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 acid 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 concentration of all thre...

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Bibliographic Details
Published in:Analytical biochemistry 1996-08, Vol.240 (1), p.48-53
Main Authors: Beckett, Philip R., Hardin, Dana S., Davis, Teresa A., Nguyen, Hanh V., Wray-Cahen, Diane, Copeland, Kenneth C.
Format: Article
Language:English
Subjects:
Amino Acid Oxidoreductases - metabolism
Amino Acids, Branched-Chain - blood
Blood Proteins - metabolism
Chromatography, High Pressure Liquid
Enzyme Stability
Humans
Hyperinsulinism - blood
Insulin - therapeutic use
Isoleucine - blood
Kinetics
Leucine - blood
Leucine Dehydrogenase
NAD - metabolism
Spectrophotometry
Valine - blood
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Summary:Plasma amino acid concentrations fall during insulin infusion. Amino acid 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 concentration of all three branched-chain amino acids in plasma within 1 min. The enzyme leucine dehydrogenase oxidatively deaminates leucine, isoleucine, and valine, with stoichiometric reduction of NAD that is measured using a spectrophotometer. The assay was developed in both a kinetic and end-point format. For the kinetic assay the buffer conditions were formulated to obtain equivalent rates with all three amino acids so that it could be used in samples containing unknown mixtures. For 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 for “clamping” the branched-chain amino acids during hyperinsulinemic euglycemic clamps in humans is demonstrated.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1996.0329