Low Erythrocyte Glutamine-to-Glutamate Ratio: A Novel Biomarker of Hemolysis and Pulmonary Hypertension in Sickle Cell Disease.
Authors: Morris Claudia R. , Suh Jung , Hagar Ward , Larkin Sandra , Bland D. Anton , Steinberg Martin , Vichinsky Elliott , Shigenaga Mark , Ames Bruce , Kuypers Frans , Klings Elizabeth 
Published: April 2020

Abstract
The erythrocyte redox environment may contribute to increased hemolysis and decreased nitric oxide (NO) bioavailability in pulmonary hypertension (PH) of sickle cell disease (SCD). Glutathione (GSH) is the principal thiol redox buffer in erythocytes and its depletion has been linked to hemolysis. Glutamine plays an additional anti-oxidant role through preservation of the intracellular nicotinamide adenine dinucleotide (NAD) levels, required for reducing GSSG back to GSH. We hypothesized that altered GSH and glutamine metabolism promotes hemolysis and contributes to PH in SCD. Glutamine, total glutathione (GSH+GSSG) and its precursors (glutamate, cysteine, glycine) were assayed in plasma and erythrocytes of 40 SCD patients and 9 controls. PH is defined by echocardiogram as a tricuspid regurgitant jet velocity (TRV) ≥ 2.5m/s. Total plasma glutathione was lower in SCD vs control patients (2.7 ± 0.3 μM vs. 4.1± 0.8 μM, p&lt;0.05). Similarly, total erythrocyte glutathione levels were decreased in SCD vs. control patients (310 ± 26 μM vs. 683 ± 110 μM, p&lt;0.0001). A trend towards higher GSH precursor levels identified in plasma and erythrocyte compartments suggests that the total glutathione (GSH+GSSG) deficit is due to heightened rate of GSH utilization rather than decreased synthesis capacity. While severity of erythrocyte GSH depletion was similar in SCD patients with and without PH, erythrocyte glutamine levels differed significantly (482±92μM, n=17 vs 934±134μM, n=23, p&lt;0.02) and values inversely correlated to TRV (r = −0.51, p&lt;0.0001). As glutamine is required for de novo synthesis of NAD(P)+ essential for GSH recycling, lower steady-state glutamine levels may reflect enhanced GSH utilization rates in the SCD erythrocytes. A significant reduction in the erythrocyte glutamine:glutamate ratio occurred in SCD patients compared with normal volunteers, with the lowest ratios observed in SCD patients with PH. The glutamine:glutamate ratio, potentially a gauge of NADPH biosynthesis and oxidative stress, was inversely correlated with TRV (r = −0.62, p&lt;0.001), implicating glutamine bioavailability as a novel factor in the pathophysiology of PH. Changes in the glutamine:glutamate ratio were predominantly caused by decreased erythrocyte glutamine levels rather than increased glutamate levels, ruling out an effect on the ratio from increased cellular glutamine uptake. Erythrocyte glutamine:glutamate ratio correlated with age in patients with SCD (r = −0.33, p=0.04), and inversely correlated with plasma arginase concentratoin (r= −0.45, p=0.012), and plasma-Hb (r= −0.41, p=0.01), linking lower glutamine bioavailability to increased red cell derived plasma arginase, hemolysis and potentially with increased mortality in PH of SCD as previously reported (Morris et al, JAMA 2005). Decreased erythrocyte total glutathione and glutamine levels contribute to alterations in the erythrocyte redox environment, which compromise erythrocyte integrity and NO bioavailability and may play a role in hemolysis and the pathogenesis of PH of SCD.