Effects of fat level, feeding period, and source of fat on lipid fluidity and physical state of rabbit plasma lipoproteins

Atherosclerosis, 48 (1983) 15-27 Elsevier Scientific Publishers Ireland,

1.5 Ltd.

ATH 3352

Effects of Fat Level, Feeding Period, and Source of Fat on Lipid Fluidity and Physical State of Rabbit Plasma Lipoproteins Elliott

Berlin

and Calvert

Young,

Jr.

Lipid Nutrition Laboratory, Beltsoille Human Nutrition Research Center, Agricultural Research Service, United States Department of Agriculture,Beltsville, MD 20705 (USA.) (Received 1 September, 1982) (Revised, received 7 February, 1983) (Accepted 7 February, 1983)

Summary Elevating fat content from 5 to 20% of diet by weight or extending the feeding period from 6 months to more than 1 year did not substantially alter the fluidity of rabbit plasma lipoprotein lipid domains. Dietary fatty acid saturation was not adequate as a predictor of lipoprotein fluidity. Rabbits fed corn oil, high in polyunsaturated fatty acid content, did not have more fluid lipoproteins than rabbits fed cocoa butter which contains a high level of saturated long chain fatty acids. Order parameters calculated from fluorescence depolarization measurements with diphenylhexatriene (DPH) showed that very low density lipoprotein (VLDL) lipids were in highly fluid or ‘liquid’ states at or below body temperature. Order parameter data showed transitions from ordered phase to isotropic liquid in low density lipoproteins (LDL) that were heretofore unnoted with DPH fluorescence depolarization measurements. The transition temperature was inversely related to the LDL triglyceride content, indicating probe intercalation between the fatty acyl chains of the core triacylglycerols in VLDL and LDL. Key words;

Dietary PUFA - Fluidity - Fluorescence depolarization Triglyceride

- Lipoprotein

-

Introduction The central role of the plasma lipoproteins in lipid transport, coupled with implications that elevated levels of certain plasma lipids are risk factors for cardio-

16

vascular disease [l] has stimulated much research into the physical chemical properties of these macromolecules. Our research on the human requirements for lipids has included continuing investigations into the effects of dietary fat composition on the fluidity of the lipid phase of the major lipoprotein classes. Earlier studies [2] demonstrated effects after feeding rabbits semipurified diets containing 5% of either corn oil, coconut oil, milkfat, or cocoa butter for periods of l-6 months. These natural fats provide variations in levels of short chain fatty acids, polyunsaturated fatty acids, and the long chain saturated fatty acids palmitic and stearic. Effects were noted both in regard to the potential for liquid crystalline type phase transitions and in the fluidities of the lipoprotein fractions, as determined by fluorescence polarization measurements with the hydrocarbon probe 1,6-diphenyl1,3,5-hexatriene (DPH). Despite the fatty acid compositions of the respective diets, the very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from cocoa butter-fed rabbits were more fluid than those from the corn oil-fed rabbits. Coconut oil-fed rabbits and in some instances milkfat-fed rabbits had VLDL fractions capable of undergoing lipid phase transitions near 30°C. Neither the LDL nor the high density lipoprotein (HDL) fractions displayed any phase transitions in the range 0-50°C in contrast with numerous reports [3-61 of LDL cholesteryl ester phase transitions observed with differential scanning calorimetry (DSC). This major difference in result with DSC and fluorescence polarization has been noted [2,4,7,8]. We have continued our work with lipoproteins to study the effects of feeding high-fat diets for periods of more than 1 year, because the development of atherosclerosis in humans is often associated with years of consuming diets containing significant amounts of fat [ 11. We have also applied newer theoretical treatments [9] to the fluorescence polarization data, thus obtaining information pertaining to the localization of the fluorescent probe and the physical state of the apolar core in VLDL and LDL, as influenced by the relative amount of cholesteryl ester and triacylglycerol. Materials and Methods * Animals and experimental diets Male New Zealand white rabbits weighing 1.5-2.0 kg purchased from laboratory-animal suppliers were fed semi-purified diets containing 5 or 20% fat by weight. Animals were housed in wire-bottom cages and ,allowed free access to food and water during the experiment. Rabbits maintained on the 5% diet were exsanguinated via cardiac puncture after 379 & 24 days, rabbits on the 20% fat diet, after 200 + 4 or 405 * 20 days. The animals were not fasted before taking blood. The low-fat diets were prepared as described [2] by using the formulation of Gaman et al. [lo] without the fat component as a basal diet and adding 50 g of

* Mention of a trade mark or proprietary product does not constitute a guarantee product by the U.S. Department of Agriculture, and does not imply its approval other products that may also be suitable.

or warranty of the to the exclusion of

17

either corn oil, coconut oil, milkfat, or cocoa butter per kg of diet. Formulations for the higher fat diets were derived from the same soy protein based diet with the fat content increased to 20% and the starch content accordingly reduced. The fats consisted of 2% corn oil plus an additional 18% of either corn oil, coconut oil, milkfat, or cocoa butter. All diets were prepared commercially to meet provided specifications. Fatty acid contents of the diets, determined by gas chromatography, are given in Table 1. Lipoprotein isolation The rabbits were exsanguinated via cardiac puncture into plastic syringes containing sufficient Na,EDTA in 0.15 M NaCl, as anticoagulant, to yield a concentration of 0.4% Na,EDTA in the whole blood. Red cells and platelets were removed by centrifugation [ 111, and the lipoproteins VLDL, LDL, and HDL were isolated from the platelet-poor plasma by ultracentrifugal flotation [ 12,131. Solvent density adjustments were made with NaCl and NaBr solutions as described [2]. Chemical analysis Cholesterol [ 143, phosphorus [ 151, and triacylglycerol [ 161 contents of the lipoprotein solutions were determined after dialysis against 0.005 M Tri-HCl buffer (pH 7.20) and extraction with isopropanol [ 121.

TABLE

1

FATTY

ACID

Acid

COMPOSITION Diet Cocoa butter

(a) 5 B far diets lo:o 12:o 14:o 16:O 16:1 18:0 18:l 18:2 18:3 20:o (b) 20 % fat diets 12:o 14:o 16:O 16:l 18:0 18:l 18:2

OF DIETS (mole %)

_ 0.43 27.39 0.39 30.92 34.31 4.94 0.35

Milkfat

Coconut

0.71 3.68 13.42 37.28 2.89 11.63 26.46 3.06 0.88

1.70 50.25 21.71 11.36

1.53 12.79 34.24 3.89

43.84 22.89 11.66 _

10.97 25.90 10.71

3.20 9.70 8.69

2.57 8.77 3.64 _

1.27

25.04 30.88 34.79 9.30

oil

Corn oil

13.59 2.20 26.58 55.45 1.61 0.57

11.29 2.33 26.24 60.15

Fluidity measurements Lipoprotein fluidity was assessed as a function of temperature from 0’ to 45°C by determining the anisotropy of fluorescence from the probe 1,6-diphenyl-1,3,5hexatriene using the methods of Shinitzky and Barenholz [ 171. Probe incorporation was accomplished by diluting 2 mM DPH in tetrahydrofuran 1000 times into the aqueous lipoprotein solutions and incubating with agitation at 35’C for 2 h. Steady state fluorescence polarization intensity was measured with an AmincoBowman spectrophotofluorometer equipped with Glan-Thompson prism polarizers. DPH was excited at 366 nm, and the fluorescence at 450 nm was detected through a Wratten 2A cut-off filter for wavelengths shorter than 415 nm. The measured anisotropies were obtained from the intensities of emission polarized parallel and perpendicular to the polarized excitation by use of standard formulae including the instrumental correction factor of Azumi and McGlynn [18] as described by Chen and Bowman [19] for polarization studies with the Aminco-Bowman spectrophotofluorometer. Light scattering errors were minimized by diluting lipoprotein solutions until the anisotropy remained constant. Scattered light intensity from unlabelled lipoprotein was less than 3% of the emitted light from DPH-labelled lipoprotein. A structural order parameter, S, for the lipid phase was calculated with the equation of Heyn [20], S2 = rm/rO, after partitioning the steady state anisotropy, rs, into dynamic, rf, and structural, r_,, components using the formulae of Van Blitterswijk et al. [9]. The value 0.362 was used for r,,, the limiting anisotropy of the probe [21]. Either the empirical approach rs = rf + r 00 and t-00= 4/3 rs - 0.1 for 0.13 < rs < 0.28 or the theoretical 2(1 + 2S)(l

treatment - S)(l -S)

rs=20(1+S)+5(1+2S)(1-S)+roS2 of Van Blitterswijk [9] was used in the appropriate rs range. Following these equations S is equal to zero for values of rs I 0.08 and corresponds to a very fluid, potentially liquid state for the lipid phase. The effects of temperature on S were determined to assess thermal influences on lipid organization and phase behavior. Statistical analysis Data were subjected to Duncan’s significant differences in anisotropies.

Multiple

Range

Test

to detect

statistically

Results Steady state fluorescence anisotropy mean values are reported in Table 2 according to diet and feeding period for all rabbit lipoprotein fractions at 37°C. Aniso-

19

TABLE

2

STEADY-STATE FLUORESCENCE ANISOTROPY VALUES, r,, FOR DPH IN THE LIPOPROTEIN FRACTIONS AT 37OC OF RABBITS FED 5% AND 20% FAT DIETS FOR 200-405 DAYS Anisotropy

values are means f SEM.

Diet/length

No. rabbits

rs VLDL

LDL

20 % fat/200 f 4 days Corn oil Coconut oil Milkfat Cocoa butter

6 6 6 6

0.096 f 0.005 a 0.097 f 0.0 11 a 0.107*0.011 = 0.084 f 0.004 ’

0.163+0.006 0.157 f0.009 0.173 f 0.009 0.149 f 0.005

20 ‘%/at/405 f 20 days Corn oil Coconut oil Milkfat Cocoa butter

3 3 3 6

0.090 f 0.009 a 0.132+0.012B 0.129~0.018’ 0.112*0.018 a

5 % fat/379 + 24 &ys Corn oil Coconut oil Milkfat Cocoa butter

3 4 3 3

0.129*0.012 0.093 f 0.008 0.134*0.027 0.150 f 0.005

ab b ab a

HDL

ab ab a b

0.197 f 0.009 0.180 f 0.006 0.194& 0.003 0.175+0.006

a ab ab b

0.144*0.009” 0.174+0.006 a 0.193 f 0.023 a 0.160+0.018a

0.170 f 0.008 0.188 + 0.007 0.204 f 0.003 0.183~0.013

a = a a

0.151+0.009 a 0.144rt0.016a 0.178 f 0.020 ’ 0.183f0.014a

0.175~o.019ab 0.162~0.011 b 0.210&0.014 = 0.209 f 0.008 a

a*b Mean values having different superscripts for a given lipoprotein fraction from animals consuming same percentage dietary fat for the same number of days are significantly different (P < 0.05).

the

tropy always increased in the sequence from VLDL to LDL to HDL, indicating more rigidly structured systems in the higher density fractions. The longer feeding period resulted in some decrease in fluidity of VLDL when rabbits were fed any 20% fat diet other than the corn oil diet. Anisotropies for the higher density fractions (LDL and HDL) changed less with time. Comparison of the present findings after 1 year on the 5% diet with data after l-6 months [2] showed that fluidity in lipoproteins from cocoa butter-fed rabbits decreased. Influence of the longer feeding period was less significant on the lipoproteins of the animals fed the other diets. It is noteworthy that these similar anisotropy data were observed either by independently examining material from each rabbit as reported here or with composite fractions isolated from pooled plasma samples of rabbits on the same diet. There were fewer rabbits (Table 2) in the longer feeding experiments, for not all of the rabbits survived for the entire period. affect lipoprotein Elevation of the dietary fat load per se did not substantially fluidity. Anisotropies from LDL and HDL are essentially the same after feeding 5 or 20% fat for approximately 1 year. Anisotropies from VLDL were lower after feeding 20% corn oil or cocoa butter, than after feeding 5% of these fats for the same interval. Results with the 20% fat diets at both intervals showed that PUFA did not

necessarily lead to more fluid lipoprotein fractions. After 200 days on diet cocoa butter feeding yielded more fluid LDL than milkfat and more fluid HDL than corn oil despite the high level of saturation in cocoa butter. There were no statistically signficant different diet effects on lipoprotein fluidities after 400 days on diet. Clearly the degree of unsaturation in the ingested fat does not necessarily control lipoprotein fluidity. Potential chemical composition effects on fluidity were assessed by linear regression analysis of the relation between DPH fluorescence anisotropy and the molar ratios of cholesterol to phosphorus or cholesterol to triglyceride in each lipoprotein class. Equations were developed in the forms: rs = m(moles

cholesterol/moles

phosphorus)

+ b

cholesterol/moles

triglyceride)

+ b

and rs = m(moles

Parameters for these equations as obtained with all the lipoprotein isolates of each class are listed in Table 3. Similar results were obtained with rs data for other temperatures as well. It is difficult to reach any consistent conclusions based on

TABLE 3 PARAMETERS FLUIDITY

FOR EQUATIONS

Diet/length

20% fat/200

days

20% fat/400

days

days

rs = m(moles cholesterol/moles 20% fat/200 days

20% fat/400

5% fat/380

days

days

THE EFFECTS

OF LIPID

COMPOSITION

Parameters

Lipoprotein

r, = m(moles cholesterol/moles

5% fat/380

DESCRIBING

m

b

Corr. coef.

0.0061 0.0174 0.0055 0.0011 - 0.0009 0.0363 0.0078 0.0043 0.0013

0.085 0.157 0.150 0.107 0.170 0.135 0.070 0.139 0.185

0.3075 0.5620 0.2379 0.0649 - 0.0217 0.5703 0.6201 0.4171 0.2567

- 0.0035 0.0122 - 0.0090 0.0204 0.007 1 -0.0107 - 0.0234 - 0.0043 -0.0155

0.098 0.155 0.193 0.103 0.157 0.199 0.138 0.167 0.196

- 0.0342 0.2446 - 0.3563 0.3249 0.2729 - 0.3643 -0.2141 - 0.0999 - 0.2261

phosphorus) + b

VLDL LDL HDL VLDL LDL HDL VLDL LDL HDL triglyceride) VLDL LDL HDL VLDL LDL HDL VLDL LDL HDL

+ b

ON

21

these equations for all lipoproteins of a particular class regardless of dietary fat. Though more significant correlations were noted in some cases when the data were analyzed separately for each diet, no consistent patterns were apparent to support the notion that cholesterol intercalation between fatty acyl chains in triglycerides or phospholipids increases DPH fluorescence anisotropy in lipoproteins. Structural order parameters, S, calculated from the anisotropy data at 37°C for the VLDL samples from each rabbit are listed in Table 4. Clearly, the most fluid lipoproteins were from rabbits fed 20% fat with cocoa butter for 200 days. VLDL from 3 cocoa butter-fed and 3 coconut oil-fed rabbits at 200 days exhibited completely unstructured or ‘liquid’ lipid phases (S = 0) at 37°C. Fewer rabbits on the other diets exhibited such liquid phases. Calculated S values for LDL and HDL did not show any liquid phases in the temperature range 0-45’C, i.e., rs always exceeded 0.08. The anisotropy data for all fractions followed exponential equations of the form rs = Aeb/T. The data consistently yielded linear graphs of In rs versus l/T, indicating monophasic behavior [ 171 in the temperature range 0-45°C. Some of the VLDL isolates did exhibit a liquid phase (S = 0) in part of this temperature range, thus suggesting that phase transitions may exist that do not necessarily induce slope changes in graphs of In rs versus l/T.

TABLE 4 STRUCTURE ORDER PARAMETER, S, VALUES VIDUAL RABBITS FED 5% OR 20% FAT DIETS

20 Q fat/405

5 % fat/379

ISOLATES

FROM

INDI-

Parameter

Diet/length

20 % /at/ZOO

AT 37’C FOR VLDL

Corn oil

Coconut

0 0.30 0.23 0.30 0.36 0.20

oil

Milkfat

Cocoa butter

0.35 0.43 0 0 0 0.44

0.18 0.37 0.54 0.16 0.25 0.35

0

0.29 0.29 0

0.35 0.50 0.5 I

0.56 0.44 0.29

0.57 0.35 0.57 0 0.26 0.10

0.48 0.33 0.52

0.32 0.35 0 0.15

0.45 0.25 0.64

0.50 0.53 0.55

f 4 days 0 0

0.30 0.15 0.18

f 20 days

+ 24 days

0.719*0.109 a 0.904+0.102 0.939 f 0.045

0.787 f 0.078 0.971 kO.034 0.996 f 0.007

0.940 f 0.038 0.992 f 0.013 1.000

VLDL LDL HDL

VLDL LDL HDL

VLDL LDL HDL

VLDL LDL HDL

Corn oil

Coconut oil

Milkfat

Cocoa butter -0.0111*0.0010 -0.0106~0.0013 - 0.0093 f 0.0011

-0.0081 f0.0016 - 0.0098 + 0.0022 - 0.0085 + 0.0008

-0.0099+0.0016 a - 0.0106 k 0.0004 - 0.0107 f 0.0007

0.742 + 0.078 0.915 kO.053 0.958 f 0.046

0.826 + 0.066 0.945 + 0.023 0.970 f 0.022

0.813 + 0.095 0.937 f 0.045 0.977 + 0.023

0.796 + 0.048 0.922 + 0.048 0.978 + 0.027

’ An ordered phase (rs 10.08) only existed for 3 of 4 animals, even to 0°C.

0.836kO.115 0.937 f0.045 0.986kO.014 -0.0108~0.0002 -0.0109+0.0002 -0.0114~0.0048

THE EQUATION

- 0.0132 + 0.0025 - 0.0105 +0.0008 - 0.0093 + 0.0006

-0.0123+0.0021 -0.0091*0.0019 - 0.0080 + O.ooO6

- 0.0135 f 0.0024 -0.0104~0.0012 -0.0094*0.0015

-0.0131*0.0007 - 0.0094 k 0.0008 -0.0082~0.0012

a

Sll

a

Lipoprotein

Diet

S0

FOLLOWING

20% fat/200 * 4 days

ORDER PARAMETERS

24 days

ON LIPOPROTEIN

5% fat/379*

EFFECT OF TEMPERATURE

TABLE 5

0.811 f0.130 0.919&0.076 0.947 kO.057

0.854 + 0.068 0.959 + 0.048 0.994 + 0.009

0.884kO.016 0.970 rf:0.009 0.987+0.012

0.716kO.045 0.865 of 0.072 0.960 f 0.0 18

S0

-0.0121 f0.0021 -0.0100+0.0018 -0.0086~0.0013

-0.0105+0.0010 -0.0083+0.0014 - 0.0082 + 0.0003

-0.0111+0.0018 - 0.0096 f 0.0008 - 0.0090 f O.COO6

- 0.0120 + 0.0009 - 0.0096 it 0.0007 - 0.0096 + 0.0011

a

20% fat/405 k 20 days

S, = S, + at IN WHICH t = “C

23

The effect of temperature on lipoprotein fluidity was assessed by linear regresion analysis of the relationship between S and temperature in the range for which S > 0. Equations of the form S, = S,, + at were developed in which S, is the order parameter at t”C and S, is the value at O’C. Mean values with standard deviations for S, and the slope, a, are listed in Table 5, by diet and time. The 0°C order parameter increased from VLDL to LDL to HDL in all groups of animals. A similar pattern with decreasing values was noted with the a values for all animals receiving 20% fat, i.e., the degree of order for VLDL was more subject to thermal effects than it was for the higher density fractions. Results with rabbits fed 5% fat after 380 days showed less consistent patterns in the a values. Discussion The effects of polyunsaturated fatty acids (PUFA) in the diet on lipoprotein fluidity have been observed by calorimetric and spectroscopic techniques. Pownall et al. [4] reported that feeding PUFA to humans increased the DPH fluorescencedetermined fluidity in all lipoprotein classes and lowered the DSC-determined transition temperature for the LDL cholesteryl ester transition between liquid crystal and isotropic liquid states. They reported that despite major changes in cholesteryl fatty acyl composition, the major determinant of LDL thermal properties was the triglyceride content. Pownall et al. [6] demonstrated a similar triglyceride level dependence for the melting temperature in swine LDL. Shepherd et al. [22] reported that feeding a diet extremely high in PUFA to 4 adult human male subjects altered lipoprotein composition and magnetic resonance probe-determined microscopic fluidity. PUFA feeding lowered the temperature of a thermotropic phase change in HDL and yielded a more fluid liquid crystalline phase at low temperature. No effects of PUFA feeding were noted, however, on HDL fluidity at 37°C. More recently, after a study with less drastic diet changes in 5 adult human females, Shepherd et al. [23] reported effects of PUFA feeding on fatty acyl composition in VLDL and LDL and on the percentage of protein in HDL. These authors assumed that the compositional differences produce corresponding changes in fluidity. Other reports [24,25] on PUFA feeding modulation of fatty acyl composition have been taken to indicate that PUFA elevates lipid core fluidity in lipoproteins; this elevation was then considered in developing mechanisms for PUFA-induced hypocholesterolemia [25]. We have shown in this work that control of lipoprotein fluidity is not strictly a function of the degree of saturation in the dietary fats. Feeding corn oil (high in PUFA) did not consistently lead to formation of more fluid lipoproteins. Feeding the highly saturated (Table 1) cocoa butter, for 200 days at the 20% level yielded more fluid LDL and HDL fractions. Safflower oil rather than corn oil was used as the source of PUFA in some of the cited studies [4,23,25]. The results of our work thus demonstrate that the effects of dietary fat are not limited to influences of dietary fatty acid saturation alone. Though only 37°C data are listed in Table 2, there were no indications of changes in relative fluidities at other temperatures in the range 0-45°C. The order parameters S, for 0°C as listed in Table 5 do not indicate a higher fluidity in HDL of PUFA-fed rabbits at low temperature, in contrast to the

24

findings of Shepherd et al. [22] with humans. Similarly, the values of a, the temperature coefficient of S, (Table 5) did not reflect a PUFA effect on HDL. Though further work is needed to determine how the fats used in this study affect lipoprotein fatty acyl groups, it is clear that dietary fatty acid composition alone cannot be used to predict lipoprotein fluidity. We used fluorescence anisotropies to describe fluidity because of the controversy [9,26,27] over the physical meaning of ‘microviscosity’ when determined from steady state fluorescence polarization rather than from nanosecond decay data. In addition, lipoprotein classes isolated from human or animal serum represent a mixture of particles that may have different physical properties due to catabolism and diet-induced heterogeneities in acyl chain composition. Van Blitterswijk et al. [9] discussed the suitability of defining membrane or lipid fluidity as the reciprocal of the DPH determined order parameter, S. We use the order parameter, S, and the steady state anisotropy, rs, to describe lipoprotein fluidity. Mtly-Goubert and Freedman [28] have expressed concerns about the partitioning of DPH between the lipid phase and the hydrophobic regions of the protein moiety in biological membranes. The high level of fluidity in all VLDL fractions and the indication of a ‘liquid’ structured VLDL with S = 0 at 37’C for a number of isolates are consistent with DPH localization in an extremely fluid lipid core rather than in the hydrophobic regions of the protein, for such a region would definitely be more ordered. The high fluidity of the VLDL most likely describes the core triglycerides in this fraction. Van Blitterswijk et al. [9] reported a value of rs = 0.083, an almost liquid state, for human milkfat globules that can be attributed to the high level of triglyceride in the apolar core of these particles. Deckelbaum et al. [5] found that the core lipids in human VLDL were liquid between 10 and 60°C. DSC work has shown phase transitions [29] in VLDL, which were usually not observed in DPH polarization measurements. Schroeder and Goh [7] using fluorescence polarization showed that previously unnoted phase transitions may exist in VLDL particles. They distinguish between their findings and results of others on experimental grounds. The S = 0 values reported here can be taken to represent liquid phases demonstrated by DPH fluorescence despite the fact that graphs of In rs versus l/T do not show any discontinuities. In the present work, no phase transitions were noted in LDL by the conventional procedure of locating slope changes in logarithmic graphs of anisotropy against the reciprocal of the absolute temperature, which contrasts with reports of phase changes observed in DSC scans [3,5]. This distinguishing feature between DPH polarization and DSC results has been noted [2,4,8] and discussed in terms of probe location. Bergeron and Scott [8] suggested that DPH partitions into the outer shell of the LDL particle or only intercalates between the alkyl chains of the core lipids rather than between steroids. Pownall et al. [4] concluded that DPH enters the lipid core but is insensitive to lipid melting because the melting is associated with cholesterol esters which occupy a relatively small fraction of the volume of LDL. Reports that an inverse relation exists between LDL melting temperature and the triglyceride content or triglyceride to cholesterol ester ratio in LDL [6,30] were taken to indicate that triglyceride and cholesteryl esters of swine LDL are in a single phase

25

t0,06=65.45-0.621

%O

I 2

I I I 4 6 8 Moles tr~acyiglycerol/mole

I 10 cblesterol

I 12 ( TG/

(“TG/“C)

I 14 C)

Fig. 1. Effect of triglyceride content, as molar ratio of triglyceride to cholesterol, temperature, t0,08, when the DPH order parameter, S, equals zero.

on the LDL transition

[6]. We propose using the concept that rS I 0.08 defines a liquid phase, thus allowing for the determination of phase transitions in lipoprotein lipids by DPH polarization that might not be recognized otherwise. Such transitions were observed with some VLDL isolates at or below 37’C. Extrapolation of LDL data yielded temperatures for which rS would be equal to 0.08. A graph of these temperatures, t,,,, as a function of triglyceride/cholesterol ratio is shown in Fig. 1. Linear regression analysis demonstrated an inverse relation, with a correlation coefficient of 0.52. We therefore postulate that the formation of a liquid phase with rS I 0.08 represents a phase transition into a less ordered state for the triglyceride portion of the LDL hydrocarbon core. These results are compatible with the view that DPH is localized between the acyl chains of the lipoprotein core triacylglycerols. In VLDL, triglycerides constitute a sufficiently large fraction of the core lipids for discernible liquid phase existence at or near body temperature, with the actual transition temperature probably under complex control of triglyceride level and fatty acyl chain spectrum. Acknowledgements The authors acknowledge and the assistance of David

the assistance of Phyllis Kliman in statistical West in experimental work with the animals.

analysis

26

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