Elsevier

Clinica Chimica Acta

Volume 465, February 2017, Pages 112-118
Clinica Chimica Acta

A rapid and precise method for measuring plasma apoE-rich HDL using polyethylene glycol and cation-exchange chromatography: a pilot study on the clinical significance of apoE-rich HDL measurements

https://doi.org/10.1016/j.cca.2016.12.016Get rights and content

Highlights

  • A PEG-column method for serum apoE-rich HDL measurement and isolation was developed

  • High reproducibility and reliability was shown for both apoE-rich and apoE-poor HDL

  • ApoE-rich HDL correlated with atherogenic lipoprotein biomarkers TG and adiponectin

  • HDL heterogeneity may be associated with adipose tissue and energy metabolism

  • PEG-column apoE-HDL separation will facilitate biomarker and research application

Abstract

Background

High-density lipoprotein (HDL) containing apolipoprotein E (apoE-rich HDL) represents only a small portion of plasma HDL. Reliable methods for determining and isolating apoE-rich HDL have not been well studied.

Methods

We established a novel analytical method for apoE-rich HDL using polyethylene glycol and a cation-exchange column (PEG-column method). Furthermore, we examined biochemical correlates of apoE-rich HDL-cholesterol (HDL-C) in 36 patients who underwent coronary computed tomographic angiography.

Results

Our PEG-column method demonstrated high reproducibility (coefficient of variation < 3.52%) and linearity up to 15 mg/dl for apoE-rich HDL-C concentrations. Isolated apoE-rich HDL exhibited a larger diameter (14.8 nm) than apoE-poor HDL (10.8 nm) and contained both apoE and apoA-I. ApoE-rich HDL-C concentrations correlated significantly with triglycerides (rs =  0.646), LDL size (rs = 0.472), adiponectin (rs = 0.476), and other lipoprotein components. No significant correlation was obtained with the coronary calcium score. Multiple regression analysis revealed that plasma triglycerides and adiponectin concentrations remained significant independent predictors of apoE-rich (adjusted R2 = 0.486) but not apoE-poor HDL-C.

Conclusions

The PEG-column method demonstrated, to various degrees, significant correlations between HDL subfractions and several lipid-related biomarkers involved in an atherogenic lipoprotein profile. Our separation technique for apoE-rich HDL is useful to clarify the role of apoE-rich HDL in atherosclerosis.

Introduction

Numerous clinical and epidemiological studies have shown that decreased concentrations of HDL-cholesterol (HDL-C) represent an independent risk factor for coronary artery disease [1], [2], [3]. HDL has been recently proposed to possess multiple biological functions including anti-inflammatory, anti-oxidative, and vasodilatory activities, as well as its traditional role in reverse cholesterol transport [4], [5], [6], [7], [8], [9]. HDLs are, however, a highly heterogeneous group and differ in lipid and protein composition, size, hydrated density, and electrophoretic mobility [3], [6], [10]. Therefore, individual HDL subpopulations might play specific roles in the multiple anti-atherogenic activities associated with HDL overall.

HDLs containing apolipoprotein E (apoE-rich HDL) represent an HDL subclass that accounts for up to 10% of the total HDL in adult human plasma [11], [12]. In animal models, apoE-rich HDL is referred to as HDL1 or HDLc induced by dietary cholesterol and can deliver cholesterol to the liver directly via interaction with the LDL receptor [10], [13], [14]. ApoE-rich HDL also exhibits several atheroprotective properties including promotion of cholesterol efflux [5], stimulation of endothelial heparin sulfate synthesis [15], inhibition of platelet aggregation [16], [17], and maintenance of arterial elasticity [18]. These fundamental findings suggest that apoE-rich HDL might serve as a useful biomarker for cardiovascular risk estimation.

In a clinical study reported by Wilson et al., the concentration of plasma apoE-rich HDL was decreased in patients with coronary heart disease compared to control subjects [19]. Proteomic analysis revealed an increased concentration of apoE in an HDL3 subfraction isolated from subjects with established coronary artery disease [20]. However, there are relatively few studies that have investigated the clinical significance of apoE-rich HDL concentrations, mainly because no conventional analytical methods are currently available for its assessment. Simple and rapid methods are therefore required for lipoprotein analysis in clinical settings.

A method used widely for the separation and fractionation of plasma lipoproteins involves ultracentrifugation; however, it has been noted that some apoE could easily dissociate from lipoprotein particles during this process [21], [22]. In addition, ultracentrifugation is time-consuming and requires a large sample volume as well as great care to precisely recover lipoproteins from the centrifuge tube. Another critical problem with ultracentrifugation is that the density range of apoE-rich HDL overlaps that of dense LDL, lipoprotein(a), and other HDLs [23]. These findings indicate that ultracentrifugation has marked limitations as a tool for plasma lipoprotein analyses.

In contrast, Chiba et al. reported a useful method for determining the serum concentration of apoE-rich HDL-C, which is calculated as the difference in the cholesterol concentrations of total and apoE-deficient HDL [11]. This method is based on double precipitation procedures wherein the total HDL fraction is separated from whole serum using polyethylene glycol (PEG) precipitation and the apoE-deficient HDL is prepared using a dextran sulfate-phosphotungstate-Mg2 + reagent. More recently, Takahashi et al. established a homogeneous assay [12] allowing sequential assessment of total, apoE-containing, and apoE-deficient HDL that might potentially promote clinical and epidemiological studies on apoE-rich HDL. However, the double precipitation and the homogeneous methods are unlikely to be useful for investigating biological functions and pathophysiological roles of apoE-rich HDL because they lack the ability to isolate apoE-rich HDL.

Based on these considerations, we aimed to develop a novel method for determining the cholesterol concentrations of apoE-rich HDL and a technique for isolating apoE-rich HDL from serum or plasma using a combination of PEG precipitation with cation-exchange chromatography (PEG-column method). To further validate the utility of our method, we examined clinical and biochemical correlates of apoE-rich HDL-C concentrations in patients who underwent coronary computed tomographic (CT) angiography.

Section snippets

Precipitation method for total HDL separation

To eliminate apoB-containing lipoproteins from whole serum or plasma samples, we used the PEG method described by Chiba et al. [11]. In brief, a 13% PEG6000 (Wako Pure Chemicals) solution was mixed with the same volume of serum or plasma. The mixed solutions were left for at least 10 min at room temperature and centrifuged at 8000 × g for 5 min to obtain supernatants containing total HDL (PEG supernatant). Each of the PEG supernatants was passed through a 0.20-μm filter (Sartorius Stedim Biotech)

Effect of Mg2 + concentration in buffer A on HDL separation

We first studied the effect of Mg2 + concentration in the running buffer A on HDL separation. For cholesterol detection, almost all HDL passed through the cation-exchange column when using buffer A without Mg2 +, whereas the amount of bound HDL increased gradually and reached a plateau at 20–40 mmol/l Mg2 + (Fig. 1A, C). For apoE detection, 70% of the apoE in HDL (HDL-apoE) passed through the column at 0 mol/l Mg2 + and bound HDL reached a plateau at 20–40 mmol/l Mg2 + (Fig. 1B, D). Approximately 90%

Discussion

Our novel PEG-column method successfully isolated plasma apoE-rich HDL and determined its cholesterol concentration precisely and rapidly. This meets quality specifications for the clinical evaluation of apoE-rich HDL as well as for basic research on plasma lipoproteins.

Several previous studies reported separation techniques of apoE-rich HDL using approaches based on heparin- [14], [15], [16], [17], [18], [19] or immunoaffinity [22], [29], reliant upon the well-known apoE heparin-binding domain

Acknowledgements

The authors gratefully thank all the volunteers who participated in the study. This work was supported by JSPS KAKENHI Grant Numbers JP15H00506 and JP15K01715.

References (35)

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