title: Characterization of enzymatic deficiencies of branched chain amino-acid catabolism in human fibroblasts by genetic complementation.
abstract: Leucine and Isoleucine metabolism in cultured skin fibroblasts from patients with leucinosis, beta-Ketothiolase deficiency, propionic, methylmalonic and isovaleric acidemia, was compared with that in normal fibroblasts. A simple assay was developed using (U14C) Isoleucine and (U14C) Leucine as substrates. Radioactive incorporation into protein aminoacids were measured. The (U14C) branched chain aminoacid incorporation into proteins provides an estimation of the protein synthesis and the incorporation ratio (14C) Aspartate + (14C) Glutamate/(14C) branched chain aminoacid, measures the integrity of the metabolic pathway. Complementation tests permits to characterize the genetic defect. The incorporation ratio was significantly decreased in blocked pathways, namely in leucinosis and isovaleric acidemia in the presence of (U14C) Leucine and in Leucinosis, beta-Ketothiolase deficiency, propionic and methylmalonic acidemia in the presence of (U14C) Isoleucine. There was a significant restoration of activity in mutant strains with distinct genetic defects after polyethylene-glycol fusion. This assay provides a valuable tool to screen for enzymatic deficiencies of branched chain aminoacid catabolism.
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000172:deficiency; D000592:Amino Acid Metabolism, Inborn Errors / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D007532:Isoleucine / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism*; D008661:Metabolism, Inborn Errors / Q000235:genetics; D009154:Mutation; D012867:Skin / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D007532:Isoleucine; D000102:Acetyl-CoA C-Acyltransferase; D007930:Leucine