleation seed particles in use by biological systems are currently unknown. A
possible silica particle role in this function, however, seems credible. Si-containing particles (putatively amorphous SiO2
sols similar to those discussed by D. Grant et al. 6b-2-2) seem always to be a characteristic component of anionic
polysaccharides (K.A. Schwarz6). Their presence in such an association to fulfill a nucleation of phase change role might
account at least in part for the fact that Si is an essential nutrient for animals and other species.

8-3 Nucleation events (subject to inhibiton by H/HS) may determine the progression of amyloidoses**

H/HS exhibits a general ability to inhibit the formation of pathological plaques which includes those containing both
mineral and protein-based plaques. This phenomenon could be, at least in part, why such H-like drugs as petnosan
polysulfate are uniquely useful therapeutic agents for the inhibition of prion diseases and putatively also for other
amyloidoses.
The correct H/HS microstructure has been indicated by in vitro studies to be needed to faciltate the effective inhibition of
pathological seeding; e.g., whereas H was highly effective for CaCO3 seed particle inhibition, de-N sulphonated H
enhanced rather than inhibited the activity of seed particles.
Such a dependence on correct microstructure of plaque seeding inhibition by H-like molecules could suggest that a diesase
or an age-related alteration in the microstructures of anti-seed H/HS polysaccharides, so as to diminish their surface
adsoption and deactivating effectivness, might be the ultimate origin of amyloidoses. A corollary to this is that therapeutic
iontervention in amyloidoses should be achievable by administration of correctly-microstructured anti-seed agents such as
parmaceutical H or H-mimetics (such a possiblilty seems to be supported by in vivo studies). The putative age-or
pathological related diminution of the anti-plaque effectiveness of HS by diminusion of its N-SO3- content might be subject
to redox metal ion catlaysis (as has been suggested by in vitro observations). The presence of inappropriate amounts of
redox metal ions which are capable of such catalysis might be promoted by the increasing tendency for serum ferritin to
increase with age (cf. Jarrett1). The apparent association of excess manganese under conditions of
depletion of inorganic elements invovled in antioxidant protection or the presence of elevated amounts of silver (occurring
together with strontium and barium in a suggested piezolelectric particle system) which has also been suggested to promote
scrapie-like illnesses2 may do so at least in part via promotion of the putative redox metal ion catlalysis of desulphonation of
HS.

9. Could Systemic HS Signaling Generate a Biological Clock which is Speeded up

During Degenerative Disease Processes?

HS protoglycans (in concert with Na+, K+, Ca2+, Mg2+ other metal ions and nitric oxide) may empower

temporal control systems which include those directing cellular assembly during embryogenesis as well as

the controlled alterations of HS microstructure which influences the course of the normal ageing processes

in individual tissues and putatively also in the entire animal organism. Human ageing was suggested by

results tabulated by K. Murata & Y. Yokoyama1 to be accompanied by a relative diminution of the relative

amounts HS at arterial surfaces (this diminution was accurately linearly dependent on age) ; E. Feyzi et

al.1a-5, in related groundbreaking studies, found a similar linear age-related alteration of HS microstructure

(this effect of age on HS [or vice versa] was suggested by these reseachers to show that HS as being the

only such age-variable biopolymer system). Age-altered-HS-microstructure could be ultimately

responsible for the age-related increase in atheroma at vascular surfaces; cf. ‘correctly’ microstructured

HS (but not, e.g., HS depleted of NSO3- groups, cf. Grant et al., 1(1992) ) which is known to strongly inhibit

the formation of sparingly soluble Ca salt deposits which further suggests that altered an age-determined

alteration of HS microstructures could act as some kind of long-time-span biological clock which could

ultimately determine lifespan. A related short time span HS clock may also influence the cell cycle (cf. HS

has been reported by B. Roussel et al. 1, to occur in the mitotic spindle and an HS-microstructural alteration

has been associated with the absorption of light (cf. circadian rhythm) by the pineal gland1a-6. HS

instrument or clock-like actions, may be also occur by a related mechanism to that by which vascular

surface HS metal ion binding alteration of conformation has been suggested to act as a servo feedback

sensor for the blood flow at endothelial surfaces (experimental evidence for which has been presented by A.

Siegel et al.1 and others).

The dynamic regulation of HS microstructure and generation of small oligomers of HS by nitrosative

scission may occur in concert with a similar but apparently separate mechanism of post-synthetic alteration

of HS microstructure achieved by 6-O sulfatase (Sulf) actions both of which could occur in concert with

inorganic cofactors (especially Ca2+) with input from the water structures elicited by entire metallome to

create temporal and spatial patterns which act to control intracellular signals involved in the ageing

processes but which seem to be capable of being accelerated during degenerative disease processes.
10. HS as a Mimetic of Humic Polymer Buffers in Natural Waters**

The notion that H and hair multielements represents a systematic perturbation of the amounts of inorganic elements present
in pure seawater also applies to exchange of inorganic ions between two types of polymer substituted sulphate or sulphonate
ion exchanger systems and applies to the exchange of multielement containing heparin dispersions with a single counterion
substituted polystyrene polysulphonate ion exchange resin column (a routine method of purifying heparin). The binding
and redistribution of the inorganic elements by an analogous natural polyuronic acid ion exchange system may also be an
important determinant of the observed distribution of inorganic elements in seawater (which contains ion exchanger organic
matter as ca. 2µ g /ml polyuronate-like humic acid containing humic–C(O)O- anionic groups which are analogous to such
groups in animal and plant anionic polysaccharides). Such a structured humate system in seawater evidently is part of a
system of natural anionic inorganic ion homeostasis agents (a possible mechanism by which this occurs in seawater (or
seawater-like biological fluids in primitive and perhaps also in more highly evolved animal organisms) is that such
polyanonic molecules act as highly efficient inhibitors of crystallization enabling them to promote the sort of long-term
supersaturated solutions which exist both in seawater and biological fluids (as clearly shown by studies of the comparative
rates of seeded crystallization of e.g. CaCO3 and BaSO4 in the presence of such anionic materials (i.e., humic and fulvic
acids from marine and land humified organic matter as well as the more accurately molecular structure defined polyanionic
polysaccharides).
Ca2+ is the most abundant multi-valent ion present in the proposed series of materials and is also is most easily exchanged (viz. the exchange of Tl+ for Ca2+
occurred in heparin with an efficiency of 103 while Ce(III/IV) was exchanged by Tl+ (the least efficient exchange) occurred at an efficiency of 0.5 (cf.
Table IV)). The ‘residual’ Ca in each of the samples defines the ‘purity’ or the degree of ion exchanged purification. This gives a simple, but apparently
also a scientifically robust, method for classifying various commercial Hs which have been purified to different extents starting from a similar multi-
element containing crude tissue extract giving rise, following extensive purification (during which the least strongly held countercations such as Mn and
Mg are lost) to the sort of Na H listed in Table IV (arising from natural buffer systems supporting multi-element solutions like biological fluids or
uncontaminated seawater). It should be noted that the same method of classifying index does not seem to apply to algal polysaccharides affected by highly
polluted waters but it does apply to such substances obtained from organisms grown in natural seawaters).

The results presented for what seems to be a unrefined Na H (Table IV) suggest that an apparent systematic enrichment of
trace and ultratrace multi- inorganic elements from some seawater(-like) animal tissue bathing fluid or perhaps brine used
during manufacture (which requires further research to confirm the relative importance of each route) similar to the
enrichment of seawater elements by marine algal polysaccharides.

Natural polyanionic systems (e.g. the complex anionic polysaccharides systems of heparin, pectin, alginates as well as
the anionic humic materials in soils and natural waters) can apparently simultaneously sequester the full range of
inorganic ions which occur in the natural bathing solutions by mechanisms which tend to favour the preferential
uptake of the least abundant elements present.

11. References

Proteoglycans containing HS (e.g. the syndecans with transmembrane core proteins and the glypicans
with phosphidylinositol membrane anchors at cell surfaces and pelican and agrin at basement membranes) contain
highly anionic, variously sulphated linear uronic acid-glucosamine-disaccharide-based polysaccharide side chains
which engage a wide variety of biochemical functions. Supramolecularly structured H/HS proteoglycan-inorganic-
glycocalyxes can be predicted to be formed at all adherent animal cell surfaces and extracellular matrices.
Such organometallic matrices are suggested to potentiate H/HS participatation inter alia, in
blood anticoagulation, antioxidant protection, protection against pathogen insult, energy metabolism
including the modulation of lipoprotein lipase activities as well as complex cellular activities such as
embryogenesis, wound healing and immune surveillance.
11-1 Reference 1 (alphabetically arranged according to the first named author)
11-1-1 General reviews of H/HS 11-1-1General reviews of H/HS
General Reviews of H/HS
Bernfield, M. et al., Ann. Rev. Biochem., 1999, 68, 729-777 cf., also J. Turnbull et al., Trends Cell Biol., 2001, 11, 75-82;
 cf., P.W. Park et al., J. Biol. Chem., 2000, 275, 29923-29926; M. Lyon and J.T. Gallagher, Matrix Biol., 1998, 17, 485-493;
K. Mishra-Gorul et al with JJ Castellot Jr., Trends Glycosci. Glycobiol., 1998, 10 (52) 193-200
N. Perrimon & M Bernfield Nature. 2000, 404, 725-728
J.D. Esko & U. Lindahl have listed in a J Chem Invest. Supplement) “A Suggested reading list” entitled “Molecular diversity
of heparan sulfate” (available at http://www.jci.org/content/full/108/2/169/DC1)
Cf. also Editorial in The New England Journal of Medicine. 2001, 344; 673-675;
B. Mullow, An. Acad. Bras. Cienc. 2005 ,77 (4) 651-664,
Habuchi Glycoconjugate J., 2004, 21, 47-52; A.K. Powell et al., Glycobiology 14, 17R-30R

M. Yanagishita & V.C. Hascall (Cell surface HS protoglycans; Minireview) J. Biol. Chem. 1992, 267, 9451-9454 was cited as a key introductory paper
(which intimated a key activity of HS is that cell surface GAGs turn over within one eighth to one third of a cell cycle and hence as noted by L.M.
McDowell (ibid., 2006, 282 (11) 6924-6930) the quantity and the fine structure of GAGs
(especially of HSs) may alter in response to the presence of a variety of stimuli.
6-O-Endosulphatase (Sulf) Action and ‘Smart’ HS Microstructure Modulation
How Environemental Stimulae Change HS Microstructure Via Sulf Actions.
Sulfatase (Sulf) activity putatively involves EDITING of HS information and associated cellular feedback loops
Extracellular Postsynthetic Sulphatase Activities can systematically alter HS microstructure
Sulf-1 and Sulf-2, HS 6-O-endosulphatases, are now known to regulate the activity of multiple growth factors such as FGF and Wnt
Sulf-1 and Sulf-2 are also potent inhibitors of myeloma tumour growth in vivo,
(Y. Dai et al., J. Biol. Chem. 2005, 280 (48) 40066-40073
Cf. X. Yue et al., J. Biol. Chem 2008, 283 (29) 20397-20407
W.C. Lamanna et al., ibid. 2008, 283 (41) 27724-27735 discussed how
Sulf loss influences N-. 2-O, and 6-O- sulphation of multiple HS proteoglycans and modulates FGF signalling;
and HS 6-O-endosulphatases (Sulfs) seem to be ESSENTIAL FOR MAMMLIAN DEVELOPMENT AND SURVIVAL,
being putatively involved in a cellular SERVO-FEED-BACK mechanisms which include the regulation of cellular signalling, and apparently the
EDITING of the sulphation patterns of numerous HS proteoglycans, and modulation of the expression of various HS biosynthetic enzymes.
(Cf. G.K. Dhoot et al Science 2001, 293, 1663-1666 who drew attention to the how developmental biology was dependent upon not –direct-genetically-
controlled extracellular post synthetic alteration of HS microstructures by actions of 6-O-endosulphatase).

----------------------------------------------------------------------------------------------------------------------------------
Adiemian K.T. et al., J. Electrochem Soc., 2002, 149, A256-261
(Silicon oxide Nafion composite for proton-excahnge membrane fuel cell operation at 80-140oC)

Albertini R. et al., Int. J. Mol Med. 2000, 6, 129-136

(The effect of glycosaminoglycans on lipid peroxidation)

Bhattacharya A, Chem. World, 2008, 5 (10) 8

Bohrer, D. et al RBAC, 2004, 36, 99-103

Table II (p,102) of this article reports the aluminium contents of some commercial H : viz. in µ g/L a selection of H samples had
732, 783 (ca. 35ppm), 72 ,436, 137, 168 and 190 which should be compared with the amounts of this element found to be present in commercial samples
of EDTA (Na) (3,37) sodium tungstate (26.)5 and sodium sulphate (26.6).

Boyd J, F.B. Williamson and J Gettins, J. Mol. Biol., 1980, 137, 175-190

Bourin, M.C. & U. Lindahl, Biochem. J. 1993, 289, 313

(Role of H as a Blood Anticoagulant)

Brown, D.R. et al., Nature, 1997, 390, 684-686

(The cellular prion protein binds copper in vivo)
Brown, D.R., R.Besinger, Biochem. J., 1998, 334, 423-429
(PrPC may regulate some aspects of copper metabolism and Cu,Zn superoxide dismutase (SOD) activity by influencing Cu
incorporation into the molecule)
Cf., Brown D.R. Trend. Nurosci. 2001, 24 (2) 69-127
Cf. also C. Treiber, A. Simons & A. Multhaup, Biochemistry, 2006, 45, 6674-6680
(Mn ions and less severely Cu ions can re-fold normal prions to the pathological misfolded form;
these authors used a yeast cellular vector).
[Cf. also N.G. Rainov et al. (with K. Doh-ura) Expert Opin. Biol. Therapy, 2007, 7 (5) 714]
A putative role of HS-nitric oxide signaling in TSEs (Depletion of both nitric oxide and copper invovled?)
The putative copper-centred prion scenario further sugges a central role of nitric oxide biochemistry TSEs: cf.,
scrapie infection of neuroblastoma cells precludes nitric oxide production when the cells are challenged with
lipopolysaccharides (this was reported by H. Lindegren et al. J. Neurosci. Res. 2003, 71, 291-299); diminution of nitric oxide
(as noted by K. Mani et al., Glycobiology, 2004, 14, 599-607) will affect the extent of S-nitrosylation of HS glypican and the
subsequent generation of HS fragments used as hormone-like signalling messenger molecules (these may also be involved in
cognition processes as well as tissue protection and ‘wound helaing’); to probe such ideas further studies are needed to establish
the variation between healthy and diseased (misfolded prion affected) brain tissues of the nitrosative cleavage
markers (anhydro mannose-containing degradative products produced by nitric oxide metabolite cleavage of HS) ( [K. Mani et
al. further note in this paper that the HS-specific epitope 10E4 is nitric oxide sensitive and partly inaccessible in glypican-1 HS.
N.b., it had also been established by these authors that glypican-1 core protein nitric oxide recycling for nitrosative cleavage is
dependent on the Cu2+,óCu+ system and Cu deficiency has been demonstrated by others to occur in brain tissues of humans
afflicted with neurodegenerative diseases).
How the infectious prion theory has been modified by the finding of later workers to include effects of metal ion dyshomeostasis in TSEs Cf the work of
Supattapone S. et al. [Supattopone S et al., J. Biol. Chem. 2004, 279, (39) 40788-40794 {Ionic strength and transition metals control PrPSc proteoase
resistance and conversion-inducing activity}] and other studies [Delaut N.R. et al., Proc. Nat. Acad. Sci. USA 2007, 104, (23) 9741-9746 (Formation of
native prions from minimal components in vitro; Saa P. et al., J Biol Chem. 2006, 281, 35245-35252 employed sonication technology in a cyclic
amplification loop (PCMA ‘cyclic amplification’) which was critically dependent on the presence of a polyanion (this could be polyA, polyT, DNA or HS)
cofactor to achieve a greatly enhanced yield of PrPSc; related studies further suggested that the active infetious agent (an aggregated form of PrPSc)
generated by PCMA was held together by metal ions. Prior to the PCMA work the prion-only hypothesis of TSEs had lacked a suitable mechanism to
explain how the very low primary misfolded infectious particle (originally defined as a slow virus) titre could realistically be demonstrated to achieve the
observed pathology in vivo].
Cf ., also ref 2 (vide infra)

Cairns-Smith A.G. “Genetic Takeover and the Mineral Origins of Life”, Cambridge University Press, Cambridge, 1982

Capila I. et al., Structure (Camb.) 2001, 9, 57-64

(Ca2+ ions crosslink H to annexin-V suggesting that a similar crosslinking is needed for apoptosis)

Cappai R., et al., J Biol. Chem. 2005, 280, 13913

Challis B.C. et al. See Ref 1a-14

Dai Y et al., J Biol Chem., 2005, 280 (48) 40066-40073

Dawson P.A. & D. Marcovich, Pflugers Arch-Eur. J. Physiol., 2002, 444, 353-350

Ding, K et al., ibid., 2002, 277, 33353-33360, (Nitrosative signaling catalyzed by metal ions) cf
K. Mani et al., Glycobiology, 2006, 16 (8) 711-718 and refs.cited
K. Mani et al.. J. Biol Chem., 2007, 282, 21934-21940
R. Cappai et al., J. Biol. Chem. 2005, 289; 13913-13920;
K. Mani et al., ibid., 2004, 279, 12918-12923;
K. Mani et al., J. Biol. Chem. 2003, 278, 38956-38965
K. Mani et al., Glycobiology, 2000, 10 (6) 577-586
Cf. M.Belting et al., J. Biol. Chem., 2003, 278 (47) 47181-47189
F. Cheng et al., ibid., 2002, 277, (46) 44431- 44439
Cf. D.Grant, Chemistry Preprint Archive 2000 Oct 94-104

Fransson, L-A, B. Havsmark, Carbohydr. Res. 1982, 105, 215-225

Fransson, L-A, ibid., 1982, 110, 127-133; Eur. J. Biochem., 1981, 120, 251-255

R. Gabizone, R. et al., J Cell. Physiol. 1993, 157, (23) 317; Chem. Abs. 123, 249546j
(H-like molecules bind differentially to prion proteins and change their metabolic fate)

Gould S.H. et al. (with D.A Rees) J. Chem. Soc. Perkin Trans. II, 1975, 237-242

Grant, D. (1977) Nature, 279, 709-710

(The chemical structure of humic substances)

Grant D. et al., (1984) Biochem. Soc. Trans.,12, 302

(Altered water structure in mixtures of H and metal ions)

Grant D et al. (1987a) Biochem J., 244, 143-149

Biochem Soc. Trans., 1990, 18 (6) 1283-1284
(Infrared spectroscopy of H-cation complexes)

Grant, D. (1986, 1987b) (with W.F. Long & F.B. Williamson) Thrombosis Research, 1986; Cf., Supplement VI, 92; Cell Biol. Int.
Reports, 1987, 11, 220

(Cation interaction with H at antithrombin-binding sites differ from that occurring elsewhere in the polymer. It was
noted that especially larger shifts in NMR absorptions due to metal ion interaction were observed close to the
hypersulphated region which occurs in the centre of this epitope;

Grant D. et al., (1988) Biochem. Soc. Trans., 16 (6) 1030-1031

(Effect of H on dismutation of superoxide ions)

Grant D et al (1988a) ibid., 16, 1029-1030

(Polystyrene surfaces may require structured water for effective cell adhesion)

Grant D., et al., (1989) Biochem J. 259, 41-45

(Inhibition of glycosaminoglycans of CaCO3 (calcite) crystallization

Grant D. et al. (1990) Biochem. J. 1989, 259, 41-45

(The dependence on counter-ion of the degree of hydration of H)
Cf., Biochem Soc. Trans., 1984, 12, 302
Grant D. et al., (1991) 277, 569-571
(Infrared overtone study using an hydrophobic medium of the glue –like action of water molecules at H surfaces for
poly-L lysine and poly-L arginine binding)

Cf . Grant D. et al., (1992) Med. Hypoth., 38, 49-55 (Degenerative and inflammatory diseases may result in effects of anitmineralization mechanisms
afforded by glycosaminoglycans; cf. ibid., 38, 46-48 (A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to
modern pathological afflictions).
(In the first of these article a review of antimineralization actions of glycosaminoglycans it was suggested that defects in this protection might contribute to
degenerative diseases and also (in the second article mentioned) that the early evolution of life might have depended on polyanions such a polyphosphates
and the precursors of glycosaminoglycans in cooperative interactions with metal ions in minerals related to apatite; it should be noted that the interaction
of Ca2+ with H and HS is greatly diminished following de-N-sulphation (cf. Laing T.N. et al., Carbohydr. Res. 1982, 106, 101-109 Ayotte L & A.S. Perlin,
ibid., 1986, 145, 267-277 and Grant D. et al., (1992b) Biochem. J. 282, 601-604. Such loss of NSO3- groups could arise by actions of unliganded redox
metals**).
Ca2+ H complex formation studied by polarimetry) and Grant D et al. (1988b) Biochem Soc. Trans. (1988c), 1028-1029 (NMR data suggested that under
0.15M physiological NaCl concentration conditions Na+ and Ca2+ reacted with H at similar affinities being bound stochastically with some compensation
effect associated with cooperativity between adjacent binding sites for Ca2+)
Grant D. et al. (1992c) Biochem. J.. 285 477-480 (A potentiometric titration study of the interaction of H with metal cations)

Grant D. et al. (1992d) ibid., 287, 849-853 (Zn2+ H interaction studies by potentiometric titration);
(Unpublished similar researches into Mg2+. Mn2+ and Al3+ H binding studied by potentiometric titration indicated that the
relative strength of complexation suggested by pH changes effected under standard conditions by binding of metal ions to Li H was
Mg2+ 1.0, Mn2+1.14, Fe2+ 1.34, Ca2+,1.75, Cu2+ 3.0, Fe3+ 3.24, and Al3+ 4.54 (this cation differed greatly from the other metal ion by its creation a pH 4.5
buffer system following saturated binding to H whereas the other cations studied created a near physiological pH buffer system under these conditions)

Grant D et al. (1992e) Biochem. J. 283, 243-246

(Cu2+ H interaction studied by polarimetry)
Cf., Biochem. Soc Trans., 20; 214S
(13C-nmr spectroscopy of Cu2+-H suggests phase separation of the complex from Cu2+ in aqueous solution)

Grant D (2000b)
Discussion Documents (Internet)
Nitrosative H/HS Signaling
Nitrosative H/HS signaling catalyzed by metal ions putatively constitutes a major inorganic ion assisted H/HS information processing and control system
in animals which seems to have been highly conserved throughout animal evolution (cf. 1a-1,2) and which could have enabled environmental inputs to
influence the course of such evolution. This system might have arisen from an earlier system where HS evolved to mimic the natural humic (poly
carboxylic acid) polyanionic buffer systems of natural waters. This notion is in keeping with the report that concentration of dispersed or soluble HS in
blood (cf., W. Manley et al., 1) is similar to that of the poly-carboxylated poly-counterion salt containing natural (humic) polymers in natural waters
including the sea where they putatively contribute to inorganic ion homeostasis (including by the potent inhibition of crystallization of sparingly soluble
salts such as CaCO3).

Internet
Grant D (2000a)
Multi-Ion Content of H
Chemistry Preprint Server CPS biochem/0010002
Cf. also refs 3b1 – 1b-10 (vide infra)

Internet
Grant D (2000)
Ascorbate & nitric oxide in redox control of HS structure
dgrant@ukonline.co.uk/dgrant/

files ( through Yahoo)

from
http://web.ukonline.co.uk/dgrant/dg1/
http://web.ukonline.co.uk/dgrant/dg2/
http://web.ukonline.co.uk/dgrant/dg3/
http://web.ukonline.co.uk/dgrant/dg4
http://web.ukonline,co.uk/dgrant/dg8
[the latter file is a list of the references cited in the previous files]

D Grant
Discussions with F.B. Williamson (Aberdeen)
HS hypothesis of animal evolution.
HS by its sensitivity vial nitrosative processes to environmental changes can also signal systemically for nitrosative selective genetic mutation. These
events can link to create systemic response to altered food availability and other environmental changes.
(Cf. also A Varki, Cell, 2006, 126, 841-845 (Nothing in glycobiology makes sense except in the light of evolution) has suggested that polysaccharides in
general might be centrally involved in evolutionary pathways)

D. Grant
Discussions with Mark Purdey (Taunton)
Cf. M. Purdey, Med. Hypoth., 2004, 62, 746-754;
and on Roles of H/HS in Prion Dysfunction Diseases
(continued with Graham Steel)
HS has been indicated to determine the metabolic fate of prions which also seem to bind and can be misfolded by bound metal ions (for which Mnn+ ions
cf. Treiber et al.1, seem to be of especial relevance for the possible augmentation of prion misfolding under the conditions of Cu depletion and Mn
intoxication. Cf. also the putative antioxidant roles of Cu ions in prion biochemistry discussed by D.R. Brown et al. 1). The H/HS-mimetic pentosan
polysulfate (PPS) which can apparently prevent or inhibit misfolded prion diseases may do so, at least in part, by influencing the putative redox metal ion-
HS-prion-nitric oxide signaling system, the efficiency of which could influence how prions are normally prevented from misfolding in vivo.
----------------------------------------------------------------------------------------------------------------------------------------
Discussion on Mark Purdey’s HS wound healing perturbation hypothesis of the etiology of multiple sclerosis indicated that the efficiency of a sulphate
transporter which was implicated in HS biosynthesis depended on a vitamin D isoform availability (with an associated input from UV B exposure)
suggesting an explanation for the latitudinal dependence of the prevalence of multiple sclerosis and especially the more exact latitudinal variation of the
prevalence
which is especially marked in Australia and the USA;
cf., also the internet discussion at
http://www.medicine.ox.ac.uk/bandolier/booth/neural/MSgeog.html
which includes the results reported by
I.A.F. van der Mei et al., Neuroepidemiol. 2001, 20, 168-174

Grant, G.T. et al., (with D.A. Rees), FEBS Lett. 1973, 32 (1) 195-198

Grenne T.S. et al., Geology, 2003, 31, 319-322

(Paleozoic and Mesozoic silica-rich seawater: evidence from hematic chert (jasper) deposits)

Haraguchi, H., J Anal. At. Spectrom., 2004, 19, 5-14

(Metallomics as integrated biometal science)
This paper restricted for biopolymer to a discussion of the relevance of metallomics for biopolymers to proteins. Polysaccharides, especially the
polyuronates and highly anionic glycosaminoglycans seem also, however, to be especially designed to act as multi-element metallomic ligands. The
inorganic element reservoirs in anionic polysaccharides may participate in evolved signaling mechanisms. Inorganic components may participate
in the transmission of information encoded within anionic patterns of the ultra anionic animal polysaccharides H and HS to enable these
polysaccharides to control protein activities.

Iozzo R.V. J. Clin. Invest., 2001, 109 (2) 165-167

Jaques L.B. ref. 6a (vide infra)

James, P.L. et al J. Materials Sci., 2000, 35, 5111-51119

(Hydraton of Nafion(® studied by AFMand X-ray scattering)

Jarrett D.J. R. et al., J Clin Exp Gerontol., 1989, 11 (3-4) 145-154

Jones L.S, et al, Mol. Cell. Proteomics, 2004, 3, 746-769

Polyanons and the proteome
Emerging evidence for the ubiquitous nature of polyanionc/protein interactions perhaps traceable to early (pre-biotic) pre-cellullar interactions
This review also discusses use of GAG-mimetics as therapeutic agents, e.g. for Alzheimer’s disease

Kojima S. et al., e.g. in Eur. J. Nucl. Med., 1983, 8, 52-50; (Elevated uptake of 67Ga and increased HS content in liver-damaged rats)
Cf.., ibid.; 1984, 9, 51-56
[These authors showed that cations which have a high affinity for HS in vitro and in vivo include Al3+, La3+, Ce3+, Er3+, Sm3+, Yb3+, In3+ , Ru3+, Eu3+ as well
as Fe3+ and Fe2+]

Kraemer, P.M. J Cell Physiol. 1968, 71, 109-120

(Production of H related glycosaminoglycans by an established mammalian cell line)
[This was the first report of the occurrence of free cell cytosol HS, not linked to protein cores; the author had also noted that N-linked –SO3- goups were
extremely labile in comparison with tahe O- linked –SO3- groups; this phenomenon assumed greater importance following the discovery that such lability
is of possible critical relevance to how inappropriate HS microstructural alteration could occur in degenerative disease processes since the hydrolysis of N-
linked SO3- produces glucosamine-NH2 groups which are primed for rapid reaction with nitric oxide metabolites such as nitrite which is characteristically
found to be associated with degenerative disease conditions which are also characterized by the nitration of tyrosine groups in proteins, cf., Ischiropoulos
H, ref. 1a-8, vide infra]

Kreuger, J, P. Jemeth, E. Sanders-Lindberg, L.Eliahu, D. Ron, C. Salmivirta and U. Lindahl, Biochem J. 2005; 389, 145-150;
Cf., Kreuger, J. et al., J Cell. Biol. 2006, 34 (3) 461-469
{Difficulty with a “DNA-analogy signaling system model” for HS}.
The smart information-encoded system manager modus operandi where dedicated patterns of anionic sugar
sequences is supposed to act as specific sites for protein binding has recently been questioned here by Kreuger
et al., who propose that the antithrombin pentasaccharide sequence is only true type of unique H/HS
epitope.
This idea is contradicted by e.g., the more recent report by Vanpouille et al. 1
that Cyclophilin A binds to H/HS via a specific type of microstructure consisting of rare sites containing
3-O-sulphated, N-unsubstituted glucosamine groups;
similarly it had been earlier indicated that differently structured H/HS fragments differentially inhibit the
binding of lipoproteinase to α 2macroglobulin (cf., Larnkjaer et al. 1).
It is now suggested that this observed lack of sensitivity found by Kreuger et al. could have arisen from a diminution of the
essential inorganic cofactors which had been present in intact H/HS polymers during the oligomer
preparation and fractionation procedures. This idea is consistent with the findings (e.g. Grant et al.1 and
Rovo & Morava1) that fragments of H generally bind metal ions less effectively that do intact polymers.

Larnkjaer, A. et al., Biochem J. , 1995, 307 (1) 205; Chem. Abs. 122, 285358v
(Structure of H fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to α 2macroglobulin-receptor/low density
lipoprotein receptor –related protein by heparin fragments)

Liu, D et al., J. Biol. Chem. 1999, 274, 4089-4095. (Roles of Metal Ions in Bacterial Heparanase)
(The Ca binding site of bacterial heparanase I is essential for its activity)
Cf., also Mihailescu, G, K.J. Nitelea, Rom. Pat. RO 76, 508; Chem Abs., 99, 93718x.
(The addition of zinc acetate was indicated to prevent loss of H due to heparinase activity during
extraction of H from animal tissues)
Shaya, D et al., J Biol. Chem. 2006, 261, (22) 15525-15535 reported that
a Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one
wall of the substrate-binding site (from studies of heparinase II form Pedobacter heparinus (Flavobacterium) and its complex
with HS-related disaccharide;

Long, W.F., & F.B. Williamson, IRCS J. Med. Sci. (Library Compendium) 1979, 7, 429-434;
Ideas arising from experimentation coupled with an extensive literature review suggested that
HS evolved from its evolutionary precursor (similar to bacterial heparanosan (cf. Bhattacharya1 this starting material is retained for Golgi apparatus
generation of animal HS polysaccharides). Isomerization of most of the precursor glucuronate to iduronate achieves a more flexible system of Ca2+
buffering which could permit HS to more effectively act as a feedback control loop including those facilitated by a range of metal ions.

Long W.F. (2003) http://www.abdn. ac.uk/~bch118/publications2003march.doc

Luck, W. Ber. Bunsengesel. Phys. Chem. 69 (1) 69 (cf. ibid., 626)

(Salt effects on the association of water)
(This and related work established that the origins of the Hofmeister effect were probably the effects of
Hofmeister agents on the supermolecular structure of liquid water)
Cf. Kleeberg E. Ed. (Proc. Symp. (2-3 Apr 1987 Marburg in Honor of the 65th Birthday of WAP Luck)
“Interaction of Water in Ionic and Nonoionic Hydrates”. Springer Verlag, Berlin, 1987;
Luck W.A.P. Top. Curr. Chem. , 1975, 5, 115-180 (Is life mainly inorganic chemistry?)

Merce, A.L. et al., J. Inorg. Biochem. 2002, 89, 212-218

(Complexes of Fe(III) with hyaluronan)

Moffat, Colin. F., Ph.D. Thesis University of Aberdeen 1987 “Synthesis, characterization and applications of chemically modified heparins”; the
multielement data for ‘Na’H are given on p.187-189 and for Tl H on p.195-196.
The procine sodium H which was selected for more detailed inorganic analysis had been donated (apparently to several academic research laboratories) by
Dr W.E. Lewis, a scientist involved in the commercial manufacture of H at a major (Runcorn, Lancs.) UK pharmaceutical industry facility.

Morissete C. et al., Potential application of glycosaminoglycan-related compounds in Alzherimer’s disease

Pharmacological Mechanisms in Alzheimer’s Disease
Springer New York, 2008, p255-272

Multhaup G et al., Biochemie, 1994, 76 (3-4) 304

Murata K & Y. Tokayama, Atherosclerosis, 1989, 78, 69-79

(Acidic glycosaminoglycans in human atherosclerotic cerebral arterial tissue)

Nader H.B et al., Int. J. Biol. Macromol., 1981, 3 (6) 356-360

Nader H.B. et al (1983) see ref. 5 (vide infra)

Ouida, M.-O., et al., Biochem. Biophys. Res. Commun. 2007, DATA

(Heparanase I generated HS polyanonic sequences by scission at highly sulphated regions which promoted tumour growth while heparnase III generated
HS sequences which inhibited tumour growth via scission at less sulphated regions.
The modus operandi is believed to be HS microstructure-dependent modulation of basic fibroblast growth factor activity)

Panov V.P. & A.M. Ovsepyan, Visokomolekularnie Soedineniya 1984 26, (9) 1963-1970
(Study of H salts by spectroscopic methods)
[This elegant study reports how counterions affect the infrared and NMR spectra of H]

Parrish R.F. & W.R. Fair, Biochem J., 1981, 193, (2) 407-410
(Cf D. Grant et al. ibid., 1992, 287 (3) 849-853 and Woodhead N.E. et al., ibid., 237 (1) 231-4

Pedersen, L.C. & M. Jorgensen, Biochem J. 1983; 211; 91-97

Kinetic rate constants obtained in LiCl, NaCl or KCl electrolytes at physiologically relevant ionic strengths, suggested that the rate determining processes
for the principal H-mediated anticoagulation mechanism were highly sensitive to the chemical nature of the electrolyte solution, suggesting that the
presence of different alkali metal cations could markedly influence transition state reactivities.
Purdey, M., Med Hypotheses, 2004, 54, 278-306
[See also related discussions and literature search by D. Grant]

Rabenstein D.L. et al., Carbohydr. Res. 1995, 278. 239-256

Multinuclear magnetic resonance studies of the interaction of inorganic cations with H

Ricard-Blum, S et al. J. Biol. Chem. 2004, 279, 2927-2936

(The binding of endostatin to H requires the presence of Zn2+ ions)
Cf. ibid., 2006, 281 (35) 25195-25209
(The binding of colagen V to H/HS requires Ca2+ ions {Zn2+ ions cannot substitute for Ca2+ here})

Roussel, B. et al., Clin. Exp. Immunol. 82 (2) 294-299; Medline 91055393

(HS could be an essential part of all membrane structures where its could modulate their formation and activities)

Rovo J. & E. Morava, Kiserl Orvostud., 1981 33 (5) 528; Chem. Abs. 96, 24695p

Salonen, J.T et al., Circulation, 1992, 86 (3) 803-811

(High stored iron levels are associated with excess risk of myocardial infarction in eastern Finnish men)

Sarrazin, S et al. J Biol. Chem., 2005, 280 (45) 37558-37564

(HS mimicry. A synthetic glycoconjugate that recognizes the H binding domain of interferon-γ inhibits the cytokine activity)

Schwarz K.A. Proc Natl. Acad. Sci. USA 1973, 70, 1608-1612
(Bound form of silicon in glycosaminoglycans and polyuronides)
Cf. McCarty M.F. Med Hypoth., 1997, 49, 177-179
(Reported antiatherosclerotic acrivity of silicon may reflect increased endothelial synthesis of HS proteoglycans)

Seale, R., E.R Morris and D.A. Rees, Carbohydr. Res. 1982, 110, 101-112

Shinko, K., et al. (with C.P. Dietrich and H.B. Nader (H/HS Disaccharides obtained by bacterial heparinase could control cytosolic Ca2+ by inhibition of
Na+/Ca2+exchanger) (H and HS disaccharides bind to the exchanger inhibitor peptide region of Na+/Ca2+ exchanger and reduce the cytosolic calcium of
smooth muscle cell lines (oligosaccharides need C4-C5 unsaturation and 1->4 glycosidic linkage for activity) J. Biol. Chem. 2002, 277, (50) 48227-48233

Shepherd, David Ian, M.D. Thesis “Multiple Sclerosis in North-East Scotland” Volumes I & II, University of Aberdeen 1976

Siegel, S. et. al.. Colloids & Surfaces A: Physiol. & Energy Aspects, 1998, 138, -351
(HS may act as a biosensor and act as a flow sensor for flow control via biofeedback at blood vessel walls via conformation determined by bound Na/Ca)

Sipos, P. et al., J. Inorg. Biochem., 2003, 95, 55-63

(Formation of spheroidal iron(III) oxyhydroxide nanoparticles sterically stabilized by chitosan in aqueous solution)

Snow, A.D. et al. Am J.Pathol., 1988, 133,(3) 456-463

(The presence of HS proteoglycans in neurite plaques and congophilic angiopathy in Alzheimer’s disease)
Cf., also Ibid., 1990, 137 (5) 1253-1270; 1994, 144 (2) 337-347 and M.M. Verbeek et al., ibid., 1999, 155 (6) 2115-2125 (which identified agrin as the
principal HS proteoglycan in Alzheimer’s disease plaques)
Zn dyshomeostasis might be involved in Alzhiemr’s disease
Zn2+ amyloid protein precursor was indicated by G. Multhaup et al., (Biochemie, 1994, 76 (3-4) 304) to be mediated by HS proteoglycans in Alzheimer’s
disease
Al3+& HS (are putatively mutually strongly interactive sytems) D. Grant**
Cf., M.E. Ferrero et al., Biochem. Soc. Trans. 1989, 360
(Ateroid, a HS-containing mixture was indicated to be a possible therapeutically effective inhibitor of Alzheimer’s disease)
Cf. also S.A. Lorens et al,. Seminar Thromb. Hemost. 1991, 17 (Suppl.2) 164
{Other related studies [Canadian work]: HS mimetics as therapeutic agents for Alzheimer’s disease cf. R Kisilevsky et al., Nature Medicine, 1995, 1, 143-
148}
Other authors have suggested that Al3+ intoxication (perhaps related to Si ingestion) might induce Alzheimer’s disease plaques
e.g. G.A. Taylor et al J. Epidemiol. Community Health, 1995, 49 (3) 323-324;
Preliminary studies by D. Grant et al., have suggested that Al3+ uniquely binds and alters the pH buffering capacity of H/HS.

Stichtenoth D.O. & J.C.Frolich, Br. J. Rheum. 1998, 37, 246-257

(Nitric oxide and inflammatory joint diseases)
(It would be of interest to compare (the effect of metal ions) in the reactivity towards nitric oxide of normally and abnormally synthesisied HS: this was
discussed by the author in a presentation made to a Glasgow University seminar arranged by Prof. KE. McCall in 1999; this idea was posted on the
internet (Grant, 2000)

Treiber, C (with G. Multhaup), Biochemistry 45, 6674

Vanpouille, C. (with M. Lyon and D.G. Fernig) et al., J. Biol Chem. 2007, 282 (33) 24416-24429
(Rare HS motifs facilitate selective binding of target proteins)

Wiggins P Life depends on two kinds of water PloS One 2008 Jan 9 3(1) e1406;
Cf. also
http://www.Isbu.ac.uk/water/monograph200904pw.pdf
Physica A, 2002, 414, 458
Wiggins P.M. Prog. Polym. Sci., 1995, 20, 1121-1161
(High and low-density water in gels)
Cf., Robinson G.W. & C.B. Cho, Biophys. J. 1999, 77, 3311-3318
(Role of hydration water in protein unfolding)

Wise, E.R. et al., Chem. Mater. 2007, 19, 5055-5057

(Bone formation: role ofsurface HS-like molecules)

(Overpurification of polysaccharides used for in vitro studies could have removed the necessary inorganic cofactor to achieve HS-protein interaction)
(An idea suggested by published as well as unpublished concepts suggested from Aberdeen University polysaccharide laboratory studies).
It can be suggested that the actual in vivo discrimination process must require cofactors (putatively multi-inorganic element containing) suggesting the use
of a more fuzzy logic, ‘analogue’ recognition system rather than an exact ‘digital’ type of organic core polysaccharide microstructure electrostatic
recognition similar to the hydrogen-bonding etc. method with DNA. The function of a polymer rather than oligomer-associated metallomic array might be
worth establishing experimentally in this context.

11.2 Further References (and Notes) Referred to by Number and

also Arranged According to Subject Matter

11.2-1 The Metallome-Heparanome (References : Metal Ions in H/HS Signaling)

The inorganic components of H and putatively HS may also normally find use in animal tissues to link specific
parts of H/HS to proteins as part of a cooperative inorganic ion/H/HS manner, is suggestive of a “metallomic-
heparanome” nature of the inorganic biochemistry of polysaccharides. A possible function of small colloidal
particles containing e.g. Ca, Sr, Ba, P, Si, Fe, V etc. in H/HS could be to provide links similar to the borate
crosslinks in plant polysaccharides (in a similar manner to how hyaluronan2a and chitosan act as a ligand for
Fe-rich particles2b).
Currently there is fairly strong evidence that single Ca, Mg, Sr, Mn, Zn and Cu ions have been reported to be required to allow a number of specific protein
HS microstructures to interlink (cf. Table I). Exogenously administered H (which it is now pointed out also includes multi-inorganic elements) is also
reported to induce an increased secretion of H-like HS fragments from the endothelium11, the mechanism of which may at least perhaps partly be due to (a
now established) Cu (redox-ascorbate)/Zn dependent nitric oxide/ HS oligosaccharide generation12 process as well as the effects of such inorganic ions on
the Golgi apparatus exemplified by the effect of the presence of Ca2+ (perhaps Sr2+), Mg2+ and Mn2+ in cell culture experiments which boost HS
biosynthesis (toxic elements, which can also occur in H (e.g., Pb2+ ,Cd2+ , Hg(II) and F- (cf. Table II) were found in similar experiments to inhibit this).

Key Findings of Inorganic Ion Assisted HS Signaling

An HS-dependent basic FGF receptor dimerization activity has been reported (Kan et al. 1) to require the
absolute presence of divalent cations. Redox metal ions, especially Cu2+/Cu+ are apparently absolutely required (Ding et al. 1) for nitric oxide-dependent
H/HS oligomer signaling/messenger molecule generation.

Other likely inorganic ion inputs into HS biochemistry include: core protein proteolytic shedding actions of Zn-dependent metalloproteinases (induced by
cellular stress, mechanical, heat shock and hyperosmolality1) induction or seeding of H/HS or H/HS-protein supramolecular structure or aggregate
formation; effects of inorganic cofactors in DNA expression, m-RNA production or activity and the modulation of the intracellular Golgi apparatus
assembly process and putatively also the roles of metal ions for the modulation of enzymic post-synthetic HS processing by bacterial heparanase (Lui et al.
1
), and other GAG structure processing enzyme activites1).

11.2-2

Ref. 1a-1
Published Discussions in Peer-Reviewed Articles of How Inorganic Ions Might Participate in H/HS Signaling
A review article which covers (inter alia) inorganic ions in HS signaling
D.R. Coombe & W.C. Kett. Cellular & Molecular Life Sciences, 2005, 62 (4) 410-4249d cf.,
Sasabi et al., EMBO J. web//emboj.oupjournals.org/cgi/ content/full/18/22/6240
and the Discussion Section of a paper reporting the structural requirements for H/HS binding to type V collagen
by S. Ricard-Blum et al., J Biol Chem., 2006, 281 (35) 25195-25204
Putative Example of an Absolute Requirement for Bivalent Metal Ions for HS Signaling
M. Kan et al., J. Biol. Chem., 1996, 271, 26143-26148

Although pharmaceutical H is essentially protein-and nucleic acid free, it is not a single stoicheiometric
molecule, but a library of at least 120 chemically distinct molecular species (cf., Nader et al. 1) to which a further level
of complexity should be added, namely, that this library supports a metallomic multielement, organometallic array
containing some 60+ inorganic elements as normal constituents, selectively highly enriched in elements which occur
only in ultratrace amounts in normal biological fluids

Ref. 1a-2 How Metal Ions Are Currently Known To Assist H/HS Signaling
Nitrosative cleavage of H/HS is believed to constitute a major signaling pathway involving oligosaccharide
messengers the formation of which depends on redox cycling by Cu2+ / + apparently assisted by Zn2+ ions
Examples of another, perhaps more general, type of non-redox-metal ion-determined-H/HS-“signaling”
include inter alia, the absolute requirements for divalent cations (e.g. Ca2+) for the facilitation of basic
fibroblast growth factor receptor assembly (cf., Kan et al. 1) the inhibition of inositol trisphosphate Ca2+
ion release by H (cf., Hill et al.1a4) and the binding of specifically unsaturated sugar H dimer to
membrane Na+/Ca2+ ion exchanger allows bacterial heparanase to inhibit this activity (Shinjo et al., ).

1a-3 Degenerative Diseases: Putative Roles of Inorganic Elements in H/HS Signaling

(H and Cancer)
H Engelberg,
Cancer (New York) 1999, 85 (23), 257-272.
S.M. Smorenburg & C.J. Van Norden Pharmacol. Rev. 2001, 53 (1) 93-105
[This study suggested that low molecular weight heparin might have therapeutically verifiable anti-cancer therapeutic
value but the results from unfractionated H were less certain. The lack of awareness of variation in the inorganic
cofactors present in Hs some of which (e.g. Ga3+ and Pt complexes) are known to inhibit tumour growth and which
differ in amounts present between different brands of H (cf .Table IV) and also probably between in molecular weight
and unfractionated forms of H, might have contributed to the confusion regarding the anti-tumour effectiveness of H];

Cf., also K. Ono et al., Br J Cancer, 2002, 86, 1803-1812,

R.J. Linhardt (H-induced cancer cell death) Chemistry & Biology, 2004, 11, 420-422
10 Cf. Vandewalle et al., (Ca2+ regulation of HS proteoglycans in breast cancer cells),
J. Cancer Res. Clin. Oncol., 1994, 120 (7) 389 Chem Abs.121, 105487j
Cf., R. Dziarski (Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by H)
J. Immunol., 143 (1) 356-365 and P.A. Steck et al., Cancer Res., 1989, 49, 2096-2103 (altered expression and
distribution of HS PGs in human gliomas
K. Mani et al. Glycobiology, 2004, 14, (5) 387-394 (Tumour attenuation by 2(hydroxynapthyl)-β − D-xylopyronoside
requires priming of HS and nuclear targeting of the products; cf., ibid., 2006, 16 (12) 1251-61 Constitutive and vitamin C
induced nitric oxide catalysed release of HS from recycling glypican-1 in late endosomes;
[The tumour cells expressed about three times more HS and hyaluronan in their growth media and on their cell surfaces than did the normal cells thus
indicating that the cellular transformation had been accompanied by an augmented biosynthesis of HS., however a lower production of HS of decreased
degree of sulphation is the usual effect of oncogenic cellular transformation {cf. H. Rahmoune J.E. Turnbull J.T. Gallagher et al., Biochem. Soc. Trans.
1996, 24 (3) 355S}].
The putative
tumour suppressors
EXT1 and EXT2 are glycosyltransferases required for the biosynthesis of HS.
T. Lind. et al., J. Biol. Chem., 1998, 273 (41) 26265-26268;
EXT1 alters the expression of cell surface HS
C. McCormick et al., Nature genetics 1998, 19, 158-161

Anti-cancer metal ions (which could be sequestered from biological fluids) by H

The presence of Ga3+ in some H samples (cf. Table III) could confer associated anti-cancer activities.
[Cf., gallium nitrate and other salts show potential anti-tumour activity
(cf., e.g., C.R. Chitamba et al., J. Pharmacol Exp. Ther. 2007, 322 (3) 1228-1236];
This possibility suggests further targeted research.

Pt may also naturally occur in ultratrace amounts in H.

Pt ions are known to bind to H (Cf. D. Grant et al. Biochem Soc. Trans. 1996, 24, 204S);
such adducts should be evaluated for anti-tumour activity.

(HS and Cancer, cont.) HS can be both pro-cancer and anti-cancer. The difference is likely to be related to HS microstructure
In a liver tumour metastasis model attenuation of HS by GAG biosynthesis inhibitors produced an anti-cancer effects relating to surface GAGs especially
pro-metastasis HS variants; anti-HS agents were therefore suggested to potentially useful for anti-metastasis therapy. J. Timar et al., Int J Cancer, 1995,
62 (6) 755-761;
On the other hand EXT-2 and EXT- 2 putative anti-tumour agents are enzymes required for HS biosynthesis.

Tumor metastasis was found by other workers to be promoted by tumour cell heparanase which can be inhibited e.g. by H and H-like agents. Angiogenesis
inhibitors (including HS-related) are also in general anti cancer. (Cf. internet discussion D Grant (2000)1
HS degradation by heparanase is believed to promote cancer especially in tumours which home to or grow within bones
(cf., R.D. Sanderson et al., Matrix Biol. 2004, 23 (6) 341-352)
Dowregulation of the expression of heparanase inhibits the invasiveness, angiogenesis and metastasis of human hepatocellular carcinoma (Y. Zhang et al.,
Biochem. Biophys. Res. Commun. 2007, 385 (1) 124-129); FGF-2 binding, signalling and angiogenesis believed to be modulated by heparanase in
metastatic melanoma cells (J. Reiland et al., Neoplasia, 2006, 8 (7) 596-606)

The sulphated oligosaccharide P188 is a promising H-mimetic anti-cancer agent (which inhibits heparanase activity as well as modulates growth factor
activities invloved in tumour growth; cf. E.A. McKenzie Br. J Pharmacol., 2007, 151, 1-14.
1a-4
(H and Atherosclerosis)
Cf. K. Murata & Y. Yokoyama Atherosclerosis
H. Engelberg, Pharmacol. Rev. 1984, 35, (2) 91-104
Cf. H. Engelberg Circulation, 1961,23, 573-577 (Cf. role of natural serum H)
Cf. D. Grant, W.F. Long & F.B. Williamson (Degenerative and inflammatory diseases may result from defects in anti-
mineralization mechanisms afforded by glycosaminoglycans) Med. Hypoth. 1992, 38, 49-55,
Cf. Biochem J.,1989, 259, 41-45
1a-5
(H and Dementia)
H. Engleberg, Dement. Geriat. Cogn. Disord., 2004, 18, 3-4
Cf. J.D. Snow et al. (vide supra)
----------------------------------------------------------------------------------------------
1a-6
Antioxidant & Antinitrant Activity (Related to Modulation of Superoxide Dismutase by H/HS) and
Sequestration and Deactivation of Redox Active Iron, Copper & Silver Ions, Etc. by H/HS)

The following articles discuss this topic:

H/HS-Superoxide Dismutase
T. Adachi & S.L. Marklund J. Biol. Chem. 1989, 264 (15) 8557
J. Biochem. (Tokyo) 1995, 117 (3) 586-590
Biol. Pharm. Bull. 1998, 21 (10) 1090; Chem. Abs. 130. 64588h

G.E. Arteel et al., Biol Chem. 381, (3) 265-268, Chem. Abs., 133, 85803f
(Selenium containing antioxidant which probably binds to endothelial wall HS)

-------------------------------------------------------------------------------------------------------------------
Cf. also,
D. Grant et al., Biochem. Soc. Trans., 1996, 24, 194s (Hs as essential antioxidants)
Ibid., 1988, 16, 1030-1031 (Effect of H on dismutation of superoxide anion)
Ibid., 1992, 20, 361s (Complexation of Fe2+ based ions by H)

D. Grant et al., Med. Hypoth. 1987, 23, 67-71 (Review article: Pericellular heparans may contribute to the protection of cells from free radicals)
D. Grant et al. had found the absence of NMR detected paramagnetic effects expected for high levels of Fe and Cu in some H samples; this was
rationalized as due to occurrence of these elements within the H supramolecular structure in a separate non-solution phase attached to H.
Cf., G. Mackintosh, Ph.D. Thesis “Heparin-Iron Interaction and its Possible Relevance to Antioxidant Activity” University of Aberdeen, 1995
[This study involved in vitro and in vivo investigations into possible inflammatory actions of Fe(II) ions which are putatively inhibited by binding of such
ions to H/HS or to H-mimetics such as pentosan polysulphate]
Cf. also M.A. Ross, M.Sc. Thesis, “Heparin as an Antioxidant”, University of Aberdeen 1992
M.A. Ross et al. Biochem J. 1992, 287, 849-853
Cf.,
R. Albertini et al., Int. J. Mol. Med. 2000, 6, 129-136; FEBS Lett., 1995, 377, 240-242

F.B. Williamson et al. (unpublished, personal communication) showed that Fe(II) heparin undergoes auto-oxidation to generate
Fe(III) (O.OH) akagaenite crystals detectable by their characteristic X-ray diffraction pattern.
Incompleted studies had suggested that the multi-inorgnaic element content of H was associated with ferroxidase catalytic property
Other related unpublished studies have indicated that iron ions can promote pathological calcification and fibrin aggregate formation which is upon
ingestion by macrophages and resultant inappropriate cytokine induction, promotes degenerative disease processes .

(Related solid phase, antioxidant function indicated by removal of potentially damaging redox iron ions by polysaccharides
was reported by Merce et al.2a and Sipos et al. 2b for anionic polysaccharides)
[P. Sipos, O. Berkesi, E. Tombacz, T.G. St Pierre & J. Webb, J Inorg. Biochem. 203; 95: 55-63
A.L. Merce, L.C. Marques Carrera, L.K. Santos Romanholi & M.A. Lobo Reico, J Inorg Biochem., 2002; 89: 212-218]

D.J.R. Jarrett et al. J. Clin. Exp. Gerontol., 1989, 11, (3-4) 145-154
Ageing as a cause of raised serum feritin in the absence of disease.

J.T. Salonen et al. (Circulation, 1992, 86, (3) 803-811) found an epidemiological correlation between mortality in Eastern Finnish men with appropriately
corrected serum ferritin values. This study supported a possible role of redox active iron ions in degenerative cardiovascular dysfunction. This can be
further suggested to include a redox iron related alteration of HS signaling.

Cf. also
T. Aoyagi et al., Biol. Pharm. Bull. 1994, 17 (2) 340, Chem. Abs., 120, 294925b
Age dependent decreases in fibrinolytic enzyme activity in serum of healthy subjects.

11.3
Further References
(1a 1-14; 1b-1h)

1a -1 H.B. Nader, T.M.P.C. Ferreira, L. Toma, S.F. Chavanto, C.P. Dietrich, B. Casu and G. Torri,
Carbohydr Res. 1988; 236,165-180

1a -2 H.B. Nader, H.K. Takahasi, J.A. Guimaraes and C.P. Dietrich, Int. J. Biol. Macromols. 1981; 356-360

1a-3 D. Grant, W.F. Long and F.B. Williamson, Med. Hypoth. 1987, 23, 67-73; cf. ibid.29, 245-253

1a-4 D. Grant, W.F. Long and F.B. Williamson Biochem J. 1989; 259; 41-45; cf. also Med Hypoth. 38, 49-55

T.D. Hill et al., Biochem Biophys. Res. Commun., 1987, 149 (3) 897-901

1a-5 E. Feyzi, T. Saldeen, E. Larsson, U. Lindahl & M. Salmivirta, J Biol Chem., 1998; 273: 13395-13398

1a-6 B. Kuberan et al. (with R.D. Rosenberg) J. Biol. Chem. 2004, 279, 5053-5054 (Light induced 3-O-sulfotransferase expression alters pineal HS fine
structure-a surprising link to circadian rhythm) (i.e. HS microstructure is subject to alteration by light).
1a-7 H/HS, alginates and sulphated marine anionic polysaccharides, e.g,. carageenenans and soluble humic acids (fulvic acids)
act as potent modulators of the crystallization of sparingly soluble e.g. CaCO3 and BaSO4 and hence influence the amounts of Ca2+,
Ba2+, CO3- and SO4- present in solution.

The naturally occurring amount of HS and fulvic acid

to achieve such buffering activity seems to correspond with that reported [1.6mg/L] for HS to occur in vivo by
W.C. Manley et al., Clin. Chim. Actam 1984, 137, (3) 315

1a-8 H. Ischiropoulos, Arch. Biochem. Biophys., 198, 356, 1-11

Cf. H. Tohgi et al. Ann. Neurol,1999, 46, 129-131
(Remarkable increase in cerebrospinal fluid 3-nitrotyrosine in patients with sporadic amylotrophic lateral sclerosis)

1a-9 Cf., K Mani F Cheng B Havsmark, M Jonsson, M Belting & L-A Fransson, J. Biol. Chem. 2003: 278: 38956-38965

1a-10 F. Baluska, J. Samaj, P. Wojraszek, D. Volkman and D Menzel, Plant Physiol. 2003: 133: 482-491
E . Melotto & J.M. Labovitch, R Bras Fisiol Bras Veg. 1994; 6: 75-82
M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Ann Rev Biochem. 1984; 6: 75-82
cf., T. Matsunaga & T Ishii, Anal Sci. 2004.; 20: 1389-1393;
(cf., borate crosslinks in plant rhamnogalacturonan II)
[In plants pectin-like oligosaccharides were originally supposed to engage in hormone-like activities but are now thought to
influence Ca2+ second messenger actions]
The system of plant anionic polysaccharide, pectin generated oligosaccharide dependent Ca2+ second messenger action could serve as a provisional
model for heparan sulphate signalling in animals which is known, at least in part, to require Ca2+ and Zn2+ as well as Cu+/Cu2+( for nitric oxide/
(ascorbate) dependent oligosaccharide generation).

1a-11 D. Lagunov & G. Warren, Arch. Biochem. Biophys., 1962, 99, 396-400; J.E.Shivley & H.E. Conrad, Biochemistry, 1970, 8
(1) 33-43

1a-12 Cf. C. Leteux et al., J. Biol Chem.,276 (16) 12539-12545

[Scrapie antigen indicated co- occurrence with HS with GlcNH2, unsulphated motif: this is the site which is targeted by NO
metabolites to create HS oligosaccharides]

1a-13 H. Diringer & B. Ehlers, J. Gen Virol. 1991, 72, 457

[Initial communication dealing with the inhibition of scrapie by sulphated polysaccharides]

1a-13-1 Communication from Graham Steel, Glasgow (2007) (including Professor Ian Bone Report)
The H-mimetic PPS has a stimulatory effect of proteoglycan synthesis and a potent suppressor of angiogeneis and tumour
growth.
In a pivotal study by Doh-ura (2004) PPS was confirmed to suppress CJD attributable to misfolded PrP. The drug was
directly infused into the brain using Tg7 mice expressing hamster than endogenous mouse PrP). Later work on some 20+
human patients worldwide corroborated the effect in humans.

1a-13-2 PPS-like molecules produced by normal stress-related cleavage of HS may act as cofactors for clearance of misfolded prions from cells.
Cf. K. Mani et al., J. Biol. Chem. 2007, 282, (30) 21934-21944
(In Niemann-Pick fibroblasts where nitrosative degradative processing of HS is defective carboxylated proteins were abundant
when glypican-1 was silenced in normal fibroblasts the level of such proteins increased suggesting that the deaminative cleavage of HS is involved in the
clearance of such abnormal including misfolded proteins)

1a-14 B.C. Challis et al., IARC Sci. Publ., 1978, 19 (Environ Aspects N-Nitroso Compd.) 127-142; Chem. Abs. 89, 196583x
Cf. E. Boyland & S.A. Walker, Nature, 1974, 601-602;
T.Y. Fan & S.R. Tannenbaum, J. Agr. Food Chem. 1973, 21(2) 237-240
B.C. Challis & J.R. Outram JCS Chem. Commun. 1978, (16) 707-708 (article highlighting silver catalysis of nitrosative
processes)

1b Cf., Grant, D., WF Long FB Williamson, Biochem J. 1987; 244: 143-149

(The infrared spectra of different H salts show individual counterion input into H structure as detected by vibrational
spectroscopy in solution and solid phases which are different from a purely electrostatic effects)

1c Z. Liu & A.S. Perlin, Carbohydr. Res. , 1994, 255, 183-191

1d R.N. Rej et al. ibid., 1990, 207, 143-152

1e R.E. Vilar et al., Biochem. J. 1997, 324, 473-497

1f J. Van den Born et al. J. Biol Chem., 270 (52) 31303

1g F. Cheng et al., J Neurochem 2006 98,1445-1457; Mani et al., J Biol Chem 2003 278 38956-38965
and refs.cited

1h D. Grant et al. unpublished observations Marischal College, University of Aberdeen

2 M. Purdey Med. Hypothes. 2004, 62 (5) 746-754
(Chronic barium intoxication disrupts sulphated proteoglycan synthesis: a hypothesis for the origins of multiple sclerosis);
Cf., Ibid., 2000, 54, 278-306
(Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical-generting divalent cation manganese and deficienceies of
antioxidant co-factors Cu. Se, Fe, Zn. Does a foreign cation substitution at prion ptotein’s Cu domain initiate TSE?)
Ibid., 2001 56 (1) 29-45
(Does an ultraviolet photooxidation of the manganese-loaded/copper depleted peion protein in the retina initiate the pathogenesis of TSE?);
Cf., Ibid., 2003, 60 (6) 797-820
(Does an infrasonic acoustic shock wave resonance of the manganese 3+ loaded/copper depleted prion protein intiatie the pathogenesis of
TSE?);
Ibid., 2004, 63 793-809
(Elevated levels of ferromagnetic metals in food chains supporting the Guam cluster of nurodegeneration: Do metal nucleated crystal
contaminanet evoke magnetic fileds that initiate the progressive pathogenesis of neurodegeneration)
Ibid., 2004 http://intl.elsevierhealth.com
(Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: Do Ag/Ba/Sr piezoelectric crystals
represent the transimissible pathogenic agent in TSEs);
http://www.purdeyenvironment.com/floodstudy.htm
(Fresh research report of a trace metal-CWD study at Saskatoon University is being exploited to discredit a misrepresented version of Purdey’s
theory)
Cf., also M. Purdey, Draft Abstract for Chapter in “Trends in Prion Research” [communicated to the author by Graham Steel, formerly CJD
Alliance press officer etcbased in Glasgow] PrP-ferritin-‘fibril’ crystals were suggested to be nucleators of solid phases which were proposed to
be the true pathological entities responsible for prion diseases.
Cf also Brown ref 1

2a A.L. Merce et al., J. Inorg Biochem. 2002, 89, 212-218

2b P. Sipos et al., ibid., 2003, 5,55-63 (Spheridal iron(III) oxyhydroxide nanopartilces sterically stabilised by chitosan in

aqueous solution)

3 A. Wassermann, Ann. Bot. 1949 NS 13 (49) 79-88;

Cf. W.A.P. Black & R.L. Mitchell. J. Mar. Biol. Assoc. U.K .,1952, 30 (3) 575-584
Cf. brief mention by F. Mo et al., Carbohydr. Res., 1985, 145, 13-24 (cf. also refs. cited herein ).
3a-1 E.g., The presence of inorganic elements in ALGINATE have been reported by
Ascophylum nodosum polysacchride-based ion exchanger kelp elements are tabulated by several internet sites, e.g.
http://www/alginure.co.uk/ascophylum-nodosum.html
3a-2 for Ecklonia maxima at http://www.gairesearch.co.za/kelp.html.
3a-3 Cf., also T.A. David et al., Appl. Biochem. Biotechnol.,2003, 110, 75-909 Su
3a-4 K Truus et al., Proc. Eston. Acad. Sci., Chem., 2001, 50, 95-103 for apparently anthropogenically polluted seaweed from
the Baltic). Data from this paper are listed in Table I which also lists similar multi-element analysis (which was
apparently provided by the Norwegian Seaweed Research Institute) of less polluted polysaccharide from
.
3b-1 The presence of inorganic elements in
H have been reported by
H.J.M. Bowen in Trace Elements in Biochemistry, Academic Press, London, 1966,
cf., p.63 noted that since heparin contains appreciable amounts of Ba, Ca, Cu , Mn, Sr and Zn; (where serious
interference problems for estimation of normal blood were caused by Mn in heparin as an anticoagulant in the
solutions to be analysed);
Cf. also Katz, S.A. Amer. Biotechnol. Lab. 1984, 2, 24-30

3b-2 (for Ca, Sr, Ba) by G.E. Harrison & A. Sutton, Nature, 1963, (4869), 809
Cf. M.E. Lyon, Clin. Biochem. 1995; 28: 79

3b-3 The presence of paramagnetic metals in natural H can also affect NMR resonances
cf. G. Gatti et al., Macromoleucles,1979, 12 (5) 1001-1007
Cf also the presence of the metal ion chelator EDTA in commercial heparin
(B. Casu et al European Patent 1987 0245813) suggests its industrial employment for removal of
metals thought to be undesirable for commercial medical applications.

3b-4 (for V in H ) G. Heineman & W. Vogt, Biol. Trace Elem. Res. 2000, 75, 227-234

3b-5 (for Cr, Mn in H) N.W. Alcock Serum versus plasma for trace metal analysis, Elem. Metab. Man, Anim. Proc. Int.
Symp. 4th, 1981 (Pub 1982). Eds. J.M. Gawthorme, J.M.M.M.C. Howell, C.L. White, p. 6578-680; Springer, Berlin;
Chem. Abs., 96, 213646

3b-6 (for Si in H/HS) K.A. Schwarz., Proc. Natl. Acad. Sci. USA, 1973; 70: 1608-16124
Cf. RK Iler The Chemistry of Silica ,Wiley, New York, 1979
and D. Grant, W.F. Long and F.B. Williamson, Med Hypoth. 1992; 38: 49-55

3b-7 (for As in H) D. Bohrer et al., J Parenteral Enteral Nutrition, 2005; 29 (1) 1-7;
(Unacceptably high levels of Al and As still occur in some commercial heparins
 alginate, carageenan etc., and calcified skeletal matrices
forming crosslinks analogous to borate in plant rhamnogalacturonan II) 4

3b-8 (for Al in H) D. Bohrer et al., RBAC. 2004; 36(2) 99-103

3b-8-1 Be2+ ions interact strongly with H

J.G. Hall, Immunology, 1988, 64, 345-351

3b-9 The presence of 38 seawater multielements in H was confirmed in Na and Tl H of the elements listed at a-f (except Al
added during the spark source mass spectrosocpic (SSMS) analysis of Mg, K, Rb, Cs, Tl, Fe, Ni, Co, Sn, Mo,Ti, Zr,Ce,
Ag, Nd, W, La, Pb, Ga, Sb, Cd, B and I in Na H and Mg, K, Rb, Cs, Cr, Fe, Ni, Co, Sn, Mo, Ti, Zr,Ce, Ag, Nd,W, La,
Pb,Ga, Sb, Cd, P, B, I, Cl, F and Br in Tl H (D. Grant, Chemistry Preprint Archive, 2000 Oct., 94-104; available at
http://preprint.chemweb.com/biochem/0010002; cf. also a preliminary summary of these data was given by D., Grant,
et al., Biochem J.,1987, 244: 143-149) and more completely for Na and Tl Hs in
C.F. Moffat, Ph.D. Thesis University of Aberdeen, 1987, briefly discussed the 205Tl NMR spectrum of Tl H (on p.144)
and the possible lack of toxicity of 8 ppm Tl in H is also discussed on p. 189.
The relevance of larger amounts of possibly toxic or therapeutic elements (such as Ga which has demonstrated anti-
tumour activities) are worth pursuing.

3b-10 web.alsglobal.se/hem2005/pdf/blood_collection_tubes_eng.pdf (2005) tabulated multi-element data obtained by ICP-

MS for types of H commonly present as additives in blood assay tubes which were extracted with 0.01M HNO3 . This
study also an assessment of the possible inorganic element inputs from empty tubes and needles employed for blood
sampling which can serve as blanks for correction of the H analyses for background contamination; the data also
reported ICP-MS data substances commonly added to blood collection tubes (EDTA and a phase boundary enhancing
agent which is rich Si, Al and in lanthanides etc). The data for extracts from six types of heparinized blood collection
tubes confirm the seawater multielement pattern of inorganic cofactors present in H previously identified by the author.
This data can be extracted from the tabulated information (which included the mainly H-derived –SO3- group inorganic
S contents of extracts, as well as those of the principal counterion for the employed single counterion-enriched forms of
H.

11 H. Haraguchi, J. Anal. At. Spectrom., 2004, 19, 5-14

5 H.B. Nader, M.G.L. Medeiros, J.F. Paiva, V.M.P. Paiva, S.M.B. Jeronimo, T.M.P.C. Ferreira & C.P. Dietrich
Comp. Biochem. Physiol., 1983, 76, 433-436
This study established that an exact mathematical relationship exists between the bathing habitat salt concentrations {in fifteen
species of Crustacea, Pelecypoda and Gastropoda} and the average total sulphated glycosaminoglycans (GAGs) present in these
organisms increased from 125 to 5265 µ g/g dry weight with increased habitat salinity. This suggests that differing salinity
induces evolution/biosynthesis of the exactly balancing amounts of GAG- SO3- groups required to titrate against Na+ (the
results of ref.1h suggest that the sum of the –SO3-(H2O)n groups present i.e. the total GAG-(O)-SO3- + GAG- (N)-SO3- rather
than the GAG-C(O)O- contents correlated with how individual GAG types were ‘induced’ by different habitats with a range of
different natural saline, principally NaCl habitats (i.e. seawater and brackish seawater etc.)

7 The work of S. Kodima et al., ( e.g. Eur J. Nucl. Med., 1983, 8, 52-59, cf., Y. Hama et al., ibid., 1984, 9, 51-56 who by in vivo and in vitro multivalent
ion binding studies e.g. of to 45Ca labelled HS (relating to the scintigraphic observation of tumours via 67Ga binding which were indicated to occur at
HS sites) support the notion that HS will occur in vivo as multi-inorganic element substituted systems.

Mast cells in vivo histochemicaly stain with ruthenium red by binding to heparin in the mast cell granules
(E. Phil, Histochem. Cell. Biol., 1970, 22, 302-315)

Cf., also: he formation of the adduct 99Tc-H enabled the bio-distribution of H to be achieved in the rat
N. Vigneron et al., J. Biophys. Med. Nucl. 1984, 8 (2-3) 192;
Cf., M. Decousus et al., ibid., 1984, 8 (2-3) 180; cf.,
99m
Tc-H studies by Mosberk et al., Nuclearmedizin (Stuttgart), 1981, 18, 343; Chem. Abs., 96, 30896w;
111
InCl3-H for visualisation of thrombosed arteries, Chem Abs., 97, P168866w;
cf. related 169Yb and 161Yb H binding studies Chem. Abs., 98, 194186g;
167Tm has strong affinity for tumour tissues especially to HS and other glycosaminoglycans:
A. Ando et al., Eur. J. Nucl. Med. Mol. Imaging, 1983, 8 (10), 440-446

Eu(III)-H complex formation enables H to be spectrophotometrically assayed:

M. Rizk, Spectroscop. Lett., 1995, 28 (8) 1235 (Eu3+ -H formation enhanced the emission of a forbidden Eu3+ band at
616nm allowing ratio of complexed/uncomplexed Eu3+ to be determined; Log10Kassoc. for H-Eu3+ -H was reported as 5.079;
cf., the log10Kassoc.value for La3+ was found by Rabenstein et al.(cf. ref 6b-2-1) to be 4.5)

11-3
Refs 6, 6a, 6a-1, 6b-1, 6b-2. 6b2-2, 6b2-3; 7-9. 9b-3. 9b3-1. 9c, 10-13, 13a; 14-17

6a
Reviews of the binding of inorganic elements include:
 LB Jaques, Science. 1978; 206: 528-533;
and in Ions in Polymers (Based on a symposium by the Division of Polymer Chemistry at the 176th Meeting of the American
Chemical Society, Miami Beach Fla., Sept 11-15, 1978) Amer. Chem. Soc. Adv. Chem. Ser. 187 (Pub 1980) p. 350-360
These perceptive articles remain highly relevant.

J.R. Dunstone, Biochem. J., 1962, 85, 336-351 and

J.E. Scott, Ch. 12, p 171-187 in
The Chemical Physiology of Mucopolysaccharides, Ed., G. Quintarelli, Churchill, London, 1968

The formation of adducts with a range of inorganic salts (e.g. iodides) with H was discussed in a Patent
(Fo-We Forschung und Verweltungs-Anstalt [Therapeutically active product ] Brit. Pat. 890,622, 1959)

6a-1

The Binding of Inorganic Sulphate to H

K. Simon Naturwiss. Rundschau 1982, 35 (11) 452-453

discussed the occurrence of ca. 12% of total S in H in the form bound inorganic SO42-

J.R. Helbert & M.A. Marini, Biochem. Biophys. Acta 1964, 83,122-134
Studied the binding of inorganic sulphate anions to H

6b-1 K.A. Schwarz, Proc. Natl. Acad. Sci., USA, 1973, 70, 1608-1612
(Bound form of silicon in glycosaminoglycans and polyuronides)

6b-2

A list of published studies from the former Aberdeen polysaccharide group (WF Long FB Williamson et al.) which includes
reports of studies by a variety of physical chemistry methods of metal ion and polyamine binding to heparin and related roles
of hydration and phase separation, was posted on the internet by W.F. Long at
http://www.abdn.ac.uk/~bch118/publications 2003march.doc

6b-2-1
Articles which deal with the binding of metal ions by H include:
D. Grant et al., Biochem. J., 1991, 275, 193-197; ibid., 1992, 287, 849-853; ibid., 1992, 282, 601-604; ibid., 1992, 283, 243-
246; ibid., 1992, 285, 477-480; Biochem. Soc. Trans., 1991, 18, 1281-1282; ibid., 18, 1282-1283; ibid., 1992, 20, 361S;
ibid., 1991,; ibid., 1991, 19, 390S; ibid, 1996, 24, 203S; ibid., 1996, 24, 204S;
D.L. Rabenstein et al., Carbohydr. Res., 1995, 278, 239-256
L. Herwats, et al., Nov. J. Chem. 1977, 1, 173-176

6b-2-2

D. Grant et al., Med Hypoth. 1992, 38, 46-48 (A putative role for colloidal silicates in primitive evolution deduced in part from their relevance to modern
pathological afflictions).
Silicate anions in aqueous solution above a threshold concentration (ca. 50 ppm) undergo self-association which is likely to be enhanced at H/HS surfaces
in keeping with the strong association of polyuronide with inorganic silicon in biological tissues where they are believed to contribute to the mechanism of
nucleation of bone formation. There is also some evidence that insufficiency of dietary Si is associated with augmented oxidative damage to tissue
(soluble silicate in drinking water has been negatively correlated with the incidence of degenerative diseases such as atherosclerosis (cf., M. F. McCarty,
Med Hypoth.,1997, 49, 177-179 ).

Both HS and redox metal dyshomeostasis may, however, be to be implicated in this effect of Si where a putative role of silicate enhanced redox metal ion
sequestration and pacification can be suggested.

A variety of types of SiO2 sols and gels have been form many years available from the chemical industry; during research into the self assembly of such
materials it became evident to the author that a form of nucleation behaviour which mimicked biological activity was apparent. A hypothesis of silicic acid
generated seeding/Darwinian evolution is rationally derivable from these observations. This type of structural seeding could continue to be of importance
in present day organisms especially in how HS supramolecular inorganic composite material structures are nucleated.

6b-2-3

Some Aberdeen Polysaccharide Group Studies

These had confirmed the results of previous workers (as well as data from industrial use of sulphonated organic polymer membranes) that H contains a
similar system of interacting -SO3- - associated water cluster structures (the details of which vary between individual metal counterions, but the greatest
degree of hydration, 6H2O/-SO3-, is found for Na+ counterions and the least for quaternary N+ counterions (D. Grant et al. Biochem. Soc. Trans. 1990, 18,
1293-1294; ibid., 1983, 11, 96; ibid., 1984; 12: 302. The heparin-associated water clusters showed a systematic change in their near infrared water cluster
absorptions upon poly-L-lysine or poly-L-arginine binding. (Glue-like surface clusters of H-metal ion associated of water structure (cf. 9d ) become more
obvious in a hydrophobic medium. (D. Grant et al., 1991, Biochem J. 1991; 277: 569-572). Such types of interactions are reminiscent of the reversible
adsorptions thought to involve both the protein core and polysaccharide side chains of cartilage proteoglycans with chromatographic media9d which have
been described as “non-specific”).
New research is required to search for ion conducting properties of GAG hydration superstructures which can be predicted to exist since this is how man-
made organic polymer-SO3-(H2O)n membrane proton conductors are employed in commercial use, their mechanism of action is believed to depend on the
properties of such water clusters (cf., e.g., James et al., J Materials Sci. 2000; 35: 5111-5119); these man-made sulphonated polymers also find use in
commercial fuel cell membranes which show improved thermal stability when the membrane contains additional SiO2 particles (Adjemian et al., J
Electrochem Soc. 2002, 149; A256-261) suggesting a related role for the presence of Si (e.g., as SiO2 particles) in GAGs (Schwarz, 1973, loc. cit., cf.
Grant et al. Med Hypoth. 1992; 38: 36-48). Further hints of how interaction between SiO2 and GAGs, especially, H/HS, may be physiologically relevant
comes from the report by Takeuchi et al . (Analysis. 1998; 28: 61-64) that a heparinised SiO2 chromatographic medium separated both cations and anions
on the same column.
Si(OH)4 perhaps forms crosslinks analogous to B(OH)3 in plant rhamnogalacturonan II.

Anionic Polysaccharides Resemble Humic Polymers as Multi-Element Matrices

In vitro studies of H indicates an ability, which it seems to share with other anionic polysacharides such as the alginate-rich biomass of kelp, to simultaneously sequester a wide range non-physiological (and

therefore potentially harmful) ions from the full range of trace and ultratrace ions etc. which occur in natural waters and biological fluids including blood serum this property is also shared with soil humic acids.
The perturbation of NMR resonance due to the presence of abundant Mn and Fe in humic acids for a long time prevented the true chemical nature of these polymers to be determined; a commonly held view was

that they were aromatic compounds related to lignin; D. Grant, Nature, 1977, 270, 709-710 suggested a procedure by which the paramagnetic ions could be removed by placing sparingly soluble detergent
formulated pyrophosphate granules in solutions of humic substances. This caused a slow removal of the paramgnetic ions into the solid chelator and a gradual narrowing of the solution phase NMR peaks. In the
case of H containing 1100ppm, Fe and 730 ppm Cu there seemed to be no detectable paramagnetc broadening. H evidently can coordinate these metals suffiencltly strongly to prevent NMR resonance
perturbation. This is indirect evidence that H can behave as an antioxidant and antinitrant (by blocking of the promotion of reactive free radical formation Cu and Fe).

Review articles are in preparation by. D Grant reporting on published articles which suggest that a range of cytokines, dietary and physical factors greatly influence heparan sulphate biosynthesis.

7 J. Folkman, Science. 1983, 221, 719-725 cf. p722-3

Cf. also J. Folkman & M Klagsburn, Science, 1987, 235, 442-447
J. Folkman et al. Nature, 1989, 339, 58-61
(steroid induced H-related anti-angiogenesis effects differed between Hs from different manufacturers)

J.-M. Herbert & J.-P. Maffand, J. Cell. Physiol., 1989, 138, 424-432
(similar HS oligosaccharides generated non-enzymically from H from different manufacturers differed markedly in their
antiproliferative properties for vascular endothelia and smooth muscle cells)

8
F.T. Borges et al., Kidney Int. 2005, 68, 1630-1642

Cf. G. Embery et al. Scand. J. Dent. Res. 1979, 87, 318-324

(Interaction of acid glycosaminoglycans with hydroxyapatite)(The order of binding was H>HS>dermatan sulphate/chondrotin
sulphate>hyaluronan. Ca2+ (and saliva) a pretreatments led to an increase in hyaluron and chondrotin 4 and 6 sulphate
adsoption, but a slight decrease in H adsorption; the presence of F- led to decreased adsorption of hyaluronan and chondrotin 4
sulphate.

Cf., J.L. Arias et al. J Mater Chem. 2004, 14 (14) 2154-2160

(Sulphated polymers inbiological mineralzation: a plausible source for bio-inspired engineering)

12 A communication entitled “Microstructure-dependent modulation of crystallization by alginates” (by D. Grant, M.I. Tait, W.F. Long & F.B.
Williamson) was presented in the form of a poster by M.I. Tait at the International Seaweed Symposium, Vancouver British Columbia, Canada in
1989. This reported that the rate constants of BaSO4 crystal growth on seed crystals (obtained in a similar manner to that described by Grant et al.,
Biochem J. 1989; 259: 41-45) for a range of alginates (detailed by J. Boyd, “Enzymic approach to the structure and function of alginic acid”, Ph.D.
Thesis, University College of North Wales, 1975, cf., J. Boyd & J.R. Turvey, Carbohydr. Res. 1978, 66, 187-194 and A.J. Currie in “The structure
and biosynthesis of alginic acid”, Ph.D. Thesis University College of North Wales, 1983) were dependent on the microstructure of M (mannuronate)
and G (guluronate) environments rather than degree of polymerisation; individual alginates: (1) “SS/DJ” , mainly a random structure with some
preponderance of polyguluronate units; (2) “F387” , an industrial product obtained from Ascophyllum nodosum also studied by Grasdalen et al. (cf.
Carbohydr. Res. 1981; 89;: 179-11) which contained proportions of M =0.58, G =0.42 of which were in diads with MM = 0.40, GG = 0.24, MG and
GM = 0.18, triad MMM = 0.08, MMG = 0.08, GMM = 0.08, GMG = 0.10, GGG = 0.18, GGM = 0.06, MGG = 0.06 and MGM = 0.12}; (3) was
polymannuronic acid from fruiting bodies of Ascophyllum nodosum; (4) was poly M blocks (5) was poly alternating blocks and (6) was poly G
blocks; (4)-(6 )were obtained from commercial samples by enzymic degradation followed by fractionation according to the methods of Haug et al.
(Acta. Chem. Scand. 1967, 21; 691-704). Second order rate constants were for (1)-(6) respectively 0, 55, 55, 54, 62 and 81 for the primary and 59.6,
18.8, 15.2, 14.6. 6.0 and 36.6 for the (principal) secondary reaction processes. The relative potency of the alginates was further correlated with
infrared spectrocopic variation in the 630-1190cm-1 region which was indicative of the microstructural differences.
This is a simple illustration of how different microstructures in complex polysaccharides such as alginates which (analogously to HS) potentially contain
complex information encoding sequences can control complex chemical activities, is that the alginate-inhibited seeded crystallization of BaSO4 which
occurs by consecutive second order rate process, shows a systematic variation of the observed rate constants as a function of the alginate microstructure
(described in terms of guluronate and mannuronate blockiness).

9b-3 D. Grant W.F. Long & F.B. Williamson, Biochem J., 1991, 277, 569-571
9b-3-1 M. Kan, F. Wang, M. Kan, B. To, J.L. Gabriel & W.L. McKeehan, J Biol Chem. 1996; 271: 26143-26148
(Although the interaction of H with FGF was not affected by divalent cations, the presence of such cations especially Ca2+ or Mg2+ 9b-3-1is reported
absolutely to be required for high affinity (Kd= 10nM) binding of H to FGFR).

9c L.J.J. Hronowski & T.P., Anastassiades, Biochem. Biophys. Res. Commun. 1990; 167; 81-88;
Anal. Biochem. 1990, 191 (1) 50-57

10 A. Lima de Faria “Evolution without selection : form and function by autoevolution” Elsevier Science Publishers B.V. (Biomedical Division)
Amsterdam, & New York 1988

11 M.A.S Pinal et al., Braz J. Med Biol Res. 1994, 27p, 2191-2195
H.B. Nader , V. Buonassisi, P. Colburn & C.P. Dietrich, Cell. Physiol. 1989; 140: 305-310

12 R. Cappai, F. Cheng, G.D. Ciccotosto, B.E. Needham, C.L. Masters, G.Multhaup. L.-A. Fransson and K. Mani, J Biol.
Chem. 2005; 280: 13913; cf., K Mani et al., ibid., 2004; 279, 12918-12923; ibid., 2003, 278: 38956-38965;

13 D Grant suggested in 2000 a similar mechanism of redox metal ion and ascorbate nitric oxide metabolite dependent HS
Oligosacchride signalling in preliminary draft discussion articles entitled “Ascorbate and Nitric Oxide in Redox Control of Heparan Sulphate and
“Ascorbate & Cancer” originally posted on web.ukonline.co.uk/dgrant/dg4/- etc are now available as cached files from Yahoo (but not now Google) using
the search terms /dgrant/dg4- and /dgrant/dg5- (with the references at /dgrant/dg8/-) cf., also /dgrant/dg2- and /dgrant/dg5- )

13a R.A.A. Muzzarelli, Polymer Sci. Technol. 1983; 23: 359-374

(which describes the preparation of heparin-like derivatives of chitosan which are evidetly highly ‘contaminated’ with iron

14 H.J.M. Bowen, J. Mar. Bio. Assoc. 1956; 35: 451-458

15 a V.N Senofonte and S Caroli, J. Trace Elem. Med. Biol., 2000, 14, 6-13

b I. Rodushkin and M.D. Axelsson, Sci Total Environ., 2000, 250, 83-100;

c I. Rodushkin and M.D. Axelsson, ibid., 2000, 262, 21-36;

[cf. also http://www.analytica.se/hem2001/human/research.asp; cf., D.A. Bass et al., Altern. Med. Rev., 2001, 6, 472-481 and
A.R. Bleise et al. Analytical Qualtity Control Services, (Hair Elements Reference Sheet), International Atomic Energy Agency,
A-1400,Vienna, Austria RAA

16 A. Kornberg (with M.R.W. Brown) PNAS USA, 2004, 46, 16095

17 J.M. Somers, M.L. Tait, W..F Long and F.B, Williamson, Hydrobiologica, 1990; 204/205: 491-497

12 Concluding Remarks
Polysaccharides apart from consisting of complex information-containing sequences of differently sulphated
polysaccharides (the heparanome) also co-exist with an array of inorganic ions and particles including alkali metal
and alkaline earth counterions present in biological fluids as well as trace and ultratrace elements also present in
such fluids.
Unfractionated H is apparently the most optimized known example of any biological metallomic matrix system
which, if this is confirmed, could suggest that the evolution of H-like structures in HS present in all
animal species and mast cell H present in many animal species must have been, at least in part, driven by
the potential advantages conferred by the presence (especially in extracellular glycocalyx environments) of
such potential reservoir matrices containing arrayed (and largely available) inorganic elements.

The complex patterns of sulphated anionic sugar HS proteoglycans, perhaps partly as flexible metallo-polysaccharide complexes can
logically be proposed to be of sufficient functional complexity to fulfill the sought after role of a non-genetic biochemical information
handing and storage system suggested to be required for the post-human genome era The information-encoded H/HS system is that it is
known to be involved in a systemic signaling and could explain why the human geneome is much smaller than expected.

It is suggested that the modulation of the activity of HS sequences by metal ions could act as an amplification loop to extend the
information processing abilities of otherwise inadequate, limited size genomes may contribute to the development and perhaps also the
evolution of complex animal organisms

HS- associated multi-inorganic ion arrays are now suggested to promote the formation of functionally relevant HS supramolecular
structures, which, further subject to the efficiency of probably required nucleation agents, and perturbation by pathological and
anthropgenic factors, are suggested to be highly relevant to the etiologies of those degenerative diseases in which HS biochemistry has
been implicated.
Sequence patterns in HS arise via servo feedback controlled alterations of the sequential utilization of the enzymic modifications during their
Gogli apparatus assembly as well as via related postsynthetic modifications which also include an important non-enzymic (environment-linked)
putatively metallomic input.
H and HS are now known able to bind and modulate the activities of such a wide range of proteins, many of which are known to require inorganic
cofactors, that this circumstance can be putatively be extrapolated to suggest that H/HS is involved, in addition to embryogenesis and ageing, also, in
virtually all physiological and pathological processes; this further could suggest that the complex inorganic multi-element metallomic biochemistry of
H/HS could contriubte to the mechanism of evolutionary change in animal genetics, probably by servo control feedback processes enabling
environmental factors to influence genetic factors via the intermediary alteration of HS signals which are known to inteconnect extracelular spce
with the geneome
12.1 Suggested Further Research

Further research is required to establish the optimal inorganic ion array to allow correct in vitro H
modelling of HS in vivo behaviour and also determine how different types of final purification procedures
adopted by different manufacturers alters the inorganic array associated with
H.

The single counterion diluted forms of H exhibit skewed inorganic ion distributions relative to the less purified
form a distribution which evidently can vary between different manufacturers, depending on the details of the
purification procedures used which is a source of potential often ignored variation in the activities of H used to
provide a convenient molecular structural model library system for biochemical investigations of HS
activities.

Purified oligosaccharide epitopes: do such preparations lack the natural multi-inorganic ions normally associated with
the intact polysaccharide molecules and which, can be suggested to be essential cofactors to allow the necessary
discrimination to accomplish developmental regulation?

*Compiled by the author (from Turriff, Scotland, UK, AB53 6SX) who was previously a senior industrial research chemist (with
Monsanto, ICI and ISR) who during later fixed-term research studies with the polysaccharide groups at Marischal College, University of
Aberdeen, U.K. the management of which (FB Williamson and WF Long) are thanked for their support which started in 1981 when this
literature survey on H/HS was also commenced and the outcome of which suggests that which this polysaccharide system is putatively a
master system manger in animal biology. Professor RJP Williams of Oxford University is also warmly thanked for conducting a long
correspondence offering advice and criticism of the subject matter of this communication.

** Review of these data is outwith the scope of this communication [but further communications are in preparation]