Warnick G R, Albers J J
J Lipid Res. 1978 Jan;19(1):65-76.
The accurate quantitation of high density lipoproteins has recently assumed greater importance in view of studies suggesting their negative correlation with coronary heart disease. High density lipoproteins may be estimated by measuring cholesterol in the plasma fraction of d > 1.063 g/ml. A more practical approach is the specific precipitation of apolipoprotein B (apoB)-containing lipoproteins by sulfated polysaccharides and divalent cations, heparin-Mn(2+) being the most commonly used combination. The present heparin-Mn(2+) procedure was found to be reasonably specific and not often subject to large errors; however, 9% (primarily hypertriglyceridemic samples) of the 966 plasma samples treated with heparin-Mn(2+) had obvious supernatant turbidity, indicating incomplete sedimentation of apoB-associated lipoproteins. Furthermore, 48% of the nonturbid supernates contained more than 1 mg/dl (mean 2.5 mg/dl) of apoB-associated cholesterol when measured by a radial immunodiffusion procedure, indicating slight overestimation of HDL cholesterol. Determination of the extent of the unprecipitated apoB-associated lipoproteins by sensitive radioimmunoassay and of the amount of precipitated high density lipoprotein by radial immunodiffusion assay of apolipoproteins A-I and A-II at various heparin and Mn(2+) concentrations indicated that the usual heparin level (approximately 1.3 mg/ml) was adequate. However, a twofold increase in Mn(2+) concentration to 0.092 M improved precipitation of the apoB-associated lipoproteins without excessive precipitation of high density lipoprotein from plasma. This increased Mn(2+) level also provided improved sedimentation of the apoB-associated lipoproteins from hypertriglyceridemic plasma. Additional observations suggested that, for convenience, the heparin and Mn(2+) can be added simultaneously as a combined reagent, that samples can be incubated for 10 minutes at room temperature before centrifugation, and that turbid supernates from hypertriglyceridemic samples can usually be made free of apoB-associated lipoproteins by centrifugation at 12,000 g for 10 minutes.
鉴于一些研究表明高密度脂蛋白与冠心病呈负相关，其准确定量最近变得更加重要。高密度脂蛋白可通过测量密度大于1.063 g/ml的血浆部分中的胆固醇来估算。一种更实用的方法是用硫酸化多糖和二价阳离子特异性沉淀含载脂蛋白B（apoB）的脂蛋白，肝素 - 锰（2+）是最常用的组合。发现目前的肝素 - 锰（2+）方法具有合理的特异性，且不常出现大的误差；然而，在用肝素 - 锰（2+）处理的966份血浆样本中，有9%（主要是高甘油三酯血症样本）的上清液有明显浑浊，表明与apoB相关的脂蛋白沉淀不完全。此外，通过放射免疫扩散法测量，48%的非浑浊上清液中含有的与apoB相关的胆固醇超过1 mg/dl（平均2.5 mg/dl），这表明对高密度脂蛋白胆固醇略有高估。通过敏感的放射免疫测定法测定未沉淀的与apoB相关的脂蛋白的程度，并通过在不同肝素和锰（2+）浓度下对载脂蛋白A - I和A - II进行放射免疫扩散测定来测定沉淀的高密度脂蛋白的量，结果表明通常的肝素水平（约1.3 mg/ml）是足够的。然而，将锰（2+）浓度增加两倍至0.092 M可改善与apoB相关的脂蛋白的沉淀，而不会使血浆中的高密度脂蛋白过度沉淀。这种增加的锰（2+）水平也改善了高甘油三酯血症血浆中与apoB相关的脂蛋白的沉淀。其他观察结果表明，为方便起见，肝素和锰（2+）可作为组合试剂同时添加，样本在离心前可在室温下孵育10分钟，高甘油三酯血症样本的浑浊上清液通常可通过在12,000 g下离心10分钟去除与apoB相关的脂蛋白。
