Search

Oxidation of straight-chain fatty acids in mitochondria involves the complicated interaction between a large variety of different enzymes. So far four different mitochondrial straight-chain acyl-CoA dehydrogenases have been identified. The physiological function of three of the four acyl-CoA dehydrogenases has been resolved in recent years especially from studies on patients suffering from certain inborn errors of mitochondrial fatty acid beta-oxidation. The physiological role of long-chain acyl-CoA dehydrogenase (LCAD) has remained obscure, however. The results described in this paper provide strong evidence suggesting that LCAD plays a central role in branched-chain fatty acid metabolism since it turns out to be the major acyl-CoA dehydrogenase reacting with 2,6-dimethylheptanoyl-CoA, a metabolite of pristanic acid, which itself is the alpha-oxidation product of phytanic acid.

Prolonged treatment of cultured cells with ethidium bromide results in loss of the capacity for oxidative phosphorylation. Because of the tight coupling between mitochondrial beta-oxidation of fatty acids and the activity of the respiratory chain, such cells may be used to study the contribution of mitochondria and peroxisomes to fatty acid beta-oxidation. To investigate this, human skin fibroblasts were cultured in the presence of ethidium bromide for at least 10 cell generations, resulting in a virtually complete absence of oxidative phosphorylation as demonstrated directly in digitonin-permeabilized fibroblasts. The cells showed a lowered ATP/ADP ratio, most likely as the consequence of the inability to generate ATP via oxidative phosphorylation. The loss of the capacity for oxidative phosphorylation was also reflected in an increased cytosolic NADH/NAD+ ratio: the cells showed a highly elevated lactate/pyruvate ratio in the suspending medium when incubated with glucose. The beta-oxidation of octanoic and palmitic acid was dramatically decreased, suggesting that the beta-oxidation of these fatty acids takes place predominantly (> 90%) in mitochondria, at least in the cells studied. In contrast, the rates of pristanic and cerotic acid beta-oxidation were only slightly decreased, suggesting that this is mainly a peroxisomal process. The reduction of beta-oxidation of cerotic and pristanic acid, 27% and 15%, respectively, is most likely due to a lowered ATP level and an increased NADH/NAD(+)-redoxstate in these cells. We conclude that fibroblasts subjected to prolonged treatment with ethidium bromide can be used as a model system to study the substrate specificity and functional characteristics of the peroxisomal beta-oxidation system.