Functional characterization of HDL in patients with type 2 diabetes using simple separation method of plasma HDL fraction

我们开发了一种从人血浆中分离HDL的简单方法，通过该方法可以同时评估2型糖尿病患者血浆中HDL功能的几种特性。研究对象为我科收治的14例2型糖尿病患者（男9例，女5例，年龄59.3±9.8岁）。实验室数据：HbA1c 8.4±1.2%,TC 181.6±26.2mg/dl, LDL-C 110.4±25.7 mg/dl, HDL-C 41.9±8.6mg/dl, TG 114.4±54.4mg/dl。空腹血浆样品应用聚丙烯酰胺凝胶电泳（PAGE）；Lipophor, Jokoh，日本，然后使用Phoreto-yield蛋白质收集装置（Jokoh，日本）回收尖锐的HDL部分，该装置配备了截止MW 15,000。在此过程中，在1小时内获得天然HDL分数。我们同时分析了几种天然HDL分数的功能标记：(1)葡萄糖氧化的羧甲基溶解酶（CML），(2)抗氧化的对氧磷酶-1 （PON-1）活性，(3)血清淀粉样蛋白A （SAA）的炎症。我们评估了每种分析方法的准确性。HDL分数的值与血浆水平的值相补偿。用ApoA-I conc校正血浆中HDL的回收率。大约70%。HDL恢复、血浆PON-1活性、血浆SAA及HDL分数的CV（变异系数）%均在25%左右。1) HDL分数中CML水平与血浆无差异（0.0574±0.0331 mg/mL）。2) HDL组PON-1活性（19.5±5.78 x104 U/L/min）高于血浆组（4.14±2.36 x104 U/L/min）。3) HDL分数中SAA水平（15.8±31.6 ng/mL）与血浆中SAA水平（18.1±21.6 ng/mL）相同。这些发现表明HDL抑制糖基化和脂质氧化的能力可能反映了HDL颗粒中特定酶和蛋白质成分的变化。

We developed a simple method to separate HDL fraction from human plasma, by which several properties of HDL function were possible to be evaluated simultaneously from plasma in type 2 diabetic patients.Subjects were 14 patients with type 2 diabetes mellitus (9 males, 5 females, age 59.3 ±9.8 y.o.) who were admitted to our department. Laboratory data were HbA1c 8.4±1.2%, TC 181.6±26.2mg/dl, LDL-C 110.4±25.7 mg/dl, HDL-C 41.9±8.6mg/dl and TG 114.4±54.4mg/dl. Fasting plasma sample was applied to polyacrylamide gel electrophoresis (PAGE); Lipophor, Jokoh, Japan, then sharp HDL fraction was recovered using Phoreto-yield protein collection device (Jokoh, Japan) equipped with cut-off MW 15,000. In this procedure, native HDL fraction was obtained within 1 hour. We analyzed several functional markers as follows in native HDL fraction simultaneously :(1) carboxymethyl-lysin (CML) for glucoxidation, (2) paraoxonase-1 (PON-1) activity for anti-oxidation (3) serum amyroid A protein (SAA) for inflmmation.We evaluated the accuracy of each analytical method. The values in HDL fraction were compensated with those in plasma levels. Recovery rate of HDL from plasma was corrected with ApoA-I conc. and about 70%. CV (coefficient of variation) % in HDL recovery, PON-1 activity and SAA in plasma and HDL fraction were about 25 %.1) CML level in HDL fraction was not detected different from plasma (0.0574±0.0331 mg/mL).2) PON-1 activities in HDL fraction (19.5±5.78 x104 U/L/min)were higher than those in plasma (4.14±2.36 x104 U/L/min).3) SAA levels in HDL fraction (15.8±31.6 ng/mL) were the same as those in plasma (18.1±21.6 ng/mL).These findings demonstrated that the ability of HDL to inhibit glycation and lipid oxidation may reflect changes in specific enzyme and protein components in HDL particles.