Atherosclerosis, 88 (1991) 193-201 0 1991 Elsevier Scientific Publishers ADONIS 0021915091001237
ATHERO
193 Ireland,
Ltd. 0021-9150/91/$03.50
04642
Effects of dietary fish oil supplementation on platelet aggregability and platelet membrane fluidity in normolipemic subjects with and without high plasma Lp( a) concentrations E. Malle I,*, W. Sattler I, E. Prenner 2, H.J. Leis 3, A. Herrnetter and G.M. Kostner ’ ’Institute of Medical Biochemistry, Department
of Mass Spectrometry,
2, A. Gries
’
-‘Institute of Pediatrics, 2 Institute of Biochemistry and Foodchemistry, Karl-Franzens University and Technical Unkersity, Graz (Austria)
(Received 31 May, 1990) (Revised, received 14 February, 1991) (Accepted 15 February, 1991)
Summary
The purpose of this study was to compare the relative effect of n - 3 fatty acids on plasma lipids and platelet function in normolipemic subjects (n = 8) with plasma Lp(a) levels > 30 mg/dl and normolipemic subjects (n = 7) without detectable plasma Lp(a) concentrations. Six weeks of dietary supplementation (3.8 g EPA and 2.9 g DHA/d) significantly reduced (P < 0.005) plasma TGs in both groups whereas no changes of plasma TC, LDL-C, HDL-C, and Lp(a), respectively, were found. Collagen- or thrombin-stimulated platelet aggregation and collagen- or thrombin-induced TXB, generation from platelets decreased by approx. 45% in Lp(a)-negative and Lp(a)-positive platelet donors after a 6 week dietary intake. Four more weeks without n - 3 supplementation restored the pretreatment values of TGs, platelet aggregability and TXB, release. The biophysical properties of platelets from normolipemics with and without high plasma Lp(a) concentrations revealed a similar structural order of platelets at 37°C using DPH, TMA-DPH, or 6-AS as fluorescent probes. Also similar temperature-dependent changes in platelet fluidity from 37°C to 17°C were observed in platelet preparations from Lp(a)-positive and Lp(a)-negative subjects. However, no subtle changes in the structural order of platelets due to nutrient intakes were found in all subjects (n = 15, 19-28 yrs) using fluorescence polarization technique. The present data suggest a similar in vitro platelet behaviour from normolipemic
Correspondence to: Dr. E. Malle, Institute of Medical Biochemistry, Karl-Franzens University, Harrachgasse 21, A8010 Graz. Austria. Tel.: (316) 380-4208; Fax: (316) 38442X.
194 subjects with and without high plasma levels of Lp(a) (which is considered a risk for premature atherosclerosis) in contrast to platelet aggregability and platelet fluidity in certain hyperlipidemic stages.
Key words: II - 3 polyunsaturated fatty acids; Platelet aggregation; Fluorescence fluidity; Lipoprotein(a)
Introduction
Populations that consume a diet rich in marine lipids containing considerable amounts of n - 3 polyunsaturated fatty acids (PUFAS), may have a lower risk of vascular complications [1,2]. As blood platelets are involved in the development and complications of atherosclerosis [3], a decrease of platelet aggregability can, hence, contribute to a reduced formation of atherosclerotic lesions. Both, (i) changes in the platelet membrane lipid composition and in the structural order (commonly defined as lipid fluidity), (ii) an imbalance of bis/trienoic platelet eicosanoids, and (iii) alterations in the fibrinolytic system contribute to changes in platelet function after fish oil consumption. In addition to these findings, a large body of data has reported a hypertriglyceridemic effect in normolipemic and hyperlipidemic subjects [1,2] after dietary intake of unsaturated fatty acids (FAs) such as timnodonic acid (EPA, C20 : 5 [allcis], n - 3) and cervonic acid (DHA, C22: 6 [allcis], n - 3). Little effect or even adverse influence on plasma cholesterol levels due to nutrient intake, however, was also reported [1,2,4]. The influence of IZ- 3 FAs upon plasma lipoprotein(a), Lp(a), is poorly investigated so far. Numerous clinical studies revealed a strong correlation between elevated plasma Lp(a> concentrations (> 30 mg/dl) and cardiovascular disease [5-71. The atherogenic properties of Lp(a) seem not to be restricted to apolipoprotein B-100 (ape B-100) but to the structural homology of its apoprotein, ape(a) to plasminogen [8,91. Based on these findings striking evidence has been put forward that Lp(a1 may play a subtle role in the fibrinolytic system [lo]. As n - 3 PUFA supplementation led to dis-
anisotropy; Membrane
tinct results with respect to changes in plasma lipids and platelet function in both normolipemic and hyperlipidemic subjects, the present study was carried out to compare these changes in normolipemic subjects with and without * high plasma Lp(a) concentrations. To address this subject further we studied platelet membrane fluidity of intact, resting platelets, that is entire platelets from Lp(a)-positive and Lp(a)-negative subjects due to nutrient intakes of PUFAs. Fluorescence anisotropy, generally considered a measure for the lipid order and, hence, inversely related to membrane fluidity [ill was estimated using DPH (1,6-diphenyl-1,3,Shexatriene), TMA-DPH (l[4-trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene) and 6-AS (6-(9-anthroyloxy) stearic acid). These fluorescent probes are of different chemical structure and charge [ll-131 and, hence, considered to label specific membrane domains of living cell systems. Materials
and methods
Subjects and dietary protocol
The subjects were 15 healthy normolipemic male subjects (19-28 yrs, nonsmokers) who had not taken medication or fish 4 weeks prior to and throughout the 10 week study period. In addition to maintaining their normal dietary habits, volunteers consumed fish oil capsules (EPAX-5000 EE, 3.8 g EPA and 2.9 g DHA/d, randomly distributed at meal times) for 6 weeks. The dietary period was followed by 4 weeks without fish oil supplementation. The chemical composition of capsules is presented in Table 1.
*
Lp(a)-negative than 4 mg/dl
subjects refers to plasma Lp(a) levels lower according to Laurel1 electrophoresis.
195 TABLE 1 COMPOSITION AND CHARACTERIZATION CAPSULATED FISH OIL Fatty acid composition total n - 3 FAs EPA DHA monounsaturated FAs Vitamins A D E Various cholesterol peroxide value
OF EN-
65% 32% 24% 5% 1 IU/g 1 IU/g 2 mg/g 0.9 mg/g 6 m&/g
Blood samples
Plastic syringes, tubes and pipettes, or siliconized glass ware were used. Blood samples were taken after an overnight fast at week 0, 2, 6 and 10 discarding the first 3 ml. For analysis of plasma lipids blood was collected in EDTAcoated tubes (Greiner, Austria) and centrifuged for 20 min (1500 x g) at 4°C. For measurements of platelet aggregation and fluorescence anisotropy, blood was collected in l/7 volume acid citrate/glucose solution (93 mM Na, citrate, 7 mM citric acid, 0.14 M o-glucose, pH 6.5). Platelet-rich plasma (PRP) was prepared by centrifugation at 250 X g for 10 min at room temperature [14]. Assays
PRP (8 ml) was applied on a Sepharose 2B column (26 X 2 cm) equilibrated with freshly prepared Ca”-free Tyrode’s solution (137 mM NaCl, 2.68 mM KCI, 0.42 mM NaH,PO,, 1.7 mM MgCl,, 5.6 mM o-glucose, pH 7.35, containing 0.2% human serum albumin). The resulting gelfiltered platelet (GFP) suspension was adjusted to 2 x 10” GFPs/ml using a Coulter Thrombocounter-C system (Coulter Electronics, Ltd., England). Platelet aggregation was performed on an Elvi Logos dual-channel aggregometer (Elvi Logos, Milan, Italy) according to techniques described elsewhere [14]. Collagen (1 and 2 pg/ml GFPs; Hormon Chemie, Munich, F.R.G.) or thrombin (0.1 and 0.2 U/ml GFPs, Sigma, Munich, F.R.G.) were used as agonists.
Platelet TXB, was measured by negative ion chemical ionization-gas chromatography/mass spectrometry as pentafluorobenzyl ester trimethylsilyl ether derivate using ‘*O,-labelled TXB, (prepared as described [153>as internal standard [14]. A Finnigan GC 9610 coupled to a Finnigan 4500 MS with an INCOS data system was used [14,151. The overall steady-state fluorescence anisotropy (r[s]) of intact, resting GFPs was measured in siliconized quartz cuvettes by means of a fluorescence spectrometer (Shimadzu RF 540, Japan) connected to an IBM computer. Platelets prepared by gel-filtration on a Sepharose 2B column (6 x 1.2 cm, equilibrated with albumin-free Tyrode’s solution, pH 7.35) were incubated with DPH (Sigma, Munich, F.R.G.), TMA-DPH or 6-AS (Molecular Probes, Oregon, USA) at 37°C for 1 h. The final concentrations were: fluorophores, 1 PM; platelets, 5 X lo6 GFPs/ml [16,17]. Controls were incubated with a volume of buffer equal to that of the fluorophore solutions added to the test samples. r[s]-Values were calculated from [ll]: Y[S] = fw -
-
GI”” 2GIVH
GE? WH
First and second subscripts of fluorescence intensities (1) refer to the vertical 0’) or horizontal (HI position of the excitation and emission polarizers, respectively. For excitation, a polarized 360 nm (DPH and TMA-DPH) or 380 nm (6-AS) band was used. For each polarizer position 20 readings were automatically recorded. Fluorescence intensities were measured at 430 nm (DPH and TMA-DPH) or 440 nm (6-AS). Monochromator slits were 10 and 30 nm, respectively. Background corrections were made although contribution of light scattering was lower than 3%. Plasma total cholesterol (TC) and triglycerides (TGs) were measured enzymatically with commercial test kits (Biotrol and Biomerieux, France) [7,16]. Low density lipoprotein-cholesterol (LDLC> and high density lipoprotein-cholesterol
196 (HDL-Cl were determined by specific precipitation method (Quantolip-LDL and QuantolipHDL, Immuno, Austria) [7,16]. Plasma Lp(a) concentrations were quantitated by Laurel1 electrophoresis as described [7]. Ape(a) phenotyping was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE, 150 V, 90 min) using discontinuous buffer and gel systems as described [Ml. Proteins were transfered to nitrocellulose (150 mA, 1.5 h) and further incubated with monoclonal anti ape(a) followed by peroxidase-conjugated rabbit anti mouse immunoglobulins (Sigma, F.R.G.). Statistics
Student’s t-test was used to compare Lp(a)negative and Lp(a)-positive subjects. Changes in plasma lipid concentrations were subjected to ANOVA analysis. Results The subjects investigated (n = 15) represented a very uniform group with respect to sex and age, and plasma lipid concentrations of TGs, TC, LDL-C, and HDL-C (Table 2). Plasma Lp(a) concentrations in Lp(a)-positive subjects ranged
TABLE
2
CHANGES IN PLASMA LIPIDS DURING 6 WEEKS OF DIETARY SUPPLEMENTATION (3.8 g EPA AND 2.9 g DNA/D) AND 4 WEEKS WITHOUT DIETARY INTAKE Week 0 A(n=7) TGs TC LDL-C HDL-C B(n = 8) TGs TC LDL-C HDL-C Lp(a)
96.3 k 189.2+ 125.3 + 49.6k
22.6 17.1 13.7 8.3
99.4 f 19.6 184.3k21.1 127.5 * 14.6 50.8+ 11.0 45.1+ 12.2
6
10
73.8 f 19.9 * 191.2+ 19.5 128.3+ 11.9 51.2+ 9.4
95.2 f 186.9 + 122.7+ 52.6+
72.5 f 189.2 k 123.0 k 49.5+ 45.8+
96 182.8 126.4 52.1 45.3
A = Lp(a)-negative platelet donors; donors; all values are expressed * Week 6 vs. week 0: P < 0.005.
20.6 * 19.3 15.2 7.9 11.2
26.5 18.5 15.3 7.9
+ 18.4 k 17.2 & 16.4 f 13.1 rt 12.0
B = Lpfal-positive platelet in mg/lOO ml plasma.
123456709 Fig. 1. Immunoblot of plasma Lp(a) samples from Lp(a)-positive platelet donors (n = 8) after SDS-PAGE (3.75% slab gels) treated with mouse monoclonal anti apofa), followed by peroxidase-conjugated rabbit anti mouse immunoglobulins. One Lp(a)-positive platelet donor (lane 5) could be phenotyped as B-type according to Utermann [18], whereas others (lanes 1-3, 6-9) correspond to different S-types. Lane 4 displays apo(a1 standard from Immuno (Austria) corresponding to different apofa) isoforms (F, Sl, S2, S3).
from 35 to 73 mg/dl. Phenotyping of ape(a) isoforms in this group revealed one B-type and seven S-types (Fig. 1) according to Utermann [ 181. A hypotriglyceridemic effect after 6 weeks of dietary supplementation was observed in all the subjects studied whereas no changes in plasma TC, LDL-C, and HDL-C were found during the 10 week study period (Table 2). In addition, no significant changes of plasma Lp(a) concentrations due to nutrient intakes were observed. Control studies without fish oil consumption showed that plasma Lp(a1 concentrations varied about 8 f 1% in 6 weeks in Lp(a)-positive subjects. Platelet aggregability using collagen (2 pg/ml, Fig. 2A, B) or thrombin (0.2 U/ml, Fig. 2 C, D) as stimulating agents was similar in Lp(a)-negative and Lp(a)-positive subjects prior to supplementation (week 01. Also, the production of TXB, from collagen- or thrombin-stimulated platelets was approx. the same in Lp(a)-positive and Lp(a)-negative subjects (Fig. 3, week 0). These data agree well with our previous findings in normolipemics [14,19] and other reports [20,21]. Platelet aggregability and platelet TXB, release after 2 weeks of dietary intake revealed no significant changes of platelet function in all the subjects studied (Figs. 2 and 3, week 2) indicating no drastic changes in the platelet fatty acid composi-
25
4
weeks
6
weeks
25
4
6
4
weeks Fig. 2. Platelet aggregability after stimulation from Lp(a)-negative (A, C) and Lp(a)-positive 0), after 2 and 6 weeks of n -3 FA intake transmission
6
weeks of 2X 10” GFPs/ml with collagen (2 pg/ml; A, B) or thrombin (0.2 U/ml; C, D) platelet donors (B, D). Aggregations were performed prior to supplementation (week and 4 subsequent weeks without supplementation. Values expressed as 9% of light [14] are given as means of duplicate determinations.
tion with respect to eicosanoid metabolism. However, 6 weeks of IZ- 3 PUFA supplementation led to a pronounced reduction of collagen- or thrombin-induced platelet aggregation by approx. 45% (Fig. 2) and a decrease of collagen- or thrombin-stimulated TXB, generation by approx. 40% (Fig. 3) in Lp(a)-negative and Lp(a)-positive platelet donors. Platelets stimulated with lower concentrations of collagen (0.1 pg/ml) or thrombin (0.1 U/ml) reaveled a similar aggregation pattern in all platelet preparations (data not shown). Four more weeks without dietary supplementation restored the pretreatment values of platelet aggregability (Fig. 2A-D, week 0) and TXB, release (Fig. 3A-D, week 10) in Lp(a)-negative and Lp(a)-positive subjects.
Measuring the overall steady-state fluorescence anisotropy of intact, resting GFPs, no differences in r[s]-values of DPH-, TMA-DPH-, or 6-AS-labelled platelets between Lp(a)-negative and Lp(a)-positive platelet donors were found (Table 3, week 0). Plotting r[s]-DPH values as a function of temperature a striking relation between membrane fluidity and temperature was observed in Lp(a)-positive (n = 4) and Lp(a)negative subjects (n = 5). Fig. 4 displays a typical example of only one platelet donor. Analysis of the data could be interpreted in terms of a straight line with a correlation coefficient r = 0.99. The r[ s]-values of DPH-labelled GFPs measured from 37°C to 17°C revealed slope values ranging from 1.943ep3” to 2.564e-3” for the above mentioned platelet donors (n = 9). Using TMA-DPH or 6-AS
4
6
4
weeks
4
6 weeks
6
4
weeks
6 weeks
Fig. 3. TXB, formation in Lp(a)-negative (A, CI and Lp(a)-positive platelet donors (B, D) after stimulation of 2 X 10’ GFPs/ml with collagen (2 pg/mI; A, B) or thrombin (0.2 U/ml; C, D). Aggregations were performed prior to supplementation (week O), after 2 and 6 weeks of n - 3 FA intake and 4 subsequent weeks without FA supplementation. Values are expressed in rig/l x lo* GFPs.
TABLE 3 FLUORESCENCE ANISOTROPY THE 10 WEEK STUDY PERIOD
VALUES
DURING
T[S]values week 0
week 6
week 10
A DPH TMA-DPH 6-AS
0.1041+ 0.0075 0.1045 f 0.0089 0.1046 + 0.0095 0.2239 f 0.0141 0.2304 + 0.0112 0.2247 f 0.0139 0.0690 + 0.0062 0.0687 f 0.0034 0.0689 + 0.0036
B DPH TMA-DPH 6-AS
0.1048 + 0.0071 0.1050+ 0.0061 0.1049 kO.0041 0.2214 + 0.0119 0.2194 f 0.0124 0.2205 f 0.0134 0.0686 rf-0.0043 0.0683 + 0.0054 0.0686 + 0.0031
0,ofl 10
I 15
.
I 20
-
, 25
.
, 30
temperature
A = Lp(a)-negative platelet donors (n = 7); B = Lp(a)-positive platelet donors (n = 8).
.I., 35
40
OC
Fig. 4. Plotting r[s]-DPH values from intact, resting GFPs (5 X 106/mlI as a function of temperature (slope of T[S] vs temperature: 2.2542em3”). For detailed measurement and incubation procedure see Methods. Values are given as means of 5-7 determinations within 3-4 min.
199 as fluorescent probes, no temperature dependency of membrane fluidity was found (data not shown). In addition, no subtle changes of platelet fluidity after 2 weeks (data not shown) and 6 weeks of dietary supplementation, and subsequent 4 weeks without n - 3 FA intake could be observed in all the subjects studied (n = 15) using DPH, TMA-DPH or 6-AS as fluorescent dyes (Table 3). Discussion
The major findings of the present study were that (1) platelet aggregation and platelet TXB, formation was similar in Lp(a)-negative and Lp(a)-positive platelet donors before and after dietary supplementation, (2) the shift from IZ- 6 (arachidonic acid, AA, C20: 5 [all-&]) to n - 3 (EPA, C20 : 4 [all-cis]), i.e. one additional double bond in acyl chains of platelet membrane phospholipids, had only marginal influence on platelet fluidity, and that (3) plasma Lp(a) concentrations were not affected due to y1- 3 nutrient intakes. The latter finding is obviously the facet that EPA inhibits more likely (1) hepatic production of TGs, (2) secretion of very low density lipoproteins, and (3) post heparin lipolytic activity [1,2], than the formation of apo B-100 rich lipoproteins, i.e. LDL and Lp(a). The amount of fish oil taken per day, the source of n - 3 FAs, and the study duration led to distinct results with respect to platelet function in normolipidemic and hyperlipidemic subjects [20-321. Studies similarly designed to our dietary protocol revealed either reduced or unchanged platelet aggregation patterns using different concentrations of the stimulating agents, i.e. collagen, thrombin, ADP, AA, or platelet-activating factor [20-321. Most of the studies performed have investigated platelet function in normolipemic [27-321 or hyperlipidemic subjects [20,21,25,26] whereas fewer studies compared both 121,261. We therefore have investigated dietary-induced changes in platelet behaviour and plasma lipids in 15 normolipemics of similar age including 8 subjects with a possible risk for premature atherosclerosis [5-71. As gel-filtration of platelets avoids morphological alterations during platelet
isolation [33], and cis-PUFAs (present in PRP during dietary supplementation) are further known to modulate (i) platelet function, (ii> receptor activity and (iii) membrane fluidity [34,351 we have studied aggregability and fluidity of platelets prepared by the same technique. Neither spontaneous platelet aggregability nor enhanced TXB,-release was observed in platelets from Lp(a)-positive subjects in the present study in contrast to in vitro platelet function in hyperlipidemic patients [36]. Although II - 3 dietary intakes lead to changes in the FA acyl chains of platelet membrane phospholipids [28,29] located at the outer and inner platelet membrane leaflet, TMA-DPH (remaining anchored at the cell surface during labelling experiments), 6-AS (located in the membrane bilayer centre), and DPH (embedded more deeply in the lipid core> showed no subtle changes in platelet fluidity during dietary supplementation in humans (Table 3) [29]. Even membrane fluidity of human erythrocytes, measured by fluorescence polarization and electron spin resonance technique was not altered during n - 3 dietary intake [37]. Feeding animals various diets, platelet fluidity was also not uniformly related to the degree of unsaturated FAs of membrane phospholipids 138-411. All these reports [29,36-401 and our present findings using 3 different fluorescent probes indicate that fluorescence spectroscopy is a less sensitive technique compared to platelet aggregability for revealing changes in membrane dynamics due to nutrient intakes. However, the same fluorescent probes (TMADPH and DPH) are able to reveal subtle differences in the overall steady-state fluorescence anisotropy from both unsealed and resealed platelet membranes as well as resting, intact GFPs from hyperlipidemic and normolipemic subjects [17,42]. Plotting r[s]-DPH values as a function of temperature, slope-values of DPH-labelled platelets from type IIB and type IV patients were slightly but definitively different in comparison to slope values observed in normolipemic controls or type IIA patients [171. In the present study we found a similar platelet membrane fluidity (Table 3) and similar slope values (Fig. 4) of DPHlabelled platelets from Lp(a)-positive platelet donors and normolipemic controls indicating that
200 platelet structural order in Lp(a)-positive subjects contrasts to platelet structural order observed in hyperlipidemic patients [ 171. In addition, the similar in vitro platelet aggregability of platelets from Lp(a)-positive subjects compared to normolipemic platelet donors without measurable plasma Lp(a) levels (Figs. 2, 3) confirm that in vitro platelet function in Lp(a)-positive subjects is more likely comparable to platelet function in normolipemics than platelet function in hyperlipemic patients [20,21,25,26]. Whether Lp(a) can exert subtle changes in platelet activation as reported for atherogenic and antiatherogenic lipoproteins [36] and whether Lp(a) is able to bind to platelets is the subject of current investigation. Acknowledgements This work was supported by grants from FWF (8249, S46-07, S46-15, 8013) and the F. Lanyar Foundation, Austria. The authors wish to thank H. Dieplinger (Innsbruck) and Immuno AG (Austria) for providing monoclonal anti ape(a) and ape(a) phenotyping standard, and M. Jacob (Hofphar, Sulzbach) for providing fish oil capsules. References Harris, W.S., Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review. J. Lipid Res., 30 (1989) 785. Grundy, S.M. and Denke, M.A., Dietary influences on serum lipids and lipoproteins. J. Lipid Res., 31, (1990) 1149. Packham, M.A. and Mustard, J.F., The role of platelets in the development and complications of atherosclerosis. Sem. Hematol., 23 (1986) 8. Dart, A.M., Riemersma, R.A. and Oliver, M.F., Effects of Maxepa on serum lipids in hypercholesterolaemic subjects. Atherosclerosis, 80 (1990) 119. Kostner, G.M., Avogaro, P., Cazzolato, G., Marth, E., Bittolo-Bon, G. and Quinci, R.B., Lipoprotein Lp(a) and the risk for myocardial infarction. Atherosclerosis, 38 (1982) 51. 6 Rhoads, G.G., Dahlen, G., Berg, K., Morton, N.E. and Dannenberg, A.L., Lp(a) lipoprotein as a risk factor for myocardial infarction. J. Am. Med. Assoc., 256 (1986) 2540. 7 Hiifler, G., Harnoncourt, F., Paschke, E., Mirtl, W., Pfeiffer, K.H. and Kostner, G.M., Lipoprotein Lp[a]: a risk
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and eicosapentaenoic acids on prostaglandin biosynthesis and platelet function in man. Klin. Wochenschr.. 64 (1986) 274. Groscurth, P., Cheng, S., Vollenweider, I. and von Felton, A., Effects of washing and gel filtration on the ultrastructure of human platelets. Acta Haematol., 77 (1987) 150. Kitagawa, S., Kotani, K. and Kametani, F.. Inhibitory mechanism of c&-polyunsaturated fatty acids on platelet aggregation: the relation with their effects on Ca*+ mobicyclic AMP levels and membrane fluidity. lization, Biochim. Biophys. Acta, 1054 (1990) 114. Swann, P.G., Venton, D.L. and Le Breton, G.C., Eicosapentaenioc acid and docosahexaenoic acid are antagonists at the thromboxane A,/prostaglandin H, receptor in human platelets. FEBS Lett., 243 (1990) 244. Bruckdorfer, K.R.. The effect of plasma lipoproteins on platelet responsiveness and on platelet and vascular prostanoid synthesis. Prostaglandins Leukotrienes Essent. Fatty Acids, 38 (1989) 247. Popp-Snijders, C., Schouten, J.A., van Blitterswijk, W.J. and van der Veen, E.A., Changes in membrane lipid composition of human erythrocytes after dietary supplementation of (n - 3)poIyunsaturated fatty acids. Maintenance of membrane fluidity. Biochim. Biophys. Acta, 854 (1986) 31. Berlin, E., Shapiro, S.G. and Friedland, M., Platelet membrane fluidity and aggregation of rabbit platelets. Atherosclerosis, 51 (1984) 223. Berlin, E., Shapiro, S.G. and Kliman, P.G., Influence of saturated and unsaturated fats on platelet fatty acids in cholesterol-fed rabbits. Atherosclerosis, 63 (19871 85. Rand, M.L, Hennissen, A.A.H.M. and Hornstra, G., Effects of dietary palm oil on arterial thrombosis, platelet responses and platelet membrane fluidity in rats. Lipids, 23 (1988) 1019. Heemskerk, J.W.M., Feijge, M.A.H., Kalafusz, R. and Hornstra, G., Influence of dietary fatty acids on membrane fluidity and activation of rat platelets. Biochim. Biophys. Acta, 1004 (1989) 252. Muller, S., Ziegler, O., Donner, M., Drouin, P. and Stoltz, J.F., Rheological properties and membrane fluidity of red blood cells and platelets in hyperlipoproteinemia. Atherosclerosis, 83 (1990) 231.
ATHERO
193 Ireland,
Ltd. 0021-9150/91/$03.50
04642
Effects of dietary fish oil supplementation on platelet aggregability and platelet membrane fluidity in normolipemic subjects with and without high plasma Lp( a) concentrations E. Malle I,*, W. Sattler I, E. Prenner 2, H.J. Leis 3, A. Herrnetter and G.M. Kostner ’ ’Institute of Medical Biochemistry, Department
of Mass Spectrometry,
2, A. Gries
’
-‘Institute of Pediatrics, 2 Institute of Biochemistry and Foodchemistry, Karl-Franzens University and Technical Unkersity, Graz (Austria)
(Received 31 May, 1990) (Revised, received 14 February, 1991) (Accepted 15 February, 1991)
Summary
The purpose of this study was to compare the relative effect of n - 3 fatty acids on plasma lipids and platelet function in normolipemic subjects (n = 8) with plasma Lp(a) levels > 30 mg/dl and normolipemic subjects (n = 7) without detectable plasma Lp(a) concentrations. Six weeks of dietary supplementation (3.8 g EPA and 2.9 g DHA/d) significantly reduced (P < 0.005) plasma TGs in both groups whereas no changes of plasma TC, LDL-C, HDL-C, and Lp(a), respectively, were found. Collagen- or thrombin-stimulated platelet aggregation and collagen- or thrombin-induced TXB, generation from platelets decreased by approx. 45% in Lp(a)-negative and Lp(a)-positive platelet donors after a 6 week dietary intake. Four more weeks without n - 3 supplementation restored the pretreatment values of TGs, platelet aggregability and TXB, release. The biophysical properties of platelets from normolipemics with and without high plasma Lp(a) concentrations revealed a similar structural order of platelets at 37°C using DPH, TMA-DPH, or 6-AS as fluorescent probes. Also similar temperature-dependent changes in platelet fluidity from 37°C to 17°C were observed in platelet preparations from Lp(a)-positive and Lp(a)-negative subjects. However, no subtle changes in the structural order of platelets due to nutrient intakes were found in all subjects (n = 15, 19-28 yrs) using fluorescence polarization technique. The present data suggest a similar in vitro platelet behaviour from normolipemic
Correspondence to: Dr. E. Malle, Institute of Medical Biochemistry, Karl-Franzens University, Harrachgasse 21, A8010 Graz. Austria. Tel.: (316) 380-4208; Fax: (316) 38442X.
194 subjects with and without high plasma levels of Lp(a) (which is considered a risk for premature atherosclerosis) in contrast to platelet aggregability and platelet fluidity in certain hyperlipidemic stages.
Key words: II - 3 polyunsaturated fatty acids; Platelet aggregation; Fluorescence fluidity; Lipoprotein(a)
Introduction
Populations that consume a diet rich in marine lipids containing considerable amounts of n - 3 polyunsaturated fatty acids (PUFAS), may have a lower risk of vascular complications [1,2]. As blood platelets are involved in the development and complications of atherosclerosis [3], a decrease of platelet aggregability can, hence, contribute to a reduced formation of atherosclerotic lesions. Both, (i) changes in the platelet membrane lipid composition and in the structural order (commonly defined as lipid fluidity), (ii) an imbalance of bis/trienoic platelet eicosanoids, and (iii) alterations in the fibrinolytic system contribute to changes in platelet function after fish oil consumption. In addition to these findings, a large body of data has reported a hypertriglyceridemic effect in normolipemic and hyperlipidemic subjects [1,2] after dietary intake of unsaturated fatty acids (FAs) such as timnodonic acid (EPA, C20 : 5 [allcis], n - 3) and cervonic acid (DHA, C22: 6 [allcis], n - 3). Little effect or even adverse influence on plasma cholesterol levels due to nutrient intake, however, was also reported [1,2,4]. The influence of IZ- 3 FAs upon plasma lipoprotein(a), Lp(a), is poorly investigated so far. Numerous clinical studies revealed a strong correlation between elevated plasma Lp(a> concentrations (> 30 mg/dl) and cardiovascular disease [5-71. The atherogenic properties of Lp(a) seem not to be restricted to apolipoprotein B-100 (ape B-100) but to the structural homology of its apoprotein, ape(a) to plasminogen [8,91. Based on these findings striking evidence has been put forward that Lp(a1 may play a subtle role in the fibrinolytic system [lo]. As n - 3 PUFA supplementation led to dis-
anisotropy; Membrane
tinct results with respect to changes in plasma lipids and platelet function in both normolipemic and hyperlipidemic subjects, the present study was carried out to compare these changes in normolipemic subjects with and without * high plasma Lp(a) concentrations. To address this subject further we studied platelet membrane fluidity of intact, resting platelets, that is entire platelets from Lp(a)-positive and Lp(a)-negative subjects due to nutrient intakes of PUFAs. Fluorescence anisotropy, generally considered a measure for the lipid order and, hence, inversely related to membrane fluidity [ill was estimated using DPH (1,6-diphenyl-1,3,Shexatriene), TMA-DPH (l[4-trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene) and 6-AS (6-(9-anthroyloxy) stearic acid). These fluorescent probes are of different chemical structure and charge [ll-131 and, hence, considered to label specific membrane domains of living cell systems. Materials
and methods
Subjects and dietary protocol
The subjects were 15 healthy normolipemic male subjects (19-28 yrs, nonsmokers) who had not taken medication or fish 4 weeks prior to and throughout the 10 week study period. In addition to maintaining their normal dietary habits, volunteers consumed fish oil capsules (EPAX-5000 EE, 3.8 g EPA and 2.9 g DHA/d, randomly distributed at meal times) for 6 weeks. The dietary period was followed by 4 weeks without fish oil supplementation. The chemical composition of capsules is presented in Table 1.
*
Lp(a)-negative than 4 mg/dl
subjects refers to plasma Lp(a) levels lower according to Laurel1 electrophoresis.
195 TABLE 1 COMPOSITION AND CHARACTERIZATION CAPSULATED FISH OIL Fatty acid composition total n - 3 FAs EPA DHA monounsaturated FAs Vitamins A D E Various cholesterol peroxide value
OF EN-
65% 32% 24% 5% 1 IU/g 1 IU/g 2 mg/g 0.9 mg/g 6 m&/g
Blood samples
Plastic syringes, tubes and pipettes, or siliconized glass ware were used. Blood samples were taken after an overnight fast at week 0, 2, 6 and 10 discarding the first 3 ml. For analysis of plasma lipids blood was collected in EDTAcoated tubes (Greiner, Austria) and centrifuged for 20 min (1500 x g) at 4°C. For measurements of platelet aggregation and fluorescence anisotropy, blood was collected in l/7 volume acid citrate/glucose solution (93 mM Na, citrate, 7 mM citric acid, 0.14 M o-glucose, pH 6.5). Platelet-rich plasma (PRP) was prepared by centrifugation at 250 X g for 10 min at room temperature [14]. Assays
PRP (8 ml) was applied on a Sepharose 2B column (26 X 2 cm) equilibrated with freshly prepared Ca”-free Tyrode’s solution (137 mM NaCl, 2.68 mM KCI, 0.42 mM NaH,PO,, 1.7 mM MgCl,, 5.6 mM o-glucose, pH 7.35, containing 0.2% human serum albumin). The resulting gelfiltered platelet (GFP) suspension was adjusted to 2 x 10” GFPs/ml using a Coulter Thrombocounter-C system (Coulter Electronics, Ltd., England). Platelet aggregation was performed on an Elvi Logos dual-channel aggregometer (Elvi Logos, Milan, Italy) according to techniques described elsewhere [14]. Collagen (1 and 2 pg/ml GFPs; Hormon Chemie, Munich, F.R.G.) or thrombin (0.1 and 0.2 U/ml GFPs, Sigma, Munich, F.R.G.) were used as agonists.
Platelet TXB, was measured by negative ion chemical ionization-gas chromatography/mass spectrometry as pentafluorobenzyl ester trimethylsilyl ether derivate using ‘*O,-labelled TXB, (prepared as described [153>as internal standard [14]. A Finnigan GC 9610 coupled to a Finnigan 4500 MS with an INCOS data system was used [14,151. The overall steady-state fluorescence anisotropy (r[s]) of intact, resting GFPs was measured in siliconized quartz cuvettes by means of a fluorescence spectrometer (Shimadzu RF 540, Japan) connected to an IBM computer. Platelets prepared by gel-filtration on a Sepharose 2B column (6 x 1.2 cm, equilibrated with albumin-free Tyrode’s solution, pH 7.35) were incubated with DPH (Sigma, Munich, F.R.G.), TMA-DPH or 6-AS (Molecular Probes, Oregon, USA) at 37°C for 1 h. The final concentrations were: fluorophores, 1 PM; platelets, 5 X lo6 GFPs/ml [16,17]. Controls were incubated with a volume of buffer equal to that of the fluorophore solutions added to the test samples. r[s]-Values were calculated from [ll]: Y[S] = fw -
-
GI”” 2GIVH
GE? WH
First and second subscripts of fluorescence intensities (1) refer to the vertical 0’) or horizontal (HI position of the excitation and emission polarizers, respectively. For excitation, a polarized 360 nm (DPH and TMA-DPH) or 380 nm (6-AS) band was used. For each polarizer position 20 readings were automatically recorded. Fluorescence intensities were measured at 430 nm (DPH and TMA-DPH) or 440 nm (6-AS). Monochromator slits were 10 and 30 nm, respectively. Background corrections were made although contribution of light scattering was lower than 3%. Plasma total cholesterol (TC) and triglycerides (TGs) were measured enzymatically with commercial test kits (Biotrol and Biomerieux, France) [7,16]. Low density lipoprotein-cholesterol (LDLC> and high density lipoprotein-cholesterol
196 (HDL-Cl were determined by specific precipitation method (Quantolip-LDL and QuantolipHDL, Immuno, Austria) [7,16]. Plasma Lp(a) concentrations were quantitated by Laurel1 electrophoresis as described [7]. Ape(a) phenotyping was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE, 150 V, 90 min) using discontinuous buffer and gel systems as described [Ml. Proteins were transfered to nitrocellulose (150 mA, 1.5 h) and further incubated with monoclonal anti ape(a) followed by peroxidase-conjugated rabbit anti mouse immunoglobulins (Sigma, F.R.G.). Statistics
Student’s t-test was used to compare Lp(a)negative and Lp(a)-positive subjects. Changes in plasma lipid concentrations were subjected to ANOVA analysis. Results The subjects investigated (n = 15) represented a very uniform group with respect to sex and age, and plasma lipid concentrations of TGs, TC, LDL-C, and HDL-C (Table 2). Plasma Lp(a) concentrations in Lp(a)-positive subjects ranged
TABLE
2
CHANGES IN PLASMA LIPIDS DURING 6 WEEKS OF DIETARY SUPPLEMENTATION (3.8 g EPA AND 2.9 g DNA/D) AND 4 WEEKS WITHOUT DIETARY INTAKE Week 0 A(n=7) TGs TC LDL-C HDL-C B(n = 8) TGs TC LDL-C HDL-C Lp(a)
96.3 k 189.2+ 125.3 + 49.6k
22.6 17.1 13.7 8.3
99.4 f 19.6 184.3k21.1 127.5 * 14.6 50.8+ 11.0 45.1+ 12.2
6
10
73.8 f 19.9 * 191.2+ 19.5 128.3+ 11.9 51.2+ 9.4
95.2 f 186.9 + 122.7+ 52.6+
72.5 f 189.2 k 123.0 k 49.5+ 45.8+
96 182.8 126.4 52.1 45.3
A = Lp(a)-negative platelet donors; donors; all values are expressed * Week 6 vs. week 0: P < 0.005.
20.6 * 19.3 15.2 7.9 11.2
26.5 18.5 15.3 7.9
+ 18.4 k 17.2 & 16.4 f 13.1 rt 12.0
B = Lpfal-positive platelet in mg/lOO ml plasma.
123456709 Fig. 1. Immunoblot of plasma Lp(a) samples from Lp(a)-positive platelet donors (n = 8) after SDS-PAGE (3.75% slab gels) treated with mouse monoclonal anti apofa), followed by peroxidase-conjugated rabbit anti mouse immunoglobulins. One Lp(a)-positive platelet donor (lane 5) could be phenotyped as B-type according to Utermann [18], whereas others (lanes 1-3, 6-9) correspond to different S-types. Lane 4 displays apo(a1 standard from Immuno (Austria) corresponding to different apofa) isoforms (F, Sl, S2, S3).
from 35 to 73 mg/dl. Phenotyping of ape(a) isoforms in this group revealed one B-type and seven S-types (Fig. 1) according to Utermann [ 181. A hypotriglyceridemic effect after 6 weeks of dietary supplementation was observed in all the subjects studied whereas no changes in plasma TC, LDL-C, and HDL-C were found during the 10 week study period (Table 2). In addition, no significant changes of plasma Lp(a) concentrations due to nutrient intakes were observed. Control studies without fish oil consumption showed that plasma Lp(a1 concentrations varied about 8 f 1% in 6 weeks in Lp(a)-positive subjects. Platelet aggregability using collagen (2 pg/ml, Fig. 2A, B) or thrombin (0.2 U/ml, Fig. 2 C, D) as stimulating agents was similar in Lp(a)-negative and Lp(a)-positive subjects prior to supplementation (week 01. Also, the production of TXB, from collagen- or thrombin-stimulated platelets was approx. the same in Lp(a)-positive and Lp(a)-negative subjects (Fig. 3, week 0). These data agree well with our previous findings in normolipemics [14,19] and other reports [20,21]. Platelet aggregability and platelet TXB, release after 2 weeks of dietary intake revealed no significant changes of platelet function in all the subjects studied (Figs. 2 and 3, week 2) indicating no drastic changes in the platelet fatty acid composi-
25
4
weeks
6
weeks
25
4
6
4
weeks Fig. 2. Platelet aggregability after stimulation from Lp(a)-negative (A, C) and Lp(a)-positive 0), after 2 and 6 weeks of n -3 FA intake transmission
6
weeks of 2X 10” GFPs/ml with collagen (2 pg/ml; A, B) or thrombin (0.2 U/ml; C, D) platelet donors (B, D). Aggregations were performed prior to supplementation (week and 4 subsequent weeks without supplementation. Values expressed as 9% of light [14] are given as means of duplicate determinations.
tion with respect to eicosanoid metabolism. However, 6 weeks of IZ- 3 PUFA supplementation led to a pronounced reduction of collagen- or thrombin-induced platelet aggregation by approx. 45% (Fig. 2) and a decrease of collagen- or thrombin-stimulated TXB, generation by approx. 40% (Fig. 3) in Lp(a)-negative and Lp(a)-positive platelet donors. Platelets stimulated with lower concentrations of collagen (0.1 pg/ml) or thrombin (0.1 U/ml) reaveled a similar aggregation pattern in all platelet preparations (data not shown). Four more weeks without dietary supplementation restored the pretreatment values of platelet aggregability (Fig. 2A-D, week 0) and TXB, release (Fig. 3A-D, week 10) in Lp(a)-negative and Lp(a)-positive subjects.
Measuring the overall steady-state fluorescence anisotropy of intact, resting GFPs, no differences in r[s]-values of DPH-, TMA-DPH-, or 6-AS-labelled platelets between Lp(a)-negative and Lp(a)-positive platelet donors were found (Table 3, week 0). Plotting r[s]-DPH values as a function of temperature a striking relation between membrane fluidity and temperature was observed in Lp(a)-positive (n = 4) and Lp(a)negative subjects (n = 5). Fig. 4 displays a typical example of only one platelet donor. Analysis of the data could be interpreted in terms of a straight line with a correlation coefficient r = 0.99. The r[ s]-values of DPH-labelled GFPs measured from 37°C to 17°C revealed slope values ranging from 1.943ep3” to 2.564e-3” for the above mentioned platelet donors (n = 9). Using TMA-DPH or 6-AS
4
6
4
weeks
4
6 weeks
6
4
weeks
6 weeks
Fig. 3. TXB, formation in Lp(a)-negative (A, CI and Lp(a)-positive platelet donors (B, D) after stimulation of 2 X 10’ GFPs/ml with collagen (2 pg/mI; A, B) or thrombin (0.2 U/ml; C, D). Aggregations were performed prior to supplementation (week O), after 2 and 6 weeks of n - 3 FA intake and 4 subsequent weeks without FA supplementation. Values are expressed in rig/l x lo* GFPs.
TABLE 3 FLUORESCENCE ANISOTROPY THE 10 WEEK STUDY PERIOD
VALUES
DURING
T[S]values week 0
week 6
week 10
A DPH TMA-DPH 6-AS
0.1041+ 0.0075 0.1045 f 0.0089 0.1046 + 0.0095 0.2239 f 0.0141 0.2304 + 0.0112 0.2247 f 0.0139 0.0690 + 0.0062 0.0687 f 0.0034 0.0689 + 0.0036
B DPH TMA-DPH 6-AS
0.1048 + 0.0071 0.1050+ 0.0061 0.1049 kO.0041 0.2214 + 0.0119 0.2194 f 0.0124 0.2205 f 0.0134 0.0686 rf-0.0043 0.0683 + 0.0054 0.0686 + 0.0031
0,ofl 10
I 15
.
I 20
-
, 25
.
, 30
temperature
A = Lp(a)-negative platelet donors (n = 7); B = Lp(a)-positive platelet donors (n = 8).
.I., 35
40
OC
Fig. 4. Plotting r[s]-DPH values from intact, resting GFPs (5 X 106/mlI as a function of temperature (slope of T[S] vs temperature: 2.2542em3”). For detailed measurement and incubation procedure see Methods. Values are given as means of 5-7 determinations within 3-4 min.
199 as fluorescent probes, no temperature dependency of membrane fluidity was found (data not shown). In addition, no subtle changes of platelet fluidity after 2 weeks (data not shown) and 6 weeks of dietary supplementation, and subsequent 4 weeks without n - 3 FA intake could be observed in all the subjects studied (n = 15) using DPH, TMA-DPH or 6-AS as fluorescent dyes (Table 3). Discussion
The major findings of the present study were that (1) platelet aggregation and platelet TXB, formation was similar in Lp(a)-negative and Lp(a)-positive platelet donors before and after dietary supplementation, (2) the shift from IZ- 6 (arachidonic acid, AA, C20: 5 [all-&]) to n - 3 (EPA, C20 : 4 [all-cis]), i.e. one additional double bond in acyl chains of platelet membrane phospholipids, had only marginal influence on platelet fluidity, and that (3) plasma Lp(a) concentrations were not affected due to y1- 3 nutrient intakes. The latter finding is obviously the facet that EPA inhibits more likely (1) hepatic production of TGs, (2) secretion of very low density lipoproteins, and (3) post heparin lipolytic activity [1,2], than the formation of apo B-100 rich lipoproteins, i.e. LDL and Lp(a). The amount of fish oil taken per day, the source of n - 3 FAs, and the study duration led to distinct results with respect to platelet function in normolipidemic and hyperlipidemic subjects [20-321. Studies similarly designed to our dietary protocol revealed either reduced or unchanged platelet aggregation patterns using different concentrations of the stimulating agents, i.e. collagen, thrombin, ADP, AA, or platelet-activating factor [20-321. Most of the studies performed have investigated platelet function in normolipemic [27-321 or hyperlipidemic subjects [20,21,25,26] whereas fewer studies compared both 121,261. We therefore have investigated dietary-induced changes in platelet behaviour and plasma lipids in 15 normolipemics of similar age including 8 subjects with a possible risk for premature atherosclerosis [5-71. As gel-filtration of platelets avoids morphological alterations during platelet
isolation [33], and cis-PUFAs (present in PRP during dietary supplementation) are further known to modulate (i) platelet function, (ii> receptor activity and (iii) membrane fluidity [34,351 we have studied aggregability and fluidity of platelets prepared by the same technique. Neither spontaneous platelet aggregability nor enhanced TXB,-release was observed in platelets from Lp(a)-positive subjects in the present study in contrast to in vitro platelet function in hyperlipidemic patients [36]. Although II - 3 dietary intakes lead to changes in the FA acyl chains of platelet membrane phospholipids [28,29] located at the outer and inner platelet membrane leaflet, TMA-DPH (remaining anchored at the cell surface during labelling experiments), 6-AS (located in the membrane bilayer centre), and DPH (embedded more deeply in the lipid core> showed no subtle changes in platelet fluidity during dietary supplementation in humans (Table 3) [29]. Even membrane fluidity of human erythrocytes, measured by fluorescence polarization and electron spin resonance technique was not altered during n - 3 dietary intake [37]. Feeding animals various diets, platelet fluidity was also not uniformly related to the degree of unsaturated FAs of membrane phospholipids 138-411. All these reports [29,36-401 and our present findings using 3 different fluorescent probes indicate that fluorescence spectroscopy is a less sensitive technique compared to platelet aggregability for revealing changes in membrane dynamics due to nutrient intakes. However, the same fluorescent probes (TMADPH and DPH) are able to reveal subtle differences in the overall steady-state fluorescence anisotropy from both unsealed and resealed platelet membranes as well as resting, intact GFPs from hyperlipidemic and normolipemic subjects [17,42]. Plotting r[s]-DPH values as a function of temperature, slope-values of DPH-labelled platelets from type IIB and type IV patients were slightly but definitively different in comparison to slope values observed in normolipemic controls or type IIA patients [171. In the present study we found a similar platelet membrane fluidity (Table 3) and similar slope values (Fig. 4) of DPHlabelled platelets from Lp(a)-positive platelet donors and normolipemic controls indicating that
200 platelet structural order in Lp(a)-positive subjects contrasts to platelet structural order observed in hyperlipidemic patients [ 171. In addition, the similar in vitro platelet aggregability of platelets from Lp(a)-positive subjects compared to normolipemic platelet donors without measurable plasma Lp(a) levels (Figs. 2, 3) confirm that in vitro platelet function in Lp(a)-positive subjects is more likely comparable to platelet function in normolipemics than platelet function in hyperlipemic patients [20,21,25,26]. Whether Lp(a) can exert subtle changes in platelet activation as reported for atherogenic and antiatherogenic lipoproteins [36] and whether Lp(a) is able to bind to platelets is the subject of current investigation. Acknowledgements This work was supported by grants from FWF (8249, S46-07, S46-15, 8013) and the F. Lanyar Foundation, Austria. The authors wish to thank H. Dieplinger (Innsbruck) and Immuno AG (Austria) for providing monoclonal anti ape(a) and ape(a) phenotyping standard, and M. Jacob (Hofphar, Sulzbach) for providing fish oil capsules. References Harris, W.S., Fish oils and plasma lipid and lipoprotein metabolism in humans: a critical review. J. Lipid Res., 30 (1989) 785. Grundy, S.M. and Denke, M.A., Dietary influences on serum lipids and lipoproteins. J. Lipid Res., 31, (1990) 1149. Packham, M.A. and Mustard, J.F., The role of platelets in the development and complications of atherosclerosis. Sem. Hematol., 23 (1986) 8. Dart, A.M., Riemersma, R.A. and Oliver, M.F., Effects of Maxepa on serum lipids in hypercholesterolaemic subjects. Atherosclerosis, 80 (1990) 119. Kostner, G.M., Avogaro, P., Cazzolato, G., Marth, E., Bittolo-Bon, G. and Quinci, R.B., Lipoprotein Lp(a) and the risk for myocardial infarction. Atherosclerosis, 38 (1982) 51. 6 Rhoads, G.G., Dahlen, G., Berg, K., Morton, N.E. and Dannenberg, A.L., Lp(a) lipoprotein as a risk factor for myocardial infarction. J. Am. Med. Assoc., 256 (1986) 2540. 7 Hiifler, G., Harnoncourt, F., Paschke, E., Mirtl, W., Pfeiffer, K.H. and Kostner, G.M., Lipoprotein Lp[a]: a risk
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