A rapid and simple method for the analysis of 1-hydroxypyrene glucuronide: a potential biomarker for poly cyclic aromatic hydrocarbon exposure

The present study describes a simple method of analyzing metabolitesof pyrene in urine. This method is capable of detecting theglucuronic acid and sulfate conjugates of pyrene as well asfree 1-hydroxypyrene in a single analysis. In comparison toother analytical methods for detecting pyrene metabolites, thisnew method does not require an overnight enzymatic hydrolosisstep and is a much more rapid method of analysis. The newlydeveloped procedure involves solid phase extraction of pyrenemetabolites followed by separation using HPLC with a phenylmodified reverse phase column and an acidic buffer and acetonitrilegradient elution system. Metabolites were detected using a fluorescencedetector with wavelength conditions optimized for each metabolite.This method resulted in baseline separation of the glucuronicacid (1-OH P-GlcUA) and sulfate conjugate (1-OH P-Sul) of 1-hydroxypyreneand free 1-hydroxypyrene (1-OH P). The potential of this methodfor use in monitoring human exposure to mixtures of PAHs wasevaluated by analyzing urine obtained from five individualsworking in a coal gasification plant. 1-OH P-GlcUA was detectedas the major metabolite in the urine of all the five workers.This metabolite accounted for 80–100% of the total pyrenemetabolites excreted in urine. 1-OH P-GlcUA levels ranged from0.31–0.94 µg/g creatinine. Low levels of the sulfateconjugate (0.002–0.06 µg/g creatinine) were detectedin four of the samples while free 1-hydroxypyrene (0.07–0.2.µg/g creatinine) was detected in two of the five urinesamples. Urine from occupationally exposed workers was alsoanalyzed for 1-hydroxypyrene following enzymatic hydrolysisusing the standard approach. Levels of 1-hydroxypyrene rangedfrom 0.51–1.17 µg/g creatinine. Comparison of thefluorescence intensities of 1-OH P-GlcUA and 1-OH P-Sul to 1-hydroxypyrenedemonstrated that the glucuronide conjugate is 3-fold more fluorescentand the sulfate conjugate is 4-fold more fluorescent than 1-hydroxypyrene.These results indicate that conjugates of pyrene, specifically,1-OH P-GlcUA can potentially be used as a more sensitive biomarkerof exposure to PAHs.     

Effect of early allograft dysfunction on outcomes following liver transplantation

Early allograft dysfunction (EAD) following liver transplantation (LT) remains a challenge for patients and clinicians. We retrospectively analyzed the effect of pre‐defined EAD on outcomes in a 10‐year cohort of deceased‐donor LT recipients with clearly defined exclusion criteria. EAD was defined by at least one of the following: AST or ALT >2000 IU/L within first‐week post‐LT, total bilirubin ≥10 mg/dL, and/or INR ≥1.6 on post‐operative day 7. Ten patients developed primary graft failure and were analyzed separately. EAD occurred in 86 (36%) recipients in a final cohort of 239 patients. In univariate and multivariate analyses, EAD was significantly associated with mechanical ventilation ≥2 days or death on days 0, 1, PACU/SICU stay >2 days or death on days 0‐2 and renal failure (RF) at time of hospital discharge (all P<.05). EAD was also significantly associated with higher one‐year graft loss in both uni‐ and multivariate Cox hazard analyses (P=.0203 and .0248, respectively). There was no difference in patient mortality between groups in either of the Cox proportional hazard models. In conclusion, we observed significant effects of EAD on short‐term post‐LT outcomes and lower graft survival.     

Isoprenoids responsible for protein prenylation modulate the biological effects of statins on pancreatic cancer cells

Background

Statin treatment of hypercholesterolemia is accompanied also with depletion of the mevalonate intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) necessary for proper function of small GTPases. These include Ras proteins, prevalently mutated in pancreatic cancer. In our study, we evaluated the effect of three key intermediates of the mevalonate pathway on GFP-K-Ras protein localization and the gene expression profile in pancreatic cancer cells after exposure to individual statins.

Methods

These effects were tested on MiaPaCa-2 human pancreatic cancer cells carrying a K-Ras activating mutation (G12C) after exposure to individual statins (20 μM). The effect of statins (atorvastatin, lovastatin, simvastatin, fluvastatin, cerivastatin, rosuvastatin, and pitavastatin) and mevalonate intermediates on GFP-K-Ras protein translocation was analyzed using fluorescence microscopy. The changes in gene expression induced in MiaPaCa-2 cells treated with simvastatin, FPP, GGPP, and their combinations with simvastatin were examined by whole genome DNA microarray analysis.

Results

All tested statins efficiently inhibited K-Ras protein trafficking from cytoplasm to the cell membrane of the MiaPaCa-2 cells. The inhibitory effect of statins on GFP-K-Ras protein trafficking was partially prevented by addition of any of the mevalonate pathway’s intermediates tested. Expressions of genes involved in metabolic and signaling pathways modulated by simvastatin treatment was normalized by the concurrent addition of FPP or GGPP. K-Ras protein trafficking within the pancreatic cancer cells is effectively inhibited by the majority of statins; the inhibition is eliminated by isoprenoid intermediates of the mevalonate pathway.

Conclusions

Our data indicate that the anticancer effects of statins observed in numerous studies to a large extent are mediated through isoprenoid intermediates of the mevalonate pathway, as they influence expression of genes involved in multiple intracellular pathways.

     

Development and pre-clinical analysis of a Plasmodium falciparum Merozoite Surface Protein-1(42) malaria vaccine

Merozoite Surface Protein-1(42) (MSP-1(42)) is a leading vaccine candidate against erythrocytic malaria parasites. We cloned and expressed Plasmodium falciparum MSP-1(42) (3D7 clone) in Escherichia coli. The antigen was purified to greater than 95% homogeneity by using nickel-, Q- and carboxy-methyl (CM)-substituted resins. The final product, designated Falciparum Merozoite Protein-1 (FMP1), had endotoxin levels significantly lower than FDA standards. It was structurally correct based on binding conformation-dependent mAbs, and was stable. Functional antibodies from rabbits vaccinated with FMP1 in Freund's adjuvant inhibited parasite growth in vitro and also inhibited secondary processing of MSP-1(42). FMP1 formulated with GlaxoSmithKline Biologicals (GSK) adjuvant, AS02A or alum was safe and immunogenic in rhesus (Macaca mulatta) monkeys.     

Top 3 Differentials in Pediatric Radiology: A Case Review – Rebecca Stein-Wexler (ed)

Indian Journal of Pediatrics -     

Individuelle Gesundheitskompetenz von Lehrkräften und deren (Un)Sicherheit im Umgang mit chronisch erkrankten Schulkindern und Notfallsituationen

Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz - Gesundheitskompetenz (GK) gilt als eine Schlüsseldeterminante im Hinblick auf die Gesundheitssicherung. Die Förderung...