Erythrocyte Glutathione Depletion Is Associated with Severity of Anemia and Pulmonary Hypertension in Patients with Sickle Cell Disease.

Background: Glutathione (GSH) is the most abundant intracellular non-protein thiol and is an anti-oxidant present in high millimolar concentration in erythrocytes. Additionally, GSH plays an important role in signal transduction, gene expression, apoptosis, protein glutathionylation, nitric oxide metabolism, and is the principal thiol redox buffer in erythrocytes. We hypothesized that, in sickle cell disease (SCD), GSH deficiency contributes to oxidative stress and pre-disposes the sickle erythrocyte to hemolysis.

Methods: A sensitive liquid chromatography coupled to tandem mass spectrometric technique was used to examine the availability of GSH and its precursors (glutamate, cysteine, glycine and glutamine) in plasma and within the erythrocytes of 25 patients with SCD and 7 ethnically-matched normal volunteers. Given the association of hemolysis with pulmonary artery hypertension (PAH) in this population, tricuspid regurgitant jet velocity (TRV) by Doppler echocardiography was also determined in all patients.

Results: Despite an increase in the amino acid precursors of GSH in the plasma of SCD patients compared with controls, total plasma GSH was significantly decreased (2.8 ± 1.5 vs. 4.1 ± 2.1 mM, p<0.05, SCD vs. controls). Additionally, a striking 61% depletion of GSH was found within the erythrocytes of SCD patients compared with normal volunteers (308.1 ± 112 vs. 790.8 ± 292 mM, p<0.0001). Erythrocyte glutamine concentrations were also decreased in SCD patients compared with controls (404.6 ± 252 vs. 1061 ± 277 mM, p<0.0001), while cysteine, glycine and glutamate levels trend higher. This indicates a potential loss of GSH synthesis capacity in SCD patients. The degree of erythrocyte GSH depletion correlated strongly with TRV (r = −0.64, p<0.0001), which is of significant clinical interest given the increased mortality risk of PAH in patients with SCD. While erythrocyte glutamine concentration also correlated inversely with TRV (r = −0.59, p < 0.001), erythrocyte glutamate and cysteine levels as well as plasma GSH and its precursors were not associated with TRV. Erythrocyte GSH depletion strongly associated with hematocrit (r = 0.60, p<0.001), suggesting a link to chronic anemia. Plasma arginase concentration strongly correlated with plasma-free Hb (r=0.83, p<0.0001) and inversely correlated with erythrocyte GSH (r=−0.41, p=0.02), linking lower erythrocyte GSH levels to increased red cell derived plasma arginase and therefore potentially an increased risk of mortality in SCD & PAH as we have previously reported (Morris et al, JAMA 2005).

Conclusions: Lower levels of erythrocyte GSH may contribute to the development of PAH possibly by exacerbating oxidative stress and triggering hemolysis, resulting in release of erythrocyte arginase and dysregulation of arginine and NO metabolism. The role of GSH metabolism in the pathophysiology of SCD and its link to PAH warrants further investigation.