Aktive edilmiş polietilen glikol ile modifiye edilen kolesterol esteraz ve kolesterol oksidaz kullanarak serumda doğrudan HDL kolesterol ölçümü

Abstract

SUMMARY Atherosclerotic disease is the major cause of death in our country and the world. Monitoring of HDL-C in serum is of a clinical importance, since an inverse correlation exits between serum HDL-C concentrations and the risk of these diseases. Several tecniques to determine HDL-C in serum have been described. Of these, ultrasantrifugation is time-consuming and require expensive materials, precipitation- based methods require pretreatment and are not amenable to automated analysis. Thus there is a great clinical need for developing a convenient and reliable method for measuring HDL-C in serum. In our study, we have automated method which have developed for measuring HDL-C in serum without any pretreatment for precipitating apolipoprotein B, using PEG-modified enzymes and sulfated a-CD. When cholesterol esterase and cholesterol oxidase enzymes were modified with PEG, they showed selective catalytic activities towards lipoprotein fractions (LDL< VLDL«chylomicron<HDL). In the precence of Mg2+ ions and dextran sülfat, a-CD sulfate reduced the reactivity of cholesterol especially in chylomicrons and VLDL. The combination of PEG-modified enzymes with a-CD sulfate provided selectivity for the determination of HDL-C in serum without the need for precipitation of those lipoprotein fractions. PEG used for CHER and CHOD modifications after it was activated by N- hydroxysuccinimide and dicyclohexylcarbodiimide. a-CD was sulfated by trimethylamine sulfate and dimethyl formamide. In the final format, reagent 1 was composed of a-CD sulfate (0.5 mmol/L), dextran sulfate (l^imol/L), MgCİ2 (2 mmol/L) and phenol (26 mmol/L) in 30 mmol/L MOPS buffer (pH 7.0); reagent 2 was composed of PEG-CHER (1kU/L), PEG-CHOD 62(5kU/L), peroxidase (30 kU/L) and 4-aminoantypyrine (2.5 mmol/L) in 30 mmol/L MOPS buffer (pH 7.0). The principle of this method which adaptated to autoanalyzer with end-point parameters in 1 st incubation step is; in the precence of MgCİ2, sulfated a-CD and dextran sulfate form water soluble complexes selectively with LDL, VLDL and chylomicrons, which are resistant to PEG-modified enzymes. In 2nd incubation step; the cholesterol concentration of HDL-C is determined enzymatically by PEG-CHER and PEG-CHOD. When the presence of modified enzymes, reduced the reactivities of cholesterol in LDL, VLDL and cyhilomicrons to, respectively, 10%, 50%, 80%. In the presence of MgCİ2, a-CD sulfate reduced the reactivities of cholesterol in VLDL to 25% and cyhlomicrons to 35%. Finally, the residual reactivities of cholesterol in the lipoprotein fractions other than HDL were completely abolished by adding of dekstran sulfate to the reagent system. With run impresicion (CV) of the HDL-C assay was (n=20), 1.44%, 0.72% and 0.98% at 220, 570, 850 mg/L HDL-C solutions in pooled sera. The between assay CV was 3.5% (n=20) and mean recovery was 102%. The established reference values (mean±2SD) were 506+1 04 mg/L. When the results of our assay (y) were compared with that of an utrasantrifugation (x<) and a NAP precipitation method (X2) for serum samples from with healthy, lipemic, icteric, the observed correlations were excellent (y=0. 85+1. 00xi, r=0.995, n=50; y= 1.1 9+0. 97x2, r=0.994, n=150). The method, we automated in our laboratory is simple and reliable for monitoring HDL-C in serum without the need for prior seperation of other fractions. 63