Exploring the metabolic fate of medium-chain triglycerides in healthy individuals using a stable isotope tracer.

BACKGROUND & AIMS: Medium chain triglyceride (MCT) supplementation is often recommended as treatment for patients with long-chain fatty acid ß-oxidation (lcFAO) disorders, since they can be utilized as an energy source without the use of the defective enzyme. However, studies in mice and preterm infants suggest that not all medium-chain fatty acids (MCFA) are oxidized and may undergo elongation to long-chain fatty acids (LCFA). In this single blinded study, we explored the metabolic fates of MCT in healthy individuals using a 13C-labeled MCT tracer. METHOD: Three healthy males in rest received on two test days a primed continuous infusion of glyceryl tri[1,2,3,4-13C4]-octanoate with either an isocaloric supplementation of 1) exclusively MCT (MCT-only) or 2) a mixture of MCT, proteins and carbohydrates (MCT-mix). Gas chromatography - combustion - isotope ratio mass spectrometry (GC-C-IRMS) was used to determine 13C-enrichment of long-chain fatty acids in plasma and of 13CO2 in exhaled air. RESULTS: When provided as single energy source, an estimated 42% of administered MCT was converted to CO2. In combination with carbohydrates and proteins in the diet, oxidation of MCT was higher (62%). In both diets <1% of 13C-label was incorporated in LCFA in plasma, indicating that administered MCT underwent chain-elongation to LCT. CONCLUSIONS: Although the relative MCT oxidation rate was higher when combined with carbohydrates and protein, quantitatively more MCT was oxidized when given an isocaloric meal with solely MCT. As these results were obtained in the resting state opposed to during exercise, it is too early to give a recommendation concerning the use of MCT in lcFAO disorders. The data show that in resting healthy individuals only a very small part of the MCT is traced back as LCFA in plasma, suggesting that MCT treatment does not result in a large LCFA burden, however further research on storage of MCT in tissues is warranted. REGISTRATION: The study was registered in the Nederlands Trialregister. Protocol ID: Trial NL7417 (NTR7650).

Nutritional ketosis improves exercise metabolism in patients with very long-chain acyl-CoA dehydrogenase deficiency.

A maladaptive shift from fat to carbohydrate (CHO) oxidation during exercise is thought to underlie myopathy and exercise-induced rhabdomyolysis in patients with fatty acid oxidation (FAO) disorders. We hypothesised that ingestion of a ketone ester (KE) drink prior to exercise could serve as an alternative oxidative substrate supply to boost muscular ATP homeostasis. To establish a rational basis for therapeutic use of KE supplementation in FAO, we tested this hypothesis in patients deficient in Very Long-Chain acyl-CoA Dehydrogenase (VLCAD). Five patients (range 17-45 y; 4 M/1F) patients were included in an investigator-initiated, randomised, blinded, placebo-controlled, 2-way cross-over study. Patients drank either a KE + CHO mix or an isocaloric CHO equivalent and performed 35 minutes upright cycling followed by 10 minutes supine cycling inside a Magnetic Resonance scanner at individual maximal FAO work rate (fatmax; approximately 40% VO2 max). The protocol was repeated after a 1-week interval with the alternate drink. Primary outcome measures were quadriceps phosphocreatine (PCr), Pi and pH dynamics during exercise and recovery assayed by in vivo 31 P-MR spectroscopy. Secondary outcomes included plasma and muscle metabolites and respiratory gas exchange recordings. Ingestion of KE rapidly induced mild ketosis and increased muscle BHB content. During exercise at FATMAX, VLCADD-specific plasma acylcarnitine levels, quadriceps glycolytic intermediate levels and in vivo Pi/PCr ratio were all lower in KE + CHO than CHO. These results provide a rational basis for future clinical trials of synthetic ketone ester supplementation therapy in patients with FAO disorders. Trial registration: ClinicalTrials.gov. Protocol ID: NCT03531554; METC2014.492; ABR51222.042.14.

Electrophysiological Abnormalities in VLCAD Deficient hiPSC-Cardiomyocytes Do not Improve with Carnitine Supplementation.

Patients with a deficiency in very long-chain acyl-CoA dehydrogenase (VLCAD), an enzyme that is involved in the mitochondrial beta-oxidation of long-chain fatty acids, are at risk for developing cardiac arrhythmias. In human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs), VLCAD deficiency (VLCADD) results in a series of abnormalities, including: 1) accumulation of long-chain acylcarnitines, 2) action potential shortening, 3) higher systolic and diastolic intracellular Ca2+ concentrations, and 4) development of delayed afterdepolarizations. In the fatty acid oxidation process, carnitine is required for bidirectional transport of acyl groups across the mitochondrial membrane. Supplementation has been suggested as potential therapeutic approach in VLCADD, but its benefits are debated. Here, we studied the effects of carnitine supplementation on the long-chain acylcarnitine levels and performed electrophysiological analyses in VLCADD patient-derived hiPSC-CMs with a ACADVL gene mutation (p.Val283Ala/p.Glu381del). Under standard culture conditions, VLCADD hiPSC-CMs showed high concentrations of long-chain acylcarnitines, short action potentials, and high delayed afterdepolarizations occurrence. Incubation of the hiPSC-CMs with 400 µM L-carnitine for 48 h led to increased long-chain acylcarnitine levels both in medium and cells. In addition, carnitine supplementation neither restored abnormal action potential parameters nor the increased occurrence of delayed afterdepolarizations in VLCADD hiPSC-CMs. We conclude that long-chain acylcarnitine accumulation and electrophysiological abnormalities in VLCADD hiPSC-CMs are not normalized by carnitine supplementation, indicating that this treatment is unlikely to be beneficial against cardiac arrhythmias in VLCADD patients.

Proposal for an individualized dietary strategy in patients with very long-chain acyl-CoA dehydrogenase deficiency.

BACKGROUND: Patients with very long chain acyl-CoA dehydrogenase deficiency (VLCADD), a long chain fatty acid oxidation disorder, are traditionally treated with a long chain triglyceride (LCT) restricted and medium chain triglyceride (MCT) supplemented diet. Introduction of VLCADD in newborn screening (NBS) programs has led to the identification of asymptomatic newborns with VLCADD, who may have a more attenuated phenotype and may not need dietary adjustments. OBJECTIVE: To define dietary strategies for individuals with VLCADD based on the predicted phenotype. METHOD: We evaluated long-term dietary histories of a cohort of individuals diagnosed with VLCADD identified before the introduction of VLCADD in NBS and their beta-oxidation (LC-FAO) flux score (rate of oleate oxidation) in cultured skin fibroblasts in relation to the clinical outcome. Based on these results a dietary strategy is proposed. RESULTS: Sixteen individuals with VLCADD were included. One had an LC-FAO flux score >90%, was not on a restricted diet and is asymptomatic to date. Four patients had an LC-FAO flux score <10%, and significant VLCADD related symptoms despite the use of strict diets including LCT restriction, MCT supplementation and nocturnal gastric drip feeding. Patients with an LC-FAO flux score between 10 and 90% (n = 11) showed a more heterogeneous phenotype. CONCLUSIONS: This study shows that a strict diet cannot prevent poor clinical outcome in severely affected patients and that the LC-FAO flux is a good predictor of clinical outcome in individuals with VLCADD identified before its introduction in NBS. Hereby, we propose an individualized dietary strategy based on the LC-FAO flux score.