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A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase
Reynold Homan1, Nadia Esmaeil, Laurel Mendelsohn
1AlphaCore Pharma, Ann Arbor, MI 48103, USA. reynh@comcast.net
Analytical Biochemistry
|July 16, 2013
Summary
A new fluorescence assay accurately measures lecithin:cholesterol acyltransferase (LCAT) activity using a fluorescent sterol and peptide activator. This sensitive method simplifies LCAT detection in plasma, aiding research in conditions like sickle cell disease.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Clinical Chemistry
Background:
- Lecithin:cholesterol acyltransferase (LCAT) plays a crucial role in high-density lipoprotein (HDL) metabolism and cholesterol reverse transport.
- Accurate measurement of LCAT activity is essential for understanding lipid disorders and related diseases.
- Existing LCAT assays can be complex, time-consuming, or lack sensitivity.
Purpose of the Study:
- To develop a simple, sensitive, and accurate fluorescence-based assay for measuring LCAT esterification activity.
- To utilize a fluorescent sterol (dehydroergosterol, DHE) and an amphiphilic peptide as replacements for traditional substrates and activators.
- To validate the assay's performance in buffer and plasma samples, including those from patients with sickle cell disease (SCD).
Main Methods:
- Employed dehydroergosterol (DHE) as a fluorescent substrate for LCAT.
- Used an amphiphilic peptide as a lipid emulsifier and LCAT activator, replacing apolipoprotein A-I.
- Incorporated cholesterol oxidase to selectively quench fluorescence of unesterified DHE, enabling specific detection of DHE esters.
- Tested the assay in buffer and in plasma depleted of apolipoprotein B lipoproteins.
Main Results:
- The developed fluorescence assay accurately quantifies LCAT esterification activity.
- The assay demonstrated high sensitivity, comparable to radiochemical methods, with improved simplicity.
- LCAT activity was accurately measured in both buffer and prepared plasma samples.
- Analysis of plasma from control subjects and SCD patients confirmed reduced LCAT activity in SCD and showed a strong correlation between LCAT activity and LCAT content.
Conclusions:
- The novel fluorescence assay provides a robust and convenient method for measuring LCAT activity.
- This assay simplifies LCAT activity assessment, making it accessible for broader research applications.
- The findings support the utility of this assay in clinical studies, particularly for investigating lipid metabolism in diseases like SCD.
