Assays for cholesterol 7α-hydroxylase and 12α-hydroxylase using high performance liquid chromatography

Mitsuhide Noshiro, Hiroko Ishida, Shin ichi Hayashi, Kyuichiro Okuda

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Rapid and accurate assay methods for cholesterol:NADPH oxidoreductase (EC 1.14.13.17, 7α-hydroxylating) and 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase (enzyme not yet registered) are described. 7α-Hydroxylase utilizes the endogenous cholesterol of liver microsomes as substrate. The reaction products were separated by high performance liquid chromatography monitored at 214 nm. Much higher activity was obtained with the method compared to literature values, which were obtained using externally added radioactive cholesterol as the substrate. The 12α-hydroxylase activity was measured using non-radioactive steroid as the substrate. The reaction products were separated by the chromatography and detected at 240 nm. Comparable activities were obtained by this method compared to those that were obtained using radioactive substrate.

Original languageEnglish
Pages (from-to)539-550
Number of pages12
JournalSteroids
Volume45
Issue number6
DOIs
Publication statusPublished - 1985 Jun
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology
  • Pharmacology
  • Clinical Biochemistry
  • Organic Chemistry

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