Disruption of glutathione homeostasis causes accumulation of S-glutathionyl proteins in response to exposure to reactive oxygen species in human erythrocytes.

Oxidation of protein cysteine residues by disulfide bond formation with glutathione (GSH) is a reversible posttranslational modification following oxidative stress. Although S-glutathionylation seems to play a key role in cellular regulation and protect protein thiols from hyperoxidation, the molecular mechanism that mediates the glutathionyl protein is still unclear. We investigated the effect of disrupting GSH homeostasis on the S-glutathionylation of proteins via exposure to tert-butyl hydroperoxide (BHP) to study the formation of glutathionyl protein in human red blood cells (RBCs). Two independent treatments aimed at disrupting GSH homeostasis were devised to examine the influences of S-glutathionylation on RBC proteins. Glutathionyl proteins were detected transiently in intact RBCs during BHP exposure. Although glutathionyl proteins (220-240 kDa) disappeared immediately in the presence of glucose, they remained for a long time after BHP exposure in RBCs when the GSH-dependent system was disrupted. Furthermore, we identified that the high molecular weight glutathionyl protein is erythroid spectrin using immunodetection. Thus, it was indicated that the protein-bound GSH produced by peroxide exposure is immediately released by the GSH-dependent system in normal RBCs. However, disruption of GSH homeostasis causes accumulation of the glutathionyl protein. We propose that S-glutathionyl spectrin may be a useful biomarker for dysfunctions in GSH homeostasis and oxidative stress in human RBCs.

Determination of reduced nicotinamide adenine dinucleotide phosphate concentration using high-performance liquid chromatography with fluorescence detection: ratio of the reduced form as a biomarker of oxidative stress.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is the principal source of reducing power in numerous processes of physiological importance. We examined the influence of oxidative stress on the relative amounts of NADPH in human red blood cells (RBCs). To determine the homeostasis of the NADPH existing in the reduced form following oxidation, we developed an improved method for measurement of NADPH in human RBCs using high-performance liquid chromatography (HPLC). The present method with fluorescent detection is reproducible and selective than enzymatic cycling method and HPLC methods with spectrometric detection. The calibration curve is linear over the range of 0.1-5.0 muM with a correlation coefficient of 0.999. The within-run precision of the assays for total NADPH (NADPH+NADP(+)) and NADPH concentrations in human RBC were 2.4% and 8.6%, respectively (n=5). After the RBC suspension was exposed to tert-butyl hydroperoxide (t-BHP), which is scavenged by glutathione peroxidase (GPX) along with NADPH consumption, a significant decrease in the NADPH ratio [(NADPH/(NADPH+NADP(+))] was observed after a transient decrease and rapid recovery of the reduced form of glutathione. The marked decrease in the NADPH ratio induced by t-BHP exposure was observed in the absence of glucose. However, the NADPH ratio was not decreased by t-BHP exposure after pre-treatment with a glutathione reductase inhibitor. This method may be useful for the measurement of small amounts of NADPH from various biological sources.

Pyruvate kinase is protected by glutathione-dependent redox balance in human red blood cells exposed to reactive oxygen species.

To determine the antioxidant role of glutathione (GSH) in human red blood cells (RBCs), we investigated the effect of disrupting GSH homeostasis on the oxidative modification of thiol-dependent enzymes by exposure to tert-butyl hydroperoxide (BHP). When hemolysate was incubated with BHP, significant decreases in enzyme activity were observed. However, the inactivation did not occur in intact RBC suspensions that were exposed to BHP. In this study, we used two independent treatments aimed at decreasing the level of reduced form of GSH, pre-incubation with a glutathione reductase inhibitor or glucose-free medium to examine the influences of preventing GSH-dependent antioxidant and reactivation activity on thiol-dependent enzyme. Pyruvate kinase (PK) activity clearly decreased along with depletion of GSH compared to other glycolytic enzyme activities by BHP exposure in RBCs. The addition of GSH, but not glucose, before BHP exposure completely prevented the inactivation of PK in hemolysate; however, partial reactivation of inactivated PK was observed by post-addition of both GSH and glutaredoxin at an early stage during BHP exposure. Moreover, hydroxyl radicals but not hydrogen peroxide inactivated PK. These results suggest that PK is highly susceptible to radicals and that GSH is essential to protect PK activity by not only directly scavenging radicals but also by systematically reactivating oxidized enzyme in human RBCs.

Glucose metabolism is accelerated by exposure to t-butylhydroperoxide during NADH consumption in human erythrocytes.

Several mechanisms have been proposed to underlie the events that occur during oxidative damage in red blood cells (RBCs) exposed to reactive oxygen species. This work explores what happens when metabolites related to redox regulation in human RBCs are oxidized to form alkoxyl radical and peroxyl radical as a result of exposure to tert-buthylhydroperoxide (BHP). During exposure to BHP, the glutathione level and the ratio of NADPH to total nicotinamide adenine dinucleotide phosphate (NADPH plus NADP(+)) were significantly decreased. Although alteration in the concentration of monosaccharides metabolized in the pentose phosphate pathway (PPP) was not observed, exposing RBCs to BHP caused the formation of methemoglobin (metHb) and a significant decrease in NADH. Moreover, we detected a significant increase in one of the peaks during BHP exposure by using HPLC with dansyl hydrazine as a prelabel reagent. A complete enzymatic conversion procedure was used to identify the peak as pyruvate based on comparison with standards. These results suggest that the rapid recovery in the level of glutathione and the formation of metHb by BHP require NADPH and NADH consumption. Subsequently, glucose metabolism accelerates to reproduce NADPH and NADH, which results in pyruvate accumulation. Our findings indicate that the level of pyruvate markedly increases upon exposure to a radical-generating oxidant capable of forming metHb. Methemoglobin reductase requires NADH as a co-factor, and oxidized form (NHADP(+)) is reduced via the glycolytic reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase. Thus, the overall acceleration of glycolysis induced by BHP is strongly dependent on the NADH reproducing pathway. In addition, the decrease in NADH enhances the increase in pyruvate by inhibiting the conversion of pyruvate to lactate in the presence of lactate dehydrogenase.