Impact of arginine therapy on mitochondrial function in children with sickle cell disease during vaso-occlusive pain

Abstract

Altered mitochondrial function occurs in sickle cell disease (SCD), due in part to low nitric oxide (NO) bioavailability. Arginine, the substrate for NO production, becomes acutely deficient in SCD patients with vaso-occlusive pain episodes (VOE). To determine if arginine improves mitochondrial function, 12 children with SCD-VOE (13.6 ± 3 years; 67% male; 75% hemoglobin-SS) were randomized to 1 of 3 arginine doses: (1) 100 mg/kg IV 3 times/day (TID); (2) loading dose (200 mg/kg) then 100 mg/kg TID; or (3) loading dose (200 mg/kg) followed by continuous infusion (300 mg/kg per day) until discharge. Platelet-rich plasma mitochondrial activity, protein expression, and protein-carbonyls were measured from emergency department (ED) presentation vs discharge. All VOE subjects at ED presentation had significantly decreased complex-V activity compared to a steady-state cohort. Notably, complex-V activity was increased at discharge in subjects from all 3 arginine-dosing schemes; greatest increase occurred with a loading dose (P < .001). Although complex-IV and citrate synthase activities were similar in VOE platelets vs steady state, enzyme activities were significantly increased in VOE subjects after arginine-loading dose treatment. Arginine also decreased protein-carbonyl levels across all treatment doses (P < .01), suggesting a decrease in oxidative stress. Arginine therapy increases mitochondrial activity and reduces oxidative stress in children with SCD/VOE. This trial was registered at www.clinicaltrials.gov as #NCT02536170.

Conflict of interest statement

Conflict-of-interest disclosure: C.R.M. is the inventor or coinventor of several University of California San Francisco-Benioff Children’s Hospital Oakland patents/patent-pending applications that include nutritional supplements and biomarkers of cardiovascular disease related to arginine bioavailability, is an inventor of an Emory University School of Medicine patent application for a nutritional supplement, is a consultant for Pfizer, and has received research support from MAST Therapeutics, the US Food and Drug Administration, and the National Institutes of Health. C.D.D. has received research support from Pfizer. The remaining authors declare no competing financial interests.

© 2020 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Differences in mitochondrial activity in patients with sickle cell disease at steady state compared to patients experiencing acute pain. (A) Complex V and (B) complex IV activity in patients with sickle cell disease at steady-state (SS, no pain) compared with patients experiencing a moderate-to-severe VOE on the day of an ED visit. Compared with a cohort of SCD patients in SS, all VOE subjects had significantly decreased complex V activity; however, complex IV activities were similar in SS platelets vs VOE.

Figure 2.

Impact of parenteral arginine therapy on mitochondrial activity and biomarkers of oxidative stress in children with sickle cell disease during acute vaso-occlusive pain episodes at admission compared to discharge. (A) Enzymatic activities of complex V, (B) complex IV, and (C) citrate synthase in platelet-rich plasma on the day of ED presentation for pain (VOE-baseline) compared with the day of hospital discharge (discharge) across 3 IV arginine dosing schemes: 100 mg/kg per dose TID vs a loading dose (200 mg/kg) followed by 10 0mg/kg per dose TID (loading + TID) vs a loading dose followed by a continuous infusion (300 mg/kg per day) (loading + CI). (D) Mitochondrial DNA, (E) protein carbonyl levels, and (F) malondialdehyde (MDA) levels across 3 arginine dosing schemes at VOE-Baseline compared with Discharge. Notably, complex-V activity, low at VOE-baseline compared with steady-state, was increased at discharge in subjects with VOE treated with arginine across all 3 dosing schemes, with greatest increase noted when using a loading dose (P < .001). Although complex IV and citrate synthase activities were not changed in VOE platelets vs steady state, the activities of these enzymes were significantly increased in VOE subjects after arginine treatment when using a loading dose (P < .01). These changes are not due to increased mitochondrial number because quantification of mitochondrial DNA before and after IV arginine was not different. Arginine therapy also significantly decrease levels of protein carbonyls and malondialdehyde in platelet-rich plasma across all treatment doses (P < .01), suggesting a decrease in oxidative stress. All panels show n = 4 subjects for each treatment arm. (A) There is substantial overlap between 2 subjects in both the TID and load + TID arms.