title: A sensitive method for the analysis of glutathione in porcine hepatocytes.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Surgical Research, Institute of Clinical Medicine, University of Tromsø , Tromsø , Norway.
authors.forename: Åse
authors.initials: Å
authors.lastname: Florholmen-Kjær
authors.forename: Roy Andre
authors.initials: RA
authors.lastname: Lyså
authors.forename: Ole-Martin
authors.initials: OM
authors.lastname: Fuskevåg
authors.forename: Rasmus
authors.initials: R
authors.lastname: Goll
authors.forename: Arthur
authors.initials: A
authors.lastname: Revhaug
authors.forename: Kim Erlend
authors.initials: KE
authors.lastname: Mortensen
mesh_terms: D000818:Animals; D004195:Disease Models, Animal; D005260:Female; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism*; D008107:Liver Diseases / Q000175:diagnosis* / Q000378:metabolism; D018384:Oxidative Stress; D012372:ROC Curve; D015203:Reproducibility of Results; D013552:Swine; D053719:Tandem Mass Spectrometry
chemical_list: D005978:Glutathione
keywords: HPLC; N-ethylmaleimide; cell suspension; glutathione; hepatocyte; liver; porcine; soft tissue; tandem mass spectrometry