Biochemistry - A Case Oriented Approach For Medical Students

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Biochemistry- A Case Oriented Approach for Medical Students
Book · June 2012
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BIOCHEMISTRY- A CASE ORIENTED
APPROACH FOR MEDICAL STUDENTS
1
Dedicated
to my wife Sumi
and
my daughter Brinda
2
ACKNOWLEDGEMENTS
Any work is not possible without the guidance of teachers, support of parents, enthusiasm by
kith and kins and undoubtedly by the Grace of Almighty God. It would be my proud privilege
to pay my sincere and profound thanks to Prof. Utpal Kumar Biswas, MD Prof, Head
Department of Biochemistry, Nilratan Sarkar Medical College and Teaching Hospital, Kolkata
under whose inspiration, guidance and support to write this book on Biochemistry a case
oriented approach for medical students. He has been constant source of encouragement for me
in all odds. I am also thankful to Prof. Susil Gunasekera Wickramashinghe, PhD, Department
of Biochemistry, Faculty of Medicine, University of Peradeniya, Sri Lanka for rendering me his
support and advice to introduce case studies in this book. I am utmost grateful to laboratory
staffs of Biochemistry department of Manipal College of Medical Sciences, Pokhara, Nepal and
Faculty of Medicine, University of Peradeniya, Srilanka for being with me and have been
supportive to me during scripting this book.
My limited vocabulary prevents me to use all the superlatives which the members of family
deserve for their association and co-operation. I thank my parents and my in-laws were very
much supportive to me at all times. I am grateful to God Almighty because of helping us in all
odds that came to my way during drafting this book. I am also grateful to my daughter Miss
Brinda who has been blissful to me at the time when I was under stressful situation. She came
to my life and filled my heart with happiness and enthusiasm.
Last but not the least, I thank to God Almighty for giving me such a caring wife, who has been
with me through all circumstances and always stood by side at the time of adversity. She has
3
been supportive and friendly even though she sometimes used to say that your computer is your
second wife, without her support I would not be able to complete scripting this book.
4
TABLE OF CONTENTS
Chapters Page No
1. Chemistry of Carbohydrates- Viva and Revision 7

2. Chemistry of Lipids and Eicosanoids Viva Questions 15
3. Chemistry of Lipids and Eicosanoids MCQs 22
4. Amino acid Metabolism- MCQs 27
5. Acid base balance- MCQs 33
6. Examination of Blood Urea- Viva Questions 37
7. Heme synthesis and degradation- MCQs 42
8. AIDS – MCQs 46
9. Liver function tests- Choose the correct answer 50
10. Liver function tests- A brief summary 52
11. Estimation of Serum total proteins – Viva Questions 53
12. Case Study- Jaundice 58
13. Case Study- Acid Base Balance 66
14. Case Study for self evaluation- Amino acid metabolism 74
15. Case Study- Orotic aciduria 78
16. Case Study – Starvation 81
17. Case Study- AIDS 86
18. Case Study- Carnitine deficiency 103
19. Case Study- Cystinosis 107
20. Case Study- Diabetic ketoacidosis 111
21. Case Study- Diet and Nutrition 122
22. Case Study- Gout 127
23. Case Study- Hypercalcemia 136
24. Case Study- Impaired liver function tests 138
25. Case Study- Metabolism of Carbohydrate 140
26. Case Study- Obesity 149
27. Case Study- Urea Cycle disorders 152
28. Case Study- Lesch Nyhan syndrome 161
29. Case Study- Porphyria 165
30. Case Study- Refsum’s disease 177
31. Case Study- Lipid Disorders- Self Assessments 181
32. Case Study- Thyroid Disorders- Self Assessments 184
33. Case Study- Mechanism of action of Heparin 186
34. Biological Oxidation 189
35. Isoenzymes and their clinical significance 194
36. Mechanism of Iron Absorption 200
37. Electrophoresis – A Brief review 202
38. Mutation – An Overview 209
39. UDP-Glucuronyl transferase catalyzed and significance 223
40. Vitamin E deficiency and functions 226
5
41. Renal Clearance 231
42. Subjective Questions- Acid base balance and imbalance 234
43. Subjective Questions- Chemistry of Nucleotides and Nucleic acids 237
44. Subjective Questions- Enzymes 239
45. Practice Questions Subjective- Amino acid metabolism 242
46. Solved Questions- Serum Creatinine and Creatinine clearance 245
47. Solution to Practice Questions 249
48. Solution to Multiple choice questions- Enzymes 253
49. Normal Laboratory reference range 258
6
CHEMISTRY OF CARBHYDRATES- VIVA AND REVISION
Q.1- Choose the odd one out-
Heparin, Heparan, Dermatan, Dextran

(Dextran)
Starch, Glycogen, Chitin, Inulin

(Chitin)
Q.3- Which out of the following dextrins does not give color with Iodine-
Achrodextrins, Erythrodextrins, Amylodextrins

(Achrodextrins)
Q.4- Name a non sulfated Heteropolysaccharide

(Hyaluronic acid)
Q.5- Which heteropolysaccharide is used as an anticoagulant

(Heparin)
Q.6- Which sugar acid is used for the detoxification of the foreign compounds?
(Glucuronic acid)
Q.7- Which monosaccharide is used as the preferred source of energy for the brain cells?
(Glucose)
Q.8-Which monosaccharide is used as a source of energy for the spermatozoa?

(Fructose)
Q.9- Which disaccharide is an intermediate in the hydrolysis of starch?

(Maltose)
Q.10- Which monosaccharide is optically inactive?

(Dihydroxy acetone)
Q.11- What is odd out of the following four-

Glucose, Galactose, Mannose and Fructose
(Fructose)
Q.12- Choose the ketopentose-

Ribose, Xylose, Xylulose and Arabinose (Xylulose)
7
Q.13- Name a six membered sugar alcohol
(Sorbitol, Mannitol or Galacitol)
Q.14- Name a sugar acid

(Gluconic acid)
Q.15- Name an amino sugar acid

(Neuraminic acid)
Q.16- Name an intracellular polysaccharide

(Heparin)
Q.17- D and L isomers differ from each other by orientation around which C atom?
(Penultimate carbon, farthest from the most oxidized C atom)
Q.18- Alpha and Beta anomers differ in orientation around 5 th carbon atom in a hexose-
True or false ? (False)
Q.19- Malt sugar is------------------------ ?

(Maltose)
Q.20- Out of Lactase and Cellulase which enzyme is absent in human beings?
(Cellulase)
Q.21- Mucic acid is produced from---- ?

(Galactose)
Q.22- Give an example of Glycosylamine

(Ribosylamine)
Q.23- Name a sugar alcohol with five carbon atoms

(Ribitol)
Q.24- Powder-puff shaped crystals are formed by------

(Lactose)
Q.25- Name two non reducing sugars

(Sucrose and Trehalose)
Q.26- Which test is used to differentiate between aldohexose and ketohexose?

(Seliwanoff test)
Q.27- Benedict’s test is more sensitive than Fehling test. True or false?
(True)
8
Q.28- Glycogen is stored mainly in muscles. True or false?
(False)
Q.29- Name a cardiac Glycoside

(Digitalis)
Q.30- What is milk sugar?

(Lactose)
Q.31- Name the product of reaction of a strong acid on a pentose

(Furfural)
Q.32- What are enediols?

(Double bonded carbon atoms each having OH group attached)
Q.33- Name a 7 Carbon atoms containing ketose sugar

(Sedoheptulose)
Q.34- Name the alcohol produced from the reduction of Glyceraldehyde.

(Glycerol)
Q.35- How many isomers of Glucose are found in the biological system?
(32, including anomers)
Q.36- Glucuronic acid produced from the reduction or oxidation of Glucose?

(Oxidation)
Q.37- Dextrin or Dextran, which out of the two is used as plasma expander?
(Dextran)
Q.38- Which one is a branched polymer out of the two-

Amylose or Amylopectin?
(Amylopectin)
Q.39- Reddish brown colour with iodine is given by which carbohydrate?

(Glycogen)
Q.40- Which sugar is called as Invert sugar?

(Sucrose)
Q.41- Agar is a homo or hetero polysaccharide?

(Homopoysaccharide)
9
Q.42- Which of the two does not contain a sugar acid -Keratan or Heparan Sulfate?
(Keratan sulfate)
Q.43- Name an epimer of Sorbitol

(Mannitol)
Q.44- Name an epimer of Glucuronic acid

(Iduronic acid)
Q.45- How galactose and fructose are related to each other?

(Isomers)
Q.46- What is Aglycon?

(Non carbohydrate component in a glycoside)
Q.47- Maltose is composed of what kind of monosaccharides?

(Glucose – glucose)
Q.48- Name a pentose sugar present abundantly in heart muscle

(Lyxose)
Q.49- Name a deoxy sugar

(Deoxy ribose)
Q.50- Name the polysaccharide present in the exoskeleton of insects

(Chitin)
Q.51- What type of linkage is present between Galactose and Glucose in Lactose?
ȕ (1ĺ4) glycosidic linkage
Q.52- The compounds having same structural formula but differing in configuration around
one carbon atom are called-
(Epimers)
Q.53- What type of linkages are present in Glycogen?

(Į(1ĺ4) in the chain and Į(1ĺ6) at the branch point
Q.54- Name a fructosan

(Inulin)
Q.55- Name a Galactosan

(Agar)
10
Q.56- Name the test for detection of carbohydrates in a solution
(Molisch test)
Q.57- When a hexose is made to react with a strong acid, what is the product called?
(Hydroxy methyl furfural)
Q.58- How are Mannose and Glucose related to each other?

(C-2 epimers)
Q.59- When both aldehyde and primary alcoholic groups are oxidized in mannose, the
product formed is?
(Mannaric acid)
Q.60- Dulcitol is produced from the reduction of which sugar?

(Galactose)
Q.61- Name two amino sugars

(Glucosamine and Galactosamine)
Q.62- Out of Mucic acid and Muramic acid which one is an amino sugar acid?
(Muramic acid)
Q.63- Name a sugar ester

(Glucose 6 phosphate)
Q- 64- Which out of the following will give Bial’s test positive
Glucose, Fructose, ribose
(Ribose)
Q.65- All except one will exhibit mutarotation?

Sucrose, Maltose, Glucose, Galactose
(Sucrose)
Q.66- Out of Pyranose and Furanose ring which one is commonly formed by Fructose?
(Furanose)
Q.67- Which out of the two has more carbohydrate content?

Proteoglycan or Glycoproteins
(Proteoglycan)
Q.68- Name the storage polysaccharides

(Glycogen, Starch, Inulin etc)
11
Q.69- Cornea is rich in which type of mucopolysaccharides?
(Keratan sulfate)
Q.70 - Name C-4 epimers

(Glucose and Galactose)
Q.71- Name a keto triose

(Dihydroxy acetone)
Q.72- Name the alcohol produced from the reduction of Fructose

(Sorbitol and Mannitol)
Q. 73- Glucose is the only source of energy for what kind of cells?
(Red blood cells and the cells which lack mitochondria)
Q.74- How is Aldonic acid produced from a monosaccharide

(By the oxidation of aldehyde group of an aldose sugar)
Q.75- What type of linkages are present between glucose residues in Cellulose?
(ȕ 1, 4 Glycosidic linkages)
Q.76- Out of all the biologically important mucopolysaccharides which one is the most
negatively charged?
(Heparin)
Q.77- Which monosaccharide is present as a structural component of RNA?

(Ribose)
Q.78- What is dextrose?

(D- Glucose)
Q.79- What is table sugar?

(Sucrose)
Q.80- What is animal starch?

( Glycogen )
Q.81- What is Muta rotation?

Carbohydrates can change spontaneously between alpha and beta configurations
through intermediate open chain formation, this leads to a process known as
Mutarotation.
12
Q.82- Which hexose is an important component of glycoproteins?
(Mannose)
Q.83- When equal amount of dextrorotatory and levorotatory isomers are present in a
mixture, the mixture is said to be ------ ?
(Racemic )
Q.84- Glucose when treated with bromine water produces ------ ?

(Gluconic acid)
Q.85- Name a glycoside which is an inhibitor of Sodium Potassium ATPase pump.

(Oubain)
Q.86- What is the storage form of glucose in plants?

(Starch)
Q.87 – Name an amino sugar acid which is present in gangliosides.

(NANA- N -acetyl Neuraminic acid)
Q.88- Deoxy ribose is synthesized from ribose by removal of oxygen around which carbon
atom?
(C-2)
Q.89- The carbohydrate of blood group substance is ----- ?

(Fucose)
Q.90- Which of the following is not a polymer of Glucose?

Cellulose, Inulin. Glycogen, Dextrins
(Inulin)
Q.91- Which of following is an anomeric pair?

a) D-glucose and L-glucose b) Į-D-glucose and ȕ-D-glucose
(Į-D-glucose and ȕ-D-glucose)

Muramic acid, Mucic acid. Mannaric acid, Mannonic acid
(Muramic acid)
Q.93- The cyclical structure of Glucose is represented by-

Glucopyranose, Glucofuranose or Glucoside
(Glucopyranose)
Q.94- What kind of monosaccharides will be produced by lactose hydrolysis?

(Glucose and Galactose)
13
Q.95- Name a keto hexose
(Fructose)
Q.96- What is an asymmetric carbon atom?

A carbon atom with all the four different attachments is called as an asymmetric
carbon atom
Q.97- How many isomers of glyceraldehyde are possible?

(D and L)
Q.98- How is Ribose and Ribulose related to each other?

(Aldose, ketose isomers)
Q.99- What is the repeating disaccharide unit in Hyaluronic acid?

(D-glucuronate + GlcNAc) n
Q.100- Name an Aldotetrose which is an intermediate of HMP pathway?

(Erythrose-4 P )
14
CHEMISTRY OF LIPIDS AND EICOSANOIDS- VIVA QUESTIONS (SOLVED)
Q.1- Name the fused ring system present in cholesterol.

Answer-Cyclo-pentano-per hydro-phenanthrene ring.
Q.2- Which alcohol is generally present in waxes?

Answer-Cetyl alcohol.
Q.3- Name the vitamins which act as alcohols to esterify fatty acids.
Answer- Vitamin A and D.
Q.4- Spontaneous oxidation of polyunsaturated fatty acids present in the biological

membranes is called ----
Answer- Lipid peroxidation..
Q.5- Which glycolipid is abundantly present in the white matter of brain?

Answer- Galactosyl ceramide
Q.6- Which lipo protein transports cholesterol from liver to peripheral tissues?
Answer-LDL (Low density Lipoprotein).
Q.7- Which reaction is catalyzed by LCAT?

Answer-Esterification of cholesterol
Lecithin+ Cholesterol---- Lysolecithin +Ester cholesterol.
Q.8- Name two tri-enoic fatty acids.

Answer-Alpha and gamma Linolenic acids.
Q.9- Give two biologically significant features of Phospholipase A2

Answer-Required for release of Arachidonic acid for the formation of Eicosanoids and
provides fatty acid for the esterification of cholesterol in reverse cholesterol transport.
Q.10- Low dose of Aspirin promotes the relative increase in the synthesis of
prostacyclins, Is it true or false ?
Answer- True
Q.11-The surface tension of dietary fat droplets in the intestine is decreased by -- ?

Answer- Phospholipids and bile salts.
Q.12- What is the advantage of taking low dose Aspirin?

Answer- Prevents thrombus formation and is recommended to high risk patients or to those
who have a family history of IHD.
15
Q.13-In which form are the fats stored in the body for long term storage of energy,
Answer- Triacyl glycerol.
Q.14- While comparing the potential energy of lipids and carbohydrates on weight
basis, is it correct to say that lipids provide considerably more energy than
carbohydrates?
Answer- yes, it is correct. Lipids provided 9.5 K. cal/G energy as compared to 4.0 K. cal/G
produced from the complete oxidation of carbohydrates.
Q.15- A child presented with hyperacusis, regression of mile stones and progressive
blindness. What could be the possible defect?
Answer-Tay Sach disease- GM2 Gangliosidosis, deficiency of Hexosaminidase A enzyme.
Q.16- The fluidity of the biological membranes is increased by the increase/ decrease
in the degree of unsaturation of the component fatty acids.
Answer- Fluidity of the biological membranes is increased by increase in the degree of
unsaturation of component fatty acids.
Q.17- What are the uses of Liposomes?

Answer- They act as drug carriers for the specific target sites without causing side effects.
Also used for gene therapy.
Q.18- Which phospholipid in a reservoir for second messenger?

Answer-Phosphatidyl Inositol
Q.19- Give two examples of unsaturated fatty acids

Answer-Oleic. Linoleic, Linolenic acid etc.
Q.20-What are sulfolipids and where are they present in the body?
Answer-Sulfated Galactosyl ceramides are sulfolipids and they are present abundantly in
the nervous tissue.
Q.21- What does the notation 18:0 signify?

Answer- Saturated fatty acid with 18 carbon atoms.
Q.22- What are the components of a Glycero phospholipid?

Answer-Glycerol, two fatty acids, phosphoric acid, a nitrogenous base or other components
Q.23- What are neutral lipids?

Answer- Because they are uncharged, Acylglycerols (glycerides), cholesterol, and
cholesteryl esters are termed neutral lipids.
Q.24- What is an alpha carbon in a fatty acid?

Answer-The carbon atoms adjacent to the carboxyl carbon.
16
Q.25- Which fatty acid is present in Oxynervon?
Answer-Hydroxy Nervonic acid.
Q.26- What is the difference between Cervonic acid and Cerebronic acid?
Answer- Cervonic acid is polyunsaturated fatty acid while Cerebronic acid is Hydroxy fatty
acid.
Q.27- Which of the following is an animal sterol?

Ergo sterol, Stigma sterol, Sitosterol, Cholesterol
Answer- Cholesterol.
Q.28- Which of the following contains a five membered ring-?

Plasmalogen, Glycolipids, Sphingomyelin and Prostaglandins
Answer-Prostaglandins.
Q.29- Prostacyclins increase the concentration of c- AMP, is it true or false?

Answer- It is true.
Q.30- What is the cause of osmotic diarrhea after PG( Prostaglandin) administration?
Answer -There is PG induced increase in the volume of pancreatic and Intestinal secretions
resulting in osmotic diarrhea.
Q.31- What are the components of a triacylglycerol?

Answer-Glycerol+ 3 fatty acids.
Q.32- Name the different kinds of lipases present in the human body ?
Answer- Lingual, gastric, pancreatic and intestinal lipases are digestive lipases for the
digestion of triacylglycerols, while there are phospholipases of different kinds besides
hormone sensitive lipase present in the adipose tissue and Lipoprotein lipase for the
digestion of lipoproteins.
Q.33- What are the sources of fatty acids?

Answer- Diet, Endogenous synthesis and derived from adipose tissue by adipolysis.
Q.34- At which position is the fatty acid attached to the cholesterol ring,?
Answer- 3 rd position (Esterified to OH group present at the 3rd position).
Q.35- What is the product of a reaction of a fatty acid with alkali ?

Answer- Salt. This property is used for soap formation and for cleaning the chocked
drains using an alkali.
Q.36- Define rancidity.

Answer-The unpleasant taste and odour developed by fats on ageing is called rancidity.
17
Q.37- What are the components of Slow releasing/Reacting substance of Anaphylaxis
?
Answer-LTC4, LTD4 and LTE4.
Q.38- What is the site of cleavage of Sphingomyelinase enzyme?

Answer-Removal of phosphoryl choline from ceramide
Q.39 -Based on the nature of fatty acids present, how many types of Cerebrosides are
there in the human system?
Answer- 4 types- Kerasin (Lignoceric acid), Cerebron (Cerebronic acid), Nervon (Nervonic
acid) and Oxynervon (Hydroxy derivative of Nervonic acid ).
Q.40- Which out of the two COX enzymes I and II is inducible?

Answer- It is COX II.
Q.41- Name a sphingolipid

Answer-Sphingomyelin.
Q.42- All are conditions of hypercholesterolemia except-

Anemia, Diabetes Mellitus, Hypothyroidism, Nephrotic syndrome
Answer- Anemia, there is low cholesterol level in blood.
Q.43- Name a selective COX inhibitor

Answer- Celecoxib and Rofecoxib.
Q.44- Name the drugs which can inhibit Prostaglandin synthesis,

Answer- Steroids and NSAIDs.
Q.45- Give the therapeutic uses of PGs

Answer-Termination of pregnancy, Treatment of asthma hypertension etc.
Q.46- Why do oils float on the surface of water?

Answer-Oils have lesser specific gravity than water.
Q.47- What is the systematic name of Alpha Linolenic acid

Answer- all cis- octadectrienoic acid-18;¨3, 9,12,15.
Q.48- What is the main function of HDL?

Answer- Transportation of cholesterol from peripheral tissues to liver, It is called Good
cholesterol.
Q.49- Butter is rich in short and medium chain fatty acids, Is it True or false?
Answer- It is true.
18
Q.50-Name the disease caused due to deficiency of Beta Glucosidase enzyme .
Answer-Gaucher’s disease.
Q.51- What is the difference between Cerebrosides and Globosides?

Answer-Cerebrosides contain a single monosaccharide while Globosides contain more than
one monosaccharide; it may be Lactosyl or oligosaccharide ceramide.
Q.52- Which phospholipid is present in the mitochondrial membrane?

Answer-Cardiolipin.
Q.53- Which phospholipid has unsaturated long chain alcohol in ether linkage with
the first hydroxyl group of glycerol ?
Answer- Plasmalogen.
Q.54- What is the basis of removing grease stains with petrol?

Answer-Petrol is an organic solvent, and grease is a wax. Petrol makes a soluble complex
with grease.
Q.55- What are the clinical manifestations in Essential fatty acids deficiency?
Answer- Growth retardation, dermatitis, fatty liver and impaired vision.
Q.56- Why are prostaglandins not conventionally used as drugs?

Answer-Short duration of action, rapidly destroyed and non specific in action.
Q.57- Name the compound lipids.

Answer-Phospholipids, glycolipids, sulfolipids, amino lipids and lipoproteins.
Q.58- Name any odd chain fatty acid.

Answer-Propionic acid, Valeric acid.
Q.59- Why is alkali used for opening the choked drains?

Answer- Alkali causes Saponification of fats forming water soluble soaps resulting in
reopening of the blocked drains.
Q.60- Name the phospholipids which act as lipotropic agents

Answer- All but most importantly, Phosphatidyl choline, Phosphatidyl ethanolamine and
Phosphatidyl Inositol.
Q.61- What is the defect in Niemann pick’s disease?

Answer-Deficiency of Sphingomyelinase enzyme.
Q.62- What is the diagnostic hall mark of Gaucher disease ?

Answer-Presence of Gaucher cells in bone marrow aspiration biopsy
19
Q.63- Infants placed on low fat diet due to a variety of reasons generally develop skin
rashes and other symptoms. What is the reason for it?
Answer- Essential fatty acid deficiency.
Q.64- Which out of the following is an inter mediate both for the synthesis of
phospholipids and Triacylglycerols-
Diacyl glycerol, Cholesterol, Choline , Inositol
Answer- Diacyl glycerol.
Q.65- What is the risk associated with increased levels of serum total cholesterol?
Answer-Atherosclerosis.
Q.66- Why is cyclo-oxygenase enzyme called the suicidal enzyme?

Answer-It catalyzes its self destruction.
Q.67-Name a saturated fatty acid with 18 carbon atoms abundantly present in the
body tissues.
Answer- Stearic acid.
Q.68-Which fatty acid should have the least melting point out of the followings-?
Stearic acid, Arachidonic acid, Timnodonic acid
Answer-Timnodonic acid, since it has five double bonds; more the degree of unsaturation,
lesser is the melting point.
Q.69- A 3 year child was brought with hepatosplenomegaly and mental retardation.
Biopsy revealed accumulation of sphingomyelin. What is the defect?
Answer The child is suffering from -Niemann Pick’s disease.
Q.70-Name the polar derivative of cholesterol

Answer- Bile salts.
Q.71- A female patient with 34 weeks of pregnancy has to under go Emergency

caesarian section for the delivery of the baby but the L:S ratio of amniotic fluid is 1:1 .
What is the significance of this ratio and what is recommended to this female?
Answer- L/S ratio should be >2-5:1 for adequate fetal lung maturity. In the given patient
L/S ratio of1: 1 indicates fetal lung immaturity, Injections of Gluco-corticoids are
recommended for her..
Q.72- What is meant by Total cholesterol?

Answer-Free cholesterol+ Esterified cholesterol.
Q.73- Why can’t essential fatty acids be synthesized by the human body?
20
Answer- Humans lacks the enzyme to incorporate double bond between the existing double
bond and the methyl end (Ȧ end).
Q.74- Name a fatty acid with 18 carbon atoms and a single double bond in trans
configuration
Answer-Elaidic acid.
Q.75- Why IS LDLc called a bad cholesterol?

Answer- Since it transports cholesterol from liver to peripheral tissues and excess of LDL
can result in atheroma formation increasing the risk for IHD, Stroke or Peripheral vascular
disease.
21
CHEMISTRY OF LIPIDS AND EICOSANOIDS- MCQ (SOLVED)
Q.1- Endogenously synthesized triacylglycerols are transported from liver to extra

hepatic tissues by which of the following lipoproteins?
a) Chylomicrons
b) VLDL
c) LDL
d) HDL
Q.2- All of the followings have 18 carbon atoms except –

a) Linoleic acid
b) Palmitic acid
c) Linolenic acid
d) Stearic acid
Q.3- Sphingosine is not present in-

a) Cerebrosides
b) Gangliosides
c) sphigomyelin
d) Plasmalogen
Q.4- Triacylglycerols are-

a) Energy rich compounds
b) Nonpolar in nature
c) Can be stored in unlimited amounts
d) All of the above
Q.5- All are essential fatty acids except-

a)Linoleic,
b) Linolenic
c) Arachidonic acid
d) Stearic acid
Q.6- The deficiency of Lung surfactant, Dipalmitoyl lecithin (DPL) causes,

Respiratory Distress Syndrome. DPL is a –
a) Cerebroside
b) Ganglioside
c) Phospholipid
d) Lipoprotein
Q.7- Choose the correct statement-

a) The melting point of a fatty acid increases with the increasing degree of unsaturation in
the hydrophobic chain
22
b) Most of the naturally fatty acids have trans double bonds
c) Arachidonic acid is a relatively non essential fatty acid
d) The membrane lipids are rich in saturated fatty acids.
Q.8- Which out of the following fatty acids is a precursor of series -1 Eicosanoids?
a) Linoleic acid
b) Arachidonic acid
c) Eicosapentaenoic acid
d) Linolenic acid
Q.9- What is the cause of hyper acidity on long term usage of Aspirin?
a) Inhibition of cyclo oxygenase
b) Increased synthesis of PGs
c) Inhibition of Phospholipase A2
d) All of the above
Q.10- Which nitrogenous base out of the followings is present in lecithin ---?
a)Choline
b) Adenine
c) Ethanolamine
d) Any of the above
Q.11- Cholesterol is a precursor of all except-

a)Bile salts,
b)Bilirubin
c) Steroids
d) vitamin D
Q.12- Glycerol is used for the synthesis of all except-

a) Glucose.,
b) Phospholipids,
c) Glycolipids,
d) Triacylglycerol
Q.13- Which out of the followings is a fatty acid with 16 carbon atoms and one double
bond?
a) Palmitoleic acid
b) Oleic acid
c) Erucic acid
d) Elaidic acid
Q.14-Which out of the followings is an ȫ 3 fatty acid?

a) Į Linolenic acid
b) Linoleic acid
23
c) Palmitic acid
d) Arachidonic acid
Q.15- Fats on keeping for a long time under go spontaneous hydrolysis, what is this
process called?
a) Saponification
b) Hydrolytic Rancidity
c) Decomposition
d) All of the above
Q.16- Which out of the following enzymes is deficient in Gaucher’s disease?

a) Beta Glucosidase
b) Beta Galactosidase
c) Hexosaminidase A
d) Neuraminidase
Q.17- Prostcyclins are synthesized in- ---------?

a) Platelets
b) Endothelial cells
c) Gastric mucosa
d) Basophils
Q.18- Cyclo-oxygenase is inhibited by all except--------?

a) Aspirin
b) Indomethacin
c) Brufen
d) Zileuton
Q.19- The normal level of serum Total cholesterol is-----------?

a) 150-220 mg/dl
b) 100-200 mg/dl
c) 1.5-2.5g/dl
d) 20-40 mg/dl
Q.20- Choose out of the followings, a fatty acid with 20 carbon atoms and five double
bonds-
a) Timnodonic acid
b) Arachidonic acid
c) Clupanodonic acid
d) Nervonic acid
Q.21- Which type of lipid is a receptor for cholera toxin in the intestine?
a) GM2 Ganglioside
b) GM1Ganglioside
24
c) Sphingomyelin
d) Galactocerebroside
Q.22- The significance of estimating L: S ratio of amniotic fluid in a pregnant female

lies in evaluating-
a) Fetal heart rate
b) Fetal lung maturity
c) Fetal head size
d) Expected date of delivery
Q.23- Iodine number is a measure of-

a) Degree of unsaturation of a fat
b) Degree of rancidity of a fat
c) Measure of volatile fatty acids in a fat
d) Measure of number of –OH groups in a fat
Q.24-Which phospholipid out of the following is antigenic in nature -?

a) Cardiolipin
b) Lecithin
c) Plasmalogen
d) Cephalin
Q.25- Which out of the followings is not a derived lipid?

a) Ketone body
b) PGE2
c) Diacylglycerol,
d) Galactosyl ceramide
Q.26- What are the components of a ceramide?

a) Sphingosine+ fatty acid
b) Glycerol+Fatty acids+Phosphoric acid
c) Glycerol+Fatty acids+Phosphoric acid+Nitoregenous base
d) Sphingosine+ fatty acids+Phosphoric acid
Q.27- Choose the incorrect statement-

a) The chemical name of Arachidonic acid is Eicosa penta enoic acid
b) Cyclo-oxygenase and peroxidase are the components of PG-H synthase complex
c) Oleic acid is represented by 18;1,¨9
d) NSAIDs act by inhibiting Phospholipase A2 enzyme.
Answers-
1-(b)- VLDL
2-(b)- Palmitic acid
3-(d)- Plasmalogen
25
4-(d) All of the above
5-(d) Stearic acid
6-(c) Phospholipid
7-(c) Arachidonic acid
8-(a) Linoleic acid
9-(a)- Inhibition of cyclo-oxygenase enzyme
10-(a) Choline
11-(b) Bilirubin
12-(c) Glycolipids
13-(a) –Palmitoleic acid
14-(a)-Į- Linolenic acid
15-(b)-Hydrolytic Rancidity
16-(a) Beta Glucosidase
17-(b)-Endothelial cells
18-(d)-Zileuton
19-(a) 150-220 mg/dL
20-(a) Timnodonic acid
21-(b) GM1- Ganglioside
22-(b) Fetal lung maturity
23-(a)- Degree of unsaturation
24-(a)- Cardiolipin
25-(a)- Galactosyl Ceramide
26-(a)- Sphingosine+Fatty acid
27-(d) NSAIDs act by inhibiting PhospholipaseA2 enzyme
26
MULTIPLE CHOICE QUESTIONS- AMINO ACID METABOLISM
(SOLVED)
Q.1- Which of the following is a common compound shared by the TCA cycle and the
Urea cycle?
a) Į- Keto glutarate
b) Succinyl co A
c) Oxalo acetate
d) Fumarate
Q.2-Which of the followings is a common nitrogen acceptor for all reactions involving
transaminases?
a) Į- Keto glutarate
b) Pyruvate
c) Oxaloacetate
d) Acetoacetate
Q.3- In a 55- year-old man, who has been diagnosed with cirrhosis of liver, Ammonia
is not getting detoxified and can damage brain. Which of the following amino acids
can covalently bind ammonia, transport and store in a non toxic form?
a) Aspartate
b) Glutamate
c) Serine
d) Cysteine
Q.4- In a new born presenting with refusal to feeds and irritability, a deficiency of
Cystathionine –ȕ- synthase has been diagnosed, which of the following compounds is
expected to be elevated in blood?
a) Serine
b) Glutamate
c) Homocysteine
d) Valine
Q.5 -A 3- month-old child is being evaluated for vomiting and an episode of

convulsions, Laboratory results show hyperammonemia and Orotic aciduria. Which
of the following enzyme defect is likely to be there?
a) Glutaminase
b) Arginase
c) Argino succinic acid synthase
d) Ornithine Transcarbamoylase
Q.6- Which out of the following amino acids is not converted to Succinyl co A?
a) Methionine
27
b) Valine
c) Isoleucine
d) Histidine
Q.7-All of the following compounds are synthesized by transmethylation reactions,

except-
a) Choline
b) Epinephrine
c) Creatine
d) Ethanolamine
Q.8- A patient diagnosed with Hart Nup disease, (due to deficiency of transporter
required for the absorption of amino acid tryptophan), has been brought with skin
rashes and suicidal tendencies. Tryptophan is a precursor for many compounds, the
deficiencies of which can cause the said symptoms. Which out of the following
compounds is not synthesized by tryptophan?
a) Serotonin
b) Epinephrine
c) Melatonin
d) Niacin
Q.9- Histamine, a chemical mediator of allergies and anaphylaxis, is synthesized from

amino acid Histidine by which of the following processes?
a) Deamination
b) Decarboxylation
c) Transamination
d) Dehydrogenation
Q.10- The synthesis of all of the following compounds except one is deficient in a
patient suffering from Phenylketonuria-
a) Melanin
b) Melatonin
c) Catecholamines
d) Thyroid hormone
Q.11- The diet of a child suffering from Maple syrup urine disease (an amino acid
disorder), should be low, in which out of the following amino acids content?
a) Branched chain amino acids
b) Phenylalanine Alanine
c) Methionine
d) Tryptophan
Q.12- Which out of the following amino acids in not required for creatine synthesis?
a) Methionine
28
b) Serine
c) Glycine
d) Arginine
Q.13- All of the following substances are synthesized from Cysteine, except-
a) Taurine
b) Mercaptoethanolamine
c) Melanin
d) Pyruvate
Q.14- Urea is synthesized in -

a) Cytoplasm
b) Mitochondria
c) Both cytoplasm and mitochondria
d) In lysosomes
Q.15-Blood urea decreases in all of the following conditions, except-

a) Liver cirrhosis
b) Pregnancy
c) Renal failure
d) Urea cycle disorders
Q.16- All of the following amino acids are donors of one carbon compounds except-
a) Histidine
b) Tyrosine
c) Tryptophan
d) Serine
Q.17- The two nitrogen of urea are derived from-

a) Aspartate and Ammonia
b) Glutamate and ammonia
c) Argino succinate and ammonia
d) Alanine and ammonia
Q.18- Which out of the following amino acids is not required for the synthesis of
Glutathione?
a) Serine
b) Cysteine
c) Glutamic acid
d) Glycine
Q.19- The first line of defence in brain in conditions of hyperammonemia is-

a) Urea formation
b) Glutamine synthesis
29
c) Glutamate synthesis
d) Asparagine formation
Q.20- Which coenzyme out of the followings is required for the oxidative deamination
of most of amino acids?
a) Folic acid
b) Pyridoxal- P
c) FMN
d) FAD
Q.21-Chose the incorrect statement about amino acid Glycine-

a) One carbon donor
b) Required for the synthesis of haem
c) Forms oxalates upon catabolism
d) Both glucogenic as well as ketogenic
Q.22- Which out of the followings is required as a coenzyme for the transamination
reactions?
a) Coenzyme A
b) Pyridoxal-P
c) Folic acid
d) Cobalamine
Q.23- A patient diagnosed with Homocystinuria should be supplemented with all of

the following vitamins except-
a) Vitamin C
b) Folic acid
c) Vitamin B12
d) Pyridoxal- P
Q.24- In a patient suffering from Cystinuria, which out of the following amino acids is
not seen in urine of affected patients?
a) Arginine
b) Methionine
c) Lysine
d) Ornithine
Q.25- Positive nitrogen balance is seen in all of the following conditions except-
a) Pregnancy
b) Growth
c) Fever
d) Convalescence
30
Q.26- The L-amino acids are absorbed from intestine by-
a) Active transport
b) Passive diffusion
c) Pinocytosis
d) Facilitated diffusion
Q.27- A child presented with increased frequency of urination, photophobia and

impairment of vision. Which out of the following defects could be responsible for the
said symptoms?
a) Tyrosinosis
b) Cystinosis
c) Alkaptonuria
d) Albinism
Q.28- Which out of the following statements about Glutamate dehydrogenase is

correct?
a) Required for transamination reactions
b) Universally present in all the cells of the body
c) Can utilize either of NAD+ /NADP+
d) Catalyzes conversion of glutamate to glutamine
Q.-29-A child was brought to pediatric OPD with complaint of passage of black
colored urine. A disorder of Phenylalanine metabolism was diagnosed. A low
phenylalanine diet and a supplementation of vitamin C were recommended. Which
enzyme defect is expected in this child?
a) Phenyl alanine hydroxylase
b) Tyrosine transaminase
c) Homogentisic acid oxidase
d) Hydrolase
Q.30- Dopamine is synthesized from which of the following amino acids?

a) Tyrosine
b) Tryptophan
c) Histidine
d) Methionine
Q.31- In mammalian tissue serine can be a biosynthetic precursor for which amino
acid?
a) Methionine
b) Glycine
c) Arginine
d) Lysine
31
Q.32- Hydroxylation of Phenyl Alanine to Tyrosine requires all except-
a) Glutathione
b) Tetra hydrobiopterin
c) Molecular oxygen
d) NADPH
Q.33- The amino acid that undergoes oxidative deamination at a highest rate is-
a) Glutamine
b) Glutamate
c) Aspartate
d) Alanine
Q.34- All of the following statements regarding serotonin are true except-
a) Causes vasodilatation
b) Causes broncho constriction
c) Metabolized to 5-hydroxy Indole acetic acid
d) Causes diarrhea
Q.35- Choose the incorrect statement about cysteine-

a) Carbon skeleton is provided by serine
b) Sulfur group is provided by Methionine
c) Forms Hippuric acid for detoxification of xenobiotics
d) Required for Bile salt formation
Answers-
1)-d, 2)-a, 3)- b, 4) -c, 5)-d, 6) -d, 7)-d, 8)-b, 9) -b, 10)-b,
11)-a), 12)-b, 13)-c, 14)-c, 15)-c,16)-b, 17)-a, 18)-a, 19)-b, 20)-c
21)-d, 22)-b,23)-a,24)-b, 25)-c, 26)-a, 27)-b,28)-c, 29)-c, 30)-a
31)-b,32)-a,33)-b, 34)-a,35)-c
32
MULTIPLE CHOICE QUESTIONS- ACID BASE BALANCE
Q.1- A person was admitted in a coma. Analysis of the arterial blood gave the
following values: PCO2 16 mm Hg, HCO3- 5 mmol/l and pH 7.1. What is the
underlying acid-base disorder?
a) Metabolic Acidosis
b) Metabolic Alkalosis
c) Respiratory Acidosis
d) Respiratory Alkalosis
Q.2- In a man undergoing surgery, it was necessary to aspirate the contents of the
upper gastro-intestinal tract. After surgery, the following values were obtained from
an arterial blood sample: pH 7.55, PCO2 52 mm Hg and HCO3- 40 mmol/l. What is the
underlying disorder?
Q.3- A young woman is found comatose, having taken an unknown number of

sleeping pills an unknown time before. An arterial blood sample yields the following
values: pH: 6.90, HCO3-: 13 meq/liter, PaCO2: 68 mmHg. This patient's acid-base
status is most accurately described as
a) Uncompensated metabolic acidosis

b) uncompensated respiratory acidosis
c) simultaneous respiratory and metabolic acidosis
d) respiratory acidosis with partial renal compensation
Q.4- A student is nervous for a big exam and is breathing rapidly, what do you expect
out of the followings-
Q.5- A 45- year-old female with renal failure, missed her dialysis and was feeling sick,
what could be the reason ?
33
Q.6- An 80 year old man had a bad cold. After two weeks he said, “It went in to my
chest, I am feeling tightness in my chest, I am coughing, suffocated and unable to
breathe!” What could be the possible reason?
Q.7- A post operative surgical patient had a naso gastric tube in for three days. The
nurse caring for the patient stated that there was much drainage from the tube that is
why she felt so sick. What could be the reason?
Q.8- The p H of the body fluids is stabilized by buffer systems. Which of the following
compounds is the most effective buffer system at physiological pH?
a) Bicarbonate buffer
b) Phosphate buffer
c) Protein buffer
d) All of the above
Q.9- Which of the following laboratory results below indicates compensated metabolic
alkalosis?
a) Low p CO2, normal bicarbonate and, high pH

b) Low p CO2, low bicarbonate, low pH
c) High p CO2, normal bicarbonate and, low p H
d) High pCO2, high bicarbonate and High pH
34
Q.10- The greatest buffering capacity at physiological p H would be provided by a
protein rich in which of the following amino acids?
a) Lysine
b) Histidine
c) Aspartic acid
d) Leucine
Q.11- Which of the following is most appropriate for a female suffering from Insulin
dependent diabetes mellitus with a pH of 7.2, HCO3-17 mmol/L and pCO2-20 mm
HG
Q.12- Causes of metabolic alkalosis include all the following except.
a) Mineralocorticoid deficiency.
b) Hypokalemia
c) Thiazide diuretic therapy.
d) Recurrent vomiting.
Q.13- Renal Glutaminase activity is increased in-

a) Metabolic acidosis
b) Respiratory Acidosis
c) Both of the above
d) None of the above
Q.14- Causes of lactic acidosis include all except-

a) Acute Myocardial infarction
b) Hypoxia
c) Circulatory failure
d) Infections
Q.15- Which out of the following conditions will not cause respiratory alkalosis?
a) Fever
b) Anxiety
c) Laryngeal obstruction
d) Salicylate toxicity
35
Q.16- All are true about metabolic alkalosis except one-
a) Associated with hyperkalemia
b) Associated with decreased ionic calcium concentration
c) Can be caused due to Primary hyperaldosteronism
d) Can be caused due to Renin secreting tumor
Q.17- Choose the incorrect statement out of the followings-

a) Deoxy hemoglobin is a weak base
b) Oxyhemoglobin is a relatively strong acid
c) The buffering capacity of hemoglobin is lesser than plasma protein
d) The buffering capacity of Hemoglobin is due to histidine residues.
Q.18- Carbonic anhydrase is present at all places except-

a) Gastric parietal cells
b) Red blood cells
c) Renal tubular cells
d) Plasma
Q.19- All are true for renal handling of acids in metabolic acidosis except-
a) Hydrogen ion secretion is increased
b) Bicarbonate reabsorption is decreased
c) Urinary acidity is increased
d) Urinary ammonia is increased.
Q.20- Choose the incorrect statement about anion gap out of the followings-
a) In lactic acidosis anion gap is increased
b) Anion gap is decreased in Hypercalcemia
c) Anion gap is decreased in Lithium toxicity
d) Anion gap is decreased in ketoacidosis.
Q.21- Excessive citrate in transfused blood can cause which of the following
abnormalities?
a) Metabolic alkalosis
b) Metabolic acidosis
c) Respiratory alkalosis
d) Respiratory acidosis
Answers- 1-a, 2-b, 3-c, 4-d, 5-a, 6-c, 7-b, 8-a, 9-d, 10-b, 11-a, 12-a, 13-c, 14-d, 15-c,
16-a, 17-c, 18-d, 19-b, 20-d, 21-a
36
ESTIMATION OF BLOD UREA- VIVA QUESTIONS
Q.1- What is the normal range of blood urea?
Answer- Blood urea in normal health ranges between 20-40mg/dl.
Q.2- How is urea formed in the body?
Answer- Urea is the end product of protein metabolism. It is formed in the liver from
ammonia and carbon dioxide; both of them are considered to be waste products of the body.
Ammonia is highly toxic, it is detoxified through conversion to urea, which is non toxic and
water soluble and is excreted through urine by the kidneys. In higher concentration urea is
also toxic. Hence formation and excretion of urea is dependent on liver and kidney
functions. In liver disorders urea formation is impaired hence blood urea decreases while in
disorders of kidney; excretion of urea is impaired resulting in high blood urea levels. Hence
blood urea level can be considered a predictor of hepatic or renal functional status.
Q. 3-What is the difference between Blood Urea and Blood Urea Nitrogen (BUN)?
Answer-Blood urea is sometimes expressed in terms of nitrogen. Such expression is very

common in clinical practice. Molecular weight of urea is 60 and each gram mol of urea
contains 28 gram of nitrogen. Thus a serum concentration of 28 mg/dl of BUN is
equivalent to 60mg/dl of urea. Any value of BUN can be converted to urea by multiplying
the figure by 2.14.
Blood urea= BUN x2.14
For example if BUN for a patient is 30 mg/dl,
Blood urea = 30x 2.14=64.20 mg/dl.
Q.4- What is meant by NPN (Non protein Nitrogen)?
Answer- NPN includes- Urea, uric acid and Creatinine. The major route for excretion of
these compounds is urine. In kidney dysfunction the levels of these compounds are elevated
in plasma. Of the three, creatinine estimation is the most specific index of renal function.
Urea level depends on the protein intake and protein catabolism and also on age of an
individual. It is also affected by volume of plasma.
Other minor components of NPN are urobilinogen, Indican, ammonia and amino acids.
Q.5- What is Uremia? What are the conditions causing uremia?
Answer- The clinical state with blood urea higher than normal is called Uremia. The
conditions causing high urea level are as follows-
A) Physiological – Advancing age and high protein diet.
37
B) Pathological-Classified in to three categories-
a) Pre Renal- Primarily there is reduction of plasma volume, leading to lowering of blood
pressure with consequent reduction of renal blood flow and Glomerular filtration
rate(GFR). This leads to urea retention. Reduced plasma volume is seen in
i) Dehydration- as in severe vomiting, intestinal obstruction, pyloric stenosis, severe
prolonged diarrhea, fluid depletion associated with Diabetic keto acidosis, shock, burns and
hemorrhages.
ii) Increased protein catabolism- as in High fever, toxic state, metabolic response to injury,
hemorrhage in to the alimentary tract, digestion of protein passing along the intestine and
later deamination of amino acids.
b) Renal Uremia-In renal diseases there is reduction of GFR resulting in urea retention.
Acute renal failure, acute glomerulonephritis, Malignant Hypertension and Pyelonephritis,
all of them produce increase in the blood urea levels.
c) Post Renal Uremia- Obstruction to the out flow of urine after it leaves the kidney leads
to back pressure on the renal pelvis, diminished glomerular filtration of urea with
consequent increase in the blood urea level.
Renal stone, stricture, Enlargement of prostate and malignant tumors may produce post
renal uremia.
Q.6- Under what conditions blood urea is lower than normal?
Answer- Blood urea level is low in liver diseases (Since urea is synthesized in liver),in
pregnancy, growing period , recovery from illness and in tissue healing, since in all these
later conditions, there is positive nitrogen balance and amino groups of the amino acids are
not available for urea formation. In disorders of urea cycle also there is impaired urea
formation, hence blood urea is low.
Q.7-What is nitrogen balance?
Answer- A normal healthy adult is said to be in nitrogen balance, because the dietary
intake equals the loss through urine, feces and skin. When the excretion exceeds intake, it is
negative nitrogen balance. When the intake exceeds excretion, it is said to be positive
nitrogen balance.
a)Positive nitrogen balance is observed in- Pregnancy- due to enlargement of uterus and
fetal growth, growing period, during convalescence(recovery from illness or surgery due to
active regeneration of tissues) and under the influence of Growth hormone, Insulin and
androgens.
38
b) Negative nitrogen Balance is observed in- acute illness, surgery, trauma, burns,
malignancy, diabetes mellitus and chronic, debilitating diseases and in protein energy
malnutrition. Corticosteroids cause a negative nitrogen balance.
Q.8- Define clearance, what is its significance?
Answer- Clearance is defined as the volume of plasma completely cleared of a substance

per unit time and is expressed in ml/minute. In other words it is the ml of plasma which
contains the amount of a substance excreted by the kidney in one minute. For example Urea
clearance of 75 ml/minute means, 75 ml of plasma gets completely cleared of urea in one
minute by excretion of urea through urine by kidney.
Q.9- If a person has blood urea as 54 mg/dl and urea clearance 25 ml/minute,
comment on the functional status of the kidney.
Answer- Urea clearance is affected by volume of urine excreted per minute. If the
excretion of urine is at the rate of 2 ml or more the clearance is designated as Maximum
urea clearance (Cm) and if the rate of excretion is less than 2ml/minute, the clearance is
designated as Standard urea clearance (Cs). The average normal values are 75 ml/minute
for Maximum urea clearance (Cm)and 54 ml/minute for Standard urea clearance (Cs).
Now in the given patient, the urea clearance is 25 ml/minute means only 25 ml of plasma is
getting completely cleared of urea per minute, this is very low in comparison to the normal
clearance values, that means kidney is failing to excrete urea in urine , hence it is getting
accumulated in blood to cause high urea level, as is apparent from the high level of 54
mg/dl in this patient. So overall interpretation is that there is functional impairment of the
kidney, this deficit seems to be a mild to moderate functional deficit.
Q.10- Calculate the urea clearance for the following three patients from the values
given below and comment on the functional status of the kidney.
Patient Blood urea Urinary Urea Volume of urine excreted/

minute
(mg/dl) (G/L) (ml/24 hours)
1) 40 12 1500
2) 80 10 1000
3) 64 18 1200
Answer-
Patient 1) Blood urea (P) = 40 mg/dl
Urinary urea (U) = 12 G/L =12,000mg/L = 1200 mg/dl
(Value should be in mg/dl)
Volume of urine excreted/day- 1500 ml/24 hours
39
Convert it into ml/minute = 1500/24x60 ml/minute
Or 1500/1440= 1.04 ml/minute
Since V is less than 2 ml/minute, hence standard urea clearance is to be considered
Cs = U¥V
--------
P
= 1200 x¥1.04
----------------
40
= 30.6 ml/min (approx)
The value of urea clearance is expressed in terms of percentage of the normal (Which is 54
ml/min – normal Cs)
Hence Percentage urea clearance = 30.6 x 100

----------------
54
= 56.6 %
It is mild renal functional deficit (The value between 50-70% of the normal is considered
mild renal deficit)
(Calculate for the other two patients- self exercise)
Q.11- If blood urea is high and serum Creatinine is normal what is the probable
diagnosis?
Answer- In renal failure both blood urea and creatinine should be high, but in the given
case normal creatinine rules out the possibility of renal failure. High blood urea can be due
to physiological factors like high protein diet or advancing age or it could be pre renal
uremia. In post renal uremia if the obstruction is not relieved renal damage does occur and
in that case creatinine also rises after a time.
Q.12- In a patient suffering from cirrhosis of liver, what results you expect from the
following biochemical parameters-
i) Blood Urea ii) Blood Ammonia iii) CSF Glutamine iv) Urinary Urea
Answer- In Cirrhosis of liver, blood urea is low ( since urea is formed in the liver), blood
ammonia is high(since ammonia is not getting converted to urea), CSF glutamine is high
40
(Ammonia couples with glutamate to form Glutamine) and urinary urea is low since blood
urea is also low.
Q.13- A known diabetic patient has been brought to emergency with nausea, vomiting,
extreme weakness, puffiness of face and mental confusion. His biochemical profile is
as follows-
Blood urea-80 mg/dl
Serum Creatinine- 3.4 mg/dl
Random blood glucose- 234 mg/dl
Serum total proteins- 4.5 G/dl
Serum Total Cholesterol- 335 mg/dl
Urine analysis – Proteins and glucose are present
Make a probable diagnosis.
Answer- The patient is suffering from Diabetic nephropathy which has progressed to renal
failure, High blood glucose, cholesterol, low serum total proteins, proteinuria and
glycosuria go in favor of diagnosis of diabetic nephropathy and high blood urea and
creatinine signify Renal failure.
41
MULTIPLE CHOICE QUESTIONS- HEME SYNTHESIS AND DEGRADATION
Q.1- Which out of the followings is not a haemo protein?

a) Tryptophan pyrrolase
b) Tyrosinase
c) Myoglobin
d) Cytochrome P450
Q.2- Which out of the following enzymes catalyzes a rate limiting step in the pathway
of haem biosynthesis?
a) ALA synthase
b) ALA dehydratase
c) PBG deaminase
d) Coproporphrinogen oxidase
Q.3- High levels of lead can affect heme metabolism by combining with SH groups of
which out the following enzymes?
a) ALA synthase
b) ALA dehydratase
c) PBG deaminase
d) Coproporphrinogen oxidase
Q.4- Pyridoxal phosphate is necessary in the pathway of Haem biosynthesis, which out
of the following enzymes requires Pyridoxal –P as a coenzyme?
a) ALA synthase
b) ALA dehydratase
c) PBG deaminase
d) Ferrochelatase
Q.5- In general, the porphyrias are inherited in an autosomal dominant manner, with
the exception of
a) Acute intermittent porphyria
b) Porphyria Cutanea Tarda
c) Variegate Porphyria
d) Congenital Erythropoietic porphyria
Q.6- Choose the incorrect statement out of the followings-

a) Synthesis of ALA occurs in the mitochondria
b) Uroporphyrinogen formed is almost exclusively the III isomer
c) A porphyrin with symmetric substitution of side chains is classified as a type III
porphyrin
d) Coproporphyrinogen oxidase is able to act only on type III isomers
42
Q.7- In which of the following porphyrias, cutaneous hypersensitivity is not observed?
a) Variegate porphyria
b) Acute intermittent porphyria
c) Congenital Erythropoietic porphyria
d) Hereditary Coproporphyria
Q.8-Which of the following statements describes the basis of giving I/V Hemin
infusion?
a) Haem/ Hemin acts as a negative regulator of the synthesis of ALA synthase
b) Heme affects translation of the enzyme
c) Heme affects transfer of enzyme from the cytosol to the mitochondrion
d) All of the above
Q.9- Porphyrins are deposited in teeth and in bones, as a result, the teeth are reddish-
brown and fluoresce on exposure to long-wave ultraviolet light, so called
‘Erythtrodontia ‘, is a sign of which porphyria ?
Q.10-A 24- year- old patient was brought to medical OPD with acute abdominal pain,
depression and extreme weakness. Urine analysis revealed the presence of ALA and
PBG (Delta amino Levulinic acid and Porphobilinogen). The patient was diagnosed
with acute intermittent porphyria, which of the following enzyme deficiencies is
expected in this patient?
a) Uroporphyrinogen III cosynthase
b) Uroporphyrinogen decarboxylase
c) Porphobilinogen decarboxylase
d) None of the above.
Q.11-An 8 year old boy was brought to a dermatologist as he had developed vesicles
and bullae on his face and arms that appeared after a week long football practice in
sun. His father had a similar condition. A diagnosis of Porphyria cutanea tarda was
confirmed by finding elevated levels of porphyrins in his serum. His disease is due to a
deficiency of which of the following enzymes?
a) ALA dehydratase
b) Ferrochelatase
c) PBG deaminase
d) Uroporphyrinogen decarboxylase.
Q.12- A 23 –year-old young woman, who recently began taking birth control pills,
presents to emergency room with cramping abdominal pain, anxiety, hallucinations
and paranoid behavior. A surgical evaluation, including Ultrasound and computed
43
tomography (CT) scan have failed to demonstrate any abdominal process.
Examination reveals vesicles and bullae on the skin of arms and face. Urine analysis
reveals the presence of porphyrins (ALA, PBG, Uro and Coproporphyrins). What is
the possible diagnosis for this patient?
Q.13- A patient presents with dull right sided abdominal pain, fever from the 7 days,
loss of appetite, pale stool and jaundice. Blood biochemistry reveals, mixed
hyperbilirubinemia, high SGPT but near normal alkaline phosphatase levels. What is
the cause of jaundice?
a) Viral hepatitis
b) Post hepatic jaundice
c) Hemolytic jaundice
Q.14- Impaired Glucuronyl transferase activity is observed in all of the followings

except-
a) Breast milk jaundice
b) Physiological jaundice of the new born
c) Crigler Najjar syndrome
d) Dubin Johnson syndrome
Q.15- Which out of the following conditions is not associated with excessive bilirubin
formation from hemolysis –
a) Sickle cell anemia
b) Thalassemia
c) Malaria
d) Rotor syndrome
Q.16-What is expected out of Van den Bergh reaction in hepatic jaundice?

a) Direct positive
b) Indirect positive
c) Biphasic
Q.17- Which serum enzyme elevation is most diagnostic in obstructive jaundice?

a) ALT(Alanine amino transferase)
b) AST (Aspartate amino transferase)
c) LDH (Lactate dehydrogenase)
d) ALP (Alkaline phosphatase).
44
Q.18- Acholuric jaundice is –
a) Hemolytic jaundice
b) Hepatic jaundice
c) Post hepatic jaundice
Q.19- Urine analysis of a patient reveals the presence of Bilirubin and urobilinogen,
which serum enzyme is expected to be elevated much higher than normal?
a) ALT (Alanine amino transferase)
c) 5’ Nucleotidase
Q.20-A 65- year –old patient presents with weight loss, loss of appetite, dull dragging
pain in the right hypochondrium and jaundice from the last 1 month. Stool is
reported to be clay colored from the same duration. Blood biochemistry reveals
Conjugated hyperbilirubinemia. Urine shows the presence of bilirubin. The patient
has been diagnosed with carcinoma of head of the pancreas. Which serum enzyme is
expected to be much higher than normal for this patient?
a) ALT (Alanine amino transferase)
c) LDH (Lactate dehydrogenase)
Answers-
Q-1-b Q.2- a Q-3-b
Q-4-a Q-5-d Q-6-c
Q-7-c Q-8-d Q-9-c,
Q-10-c Q11-d Q12-a
Q-13-a Q14-d Q-15-d
Q.16-c Q-17-d Q-18-a
Q-19-a Q-20-d
45
MULTIPLE CHOICE QUESTIONS- AIDS
Q.1- Choose the incorrect statement out of the followings for human immune
deficiency virus-
a) The reverse transcriptase enzyme is characteristic feature of retroviruses

b) p10 is a protease that cleaves gag precursor
c) p32 is an Integrase
d) gag encodes for the lipid bilayer of the virus.
Q.2- The specific binding of HIV to the CD 4 surface molecules of the host cell
membrane is brought about by-
a) gp 120
b) gp 41
c) p32
d) p55
Q.3- All of the followings except one are CD 4 + cells.
a) Monocytes
b) T –helper cells
c) T-Cytotoxic cells
d) Macrophages
Q.4- Which immune marker is present during the window period of HIV infection?
a) p24 antigen
b) Antibodies to gp 120
c) Antibodies to gp 41
d) p17 antigens
Q.5. Which out of the followings is a preferred mode of transmission of HIV infection
from mother to child?
a) During pregnancy through placenta

b) During delivery through mixing of blood
c) Through breast milk during lactation
d) All of the above.
Q.6-Which out of the followings are the high risk subjects for acquiring HIV
infection?
a)Professional sex workers
46
b) Drug addicts
c) Persons getting repeated blood transfusions
d) All of the above
Q.7- The pol gene does not encode for which of the following enzymes?
a) Protease
b) Integrase
c) Reverse Transcriptase
d) RNA polymerase
Q.8-Which of the followings best describes the role of Reverse transcriptase?
a) For the synthesis of RNA from genomic RNA

b) For the synthesis of DNA from genomic DNA
c) For the synthesis of DNA from genomic RNA
d) For the synthesis of mRNA from Host DNA.
Q.9- Choose the odd one out –
a) gag
b) tat
c) nif
d) rev
Q.10-Which statement best describes the basis for failure to produce a vaccine against
HIV-
a) HIV is a highly mutable virus

b) HIV Integrase its genome in to the host DNA, hence protected from immune system
c) HIV is not killed only by humoral response, cellular immunity is also required
Q.11-A gynaecologist while doing a cesarean section for an HIV positive female got
accidentally pricked by a needle. After 2 weeks of acquiring the infection the serum
sample was sent for analysis, which of the following markers might have been positive
for confirmation of diagnosis?
a) p24
b) viral RNA
c) Free virus
47
Q.12-An HIV positive male presented to the emergency with fever, pneumonia and
oral thrush. The attending physician described that all the presenting symptoms are
due to underlying collapsed immune system. What is the possible cause for immune
incompetence?
a) T-c cells remain inactive cell mediated immunity is compromised

b) NK cells are depleted; cancer and virally infected cells are not removed
c)T-helper cells are depleted, all components of immune system are paralysed
Q.13- At present the reasonable approach to initiate antiretroviral therapy to any one
is–
a)All symptomatic patients

b) Pregnant women
c) CD 4 count below 350U/L
d) Any of the above.
Q.14- Vital nucleic acids can be detected by which of the following techniques?
a) ELISA
b) Western Blotting
c) PCR
d) viral isolation
Q.15- Which of the followings is not a rapid test for the diagnosis of HIV infection?
a) Dot Blot Assay

b) Particle agglutination test
c) Western Blotting
d) HIV spot and comb test
Q.16- Western Blotting is considered a gold standard for the confirmation of HIV
infection. Which statement best describes the basis?
a) It is a rapid and sensitive test

b) It can detect antigen and antibodies simultaneously
c) Antibodies against gag and env are simultaneously detected
Q.17-The best treatment for HIV infection is-
a) Reverse transcriptase inhibitors

b) Protease Inhibitors
48
c) Integrase inhibitors
d) Highly active antiretroviral therapy (HAART)
Q.18- Which structural component of HIV is required for cell –cell fusion?
a) p 24
b) gp 41
c) gp 120
d) p32
Answers-
1)-d 2)- a 3)- c 4)- a 5)- d 6)- d
7)- d 8)- c 9)- a 10)-d 11)- d 12)- d
13)- d 14)- c 15)-c 16)-c 17)-d 18)-b
49
LIVER FUNCTION TESTS
Choose the correct answer

1) In prehepatic jaundice Bilirubin/Urobilinogen is detected in urine. (Urobilinogen)
2) In post hepatic jaundice Bilirubin/Urobilinogen is detected in urine. (Bilirubin)
3) Bilirubin/Biliverdin is the first pigment to be formed. (Biliverdin)
4) Both Bilirubin and urobilinogen are detected in urine in hepatic/post hepatic jaundice.
(Hepatic jaundice)
5) Vitamin K deficiency is not encountered in prehepatic/post hepatic jaundice.

(Prehepatic jaundice)
6) Serum Albumin/Globulin levels are increased in chronic liver disorders.

(Globulin)
7) Prothrombin time is decreased /increased in liver disorders.

(Increased)
8) Blood glucose/Galactose level would increase in Galactose Tolerance test in a normal

individual. (Glucose)
9) Blood glucose/Galactose level would increase in Galactose Tolerance test in a patient

with liver disorder. (Galactose)
10) Serum total cholesterol level increases /decreases in obstructive jaundice.

(Increases)
11) Benzoic acid conjugates with Cysteine/ Glycine for the formation of Hippuric acid.
(Glycine)
12) ALP/ALT rises in obstructive liver disorders.

(ALP-Alkaline Phosphatase)
13) ALP/ALT rises in viral hepatitis.

(ALT- Alanine amino transferase)
14) AST/ALT would rise more in alcoholism.

(AST-Aspartate amino transferase)
50
15) Pale/Dark colored stools are observed in hemolytic jaundice.
(Dark colored)
16) Pale/Dark colored stools are observed in obstructive jaundice.

(Pale- In fact Clay colored)
17) Vitamin B2/B12 is stored in liver.

(B12)
18) Normal blood ammonia level should be 40-70 µ g/dl /10-40 µ g/dl.
(40-70 µg/dl)
19) Indirect/Direct positive Van den Bergh reaction is observed in hemolytic jaundice.
(Indirect positive)
20) Indirect/Direct positive Van den Bergh reaction is observed in post hepatic jaundice.
(Direct positive)
21) In cirrhosis of liver blood urea/ammonia would be higher than normal.

(Blood Ammonia)
22) Normal prothrombin time is 14 seconds/14 minutes.

(14 seconds)
23) Plasma fibrinogen level decreases/increases in advanced liver cirrhosis

(Decreases)
51
LIVER FUNCTION TEST- A BRIEF SUMMARY
Figure showing functions of liver
52
ESTIMATION OF SERUM TOTAL PROTEINS- VIVA QUESTIONS
Q.1- What is the normal range of serum total proteins? What is A : G ratio?
Answer- The total plasma protein concentration normally ranges between 6.0 to 8.0 G/dl.
Albumin ranges between 3.5 to 5.5 G/dl and Globulins range between 2.5-3.5G/dl. The
ratio of albumin to Globulin concentration is called A: G ratio and it varies from 1.5-2.5:1.
Q.2 – What are the causes of hypoproteinemia?
Answer- Hypoproteinemia, i.e., a generalized decrease in plasma proteins occurs in –
(1) Liver diseases, because hepatic protein synthesis is depressed (Mainly albumin is low)
(2) Renal disorders like the Nephrotic syndrome, in which glomerular membrane
permeability increases markedly.
(3) Malnutrition and starvation- reduced dietary availability
(4) Protein-losing enteropathy- excessive loss through intestine
(5) Wide spread burns- loss from skin
(6) Severe hemorrhage- increased protein catabolism
(7) Defective digestion or malabsorption as in carcinoma of stomach or pancreas, peptic

ulcer and steatorrhea.
(8) Fever- increased protein catabolism
(9) Pregnancy-hemodilution and increased requirement
(10) Acute infections in-untreated diabetes mellitus and hyperthyroidism
Q.3- What are the causes of hyperproteinemia?
Answer- Increase in plasma proteins are seen in
(1) Acute inflammatory states. Several plasma proteins increase sharply during any acute
inflammation. These are called acute phase proteins. They include C-reactive proteins
(CRP), so called because it reacts with C- polysaccharide of pneumococci.
(2) Multiple myeloma. In this condition, the plasma cells secrete large amounts of
immunoglobulin resulting in hypergammaglobulinemia
53
(3) Dehydration – due to hemoconcentration
(4) Chronic infections
(5) Leukemias
(6) Lymphomas
(7) Tuberculosis
(8) Kala azar
(9) HIV infection
Increase in plasma protein concentration is generally due to an increase in total globulins

(Gamma globulins) and the concentration of albumin remains the same or decreases
marginally. A decrease in total protein concentration is due to fall in albumin and some
times globulins. In these conditions A:G ratio changes due to either reduction of Albumin
or increase of Globulins.
Q.4-What is the effect of plasma volume on total protein concentration?
Answer- Decrease in the volume of plasma water –Hemoconcentration, is reflected as

relative hyperproteinemia and the concentration of all individual plasma proteins are
increased to the same degree.
Hyperproteinemia is observed in dehydration due to- inadequate water intake or due to
excessive water loss as in –Diarrhea, vomiting, Addison’s disease, diabetic ketoacidosis
and diabetes Insipidus.
Hemodilution occurs with water intoxication and salt retention syndromes, during
massive intravenous infusions and physiologically when a recumbent position is
achieved. A recumbent position decreases plasma protein concentration by 03-0.5 G/dl. In
hemodilution individual plasma proteins are decreased by the same degree.
Q.5- What are the functions of plasma proteins?
Answer- Functions of the plasma proteins include:
• Intravascular osmotic effect for maintaining fluid and electrolyte balance

• Contribute to the viscosity of the plasma
54
• Transport of insoluble substances around the body by allowing them to bind to
protein molecules. Blood plasma proteins like albumin functions as carrier proteins
that help in the translocation of different biomolecules in body.
• Protein reserve for the body
• Clotting of blood
• Inflammatory response
• Protection from infection- The gamma globulins function as antibodies
• Maintenance of the acid-base balance
• Blood plasma contains the protease inhibitor enzymes like alpha-1 antitrypsin that
help in the reduced proteolytic activity in the blood.
Q.6-What is the cause of hyperproteinemia in cirrhosis of liver or other liver

disorders?
Answer-Although the concentration of serum Albumin is reduced in severe liver diseases

(Since albumin is synthesized in liver), that of globulins is usually increased (For
compensation as they are synthesized in spleen and bone marrow) so that the total plasma
protein concentration is rarely low and is often high.
Q.7- What is the cause of edema in hypoproteinemia?
Answer- Plasma proteins contribute to plasma colloidal osmotic pressure, counteracting the
effects of capillary blood pressure, which tends to force water in to tissue spaces. The
lowered plasma protein concentration causes a decrease in the plasma osmotic pressure and
water is forced in to tissue spaces resulting in edema. Edema is probable when the albumin
concentration falls below 2G/dl.
Q.8- State your diagnosis from the following Blood and Urinary findings
Biochemical Parameter Pateint1 Patient2
Blood Hypoproteinemia Hypoproteinemia
Urine Proteinuria Normal Urine
Answer-Patient 1- With Hypoproteinemia and proteinuria is most probably suffering from

Nephrotic syndrome, while Patient 2 with hypoproteinemia without proteinuria might be
suffering from Kwashiorkor.
Q.9- What is Nephrotic syndrome? What are the laboratory findings in this
syndrome?
Answer- Nephrotic syndrome (NS), also known as Nephrosis, is defined by the presence of
nephrotic-range proteinuria, edema, hyperlipidemia, and hypoalbuminemia.
55
In a healthy individual, less than 0.1% of plasma albumin may traverse the glomerular
filtration barrier.Nephrotic-range proteinuria in adults is characterized by protein excretion
of 3.5 g or more per day.
Biochemical basis
1) An increase in glomerular permeability leads to albuminuria and eventually to
hypoalbuminemia. In turn, hypoalbuminemia lowers the plasma colloid osmotic pressure,
causing greater trans capillary filtration of water throughout the body and thus the
development of edema.
2) In the nephrotic syndrome, levels of serum lipids are usually elevated. This can occur via
(i) hypoproteinemia that stimulates protein, including lipoprotein, synthesis by the liver,
and (ii) diminution of lipid catabolism caused by reduced plasma levels of lipoprotein
lipase.
Laboratory findings
1) Urinalysis- Proteinuria is observed

2) Blood chemistries
i) Serum total and differential protein- A decreased serum albumin (< 3 g/dL) and total
serum protein less than 6 g/dL may be observed.
ii)-Lipid Profile-Hyperlipidemia occurs in over 50% of those with early nephrotic
syndrome. The degree of hyperlipidemia increases with the degree of protein loss.
iii) ESR and Serum Fibrinogen levels-Patients can have an elevated erythrocyte
sedimentation rate as a result of alterations in some plasma components such as increased
levels of fibrinogen.
iv) Renal Function Tests- BUN (Blood urea nitrogen) and serum creatinine rise in the
setting in of renal failure.
v) Renal biopsy- For confirmation of diagnosis
56
THE USEFULNESS OF CASE STUDIES IN BIOCHEMISTRY FOR MEDICAL
STUDENTS
As a sophomore medical Student, when they embark themselves in MBBS curriculum, the
students’ interests towards the Biochemistry as a subject is very minimal as it requires more
of correlations and understanding unlike Anatomy and Physiology. The students loose
interests in this subject as they think it comprise of cycles and recall memory, but the real
understanding comes when this subject is taught well by an experienced teachers trained for
teaching medical biochemistry.
They fail to realize that this subject if read and understood well it becomes easy for them to
understand Medicine in final year. In this book the author has given more emphasis to case
studies which would create likeness and the usefulness in diagnosis of various diseases
when they continue with medical curriculum.
Usually Case study exercises must be introduced in their biochemistry course in medical
curriculum so that it would help students master the content of the course while at the same
time help build critical thinking and problem-solving skills.
We can use case studies in a variety of forms from as in-class group activities, to either
augment or replace content covered in lecture or as homework assignments, completed
either individually or in groups, to assess mastery of content. It could also be used as exam
questions, to assess the students’ ability to apply their biochemistry knowledge to a larger
context and to discourage rote memorization.
A case oriented approach would help the teachers and instructors to focus student attention
on the key content areas that the instructor wishes to cover, doing so in a manner that is
interesting and engaging to the students.
Since we all are switched from teacher- centered learning to students’ centered learning, the
cases covered in this book would generate interests among students and they would further
dig deep into the subject and learn the art of correlations and diagnosis in their sophomore
stage so that it would enable them to have a better understanding of medicine in their
curriculum.
57
CASE STUDY- JAUNDICE
Case study-I
A 24 –year –old male suffering from Malaria was put on Primaquine. He developed
malaise, fatigue and yellow discoloration of sclera and skin.
On examination – There was pallor- ++, icterus ++, Pulse – 100/min., Temperature 102°F.
Liver and spleen were palpable.
The investigation report was as follows-

Hb-5gm%
TLC-13000 cmm esp. polymorphs
Serum bilirubin- 6 mg%
Van den Bergh- Indirect positive.
Urine- Hemoglobin + and Urobilinogen +
The color of the urine was brownish black
What is the probable diagnosis?

What is the relation ship of primaquine intake and the present manifestations?
Case Details- This is a case of primaquine induced Hemolytic anemia, progressing to

jaundice. Glucose-6-P dehydrogenase deficiency seems to be the underlying defect. High
fever is due to malaria, while pallor and icterus are due to hemolytic anemia and underlying
jaundice as apparent from low Hb and high bilirubin levels. Indirect positive Van Den
Bergh indicates Uncinjugated Bilirubinemia. Urine is positive for hemoglobin and
urobilinogen indicating the underlying hemoglobinuria and hemolytic jaundice
Primaquine being an oxidant drug precipitates the underlying defect to induce hemolysis.
(See the details below).The liver has the capacity to conjugate and excrete over 3000 mg of
bilirubin per day, whereas the normal production of bilirubin is only 300mg/day. The
excess capacity allows the liver to respond to increased haem degradation with a
corresponding increase in conjugation and secretion of bilirubin diglucuronide.
However massive lysis of red blood cells, as in Glucose-6 –phosphate dehydrogenase

deficiency, may produce bilirubin faster than it can be conjugated. More bilirubin is
excreted in to the bile, the amount of urobilinogen entering the enterohepatic circulation is
increased and urinary urobilinogen is also increased. Unconjugated bilirubin levels become
elevated in the blood causing jaundice.
Case study-2
A 10 –year- old boy received a sulfonamide antibiotic as prophylaxis for recurrent

urinary tract infections. Although he was previously healthy and well nourished, he
58
became progressively ill and presented with pallor and irritability. A blood count
revealed that he was severely anaemic with jaundice due to hemolysis of the red blood
cells.
What is the problem with the boy?
What is the cause of anemia and jaundice in this boy?
What is the simplest way for the diagnosis of this problem?
Case details- The child is suffering from Glucose-6-phosphate dehydrogenase deficiency.

The individuals with G-6-P-D deficiency present with excessive hemolysis on exposure to
certain drugs like antibiotics, analgesics and Antimalarials. Acute HA can develop as a
result of three types of triggers: (1) fava beans, (2) infections, and (3) drugs. Glucose 6-
phosphate dehydrogenase (G6PD) is an enzyme critical in the redox metabolism of all
aerobic cells .In red cells, its role is even more critical because it is the only source of
reduced nicotinamide adenine dinucleotide phosphate (NADPH), which, directly and via
reduced glutathione (GSH), defends these cells against oxidative stress.
Figure- showing the role of G-6-P-Dehydrogenase in Glucose metabolism.
NADPH is a required cofactor in many biosynthetic reactions which also maintains

glutathione in its reduced form. Reduced glutathione acts as a scavenger for dangerous
oxidative metabolites in the cell. With the help of the enzyme glutathione peroxidase,
reduced glutathione converts harmful hydrogen peroxide to water. The inability to
decompose hydrogen peroxide results in free radical induced membrane disruption and
reduced life span as a result of methaemoglobin formation.G6PD deficiency is a prime
example of a hemolytic anemia due to interaction between an intracorpuscular and an
extracorpuscular cause, because in the majority of cases hemolysis is triggered by an
exogenous agent. People deficient in glucose-6-phosphate dehydrogenase (G6PD) are not
prescribed oxidative drugs, because their red blood cells undergo rapid hemolysis under
this stress. Although in G6PD-deficient subjects there is a decrease in G6PD activity in
most tissues, this is less marked than in red cells, and it does not seem to produce
symptoms.
Clinical manifestations- The vast majority of people with G6PD deficiency remain
clinically asymptomatic throughout their lifetime.
59
However, all of them have an increased risk of developing neonatal jaundice (NNJ) and a
risk of developing acute HA when challenged by a number of oxidative agents. Typically,
a hemolytic attack starts with malaise, weakness, and abdominal or lumbar pain. After an
interval of several hours to 2–3 days, the patient develops jaundice and often dark urine,
due to hemoglobinuria. The onset can be extremely abrupt, especially with favism in
children. The anemia is moderate to extremely severe, usually normocytic and
normochromic, and due partly to intravascular hemolysis; hence, it is associated with
haemogobinemia, hemoglobinuria, and low or absent plasma Haptoglobin. Jaundice is
prehepatic.
The laboratory workup for glucose-6-phosphate dehydrogenase (G6PD) deficiency
includes the following:
• Measure the actual enzyme activity of G6PD rather than the amount of glucose-6-
phosphatase dehydrogenase (G6PD) protein.
• Obtain a complete blood cell (CBC) count with the reticulocyte count to determine
the level of anemia and bone marrow function.
• Indirect bilirubinemia occurs with excessive hemoglobin degradation and can
produce clinical jaundice.
• Urinary urobilinogen is high
Treatment- Identification and discontinuation of the precipitating agent is critical in cases
of glucose-6-phosphate dehydrogenase (G6PD) deficiency. Affected individuals are treated
with oxygen and bed rest, which may afford symptomatic relief. Prevention of drug-
induced hemolysis is possible in most cases by choosing alternative drugs. When acute HA
develops and once its cause is recognized, no specific treatment is needed in most cases.
However, if the anemia is severe, it may be a medical emergency, especially in children,
requiring immediate action, including blood transfusion.
Case study-3
A 50 –year-old woman had 8 day history of loss of appetite, nausea and flu like
symptoms. She had noticed that her urine had been dark in color over the past two
days. On examination she had tenderness in the right upper quadrant.
Laboratory investigations showed;

Serum Total Bilirubin- 4.5 mg%
Direct Bilirubin- 2.5 mg%
Indirect Bilirubin- 2.0 mg%
Serum AST- 40 IU/L
Serum ALT-115 IU/L
Serum ALP- 20 Units (KA)

What will be the observation regarding bile pigments in urine?
60
Case details- Flu like symptoms are indicative of viral hepatitis. Damage to liver cells can
cause unconjugated bilirubin to increase in the blood as a result of decreased conjugation.
The bilirubin that is conjugated is not efficiently secreted in to the bile, but instead diffuses
in to the blood. Urobilinogen is increased in urine because hepatic damage decreases the
enterohepatic circulation of this compound allowing more to enter blood, from which it is
filtered in to the urine. The urine thus becomes dark in color, whereas stools are pale
colored. Plasma levels of AST and ALT are elevated. This is a case of hepatic jaundice.
Case study-4
Based on the following clinical laboratory data, give the most probable diagnosis-
Serum Bilirubin- 4 mg%
Direct Bilirubin- 0.2 mg%
Serum Alkaline phosphatase- 6 units( KA)
SGOT- 30 IU/L
SGPT- 26 IU/L
Urine Bilirubin- Negative
Urine urobilinogen-Positive
Urine Bile Salts- Negative
Case details- Normal enzyme profile, Hyperbilirubinemia, absence of urinary bilirubin and
positive urobilinogen are indicative of Hemolytic jaundice.
Case study-5
A 40 –year- old, fat female, presents with intolerance to fatty foods, pain in the right
side of abdomen, yellowness of eyes and passage of clay colored stools.
Laboratory Investigations revealed
Serum
Total Bilirubin – 20 mg%
Direct Bilirubin- 16 mg%
ALP- 800 U(KA)
SGPT- 90 IU/L
Urine
Color- deep yellow
Bilirubin- ++
Urobilinogen- absent
Stools
Clay colored
Stercobilnogen- absent
What is the likely diagnosis?

Which other enzymes are likely to increase?
61
Case details- This is a case of obstructive jaundice due to gall stones. This patient fits the
“classic” criteria of gallbladder disease: female, middle-aged, overweight. Gallstones are
collections of solid material (predominantly crystals of cholesterol) in the gallbladder.
Gallstones may cause pain. Pain develops when the stones pass from the gallbladder into
the cystic duct, common bile duct, or ampulla of Vater and block the duct. Then the
gallbladder dilates, causing pain called biliary colic. The pain is felt in the upper abdomen,
usually on the right side. Eating a heavy meal can trigger biliary colic, but simply eating
fatty foods does not.
In this instance jaundice is not due caused due to overproduction of bilirubin, but instead
results from obstruction of the bile duct from the gall stones. The liver regurgitates
conjugated bilirubin in to the blood (Hyperbilirubinemia)
High direct bilirubin (Conjugated hyperbilirubinemia), high alkaline phosphatase (marker
of cholestasis),slightly increased SGPT level are suggestive of post hepatic or obstructive
jaundice. Furthermore the diagnosis is supported by the presence of bilirubin (since it is
conjugated) and absence of urobilinogen (Since there is obstruction to the out flow of bile)
in urine. Due to the same reason of obstruction stool is clay colored as stercobilnogen is
absent.
Treatment is based on the relieving the obstruction surgically. Prolonged obstruction of the
bile duct can lead to liver damage and a subsequent rise in unconjugated
hyperbilirubinemia and a rise in SGPT levels.
Case Study- 6
An Rh negative mother delivers a baby who develops jaundice immediately after

birth.
General Examination reveals

Heart Rate 80/min
Icterus +
Irritability +
Liver Palpable
Laboratory Investigations
Serum
Bilirubin
• Total 10 mg%
• Indirect 7 mg%
• Direct 3 mg%
Alkaline phosphatase 50 U/L
Urine
62
Urobilinogen +++
Feces
Stercobilnogen +++
What is your likely diagnosis?
Case details- This is a case of hemolytic jaundice due to Rh incompatibility. Indirect hyper
bilirubinemia (Unconjugated hyperbilirubinemia), high urinary urobilinogen and fecal
stercobilnogen are indicative of hemolytic jaundice. Typically bilirubin is absent in urine
since unconjugated bilirubin being water insoluble and albumin bound (macromolecule),
can not pass through glomeruli to appear in urine.
Case Study- 7
A 65 –year-old man reported with visible Jaundice which he had noticed to be deepening in
color. There was no history of pain, fever or any drug intake, but he complained of some
weight loss and pale stools from the past few days. He was a moderate drinker. There was
no history of such like episode before.
Blood Biochemistry revealed-
Serum Total Bilirubin- 20mg/dl
AST-87 U/L
ALT- 92 U/L
ALP-350 U/L
Case discussion- In this case, by far the most likely diagnosis is Obstructive Jaundice
which might be due to carcinoma of the head of the head of pancreas, obstructing the
common bile duct. This classically gives rise to severe, painless, deep jaundice which is in
keeping with a Bilirubin of 20 mg/dL. In this case obstructive Jaundice is characterized by
high Alkaline phosphatase activity that is more than three times the upper limit of the
reference range. The Aspartate and Alanine aminotransferase activities in the given case do
not indicate severe hepatocellular damage.
1) The aminotransferase (transaminases) are sensitive indicators of liver cell injury and
are most helpful in recognizing acute hepatocellular diseases such as hepatitis. The
aminotransferases are normally present in the serum in low concentrations. These enzymes
are released into the blood in greater amounts when there is damage to the liver cell
membrane resulting in increased permeability. Liver cell necrosis is not required for the
release of the aminotransferases, Levels of up to 300 U/L are nonspecific and may be found
in any type of liver disorder. Striking elevations—i.e., aminotransferases > 1000 U/L—
occur almost exclusively in disorders associated with extensive hepatocellular injury such
as (i) viral hepatitis, (ii) ischemic liver injury (prolonged hypotension or acute heart
63
failure), or (iii) toxin- or drug-induced liver injury. In most acute hepatocellular disorders,
the ALT is higher than or equal to the AST. An AST:ALT ratio > 2:1 is suggestive while a
ratio > 3:1 is highly suggestive of alcoholic liver disease. In obstructive jaundice the
aminotransferases are usually not greatly elevated.
2) Alkaline phosphatase The activities of three enzymes—alkaline phosphatase, 5'-

nucleotidase, and Ȗ-glutamyl transpeptidase (GGT)—are usually elevated in Obstructive
liver diseases(Cholestasis). GGT elevation in serum is less specific for cholestasis than are
elevations of alkaline phosphatase or 5'-nucleotidase. GGT estimation is done to identify
patients with occult alcohol use. The normal serum alkaline phosphatase consists of many
distinct isoenzymes found in the liver, bone, placenta, and, less commonly, small intestine.
Elevation of liver-derived alkaline phosphatase is not totally specific for cholestasis, and a
less than threefold elevation can be seen in almost any type of liver disease. Alkaline
phosphatase elevations greater than four times normal occur primarily in patients with
cholestatic liver disorders, infiltrative liver diseases such as cancer and bone
conditions characterized by rapid bone turnover (e.g., Paget's disease). In bone
diseases, the elevation is due to increased amounts of the bone isoenzymes.In liver diseases,
the elevation is almost always due to increased amounts of the liver isoenzyme. In
intratrahepatic obstruction values are increased as in drug-induced hepatitis and primary
biliary cirrhosis. Very high values are found in Extrahepatic obstructive due to cancer,
common duct stone, or bile duct stricture.The level of serum alkaline phosphatase elevation
is not helpful in distinguishing between intrahepatic and extrahepatic cholestasis. Values
are also greatly elevated in hepatobiliary disorders seen in patients with AIDS
Some Practice Case Studies-
A fourteen year old boy, who was a resident of a boarding school, was admitted to the
hospital. He was ill looking and frankly jaundiced. On the day prior to the
development of jaundice, he noticed that his urine was dark and frothy.
The laboratory serum analysis results were as follows:
Parameter Value Reference Range

Serum total protein : 7.7 g/dl (6-8 g/dl)
Serum Albumin : 4.4 g/dl (3.5-5.5 g/dl)
Serum ALP : 150 KA Units (3-13 KAUnits)
Serum ALT : 4000 U/L ( <35 U/L)
Serum bilirubin : 4 mg/dl ( <1.0 mg/dl)
Urine bilirubin : ++ (negative)
What is the most probable diagnosis?

What further investigations do you suggest?
64
A fourteen year old obese female present with intolerance to fatty food, pain in the
right abdomen, yellowness of the eyes and clay colored stool.

Serum bilirubin : 10 mg/dl (<1.0 mg/dl)
Serum conjugated bilirubin : 8.5 mg/dl (0.1-0.4 mg/dl)
Serum unconjugated bilirubin : 1.5 mg/dl (0.2-0.7 mg/dl)
Serum ALP : 40 KA Units (3-13 KA Units)
Serum AST : 80 Units (<35 Units)
Serum ALT : 90 Units (<40 Units)
Urine bile salts : ++ (negative)
Urine bile pigments : ++ (negative)
Urobilinogen : negative (negative)
Stercobilinogen : negative (negative)
A twenty year old boy was admitted to the hospital with symptoms of headache, pain
in the flanks, anorexia. His blood pressure was found to be 170/110 mmHg. He passed
red colored urine and had oedema around his eyes.
Serum Sodium : 155 mmol/L (135-145 mmol/L)

Serum Potassium : 5.2 mmol/L ( 3.5-5.0 mmol/L)
Serum Calcium : 7.5 mg/dl (8.4 – 10.4 mg/dl)
Serum Phosphate : 5.5 mg/dl (2.5-3.5 mg/dl)
Serum total protein : 7.0 g/dl (6.0-8.0 g/dl)
Serum Albumin : 4.4 g/dl (3.5-5.5 g/dl)
Serum Globulin : 2.5 g/dl ( 2.5-3.5 g/dl)
BUN : 45 mg/dl (8-25 mg/dl)
Serum creatinine : 3.0 mg/dl (0.6-8.0 mg/dl)
Hemoglobin : 9 gm/dl (14-18 g/dl)
Creatinine clearance : 50 ml/min (110-150 ml/min)
65
CASE STUDY- ACID BASE BALANCE
Case Study-1
A 45 year-old-female suffering from bronchial asthma was brought to emergency in a

critical state with extreme difficulty in breathing.
The blood gas analysis revealed the followings-

pH- 7.3
PCO2- 46 mm Hg
PO2- 55 mm Hg
HCO3- 24meq/L
What is your Interpretation?
Case details-
Low p H – acidosis
Low PO2 and PCO2 excess signify Primary respiratory problem
HCO3:24 -normal
Thus, the patient is suffering from Acute respiratory acidosis.
Case Study -2
A 4 day old girl neonate became lethargic and uninterested in breast feeding. Physical
examination revealed tachypnea (rapid breathing) with a normal heart beat and
breath sounds. Initial blood chemistry values included normal glucose, sodium,
potassium, chloride, and bicarbonate (HCO3-) levels.
Blood gas values revealed a pH of 7.53, partial pressure of oxygen (PO2) was normal
(103 mm Hg) but PCO2 was 27 mmHg.
Case details-
The baby is suffering from Respiratory Alkalosis

Tachypnea in term infants may be due to brain injuries and metabolic diseases that irritate
the respiratory center. The increased respiratory rate removes carbon dioxide from the lung
alveoli and lowers blood CO2, forcing a shift in the indicated equilibrium towards left
CO2 + H2O ĺ H2CO3 ĺ H+ + HCO3-
66
Carbonic acid (H2CO3) can be ignored because negligible amounts are present at
physiological pH, leaving the equilibrium
CO2 + H2Oĺ H+ + HCO3-
The leftward shift to replenish exhaled CO2 decreases the hydrogen ion (H+) concentration
and increases the pH to produce alkalosis. This respiratory alkalosis is best treated by
diminishing the respiratory rate to elevate the blood [CO2], to force the above equilibrium
to the right, elevate the [H+], and decrease the pH.
Case study-3
A new born with tachypnea and cyanosis (bluish color) is found to have a blood pH of
7.1. Serum bicarbonate is measured as 12 mM while pCO2 is 40 mm Hg.
Case details-
Low p H and low bicarbonate indicate metabolic acidosis. Since p CO2 is normal it can not
be compensatory respiratory acidosis ( If the baby had respiratory acidosis, the PCO2
would have been elevated).This is a hypoxia related metabolic acidosis. Hyperventilation
is as a compensation to metabolic acidosis.
This condition can be treated administration of oxygen to improve tissue perfusion and
decrease metabolic acidosis.
Case study -4
A 60 year old man was brought to hospital in a very serious condition. The patient
complained of constant vomiting containing several hundred mL of dark brown fluid
from the previous two days plus several episodes of melaena. Past history of
alcoholism, cirrhosis, portal hypertension and a previous episode of bleeding varices
were there.
Arterial Blood Gases revealed-
pH - 7.10
pCO2 - 13.8 mmHg
pO2- 103 mmHg
HCO3- 14.1 mmol/l
Na+ 131 mmol/l., Cl- 85 mmol/l. K+ 4.2 mmol/l., "total CO2" 5.1, glucose 52mg/dl, urea
38.6mg/dl, creatinine1.24mg/dl, lactate 20.3 mmol/l Hb 6.2 G%, and WBC- 18 x103/mm3
67
Case details-
The patient is severely ill with circulatory failure and GI bleeding on a background of
known cirrhosis with portal hypertension.
The very low pH indicates a severe acidosis. The combination of a low pCO2 and low
bicarbonate indicates either a metabolic acidosis or a compensatory respiratory alkalosis (or
both). As this patient has a severe acidosis, so the most probable diagnosis is metabolic
acidosis. The anion gap is 31 indicating the presence of a high anion gap disorder. The
lactate level of 20.3mmol/l is extremely high and this confirms the diagnosis of a severe
lactic acidosis. Hb is very low consistent with the history of bleeding and hypovolemia.
Urea & creatinine are elevated (renal failure) but at these levels there would not be
retention of anions sufficient to result in a renal acidosis. Hence,
Lactic acidosis can be suspected. The respiratory efforts may be due to the distress or as a
consequence of a metabolic acidosis (ie compensatory).
Case study-5
A 56- year -old man who smoked heavily for many years developed worsening cough
with purulent sputum and was admitted to the hospital because of difficulty in
breathing. He was drowsy and cyanosed. His arterial blood gas analysis was as
follows;
pH - 7.2
p CO2 - 70 mm Hg
HCO3 - 26 mmol/L
P O2 - 50 mm Hg
Case details
The patient is suffering from Respiratory acidosis. Difficulty in breathing, cough and
purulent sputum signify the underlying lung pathology. Low p H and raised pCO2 indicate
respiratory acidosis. Slightly high HCO3- may be due to compensation as a result of
increased reabsorption from the kidney. The low pO2 is due to associated hypoxia. The
treatment is based on the treating the primary cause.O2 and mechanical ventilation are
often needed.
Case study-6
A 5year old girl displayed increased appetite, increased urinary frequency, and thirst.
Her physician suspected new onset diabetes mellitus and confirmed that she had
elevated urine glucose and ketones.
68
Blood gas analysis revealed
pH-7.33
Bicarbonate-12.0 mmol/L
Arterial PCO2= 21
Case details
The patient is suffering from Diabetic ketoacidosis

In the presence of insulin deficiency, a shift to fatty acid oxidation produces the ketones
that cause metabolic acidosis. The pH and bicarbonate are low, and there is frequently some
respiratory compensation (hyperventilation with deep breaths) to lower the PCO2. A low
pH with high PCO2 would have represented respiratory acidosis which is not there in the
given case.
Case study-7
A19-year-old boy was brought to the emergency department with loss of consciousness.
Apparently the patient was a homeless found on the street.
Arterial blood gases revealed-

pH - 7.33,
pCO2 - 28 mm Hg,
pO2- 117 mmHg and
HCO3- 14 mmol/L
The blood level of methanol was 0.4 mg/dl.
What is your diagnosis?
Case details-
The patient is suffering from metabolic acidosis as evident from the low p H and low
bicarbonate levels. Low p CO2 and high p O2 signify that the patient is in a state of
respiratory compensation. Blood methanol level is high, so it might be case of Methanol
poisoning producing metabolic acidosis.
Case study-8
A 66 year old man had a postoperative cardiac arrest. Past history of hypertension treated
with an ACE inhibitor was there. There was no past history of Ischemic heart disease.
Following reversal and extubation, myocardial ischemia was noticed on ECG. He was
transferred to ICU for overnight monitoring. On arrival in ICU, BP was 90/50, pulse
80/min, respiratory rate was 16/min and SpO2 99%. During handover to ICU staff, he
69
developed ventricular fibrillation which reverted to sinus rhythm with a single 200J
counter shock. Soon after, blood gases were obtained from a radial arterial puncture:
Arterial Blood Gases
pH 7.27
pCO2 55.4 mmHg
pO2 144 mmHg
HCO3 24.3 mmol/l
Biochemistry Results (all in mmol/l): Na+ 138, K+ 4.7, Cl- 103, urea 6.4 & creatinine 0.07
Acid-base Diagnosis
1) pH- low , Acidosis is present.
2) pCO2- high, hypoventilation(The residual depressant effect of the Anesthetic agents is
considered the most likely cause)
3) Bicarbonate- near normal
4) pO2- high- This is because the patient is breathing a high inspired oxygen concentration.
If the patient had been breathing room air (FIO2 = 0.21), then a depression of alveolar pO2
must have occurred. Most ill patients in hospital breathe supplemental oxygen so it is
common for the pO2 to be elevated on blood gas results.
5) An acidemia with the pattern of elevated pCO2 and normal HCO3 is consistent with an
acute respiratory acidosis.
6) Anion gap- The anion gap is about 11 which is normal so no evidence of a high anion
gap acidosis.
Diagnosis- Acute respiratory acidosis
Cause- Resuscitation from postoperative ventricular fibrillation
Case study -9
A 72 year old male with diabetes mellitus is evaluated in the emergency room because
of lethargy, disorientation, and long, deep breaths (Kussmaul respiration). Initial
chemistries on venous blood demonstrate high glucose level of 380 mg/dl (normal up
to 120 mg/dl) and pH of 7.3. Bicarbonate 15mM and PCO2 30mmHg, What is the
probable diagnosis ?
Case details-
The man is acidotic as defined by pH lower than normal 7.4. His hyperventilation with
Kussmaul respiration can be interpreted as compensation by lungs to blow off CO2 to
lower PCO2, to increase [HCO3-]/[CO2] ratio, and to raise pH. Thus the patient has
metabolic acidosis due to underlying Diabetic ketoacidosis.
Case study-10
A 24 year female with broken ankle was brought to emergency with acute pain.
Blood gas analysis revealed the followings-
70
pH- 7.55
PCO2- 27
PO- 105,
HCO3- 23
Case details-
pH:- 7.55 – indicates Alkalosis

PCO2: 27 -low, it is a Primary respiratory disturbance
PCO2 Deficit = 40-27 = 13
HCO3 = 23 (Normal)
Interpretation:
It is Respiratory alkalosis due to pain related hyperventilation.
1. A patient suffering from acute severe asthma was brought to hospital. His blood
sample was taken and sent for varios investigations.
Some of the biochemical findings are as follows:
pH : 7.19
PCO2 : 79 mm of Hg
Plasma bicarbonate : 25 mmol/L
Interpret the results.
2. Name the acid-base status of the patient from given data in the following cases:
pH : 7.2
PCO2 : 40 mm of Hg
Carbonic acid : 1.35 mEq/L
3. A woman was admitted to hospital following a head injury. A skull fracture was
demonstrated on radiography and computerized tomography (CT) scan revealed
extensive cerebral contusions.
The respiratory rate was 38/min. Three days after admission, the patient’s
condition was unchanged and arterial blood was analyzed.
Arterial blood pH : 7.59
PCO2 : 29.3 mm of Hg
71
4. A twenty five year old lady was brought to hospital. She was over breathing (rate
and depth of inspiration and expiration) was increased.
The lady has given birth to a child recently. Her blood was sent to acid base
laboratory.

5. The following are the blood gas result of patient who has been on a respirator
for the past week.
PO2 : 88 mm of Hg
1. What is the acid-base status of the patient?

2. What is the nature of compensatory change?
6. Arterial blood sample sent from the emergency department for acid base analysis.
The biochemical findings are as follows:

H2CO3 : 1.35 mEq/L
Give your comments and conclusion.
7. A seventy year old man, known to suffer from chronic obstructive airway disease,
was admitted to hospital with an acute exacerbation of his illness.
Arterial blood analysis was carried out on admission.

72
8. Arterial blood sample sent from the emergency department for acid base analysis.
PO2 : 60.0 mm of Hg
What is the nature of disturbance?
How it is compensated?
9. The acid base balance parameters of a patient are as follows.

Explain the disturbance in the acid base balance, if any.

PO2 : 85.0 mm of Hg
10. An Arterial blood sample sent from MTH emergency department for acid base
analysis.

Arterial blood pH : 7.2 (7.35-7.45)
PCO2 : 40.0 mm of Hg (35-45 mmHg)
Plasma bicarbonate : 15 mEq/L (20-25 mEq/L)
73
CASE STUDIES FOR SELF EVALUATION- AMINO ACID METABOLISM
Case study-1
A 2 -week –old child was brought to the emergency. The parents were fearful that the child
had been given some poison as they noted black discoloration on the diaper. They had
delayed disposing one of the child’s diapers and noted black discoloration where the urine
had collected. Later, they realized that all of the child’s diapers would turn black if kept
unwashed for a longer time.
The attending pediatrician examined the child and explained them that it was due to an
amino acid disorder.
Which amino acid pathway is implicated in this phenomenon?
What is the cause of blackening of the diapers?
How can this defect be treated?
Case Study-2
A 12-year-old boy was admitted to the hospital with a red scaly rash and mild cerebellar
ataxia. His mother thought that the boy is suffering from Pellagra because the same
symptoms in her older daughter had been so diagnosed earlier. The boy did not have the
usual dietary deficiency form of Pellagra, but large amounts of free amino acids were found
in his urine. When the older daughter had a recurrent attack of ataxia, it was found that her
urine also contained excessive amount of amino acids. Two other siblings had the sane
aminoaciduria; however four others were normal. The parents were asymptomatic, but a
family history revealed that they were first cousins.
Assuming that this defect is inherited, what abnormality could account for the
unusual amount of amino acids in urine and what is its relationship with pellagra like
rashes?
How can this defect be treated?
Case study-3
A 2-week –old infant with refusal to feeds lethargy, excessive cry and irritability,
responded positively to a test for Phenylketonuria, A diagnosis of Classical
Phenylketonuria (PKU) was made and the child was maintained on a low Phenyl Alanine
diet.
Serum Phenyl Alanine was 30 mg/dl and Tyrosine was 2mg/dl. Ferric chloride test was
positive for urine. A diagnosis of Classical Phenylketonuria (PKU) was made and the child
was maintained on a low Phenyl Alanine diet.
What enzymatic reactions are defective in the patient with PKU?
What are the physiological consequences of PKU and why it should be detected as
early as possible?
What is the treatment for this disease?
74
Case study-4
A child presented with severe vomiting, dehydration and fever. History revealed that the
child was born normal but was not growing well from the last few months. There was
progressive mental retardation. Urine analysis revealed the presence of branched amino
acids and their Keto acids in high amount. Preliminary results from the blood amino acid
screen showed two elevated amino acids with non polar side chains.
Blood studies showed acidosis with a low bicarbonate concentration. The urine of the
patient had a smell of burnt sugar. Urine analysis revealed the presence of branched amino
acids and their Keto acids in high amount. Preliminary results from the blood amino acid
screen showed two elevated amino acids with non polar side chains.
What is the probable defect?
What is the basis for these symptoms?
Case study-5
A 38- year- old female reported with a dull pain in the left flank. The pain was radiating
towards left leg. She was in real agony. She reported that there was fever and inability to
pass urine from the last few days. History revealed that she was admitted twice with the
similar symptoms in the previous six months. .
On general physical examination, she was found to be anemic, pulse was 80/minute, B.P.
was 130/90 mm Hg and the abdomen was tender to touch. The patient was admitted for
observation and treatment. There was Costovertrebral angle tenderness on Murphy’s punch.
Routine urine analysis revealed the presence of RBCs, pus cells, WBC casts, characteristic
hexagonal crystals and amino acids. A diagnosis of aminoaciduria was made.
What is the probable defect? Which amino acid is expected to be there in urine?
What is the possible treatment?
Case study-6
A 54-year-old postmenopausal woman, presented to internist for care of hot flashes that
have returned 2 years after menopause. The symptoms occurred mostly after meals, when
she drank wine, or when she went for running. There was history of hypertension and
frequent diarrheas also. The patient was referred to gynecologist. The general physical
examination was entirely normal. Hormone-replacement therapy (HRT) was prescribed to
the patient and she was referred to gastroenterologist (GI) for Irritable Bowel Syndrome
(IBS) treatment. She experienced 2 hot flashes with no sweating during the examination.
Patient returned 3 months later with no improvement in hot flashes. A different formulation
of HRT was prescribed. Patient returned to GI doctor 1 year later with worsening of her
IBS. Patient reported cramping, abdominal pain, and increased episodes of diarrhea. She
had to wake up at night with diarrhea. Patient discontinued her HRT although they were
ineffective. After 1 year, patient was in need of emergency care. She presented with acute
bowel obstruction and was taken to the operating room.
75
There was marked fibrosis of the terminal ileum with multiple hairpin turns of the bowel
and a small tumor in the terminal ileum. Pathological report concluded that the patient had
Carcinoid syndrome.
What is the probable defect in Carcinoid syndrome?
How can this be diagnosed at the earliest possible? What is the biochemical basis for
all the clinical manifestations?
What is the prognosis of this disease?
Case Study-7
A fair chubby boy was brought to the hospital with the complaint that he has mental
retardation. Blood Chemistry revealed that serum phenylalanine was abnormally
high. Phenyl pyruvate and phenyl lactate were present in appreciable amounts in
urine.
Case Study-8
The case findings of a 2 week old female infant are given below:
Test for phenylketonuria : positive

Serum phenylalanine : 30 mg/dl (1.2-3.4 mg/dl)
From the result, answer the following questions.
1. What is the possible diagnosis?

2. What enzymatic reactions are defective in the patients with the above
disorders?
3. What are the physiological consequences in such conditions?
4. Why this disease should be detected as early as possible?
5. What is the incidence of the above disorder in the populations studied?
6. What treatment do you recommend?
Case Study-9
The case findings of a 2 week old female infant are given below:
Test for phenylketonuria : positive

Serum phenylalanine : 30 mg/dl (1.2-3.4 mg/dl)
Symptoms present – mental retartadation
Give your comments
76
Case Study-10
An infant is born without compliations but becomes extremely lethargic and begins to
hyperventilate beginning 24 hours after birth.
Blood analysis indicated a below-normal blood urea nitrogen (BUN) level, a slightly
alkaline pH, and a below normal level of partial pressure of CO2. A chest radiograph was
normal.
Further blood analysis reveals very high levels of ammonia, a high level of glutamine and
no detectable citrulline.
Urine analysis revealed extremely high level of orotic acid.
What is your diagnosis?
77
CASE STUDY-OROTIC ACIDURIA
A 4 year old girl presented to the clinic with Megaloblastic anemia and failure to
thrive. History revealed that the child was born normally. The red blood cell count
was 2.55 millions/cmm and hemoglobin was 6g/dl. He was given antibiotics and
transfusions. Despite that the anemia worsened. There was no response following
treatment with B12, Folic acid, or pyridoxine.
A prominent feature of the child’s urine was a crystalline sediment, which was found
to be orotic acid. Orotic acid in amounts as high as 1500 mg (9.6 mmol) was excreted
daily (Normal 1.4 mg/day, 9 ȝmol). Enzyme measurements of white blood cells
revealed a deficiency of the pyrimidine biosynthesis enzyme orotate
phosphoribosyltransferase and abnormally high activity of enzyme Aspartate
Transcarbamoylase.
What is the nature of the disease?
How can this be treated?
Case Discussion
The child is suffering from Orotic aciduria.
Orotic aciduria
Basic concept
Orotic aciduria refers to an excessive excretion of Orotic acid in urine.This is a disorder

of pathway of pyrimidine biosynthesis.
Biochemical Defect
Orotic aciduria is a rare Autosomal recessive disorder. The usual form of hereditary Orotic
aciduria is the build up of Orotic acid due to the deficiency in one or both of enzymes that
convert it to UMP. Either orotate phosphoribosyltransferase and orotidylate
decarboxylase both are defective, or the decarboxylase alone is defective. It can also
arise secondary to blockage of the urea cycle, particularly ornithine
Transcarbamoylase deficiency.
Pathway of pyrimidine biosynthesis
78
Figure-showing the steps of de novo pyrimidine nucleotide biosynthesis
The first step in de novo pyrimidine biosynthesis is the synthesis of Carbamoyl phosphate
from bicarbonate and glutamine in a multistep process, requiring the cleavage of two
molecules of ATP. This reaction is catalyzed by Carbamoyl phosphate synthetase -II (CPS-
II ). Carbamoyl phosphate synthetase-II primarily uses glutamine as a source of ammonia.
Carbamoyl phosphate reacts with aspartate to form Carbamoyl aspartate in a reaction

catalyzed by aspartate Transcarbamoylase. Carbamoyl aspartate cyclizes to form Dihydro
orotate, which then gets oxidized by Dihydro orotate dehydrogenase in the presence of
NAD+ to form orotate. At this stage, orotate couples to ribose, in the form of 5-
phosphoribosyl-1-pyrophosphate (PRPP), a form of ribose activated to accept nucleotide
bases. Orotate reacts with PRPP to form orotidylate (Orotate mono phosphate), a
pyrimidine nucleotide. This reaction is driven by the hydrolysis of pyrophosphate. The
enzyme that catalyzes this addition, pyrimidine phosphoribosyl transferase, is homologous
to a number of other phosphoribosyl transferases that add different groups to PRPP to form
the other nucleotides.
79
Orotidylate is then decarboxylated to form uridylate (UMP), a major pyrimidine nucleotide
that is a precursor to RNA. This reaction is catalyzed by orotidylate decarboxylase. This
enzyme is one of the most proficient enzymes known. In its absence, decarboxylation is
extremely slow and is estimated to take place once every 78 million years; with the enzyme
present, it takes place approximately once per second, a rate enhancement of many folds!
UMP is the parent nucleotide; the other pyrimidine nucleotides are formed from UMP.
Clinical manifestations
This disorder usually appears in the first year of life and is characterized by growth failure,
developmental retardation, megaloblastic anemia, and increased urinary excretion of
Orotic acid.
UMP, The end product of this pathway, is the precursor of UTP, CTP and TMP. All of
these end products normally act in some way to feedback inhibit the initial reactions of
pyrimidine synthesis. Specially, the lack of CTP inhibition allows Aspartate
Transcarbamoylase to remain highly active. This results in more and more production of
Orotic acid which gets accumulated and is excreted in urine excessively.
Lack of CTP, TMP, and UTP leads to a decreased nucleic acid synthesis and decreased
erythrocyte formation resulting in Megaloblastic anemia.
Physical and mental retardation are frequently present. The anemia is refractory to
vitamin B12 or folic acid.
Laboratory Diagnosis
The diagnosis of this disorder is suggested by the presence of severe Megaloblastic anemia
with normal serum B12 and Folate levels and no evidence of TC-II deficiency
( Transcobalamine- II).A presumptive diagnosis is made by finding increased urinary orotic
acid. Confirmation of the diagnosis, however, requires assay of the Transferase and
decarboxylase enzymes in the patient's erythrocytes
Treatment
Uridine treatment is effective because Uridine can easily be converted into UMP by
omnipresent tissue kinase, thus allowing UTP, CTP, and TMP to be synthesized and
feedback inhibit further Orotic acid production.
80
CASE STUDY-STARVATION
A 35 –year-old woman became severely depressed after the sudden death of her husband.
Two months later, she was brought to emergency room by her friend because of extreme
weakness and lethargy. She appeared thin and pale. Questioning revealed that she had not
eaten for several weeks.
Analysis of a plasma sample indicated elevated levels of Alanine, Acetoacetate, ȕ hydroxy
butyrate, and blood urea nitrogen (BUN). However her plasma glucose concentration was
low (55mg/dL). She was hospitalized, given intravenous feeding, antidepressant
medications and subsequently shifted to an 1800 Cal (7500kJ) diet. Her recovery was
uneventful.
How was the patient obtaining energy during the time when she was not eating?
How could patient maintain her plasma glucose within normal limits even though she
was not eating?
What is the significance of elevated plasma Alanine level?
Why is BUN elevated?
What is indicated by the fact that the plasma Acetoacetate and ȕ- hydroxy butyrate
levels are elevated?
It is a case of starvation. The high blood Alanine level signifies the catabolic state. Alanine
in excess is released during starvation from muscle to serve as a substrate for glucose
production in liver. Acetoacetate and ȕ hydroxy butyrate are ketone bodies which are used
as alternative fuel during conditions of glucose deprivation. High BUN signifies protein
degradation; the carbon skeletons of amino acids are utilized for glucose production while
amino groups are converted to urea.
Starvation
Prolonged fasting may result from an inability to obtain food, from the desire to lose weight
rapidly, or in clinical situations in which an individual can not eat because of trauma,
surgery, neoplasms, burns etc or even in depression (As in the given case) . In the absence
of food the plasma levels of glucose, amino acids and triacylglycerols fall, triggering a
decline in insulin secretion and an increase in glucagon release. The decreased insulin to
glucagon ratio, and the decreased availability of circulating substrates, make this period of
nutritional deprivation a catabolic state, characterized by degradation of glycogen,
triacylglycerol and protein. This sets in to motion an exchange of substrates between liver,
adipose tissue, muscle and brain that is guided by two priorities (i) the need to maintain
glucose level to sustain the energy metabolism of brain ,red blood cells and other glucose
requiring cells and (ii) to supply energy to other tissues by mobilizing fatty acids from
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adipose tissues and converting them to ketone bodies to supply energy to other cells of the
body.
Fuel Stores
A typical well-nourished 70-kg man has fuel reserves totaling about 161,000 kcal (670,000
kJ). The energy need for a 24-hour period ranges from about 1600 kcal (6700 kJ) to 6000
kcal (25,000 kJ), depending on the extent of activity. Thus, stored fuels suffice to meet
caloric needs in starvation for 1 to 3 months. However, the carbohydrate reserves are
exhausted in only a day.
Energy supply during starvation
During starvation the energy needs are fulfilled by three types of fuels, glucose, fatty acids
and ketone bodies.
a) Glucose supply during starvation (Gluconeogenesis)
Energy needs of brain and RBCs
Even under conditions of starvation, the blood-glucose level has been maintained above 2.2
mM (40 mg/dl). The first priority of metabolism in starvation is to provide sufficient
glucose to the brain and other tissues (such as red blood cells) that are absolutely dependent
on this fuel. However, precursors of glucose are not abundant. Most energy is stored in the
fatty acyl moieties of triacylglycerols. Fatty acids cannot be converted into glucose,
because acetyl CoA cannot be transformed into pyruvate. The glycerol moiety of
triacylglycerol can be converted into glucose, but only a limited amount is available. The
only other potential source of glucose is amino acids derived from the breakdown of
proteins. However, proteins are not stored, and so any breakdown will necessitate a loss of
function.
Thus, the second priority of metabolism in starvation is to preserve protein, which is
accomplished by shifting the fuel being used from glucose to fatty acids and ketone bodies
by cells other than brain cells and the cells lacking mitochondria.
It is a biological compromise to provide glucose to these cells as a priority. During
prolonged starvation, when the gluconeogenic precursors are not available, proteins are
however broken down to use carbon skeleton of glucogenic amino acids for glucose
production.
b) Fatty acid oxidation
Energy need of liver

The low blood-sugar level leads to decreased secretion of insulin and increased secretion of
glucagon. Glucagon stimulates the mobilization of triacylglycerols in adipose tissue and
gluconeogenesis in the liver. The liver obtains energy for its own needs by oxidizing fatty
acids released from adipose tissue. The concentrations of acetyl CoA and citrate
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consequently increase, which switch off glycolysis. Thus glucose utilization is stopped in
liver cells to preserve glucose for priority cells
Energy need of muscles
The uptake of glucose by muscle is markedly diminished because of the low insulin level,
whereas fatty acids enter freely. Consequently, muscle shifts almost entirely from glucose
to fatty acids for fuel. The beta-oxidation of fatty acids by muscle halts the conversion of
pyruvate into acetyl CoA, because acetyl CoA stimulates the phosphorylation of the
pyruvate dehydrogenase complex, which renders it inactive. Most of the pyruvate is
transaminated to alanine, at the expense of amino acids arising from breakdown of "labile"
protein reserves synthesized in the fed state. The alanine, lactate and much of the keto-acids
resulting from this transamination are exported from muscle, and taken up by the liver,
where the alanine is transaminated to yield pyruvate. Pyruvate is a major substrate for
gluconeogenesis in the liver.
Figure- showing Glucose Alanine and Cori’s cycle
In adipose tissue the decrease in insulin and increase in glucagon results in activation of
intracellular hormone-sensitive lipase. This leads to release from adipose tissue of increased
amounts of glycerol (which is a substrate for gluconeogenesis in the liver) and free fatty
83
acids, which are used by liver, heart, and skeletal muscle as their preferred metabolic fuel,
therefore sparing glucose.
Loss of muscle mass

During starvation, degraded proteins are not replenished and serve as carbon sources for
glucose synthesis. Initial sources of protein are those that turn over rapidly, such as proteins
of the intestinal epithelium and the secretions of the pancreas. Proteolysis of muscle protein
provides some of three-carbon precursors of glucose. The nitrogen part of the amino acids
is converted to urea (BUN)
c) Ketosis
Energy need of peripheral tissues
After about 3 days of starvation, the liver forms large amounts of acetoacetate and beta-
hydroxybutyrate. Their synthesis from acetyl CoA increases markedly because the citric
acid cycle is unable to oxidize all the acetyl units generated by the degradation of fatty
acids. Gluconeogenesis depletes the supply of oxaloacetate, which is essential for the entry
of acetyl CoA into the citric acid cycle. Consequently, the liver produces large quantities of
ketone bodies, which are released into the blood. At this time, the brain begins to consume
appreciable amounts of acetoacetate in place of glucose. After 3 days of starvation, about a
third of the energy needs of the brain are met by ketone bodies. The heart also uses ketone
bodies as fuel. After several weeks of starvation, ketone bodies become the major fuel of
the brain.
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Figure- fatty acid oxidation and ketosis during starvation.
In essence, ketone bodies are equivalents of fatty acids that can pass through the blood-
brain barrier. Only 40 g of glucose is then needed per day for the brain, compared with
about 120 g in the first day of starvation. The effective conversion of fatty acids into ketone
bodies by the liver and their use by the brain markedly diminishes the need for glucose.
Hence, less muscle is degraded than in the first days of starvation. The breakdown of 20 g
of muscle daily compared with 75 g early in starvation is most important for survival.
A person's survival time is mainly determined by the size of the triacylglycerol depot.
What happens after depletion of the triacylglycerol stores? The only source of fuel that
remains is proteins. Protein degradation accelerates, and death inevitably results from a loss
of heart, liver, or kidney function.
85
CASE STUDY-AIDS
A- 29 year- old officer presented to emergency department complaining of chills and

breathing difficulty. He was a heroin abuser who was admitted in the same hospital 7
years ago because of drug over dosage. He had lost 10 kg of body weight since his last
clinical visit.
On examination, he had multiple enlarged lymph nodes. Several red nodules were
there on the chest and arms. His body temperature was 40oC, B.P. 170/50 mm Hg and
respiration was shallow with a respiratory rate of 40 breaths per minute. The chest
radiograph showed diffuse pneumonia.
Sputum smear revealed numerous Pneumocystis organisms while skin biopsy revealed
Kaposi’s sarcoma.
What is your presumptive diagnosis for this patient?

What is the biochemical basis for all the clinical manifestations seen in this patient?
How can the diagnosis be confirmed?
Case discussion
The Patient is suffering from AIDS. Intravenous drug usage seems to be the major
causative factor. Patient‘s immune system has been collapsed to the extent of loss of
resistance to ordinary viral, fungal and protozoal infections which could otherwise have
been controlled easily. Kaposi sarcoma and Pneumocystis pneumonia are considered
markers of AIDS since they signify the underlying collapsed immune system. Non specific
Fever and weight loss of more than 10% body weight are also diagnostic of AIDS.
AIDS
Acquired Immuno deficiency syndrome or AIDS, is a collection of symptoms due to

underlying infections and malignancies resulting from specific damage to immune system
caused by human immunodeficiency virus (HIV).
Incidence
HIV (human immunodeficiency virus) infection has now spread to every country in the
world. Approximately 40 million people are currently living with HIV infection, and an
estimated 25 million have died from this disease. The scourge of HIV has been particularly
devastating in sub-Saharan Africa, but infection rate in other countries remain high. In the
United States, approximately 1 million people are currently infected.
The first indication of this new syndrome came in 1981 in homosexual drug addict males;
they had two things in common- Pneumocystis pneumonia and Kaposi’s sarcoma. Both
these are markers of collapsed immune system, The affected patients appeared to have lost
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their immune competence, rendering them vulnerable to overwhelming and fatal infections
with relatively avirulent micro-organisms, as well as to lymphoid and other malignancies.
This condition was given the name Acquired Immuno deficiency Syndrome (AIDS).
In 1986, The International Committee on virus Nomenclature decided on the generic name
of the causative virus as the Human Immunodeficiency Virus.
Human Immunodeficiency Virus
HIV, the etiological agent of AIDS, belongs to lentivirus subgroup of the retroviridae
family. This family of viruses is known for latency, persistent viremia, infection of the
nervous system, and weak host immune responses. HIV has high affinity for CD4 T
lymphocytes and monocytes. HIV binds to CD4 cells and becomes internalized. The virus
replicates itself by generating a DNA copy by reverse transcriptase. Viral DNA becomes
incorporated into the host DNA, enabling further replication. Besides HIV, the related
animal immunodeficiency viruses are also assigned to this group.
Structural Characteristics of HIV
HIV is a spherical enveloped virus of about 90-120 nm in diameter.(See Figure-1 below).

There is a lipoprotein envelop, which consists of lipids derived from the host cell
membrane and glycoproteins which are viral coded. The major virus coded envelop
proteins are the projecting knob like spikes on the surface and the anchoring
transmembrane pedicles. The spikes, gp 120 constitute the major surface component of the
virus which binds to the cell CD4 receptors on susceptible host cells. These specific
receptors, known as cluster of differentiation- CD4 are present on certain cells in the
body, the cells possessing these receptors are called CD 4 + cells and these are -T helper
cells, B lymphocytes ,macrophages, monocytes and dendritic cells. Transmembrane
pedicles gp 41 cause cell to cell fusion.
Interior to the envelope is an outer icosahedral nuclear capsid shell and an inner cone
shaped core containing ribonucleoproteins.The enzymes integrase p32, protease p10,
reverse transcriptase p55/66 and 2 copies of single stranded genomic RNA are present
inside the core. (The proteins and glycoproteins are indicated by their mass expressed as
kilo Daltons)
The reverse transcriptase enzyme is characteristic feature of retroviruses. When the virus
infects a cell, the viral RNA is transcribed by the enzyme first in to single stranded DNA
and then in to double stranded DNA (provirus), which is integrated in to the host cell
chromosome. The provirus can remain latent for long period though it influences the host
cell functions. At times in response to viral promoters, the provirus initiates viral
replication by directing the synthesis of viral RNA and other components.
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Figure-1-A cross sectional schematic diagram of HIV virion, showing lipid bilayer in the
form of viral envelop, Nucleocapsid core, which includes a layer of a protein called p17
and an inner layer of a protein called p24. The HIV genome consists of two copies of the
single stranded RNA, which are associated with two molecules of Reverse transcriptase
p64, protease p10 and integrase p32. The outer viral proteins are gp 120 and gp 41.
Genome of HIV
There are two types of genes analyzed,(See figure-2)
a) Structural genes encode for products which participate in formation of functional

structure of virus-
1) gag gene - encodes for core and shell of virus. The gene product is a precursor protein
p55, which is cleaved into p17, p24 and p15. The p24 antigen (major core antigen) can
be detected in serum during the early stages of infection till the appearance of the
antibodies.
2) pol gene- encodes for the polymerase reverse transcriptase and other viral enzymes such
as protease and integrase. It is expressed as a precursor protein, which is cleaved in to
components like p64 which has reverse transcriptase and RNAse activity: p51 which has
only reverse transcriptase activity: p10 is a protease that cleaves gag precursor and p32 is
an integrase.
3) env gene - determines the synthesis of envelop glycoprotein gp 160 which is cleaved
into gp 120 and gp 41 .Glycosylation occurs after cleavage. The antibodies to gp 120 are
the first to appear after HIV infection and are present in circulation till the terminal stage of
infection.
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b) Non structural and Regulatory genes
1) vif (Viral infectivity factor gene) influences infectivity of viral particles.
2) vpr-stimulates promoter region of the virus
3) vpu(only in HIV-1 ) and vpx (only in HIV-2) enhance maturation and release of
progeny virus from cells. Detection of the type specific sequences vpu and vpx is useful in
distinguishing between infection by HIV type 1 and 2.
4) tat –( transactivating gene) – (2 copies) having stimulatory effect on synthesis of all
viral proteins .
5) rev - (Regulator of viral genes) –(2 copies )–required for expression of structural genes.
6) nef ( negative factor gene) down regulates viral replication. It may be responsible for the
regulation of latent state of virus.
7) LTR - (long terminal repeat) sequences flanking on both sides giving promoter,
enhancer and integration signals.
Figure-2 schematic representation of HIV genome
Antigenic variations in HIV
Based on molecular and antigenic differences, two types of HIV have been recognized. The
original isolates of HIV and the related strains present all over the world belong to HIV
type 1.The HIV strains, first isolated from West Africa, which react with HIV type I
antiserum very weakly or not at all have been termed as HIV type 2. It has 40 % genetic
similarity and is more closely related to Simian immunodeficiency virus than to HIV-1.It
can cause AIDS but is less pathogenic and is less common. It infects mainly monkeys and
other similar species and is largely confined to West Africa, though isolations have been
reported from some other areas, including Western and Southern India. The envelop
antigens of the two types are different though their core peptides show some cross
reactivity.
HIV is a highly mutable virus and exhibits frequent antigenic variations as well as
differences in other features such as nucleotide sequences, cell tropism, growth
characteristics and cytopathology. Not only are there differences between isolates of HIV
89
from different races or persons but also between sequential isolates from the same person,
and even between those obtained from different sites of the same person at the same time.
This great variability is believed to be due to error prone nature of reverse transcription.
Antigenic variation is most frequent in respect of the envelop proteins, but is also seen with
other antigens.
HIV-1 strains have been classified in to at least ten subtypes base on sequence analysis of
their gag and env genes. These subtypes have been are designated as A to J and constitute
the Group M (For major), which cause the large majority of HIV-1 infections worldwide. A
few HIV-1 strains isolated from West Africa do not fall within the major group and have
been designated as group O ( For Outlier) Some later isolates of HIV-1 distinct from M
and O groups have been called Group N (for new)
Modes of transmission
HIV is transmitted when the virus enters the body, usually by injecting infected cells or
semen. There are several possible ways in which the virus can enter.
1) Sexual contact- In 75 % cases , transmission is by sexual contact. Most commonly,

HIV infection is spread by having sex with an infected partner. The virus can enter the
body through the lining of the vagina, vulva, penis, rectum, or mouth during sex. People
who already have a sexually transmitted disease, such as syphilis, genital herpes,
chlamydial infection, gonorrhea, or bacterial vaginitis, are more likely to acquire HIV
infection during sex with an infected partner.
2) Parenteral- In 15 % cases, it is by blood transfusion or blood product transfusion. Blood

products are now tested to minimize this risk. Sharing of unsterilized needles or syringes
in drug addicts contaminated with blood from an infected person can also spread virus. HIV
can be spread in health-care settings through accidental needle sticks or contact with
contaminated fluids. HIV can also spread through organ transplantation. If tissues or
organs from an infected person are transplanted, the recipient may acquire HIV. Donors are
now tested for HIV to minimize this risk.
3) From mother to child : Women can transmit HIV to their babies during pregnancy or
birth, when infected maternal cells enter the baby's circulation.30% of children born to
infected mothers have the acquired infection unless virus is treated by antiviral drugs before
pregnancy. In nursing mothers transmission can occur through breast milk.
Mortality/Morbidity
The course of HIV infection is characterized primarily by latency. Unfortunately, profound

immune suppression eventually develops and the illness appears to be almost uniformly
lethal. More than 500,000 persons have died of AIDS in the United States.
Progression from HIV infection to AIDS occurs 8-10 years after infection without
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antiretroviral treatment. In the recent past, most patients would not survive more than 1-2
years following diagnosis of AIDS. However, since the introduction of highly active
antiretroviral therapy (HAART) and prophylaxis against opportunistic pathogens, death
rates from AIDS have declined significantly. An HIV-positive patient older than 50 years
with a nearly undetectable viral load and a CD4 count more than 350 now has less than a
5% chance of dying or progressing to full blown AIDS within 3 years.
Age
Most AIDS cases occur in adults aged 25-49 years (70% of cases). Adolescents and young
adults (aged 13-24 y) represent 25% of new cases. Young children represent fewer than 1%
of AIDS cases in the United States. Internationally, children younger than 15 years are
estimated to account for close to 10% of all HIV cases.
Pathogenesis
Infection is transmitted when virus enters the blood or tissues of a person and comes in to
contact with a suitable host cell, principally the CD4 lymphocytes. The virus may infect
any cell bearing the CD4 antigen on the surface. Primarily these are the CD4 + helper T
lymphocytes. Some other immune cells possessing CD4 antigens are also susceptible to
infection, like B lymphocytes, monocytes and macrophages including specialized
macrophages such as Alveolar macrophages in the lungs and Langerhans cells in the
dermis. Glial cells and microglia cells are also susceptible. Follicular dendritic cells from
tonsils can be infected by HIV without the involvement of CD4. The steps of viral entry in
to the host cell are as follows-
1. Attachment of virus into the host cell –Specific binding of the virus to the CD4
receptors is by the envelop glycoprotein gp120.
2. Cell to cell fusion – For infection to take place the cell fusion is essential. This is
brought about by the transmembrane glycoprotein gp 41. HIV-1 utilizes two major co-
receptors along with CD4 to bind to, fuse with, and enter target cells; these co-receptors are
CCR5 and CXCR4, which are also receptors for certain endogenous chemokines. Strains of
HIV that utilize CCR5 as a co-receptor are referred to as macrophage tropic viruses.
Strains of HIV that utilize CXCR4 are referred to as T cell- tropic viruses. Many virus
strains are dual tropic in that they utilize both CCR5 and CXCR4.
The infected CD4 cells express a high level of gp 120 on their surface. The gp 120 on the
surface of infected cells leads to fusion of these cells with CD4 protein of uninfected
neighboring cells with formation of multinucleated syncytial cells. The lysis of fused cells
finally occurs resulting in depletion of large number of uninfected cells from the
circulation.
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3. Uncoating of the viral envelope and entry of nuclear capsid core into the cell -After
fusion of virus with the host cell membrane, HIV genome is uncoated and internalized in to
cell. Viral RNA is released into the core cytoplasm.
4. Viral transcription- viral reverse transcriptase mediates transcription of its RNA; RNA-
DNA hybrid is formed. Original RNA strand is degraded by ribonuclease H, followed by
synthesis of second strand of DNA to yield double strand HIV DNA
5. Integration into the host DNA as provirus- The double stranded DNA is integrated in
to the genome of the infected host cell through the action of the viral integrase enzyme,
causing a latent infection.
6. Fate of provirus-From time to time, lytic infection is initiated releasing progeny virions,
which infect other cells. The long and variable incubation period of HIV is because of the
latency. In an infected individual the virus can be isolated from the blood, lymphocytes,
cell free plasma, semen, cervical secretions, saliva, urine and breast milk.
7. Transcription back into RNA - The viral DNA is transcribed into RNA and multiple
copies of viral RNA are produced. There are only nine genes in HIV RNA and these codes
for the production of structural proteins, accessory proteins, and enzymes essential for the
virus's replicative cycle.
8. Virion assembly - With the help of viral protease, the new virions are assembled into the
polypeptide sequences needed for HIV virion formation and infectivity.
9. Cell lysis. The infected cell is made to burst open, presumably by the action of cellular
proteins.(See figure-3)
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Figure-3 showing the life cycle of HIV (Integration and excision of viral genome)
Causes of immune deficiency
The primary pathogenic mechanism in HIV infection is the damage caused to the
CD4+ T lymphocytes- The T4 cells decrease in numbers and the T4:T8 cell ratio is
reversed. The infected cells do not release cytokines. This has a marked damping effect in
the cell mediated immune response. Though the major damage is to cellular immunity, the
humoral mechanisms are also affected. AIDS patients are unable to respond to new
antigens. There is polyclonal activation of B lymphocytes leading to
hypergammaglobulinemia. These are non specific antibodies and are irrelevant to antigens.
Monocyte, macrophage system is also affected apparently due to the lack of the activating
factors by the T4 lymphocytes. The activity of NK cells and Tc (T Cytotoxic) cells are also
affected. The clinical manifestations are due to failure of the immune responses. This
renders the patient susceptible to life threatening opportunistic infections and malignancies.
The exceptions to this may be the neurological lesions seen in AIDS. Dementia and other
93
degenerative neurological lesions seen in AIDS are due to direct toxic effects of HIV on the
central nervous system.
Clinical Manifestations
AIDS is only the last stage in the wide spectrum of clinical features in HIV infection. The
center for disease control (USA) has classified the clinical course of HIV infection under
various groups.
1. Acute HIV infection
2. Asymptomatic or Latent infection
3. Persistent generalized lymphadenopathy (PGL)
4. AIDS related complex
5. Full blown AIDS (Last stage)
1. Acute HIV infection

Many people do not develop symptoms after they first get infected with HIV. Others have a
flu-like illness with fever, sore throat, headache, tiredness, skin rashes and enlarged
lymph nodes in the neck within several days to weeks after exposure to virus. These
symptoms usually disappear of their own within a few weeks. The test for HIV antibodies
appears negative while HIV antigenemia (p24 antigen) and viral nucleic acids can be
demonstrated at the beginning of the phase. This phase is also called window period or
phase of Sero conversion.
2. Asymptomatic or Latent infection

All persons infected with HIV, pass through a phase of symptomless infection (Clinical
latency), which may last up to several years. The progression of disease varies widely
among individuals. This state may last from a few months to more than 10 years. During
this period, the virus continues to multiply actively and infects and kills the cells of the
immune system. The virus destroys the CD4 cells that are the primary infection fighters.
Even though the person has no symptoms, he or she is contagious and can pass HIV to
others through the routes listed above. The patients show positive antibody tests during
this phase. The infection progresses in course of time through various stages, CD4
lymphocytopenia, minor opportunistic infections, persistent generalized lymphadenopathy,
AIDS related complex, ultimately terminating in full blown AIDS.(Figure-5) The median
time between primary HIV infection and development of AIDS has been stated as
approximately 10 years. About 5-10 % percent of the infected appear to escape clinical
AIDS for 15 years or more. They have been ‘long term survivors” or “long term non
progressors”. The mechanism for such long time survivors is not exactly known, though
many viral and host determinants are responsible.
During this period the host mounts an immune response against the virus, both humoral and
cellular, which can only limit the viral load but can not clear it completely. A chronic
persistent infection with varying degree of multiplication is the result.
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3. Persistent generalized lymphadenopathy (PGL)
This has been defined by presence of enlarged lymph nodes, at least I cm in diameter, in
two or more non contiguous extra inguinal sites, that persist for at least three months, in the
absence of any current illness or medication that may cause lymphadenopathy. These are
diagnostic of HIV when blood tests are positive for antibodies.
4. AIDS related complex
This group includes patients with considerable Immuno deficiency suffering from various
constitutional symptoms of minor opportunistic infections. The patients present with
weight loss – of more than 10% of body weight, persistent fever, diarrhea, generalized
fatigue and signs of other opportunistic infections may be apparent. The opportunistic
infections are oral candidiasis, herpes zoster, salmonellosis or Tuberculosis and hairy
cell leucoplakia. The patients are usually severely ill and many of them progress to AIDS
in few months. The CD4 cell count decreases steadily when the count falls to 200, or
less, clinical AIDS usually sets in. For this reason the case definition by CDC includes
all HIV infected cases with CD4 + T cell counts of 200 or less, irrespective of clinical
condition.
5. Full blown AIDS
This is the end stage disease representing the irreversible break down of immune defense
mechanisms. In addition to the opportunistic infections the patient may develop primary
CNS lymphomas and progressive multifocal leukoencephalopathy, dementia and other
neurological abnormalities. Kaposi sarcoma (Figure-4) and Pneumocystis pneumonia are
almost always observed in a majority of patients.
Figure- 4-showing Kaposi’s sarcoma- it is an indolent, multifocal non metastasizing

mucosal or cutaneous tumor probably of endothelial origin, represented in the form of
purple spots in the skin.
95
Figure-5 showing the disease progression through different phases in HIV infected
cases.CD4 count comes down while the viral count goes high with the passage of time.
Laboratory Diagnosis of HIV infection
Laboratory procedures for the diagnosis of HIV infection include tests for
immunodeficiency as well as specific tests for HIV.
1) Non Specific Tests- The following tests help to establish the immunodeficiency in HIV
infection.
a) Total Leukocyte and lymphocyte count- to demonstrate leucopenia and lymphopenia.
The lymphocytic count is usually below 2000/mm3
b) T cell subset Assays- Absolute CD4+ cell count is less than 200 /L.T4 T8 ratio is
reversed.. The decrease in CD4 is the hall mark for AIDS.
c) Platelet count- shows Thrombocytopenia.
d) IgA and Ig G levels are raised
e) Diminished cell mediated Immunity as indicated by skin tests
f) Lymph node biopsy shows profound abnormalities.
2 .Specific Tests for HIV infection- These include demonstration of HIV antigen,
antibodies. Viral nucleic acids or other components and isolation of virus-
i) Detection of antigen- Following a contact, as by blood transfusion, the viral antigen may
be detectable in blood after about 2 weeks. The major core antigen p24 is the earliest virus
marker to appear in blood. IgM antibodies appear in about 4-6 weeks, to be followed by
IgG antibodies
If the infecting dose is small, as following a needle stick injury, the process may be
considerably delayed. Afterwards free p24 antigen disappears from circulation and remains
absent during the long asymptomatic phase to reappear only when severe clinical disease
96
sets in. However antibody bound p24 antigen continues to be demonstrable, after
dissociation.
The p24 Capture ELISA assay, which uses anti p24 antibody as the solid phase can be used
for this. This test is positive in about 30% of the infected persons. In the first few weeks
after infection and in the terminal phase, the test is uniformly positive. This test along with
HIV ELISA is currently used for screening blood donors.
2. Detection of antibodies
Demonstration of antibodies is the simplest and most widely employed technique for the
diagnosis of HIV infection. However it takes 2-8 weeks to months for the antibodies to
appear in circulation and during that period the person may be highly infectious. Once
antibodies appear they increase in titer for the next several months. IgM antibodies
disappear in 8-10weeks while IgG antibodies remain through out. When immunodeficiency
becomes severe following clinical AIDS some components of anti p24 may disappear.
There are two types of serological tests- Screening tests and supplemental tests.
i) Screening tests include-
ELISA- ELISA is the most frequently used method for screening of blood samples for
HIV antibody. The sensitivity and specificity of the presently available commercial systems
approaches 100% but false positive and false negative reactions occur.
1) First generation - whole viral lysates
2) Second generation - recombinant antigen
3) Third generation - synthetic peptide
4) Fourth generation - antigen + antibody (Simultaneous detection of HIV antigen and
antibody) - HIV duo
Antibody can be detected in a majority of individuals within 6-12 weeks after infection
using the earlier generation of assays. But it can be detected within 3-4 weeks when using
the newer third generation ELISA. Due to their ability to detect p24 antigen, the fourth-
generation ELISA will be of value in detecting early infection. The window period can be
shortened to two weeks using p24-antigen assay.
ii) Supplemental Tests
a)Western Blot Test

b) Indirect Immunoflorescence test
c)Radio ImmunoPrecipitaion Assay
iii) Rapid Tests

a) Dot Blot assay
b) Particle Agglutination tests
c) HIV spot and comb test
97
d)Fluorimetric micro particle technologies
Western blotting- Western blots are regarded as the gold standard and seropositivity is
diagnosed when antibodies against both the env and the gag proteins are detected. The
sensitivity of the test systems are currently being improved by the use of recombinant
antigens.
Rapid tests - These are tests that can yield results in < 30 min. The results are read by
naked eye.
Rapid HIV assays have proven particularly useful for testing pregnant women in labor who
have not received prenatal care. These are also helpful in detecting HIV-2 infection which
can not be detected by ELISA.
3. Demonstration of viral Nucleic acid - this can be accomplished by probes or by PCR

techniques. The latter may be useful because of its extremely high sensitivity. Although
standard tests that measure antibody response to the HIV virus have become increasingly
sensitive, cases of HIV are occasionally missed because individuals can have negative
antibody tests during the early stages of infection. Also, a few people with long-term HIV
infection may have false negative antibody tests or may be chronic carriers who are
clinically asymptomatic.
PCR -In this the target HIV RNA or proviral DNA is amplified enzymatically in vitro by
chemical reaction. It is an extremely sensitive assay because a single copy of proviral DNA
can be amplified. Qualitative PCR is useful for diagnostic purposes.
Three different techniques namely RT-PCR, nucleic acid sequence based amplification
(NASBA) and branched-DNA (b-DNA) assay have been employed to develop commercial
kits.
4. Virus isolation - virus isolation is accomplished by the co cultivation of the patient's

lymphocytes with fresh peripheral blood cells of healthy donors or with suitable culture
lines such as T-lymphomas. The presence of the virus can be confirmed by reverse
transcriptase assays, serological tests, or by changes in growth pattern of the indicator cells.
However virus isolation is tedious and time consuming (weeks) and is successful in only 70
to 90% of cases. Therefore virus isolation is mainly used for the characterization of the
virus.
5. Alternative to classical tests
a) Oral fluid (saliva) HIV tests

b) Urine tests
Treatment
98
Virtually all treatment for HIV infection and AIDS today focuses on arresting the
progression of the disease within the body as measured by T cell counts and tests for viral
load.
At present, a reasonable course of action is to initiate ARV( Antiretroviral)therapy in
anyone with the acute HIV syndrome; all pregnant women; patients with symptomatic
disease; and patients with asymptomatic disease with CD4+ T cell counts <350/L. In
addition, one may wish to administer a 6-week course of therapy to uninfected individuals
immediately following a high-risk exposure to HIV.
There are two principal approaches to treatment: immunotherapy and anti-HIV drug
treatments
a)Immunotherapy- Immunotherapy is transfusion based treatment designed to replace lost

immunoglobulins needed to fight HIV infection (passive immunotherapy), to provide
cellular factors such as interleukins (IL-2) or to introduce selected or altered immune cells
to attack cells harboring the virus (adoptive immunotherapy). The results of trials using this
latter approach, however, have been inconclusive, and no group has yet shown a survival
benefit.
b) Anti-HIV drug treatment -Treatment with anti-HIV drugs attempts to reduce viral load
by blocking new infection in the host cell. The drugs used target two major enzymes of
HIV which are needed for the infection cycle: reverse transcriptase and protease.
I) Reverse transcriptase inhibitors - Reverse transcriptase inhibitors act at the pre-

integration stage - before the viral RNA has been converted to DNA and enters the host cell
nucleus to integrate into the cell chromosome. These drugs block the reverse transcription
of viral RNA into viral DNA. There are two types of reverse transcriptase inhibitors, both
of which accomplish the same objective: nucleoside and nucleotide analogues, and non-
nucleoside reverse transcriptase inhibitors.
i) Nucleoside analogues, which constitute the most effective family of antiretroviral drugs,
operate by mimicking nucleic acids normally incorporated into viral DNA. They interfere
with reverse transcriptase and thus prevent infection. Nucleoside analogues can be placed
in two groups, those replacing thymidine, such as AZT and d4T, which protect activated T
cells from infection, and non-thymidine analogues such as ddI and 3TC, which protect
resting T cells.
Nucleotide analogues have the same action but are based around a different sugar.
ii) Non-nucleoside reverse transcriptase inhibitors directly inhibit reverse transcription

and, unlike nucleoside analogues, do not have to go through chemical changes in the
infected cell before beginning their action. The two main non-nucleoside reverse
transcriptase inhibitors are nivirapine, which can show toxicity and early resistance
problems, and delavirdine, with which there is less clinical experience as yet.
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2) Protease inhibitors -Protease inhibitors block HIV replication after integration. These
drugs inhibit the function of the protease by acting as analogues for the peptide and non-
peptide protease substrates needed to process the ‘gag-pol’ polypeptide into proteins. As a
result, no infectious virus can be produced.
Since protease inhibitors act after integration, they can obstruct infectious HIV production
in both acutely and chronically infected T cells and macrophages. (In contrast, reverse
transcriptase inhibitors can only inhibit the acute infection of cells.). Protease inhibitors
have also been shown to be less toxic than nucleoside analogues. So far only a few have
been licensed for use, including saquinavir, ritonavir, indinavir, and, more recently,
nelfinavir, although others are expected. (Figure-6)
3) Combination treatment -Because reverse transcriptase inhibitors and protease

inhibitors address different stages of viral replication, using both families of drugs in
combination has been shown to be more effective than monotherapy in impeding the spread
of HIV in the body and reducing viral loads. Although the optimum combination of anti-
HIV drugs is as yet unknown, AZT, 3TC, and a protease inhibitor are usually the first drugs
given. This combination of two reverse transcriptase inhibitors, one of which is a thymidine
analogue, and a protease inhibitor, blocks infection both before and after integration and in
both activated and resting T cells. The aim of anti-HIV therapy has now shifted from
simply delaying the progression of disease to finding a permanent cure. This combinational
therapy is termed as highly active anti-retroviral therapy (HAART).
The current consensus is that one should give a potent combination of agents, HAART
right from the start when treatment is indicated.
4) The future- In addition to the positive results shown by combination therapy trials, a
number of developments may hold promise for the near future. These include genetically
engineered killer T cells which attack HIV before it reproduces, and research into
genetically deactivating the CXCR4 and CCR5 T cell co-receptors, which are a path for
HIV entry into cells.
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Figure-6 showing the mechanism of action of different drugs used for the treatment of HIV
infection
Prevention
The risk of contracting HIV increases with the number of sexual partners. A change in the
lifestyle would obviously reduce the risk.
HIV-infected mothers are not recommended to have children at present and pregnancy
itself would appear to be a risk factor for seropositive mothers. A recent clinical trial
demonstrated the efficacy of AZT in preventing transmission of HIV from the mother to the
fetus.
The spread of HIV through blood transfusion had virtually been eliminated since the
introduction of blood donor screening in many countries. Blood products such as factor
VIII now undergo routine treatment which appears to inactivate any HIV present
effectively.
Development of vaccine
Development of vaccine is fraught with several problems unique to this virus. These
include-
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1) HIV can mutate rapidly, thus, it is not possible to design antibodies against all antigens.
2) Antibody alone is not sufficient, cell mediated immunity may also be necessary.
3) Virus enters the body not as free virions but also as infected cells, in which the virus or
the provirus is protected against antibody or cell mediated lysis.
4) Virus readily establishes life long latent infection hiding from antibodies.
The main types of approaches to an AIDS vaccine are as follows:
1. Live attenuated virus

2. Inactivated virus
3. Live recombinant viruses
4. Synthetic peptides
5. Recombinant DNA products (gp120, gp160)
6. Native envelope and/or core proteins
7. Anti-idiotypes antibodies
8. Passive immunization
To-date, the best hope lies in an inactivated vaccine

Despite progress in dealing directly with HIV, however, the virus, by impairing the
immune system, exposes the infected person to a range of opportunistic viral, bacterial, and
parasitic infections and malignancies. It is these which are the actual cause of death in most
patients with AIDS, it is still unclear whether HIV induced damage to the immune system
can be reversed. New strains of HIV undetectable by current screening methods and
resistant to the best antiretroviral drugs currently available have also now been discovered.
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CASE STUDY- CARNITINE DEFICIENCY
A teenage girl was brought to the medical centre because of her complaints that she
used to get too tired when asked to participate in gym classes. A consulting neurologist
found muscle weakness in girl’s arms and legs. When no obvious diagnosis could be
made, biopsies of her muscles were taken for test.
Biochemistry revealed greatly elevated amounts of triglycerides esterified with
primary long chain fatty acids. Pathology reported the presence of significant
numbers of lipid vacuoles in the muscle biopsy

What is the cause for these symptoms?
Case details
The most likely cause of these symptoms is carnitine deficiency.
Carnitine deficiency
The amino acid carnitine is required for the transport of long-chain fatty acyl coenzyme
esters into myocyte mitochondria, where they are oxidised for energy. Carnitine is obtained
from foods, particularly animal-based foods, and via endogenous synthesis.
Carnitine deficiency results from inadequate intake of or inability to metabolise the amino
acid carnitine. It can cause a heterogeneous group of disorders. Muscle metabolism is
impaired, causing myopathy, hypoglycemia, or cardiomyopathy. Infants typically present
with hypoglycemic, hypoketotic encephalopathy. Most often, treatment consists of dietary
l-carnitine.
Carnitine
Basic concept
Fatty acids are activated on the outer mitochondrial membrane, whereas they are oxidised
in the mitochondrial matrix. The activation is brought by converting the fatty acid into Acyl
co A ester under the activity of Acyl co A synthetase (1). A special transport mechanism is
needed to carry long-chain acyl CoA molecules across the inner mitochondrial membrane.
Activated long-chain fatty acids are transported across the membrane by conjugating them
to carnitine. The acyl group is transferred from the sulphur atom of CoA to the hydroxyl
group of carnitine to form acyl carnitine. This reaction is catalysed by carnitine acyl
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transferase I (also called carnitine palmitoyl transferase I), which is bound to the outer
mitochondrial membrane.(2)
Acyl carnitine is then shuttled across the inner mitochondrial membrane by a translocase.
(3)
The acyl group is transferred back to CoA on the matrix side of the membrane. This
reaction, which is catalyzed by carnitine acyl transferase II (carnitine palmitoyl transferase
II) (4), is simply the reverse of the reaction that takes place in the cytosol.
Finally, the translocase returns carnitine to the cytosolic side in exchange for an incoming
acyl carnitine (See figure).
Figure -showing the role of carnitine in transporting the activated fatty acids in to the
mitochondria.
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Pathogenesis
A number of diseases have been traced to a deficiency of carnitine, the transferase or the
translocase. The symptoms of carnitine deficiency range from mild muscle cramping to
severe weakness and even death. The muscle, kidney, and heart are the tissues primarily
affected. Muscle weakness during prolonged exercise is an important characteristic of a
deficiency of carnitine acyl transferase because muscle relies on fatty acids as a long-term
source of energy. Medium-chain (C8-C10) fatty acids, which do not require carnitine to
enter the mitochondria, are oxidised normally in these patients. These diseases illustrate
that the impaired flow of a metabolite from one compartment of a cell to another can lead
to a pathological condition.
Causes of carnitine deficiency
Causes of carnitine deficiency include the following:

• Inadequate intake (e.g., due to fad diets, lack of access, or long-term TPN)
• Inability to metabolise carnitine due to enzyme deficiencies (e.g., carnitine
palmitoyl Transferase deficiency.
• Decreased endogenous synthesis of carnitine due to a severe liver disorder
• Excess loss of carnitine due to diarrhoea, diuresis, or hemodialysis
• A hereditary disorder in which carnitine leaks from renal tubules (Primary
carnitine deficiency)
• Increased requirements for carnitine when ketosis is present or demand for fat
oxidation is high (eg, during a critical illness such as sepsis or major burns; after
major surgery of the GI tract)
• Decreased muscle carnitine levels due to mitochondrial impairment
Primary Carnitine deficiency- The underlying defect involves the plasma membrane
sodium gradient–dependent carnitine transporter that is present in heart, muscle, and
kidney. This transporter is responsible both for maintaining intracellular carnitine
concentrations 20- to 50-fold higher than plasma concentrations and for renal conservation
of carnitine. Primary carnitine deficiency has an Autosomal recessive pattern of
inheritance. Mutations in the gene lead to the production of defective carnitine transporters.
As a result of reduced transport function, carnitine is lost from the body and cells are not
supplied with an adequate amount of carnitine.
Clinical manifestations of Carnitine deficiency
Symptoms and the age at which symptoms appear depend on the cause. Carnitine
deficiency may cause muscle necrosis, myoglobinuria, hypoglycemia, fatty liver, muscle
aches, fatigue, and cardiomyopathy.
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1) The most common presentation is progressive cardiomyopathy with or without skeletal
muscle weakness beginning at 2–4 yr of age.Energy deprived muscle cells are damaged.
2) A smaller number of patients may present with fasting hypoketotic hypoglycemia

during the 1st yr of life before the cardiomyopathy becomes symptomatic. Blockage of the
transport of long chain fatty acids into mitochondria deprives the patient of energy
production, as the fatty acid oxidation is impaired; all the energy needs are fulfilled by
glucose oxidation. The resultant imbalance between demand and supply causes
hypoglycemia. The compensatory ketosis in carnitine induced hypoglycemia is not
observed as the precursor, Acetyl co A is not available for ketone body production. The
main source of Acetyl co Is fatty acid oxidation and that is impaired in carnitine
deficiency.
3) Serious complications such as heart failure, liver problems, coma, and sudden
unexpected death are also a risk.
Deficiencies in the carnitine acyl Transferase enzymes I and II can cause similar symptoms.
Diagnosis
1) Diagnosis of the carnitine transporter defect is aided by the fact that patients have
extremely reduced carnitine levels in plasma and muscle (1–2% of normal).
2) Fasting ketogenesis may be normal if liver carnitine transport is normal, but it may be
impaired if dietary carnitine intake is interrupted.
3) Hypoglycemia is a common finding. It is precipitated by fasting and strenuous exercise.
4) Muscle biopsy reveals significant lipid vacuoles.
Treatment
Treatment of this disorder with pharmacological doses of oral carnitine is highly effective
in correcting the cardiomyopathy and muscle weakness as well as any impairment in
fasting ketogenesis. All patients must avoid fasting and strenuous exercise. Some patients
require supplementation with medium-chain triglycerides and essential fatty acids (eg,
Linoleic acid, Linolenic acid). Patients with a fatty acid oxidation disorder require a high-
carbohydrate, low-fat diet.
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CASE STUDY-CYSTINOSIS
A 2 –year-old child was brought to pediatrics department for consultation. Mother of

child reported that the child was born normally; there was no complication during
delivery, and her other two children were also normal. The child was not taking feeds
properly and was also not growing well.
There was history of increased frequency of urination from the previous few months
for which the mother was very much apprehensive. The child avoided going to sun
and there was apparent visual impairment.
On general physical examination, the child was found pale, thin and small for his age.
There was slight hepatomegaly and he looked slightly dehydrated.
A urine sample was sent for complete analysis. The urine examination revealed
presence of excessive amount of phosphates and amino acids.
A thorough examination of eye was also done to know the cause of visual impairment.
On slit lamp microscopic examination, characteristic crystals were found deposited in
the cornea.
What is the possible defect?

What is the biochemical basis for phosphate and amino acids in urine?
Case discussion- The child is suffering from Cystinosis. Cystinosis is a Lysosomal

storage disease characterized by the abnormal accumulation of the amino acid Cystine.
Excess cystine forms crystals that can build up and damage cells. These crystals negatively
affect many systems in the body, especially the kidneys and eyes. Excessive urination,
phosphaturia and aminoaciduria in the given patient are due to renal tubular damage while
the visual impairment is due to deposition of cystine crystals in cornea.
Cystinosis
Inheritance- Cystinosis is an autosomal recessive genetic disease.
Biochemical defect-
Endogenous protein enters the lysosome, where acid hydrolases degrade it to its component
amino acids, including cysteine. Within the lysosome, cysteine is readily oxidized to
cystine (a disulfide of the amino acid cysteine). In healthy individuals, both cystine and
cysteine can normally enter the cytoplasm, where cystine is rapidly converted to cysteine
by the reducing agent glutathione under the activity of oxidoreductase enzyme.
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Cytoplasmic cysteine is incorporated into protein or degraded to inorganic sulfate for
excretion.
Cystinosis is caused by one of several mutations in the gene that encodes cystinosin, the
cystine-lysosomal exporter. Because of the defect in cystinosin, cystine cannot leave the
lysosomes and is accumulated there as birefringent, hexagonal, or rectangular crystals
within cells of various organ systems.
Clinical Manifestations- There are three distinct types of cystinosis. In order of decreasing
severity, they are –
• Nephropathic Cystinosis (Infantile)
• Intermediate Cystinosis (Juvenile)
• Non-Nephropathic or ocular Cystinosis (Adult)
1) Nephropathic Cystinosis- In the infantile nephropathic form of cystinosis, the kidney is

affected early in life by cystine crystals deposited in proximal tubule cells. This leads
eventually to a Fanconi syndrome, characterized by wasting of substances reabsorbed in
this nephron segment, including sodium, potassium, phosphate, calcium, magnesium,
bicarbonate, and others. Metabolic acidosis and electrolyte disturbances ensue and
contribute to the stunting of growth in children with cystinosis. Cystinosis is the most
common inherited cause of Fanconi syndrome.
Patients usually present during the first year of life with polyuria, polydipsia, dehydration,
metabolic acidosis (normal anion gap hyperchloremic acidosis), hypophosphatemic rickets,
failure to thrive, and laboratory findings consistent with Fanconi’s syndrome. If untreated,
renal failure develops by age 7-10 years.
Cystine continues to accumulate in other tissues, resulting in such complications as eye

disease (eg, severe photophobia, corneal ulcerations, and retinal blindness), delayed
puberty, hypothyroidism, pancreatic disease (eg, exocrine insufficiency, insulin-dependent
diabetes mellitus), liver disease, swallowing difficulties, and CNS involvement
2) Intermediate Cystinosis-The signs and symptoms of intermediate cystinosis are the

same as Nephropathic Cystinosis, but they occur at a later age. Intermediate cystinosis
typically becomes apparent in affected individuals in adolescence. Malfunctioning kidneys
and corneal crystals are the main initial features of this disorder. If intermediate Cystinosis
is left untreated, complete kidney failure will occur, but usually not until the late teens to
mid-twenties.
3) Nonnephropathic Cystinosis is considered a benign variant and is usually diagnosed by

an ophthalmologist treating patients for photophobia. Photophobia may not begin until
middle age and is not usually as debilitating as in the nephropathic form of the disease. Slit-
lamp examination reveals corneal crystal deposits. In addition to the eye, cystine crystals
are present in the bone marrow and leukocytes but are absent in the kidney and the retina.
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Diagnosis-
1) Serum electrolyte measurements are used to detect the presence of acidosis

(hyperchloremic, normal anion gap) and severity of hypokalemia, hyponatremia,
hypophosphatemia, and low bicarbonate concentration in patients with cystinosis.
2) Blood gases may be used to detect metabolic acidosis and the degree of respiratory
compensation.
3) Urine testing reveals low osmolality, glucosuria, and tubular proteinuria (including
generalized amino aciduria).
4) Measurements of urine electrolytes serve to detect the loss of bicarbonate and

phosphaturia.
Definitive diagnosis and treatment monitoring are most often performed through
measurement of white blood cell cystine level.
Other Investigations
• Renal ultrasonography, Radiography for kidneys, ureters, and bladder (KUB) may
be needed to evaluate possible urinary tract calcifications.
• CT scanning and MRI are used to evaluate adult patients with infantile nephropathic
cystinosis who have CNS symptoms.
• Slit-lamp examination of the eyes reveals corneal and conjunctival cystine crystals
(pathognomonic for cystinosis) as early as age 1 year, although photophobia does
not usually become apparent until age 3-6 years.
• Examination of the eye fundi may reveal the presence of peripheral retinopathy. In
some patients, retinopathy may lead to blindness.
Treatment- Cystinosis is a common cause of the Fanconi Syndrome, a renal tubular

disease. By about one year of age, patients eliminate large volumes of urine and lose large
amounts of salt and other minerals in their urine.
Replacement of urinary losses: The child must be well-hydrated and administered

supplements of potassium and bicarbonate, as needed. Rickets should be treated with
vitamin D and phosphate supplementation.
Without specific treatment, these children progress to end-stage renal failure by an average
age of nine years. These patients can receive renal dialysis or transplantation, but even
with successful transplantations, they develop abnormalities in other organs.
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The drug cysteamine slows the progression of cystinosis by removing the cystine from
cells, but for the drug treatment to be effective, it must be taken every six hours. When
administered regularly, cysteamine decreases the amount of cystine stored in lysosomes and
correlates with conservation of renal function and improved growth. Cysteamine eye drops
remove the cystine crystals in the cornea that can cause photophobia if left unchecked.
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CASE STUDY- DIABETIC KETOCIDOSIS
A 22- year-old diabetic comes to the Accident and Emergency department. She gives a
2-day history of vomiting and abdominal pain. She is drowsy and her breathing is
deep and rapid. There is distinctive smell from her breath
What is the most likely diagnosis?
What is the biochemical basis for all the presenting symptoms?
Which laboratory test would you request?
Case details-The patient is most probably suffering from diabetic ketoacidosis. She is a
known diabetic and the presenting symptoms like abdominal pain, vomiting, rapid
breathing and distinctive smell of breath, all indicate associated ketoacidosis.
Basic concept- Diabetic Ketoacidosis (DKA) is a state of inadequate insulin levels

resulting in high blood sugar and accumulation of organic acids and ketones in the blood.
It is a potentially life-threatening complication in patients with diabetes mellitus. It happens
predominantly in type 1 diabetes mellitus, but it can also occur in type 2 diabetes mellitus
under certain circumstances.
Causes- DKA occurs most frequently in known Diabetics. It may also be the first
presentation in patients who had not been previously diagnosed as diabetics. There is often
a particular underlying problem that has led to DKA episode. This may be-
1) Inter current illness- such as Pneumonia, Influenza, Gastroenteritis, Urinary tract
infection or pregnancy.
2) Inadequate Insulin administration- may be due to defective insulin pen device or in
young patient intentional missing of dose due to fear of weight gain.
3) Associated myocardial infarction, stroke or use of cocaine
4) Inadequate food intake- may be due to anorexia associated with infective process or
due to eating disorder in children.
Diabetic ketoacidosis may occur in those previously known to have diabetes mellitus type 2
or in those who on further investigations turn out to have features of type 2 diabetes (e.g.
obesity, strong family history); this is more common in African, African-American and
Hispanic people. Their condition is then labelled "ketosis-prone type 2 diabetes".
Pathophysiology
DKA results from relative or absolute insulin deficiency combined with counter
regulatory hormone excess (Glucagon, Catecholamines, cortisol, and growth hormone).
The decreased ratio of insulin to Glucagon promotes Gluconeogenesis, glycogenolysis,
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and Ketone body formation in the liver, as well as increases in substrate delivery from
fat and muscle (free fatty acids, amino acids) to the liver.
a) Cause of hyperglycemia- Uncontrolled IDDM leads to increased hepatic glucose

output. First, liver glycogen stores are mobilized then hepatic gluconeogenesis is used to
produce glucose. Insulin deficiency also impairs non-hepatic tissue utilization of glucose.
In particular in adipose tissue and skeletal muscle, insulin stimulates glucose uptake. This is
accomplished by insulin-mediated movement of glucose transporter proteins to the plasma
membrane of these tissues. Reduced glucose uptake by peripheral tissues in turn leads to a
reduced rate of glucose metabolism. In addition, the level of hepatic Glucokinase is
regulated by insulin. Therefore, a reduced rate of glucose phosphorylation in hepatocytes
leads to increased delivery to the blood. Other enzymes involved in anabolic metabolism of
glucose are affected by insulin (primarily through covalent modifications). The
combination of increased hepatic glucose production and reduced peripheral tissues
metabolism leads to elevated plasma glucose levels.
b) Cause of ketosis- One major role of insulin is to stimulate the storage of food energy
following the consumption of a meal. This energy storage is in the form of glycogen in
hepatocytes and skeletal muscle. Additionally, insulin stimulates hepatocytes to synthesize
triglycerides and storage of triglycerides in adipose tissue. In opposition to increased
adipocyte storage of triglycerides is insulin-mediated inhibition of lipolysis. In uncontrolled
IDDM there is a rapid mobilization of triglycerides leading to increased levels of plasma
free fatty acids.
The free fatty acids are taken up by numerous tissues (however, not the brain) and
metabolized to provide energy. Free fatty acids are also taken up by the liver. Normally, the
levels of malonyl-CoA are high in the presence of insulin. These high levels of malonyl-
CoA inhibit carnitine palmitoyl Transferase I, the enzyme required for the transport of fatty
acyl-CoA's into the mitochondria where they are subject to oxidation for energy production.
Thus, in the absence of insulin, malonyl-CoA levels fall and transport of fatty acyl-CoA's
into the mitochondria increases. Mitochondrial oxidation of fatty acids generates acetyl-
CoA which can be further oxidized in the TCA cycle. However, in hepatocytes the majority
of the acetyl-CoA is not oxidized by the TCA cycle but is metabolized into the ketone
bodies, Acetoacetate and ȕ-hydroxybutyrate. TCA cycle is in a state of suppression due to
non availability of oxaloacetate which is channeled towards pathway of gluconeogenesis in
the absence of Insulin.
These ketone bodies leave the liver and are used for energy production by the brain, heart
and skeletal muscle. In IDDM, the increased availability of free fatty acids and ketone
bodies exacerbates the reduced utilization of glucose furthering the ensuing hyperglycemia.
Production of ketone bodies, in excess of the body’s ability to utilize them leads to
ketoacidosis. In diabetics, this can be easily diagnosed by smelling the breath. A
spontaneous breakdown product of Acetoacetate is acetone which is volatilized by the
lungs producing a distinctive odor.
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c) Causes of Acidosis and hyperventilation-The ketone bodies, however, have a low pH
and therefore turn the blood acidic (metabolic acidosis). The body initially buffers this with
the bicarbonate buffering system, but this is quickly overwhelmed and other mechanisms to
compensate for the acidosis, such as hyperventilation to lower the blood carbon dioxide
levels. This hyperventilation, in its extreme form, may be observed as Kussmaul
respiration. Ketones, too, participate in osmotic diuresis and lead to further electrolyte
losses. As a result of the above mechanisms, the average adult DKA patient has a total body
water shortage of about 6 liters (or 100 ml/kg), in addition to substantial shortages in
sodium, potassium, chloride, phosphate, magnesium and calcium. Glucose levels usually
exceed 13.8 mmol/l or 250 mg/dl.
Increased lactic acid production also contributes to the acidosis. The increased free fatty
acids increase triglyceride and VLDL production. VLDL clearance is also reduced because
the activity of insulin-sensitive lipoprotein lipase in muscle and fat is decreased. Most
commonly, DKA is precipitated by increased insulin requirements, as might occur during a
concurrent illness. Occasionally, complete omission of insulin by the patient with type 1
DM precipitates DKA.
Clinical manifestations- The symptoms of an episode of diabetic ketoacidosis usually

evolve over the period of about 24 hours. Predominant symptoms are nausea and
vomiting, pronounced thirst, excessive urine production and abdominal pain that may
be severe. Hyperglycemia is always present .In severe DKA, breathing becomes labored
and of a deep, gasping character (a state referred to as "Kussmaul respiration"). The
abdomen may be tender to the point that an acute abdomen may be suspected, such as
acute pancreatitis, appendicitis or gastrointestinal perforation.
Coffee ground vomiting (vomiting of altered blood) occurs in a minority of patients; this
tends to originate from erosions of the esophagus. In severe DKA, there may be confusion,
lethargy, stupor or even coma (a marked decrease in the level of consciousness).
On physical examination -there is usually clinical evidence of dehydration, such as a

dry mouth and decreased skin turgor. If the dehydration is profound enough to cause a
decrease in the circulating blood volume, tachycardia (a fast heart rate) and low blood
pressure may be observed. Often, a "ketotic" odor is present, which is often described as
"fruity". If Kussmaul respiration is present, this is reflected in an increased respiratory rate.
Small children with DKA are relatively prone to cerebral edema (swelling of the brain
tissue), which may cause headache, coma, loss of the pupillary light reflex, and progress to
death. It occurs in 0.7–1.0% of children with DKA, and has been described in young adults,
but is overall very rare in adults. It carries 20–50% mortality.
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Figure showing causes and consequences of DKA
Diagnosis
Investigations
Diabetic Ketoacidosis may be diagnosed when the combination of hyperglycemia (high

blood sugars), ketones on urinalysis and acidosis are demonstrated. Arterial blood gas
measurement is usually performed to demonstrate the acidosis; this requires taking a blood
sample from an artery. In addition to the above, blood samples are usually taken to measure
urea and creatinine (measures of kidney function, which may be impaired in DKA as a
result of dehydration) and electrolytes. Furthermore, markers of infection (complete
blood count, C-reactive protein) and acute pancreatitis (amylase and lipase) may be
measured. Given the need to exclude infection, chest radiography and urinalysis are
usually performed.
If cerebral edema is suspected because of confusion, recurrent vomiting or other symptoms,
computed tomography may be performed to assess its severity and to exclude other
causes such as stroke.
Management
The main aims in the treatment of diabetic ketoacidosis are replacing the lost fluids and
electrolytes while suppressing the high blood sugars and ketone production with insulin.
a) Fluid replacement- The amount of fluid depends on the estimated degree of

dehydration. If dehydration is so severe, rapid infusion of saline is recommended to restore
circulating volume.
b) Insulin is usually given continuously.

114
c) Potassium levels can fluctuate severely during the treatment of DKA, because insulin
decreases potassium levels in the blood by redistributing it into cells. Serum potassium
levels are initially often mildly raised even though total body potassium is depleted.
Hypokalemia often follows treatment. This increases the risk of irregularities in the heart
rate. Therefore, continuous observation of the heart rate is recommended, as well as
repeated measurement of the potassium levels and addition of potassium to the intravenous
fluids once levels fall below 5.3 mmol/l. If potassium levels fall below 3.3 mmol/l, insulin
administration may need to be interrupted to allow correction of the hypokalemia.
d) Bicarbonate-
Sodium bicarbonate solution is administered to rapidly improve the acid levels in the blood.
Cerebral edema- administration of fluids is slowed; intravenous Mannitol and hypertonic

saline (3%) are used.
Prognosis
With appropriate therapy, the mortality of DKA is low (<5%) and is related more to the
underlying or precipitating event, such as infection or myocardial infarction. The major
nonmetabolic complication of DKA therapy is cerebral edema, which most often
develops in children as DKA is resolving. The etiology of and optimal therapy for cerebral
edema are not well established, but over replacement of free water should be avoided. The
other known complications of DKA therapy are, Hypoglycemia, hypokalemia and
hypophosphatemia.
Venous thrombosis, upper gastrointestinal bleeding, and acute respiratory distress
syndrome occasionally complicate DKA.
Prevention of DKA
Following treatment, the physician and patient should review the sequence of events that
led to DKA to prevent future recurrences. Foremost is patient education about the
symptoms of DKA, its precipitating factors, and the management of diabetes during a
concurrent illness. During illness or when oral intake is compromised, patients should: (1)
frequently measure the capillary blood glucose; (2) measure urinary ketones when the
serum glucose > 16.5 mmol/L (300 mg/dL); (3) drink fluids to maintain hydration; (4)
continue or increase insulin; and (5) seek medical attention if dehydration, persistent
vomiting, or uncontrolled hyperglycemia develop. Using these strategies, early DKA can be
prevented or detected and treated appropriately on an outpatient basis.
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DKA IN PREGNANCY
DKA in pregnancy is of special concern. It tends to occur at lower plasma glucose levels
and more rapidly than in non-pregnant patients and usually occurs in the second and third
trimesters because of increasing insulin resistance. Fetal mortality rates have previously
been reported as high as 30% rising to over 60% in DKA with coma. However with
improvements in diabetic care the figure for fetal loss has been reported as low as 9% in
some countries. Prevention, early recognition and aggressive management are vitally
important to minimize fetal mortality.
Some Practice Questions-
A known diabetic patient was admitted in a semi comatose state. He had cellulitis of
the right foot. The laboratory result of a blood sample drawn at the time of admission
was:
Blood Glucose : 385 mg/dl

Blood Urea : 80 mg/dl
Serum Creatinine : 2 mg/dl
Serum Sodium : 134 mmol/l
Serum Potassium : 5.8 mmol/l
Serum Bicarbonate : 18 mmol/l
Serum Chloride : 98 mmol/l
Glycohemoglobin : 18%
Urine glucose : Positive
Urine Ketone bodies : Positive
(A) What is the most probable cause?
(B) What is the biochemical explanation for this condition?
Oral glucose tolerance test was performed with a 48 year old person. Interpret the
results given below:
Time (hrs) Blood glucose (mg/dl) Urine Sugar

(Benedict’s test) (Benedict’s test)
0 (fasting) 250 blue

0.5 340 red
1.0 450 red
1.5 435 red
2.0 400 red
Give your conclusion

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An young man had a complaint of arthritis. The laboratory findings are given below
1. Urine benedict’s test : POSITIVE

2. Fasting blood glucose : 90 mg/dl
3. Post-prandial blood glucose : 120 mg/dl
4. Serum Uric acid : 4 mg/dl
5. Urine, on standing turned to blackish colour
From the above biochemical tests, explain the defect and the possible diagnosis
One evening, a smart 40 year old male businessman entertained a party in which
much food and alcohol has been consumed. In the next early morning, he had fever,
his ankle joint was swollen, red, felt hot to touch, was very tender ad stiff. No other
joints were involved. Lymph glands were normal and non tender. The laboratory data
were:
Blood glucose : 130 mg/dl

Blood uric acid : 38 mg/dl
A boy aged 5 years was brought to the hospital with the complaints of swelling in the
abdomen and with a history of reeling sensation. On examination, liver was found to
be enlarged, there was increased uric acid and free fatty acid level associated with
hypoglycemia. No increase in blood glucose was found even after intravenous
administration of glucagons. What is the likely diagnosis of the case?
A forty five year old patient presented with the complaints of polyuria and polyphagia
to the district hospital. History revealed that he ad recurrent boils and skin infections
since last few months. He told that wounds take long time to heal.
His biochemical findings were as follows:
Fasting blood sugar : 260 mg/dl

Serum bicarbonate : 10 mEq/L
Plasma pH : 7.25
Urine for sugar (benedict’s test) : orange colour
Urine for ketone bodies : present
Give your comments on the findings and interpretation of the case.

A fifty year old man came to Manipal Teaching Hospital to consult the physician. He
complained of excess thirst and increased frequency and volume of urine.
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The physician asked him to get various laboratory investigations done.
The biochemical findings were as follows:
Fasting blood glucose : 390 mg/dl

Urine sugar (benedict’s test) : ++++ ( brick red colour)
A known diabetic patient was admitted in a semi comatose state. He had cellulitis of
the right foot.
The laboratory findings at the time of admission were as follows:

Blood urea : 83 mg/dl
Serum creatinine : 2 mg/dl
Serum Sodium : 134 mmol/L
Serum Potassium : 5.8 mmol/L
Serum bicarbonate : 18 mmol/L
Serum Chloride : 98 mmol/L
Glycated hemoglobin : 18%
Urine for sugar (benedict’s test) : brick red colour
Urine for ketone bodies : positive

Explain the clinical condition biochemically.
During a period of a textile mill strike, one of the employees was brought to the
hospital in an unconscious state.
The laboratory findings at the time of admission were as follows:
Blood pH : 7.25
Serum bicarbonate : 15 mmol/L
Urine for ketone bodies : positive
Give your conclusion and comments on the laboratory reports
118
A sixty years old diabetic patient who had been on insulin for the past 5 years, claimed
that she was following the dietary regimen strictly and had never missed the insulin
injection.
Results of the routine laboratory investigations done are given below:
Fasting blood glucose : 100 mg/dl (70-110 mg/dl)

Post-prandial blood glucose : 140 mg/dl (<140 mg/dl)
Blood urea : 25 mg/dl (15-40 mg/dl)
Glycated hemoglobin (HbA1c) : 12% (3-5%)
Urinary reducing sugar : Absent (Absent)
Urinary ketone bodies : Absent (Absent)
Interpret the laboratory results and diagnose the clinical case
A fifty years old man with long standing diabetes mellitus presented with fever,
pruritis, delirium and oliguria.
Investigations reports are given below:
Blood urea : 70 mg/dl (15-40 mg/dl)

Urea clearance : 40 ml/min
What could be the most probable diagnosis?

0 (fasting) 80 blue
0.5 130 blue
1.0 160 blue
1.5 142 blue
2.0 120 blue
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0 (fasting) 60 blue
0.5 100 blue
1.0 130 blue
1.5 100 blue
2.0 55 blue
The laboratory findings of a fourteen year old girl admitted to children’s hospital are
given below. Her mother stated that her daughter had been in good health until
approximately weels previously, when she developed a sore throat and moderate
fever. She subsequently lost appetite and began to complain of undue thirst.
On examination in the hospital, she was dehydrated and was having kussumaul
respiration.
The biochemical reports were as follows:
Plasma glucose : 630 mg/dl (70-100 mg/dl)

ȕ-OH butyrate : 13.0 mmol/L (<0.25 mmol/L)
Acetoacetate : 2.8 mmol/L (<0.2 mmol/L)
Serum bicarbonate : 5 mmol/L ( 20-25 mmol/L)
Blood urea nitrogen : 12 mmol/L (2.9 -8.9 mmol/L)
Blood pH : 7.05
Urinary sugar : ++++
Urinary ketone bodies : ++++

Explain the biochemical basis of the diagnosis.
An eighteen year old girl consulted her family physician because of tiredness and
weigh loss. On interrogation, she admitted of feeling thirsty and passing more urine
than normal. Next day her physician examined and found that she had deep, sighing
respiration (Kussmaul’s respiration) and her breath had fruity odor.
Blood sample was collected and sent to laboratory for investigations.
His biochemical findings were as follows:
120
Random plasma glucose : 411 mg/dl
Serum Urea : 60 mg/dl
Serum Creatinine : 1.7 mg/dl
Serum Sodium : 130 mmol/L
Serum potassium : 5.8 mmol/L
Urine for sugar (benedict’s test) : orange colour
Blood pressure : 95/60 mmHg
Pulse rate : 112/min
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CASE STUDY- DIET AND NUTRITION
A 35 –year-old woman became severely depressed after the sudden death of her husband.
Two months later, she was brought to emergency room by her friend because of extreme
weakness and lethargy. She appeared thin and pale. Questioning revealed that she had not
eaten for several weeks.
Analysis of a plasma sample indicated elevated levels of Alanine, Acetoacetate, ȕ hydroxy
butyrate, and blood urea nitrogen (BUN). However her plasma glucose concentration was
low (55mg/dL). She was hospitalized, given intravenous feeding, antidepressant
medications and subsequently shifted to an 1800 Cal (7500kJ) diet. Her recovery was
uneventful.
How was the patient obtaining energy during the time when she was not eating?
How could patient maintain her plasma glucose within normal limits even though she
was not eating?
What is the significance of elevated plasma Alanine level?
Why is BUN elevated?
What is indicated by the fact that the plasma Acetoacetate and ȕ- hydroxy butyrate
levels are elevated?
It is a case of starvation. The high blood Alanine level signifies the catabolic state. Alanine
in excess is released during starvation from muscle to serve as a substrate for glucose
production in liver. Acetoacetate and ȕ hydroxy butyrate are ketone bodies which are used
as alternative fuel during conditions of glucose deprivation. High BUN signifies protein
degradation; the carbon skeletons of amino acids are utilized for glucose production while
amino groups are converted to urea.
Starvation
Prolonged fasting may result from an inability to obtain food, from the desire to lose weight
rapidly, or in clinical situations in which an individual can not eat because of trauma,
surgery, neoplasms, burns etc or even in depression (As in the given case) . In the absence
of food the plasma levels of glucose, amino acids and triacylglycerols fall, triggering a
decline in insulin secretion and an increase in glucagon release. The decreased insulin to
glucagon ratio, and the decreased availability of circulating substrates, make this period of
nutritional deprivation a catabolic state, characterized by degradation of glycogen,
triacylglycerol and protein. This sets in to motion an exchange of substrates between liver,
adipose tissue, muscle and brain that is guided by two priorities (i) the need to maintain
glucose level to sustain the energy metabolism of brain ,red blood cells and other glucose
requiring cells and (ii) to supply energy to other tissues by mobilizing fatty acids from
adipose tissues and converting them to ketone bodies to supply energy to other cells of the
body.
Fuel Stores
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A typical well-nourished 70-kg man has fuel reserves totaling about 161,000 kcal (670,000
kJ). The energy need for a 24-hour period ranges from about 1600 kcal (6700 kJ) to 6000
kcal (25,000 kJ), depending on the extent of activity. Thus, stored fuels suffice to meet
caloric needs in starvation for 1 to 3 months. However, the carbohydrate reserves are
exhausted in only a day.
Energy supply during starvation
During starvation the energy needs are fulfilled by three types of fuels, glucose, fatty acids
and ketone bodies.
a) Glucose supply during starvation (Gluconeogenesis)
Energy needs of brain and RBCs
Even under conditions of starvation, the blood-glucose level has been maintained above 2.2
mM (40 mg/dl). The first priority of metabolism in starvation is to provide sufficient
glucose to the brain and other tissues (such as red blood cells) that are absolutely dependent
on this fuel. However, precursors of glucose are not abundant. Most energy is stored in the
fatty acyl moieties of triacylglycerols. Fatty acids cannot be converted into glucose,
because acetyl CoA cannot be transformed into pyruvate. The glycerol moiety of
triacylglycerol can be converted into glucose, but only a limited amount is available. The
only other potential source of glucose is amino acids derived from the breakdown of
proteins. However, proteins are not stored, and so any breakdown will necessitate a loss of
function.
Thus, the second priority of metabolism in starvation is to preserve protein, which is
accomplished by shifting the fuel being used from glucose to fatty acids and ketone bodies
by cells other than brain cells and the cells lacking mitochondria.
It is a biological compromise to provide glucose to these cells as a priority. During
prolonged starvation , when the gluconeogenic precursors are not available, proteins are
however broken down to use carbon skeleton of glucogenic amino acids for glucose
production.
b) Fatty acid oxidation
Energy need of liver

The low blood-sugar level leads to decreased secretion of insulin and increased secretion of
glucagon. Glucagon stimulates the mobilization of triacylglycerols in adipose tissue and
gluconeogenesis in the liver. The liver obtains energy for its own needs by oxidizing fatty
acids released from adipose tissue. The concentrations of acetyl CoA and citrate
consequently increase, which switch off glycolysis. Thus glucose utilization is stopped in
liver cells to preserve glucose for priority cells
Energy need of muscles
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The uptake of glucose by muscle is markedly diminished because of the low insulin level,
whereas fatty acids enter freely. Consequently, muscle shifts almost entirely from glucose
to fatty acids for fuel. The beta-oxidation of fatty acids by muscle halts the conversion of
pyruvate into acetyl CoA, because acetyl CoA stimulates the phosphorylation of the
pyruvate dehydrogenase complex, which renders it inactive. Most of the pyruvate is
transaminated to alanine, at the expense of amino acids arising from breakdown of "labile"
protein reserves synthesized in the fed state. The alanine, lactate and much of the keto-acids
resulting from this transamination are exported from muscle, and taken up by the liver,
where the alanine is transaminated to yield pyruvate. Pyruvate is a major substrate for
gluconeogenesis in the liver.
Figure- showing Glucose Alanine and Cori’s cycle
In adipose tissue the decrease in insulin and increase in glucagon results in activation of
intracellular hormone-sensitive lipase. This leads to release from adipose tissue of increased
amounts of glycerol (which is a substrate for gluconeogenesis in the liver) and free fatty
acids, which are used by liver, heart, and skeletal muscle as their preferred metabolic fuel,
therefore sparing glucose.
Loss of muscle mass

During starvation, degraded proteins are not replenished and serve as carbon sources for
glucose synthesis. Initial sources of protein are those that turn over rapidly, such as proteins
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of the intestinal epithelium and the secretions of the pancreas. Proteolysis of muscle protein
provides some of three-carbon precursors of glucose. The nitrogen part of the amino acids
is converted to urea (BUN)
c) Ketosis
Energy need of peripheral tissues
After about 3 days of starvation, the liver forms large amounts of acetoacetate and beta-
hydroxybutyrate. Their synthesis from acetyl CoA increases markedly because the citric
acid cycle is unable to oxidize all the acetyl units generated by the degradation of fatty
acids. Gluconeogenesis depletes the supply of oxaloacetate, which is essential for the entry
of acetyl CoA into the citric acid cycle. Consequently, the liver produces large quantities of
ketone bodies, which are released into the blood. At this time, the brain begins to consume
appreciable amounts of acetoacetate in place of glucose. After 3 days of starvation, about a
third of the energy needs of the brain are met by ketone bodies. The heart also uses ketone
bodies as fuel. After several weeks of starvation, ketone bodies become the major fuel of
the brain.
Figure- fatty acid oxidation and ketosis during starvation.

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In essence, ketone bodies are equivalents of fatty acids that can pass through the blood-
brain barrier. Only 40 g of glucose is then needed per day for the brain, compared with
about 120 g in the first day of starvation. The effective conversion of fatty acids into ketone
bodies by the liver and their use by the brain markedly diminishes the need for glucose.
Hence, less muscle is degraded than in the first days of starvation. The breakdown of 20 g
of muscle daily compared with 75 g early in starvation is most important for survival.
A person's survival time is mainly determined by the size of the triacylglycerol depot.
What happens after depletion of the triacylglycerol stores? The only source of fuel that
remains is proteins. Protein degradation accelerates, and death inevitably results from a loss
of heart, liver, or kidney function.
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CASE STUDY- GOUT
A 46-year-old male presented to the emergency department with severe right toe pain.
The patient was in usual state of health until early in the morning when he woke up
with severe pain in his right big toe. The patient denied any trauma to the toe and no
previous history of such pain in other joints. He did say that he had a “few too many”
beers with the guys last night. Patient's past medical history was significant for
hypertension, diabetes mellitus, chronic Alcoholism and renal stones for which he
underwent left nephrectomy about 25 years ago. Family history was non-
contributory.
On examination, he was found to have a temperature of 38.2°C (100.8°F) and in
moderate distress secondary to the pain in his right toe. The right big toe was swollen,
warm, red, and exquisitely tender. The remainder of the examination was normal.
Synovial fluid was obtained and revealed rod- or needle-shaped crystals that were
negatively birefringent under polarizing microscope. The laboratory investigation
report revealed;
Hemoglobin - 8.9gm/dl,
ESR -124 mm at the end of first hour,
Leucocyte count -7400/cmm with normal differential count
Random blood sugar-139 mg/dl
Creatinine- 1.6 mg/dl
Serum uric acid level- 10.9 mg/dl
His 24 hour urinary uric acid excretion was 446 mg/dl.
Serum calcium, phosphorus, LFT, electrolytes and lipid profile were normal.
How would you make a definite diagnosis?
What is the Pathophysiology of this disorder?
Case Details
The patient is suffering from Gouty Arthritis. The patient reported with pain in the big toe.
Pain in the big toe precipitated by alcohol is very typical of history of gout. The patient had
past history of alcoholism and renal stones. High serum urate levels and synovial fluid
analysis are diagnostic of gout.
Gout is a metabolic disease most often affecting middle-aged to elderly men and
postmenopausal women. It is the result of an increased body pool of urate with
hyperuricemia. It is typically characterized by episodic acute and chronic arthritis, due to
deposition of Mono Sodium Urate crystals in joints and connective tissues with the risk for
deposition in kidney interstitium or uric acid nephrolithiasis.
Acute arthritis is initially monarticular and often involves the first metatarsophalangeal
joint. Symptoms include acute pain, tenderness, warmth, redness, and swelling.
Diagnosis requires identification of crystals in synovial fluid. Treatment of acute attacks is
with anti-inflammatory drugs. The frequency of attacks can be reduced by regular use of
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NSAIDs, colchicine, or both and by treating hyperuricemia with Allopurinol or uricosuric
drugs.
Gout
Overview of Uric Acid Metabolism
Uric acid is the final breakdown product of purine degradation in humans.

Purine bases are used in many important biological processes including the formation
of nucleic acids (ribonucleic acid [RNA] and deoxyribonucleic acid [DNA]), energy
currency (adenosine triphosphate [ATP]), cofactors (nicotinamide adenine dinucleotide
[NAD], flavin adenine dinucleotide [FAD]), and cellular signalling (cAMP and cGMP).
Purines are both synthesized de novo and taken in through the diet. Their degradation is a
ubiquitous process; however, increased levels of the enzymes that carry out the metabolism
of purine bases suggest that purine catabolism is higher in the liver and the gastrointestinal
tract. Abnormalities in purine biosynthesis and degradation are associated with numerous
disorders suggesting that the regulation of purine levels is essential.
Degradation of purine nucleotides, nucleosides and bases follow a common pathway.
During purine catabolism, the purine nucleotides
128
Figure showing purine nucleotide catabolism.
Adenosine monophosphate (AMP) and GMP are generated from the dephosphorylation of
ATP and GTP, respectively. AMP is then deaminated to IMP by AMP deaminase.
Subsequently, GMP and IMP are dephosphorylated by specific 5’Nucleotidase to produce
the nucleosides guanosine and Inosine respectively.
129
Alternatively, AMP can be dephosphorylated to form adenosine, which is then deaminated
by adenosine deaminase (ADA) to form Inosine. Inosine and guanosine are further broken
down by the cleavage of the purine base from the ribose sugar to yield ribose 1-phosphate
and hypoxanthine and guanine, respectively. Similar reactions are carried out for the
degradation of purine deoxy Ribonucleotides and deoxyribonucleoside. Guanine is
deaminated to form Xanthine, whereas hypoxanthine is oxidized to form Xanthine by the
enzyme Xanthine oxidase. Xanthine is further oxidized, again by Xanthine oxidase, to form
uric acid, which is excreted in the urine.
Uric acid has a pKa of 5.4 and is in the ionized urate form at physiologic pH. Urate is not
very soluble in an aqueous environment and the concentration of urate in human blood is
very close to saturation. Therefore, conditions that lead to excessive degradation of purine
bases can lead to the formation of urate crystals.
Hyperuricemia
Hyperuricemia can result from increased production or decreased excretion of uric acid or
from a combination of the two processes. Sustained hyperuricemia predisposes some
individuals to develop clinical manifestations including gouty arthritis, urolithiasis, and
renal dysfunction.
Hyperuricemia is defined as a plasma (or serum) urate concentration >408 mol/L (6.8
mg/dL). The risk of developing gouty arthritis or urolithiasis increases with higher urate
levels and escalates in proportion to the degree of elevation.
Incidence
Hyperuricemia is present in between 2.0 and 13.2% of ambulatory adults and is even more
frequent in hospitalized individuals.
Causes of Hyperuricemia
Hyperuricemia may be classified as primary or secondary depending on whether the

cause is innate or is the result of an acquired disorder. However, it is more useful to classify
hyperuricemia in relation to the underlying pathophysiology, i.e., whether it results from
increased production, decreased excretion, or a combination of the two.
A) Increased Urate Production-
The common causes are as follows-
1) Diet contributes to the serum urate in proportion to its purine content. Foods high in
nucleic acid content include liver, "sweetbreads" (i.e., thymus and pancreas), kidney, and
anchovy. Excessive consumption leads to hyperuricemia.
130
2) Endogenous sources of purine production also influence the serum urate level. De
novo purine biosynthesis is an 11-step process that forms Inosine monophosphate (IMP).
The rates of purine biosynthesis and urate production are determined, for the most part, by
amidotransferase, which combines phosphoribosylpyrophosphate (PRPP) and glutamine.
Metabolic abnormalities that lead to the overproduction of purine nucleotides through the
de novo pathway lead to increased purine degradation and subsequent hyperuricemia. An
example of this is an increase in the activity of 5-phosphoribosyl-1-pyrophosphate
(PRPP) synthetase. This enzyme is responsible for the production of PRPP, which is an
important precursor of both purine and pyrimidine de novo biosynthesis. Elevations in
PRPP lead to increased purine nucleotide production that can in turn increase the rate of
degradation and hence increased uric acid production. Similarly increased activity of
amidotransferase also leads to similar effects of increased uric acid production.
3) Hyperuricemia can also result from defects in the purine salvage pathway. The
enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is responsible for
reforming IMP and GMP from hypoxanthine and guanine, respectively. In this manner
purine bases are salvaged back into the purine nucleotide pool. Lesch-Nyhan syndrome
results from an inherited deficiency in HGPRT. This syndrome is associated with mental
retardation and self-destructive behavior, which may be associated with inadequate
production of purine nucleotides through the salvage pathway in certain neuronal cells. In
addition, Lesch-Nyhan patients have gout resulting from the inability to salvage purine
bases, which leads to increased levels of uric acid.
4) Over production of urate may also be due to glucose-6-phosphatase deficiency (von

Gierke disease) , In von Gierke disease its both overproduction and decreased excretion
responsible for hyperuricemia. Overproduction is due to over active HMP pathway to
utilize the excess load of glucose-6 phosphate and decreased excretion is due to lactate
accumulation as a result of anaerobic Glycolysis in muscles. Lactate is excreted while uric
acid reabsorbed through anion exchange transporters.
5) Accelerated purine nucleotide degradation can also cause hyperuricemia, i.e., with
conditions of rapid cell turnover, proliferation, or cell death, as in leukemia, Cytotoxic
therapy for malignancy, hemolysis, or rhabdomyolysis.
6) Overproduction of uric acid may also occur, hemolytic anemias, pernicious anemia,
ineffective erythropoiesis (as in B-12 deficiency) and obesity.
B) Decreased Uric Acid Excretion
Over 90% of individuals with sustained hyperuricemia have a defect in the renal handling
of uric acid.
Common causes of secondary gout due to under excretion of uric acid include-
131
• Primary idiopathic
• Renal insufficiency
• Polycystic kidney disease
• Diabetes insipidus
• Hypertension
• Acidosis
• Lactic acidosis
• Diabetic ketoacidosis
• Starvation ketosis
• Lead intoxication
• Hyperthyroidism
• Hypothyroidism
• Toxemia of pregnancy
• Down syndrome
• Low dose salicylates
• Diuretic
• Alcohol
• Nicotinic acid
C) Combined Mechanism
• Alcohol
• Glucose- 6 phosphatase deficiency
• Shock
Alcohol promotes hyperuricemia because of increased urate production and

decreased uric acid excretion. Excessive alcohol consumption accelerates hepatic
breakdown of ATP to increase urate production. Alcohol consumption can also induce
hyperlacticacidemia, which blocks uric acid secretion. The higher purine content in some
alcoholic beverages such as beer may also be a factor.
Decreased renal excretion is by far the most common cause of hyperuricemia.
Pathophysiology
Urate precipitates as needle-shaped monosodium urate (MSU) crystals, which are deposited
extracellularly in cartilage tendons, tendon sheaths, ligaments, walls of bursae and skin
around cooler distal joints and tissues (eg, ears). In severe, long-standing hyperuricemia,
MSU crystals may be deposited in larger central joints and in the parenchyma of organs
such as the kidney. At the acid pH of urine, urate precipitates readily as small plate like or
irregular crystals that may aggregate to form gravel or stones, which may cause obstruction.
Tophi are MSU crystal aggregates that most often develop in joint and cutaneous
tissue.
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Symptoms and Signs
Acute gouty arthritis usually begins with sudden onset of pain (often nocturnal). The
metatarsophalangeal joint of a great toe is most often involved, but the ankle, knee, wrist,
and elbow are also common sites. Rarely, the hip, shoulder, sacroiliac, sternoclavicular, or
cervical spine joints are involved. The pain becomes progressively more severe, usually
over a few hours, and is often excruciating. Swelling, warmth, redness, and exquisite
tenderness may suggest infection. The overlying skin may become tense, warm, shiny,
and red or purplish. Fever, tachycardia, chills, and malaise sometimes occur. Coexisting
hypertension, hyperlipidemia, and obesity are common.
Gout in 1st Metatarsophalangeal Joint
Course: The first few attacks usually affect only a single joint and last only a few days.
Later attacks may affect several joints simultaneously or sequentially and persist up to 3 wk
if untreated. Subsequent attacks develop after progressively shorter symptom-free intervals.
Eventually, several attacks may occur each year.
Tophi: They are usually firm yellow or white papules or nodules, single or multiple. They
can develop in various locations, commonly the fingers, hands, feet, and around the
olecranon or Achilles tendon. Tophi can also develop in the kidney and other organs and
under the skin on the ears. Tophi may even erupt through the skin, discharging chalky
masses of urate crystals. Tophi may eventually cause deformities.
Figure showing Tophi
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Chronic gout: Chronic gouty arthritis can cause pain, deformity, and limited joint motion.
Inflammation can be flaring in some joints while subsiding in others. About 20% of
patients with gout develop urolithiasis with uric acid stones or Ca oxalate stonese.
Untreated progressive renal dysfunction, most often related to coexisting hypertension or,
less often, some other cause of nephropathy, further impairs excretion of urate, accelerating
crystal deposition in tissues.
Cardiovascular disease and the metabolic syndrome are common among patients with gout.
i) Diagnosis of Acute gouty arthritis
• Clinical criteria
• Synovial fluid analysis
• Serum uric acid level
Gout should be suspected in patients with acute single joint involvement, particularly older
adults or those with other risk factors.
Synovial fluid analysis: Synovial fluid analysis can confirm the diagnosis by identifying
needle-shaped, strongly negatively birefringent urate crystals that are free in the fluid or
engulfed by phagocytes.
Serum urate level: An elevated serum urate level supports the diagnosis of gout but is
neither specific nor sensitive; at least 30% of patients have normal serum urate at the time
of an acute attack. However, the serum urate level reflects the size of the extracellular
miscible urate pool. The level should be measured on 2 or 3 occasions in patients with
newly proven gout to establish a baseline; if elevated (> 7 mg/dL [> 0.41 mmol/L]), 24-h
urinary urate excretion can also be measured. Normal 24-h excretion is about 600 to 900
mg on a regular diet. Quantification of urinary uric acid can indicate whether hyperuricemia
results from impaired excretion or increased production and help guide any serum urate–
lowering therapy. Patients with elevated urine excretion of urate are at increased risk of
urolithiasis.
X-rays: X-rays of the affected joint may be taken to look for bony tophi but are probably
unnecessary if the diagnosis has been established by synovial fluid analysis.
ii) Diagnosis of chronic gouty arthritis: Chronic gouty arthritis should be suspected in
patients with persistent joint disease or subcutaneous or bony tophi. Plain x-rays of the first
metatarsophalangeal joint or other affected joint may be useful. Bony lesions are not
specific or diagnostic but nearly always precede the appearance of subcutaneous tophi.
Prognosis
With early diagnosis, therapy enables most patients to live a normal life. Gout is generally
more severe in patients whose initial symptoms appear before age 30.
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Treatment
• Termination of an acute attack with NSAIDs(Non steroidal anti inflammatory

drugs) or corticosteroids
• Prevention of recurrent acute attacks with daily colchicine or an NSAID
• Prevention of further deposition of MSU crystals and resolution of existing tophi by
lowering the serum urate level
• Treatment of coexisting hypertension, hyperlipidemia, and obesity.
Lowering the serum urate level:
Neither colchicine, NSAIDs, nor corticosteroids, retard the progressive joint damage
caused by tophi. Such damage can be prevented and, if present, reversed with urate-
lowering drugs. Tophaceous deposits are resorbed by lowering serum urate. Lowering
serum urate may also decrease the frequency of acute arthritic attacks. This decrease is
accomplished by
• Blocking urate production with allopurinol

• Increasing urate excretion with a uricosuric drug
• Using both types of drugs together in severe tophaceous gout
Uricase can also be used but not yet routinely. Uricase is an enzyme that converts urate to
allantoin, which is more soluble.
Other treatments:
• Fluid intake 3 L/ day is desirable for all patients, especially those who chronically
pass urate gravel or stones.
• Alkalinization of urine (with K citrate, or acetazolamide) is also occasionally effective
for those with persistent uric acid urolithiasis despite Hypouricemic therapy and
adequate hydration.
• Extracorporeal shock wave lithotripsy may be needed to disintegrate renal stones.
• Large tophi in areas with healthy skin may be removed surgically.
• Dietary restriction of purines is less effective, but high intake of high-purine food and
alcohol (beer in particular) should be avoided.
• Carbohydrate restriction and weight loss can lower serum urate in patients with
insulin resistance because high insulin levels suppress urate excretion.
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CASE STUDY- HYPERCALCEMIA
A 35 -year -old female reported to emergency with severe pain in the left flank region,
which was radiating towards lower leg and back. The patient was in acute distress and
agony. History revealed that she frequently suffered from urinary tract infections and had
several such episodes of pain. She further reported that she constantly felt weakness,
fatigue and bone pains from the previous few months. There was no history of fever and
there was no personal or family history of medical problems.
Her physical examination was normal except for tenderness in the left renal region.
The attending physician ordered for complete blood count, electrolytes and a complete
urine analysis.
The laboratory investigation report revealed a normal complete blood count (CBC), and
significantly elevated calcium level and low phosphorus level. Urine was cloudy and had
plenty of pus cells. The patient was admitted and treated for renal colic.
What is the underlying cause for repeated episodes of renal colic?
What is the most likely diagnosis?
What is the relationship of bone pains and frequent urinary tract infections in this
patient?
What is the cause for high serum calcium and low phosphorus level in this patient?
Case details- Hypercalcemia, hypophosphatemia, recurrent urinary tract infections, renal

stones and bone pains all signify underlying hyperparathyroidism. (Cloudy urine and pus
cells are indicative of urinary tract infection).
Hyperparathyroidism is over activity of the parathyroid glands resulting in excess

production of parathyroid hormone (PTH). The parathyroid hormone regulates calcium and
phosphate levels. Hyperparathyroidism is classified in three categories-
1) Primary hyperparathyroidism- Primary hyperparathyroidism results from a hyper

function of the parathyroid glands themselves. There is over secretion of PTH due to
adenoma, hyperplasia or, rarely, carcinoma of the parathyroid glands.
2) Secondary hyperparathyroidism-Secondary hyperparathyroidism is the reaction of the

parathyroid glands to a hypocalcaemia caused by something other than a parathyroid
pathology, e.g. chronic renal failure or vitamin D deficiency.
3)Tertiary hyperparathyroidism- Tertiary hyperparathyroidism results from hyperplasia

of the parathyroid glands and a loss of response to serum calcium levels. In cases of long-
standing secondary hyperparathyroidism, the hypertrophied parathyroid glands can become
autonomously functioning and continue to secrete PTH independent of whether the original
stimuli to secrete PTH are still present.
In all cases, the raised PTH levels are harmful to bone, and treatment is often needed.
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Serum calcium- In cases of primary hyperparathyroidism or tertiary hyperparathyroidism
heightened PTH leads to increased serum calcium (Hypercalcemia) due to:
1. increased bone resorption, allowing flow of calcium from bone to blood

2. reduced renal clearance of calcium
3. increased intestinal calcium absorption
By contrast, in secondary hyperparathyroidism effectiveness of PTH is reduced.
Serum phosphate
In primary hyperparathyroidism, serum phosphate levels are abnormally low as a result of

decreased renal tubular phosphate reabsorption. However, this is only present in about 50%
of cases. This contrasts with secondary hyperparathyroidism, in which serum phosphate
levels are generally elevated because of renal disease.
Manifestations of hyperparathyroidism involve primarily the kidneys and the skeletal

system. Kidney involvement is due to either deposition of calcium in the renal parenchyma
or to recurrent nephrolithiasis. Renal stones are usually composed of either calcium oxalate
or calcium phosphate. In occasional patients, repeated episodes of nephrolithiasis or the
formation of large calculi may lead to urinary tract obstruction, infection, and loss of renal
function. Nephrocalcinosis may also cause decreased renal function and phosphate
retention.
There are great variations in the manifestations. Patients may present with multiple signs
and symptoms, including recurrent nephrolithiasis, peptic ulcers, mental changes, and, less
frequently, extensive bone resorption.
Treatment and monitoring- Treatment depends upon the severity and cause of the
condition. If there is mildly increased calcium levels due to primary hyperparathyroidism
and no symptoms, just regular check ups are needed. If symptoms are present or calcium
level is very high, surgery may be needed to remove the parathyroid gland that is
overproducing the hormone. Treatment of secondary hyperparathyroidism depends on the
underlying cause.Vitamin D and Phosphorus supplementations can also be done.
Calcimimetics
A Calcimimetics (cinacalcet) is a new type of drug for people with primary and secondary
hyperparathyroidism on dialysis. It mimics the effect of calcium in tissues. This reduces
PTH release from parathyroid glands, leading to lower calcium and phosphorus levels in
blood.
Surgery for hyperparathyroidism may lead to low blood calcium levels, which causes
tingling and muscle twitching. This requires immediate treatment.
137
CASE STUDY- IMPAIRED LIVER FUNCTION TESTS
Case Study
A 28-year-old man with a long history of intravenous drug abuse and chronic hepatitis B
presented with jaundice. Physical examination revealed an anemic, malnourished man with
dependent pitting edema and ascites. He had the following profile-
A) Blood biochemistry-:
1) Total protein 8.2 g./dL
2) Albumin 2.6 g/dl
3) Globulins 5.6 g/dL
4) Calcium 6.8 mg/dL,
5) BUN 6 mg/dL
6) Creatinine 0.9 mg/dL
7) Total Bilirubin 6.0 mg/dL
8) AST (Aspartate amino transferase) 200 U/L
9) ALT (Alanine amino transferase) 350 U/L
10) ALP(Alkaline Phosphatase) 180 U/L
11) LDH (Lactate Dehydrogenase) 300 U/L
12) CBC: macrocytic anemia with hypersegmented neutrophils, mild neutropenia, and
mild thrombocytopenia
13) Prothrombin time (PT) was prolonged and did not correct with intramuscular vitamin
K administration.
B) Urinalysis: Positive for Bilirubin
I. What is the clinical significance of his abnormal liver function tests,

hypoalbuminemia, and prolonged prothrombin time that does not correct
with intramuscular vitamin K?
II. What is the clinical significance of hypocalcemia in this patient?
III. Why does he have a low serum BUN?
IV. What is the most likely cause of the macrocytic anemia?
Case discussion
Cause for Abnormal liver function tests
The patient is most probably suffering from chronic end-stage liver disease secondary, most
likely, to chronic hepatitis B.
138
1) Transaminases
The transaminases are moderately elevated, and ALT is higher than AST. In acute
hepatitis, the transaminases would be higher owing to the presence of more liver tissue;
however, in chronic hepatitis, the loss of hepatocytes and replacement by fibrosis results in
less of an increase.
2) Hypoalbuminemia and a prolonged prothrombin time (PT) are markers of
increased severity of liver disease, since both depend on the ability of the liver to synthesize
proteins (albumin and coagulation factors). Inability to correct the PT with vitamin K
signifies that the liver is unable to synthesize the precursor vitamin K-dependent factors II,
VII, IX, and X for (Ȗ-carboxylation by vitamin K into functional coagulation factors). So
the synthetic functions of liver are impaired.
3) Hypocalcemia- The total serum calcium represents the calcium that is bound to
albumin (40%), other anions (13%), and free (ionized calcium; 47%). Low ionized calcium
may result in tetany. Therefore, in the presence of hypoalbuminemia, the most common
cause of hypocalcemia, the total calcium is decreased while the ionized calcium is normal,
so tetany is not present. Hypocalcaemia is partly due to hypoalbuminemia and to
hypovitaminosis D secondary to his liver disease. The liver is responsible for the first
hydroxylation step in vitamin D metabolism after its reabsorption from the small bowel.
Hypocalcemia is a stimulus for secondary hyperparathyroidism, which is likely, to be
present in this patient as well.
4) Low BUN level-owing to location of the urea cycle in the liver, the presence of liver
disease seriously hampers the normal function of disposing of ammonia in the urea cycle.
Hence, in chronic liver disease, the serum BUN is low while the ammonia level is
increased.
5) Macrocytic anemia-The macrocytic anemia with hypersegmented neutrophils in this
patient is more likely secondary to folate rather than to B12 deficiency owing to the 3- to 4-
month supply of folate in the liver versus the 6- to 9-year supply of B12. This is easily
verified by measurement of serum folate, RBC folate, and B12.
6) Elevated LDH-The elevated serum LDH is most likely secondary to destruction of the
macrocytic RBCs (which contain increased LDH1 isoenzyme) in the bone marrow by
macrophages (ineffective erythropoiesis).
139
CASE STUDY- METABOLISM OF CARBOHYDRATE
A 12- year-old girl who had a grossly enlarged abdomen reported to OPD . She had a
history of frequent episodes of weakness, sweating and pallor that were eliminated by
eating. Her development had been slow; she sat at the age of 1 year, walked unassisted
at the age of 2 years, and was doing poorly in the school.
Physical examination revealed normal blood pressure, temperature and a normal

pulse rate but a sub normal weight (23 Kg).The liver was enlarged, firm and was
descended in to pelvis. The spleen was not palpable, nor was the kidneys. The
remainder of the physical examination was within the normal limits.
Laboratory investigation report revealed, low blood glucose, low p H, high lactate,
triglycerides, ketones and high free fatty acids. The liver biopsy revealed high
glycogen content. Hepatic glycogen structure was normal. The enzyme assay
performed on the biopsy tissue revealed very low glucose-6- phosphatase levels.

What is the possible treatment for this patient?
Case details
The girl is suffering from Von –Gierke’s disease. The clinical picture, biochemical
findings, hypoglycemia and increased Hepatic Glycogen stores are all characteristic of
Von –Gierke’s disease.
Von –Gierke’s disease
Glycogen storage disease (GSD) type I, is also known as Von Gierke’s disease or
hepatorenal Glycogenesis. Von Gierke described the first patient with GSD type I in 1929.
Basic concept- Glycogen is a readily mobilised storage form of glucose. It is a very large,
branched polymer of glucose residues that can be broken down to yield glucose molecules
when energy is needed. Most of the glucose residues in glycogen are linked by Į-1, 4-
glycosidic bonds. Branches at about every tenth residue are created by Į-1, 6-glycosidic
bonds.
140
Figure-1- showing structure of Glycogen
Glycogen is not as reduced as fatty acids are and consequently not as energy rich.
Why do animals store any energy as glycogen? Why not convert all excess fuel into fatty
acids?
Glycogen is an important fuel reserve for several reasons-

1) The controlled breakdown of glycogen and release of glucose increase the amount of
glucose that is available between meals. Hence, glycogen serves as a buffer to maintain
blood-glucose levels.
2) Glycogen's role in maintaining blood-glucose levels is especially important because
glucose is virtually the only fuel used by the brain, except during prolonged starvation.
3) Moreover, the glucose from glycogen is readily mobilised and is therefore a good
source of energy for sudden, strenuous activity.
4) Unlike fatty acids, the released glucose can provide energy in the absence of oxygen
and can thus supply energy for anaerobic activity.
The two major sites of glycogen storage are the liver and skeletal muscle. The
concentration of glycogen is higher in the liver than in muscle (10% versus 2% by weight),
but more glycogen is stored in skeletal muscle overall because of its much greater mass.
Glycogen is present in the cytosol in the form of granules ranging in-
Figure- showing glycogen granules
141
-diameter from 10 to 40 nm .In the liver, glycogen synthesis and degradation are regulated
to maintain blood-glucose levels as required to meet the needs of the organism as a whole.
In contrast, in muscle, these processes are regulated to meet the energy needs of the muscle
itself.
An Overview of Glycogen Metabolism
Glycogen degradation and synthesis are relatively simple biochemical processes. Glycogen
degradation consists of three steps:
(1) The release of glucose 1-phosphate from glycogen,
(2) The remodelling of the glycogen substrate to permit further degradation, and
(3) The conversion of glucose 1-phosphate into glucose 6-phosphate for further
metabolism. (See Figure-2)
The glucose 6-phosphate derived from the breakdown of glycogen has three fates –
(a) It is the initial substrate for Glycolysis,
(b) It can be processed by the pentose phosphate pathway to yield NADPH and ribose
derivatives; and
(c) It can be converted into free glucose for release into the bloodstream.
This conversion takes place mainly in the liver and to a lesser extent in the intestines and
kidneys.
142
Figure – Showing glycogen degradation and the fate of glucose-6- phosphate.
Glycogen synthesis -requires an activated form of glucose, uridine diphosphate glucose

(UDP-glucose), which is formed by the reaction of UTP and glucose 1-phosphate. UDP-
glucose is added to the nonreducing end of glycogen molecules. Branching takes place after
the addition of at least 12 glucose residues. As is the case for glycogen degradation, the
glycogen molecule must be remodeled for continued synthesis.
The regulation of these processes is quite complex. Several enzymes taking part in
glycogen metabolism allosterically respond to metabolites that signal the energy needs of
the cell. These allosteric responses allow the adjustment of enzyme activity to meet the
needs of the cell in which the enzymes are expressed. Glycogen metabolism is also
regulated by hormonally stimulated cascades that lead to the reversible phosphorylation of
enzymes, which alters their kinetic properties. Regulation by hormones allows glycogen
metabolism to adjust to the needs of the entire organism. By both these mechanisms,
glycogen degradation is integrated with glycogen synthesis.
143
Figure – showing an overview of glycogen metabolism
Pathophysiology of Von –Gierke’s disease
Because of insufficient G6Pase activity, G6P cannot be converted into free glucose, but
G6P is metabolised to lactic acid or incorporated into glycogen. In this way, large quantities
of glycogen are formed and stored as molecules with normal structure in the cytoplasm of
hepatocytes and renal and intestinal mucosa cells; therefore, enlarged liver and kidneys
dominate the clinical presentation of the disease.
The chief biochemical alteration is hypoglycemia, while secondary abnormalities are

hyperlactatemia, metabolic acidosis, hyperlipidemia, and hyperuricemia.
144
Hypoglycemia- The deficiency of G6Pase blocks the process of glycogen degradation and
gluconeogenesis in the liver, preventing the production of free glucose molecules. As a
consequence, patients with GSD type I have fasting hypoglycemia. Hypoglycemia inhibits
insulin secretion and stimulates glucagon and cortisol release.
Hyperlactatemia and acidosis- Undegraded G6P is metabolised to lactate, which is used

in the brain as an alternative source of energy. The elevated blood lactate levels cause
metabolic acidosis.
Hyperuricemia- Blood uric acid levels are raised because of the increased endogenous
production and reduced urinary elimination caused by competition with the elevated
concentrations of lactate, which should be excreted.
Hyperlipidemia- Elevated endogenous triglyceride synthesis and diminished lipolysis

causes hyperlipidemia. Triglycerides increase the risk of fatty liver infiltration, which
contributes to the enormous amount of liver enlargement. Despite significantly elevated
serum triglyceride levels in patients, vascular lesions and atherosclerosis are rare
complications.
Incidence
Patients with GSD type I account for 24.6% of all patients with GSD.
Inheritance
Type I glycogen storage disease is an autosomal recessive disorder .As with other
genetically determined diseases, GSD type 1develops during conception, yet the first signs
of the disease may appear at birth or later.
• The earliest signs of the disease may develop shortly after birth and are caused by
hypoglycemia and lactic acidosis.
• Convulsions are a leading sign of disease.
• Frequently, symptoms of moderate hypoglycemia, such as irritability, pallor,
cyanosis, hypotonia, tremors, loss of consciousness, and apnoea, are present.
• A leading sign of GSD type I is enlargement of the liver and kidneys. During the
first weeks of life, the liver is normal size. It enlarges gradually thereafter, and in
some patients, it even reaches the pubic symphysis. Enlargement of the abdomen
due to hepatomegaly can be the first sign noted by the patient's mother.
145
• The patient's face is characteristically reminiscent of a doll's face (rounded cheeks
due to fat deposition).
• Mental development proceeds normally.
• Growth is retarded and children affected with GSD type I never gain the height
otherwise expected from the genetically determined potential of their families. The
patient's height is usually below the third percentile for their age. The onset of
puberty is delayed.
• Late complications of disease are renal function disturbance, renal stones, tubular
defects, and hypertension, mainly in patients older than 20 years. Renal function
deterioration progresses to terminal insufficiency, requiring dialysis and
transplantation.
• Skin and mucous membrane changes include the following:
Eruptive xanthomas develop on the extensor surfaces of the extremities.

Tophi or gouty arthritis may occur. Uric tophi often have the same distribution as
xanthomas.
Many patients bleed easily, particularly from the nose. This tendency is a result of
altered platelet function due to the platelets' lower adhesiveness. Frequent and,
occasionally, prolonged epistaxis may cause anaemia. At times, the bleeding may be
so severe that blood transfusions are required.
GSD type I: Serum glucose and blood pH levels are frequently decreased, while the serum
lactate, uric acid, triglyceride, and cholesterol levels are elevated. Urea and creatinine levels
might be elevated when renal function is impaired. The following laboratory values should
be obtained:
o Serum glucose and electrolyte levels (Higher anion gap may suggest lactic
acidosis.)
o Serum lactate level
o Blood pH
o Serum uric acid level
o Serum triglyceride and cholesterol levels
o Gamma glutamyltransferase level (Liver dysfunction)
o CBC and differential (eg, anaemia, leucopenia, neutropenia)
o Coagulation- Bleeding and clotting time
o Urinalysis for aminoaciduria, proteinuria, and microalbuminuria in older
patients
o Urinary excretion levels of uric acid and calcium
o Serum alkaline phosphatase, calcium, phosphorus, urea, and creatinine
levels.
Imaging Studies
146
• In GSD type I, liver and kidney ultrasonography should be performed for follow-up
of organomegaly.
• Abdominal CT scanning or MRI is advised whenever the lesions are large, poorly
defined, or are growing rapidly.
Other Tests
o Glucagon and epinephrine tests do not cause a rise in glucose levels, but
plasma levels of lactic acid are raised.
o Orally administered galactose and fructose (1.75 g/kg) do not increase
glucose levels, but plasma lactic acid levels do increase.
o Glucose tolerance test (1.75 g/kg PO) progressively lowers lactic acid levels
over several hours after the administration of glucose.
Treatment
Most children with GSD type I are admitted to the hospital to make a final diagnosis, to
manage hepatomegaly or hypoglycemia.
Because no specific treatment is available, symptomatic therapy is very important.
Diet
The primary goal of treatment is to correct hypoglycemia and maintain a normoglycemic

state. The normoglycemic state can be achieved with overnight nasogastric infusion of
glucose, parental nutrition, or per oral administration of raw corn starch. Glucose molecules
are continuously released by hydrolysis of corn starch in the digestive tract over 4 hours
following its intake. The intake of fructose and galactose should be restricted because it has
been shown that they can not be converted to glucose but they do increase lactic acid
production. Limited intake of lipids is advisable for the existing hyperlipidemia.
Medication
No specific drug treatment is recommended for GSD type I. Appropriately treat

concurrent infections with antibiotics.
Allopurinol (Zyloprim), a xanthine oxidase inhibitor, therapy can reduce uric acid
levels in the blood and prevent occurrence of gout and kidney stones in adult life.
Hyperlipidemia can be reduced by lipid-lowering drugs (eg, 3-hydroxy-3-
methylglutaryl coenzyme A [HMG-CoA] reductase inhibitors, fibric acid derivatives).
In patients with renal lesions, microalbuminuria can be reduced with Angiotensin-
converting enzyme (ACE) inhibitor therapy. In addition to their antihypertensive effects,
ACE inhibitors are renoprotective and reduce albuminuria. Nephrocalcinosis and renal
calculi can be prevented with citrate therapy.
147
Additionally, for patients with GSD type I, the future may bring Adeno-associated
virus vector – mediated gene therapy, which may result in curative therapy,
Complications
o Bacterial infections and cerebral oedema are caused by prolonged hypoglycemia

and metabolic acidosis.
o Long-term complications encompass growth retardation, hepatic adenomas
with a high rate of malignant change, xanthomas, gout, and renal
dysfunction. Long-term complications result from metabolic disturbances,
mostly hypoglycemia.
o Acute hypoglycemia may be fatal, and long-term complications include
irreversible damage to the CNS.
o Early death usually caused by acute metabolic complications (eg, hypoglycemia,
acidosis) or bleeding in the course of various surgical procedures
Prognosis
The prognosis is better than in the past provided that all the available dietary and medical
measures are implemented.
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CASE STUDY- OBESITY
A 40 –year- old woman, 5 feet 1 inch tall and weighing 188 pounds came for
consultation to a physician complaining of frequent episodes of dizziness and
numbness in her legs. She was too worried for her weight. Her waist measured 41
inches and hip measured 39 inches. Her only child who was 15 year old, her sister and
both of the parents were over weight. The patient recalled that she had been obese
throughout her childhood and adolescence. Over the past 6 years she had been on
seven different diets for periods of two weeks to three months, losing from 5 to 25
pounds. On discontinuation of each diet, she regained weight returning to 185 to 190
pounds.
During routine physical examination the patient was observed to be hypertensive

(blood pressure of 200/120 mm Hg) but no abnormality was detected upon
examination of Chest, CNS and Abdomen .
The patient was asked to return to the clinic a week later in the fasting state, during
which time a blood specimen was obtained. Blood Biochemistry revealed fasting
hyperglycemia, hyperinsulinemia, Dyslipidemia, and glucose intolerance.
What other investigations should be carried out to confirm the diagnosis?
Calculate the BMI for this woman and comment on the grade of obesity
Case details- The patient is obese and is also suffering from “Metabolic syndrome”.
She has a history of obesity dating to early child hood and also has a positive family
history. Her symptoms are suggestive of metabolic syndrome, a common complication of
Obesity. Some of her features can be discussed as -
1) Obesity -She has an apple (android) pattern of fat distribution. Her waist to hip ratio is
41/39=1.05. Apple shape is defined as a waist to hip ratio of more than 0.8 in women, and
more than 1.0 in men. She has therefore apple pattern of fat distribution which is common
in males. Compared with other women of same body weight who have gynoid fat pattern,
the presence of increased visceral or intra abdominal adipose tissue places her at greater
risk for diabetes, hypertension, dyslipidemia and coronary heart disease. (The gynoid,
“pear- shaped” or lower body obesity is defined as a waist to hip ratio of less than 0.8 for
women and less than 1.0 for men. The pear shape is relatively benign health wise and is
commonly found in females).
2) BMI (Body Mass Index)
BMI= Weight (kg)/height (m2).
For this patient
149
188 Pounds=85.5 kg (Approximately)
5 feet 1 inch height =1.55meters (154.94cm)
= 85.5/(1.55) 2
= 35.6 kg/ m2
World Health Organization (WHO) criteria based on BMI Under this convention for
adults,
• Grade 1 overweight (commonly and simply called overweight) is a BMI of 25-29.9

kg/m2
• Grade 2 overweight (commonly called obesity) is a BMI of 30-39.9 kg/m2.
• Grade 3 overweight (commonly called severe or morbid obesity) is a BMI greater than
or equal to 40 kg/m2.
From the result calculated for the given patient, it is indicated that the patient is obese
(Grade 2 overweight).
3) Metabolic syndrome- The patient is also suffering from ‘Insulin resistance

syndrome’. She has hypertension, dyslipidemia, Hyperinsulinemia and impaired glucose
tolerance.
Metabolic syndrome also referred to as Syndrome X or insulin resistance syndrome consists
of a number of metabolic risk factors that increase the risk for atherosclerotic
cardiovascular disease (CVD) and other cardiovascular complications such as cardiac
arrhythmias, heart failure, and thrombotic events.
a) Criteria for diagnosis- According to the National Cholesterol Education Program

(NCEP) Adult Treatment Panel III (ATP III) report, there are six major components of
metabolic syndrome relating to the development of CVD: 1) abdominal obesity, 2)
atherogenic dyslipidemia, 3) elevated blood pressure, 4) insulin resistance (with or without
the presence of glucose intolerance), 5) proinflammatory state, and 6) prothrombotic state.
Note- A prothrombotic state is characterized by abnormalities, specifically elevations, in

procoagulant factors, antifibrinolytic factors, platelet alterations, and endothelial
dysfunction. A proinflammatory state is characterized by elevations of circulating
inflammatory molecules such as C-reactive protein (CRP), tumor necrosis factor-alpha,
plasma resistin, interleukin (IL)-6, and IL-18. CRP is a general marker of inflammation that
has been linked to CVD in patients with metabolic syndrome.
b)Associated diseases- Cardiovascular diseases and type 2 diabetes mellitus can be

present in association with metabolic syndrome. The relative risk for new-onset CVD in
patients with the metabolic syndrome, in the absence of diabetes, averages between 1.5-
and threefold. Overall, the risk for type 2 diabetes in patients with the metabolic
syndrome is increased three- to fivefold. Patients with metabolic syndrome are also at
increased risk for peripheral vascular disease. In addition to the features specifically
associated with metabolic syndrome, insulin resistance is accompanied by other metabolic
150
alterations. These include increases in uric acid, microalbuminuria, nonalcoholic fatty
liver disease (NAFLD) and/or polycystic ovarian disease (PCOS), and obstructive
sleep apnea (OSA).
A person suspected of having this syndrome should have a through history taken especially
with regard to family history and presence of other cardiovascular risk factors.
c) An examination should include:-
• Recording the body weight
• Calculating the BMI
• Measurement of the waist circumference in inches
• Calculating the hip-waist ratio
• Measurement of the subcutaneous fat at 4 sites-biceps, triceps, sub scapular and supra-
iliac
• Blood pressure measurement.
d) Laboratory Tests
1) Fasting lipids and glucose estimations are needed to determine if the metabolic
syndrome is present.
2) The measurement of additional biomarkers associated with insulin resistance must be

individualized. Such tests might include apo B, high-sensitivity CRP, fibrinogen, uric
acid, urinary micro albumin, and liver function tests.
3) A sleep study should be performed if symptoms of OSA are present.

4) If PCOS is suspected based on clinical features and an ovulation, testosterone,
luteinizing hormone, and follicle-stimulating hormone should be measured.
e) Management of metabolic syndrome - is highly dependent on the control of all of the

contributing factors. This includes both underlying risk factors as well as metabolic risk
factors. Lifestyle modifications should be implemented immediately for all patients
diagnosed with metabolic syndrome. Lifestyle modifications include weight reduction,
increased physical activity and nutritional therapy. Additional risk assessments should be
performed in patients to assure appropriate goals of therapy throughout the course of the
syndrome.
The key to preventing metabolic syndrome, however, remains diet and exercise. Any
person with a strong family history of metabolic syndrome or type 2 diabetes should be
especially careful to maintain a healthy lifestyle.
151
CASE STUDY- UREA CYCLE DISORDERS
A 6 month- old Infant began to vomit occasionally and ceased to gain weight. At 9
months of age he was readmitted to the hospital.
Routine examination and laboratory tests were normal but after one week he became
drowsy, his temperature rose to 39.4 ° C, his pulse was elevated, and his liver was
enlarged. The Electro Encephalogram (EEG) was grossly abnormal. Since the infant
could not retain milk by tube feeding, Intravenous glucose was administered. Urine
analysis showed abnormally high amount of Glutamine and Uracil. This suggested
high amount of Ammonia concentration, which was confirmed by laboratory test.
What is the cause of hyper ammonemia in this patient?

Why were the urine glutamine and Uracil levels elevated?
How can such a patient be treated?
Case Details- The child is most probably suffering from hyperammonemia due to
impaired urea formation. Hyperammonemia in a new born or very young infant is the
characteristic sign of inherited defect in a gene for urea cycle enzymes. The enzyme
affected in this patient seems to be Ornithine Transcarbamoylase as apparent from the
enhanced excretion of Uracil. Excessive excretion of Uracil or its precursor Orotic acid,
results from an accumulation of Carbamoyl phosphate in the mitochondria. In the absence
of Ornithine Transcarbamoylase, Carbamoyl phosphate accumulates and leaks in to the
cytoplasm, where it can be used to make Carbamoyl Aspartate, the first intermediate in the
pathway of pyrimidine nucleotide biosynthesis. This case is unusual in that the symptoms
took longer to appear. Urine glutamine excretion has increased because it is excreted in
compensation for the inoperative urea cycle. Free ammonia is toxic to brain. It is
detoxified by conversion of Glutamate to glutamine. High Glutamine level indicates
hyperammonemia that may be due to any liver pathology or may be due to defective urea
cycle enzymes.
Ornithine Transcarbamoylase deficiency
The disease is characterized as X linked dominant because most females are also somewhat
affected. Females usually respond well to treatment. A significant number of carrier
females have hyperammonemia and neurologic compromise. The risk for hyperammonemia
is particularly high in pregnancy and the postpartum period. The disease is much more
severe in males than in females. The enzyme activity can range from 0% to 30% of the
normal.
152
Urea formation
(Urea cycle)
The urea cycle is the sole source of endogenous production of arginine and it is the
principal mechanism for the clearance of waste nitrogen resulting from protein turnover and
dietary intake. This extra nitrogen is converted into ammonia (NH3) and transported to the
liver where it is processed. The urea cycle disorders (UCD) result from inherited molecular
defects which compromise this clearance.
Reactions of urea cycle
Synthesis of 1 mol of urea requires 3 mol of ATP plus 1 mol each of ammonium ion and of
the Į-amino nitrogen of aspartate. Five enzymes catalyze the reactions of urea cycle (See
Figure). Of the six participating amino acids, N-acetyl glutamate functions solely as an
enzyme activator. The others serve as carriers of the atoms that ultimately become urea.
The major metabolic role of Ornithine, Citrulline, and argininosuccinate in mammals is
urea synthesis. Urea synthesis is a cyclic process. Since the Ornithine consumed in 2nd
reaction and is regenerated in last reaction, so there is no net loss or gain of Ornithine,
Citrulline, argininosuccinate, or arginine. Ammonium ion, CO2, ATP, and aspartate are,
however, consumed. Some reactions of urea synthesis occur in the matrix of the
mitochondrion, other reactions in the cytosol (See Figure).
Reaction 1- Carbamoyl Phosphate Synthase I Initiates Urea Biosynthesis

Condensation of CO2, ammonia, and ATP to form Carbamoyl phosphate is catalyzed by
mitochondrial Carbamoyl phosphate synthase I (CPS-1), A cytosolic form of this
enzyme, Carbamoyl phosphate synthase II, uses glutamine rather than ammonia as the
nitrogen donor and functions in pyrimidine biosynthesis. Carbamoyl phosphate synthase I,
the rate-limiting enzyme of the urea cycle, is active only in the presence of its allosteric
activator N-acetyl glutamate, which enhances the affinity of the synthase for ATP.
Formation of Carbamoyl phosphate requires 2 mol of ATP, one of which serves as a
phosphoryl donor.
Reaction -2-Carbamoyl Phosphate Plus Ornithine Forms Citrulline

L-Ornithine Transcarbamoylase (OTC) catalyzes transfer of the Carbamoyl group of
Carbamoyl phosphate to Ornithine, forming Citrulline and orthophosphate. While the
reaction occurs in the mitochondrial matrix, both the formation of Ornithine and the
subsequent metabolism of Citrulline take place in the cytosol. Entry of Ornithine into
mitochondria and exodus of Citrulline from mitochondria therefore involve mitochondrial
inner membrane transport systems.
Reaction -3 Citrulline plus Aspartate Forms Argininosuccinate

Argininosuccinate synthase (ASS) links L- Aspartate and Citrulline via the amino group
of aspartate and provides the second nitrogen of urea. The reaction requires ATP and
involves intermediate formation of citrullyl-AMP. Subsequent displacement of AMP by
aspartate then forms Argininosuccinate.
153
Reaction -4-Cleavage of Argininosuccinate Forms Arginine & Fumarate
Cleavage of argininosuccinate catalyzed by argininosuccinate lyase (ASL), proceeds with
retention of nitrogen in arginine and release of the aspartate skeleton as fumarate. Addition
of water to fumarate forms L-malate, and subsequent NAD+-dependent oxidation of malate
forms oxaloacetate. These two reactions are analogous to reactions of the citric acid cycle
but are catalyzed by cytosolic Fumarase and malate dehydrogenase. Transamination of
oxaloacetate by glutamate aminotransferase then re-forms aspartate. (See Figure 2)The
carbon skeleton of aspartate-fumarate thus acts as a carrier of the nitrogen of glutamate into
a precursor of urea.
Reaction -5-Cleavage of Arginine Releases Urea & Re-Forms Ornithine

Hydrolytic cleavage of the guanidino group of arginine, catalyzed by liver arginase
(ARG1) releases urea, the other product, Ornithine, reenters liver mitochondria for
additional rounds of urea synthesis. Ornithine and lysine are potent inhibitors of arginase,
competitive with arginine. Arginine also serves as the precursor of the potent muscle
relaxant nitric oxide (NO) in a Ca2+-dependent reaction catalyzed by NO synthase.
Regulation of Urea formation
Carbamoyl Phosphate Synthase I Is the Pacemaker Enzyme of the Urea Cycle

The activity of Carbamoyl phosphate synthase I is determined by N-acetyl glutamate,
whose steady-state level is dictated by its rate of synthesis from acetyl-CoA and glutamate
and its rate of hydrolysis to acetate and glutamate. These reactions are catalyzed by N-
acetyl glutamate synthase and N-acetyl glutamate Hydrolase, respectively. Major changes
in diet can increase the concentrations of individual urea cycle enzymes 10- to 20-fold.
Starvation, for example, elevates enzyme levels, presumably to cope with the increased
production of ammonia that accompanies enhanced protein degradation.
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Figure1- showing reactions of urea cycle
Figure -2 showing the relationship of Urea cycle to TCA
Fate of Urea- Urea formed in the liver is transported through circulation to kidneys for
excretion through urine. It is also transported to intestine where it is decomposed by Urease
produced by microbial action. Ammonia liberated by this activity is transported by portal
155
circulation to liver where it is detoxified back to urea. A fraction of ammonia goes to
systemic circulation. (See figure -3)
figure-3 Showing the fate of urea
Urea cycle disorders
1) Carbamoyl Phosphate synthetase (CPS-1) deficiency
Along with OTC deficiency, deficiency of CPSI is the most severe of the urea cycle
disorders. Individuals with complete CPS-I deficiency rapidly develop hyperammonemia in
the newborn period. Children who are successfully rescued from crisis are chronically at
risk for repeated bouts of hyperammonemia.
2) Ornithine Transcarbamoylase deficiency (OTC deficiency)

Absence of OTC activity in males is as severe as CPSI deficiency. Approximately 15% of
carrier females develop hyperammonemia during their lifetime and many require chronic
medical management
3) Citrullinemia type I (ASS deficiency)

The hyperammonemia in this disorder is quite severe. Affected individuals are able to
incorporate some waste nitrogen into urea cycle intermediates, which makes treatment
slightly easier.
4) Argininosuccinic aciduria (ASL deficiency)

This disorder also presents with rapid-onset hyperammonemia in the newborn period. This
enzyme defect is past the point in the metabolic pathway at which all the waste nitrogen has
been incorporated into the cycle. Treatment of affected individuals often requires only
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supplementation of arginine. ASL deficiency is marked by chronic hepatic enlargement and
elevation of transaminases. Biopsy of the liver shows enlarged hepatocytes, which may
over time progress to fibrosis, the etiology of which is unclear. Affected individuals can
also develop trichorrhexis nodosa, a node-like appearance of fragile hair, which usually
responds to arginine supplementation. Affected individuals who have never had prolonged
coma but nevertheless have significant developmental disabilities have been reported.
5) Arginase deficiency (hyperargininemia; ARG deficiency)

This disorder is not typically characterized by rapid-onset hyperammonemia. Affected
individuals develop progressive spasticity and can also develop tremor, ataxia, and
choreoathetosis. Growth is affected
6) NAG Synthase deficiency. Deficiency of this enzyme has been described in a number
of affected individuals. Symptoms mimic those of CPSI deficiency; since CPSI is rendered
inactive in the absence of NAG
Incidence
The incidence of UCDs (Urea cycle disorders)is estimated to be at least 1:25,000 births;
partial defects may make the number much higher.
Infants with a urea cycle disorder often appear normal initially but rapidly develop
cerebral edema and the related signs of lethargy, anorexia, hyperventilation or hypo
ventilation, hypothermia, Slurring of the speech, Blurring of vision, seizures,
neurological posturing, and coma. In milder (or partial) urea cycle enzyme deficiencies,
ammonia accumulation may be triggered by illness or stress at almost any time of life,
resulting in multiple mild elevations of plasma ammonia concentration; the
hyperammonemia is less severe and the symptoms more subtle. In individuals with partial
enzyme deficiencies, the first recognized clinical episode may be delayed for months or
years.
Laboratory diagnosis
The diagnosis of a urea cycle disorder is based on evaluation of clinical, biochemical, and
molecular genetic data.
• A plasma ammonia concentration of 150 mmol/L or higher is a strong indication
for the presence of a UCD.
• Plasma quantitative amino acid analysis can be used to diagnose a specific urea
cycle disorder: plasma concentration of arginine may be reduced in all urea cycle
disorders, except ARG(Arginase) deficiency, in which it is elevated five- to
sevenfold.
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• Plasma concentration of Citrulline helps discriminate between the proximal and
distal urea cycle defects, as Citrulline is the product of the proximal enzymes (OTC
and CPSI) and a substrate for the distal enzymes (ASS, ASL, ARG).
• Urinary Orotic acid is measured to distinguish CPSI deficiency and NAGS (N-
Acetyl Glutamate Synthase) deficiency from OTC deficiency.
• A definitive diagnosis of CPS-I deficiency, OTC deficiency, or NAGS deficiency
depends on determination of enzyme activity from a liver biopsy specimen.
• However, the combination of family history, clinical presentation, amino acid and
Orotic acid testing, and, in some cases, molecular genetic testing is often sufficient for
diagnostic confirmation, eliminating the risks of liver biopsy.
Treatment
The mainstays of treatment for urea cycle disorders include-

• Dialysis to reduce plasma ammonia concentration.
• Intravenous administration of arginine chloride and nitrogen scavenger drugs to
allow alternative pathway excretion of excess nitrogen, Excess nitrogen is removed
by intravenous phenyl acetate and that conjugate with glutamine and glycine,
respectively, to form phenylacetylglutamine and Hippuric acid, water-soluble
molecules efficiently excreted in urine.
• Arginine becomes an essential amino acid (except in arginase deficiency) and should
be provided intravenously to resume protein synthesis. If these measures fail to reduce
ammonia, hemodialysis should be initiated promptly.
• Restriction of protein for 24-48 hours to reduce the amount of nitrogen in the diet,
providing calories as carbohydrates (intravenously as glucose) and fat (intralipid or as
protein-free formula) to reduce catabolism,
• Physiologic stabilization with intravenous fluids
• Chronic therapy consists of a protein-restricted diet, phenyl butyrate (a more palatable
precursor of phenyl acetate), arginine, or Citrulline supplements, depending on the
specific diagnosis.
• Liver transplantation should be considered in patients with severe urea cycle defects
that are difficult to control medically.
Genetic counselling
Deficiencies of CPSI, ASS, ASL, NAGS, and ARG are inherited in an autosomal recessive
manner. OTC deficiency is inherited in an X-linked manner. Prenatal testing using
molecular genetic testing is available for five of the six urea cycle disorders
Differential diagnosis
A number of other disorders that perturb the liver can result in hyperammonemia and
mimic the effects of a urea cycle disorder. The most common/significant ones are viral
infection of the liver and vascular bypass of the liver.
158
Figure-4 showing the mechanism of Nitrogen removal by Glycine, phenyl acetate
and Arginine
159
Figure- 5 showing an overview of Urea cycle. Glutamate is the ultimate precursor for both
nitrogen atoms of urea. One through ammonia by oxidative deamination of Glutamate by
glutamate dehydrogenase and the second through the activity of transaminase whereby
aspartate is formed from Glutamate, hence both nitrogen actually come from Glutamate
only.
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CASE STUDY- LESCH NYHAN SYNDROME
At the age of 11 months, a boy showed signs of delayed motor development and was
brought for consultation. His mother had noticed sand like crystals on the diaper of
the baby but reported only when asked particularly about it. History revealed that the
child had a compulsive urge to bite his lips and fingers. Upon questioning the mother
revealed that she had a brother with similar symptoms.
The Lesch Nyhan syndrome was suspected, urinary and serum uric acid levels were
estimated. Both were abnormally high for the boy’s age. The diagnosis was confirmed
by estimating enzyme levels in skin fibroblasts; the enzyme activity was 50 % of the
normal.
Which enzyme is deficient in Lesch Nyhan Syndrome?
What is the basis for Hyperuricemia, hyperuricosuria and self mutilation in this
disorder?
Lesch–Nyhan syndrome (LNS)
Case discussion
Lesch–Nyhan syndrome (LNS), also known as Nyhan’s syndrome and Juvenile gout, is
a rare inherited disorder.
Biochemical Defect
LNS is an X-linked recessive disorder, caused by a deficiency of the enzyme hypoxanthine-

guanine phosphoribosyltransferase (HGPRT), and produced by mutations in the HGPRT
gene. This enzyme is normally present in each cell in the body, but its highest concentration
is in the brain, especially in the basal ganglia. The HGPRT gene has been localized to the
long arm of the X chromosome (q26-q27). The disorder appears in males; occurrence in
females is extremely rare.
Pathogenesis
Formation of DNA (during cell division) requires nucleotides, molecules that are the
building blocks for DNA. The purine bases (adenine and guanine) and Pyrimidine bases
(thymine and cytosine) are bound to deoxyribose and phosphate is incorporated as
necessary. Normally, the nucleotides are synthesized de novo from amino acids and other
precursors. A small part, however, is 'recycled' from degraded DNA of broken-down cells.
This is termed the "salvage pathway".(Figure)
HGPRT is the "salvage enzyme" for the purines: it channels hypoxanthine and guanine
back into DNA synthesis. Failure of this enzyme has two results:
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• Cell breakdown products cannot be reused, and are therefore degraded. This gives
rise to increased uric acid, a purine breakdown product.
• The de novo pathway is stimulated due to an excess of PRPP (5-phospho-D-ribosyl-
1-pyrophosphate or simply phosphoribosyl-pyrophosphate).
In the absence of HGPRT, these purine bases cannot be salvaged, and instead are degraded
and excreted as uric acid. In addition to the failure of purine recycling, the synthetic rate for
purines is accelerated, presumably to compensate for purines lost by the failure of the
salvage process. The failure of recycling together with the increased synthesis of purines is
the basis for the overproduction of uric acid.
Figure-1 showing Salvage pathway.
The increased production of uric acid leads to hyperuricemia. Since uric acid is near its
physiological limit of solubility in the body, the persistent hyperuricemia increases the risk
of uric acid crystal precipitation in the tissues. Uric acid crystal deposition in the joints
produces an inflammatory reaction and gouty arthritis. The kidneys respond to the
hyperuricemia by increasing its excretion into the urinary tract, increasing the risk of
forming urate stones in the urinary collecting system. These stones may be passed as a
sandy sludge or as larger particles that may obstruct urine flow and increase the risk for
hematuria and urinary tract infections.
Frequency
162
The reported worldwide prevalence is 1 case per 380,000 population.
LNS is characterized by three major hallmarks: neurological dysfunction, cognitive and

behavioral disturbances including self-mutilation, and uric acid overproduction
(hyperuricemia). Some may also be afflicted with macrocytic anemia. Virtually all
patients are males; males suffer delayed growth and puberty, and most develop shrunken
testicles or testicular atrophy. Female carriers are at an increased risk for gouty arthritis,
but are usually otherwise unaffected.
1) Overproduction of uric acid
One of the first symptoms of the disease is the presence of sand-like crystals of uric acid
in the diapers of the affected infant.
The overproduction of uric acid is present at birth, but may not be recognized by routine
clinical laboratory testing methods. The serum uric acid concentration is often normal
initially, as the excess purines are promptly eliminated in the urine. But as the disease
progresses the hyperuricemia may be observed.
The crystals usually appear as an orange grainy material, or they may coalesce to form
either multiple tiny stones or distinct large stones that are difficult to pass. The stones, or
calculi, usually cause hematuria (blood in the urine) and increase the risk of urinary tract
infection. Some victims suffer kidney damage due to such kidney stones. Stones may be
the presenting feature of the disease, but can go undetected for months or even years.
2) Nervous system impairment
The most common presenting features are abnormally decreased muscle tone (hypotonia)
and developmental delay, which are evident by three to six months of age. Lack of speech
is also a very common trait associated with LNS.
Irritability, loss of motor control, involuntary movements and arching of the spine
(opisthotonus) are also there
3) Self-injuring behavior
The age at onset of self-injury may be as early as 1 yr and occasionally as late as the teens.
The self-injury begins with biting of the lips and tongue; as the disease progresses, affected
individuals frequently develop finger biting and head banging. The self-injury can increase
during times of stress. Self-mutilation is a distinguishing characteristic of the disease and is
apparent in 85% of affected males.
Diagnosis
The gross overproduction of uric acid is often evident in routine blood and urine studies.
1) Uric acid levels in the blood typically are elevated,
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2) Urinary uric acid excretion also is increased typically.
3) Definitive diagnosis is obtained most often by measurement of HGPRT enzyme
activity in blood or tissue
Diagnosis is confirmed by identifying a molecular genetic mutation in the HGPRT
gene. Molecular genetic diagnosis provides an ideal tool for carrier detection and prenatal
screening of at-risk pregnancies.
4) Macrocytic anemia, sometimes profound, is relatively common. Vitamin B-12, folate,
and iron results are typically normal.
Other Tests
Noninvasive imaging studies of the kidneys and other parts of the urogenital system are
warranted because of the marked increase in the risk for kidney stones.
Treatment
Treatment for LNS is symptomatic. Gout can be treated with Allopurinol to control
excessive amounts of uric acid. Kidney stones may be treated with lithotripsy, a technique
for breaking up kidney stones using shock waves or laser beams. There is no standard
treatment for the neurological symptoms of LNS
Prognosis
The prognosis for individuals with severe LNS is poor. Death is usually due to renal failure
or complications from hypotonia, in the first or second decade of life. Less severe forms
have better prognoses.
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CASE STUDY- PORPHYRIA
A 30-year-old woman had severe abdominal pain, nausea, vomiting and diarrhea.
Evaluations including upper and lower endoscopies did not establish any intestinal
infection. She gradually improved and was discharged after 2 weeks. 2 years later she was
admitted to a psychiatric unit with acute mental changes and hallucinations, she had to be
transferred to the emergency department due to abdominal pain, a grand mal seizure and
hyponatremia.
Her pulse was 120 and BP 174/114 mm Hg. She was disoriented but had no focal
neurological signs. MRI showed sub cortical abnormalities, and the spinal fluid was
normal. After cholecystectomy for a distended gallbladder, she was discharged but she
stayed with a family member in another city because her symptoms were worse and muscle
weakness had developed. She was hospitalized and progressed to quadriparesis, respiratory
failure and aspiration pneumonia. Urinary porphobilinogen (PBG) was reported as 44
mg/24 hours (reference range 0-4).
What is the diagnosis and defect in this disease?

How is the diagnosis done and what is its prognosis?
What is the genetic basis of this disease?
Case Discussion
The patient is suffering from acute intermittent Porphyria as evident from the typical
combination of abdominal pain, motor neuropathy, psychiatric symptoms and increased
amounts of urinary Porphyrins and their precursors. Acute intermittent Porphyria (AIP) is
a rare autosomal dominant metabolic disorder affecting the production of heme. It is
characterized by deficiency of enzyme porphobilinogen deaminase.
Basic concept
Haem synthesis
Heme is required for a variety of haemoproteins such as hemoglobin, myoglobin,

respiratory Cytochromes, and the cytochrome P450 enzymes (CYPs). Hemoglobin
synthesis in erythroid precursor cells accounts for approximately 85% of daily heme
synthesis in humans. Hepatocytes account for most of the rest, primarily for synthesis of
CYPs, which are especially abundant in the liver endoplasmic reticulum, and turn over
more rapidly than many other haemoproteins, such as the mitochondrial respiratory
cytochromes.
Heme biosynthesis involves eight enzymatic steps in the conversion of glycine and
succinyl-CoA to heme (Fig.1). These eight enzymes are encoded by nine genes, as the first
enzyme in the pathway, 5'-aminolevulinate synthase (ALA-synthase), has two genes that
encode unique housekeeping (ALAS1) and erythroid-specific (ALAS2) isozymes. The first
165
and last three enzymes in the pathway are located in the mitochondrion, whereas the other
four are in the cytosol. As shown in Fig.1, pathway intermediates are the porphyrin
precursors, ALA and PBG, and porphyrins (mostly in their reduced forms, known as
porphyrinogens). At least in humans, these intermediates do not accumulate in significant
amounts under normal conditions or have important physiologic functions.
Liver Bone Marrow
1
Pb Ĺ ALA
ņ 2
Neurological disturbance
3
& Abdominal Pain
4
Photosensitivity 5 Inherited Enzyme

Inherited Enzyme deficiencies in Bone
deficiencies in Liver 6 Marrow
HEPATIC PORPHYRIA ERYTHROPOIETIC PORPHYRIA
3 Acute Intermitent 7
5 4
Porphyria Cutanea Tarda 8 Con E Porphyria
6 5
Hereditary Cooproporphyria Por: Cu. Tarda
7 8
Variegata Porphyria Erythropoietic protoporphyria
Figure-1- showing the steps of Haem synthesis
Steps of Haem synthesis
1) The first and rate-controlling step is the condensation of glycine and succinyl–coenzyme
A (CoA) to form į-aminolevulinic acid (ALA). The enzyme, ALA-synthase is activated by
Pyridoxal phosphate. In the liver, this rate-limiting enzyme can be induced by a variety of
drugs, steroids, and other chemicals. Defects in the erythroid gene, cause X-linked
Sideroblastic anemia (XLSA).
166
2) The ALA formed is transported into the cytoplasm, where the second enzyme, ALA
dehydratase (also known as porphobilinogen synthase), condenses two molecules of
ALA to form the monopyrrole porphobilinogen.
3) The third enzyme, porphobilinogen deaminase (also known as hydroxymethylbilane

synthase), forms a linear tetrapyrrole, hydroxymethylbilane, which is normally rapidly
converted, mainly to the cyclic intermediate Uroporphyrinogen III, by the enzyme
Uroporphyrinogen III synthase (also known as Uroporphyrinogen cosynthase). When
Uroporphyrinogen III synthase is deficient, as in congenital erythropoietin Porphyria
(Guenther's disease), hydroxymethylbilane rapidly undergoes nonenzymatic ring closure to
form Uroporphyrinogen I.
4) The enzyme Uroporphyrinogen decarboxylase carries out the stepwise

decarboxylation of Uroporphyrinogen I or III to form intermediates with 7-, 6-, 5-, and 4-
carboxyl groups. Coproporphyrinogen is the common name for the 4-carboxyl–containing
intermediate.
5) Coproporphyrinogen III is transported back into mitochondria, where the enzyme

Coproporphyrinogen III oxidase carries out the stepwise oxidative decarboxylation of
two of the remaining propionate beta side chains, at positions 2 and 4 (on rings A and B,
respectively), to vinyl groups, forming protoporphyrinogen IX.
6) Next, the enzyme protoporphyrinogen oxidase carries out the oxidation of

protoporphyrinogen IX to form protoporphyrin IX, after which the enzyme Ferrochelatase
(also called heme synthase) inserts ferrous iron into the protoporphyrin IX macrocycle to
form the end product heme. (See figure-1)
Regulation of Heme Biosynthesis
Regulation of heme synthesis differs in the two major heme-forming tissues, the liver and
erythron. In the liver, "free" heme regulates the synthesis and mitochondrial translocation
of the housekeeping form of ALA-synthase. Heme represses the synthesis of the ALA-
synthase mRNA and interferes with the transport of the enzyme from the cytosol into
mitochondria. Hepatic ALA-synthase is increased by many of the same chemicals that
induce the cytochrome P450 enzymes in the endoplasmic reticulum of the liver. Because
most of the heme in the liver is used for the synthesis of cytochrome P450 enzymes, hepatic
ALA-synthase and the cytochrome P450s are regulated in a coordinated fashion, and many
drugs that induce hepatic ALA-synthase also induce CYPs. The other hepatic heme
biosynthetic enzymes are presumably expressed at constant levels, although their relative
activities and kinetic properties differ. For example, normal individuals have high activities
of ALA-dehydratase but low activities of HMB-synthase, the latter being the second rate-
limiting step in the pathway.
167
In the erythron, novel regulatory mechanisms allow for the production of the very large
amounts of heme needed for hemoglobin synthesis. The response to stimuli for hemoglobin
synthesis occurs during cell differentiation, leading to an increase in cell number. The
erythroid-specific ALA-synthase is expressed at higher levels than the housekeeping
enzyme, and erythroid-specific control mechanisms regulate other pathway enzymes as
well as iron transport into erythroid cells.
PORPHYRIAS
The Porphyrias are a group of rare metabolic disorders arising from reduced activity of any
of the enzymes in the heme biosynthetic pathway. The disorders may be either acquired or
inherited through a genetic defect in a gene encoding these enzymes. These deficiencies
disrupt normal heme production, and produce symptoms when increased heme is required.
Porphyrin precursors, overproduced in response to synthetic pathway blockages,
accumulate in the body and cause diverse pathologic changes thereby becoming the basis
for diagnostic tests.
The diagnosis of acute porphyrias can be confirmed by repeating the quantitation of urinary
porphyrin during an acute episode and finding elevated levels (2–5 times of normal) of
porphobilinogen.
Incidence
The prevalence of this condition is unknown, but probably ranges from 1 in 500 to 50,000
worldwide. Certain types of porphyrias are more common in specific populations, such as
whites in South Africa and Scandinavians.
Classification
The Porphyrias can be classified as either hepatic or erythropoietic, depending on whether

the heme biosynthetic intermediates that accumulate arise initially from the liver or
developing erythrocytes, or as acute or cutaneous, based on their clinical severity.
Inheritance
Most of the Porphyrias are inherited. Inheritance patterns depend on the type of porphyria.
Some forms of the condition are inherited in an autosomal dominant pattern, which means
one copy of the altered gene is sufficient to cause the disorder. Other Porphyrias are
inherited in an autosomal recessive pattern, which means two copies of the gene must be
altered for a person to be affected by the condition.
Typically, patients with the autosomal dominant varieties present initially in adulthood, and
those with homozygous variants present in early childhood. Symptomatic porphyria is
thought to be more common in female than male patients with a female-male ratio of 5 to 1.
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A) The Hepatic Porphyrias
1) ALA-Dehydratase Deficient Porphyria (ADP)
ADP is a rare autosomal recessive acute hepatic porphyria caused by a severe deficiency of
ALA-dehydratase activity. The clinical presentation depends on the amount of residual
ALA-dehydratase activity. Symptoms resemble those of AIP (Acute intermittent Porphyria)
including abdominal pain and neuropathy. Infant with more severe disease manifest failure
to thrive beginning at birth. Diagnosis is confirmed by significantly elevated levels of
plasma and urinary ALA, urinary coproporphyrin III and ALAD activity in erythrocytes
<10% of normal.
The treatment of ADP acute attacks is similar to that of AIP.
2) Acute Intermittent Porphyria (AIP)
Acute intermittent porphyria is inherited as an autosomal dominant, though it remains

clinically silent in most patients who carry the trait. Clinical illness usually develops in
women. Symptoms begin in the teens or 20s, but onset can begin after menopause in rare
cases. The disorder is caused by partial deficiency of porphobilinogen deaminase activity,
leading to increased excretion of aminolevulinic acid and porphobilinogen in the urine. The
diagnosis may be elusive if not specifically considered. The characteristic abdominal pain
may be due to abnormalities in autonomic innervations in the gut. In contrast to other forms
of porphyria, cutaneous photosensitivity is absent in acute intermittent porphyria. Attacks
are precipitated by numerous factors, including drugs and intercurrent infections.
Hyponatremia may be seen, due in part to inappropriate release of antidiuretic hormone,
though gastrointestinal loss of sodium in some patients may be a contributing factor.
Clinical Findings
Symptoms and Signs
Patients show intermittent abdominal pain of varying severity, and in some instances it may
so simulate acute abdomen as to lead to exploratory laparotomy. Complete recovery
between attacks is usual. Any part of the nervous system may be involved, with evidence
for autonomic and peripheral neuropathy. Peripheral neuropathy may be symmetric or
asymmetric and mild or profound; in the latter instance, it can even lead to quadriplegia
with respiratory paralysis. Other central nervous system manifestations include seizures,
psychosis, and abnormalities of the basal ganglia. Hyponatremia may further cause or
exacerbate central nervous system manifestations.
Laboratory Findings
169
Often there is profound hyponatremia. The diagnosis can be confirmed by demonstrating an
increased amount of porphobilinogen in the urine during an acute attack. Freshly voided
urine is of normal color but may turn dark upon standing in light and air.
Prevention
Avoidance of factors known to precipitate attacks of acute intermittent porphyria—
especially drugs can reduce morbidity. Starvation diets also cause attacks and so must be
avoided.
Treatment
Treatment with a high-carbohydrate diet diminishes the number of attacks in some patients
and is a reasonable empiric gesture considering its benignity. Acute attacks may be life-
threatening and require prompt diagnosis, withdrawal of the inciting agent (if possible), and
treatment with analgesics and intravenous glucose and hematin. Electrolyte balance
requires close attention. Liver transplantation may provide an option for patients with
disease poorly controlled by medical therapy.
3) Porphyria Cutanea Tarda
PCT, the most common of the Porphyrias, can be either sporadic (type 1) or familial (types
2 and 3) and can also develop after exposure to halogenated aromatic hydrocarbons.
Hepatic URO-decarboxylase is deficient in all types of PCT, and for clinical symptoms to
manifest, this enzyme deficiency must be substantial (~20% of normal activity or less).
Clinical Features
Blistering skin lesions that appear most commonly on the backs of the hands are the major
clinical feature .These rupture and crust over, leaving areas of atrophy and scarring. Lesions
may also occur on the forearms, face, legs, and feet. Occasionally, the skin over sun-
exposed areas becomes severely thickened, with scarring and calcification that resembles
systemic sclerosis. Neurologic features are absent.
A number of susceptibility factors, can be recognized clinically and can affect management.
These include hepatitis C, HIV, excess alcohol, elevated iron levels, and estrogens. Excess
alcohol is a long-recognized contributor, as is estrogen use in women. Multiple
susceptibility factors that appear to act synergistically can be identified in the individual
patient with PCT. Patients with PCT characteristically have chronic liver disease and
sometimes cirrhosis and are at risk for hepatocellular carcinoma.
Diagnosis
170
Porphyrins are increased in the liver, plasma, urine, and stool. The urinary ALA level may
be slightly increased, but the PBG level is normal. Urinary porphyrins consist mostly of
uroporphyrin with lesser amounts of coproporphyrin. Plasma porphyrins are also increased.
Porphyria Cutanea Tarda: Treatment
Alcohol, estrogens, iron supplements, and, if possible, any drugs that may exacerbate the
disease should be discontinued, but this step does not always lead to improvement. A
complete response can almost always be achieved by the standard therapy, repeated
phlebotomy, to reduce hepatic iron. A unit (450 mL) of blood can be removed every 1–2
weeks. The aim is to gradually reduce excess hepatic iron until the serum ferritin reaches
the lower limits of normal. Because iron overload is not marked in most cases, remission
may occur after only five or six phlebotomies; however, PCT patients with
hemochromatosis may require more treatments to bring their iron levels down to the normal
range.
An alternative when phlebotomy is contraindicated or poorly tolerated is a low-dose
regimen of chloroquine or hydroxychloroquine, both of which complex with the excess
porphyrins and promote their excretion. Hepatic imaging can diagnose or exclude
complicating hepatocellular carcinoma. Treatment of PCT in patients with end-stage renal
disease is facilitated by administration of erythropoietin.
Sun screen lotions and beta carotene are recommended to prevent skin damage caused by
sun light.
4 ) Hereditary Coproporphyria
HCP is an autosomal dominant hepatic porphyria that results from the half-normal activity
of COPRO-oxidase. The disease presents with acute attacks, as in AIP. Cutaneous
photosensitivity also may occur, but much less commonly than in VP. HCP patients may
have acute attacks and cutaneous photosensitivity together or separately. HCP is less
common than AIP and VP.
Clinical Features
HCP is influenced by the same factors that cause attacks in AIP. The disease is latent
before puberty, and symptoms, which are virtually identical to those of AIP, are more
common in women. HCP is generally less severe than AIP. Blistering skin lesions are
identical to PCT and VP.
Diagnosis
COPRO III is markedly increased in the urine and feces in symptomatic disease and often
persists, especially in feces, when there are no symptoms. Urinary ALA and PBG levels are
increased (but less than in AIP) during acute attacks but may revert to normal more quickly
171
than in AIP when symptoms resolve. Plasma porphyrins are usually normal or only slightly
increased, but they may be higher in cases with skin lesions. The diagnosis of HCP is
readily confirmed by increased fecal porphyrins consisting almost entirely of COPRO III,
which distinguishes it from other porphyrias. An increase in the fecal COPRO III/COPRO I
ratio is useful for detecting latent cases.
Although the diagnosis can be confirmed by measuring COPRO-oxidase activity, the
assays for this mitochondrial enzyme are not widely available and require cells other than
erythrocytes.
Hereditary Coproporphyria: Treatment
Neurologic symptoms are treated as in AIP .Phlebotomy and chloroquines are ineffective
when cutaneous lesions are present.
5) Variegate Porphyria
VP is an autosomal dominant hepatic porphyria that results from the deficient activity of
PROTO-oxidase, the seventh enzyme in the heme pathway, and can present with
neurologic symptoms, photosensitivity, or both. VP is particularly common in South
Africa, where 3 of every 1000 whites have the disorder.
Clinical Features
VP can present with skin photosensitivity, acute neurovisceral crises, or both. Acute attacks
are identical to those in AIP and are precipitated by the same factors as AIP. Blistering skin
manifestations are similar to those in PCT but are more difficult to treat and usually are of
longer duration. Homozygous VP is associated with photosensitivity, neurologic
symptoms, and developmental disturbances, including growth retardation, in infancy or
childhood.
Diagnosis
Urinary ALA and PBG levels are increased during acute attacks but may return to normal
more quickly than in AIP. Increases in fecal protoporphyrin and COPRO III and in urinary
COPRO III are more persistent. Plasma porphyrin levels also are increased, particularly
when there are cutaneous lesions. VP can be distinguished rapidly from all other porphyrias
by examining the fluorescence emission spectrum of porphyrins in plasma at neutral pH
since VP has a unique fluorescence peak at neutral pH.
Assays of PROTO-oxidase activity in cultured fibroblasts or lymphocytes are not widely
available.
Variegate Porphyria: Treatment
172
Acute attacks are treated as in AIP, and hemin should be started early in most cases. Other
than avoiding sun exposure, there are few effective measures for treating the skin lesions.
Beta-Carotene, phlebotomy, and chloroquine are not helpful.
B) The Erythropoietic Porphyrias
In the erythropoietic porphyrias, excess porphyrins from bone marrow erythrocyte

precursors are transported via the plasma to the skin and lead to cutaneous photosensitivity.
1) X-Linked Sideroblastic Anemia

XLSA results from the deficient activity of the erythroid form of ALA-synthase and is
associated with ineffective erythropoiesis, weakness, and pallor.
Clinical Features
Typically, males with XLSA develop refractory hemolytic anemia, pallor, and weakness
during infancy. They have secondary hypersplenism, become iron overloaded, and can
develop hemosiderosis. The severity depends on the level of residual erythroid ALA-
synthase activity and on the responsiveness of the specific mutation to Pyridoxal 5´-
phosphate supplementation.
Diagnosis
Peripheral blood smear reveals a hypochromic, microcytic anemia with striking
anisocytosis, poikilocytosis, and polychromasia; the leukocytes and platelets appear
normal. Hemoglobin content is reduced, and the mean corpuscular volume and mean
corpuscular hemoglobin concentration are decreased.
Bone marrow examination reveals hypercellularity with a left shift ,megaloblastic
erythropoiesis with an abnormal maturation. A variety of Prussian blue–staining
sideroblasts are observed.
Levels of urinary porphyrin precursors and of both urinary and fecal porphyrins are normal.
The level of erythroid ALA-synthase is decreased in bone marrow, but this enzyme is
difficult to measure in the presence of the normal ALA-synthase housekeeping enzyme.
Definitive diagnosis requires the demonstration of mutations in the erythroid ALAS gene.
X-Linked Sideroblastic Anemia: Treatment
The severe anemia may respond to pyridoxine supplementation. This cofactor is essential
for ALA-synthase activity, and mutations in the pyridoxine binding site of the enzyme have
been found in several responsive patients. Cofactor supplementation may make it possible
to eliminate or reduce the frequency of transfusion. Unresponsive patients may be
transfusion-dependent and require chelation therapy.
2) Congenital Erythropoietic Porphyria
173
CEP, also known as Günther disease, is an autosomal recessive disorder. It is due to the
markedly deficient, but not absent, activity of URO-synthase and the resultant
accumulation of uroporphyrin I and coproporphyrin I isomers. CEP is associated with
hemolytic anemia and cutaneous lesions.
Clinical Features
Severe cutaneous photosensitivity begins in early infancy. The skin over light-exposed
areas is friable, and bullae and vesicles are prone to rupture and infection. Skin thickening,
hypo- and hyperpigmentation, and hypertrichosis of the face and extremities are
characteristic. Secondary infection of the cutaneous lesions can lead to disfigurement of the
face and hands. Porphyrins are deposited in teeth and in bones. As a result, the teeth are
reddish-brown and fluoresce on exposure to long-wave ultraviolet light. (Erythrodontia)-
See figure Hemolysis is probably due to the marked increase in erythrocyte porphyrins and
leads to splenomegaly. Adults with a milder form of the disease also have been described.
Diagnosis
Uroporphyrin and coproporphyrin (mostly type I isomers) accumulate in the bone marrow,
erythrocytes, plasma, urine, and feces. The predominant porphyrin in feces is
coproporphyrin I. The diagnosis of CEP can be confirmed by demonstration of markedly
deficient URO-synthase activity and/or the identification of specific mutations in the UROS
gene. The disease can be detected in utero by measuring porphyrins in amniotic fluid and
URO-synthase activity in cultured amniotic cells or chorionic villi, or by the detection of
the family's specific gene mutations.
Congenital Erythropoietic Porphyria: Treatment
Severe cases often require transfusions for anemia. Chronic transfusions of sufficient blood
to suppress erythropoiesis are effective in reducing porphyrin production but results in iron
overload. Splenectomy may reduce hemolysis and decrease transfusion requirements.
Protection from sunlight and from minor skin trauma is important. Beta Carotene may be of
some value. Complicating bacterial infections should be treated promptly. Recently, bone
marrow and cord blood transplantation has proven effective in several transfusion-
dependent children, providing the rationale for stem-cell gene therapy.
3) Erythropoietic Protoporphyria
EPP is an inherited disorder resulting from the partial deficiency of ferrochelatase activity,
the last enzyme in the heme biosynthetic pathway. EPP is the most common erythropoietic
porphyria in children and, after PCT, the second most common porphyria in adults. EPP
patients have ferrochelatase activities as low as 15–25% in lymphocytes and cultured
fibroblasts. Protoporphyrin accumulates in bone marrow reticulocytes and then appears in
plasma, taken up in the liver, and excreted in bile and feces.
174
Clinical Features
Skin photosensitivity, which differs from that in other porphyrias, usually begins in
childhood and consists of pain, redness, and itching occurring within minutes of sunlight
exposure .Photosensitivity is associated with substantial elevations in erythrocyte
protoporphyrin and occurs only in patients with genotypes that result in Ferrochelatase
activities below ~35% of normal. Redness, swelling, burning, and itching can develop
shortly after sun exposure Symptoms may seem out of proportion to the visible skin
lesions.
Although EPP is an erythropoietic porphyria, up to 20% of EPP patients may have minor
abnormalities of liver function, and in about 5% of these patients the accumulation of
protoporphyrins causes chronic liver disease that can progress to liver failure and death.
Diagnosis
A substantial increase in erythrocyte protoporphyrin, which is predominantly free and not

complexed with zinc, is the hallmark of this disease. Protoporphyrin levels are also variably
increased in bone marrow, plasma, bile, and feces.
Erythropoietic Protoporphyria: Treatment
Avoiding sunlight exposure and wearing clothing designed to provide protection for
conditions with chronic photosensitivity are essential. Oral Beta-carotene improves
tolerance to sunlight in many patients. The beneficial effects of -carotene may involve
quenching of singlet oxygen or free radicals.
Treatment of hepatic complications, which may be accompanied by motor neuropathy, is

difficult. Cholestyramine and other porphyrin absorbents such as activated charcoal may
interrupt the enterohepatic circulation of protoporphyrin and promote its fecal excretion,
leading to some improvement. Splenectomy may be helpful when the disease is
accompanied by hemolysis and significant splenomegaly. Plasmapheresis and intravenous
hemin are sometimes beneficial.
Liver transplantation has been carried out in some EPP patients with severe liver
complications and is often successful in the short term. Bone marrow transplantation is
considered after the liver
175
Figure-2 showing Erythtrodontia in Congenital Erythropoietic porphyria
176
CASE STUDY- REFSUM’S DISEASE
A 6-year- old child with progressive hearing loss was brought for consultation.
History revealed that the child was born normal but progressively developed loss of
hearing and loss of smell. From the past few months the child was finding it difficult
to locate the things at night time.
The child had dysmorphic features, a flat bridge of nose, and low-set ears.
On examination, pulse was irregular and the liver was enlarged. Laboratory
investigations revealed low levels of plasma cholesterol, HDL and LDL. A diagnosis of
Refsum disease was made.
What is the defect in this disease?
Refsum disease
Refsum disease (RD) is a neurocutaneous syndrome that is characterized biochemically by
the accumulation of phytanic acid in plasma and tissues. Refsum first described this
disease. Patients with Refsum disease are unable to degrade phytanic acid because of a
deficient activity of Phytanic acid oxidase enzyme catalyzing the phytanic acid alpha-
oxidation.
Peripheral polyneuropathy, cerebellar ataxia, retinitis pigmentosa, and
Ichthyosis (rough, dry and scaly skin) are the major clinical components. The
symptoms evolve slowly and insidiously from childhood through adolescence and early
adulthood.
Biochemical defect
Refsum disease is an Autosomal recessive disorder characterized by defective alpha-

oxidation of phytanic acid. Consequently, this unusual, exogenous C20-branched-chain (3,
7, 11, 15-tetramethylhexadecanoic acid) fatty acid accumulates in brain, blood and other
tissues. It is almost exclusively of exogenous origin and is delivered mainly from dietary
plant chlorophyll and, to a lesser extent, from animal sources. Blood levels of phytanic acid
are increased in patients with Refsum disease. These levels are 10-50 mg/dL, whereas
normal values are less than or equal to 0.2 mg/dL, and account for 5-30% of serum lipids.
Phytanic acid replaces other fatty acids, including such essential ones as Linoleic and
Arachidonic acids, in lipid moieties of various tissues. This situation leads to an essential
fatty acid deficiency, which is associated with the development of ichthyosis.
Reactions-
Phytanic acid
177
This process involves hydroxylation of the alpha carbon, removal of the terminal carboxyl
group and concomitant conversion of the alpha hydroxyl group to a terminal carboxyl
group, and linkage of CoA to the terminal carboxyl group. This branched substrate will
function in the beta-oxidation process, ultimately yielding propionyl-CoA, acetyl Co As
and, in the case of phytanic acid, 2-methyl propionyl CoA (Iso butyryl Co A).
Figure- showing steps of oxidation of phytanic acid. Pristanic acid is formed by alpha
oxidation which subsequently undergoes beta oxidation to yield the final products.
178
Refsum disease is rare, with just 60 cases observed so far.
Classic Refsum disease manifests in children aged 2-7 years; however, diagnosis usually is
delayed until early adulthood. Infantile Refsum disease makes its appearance in early
infancy. Symptoms develop progressively and slowly with neurologic and ophthalmic
manifestations. The disease is characterized by
• Night blindness due to degeneration of the retina (retinitis pigmentosum)
• Loss of the sense of smell (anosmia)
• Deafness
• Concentric constriction of the visual fields
• Cataract
• Signs resulting from cerebellar ataxia –

o Progressive weakness
o Foot drop
o Loss of balance
• Cardiac arrhythmias
• Some individuals will have shortened bones in their fingers or toes.
• The children usually have moderately dysmorphic features that may include epicanthal
folds, a flat bridge of the nose, and low-set ears.
Laboratory Diagnosis
• Levels of plasma cholesterol and high- and low-density lipoprotein are often
moderately reduced.
• Blood phytanic acid levels are elevated.
• Cerebrospinal fluid (CSF) shows a protein level of 100-600 mg/dl.
• Routine laboratory investigations of blood and urine do not reveal any consistent
diagnostic abnormalities.
• Phytanic oxidase activity estimation in skin fibroblast cultures is important
Imaging
Skeletal radiography is required to estimate bone changes.
179
Treatment
• Eliminate all sources of chlorophyll from the diet.

o The major dietary exclusions are green vegetables (source of phytanic acid)
and animal fat (phytol).
o The aim of such dietary treatment is to reduce daily intake of phytanic acid
from the usual level of 50 mg/d to less than 5 mg/d.
• Plasmapheresis - Patients may also require plasma exchange (Plasmapheresis) in
which blood is drawn, filtered, and reinfused back into the body, to control the
buildup of phytanic acid.
o The main indication for Plasmapheresis in patients with Refsum disease is a

severe or rapidly worsening clinical condition.
o A minor indication is failure of dietary management to reduce a high plasma
phytanic acid level.
Prognosis
Prognosis in untreated patients generally is poor. Dysfunction of myelinated nerve fibers
and the cardiac conduction system leads to central and peripheral neuropathic symptoms,
impaired vision, and cardiac arrhythmias. The latter frequently are the cause of death.
In early diagnosed and treated cases, phytanic acid decreases slowly, followed by
improvement of the skin scaling and, to a variable degree, reversal of recent neurological
signs. Retention of vision and hearing are reported.
Pharmacological up regulation of the omega-oxidation of phytanic acid may form the basis
of the new treatment strategy for adult Refsum disease in the near future.
180
CASE STUDY- LIPID DISORDERS – SELF ASSESSMENTS
A thiry six year old man consulted an optician to obtain a prescription for reading
glasses. The optician noticed that the patient had bilateral arcus senilis, and
recommended that he consult to general medical practitioner. The general
practitioner found that he also had tendon xanthoma, arising from the Achilles
tendons. Blood pressure was normal; his father had died of a heart attack at the age of
forty years. An ECG taken at rest was normal but ischemic changes developed on
exercise. Analysis of fasting blood for lipids showed the following
Serum Cholesterol : 725 mg/dl

Serum Triglyceride : 149 mg/dl
LDL-cholesterol : 538 mg/dl
HDL-cholesterol : 40 mg/dl
Serum electrophoresis had shown excess of beta lipoprotein.
Give your comments on the findings and interpret the case.
A forty year old male was found to have a plasma cholesterol level of 10.6 mmol/l
(normal is <5.5mmol/l). The doctor recommended a cholesterol free formula for 3
months, at the end of which his plasma cholesterol level was decreased to only 7.7
mmol/l.
In an effort to decrease his plasma cholesterol level further he was treated with
cholestepol hydrochloride, a bile acid binding resin. The resin is not absorbed and
remains in the intestinal lumen, where it binds bile acids, causing increased amounts
to be excreted in the faeces.
Following the drug treatment, plasma cholesterol concentration was lowered to 5.8
mmol/l, an acceptable value for this patient.
181
1. Why does this patient’s cholesterol level remain high despite being on cholesterol
free diet for three months? Other than restricting the cholesterol intake what other dietary
precautions should the patient take?
Explain the biochemical reasons for your answer.
2. How is the synthesis of cholesterol regulated?
3. How does cholesterol hydrochloride lower the plasma cholesterol concentration?

Would treatment with lovastatin have a similar effect on the plasma cholesterol level?
Explain your answer.
A patient attended the general out patient department of a hospital with the following
symptoms, i.e., puffiness of face, oedema of feet and generalized weakness. His
biochemical findings were:

Serum Albumin : 2.5 g/dl
Serum Globulin : 3.0 g/dl
Urinary protein : 4 gm/24 hours
Interpret the report and comment on it
A middle aged man was referred by his family doctor to a dermatologist because of
yellowish papules, with erythematous bases, on his buttocks and elbow. The
dermatologist recognized these as eruptive xanthomata and noticed hat there were
yellow, fatty streaks in the palmer creases. Blood was drawn after an overnight fat for
lipid analysis and the serum was seen to be slightly turbid.

Serum Triglyceride : 670 mg/dl
182
Serum electrophoresis had shown broad beta and a trace of chylomicron.
Give your comments on the findings and interpret the case.
183
CASE STUDY- THYROID DISORDERS – SELF ASSESSMENTS
A thirty years old woman married 5 years ago had no children complained of
tiredness, weight gain, cold intolerance, neuromuscular pains and constipation. She
was found to be anemic.
The laboratory report is given below

T3 0.3 ng/ml (0.5 – 1.85 ng/ml)
T4 2.4 ȝg/dl (4.4 - 10.8 ȝg/dl)
TSH 7.9 ȝIU/ml (0.4 - 6.2 ȝIU/ml)
Comment on the laboratory reports
A fifteen years old girl complained of weight loss, nervousness, anxiety, sensitivity to
heat, increased pulse rate (tachycardia).
The laboratory findings are given below:

T3 2.3 ng/ml (0.5 – 1.85 ng/ml)
T4 14.0 ȝg/dl (4.4 - 10.8 ȝg/dl)
TSH 0.3 ȝIU/ml (0.4 - 6.2 ȝIU/ml)
Comment on the laboratory findings
A forty nine years old female had the following baseline laboratory results

TSH 17.8 ȝIU/ml (0.4 - 6.2 ȝIU/ml)
T4 2.4 ȝg/dl (4.4 - 10.8 ȝg/dl)
What is the original diagnosis?

Why her TSH value is elevated?
A thirty years old woman married 5 years ago had no children complained of
tiredness, weight gain, cold intolerance, neuromuscular pains and constipation. She
was found to be anemic.

T3 0.3 ng/ml (0.5 – 1.85 ng/ml)
T4 2.4 ȝg/dl (4.4 - 10.8 ȝg/dl)
TSH 7.9 ȝIU/ml (0.4 - 6.2 ȝIU/ml)
Cholesterol 270 mg/dl (150-240 mg/dl)
184
Comment on the laboratory reports
A forty years old female had the following baseline laboratory results:

T4 5.8 ȝg/dl (4.4 - 10.8 ȝg/dl)
TSH 7.9 ȝIU/ml (0.4 - 6.2 ȝIU/ml)
What is the original diagnosis?

Why her TSH value is elevated?
A twenty-four year old physiotherapist consulted her physician because she had
excessive moistness of her skin and was causing embarrassment at work. She was also
concerned that her eyes seemed to have become more prominent, her doctor observed
that her pulse rate was 92 /minute and she has a slightly enlarged thyroid gland.

fT3 12.0 pmol/L (3.0 – 8.8 pmol/L)
fT4 34 pmol/L (9 - 26 pmol/L)
TSH <0.1 ȝIU/ml (0.4 - 6.2 ȝIU/ml)
Autoantibodies TSIg were detected in her titer
Interpret on the laboratory reports
A fifty year old lady teacher complains of hoarseness of voice and feeling of tiredness.
She admitted that she had started putting on weight and feeling comfortable in warm
weather. After initial examination by doctor she was referred to the endocrinologist.

RBS 95 mg/dl (<126 mg/dl)
Cholesterol 285 mg/dl (150-240 mg/dl)
T3 7.0 ng/ml (0.5 – 1.85 ng/ml)
T4 4.0 ȝg/dl (4.4 - 10.8 ȝg/dl)
TSH 7.2 ȝIU/ml (0.4 - 6.2 ȝIU/ml)
Interpret on the laboratory reports
Why TSH has increased while T4 is lowered?
185
CASE STUDY- MECHANIMS OF ACTION OF HEPARIN
A 54 –year- old woman who was bed bound in a nursing home began to develop
swelling of her left leg. She was evaluated with venous Doppler ultrasound and was
found to have a deep vein thrombosis. She was immediately started on heparin to
prevent the clot from further enlarging.
What is the chemical nature of Heparin?
How will it help in preventing the clot formation?
Case Discussion-
Heparin
Heparin also called Į Heparin, is a highly sulfated GAG (Glycosaminoglycans). It is an

anticoagulant widely used in clinical practice. It is present in liver, lungs, spleen and
monocytes. Commercial preparations are from animal lung tissues. It contains repeating
units of sulphated Glucosamine and either of the two uronic acids-D-Glucuronic acid and
L-Iduronic acid. In fully formed Heparin molecules 90% or more of uronic acid residues
are L-Iduronic acid. It is strongly acidic due to sulphuric acid groups and readily forms
salts.
Clinical role of Heparin
In vitro Heparin is used as an anticoagulant while taking blood samples, 2 mg/10 ml of

blood is used. It is considered the most satisfactory anti coagulant as it does not produce a
change in red cell volume or interfere with subsequent determinations.
In vivo Heparin is used in suspected thromboembolic conditions to prevent intravascular
coagulation. Heparin is used for anticoagulation for the following conditions:
• Acute coronary syndrome,

• Atrial fibrillation
• Deep-vein thrombosis and pulmonary embolism.
• Cardiopulmonary bypass for heart surgery.
Mechanism of action
Role of heparin as an anti-coagulant

It produces its major anticoagulant effect by inactivating thrombin and activated factor X
(factor Xa) through an antithrombin (AT)-dependent mechanism. Heparin binds to AT
through a high-affinity pentasaccharide.(See figure-1)
186
Figure-1- showing the binding of heparin to antithrombin. To potentiate thrombin
inhibition, heparin must simultaneously bind to antithrombin and thrombin.
Binding of Heparin to lysine residues in antithrombin produces conformational changes

which promote the binding of the latter to serine protease “thrombin” which is inhibited,
thus fibrinogen is not converted to fibrin and the coagulation is inhibited.(See figure -2)
Figure-2- showing the mechanism of action of Heparin. Heparin has a multitude of effects
on the clotting cascade; however, the primary sites of action are the inhibition of factor II,
also called thrombin, and factor X.
187
Role of Heparin as a coenzyme
Heparin acts in the body to potentiate the activity of the enzyme “Lipoprotein lipase”.
Heparin binds specifically to the enzyme present in capillary walls causing its release in to
the circulation. Hence it is also called “releasing factor”.
Administration of Heparin
Heparin is given parenterally, as it is degraded when taken by mouth. It can be injected

intravenously or subcutaneously. Intramuscular injections are avoided because of the
potential for forming hematomas.
Because of its short biologic half-life of approximately one hour, heparin must be given
frequently or as a continuous infusion. However, the use of low-molecular-weight heparin
(LMWH) has allowed once-daily dosing, thus not requiring a continuous infusion of the
drug. If long-term anticoagulation is required, heparin is often used only to commence
anticoagulation therapy until the oral anticoagulant Warfarin takes effect.
Adverse effects
The most common side effect is bleeding .The risk of bleeding increases with higher
dosage.
A serious side-effect of heparin is heparin-induced thrombocytopenia (HIT). HIT is
caused by an immunological reaction that makes platelets a target of immunological
response, resulting in the degradation of platelets. This is what causes thrombocytopenia.
This condition is usually reversed on discontinuation, and can generally be avoided with the
use of synthetic heparins. There is also a benign form of thrombocytopenia associated with
early heparin use, which resolves without stopping heparin.
There are two nonhemorrhagic side-effects of heparin treatment. The first is elevation of
serum Aminotransferases levels, which has been reported in as many as 80% of patients
receiving heparin. This abnormality is not associated with liver dysfunction, and it
disappears after the drug is discontinued. The other complication is hyperkalemia, which
occurs in 5 to 10% of patients receiving heparin, and is the result of heparin-induced
aldosterone suppression. The hyperkalemia can appear within a few days after the onset of
heparin therapy.
Osteoporosis - has also been reported with long term Heparin therapy, since Heparin
causes bone loss both by decreasing bone formation and by enhancing bone resorption.
188
BIOLOGICAL OXIDATION
Complete oxidation of glucose in skeletal muscle yields 36 ATPs whie in other tissues
the yield is 38- What is the reason?
Under aerobic conditions regeneration of cytosolic NAD+ from cytosolic NADH is

accomplished by transferring electrons across the mitochondrial membrane barrier to the
electron transport chain where the electrons are transferred to oxygen.
There are two different shuttle mechanisms whereby this transfer of electrons across the
membrane to regenerate cytosolic NAD+ can be accomplished, the glycerol 3-phosphate
shuttle and the malate-aspartate shuttle.
1) The glycerol 3-phosphate shuttle (Figure-1) functions primarily in skeletal muscle and
brain. The shuttle takes advantage of the fact that the enzyme glycerol-3-phosphate
dehydrogenase exists in two forms, a cytosolic form that uses NAD+ as cofactor and a
mitochondrial FAD-linked form.
Figure-1- showing glycerol-3-Phosphate shuttle. G3P- glycerol-3-P, DHAP- Dihydroxy

acetone-Phosphate
189
Cytosolic glycerol-3-phosphate dehydrogenase uses electrons from cytosolic
NADH to reduce the glycolytic intermediate dihydroxyacetone phosphate to glycerol 3-
phosphate, thereby regenerating cytosolic NAD+. The newly formed
glycerol 3-phosphate is released from the cytosolic form of the enzyme and crosses to and
is bound to the mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase, which is
bound to the cytosolic side of the mitochondrial inner membrane. There the mitochondrial
glycerol-3-phosphate dehydrogenase reoxidizes glycerol 3-phosphate to dihydroxyacetone
phosphate (preserving mass balance) reducing its FAD cofactor to FADH2. Electrons are
then passed through complex II to coenzyme Q of the electron transport chain and on to
oxygen generating two ATP molecules per electron pair and therefore per glycerol -3-
phosphate.
2) The malate-aspartate shuttle, however, functions primarily in the heart, liver, and
kidney (Figure 2). This shuttle requires cytosolic and mitochondrial forms of malate
dehydrogenase and glutamate-oxaloacetate transaminase and two antiporters, the malate-Į-
ketoglutarate antiporter and the glutamate aspartate antiporter, which are both localized in
the mitochondrial inner membrane.
In this shuttle cytosolic NADH is oxidized to regenerate cytosolic

NAD+ by reducing oxaloacetate to malate by cytosolic malate dehydrogenase.(1)
Figure-2-Showing Malate Aspartate shuttle

190
Malate is transported into the mitochondrial matrix while Į-ketoglutarate is transported out
by the malate-Į-ketoglutarate antiporter, a seeming mass unbalance(2).
Next malate is oxidized back to oxaloacetate producing NADH from NAD+ in the
mitochondrial matrix by mitochondrial malate dehydrogenase (3).
Oxaloacetate cannot be transported per se across the mitochondrial membrane. It is, instead
transaminated to aspartate from the NH3 donor glutamate by mitochondrial glutamate-
oxaloacetate transaminase (4). Aspartate is transported out of the matrix whereas glutamate
is transported in by the glutamate-aspartate antiporter (5)in the mitochondrial membrane,
obviating the apparent mass unbalance noted above.
The last step of the shuttle is catalyzed by cytosolic glutamate-oxaloacetate transaminase

regenerating cytosolic oxaloacetate from aspartate and cytosolic glutamate from Į-
ketoglutarate(6) both of which were earlier transported in opposing directions by the
malate- Į- ketoglutarate antiporter.
The net effect of this shuttle is to transport electrons from cytosolic NADH to
mitochondrial NAD+. Therefore, those electrons can be presented by the newly formed
NADH to electron transport system complex I thereby producing three ATPs by oxidative
phosphorylation.
Note that depending on which shuttle is used (i.e., which tissue is catalyzing glycolysis)
either two or three ATPs are produced by oxidative phosphorylation per triose phosphate
going through the latter steps of glycolysis.
191
ATP Formation in the Catabolism of Glucose
Pathway Reaction Catalyzed by Method of Formation ATP per Mol
ATP of Glucose
Glycolysis Glyceraldehyde 3- Respiratory chain 6*
phosphate dehydrogenase oxidation of 2 NADH
Phosphoglycerate kinase Substrate level 2
phosphorylation
Pyruvate kinase Substrate level 2
phosphorylation
Total yield 10
Consumption of ATP for reactions of hexokinase and –2
phosphofructokinase
Net 8
Citric acid Pyruvate dehydrogenase Respiratory chain 6
cycle oxidation of 2 NADH
Isocitrate dehydrogenase Respiratory chain 6
oxidation of 2 NADH
Į-Ketoglutarate Respiratory chain 6
dehydrogenase oxidation of 2 NADH
Succinate thiokinase Substrate level 2
phosphorylation
Succinate dehydrogenase Respiratory chain 4
oxidation of 2 FADH2
Malate dehydrogenase Respiratory chain 6

oxidation of 2 NADH
Net 30
Total per mol of glucose under aerobic conditions 38
Total per mol of glucose under anaerobic conditions 2
This assumes that NADH formed in glycolysis is transported into mitochondria by

the malate shuttle.
If the Glycerol-phosphate shuttle is used, then only 2 ATP will be formed per mol
of NADH. At the step of glyceraldehyde-3-P dehydrogenase-4 ATP will be produced.
Hence the total will be-6+30= 36 ATP. ( 6 from Glycolysis and 30 from PDH complex and
TCA cycle. Since this shuttle operates. in skeletal muscle and brain, hence the total yield
per glucose mol will be 2 ATP less as compared to other tissues.
Clinical Significance
In nonaerobic glycolysis, as in the case when a tissue is subjected to an ischemic episode

(i.e., myocardial infarction), neither the ATP produced by the shuttle nor the ATPs
192
produced by normal passage of electrons through the electron transport chain are produced
because of oxygen insufficiency.
Therefore glycolysis must increase in rate to meet the energy demand. In damaged
tissue this increased rate is compromised. Moreover the shuttle mechanisms to regenerate
NAD+ from NADH formed by glycolysis are unavailable.
Glycolysis under ischemic conditions satisfies the requirement for NAD+ by reducing
pyruvate, the glycolytic end product under normal conditions, to lactate with the reducing
equivalents of NADH.
The new end product lactate accumulates in muscle cells under ischemic conditions and
damages cell walls with its low pH causing rupture and loss of cell contents such as
myoglobin and troponin I. These compounds as well as other end products combine to
cause increased cell rupture and pain.
193
ISOENZYMES AND THEIR CLINICAL SIGNIFICANCE
Isoenzymes are enzymes that catalyze identical chemical reactions but are composed of
different amino acid sequences. They are sometimes referred to as isozymes. Isoenzymes
are produced by different genes and are not redundant despite their similar functions. They
occur in many tissues throughout the body and are important for different developmental
and metabolic processes.
Isoenzymes are useful biochemical markers and can be measured in the bloodstream to
diagnose medical conditions. Isoenzymes can be differentiated from one another using gel
electrophoresis. In gel electrophoresis, isoenzyme fragments are drawn through a thick gel
by an electric charge. Each isoenzyme has a distinct charge of its own because of its unique
amino acid sequence. This enables gel electrophoresis to separate the fragments into bands
for identification. Some clinically important isoenzymes are as follows-
1) Creatine Kinase (CK, CPK) is an enzyme found primarily in the heart and skeletal
muscles, and to a lesser extent in the brain but not found at all in liver and kidney. Small
amounts are also found in lungs, thyroid and adrenal glands .Significant injury to any of
these structures will lead to a measurable increase in CK levels. It is not found in red blood
cells and its level is not affected by hemolysis.
Normal Value- serum activity varies from 10-50 IU/L at 30°C.
Elevations are found in:
• Myocardial infarction
• Crushing muscular trauma
• Any cardiac or muscle disease, but not myasthenia gravis or multiple sclerosis
• Brain injury
• Hypothyroidism
• Hypokalemia
After myocardial infarction- serum value is found to increase within 3-6 hours, reaches a
peak level in 24- 30 hours and returns to normal level in 2-4 days (usually in 72 hours). CK
is a sensitive indicator in the early stages of myocardial ischemia. No increase in activity is
found in heart failure and coronary insufficiency. In acute MI, CPK usually rises faster than
SGOT and returns to normal faster than the SGOT.
CK/CPK Isoenzymes
There are three Isoenzymes. Measuring them is of value in the presence of elevated levels
of CK or CPK to determine the source of the elevation. Each iso enzyme is a dimer
194
composed of two protomers ‘M’ (for muscles) and ‘B’( for Brain). These isoenzymes can
be separated by, Electrophoresis or by Ion exchange Chromatography. The three possible
iso enzymes are;
Isoenzyme Electrophoretic mobility Tissue of origin Mean percentage in

blood
MM(CK3) Least Skeletal muscle 97-100%
Heart muscle
MB(CK2) Intermediate Heart muscle 0-3%
BB(CK1) Maximum Brain 0%
• Normal levels of CK/CPK are almost entirely MM, from skeletal muscle.
• Elevated levels of CK/CPK resulting from acute myocardial infarction are about
half MM and half MB. Myocardial muscle is the only tissue that contains more than
five percent of the total CK activity as the CK2 (MB) isoenzyme.
• Following an attack of acute myocardial infarction, this isoenzyme appears within 4
hours following onset of chest pain, reaches a peak of activity at approximately 24
hours and falls rapidly. MB accounts for 4.5- 20 % of the total CK activity in the
plasma of the patients with recent myocardial infarction and the total isoenzyme is
elevated up to 20-folds above the normal.
• Atypical Isoenzymes- In addition to the above three isoenzymes two atypical iso
enzymes of CPK have been reported. They are; Macro CK(CK-Macro) and
Mitochondrial CK(CK-Mi).
o Macro CK(CK-Macro)- It is formed by the aggregation of CK-BB with

immunoglobulins usually with IgG but sometimes Ig A. It may also be formed by
complexing CK-MM with lipoproteins. No specific disease has been found to be associated
with this isoenzyme.
o Mitochondrial CK(CK-Mi)- It is present bound to the exterior surface of the inner
mitochondrial membrane of muscle, liver and brain. It can exist in dimeric form or as
oligomeric aggregates having molecular weight of approximately 35,000. It is only present
in serum when there is extensive tissue damage causing breakdown of mitochondrial
membrane and cell membrane. Thus its presence in serum indicates severe illness and
cellular damage. It is not related with any specific disease states but it has been detected in
certain cases of malignant tumors.
2) Aspartate amino Transferase (AST)

It is also called as Serum Glutamate Oxalo acetate Transaminase (SGOT). The level is
significantly elevated in Acute MI.
195
Normal Value- 0-41 IU/L at 37°C
In acute MI- Serum activity rises sharply within the first 12 hours, with a peak level at 24
hours or over and returns to normal within 3-5 days. The rise depends on the extent of
infarction. Re- infarction results in secondary rise of SGOT.
Prognostic significance- Levels> 350 IU/L are due to massive Infarction (Fatal),
> 150 IU/L are associated with high mortality and levels < 50 IU/L are associated with low
mortality.
Other diseases- The rise in activity is also observed in muscle and hepatic diseases. These
can be well differentiated from simultaneous estimations of other enzyme activities like
SGPT etc, which do not show and rise in activity in Acute MI.
3) Alanine amino transferase (ALT)- Also called serum Glutamate pyruvate

transaminase.
Normal serum level ranges between 0-45 IU/L at 37oC.

Very high values are seen in Acute hepatitis, toxic or viral in origin. Both ALT and AST
rise but ALT> AST. Moderate increase may be seen in chronic liver diseases such as
Cirrhosis and Malignancy in liver. A sudden fall in AST level in hepatitis signifies bad
prognosis.
4) Lactate dehydrogenase (LDH)

Lactate dehydrogenase catalyzes the reversible conversion of pyruvate and lactate. LDH is
essential for anaerobic respiration. When oxygen levels are low, LDH converts pyruvate to
lactate, providing a source of muscular energy.
Normal level- 55-140 IU/L at 30°C. The levels in the upper range are generally seen in
children. LDH level is 100 times more inside the RBCs than in plasma, and therefore minor
amount of hemolysis results in false positive result.
In Acute MI-The serum activity rises within 12 to 24 hours, attains a peak at 48 hours (2 to
4 days) reaching about 1000 IU/L and then returns gradually to normal from 8 th to 14 th
day. The magnitude of rise is proportional to the extent of myocardial infarction. Serum
LDH elevation may persist for more than a week after CPK and SGOT levels have returned
to normal levels.
Other diseases-The increase in serum activity of LDH is also seen in hemolytic anemias,
hepatocellular damage, muscular dystrophies, carcinoma, leukemias, and any condition
196
which causes necrosis of the body cells. Since the total LDH is increased in many diseases,
so the study of Iso enzymes of LDH is of more significance.
Iso enzymes of LDH
LDH enzyme is tetramer with 4 subunits. The subunit may be either H(Heart) or
M(Muscle) polypeptide chains. These two chains are the product of 2 different genes.
Although both of them have the same molecular weight, there are minor amino acid
variations. There can be 5 possible combinations; H4, H3M1, H2M2, H1M3. M4, these are
5 different types of isoenzymes seen in all individuals.
No. of Subunit Electrophoretic Activity at Tissue Percentage in

Isoenzyme make up of mobility at 60°for 30 origin human
isoenzyme pH8.6 minutes serum(Mean)
LDH-1 H4 Fastest Not Heart 30%
destroyed muscle
LDH-2 H3M1 Faster Not RBC 35%
destroyed.
LDH-3 H2M2 Fast Partly Brain 20%
destroyed
LDH-4 H1M3 Slow Destroyed Liver 10%
LDH-5 M4 Slowest Destroyed Skeletal 5%
Muscles
Normally LDH- 2(H3M1) level in blood is greater than LDH-1, but this pattern is reversed
in myocardial infarction, this is called ‘flipped pattern’. These iso enzymes are separated
by cellulose acetate electrophoresis at pH 8.6.
5) Alkaline phosphatase (ALP)-is an enzyme that removes phosphate groups from organic
or inorganic compounds in the body. It is present in a number of tissues including liver,
bone, intestine, and placenta. The activity of ALP found in serum is a composite of
isoenzymes from those sites and, in some circumstances, placental or Regan isoenzymes.
The optimum pH for enzyme action varies between 9-10. It is a zinc containing
metalloenzyme and is localized in the cell membranes (ectoenzyme). It is associated with
transport mechanism in the liver, kidney and intestinal mucosa.
Normal serum Level- of ALP ranges between 40-125 IU/L. In children the upper level of
normal value may be more, because of increased osteoblastic activity.
Total Alkaline Phosphatase (ALP)
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Serum ALP is of interest in the diagnosis of 2 main groups of conditions-hepatobiliary
diseases and bone diseases associated with increased osteoblastic activity.
Mild increase is observed in pregnancy, due to production of placental enzyme.
A moderate rise in ALP activity occurs in hepatic diseases such ss infective hepatitis,
alcoholic hepatitis or hepatocellular carcinoma. Moderate elevation of ALP may also be
seen in other disorders such as Hodgkin's disease, congestive heart failure, ulcerative
colitis, regional enteritis, and intra-abdominal bacterial infections.
ALP elevations tend to be more marked (more than 3-fold) in extra hepatic biliary
obstructions (eg, by stone or cancer of the head of the pancreas) than in intrahepatic
obstructions, and the more complete the obstruction, the greater the elevation. With
obstruction, serum ALP activities may reach 10 to 12 times the upper limit of normal,
returning to normal upon surgical removal of the obstruction. The ALP response to
cholestatic liver disease is similar to the response of gamma-glutamyltransferase (GGT),
but more blunted. If both GGT and ALP are elevated, a liver source of the ALP is
likely.The response of the liver to any form of biliary tree obstruction is to synthesize more
ALP. The main site of new enzyme synthesis is the hepatocytes adjacent to the biliary
canaliculi.
ALP also is elevated in disorders of the skeletal system that involve osteoblast
hyperactivity and bone remodeling, such as Paget's disease rickets and osteomalacia,
fractures, and malignant tumors.
Among bone diseases, the highest level of ALP activity is encountered in Paget's disease,
as a result of the action of the osteoblastic cells as they try to rebuild bone that is being
resorbed by the uncontrolled activity of osteoclasts. Values from 10 to 25 times the upper
limit of normal are not unusual.
Only moderate rises are observed in osteomalacia, while levels are generally normal in
osteoporosis. In rickets, levels 2 to 4 times normal may be observed. Primary and
secondary hyperparathyroidism are associated with slight to moderate elevations of ALP;
the existence and degree of elevation reflects the presence and extent of skeletal
involvement.
Very high enzyme levels are present in patients with osteogenic bone cancer.
A considerable rise in ALP is seen in children following accelerated bone growth.

Patients over age 60 can have a mildly elevated alkaline phosphatase (1–1½ times normal),
while individuals with blood types O and B can have an elevation of the serum alkaline
phosphatase after eating a fatty meal due to the influx of intestinal alkaline phosphatase into
the blood.
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ALP Isoenzymes
Electrophoresis is considered the most useful single technique for ALP isoenzyme analysis.
By starch gel electrophoresis at pH 8.6 , at least 6 isoenzyme bands can be visualized.
1) Hepatic ALP isoenzyme- moves fastest towards the anode and occupies the same
position as Alpha 2 globulins. It is associated with biliary epithelium and is elevated in
cholestatic processes. Various liver diseases (primary or secondary cancer, biliary
obstruction) increase the liver isoenzyme.
2) Bone isoenzyme- It closely follows the hepatic enzyme and occupies the beta region.
Osteoblastic bone tumors and hyperactivity of osteoblasts involved in bone remodeling (eg,
Paget's disease) increase the bone isoenzyme. Paget's disease leads to a striking, solitary
elevation of bone ALP.
3) Placental isoenzyme follows bone isoenzyme. It is heat stable isoenzyme and increases
during last six weeks of pregnancy.
4) The intestinal isoenzyme-is the slowly moving band and follows the placental
isoenzyme. It may be increased in patients with cirrhosis and in individuals who are blood
group O or B secretors. Increased levels are also sen in patients undergoing hemodialysis.
Atypical ALP isoenzymes (Oncogenic markers)- In addition to 4 major isoenzymes, 2

more abnormal fractions are seen associated with tumors. These are Regan and Nagao
isoenzymes. They are also called “Carcino placental ALP”, as they resemble placental
isoenzymes.
Regan isoenzyme is elevated in various carcinomas of breast, lungs, colon and ovaries.
Highest positivity is observed in carcinoma of ovary and uterus.
Rise in Nagao isoenzyme is observed in metastatic carcinoma of pleural surfaces and

adenocarcinoma of pancreas and bile duct.
199
MECHANISM OF IRON ABSORPTION
Iron absorption takes place largely in the proximal small intestine and is a carefully
regulated process. In general, there is no regulation of the amounts of nutrients absorbed
from the gastro intestinal tract. A notable exception is iron, the reason that absorption must
be carefully regulated is that the body does not possess a physiological mechanism to
eliminate much iron from the body. The small amount of iron that is lost each day (about 1-
2 mg) is matched by dietary absorption of iron.
Mechanism of iron absorption
Iron is found in the diet as ionic (non-haem) iron and haem iron. Absorption of these two
forms of iron occurs by different mechanisms. Absorption is a multistep process involving
the uptake of iron from the intestinal lumen across the apical cell surface of the villus
enterocytes and the transfer out of the enterocyte across the basolateral membrane to the
plasma. Ionic iron is present in the reduced (ferrous) or oxidised (ferric) state in the diet and
the first step in the uptake of ionic iron involves the reduction of iron. Recently, a reductase
that is capable of reducing iron from its ferric to ferrous state has been identified. It is a
membrane bound haem protein called Dcytb that is expressed in the brush border of the
duodenum. Next, ferrous ion is transported across the lumen cell surface by a transporter
called divalent metal transporter 1 (DMT1) that can transport a number of other metal ions
including copper, cobalt, zinc, and lead.
Figure- Showing the mechanism of iron absorption
Once inside the gut cell, iron may be stored as ferritin or transported through the cell to be
released at the basolateral surface to plasma transferrin through the membrane-embedded
200
iron exporter, ferroportin. The function of ferroportin is negatively regulated by hepcidin,
the principal iron regulatory hormone. More the Hepcidin levels lesser is the iron
absorption and vice versa. In the process of release, iron interacts with another ferroxidase,
hephaestin, which oxidizes the iron to the ferric form for transferrin binding. Hephaestin is
similar to ceruloplasmin, the copper-carrying protein.
The mechanism of absorption of haem iron has yet to be elucidated. Transfer across the
brush border membrane is probably mediated by an unidentified haem receptor. Once
inside, enterocyte iron is released from haem by haem oxygenase and either stored or
transferred out of the enterocyte by a mechanism that is likely to be similar to that for ionic
iron
Factors affecting iron absorption

Iron absorption is influenced by a number of physiologic states.
1) Erythroid hyperplasia stimulates iron absorption, even in the face of normal or
increased iron stores, and in this state hepcidin levels are inappropriately low. The
molecular mechanism underlying this relationship is not known. Thus, patients with
anemias associated with high levels of ineffective erythropoiesis absorb excess amounts of
dietary iron. Over time, this may lead to iron overload and tissue damage. In iron
deficiency, hepcidin levels are low and iron is much more efficiently absorbed from a given
diet; the contrary is true in states of secondary iron overload.
2) Hypoxia-Both the rate of erythropoiesis and hypoxia regulate iron absorption.
Expression of ferroportin and Dcytb are increased in hypoxia, resulting in more iron
absorption.
3) Body Stores- Iron absorption is stimulated if the levels of body stores are low. On the
contrary, Hepcidin is produced excessively by hepatocytes when iron stores are full,
hepcidin makes a complex with ferroportin promoting its degradation and thus iron is not
transported out of the enterocyte in to the blood. Iron remains inside the cell in the form of
ferritin till the life span of the cell
Figure- showing influence of body iron stores on iron absorption
4) Inflammation can also stimulate hepcidin production resulting in lowered iron

absorption.
201
ELECTROPHORESIS- A BRIEF REVIEW
Electrophoresis is the movement of charged particles through an electrolyte when

subjected to an electric Field
• Cations move towards cathode

• Anions move towards anode
• By this technique solutes are separated by their different rates of travel through an
electric field.
• Commonly used in biological analysis, particularly in the separations of proteins,
peptides and nucleic acids
Factors affecting Electrophoresis
The rate of migration of a solute in an electric field depends on the following factors-
1) Net charge on the particle
2) Mass and shape of the particles
3) p H of the medium
4) Strength of electric field
5) Properties of supporting medium
6) Temperature
Electrophoretic Mobility
Electrophoretic mobility is defined as the rate of migration (cm/sec) per unit field strength
(Volts/cm)
µ=Q/6ʌrȘ
Where µ- Electrophoretic mobility
Q-Net charge on the ion
r- Ionic radius of the solute
Ș- Viscosity of the medium
o The electrophoretic mobility is directly proportional to net charge and inversely

proportional to molecular size and viscosity of the electrophoresis medium
o The pH of solution affects the mobility of the ion by determining the amount and
nature of charge
o Proteins, nucleic acids, nucleotides and amino acids bear charged polar groups
making them suitable groups for electrophoresis
o Carbohydrates carrying no charged groups are first bound to charged groups like
Borate or Sulfite ions and then electrophoresis is carried out
o Lipids are not electrophoresed because electrophoretic current requires polar solvents
in which most lipids are insoluble
202
Types of Electrophoresis
1) Horizontal
2) Vertical
Vertical electrophoresis is mainly used for Polyacrylamide gel electrophoresis.
Electrophoresis Apparatus
Electrophoresis apparatus consists of-

1) Buffer tank -to hold the buffer
2) Buffer
3) Electrodes- made of platinum or carbon
4) Power supply
5) Support media
Figure showing -Electrophoresis Apparatus
Note-Choice of buffer depends on the nature of substance to be separated and the electricity
is supplied at a constant current and voltage.
The electrophoresis support on which separation takes place may contact the buffer directly
or by means of wicks.
The entire apparatus is covered to minimize separation
Support media for electrophoresis
1) Filter Paper
2) Cellulose acetate membrane
3) Agar or Agarose gel
4) Starch Gel
203
5) Polyacrylamide gel
A) Paper Electrophoresis
The support medium is a filter paper
Frequently used in isolating proteins, amino acids and oligopeptides.
Procedure-
1) A long strip of filter paper is moistened with a suitable buffer solution of the desired pH
and the sample is applied transversely across the central part of the strip
2) Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes
3) Electric field of about 20 volts/cm is established
4) The charged particles of sample migrate along the strip towards respective electrodes of
opposite polarity, according to net charges, sizes and interactions with the solid matrix
5) Homogeneous group of particles migrate as a separate band
6) The electrophoresis is carried out for 16-18 hours
7) Separated Proteins are fixed to a solid support using a fixative such as Acetone or
Methanol
8) Proteins are stained to make them visible
9) The separated proteins appear as distinct bands
10) Drawback-long time interval and blurring of margins
B) Cellulose Acetate Membrane Electrophoresis
• Preferred solid support medium

• Less time consuming
• Excellent separation
• Membranes can be stored for a longer time
• Widely used for separation of lipo proteins, isoenzymes and hemoglobin
C) Gel Electrophoresis
204
• The term "gel" in this instance refers to the matrix used for containing, and then
separating the target molecules
• In most cases the gel is a cross linked polymer whose composition and porosity is
chosen based on the specific weight and composition of the target to be analyzed.
• A gel block made of Polyacrylamide, Agarose or substituted starch gel is used in
this method as the solid support
• Agar gel is used for separation of different types of protein mixtures as well as
nucleic acids
• Polyacrylamide is most suitable for separation of nucleic acids. It is also
frequently used in separating proteins, peptides and amino acids from microgram
quantities of mixed samples
a) Agarose gel electrophoresis
• Commonly used support medium

• Less expensive than cellulose acetate
• Equally good separation
• Agar is a complex acidic polysaccharide containing monomers of sulfated
galactose
• Agarose is a sulfate free fraction of Agar
• Gel is prepared in buffer and spread over a microscopic slide
• A small sample of serum or biological fluid is applied by cutting in to the gel with
a sharp edge
• The electrophoretic run takes about 90 minutes
b) Poly Acrylamide Gel Electrophoresis-PAGE
• Most popular type

• Polyacrylamide is a polymer formed when acrylamide is heated with a variety
of catalysts with or without cross linking agents
• It is thermostable, transparent, strong and relatively chemically inert
• Gels are uncharged and are prepared in a variety of pore sizes
• Proteins are separated on the basis of charge to mass ratio and molecular size,
a phenomenon called Molecular sieving
Types of PAGE
PAGE can be classified according the separation conditions into:
Native-PAGE:
205
• Separation is based upon charge, size, and shape of macromolecules
• Useful for separation and/or purification of mixture of proteins
• This was the original mode of electrophoresis.
Denatured-PAGE or SDS-PAGE
• Separation is based upon the molecular weight of proteins

• The most common method for determining MW of proteins
• Very useful for checking purity of protein samples
PAGE-Procedure
1. The gel of different pore sizes is cast in to a column inside a vertical tube, often
with large pore gel at the top and small pore gel at the bottom
2. Micrograms quantity of the sample is placed over the top of the gel column and
covered by a buffer solution having such a p H so as to change sample components
into anions
3. The foot of the gel column is made to dip in the same buffer in the bottom reservoir
4. Cathode and anode are kept above and below the column to impose an electric field
through the column
5 Macromolecular anions move towards the anode down the gel column
6 There is no external solvent space, all the migratory particles have to pass through
the gel pores
7. Rate of migration depends on the charge to mass ratio
8 Different sample components get separated in to discrete migratory bands along the
gel column on the basis of electrophoretic mobility and gel filtration effect
9 PAGE may yield 20 or more fractions and may be used to study individual proteins
and nucleic acids in serum especially genetic variants and isoenzymes
Slab PAGE
• The Polyacrylamide gel is cast as thin rectangular slab inside a plastic frame and
this slab is placed vertically on a buffer solution taken in a reservoir
• Several samples dissolved in dense sucrose solution or glycerol are placed in
separate wells cut in to the upper edge of the slab and are covered by the same
buffer solution. Cathode and anode are above and below to produce electric field
effect. Different components migrate simultaneously down parallel lanes in the slab
and get separated in to bands
206
Figure showing Slab gel electrophoresis
SDS PAGE
• SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is

technique widely used in biochemistry, forensics, genetics and molecular biology to
separate proteins according to their electrophoretic mobility
• The SDS gel electrophoresis of samples having identical charge to mass ratios
results in fractionation by size and is probably the world's most widely used
biochemical method
• When a detergent SDS(Sodium -Dodecyl-Sulfate)is added to PAGE the
combined procedure is termed as SDS PAGE SDS coats protein molecules
giving all proteins a constant charge-mass ratio
• Due to masking of charges of proteins by the large negative charge on SDS
binding with them, the proteins migrate along the gel in order of increasing
sizes or molecular weights
• Molecular weight of a given protein can be determined by comparing the
relative electrophoretic mobility of sample with that of standard protein of
known molecular weight, when both the sample and the standard proteins are
electrophoresed side by side in the same gel slab
• In oligomeric proteins, SDS PAGE usually gives the molecular weight of separated
monomer chains of the proteins because SDS cleaves the non covalent bonds
interlinking the monomer chains in the intact molecule.
VISUALIZATION
• After the electrophoresis is complete, the molecules in the gel can be stained to
make them visible
• Ethidium bromide, silver, or coomassie blue dye may be used for this process
• Other methods may also be used to visualize the separation of the mixture's
components on the geL
• If the analyte molecules fluoresce under ultraviolet light, a photograph can be
taken of the gel under ultraviolet lighting conditions. If the molecules to be
207
separated contain radioactivity added for visibility, an autoradiogram can be
recorded of the gel.
OTHER TYPES OF ELECTROPHORESIS

Capillary Electrophoresis
Immuno electrophoresis
Isoelectric focusing(Isoelectrophoresis)
208
MUTATIONS –AN OVERVIEW
Mutations
A mutation is a permanent change in the nucleotide sequence of a gene. Mutations may be

either gross, so that large area of chromosome is changed, or may be subtle with a change
in one or a few nucleotides.
Causes of Mutations
1) Spontaneous
Spontaneous mutations on the molecular level include:
• Tautomerism - A base is changed by the repositioning of a hydrogen atom.

• Depurination - Loss of a purine base (A or G).
• Deamination - Changes a normal base to an atypical base; C ĺ U, (which can be
corrected by DNA repair mechanisms), or spontaneous deamination of 5-
methycytosine (irreparable), or A ĺ HX (hypoxanthine).
• Transition - A purine changes to another purine, or a pyrimidine to a pyrimidine.
• Transversion - A purine becomes a pyrimidine, or vice versa.
2) Induced by Mutagens
Induced mutations on the molecular level can be caused by:
• Chemicals
o Nitroso compounds
o Hydroxylamine NH2OH
o Base analogs
o Simple chemicals (e.g. acids)
o Alkylating agents (e.g. N-ethyl-N-nitrosourea (ENU)) These agents can
mutate both replicating and non-replicating DNA. In contrast, a base analog
can only mutate the DNA when the analog is incorporated in replicating the
DNA.
o Methylating agents
o Polycyclic aromatic hydrocarbons e.g. benzopyrenes
o DNA intercalating agents (e.g. ethidium bromide)
o DNA cross linker (e.g. platinum)
o Oxidative damage caused by oxygen(O)] radicals
• Radiation
o Ultraviolet radiation (nonionizing radiation) - excites electrons to a higher
energy level. DNA absorbs ultraviolet light. Two nucleotide bases in DNA -
cytosine and thymine-are most vulnerable to excitation that can change base-
209
pairing properties. UV light can induce adjacent thymine bases in a DNA
strand to pair with each other, as a bulky dimer.
o Ionizing radiation
• Biological- Viruses
DNA has so-called hotspots, where mutations occur up to 100 times more frequently than
the normal mutation rate. A hotspot can be at an unusual base, e.g., 5-methylcytosine.
Classification of mutations-
Structurally, mutations can be classified as:
A) Point mutations, often caused by chemicals or malfunction of DNA replication, and

include single nucleotide changes-
Substitution-exchange a single nucleotide for another. Most common is the transition
that exchanges a purine for a purine (A ļ G) or a pyrimidine for a pyrimidine, (C ļ T). A
transition can be caused by nitrous acid, base mis-pairing, or mutagenic base analogs. Less
common is a transversion, which exchanges a purine for a pyrimidine or a pyrimidine for a
purine (C/T ļ A/G). An example of a transversion is adenine (A) being converted into a
cytosine (C).
Insertions add one nucleotide into the DNA. They are usually caused by transposable
elements or errors during replication of repeating elements (e.g. AT repeats). Insertions in
the coding region of a gene may alter splicing of the mRNA (splice site mutation), or cause
a shift in the reading frame (frame shift), both of which can significantly alter the gene
product.
Deletions remove one nucleotide from the DNA. Like insertions, these mutations can
alter the reading frame of the gene.
B ) Large-scale mutations in chromosomal structure, including:
a) Amplifications (or gene duplications) leading to multiple copies of all chromosomal

regions, increasing the dosage of the genes located within them.
b) Deletions of large chromosomal regions, leading to loss of the genes within those
regions.
c) Mutations whose effect is to juxtapose previously separate pieces of DNA, potentially
bringing together separate genes to form functionally distinct fusion genes (e.g. bcr-abl).
These include:
Chromosomal translocations: interchange of genetic parts from nonhomologous
chromosomes.
Interstitial deletions: an intra-chromosomal deletion that removes a segment of DNA
from a single chromosome, thereby apposing previously distant genes.
Chromosomal inversions: reversing the orientation of a chromosomal segment.
210
Effects of mutations
Although the initial change may not occur in the template strand of the double-stranded
DNA molecule for that gene, after replication, daughter DNA molecules with mutations in
the template strand will segregate and appear in the population of organisms.
If the nucleotide sequence of the gene containing the mutation is transcribed into an RNA
molecule, then the RNA molecule will possess a complementary base change at this
corresponding locus.(See figure)
Single-base changes in the mRNA molecules may have one of several effects when
translated into protein:
(1) There may be no detectable effect because of the degeneracy of the code; such
mutations are often referred to as silent mutations. This would be more likely if the
changed base in the mRNA molecule were to be at the third nucleotide of a codon. Because
of wobble, the translation of a codon is least sensitive to a change at the third position. E.g.
valine has 4 codons GUU, GUC, GUA, or GUG, the change in the third nucleotide will
have the incorporation of same amino acid, thus there will not be any effect on the
functional capacity of the protein.
(2) A missense effect will occur when a different amino acid is incorporated at the
corresponding site in the protein molecule. This mistaken amino acid—or missense,
depending upon its location in the specific protein—might be acceptable, partially
acceptable, or unacceptable to the function of that protein molecule. From a careful
examination of the genetic code, one can conclude that most single-base changes would
result in the replacement of one amino acid by another with rather similar functional
groups. This is an effective mechanism to avoid drastic change in the physical properties of
a protein molecule. If an acceptable missense effect occurs, the resulting protein molecule
may not be distinguishable from the normal one. A partially acceptable missense will result
in a protein molecule with partial but abnormal function. If an unacceptable missense effect
occurs, then the protein molecule will not be capable of functioning in its assigned role.
a) Acceptable Missense mutations- The sequencing of a large number of hemoglobin

mRNAs and genes from many individuals has shown that the codon for valine at position
67 of the beta chain of hemoglobin is not identical in all persons who possess a normally
functional bets chain of hemoglobin. The codon changes by point mutation from GUU (Of
valine) to GAU of Aspartic acid in Hb Bristol. Similarly in Hb Sydney the codon changes
from GUU to GCU for Alanine. Both Hb Bristol and Hb Sydney are normal Hb variants
with normal oxygen carrying capacity. Thus these are acceptable mutations. Hemoglobin
Hikari has been found in at least two families of Japanese people. This hemoglobin has
asparagine substituted for lysine at the 61 position in the beta chain. The corresponding
transversion might be either AAA or AAG changed to either AAU or AAC. The
replacement of the specific lysine with asparagine apparently does not alter the normal
function of the beta chain in these individuals.
211
b) Partially acceptable Missense mutations
A partially acceptable missense mutation is best exemplified by hemoglobin S, which is
found in sickle cell anemia. Here glutamic acid, the normal amino acid in position 6 of the
beta chain, has been replaced by valine. The corresponding single nucleotide change within
the codon would be GAA or GAG of glutamic acid to GUA or GUG of valine. Clearly, this
missense mutation hinders normal function and results in sickle cell anemia when the
mutant gene is present in the homozygous state. The glutamate-to-valine change may be
considered to be partially acceptable because hemoglobin S does bind and release oxygen,
although abnormally.
c) Unacceptable Missense Mutations For example, the hemoglobin M mutations generate

molecules that allow the Fe2+ of the heme moiety to be oxidized to Fe3+, producing met
hemoglobin. Here the single nucleotide change alters the properties of a protein to such an
extent that it becomes non functional. Hb M results from histidine to tyrosine substitution.
Distal Histidine of alpha chain of Globin is replaced by Tyrosine. The codon CAU is
changed to UAU with the resultant incorporation of Tyrosine and formation of Met Hb.
Met hemoglobin cannot transport oxygen.
(3) A nonsense codon may appear that would then result in the premature termination of
a peptide chain and the production of only a fragment of the intended protein molecule. The
probability is high that a prematurely terminated protein molecule or peptide fragment will
not function in its assigned role.e.g. The codon UAC for Tyrosine may be mutated to UAA
or UAG, both are stop codons. Beta Thalassemia is an example of non sense mutation.
In certain conditions as a result of mutational event the stop codon may be changed to
normal codon (UAA to CAA) . This results in the elongation of protein to produce “Run on
polypeptides”. The resultant protein is a functionally abnormal protein.
Frame shift Mutations
A frame shift mutation is a mutation caused by inserts or deletes of a number of nucleotides

from a DNA sequence. Due to the triplet nature of gene expression by codons, the insertion
or deletion can disrupt the reading frame, or the grouping of the codons, resulting in a
completely different translation from the original. The earlier in the sequence the deletion
or insertion occurs, the more altered the protein produced is.
If three nucleotides or a multiple of three are deleted from a coding region, the
corresponding mRNA when translated will provide a protein from which is missing the
corresponding number of amino acids. Because the reading frame is a triplet, the reading
phase will not be disturbed for those codons distal to the deletion.
Triplet deletion
A triplet deletion removes exactly one amino acid from the polypeptide ,the most common
mutation in cystic fibrosis is Delta F508 (i.e. deletion of amino acid number 508 (a
phenylalanine, F)).
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Trinucleotide expansion
The commonest inherited cause of mental retardation is a syndrome originally known as

Martin-Bell syndrome. Patients are most usually male, have a characteristic elongated face
and numerous other abnormalities including greatly enlarged testes. In 1969 the name of
the syndrome was changed to the fragile X syndrome. The mutation was tracked down to a
trinucleotide expansion in the gene now named FMR1 (Fragile site with Mental
Retardation). A number of diseases have now been ascribed to trinucleotide expansions.
These include Huntington's disease and Myotonic dystrophy.
Gene deletions- Alpha Thalassemia is an example of Gene deletion. The clinical

manifestations are as per the number of genes deleted.
Consequences of Mutations
Harmful mutations
Changes in DNA caused by mutation can cause errors in protein sequence, creating
partially or completely non-functional proteins. To function correctly, each cell depends on
thousands of proteins to function in the right places at the right times. When a mutation
alters a protein that plays a critical role in the body, a medical condition can result. A
condition caused by mutations in one or more genes is called a genetic disorder. However,
only a small percentage of mutations cause genetic disorders; most have no impact on
health. For example, some mutations alter a gene's DNA base sequence but don’t change
the function of the protein made by the gene.
If a mutation is present in a germ cell, it can give rise to offspring that carries the mutation
in all of its cells. This is the case in hereditary diseases. On the other hand, a mutation can
occur in a somatic cell of an organism. Such mutations will be present in all descendants of
this cell, and certain mutations can cause the cell to become malignant, and thus cause
cancer.
Often, gene mutations that could cause a genetic disorder are repaired by the DNA repair
system of the cell. Each cell has a number of pathways through which enzymes recognise
and repair mistakes in DNA. Because DNA can be damaged or mutated in many ways, the
process of DNA repair is an important way in which the body protects itself from disease.
Beneficial mutations
A very small percentage of all mutations actually have a positive effect. These mutations
lead to new versions of proteins that help an organism and its future generations better
adapt to changes in their environment. For example, a specific 32 base pair deletion in
human CCR5 (CCR5-ǻ32) confers HIV resistance to homozygotes and delays AIDS onset
in heterozygotes. The CCR5 mutation is more common in those of European descent.
Summary of Mutations
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Translation
The pathway of protein synthesis is called Translation because the ‘language’ of the
nucleotide sequence on the mRNA is translated into the language of the amino acid
sequence. The m RNA is translated from its 5’end to its 3’end, producing a protein
synthesized from its amino terminal end to its carboxyl terminal end.
Prokaryotic Translation
Components required for Translation-

Amino acids
Transfer RNA
Messenger RNA
Aminoacyl t RNA synthetase
Functionally competent ribosomes
Protein factors
ATP and GTP as a source of energy
Ribosomes
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Ribosomes are large complexes of protein and r RNA. They consist of two subunits- one
large (Heavy) and one small (Light) whose relative sizes are generally given in terms of
their sedimentation coefficients or S (Svedberg) values. The S values are determined by the
shape as well as by the molecular mass; their numerical values are strictly not additive. The
prokaryotic 50S and 30S ribosomal subunits together form a ribosome with an S value of
70.
Figure-1
Figure-2
The ribosome has two binding sites for t RNA molecule A and P sites, each of which
extends over both subunits. Together they cover the neighboring codons. (See figure-2)
During translation, The A site binds an incoming Aminoacyl t RNA as directed by the
codon currently occupying the site. This codon specifies the next amino acid to be added to
the growing peptide chain.
The P site codon is occupied by the Peptidyl-t RNA. This t RNA carries the chain of
amino acids that has already been synthesized.
An E site is also there that is occupied by the empty t RNA that is about to exit the
ribosome
Transfer RNA
At least one specific type of t RNA is required per amino acid. Two sites are important, one
Amino acid attachment site the other is Anticodon site. Because of their ability to carry a
specific amino acid and to recognize the codons for that amino acid, t RNAs are called the
adapter molecules.
215
(Figure-3 )
Amino acids are activated before incorporation and this activation is brought about by
amino acyl t RNA synthetase in the presence of ATP. There is at least one amino acyl t
RNA synthetase per amino acid. The carboxyl group of the amino acid is esterified to the
3’hydroxyl group of the t RNA.
Steps of Protein Synthesis
The process of protein synthesis is divided into 3 stages-

i) Initiation
ii) Elongation
iii) Termination
i) Initiation-Initiation of the protein synthesis involves the assembly of the components

of the translation system before the peptide bond formation occurs. These components
include-
o Two ribosomal subunits
o m RNA
o Aminoacyl tRNA specified by the codons in the message
o GTP and
o Initiation factors that facilitate the assembly of this initiation complex. In prokaryotes
three initiation factors are known (IF-1,IF-2 and IF-3) while in eukaryotes there are at least
9 designated as e- IF to indicate the eukaryotic origin
Ribosomal assembly and formation of Initiation complex-The small ribosomal subunit

binds to Initiation Factor 3 (IF3). The small subunit/IF3 complex binds to the mRNA.
Specifically, it binds to the sequence AGGAGG, known as the Shine-Delgarno sequence,
which is found in all prokaryotic mRNAs. (Figure 4).
Meanwhile, the fmet tRNA binds to Initiation Factor 2 (IF2), which promotes binding of
the tRNA to the start codon. (Figure 5)
216
(Figure-4)
(Figure-5)
The small subunit/IF3 complex scans along the mRNA until it encounters the start codon.
The tRNA/IF2 complex also binds to the start codon. This complex of the small ribosomal
subunit, IF3, initiator tRNA, and IF2 is called the initiation complex. (Figure-6)
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(Figure-6)
At this point, the large ribosomal subunit joins in. A molecule of GTP is hydrolyzed, and
the initiation factors are released. The ribosomal complex is now ready for protein
synthesis.(Figure-7)
(Figure-7)
When the ribosome is assembled, two tRNA binding sites are created; these are designated
'P' and 'A' (P stands for Peptidyl, A stands for Aminoacyl). The initiator tRNA is in the P
site, and the A site will be filled by the tRNA with the anticodon that is complementary to
the codon next to the start. (In this case, it is the tRNA that binds proline.) Figure-8
(Figure-8)
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ii) Elongation
When the second tRNA base pairs with the appropriate codon in the mRNA, an enzyme
called Peptidyl transferase catalyzes the formation of a peptide bond between the two
amino acids present (while breaking the bond between fmet and its tRNA).This activity is
intrinsic to the 23S r RNA found in the large subunit. Since the r RNA catalyzes this
process, it is referred to as the Ribozyme. At this point, the whole ribosome shifts over one
codon. This shift requires several elongation factors (not shown) and energy from the
hydrolysis of GTP. The result of the shift is that the uncharged tRNA that was in the P site
is ejected, and the tRNA that was in the A site is now in the P site. The A site is free to
accept the tRNA molecule with the appropriate anticodon for the next codon in the
mRNA.(Figure 9)
(Figure-9)
The next tRNA base pairs with the next codon, and Peptidyl transferase catalyzes the
formation of a peptide bond between the new amino acid and the growing peptide chain.
Once again, the ribosome shifts over, so that the uncharged tRNA is expelled, and the
tRNA with the peptide chain occupies the P site. (This is why this site is called the
'Peptidyl' site - after the shift, it contains the tRNA with the growing peptide chain. The
other site will accept a tRNA with an amino acid, hence the name 'Aminoacyl' site.) The
process of shifting and peptide bond formation continues over and over until a termination
codon is encountered.(Figure-10)
(Figure-10)
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The elongation process is fairly rapid, with prokaryotic ribosomes able to add 15 amino
acids to the growing polypeptide every second. The process is also relatively error-free.
Only one mistake is made every 10,000 amino acids. For large proteins of 1000 amino
acids, that would mean one wrong amino acid in every 10 polypeptides.
iii) Termination
When a termination codon enters the A site, translation halts. This is because there is no
tRNA with an anticodon that is complementary to any of the stop codons. The release
factor causes the translation complex to fall apart, and cleaves the polypeptide from the
final tRNA.(Figure-11)
(Figure 11)
The polypeptide product is now free to function in the cell. The mRNA molecule is now
available to be translated again. Very often, more than one ribosome will translate a single
mRNA at the same time. One ribosome will initiate translation, and after it moves down the
mRNA a bit, another ribosome will initiate, then another, and so on. The structure
consisting of multiple ribosomes translating a single mRNA molecule is called a polysome.
Eventually, the mRNA is degraded, and translation of that particular message will cease.
Eukaryotic Translation
Eukaryotic translation is very similar overall to prokaryotic translation. There are a few
notable differences, These include the following:
Eukaryotic mRNAs do not contain a Shine-Delgarno sequence. Instead, ribosomal subunits

recognize and bind to the 5' cap of eukaryotic mRNAs. In other words, the 5' cap takes the
place of the Shine-Delgarno sequence.
Eukaryotes do not use formyl methionine as the first amino acid in every polypeptide;
ordinary methionine is used. Eukaryotes do have a specific initiator tRNA,
however, Eukaryotic translation involves many more protein factors than prokaryotic
translation (For example, eukaryotic initiation involves at least 10 factors, instead of the 3
in prokaryotes).
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Inhibitors of protein synthesis
The tetracyclines (tetracycline, doxycycline, demeclocycline, minocycline, etc.) block
bacterial translation by binding reversibly to the 30S subunit and distorting it in such a way
that the anticodon of the charged tRNAs cannot align properly with the codons of the
mRNA.
Puromycin structurally binds to the amino acyl t RNA and becomes incorporated into
the growing peptide chain thus causing inhibition of the further elongation.
Chloramphenicol inhibits prokaryotic Peptidyl Transferase
Clindamycin and Erythromycin bind irreversibly to a site on the 50 s subunit of the
bacterial ribosome thus inhibit translocation.
Diphtheria toxin inactivates the eukaryotic elongation factors thus prevent
translocation.
Post Translational Modifications
The newly synthesized protein is modified to become functionally active. The various post
translational modifications are as follows-
1) Trimming- Trimming removes excess amino acids.
2) Covalent Modifications
a)Phosphorylation
b) Glycosylation
c) Hydroxylation
d) Gamma carboxylation
e) Isoprenylation
f) Methylation
g) Acetylation
h) Protein degradation
Phosphorylation may activate or inactivate the protein.e.g .Glycogen Phosphorylase

becomes active while glycogen synthase becomes in active on phosphorylation.
Glycosylation targets a protein to become a part of the plasma membrane, or lysosomes or
be secreted out of the cell
Hydroxylation such as seen in collagen is required for acquiring the three dimensional
structure and for imparting strength.
Gamma carboxylation of glutamic acid residues of prothrombin takes place in the
presence of vitamin K.
Methylation or Acetylation of histones takes place for gene expression.
Defective proteins or destined for turn over are marked for destruction by attachment of a
Ubiquitin protein. Proteins marked in this way are degraded by a cellular component
known as the Proteasome.
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3) Subunit Aggregation- Examples are immunoglobulins, hemoglobin and maturation of
collagen. Failure of post translational modifications affects the functional capacity of the
proteins
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UDP-GLUCURONYL TRANSFERASE- REACTION CATALYZED
AND SIGNIFICANCE
This is an enzyme of Uronic acid pathway. Uronic acid pathway is an alternative pathway
for the oxidation of glucose that does not provide a means of producing ATP, but is utilized
for the generation of the activated form of glucuronate, UDP-glucuronate which is mainly
used for detoxification of foreign chemicals, some endogenous compounds like bilirubin,
certain hormones, metabolites and for the synthesis of mucopolysaccharides. This pathway
also produces Ascorbic acid in certain animals.
The unutilized Glucuronate produced in this pathway is converted to Xylulose-5 P which is
further metabolized through HMP pathway
Figure1- showing the overview of Uronic acid pathway
Biological significance of UDP –Glucuronyl Transferase
UDP glucuronate the active form of Glucuronic acid, can readily donate the Glucuronic
acid component under the catalytic activity of UDP –Glucuronyl Transferase for the
following functions-
1) Detoxification of foreign compounds and drugs- During detoxification, the glucuronate
residues are covalently attached to lipid soluble substances. Since glucuronate residues are
strongly polar, their attachment imparts polar character to these substances, making them
water soluble and readily excretable. Bilirubin, certain hormones and drugs are made more
polar for renal excretion in this manner. UDP-Glucuronic acid is the Glucuronyl donor, and
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a variety of glucuronosyl transferases, present in both the endoplasmic reticulum and
cytosol, are the catalysts. Molecules such as 2-acetylaminofluorene (a carcinogen), aniline,
benzoic acid, meprobamate (a tranquilizer), phenol, and many steroids are excreted as
glucuronides. The glucuronide may be attached to oxygen, nitrogen, or sulfur groups of the
substrates. Glucuronidation is probably the most frequent conjugation reaction.
2) Synthesis of Mucopolysaccharides-UDP Glucuronic acid is an essential component
of Hyaluronic acid and heparin.
3) Conjugation of Bilirubin-Bilirubin is nonpolar and would persist in cells (eg, bound
to lipids) if not rendered water-soluble. Hepatocytes convert bilirubin to a polar form,
which is readily excreted in the bile, by adding Glucuronic acid molecules to it. This
process is called conjugation .The conjugation of bilirubin is catalyzed by a specific
Glucuronyl transferase. The enzyme is mainly located in the endoplasmic reticulum, uses
UDP-Glucuronic acid as the glucuronosyl donor, and is referred to as bilirubin-UGT.
Bilirubin Monoglucuronide is an intermediate and is subsequently converted to the
diglucuronide. Most of the bilirubin excreted in the bile of mammals is in the form of
bilirubin diglucuronide.
Figure- showing the conjugation of bilirubin
Clinical Significance- Diminished activity of Bilirubin UDP Glucuronyl Transferase

(UGT)
1) Neonatal "Physiologic Jaundice"
This transient condition is the most common cause of unconjugated hyperbilirubinemia. It

results from an accelerated hemolysis around the time of birth and an immature hepatic
system for the uptake, conjugation, and secretion of bilirubin. Not only is the bilirubin-
UGT activity reduced, but there probably is reduced synthesis of the substrate for that
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enzyme, UDP-glucuronic acid. Since the increased amount of bilirubin is unconjugated, it
is capable of penetrating the blood-brain barrier when its concentration in plasma exceeds
that which can be tightly bound by albumin (20–25 mg/dL). This can result in a
hyperbilirubinemic toxic encephalopathy, or kernicterus, which can cause mental
retardation. Because of the recognized inducibility of this bilirubin UGT enzyme system,
phenobarbital has been administered to jaundiced neonates and is effective in this disorder.
In addition, exposure to blue light (phototherapy) promotes the hepatic excretion of
unconjugated bilirubin by converting some of the bilirubin to other derivatives such as
maleimide fragments and geometric isomers that are excreted in the bile.
2) Crigler-Najjar Syndrome, Type I; Congenital Nonhemolytic Jaundice
a) Type I Crigler-Najjar syndrome is a rare autosomal recessive disorder. It is

characterized by severe congenital jaundice (serum bilirubin usually exceeds 20 mg/dL)
due to mutations in the gene encoding bilirubin-UGT activity in hepatic tissues. The disease
is often fatal within the first 15 months of life. Children with this condition have been
treated with phototherapy, resulting in some reduction in plasma bilirubin levels.
Phenobarbital has no effect on the formation of bilirubin glucuronides in patients with type
I Crigler-Najjar syndrome. A liver transplant may be curative.
It should be noted that the gene encoding human bilirubin-UGT is part of a large UGT gene
complex situated on chromosome 2. Many different substrates are subjected to
glucuronosylation, so many glucuronosyltransferases are required.
b) Crigler-Najjar Syndrome, Type II

This rare inherited disorder also results from mutations in the gene encoding bilirubin-
UGT, but some activity of the enzyme is retained and the condition has a more benign
course than type I. Serum bilirubin concentrations usually do not exceed 20 mg/dL. Patients
with this condition can respond to treatment with large doses of phenobarbital.
3) Gilbert Syndrome
Again, this relatively prevalent condition is caused by mutations in the gene encoding
bilirubin-UGT. It is more common among males. Approximately 30% of the enzyme's
activity is preserved and the condition is entirely harmless.
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VITAMIN E FUNCTIONS AND DEFICIENCY
Vitamin E is a collective name for all stereoisomers of tocopherols and tocotrienols. The
most biologically active form is Į-tocopherol, but ȕ-, Ȗ-, į-tocopherols, 4 tocotrienols, and
several stereoisomers may also have important biological activity. Vitamin E acts as a
chain-breaking antioxidant and is an efficient free radical scavenger, to protect low-
density lipoproteins (LDLs) and polyunsaturated fats in membranes from oxidation. A
network of other antioxidants (e.g., vitamin C, glutathione) and enzymes maintain vitamin
E in its reduced state.
Absorption and Metabolism
After absorption, vitamin E is taken up from chylomicrons by the liver, and a hepatic Į -
tocopherol transport protein mediates intracellular vitamin E transport and incorporation
into very low-density lipoprotein (VLDL). The transport protein has particular affinity for Į
-tocopherol; thus this natural isomer has the most biologic activity.
Requirement
Vitamin E is widely distributed in the food supply and is particularly high in sunflower oil,
safflower oil, and wheat germ oil; Ȗ tocotrienols are notably present in soybean and corn
oils. Vitamin E is also found in meats, nuts, and cereal grains, and small amounts are
present in fruits and vegetables. The RDA for vitamin E is 15 mg/d (34.9 ȝmol or 22.5 IU)
for all adults. Diets high in polyunsaturated fats may necessitate a slightly higher
requirement for vitamin E.
Functions of vitamin E
1) It acts as a lipid-soluble antioxidant in cell membranes, and is important in maintaining

the fluidity of cell membranes.
Antioxidant role of vitamin E
Reactive oxygen species (ROS) are molecular oxygen metabolites that are highly reactive
with lipids, proteins, and DNA, causing oxidative damage to these cellular
macromolecules. This damage, termed oxidative stress, accumulates over time and is
thought to contribute to both disease pathology and the aging process. Cellular mechanisms
that exist to counteract ROS include stabilization by enzymes such as superoxide dismutase
and Catalase, and direct scavenging by antioxidant molecules such as glutathione (GSH)
and a major extracellular antioxidant in plasma; vitamin E, a major lipid soluble
antioxidant; and ascorbate, a critical intracellular and extracellular antioxidant.
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The main function of vitamin E is as a chain-breaking, free-radical trapping antioxidant
in cell membranes and plasma lipoproteins.
By reacting with the lipid peroxide radicals formed by peroxidation of polyunsaturated fatty
acids, it gets converted to tocopheroxyl radical. The resultant radical (Oxidized form) is
relatively unreactive, and ultimately forms nonradical compounds. Commonly, the
tocopheroxyl radical is reduced back to tocopherol by reaction with vitamin C from plasma.
(See Figure)
Ascorbate (Vitamin C) is essential for maintaining vitamin E in its reduced, active form.
Ascorbate is oxidized to dehydroascorbate in plasma and that is recycled back to ascorbate
by GSH as well as by several enzyme systems in erythrocytes, neutrophils, endothelial cells
and hepatocytes (See figure).
GSH itself gets oxidized during this process and is converted back to its reduced form by
Glutathione reductase utilizing NADPH as the reductant. GSH is also required by
Selenium containing Glutathione Peroxidase enzyme for decomposing H2O2.
A synergism is observed between selenium and vitamin E .The synergism is related to the
process of antioxidation, wherein tocopherols tend to prevent oxidative damage to
polyunsaturated fats in cell membranes, whereas selenium, as part of seleno-enzyme
glutathione peroxidase, catalyzes the destruction of lipid hydro peroxides. This explains
how these two nutrients play separate but interrelated roles in the cellular defense system
against oxidative damage. Vitamin E deficiency results in failure to scavenge free radicals
and as a consequence there is membrane disruption especially of red blood cells.(See the
details below)
As an antioxidant, vitamin E plays a protective role in many organs and systems. Vitamin E
is necessary for maintaining a healthy immune system, and it protects the thymus and
circulating white blood cells from oxidative damage. Also, it may work synergistically with
vitamin C in enhancing immune function. Recent research evidence indicates that the
combined use of high doses of vitamin C and vitamin E helps prevent Alzheimer’s disease.
In eyes, vitamin E is needed for the development of the retina and protects against cataracts
and macular degeneration.
227
Figure- showing the anti oxidant role of vitamin E, A synergism is observed between
Vitamin E, C and G-SH dependent Glutathione peroxidase.
2) Other functions of vitamin E- It also has a (relatively poorly defined) role in cell
signaling. Besides that, Vitamin E inhibits prostaglandin synthesis and the activities of
protein kinase C and Phospholipase A2.
3) In high concentration, the tocopheroxyl free radical can penetrate further into cells and,
potentially, propagate a chain reaction. Therefore, vitamin E may, like other antioxidants,
also have pro-oxidant actions, especially at high concentrations. This explains the
bleeding observed in vitamin E toxicity.
Vitamin E Deficiency
Dietary vitamin E deficiency is common in developing countries; deficiency among adults

in developed countries is uncommon and is usually due to fat malabsorption. The main
symptoms are hemolytic anemia and neurologic deficits.
Etiology
Absorption of vitamin E depends on normal pancreatic biliary function, biliary secretion,

micelle formation, and penetration across intestinal membranes. Interference with any of
these processes could result in a deficiency state.
In developing countries, the most common cause is inadequate intake of vitamin E. In

developed countries, the most common causes are disorders that cause fat malabsorption,
including Abetalipoproteinemia (genetic absence of Apo lipoprotein B), chronic
cholestatic hepatobiliary disease, pancreatitis, short bowel syndrome, and cystic
fibrosis.
Vitamin E deficiency is seen in only severe and prolonged malabsorptive diseases, such as
celiac disease, or after small-intestinal resection. Children with cystic fibrosis or prolonged
228
cholestasis may develop vitamin E deficiency characterized by areflexia and hemolytic
anemia.
Children with Abetalipoproteinemia cannot absorb or transport vitamin E and become

deficient quite rapidly.(Apo B48 is required for chylomicron formation and that is needed
for transportation of vitamin E from gut to liver)
Isolated vitamin E deficiency syndrome - Developing in the absence of fat

malabsorption, this syndrome is caused by an autosomal-recessive genetic disorder.
Neurologic findings develop within the first decade of life. It is due to a defect in the Į-
tocopherol transport protein.
The main symptoms are-

Mild hemolytic anemia and nonspecific neurological deficits
Biochemical basis of hemolytic anemia- Vitamin E deficiency results in oxidative damage

to the red cell membrane, with the resultant altered permeability and osmolysis.(See the
flowchart below)
Flow chart- showing the biochemical basis of hemolytic anemia observed in vitamin E
deficiency
Biochemical basis of Ataxia and neurological symptoms- Vitamin E deficiency causes

axonal degeneration of the large myelinated axons and results in posterior column and
spinocerebellar symptoms. Peripheral neuropathy is initially characterized by areflexia,
with progression to an ataxic gait, and by decreased vibration and position sensations.
In adults with malabsorption, vitamin E deficiency very rarely causes spinocerebellar ataxia
because adults have large vitamin E stores in adipose tissue.
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Diagnosis
Low Į-tocopherol level or low ratio of plasma Į-tocopherol to plasma lipids

Measuring the plasma Į-tocopherol level is the most direct method of diagnosis. In adults,
vitamin E deficiency is suggested if the Į-tocopherol level is < 5 ȝg/mL (< 11.6 µmol/L).
Because abnormal plasma lipid levels can affect vitamin E status, a low ratio of plasma Į-
tocopherol to plasma lipids (< 0.8 mg/g total lipid) is the most accurate indicator in adults
with hyperlipidemia.
In children and adults with Abetalipoproteinemia, plasma Į-tocopherol levels are usually
undetectable.
Prevention
Although premature neonates may require supplementation, human milk and commercial
formulas have enough vitamin E for full-term neonates.
Treatment
Supplemental Į-tocopherol
If malabsorption causes clinically evident deficiency, Į-tocopherol 15 to 25 mg/kg orally
once/day should be given. However, larger doses given by injection are required to treat
neuropathy during its early stages or to overcome the defect of absorption and transport in
Abetalipoproteinemia.
Vitamin E Toxicity
Many adults take relatively large amounts of vitamin E (Į-tocopherol 400 to 800 mg/day)
for months to years without any apparent harm. Occasionally, muscle weakness, fatigue,
nausea, and diarrhea occur. The most significant risk is bleeding. However, bleeding is
uncommon unless the dose is > 1000 mg/day or the patient takes oral coumarin or warfarin.
230
RENAL CLEARANCE
Renal clearance is a measurement to determine the functional status of the kidney. By

definition clearance is the volume of plasma from which a substance is completely
removed through excretion by the kidney in a given amount of time (usually a minute).
For example, the clearance for urea is 75 ml/min. This means that the kidney removes
all of the urea in 75 ml of plasma in one minute.
Of the 625 ml/min of plasma that goes to the glomerulus, 125 ml/min is filtered into
Bowman's capsule forming the filtrate (The rate of filtration is known as the Glomerular
filtration rate- GFR). The remaining 500 ml/min enters into the peritubular capillaries. Of
the 125 ml/min filtered, almost all of the water in this fluid is reabsorbed back into the
blood. The composition of the filtrate in Bowman's capsule is identical to the composition
of the plasma except that the filtrate has no or very little amount of proteins.
Any substance, which is freely filtered by the glomerulus and is neither reabsorbed nor
secreted, ends up in the urine. Thus all the plasma that gets filtered is cleared of that
substance (that is, all the substance in the filtrate gets excreted) while the substance that
that is not filtered (and thus remains in the plasma) is not excreted. Since clearance is
defined as the volume of plasma 'cleared' of a substance in 1 min, the clearance for that
substance would be 125 ml/min. This means that out of the 625 ml of plasma that come to
the kidney in one minute, 125 ml (the fraction that is filtered) has all of the substance
removed from it in that minute, the other 500 ml (the fraction that is not filtered) keeps it as
there is no way for the substance get into the urine as it is not secreted.
The GFR is typically recorded in units of volume per time, e.g., milliliters per minute
ml/min.
The compound inulin is cleared in the same way as mentioned above. All of the plasma
that is filtered is cleared of inulin so that if one has to measure the clearance of inulin, it
would be equal the amount of plasma filtered in a minute, the glomerular filtration rate.
Therefore, the clearance of inulin is equal to the glomerular filtration rate, the volume of
plasma filtered in one minute. Inulin is not a normal metabolite of the body; it is in fact
administered to determine the functional status of the kidney
The clearance of any other substance is not similar to clearance of inulin. For example-
Glucose, like inulin, is freely filtered. Thus glucose is present in Bowman's Capsule.
However, glucose does not appear in urine because glucose is completely reabsorbed as it
passes through the tubules. Inulin is not reabsorbed. This means all of the glucose that
comes to the kidney is saved and leaves the kidney in the plasma and that no glucose is
excreted into the urine. The clearance of glucose is therefore 0 ml/min as no plasma has its
glucose removed as it passes through the kidney. This would be true for any substance that
is completely reabsorbed. Hence if the clearance of Tryptophan (an amino acid) is 0
ml/min, it can be inferred that Tryptophan must be completely reabsorbed (as long as it is
freely filtered).
Taking the example of another substance, Para amino Hippuric acid (PAH), It is freely
filtered, not reabsorbed and is completely secreted by the kidney. Thus all of the PAH
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entering the kidney ends up in the urine, both the PAH that is filtered and that that is not
filtered. This means that all the plasma entering the kidneys would be cleared of PAH.
Since the renal plasma flow is about 625 ml/min in a 'normal' kidney, the clearance of
PAH must be 625 ml/min. Therefore, the PAH clearance is equal to the renal plasma flow.
PAH clearance is used to determine whether the kidneys have an adequate plasma flow.
Now, if the clearance of a substance is 625 ml/min, this would suggest that the kidney
completely secretes this substance (that is, the kidney 'treats' this substance the same as
PAH which is known to be completely secreted). Using similar logic, a clearance value of
125 would suggest that the kidney neither reabsorbs nor secretes the substance and a
clearance value of 0 suggests that the kidney completely reabsorbs the substance (assuming
that the substance is freely filterable in the glomerulus).
The urea clearance has been measured to be 75 ml/min. What does the kidney 'do' with urea
(does it reabsorb, secrete or neither)? Well if urea is completely reabsorbed, its clearance
should be like that of glucose (0 ml/min) and if urea is not reabsorbed at all (and not
secreted), its clearance should be 125 ml/min. Since the value of urea clearance is 75
ml/minute, which means urea is partially reabsorbed. Note that the common belief
concerning kidney function is that it removes urea from the blood yet the nephron partially
reabsorbs urea! Thus urea clearance is not a true predictor of Glomerular filtration rate as is
Inulin clearance.
In clinical practice, however, creatinine clearance or estimates of creatinine clearance
based on the serum creatinine level are used to measure GFR. Creatinine is produced
naturally by the body (creatinine is a break-down product of creatine phosphate, which is
found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the
peritubular capillaries in very small amounts such that creatinine clearance overestimates
actual GFR by 10-20%. This margin of error is acceptable, considering the ease with which
creatinine clearance is measured. Unlike precise GFR measurements involving constant
infusions of inulin, creatinine is already at a steady-state concentration in the blood, and so
measuring creatinine clearance is much less cumbersome.
The laboratory findings of a 60 year old male patient are given below.
Calculate the creatinine clearance from the data and interpret the results.
24 hour urinary output : 1.2 L

Urinary creatinine : 90 mg/dl
Plasma creatinine : 1.0 mg/dl
The following are some of the biochemical findings in a patient. What is your
probable diagnosis?
232
Serum Uric acid : 8.8 mg/dl
Serum Inorganic phosphorous : 6.2 mg/dl
A ten year old boy was referred to the nephrologists with the following laboratory
result

Serum Sodium : 125 mg/dl
Serum Pottassium : 5.2 mg/dl
Urinary protein : 4 g/dl
Interpret the report
233
SUBJECTIVE QUESTIONS ACID BASE BALANCE AND IMBALANCE
Q.1- Explain clearly how hyperventilation and hypoventilation affect blood p H ? Give
suitable examples in support of your answer.
Q.2- Explain the role of hemoglobin as a buffer in the maintenance of acid base balance in
the body.
Q.3-The maintenance of intracellular pH within narrow limits is essential for life processes.
Briefly discuss why this is so and describe the mechanism by which the human body
maintains a relatively constant pH despite continuous acid production from cellular
metabolism.
Q.4- Name 3 physiological buffer systems, and explain the mode of action of any one of
them.
Q.5-A person was brought to the hospital after ingesting a large amount of ammonium
chloride. His arterial blood pH was found to be 7.29. Calculate the ratio of [HC03] to
[dissolved CO2] in the blood.
Dissolved CO2 + H20 H2CO3 H+ + HCO3- (pKa = 6.1) How might changes in the
pulmonary ventilation help to minimize the fall in pH?
Q.6- Discuss the role of kidneys in the maintenance of acid base balance of the body.
Support your answer with flow charts showing the details of the mechanisms.
Q.7- What is anion gap? State all the conditions of variations of anion gap in the body?
Q.8- Calculate the anion gap for a patient who has reported to emergency in a state of shock
with following blood reports-
p H- 7.2
PCO2- 45 mm Hg
HCO3—12 meq/L
Serum Na + 135 meq/L
Cl - - -85 meq/L
234
Q.9-A 14-year-old girl with cystic fibrosis has complained of an increased cough
productive of green sputum over the last week. She also complained of being increasingly
short of breath, and she is noticeably wheezing on physical examination. Arterial blood was
drawn and sampled, revealing the following values:
pH 7.30
pCO2 50 mm Hg
pO2 55 mm Hg
Hemoglobin -45 %
O2 saturation
[HCO3-] 24 meq / liter
What is the acid base status of the girl? Discuss in detail about the imbalance
How would the kidneys try to compensate for the girl's acid-base imbalance?
Q.10- A 76-year-old man complained to his wife of severe sub-sternal chest pain that
radiated down the inside of his left arm. Shortly afterward, he collapsed on the living room
floor. Paramedics arriving at his house just minutes later found him unresponsive, not
breathing, and without a pulse. CPR and electroconvulsive shock were required to start his
heart beating again. Upon arrival at the Emergency Room, the man started to regain
consciousness, complaining of severe shortness of breath (dyspnea) and continued chest
pain. On physical examination, his vital signs were as follows:
Systemic blood pressure 85 mm Hg / 50 mm

Hg
Heart rate 175 beats / minute
Respiratory rate 32 breaths / minute
Temperature 99.2oF
His breathing was labored, his pulses were rapid and weak every where, and his skin was
cold and clammy. An ECG was done, revealing significant "Q" waves in most of the leads.
Blood testing revealed markedly elevated creatine phosphokinase (CPK) levels of cardiac
muscle origin. Arterial blood was sampled and revealed the following:
pH 7.22
pCO2 30 mm Hg
pO2 70 mm Hg
Hemoglobin - O2 saturation 88 %
What is the diagnosis? What evidence supports your diagnosis?
How would you classify his acid-base status? What specifically caused this acid-base
disturbance?
How has his body started to compensate for this acid-base disturbance?
235
What would his blood pH be if his body had not started compensating for the acid-base
disturbance? Show your work.
List some other causes of this type of acid-base disturbance.
Q.11-An elderly gentleman is in a coma after suffering a severe stroke. He is in the

intensive care unit and has been placed on a ventilator. Arterial blood gas measurements
from the patient reveal the following:
pH 7.50
pCO2 30 mm Hg
pO2 100 mm Hg
Hemoglobin - O2 saturation 98%
How would you classify this patient's acid-base status?
How does this patient's hyperventilation pattern raise the pH of the blood?
How might the kidneys respond to this acid-base disturbance?
List some other causes of this type of acid-base disturbance.
Q.12-A 28-year-old woman has been sick with the flu for the past week, vomiting several
times every day. She is having a difficult time keeping solids and liquids down, and has
become severely dehydrated. After fainting at work, she was taken to a walk-in clinic,
where an IV was placed to help rehydrate her. Arterial blood was drawn first, revealing the
following:
pH 7.50
pCO2 40 mm Hg
pO2 95 mm Hg
Hemoglobin - O2 saturation 97%
1) How would you classify her acid-base disturbance?
2) Why might excessive vomiting cause her particular acid-base disturbance?
3) How would the kidneys compensate for this acid-base disturbance?
4) List some other causes of this type of acid-base disturbance.
236
SUBJECTIVE QUESTIONS- CHEMISTRY OF NUCLEOTIDES AND NUCLEIC
ACIDS
Q.1- Give a brief description of functions of c AMP? Justify its role as a second messenger
in hormonal action.
Q.2- Distinguish between:

a) Nucleoside and Nucleotide
b) Denaturation and Renaturation
c) Ribonucleotide and Deoxyribonucleotide
d) Uridine and Pseudouridine
e) Nucleotides in RNA and DNA
Q.3- Name the components of a nucleotide and show the order in which they are linked
together.
Q.4-Why ATP is called the “energy currency of a cell”? Support your answer giving
suitable examples.
Q.5- Name base and nucleoside analogs used as anticancer drugs.
Q,6- What is meant by hyperchromicity of denaturation?
Q.7- a) “Uracil is not present in DNA”, suggest the possible reason?

b) ‘Thymine nucleotides are not present in RNA but exception to the rule is there’,
give example in support of the statement.
Q.8- Discuss the biological significance of nucleotides?
Q.9- Compare and contrast the B and Z forms of DNA.
Q.10- Explain the extent to which the Watson-Crick structure of DNA is compatible with
Chargaff's rule.
Q-11- Explain how base-paired segments may occur in a single strand of RNA.
Q.12- Explain the following terms in the context of RNA structure: (a) poly A tail (b) cap.
Q.13- The following base sequence represents part of the transcribing strand of DNA
5’TACCATGGGCCC.3’
(a) Give the orientation and base sequence of the complementary strand.
(b) Give the orientation and base sequence of the RNA that is synthesized from it.
Q.14-Enlist the important differences between DNA and RNA
237
Q.15-Draw a well labeled diagram of secondary structure of t RNA and describe the
significance of each of its arm. Why is t RNA called an adapter molecule?
Q.16- Discuss the functions of different types of RNAs present in a cell?
Q.17- Discuss the role played by Histones in DNA packaging?
Q.18- Draw a well labeled diagram showing the secondary structure of DNA. Discuss the
salient features of Watson and Crick model of double stranded structure of DNA.
Q.19-Give a brief account of the small RNA s present in a cell. Discuss the significance of
each of them.
Q.20-What is meant by polarity of DNA? What is its significance in replication or

transcription mechanisms?
238
SUBJECTIVE QUESTIONS - ENZYMES
1- What is IUBMB system of nomenclature of enzymes? What is E.C.code number? What

is its significance?
2. - Urease acts upon urea to form CO2 and NH3. How will you comment upon the
specificity of Urease enzyme for its substrate? Give a brief account of enzyme specificity
giving various examples.
3. - What is active site? How does it participate in enzyme catalysis?
4. - What are cofactors? How can you differentiate between cofactor, coenzyme and
prosthetic group?
5. - Enzymes are known to increase the rate of catalysis by 100- 1000 folds or more. What
are the processes involved in enzyme catalyzed reactions?
6.-What is activational barrier? State your answer in terms of enzyme catalyzed reactions.
7.-The nature works in a conservative manner. How are enzyme activities regulated as per
the needs of the cells? Elaborate your answer giving suitable examples.
8. - What are the various factors which affect the rate of enzyme catalyzed reactions?
Or
Express the significance of-
a) Rising temperature on enzyme activity
b) Variation of p H on enzyme activity
c) Rising substrate concentration for a given amount of enzyme
d) Rising enzyme concentration for a given amount of substrate
9. - What is the effect of compartmentalization on enzyme activity?

Or
In urea cycle and in haem synthetic pathway, some of the reactions are cytoplasmic while
some are mitochondrial, what is the significance of this biological compartmentalization?
10- Classify enzyme inhibitors based on their mechanisms of actions.

Or
Enlist the commercially used enzyme inhibitors that are used as poisons
Or
Enlist the enzyme inhibitors used as pharmaceutical agents.
11.-What is suicidal inhibition? Support your answer giving suitable examples
12.- Differentiate between competitive and non competitive inhibition.
239
13- Differentiate between non competitive and un-competitive inhibition.
14- What is feed back inhibition? How does it differ from feed back regulation? Give
suitable examples in support of your answer.
15-What do you understand by allosteric modification? What is the ultimate mechanism

involved in this mode of regulation. Give suitable examples and support your answer with
diagrams.
16- Methotrexate an anticancer drug is known to increase km of the enzyme dihydrofolate

reductase (DHFR), for its substrate, dihydrofolate. The Vmax remains constant. What could
be the possible mechanism of action of this drug? Give examples of some inhibitors which
act similarly.
17- What is the relationship of km with the rate of reaction and substrate concentration?
Write an equation representing their mutual relationship.
18-What are isoenzymes? Explain your answer giving suitable examples of clinically
important isoenzymes.
19-Discuss the diagnostic significance of LDH (Lactate dehydrogenase), ALP(Alkaline

phosphatase) and Transaminases ?
20- Enlist the significance of enzyme estimations in –

a) Liver disorders
b) Alcoholism
c) Acute MI
d) Bony disorders
e) Cancers
21- Enzymes can be used as diagnostic reagents. Mention the names of the tests where
enzymes participate as diagnostic reagents.
22- A patient with carcinoma bladder has reported with wide spread secondaries in the
body. Which enzyme estimation would be of help in framing the diagnosis?
23- Discuss the therapeutic significance of enzymes. Elaborate your answer giving
examples.
24- Explain how investigation of the isozymes of lactate dehydrogenase and creatine kinase
in the serum of a person who had collapsed during a marathon would help to determine
whether the problem was a myocardial infarction or severe skeletal muscle damage?
240
25- Name the enzymes and coenzymes required for (a) the oxidation of ethanol to
acetaldehyde and (b) the removal and replacement of the amino group from the alpha-
carbon of amino acids?
26-Name two coenzymes which are required for carboxylation reactions, Point out how
they differ and write a reaction to illustrate the action of each coenzyme.
27- What is meant by rate limiting enzyme? Give two examples of such like enzymes in
support of your answer giving the reactions catalyzed by them.
28- What is the unit of measuring enzyme activity? What is turn over number?
29- What are zymogens? Give suitable examples.
30- Enlist functional enzymes of plasma. What is the significance of measuring non
functional plasma enzymes ?
241
PRACTICE QUESTIONS (SUBJECTIVE)- AMINO ACID
METABOLISM
Q.1- What is the biological advantage of secretion of proteolytic enzymes in the zymogen
forms in the gut?
Q.2- What is the role played by Glutathione in the absorption of amino acids?
Q.3- Discuss the disorders associated with the absorption of amino acids.
Q.4- Justify the reasoning that glutamic acid plays a pivotal role in the metabolism of
amino acids.
Q.5- Alpha Methyldopa is a drug used in the treatment of hypertension. Explain its possible
mode of action. (Hint- It is an inhibitor of DOPA Decarboxylase enzyme)
Q.6- Discuss the mechanism by which Ammonia is detoxified in the body.
Q.7-Describe the glucose-alanine cycle and explain its role in amino acid metabolism.
Q.8-Decarboxylation of some amino acids can lead to synthesis of physiologically

important compounds. Give evidences in support of this statement.
Q.9- What is the significance of urea cycle apart from urea formation?
Q.10- Give the reactions of the pathway of urea synthesis that involve the participation of
ATP
Q.11-What is oxidative deamination of amino acids? Give examples in support of your

answer.
Q.12- Name two neurotransmitters that are derived from the metabolism of amino acids
Show by means of reactions the mechanism of synthesis of each of them.
Q.13- What is transdeamination? State its importance and illustrate the answer giving
reactions in support of your answer.
Q.14-Discuss the biochemical roles of glutamate and glutamine in cell metabolism
Q.15- How will you define a nonessential amino acid? Under what condition can a non-
essential amino acid become essential? Explain clearly and illustrate your answer
giving suitable example.
242
Q.16-Discuss briefly about the metabolic role of Tyrosine, giving examples and suitable
reactions
Q.17-Describe transmethylation reactions giving suitable examples
Q.18-Show, by means of a diagram, the relationship between the urea cycle and the citric
acid cycle
Q.19-What is meant by (a) ketogenic amino acid and (b) glucogenic amino acid? Illustrate
your answer with a named example of each.
Q.20-Explain why phenylketonurics are warned against eating products containing the
artificial sweetener aspartame (Nutrasweet; chemical name L-Aspartyl-L-
Phenylalanine methyl ester)?
Q.21-Why are polyamines important in mammalian metabolism? Write the reactions of

polyamine biosynthesis and catabolism
Q.22-Describe the importance of glutamic acid in the synthesis and catabolism of other
amino acids
Q.23-Name the immediate precursor and the enzyme catalyzing the formation of: (a)
GABA (gamma-amino butyric acid) (b) Histamine and (c) DOPA
(dihydroxyphenylalanine).
Q.24-Explain briefly why ammonia is highly toxic to brain cells?
Q.25-A diet containing very little phenylalanine is used in the treatment of

Phenylketonuria, what is the reason? Explain why it is necessary to supplement
tyrosine in this diet.
Q.26-Certain amino acid are described as glucogenic. Explain briefly what is meant by the
term "glucogenic", illustrating your answer with the metabolic reactions of three
named glucogenic amino acid.
Q.27-Outline the metabolic processes by Tryptophan is converted into hormones and

neurotransmitters. Describe briefly the clinical condition produced by deficiencies in
these processes.
Q.28-Discuss the significance of Xanthurenic acid excretion test.
Q.29-Show by means of a diagram the point of entry of phenylalanine, glutamine and

methionine into the citric acid cycle
243
Q.30- Outline the steps of urea cycle and state its importance.
Q.31-Give the reaction catalyzed by a named (a) amino acid Decarboxylase and (b)
aminotransferase
Q.32-What is the origin of the nitrogen atoms in urea formation ? Discuss the reason that
deficiency of urea cycle enzymes especially Ornithine Trans Carbamoylase leads to
Orotic aciduria
Q.33-What is the P:O ratio when glutamate is oxidized by the glutamate dehydrogenase
reaction? Show the reaction in support of your answer.
Q.34- Outline the metabolic role of glycine, justifying the fact that it is nutritionally non
essential but functionally very essential.
Q.35-Trace the metabolic origin of the following urinary constituents: (a) creatinine (b)
urea and (c) ammonia. Discuss the significance of their altered excretion with
suitable examples.
Q.36-Account for the biochemical changes in the blood of a phenylketonuric subject.
Q.37- What is the biochemical basis for pellagra like rashes in Hart nup disease ?
Q.38- What is the defect in Carcinoid syndrome? What is the biochemical basis of
increased HIAA (Hydroxy Indole Acetic acid excretion) in Carcinoid syndrome ?
Q.39- Metabolism of which amino acid is associated with FIGLU excretion test for the
detection of underlying folic acid deficiency?
Q.40- What is the defect in Maple syrup urine disease? Discuss in brief about the
symptoms, laboratory diagnosis and its treatment.
Q.41- What is the cause of increased risk for ischemic heart disease in patients of
Homocystinuria Discuss in brief about the classification, clinical manifestations and
laboratory diagnosis of Homocystinuria.
Q.42- Discuss the functions and therapeutic uses of nitric oxide
Q.43 - Discuss briefly about the biological and clinical significance of Transaminases.
Q.44- What is the defect in Cystinuria? Why is it associated with renal stone formation?
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PRACTICE QUESTIONS- SERUM CREATININE AND CREATININE
CLEARANCE ESTIMATION
Q.1- What is the range of serum creatinine in normal health?

Answer- The serum creatinine ranges between-
1) In children (<12 years) 0.25-0.85 mg/dl
2) Adult male- 0.7-1.5 mg/dl
3) Adult female- 0.4-1.2 mg/dl
Q.2- What are the conditions of high serum creatinine levels?

Answer- Higher levels are observed in - Renal failure (All causes) and in muscular
dystrophies. Falsely high levels are observed in diabetic ketoacidosis.
Q.3- If in a patient, serum creatinine has been found to be higher than normal but
blood urea is within the normal range, what is the likely possibility?
Answer- It can not be renal failure because in such a state both blood urea and serum
creatinine should have been higher, since both are excreted by kidney through urine in
normal health. But since blood urea is normal, it could be any other reason and the most
likely cause is muscular dystrophy. The diagnosis can be made from the history and clinical
symptoms. The other possibility can be of false high value as in diabetic keto acidosis.
Q.4- In a patient with normal serum creatinine level, blood urea has been found to be
much higher than normal, what could be the possibility?
Answer- Both urea and creatinine should be higher than normal in renal failure, if
creatinine is normal the possibility of renal failure can be ruled out. Blood urea can be
higher than normal in conditions of- Advancing age, high protein diet, dehydration,
catabolic state and in post renal obstructive conditions (Stone, stricture, growth etc)
Q.5- What is the principle of alkaline picrate method (Jaffe’s reaction) for the
estimation of serum creatinine?
Answer- Creatinine under alkaline conditions reacts with Picric acid to form Creatinine
picrate (An orange red colored complex), the intensity of which is measured at 520 nm.
Q.- 6- Alkaline picrate method is considered a less sensitive method for creatinine
estimation, what are the other substances which can give a positive reaction with
alkaline picrate?
Answer- Jaffe’s reaction is not specific for creatinine. In serum up to 20% of the total
chromogens (Color forming substances) can be substances other than creatinine which give
a positive reaction with alkaline picrate, while in urine these are only 5%. Other non
specific chromogens that react with Picric acid are – proteins, ketone bodies, pyruvate,
glucose and Ascorbate.
Q.7- What is the difference between creatine and creatinine?
245
Answer- Creatinine is the anhydrous product of creatine. Creatine is converted to creatinine
non enzymatically by the loss of one molecule of water. About 2 % of creatine is converted
to Creatinine daily.
Q.8- Which form out of creatine and creatinine is present in urine in normal health?
Answer- In normal urine creatinine is mainly present, creatine is present only in trace
amounts.
Q.9-Name the amino acids that contribute towards creatine synthesis

Answer- Creatine is synthesized from Glycine, Arginine and Methionine. In the first step,
Glycine and Arginine combine together to form Guanido Acetic acid, this reaction takes
place in kidney. In the second step, Guanido acetic acid is methylated by Methionine to
form Methyl Guanido acetic acid (Creatine). This reaction takes place in liver. Creatine is
transported to muscles, where it is phosphorylated and stored in the form of creatine-P.
98% of the total amount of creatine is present in muscles.
Q.10- What is Lohmann reaction?

Answer- Creatine is phosphorylated to creatine-P by the enzyme Creatine kinase, present in
muscle, brain and myocardium. The stored creatine phosphate in the muscle serves as an
immediate source of energy. During muscle contraction, the energy is first derived from
ATP hydrolysis. Thereafter, the ATP is generated by the -hydrolysis of creatine-P. The high
energy phosphate is transferred to ADP to form ATP. This reaction is called Lohmann
reaction. In the resting muscles the creatine-P is restored at the expense of ATP provided
from glycolysis.
Q.11- What are the common causes of creatinuria?

Answer- Excretion of creatine in urine is called Creatinuria, which is observed under the
following conditions-
1) In children- Probably due to impaired conversion of creatine to creatinine
2) Pregnancy
3) Febrile conditions
4) Thyrotoxicosis
5) Muscular dystrophies, myositis and Myasthenia gravis
6) Uncontrolled diabetes mellitus
7) Starvation
8) Wasting diseases- such as Malignancies.
Q.12- What is creatinine co-efficient? What is its significance?

Answer- It is the ratio of- mg of creatinine in urine in 24 hours/ Body weight in kg.
The value is 20-26 for males and 14 to 22 in females.
Significance- It depends on muscle mass and remains fairly constant. Since muscle mass
remains constant in a given individual, the creatine coefficient serves as a reliable index of
the adequacy of a 24 hour urine collection
246
Q.13- What is the reason for high creatinine level in males in normal health?
Answer- Creatine is synthesized in liver, passes in to circulation and is almost taken
entirely by the skeletal muscle for conversion to creatine-P, which serves as a storage form
of energy in skeletal muscles. About 2% of creatine is converted to Creatinine daily. Since
its concentration is related to the muscle mass and males have more muscle mass that is
why the level of serum creatinine is higher in males in normal health.
Q.14- What is the normal range of creatinine clearance? What is the significance of
measuring creatinine clearance?
Answer- The normal values of creatinine clearance are-
Males- 95-140 ml/minute
Females-85-125 ml/minute
These values are close to GFR (Glomerular filtration rate). Clearance values are decreased
in impaired renal functions and so provide a rough measure of renal damage.
Q.15- Out of urea and creatinine clearance, the estimation of which clearance is
preferred to assess renal functional status and why?
Answer- Unlike urea, serum Creatinine level is not affected by diet, age, dietary factors or
by fluid depletion. Creatinine is filtered but is not absorbed by the tubules (unlike urea),
hence it is a better predictor of GFR. (The values are slightly higher than GFR due to
tubular secretion). The methodology is also simple, due to all these reasons; creatinine
clearance is preferred over urea clearance for determining the functional status of the
kidney.
Q.16- Calculate the creatinine clearance of a patient with serum creatinine of 3

mg/dL, volume of urine excreted 1500 ml/ day and urinary creatinine of 0.75 G/L
Answer- Creatinine clearance(C)= UV/P
Where U= Urinary creatinine (mg/dl)
V= Volume of urine excreted (ml/day)
P= Serum Creatinine (mg/dl)
Thus applying the values-
V= 1500 ml/day, convert to ml/ minutes
i.e. = 1500/24x60= 1.1 ml/minute (Approximately)
U= 0.75 G/L, Convert it to mg/dl
i.e. - 75 mg/dl
Creatinine clearance (C) = 75x1.1/3
= 27.5 ml/minute
It is much below the physiological range; hence it is a case of impaired renal functions.
Q.17- Comment upon the functional status of the kidney, if the serum creatinine is 4.5
mg/dL, blood urea- 86 mg/dL and serum uric acid as 12 mg/dl.
Answer- Urea, creatinine and uric acid are normally excreted by kidney through urine,
since the levels of three of them are higher than normal in the given case that means kidney
247
is failing to clear out these substances from blood that is why they are accumulating in
blood, hence it is a case of impaired renal functions, possibly renal failure.
Q.18- A patient with long standing diabetes mellitus has reported to emergency with
generalized swelling of the body. Blood biochemistry reveals-
Hb- 8 G/dL
F.B.S- 260 mg/dl
Blood urea- 98 mg/dl
Serum creatinine- 3.4 mg/dL
Serum Uric acid- 10.8 mg/Dl
Urine analysis- Sugar ++++
Protein-Present
Urea clearance 32 ml/minute
Creatinine clearance- 68 ml/minute
Comment on the findings and provide a provisional diagnosis.
Answer- It is a case of renal failure due to long standing diabetes mellitus. Blood urea,
serum creatinine and uric acid are high, clearance values are low, Hb is low due to
decreased Erythropoietin, sugar and protein in urine are suggestive of Diabetic nephropathy
which has progressed to renal failure.
Q.19- Out of serum creatinine and blood urea, which is more sensitive indicator of
falling renal functions?
Answer- Serum creatinine is more sensitive indicator of falling renal functions than blood
urea. Urea level is affected by non renal causes also, while creatinine is a relatively a stable
parameter, hence its measurement carries more significance to assess falling functional
status of the kidney.
Q.20- In a patient with diabetic ketoacidosis, creatinine is high while blood urea is
normal, what is the possibility?
Answer- Jaffe’s reaction for estimation of serum creatinine gives positive reaction with
Glucose and ketone bodies also, which are high in diabetic ketoacidosis. It is falsely high
level due to other chromogens and not due to creatinine. Normal blood urea level indicates
normal renal functions.
248
SOLUTION TO PRACTICE QUESTIONS BIOCHEMISTRY
Q.-1- All amino acids except one participate in phase 2 reactions of detoxification-
a) Serine
b) Glycine
c) Glutamine
d) Cysteine
(a)
Q.2- Which out of the following pathways helps in reductive biosynthesis-
a) Uronic acid pathway
b) HMP pathway
c) Glycolysis
d) All of the above
(b)
Q.3- All of the following clinical manifestations except one are present in
hemochromatosis-
a) Bronze discoloration of skin
b) Diabetes mellitus
c) Iron overload
d) Hemolytic anemia
(d)
Q.4- A person on ingestion of Primaquine develops hemolytic anemia, what is the possible
defect?
a) Deficiency of Iron
b) Vitamin K deficiency
c) Glucose-6-P dehydrogenase deficiency
d) Vitamin C deficiency
(c)
Q.5- The enzyme responsible for conversion of Biliverdin to Bilirubin is-
a) Bilirubin esterase
b) Bilirubin oxidase
c) Glucuronyl transferase
d) Biliverdin reductase
(d)
Q.6-Biotin is involved in which of the following types of reactions-
a) Deamination
b) Decarboxylation
c) Carboxylation
d) Transamination
(c)
Q.7-The Xanthurenic acid test (Xanthurenic index) can be used to measure pyridoxine
deficiency, it involves the metabolism of-
a) Glycine
249
b) Histidine
c) Tryptophan
d) Tyrosine
(c)
Q.8-McArdle’s disease is characterized by the deficiency of-
a) Muscle phosphorylase
b) Liver phosphorylase
c) Glucose-6-phosphatse
d) Phosphofructokinase
(a)
Q.9-Out of 24 mols of ATP formed in citric acid cycle, 2 mols of ATP can be formed at
substrate level by which of the following reaction?
a) Citrate------ Isocitrate
b) Isocitrate-------- Oxalo succinate
c) Succinate------- Fumarate
d)Succinyl co A-- Succinate
(d)
Q.10- All of the following metabolic abnormalities are observed in Diabetes mellitus,
except-
a) Increase plasma free fatty acids
b) Increased pyruvate carboxylase activity
c) Decreased PDH complex activity
d) Increased lipoprotein lipase activity
(d)
Q.11-Beta oxidation of odd chain fatty acids yields-
a) Succinyl co A
b) Propionyl co A
c) Acetoacetyl co A
d) Dicarboxylic acids
(b)
Q.12-Iron therapy is ineffective in which of the following conditions-

a) Chronic blood loss
b) Inadequate iron intake
c) Thalassemia major
d)Acute blood loss
(c)
Q.13-Which of the following vitamins is not a component of electron transport chain-

a) Nicotinamide
b) Ubiquinone
c) Biotin
250
d) Riboflavin
(c)
Q.14-Which of the following enzymes does not have an impaired activity in Vitamin B1
deficiency?
a) Succinate dehydrogenase
b) Pyruvate dehydrogenase
c) Transketolase
d) Alpha keto glutarate dehydrogenase
(a)
Q.15- Considering the citric acid cycle steps between alpha keto glutarate and Malate, how
many high-energy phosphate bonds or net ATP molecules can be generated?
a) 5
b) 6
c) 8
d) 10
(b)
Q.16- The standard free energy change (in terms of net ATP production) when glucose is
converted to 6CO2 and 6H2O is about how many times as great as the free energy change
when glucose is converted to two lactate molecules ?
a) 2
b) 4
c) 19
d) 10
(c)
Q.17- The rate of flow of electrons through the electron transport chain is regulated by
a)ATP:ADP ratio
b) Concentration of Acetyl co A
c) Feed back inhibition by H2O
d) Catalytic rate of cytochrome oxidase
(a)
Q.18- The major product of fatty acid synthase complex is-

a) Oleate
b) Palmitate
c) Palmityl co A
d) Stearoyl co A
(b)
Q.19- The primary enzyme for utilization of ketone bodies is-
a) Thiokinase
b) Thioesterase
c) Thiophorase
251
d) Thiolase
(c)
Q.20-All of the following processes except one are mitochondrial-
a) Glycolysis
b) TCA cycle
c) Beta oxidation of fatty acids
d) Ketogenesis
(a)
252
SOLUTION TO MULTIPLE CHOICE QUESTIONS-ENZYMES
Q.1- Glycogen phosphorylase, which mobilizes glycogen for energy, requires which of the
followings as a cofactor?
a) Pyridoxal phosphate
b) Tetra hydro folate
c) Adenosyl Cobalamine
d) Coenzyme A (a)
Q.2- Choose the incorrect statement about Active Site of an enzyme-

a) The active site is a three-dimensional cleft
b) The active site takes up a large part of the total volume of an enzyme
c) Substrates are bound to enzymes by multiple weak attractions
d) The specificity of binding depends on the precisely defined arrangement of atoms in an
active site.
(b)
Q.3- Any of the following processes except one are involved at the active site of an enzyme
to accelerate the rate of reaction-
a) Catalysis by Bond Strain
b) Catalysis by Proximity and Orientation
c) Non covalent catalysis
d) Acid base catalysis (c)
Q.4-A given substrate may be acted upon by a number of different enzymes, each of which
uses the same substrate(s) and produces the same product(s). The individual members of a
set of enzymes sharing such characteristics are known as-
a) Group specific enzymes
b) Isoenzymes
c) Substrate specific enzymes
d) Allosteric enzymes
(b)
Q.5- A recently diagnosed hypertensive patient has been prescribed an ACE inhibitor
(Angiotensin convertase inhibitor) which is known to act by lowering V max, what is the
possible mechanism of inhibition of this drug?
a) Competitive
b) Non Competitive
c) Uncompetitive
(a)
253
Q.6- Which statement out of the followings is incorrect about the effect of increasing
temperature on enzyme activity-
a) Raising the temperature increases the kinetic energy of molecules
b) A ten degree Centigrade rise in temperature will increase the activity of most enzymes
by 50 to 100%.
c) Most animal enzymes rapidly become denatured at temperatures above 40oC
d) Storage of enzymes at 5°C or below is generally not suitable.
(d)
Q.7-A 54-year –old male was rushed to emergency when he collapsed in the middle of a
business meeting. Examination revealed excessive sweating and high blood pressure.ECG
chest was conclusive of Acute Myocardial infarction. Which biochemical investigation out
of the followings would be of no help in the confirmation of diagnosis?
a) Cardiac Troponins
b) Serum myoglobin
c) Lactate dehydrogenase
d) Creatine Phospho kinase-MB(CPK-MB)
(c)
Q.8- A coal mine worker was brought in an unconscious state to emergency room after a
blast in the mine. His blood Carboxy hemoglobin level was high and he was diagnosed
with CO poisoning. CO is a known inhibitor of electron transport chain. Which complex of
electron transport chain is inhibited by CO?
a) Complex I
b) Complex II
c) Complex III
d) Complex IV
(d)
Q.9- A 42-year-old obese female presented to the emergency center with complaints of
worsening nausea, vomiting, and abdominal pain. Her pain was located in the midepigastric
area and right upper quadrant. Blood biochemistry revealed high serum amylase level.
What is the probable diagnosis for this patient?
a) Viral hepatitis
b) Acute Pancreatitis
c) Renal colic
d) Acute gastritis
(b)
254
Q.10- A 60 year old chronic alcoholic was brought to the hospital with complaints of
protuberant abdomen (ascites) and edema feet. He also had history of hemorrhages. Blood
biochemistry revealed – High serum transaminases, low Serum total proteins, Albumin and
a prolonged prothrombin time. Urine analysis was normal. What could be the possible
diagnosis?
a) Renal failure
b) Protein malnutrition
c) Cirrhosis of liver
d) Heart failure
(c)
Q.11- A 2 -week –old child was brought to the emergency. The parents were fearful that the
child had been given some poison as they noted black discoloration on the diaper. A
diagnosis of Alkaptonuria was made and the child was given Vitamin C as a supplement.
Alkaptonuria occurs due to reduced activity of Homogentisic acid oxidase enzyme. What is
the role played by vitamin C in this defect?
a) Acts as an oxidant
b) Acts as a coenzyme
c) Acts as an inducer
d) Acts as a positive allosteric modifier
(b)
Q.12-A 67- year-old army officer in good health previously presented with sudden pain in
the great toe. Serum uric acid level was high, and a diagnosis of gouty arthritis was made
He was advised bed rest, pain killers and Allopurinol. What is the mechanism of action of
Allopurinol in lowering serum uric acid levels?
a) Suicidal inhibition
b) Non competitive inhibition
c) Allosteric inhibition
d) Feed back inhibition
(a)
Q.13-One out of the following enzymes has absolute specificity for its substrate; choose the
correct option-
a) Urease
b) Carboxy peptidase
c) Pancreatic lipase
d)Lipoprotein lipase
(a)
Q.14- Which out of the followings is a substrate-specific enzyme?

a) Hexokinase
255
b)Thiokinase
c) Lactase
d) Decarboxylase
(c)
Q.15-Which out of the followings is not a substrate-specific enzyme?

a)Glucokinase
b)Fructokinase
c) Hexokinase
d)Phosphofructokinase
(c)
Q.16-Group I Co enzymes participate in which of the following reactions-
a) Oxidation-reduction
b)Transamination
c) Phosphorylation
d) All of the above
(a)
Q.17-Which out of the following co enzymes takes part in hydrogen transfer reactions in
the electron transport chain-
a) Tetrahydrofolate
b) Methyl Cobalamine
c) Co enzyme Q
d) Biotin
(c)
Q.18.The conversion of Pyruvate to oxaloacetate involves the participation of---- as a

coenzyme -
a) NAD+
b) NADPH
c) Biotin
d) All of the above (c)
Q.19- The drug Fluorouracil is recommended for the treatment of cancers. It undergoes a
series of changes and then binds to Thymidylate synthase enzyme resulting in its inhibition
and blockage of cell division. This mode of inhibition is most probably due to-
a) Allosteric inhibition
b) Competitive inhibition
c) Noncompetitive Inhibition
d) Suicidal inhibition (d)
256
Q.20-The activities of many enzymes, membrane transporters and other proteins can be
quickly activated or inactivated by phosphorylation of specific amino acid residues. This
regulation is called-
a) Allosteric modification
b) Covalent modification
c) Induction
d) Repression
(b)
257
NORMAL LABORATORY REFERENCE RANGE
Blood (B), Serum(S), Plasma (P), Urine (U)
Blood Gases
pH (B- Arterial) 7.35 - 7.45 (H+44.7-45.5 nmol/L)
Partial pressure of CO2 (B-Arterial) 35 – 45mm Hg(4.7-6 kPa
( PaCO2)
Partial pressure of O2(PaO2 ) (B-Arterial 80) - 100mm Hg(10.67-13.33kPa)
Bicarbonate (HCO3-) 24-28 meq/L(24-28 mmol/L)
O2 Sat %( Arterial) 90-95
O2 Sat %( Venous) 40-70
CO2 content Total Serum 21-30 mmol/L
CO <5% of Hemoglobin
Anion gap 7–16 mmol/L
Hematology
Hemoglobin (B)Men-14-18g/dl (2.09-2.79mmol/L)
Women-12 – 16 g/dL (1.86-2.48 mmol/L)
(S) 2-3mg/dl
Haemtocrit (PCV) Men 40 – 52% (0.4-0.52)
Women 37 – 47%(0.37-0.47)
Red Blood Count (RBC) Men-4.5-6.2million/ȝl(4.5-6.2x1012/L)
Women-4-5.5million/ȝl(4-5.5x1012/L)
Reticulocytes 0.2-2% of red cells
White blood Count(WBC) 5000-10,000/ ȝl (5-10x109/L)
• Polymorph nuclear (PMN): 35-80%

• Immature Polys (Bands): 0-10%
• Lymphocytes (Lymp): 20-50%
• Monocytes (Mono): 2-12%
• Eosinophils (Eos): 0-7%
• Basophils (Bas): 0-2%
Platelets 150,000-400,000/ ȝL(0.15-0.4x1012/L)

Mean Corpuscular Men-80-94f L
volume(MCV) Women-81-99f L (By coulter counter)
Mean Corpuscular 27-32pg
Hemoglobin(MCH)
Mean Corpuscular Hemoglobin 32-36g/dl, red blood cells (32-36%)
Concentration(MCHC)
Average diameter of red cell 7.3ȝm(5.5-8.8 ȝm)
Bleeding time Ivy method, 1-7 minutes(60-420 seconds)
Template method, 3-9 minutes (180-540seconds).
258
Clot retraction Begins in 1-3 hrs: complete in 6-24 hrs. No clot lysis
in 24 hours.
Fragility of red cells Begins at 0.45-0.38%NaCl, complete at 0.36-0.3%
NaCl
Partial Thromboplastin time Activated, 25-37seconds
Prothrombin Time (P)11-14.5 seconds, International Normalized
Ratio(INR)- (P)2.0-3.0
Erythrocyte Sedimentation Male: 1 - 13 mm/hr
Rate (ESR or Sed-Rate) Female: 1 - 20 mm/hr
Blood Volume 8.5 - 9.1% of total body weight
Endocrines
Adrenals hormones
Cortisol (P) 8.00 AM, 5-25ȝg/dl(138-690nmol/L)
8.00 PM<10 ȝg/dl(275 nmol/l)
Aldosterone (P) Supine 2-9 ng/dl (56-250 pmol/L)Increased when
upright
Dopamine (P) <135 pg/ml
Epinephrine (P)Supine,<100 pg/ml(<550pmol/l)
Nor Epinephrine (P)Supine,<500 pg/ml(<3nmol/l)
Adrenal hormones and
metabolites
Aldosterone (U)2-26ȝg/24 hrs (5.5-72 nmol/d), values vary with
sodium and potassium intake
Catecholamines (U)Total,<100 ȝg//24 hrs
Epinephrine (U)<10 ȝg//24 hrs(<100 nmol/d)
Nor Epinephrine (U)<100 ȝg//24 hrs(<590nmol/d)

Cortisol Free(U)-20-100 ȝg//24 hrs(0.55-2.76ȝmol/d)
11,17 OH corticoids (U)Men-4-12mg/24 hrs
Women-4-8mg/24 hrs
17- keto steroids (U)< 8 Years 0-2mg/24 hrs
Adolescents-2-20mg/24 hrs(1mg=3.5ȝmol)
Metanephrine (U)<1.3 mg/24 hrs(<6.6 ȝmol/d) or <2.2ȝg/mg
creatinine
Vanillyl Mandelic acid(VMA) (U)Up to 7 mg/24 hrs(< 35 ȝmol/d)
Pituitary
Growth hormone(GH) (S) Adults, 1-10 ng/ml, (46-465 pmol/L) by RIA
Thyroid Stimulating (S) < 10ȝU/ ml
hormone(TSH)
Follicle stimulating hormone (S) Pre pubertal-, 2-12 m IU/ml
(FSH) Adult men- 1-15 m IU/ml
Adult Women-1-30 m IU/ml
259
Luteinizing hormone(LH) (S) Pre pubertal-, 2-12 m IU/ml
Adult men- 1-15 m IU/ml
Adult Women-< 30 m IU/ml
Corticotropin (ACTH) (P) 8.00-10.00 AM, up to 100pg/ml, (22pmol/L)

Prolactin (S) 1-25ng/ml (0.4-10 nmol/L)
Somatomedin C (P) 0.4-2U/ml
Anti diuretic Hormone (ADH, (P)Serum osmolality285 mosm/kg, 0-2 pg/ml; >290
Vasopressin) mosm/kg. 2-12 + pg/ml
Placenta
Estriol(E3) (S) Men and non pregnant women <0.2ȝg/dl
(<7nmol/L) by RIA
Chorionic Gonadotropin (S) Beta subunit; Men-< 9 mIU/ mL
Pregnant Women->10 mIU/ mL
Gonads
Testosterone, free (S) Men, 10-30 ng/dl; Women 0.3-2 ng/dl.
(1ng/dl=0.035nmol/L)
Testosterone, Total (S) Pre pubertal , < 100 ng/dl ; Adult men, 300-1000
ng/dl; Adult women 20-80 ng/dl, luteal phase up to
120 ng/dl
Estradiol(E2) (S) Men- 12-34 pg/ml;
women, menstrual cycle 1-10 days 24-68 pg/ml
11-20 days, 50-300 pg/ml
21-30 days, 73-149 pg/ml by RIA, (Ipg/ml=3.6
pmol/L)
Progesterone (S) Follicular phase, 0.2-1.5 ng/ml
luteal phase, 6-32 ng/ml;
Pregnancy,>24 ng/ml,
Men, < 1ng/ml= 3.2 nmol/L
Thyroid
Thyroxin, free T4 (FT4) (S) 0.8-2.4ng/dl (10-30 p mol/L)
Thyroxin, Total (TT4) (S) 5-12 ȝg/dl(65-156nmol/L) by RIA
Thyroxin binding globulin (S) 12-28 ȝg T4/dl (150-360 nmol T4/dl)
capacity
Tri iodo thyronine (T3) (S) 80-220 ng/dl (1.2-3.3 nmol/L)
Reverse Tri iodo thyronine (r (S) 30-80 ng/dl (0.45-1.2 nmol/L)
T3)
Tri iodo thyronine (S) 25-36% as TBG assessment(RT3U ratio) 0.85-1.15
uptake(RT3U)
Calcitonin (S)<100 pg/ml (< 29.2pmol/L)
Islets
260
Insulin (S) 4-25ȝU/mL(29-181pmol/L)
C- peptide (S) 0.9-4.2ng/mL
Glucagon (S), fasting) 20-100pg/mL
Parathyroid
Parathyroid hormone (intact) (S)8–51 pg/mL.
Stomach
Gastrin (S) up to 100 pg/ml (47 p mol/L).
Elevated, > 200 pg/ml
Pepsinogen I (S) 25-100 ng/ml
Iron Studies
Ferritin Adult Women- 20-120ng/ml

Men-30-300ng/ml
Child-15 years-7-140ng/ml
Iron (S) 50-175 ȝg/dl(9-31.3 ȝmol/L)
Iron Binding capacity (S)Total- 250-410 ȝg/dl(44.7-73.4ȝmol/L)
% saturation- 20-55%
Transferrin (S) 200-400 mg/dl(23-45ȝmol/L)

Haptoglobin (S) 40-170mg of Hb binding capacity
Blood Chemistry
Alanine Aminotransferase 0-45 IU/L at 37°C
(ALT) SGPT
Aspartate Aminotransferase 0-41 IU/L at 37°C
(AST) SGOT
Alkaline Phosphatase (ALP) (S) Adults- 5-13 units(KA), 0.8-2.3(Bessey-
Lowry):SMA 30-85 IU/L at 37°C: SMAC 30-115IU/L
at 37°C
Amylase (S)- 80-180 units/dl (Somogyi)

Gamma Glutamyl (S) <30 units/L at 30°C
Transpeptidase
Lipase (S) <150 units/L
Acid phosphatase (S)- 1-5 U(KA),0.1-0.63U(Bessey- Lowry)
Total Creatine Kinase (Total (S)-10-50 IU/L at 30°C
CK)
Creatine Kinase-MB (CK-MB) 0-3%
Creatine Kinase-MM (CK- 97-100%
MM)
Creatine Kinase-BB (CK-BB) 0%
Lactate dehydrogenase (S)-55-140 IU/L at 30°C
Aldolase (S) 1.5-2.0 U/L
261
Troponin <0.4 ng/ml
5' Nucleotidase 0–11 U/L
Anti-streptolysin O Titer Adult: <125
(ASO)
Child: <250
Glucose (S or P) 65-110 mg/dl(3.6-6.1 mmol/l)
Impaired glucose tolerance 111–125 mg/dL (6.2–6.9 mmol/L)
Diabetes mellitus >125 mg/dL(>7.0 mmol/L)
Glucose, 2 h postprandial 70–120 mg/dL(3.9–6.7 mmol/L)
Fructosamine <285 ȝ mol/L
Hemoglobin Alc 4.0–6.0%
Urea Nitrogen (S or P) 8-25 mg/dl (2.9-8.9 mmol/)
Creatinine (S or P) 0.7-1.5 mg/dl (62-132ȝmol/)
Uric acid (S or P) Men-3-9 mg/dl(0.18-0.54 mmol/)
Women-2.5-7.5 mg/dl(0.15-0.45 mmol/)
Bicarbonate (S) 24-28 meq/L(24-28mmol/L)

Potassium (S or P) 3.5-5 meq/L (3.5-5 mmol/L)
Sodium (S or P) 136-145 meq/L (136-145mmol/L)
Chloride (S or P) 96-106 meq/L( 96-106mmol/L)
Magnesium (S or P)1.8-3 mg/dl(0.75- 1.25mmol/L)
Copper (S or P) 100-200ȝg/dl(16-31 ȝmol/L)
Zinc (S) 50-150 ȝg/dl (7.65-22.95 ȝmol/L)
Phosphorus, inorganic (S –fasting) 3-4.5 mg/dl (1-1.5mmol/L)
Calcium (S) 8.5-10.3 mg/dl (2.1-2.6mmol/L)
Calcium (ionized) (S) 4.25-5.25 mg/dl; 2.1-2.6 meq/L (1.05-1.3 mmol/L)
Lead (U)< 80 ȝg/24 hrs (< 0.4ȝmol/d)
Pyruvate (B) 0.6-1 mg/dl (70-114 ȝmol/L)
Lactate (B) Venous-, 4-16 mg/dl, (0.44-1.8mmol/L)
Ketone (acetone) (S)Negative
Acetone and Acetoacetate (S) 0.3-2 mg/dl (3-20 mg/L)
ȕ-Hydroxybutyrate (S)0–3 mg/dL(0–290 ȝmol/L)
Serum Total Protein (S) 6-8g/dl (60-80 g/L)
Albumin (S) 3.5 -5.5 g/dl (35-55 g/L)
Prealbumin 170–340 mg/L
Globulins (S)2-3.6 g/dl ( 20-36 g/L)
Fibrinogen (P) 0.2-0.6 g/dl (2-6 g/L)
Homocysteine 4.4–10.8 ȝmol/L
Į1 –Antitrypsin (S)> 180 mg/dl
C-reactive protein 0.2–3.0 mg/L
Prostate-Specific Antigen 0 - 4 ng/mL (likely higher with age)
262
(PSA)
Ceruloplasmin (S) 25-43mg/dl (1.7-2.9ȝ mol/L)
Bilirubin (S) Total, 0.2-1.2 mg/dl (3.5-20.5 ȝ mol/L)
Direct- (Conjugated), 0.1-0.4 mg/dl (<7 ȝ mol/L)
Indirect, 0.2-0.7 mg/dl (<12 ȝ mol/L)
Urobilinogen (U) 0-2.5mg/24 hrs (70-470 ȝ mol/d)
Urobilinogen(Fecal) 40-280 mg/24 hrs (70-470 ȝ mol/d)
Porphyrins 1.5-7.5 mg/24 hrs (11-57 ȝ mol/d)
Delta Amino Levulinic acid (U)
Prophobilinogen (U) < 2 mg/24 hrs (<8.8 ȝ mol/d)
Ammonia (P) 10-80 ȝg/dl
Osmolality (S) 280-296mosm/kgwater (280-296 mmol/kg water)
Specific Gravity (B) 1.056(Varies with Hb and protein concentration)
(S) 1.0254- 1.0288(varies with protein concentration)
Fecal Fat <30% dry weight
Lipid profile
Cholesterol mg/dL (mmol/L) (S or P) 150-250 mg/dl (3.9- 5.72 mmol/L)
<200 (<5.17)- desirable
200–239 (5.17–6.18) –borderline high
>240 (>6.21) - high
Triglycerides (S)< 165 mg/dl (1.9 mmol/L)

Lipid fractions Desirable levels
HDL Cholesterol <40 (<1.03)- low, >60 (>1.55)- high
mg/dL(mmol/L)
LDL Cholesterol i) <70 (<1.81) -Therapeutic option for very
mg/dL(mmol/L) high risk patients
ii) <100 (<2.59) -Optimal
iii) 100–129 - Near optimal/above
(2.59 –3.34) optimal
iv) 130–159 -Borderline high
(3.36– 4.11)
v) 160–189 -High
(4.14–4.89)
vi) >190 (4.91) -Very high
VLDL Cholesterol <40 mg/dl
Free fatty acid (P) 200-800ȝmol/L
Lipoprotein (a) 0–30 mg/dL
Vitamins
Vitamin A (S) 15-60 ȝg/dl (0.53-2.1 ȝmol/L)
ȕ Carotene (S) Fasting) 50-300 ȝg/dl
263
Vitamin B12 (S)> 200 pg/ml (>148 pmol/L)
Vitamin D (S) Cholecalciferol (D3); 25- hydroxy cholecalciferol,
8- 55 ng/ml (19.4-137 nmol/L);
1,25 dihydroxy cholecalciferol, 26-65 pg/ml (62-
155pmol/L);24,25-dihydroxy cholecalciferol,
1-5ng/ml (2.4-12 nmol/L).
Vitamin C (Ascorbic acid) (P) 0.4-1.5 mg/dl (23-85ȝmol/L)

Folic acid (S) 14-34 nmol/L
Vitamin E (S) 5–18 ȝg/mL(12–42ȝ mol/L)
Vitamin K (S) 0.13–1.19 ng/mL(0.29–2.64 nmol/L)
Vitamin B6 (P) 5–30 ng/mL(20–121 nmol/L)
Immunoglobulins
Ig A 9.0-33 g/L
Ig G 7.2-15.0 g/L
Ig M 0.5-2.5 g/L
Ig D 0-0.4 g/L
Ig E 100-200 ȝg/L
Renal function Tests
p- Amino Hippurate(PAH) Men, 560-830 ml/min,
clearance (RPF) Women, 490-700 ml/min.
Creatinine Clearance, Men, 110-150 ml/min.
endogenous (GFR) Women, 105-132 ml/min.
Inulin clearance Approximately same as creatinine( corrected to 1.73
m2 surface area
Filtration fraction(FF) Men-17-21%; women, 17-23 %(FF=GFR/RPF)
Osmolality (U)On normal diet and fluid intake: Range 500-850
mOsm/kg water. Achievable range, normal kidney:
Dilution 40-80 mOsm; concentration(dehydration) up
to 1400 mOsm/kg water(At least three to four times
plasma osmolality)
Specific gravity of urine 1.003-1.030
Urinalysis
Urine volume 0.4 -2.0 L/day.
Urine pH 5-7
Acidity, titratable 20–40 meq/d(20–40 mmol/d)
Ammonia 30–50 meq/d(30–50 mmol/d)
Specific Gravity 1.002-1.030
Urine Ketone Negative
Urine blood Negative
Urine Proteins Negative
264
Urine Nitrites Negative, Traces
Urine Bilirubin Negative
Urobilinogen 0-2.5mg/24 hrs
Urine Micro • RBCs: 0-2/HPF
• WBCs: 0-2/HPF
• RBC Casts: 0/HPF
Urine Glucose < 250mg/dl

Urine Creatinine Men-1.0-2.0 g/d
Women-0.8-1.8 g/d
Urea nitrogen 6–17 g/d(214–607 mmol/d)
Uric acid (normal diet) 250–800 mg/d(214–607 mmol/d)
Sodium (varies with intake) 100–260 meq/d(100–260 mmol/d)
Potassium (varies with intake) 25–100 meq/d(25–100 mmol/d)
Phosphate (phosphorus) (varies 400–1300 mg/d(12.9–42.0 mmol/d)
with intake)
Micro albumin
Normal 0–30 mg/d(0.0–0.03 g/d)
Microalbuminuria 30–300 mg/d(0.03–0.30 g/d)
Clinical albuminuria >300 mg/d(>0.3 g/d)
Micro albumin/creatinine ratio

Normal 0–30 ȝg/mg creatinine(0–3.4 g/mol creatinine)
Microalbuminuria 30–300 ȝg/mg creatinine(3.4–34 g/mol creatinine)
Clinical albuminuria >300ȝg/mg creatinine(>34 g/mol creatinine)
Cerebral Spinal Fluid
Pressure 60-150 mm Hg
Specific gravity 1.006 to 1.007
pH 7.3
CSF glucose 45-100 mg/dl
CSF Proteins 10-45 mg/dl
CSF Chlorides 700-760 mg/dl (120-130 meq/L) as NaCl
CSF urea 20-40 mg/dl
CSF Calcium 5.5-6 mg /dl
CSF cells 0-4 mononuclear per C.mm
265
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Biochemistry- A Case Oriented Approach for Medical Students

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1. Chemistry of Carbohydrates- Viva and Revision 7

Q.1- Choose the odd one out-

Heparin, Heparan, Dermatan, Dextran

Q.2- Choose the odd one out-

Starch, Glycogen, Chitin, Inulin

Achrodextrins, Erythrodextrins, Amylodextrins

Q.4- Name a non sulfated Heteropolysaccharide

Q.5- Which heteropolysaccharide is used as an anticoagulant

Q.8-Which monosaccharide is used as a source of energy for the spermatozoa?

Q.9- Which disaccharide is an intermediate in the hydrolysis of starch?

Q.10- Which monosaccharide is optically inactive?

Q.11- What is odd out of the following four-

Q.12- Choose the ketopentose-

Q.14- Name a sugar acid

Q.15- Name an amino sugar acid

Q.16- Name an intracellular polysaccharide

Q.19- Malt sugar is------------------------ ?

Q.21- Mucic acid is produced from---- ?

Q.22- Give an example of Glycosylamine

Q.23- Name a sugar alcohol with five carbon atoms

Q.24- Powder-puff shaped crystals are formed by------

Q.25- Name two non reducing sugars

Q.26- Which test is used to differentiate between aldohexose and ketohexose?

Q.29- Name a cardiac Glycoside

Q.30- What is milk sugar?

Q.31- Name the product of reaction of a strong acid on a pentose

Q.32- What are enediols?

Q.33- Name a 7 Carbon atoms containing ketose sugar

Q.34- Name the alcohol produced from the reduction of Glyceraldehyde.

Q.36- Glucuronic acid produced from the reduction or oxidation of Glucose?

Q.38- Which one is a branched polymer out of the two-

Q.39- Reddish brown colour with iodine is given by which carbohydrate?

Q.40- Which sugar is called as Invert sugar?

Q.41- Agar is a homo or hetero polysaccharide?

Q.43- Name an epimer of Sorbitol

Q.44- Name an epimer of Glucuronic acid

Q.45- How galactose and fructose are related to each other?

Q.46- What is Aglycon?

Q.47- Maltose is composed of what kind of monosaccharides?

Q.48- Name a pentose sugar present abundantly in heart muscle

Q.49- Name a deoxy sugar

Q.50- Name the polysaccharide present in the exoskeleton of insects

Q.53- What type of linkages are present in Glycogen?

Q.54- Name a fructosan

Q.55- Name a Galactosan

Q.58- How are Mannose and Glucose related to each other?

Q.60- Dulcitol is produced from the reduction of which sugar?

Q.61- Name two amino sugars

Q.63- Name a sugar ester

Q.65- All except one will exhibit mutarotation?

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