Evaluation of metabolic pathway activity in cultured skin fibroblasts and blood leukocytes

Abstract

Screening for a variety of blocked catabolic mutants can be achieved in cultured skin fibroblasts and in peripheral blood leukocytes with a simple radioisotope method that is reliable and convenient. The method measures the incorporation into trichloroacetic acid (TCA) insoluble macromolecules of a 14C-labelled intermediate in the pathway under question; incorporation of a 3H-labelled metabolite, not in the same pathway, is used as an internal control of metabolism in the cell population. The 14C/3H incorporation ratio is decreased in blocked pathways; use of the ratio method eliminates the delay required by radioautography (used in earlier adaptations of this method), the problems involved in CO2 collection, and the need to standardize cultures for cell number. This method has been used to identify cells with biochemical lesions in the oxidation of propionate, galactose, hypoxanthine and pyruvate; it has allowed us to identify a new variant of methylmalonicaciduria; we believe it can be extended to include other metabolites and pathways.