Effect of saturated and unsaturated fat diets on lipid profiles of plasma lipoproteins

221

Atherosclerosis, 41 (1982) 221-240 0 Elsevier/North-Holland Scientific Publishers, Ltd.

EFFECT OF SATURATED AND UNSATURATED PROFILES OF PLASMA LIPOPROTEINS

FAT DIETS ON LIPID

A. KUKSIS l, J.J. MYHER ‘, K. GEHER l, G.J.L. JONES l, J. SHEPHERD 2, C.J. PACKARD 2, J.D. MORRISETT 2, O.D. TAUNTON 2 and A.M. GOTTO 2

’ Banting and Best Department of Medical Research and Toronto-McMaster Lipid Research Clinic, University of Toronto, Toronto (Canada) and 2 Division of Atherosclerosis and Lipo protein Research, Baylor College of Medicine and Methodist Hospital, Houston, TX (U.S.A.) (Received 3 March, 1981) (Revised, received 18 May, 1981) (Accepted 16 June, 1981)

Summary Four to five healthy normolipidemic men aged 21-23 years were maintained for 5 weeks on controlled diets containing 40% of calories from either unsaturated (unsaturated/saturated fatty acid ratio 4) or saturated (unsaturated/saturated fatty acid ratio 0.25) fat, with a 5-week period of ad libitum diet in between. The effect of the diets on the total lipid profiles of the very low (VLDL), low (LDL) and high (HDL3) density lipoproteins was determined by high temperature gas-liquid chromatography of the intact fatty esters and free cholesterol. When compared with the saturated, the unsaturated fat died caused a significant decrease (25%) in the protein content in HDLJ and to a lesser extent (maximum 10%) in LDL, which were compensated for by a proportional increase in all lipid classes, resulting in essentially similar lipid class proportions on both high fat diets. Furthermore, there were no significant alterations induced by the diet in the neutral lipid/polar lipid ratios, so that the radii of the particles calculated on the basis of the surface and core component content

These studies were supported by funds from the Heart and Lung Institute, NIH-NHLI-72-917, Bethesda, MD, U.S.A., the Ontario Heart Foundation, and the Medical Research Council of Canada. Address correspondence to: Dr. A. Kuksis. Banting and Best Department of Medical Research. University of Toronto, 112 College Street, Toronto, Canada M5G lL6. * Present address: Department of Pathological Chemistry, Royal Infirmary, Glasgow G4 OSF, Scotland. Abbreviations: VLDL. very low density lipoprotein, d < 1.006 g/ml; LDL. low density lipoprotein. d = 1.019-1.063 g/ml; HDL3, high density lipoprotein. d = 1.125-1.21 g/ml; EDTA, ethylene diamine tetraacetate: LCAT, lecithin-cholesterol acyltransferase; DPH, diphenylhexatriene: PTFE, polytetrafluoroethylene.

0021-9150/82/0000-0000/$02.7501982

Elsevier/North-Holland

Scientific Publishers, Ltd.

222

gave comparable values for corresponding lipoprotein classes on both diets. There was a minor relative increase in the proportion of triacylglycerols and a decrease in the proportion of cholesteryl esters in the LDL fractions from the unsaturated fat diet. The opposite effect was observed for VLDL, while the HDL3 showed no change on these diets. The extreme dietary variations resulted in significant changes in the molecular weight or carbon number profiles of the cholesteryl esters, phosphatidylcholines and triacylglycerols. There was a decrease in the lower molecular weight species and an increase in the higher molecular weight species on the unsaturated fat diet and vita versa on the saturated fat diet for all lipoprotein classes. In comparison to the two controlled high fat diets, the ad libitum diet produced a significantly lower free cholesterol/total phospholipid ratio in VLDL and of sphingomyelin/phosphatidylcholine ratio in LDL, with the alterations in the composition of the molecular species of the lipid classes falling between the two extremes observed on the saturated and unsaturated fat diets. The present results suggest that the decreases in plasma cholesterol and triacylglycerols commonly observed on unsaturated fat diets are due to a decrease in number of plasma lipoprotein particles rather than to a change in the particle size or composition as suggested previously. -Key words:

Lipid profiles

-Plasma

lipoproteins

-Saturated

fat diet - Unsaturated

fat diet

Introduction The ingestion of unsaturated fat by man is known to increase the proportion of unsaturated fatty acids in the lipids of plasma lipoproteins [l---3] and to bring about alteration in the positional distribution of acyl groups in plasma glycerophospholipids [4,5] and triacylglycerols [ 5,6]. In addition, there is a decrease in plasma total cholesterol [7-111 and triacylglycerol [lo--121 concentration. Similar effects have been observed [13-151 in experimental animals. Morrisett et al. [3] and Shepherd et al. [ll] have shown that these dietary alterations result in profound changes in the thermotropic properties of all major plasma lipoproteins and have discussed the possible relationship between lipoprotein fluidity and metabolism. Furthermore, Stange et al. [16] have shown that the feeding of unsaturated fats to rabbits leads to alterations in the electrophoretic properties of the plasma lipoproteins and in an appearance of new apoprotein bands on polyacrylamide gel electrophoresis not found during feeding of saturated fats. Hojnacki et al. [ 171 observed that feeding of corn oil to cebus monkeys led to a larger diameter size in VLDL than coconut oil feeding. Consumption of polyunsaturated phospholipid-rich meals by chimpanzees gave changes in the fatty acid composition of VLDL, LDL and HDL3 fractions, but the fluidity of the lipoproteins measured after DPH labelling was not significantly changed [ 151. The implication from these studies is that lipoprotein structure becomes profoundly changed and that these changes may be brought about by specific alterations in the lipid class and molecular species composition of the lipoproteins accompanying the dietary manipulations. In a controlled dietary study we have reexamined the qualitative and quantitative changes in the composition of lipid classes and major molecular species

223

of plasma lipoproteins and have assessed their statistical significance. The characteristic alterations observed in the total lipid profiles have been documented as a background for assessing the potential effect of diet upon the recognition of normolipemic and hyperlipemic subjects in a free living population. Materials and Methods The subjects and the dietary regimes for this study were as previously described [3]. Briefly, 4-5 healthy males, 21-23 years old, with normal lipoprotein patterns and plasma lipid concentrations were voluntarily subjected to 3 diets of 5 weeks duration in the following order: I, highly saturated fat diet with P : S = 0.25; II, ad libitum diet; III, highly unsaturated fat diet with P : S = 4. All meals except for the ad libitum diets were prepared in the metabolic kitchen of the Methodist Hospital General Clinical Research Center and were designed to maintain body weight and provide 400 mg cholesterol daily (Table 1). After 35 days of diet therapy, 200 ml of blood was drawn from each of the 4 fasting study subjects. Immediately after collection of blood in vacuum bottles containing EDTA (1 mg/ml) red cells were sedimented by low-speed centrifugation and the supernatant plasma was subjected to high-speed ultracentrifugation [18] for isolation of the different lipoprotein classes. Each isolated class was further purified by an additional centrifugation step at the appropriate density. The purified VLDL, LDL and HDL, were dialized extensively against 150 mM NaCl, 1 mM NaN3 and 1 mM EDTA, pH 7.5, and were stored frozen until subjected to total lipid profile analyses as outlined below. HDL3 did not form detectable precipitin lines on Ouchterlony plates with antiserum to either LDL or albumin. LDL did not form detectable precipitin lines with antisera prepared against HDL. Protein concentration was determined by the method of Lowry et al. [19]. Phospholipid concentration was determined by measuring the phosphorus concentration and multiplying it by 25 [ 201.

TABLE

1

COMPOSITION

OF SATURATED

AND UNSATURATED Saturated

Protein (%l Carbohydrate (W) Fat (X) Cholesterol (mg/day) Calories Unsaturated/saturated

fatty acids

20 40 40 400 Isocaloric 1:4

diet

a

DIETS

a Unsaturated 20 40 40 400 Isocaloric 4:l

diet

a

a The saturated fat was presented in the form of dairy products (e.g. butter and processed cheese). The unsaturated fat was safflower oil. The protein was supplied in the form of chicken, fish and specially prepared lean beef. The carbohydrate was made up of cereals, potatoes and sugar. The fatty acid composition of a similar diet has been presented [31. Both diets contained 400 mg cholesterol/day and were isocaloric. The subjects on such diets remained within 3 pounds of their starting weight.

224

Determination

of lipid profiles

The total lipid profiles of the individual lipoprotein classes were determined by means of automated high temperature GLC on a Hewlett---Packard Model 5700A automated gas chromatograph equipped with dual stainless-steel columns (50 cm X 2 m I.D.) containing 3% OV-1 on 100-120 mesh Gas Chrom Q (Applied Science Laboratories, State College, PA) and an automatic liquid sample injector, as previously described [ 21,221. For this purpose a neutral lipid extract was prepared as follows. An aliquot of the lipoprotein fractions equivalent to about 0.2 ml of plasma was added to a PTFE lined screw-cap centrifuge tube containing 0.2-0.4 mg phospholipase C in 4 ml of 17.5 mM Tris buffer, pH 7.3, along with 1.3 ml of 1% CaCl, and 1 ml of diethyl ether, and the mixture incubated with shaking for 2 h at 30°C. The reaction mixture was then treated with 5 drops of 0.1 N HCI and extracted once by vigorous shaking with 10 ml of chloroform--methanol 2 : 1 containing 150-250 pg tridecanoylglycerol as internal standard. The solvent phases were immediately separated by centrifugation for 10 min at 200 X g. The clear chloroform phase was removed from the bottom of the tube and was dried by passing through a Pasteur pipet containing 2 g of anhydrous sodium sulfate. The efluent was evaporated under nitrogen and the residue dissolved in TRISIL-BSA (150-250 ~1) and transferred to a sampling vial and the vial sealed. One 1_t1 of the solution was automatically injected onto the gas-chromatographic column without the benefit of a flash evaporator and the temperature programmed from 175”C-350°C at S”C/min. The various peaks were identified using appropriate retention time windows and the peak areas were calculated using appropriate calibration factors. Calibration

and statistical analyses

Due to a partial overlap of the higher molecular weight diacylglycerols, ceramides and the lower molecular weight cholesteryl esters in the C&--C& carbon number range the content of the phosphatidylcholine and sphingomyelin of the lipoprotein fractions was calculated from the peak areas of carbon numbers C&-&s, and of sphingomyelin from the peak areas of carbon number C&, as previously described [22]. Total PC = total DG X 1.28, where total DG = corrected (Cs6 + C&)/0.81, and corrected C36 + C& = (C& + C,,) -k& ceramide; and where C& ceramide = C&,- 0.051 X C&. The total sphingomyelin is determined as follows: Total sphingomyelin = total ceramide X 1.28, where total ceramide = Cs4 ceramide X 0.758/0.30. The factor 0.758 is the ratio of the response for ceramide and diacylglycerol TMS ether (0.758 = 0.681/ 0.898). The factors 0.81 and 0.30 represent the measured diacylglycerol and ceramide species of plasma phosphatidylcholine and sphingomyelin as derived from analyses of large plasma pools. The multiplication factor 1.28 converts the ceramides and diacylglycerols into the corresponding phosphorylcholine derivatives. Significance testing of differences between means was made using established statistical procedures. The error term for these comparisons was obtained by considering the experiment (for each component) as a nested factorial, with the

225

main effects being diets and lipoproteins, the interaction term being diets X lipoproteins, and the nested effect being subjects within diets (removing the subject to subject variability factor). The remaining effect was considered to be random error, and was used for the Duncan multiple range comparisons among means [ 231. In general, the diet X lipoprotein interactions were significant and led to comparisons of individual diet-lipoprotein means. In certain instances where the interaction terms were not significant, pooled means were compared. The large magnitude of differences between lipoproteins for the lipid classes resulted in non-homogeneity of variances, and the variances being proportional to the means. The analyses of variance for these components were computed on the logarithm of the values in order to eliminate this effect and equalize variances. Results Total lipid profiles

of VLDL

Figure 1 shows the total lipid profiles of VLDL of 2 subjects following 5 weeks on either saturated or unsaturated fat regimen. These chromatograms were selected to illustrate the minimum and maximum differences observed in the patterns in a total of 5 normolipemic subjects studied. The quantitative results obtained for the lipid class composition with all the subjects are given in Table 2 along with those obtained for the same subjects on a free choice diet. The protein/lipid ratios and the relative composition of the major lipid classes are in general similar to those reported in the literature on free choice diets [24] or on diets rich in saturated or unsaturated [25] fats. In order to obtain a more direct comparison of the composition of the VLDL among the 3 diets, the lipid class content has been expressed as mg/lOO mg protein. On this basis no significant differences were seen among the diets in total cholesterol, free cholesterol, cholesteryl ester, or sphingomyelin content. Furthermore, there was no significant difference in the total lipid, phosphatidylcholine or total phospholipid content of the VLDL between saturated and unsaturated diets, while VLDL from free choice diets possessed a significantly higher content of total lipid, phosphatidylcholine and total phospholipid. The VLDL from the 3 different diets differed significantly in the content of total triacylglycerols, that from the free choice diet containing the most and that from the unsaturated diet the least of this neutral lipid class. When expressed as per cent total lipid the subjects on the unsaturated fat diet contained significantly less triacylglycerols and significantly more cholesteryl esters than the subjects on either free choice or saturated fat diets. The subjects on free choice diet also contained significantly less free cholesterol than those on the saturated or unsaturated fat diets. The molar ratios of FC/PL are significantly lower on the free-choice diet, than on the other fat diets, which did not differ between them in this respect. There were no significant differences seen in the FC/TC or SPH/PC ratios among the diets. The FC/TC ratios’(O.42-0.45) found for VLDL in the present study are somewhat higher than those (0.37) calculated from the data reviewed by Skipski [26]. The FC/PL ratios (0.53-0.67) are within the range (0.62-0.67) cal-

226

a 52

52

I 27

45

L I

363a /I

5

v

3

!!!lt&& i

%n

4

18 20

54

2

d

k/J

2%

355”

27

Fig. 1. GLC profiles of VLDL lipids from subjects on saturated and unsaturated fat diets. (a) subject 1 on saturated fat; (b) subject 1 on unsaturated fat; (c) subject 2 on saturated fat; (d) subject 2 on unsaturated fat. Peaks 16 and 18. TMS esters of free fatty acids with 16 and 18 acyl carbons; peaks 22-24, di-TMS ethers of monoacylglycerols with 16 and 18 acyl carbons; peak 27, TMS ether of cholesterol; peak 30. tridecanoylglycerol internal standard; peak 34, TMS ether of pahnitoylsphingosine; peaks 3642. TMS ethers of diacylglycerols of a total number of 3440 acyl carbons; peaks 43-47, cholesteryl esters of fatty acids with a total number of 16-20 acyl carbons: peaks 48-54, triacylglycerols with a total number of 48-54 acyl carbons. Sample size 1 ~1 of an approximately 1% solution in silylation mixture. Attenuation 100 times full sensitivity. Temperature program as shown. Other GLC conditions as given in text.

221

TABLE

2

COMPOSITION

Chemical

OF

VERY

LOW

DENSITY

component

LIPOPROTEINS

a

Diets Ad

libitum

(n = 4) Ouerall

(VLDL)

Saturated

Unsaturated

(n = 5)

(n = 4)

(wt.%)

composition

Total

protein

12.0

f

1.2

(G)b

13.6

r

1.7

(F)

15.0

+

1.8

(F)

Total

lipid

88.0

k

1.2

(A)

86.4

f

1.7

(A)

85.0

+

1.8

(A)

f 18.5

(A)

107.0

f 16.0

(B)

106.4

+

7.8

(B)

f

1.7

(D)

f

(D)

16.2

+

2.5

(D)

f 57.4

(A)

374.6

t 55.7

(B)

317.8

92.5

f

2.9

(C)

83.2

+ 16.6

(C)

40.5

f

2.9

(B)

41.8

f

(A)

623.7

Lipid

content

(mg/lOO

mg protein)

Lecithins

138.9

Sphingomyelins

14.8

Triacylglycerols Cholesteryl Free

470.0

esters

cholesterol

Total

756.7

Total

phospholipid

Total

cholesterol

Lipid

composition

(% total

+ 88.8

17.1

5.0

+ 62.5

(C)

96.3

+

6.7

(C)

(B)

41.3

?;

4.6

(B)

f 97.9

(B)

577.9

f 79.4

(B)

8.0

153.7

+ 18.6

(A)

124.1

f: 20.8

(B)

122.6

f

9.8

(B)

96.0

+ 15.6

(D)

91.6

f 17.1

(D)

98.8

c

7.9

(D)

lipid)

Phospholipid

20.3

f

0.5

(E)

19.9

f

0.0

(E)

21.2

f

1.6

(E)

Triacylglycerols

62.1

f

2.3

(A)

60.1

+

1.0

(A)

55.0

f

3.6

(B)

Cholesteryl

12.2

f

2.9

(E)

13.3

c

1.3

(E)

16.6

+

2.2

(D)

5.4

f

0.5

(C)

6.7

+

0.5

(D)

7.1

f

0.5

(D)

FC/TC

0.43

f

0.06

(A)

0.45

+

0.03

(A)

0.42

c

0.03

(A)

FC /PL

0.53

f

0.04

(E)

0.67

+

0.03

(D)

0.67

+

0.06

(D)

SPH /PC

0.11

f

0.02

(B)

0.17

+

0.02

(C)

0.16

+

0.02

(C)

Free Lipid

a Mean

esters

cholesterol

class

ratios

(moles/moles)

f SD.

b Identical

letters

on horizontal

lines

indicate

that

the values

are not

significantly

different.

culated from the data given by Packard et al. [25] while the SPH/PC ratios (0.11-0.17) were not unlike those (0.21) calculated from the data of Skipski et al. [27]. Table 3 gives the relative distribution of the carbon numbers of the cholesteryl esters, phosphatidylcholines and triacylglycerols of VLDL as obtained on the 3 different diets. Statistically significant changes are seen in the relative content of CQ3cholesteryl ester, the contribution of which decreased on the unsaturated diet compared to the saturated and free choice diet. There was a corresponding significant increase in the proportion of the C& cholesteryl esters. These changes represent essentially the relative decrease of palmitic and an increase in linoleic acid esters of cholesterol on the polyunsaturated fat diet. There was relatively little change in the carbon number distribution of the phosphatidylcholines of the VLDL. There was, however, a small but statistically significant increase in the proportion of C& species on the polyunsaturated diet presumably due to a relative increase in the Cl8 fatty acids in the diet. In comparison to the saturated diet, the unsaturated diet caused a significant decrease in the proportion of the C5,, and a marked increase in the proportion of the Cs4 components. The alterations in the carbon numbers of the triacylglycerols following consumption of the free choice diet were intermediate between those observed on the saturated and the unsaturated diets.

228

TABLE

3

CARBON

NUMBERS

Chemical

a OF

LIPID

CLASSES

component

OF

VERY

LOW

libitum

(n = 4) esters

LIPOPROTEINS

(VLDL)

b

Diets Ad

~holesteryl

DENSITY

(% lipid

Saturated

Unsaturated

(n=

(n = 4)

5)

class)

43

22.3

f 0.0

(A)

22.4

f 0.8

(A)

17.4

f 0.6

(C)

45

77.7

_+ 0.5

(C)

77.6

f 1.8

(C!)

82.6

f 1.7

(A)

47

N.D.

=

Lecithins

N.D.

N.D.

(% lipid class)

36

35.3

f 1.9

(B)

37.0

f 1.9

(B)

34.5

f 3.4

(B)

38

40.5

+ 1.7

(D)

39.6

C 0.9

(D)

43.3

f 1.3

(B)

24.3

f 2.5

(A)

23.4

+ 1.5

(A)

22.3

+ 2.5

(A)

48

3.9

f 0.2

(A)

7.2

i- 1.0

(B)

3.6

f 0.2

(A)

50

17.2

+ 1.7

(D)

k 2.9

(C)

11.8

f 0.9

(E)

40

Triglycerides

(‘36lipid class) 23.2

52

56.4

f 2.0

(A)

52.1

+ 2.3

(A)

45.4

+ 2.0

(E)

54

22.5

+ 1.1

(D)

17.5

? 2.7

(D)

39.2

f 2.9

(A)

a Carbon b Means C N.D.,

numbers

as explained

in Fig.

1.

+ SD. not

determined;

Total lipid profiles

but

may

contribute

up to 10%

of total

cholesteryi

ester.

of LDL

Figure 2 shows the total profiles of LDL of the same 2 subjects following 5 weeks on saturated and unsaturated fat diets. The quantitative results obtained with all the subjects for the lipid class composition are given in Table 4. Again the protein/lipid ratios and the relative composition of the lipid classes are similar to those reported for the LDL of normal subjects on free choice diets [24,26,27]. For the purpose of more detailed comparison, the present data is expressed as mg/lOO mg protein. In comparison to the saturated, the unsaturated diet contained significantly more sphingomyelin and free cholesterol in LDL. On the free choice diet the sphingomyelin content was the lowest. There was a statistically significant lower content of triacylglycerols in the LDL from the saturated fat diet, when compared to the unsaturated fat and ad libitum diets. Likewise, the cholesteryl esters were higher on the unsaturated diet than on the other 2 diets, which did not differ between them in this respect. The total cholesterol levels were significantly lower on the ad libitum, but did not differ between the saturated and the unsaturated fat diet. The 3 diets did not differ significantly in content of total phospholipid or of phosphatidylcholine. The FC/TC ratios of LDL (0.28--0.29) which did not differ among the diets were closely similar to those (0.24) calculated from the data reported by Skipski et al. [26]. Likewise, the FC/PL ratios (0.87+.92), which also did not differ among the diets were identical to those (0.85) calculated from the data given by Skipski et al. [ 261. The LDL from the saturated and unsaturated fat diet had a slightly higher SPH/PC ratio than that of the LDL from the free choice diet. The SPH/PC ratios for LDL found in this study (0.384.47) are

229

27

d

45

1

i 1 27

27

2 Fig. 2. GLC profiles of LDL lipids from subjecp on saturated and unsaturated fat diets. (a) subject 1 on saturated fat: (b) subject 1 on unsaturated fat; (c) subject 2 on saturated fat; (d) subject 2 on unsaturated fat. Other legends as in Fig. 1.

closely similar to those (0.39 and 0.38, respectively) found by Skipski et al. [ 261 and Phillips [ 281 on free choice diets. Table 5 gives the relative distribution of the carbon numbers of the cholesteryl esters, phosphatidylcholines and triacylglycerols of LDL as obtained on 3 different diets. The relative proportion of the C43 species of cholesteryl esters is significantly higher on the saturated diet than on the unsaturated diet, with

230 TABLE

4

COMPOSITION

Chemical

Overall

OF

LOW

DENSITY

component

LIPOPROTEINS

(LDL)

a

Diets

(weight

composition

As libitum

Saturated

Unsaturated

(n = 4)

(n=

(n = 4)

5)

o/o)

Total

protein

21.1

_+

1.0

(D)

19.2

f

0.7

(D)

18.5

f

0.6

(D)

Total

lipid

78.9

f

0.9

(C)

80.8

+

0.7

(C)

81.5

+

0.6

(C)

Lecithins

80.0

+

3.2

(C)

80.8

f

4.7

(C)

76.2

+

3.3

(C)

Sphingomyelins

28.4

f

2.2

(C)

35.6

+

3.6

(B)

42.2

+

4.5

(A)

Triacylglycerols

36.6

f

3.7

(D)

21.4

f

3.0

(E)

40.5

_+

6.2

(D)

190.9

+

8.1

(B)

219.0

+ 12.3

(A)

217.7

_+ 13.8

(A)

47.1

f

1.3

(B)

50.1

f

1.1

(B)

_+

(A)

383.0

f 13.3

(C)

407.6

c 20.1

(C)

432.1

_+ 16.1

(C)

108.5

+

4.2

(D)

122.4

f

6.7

(D)

116.0

+

2.2

(D)

161.2

r

6.5

(C)

184.6

?-

8.1

(B)

186.0

f 10.3

(B)

28.3

f

0.6

(C)

28.0

+

0.6

(C)

27.8

9.6

f

1.0

(C)

5.3

2

0.6

(D)

49.8

f

1.4

(B)

54.1

_+ 0.7

(A)

12.3

_+ 0.0

(A)

12.5

+

0.6

(A)

Lipid content

Cholesteryl Free

(mg/lOO

mg protein)

esters

cholesterol

Total Total

phospholipid

Total

cholesterol

Lipid composition

(%

total

Triacylglycerols Free

esters

cholesterol

Lipid class ratios

2.6

lipid)

Phospholipid Cholesteryl

55.7

+

1.0

(C)

+

1.7

(C)

50.1

f

2.2

(B)

12.7

_+ 0.0

(A)

9.3

(moles/moles)

FC/TC

0.29 f 0.00 (B)

0.28

+

0.01

(B)

0.29

_+ 0.01

(B)

FCjPL

0.87

+

0.03

(A)

0.89

+

0.04

(A)

0.92

f

0.03

(A)

SPHIPC

0.38

f

0.03

(B)

0.42

i

0.06

(A)

0.47

k

0.06

(A)

OF

LOW

DENSITY

LIPOPROTEINS

a See legends

TABLE

to Table

2.

5

CARBON

Chemical

NUMBERS

OF.LIPID

component

CLASSES

libitum

(n = 4) esters

a

Diets Ad

Cholesteryl

(LDL)

(W lipid

Saturated

Unsaturated

(n = 5)

(n = 4)

class)

43

19.0

_+ 1.2

(C)

21.2

f 2.6

(A)

16.5

f 1.3

(D)

45

71.5

+ 1.9

(C)

70.2

+ 0.9

(C)

74.0

_+ 1.2

(A)

9.5

+ 1.9

(A)

8.0

f 2.8

(B)

9.5

f 0.6

(A)

47 Lecithins

(% lipid

class)

36

44.0

_+ 1.8

(A)

48.4

_+ 1.5

(C)

43.0

k 2.9

(A)

38

42.3

f 0.5

(B)

41.6

+ 1.9

(B)

45.8

c 1.7

(A)

13.8

f 1.7

(B)

14.0

f 2.7

(B)

11.3

+ 1.3

(C)

40

Triglycerides

(76 lipid

class)

50

19.5

f 1.0

(D)

31.4

_+ 3.5

(A)

18.8

+ 1.0

(D)

52

51.8

+ 1.0

(C)

51.0

+ 2.0

(C)

46.8

? 3.6

(E)

54

28.8

f 1.3

(B)

17.6

f 2.3

(E)

32.0

f 4.7

(B)

a Legends

as in Table

3.

231

the free choice diet showing an intermediate value. The relative proportion of the C& species is significantly higher on the unsaturated diet, with the saturated and free choice diets showing no difference. The saturated diet gave the lowest relative CJ7 cholesteryl ester content. There was also a significant relative increase in the proportion of the C& species of phosphatidylcholine on the unsaturated fat diet, with the saturated and free choice diets showing no significant differences. Statistically significant also was the small relative increase in the C& component on the saturated fat diet. Surprisingly the CQOspecies was significantly lower on the unsaturated fat diet than on the other diets. There was an increase in the relative proportion of the C+, triacylglycerols on the unsaturated fat diet, and a relative increase in the C5,, species on the saturated diet. These alterations in the relative proportions of the carbon numbers of the corresponding lipid classes presumably reflect changes in the relative proportions of the Cl6 and Cl8 fatty acids in the corresponding diets as already claimed in the literature [3] on the basis of fatty acid analyses. The dietinduced changes in the composition of the triacylglycerols are greater than those in the cholesteryl esters of in the phosphatidylcholines. Morrisett et al. [ 31 had observed a 28% increase in linoleic acid of the triacylglycerols of LDL in subjects on the unsaturated fat diet. However, these changes in the LDL lipids are relatively smaller than those in the corresponding lipid classes of the VLDL. Total lipid profiles of HDL Figure 3 shows the total lipid profiles of HDL3 of the same 2 subjects following 5 weeks on saturated and 5 weeks on unsaturated fat fiets. The quantitative results obtained from these chromatograms and from similar ones recorded for the other subjects for the various lipid classes are given in Table 6. The overall lipid/protein ratios from the HDL3 fraction are in the general range reported in the literature for total HDL and its subfractions [11,26,27]. There are significant differences in the lipid/protein ratios among the 3 diets with the unsaturated diet containing the least and the saturated the most protein in their HDL3. The overall GLC estimates of the various lipid classes in the HDLJ fraction are in close agreement with the values reported for total HDL and for HDL3 in the literature [ 26,271 based on conventional analyses. In general the lipid class profiles of HDL3 from all diets are similar to each other. However, the unsaturated diet contains a significantly higher proportion of phosphatidylcholine, total phospholipid, cholesteryl ester, triacylglycerol, total lipid and total cholesterol than the other 2 diets, which do not differ significantly between them in these respects. When compared as percent total lipid, the lipoproteins from the various diets contained the same relative amounts of the neutral and polar lipid classes. There were no significant differences in the relative contents of the other lipid classes among these diets. The FC/TC ratio in the HDL3 fraction (0.19) corresponded closely to the value (0.16) calculated from the data of Skipski et al. [27] for HDL3 of fasting females on free choice diet. The FC/PL ratios of 0.21-0.24 also corresponded closely to that (0.23) calculated from the data of Skipski et al. [ 271, as did the SPH/PC ratios (0.09-0.13) when compared to that (0.12) calculated from the data of Skipski et al. [ 271.

232

I

a

4$ci

27

36

Fig, 3. GLC profiles of HDL3 lipids from subjects on saturated and unsaturated fat diets. (a) subject 1 on saturated fat; (b) subject 1 on unsaturated fat; (c) subject 2 on saturated fat; (d) subject 2 on unsaturated fat. Other legends as in Fig. 1.

Table 7 gives the carbon number composition of the cholesteryl esters, phosphatidylcholines and triacylglycerols of the HDLJ fraction from the same 4 subjects on the 3 different diets. There are statistically significant differences among the relative proportions of the cholesteryl esters from the 3 diets. The HDL3 from the unsaturated diet contained less of the Cg3 and more of the Cq5 species than that from the saturated diet, with the HDL3 from the free choice

233

TABLE

6

COMPOSITION

Chemical

OF

HIGH

DENSITY

component

LIPOPROTEINS

(HDL)s

a

Diets Ad

libitum

(n = 4)

Overall composition

(weight

Saturated

Unsaturated

(n=

(n = 4)

5)

%)

Total

protein

46.3

+

3.2

(B)

50.0

+ 2.1

(A)

37.3

i

2.4

(C)

Total

lipid

53.8

f

3.2

(F)

50.0

f 2.1

(G)

62.8

?;

2.4

(E)

41.3

(C)

Lipid content (mg/lOO

mg protein) f

5.9

(E)

+ 3.5

(E)

69.9

t

6.9

Sphingomyelins

55.8 4.7

+

1.3

(E)

4.7

it 0.8

(E)

8.5

t

2.9

(E)

Triacylglycerols

8.5

f

2.1

(D)

7.0

+ 0.8

(D)

14.8

f

2.4

(E)

f

6.4

(E)

+ 3.6

(E)

66.5

t

6.6

(D)

c

1.3

(C)

c 0.8

(C)

+

1.7

Lecithins

Cholesteryl Free

esters

43.5

cholesterol

6.5 119.1

Total

38.8 5.2

9.5

+ 14.2

(D)

97.0

f 8.8

(D)

169.1

(C)

? 17.0

(E)

Total

phospholipid

60.6

+

7.1

(F)

46.0

f 4.2

(G)

78.3

f

9.1

(E)

Total

cholesterol

32.2

f

4.9

(F)

28.6

f 2.6

(F)

49.5

f

6.0

(E)

46.4

Lipid composition

(76 total

lipid)

Phospholipid Triacylglycerols Cholesteryl Free

50.9

f

1.6

(A)

47.4

f 0.0

(A)

7.1

f

1.9

(C)

7.2

f 0.5

(C)

8.7

f 1.0

(C)

39.3

+ 0.7

(D)

5.6

36.5

esters

5.5

cholesterol

C

1.4

(C)

+

0.6

(D)

5.4

40.0

f

1.0

(A)

+

1.5

(C)

f

0.0

(C)

+

0.6

(D)

Lipid class ratios (moles/moles) FCITC

0.19

+* 0.01

(C)

0.19

+ 0.02

(C)

0.18

f

0.01

(C)

FC/PL

0.21

f

0.02

(F)

0.23

+ 0.02

(F)

0.24

f

0.02

(F)

SPH/PC

0.09

_+ 0.02

(E)

0.12

f 0.01

(E)

0.13

f

0.04

(E)

a See legends

TABLE CARBON

Chemical

to Table

2.

7 NUMBERS

OF

LIPID

CLASSES

component

OF

HIGH

DENSITY

LIPOPROTEINS

(HDL3)

a

Diets Ad

libitum

(n = 4)

Saturated

Unsaturated

(n=

(n = 4)

5)

Cholesteryl esters (‘3 lipid class) 43

18.3

f 0.5

(C)

19.6

f 0.6

(C)

16.0

+ 1.0

45

70.5

c 1.0

(C)

69.6

f 0.6

(C)

74.0

f 0.0

(D) (A)

47

11.3

+ 1.0

(A)

10.8

f 0.5

(A)

10.8

+ 1.0

(A)

36

35.8

k 1.7

(B)

35.8

+ 2.5

(B)

33.0

+ 2.9

(B)

38

41.3

+ 0.5

(D)

40.8

k 1.1

(D)

43.0

t 1.4

(B)

40

23.0

f 1.4

(A)

23.4

f 2.4

(A)

24.0

+ 2.2

(A)

50

22.5

+ 2.4

(C)

27.2

f 0.8

(B)

16.5

f 2.7

(D)

52

56.0

+ 5.5

(B)

53.4

f 3.8

(B)

48.0

+ 2.9

(D)

54

20.8

+ 4.2

(D)

20.8

c 0.5

(D)

35.5

5 5.5

(B)

Lecithins (Q lipid class)

Triglycerides (7%lipid class)

a See legends

to Table

2 and

Fig.

1.

234

diet not differing significantly from that of the saturated diet. There were no differences among the Ce7 proportions on the 3 diets. On the unsaturated diet the HDL3 triacylglycerols had a significantly lower proportion of CSOand CS2 and a higher proportion of CS4 species than on the saturated diet, which differed from the free choice diet only in a higher proportion of C,, triacylglycerol component. There were no statistically significant differences in the relative proportions of the carbon numbers of the phosphatidylcholines of HDL3 from the 3 diets. The shifts in carbon number profiles of the cholesteryl esters and triacylglycerols are consistent with the 18% increase in the 18 : 2 content of HDL3 observed on this diet on the basis of fatty acid analyses [3]. The above noted changes in the carbon number profiles are much greater than those expected from the report of Gordon et al. [5], who noted only a 2% increase in while the VLDL and LDL triacylthe 18 : 2 content of HDL triacylglycerols, glycerols increased 4 and 6%, respectively, when a subject was fed unsaturated fat. The carbon numbers of the triacylglycerols, however, cannot be related directly to the carbon numbers of the fatty acids because the molecular association of different chain length acids must also be taken into consideration. Relative distribution

of molecular species of lipids among plasma lipoproteins

An evaluation of the differences in the relative content of the major carbon numbers of cholesteryl esters, phosphatidylcholines and triacylglycerols among the different lipoprotein classes isolated on each diet showed statistically significant differences. Thus on the saturated diet, the LDL contained significantly more C& and less C4, species than the HDL3, but the differences in the relative content of C& species of the lipoproteins were not significant on any of the diets. On all diets there were significantly higher relative proportions of the CS6 and lower relative proportion of the C 40 species of phosphatidylcholine in the LDL than in VLDL or HDL, which did not differ between them in the relative content of these species. On the unsaturated diet the relative proportion of Cd0 species was markedly lower in LDL than in VLDL or HDL, which again did not differ in the relative content of these species. On the saturated diet, the relative proportion of the C&, species was significantly lower in LDL than in HDL, or in VLDL, which did not differ significantly from HDL. Likewise, there were differences in relative proportions of the major triacylglycerol species in the different classes of lipoproteins within each diet. Thus, on the saturated fat diet there was a relative increase in the CSOspecies in the LDL, when compared to HDL and especially to VLDL, which also differed from each other in the relative proportions of this species. The corresponding decrease in the C+, species in these lipoproteins on the saturated diet spas more marked in LDL than in the VLDL or HDL, which did not differ significantly. On the unsaturated fat diet the relative CSOtriacylglycerol content was significantly higher and the CS4 content significantly lower in the LDL than in VLDL or HDL. Furthermore, significant differences in the molecular species composition were seen among the lipoproteins when the dietary differences were ignored (data pooled from all diets; interaction between lipoproteins and diets not significant). Thus, LDL still contained relatively more of the C43 and less C4, spe-

235

ties than VLDL or HDL. Likewise, LDL contained relatively more of the Cj6 and C& species but much less C,, species of phosphatidylcholines than VLDL or HDL. Of the triacylglycerol species, VLDL contained significantly less CsO and the LDL significantly less Cs2 species than the corresponding other lipoprotein classes, no significant differences being seen in the relative proportions of the C+, species among these lipoprotein classes. A significant diet X lipoprotein interaction was present for CsOand C 54, and general conclusions about triacylglycerol differences among the lipoproteins cannot be made without considering the two factors together. Calculation

of particle size

Assuming that all the lipoprotein particles have a spherical shape and contain the non-polar lipids (cholesteryl esters and triacylglycerols) in the core and the polar lipids (phosphatidylcholine, sphingomyelin and free cholesterol) in the surface monolayer, it is possible to calculate the diameter of the particles from the lipid composition and the knowledge of the thickness of the lipid-protein monolayer [29,30]. Assuming a volume of 1600 A3 for an average molecule of triacylglycerol and of 1090 A for an average molecule of cholesteryl ester, along with a cross-sectional area of 71 A” for an average molecule of phosphatidylcholine and of 41 A.’ for free cholesterol, it is possible to calculate the radius of the neutral lipid core by dividing the volume of a sphere by the surface area and multiplying the result by 3. The total radius of each particle is then obtained by adding the thickness of the surface monolayer previously demonstrated to be about 21 A [31]. Table 8 gives the radii (A) of the average particles for the VLDL, LDL, and HDL3 fractions from the free choice, and the saturated and unsaturated fat diets. It can be seen that in all instances the calculated radii are close to the range of radii obtained for these particles by means of independent physico-chemical measurements [26,32]. These findings are similar to those of Shen et al. [29] who demonstrated that the measured particle size was consistent with the lipid content and composition data. The

TABLE

8

CALCULATED

AVERAGE

RADII

(A)

OF

LIPOPROTEIN

PARTICLES

ASSUMING

LIPID

CORE

STRUCTURE

Lipoprotein

classes

Lit.

a

Diets Ad

Saturated

Unsaturated

(n = 4)

libitum

(n=

(n = 4)

5)

150-350

192.4

183.0

168.7

LDL

85-130

101.7

101.3

101.5

HDL3

20-

65.1

71.8

73.6

VLDL

r = K + 3(4/3m3)/(4nr2)

[(mole

= 3

[(mole where

VTG

representing

a Skipski

and

VCE

are

the thickness [ZSI.

70

1600

% TG) (VTG) ______ % PL)

and

(APL)

1 090A3

of the phospholipid-protein

+ (mole

+ (mole and

ApL

% CE)

% CHOL) and

monolayer

(VCE)]

(ACHOL)I

AcHDL

828 71

of each

and

particle

41

!I’,

1301.

and

K = 21

a

236

present results therefore support the hypothesis that the structure of circulating normal lipoproteins may be represented by a neutral lipid core which is surrounded by a polar lipid-protein monolayer, with a possible exception of HDL3 [22]. In any event there would appear to have been no change in the particle size of these lipoproteins as a result of the dietary alterations. Discussion Effect of diet on component class ratios It has been shown elsewhere [3,11] that unsaturated fat diets comparable to that employed in the present study produce significant reductions in total plasma cholesterol and triacylglycerol levels and also reduce the plasma levels of cholesterol associated with VLDL, LDL and HDL. In case of HDL, it was shown that the unsaturated fat diet induced a relative decrease in the protein content apparently resulting from a decreased synthesis and/or secretion of apo A-l peptide [ll]. The present work confirms the large decrease in the HDL protein/lipid ratio claimed by Shepherd et al. [ll], but indicates no significant change in the protein/lipid ratio in LDL or in VLDL. The decrease in the HDL3 protein observed in our study is about twice that noted by Shepherd et al. [ll] for total HDL. Shepherd et al. [ll] found a fall in HDL2/HDL3 ratio, but the absolute concentration of HDLl was very small even on the saturated diet. The present decrease (25%) in HDL3 protein content, however, corresponds closely to the decrease (25%) in the total plasma apo A-l concentration when compared to that obtained on the saturated fat diet. The total phospholipid/protein ratio on the unsaturated fat diet for HDL3 (0.79) was identical to that (0.79) found by Shepherd et al. [ll] for total,HDL. On the other hand, the phospholipid/protein ratio for HDL3 (0.50) on the saturated fat diet was somewhat lower than that (0.64) observed by Shepherd et al. [ll] for total HDL. There were no significant changes in the relative proportions of any of the lipid classes between the HDL3 fractions obtained from subjects on saturated fat and free choice diets. There were no significant differences in the lipid class ratios of the VLDL fraction between the saturated and unsaturated fat diets, while those in the LDL were significant. These findings extend to the ratios of the polar (free cholesterol and total phospholipid) and non-polar (cholesteryl esters and triacylglycerols) lipids in these lipoproteins and suggest that the particle radii did not significantly change on the different diets, and this conclusion was confirmed by calculation. In such a case the decrease in the plasma total cholesterol and total lipid content [3,11] must have been due largely to decreases in the total number of the VLDL, LDL and HDL particles on the unsaturated, when compared to the saturated fat diet. It is pertinent and instructive to note that some of the above described changes in human subjects have been previously demonstrated in chimpanzees [15] and cebus monkeys [17]. However, the VLDL from corn oil-fed cebus monkeys [17] had significantly larger particle size than VLDL from control animals receiving coconut oil. This difference was reflected in a correspondingly altered surface and core lipid ratio. In chimpanzees [15] feeding of polyunsaturated lecithin decreased the proportion of the protein moiety in VLDL

237

and HDL, and to a lesser extent in LDL, with a corresponding increase in the content of cholesteryl esters and lysophosphatidylcholines. The latter increase was apparently due to the excessive intake of lysophosphatidylcholine from the lumen. Similarities were also found in the SPH/PC ratios in the VLDL (0.1) and HDL (0.1) but not in the SPH/PC ratio in the LDL (0.2) which was only about one half that seen in the LDL of man (0.38-0.47). Effect

of diet on molecular

species composition

Morrisett et al. [3] and Shepherd et al. [ll] have shown that the fatty acid composition of plasma VLDL, LDL and HDL was directly correlated with the saturation level of the ingested fat. According to Morrisett et al. [3] the most dramatic change took place in the triacylglycerols of LDL and the least in HDL, with the VLDL registering an intermediate change in its fatty acid composition. The major alteration in all instances was the substitution of 18 : 2 for 16 : 0 acids. There were smaller decreases in the 18 : 0 and 18 : 1 content with a reciprocal increase in 18 : 2 proportions. In keeping with these observations there were, in the present study, marked decreases in those triacylglycerols expected to contain palmitic acid (C,,) in the LDL, but VLDL and HDL from the subjects on the unsaturated fat diet also showed a decrease with a corresponding increase in the triacylglycerols (C,,) expected to contain the bulk of the linoleic acid. This finding contrasts that of Morrisett et al. [3], who noted only a limited change (5%) taking place in the fatty acids of the HDL triacylglycerols. However, Shepherd et al. [ll] did find a change in 18 : 2 content of HDL comparable to that indicated by the change in the carbon numbers of the triacylglycerols. Detailed chemical and stereochemical analyses of the triacylglycerols of plasma lipoproteins have demonstrated that distinct differences may occur in the composition of the molecular species of triacylglycerols among the lipoproteins [ 61. In the cholesteryl esters the changes were confined essentially to a decrease in the C& (cholesteryl palmitate) and an increase in the C& (cholesteryl oleate + cholesteryl linoleate) components and appeared to affect equally the cholesteryl esters of the VLDL, LDL and HDL fractions. Previously, Morrisett et al. [3] had observed that the fatty acid differences were most obvious in the cholesteryl esters of the VLDL. Much of this change involved a substitution of 18 : 2 for 18 : 1 fatty acid, which could not be observed directly in this study because of an overlap of these two species of cholesteryl esters on GLC. In the present study the least change was observed in the molecular species (carbon number distribution) of the diacylglycerophospholipids (essentially phosphatidylcholine). The change was limited to a statistically significant increase in the proportion of the C 38 component, which represents largely molecular species consisting of two C 18 fatty acid esters. The anticipated decrease in the species rich in 16 : 0, however, was not statistically significant. All of the lipoprotein classes appeared to be equally affected. We have shown elsewhere [33] that marked changes take place in the composition of the molecular species of the phosphatidylcholines when an unsaturated diet is substituted for a saturated diet, but that the differences among the various lipoproteins on any one diet are not very significant. Closely similar carbon number

238

distributions for all lipoproteins were also noted for the subject on the free choice diets. Previous analyses of fatty acids of the phospholipids had shown the greatest change in the VLDL [3]. However, the fatty acid changes in the phospholipids did not parallel those in the triacylglycerols or in the cholesteryl esters. The latter point is surprising since much of the plasma cholesteryl ester is derived by LCAT action on the plasma phosphatidylcholine [ 341. The nature of fatty acid pairings responsible for the resistance to the change in the carbon number distribution of the phosphatidylcholines is reported elsewhere [33] along with the complete account of the molecular species of individual plasma lipoproteins. Physico-chemical

and metabolic

consequences

One result of these compositional alterations is the change in the thermotropic properties of the lipoproteins. VLDL and LDL from the unsaturated fat diet gave plots, the line slopes of which were considerably elevated, indicating that the particles from this diet suffered a greater increase in fluidity with increasing temperature than those isolated from the saturated fat diet [3]. Similar changes were observed in the HDL behaviour in the study of Shepherd et al. [ll], although Morrisett et al. [3] had not been able to show a clear-cut difference between saturated and unsaturated fat diets for HDL. Another effect is the relative loss of protein from the HDL and to a lesser extent from the LDL particles. It may be postulated that this decrease in the relative proportion of protein in these lipoproteins is due to a shortage of apopeptides in the plasma resulting from an inhibition of protein synthesis and/or secretion by the liver and/or intestine on the unsaturated fat diet. Shepherd et al. [ll] have already demonstrated that the synthesis and/or secretion of apoprotein A-l is reduced as a result of feeding unsaturated fat. A similar inhibition of synthesis may take place for other apoproteins and may lead not only to a decreased apoprotein/lipid ratio in the lipoproteins but also to a decreased level of the various plasma lipoproteins, which would then account for the cholesterol- and lipid-lowering effect of an unsaturated fat diet. Mutto and Gibson [35] have shown that dietary polyunsaturated fatty acids exert a significant inhibitory effect on various lipogenic enzymes of the liver which conceivably could reflect an inhibition of protein synthesis as well as result in decreasing plasma lipoprotein formation and secretion. The possibility that increased fluidity of cell membranes resulting from a substitution of linoleic acid for palmitic and oleic acids in membrane phospholipids may lead to a significant inhibition of certain synthetic and transport activities is supported by the observations that Mg’+ ATPase activity of the hepatocyte and lymphoblast plasma membrane is inversely related to membrane fluidity [36]. There are no reports in the literature on the effect of saturated and unsaturated fat feeding on protein synthesis, but a general decrease in total liver and plasma proteins has been claimed for rats on high corn oil diet

r141. Furthermore, the changes in the ratios of FC/TC, FC/PL and CE/TG have been correlated with the fluidity, permeability and overall stability of natural and artificial membranes and lipoprotein particles and their relative activity as

239

supports for certain enzymic activities including LCAT and lipoprotein lipase [ 371. Deckelbaum et al. [38] have shown that the core fluidity of a lipoprotein can affect its surface coat properties and therefore probably its metabolism. At the present time the optimum ratios of the various surface and core components are not known and conclusions about their relative significance cannot be made. It must be pointed out that the diets in this study and in the studies of Morrisett et al. [3] and Shepherd et al. [ll] are examples of extreme dietary variation not usually encountered in nutritional or clinical practice. The demonstration of a dietary suppression of selective plasma lipoprotein classes by the unsaturated fat suggests that other dietary regimens may be found that might bring about a selective increase in certain plasma lipoprotein levels (HDL) which would be of nutritional interest because of a potential therapeutic benefit [39]. References 1 Bragdon. J.H. and Karmen, A., Effect of ingested fat on fatty acid composition of serum lipoproteins. J. Lipid Res., 2 (1961) 400. 2 Kayden. H.J. Karmen, A. and Dumont, A., Alterations in the fatty acid composition of human lymph and serum lipoproteins by single feedings, J. Clin. Invest., 42 (1963) 1373. 3 Morris&t, J.D.. Pownall, H.J., Jackson, R.L., Segura. R., Gotto. Jr., A.M. and Taunton, O.D., Effects of polyunsaturated and saturated fatsdiets on the chemical composition and thennotropic properties of human plasma lipoproteins. In: R.T. Holamn and W.H. Kunau (Eds.), Polyunsaturated Fatty Acids (American Oil Chemists’ Society, Monograph No. 4). A.O.C.S. Publishers, Champaign, IL, 1977. pp. 139-162. 4 Spritz, N. and Mishkel. M.A., Effects of dietary fats on plasma lipids and lipoproteins - An hypothesis for the lipid-lowering effect of unsaturated fatty acids, J. Clin. Invest., 48 (1969) 78. 5 Gordon, D.T., Pitas, R.E. and Jensen, R.G.. Effects of diet and type Ha hyperlipoproteinemia upon structure of triacylglycerols and phosphatidylcholines from human plasma lipoproteins, Lipids, 10 (1975) 270. 6 Parijs, J., De Weerdt, G.A.. Beke, R. and Barbier, F.. Stereospecific distribution of fatty acids in human plasma triglycerides, Clin. Chim. Acta, 66 (1976) 43. 7 Beveridge. J.M.R., Connell. W.F. and Mayer, G.A., The nature of the substances in dietary fat affecting the level of plasma cholesterol in humans. Can. J. Biochem.. 35 (1957) 257. 8 Keys, A., Anderson, J.T. and Grande. F., Serum cholesterol response to dietary fat, Lancet, i (1957) 787. 9 Spritz, N.. Grundy, S.M. and Ahrens. Jr., E.H., Sterol balance in man as plasma cholesterol concentrations are altered by exchanges of dietary fat. J. Clin. Invest., 44 (1965) 1482. 10 Grundy. S.M., Effects of polyunsaturated fats on lipid metabolism in patients with hypertriglyceridemia, J. Clin. Invest., 55 (1975) 269. 11 Shepherd, J., Packard, C.J., Patsch, J.R., Gotto, Jr.. A.M. and Taunton, D.O., Effects of dietary polyunsaturated and saturated fat on the properties of high density lipoproteins and the metabolism of apolipoprotein A-l, J. Clin. Invest., 61 (1978) 1582. 12 Engelberg. H., Mechanisms involved ln the reduction of serum triglycerides in man upon adding unsaturated fats to the normal diet, Metabolism, 15 (1966) 796. 13 Wigand, G., Production of hypercholesterolemia and atherosclerosis in rabbits by feeding different diets without supplementary cholesterol, Acta Med. &and. 166 (Suppl. 351) (1960) 1. 14 Narayan. K.A.. McMullen, J.J., Butler, D.P.. Wakefield, T. and Calhoun, W.K., The influence of a high level of dietary corn oil on rat serum and liver lipids, Nutr. Rep. Intern., 10 (1974) 25. 15 Rosseneu. M.. Declercs, B.. Vandamme, D.. Vercaemst, R.. Soetewey, F., Peeters, H. and Blaton, V., Influence of oral polyunsaturated and saturated phospholipid treatment on the lipid composition and fatty acid profile of chimpanzee lipoproteins. Atherosclerosis, 32 (1979) 141. 16 Stange. E., Augustini, B. and Papenberg, J., Changes in rabbit lipoprotein properties by dietary cholesterol and polyunsaturated fat, Atherosclerosis, 22 (1975) 125. 17 Hojnacki, J.L., Nicolosi. R.J., Hoover, G., Llansa. N.. el Lazy. M. and Hayes, K.C., Effects of dietary fat on the composition and size of primate very low density lipoproteins, Artery, 3 (1977) 409. 18 Havel, R.J.. Eder, H.A. and Bragdon, J.H.. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest.. 34 (1955) 1345.

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Lowry, O.H.. Rosebrough, N.J.. Farr, A.L. and Randall, R.J., Protein measurements with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 20 Bartlett, G.R., Phosphorus assay in column chromatography, J. Biol. Chem., 234 (1959) 466. 21 Kuksis. A., Myher, J.J., Geher, K.. Hoffman, A.G.D., Breckenridge. W.C., Jones, G.J.L. and Little, J.A., Comparative determination of plasma cholesterol and triacylglycerol levels by automated gasliquid chromatographic and Autoanalyzer methods, J. Chromatogr. Biomed. Applic., 146 (1978) 393. 22 Kuksis. A., Myher. J.J., Geher, K., Breckenridge, W.C.. Jones, G.J.L. and Little, J.A., Lipid class and molecular species interrelationships among plasma lipoproteins of normolipemic subjects, J. Chromatogr. Biomed. Applic.. 224 (1981) 1. 23 Steel, R.G.D. and Torrie, J.H.. Principles and Procedures of Statistics, McGraw-Hill, New York, pp. 107-109. 24 Granada, J.L. and Scanu, A., Solubilization and properties of the apoproteins of the very low- and low-density lipoproteins of human serum. Biochemistry, 5 (1966) 3301. 25 Packard, C.J., Shepherd, J., Joems, S., Gotto, Jr., A.M. and Taunton, O.D.. Very low density and low density lipoprotein subfractions in type III and type IV hyperlipoproteinemia. Biochim. Biophys. Acta. 572 (1979) 269. 26 Skipski, V.P., Lipid composition of lipoproteins in normal and diseases states. In: G.J. Nelson (Ed.), Blood Lipids and Lipoproteins, Wiley-Interscience, New York, NY, 1972, pp. 471-583. 27 Skipski, V.P., Barclay. M., Barclay. R.K., Fetzer, V.A., Good, J.J. and Archibald, F.M.. Lipid composition of human serum lipoproteins, Biochem. J., 104 (1967) 340. 28 Phillips, G.B., The phospholipid composition of human serum lipoprotein fractions separated by ultracentrifugation, J. Clin. Invest.. 38 (1959) 489. 29 Shen, B.W.. Scanu, A.M. and Kezdy, F.J., Structure of human serum lipoproteins inferred from compositional analysis, Proc. Nat. Acad. Sci. (USA), 74 (1977) 837. 30 Kuksis. A., Breckenridge, W.C.. Myher, J.J. and Kakis. G.. Replacement of endogenous phospholipids in rat plasma lipoproteins during intravenous infusion of an artificial lipid emulsion, Can. J. Biochem., 56 (1978) 630. 31 Sata, T., Havel, R.J. and Jones, A.L., Characterization of subfractions of triglyceride-rich lipoproteins separated by gel chromatography from blood plasma of normolipemic and hyperlipemic humans, J. Lipid Res., 13 (1972) 757. 32 Scanu, A.M., Structural studies on serum lipoproteins, Biochim. Biophys. Acta. 265 (1972) 471. 33 Myher, J.J., Kuksis, A., Shepherd, J., Packard, C.J., Morris&t, J.D., Taunton, O.D. and Gotto, A.M., Effect of saturated and unsaturated fat diets on the molecular species composition of phosphatidylcholines and sphingomyelins of human plasma lipoproteins, Biochim. Biophys. Acta, In press. ‘. 34 35 36 37 38 39

Glomset, J.A.. The plasma lecithin : cholesterol acyltransferase reaction, J. Lipid Res., 9 (1968) 155. Muto, Y. and Gibson, D.M., Selective dampening of lipogenic enzymes of liver by exogenous PolYuosaturated fatty acids, Biochem. Biophys. Res. Commun., 38 (1970) 9. Riordan, J.R., Plasma membrane MG* ATPase activity is inversely related to lipid fluidity. In: M. Katers and A. Kuksis (Eds.), Membrane Fluidity, Humana Press, Clifton, NJ, 1980, PP. 119-129. Casu, A., The ratio glycerophospholipids/free cholesterol of human serum -A possible Parameter of asymmetric fluidity of lipoprotein molecules, Ital. J. Biochem.. 28 (1979) 26. Deckelbaum, R.J., Shipley, G.G., Tall, A.R. and Small, D.M., In: H. Peeters (Ed.). Protides of the Biological Fluids, Pergamon Press, New York, NY. 1977, PP. 91-98. Heiss, G. and Tyroler, H., Epidemiology of h&b density lipoprotein - A review. In: K. Lippel (Ed.), Report of the High Density Lipoprotein Methodology Workshop, NIH Publication NO. 79-1661. 1979. pp. 7-27.