A pathway map of glutamate metabolism.

Glutamate metabolism plays a vital role in biosynthesis of nucleic acids and proteins. It is also associated with a number of different stress responses. Deficiency of enzymes involved in glutamate metabolism is associated with various disorders including gyrate atrophy, hyperammonemia, hemolytic anemia, γ-hydoxybutyric aciduria and 5-oxoprolinuria. Here, we present a pathway map of glutamate metabolism representing metabolic intermediates in the pathway, 107 regulator molecules, 9 interactors and 3 types of post-translational modifications. This pathway map provides detailed information about enzyme regulation, protein-enzyme interactions, post-translational modifications of enzymes and disorders due to enzyme deficiency. The information included in the map was based on published experimental evidence reported from mammalian systems.

LC-MS-based serum metabolomic analysis reveals dysregulation of phosphatidylcholines in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers with poor prognosis. Here, we carried out liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS)-based untargeted metabolomic analysis of ESCC serum samples. Statistical analysis resulted in the identification of 652 significantly dysregulated molecular features in serum from ESCC patients as compared to the healthy subjects. Phosphatidylcholines were identified as a major class of dysregulated metabolites in this study suggesting potential perturbation of phosphocholine metabolism in ESCC. By using a targeted MS/MS approach both in positive and negative mode, we were able to characterize and confirm the structure of seven metabolites. Our study describes a quantitative LC-MS approach for characterizing dysregulated lipid metabolism in ESCC. BIOLOGICAL SIGNIFICANCE: Altered metabolism is a hallmark of cancer. We carried out (LC-MS)-based untargeted metabolomic profiling of serum from esophageal squamous cell carcinoma (ESCC) patients to characterize dysregulated metabolites. Phosphatidylcholine metabolism was found to be significantly altered in ESCC. Our study illustrates the use of mass spectrometry-based metabolomic analysis to characterize molecular alterations associated with ESCC. This article is part of a Special Issue entitled: Proteomics in India.

Anticancer Effect of dl-Glyceraldehyde and 2-Deoxyglucose in Ehrlich Ascites Carcinoma Bearing Mice and Their Effect on Liver, Kidney and Haematological Parameters.

Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect). Compounds that inhibit glycolysis have potential applications in cancer treatment. dl-glyceraldehyde (DLG) and 2-Deoxyglucose (2-DG) have been proven effective in the inhibition of glucose metabolism. Ehrlich ascites carcinoma (EAC) cells were injected intraperitoneally (i.p) in 10-12 weeks old Swiss albino mice, weighing between 20 and 30 g. The anticancer activity of DLG and 2-DG were determined by tumor volume, tumor weight, viable and nonviable tumor cell count, average survival time, percentage increase in life span and tumor inhibition ratio. The blood samples were obtained for biochemical analysis after 9 days of treatment to study the effect on liver, kidney and haematological parameters. Histopathological examination of liver and kidney was also performed. One-way ANOVA test and Dunnett's test were used for comparisons of parameters in study groups. Both DLG and 2-DG individually decreased the tumor weight, tumor volume, viable tumor cell count and significantly increased the life span of treated mice, however the combination was found to be better. The biochemical parameters of liver and kidney functions and haematological parameters were restored close to control group as compared with the EAC bearing mice. Histopathological examination of liver and kidney in EAC control group showed large areas of necrosis, congestion and mononuclear cell infiltration but such changes were not observed in liver and kidney sections observed after i.p injection of DLG and 2-DG for 9 days. Improvement was much better in the group where combination of these two drugs were used.

Serum urea:albumin ratio as a prognostic marker in critical patients with non-chronic kidney disease.

Routine laboratory investigations play an important role in estimating the risk of mortality in intensive care unit (ICU) patients. The significance of urea:albumin ratio (UAR) in predicting the stay and mortality of ICU patients is not known. It is a retrospective study of patients admitted to ICU (n = 412) with non-chronic kidney disease (non-CKD). Receiver-operating characteristics (ROC) analysis for predicting mortality was carried out to find area under curve (AUC) and threshold levels. Analysis of survival probability was carried out by Kaplan-Meier method and Log-rank test. The AUC to predict mortality were 0.695, 0.767 and 0.791 for serum albumin, urea and UAR, respectively. The threshold levels for albumin, urea and UAR were 2.8 g/dL, 53 mg/dL, and 23.44 mg/g, respectively. The highest odds ratio (OR) of 9.75 to predict mortality at threshold level was observed for UAR, while OR were 7.0 and 3.62 for serum urea and albumin, respectively. The serum urea above and albumin below threshold level were associated with increase in ICU stay of >3 days but the highest OR of 4.73 to predict stay of >3 days was observed for UAR. Kaplan-Meier survival analysis shows significant (p < 0.001) difference at the threshold value of UAR. Serum urea and albumin are found to be an independent predictor for the mortality and stay; however an increased UAR value is the best parameter in predicting mortality and stay in ICU patients with non-CKD illness.

Prevalence of unsuspected glucose intolerance in coronary artery disease (CAD) patients: Importance of HbA1c.

BACKGROUND: The mortality and morbidity rates are two to fourfold higher among Coronary Artery Disease (CAD) patients with type 2 diabetes mellitus (DM). American Diabetes Association (ADA) and World Health Organization (WHO) define different criteria for the diagnosis of glucose intolerance. This study compares the available diagnostic criteria for DM in Indian men and their importance in CAD patients. METHODS: This cross-sectional study was done on 794 male volunteers; 483 individuals from general population and 311 patients undergoing angiography for evaluation of CAD. Individuals with previous clinical history of diabetes mellitus were excluded. RESULTS: More than 90% of diabetics by ADA criteria could be diagnosed by Fasting plasma glucose (FPG) and HbA1c criteria while FPG and pg2h plasma glucose (WHO criteria) could detect only 74%. Impaired Fasting Glucose (IFG) or Impaired Glucose Tolerance (IGT) was present in 36.7% of individuals diagnosed to be diabetic based on HbA1c; more in CAD +ve group (53.8%) than in general population (23.6%). ROC analysis suggests >121 mg/dl of FPG or >6.2% of HbA1c as optimum cut-off for the diagnosis of DM. FPG and HbA1c criteria have higher Relative Risk for presence of coronary artery occlusion and HOMA-IR. CONCLUSION: Inclusion of HbA1c in the criteria for diagnosis of DM (ADA criteria) can detect large number of cases with persistent hyperglycemia in the non-diagnostic range of DM (IFG or IGT) among general population and CAD patients. This has special relevance to epidemiological studies as the diagnosis of DM can be made on single fasting blood sample.

Umbilical cord blood plasma vitamin A levels in low birth weight (LBW) babies.

BACKGROUND: Role of vitamin A in reducing the mortality in infants more than six months of age is well known. Supplementing newborn infants with vitamin A within 48 hours of birth reduces infant mortality by almost a quarter, with the greatest benefit to those of low birth weight (LBW). Studies that could highlight deficiency states in neonates, particularly LBW babies by objective measurement of vitamin A levels would help in formulating the recommendations to supplement these babies with vitamin A. METHODS: Cord blood plasma vitamin A levels of 154 LBW babies with birth weight in the range of 1505-2455 were analysed for plasma vitamin A (retinol) levels by HPLC method. Samples of 55 babies with normal birth weight were also analysed. LBW babies were divided into two subgroups of preterm LBW and LBW-term small for gestational age (SGA). RESULTS: Of the 154 babies with LBW, 92 were preterm LBW and 52 were LBW-term SGA. Mean cord blood plasma vitamin A levels were significantly lower in the preterm LBW group (n = 92) compared to levels observed in babies with normal birth weight (n = 55) and LBW-term SGA subgroups (n = 62). There was no significant difference in the mean vitamin A values between the normal birth weight babies and LBW-term SGA group. There was significant positive correlation of cord blood vitamin A levels with birth weight in the entire set of (n = 154) LBW babies (r=0.37, P < 0.0001). CONCLUSION: This study revealed significantly lower cord blood vitamin A levels in the preterm LBW babies. The level of vitamin A in LBW babies also correlated with their birth weight. There are enough evidence to support causative association between vitamin A deficiency state and neonatal morbidity. Simple interventions like vitamin A supplementation during a crucial stage of an infant's life may be beneficial in the long run. There is a need to establish norms for vitamin A levels and seriously examine the role of vitamin A supplementation for LBW babies during the immediate postnatal period.

Adrenaline, Noradrenaline and Dopamine Level Estimation in Depression : Does it Help?

BACKGROUND: Abnormalities of catecholaminergic function have been hypothesised in causation of depressive illness. Electroconvulsive therapy (ECT) is postulated to have noradrenergic mechanism of action. We studied the clinical utility of estimating catecholamines level changes after ECT. METHODS: Plasma adrenaline, noradrenaline and dopamine in healthy controls and depressed patients were estimated by high performance liquid chromatography with electrochemical detection method before and after ECT. RESULT: Mean ± standard deviation of plasma adrenaline, noradrenaline and dopamine in controls was 36.7 ± 13.2, 209.3 ± 76, 21.8 ± 9.5 ng/L respectively, while in depressed patients before and after ECT it was found to be 32.5 ± 12.0, 419.3 ± 167.7, 22.1 ± 16.0ng/ L and 37.2 ± 19.6, 386.1 ± 168.4, 22.3 ± 15.5ng/L respectively. Correlation of adrenaline, noradrenaline and dopamine concentration with scores of Beck Depression Inventory, Suicidal Ideation Scale and Melancholia Inventory was positive but statistically not significant and poor. Based on the cut off values of noradrenaline, only 62% cases could be categorized as abnormal, which after ECT reduced to 50%, whereas post ECT psychiatric ratings was normal in about 78% cases. CONCLUSION: There is no clinical significance of estimating adrenaline, noradrenaline and dopamine in depressed patients.

Incidence of microalbuminuria in hypertensive patients.

The prevalence of microalbuminuria was assessed in 174 albustix negative hypertensive patients by estimating albumin in the morning random urine samples by immunoturbidimetric method within four hours of voiding of urine. The urine samples were not stored and collected without any preservatives. The urinary albumin was calculated in terms of ratio with respect to urinary creatinine and expressed as albumin creatinine ratio (mg/g). Out of 174 albustix negative hypertensives, 58 (33.3%) patients were found to have microalbuminuria. The prevalence of microalbuminuria in males and females was found to be 34% and 30.7% respectively. No correlation was found between the Body Mass Index (BMI) and albumin excretion (r(2) = 0.0271) and between duration of hypertension and urinary albumin excretion (r(2) = 0.0042). Prevalence of microalbuminuria in nonsmokers and non-alcoholic hypertensives was 20%. The prevalence in alcoholics, smokers and both smokers and alcoholics was found to be 35%, 42% and 41% respectively. The high prevalence of microalbuminuria than the various reported studies on the subject demands establishment of a screening programme for microalbuminuria, implementation of specific intervention methods and education of hypertensive patients about the consequences of smoking and alcohol on possible involvement of renal system.

Study of Clinicobiochemical Spectrum of Hypothyroidism.

BACKGROUND: The aim of this study was to assess the clinico biochemical spectrum of hypothyroidism and the relative importance of thyroid function tests, clinical symptoms and signs in thyroid dysfunction. METHODS: A retrospective study was done and 1702 requisitions for screening of hypothyroidism were analysed. The clinical presentation of cases was correlated with the results of thyroid profile tests. RESULTS: 31.5% of the 1702 cases referred had thyroid dysfunction in the form of subclinical or overt hypothyroidism. In the hypothyroid group generalized weakness, weight gain and myxoedema was common. In cases of primary infertility and depression, subclinical and overt hypothyroidism was high (40% and 45.8% respectively). The average age of females with subclinical hypothyroidism was 30.8 years, 5.4 years less than females with overt hypothyroidism. CONCLUSION: We conclude that hypothyroidism is common and often under-diagnosed. Therefore routine evaluation of female patients with weight gain, generalized weakness, infertility, depression and mood changes should include thyroid profile.

Urinary N-acetyl beta glucosaminidase and gamma glutamyl transferase as early markers of diabetic nephropathy.

Albumin and enzymes-N-acetyl-beta-glucosaminidase (NAG) and gamma glutamyl transferase (GGT) were estimated in the morning random urine samples of 196 albustix negative diabetic patients to evaluate the clinical utility of these urinary enzymes as early markers of diabetic nephropathy. Albumin was estimated by immunoturbidimetric method and enzymes by linetic essay within six hours of voiding of urine. The urinary albumin and urinary enzyme concentration was calculated in terms of ratio with respect to urinary creatinine. Correlation coefficient (r) bewween urinary albumin and urinary enzymes in normoalbuminuric, microalbuminuric and overall diabetic cases was 0.23, 0.32 and 0.40 respectively for NAG, and 0.08, 0.06 and 0.18 respectively for GGT. NAG excretion was found increased in 34%, 63.7% and 49.5% of normoalbuminuric, microalbuminuric and overall diabetic cases respectively while GGT in 6.4%, 24.5% and 15.8%. The correlation coefficient between urinary albumin and NAG in normoalbuminuric, microalbuminuric, and overall diabetic patients with increased NAG excretion was found only 0.31, 0.27 and 0.35 respectively. No correlation was found between duration of diabetes and enzyme excretion. The study suggests that urinary NAG or GGT or both together do not have any clinical significance as an early marker of diabetic nephropathy.

Differentials and Determinants of Syndrome 'X' and its Role as Coronary Risk among Healthy Middle Aged Indian Army Personnel.

BACKGROUND: Syndrome 'X', a clustering of impaired glucose tolerance (IGT), raised blood pressure, raised serum triglycerides and low HDL-cholesterol, occurring under the influence of insulin resistance and resultant hyperinsulinaemia, has been hypothesised to be a major risk factor for ischaemic heart disease (IHD). However, there is a lack of research based evidence in this field, in our country. METHODS: The study was a cross-sectional analytical epidemiological design of 614 healthy Indian Army personnel, aged 35 years and above, selected by random sampling. RESULTS: The study indicated that there is a statistically significant (p < 0.001) clustering between fasting hyperinsulinaemia, raised blood pressure, IGT, raised triglycerides and low HDL. The prevalence of syndrome 'X' was 8.47% (95% CI 6.27% to 10.47%). Initial univariate and subsequent multivariate analysis using multiple logistic regression method, indicated that predictors of syndrome 'X' were increasing age, overweight, increasing central (abdominal) obesity, lack of adequate physical exercise and low level of physical fitness. Presence of syndrome 'X' increased the risk of resting ECG changes suggestive of coronary insufficiency (OR = 6.29, p < 0.001). CONCLUSION: Based on the findings, recommendations for prevention of this syndrome have been submitted.

Polyacrylamide Gel Affinity Electrophoresis for Separation of Enzyme Isoforms.

Affinity electrophoresis of differently glycosylated isoforms of enzymes using lectin as affinity ligand has been reported on support media such as cellulose acetate membrane (CAM) or agarose gel. We report a method for affinity electrophoresis in polyacrylamide gel (PAG) using wheat germ agglutinin (WGA). WGA is added to acrylamide-Bis mixture and incubated for 10 minutes at room temperature. This causes WGA to react covalently with acrylamide and Bis. Polymerization is initiated with addition of N,N,N,N-tetramethylethylene diamine (TEMED) and ammonium persulphate to give polyacrylamide gel with immobilized lectin. This gel has been found to effectively separate differently glycosylated isoforms of alkaline phosphatase (ALP). Concanavalin - A, similarly immobilized, did not give effective separation of ALP isoforms. The immobilization of lectin on polyacrylamide as support media requires less amount of lectin in comparison to CAM and agarose. Additional advantage of affinity electrophoresis on PAG is separation of biomolecules according to size.

DIAGNOSING DISSEMINATED INTRAVASCULAR COAGULATION IN ACUTE INFECTION : CAN WE DO WITHOUT FDP & D-DIMER.

Alterations in coagulation profile viz. platelet count, prothrombin time (PT), partial thromboplastin time with kaolin (PTTK), thrombin time (TT) and fibrinogen were studied in 96 patients (73 males and 23 females) of acute infections. Fibrin/fibrinogen degradation products (FDP) level >25µg fibrinogen equivalent unit (FEU)/ml along-with D-dimer >1.0µg FEU/ml was considered criteria for diagnosis of disseminated intravascular coagulation (DIC). Normal values were established using plasma from 12 healthy voluntary blood donors. Out of these 96 patients, 15 had infection with Gram positive bacteria, 23 with Gram negative bacteria and 38 with Dengue. In 20 patients, nature of infection was not defined. Mean platelet count per cubic millimetre was 2.14 lac in Gram positive infection and 1.74 lac in Gram negative infection (p=0.07). There was no significant difference in other coagulation parameters in Gram positive and Gram negative infection. Platelet counts were low in 71% of Dengue patients but there was no significant alteration in PT, PTTK and TT. None of the Dengue patients had hypofibrinogenemia or DIC though hyperfibrinogenemia was present in 21% of Dengue patients. 20 patients had features of septicemia (Gram +ve 7, Gram -ve 8, undefined 5); 10 had concomitant DIC. DIC was present in additional 4 patients of acute infection without septicemia. PTTK was raised in 60% of the septicemia patients. 20 out of 82 non-DIC acute infection patients had subnormal PTTK. Commonest alteration in 14 DIC patients was raised PTTK with a sensitivity of 78.6% and specificity of 81.7%. Low fibrinogen levels though specific for DIC, were present in only 21.4% of the DIC patients. Combinations of PTTK >38 sec with PT >15 sec or platelet count < 1.5 lac/mmm(3) were good screening tests for DIC and detected 11 and 10 patients out of 14 with three and two false positives respectively.

Serum Ornithine Carbamoyl Transferase as a Surrogate Marker in Malaria.

Ornithine carbamoyl transferase (OCT) activity and other liver function tests were studied in a total of 50 patients of clinical malaria and 15 controls. They were grouped as group I (positive for malarial parasite on peripheral blood smear, n=18), group II (negative for malarial parasite on peripheral blood smear (PBS) but responded to antimalarials, n=17) and group III (peripheral blood smear negative and did not respond to antimalarial therapy, n=15). The mean OCT levels were significantly raised in group I (6.79 ± 1.84 IU/L, p value = 0.006) and group II (5.0 ± 1.15 IU/L, p value = 0.014) as compared to controls (2.5 ± 1.13 IU/L) and returned to normal after treatment In contrast, group III had normal levels except in a case of kala azar and septicemia where OCT levels were high and increased further on treatment. Taking PBS positivity as a gold standard of diagnostic criteria, OCT had a sensitivity of 83% and specificity of 86% with a high positive predictive value of 88% as compared to ALT which had a lower sensitivity of 55% and specificity of 80%. The clinical response rate in PBS negative cases of fever having high OCT level was 83% as compared to 35% in cases with normal OCT level, making OCT a good surrogate marker of malaria. OCT levels could also be of prognostic significance as 2 cases of cerebral malaria had high OCT levels of 11.1 UAL and 10.7 IU/L, respectively.

A STUDY OF HYPERINSULINAEMIA AND DYSLIPIDAEMIA IN HYPERTENSIVE PATIENTS OF ARMED FORCES.

All patients attending the outpatient department were screened for hypertension. An attempt was made to correlate presence of hyperinsulinaemia (HI), dyslipidaemia and anthropometric characteristics in these hypertensives. Effect of angiotensin converting enzyme inhibitor (ACEI) and beta blockers on serum insulin was also studied. 85 patients with blood pressure (BP) ≥ 160/90 mm Hg and 94 controls with a BP of < 130/85 mm Hg were studied. All underwent clinical examination, anthropometric measurements (body mass index (BMI), waist hip ratio (WHR), skin fold thickness (SFT) and laboratory investigation (serum insulin, glucose, lipid profile) and post oral glucose load (POGL) for insulin and glucose. Serum insulin was estimated by I(125) radio immuno assay. Patients were randomly divided into group A (Tab enalapril) and group B (Tab atenolol). In 51 patients who completed the study, fasting and POGL insulin and fasting lipid profile were estimated two months after treatment. Mean age of cases was 38.91 years. 50% of patients had stage II hypertension. BMI was increased in 36 cases (42.35%) as compared to 9 in (9.57%) controls. Increased WHR was found in 40 cases as compared to 26 in controls. The SFT was more in cases compared to controls. 47 (55.29%) of 85 cases had abnormal lipid profile as compared to 25 (26.60%) in 94 controls. The fasting and POGL insulin levels (13.85 and 60.05 micro u/ml respectively) in cases were significantly higher than in controls (6.87 and 16.16 micro u/ml respectively). The mean POGL insulin values were much higher in obese compared to non-obese hypertensives. The decrease in mean fasting and POGL insulin values in patients taking ACEI and beta blockers were similar. Abnormal lipid profile was significantly more in cases than controls. Increased total cholesterol (TC), Low density lipoprotein cholesterol (LDLC) and total cholesterol (TC)/high density lipoprotein (HDL) ratio were the most frequent abnormality. The mean insulin (both fasting and POGL) levels were higher in obese hypertensives and those with abnormal lipid profile. Both drugs had equal efficacy in reducing the insulin values.

Elimination of superoxide dismutase interference in fructosamine assay.

OBJECTIVE: Superoxide dismutase (SOD) (EC 1.15.1.1) is reported to decrease the reduction of nitroblue tetrazolium (NBT) in the fructosamine assay. The study was undertaken to find a method to eliminate this interference. DESIGN AND METHODS: We studied the NBT reduction in the presence and absence of added bovine erythrocyte SOD during fructosamine assay. Formation of reduced NBT decreased with the increasing concentration of SOD. Various inhibitors of SOD were experimented with for effectively eliminating this interference. RESULTS: Cyanide eliminates the interference due to SOD, but is unsuitable because in it's presence glucose becomes reducing under the conditions of fructosamine assay. SOD inhibitors such as EDTA and Azide did not eliminate the effect of SOD. Guanidine. HCl gives opalescence in the reaction mixture. Addition of 2M HCl to the serum and incubation at 37 degrees C for 10 min eliminated the effect of added SOD (70 kU/L). The correlation between deltaA10-20 min of serum treated with HCI in presence and absence of added SOD is y = 0.9011x + 0.01055 with r = 0.9789 and S.E. (y) 0.007686. CONCLUSION: SOD does not interfere in globin bound fructosamine assay as acid-acetone treatment in preparation of heme free globin inhibits SOD. Pretreatment of serum with HCl can satisfactorily eliminate the interference due to SOD in fructosamine assay. The acid treatment could be used to inhibit SOD in various other reactions that are followed with NBT reduction.