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Dietary protein and fatty acid composition of liver lipids in the rat
Bioc~imi~a et B~o~hysjcu Actu, 104 Elsevier
187
(1990) 187-192
BBALIP 53400
Dietary protein and fatty acid composition of liver lipids in the rat LinnCa Sj6blom and Anders Eklund Department
ofMedico1and Physiological Chemistry
University of Uppsala, Vppsala (Sweden)
(Received 26 June 1989) (Revised manuscript received 26 January 1990)
Key words: A~a~~do~c acid; Dietary protein; Icosatrienoic acid; Fatty acid; Linoleic acid; Phosphatidyli~ositol: (Male rat); (Liver)
In order to compare the effects of different sources of dietary protein on the fatty acid composition of phosphatidylcholines (PC), p~sphatidyleth~ol~~nes (PE), phosphatidylinositols (PI), choleste~l esters and ~acyl~ycerols, male rats were fed for a 4-week period on cholesterol-free, or cholesterol-containing, diets based on casein, or soybean protein and olive oil. The most conspicuous difference observed was the occurrence of significantly higher levels of 5,8,1&eicosatrienoic acid, 20:3 (n - 9), in the different lipid classes of case&fed, compared with soybean pro&+fed, animals. In the PI fraction of livers from the groups of rats fed casein diet, this fatty acid amounted to between 9.9 and 13.3% by weight of the total fatty acids. Phospholipids from livers of casein-fed rats contained increased levels of oleic acid, 18: 1 (n - 9) (in PC and PE) and reduced levels of stearic acid (18 : 0). Moreover, in this group of rats PI contained a reduced level of arachidonic acid, 20: 4 (n - 6). A casein-related decrease in the linoleic acid, 18: 2 (n - 6), content of PC and PE was observed only in the rats fed on cholesterol-free diet. Effects on the fatty acid composition were also observed in the triacylglycerol and cholesteryl ester fractions, in which the rats fed casein diet showed higher levels of palmitoleic acid, 16: 1 (n - 7) (cholesterol-supplemented diet) and lower values for linoleic acid, than the soybean p~tein-fed rats.
Introduction
it
Like in other animal species, enhanced plasma cholesterol levels can be produced in rats by dietary cholesterol administration. It has been recognized for some time, that this response to dietary cholesterol is under strong influence of the type of dietary protein [l-3]. In rats, this type of hypercholesterolemia is largely due to an increased concentration of plasma very-lowdensity lipoprotein (VLDL) [2]. In contrast to normal plasma VLDL, VLDL from hypercholesterolemic rats has the ability to promote the in vivo acc~~lation of cholesteryl esters in macrophages [4,5]. This accumulation of choiesteryl esters in macrophages has also been shown to be influenced by dietary protein-related effects on plasma VLDL [5]. Since VLDL is manufactured primarily by the liver,
is of interest to study any dietary protein-related changes in metabolic fictions of the liver, which are connected to the concentration and properties of plasma VLDL. Previous studies have demonstrated effects on hepatic sterogenesis 161, hepatic secretion of VLDL [7] and hepatic expression of LDL-receptors [Sl. Recently, Huang et al. [9] and Sugano et al. [lo] have shown effects of casein and soybean protein on the fatty acid composition of plasma and liver. In these papers, the hepatic phospholipids studied were total phospholipids [9], or phosphatidylcholines [lo]. In contrast, the present studies were aimed at unravelling dietary proteindependent effects on mass proportions and fatty acid composition of individual phospholipid classes in the liver of male rats fed on cholesterol-free as well as cholesterol-supplemented diets.
Materials and Methods Abbreviations: PC, p~osphatidyl~holines; PE, phosphatidylethanolamines; PI, phosphatidylinositols; VLDL, very-low-density lipoprotein; EFA, essential fatty acid. Correspondence: A. Eklund, Department of Medical and Physiological Chemistry, U~versity of Uppsala, P.Q. Box 575, S-751 23 Up. psala, Sweden. OOW2760/90/$63.50
Feeding experiments. The composition of the diets was by percentage: protein fcasein (dry wt. composition: 95% protein, 0.4% fat, 3% ash), Cat. No. 1.2600 (Kebo-Grave, Stockholm, Sweden); or soybean protein isolate, Purina Protein 590 E (dry wt. composition: 97% protein, 0.6% fat, 4% ash) supplied by Karlshamns
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
188 TABLE
I
Mass proportions of liver lipids Mean value f SD. for eight rats unless otherwise I tern
specified.
0% Cholesterol casein
Liver weight (g) (g/100 g body wt.) Protein (mg/g) Total lipid (mg/g) free cholesterol (mg/g) cholesteryl ester (mg/g) phospholipids (mg/g)
11.Ok1.3
0.5% Cholesterol soybean
a.b
3.9 f 0.2 h 183 +7 56 +6 a,’ 2.3 : 0.3 ’ 1.4kO.6’ 15.7* 1.9
protein
9.0+ 0.9 3.6k 0.3 178 +11 71 f8h 2.8k 0.5 ’ 1.7+ 0.8 b 16.9* 2.7
casein ’
soybean
13.0+ 1.5 a 4.8* 0.3 a 175 *20 133 *20 5.6+ 2.5 12.9* 3.5 15.5* 2.2
9.3+ 1.4 3.9k 0.5 184 *26 126 k16 4.4* 1.4 11.1 f 3.3 16.0+ 3.1
a Same level of dietary cholesterol, comparison between casein and soybean protein, P i 0.01. h Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented ’ Values for ten rats.
Oljefabriker (Karlshamn, Sweden), 20; olive oil, 10; cholesterol (EGA Cat. No. C7, 520-9, dissolved in olive oil before inclusion in diet), 0 and 0.5, respectively; vitamin premix [2], 2; mineral mixture (Williams & Briggs salt mixture [ll]), 3.5; cellulose powder (Ricenta, Stockholm, Sweden) 5.3; and corn starch to 100. The percentage of calories provided by protein, fat and carbohydrates, respectively, was 21, 23 and 56 energy %, in all diets. In a first experiment six rats were fed on each of the diets supplemented with 0.5% cholesterol. In a second experiment, four rats were fed on each of the cholesterol-supplemented diets and eight rats were fed on each of the cholesterol-free diets. Since the results from the corresponding groups of the two different experiments were in excellent agreement, the results were combined. In each experiment, 21-day-old, outbred Sprague-Dawley strain rats (Alab, Sollentuna, Sweden), weighing about 65 g, were housed in subgroups of three or four in macrolon cages at 25 o C and 50% relative humidity in rooms with a 12 h (06.00 to 18.00 h) light-dark cycle. They were allowed free access to food and fresh tap water. After 4 weeks on the respective regimen, they were anaesthetized with diethyl ether and killed by terminal exsanguination. All animals were fasted for 4 h before sampling between 10.00-11.00 h. Livers were removed, weighed and lyophilized. Protein content was calculated from nitrogen analysis [2]. Extraction of liver lipids. Total lipid content of livers was determined as previously described [12]. Cholesterof analysis. The cholesterol level in the total lipid fraction of the liver was determined using an enzymatic method (Boehringer, Mannheim, F.R.G., Cat.No. 237574). Thin-layer chromatography. Lipid classes were separated by thin-layer chromatography in a two-step procedure using Silica gel plates (20 X 20 cm, Merck DCFertigplatten cat. No. 5721, E. Merck, Darmstadt,
protein’
diet. P < 0.01.
F.R.G.). The first step was performed as described by Arvidson [13] for separation of subclasses of phospholipids. In a second step, on the same plate, the neutral lipids at the front were developed in hexane/ diethyl ether/acetic acid, (80: 20: 1, v/v). The spots were identified by comparison with authentic standards after visualization with, 2’,7’-dichlorofluorescein (Merck, Cat. No. 9677, E. Merck, Darmstadt, F.R.G.). All solvents system used contained 0.002% (w/v) butylated hydroxyanisole (BHA). By this procedure triacylglycerols, cholesteryl esters and different phospholipid fractions were isolated, extracted from the plates [ 131 and dried under a stream of N,. Phosphorus analysis. Phospholipid determination was performed by phosphorus analysis of the liver total lipid extract and separated fractions according to a modification of the method of Chen., Jr. et al. [14]. Gas-liquid chromatography. Methyl esters of fatty acids were formed by incubation in 0.5 ml 1 M sodium hydroxide in anhydrous methanol/ benzene (60 : 40, v/v) for 15 min at room temperature. After addition of 0.5 ml 1.4 M hydrochloric acid, 2 ml chloroform and 1.2 ml water, the mixture was centrifuged at 500 x g for 30 min. The aqueous layer was removed by vacuum suction and the solvents were evaporated under N,. The fatty acid methyl esters were isolated from the dried residues by thin-layer chromatography on Silica gel plates (20 X 20 cm, Merck DC-Fertigplatten cat. No. 5721, E. Merck, Darmstadt, F.R.G.) developed in hexane/ toluol (1 : 1, v/v). The dried fatty acid methyl esters were dissolved in isooctan and separated by gasliquid chromatography using a 25 m capillary column, i.d. 0.32 mm, coated with a 0.25 pm layer of cross-linked FFAP from Quadrex. The relative mass contribution of fatty acid methyl esters was monitored by a flame ionization detector and further processed by means of a Shimadzu Chromatopack integrator (Shimadzu, Kyoto, Japan). Known amounts of pure standard fatty acid methyl esters, purchased from Nu-
189 TABLE
II
Livers: phospholipid composition Mean values f S.D. for eight rats unless otherwise
specified.
Phospholipid classes (mg/g fresh liver)
0% Cholesterol casein
soybean
0.5% Cholesterol
Phosphatidylcholine Phosphatidyletanolamine Phosphatidylinositol Phosphatidylserine Sphingomyelin and lyso-phosphatidylethanolamine Lyso-phophatidylcholine
5.9+ 1.3 5050.7 b 1.7*0.1 b 0.5+0.2 0.9*0.4 0.9+0.2 a
6.3+1.3 5.2kO.4 b 1.6+0.4 b 0.4*0.1 0.8 + 0.3 0.6 f 0.2
protein
a Same level of dietary cholesterol, comparison between casein and soybean protein, P < 0.01. b Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented ’ Values for four rats, except phosphatidylcholine and phosphatidylethanolamine which represent
check-Prep, (Elysian, MN) were used for the purpose of identification and quantification. Statistical methods. Statistical analysis was performed by the Student’s f-test method [15]. Results and Discussion Data showing the growth, food consumption and plasma lipid levels of the rats are not given, since all values agreed very well with what has been previously reported from studies with rats of the same type and age-group maintained under corresponding environmental and dietary conditions [2]. Mass proportions
of liver lipids
The contents of protein, total lipids, cholesterol and phospholipids in the livers are shown in Table I. In agreement with previous studies, the total lipid content of the liver was increased by cholesterol supplementation. In rats on cholesterol-free diet, the total lipid concentration (mg/g liver) was higher in the group fed soybean protein than in the group fed casein (P < 0.01). However, the amount of total lipid per whole liver (values not shown) was not significantly different between these groups. In contrast, in groups fed cholesterol-supplemented diet there was no difference in total lipid concentration (mg/g liver) but a significantly increased total lipid mass (mg/liver) in casein-fed versus soybean protein-fed rats (P < 0.01). Both the concentration (mg/g) and total liver content (mg) of free cholesterol and cholesteryl esters, were increased by cholesterol supplementation. Since the liver weight of rats fed cholesterol-supplemented casein diet was increased by 40% compared with that of soybean proteinfed rats, values for protein (P -C O.Ol), free cholesterol (P < 0.05), cholesteryl ester (P < 0.01) and phospholipids (P < 0.01) calculated per total liver were higher in
casein ’
soybean
5.4k1.2 3.3 + 0.7 1.2kO.3 0.5+0.1 l.OkO.3 0.9+0.2
5.4+1.5 3.8 * 0.8 1.1 kO.3 0.5 i 0.2 0.9 f 0.3 0.9 f 0.5
protein ’
diet, P c 0.01. ten rats.
the casein-fed rats, despite a similar concentration (mg/g liver) in the two groups. Sugano [lo], recently reported a huge increase in hepatic cholesterol and triacylglycerol contents (mg/g liver) in rats fed casein, especially in conjunction with palm olein, compared with rats fed on soybean protein. This type of differential effect between casein and soybean protein was not observed in the present studies. The reason for this discrepancy is unclear. As shown in Table II, no dietary protein-related differences in the concentrations of individual phospholipid classes were observed, except for a lower content of lysophosphatidylcholines in rats fed soybean protein in a cholesterol-free diet. This difference still exists when the values are expressed as mg per liver. In that case, there is also a difference between cholesterol-supplemented casein and soybean protein diets with respect to the total amount of liver phosphatidylcholine (PC) recorded (P < 0.01). Dietary cholesterol caused a decreased hepatic concentration (mg/g liver) of phosphatidylethanolamines (PE) and phosphatidylinositols (PI) irrespective of the source of dietary protein. Fatty acid composition
of liver lipids
Triacylglycerols and cholesteryl esters Effects due to cholesterol supplementation. The main effect of cholesterol supplementation seen in both of the two protein groups were increased palmitoleic acid, 16 : 1 (n - 7) and decreased stearic acid, 18 : 0, in both triacylglycerols and cholesteryl esters (Table III). In addition, palmitic acid, 16 : 0, was increased by cholesterol in cholesteryl esters of casein-fed rats. Effect due to dietary protein. In casein-fed rats, compared with soybean-protein-fed rats, the content of linoleic acid, 18 : 2 (n - 6), was reduced by 30-40% in cholesteryl esters, and, in close agreement with the results of Huang et al. [9], by 50-60% in tri-
190 TABLE
III
Fatty acid composition of cholesteryl esters and triacylglycerols Mean value+ Fatty acid
SD. for eight rats unless otherwise Cholesteryl
ester
Triacylglycerol
0% cholesterol casein
16:0 16:1(n-7) 17:o 18:O 18:l (n-9) lS:l(n-7) 18:2(n-6) 2O:l 20:2(n-6) 20:3(n-9) 20:3(n-6) 20:4(n-6) 22:4(n-6) 22:5(n-6) 22:6(n-3)
7.6* 1.0 2.9 + 0.8 0.2*0.2 2.3+1.1 75.9k4.8 0 3.6il.O 0.7 * 0.1 2.1+ 1.9 0.7 f 0.1 0 1.9*1.3 0 0.1 f 0.2 0.4*0.4
in rat liver (% by wt. of total fatty acia3)
specified.
b b b
b ?xb
b
0.5% cholesterol soybean protein
casein ’
7.4+ 1.3 2.1 f 0.3 b 0.1+ 0.2 2.8+ 1.0 b 77.8 + 5.1 0 5.2 f 1.5 0.7*0.1 1.3k1.6 0.3 + 0.2 a,b 0 1.2*1.0 0 0.1& 0.2 0.2kO.3
10.1 f 1.3 8.5 f 1.2 0.2+0.3 1.0*0.2 73.7 f 2.8 0 3.3kO.5 0.7 f 0.1 0 0.1 + 0.2 0 0.8 + 0.1 0 0 0”
a a
a a
=
0% cholesterol
0.5% cholesterol
soybean protein ’
casein
soybean protein
casein ’
8.2 f 0.6 4.3 + 0.9 0.2+0.6 1.1 kO.4 78.1_+ 2.0 0 5.4kO.8 0.7*0.3 0 0 0 1.0*0.1 0 0 0.2 * 0.2
26.0 f 1.4 1.7f0.8 b Ob 1.4*0.7 62.8 f 4.0 0 3.6k1.6 a 0.6+0.1 b 0.6kO.7 0.2kO.3 0 1.6k1.5 0 0 0.3 f 0.4
24.4 + 1.9 1.5ItO.6’ 0
25.4+1.9 4.8fl.O 0.1 kO.1 0.9 + 0.1 63.7 rf:4.3 l.Of2.0 2.9 f 0.4 0.3kO.2 0 0 0 0.3+0.4 0 0 0
’ Same level of dietary cholesterol, comparison between casein and soybean protein, P < 0.01. ’ Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented ’ Values for ten rats.
acylglycerols, respectively. This difference may indicate a reduced availability of linoleic acid in the free fatty acid pool of the liver of casein-fed rats. In the case of cholesterol-supplemented diets, casein produced increased levels of palmitic and palmitoleic acids in the cholesteryl ester fraction; and of palmitoleic acid in triacylglycerols. In addition, a 6% decline in oleic acid, 18 : 1 (n - 9), content was seen in the cholesteryl ester fraction and a 31% reduction of stearic acid in the triacylglycerol fraction of livers from rats fed casein in combination with cholesterol. Phospholipids Effects due to cholesterol supplementation. AS expected, cholesterol supplementation of the diet also led to significant changes in the fatty acyl composition of the phospholipid fractions. Some marked changes were increased oleic acid and reduced stearic acid in all phospholipid fractions and reduced arachidonic acid, 20 : 4 (n - 6) in phosphatidylcholines and phosphatidylinositols. In addition, cholesterol supplementation led to an increased level of palmitic, vaccenic, 18 : 1 (n - 7) and linoleic acids in the phosphatidylinositol fraction. Effects due to dietary protein. In all three hepatic phospholipid fractions studied, the most conspicuous difference was a 2- to 3-fold increase in 5,8,11eicosatrienoic acid, 20 : 3 (n - 9), in casein-fed vis-a-vis soybean protein-fed rats (Table IV). This effect was observed independently of the dietary cholesterol level.
2.2k0.3 b 61.8 f 2.5 0 7.2 k 0.8 0.8 + 0.2 0.1 fO.l 0.1+ 0.2 0 1.2*0.5 b 0 0 0.2 f 0.2
soybean protein’
a =
a a
24.1 i-o.9 2.5 + 0.7 0 1.3kO.4 64.1 k 1.0 0 6.7 + 1.1 0.7 i 0.1 0 0 0 0.3 f 0.2 0 0 0.1*0.2
diet, P < 0.01
Normally, this fatty acid is present in very low amounts in rat liver lipids, but increases to high levels in conditions of essential fatty acid (EFA) deficiency [16]. The linoleic acid content of the diets used in the present experiments was estimated to be about 2 energy 5%.At this level of linoleic acid intake, EFA-deficiency would not be expected to occur [17]. Moreover, there were no visual signs of EFA-deficiency in the rats. Therefore, the comparatively high level of 20 : 3 (n - 9) occurring in hepatic phospholipids of casein-fed rats, not least in the phosphatidylinositol fraction (see below), is a most interesting finding indicating dietary protein-related changes in the metabolic turnover of polyunsaturated fatty acids. In agreement with the result of Huang et al. [9] and Sugano et al. [lo], the ratio of arachidonate to linoleate in rat liver tissue phospholipids (PC and PE) was higher, using cholesterol-free diets, when the dietary protein was casein than when it was soybean protein, suggesting a protein-dependent modulation of the desaturation of linoleate. It remains to be seen whether these changes bear any causal relationship to a reduced availability of linoleic acid in the liver of casein-fed rats as discussed above. In the phosphatidylinositol fraction, 20 : 3 (n - 9) amounted to as much as about 13% of the fatty acid mass in casein-fed rats on cholesterol-free diet. As appears from the values in Table IV, the ratio of 20 : 3 (n - 9) to arachidonic acid in the phosphatidylinositol fraction of casein-fed rats is close to 0.6, compared with a ratio of about 0.2 in soybean protein-fed rats. The
21.4rt2.3 0.9kO.2 b 0.3 + 0.2 21.9+ 1.0 b 15.9* 1.7 b 3.7 f 0.5 a 8.4* 1.2 4b 0.6rtO.2 0 3.4*0.7 a 1.3*0.1 b 17.2k 2.3 b 0 0.7 + 0.2 = 3.3 f 0.8
21.0* 1.1 0.7*0.2 0.1+0.2 23.6 f 1.5 b 14.1 f 1.3 b 2.4 f 0.3 11.1 f0.8 0.4kO.2 0.0*0.1 1.0*0.1 1.4fO.lb 19.1 f 1.9 b 0 0.3 *0.2 3.9 f 0.8 22.6 f 2.1 2.4kO.9 0.1 f 0.2 14.9 + 0.6 23.6 + 2.7 2.9 + 3.3 11.5fl.l 0.7*0.3 0.3 + 0.6 2.6& 1.0 2.OkO.5 12.2 f 2.8 0 0.7 * 0.3 2.6 k 0.5 a
=
a a
a
23.0+ 1.8 0.9 f 0.6 0.0+0.1 18.6* 1.3 20.1+ 1.9 1.6+2.0 12.4+ 1.9 0.4 f 0.2 0.2 * 0.3 0.9 * 0.5 2.0 f 0.5 15.5 + 2.5 0 0.6 + 0.3 3.4 + 0.6
12.6 f 0.6 0.5+0.3 0.1+ 0.1 24.6 + 0.7 7.7 + 0.6 4.7 f 0.7 11.0+1.3 0.5 f 0.2 0.0 * 0.1 1.8*0.4’ 1.0*0.1 24.8k1.3 0.1+0.1 l.OkO.4 8.5k1.3
casein
a
b
a*b a.b B a
11.5+1.0 0.2 + 0.2 0.2*0.1 27.7* 1.0 5.3kO.8 b 2.9 kO.4 13.1*0.9 b 0.3 f 0.2 0.0+0.1 0.7kO.l 1.0*0.1 b 24.3 + 0.8 0.5 f 0.4 b 0.7 + 0.2 b 10.6 + 2.0
soybean protein
0% cholesterol
casein soybean protein ’
0.5% cholesterol casein ’
0% cholesterol
soybean protein
Phosphatidyletanolamine
Phosphatidylcholine
12.5 +0.5 0.8 +0.7 0.1 f 0.2 21.4k2.3 = 11.2k2.4 ’ 3.4+ 3.6 9.5 f 2.8 0.5 f 0.3 0 1.9kO.8 a 1.4+0.1 23.2 + 2.5 0.1+ 0.2 1.5 *0.3 8.6 + 1.1 a
casein ’
d Values for four rats.
12.3+1.0 0.4 + 0.3 0.1 f 0.1 25.5 f 3.4 7.9* 1.2 1.7* 1.8 9.8 + 3.0 0.5 f 0.1 0.1 f 0.2 0.8 + 0.4 1.3+0.2 24.9 + 2.6 0.2 + 0.2 1.6kO.4 11.2+1.7
soybean protein ’
0.5% cholesterol
a Same level of dietary cholesterol, comparison between casein and soybean protein, P -c0.01. b Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented diet, P < 0.01. ' Values for ten rats.
16:O 16:1(n-7) 17:o 18:O 18:1(n-9) 18:1(n-7) 18:2(n-6) 2O:l 20:2(n-6) 20:3(n-9) 20:3(n-6) 20:4(n-6) 22:4(n-6) 22:5(n-6) 22:6(n-3)
Fatty acid
Mean vahtef SD. for eight rats unless otherwise specified.
Fatty acid composition of phospholipidr in rat Iiver (5%by wt. of total fatty acids)
TABLE IV
3.8f0.6 b 0.3 f 0.2 b 0.2 * 0.2 40.3f1.4b 4.3 f 1.2 b 2.3 + 0.8 b 3.8k1.6 b 0.5 + 0.1 a 0.1+ 0.1 b 13.3k2.7 a 1.9+0.2 a 22.1+ 1.9 a.b 0.4f0.2 a 1.6kO.4 2.7 f 0.6
casein
0% cholesterol
4.3 + 0.8 b 0.2+0.1 b 0.1 f 0.1 b 41.7+ 1.8 b 3.OkO.6 b 1.6kO.6 b 3.8kl.l b 0.2 + 0.1 b 0.2 + 0.2 5.2kO.9 2.7 f 0.3 30.3+1.7 b 0.6 + 0.1 b 1.2*0.2 3.3 + 0.6
soybean protein
Phosphatidylinositol
s.9*1.1 0.9 * 0.2 0.4*0.1 29.0 f 1.4 7.6&-0.7 5.9 * 0.4 6.9kl.O 0.6kO.l 1.8kO.8 9.9k1.2 2.2 f 0.2 16.9+ 1.4 0.4+0.0 1.3kO.l 3.1+ 0.4
casein d
=
a
a
a
= a a
9.3 f 1.9 0.5 f 0.1 0.3 f 0.0 34.4* 1.1 6.2 f 0.7 3.5 f 0.8 7.4 f 0.9 0.4*0.1 0.7 f 0.8 5.5 *0.4 2.5 f 0.2 22.2 f 1.7 0.4*0.0 1.5 rto.3 3.3kO.3
soybean protein d
0.5% cholesterol
192 possible biological implications of this comparatively high level of 20 : 3 (n - 9) are unclear. Recent studies suggest that the liver may have an important role in supplying extrahepatic cells such as platelets and arterial endothelial cells with eicosapolyenoic acids for the production of biologically active mediators such as prostaglandins, thromboxanes and leukotrienes [l&19]. Incubation of platelets with 20 : 3 (n - 9) leads to the production of a monohydroxy derivative capable of stimulating platelet aggregation [20]. Lefkowith et al. [21] recently reported that 20 : 3 (n - 9) leads to decreased leukotriene C, and B4 synthesis from arachidonate when macrophages are incubated with exogenous arachidonate and 20 : 3 (n - 9). Thus, increased hepatic production of 20 : 3 (n - 9) may influence atherogenic processes provided that this fatty acid is exported from the liver by way of plasma lipoproteins. Another aspect of interest in this connection is the proposed role for phosphatidylinositols in secretary processes of the liver [22]. It is an interesting possibility that these changes in fatty acid composition of the phosphatidylinositol fraction may be causally related to the increased rate of lipoprotein secretion known to occur in casein-fed rats [3]. In addition to the difference in the proportions of 20 : 3 (n - 9), other protein-related discrepancies were observed. The casein diet, in comparison with the soybean protein diet, led to a higher level of oleic acid (in PC and PE) and to a reduced level of stearic acid in all three phospholipid fractions studied, especially in rats fed cholesterol-supplemented diet. Moreover, the casein diet caused a reduced level of arachidonic acid in the phosphatidylinositol fraction and of linoleic acid in phosphatidylcholines and phosphatidylethanolamines in rats fed cholesterol-free diet. Congruously, a casein-dependent reduction in the level of linoleic acid in total phospholipids [9] and the phosphatidylcholine fraction [lo], has recently been reported. In conclusion, the present studies have provided evidence, that the source of dietary protein affects the fatty acid composition of hepatic phospholipids and, especially, the phosphatidylinositol fraction. Since it involves the levels of linoleic acid, arachidonic acid and 20 : 3 (n - 9), the phenomenon may have wider implications concerning biochemical and physiological processes. Future studies will be aimed at determining, if there is any direct association between the changes in fatty acid pattern of liver lipids, and the hyperlipidemic response of rats to casein, vis-a-vis soybean protein.
Furthermore, the possible impact of an increased hepatic 20 : 3 (n - 9) to arachidonic acid ratio to atherogenic mechanisms will be studied. Acknowledgements This investigation was supported by grants from the Swedish Medical Research Council (Projekt No. 03X5172), from the Swedish Nutrition Foundation, from Hassle AB, Molndal and from the Faculty of Medicine at Uppsala University. The authors thank Ms. Elwy Wallin and Mr. Bengt Ejdesjo for skilful technical assistance. Thanks are also due to Dr. Sten Johnsson, managing director at AB Karlshamns Cirkelprodukter, AB Karlshamns Oljefabriker, Karlshamn, for the gift of soybean protein isolate. We are indebted to Dr. LarsBbrje Croon, The National Food Administration, Uppsala, for valuable help in performing fatty acid analysis by capillary gas-liquid chromatography. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
Terpstra, A.H.M.,
Hermus, R.J.J. and West, C.E. (1983) World Rev. Nutr. Diet 42, l-55. Eklund, A. and Sjoblom, L. (1986) Biochim. Biophys. Acta 877, 127-134. Pfeuffer, M. (1989) Atherosclerosis 76, 89-91. Cole, T.G., Kuisk, I., Patsch, W. and Schonfelt, G. (1984) J. Lipid Res. 25, 593-603. EkIund, A., Sjoblom, L. and Gstlund-Lindqvist, A.-M. (1986) Atherosclerosis 62, 129-136. Nagata, Y., Isbiwaka, N. and Sugano, M. (1982) J. Nutr. 112, 1614-1625. Pfeuffer, M. and Barth, C.A. (1986) Annl. Nutr. Metab. 30, 281-288. Chao, Y.-S., Yamin, T.-T. and Alberts, A.W. (1982) J. Biol. Chem. 257, 3623-3627. Huang, Y.S., Cuanne, SC. and Horrobin, D.F. (1986) Proc. Sot. Exp. Biol. Med. 181, 399-403. Sugano, M., Ishida, T. and Koba, K. (1988) J. Nutr. 118,548-554. Williams, M.A. and Briggs, G.M. (1963) Fed. Proc. 22, 261. Eklund, A. (1980) Annl. Nutr. Metab. 24, 91-101. Arvidson, G.A.E. (1968) European J. B&hem. 4, 478-486. Chen, P.S., Jr., Toribara, T.Y. and Warner, H. (1956) Anal. Chem. 28, 1756-1758. Snedecor, G.W. and Cochran, W.G. (1967) Statistical Methods, 6th Edn., Iowa State University Press, Ames. Weiner, T.W. and Sprecher, H. (1984) Biochim. Biophys. Acta 792, 293-303. Hansen, H.S. (1983) World Rev. Nutr. Diet. 42, 102-134. Spector, A.A., Kaduce, T.L., Hoak, J.C. and Fry, G.L. (1981) J. Clin. Invest. 68, 1003-1011. Needleman, SW., Spector, A.A. and Hoak, J.C. (1982) Prostaglandins 24, 607-622. Lagarde, M., Burtin, M., Sprecher, H., Dechavanne, M. and Renaud, S. (1983) Lipids 18, 291-294. Lefkowith, J.B., Jakschik, B.A., Stahl, P. and Needleman, P. (1987) J. Biol. Chem. 262, 6668-6675. Holub, B.J. (1986) Annu. Rev. Nutr. 6, 563-597.
187
(1990) 187-192
BBALIP 53400
Dietary protein and fatty acid composition of liver lipids in the rat LinnCa Sj6blom and Anders Eklund Department
ofMedico1and Physiological Chemistry
University of Uppsala, Vppsala (Sweden)
(Received 26 June 1989) (Revised manuscript received 26 January 1990)
Key words: A~a~~do~c acid; Dietary protein; Icosatrienoic acid; Fatty acid; Linoleic acid; Phosphatidyli~ositol: (Male rat); (Liver)
In order to compare the effects of different sources of dietary protein on the fatty acid composition of phosphatidylcholines (PC), p~sphatidyleth~ol~~nes (PE), phosphatidylinositols (PI), choleste~l esters and ~acyl~ycerols, male rats were fed for a 4-week period on cholesterol-free, or cholesterol-containing, diets based on casein, or soybean protein and olive oil. The most conspicuous difference observed was the occurrence of significantly higher levels of 5,8,1&eicosatrienoic acid, 20:3 (n - 9), in the different lipid classes of case&fed, compared with soybean pro&+fed, animals. In the PI fraction of livers from the groups of rats fed casein diet, this fatty acid amounted to between 9.9 and 13.3% by weight of the total fatty acids. Phospholipids from livers of casein-fed rats contained increased levels of oleic acid, 18: 1 (n - 9) (in PC and PE) and reduced levels of stearic acid (18 : 0). Moreover, in this group of rats PI contained a reduced level of arachidonic acid, 20: 4 (n - 6). A casein-related decrease in the linoleic acid, 18: 2 (n - 6), content of PC and PE was observed only in the rats fed on cholesterol-free diet. Effects on the fatty acid composition were also observed in the triacylglycerol and cholesteryl ester fractions, in which the rats fed casein diet showed higher levels of palmitoleic acid, 16: 1 (n - 7) (cholesterol-supplemented diet) and lower values for linoleic acid, than the soybean p~tein-fed rats.
Introduction
it
Like in other animal species, enhanced plasma cholesterol levels can be produced in rats by dietary cholesterol administration. It has been recognized for some time, that this response to dietary cholesterol is under strong influence of the type of dietary protein [l-3]. In rats, this type of hypercholesterolemia is largely due to an increased concentration of plasma very-lowdensity lipoprotein (VLDL) [2]. In contrast to normal plasma VLDL, VLDL from hypercholesterolemic rats has the ability to promote the in vivo acc~~lation of cholesteryl esters in macrophages [4,5]. This accumulation of choiesteryl esters in macrophages has also been shown to be influenced by dietary protein-related effects on plasma VLDL [5]. Since VLDL is manufactured primarily by the liver,
is of interest to study any dietary protein-related changes in metabolic fictions of the liver, which are connected to the concentration and properties of plasma VLDL. Previous studies have demonstrated effects on hepatic sterogenesis 161, hepatic secretion of VLDL [7] and hepatic expression of LDL-receptors [Sl. Recently, Huang et al. [9] and Sugano et al. [lo] have shown effects of casein and soybean protein on the fatty acid composition of plasma and liver. In these papers, the hepatic phospholipids studied were total phospholipids [9], or phosphatidylcholines [lo]. In contrast, the present studies were aimed at unravelling dietary proteindependent effects on mass proportions and fatty acid composition of individual phospholipid classes in the liver of male rats fed on cholesterol-free as well as cholesterol-supplemented diets.
Materials and Methods Abbreviations: PC, p~osphatidyl~holines; PE, phosphatidylethanolamines; PI, phosphatidylinositols; VLDL, very-low-density lipoprotein; EFA, essential fatty acid. Correspondence: A. Eklund, Department of Medical and Physiological Chemistry, U~versity of Uppsala, P.Q. Box 575, S-751 23 Up. psala, Sweden. OOW2760/90/$63.50
Feeding experiments. The composition of the diets was by percentage: protein fcasein (dry wt. composition: 95% protein, 0.4% fat, 3% ash), Cat. No. 1.2600 (Kebo-Grave, Stockholm, Sweden); or soybean protein isolate, Purina Protein 590 E (dry wt. composition: 97% protein, 0.6% fat, 4% ash) supplied by Karlshamns
0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
188 TABLE
I
Mass proportions of liver lipids Mean value f SD. for eight rats unless otherwise I tern
specified.
0% Cholesterol casein
Liver weight (g) (g/100 g body wt.) Protein (mg/g) Total lipid (mg/g) free cholesterol (mg/g) cholesteryl ester (mg/g) phospholipids (mg/g)
11.Ok1.3
0.5% Cholesterol soybean
a.b
3.9 f 0.2 h 183 +7 56 +6 a,’ 2.3 : 0.3 ’ 1.4kO.6’ 15.7* 1.9
protein
9.0+ 0.9 3.6k 0.3 178 +11 71 f8h 2.8k 0.5 ’ 1.7+ 0.8 b 16.9* 2.7
casein ’
soybean
13.0+ 1.5 a 4.8* 0.3 a 175 *20 133 *20 5.6+ 2.5 12.9* 3.5 15.5* 2.2
9.3+ 1.4 3.9k 0.5 184 *26 126 k16 4.4* 1.4 11.1 f 3.3 16.0+ 3.1
a Same level of dietary cholesterol, comparison between casein and soybean protein, P i 0.01. h Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented ’ Values for ten rats.
Oljefabriker (Karlshamn, Sweden), 20; olive oil, 10; cholesterol (EGA Cat. No. C7, 520-9, dissolved in olive oil before inclusion in diet), 0 and 0.5, respectively; vitamin premix [2], 2; mineral mixture (Williams & Briggs salt mixture [ll]), 3.5; cellulose powder (Ricenta, Stockholm, Sweden) 5.3; and corn starch to 100. The percentage of calories provided by protein, fat and carbohydrates, respectively, was 21, 23 and 56 energy %, in all diets. In a first experiment six rats were fed on each of the diets supplemented with 0.5% cholesterol. In a second experiment, four rats were fed on each of the cholesterol-supplemented diets and eight rats were fed on each of the cholesterol-free diets. Since the results from the corresponding groups of the two different experiments were in excellent agreement, the results were combined. In each experiment, 21-day-old, outbred Sprague-Dawley strain rats (Alab, Sollentuna, Sweden), weighing about 65 g, were housed in subgroups of three or four in macrolon cages at 25 o C and 50% relative humidity in rooms with a 12 h (06.00 to 18.00 h) light-dark cycle. They were allowed free access to food and fresh tap water. After 4 weeks on the respective regimen, they were anaesthetized with diethyl ether and killed by terminal exsanguination. All animals were fasted for 4 h before sampling between 10.00-11.00 h. Livers were removed, weighed and lyophilized. Protein content was calculated from nitrogen analysis [2]. Extraction of liver lipids. Total lipid content of livers was determined as previously described [12]. Cholesterof analysis. The cholesterol level in the total lipid fraction of the liver was determined using an enzymatic method (Boehringer, Mannheim, F.R.G., Cat.No. 237574). Thin-layer chromatography. Lipid classes were separated by thin-layer chromatography in a two-step procedure using Silica gel plates (20 X 20 cm, Merck DCFertigplatten cat. No. 5721, E. Merck, Darmstadt,
protein’
diet. P < 0.01.
F.R.G.). The first step was performed as described by Arvidson [13] for separation of subclasses of phospholipids. In a second step, on the same plate, the neutral lipids at the front were developed in hexane/ diethyl ether/acetic acid, (80: 20: 1, v/v). The spots were identified by comparison with authentic standards after visualization with, 2’,7’-dichlorofluorescein (Merck, Cat. No. 9677, E. Merck, Darmstadt, F.R.G.). All solvents system used contained 0.002% (w/v) butylated hydroxyanisole (BHA). By this procedure triacylglycerols, cholesteryl esters and different phospholipid fractions were isolated, extracted from the plates [ 131 and dried under a stream of N,. Phosphorus analysis. Phospholipid determination was performed by phosphorus analysis of the liver total lipid extract and separated fractions according to a modification of the method of Chen., Jr. et al. [14]. Gas-liquid chromatography. Methyl esters of fatty acids were formed by incubation in 0.5 ml 1 M sodium hydroxide in anhydrous methanol/ benzene (60 : 40, v/v) for 15 min at room temperature. After addition of 0.5 ml 1.4 M hydrochloric acid, 2 ml chloroform and 1.2 ml water, the mixture was centrifuged at 500 x g for 30 min. The aqueous layer was removed by vacuum suction and the solvents were evaporated under N,. The fatty acid methyl esters were isolated from the dried residues by thin-layer chromatography on Silica gel plates (20 X 20 cm, Merck DC-Fertigplatten cat. No. 5721, E. Merck, Darmstadt, F.R.G.) developed in hexane/ toluol (1 : 1, v/v). The dried fatty acid methyl esters were dissolved in isooctan and separated by gasliquid chromatography using a 25 m capillary column, i.d. 0.32 mm, coated with a 0.25 pm layer of cross-linked FFAP from Quadrex. The relative mass contribution of fatty acid methyl esters was monitored by a flame ionization detector and further processed by means of a Shimadzu Chromatopack integrator (Shimadzu, Kyoto, Japan). Known amounts of pure standard fatty acid methyl esters, purchased from Nu-
189 TABLE
II
Livers: phospholipid composition Mean values f S.D. for eight rats unless otherwise
specified.
Phospholipid classes (mg/g fresh liver)
0% Cholesterol casein
soybean
0.5% Cholesterol
Phosphatidylcholine Phosphatidyletanolamine Phosphatidylinositol Phosphatidylserine Sphingomyelin and lyso-phosphatidylethanolamine Lyso-phophatidylcholine
5.9+ 1.3 5050.7 b 1.7*0.1 b 0.5+0.2 0.9*0.4 0.9+0.2 a
6.3+1.3 5.2kO.4 b 1.6+0.4 b 0.4*0.1 0.8 + 0.3 0.6 f 0.2
protein
a Same level of dietary cholesterol, comparison between casein and soybean protein, P < 0.01. b Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented ’ Values for four rats, except phosphatidylcholine and phosphatidylethanolamine which represent
check-Prep, (Elysian, MN) were used for the purpose of identification and quantification. Statistical methods. Statistical analysis was performed by the Student’s f-test method [15]. Results and Discussion Data showing the growth, food consumption and plasma lipid levels of the rats are not given, since all values agreed very well with what has been previously reported from studies with rats of the same type and age-group maintained under corresponding environmental and dietary conditions [2]. Mass proportions
of liver lipids
The contents of protein, total lipids, cholesterol and phospholipids in the livers are shown in Table I. In agreement with previous studies, the total lipid content of the liver was increased by cholesterol supplementation. In rats on cholesterol-free diet, the total lipid concentration (mg/g liver) was higher in the group fed soybean protein than in the group fed casein (P < 0.01). However, the amount of total lipid per whole liver (values not shown) was not significantly different between these groups. In contrast, in groups fed cholesterol-supplemented diet there was no difference in total lipid concentration (mg/g liver) but a significantly increased total lipid mass (mg/liver) in casein-fed versus soybean protein-fed rats (P < 0.01). Both the concentration (mg/g) and total liver content (mg) of free cholesterol and cholesteryl esters, were increased by cholesterol supplementation. Since the liver weight of rats fed cholesterol-supplemented casein diet was increased by 40% compared with that of soybean proteinfed rats, values for protein (P -C O.Ol), free cholesterol (P < 0.05), cholesteryl ester (P < 0.01) and phospholipids (P < 0.01) calculated per total liver were higher in
casein ’
soybean
5.4k1.2 3.3 + 0.7 1.2kO.3 0.5+0.1 l.OkO.3 0.9+0.2
5.4+1.5 3.8 * 0.8 1.1 kO.3 0.5 i 0.2 0.9 f 0.3 0.9 f 0.5
protein ’
diet, P c 0.01. ten rats.
the casein-fed rats, despite a similar concentration (mg/g liver) in the two groups. Sugano [lo], recently reported a huge increase in hepatic cholesterol and triacylglycerol contents (mg/g liver) in rats fed casein, especially in conjunction with palm olein, compared with rats fed on soybean protein. This type of differential effect between casein and soybean protein was not observed in the present studies. The reason for this discrepancy is unclear. As shown in Table II, no dietary protein-related differences in the concentrations of individual phospholipid classes were observed, except for a lower content of lysophosphatidylcholines in rats fed soybean protein in a cholesterol-free diet. This difference still exists when the values are expressed as mg per liver. In that case, there is also a difference between cholesterol-supplemented casein and soybean protein diets with respect to the total amount of liver phosphatidylcholine (PC) recorded (P < 0.01). Dietary cholesterol caused a decreased hepatic concentration (mg/g liver) of phosphatidylethanolamines (PE) and phosphatidylinositols (PI) irrespective of the source of dietary protein. Fatty acid composition
of liver lipids
Triacylglycerols and cholesteryl esters Effects due to cholesterol supplementation. The main effect of cholesterol supplementation seen in both of the two protein groups were increased palmitoleic acid, 16 : 1 (n - 7) and decreased stearic acid, 18 : 0, in both triacylglycerols and cholesteryl esters (Table III). In addition, palmitic acid, 16 : 0, was increased by cholesterol in cholesteryl esters of casein-fed rats. Effect due to dietary protein. In casein-fed rats, compared with soybean-protein-fed rats, the content of linoleic acid, 18 : 2 (n - 6), was reduced by 30-40% in cholesteryl esters, and, in close agreement with the results of Huang et al. [9], by 50-60% in tri-
190 TABLE
III
Fatty acid composition of cholesteryl esters and triacylglycerols Mean value+ Fatty acid
SD. for eight rats unless otherwise Cholesteryl
ester
Triacylglycerol
0% cholesterol casein
16:0 16:1(n-7) 17:o 18:O 18:l (n-9) lS:l(n-7) 18:2(n-6) 2O:l 20:2(n-6) 20:3(n-9) 20:3(n-6) 20:4(n-6) 22:4(n-6) 22:5(n-6) 22:6(n-3)
7.6* 1.0 2.9 + 0.8 0.2*0.2 2.3+1.1 75.9k4.8 0 3.6il.O 0.7 * 0.1 2.1+ 1.9 0.7 f 0.1 0 1.9*1.3 0 0.1 f 0.2 0.4*0.4
in rat liver (% by wt. of total fatty acia3)
specified.
b b b
b ?xb
b
0.5% cholesterol soybean protein
casein ’
7.4+ 1.3 2.1 f 0.3 b 0.1+ 0.2 2.8+ 1.0 b 77.8 + 5.1 0 5.2 f 1.5 0.7*0.1 1.3k1.6 0.3 + 0.2 a,b 0 1.2*1.0 0 0.1& 0.2 0.2kO.3
10.1 f 1.3 8.5 f 1.2 0.2+0.3 1.0*0.2 73.7 f 2.8 0 3.3kO.5 0.7 f 0.1 0 0.1 + 0.2 0 0.8 + 0.1 0 0 0”
a a
a a
=
0% cholesterol
0.5% cholesterol
soybean protein ’
casein
soybean protein
casein ’
8.2 f 0.6 4.3 + 0.9 0.2+0.6 1.1 kO.4 78.1_+ 2.0 0 5.4kO.8 0.7*0.3 0 0 0 1.0*0.1 0 0 0.2 * 0.2
26.0 f 1.4 1.7f0.8 b Ob 1.4*0.7 62.8 f 4.0 0 3.6k1.6 a 0.6+0.1 b 0.6kO.7 0.2kO.3 0 1.6k1.5 0 0 0.3 f 0.4
24.4 + 1.9 1.5ItO.6’ 0
25.4+1.9 4.8fl.O 0.1 kO.1 0.9 + 0.1 63.7 rf:4.3 l.Of2.0 2.9 f 0.4 0.3kO.2 0 0 0 0.3+0.4 0 0 0
’ Same level of dietary cholesterol, comparison between casein and soybean protein, P < 0.01. ’ Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented ’ Values for ten rats.
acylglycerols, respectively. This difference may indicate a reduced availability of linoleic acid in the free fatty acid pool of the liver of casein-fed rats. In the case of cholesterol-supplemented diets, casein produced increased levels of palmitic and palmitoleic acids in the cholesteryl ester fraction; and of palmitoleic acid in triacylglycerols. In addition, a 6% decline in oleic acid, 18 : 1 (n - 9), content was seen in the cholesteryl ester fraction and a 31% reduction of stearic acid in the triacylglycerol fraction of livers from rats fed casein in combination with cholesterol. Phospholipids Effects due to cholesterol supplementation. AS expected, cholesterol supplementation of the diet also led to significant changes in the fatty acyl composition of the phospholipid fractions. Some marked changes were increased oleic acid and reduced stearic acid in all phospholipid fractions and reduced arachidonic acid, 20 : 4 (n - 6) in phosphatidylcholines and phosphatidylinositols. In addition, cholesterol supplementation led to an increased level of palmitic, vaccenic, 18 : 1 (n - 7) and linoleic acids in the phosphatidylinositol fraction. Effects due to dietary protein. In all three hepatic phospholipid fractions studied, the most conspicuous difference was a 2- to 3-fold increase in 5,8,11eicosatrienoic acid, 20 : 3 (n - 9), in casein-fed vis-a-vis soybean protein-fed rats (Table IV). This effect was observed independently of the dietary cholesterol level.
2.2k0.3 b 61.8 f 2.5 0 7.2 k 0.8 0.8 + 0.2 0.1 fO.l 0.1+ 0.2 0 1.2*0.5 b 0 0 0.2 f 0.2
soybean protein’
a =
a a
24.1 i-o.9 2.5 + 0.7 0 1.3kO.4 64.1 k 1.0 0 6.7 + 1.1 0.7 i 0.1 0 0 0 0.3 f 0.2 0 0 0.1*0.2
diet, P < 0.01
Normally, this fatty acid is present in very low amounts in rat liver lipids, but increases to high levels in conditions of essential fatty acid (EFA) deficiency [16]. The linoleic acid content of the diets used in the present experiments was estimated to be about 2 energy 5%.At this level of linoleic acid intake, EFA-deficiency would not be expected to occur [17]. Moreover, there were no visual signs of EFA-deficiency in the rats. Therefore, the comparatively high level of 20 : 3 (n - 9) occurring in hepatic phospholipids of casein-fed rats, not least in the phosphatidylinositol fraction (see below), is a most interesting finding indicating dietary protein-related changes in the metabolic turnover of polyunsaturated fatty acids. In agreement with the result of Huang et al. [9] and Sugano et al. [lo], the ratio of arachidonate to linoleate in rat liver tissue phospholipids (PC and PE) was higher, using cholesterol-free diets, when the dietary protein was casein than when it was soybean protein, suggesting a protein-dependent modulation of the desaturation of linoleate. It remains to be seen whether these changes bear any causal relationship to a reduced availability of linoleic acid in the liver of casein-fed rats as discussed above. In the phosphatidylinositol fraction, 20 : 3 (n - 9) amounted to as much as about 13% of the fatty acid mass in casein-fed rats on cholesterol-free diet. As appears from the values in Table IV, the ratio of 20 : 3 (n - 9) to arachidonic acid in the phosphatidylinositol fraction of casein-fed rats is close to 0.6, compared with a ratio of about 0.2 in soybean protein-fed rats. The
21.4rt2.3 0.9kO.2 b 0.3 + 0.2 21.9+ 1.0 b 15.9* 1.7 b 3.7 f 0.5 a 8.4* 1.2 4b 0.6rtO.2 0 3.4*0.7 a 1.3*0.1 b 17.2k 2.3 b 0 0.7 + 0.2 = 3.3 f 0.8
21.0* 1.1 0.7*0.2 0.1+0.2 23.6 f 1.5 b 14.1 f 1.3 b 2.4 f 0.3 11.1 f0.8 0.4kO.2 0.0*0.1 1.0*0.1 1.4fO.lb 19.1 f 1.9 b 0 0.3 *0.2 3.9 f 0.8 22.6 f 2.1 2.4kO.9 0.1 f 0.2 14.9 + 0.6 23.6 + 2.7 2.9 + 3.3 11.5fl.l 0.7*0.3 0.3 + 0.6 2.6& 1.0 2.OkO.5 12.2 f 2.8 0 0.7 * 0.3 2.6 k 0.5 a
=
a a
a
23.0+ 1.8 0.9 f 0.6 0.0+0.1 18.6* 1.3 20.1+ 1.9 1.6+2.0 12.4+ 1.9 0.4 f 0.2 0.2 * 0.3 0.9 * 0.5 2.0 f 0.5 15.5 + 2.5 0 0.6 + 0.3 3.4 + 0.6
12.6 f 0.6 0.5+0.3 0.1+ 0.1 24.6 + 0.7 7.7 + 0.6 4.7 f 0.7 11.0+1.3 0.5 f 0.2 0.0 * 0.1 1.8*0.4’ 1.0*0.1 24.8k1.3 0.1+0.1 l.OkO.4 8.5k1.3
casein
a
b
a*b a.b B a
11.5+1.0 0.2 + 0.2 0.2*0.1 27.7* 1.0 5.3kO.8 b 2.9 kO.4 13.1*0.9 b 0.3 f 0.2 0.0+0.1 0.7kO.l 1.0*0.1 b 24.3 + 0.8 0.5 f 0.4 b 0.7 + 0.2 b 10.6 + 2.0
soybean protein
0% cholesterol
casein soybean protein ’
0.5% cholesterol casein ’
0% cholesterol
soybean protein
Phosphatidyletanolamine
Phosphatidylcholine
12.5 +0.5 0.8 +0.7 0.1 f 0.2 21.4k2.3 = 11.2k2.4 ’ 3.4+ 3.6 9.5 f 2.8 0.5 f 0.3 0 1.9kO.8 a 1.4+0.1 23.2 + 2.5 0.1+ 0.2 1.5 *0.3 8.6 + 1.1 a
casein ’
d Values for four rats.
12.3+1.0 0.4 + 0.3 0.1 f 0.1 25.5 f 3.4 7.9* 1.2 1.7* 1.8 9.8 + 3.0 0.5 f 0.1 0.1 f 0.2 0.8 + 0.4 1.3+0.2 24.9 + 2.6 0.2 + 0.2 1.6kO.4 11.2+1.7
soybean protein ’
0.5% cholesterol
a Same level of dietary cholesterol, comparison between casein and soybean protein, P -c0.01. b Same source of dietary protein, comparison between cholesterol-free and cholesterol-supplemented diet, P < 0.01. ' Values for ten rats.
16:O 16:1(n-7) 17:o 18:O 18:1(n-9) 18:1(n-7) 18:2(n-6) 2O:l 20:2(n-6) 20:3(n-9) 20:3(n-6) 20:4(n-6) 22:4(n-6) 22:5(n-6) 22:6(n-3)
Fatty acid
Mean vahtef SD. for eight rats unless otherwise specified.
Fatty acid composition of phospholipidr in rat Iiver (5%by wt. of total fatty acids)
TABLE IV
3.8f0.6 b 0.3 f 0.2 b 0.2 * 0.2 40.3f1.4b 4.3 f 1.2 b 2.3 + 0.8 b 3.8k1.6 b 0.5 + 0.1 a 0.1+ 0.1 b 13.3k2.7 a 1.9+0.2 a 22.1+ 1.9 a.b 0.4f0.2 a 1.6kO.4 2.7 f 0.6
casein
0% cholesterol
4.3 + 0.8 b 0.2+0.1 b 0.1 f 0.1 b 41.7+ 1.8 b 3.OkO.6 b 1.6kO.6 b 3.8kl.l b 0.2 + 0.1 b 0.2 + 0.2 5.2kO.9 2.7 f 0.3 30.3+1.7 b 0.6 + 0.1 b 1.2*0.2 3.3 + 0.6
soybean protein
Phosphatidylinositol
s.9*1.1 0.9 * 0.2 0.4*0.1 29.0 f 1.4 7.6&-0.7 5.9 * 0.4 6.9kl.O 0.6kO.l 1.8kO.8 9.9k1.2 2.2 f 0.2 16.9+ 1.4 0.4+0.0 1.3kO.l 3.1+ 0.4
casein d
=
a
a
a
= a a
9.3 f 1.9 0.5 f 0.1 0.3 f 0.0 34.4* 1.1 6.2 f 0.7 3.5 f 0.8 7.4 f 0.9 0.4*0.1 0.7 f 0.8 5.5 *0.4 2.5 f 0.2 22.2 f 1.7 0.4*0.0 1.5 rto.3 3.3kO.3
soybean protein d
0.5% cholesterol
192 possible biological implications of this comparatively high level of 20 : 3 (n - 9) are unclear. Recent studies suggest that the liver may have an important role in supplying extrahepatic cells such as platelets and arterial endothelial cells with eicosapolyenoic acids for the production of biologically active mediators such as prostaglandins, thromboxanes and leukotrienes [l&19]. Incubation of platelets with 20 : 3 (n - 9) leads to the production of a monohydroxy derivative capable of stimulating platelet aggregation [20]. Lefkowith et al. [21] recently reported that 20 : 3 (n - 9) leads to decreased leukotriene C, and B4 synthesis from arachidonate when macrophages are incubated with exogenous arachidonate and 20 : 3 (n - 9). Thus, increased hepatic production of 20 : 3 (n - 9) may influence atherogenic processes provided that this fatty acid is exported from the liver by way of plasma lipoproteins. Another aspect of interest in this connection is the proposed role for phosphatidylinositols in secretary processes of the liver [22]. It is an interesting possibility that these changes in fatty acid composition of the phosphatidylinositol fraction may be causally related to the increased rate of lipoprotein secretion known to occur in casein-fed rats [3]. In addition to the difference in the proportions of 20 : 3 (n - 9), other protein-related discrepancies were observed. The casein diet, in comparison with the soybean protein diet, led to a higher level of oleic acid (in PC and PE) and to a reduced level of stearic acid in all three phospholipid fractions studied, especially in rats fed cholesterol-supplemented diet. Moreover, the casein diet caused a reduced level of arachidonic acid in the phosphatidylinositol fraction and of linoleic acid in phosphatidylcholines and phosphatidylethanolamines in rats fed cholesterol-free diet. Congruously, a casein-dependent reduction in the level of linoleic acid in total phospholipids [9] and the phosphatidylcholine fraction [lo], has recently been reported. In conclusion, the present studies have provided evidence, that the source of dietary protein affects the fatty acid composition of hepatic phospholipids and, especially, the phosphatidylinositol fraction. Since it involves the levels of linoleic acid, arachidonic acid and 20 : 3 (n - 9), the phenomenon may have wider implications concerning biochemical and physiological processes. Future studies will be aimed at determining, if there is any direct association between the changes in fatty acid pattern of liver lipids, and the hyperlipidemic response of rats to casein, vis-a-vis soybean protein.
Furthermore, the possible impact of an increased hepatic 20 : 3 (n - 9) to arachidonic acid ratio to atherogenic mechanisms will be studied. Acknowledgements This investigation was supported by grants from the Swedish Medical Research Council (Projekt No. 03X5172), from the Swedish Nutrition Foundation, from Hassle AB, Molndal and from the Faculty of Medicine at Uppsala University. The authors thank Ms. Elwy Wallin and Mr. Bengt Ejdesjo for skilful technical assistance. Thanks are also due to Dr. Sten Johnsson, managing director at AB Karlshamns Cirkelprodukter, AB Karlshamns Oljefabriker, Karlshamn, for the gift of soybean protein isolate. We are indebted to Dr. LarsBbrje Croon, The National Food Administration, Uppsala, for valuable help in performing fatty acid analysis by capillary gas-liquid chromatography. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
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