Quantification of reduced glutathione by HPLC‐UV in erythrocytes of hemodialysis patients

Abstract

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 RP18 gradient elution, lambda=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 mm (r2>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 degrees 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.

Ultraviolet derivatization of low-molecular-mass thiols for high performance liquid chromatography and capillary electrophoresis analysis

“…4J-L), invented by Ellman in 1959 [108], 5,5-dithio-bis-2-nitrobenzoic acid (DTNB, Fig. 4L) is still in use in liquid phase separation methods [109][110][111][112]. This symmetric aryl disulfide, reacts with reduced thiol to produce a mixed disulfide (RS-TNB) and one equivalent of 5-thio-2-nitrobenzoate (TNB) anion (Fig.…”

Chromatographic and mass spectrometric analysis of glutathione in biological samples

“…Recently, Garcia et al reported a DTNB-based HPLC-UV method for erythrocytes [142]. Reed et al [143] developed a new method which allows the simultaneous determination of GSH and GSSG by blocking free thiols with IAA and derivatizing amino groups by 2,4-dinitrofluorobenzene (FDNB).…”

Treatment with N-acetylcysteine does not alter blood glucose levels and the oxidative stress status in diabetic rats

“…In this sense, the measurement of plasmatic GSH would not reflect the real GSH status, once red blood cells represent 99.5% of the circulating GSH, being responsible for the erythrocyte homeostasis against oxidative damage. [25,42,43] All these aspects could be involved in the decrease of erythrocyte GSH levels and consequently high levels of MDA, in spite of NAC treatment.…”

“…[25] GSH levels were expressed as micromole per gram of hemoglobin (μmol/g Hb). Malondialdehyde (MDA) assay was determined in plasma-EDTA by HPLC.…”