High-density lipoprotein (HDL) is regarded as atheroprotective because it provides antioxidant and anti-inflammatory benefits and plays an important role in reverse cholesterol transport. and H5. Lipoprotein isoforms were analyzed in each subfraction by using matrix-assisted laser desorption-time of flight mass spectrometry. To quantify other proteins in the HDL subfractions we used the isobaric tags for relative and absolute quantitation approach followed by nanoflow liquid chromatography-tandem mass spectrometry analysis. Most antioxidant proteins detected were found in H4 and H5. The ability of each subfraction to induce cholesterol efflux from macrophages increased with increasing HDL electronegativity with the exception of H5 which promoted the least efflux activity. In conclusion anion-exchange chromatography is an attractive method for separating HDL into subfractions with distinct lipoprotein compositions and biologic activities. By comparing the properties of these subfractions it may be possible to uncover HDL-specific proteins that play a role in disease. INTRODUCTION Epidemiologic studies Rabbit polyclonal to AGAP. and prospective randomized trials have consistently shown a powerful inverse association between high-density lipoprotein (HDL) cholesterol levels and the risk of coronary heart disease; the risk is increased by approximately 3% in women and 2% in men for each 1 mg/dL decrement in HDL cholesterol level.1-3 Although low plasma HDL levels have SU6668 been correlated with an increased risk for cardiovascular diseases clinical trials aimed at increasing plasma HDL levels have failed to prove any benefit suggesting that the quality of HDL particles is SU6668 more important than the quantity of total HDL.4 HDL is heterogeneous composed of 50% protein and 50% lipid by mass. The protein composition of HDL is complex and includes multiple acute-phase response proteins protease inhibitors and complement regulatory proteins.5-6 The primary protein components of HDL are apolipoprotein (apo) AI (70%) SU6668 apoAII (20%) and to a lesser extent apoE clusterin (apoJ) paraoxanase (PON) haptoglobin 2 and lecithin-cholesterol acyltransferase (LCAT).7 The lipid portion of HDL is composed of a phosphatidylcholine shell a cholesteryl ester core and small amounts of free cholesterol and triglycerides. Because of the involvement of HDL in reverse cholesterol transport (RCT)8-9 and its anti-apoptotic 10 anti-inflammatory and antioxidant properties 6 11 HDL helps protect against the development of atherosclerosis. The methodology most commonly used to separate HDL into subclasses is density-gradient ultracentrifugation which divides whole HDL into subclasses with increasing density known as HDL2 and HDL3.12-13 In a prospective study HDL2 showed a stronger inverse association with ischemic heart disease risk than did HDL3.12 In addition Salonen and colleagues14 reported that levels of both HDL2 and total HDL were inversely associated with the risk of acute myocardial infarction suggesting that these forms of HDL may play a protective role in ischemic heart disease. The role of HDL3 remains equivocal although small dense HDL3 has been shown to protect low-density lipoprotein (LDL) from oxidative stress.4 15 HDL SU6668 has also been separated by using immunoaffinity chromatography and classified into 2 types of apoAI-containing lipoprotein: apoAI-containing lipoprotein with apoAII (LpA-I:A-II) SU6668 and apoAI-containing lipoprotein without apoAII (LpA-I).16-17 Nuclear magnetic resonance spectroscopy has been used to characterize HDL with small medium and large particle sizes.18 In addition pre-β and α forms of HDL have been characterized according to their electrophoretic mobility and particle size by using 2-dimensional electrophoresis.19 In several studies researchers have reported the separation of HDL LDL or very-low density lipoprotein (VLDL) by using anion exchange chromatography.20-22 HDL separated by using anion-exchange chromatography has been subclassified into 2 or 3 3 SU6668 subfractions.23 24 We previously used anion-exchange chromatography to separate LDL into subfractions according to charge and found that the most electronegative LDL subfraction (L5) from diabetic and hypercholesterolemic patients exhibits anti-proliferative and pro-apoptotic properties in vascular.