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Quantification of reduced glutathione by HPLC‐UV in erythrocytes of hemodialysis patients
Author(s) -
Garcia Solange Cristina,
Schott Karen,
Charão Mariele,
Moro Angela,
Bulcão Rachel,
Grotto Denise,
Valentini Juliana,
Bohrer Denise,
Cardoso Simone,
Pomblum Valdeci
Publication year - 2008
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.954
Subject(s) - chemistry , chromatography , trichloroacetic acid , glutathione , high performance liquid chromatography , gradient elution , hemodialysis , phosphate buffered saline , elution , biochemistry , surgery , enzyme , medicine
Reduced glutathione (GSH) is a well‐known multifunctional antioxidant. Its depletion is linked to a number of pathologies, such as renal insufficiency. Feasible methodologies in clinical chemistry are vital. Therefore a methodology for GSH quantification was optimized and validated by HPLC‐UV. Important aspects such as acid deproteinization and GSH stability were established. The erythrocytes were hemolyzed, deproteinized, derivatized with 5,5‐dithio‐bis (2‐nitrobenzoic) acid and analyzed using HPLC, on an RP 18 gradient elution, λ = 330 nm. The method was applied to hemodialysis patients ( n = 75) compared with healthy subjects ( n = 40). The assay was linear from 0.5 to 3.0 m m ( r 2 > 0.99). The intra‐ and inter‐run reproducibilities were obtained with CV% < 10%. The accuracy (bias %) ranged from 1.32 to –6.38%, and the recovery was >94%. The derivatized sample was stable for 60 days at –20°C. The GSH levels in hemodialysis patients showed a significant increase compared with healthy subjects ( p < 0.05) and an inverse correlation with age ( r = –0.286; p = 0.013) was found. This method used UV detection, reduction of the phosphate concentration in the mobile phase and effective protein removal with trichloroacetic acid. The method proved to be reproducible, precise, accurate and stable. Thus, it can be suggested for routine laboratory tests for the verification of physiopathological conditions. Copyright © 2007 John Wiley & Sons, Ltd.