Development and optimization of an in-house heterologous ELISA for detection of prednisolone drug in enzyme conjugates using spacers

The introduction of spacers in coating steroid protein complexes and/or enzyme conjugates or immunogens is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We investigated the impact of different homobifunctional spacers, ranging in atomic length from 3 to 10, on the sensitivity and specificity of prednisolone (PSL) enzyme immunoassays. In this study, four homo-bifunctional spacers, namely, carbohydrazide (CH), adipic acid dihydrazide (ADH), ethylene diamine (EDA), and urea (U), were incorporated between PSL and horseradish peroxidase (HRP) for preparing the enzyme conjugate with an aim to improve the sensitivity of the assay without compromising assay specificity. The assays were developed using these enzymes conjugated with antibodies raised against the PSL-21-HS-BSA immunogen. The sensitivity of the PSL assays after insertion of a bridge in the enzyme conjugate was 1.22 ng/mL, 0.59 ng/mL, 0.48 ng/mL, and 0.018 ng/mL with ADH, CH, EDA, and urea as a spacer, respectively. Among the four combinations, the PSL-21-HS-BSA-antibody with PSL-21-HS-U-HRP-enzyme conjugate gave better sensitivity and less cross-reaction. The percent recovery of PSL from the exogenously spiked human serum pools was in the range of 88.32%-102.50%. The intra and inter-assay CV% was< 8.46%. The PSL concentration was estimated in the serum samples of patients on PSL treatment. The serum PSL values obtained by this method correlated well with the commercially available kit (r2 = 0.98). The present study suggests that the nature of the spacer is related to assay sensitivity and not the spacer length

Gender variability in performance of multiple-choice question-based assessment on medical biochemistry topics among 1st year MBBS students

BACKGROUND: Owing to its popularity and ubiquity, multiple-choice questions (MCQs) are now a commonly used assessment means used in medical education. However, the gender variability in the performance of students in MCQ-based examinations has been a notable topic of exploration in educational research. AIM: To discern if there are any gender-based differences in the performance as well as the risk-taking tendency in MCQ-based assessment among the 1st year medical students in Biochemistry. MATERIALS AND METHODS: This observational study was carried out on 237 1st year MBBS students as an end-of-module assessment test on “Miscellaneous topics in Biochemistry.” Six different types of formats of MCQs: single best response, multiple responses, true and false, reason assertion, pictorial, and matching types were administered to them. The performances of the students were compared among the different types of MCQs after stratifying the students according to gender and academic standing. The percentages of students who attempted and who correctly marked a particular type of MCQ were compared using appropriate statistical tests. RESULTS: No significant differences were observed in the pattern of scoring, attempt, or skipped questions in between the two genders in any of the six types of MCQs. The difference was observed in the type of MCQ formats preferred and the risk aversion pattern between the two genders. CONCLUSION: To affirm the gender neutrality and fairness of MCQ-based examinations, all the formats of MCQs must be administered in MCQ-based assessments

Assay of urinary protein-bound sialic acid can differentiate steroidsensitive nephrotic syndrome from steroid-resistant cases

The protein selectivity index as measured from the ratio of urinary immunoglobulin to albumin failed to differentiate between steroid-sensitive (SS) and steroid-resistant (SR) cases of nephrotic syndrome (NS). Sialic acid contributes negative charges to many plasma proteins. The negative charge is a determinant of protein excretion rate. The prognostic significance of assay of urinary excretion of protein-bound sialic acid in NS has not been evaluated. Hence, the present study was designed to evaluate whether measurement of urinary protein bound sialic acid (UPBSA) can be used as a marker to differentiate SS from SR cases of NS. The urine samples of 70 (47 SS and 23 SR) pediatric NS children were assayed for UPBSA by Aminoff′s method. The levels were compared and the receiver-operator curve was drawn to determine the optimum cutoff point to differentiate among the groups before starting the therapy. The excretion of UPBSA in SR cases of NS was significantly higher than that of SS cases (P<0.05). The optimum cutoff limit for UPBSA was 2.71 μg/mg of proteins with 75% sensitivity and 75.5% specificity for differentiating SS cases from SR cases (area under the plasma- concentration time curve = 0.814, P = 0.009). We conclude that UPBSA can differentiate SR cases from SS cases of NS in pediatric patients and may help in predicting the response to steroid therapy

Environmental contaminants in pathogenesis of breast cancer

597-617This review is an attempt to comprehend the diverse groups of environmental chemical contaminants with a potential for pathogenesis of breast cancer, their probable sources and the possible mechanisms by which these environmental contaminants act and interplay with other risk factors. Estrogens are closely related to the pathogenesis of breast cancer. Oxidative catabolism of estrogen, mediated by various cytochrome P450 enzymes, generates reactive free radicals that can cause oxidative damage. The same enzymes of estrogenic metabolic pathways catalyze biological activation of several environmental (xenobiotic) chemicals. Xenobiotic chemicals may exert their pathological effects through generation of reactive free radicals. Breast tissue can be a target of several xenobiotic agents. DNA-reactive metabolites of different xenobiotic compounds have been detected in breast tissue. Many phase I and II xenobiotic metabolizing enzymes are expressed in both normal and cancerous breast tissues. These enzymes play a significant role in the activation/detoxification of xenobiotic and endogenous compounds including estrogens. More than 30 carcinogenic chemicals are present in tobacco smoke; many of them are fat-soluble, resistant to metabolism and can be stored in breast adipose tissue. Similarly, pesticides are also known to cause oxidative stress; while some act as endocrine disruptor, some are shown to suppress apoptosis in estrogen sensitive cell lines. Reports have shown an association of smoking (both active and passive) and pesticides with breast cancer risk. However, the issues have remained controversial. Different mutagenic substances that are generated in the cooking process e.g., heterocyclic amines and polycyclic aromatic hydrocarbons (PAHs) can be a threat to breast tissue. PAHs and dioxins exert their adverse effects through the aryl hydrocarbon receptor (AhR), which activates several genes involved in the metabolisms of xenobiotic compounds and endogenous estrogens. These chemicals also induce AhR-dependent mitochondrial dysfunction. Many of the environmental pollutants suppress the immune system, which are implicated to risk. A better understanding about the biological effects of different environmental carcinogenic compounds and determination of their impact on rising incidence of breast cancer will be beneficial in improving preventive policy against breast cancer

Hepatoprotective activity of six polyherbal formulations in CCl₄-induced liver toxicity in mice

257-263To evaluate pretreatment of six polyherbal liquid formulations (PLFs) commercially available in India, on CCl4-induced liver injury, Swiss albino mice were treated for 7 days with distilled water or PLFs (2.6 and 5.2 ml/kg body weight/day, po) followed by single sc injection of 50% (v/v) CCl4 in arachis oil at a dose of 1ml/kg. The serum biochemical parameters such as alanine transaminases, aspartate transaminases and alkaline phosphatase were estimated. Phenobarbitone-induced sleeping time and liver histopathology were also carried out. CCl4-treated animals showed significant increase in the levels of liver enzymes, phenobarbitone-induced sleeping time and revealed fatty changes and centrizonal necrosis on histological examination of liver indicating hepatic damage. When pretreated with PLFs at a dose of 5.2 ml/kg body weight/day, the CCl4-induced changes were significantly reversed. The pretreatment with PLFs can prevent acute liver damage induced by CCl4 only at a higher dose. Therefore, it is suggested that a dose adjustment of these PLFs may be necessary for their optimal effects in human liver diseases

Effect of consumption of unheated and thermally-modified sesame and coconut oils on inflammation mediated metabolic disorders in Wistar rats

251-255Coconut oil and sesame oil are commonly used in South India for frying foods. On heating, edible oils form hazardous chemicals. This study explores the effect of consumption of unheated and thermally-altered sesame and coconut oils on coronary artery disease (CAD) risk factors in Wistar rats. Thirty Wistar albino rats were randomly divided into five groups (n=6/group). Group I (Control) was fed only chow, Group II: chow + unheated sesame oil, Group III: chow + heated sesame oil, Group IV: Chow + unheated coconut oil, Group V: chow + heated coconut oil. After eight weeks of treatment, serum lipid profile, hs-CRP, leptin, glucose, insulin, HOMA-IR, TNF-α, IL-6 and plasma homocysteine and fibrinogen levels were estimated. Rats in Group II showed a significant decrease in serum cholesterol, LDL-c, TNF-α, hs-CRP, insulin, and HOMA-IR but a significant increase in HDL-c, Group III showed opposite effects on these parameters, except that it decreased serum triglycerides level. Group IV and V did not show any significant effect on stated parameters. We conclude that consumption of unheated sesame oil gives protective effects against the CAD. Thermally altered sesame oil increases the CAD risk. Unheated and thermally altered coconut oil did not show any significant effect. Hence, we recommend that sesame oil better be used for dressing the food and coconut oil for frying