A spectrophotometric, enzymatic assay for d-3-hydroxybutyrate that is not dependent on hydrazine

Allison Brashear, George Cook

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for d-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of d-3-hydroxybutyrate to acetoacetate by d-3-hydroxybutyrate dehydrogenase even though the reaction favors d-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of d-3-hydroxybutyrate added to liver samples was 98 ± 1% (mean ± SEM, n = 6). The assay was found to be suitable for the measurement of d-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to d-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of d-3-hydroxybutyrate in a variety of physiological and experimental samples.

Original languageEnglish (US)
Pages (from-to)478-482
Number of pages5
JournalAnalytical Biochemistry
Volume131
Issue number2
DOIs
StatePublished - Jan 1 1983

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hydrazine
3-Hydroxybutyric Acid
Enzyme Assays
Assays
Hydroxybutyrate Dehydrogenase
Health hazards
Liver
NAD
Hepatocytes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

A spectrophotometric, enzymatic assay for d-3-hydroxybutyrate that is not dependent on hydrazine. / Brashear, Allison; Cook, George.

In: Analytical Biochemistry, Vol. 131, No. 2, 01.01.1983, p. 478-482.

Research output: Contribution to journalArticle

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