- Email: [email protected]
SMALL EFFECTS OF LINSEED OIL OR FISH OIL SUPPLEMENTATION ON POSTPRANDIAL CHANGES IN HEMOSTATIC FACTORS
ThrombosisResearch,Vol.85,No.2, pp. 147–152,1997 Copyright@ 1997ElsevierScirnceLtd PrirdedintheUSA. AUrightsmwrved 0049-3848/97$17.00+ .WJ
P
PII S0049-3848(96)00231-9
BRIEF COMMUNICATION
SMALL EFFECTS OF LINSEED OIL OR FISH OIL SUPPLEMENTATION ON POSTPRANDIAL CHANGES IN HEMOSTATIC FACTORS RiittaFreese and Marja Mutanen Divisionof Nutrition,Universityof Helsinki,Helsinki,Finland
(Received2September1996by EditorB. Dahlb~ck;revised/accepted14November1996)
Fish oils decrease alimentarylipemia(1,2). This effectmaybe one of the protectivemechanisms of fish or fish oils against coronary heart disease because postprandial lipid metabolism is involvedin atherogenesis(3). The modulationof lipemiahas been attributedto the long-chainn-3 polyunsaturatedfatty acids, eicosapentaenoicacid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). To our knowledge, the effects of a-linolenic acid (ALA, 18:3 n-3), the precursorof EPA, havenot been studied. Postprandial lipemiaaffects the hemostatic system. Coagulationfactor VII coagulant activity (FVII:C) increasesafter high-fatmeal(4-9) whilethe activityof plasminogenactivatorinhibitor1 (PAI-1) has been shown to decrease postprandially(4,5). Platelet aggregation is also altered during lipemia(see 9,10). Reports on the influenceof the backgrounddiet on the postprandial changes in hemostatic factors are few. In the present study, we investigatedthe postprandial responses of FWI:C, PAI-1 activity and platelet function in healthy normolipidemicsubjects before and afier supplementationof ALA in linseedoil or EPA+DHAin fish oil. MATERIAL AND METHODS The subjects were 30 healthy volunteers (age 22-42 years, BMI 17.6-29.8 kg/m’2)who were supplementedfor 4 weeks eitherwith encapsulatedlinseedoil (LO-group;8 females,6 males)or fish oil (Pikasol@,Lube A/S, Denmark;FO-group;7 females,9 males).The subjectsin the FOgroup took also a smallamount(2.0-3,5g) of sunfloweroil to balancethe intakeof Iinoleicacidin the two groups. Total supplementedamount of oil was 1#200 kcal of the calculated energy expenditure.The mean intakesof the supplementsand majbr fatty acids were in the LO-group: 11.9 g/day (18: 1 1.89 g; 18:2n-6 1.74 g; and ALA 6.21 g; n-3/n-6-ratio 3.6), and in the FOKey words: n-3 fatty acids, coagulation,fibrinolysis,platelettimction,postprandial,human Corresponding author: Riitta Freese, Department of Applied Chemistry and Microbiology, P.O.Box 27 (Viikki,Nutrition,BuildingB), FIN-00014Universityof Helsinki,Finland tel. +358 97085202 fax +358 97085269 E-mail:[email protected]
147
148
HEMOSTATICFACTORSAND LIPEMIA
vol.
85, No. 2
group: 12.2g/day (18:1 1.04g; 18:2n-61.58g; EPA 3.04 g; and DHA 2.45 g; n-3/n-6-ratio4.0). Five females in both groups used oral contraceptivesand one male in the LO-group and two males in the FO-group smoked 2 to 10 cigarettes/day.The habitualdiets were monitored by repeated 48-hour dietary recalls and they did not changeduring the supplementation(data not shown). Oral fat load tests were carriedout before and after the 4-week supplementationperiod. Baseline blood sampleswere taken after an overnightfast in the morningduring 7.30 and 9.30 am. The subjectswere then givena liquidmealcontainingsunfloweroil 1 gkg bodyweight(9). Additional blood sampleswere taken 3 and 5 hours after the meal. During this 5-hour period the subjects were not allowed to eat, drink, or smoke, and strenuous physicalactivitywas also forbidden. Blood sampleswere drawn in vacuumtubes from the antecubitalvein alwaysin the same order and by the samelaboratorytechnician.Subjectswere in supinepositionduringblood sampling. Samplesfor analysisof hemostaticfactors were taken in 3.2% trisodium citrate (Vacutainer, USA). Blood for FVII:C and PAI-I activityanalyseswas centrifugedwithin 20 minutes tlom venejectionat 3000 x g for 15 min.Plasma sampleswere snap-frozenand kept at -40”C. The analyseswere done in the CoagulationLaboratoryof the FinnishRed Cross Blood Transfusion Service,Helsinki.F VII:C was analyzedas describedearlier(9). Measurementof PAI-I activity was carriedout with CoatestPAI (ChromogenicAB, Sweden).The detectionlimitwas 5 AU/mL and results below this were coded 5 AU/mL. Results from subjectswith all values below the detection limit were excluded from statisticalanalyses(final number of subjects 8 in the LOgroup and 9 in the FO-group).Plateletaggregationwas measuredin plateletrich plasma(PRP) as described(11). PRP sampleswere kept at room temperature.The aggregatingagentswere ADP (1, 2, and 3 ~mol/L; Boehringer Mannheim, Germany) and collagen (0.5, 1 and 3 pg/mL; Nycomed,Germany).Aggregationswere followedfor 3 minutesand the velocityis expressedas the slope (O/O/rein) of the aggregationcurve. Samplesfor thromboxaneB2 analysis(TXB2)were taken from PRP activated with 3 pg/mL collagen and handled as described (11). TXB2 was analyzed with enzymoimmunoassay(12). Samples for plasma glucose analysiswere taken in tubes containingpotassiumoxalateand sodiumfluoride(Venoject,USA). Glucose,triglycerides and cholesterolwere measuredfrom samplesstored at -20”Cwith commercialkits (Merck and BohringerMannheim,Germany).Hematocritwas measuredwith the centrifhgationmethod. Statisticalanalysesof the fastingdata (PAI-1 excluded)were carried out with paired (before vs. after supplementation) and independent (LO- vs. FO-group) t-tests. Because fish oil supplementationchangedthe fastingvalues, the postprandialdata was standardizedby dividing the postprandialvalues by the respectivefasting value. These ratios were largely not normally distributed and were analyzed with nonparametric tests (Wilcoxon signed ranks test and Kolmogorov-Smirnovtwo-sampletest). Nonparametrictests were used also with fastingPAI-1 values.In alltests, p-valuessmallerthan 0.05 were consideredsignificant. RESULTS The supplementsand the test-meal were well tolerated and no major gastric side-effectswere reported. The increasedintake of n-3 polyunsaturateswas seen in platelettotal lipids:ALA and 22:5 n-3 (DPA) increasedin the LO-group and EPA, DPA and DHA in the FO-group (data not shown).
vol. 85, No. 2
149
HEMOSTATICFACTORSAND LIPEMIA
TABLE I. Fasting Values of the MeasuredVariablesBefore and After Supplementationin the Study Groups [mean(SD)]. LO-GROUP1 Pr& Exp F VII:C (~,) 87.2 (18.1) PAI-1 (AU/mL) 9.3 (6.0) ADP aggregation(%/rein) 1 pmol/L 34.8 (12.5) 2 pmol/L 56.3 (10.8) 3 pmol/L 68.8 (10.0) Collagenaggregation(%/rein) 0.5 pghm 1 l.lght 3 pg/mL TXB2 (ng/mLPRP)
44.8 (28.5) 78.6 (23,4) 94.3 (13.1)
FO-GROUP Exp Pre
89.3 (16.7) 10.2(7.7)
85.6 (16.2) 7.1 (2.3)
94.3(16.6)$ 10.6(2.3)$
34.1 (17.4) 54.7 (18.1) 63.8 (16.6)
34.4 (13.3) 60.0 (12.0) 72.3 (13.4)
38.2 (10.5) 64.6 (7.5)$ 71.6 (9.0)
35.2 (31.8) 76.8 (24.1) 90.3 (15.3) 92.2 (38.9) 98.4 (41.2) Triglycer.(mmol/L) 0.97 (0.33) 0.91 (0.31) Cholesterol(mmol/L) 4.94 (0,71) 4.76 (0,58) 4.71 (0.39) 4.75 (0.41) Glucose(mmol/L) Hematocrit(%) 42.3 (4,3) 42.9 (3.1)
31.1 (20.2)$ 53.3(30.3) 81.2(15.0) 79.0 (20.4) 96.2 (10.9) 99,6 (12.8) 124.4(67.1) 113.2(48.3) 0.68 (0.28)$~ 0.92 (0.34) 4.90 (0,76)$ 5.37 (0.77) 4.95 (0.30)$ 4.69 (0.32) 44.0 (3.3) 42.7 (2.8)
1LO-group was supplementedwith linseedoil and FO-group with fish oil and sunflower oil. ‘2Pre=before supplementation,Exp=after supplementation. $ Significantlydifferent fromthe respectivePre value. ~ Significantdifferencebetweenthe LO and FO-groups. No differences between the groups were seen in the fasting levels of any varible before supplementation(Table 1), Linseedoil supplementationhad no effects on the fastingvalues but in the FO-group,severalvaluesdecreased(aggregationto 0.5 pg/mLcollagen,serumtriglycerides and cholesterol) or increased (FVII:C, PAI-I activity, aggregation to ADP 2 pmol/mL, and plasma glucose). Serum triglycerideswas, however, the only variable which was on different levelsin the groupsafter supplementation(TableI). Except for the collagen-inducedaggregation and FVII:C the fastinglevelsin the FO-group returnedto the baselineduringa 12-weekfollowUPperiod (data not shown). Postprandialincreasein FVII:C was seen in both groups before and after supplementation.This increase was higher at 5 hours than at 3 hours after the meal in both groups at the end of the supplementationperiod (TableII). PAI-1 activitydecreasedin the LO-group duringpostprandial phase before and afler supplementation.In the FO-group, this decrease was seen only atler supplementation(Table H). Platelet aggregationto ADP and collagen decreased or tended to decrease postprandially(Table 11).Flatteningin the responseto ADP was seen after linseedoil supplementation.The onlydifferencebetweenthe postprandialresponsesin the two groups was seen in aggregation to 0.5 pg/mL collagen before supplementation.The results with ADP 3 ~mol/Land collagen3 p#mL paralleledthe resultsobtainedwith ADP 2 pmol/L and collagen1 pg/mL (data not shown). TXB2 productionwas not affectedby postprandiallipemia(data not shown).
150
HEMOSTATICFACTORSAND LIPEMIA
Voi.85, No. 2
TABLE II. Standardized Postprandial Valuesl 3 and 5 Hours After the Test Meal Before and After Supplementationin the StudyGroups [mean(SD)]. LO-GROUP2 Preq Exp
.—.—-———--——---—-——--.— 3h 1.04 (0.04)* F VII:C 5h 1.03(0.04)* 0.74 (0.25)* PM-1 3h 0.66 (0,22)* 5h 0.82 (0.29) ADP 1 pmol/L 3h 5h 0.84 (0.33) 0.85 (0.25) 2 pmol/L 3h 0.82 (0.21)* 5h 1.04(0,40) Coil. 0.5 j,@mL 3h 5h 0.67 (0.31)* 0.72 (0.25)* 1 p@nL 3h 5h 0.72 (0.28)* 2.00 (0,46)” Triglycerides 3h 5h 1,71(0.53)* 1.03(0.08) Cholesterol 3h 1.02(0.08) 5h 3h 1.03(0.07) Glucose 5h 1.05(0.07)*
1.02 (0.04) 1.05(0.05)*7 0.71 (0.30)* 0.64 (0.26)* 1.20(0.62)$ 1.05(0.65) 1.05(0.36) 1.03(0.36) 1.31(1,49) 0.54 (o.31)*~ 0.82 (0.35)* 0.79 (0.35) 2.02 (0.56)* 1,70(0.37)* 1.03(0,04)* 1.05(0.05)* 1.05(0.09) 1.03(0.07)
Pre
FO-GROUP Exp
1.02(0.04) 1.03(0.05) 1.04(0.06)* 1.05(0.05)*t 0.70 (0.26)*fj 0.96 (0.27) 0.70 (0.36) 0.87 (0.29) 0.85 (0.42)” 0.91 (0.22) 0.85 (0.48)* 0.86 (0.25)* 0.76 (0.14)* 0.83 (0.16)* 0.79 (0.19)* 0,90 (0.14)* 0.56(0.23)* 0.73(0.64)* 0.56 (0.35)*# 0.51 (0.30)* 0.70(0.17)* 0.92(0.38) 0,71(0.19)* 0.84(0.41) 2.14(0.70)* 2.06(0.59)* 1.89(0.67)* 1.81(0.55)* 1,05(0.06)* 1.04(0.08) 1.04(0.06)* 1.06(0.07)* 0.98 (0.07)$ 1.04(0.09) 1.O6(O.1O)* 1.00(0.06) -— ——--——— ——-————— -— ————-—— ——-————-—-—— -
1Value greater than 1 indicatesan increaseand value smallerthan 1 indicatesan decrease from the fasting level. z Groups are indicatedas in Table I. 3Periods are indicatedas in Table II. * Significantlydifferentfrom 1 (=fastingvalue). t Significantlydifferent from the respective 3-hour value. $ Significantlydifferentfrom the respective Pre value. # Significantdifferencebetweenthe LO and FO-groups. The postprandialcholesteroland triglycerideresponseswere not altered by linseedoil or fish oil supplementation.Glucose responseremainedalso unchangedin the LO-group but was flattened afler supplementationin the FO-group (Table II). Hematocrit increased or tended to increase slightlyin both groups during postprandialphase before and after supplementation (data not shown). DISCUSSION The mean intake of the supplemented ALA was 6.2 g/day in the LO-group and that of EPA+DHA 5.5 g/day in the FO-group. These intakes are much higher than in young Finnish adults in average (ALA 1.5 g/day and EPA+DHA 0.4 g/day) (13) and were reflected in the changesin plateletlipids. FVII:C increased afler high-fatmeal which is a well-knownphenomenon.The peaking time of FVII:C has been reported to be delayedfrom that of triglycerides(4,6,8). It has been suggested that free fatty acids released from chylomicronsand VLDL during lipoprotein lipase-catalyzed
Vol.85, No. 2
HEMOSTATICFACTORSAND LIPEMIA
151
lipolysisactivate the contact system of coagulation,This leads to activationof factor VII and enhancementof FVII:C (6,7). The increasein FVII:C has been shownto be similarafter meals rich in dairy fat or vegetable oils (4,8,9) but different saturated fatty acids may differ in this respect (14). Our results suggest that n-3 fatty acids in the diet modify the peaking time of FVII:C because the 5-hour value was higher than the 3-hour value in both groups after supplementation. The pattern of this change and the underlying mechanisms remain to be unraveled becauseno data on the time and levelof the peak valuesof activatedFVII, FVII:C or chylomicronand VLDL triglycerides,or the fatty acid compositionof the lipoproteinparticlesis availablefrom the present study. The altered PAI-I response in the FO-group is at least partly an artefact because the fasting levelsbefore supplementationwere low in the FO-group and the detection limit 5 AU/mL was easilyachievedduringthe postprandialphase,PAI-1 activityhas earlierbeen shown to decrease in a similarway after low-fatand high-fatmeals(4,5), and afler mealsrich in either myristicacid or stearicacid(14). Platelet aggregation to ADP was not decreased postprandially in the LO-group after supplementation.Lipemiawas not altered duringlinseedoil supplementationwhich excludesan artefact caused by differentlylipemicsamples,Chylomicronsand VLDL have been reported to decrease platelet aggregation measured by particle-counting method (10) and linseed oil supplementationhas been shownto increasethe proportionof ALA in VLDL triglycerides(15). One could speculate that the ALA supplementation modifies the postprandial plasma environmentand preservesADP-inducedplateletaggregationon the fastinglevel. Fasting glucose concentrationincreased during supplementationperiod in the FO-group. This change may be due to increasedoxidationbecausein one study (16) the increase was seen with capsulescontaining1.5 IU/g of vitaminE but not with capsulescontaining4.5 IU/g of vitaminE. In the present studytocopherolswere added 1.5 mg/g in the linseedoil capsulesand 3.3 mg/g in the fishoil capsulesand a-tocopherollevelsin serumwere not changed(data not shown). In summary,the presentdatademonstratesthatwhen the n-3 fatty acid statusof healthysubjectsis modified either with ALA or EPA+DHA, postprandialresponsesof FVII:C and ADP-induced platelet aggregationare affected.The pattern of the alteredFVII:C responseafler n-3 fatty acid supplementationand the mechansimunderlyingthe changesin platelet aggregationin the LOgroup shouldbe studiedin more detail, Acknowledgements We thank Elina Vahtera, Phil. Lie., and Tom Krusius,MD, from the FinnishRed Cross Blood Transfusion Service for FVII:C and PAI-I activity anaIyses;Lube A/S for the encapsulated supplements and Mildola Ltd., Finland for the sunflower oil. This study was supported by Jenny and Antti WihuriFoundation,The 350th AnniversaryFund@fthelJnivemity of I-Ielsinki, and the Academyof Finland. REFERENCES 1. HARRIS, W.S., CONNOR, W.E., ALAM, N. and ILLINGWORTH, D.R. Reduction of postprandialtriglyceridemiain humansby dietary n-3 fatty acids. J Lipid Res 29, 1451-1460, 1988.
152
HEMOSTATICFACTORSAND LIPEMIA
vol.
85, No. 2
WEINTRAUB, M.S., ZECHNER, R., BROWN, A., EISENBERG, S. and BREWSLOW, J.L. Dietary polyunsaturatedfats of the w-6 and w-3 series reduce postprandial lipoprotein levels.J ClinInvest82, 1884-1893,1988. 3. SILVERSMITH, D.B. Atherogenesis:a postprandialphenomenon.Circulation60, 473-485, 1979. 4. SALOMAA, V., RASI, V., PEKKANEN, J., JAUHIAINEN, M., VAHTERA, E., PIETINEN, P., KORHONEN, H., KUULASMAA, K. and EHNHOLM, C. The effects of saturated fat and n-6 polyunsaturated fat on postprandial Iipemia and hemostatic activity. Atherosclerosis103, 1-11, 1993. 5. MARCKMANN, P., SANDSTROM,B. and JESPERSEN J, Dietary effects on circadian fluctuationin humanblood coagulationfactor VII and fibrinolysis.Atherosclerosis101,225-234, 1993. 6. SILVEIRA, A., KARPE, F,, BLOMBACK, M., STEINER, G., WALLDIUS, G. and HAMSTEN, A. Activation of coagulationfactor VII during alimentarylipemia. Arterioscler Thromb14, 60-69, 1994. 7. MITROPOULOS, K.A., MILLER, G.J., MARTIN, J,C., REEVES, B.E.A. and COOPER, J. Dietary fat induces changes in factor VII coagulant activitythrough effects on plasma free stearicacidconcentration.ArteriosclerThromb1~, 214-222, 1994. 8. MILLER, G.J., MARTIN, J.C., MITROPOULOS,K.A., REEVES, B.E.A., THOMPSON, R.L., MEADE, T.W., COOPER, J.A, and CRUICKSHANK,J.K, Plasmafactor VII is activated by postprandialtriglyceridemia,irrespectiveof dietaryfat composition.Atherosclerosis86, 163171, 1991. 9. FREESE, R. and MUTANEN,M. Postprandialchangesin plateletfinction and coagulation factors after high-fatmealswith differentfatty acid compositions.Eur J ClinNutr 49, 658-664, 1995. 10. ORTH, M., LULEY, C., MAYER, H. and WIELAND,H. Effects of VLDL, chylomicrons, and chylomicronremnantson plateletaggregability.ThrombRes 79,297-305, 1995. 11. MUTANEN, M., FREESE, R., VALSTA, L.M., AHOLA, I. and AHLSTROM, A. Rapeseed oil and sunflower oil diets enhance platelet in vitro aggregation and thromboxane production in healthy men when comparedwith milk fat or habitual diets. Thromb Haemostas 2.
67,352-356,1992. 12. PRADELLES, P., GRASSI, J. and MACLOUF, J. Enzyme immunoassay of eicosanoids
using acetylcholineesterase as label:an alternativeto radioimmunoassay.Anal Chem57, 11701173,1985. 13. KLEEMOL~ P., VIRTANEN, M. and PIETINEN, P. The 1992 dietary survey of finnish adults.Publicationsof the NationalPublicHealth InstituteNo.: B2/1994,Helsinki,1994. 14. THOLSTRUP,T., ANDREASEN,K. and SANDSTROM,B. Acute effect ofhigh-fat meals rich in either stearic and myristicacid on hemostaticfactors in healthyyoung men. Am J Clin Nutr 64, 168-176,1996. 15. LAYNE, K.S., GOH, Y.K., JUMPSEN, J.A., RYAN, E.A., CHOW, P. and CLANDININ, M.T. Normal subjectsconsumingphysiologicallevelsof 18:3(n-3)and 20:5(n-3) from flaxseed and fish oils have characteristicdifferencesin plasma lipid and lipoproteinfatty acid levels. J Nutr 126,2130-2140, 1996. 16. LUOSTARINEN, R., WALLIN, R., WIBELL, L. and SALDEEN T. Vitamin E supplementationcounteractsthe fish oil-inducedincreaseof blood glucosein humans.Nutr Res 15, 953-968, 1995.
P
PII S0049-3848(96)00231-9
BRIEF COMMUNICATION
SMALL EFFECTS OF LINSEED OIL OR FISH OIL SUPPLEMENTATION ON POSTPRANDIAL CHANGES IN HEMOSTATIC FACTORS RiittaFreese and Marja Mutanen Divisionof Nutrition,Universityof Helsinki,Helsinki,Finland
(Received2September1996by EditorB. Dahlb~ck;revised/accepted14November1996)
Fish oils decrease alimentarylipemia(1,2). This effectmaybe one of the protectivemechanisms of fish or fish oils against coronary heart disease because postprandial lipid metabolism is involvedin atherogenesis(3). The modulationof lipemiahas been attributedto the long-chainn-3 polyunsaturatedfatty acids, eicosapentaenoicacid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). To our knowledge, the effects of a-linolenic acid (ALA, 18:3 n-3), the precursorof EPA, havenot been studied. Postprandial lipemiaaffects the hemostatic system. Coagulationfactor VII coagulant activity (FVII:C) increasesafter high-fatmeal(4-9) whilethe activityof plasminogenactivatorinhibitor1 (PAI-1) has been shown to decrease postprandially(4,5). Platelet aggregation is also altered during lipemia(see 9,10). Reports on the influenceof the backgrounddiet on the postprandial changes in hemostatic factors are few. In the present study, we investigatedthe postprandial responses of FWI:C, PAI-1 activity and platelet function in healthy normolipidemicsubjects before and afier supplementationof ALA in linseedoil or EPA+DHAin fish oil. MATERIAL AND METHODS The subjects were 30 healthy volunteers (age 22-42 years, BMI 17.6-29.8 kg/m’2)who were supplementedfor 4 weeks eitherwith encapsulatedlinseedoil (LO-group;8 females,6 males)or fish oil (Pikasol@,Lube A/S, Denmark;FO-group;7 females,9 males).The subjectsin the FOgroup took also a smallamount(2.0-3,5g) of sunfloweroil to balancethe intakeof Iinoleicacidin the two groups. Total supplementedamount of oil was 1#200 kcal of the calculated energy expenditure.The mean intakesof the supplementsand majbr fatty acids were in the LO-group: 11.9 g/day (18: 1 1.89 g; 18:2n-6 1.74 g; and ALA 6.21 g; n-3/n-6-ratio 3.6), and in the FOKey words: n-3 fatty acids, coagulation,fibrinolysis,platelettimction,postprandial,human Corresponding author: Riitta Freese, Department of Applied Chemistry and Microbiology, P.O.Box 27 (Viikki,Nutrition,BuildingB), FIN-00014Universityof Helsinki,Finland tel. +358 97085202 fax +358 97085269 E-mail:[email protected]
147
148
HEMOSTATICFACTORSAND LIPEMIA
vol.
85, No. 2
group: 12.2g/day (18:1 1.04g; 18:2n-61.58g; EPA 3.04 g; and DHA 2.45 g; n-3/n-6-ratio4.0). Five females in both groups used oral contraceptivesand one male in the LO-group and two males in the FO-group smoked 2 to 10 cigarettes/day.The habitualdiets were monitored by repeated 48-hour dietary recalls and they did not changeduring the supplementation(data not shown). Oral fat load tests were carriedout before and after the 4-week supplementationperiod. Baseline blood sampleswere taken after an overnightfast in the morningduring 7.30 and 9.30 am. The subjectswere then givena liquidmealcontainingsunfloweroil 1 gkg bodyweight(9). Additional blood sampleswere taken 3 and 5 hours after the meal. During this 5-hour period the subjects were not allowed to eat, drink, or smoke, and strenuous physicalactivitywas also forbidden. Blood sampleswere drawn in vacuumtubes from the antecubitalvein alwaysin the same order and by the samelaboratorytechnician.Subjectswere in supinepositionduringblood sampling. Samplesfor analysisof hemostaticfactors were taken in 3.2% trisodium citrate (Vacutainer, USA). Blood for FVII:C and PAI-I activityanalyseswas centrifugedwithin 20 minutes tlom venejectionat 3000 x g for 15 min.Plasma sampleswere snap-frozenand kept at -40”C. The analyseswere done in the CoagulationLaboratoryof the FinnishRed Cross Blood Transfusion Service,Helsinki.F VII:C was analyzedas describedearlier(9). Measurementof PAI-I activity was carriedout with CoatestPAI (ChromogenicAB, Sweden).The detectionlimitwas 5 AU/mL and results below this were coded 5 AU/mL. Results from subjectswith all values below the detection limit were excluded from statisticalanalyses(final number of subjects 8 in the LOgroup and 9 in the FO-group).Plateletaggregationwas measuredin plateletrich plasma(PRP) as described(11). PRP sampleswere kept at room temperature.The aggregatingagentswere ADP (1, 2, and 3 ~mol/L; Boehringer Mannheim, Germany) and collagen (0.5, 1 and 3 pg/mL; Nycomed,Germany).Aggregationswere followedfor 3 minutesand the velocityis expressedas the slope (O/O/rein) of the aggregationcurve. Samplesfor thromboxaneB2 analysis(TXB2)were taken from PRP activated with 3 pg/mL collagen and handled as described (11). TXB2 was analyzed with enzymoimmunoassay(12). Samples for plasma glucose analysiswere taken in tubes containingpotassiumoxalateand sodiumfluoride(Venoject,USA). Glucose,triglycerides and cholesterolwere measuredfrom samplesstored at -20”Cwith commercialkits (Merck and BohringerMannheim,Germany).Hematocritwas measuredwith the centrifhgationmethod. Statisticalanalysesof the fastingdata (PAI-1 excluded)were carried out with paired (before vs. after supplementation) and independent (LO- vs. FO-group) t-tests. Because fish oil supplementationchangedthe fastingvalues, the postprandialdata was standardizedby dividing the postprandialvalues by the respectivefasting value. These ratios were largely not normally distributed and were analyzed with nonparametric tests (Wilcoxon signed ranks test and Kolmogorov-Smirnovtwo-sampletest). Nonparametrictests were used also with fastingPAI-1 values.In alltests, p-valuessmallerthan 0.05 were consideredsignificant. RESULTS The supplementsand the test-meal were well tolerated and no major gastric side-effectswere reported. The increasedintake of n-3 polyunsaturateswas seen in platelettotal lipids:ALA and 22:5 n-3 (DPA) increasedin the LO-group and EPA, DPA and DHA in the FO-group (data not shown).
vol. 85, No. 2
149
HEMOSTATICFACTORSAND LIPEMIA
TABLE I. Fasting Values of the MeasuredVariablesBefore and After Supplementationin the Study Groups [mean(SD)]. LO-GROUP1 Pr& Exp F VII:C (~,) 87.2 (18.1) PAI-1 (AU/mL) 9.3 (6.0) ADP aggregation(%/rein) 1 pmol/L 34.8 (12.5) 2 pmol/L 56.3 (10.8) 3 pmol/L 68.8 (10.0) Collagenaggregation(%/rein) 0.5 pghm 1 l.lght 3 pg/mL TXB2 (ng/mLPRP)
44.8 (28.5) 78.6 (23,4) 94.3 (13.1)
FO-GROUP Exp Pre
89.3 (16.7) 10.2(7.7)
85.6 (16.2) 7.1 (2.3)
94.3(16.6)$ 10.6(2.3)$
34.1 (17.4) 54.7 (18.1) 63.8 (16.6)
34.4 (13.3) 60.0 (12.0) 72.3 (13.4)
38.2 (10.5) 64.6 (7.5)$ 71.6 (9.0)
35.2 (31.8) 76.8 (24.1) 90.3 (15.3) 92.2 (38.9) 98.4 (41.2) Triglycer.(mmol/L) 0.97 (0.33) 0.91 (0.31) Cholesterol(mmol/L) 4.94 (0,71) 4.76 (0,58) 4.71 (0.39) 4.75 (0.41) Glucose(mmol/L) Hematocrit(%) 42.3 (4,3) 42.9 (3.1)
31.1 (20.2)$ 53.3(30.3) 81.2(15.0) 79.0 (20.4) 96.2 (10.9) 99,6 (12.8) 124.4(67.1) 113.2(48.3) 0.68 (0.28)$~ 0.92 (0.34) 4.90 (0,76)$ 5.37 (0.77) 4.95 (0.30)$ 4.69 (0.32) 44.0 (3.3) 42.7 (2.8)
1LO-group was supplementedwith linseedoil and FO-group with fish oil and sunflower oil. ‘2Pre=before supplementation,Exp=after supplementation. $ Significantlydifferent fromthe respectivePre value. ~ Significantdifferencebetweenthe LO and FO-groups. No differences between the groups were seen in the fasting levels of any varible before supplementation(Table 1), Linseedoil supplementationhad no effects on the fastingvalues but in the FO-group,severalvaluesdecreased(aggregationto 0.5 pg/mLcollagen,serumtriglycerides and cholesterol) or increased (FVII:C, PAI-I activity, aggregation to ADP 2 pmol/mL, and plasma glucose). Serum triglycerideswas, however, the only variable which was on different levelsin the groupsafter supplementation(TableI). Except for the collagen-inducedaggregation and FVII:C the fastinglevelsin the FO-group returnedto the baselineduringa 12-weekfollowUPperiod (data not shown). Postprandialincreasein FVII:C was seen in both groups before and after supplementation.This increase was higher at 5 hours than at 3 hours after the meal in both groups at the end of the supplementationperiod (TableII). PAI-1 activitydecreasedin the LO-group duringpostprandial phase before and afler supplementation.In the FO-group, this decrease was seen only atler supplementation(Table H). Platelet aggregationto ADP and collagen decreased or tended to decrease postprandially(Table 11).Flatteningin the responseto ADP was seen after linseedoil supplementation.The onlydifferencebetweenthe postprandialresponsesin the two groups was seen in aggregation to 0.5 pg/mL collagen before supplementation.The results with ADP 3 ~mol/Land collagen3 p#mL paralleledthe resultsobtainedwith ADP 2 pmol/L and collagen1 pg/mL (data not shown). TXB2 productionwas not affectedby postprandiallipemia(data not shown).
150
HEMOSTATICFACTORSAND LIPEMIA
Voi.85, No. 2
TABLE II. Standardized Postprandial Valuesl 3 and 5 Hours After the Test Meal Before and After Supplementationin the StudyGroups [mean(SD)]. LO-GROUP2 Preq Exp
.—.—-———--——---—-——--.— 3h 1.04 (0.04)* F VII:C 5h 1.03(0.04)* 0.74 (0.25)* PM-1 3h 0.66 (0,22)* 5h 0.82 (0.29) ADP 1 pmol/L 3h 5h 0.84 (0.33) 0.85 (0.25) 2 pmol/L 3h 0.82 (0.21)* 5h 1.04(0,40) Coil. 0.5 j,@mL 3h 5h 0.67 (0.31)* 0.72 (0.25)* 1 p@nL 3h 5h 0.72 (0.28)* 2.00 (0,46)” Triglycerides 3h 5h 1,71(0.53)* 1.03(0.08) Cholesterol 3h 1.02(0.08) 5h 3h 1.03(0.07) Glucose 5h 1.05(0.07)*
1.02 (0.04) 1.05(0.05)*7 0.71 (0.30)* 0.64 (0.26)* 1.20(0.62)$ 1.05(0.65) 1.05(0.36) 1.03(0.36) 1.31(1,49) 0.54 (o.31)*~ 0.82 (0.35)* 0.79 (0.35) 2.02 (0.56)* 1,70(0.37)* 1.03(0,04)* 1.05(0.05)* 1.05(0.09) 1.03(0.07)
Pre
FO-GROUP Exp
1.02(0.04) 1.03(0.05) 1.04(0.06)* 1.05(0.05)*t 0.70 (0.26)*fj 0.96 (0.27) 0.70 (0.36) 0.87 (0.29) 0.85 (0.42)” 0.91 (0.22) 0.85 (0.48)* 0.86 (0.25)* 0.76 (0.14)* 0.83 (0.16)* 0.79 (0.19)* 0,90 (0.14)* 0.56(0.23)* 0.73(0.64)* 0.56 (0.35)*# 0.51 (0.30)* 0.70(0.17)* 0.92(0.38) 0,71(0.19)* 0.84(0.41) 2.14(0.70)* 2.06(0.59)* 1.89(0.67)* 1.81(0.55)* 1,05(0.06)* 1.04(0.08) 1.04(0.06)* 1.06(0.07)* 0.98 (0.07)$ 1.04(0.09) 1.O6(O.1O)* 1.00(0.06) -— ——--——— ——-————— -— ————-—— ——-————-—-—— -
1Value greater than 1 indicatesan increaseand value smallerthan 1 indicatesan decrease from the fasting level. z Groups are indicatedas in Table I. 3Periods are indicatedas in Table II. * Significantlydifferentfrom 1 (=fastingvalue). t Significantlydifferent from the respective 3-hour value. $ Significantlydifferentfrom the respective Pre value. # Significantdifferencebetweenthe LO and FO-groups. The postprandialcholesteroland triglycerideresponseswere not altered by linseedoil or fish oil supplementation.Glucose responseremainedalso unchangedin the LO-group but was flattened afler supplementationin the FO-group (Table II). Hematocrit increased or tended to increase slightlyin both groups during postprandialphase before and after supplementation (data not shown). DISCUSSION The mean intake of the supplemented ALA was 6.2 g/day in the LO-group and that of EPA+DHA 5.5 g/day in the FO-group. These intakes are much higher than in young Finnish adults in average (ALA 1.5 g/day and EPA+DHA 0.4 g/day) (13) and were reflected in the changesin plateletlipids. FVII:C increased afler high-fatmeal which is a well-knownphenomenon.The peaking time of FVII:C has been reported to be delayedfrom that of triglycerides(4,6,8). It has been suggested that free fatty acids released from chylomicronsand VLDL during lipoprotein lipase-catalyzed
Vol.85, No. 2
HEMOSTATICFACTORSAND LIPEMIA
151
lipolysisactivate the contact system of coagulation,This leads to activationof factor VII and enhancementof FVII:C (6,7). The increasein FVII:C has been shownto be similarafter meals rich in dairy fat or vegetable oils (4,8,9) but different saturated fatty acids may differ in this respect (14). Our results suggest that n-3 fatty acids in the diet modify the peaking time of FVII:C because the 5-hour value was higher than the 3-hour value in both groups after supplementation. The pattern of this change and the underlying mechanisms remain to be unraveled becauseno data on the time and levelof the peak valuesof activatedFVII, FVII:C or chylomicronand VLDL triglycerides,or the fatty acid compositionof the lipoproteinparticlesis availablefrom the present study. The altered PAI-I response in the FO-group is at least partly an artefact because the fasting levelsbefore supplementationwere low in the FO-group and the detection limit 5 AU/mL was easilyachievedduringthe postprandialphase,PAI-1 activityhas earlierbeen shown to decrease in a similarway after low-fatand high-fatmeals(4,5), and afler mealsrich in either myristicacid or stearicacid(14). Platelet aggregation to ADP was not decreased postprandially in the LO-group after supplementation.Lipemiawas not altered duringlinseedoil supplementationwhich excludesan artefact caused by differentlylipemicsamples,Chylomicronsand VLDL have been reported to decrease platelet aggregation measured by particle-counting method (10) and linseed oil supplementationhas been shownto increasethe proportionof ALA in VLDL triglycerides(15). One could speculate that the ALA supplementation modifies the postprandial plasma environmentand preservesADP-inducedplateletaggregationon the fastinglevel. Fasting glucose concentrationincreased during supplementationperiod in the FO-group. This change may be due to increasedoxidationbecausein one study (16) the increase was seen with capsulescontaining1.5 IU/g of vitaminE but not with capsulescontaining4.5 IU/g of vitaminE. In the present studytocopherolswere added 1.5 mg/g in the linseedoil capsulesand 3.3 mg/g in the fishoil capsulesand a-tocopherollevelsin serumwere not changed(data not shown). In summary,the presentdatademonstratesthatwhen the n-3 fatty acid statusof healthysubjectsis modified either with ALA or EPA+DHA, postprandialresponsesof FVII:C and ADP-induced platelet aggregationare affected.The pattern of the alteredFVII:C responseafler n-3 fatty acid supplementationand the mechansimunderlyingthe changesin platelet aggregationin the LOgroup shouldbe studiedin more detail, Acknowledgements We thank Elina Vahtera, Phil. Lie., and Tom Krusius,MD, from the FinnishRed Cross Blood Transfusion Service for FVII:C and PAI-I activity anaIyses;Lube A/S for the encapsulated supplements and Mildola Ltd., Finland for the sunflower oil. This study was supported by Jenny and Antti WihuriFoundation,The 350th AnniversaryFund@fthelJnivemity of I-Ielsinki, and the Academyof Finland. REFERENCES 1. HARRIS, W.S., CONNOR, W.E., ALAM, N. and ILLINGWORTH, D.R. Reduction of postprandialtriglyceridemiain humansby dietary n-3 fatty acids. J Lipid Res 29, 1451-1460, 1988.
152
HEMOSTATICFACTORSAND LIPEMIA
vol.
85, No. 2
WEINTRAUB, M.S., ZECHNER, R., BROWN, A., EISENBERG, S. and BREWSLOW, J.L. Dietary polyunsaturatedfats of the w-6 and w-3 series reduce postprandial lipoprotein levels.J ClinInvest82, 1884-1893,1988. 3. SILVERSMITH, D.B. Atherogenesis:a postprandialphenomenon.Circulation60, 473-485, 1979. 4. SALOMAA, V., RASI, V., PEKKANEN, J., JAUHIAINEN, M., VAHTERA, E., PIETINEN, P., KORHONEN, H., KUULASMAA, K. and EHNHOLM, C. The effects of saturated fat and n-6 polyunsaturated fat on postprandial Iipemia and hemostatic activity. Atherosclerosis103, 1-11, 1993. 5. MARCKMANN, P., SANDSTROM,B. and JESPERSEN J, Dietary effects on circadian fluctuationin humanblood coagulationfactor VII and fibrinolysis.Atherosclerosis101,225-234, 1993. 6. SILVEIRA, A., KARPE, F,, BLOMBACK, M., STEINER, G., WALLDIUS, G. and HAMSTEN, A. Activation of coagulationfactor VII during alimentarylipemia. Arterioscler Thromb14, 60-69, 1994. 7. MITROPOULOS, K.A., MILLER, G.J., MARTIN, J,C., REEVES, B.E.A. and COOPER, J. Dietary fat induces changes in factor VII coagulant activitythrough effects on plasma free stearicacidconcentration.ArteriosclerThromb1~, 214-222, 1994. 8. MILLER, G.J., MARTIN, J.C., MITROPOULOS,K.A., REEVES, B.E.A., THOMPSON, R.L., MEADE, T.W., COOPER, J.A, and CRUICKSHANK,J.K, Plasmafactor VII is activated by postprandialtriglyceridemia,irrespectiveof dietaryfat composition.Atherosclerosis86, 163171, 1991. 9. FREESE, R. and MUTANEN,M. Postprandialchangesin plateletfinction and coagulation factors after high-fatmealswith differentfatty acid compositions.Eur J ClinNutr 49, 658-664, 1995. 10. ORTH, M., LULEY, C., MAYER, H. and WIELAND,H. Effects of VLDL, chylomicrons, and chylomicronremnantson plateletaggregability.ThrombRes 79,297-305, 1995. 11. MUTANEN, M., FREESE, R., VALSTA, L.M., AHOLA, I. and AHLSTROM, A. Rapeseed oil and sunflower oil diets enhance platelet in vitro aggregation and thromboxane production in healthy men when comparedwith milk fat or habitual diets. Thromb Haemostas 2.
67,352-356,1992. 12. PRADELLES, P., GRASSI, J. and MACLOUF, J. Enzyme immunoassay of eicosanoids
using acetylcholineesterase as label:an alternativeto radioimmunoassay.Anal Chem57, 11701173,1985. 13. KLEEMOL~ P., VIRTANEN, M. and PIETINEN, P. The 1992 dietary survey of finnish adults.Publicationsof the NationalPublicHealth InstituteNo.: B2/1994,Helsinki,1994. 14. THOLSTRUP,T., ANDREASEN,K. and SANDSTROM,B. Acute effect ofhigh-fat meals rich in either stearic and myristicacid on hemostaticfactors in healthyyoung men. Am J Clin Nutr 64, 168-176,1996. 15. LAYNE, K.S., GOH, Y.K., JUMPSEN, J.A., RYAN, E.A., CHOW, P. and CLANDININ, M.T. Normal subjectsconsumingphysiologicallevelsof 18:3(n-3)and 20:5(n-3) from flaxseed and fish oils have characteristicdifferencesin plasma lipid and lipoproteinfatty acid levels. J Nutr 126,2130-2140, 1996. 16. LUOSTARINEN, R., WALLIN, R., WIBELL, L. and SALDEEN T. Vitamin E supplementationcounteractsthe fish oil-inducedincreaseof blood glucosein humans.Nutr Res 15, 953-968, 1995.