Innovatively Established Analysis Method for Lipoprotein Profiles Based on High-Performance Anion-Exchange Liquid Chromatography

摘要

Separation analysis of lipoprotein classes have various methods, including ultracentrifugation, electrophoresis, and gel permeation chromatography (GPC). All major lipoprotein classes can be separated via ultracentrifugation, but performing the analysis takes a long time. Low-density lipoprotein (LDL), intermediate-density lipoprotein (IDL), and very low-density lipoprotein (VLDL) in patient samples cannot be sufficiently separated via electrophoresis or GPC. Thus, we established a new method [anion-exchange high-performance liquid chromatography (AEX-HPLC)] by using HPLC with an AEX column containing nonporous gel and an eluent containing chaotropic ions. AEX-HPLC can separate five lipoprotein fractions of high-density lipoprotein (HDL), LDL, IDL, VLDL, and others in human serum, which can be used in substitution for ultracentrifugation method. The method was also approved for clinical use in the public health-care insurance in Japan in 2014. Furthermore, we developed an additional method to measure cholesterol levels of the four leading lipoprotein fractions and two subsequent fractions (i.e., chylomicron and lipoprotein(a)). We evaluated the clinical usefulness of AEX-HPLC in patients with coronary heart disease (CHD), diabetes, and kidney disease and in healthy volunteers. Results indicate that the cholesterol levels in IDL and VLDL measured by AEX-HPLC may be useful risk markers of CHD or diabetes. Furthermore, we developed another new method for the determination of alpha-tocopherol (AT) in lipoprotein classes, and this method is composed of AEX-HPLC for the separation of lipoprotein classes and reverse-phase chromatography to separate AT in each lipoprotein class. The AT levels in LDL were significantly correlated with the lag time to copper ion-induced LDL oxidation, which is an index of oxidation resistance. The application of AEX-HPLC to measure various substances in lipoproteins will be clinically expected in the future.