A fluorescence method to detect and quantitate sterol esterification by lecithin:Cholesterol acyltransferase

Reynold Homan, Nadia Esmaeil, Laurel Mendelsohn, Gregory J. Kato

Research output: Contribution to journalArticlepeer-review

Abstract

We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:Cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate, and measure scheme. This is possible by using the fluorescent sterol dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE nonfluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of nonradiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.

Original languageEnglish (US)
Pages (from-to)80-86
Number of pages7
JournalAnalytical biochemistry
Volume441
Issue number1
DOIs
StatePublished - Jan 1 2013

Keywords

  • Amphipathic peptide
  • Apolipoprotein A-I mimetic
  • Cholesterol oxidase
  • Dehydroergosterol
  • Dehydroergosteryl ester
  • Sickle cell disease

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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