Microphase Separation in Low Density Lipoproteins
1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês
10.1074/jbc.274.3.1334
ISSN1083-351X
AutoresMagdalena Pregetter, Ruth Prassl, Bernhard Schuster, Manfred Kriechbaum, Fabienne Nigon, John Chapman, Peter Laggner,
Tópico(s)Metabolomics and Mass Spectrometry Studies
ResumoThe structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1.0244–1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32 °C, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core. The structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1.0244–1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32 °C, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core. Low density lipoproteins (LDL) 1The abbreviations used are: LDL, low density lipoprotein(s); CE, cholesteryl ester(s); TG, triglyceride(s); DSC, differential scanning calorimetry; 16-CE, (2-(14-carboxytetradecyl)2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl)cholesterylester; 5-DSA, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl; 12-DSA, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, 16-DSA, 2-(14-carboxytetradecyl)2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl); 16-TG, (2-(14-carboxytetradecyl)2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl)dioleylglycerol; T m, peak transition temperature of the LDL core; τc, rotational correlation time. 1The abbreviations used are: LDL, low density lipoprotein(s); CE, cholesteryl ester(s); TG, triglyceride(s); DSC, differential scanning calorimetry; 16-CE, (2-(14-carboxytetradecyl)2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl)cholesterylester; 5-DSA, 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl; 12-DSA, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, 16-DSA, 2-(14-carboxytetradecyl)2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl); 16-TG, (2-(14-carboxytetradecyl)2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl)dioleylglycerol; T m, peak transition temperature of the LDL core; τc, rotational correlation time. are the major carriers of cholesterol in the circulation and are intimately involved in atherogenesis (1 and references therein). These particles represent complex supramolecular assemblies of phospholipids (∼20% of the total mass), free (∼12%) and esterified (∼40%) cholesterol, triglycerides (∼5–10%), and a single copy of apolipoprotein B-100, a glycoprotein of 4,536 amino acids (2Knott T.J. Pease R.J. Powell L.M. Wallis S.C. Rall S.C. Innerarity T.L. Blackhart B. Taylor W.H. Marcel Y. Milne R. Johnson D. Fuller M. Lusis A.J. McCarthy B.J. Mahley R.W. Levy-Wilson B. Scott J. Nature. 1986; 323: 734-738Crossref PubMed Scopus (399) Google Scholar, 3Yang C.-Y. Chen S.-H. Gianturco S.H. Bradley W.A. Sparrow J.T. Tanimura M. Li W.-H. Sparrow D.A. DeLoof H. Rosseneu M. Lee F.-S. Gu Z.-W. Gotto Jr., A.M. Chan L. Nature. 1986; 323: 738-742Crossref PubMed Scopus (269) Google Scholar, 4Kostner G.M. Laggner P. Fruchart J.C. Shepherd J. Human Plasma Lipoproteins : Clinical Biochemistry, Principles, Methods, Applications 3. Walter de Gruyter, Berlin1989: 23-54Crossref Google Scholar). In addition, LDL transports minor amounts of lipophilic vitamins and drugs (5Esterbauer H. Gebicki J. Puhl H. Jürgens G. Free Rad. Biol. Med. 1992; 13: 341-390Crossref PubMed Scopus (2125) Google Scholar). The structure of LDL can be described, in general terms, by a quasispherical core-shell model, in which the apolar constituents (cholesteryl esters (CE) and triglycerides (TG)) form a hydrophobic core of about 150 Å diameter, whereas the phospholipids, most of the unesterified cholesterol, and the apoprotein form an outer surface monolayer with a thickness of about 30 Å (6Laggner P. Müller K. Q. Rev. Biophys. 1978; 11: 371-425Crossref PubMed Scopus (59) Google Scholar, 7Atkinson D. Small D.M. Annu. Biophys. Chem. 1986; 15: 403Crossref PubMed Scopus (135) Google Scholar, 8Laggner P. Brumberger H. Modern Aspects of Small-angle Scattering. Kluwer Academic Publishers, Norwell, MA1995: 371-386Crossref Google Scholar). In this general sense, LDL would appear to fit readily into the structural core-shell scheme of circulating lipoproteins (9Shen B.W. Scanu A.M. Kézdy F. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 837-841Crossref PubMed Scopus (284) Google Scholar). However, LDL exhibits a distinct physical feature that makes it unique among all structural elements of blood: it is the only component to undergo a major structural transition just below physiological body temperature, in the range between 15 and 32 °C (10Deckelbaum R.J. Shipley G.G. Small D.M. Lees R.S. George P.K. Science. 1975; 190: 392-394Crossref PubMed Scopus (134) Google Scholar, 11Deckelbaum R.J. Shipley G.G. Small D.M. J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar). The transition temperature varies among individual donors and is correlated to the core lipid composition. This lipid-melting transition directly affects the molecular packing in the core of LDL, i.e. approximately half of the particle mass (12Laggner P. Degovics G. Müller K.W. Glatter O. Kostner G.M. Holasek A. Hoppe-Seyler's Z. Physiol. Chem. 1977; 358: 771-778Crossref PubMed Scopus (41) Google Scholar, 13Atkinson D. Deckelbaum R.J. Small D.M. Shipley G.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1042-1046Crossref PubMed Scopus (101) Google Scholar). The local molecular dynamics and polarities in the surface monolayer, including apolipoprotein B, are affected indirectly, such that the transition can be recognized from the particle surface (14Laggner P. Kostner G.M. Eur. J. Biochem. 1978; 84: 227-232Crossref PubMed Scopus (34) Google Scholar, 15Herak J.N. Pifat G. Brnjas-Kraljevic J. Lipka G. Müller K. Knipping G. Chem. Phys. Lipids. 1988; 48: 135-139Crossref PubMed Scopus (5) Google Scholar). As to the actual transition process, it is generally assumed that it occurs between a low temperature state, in which CE and TG molecules are rigidly packed with their long molecular axes radially arranged in two concentric shells of approximately 36 Å radii each, and a fluid, oily droplet state above the transition (12Laggner P. Degovics G. Müller K.W. Glatter O. Kostner G.M. Holasek A. Hoppe-Seyler's Z. Physiol. Chem. 1977; 358: 771-778Crossref PubMed Scopus (41) Google Scholar,13Atkinson D. Deckelbaum R.J. Small D.M. Shipley G.G. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 1042-1046Crossref PubMed Scopus (101) Google Scholar). A quantitative description of the two states and of the processes involved in the transition is still lacking. However, more detailed structural information is required to understand the biochemical effects of the transition. Several important biological activities of LDL have been shown to be tightly controlled by the physical state of the core lipids: the activity of lecithin-cholesterol acyltransferase (16Zechner R. Kostner G.M. Dieplinger H. Degovics G. Laggner P. Chem. Phys. Lipids. 1984; 36: 111-119Crossref PubMed Scopus (12) Google Scholar) and of CE transfer protein (17Morton R.E. Parks J.S. J. Lipid Res. 1996; 37: 1915-1923PubMed Google Scholar) and the susceptibility of LDL to copper-induced lipid peroxidation (18Schuster B. Prassl R. Nigon F. Chapman M.J. Laggner P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2509-2513Crossref PubMed Scopus (38) Google Scholar). The present study was performed to obtain detailed information on the relationship between core lipid composition and the physical states involved in the transition, by differential scanning calorimetry (DSC) analyses of a large number (n > 60) of LDL subspecies. Defined LDL particle subspecies have the advantage over the broad continuum of total LDL of possessing a high degree of structural homogeneity with respect to hydrated density, chemical composition, and physicochemical properties (19Chapman M.J. Laplaud P.M. Luc G. Forgez P. Bruckert E. Goulinet S. Lagrange D. J. Lipid Res. 1988; 29: 442-458Abstract Full Text PDF PubMed Google Scholar). The range of chemical compositions of the core lipids covered in this study exceeds that of previous studies, particularly with respect to the upper limits of triglyceride content. The composition dependence of the calorimetric core melting transition temperature revealed a hitherto undetected break at CE/TG ratios (w/w) of about 7:1, which does not occur in isolated lipid systems of the same composition (20Small D.M. The Physical Chemistry of Lipids : From Alkanes to Phospholipids (Anonymous). Plenum Press, New York1986: 395-473Crossref Google Scholar, 21Ekman S. Lundberg B. Acta Chem. Scand. B. 1976; 30: 825-830Crossref Scopus (14) Google Scholar, 22Lundberg B. Acta Chem. Scand. B. 1976; 30: 150-156Crossref PubMed Scopus (19) Google Scholar). This discontinuity suggests that a microphase separation occurs at this point, i.e. a partial demixing of CE and TG molecules, a phenomenon that was investigated further by electron spin resonance spectroscopy (ESR) using CE and TG spin labels. Our results demonstrate that the triglycerides remain in a fluid state even below the lipid transition temperature. By combining our present findings with earlier small angle x-ray and neutron scattering data, we now propose a substantially refined structural model of the LDL particle. 16-DSA (2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl), 5-DSA (2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl), cholesterol, dioleylglycerol (mixed isomeres), egg yolk phosphatidylcholine, and triolein were obtained from Sigma (Deisenhofen, Germany). Pefablock and trypsin inhibitor (both for biochemical purposes) were from Merck (Darmstadt, Germany), gentamycin (research grade) from Serva (Heidelberg, Germany), and all other chemicals were of analytical grade. Distinct homogeneous LDL subspecies were used for all experiments. LDL was isolated from human plasma, and discontinuous density gradients were fractionated as described earlier (19Chapman M.J. Laplaud P.M. Luc G. Forgez P. Bruckert E. Goulinet S. Lagrange D. J. Lipid Res. 1988; 29: 442-458Abstract Full Text PDF PubMed Google Scholar). The subspecies used for this study were in a density range of 1.0244–1.0435 g/ml; 90% of LDL in normolipidemic human plasma possess hydrated densities in this range. The LDL preparations were dialyzed exhaustively in the dark at 4 °C against phosphate-buffered saline, pH 7.4, i.e.10 mm phosphate buffer (Na2HPO4/KH2PO4), 150 mm NaCl, 270 μm EDTA, and 50 mg/liter gentamycin. Phosphate-buffered saline was deoxygenated in a vacuum followed by purging with nitrogen gas. To prevent oxidation, LDL was stored in the dark at 4 °C in an argon atmosphere. Total and free cholesterol were determined with CHOD-PAP enzymatic test kits (Boehringer Mannheim, Germany). Cholesteryl ester content was calculated as (total cholesterol − free cholesterol) × 1.67. This factor represents the ratio of the average molecular weight of cholesteryl ester to free cholesterol. Phospholipids and triglycerides were assayed by two enzymatic kit tests (bioMérieux, France). Protein was measured by the bicinchoninic acid assay (BCA assay, Pierce, The Netherlands). The spin-labeled lipids were synthesized by the pyrrolidinopyridine method (23Patel K.M. Sklar L.A. Currie R. Pownall H.J. Morrisett J.D. Sparrow J.T. Lipids. 1979; 14: 816-818Crossref PubMed Scopus (27) Google Scholar). Either dioleylglycerol or cholesterol was brought to reaction at room temperature with 16-DSA in the presence of pyrrolidinopyridine andN,N′-dicyclohexylcarbodiimide. 16-Doxylstearoylcholesterylester (16-CE) and (16-doxylstearoly)dioleylglycerol (16-TG) were separated by chromatography on a Silicacolumn (Mega Bond Elut-SI, 1 g, Varian). The purity of the products was checked by thin layer chromatography (for 16-CE/ethyl acetate/hexane (9:3, v/v); for 16-TG: chloroform/acetone/acetic acid (96:4:1, v/v/v)). Microemulsions were prepared by codissolving egg yolk phosphatidylcholine, cholesterol, and the desired amount of spin probe (5:1:5 molar ratio) in chloroform (24Craig I.F. Via D.P. Sherrill B.C. Sklar L.A. Mantulin W.W. Gotto Jr., A.M. Smith L.C. J. Biol. Chem. 1982; 257: 330-335Abstract Full Text PDF PubMed Google Scholar). The chloroform was evaporated in a nitrogen stream and the dry lipid film redissolved in 15–30 μl of 2-propanol at 37 °C. The 2-propanol solution was injected rapidly into phosphate-buffered saline. This method to prepare microemulsions does not lead to particles of defined composition and size, which are not required in this case as they only serve as a substrate for TG or CE transfer. In a typical transfer preparation, 12 mg of LDL protein was incubated with microemulsions containing 3 mg of spin label (the amount of 2-propanol did not exceed 0.5% of the total incubation mixture) and 300–400 mg of lipoprotein-deficient plasma (d > 1.21 g/ml), a source of CE transfer protein at 37 °C (25Granot E. Deckelbaum R.J. Eisenberg S. Oschry Y. Bengtsson-Olivecrona G. Biochim. Biophys. Acta. 1985; 833: 308-315Crossref PubMed Scopus (48) Google Scholar). The total volume of the mixture was 6 ml. The tubes were flushed with argon and placed in a water bath at 37 °C for 5 h (16-CE), or for 24 h (16-TG). After incubation, the labeled LDL was reisolated using the following procedure. The density of the incubation mixture was adjusted to 1.41 g/ml. About 1.2 ml of mixture was transferred to a centrifugation tube and overlayered with solutions of 1.080, 1.050, and 1.000 g/ml (1.2 ml each). After centrifugation (Sorvall OTD 65B centrifuge, AH-650 rotor) at 4 °C for 24 h at 40,000 rpm, the LDL fraction was removed using a bent needle. Spin-labeled LDL was concentrated using Centricon 10 concentrators (Amicon, MA) to a final concentration of 7 mg/ml protein. For preparations of spin labels in pure triolein or olive oil, lipids were dissolved in chloroform and deposited as a dry, thin film on the glass tubing. Triolein or olive oil was added and incubated with moderate mixing for 10 min at room temperature. For ascorbate quenching, the solutions of ascorbic acid 3*10−3m were prepared immediately before use (phosphate-buffered saline with 0.1 g/liter EDTA, pH 7.4) and added to the sample at a ratio of 1:10 (v/v). The ESR signal was measured as quickly as possible after mixing (after approximately 4 min). All ascorbate-quenching measurements were performed at 37 °C. Calorimetry experiments were performed on the high sensitivity, differential adiabatic scanning microcalorimeter DASM-4 (Biobribor, Pushtchino, Russia) designed by Privalov (26Privalov P.L. Pure Appl. Chem. 1980; 52: 479-497Crossref Scopus (170) Google Scholar). The scanning rate was 1 °C/min; samples (1–2 mg/ml total LDL cholesterol) were loaded into the calorimeter sample cell at room temperature, and an equal amount of buffer was loaded into the reference cell. The cells were pressurized with nitrogen to 250 kilopascals (2.5 bar). Heat capacity functions were obtained after base-line subtraction and normalization of experimental data, given in μW, to heat capacity units (J/°C/g of CE). Data were collected on heating runs from 1 °C to 45 °C. Each sample was scanned two or three times and held at 1 °C for approximately 30 min between runs. The transition temperature (T m) was taken as the point of maximum change in heat capacity in the heat capacity versustemperature curves. ESR measurements were performed in glass capillaries (1-mm inner diameter) on an X-band Bruker ECS 106 ESR spectrometer (Bruker, Rheinstetten, Germany). The following settings were used for determination of the rotational correlation times: microwave power, 2 mW; modulation amplitude, 1 G; modulation frequency, 100 KHz; scan width, 100 G. The temperature was controlled to within ± 0.1 °C by a Bruker temperature controller B-VT 2000. The initial temperature was 4 °C and was increased stepwise to 40 °C. The preparations were allowed to equilibrate for at least 5 min at each temperature before data acquisition. 10–20 scans were accumulated per temperature point. The determination of the spectral parameters used in the evaluation of ESR data is shown in Fig. 3 A. Correlation times (τc) for the approximately isotropic motion were calculated from line widths W 0 as described by Kivelson (27Kivelson D. J. Chem. Phys. 1967; 46: 3048-3052Crossref Scopus (70) Google Scholar) τc=(6.5×10−10)W0((h0/h−1)½−1)Equation 1 where h 0 and h −1refer to first derivative line heights of the midfield line and the highfield line, respectively. For the sample of 16-CE in LDL displaying a high CE/TG weight ratio, it was not possible to determine the rotational correlation time because of the more anisotropic motion of this spin label. Equation 1 cannot be used for anisotropic spectra with relatively slow motion. Thus, as a parameter related to the motional characteristics, the ratioh 0/h −1 has to be used (28Smith I.C.P. Swartz H.M. Bolton R.B. Borg D.C. Biological Applications of Electron Spin Resonance. John Wiley & Sons, New York1972: 483-539Google Scholar, 29Krieger M. Peterson J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1980; 255: 3330-3333Abstract Full Text PDF PubMed Google Scholar). To estimate the polarity of the environment of the spin probes, the isotropic hyperfine couplinga n′ was used: aN′=1/3(A∥+2A⊥)Equation 2 where 2A ∥ and 2A ⊥ are the outer and the inner hyperfine splitting of the spin probe, respectively (30Hubbell W.L. McConnell H.M. J. Am. Chem. Soc. 1971; 93: 314-325Crossref PubMed Scopus (1425) Google Scholar, 31Seelig J. J. Am. Chem. Soc. 1970; 92: 3881-3887Crossref Scopus (285) Google Scholar). We first present the calorimetric data that lead to the postulation of a microphase separation of apolar lipids in the LDL core. DSC analysis and determination of the actualT m were performed on a large number (n > 60) of discrete LDL subspecies isolated from several single donors. The average composition of the subspecies that were analyzed in this study is presented in Fig.1. The use of LDL subspecies, which are highly homogeneous and well defined with respect to their physicochemical properties (19Chapman M.J. Laplaud P.M. Luc G. Forgez P. Bruckert E. Goulinet S. Lagrange D. J. Lipid Res. 1988; 29: 442-458Abstract Full Text PDF PubMed Google Scholar, 32Dejager S. Bruckert E. Chapman M.J. J. Lipid Res. 1993; 34: 295-308Abstract Full Text PDF PubMed Google Scholar, 33McNamara J.R. Small D.M. Li Z.L. Schaefer E.J. J. Lipid Res. 1996; 37: 1924-1935Abstract Full Text PDF PubMed Google Scholar), presents a significant and major advantage and is absolutely necessary for such experiments. Unfractionated LDL, or even worse, LDL isolated from plasma pools of many donors, can obscure findings as a result of its marked heterogeneity. The presence of various transition temperatures and chemical compositions in the same preparation would make it impossible to discuss differences in thermotropic behavior reliably. The transition temperatures of the LDL core varied between 15 and 32 °C, although most samples fell into the range of 24–30 °C. The weight ratio of CE to TG was chosen as chemical parameter because CE and TG are the principal LDL core lipids. When free cholesterol was included in the calculations, the results were unaffected. PlottingT m versus the weight ratio of CE/TG (Fig. 2) led to the observation that a single linear approximation was insufficient to fit the data points. Rather, it appeared that a discontinuity occurred at CE/TG ratios of approximately 7. Such a discontinuity in the concentration dependence of a transition temperature in a mixed lipid system is a clear indicator of a phase separation at this point. To understand the molecular basis of this nonlinear behavior further and to obtain additional evidence concerning the hypothesis that a microphase separation occurs within the core lipids, spin probes were introduced into the LDL core, and the mobility and polarity of their environment were investigated (Fig.3). Temperature series of ESR measurements were performed on various LDL subspecies labeled either with 16-CE or with 16-TG. To characterize the situation above and below the break point in the plot ofT m versus CE/TG ratio (see Fig. 2), some representative measurements are introduced. At high CE/TG ratios, the 16-CE label incorporated into the LDL core displayed two distinctly different types of spectrum. ESR spectra recorded at T < T m showed line shapes that are characteristic for anisotropic motion, as is most clearly reflected in the splitting of the low field line (seearrows in Fig. 4 A). At T > T m, the motion of the spin-labeled CE became a more isotropic one but still remained restricted as seen by the relatively broad spectral lines. Because of the anisotropic slow motion of the label, the line/height ratio was used to follow the temperature course (Fig. 4 C). In contrast, the recorded spectra for 16-TG-labeled subspecies with a high CE/TG ratio display a rather isotropic behavior (Fig.4 B). The temperature dependence of the rotational correlation time τc showed no change aroundT m (Fig. 4 D), contrary to the temperature profile of 16-CE. At the other extreme of the CE/TG range, one has to examine the situation at a CE/TG ratio below 7. In this case, the behavior of 16-CE and 16-TG labels in the LDL core are almost identical. One cannot distinguish between LDL subspecies labeled with 16-CE or with 16-TG on the basis of their spectral parameters (see Fig.5, A and B). Neither the spectra of 16-CE nor of 16-TG reveal the degree of immobilization displayed with 16-CE in LDL with a high CE/TG ratio. Also, at T > T m, a highly mobile situation is reached. Plotting τc versus T results in essentially congruent plots (Fig. 5, C and D). To compare the behavior of 16-TG in LDL with that in a TG-rich environment, the ESR spectra of 16-TG were measured in olive oil and triolein. The spectra of 16-TG in LDL of CE/TG ratio > 7 and those in olive oil or triolein showed the same spectral characteristics but were shifted by about 20 °C (Fig.6). Results for 16-TG in triolein and in olive oil were identical. The notion that 16-TG within LDL with a CE/TG ratio > 7 is located in a TG-rich domain is supported by comparing τcfor 16-TG in olive oil and LDL. In this case, the same temperature-dependent decrease with an almost identical decay constant of the regression fits was observed. Furthermore, the isotropic hyperfine couplinga n ′was calculated to obtain information on the polarity of the 16-TG environment (data not shown). These calculations clearly reveal that 16-TG is located in a low polarity environment. Somewhat surprising is the fact that the polarity of 16-TG in olive oil is higher than the polarity of 16-TG in LDL, despite the presence of groups that increase the polarity of LDL (hydroxyl group of cholesterol, ester groups, etc.). The degree of unsaturation in LDL is similar to that in olive oil. We tend to assume that mobility effects play a role in the determination of polarity through a n′. In any case, however, these results confirm that 16-TG is located in an extremely apolar environment in LDL. To assure the validity of the results, it was essential to confirm that the lipid spin labels were intimately associated with and situated in the core of LDL particles. This question was approached by use of ascorbate quenching. Ascorbate is one of the most efficient water-soluble reducing agents for nitroxide radicals (34Gotoh N. Noguchi N. Tsuchiya J. Morita K. Sakai H. Shimasaki H. Niki E. Free Rad. Res. 1996; 24: 123-134Crossref PubMed Scopus (70) Google Scholar, 35Kveder M. Pifat G. Pecar S. Schara M. Chem. Phys. Lipids. 1994; 70: 101-108Crossref PubMed Scopus (10) Google Scholar). To determine the accessibility of each spin-labeled lipid type within LDL to the aqueous phase and to verify the location of the spin labels in the LDL core, the rates of ascorbate-induced reduction of the nitroxyl moiety in the LDL core and in the microemulsions labeled with either 16-CE or 16-TG were examined by monitoring the loss of the ESR signal. Adding ascorbate solution to LDL preparations containing 16-TG or 16-CE resulted in a very slow loss of ESR signal intensity. The ESR signal remained stable for a period of more than 2 h. On the contrary, the addition of ascorbate solution to the microemulsion containing either large quantities of 16-CE or 16-TG led to an almost immediate (approximately 5 min) loss of paramagnetism. Comparing the rate of reduction with that of 5-DSA and 16-DSA in LDL, it can be stated that 16-TG and 16-CE in LDL were reduced by a lower rate (Fig.7). These observations confirm the notion that the 16-CE and 16-TG spin probes were localized in the core of the LDL particle (see Fig. 8).Figure 8Cross-sectional model for the proposed arrangement of the CE and TG in the LDL core at two representative CE/TG ratios. A and B show the situation below T m and C, aboveT m. The symbols used for CE, TG, free cholesterol (FC), phospholipids (PL), and apolipoprotein B (apoB) are indicated in the figure.View Large Image Figure ViewerDownload (PPT) To ensure that the integrity of LDL was not impaired by the incubation and reisolation processes, polyacrylamide gel electrophoresis was performed. The labeled LDL behaved in the same manner as the control preparation. The present results strongly suggest that the apolar lipids TG and CE, in the core of LDL, undergo phase separation below the core lipid transition. This finding is at variance with most of the models proposed in the literature, and it is therefore necessary to recall the evidence that has led to these models. Before that, however, a more general feature of a quasispherical core-shell model, as is consistent with LDL, has to be considered. The relative weight of the shells varies with the third power of the radius, such that the innermost parts are only a very minor fraction. For instance, in a three-shell structure with radii 1:2:3, the core contributes only 3.7% to the total volume. Therefore, any mass-dependent measurement, ase.g. SAXS, SANS, or NMR, will attain the limits of resolution in terms of the definition of the details of core structure. On the other hand, molecular probe methods such as spin probe ESR, which rely inherently on the exclusive signal from the probe, are not limited by such restraints as long as the probe can be considered nonperturbing. Similarly, DSC signals from a melting transition are sensitive to minor contamination of the melting matrix, which would hardly be detectable by scattering or NMR methods. This feature makes the methods chosen in this study well suited for studies of questions concerning the state of a minority component (TG) in the excess of a second component (CE). Key evidence for the presently most widely accepted model of the neutral lipid core in LDL has come from the combined x-ray and calorimetric studies by Deckelbaum et al. (10Deckelbaum R.J. Shipley G.G. Small D.M. Lees R.S. George P.K. Science. 1975; 190: 392-394Crossref PubMed Scopus (134) Google Scholar, 11Deckelbaum R.J. Shipley G.G. Small D.M. J. Biol. Chem. 1977; 252: 744-754Abstract Full Text PDF PubMed Google Scholar), which was confirmed
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