Biochemistry Key Answers
Biochemistry- Paper II Feb 2007
I. Essay
1. What is the active form of methionine? How it is formed? What are its functions? Enumerate the steps of methionine metabolism and write the disorders associated with its metabolism.(20)
Ans.
Active form of methionine is S-Adenosyl Methionine(SAM).
SAM is obtained by accepting adenosyl group from ATP by methionine by methionine adenosyl transferase.
The function of SAM is transmethylation reactions. Transmethylation reaction is acceptance of a methyl group from a donor like S-adenosyl methionine(SAM) by a compound resulting in another compound.
The transmethylation reactions are
Methyl acceptor Methylated product Guanidoacetic acid Creatine Serine Choline Epinephrine Metanephrine Nor epinephrine Epinephrine tRNA Methylated tRNA
Steps of methionine metabolism:
1. methionine converts to SAM
2. SAM donates methyl group to methyl acceptors by methyl transferases to form S-adenosyl homocysteine(SAH)
3. SAH loses adenosine using adenosine homocysteinase to form homocysteine.
4. Homocysteine forms methionine by homocysteine methyl transferase. This step uses methyl tetra hydrofolate which becomes THFA using B12. 5. Homocysteine combines with serine to form Cystathionine using Cystathionine synthase and Cystathionine is hydrolyzed to cysteine and homoserine by cystathioninase.
Diseases in methionine metabolism:
Homocystinurias are autosomal recessive disorders of about 1:200000 child births.
Hyperhomocysteinemia is a risk factor for coronary heart disease. It is seen in smokers, alcoholics, and hypothyroidism.
Homocystinurias are seen in the following:
1. Cystathionine beta synthase deficiency: - methionine and homocysteine level increase and seen in urine.
- Charlie Chaplin gait, mental retardation, ectopia lentis are seen
- homocysteine activates Hageman’s factor leading to thrombosis
Cyanide Nitroprusside test will be positive. Urinary homocysteine levels are elevated.
- Treatment is a diet low in methionine and rich in cysteine.
2. Cobalamin deficiency:
-N5 methyl THFA homocysteine methyl transferase is dependent on B12. Hyperhomocysteinemia occurs
3. Deficient N5, N10 methylene THFA reductase
4. Cystathioninuria
- it is due to cystathioninase deficiency.
- mental retardation, anemia, thrombocytopenia and endocrinopathies.
- acquired is seen in pyridoxine deficiency
5. Acquired hyper homocysteinemias
- Nutritional deficiency of vitamins like Cobalamin, folic acid and pyridoxine.
- metabolic: chronic renal diseases, hypothyroidism
- folate agonists, Vit B12 antagonists, pyridoxine agonists, estrogen antagonists
2. What is the normal pH of blood. Discuss the mechanism involved in its regulation.(15)
Ans.
Normal blood pH is btween7.38-7.42. it is maintained by:
I. Role of buffers in body fluids:
Buffers resist changes in pH when small quantities of an acid or an alkali are added. Various buffers in body are:
1. Bicarbonate buffer system
it is the most important buffer in plasma and is formed by (NaHCO3/H2CO3)
- the base HCO3 is the metabolic component as it is regulated by kidney and the acid H2CO3 is called respiratory component since it is regulated by the lungs.
The normal bicarbonate level in plasma is 24mmol/L. It has a pKa of 6.1-so it is a poor buffer. But the high blood concentration and the ratio of base to salt is high(20:1), which makes it an effective buffer.
When acid(H+) is added
+ - o H + HCO3 H2CO3 H20 + CO2 excreted by lungs and kidney.
When alkali is (HCO3-) added
+ - o H + HCO3 H2CO3 H20 + CO2 excreted by lungs and kidney.
2. Phosphate buffer system
It is made of NaHPO4/NaH2PO4. It has a pKa of 6.8.
+ In acidosis NaHPO4 + H NaH2PO4 - excreted by the kidneys
+ In alkalosis NaH2PO4 - NaHPO4 + H
3. Protein buffer system
The Histidine molecules in albumin acts as a buffer.
In acidosis H+ + Pr- - HPr
In alkalosis HPr H+ + Pr- II. Kidneys regulate acid base balance by:
+ 1. Excretion of H -In PCT cells CO2 combines with water to form carbonic acid using carbonic + - + anhydrase. Then it becomes H and HCO3 . this H is then excreted into lumen in exchange for Na+ .
- 2. Reabsorption of HCO3 - sodium bicarbonate in the lumen becomes sodium and bicarbonate. + - Sodium is taken up by PCT cell in exchange of hydrogen ions. H combines with HCO3 to form carbonic acid, which forms CO2 and water and both are reclaimed into the cell and converted + - - back to carbonic acid and again to H and HCO3 . HCO3 is taken into blood with sodium.
+ - Fig. Excretion of H fig. Reabsorption of HCO3
+ - 3. Excretion of titrable acid- The Na2 HPO4 becomes Na and NaHPO4 . sodium is exchanged + + – with H ions and H combines with NaHPO4 to become Na H2PO4 and gets excreted.
Fig. Excretion of titrable acid and ammonium ions
+ 4. Excretion of NH4 - Glutamine in DCT becomes glutamate and ammonia. This ammonia is secreted into the lumen which combines with hydrogen ions to become ammonium ions and gets excreted. III. Role of lungs in Acid base balance
When there is fall in pH the respiratory rate is stimulated resulting in hyperventilation.
This would eliminate more CO2 thereby lowering H2CO3 .
In tissues pCO2 is high and pH is low to the formation of acids by the cells like lactate and production of CO2 by cells. CO2 diffuses into RBC. It combines with water to form carbonic + - + acid by carbonic anhydrase. And dissociates into H and HCO3 . So RBC traps H from the tissues. - Some of the HCO3 diffuses out of the cell in exchange for chloride.
+ - In the lungs H combines with HCO3 to form H2CO3 which becomes H2O and CO2. This
CO2 is released into the lungs. So lungs reduce the acid load of H2CO3 by excretion of CO2.
In metabolic acidosis lungs hyperventilate to excrete more acid. In Metabolic alkalosis the reverse happens.
Fig. Reactions in tissues fig. Reaction in lungs
3. Mention the sources, Daily requirement, functions and deficiency symptoms of Calcium. Explain how serum level of calcium is regulated.(15)
Ans.
Parathyroid hormone(PTH) is secreted by four parathyroid glands in the thyroid tissue. Decreased serum calcium leads to release of PTH from parathyroids. PTH activates adenylyl cyclase in target cells and increases intracellular calcium concentration. A protein kinase is activated which activates enzyme systems. PTH acts on
1. PTH and bones- PTH causes demineralization in bones. It activates pyrophosphatase in osteoclasts leading to bone resorption and solubilising calcium. Calcium is released into the blood stream and increases blood calcium level. This leads to loss of bone matrix.
2. PTH and kidneys: PTH causes decreased renal excretion of calcium and increased excretion of phosphates and increased reabsorption of calcium leading to increased blood calcium level.
3. PTH and intestines: PTH stimulates increased production of VIT D3 which acts on intestine to absorb more calcium leading to increased calcium level in blood.
II. a. Oncogenes
Ans.
Functions of Oncogenes:
Oncogenes are normal genes, whose products perform various functions in the cell.
Products of many Oncogenes are polypeptide growth factors.
Some of the products act as receptors for growth factors. Eg. Erb-B
Some oncogene products act on key intracellular pathways involved in growth control. Eg. Src product
The c-Oncogenes are under the control of regulatory genes and are expressed only when required.
Activation of Oncogenes:
Viruses, chemical carcinogens, chromosome translocations, gamma rays, spontaneous mutations and all such factors may converge into one biochemical abnormality, the activation of Oncogenes which leads to malignancy.
Examples for Oncogenes causing cancer:
erb-B1- lung cancer
erb-B2-gastric tumors
erb-B3-breast cancer
sis- osteosarcoma by activating PDGF
abl- leukemia
ras- leukemias, lung cancer, pancreatic and colon cancer
b. Electrophoresis
Ans.
The term electrophoresis refers to the movement of charged particles through an electrolyte when subjected to an electric field. Cations(positively charged ions) move towards cathode and anions(negative) to anode.
When a biological mixture is subjected to electrophoresis, the compounds in the mixture move in relation to their net charge, size, molecular weight and mass and gets separated according to these characteristics, so that the desired compound can be identified and isolated.
Factors affecting electrophoresis: rate of migration will depend on:
Net charge of the particles (eg. more negative particles move faster than less negative)
Mass and shape of particles(larger sized particles move slowly)
pH of medium(particles move better in a pH in which they are more ionized)
strength of electrical field(eg. Higher the voltage-faster the movement)
properties of supporting medium
temperature-increased temperature solidifies the support and impairs migration)
The electrophoresis apparatus consists if a tank which contains electrodes connected to a power supply and buffer. The pH of buffer is selected so that it imparts maximum charge to the electrophoresed substances(eg. Proteins get separated well in a buffer pH of 8.6
Supporting medium is the surface on which electrophoresis is carried out. It may be agar gel, agarose gel, cellulose acetate, paper, etc.,
After the run the bands are visualized using naked eye or if needed to be quantified a densitometer can be used.
Clinical applications:
1. serum protein electrophoresis:
- in nephrotic syndrome – globulin is produced more by liver in compensation of renal loss of albumin. So alpha 2 band is prominent
- cirrhosis- albumin band is less prominent - multiple myeloma- light chain immunoglobulins are produced more so there will be a prominence in gamma globulin region(M band)
Fig. serum protein electrophoresis in health and disease
2. hemoglobin electrophoresis
- S band is seen in sickle cell anemia
- various hemoglobinopathies and thalasemias can be diagnosed
c. Genetic code
Ans.
The letters A, G, T, and C correspond to the nucleotides found in DNA. Within the protein coding genes these nucleotides are organized into three-letter code words called codons, and the collection of these codons makes up the genetic code. The code provides a foundation for explaining the way in which protein defects may cause genetic disease and for the diagnosis and perhaps the treatment of these disorders. A triplet sequence of nucleotide on the mRNA is the codon for each aminoacid. There are four different bases they can generate 64 codons.
SALIENT FEATURES:
TRIPLE CODONS: Each codon is consecutive sequence of three bases on the mRNA e.g. UUU codes for phenyl alanine.
NON OVERLAPPING: The codes are read one after another in a continous manner. E.g. AUG, GAU, GCA etc.
NONPUNCTUATED: There is no punctuation inbetween codons.
DEGENERATE: 61 codon codes for 20 amino acids so one aminoacid has more than one codons e.g. serine has 6 codons and glycine has 4 codons this is called degeneracy of code.
UNAMBIGOUS: Codons are unambiguous that means one codon stands only for one amino acids.
UNIVERSAL: The codons are same for same aminoacids in all species.
WOBBLING HYPOTHESIS: Reduced stringency between the third base and the complementary nucleotide in anticodon is wobbling. E.g. GGU, GGC, and GGA codes for glycine all three pair with anticodon
TERMINATOR CODON: There are three codon do not code for any aminoacids. They are nonsense codons. The three codons are UAA, UAG, UGA
INITIATOR CODON: AUG acts as initiator codon.
d. Insulin
Ans.
Structure :
Insulin is a 51 aminoacid protein. It contains 2 chains, A and B. A contains 21 aminoacids, B chain contains 20 aminoacids. It is produced as prepro insulin in beta islets of Pancreas and the sid3 chain cleavd and released as insulin. It has two inter chain Disulphide bridges- 7th aa of A chain bonds with 7th aa of B chain, 20th aa of A chain bonds with 19thy aa of B chain. There is one intra chain bonding between 6th and 11th aa of A chain.
Functions:
Insulin plays a central role in regulation of the metabolism of carbohydrates, lipids, and proteins.
Insulin facilitates the membrane transport of glucose.
Glycolysis is stimulated by insulin.
Glycogen synthesis and fatty acid formation increased
Insulin lowers the blood glucose level by promoting utilization and storage.
Gluconeogenesis ang glycogenolysis are inhibited. The blood glucose level is lowered.
Insulin inhibits lipolysis in adipose tissue due to inhibition of hormone sensitive lipase.
Insulin suppresses the HMGCOA synthase and so ketogenesis is decreased. e. Gout
Ans.
When uric acid levels increase in the blood it tends to get deposited as crystals in synovial fluid of joints leading to inflammation and acute arthritis. This disease is called Gout
Etiology:
Primary gout
o 5-phosphoribosyl amidotransferase- there will be increased production of purines due to absence of regulation on this enzyme. It’s a genetic defect
o Abnormal PRPP synthase- there will be increased production of PRPP due to absence of regulation on PRPP synthase. It’s a genetic defect.
o Salvage pathway enzyme deficiencies-there would be more availability of PRPP leading to production of purines uric acid
o Von Gierke’s Disease- due to G-6-Pas deficiency, G-6-P is not converted to glucose. So it goes through HMP shunt resulting in more nucleotide bases, increasing urate production.
Secondary Gout:
o Increased production of uric acid- malignancy- lymphomas, leukemias; after treatment of cancer, cancer cells breakdown, leading to hyperuricemia; trauma- tissue damage; starvation-where catabolism is increased
o Reduced excretion- renal failure, thiazide diuretics- which inhibits urate secretion, lactic acidosis and Ketoacidosis- interferes with urate secretion
Clinical features:
Uric acid gets deposited in the cooler areas of body like distal joints to form tophi. Hypeuricemia leads to increased excretion of uric acid through the kidneys, so uric acid crystals gets deposited in the urinary tract leading to renal calculi.
Treatment:
1. Dietary Purine intake should be reduced, alcohol should be restricted
2. Uricosuric drugs which increases the excretion of uric acid like probenecid should be used 3. For calculi Allopurinol can be used. It inhibits xanthine oxidase and reduces the formation of uric acid. It’s a type of suicide inhibition like aspirin, where the enzyme becomes completely functionless.
4. Colchicine, an anti-inflammatory drug used in RA can be used to reduce inflammation in joints.
Lysch Nyhan syndrome
it is a X-linked inborn error of purine metabolism, incidence 1:10,000
deficiency of HGPRTase which acts in salvage pathway
so the salvage pathway is stopped and PRPP accumulates which will go for catabolism to uric acid
hyperuricemia leads to nephrolithiasis and gout
it is also characterized by self mutilation, mental retardation
f. Detoxification
Ans.
Xenobiotics are compounds which may be accidentally ingested or taken as drugs or compounds produced in the body by bacterial metabolism.
Various xenobiotics:
Compounds accidentally ingested like preservatives, food additives and adulterants
Drugs taken for therapeutic purposes
Endogenous compounds which has to be eliminated by body like bilirubin, steroids
Compounds produced by bacterial metabolism
o Histidinehistamine
o Lysinecadaverine
o Ornithineputrescine
Phases of detoxification:
1. Phase 1 reactions: it is the alteration of foreign molecule by adding a functional group like hydroxylation, oxidation, hydrolysis, dealkylation Epoxidation, etc., the main function of phase 1 is to convert it into anon toxic metabolite.
Eg. Toluene Benzyl alcohol by oxidation
Benzene phenol by oxidation
Picric acid picramic acid by reduction
Aspirin acetic acid and salicylic acid by hydrolysis
Sometimes phase one reaction will to production of a toxic product.
Eg. Methanol formic acid
2. phase 2 reactions: conjugation
A xenobiotic that undergone a phase 1 reaction is now a new metabolite that contains a chemical group like OH, NH2 , COOH groups. Phase 2 reactions lead to conjugation(addition) of conjugating agents like: Glucuronic acid: Bilirubin conjugated with glucuronic acid to form Bilirubin Diglucuronide and excreted in bile.
Sulfate conjugation: phenol converted to phenol sulphate using PAPS(phosphor adenosyl phosphor sulphate-active sulphate)
Cysteine and Glutathione: alkyl or aryl halide, epoxide are detoxified in this manner.
Acetylation: acetic acid s is conjugated to sulfanilamide, INH
Glycine: Benzoic acid is conjugated with glycine to form hippuric acid
Glutamine: phenyl acetic acid is conjugated to form Phenyl acetyl glutamine