Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp. 4519-4523, September 1978
Medical Sciences
Formation of high density lipoprotein2-like particles during lipolysis of very low density lipoproteins in vitro (lipoprotein lipase/apolipoproteins/lipoprotein lipids/ultracentrifugation)
JOSEF R. PATSCH, ANTONIO M. GOTTO, JR., THOMAS OLIVECRONA*, AND SHLOMO EISENBERGt Department of Medicine, Baylor College of Medicine and The Methodist Hospital, Houston, Texas 77030
Communicated by William E. Gordon, June 22, 1978
The effects of lipolysis of human plasma very ABSTRACT low density lipoprotein (VLDL) on the structure and composition of high density lipoproteins (HDL) have been investigated. Lipolysis was performed in a controlled system in vitro containing VLDL (d < 1.006 g/ml) and HDL3 (d = 1.125-1.210 g/ml) from human plasma and lipoprotein lipase (EC 3.1.1.34) purified from bovine milk. Li lysis of VLDL caused profound changes in HDL3. Protein, phospholipid, and cholesterol liberated from VLDL during its lipolysis were transferred to the HDL3 particles. As a consequence of this in vitro transfer, the chemical composition and biophysical properties of HDL3 were substantially altered. The newly formed Particles exhibited a flotation rate (i41.") of 6.7 and a hydrated density of 1.110 g/ml. The chemical composition closely resembled that of native HDL2, and their size was slightly larger than that of the precursor HDL3. When HDL3 and postlipolysis HDL2 were subjected to ultracentrifugation under flotation velocity and equilibrium conditions, both proved to be stable particles. These results, when extrapolated to in vivo conditions, suggest an important metabolic relationship between the levels of circulating VLDL and HDL2 in plasma. This relationship now permits a reasonable explanation for numerous in vivo observations in which the levels of VLDL and HDL2 change reciprocally.
The high density lipoproteins (HDL) represent one of the four major families of plasma lipoproteins that circulate in human plasma. The HDL have become the focus of much interest since their plasma concentrations have been shown to be inversely correlated with the risk of coronary heart disease (1-3). This correlation is an epidemiologic observation; the mechanism(s) by which HDL protect against atherosclerosis is unknown although several have been suggested (1, 4). A detailed knowledge of the metabolism of HDL is essential for elucidating this mechanism. The pathways of HDL synthesis and degradation in human beings are poorly understood. In animal experiments with isolated perfused rat liver (5) and with rat intestinal lymph (6) a type of HDL resembling a bilayered disc has been isolated. This particle has been called nascent HDL. Hamilton et al. (5) have suggested that the discoidal nascent HDL are transformed to spherical particles characteristic of HDL by the intravascular action of lysolecithin acyltransferase (EC 2.3.1.23). ApoA-I, the major protein of HDL, can be synthesized in rat intestine (6) and is found in the lymph associated both with chylomicrons and nascent HDL. When chylomicrons enter the plasma, apoA-I is transferred to circulating HDL (7). The source of the lipids necessary to form the HDL particles with apoA-I has not been elucidated. HDL can be divided into at least two subfractions, HDL2 and HDL3, which are traditionally isolated at density intervals of 1.063-1.125 and 1.125-1.210 g/ml, respectively (8). HDL2 particles are larger and contain a larger proportion of lipids than
do the HDL3 (9, 10). Whether HDL2 and HDL3 are metabolically interrelated is not known. The present study was undertaken to test the hypothesis that the lipid and apoprotein constituents formed in the degradation of triglyceride-rich lipoproteins may contribute to the mass of HDL. We now report the transformation of human plasma HDL3 to an HDL2-like particle in a controlled lipolysis system in vitro when very low density lipoproteins (VLDL) and HDL3 are incubated with lipoprotein lipase (EC 3.1.1.34). MATERIALS AND METHODS Preparation of Lipoproteins and Labeled Lipoproteins. Plasma lipoproteins were isolated from normal human subjects. The plasma was obtained by plasmapheresis after a 12- to 14-hr fast of individuals whose plasma cholesterol and triglyceride concentrations ranged from 160 to 200 mg/dl and 150 to 200 mg/dl, respectively. VLDL were isolated at plasma density by ultracentrifugation in a Beckman L2-65B ultracentrifuge with a Beckman 6OTi rotor at 50,000 rpm for 18 hr at 40 followed by ultracentrifugation in a Beckman SW41 swinging bucket rotor at 40,000 rpm for 16 hr at d = 1.006 g/ml. HDL3 were isolated from the same plasma by ultracentrifugation in a Beckman 6OTi rotor at 55,000 rpm for 48 hr between densities 1.125 and 1.210 g/ml. The HDL3 was further purified by rate zonal ultracentrifugation as described below. Purity of the isolated VLDL and HDL3 was determined by their flotation properties in the zonal rotor (10), their lipid and protein percentage compositions, lipoprotein electrophoresis, and analysis of the apoproteins with polyacrylamide gel electrophoresis. HDL3 was labeled in its protein moiety with '3'I by a modification of the IC1 technique (11). Approximately 40% of the radioactivity was associated with apoA-I, 45% with apoA-II, and 5% with the apoC proteins. In certain experiments, purified apoC-II was labeled with '25I by the same labeling procedure as for HDL3. For all iodination experiments, the molar ratio of iodine to apoprotein was less than 0.5. Incubation Studies. All incubations were at 370 in a final volume of 25 ml. The incubation mixture contained VLDL and HDL3 at a protein ratio (wt/wt) of approximately 1 in 20 mM Tris buffer, pH 8.2. In order to simulate plasma conditions as much as possible, the plasma fraction of d > 1.210 g/ml was added to the incubation mixture at a final concentration of 30% (vol/vol) of that of plasma. Bovine albumin (Sigma Laborato-
Abbreviations: HDL, high density lipoproteins (d = 1.063-1.210 g/ml); HDL2, high density lipoproteins2 (d = 1.063-1.125 g/ml); HDL3, high density lipoproteinss (d = 1.125-1.210 g/ml); VLDL, very low density lipoproteins (d < 1.006 g/ml); apoA-I, apoA-II, apoB, apoC-I, apoC-II, apoC-III, and apoE, apolipoprotein constituents of human plasma lipoproteins. * Department of Medical Chemistry, University of Umea, Umea, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adSweden. t On sabbatical leave from Hadassah University Hospital, Jerusalem, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Israel. 4519
4520
Proc. Nati. Acad. Sci. USA 75 (1978)
Medical Sciences: Patsch et al.
ries, St. Louis, MO) was added to the incubation mixture to a final concentration of 4 g/dl to trap fatty acids released during lipolysis. Lipoprotein lipase was purified from bovine milk by chromatography on heparin-Sepharose (12). In control experiments, Tris buffer was substituted for the lipase. After 4 hr of incubation, the reaction mixture was divided into two portions, one containing 2.5 ml and the other 22.5 ml. These fractions were immediately subjected to rate zonal ultracentrifugation. Analysis and Isolation of Lipoproteins from Lipolysis Incubation Mixtures. The smaller 2.5-ml aliquot from the incubation mixture was subjected to zonal ultracentrifugation to analyze for distribution of VLDL subclasses (13). Ultracentrifugation was performed with a Beckman L2-65B ultracentrifuge and a Beckman Ti-14 rotor at 42,000 rpm for 45 min at 14'C. Procedures for loading and unloading the rotor and for determining the densities of various gradient fractions have been described (10). The effluent was continuously monitored by absorbance at 280 nm. The larger 22.5-ml fraction from the incubation mixture was subjected to rate zonal ultracentrifugation in a nonlinear NaBr gradient (10). Ultracentrifugation was at 41,000 rpm for 22 hr at 10'C. With this procedure, lipoproteins of d < 1.063 g/ml emerge in the first 50 ml of the rotor effluent, while lipoproteins of greater density, i.e., HDL, emerge at rotor volumes of 80-370 ml (10). Fractions containing either VLDL or HDL were pooled and dialyzed exhaustively against 100 mM NaCI/10 mM sodium azide/1 mM EDTA, adjusted to pH 7.4. After dialysis, the VLDL and HDL fractions were ultrafiltered with an Amicon cell containing a UM-10 membrane, to a concentration of 0.2-0.4 mg.of lipoprotein protein per ml. The recoveries of labeled VLDL and HDL averaged, respectively, 102.3 and 103.1% after dialysis and 92.2 and 92.1% after concentration. Analytical ultracentrifugation was performed with a Beckman model E using an AN-D rotor, Schlieren optics, and double sector cells. Flotation coefficients were calculated as described (10). Apparent flotation rates were corrected for concentration dependence (8) and expressed as F?.21 values. Zonal ultracentrifugation of HDL under isopycnic conditions was performed in a Beckman Ti-15 zonal rotor (14). Analytical gel filtration chromatography was performed with Bio-Gel A-SM. Immunochemical, Electrophoretic, and Chemical Analyses. The lipoproteins were characterized immunologically by double-diffusion experiments in Ouchterlony plates. Details of these procedures involving antisera specific for apoA-I, apoA-II, apoB, the apoC proteins, and apoE have been described (14). Lipoprotein electrophoresis in agarose-agar gel was as described by Noble (15). Apoproteins were identified by polyacrylamide gel electrophoresis on 7.5% gels in 0.1% sodium dodecyl sulfate or on 10% gels in 8 M urea at pH 8.9. Proteins were visualized by staining with Coomassie blue. Protein concentrations were determined by the method of Lowry et al. (16), phospholipid by that of Bartlett (17), cholesterol by an enzymatic procedure (18), and triglycerides by the Technicon AutoAnalyzer. RESULTS In control experiments in which lipoprotein lipase was omitted from the incubation mixture, the VLDL contained the full range of subfractions as observed with normal plasma (13). The median Sf value for our substrate VLDL was about 100, and more than 95% of the VLDL exhibited So values between 60 and 400 (Fig. 1A). By contrast, after lipolysis a new product was formed which was more dense than the original VLDL (Fig. 1B). The flotation rate of the VLDL lipolysis product had a median So of 30 and ranged from slightly less than S? 20 to 60.
Density, g/ml
1.05
1.10
1.15
E C
0
co Co
0) U
.0 c
.m Q
proteins 500 600
200 300 400 Zonal rotor effluent, ml FIG. 1. Zonal ultracentrifpgal analysis of the incubation mixture for VLDL subclasses (A) without and (B) with lipoprotein lipase. Incubation mixtures (25 ml) contained VLDL (7.9 mg of protein) and HDL3 (7.7 mg of protein) in 20 mM Tris buffer, pH 8.2. The plasma fraction of d > 1.210 g/ml and bovine serum albumin were added at a final concentration of 30% (vol/vol) of that of plasma and 4 g/dl, respectively. After 4 hr of incubation at 370, 2.5 ml of each mixture was centrifuged in a Beckman Ti-14 zonal rotor. Effluent volumes for lipoproteins with SI values of 100, 30, and 20 are indicated (13). The ordinate has been corrected for the total volume of the incubation mixture (25 ml). 100
Substantial hydrolysis (>80%) of VLDL triglyceride occurred during the 4-hr incubation with lipoprotein lipase (Table 1). Approximately 45% of both the VLDL protein and phospholipid and 15-20% of the VLDL cholesterol disappeared during
lipolysis. Substantial changes also occurred in HDL3 concomitant with the lipolysis of VLDL. During the 4-hr incubation without the enzyme, no changes of the HDL3 occurred (Fig. 2A). When lipase was present, the HDL3 disappeared completely and at the end of the incubation was replaced by a less dense lipo-
protein which exhibited the flotation characteristics of HDL2 (Fig. 2B).t The HDL2 was not produced in the control experiment. During lipolysis, apoproteins, phospholipids, and cholesterol were transferred from VL'DL to the HDL3, resulting in a 50% increase of the total mass of the lipoprotein (Table
1).
The HDL formed during the lipolysis of VLDL has been designated as "HDL2." The chemical composition of "HDL2" (Table 1) closely resembled that of native HDL2 from human plasma. Double diffusion on Ouchterlony plates showed apoC in this "HDL2." Polyacrylamide gel electrophoresis showed that "HDL2" contained a greater proportion of apoC than HDL3 (Fig. 3). apoE was found in both HDL3 and "HDL2," with no obvious difference in abundance. Neither HDL3 nor "HDL2"
contained apoB. The F?.21 rate of HDL3 was 4.1; that of "HDL2" was 7.4. The Schlieren patterns of HDL incubated without and with lipoprotein lipase are presented in Fig. 4. The hydrated density of the HDL3 and "HDL2," as determined by
t When d > 1.210 g/ml plasma fraction was omitted from the incubation mixtures, the zonal rotor effluent pattern (mass and radioactivity) was similar to those shown in Figs. 2B and 7B. Similarly, addition of 2 mM p-chloromercuriphenylsulfonic acid to inhibit any lysolecithin acyltransferase activity had no effects on the results.
Medical Sciences: Patsch et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
Table 1. Mass distribution and composition of VLDL and HDL incubated in the absence and presence of lipoprotein lipase LP
VLDL
VLDL HDL
HDL
VLDL VLDL VLDL HDL HDL HDL
LPL PROT
PL
EC
FC
LP lipid and LP protein, mg* 7.9 15.0 5.Ot 4.3 (1.4) (0.7) (0.5) + 4.5 4.3t 8.0 3.3 (1.2) (0.8) (0.4) (0.3) 7.7 3.9 1.Lt 0.4 (0.4) (0.2) (0.2) + 9.5 7.6 1.4t 1.0 (0.2) (0.5) (0.6) (0.2) LP lipid and LP protein, mg/100 mg of LP 9.5 19.5 9.5§ 5.6 9.5 18.2 9.8§ 5.2 + 14.7 26.4 23.2§ 10.8 54.6 25.1 13.6§ 2.3 11.7§ 50.7 25.7 2.7 + 43.4 34.8 10.4§ 4.6
-
TG
Total
47.2
79.4 27.7
1.4 (0.1) 1.5 (0.2)
14.5
,.11 | _rv I~
~
A-I
_0
I Odom
4- A-TI
-
55.9 57.3 24.9 4.4 9.2 6.8
A-I
c-lI C-fIT-I C-m-2
-
A
S0
C
zonal ultracentrifugation under equilibrium conditions (Fig. 5), was 1.138 and 1. 1 10 g/ml, respectively. The ultracentrifugation experiments demonstrated that HDL3 and "HDL2" were distinct, stable lipoprotein species. Gel filtration experiments indicated that the Stokes radii of HDL3 and "HDL2" were very similar, with the qualification that "HDL2" is slightly larger (Fig. 6), as is native HDL2 from human plasma. Density, g/ml
1.24
up'. I'
21.0
LP, lipoprotein; LPL, lipoprotein lipase; PROT, protein; PL, phospholipid; EC, esterified cholesterol; FC, free cholesterol; TG, triglyceride. * Values are means ± 1 SD of five experiments; SDs are given in parentheses. Values obtained from the different experiments were normalized to 7.9 mg of VLDL protein and to 7.7 mg of HDL protein. The amounts of VLDL and HDL protein varied in the different experiments between 5.0 and 7.9 mg. t Expressed as cholesterol (molecular weight 387). 1 Composition of VLDL and HDL3 before incubation. § Expressed as cholesteryl esters (molecular weight 625).
1.18
(-)
(2.9) 7.6 (3.7)
4521
1.31
( + )
HDL3 'HDL2 HDL3 HDL2
FIG. 3. Electrophoretic behavior of ether/ethanol delipidated "HDL2" and HDL3 on 7.5% polyacrylamide gels in 0.1% sodium dodecyl sulfate (Left) and on 10% polyacrylamide gels in 8 M urea (Right), pH 8.4.
To determine whether apoA-I or apoA-II was displaced from HDL3 during lipolysis, we labeled HDL3 in its protein moiety with 131I prior to incubation. An aliquot of iodinated HDL3 (131I-HDL3) was added to unlabeled HDL3 (mass ratio of 131I-HDL3:HDL3 < 1:50). After lipolysis of VLDL, the labeled apoA-I and apoA-II originally associated with HDL3 were now found in "HDL2" (Fig. 7). The transfers of mass (unlabeled lipoprotein) and of radioactivity were quantitative and indistinguishable, indicating that iodination did not significantly alter the properties of HDL3. No transfer of apoA-I or apoA-II from HDL3 to VLDL or to the d > 1.210 g/ml fraction could be detected. We conclude, therefore, that HDL3 was modified to "HDL2" by a transfer of VLDL constituents during lipolysis. This premise was further supported by the observation that 125I-apoC-II was transferred from VLDL to HDL during lipolysis. The radioactivity profile of the 1311-labeled apoA-I and
E c
0
co Cu
.0 .0
.0
0
100
200 300 400 500 Zonal rotor effluent, ml
600
700
FIG. 2. Zonal ultracentrifugal analysis of the incubation mixture for HDL subclasses (A) without and (B) with lipoprotein lipase. Same experiment as detailed in Fig. 1. A 22.5-ml aliquot of each incubation mixture was centrifuged in a Beckman Ti-14 zonal rotor. Solid blocks indicate fractions that were pooled and then used for firther characterization.
FIG. 4. Flotation of HDL3 (upper curves) and "HDL2" (lower curves) in the analytical ultracentrifuge. Schlieren patterns were obtained at 42,040 rpm, 2600 in a NaBr solution of d = 1.210 g/ml. Lipoprotein concentrations were both 2.4 mg/ml. Photographs were taken after 47min (Right) and 79min (Left). Arrows indicate direction of flotation.
Proc. Natl. Acad. Sci. USA 75 (1978)
Medical Sciences: Patsch et al.
4522
Density, g/ml 1 lb
Density, gfml 24
__
0
E E
1
-2
_
I
C
a
I
-Er
0
Q
a
C
0
-_
n-
I
-3
C-
I
C-
ii
I'
0~ ai
Effluent volume, ml
FIG. 5. Ultracentrifugal behavior of HDL3 (0) and "HDL2" (0) under equilibrium conditions. HDL3 and "HDL2" (about 2 mg of lipoprotein) were centrifuged separately using a linear gradient of NaBr (d = 1.00-1.20 g/ml) in a Beckman Ti-15 rotor under identical conditions: 30,000 rpm, 20'C, 73 hr. Fractions of-25 ml were collected and analyzed for the radioactively 1311-labeled HDL in a Packard autogamma
spectrometer.
apoA-II originating in HDL3 coincided precisely with that of '2I-apoC-II transferred from VLDL (Fig. 7).
Zonal rotor effluent, ml
FIG. 7. Zonal ultracentrifugal analyses of incubation mixtures for HDL subclasses (A) without and (B) with lipoprotein lipase. Conditions were identical to those described for Fig. 2. Shaded areas represent the mass as monitored by the absorbance at 280 nm. 0, Profile of 131I associated originally with HDL3. 0, Distribution of 125I-labeled apoC-Il added to the mixtures before incubation.
the constituents of VLDL that are transferred during lipolysis the HDL density range associate with pre-existing HDL particles or form a separate lipoprotein family. Kostner and Alaupovic have suggested (24) that the HDL density range contains several discernable lipoprotein families, designated LpA, LpB, and LpC. Others have found that bilayer disc structures are formed and float in the density range of 1.040-1.210 g/ml in in vitro systems when VLDL from rats or humans are subjected to lipolysis by lipoprotein lipase in the absence of plasma HDL (25, 26). The principal constituents of these discoidal particles are the apoC proteins, phospholipids, and unesterified cholesterol which could conceivably form a distinct LpC family floating in the HDL density range. However, the results obtained in the present study do not support this hypothesis. We did not find independent LpC particles generated during the lipolysis of VLDL when HDL3 was present. Instead, the apoC proteins, phospholipids, and cholesterol displaced from VLDL during lipolysis were assimilated by pre-existing HDL3, resulting in the formation of HDL2-like particles. These HDL2 produced in vitro are stable particles, do not disintegrate upon recentrifugation, are slightly larger than HDL3, and contain the apoA-I and apoA-II originally associated with HDL3. These data have led us to the hypothesis that this mechanism plays a significant role in vivo. In support of this hypothesis, the HDL2 populations isolated from normal human subjects (9, 10,27) closely resemble the "HDL2" formed in vitro in our experiments (Table 2). Our hypothesis is further supported by studies in which a correlation was established between the total concentration of HDL or HDL2 and the rate of lipolysis in human subjects. A few examples of these correlations are cited here. Administration of clofibrate, a drug that enhances lipolysis (28), causes an increase in the concentration of HDL2 and of HDL cholesterol in patients with broad beta disease or type III hyperlipoproteinemia (29). Concentrations of HDL2 are increased during to
DISCUSSION The work described in this manuscript began with the premise that during lipolysis, triglyceride-rich lipoproteins can provide a potential source of lipids and apoproteins for HDL. Initiation of lipolysis by intravenous injection of heparin (19) or during clearance of alimentary lipemia (20) is associated with the transfer of apoC proteins to HDL. A similar transfer occurs for phospholipids (20, 21) and for cholesterol (21, 22). The metabolic interrelationship between HDL and VLDL is further emphasized by the fact that in population studies, the concentrations of these two lipoprotein families are related to each other in an inverse fashion (23). The nature of the precise metabolic interrelationship between HDL and VLDL, however, remains to be elucidated. For example, it is not known if
x14 12
V
E10-
x
6
0
10
15 20 Fraction
25
30
FIG. 6. Chromatographic behavior of HDL3 (0) and "HDL2" (0). Conditions: 0.9 X 55 cm column of BioGel A-5M at 5.5 ml/hr. Vo, excluded volume as determined by using plasma VLDL.
Medical Sciences: Patsch et al.
Proc. Nati. Acad. Sca. USA 75 (1978)
Table 2. Comparison of some characteristics of HDL2 produced in vitro with those reported for native human HDL2 HDL, size In vitro HDL2 HDL2* HDL2t range It
TF'.21
7.4
6.5-8.7
6.0
5.3 6.7§ 5.9-7.9§ F?.20 1.090 1.110 1.110 Hydrated density Weight, mg/100 mg lipoprotein 37.8 40.6 40.1 43.4 Protein 39.8 35.0 34.8 29.9 Phospholipids 6.0 4.6 Free cholesterol 17.0 23.3 14.1 17. 10.4 3 Cholesteryl ester 3.8 2.6 6.8 6.6 Triglyceride * Patsch et al. (10). t Scanu and Granda (9). t Anderson et al. (27). § 1.20 rates were obtained from F.21 rates by calculation (8).
estrogen treatment.§ This hormone also increases triglyceride
production (30) and the activity of adipose tissue lipoprotein lipase (31). Premenopausal women have high concentrations of HDL and HDL2 as compared to men (32), while their HDL3 levels are lower by 31% (27), presumably due to estrogen effects. Nikkili et al. have observed a strongly positive correlation (r = 0.68, P < 0.001) between the concentration of HDL cholesterol and postheparin lipolytic activity in 91 diabetic subjects treated with insulin (33). Physical exercise has been reported to increase the concentration of HDL2, but not of HDL3 (34). This rise in HDL is associated with a 40-50% decrease in the concentration of plasma triglyceride and a 60-70% decrease in the concentration of VLDL cholesterol (35). In a contrasting situation, the plasma of patients with low levels of lipoprotein lipase activity (type I hyperlipoproteinemia) exhibit low concentrations of HDL3 and a complete absence of HDL2 (36). We propose that the transformation of HDL3 to HDL2 through the assimilation of constituents freed from VLDL during its lipolysis occurs in vivo and is a common metabolic pathway reflected in the examples cited above and in other physiological and pathological situations. We are grateful for the excellent technical assistance of Ms. Debbie Ehler and Mr. Inderjit Singh Thandi and for the help of Ms. Debbie Mason and Ms. Kaye Shewmaker in preparation of the manuscript and the figures. This material was developed by the Atherosclerosis, Lipids and Lipoproteins section of the National Heart and Blood Vessel Research and Demonstration Center, Baylor College of Medicine; a grant-supported research project of the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL 17269; and by National Institutes of Health Lipid Research Clinic Contract 712156. J., Levy, R. I., Jenkins, L. L. & Brewer, H. B. (1977) Proceedings of the Sixth International Symposium on Drugs Affecting Lipid Metabolism, 82 (abst.).
§ Schaefer, E.
3. Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W. B. & Dawber, T. R. (1977) Am. J. Med. 62,707-714. 4. Carew, Th. E., Koschinsky, Th., Hayes, S. B. & Steinberg, D. (1976) Lancet i, 1315-1317. 5. Hamilton, R. L., Williams, M. C., Fielding, C. J. & Havel, R. J. (1976) J. Clin. Invest. 58,667-680. 6. Green, P. H. R., Tall, A. R. & Glickman, R. M. (1978) J. Clin. Invest. 61, 528-534. 7. Schaefer, E. J., Jenkins, L. L. & Brewer, H. B. (1978) Biochem. Biophys. Res. Commun. 80,405-412. 8. Lindgren, F. T., Jensen, L. C. & Hatch, F. T. (1972) in Blood Lipids and Lipoproteins: Quantitation, Composition, and Metabolism, ed. Nelson, G. J. (Wiley-Interscience, New York), pp. 181-274. 9. Scanu, A. & Granda, J. L. (1966) Biochemistry 5,446-455. 10. Patsch, J. R., Sailer, S., Kostner, G., Sandhofer, F., Holasek, A. & Braunsteiner, H. (1974) J. Lipid Res. 15, 356-366. 11. Bilheimer, D. W., Eisenberg, S. & Levy, R. I. (1972) Biochim. Biophys. Acta 260,212-221. 12. Bengtsson, G. & Olivecrona, T. (1977) Biochem. J. 167, 109120. 13. Patsch, W., Patsch, J. R., Kostner, G. M., Sailer, S. & Braunsteiner, H. (1978) J. Biol. Chem. 253, 4911-4915. 14. Patsch, J. R., Aune, K. C., Gotto, A. M., Jr. & Morrisett, J. D. (1977) J. Biol. Chem. 252,2113-2120. 15. Noble, R. P. (1968) J. Lipid Res. 9,693-700. 16. Lowry, 0. H., Rosebrough, N. J., Farm, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 17. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468. 18. Patsch, W., Sailer, S. & Braunsteiner, H. (1976) J. Lipid Res. 17, 182-185. 19. Eisenberg, S., Bilheimer, D. W., Levy, R. I. & Lindgren, F. T. (1973) Biochim. Biophys. Acta 326,361-377. 20. Havel, R. J., Kane, J. P. & Kashyap, M. L. (1973) J. Clin. Invest.
52,32-38.
21. Eisenberg, S. & Schurr, D. (1976) J. Lipid Res. 17,578-587. 22. La Rosa, J. C., Levy, R. I., Brown, W. V. & Fredrickson, D. S. (1971) Am. J. Physiol. 220,785-791. 23. Gofman, J. W., de Lalla, O., Glazier, F., Freeman, N. K., Lindgren, F. T., Nichols, A. V., Strisower, B. & Tamplin, A. R.
(1954) Plasma 2,413-484.
24. Kostner, G. M. & Alaupovic, P. (1972) Biochemistry 11,
3419-3428. 25. Chajek, T. & Eisenberg, S. (1978) J. Clin. Invest. 62, 16541665. 26. Deckelbaum, R., Eisenberg, S., Barenholz, T. & Olivecrona, T. (1977) Circulation Suppl. 3, 56,56 (abstr.). 27. Anderson, D. W., Nichols, A. V., Forte, T. M. & Lindgren, F. T. (1977) Biochim. Biophys. Acta 493,55-68. 28. Wolfe, B. M., Kane, J. P., Havel, R. J. & Brewster, H. P. (1973) J. Clin. Invest. 52,2146-2159. 29. Patsch, J. R., Yeshurun, D., Jackson, R. L. & Gotto, A. M. (1977) Am. J. Med. 63,1001-1009. 30. Glueck, C. J., Fallat, R. W. & Schal, D. (1975) Metabolism 24, 537-545. 31. Appelbaum, D. M., Goldberg, A. P., Pykalisto, 0. J., Brunzell, J. D. & Hazzard, W. R. (1977) J. Clin. Invest. 59,601-608. 32. Nichols, A. V. (1967) Adv. Biol. Med. Phys. 11, 110-158. 33. NikkiJd, E. A., Hamilton, P. & Huttunen, J. E. (1977) Circulation Suppl. 3, 56, 23 (abstr.). 34. Krauss, R. M., Lindgren, F. T., Wood, P. O., Haskell, W. L., Albers, J/ J. & Cheung, M. C. (1977) Circulation Suppl. 3, 56, 4
(abstr.).
1. Miller, G. E. & Miller, J. E. (1975) Lancet i, 16-19. 2. Rhodas, G., Gulbrandsen, C. L. & Kagan, A. (1976) N. Engl. J. Med. 294, 293-295.
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35. Wood, P. O., Haskell, W., Klein, H., Lewis, S., Stern, M. P. & Farquhar, J. W. (1976) Metabolism 25, 1249-1257. 36. Fredrickson, D. S., Levy, R. I. & Lindgren, F. T. (1968) J. Clin. Invest. 47, 2446-2457.
Medical Sciences
Formation of high density lipoprotein2-like particles during lipolysis of very low density lipoproteins in vitro (lipoprotein lipase/apolipoproteins/lipoprotein lipids/ultracentrifugation)
JOSEF R. PATSCH, ANTONIO M. GOTTO, JR., THOMAS OLIVECRONA*, AND SHLOMO EISENBERGt Department of Medicine, Baylor College of Medicine and The Methodist Hospital, Houston, Texas 77030
Communicated by William E. Gordon, June 22, 1978
The effects of lipolysis of human plasma very ABSTRACT low density lipoprotein (VLDL) on the structure and composition of high density lipoproteins (HDL) have been investigated. Lipolysis was performed in a controlled system in vitro containing VLDL (d < 1.006 g/ml) and HDL3 (d = 1.125-1.210 g/ml) from human plasma and lipoprotein lipase (EC 3.1.1.34) purified from bovine milk. Li lysis of VLDL caused profound changes in HDL3. Protein, phospholipid, and cholesterol liberated from VLDL during its lipolysis were transferred to the HDL3 particles. As a consequence of this in vitro transfer, the chemical composition and biophysical properties of HDL3 were substantially altered. The newly formed Particles exhibited a flotation rate (i41.") of 6.7 and a hydrated density of 1.110 g/ml. The chemical composition closely resembled that of native HDL2, and their size was slightly larger than that of the precursor HDL3. When HDL3 and postlipolysis HDL2 were subjected to ultracentrifugation under flotation velocity and equilibrium conditions, both proved to be stable particles. These results, when extrapolated to in vivo conditions, suggest an important metabolic relationship between the levels of circulating VLDL and HDL2 in plasma. This relationship now permits a reasonable explanation for numerous in vivo observations in which the levels of VLDL and HDL2 change reciprocally.
The high density lipoproteins (HDL) represent one of the four major families of plasma lipoproteins that circulate in human plasma. The HDL have become the focus of much interest since their plasma concentrations have been shown to be inversely correlated with the risk of coronary heart disease (1-3). This correlation is an epidemiologic observation; the mechanism(s) by which HDL protect against atherosclerosis is unknown although several have been suggested (1, 4). A detailed knowledge of the metabolism of HDL is essential for elucidating this mechanism. The pathways of HDL synthesis and degradation in human beings are poorly understood. In animal experiments with isolated perfused rat liver (5) and with rat intestinal lymph (6) a type of HDL resembling a bilayered disc has been isolated. This particle has been called nascent HDL. Hamilton et al. (5) have suggested that the discoidal nascent HDL are transformed to spherical particles characteristic of HDL by the intravascular action of lysolecithin acyltransferase (EC 2.3.1.23). ApoA-I, the major protein of HDL, can be synthesized in rat intestine (6) and is found in the lymph associated both with chylomicrons and nascent HDL. When chylomicrons enter the plasma, apoA-I is transferred to circulating HDL (7). The source of the lipids necessary to form the HDL particles with apoA-I has not been elucidated. HDL can be divided into at least two subfractions, HDL2 and HDL3, which are traditionally isolated at density intervals of 1.063-1.125 and 1.125-1.210 g/ml, respectively (8). HDL2 particles are larger and contain a larger proportion of lipids than
do the HDL3 (9, 10). Whether HDL2 and HDL3 are metabolically interrelated is not known. The present study was undertaken to test the hypothesis that the lipid and apoprotein constituents formed in the degradation of triglyceride-rich lipoproteins may contribute to the mass of HDL. We now report the transformation of human plasma HDL3 to an HDL2-like particle in a controlled lipolysis system in vitro when very low density lipoproteins (VLDL) and HDL3 are incubated with lipoprotein lipase (EC 3.1.1.34). MATERIALS AND METHODS Preparation of Lipoproteins and Labeled Lipoproteins. Plasma lipoproteins were isolated from normal human subjects. The plasma was obtained by plasmapheresis after a 12- to 14-hr fast of individuals whose plasma cholesterol and triglyceride concentrations ranged from 160 to 200 mg/dl and 150 to 200 mg/dl, respectively. VLDL were isolated at plasma density by ultracentrifugation in a Beckman L2-65B ultracentrifuge with a Beckman 6OTi rotor at 50,000 rpm for 18 hr at 40 followed by ultracentrifugation in a Beckman SW41 swinging bucket rotor at 40,000 rpm for 16 hr at d = 1.006 g/ml. HDL3 were isolated from the same plasma by ultracentrifugation in a Beckman 6OTi rotor at 55,000 rpm for 48 hr between densities 1.125 and 1.210 g/ml. The HDL3 was further purified by rate zonal ultracentrifugation as described below. Purity of the isolated VLDL and HDL3 was determined by their flotation properties in the zonal rotor (10), their lipid and protein percentage compositions, lipoprotein electrophoresis, and analysis of the apoproteins with polyacrylamide gel electrophoresis. HDL3 was labeled in its protein moiety with '3'I by a modification of the IC1 technique (11). Approximately 40% of the radioactivity was associated with apoA-I, 45% with apoA-II, and 5% with the apoC proteins. In certain experiments, purified apoC-II was labeled with '25I by the same labeling procedure as for HDL3. For all iodination experiments, the molar ratio of iodine to apoprotein was less than 0.5. Incubation Studies. All incubations were at 370 in a final volume of 25 ml. The incubation mixture contained VLDL and HDL3 at a protein ratio (wt/wt) of approximately 1 in 20 mM Tris buffer, pH 8.2. In order to simulate plasma conditions as much as possible, the plasma fraction of d > 1.210 g/ml was added to the incubation mixture at a final concentration of 30% (vol/vol) of that of plasma. Bovine albumin (Sigma Laborato-
Abbreviations: HDL, high density lipoproteins (d = 1.063-1.210 g/ml); HDL2, high density lipoproteins2 (d = 1.063-1.125 g/ml); HDL3, high density lipoproteinss (d = 1.125-1.210 g/ml); VLDL, very low density lipoproteins (d < 1.006 g/ml); apoA-I, apoA-II, apoB, apoC-I, apoC-II, apoC-III, and apoE, apolipoprotein constituents of human plasma lipoproteins. * Department of Medical Chemistry, University of Umea, Umea, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adSweden. t On sabbatical leave from Hadassah University Hospital, Jerusalem, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Israel. 4519
4520
Proc. Nati. Acad. Sci. USA 75 (1978)
Medical Sciences: Patsch et al.
ries, St. Louis, MO) was added to the incubation mixture to a final concentration of 4 g/dl to trap fatty acids released during lipolysis. Lipoprotein lipase was purified from bovine milk by chromatography on heparin-Sepharose (12). In control experiments, Tris buffer was substituted for the lipase. After 4 hr of incubation, the reaction mixture was divided into two portions, one containing 2.5 ml and the other 22.5 ml. These fractions were immediately subjected to rate zonal ultracentrifugation. Analysis and Isolation of Lipoproteins from Lipolysis Incubation Mixtures. The smaller 2.5-ml aliquot from the incubation mixture was subjected to zonal ultracentrifugation to analyze for distribution of VLDL subclasses (13). Ultracentrifugation was performed with a Beckman L2-65B ultracentrifuge and a Beckman Ti-14 rotor at 42,000 rpm for 45 min at 14'C. Procedures for loading and unloading the rotor and for determining the densities of various gradient fractions have been described (10). The effluent was continuously monitored by absorbance at 280 nm. The larger 22.5-ml fraction from the incubation mixture was subjected to rate zonal ultracentrifugation in a nonlinear NaBr gradient (10). Ultracentrifugation was at 41,000 rpm for 22 hr at 10'C. With this procedure, lipoproteins of d < 1.063 g/ml emerge in the first 50 ml of the rotor effluent, while lipoproteins of greater density, i.e., HDL, emerge at rotor volumes of 80-370 ml (10). Fractions containing either VLDL or HDL were pooled and dialyzed exhaustively against 100 mM NaCI/10 mM sodium azide/1 mM EDTA, adjusted to pH 7.4. After dialysis, the VLDL and HDL fractions were ultrafiltered with an Amicon cell containing a UM-10 membrane, to a concentration of 0.2-0.4 mg.of lipoprotein protein per ml. The recoveries of labeled VLDL and HDL averaged, respectively, 102.3 and 103.1% after dialysis and 92.2 and 92.1% after concentration. Analytical ultracentrifugation was performed with a Beckman model E using an AN-D rotor, Schlieren optics, and double sector cells. Flotation coefficients were calculated as described (10). Apparent flotation rates were corrected for concentration dependence (8) and expressed as F?.21 values. Zonal ultracentrifugation of HDL under isopycnic conditions was performed in a Beckman Ti-15 zonal rotor (14). Analytical gel filtration chromatography was performed with Bio-Gel A-SM. Immunochemical, Electrophoretic, and Chemical Analyses. The lipoproteins were characterized immunologically by double-diffusion experiments in Ouchterlony plates. Details of these procedures involving antisera specific for apoA-I, apoA-II, apoB, the apoC proteins, and apoE have been described (14). Lipoprotein electrophoresis in agarose-agar gel was as described by Noble (15). Apoproteins were identified by polyacrylamide gel electrophoresis on 7.5% gels in 0.1% sodium dodecyl sulfate or on 10% gels in 8 M urea at pH 8.9. Proteins were visualized by staining with Coomassie blue. Protein concentrations were determined by the method of Lowry et al. (16), phospholipid by that of Bartlett (17), cholesterol by an enzymatic procedure (18), and triglycerides by the Technicon AutoAnalyzer. RESULTS In control experiments in which lipoprotein lipase was omitted from the incubation mixture, the VLDL contained the full range of subfractions as observed with normal plasma (13). The median Sf value for our substrate VLDL was about 100, and more than 95% of the VLDL exhibited So values between 60 and 400 (Fig. 1A). By contrast, after lipolysis a new product was formed which was more dense than the original VLDL (Fig. 1B). The flotation rate of the VLDL lipolysis product had a median So of 30 and ranged from slightly less than S? 20 to 60.
Density, g/ml
1.05
1.10
1.15
E C
0
co Co
0) U
.0 c
.m Q
proteins 500 600
200 300 400 Zonal rotor effluent, ml FIG. 1. Zonal ultracentrifpgal analysis of the incubation mixture for VLDL subclasses (A) without and (B) with lipoprotein lipase. Incubation mixtures (25 ml) contained VLDL (7.9 mg of protein) and HDL3 (7.7 mg of protein) in 20 mM Tris buffer, pH 8.2. The plasma fraction of d > 1.210 g/ml and bovine serum albumin were added at a final concentration of 30% (vol/vol) of that of plasma and 4 g/dl, respectively. After 4 hr of incubation at 370, 2.5 ml of each mixture was centrifuged in a Beckman Ti-14 zonal rotor. Effluent volumes for lipoproteins with SI values of 100, 30, and 20 are indicated (13). The ordinate has been corrected for the total volume of the incubation mixture (25 ml). 100
Substantial hydrolysis (>80%) of VLDL triglyceride occurred during the 4-hr incubation with lipoprotein lipase (Table 1). Approximately 45% of both the VLDL protein and phospholipid and 15-20% of the VLDL cholesterol disappeared during
lipolysis. Substantial changes also occurred in HDL3 concomitant with the lipolysis of VLDL. During the 4-hr incubation without the enzyme, no changes of the HDL3 occurred (Fig. 2A). When lipase was present, the HDL3 disappeared completely and at the end of the incubation was replaced by a less dense lipo-
protein which exhibited the flotation characteristics of HDL2 (Fig. 2B).t The HDL2 was not produced in the control experiment. During lipolysis, apoproteins, phospholipids, and cholesterol were transferred from VL'DL to the HDL3, resulting in a 50% increase of the total mass of the lipoprotein (Table
1).
The HDL formed during the lipolysis of VLDL has been designated as "HDL2." The chemical composition of "HDL2" (Table 1) closely resembled that of native HDL2 from human plasma. Double diffusion on Ouchterlony plates showed apoC in this "HDL2." Polyacrylamide gel electrophoresis showed that "HDL2" contained a greater proportion of apoC than HDL3 (Fig. 3). apoE was found in both HDL3 and "HDL2," with no obvious difference in abundance. Neither HDL3 nor "HDL2"
contained apoB. The F?.21 rate of HDL3 was 4.1; that of "HDL2" was 7.4. The Schlieren patterns of HDL incubated without and with lipoprotein lipase are presented in Fig. 4. The hydrated density of the HDL3 and "HDL2," as determined by
t When d > 1.210 g/ml plasma fraction was omitted from the incubation mixtures, the zonal rotor effluent pattern (mass and radioactivity) was similar to those shown in Figs. 2B and 7B. Similarly, addition of 2 mM p-chloromercuriphenylsulfonic acid to inhibit any lysolecithin acyltransferase activity had no effects on the results.
Medical Sciences: Patsch et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
Table 1. Mass distribution and composition of VLDL and HDL incubated in the absence and presence of lipoprotein lipase LP
VLDL
VLDL HDL
HDL
VLDL VLDL VLDL HDL HDL HDL
LPL PROT
PL
EC
FC
LP lipid and LP protein, mg* 7.9 15.0 5.Ot 4.3 (1.4) (0.7) (0.5) + 4.5 4.3t 8.0 3.3 (1.2) (0.8) (0.4) (0.3) 7.7 3.9 1.Lt 0.4 (0.4) (0.2) (0.2) + 9.5 7.6 1.4t 1.0 (0.2) (0.5) (0.6) (0.2) LP lipid and LP protein, mg/100 mg of LP 9.5 19.5 9.5§ 5.6 9.5 18.2 9.8§ 5.2 + 14.7 26.4 23.2§ 10.8 54.6 25.1 13.6§ 2.3 11.7§ 50.7 25.7 2.7 + 43.4 34.8 10.4§ 4.6
-
TG
Total
47.2
79.4 27.7
1.4 (0.1) 1.5 (0.2)
14.5
,.11 | _rv I~
~
A-I
_0
I Odom
4- A-TI
-
55.9 57.3 24.9 4.4 9.2 6.8
A-I
c-lI C-fIT-I C-m-2
-
A
S0
C
zonal ultracentrifugation under equilibrium conditions (Fig. 5), was 1.138 and 1. 1 10 g/ml, respectively. The ultracentrifugation experiments demonstrated that HDL3 and "HDL2" were distinct, stable lipoprotein species. Gel filtration experiments indicated that the Stokes radii of HDL3 and "HDL2" were very similar, with the qualification that "HDL2" is slightly larger (Fig. 6), as is native HDL2 from human plasma. Density, g/ml
1.24
up'. I'
21.0
LP, lipoprotein; LPL, lipoprotein lipase; PROT, protein; PL, phospholipid; EC, esterified cholesterol; FC, free cholesterol; TG, triglyceride. * Values are means ± 1 SD of five experiments; SDs are given in parentheses. Values obtained from the different experiments were normalized to 7.9 mg of VLDL protein and to 7.7 mg of HDL protein. The amounts of VLDL and HDL protein varied in the different experiments between 5.0 and 7.9 mg. t Expressed as cholesterol (molecular weight 387). 1 Composition of VLDL and HDL3 before incubation. § Expressed as cholesteryl esters (molecular weight 625).
1.18
(-)
(2.9) 7.6 (3.7)
4521
1.31
( + )
HDL3 'HDL2 HDL3 HDL2
FIG. 3. Electrophoretic behavior of ether/ethanol delipidated "HDL2" and HDL3 on 7.5% polyacrylamide gels in 0.1% sodium dodecyl sulfate (Left) and on 10% polyacrylamide gels in 8 M urea (Right), pH 8.4.
To determine whether apoA-I or apoA-II was displaced from HDL3 during lipolysis, we labeled HDL3 in its protein moiety with 131I prior to incubation. An aliquot of iodinated HDL3 (131I-HDL3) was added to unlabeled HDL3 (mass ratio of 131I-HDL3:HDL3 < 1:50). After lipolysis of VLDL, the labeled apoA-I and apoA-II originally associated with HDL3 were now found in "HDL2" (Fig. 7). The transfers of mass (unlabeled lipoprotein) and of radioactivity were quantitative and indistinguishable, indicating that iodination did not significantly alter the properties of HDL3. No transfer of apoA-I or apoA-II from HDL3 to VLDL or to the d > 1.210 g/ml fraction could be detected. We conclude, therefore, that HDL3 was modified to "HDL2" by a transfer of VLDL constituents during lipolysis. This premise was further supported by the observation that 125I-apoC-II was transferred from VLDL to HDL during lipolysis. The radioactivity profile of the 1311-labeled apoA-I and
E c
0
co Cu
.0 .0
.0
0
100
200 300 400 500 Zonal rotor effluent, ml
600
700
FIG. 2. Zonal ultracentrifugal analysis of the incubation mixture for HDL subclasses (A) without and (B) with lipoprotein lipase. Same experiment as detailed in Fig. 1. A 22.5-ml aliquot of each incubation mixture was centrifuged in a Beckman Ti-14 zonal rotor. Solid blocks indicate fractions that were pooled and then used for firther characterization.
FIG. 4. Flotation of HDL3 (upper curves) and "HDL2" (lower curves) in the analytical ultracentrifuge. Schlieren patterns were obtained at 42,040 rpm, 2600 in a NaBr solution of d = 1.210 g/ml. Lipoprotein concentrations were both 2.4 mg/ml. Photographs were taken after 47min (Right) and 79min (Left). Arrows indicate direction of flotation.
Proc. Natl. Acad. Sci. USA 75 (1978)
Medical Sciences: Patsch et al.
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Density, g/ml 1 lb
Density, gfml 24
__
0
E E
1
-2
_
I
C
a
I
-Er
0
Q
a
C
0
-_
n-
I
-3
C-
I
C-
ii
I'
0~ ai
Effluent volume, ml
FIG. 5. Ultracentrifugal behavior of HDL3 (0) and "HDL2" (0) under equilibrium conditions. HDL3 and "HDL2" (about 2 mg of lipoprotein) were centrifuged separately using a linear gradient of NaBr (d = 1.00-1.20 g/ml) in a Beckman Ti-15 rotor under identical conditions: 30,000 rpm, 20'C, 73 hr. Fractions of-25 ml were collected and analyzed for the radioactively 1311-labeled HDL in a Packard autogamma
spectrometer.
apoA-II originating in HDL3 coincided precisely with that of '2I-apoC-II transferred from VLDL (Fig. 7).
Zonal rotor effluent, ml
FIG. 7. Zonal ultracentrifugal analyses of incubation mixtures for HDL subclasses (A) without and (B) with lipoprotein lipase. Conditions were identical to those described for Fig. 2. Shaded areas represent the mass as monitored by the absorbance at 280 nm. 0, Profile of 131I associated originally with HDL3. 0, Distribution of 125I-labeled apoC-Il added to the mixtures before incubation.
the constituents of VLDL that are transferred during lipolysis the HDL density range associate with pre-existing HDL particles or form a separate lipoprotein family. Kostner and Alaupovic have suggested (24) that the HDL density range contains several discernable lipoprotein families, designated LpA, LpB, and LpC. Others have found that bilayer disc structures are formed and float in the density range of 1.040-1.210 g/ml in in vitro systems when VLDL from rats or humans are subjected to lipolysis by lipoprotein lipase in the absence of plasma HDL (25, 26). The principal constituents of these discoidal particles are the apoC proteins, phospholipids, and unesterified cholesterol which could conceivably form a distinct LpC family floating in the HDL density range. However, the results obtained in the present study do not support this hypothesis. We did not find independent LpC particles generated during the lipolysis of VLDL when HDL3 was present. Instead, the apoC proteins, phospholipids, and cholesterol displaced from VLDL during lipolysis were assimilated by pre-existing HDL3, resulting in the formation of HDL2-like particles. These HDL2 produced in vitro are stable particles, do not disintegrate upon recentrifugation, are slightly larger than HDL3, and contain the apoA-I and apoA-II originally associated with HDL3. These data have led us to the hypothesis that this mechanism plays a significant role in vivo. In support of this hypothesis, the HDL2 populations isolated from normal human subjects (9, 10,27) closely resemble the "HDL2" formed in vitro in our experiments (Table 2). Our hypothesis is further supported by studies in which a correlation was established between the total concentration of HDL or HDL2 and the rate of lipolysis in human subjects. A few examples of these correlations are cited here. Administration of clofibrate, a drug that enhances lipolysis (28), causes an increase in the concentration of HDL2 and of HDL cholesterol in patients with broad beta disease or type III hyperlipoproteinemia (29). Concentrations of HDL2 are increased during to
DISCUSSION The work described in this manuscript began with the premise that during lipolysis, triglyceride-rich lipoproteins can provide a potential source of lipids and apoproteins for HDL. Initiation of lipolysis by intravenous injection of heparin (19) or during clearance of alimentary lipemia (20) is associated with the transfer of apoC proteins to HDL. A similar transfer occurs for phospholipids (20, 21) and for cholesterol (21, 22). The metabolic interrelationship between HDL and VLDL is further emphasized by the fact that in population studies, the concentrations of these two lipoprotein families are related to each other in an inverse fashion (23). The nature of the precise metabolic interrelationship between HDL and VLDL, however, remains to be elucidated. For example, it is not known if
x14 12
V
E10-
x
6
0
10
15 20 Fraction
25
30
FIG. 6. Chromatographic behavior of HDL3 (0) and "HDL2" (0). Conditions: 0.9 X 55 cm column of BioGel A-5M at 5.5 ml/hr. Vo, excluded volume as determined by using plasma VLDL.
Medical Sciences: Patsch et al.
Proc. Nati. Acad. Sca. USA 75 (1978)
Table 2. Comparison of some characteristics of HDL2 produced in vitro with those reported for native human HDL2 HDL, size In vitro HDL2 HDL2* HDL2t range It
TF'.21
7.4
6.5-8.7
6.0
5.3 6.7§ 5.9-7.9§ F?.20 1.090 1.110 1.110 Hydrated density Weight, mg/100 mg lipoprotein 37.8 40.6 40.1 43.4 Protein 39.8 35.0 34.8 29.9 Phospholipids 6.0 4.6 Free cholesterol 17.0 23.3 14.1 17. 10.4 3 Cholesteryl ester 3.8 2.6 6.8 6.6 Triglyceride * Patsch et al. (10). t Scanu and Granda (9). t Anderson et al. (27). § 1.20 rates were obtained from F.21 rates by calculation (8).
estrogen treatment.§ This hormone also increases triglyceride
production (30) and the activity of adipose tissue lipoprotein lipase (31). Premenopausal women have high concentrations of HDL and HDL2 as compared to men (32), while their HDL3 levels are lower by 31% (27), presumably due to estrogen effects. Nikkili et al. have observed a strongly positive correlation (r = 0.68, P < 0.001) between the concentration of HDL cholesterol and postheparin lipolytic activity in 91 diabetic subjects treated with insulin (33). Physical exercise has been reported to increase the concentration of HDL2, but not of HDL3 (34). This rise in HDL is associated with a 40-50% decrease in the concentration of plasma triglyceride and a 60-70% decrease in the concentration of VLDL cholesterol (35). In a contrasting situation, the plasma of patients with low levels of lipoprotein lipase activity (type I hyperlipoproteinemia) exhibit low concentrations of HDL3 and a complete absence of HDL2 (36). We propose that the transformation of HDL3 to HDL2 through the assimilation of constituents freed from VLDL during its lipolysis occurs in vivo and is a common metabolic pathway reflected in the examples cited above and in other physiological and pathological situations. We are grateful for the excellent technical assistance of Ms. Debbie Ehler and Mr. Inderjit Singh Thandi and for the help of Ms. Debbie Mason and Ms. Kaye Shewmaker in preparation of the manuscript and the figures. This material was developed by the Atherosclerosis, Lipids and Lipoproteins section of the National Heart and Blood Vessel Research and Demonstration Center, Baylor College of Medicine; a grant-supported research project of the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL 17269; and by National Institutes of Health Lipid Research Clinic Contract 712156. J., Levy, R. I., Jenkins, L. L. & Brewer, H. B. (1977) Proceedings of the Sixth International Symposium on Drugs Affecting Lipid Metabolism, 82 (abst.).
§ Schaefer, E.
3. Gordon, T., Castelli, W. P., Hjortland, M. C., Kannel, W. B. & Dawber, T. R. (1977) Am. J. Med. 62,707-714. 4. Carew, Th. E., Koschinsky, Th., Hayes, S. B. & Steinberg, D. (1976) Lancet i, 1315-1317. 5. Hamilton, R. L., Williams, M. C., Fielding, C. J. & Havel, R. J. (1976) J. Clin. Invest. 58,667-680. 6. Green, P. H. R., Tall, A. R. & Glickman, R. M. (1978) J. Clin. Invest. 61, 528-534. 7. Schaefer, E. J., Jenkins, L. L. & Brewer, H. B. (1978) Biochem. Biophys. Res. Commun. 80,405-412. 8. Lindgren, F. T., Jensen, L. C. & Hatch, F. T. (1972) in Blood Lipids and Lipoproteins: Quantitation, Composition, and Metabolism, ed. Nelson, G. J. (Wiley-Interscience, New York), pp. 181-274. 9. Scanu, A. & Granda, J. L. (1966) Biochemistry 5,446-455. 10. Patsch, J. R., Sailer, S., Kostner, G., Sandhofer, F., Holasek, A. & Braunsteiner, H. (1974) J. Lipid Res. 15, 356-366. 11. Bilheimer, D. W., Eisenberg, S. & Levy, R. I. (1972) Biochim. Biophys. Acta 260,212-221. 12. Bengtsson, G. & Olivecrona, T. (1977) Biochem. J. 167, 109120. 13. Patsch, W., Patsch, J. R., Kostner, G. M., Sailer, S. & Braunsteiner, H. (1978) J. Biol. Chem. 253, 4911-4915. 14. Patsch, J. R., Aune, K. C., Gotto, A. M., Jr. & Morrisett, J. D. (1977) J. Biol. Chem. 252,2113-2120. 15. Noble, R. P. (1968) J. Lipid Res. 9,693-700. 16. Lowry, 0. H., Rosebrough, N. J., Farm, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193,265-275. 17. Bartlett, G. R. (1959) J. Biol. Chem. 234, 466-468. 18. Patsch, W., Sailer, S. & Braunsteiner, H. (1976) J. Lipid Res. 17, 182-185. 19. Eisenberg, S., Bilheimer, D. W., Levy, R. I. & Lindgren, F. T. (1973) Biochim. Biophys. Acta 326,361-377. 20. Havel, R. J., Kane, J. P. & Kashyap, M. L. (1973) J. Clin. Invest.
52,32-38.
21. Eisenberg, S. & Schurr, D. (1976) J. Lipid Res. 17,578-587. 22. La Rosa, J. C., Levy, R. I., Brown, W. V. & Fredrickson, D. S. (1971) Am. J. Physiol. 220,785-791. 23. Gofman, J. W., de Lalla, O., Glazier, F., Freeman, N. K., Lindgren, F. T., Nichols, A. V., Strisower, B. & Tamplin, A. R.
(1954) Plasma 2,413-484.
24. Kostner, G. M. & Alaupovic, P. (1972) Biochemistry 11,
3419-3428. 25. Chajek, T. & Eisenberg, S. (1978) J. Clin. Invest. 62, 16541665. 26. Deckelbaum, R., Eisenberg, S., Barenholz, T. & Olivecrona, T. (1977) Circulation Suppl. 3, 56,56 (abstr.). 27. Anderson, D. W., Nichols, A. V., Forte, T. M. & Lindgren, F. T. (1977) Biochim. Biophys. Acta 493,55-68. 28. Wolfe, B. M., Kane, J. P., Havel, R. J. & Brewster, H. P. (1973) J. Clin. Invest. 52,2146-2159. 29. Patsch, J. R., Yeshurun, D., Jackson, R. L. & Gotto, A. M. (1977) Am. J. Med. 63,1001-1009. 30. Glueck, C. J., Fallat, R. W. & Schal, D. (1975) Metabolism 24, 537-545. 31. Appelbaum, D. M., Goldberg, A. P., Pykalisto, 0. J., Brunzell, J. D. & Hazzard, W. R. (1977) J. Clin. Invest. 59,601-608. 32. Nichols, A. V. (1967) Adv. Biol. Med. Phys. 11, 110-158. 33. NikkiJd, E. A., Hamilton, P. & Huttunen, J. E. (1977) Circulation Suppl. 3, 56, 23 (abstr.). 34. Krauss, R. M., Lindgren, F. T., Wood, P. O., Haskell, W. L., Albers, J/ J. & Cheung, M. C. (1977) Circulation Suppl. 3, 56, 4
(abstr.).
1. Miller, G. E. & Miller, J. E. (1975) Lancet i, 16-19. 2. Rhodas, G., Gulbrandsen, C. L. & Kagan, A. (1976) N. Engl. J. Med. 294, 293-295.
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35. Wood, P. O., Haskell, W., Klein, H., Lewis, S., Stern, M. P. & Farquhar, J. W. (1976) Metabolism 25, 1249-1257. 36. Fredrickson, D. S., Levy, R. I. & Lindgren, F. T. (1968) J. Clin. Invest. 47, 2446-2457.
