Fish Oil: A Potent Inhibitor of Platelet Adhesiveness - Blood Journal

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1 From by guest on June 13, 2017. For personal use only. Fish Oil: A Potent Inhibitor of Platelet Adhesiveness By Xiaolin Li and Manfred Steiner The effect of fish oil administration on platelet function on fish oil administration. The profile of the fatty acids was studied in eight normal individuals, four men and four extracted from plasma confirmed compliance of the volun- women, who received fish oil equivalent t o 6 g eicosapen- teers with their dietary supplements. Analysis of phospho- taenoic acid per day for a period of 25 days. Platelet lipids showed changes in sphingomyelin, lysophosphatidyl- aggregation, platelet adhesion, phospholipid and fatty acid choline, and phosphatidylcholine between pseudopodia and distribution were measured at periodic intervals before, platelet cell bodies. Fish oil administration did not affect during, and after the period of fish oil administration. their overall distribution except for a moderate decrease in Platelet aggregation induced by arachidonic acid, adeno- phosphatidylethanolamine in platelet pseudopodia. Changes sine diphosphate, and collagen showed a moderate in- were also recognized in the total fatty acids extracted from crease in ED 50 in response t o the administration of fish oil. platelets, affecting primarily arachidonic acid, eicosapen- Conversely, platelet adhesion t o fibrinogen and collagen 1, taenoic acid, and docosahexaenoic acid. There were no which was studied at low shear rates in a laminar flow changes in platelet adhesiveness in a group of five normal chamber, showed a striking 60% t o 65% decrease after fish individuals who received a vegetable oil supplement of oil supplementation of the diet. The change in adhesive- equal dose and duration as that of the fish oil. We conclude ness could be correlated with the pseudopodia formed in from these studies that fish oil. at least when administered response t o agonistic stimulation. Scanning electron micro- over a limited period of time, is an effective inhibitor of scopic examination of adherent platelets showed an overall platelet adhesiveness. reduction of pseudopodia that appeared short and stubby 0 1990 by The American Society of Hematology. 0 BSERVATIONS I N Greenland Eskimos led Dyerberg et all to conclude that a diet rich in (n-3) long-chain fatty acids has profound effects on hemostasis and lipopro- administration of moderate to high doses of (n-3) polyunsat- urated fatty acids.6-8For this reason we have investigated the adhesive behavior of platelets. A group of eight normal tein metabolism. They postulated that such effects accounted individuals was tested before and after supplementation of for the apparent rarity of thrombo-occlusive vascular disease their usual diet with fish oil equivalent to 6 g EPA/d. The in the aforementioned population Controlled exper- results of our study show a dramatic reduction in platelet iments in many have subsequently provided adhesiveness to collagen and fibrinogen when tested in a direct proof that fish oils affect platelet number, composition, laminar flow chamber under controlled flow conditions. This and function. Although there have been some discrepant may be due to a fish oil induced decrease of pseudopodial results, it appears that platelet aggregation is only mildly processes in stimulated platelets. A delayed onset and pro- affected by the ingestion of 5,8,11,14,17-eicosapentaenoic longed washout period characterized the response. acid (20:5 [n-3], EPA) in various forms of purity (up to 85%).14 Doses of less than 3 to 4 g EPA/d were r e p ~ r t e d ~ . ' ~ . ' ~ . ' ~ MATERIALS AND METHODS to produce some decrease in platelet aggregation, especially Experimental design. A total of eight normal, healthy volun- with collagen as the stimulant. Higher doses of fish oil, teers (four men and four women), all nonsmokers, aged 25 to 56 equivalent to 6 g EPA or m ~ r e , ~are , ~ needed .'~ to affect more years were studied. They abstained from all medications for the definite changes in the aggregatory response of platelets. But entire 37-day period of the investigation. All participants in the even at very high intake levels of EPA, the effect of fish oil study were advised to continue their regular (average American, never equals or comes close to the inhibition that acetylsali- mixed) diet and to refrain from alcoholic beverages for the duration cylic acid produces. of the study. A dietician carefully interviewed the volunteers at the time they donated blood to ensure that the individuals were continu- There are as yet no studies on the effect of dietary fish oil ing their usual dietary habits while taking the supplementation. Fish supplementation on platelet adhesiveness. The bleeding time oil was administered in the form of 1 g capsules (Max EPA). The that has been considered to be closely related to platelet total amount was equivalent to 6 g EPA. Analysis of representative adhesiveness has been prolonged to a variable degree by the examples of the fish oil capsules showed an EPA content between 17.4% and 18.3% and a DHA content (4,7,10,13,16,19-docosa- hexaenoic acid, C22: 6 [n-3]) varying between 11.2% and 12.3% of From the Division of HematologyfOncology. Memorial Hospital the total lipids present. Baseline investigations included complete of Rhode Island; and Brown University, Pawtucket. RI. blood and platelet counts, platelet aggregation and adhesion measure- Submitted November 28,1989; accepted May 7,1990. ments, quantification of platelet pseudopodia and lipid analyses, Supported by Grant HL 39192 from the National Heart. Lung including distribution of phospholipids and fatty acids. Measure- and Blood Institute. ments of these parameters were repeated at intervals during the Address reprint requests to Manfred Steiner, PhD, Division of 25-day period of fish oil supplementation and the week after Hematology/Oncology. Memorial Hospital of Rhode Island, 1I I discontinuation (usually day 30) of the fish oil regimen. In addition, Brewster St, Pawtucket. RI 02860. a group of five normal individuals (three men and two women) aged The publication costs ofihis article were defrayed in part by page 36 to 62 years were placed on a vegetable oil supplement for the same charge payment. This article must therefore be hereby marked period of time and in the same amount as the fish oil was given. The "advertisement" in accordance with 18 U.S.C.section I734 solely to vegetable oil consisted of a 1:l mixture of palm oil and corn oil and indicate this fact. was administered in liquid form. Both fish and vegetable oil o I990 by The American Society of Hematology. contained 1 mg or-tocopherollg of oil. Informed consent was ob- 0006-4971/90/7605-0017$3.00/0 tained from all volunteers participating in this study. 938 Blood, Vol 76, No 5 (September l ) , 1990: pp 938-945

2 From by guest on June 13, 2017. For personal use only. FISH on AND PLATELET ADHESIVENESS 939 Fig 1. TrommMon doc- tron mkrogroph~of pudopo- did (A) ond cell body fBI frac- tions of pletelets (original magnHiution x l7,OOOl. Prepurution o/p/ufe/efs. Blood was obtained from the antccu- butkr. the platelets were muspended in MES b u l k and their bital vein and collected into 1/10 vol of 3.8% sodium citrate. numberadjusted to I x lO'/mL. Platelet-rich plasma (PRP) was prepared as previously described." Evaluation of pluirlef functions. Platelet aggregation was per- The platelet count of the PRP was adjusted to 300.000 to 3S0.000/ formed as previously described." Minimal erective concentrations PI.. to achieve complete aggregation were determined for arachidonic For the measurement of platelet pseudopodia. platelets were acid. adenosine diphosphate (ADP), epinephrine. and collagen 1. separated from PRP by centrihgation at 8OOg and the platelet pellet Platelet aggregation was evaluated with rapect to the initial slope was muspended in 3 mmol/L (N-morpholino) ethane sulfonic acid and the maximal height of the aggregation curve achieved (percent containing 0.147 mmol/L NaCI. 3 mmol/L KCI. and 4.8 mmol/L optical transmission: 0% PRP. 100% platelet-poor plasma IPPP]). D-glucose (MFSbulfer) containing 10% ACD. The pH of this bulfer The dose-raponse relation ktween concentration of platelet agonist was adjusted with Tris base to pH 7.2. After 2 washes with this and aggregation response was evaluated by plotting the log dose

3 From by guest on June 13, 2017. For personal use only. 940 LI AND STEINER cumulative sites versus time, and the reuse of sites index represented by the slope of the least square linear regression line for a plot of the natural logarithm of the total number of sites occupied by platelets versus the different number of times such occupations occurred. Isolation of platelet pseudopodia. After careful elimination of all contaminating red blood cells by repeated centrifugation (58Og), the platelets were sedimented from the PRP, washed twice with MES buffer containing 10%ACD, and finally resuspended in a small volume of this buffer without ACD. The platelet concentration was adjusted to approximately 3 x 109/mL, and the pH of the platelet suspension was brought up to pH 7.2 using Tris base. The platelets were allowed to remain for 15 minutes at 37OC and were then stimulated with bovine thrombin (0.5 U/mL). During the next 5 minutes, the platelets were gently inverted x 2. Shape change was 1 : verified by phase microscopy. The platelet suspension was then subjected to homogenization in a glass/Teflon tissue grinder with a TIME (MINUTES) clearance of 0.056 mm. After 5 up and down strokes, the platelet suspension was centrifuged at 950g for 25 minutes. The supernatant Fig 2. Platelet adhesion to fibrinogen. Cumulative number of was taken off and centrifuged at 2,300g for 25 minutes. The pellets adhesion sites over a 13-minute period are shown. Platelet of the two centrifugations, representing cell bodies and pseudopodia, adhesion was measured at baseline (01,after 2 weeks ( 6 1, after 25 days (0). and on day 30 (0)after fish oil was started. Each point respectively, were resuspended in a small volume of MES buffer and represents the mean of the results obtained from eight individuals were then ready for lipid extraction, protein analysis, or inorganic whose platelets were studied. The differences in the cumulative phosphorus measurement. The purity of the separated fractions was number of adhesion sites between baseline and study period also determined by electron microscopy (Fig I). The pseudopodia measurements were significant at P < .025 at 6 minutes. The preparations (Fig 1A) showed a fairly uniform picture of small significance increased to P < .005 at 12 minutes. All differences vesicle-like structures, most of which contained material of similar were calculated by paired t-tests. appearance as that of the cytoplasm of platelets. The platelet bodies (Fig 2) by and large displayed rounded shapes with subcellular versus the percent response and is reported as the dose producing a granules dispersed throughout the cell. 50% response (ED 50).* Each individual served as his/her own Lipid analysis. Washed suspensions of platelets or PPP were control. extracted with chloroform/methanol (1:2, vol/vol) containing buty- Platelet adhesion was measured in a laminar flow chamber as lated hydroxytoluene (BHT), 50 pg/mL. To l vol cell extract or PPP previously described. Two adhesive surfaces were examined, hu- were added 1/10 vol0.2 mol/L EDTA, 1 drop 88% formic acid, and man fibrinogen and collagen I. Time resolved measurements were 3.7 vol chloroform/methanol. An additional 1.25 vol 2 mol/L KCl made over a 13-minute period at 30-second intervals in a 71,000 and 1.25 vol chloroform with BHT were added. The lipid extract was p(mol/L)area at a shear rate ranging between 20 and 25 s- I. Shear recovered and the remainder was reextracted with 1.5 vol chloroform rates were calculated according to methods described by Batchelor containing BHT. The combined extracts were concentrated under for Hele-Shaw cells. N, and redissolved in a small amount (usually 0.2 mL) chloroform/ Computer-aided analysis of the data was performed on a series of methanol. Platelet phospholipidswere analyzed by thin layer chroma- developed photomicrographs resulting from each experiment. For tography as previously described. Lipid phosphorus was measured each discrete time period, the following parameters were evaluated: according to Chen et aLZ2A standard curve was prepared using (1) Occupied sites, ie, the total number of sites presently occupied by sphingomyelin. platelets; (2) fresh adhesion sites, ie, the number of currently To analyze fatty acids from whole platelets or PPP, an internal occupied sites that were not occupied during the immediately standard, 5 pg heptadecanoic acid, was added to each lipid extract. preceding time period; (3) new sites, ie, the number of currently Fatty acids were transesterified with boron trifluoride (at 100C for occupied sites that had never been occupied until the present time; 90 minutes). The fatty acid methyl esters were extracted twice with and (4) cumulative sites giving a running total of the fresh adhesion petroleum ether. The extracts were combined, evaporated, and sites. dissolved in 25 to 50 pL methylene chloride containing BHT (5 From these data we determined the adhesion rate, which is mg/mL). Gas chromatography was performed in a model 8500 represented by the slope of the linear regression of a plot relating Perkin-Elmer gas chromatograph using a capillary column SP-2230 Table 1. ED 50 of Platelet Aggregation Before and After Fish Oil Supplementation Study Day. Log of Agonist Concentration at 50% Maximal Deviation of Aggregation Curve Agonist Baseline Day 14 Day 25 Day 30 Arachidonic acid (MI -3.34 j; 0.12 -3.29 f 0.08 -2.56 f 0.29t -3.2 f 0.07 Epinephrine (MI -5.85 f 0.62 -5.87 f 0.66 -4.94 f 0.57 -5.86 f 0.71 ADP (MI -5.78 f 0.38 -5.66 k 0.28 -4.52 f 0.34$ -5.65 f 0.32 Collagen (g/mLI -6.04 f 0.35 -6.00 f 0.26 -4.74 f 0.23$ -5.99 f 0.18 *Mean f 1 SD of eight individuals tested. t P < .05. $ P < ,025.

4 From by guest on June 13, 2017. For personal use only. FISH OIL AND PLATELET ADHESIVENESS 94 1 Table 2. Effect of Fish Oil Administration on Total Number of Adhesion Sites ~~ Study Day. Adhesive Protein Baseline Dav 4 Day 14 Day 25 Day 30 ~ Fibrinogen 560 f 120 - 152.3 f 7 5 t 95.3 f 3 9 t 112.1 k 42t Collagen I 277.4 k 125 212.7 f 93 140.6 k 82.8$ 70.8 k 19.2t 62.6 k 26.6t *Mean f 1 SD of eight individuals. t P < ,0005. $ P < .01. (Supelco, Bellefonte, PA). The injector had a 1/10 split ratio. Bleed showed no statistically significant difference either compar- calibration of a blank run was automatically subtracted from the ing population means or performing individual paired t-tests. experimental run. After an initial isothermal period of 5 minutes at However, when ED 50 values were determined for each 85OC, the temperature was raised to 185C at 20C/min. The latter agonist (Table l), moderate increases of the threshold temperature was maintained for 20 minutes and then raised to concentrations were noted in response to fish oil administra- 200C again at 20C/min, and remained at the final temperature for tion. The changes for arachidonic acid, ADP, and collagen an additional 6 minutes. With this temperature program, we were able to elute all the fatty acid methyl esters within a 38-minute attained statistical significance at day 25 of the study. period. Most of the fatty acid methyl esters could be identified by In contrast to the aggregation studies, platelet adhesion comparison of their retention times with those of authentic stan- was found to be drastically affected by the administration of dards. However, certain fatty acid methyl esters were also identified fish oil. Measurement of platelet adhesivity to fibrinogen and by mass spectrometry (Finnegan mass spectrometer). collagen a t low shear rate showed an impressive reduction of Materials. Human fibrinogen was purchased from Kabi Diagnos- the cumulative number of adhesion sites (Table 2) and of the tics (Uppsala, Sweden). Rat tail collagen, type I was obtained from rate of adhesion (Figs 2 and 3). A 60% to 65% decrease in Serva Biochemicals (Westbury, NY). Fish oil was administered in 1 both of these parameters was noted within 14 days after the g gelatin capsules obtained from Scherer Corp (Clearwater, FL) initiation of the fish oil administration. As previously noted (Max EPA). Palm oil was obtained from Palmco, Inc (Portland, OR) and corn oil from Best Foods (Union, NJ). Boron trifluoride in by other investigators,8 the effect of the dietary supplement 14% methanol was obtained from Analabs (Norwalk, CT). All other has a delayed onset and requires a washout time that exceeds chemicals were of highest purity available. 1 week. The reuse of site index also decreased (Figs 4 and 5 ) , RESULTS Platelet aggregation performed with a set of fixed concen- trations of agonists, (624 pmol/L arachidonic acid, 5 pmol/L epinephrine, 2.5 pmol/L ADP, and 1.6 pg/mL collagen) TIME (MINUTES) NUMBER OF TIMES SITE WAS OCCUPIED Fig 3. Platelet adhesion t o collagen 1. Cumulative number of Fig 4. Reuse of adhesion sites plotted against the number of adhesion sites over a 13-minute period are shown. Platelet times a specific adhesion site was occupied during the adhesion adhesion was studied at baseline (0)after 2 weeks ( + 1, after 25 assay. Adhesion t o fibrinogen coated surfaces was studied. Mea- days (0).and on day 30 ( 0 )after fish oil was started. Each point surements before fish oil administration (0),2 weeks after fish oil represents the mean of the results obtained in eight individuals. administration (+I, 25 days after fish oil was begun (0).and on day The differences in the cumulative number of adhesion sites 30 of the study period ( 0 ) .Each point represents the mean of the between baseline and study period measurements were signifi- results obtained from eight individuals. The differences between cant at P < .025 at 6 minutes. The significance increased t o P < baseline and study period measurements were signiticant for 2 .005 at 12 minutes. All differences were calculated by paired ( P< .025) and 3 to 5 revisitations ( P< .005). All differences were t-tests. calculated by paired t-tests.

5 From by guest on June 13, 2017. For personal use only. 942 LI AND STEINER In addition, the pseudopodia were shorter, wider at the base, and more rounded. To ascertain whether these differences h v) were representative of the majority of the platelets, we w obtained quantitative measurements of the pseudopodia that c v) formed in fish oil supplemented platelets on stimulation with LL thrombin (Table 6). A progressive decrease in the ratio of 0 pseudopodia to platelet cell bodies was noticeable over the a entire period of the study. It is interesting to note that the W 2 3 magnitude of this reduction was comparable with that affecting platelet adhesiveness. z v The distribution of phospholipids between pseudopodia C and cell bodies showed differences in lysophosphatidylcho- J line, sphingomyelin, and phosphatidylcholine (Table 7). These changes were not influenced by the administration of fish oil. Only phosphatidylethanolamine decreased slightly in pseudopodia and increased somewhat in platelet cell bodies. 2 13 4 5 6 DISCUSSION NUMBER OF TIMES SITE WAS OCCUPIED Our aggregation studies were able to show only a minor Fig 5. Reuse of adhesion sites plotted against the number of increase of the threshold concentrations of various platelet times a specific adhesion site was occupied during the adhesion agonists after fish oil consumption. Other investiga- assay. Adhesion to collagen I coated surfaces was studied. tors5. have obtained more pronounced fish oil-induced Measurements before fish oil administration (W,2 weeks after fish oil administration ( + I , 2 5 days after fish oil was begun ( 0 ) .and on reductions in platelet aggregability even though the supple- day 30 of the study period (0).Each point represents the mean of mentation level of our study was similar to that used in other the results obtained from eight individuals. The differences be- studies. The reason for the discrepancy is not immediately tween baseline and study period measurements were significant apparent. Differences in the populations examined in the for 2, 3, 4, and 5 revisitations of adhesion sites at the 25-day various studies could be one possible explanation. Noncompli- sampling ( PiW), and for 3, 4, and 5 revisitations of adhesion sites at the 30-day sampling ( P i .025). All differences were ance of the volunteers with the fish oil administration can be calculated by paired t-tests. ruled out because of the increase of DHA and EPA in platelets and plasma. but not as much as the other adhesion indices. Nevertheless, Our studies showed a fish oil-induced decrease in all the the number of multiple reoccupations of adhesion sites was adhesion parameters that we determined. Measurement of strikingly decreased. Adhesion studies in a group of five adhesion in a laminar flow chamber has been well- normal individuals on a vegetable oil supplement, given in ~ h a r a c t e r i z e dTime-resolved .~~ analyses of platelet adhesion equal amounts and for the same length of time as the fish oil, give important information on the reutilization of sites that failed to show a difference between pre- (baseline) and had been used before for adhesion of platelets. It is surprising postsupplementation samples (Table 3). that more than half of the adhesion events take place at sites Compliance of the study subjects could be verified by the that have been previously occupied by platelets.24 A fish changes in the fatty acid distribution in plasma and platelets oil-induced reduction of the reutilization index was clearly (Tables 4 and 5). There was a very marked increase in C20:S evident in our studies. As we have not yet found a satisfactory (n-3) and C22:6 (n-3). There was also an increase in C16:l explanation for the preferred utilization of previously OCCU- and C24:l concomitant with the fish oil administration. On pied adhesion sites, it may be presumptuous to offer an the other hand, C18:2 and C20:4 showed a decrease over the explanation of the change in this behavior brought about by same time interval. After fish oil was discontinued on day 25 the administration of fish oil. However, the ultrastructural of the study, these fatty acids began to return toward morphology of platelets enriched with (n-3) polyunsaturated baseline, a process that was not yet completed on day 30. fatty acids is very persuasive evidence for the importance of In an effort to determine the reason for the reduced pseudopodia in this phenomenon. platelet adhesiveness after fish oil administration, we per- The overall adhesion represented by the cumulative adhe- formed scanning electron microscopy of the adherent plate- sion count and the rate of adhesion showed drastic reductions lets. As shown in Fig 6, fish oil supplemented platelets when fish oil was administered. These results are very similar exhibited a striking decrease in the number of pseudopodia. to those obtained with platelets of individuals on supplemen- Table 3 . Effect of Vegetable Oil Administration on Total Number of Adhesion Sites Study Day' Adhesive Protein Baseline Day 14 Day 25 Day 30 Fibrinogen 548.8 + 67.7 493.2 t 45.4 532.6 + 59.2 529.0 + 70.2 Collagen I 344.4 ? 92.4 351.4 k 55.1 349.1 t 68.7 338.5 + 52.6 *Mean * 1 SD of five individuals.

6 From by guest on June 13, 2017. For personal use only. FISH OIL AND PLATELET ADHESIVENESS 943 Table 4. Total Fatty Acid Distribution (%I of Plasma Before and After Fish Oil Administration Study Day* ~ Fany Acid Baseline Day 10 Day 17 Day 25 Day 28 C 16:O 26.1 f 2.5 26.2 i 2.8 24.8 2 2.8 25.3 f 3.1 26.2 f 3.0 C 16:l 1.1 f 0.4 3.6 f 1.5t 3.5 f o.9t 3.1 f 0.8$ 1.8 f 0.8 C 18:O 11.0 f 1.2 11.3 ? 1.6 10.1 f 1.8 11.5 f 1.9 11.0 f 1.8 C 18:l 19.3 f 1.8 20.1 f 2.7 21.8 i 3.0 20.8 i 3.3 18.6 i 3.2 C 18:2 26.8 f 3.6 18.6 f 4.2$ 16.8 f 2.8t 17.9 f 3.0t 21.6 f 3.45 C 18:3 0.9 f 0.2 1.5 0.811 1.6 f 0.911 1.5 1.811 1.2 f 0.7 c 20:o 0.3 i 0.1 * 0.6 0.2% 0.5 f 0.27 0.2 i 0.1 0.3 f 0.1 c 20:l 1.1 f 0.3 1.1 f 0.3 1.0 i 0.3 1.2 f 0.3 1.1 f 0.4 C 20:3 1.3 f 0.5 2.1 f 0.55 2.5 f 0.6$ 2.2 f 0.75 1.7 f 0.4 C 20:4 5.5 f 0.9 3.7 f 0.8$ 3.5 f 0.8t 3.4 f 0.8t 4.6 f 0.911 c 22:o 0.3 f 0.1 0.3 f 0.1 0.5 i 0.2 0.4 f 0.1 0.4 f 0.1 C22:l 0.1 f 0.05 0.2 f 0.05 0.1 f 0.05 0.2 f 0.1 0.1 f 0.05 C 20:5 0.1 f 0.05 3.4 f 1.5t 4.8 i 1.7t 4.3 i 1.6t 0.9 f 0.3t C 24:O 1.0 f 0.2 0.8 f 0.2 0.7 f 0.2 0.8 f 0.2 1.0 f 0.2 C 24: 1 0.4 i 0.1 1.1 f 0.3t 2.8 i 0.9t 2.7 f 0.8t 1.8 f 0.7t C 22:6 1.6 f 0.4 4.7 f 1.8t 4.9 f 1.9t 4.1 i 1.7$ 3.9 f 1.0$ *Mean f 1 SD of eight individuals. t P < ,0005. $P < ,005. 5P < .01. IIP < .05. VP < .025. tal a - t o ~ o p h e r o l . ~ ~Both * * ~ nutritional additives reduced of the platelet cell body confirmed that the observations pseudopodia formation when stimulated by agonists. The made by scanning electron microscopy did not reflect unique platelets from fish oil-supplemented individuals displayed and isolated examples, but rather were indicative of the short, stubby pseudopodia that are identical in appearance to overall changes induced by fish oil. The increase in inorganic those observed in a-tocopherol-enriched platelets.24 Al- phosphorus of the pseudopodial and the platelet cell body though in the present study only two adhesive surfaces were fraction indicates an increase in platelet size in response to investigated, ie, fibrinogen and collagen I, we believe that the fish oil supplementation. Evidence of a prolonged wash- similar observations will hold true for other adhesive sur- out time of these changes was also apparent in the ratio of faces, as could be shown when we studied the effect of pseudopodia/platelet cell bodies. In fact, at day 30 of our a-tocopherol supplementation. study this ratio was lower than before fish oil supplementa- The quantification of pseudopodia as a relative proportion tion was begun. We believe that this is due primarily to a Table 5. Total Fatty Acid Distribution (%) of Platelets Before and After Fish Oil Administration Study Day' Fatty Acid Baseline Day 14 Day 25 Day 30 C 16:O 19.8 i 2.9 18.9 f 2.6 21.3 f 2.8 20.1 f 2.8 C 16:l 1.2 f 0.2 2.4 i 0.5 2.3 f 0.5 1.6 f 0.4 C 18:O 17.6 f 2.1 14.1 f 2.2t 13.4 f 2.3t 16.2 f 1.9 C 18:l 17.4 i 2.1 16.8 f 2.3 17.0 f 2.9 17.6 f 2.3 C 18:2 8.1 f 1.4 5.4 f 1.3t 4.2 i 1.2$ 6.9 f 1.8 C 18:3 0.8 f 0.2 0.9 f 0.3 0.8 2 0.3 0.8 f 0.3 c 20:o 1.6 f 0.4 1.3 f 0.4 1.0 f 0.3t 1.5 f 0.3 c 20: 1 1.1 f 0.3 1.8 f 0.5t 2.5 f 0.5t 1.8 f 0.4t C 20:3 0.4 f 0.1 0.6 f 0.2 0.8 f 0.3t 0.5 f 0.1 C 20:4 24.1 + 3.1 20.2 f 3.05 17.1 f 2.9$ 23.3 i 3.0 c 22:o 3.2 i 0.4 2.5 f 0.3t 2.2 f 0.3$ 3.1 f 0.4 c 22:l 0.8 f 0.2 1.4 f 0.3$ 1.9 f 0.4$ 0.9 f 0.2 C 20:5 0.1 f 0.05 4.8 f 0.5$ 4.9 f 0.5$ 1.2 f 0.3$ C 24:O 1.2 f 0.3 0.9 f 0.2 0.6 f 0.1$ 1.0 f 0.2 C 24:l 1.4 f 0.2 2.3 f 0.5$ 3.4 f 0.6$ 1.5 f 0.4 C 22:6 0.9 f 0.2 5.8 f 1.1$ 6.0 f 1.0$ 2.0 f 0.4$ *Mean f 1 SD of eight individuals. t P < .005. $P < .0005. 5P < ,025.

7 From by guest on June 13, 2017. For personal use only. 944 LI AND SEINER Fig 6. &mingehctron p p h s o l p ( -ot- S a t badlna (A) m d .*.r 3 rm*rolIhh d( .bnwmnion(E) falgkulnugnwlutlon x 10,Ooo). disproportionately slow return of cell bodies to baseline The effcc~of fish oil on platelet adhesion appears to be values. These findings suggest that the changes affect not specific. An equally high intake of vegetable oil (a I:I only the platelets in the peripheral circulation but also the mixture of palm oil and corn oil) did not alter platelet megakaryocytes, thus leading to a very delayed return to adhesion. This rules out that the increase in dietary fat was presupplementation conditions. responsible for the observed results. In fact. marine and T.bk6.Wod-- (Po) md Cell Bodk.(CB) Boforoand A f t u Fbh OH Adminbmtkn w D . v o r ~ P J lY t 0 9 P t BrJlnr b v 14 Ow 26 ow 30 PI 36.9 2 2.4 40.6 * 4.6t 42.3 I6.8: 31.6 t 3.3 CB 162.1 t 9.7 242.3 I26.14 236.7 t 29.89 222.6 t 26.99 P8JC0 21.8 t 1.9 17.1 t 2.19 16.6 t 2.49 14.3 t 2.69 . . ~ P h Q B n k p h W p h W 8 . + oMea 1 [email protected] hlivkh&. For this nudy. pbtdsawamsrhdmdwith tl * ,0.06 U J m i . - tP c .026. :Pc .01 4P < .ooo6. 1.bk 7. Mmiknkn -lo B.,p(mkt p..udoeodb (Po) and c.))Bodkr (CB) B.toroand A l t u Fhh OH S W Dove 96 DbndLbldP. phaohopid BrJh. 0.v 14 D.v 26 ow30 L M PI 8.8 f 2.q 10.0 t 3.2: 8.1 I2.49 7.8 t 1.6t C8 6.7 t 2.6 6.7 * 1.7 6.3 f 1.6 6.9 t 1.4 SM PI 11.4 t 3.1t 10.8 t 3.74 11.1 I 1.6 9.3 t 2.9t C8 6.8 t 1.8 7.1 t 2.9 7.1 f 2.3 6.6 t 1.0 Pc PI 26.6 t 6.0t 32.6 t 6.6 29.8 t 7.34 27.8 t 10.2t C8 33.7 t 7.6 36.1 t 6.1 35.0 t 2.2 36.1 t 3.6 PI PI 10.8 t 2.6 11.6 t 4.0 12.4 t 1.6 11.1 t 1.6 C8 10.6 t 4.2 11.6 t 2.3 11.2 I2.4 9.6 I 1.8 ps PI 12.1 t 4.1 11.7 t 3.8 13.6 t 2.6 12.8 t 2.4 C8 13.3 t 4.9 9.6 t 6.1 11.4 t 2.7 * 11.3 2.8 P P. 24.9 t 4.2 21.6 t 2.49 21.6 I 1.2: 24.1 t 3.2 C8 23.1 t 3.1 26.6 t 3.4 26.3 t 3.2 26.4 t 1.9 *Memt1SDddghlinbvidud.. t P < .026. SP c .m5. P s .05.

8 From by guest on June 13, 2017. For personal use only. FISH OIL AND PLATELET ADHESIVENESS 945 vegetable oil were similar in degree of saturated, monounsat- number of individuals, we are certain that our observations urated, and polyunsaturated fatty acid content (30%, 28%, can be generalized since the individuals we examined com- and 42%, respectively, for fish oil and 29%, 35%, and 36% for prised both sexes and a wide range of age groups. Because the vegetable oil). Therefore, the difference is narrowed to their original dietary habits remained undisturbed, only the the (n-3) polyunsaturated fatty acids, although it should be effect of fish oil supplementation appears to be reflected in pointed out that fish oil contained cholesterol (4.5 mg/g) the results. Our study, similar to previous investigations of whereas the vegetable oil mixture did not. platelet function, used a relatively short period of dietary We have investigated the distribution of phospholipids supplementation with fish oil. We realize that it will be between pseudopodia and platelet cell bodies to find out important to show that long-term administration maintains whether fish oil induces changes in their proportion. O u r the beneficial effect that fish oil has on platelet adhesiveness. results have only shown a change of phosphatidylethanola- Based on the present and our previous investigations of mine in response to fish oil ingestion. Pseudopodia usually a-to~opherol,~ we~ *believe ~~ that the combined administra- have a somewhat higher proportion of lysophosphatidylcho- tion of platelet anti-aggregating agents, eg, aspirin, and line and sphingomyelin, but show a moderate decrease in substances with anti-adhesive properties, such as fish oil or phosphatidylcholine compared with platelet cell bodies. The a-tocopherol, could have a very potent antithrombotic effect. question arises whether this change may be related to the fish However, further studies will have to be performed to oil-induced abnormalities in platelet pseudopodia. No infor- establish that such a combination therapy has no undesirable mation is available to form a n opinion. side effects. Although our study was based on a relatively small REFERENCES 1. Dyerberg J, Bang HO, Stofferson E, Moncada S, Vane JR: kinetics in patients with ischaemic heart disease. Lancet 1:1269, Eicosapentaenoic acid and prevention of thrombosis and atheroscle- 1982 rosis? Lancet 2:117, 1978 14. Schacky CV, Weber PC: Metabolism and effects on platelet 2. Dyerberg J, Bang HO: Atherogenesis and haemostasis in function of the purified eicosapentaenoic and docosahexaenoic acids Eskimos. The role of the prostaglandin 3 family. Haemostasis 8:227, in humans. J Clin Invest 76:2446,1985 1979 15. Hirai A, Terano T, Hamazaki T, Sajiki J, Kondo S, Ozawa A, 3. Dyerberg J, Bang HO: Haemostatic function and platelet Fujita T, Miyamoto T, Tamura Y,Kumagai K. The effects of the polyunsaturated fatty acids in Eskimos. Lancet 2:433, 1979 oral administration of fish oil concentrate on the release and the 4. Bang HO, Dyerberg J: Lipid metabolism and ischemic heart metabolism of [C] aracidonic acid and [C] eicosapentaenoic acid disease in Greenland Eskimos. Adv Nutr Res 3:1, 1980 by human platelets. Thromb Res 28:285, 1982 5. Siess W, Roth P, Scherer B, Kurzman I, Bohlig B, Weber PC: 16. Ahmed AA, Holub BJ: Alteration and recovery of bleeding Platelet membrane, fatty acids, platelet aggregation, and thrombox- times, platelet aggregation and fatty acid composition of individual ane formation during a mackerel diet. Lancet 1:441, 1980 phospholipids in platelets of human subjects receiving a supplement 6. Goodnight SH, Harris WS, Connor WE: The effects of dietary of cod-liver oil. Lipids 19:617, 1984 omega-3 fatty acids on platelet composition and function in man: A 17. Landolfi R, Mower RL, Steiner M: Modification of platelet prospective, controlled study. Blood 58:880, 1981 function and arachidonic acid metabolism by biotlavanoids. Biochem 7. Brox JH, Killie J, Gumes S, Nordoy A: The effect of cod liver Pharmacol33:1525,1984 oil and corn oil on platelets and vessel wall in man. Thromb Haemost 18. Tallarida RJ, Murray RB: Manual of pharmacologic calcula- 46:604, 1981 tions with computer programs. New York, NY, Springer, 1984, p 14 8. Thorngren M, Gustafson A: Effects of 11-week increase in 19. Jandak J, Steiner M, Richardson PD: Alpha-tocopherol, an dietary eicosapentaenoic acid on bleeding time, lipids and platelet effective inhibitor of platelet adhesion. Blood 73:141, 1989 aggregation. Lancet 2:1190, 1981 20. Batchelor GK: An Introduction to Fluid Dynamics. Cam- 9. Sanders TAB, Vickers M, Haines AP: Effect on blood lipids bridge, UK, Cambridge University, 1967, p 222 and haemostasis of a supplement of cod-liver oil, rich in eicosapen- taenoic and docosahexaenoic acids, in healthy young men. Clin Sci 21. Okuma M, Steiner M, Baldini M: Lipid content and in vitro 61:317, 1981 incorporation of free fatty acids into lipids of human platelets: The 10. Goodnight SH, Harris WS, Connor WE, Illingworth DR: effect of storage at 4OC. Blood 38:27,1971 Polyunsaturated fatty acids, hyperlipidemia and thrombosis. Arterio- 22. Chen PS Jr, Toribara TY, Warner H: Microdetermination of sclerosis 2:87, 1982 phosphorus. Anal Chem 28:1756,1956 11. Sanders TAB, Younger K: The effect of dietary supplements 23. Richardson PD, Mohammed SF, Mason RG: Flow chamber of omega 3 polyunsaturated fatty acids on the fatty acid composition studies of platelet adhesion at controlled spatially varied shear rates. of platelets and plasma choline phosphoglycerides.Br J Nutr 45:613, Proc Eur SOC Artif Organs 4:175,1977 1981 24. Jandak J, Steiner M, Richardson PD: Alpha-tocopherol, an 12. Fischer S, Weber PC: Thromboxane A, (TXA,) is formed in effective inhibitor of platelet adhesion. Blood 73:141, 1989 human platelets after dietary eicosapentaenoic acid (C20:5 3). 25. Jandak J, Steiner M, Richardson PD: Reduction of platelet Biochem Biophys Res Commun 116:1091, 1983 adhesiveness by vitamin E supplementation in humans. Thromb Res 13. Hay CRM, Durber AP, Saynor R: Effect of fish oil on platelet 49:393, 1988

9 From by guest on June 13, 2017. For personal use only. 1990 76: 938-945 Fish oil: a potent inhibitor of platelet adhesiveness [see comments] XL Li and M Steiner Updated information and services can be found at: Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: Information about ordering reprints may be found online at: Information about subscriptions and ASH membership may be found online at: Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved.

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