Determination of leucine and α-ketoisocaproic acid concentrations and specific activity in plasma and leucine specific activities in proteins using high-performance liquid chromatography

Abstract

Plasma concentrations and 3H and 14C specific activities (specific radioactivities) of leucine and α-ketoisocaproate (KIC) and leucine specific radioactivity in hydrolyzed tissue and plasma proteins were determined using an automated isocratic high-performance liquid chromatographic (HPLC) system. Within-day variability of leucine and KIC specific radioactivity in plasma was ⋍1%, whereas that observed for leucine derived from protein hydrolysis was ⋍5%. Day-to-day variability of leucine and KIC specific radioactivity in plasma was ⋍5% and in protein-derived leucine ⋍6%. In addition, an indirect method is described to measure low specific activities of [ 3H]- and [ 14C]leucine derived from the hydrolysis of in vivo labeled proteins with low turnover rates (skeletal muscle and diaphragm). In proteins with higher turnover rates (fibrin, kidney, liver, jejunum and heart muscle), this indirect method gave similar results to the direct HPLC method. Using these methods, fractional protein synthetic rates in a variety of tissues can be accurately determined using radioisotopes of KIC and/or leucine.