SPECTROPHOMETRIC ASSAY FOR MEASURING BRANCHED-CHAIN AMINO-ACID-CONCENTRATIONS - APPLICATION FOR MEASURING THE SENSITIVITY OF PROTEIN-METABOLISM TO INSULIN

Plasma amino acid concentrations fall during insulin infusion. Amino a cid concentrations can be maintained using an infusion of amino acids if their plasma concentration can be determined within a few minutes. We developed a spectrophometric assay which determines the total conce ntration of all three branched-chain amino acids in plasma within 1 mi n. The enzyme leucine dehydrogenase oxidatively deami nates leucine, i soleucine, and valine, with stoichiometric reduction of NAD that is me asured using a spectro photometer. The assay was developed in both a k inetic and end-point format. For the kinetic assay the buffer conditio ns were formulated to obtain equivalent rates with all three amino aci ds so that it could be used in samples containing unknown mixtures. Fo r the end-point assay additional enzyme was added so that an end-point could be reached within 1 min. The application of the kinetic assay f or ''clamping'' the branched-chain amino acids during hyperinsulinemic euglycemic clamps in humans is demonstrated. (C) 1996 Academic Press, Inc.