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. 2011 Feb;52(2):383-8.
doi: 10.1194/jlr.D008979. Epub 2010 Nov 1.

Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC

Jun Dong  1 , Hanbang Guo, Ruiyue Yang, Hongxia Li, Shu Wang, Jiangtao Zhang, Wenxiang Chen
Affiliations

Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC

Jun Dong et al. J Lipid Res. 2011 Feb.

Abstract

Simple and precise methods for LDL-cholesterol (LDL-C) and HDL-cholesterol (HDL-C) measurements are essential for assessment of cardiovascular disease (CVD) risks and for lipid and lipoprotein studies. We report here an ultracentrifugation (UC) and HPLC method that requires substantially less specimen volume and provides the necessary reliability and throughput required by large-volume, high-quality research and clinical studies. 2-Mercaptoethanol (ME) was used to dissociate serum lipoprotein [a] (Lp[a]) into apolipoprotein [a] and Lp[a] remnant (Lp[a-]) and eliminated the contamination of Lp[a] in HDL separated by UC. Serum aliquots were centrifuged at a density of 1.006 kg/l for the separation of HDL plus LDL, and in the presence of ME at a density of 1.063 kg/l for the separation of HDL. Cholesterol concentrations of the bottom fractions were analyzed by HPLC. LDL-C and HDL-C determined using this method were equivalent to those with β-quantification and the designated comparison method of the Centers for Disease Control. The total coefficient of variations for LDL-C and HDL-C were 0.65-1.12% and 0.96-2.07%, respectively. This method requires a small amount of specimen and is easy to operate. This method may be used in research or in clinical laboratories where precise and specific lipoprotein cholesterol analysis is needed.

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Figures

Fig. 1.
Fig. 1.
Correlation between 2-mercaptoethanol (ME) concentrations versus d = 1.063 kg/l ultracentrifuged bottom fraction cholesterol (BFC) concentrations (n = 6). Aliquots of mixed serum with Lp[a] mass of about 50 mg/dl were centrifuged in the presence of 0, 0.01, 0.02, 0.04, and 0.08 M of ME at a density of 1.063 kg/l. Serum was spun three times in duplicate at 23,000 rpm for 18.5 h (78,196 g) at 20°C, and BFC concentrations were measured by HPLC.
Fig. 2.
Fig. 2.
Differences between BFC1.063ME(−) and BFC1.063ME(+) versus Lp[a] mass. Serum samples from 49 individuals were ultracentrifuged in the presence and absence of 2-mercaptoethanol (ME) at a density of 1.063 kg/l. Bottom fraction cholesterol [BFC, BFC1.063ME(−), and BFC1.063ME(+)] was determined by HPLC. Serum Lp[a] mass was measured by immunoturbidimetric assay.
Fig. 3.
Fig. 3.
Western blot of apolipoprotein B (apoB) in centrifuged bottom fractions. Individual serum samples were spun at a background density of 1.063 kg/l in the presence and absence of 2-mercaptoethanol (ME). Aliquots of 50 μl of each bottom fraction were fractionated by 6% SDS-PAGE and immunoblotted with a sheep anti-human apoB polyclonal antibody. Lp[a] concentrations of samples 1, 2, 3, and 4 were 45.3, 50.5, 60.7, and 109.2 mg/dl, respectively.
Fig. 4.
Fig. 4.
Correlation (A) and relative difference (B) plots between ultracentrifugation/HPLC (UC/HPLC) and the modified β-quantification for the measurement of bottom fraction cholesterol (BFC) (LDL-C plus HDL-C) levels in 67 serum samples. For the UC/HPLC method, 50 μl serum samples were diluted with NaBr solutions and centrifuged at a density of 1.006. For the modified β-quantification, 0.8 ml serum samples were gravimetrically transferred into tubes and centrifuged. After UC, the bottom fractions were reconstituted gravimetrically. BFC was analyzed by HPLC. Relative difference is defined as the difference between UC/HPLC BFC and β-quantification BFC divided by β-quantification BFC.
Fig. 5.
Fig. 5.
Correlation (A) and relative difference (B) plots between ultracentrifugation/HPLC (UC/HPLC) and the designated comparison method (DCM) for the measurement of HDL-C in 124 serum samples. For the UC/HPLC method, serum solutions were centrifuged at a density of 1.063 kg/l in the presence of 0.05 M ME. For the DCM, the precipitation of apoB-containing lipoproteins was performed with dextran-sulfate. HDL-C concentrations were analyzed by the HPLC method. Relative difference is defined as the difference between UC/HPLC HDL-C and DCM HDL-C divided by DCM HDL-C. The total analytical error limits specified by the NCEP (<13%) are displayed (B).
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