The effects of glutamine supplementation on markers of apoptosis and autophagy in sickle cell disease peripheral blood mononuclear cells.

OBJECTIVES: L-Glutamine was FDA-approved for sickle cell disease (SCD) in 2017, yet the mechanism(s)-of-action are poorly understood. This study investigates the potential activation of autophagy as a previously unexplored mechanism-of-benefit. DESIGN: Prospective, open-label, 8-week, phase-2 trial of oral L-glutamine (10 g TID) in patients with SCD at risk for pulmonary hypertension identified by Doppler-echocardiography by an elevated tricuspid-regurgitant-jet-velocity (TRV)≥ 2.5 m/s. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples taken from SCD patients at baseline, two, four, six and eight weeks of glutamine therapy, and from controls at baseline; BAX (pro-apoptotic marker) and LC3-II/LC3-I (autophagy marker) were measured via western blot analysis to assess apoptosis and autophagy respectively. SETTING: Comprehensive SCD Center in Oakland, California. RESULTS: Patients with SCD (n = 8) had a mean age of 44 ± 16, 50% were male; 63% Hb-SS, and mean TRV= 3.1 ± 0.7 m/s. Controls' mean age (n = 5) was 32 ± 12% and 57% were male; all were Hb-AA with a mean TRV= 1.8 ± 0.6. At baseline, SCD-PBMCs had 2-times higher levels of BAX and LC3-I versus controls (both p = 0.03). Levels of BAX expression increased by 300% after 8-weeks of glutamine supplementation (p = 0.005); LC3-I protein levels decreased while LC3-II levels increased by 70%, giving a significant increase in the LC3-II/LC3-I ratio (p = 0.02). CONCLUSION: PBMCs from glutamine-supplemented SCD patients have upregulated apoptotic and autophagy proteins. The parallel increase in BAX and the LC3-II / LC3-I ratio with glutamine supplementation suggest a possible role of autophagic cell death. The increase in apoptotic markers provide insight into a possible mechanism used by peripheral PBMCs during glutamine supplementation in patients with SCD.

Implications for the metabolic fate of oral glutamine supplementation within plasma and erythrocytes of patients with sickle cell disease: A pharmacokinetics study.

OBJECTIVES: L-Glutamine is FDA-approved for sickle cell disease (SCD), yet the mechanism(s)-of-action are poorly understood. We performed a pharmacokinetics (pK) study to determine the metabolic fate of glutamine supplementation on plasma and erythrocyte amino acids in patients with SCD. DESIGN: A pK study was performed where patients with SCD fasting for > 8 h received oral L-glutamine (10 g). Blood was analyzed at baseline, 30/60/90 min/2/3/4/8 hrs. A standardized diet was administered to all participants at 3 established time-points (after 2/5/7hrs). A subset of patients also had pK studies performed without glutamine supplementation to follow normal diurnal fluctuations in amino acids. SETTING: Comprehensive SCD Center in Oakland, California RESULTS: Five patients with SCD were included, three of whom performed pK studies both with and without glutamine supplementation. Average age was 50.6 ± 5.6 years, 60% were female, 40% SS, 60% SC. Plasma glutamine levels increased significantly after oral glutamine supplementation, compared to minimal fluctuations with diet. Plasma glutamine concentration peaked within 30-min of ingestion (p = 0.01) before decreasing to a plateau by 2-h that remained higher than baseline by 8 h. Oral glutamine also increased plasma arginine concentration, which peaked by 4-h (p = 0.03) and remained elevated through 8-h. Erythrocyte glutamine levels began to increase by 8-h, while erythrocyte arginine concentration peaked at 4-h. CONCLUSIONS: Oral glutamine supplementation acutely improved glutamine and arginine bioavailability in both plasma and erythrocytes. This is the first study to demonstrate that glutamine therapy increases arginine bioavailability and may provide insight into shared mechanisms-of-action between these conditionally-essential amino acids.

A randomized, placebo-controlled trial of arginine therapy for the treatment of children with sickle cell disease hospitalized with vaso-occlusive pain episodes.

Painful episodes of vaso-occlusion are the leading cause of hospitalizations and emergency department visits in sickle cell disease, and are associated with increased mortality. Low nitric oxide bioavailability contributes to vasculopathy in sickle cell disease. Since arginine is the obligate substrate for nitric oxide production, and an acute deficiency is associated with pain, we hypothesized that arginine may be a beneficial treatment for pain related to sickle cell disease. Thirty-eight children with sickle cell disease hospitalized for 56 episodes of pain were randomized into this double-blinded placebo-controlled trial. Patients received L-arginine (100 mg/kg tid) or placebo for 5 days or until discharge. A significant reduction in total parenteral opioid use by 54% (1.9 ± 2.0 mg/kg versus 4.1 ± 4.1 mg/kg, P=0.02) and lower pain scores at discharge (1.9 ± 2.4 versus 3.9 ± 2.9, P=0.01) were observed in the treatment arm compared to the placebo one. There was no significant difference in hospital length of stay (4.1 ± 01.8 versus 4.8 ± 2.5 days, P=0.34), although a trend favored the arginine arm, and total opioid use was strongly correlated with the duration of the admission (r=0.86, P<0.0001). No drug-related adverse events were observed. Arginine therapy represents a novel intervention for painful vaso-occlusive episodes. A reduction of narcotic use by >50% is remarkable. Arginine is a safe and inexpensive intervention with narcotic-sparing effects that may be a beneficial adjunct to standard therapy for sickle cell-related pain in children. A large multi-center trial is warranted in order to confirm these observations.

Hemolysis-associated pulmonary hypertension in thalassemia.

Accumulating evidence supports the existence of a condition involving hemolysis-associated pulmonary hypertension (PHT). Hemolysis-induced release of cell-free hemoglobin and red blood cell arginase, resulting in impaired nitric oxide bioavailability, endothelial dysfunction, and PHT, has been reported in sickle cell disease. Since thalassemia is also a condition of chronic hemolysis, these patients are at risk. The data demonstrate that hemolysis-induced dysregulation of arginine metabolism and PHT also occurs in thalassemia. Erythrocyte release of arginase during hemolysis contributes to the development of PHT. Therapies that maximize arginine and nitric oxide bioavailability may benefit patients with thalassemia.

Decreased arginine bioavailability and increased serum arginase activity in asthma.

Recent studies suggest that a nitric oxide (NO) deficiency and elevated arginase activity may play a role in the pathogenesis of asthma. Although much attention has been directed toward measurements of exhaled NO in asthma, no studies to date have evaluated levels of plasma arginase or arginine, the substrate for NO production, in patients with asthma. This study, therefore, measured amino acid levels, arginase activity, and nitric oxide metabolites in the blood of patients with asthma, as well as NO in exhaled breath. Although levels of virtually all amino acids were reduced, patients with asthma exhibited a striking reduction in plasma arginine levels compared with normal control subjects without asthma (45 +/- 22 vs. 94 +/- 29 microM, p < 0.0001), and serum arginase activity was elevated (1.6 +/- 0.8 vs. 0.5 +/- 0.3 micromol/ml/hour, asthma vs. control, p < 0.0001). High arginase activity in patients with asthma may contribute to low circulating arginine levels, thereby limiting arginine bioavailability and creating a NO deficiency that induces hyperreactive airways. Addressing the alterations in arginine metabolism may result in new strategies for treatment of asthma.