Vivek Ambade

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.

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 (r2 = 0.0271) and between duration of hypertension and urinary albumin excretion (r2 = 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.

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.

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 its 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.

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.

Serum ferroxidase albumin ratio as a marken in pulmonary tuberculosis

Serum ferroxidase and albumin levels were determined in 98 patients of tubercuiosis, of whom 49 were freshly diagnosed, sputum positive (group-I) & 49 were completely treated patients (group-II). Forty nine age and sex matched healthy individuals were taken as controls. Mean±SD of serum ferroxidase and albumin levels in controls, group-I and group-II was found to be 864.35±106.35 IU/L & 3.91±0.234 g/dL, 1603.76±222.65 IU/L & 3.24±0.518 g/dL and 1001.78±201.63 IU/L & 3.82±0.43 g/dL, respectively. Serum ferroxidase in group I was significantly higher as compared to controls and group-II (p<0.01). The decreased levels of serum albumin in group I, as compared to control and group-II was statistically significant (p<0.01). Serum ferroxidase: albumin ratio (Ferroxidase in International Unit per gram of albumin) in group I (50.47±10.36 IU/g) was significantly higher than controls (22.22±3.3 IU/g), (p<0.001) while in group II it was significantly lower (26.72±7.18 IU/g, p<0.001) than group-I and close to control values. Serum ferroxidase: albumin ratio (IU/g) can therefore be incorporated as a surrogate marker to assist in diagnosis and prognosis of pulmonary tuberculosis.

Markers for differentiation of tubercular pleural effusion from non-tubercular effusion

BACKGROUND The limitations of the conventional methods for diagnosing tuberculosis (TB) have spurred multi-faceted research activities throughout the world. This study aims to explore the levels of adenosine deaminase (ADA) and interleukins in pleural effusion of tuberculous, malignant, and miscellaneous origin for differential diagnosis of tubercular and non-tubercular effusion. METHOD Adenosine deaminase was estimated by kinetic method employing xanthine oxidase while interleukins were measured using commercially available ELISA kits in pleural fluids of tubercular and non-tubercular origin. RESULTS Pleural fluids INF-γ, sIL-2R, TNF-α and ADA were significantly higher in TB group (n = 48) as compared to the non-TB group (n = 33) (mean ± SD: INF-γ; 1,958.7 ± 896.5 pg/mL vs 356.9 ± 733.6 pg/mL, sIL-2R; 6,101 ± 1,753.8 pg/mL vs 3,166 ± 2,611.1 ± pg/mL, TNF-α; 195.5 ± 292.1 pg/mL vs 59.7 ± 128.9 pg/mL, ADA; 123.6 ± 81.8 IU/L vs 48 ± 48.5 IU/L, P < 0.01). CONCLUSION INF-(is more sensitive and specific than ADA for the diagnosis of TB and should be added to the armamentarium of the diagnostic workup of pleural fluids for timely and accurate diagnosis of TB and differentiation of tubercular pleural effusion from non-tubercular effusion.

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.

METHODS FOR ESTIMATION OF BLOOD GLUCOSE : A COMPARATIVE EVALUATION

Glucose in blood is the most frequent analyzed parameter in a clinical chemistry laboratory. In Armed Forces Laboratories, copper reduction method (Modified Folin Wu) is commonly used. Here we have compared this method as well as O-Toluidine and GOD-POD method with reference UV-Hexokinase method. Both Modified Folin Wu and O-Toluidine showed upward deviation with substantial imprecision (CV=4.9% - 3.5% and 6% - 5.8% respectively) and inaccuracy (Average deviation = 5.76% and 10.68% respectively). GOD-POD method was found linear (up to 500 mg/dl), with good precision (CV=0.7% to 1.4%) and accuracy (Average deviation= -0.97%). This method is simple, rapid, economical and sensitive, and can be adopted to a routine colorimeter.