A micro-enzymatic method to measure cholesterol and triglyceride in lipoprotein subfractions separated by density gradient ultracentrifugation

A micro-enzymatic method was developed to measure total cholesterol (CHOL) and triglyceride (TG) in lipoproteins and their subfractions separated by density gradient ultracentrifugation. This method had a detection limit and sensitivity below 2 mg/dl and accuracy (bias to reference sera) and imprecision (coefficient of variation) of less than 3% between 2 and 30 mg/dl for both CHOL and TG. In addition, the method was in good agreement with standardized Abell-Kendall CHOL (T = 0.98) and enzymatic TG (r = 0.99) methods. Lipoproteins from 200 pl of plasma or serum were separated by either equilibrium (EQor rate zonal (RZ)-density gradient ultracentrifugation and the resulting fractions were analyzed for CHOL and TG by the micro-enzymatic method. Lipoprotein measurements by these micro-enzymatiddensity gradient methods were highly correlated with standardized Lipid Research Clinic (LRC) procedures and preparative ultracentrifugation. The EQ-density gradient procedure also allowed determination of CHOL and TG in LDL and HDL subfractions within any desired density interval. These methods will facilitate the measurement and study of lipoproteins and their subfractions especially in infants, children, the elderly, and small animals. In addition, the micro-enzymatic method may be adapted to other modes of lipoprotein separation such as liquid chromatography, electrophoresis, and precipitation. CHOL or TG determinations could be made on approximately 500 density gradient fractions per hour. -Belcher, J. D., J. 0. Egan, G . Bridgman, R. Baker, and J. M. Flack. A micro-enzymatic method to measure cholesterol and triglyceride in lipoprotein subfractions separated by density gradient ultracentrifugation from 200 microliters of plasma or serum.J. Lipid Res. 1991. 32: 359-370. Supplementary key words low density lipoproteins high density lipoproteins particles has been associated with a greater risk for nonfatal myocardial infarction (14) and CAD (8). Also, changes in levels of large, less dense LDL (IDL or LDLJ have been positively associated with progression of CAD (10). Levels of both large and small LDL subspecies appear to be positively linked to plasma TG levels (9, 14). Likewise, the HDL2 subfraction has been reported to be more correlated with protection against CAD than the smaller, more dense HDL3 subfraction (18). HDL, appears to be negatively linked to plasma TG levels (20). LDL and HDL subspecies have been characterized by techniques such as polyacrylamide gradient gel electrophoresis (7, 9), density gradient ultracentrifugation (6, 7, 11, 12), precipitation (21), and preparative and analytical ultracentrifugation (2, 22). Some of these methods (particularly those used for measuring LDL subspecies) quantitate subspecies by indirectly measuring particle mass with densitometry or schlieren optical patterns. Measurement of particle mass is informative, but routine measurement of particle composition would be desirable. Further metabolic understanding of these subspecies has been hindered by the lack of easy and rapid micromethods for measuring their composition. This paper describes a n accurate and precise micro-enzymatic method to measure the CHOL and TG concentrations of dilute lipoproteins and their subfractions. Abbreviations: VLDL. very low densitv lipoproteins: IDL. interme. I , 1 . Lipoprotein subfractions or subspecies exist within diate density lipoproteins; LDL, low density lipoproteins; Lp[a], lipoprotein[a]; HDL, high density lipoproteins; VHDL, very high density lipoproteins: CHOL. cholesterol; 'E. trielvceride: LRC. Lipid ReVLDL, LDL, and HDL (l). These subspecies have been _ . . . . . . shown to differ in size, density, charge, composition, search Clinics; EDTA, ethylenediamine tetraacetic acid; CHD, coronary metabolism, and risk for CHD (2-19). Differences in the termining CHD risk. An increase of small, dense LDL heart disease; MI, myocardial infarction; CAD, coronary artery disease; EQ, equilibrium or isopycnic; RZ, rate zonal; CDC, Centers for Discient of variation. concentration Of these subspecies may be important in deease Control; ELISA, enzyme-linked immunosorbent assay; CV, co&Journal of Lipid Research Volume 32, 1991 359 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom MATERIALS AND METHODS