AN OPTIMIZED METHOD FOR MEASURING LECITHIN - CHOLESTEROL ACYLTRANSFERASE ACTIVITY, INDEPENDENT OF THE CONCENTRATION AND QUALITY OF THE PHYSIOLOGICAL SUBSTRATE
AN OPTIMIZED METHOD FOR MEASURING LECITHIN - CHOLESTEROL ACYLTRANSFERASE ACTIVITY, INDEPENDENT OF THE CONCENTRATION AND QUALITY OF THE PHYSIOLOGICAL SUBSTRATE
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DOI：
10.1016/0005-2760(81)90055-2
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
SEIDEL, D
中科院分区：
文献类型：
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作者：
BARTHOLOME, M;NIEDMANN, D;SEIDEL, D
该研究的目的是实现卵磷脂：胆固醇酰基转移酶（EC 2.3.1.43）（LCAT）活性的测量，该活性独立于不同脂蛋白之间的脂质平衡，不受其摩尔比或其底物质量可能差异的影响。将磷钨酸钠/MgCl 2的混合物加入血清样品中，以实现所有血浆脂蛋白的完全沉淀。将含有总血浆LCAT活性、载脂蛋白A-I和主要血浆蛋白的滤液用于测定。以磷脂酰胆碱、胆固醇和磷酸二鲸蜡酯组成的脂质体为底物。在37 ℃温育60分钟后，用酶法测定游离胆固醇的减少。C.该测定遵循零级动力学，线性超过60分钟。以下Km值为各种底物获得：脂质体，0.43 mM; HDL [高密度脂蛋白]，0.63 mM; LDL [低密度脂蛋白]，0.0; VLDL [极低密度脂蛋白]，0.0;异常脂蛋白，发现胆汁淤积，（LP-X），0.0。与通常使用的LCAT测定方法的比较显示，健康对照组的活性相似，但是，在不同形式的血脂异常中，用这种方法获得了更高的值，在特殊情况下，通过加入载脂蛋白A-I可以增强活性。
The aim of the study was to achieve the measurement of lecithin:cholesterol acyltransferase (EC 2.3.1.43) (LCAT) activity that is independent of and uninfluenced by equilibration of lipids between different lipoproteins, by their molar ratios, or by possible differences in their substrate quality. A mixture of sodium phosphotungstic acid/MgCl2 was added to serum samples to achieve total precipitation of all plasma lipoproteins. The filtrate containing the total plasma LCAT activity, apolipoprotein A-I and major plasma proteins was used for the assay. Liposomes comprised of phosphatidylcholine, cholesterol and dicetylphosphate served as substrate. The decrease in free cholesterol was determined enzymatically after incubation for 60 min at 37.degree. C. The assay followed zero-order kinetics and was linear for more than 60 min. The following Km values for various substrates were obtained: liposomes, 0.43 mM; HDL [high density lipoprotein], 0.63 mM; LDL [low density lipoprotein], 0.0; VLDL [very low density lipoprotein], 0.0; abnormal lipoprotein, found in cholestasis, (LP-X), 0.0. Comparison with generally used LCAT assay methods revealed similar activities for healthy controls, but, in different forms of dyslipoproteinemia, higher values were obtained with this method and, in special cases, the activity could be enhanced by addition of apolipoprotein A-I.