Effects of gamma-linolenic acid on fatty acid profiles and eicosanoid production of the hamster

NUTRITION RESEARCH, Vol. 7, pp. 1085-1092, 1987 0271-5317/87 $3.00 + .00 Printed in the USA. Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved.

EFFECTS OF GAMMA-LINOLENIC ACID ON FATTY ACID PROFILES AND EICOSANOID PRODUCTION OF THE HAMSTER I

Takashi Ide 2,3, Michihiro Sugano2,% Takahiro Ishida 2 Motohiro Niwa s , Masahiro Arima 5 and Akihito Morita s 2Laboratory of Nutrition Chemistry, Kyushu University School of Agriculture 46-09, Fukuoka 812, Japan SBrewing Science Laboratory, Kirin Brewery Co., Ltd,, Takasaki 370-12, Japan

ABSTRACT

Male young hamsters were fed for 30 days cholesterol-free purified diets containing two different fats with an essentially comparable fatty acid composition except for polyunsaturated fatty acids; either the mold oil produced by Mortierella sp. containing 13.5% linoleic acid (LA) and 10.1% y-linolenic acid (GLA) or the blend (23.6% LA) of palm oil and two types of safflower oils. Growth parameters were comparable in both dietary regimens. The effects on plasma and liver cholesterol of GLA were not evident as the levels were kept low in the two groups. The liver cholesterol and triacylglycerol contents were unaffected by dietary treatment. In addition to a significantly higher proportion of arachidonic acid (AA), a considerable amount of dihomo-GLA was detected in liver phosphatidylcholine from the hamster fed GLA-containing oil. The fatty acid compositions of adipose tissue and liver triacylglycerol reflected those of dieatry fats and the changes seen in the mold-oil fed rats paralleled with those of liver phosphatidylcholine. The plasma concentration of thromboxane B 2 was comparable, while the aortic production of prostacyclin was significantly greater in hamsters fed the mold oil. Thus, prostacyclin generating capacity may be Changed by an increased GLA intake even in animal species with limited A6- and A5-desaturase activities.

KEY WORD: y-Linolenic acid, linoleic acid, thromboxane A2, prostacyclin, phosphatidylcholine, hamster

ISupported by Grant-in-Aid for Scientific Research (No. 6156101) from the Ministry of Education, Science and Culture, Japan. 3Present address: National Food Research Institute, Tsukuba 305, Japan. ~To whom requests for reprints should be addressed.

1085

1086

T. IDE et al. INTRODUCTION

The reaction catalyzed by the A6-desaturase is the rate-limiting step leading linoleic acid (LA) to arachidonic acid (AA) (1,2) and hence possibly governs the rate of eicosanoid production. The enzyme activity is influenced by various factors (1-5). Because of the specific role of the eicosanoids in the etiology of thrombosis and hence atherosclerosis, the effects of dietary manipulations on the production of thromboxane and prostacyclin have attracted much attention (6,7). Like the human, the hamster appears to have s limited ability to desaturate LA to AA through y-linolenic acid (GLA) (7,8). For that reason, this species may be a better model for examining the effects of an GLA-enriched intake on tissue lipid composition and hence elcosanoid production than in the rat which has a high A6-desaturase activity (9,10). In the preceding experiment (ii), we have evaluated the effects of the mold (Mortierella sp.) oil containing GLA on the production of eicosanoids in rats, and found that this oil is of particular use as a hypocholesterolemic fat source similar to evening primrose oil which contains a comparable amount of GLA but quite a low level of palmitic acid and a high level of LA as compared to the mold oil (ll-13). The present paper deals with the effects of mold oil on the eicosanoid levels and fatty acid profiles of the hamster, an animal model which resembles humans with respect to the patterns of polyunsaturated fatty acids (14). Table 1 Diet Compositions Ingredients

Groups

Casein Mold oil Palm oil High oleic safflower oil Safflower ail B-Sitosterol Mineral mixture a Vitamin mixture a Choline chloride Inositol ~-Aminobenzoic acid DL-Methionine Cellulose Corn starch Sucrose Fatty acids

MATERIALS AND METHODS

Mold oil

Fat blend

20 i0

2O

3.5 1 0.2 0.25 0.I 0.3 5 44.65 15

3.72 4.72 1.56 0.13 3.5 1 0.2 0.25 0.I 0.3 5 44.52 15

Compositions (weight %)

16:0 16:1 18:0 18:1 18:2(~-6) ~-18:3(w-3) y-18:3(m-6) aAIN-76 TM mixture (16).

19.0 2.0 3.1 50.4 13.5 I0.i

19.0 0.2 3.0 52.5 23.6 0.3

Animals and Diets: Male hamsters, 4 weeks of age, were purchased from Kyudo Co., Ltd., Kumamoto and were housed individually in an airconditioned room (20-23 ~ lights on 0800 to 2000 hours). After acclimation for a few days, they were fed ad libitum purified diets (Table 1)(15,16) for 30 days. The mold oil with more then 98% triacylglycerol was harvested from Mortierella rammanniana var. angulispora IFO 8187 (ll)(kindly provided by Kirin Brewery Co., Ltd., Tokyo). Fatty acid compositions of the two diets were adjusted to be comparable except for the profiles of polyunsaturated fatty acids; thus mold oil contained GLA at the expense of LA. The phytosterol contents of both diets were also made the same by adding B-sitosterol (ICN Pharmaceuticals Inc., Cleveland OH) to the fat blend (II). Body weight and food intake were recorded every other day.

FATTY ACID METABOLISM

1087

Lipid and Eicosanoid Analyses: Non-fasting blood samples were withdrawn from the abdominal aorta in a syringe containing Na3-citrate and indomethacin under light ether anesthesia and analyzed for thromboxane B 2 (TXB2)(13). The abdominal aorta was excised immediately and incubated for production of prostacyclin (measured as 6-keto-PGF1~(13, 17). Care was taken to evade the difference in the amount of time to correct blood and to excise aortic tissue. These eicosanoids were measured using commercial radioimmunoassay kits (New England Nuclear, Boston MA) as described elsewhere (13). Plasma and liver lipids were extracted and analyzed for cholesterol, triacylglycerol and phospholipid (11,12). Lipids from the epididymal adipose tissue and liver were separated by thin-layer chromatography and their fatty acid compositions were determined by gas-liquid chromatography (18). Statistical Analysis: The data were analyzed by Student's t--test and the differences between means greater than p
RESULTS

Growth Parameters: In accordance with the experiment with rats (ii), there were no untoward effects of mold oil on food intakes and body weight gain. These parameters were all comparable between the two groups (Table 2). Liver weight was also the same in both groups. Table 2 Growth Parameters and Liver Weight Body weight (g) Groups (n)

Mold oil (5) Fat blend (6)

Initial

gain/30 days

59• 57•

67• 66•

Food intake (g/day)

Liver weight (g/lO0 g body weight)

9.7• 9.6•

3.98• 3.89•

MeaniSE. Plasma and Liver Lipids: As shown in Table 3, there was a trend toward lowering the plasma cholesterol level in the hamsters fed on the mold oil diet, but the difference between the two groups was not statistically significant. The concentrations of liver cholesterol and triacylglycerol were comparable, but that of phospholipid was slightly but significantly higher in the mold oil group.

Table 3 Concentrations of Plasma and Liver Lipids

Groups (n)

Mold oil (5) Fat blend (6) MeaniSE.

Plasma cholesterol (mg/dl) 105• 116•

Liver lipids (mg/g) Cholesterol Triacylglycerol Phospholipid 2.25• 2.35•

Significant difference.

10.7 • 9.69•

35.4• 32.7•

1088.

T. IDE et al.

Fatty Acid Compositions of Tissue Lipids: Table 4 summarizes fatty acid compositions of the adipose tissue and liver triacylglycerol, and liver phosphatidylcholine. Fatty acid compositions of adipose tissue triacylglycerol reflected those of the dietary fats. Thus, the major differences were observed with LA and GLA; the sum of polyunsaturated fatty acids was comparable between the two groups, whereas a significant amount of GLA was detected only in the hamsters fed mold oil. Percentages of AA and dihomo-GLA were both clearly higher in the mold oil group although these were not the major components. Similar trends were observed with liver triacylglycerol. Fatty acid compositions of liver phosphatidylcholine from the mold oil-fed hamsters, as compared to those fed the fat-blend, were characterized by the markedly high percentage of dihomo-GLA. In addition, the percentage of LA was significantly lower and that of AA was significantly higher in the mold oil group. Due to these alterations, the proportion of AA to the parent molecules, either LA or LA+GLA, was markedly high in the mold oil group. The responses by the adipose tissue and liver triacylglycerols and liver phosphatidylcholine were remarkably similar in the mold oil-fed animals. Table 5 Eicosanoid Levels

Groups (n)

Aortic PGI 2 production (pg/mg) a

Mold oil (5) Fat blend (6)

87.0• 48.0 • 7.6

Mean•

Plasma thromboxa~e A 2 (pg/ml) u llO• 128•

Significant difference.

aMeasured as 6-keto-PGFz~. thromboxane B .

Mesured as

Aortic PGI 2 Production and Plasma TXA 2 Concentration: Table 5 shows that the aorta from hamsters fed GLA had a significantly greater ability to produce prostacyclin, whereas the level of plasma TXB 2 was virtually the same as compared to that in the animals fed the fat blend containing LA as a sole source of polyunsaturated fatty acids.

DISCUSSION

Since a variety of pathological, physiological and nutritional factors interferes with the activity of A6-desaturase (1-5), ingestion of the reaction product, GLA, may promote the availability of substrates for the eicosanoids, in spite of the second rate-limiting step catalyzed by A5-desaturase, conversion of dihomo-GLA to AA (8). In fact, the arachidonic acid contents increased in the mold oil-fed animals. Since this change was more pronounced than that observed in the rats (ii), the effect of the ~6-desaturase product seems to be greater in the animal species whose desaturation ability is relatively limited. The present results showed that GLA enhanced the aortic production of PGI 2 without apparently influencing the plasma level of TXB 2 even in an animal model whose capacity to desaturate LA to GLA and dihomo-GLA to AA is relatively low (8-10). Thus, although still preliminary, the present study indicated a possibly favorable effect of GLA as mold oil in the prevention of thrombosis and hence atherosclerosis. These effects were comparable to those observed in rats fed evening primrose oil in spite of a relatively high level of palmitic acid and a low level of LA in the mold oil (13,19). In addition, feeding the mold oil containing GLA caused a deposition of a considerable amount of dihomo-GLA in the liver phosphatidylcholine, the substrate for the 1-series eicosanoids. This phenomenon appears to be specific to the hamster, as no such definite deposition has been observed even in guinea pigs given evening primrose oil (i0) whose desaturation activity seems to be

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also low (14). Alternately, there is a possibility that the characteristic fatty acid patterns of mold oil may be responsible for the deposition of dihomo-GLA. In this context, Nassar et a1.(20,21) recently observed that marine oil in combination with evening primrose oil caused an increase in dihomo-GLA concentration and a decrease in AA concentration of tissue phospholipid of rats and suggested that eicosapentaenoic acid (~0-3)is responsible. It seems reasonable to suggest that in the hamster and perhaps in humans, in which AS-desaturase reaction proceeds less rapidly (8,22,23), dietary GLA may increase the production of the 1-series prostaglandins. Stone et al.(8) observed in man that dihomo-GLA enhanced PGE l production by the platelet. Theameliorative effects of PGE I on hypercholesterolemia and atherogenesis have been reported (24,25). The failure to detect significant effects of GLA on the plasma cholesterol level is due presumably to the use of a cholesterol-free diet, as in the rat the effect of the mold oil on the plasma cholesterol level was more marked when cholesterol-enriched diets rather than when cholesterol-free diets were fed (ii). The concept that GLA has a greater beneficial effect than LA is based in part on the assumption that by-passing the reaction catalyzed by A6-desaturase causes stimulation of the production of several eicosanoids, especially 1series PGs, to desirable levels. Thus, the use of GLA may be of particular importance in situations where desaturation activity is limited or largely inhibited as in the aged or in disease states such as diabetes or atherosclerosis (3-5).

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Accepted for publication June 16, 1987