A homogeneous assay to determine high-density lipoprotein subclass cholesterol in serum.

Existing methods to measure high-density lipoprotein cholesterol (HDL-C) subclasses (HDL2-C and HDL3-C) are complex and require proficiency, and thus there is a need for a convenient, homogeneous assay to determine HDL-C subclasses in serum. Here, cholesterol reactivities in lipoprotein fractions [HDL2, HDL3, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL)] toward polyethylene glycol (PEG)-modified enzymes were determined in the presence of varying concentrations of dextran sulfate and magnesium nitrate. Particle sizes formed in the lipoprotein fractions were measured by dynamic light scattering. We optimized the concentrations of dextran sulfate and magnesium nitrate before assay with PEG-modified enzymes to provide selectivity for HDL3-C. On addition of dextran sulfate and magnesium nitrate, the sizes of particles of HDL2, LDL, and VLDL increased, but the size of HDL3 fraction particles remained constant, allowing only HDL3-C to participate in coupled reactions with the PEG-modified enzymes. In serum from both healthy volunteers and patients with type 2 diabetes, a good correlation was observed between the proposed assay and ultracentrifugation in the determination of HDL-C subclasses. The assay proposed here enables convenient and accurate determination of HDL-C subclasses in serum on a general automatic analyzer and enables low-cost routine diagnosis without preprocessing.

Chemical modification of high density lipoprotein subfractions - HDL2 and HDL3 - after use of atorvastatin.

Regardless of its effect on the concentrations of serum cholesterol, statins exert pleiotropic effects, including the regulation of endothelial function, reduced oxidative stress and inflammation, as well as a slight improvement in the concentrations of high density lipoprotein (HDL). However, its role on the composition of HDL is not yet established. The aim of this study was to evaluate the composition of HDL subfractions, HDLsub>2 and HDL3, after 14 days of placebo and atorvastatin (10 mg/day) use in 30 asymptomatic volunteers. The serum parameters and the HDL subfractions compositions were determined using radiometric, nephelometric and biochemical enzymatic methods. We observed significant reductions of total cholesterol, low density lipoprotein (LDL) and apolipoprotein B-100 by 28%, 40% and 38%, respectively. The analyses of chemical composition of the subfractions revealed a lower lipid protein ratio in HDL2, suggesting enrichment in proteins, and also lower in HDL3, probably by an increase in the number of particles. Several mechanisms can be suggested for the effects observed after the use of atorvastatin, such as a possible action on the reverse cholesterol transport (decreased activity of hepatic lipase and increased phospholipid transfer protein, PLTP), which would explain the enrichment of HDL. The results suggest that statin use may be relevant in the primary prevention of atherosclerosis not only by its lowering effect on LDLcholesterol and its anti-inflammatory effect but also by beneficial changes in HDL subfractions.

FT-IR spectroscopy of lipoproteins--a comparative study.

FT-IR spectra, in the frequency region 4000-600 cm(-1), of four major lipoprotein classes: very low density lipoprotein (VLDL), low density lipoprotein (LDL) and two subclasses of high density lipoproteins (HDL(2) and HDL(3)) were analyzed to obtain their detailed spectral characterization. Information about the protein domain of particle was obtained from the analysis of amide I band. The procedure of decomposition and curve fitting of this band confirms the data already known about the secondary structure of two different apolipoproteins: apo A-I in HDL(2) and HDL(3) and apo B-100 in LDL and VLDL. For information about the lipid composition and packing of the particular lipoprotein the well expressed lipid bands in the spectra were analyzed. Characterization of spectral details in the FT-IR spectrum of natural lipoprotein is necessary to study the influence of external compounds on its structure.