The Effects of Glutamine Supplementation on Markers of Autophagy and Apoptosis in Peripheral Blood Mononuclear Cells from Patients with Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is a hemoglobinopathy associated with an increased risk of pulmonary hypertension (PH) due to a number of mechanisms that includes iron overload, hemolysis, erythrocyte-derived arginase (which limits both nitric oxide and arginine bioavailability), functional splenectomy, and a hypercoagulable state among others. Glutathione (GSH, and its oxidized pair glutathione disulfide GSSG) is the principal thiol redox buffer in erythrocytes, which has been linked to hemolysis when depleted. Glutamine is not only a precursor to GSH, but also plays an anti-oxidant role through preservation of the intracellular nicotinamide adenine dinucleotide (NAD) levels, required for reducing GSSG back to GSH, thus decreasing the risk for hemolysis. Low erythrocyte glutamine levels are associated with risk of PH as defined by a tricuspid regurgitant jet velocity of (TRV) ≥2.5 m/s measured by Doppler echocardiography. SCD also exhibits an elevated level of circulating leukocytes, known as leukocytosis, which may contribute to vascular occlusion. The mechanism by which leukocytosis occurs is currently unknown. However, leukocyte cell death can be informative on the regulation of leukocyte cell numbers and measurement of mitochondrial BAX and caspase 9, are classic indicators of an active intrinsic cell death pathway. Autophagy is responsible for the turnover of macromolecules and organelles via the lysosomal degradative pathway. In this pathway, LC3 is important for the maturation and transport of autophagosomes and therefore, a reflection of autophagic activity. Autophagy ensures cell survival under certain conditions of nutrient deprivation or growth factor withdrawal and has also been implicated in innate and adaptive immune responses. In this study, the mitochondrial apoptotic marker BAX and the autophagy marker LC3 were examined in a SCD trial of glutamine therapy in patients at risk for PH.

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples taken from SCD (n=13) and control patients (n=7) and BAX and LC3 were measured via western blot analysis. Western blot results were evaluated via densitometry. SCD patients with PH-risk were treated with oral L-glutamine supplementation (10 mg TID) with the objective of estimating the level of cell death and autophagy proteins in circulating PBMCs from SCD patients at baseline and after glutamine supplementation. SCD patients were sampled at baseline (BL),and then at two weeks (W2), four weeks (W4), six weeks (W6), and eight weeks (W8) during the glutamine therapy.

Results: Mean age for patients with SCD was 46±14; 39% were male with 54% having Hb-SS, while 46% had Hb-SC. Mean TRV was 3.0±0.6 m/s. The mean age for controls was 32± 12 and 57% were male; all controls were Hb-AA with a mean TRV of 1.8±0.6. At baseline there was no statistical difference in BAX expression between control and SCD patients. In comparison to baseline, however, supplementation with glutamine in SCD patients resulted in significantly increased expression of BAX in PMBCs by 15% over the 8 weeks of therapy; potentially indicating a restorative effect of glutamine on the intrinsic mitochondrial apoptotic pathway, which may ultimately reduce leukocytosis. In contrast, glutamine supplementation over 8 weeks, significantly reduced LC3 expression by 42% in PBMCs, suggesting a decrease in cellular autophagy, thus reducing the ability for PBMCs to remain in circulation.

Conclusions: At baseline there was no difference in BAX expression between control and SCD patients, however, after 8 weeks of glutamine supplementation, PBMCs had an increased BAX expression and a decreased LC3 expression. This suggests that PBMCs from glutamine supplemented SCD patients may lose their ability to remain in circulation via apoptosis upregulation and autophagy downregulation