Biosynthesis of prostanoids, tissue fatty acid composition and thrombotic parameters in rats fed diets enriched with docosahexaenoic (22:6n3) or eicosapentaenoic (20:5n3) acids

THROMBOSIS RESEARCH 34; 479-497, 1984 0049-3848/84 $3.00 + .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.

BIOSYNTHESIS OF PROSTANOIDS, TISSUE FATTY ACID COMPOSITION AND THROMBOTIC PARAMETERS IN RATS FED DIETS ENRICHED WITH DOCOSAHEXAENOIC (22:6n3) OR EICOSAPENTAENOIC (20:5n3) ACIDS

Geza G. Bruckner, Bruce German,

*, Belur Lokesh, and John E. Kinsella,

*Current Address: Department of Clinical Nutrition, University of Kentucky, Lexington, Kentucky 40536; Department of Food Science, Cornell University, Ithaca, New York 14853 (Received 19.12.1983; Accepted in revised form 27.3.1984 by Editor K.M. Brinkhous) ABSTRACT The objective of this experiment was to elucidate the effect(s) of eicosapentaenoic (20:5n3) vs docosahexaenoic (22:6n3) acid on prostaglandin biosynthesis and related thrombotic parameters. Diets were formulated to contain oils absent in (control) or enriched with either 20:5n3 (EPA) or 22:6n3 (DHA). The diets were fed to rats for three weeks and the following evaluated: 1) bleeding time; 2) blood viscosity; 3) platelet aggregation; 4) tissue fatty acids; 5) serum thromboxane (TXB2), aortic prostacylin (6-keto) and 6) arachidonic acid conversion to eicosanoids by lung microsomes. There were no significant differences between treatments for bleeding time, red blood cell viscosity or platelet aggregation. In EPA fed rats 20:5n3 increased significantly in platelet and aorta phospholipids. In platelets and aorta 20:4n6 was slightly decreased in EPA and DHA animals. Platelet 22:6n3 levels were not altered by treatment, but 22:6n3 increased in the aorta of EPA and DHA fed rats. Similar changes were noted in lung and liver fatty acid composition. Serum TXB2 levels were significantly decreased only in the EPA vs control group. No differences were noted for aortic 6-keto levels or in the amount of hydroxy fatty acids, PGE, TXB2 or PGF2u produced by lung microsomes. While fish oils have been shown to alter hematologic parameters in humans this study suggests that the rat is not similarly affected. Furthermore, it is evident that in the rat, 20:5n3 and not 22:6n3 is responsible for the alterations in platelet prostaglandin biosynthesis; however, these observations may not be directly applicable to other species.

Key Words: Docosahexaeno ic, eicosapentaenoic, platelet, prostacyclin, 479

fish oil, thromboxane,

480

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

INTRODUCTION Greenland Eskimos have a low incidence of coronary heart disease, exhibit increased bleeding tendency, show alterations in plasma lipoproteins, and demonstrate marked changes in platelet aggregation responses compared to their Danish counterparts (1,2,3). These alterations in hematologic functions have been attributed to increased consumption of fish by the Greenland Eskimos (4). Fish lipids contain large amounts of n3 fatty acids in their membrane phospholipids and triacylglycerides compared to mammals (5). These fish oils may contain among other fatty acids lo-20% of the n-3 fatty acids as eicosapentaenoic (EPA - 20:5n3) or docosahexaenoic (DHA 22:6n3) acids (5). Greenland Eskimos (4) and Japanese fishermen (6,7) consuming a largely fish based diet accumulate EPA in plasma and platelet phospholipids; e.g. Eskimo platelet phospholipids contain 8% EPA compared to 0.5% in Eskimos living in Denmark (8). EPA is apparently incorporated into platelet phospholipids at the expense of arachidonic acid (AA) and is thereby, in part, thought to elicit its beneficial antithrombotic effect by decreasing the substrate level for proaggregatory prostanoids (9‘11). Indeed, as the concentration of fish oil is increased in the diet there is an apparent decrease in platelet aggregability (10,12). Administration of fish oils to dogs (13,52), cats (14) and humans is associated with a decreased incidence of infarcts (3). To date, the beneficial effects of fish oils on hemostatic functions have been mainly attributed to EPA; however, many of the fish oils used contain substantial amounts of other fatty acids. Of particular interest in this context is DHA which may constitute up to 20 wt% of some oils. This fatty acid is a more effective competitive inhibitor of cyclooxygenase than EPA -in vitro (15). The competition of the n-3 with the n-6 fatty acids for the cyclooxygenase active site and the subsequent decrease in proaggregatory prostanoid biosynthesis has been suggested as a possible mechanism for the observed beneficial effects of fish oils (8,12). The relative effectiveness of DHA in vivo biochemical and physiological parameters has not been vs. EPA on -studied. Therefore, this investigation was conducted to assess the effects of diets enriched in either DHA or EPA on the following specific parameters in rats: 1) bleeding time; 2) blood viscosity; 3) platelet aggregation; 4) tissue phospholipid fatty acid composition; 5) serum thromboxane and aortic prostacyclin biosynthesis; measured as TXB2 and 6-keto PFGld, respectively, and 6) arachidonic acid conversion to eiconsanoids by lung microsomes. METHODS Diets and Enrichment of n-3 PUFA A semi-synthetic diet was formulated as depicted in Tables I and II to meet the known nutrient requirements of rats (16). The diets were mixed to contain sufficient amounts of essential fatty acid (EFA) as 18:2n6 (2 energy%). Fish oils containing high concentrations of either EPA (menhaden oil) or DHA (shark liver oil) were further enriched by n3 fatty acids to accentuate the possibly different effects of these fatty acids on thrombotic parameters as shown in Table III and Fig. 1 using fractionation methods based on lithium hydroxide crystallization and urea adduct formation as basically described by Hilditch and Maddison (17) and Haagama et al. (18). By this protocol EPA and DHA were enriched in the menhaden

Vol.34,

481

EPA-DHA AND PROSTAGLANDIN

No.6

oil from 17.9 to 29.2 and 7.6 to 31.9%, respectively; EPA and DHA in shark oil were enriched from 6.5 to 16.7 and 19.7 to 60.1, respectively. This

fractionation procedure permitted the removal of monoenoic and saturated fatty acids and resulted in a more favorable fatty acid mixture by removal of cetoleic acid (22:lnil) which is known for its cardiopathic effects (19). The enriched n-3 fatty acids were then ethylated and the ethyl esters reacted with glycerol (1:4 molar ratio) in the presence of 0.5% NaOH to form a triglyceride (TG), diglyceride (DG), monoglyceride (MG), ethyl ester (EE) mixture as previously described (20). The percent distribution of these fractions were as follows: TG (41%), DG and MG (25%), EE (29%) and polymerized FA (5%). The enriched n-3 mixture was solvent extracted, washed 3X with H20, BHT (0.02%)added and stored at -70 degrees C under N, prior to use. The final fatty acid composition of the diets as analyzed is presented in Table 2 (see Methods, Lipid Analysis). TABLE I. Composition _--of Diet Used in Studying the Effects of Eicosapentaenoic and Docosahexaenoic Acids in Rat;;--Weight % Casein* Cornstarch Cellulose AIN 76 Mineral Mix** AIN 76 Vitamin Mix** Methionine Choline Fat (.02% BHT and 5 IU Vitamin E acetate+/g oil)

Fat Components Safflower Oil Tristearin Triolein Mix Enriched Shark Oil Enriched Menhaden Oil Amount of: 20:5n-3 22:6n-3

Kcal/Kg

20.0 65.0

10400 33800

5.0 3.5

1.0 0.3 0.2 5.0

4095

Control EPA DHA __--___wt%__-__.Y1.22

1.22

1.22

3.78

0 0

2.28 0 1.50

2.70 1.09 0

0 0

.38

-41

.16 .56

"Casein-vitamin free (ICN Nutritional Biochemical, Cleveland, OH) **J. Nutr. 1977. 107(7):1340-1348. +Vitamin E acetate - Eastman Kodak, Rochester, NY. The diets were mixed and stored in plastic containers (-250 g portions) under N2 at -20 degrees C. Diets were replaced daily (24 hrs) and any unconsumed diet was discarded. The diets were tested for lipid peroxidation by assessing the amount of malondialdehyde (MDA) present using thiobarbituric acid reagent (21,22). The initial MDA content of the control, EPA and DHA diets was as follows: 65.9, 92.0 and 78.7 ug/lOO g. After 24 hr exposure, there was an insignificant increase in MDA content

482

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

(70.2, 91.6, and 88.6 ug/lOO g, respectively). MDA values of the DHA diet (most highly unsaturated) increased approximately 251 during the 21 day feeding trial; however, the amount of XDA present (range 48-92 ug/lOO g diet) yas far below the values reported by others in diets containing fish oils (23). TABLE II. Fatty Acid Composition -of the Dietary --__-Fats as Fed to Rats Control

EPA*

DHA*

_________wt&____16:O 16:1 18:O 18:l 18:2 18:3 18:4 20:4 20:5 21:5 22:4 22~5 22:6

7.2 3.9 8.1 42.5 36.9 1.5

4.9 2.7 5.3 29.6 33.8 1.1 3.9 8.2 1.1 0.1 8.9

6.2 3.4 5.8 34.5 35.0 1.0 -2 .9 3.7 0.8 0.6 14.9

*See Table I and Text for Definition Animals Sprague Dawley (Charles River, CDSDBR, Wilmington, MA) male rats, 250300 g, were randomly assigned to the various dietary regimens (10 rats/group) and housed individually in wire bottom galvanized steel cages. Food and water were provided ad libitum and feed consumption and body weight gains recorded. Room temperature was maintained at 23 + 2 degrees C and a 12 hour dark-light cycle automatically controlled. Bleeding Time To determine if dietary EPA or DHA affected hemostatic parameters in rats, the bleeding time was determined via the right saphenous vein of the rats following Na pentobarbitol anesthesia (35 mg/Kg body weight) as previously described (24). This method is a measure of the time (seconds) required for cessation of blood flow through a 27 gauge needle. Blood and Tissue Sample Preparation Following the induction of anesthesia an incision along the -linea alba Arterial blood was was performed and the abdominal aorta exposed. withdrawn through a siliconized 22 gauge needle into a plastic syringe (-79 ml) and a portion quickly dispensed into a plastic centrifuge tube containing 3.8% sodium citrate to result in a 0.8:9.2 citrate to blood volume (total‘volume 5 ml). The remaining blood was placed into a glass centrifuge tube and allowed to clot for 30 min at room temperature (25

483

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

degrees C). The clotted blood was centrifuged and the serum removed and acLi. blood was acldLfLed to pH 4.0-4.5 with formic The titrated centrifuged at 15Oxg for 5 min and the platelet rlzh plasma (PRP) removed and the number of platelets per microliter determined following dilution via Dacies fluid (24). The PRP (five animals/group) was used for pLatelet aggregation studies using collagen and ADP via standard spectrophotometric measurements as previously described (25). PRP from the remaining animals was centrifuged, the plasma removed and the platelets washed twice with a solution consisting of 0.85% NaC1, 2 mM EDTA, 25 mM Tris at pH 7.5 for lipid analysis. Red blood cells remaining after separation of PRP were centrifuged (1500 Xg, 10 min) and the packed red blood cells (PRBC) used for viscosity measurements. The lungs and livers xere removed from the animals, placed on ice, and weighed for subsequent fatty acid analysis or isolation of microsomes. Aortas were carefully excised, meticulously cleaned of extraneous material and a 1.5 cm segment placed in 1 ml KrebsHenseleit solution and gently agitated at room teaperature for 30 min The aorta was then removed and the incubation media acidified and (24,25). extracted twice with two volumes of ethyl acetate to recover prostanoids (26). Another portion of the excised, cleaned aorta ( 1.5 cm) was cut longitudinally and stored at - 70 degrees C in hexane isopropanol (.02 % BHT) for lipid analysis. TABLE III. Fatty Following

Acid Composition of Menhaden ---and Shark Liver Oil Cry sta%zation Fractional and Urea Adduct Formation --Shark ---

Menhaden Oil Fatty

Acid

Original

Enriched -$

14:o 16:0 14:2 16 : 1 16:3 16:4 18:0 18:l 18:3n3 18:4n3 2O:l 20:2 20:4n6 20:5n3 21:5 22:l 22:4 24:l 22:5n3 22:6n3

7.0 16.2 3.2 8.0 11.1 4.6 2.3 7.6 3.6 3.8 0 0 2.5 17.9 1.5 0 0 0 2.0 7.6

1.1

13.2

2.3

31.9

Liver

Original of

Oil Enriched

Total-

3.5 19.5 1.0 2.5 3.2 0 4.8 16.0 1.8 0.4 9.2 0.4 2.8 6.5 0 4.7 0.6 0.9 1.2 19.7

0.6 1.4

7.8 16.7

6.2 2.8 _60.1

484

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

Lipid and Fatty Acid Analysis Platelets, plasma and aortic tissues were homogenized and the lipids extracted with hexane : isopropanol (5:2) as described by Hara and RadLn (27). The lung and liver samples were homogenized in 0.25 M sucrose, 2 mM EDTA in 10 mM Tris buffer at pH 7.4, and the microsomal fraction prepared as described by Kamp and Wirtz (28). The lipids were extracted from these microsomal fractions according to the method of Bligh and Dyer (29). Tissue lipids were fractionated by thin-layer chromatography (TLC) into phospholipid and neutral lipid classes using chloroform : methanol (8:l) as the solvent system. The phospholipid and neutral lipid TLC bands were then scraped, pentadecenoic acid was added as the internal standard, and the samples were methylated with either 14% boron trifluoride in methanol (30) or R2S04/methanol as previously described (31). Total phospholipid and cholesterol concentration was assessed using previously established methods (32,38). The fatty acid methyl esters were quantified by gas-liquid chromatography (5880A. Hewlett-Packard, Avondale, PA). The column packing material was Silar 10-C (Supelco, Bellefonte, PA), and the temperature program was set at 3 C/min (175 -205). Nitrogen flow was 15 ml/min. Arachidonic Acid (AA) Conversion to Eicosanoids by Lung Microsomes To assess the effects of dietary n-3 fatty acids on eicosanoid synthesis, microsomes derived from the DHA, EPA and control lungs were resuspended in 0.05 M Tris-HCl buffer, pH 7.4 ("2.5-3 mg protein/ml). Labeled arachidonic acid (14C, -05 p ci/n mole in 10 ~1 ETOH) was added to the microsomal suspension and incubated at 37 C for 15 min. The reaction was terminated (3% formic acid to pH 3.5) and eicosanoids extacted with ethyl acetate as previously described (26). The ethyl acetate was evaporated under N2 and the labeled products along with standard eicosanoids (Upjohn Co., Kalamazoo, MI) spotted on silica gel thin layer plates (60-G Merck) and the plates were developed twice using ChC13 : MeOH After : HOAc : H20 (90:8:1:0.8 v/v) as the solvent system (33). development the plates were sprayed with 10% phosphomolybdic acid and heated to 110 C for 10 min to visualize the eicosanoid bands. Individual bands were scraped and the amount of radioactivity present was measured in Protein3 were a liquid scintillation spectrometer (Packard Model 3385). quantified by the method of Lowry et al. (34) using bovine serum albumin as the reference standard. Radioimmunoassay (RIA) The ethyl acetate extracts of serum and aortic incubation samples were evaporated under ~2 and the residue resuspended in phosphate-buffered gelatinized saline. Thromboxane B2 (TXB2) and 6-keto-PGFld (6-keto) (the inactive metabolites of thromboxane A2 and prostacyclin) were determined using TXB2 (Upjohn Diagnostic, Kalamazoo, MI) and 6-keto (Wellcome Research Laboratories, Kent, England) antibodies as previously described (25). All RIA data were analyzed using a computer program based on least square linear regression analysis developed at the Cornell Diagnostic Services and made available to us by Dr. T. J. Reimers and Robert Cowan. This analysis is a modification of the program as described by Duddelson et al (35).

485

EPA-DHA AND PROSTAGLANDIN

Vo1.34, No.6

SELECTIVE

ENRICHENT

OF

N-3 FATTY ACIDS

m Homogenize

(1OOg)

I

in Hexane:Is0propan0l:NdSO4

Fish Liver

Saponify

Hz0 (6.72)

Oil

1 (NaOH:Ethanol)

Acidify Non-Saponifiables Discarded Free Fatty Acids{

(50 g)

4 Reflux with Li.OH:Acetone \r

Discard Lithium

insoluble Soaps

(30 g)

lJrea:Methanol

Adduct

Enriched

Formation

N-3 Fatty Acid

(15 9)

Scheme showing the methods used for selectively enriching the fish oils in the n-3 polyunsaturated fatty acids. Blood Viscosity Packed red blood cells (1.5 ml) were diluted with an equal volume of titrated saline (0.9% NaC1, 0.38% Na citrate). The diluted red blood cells were placed in a capillary viscosimeter (Cannon-Fenske, size 25) at 37 C. The viscosimeter was attached to a manometer and a constant head pressure maintained on the sample via an' adjustable reservoir of irater. The

486

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

viscosity of each blood sample was deternlned at 75, 51 and 10 mm fighead Water was used as the reference standard and viscosity is pressure. expressed relative to water. Statistical Analysis Significant differences between data were tested for by one way ANOVA and LSD (36). RESULTS There were no significant differences noted for feed consumption, water consumption or weight gains between the control, EPA or DHA groups during the three week feeding period (data not shown). Bleeding times and red blood cell viscosity measurements are presented in Table IV. There were no significant differences noted in bleeding time or relative red blood cell viscosity between any treatment group. The fish oil supplemented diets did not alter -in vivo clotting time in rats nor did they decrease red blood cell viscosity at the levels fed. Platelet aggregation in response to challenge with ADP or collagen is depicted in Fig. 2. PRP samples from control, EPA and DHA fed animals were challenged with incremental concentrations of ADP (5~10-~-5xlO-~M) and collagen (.08-0.5mg/ml). No differences between treatments were noted at any concentration of agonist administered. Platelet and aorta phospholipid fatty acid composition was markedly affected by the different levels of n-3 fatty acids consumed (Table V). A significant increase in 20:5n5 was noted in platelet and aortas from the EPA fed vs. the control group. While there was also an increase in the 20:5 levels for the DHA group this difference was not significant in platelets. The levels of 20:4n6 in platelets and aorta were decreased in both EPA and DHA vs. control; however, this difference was only significant for platelets at P<.lO. There was no difference in 20:4n6 between EPA and DHA fed animals. The decreases noted for 20:4n6 and the increases in 20:5n3 after fish oil consumption have been reported by others (6,7,8,37). In spite of the almost two fold higher dietary levels of 22:6n3 in the DHA compared to the EPA group no increases in 22:6n3 were noted in the platelet phospholipids. The levels of 22:6n3 in the aorta were increased in both the DHA and EPA vs. control group; however, no differences were noted between the n-3 fed groups. Plasma total fatty acids reflected increases in both 20:5n5 and 22:6n3 in both the EPA and DHA animals (data not shown). Rat liver microsomal phospholipid fatty acids were altered more dramatically than the platelet phospholipid fatty acids following consumption of the n-3 fatty acids (Table VI). There were marked decreases in 20:4n6 in the EPA and DHA compared to control. The EPA diet seemed to decrease 20:4n6 levels more than the DHA diet in rat liver phospholipid microsomes although this difference was not significant. The amount of to control animals. 20:5n3 increased in the n-3 fed as compared Furthermore, the levels of 22:6nfiwere increased approximately three fold in the microsomes of animals receiving either dietary EPA or DHA. The amount of phospholipid and cholesterol in the liver microsomes was not altered by the various dietary regimes.

487

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

TABLE IV. Bleeding ----Time and Red Blood Cell Viscosity _-of Rats Fed EPA* or DHA* Diets for Three Weeks -------

Bleeding Time (seconds) Relative Viscosity of RBC

68.0 + 15.0+ 3.50'

DHA

EPA

Control

Dietary Group

.29

69.4 + 9.6 3.545 .34

70.7 + 11.6 .18 3.455

(40 mm Hg) *See Tables I & II for EPA, DHA Composition +Mean -+ SD Lung phospholipid microsoma 1 fatty acid composition from rats fed DHA and EPA enriched diet is shown in Table VII. The inclusion of n-3 fatty acids in the diets decreased the amount of 20:4n6 and 22:4n6 while concommitantly elevating the levels of 20:5n3, 22:5n3 and 22:6n3 in the phospholipids from lungs of DHA and EPA animals. The amount of 20:5n3 was slightly higher in the EPA vs DHA animals while the inverse appeared to be true for 22:6n3. No changes were noted in phospholipid or cholesterol concentration in any treatment group.

TABLE V. Fatty Acid Composition of Platelet and Aorta Phospholipids from Rats Fed EPA* and DHA* Diets for Three Weeks ---------Fatty Acid wt$+

Aorta Fatty Acid

lb:0 18:0 18:l 18:2n6 20:4n6 20:5n3 22:4n6 22:5n3 22:6n3

CON EPA ------

Platelet DHA

CON

EPA

DHA

(n=4)

(n=3)

(n=3)

(n=l)

(n=2)

(n=2)

23.5

23.4

24.4

45.8+

43.4

46.5

18.6 19.2 7.9 25.2 0.9 2.9

18.7

18.0 16.1

15.9 7.2

19.1

19.1 9.1


1.7

15.5

7.8 19.8 3.5

1.8 1.6 7.6

6.7

4.3

7.3 5.0

20.8 2.3 2.5 1.0 7.9

16.8

10.9

l.7a 7.3

<1 <1

5.5b 6.1

2.3
4.0

10.9 3.5ab 5.5


*See Tables I and II for dietary regimens. +Means ab Values having different superscripts are significantly different at P<.O5

488

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

FIGURE 2 PLATELZT AGGREGATION

COLLAGEN

ADP

5

x

10

.3

mglml

-6 M 75-

50-

96 AGG 50’ 25-

96 AGG

.A EPA

CON

DHA

L EPA

CON

DHA

Platelet aggregation in response to ADP and collagen challenge. Platelets were derived from control, EPA and DHA fed rats.

TABLE VI. Effect of EPA* and DHA* Enriched Diets on the Fatty Acid --------Composition --of Rat Liver Microsomes Phospholipid Fatty Acids Fatty Acid --

Control (n=3)

16:0 16:l 18:0 18:l 18:2 20:4 20:5 22:6 Phospholipid fig/mg protein Cholesterol pg/mg protein

EPA (n=4) _---

DHA (n=4)

_________---_-

wt%**__________-__

21.4 2.8

28.0

35.0 11.7 8.5 14.5 1.5

1.8 417

20.1

3.0 32.4 9.2 9.3 7.5a 3.5a 5.4a 456

19.1

26.0 3::; 9.5 9.7 9.6a

2.6a 7.3a 426 19.1

*See Tables I and II for dietary regimen =Mean a-Values differ significantly from control PC.05 Thromboxane B2 synthesis was significantly decreased in the serum of animals maintained on the EPA vs. control diets (Table VIII). While there

489

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

was a slight decrease in TXB2 in the DHA fed group it was not significantly different from the control or ETA group. No significant differences were noted for the 6-keto synthesizing capacity of the aortic segments from any treatment group, although there was an indication that EPA decreased 6-keto compared to DHA fed animals. The conversion of exogenous C14AA to eicosanoids by lung microsomal fractions from the rats fed n-3 enriched fatty acids is presented in Fig. 3. AA was converted into both lipoxygenase and cyclooxygensae products in similar amounts for all the animals tested. No differences were noted betsreen the amount of hydroxy fatty acids, PGE, TXB2, or PGF2, produced in any of the treatment groups. TABLE VII. Effect of EPA and DHA* Enriched --Diet on Rat Lung Microsomal ----Fatty Acid Composition Lung Phospholipid Fatty Acids Fatty Acid

Control(n=4)

EPA(n=4)

DHA (n=4) --

d+*___________ __-________wtp 14:o 16:0 16:1 1a:o 18:l 18:2 20:4 20:5 22:4 22:5 22:6 Phospholipid

bg/mg protein) Cholesterol (pglmg protein)

1.6 38.6

1.9

1.9

40.1

42.6

7.3

6.5

7.2

11.4 14.1 4.5 13.1 c.5 3.2 c.5 0.8

11.6 13.0 6.1 8.1a 3.3a 1.3a 2.1a 3.5a

10.5 13.4 5.4 8.3a 2.0a 1.4a 1.5a 4.0a

268

256

248

36

41

36

*See Table I and II for Dietary Regimen **Mean a-Values with different superscripts are significantly different at P<.O5 DISCUSSION The suggestion that dietary n-3 fatty acids may alter thrombotic events _in vivo was first proposed by Bang and Dyerberg (39). Subsequent studies demonstrated both beneficial as well as detrimental effects of fish oil consumption (10,12,14,40,41). The observation that Greenland Eskimos accumulate eicosapentaenoic acid (20:5n3) in their platelet phospholipid membranes suggested that this fatty acid may be involved in the observed alteration of thrombosis tendency, i.e., decreased platelet aggregabiity and increased bleeding times (8). Dyerberg (42,46) and others (43) showed that 20:5n3 could give rise to prostaglandins of the 3 series -in vitro.

EPA-OHA AND PROSTAGLANDIN

490

vo1.34, No.6

Thromboxsne A3 was found not to be a potent sggregatory agent, whereas ?“,I< to be a good antiaggregatory prostanai? (44). Subsequent appeared feedin; trials, hoyever, have demonstrated that while 20:5nj was accumulated in lipids it was not converted to the 3 series prostanoids olatalet and aortic in viva (12,45). This may be due to species differences (54). Therefore, it was proposed that 23:5n3 exerts its beneficial effects by competing with 20:4n6 for the active enzyme site (cyclooxygensase) and thereby reducing overall prostanoid synthesis (12,45). docosahexaenoic In this context, acid (22:6n3) has been shown to be a better competitive inhibitor of cyclooxygenase in vitro than 20:5n3 (15). -TABLE VIII. Prostanoid

Levels in Serum and Aortic Extracts ----from EPA* and DHA Fed Rats ------

Aorta

Serum TXB2 ng/ml+ Control EPA DHA *See Tables I and II for +Mean a,b-Values with different different at P<.O5

fatty assess study

6-Keto

38.3a 19.ga 26.4ab dietary

Derived

Synthesis

PGFld ng/ml+ 43.3 34.2 43.0

regimen

superscripts

are

significantly

Because fish oils contain numerous n-3 fatty acids and because these acids may modulate different physiological events it is important to Our DHA, EPA the individual effects of the different n-3 PUFA. was conducted to elucidate some of these interactions.

It is apparent from the observed results that diets enriched in either DHA or EPA did not alter bleeding time or platelet aggregation responses to These results are in agreement ADP or collagen in rats at the levels fed. with others (47) but are contrary to data reported by Hornstra et al. (45) who reported increased bleeding times in rats fed fish as compared to However, there are several differences between sunflower oil based diets. their experimental protocol (45) and that used in this study; i.e. the amount of fish oil fed in their diets was 20 wt%, which is approximately 10 bleeding time in their experiment was fold greater than in our diet; the measured via the rat tail while we utilized the saphenous vein; duration of their experiment was 10 weeks vs. our 3 week study. The fatty acid composition of platelet phospholipids was simi.lar following EPA or DHA feeding vs. control, i.e., 20:4n6 and 22:4n6 decreased The slight decreases in while 20:5n6 increased; 22:6n3 was not altered. The aorta fatty acid 20:4n6 were the same for EPA vs DHA fed animals. composition also reflected the dietary changes in that 20:5n3 and 22:6n3 It is increased while 20:4n6 levels were not significanty decreased. apparent that 22:6n3 is not readily incorporated by rat platelet The increases noted for 20:5nJ phospholipids but is taken up by the aorta. in platelet and aortic phospholipids were similar in magnitude; however,

491

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

the 20:5n3 levels were phospholipids (Table V).

consistently

higher

In 3PA

vs.

DHA

tissue

FIGURE 3

LUNG MICROSOMES

40

C-CONTROL (OH)-FA

t

Cl-DHA

enriched

E-EPA

enriched

TX6 al

z

20

E a

PGF2a

The EPA diet significantly decreased platelet thromboxane production; this has been noted by others (10,12,45,48). However, at the level fed neither EPA nor DHA significanty altered prostacyclin production by the aorta although there was a slight decrease in 6-keto levels noted for the EPA animals. This is contrary to data reported by others (37,45) and is most likely due to the low levels of 20:5n3 in our diet (higher levels of dietary fish oil 10 wt$, decreased 6-keto, unpublished data). Our data indicate that an EPA enriched diet fed to rats, apparently results in a favorable thromboxane/prostacyclin (TXA/6-keto) ratio. This shift in TXA/6-keto ratio does not result in a noticable modification of platelet aggregation nor bleeding time. Higher levels of fish oil feeding have been shown by others to decrease both TXA and 6-keto by the same magnitude and thereby not alter the TXA/6-keto ratio; however, while the ratio was apparently unaltered the thrombotic tendency was improved, i.e. increased bleeding and arterial obstruction times were noted (45). Furthermore, rats fed linoelaidate had a favorable TXA/6-keto ratio but no differences were noted in platelet aggregation or bleeding times (24,25). The importance of TXA/6-keto ratios in regulating thrombotic events in rats is therefore

492

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

questionable. It is also evident from the data that 22:6n3 does not inhibit platelet TXA production nor does it inhibit subsequent platelet aggregatory responses (Table VIII, Fig. 2). While 22:6n3 may compete for the cyclooxygenase active site -in vitro it does not accumulate in rat platelet phospholipids, and therefore its role as a competitive inhibitor in vivo is negligible. The apparent exclusion of 22:6n3 from rat platelet -phospholipids has been shown by others (37). However, 22:6n3 is apparently incorporated into the platelet phospholipids of other species, i.e., dog (52), rabbit (37), human (10,48), and therefore the results from our experiment cannot be extrapolated directly to these species. It is interesting to note, however, that Culp et al. (52) noted marked increased in 20:5n3 and 22:6n3 in dog platelet phospholipids after fish oil supplementation with a subsequent decrease in infarct size; however, platelet aggregability to ADP, collagen and AA remained unaltered. Blood viscosity in humans following fish oil consumption has been shown to decrease (50). While, in this experiment, blood viscosity was not altered by dietary EPA or DHA we have noted in subsequent rat feeding trials with higher levels of fish oil (10 wt$) a decrease in whole blood viscosity (unpublished data). It is possible that changes in bleeding tendency (37) result from decreased blood viscosity or platelet numbers more than alterations in prostanoid biosynthesis when high amounts of fish oil are fed to rats. This hypothesis is being further investigated in our laboratory. In the rat lung microsomes the levels of 20:4n6 and 22:4n6 were decreased while 20:'jn3and 22:6n3 were elevated (Table VII). However, this change in fatty acid composition did not affect lung microsomal eicosanoid production from exogenously added substrate (AA) in the EPA and DHA fed animals (Fig. 3). While hydroxy fatty acid (OHFA) biosynthesis in lung microsomes has not been studied following administration of fish oils it has been noted that platelets from rats fed fish oil produce less OH-FA and prostanoids than sunflower oil fed controls (45). When higher amounts of fish oil were fed to rats (5 or 10 wt%), decreased OH-FA and prostanoid production by rat lung microsomes was observed (unpublished data). Fish oils appear to modulate eicosanoid biosynthesis not by decreasing the amount of substrate available (12) (20:4n6 levels are not different in the EPA vs DHA fed rats) but rather by either competing for the enzyme active site and/or by altering general membrane properties; i.e., fluidity. The evidence which suggests the latter as a likely mechanism is that the amount of TXB2 produced is significantly decreased in the EPA group but only slightly decreased in the DHA fed animals. Alteration in membrane fatty acid composition may influence phospholipase A2 activity after fish oil feeding as suggested by others (12). Docosahexaenoic acid apparently does not modulate prostanoid biosynthesis in rat platelets because its incorporation is not altered by increased dietary levels. In conclusion, while fish oils have been shown to alter various hemotologic parameters in humans (6,8,10,12,48)our data from short term studies suggest that the rat is not similarly affected. While the dietary n-3 fatty acids are incorporated into various rat tissues it is apparent that there are species differences, i.e., 22:6n3 is excluded from rat platelet phospholipids but is apparently incorporated by other species (10,13,37,48).

Vol.34,

No.6

EPA-DHA

493

AND PROSTAGLANDIN

The amount of dietary EPA fed to rats and its subsequent incorporation into platelet membrane phospholipids seems to be more directly involved in regulation of thromboxane production than decreases in 20:4n6 levels (no differences in 20:4n6 were noted for the DHA vs. ETA group, but TXB2 was markedly decreased in EPA) (12). From the data presented here and by others (47) it appears that the rat is not the most suitable model for evaluating thrombotic parameters as they relate to humans. While dietary fatty acids do influence prostanoid biosynthesis in the rat (25,37,45), these alterations differ in many ways from other animal models (51,53). Of the fish oil fatty acids it is evident that in the rat platelets 20:5n3 and not 22:6n3 is responsible for the precipitated alterations in prostaglandin biosynthesis; however, these observations may not be directly applicable to other species, i.e., human, dog. Further work is needed to clarify these observations. Acknowledgements This research was supported by USDA Nutrition Grant #82-CRCR-l-1036 and New York Sea Grant Program. The technical assistance of Susan Leung and Patreena Deegan are greatly appreciated. A special thanks to Rudy Hsieh for analysis of lipid peroxidation (MDA) and to Nancy King and Kay Ragland for text preparation. REFERENCES 1.

BANG, H.O., and DYERBERG, J. Plasma lipids and lipoproteins Greenland West Coast Eskimos. Acta Med Stand 1972,=,85-94. _~_.___

2.

BANG, H.O., and DYERBERG, J. The bleeding tendency in Greenland Eskimos. Dan Med Bull 1980,3,202-205.

3.

DYERBERG, J. and BANG, H.O. A hypothesis on the development of acute myocardial infarction in Greenlanders. Stand J Clin Lab Invest 1982.42(161).7-13. -

4.

BANG, H.O., DYERBERG, J. and SINCLAIR, H.M. The composition of the Eskimo food in North-Western Greenland. Am J Clin Nutr --.--_---__ 1980.33.2657-2661. -

5.

ACKMAN, R.G. Fatty acid composition of fish oils. In: Barlow, S.M. Stansby, M.E., eds. Nutritional Evaluation of Long Chain FatQ, ___________. .._ Acids _--__ in Fish ~ Oil. Academic Press, NY 1982.25-87.

6.

SHIBA, T., YAMAGUCHI, R. and IGARASHI, M. Effect of eicosapentaenoic acid on platelet aggregation induced by arachidonic acid. J Med Sot, Toho, Japan 1981.22(1-2).194-198.

7.

SHIBA, T., URITA, Y., TAKATSLJKA, J., TAKEUCHI, S., IGARASHI, M., ASADA, T., ASANO, T., KANAI, A., and OMOTO, M. Effect of dietary habits on eicosapentaenoic acid and arachidonic acid contents in human plasma. J Med Sot, Toho, Japan 1980.27(4).404-407. -

in

EPA-DHA AND PROSTAGLANDIN

494

Vo1.34, No.6

a. DYERBERG, J.

Dietary manipulation of prostaglandln synthesis: Benefici al or detrimental? In: Herman, A-G., Vanholutte, T.M., Denolin, H., Goosens, A., eds. Cardiovascular Pharmacology of the --_-.---__~_~~ Prostaglandins. Raven Press, NY, 1982, p. 233-244.

9.

GRYGLEWSKI, R-J., SALMON, J.A., UBATUBA, F.B., WEATHERLY, B.C., MONCADA, S., and VANE, J.R. Effects of all cis 5,8,11,14,17 eicosapentaenoic acid and PGH3 on platelet aggregation. Prostaglandins 1979,12(3),453-478.

10.

SEISS, W., ROTH, P., SCHERER, B., KURZMANN, I., BOHIG, B., AND WEBER, P.C. Platelet-membrane fatty acids, platelet aggregation and thromboxane formation during a mackeral diet. Lancet 19ao,A,441444.

11.

NEUFELD, E.J., WILSON, D.B., SPRECHER, H. and MAJERUS, P.W. High affinity esterification of eicosanoid precursor fatty acids by Clin Invest 1983,72,214-220. platelets. J __.__~ -

12.

HIRAI, A., TERANO, T., HAMAJAKI, T., SAJIKI, J., KONDO, S., OZAWA, A., FUJITA, T., MIYAMOTO, T., TAMURA, Y., and KUMAGAI, A. The effects of the oral administration of fish oil concentrate on the release and the metabolism of [14c] arachidonic acid and [14c] eicosapentaenoic acid by human platelets. Thrombosis Research --_~-_--1982,22,285-298.

13.

W.E.M. Inhibition of CULP, B.R., TITUS, B.G. and LANDS, prostaglandin biosynthesis by eicosapentaenoic acid. Prostaglanda _-Med 1979,,3,269-278. -_

14.

BLACK, K.L., CULP, B., MADISON, D., RANDALL, O.S. and LANDS, W.E.M. The protective effects of dietary fish oil on focal cerebral infarction. Prostaglandins Med 1979,2,257-268. -

15.

LANDS, W.E.M., LeTELLIER, P.R., ROME, L.H., and VANDERHOCK, J.Y. Adv Bio Sci 1973,2,15-28. Inhibition of prostaglandin biosynthesis. ~___

16.

Report of the American Institute of Nutrition Ad Hoc Committee on Standards for Nutritional Studies. J Nutr 1977,107(7),1340-1348. -

17.

The mixed unsaturated glycerides of HILDITCH, T.R. and MADDISON, M. liquid fats. J Sot Chem Ind 1942,%,169-174.

18.

HAAGSMA, N., VAN GENT, C-M., LUTEN, J.B., DE JONG, R.W. and VAN DOORN, E. Preparation of an w-3 fatty acid concentrate from codliver oil. J Am Oil Chem Sot 1982,2(3),117-118.

19.

CHRISTOPHERSEN, B.O., NORSEHT, J., THOMASSEN, M.S., CHRISTIANSEN, E.N., NORUM, K.R., OSMUNDSEN, H., and BREMER, J. Metabolism and metabolic effects of C22:l fatty acids with special reference to eds. ME., Barlow, S.M., Stansby, cardiac linodosis. In: Nutritional- Evaluation of Long Chain Fatty Acids in Fish Oil, --__---Academic Press,NY, 1982,89-139.

20.

BRUCKNER G., SHIMP, J., GOSWAMI, S., MAI, J., and KINSELLA, J.E. Dietary trilinoelaidate: Effects on metabolic parameters related to

vo1.34, No.6

EPA-DHA AND PROSTAGLANDIN

J Nutr 19827-3 112 126-135. EFA metabolism in rats. -__21.

New Series LEMON, D.W. An improved TBA test for rancidity. Circular #5. Halifax Laboratory, Fisheries and Marine Service, Canada, 1975.

22.

MAI, J. and KINSELLA, J.E. Changes in lipid composition of cooked minced carp (Cyprinus capio) during frozen storage. J Food Sci 1979,44,1609-1624.

23.

HUNG, S.S.O.,CHO, C.Y. and SLINGER, S.J. Measurement of oxidation in fish oil and its effect on vitamin E nutrition of rainbow trout. Can _- J Fish Aquat Sci 1980,x,1248-1253.

24.

BRUCKNER, G., TRIMBO, S., GOSWAMI, S., and KINSELLA, J.E. Dietary trilinoelaidate: Effects on hematological parameters, serum eicosanoids and tissue fatty acid composition in rats. J Nutr 1983,12,704-713.

25.

BRUCKNER, G., GOSWAMI, J.E. S. and KINSELLA, Dietary trilinoeladate: Effects on organ fatty acid composition, prostanoid biosynthesis and platelet function in rats. J Nutr 1984,1>,58-67.

26.

GOSWAMI, S., MAI, J., BRUCKNER, G. and KINSELLA, J.E. Extraction and purification of prostaglandin and thromboxane from biological samples for gas chromatographic analysis. Prostaglandins --~--_ 1981,22(5),693-702.

27.

HARA, A. and RADIN, N.S. Lipid extraction of tissues with low toxicity solvent. __ Anal Biochem 1978,90,420-426.

28.

KAMP, H.H. and WIRTZ, K.W.A. Phosphatidyl-choline exchange protein from beef liver. Methods Enzymol 1974,32(B),140-146. -

29.

BLIGH, E.G. and DYER, W.J. A rapid method of total lipid extraction and purificiation. Can J Biochem Physiol 1959,17_.,911-920.

30.

MORRISON, W.R. and SMITH, L.M. Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron trifluoride methanol. J Lipid Res 1964,5,600-609.

31.

BOWYER, D.E., LEAT, W.M.F., HOWARD, A.N. and GRESHAM, G.A. The determination of the fatty acid composition of serum lipids separated by thin layer chromatography; and a comparison with column chromatography. Biohem Biophys Acta 1963,70,423-431. -

32.

STEWART, J.C.M. Calorimetric determination of phospholipids with ammonium ferrothiocynate. Anal Biochem 1980,1~,10-14.

33.

SALMON, J.A. and FLOWER, R.J. Extraction and thin layer chromatography of arachidonic acid metabolites. Methods Enzymol 1982,86,477-493.

34.

LOWRY, O.H., FARR, A.L., ROSENBROUGH, N.J. and RANDALL, R. Protein measurement with Folin-Phenol reagent. J Biol Chem 1951,193,265.274.

495

496

35.

EPA-DHA AND PROSTAGLANDIN

vo1.34, No.6

DUDDLESON, W.G., MIDGLZY, A.R., JR., and NES'dENDER,G.D. Conputer program sequence for analysis and summary of RIA data. Biomed Res 1972,2,205-217.

Computers

36.

SNEDECOR, G.W. and COCHRAN, W.G. Statistical Methods 6th Ed., Iowa State University Press, Ames, IA, 1967.

37.

HORNSTRA, G., HADDEMAN, E., KLOEZE, J. and VERSCHUREN, P.M. Dietary-Fat-Induced changes in the formation of prostanoids of the 2 and 3 series in relation to arterial thrombosis (rat) and atherosclerosis (rabbit). In: Samuelsson, B., Paoletti, R. and Ramwell, P., eds. Adv. Prostaglandin, Thromboxane and Leukotriene -__~-___-__-._I-_ Rg, Raven Press, NY 1983,12,193-202.

38.

RUDEL, L.L. and MORRIS, M.D. Determination of cholesterol using ophthaladehyde. J Lipid Res 1973,1_4,364-366.

39.

BANG, H.O. and DYERBERG, J. Lipid metabolism and ischemic heart disease in Greenland Eskimos. Adv Lipid Res 1980,&l-22.

40.

SOCINI, A., TREMOLI, E., COLUMBO, C. and GALLI, C. Fish oil administration lowers prostacyclin production and the antiaggregatory activity of aortic walls more effectively than platelet aggregation and thromboxane in the rat. In: Samuelsson, Paoletti, R. and Ramwell, P., eds. Adv. Prostaglandin, B Thtromoxaneand-_____-Leukotriene Res., Raven Press, NY, 1983,12,185-191.

41.

SCHERHAG, R., KRAMER, H.J. and DUSING, R. Dietary administration of eicosapentaenoic and linolenic acid increases arterial blood pressure and suppresses vascular prostacyclin synthesis in rats. Prostaglandins 1982,2?(3),369-382. .---

42.

DYERBERG, J. and JORGENSEN, K.A. The effect of arachidonic and eicosapentaenoic acid on the synthesis of prostacylin-like material in human umbilical vasculature. Artery 1980,8_(1),12-17.

43.

WHITAKER, M.O., WYCHE, A., FITZPATRICK, F., SPRECHER, H. and Prostaglandin D3 and NEEDLEMAN, P. Triene prostaglandins: icosapentaenoic acid as potential antithombotic substances. Proc Nat1 Acad Si 1979,72(11),5919-5923.

44.

NEEDLEMAN, P., RAZ, A., MINKES, M.S., FERRENDALLI, J.A. and SPRECHER, H. Triene prostaglandins: Prostacyclin and thromboxane Proc Nat1 Acad Sci biosynthesis and unique biological properties. --~1979,7&944-948.

45.

HORNSTRA, G., CHRIST-HAZELHOF, E., HADDEMAN, E., TEN HOOR, F., and 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 13. Prostaglandins 1981,21(5),727-738. .-

46.

DYERBERG, J., JORGENSEN, K.A. and ARNFRED, T. Human umbilical blood vessel converts all cis-5,8,11,14,17 eicosapentaenoic acid to prostaglandin 13. Prostaglandins 1981,22(6),857-863. -

vo1.34, No.6

EPA-DHA AND PROSTAGLANDIN

47.

Effects of MORITA, I., SAITO, Y., CHANG, W.C. and MUROTA, S. purified eicosapentaenoic acid on arachidonic acid metabolism in cultured murine aortic smooth muscle cells, vessel walls and platelets. Lipids 1983,12(1),42-49.

40.

LORENZ, R., SPENGLER, U., FISHER, S., DUHM, J. and WEBER, P.G. Platelet function, thromboxane formation and blood pressure control during supplementation of the western diet with cod liver oil. Circulation 1983,6_1(3),504-511. __

49.

VAN DIAS, F.W., GIBNEY, M.J. and TAYLOR, T.G. The effect of polyunsaturated fatty acids of the n-5 and n-6 series on platelet aggregation and platelet and aortic fatty acid composition in rabbits. Atherosclerosis 1982,4>,245-257.

50.

KOBAYASHI, S., HIRAI, A., TERANO, T., HAMAZAKI, T., TAMURA, Y. and KUMAGAI, A. Reduction in blood viscosity by eicosapentaenoic acid. Lancet 1981,g,197.

51.

LEACH, C.M. and THORBURN, G.D. A comparative study of collagen induced thromboxane release from platelets of different species: Implications for human atherosclerosis models. Prostaglandins

497

1982,2_4(1),47-59. 52.

CULP, B.R., LANDS, W.E.M., LUCCHESI, B.R., PITT, B. and ROMSON, J. The effect of dietary supplementation of fish oil on experimental myocardial infarction. Prostaglandins 1980,2~(6),1021-1051.

53.

ROSENBLUM, W-I., NELSON, G.H., COCKRELL, C.S., and ELLIS, E.F. Some properties of mouse platelets. Thrombosis Research 1983;3~,347-355.

54.

MORITA, I., TAKAHASI, R., SAITO, Y. and MUROTA, S. Effects of eicosapentaenoic acid on arachidonic acid metabolism in cultured vascular cells and platelets, species differences. Thrombosis ----Research 1983, 3_1,211-217.