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The prostaglandin outflow from perfused mesenteric vasculature of rats fed different fats
Prostaglandins Leukotrienes and Essential Fatty Acids (1969) 35.73-79 0 Longman Group UK Ltd 1989
The Prostaglandin Outflow From Perfused Mesenteric Vasculature of Rats Fed Different Y.-S.‘HUANG,
B. A. NASSAR*
Fats
and D. F. HORROBIN
Efamol Research Institute, Kentville,
Nova Scotia, Canada B4N 4H8 (reprint requests to YSH)
Abstract - The effects of dietary n-6 polyunsaturated fatty acids and replacement with saturated fat or fish oil on the prostaglandin outflow from perfused mesenteric vasculature in rats were studied. Seventy-two weanling male rats were fed ad libitum a semi-synthetic diet supplemented with 10% by weight of oil, composed wholly of n-6 fatty acid-rich evening primrose oil, or replaced partly or completely (25, 50, 75 or 100%) by n-6 fatty acid-deficient fish oil or hydrogenated coconut oil for 8 weeks. The outflows of 6-ketoPGF,,, thromboxane Bz, and prostaglandin E from the perfused mesenteric vasculature were measured at 60 min-time point after starting the perfusion. In general, the release of prostanoids from the mesenteric vasculature was significantly reduced in rats fed a diet in which evening primrose oil was partly or completely replaced by either hydrogenated coconut or fish oil. This was probably due to the insufficient conversion of linoleic acid to arachidonic acid. The extent of reduction was greater in fish oil-fed than in hydrogenated coconut oil-fed rats, while the levels of arachidonic acid in aortic phospholipids were similar between these two groups. This result implies that the greater reduction of prostaglandin synthesis in rats fed fish oil was due to the inhibitory effect of eicosapentaenoic and docosahexaenoic acids in fish oil on the conversion of arachidonate to eicosanoids.
Introduction
rapidly elongated. to dihomo-gamma-linolenic acid (DGLA, 20:3n-6). DGLA is in turn desaturated to arachidonic acid (AA, 20:4n-6) (4). DGLA and AA serve as the immediate precursors for synthesis of 1 and 2 series of prostaglandins (PGs) and thromboxane (TX) via the cyclooxygenase pathway (5). Thus, reducing the dietary supply of LA can significantly diminish PG synthesis (6-8). Increasing fish oil intake also reduces the formation of TX (9, 10). Previously, we have demonstrated that the levels of dietary
Dietary polyunsaturated fatty acids, mainly linoleic acid (LA, 18:2n-6), play an important role in regulating the amount and type of eicosanoids synthesized (l-3). In the animal body, LA is desaturated by delta-6-desaturase .to gamma-linolenic acid (GLA, 18:3n-6), which is * Present Address: Department of Pathology, Division of Clinical Chemistry, Victoria General Hospital, Dalhousie University, Halifax, Nova Scotia, Canada B3H lV8. 73
74
PROSTAGLANDINS
LEUKOTRIENES
AND ESSENTIAL FATTY ACIDS
fish oil significantly affected the compositions of each. Animals were fed ad libitum a fat free (FF) n-3 and n-6 fatty acids in various rat tissue phos- semi-synthetic diet (Teklad Test Diets, Madison, pholipids (11, 12). Oils of sea water fish are WI) supplemented with 10% by weight oil, usually rich in n-3 polyunsaturated fatty acids; composed wholly of n-6 fatty acid-rich EPO (containing 74.1% 18:2n-6 and 9.2% 18:3n-6, whereas they are .deficient in n-6 fatty acids. Since dietary fish 011n-3 fatty acids compete with group A), or replaced partly or completely (25, n-6 fatty acids in the body for incorporation into 50, 75 or lOO%, represent groups B, C, D and E respectively) by n-6 fatty acid-deficient fish oil the tissue phospholipids as well as suppressing acid the formation of prostanoids from AA (2, 9, 13, (FO, containing 16.9% eicosapentaenoic 14), we have thus cautioned that prolonged high (EPA, 20:5n-3) and 12% docosahexaenoic acid level fish oil intake may induce a metabolic (DHA, 22:6n-3)) or hydrogenated coconut oil (HCO, containing 96.9% C8-Cl8 saturated fatty deficiency of n-6 fatty acids, particularly when acids) for 8 weeks. Animals were given fresh diet dietary intake of the n-6 fatty acids is not every day to minimize the oxidation of dietary adequate (11). Effects of fish oil on PG metabfats. Both FO and HCO contain very low olism have been frequently studied in rat aortic amounts of n-6 fatty acids. The detailed fatty tissues (15117), but little information is available acid compositions of dietary fats are shown in concerning the effects of diet on PG metabolism Table 1. The composition of the FF diet in the peripheral vessel system (18). Since the (containing 20% casein, 70.2% sucrose, 5% behaviour of different vascular systems can vary cellulose, 3.5% AINmineral mix, 1.0% significantly, the production of PGs from the vitamin mix, and 0.3% DL-methionine) has been peripheral vasculature in response to dietary described previously (23). manipulation may not be the same as that from the aorta. _ In the present study, we examined the outflow I Table 1 Fatty Acid Composition of Diet Fats of PGs and TxB2 from the isolated mesenteric % Weight vascular bed of the rat following administration Fatty EPO FO HCO of fish oil n-3 fatty acids in association with Acid partial or complete depletion of dietary n-6 fatty 8:O 7.1 acids. It has been demonstrated that n-3 fatty 1o:o 6.0 acids suppress the activity of delta-6-desaturation 12:o 44.9 0.1 0.1 14:o 17.2 7.1 (4, 20-22). We fed animals a diet supplemented 16.4 6.2 9.1 with evening primrose oil (EPO) which is rich in 16:O 16:1 0.1 8.6 both 18:2n-6 and 18:3n-6. The presence of the 16:2+ 16:3 1.7 latter acid, which by-passes the rate-limiting step 18:0 1.9 3.2 12.6 of delta-6-desaturation (4) facilitates the forma18:l 14.5 8.1 3.0 74.1 0.1 1.2 18:2n-6 tion of AA and provides the precursors for 9.1 18:3n-6 subsequent PG and TX biosynthesis. Materials and Methods Chemicals
All PGs and thromboxane B2 (TxB2) were purchased from Upjohn Co. (Kalamazoo, Michigan). Antisera to 6-keto-PGFi, and TxB2 were obtained from Seragen Inc. (Boston, MA), and antiserum to PGE2 from Institut Pasteur Production (Paris, France). Tritium labelled prostanoids were supplied by New England Nuclear (Boston, MA). Animal;
and diets
Seventy-two male Sprague-Dawley rats (Charles River, Montreal, Quebec), weighing 60-70 g, were randomly separated into 9 groups of 8 rats
0.4
18:3n-3 18:4n-3 20: 1 22: 1 20:4n-3 20:5n-3 22:4n-3 22:5n-3 22:6n-3 24:l
Minor
-
-
-
-
-
-
-
fatty
acids (< 0.1%)
2.1 2.8 2.6 3.0 1.0 16.9 0.5 2.6 12.0 1.4
are not included.
The superior mesenteric vascular bed (MVB) was prepared by cannulating the superior mesenteric artery 0.7 cm distal to the aorta as previously described (24, 25). The isolated MVB was then mounted in an organ bath at 37”C, and perfused with Krebs-Henseleit solution gassed
PG OUTFLOW FROM PERFUSED MESENTERIC VASCULATURE
7s
OF RATS
fatty acids of total phospholipids were then methylated and analyzed on a 6-ft glass column packed with 10% Silar 1OC on Gas Chrom Q, 100/120 mesh (Applied Science Laboratories, State College, PA) using a Hewlett-Packard 588OA gas chromatograph as described previously (26).
with ‘5% CO;! in oxygen at a flow rate of 3 ml/min using a Watson-Marlow peristaltic pump flow inducer. The perfusion pressure was monitored via a side arm off the arterial cannula using a pressure transducer (Model 13-4615-52, Gould). Aliquots of the effluent were collected for 1 minute at 30, 60 and 90 minutes after starting the perfusion.
Statistical analysis Radioimmunoassay
Results are expressed as means + SEM. The significance of differences between HCO- and FO-fed rats (with the same proportion of oil replacement) were compared using Student’s ttest. The significance of variations between different dietary groups was assessed using oneway analysis of variance by a computer.
The levels of chemically unstable PGI2 and TxA2 were assessed by measuring the concentrations of their respective stable metabolites, 6-ketoPGFi, and TxB2. All PGs and TxB2 were measured by direct radio-immunoassay without extraction (19). The cross-reactivities of 6-ketoPGFI,, TxB2 and PGE:! antisera to 3-series PGs and thromboxane have not been determined, but to other eicosanoids and essential fatty acids, e.g., PGs, AA, EPA and DHA were less than 0.1% and were described previously (25).
Results and Discussion During the study, no significant differences were found for food consumption. At the end of the feeding, the body weight gains were significantly lower in rats fed the 10% HCO supplemented diet than in rats fed a diet supplemented wholly or partly with either EPO or FO (data not shown). No differences were noted among the latter groups. The analyses of plasma and liver fatty acids in rats fed the 10% HCO supplemented diet showed high levels of 20:3n-9 and high
Fatty acid analysis of aortic phospholipids
Aortae were excised, homogenized and extracted with chloroform-methanol, 2: 1 (v/v) containing 0.025% butylated hydroxytoulene as antioxidant. Lipids were fractionated by silica gel thin layer chromatoplate using hexane-diethyl ether-acetic acid, 80:20:1 (v/v/v) as developing solvent. The
M-T
PCE2
llO-
TxB,
loo-
--l m-
l
\ \ \ \ I , 60\ \ \ 50! , \I 40l. w-
M-
I
P-
.\
-\ T ‘. ‘\
\
I “q
-
10 o0
30
PERFWON
60
TIME
90
0
30
,
II
so
90
(mln)
Fig 1 The levels of 6-keto-PGF,,, TxBz and PGEz in the perfusate (O-90 min) from the mesenteric vascular a FF diet supplemented with 10% by weight of evening primrose oil for 8 weeks. Each data point represents of 8 aminals.
bed of rats fed mean f SEM
76
PROSTAGLANDINS
trieneitetraene ratios indicating the presence of essential fatty acid (EFA) deficiency (27). This was not observed in any other rats supplemented with EPO or FO (wholly or partly) indicating the absence of EFA deficiency. There were also no significant differences between the weights of the prepared mesenteric vascular bed among groups. Nevertheless, the outputs of prostaglandins (PGE2 and 6-keto-PGF1,) and thromboxane (TxB2) were adjusted for tissue weights for the comparison. Values were expressed as pg/ml/g perfused mesenteric tissue. In the present study, the most abundant prostanoids in the perfusates from the MVB of rats fed diets supplemented wholly with EPO, were followed by TxB2 and PGE2 6-keto-PGFi,, (Figure 1). In general, the patterns of release were similar and the initial levels of 6-ketoPGFI,, TxBz and PGE2 were very high. The levels fell rapidly during the first 30 minutes of perfusion. The decline continued during the next 60 minutes but at a slower pace. We have previously suggested that the high initial release of PGs and TxB2 may be caused by the trauma of the MVB preparation as well as the ample supply of PG precursor (AA). We have also suggested that the levels between 30 and 90 minute-perfusion best represent the physiological situation (28). Hence, the responses of PGs and TxBz outflow to dietary manipulations in this study, were reported at one selected time point - 60 min after starting the perfusion. The effects of dietary change on the release of prostanoids are shown in Figure 2. The levels of (PG12 metabolite), which is 6-keto-PGF1, synthesized and secreted by endothelial cells of the arteries (29) were significantly reduced from MVB of rats fed a diet in which EPO was partly or completely replaced by either FO or HCO (Figure 2, panel A). The reduction was accentuated as the proportion of the replacement increased. The extent of reduction was greater in FO-fed than in HCO-fed rats. Although EPA has been shown to form PGI3, which is as effective as PGI2, in humans (30), this has not been confirmed in rats (31, 32). Thus, the 6-ketoPGFi,-like activities measured in the present study were probably solely contributed by AA. A reduction of 6-keto-PGFi, outflows was likely the result of suppressed conversion of AA by fish oil n-3 fatty acids. There is evidence that indicates TxAz can be synthesized by various vascular systems (33-35). When dietary EPO was replaced partly or
LEUKOTRIENES
I
AND ESSENTIAL FAT-I-Y ACIDS
6-keto-PCFta
.
0 A
, B
1 C
I 0
I E
Fig 2 The levels of 6-keto-PGF,,, TxB, and PGEl in the effluents (at the 60-minute perfusion time) of mesenteric vascular bed isolated from rats fed a diet supplemented with 10% by weight of oil, composed wholly evening primrose oil (group A, o), or partly and completely (25, 50,75 and lOO%, represent respectively groups B, C. D and E) replaced by either hydrogenated coconut oil (O----O) or fish oil (A-..A). Each point represents means f SEM of 8 determinations. *p < 0.05.
completely by FO or HCO, the levels of TxBz in effluents from MVBs were also significantly reduced. The reduction was greater in FO-fed than in HCO-fed rats (Figure 2, panel B). The activities of TxB2 were nearly abolished when FO replacement reached 50% or more of the dietary fat. A relatively greater reduction of TxB2 secretion in FO-fed rats could be due to a reduction of TxB2 biosynthesis by mesenteric vessel wall as EPA in fish oil not only competes with AA for PL incorporation (14), but also suppresses the conversion of AA to thromboxanes (9, 10, 17). Although it has been shown that
PG OUTFLOW
FROM PERFUSED
MESENTERIC
VASCULATURE
EPA can be converted to TxAs which was less effective in comparison with TxA2 in human platelets (36), this was not observed in rats (31). Therefore, the reduction of TX-like activities was probably not due to the formation of inactive TxA3 but to the reduction of TxA2. The pattern of the release of PGE2 into the perfusate was similar to that of 6-keto-PGF,,, although the levels of PGE2 were significantly lower. The levels of PGEz were further reduced in animals fed a diet in which EPO was partly or completely replaced with FO or HCO. Unlike the responses of either 6-keto-PGFi, or TxB2, the levels of PGE2 were slightly higher in rats fed a diet in which 25% of the supplemented fat was replaced with FO than with HCO. This difference was not observed when the replacement of either FO or HCO exceeded 25%. In our previous study (25) we observed an increase of PGEz in animals fed a similar diet supplemented with 10% by weight oil of which 80% was EPO and 20% was fish oil. It is not known why the levels of PGE2 in MVB from rats fed a diet of which 20-25% EPO was replaced with FO should be greater than when replaced by HCO. Perhaps at this combination, the AA levels although reduced remain sufficient to provide the substrate for PGEz biosynthesis, whereas the level of EPA was not great enough to suppress that PGE2 synthesis. In any case, the biosynthesis of PGE2 appeared to be regulated differently from that of 6-keto-PGFi, or TxB2. Overall our study indicates that a partial or complete depletion of dietary n-6 fatty acids significantly reduced the outflow of PGs from isolated MVB indicating that the outflows of PGs and TxB2 depend on the availability of AA. The presence of n-3 fatty acids further accentuated this effect, and is consistent with the previous observation that dietary fish oil suppressed the formation of PGs (37). Replacing only 25% of the dietary oil with FO (group B) nearly abolished the outputs of PGIZ, but to achieve the same extent of effect for TxB2 and PGE2, 50% replacement was required (Figure 2). This suggests that the long chain n-3 fatty acids differentially affect the outflows of PGs and TxB2. in the present study, the isolated measenteric vasculature were perfused for at least 1 l/2 hour. It is possible that the phospholipid fatty acid composition of the tissue at the end of the perfusion might not accurately reflect the initial physiological condition. We elected to examine the fatty acid composition of aortic tissue in
77
OF RATS
order to provide some reference data. Since the response of mesenteric vasculature to FO feeding appeared to be in accordance with that in aortic segments (18), we believe it is justifiable to extrapolate the changes of aortic PL composition to PG release from the mesenteric vasculature in response to dietary manipulation. It is interesting to notice that the levels of 18:3n6 in animals fed the diet supplemented with soly 18:3n-6 rich-EPO were not significantly elevated. Perhaps, the absorbed GLA was rapidly metabolized to its metabolites when the metabolic process was not interfered through the competition and suppression by the presence of other fatty acids. In animals fed the HCO-supplemented diet, the levels of 20:3n-9 were significantly elevated, whereas those of 18:2n-6 and 20:4n-6 were reduced in aortic phospholipids indicating the presence of EFA deficiency (Figure 3). However, the ratio of 20:4n-6/18:2n-6 (2.03) was not significantly different from that in rats fed a wholly EPO-supplemented diet (1.92). This result suggests that the reduction of PG production in EFA deficient rats was due to the insufficiency of supply of 18:2n-6 and its metabolite 20:4n-6. On the other hand, dietary fish oil which elevated the content of EPA, had no effect on the
20 8 -0 z 16 6 > z m 12 LL
I
Fig 3 The percent composition (mean f SD of 8 determinations) of 20:3n-9, 18:2n-6, 20:4n-6 and 20:5n-3 in aortic phospholipids in rats fed a semi-synthetic diet supplemented with 10% by weight of evening primrose oil (CI), fish oil (@) or hydrogenated coconut oil ia) for 8 weeks: *Significahtlv different from that in EPO-fed rats at D < 0.05. +Significantly different from that in FO-fed rats ai p < 0.05.
78
PROSTAGLANDINS
levels of 18:2n-6 but significantly reduced those of 20:4n-6 in the aortic tissues. The ratio of 20:4n-6/18:n-6 (0.85) was significantly reduced in the aortic PL of FO-fed rats. Thus, our results indicate that the reduction of PG production in FO-fed rats was due to the insufficient conversion of LA to AA. An interesting observation is that feeding HCO also resulted in an increase in EPA, but not DHA (Figure 3). Since, AA levels were similar in animals fed HCO and FO, while PG synthesis was consistently lower in the FO group, this probably indicates inhibition of conversion of AA to eicosanoids by the high contents of EPA and DHA resulted in fish oilfeeding. Acknowledgement
12.
13.
14.
15.
16.
The authors wish to thank Mrs. Valerie Billard-Simmons and Mr. Ken Jenkins for their excellent technical assistance.
References of prostaglandin production in tussues by dietary essential fatty acids. Acad Med Stand 642 (Suppl): 171-179, 1980 2. ten Hoor F, de Deckere E A M, Haddeman E, Hornstra G, Quandt JFA. Dietary manipulation of prostaglandin and thromboxane synthesis in heart, aorta and blood platelets of the rat. Adv Prostaglandin Thromboxane Res 8: 1771-1781, 1980. 3. Croft K D, Codde J P, Beilin L J, Vandongen R. Dietary modification of eicosanoid synthesis - a time course study. Prog Lipid Res 25: 181-184, 1986. 4. Bernert J T, Jr, Sprecher H. Studies to determine the role rates of chain elongation and desaturation play in regulating the unsaturated fatty acid composition of rat liver lipids. Biochim Biophys Acta 398: 354-363, 1976. 5. Willis A L. Nutritional and pharmacological factors in eicosanoid biology. Nutr Rev 39: 289-300, 1981. 6. Crawford M A. Denton J P, Hassam A G, Lynn L. Marples P, Stevens A, Willis A L. Levels of prostaglandins and their precursors in EFA-deficient rabbits - a new concept of prostaglandin biosynthesis. Br J Pharmacol 63: 363P-364P, 1978. 7. Willis A L, Hassam A G, Crawford M A, Stevens P, Denton J P. Relationships between nrostaglandins. prostacyclin and EFA precursors in rabbit maintained on EFA-deficient diets. Pror! Linid Res 20: 161-167. 1981. 8. Huang Y-S, Mitchell J, J&kin K, Manku M S, Horrobin D F. Effect of dietary depletion and repletion of linoleic acid on renal fatty acid composition and urinary prostaglandin excretion. Prostaglandins Leukotrienes Med 15: 223-228, 1984. 9. Needleman P, Raz A, Minkes M S, Ferrendelli J A, Sprecher H. Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties. Proc Nat1 Acad Sci USA 76: 944-948, 1979. 10. Gulp B R, Titus B G, Lands W E M. Inhibition of prostaglandin biosynthesis by eicosapentaenoic acid. Prostaglandins Med 3: 269-278, 1979. 11. Huang Y-S, Nassar B A, Horrobin D F. Changes of
17.
1. Galli C, Agradi E, Petroni A, Socini A. Modulation
18.
19.
20.
21.
22. 23.
24. 25.
26. 27
LEUKOTRIENES
AND ESSENTIAL FATI’Y ACIDS
plasma lipids and long-chain n-3 and n-6 fatty acids in plasma, liver, heart and kidney phospholipids of rats fed varable levels of fish oil with or without cholesterol supplementation. Biochim Biophys Acta 879: 22-27. 1986. Nassar B A. Huang Y-S, Horrobin D F. Response of tissue phospholipid fatty acid composition to dietary (n6) and replacement with marine (n-3) and saturated fatty acids in the rat. Nutr Res 6: 1397-1409, 1986. Nassar B A, Huang Y-S, Manku M S. Das U N, Mores N, Horrobin D F. The influence of dietary manipulation with n-3 and n-6 fatty acids on liver and plasma phospholipid fatty acids in rats. Lipids 21: 652-656, 1986. Iritani N, Fujikawa S. Competitive incorporation of dietary omega-3 and omega-6 polyunsaturated fatty acids into tissue phospholipids. J Nutr Sci Vitamin01 28: 621-629, 1982. Lands W E M, LeTeilier P R, Rome L H, Vanderhoek J Y. Inhibition of prostaglandin biosynthesis. Adv Biosci 9: 15-28, 1973. Hoffman P, Taube C, Ponicke K, Zehl U, Beitz J, Forster W, Sonova S, Orbetzova V, Davidson F. Alterations in renal and aortic prostaglandin E and F formation correlate with blood pressure increase in salt loaded rats after dietary linoleate deficiency. Arch Int Pharmacodyn Therap 259: 40-58, 1982. Hornstra G. In: Dietary Fats, Prostanoids and Arterial Thrombosis. Boston, M. Nijhoff Publishers, 1982. Socini A, Galli C. Columbo C. Tremoli E. Fish oil administration as a supplement to corn oil containing diet affects arterial prostacyclin production more than platelet thromboxane formation in the rat. Prostaglandins 25: 693-709, 1983. Soma M, Mnaku M S, Jenkins D K. Horrobin D F. Prostaglandin and thromboxane outflow from the perfused mesenteric vascular bed in spontaneously hypertensive rats. Prostaglandins 29: 323-332, 1985. _ Brenner R P. De Thomas M E. Peluffo R 0. Effect of polyunsaturated fatty acids on the desaturation in vitro of linoleic acid and gamma-linolenic acid. Biochim Biophys Acta 106: 640~-642. Garcia P T. Holman R R. Comoetitive inhibitions in the metabolism of polyunsaturated fatty acids studied via the composition of phospholipids, triglycerides, and cholesteryl esters of rat tissues. J Am Oil Chem Sot 42: 1137-1141. Sprecher H. Biochemistry of essential fatty acids. Prog Lipid Res 20: 13-22. Huang Y-S, Horrobin D F. Responses of the ratio of 20:3n-9/total n-6 fatty acids in tissue phospholipids to dietary linoleate in the essential fatty acid deficient rats. Biochem Arch 2: 267-277. 1986. cGregor D D. The effect of sympathetic nerve stimulation on vascoconstrictor responses in perfused mesenteric blood vessels of the rat. J Physiol 177: 21-30, 1965. Nassar B A, Manku M S, Huang Y-S, Jenkins D K. Horrobin D F. The influence of dietary marine oil (POLEPA) and evening primrose oil (Efamol) on prostaglandin production by the rat mesenteric vasculare. Prostaglanhins Leukotrienes Med 26: 253-263, 1987. Manku M S, Horrobin D F, Huang Y-S, Morse N. Fatty acids in plasma and red cell membranes of normal humans. Lipids 18: 906908, 1983. Holman R T. The ratio of trienoic:tetraenoic acids in tissue lipids as a measure of essential fatty acid requirement. J Nutr 70: 405-410, 1960.
PC OUTFLOW FROM PERFUSED MESENTERIC VASCULATURE 28. Fujii K, Soma M, Huang Y-S, Manku M S, Horrobin D F. Increased release of prostaglandins from the mesenteric vascular bed of diabetic animals: The effects of glucose and insulin. Prostaglandins’Leukotrienes Med 24: 151-161, 1986. 29 Raz A, Isakson P C, Minkes M S, Needleman P. Characterization of a novel metabolic pathway of arachidonate in coronary arteries which generates a potent endogenous coronary vasodilator. J Biol Chem 252: 1123-1126, 1977. 30 Fischer S. Weber P C. Prostaglandin Ii is formed in vivo in man after dietary eicosapentaenoic acid. Nature 307: 165-168, 1984. 31 Hornstra G, Christ-Hazelhof E, Haddeman F, ten Hoor F. Nugteren D H. Fish oil feeding lowers thromboxane and prostacyclin production by rat platelets and aorta and does not result in the formation of prostaglandin I+ Prostaglandins 21: 727-738, 1981. 32 Hamazaki T, Hirai A, Terano T. Sajiki J, Kondo S, Fujita T. Tamura Y, Kumaga A. Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA. 20:5 w-3) on PGIr-like substance production by rat aorta. Prostaglandins 23: 557-567, 1982.
OF RATS
79
33. Levine L, Alam I. Arachidonic acid metabolism by cells in culture: analyses of culture fluids for cycle-oxygenase products by radioimmunoassay before and after separation by high pressure liquid chromatography. Prostaglandins Med 3: 295-304, 1979. 34. Ally A I, Horrobin D F. Thromboxane A, in blood vessel wall and its physiological significance: relevance to thrombosis and hypertension. Prostaglandins Med 4: 431-438. 1980. 35. Maurer P, Moskovitz M A, Levine L. Melamed E. The synthesis of prostaglandins by bovine cerebral microvesskis. Prostaglandins Med 4: j53-162, 1980. 36. Fischer S. Weber P C. Thromboxane A, (TxA,b is formed in human platelets after dietary eicosapentaenoic acid (C20:5w3). Biochem Biophys Res Commun 116: 1091-1099. 1983. 37. Soma M, Manku MS. Jenkins D K, Huang Y-S, Horrobin D F. Effects of dietary saturated, n-6 and n-3 polyunsaturated fats on blood pressure and prostaglandins outflow from perfused mesenteric vascular bed in rats. Prostaglandins 30: 969-980, 1985.
The Prostaglandin Outflow From Perfused Mesenteric Vasculature of Rats Fed Different Y.-S.‘HUANG,
B. A. NASSAR*
Fats
and D. F. HORROBIN
Efamol Research Institute, Kentville,
Nova Scotia, Canada B4N 4H8 (reprint requests to YSH)
Abstract - The effects of dietary n-6 polyunsaturated fatty acids and replacement with saturated fat or fish oil on the prostaglandin outflow from perfused mesenteric vasculature in rats were studied. Seventy-two weanling male rats were fed ad libitum a semi-synthetic diet supplemented with 10% by weight of oil, composed wholly of n-6 fatty acid-rich evening primrose oil, or replaced partly or completely (25, 50, 75 or 100%) by n-6 fatty acid-deficient fish oil or hydrogenated coconut oil for 8 weeks. The outflows of 6-ketoPGF,,, thromboxane Bz, and prostaglandin E from the perfused mesenteric vasculature were measured at 60 min-time point after starting the perfusion. In general, the release of prostanoids from the mesenteric vasculature was significantly reduced in rats fed a diet in which evening primrose oil was partly or completely replaced by either hydrogenated coconut or fish oil. This was probably due to the insufficient conversion of linoleic acid to arachidonic acid. The extent of reduction was greater in fish oil-fed than in hydrogenated coconut oil-fed rats, while the levels of arachidonic acid in aortic phospholipids were similar between these two groups. This result implies that the greater reduction of prostaglandin synthesis in rats fed fish oil was due to the inhibitory effect of eicosapentaenoic and docosahexaenoic acids in fish oil on the conversion of arachidonate to eicosanoids.
Introduction
rapidly elongated. to dihomo-gamma-linolenic acid (DGLA, 20:3n-6). DGLA is in turn desaturated to arachidonic acid (AA, 20:4n-6) (4). DGLA and AA serve as the immediate precursors for synthesis of 1 and 2 series of prostaglandins (PGs) and thromboxane (TX) via the cyclooxygenase pathway (5). Thus, reducing the dietary supply of LA can significantly diminish PG synthesis (6-8). Increasing fish oil intake also reduces the formation of TX (9, 10). Previously, we have demonstrated that the levels of dietary
Dietary polyunsaturated fatty acids, mainly linoleic acid (LA, 18:2n-6), play an important role in regulating the amount and type of eicosanoids synthesized (l-3). In the animal body, LA is desaturated by delta-6-desaturase .to gamma-linolenic acid (GLA, 18:3n-6), which is * Present Address: Department of Pathology, Division of Clinical Chemistry, Victoria General Hospital, Dalhousie University, Halifax, Nova Scotia, Canada B3H lV8. 73
74
PROSTAGLANDINS
LEUKOTRIENES
AND ESSENTIAL FATTY ACIDS
fish oil significantly affected the compositions of each. Animals were fed ad libitum a fat free (FF) n-3 and n-6 fatty acids in various rat tissue phos- semi-synthetic diet (Teklad Test Diets, Madison, pholipids (11, 12). Oils of sea water fish are WI) supplemented with 10% by weight oil, usually rich in n-3 polyunsaturated fatty acids; composed wholly of n-6 fatty acid-rich EPO (containing 74.1% 18:2n-6 and 9.2% 18:3n-6, whereas they are .deficient in n-6 fatty acids. Since dietary fish 011n-3 fatty acids compete with group A), or replaced partly or completely (25, n-6 fatty acids in the body for incorporation into 50, 75 or lOO%, represent groups B, C, D and E respectively) by n-6 fatty acid-deficient fish oil the tissue phospholipids as well as suppressing acid the formation of prostanoids from AA (2, 9, 13, (FO, containing 16.9% eicosapentaenoic 14), we have thus cautioned that prolonged high (EPA, 20:5n-3) and 12% docosahexaenoic acid level fish oil intake may induce a metabolic (DHA, 22:6n-3)) or hydrogenated coconut oil (HCO, containing 96.9% C8-Cl8 saturated fatty deficiency of n-6 fatty acids, particularly when acids) for 8 weeks. Animals were given fresh diet dietary intake of the n-6 fatty acids is not every day to minimize the oxidation of dietary adequate (11). Effects of fish oil on PG metabfats. Both FO and HCO contain very low olism have been frequently studied in rat aortic amounts of n-6 fatty acids. The detailed fatty tissues (15117), but little information is available acid compositions of dietary fats are shown in concerning the effects of diet on PG metabolism Table 1. The composition of the FF diet in the peripheral vessel system (18). Since the (containing 20% casein, 70.2% sucrose, 5% behaviour of different vascular systems can vary cellulose, 3.5% AINmineral mix, 1.0% significantly, the production of PGs from the vitamin mix, and 0.3% DL-methionine) has been peripheral vasculature in response to dietary described previously (23). manipulation may not be the same as that from the aorta. _ In the present study, we examined the outflow I Table 1 Fatty Acid Composition of Diet Fats of PGs and TxB2 from the isolated mesenteric % Weight vascular bed of the rat following administration Fatty EPO FO HCO of fish oil n-3 fatty acids in association with Acid partial or complete depletion of dietary n-6 fatty 8:O 7.1 acids. It has been demonstrated that n-3 fatty 1o:o 6.0 acids suppress the activity of delta-6-desaturation 12:o 44.9 0.1 0.1 14:o 17.2 7.1 (4, 20-22). We fed animals a diet supplemented 16.4 6.2 9.1 with evening primrose oil (EPO) which is rich in 16:O 16:1 0.1 8.6 both 18:2n-6 and 18:3n-6. The presence of the 16:2+ 16:3 1.7 latter acid, which by-passes the rate-limiting step 18:0 1.9 3.2 12.6 of delta-6-desaturation (4) facilitates the forma18:l 14.5 8.1 3.0 74.1 0.1 1.2 18:2n-6 tion of AA and provides the precursors for 9.1 18:3n-6 subsequent PG and TX biosynthesis. Materials and Methods Chemicals
All PGs and thromboxane B2 (TxB2) were purchased from Upjohn Co. (Kalamazoo, Michigan). Antisera to 6-keto-PGFi, and TxB2 were obtained from Seragen Inc. (Boston, MA), and antiserum to PGE2 from Institut Pasteur Production (Paris, France). Tritium labelled prostanoids were supplied by New England Nuclear (Boston, MA). Animal;
and diets
Seventy-two male Sprague-Dawley rats (Charles River, Montreal, Quebec), weighing 60-70 g, were randomly separated into 9 groups of 8 rats
0.4
18:3n-3 18:4n-3 20: 1 22: 1 20:4n-3 20:5n-3 22:4n-3 22:5n-3 22:6n-3 24:l
Minor
-
-
-
-
-
-
-
fatty
acids (< 0.1%)
2.1 2.8 2.6 3.0 1.0 16.9 0.5 2.6 12.0 1.4
are not included.
The superior mesenteric vascular bed (MVB) was prepared by cannulating the superior mesenteric artery 0.7 cm distal to the aorta as previously described (24, 25). The isolated MVB was then mounted in an organ bath at 37”C, and perfused with Krebs-Henseleit solution gassed
PG OUTFLOW FROM PERFUSED MESENTERIC VASCULATURE
7s
OF RATS
fatty acids of total phospholipids were then methylated and analyzed on a 6-ft glass column packed with 10% Silar 1OC on Gas Chrom Q, 100/120 mesh (Applied Science Laboratories, State College, PA) using a Hewlett-Packard 588OA gas chromatograph as described previously (26).
with ‘5% CO;! in oxygen at a flow rate of 3 ml/min using a Watson-Marlow peristaltic pump flow inducer. The perfusion pressure was monitored via a side arm off the arterial cannula using a pressure transducer (Model 13-4615-52, Gould). Aliquots of the effluent were collected for 1 minute at 30, 60 and 90 minutes after starting the perfusion.
Statistical analysis Radioimmunoassay
Results are expressed as means + SEM. The significance of differences between HCO- and FO-fed rats (with the same proportion of oil replacement) were compared using Student’s ttest. The significance of variations between different dietary groups was assessed using oneway analysis of variance by a computer.
The levels of chemically unstable PGI2 and TxA2 were assessed by measuring the concentrations of their respective stable metabolites, 6-ketoPGFi, and TxB2. All PGs and TxB2 were measured by direct radio-immunoassay without extraction (19). The cross-reactivities of 6-ketoPGFI,, TxB2 and PGE:! antisera to 3-series PGs and thromboxane have not been determined, but to other eicosanoids and essential fatty acids, e.g., PGs, AA, EPA and DHA were less than 0.1% and were described previously (25).
Results and Discussion During the study, no significant differences were found for food consumption. At the end of the feeding, the body weight gains were significantly lower in rats fed the 10% HCO supplemented diet than in rats fed a diet supplemented wholly or partly with either EPO or FO (data not shown). No differences were noted among the latter groups. The analyses of plasma and liver fatty acids in rats fed the 10% HCO supplemented diet showed high levels of 20:3n-9 and high
Fatty acid analysis of aortic phospholipids
Aortae were excised, homogenized and extracted with chloroform-methanol, 2: 1 (v/v) containing 0.025% butylated hydroxytoulene as antioxidant. Lipids were fractionated by silica gel thin layer chromatoplate using hexane-diethyl ether-acetic acid, 80:20:1 (v/v/v) as developing solvent. The
M-T
PCE2
llO-
TxB,
loo-
--l m-
l
\ \ \ \ I , 60\ \ \ 50! , \I 40l. w-
M-
I
P-
.\
-\ T ‘. ‘\
\
I “q
-
10 o0
30
PERFWON
60
TIME
90
0
30
,
II
so
90
(mln)
Fig 1 The levels of 6-keto-PGF,,, TxBz and PGEz in the perfusate (O-90 min) from the mesenteric vascular a FF diet supplemented with 10% by weight of evening primrose oil for 8 weeks. Each data point represents of 8 aminals.
bed of rats fed mean f SEM
76
PROSTAGLANDINS
trieneitetraene ratios indicating the presence of essential fatty acid (EFA) deficiency (27). This was not observed in any other rats supplemented with EPO or FO (wholly or partly) indicating the absence of EFA deficiency. There were also no significant differences between the weights of the prepared mesenteric vascular bed among groups. Nevertheless, the outputs of prostaglandins (PGE2 and 6-keto-PGF1,) and thromboxane (TxB2) were adjusted for tissue weights for the comparison. Values were expressed as pg/ml/g perfused mesenteric tissue. In the present study, the most abundant prostanoids in the perfusates from the MVB of rats fed diets supplemented wholly with EPO, were followed by TxB2 and PGE2 6-keto-PGFi,, (Figure 1). In general, the patterns of release were similar and the initial levels of 6-ketoPGFI,, TxBz and PGE2 were very high. The levels fell rapidly during the first 30 minutes of perfusion. The decline continued during the next 60 minutes but at a slower pace. We have previously suggested that the high initial release of PGs and TxB2 may be caused by the trauma of the MVB preparation as well as the ample supply of PG precursor (AA). We have also suggested that the levels between 30 and 90 minute-perfusion best represent the physiological situation (28). Hence, the responses of PGs and TxBz outflow to dietary manipulations in this study, were reported at one selected time point - 60 min after starting the perfusion. The effects of dietary change on the release of prostanoids are shown in Figure 2. The levels of (PG12 metabolite), which is 6-keto-PGF1, synthesized and secreted by endothelial cells of the arteries (29) were significantly reduced from MVB of rats fed a diet in which EPO was partly or completely replaced by either FO or HCO (Figure 2, panel A). The reduction was accentuated as the proportion of the replacement increased. The extent of reduction was greater in FO-fed than in HCO-fed rats. Although EPA has been shown to form PGI3, which is as effective as PGI2, in humans (30), this has not been confirmed in rats (31, 32). Thus, the 6-ketoPGFi,-like activities measured in the present study were probably solely contributed by AA. A reduction of 6-keto-PGFi, outflows was likely the result of suppressed conversion of AA by fish oil n-3 fatty acids. There is evidence that indicates TxAz can be synthesized by various vascular systems (33-35). When dietary EPO was replaced partly or
LEUKOTRIENES
I
AND ESSENTIAL FAT-I-Y ACIDS
6-keto-PCFta
.
0 A
, B
1 C
I 0
I E
Fig 2 The levels of 6-keto-PGF,,, TxB, and PGEl in the effluents (at the 60-minute perfusion time) of mesenteric vascular bed isolated from rats fed a diet supplemented with 10% by weight of oil, composed wholly evening primrose oil (group A, o), or partly and completely (25, 50,75 and lOO%, represent respectively groups B, C. D and E) replaced by either hydrogenated coconut oil (O----O) or fish oil (A-..A). Each point represents means f SEM of 8 determinations. *p < 0.05.
completely by FO or HCO, the levels of TxBz in effluents from MVBs were also significantly reduced. The reduction was greater in FO-fed than in HCO-fed rats (Figure 2, panel B). The activities of TxB2 were nearly abolished when FO replacement reached 50% or more of the dietary fat. A relatively greater reduction of TxB2 secretion in FO-fed rats could be due to a reduction of TxB2 biosynthesis by mesenteric vessel wall as EPA in fish oil not only competes with AA for PL incorporation (14), but also suppresses the conversion of AA to thromboxanes (9, 10, 17). Although it has been shown that
PG OUTFLOW
FROM PERFUSED
MESENTERIC
VASCULATURE
EPA can be converted to TxAs which was less effective in comparison with TxA2 in human platelets (36), this was not observed in rats (31). Therefore, the reduction of TX-like activities was probably not due to the formation of inactive TxA3 but to the reduction of TxA2. The pattern of the release of PGE2 into the perfusate was similar to that of 6-keto-PGF,,, although the levels of PGE2 were significantly lower. The levels of PGEz were further reduced in animals fed a diet in which EPO was partly or completely replaced with FO or HCO. Unlike the responses of either 6-keto-PGFi, or TxB2, the levels of PGE2 were slightly higher in rats fed a diet in which 25% of the supplemented fat was replaced with FO than with HCO. This difference was not observed when the replacement of either FO or HCO exceeded 25%. In our previous study (25) we observed an increase of PGEz in animals fed a similar diet supplemented with 10% by weight oil of which 80% was EPO and 20% was fish oil. It is not known why the levels of PGE2 in MVB from rats fed a diet of which 20-25% EPO was replaced with FO should be greater than when replaced by HCO. Perhaps at this combination, the AA levels although reduced remain sufficient to provide the substrate for PGEz biosynthesis, whereas the level of EPA was not great enough to suppress that PGE2 synthesis. In any case, the biosynthesis of PGE2 appeared to be regulated differently from that of 6-keto-PGFi, or TxB2. Overall our study indicates that a partial or complete depletion of dietary n-6 fatty acids significantly reduced the outflow of PGs from isolated MVB indicating that the outflows of PGs and TxB2 depend on the availability of AA. The presence of n-3 fatty acids further accentuated this effect, and is consistent with the previous observation that dietary fish oil suppressed the formation of PGs (37). Replacing only 25% of the dietary oil with FO (group B) nearly abolished the outputs of PGIZ, but to achieve the same extent of effect for TxB2 and PGE2, 50% replacement was required (Figure 2). This suggests that the long chain n-3 fatty acids differentially affect the outflows of PGs and TxB2. in the present study, the isolated measenteric vasculature were perfused for at least 1 l/2 hour. It is possible that the phospholipid fatty acid composition of the tissue at the end of the perfusion might not accurately reflect the initial physiological condition. We elected to examine the fatty acid composition of aortic tissue in
77
OF RATS
order to provide some reference data. Since the response of mesenteric vasculature to FO feeding appeared to be in accordance with that in aortic segments (18), we believe it is justifiable to extrapolate the changes of aortic PL composition to PG release from the mesenteric vasculature in response to dietary manipulation. It is interesting to notice that the levels of 18:3n6 in animals fed the diet supplemented with soly 18:3n-6 rich-EPO were not significantly elevated. Perhaps, the absorbed GLA was rapidly metabolized to its metabolites when the metabolic process was not interfered through the competition and suppression by the presence of other fatty acids. In animals fed the HCO-supplemented diet, the levels of 20:3n-9 were significantly elevated, whereas those of 18:2n-6 and 20:4n-6 were reduced in aortic phospholipids indicating the presence of EFA deficiency (Figure 3). However, the ratio of 20:4n-6/18:2n-6 (2.03) was not significantly different from that in rats fed a wholly EPO-supplemented diet (1.92). This result suggests that the reduction of PG production in EFA deficient rats was due to the insufficiency of supply of 18:2n-6 and its metabolite 20:4n-6. On the other hand, dietary fish oil which elevated the content of EPA, had no effect on the
20 8 -0 z 16 6 > z m 12 LL
I
Fig 3 The percent composition (mean f SD of 8 determinations) of 20:3n-9, 18:2n-6, 20:4n-6 and 20:5n-3 in aortic phospholipids in rats fed a semi-synthetic diet supplemented with 10% by weight of evening primrose oil (CI), fish oil (@) or hydrogenated coconut oil ia) for 8 weeks: *Significahtlv different from that in EPO-fed rats at D < 0.05. +Significantly different from that in FO-fed rats ai p < 0.05.
78
PROSTAGLANDINS
levels of 18:2n-6 but significantly reduced those of 20:4n-6 in the aortic tissues. The ratio of 20:4n-6/18:n-6 (0.85) was significantly reduced in the aortic PL of FO-fed rats. Thus, our results indicate that the reduction of PG production in FO-fed rats was due to the insufficient conversion of LA to AA. An interesting observation is that feeding HCO also resulted in an increase in EPA, but not DHA (Figure 3). Since, AA levels were similar in animals fed HCO and FO, while PG synthesis was consistently lower in the FO group, this probably indicates inhibition of conversion of AA to eicosanoids by the high contents of EPA and DHA resulted in fish oilfeeding. Acknowledgement
12.
13.
14.
15.
16.
The authors wish to thank Mrs. Valerie Billard-Simmons and Mr. Ken Jenkins for their excellent technical assistance.
References of prostaglandin production in tussues by dietary essential fatty acids. Acad Med Stand 642 (Suppl): 171-179, 1980 2. ten Hoor F, de Deckere E A M, Haddeman E, Hornstra G, Quandt JFA. Dietary manipulation of prostaglandin and thromboxane synthesis in heart, aorta and blood platelets of the rat. Adv Prostaglandin Thromboxane Res 8: 1771-1781, 1980. 3. Croft K D, Codde J P, Beilin L J, Vandongen R. Dietary modification of eicosanoid synthesis - a time course study. Prog Lipid Res 25: 181-184, 1986. 4. Bernert J T, Jr, Sprecher H. Studies to determine the role rates of chain elongation and desaturation play in regulating the unsaturated fatty acid composition of rat liver lipids. Biochim Biophys Acta 398: 354-363, 1976. 5. Willis A L. Nutritional and pharmacological factors in eicosanoid biology. Nutr Rev 39: 289-300, 1981. 6. Crawford M A. Denton J P, Hassam A G, Lynn L. Marples P, Stevens A, Willis A L. Levels of prostaglandins and their precursors in EFA-deficient rabbits - a new concept of prostaglandin biosynthesis. Br J Pharmacol 63: 363P-364P, 1978. 7. Willis A L, Hassam A G, Crawford M A, Stevens P, Denton J P. Relationships between nrostaglandins. prostacyclin and EFA precursors in rabbit maintained on EFA-deficient diets. Pror! Linid Res 20: 161-167. 1981. 8. Huang Y-S, Mitchell J, J&kin K, Manku M S, Horrobin D F. Effect of dietary depletion and repletion of linoleic acid on renal fatty acid composition and urinary prostaglandin excretion. Prostaglandins Leukotrienes Med 15: 223-228, 1984. 9. Needleman P, Raz A, Minkes M S, Ferrendelli J A, Sprecher H. Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties. Proc Nat1 Acad Sci USA 76: 944-948, 1979. 10. Gulp B R, Titus B G, Lands W E M. Inhibition of prostaglandin biosynthesis by eicosapentaenoic acid. Prostaglandins Med 3: 269-278, 1979. 11. Huang Y-S, Nassar B A, Horrobin D F. Changes of
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LEUKOTRIENES
AND ESSENTIAL FATI’Y ACIDS
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PC OUTFLOW FROM PERFUSED MESENTERIC VASCULATURE 28. Fujii K, Soma M, Huang Y-S, Manku M S, Horrobin D F. Increased release of prostaglandins from the mesenteric vascular bed of diabetic animals: The effects of glucose and insulin. Prostaglandins’Leukotrienes Med 24: 151-161, 1986. 29 Raz A, Isakson P C, Minkes M S, Needleman P. Characterization of a novel metabolic pathway of arachidonate in coronary arteries which generates a potent endogenous coronary vasodilator. J Biol Chem 252: 1123-1126, 1977. 30 Fischer S. Weber P C. Prostaglandin Ii is formed in vivo in man after dietary eicosapentaenoic acid. Nature 307: 165-168, 1984. 31 Hornstra G, Christ-Hazelhof E, Haddeman F, ten Hoor F. Nugteren D H. Fish oil feeding lowers thromboxane and prostacyclin production by rat platelets and aorta and does not result in the formation of prostaglandin I+ Prostaglandins 21: 727-738, 1981. 32 Hamazaki T, Hirai A, Terano T. Sajiki J, Kondo S, Fujita T. Tamura Y, Kumaga A. Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA. 20:5 w-3) on PGIr-like substance production by rat aorta. Prostaglandins 23: 557-567, 1982.
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79
33. Levine L, Alam I. Arachidonic acid metabolism by cells in culture: analyses of culture fluids for cycle-oxygenase products by radioimmunoassay before and after separation by high pressure liquid chromatography. Prostaglandins Med 3: 295-304, 1979. 34. Ally A I, Horrobin D F. Thromboxane A, in blood vessel wall and its physiological significance: relevance to thrombosis and hypertension. Prostaglandins Med 4: 431-438. 1980. 35. Maurer P, Moskovitz M A, Levine L. Melamed E. The synthesis of prostaglandins by bovine cerebral microvesskis. Prostaglandins Med 4: j53-162, 1980. 36. Fischer S. Weber P C. Thromboxane A, (TxA,b is formed in human platelets after dietary eicosapentaenoic acid (C20:5w3). Biochem Biophys Res Commun 116: 1091-1099. 1983. 37. Soma M, Manku MS. Jenkins D K, Huang Y-S, Horrobin D F. Effects of dietary saturated, n-6 and n-3 polyunsaturated fats on blood pressure and prostaglandins outflow from perfused mesenteric vascular bed in rats. Prostaglandins 30: 969-980, 1985.