Dual-precipitation method evaluated for determination of high-density lipoprotein (HDL), HDL2, and HDL3 cholesterol concentrations

We evaluated a dual-precipitation method for determining cholesterol in high-density lipoprotein (HDL) and its subfractions HDL2 and HDL3. After total HDL was isolated by precipitation of very-low-density (VLDL) and low-density (LDL) lipoproteins with polyethylene glycol (Mr 8000), HDL2 was isolated from total HDL by precipitation with dextran sulfate (Mr 15,000), leaving HDL3 in the supernate. Concentration of total HDL cholesterol after precipitation of VLDL and LDL with PEG showed significant proportional and constant biases of -3.8% and 0.04 mmol/L, respectively, when compared with a phosphotungstic acid-based comparison method, although results by the two methods were correlated highly (r = 0.99, P less than 0.001). HDL2 and HDL3 cholesterol concentrations measured with the present technique were not different from those obtained by density-gradient ultracentrifugation or by combined precipitation-ultracentrifugation.     

Measurement of total HDL, HDL2 and HDL3 by dextran sulfate-MgCl2 precipitation technique in human serum

We describe a simple and reliable method for determination of total HDL, HDL2, and HDL3 by a precipitation technique using dextran-sulfate (Mr 50,000)-Mg2+. A combined solution of dextran sulfate and Mg2+ at their respective final concentration of 0.9 g/l and 27 mmol/l was optimal for separating total HDL from the other lipoproteins. The present method compares favorably with a heparin-MnCl method (r = 0.998). The HDL was further resolved into HDL2 and HDL3 by addition of a combined solution of dextran sulfate and Mg2+ (1.5 m/l and 10 g/l) to the total HDL solution. Comparison of this precipitation method with the well-established ultracentrifugation method yielded the mean correlation coefficient of 0.941 and 0.869 for HDL2 and HDL3, respectively.     

Serum paraoxonase-1 activity is more closely related to HDL particle concentration and large HDL particles than to HDL cholesterol in Type 2 diabetic and non-diabetic subjects

Objectives

We determined relationships of the anti-oxidative enzyme, paraoxonase-1 (PON-1), with high density lipoprotein (HDL) subfractions, and tested whether these relationships are stronger than those with HDL cholesterol and apolipoprotein A-I (apoA-I) in subjects with and without type 2 diabetes mellitus (T2DM).

Design and methods

Serum PON-1 (arylesterase activity) and HDL subfractions (nuclear magnetic resonance spectroscopy) were determined in 67 T2DM patients and in 56 non-diabetic subjects.

Results

PON-1 activity, HDL cholesterol and apoA-I were decreased in T2DM (all p < 0.05). The HDL particle concentration was unaltered, but large HDL particles, medium HDL particles and HDL particle size were decreased, whereas small HDL particles were increased in T2DM (all p < 0.05). PON-1 was more closely related to HDL cholesterol than to apoA-I (p = 0.001). In turn, the positive relationship of PON-1 with the HDL particle concentration and with large HDL particles was stronger than that with HDL cholesterol (both p < 0.01). The inverse relationship of PON-1 with T2DM was only modestly attenuated by HDL cholesterol or HDL particle characteristics.

Conclusions

PON-1 activity is more closely related to the HDL particle concentration or large HDL particles than to HDL cholesterol. Impaired PON-1 activity in T2DM is not to a considerable extent explained by altered HDL subfraction levels.     

Problems with PEG-based precipitation methods in the determination of HDL2- and HDL3-cholesterol

In this study a comparison of two polyethylene glycol (PEG)-based precipitation methods, that is, one using PEG-6000/DS-15000 and the other using two different concentrations of PEG-20000 are presented. The total HDL-cholesterol values measured with the PEG-6000 procedure were higher (13%) than those measured with PEG-20000 (r=0.91) in 16 pooled serum samples tested. The HDL₂- and HDL₃-cholesterol values measured with the two methods differed markedly from each other in spite of the moderate correlation (HDL₂: r=0.74; HDL₃: r=0.26) observed. The advantage of the PEG-20000 method as compared with the other method was its simplicity and rapidity. The separation of HDL₂/HDL₃ is more or less arbitrary in both methods used and further studies are needed for the estimation of optimal conditions for subclass separation.     

Precipitation with polyethylene glycol and density-gradient ultracentrifugation compared for determining high-density lipoprotein subclasses HDL2 and HDL3

The purpose of this study was to compare quantification of cholesterol in high-density lipoprotein subfractions HDL2 and HDL3 by precipitation with polyethylene glycol (PEG) with that by density-gradient ultracentrifugation. Fresh serum samples from 32 fasting, obese children were analyzed with precipitation reagent "Quantolip" (Immuno AG), and then fractionated with a Beckman TL 100 ultracentrifuge with a swinging-bucket rotor. After centrifugation we carefully removed the supernate with a syringe and measured the cholesterol from each fraction enzymatically with CHOD-PAP reagent (Boehringer Mannheim). The low-density lipoprotein (LDL-), HDL2-, and HDL3-cholesterol values measured by ultracentrifugation did not differ significantly from those obtained by precipitation; the correlation coefficients (r) between the two methods were 0.96 for LDL, 0.75 for HDL2, and 0.96 for HDL3. The relatively simple PEG precipitation method used in this study measures total HDL and its major subclasses HDL2 and HDL3 with accuracy and precision comparable with those of the well-established ultracentrifugation method.     

Problems with PEG-based precipitation methods in the determination of HDL2- and HDL3-cholesterol

In this study a comparison of two polyethylene glycol (PEG)-based precipitation methods, that is, one using PEG-6000/DS-15000 and the other using two different concentrations of PEG-20000 are presented. The total HDL-cholesterol values measured with the PEG-6000 procedure were higher (13%) than those measured with PEG-20000 (r = 0.91) in 16 pooled serum samples tested. The HDL2- and HDL3-cholesterol values measured with the two methods differed markedly from each other in spite of the moderate correlation (HDL2: r = 0.74; HDL3: r = 0.26) observed. The advantage of the PEG-20000 method as compared with the other method was its simplicity and rapidity. The separation of HDL2/HDL3 is more or less arbitrary in both methods used and further studies are needed for the estimation of optimal conditions for subclass separation.     

Laboratory assessment of HDL heterogeneity and function

BACKGROUND: Plasma concentrations of HDL cholesterol (HDL-C) and its major protein component apolipoprotein (apo) A-I are strongly inversely associated with cardiovascular risk, leading to the concept that therapy to increase HDL-C and apoA-I concentrations would be antiatherosclerotic and protective against cardiovascular events. The recent failure of the drug torcetrapib, a cholesteryl ester transfer protein inhibitor that substantially increased HDL-C concentrations, has brought focus on the issues of HDL heterogeneity and function as distinct from HDL-C concentrations. CONTENT: This review addresses the current state of knowledge regarding assays of HDL heterogeneity and function and their relationship to cardiovascular disease. HDL is highly heterogeneous, with subfractions that can be identified on the basis of density, size, charge, and protein composition, and the concept that certain subfractions of HDL may be better predictors of cardiovascular risk is attractive. In addition, HDL has been shown to have a variety of functions that may contribute to its cardiovascular protective effects, including promotion of macrophage cholesterol efflux and reverse cholesterol transport and antiinflammatory and nitric oxide-promoting effects. SUMMARY: Robust laboratory assays of HDL subfractions and functions and validation of the usefulness of these assays for predicting cardiovascular risk and assessing response to therapeutic interventions are critically important and of great interest to cardiovascular clinicians and investigators and clinical chemists.     

The double jeopardy of HDL

The ability of high-density lipoprotein (HDL) to promote cholesterol efflux is thought to be important in its protection against cardiovascular disease. Anti-inflammatory properties of HDL have emerged as additional properties that may also be important. HDL appears to have evolved as part of the innate immune system functioning to inhibit inflammation in the absence of an acute phase response (APR) but functioning to increase inflammation in the presence of an APR. Inbred strains of mice that are genetically susceptible to atherosclerosis have pro-inflammatory HDL, while inbred strains that are resistant to atherosclerosis have anti-inflammatory HDL. In one small study, humans with coronary heart disease (CHD) or CHD equivalents had pro-inflammatory HDL prior to statin therapy and about half continued to have pro-inflammatory HDL after statin therapy despite a profound decrease in plasma lipids. Pro-inflammatory HDL was relatively weak in its ability to promote cholesterol efflux while anti-inflammatory HDL was better in promoting cholesterol efflux. In other studies, oxidative alterations of the major protein of HDL, apolipoprotein A-I (apoA-I), impaired the ability of the apoA-I to promote cholesterol efflux. Thus, HDL structure and function may be more important than HDL-cholesterol levels in predicting risk for cardiovascular disease.     

Fatty acyl composition of the major lipid classes of HDL2 and HDL3 of human serum

High density lipoprotein subfractions 2 and 3 were isolated from the sera of normolipidaemic males by rate zonal ultracentrifugation. The fatty acyl compositions of their major lipid classes--phospholipids, cholesteryl esters and triacylglycerols--were determined. The average chain length and degree of unsaturation of the fatty acyl components of the phospholipids and cholesteryl esters of the HDL3 subfraction were found to be significantly higher than those of the HDL2 subfraction. The triacylglycerol moieties of the HDL3 subfraction had a higher proportion of unsaturated fatty acyl components than HDL2, but there was no difference in the average chain length between the two subfractions. These results are not consistent with an equilibration of the lipid components between the two HDL subfractions, as would be expected from the results of tracer studies. A role for the fatty acyl composition of high density lipoproteins in the determination of high density lipoprotein subfraction distribution is proposed.     

HDL apolipoprotein A-I and HDL apolipoprotein A-II concentrations in male company employees in Westphalia aged 40 years and older

The major structural components of high density lipoproteins were determined in the sera of 638 male employees aged 40 years and older. It was demonstrated that the HDL apolipoprotein A-I/HDL cholesterol ratio as well as the HDL apolipoprotein A-II/HDL cholesterol ratio are similarly correlated to a cumulative score of established risk factors for atherosclerosis. Most important, however, is the finding that the correlation of these ratios to the risk factor rating of atherosclerosis is found in subgroups with normal or elevated HDL cholesterol values. Furthermore, it is shown that the relative content of apolipoproteins A-I and A-II in individual HDL is partly dependent on the plasma concentration of HDL cholesterol and triglycerides. It is concluded that HDL composition may have an additional predictive significance for the development of atherosclerosis.     

The double jeopardy of HDL

The ability of high‐density lipoprotein (HDL) to promote cholesterol efflux is thought to be important in its protection against cardiovascular disease. Anti‐inflammatory properties of HDL have emerged as additional properties that may also be important. HDL appears to have evolved as part of the innate immune system functioning to inhibit inflammation in the absence of an acute phase response (APR) but functioning to increase inflammation in the presence of an APR. Inbred strains of mice that are genetically susceptible to atherosclerosis have pro‐inflammatory HDL, while inbred strains that are resistant to atherosclerosis have anti‐inflammatory HDL. In one small study, humans with coronary heart disease (CHD) or CHD equivalents had pro‐inflammatory HDL prior to statin therapy and about half continued to have pro‐inflammatory HDL after statin therapy despite a profound decrease in plasma lipids. Pro‐inflammatory HDL was relatively weak in its ability to promote cholesterol efflux while anti‐inflammatory HDL was better in promoting cholesterol efflux. In other studies, oxidative alterations of the major protein of HDL, apolipoprotein A‐I (apoA‐I), impaired the ability of the apoA‐I to promote cholesterol efflux. Thus, HDL structure and function may be more important than HDL‐cholesterol levels in predicting risk for cardiovascular disease.     

Ultracentrifugation in swinging-bucket and fixed-angle rotors evaluated for isolation and determination of high-density lipoprotein subfractions HDL2 and HDL3

We evaluated the density-gradient ultracentrifugation method in a swinging-bucket rotor (Anal Biochem 111, 149-157, 1981) in a slightly modified version for isolation and determination of high-density lipoprotein (HDL) subfractions. We prestained the serum with Coomassie Brilliant Blue R, which did not change the hydrated densities of the lipoproteins, and after only 2.2 X 10(8) gav . min obtained an equilibrium distribution of the lipoproteins along the gradient. The density distribution of the HDL of 120 sera obtained from apparently healthy persons and from patients with different types of hyperlipoproteinemia was bimodal. The HDL2 could be isolated in the density range 1.072-1.098 kg/L and the HDL3 at 1.100-1.176 kg/L, the latter fraction being more heterogeneous. At a solvent density of 1.100 we obtained similar results for HDL2-and HDL3-cholesterol by ultracentrifugation in two different fixed-angle rotors with tube angles of 15 degrees or 35 degrees. Independent of the rotor and the ultracentrifugation technique, subfractionation at d = 1.100 resulted in more distinct stained entities than ultracentrifugation at d = 1.125. In the swinging-bucket rotor procedure, interference by sinking pre-beta-lipoproteins was minimized because, having hydrated densities between 1.058 and 1.075, they could be removed without aspirating the HDL2. The method is both accurate and precise. For HDL2- and HDL3-cholesterol determined in a thawed frozen serum pool, CVs were 8.8 and 6.3%, respectively (n = 18).     

Effects of bezafibrate on HDL2/HDL3 ratio in postmenopausal hypertriglyceridemic women

The short-term effects of bezafibrate on high-density lipoprotein cholesterol quality and triglyceride-rich lipoprotein metabolism in 186 postmenopausal hypertriglyceridemic women were investigated. Patients were randomized to an untreated group and to bezafibrate (400 mg/d) for 6 months. Fasting lipid concentrations, high-density lipoprotein 2, and high-density lipoprotein 3 levels were measured at baseline and after 3 and 6 months. At 3 months, bezafibrate had significantly decreased mean serum triglycerides and remnant-like particle cholesterol levels (105.7 +/- 43.4 mg/dL and 5.33 +/- 2.1 mg/dL, P < .001, respectively) from baseline values (232.5 +/- 63.9 mg/dL and 9.69 +/- 3.8 mg/dL, respectively). It also maintained lower total cholesterol, low-density lipoprotein cholesterol, triglycerides, and remnant-like particle cholesterol concentrations to 6 months. After 3 months, it significantly increased mean serum high-density lipoprotein cholesterol (55.1 +/- 14.7 vs 64.8 +/- 12.1 mg/dL; P < .0001) and maintained higher high-density lipoprotein cholesterol at 6 months. The high-density lipoprotein 2-high-density lipoprotein 3 ratio was decreased after 3 months of therapy with bezafibrate (2.13 +/- 0.68) from the baseline (2.42 +/- 0.71) (P < .01).