Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A2
2006; Elsevier BV; Volume: 48; Issue: 2 Linguagem: Inglês
10.1194/jlr.m600249-jlr200
ISSN1539-7262
AutoresJohn W. Gaubatz, Baiba K. Gillard, John B. Massey, Ron C. Hoogeveen, Max T. Huang, Eric E. Lloyd, Joe L. Raya, Chao-yuh Yang, Henry J. Pownall,
Tópico(s)Cholesterol and Lipid Metabolism
ResumoSmall, dense, electronegative low density lipoprotein [LDL(−)] is increased in patients with familial hypercholesterolemia and diabetes, populations at increased risk for coronary artery disease. It is present to a lesser extent in normolipidemic subjects. The mechanistic link between small, dense LDL(−) and atherogenesis is not known. To begin to address this, we studied the composition and dynamics of small, dense LDL(−) from normolipidemic subjects. NEFA levels, which correlate with triglyceride content, are quantitatively linked to LDL electronegativity. Oxidized LDL is not specific to small, dense LDL(−) or lipoprotein [a] (i.e., abnormal lipoprotein). Apolipoprotein C-III is excluded from the most abundant LDL (i.e., that of intermediate density: 1.034 < d < 1.050 g/ml) but associated with both small and large LDL(−). In contrast, lipoprotein-associated phospholipase A2 (LpPLA2) is highly enriched only in small, dense LDL(−). The association of LpPLA2 with LDL may occur through amphipathic helical domains that are displaced from the LDL surface by contraction of the neutral lipid core. Small, dense, electronegative low density lipoprotein [LDL(−)] is increased in patients with familial hypercholesterolemia and diabetes, populations at increased risk for coronary artery disease. It is present to a lesser extent in normolipidemic subjects. The mechanistic link between small, dense LDL(−) and atherogenesis is not known. To begin to address this, we studied the composition and dynamics of small, dense LDL(−) from normolipidemic subjects. NEFA levels, which correlate with triglyceride content, are quantitatively linked to LDL electronegativity. Oxidized LDL is not specific to small, dense LDL(−) or lipoprotein [a] (i.e., abnormal lipoprotein). Apolipoprotein C-III is excluded from the most abundant LDL (i.e., that of intermediate density: 1.034 < d < 1.050 g/ml) but associated with both small and large LDL(−). In contrast, lipoprotein-associated phospholipase A2 (LpPLA2) is highly enriched only in small, dense LDL(−). The association of LpPLA2 with LDL may occur through amphipathic helical domains that are displaced from the LDL surface by contraction of the neutral lipid core. Although low density lipoprotein-cholesterol is a risk factor for cardiovascular disease, an expanding body of evidence suggests that one or more minor LDL subfractions serve as the atherogenic agent. One hypothesis is that LDL enters the arterial wall, where its lipids are oxidatively modified, thereby initiating a series of biochemical processes that culminate in lesion formation (1Navab M. Berliner J.A. Watson A.D. Hama S.Y. Territo M.C. Lusis A.J. Shih D.M. Van Lenten B.J. Frank J.S. Demer L.L. The yin and yang of oxidation in the development of the fatty streak. Arterioscler. Thromb. Vasc. Biol. 1996; 16 (et al.): 831-842Google Scholar). Nonetheless, modified LDL present in the circulation also has atherogenic properties (2Kovanen P.T. Pentikäinen M.O. Circulating lipoproteins as proinflammatory and anti-inflammatory particles in atherogenesis. Curr. Opin. Lipidol. 2003; 14: 411-419Google Scholar), as indicated by the correlation of atherosclerosis with levels of oxidized plasma LDL (3Holvoet P. Vanhaecke J. Janssens J. Van de Werf F. Collen D. Oxidized LDL and malondialdehyde-modified LDL in patients with acute coronary syndromes and stable coronary artery disease. Circulation. 1998; 98: 1487-1494Google Scholar, 4Ehara S. Ueda M. Nakuro T. Haze K. Itoh A. Otsuka M. Komatsu R. Matsuo T. Itabe H. Takano T. Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. Circulation. 2001; 103 (et al.): 1955-1960Google Scholar, 5Nishi K. Itabe H. Uno M. Kitazato K.T. Horiguchi H. Shinno K. Nagahiro S. Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1649-1654Crossref PubMed Scopus (330) Google Scholar). Plasma contains several forms of modified LDL, including heterogeneously oxidized LDL (6Schuh J. Fairclough G.F. Haschemeyer R.H. Oxygen-mediated heterogeneity of apo-low-density lipoprotein. Proc. Natl. Acad. Sci. USA. 1978; 75: 3173-3177Crossref PubMed Scopus (256) Google Scholar), glycated LDL from diabetic subjects (7Kim H.J. Kurup I.V. Nonenzymatic glycosylation of human low density lipoprotein: evidence for in vivo and in vitro glucosylation. Metabolism. 1982; 31: 348-352Google Scholar), and desialylated LDL (8Orekhov A.N. Tertov V.V. Mukhin D.N. Mikhailenko I.A. Modification of low density lipoprotein by desialylation causes lipid accumulation in cultured cells: discovery of desialyated lipoprotein with altered cellular metabolism in the blood of atherosclerotic patients. Biochem. Biophys. Res. Commun. 1989; 162: 206-211Google Scholar). Importantly, plasma from both normal and dyslipidemic patients contains electronegative low density lipoprotein [LDL(−)] (9Avogaro P. Bittolo-Bon G. Cazzolato G. Presence of a modified low density lipoprotein in humans. Arteriosclerosis. 1988; 8: 79-87Google Scholar); however, there have been few comprehensive studies of LDL(−) in normolipidemic subjects.Plasma LDL(−) is increased in subjects at high risk for cardiovascular disease as a result of hypercholesterolemia (10Bittolo-Bon G. Cazzolato G. Avogaro P. Probucol protects low-density lipoproteins from in vitro and vivo oxidation. Pharmacol. Res. 1994; 29: 337-344Google Scholar, 11Vedie B. Jeunemaitre X Mégnien J.L. Myara I. Trebeden H. Simon A. Moatti N. Charge heterogeneity of LDL in asymptomatic hypercholesterolemic men is related to lipid parameters and variations in the apo B and CIII genes. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1780-1789Google Scholar, 12Sánchez-Quesada J.L. Otal-Entraigas C. Franco M. Jorba O. Gonzalez-Sastre F. Blanco-Vaca F. Ordonez-Llanos J. Effect of simvastatin treatment on the electronegative low-density lipoprotein present in patients with familial hypercholesterolemia. Am. J. Cardiol. 1999; 84: 655-659Google Scholar), hypertriglyceridemia (11Vedie B. Jeunemaitre X Mégnien J.L. Myara I. Trebeden H. Simon A. Moatti N. Charge heterogeneity of LDL in asymptomatic hypercholesterolemic men is related to lipid parameters and variations in the apo B and CIII genes. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 1780-1789Google Scholar, 13Sánchez-Quesada J.L. Benítez S. Otal C. Franco M. Blanco-Vaca F. Ordonez-Llanos J. Density distribution of electronegative LDL (LDL(−)) in normolipemic and hyperlipemic subjects. J. Lipid Res. 2002; 43: 699-705Google Scholar), diabetes (14Sánchez-Quesada J.L. Pérez A. Caixàs A. Ordonmez-Llanos J. Carreras G. Payes A. Gonzalez-Sastre F. de Leiva A. Electronegative low-density lipoprotein subform is increased in patients with short-duration IDDM and is closely related to glycaemic control. Diabetologia. 1996; 39: 1469-1476Google Scholar, 15Sánchez-Quesada J.L. Pérez A. Caixàs A. Rigla M. Payes A. Benitez S. Ordonez-Llanos J. Effect of glycemic optimization on electronegative low-density lipoprotein in diabetes: relation to nonenzymatic glycosylation and oxidative modification. J. Clin. Endocrinol. Metab. 2001; 86: 3243-3249Google Scholar, 16Moro E. Zambón C. Pianetti S. Cazzolato G. Pais M. Bittolo Bon G. Electronegative low density lipoprotein subform is increased in type 2 (non-insulin-dependent) microalbuminuric patients and is closely associated with LDL susceptibility to oxidation. Acta. Diabetol. 1998; 35: 161-164Google Scholar, 17Moro E. Alessandrini P Zambón C. Pianetti S. Pais M. Cazzolato G. Bon G.B. Is glycation of low density lipoproteins in patients with type 2 diabetes mellitus an LDL pre-oxidative condition? Diabet. Med. 1999; 16: 663-669Google Scholar), or coronary artery disease (18Tomasik A. Jache W Skrzep-Poloczek B. Widera-Romuk E. Wodniecki J. Wojciechowska C. Circulating electronegatively-charged low-density lipoprotein in patients with angiographically documented coronary artery disease. Scand. J. Clin. Lab. Med. 2003; 63: 259-266Google Scholar). Simvastatin therapy reduces the proportion of LDL(−) without modifying its oxidizability (12Sánchez-Quesada J.L. Otal-Entraigas C. Franco M. Jorba O. Gonzalez-Sastre F. Blanco-Vaca F. Ordonez-Llanos J. Effect of simvastatin treatment on the electronegative low-density lipoprotein present in patients with familial hypercholesterolemia. Am. J. Cardiol. 1999; 84: 655-659Google Scholar). Relative to normal LDL, LDL(−) has 1) lower vitamin E content; 2) increased lipid peroxidation, as assessed by thiobarbituric acid-reactive substances and conjugated dienes; 3) more highly aggregated apolipoprotein B (apoB); 4) reduced affinity for the LDL receptor; 5) greater density and is more readily oxidized; and 6) higher lipoperoxide content (9Avogaro P. Bittolo-Bon G. Cazzolato G. Presence of a modified low density lipoprotein in humans. Arteriosclerosis. 1988; 8: 79-87Google Scholar, 19Cazzolato G. Avogaro P Bittolo-Bon G. Characterization of a more electronegatively-charged LDL subfraction by ion exchange HPLC. Free Radic. Biol. Med. 1991; 11: 247-253Crossref PubMed Scopus (99) Google Scholar, 20Avogaro P. Cazzolatto G Bittolo-Bon G. Some questions concerning a small, more electronegative LDL circulating in human plasma. Atherosclerosis. 1991; 91: 163-171Google Scholar, 21Hodis H.N. Kramsch D.M. Avogaro P. Bittolo-Bon G. Cazzolato G. Hwang J. Peterson H. Sevanian A. Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low-density lipoprotein (electronegative LDL). J. Lipid Res. 1994; 35: 669-677Google Scholar, 22Sevanian A. Hwang J. Hodis H. Cazzolato G. Avogaro P. Bittolo-Bon G. Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subpopulations. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 784-793Google Scholar). Other studies have shown that the cytotoxicity of LDL(−) to endothelial cells is independent of its oxidation and peroxidation (23Shimano H. Yamada N. Ishibashi S. Mokuno H. Mori N. Gotoda T. Harada K. Akanuma Y. Murase T. Yazaki Y. Oxidation-labile subfraction of human plasma low-density lipoprotein isolated by ion-exchange chromatography. J. Lipid Res. 1991; 32 (et al.): 763-773Google Scholar, 24Chappey B. Myara I. Benoit M.O. Maziere C. Maziere J.C. Moatti N. Characteristics of ten charge-differing subfractions isolated from human native low-density lipoprotein (LDL). No evidence of peroxidative modifications. Biochim. Biophys. Acta. 1995; 1259: 261-270Google Scholar, 25Demuth K. Myara I. Chappey B. Vedie B. Pech-Amsellem M.A. Haberland M.E. Moatti N. A cytotoxic electronegative LDL subfraction is present in human plasma. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 773-783Crossref PubMed Scopus (95) Google Scholar, 26Nyyssönen K. Kaikkonen J. Salonen J.T. Characterization and determinants of an electronegatively-charged low-density lipoprotein in human plasma. Scand. J. Clin. Lab. Invest. 1996; 56: 681-689Google Scholar, 27de Castellarnau C. Sánchez-Quesada J.L. Benítez S. Rosa R. Caveda L. Vila L. Ordonez-Llanos J. Electronegative LDL from normolipemic subjects induces IL-8 and monocyte chemotactic protein secretion by human endothelial cells. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2281-2287Google Scholar, 28Sánchez-Quesada J.L. Camacho M Antón R. Benitez S. Vila L. Ordonez-Llanos J. Electronegative LDL of FH subjects. Chemical characterization and induction of chemokine release from human endothelial cells. Atherosclerosis. 2003; 166: 261-270Google Scholar). Thus, questions remain regarding the composition and properties of LDL(−) and their relationship to pathogenicity. Although there have been a few studies of LDL(−) in normolipidemic subjects, none has elucidated LDL structure in situations of minimal lipoprotein modification, a condition potentially preceding the formation of more severely modified, and patently atherogenic, particles. Given the association of LDL(−) with several pathological states, it is important to fully characterize the properties of LDL(−). Here, we provide a detailed physical and chemical analysis of LDL(−) derived from normolipidemic subjects.METHODSLDL subfractionation by densityPlasma was isolated from blood obtained through the Methodist Hospital Blood Donor Center, where each participant provided informed consent. All patient protocols were approved by the institutional review board. The Lipid Laboratory of Baylor College of Medicine used accepted methodologies to analyze plasma triglyceride and cholesterol. None of the volunteers was overtly dyslipidemic. The respective means and (SEM) for total cholesterol and triglyceride were 138 (5Nishi K. Itabe H. Uno M. Kitazato K.T. Horiguchi H. Shinno K. Nagahiro S. Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1649-1654Crossref PubMed Scopus (330) Google Scholar) and 118 (12Sánchez-Quesada J.L. Otal-Entraigas C. Franco M. Jorba O. Gonzalez-Sastre F. Blanco-Vaca F. Ordonez-Llanos J. Effect of simvastatin treatment on the electronegative low-density lipoprotein present in patients with familial hypercholesterolemia. Am. J. Cardiol. 1999; 84: 655-659Google Scholar) mg/dl; ranges were 112–193 and 44–255 mg/dl.LDL was isolated from donor plasma via sequential flotation in solutions adjusted to the respective densities of 1.006, 1.019, and 1.063 g/ml by the addition of KBr (29Schumaker V.N. Puppione D.L. Sequential flotation ultracentrifugation. Methods Enzymol. 1986; 128: 155-170Crossref PubMed Scopus (467) Google Scholar). LDL (4 ml) from the flotation at d = 1.063 g/ml was transferred to the bottom of 14 × 95 mm SW40 centrifuge tubes (Beckman Coulter, Fullerton CA). LDL was sequentially overlaid with 2 ml each of saline adjusted with KBr to densities of 1.055, 1.050, 1.040, and 1.030. LDL was ultracentrifuged in a Beckman SW40 Ti swinging-bucket rotor at 35,000 rpm for 48 h at 4°C, after which each tube was marked along its length using a template to guide fractionation. Guided by the marks, fractions were collected by aspiration with a Pasteur pipette. The top 0.5 ml was discarded, and seven fractions (1.4 ml) increasing in density from D1 to D7 were collected. The bottom 1.0 ml (i.e., infranatant) was collected but not included in the LDL fractions. The densities of each fraction were determined by pycnometry of similarly treated tube contents (n = 3), which reflected the same gradient of KBr without LDL.Agarose gel electrophoresisThe LDL density subfractions were analyzed by electrophoresis in 0.7% agarose (90 mM Tris, 80 mM borate, pH 8.2). LDL samples (2–10 μg of protein in <20 μl) were loaded onto the gels, and electrophoresis was performed at 4°C at 90 V for 90 min.Anion-exchange analysis of LDLUsing an Amersham/Pharmacia ÄKTA Chromatography System and a Mono Q 5/50 GL column (Amersham/Pharmacia), LDL subfractions were isolated according to density and separated on the basis of particle charge (13Sánchez-Quesada J.L. Benítez S. Otal C. Franco M. Blanco-Vaca F. Ordonez-Llanos J. Density distribution of electronegative LDL (LDL(−)) in normolipemic and hyperlipemic subjects. J. Lipid Res. 2002; 43: 699-705Google Scholar, 30Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1083-1090Google Scholar). Fractionation was achieved using a flow rate of 1 ml/min and a linear gradient of 0–0.1 M NaCl (10 min) followed by 0.2, 0.3, and 1 M, then reequilibrated to 0 M NaCl in 10 mM Tris, 0.5 mM EDTA (pH 7.4) for 8, 6, 5, and 6 min, respectively. Taking absorbance values at 280 nm, this program yielded electropositive and electronegative fractions [LDL(+) and LDL(−)] at 0.2 and 0.3 M NaCl, respectively; their relative abundance was determined via peak integration software supplied with the system. In some experiments, LDL(+) and LDL(−) were collected in 1 ml fractions and concentrated with Centriprep filters (YM-100; Millipore). Isolated fractions were stored at 4°C until needed for further analysis.SDS-PAGELDL subfractions were delipidated with ethyl acetate-ethanol (1:1), solubilized with 10% SDS, and separated on 4–15% SDS gels (Bio-Rad Laboratories) at 4°C at 100 V for 2 h.Lipid-free human serum albuminScavenging of monoacyl lipids required punctiliously NEFA-free human serum albumin (HSA). Nominally NEFA-free HSA (Sigma-Aldrich, St. Louis, MO) was additionally extracted with Folch reagent (31Folch J. Lees M Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar) to remove adventitious lipid.Immunoanalysis of lipoprotein-associated phospholipase A2 and apoC-IIIAfter electrophoresis of LDL subfractions on agarose or SDS-polyacrylamide gels and electrotransfer onto polyvinylidene difluoride membranes (Bio-Rad), blots were blocked with Tris-buffered saline containing 0.05% (v/v) Tween-20 and 3% (w/v) nonfat dry milk. Probing was performed overnight at 4°C with polyclonal goat antibody to apoC-III (1:1,000 dilution; Academy Biomedical Co., Houston, TX) or for 1 h at room temperature with polyclonal rabbit anti-human lipoprotein-associated phospholipase A2 (LpPLA2) (1:2,500 dilution; Cayman Chemical, Ann Arbor, MI). After washing the membranes four times in 10 mmol/l Tris-HCl, 150 mM NaCl, and 0.05% Tween-20 (pH 7.5), a secondary HRP-conjugated donkey anti-goat antibody (Jackson ImmunoResearch Laboratories; apoC-III) or HRP-conjugated goat anti-rabbit antibody (Bio-Rad; LpPLA2) was incubated at 1:1,000 at room temperature for 1 h. The membrane was washed and immunoreactive bands were detected via chemiluminescence using the ECL Plus Western blotting detection system (GE Healthcare). The blots were exposed to CL-X film (Pierce) for 1–10 min. LpPLA2 mass was also quantified using a commercial PLAC™ kit (diaDexus, San Francisco, CA) that is based on a sandwich ELISA using two monoclonal antibodies (32Hoogeveen R.C. Ballantyne C.M. PLAC test for identification of individuals at increased risk for coronary heart disease. Expert Rev. Mol. Diagn. 2005; 5: 9-14Google Scholar).Lipid analysisLipids were extracted from LDL in accordance with Folch, Lees, and Sloane Stanley (31Folch J. Lees M Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar). The free cholesterol (FC), phospholipid, NEFA, and triglyceride were determined using enzyme-based kits (Wako Chemicals USA, Richmond, VA). Total cholesterol was determined after treating samples with a cholesteryl esterase. Cholesteryl ester (CE) was measured by calculating the difference between total cholesterol and FC multiplied by 1.68. Protein was determined as described by Lowry et al. (33Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Oxidized LDL levels (n = 4) were measured via direct sandwich ELISA (Mercodia, Winston-Salem, NC) based on the capture of monoclonal antibody 4E6, which detects oxidatively modified apoB. The values were normalized to protein concentrations of 1 mg/ml (34Holvoet P. Macy E. Landeloos M. Jones D. Nancy J.S. Van de Werf F. Tracy R.P. Analytical performance and diagnostic accuracy of immunometric assays for the measurement of circulating oxidized LDL. Clin. Chem. 2006; 52: 760-764Google Scholar). LDL size was calculated in accordance with Sherman et al. (35Sherman M.B. Orlova E.V. Decker G.L. Chiu W. Pownall H.J. Structure of triglyceride-rich human low-density lipoproteins according to cryoelectron microscopy. Biochemistry. 2003; 42: 14988-14993Google Scholar) using the partial specific volumes of each of the components, as reported by Tardieu et al. (36Tardieu A. Mateu L. Sardet C. Weiss B. Luzzati V. Aggerbeck L. Scanu A.M. Structure of human serum lipoproteins in solution. II. Small-angle x-ray scattering study of HDL3 and LDL. J. Mol. Biol. 1976; 105: 459-460Google Scholar), and their respective molar ratios relative to apoB-100, which is assumed to have a partial specific volume of 0.73 ml/g and to occur in LDL at only one copy per particle.NEFA acyl chain compositions were determined by mass spectrometry of their fatty acid methyl esters, after separation by gas chromatography (37Lepage G. Roy C.C. Specific methylation of plasma nonesterified fatty acids in one-step reaction. J. Lipid Res. 1988; 29: 227-235Google Scholar). As an internal standard, heptadecanoic acid (1 μg) was dissolved in 5 ml of methylating reagent (2% acetyl chloride in methanol), and 150 μl of LDL was added and mixed in a tightly sealed glass tube at 24–29°C for 45 min. The reaction was quenched with 3 ml of 6% K2CO3. Fatty acid methyl esters were extracted with 300 μl of hexane, transferred to a test tube, and evaporated. The residue was dissolved in 100 μl of decane and transferred to vials for a Hewlett-Packard 7683 injector. Fatty acid methyl esters were separated on a 30 m × 0.25 mm fused silica AT-255 column via 50% cyanopropylphenyl-dimethylpolysiloxan stationary phase (Alltech Associates, Deerfield, IL). Analyses were performed on a Hewlett-Packard 6890 gas chromatograph coupled to a Hewlett-Packard 5973 mass selective detector operated in the electron-impact mode (70 eV) with full-scan monitoring. The elution program consisted of 2 min at 70°C, increase to 180°C at 20°C/min and to 220°C at 3.0°C/min, and hold for 16 min. Injector and detector temperatures were 250°C. Peaks were identified on the basis of retention times and mass spectral analysis of standards (Sigma-Aldrich) from the National Institute of Standards and Technology (standard reference database number 69). For quantification, the peak area of each sample was compared with that of the internal standard.Thin-layer chromatography of LDL lipids and lipids scavenged by HSALDL subfractions (1 mg of protein) were incubated with and without HSA (10 mg) for 60 min at 4°C and separated by ultracentrifugation (d = 1.12 g/ml). The floated LDL and sedimented HSA were collected and extracted in accordance with Folch, Lees, and Sloane Stanley (31Folch J. Lees M Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 1957; 226: 497-509Google Scholar), and the dried residue was analyzed by high-performance TLC, as described by White et al. (38White T. Bursten S. Federighi D. Lewis R.A. Nudelman E. High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian cells and sera by multi-one-dimensional thin-layer chromatography. Anal. Biochem. 1998; 258: 109-117Google Scholar), and compared with lipid standards. Lipids were visualized with primulin (Sigma-Aldrich); lipid standards were from Avanti Polar Lipids (Alabaster, AL). The eluting solvents were of HPLC grade. The primulin-stained lipids on the TLC plates were scanned with a Molecular Dynamics Storm imaging system. Subsequently, the lipid bands on the scanned images were quantified using Molecular Dynamics ImageQuant software (version 5.2). All pixels inside the rectangle enclosing each band were integrated; background correction was performed by subtracting the intensity of a duplicate rectangle in a proximal region without visible staining. The calculation of lipid composition was based on integrated values, assuming no lipid-specific differences in relative staining intensity. Lipids associated with LDL protein were applied to each lane in amounts corresponding to equal protein.NEFA loading of LDLAssuming molecular weights of 282 and 512,000, respectively, for oleic acid and apoB, oleic acid in ethanol (50 mM) was added to 1–2 mg of LDL at an oleate-to-apoB-100 molar ratio of 125 and incubated for 15 min at 37°C. The final ethanol and oleate concentrations were ⩽1% and 0.5 mM, respectively. After exhaustive dialysis against TBS, changes in LDL charge were determined by agarose gel electrophoresis.Statistical analysisThe significance of trends in LDL properties was determined via one-way ANOVA.RESULTSLDL subfractionation according to densityLDL isolated by density gradient ultracentrifugation was collected into seven fractions that were analyzed for charge, composition, and lipid transfer dynamics (Fig. 1 ). The majority of the LDL protein (∼85%) was confined to fractions D2 through D5 (Fig. 1A). The remainder appeared in fractions D1, D6, and D7; D7 is the least abundant fraction, comprising only 1.7% of the total protein. An infranatant fraction of lipid-poor material that sediments during ultracentrifugation (data not shown) typically contains approximately half of the amount of protein found in fraction D7. According to the criteria of Berg et al. (39Berg G. Muzzio M.L. Wikinski R. Schreier L. A new approach to the quantitative measurement of dense LDL subfractions. Nutr. Metab. Cardiovasc. Dis. 2004; 14: 73-80Abstract Full Text PDF PubMed Scopus (9) Google Scholar), D5, D6, and D7 are small, dense LDLs (d = 1.048–1.063 g/dl). These subfractions account for ∼25% of the total LDL protein.Analysis of LDL chargeAnalysis of the LDL by agarose gel electrophoresis showed fraction-specific differences in particle charge (Fig. 1B); D7 was the most electronegative, followed by two other minor fractions, D1 and D6. In each instance, agarose gel electrophoresis of each LDL subfraction revealed a single broad band. In contrast, anion-exchange chromatography of each LDL subfraction resolved the sample into two major fractions (Fig. 1C), an early-eluting electropositive particle, LDL(+), and a late-eluting electronegative particle, LDL(−). Fractions D2 through D5 contained relatively little (4–13%) LDL(−). However, minor fractions D1, D6, and D7 were rich in LDL(−), 33–85%. In sum, an examination of the agarose gel electrophoresis data shows that the LDL subfractions with the highest electronegativity have the highest content of LDL(−).LDL compositionFigure 2 shows the composition of different density subfractions, with respect to surface (A) and core (B) composition and NEFA content (C). As expected, this analysis revealed trends in composition, based on differences in density and size. For example, with D1 through D7, there was a significant increase in protein content with increasing density (20–35%; Fig. 2A) (one- way ANOVA, P < 0.0001). PL content decreased slightly with increasing density, and, as expected, FC, which associates mainly with PL, demonstrated a similar pattern (Fig. 2A). Although total neutral lipids [CE + triglyceride (TG)] declined from 45% to 40% (Fig. 2B), the specific contributions of CE and TG varied according to the subfraction. The highest CE and lowest TG contents were observed in the middle fractions, whereas D1 and D7, at opposite ends of the density continuum, had the lowest CE and highest TG contents. Based on LDL composition, known partial specific volumes of the components, and the assumption of one copy of apoB-100 per particle, the calculated diameters ranged from 20.71 nm for D1 to 16.95 nm for D7 (Table 1 ). Using the known densities, we determined that these dimensions correspond to relative particle masses of 100, 95.8, 87.4, 79.9, 72.7, 66.7, and 56.9% for D1 through D7, respectively. The composition of the infranatant (i.e., ∼54% protein, ∼15% TG, ∼4% FC, 14% CE, and 13% PL), which did not correspond to a normal lipoprotein class, is likely a mixture of lipid-free protein, precipitated LDL, HDL, and lipoprotein [a] (Lp[a]). Others have used the LDL fractions of 1.02 < d < 1.05 g/ml to identify differences between LDL(+) and LDL(−). Although this eliminates the possible confounding effects of Lp[a] that can exist in fractions with greater density, it also excludes the fractions corresponding to D6 and D7 in our study, which are profoundly different from the more electropositive fractions, including those with the highest affinity for LpPLA2. According to ELISA analysis (40Gaubatz J.W. Cushing G.L. Morrisett J.D. Quantitation, isolation, and characterization of human lipoprotein (a). Methods Enzymol. 1986; 129: 167-186Google Scholar), Lp[a] constituted <3% of any LDL subfraction (data not shown).Fig. 2.Compositions of LDL subfractions. A: Surface components. FC, free cholesterol; PL, phospholipid. B: Core lipids. CE, cholesteryl ester; NL, total neutral lipid (CE + TG); TG, triglyceride. Core and surface compositions are expressed as percentage of total LDL weight. C: Percentage of neutral lipid occurring as TG (open circles) and number of NEFA molecules per LDL particle (closed circles), assuming that apolipoprotein B-100 (apoB-100) is the sole protein (512 kDa) and that there is one copy of apoB-100 per LDL particle. The NEFA per particle and percentage neutral lipid as TG were linearly correlated (inset; r2 = 0.90). Data are means ± SEM (n = 9). Error bars represent SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Properties of LDL subfractions isolated according to densityFractiondDaDiameter calculated from partial specific volumes and compositions.MassbLDL mass = 100 × 512,000/% protein (mass relative to D1).ΔG (LpPLA2)cΔG = −RT ln K, where K = (LpPLA2)D7/(LpPLA2)Di and (LpPLA2)D7 and (LpPLA2)Di are the amounts of LpPLA2 associated with each fraction (i) relative to apoB-100.ApoC-IIINEFA/nm2dNumber of NEFA molecules per surface area of a spherical particle given by A = 4πr2; a plot of this is overlaid on the agarose gel in Fig. 1B.g/mlnmkDakcalD11.02920.712,640 (
Referência(s)