Thermo-sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy.

Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.

Characterization of L-aminocarnitine, an inhibitor of fatty acid oxidation.

The pathogenesis of hypoketotic hypoglycemia and cardiomyopathy in patients with fatty acid oxidation (FAO) disorders is still poorly understood. In vitro studies are hampered by the lack of natural mutants to asses the effect of FAO inhibition. In addition, only a few inhibitors of FAO are known. Furthermore, most inhibitors of FAO are activating ligands of peroxisome proliferator-activated receptors (PPARs). We show that l-aminocarnitine (L-AC), a carnitine analog, inhibits FAO efficiently, but does not activate PPAR. L-AC inhibits carnitine palmitoyltransferase (CPT) with different sensitivities towards CPT1 and CPT2, as well as carnitine acylcarnitine translocase (CACT). We further characterized L-AC using fibroblasts cell lines from controls and patients with different FAO defects. In these cell lines acylcarnitine profiles were determined in culture medium after loading with [U-(13)C]palmitic acid. In control fibroblasts, L-AC inhibits FAO leading to a reduction of C2-acylcarnitine and elevation of C16-acylcarnitine. In very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts, L-AC decreased the elevated C14-acylcarnitine and increased C16-acylcarnitine. In CACT and CPT2-deficient cell lines, L-AC did not change the already elevated C16-acylcarnitine level, showing that CPT1 is not inhibited. Oxidation of pristanic acid was only partly inhibited at high L-AC concentrations, indicating minimal CACT inhibition. Therefore, we conclude that in intact cells L-AC inhibits CPT2. Combined with our observation that l-AC does not activate PPAR, we suggest that L-AC is useful to simulate a FAO defect in cells from different origin.

An improved enzyme assay for carnitine palmitoyl transferase I in fibroblasts using tandem mass spectrometry.

Carnitine palmitoyl transferase I (CPTI), which converts acyl-CoA and carnitine into acyl-carnitine and free CoASH, is the rate limiting enzyme of hepatic mitochondrial beta-oxidation. CPTI-deficiency is a severe disorder characterized by Reye-like attacks with hypoketotic hypoglycemia, hepatomegaly, elevated liver enzymes and hyperammonemia. We developed a simple tandem-MS-based assay to measure CPTI activity in human fibroblasts. Surprisingly, a large part of the palmitoyl-carnitine formed in our assay by CPTI was degraded into C14- to C2-acyl-carnitines. Degradation of the product of CPTI leads to under estimation of the CPTI activity. When we used potassium cyanide to inhibit enzymes downstream of CPTI and thereby degradation of the product, we measured four times more CPTI activity than the previous methods. This inhibition is essential for correct calculation of CPTI activity. In fibroblasts of CPTI-deficient patients, CPTI activity was not detectable and this assay can be used for the diagnosis of CPTI-deficiency.