Glutathione Stability in Whole Blood: Effects of Various Deproteinizing Acids

Abstract

High-performance liquid chromatography separation of reduced and oxidized glutathione (GSH and GSSG) in biologic samples using electrochemical detection offers the convenience of both simultaneous quantitation and simple sample preparation. Rapid acidification is required to prevent GSH autooxidation, GSH and GSSG degradation, and precipitate proteins that interfere with analysis. Currently, little consistency exists in the literature regarding acid selection or the feasibility of sample storage before analysis. The purpose of this work was to examine the effects of perchloric (PCA), trichloroacetic (TCA), metaphosphoric (MPA), and 5-sulfosalicylic (SSA) acids on the short-term stability of GSH and GSSG measurements in whole blood. Samples were collected from adult volunteers and treated with multiple concentrations of each acid. The samples were analyzed immediately and aliquots were stored at -80 degrees C for up to 28 days. The suitability of each acid was assessed by percentage change of GSH and GSSG from baseline, efficiency of protein removal, and alteration of chromatogram characteristics. In general, increasing the acid concentration improved sample stability. Nevertheless, SSA did not achieve acceptable sample stability at any concentration tested. MPA was found to leave substantial amounts of protein in the samples, and TCA may interfere with the peaks of interest. Based on these results, a final concentration of 15% PCA is suggested for analysis of glutathione in whole blood. Although immediate sample preparation is preferred, 15% PCA can maintain sample integrity for 4 weeks after storage at -80 degrees C.

Functional Expression of the Multidrug Resistance Protein 1 in Microglia

“…This was a greater decrease than previously reported for MRP1-over-expressing cells treated in a similar manner, where intracellular GSH was depleted by 80 -87% (Feller et al, 1995;Zaman et al, 1995). We used a highly sensitive and specific HPLC assay coupled to electrochemical detection, which we have recently shown simultaneously detects GSH and other nonprotein thiols (Stempak et al, 2001). In BSOtreated MLS-9 cells, vincristine accumulation consistently increased at several time intervals, suggesting that GSHdependent drug efflux was decreased.…”

“…The R2 sensitivity (gain range) for E2 was set at 50 to 100 A. GSH peak height values were corrected per 1 million cells and BSO-or GSH-treated cells were compared with control cells and expressed as a percent value. The intraday and interday variability of the assay is less than 10% (Stempak et al, 2001 …”

“…Intracellular concentrations of GSH after GSH depletion (BSO-treated) or augmentation (GSH-treated) were determined using a slightly modified HPLC method previously described and validated by our group (Stempak et al, 2001). MLS-9 and CH R C5 cells were cultured to ϳ90% confluence in 75-cm 2 tissue culture flasks as described above.…”

Influência de desproteinizantes ácidos na quantificação da glutationa reduzida eritrocitária por CLAE-UV

“…Apesar das desvantagens descritas em alguns trabalhos sobre a utilização de ácidos para remover proteínas de amostras complexas, este procedimento é de simples execução e ainda muito utilizado, uma vez comprovada sua eficiên-cia. A avaliação sistemática do uso do ácido desproteinizante é importante porque proteínas residuais podem interferir na análise, além de degradar o sistema cromatográfico 17,18 . Com o objetivo de evitar interferência com resultados artefatuais de GSH 7,11 e dano ao sistema cromatográfico, avaliou-se a eficiência desproteinizante dos ácidos em várias concentrações.…”

“…Observou-se que soluções contendo MPA 5 a 50%, SSA 5 a 10% e TCA 5 a 10% utilizadas para desproteinização de eritrócitos resultaram em sobrenadantes não límpidos, insatisfatórios para as análises cromatográficas. Os resultados obtidos no presente trabalho referentes à eficiência do MPA na desproteinização podem ser associados ao estudo realizado por Stempak et al 17 que demonstraram que 1 volume de MPA 5 a 15% foi insuficiente para remover proteínas em 1 volume de amostras de sangue total. Recentemente, o efeito pró-oxidante do MPA em amostras contendo eritrócitos foi demonstrado através da quantificação de GSH e GSSG por eletroforese capilar, onde foi observada perda de GSH por oxidação a GSSG quando comparada à desproteinização realizada com o solvente orgânico acetonitrila 19 .…”