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Atherogenic diets and neutral-lipid organization in plasma low density lipoproteins
59
Atherosclerosis, 33 (1979) 59-70 @ Elsevier/North-Holland Scientific
Publishers,
Ltd.
ATHEROGENIC DIETS AND NEUTRAL-LIPID ORGANIZATION PLASMA LOW DENSITY LIPOPROTEINS
TOMAS KIRCHHAUSEN, M. SCANU
STEVEN
H. UNTRACHT,
GUNTHER
IN
M. FLESS and ANGELO
Departments of Medicine, Biophysics and Theoretical Biology, and Biochemistry, The University of Chicago Pritzker School of Medicine, and The Franklin McLean Memorial Research Institute, Chicago, IL 60637 (U.S.A.) (Received 11 September, 1978) (Revised, received 27 November, (Accepted 29 November, 1978)
1978)
summary The plasma low density lipoproteins (LDL) of rhesus monkeys fed 3 atherogenie diets exhibited thermal transitions at temperatures much higher (3743°C) than those observed in control animals or in normal humans (20-33°C). The same differences were noted in the neutral lipids (cholesteryl esters and triglycerides) which were isolated from the respective lipoproteins. In particular, the difference in thermal properties between the normal and abnormal LDLs was attributable to subtle differences in their cholesteryl ester compositions (mainly an increase in the saturated and monounsaturated fatty acid moieties), with altered triglyceride contents playing only a minor role. Thus, at body temperature, the hyperlipidemia that follows the administration of atherogenic diets is associated with a high degree of order of the neutral lipids in the core of the LDL particle. This, in turn, may be related to the atherogenicity of the abnormal lipoprotein species. Key words:
Atherogenic diets - Atherogenic lipoproteins -Differential scanning calorimetry - LDL - NeutraNipid core - Order-disorder thermal transition Saturated fats
This work was supported by Grants USPHS HG18577 and USPHS Ht15062 from the United States Public Health Service. Thomas Kirchbaullen is recipient of USPHS cardiovascular training grant HL-7237. Steven H. Untracht is recipient of Medical Scientist Training Program Grant 6 T32 GM07281-03 from the National Institute of General Medical Sciences. United States Public Health Service. Gunther M. Fless received partial support from Grant A 77-19 from the Chicago Heart Association. The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the United States Department of Energy under Contract No. EY-76-C-02-0069.
60
Introduction
Previous work has shown that monkeys of several species readily develop hyperlipidemia and atherosclerosis when they are fed either various oils supplemented with cholesterol or an “average American” diet [l-7]. It has also been shown that the plasma low density lipoproteins (LDL) from hyperlipidemit rhesus monkeys stimulate the proliferation of cultured aortic medial smooth muscle cells [8] and enhance the intracellular accumulation of cholesteryl esters [9] ; these events are thought to initiate the formation of the atheromatous lesion [lo]. Furthermore, the onset of hyperlipidemia is attended by significant alterations in the chemical composition and in the bulk physical properties of the LDL, expressed mainly by an increased cholesteryl ester content, a decrease in the relative quantity of triglycerides, and an increase in the size of the lipoprotein particle [ 111. Based on these observations, it appeared of interest to investigate the ultrastructure of LDL during diet-induced hyperlipidemia, with the rhesus monkey used as an experimental model. To this end, we correlated the changes in the chemical composition of these particles with the physical organization of their neutral lipids. We used the technique of differential scanning calorimetry (DSC), first applied successfully by Deckelbaum and his colleagues [12,13] to the study of human LDL. This method, which is capable of revealing thermal transitions that occur within cooperatively ordered arrays, has also provided new information on biological membranes [ 14-171, membrane lipids [14,X321], artificial lipid-protein complexes [22,23 1, and a wide variety of soluble proteins [24-261. Materials and Methods Monkeys, diets, and lipoproteins
Adult male rhesus monkeys (Macaca mulatta) were divided into 4 groups. Three animals serving as controls were fed a regular Purina primate chow diet, and those with induced hyperlipidemia were placed on one of the following 3 atherogenic regimens: (a) a diet consisting of 70.5% of a modified low-fat Purina primate chow supplemented with 25% coconut oil, 2% cholesterol, 1% vitamin mix, and 1.5% gelatin [27] (2 animals); (b) a diet identical to (a), except that peanut oil was substituted for coconut oil [3] (1 animal); and (c) a human diet which represented an “average American” diet as described by Wissler and Vesselinovitch [ 21 (1 animal). The serum cholesterol levels of control monkeys ranged from 100 to 150 mg/lOO ml, while that for donors on the coconut oil, peanut oil, and average American diets were between 500 and 1000, 300 and 1100, and 250 and 450 mg/lOO ml, respectively. In all cases, the serum triglyceride levels were less than 100 mg/lOO ml. From each of the animals, plasma was collected by plasmapheresis after 1618 h of fasting, Human serum was obtained from 5 fasting male donors (type A+). Low density lipoprotein of 1.019 < d < 1.050 g/ml was prepared from unpooled plasma or serum by sequential ultracentrifugal flotation, as previously described [ 281. Before DSC analysis, each sample was concentrated to approximately 200 mg of lipoprotein/ml either by ultracentrifugation or by
61
ultrafiltration with Minicon A-25 concentrating cells (Amicon, Lexington, MA). Lipids were extracted from aliquots of each of the LDL samples according to the method of Folch et al. [ 291. The total lipid extract was dissolved in chloroform, and the various neutral lipid classes were separated by thin-layer chromatography on freshly activated, diethyl ether-washed, precoated Silica Gel G plates (Analtech), with the solvent system of hexane/diethyl ether/glacial acetic acid (90/20/2, v/v/v). Upon visualization with iodine vapor, the areas corresponding to cholesteryl esters and triglycerides were scraped off the plates and were dissolved in chloroform for quantitative analyses and studies by DSC. Cholesteryl esters were quantitated as described by Zak [30], with a factor of 1.65 to account for the fatty acid chain. Triglycerides were determined by the method of Kritchevsky et al. [ 311, and the acyl chains of the cholesteryl esters were separated and measured by gas chromatography as described by Fless et al. [ll]. Differential scanning calorimetry studies
Samples to be studied by DSC were sealed in 75 pl-capacity stainless steel pans. In the case of native LDL, each sample holder contained between 15 and 20 mg of lipoprotein. Calorimetry was performed on duplicate or triplicate aliquots of most preparations with a Perkin-Elmer (Norwalk, CT) DSC-2 unit equipped with a Stage I Intracooler. The temperature and enthalpy scales of the instrument were calibrated with indium, potassium chromate, and cholesteryl linoleate standards. Thermal transitions were recorded at ordinate sensitivities as high as 0.25 mcal/sec, at heating or cooling rates of 2.5, 5, and 10” C/min. In all cases, the temperatures of the peak maxima (which had a precision of 0.5”C in multiple sample aliquots) were extrapolated to zero heating rate. Results The lipoproteins obtained from the three groups of hyperlipidemic rhesus monkeys were relatively poor in triglycerides, which comprised less than 3% by weight of the neutral lipids (Table 1). In contrast, the LDL obtained from the control monkeys and from the normal human donors contained more than 7% of the neutral lipids in the form of triglycerides *. The average fatty acid composition of the cholesteryl esters of the various LDL preparations are shown in Table 2. The normal human and rhesus lipoproteins had similar cholesteryl ester distributions. In contrast, the LDLs isolated from monkeys maintained on each of the 3 atherogenic diets differed among each other and from the controls. In the case of the “average American” diet, LDL exhibited a
* By lsopycnlc density ultracentrifugation. the LDL from the control animals can be resolved into three distinct subpopulations of particles: LDL-I. LDL-II, and LDL-III having hydrated densities of 1.027, 1.036 and 1.050 g/ml, respectively (G.M. Fless and A.M. Scanu, manuscrlgt in preparation).
62 TABLE
1
CHARACTERISTICS
OF THE LDLs EXAMINED Control diet
FROM
RHESUS
Average American
diet
MONKEYS
FED DIFFERENT
Coconut oil diet
DIETS
Peanut oil diet a
Weight percent Protein Phospholipid Free cholesterol Cholesterol esters Triglyceride Molecular
23.2? 25.2 + 11.0 + 35.7 + 4.8 +
weight b
1.0 1.0 1.2 2.0 0.2
3.0 x 106
19.8 25.9 9.6 44.0 0.9
+ + + + f
0.7 1.0 1.2 2.3 0.2
3.5 x 106
a L. Lusk (unpublished observation, one determination). b Data taken from Fless et al. [ll]. c G.M. Flew (unpublished observation): a minor component also observed.
19.0 23.6 9.1 47.0 1.2
f + t + f
1.3 1.5 1.4 2.5 0.2
3.5 x 106
with a molecular
weight
18 24 13 46 Trace 4.8 X 106 c
of 2.9 X 106 was
significant decrease in chOle8~~1 linoleate (18 : 2) and a corresponding increase in cholesteryl oleate (18:1). The coconut oil-cholesterol-fed monkey8 had a modest increase in the total amount of saturated cholesteryl esters (mainly 12 :0 and 14 :0) in their LDL at the expense of the less saturated components. In turn, the animals on the peanut oil-cholesterol diet also exhibited elevated levels of LDL cholesteryl laurate (12 :0) and myristate
b /
coconut oildiet I
20
,
I
40
I
,
60
,
,
80
I
,
100
Fig. 1. Examples of DSC heating runs of LDL from control (a, b) and hyperllpidemic (in this case coconutoil diet) monkeys (c,d). c and c show the results of initial heating of the native lipoproteins from 0 to 110°C. b and d are the respective heating runs of the LDLs after thermal disruption by cooling from 110 to 0°C. The arrows indicate the shoulder appearing on the neutral-lipid transition after denaturation of the lipoproteins. In this and the other figures, the curves were corrected for thermal lag by extrapolation of the peak temperatures to zero heating rate and by shifting the abscissa appropriately.
63 TABLE 2 FATTY ACID ANALYSES OF LDL CHOLESTERYL ESTERS RHESUS, AND VARIOUS HYPERLIPIDEMIC MONKEYS * Fatty acid
Human
FROM NORMAL
HUMAN,
NORMAL
Rhesus monkey LDL
LDL Control diet
Average American diet
coconut diet
oil
Peanut oil diet
Weight % of total cholesteryl ester fatty acids 12: 0 14 : 0 16 : 0 18: 0 20 : 0 22 : 0
1.6 2.2 9.8 1.4 1.4 0.3
1.5 14.4 4.9 -
0.8 13.1 4.6 -
3.3 8.2 16.6 2.4 -
3.7 7.3 13.3 4.6 1.5 2.3
16.7
20.8
18.5
30.5
32.7
3.3
4.0
4.1
4.5
1.5 -
17.2 0.1
23.9 -
37.0 -
21.6 0.8
24.4 2.4 -
Total monounsaturated
20.6
27.9
41 .l
26.9
28.3
12: 14 16 18: 20 22
1.2 0.6 48.8 -
2.5 44.6 -
0.9 0.6 35.5 -
42.7 -
1.5 1.1 1.4 12.3 1.0 0.4
50.6
Total saturated 12: 14 16 18: 20 22
: : : :
1 1 1 1 1 1
2
:2 :2 : :
2 2 2
Total diunsaturated 10 12: 14 16 18: 20 22
47.0
37.0
42.7
17.7
:3
0.7
-
-
-
-
3 3 3 3 3 3
0.6 1.6 -
-
-
-
2.0 1.0 1.0 3.1 0.6 6.6
2.9
0
0
0
4.4
3.4
-
: : : :
Total triunsaturated 14 16 20
-
:4 :4 :4
Total tetraunsaturated Total saturated plus monounsaturated
0.9 8.3 9.2
4.4
3.4
37.3
48.7
59.6
14.3 2.7 3.0 1.3
57.4
7.0 61.0
a The values are averages of three determinations. In all cases. the variation was less than 5%.
: 0), with a large decrease in cholesteryl linoleate. One feature common to the LDLs of the 3 groups of hyperlipidemic monkeys was the large increase in both saturated and monounsaturated cholesteryl esters (Table 2). Representative differential scanning calorimetry heating endotherms are shown in Fig. 1. Each of the native lipoproteins displayed two thermal transi-
(14
64
tions. The major transition was reversible when heating and cooling were confined to the temperature range between -20 and +6O”C (Figs. la, c). This endothermic peak had previously been shown by Deckelbaum et al. [12,13] to correspond to a “smectic’‘-disorder transition of the cholesteryl esters located in the core of human LDL. The second transition, corresponding to the disruption of the lipoprotein [12], occurred at approximately 85°C in all of the LDLs and was irreversible, as indicated by its absence on subsequent heating (Figs. lb, d). Thermal disruption of the lipoprotein caused no change in the temperature of the reversible cholesteryl ester endotherm (Figs. la, b and c, d). However, denaturation of the LDL particle was associated with two significant differences: first, the relative enthalpy of the transition increased by lo-20% in most cases; second, the shape of the peak was more complex, showing a prominent shoulder on the high-temperature side of the main endotherm (Figs. lb, d, arrows). In monkeys with hyperlipidemia, the cholesteryl ester transition in the LDL exhibited striking alterations. Multiple samples of unpooled lipoprotein preparations from a total of 4 experimental animals which had been maintained on the various atherogenic diets showed transitions having a peak maximum above body temperature (Table 3). For example, Figs. 2c-e show the reversible endotherms displayed by the LDLs of 3 hyperlipidemic animals, each subjected to a different atherogenic diet. As indicated by the shaded area, in each case more than 60% of the thermal transition occurred above the physiological temperature. In contrast, in the LDL of normal controls, the thermal disordering of the neutral lipids was essentially complete at body temperature (Figs. 2a, b). To clarify the origin of this thermal behavior, we performed experiments with the neutral lipids isolated from each LDL preparation. In these experiments, chloroform solutions of the LDL cholesteryl esters, or of the cholesTABLE 3 TEMPERATURES
(“C) OF ENDOTHERMIC
Transition
Humail LDL
TRANSITIONS a Rhesus monkey LDL Control diet
Average American diet
Coconut oil diet
Peanut oil diet
Native LDL “Smectbdisorder” Particle denaturation
29 (20-31) 76
29 (28-33) 63
43 67
41 (3742) 70
38 60
Heat-denatured LDL Smectic-holesteric Cholesteric-liquid
25 33
29 33
39 43
39 43
37 41
Cholesteryl esters from LDL Smectic--cholesteric Cholesteric-liquid
31 36
29 ?
42 49
42 47
-
a The transition temperatures correspond to the same single LDL samples whosa analyses are given in Table 2. The values in parentheses indicate the range of transition temperatures for multiple donors and several bleedings.
65 LDL
TEMPERATURE (‘C)
Fig. 2. Typical DSC endotherms obtained for (I: normal human LDL; b: normal rhesus LDL; and LDL obtained from hyperlipidemic monkeys mafntafned on the following diets: c: “average American”,; d: coconut oil-cholesterol; e: peanut oil-cholesterol. Curves f to i rue the corresponding DSC endotherms of the cholesterol esters isolated from the LDLs whose core transitionsare shown in a through d. respec tiveb. The shaded areas indicate the portion of the transition occurring above body temperature. The quantities of LDL from the monkey fed peanut oil diet were insufficfent to allow isolation of cholesteryl esters for study by DSC. For comparison with the other curves, j shows the transition of the thermally dkupted LDL.
teryl esters and triglycerides mixed in the same proportions found in the original lipoprotein sample, were added to DSC pans. After removal of the solvent in vacua, the sample holders, which contained between 2 and 10 mg of dry lipid, were sealed under nitrogen. At the end of the experiment, these samples were redissolved in chloroform and rechromatographed to assure that degradation of the lipids had not occurred. The resulting DSC heating curves are shown in Figs. 2f-i. In most cases, a double-peaked endotherm was observed *. Studies with various synthetic cholesteryl esters [32] have shown that the peak which occurs at lower temperatures corresponds to a transition from smectic to cholesteric * * structures, whereas the higher-temperature endotherm corresponds to a transition from cholesteric to liquid isotropic phases. In all cases examined, the lower-temperature component of the cholesteryl ester endotherm occurred within l-3°C of that of the corresponding native lipoprotein (e.g., compare curves a-d and f-i, respectively, of Fig. 2). Thus, the diet-induced elevation of the LDL neutral-lipid transition was reflected in the thermal behavior of the isolated cholesteryl esters. Furthermore, as shown * The cholesteryl esters from the control rhesus LDL were the only exception (Fig. %). On both heating and cooling, only a sfngle transition was observed. ** In the cholesteric phase. the lipids are arranged with their molecular long axes orfented m one general direction, but without the formation of discrete layers.
66 coconut
oil diet
Fig. 3. Effects of triglycerides on the endotherm of cholesterol esters isolated from LDL. a: Core tranaition of native LDL from a hyperlipidemic (coconut oil-fed) monkey. b: Corresponding neutral-lipid transition of the thermally disrupted lipoprotein. c: DSC tracing of the isolated neat cholesterol esters. d: Endotherm for the cholesterol esters and triglycerides obtained from the lipoprotein when these were mixed in the proportions present in the original LDL sample. The vertical line indicates the body temperature of the monkey.
in Fig. 3 for the coconut oil diet LDL, the in vitro addition of the LDL triglycerides to the cholesteryl esters, in the proportion occurring in the lipoprotein, resulted in several changes in the endotherms. In particular, both peaks were broadened and were reduced in temperature by 3-4°C. Moreover, the higher-temperature cholesteric-isotropic transition appeared as a shoulder on the main peak. The configuration of this latter transition very closely resembles that of the denatured LDL (cf. Figs. 3b and d). Discussion and Conclusions Deckelbaum and his co-workers have established that, the reversible thermal transition, occurring in human plasma LDL at approximately 3O”C, can be ascribed to a cooperative order-disorder transformation of a core of cholesteryl esters and triglycerides from multilayered smectic to more disorganized conformations [ 12,13,33,34]. The major finding in the present study is that the administration of different types of atherogenic diets to rhesus monkeys induces changes in this core of cholesteryl esters and triglycerides; these compositional changes cause a retention of structural order in the neutral lipid core at body temperature and permit disordering only at significantly higher temperatures. This property is in striking contrast to that of the normal rhesus and human controls in which the.interior of the lipoprotein exists in a disordered, liquidlike state at body temperature. Thus, at physiological temperature, the neutral
67
lipids in LDL from the hyperlipidemic monkeys are arranged in an ordered, possibly multilayered [ 13,33,34] core. In previous studies, the LDLs prepared from a large number of individual human donors were found to have neutral-lipid transition temperatures ranging from 20 to 34”C 113,351. When the neutral-lipid composition in each of these LDLs was determined, the transition temperature showed a strong inverse correlation with the relative triglyceride content. In our studies, although the LDLs obtained from the hyperlipidemic monkeys were relatively depleted of triglycerides, several observations indicated that this factor is not responsible for the abnormally high transition temperature. First, the neutral-lipid transition in these abnormal rhesus LDLs occurred at temperatures outside the range obtained for the normal human and monkey lipoproteins, even when corrections were made for differences in relative triglyceride content (Fig. 7 in [ 131). Secondly, as shown in Fig. 2, the differences in thermal properties exhibited by the normal and abnormal LDLs were also observed in the pure cholesteryl esters isolated from the same lipoproteins; moreover, readdition of the triglycerides in the original proportions only caused minor broadening of the peaks and a slight reduction in their transition temperatures (Fig. 3). It follows that, whereas the relative triglyceride content may modulate transition temperatures within separate groups of normal or abnormal LDLs, the basic differences in thermal properties between these two groups of lipoproteins are determined by their cholesteryl esters. Although each of the 3 atherogenic diets was associated with a different distribution of cholesteryl ester composition, in all cases there was a general increase in saturated and monounsaturated fatty acids at the expense of more polyunsaturated components. This last fact is likely to account for the elevated transition temperatures in the LDLs of the hyperlipidemic animals. After denaturation of both the control and abnormal lipoproteins, the cholesteryl ester transition exhibited a shoulder on the high-temperature side of the main endotherm, a result also reported for the LDL and HDL, of atherosclerotic swine [ 361. This shoulder is likely to be the residual of the cholestericisotropic transition, similar to that occurring in cholesteryl ester-triglyceride mixtures (Fig. 3), whereas the main peak represents the smectic-cholesteric transition. This shoulder was absent in the DSC tracings of native LDLs, suggesting that, in the intact lipoprotein, the neutral lipids remain in a cholestericlike liquid-crystal phase (i.e., the molecules retain a generally radial orientation) at high temperatures. Alternatively, the core of the lipoprotein may gradually progress from cholesteric-like to isotropic phases in a noncooperative fashion. In either case, the surface components of LDL (apo B, phospholipids, and unesterified cholesterol), together with the relatively small size of the lipoprotein particle, are likely to impose some restriction on the behavior of the core components. These features do not seem to be influenced by the increase in the size of the lipoprotein particle noted previously [ll], or by the changes in its neutral-lipid composition following the feeding of atherogenic diets. Two important facts raise the possibility that, in the rhesus monkey, the ordered LDL core is causally related to accelerated atherosclerosis: (a) the diets that lead to the abnormal LDL are also those which cause atherosclerosis; and (b) these abnormal LDLs stimulate the proliferation of cultured aortic medial
68
smooth muscle cells and enhance intracellular accumulation of cholesteryl esters [8,9]. Regardless of whether or not the elevated LDL transition temperature is ultimately responsible for the excessive accumulation of cholesterol in the arterial wall, it is nevertheless interesting to speculate on how such a hypothetical process might be mediated. For example, the unusual degree of order displayed by the LDL core may result in crucial changes in the conformation of the adjacent surface components. These changes may, in turn, grossly influence the effects of the lipoprotein on cellular receptors in the arterial wall [8,9,37, 381. Alternatively, or in addition, the relative rigidity of the LDL core may render the cholesteryl esters inaccessible to degradation by the appropriate lysosomal enzymes [37] and thus result in continual intracellular accumulation of the steroid and in formation of fatty streaks. A word of caution is required, however. Even if these abnormal lipoproteins are in fact “atherogenic”, this may be due to molecular properties unrelated to the elevated transition temperature. Moreover, aside from their effects on LDL, all of the atherogenic diets cause profound hyperlipidemia, a condition which alone could be responsible for the accelerated atherosclerosis. In this latter case, however, the high levels of circulating LDL could be related to reduced clearance of the lipoprotein, thus again raising the question of the possible role played by the organized core. Finally, with regard to the significance of the current findings to human atherosclerosis, it is important to note that studies on the LDL of individuals with acute diet-induced hyperlipidemia *, patients with Type IIa familial hypercholesterolemia [13], and patients with acute ischemic heart disease [35] thus far have not shown thermal transitions occurring above body temperature. However, particularly in the studies of ‘Iype IIa patients, who were maintained on a low-fat, low-cholesterol. diet [13], and in whom the biochemical defect is likely to be associated with the LDL receptor [37], it is not surprising that their lipoproteins appeared normal. Thus, it is clear that more rigorous studies, under conditions more closely approximating those used in the present work, are required before the role of the ordered core in atherosclerosis can be assessed. Nevertheless, our results have clearly shown that a physical method such as differential scanning calorimetry can detect important structural abnormalities in a lipoprotein which has been associated for a long time with the genesis of this disease. Thus, the current observations open new avenues of exploration of the possible relationships between structural alterations in lipoproteins and pathophysiological processes. Acknowledgements The authors wish to thank Mr. Lance Lusk and Mr. Roger Franz for providing samples of rhesus lipoproteins, and for chemical analysis of the cholesteryl esters. They also wish to thank Dr. Robert W. Wissler for his interest in this work. Mrs. Rose Scott and Mrs. Karen Kosakowski deserve special thanks for helping with the preparation of the manuscript and Mrs. Elisabeth Lanzl for its editing. * S.H.
Untracht, T. Kircbhausen. G.M. F&s.
and A.M. Scanu (unpublished observations).
69
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J.D., Small, D.M. and Shipley, G.G., Protein-lipid interactions - Recombinants of the proteolipid apoprotein of myelin with dimyrlstoyllecithm. Biochemistry, 16 (19771 2313. 23 Tall, A.R.. Small, D.M., Deckelbaum, R.J. and Shipley, G.G.. Structure and thermodynamic proper ties of high-density lipoprotein recombinants. J. Biol. Chem.. 252 (1977) 4701. 24 Tsong. T.Y., Hearn, R.P.. Wrathall. D.P. and Sturtevant. J.M., A calorimetric study of thermally induced conformational transitions of Ribonuclease A and certain of its derivatives, Biochemistry, 9 (1970) 2666. 25 Privalov, P.L. and Khechinashvlli, N.N., A thermodynamic approach to the problem of stabilization of globular protein structure - A calorimetric study, J. Mol. Biol., 86 (19741 665. 26 Tall, A.R., Shipley, G.G. and Small, D.M.. Conformational and thermodynamic Properties of aPo A-I of human plasma high-density lipoproteins. J. Biol. Chem., 251 (1976) 3749. 27 Wissler, R.W., Frazier. L.E., Hughes, R.H. and Rasmussen, R.A.. Atherogenesls in the cebus monkey, Arch. Path., 74 (1972) 312. 28 Fleas, G.M. and Scanu, A.M., Physicochemical characterization of rhesus low-density lipoproteins, Biochemistry, 14 (1976) 1783. 29 Folch, J.. Lees, M. and Sloane-Stanley, G.H., A simple method for the isolation and purification of
70 total lipids from animal tissue, J. Biol. Chem., 226 (1967) 497. 30 Zak, B.. Dickenman. R.C.. White, E.G., Burnett. H. and Chervey, P.J.. Rapid estimation of free and total cholesterol, Amer. J. Clin. Path., 24 (1964) 1307. 31 Kritchevsky, D.. Davidson, L.M., Kim, ILK. and Malhotra. S.. Quantitation of serum lipids by a simple TLC-charring method, Clin. Chim. Acta. 46 (1973) 63. 32 Small, D.M.. The physical state of lipids of biological importance - Cholesterol esters, cholesterol, triglyceride. In: M. Blank (Ed.). Surface Chemistry of Biological Systems, Plenum Publishing Co., New York, NY, 1970. PP. 65-83. 33 Atkinson, D.. Deckelbaum. R.J.. Small, D.M. and Shipley, G.G.. Structure of human plasma lowdensity lipoproteins-Molecular organization of the central core, Proc. Nat. Acad. Sci. U.S.A., 74 (1977) 1042. 34 Sears, B., Deckelbaum, R.J.. Janiak, M.J.. Shipley, G.G. and Small, D.M., Temperature-dependent 13C nuclear magnetic resonance studies of human serum low-density lipoproteins, Biochemistry, 15 (1976) 4161. 36 Mateu. K.. Kfrchhausen, T.. Padron, R. and Camejo, G., Small-angle x-ray scattering study of human plasma low-density lipoproteins with differential reactivity for an arterial proteoglycan. J. Supramolec. Strut., 7 (1977) 435. 36 Tall, A.R., Atkinson, D.. Small, D.M. and Mahley. R.W.. Characterization of the lipoproteins of atherosclerotic swine, J. Biol. Chem.. 262 (19’77) 7288. 37 Brown, M.S. and Goldstein, J.L.. Receptor-mediated control of cholesterol metabolism, Science, 191 (1976) 160. 38 Weinstein, D.B.. Carew, T.E. and Steinberg, D.. Uptake and degradation of low-density lipoprotein by swine arterial smooth muscle cells with inhibition of cholesterol biosynthesis, Biochim. Biophys. Acta. 424 (1976) 404.
Atherosclerosis, 33 (1979) 59-70 @ Elsevier/North-Holland Scientific
Publishers,
Ltd.
ATHEROGENIC DIETS AND NEUTRAL-LIPID ORGANIZATION PLASMA LOW DENSITY LIPOPROTEINS
TOMAS KIRCHHAUSEN, M. SCANU
STEVEN
H. UNTRACHT,
GUNTHER
IN
M. FLESS and ANGELO
Departments of Medicine, Biophysics and Theoretical Biology, and Biochemistry, The University of Chicago Pritzker School of Medicine, and The Franklin McLean Memorial Research Institute, Chicago, IL 60637 (U.S.A.) (Received 11 September, 1978) (Revised, received 27 November, (Accepted 29 November, 1978)
1978)
summary The plasma low density lipoproteins (LDL) of rhesus monkeys fed 3 atherogenie diets exhibited thermal transitions at temperatures much higher (3743°C) than those observed in control animals or in normal humans (20-33°C). The same differences were noted in the neutral lipids (cholesteryl esters and triglycerides) which were isolated from the respective lipoproteins. In particular, the difference in thermal properties between the normal and abnormal LDLs was attributable to subtle differences in their cholesteryl ester compositions (mainly an increase in the saturated and monounsaturated fatty acid moieties), with altered triglyceride contents playing only a minor role. Thus, at body temperature, the hyperlipidemia that follows the administration of atherogenic diets is associated with a high degree of order of the neutral lipids in the core of the LDL particle. This, in turn, may be related to the atherogenicity of the abnormal lipoprotein species. Key words:
Atherogenic diets - Atherogenic lipoproteins -Differential scanning calorimetry - LDL - NeutraNipid core - Order-disorder thermal transition Saturated fats
This work was supported by Grants USPHS HG18577 and USPHS Ht15062 from the United States Public Health Service. Thomas Kirchbaullen is recipient of USPHS cardiovascular training grant HL-7237. Steven H. Untracht is recipient of Medical Scientist Training Program Grant 6 T32 GM07281-03 from the National Institute of General Medical Sciences. United States Public Health Service. Gunther M. Fless received partial support from Grant A 77-19 from the Chicago Heart Association. The Franklin McLean Memorial Research Institute is operated by The University of Chicago for the United States Department of Energy under Contract No. EY-76-C-02-0069.
60
Introduction
Previous work has shown that monkeys of several species readily develop hyperlipidemia and atherosclerosis when they are fed either various oils supplemented with cholesterol or an “average American” diet [l-7]. It has also been shown that the plasma low density lipoproteins (LDL) from hyperlipidemit rhesus monkeys stimulate the proliferation of cultured aortic medial smooth muscle cells [8] and enhance the intracellular accumulation of cholesteryl esters [9] ; these events are thought to initiate the formation of the atheromatous lesion [lo]. Furthermore, the onset of hyperlipidemia is attended by significant alterations in the chemical composition and in the bulk physical properties of the LDL, expressed mainly by an increased cholesteryl ester content, a decrease in the relative quantity of triglycerides, and an increase in the size of the lipoprotein particle [ 111. Based on these observations, it appeared of interest to investigate the ultrastructure of LDL during diet-induced hyperlipidemia, with the rhesus monkey used as an experimental model. To this end, we correlated the changes in the chemical composition of these particles with the physical organization of their neutral lipids. We used the technique of differential scanning calorimetry (DSC), first applied successfully by Deckelbaum and his colleagues [12,13] to the study of human LDL. This method, which is capable of revealing thermal transitions that occur within cooperatively ordered arrays, has also provided new information on biological membranes [ 14-171, membrane lipids [14,X321], artificial lipid-protein complexes [22,23 1, and a wide variety of soluble proteins [24-261. Materials and Methods Monkeys, diets, and lipoproteins
Adult male rhesus monkeys (Macaca mulatta) were divided into 4 groups. Three animals serving as controls were fed a regular Purina primate chow diet, and those with induced hyperlipidemia were placed on one of the following 3 atherogenic regimens: (a) a diet consisting of 70.5% of a modified low-fat Purina primate chow supplemented with 25% coconut oil, 2% cholesterol, 1% vitamin mix, and 1.5% gelatin [27] (2 animals); (b) a diet identical to (a), except that peanut oil was substituted for coconut oil [3] (1 animal); and (c) a human diet which represented an “average American” diet as described by Wissler and Vesselinovitch [ 21 (1 animal). The serum cholesterol levels of control monkeys ranged from 100 to 150 mg/lOO ml, while that for donors on the coconut oil, peanut oil, and average American diets were between 500 and 1000, 300 and 1100, and 250 and 450 mg/lOO ml, respectively. In all cases, the serum triglyceride levels were less than 100 mg/lOO ml. From each of the animals, plasma was collected by plasmapheresis after 1618 h of fasting, Human serum was obtained from 5 fasting male donors (type A+). Low density lipoprotein of 1.019 < d < 1.050 g/ml was prepared from unpooled plasma or serum by sequential ultracentrifugal flotation, as previously described [ 281. Before DSC analysis, each sample was concentrated to approximately 200 mg of lipoprotein/ml either by ultracentrifugation or by
61
ultrafiltration with Minicon A-25 concentrating cells (Amicon, Lexington, MA). Lipids were extracted from aliquots of each of the LDL samples according to the method of Folch et al. [ 291. The total lipid extract was dissolved in chloroform, and the various neutral lipid classes were separated by thin-layer chromatography on freshly activated, diethyl ether-washed, precoated Silica Gel G plates (Analtech), with the solvent system of hexane/diethyl ether/glacial acetic acid (90/20/2, v/v/v). Upon visualization with iodine vapor, the areas corresponding to cholesteryl esters and triglycerides were scraped off the plates and were dissolved in chloroform for quantitative analyses and studies by DSC. Cholesteryl esters were quantitated as described by Zak [30], with a factor of 1.65 to account for the fatty acid chain. Triglycerides were determined by the method of Kritchevsky et al. [ 311, and the acyl chains of the cholesteryl esters were separated and measured by gas chromatography as described by Fless et al. [ll]. Differential scanning calorimetry studies
Samples to be studied by DSC were sealed in 75 pl-capacity stainless steel pans. In the case of native LDL, each sample holder contained between 15 and 20 mg of lipoprotein. Calorimetry was performed on duplicate or triplicate aliquots of most preparations with a Perkin-Elmer (Norwalk, CT) DSC-2 unit equipped with a Stage I Intracooler. The temperature and enthalpy scales of the instrument were calibrated with indium, potassium chromate, and cholesteryl linoleate standards. Thermal transitions were recorded at ordinate sensitivities as high as 0.25 mcal/sec, at heating or cooling rates of 2.5, 5, and 10” C/min. In all cases, the temperatures of the peak maxima (which had a precision of 0.5”C in multiple sample aliquots) were extrapolated to zero heating rate. Results The lipoproteins obtained from the three groups of hyperlipidemic rhesus monkeys were relatively poor in triglycerides, which comprised less than 3% by weight of the neutral lipids (Table 1). In contrast, the LDL obtained from the control monkeys and from the normal human donors contained more than 7% of the neutral lipids in the form of triglycerides *. The average fatty acid composition of the cholesteryl esters of the various LDL preparations are shown in Table 2. The normal human and rhesus lipoproteins had similar cholesteryl ester distributions. In contrast, the LDLs isolated from monkeys maintained on each of the 3 atherogenic diets differed among each other and from the controls. In the case of the “average American” diet, LDL exhibited a
* By lsopycnlc density ultracentrifugation. the LDL from the control animals can be resolved into three distinct subpopulations of particles: LDL-I. LDL-II, and LDL-III having hydrated densities of 1.027, 1.036 and 1.050 g/ml, respectively (G.M. Fless and A.M. Scanu, manuscrlgt in preparation).
62 TABLE
1
CHARACTERISTICS
OF THE LDLs EXAMINED Control diet
FROM
RHESUS
Average American
diet
MONKEYS
FED DIFFERENT
Coconut oil diet
DIETS
Peanut oil diet a
Weight percent Protein Phospholipid Free cholesterol Cholesterol esters Triglyceride Molecular
23.2? 25.2 + 11.0 + 35.7 + 4.8 +
weight b
1.0 1.0 1.2 2.0 0.2
3.0 x 106
19.8 25.9 9.6 44.0 0.9
+ + + + f
0.7 1.0 1.2 2.3 0.2
3.5 x 106
a L. Lusk (unpublished observation, one determination). b Data taken from Fless et al. [ll]. c G.M. Flew (unpublished observation): a minor component also observed.
19.0 23.6 9.1 47.0 1.2
f + t + f
1.3 1.5 1.4 2.5 0.2
3.5 x 106
with a molecular
weight
18 24 13 46 Trace 4.8 X 106 c
of 2.9 X 106 was
significant decrease in chOle8~~1 linoleate (18 : 2) and a corresponding increase in cholesteryl oleate (18:1). The coconut oil-cholesterol-fed monkey8 had a modest increase in the total amount of saturated cholesteryl esters (mainly 12 :0 and 14 :0) in their LDL at the expense of the less saturated components. In turn, the animals on the peanut oil-cholesterol diet also exhibited elevated levels of LDL cholesteryl laurate (12 :0) and myristate
b /
coconut oildiet I
20
,
I
40
I
,
60
,
,
80
I
,
100
Fig. 1. Examples of DSC heating runs of LDL from control (a, b) and hyperllpidemic (in this case coconutoil diet) monkeys (c,d). c and c show the results of initial heating of the native lipoproteins from 0 to 110°C. b and d are the respective heating runs of the LDLs after thermal disruption by cooling from 110 to 0°C. The arrows indicate the shoulder appearing on the neutral-lipid transition after denaturation of the lipoproteins. In this and the other figures, the curves were corrected for thermal lag by extrapolation of the peak temperatures to zero heating rate and by shifting the abscissa appropriately.
63 TABLE 2 FATTY ACID ANALYSES OF LDL CHOLESTERYL ESTERS RHESUS, AND VARIOUS HYPERLIPIDEMIC MONKEYS * Fatty acid
Human
FROM NORMAL
HUMAN,
NORMAL
Rhesus monkey LDL
LDL Control diet
Average American diet
coconut diet
oil
Peanut oil diet
Weight % of total cholesteryl ester fatty acids 12: 0 14 : 0 16 : 0 18: 0 20 : 0 22 : 0
1.6 2.2 9.8 1.4 1.4 0.3
1.5 14.4 4.9 -
0.8 13.1 4.6 -
3.3 8.2 16.6 2.4 -
3.7 7.3 13.3 4.6 1.5 2.3
16.7
20.8
18.5
30.5
32.7
3.3
4.0
4.1
4.5
1.5 -
17.2 0.1
23.9 -
37.0 -
21.6 0.8
24.4 2.4 -
Total monounsaturated
20.6
27.9
41 .l
26.9
28.3
12: 14 16 18: 20 22
1.2 0.6 48.8 -
2.5 44.6 -
0.9 0.6 35.5 -
42.7 -
1.5 1.1 1.4 12.3 1.0 0.4
50.6
Total saturated 12: 14 16 18: 20 22
: : : :
1 1 1 1 1 1
2
:2 :2 : :
2 2 2
Total diunsaturated 10 12: 14 16 18: 20 22
47.0
37.0
42.7
17.7
:3
0.7
-
-
-
-
3 3 3 3 3 3
0.6 1.6 -
-
-
-
2.0 1.0 1.0 3.1 0.6 6.6
2.9
0
0
0
4.4
3.4
-
: : : :
Total triunsaturated 14 16 20
-
:4 :4 :4
Total tetraunsaturated Total saturated plus monounsaturated
0.9 8.3 9.2
4.4
3.4
37.3
48.7
59.6
14.3 2.7 3.0 1.3
57.4
7.0 61.0
a The values are averages of three determinations. In all cases. the variation was less than 5%.
: 0), with a large decrease in cholesteryl linoleate. One feature common to the LDLs of the 3 groups of hyperlipidemic monkeys was the large increase in both saturated and monounsaturated cholesteryl esters (Table 2). Representative differential scanning calorimetry heating endotherms are shown in Fig. 1. Each of the native lipoproteins displayed two thermal transi-
(14
64
tions. The major transition was reversible when heating and cooling were confined to the temperature range between -20 and +6O”C (Figs. la, c). This endothermic peak had previously been shown by Deckelbaum et al. [12,13] to correspond to a “smectic’‘-disorder transition of the cholesteryl esters located in the core of human LDL. The second transition, corresponding to the disruption of the lipoprotein [12], occurred at approximately 85°C in all of the LDLs and was irreversible, as indicated by its absence on subsequent heating (Figs. lb, d). Thermal disruption of the lipoprotein caused no change in the temperature of the reversible cholesteryl ester endotherm (Figs. la, b and c, d). However, denaturation of the LDL particle was associated with two significant differences: first, the relative enthalpy of the transition increased by lo-20% in most cases; second, the shape of the peak was more complex, showing a prominent shoulder on the high-temperature side of the main endotherm (Figs. lb, d, arrows). In monkeys with hyperlipidemia, the cholesteryl ester transition in the LDL exhibited striking alterations. Multiple samples of unpooled lipoprotein preparations from a total of 4 experimental animals which had been maintained on the various atherogenic diets showed transitions having a peak maximum above body temperature (Table 3). For example, Figs. 2c-e show the reversible endotherms displayed by the LDLs of 3 hyperlipidemic animals, each subjected to a different atherogenic diet. As indicated by the shaded area, in each case more than 60% of the thermal transition occurred above the physiological temperature. In contrast, in the LDL of normal controls, the thermal disordering of the neutral lipids was essentially complete at body temperature (Figs. 2a, b). To clarify the origin of this thermal behavior, we performed experiments with the neutral lipids isolated from each LDL preparation. In these experiments, chloroform solutions of the LDL cholesteryl esters, or of the cholesTABLE 3 TEMPERATURES
(“C) OF ENDOTHERMIC
Transition
Humail LDL
TRANSITIONS a Rhesus monkey LDL Control diet
Average American diet
Coconut oil diet
Peanut oil diet
Native LDL “Smectbdisorder” Particle denaturation
29 (20-31) 76
29 (28-33) 63
43 67
41 (3742) 70
38 60
Heat-denatured LDL Smectic-holesteric Cholesteric-liquid
25 33
29 33
39 43
39 43
37 41
Cholesteryl esters from LDL Smectic--cholesteric Cholesteric-liquid
31 36
29 ?
42 49
42 47
-
a The transition temperatures correspond to the same single LDL samples whosa analyses are given in Table 2. The values in parentheses indicate the range of transition temperatures for multiple donors and several bleedings.
65 LDL
TEMPERATURE (‘C)
Fig. 2. Typical DSC endotherms obtained for (I: normal human LDL; b: normal rhesus LDL; and LDL obtained from hyperlipidemic monkeys mafntafned on the following diets: c: “average American”,; d: coconut oil-cholesterol; e: peanut oil-cholesterol. Curves f to i rue the corresponding DSC endotherms of the cholesterol esters isolated from the LDLs whose core transitionsare shown in a through d. respec tiveb. The shaded areas indicate the portion of the transition occurring above body temperature. The quantities of LDL from the monkey fed peanut oil diet were insufficfent to allow isolation of cholesteryl esters for study by DSC. For comparison with the other curves, j shows the transition of the thermally dkupted LDL.
teryl esters and triglycerides mixed in the same proportions found in the original lipoprotein sample, were added to DSC pans. After removal of the solvent in vacua, the sample holders, which contained between 2 and 10 mg of dry lipid, were sealed under nitrogen. At the end of the experiment, these samples were redissolved in chloroform and rechromatographed to assure that degradation of the lipids had not occurred. The resulting DSC heating curves are shown in Figs. 2f-i. In most cases, a double-peaked endotherm was observed *. Studies with various synthetic cholesteryl esters [32] have shown that the peak which occurs at lower temperatures corresponds to a transition from smectic to cholesteric * * structures, whereas the higher-temperature endotherm corresponds to a transition from cholesteric to liquid isotropic phases. In all cases examined, the lower-temperature component of the cholesteryl ester endotherm occurred within l-3°C of that of the corresponding native lipoprotein (e.g., compare curves a-d and f-i, respectively, of Fig. 2). Thus, the diet-induced elevation of the LDL neutral-lipid transition was reflected in the thermal behavior of the isolated cholesteryl esters. Furthermore, as shown * The cholesteryl esters from the control rhesus LDL were the only exception (Fig. %). On both heating and cooling, only a sfngle transition was observed. ** In the cholesteric phase. the lipids are arranged with their molecular long axes orfented m one general direction, but without the formation of discrete layers.
66 coconut
oil diet
Fig. 3. Effects of triglycerides on the endotherm of cholesterol esters isolated from LDL. a: Core tranaition of native LDL from a hyperlipidemic (coconut oil-fed) monkey. b: Corresponding neutral-lipid transition of the thermally disrupted lipoprotein. c: DSC tracing of the isolated neat cholesterol esters. d: Endotherm for the cholesterol esters and triglycerides obtained from the lipoprotein when these were mixed in the proportions present in the original LDL sample. The vertical line indicates the body temperature of the monkey.
in Fig. 3 for the coconut oil diet LDL, the in vitro addition of the LDL triglycerides to the cholesteryl esters, in the proportion occurring in the lipoprotein, resulted in several changes in the endotherms. In particular, both peaks were broadened and were reduced in temperature by 3-4°C. Moreover, the higher-temperature cholesteric-isotropic transition appeared as a shoulder on the main peak. The configuration of this latter transition very closely resembles that of the denatured LDL (cf. Figs. 3b and d). Discussion and Conclusions Deckelbaum and his co-workers have established that, the reversible thermal transition, occurring in human plasma LDL at approximately 3O”C, can be ascribed to a cooperative order-disorder transformation of a core of cholesteryl esters and triglycerides from multilayered smectic to more disorganized conformations [ 12,13,33,34]. The major finding in the present study is that the administration of different types of atherogenic diets to rhesus monkeys induces changes in this core of cholesteryl esters and triglycerides; these compositional changes cause a retention of structural order in the neutral lipid core at body temperature and permit disordering only at significantly higher temperatures. This property is in striking contrast to that of the normal rhesus and human controls in which the.interior of the lipoprotein exists in a disordered, liquidlike state at body temperature. Thus, at physiological temperature, the neutral
67
lipids in LDL from the hyperlipidemic monkeys are arranged in an ordered, possibly multilayered [ 13,33,34] core. In previous studies, the LDLs prepared from a large number of individual human donors were found to have neutral-lipid transition temperatures ranging from 20 to 34”C 113,351. When the neutral-lipid composition in each of these LDLs was determined, the transition temperature showed a strong inverse correlation with the relative triglyceride content. In our studies, although the LDLs obtained from the hyperlipidemic monkeys were relatively depleted of triglycerides, several observations indicated that this factor is not responsible for the abnormally high transition temperature. First, the neutral-lipid transition in these abnormal rhesus LDLs occurred at temperatures outside the range obtained for the normal human and monkey lipoproteins, even when corrections were made for differences in relative triglyceride content (Fig. 7 in [ 131). Secondly, as shown in Fig. 2, the differences in thermal properties exhibited by the normal and abnormal LDLs were also observed in the pure cholesteryl esters isolated from the same lipoproteins; moreover, readdition of the triglycerides in the original proportions only caused minor broadening of the peaks and a slight reduction in their transition temperatures (Fig. 3). It follows that, whereas the relative triglyceride content may modulate transition temperatures within separate groups of normal or abnormal LDLs, the basic differences in thermal properties between these two groups of lipoproteins are determined by their cholesteryl esters. Although each of the 3 atherogenic diets was associated with a different distribution of cholesteryl ester composition, in all cases there was a general increase in saturated and monounsaturated fatty acids at the expense of more polyunsaturated components. This last fact is likely to account for the elevated transition temperatures in the LDLs of the hyperlipidemic animals. After denaturation of both the control and abnormal lipoproteins, the cholesteryl ester transition exhibited a shoulder on the high-temperature side of the main endotherm, a result also reported for the LDL and HDL, of atherosclerotic swine [ 361. This shoulder is likely to be the residual of the cholestericisotropic transition, similar to that occurring in cholesteryl ester-triglyceride mixtures (Fig. 3), whereas the main peak represents the smectic-cholesteric transition. This shoulder was absent in the DSC tracings of native LDLs, suggesting that, in the intact lipoprotein, the neutral lipids remain in a cholestericlike liquid-crystal phase (i.e., the molecules retain a generally radial orientation) at high temperatures. Alternatively, the core of the lipoprotein may gradually progress from cholesteric-like to isotropic phases in a noncooperative fashion. In either case, the surface components of LDL (apo B, phospholipids, and unesterified cholesterol), together with the relatively small size of the lipoprotein particle, are likely to impose some restriction on the behavior of the core components. These features do not seem to be influenced by the increase in the size of the lipoprotein particle noted previously [ll], or by the changes in its neutral-lipid composition following the feeding of atherogenic diets. Two important facts raise the possibility that, in the rhesus monkey, the ordered LDL core is causally related to accelerated atherosclerosis: (a) the diets that lead to the abnormal LDL are also those which cause atherosclerosis; and (b) these abnormal LDLs stimulate the proliferation of cultured aortic medial
68
smooth muscle cells and enhance intracellular accumulation of cholesteryl esters [8,9]. Regardless of whether or not the elevated LDL transition temperature is ultimately responsible for the excessive accumulation of cholesterol in the arterial wall, it is nevertheless interesting to speculate on how such a hypothetical process might be mediated. For example, the unusual degree of order displayed by the LDL core may result in crucial changes in the conformation of the adjacent surface components. These changes may, in turn, grossly influence the effects of the lipoprotein on cellular receptors in the arterial wall [8,9,37, 381. Alternatively, or in addition, the relative rigidity of the LDL core may render the cholesteryl esters inaccessible to degradation by the appropriate lysosomal enzymes [37] and thus result in continual intracellular accumulation of the steroid and in formation of fatty streaks. A word of caution is required, however. Even if these abnormal lipoproteins are in fact “atherogenic”, this may be due to molecular properties unrelated to the elevated transition temperature. Moreover, aside from their effects on LDL, all of the atherogenic diets cause profound hyperlipidemia, a condition which alone could be responsible for the accelerated atherosclerosis. In this latter case, however, the high levels of circulating LDL could be related to reduced clearance of the lipoprotein, thus again raising the question of the possible role played by the organized core. Finally, with regard to the significance of the current findings to human atherosclerosis, it is important to note that studies on the LDL of individuals with acute diet-induced hyperlipidemia *, patients with Type IIa familial hypercholesterolemia [13], and patients with acute ischemic heart disease [35] thus far have not shown thermal transitions occurring above body temperature. However, particularly in the studies of ‘Iype IIa patients, who were maintained on a low-fat, low-cholesterol. diet [13], and in whom the biochemical defect is likely to be associated with the LDL receptor [37], it is not surprising that their lipoproteins appeared normal. Thus, it is clear that more rigorous studies, under conditions more closely approximating those used in the present work, are required before the role of the ordered core in atherosclerosis can be assessed. Nevertheless, our results have clearly shown that a physical method such as differential scanning calorimetry can detect important structural abnormalities in a lipoprotein which has been associated for a long time with the genesis of this disease. Thus, the current observations open new avenues of exploration of the possible relationships between structural alterations in lipoproteins and pathophysiological processes. Acknowledgements The authors wish to thank Mr. Lance Lusk and Mr. Roger Franz for providing samples of rhesus lipoproteins, and for chemical analysis of the cholesteryl esters. They also wish to thank Dr. Robert W. Wissler for his interest in this work. Mrs. Rose Scott and Mrs. Karen Kosakowski deserve special thanks for helping with the preparation of the manuscript and Mrs. Elisabeth Lanzl for its editing. * S.H.
Untracht, T. Kircbhausen. G.M. F&s.
and A.M. Scanu (unpublished observations).
69
References 1 Wissler, R.W. and Vesselinovitch, D., Atherosclerosis in nonhuman primates, Adv. Vet. Sci. ComP. Med., 21 (1977) 351. 2 Wissler, R.W. and Vesselinovltch, D., The effects of feeding various dietary fats on the development and regression of hypercholesterolemia and atherosclerosis. In: C. Slrtorl, G. Ricci and S. Gomi (Eds.), Diet and Atherosclerosis, Plenum Publishing Corp., New York. NY, 1975, PP. 65-76. 3 Vesselinovitch. D., Getz. G.S.. Hughes, R.H. and Wissler. R.W.. Atherosclerosis ln the rhesus monkey fed three food fats, Atherosclerosis. 20 (1974) 303. 4 Wissler, R.W. and Vessellnovitch, D., Studies of regression of advanced atherosclerosis in experimental animals and man, Ann. N.Y. Acad Sci.. 275 (1976) 363. 5 Rudel, L.L., Pitt, L.L. and Nelson, C.A.. Characterization of plasma low density lipoproteins of nonhuman primates fed dietary cholesterol, J. Lipid Res.. 18 (1977) 211. 6 Srinivasan. S.R., Smith, C.C., Radhakrlshnamurthy. B., Wolf. 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