Influence of polyunsaturated fats and fat restriction on serum lipoproteins in humans

Influence

of Polyunsaturated Fats and Fat Restriction Serum Lipoproteins in Humans Peter Weisweiler,

Peter Janetschek,

on

and Peter Schwandt

This study was designed to determine the effects of polyunsaturated fats and of reducing intake of total fat on serum lipids, lipoproteins, and apolipoproteins. Twenty-two normolipidemic women living in a nunnery were given a reference diet (fat/carbohydrate 42/46% of energy, P/S ratio 0.16). a polyunsaturated diet W/46%. P/S 1.0). and a low-fat, polyunsaturated diet (32/56%, P/S 1.0) for 6 weeks each. Serum and lipoprotein lipids were determined by standard procedures, apolipoproteins either by laser immunonephelometry or by rocket immunoelectrophoresis. Consumption of the polyunsaturated diet decreased cholesterol and apolipoprotein 6 levels in VLDL (-33.1% and -23.8%) end in LDL (-13.5% and -6.6%) without affecting HDL. Consumption of the low-fat, polyunsaturated diet resulted in a reincrease of VLDL triglycerides, but not of VLDL cholesterol. Concentration of VLDL apolipoprotein B further fell (-41.6%) and that of apolipoprotein E decreased ( -25.9%). resulting in an increased VLDL lipid/apolipoprotein mass ratio. This study indicates that responses to therapeutic polyunsaturated diet are lowered levels of VLDL and LDL, but unchanged levels of HDL. Additional restriction of dietary fat intake alters the VLDL composition with a decrement in apolipoprotein E enriched VLDL particles.

E

PIDEMIOI ,OGICAL DATA suggest that there is a causal relationship between the consumption of dietary saturated fat and the eventual development of coronary heart disease.‘” Nutrition is a major factor affecting serum cholesterol and its distribution among lipoproteins. However, the relative effects of fat saturation and changes in the fat intake in favor of carbohydrates have been debated: some have suggested that the replacement with polyunsaturated fats plays the major role,4 while others have suggested that the reduction in saturated fats is most important.s,6 Diets with different ratios of polyunsaturated to saturated fatty acids (P/S ratio) may lower not only the concentration of low density lipoproteins (LDL)7-9 that are considered atherogenic,“.” but also the concentration of high density lipoproteins (HDL)“,13 that are held to be an antiatherogenic factor.“3’4 Some nutritional studies have shown that modified fat diets lower, furthermore, the concentration of very low-density lipoproteins (VLDL),‘2v’3 in particular that of cholesterol enriched VLDL” that are considered atherogenic just as LDL.‘6*‘7 The current study was undertaken in order to assess the effects of polyunsaturated fats and changes in the relative proportion of dietary total fats and carbohydrates on the levels of serum lipids, lipoproteins, and apolipoproteins in normolipidemic women, by using therapeutic diets with defined lipid and other nutrient composition. MATERIALS

AND

METHODS

Subjects Twenty-two healthy women participated in the study. They ranged in age from 22 to 55 years (mean + SD 36 f 12) and weighed from 50.5 to 69.0 kg (60.1 5 6.0). Six women were premenopausal. The mean percent of ideal body weight was 105 + 11%. Serum cholesterol levels at the entry of the study ranged from 145 to 275 mg/dL (mean 224 + 44) and triglyceride levels from 38 to 174 Merabdism, Vol34,

No

1

(January),

1985

mg/dL (mean 83 + 35). All subjects were nuns living in a nunnery and consuming the same diet for several months. Physical activity levels were moderate and remained constant throughout the study. Informed consent was given by each subject prior to the entry into the study in accordance with the requirements of our Committee on Human Research.

Study Design Every participant consumed a reference diet followed by two modified fat diets (Table 1). Diets were prepared by a dietitian in the nunnery kitchen. All meals were eaten in the dining room under close control. Compliance was estimated by the stability of body weight, by daily personal contact between dietitian and subjects, and by 24-hour dietary recalls every week. The three diets were fed for 6 weeks each. The reference diet (P/S ratio 0.16, fat 42% and carbohydrate 46% of energy, respectively) was designed to simulate the composition of the previous diet and was made up of whole foods. Saturated fat and cholesterol came from meat and sausage, poultry, fish, milk, and cheese. Compared with the reference diet the first therapeutic diet (polyunsaturated diet) differed in fat saturation (P/S ratio 1.O). Polyunsaturated fatty acids were provided by sunflower oil and diet margarine. The second therapeutic diet (low-fat, polyunsaturated diet) differed in fat saturation (P/S ratio 1.0) and in the relative proportion of fats and carbohydrates (32% and 56% of energy, respectively). This diet included less whole milk and diet margarine, but more bread and potatoes, than did the polyunsaturated diet. All diets were planned so as to be precisely isocaloric with the estimated energy intake of the participants as assessed by four 24-hour dietary recalls prior to the study.

Laboratory

Methods

Venous blood samples were drawn with minimal hemostasis sitting position. All examinations were made in the morning

in the in the

From the Medical Department II. Grosshadern Clinic, University of Munich, Munich, FRG. Supported by the Deutsche Forschungsgemeinschaft (We 9551 1-3 and by the European Economic Community (723178 B/09.10). Address reprint requests to Priv. Doz. Dr. med. Peter Weisweiler. Medical Department II, Grosshadern Clinic, University of Munich. Marchioninistrasse IS. D-8000 Munich 70. FRG. 0 I985 by Grune & Stratton, Inc. 0026-0495/85/3401-00I5$0I.00/0 a3

a4

WEISWEILER, JANETSCHEK. AND SCHWANDT

Table 1. Nutrient

Composition

of the Reference

Diet and the Therapeutic

Reference Diet Calories, Kcal/d

Low-Fat, Polyunsaturated Diet

Polyunsaturated Diet

2000-2200

Protein, % of energy

Diets and Body Weight

2000-2200

12

2000-2200

12

12

Vegetable protein (% of protein)

35

35

35

42

42

32

Saturated (% of energy)

21.4

13.2

10.1

Monounsaturated (% of energy)

17.2

15.6

11.8

Polyunsaturated (% of energy)

3.4

13.2

10.1

P/S ratio

0.16

Fat (% of energy)

1 .o

1.0

400

400

Carbohydrates* (% of energy)

46

46

56

Dietary fiber (g/d)

21

19

25

Cholesterol (mg/d)

Body weight (kg r SD)

60.1

? 6.0

59.8

400

f 6.1

60.2

+ 6.2

*Monosaccharides and disaccharides contributed 2 1% to 25% of energy in all three diets.

fasting state after each dietary period. Specimens were centrifuged and transported immediately to the laboratory. VLDL were isolated from serum by ultracentrifugation at 1.006 g/mL at 40.000 rpm for 22 hours at 4 “C in Beckman 40.3 rotors, and the lipoprotein fractions were recovered by tube slicing.” HDL in the infranate were separated from LDL by precipitation of LDL using a commercially available combination of sodium phosphotungstate and magnesium chloride according to the described method (Boehringer-kit, Mannheim, FRG). The mass of cholesterol and triglycerides in whole serum and separated lipoproteins was determined in an automated analyzer by enzymatic techniques (Boehringer-kits. Mannheim, FRG). LDL cholesterol was calculated as the difference in the mass of cholesterol in the infranate and in HDL. Concentrations of apolipoproteins A-I, 9, and E in serum(all apolipoproteins), VLDL, and infranate (apolipoproteins B and E) were measured by laser Serum apolipoprotein A-II levels were immunonephelometry.‘9~zo determined by rocket immunoelectrophoresis.” The detergent hydroxypolyethoxydodecane (Thesit, Desitinwerke Karl Klinke, Hamburg, FRG) was added to a final concentration of 0.33 g/L in the samples and standards. Standard curves were obtained by dilution of isolated apolipoproteins and LDL and were run simultaneously with the samples. The coefficients of variation of these assays ranged from 2% to 5%.

Statistical

Analysis

Because each subject served as her own control, statistical analysis (P < 0.01) was made using the paired Wilcoxon rank test for comparisons among two diets.‘*

polyunsaturated diet resulted in a decrease of the mean serum, VLDL and LDL cholesterol concentrations by 11.8%, 26.8%, and 15.4%, respectively. Serum and VLDL triglyceride levels and HDL cholesterol levels remained statistically unchanged. However, the mean cholesterol/triglyceride mass ratio within VLDL declined from 0.24 + 0.12 to 0.19 f 0.08 during this diet. In order to study the difference between both therapeutic diets, the effects of the polyunsaturated diet were compared with those of the low-fat, polyunsaturated diet. Serum and LDL cholesterol levels were reduced in a similar extent. However, the low-fat, polyunsaturated diet had an additional hypertriglyceridemic effect (+ 17.1%). This finding was reflected in changes within VLDL: due to the increase of VLDL triglyceride levels by 41.8% the mean cholesterol/ triglyceride mass ratio declined from 0.23 c 0.15 to 0.19 k 0.08 during the low-fat, polyunsaturated diet. HDL cholesterol levels remained statistically unchanged from either therapeutic diet. Apolipoprotein Levels Apolipoprotein B changes during the polyunsaturated diet paralleled those of cholesterol, decreasing by

RESULTS Serum and Lipoprotein Lipid Levels

Table 2. Response to Therapeutic

Diets: Serum and Lipoprotein

Lipid Levels

Because the serum lipoprotein responses of individuals to diets were similar, the results from all subjects were pooled for analysis (Table 2). Compared with the reference diet consumption of the polyunsaturated diet decreased the mean serum cholesterol and triglyceride concentrations by 11.4% and 19.8%, respectively. VLDL cholesterol and triglyceride levels fell by 33.1% and 33.9%, respectively, while LDL cholesterol levels decreased by 13.5%. HDL cholesterol levels were statistically not affected by this diet. In comparison with the reference diet consumption of the low-fat,

Reference Diet

Polyunsaturated Diet

Low-Fat. Pdyunsaturated Diet

219 + 43

194 f 40’

193 + 28’

Triglycerides

80 & 24

64 + 18.

75 + 23t

VLDL cholesterol

12 + 6

8 + 3*

9 + 4.

VLDL triglycerides

50+

18

33 + 13*

47 + 13t

LDL cholesterol

156 + 43

135 + 38.

142 + 33.

HDL cholesterol

47 f 9

49 + 8

Cholesterol

Results in mg/dL are mean + SD, n = 22,

l,tP

48 * 9 < 0.01 (’ reference

diet Y therapeutic diets, tpolyunsatureted diet v low-fat, polyunsaturated diet).

85

DIETARY FAT AND SERUM LIPOPROTEINS

rated fats is one characteristic feature of a modified fat diet. The first therapeutic diet was typical of a conventional polyunsaturated diet, by using a moderately increased P/S ratio of 1.0. This diet was designed to answer the questions of how far serum, VLDL, and LDL cholesterol levels can be reduced and whether adverse effects on HDL can be observed during this diet. The second therapeutic diet takes account the recommendations23’24 to replace part of dietary fats by complex carbohydrates. This diet was, therefore, designed to point out the additive effect of changes in the relative proportion of dietary fats and carbohydrates on serum lipoproteins. The lower concentrations of both VLDL and LDL seen when the first therapeutic diet was consumed has been associated to a reduced rate of synthesis of both classes of lipoproteins.25 Another investigation found an increased fractional catabolic rate of LDL during the polyunsaturated diet.26 The simultaneous decrease of cholesterol and apolipoprotein B in VLDL and LDL confirms the data of Vega et a127that polyunsaturated fats appear to have a uniform decrease in lipoprotein constituents. The reported lowered HDL values after diets high in polyunsaturated fats7,8,‘zmay be due to unusually high P/S ratios (2.0 to 4.0). The more moderate the change in fat saturation the greater the change in LDL relative to HDL, ie, a P/S ratio above 1.5 might be crucial for the dietary effect on HDL.28 Replacing as little as 10% fats with carbohydrates during the second therapeutic diet we found a reincrease of serum and VLDL triglyceride levels. Nevertheless, values were still below the values from the reference diet. It is well known that diets low in fats and high in carbohydrates have the potential to raise VLDL secreted by the liver.29 Carbohydrate-induced VLDL that are larger in size than normal VLDL3’ are enriched in triglycerides relative to apolipoproteins.3’ Furthermore, apolipoproteins C are increased relative to apolipoproteins B and E.32 In similar fashion, we could ascertain an increase of the lipid/apolipoprotein mass ratio in VLDL during the low-fat, polyunsaturated diet. The increase in VLDL triglycerides and the decrease in VLDL apolipoprotein B can be explained by an increased VLDL triglyceride secretion rate, but

Table 3. Response to Therapeutic Diets: Apolipoprotein Levels

Reference Diet Apolipoprotein A-l Apolipoprotein A-II

137.8

2 10.3

39.5

Low-fat, Polyunsaturated Polyunsaturated Diet Diet 133.1

+ 5.8

140.1

+ 6.5

+ 2.2

38.9

k 1.7

40.1

k 1.4

+ 18.2

95.6

k 14.6’

92.3

+ 11.7’

Apolipoprotetn 0 Total

105.6

VLDL

12.1 + 3.5

lnfranate

92.9

2 13.8

9.2 + 3.8’ 85.5

k 12.6’

7.0 2 2.3’,T 85.2

+ 12.1’

Apolipoprotein E 9.3 + 0.7

Total

10.0 + 0.9

9.8 -t 0.6

VLDL

3.4 -r 0.4

3.3 + 0.2

2.1 + 0.3y

lnfranate

6.4 k 0.6

6.4 f 0.7

6.9 k 0.8

Results in mg/dL are mean + SD, n = 22, “T P < 0.01 (’ reference diet Y therapeutic diets, t polyunsaturated diet Y low-fat, polyunsaturated diet).

9.5% in serum, 23.8% in VLDL, and 8.8% in the infranate (= LDL apolipoprotein B). Mean serum apolipoprotein A-I, A-II, and E concentrations and the apolipoprotein E distribution in lipoproteins did not change significantly (Table 3). Mass ratios of lipids to apolipoproteins were statistically not altered by this diet (Table 4). In similar fashion, the low-fat, polyunsaturated diet resulted in a decrease of apolipoprotein B levels by 12.5%, 41.6%, and 8.7% in serum, VLDL, and infranate, respectively, while the mean serum concentrations of apolipoproteins A-I, A-II, and E were statistically unchanged. However, the apolipoprotein E distribution in lipoproteins changed: VLDL apolipoprotein E levels fell by 25.9%. The lipid/apolipoprotein mass ratio in VLDL increased by 43.9%. When compared to the polyunsaturated diet the low-fat, polyunsaturated diet had reduced apolipoprotein B and E contents ( - 23.9% and - 36.1%, respectively) in VLDL. In VLDL, the mean lipid/apolipoprotein mass ratio rose, therefore, by 67.4% during the low-fat, polyunsaturated diet. DISCUSSION

The purpose of the present work was to separate the effects of changes of dietary fat saturation from changes of the dietary fat intake in normolipidemic subjects. Substituting polyunsaturated fats for satu-

Table 4. Response to Therapeutic Diets: LipidIApolipoprotein Mass Ratios

Reference Diet

Polyunsaturated Diet

Low-Fat, Polyunsaturated Dtet

VLDL-cholesterol + VLDL triglycerides VLDL-apolipoprotein 8 + VLDL-apolipoprotein E

3.96

+ 0.79

3.49

+ 0.80

5.91

t

1.07’T

LDL-cholesterol LDL apolipoprotein 8

1.63 + 0.41

1.58 + 0.35

1.56 + 0.30

0.34

0.36

0.35

HDL cholesterol HDL apolipoprotein A-l

+ 0.07

+ 0.05

+ 0.03

Results are mean + SD, n = 22, +’ tP < 0.01 (*reference diet Y therapeutic diets, tpolyunsaturated diet Y low-fat, polyunsaturated diet).

86

WEISWEILER, JANETSCHEK, AND SCHWANDT

an unchanged VLDL apolipoprotein B production rate,33 furthermore by significant alterations in the pathway of VLDL apolipoprotein B degradation.34 Because no further influences of exchanging carbohydrates for fats on LDL or HDL have been observed in our study, the importance of dietary fat intake for the VLDL composition must be emphasized. In humans, high cholesterol and/or saturated fat diets cause an increase in VLDL cholesterol and VLDL apolipoprotein E.35-39Fisher et al39 reported that only the high dietary saturated fat intake was responsible for the increase of the apolipoprotein E concentration in VLDL. In contrast to these studies our data demonstrate that additional restriction in dietary fat intake resulted in opposite changes, ie, in a relative decrement in the VLDL cholesterol content and in an absolute decrement in the VLDL apolipoprotein E content. These findings confirm previous results obtained from normal male subjects on a modified fat dietI and can be associated with the reported lowered

serum apolipoprotein E levels in vegetarians on a low-fat, polyunsaturated diet.40 In animals, elevated concentrations of cholesterol and apolipoprotein E enriched VLDL are associated with an accelerated development of atherosclerosist6v4 These VLDL particles may be partially degraded (remnant) lipoproteins of intestinal and hepatic origin.42,43Whether the findings presented here substantiates the evidence for atherogenic remnants in the VLDL density range which has been eliminated by the low-fat, polyunsaturated diet, needs further investigation. Besides the effects on LDL the apparent modification of VLDL may be important in mediating possible effects of therapeutically used diets on atherogenesis. ACKNOWLEDGMENT The authors wish to thank all participants and the dietary staff for their fine assistance in carrying out this study, C. Fried1 and M. Ungar for their excellent laboratory work.

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