Micro-method for the determination of glutathione in human blood

Abstract

A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 μL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5'-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with -1.4% to 2.3% intra-day relative error (RE) and -2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.

Keywords: Blood; DTNB; Fingerstick; Glutathione; Oxidative stress.

Fig. 1

Spectrophotometric analysis of the GSH content of a 2-μL aliquot of human blood. Kinetic analysis of TNB appearance resulting from the reaction of DTNB reagent with either a 2 μL whole blood sample (upper line) or acidified stabilizing solution (blank sample, lower line), as detailed in the Experimental section.

Fig. 2

Stability of GSH in blood. Three milliliter aliquots of whole blood (downward triangle), haemolyzed blood (1:300, v/v, dilution; white circle) and deproteinized supernatant of haemolyzed blood (dark circle) were stored at 4°C for 96 h, and GSH concentration was analyzed in aliquots at the indicated time-points by spectrophotometry. Blood was obtained from healthy donors by venipuncture. Each point represents the mean ± SD of four analyses.

Figure 3

Bland-Altman plot of GSH as measured by the present DTNB-based spectrophotometric method of GSH in fingerstick capillary blood from 23 healthy subjects and by a reference HPLC method for GSH in NEM-spiked peripheral blood from the same subjects using a HPLC method, as described in Experimental section.