A stable-isotope HPLC-MS/MS method to simplify storage of human whole blood samples for glutathione assay.

BACKGROUND: Glutathione is the principal non-protein tripeptide thiol present in most mammalian cells and plays an important role in the redox status of biological systems. The accurate assessment of reduced glutathione (GSH) status as a reliable index of oxidative stress is of research and clinical significance. GSH undergoes rapid oxidation after sample collection and this presents a challenge. METHODS: Validation of an HPLC-MS/MS assay is reported. Storage stability using four variants of a methanolic precipitation with addition of stable isotope internal standard at collection is compared to L-serine borate/EDTA with perchloric acid precipitation (SBPE). RESULTS: Precipitation with methanol and addition of stable isotope on sample collection, combined with storage in solution at -70 °C showed superior storage stability to SBPE and other variants of the methanolic precipitation method up to 99 days. CONCLUSIONS: The combination of stable isotope with methanolic precipitation at collection, with assay by HPLC-MS/MS provides superior results after storage of whole blood samples for at least 99 days.

Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse.

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24h. The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity. Conversely, pre-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action.