4.5 Article

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

Journal

ANALYTICAL BIOCHEMISTRY
Volume 441, Issue 1, Pages 80-86

Publisher

ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2013.06.018

Keywords

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

Funding

  1. Small Business Innovation Research (SBIR) from the National Heart, Lung, and Blood Institute, National Institutes of Health [1 R43 HL092656-01A1]
  2. National Institutes of Health Division of Intramural Research [1 ZIA HL006013-04]

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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. (c) 2013 Elsevier Inc. All rights reserved.

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