Metabolomics to Assess Treatment Responses to a Single Large Bolus of Enteral Cholecalciferol in Vitamin D Deficient Critically Ill Children: Insights for Precision Nutrition

Vitamin D deficiency (VDD) is prevalent globally and in pediatric intensive care units, where it represents a modifiable risk factor that may impact patient recovery during hospitalization. However, few studies have investigated the dynamic metabolic effects from a single large bolus of enteral cholecalciferol to rapidly restore vitamin D status in critically ill children. Herein, we performed a retrospective analysis of serum samples collected from the VITdALIZE-KIDS pilot study, a phase-II randomized placebo-controlled trial, to elucidate treatment response variations following the intake of 10,000 IU/kg vitamin D3 (up to a maximum of 400,000 IU or 10 mg) by critically ill children with VDD (25-OH-D < 50 nmol/L). Comprehensive vitamin D metabolite and lipidomic studies were performed by reversed-phase ultra-high performance liquid chromatography-mass spectrometry, whereas serum metabolomics and a targeted electrolyte panel were acquired by multisegment injection-capillary electrophoresis-mass spectrometry and capillary electrophoresis with indirect UV detection, respectively. Overall, 6 vitamin D species, 239 lipids, 68 metabolites and 4 electrolytes were measured in most serum samples (> 70%) with adequate precision (CV < 40%) from pooled quality control samples using a multi-step data workflow for molecular feature selection and metabolite authentication. Complementary statistical methods were used to classify circulating metabolites/lipids associated with treatment induced vitamin D sufficiency on day 7 (25-OH-D > 75 nmol/L) following high-dose vitamin D3 intake (n=20) or placebo (n=11) who received a daily maintenance of vitamin D3 (< 1000 IU/day). There was a striking increase in median serum concentrations of 25-OH-D3 (4.7-fold, p = 2.39 x 10-8), 3-epi-25-OH-D3 (24-fold, p = 8.27 x 10-10) and their C3-epimer ratio (6.7-fold, p = 6.22 x 10-5) in treated patients on day 3, whereas serum vitamin D3 levels peaked on day 1 (128-fold, p = 9.11 x 10-8) relative to the standard-of-care. Treatment response differences were largely attributed to differences in oral D3 bioavailability and C3-epimerase activity, but not 25-hydroxylation activity nor baseline calcium or magnesium status. Also, two treated participants were identified as poor responders likely due to malabsorption, and there was no evidence of hypercalcemia in this study. For the first time, we report the detection of circulating 3-epi-D3 that was strongly correlated with vitamin D3 uptake (r = 0.898, p = 3.27 x 10-53), however, its C3-epimer ratio did not change when compared to the daily maintenance therapy as placebo. The systemic metabolic effects of vitamin D sufficiency (serum 25-OH-D > 75 nmol/L on days 2 or 3) corresponded to lower circulating levels of 3-methylhistidine, cystine, S-methylcysteine, uric acid, and two lysophosphatidylcholines (LPC 20:0; LPC 20:2) measured after 7 days with covariate adjustments (p < 0.05; age, sex, weight, and season) relative to vitamin D insufficient patients (serum 25-OH-D < 75 nmol/L). The former three polar metabolites were also significant after adjusting for baseline 25-OH-D status. Overall, serum cystine trajectories were the most sensitive indicator of vitamin D repletion after the first week. Rapid correction of VDD was associated with a metabolic phenotype associated with lower oxidative stress, inflammation, and muscle protein turn-over that may impart clinical benefits.