The impact of partial hepatectomy on oxidative state in the liver remnant - An in vivo swine model.

BACKGROUND: Previous studies on oxidative state after partial hepatectomy (PHx) report conflicting data on levels of glutathione (GSH) and are mainly presented in rodent models by methodology less sensitive than the present technologies. The current swine model presents GSH levels and the following genetic response post-PHx, utilizing an analytical platform more sensitive and precise than earlier available. METHOD: Twelve pigs were randomized to a PHx- and a control group (n=6 in each). The PHx group had a 60% hepatectomy. Serial in vivo liver biopsies during 12h of anaesthesia post-PHx were analyzed for GSH by liquid chromatography mass spectrometry (LC-MS/MS). Transcriptional alterations of genes (GS, GCLM, GCLC, GR, HGF, NFE2L2, TGFß1) regulating GSH synthesis were measured by real-time PCR. RESULTS: No difference was detected between the GSH levels in the PHx- and the control group during the experiment (P=0.247). Still, decreased gene expression of GS (P=0.026) and NFE2L2 (P=0.014) the first nine hours, and a decrease of TGFß1 (P=0.029) the first seven hours post-PHx was seen in the liver remnant. CONCLUSION: The results show that the liver has an extended capacity to maintain GSH homeostasis during major stress and parenchymal loss, even at the early onset of such trauma. This observation was not explained by increased expression of key genes in GSH pathways. Consequently, the results indicate an inherent compensatory capacity to maintain GSH homeostasis in the reduced organ.

A sensitive method for the analysis of glutathione in porcine hepatocytes.

BACKGROUND: Reduced glutathione (γ-glutamylcysteinylglycine), GSH, is essential when protecting cells from oxidative stress and also an indicator of disease risk. Reported concentrations of GSH and its oxidized form, glutathione disulfide (GSSG), varies considerably, primarily due to the instability of GSH and various analytical methods. METHODS: We designed a sensitive method to analyze GSH and GSSG in porcine hepatocytes using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after stabilization with N-ethylmaleimide (NEM). This method includes stable isotope labeled internal standards and simple synthesis of labeled GSSG which commercial sources rarely offer. GSH and GSSG were analyzed in porcine liver biopsies giving a reference interval based on a large number of samples (26 pigs; 3 parallels). RESULTS: The LC-MS/MS results revealed excellent linearity for both GSH and GSSG, with interday coefficient of variation (%CV) for GSH-NEM and GSSG <10 %. Accuracy for recovery tests was between 95.6% and 106.7% (n = 3) for GSH and between 92.3% and 107.7% (n = 3) for GSSG. The limits of quantification were 0.1 µM for GSH-NEM and 0.08 µM for GSSG. The mean concentration of GSH was 3.5 (95% CI = 1.5-8.1) mmol/liter and of GSSG 0.0023 (95% CI = 0.0003-0.019) mmol/liter. CONCLUSION: For the first time GSH and GSSG are analyzed in porcine hepatocytes by LC-MS/MS yielding a reference level of GSH and GSSG. The method is reproducible in any laboratory with LC-MS/MS service and will probably be applicable in all soft tissues and cell suspensions, essentially with no modification.