digitonin and Metabolism--Inborn-Errors

We have used complementation analysis after somatic cell fusion to investigate the genetic relationships among various genetic diseases in humans in which there is a simultaneous impairment of several peroxisomal functions. The activity of acyl-coenzyme A:dihydroxyacetonephosphate acyltransferase, which is deficient in these diseases, was used as an index of complementation. In some of these diseases peroxisomes are deficient and catalase is present in the cytosol, so that the appearance of particle-bound catalase could be used as an index of complementation. The cell lines studied can be divided into at least five complementation groups. Group 1 is represented by a cell line from a patient with the rhizomelic form of chondrodysplasia punctata. Group 2 consists of cell lines from four patients with the Zellweger syndrome, a patient with the infantile form of Refsum disease and a patient with hyperpipecolic acidemia. Group 3 comprises one cel line from a patient with the Zellweger syndrome, group 4 one cell line from a patient with the neonatal form of adrenoleukodystrophy, and group 5 one cell line from a patient with the Zellweger syndrome. We conclude that at least five genes are required for the assembly of a functional peroxisome.

Topics: Acyltransferases; Adrenoleukodystrophy; Catalase; Cell Fusion; Cell Line; Centrifugation, Density Gradient; Chondrodysplasia Punctata; Digitonin; Fibroblasts; Genetic Complementation Test; Humans; Metabolism, Inborn Errors; Microbodies; Refsum Disease; Syndrome

A small-for-gestational-age female infant born at term developed severe lactic acidosis and died on day 13 of life. Two previous sibs had also died of overwhelming lactic acidosis in the neonatal period. The lactate-to-pyruvate and 3-hydroxybutyrate-to-acetoacetate ratios were elevated at 136 and 42 to one, respectively. The activities of the pyruvate dehydrogenase complex and pyruvate carboxylase in cultured skin fibroblasts were normal but a defect in respiration was indicated by the low rates of conversion of 1-[14C]pyruvate, glutamate, and lactate to 14CO2 in these cells. Skin fibroblast cultures also displayed an elevated lactate-to-pyruvate ratio (72:1) when incubated with glucose as substrate compared to control cell cultures (20:1). When mitochondrial preparations of skin fibroblasts (prepared by digitonin extraction) were tested for their ability to synthesize ATP from a variety of substrates, it was found that those of the patient made adequate amounts of ATP with either succinate or ascorbate/tetramethyl-phenylenediamine as substrate but not with the NAD-linked substrates pyruvate, isocitrate, and palmitoyl carnitine. We propose that this is indicative of a defect in the respiratory chain between NADH and coenzyme Q, for the first time demonstrable in cultured skin fibroblasts.

Topics: Acidosis; Adenosine Triphosphate; Cells, Cultured; Digitonin; Female; Fibroblasts; Humans; Infant, Newborn; Lactates; Metabolism, Inborn Errors; Mitochondria; NAD; Oxidation-Reduction; Oxygen Consumption; Pyruvates; Skin; Substrate Specificity

Topics: Acetates; Animals; Carbon Dioxide; Carbon Isotopes; Catalase; Cholesterol; Digitonin; Heterozygote; Homozygote; In Vitro Techniques; Kinetics; Lipids; Liver; Metabolism, Inborn Errors; Mice; Mutation; Sodium; Sterols; Triglycerides