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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK033289-04
Application #
3231663
Study Section
Biochemistry Study Section (BIO)
Project Start
1984-07-01
Project End
1992-06-30
Budget Start
1987-07-01
Budget End
1988-06-30
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Nada, M A; Rhead, W J; Sprecher, H et al. (1995) Evidence for intermediate channeling in mitochondrial beta-oxidation. J Biol Chem 270:530-5
Gordon, J A; Heller, S K; Rhead, W J et al. (1995) Formation of a novel arachidonic acid metabolite in peroxisomes. Prostaglandins Leukot Essent Fatty Acids 52:77-81
Rhead, W; Roettger, V; Marshall, T et al. (1993) Multiple acyl-coenzyme A dehydrogenation disorder responsive to riboflavin: substrate oxidation, flavin metabolism, and flavoenzyme activities in fibroblasts. Pediatr Res 33:129-35
Freneaux, E; Sheffield, V C; Molin, L et al. (1992) Glutaric acidemia type II. Heterogeneity in beta-oxidation flux, polypeptide synthesis, and complementary DNA mutations in the alpha subunit of electron transfer flavoprotein in eight patients. J Clin Invest 90:1679-86
Amendt, B A; Freneaux, E; Reece, C et al. (1992) Short-chain acyl-coenzyme A dehydrogenase activity, antigen, and biosynthesis are absent in the BALB/cByJ mouse. Pediatr Res 31:552-6
Triggs, W J; Roe, C R; Rhead, W J et al. (1992) Neuropsychiatric manifestations of defect in mitochondrial beta oxidation response to riboflavin. J Neurol Neurosurg Psychiatry 55:209-11
Rhead, W J (1991) Inborn errors of fatty acid oxidation in man. Clin Biochem 24:319-29
Lutz, R; Garnica, A; Shires, A et al. (1991) An atypical case of cytochrome c oxidase deficiency with biochemical heterogeneity in fibroblasts. Neurology 41:1957-60
Medlock, M D; Rhead, W J; Pollack, L et al. (1991) A case of glutaric acidemia type II (severe multiple acyl-CoA dehydrogenation disorder) with subsequent prenatal exclusion in a sibling. J Perinatol 11:227-30
Mihalik, S J; Rhead, W J (1991) Species variation in organellar location and activity of L-pipecolic acid oxidation in mammals. J Comp Physiol B 160:671-6

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