Increased Efflux of Oxidized Glutathione (GSSG) Causes Glutathione Depletion in Sickle Erythrocytes.

Sickle cell disease (SCD) is characterized by increased oxidative stress playing an important role in the pathophysiology of hemolysis, vascular occlusion and organ damage in sickle cell patients. Sickle erythrocytes are both an important source and target of reactive oxygen species (ROS). Levels of both total and reduced form of glutathione (GSH), a major intracellular anti-oxidant, have been demonstrated to be decreased in sickle erythrocytes, despite the increased de novo synthesis of GSH in these cells. The mechanism leading to this depletion of intracellular glutathione in sickle erythrocytes is not known yet. After reaction with ROS, GSH is oxidized into its oxidized form (GSSG) and can be transported actively out of the erythrocyte. We questioned whether, during episodes of increased oxidative stress, GSSG efflux in sickle erythrocytes is higher than in normal erythrocytes.

Erythrocytes of 10 homozygous sickle cell patients and 9 race-matched healthy controls were stimulated with 2,3-dimethoxy-l,4-naphthoquinone (DMNQ), which induces intracellular ROS generation, and hydrogen peroxide (H₂O₂) to stimulate GSH consumption. Intra- and extracellular levels of GSH and GSSG were measured at baseline and after 210 minutes of DMNQ and H₂O₂ stimulation.

While both intra- and extracellular GSSG levels (μM) at baseline were comparable in sickle and control erythrocytes (14.5(11.5–22.7) vs. 14.3(11.6–16.3) and 0.05(0.00–0.19) vs. 0.07(0.00–0.20) respectively), GSSG levels were significantly higher in sickle erythrocytes after 210 minutes DMNQ stimulation (intracellular: 74.4(52.9–93.1) vs. 45.3(40.8–66.7),P=0.005; extracellular: 23.3(18.2–37.3) vs. 13.2(11.1–14.6),P=0.001) which suggests an increased generation of GSSG intracellularly and a resulting elevated efflux to the extracellular environment. These observations were confirmed with H₂O₂ stimulation of erythrocytes, showing that, while comparable at baseline, the GSSG levels were higher in sickle erythrocytes after 210 minutes stimulation (intracellular: 26.1(22.8–30.1) vs. 17.5(14.2–20.1),P=0.043; extracellular: 6.9(2.3–16.6) vs.1.2(0.6–1.6),P=0.008). In contrast to the control erythrocytes, where intracellular GSH levels remained unchanged, GSH levels decreased significantly in sickle erythrocytes during DMNQ stimulation, suggesting a limited anti-oxidative reserve capacity in SCD.

GSSG efflux in sickle erythrocytes is increased and results in net loss of intracellular glutathione, rendering sickle erythrocytes more susceptible to oxidative damage. The higher rate of GSH consumption during an episode of oxidative exacerbation in sickle erythrocytes suggests a reduced anti-oxidative reserve capacity in SCD.

No relevant conflicts of interest to declare.