In recent years, our laboratory and other biochemical genetics units have identified a number of patients who have persistent 3-methylglutaconic aciduria but who do not have an identifiable disturbance in their metabolism of leucine, the presumed precursor of 3-methylglutaconic acid in man. Our further studies of these patients suggest instead that their urinary 3-methylglutaconate derives from the """"""""trans-methyl-glutaconate (mevalonate) shunt, """"""""a little known isoprenoid-related pathway that may be of fundamental importance to our understanding of not only these patients but also regulation of polyisoprenoid and cholesterol metabolism in man. This proposal addresses several questions we seek to answer to be able to understand better the different pathways of 3-methylglutaconic acid metabolism in man. 1) What is the activity of the mevalonate shunt in cultured human fibroblasts, lymphoblasts, and myoblasts? Methods will be developed for measuring overall flux through the shunt and then applied to the study of the activity of the methyl-glutaconate shunt in cultured cells. 2) What is the subcellular localization of the mevalonate shunt? Are there multiple enzymes with the ability to hydrate 3-methylglutaconate or 3- methylglutaconyl-CoA with different subcellular localizations? We will assay subcellular fractions (rat liver, muscle, and fibroblast) for hydratase activity and compare the activities in normal cells with those found in cells from patients with 3-methylglutaconic aciduria. 3) What is the metabolic pathway for the production of 3-methylglutaconate independent of leucine catabolism and the diversion of mevalonate carbon to the synthesis of n-fatty acids? The metabolism of leucine and mevalonate in cultured cells will be traced using various labelling strategies to identify the level(s), synthetic or catabolic, at which isoprenoid carbon is diverted to 3-methylglutaconate. Identified pathways will then be studied in cell lines from patients with leucine-independent 3- methylglutaconic aciduria.
