In Vitro Production of Human Plasma Low Density Lipoprotein-like Particles

Transcription

1 THE <JUURNAL OF B~LOGICA~ CHEMISTRY Vol 254, No. 13, Issue of duly 10, pp , 1979 Printed in USA. In Vitro Production of Human Plasma Low Density Lipoprotein-like Particles A MODEL FOR VERY LOW DENSITY LIPOPROTEIN CATABOLISM* (Received for publirntion, August 17, 1978, and in revised form, December 1, 1978) Richard J. Deckelbaum,@ Shlomo Eisenberg,l Menachem Fainaru,l Yechezkel Barenholz,ll and Thomas Olivecrona** From the Departments of +Pediatrics, IMedicine B, and IIBiochemistry, Hadassah University Hospital, Hebrew University- Hadassah Medical School, Jerusalem, Israel, and the **Department of Medical Chemistry, University of Umea, Umea, Sweden To test whether human plasma low density lipoprotein (LDL) can be formed from very low density lipoprotein (VLDL) entirely in the plasma compartment, VLDL was incubated in vitro with purified bovine milk lipoprotein lipase and albumin. After a l-h incubation, over 97% of VLDL triglyceride is hydrolyzed. Lipoproteins of density <1.019 g/ml, to g/ml (in vitro LDL ), and to 1.21 g/ml are then isolated by ultracentrifugation. In vitro LDL contains almost 80% of recovered cholesterol ester originally associated with VLDL, and 54 to 63% of the other recovered VLDL constituents. Electrophoretic mobility is slightly faster than native LDL. Differential scanning calorimetry, fluorescence polarization, and lipoprotein compositions suggest very similar organization for the cholesterol ester-rich core, and phospholipid surface of native and in vitro particles. Analytical ultracentrifugation and electron microscopy show that in vitro LDL is less homogeneous and larger than native LDL (diameter 270 A uersus 215 A, M, = 5.0 x lo6 uersus 2.5 x 106). Excess phospholipid and apoproteins ( surface remnants ) removed from VLDL upon core triglyceride hydrolysis are present in the to g/ml density range, mainly as sac-like unilamellar liposomes, and in the to 1.21 g/ml density range as discoidal particles. Therefore, lipolysis of VLDL using only an extrahepatic lipoprotein lipase can produce an apoprotein B-cholesterol ester-rich particle having many features in common with, but not identical to plasma LDL. Additional pathways must operate in uiuo to form more homogeneous and smaller circulating LDL. In man, plasma low density lipoprotein are the major carriers of plasma cholesterol, while very low density lipoproteins and chylomicrons are the major transport vehicles for triglycerides. Through the activity of extrahepatic lipoprotein li- * This work was supported by the Herbert M. and Nell Singer Medical Research Fund of the Joint Research Fund of Hadassah University Hospital-Hebrew University; Israel Commission for Basic Research; The Center for Absorption in Science, the Ministry for Immigrant Absorption, State of Israel; The United States-Israel Binational Science Foundation (Research Grant 219); United States Public Health Service (HL 17576); Swedish Medical Research Council (13X-00727). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 8 To whom correspondence should be addressed. pases, both chylomicrons and VLDL lose triglyceride and form particles poorer in triglyceride and richer in cholesterol ester, that is, intermediate density lipoprotein (IDL) or remnant particles (1). Formation of IDL occurs in the plasma compartment (2, 3) and IDL obtained from either chylomicrons or VLDL cannot be differentiated in terms of size, lipid, or protein composition (4). Recent studies suggest that almost all of the protein moiety of plasma LDL originates from VLDL via the intermediate stage of IDL formation (2,3,5), although the relative transfer of protein from VLDL to LDL is much greater in man (3, 5) than in other mammals such as the rat (6-8). Triglyceride and cholesterol ester in the triglyceride-rich lipoproteins are catabolized by dissimilar pathways, at least in some animals (9-12). Initially, triglyceride is removed from VLDL in plasma or extrahepatic tissues forming intermediate (remnant) particles. Formation of LDL has been postulated to follow the uptake of IDL by the liver, with subsequent release of apoprotein B-cholesterol ester-rich particles, LDL. Uptake of IDL by the liver, however, has only been demonstrated in animal species (6, 8, 9) but not in man. Man, which has the highest LDL concentration of all mammals (13) is the only species in which almost all of the VLDLlabeled apoprotein B can be recovered in LDL (3, 5). Hence, it is likely that VLDL catabolism to IDL and then LDL differs in man compared to the animals upon which were based the experiments suggesting a role for the liver in formation of plasma LDL. Is it possible that in the human, LDL may be formed from VLDL entirely in the plasma compartment and independently of the liver? We have investigated this hypothesis by incubating plasma VLDL with an extrahepatic purified lipoprotein lipase in a model system free of any hepatic component. Our results show that solely by the action of lipoprotein lipase on VLDL, lipoprotein particles floating in the LDL density range can be produced in the test tube. These in vitro produced low density lipoprotein-like particles ( LDL ) have many features in common with, but are not identical to, native plasma LDL. METHODS Isolation of Native Plasma Lipoproteins-Blood was collected in vacuum packs containing 1 mg/ml of disodium EDTA from fasting I The abbreviations used are: VLDL, very low density lipoprotein(s); LDL, low density lipoprotein(s); IDL, intermediate density lipoprotein(s); HDL, high density lipoprotein(s); DSC, differential scanning calorimetry. In vitro produced particles floating in the IDL, LDL, and HDL density ranges are enclosed in quotation marks as IDL, LDL, and HDL, respectively. Native lipoproteins isolated from plasma are identified without quotation marks. 6079

2 Production of LDL in Vitro (14 to 16 h) normolipemic or mild type IV hypertriglyceridemic males. Plasma was immediately separated, and individual lipoprotein classes were prepared by repetitive salt density ultracentrifugation using density solutions of NaCl and KBr, or solid KBr as previously described (14). VLDL was isolated at plasma density d = g/ml, IDL between and g/ml, LDL between and g/ ml, and HDL between salt densities and g/ml. Isolated lipoproteins were washed once in NaCl/KBr solutions of appropriate density. All solutions contained 1 mm EDTA and were adjusted to ph 8.5 using NH,OH. Densities of solutions were verified by densimetry. All centrifuge runs were at 4 C for at least 18 h, (HDL was isolated over 48-h runs) in Beckman 40.3 or 50.1 rotors, at 40,000 rpm and 50,000 rpm, respectively. Chylomicrons (S, > 400) were removed from VLDL by a single spin of the VLDL in a Beckman SW-41 rotor at 40,000 rpm for 30 min, at 10 C. After isolation, all lipoproteins were dialyzed in.the dark for 24 h against normal saline (0.15 M NaCI, 1 mm EDTA, ph 8.5,1:40 v/v) at 4 C with a minimum of three changes of dialysate. For storage, lipoproteins were always layered with nitrogen and stored in the dark at 4 C. Preparation of in Vitro-Produced Lipoproteins-VLDL was obtained as described in the preceding section. Purified bovine skim milk lipoprotein lipase was isolated by heparin-sepharose affinity chromatography using methods described in detail elsewhere (15). The enzyme was stored frozen in 1.5 M NaCl at a concentration of 300 pg/ml. The activity of the purified enzyme in the incubation buffer described below was 300 to 400 units of lipolytic activity/mg of protein (1 unit = 1 pmol of fatty acid released/min at 24 C). Enzyme was stored at -70 C and small aliquots were thawed for use immediately prior to each experiment. Incubation of VLDL with lipase was performed in precleaned Beckman 40.3 cellulose nitrate centrifuge tubes. Each 5 ml contained 6% bovine albumin (fatty acid poor Pentex albumin, Miles Laboratories, Kankakee, Ill.); 0.2 M Tris/HCl buffer ph 8.4 in 0.08 M NaCl (Sigma Chemical Co., St. Louis, MO.); 10 units of heparin (Evans Medical Ltd., Liverpool, England); 5 mg of VLDL triglyceride, and 10 ~1 of purified lipoprotein lipase solution. (VLDL and lipase were added only after the previous components were mixed.) The tubes were incubated at 37 C for 1 h in a shaking water bath. At 60 min, enzyme activity was inhibited (16) by addition of 0.67 ml of d = g/ml NaCl solution which raised the salt density of the incubation mixture to g/ml. Particles produced following VLDL lipolysis were then separated by repetitive salt density ultracentrifugation in a Beckman 40.3 rotor by methods identical to those described in the preceding section. In uitro-produced lipoproteins were isolated at density ranges corresponding to their native plasma counterparts; i.e., in uitro IDL at d < g/ml, in vitro LDL at d = to g/ml, and in vitro HDL at d = to g/ml. In uitroproduced lipoproteins were dialyzed and stored as described above and experiments were performed within 7 days of isolation. Analysis-For lipid analysis, lipids were extracted from lipoprotein fractions in chloroform:methanol, 2:1, v/v (17). Lipoprotein phospholipid content was determined using the procedure of Bartlett (18), total cholesterol by the method of Chiamori and Henry (19) and triglycerides by the Auto Analyzer method (26). Content of free fatty acids was measured in extracts prepared by Dole s method (21) and by radiochemical assay (22). The relative contribution of individual phospholipids to total phospholipid, and free cholesterol and cholesterol ester to total cholesterol, were determined after separation of individual lipid classes by thin layer chromatography using methods described previously (23). When only small amounts of sample were available, full lipid analysis was kindly performed by Dr. Saul Katz, Department of Medicine, McGill University, Montreal, Canada, using quantitative thin layer chromatography (24). Lipoprotein protein composition was determined by the method of Lowry (25) using bovine serum albumin as a standard. Content of tetramethylureainsoluble protein which is essentially B apoprotein, and tetramethylurea-soluble apoprotein which measures non-b apoproteins were measured by the method of Kane (26, 27). Polyacrylamide gel electrophoresis in 8 M urea of tetramethylurea-delipidated lipoprotein was performed according to the procedure described by Kane (26). Agarose gel electrophoresis of lipoproteins was performed on precast agarose slides in 0.05 M barbital buffer, ph 8.6 (Bio-Gram A, Bio-Rad Laboratories, Richmond, Calif.). Differential Scanning Calorimetry-Calorimetry studies were performed on a Perkin-Elmer DSC-2 differential scanning calorimeter at a full scale sensitivity of 0.1 to 0.2 meal/s with heating and cooling rates of 5 C/min, as previously described in detail (28). Samples were hermetically sealed in stainless steel (70 ~1) sample pans (Perkin- Elmer, Norwalk, Conn.). Native LDL and in uitro-produced LDL samples contained about 1 to 2 mg of lipoprotein and were run with equivalent amounts of solvent in the reference pan. Fluorescence Polarization-Fluorescence anisotropy of 1,6-diphenylhexatriene was determined from fluorescence depolarization of the fluorophore incorporated into individual lipoprotein fractions. The general theory of this technique is described elsewhere (29). Fluorescence depolarization measurements with diphenylhexatriene were performed as described by Shinitzky and Barenholz (30) using the instrument previously described (31). All measurements were obtained by simultaneous measurement of Ill/IL and I, which are the fluorescent intensities detected through a polarizer oriented parallel (11) and perpendicular (I) to the direction of polarization of the excitation beam. Four milliliters of lipoprotein solution in 0.15 M NaCl (with 1 mm EDTA, ph 8.5) were incubated at 25 C following injection and vigorous 10-s mixing of 1 ~1 of 0.5 mm diphenylhexatriene in tetrahydrofuran. For these experiments, about 1 mol of diphenylhexatriene/1500 mol of lipoprotein phospholipid was used. Measurements were done exactly as detailed previously by Barenholz et al. (32). Apparent microviscosities were calculated from the Perrin equation, which describes the effect of viscosity on the rotation depolarization of a fluorophore (30). Fluorescence decay measurements (kindly performed by Dr. A. Gafni, Weizman Institute of Science, Rehovot, Israel) were done precisely as described previously (32), incorporating modifications to overcome problems due to drift associated with repeated averaging procedures used in fluorescence decay measurements (33). Analytical Ultracentrifugation-Analytical ultracentrifugation was performed in a Spinco model E ultracentrifuge with schlieren optics and double-sectored analytical cells. The runs were made at 21 C at 40,000 rpm. Standard equations were used to calculate flotation values (S, rates) at salt density of d = g/ml (34). Samples were studied at concentrations of about 0.5 to 1.0 mg of lipoprotein protein/ml, and were run against the solvent buffer as a blank reference. Electron Microscopy-Electron microscopy was kindly performed bv Dr. S. Huana, Denartment of Patholonv. McGill Universitv. Montreal, Canada. Lipoproteins were negatively stained with B% sodium phosphotungstate, ph 7.4, on Formvar-coated grids. Electron micrographs were obtained with a Phillips 300 electron microscope, calibrated with a catalase standard, at instrument magnification of 71,000. For sizing of lipoproteins, at least three different grids were employed with each preparation. A histogram for particle size distribution was tabulated only after at least 400 particles had been sized for an individual preparation. Samples were examined at a concentration of 0.1 to 0.5 mg of protein/ml. RESULTS Fate of VLDL Constituents Postlipolysis Following incubation2 of VLDL with lipoprotein lipase for 1 h, more than 97% of the VLDL triglyceride is hydrolyzed. In 11 incubations with VLDL obtained from different donors, only % (mean + S.E.) of the original triglyceride in the incubation mixture remained at the end of the incubation (assuming the original amount of VLDL triglyceride in the incubation mixture to be 100%). 1.4 f 0.3% of triglyceride was recovered as the IDL density range, and % in the LDL density range. In only one incubation was any triglyceride detected in the HDL density range, and it represented less than 0.05% of the original triglyceride. The relative redistribution of the other VLDL lipids in the in vitro lipoproteins is outlined in Table I. After incubation, a Serial sampling of incubation mixtures with time showed that in every case free fatty acid release reached maximum values and triglyceride minimum values by 30 min after addition of lipoprotein lipase. Increasing incubation time to 120 min, or doubling the amount of albumin in the incubation mixture did not increase triglyceride lipolysis as judged by free fatty acid release. Experiments performed without addition of enzyme resulted in no increase in free fatty acid in the incubation mixture over control values. The enzyme did not hydrolyze cholesterol ester as assayed by the total content of free cholesterol and cholesterol ester, and the free cholesterol to cholesterol ester ratios, in the incubation mixture before and after incubation.

3 Production of LDL in Vitro 6081 Lipoprotein TABLE Relative redistribution of VLDL constituents into in vitro density classes after VLDL triglyceride hydrolysis Cholesterol, es- Cholesterol, ter free Lipids I Proteir? Non-triglyceride Lecithin Sphingomyelin Apoprotein B Non-B (TMLJ- VLDL constitu- (TMU-insolu- ents ble) soluble) IDL 12.9 f f t f f 1.8 LDL f f f HDL f f n Based on analysis of lipoproteins produced in vitro from 11 incubations from separate VLDL donors. b Based on analysis from eight incubations from separate VLDL donors. TMU = tetramethylurea. Per cent of total recovered in in vitro produced lipoproteins following VLDL incubation with lipoprotein lipase in vitro. Results are expressed as mean f SE. TABLE Composition analysis of substrate VLDL, native LDL, and in vitro LDL All results are obtained from 11 separate incubations, except for per cent tetrarnethylurea soluble protein where analysis was carried out on data from 8 VLDL incubations. Lipoprotein Triglyceride II Cholesterol, es- Protein, TMU- Cholesterol/ Lecithin/sphinter Cholesterol, free Phospholipid Protein, total soluble, % phospholipid gomyelin molar molar ratioh ratio VLDL f * f Native k f LDL In vitro f f 0.1 LDL n Per cent of total protein + SE.; TMU = tetramethylurea. Ratio calculated from means of free cholesterol and phospholipid listed on left hand side of this table with molecular weight of free cholesterol and phospholipid being 387 and 760 daltons, respectively. Composition is expressed as relative weight composition (% of total lipoprotein mass). Results are mean + SE. cholesterol ester partitions mainly to in vitro LDL which accounts for almost 80% of recovered cholesterol ester. In contrast, only two-thirds or less of the VLDL lipids are found in in vitro LDL, showing a relative concentration of cholesterol ester in in vitro LDL. During VLDL lipolysis, between 24 and 29% of the lecithin was hydrolyzed to lysolecithin, and this was recovered almost exclusively with the albumin in the g/ml infranate. Although the IDL and especially HDL density ranges contain significant amounts of the original VLDL lipids, in vitro LDL remains the major catabolic product of in vitro VLDL lipolysis. Table I also gives the values for postlipolytic apoprotein distribution in the in vitro lipoprotein density ranges. Tetramethylurea-insoluble apoprotein, like cholesterol ester, concentrates mainly in in vitro LDL but to a lesser degree. Of the original VLDL constituents, the non-b proteins (tetramethylurea-soluble) are unique in that these redistribute largely to the in vitro HDL density range, but with a significant fraction also partitioning with in vitro LDL. When the relative distribution of all non-triglyceride VLDL components in in uitro lipoproteins is summed after incubation (Table I), LDL accounts for almost two-thirds of the non-triglyceride VLDL constituents, while in vitro HDL and to a lesser degree IDL account for the remainder. Composition of Lipoproteins The relative weight composition of the VLDL used for incubation with lipoprotein lipase, and native LDL obtained from the same donors, are compared with the particles isolated in the in vitro LDL density range in Table II. With the loss of triglyceride from VLDL, cholesterol ester becomes the major component of both in vitro and native LDL. Of all constituents, only the contribution of triglyceride significantly differs (p < 0.005) making up only 3% of the particle weight of in vitro particles, in contrast to 7% in native LDL. The ratio of lecithin to sphingomyelin in VLDL is shown in Table II as decreasing to the same degree on the production of LDL in the test tube or in native human plasma. This equivalent relative enrichment of sphingomyelin suggests similar factors may be controlling this ratio in both in vitro and in uiuo systems. Both Table II and Fig. 1 show that after lipolysis, the apoprotein composition of in uitro-produced LDL is changed markedly relative to VLDL, but it differs from native LDL. Half of VLDL apoprotein is tetramethylurea-insoluble or apoprotein B. Plasma LDL contains almost entirely apoprotein B; but the apoprotein of in vitro LDL contains a significant fraction, over 20%, as non-b apoprotein. Tetramethylurea-polyacrylamide gel electrophoresis (Fig. 1) illustrates that in VLDL the non-b apoprotein fraction consists mainly of the C apoproteins and apoprotein E. In in uitro LDL, apoprotein B remains predominant as in native LDL but also present are C apoproteins, albumin, and traces of apoprotein E. Densitometric scanning of the in vitro LDL gels gives an approximate ratio of the content of C apoproteins to albumin to be 1:l. Characterization of in Vitro LDL Electrophoretic Mobility-Fig. 2 compares the electrophoretie mobility of in vitro LDL with VLDL and native LDL, obtained from the corresponding donor. The mobility of in vitro LDL is intermediate between VLDL and LDL. While the band obtained for native LDL is always sharp and well defined, in vitro LDL shows wider and more diffuse staining suggesting a less homogenous particle population for the latter. Analytical Ultracentrifugation-Representative schlieren flotation photographs of native LDL and in vitro LDL are portrayed in Fig. 3. The peak flotation rates of in vitro particles are consistently greater than native LDL, with a The identification of individual apoprotein bands by polyacrylamide gel electrophoresis was done by co-electrophoresis of the purified isolated apoproteins, after their isolation by column chromatography.

4 6082 b Production of LDL in Vitro ALBUMIN- FIG. 3. Analytical ultracentrifugation schlieren flotation photographs of (a) native LDL (concentration 3.9 mg/ml) and ( b) in uztro LDL (concentration 3.1 mg/ml). Photographs are at 16 min after reaching speed of 40,000 rpm. FIG. 1. Polyacrylamide gel electrophoresis of tetramethylurea-delipidated lipoproteins in 8 M urea, (a) substrate VLDL, (6) native LDL, and (c) in vitro LDL. In each case, 0.10 to 0.11 mg of total apoprotein was applied. Apoprotein bands are identified on the basis of their mobilities relative to purified apoproteins isolated by column chromatography. 0 + a b c ORIGIN- FIG. 2. Agarose gel electrophoresis on precast agarose slides of (a) substrate VLDL, (b) in vitro LDL, and (c) native LDL. mean S:$j? of uersus , respectively (n = 3). (Mean peak flotation rate for substrate VLDL under the same conditions was 49.0 rt 3.5 Svedberg units.) Since the relative weight composition of each particle is similar, this suggests that in vitro LDL may be larger than native LDL. In addition to moving faster, the pattern obtained for in vitro LDL is broader than its native plasma counterpart suggesting a less homogenous particle population. Electron Microscopy-Studies of electron micrographs of native LDL and in vitro LDL (Fig. 4) visually confirm that in vitro LDL is not only more heterogenous but also larger than plasma LDL. The mean diameter of native LDL is 215 DIAMETER (i, FIG. 4. Top, electron micrographs of negatively stained: (a) native LDL and (b) in vitro LDL. Bottom, the frequency distribution of the diameters of native LDL (---) and in vitro LDL (--). In a and b, the bar indicates 1000 A. Magnification of original was x 71,000. In b, the solid arrows point to projections on spherical particles and the inset shows electron-lucent sac-like liposomes and a discoidal structure (hatched arrow). Samples contained about 0.2 mg of protein/ml. f 15 (S.D.) A compared to A for in vitro LDL. This comparatively small difference in diameter, however, represents an almost 2-fold difference in particle volume. Hence, the molecular weight of in vitro LDL would also be about twice that of plasma LDL. On about 1.5% of the spher-

5 Production of LDL in Vitro 6083 ical particles, projections from the surface are observed, suggesting the occasional presence of excess surface layer for the now reduced core. These protrusions are up to 100 A in length, and 70 to 90 A in width, which is thicker than a lipid bilayer, suggesting that some core material may extend into the surface projection. In addition to spherical particles which make up most of the population, a small fraction of in vitro LDL appears as sac-like liposomes which appear electron-lucent, and rare discoidal structures. The electron-lucent sacs do not show repetitive spacing at their perimeter and thus can be considered to be unilamellar liposomes. They measure between 350 and 800 A in their widest diameter. The discoidal structures are about 50 to 55 8, thick, with diameter ranging from 275 to 350 A. Thus, following in vitro lipolysis of VLDL, in the model system described, two distinct particle populations are formed, a major group of spherical particles, similar to native LDL although larger, and a minor group of sac-like liposomes and discoidal particles. The latter group seems relatively devoid of core material, and hence is likely protein and phospholipidrich relative to the spherical particles, which would be cholesterol ester-rich. Fig. 5 illustrates that the material isolated in the in vitro HDL density range, after VLDL lipolysis, contains discoidal particles measuring between 150 and 350 A in diameter and 55 A in thickness. Analysis of the material isolated in the HDL density range shows a protein/lipid ratio of 1:l suggesting that with increasing amounts of protein the larger phospholipid-rich particles isolated with in vitro LDL will partition to a heavier range, i.e., HDL. Differential Scanning Calorimetry-To determine if the neutral lipid of in vitro LDL is organized in a cholesterol ester-rich domain in the particle core, as previously demonstrated for native LDL (28,35), in vitro LDL was compared with native LDL, obtained from the same donors who pro- vided the substrate VLDL (Fig. 6). On heating native LDL from 0 to 45 C an endothermic transition is observed between 20 and 45 C (Fig. 6a). This transition has recently been proven to be associated with a liquid crystalline+ liquid phase change of the cholesterol esters in the core of native LDL (28, 35). A similar transition is observed with in uitro LDL (Fig. 6b). As in native LDL (28,35), the transitions in in vitro LDL are perfectly reversible on cooling, and are unchanged on repeated runs between 0 and 45 C, or after TEMPERATURE ( Cl I I I I I ENDOTHERMIC FIG. 6. Differential scanning calorimetry curves of solutions of native LDL and in vitro LDL. a, native LDL heated from 0 to 45 C; b, in vitro LDL heated from 0 to 45 C; and c, denatured in uitro LDL heated from -60 to 45 C after cooling from 100 C (only the curve obtained after the ice melted is shown). Native LDL contained 2 mg of lipoprotein/70 al and was run at a full scale sensitivity of 0.2 meal/s at 5 C/min. In vitro LDL contained about 1 mg of lipoprotein/70 ~1 and was run at a full scale sensitivity of 0.1 meal/s at 5 C/min. TEMPERATURE ( C) a 1 a t FIG. 5. Electron micrograph of the negatively stained particles isolated in the in vitro HDL density range (1.063 to g/ml). The bar represents 1000 A. Magnification of original was x 71,000. Sample contained 0.4 mg of protein/ml. I I I 1 / I / 0.3 I o 38 I/T x IO FIG. 7. Arrhenius plot of apparent microviscosity as a function of the reciprocal of the absolute temperature (l/t) of: (a) native LDL (0), (b) in vitro LDL (A), and (c) substrate VLDL (0). Calculations are based on lifetime values listed in Table III. Lipoprotein solutions were studied at a concentration of 0.25 mg of phospholipid/ml. Points plotted are from cooling runs with cooling rate of O.Y C/min. Identical values were obtained at corresponding temperatures on heating. ( K- 1

6 6084 Production of LDL in Vitro TABLE III Fluorescence polarization parameters of substrate VLDL, native LDL, and in vitro LDL Values shown are from experiments on lipoproteins obtained from The variation in the apparent microviscosity for three different pairs a single donor, and are from the same sample used to plot Fig. 1. of native LDL and in vitro LDL were in the range of 6.5 to 10.5 Apparent microviscosity varied between donors although for a specific poise and for VLDL 0.92 to 1.15 poise. The values reported by Jonas VLDL donor, the apparent microviscosity of native LDL was always for lifetime, apparent microviscosity, and flow activation energy agree extremely close to that of in vitro LDL at a given temperature. well with this range (36). Lipoprotein Lifetime at 25 C Anisotropy 25OC 37 C Apparent microviscosity Anisotropy Flow activation Correlation coef- Apparent mi- energy, E ficienth croviscosity VLDL Native LDL In vitro LDL 9.1; Lifetime of diphenylhexatriene probe incorporated into lipoprotein. Correlation coefficient calculated by linear regression analysis of points plotted in Fig. 7. cooling the sample to -60 C and reheating. Following denaturation of in vitro LDL by heating to 100 C in the calorimeter, cooling to -60 C followed by reheating to 45 C now results in a transition between 5 and 40 C about 5 to 6 times larger than the initial transition in intact native and in vitro LDL (Fig. 6~). Similar behavior has been described for native LDL where, after denaturation of the particle, released cholesterol ester is free to crystallize on cooling to -60 C (which does not occur in the intact particle), and on reheating these crystallized esters now undergo the relatively large crystal + liquid cholesterol-ester melting transition (28). Fluorescence Polarization-Fig. 7 and Table III compare the behavior of the hydrophobic fluorophore diphenylhexatriene in substrate VLDL, native LDL, and in vitro LDL. Results obtained with both native and in vitro LDL are very similar. Both show almost identical values for apparent microviscosity as a function of temperature, with considerably greater values than that obtained for VLDL. Using this technique, no lipoprotein fraction shows a phase transition (Fig. 7). The fluorescence intensity decay measurements (Table III) indicate only a single population of diphenylhexatriene molecules in VLDL and native LDL. In contrast, the best fit for decay measurements in in vitro LDL is obtained for two populations of fluorophore molecules. Based on relative amplitudes, the major population includes more than 75% of the fluorophore molecules and is very similar to native LDL. The minor population includes less than 25% of the probe molecules which have a very short lifetime (2.3 ns) which may represent quenching due to interactions (such as energy transfer) with protein. DISCUSSION The present study was initiated to find out whether LDL can be formed directly from VLDL during the course of VLDL lipolysis. The experiments have indeed demonstrated that an apoprotein B-cholesterol ester-rich particle can be isolated at the LDL density range following an almost complete (>97%) lipolysis of VLDL. These particles were produced in a very simple model system. The experiments thus provide a unique opportunity to study the relative importance of factors presumably involved in LDL formation and the overall process of conversion of VLDL to LDL. With this aim in mind, a 4 The fluorescence decay curves were analyzed using the method of nonlinear least squares (32). The root mean square of the deviations between the calculated and experimental decay curves of the native lipoproteins for single exponential decay were good, to , and did not improve significantly with analysis for two exponential decays. The root mean square for single exponential decay of in vitro LDL, however, was high, , and improved markedly to when analyzed for two exponential decays. detailed comparison was carried out between the in vitro produced LDL and the circulating LDL isolated from the blood plasma of the same VLDL donors and presumably formed from VLDL identical to that used for the in vitro incubation. The incubation mixture employed here allowed for maximal!ipolysis in a system containing only VLDL, buffer, albumin (to trap fatty acids) and lipoprotein lipase purified from bovine milk. The amount of fatty acid-poor albumin included (1 mol/ every 5 mol of triglyceride fatty acids) provided sufficient sites to bind all the fatty acids released (37). With this system, LDL was produced in the test tube within 30 to 60 min as compared to 8 to 12 h needed for a complete conversion of circulating VLDL apoprotein B to LDL apoprotein B (3, 5). The in vitro LDL contained less triglycerides than the native LDL. We therefore conclude that the activities of other triglyceride-hydrolyzing enzymes (i.e. the heparin-releasable triglyceride hydrolase of hepatic origin (38)) are not essential for the formation of LDL and VLDL. Similar conclusions were suggested in the studies of Chajek and Eisenberg (39) and Dory et al. (40) while using VLDL and a membranesupported lipoprotein lipase present in the isolated perfused rat heart. They are moreover consonant with the observation that, whereas plasma triglycerides and lipoprotein lipase are strongly correlated in men, no correlation exists between the levels of the hepatic triglyceride hydrolase and plasma triglycerides (41). But these observations do not rule out the possibility that the hepatic triglyceride hydrolase, when present, plays a role in the metabolism of triglyceride-rich lipoproteins. To elucidate the features of LDL formation that may be entirely dependent on triglyceride hydrolysis and those that may require the presence of cells, tissues, other lipoproteins and other enzymes, we have further characterized in vitroproduced and circulating native LDL. The two were similar (although not identical) in lipid composition, relative protein content, and, as shown by DSC and fluorescence polarization, organization of the component lipids. In vitro-produced LDL however differed from the native lipoprotein in the proportions of polar to apolar constituents, apoprotein composition, particle homogeneity, and size. The organization of lipids in the core of LDL has been studied in detail (28, 35, 42, 43). Cholesterol ester constitutes the major core lipid in which LDL triglyceride and some of the free cholesterol is distributed. Since in VLDL the cholesterol esters are completely dissolved in a predominantly triglyceride-rich core and are not sequestered in a separate domain, as in LDL, it has been suggested that LDL formation is due to removal of triglyceride leaving behind the cholesterol ester moiety together with apoprotein B and surface lipids

7 Production of LDL in Vitro 6085 (44). Apparently no factor other than triglyceride hydrolysis by an extrahepatic lipoprotein lipase is necessary for this process to occur. Using differential scanning calorimetry, the behavior and organization of cholesterol ester in in vitro LDL is shown to be similar to plasma LDL. As previously reported, the transitions observed in LDL about body temperature are attributable only to cholesterol esters, as neither proteins of LDL nor the other lipid classes undergo phase transition in the same temperature range as the cholesterol esters (28). Thus, the transition observed by DSC could be derived only from cholesterol ester-rich domains in in vitro LDL particles. The higher peak melting temperature in in vitro LDL (Fig. 6) of the liquid crystal + liquid cholesterol ester phase transition likely reflects the smaller amount of triglyceride relative to native LDL. Decreasing amounts of triglyceride relative to cholesterol ester in LDL have been reported to increase the temperature of this transition (28). Furthermore, like in native LDL (28), the cholesterol esters in in vitro LDL are in a constrained state as evidenced by their inability to undergo crystallization in the intact particle. Only after lipoprotein denaturation are the released cholesterol esters free to form crystals and undergo crystal -+ liquid transitions (Fig. 6c) as has been demonstrated in native LDL (28). Let us now consider the surface constituents of LDL. It has been demonstrated that the amount of surface constituents of lipoproteins of different diameters change such that in all particles a monomolecular film of about 20 A in width can be formed (45). During VLDL lipolysis, it has been shown that, as the core of the lipoprotein is depleted, surplus surface constituents are removed from the particle (2, 4, 23, 39, 46). This process is presumably independent of the presence of other lipoproteins in the lipolysis system (39, 40, 47). Our results agree with these concepts. The majority of particles isolated in the LDL density range were of spherical shape and only in a few, evidence of excess surface could be identified (see Fig. 4). Also, we found that surface constituents freed from VLDL concomitantly with triglyceride hydrolysis were isolated at least in part at the density interval of to g/ml. As predicted from the chemical composition, the lipids and proteins in these HDL particles were associated and exhibited discoidal shape. Morphologically, the discoidal structures are similar to nascent HDL isolated from rat liver perfusates (48) and intestinal lymph (49). They are also similar to discoidal shaped lipoproteins isolated in the HDL density range during lipopolysis of rat plasma VLDL in the perfused rat heart (39). On morphological grounds then, it can be speculated that some, or almost all, of the HDL precursors reflect phospholipid. cholesterol. apoprotein complexes originating from the surface of lipolyzed triglyceride-rich lipoproteins. This speculation provides an explanation for the observed relationships of lipoprotein lipase and HDL in man (50), and the extremely low HDL levels found in the plasma of patients with lipoprotein lipase deficiency (51) or apoprotein C-II deficiency (52); it however remains to be critically evaluated in the future. The removal of surplus surface concomitant with the formation of a cholesterol ester-rich core, is probably responsible for the observation that the microviscosity of in vitro LDL as measured with diphenylhexatriene, is very different from the precursor lipoprotein, VLDL, and is almost identical with that of circulating LDL. Although the location of diphenylhexatriene probe in serum lipoproteins remains undefined (36, 53), this similar behavior of the probe in both native and in uitro-produced LDL particles suggest similar structural interrelationships of lipids in both lipoproteins. In vitro LDL was isolated at an operational density range of to g/ml. In addition to spherical LDL and rare discoidal structures, we were able to occasionally identify larger, liposome-like particles of 350 to 800 A diameter, at this density range. As pure phospholipid liposomes would be expected to float above density of g/ml (54), it is likely that some protein is interacting with the phospholipids to bring it into the LDL density range. It is interesting to note that similar structures, both discoidal and liposome-like, have been described in the plasma of human subjects with either Tangier disease (55) or 1ecithin:cholesterol acyltransferase deficiency (56), and that these structures seem to decrease in number on fat-free diets. More recently, similar structures were observed in plasma of hypertriglyceridemic humans after the injection of heparin (57). The mechanisms involved in dissociation of excess surface components from VLDL with lipolysis remain undefined. Shrinkage of the neutral lipid core, alone, may be sufficient to destabilize the particle surface, so that redundant surface constituents simply fall off. An alternative mechanism may require formation of lysolecithin at the particle surface prior to removal of surface. The enzyme used in our experiments demonstrates significant phospholipase activity. Similar activity is seen in lipoprotein lipases of supradiaphragmatic rats (46) and rat heart (39). On the basis of our data, we cannot differentiate between these two possibilities. Since complete degradation of VLDL lecithin to lysolecithin, however, by purified phospholipase AS without concomitant triglyceride hydrolysis is insufficient for removal of surface constituents (58), we suggest that reduction of core volume in itself is sufficient to allow dissociation of excess lipid from VLDL as it undergoes lipolysis. Inasmuch as surface constituents were deleted from VLDL at an accelerated rate as compared to cholesterol esters, +he lipoproteins isolated at the density interval of to 1.c16 g/ml contained surplus phospholipids and protein, especiali, non-b proteins. According to data published by Sata et al. (45), lipoproteins of diameter 270 A should contain about 40% of the volume as surface constituents. Our data show that cholesterol, phospholipids, and apoproteins make up slightly over 55%. We believe that this discrepancy is due, at least in part, to the heterogeneity of the lipoproteins isolated at the density range of to g/ml, in particular the liposomelike particles and discoidal structures (surface-remnants?) discussed above. Such contamination also explains the excess non-b proteins found with the in vitro LDL. 7 In particular, This is evident from the change in composition of constituents in VLDL and in. vitro LDL and can be calculated from Table II. Relative cholesterol ester composition was enriched by 2.6 (35.6% in in vitro LDL and 13.6% in VLDL), phospholipid by 1.9 (26.8% and 13.9%), and protein by 2.0 (24.5% and 12.0%). Since the contribution of non-b proteins decreased from 50% to 20% of total protein, they were removed at a much higher rate. The per cent volume contribution of the components of in vitro LDL is calculated from the relative weight composition as listed in Table II, and using values for the partial specific volumes for each component as used by Sata et al. (451, i.e. protein, 0.705; triglyceride, 1.093; cholesterol ester, 1.044; free cholesterol, 0.968; and phospholipid, ml/g. The per cent volume contribution of each composition in in vitro LDL is then: triglyceride, 3.4%; cholesterol ester, 39.8%; free cholesterol, 10.5%; phospholipid, 27.8%; and protein, 18.5% the latter three being surface constituents. 7 Preliminary studies suggest that most of the non-b proteins are not on the same particles as apoprotein B. Passing in vitro LDL through concanavalin A-Sepharose columns only 18 to 20% of the original protein did not adhere to the column. As particles containing apoprotein B strongly adhere to concanavalin A (59), only particles without B apoprotein are recovered and these account for most of the tetramethylurea soluble protein of in vitro LDL. (Fainaru, M., and Deckelbaum, R. J., unpublished results.)

8 6086 Production of LDL in Vitro it may explain the contribution of albumin to particles isolated in in vitro LDL (despite repeated washing) which most likely represents albumin trapped in the sac-like liposomes during incubation. Yet some excess phospholipids and non-b proteins may be present with the particles containing the apoprotein B and cholesterol ester. Such association may cause the faster electrophoretic mobility of in vitro LDL as compared to native LDL. We cannot therefore rule out the possibility that in the presence of an avid acceptor for surface constituents (e.g. HDL), the resulting LDL particles may be more similar to native LDL than in its absence. Thus, the main fundamental differences between in vitro and circulating LDL particles are the size, weight, and content of apoprotein B and cholesterol esters. As judged from the size, composition, and Sf rates, we estimate that the molecular weight of native LDL is about 2.5 X 10 and that of irz vitro LDL, 5.0 x 10. Calculation of the mass contribution of apoprotein B to the total mass of circulating LDL and in vitro LDL yields values of 625,000 and 950,000, daltons, respectively, whereas those of cholesterol ester are 900,000 and 1,800,OOO daltons. Could these differences reflect fusion of VLDL particles during lipolysis? We do not favor this hypothesis. Even assuming a minimum mean molecular weight for substrate VLDL of 12.0 x lo, our compositional data show that fusion of two VLDL particles would result in almost 2 times more cholesterol ester and apoprotein B in each in vitro LDL particle than our results suggest. The 2-fold increase in cholesterol ester likely reflects true differences in the in vitro system as compared to the circulation. In contrast, the apparent increased mass contribution of apoprotein B to in vitro LDL may result from inadequacy of techniques. Apoprotein B is defined here as tetramethylurea-insoluble protein (26). The values of true apoprotein B may be lower than of tetramethylurea-insoluble protein, predominantly in in vitro LDL. Relative to apoprotein B, the original incubation mixture contained 600 times more albumin (by weight). Adsorption of even a minute amount of tetramethylurea-insoluble proteins from the albumin preparation to postlipolysis lipoproteins would be sufficient to explain the apparent increase of apoprotein B in the in vitro LDL. Also, if small amounts of either apoprotein E or apoprotein C remain associated with the insoluble apoprotein B, then the content of apoprotein B in the two lipoproteins becomes very similar. Alternatively, we cannot rule out the possibility that the precursor VLDL may contain surplus apoprotein B which under physiological conditions is deleted from the lipolyzed particles. Such a pathway however has not yet been demonstrated in either humans or experimental animals. Our hypothesis then is that the precursor VLDL contains about twice the number of cholesterol ester molecules than the LDL, and that during the in vitro lipolysis all are retained in the LDL. This retention of cholesterol esters is responsible for the increase in size, weight, and cholesterol ester content of the in t&o-produced LDL. Such hypothesis is consistent with previous studies demonstrating higher content of cholesterol ester in VLDL particles than in LDL (2). According to this hypothesis, cholesterol esters are removed from VLDL during the physiological process of LDL formation. Alternatively, newly formed LDL particles may indeed contain double the amount of cholesterol ester even when formed in the circulation; they, however, gradually lose cholesterol ester during the 3 to 4 days of circulation or passage through extraplasma spaces, or both, in particular through the liver (60). 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