Improved graft patency associated with altered platelet function induced by marine fatty acids in dogs

JOURNAL OF SURGICAL RESEARCH 4@6-

12 ( 1986)

Improved Graft Patency Associated with Altered Platelet Function Induced by Marine Fatty Acids in Dogs ROBERTE. CASALI,M.D., JANETA. HALE, M.D., LEROY LENARZ, M.D., FRED FAAS,M.D., AND MANFORDD. MORRIS,PH.D. Departments of Surgery, Pediatrics, and Biochemistry, University of Arkansas for Medical Sciences,Little Rock, Arkansas Submitted for publication February 9, 1984 Female mongrel dogs fed a marine fish diet rich in long-chain polyenoic fatty acids had improved patency of small-diameter arterial prosthetic grafts ascompared to controls. Also, in vivo platelet function as measured by bleeding times was significantly prolonged. Eicosapentaenoic acid, not found in the serum of control animals, was present in relatively high concentrations in both the serum and a plateletrich,fraction of the marine oil-fed group. Eicosapentaenoicacid, unlike arachidonic acid, doesnot induce platelet aggregation and this phenomonon may account for the altered platelet function demonstrated in our animals and hence the improved gratI patency. These data lend huther support to the role of platelets in determining the patency of vascular grafts. 8 1986 Academic RQS, ICC.

This study evaluates the effects of altered platelet function on the patency rate of small diameter prosthetic grafts in female mongrel dogs. Platelet function was modified by administering anti-platelet drugs (aspirin and dipyridamole) to one group and a marine fish diet supplemented with menhaden oil, an oil rich in epa, to another group of dogs. Both treatment groups were compared to control animals who were fed standard dog chow.

INTRODUCTION

Thrombosis is a frequent cause of smalldiameter prosthetic graft failure. Platelet activation, i.e., adhesion, aggregation, and reIease of secretory products, may play an important role in initiating or triggering this thrombotic process. Experimentally, antiplatelet agents, such as aspirin and dipyridamole, enhance the patency rate of small prosthetic grafts [ll, 13, 15, 16, 211. Greenland Eskimos have a decreasedincidence of atherosclerotic vascular diseaseand a bleeding tendency [8]. The hemorrhagic tendency is associatedwith a prolonged bleeding time and altered platelet function [9]. Their diet has been studied extensively [ 13, 3, 10, 231. It contains a large amount of mackerel, a marine fish with a relatively high concentration of eicosapentaenoic acid (epa), which results in increased levels of serum and possibly platelet epa [9, 81.Eicosapentaenoic acid and its metabolites do not induce platelet aggregation [5, 81. Associated with the increase in epa is a decreasein arachidonic acid (aa), the precursor compound of the proaggregatory prostaglandin endoperoxides and thromboxane A2. 00224804186 $1SO CopyAght 0 1986 by Academic Pms, Inc. All rights of reproduction in my form resuwd.

MATERIALS

AND METHODS

Subjects Thirty conditioned female mongrel dogs were selected from kennel stock and the following blood studies were performed prior to randomization. Platelet Studies Pretreatment values for platelet counts were obtained. Platelet aggregation by sodium arachidonate (Nu-Check-Prep, Elysian, Minn.) (0.6 and 0.22 m&f) was measured before and after addition of epinephrine (5.4 and 0.54 m/I& respectively), acid-soluble bovine colla6

CASALIET AL.: IMPROVEDGRAFTPATENCY gen 0.25% (Sigma Chemical Co., St. Louis, MO.), and ADP (20, 10, 5, and 2.5 mM). The threshold concentration that causedaggregation for each agent was recorded. All of the aggregationstudieswere performed according to the spectrophotometricmethod of Born [61. Bleeding times were done using a modification of the template method of Miekle [ 171. A blood pressurecuff was inflated around the proximal leg to 40 mm of mercury. The disposable “Simplate II” (General Diagnostics) was used to cut a standard incision 0.5 cm long and 1 mm deepin the lateraldistal foreleg of the dog. Other Studies

Prothrombin (PT) and partial thrombop&tin (APTT) times were performed using standard fibrometer techniques. Lipid studies andfatty acid analysis. Blood was collectedin Corvac tubes in the presence of 55”dithiobis-2-nitrobenzoic acid (DTNB) to inhibit serum lecithin cholesterol acyl transferase.The tube stood at room temperature for one-half hour. Centrifugation at 4°C allowed serum removal. An aliquot of serum was analyzed for total cholesterol (C), high density lipoprotein cholesterol(HDGC), and triglycerides(TG). Lipoprotein electrophoresis in agarosewas conducted on the serum sample. ThimerosalO.05mg/ml and EDTA (1 mg/ ml) wereadded to the remainder of the serum to prevent bacterial growth and denaturation of the lipoprotein. An aliquot of serum was extracted for determination of fatty acids by gas liquid chromatography, utilizing chloroform-methanol extraction, alkaline saponfication, and methylation by BF, in methanol for the formation of methyl estersof the fatty acids. The fatty acid methyl esterswere separated on a 10% EGSS-X column by which the long-chain polyunsaturated fatty acids were well separated.All of the above studies were performed twice for baselineevaluation except for the fatty acid analysis which was performedafterthe animalswere on diets.The animals were then randomized into three groups.

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Experimental Groups (Diets)

Group I (control) consumed regular dog chow (Canine Diet No. 5006, Ralston Purina Co.) Group II (mackerel)animals were fed only mackerel fish supplementedwith menhaden oil in an amount calculatedto meet the daily requirementsfor protein and fat with appropriate vitamin and mineral supplements.The diet was begun 10 days prior to surgery. Group III (anti-platelet)receivedregulardog chow and anti-platelet agents, dipyridamole (Boerhinger) 50 mg orally was given twice daily and aspirin (Burroughs-Wellcome) 5 grains orally, once daily. Both drugs were started 10 days prior to surgery. Operative Procedure

After baselineblood work and randomization, the dogs were allotted 10 days on diet and anti-platelet drugs. Bilateral synthetic grafts 6 cm in length and 4 mm in diameter were then usedto bypassboth ligated femoral arteries in an end-to-side fashion. The bypassedsegmentof each femoral artery was ligated adjacent to each anastomosis.Knitted velour grafts (USCI) were implanted into one femoral artery and polytetraflourorethylene grafts(PTFE = Gore Tex), into the other femoral arteryof eachanimal. All animalsreceived perioperativeprophylactic antibiotics. Evaluation GraJ2patency. GrafI patency was asses& by weekly palpations.Grafts wereremovedeither when pulses became absent or at 6 months. Histologic evaluation of the grajb. The graft

and adjacent artery were dissectedand perfused with saline and then 10%buffered formalin at arterial pressures.Six microscopic sectionsof each graft were taken through the toe area of the distal anastomosis. Tissue blocks of cross-sectionedsegmentswere dehydrated, embeddedin pan&n, and stained with Verhoff von Gieson and Masson’s Trichrome stains. Magnitied (10X) photographs

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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 1, JANUARY 1986

were made of each section and examined by a pathologist in a blind manner. A grading sale of 1 to 5 was used: Grade I, minimal intimal thickening, essentially a normal vessel; Grade II, mild intimal thickening with insigr&ant luminal compromise; Grade III, moderate intimal thickening with diameter narrowing up to 50%; Grade IV, intimal thickening with diameter narrowing greater than 50%; Grade V, severeintimal thickening and luminal narrowing with graft thrombosis.

l-

of E6c

FM

.E 1’

3

/Ai

AsA+)

A

Statistical Methods =b=4+-

Statistical analytical evaluation of bleeding times and histopathology were conducted using the Mann-Whitney U test. Lipid studies FIG. 1. Bleeding times for all animals. were analyzed statistically using the SPSS technique. < 0.001). Furthermore, these statistically significant differences between the groups were RESULTS maintained throughout the study period. Subjects The animals in each group consumed their Other Studies offered diet and gained weight. Three animals Coagulation (PT and APTT). There were died either during the operative procedure or no statistical differences among the groups at in the first 24 hr following surgery; there were any time during the entire study period (data two anesthetic deaths (one each in group I and not shown). group II) and one death in group II from heart Lipid studies and fatty acid analysis. The worms. One animal in group II was sacrificed mean total cholesterol (C) and the high density at 1 month because of infected wounds with lipoprotein cholesterol (HDGC) are shown in patent grafts. All other animals survived with- Table 1. There were no statistical differences out technical problems for an adequate time in the pretreatment values among the groups. to he studied. At 1 and 4 months, the mean C and HDGC are reduced, but these values were not statisPlatelet Studies tically significant. The results of the serum fatty acid analysis In vitro platelet aggregation. In vitro platelet aggregation studies revealed no differences are presented for four group I and two group among the three groups for any of the test re- II animals (Table 2). As a fatty acid proceduraI agents (data not shown). These findings are standard we utilized a sample of menhaden similar to the work by Culp et al. [7]. oil to document quantitatively the recovery of Mean bleeding time data. The mean bleed- long-chain polyunsaturated fatty acids. The ing time data for each group are shown in Fig. major fatty acid differences are a twofold re1. The two pretreatment values did not differ duction in the oleic and arachidonic acid and among the three groups. At one month, group a sixfold decreasein linoleic acid in the group II was statistically different from group I (P II animals. Eicosapentenoic acid constituted < 0.0007). Group II was also statistically dif- less than 0.1% of total fatty acid in the group ferent from group III (P < 0.001). Group III I dogs but accounted for greater than 25% of was significantly different from group I (P the total fatty acids in the group II dogs.Eleven

9

CASALI ET AL.: IMPROVED GRAFT PATENCY TABLE 1 b CONCENTRATION OFTOTAL AND HIGH DENSITYLIPOPROTEINCHOLESTEROL OFDOGSFED CONTROL, ASPIRIN-PERSANTIN, AND MARINE FISH-CONTAINING DIETS

Time on diet Control 2

Control I

1 Month

2 Months

4 Months

TCHL

HCHL

TCHL

HCHL

TCHL

HCHL

TCHL

HCHL

114 15

162 24

141 28

169 21

135 16

172 70

125 31

153 40

119 24

155 34

110 21

152 28

132 30

154 30

123 24

132 20

110 13

146 35

110 27

161 33

118 23

162 34

141 32

175 33

136 24

174 45

135 23

153 54

115 42

Group

T CHL”

H CHLb

Control Mean SDd

148 18

Fish Mean SD ASA-PC Mean SD

’ Total cholesterol, mg/dl. b High density lipoprotein cholesterol, m&U. c Aspirin - persantin. d Standard deviation.

percent of the total fatty acids in the group II animals were accounted for by docosahexenoic acid (22:6 omega 3). Platelet fatty acids were compared in group

I and group II dogs and revealed several significant differences. A fivefold reduction in platelet linoleic acid and 50% reduction in arachidonic acid was seen in group II. Fifteen

TABLE 2 PERCENTAGE DISTRIBUTIONOFSERUMFATTY ACIDSIN CONTROLAND FISH DIET DOGS Control

Fish

Dog No.

1

2

3

4

5

6

Fatty acids 14:o 16:O 16:l 18:O 18:1 (oleic) 18:2 (linoleic) I8:3 (linolenic) 20:4 (arachidonic) 20~5(eicosapentaenoic) 22:6 (docosahexenoic)

0.3 14.0 2.0 20.5 17.3 23.6 20.9 -

0.6 16.9 1.8 17.3 16.5 29.6 16.6 -

0.6 16.8 2.1 18.7 15.4 25.9 18.7 -

0.5 14.5 1.6 17.4 17.7 25.2 19.9 -

1.6 11.1 3.0 10.8 8.2 3.9 1.0 8.3 28.8 11.3

1.4 13.1 2.6 14.7 9.5 4.6 0.8 9.0 25.3 11.3

-

-

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percent of the total platelet fatty acids in group let function [9, 8, 141. Among these comII were eicosapentenoic acid and docosahex- pounds are the long-chain polyunsaturated enoic acid. These acids were undetectable in fatty acids belonging to the family of omega 3 fatty acids which are present in high conthe platelets in group I. centration in certain marine oils. Our data clearly demonstrate that dogs fed Graf2 Studies naturally occurring fish oil which is high in Graji patency. Eighty-six percent of the long-chain polyunsaturated fatty acids such as grafts in group II remained patent throughout epa have a marked biologic effect on in vivo the 6-month study period which is signi&antly parameters of platelet function such as bleeddifferent from the 44% patency of group I (P ing times. In addition, the statistically signif< 0.02). There was no statistical difference be- icant finding of improved patency of surgically tween the patency rates of group III as com- implanted synthetic grafts was demonstrated pared to group II. The patency rate of the when compared to dogs treated with aspirin PTFE grafts was 79 and 7 1% for the Dacron and dipyridamole or untreated animals. The grafts, which was not statistically different. histopathologic examination of the grafts reHistopathologic studies. The histopatho- vealed fewer intimal changes in the marine logic evaluation revealed a significant reduc- fish-fed group compared to the other two tion of intimal thickening in group II as com- groups. This lends further support for the role pared to the other groups (P = 0.04). Group of platelet function in graft failure. Fatty acid III had less intimal change than group I but analysis of a platelet rich fraction revealedthat these differences were not statistically signifi- 20% of the total fatty acids were present as cant. C20:5 (epa) and C22:6 polyenoic fatty acids derived from the fish diet. We presume that the presence of high amounts of epa (C20:5) DISCUSSION is the basis for the altered in vivo biologic activity of the platelets in our study. Normally, Previous studies have suggestedthat plate- platelets have a high concentration of aa (C20: lets and their releaseproducts play a signihcant 4) which is the precursor of the pro-aggregation role in determining the patency of synthetic compound, thromboxane A2. Our data demgrafts surgically implanted in animals [ 11,15, onstrated that the platelet content of fatty acid 211. These studies have all been relatively (C20:4 and C20:5) could be modified by diet short-term and involved the useof anti-platelet with a marked shift from C20:4 to C20:5. It has been shown that C20:5 effectively comagents, aspirin and dipyridamole. The mechanism by which platelets influence petes with C20:4 for platelet cycle-oxygenase the patency of grafts is unknown. It has been and lipoxygenase and thereby further suppress proposed that platelets contribute to the for- the formation of the pro-aggregation submation of anastomotic intimal hyperplasia, stances PGH2 and TXA2 [ 19, 201. Upon which narrows the lumen and ultimately may platelet stimulation the release product of cause graft failure. Aspirin and dipyridamole platelets rich in C20:5 is probably PGI3, which inhibit platelet function, the former by block- is a potent inhibitor of platelet aggregation ing cyclo-oxygenase; the later probably by in- similar to PG12. In comparison to other experimental studies creasing cyclic AMP [2,18]. This alteration of platelet function decmasesthe formation of [ 11, 15,2 1] the present study is unique in that pro-aggreatory substancessuch as thrombox- altered platelet function was accomplished by feeding a diet rich in very long-chain polyenoic ane AZ. Other substances which occur naturally acids known to have potent platelet anti-aghave also been shown to markedly alter plate- gregation activity. We presumed that these

CASALI ET AL.: IMPROVED GRAFf

fatty acids would be incorporated into the platelet membranes and therefore exert their influence by a direct mechanism. Clinically and experimentally, the ideal dose of ASA required to obtain the desired altered platelet function is unknown. This is also true of marine oil. It is likely that the concentration of marine oil in our model was excessiveand would have to be altered to be practical. Although no deleterious short-term effectson the animals were noted, we acknowledge the need to tend these studies for more precise endpoint measurements.A satisfactory responseof graft patency to polyunsaturated fatty acid feeding could be better achieved by the use of daily supplements that would be more palatable than the diet used in this study [ 11. In previous studies [ 15, 211 others have demonstrated a better effect of ASA on graft patency than we found. These differences could be related to the duration of the study (6 months in our study vs 4 months in others) and the use of female dogs in our study. It is of interest that two prospective human trials designed to study platelet associated venous thrombotic events [22] and stroke-related events [4] have shown ASA therapy not to be effective in females, but beneficial in males. Although these studies did not evaluate the sameparameters asthe present work, prcsumably the common focal point was altered platelet function. Therefore, sex could be important with the use of ASA therapy. Importantly, our marine oil-fed female dogs had altered platelet function and a statistically significant improved graft patency. The ratio of C20:4 to FGIz is important in determining platelet adhesiveness. The balance between platelet aggregation and inhibition has many modifying influences. The platelet arachidonic cascade and its metabolites are pro-aggregatory. Factors that inhibit this system, as shown by our study, will favorably enhance graft patency. Our model clearly demonstrated superior effect on platelet tinction (bleeding time) and on graft patency rate by the fish oil diet ascompared to standard chow plus ASA.

PATENCY

I1

ACKNOWLEDGMENTS We express gratitude to Anthony P. Bimbo, Director of Research,Zapata-Haynie Corporation, Reed&he, Virginia, for the generous supply of Menhaden oil used in these studies. We also express thanks to Ms. Carolyn Thompson for her participation in the statistical analysis.

REFERENCES 1. Abramowicz, M. Fish oil for prevention of atherosclerosis.Med. Lett. 24: 99, 1982. 2. Ally, A. I., Manku, M. S., and Horribin, D. F., et al. Dipyrimadole: A possible potent inhibitor of thromboxane Az synthetase in vascular smooth muscle. Prostaglandins 14: 607, 1978. 3. Bang, H. O., Dyerberg, J. and Hjorne, N. Composition of food consumed by Greenland eskimos. Actu Med. Scund. 200~69, 1976. 4. Bamett, H. J. M. A randomized trial of aspirin and sulfinpyrazone in threatened stroke. N. Engl. J. Med. 299: 53, 1978. 5. Black, K. L., Culp, B., and Madison, D., et al. The protective effects of dietary fish oil on focal cerebral infarction. Prostaglandins 3: 258, 1979. 6. Born, G. V. R., and Cross, M. J. The aggregation of blood platelets. J. Physiol. 168: 178, 1963. 7. Culp, B. R., Lands, W. E. M., Lucchesi, B. R., and Pitt, B., et al. The effect of dietary supplementation of fish oil on experimental myocardial infarction. Prostoglandins 20: 1021, 1980. 8. Dyerberg, J., Band, H. O., and Stoffemen,E. Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis.Lancet 2: 117, 1978. 9. Dyerberg, J., and Band, H. 0. Haemostatic function and platelet polyunsaturated fatty acids in eskimos. Lancet 2: 433, 1979. 10. Dyerberg, J., Bang, H. O., and Hjome, N. Fatty acid composition of the plasma lipids in Greenland eskimos. Am. J. C/in. Nutr. 28: 958, 1975. 11. Hagen, P., Wang, Z. G., and Mikat, E. M., et al. Antiplatelet therapy reduced aortic intimal hyperplasia distal to small diameter vascular prosthesis (PRTE) in nonhuman primates. Ann. Surg. 195: 328, 1982. 12. Hoepp, L. W., Elbadawi, A., and Cohn, M., ef al. Steroids and immunosuppression. Arch. Surg. 114: 273, 1979. 13. Imparato, A. M., Baumann, F. G., and Pearson, J., et al. Electron microscopic studies of experimentally produced fibromuscular arterial lesions.Surg. Gynecol. Obstet. 139: 497, 1974. 14. Lorenz, R. Platelet function thromboxane formation and blood pressure control during supplementation of the western diet with cod liver oil. Circulation 67: 504, 1983. 15. McCann, R. L., Hagen, P. O., and Fuchs, J. C. A.

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JOURNAL OF SURGICAL RESEARCH: VOL. 40, NO. 1, JANUARY 1986

Aspirin and dipyridamole decreamintimal hyperplasia in experimental vein grafts.Ann. Surg. 191: 238,198O. 16. Metke, M. P., Lie, J. T., and Fuster, V., er al. Reduction of intimal thickening in canine coronary bypass vein grafts with dipyridamole and aspirin. Amer. J. Cardiol. 43: 1144, 1979. 17. Mielke, C. H., Kaneshiro, M. M., Maher, I. A., and Weiner, J. M., et al. The standardized normal ivy bleeding time and its prolongation by aspirin. Blood 34: 204, 1969. 18. Mocada, S., and Korbut, R. Dipyridamole and other phosphodiesterase inhibitors act as antithrombotic agentsby potentiating endogenousprostacychn. Luncet 2: 1286, 1978. 19. Mocada, S., and Vane, J. R. Arachidonic acid metabolites and the intemctions between platelets and blood vesselwalIs. N. Engl. J. Med. 300: 1142, 1979.

20. Needleman, P., Raz, A., and Minkes, M. S., et al. Triene prostaglandins:Prostacyclin and thromboxane biosynthesis and unique biological properties. Proc. Nati. Acad. Sci. USA 16: 944, 1979. 21 Oblath, R. W., Buckley, F. O., Jr., and Green, R. M., et al. Prevention of platelet aggregationand adherence to prosthetic vascular grafts by aspirin and dipyridamole. Surgery 84: 37, 1980. 22. Salzman, E. W., Harris, W. H., and DeSanctis, R. W. Reduction in venous thromboembolism by agentsaffecting platelet function. N. Engl. J. Med. 284: 1287, 1971.

23. Von Lossoncyzy, T. O., Ruiter, A., BronsgeestSchoute, H. C., and Van Gent, C. M., et al. The effect of a fish diet on serum lipids in healthy human subjects. Amer. J. Clin. Nutr. 31: 1340, 1978.