Inborn errors of Beta-oxidation (EBO) identified to date include the severe (S) and mild (M) multiple acyl-CoA dehydrogenation disorders (MAD) and deficiencies of the short-(SC), medium-(MC), and long-chain (LC) acyl-CoA dehydrogenases (ADH). The MAD result from deficiencies of electron transfer flavorprotein (ETF) or ETF-dehydrogenase (ETF-DH); riboflavin-responsive (RR) MAD/M variants are known. After carnitine (CN) loading, we will measure urine and plasma organic acids and acyl-CNs in EBO patients and quantitate acyle-CoAs and -CNs in EBO fibroblasts by fast atom bombardment-mass spectroscopy. We will measure catabolism of 14C-butyrate and 3H-palmitate to screen suspected EBO cell lines. Complementation analysis and direct assay of ADHs, ETF, and ETF-DH should specify the enzymatic defect in known classes of the EBO. We will raise polyclonal and monoclonal antibodies to pig SCADH, MCADH and ETF and use immunotitration and immunoblot analysis to detect molecular weight, isoelectric or cross-reacting material-negative variants of these enzymes in the EBO. In EBO cell lines showing deficient substrate oxidation with normal activities of these enzymes, we will assay other Beta-oxidation enzymes, such as CN-acyl transferases, crotonases, 3-hydroxyacyl-CoA DHs and 3-ketoacyl- CoA thiolase. Since riboflavin depletion should accentuate the biochemical defect(s) in RR-MAD/M cells, analogous studies will be done with riboflavin-depleted and-supplemented cells, both with and without added FAD. We will also study FAD transport into both rat liver and human fibroblast mitochondria. Since clofibric acid increases ADH and ETF activities in the rat and 14 C-fatty acid oxidation in fibroblasts, we will assay and immunotitrate ADHs and ETF in EBO cells cultured in the presence and absence of this compound. The effect of CN on fatty acid oxidation in intact EBO cells will also be tested. Pipecolic acid (PIP) accumulates in certain neurodegenerative disorders, including hyperpipecolic acidemia (HPA) and Zellweger syndrome (ZS). Using L-3H-PIP as substrate, the conversion of PIP to alpha-aminoadipate will be further characterized in rabbit mitochondria and monkey peroxisomes. We will also purify the rabbit mitochondrial PIP dehydrogenase using conventional techniques. Analogous experiments will then be done with lymphoblasts and tissues obtained post mortem from normal controls and patients with HPA, ZS or other disorders characterized by PIP accumulation.
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