REACTIVITY OF LECITHIN-CHOLESTEROL ACYL TRANSFERASE (LCAT) TOWARDS GLYCATED HIGH-DENSITY-LIPOPROTEINS (HDL)
REACTIVITY OF LECITHIN-CHOLESTEROL ACYL TRANSFERASE (LCAT) TOWARDS GLYCATED HIGH-DENSITY-LIPOPROTEINS (HDL)
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DOI：
10.1016/0009-8981(94)05975-x
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发表时间：
1995-01-31
影响因子：
5
通讯作者：
MOATTI, N
中科院分区：
文献类型：
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作者：
FOURNIER, N;MYARA, I;MOATTI, N
糖尿病患者的高血糖导致血浆蛋白的非酶糖化，包括脂蛋白，例如高密度脂蛋白（HDL）。我们研究了体外 HDL 糖化对卵磷脂胆固醇酰基转移酶 (LCAT) 活性的影响，LCAT 是 HDL 血浆代谢的关键酶。 LCAT 是从非糖尿病受试者中制备的，HDL 是通过连续密度超速离心（密度范围为 1.063-1.21 g/ml）从糖尿病和非糖尿病患者中制备的。通过将脂蛋白与100mmol/l葡萄糖在37℃下以氰基硼氢化钠作为还原剂孵育不同时间，将非糖尿病患者的HDL体外糖化。 HDL 蛋白的糖化通过使用 TNBS 测定测量衍生氨基酸残基的百分比来定量。首先使用非糖尿病患者的天然 HDL 和体外糖化 HDL 测定 LCAT 的动力学参数。对于天然 HDL，K-m 和 V-max 分别为 51.1 +/- 4.2 mu mol/l (n = 8) 和 12.9 +/- 2.4 nmol/ml/h (n = 8)。酶反应性（以 V-max/K-m 比率计算）为 0.25 +/- 0.04 h(-1) (n = 8)。在中度糖化的情况下（衍生残基 < 30%；n = 19），观察到 K-m（18.2 +/- 3.4%；平均值 +/- S.D.）和 V-max（9.3 +/- 2.4%）显着增加。相反，当糖化水平较高时（衍生残基> 30%；n = 8），两个参数均下降（K-m，25 +/- 6.3%；V-max，34.1 +/- 3.3%）。此外，无论糖化水平如何，在体外糖化 HDL 存在下，酶反应性均较低。 LCAT 反应性的降低既不是由于过氧化过程，也不是由于体外糖化 HDL 的蛋白质和脂质组成的改变。然而，这可以通过载脂蛋白 A-I 中赖氨酸残基的糖化来解释，载脂蛋白 A-I 是 LCAT 最有效的激活剂。在第二系列实验中，使用天然糖尿病 HDL 制剂作为 LCAT 底物。未观察到 K-m 值发生变化，但 V-max (28%) 和酶反应性 (32%) 均显着下降。天然糖尿病 HDL 和糖化水平较低的体外糖化 HDL 之间 K-m 和 V-max 变化的差异可能是由生理修饰（糖化除外）的影响来解释的，糖化可能对糖尿病患者 HDL 的化学物理特性产生不同的影响。总之，在天然糖尿病 HDL 和体外糖化 HDL 中观察到的 LCAT 反应性降低可能影响 HDL 的反向胆固醇转运，从而有助于糖尿病患者的动脉粥样硬化过程。
Hyperglycaemia in diabetic patients results in non-enzymatic glycation of plasma proteins, including lipoproteins such as high-density lipoproteins (HDL). We studied the effects of in vitro HDL glycation on the activity of lecithin-cholesterol acyl transferase (LCAT), a key enzyme in HDL plasma metabolism. LCAT was prepared from non-diabetic subjects and HDL by sequential density ultracentrifugation (in the density range of 1.063-1.21 g/ml) from both diabetic and non-diabetic patients. HDL from non-diabetic patients were glycated in vitro by incubating lipoproteins with 100 mmol/l glucose for various times at 37 degrees C with sodium cyanoborohydride as reducing agent. Glycation of HDL protein was quantified by measuring the percentage of derived amino acid residues using the TNBS assay. Kinetic parameters of LCAT were first determined using native HDL from non-diabetic patients and in vitro glycated HDL. With native HDL, K-m and V-max were 51.1 +/- 4.2 mu mol/l (n = 8) and 12.9 +/- 2.4 nmol/ml/h (n = 8), respectively. Enzyme reactivity, calculated as the V-max/K-m, ratio, was 0.25 +/- 0.04 h(-1) (n = 8). In the case of moderate glycation (derived residues < 30%; n = 19) a significant increase in both K-m (18.2 +/- 3.4%; mean +/- S.D.) and V-max (9.3 +/- 2.4%) was observed. In contrast, with a high level of glycation (derived residues > 30%; n = 8), both parameters fell (K-m, 25 +/- 6.3%; V-max, 34.1 +/- 3.3%). In addition, whatever the level of glycation, enzyme reactivity was lower in the presence of in vitro glycated HDL. This decrease in LCAT reactivity was not due to a peroxidative process nor to an alteration of the protein and lipid composition of in vitro glycated HDL. It could, however, be explained by glycation of lysine residues in apolipoprotein A-I, which is the most potent activator of LCAT. In a second series of experiments, native diabetic HDL preparations were used as LCAT substrate. No alteration in K-m values was observed, but there was a significant decrease in both V-max (28%) and enzyme reactivity (32%). This difference in K-m and V-max alterations between native diabetic HDL and in vitro glycated HDL with low levels of glycation might be explained by the impact of physiological modifications, other than glycation, which could differently affect the chemicophysical properties of HDL in diabetic patients. In conclusion, the decrease in LCAT reactivity observed with both native diabetic HDL and in vitro glycated HDL could affect the reverse cholesterol transport of HDL and thereby contribute to the atherosclerotic process in diabetic patients.