Ready-to-use food supplement, with or without arginine and citrulline, with daily chloroquine in Tanzanian children with sickle-cell disease: a double-blind, random order crossover trial

Background: Sickle cell disease increases malnutrition risk. Low arginine and nitric oxide [NO] bioavailability are implicated in sickle-related morbidity. Simple interventions are required, especially in low-income settings. We aimed to test the hypotheses: (1) supplementary arginine, citrulline and daily chloroquine increases bioavailable arginine and flow-mediated-dilatation (FMDmax%; a measure of NO-dependent endothelial function), and (2); protein energy supplementation in the form of ready-to-use supplementary-food (RUSF) improves nutritional status in children with sickle cell disease. Methods: A random-order, double-blind, cross-over trial with two four-month intervention periods (each followed by four-months wash-out) was conducted in Dar-es-Salaam, Tanzania. 119 children aged 8-12 years, naïve to hydroxyurea, were enrolled from the Muhimbili National Hospital Sickle Cohort. The random order sequence and allocation codes were generated centrally. Two formulations of RUSF (500kcal/day) were tested: ‘basic’ with weekly chloroquine (150/225mg base, depending on weight) (RUSF-b) and ‘vascular’ (RUSF-v) fortified with arginine, citrulline designed to achieve mean intakes of 0.2g/0.1g/kg/day and daily chloroquine (max 3mg base/kg/day). The primary outcomes of the comparison of the 2 RUSF formulations were mean FMDmax%, mean plasma arginine to ornithine ratio and mean plasma arginine to asymmetric-di-methylated-arginine (ADMA) ratio. The primary outcomes of the combined effect of both RUSF interventions were mean height and body mass index for age z-scores with analysis by intention to treat. Trial registration: ISRCTN74331412 Findings: 114/119 children had complete data for all reported endpoints. There was no treatment effect of RUSF-v compared to RUSF-b on the ratio of arginine to ornithine (mean within individual difference -0.09, 95% CI -0.03/0.2, p=0.12), or on FMDmax% (-1.00 95% CI -2.47/0.47, p=0.18) but the arginine:ADMA ratio was significantly increased (-0.56, 95% CI -0.81/-0.31, P<0.001). In planned analyses using random effects models to estimate the effect of each intervention compared to baseline/washout, the arginine:ADMA ratio increased following both RUSF-v or RUSF-b (+86%, p<0.001; +41%, p<0.001). Similarly, FMDmax% was higher after 2 RUSF-v (+0.92, p<0.001) but not after RUSF-b intervention (+0.39, p=0.22). Adjusted for covariates, effect estimates for FMDmax% increased: RUSF-v (+1.19, p<0.001) and RUSF-b (+0.93, p=0.008). Following either intervention (RUSF-b and RUSF-v pooled) compared to baseline/wash-outs, body-mass-index-z-score (+0.091, P=0.001) and height-for-age-z-score (+0.013, P=0.081) increased. There were 71 and 81 adverse events of which 21 and 26 were serious during intervention and washout (P=0.31) in 83 participants, 1 of whom died in the 2nd washout period. Interpretation: RUSF providing 500kcal/day results in small weight gains in children with sickle cell disease. However, RUSF even without arginine and citrulline fortification improves arginine dysregulation and may improve endothelial function. Long-term studies are required to assess if these physiological effects translate to improved clinical outcomes and better growth and development in sickle cell disease.