Open AccessDevelopment of a novel fluorescent activity assay for lecithin:cholesterol acyltransferase
Author(s) -
Toshihiro Sakurai,
Akiko Sakurai,
Boris Vaisman,
Tamotsu Nishida,
Edward B. Neufeld,
Stephen J. Demosky,
Maureen Sampson,
Robert D. Shamburek,
Lita A. Freeman,
Alan T. Remaley
Publication year - 2017
Publication title -
annals of clinical biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.6
H-Index - 80
eISSN - 1758-1001
pISSN - 0004-5632
DOI - 10.1177/0004563217733285
Subject(s) - cholesterol , lecithin , bodipy , sterol o acyltransferase , enzyme , chemistry , fluorescence , enzyme assay , lecithin—cholesterol acyltransferase , biochemistry , acyltransferase , substrate (aquarium) , chromatography , lipoprotein , biology , ecology , physics , quantum mechanics
Background Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme that esterifies cholesterol. Recombinant human LCAT (rhLCAT) is now being developed as an enzyme replacement therapy for familial LCAT deficiency and as a possible treatment for acute coronary syndrome. The current 'gold standard' assay for LCAT activity involves the use of radioisotopes, thus making it difficult for routine clinical use. Methods We have developed a novel and more convenient LCAT activity assay using fluorescence-labelled cholesterol (BODIPY-cholesterol), which is incorporated into proteoliposomes as a substrate instead of radiolabelled cholesterol. Results The apparent K m and V max were 31.5 µmol/L and 55.8 nmol/h/nmoL, rhLCAT, respectively, for the 3 H-cholesterol method and 103.1 µmol/L and 13.4 nmol/h/nmol rhLCAT, respectively, for the BODIPY-cholesterol method. Although the two assays differed in their absolute units of LCAT activity, there was a good correlation between the two test assays ( r = 0.849, P < 1.6 × 10 -7 , y = 0.1378x + 1.106). The BODIPY-cholesterol assay had an intra-assay CV of 13.7%, which was superior to the intra-assay CV of 20.8% for the radioisotopic assay. The proteoliposome substrate made with BODIPY-cholesterol was stable to storage for at least 10 months. The reference range ( n = 20) for the fluorescent LCAT activity assay was 4.6-24.1 U/mL/h in healthy subjects. Conclusions In summary, a novel fluorescent LCAT activity assay that utilizes BODIPY-cholesterol as a substrate is described that yields comparable results to the radioisotopic method.