cytochrome-c-t and Metabolism--Inborn-Errors

Fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are key pathways involved in cellular energetics. Reducing equivalents from FAO enter OXPHOS at the level of complexes I and III. Genetic disorders of FAO and OXPHOS are among the most frequent inborn errors of metabolism. Patients with deficiencies of either FAO or OXPHOS often show clinical and/or biochemical findings indicative of a disorder of the other pathway. In this study, the physical and functional interactions between these pathways were examined. Extracts of isolated rat liver mitochondria were subjected to blue native polyacrylamide gel electrophoresis (BNGE) to separate OXPHOS complexes and supercomplexes followed by Western blotting using antisera to various FAO enzymes. Extracts were also subjected to sucrose density centrifugation and fractions analyzed by BNGE or enzymatic assays. Several FAO enzymes co-migrated with OXPHOS supercomplexes in different patterns in the gels. When palmitoyl-CoA was added to the sucrose gradient fractions containing OXPHOS supercomplexes in the presence of potassium cyanide, cytochrome c was reduced. Cytochrome c reduction was completely blocked by myxothiazol (a complex III inhibitor) and 3-mercaptopropionate (an inhibitor of the first step of FAO), but was only partially inhibited by rotenone (a complex I inhibitor). Although palmitoyl-CoA and octanoyl-CoA provided reducing equivalents to OXPHOS-containing supercomplex fractions, no accumulation of their intermediates was detected. In contrast, short branched acyl-CoA substrates were not metabolized by OXPHOS-containing supercomplex fractions. These data provide evidence of a multifunctional FAO complex within mitochondria that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling.

Topics: Animals; Cytochromes c; Electron Transport Complex I; Electron Transport Complex III; Fatty Acids; Metabolism, Inborn Errors; Mitochondria, Liver; Oxidation-Reduction; Oxidative Phosphorylation; Rats

Isolated complex I deficiency is the most common oxidative phosphorylation defect and is associated with substantial morbidity and mortality. The diagnosis is made by enzymatic analysis and for most patients the molecular pathology remains undefined. Various cofactors and vitamins are frequently administered, but their efficacy have been difficult to assess. We employed determination of ATP production in fibroblast cell lines from patients with complex I deficiency to evaluate the usefulness of therapeutic agents. The effect of each additive varied among the different patients with certain agents favorably affecting ATP production rate in some of the patients and adversely affecting it in others. The reduced nicotinamide adenine dinucleotide (NADH)-ferricyanide reductase assay in muscle mitochondria correlated better than the NADH-coenzyme Q and NADH-cytochrome c assays with ATP production rate in fibroblasts. Our results underscore the necessity of evaluation of different agents for each patient separately. The NADH-ferricyanide reductase assay play a helpful role in directing mutation analysis and identifying patients which are more likely to have their cells amenable for ATP production assessment.

Topics: Adenosine Triphosphate; Cytochromes c; Electron Transport; Electron Transport Complex I; Fibroblasts; Humans; Infant; Infant, Newborn; Metabolism, Inborn Errors; Mitochondria, Muscle; Mutation; NAD; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Ubiquinone