A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase.
A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase.
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DOI：
10.1016/j.ab.2013.06.018
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发表时间：
2013-10-01
影响因子：
2.9
通讯作者：
Kato GJ
中科院分区：
文献类型：
--
作者：
Homan R;Esmaeil N;Mendelsohn L;Kato GJ
本文介绍了一种简单而灵敏的荧光法来准确地检测卵磷脂：胆固醇酰基转移酶（LCAT）的酯化活性。新的测定方案采用方便的混合、孵育和测量方案。这是可能的，通过使用荧光甾醇，脱氢麦角甾醇（DHE）代替胆固醇作为LCAT底物。通过掺入两亲性肽代替载脂蛋白A-I作为脂质乳化剂和LCAT激活剂，进一步增强了测定方法。DHE酯合成的特异性荧光检测通过采用胆固醇氧化酶选择性地使未酯化的DHE不发荧光来实现。该检测试剂盒可准确检测缓冲液和通过选择性沉淀去除载脂蛋白B脂蛋白的血浆中的LCAT活性。对来自对照受试者和镰状细胞病（SCD）患者的血浆中的LCAT活性的分析证实了先前关于SCD中LCAT活性降低的报道，并证明了血浆LCAT活性与LCAT含量之间的强相关性。荧光测定将放射化学测定的灵敏度与非放射化学测定的简单性相结合，以获得LCAT酯化活性的准确和稳健的测量。
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 non-fluorescent. 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 non-radiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.
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影响因子：
--
作者：
Charles-Schoeman, Christina;Watanabe, Junji;Reddy, Srinivasa T.
通讯作者：
Reddy, Srinivasa T.
影响因子：
7.3
作者：
Busseuil, D.;Shi, Y.;Tardif, J-C
通讯作者：
Tardif, J-C
DOI：
10.1016/0005-2760(73)90188-4
发表时间：
1973-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
MARCEL, YL;VEZINA, C
通讯作者：
VEZINA, C
DOI：
10.1016/s0378-4347(97)00651-8
发表时间：
1998-04-24
期刊：
JOURNAL OF CHROMATOGRAPHY B
影响因子：
--
作者：
Homan, R;Anderson, MK
通讯作者：
Anderson, MK
影响因子：
3.5
作者：
POWNALL, HJ;KNAPP, RD;MASSEY, JB
通讯作者：
MASSEY, JB