Isovaleryl-CoA Dehydrogenase Activity in Isovaleric Acidemia Fibroblasts Using an Improved Tritium Release Assay

Abstract

ABSTRAm. Isovaleric acidemia is a disorder of leucine metabolism caused by a deficiency of isovaleryl-CoA dehydrogenase. At least two clinical subgroups of patients exist: a severe form, in which symptoms occur within the 1st wk of life, and a milder variant in which manifestations develop later in life. We developed a modified version of the tritium release assay to accurately measure residual isovaleryl-CoA dehydrogenase activity in fibroblasts from patients with both forms of isovaleric acidemia. In the modified assay, specific isovaleryl-CoA dehydrogenasecatalyzed tritium release from [2,3-3H]isovaleryl-CoA was determined by including an inhibitor of isovaleryl-CoA dehydrogenase, (methylenecyclopropy1)acetyl-CoA, in one of the tubes in paired assays, to determine the nonspecifically released 3H20. Residual activities of the nine isovaleric acidemia lines tested ranged from 0 to 0.67 pmol Isovaleric acidemia is an inherited disorder of leucine metabolism caused by a deficiency of the activity of isovaleryl-CoA dehydrogenase (EC. 1.3.99.10) (1, 2). Isovaleric acidemia has generally been considered to have two clinical phenotypes (3). One form (severe type) manifests as severe vomiting, lethargy, and coma, which commences within the first 2 wk of life and often causes death in the neonatal period. The other form (mild type) is characterized by recurrent milder episodes of vomiting, lethargy, and ketoacidosis. These episodes tend to begin later in life, and they are often triggered by viral or bacterial infection.

Isovaleric acidemia: New aspects of genetic and phenotypic heterogeneity

“…Fibroblasts, lymphocytes, and amniocytes all have measurable amounts of IVD activity and serve as ready sources of tissue for this purpose [Ensenauer et al, 2004;Kleij et al, 1995;Mohsen et al, 1998;Vockley et al, 1991]. While significant residual activity blunts the level of abnormal metabolites, correlation between clinical presentation and enzyme activity has been poor [Hyman and Tanaka, 1986;Ikeda et al, 1985b;Vockley et al, 1991].…”

“…Several direct and indirect methods to assay IVD activity have been published and, in addition to molecular genetic analysis, can be used to confirm a diagnosis of IVA [Hyman and Tanaka, 1986;Mohsen et al, 1998;Rhead and Tanaka, 1980;Shih et al, 1973;Tajima et al, 2005;Vockley et al, 1991;Yoshida et al, 1985]. Fibroblasts, lymphocytes, and amniocytes all have measurable amounts of IVD activity and serve as ready sources of tissue for this purpose [Ensenauer et al, 2004;Kleij et al, 1995;Mohsen et al, 1998;Vockley et al, 1991].…”

“…Urine was subsequently shown to contain isovalerylglycine and 3-hydroxyisovaleric acid as well as other metabolites Tanaka et al, 1968]. Oxidation of labeled isovaleric acid by patient white blood cells was reduced compared to control cells, and a defect of leucine metabolism was postulated [Hyman and Tanaka, 1986;Rhead and Tanaka, 1980;Shih et al, 1973;Yoshida et al, 1985]. It must be remembered that until the characterization of these patients, the oxidation of isovaleryl-CoA was assumed to be catalyzed by the newly identified short chain acyl-CoA dehydrogenase [Crane et al, 1956;Engel and Massey, 1971;Frerman et al, 1980;McKean et al, 1979].…”

The Treatment of Isovaleric Acidemia with Glycine Supplement

“…Dubiel et al (23) also found no difference in the residual activity of 12 isovaleric acidemia cell lines using macromolecular-labeling techniques. An improved tritium release assay using an acyl-CoA dehydrogenase inhibitor (methylenecyclopropylacetyl-CoA) (24) gave an unexpected finding. The acute, early-onset form had residual activity of 0.41 pmol 3H20/min/mg protein compared to a normal of 19.4 + 8.0 whereas three milder clinical forms had no enzyme activity.…”

“…The acute, early-onset form had residual activity of 0.41 pmol 3H20/min/mg protein compared to a normal of 19.4 + 8.0 whereas three milder clinical forms had no enzyme activity. The authors suggested that age of onset and the severity of clinical presentation were not caused by differences in impaired isovaleryl-CoA dehydrogenase, but by the competency of the glycine-N-acylase pathway to use accumulated isovaleric acid (24). When [2][3][4][5][6][7][8][9][10][11][12][13][14]C-leucine conversion to I4CO2 by intact cells was used as the enzyme assay, less than 2% residual activity was seen in acute isovaleric acidemia (25).…”