The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 15-24

Hydrogenated Fat High in Trans Monoenes with an Adequate Level of Linoleic Acid Has no Effect on Prostaglandin Synthesis in Rats^1,2

Mohamedain M. Mahfouz and Fred A. Kummerow³

Burnsides Research Laboratory, Urbana, IL and Harlan E. Moore Heart Research Foundation, Champaign, IL

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Our study was designed to determine whether hydrogenated fat high in trans monoenes concentration affected prostaglandin synthesis. Corn oil (CO), butter (B), hydrogenated vegetable oil (HF) and coating fat (CF) were used in this study. These fats were fed to rats for 10 wk at 10 g/100 g diet. The phospholipid (PL) fatty acid content of platelets, aorta and heart was determined by gas liquid chromatography, and the in vitro aorta production of prostacyclin (PGI₂) from exogenous or endogenous arachidonic acid (AA) was measured using the radioimmuno-assay (RIA) method. Serum thromboxane B₂ (TXB₂) released by platelets as thromboxane A₂ (TXA₂) during incubation of whole blood was also measured by this method. In the group fed CF, AA was significantly lower in the PL of aorta, platelet and heart, and the ratio 20:3(n-9)/20:4(n-6) was greater than in the groups fed CO, B or HF, indicating that the group fed CF was essential fatty acid (EFA) deficient. Although AA was significantly lower in the aorta and platelet PL of the group fed HF compared to the group fed CO, that difference did not affect the amounts of PGI₂ or TXB₂ produced in these groups. The group fed CF had significantly less PGI₂ and TXB₂ released by aorta and platelets than the other groups. This was the result of the reduced level of AA and the presence of higher amounts of 20:3(n-9) acid in the PL, which might act as a competitive inhibitor for cyclooxygenase. The aortic production of PGI₂ from exogenous AA did not differ among the groups indicating that prostaglandin synthetase was not affected by the dietary fat. We conclude that the consumption of hydrogenated fats high in trans 18:1 acids with adequate amount of linoleic acid had no effect on the amount of thromboxane or prostacyclin produced by platelet or aorta in vitro.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Isomeric unsaturated fatty acids containing one or more double bonds in the trans position are formed concomitantly with saturated fatty acids when vegetable oils are hardened by hydrogenation. These dietary trans fatty acids found in margarines, dressings and other fat products that contain hydrogenated fat are mainly isomeric trans monoenes with a very low concentration of trans 18:2 acids. These trans acids may be incorporated into membrane lipids following absorption (Privett et al. 1966) and thereby affect the physical properties of the cell membrane (Chapman et al. 1966, Wenzel and Kloepell 1980) and membrane-bound enzymes (Mahfouz et al. 1980, Shimp et al. 1982).

Previous studies showed that the conversion of linoleic acid [18:2(n-6)] to arachidonic acid (AA)⁴ [20:4(n-6)] in rats was impaired, and 6 desaturase activity was decreased when partially hydrogenated soybean oil (PHSO) was included with corn oil in the diet (dietary fat contained 35% trans 18:1 and 15% linoleic acids) compared to the control diet which contained 15% linoleic acid and 0% trans 18:1 (Mahfouz et al. 1984). This also was evident by the increase of 18:2(n-6) and decrease of 20:4(n-6) acids in tissue phospholipid (PL) of rats fed hydrogenated fat high in trans 18:1 acid (Mahfouz et al. 1984, Wahle and James 1993). Pure trans 18:1 isomeric fatty acids also acted as competitive inhibitors in vitro for 6 desaturase, the key enzyme in the metabolic conversion of 18:2(n-6) to 20:4(n-6) acid (Mahfouz et al. 1980).

Many of the reported detrimental effects of trans isomers in animals are thought to result from essential fatty acid (EFA) deficiency rather than a specific effect of trans isomers, since they can apparently be prevented by increasing EFA availability (Gurr 1983). The observation that dietary trans fatty acids, but not saturated fatty acids, exacerbate EFA deficiency in rats (Beare-Rogers 1988, Senti 1988) does suggest a specific effect of trans isomers on EFA metabolism when EFA status is low or inadequate.

Eicosatrienoic 20:3(n-6) and eicosatetraenoic 20:4(n-6) acids derived from EFA serve as precursors for a group of bioactive compounds collectively termed eicosanoids (Kinsella et al. 1981, Lands et al. 1977). The level of these substrate fatty acids are important in eicosanoid biosynthesis (Hwang et al. 1982, Kinsella et al. 1981, Lands et al. 1977) and subsequent regulations of various physiological parameters such as platelet function and thrombosis. The demonstration that some actions of these eicosanoids are relevant to the etiology of coronary heart disease and platelet function underline the importance of studying the effect of the unnatural trans isomers of dietary fatty acids on the biosynthesis of these important compounds.

Trans 18:1 acids are the major trans acids in the hydrogenated fats available in supermarkets. However, most of the published studies that dealt with the effect of trans acids on prostaglandins used trans isomers of linoleic acid. In a previous study by Kinsella et al. (1979), feeding trans, trans linoleate to rats as 50% and 100% of the dietary fat decreased the concentrations of (n-6) fatty acids such as 18:2(n-6), 20:3(n-6) and 20:4(n-6) in rat tissue and platelets. The concentrations of prostaglandins E₁, E₂, and F₂ in serum were also significantly decreased in rats. However, at lower concentrations of trans, trans 18:2 (6.3% energy) in the presence of linoleic acid (1.1% energy), no effect on prostaglandin concentration was found (Bruckner et al. 1984).

One of the few studies of the effect of trans 18:1 acids on prostaglandins was published by Craig-Schmidt et al. (1984). When they fed women hydrogenated fat high in trans monoenes such as margarine, hydrogenated soybean oil, and shortening, in alternation with a diet containing nonhydrogenated fat such as corn oil, butter and lard, no effect was found on the concentrations of PGF₂ and PGE in human milk. Similarly, when rats were fed hydrogenated fat which contained 11% of its total fatty acids as trans 18:1 with different levels of linoleic acid (energy from 3.5% to 41%), no effect was observed on the PGF₂ and PGE concentrations in rat dams milk (Wickwire et al. 1987).

The control of platelet aggregation appears to be a complex balance between prostacyclin (PGI₂), which is released by the artery wall and inhibits aggregation, and thromboxane A₂ (TXA₂), which is released by stimulated platelets and promotes aggregation (Needleman et al. 1980). Both prostaglandins are ultimately derived from arachidonic acid, 20:4(n-6) (Hemberg et al. 1975, Moncada et al. 1976). The modulation of the production of these two prostaglandins has been correlated with changes in the fatty acid composition of the PL in platelets and aortic membranes (Lagarde et al. 1985).

The aim of this study was to compare the effects of three dietary fats which are available in supermarkets including corn oil (CO, high in linoleic), butter (B, high in saturated acid), hydrogenated vegetable oil (HF, high in trans 18:1 and linoleic acids) as well as coating fat (CF, high in trans 18:1 acid with no linoleic acid) on the availability of the substrate fatty acid (AA) in tissue phospholipids and the biosynthesis of prostacyclin in aorta and thromboxane in platelets of rats. These two prostaglandins play an important role in thrombosis and hemostasis.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Male weanling Sprague-Dawley rats, divided into four groups of eight rats each, were fed a modified AIN-76 semipurified diet for 10 wk (AIN 1977 and 1980). The diet was obtained from ICN Pharmacuetical (Costa Mesa, CA). Ten percent of the diet (g/100 g) was comprised of CO, B, CF or HF. CF was obtained from Quest International (Hoffman Estates, IL). The fatty acid composition of the fat supplements is shown in Table 1. Food was prepared as needed and stored at 4°C. The rats were fed fresh food daily and allowed free access to food and water. The rats were housed individually in suspended cages with wire mesh bottoms. Room lighting consisted of 12 h periods of light and dark. All animal protocols were approved by the University of Illinois at Urbana-Champaign Animal Care and Use Committee. The rats were weighed weekly and were deprived of food overnight before being killed.

Chemicals and reagents.  1-[¹⁴C] Arachidonic acid [sp. act. 51 mCi/mmol, (1.9 GBq/mmol)] and ³[H] radioimmuno-assay (RIA) kits for 6-ketoprostaglandin F₁ (6-ketoPGF₁) and thromboxane B₂ (TXB₂) were obtained from DuPont NEN (Boston, MA). Standard 6-ketoPGF₁ and TXB₂ were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals were analytical grade.

Sample collection.  Rats were anesthetized with 0.5 mL of 2.2 mol/L urethane/100 g body wt, and blood was withdrawn by heart puncture into a plastic syringe. Three milliliters of blood were collected in an ice-cold plastic tube containing 95 µL of 54 mmol/L EDTA and 5 µL of 40 mmol/L indomethacin/L blood. Plasma was obtained by immediate centrifugation for 6-keto PGF₁ quantification. Another 3 mL of blood were allowed to clot for 2 h at room temperature and serum was obtained for TXB₂ assay.

For preparing platelets and plasma for fatty acid analysis, 8 mL of blood were collected into a plastic syringe containing one-tenth portion of 27 mmol/L EDTA and was centrifuged at 180 × g for 10 min at room temperature to obtain platelet-rich plasma. The platelets were separated from platelet-rich plasma by centrifugation at 1000 × g for 15 min and washed twice with Ca²⁺-free Tyrode-Hepes buffer (137 mmol/L NaCl, 2.7 mmol/L KCl, 1 mmol/L MgCl₂, 5.6 mmol/L glucose, 2.9 mmol/L NaH₂PO4, 3.8 mmol/L Hepes, pH 6.5) (Sato et al. 1987). Platelets were resuspended in 5 mL methanol and the plasma and platelets were stored at 80°C until used for fatty acid analysis. Rats were opened by midline incisions and their hearts and aortas were rapidly removed. The aortas were rinsed with Tris buffer (10 mmol/L, 153 mmol/L NaCl, pH 7.4) and freed of fatty tissues and adventitia while held on a petri dish containing ice-cold Tris buffer. Before use the aorta was opened longitudinally and carefully blotted so as not to remove endothelial cells. Two pieces of each aorta (1 cm length) were taken for measuring prostacyclin synthesis and the remaining aortic tissues were frozen and kept at 80°C until used for fatty acid analysis. The hearts were also rinsed with cold saline and stored at 80°C.

Separation and estimation of 6-keto prostaglandin F₁ from aorta incubated with arachidonic acid.  Assay of PGI₂ synthesis in aorta was carried out by an aqueous sampling method as described by Panganamala et al. (1981). In this method, incubation of aortic tissues with AA was carried out in Tris buffer (50 mmol/L, pH 8.0) containing 1 mmol/L EDTA and 150 mmol/L NaCl. Twenty nmol of 1-[¹⁴C] arachidonic acid was converted into sodium soap by mixing with 200 µL of Na₂Co₃ solution (150 mmol/L) and dissolved in 1 mL Tris buffer. Rat aorta (1-cm-long segments) was added to the reaction mixture and incubated at 37°C in a shaking thermostatic water bath with frequency 30/min for up to 90 min in air. Aliquots (50 µL) of the reaction mixture were removed at 30, 60 and 90 min intervals to which unlabeled carriers of AA and 6-keto PGF₁ were added and spotted on silica gel G-TLC plates under a stream of argon. The plates were developed in the organic phase of the solvent mixture containing ethyl acetate/acetic acid/2,3,4-trimethylpentane/water (110:20:50:100 v/v) (Marcus et al. 1978). Spots were visualized by iodine vapor. After iodine was removed under a stream of argon, spots corresponding to AA and 6-keto PGF₁ were scraped from the plates and mixed with 10 mL counting fluid (Biosafe II, Research Products International, Mount Prospect, IL). Radioactivity was then counted (LS 3801, Beckman). After corrections for quenching, dpm were converted to pmol of product. At the end of the incubation, the aortas were removed and dried over a sheet of filter paper and their weights were determined.

Generation of prostacyclin from aorta.  Specimens of rat aorta (1 cm in length) isolated as described above were incubated at 37°C in 1 mL of Tris-saline buffer (50 mmol/L, 150 mmol/L NaCl, pH 8.0) (Karpen et al. 1981, Panganamala et al. 1981) in a shaking water bath with a frequency of 30/min. Aliquots (100 µL) were withdrawn at 30, 60, 90 min for the estimation of 6-keto PGF₁ by RIA method after their dilution 1:1000 with the assay buffer. At the end of the incubation time the aortas were removed and dried over a sheet of filter paper and their weights were determined.

Measurement of serum thromboxane.  The concentration of TXB₂ in serum as the stable metabolic product of TXA₂ synthesized by the platelets during blood clotting was measured by the RIA method after serum dilution 1:1000 with the assay buffer. Radioimmuno-assay kit for TXB₂ (DuPont NEN, Boston, MA), which employs ³[H] as the radioactive tracer, was used.

Measurement of 6-keto F₁ in plasma.  Plasma samples that had previously been acidified with 1 mol/L HCl to pH 3.5 were extracted on Sep-Pak C₁₈ Cartridge as described by Powell (Powell 1982). 6-Keto PGF₁ was quantified by RIA 6-keto PGF₁ kit (DuPont NEN) which employs ³[H] as the radioactive tracer.

Extraction of lipids and analysis of fatty acids.  The effect of the different dietary fats on the fatty acid composition and the availability of fatty acids used as precursor for protaglandin (PG) in the PL fraction of the plasma, platelets, aorta, and hearts were studied by first extracting their total lipids according to the Folch et al. (1957). The PL fraction was then separated from other portions of the lipid extract by polysilicic acid gel impregnated glass fiber sheet (Gelman Sciences, Ann Arbor, MI), using a solvent system of petroleum ether/diethyl ether/acetic acid (80:20:1 v/v). The phospholipids were transesterified by boron triflouride methanol complex (Morrison and Smith 1964). Butylated hydroxytoluene was added as antioxidant in a concentration of 230 µmol/L to all solvents used for lipid extraction and chromatography. All operations were carried out under N₂. The fatty acid methyl esters were separated by gas liquid chromatography as previously described (Mahfouz et al. 1989). A Packard Model 4790 gas chromatograph (Hewlett Packard, Chicago, IL) equipped with an all glass splitter and flame ionization detector was used to separate methyl esters on a Supelco SP-2560 wall coated 100 m × 0.25 mm i.d. fused silica capillary column. Retention time, peak areas and peak relative area percentages were determined electronically using a Hewlett Packard Model 3390A Reporting integrator. Identification of methyl esters of fatty acids was accomplished by comparing relative retention times with authentic standards (NuChek Prep., Elysian, MN and Supelco, Bellefonte, PA).

Statistical analysis.  Data of fatty acids were analyzed by the computer program Microsoft Excel version 5.0a (Microsoft Corporation, Redmond, WA). Data on prostaglandins were analyzed by one-way ANOVA and Duncan's new multiple range test (Torrie 1980) to determine whether mean values were significantly different at P < 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

No significant differences in the mean weight gain or food intake were observed among rats fed CO, B or HF. The mean weight gain was only significantly lower in those fed CF. The mean initial weight for rats was 49.4 ± 1.7 g, and the mean final weight was 360 ± 36, 366 ± 20 and 355 ± 10 g for the groups fed CO, B or HF, respectively, and 313 ± 10 g for rats fed CF. The mean weight gain for the rats fed CO, B and HF were 4.8 ± 0.5, 4.6 ± 0.4 and 4.7 ± 0.2, respectively, while for the group fed CF it was 4.1 ± 0.2 g/(d pd rat). The lower weight gain of the CF group can be related to the essential fatty acid deficiency (EFAD) due to the absence of an adequate amount of linoleic acid in the diet. No other signs of EFAD symptoms such as dermal scores or hair loss were apparent on animals fed CF until the end of the experiment.

The levels of total (n-6) fatty acids and AA in the plasma of the different groups almost reflected the level of linoleic acid content of their diets, since the CO group had the highest and CF had the lowest concentrations in linoleic acid (Table 1). Plasma from the groups fed CO or HF were characterized by high levels of PUFA but lower levels of monounsaturated fatty acid, while in the groups fed B or CF the lower concentration of their PUFA in plasma relative to the CO group was almost replaced by monounsaturated fatty acids. The double bond index (DI) of the plasma fatty acids was highest in the group fed CO and lowest in the group fed CF (Table 2). In contrast, the PUFA content and DI of the PL fractions of platelet (Table 3), aorta (Table 4) and heart (Table 5) from the groups fed CO, B, or HF did not vary as much as in the plasma (Table 2), but there was some differences among groups. It appears that within the membrane there is a mechanism which maintains the fatty acid composition and the degree of unsaturation within a narrow range probably to preserve the membrane properties such as fluidity. In the group fed CF, the PUFA and the DI were lower than in the groups fed CO, B, and HF diet due to the EFAD.

The group fed CF was EFA deficient as indicated by the ratio 20:3(n-9)/20:4(n-6) of 1.2 in their plasma. In the CF group, the level of linoleic acid in the diet was 0.3%, yet ~12% was found in the plasma as (n-6) fatty acids and 6.5% as AA (Table 2). In the group fed B, the presence of 20:3(n-9) acid at the level of 2.8% in the plasma indicated that the desaturase enzymes were not fully occupied by linolenic and linoleic acids probably due to their low levels in the diet. Oleic acid, which was more available in the butter, was therefore able to be desaturated and elongated to 20:3(n-9) acid. The group fed B, however, was not EFA deficient as indicated by the ratio 20:3(n-9)/20:4(n-6) of 0.15 in their plasma (Table 2).

The PL fractions of platelets (Table 3), aortas (Table 4) and hearts (Table 5) of the groups fed CF or HF contained lower levels of stearic acid than the groups fed CO or B. The decrease of stearic acid was accompanied by the incorporation of an equivalent amount of trans 18:1 acids into the PL fractions. Trans 18:1 acids probably compete with stearic acid (as saturated acid with the same chain length) for incorporation into the 1-position of PL.

The amounts of trans 18:1 acids incorporated into the plasma lipids (Table 2) or the PL fractions of platelets (Table 3), aorta (Table 4) or heart (Table 5) of the groups fed CF or HF were proportional to their levels in the diet. Heart PL contained the highest amount of trans 18:1 acids which reached 5.7% in the group fed CF and 3% in the group fed HF (Table 5). The aorta PL contained the least amount of trans 18:1 acids which reached 2.5% in the group fed CF and 1.7% in the group fed HF (Table 4).

When rat aortas were incubated in the presence of EDTA with 1-[¹⁴C] arachidonic as an exogenous substrate for prostaglandin sythetase, the amount of PGI₂ biosynthesized are shown in Figure 1. No significant differences were observed among the different groups for the amount of PGI₂ synthesized at any time point. More than 90% of the radioactivity was found in the AA and 6-ketoPGF₁ fractions which indicated that PGI₂ is the major PG produced by rat aorta.

View larger version (55K): [in this window] [in a new window]   Fig 1. In vitro production of prostacyclin from exogenously added arachidonic acid to aortas from rats fed diets containing 10 g/100 g corn oil, butter, coating fat or hydrogenated vegetable oil for 10 wk. Twenty nmoles of 1-[14C]-arachidonic acid were added to a 1-cm-long segment of aorta in 1 mL of 50 mmol/L Tris-buffer containing 1 mmol/L EDTA and incubated for up to 90 min in air at 37°C. The amount of prostacyclin released was measured as described in the text and expressed a pmol 6-ketoprostaglandin (6-keto PGF1) released/mg aorta. Values are means ± SD, n = 6. Means at each time point did not significantly differ, P > 0.05.

Aortas were also incubated without addition of AA in absence of EDTA to measure the amount of PGI₂ released by aorta from the endogenous AA as substrate which is released by the action of phospholipase on the PLs. At all the time points tested, the amount of PGI₂ released was significantly lower in the group fed CF (54% inhibition) than in the CO group. No significant differences were observed for the amount of PGI₂ released by aortas from the endogenes AA among the groups fed CO, B or HF (Fig. 2).

View larger version (46K): [in this window] [in a new window]   Fig 2. In vitro production of prostacyclin from endogenous arachidonic acid in aortas from rats fed diets containing 10 g/100 g corn oil, butter, coating fat or hydrogenated vegetable oil for 10 wk. One-centimeter-long segments of aortas were incubated at 37°C in 1 mL of 50 mmol/L Tris-buffer without EDTA for up to 90 min in air. 6-Keto-PGF1 was assayed by RIA method and expressed as pmol/mg aorta. Values are mean ± SD, n = 6. Bars with no letters in common at each time point are significantly different (P < 0.05).

Figure 3 shows the amount of TXA₂ released by the platelets during blood clotting at room temperature (25°C) and measured as TXB₂, the stable metabolite of TXA₂. The serum TXB₂ concentration was significantly lower in the group fed CF (85% inhibition) compared to the group fed CO. No significant differences were observed for the amount of TXB₂ released by platelets among the groups fed CO, B or HF diets (Fig. 3). Similarly, the concentration of PGI₂ in plasma measured as 6-keto PGF₁ was significantly lower in the group fed CF compared to other groups (Fig. 4). No significant differences were observed for the level of plasma 6-keto PGF₁ among the groups fed CO, B or HF (Fig. 4).

View larger version (42K): [in this window] [in a new window]   Fig 3. In vitro production of thromboxane by platelets of rats fed diets containing 10 g/100 g corn oil, butter fat, coating fat or hydrogenated vegetable oil for 10 wk. The whole blood was incubated at room temperature for 2 h then the amount of TXA2 released by platelets during blood clotting was measured as TXB2 by RIA method after serum dilution 1:1000. Values are the means ± SD of six different rats per group and expressed as nmol thromboxane B2/L serum. Bars with no letters in common are significantly different (P < 0.05).

View larger version (54K): [in this window] [in a new window]   Fig 4. Concentration of 6-ketoprostaglandin in plasma of rats fed diets containing 10 g/100 g corn oil, butter, coating fat or hydrogenated vegetable oil for 10 wk. 6-Keto-prostaglandin was measured by RIA method after its separation from plasma on Sep-Pak C18 cartridge. Values are means ± SD of six different rats per group and expressed as nmol 6-ketoprostaglandin/L plasma. Bars with no letters in common are significantly different (P < 0.05).

The ratios between the amount of TX released by the platelets to the PGI₂ released by aorta and measured as 6-keto PGF₁ in the different groups are shown in Table 6. This ratio was not significantly different among the groups fed CO, B and HF. In the group fed CF, the TXB₂/6-keto PGF₁ ratio was significantly less than in the other groups (Table 6). Similar differences for the ratio of TXB₂ released by platelets in serum/the 6-keto PGF₁ in plasma were also observed among the different groups (Table 6).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Previous studies showed that tissues from EFAD rats exhibited increased activity of the in vitro conversion of exogenous AA to PG (Dunham et al. 1979, Kaa 1977), indicating an enhanced PG synthetase activity in EFAD. This contradicts our findings. In the present study, when aorta from the rats fed CF were incubated with exogenous AA, the amount of PGI₂ synthesized was not significantly different from the amount of PGI₂ synthesized by aorta from the groups fed CO, B or HF. The incubation was carried out in the presence of EDTA in the incubation buffer which inhibits the Ca²⁺-sensitive phospholipase A₂ and minimizes the AA liberated from aortic PL. Our results indicated that PG synthetase was not enhanced in the aorta of the group fed CF, considered to be EFAD. When linoleic acid is low in the diet, oleic acid has a better chance to be desaturated and elongated to 20:3(n-9) acid, which increases in the tissues of EFAD animals. The accumulation of 20:3(n-9) acid may compensate for the decrease of (n-6)PUFA and maintain the physical properties and the integrity of the membrane, but does not cure EFAD symptoms. In vivo (Mahfouz et al. 1984, Mahfouz 1981) and in vitro (Mahfouz et al. 1980) studies showed that isomeric trans 18:1 acids act as competitive inhibitors for 6 desaturase, the first step in the conversion of oleic acid [18:1(n-9)] to 20:3(n-9) acid. It is possible that the high level of trans 18:1 acid in the CF diet impaired the conversion of oleic to 20:3(n-9) acid through its inhibitory effect on 6 desaturase. This can decrease the 20:3(n-9) acid level in the tissues which became less available than in the case of fat-free or hydrogenated coconut oil diets (Dunham et al. 1979, Hwang and Kinsella 1979, Privett et al. 1977) which lack the trans 18:1 acids. This can cause a significant drop in the amount of PUFA and the DI of the membrane fatty acids as we found in the PL of aorta and platelets of the group fed CF. These changes can make the membrane more rigid and influence the physical properties of the membranes as well as the activities of the membrane associated enzymes (Alam et al. 1989, Champan et al. 1966) such as PG synthetase.

CF is present as 2-40% in different types of foods such as bread mixes and fried crusts, candy and frostings, cream substitutes, cereals and pudding, cookies and french fried potatoes (Enig et al. 1983).

The reduced synthesis of PGI₂ from the endogenous AA in the aorta and the significant reduction of TXB₂ concentration in serum (released by platelets as TXA₂ during blood clotting) of the group fed CF can be explained by the low level of AA in aorta and platelets PL of that group which makes AA less available for the enzymes, thereby decreasing PGI₂ and TXB₂ synthesis. In addition, the presence of 20:3(n-9) acid in aorta and platelet PL which might be released simultaneously due to the same stimuli can compete with AA for the active site of the cyclooxygenase enzyme (Dunham et al. 1979, Lands et al. 1977, Wenzel and Kloepell 1980, Ziboh et al. 1972). These two mechanisms, lack of precursors and competition for the enzyme, may explain the observed results.

In the present study the corn oil diet contained 59% linoleic acid, while HF contained 32% linoleic acid. These two levels of linoleic acid are more than adequate and in both cases the level of linoleic acid in the tissues will be high enough to fully saturate the enzymes involved in its metabolic conversion to AA. This has been shown in our previous study (Mahfouz et al. 1984) in which two groups of rats were fed either corn oil or safflower oil (as 15% of the diet) which provided 61% and 15% of linoleic acid, respectively. The level of arachidonic acid in the total lipids as well as phospholipid of the tissues of these two groups was almost identical. The same observation was reported by Kurata and Privett (1980) and Tahin et al. (1981) when they fed two groups of rats two different levels of adequate amounts of linoleic acid.

The lower level of AA in aorta PL of the group fed HF compared to the group fed CO, however, did not cause any decrease in the amount of PGI₂ produced by the aorta from endogenous AA in the group fed HF compared to the groups fed CO. This may indicate that AA was present in sufficient amounts to saturate the enzyme. DeDeckere et al. (1979) have observed that feeding rats >3.5% energy as linoleate did not cause an increase in PGI₂ product in aorta and heart. In this study, dietary hydrogenated fats which contained trans 18:1 acids had no effect on PGI₂ production by aorta, if linoleic acid is supplied in adequate amounts. The same conclusion was drawn by Bruckner et al. (1984 and 1982) who found no effects of trans 9, trans 12 linoleic acid on PG synthesis when it was consumed by rats in reasonable amounts in the presence of adequate amount of linoleic acid.

The aorta and platelet PL of the group fed CF contained almost the same level of AA. The amount of TXB₂ released from the platelets of that group was decreased to a greater extent than the decrease of PGI₂ produced by aorta of the same group compared to the groups fed CO or HF. This differential inhibition could be attributed to the presence of more 20:3(n-9) acid in the platelet PL than in aorta PL, which can have more competitive inhibition with AA for the enzyme. An alternative explanation is that PI and PS are the reservoir of the substrate (Naughton et al. 1988) for PG synthesis. If AA was differentially reduced in these particular PL in the platelets of the group fed CF, this can cause a greater reduction in the amount of TXB₂ produced by platelets. A similar observation was made by others (Bruckner et al. 1984) who found that trilinoelaidate differentially inhibited TXB₂ biosynthesis to a greater degree than PGI₂ biosynthesis in rats.

The significantly lower TXB₂/6-keto PGF₁ ratio in the group fed CF is attributed to the more dramatic decrease of TXB₂ released by platelets than the decrease of PGI₂ released by aorta in that group. The decreased production of proaggregatory (TXA₂) versus antiaggregatory (PGI₂) PG in EFAD rats has been observed and implicated as a possible cause for the increased bleeding time (Hornstra 1975). In the group fed B, the TXB₂/6-keto PGF₁ ratio was lower than in the groups fed CO or HF, however that difference was not significant (P = 0.1). This decrease is due to the higher reduction in the amount of TXB₂ released by platelets than the reduction of PGI₂ released by the aorta of the group fed B compared to the groups fed CO or HF. The lower ratio of TXB₂/6-keto PGF₁ does not necessarily indicate a prolonged bleeding time or less platelet aggregation since evidence has shown that a reduction of platelet thromboxane production is associated with a higher sensitivity of these platelets for the prothrombotic activity of thromboxane (Heemskerk 1989), which largely offsets the beneficial effect of reduced thromboxane formation. The higher monounsaturated + saturated/polyunsaturated ratio in the fatty acids of the platelet PL in the group fed B can also enhance the platelet thrombotic tendency and hypersensitivity to aggregating agents (Aznar et al. 1980).

Most of the published work which examined the effect of trans fatty acids on PGI₂ and TXA₂ synthesis in aorta and platelets used trans isomers of linoleic acid. The present study shows for the first time that the consumption of hydrogenated fats high in trans 18:1 acids in presence of adequate amounts of linoleic acid still can lower the level of AA in tissue phospholipids as previously reported (Mahfouz et al. 1984). However, this significant drop of AA content did not affect the amount of thromboxane or PGI₂ produced in vitro by platelet and aorta, respectively. Our results also agree with others who found no effect of hydrogenated fat (Craig-Schmidt et al. 1984) or trans 18:1 acids (Wickwire et al. 1987) on the concentrations of PG in the milk of nursing women and rat dams when linoleic acid was present at levels between 3.5-41.4% in the diet (Wickwire et al. 1987). The present study also showed that EFA-deficient diet which contained high level of trans 18:1 acids (CF) did not enhance the PG synthetase activity. This effect is in contrast with the observation that EFA deficiency without the presence of trans acids enhanced the PG synthetase activity (Dunham et al. 1979, Kaa 1977).

	FOOTNOTES

1

Manuscript received 21 April 1998. Initial reviews completed 24 June 1998. Revision accepted 8 September 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

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