Estimation of glycated hemoglobin by 2,6-dimethylphenol: Sulphuric acid conventional method

Glycated hemoglobin levels in hemolysate of normal and diabetic patients were determined by the 2,6-dimethylphenol:57.5% sulphuric acid conventional method and the values were 0.39±025 and 0.69±0.21 moles of hydroxymethylfurfural(HMF)/mole of globin, respectively. The mean increase in glycated hemoglobin values in diabetics (1.8fold) was highly significant (p<0.001). A good correlation (r=0.95) was found between the glycated hemoglobin values obtained by this method and the phenol:sulphuric acid method. The values obtained by former method were about 1.2–1.4 times the values by the phenol:sulphuric acid method. This study indicates that conventional 2,6-dimethylphenol: 57.5% sulphuric acid method is more sensitive for the estimation of glycated hemoglobin than any other method based on the same principle. It is less time consuming, reliable and hence can be employed for the routine laboratory estimation of glycated hemoglobin for the assessment of glycemic control.     

Changes in glycated proteins during impaired glucose tolerance

In persons with impaired glucose tolerance, serum glycated proteins (9.44±1.46μg hexose/mg protein) increased 1.69-fold (P<0.001) over control subjects compared to an increase of 1.95-fold in overt diabetics. However, correlations between serum glycated protein values and 2 h blood sugar values during the tolerance test (r=0.39) and between the former and graph area (r=0.30) were not strong. Glycated albumin (2.25 mol hexose/mol protein) and glycated haemoglobin (0.398±0.07 mol hexose/mol protein) in the study group also showed 1.67 and 1.24-fold increase respectively compared to controls. Fairly good correlations between glycated serum protein and glycated albumin (r=0.50) and glycated haemoglobin (r=0.45) were observed during this study. The data suggest that measurement of glycated proteins will be a useful index to detect impaired glucose tolerance.     

Seminal plasma levels of free 8-isoprostane and its relationship with sperm quality parameters

There is growing evidence that damage to spermatozoa by reactive oxygen species play a key role in male infertility. The aim of this study was to assess seminal plasma free 8-Isoprostane levels in men with asthenozoospermia, asthenoteratozoospermia and oligoasthenoteratozoospermia compared to normozoospermic males and its correlation with seminal parameters. The case group consisted of men with asthenozoospermia (n=15), asthenoteratozoospermia (n=16) and oligoasthenoteratozoospermia (n=15). The control group consisted of 16 males with normozoospermia. After Purification of Free 8-Isoprostane by affinity column, its concentration was measured by enzyme immunoassay method. Free 8-Isoprostane evaluation showed significantly greater values in the total case group (n=46) versus control group (18.23±3.56 vs 2.6±0.38 ng/ml). In each case group free 8-Isoprostane also showed a significant increasing compared to normozoospermic males. Free 8-Isoprostane showed an inversely significant correlation with sperm motility and sperm morphology. Lipid peroxidation could have significant role in etiology of sperm abnormalities. Measurement of 8-Isoprostane can be used as a specific biomarker for assessing lipid peoxidation in sperm.     

Pattern of urinary iodine excretion in children

Urinary iodine was estimated chemically by the kinetic method, after mineralisation of the organic iodide by alkaline ashing. The experimental conditions were optimised. The methodological modifications of an existing technique are highlighted. Urinary iodine levels were determined in 349 school children of Bombay, of whom 163 had Grade IA/IB/II goitre and the remaining 186 did not have goitre. Urinary iodine concentration (Mean±SEM) of the control (n=186) and the goitre (n=163) groups were 76.47±4.97 and 71.09±6.23 μg/g creatinine respectively. The difference in the means was statistically not significant. Urinary iodine exhibited a log-normal distribution. It is concluded that the presence of goitre in the clinically euthyroid group of children was not caused by iodine deficiency.     

Role of reactive oxygen species in pathogenesis of nephrotic syndrome

Nephrotic syndrome is the common chronic disorder characterized by alteration of permeability of the glomerular capillary wall, resulting in its inability to restrict the urinary loss of proteins. Nephrotic syndrome is characterized by heavy proteinuria, hypoalbuminemia, hyperlipidemia associated with peripheral edema. The molecular basis of glomerular permselectivity remains largely unknown. In recent years it has been proposed that Nephrotic syndrome is a consequence of an imbalance between oxidant and antioxidant activity. The present study was aimed to test that the reactive oxygen species are the mediators of excessive protein permeability and other complications of Nephrotic syndrome. For this 30 adults with Nephrotic syndrome were studied. The control group comprised 30 healthy adults matched for age. Serum levels of lipid peroxides, nitric oxide (NO⊙), α- tocopherol, ascorbic acid, erythrocyte superoxide dismutase activity, serum albumin, uric acid, cholesterol and plasma total antioxidant capacity were measured. Student’s ‘t’ test was applied for statistical analysis. There was a significant increase in lipid peroxide (1.58 ± 0.42 in controls, 3.64 ±1.3 in patients) (P<0.001) levels in study group as compared with controls. α-tocopherol (12.95 ± 1.04 in controls, 9.93 ± 1.43 in patients) (P<0.001), erythrocyte SOD activity(1.88 ± 0.9 in controls 1.07 ± 0.5 in patients) (P=0.01), serum albumin(4.06 ± 0.50 in controls, 3.04 ± 0.11 in patients) (P<0.001), and plasma total antioxidant capacity (847.33 ± 126.83 in controls, 684.00±102.94 in patients) (P<0.001) were significantly decreased. There was non-significant increase in uric acid (P>0.05), a non-significant decrease in NO⊙ (38.48 ± 15.47 in controls 37.47 ± 14.27 in patients) (P>0.05) and ascorbic acid levels ascorbic acid,( 0.95 ± 0.31in controls 0.79 ± 0.30 in patients) (P>0.05) in study group as compared with controls. Imbalance between oxidants and antioxidants may contribute to pathogenesis of proteinuria and related complications in nephrotic syndrome.     

Glycosylated hemoglobin A1 in a random group of adult onset diabetics of indian subpopulation

Glycosylated Hemoglobin (GHb) was estimated using cation exchange resin binding method in 50 non diabetic healthy controls and 500 maturity onset diabetics. Percentage of glycosylated hemoglobin levels in controls was 7.27±0.456 (range 6.0–8.04) and 9.47±0.98 (range 8.2–13.63) in diabetic group. Glycosylated hemoglobin levels remain unaffected inspite of transient rise of serum glucose levels during an oral glucose tolerance test. Significantly elevated GHb A₁. Furthermore significant elevation of GHb A₁ was noticed in diabetics with all secondary complications suggesting poor glycemic control in these patients. The diabetics having retinopathy, nephropathy or diabetic foot had relatively higher GHb levels compared to patients having coronary artery disease or hypertension.     

Hypolipidemic effect of water extract ofFicus bengalensis in alloxan induced diabetes mellitus in rabbits

Hypolipidemic effect of the water extract of the bark ofFicus bengalensis was investigated in alloxan induced diabetes mellitus in rabbits. Treatment for one month (50 mg/kg body weight/day) brought down the level of total serum cholesterol (TC) in subdiabetic and diabetic rabbits (five in each group) from 82±11 mg% and 118±10.6 mg% to 42.7±3.1 mg% and 51.7±4.7 mg% respectively. Low density lipoprotein cholesterol and very low density lipoprotein cholesterol values came down from 34±10 mg% and 95±24 mg% to 16±3 mg% and 29±4 mg% in subdiabetic and diabetic rabbits. Triacylglycerol level before treatment was 121±21.6 mg% and 416±70 mg% in subdiabetic and diabetic rabbits. Treatment brought it down to 45±5 mg% and 81±27.5 mg%. Glycosylated hemoglobin was brought down from 2.1±0.3% to 1.5±0.1% in subdiabetic group and from 4.28±0.5% to 2±0.3% in diabetic group, indicating that treatment with water extract effectively controlled blood sugar. After treatment with water extract serum lipid values were nearly equal to those in healthy controls (normal) in subdiabetic rabbits, while in diabetic group, the values were only slightly above those of normal.     

Assessment of oxidative stress and effect of antioxidant supplementation during radiotherapy in carcinoma of upper digestive tract

Oxidative stress was studied by estimating plasma levels of malondialdehyde (MDA), beta carotene, vitamin E and erythrocytic superoxide dismutase(E-SOD) activity in 50 cases of carcinoma of upper digestive tract which included carcinoma of oral cavity, pharynx and oesophagus. While plasma MDA level was found to be increased (3.5±1.0 nmole/ml), a significant decrease in beta carotene (81.2±14.5mg%), vitamin E (8.5±1.1 mg/L) level and E-SOD activity (657.0±80.6 U/G Hb) were observed in carcinoma of upper digestive tract. Patients were treated with radiotherapy which itself was toxic enough and produced its deleterious effects by generation of reactive oxygen species (ROS). As antioxdiants can detoxify ROS, beneficial effect if any, of antioxidant administration during radiotherapy was studied in two groups of patients, group A (n=5, supplemented with antioxidants) and group B (n=5, without antioxidant supplementation). Plasma MDA level was found to be elevated in both the groups but the increase in group B was significant, compared to pretreatment level. Further, body weight was found to be significantly decreased in group B patients, which was maintained in group A patients. Moreover, group A patients showed significant elevation in beta carotene concentration, thus showing beneficial effect of administration of antioxidants during radiotherapy without disturbing the desirable therapeutic effect of radiotherapy.     

Raised serum homocysteine levels in patients of coronary artery disease and the effect of vitamin B12 and folate on its concentration

Homocysteine(Hcy) has been implicated as a novel risk factor of Coronary Artery Disease (CAD) among Asian Indians, but many studies done in India failed to reveal any direct correlation. It has also been reported that Folic acid and Vitamin B12 levels inversely affect serum levels of homocysteine. In this study, we looked at the levels of homocysteine among patients with CAD. The effect of Vitamin B12, Folate and other risk factors on homocysteine levels were also evaluated. Mean homocysteine levels in cases (22.81±13.9, n=70) were significantly higher (p=<0.001) than the controls (7.77±7.3, n=70). However no statistically significant correlation could be deduced between homocysteine Vitamin B12, and Folate. Cumulative analysis have indicated an increase in homocysteine levels among patients with CAD with every additional risk factor.     

Protective role of<Emphasis Type="Italic">Phyllanthus niruri</Emphasis> against nimesulide induced hepatic damage

Present study aimed to evaluate the protective role of the aqueous extract of Phyllanthus niruri (P. niruri) against nimesulide-induced hepatic disoder in mice by determining levels of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) in serum and also by measuring the hepatic content of the antioxidant enzymes, superoxide dismitase (SOD) and catalase (CAT); the free radical scavenger, reduced glutathione (GSH) and thiobarbituric acid reacting substances (TBARS). Aqueous extract of P. niruri was administered either orally or intraperitoneally in different doses and times as needed for the experiments. Intraperitoneal of the extract (100 mg/kg body weight for seven days) reduced nimesulide (750 mg/kg body weight for 3 days) induced increased levels of GOT (37.0±1.8 units/ml in control group vs. 91.8±2.0 units/ml in nimesulide treated group vs. 35.0±1.0 units/ml in extract treated group), GPT (30.0±2.1 units/ml in control group vs. 88.4±2.9 units/ml in nimesulide treated group vs. 34.1±1.8 units/ml in extract treated group), and ALP (7.86±0.47 KA units/ml in control group vs. 23.80±0.60 KA units/ml in nimesulide treated group vs. 7.30±0.40 KA units/ml, in extract treated group) to almost nomal. In addition, P. niruri restored the nimesulide induced alterations of hepatic SOD (550±20 units/mg total protein in control group vs. 310±13 units/mg total protein in nimesulide treated group vs. 515±10 units/mg total protein in extract treated group), CAT (99.5±2 units/mg total protein in control group vs. 25.0±1.5 units/mg total protein in nimesulide treated group vs. 81.0±0.8 units/mg total protein in extract treated group), GSH (90±3 nmoles/mg total protein in control group vs. 17±4.2 nmoles/mg total protein in nimesulide treated group vs. 81±1 nmoles/mg total protein in extract treated group) and TBARS (measured as MDA, 36.6±3.0 nmoles/g liver tissue in control group vs. 96.3±5.2 nmoles/g liver tissue in nimesulide treated group vs. 41.2±1.7 nmoles/g liver tissue in extract treated group) contents. Dose-dependent studies showed that the herb could protect liver even if the nimesulide-induced injury is severe. Intraperitoneal administration of the extract showed better protective effect than oral administration. Combining all, the data suggest that P. niruri possesses hepatoprotective activity against nimesulide-induced liver toxicity and probably acts via an antioxidant defense mechanism. To the best of our knowledge, this is the first report of the hepatoprotective action of P. niruri against nimesulide induced liver damage.     

Correlation of Red Blood Cell Acetylcholinesterase Enzyme Activity with Various RBC Indices

Cholinesterases belongs to class hydrolases. There are two types acetylcholinesterase and butyryl cholinesterase. Acetylcholinesterase present in nerve endings and also in the RBC membrane. It helps to maintain the shape and size of RBCs. Any change in shape and size of RBCs may affect the activity of Acetylcholinesterase. Thus this study aimed to estimate RBCs Acetylcholinesterase enzyme activity in various types of anemias and correlate the RBCs Acetylcholinesterase enzyme activity with various hematological indices such as Erythrocyte Sedimentation Rate (ESR), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Mean Corpuscular Volume (MCV), Red cell Distribution Width (RDW) etc. After obtaining ethical approval from Institutional ethics committee total of 100 samples were collected from Clinical Biochemistry laboratory, Kasturba Medical College, Manipal, Manipal University. 25 were having normal RBC indices, 12 with hemolytic anemia, 26 with microcytic anemia and 26 with macrocytic anemia based on peripheral smear report and RBC indices. Acetylcholinesterase were measured using Ellman’s method. RBC acetylcholinesterase activity was significantly increased in microcytic anemia (58.13 ± 5.4) and macrocytic anemia (76.87 ± 6.7) than normal group (37.62 ± 2.71). Also increased RBC acetylcholinesterase was seen in hemolytic anemia (48.11 ± 5.18) but the increase is not statistically significant. RBC acetylcholinesterase correlated negatively with hemoglobin (r = ?0.356, p = 0.001) and positively with RDW (r = 0.31, p = 0.003). To conclude RBC acetylcholinesterase activity can be used as one of the potential marker for various types of anemia.     

Estimation of Ischemia Modified Albumin (IMA) Levels in Patients with Acute Coronary Syndrome

Myocardial ischemia produces free radicals that catalyze a series of oxidative reactions that damage healthy tissues. The N-terminal sequence of albumin is one of the proteins modified by these highly reactive oxygen species and forms the ischemia modified albumin (IMA). This study involves investigations undertaken in different study groups to assess the levels of IMA. Mean serum IMA levels (U/mL) in patients with ST-segment elevated myocardial infarction (92.1 ± 10.6), non-ST-segment elevated myocardial infarction (87.3 ± 5.95) and unstable angina (UA) (88.9 ± 6.16) were significantly higher than non-cardiac chest pain (77.9 ± 6.69) and also healthy subjects (54.7 ± 17.2) (p < 0.001). IMA is a highly sensitive marker and has a high predictive value, which might prove the usefulness of this biomarker for early detection of myocardial ischemia. These data indicate a possible role of the IMA test in the early triage of patients with chest pain.     

Usefulness of lipase / amylase ratio in acute pancreatitis in South Indian population

This retrospective study was conducted to assess the serum lipase / amylase ratio in acute pancreatitis for South Indian population attending the clinics of Gastroenterology and Emergency medicine in the last five years. One thousand one hundred and thirty two patients (768 males and 364 females) with acute pancreatitis (AP) were selected for the study. The diagnosis of AP was based on clinical evaluation, Computed Tomography (CT) findings and biochemical parameters such as serum lipase and amylase. Based on the etiology, the groups were divided into alcoholic, biliary and miscellaneous AP. Based on CT findings, groups were divided into mild, moderate and severe AP. Serum lipase, amylase and lipase / amylase ratio were calculated and statistically analyzed. Serum lipase levels in alcoholic group (3226.3 ± 1384.7) were significantly lower in comparison to biliary (6064.5 ± 1640.8) group though the levels were not significantly different from miscellaneous group (2132.3 ± 1210). Serum amylase values were significantly lower in alcoholic group (923.4 ± 557.5) in comparison to biliary (1736.2 ± 390.7) and there was significant difference between alcoholic and miscellaneous group (535.8 ± 477.6). The serum lipase / amylase ratio > 4 occurred in alcoholic group than with biliary and miscellaneous group. The sensitivity and specificity to predict alcoholic AP with lipase / amylase ratio at >4.0 was 84 % and 59 % respectively. In conclusion the serum lipase to amylase ratio greater than 3.0 could be used to differentiate but keeping the cut off at 4.0 would be of higher sensitivity without much change in specificity. The serum lipase to amylase ratio with a cut off of 3.0 or greater is not useful to differentiate the severe AP from milder AP. Hence, serum amylase and lipase are important for evaluation of pancreatitis though it is not a gold standard for the diagnosis or assessment of severity of acute pancreatitis.     

Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates

BackgroundChinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE.ResultsHigher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa).ConclusionsThese results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.How to cite: Pérez-Rodriguez S, Ramírez OT, Trujillo-Roldán MA et al. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates. Electron J Biotechnol 2020;48. https://doi.org/10.1016/j.ejbt.2020.09.006.     

Ascorbic acid status in third trimester of pregnancy,at delivery and in cord blood

Ascorbic acid levels in 30 subjects in their third trimester of pregnancy were estimated. Their haematological parameters (packed cell volume, haemoglobin and mean cell haemoglobin concentration) were also determined. The values were compared with that of 30 non-pregnant women (control group). The ascorbic acid status of newly born babies using cord blood were determined along with that of their mothers at delivery. The ascorbic acid level of 0.92±0.37 mg/dl was reported in third trimester. This value was not significantly (P>0.05) lower than in the control group (1.04±0.4 mg/dl). The result showed that the ascorbic acid level at delivery was significantly (P<0.05) higher than that of the control group. The ascorbic acid levels in the cord blood of the newly born babies were significantly (P<0.01) higher than the maternal level. There was insignificant difference in the haematological parameters between the pregnant and non-pregnant women.     

Standardization of homocysteine on HPLC using DTT as reductant

Determination of plasma total homocysteine by high-pressure liquid chromatography (HPLC) usually requires reduction of protein bound or free homocysteine-disulphides into thiols by a reducing agent and the liberated thiols are then derivatized by a fluorescent marker. In this study we have standardized the HPLC method for homocysteine measurement using dithiothreitol (DTT) as reductant. The results of plasma total homocysteine values obtained by HPLC were compared with IMx method. The difference between the two means was statistically insignificant [P=0.616847 (two tail)] Linear regression analysis showed strong correlation between the two methods (r=0.983). Using this method we have analyzed 132 controls and 130 Coronary Heart Disease (CHD) patients for plasma total homocysteine, wherein, the mean plasma total homocysteine levels were 10.51±8.36 and 11.51±10.06 μmol/L respectively. Our research study suggests that DTT method is a simple and inexpensive assay for homocysteine determination in human plasma for research application.     

Circadian Periodicity of Circulating Plasma Lipid Peroxides,Uric Acid and Ascorbic Acid in Renal Stone Formers

Circadian periodicity of plasma lipid peroxides and serum ascorbic acid and uric acid levels were studied in one hundred renal stone formers (55 women and 45 men; age 20–60 years) and 50 clinically healthy volunteers (21 women and 29 men; age 21–45 years) with diurnal activity from 06:00 to 22:00 and nocturnal rest. A marked circadian variation was demonstrated by population-mean-cosinor for all studied variables in stone formers and healthy subjects. By comparison to the healthy controls, parameter tests indicate that the stone formers had a higher MESOR (±SE) of MDA (2.90 ± 0.03 vs. 2.28 ± 0.06; F = 94.929, p < 0.001), a lower MESOR of serum ascorbic acid (0.722 ± 0.010 vs. 0.839 ± 0.10; F = 32.083, p < 0.001), and a similar MESOR of serum uric acid. Furthermore, the patients also differed from the healthy subjects in terms of their circadian amplitude and acrophase (tested jointly) of all three variables (p < 0.001). The demonstration herein of a circadian rhythm in MDA, serum ascorbic and uric acid suggests that these variables could also serve as markers to optimize the timing of treatment and to assess the patient’s response to treatment for further management.     

Effect of low-dose omega-3 fatty acids substitution on blood pressure, hyperinsulinemia and dyslipidemia in Indians with essential hypertension: A pilot study

The present study comprised 100 patients of essential hypertension who were screened for fasting hyperinsulinemia, which was detected in 77% cases. Twenty such hyperinsulinemic cases were subjected to 4 weeks of dietary control phase followed by 6 weeks of omega-3 fatty acids substitution [either 0.6 g/d (group 1) or 1.2 g/d (group 2)]. The mean basal fasting plasma insulin levels were significantly higher (p<0.001) in patients of hypertension when compared to normal controls (126.51±80.36 and 19.35±12.61 μU/ml respectively). At the end of 4 weeks of diet control only, no significant change was observed in any parameter. After substitution of omega-3 fatty acid, a significant reduction of fasting plasma insulin levels in both group 1 (29%) and group 2 (22.8%) was observed (p<0.001). Significant reduction of systolic and diastolic blood pressure (16.4% and 25% respectively), serum cholesterol, triglycerides and low density lipoprotein was also noted in both groups (p<0.001), while high density lipoprotein increased by ∼8 mg% in both groups. Thus omega-3 fatty acid substituion in low dose along-with curtailment of dietary omega-6 fatty acid may be used as an adjunctive measure in the management of essential hypertension.     

A specific colorimetric assay for Dapsone in biological fluids

The original Bratton and Marshall method for sulfanilamide assay has been modified for differential assay of Dapsone even in the presence of other diazotisable compounds. The property of Dapsone diazo derivative to precipitate out upon its coupling with N-(1-naphthyl) ethylene diamine has been taken advantage to differentiate this sulphone from that of other diazotisable compounds. The present assay method also enables one to assay free DDS and its acid labile metabolites in biological samples even in the presence of other diazotisable compounds. The use of the organic solvent to extract the azo colour complex renders greater sensitivity and specificity for DDS assay.