Serum Lipoprotein Cholesterol Determined By Ultracentrifugation And High-performance Liquid Chromatography

The different types of lipoproteins have been defined historically by ultracentrifugation.Lipoproteins can mainly be classified into the following fractions according to their hydrated densities:High density lipoproteins(HDL,1.063-1.12g/mL), HDL2(1.063-1.125),HDL3(1.125-1.21);Low density lipoproteins(LDL,1.006-1.063), small dense LDL(LDLb,1.044-1.063),large buoyant LDL(LDLa,1.019-1.044); intermediate density lipoproteins(IDL,1.006-1.019) and lipoprotein(a)[Lp(a),1.05-1.11]. LDL cholesterol actually contains the contributions of IDL and Lp(a) cholesterol in current clinical laboratory measurements.In ultracentrifugation,the Lp(a) density range overlaps those of both HDL and LDL.The recovered lipoprotin frations,especially LDLb and HDL2,are likely to be comtaminated with Lp(a).Lp(a) is formed by joining a lipoprotein that is structurally very similar to LDL to apo(a).In the Lp(a) particle,apo(a) is covalently linked to apoB by a single disulfide bridge.A small amount of Lp(a) or apo(a) is also noncovalently associated with apoB-rich lipoproteins.2-mercaptoethanol(ME) was used to dissociate Lp(a) in this study.The concentration of ME used,the effectiveness of the dissociation,HDL stability and the effect of proline were investigated.The results showed that 0.05mol/L ME can effectively dissociate Lp(a) into apo(a) and apo(a) depleted Lp(a)[Lp(a-)],and 0.1mol/L proline dissociate the interaction of Lp(a) with apoB-rich lipoproteins.The density range of Lp(a-) is similar to that of LDL,which enables the ultracentrifugation separation of HDL and LDL.Further studies demonstrated that there is a distinct density zone between Lp(a-) (＜1.040) and Lp(a)(1.05-1.11) and the density cut point between LDLa and LDLb (1.044) is coincidentaUy fall into this zone.Based on these findings,an ultracentrifugation method for separation of HDL,HDL2,HDL3,LDL,LDLa,LDLb and Lp(a) were established.A precise method is needed for determination of cholesterol concentrations in separated lipoprotein fractions.A modified HPLC method was developed by using merchandised stigmasterol as an internal standard and the Jones Oxidation for the oxidation of cholesterol and stigmasterol into cholest/stigmast-4-en-3,6-dione.This method is highly precise and can be used for detecting low levels of cholesterol concentrations in lipoprotein fractions.Based on the above investigations,an ultracentdfugation and HPLC method for determinations of serum lipoprotein cholesterol was established.Serum aliquots were centrifuged in the presence of proline and in the presence or absence of mercaptoethanol at densities of 1.125,1.063,1.044 and 1.006 g/mL for the separation of lipoprotein fractions. Cholesterol levels in the ultracentrifugation bottom fractions were analyzed by HPLC. Cholesterol concentrations of lipoprotein fractions were calculated as follows.HDLC = BF_1.063MECLDLC = BF_1.006C - BF_1.063MECLDLaC = BF_1.006C - BF_1.044CLDLbC = BF_1.044MEC - BF_1.063MECHDL2C = BF_1.063MEC - BF_1.125MECHDL3C = BF_1.125MECLp(a)-C = BF_1.044C - BF_1.044MECFor determination of the centrifugation bottom fraction(d=1.006,HDLC+LDLC), the results provided by ultracentrifugation and HPLC method were highly correlated with those provided by CDC reference method(r=0.998);For determination of HDLC,the results were highly correlated with those from CDC designated comparison method(DCM) (r=0.998).The total CVs for the measurement of HDLC and LDLC were 0.96%～2.07% and 0.65%～1.12%;The total CVs for the measurement of HDL2,HDL3,LDLa,LDLb and Lp(a) cholesterol were 0.85%-2.66%,0.87%-3.21%,0.86%-1.11%,2.59%-6.35%and 4.42%-12.29%,respectively.For determination of Lp(a) cholesterol,a more sensitive,two-step ultracentrifugation method was designed.Serum aliquots were centrifuged in the presence of proline at a density of 1.044 g/mL and centrifuged.The bottom fraction,which contains＞95%of Lp(a) particles,was readjusted with a density solution containing ME to a density of 1.040 to cause flotation of Lp(a-) in the next centrifugation.Cholesterol concentration in the top fraction was determined with HPLC.The two-step ultracentrifugation procedure increased the sensitivity and specificity of the method,The total CVs for the measurement of Lp(a)-C was 2.85-4.29%。In conclusion,an ultracentrifugation and HPLC method for determination of cholesterol concentrations in lipoprotein and their subfractions and Lp(a) has been developed.The new method has the following characteristics:(1) Lipoprotein and their subfractions are separated with ultracentrifugation,and the separated lipoproteins are in agreement with the widely used lipoprotein nomenclature;(2) Ultracentrifugation is a physical procedure in which the cholesterol transfers among lipoproteins are minimal.(3) Lipoprotein fractions and Lp(a) levels are all represented by their cholesterol levels,so a direct comparison is possible among these liporoteins.(4) The sample size is low,0.5mL is needed for determinations of 7 lipoprotein fractions.For each analytical run,20 samples can be analyzed.(5) This method is simple,easy to operate and precise,and may be used as a candidate reference method for determination of cholesterol concentrations in HDL, LDL,HDL and LDL subfractions and LP(a).