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Effect of polyunsaturated isocaloric fat diets on plasma lipids, apolipoproteins and fatty acids
9
Atherosclerosis, 53 (1984) 9-20 Elsevier Scientific Publishers Ireland, Ltd
ATH 03513
Effect of Polyunsaturated Isocaloric Fat Diets on Plasma Lipids, Apolipoproteins and Fatty Acids V. Blaton, Department
M. De Buyzere, B. Declercq, A. Pracetyo J. Delanghe and J. Spincemaille
*, G. Vanderkelen,
of Clinical Chemistry, A. Z. Sint -Jan, Ruddershooe IO, B - 8000 Bruges (Belgium) (Received 15 April, 1983) (Revised, received 5 January, 22 March, 1984) (Accepted 24 March, 1984)
Summary The effect of an increased polyunsaturated fatty acid concentration in the diet on the plasma lipoproteins from a normal group of healthy persons and from a group of hypercholesterolemic patients, consuming an isoenergetic and an isocholesterolemic diet, was examined and the changes in the plasma phospholipids were measured. Nine normal and 10 hypercholesterolemic patients were treated with a polyunsaturated diet for 1 month. Controls and hypercholesterolemic patients were screened on their lipid and lipoprotein profiles and their P/S ratio in the diet was calculated and increased with a factor 4. In the control group the P/S ratio was increased from 0.35 to 1.38 and in the hypercholesterolemic group from 0.46 to 1.59. They received the diet for at least 4 weeks before a second analysis of lipids and lipoproteins. The most important results are a decrease of plasma cholesterol, followed by a significant increase of HDL cholesterol. The cholesterol-lowering effect results largely from the plasma LDL decrease, especially in the patient group. Apo A-I is decreased accompanied by a significant increase of the ratio HDL-C/ape A-I. The observed changes are most pronounced in the hypercholesterolemic group. There is no change in apo B but a significant change in the linoleic acid concentration especially in the HDL cholesterol esters. The major phospholipids in plasma are
* Present address: Department of Internal Medicine, University of Brawijaya, Malang, Indonesia. Abbreuiations: HDL-C, high density lipoprotein cholesterol: LDL-C, low density lipoprotein cholesterol; TC, total plasma cholesterol; TG, plasma triglycerides; PC, phosphatidylcholine; Sphm, sphingomyelin; HDL-CE, high density lipoprotein cholesterol esters; HDL-PL, high density lipoprotein phospholipids: Plasma-CE, plasma cholesterolesters; Apo A-I, apolipoprotein A-I; apo B, apolipiprotein B; P/S, ratio of polyunsaturated to saturated fatty acids. 0021-9150/84/$03.00
0 1984 Elsevier Scientific Publishers Ireland, Ltd.
10
identical in both groups and there is an identical sphingomyelin is increased and phosphatidylcholine related to an increase of the HDL,/HDL, ratio. Key words:
change under the PUFA diet, is decreased, which may be
Fatty acids - Lipid profiles - Plasma apolipoproteins teins - Unsaturated fat diet
- Plasma
lipopro-
Introduction The ingestion of unsaturated fatty acids by man increases the proportion of unsaturated fatty acids in the lipids of plasma lipoproteins [l-3] and changes the positional distribution of acyl groups in plasma glycerophospholipids and triacylglycerols [4,5]. Although diets rich in polyunsaturated fatty acids lower the serum cholesterol [6,7], the mechanisms involved, however, have remained obscure. Spritz and Mishkel [8] proposed that plasma cholesterol is decreased because lipid esters containing polyunsaturated fatty acids require more space in lipoproteins and exclude cholesterol, but this was not confirmed by Vega et al. [9], who suggest that the major action of polyunsaturates is to alter the production or clearance of the plasma lipoproteins and not to change the relative proportions of their lipid and apolipoprotein components. Morrisett et al. [l] and Shepherd et al. [lo] have further shown that fatty acid alterations result in changes in the thermotropic and physical properties of all major plasma lipoproteins and have discussed the possible relationship between structural changes and metabolism. Similar findings were observed in chimpanzees (pantroglodytes), induced with diet type II hyperlipoproteinemia and treated with saturated and unsaturated phosphatidylcholine [11,12]. Consumption of polyunsaturated phospholipid-rich meals by chimpanzees gave changes in the fatty acid composition of VLDL, LDL and HDL, fractions, but the fluidity of the lipoproteins measured after diphenylhexatriene labeling was not significantly changed [12]. Polyunsaturated fats affected HDL, HDL-C and HDL apo A-I [2,10,13]. The implication from these studies is that the lipoprotein structure is profoundly changed by alterations in the lipid, apoprotein and fatty acid composition accompanying the dietary manipulations. As contradictory results are described on the effects of polyunsaturated fatty acids, we studied the influence of an increase in the P/S ratio under an isoenergetic and isocholesterolemic diet in a normal group of healthy persons and in a hypercholesterolemic group. Materials and Methods Nine normal persons (mean age 42 years, range 32-54 years) of both sexes (7 males, 2 females) took part in this study. Their initial serum cholesterol value was less than 240 mg/lOO ml. The hypercholesterolemic patients (n = 10; 8 males, 2 females) were screened according to clinical and biochemical parameters. The selected patients had hyperbe-
11
talipoproteinemia with a cholesterol value higher than 260 mg/lOO ml. Of the cohort members 30% showed clinical symptoms of hypertension, 56% had cardiovascular complications and 14% had peripheral atherosclerosis. Diabetics and subjects with familial hyperlipemia, overt obesity, intake of alcohol above 30 g/day, or nicotine consumption above 10 cigarettes/day were excluded. No drugs known to influence the serum lipid composition nor beta-blockers were administered The study was conducted over an g-week period. The subjects maintained their usual diet for the first 4 weeks of the study, meanwhile lipids and lipoproteins were followed and a food examination was done by a diabetician over 2 weeks at home. The average percentage composition of the diet of the control group and of the patients is given in Table 1. The main differences are higher kJ and % fat intake in the patient group. Under isocaloric and isocholesterolemic conditions unsaturated fats were increased in their diets by changing saturated through unsaturated diet components and through a supplement of unsaturated oil (corn oil, one spoon/day). The ratio of unsaturated to saturated fat (P/S) was 4 times increased individually ranging between 0.30 and 1.40 in the control group and between 0.40 and 1.60 in the patient group (Table 1). All subjects were 4 weeks on the unsaturated fat diet and were visited and controlled weekly by a dietitian. Blood samples in EDTA were taken after an overnight fast. Plasma total cholesterol (TC) were measured by the CHODCOD method [14], plasma triglycerides (TG) by the enzymatic glycerokinase method [15] and plasma phospholipids by the metavanadate method [16]. Sodium phosphotungstate magnesium chloride precipitation of LDL and VLDL [17] was performed and HDL-C assayed by the enzymatic CHOD-COD method on the HDL supernatant [18]. The apolipoproteins Apo A-I and Apo B were measured by nephelometric immunochemical methods [19,20]. For the quantitation of apo B a LDL,-fraction (1.03 < d < 1.05 g/dl) was used as standard. For apo A-I quantitation, the primary standard consisted of pure lyophylized apo A-I dissolved in 0.3 M guanidine chloride. For assay purpose, a plasma sample was standardized using the lyophylized apoprotein and was subsequently used as secondary standard, kept in sodium azide (0.1 g/l) at 4OC. TABLE
1
ISOCALORIC AND ISOCHOLESTEROLEMIC DIET COMPOSITION AND A HYPERCHOLESTEROLEMIC PATIENT GROUP
Daily energy intake (kJ) Protein (W) Fat (%) Carbohydrate (W) Alcohol (a) Cholesterol (mg/d) P/S B
OF A CONTROL
GROUP
Control group (n = 9)
Patient group (n = 10)
Basal
PUFA diet
Basal
PUFA diet
9907 16 36 40 8 288 0.35
9844 17 35 40 8 242 1.38
10550 16 41 40 3 261 0.46
10203 16 40 41 3 203 1.59
a Abbreviations: P = polyunsaturated fatty acids, S = saturated fatty acids.
12
Serum lipids and phospholipid subclasses were separated by thin-layer chromatography after extraction of 1 ml serum with 24 ml of chloroform/methanol (2 : 1, v/v) and 10 ml of 2 g/l calcium chloride solution [21]. After centrifugation at 1200 x g, the chloroform layer was removed and evaporated at 40 ‘C under nitrogen. The residue was redissolved in 0.2 ml chloroform and applied to a silicagel plate, prepared as follows: silicagel (60HR Merck) extra pure was spread as a slurry (50 g/125 ml water) with a 0.30-mm spreader on to glass plates, pre-cleaned and dried. The plates were activated just before use, by heating for 30 min at 110 ‘C. To separate serum apolar lipids we used petroleum ether/diethylether/acetic acid developed at room temperature for 45 min. (80 : 20 : 1, v/v/v) The phospholipids were separated into subclasses with chloroform/methanol/ acetic acid/water (50 : 125 : 8 : 2, v/v/v/v). The phospholipid subfractions were determined after silica gel extraction by the molybdate method [22]. The determinations of the Sphm/PC ratio were performed by the TLC method using HClO, (100 g/l) as the charring reagent. The fatty acid composition of the plasma lipids and of the plasma lipoproteins were analyzed based on an integrated procedure [23]. The lipid extracts were saponified with 3% potassium hydroxide in methanol. After acidification with 6% H,S04 the fatty acids were extracted with petroleum ether. The dried extract was esterified with BF3-CH,OH (250 mg/l) by heating for 30 min at 80 ‘C in a sealed tube. The separation was performed on a Varian gas chromatograph equipped with a 6-ft column filled with 10% diethylene-glycol succinated on Gaschrom @ and a flame ionization detector. The temperature was programmed from 120 to 210°C at 2’C/min after an isothermal period of 5 min. Statistical analysis was carried out by both one- and two-way analysis of variance. Results Plasma lipid and apoprotein changes after the PUFA diet The efficiency of the polyunsaturated fat diet in lowering plasma cholesterol and LDL-C especially in hypercholesterolemic patients is shown in Table 2 (P -C 0.01). The polyunsaturated fat produced significant reductions in the patient group in total plasma cholesterol and in the plasma level of cholesterol associated with LDL. The changes in the HDL composition are different in both groups. HDL-C is only significantly increased in the patient group and stays rather constant in the control group which does not corroborate the observation of other workers [2,20]. In the normal control group, however, there is a significant increase of plasma phospholipids (48%), mainly associated with HDL phospholipids and accompanied by a decrease of the C/PL ratio. The parallel increase of HCL-C and HDL-PL in the patients suggests an increase in the total HDL concentration. The polyunsaturated fat diet decreases slightly the apo A-I in patients as well as in controls, and those observations are in agreement with literature data for normolipemic subjects WOI. Polyunsaturated molecular species
fat ingestion, however, did cause a significant change in the of the phospholipid subclasses, especially phosphatidylcholine,
13
TABLE 2 PLASMA LIPID AND APOLIPOPROTEIN BEFORE AND AFTER PUFA DIET
LEVELS IN A CONTROL
AND A PATIENT
GROUP
Values are mean f SD, lipid and apoprotein levels are mg/dl. Patient group (n = 10)
Total cholesterol Triglycerides Phospholipids LDL cholesterol a HDL cholesterol HDL phospholipids Apo A-I Apo B HDL cholesterol/Ape A-I LDL cholesterol/Ape B Apo A-I/APO B
Control group (n = 9)
Basal
After PUFA diet
Basal
After PUFA diet
323 k33 144 +56 312 +51 259 k45 35 fl3 96 +39 117 +16 153 *15 0.30* 0.09 1.78& 0.51 0.78* 0.13
280 *37* 130 +42 309 +25 208 +38* 46 f12* 122 &29 106 +ll 153 * 14 0.43* 0.08 1.36* 0.13 * 0.71+ 0.16
210 *19 94 +37 211 k42 141 +20 52 +9 97 522 126 i 25 105 k23 ** 0.42+ 0.06 1.40* 0.35 1.2s* 0.44
200 +33 99 +33 261 k32* 131 +34 49 *8 144 *39 116 ,512 106 f25 0.46k 0.13 1.24+ 0.27 1.14+ 0.29
* P < 0.01; ** Statistical analysis control against patient group: P < 0.01. a Friedewald [35].
which are responsible for the metabolic transformations of the lipoproteins [24]. These data indicate that the capacity of an increased cholesterol uptake by HDL for the patient group is dependent on the PUFA diet and related to an increased activity of LCAT suggesting also an increased ratio of HDL,/HDL,. Apo B which is significantly higher in the patient group, in unchanged under PUFA diet in both groups, although total cholesterol and LDL-C are decreased and most pronounced in the patient group, which corroborates the data of Durrington et al. [25], but the normal group of the latter study was not on an isocholesterolemic diet. The present data are in agreement with the study of Mishkel [8] who observed no change in the protein concentration. The main effect of the unsaturated fat diet on LDL is observed in the patient group where we obtained a significant decrease of the ratio LDL-C/ape B, although in the control group a similar trend was observed. At a high P/S ratio of 4 Kuksis et al. [2] observed a 10% decrease of the LDL protein content, which was compensated by a proportional increase in all lipid classes. As we observed an unchanged apo B concentration under the described diet change, the decreased LDL-C must be related to an increased exchange of unsaturated CE or to an increased catabolic rate. The significant decrease of LDL-C by PUFA diets is an important factor reducing the atherogenic effect of the plasma lipoproteins. The decrease of the LDL-C/ape B can be related to the interconversion of LDL subclasses due to a higher removal rate of IDL-C by the HDL particle. Although main changes are observed in HDL and LDL the potential atherogenic index apo A-I/ape B is rather constant in both groups during PUFA diet treatment.
14
Changes
of the plasma
unsaturated
lipoproteins
and plasma
phospholipid
subclasses
after
the
as demonstrated
by
diet
Although
the lipoprotein
subclasses
are significantly
changed
the described data, we do not observe marked qualitative lipoprotein alteration by the standardized lipoprotein electrophoretic technique [26]. The hypercholesterolemit group has significantly
higher LDL
lipoproteins
accompanied
by lower HDL
values. Based
on
unsaturated decreased.
the diet
described
technique,
in both
groups,
Based on the described
ual phospholipid
subclasses
Phosphatidylcholine
high
similar
changes
TLC procedure,
of both groups before
and sphingomyelin,
are
observed
as well as low density we analyzed
further
and after PUFA
3). The major changes
the individ-
the major plasma phospholipids
in both groups,
and a decreased
phosphatidylcholine
concentration,
by the Sph/PC
ratio. The increased
sphingomyelin
are identi-
polyunsaturated
are an increased
sphingomyelin
which is significantly concentration
the are
diet treatment.
cal in both groups and there is a similar change under the increased diet (Table
under
lipoproteins
expressed
may be related to
an increase of HDL,, which contains more sphingomyelin than HDL, [27]. The minor phospholipid classes, lysophosphatidylcholine and phosphatidylethanolamine, identical
in both groups are unchanged
found no change in Sph/PC activity
under PUFA
concentration obtained
LDL
diet can be responsible
accompanied
with non-human
by an increased
under the high P/S ratio. Kuksis
ratio in VLDL, by an increased primates,
and HDL,.
for a decrease sphingomyelin
where a decreased
lysophosphatidylcholine
et al. [2]
The increased
LCAT
of phosphatidylcholine value. Similar data were
PC value was accompanied
[ 121.
Changes
of the fatty acid composition of plasma, of plasma
a PUFA
diet
CE and of HDL-CE
under
A comparison of the fatty acid patterns in plasma, in plasma cholesterol esters and in the cholesterol esters of HDL lipoproteins is given in Table 4 for the control group and in Table 5 for the patient group, before as well as after the PUFA diet
TABLE 3 PERCENTAGE (%) PHOSPHOLIPID PATIENT GROUP OH-PC a
Control
AFTER
PUFA
DIET OF A CONTROL
AND
A
Phospholipids Sphm a
PC”
PEa
Sphm/PC
6.1 f 1.3 6.5 f 1.6
19.2kl.l 22.4~ 2.9
68.9 f 2.0 65.8 f 1.9
5.8 + 0.8 5.3*0.9
0.28 f 0.02 0.34 f 0.05 *
6.1 f 0.8 5.9*1.5
19.3 * 0.9 22.5 f 1.8
68.5 f 0.9 65.2 f 2.8
6.0 f 0.8 6.3 f 0.9
0.29 k 0.02 0.35 + 0.04 *
ratio
group (n = 6)
Before PUFA diet After PUFA diet Patient
CHANGES
group (n = 4)
Before PUFA diet After PUFA diet
’ Abbreviations: OH-PC phosphatidylethanolamine. * P -e 0.01.
= 2-hydroxyphosphatidylcholine,
Sphm
= sphingomyelin,
PC =
HDL-CE
SUBFRACTIONS
0.7 + 0.3
0.6 + 0.4
0.9 f 0.3
0.4*0.1
1.5*0.7
8.3 + 1.6
c2o:o
C20:3
C20:4
7.3k1.4
0.8 k 0.5
55.5 * 4.7
34.0 * 3.1
C18:3
17.1 f 1.6
C18:2
1.8
1.2+0.4
20.1+
7.9 f 0.8
C18:O
3.9k1.2
11.5 + 1.4
0.6 f 0.3
C18:l
3.25 1.0
21.5k2.1
C16:O
C16:l
0.7 f0.2
c15:o
0.8 + 0.4
1.3
6.6+1.6
1.7kO.5
0.3 * 0.1
0.8 + 0.3
30.7 5 3.1
17.9+1.4
9.6+ 1.3
2.9k
25.2 + 3.1
0.9 + 0.5
l.lkO.5
7.9*
1.3
1.2kO.8
0.5 * 0.2
0.7kO.3
53.3*4.4
16.7k1.3
1.7+0.3
5.3 f 1.4
11.4+1.3
0.5 +0.3
0.8 k 0.4
8.1 k 1.0
1.7*0.5
0.5 + 0.3
1.1 f0.5
36.6 f 4.7
19.6k1.7
7.2 f 0.5
3.6k1.2
20.4 + 2.3
0.4 + 0.2
0.9 + 0.4
diet
OF A CONTROL
Plasma
HDL
PLASMA
Plasma
Plasma-CE
AND
After PUFA
group (n = 9)
OF PLASMA
Basal
Control
COMPOSITION
1.5+0.7
ACID
FATTY
c14:o
4
TABLE
1.0
7.5kl.l
0.9 * 0.5
0.6 k 0.2
0.5 + 0.2
57.0 k 4.6
15.8+1.5
l.OkO.4
3.8 * 1.4
11.7*
0.6*0.3
0.8 t 0.3
Plasma-CE
GROUP
BEFORE
AFTER
9.2 f 1.4
2.1 f 1.0
0.3fO.l
0.7 * 0.3
32.9 + 3.1
18.1 k 2.2
9.5kl.O
2.6fl.O
23.1 f 1.7
0.5 * 0.3
0.8 f 0.4
HDL
AND
DIET
8.1 f0.9
1.2kO.5
0.7kO.3
0.7 + 0.4
55.5 + 3.6
15.5+1.9
1.4kO.7
4.3k1.5
11.4k1.2
0.5 f 0.2
1.0*0.3
HDL-CE
PUFA
(%)
OF PLASMA
0.5 f0.2
10.0+ 1.1
4.1 k 0.8
59.4k 2.9
0.6 + 0.2
0.4kO.l
20.8 f 0.7
3.6 f 0.4
7.3 + 0.5
20.6 + 2.6
36.3 + 4.1
1.2 + 0.4
0.4 f 0.1
1.6+0.5
6.9k1.4
c15:o
C16:O
C16:l
C18:O
C18:l
C18:2
C18:3
c2o:o
C20:3
C20:4
7.1*
1.7
0.7kO.2
0.4 f 0.2
14.8 * 2.1
1.0+0.2
0.8 + 0.2
Plasma-CE
2.3
8.6 f 2.2
2.1 f 0.9
0.4 * 0.2
0.9+0.3
33.0& 2.6
16.7 + 2.3
9.1 k 2.5
4.3k1.4
23.4+
0.6 f 0.4
1.0+0.3
1.9
6.6 f 0.9
0.8 f 0.6
0.4 f 0.2
0.6 f 0.2
54.6 + 4.3
15.0+
7.1 f 1.6
1.6kO.6
0.3 f 0.1
1.0 * 0.3
39.0 + 4.6
18.8+2.8
3.4 f 0.6 7.5 + 0.6
1.9
2.0+ 1.8
19.9*
0.5 f 0.1
1.0+0.3
6.0 + 1.6
12.5 f 1.7
0.5 + 0.2
0.9 f 0.4
diet
OF A PATIENT
Plasma
HDL-CE
SUBFRACTIONS
Plasma
PLASMA
After PUFA HDL
AND
Basal
Patient group (n = 10)
COMPOSITION
1.0 f 0.3
ACID
FATTY
c14:o
5
TABLE
7.2k 1.8
0.8 k 0.2
0.3kO.l
0.6 k 0.3
61.Ok3.3
13.8k2.5
1.2kO.3
3.3 f 0.7
10.6 + 0.8
0.4 * 0.2
0.7 f 0.2
Plasma-CE
GROUP
BEFORE
AFTER
8.3 & 1.8
1.8kO.6
0.3+0.1
0.7 f 0.3
35.7 * 3.4
16.3 k 2.1
10.3 * 0.9
2.8~ 1.1
22.6 + 2.0
0.4 + 0.3
0.7 f 0.3
HDL
AND
DIET
6.5+1.9
1.350.7
0.6 k 0.4
0.7 + 0.4
58.6 + 4.0
13.6 f 2.3
1.7kO.7
4.1 +0.6
11.3*1.1
0.7 f 0.3
0.9 * 0.3
HDL-CE
PUFA
(%)
17
treatment. The major fatty acids in plasma and in HDL of the control group are palmitic, oleic and linoleic acid and there are only minor differences, except for a lower palmitic acid (16 : 0) in plasma and a lower arachidonic acid (20 : 4) in HDL. After PUFA diet similar observations are shown for plasma cholesterol esters and HDL and HDL-CE where oleic (18 : 1, 17%) and linoleic (18 : 2, 55%) are the major fatty acids, except for dihomo-y-linoleic acid (20 : 3) and arachidonic acid which are higher in the HDL. Comparing the control group and the hypercholesterolemic patients, the main differences obtained are higher percentages of polyunsaturated fatty acids especially in the plasma cholesterol esters of the patient group (sum of 18 : 2, 18 : 3, 20 : 3 and 20 : 4). The arachidonic level is higher in plasma of the control group and higher in the HDL fraction of the hypercholesterolemic group; there are also major differences in the ratio of 20 : 3/20 : 4 in both groups. As already shown [lo], the polyunsaturated fat diets increased the proportion of polyunsaturated fatty acids in the cholesterol esters and decreased the monounsaturated fatty acids in these lipids. The major changes in the fatty acid composition under the described diet involved, are palmitate (16 : 0), oleate (18 : 0) and linoleate (18 : 2). The most pronounced effect is observed in the HDL-CE, where we noted a significant decrease of oleate and a significant increase of linoleate. There was also a significant increase of arachidonic acid in HDL of the control group. Discussion It is well documented [1,6] that ingestion of polyunsaturated fat by normal subjects causes a reduction in plasma cholesterol. Our data confirm this observation and demonstrate a higher effect in hypercholesterolemic patients. Similar results were obtained after the administration of polyunsaturated lecithin to patients with type II hyperlipoproteinemia [28]. The cholesterol-lowering effect results largely from a decrease in plasma LDL and significantly in the patient group. A fall in HDL-C may also contribute to this effect v91. In a short-term investigation involving a diet high in polyunsaturates Shepherd et al. [lo] observed a reduction in HDL-C but long-term observations showed that HDL-C was not negatively affected [30]. In men with hypercholesterolemia kept for 4 years on an isocaloric, low-fat diet high in polyunsaturated fatty acids, HDL-C levels increased (20%) to a corresponding control group [31]. Identical results are observed in our study, where we noted a significant increase of HDL-C in the patient group against a rather constant value in the control group. Our data are also in agreement with those observed by Thompson et al. [13], who claimed that the fall in LDL and the raise in HDL/LDL ratio is a function of the percentage of the linoleate increase in plasma triglycerides. Our study design differed in several respects from that of Shepherd et al. [lo]. Our diet was iso-energetic and iso-cholesterolemic with a much lower P/S ratio. It is not easy to postulate which of these factors is responsible for the discrepancy between the obtained results, although it has been clear that very high P/S ratio diets reduce HDL-C [lo]. The present work confirms the results obtained by Spritz et al. [8], who measured serum LDL protein
18
fat in normal subjects on a high polyunsaturated fat diet and obtained a significant reduction of the LDL-C/ape B ratio. Our data on the apo B determination analyzed by immunonephelometry are not comparable with the apo B data from Durrington et al. [25] obtained by double antibody radioimmuno-assay. The essential differences in the diet composition and especially the higher P/S ratio are of possible relevance to the different results. In the 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 increased secretion of apoprotein A-l [lo]. The present work confirms a large decrease in the HDL protein/lipid ratio claimed by Shepherd [lo] and the relative decrease of apo A-I in normal men by Kuksis [2]. Under the described diet composition with an increased P/S ratio, we obtained similar changes in the control and in the hypercholesterolemic patient group. We noted a high increase of HDL-PL with a significant change of the sphingomyelin/ phosphatidylcholine ratio in both groups. The decrease of apo A-I accompanied by an increase of phospholipids and the significant changes of the Sphm/PC ratio in HDL in our experiments, confirm the increase of the HDL,/HDL, ratio under the polyunsaturated diet. Identical observations were also obtained during treatment of type II hyperlipoproteinemic patients with polyunsaturated phosphatidylcholine
P81. The fatty acid composition of plasma and of HDL is directly correlated with the unsaturation level of the ingested fat. Ingestion of unsaturated fat caused significant enrichment of linoleate in the HDL lipids accompanied by the fall in saturated fatty acids as palmitate, most pronounced in HDL-CE. One result of these compositional changes is an alteration of the thermotropic properties of the HDL lipoproteins whose microscopic fluidity is increased by polyunsaturated fat feeding influencing the rate of catabolism [l]. There are several mechanisms involved in the effect of polyunsaturated fatty acids on plasma lipids and lipoproteins. An increased ingestion of polyunsaturated fatty acids increases polyunsaturated phosphatidylcholine which is a better substrate for LCAT activity [32], increasing the cholesterol esterification rate. Polyunsaturated triglycerides are hydrolyzed faster by lipoprotein lipase, which is activated by polyunsaturated phosphatidylcholine [33]. Thompson et al. [34] demonstrated also an increased cholesterol turnover by a decrease in the LDL oleate/LDL linoleate ratio. Furthermore, the compositional alterations in the fatty acid composition change the thermotropic properties and fluidity of the plasma lipoproteins and their relative activity as support for certain enzymic activities including LCAT and lipoprotein lipase. The diets in the described studies are examples of extreme dietary variation and not usually encountered in nutrition, while our described study is based on a clinical practice. These data suggest the therapeutic efficacy of polyunsaturated fats in reducing the development of atherosclerotic heart disease. However, the demonstration of suppression of LDL-C and the selective increases of HDL-C obtained with the described diet changes, provide a way for further studies designed to quantify the effects of polyunsaturated fat diets.
19
Acknowledgements We thank Dr. Ingelaere Medicine, analysis We
for providing
of the apoproteins also
thank
Mr
of the A.Z. Sint-Lukas,
the hypercholesterolemic M.
Bruges, patients,
Department
and Mrs G. Waes for the technical Devlieghere
for
assistance
of Internal
Dr. M. Rosseneu
for the
assistance.
in the preparation
of the
manuscript.
References 1 Morrisett, J.D., Pownall, H.J., Jackson, R.L., Segura, R., Gotto, Jr., A.M. and Taunton, O.D., Effects of polyunsaturated and saturated fat diets on the chemical composition and thermotropic properties of human plasma lipoproteins. In: R.T. Holman and W.H Kunaus (Eds.), Polyunsaturated Fatty Acids (American Oil Chemists’ Society, Monograph No. 4), A.O.G.S. Publishers, Champaign, IL, 1977, p. 139. 2 Kuksis, A., Myher, J.J., Geher, K., Jones, G.J., Shepherd, J., Packard, C.J., Morrisett, J.D., Taunton, O.D. and Gotto, A.M., Effects of saturated and unsaturated fat diets on lipid profiles of plasma lipoproteins, Atherosclerosis, 41 (1982) 221. 3 Keys, A., Anderson, J.T. and Grande, F., Serum cholesterol response to dietary fat, Lancet, i (1957) 787. 4 Gordon, D.T., Pitas, R.E. and Jensen, R.G., Effects of diet and type II, hyperlipoproteinemia upon structure of triacylglycerols and phosphatidylcholine from human plasma lipoproteins, Lipids, 10 (1975) 270. 5 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. 6 Steiner, A., Varsos, A. and Samuel, P., Effect of saturated and unsaturated fats on the concentration of serum cholesterol and experimental atherosclerosis, Circ. Res., 7 (1959) 448. 7 Ahrens, F.H., Hirsch, J., Insull, W., Tsaltas, T.T., Blomstrand, R. and Peterson, T.L., The influence of dietary fats on serum lipid levels in man, Lancet, i (1957) 943. 8 Spritz, N. and Mishkel, M.A., Effects of dietary fats on plasma lipids and lipoproteins A hypothesis for the lipid-lowering effect of unsaturated fatty acids, J. Clin. Invest., 49 (1969) 78. 9 Vega, G.L., Groszek, E., Wolf, R. and Grundy, S.M., Influence of polyunsaturated fats on composition of plasma lipoproteins and apolipoproteins, J. Lipid Res., 23 (1982) 811. 10 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. 11 Blaton, V., Vercaemst, R., Vandecasteele, N., Caster, H. and Petters, H., Dietary induced hyperbetalipoproteinemia in chimpanzees - Comparison to human hyperlipoproteinemia, Exp. Mol. Path., 20 (1974) 132. 12 Rosseneu, M., Declercq, 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. 13 Thompson, G.R., Trayner, I., Damford, W., Mark, F. and Harrison, R., Plasma lipids and lipoproteins after modified fat diet, Lancet, ii (1980) 421. 14 Allain, C.C., Poon, L.S., Chan, C.S., Richmond, W. and Fu, P.C., Enzymatic determination of total serum cholesterol, Clin. Chem., 20 (1974) 470. 15 Bucolo, G. and David, H., Quantitative determination of serum triglycerides by the use of enzymes, Clin. Chem., 19 (1973) 476. 16 Michelson, O.B., A calorimetric method for the determination of serum phospholipids, Anal. Chem., 29 (1957) 60. 17 Burstein, M. and Scholinck, H.R., Lipoprotein-polyanion-metal interactions, Adv. Lipid Res., 11 (1972) 67.
20
18 Grove, T.H., Effects of reagent pH on determination of high-density lipoprotein cholesterol by precipitation with sodium phosphotungstate-magnesium, Clin. Chem., 25 (1979) 560. 19 Rosseneu, M., Vercaemst, R. and Vinaimont, N., Quantitative determination of human plasma apolipoprotein A-I by laser immunonephelometry, Clin. Chem., 27 (1981) 856. 20 Rosseneu, M., Vinaimont, N. and Vercaemst, R., Standardization of immunoassays for the quantitation of plasma apo-B protein, Anal. Biochem., 116 (1981) 204. 21 Blaton, V., De Buyzere, M., Spincemaille, J. and Declercq B., Enzymic assay for phosphatidylcholine and sphingomyelin in serum, Clin. Chem., 29 (1984) 806. 22 Rouser, G., Siakotos, A.N. and Yamamoto, A., Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots, Lipids, 1 (1966) 85. 23 Blaton, V. and Peeters, H., Integrated approach to plasma lipid and lipoprotein analysis. In: G. Nelson (Ed.), Blood Lipids and Lipoproteins, J. Wiley and Sons, New York, 1971, p. 369. 24 Myher, J.J., Kuksis, A., Shepherd, J., Packard, C.J., Morrisett, J.D. Taunton, O.D. and Gotto, A.M.. Effect of saturated and unsaturated fat diets on molecular species of phosphatidylcholine and sphingomyelin of human plasma lipoproteins, Biochim. Biophys. Acta, 666 (1981) 110. 25 Durrington, P.N., Bolton, C.H., Hartog, M., Angelinetta, R., Emmett, R. and Furnis, S., The effect of a low-cholesterol, high polyunsaturated diet on serum lipid levels, apoprotein B levels and triglyceride fatty acid composition, Atherosclerosis, 27 (1977) 465. 26 Hatch, F.R., Lindgren, F.T., Adamson, G.L., Jensen, L.C., Wang, A.W. and Levy, R.I., Quantitative agarose gel electrophoresis of plasma lipoproteins - A simple technique and two methods for standardization, J. Lab. Clin. Med., 81 (1972) 946. 27 Kuksis, A., Myher, J.J., Breckenridge, W. and Little, J.A., Lipid profiles of human plasma high-density lipoproteins. In: Report of the HDL Methodology Workshop (NIH Publication No. 79-1661) U.S. Dept. of Health, Education and Welfare, Bethesda, MD, 1979, p. 142. 28 Blaton, V., Declercq, B., Vandamme, D. and Soetewey, F., The human plasma lipids and lipoproteins under influence of EPL therapy. In: H. Peeters (Ed.), Phosphatidylcholine, Springer-Verlag, Berlin, Heidelberg, 1976, p. 125. 29 Nichaman, M.Z., Sweeley, C. and Olson, R., Plasma fatty acids in normolipemic and hyperlipemic subjects during fasting and after linoleate feeding, Amer. J. Clin. Nutr., 20 (1967) 1057. 30 Hulley, S., Ashman, P. Kuller, L., Lasser. N. and Sherwin, R., HDL cholesterol levels in multiple risk factors intervention trial (MRFIT) by the MRFIT Research Group, Lipids, 14 (1979) 119. 31 Hjermann, I., Enger, S.C., Helgeland, A., Holme, I., Lereu, P. and Tryggi, K., The effect of dietary changes on high density lipoprotein cholesterol - The Oslo Study, Amer. J. Med., 66 (1979) 119. 32 Assmann, G., LCAT lipoprotein and phospholipid substrate specificity. In: H. Peeters (Ed.), Phosphatidylcholine, Springer-Verlag, Berlin, Heidelberg, 1976, p. 34. 33 Blaton, V., Vandamme, D. and Peeters, H., Activation of lipoprotein lipase in vitro by unsaturated phospholipids, FEBS Letters, 44 (1974) 185. 34 Thompson, G.R., Jadhav, A., Nava, M. and Gotto, A., Effects of intravenous phospholipid on low density lipoprotein turnover in man, Europ. J. Clin. Invest., 6 (1976) 241. 35 Friedewald, W.T., Levy, R.I. and Frederickson, D.S., Estimation of the concentration of low density lipoprotein cholesterol in plasma without use of the preparative ultracentrifuge, Clin. Chem.. 18 (1972) 499.
Atherosclerosis, 53 (1984) 9-20 Elsevier Scientific Publishers Ireland, Ltd
ATH 03513
Effect of Polyunsaturated Isocaloric Fat Diets on Plasma Lipids, Apolipoproteins and Fatty Acids V. Blaton, Department
M. De Buyzere, B. Declercq, A. Pracetyo J. Delanghe and J. Spincemaille
*, G. Vanderkelen,
of Clinical Chemistry, A. Z. Sint -Jan, Ruddershooe IO, B - 8000 Bruges (Belgium) (Received 15 April, 1983) (Revised, received 5 January, 22 March, 1984) (Accepted 24 March, 1984)
Summary The effect of an increased polyunsaturated fatty acid concentration in the diet on the plasma lipoproteins from a normal group of healthy persons and from a group of hypercholesterolemic patients, consuming an isoenergetic and an isocholesterolemic diet, was examined and the changes in the plasma phospholipids were measured. Nine normal and 10 hypercholesterolemic patients were treated with a polyunsaturated diet for 1 month. Controls and hypercholesterolemic patients were screened on their lipid and lipoprotein profiles and their P/S ratio in the diet was calculated and increased with a factor 4. In the control group the P/S ratio was increased from 0.35 to 1.38 and in the hypercholesterolemic group from 0.46 to 1.59. They received the diet for at least 4 weeks before a second analysis of lipids and lipoproteins. The most important results are a decrease of plasma cholesterol, followed by a significant increase of HDL cholesterol. The cholesterol-lowering effect results largely from the plasma LDL decrease, especially in the patient group. Apo A-I is decreased accompanied by a significant increase of the ratio HDL-C/ape A-I. The observed changes are most pronounced in the hypercholesterolemic group. There is no change in apo B but a significant change in the linoleic acid concentration especially in the HDL cholesterol esters. The major phospholipids in plasma are
* Present address: Department of Internal Medicine, University of Brawijaya, Malang, Indonesia. Abbreuiations: HDL-C, high density lipoprotein cholesterol: LDL-C, low density lipoprotein cholesterol; TC, total plasma cholesterol; TG, plasma triglycerides; PC, phosphatidylcholine; Sphm, sphingomyelin; HDL-CE, high density lipoprotein cholesterol esters; HDL-PL, high density lipoprotein phospholipids: Plasma-CE, plasma cholesterolesters; Apo A-I, apolipoprotein A-I; apo B, apolipiprotein B; P/S, ratio of polyunsaturated to saturated fatty acids. 0021-9150/84/$03.00
0 1984 Elsevier Scientific Publishers Ireland, Ltd.
10
identical in both groups and there is an identical sphingomyelin is increased and phosphatidylcholine related to an increase of the HDL,/HDL, ratio. Key words:
change under the PUFA diet, is decreased, which may be
Fatty acids - Lipid profiles - Plasma apolipoproteins teins - Unsaturated fat diet
- Plasma
lipopro-
Introduction The ingestion of unsaturated fatty acids by man increases the proportion of unsaturated fatty acids in the lipids of plasma lipoproteins [l-3] and changes the positional distribution of acyl groups in plasma glycerophospholipids and triacylglycerols [4,5]. Although diets rich in polyunsaturated fatty acids lower the serum cholesterol [6,7], the mechanisms involved, however, have remained obscure. Spritz and Mishkel [8] proposed that plasma cholesterol is decreased because lipid esters containing polyunsaturated fatty acids require more space in lipoproteins and exclude cholesterol, but this was not confirmed by Vega et al. [9], who suggest that the major action of polyunsaturates is to alter the production or clearance of the plasma lipoproteins and not to change the relative proportions of their lipid and apolipoprotein components. Morrisett et al. [l] and Shepherd et al. [lo] have further shown that fatty acid alterations result in changes in the thermotropic and physical properties of all major plasma lipoproteins and have discussed the possible relationship between structural changes and metabolism. Similar findings were observed in chimpanzees (pantroglodytes), induced with diet type II hyperlipoproteinemia and treated with saturated and unsaturated phosphatidylcholine [11,12]. Consumption of polyunsaturated phospholipid-rich meals by chimpanzees gave changes in the fatty acid composition of VLDL, LDL and HDL, fractions, but the fluidity of the lipoproteins measured after diphenylhexatriene labeling was not significantly changed [12]. Polyunsaturated fats affected HDL, HDL-C and HDL apo A-I [2,10,13]. The implication from these studies is that the lipoprotein structure is profoundly changed by alterations in the lipid, apoprotein and fatty acid composition accompanying the dietary manipulations. As contradictory results are described on the effects of polyunsaturated fatty acids, we studied the influence of an increase in the P/S ratio under an isoenergetic and isocholesterolemic diet in a normal group of healthy persons and in a hypercholesterolemic group. Materials and Methods Nine normal persons (mean age 42 years, range 32-54 years) of both sexes (7 males, 2 females) took part in this study. Their initial serum cholesterol value was less than 240 mg/lOO ml. The hypercholesterolemic patients (n = 10; 8 males, 2 females) were screened according to clinical and biochemical parameters. The selected patients had hyperbe-
11
talipoproteinemia with a cholesterol value higher than 260 mg/lOO ml. Of the cohort members 30% showed clinical symptoms of hypertension, 56% had cardiovascular complications and 14% had peripheral atherosclerosis. Diabetics and subjects with familial hyperlipemia, overt obesity, intake of alcohol above 30 g/day, or nicotine consumption above 10 cigarettes/day were excluded. No drugs known to influence the serum lipid composition nor beta-blockers were administered The study was conducted over an g-week period. The subjects maintained their usual diet for the first 4 weeks of the study, meanwhile lipids and lipoproteins were followed and a food examination was done by a diabetician over 2 weeks at home. The average percentage composition of the diet of the control group and of the patients is given in Table 1. The main differences are higher kJ and % fat intake in the patient group. Under isocaloric and isocholesterolemic conditions unsaturated fats were increased in their diets by changing saturated through unsaturated diet components and through a supplement of unsaturated oil (corn oil, one spoon/day). The ratio of unsaturated to saturated fat (P/S) was 4 times increased individually ranging between 0.30 and 1.40 in the control group and between 0.40 and 1.60 in the patient group (Table 1). All subjects were 4 weeks on the unsaturated fat diet and were visited and controlled weekly by a dietitian. Blood samples in EDTA were taken after an overnight fast. Plasma total cholesterol (TC) were measured by the CHODCOD method [14], plasma triglycerides (TG) by the enzymatic glycerokinase method [15] and plasma phospholipids by the metavanadate method [16]. Sodium phosphotungstate magnesium chloride precipitation of LDL and VLDL [17] was performed and HDL-C assayed by the enzymatic CHOD-COD method on the HDL supernatant [18]. The apolipoproteins Apo A-I and Apo B were measured by nephelometric immunochemical methods [19,20]. For the quantitation of apo B a LDL,-fraction (1.03 < d < 1.05 g/dl) was used as standard. For apo A-I quantitation, the primary standard consisted of pure lyophylized apo A-I dissolved in 0.3 M guanidine chloride. For assay purpose, a plasma sample was standardized using the lyophylized apoprotein and was subsequently used as secondary standard, kept in sodium azide (0.1 g/l) at 4OC. TABLE
1
ISOCALORIC AND ISOCHOLESTEROLEMIC DIET COMPOSITION AND A HYPERCHOLESTEROLEMIC PATIENT GROUP
Daily energy intake (kJ) Protein (W) Fat (%) Carbohydrate (W) Alcohol (a) Cholesterol (mg/d) P/S B
OF A CONTROL
GROUP
Control group (n = 9)
Patient group (n = 10)
Basal
PUFA diet
Basal
PUFA diet
9907 16 36 40 8 288 0.35
9844 17 35 40 8 242 1.38
10550 16 41 40 3 261 0.46
10203 16 40 41 3 203 1.59
a Abbreviations: P = polyunsaturated fatty acids, S = saturated fatty acids.
12
Serum lipids and phospholipid subclasses were separated by thin-layer chromatography after extraction of 1 ml serum with 24 ml of chloroform/methanol (2 : 1, v/v) and 10 ml of 2 g/l calcium chloride solution [21]. After centrifugation at 1200 x g, the chloroform layer was removed and evaporated at 40 ‘C under nitrogen. The residue was redissolved in 0.2 ml chloroform and applied to a silicagel plate, prepared as follows: silicagel (60HR Merck) extra pure was spread as a slurry (50 g/125 ml water) with a 0.30-mm spreader on to glass plates, pre-cleaned and dried. The plates were activated just before use, by heating for 30 min at 110 ‘C. To separate serum apolar lipids we used petroleum ether/diethylether/acetic acid developed at room temperature for 45 min. (80 : 20 : 1, v/v/v) The phospholipids were separated into subclasses with chloroform/methanol/ acetic acid/water (50 : 125 : 8 : 2, v/v/v/v). The phospholipid subfractions were determined after silica gel extraction by the molybdate method [22]. The determinations of the Sphm/PC ratio were performed by the TLC method using HClO, (100 g/l) as the charring reagent. The fatty acid composition of the plasma lipids and of the plasma lipoproteins were analyzed based on an integrated procedure [23]. The lipid extracts were saponified with 3% potassium hydroxide in methanol. After acidification with 6% H,S04 the fatty acids were extracted with petroleum ether. The dried extract was esterified with BF3-CH,OH (250 mg/l) by heating for 30 min at 80 ‘C in a sealed tube. The separation was performed on a Varian gas chromatograph equipped with a 6-ft column filled with 10% diethylene-glycol succinated on Gaschrom @ and a flame ionization detector. The temperature was programmed from 120 to 210°C at 2’C/min after an isothermal period of 5 min. Statistical analysis was carried out by both one- and two-way analysis of variance. Results Plasma lipid and apoprotein changes after the PUFA diet The efficiency of the polyunsaturated fat diet in lowering plasma cholesterol and LDL-C especially in hypercholesterolemic patients is shown in Table 2 (P -C 0.01). The polyunsaturated fat produced significant reductions in the patient group in total plasma cholesterol and in the plasma level of cholesterol associated with LDL. The changes in the HDL composition are different in both groups. HDL-C is only significantly increased in the patient group and stays rather constant in the control group which does not corroborate the observation of other workers [2,20]. In the normal control group, however, there is a significant increase of plasma phospholipids (48%), mainly associated with HDL phospholipids and accompanied by a decrease of the C/PL ratio. The parallel increase of HCL-C and HDL-PL in the patients suggests an increase in the total HDL concentration. The polyunsaturated fat diet decreases slightly the apo A-I in patients as well as in controls, and those observations are in agreement with literature data for normolipemic subjects WOI. Polyunsaturated molecular species
fat ingestion, however, did cause a significant change in the of the phospholipid subclasses, especially phosphatidylcholine,
13
TABLE 2 PLASMA LIPID AND APOLIPOPROTEIN BEFORE AND AFTER PUFA DIET
LEVELS IN A CONTROL
AND A PATIENT
GROUP
Values are mean f SD, lipid and apoprotein levels are mg/dl. Patient group (n = 10)
Total cholesterol Triglycerides Phospholipids LDL cholesterol a HDL cholesterol HDL phospholipids Apo A-I Apo B HDL cholesterol/Ape A-I LDL cholesterol/Ape B Apo A-I/APO B
Control group (n = 9)
Basal
After PUFA diet
Basal
After PUFA diet
323 k33 144 +56 312 +51 259 k45 35 fl3 96 +39 117 +16 153 *15 0.30* 0.09 1.78& 0.51 0.78* 0.13
280 *37* 130 +42 309 +25 208 +38* 46 f12* 122 &29 106 +ll 153 * 14 0.43* 0.08 1.36* 0.13 * 0.71+ 0.16
210 *19 94 +37 211 k42 141 +20 52 +9 97 522 126 i 25 105 k23 ** 0.42+ 0.06 1.40* 0.35 1.2s* 0.44
200 +33 99 +33 261 k32* 131 +34 49 *8 144 *39 116 ,512 106 f25 0.46k 0.13 1.24+ 0.27 1.14+ 0.29
* P < 0.01; ** Statistical analysis control against patient group: P < 0.01. a Friedewald [35].
which are responsible for the metabolic transformations of the lipoproteins [24]. These data indicate that the capacity of an increased cholesterol uptake by HDL for the patient group is dependent on the PUFA diet and related to an increased activity of LCAT suggesting also an increased ratio of HDL,/HDL,. Apo B which is significantly higher in the patient group, in unchanged under PUFA diet in both groups, although total cholesterol and LDL-C are decreased and most pronounced in the patient group, which corroborates the data of Durrington et al. [25], but the normal group of the latter study was not on an isocholesterolemic diet. The present data are in agreement with the study of Mishkel [8] who observed no change in the protein concentration. The main effect of the unsaturated fat diet on LDL is observed in the patient group where we obtained a significant decrease of the ratio LDL-C/ape B, although in the control group a similar trend was observed. At a high P/S ratio of 4 Kuksis et al. [2] observed a 10% decrease of the LDL protein content, which was compensated by a proportional increase in all lipid classes. As we observed an unchanged apo B concentration under the described diet change, the decreased LDL-C must be related to an increased exchange of unsaturated CE or to an increased catabolic rate. The significant decrease of LDL-C by PUFA diets is an important factor reducing the atherogenic effect of the plasma lipoproteins. The decrease of the LDL-C/ape B can be related to the interconversion of LDL subclasses due to a higher removal rate of IDL-C by the HDL particle. Although main changes are observed in HDL and LDL the potential atherogenic index apo A-I/ape B is rather constant in both groups during PUFA diet treatment.
14
Changes
of the plasma
unsaturated
lipoproteins
and plasma
phospholipid
subclasses
after
the
as demonstrated
by
diet
Although
the lipoprotein
subclasses
are significantly
changed
the described data, we do not observe marked qualitative lipoprotein alteration by the standardized lipoprotein electrophoretic technique [26]. The hypercholesterolemit group has significantly
higher LDL
lipoproteins
accompanied
by lower HDL
values. Based
on
unsaturated decreased.
the diet
described
technique,
in both
groups,
Based on the described
ual phospholipid
subclasses
Phosphatidylcholine
high
similar
changes
TLC procedure,
of both groups before
and sphingomyelin,
are
observed
as well as low density we analyzed
further
and after PUFA
3). The major changes
the individ-
the major plasma phospholipids
in both groups,
and a decreased
phosphatidylcholine
concentration,
by the Sph/PC
ratio. The increased
sphingomyelin
are identi-
polyunsaturated
are an increased
sphingomyelin
which is significantly concentration
the are
diet treatment.
cal in both groups and there is a similar change under the increased diet (Table
under
lipoproteins
expressed
may be related to
an increase of HDL,, which contains more sphingomyelin than HDL, [27]. The minor phospholipid classes, lysophosphatidylcholine and phosphatidylethanolamine, identical
in both groups are unchanged
found no change in Sph/PC activity
under PUFA
concentration obtained
LDL
diet can be responsible
accompanied
with non-human
by an increased
under the high P/S ratio. Kuksis
ratio in VLDL, by an increased primates,
and HDL,.
for a decrease sphingomyelin
where a decreased
lysophosphatidylcholine
et al. [2]
The increased
LCAT
of phosphatidylcholine value. Similar data were
PC value was accompanied
[ 121.
Changes
of the fatty acid composition of plasma, of plasma
a PUFA
diet
CE and of HDL-CE
under
A comparison of the fatty acid patterns in plasma, in plasma cholesterol esters and in the cholesterol esters of HDL lipoproteins is given in Table 4 for the control group and in Table 5 for the patient group, before as well as after the PUFA diet
TABLE 3 PERCENTAGE (%) PHOSPHOLIPID PATIENT GROUP OH-PC a
Control
AFTER
PUFA
DIET OF A CONTROL
AND
A
Phospholipids Sphm a
PC”
PEa
Sphm/PC
6.1 f 1.3 6.5 f 1.6
19.2kl.l 22.4~ 2.9
68.9 f 2.0 65.8 f 1.9
5.8 + 0.8 5.3*0.9
0.28 f 0.02 0.34 f 0.05 *
6.1 f 0.8 5.9*1.5
19.3 * 0.9 22.5 f 1.8
68.5 f 0.9 65.2 f 2.8
6.0 f 0.8 6.3 f 0.9
0.29 k 0.02 0.35 + 0.04 *
ratio
group (n = 6)
Before PUFA diet After PUFA diet Patient
CHANGES
group (n = 4)
Before PUFA diet After PUFA diet
’ Abbreviations: OH-PC phosphatidylethanolamine. * P -e 0.01.
= 2-hydroxyphosphatidylcholine,
Sphm
= sphingomyelin,
PC =
HDL-CE
SUBFRACTIONS
0.7 + 0.3
0.6 + 0.4
0.9 f 0.3
0.4*0.1
1.5*0.7
8.3 + 1.6
c2o:o
C20:3
C20:4
7.3k1.4
0.8 k 0.5
55.5 * 4.7
34.0 * 3.1
C18:3
17.1 f 1.6
C18:2
1.8
1.2+0.4
20.1+
7.9 f 0.8
C18:O
3.9k1.2
11.5 + 1.4
0.6 f 0.3
C18:l
3.25 1.0
21.5k2.1
C16:O
C16:l
0.7 f0.2
c15:o
0.8 + 0.4
1.3
6.6+1.6
1.7kO.5
0.3 * 0.1
0.8 + 0.3
30.7 5 3.1
17.9+1.4
9.6+ 1.3
2.9k
25.2 + 3.1
0.9 + 0.5
l.lkO.5
7.9*
1.3
1.2kO.8
0.5 * 0.2
0.7kO.3
53.3*4.4
16.7k1.3
1.7+0.3
5.3 f 1.4
11.4+1.3
0.5 +0.3
0.8 k 0.4
8.1 k 1.0
1.7*0.5
0.5 + 0.3
1.1 f0.5
36.6 f 4.7
19.6k1.7
7.2 f 0.5
3.6k1.2
20.4 + 2.3
0.4 + 0.2
0.9 + 0.4
diet
OF A CONTROL
Plasma
HDL
PLASMA
Plasma
Plasma-CE
AND
After PUFA
group (n = 9)
OF PLASMA
Basal
Control
COMPOSITION
1.5+0.7
ACID
FATTY
c14:o
4
TABLE
1.0
7.5kl.l
0.9 * 0.5
0.6 k 0.2
0.5 + 0.2
57.0 k 4.6
15.8+1.5
l.OkO.4
3.8 * 1.4
11.7*
0.6*0.3
0.8 t 0.3
Plasma-CE
GROUP
BEFORE
AFTER
9.2 f 1.4
2.1 f 1.0
0.3fO.l
0.7 * 0.3
32.9 + 3.1
18.1 k 2.2
9.5kl.O
2.6fl.O
23.1 f 1.7
0.5 * 0.3
0.8 f 0.4
HDL
AND
DIET
8.1 f0.9
1.2kO.5
0.7kO.3
0.7 + 0.4
55.5 + 3.6
15.5+1.9
1.4kO.7
4.3k1.5
11.4k1.2
0.5 f 0.2
1.0*0.3
HDL-CE
PUFA
(%)
OF PLASMA
0.5 f0.2
10.0+ 1.1
4.1 k 0.8
59.4k 2.9
0.6 + 0.2
0.4kO.l
20.8 f 0.7
3.6 f 0.4
7.3 + 0.5
20.6 + 2.6
36.3 + 4.1
1.2 + 0.4
0.4 f 0.1
1.6+0.5
6.9k1.4
c15:o
C16:O
C16:l
C18:O
C18:l
C18:2
C18:3
c2o:o
C20:3
C20:4
7.1*
1.7
0.7kO.2
0.4 f 0.2
14.8 * 2.1
1.0+0.2
0.8 + 0.2
Plasma-CE
2.3
8.6 f 2.2
2.1 f 0.9
0.4 * 0.2
0.9+0.3
33.0& 2.6
16.7 + 2.3
9.1 k 2.5
4.3k1.4
23.4+
0.6 f 0.4
1.0+0.3
1.9
6.6 f 0.9
0.8 f 0.6
0.4 f 0.2
0.6 f 0.2
54.6 + 4.3
15.0+
7.1 f 1.6
1.6kO.6
0.3 f 0.1
1.0 * 0.3
39.0 + 4.6
18.8+2.8
3.4 f 0.6 7.5 + 0.6
1.9
2.0+ 1.8
19.9*
0.5 f 0.1
1.0+0.3
6.0 + 1.6
12.5 f 1.7
0.5 + 0.2
0.9 f 0.4
diet
OF A PATIENT
Plasma
HDL-CE
SUBFRACTIONS
Plasma
PLASMA
After PUFA HDL
AND
Basal
Patient group (n = 10)
COMPOSITION
1.0 f 0.3
ACID
FATTY
c14:o
5
TABLE
7.2k 1.8
0.8 k 0.2
0.3kO.l
0.6 k 0.3
61.Ok3.3
13.8k2.5
1.2kO.3
3.3 f 0.7
10.6 + 0.8
0.4 * 0.2
0.7 f 0.2
Plasma-CE
GROUP
BEFORE
AFTER
8.3 & 1.8
1.8kO.6
0.3+0.1
0.7 f 0.3
35.7 * 3.4
16.3 k 2.1
10.3 * 0.9
2.8~ 1.1
22.6 + 2.0
0.4 + 0.3
0.7 f 0.3
HDL
AND
DIET
6.5+1.9
1.350.7
0.6 k 0.4
0.7 + 0.4
58.6 + 4.0
13.6 f 2.3
1.7kO.7
4.1 +0.6
11.3*1.1
0.7 f 0.3
0.9 * 0.3
HDL-CE
PUFA
(%)
17
treatment. The major fatty acids in plasma and in HDL of the control group are palmitic, oleic and linoleic acid and there are only minor differences, except for a lower palmitic acid (16 : 0) in plasma and a lower arachidonic acid (20 : 4) in HDL. After PUFA diet similar observations are shown for plasma cholesterol esters and HDL and HDL-CE where oleic (18 : 1, 17%) and linoleic (18 : 2, 55%) are the major fatty acids, except for dihomo-y-linoleic acid (20 : 3) and arachidonic acid which are higher in the HDL. Comparing the control group and the hypercholesterolemic patients, the main differences obtained are higher percentages of polyunsaturated fatty acids especially in the plasma cholesterol esters of the patient group (sum of 18 : 2, 18 : 3, 20 : 3 and 20 : 4). The arachidonic level is higher in plasma of the control group and higher in the HDL fraction of the hypercholesterolemic group; there are also major differences in the ratio of 20 : 3/20 : 4 in both groups. As already shown [lo], the polyunsaturated fat diets increased the proportion of polyunsaturated fatty acids in the cholesterol esters and decreased the monounsaturated fatty acids in these lipids. The major changes in the fatty acid composition under the described diet involved, are palmitate (16 : 0), oleate (18 : 0) and linoleate (18 : 2). The most pronounced effect is observed in the HDL-CE, where we noted a significant decrease of oleate and a significant increase of linoleate. There was also a significant increase of arachidonic acid in HDL of the control group. Discussion It is well documented [1,6] that ingestion of polyunsaturated fat by normal subjects causes a reduction in plasma cholesterol. Our data confirm this observation and demonstrate a higher effect in hypercholesterolemic patients. Similar results were obtained after the administration of polyunsaturated lecithin to patients with type II hyperlipoproteinemia [28]. The cholesterol-lowering effect results largely from a decrease in plasma LDL and significantly in the patient group. A fall in HDL-C may also contribute to this effect v91. In a short-term investigation involving a diet high in polyunsaturates Shepherd et al. [lo] observed a reduction in HDL-C but long-term observations showed that HDL-C was not negatively affected [30]. In men with hypercholesterolemia kept for 4 years on an isocaloric, low-fat diet high in polyunsaturated fatty acids, HDL-C levels increased (20%) to a corresponding control group [31]. Identical results are observed in our study, where we noted a significant increase of HDL-C in the patient group against a rather constant value in the control group. Our data are also in agreement with those observed by Thompson et al. [13], who claimed that the fall in LDL and the raise in HDL/LDL ratio is a function of the percentage of the linoleate increase in plasma triglycerides. Our study design differed in several respects from that of Shepherd et al. [lo]. Our diet was iso-energetic and iso-cholesterolemic with a much lower P/S ratio. It is not easy to postulate which of these factors is responsible for the discrepancy between the obtained results, although it has been clear that very high P/S ratio diets reduce HDL-C [lo]. The present work confirms the results obtained by Spritz et al. [8], who measured serum LDL protein
18
fat in normal subjects on a high polyunsaturated fat diet and obtained a significant reduction of the LDL-C/ape B ratio. Our data on the apo B determination analyzed by immunonephelometry are not comparable with the apo B data from Durrington et al. [25] obtained by double antibody radioimmuno-assay. The essential differences in the diet composition and especially the higher P/S ratio are of possible relevance to the different results. In the 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 increased secretion of apoprotein A-l [lo]. The present work confirms a large decrease in the HDL protein/lipid ratio claimed by Shepherd [lo] and the relative decrease of apo A-I in normal men by Kuksis [2]. Under the described diet composition with an increased P/S ratio, we obtained similar changes in the control and in the hypercholesterolemic patient group. We noted a high increase of HDL-PL with a significant change of the sphingomyelin/ phosphatidylcholine ratio in both groups. The decrease of apo A-I accompanied by an increase of phospholipids and the significant changes of the Sphm/PC ratio in HDL in our experiments, confirm the increase of the HDL,/HDL, ratio under the polyunsaturated diet. Identical observations were also obtained during treatment of type II hyperlipoproteinemic patients with polyunsaturated phosphatidylcholine
P81. The fatty acid composition of plasma and of HDL is directly correlated with the unsaturation level of the ingested fat. Ingestion of unsaturated fat caused significant enrichment of linoleate in the HDL lipids accompanied by the fall in saturated fatty acids as palmitate, most pronounced in HDL-CE. One result of these compositional changes is an alteration of the thermotropic properties of the HDL lipoproteins whose microscopic fluidity is increased by polyunsaturated fat feeding influencing the rate of catabolism [l]. There are several mechanisms involved in the effect of polyunsaturated fatty acids on plasma lipids and lipoproteins. An increased ingestion of polyunsaturated fatty acids increases polyunsaturated phosphatidylcholine which is a better substrate for LCAT activity [32], increasing the cholesterol esterification rate. Polyunsaturated triglycerides are hydrolyzed faster by lipoprotein lipase, which is activated by polyunsaturated phosphatidylcholine [33]. Thompson et al. [34] demonstrated also an increased cholesterol turnover by a decrease in the LDL oleate/LDL linoleate ratio. Furthermore, the compositional alterations in the fatty acid composition change the thermotropic properties and fluidity of the plasma lipoproteins and their relative activity as support for certain enzymic activities including LCAT and lipoprotein lipase. The diets in the described studies are examples of extreme dietary variation and not usually encountered in nutrition, while our described study is based on a clinical practice. These data suggest the therapeutic efficacy of polyunsaturated fats in reducing the development of atherosclerotic heart disease. However, the demonstration of suppression of LDL-C and the selective increases of HDL-C obtained with the described diet changes, provide a way for further studies designed to quantify the effects of polyunsaturated fat diets.
19
Acknowledgements We thank Dr. Ingelaere Medicine, analysis We
for providing
of the apoproteins also
thank
Mr
of the A.Z. Sint-Lukas,
the hypercholesterolemic M.
Bruges, patients,
Department
and Mrs G. Waes for the technical Devlieghere
for
assistance
of Internal
Dr. M. Rosseneu
for the
assistance.
in the preparation
of the
manuscript.
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