Measurement of branched-chain ketoacids in plasma by high-performance liquid chromatography

Branched-chain ketoacids were isolated from plasma or serum samples by acidification, passage through a cationic exchange resin, ether extraction, and extraction of the ether layer with phosphate buffer. The recovery of 2-[1- 14C]ketoisocaproate taken through these procedures averaged 95 ± 3%. Branched-chain ketoacids were measured by high-performance liquid chromatography using a single mobile phase (sodium phosphate:acetonitrile). In normal human subjects, mean ± SD fasting levels of 2-ketoisocaproate, 2-keto-3-methylvalerate, and 2-ketoisovalerate were 29 ± 8, 18 ± 4, and 12 ± 3 μ m, respectively. In normal rats, slightly different results were found: 24 ± 10, 19 ± 7 and 17 ± 6 μ m, respectively. In both species, levels of each ketoacid expressed as fractions of total branched-chain ketoacids were much less variable.