Endogenous fats play a major role in energy homeostasis, serving as a source of fuel for skeletal and cardiac muscle, and in the liver providing substrate for ketone body production and generating acetyl CoA and reducing equivalents (NADH) needed for gluconeogenesis and ureagenesis. This role increases in importance when energy demands are high or when exogenous intake is decreased or absent. Clinically, children with defects in fatty acid oxidation have come to medical attention because of hypoglycemia without ketones, often with the history of a previous episode of hypoglycemia, or with the history of a sibling with hypoglycemia or Reye syndrome. Subsequent evaluation has revealed evidence of a defect in fasting adaptation including decreased fasting tolerance, increased urinary dicarboxylic acids (the products of cytosolic fatty acid oxidation) and increased concentrations of esterified carnitine in serum and urine. In some of these children the specific defect has been identified (e.g. the medium-chain acyl CoA dehydrogenase deficiency) while in others the site remains to be elucidated. Hence, the goals of this project are: the development: of methods for the measurement of the enzymes of mitochondrial beta-oxidation in human fibroblasts; the application of these assays in the identification of the site of defect in fibroblasts from children with clinical evidence of disordered fasting adaptation; and the characterization of the molecular mechanisms which lead to the specific enzymatic defects. Spectrophotometric methods for the measurement of the enzymes (enoyl CoA hydratases, 3-hydroxyacyl CoA dehydrogenases, 3-ketoacyl CoA thiolases, 3cis, 2trans enoyl CoA isomerase and 2,4 dienoyl CoA reductase) have been described; however, these assays must be downscaled for use with readily accessible tissues (fibroblasts, leukocytes) which contain relatively small quantities of the enzymes. The substrates needed for these assays must be synthesized since they are not commercially available. Subsequently fibroblasts from children with features suggestive of a defect in fatty acid oxidation will be evaluated. Collaborative studies to examine the molecular mechanisms, leading to the medium- chain acyl CoA dehydrogenase deficiency will be expanded to include the long-chain and short-chain acyl CoA dehydrogenase deficiencies and new enzyme defects as they are identified. From a clinical perspective, elucidation of the site of defect allows therapeutic strategies to be developed. Screening of potentially affected siblings can also be undertaken at an early age, and if the defect is present, appropriate prophylactic treatment can be instituted and catastrophic illness can be avoided.
