Phospholipase A2 group XV activity during cardiopulmonary bypass surgery.

OBJECTIVES: All patients who undergo cardiopulmonary bypass (CPB) experience some degree of ischemia reperfusion injury (IRI). Severe IRI-induced acute kidney injury (AKI) occurs in approximately 1-2% of patients undergoing CPB. Previous studies using activity-based protein profiling of urine identified group XV phospholipase A2, PLA2G15/LPLA2, as potentially associated with patients who develop AKI post CPB. The present study examined urinary PLA2G15/LPLA2 activity during CPB and in the near postoperative period for associations with subsequent AKI development. DESIGN & METHODS: Samples were collected in a nested case controlled cohort of 21 patients per group who either did (AKI) or did not (non-AKI) develop AKI post-operatively. Serum and urine samples from each patient before, during and after CPB were assayed for PLA2G15/LPLA2 activity. RESULTS: Urine activity significantly increased during the intra operative period. In contrast the activities in paired sera were markedly decreased during CPB. There was no correlation between the serum and urine activity levels of patients. There were no significant differences in activity levels of PLA2G15/LPLA2 in the urine or sera from patients that did and did not develop AKI. CONCLUSIONS: The lack of correlation between serum and urine activity levels suggests that the rapid intraoperative increases in PLA2G15/LPLA2 activity may originate from the kidney and as such offer an intraoperative indicator of early renal response to CPB associated stressors.

An easy and sensitive sandwich assay for detection of Mycobacterium tuberculosis Ag85B antigen using quantum dots and gold nanorods.

Mycobacterium tuberculosis is a serious global infectious pathogen causing tuberculosis (TB). The development of an easy and sensitive method for the detection of M. tuberculosis is in urgent need due to complex and low specificity of the current assays. Herein, we present a novel method for M. tuberculosis detection based on a sandwich assay via antigen-antibody interaction using silica-coated quantum dots (SiQDs) and gold nanorods (AuNRs). A genetically engineered recombinant antibody (GBP-50B14 and SiBP-8B3) was bound to surfaces of AuNRs and SiQDs respectively, without any surface modification. The antigen-antibody interaction was revealed using M. tuberculosis-specific secretory antigen, Ag85B. Two biocomplexes showed a quenching effect in the presence of the target antigen through a sandwich assay. The assay response was in the range of 1×10-3-1×10-10µgmL-1 (R=0.969) and the limit of detection for Ag85B was 13.0pgmL-1. The Ag85B was selectively detected using three different proteins (CFP10, and BSA), and further specifically confirmed by the use of spiked samples. Compared with existing methods, a highly sensitive and selective method for Ag85B-expressing M. tuberculosis detection has been developed for better diagnosis of TB.

Ghrelin O-acyltransferase (GOAT) enzyme is overexpressed in prostate cancer, and its levels are associated with patient's metabolic status: Potential value as a non-invasive biomarker.

Ghrelin-O-acyltransferase (GOAT) is the key enzyme regulating ghrelin activity, and has been proposed as a potential therapeutic target for obesity/diabetes and as a biomarker in some endocrine-related cancers. However, GOAT presence and putative role in prostate-cancer (PCa) is largely unknown. Here, we demonstrate, for the first time, that GOAT is overexpressed (mRNA/protein-level) in prostatic tissues (n = 52) and plasma/urine-samples (n = 85) of PCa-patients, compared with matched controls [healthy prostate tissues (n = 12) and plasma/urine-samples from BMI-matched controls (n = 28), respectively]. Interestingly, GOAT levels in PCa-patients correlated with aggressiveness and metabolic conditions (i.e. diabetes). Actually, GOAT expression was regulated by metabolic inputs (i.e. In1-ghrelin, insulin/IGF-I) in cultured normal prostate cells and PCa-cell lines. Importantly, ROC-curve analysis unveiled a valuable diagnostic potential for GOAT to discriminate PCa at the tissue/plasma/urine-level with high sensitivity/specificity, particularly in non-diabetic individuals. Moreover, we discovered that GOAT is secreted by PCa-cells, and that its levels are higher in urine samples from a stimulated post-massage vs. pre-massage prostate-test. In conclusion, plasmatic GOAT levels exhibit high specificity/sensitivity to predict PCa-presence compared with other PCa-biomarkers, especially in non-diabetic individuals, suggesting that GOAT holds potential as a novel non-invasive PCa-biomarker.

Benzoic acid metabolism in presence of Schistosoma mansoni infection in mice.

The metabolism of benzoic acid was examined in S. mansoni infected CBA mouse. The result showed that control animals dosed with 150 mg/kg benzoic acid resulted in urinary excretion of two metabolites, hippuric acid and benzoic acid glucuronide. Administration of the same dose to animal carrying S. mansoni for a period of over 6 weeks resulted in decreased formation of hippuric acid and total elimination of benzoic acid by glucuronide pathway.

Cis-diamminedichloroplatinum(II) nephrotoxicity and its relationship to renal gamma-glutamyl transpeptidase and glutathione.

To investigate the mechanism of cis-diamminedichloroplatinum(II) (cisplatin) nephrotoxicity, male Sprague-Dawley rats were given one injection of cisplatin (6 mg/kg i.v.). Urinary levels of amino acids and gamma-glutamyl transpeptidase were monitored for 8 days; kidney homogenate content of gamma-glutamyl transpeptidase was followed for 50 hr, and that of selenium-dependent glutathione peroxidase and total glutathione was followed for 4 hr. Peak urinary levels of amino acids and gamma-glutamyl transpeptidase occurred 4.5 hr after the i.v. dose. Glutamine, glycine, and ethanolamine were all elevated greater than 20 times that of the control at 4.5 hr and were still significantly elevated at 50 hr. Total renal glutathione content increased 51 +/- 17% (S.D.) of control values 20 min after cisplatin was given, before returning to base-line levels. No depletion of either renal glutathione or glutathione peroxidase was detected over the time interval studied. These results demonstrate an earlier physiological impairment than has hitherto been shown. Furthermore, depletion of glutathione and glutathione peroxidase does not occur in the rat kidney following therapeutic doses of cisplatin, in contrast to the changes observed in cardiac tissue following doxorubicin treatment.

Gamma-glutamyltransferase release by the post ischemic kidney: multiple forms and cellular pH.

The release of gamma-glutamyltransferase from renal tubule cells was studied in situ following 30 minutes of ischemia. The ischemic kidney enzyme level fell 33 percent after 15 minutes of reflow of which only 1.2 percent was recovered in the urine; none was released into the renal vein. At this time the overwhelming majority of the enzyme appears bound to membranes in both the kidney and the urine. In the subsequent 15 minutes renal levels continue to decline while urinary excretion accounts for 5 percent of that disappearing from the kidney. Interestingly the form of the enzyme present in kidney and urine shifts to a soluble form coinciding with cellular alkalosis, urinary alkalinization and a rise in ATP levels. Alkalinization of renal homogenates result in a 2-fold increase in the soluble enzyme form. The results are consonant with the immediate loss of brush border enzyme via uptake into the cell or release into the urine with the former pathway predominating; subsequent appearance of the soluble enzyme appears to reflect intracellular alkaline proteinase activity and exocytosis. The form in which the enzyme is excreted may provide a useful clinical index: membranous reflecting cellular necrosis and soluble reflecting cellular recovery.

Development of radioimmunoassay for gamma-glutamyl transferase using pancreatic enzyme.

A radioimmunoassay (RIA) for the determination of gamma-glutamyl transferase (GGT) was developed using human pancreatic enzyme as antigen. The assay allows the determination of GGT in concentrations as low as 80 ng/ml, and it is reproducible and specific. A good parallel relation was demonstrated between the standard curve and dilution curves for serum, urine, bile, and partially purified kidney GGT. In normal individuals, the mean serum concentration of GGT determined by RIA was found to be 3.43 micrograms/ml (SD +/- 1.20). Enzyme activity calculated from the GGT concentration measured by the radioimmunoassay using a regression equation was approximately twice as great as that determined by conventional enzyme assay.

Effect of castration and testosterone substitution on the urinary output of gamma-glutamyl transpeptidase and of N-acetyl-beta-D-glucosaminidase of male rats with renovascular hypertension.

The effect of castration of male rats with experimental renal hypertension ('two-kidney Goldblatt hypertension') was studied on the height of the hypertension and on the urinary output of gamma-glutamyl transpeptidase (gamma GT) and of N-acetyl-beta-D-glucosaminidase (NAG). Castration was carried out immediately after clamping one renal artery. Some of the castrates received testosterone substitution from the 3rd postoperative week onwards. Uncastrated hypertensive males served as controls. The experiments were carried out 8-18 weeks after eliciting high blood pressure. Hypertension as well as enzymuria were less expressed in castrates than in uncastrated males or in testosterone-substituted rats. In all animals studied the gamma GT excretion rate showed a positive correlation with the blood pressure. The output of gamma GT and of NAG as well as the specific gamma GT activity of the renal membrane fraction was lower in castrates than in uncastrated males or in substituted castrates. In uncastrated males and in testosterone-substituted castrates the daily NAG output showed a direct correlation with the renal hydroxyproline content. No such correlation was found in castrated males. The kidneys of castrates and of testosterone-substituted castrates contained less hydroxyproline than those of uncastrated males.