A Microarray Immunoassay for Serum Thyrotropin and Thyroglobulin Using Antibodies Immobilized on Track-Etched Membranes

Serum thyroglobulin (Tg) and thyroid stimulating hormone (TSH) measurements have evolved as important analytes for monitoring the prognosis of patients with differentiated thyroid cancer, post-thyroidectomy. Individual analyte immunoassay is the current practice in clinical pathology, but the simultaneous assay for all relevant analytes for a given disease, can reduce assay costs, improve patient compliance and give the clinician more information for an unequivocal diagnosis. Microarray immunoassay (MI) can achieve this goal and, hence, we have developed and validated a immuno-radiometric MI for quantitation of serum TSH and Tg by using highly micro-porous polycarbonate (PC) track-etched membranes (TEM) to immobilize the monoclonal anti-TSH and polyclonal anti-Tg antibodies in ~1 mm diameter spots. Non-competitive immunoassays were performed using mixture of ¹²⁵I labeled monoclonal anti-TSH and anti-Tg antibodies. Phosphorimager was used to quantify the bound radioactivity. TSH and Tg were detected with detection limit of 0.07 µIU/ml and 0.13 ng/ml respectively, which is lower than the clinically required cut-off level. The assay showed: acceptable intra-assay precision within 20 % and recovery in the range of 76–111.2 %. MI compared well with the established immunoradiometric assay (IRMA) with r = 0.98, p < 0.01 (n = 41). No cross-reactivity was seen between the immobilized antibodies. Although two hormones are addressed in this report, MI using PC TEM and isotopic/non-isotopic tracers has the potential for highly automated multiplexed analysis.     

Elimination of lipaemic interference by high-speed centrifugation

IntroductionIn order to deliver high quality results, detection and elimination of possible analytical interferences, such as lipaemia, is crucial. The aim of this study is to evaluate the efficacy of high-speed centrifugation in eliminating lipaemic interference and to define own lipaemic index (LI) for the studied biochemical analytes.Materials and methodsEvaluated analytes were: albumin, alkaline phosphatase, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), calcium, creatinine, gamma-glutamyltransferase (GGT), glucose, phosphates, total proteins, urea and total bilirubin. Those analytes and LIs have been analysed in duplicate in the Roche Diagnostics-c8000 analyser in samples centrifuged at 3000 rpm/10 minutes in the SL16 (Thermo Scientific, Waltham, USA) centrifuge and according to an own high-speed centrifugation protocol (12,900 rpm/15 minutes) in the MicroCL17R (Thermo Scientific, Waltham, USA) centrifuge. Lipaemia has been measured in each sample. The efficiency of high-speed centrifugation is verified by the Wilcoxon test (P < 0.05). In cases where significant differences are observed, our own LI is calculated. For ALT and AST, it is verified by McNemar test (P < 0.05). For creatinine, both Wilcoxon and McNemar test were applied.ResultsThere were statistically significant differences in analyte concentration before and after high-speed centrifugation for: albumin, creatinine, GGT, glucose, phosphates, urea and total bilirrubin. Own LI is calculated. McNemar test shows statistically significant diferences in the proportion of delivered results before and after high-speed centrifugation in ALT, AST and creatinine.ConclusionsThis study confirms the efficacy of high-speed centrifugation protocol for all the considered analytes, excepting calcium, alkaline phosphatase and total proteins.     

Status and Interrelationship of Zinc,Copper, Iron,Calcium and Selenium in Prostate Cancer

Deficiency or excess of certain trace elements has been considered as risk factor for prostate cancer. This study was aimed to detect differential changes and mutual correlations of selected trace elements in prostate cancer tissue versus benign prostatic hyperplasia tissue. Zinc, copper, iron, calcium and selenium were analysed in histologically proven 15 prostate cancer tissues and 15 benign prostatic hyperplasia tissues using atomic absorption spectrophotometer. Unpaired two tailed t test/Mann–Whitney U test and Pearson correlation coefficient were used to compare the level of trace elements, elemental ratios and their interrelations. As compared to benign prostatic tissue, malignant prostatic tissue had significantly lower selenium (p = 0.038) and zinc (p = 0.043) concentrations, a lower zinc/iron ratio (p = 0.04) and positive correlation of selenium with zinc (r = 0.71, p = 0.02) and iron (r = 0.76, p = 0.009). Considerably divergent interrelationship of elements and elemental ratios in prostate cancer versus benign prostatic hyperplasia was noted. Understanding of differential elemental changes and their interdependence may be useful in defining the complex metabolic alterations in prostate carcinogenesis with potential for development of element based newer diagnostic, preventive and therapeutic strategies. Further studies may be needed to elucidate this complex relationship between trace elements and prostate carcinogenesis.     

Regional Distribution of Copper,Zinc and Iron in Brain of Wistar Rat Model for Non-Wilsonian Brain Copper Toxicosis

In previous studies, we have reported first in vivo evidence of copper deposition in the choroid plexus, cognitive impairments, astrocytes swelling (Alzheimer type II cells) and astrogliosis (increase in number of astrocytes), and degenerated neurons coupled with significant increase in the hippocampus copper and zinc content in copper-intoxicated Wistar rats. Nonetheless, hippocampus iron levels were not affected by chronic copper-intoxication. Notwithstanding information on distribution of copper, zinc and iron status in different regions of brain due to chronic copper exposure remains fragmentary. In continuation with our previous study, the aim of this study was to investigate the effects of intraperitoneally injected copper lactate (0.15 mg Cu/100 g body weight) daily for 90 days on copper, zinc and iron levels in different regions of the brain using atomic absorption spectrophotometry. Copper-intoxicated group showed significantly increased cortex, cerebellum and striatum copper content (76, 46.8 and 80.7 % increase, respectively) compared to control group. However, non-significant changes were observed for the zinc and iron content in cortex, cerebellum and striatum due to chronic copper exposure. In conclusion, the current study demonstrates that chronic copper toxicity causes differential copper buildup in cortex, cerebellum and striatum region of central nervous system of male Wistar rats; signifying the critical requirement to discretely evaluate the effect of copper neurotoxicity in different brain regions, and ensuing neuropathological and cognitive dysfunctions.     

Hepatoprotective and Nephroprotective Effect of Curcumin Against Copper Toxicity in Rats

Curcumin is a natural anti-inflammatory and antioxidant with several potential health benefits. Although it has been examined in several metals toxicity studies, but its role in the protection against copper toxicity has not been investigated. In this study; the detoxification and antioxidant effect of curcumin were examined to determine its prophylactic/therapeutic role experimentally in rats. Forty albino rats were divided into five groups; control, CuSO₄ (4 mg/kg body weight), curcumin (80 mg/kg body weight), curcumin post-treatment (CuSO₄ for 15 days followed by curcumin for the next 15 days) and curcumin co-treatment (CuSO₄ plus curcumin for 30 days). All rats were treated orally by stomach tube for 30 days/once a day. Changes were observed in hepatic marker enzymes such as: aspartate aminotransferase (AST), alanine transaminase-(ALT), alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT), besides the serum total protein, urea and creatinine. Concentration of liver and kidney antioxidants such as: catalase (CAT), superoxide dismutase (SOD), reduced glutathione-(GSH) and malondialdehyde (MDA) were measured. An increased in the activities of liver marker enzymes, urea, creatinine and the MDA contents were detected after exposure to CuSO₄. Meanwhile, the activities of serum total protein, hepatic and renal antioxidants were decreased. Changes in all biochemical parameters were alleviated by the post-treatment and co-treatment of curcumin. Our finding suggests that the curcumin showed protective effects on CuSO₄-induced hepatotoxicity and nephrotoxicity.