The Development Of Homogeneous Method For Detection Of Small Dense Low-density Lipoprotein

Small dense low-density lioprotein (sd-LDL) has been established as an important indicator for the development of atherosclerosis. It is significant to detect the levels of sd-LDL for the diagnosis and judgment of the atherosclerotic cardiovascular disease. However, the existing methods for the detection of the levels of sd-LDL could not be applied in the clinical. Thus, it is urgent to explore a more simple method which could be applied in the clinical with high precision and accuracy. Recently, a homogeneous assay for sd-LDL cholesterol (sd-LDL-C) equiped with autoanalyzers was reported, while it is based on the surfactant with high capacibility of elimination of specific lipoprotein.Based on the selective dissociating interaction between surfactant and specific lipoprotein, this study was undertaken to investigate the new method for the determination the concentration of sd-LDL. First of all, we used ultracentrifugation to separate and purificate components of serum lipoprotein, and optimized the separation conditions to get the best density lipoprotein parameters for the extraction of high purity various subtypes of lipoprotein. Secondly, more than hundreds of commercial surfactants, including cationic, anionic and nonionic were choosed to research the relationship between the structural characteristics and selective dissociating effect on specific lipoprotein. The results showed that the lipoprotein disintegrating effect of non-ionic surfactant was stronger when HLB value ranges from10to14, and those surfactants EM701and JK-14, which are non-ionic surfactants with structure of polyethylene glycol, exhibited the selective dissociating effect on high-density lipoprotein cholesterol (HDL-C). Then, we designed and prepared more than30kinds of non-ionic surfactant possessing structure of polyethylene glycol, to screen the required surfactant with highly selective dissociating effect on the specific lipoprotein. The results showed that the surfactant polyethylene glycol3-benzyl oxo phenyl ether showed higher degree of dissociation of HDL, very low-density lipoprotein and chylomicron. Finally, the kit for the detection of sd-LDL was prepared with the surfactant polyethylene glycol3-benzyl oxo phenyl ether, and was applied to the determination of the high, medium and low concentration of serum sd-LDL-C, respectively. This new sd-LDL-C kit was found to be available for the medium concentration of serum sd-LDL-C, but couldn’t be used in testing the high and low concentration of serum sd-LDL-C results with low sensitivity, which remained to be further research and improvement.