Evaluation of the dual-precipitation method for determination of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 in serum.

Abstract

We compared the dual-precipitation method for measurement of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 (Gidez et al., J Lipid Res 1982;23:1206-33) with density-gradient ultracentrifugation in a swinging-bucket rotor (Demacker et al., Clin Chem 1983;29:656-63). The concentration of dextran sulfate 15,000 (DS) needed for optimal accuracy of the HDL2-chol and HDL3-chol values was established empirically. At a DS concentration of 0.87 g/L, the values for HDL2-chol as well as for HDL3-chol in 88 sera did not differ significantly from those obtained by ultracentrifugation. The precision of the method was satisfactory and was related to the concentration. Nevertheless, the dual-precipitation method lacks specificity inasmuch as it produces no fractions that contain only one HDL subfraction. HDL2 and HDL3 each contained an equivalent amount of cholesterol from the other. At increasing DS concentrations, some radiolabeled HDL3 appeared to have precipitated prior to complete precipitation of HDL2. This lack of specificity can be tolerated in large-scale epidemiological studies for screening, but not in small-scale intervention studies or in assay of clinical samples, where better accuracy is needed and ultracentrifugation is preferred.

High-density lipoprotein subpopulations as substrates for the transfer of cholesteryl esters to very-low-density lipoproteins

“…Actually, the interaction in vitro of apo E with heparin and the precipitation of apo E-rich HDL with this glycosaminglycan, which have already been shown by others [29], may explain the high precipitation rate of HDL2 fraction C. In addition to apo E, the different content of apo A-II as compared with apo A-I (higher in fraction A than in fraction B) may also be responsible for the different behaviour of these fractions, since apo A-IT has been observed to be less effective than apo A-I in the formation of polyanion complexes [30]. Independently of the responsible mechanisms, our results demonstrate that the different HDL subpopulations behave differently under the precipitative action of heparin-Mn2 , which also accords with findings by Demacker et al [27], who observed, with 125I-labelled lipoproteins, that HDL2b (d = 1.063-1.100) was more readily precipitable with dextran sulphate-Mg2+ than was HDL2a (d = 1.100-1.125).…”

“…For obvious reasons we used ultracentrifugally isolated HDL, which could be altered in some undetermined way, and the medium in which they were precipitated was not identical with plasma or serum. In this respect, it is known that the conditions for use of this kind of precipitant reagents have been highly adjusted to precipitate VLDL and LDL (but not HDL) in whole serum or plasma [21], and a minor change in the final concentrations of reagents or pH may lead to the incomplete precipitation of VLDL or LDL, or even to the precipitation of a significant amount of HDL [20,26,27]. Therefore our observations do not necessarily reflect the behaviour of intact HDL in serum under the action of the precipitant reagents.…”

Clinical chemistry

“…A variety of assays exist to determine or purify cholesterol subfractions. New modifications have recently been described and evaluated: HDL (337)(338)(339)(340)(341)(342)(343)(344), VLDL (341), LDL (341,345,346), Lp(X) (347). There are three major methods to quantitate concentrations of apolipoproteins (303,304,348,349): chromatography, isoelectric focusing and immunoassays.…”

Association of High-Density Lipoprotein Subclasses with Carotid Intima-Media Thickness: Shimane CoHRE Study

“…In spite of increasing attention to physiological roles of the HDL subclasses, a limited number of reports have so far been available on the role of the HDL-C subclasses in cardiovascular diseases probably due to methodological difficulty in quantification of the subclasses 1 , 14 – 17 ) ; it is necessary to separate the two HDL subclasses by ultracentrifugation [HDL2 (1.063–1.125) and HDL3 (1.125–1.210)] 18 – 20 ) . Measurement of plasma concentration of HDL subclasses was therefore a laborious and time-consuming process, which hampers quantification of HDL2 and HDL3 in large-scale clinical and epidemiological studies.…”