A study of the metabolism of [U-14C]3-methyl-2-oxopentanoate by rat liver mitochondria using h.p.l.c. with continuous on-line monitoring of radioactive intact acyl-coenzyme A intermediates.

A reverse-phase h.p.l.c. system for the resolution of the acyl-CoA intermediates of the degradation of 3-methyl-2-oxopentanoate is described. The validation of a method for the measurement of radioactive intermediates produced by the incubation of [U-14C]3-methyl-2-oxopentanoate with rat liver mitochondrial fractions is described. The absence of bicarbonate caused the accumulation of [14C]propionyl-CoA. The accumulation of [14C]2-methylbutyryl-CoA was observed in incubations with mitochondrial fractions derived from riboflavin-deficient animals. Studies of the accumulation of labelled intermediates with time suggest that there is regulation of the pathway of isoleucine degradation at methylmalonyl-CoA mutase, as suggested for valine [Corkey, Martin-Requero, Walajtys-Rode, Williams & Williamson (1982) J. Biol. Chem. 257, 9668-9676]. These studies demonstrate that h.p.l.c. with on-line continuous radiochemical detection is a powerful method for the investigation of the control of intermediary metabolism.