AlphaCore Pharma, Ann Arbor, MI 48103, USA.
Anal Biochem. 2013 Oct 1;441(1):80-6. doi: 10.1016/j.ab.2013.06.018. Epub 2013 Jul 12.
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.
我们描述了一种简单但灵敏的荧光法，可准确检测卵磷脂胆固醇酰基转移酶（LCAT）的酯化活性。新的测定方案采用了方便的混合、孵育和测量方案。这是通过使用荧光甾醇脱氢麦角固醇（DHE）代替胆固醇作为 LCAT 底物来实现的。通过用两性肽代替载脂蛋白 A-I 作为脂质乳化剂和 LCAT 激活剂，进一步增强了测定方法。通过胆固醇氧化酶选择性地使未酯化的 DHE 无荧光，实现了 DHE 酯合成的特异性荧光检测。该测定法可准确检测缓冲液中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 LCAT 活性的分析证实了 SCD 中 LCAT 活性降低的先前报道，并表明血浆 LCAT 活性与 LCAT 含量之间存在很强的相关性。荧光测定法将放射性化学测定法的灵敏度与非放射性化学测定法的简单性相结合，可获得 LCAT 酯化活性的准确、稳健测量。
