Method for quantitating cholesterol in subfractions of serum lipoproteins separated by gradient gel electrophoresis

Abstract

Extensive heterogeneity in particle size distribution of serum lipoproteins of baboons was resolved by a procedure that combined Sudan black B prestaining, polyacrylamide gradient gel electrophoresis (GGE), and quantitative densitometry. Each densitometric scan represented a continuous distribution of the relative amount of cholesterol in a serum sample, as a function of the lipoprotein particle size. For analytical purposes, each scan was divided into 12 fractions, representing 12 particle size ranges. The relationship between the estimated cholesterol concentrations in the summed GGE/densitometric fractions corresponding to very low-density lipoproteins (VLDL) + low-density lipoproteins (LDL) and those corresponding to high-density lipoproteins (HDL) and concentrations measured by the heparin-Mn2+ precipitation/enzymatic procedure was linear over a broad range. However, a systematic overestimation of HDL cholesterol concentration and an underestimation of VLDL + LDL cholesterol concentration was apparent. Therefore, correction factors were developed for adjusting the estimates of VLDL + LDL and HDL cholesterol concentrations obtained by the GGE/densitometric method. This analytical method is rapid, repeatable, economical, and useful for genetic and dietary research in which cholesterol concentrations in multiple particle size ranges of lipoproteins must be measured in large numbers of samples. It also is adaptable to immunoblotting procedures for detecting the distribution of specific apolipoproteins among the size-resolved lipoproteins.

A Genome Search Identifies Major Quantitative Trait Loci on Human Chromosomes 3 and 4 That Influence Cholesterol Concentrations in Small LDL Particles

“…LDL-C absorbance profiles from densitometric scans of gradient gels were fractionated into 4 subclasses as described in Methods. Several previous studies have reported a linear response of Sudan Black B staining to the amount of cholesterol in HDL and its subfractions, [33][34][35] although there appeared to be a consistent difference in chromogenicities of LDLs and HDLs (ie, amount of stain bound per unit of cholesterol). 35 Figure 1 shows the results of such comparisons for 11 pairs of samples.…”

“…Several previous studies have reported a linear response of Sudan Black B staining to the amount of cholesterol in HDL and its subfractions, [33][34][35] although there appeared to be a consistent difference in chromogenicities of LDLs and HDLs (ie, amount of stain bound per unit of cholesterol). 35 Figure 1 shows the results of such comparisons for 11 pairs of samples. The cholesterol concentrations in size fractions from the samples run separately (ie, expected) showed a strong linear relationship with those observed in the mixture (r 2 ϭ0.991), and the slope (0.93Ϯ0.01) and y intercept (0.4Ϯ0.1 nmol) were close to 1 and 0, respectively, which would be the expectation if all LDLs have identical chromogenicities.…”

Comparison of Various Electrophoretic Characteristics of LDL Particles and Their Relationship to the Risk of Ischemic Heart Disease

“…15 The absorbance profile with Sudan black staining was also assumed to closely reflect the cholesterol distribution among LDL particles of different sizes. 16 Thus, the absolute concentration of cholesterol among particles Ͻ255 Å was calculated by multiplying the total plasma LDL cholesterol levels by the relative proportion of LDL with a diameter Ͻ255 Å. A similar approach was used to assess the relative and absolute concentrations of cholesterol in particles with a diameter Ͼ260 Å.…”

Effect of type 2 diabetes on various electrophoretic characteristics of low-density lipoprotein particles in women

“…It has been documented that Sudan black stains mainly non-polar lipids [15]. The absorbance profile with Sudan black staining was also assumed to closely reflect the cholesterol distribution among LDL particles of different sizes [16]. The absolute concentration of cholesterol among particles smaller than 255 Å was calculated by multiplying plasma LDL cholesterol levels by the relative proportion of LDL with a diameter smaller than 255 Å.…”