Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate

Paul M. Rougraff, Ralph Paxton, Gary W. Goodwin, Reid G. Gibson, Robert Harris

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

An enzymatic spectrophotometric end-point assay has been developed for determination of S-3-hydroxyisobutyrate in biological fluids. The assay measures NADH production at 340 nm after initiation of the reaction with rabbit liver 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). The assay is not affected by R-3-hydroxyisobutyrate, lactate, malate, 3-hydroxybutyrate, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 3-hydroxy-n-valerate, 2-methyl-3-hydroxyvalerate, and 3-hydroxypropionate. The assay does measure 2-ethyl-3-hydroxypropionate, a minor metabolite produced by catabolism of alloisoleucine. Application of the method to measure S-3-hydroxyisobutyrate in plasma obtained from normal, 48-h starved, and mildly and severely diabetic rats gave levels of 28, 42, 112, and 155 μm, respectively.

Original languageEnglish
Pages (from-to)317-320
Number of pages4
JournalAnalytical Biochemistry
Volume184
Issue number2
DOIs
StatePublished - Feb 1 1990

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3-hydroxyisobutyrate dehydrogenase
Enzyme Assays
Assays
Endpoint Determination
Valerates
3-Hydroxybutyric Acid
Isoleucine
NAD
Lactic Acid
Rabbits
Liver
Metabolites
Rats
Plasmas
Fluids

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate. / Rougraff, Paul M.; Paxton, Ralph; Goodwin, Gary W.; Gibson, Reid G.; Harris, Robert.

In: Analytical Biochemistry, Vol. 184, No. 2, 01.02.1990, p. 317-320.

Research output: Contribution to journalArticle

Rougraff, Paul M. ; Paxton, Ralph ; Goodwin, Gary W. ; Gibson, Reid G. ; Harris, Robert. / Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate. In: Analytical Biochemistry. 1990 ; Vol. 184, No. 2. pp. 317-320.
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