Send Orders for Reprints to
[email protected] Current Medicinal Chemistry, 2014, 21, ????-????
1
Subfractions and Subpopulations of HDL: An Update M. Rizzo*,1,2, J. Otvos3, D. Nikolic1, G. Montalto1, P.P. Toth4 and M. Banach5 1
Biomedical Department of Internal Medicine and Medical Specialties, University of Palermo, Italy; 2EuroMediterranean Institute of Science and Technology, Italy; 3LipoScience, Inc., Raleigh, NC, USA; 4CGH Medical Center Sterling, Sterling, IL; University of Illinois School of Medicine, Peoria, Il, USA; 5Department of Hypertension, Chair of Nephrology and Hypertension, University of Lodz, Poland Abstract: High-density lipoproteins (HDL) are classified as atheroprotective because they are involved in transport of cholesterol to the liver, known as “reverse cholesterol transport (RCT)”exerting antioxidant and anti-inflammatory activities. There is also evidence for cytoprotective, vasodilatory, antithrombotic, and anti-infectious activities for these lipoproteins. HDLs are known by structural, metabolic and biologic heterogeneity. Thus, different methods are able to distinguish several subclasses of HDL. Different separation techniques appear to support different HDL fractions as being atheroprotective or related with lower cardiovascular (CV) risk. However, HDL particles are not always protective. Modification of constituents of HDL particles (primarily in proteins and lipids) can lead to the decrease in their activity and induce proatherogenic properties, especially when isolated from patients with augmented systemic inflammation. According to available studies, it seems that HDL functionality may be a better therapeutic target than HDL cholesterol quantity; however, it is still disputable which subfractions are most beneficial. There is mounting evidence supporting HDL subclasses as an important biomarker to predict and/or reduce CV risk. In this review we discuss recent notices on atheroprotective and functional characteristic of different HDL subfractions. Also, we provide a brief overview of the different methods used by clinicians and researchers to separate HDL subfractions. Ongoing and future investigations will yield important new information if any given separation method might represent a ‘gold standard’, and which subfractions are reliable markers of CV risk and/or potential targets of novel, more focused, and effective therapies.
Keywords: Cardiovascular risk, electrophoresis, high-density lipoprotein, nuclear magnetic resonance, proteome, subclasses, subfractions, ultracentrifugation. 1. INTRODUCTION Circulating high-density lipoproteins (HDL) represent a heterogeneous lipoproteins with a density more than 1.063 g/mL and a small size (5-12 nm). HDL particles are composed of the highest proportion of protein (apolipoprotein AI [apoA-I; 60%] and apoA-II [20% of the protein content], less quantity of apo C, E, A-IV, D and J) relative to lipid content (phospholipids, cholesteryl esters, unesterified cholesterol and triglycerides [TGs]). Free cholesterol, phospholipid, and varied apolipoproteins form an outer layer, while below is a hydrophobic part, composed mainly of TGs and the esters of cholesterol [1]. HDL particles also exert antioxidant effects carrying enzymes: paraoxonase-1 (PON1), platelet-activating factor acetyl hydrolase (PAF-AH), glutathione selenoperoxidase (GSPx), lecithin-cholesterol acyltransferase (LCAT) and phospholipid transfer protein (PLTP) [1, 2]. The quality and quantity of lipids and apolipoproteins vary resulting in different HDL subclasses, which can be distinguished by shape, charge, density and size, using different methods [2]. For instance, regarding HDL subpopulations, increased activities of anti-oxidative enzymes such as PON1, PAF-AH, LCAT, have been seen in *Address correspondence to this author at the Biomedical Department of Internal Medicine and Medical Specialties (DiBiMIS), University of Palermo, Via del Vespro, 141, 90127, Palermo, Italy; Tel/Fax: +39 (091) 6552945; E-mail:
[email protected] 0929-8673/14 $58.00+.00
small, dense HDL-3c and these particles also have a predominance of apoJ, apoL-1 and apoF proteins and PLTP, as well [3, 4]; while, apoE, apoC-I, -II and -III occur predominantly in larger, less dense HDL-2 particles. In addition, continuous intravascular remodeling of HDL particles during reverse cholesterol transport (RCT) contributes to their heterogeneity. During RCT, nascent discoidal HDL particles are progressively lipidated to form, in succession, small, dense HDL-3 and then large HDL-2 particles by LCAT [5]. It is established that the increased production of small, dense HDL could be the result of insulin resistance, through enhanced the cholesteryl ester transfer protein (CETP) activity and hydrolysis by hepatic lipase (HL) [6]. In the case of hypertriglyceridemia HDL becomes enriched in TGs leading to an increased exchanging cholesteryl esters mediated by the CETP [7], with a consequence generation of such TGrich HDL particles which can be catabolized by HL [8]. 2. DIVERSE METHODS TO CHARACTERIZE HDL SUBCLASSES Physicochemical properties of particles, including density, mobility, size, and apolipoprotein contents, are characteristics which enable several HDL subclasses to be differentiated or fractionated by different techniques (Table 1). The © 2014 Bentham Science Publishers
2 Current Medicinal Chemistry, 2014, Vol. 21, No. 1
apolipoprotein concentrations and the amount of cholesterol ester mainly lead to differences in particle size [1]. Table 1.
Classification of HDL Subpopulations by Different Methods (Modified From [33])
Separation Principle
HDL Subclasses
Density
HDL-2 (density range 1.0631.125 g/mL) HDL-3 (density range 1.1251.210 g/mL)
Electrophoretic mobility
-migrating HDL pre- migrating HDL
Electrophoretic mobility and size
HDL-2b (9.7-12 nm) HDL-2a (8.8-9.7 nm) HDL-3a (8.2-8.8 nm) HDL-3b (7.8-8.2 nm) HDL-3c (7.2-7.8 nm)
2D gel electrophoresis
Alpha 1, 2, 3, 4 Pre-1 HDL
Apolipoprotein composition
LpAI LpAI:AII
NMR and size range
Large HDL-P (9.4-14 nm) Medium HDL-P (8.2-9.4 nm) Small HDL-P (7.3-8.2 nm)
Ion mobility and size range
HDL-2b (10.5-14.5 nm) HDL-2a + 3 (7.65-10.5 nm)
Lipoprint System
Large (1-3), intermediate (4-7) and small (8-10)
By ultracentrifugation, the earliest method used for quantification of HDL, two density subfractions can be obtained: HDL-2 and HDL-3, based on their ultracentrifugal flotation rate [9]. This method was used in a prospective study where an inverse relation of plasma HDL concentration to coronary heart disease (CHD) risk was reported [10]. The authors noted the higher levels of HDL2b and HDL2a particles in women and men for every decades of the respondents' age, and levels of HDL3 were a bit higher in men compared to women, considering the whole population. HDL3 levels were negatively correlated with HDL2b levels, and HDL2b as well as HDL2a levels were closely related and showed a progressive rise with an increase of total HDL levels. Given these data, the authors suggested that HDL2b and HDL2a might present the HDL constituents defining the reported opposite relation of HDL cholesterol with CHD [10]. Vertical auto profiling (VAP) is another HDL subfractionation method based on ultracentrifugation [11] by which the two major subfractions, HDL-2 and HDL-3, can be identified [12]. Among patients in the Framingham Offspring Study (FOS; predominantly white patients), Jackson Heart Study (JHS; all black patients), and a meta-analysis of the two studies, HDL-3 was protective and related to reduced CV risk, while the association between HDL-2 and CV events was not significant. For the meta-analysis, the hazard
Rizzo et al.
ratio per standard deviation increase for both HDL-3 and HDL-2 (0.77; p
