An Assay of Total Glutathione and Glutathione Disulfide in Human Whole Blood and Plasma Using a High-Performance Liquid Chromatography With Fluorescence Detection

Abstract

& A method is described here for the determination of total glutathione and glutathione disulfide in human whole blood and plasma with a suitable sample preparation procedure to prevent glutathione oxidation and glutathione disulfide hydrolysis. Whole blood samples were obtained from a group of blood donors. After adequate sample preparation, the samples were derivatized with ortho-phthaldialdehyde to form a stable, highly fluorescent tricyclic derivative. Reverse-phase column chromatography was used for the separation and fluorimetric detection to monitor the effluent. The analytical performance of this method was satisfactory for the determination of total glutathione and glutathione disulfide. The intra-assay and inter-assay coefficients of variation were below 10%. The recoveries were as follows: 96.1% (CV 1.4%) and 104.3% (CV 2.4%) for whole blood, 92.3% (CV 2.4%) and 107.4% (CV 2.6%) for plasma. We found no significant differences in both total glutathione and glutathione disulfide concentration between the women and men. We developed a selective high-performance liquid chromatography method for the determination of total glutathione and glutathione disulfide in human whole blood and plasma. Our sophisticated sample preparation procedure prevents significant glutathione oxidation and glutathione disulfide hydrolysis.

The ratio of oxidized and reduced forms of selected antioxidants as a possible marker of oxidative stress in humans

“…GSSG is only stable when using metaphosphoric acid with NEM, but for a maximum of 1 h; the use of other acids, particularly PCA, causes rapid decrease of GSSG. It is reported that GSH–NEM is unstable and excess of NEM may result in loss of GSSG (Kanďár et al ., ). On the other hand, it is stated that NEM blocks the glutathione reductase (GR, EC 1.8.1.7), thereby preventing backward GSSG reduction to GSH (Floreani et al ., ).…”

“…It is also possible that, at room temperature, the enzyme activity of GGT is sufficient to catalyse peptide bond hydrolysis between glutamate and cysteine. That could explain the sharp decline of TGSH during 1 h at room temperature and the only slight decline at 4°C (Kanďár et al ., ). Appropriate derivatization with a suitable reagent is needed to increase sensitivity, and also the selectivity.…”

“…In the first case, we determine TGSH, and in the second case, GSSG. This is a suitable method to accurately determine GSSG (Kanďár et al ., ). One can say that similarly to in the case of ascorbic acid determination by HPLC with UV detection, GSH is also much more stable in whole blood than in the supernatant.…”

“…GSH is stable in whole blood stored at −80°C for several months and in the supernatant at 4°C for at least 10 h; and GSH‐OPA derivative at 4°C is stable for more than 24 h. The reason for speedy derivatization after deproteinization is that GSSG in the supernatant is only stable for about 1 h at 4°C. As mentioned above, NEM may be the cause (Kanďár et al ., ).…”

O-Phthalaldehyde Derivatization for the Paper-Based Fluorometric Determination of Glutathione in Nutritional Supplements

“…Under the optimu conditions, a "cumulative" regression equation was obtained by assimilating the resu from 40 standard solutions analyzed in different working days (n = 8), setting t calibration curve more representative by embracing any potential variations. Additionally, the within-day precision was calculated at two GSH concentrati values, 100 and 350 µmol L −1 , by repetitious measurements of different paper devices ( 5). The intra-day relative standard deviation (RSD) was 6.2% and 3.1%, respectively, these two concentration values, while the inter-day RSD was 7.3% and 4.4%, respective For both intra-day and inter-day precision experiments, de-ionized water was utilized the solvent for standard solutions.…”