The Department of Biochemistry has the distinction of being the first full fledged Department engaged in quality biochemical research and teaching above the Vindhyas in North India, since at that time AIIMS was non-existent and the Medical Colleges did not have separate Biochemistry Department. The Department at the start was headed by Dr. Ranganatha Rao, a medical graduate who had training in biochemical research in U.S.A. After some years he left VPCI for Hyderabad and the position was taken over by Dr. T.A.V. Subramanian who returned from having worked with well known scientists Dr. C.G. King and Dr. Jim Price at College of Physicians and Surgeons, Columbia University and University of Wisconsin Medical School, Dept. of Clinical Oncology. Dr. T.A.V. Subramanian was largely responsible for shaping the Department into a reputed Centre by introducing M.D., M.Sc. and Ph.D. in Medical Biochemistry. He was assisted by highly trained colleagues, Drs. D. Subramanyam, Bhattatiry, Sharada Subramanian, U.K. Misra, V.N. Singh, L. Viswanthan, P.S. Murthy and many others who were either permanent or project scientists. Dr. T.A.V. Subramanian initiated research work in several areas including experimental tuberculosis, biochemistry of Mycobacterium tuberculosis, vitamin A research, radiation biochemistry , nutritional biochemistry and biological aspects of aflatoxin research. Biochemistry Department of VPCI got recognition for excellent research in areas of mycobacteria and aflatoxin. Dr. V .N .Singh left VPCI in 1972 for joining Hoffman La Roche U.S.A and the position was occupied by Dr. H.G. Raj who spearheaded vigorously aflatoxin research and established a good laboratory for molecular toxicology and carcinogenesis. When Dr. Raj was promoted as Reader, Dr. S.K. Bansal was appointed. Since its inception, the main activities of the Department have been (i) routine and biochemical investigations on patients refered from Clinical Research Centre (till 1972), (ii) post-graduate teaching (D.T.C.D., D.R.M., M.D. (Chest diseases), M.D. (Medical Biochemistry), M.Sc. (Medical Biochemistry)) and (iii) research training of post-graduate students and scientists from all over India. The Department till date had produced over 200 Ph.D. and a large number of M.D. graduates. Dr. T.A.V Subramanian was Professor and Head of Medical Biochemistry Department in the Faculty of Medical Sciences and also became the first non medical graduate to become the Dean of Medical Faculty Medical Sciences of the University of Delhi. A large number of old students of the Department are doing excellent research and have occupied high academic positions in India and abroad. The Department currently headed by Prof. H.G. Raj continues doing high quality biochemical research work.

Major Activities and Achievements

1. Metabolic studies on mycobacteria

Various comparative studies between human pathogenic, non-pathogenic, saprophytic mycobacteria and atypical species have been carried out. The attributed role of mycobacterial lipids in pathogenecity of tuberculosis has led to their detailed study with respect to characterization, age of culture, environmental changes and turnover rates. The stimulating factor for fatty acid synthetase which is heat-acid stable in M smegmatis and heat abile in M. tuberculosis H₃₇ Rv, has been characterized. The fatty acid synthetase complex was purified from human pathogenic strain and saprophytic strain. A study on the effect of various antitubercular drugs on enzymes and lipids has been carried out. Various enzymes of tricarboxylic acid cycle, glycolysis, glyoxylate cycle and pentose phosphate pathway have been studied in different species of mycobacteria. The uptake and accumulation of labelled sugars and transport of amino acids and sugars have been carried out in mycobacteria. The co-enzyme forms of NAD⁺ and NADP⁺ have been estimated as also the level of adenine nucleotides. Evidence has been presented to demonstrate for the first time the occurrence of cyclic-AMP and endogenous reserve, poly-beta-hydroxybutyrate in mycobacteria. Enzymes involved in their biosynthesis and degradation are under investigation. The presence of calmodulin in mycobacteria has been demonstrated.

2. Functions and regulation of polyamines in mycobacteria

Polyamines in M. smegmatis TMC 15-46 were found to increase during the growth of the organism. This significant increase in polyamines was preceded by a rise and fall in the activity of ornithine-decarboxylase (ODC), the enzyme responsible for the synthesis of putrescine from ornithine. This is the first step in polyamine biosynthesis. ODC, in M. smegmatis was found to be regulated in a novel manner by a specific ribonucleic acid (RNA) of 0.194 Kb. The concentration of this RNA species increased four fold when induced by polyamines. In addition to ODC, putrescine was also synthesized from arginine by the action of arginine decarboxylase.

3. Molecular biology of tubercle bacilli

The current methods used to identify strains of Mycobacterium tuberculosis rely on identifying phenotypic differences between the strains. A more direct approach can be be to analyse the M. tuberculosis genome and identify the genotypic differences. The cause of mycobaterial virulence, at the genetic level, may be traced either by the plasmid DNA or chromosomal DNA. Plasmids often carry some genes that code for virulence factors apart from other properties. To study virulence associated with plasmids, plasmid DNA has to be prepared first. A method was developed for the preparation of spheroblasts of M. smegmatis to enable easy lysis of cells. Spheroplasts were prepared by making the cells osmotically sensitive by the addition of D-cycloserine, glycine, lysozyme, EDTA and LiCl to the growing cells in log phase of growth. The other approach is to study the mycobacterial genome. This would be facilitated if mycobacterial DNA can be cloned into a plasmid vector and introduced into a simple organism like E. coli, The preparation of a genomic library i.e., a set of cloned fragments that together represent the entire genome, was undertaken. The vector chosen for cloning is a plasmid expression vector, pYA626 which is derived from the plasmid pBR322 by replacing its Pst I-EcoRI fragment by a 209 bp fragment of asd gene from Streptococcus-mutans. It has a unique site for Pst I, Tet marker promoter region of asd gene and also the shine-dalgado sequence. The vector was isolated from E.coli strain HB 101 by the standard plasmid isolation protocol. It was also treated with Pst I and linearized and then dephosphorylated by treating with calf intestinal alkaline phosphatase to prevent its recirculation of self-ligation.

4. Studies on aflatoxin

Another major area of interest is biosynthesis of aflatoxin, a hepatocarcinogen produced by fungus Aspergillus parasiticus. Using hypo and hyper-vitaminotic A models in rats and chicks, studies have been carried out to find the mode of action of vitamin A. in cellular metabolism. These studies showed that the effects of excess vitamin A are mediated through adrenocorticoids and the effects appear to be different in different organs.

5. Seasonal variation of metal in coal fly ash

Seasonal variations in fly ash collected from the electrostatic precipitator of a thermal power plant over a period of one year have been studied in parameters (i) particle size, (ii) per cent silicate, (iii) haemolysis, (iv) contents of Ni, Rb, Mn, Zn, Hg, Cd, Mg, Na, Fe, Ca, K, Cu, Sr, As, Se, C and Cr. .Monthly variations were noted in the above parameters, pulmonary and hepatic glutathione levels, glutathione shuttle enzymes and lipid peroxidation in rats exposed to intratracheally administered coal fly ash.

Fly ash and fly ash residue iricreased formation of conjugated dienes and the levels of oxidised glutathione (GSSG) and reduce the levels of reduced glutathione (GSH) in lung and liver whereas fly ash extract administration had no effect on the formation of conjugated dienes and glutathione levels in lung and liver. Fly ash and fly ash residue reduced the activity of glutathione reductase both in lung and liver but did not alter the activity of glutathione peroxidase. Fly ash and fly ash extract significantly increased glucose-6-phosphate dehydrogenase activity in lung whereas in liver, fly ash and fly ash residae reduced the activity of glucose-6-phosphate dehydrogenase. Fly ash residue did not alter the activity of glucose-6-phosphate dehydrogenase in lung whereas fly ash extract was not effective in liver .

6. Cytotoxicity of intratracheally administered coal fly ash

Intratracheal administration of fly ash significantly reduced plasma total cholesterol levels. Plasma total phospholipids were reduced upto 60 days after fly ash treatment but phosphatidyl choline (PC) content showed reductions upto 30 days after treatment.

7. Biochemical and pathological effects of fly ash on lung, liver and blood of rats

The most characteristic effect of fly ash was a reduction of white blood cells (WBC) count which was dose dependent and it persisted upto 120 days after the administration of 5 mg fly ash. A dose dependent increase in blood glucose levels, the activity of glutamate pyruvate transaminase (GPT), a decrease in the activity of glutamate oxaloacetate transaminase (GOT) and alkaline phosphatase was observed in fly-ash treated rats. Fly ash significantly increased blood glucose levels and decreased uric acid levels and the activities of GOT and acid phosphate 15 days after administration.

8. Nutrition and metabolism

Studies have been carried out on the effect of dietary proteins and other nutrients on microsomal drug metabolizing enzymes of liver, kidney, adrenals and lungs. Maturation or brain of rats exposed to insecticides has also been studied. The metabolism of various molecular species of phosphatidyl choline of liver, lung and heart under various dietary manipulations has been carried out.

9. Membrane dynamics, hormone secretion and protein nutrition

Importance of protein in nutrition has been emphasized in the last three, decades with the identification of protein deficiency diseases. Protein malnutrition is a very complex phenomenon where the physiological, biochemical and pathological changes occur. On feeding animals (rats) with three diets, varying in their quality and quantity of protein, the overall metabolism of pancreas, a vital organ of the body, is significantly changed. It resulted in glucose intolerance, decreased serum insulin levels, increased pancreatic insulin and cAMP levels and decreased activity of the regulatory enzymes of the second messenger. Overall phospholipid metabolism was affected and polyphosphoinositides, which act as membrane transducing signals, were markedly affected in low protein diet conditions. Micro-filamentous microtubule network was disrupted and hence resulted in impaired insulin release from pancreas in inadequate dietary protein condition. These findings elucidate pancreatic chemistry and functions in relation to dietary protein.

10. Hepatic plasma membrane chemistry and function in relation to protein nutrition

Feeding of low quality and quantity of dietary proteins to experimental animals alters various metabolic activities of liver including the content of lipids. Total lipid profile of hepatic plasma was found to be altered. The metabolic pathway, viz., CDP -choline pathway and N-methylation pathway was affected under low protein diet feeding. The membrane protein profile of plasma membrane, mitochondria and microsomes was different from each other and both the qualitative as well as quantitative distribution of proteins in these membranes were affected by the poor quality and quantity of dietary proteins. The steady state fluorescence polarization as well as fluorescence anisotrophy in hepatic plasma membrane was significantly increased in inadequate protein diet feeding. On the basis of these results it was concluded that hepatic plasma membrane had undergone dynamic alteration and it had profoundly affected the chemistry and function of plasma membranes.

11. Transmembrane signalling, protein kinase C and protein nutrition

Ingestion of protein deficient diet is known to decrease the protein biosynthesis, enzyme load and phosphoinositide pool of the tissues. Animals maintained on protein deficient diet when challenged with xenobiotics have been reported to show marked changes in drug metabolizing enzymes and phosphatidyl choline suggesting altered cellular responses which are speculated to be associated with changes in phosphoinositide hydrolysis and protein kinase C (PKC) mediated signalling pathway. Therefore, studies were conducted in various tissues, viz. brain, lung, heart, spleen, liver and kidney of rats maintained on protein deficient (approximately 4% protein) diet for 28 days which were pair fed with casein (20%) diet and supplemented diet (deficient diet, supplemented appropriately with L-Iysine and DL-threonine). At cellular level, hepatocytes were used as cell model from these three groups. The results show that dietary protein deficiency caused a significant alteration in total PKC activity, its translocation and the extent of phosphorylation of target proteins, which may be involved in impairment of the responses of these tissues under stimulus induced conditions. These changes were associated with the alteration in the metabolism of various phosphoinositides (viz. phosphatidyl inositol 4,5 bisphosphate, phosphatidyl inositol 4, monophosphate and phosphatidyl inositol) and choline phospholipid (phosphatidyl choline). These findings suggest that dietary protein deficiency may cause changes in the enzymes of inositol phospholipid cycle, phospholipase C and phospholipase A₂. The changes in hepatocytes in total PKC, its translocation, phosphoinositides and phosphatidyl choline leyels were comparable to the changes in liver. Improvement in quality of protein deficient diet by supplementing it with limiting amino acids remarkably reversed the changes in liver, kidney and hepatocytes but in other tissues viz. brain, lung, heart and spleen the changes were partial. It can be concluded that suitable quality of protein is mainly required for normal metabolism and physiology of liver and kidney. Other tissues require both proper quality and quantity of protein for regular metabolism and hence transmission of signals across the membrane.

12. Role of endothelin in bronchial asthma. A study on membrane phospholipid turnover and protein kinase C

Endothelin is a bronchoconstricting peptide. Its raised levels have been reported in bronchoalveolar lavage fluid of asthmatic patients. Its effects were studied on alveolar macropahges (AM) which are the phagocytic cells in the alveolar spaces of the lung. The experiments were conducted on alveolar macropahges of guinea pigs to elucidate the changes induced by endothelin on the mechanism of signal transduction. On the basis of results obtained, we concluded that protein kinase C (PKC) plays an important role in the activation of AM and endothelin influences the activation of AM by two different pathways i) by activating diacylglycerol -PKC pathway involving phospholipase C (PLC) and ii) by activating phospholipase A₂ (PLA₂) dependent pathway. The first pathway seems to trigger the initiation of activation of macrophages. The activation of second pathway may possibly bring about sustained effect on macrophage activation for the long-term activation and may be subsequent to the activation of the first pathway, showing a cross-talk between PLC and PLA₂ via PKC.

13. Effect of vitamin A deficiency on signal transduction

Vitamin A deficiency is a widespread nutritional problem. Its deficiency is known to alter several membrane functions. The studies were therefore conducted to delineate the effects of feeding vitamin A deficient diet on mechanism of signal transduction in lung and liver of rats. Under the experimental conditions, feeding of vitamin A depleted diet did not develop hypovitaminosis A. However, there was an increase in total activity of protein kinase C although the translocation of the enzyme from cytosol to membrane decreased. Supplementation of diet with vitamin A caused partial reversal of this trend both in lung and liver. The data suggest that feeding of vitamin A deficient diet may impair the transmembrane signalling in rats.

14. Transmembrane signalling in bronchial asthma

Studies on mechanism of signal transduction were conducted on peripheral blood lymphocytes of asthmatic patients. The initial studies have shown that in asthmatic condition, there is an increase in total protein kinase C (PKC) activity in lymphocytes and it has a significant correlation with the degree of airway obstruction. PKC exists in 12 isomeric forms and we are now identifying the type of isoenzymes of PKC involved in the disease. In normal human lymphocytes, we have identified eight different isoenzymes of PKC viz. PKC a, b, g, e, h, m, l, and z;. To compare the pattern of the isoenzymes with the experimental animals, PKC isoenzyme profile of lymphocytes and airway smooth muscles (ASM) of guinea pig was also determined. The lymphocytes of guinea pig showed the presence of seven isoenzymes viz. PKC α ,β,γ,ε,η,μ,λ and ζ while ASM showed only five viz. PKC, γ, ε, η and ζ . These findings show that five isoenzyme viz. PKC α, γ, ε, η and ζ were common to lymphocytes of human and lymphocytes and ASM of guinea pig. The effect of asthmogens and anti asthmatic drugs on these isoenzymes is in progress.

Training and Services

The Department imparted training to scientists of other research institutions in various techniques, viz., analysis of mycotoxins in food materials, analysis of lipids from biological sources and other research techniques. The Department also had a unit through Ministry of health and Family Welfare for the analysis of mycotoxins in food materials, analysis of lipids from biological sources and the research techniques. The Department had collaborative research projects with Departments of Botany, Chemistry and Zoology of Delhi University and Department of Biochemistry of South Campus.