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A simple and precise method to detect sterol esterification activity of lecithin/cholesterol acyltransferase by high-performance liquid chromatography
Analytical and Bioanalytical Chemistry ( IF 4.3 ) Pub Date : 2018-01-06 , DOI: 10.1007/s00216-017-0834-4
Yu Wang , Siming Wang , Lijiao Zhang , Jie Zeng , Ruiyue Yang , Hongxia Li , Yueming Tang , Wenxiang Chen , Jun Dong

The measurement of lecithin: cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity is important in high-density lipoprotein (HDL) metabolism study and cardiovascular disease (CVD) risk assessment. However, current methods suffer from complex design and preparation of exogenous substrate, low reproducibility, and interference of cofactors. In this study, we developed a simple and precise high performance liquid chromatography (HPLC) method for the measurement of LCAT activity. By using 7-dehydrocholesterol (7-DHC) and 1,2-didecanoyl-sn-glycero-3-phosphocholine(10:0PC) as substrates, and an LCAT activating peptide (P642) as activator and emulsifier, the substrate reagent was easily made by vortex. The substrate reagent was mixed with serum samples (50:1, v/v) and incubated at 37 °C for 1 h. After incubation, the lipid was extracted with hexane and ethanol. With a conjugated double bond and ultraviolet absorption, 7-DHC and its esterification product could be separated and analyzed by a single HPLC run without calibration. LCAT activity was a linear function of the serum sample volume and the intra- and total assay coefficients of variation (CV) less than 2.5% were obtained under the standardized conditions. The substrate reagent was stable, and assay result accurately reflected LCAT activity. LCAT activities in 120 healthy subjects were positively correlated with triglyceride (P < 0.05), fractional esterification rate of HDL cholesterol (FERHDL) (P < 0.0001), and negatively correlated with apolipoprotein AI (apoAI) (P < 0.05) and HDL cholesterol (HDL-C) (P < 0.001). These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components and added substances, and will be a useful tool in the lipid metabolism study and the risk assessment of CVD.



中文翻译:

高效液相色谱法检测卵磷脂/胆固醇酰基转移酶固醇酯化活性的简便方法

卵磷脂:胆固醇酰基转移酶(LCAT,EC 2.3.1.43)活性的测量在高密度脂蛋白(HDL)代谢研究和心血管疾病(CVD)风险评估中很重要。然而,当前的方法遭受外源底物的复杂设计和制备,低再现性以及辅因子的干扰。在这项研究中,我们开发了一种简单而精确的高效液相色谱(HPLC)方法来测量LCAT活性。通过使用7-脱氢胆固醇(7-DHC)和1,2-二癸酰基-sn-甘油-3-磷酸胆碱(10:0PC)作为底物,并使用LCAT活化肽(P642)作为活化剂和乳化剂,底物试剂很容易由涡旋制成。将底物试剂与血清样品(50:1,v / v)并在37°C下孵育1小时。温育后,用己烷和乙醇萃取脂质。通过共轭双键和紫外线吸收,可以分离7-DHC及其酯化产物,并通过一次HPLC进行分析,而无需校准。LCAT活性是血清样品量的线性函数,在标准条件下获得的内部和总分析变异系数(CV)小于2.5%。底物试剂稳定,测定结果准确反映了LCAT活性。120名健康受试者的LCAT活性与甘油三酸酯(P  <0.05),HDL胆固醇的部分酯化率(FER HDL)正相关(P <0.0001),并且与载脂蛋白AI(apoAI)呈负相关(P  <0.05)和HDL胆固醇(HDL-C)(P  <0.001)。这些结果表明,该方法灵敏,可重现,不受血清成分和添加物质的影响很大,将成为脂质代谢研究和CVD风险评估的有用工具。

更新日期:2018-01-06
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