Platelet aggregation in pigs fed diets containing anhydrous milkfat, fish oil or hydrogenated coconut oil

Nutrition Research 22 (2002) 1281–1298 www.elsevier.com/locate/nutres

Platelet aggregation in pigs fed diets containing anhydrous milkfat, fish oil or hydrogenated coconut oil Kerry A.C. Jamesa,*, Keith G. Thompsonb, Alison J. Maccollc, Christine L. Boothc, Denis R. Bodya, Yuliy Y. Chirkovd, Ruth C. Butlera, Paul J. Moughane, Wilhelm F. Lubbef a

b

New Zealand Institute for Crop & Food Research Limited, Palmerston North, New Zealand Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand c New Zealand Dairy Research Institute, Palmerston North, New Zealand d Department of Cardiology, Queen Elizabeth Hospital, Adelaide, South Australia, Australia e Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand f Department of Medicine, School of Medicine, University of Auckland, Auckland, New Zealand Received 10 January 2002; received in revised form 20 June 2002; accepted 24 June 2002

Abstract The purpose of the study was to measure platelet aggregation and fatty acid composition for pigs fed diets containing different fats/oils, and to compare platelet aggregation with empirical indices calculated from the fatty acid compositions of the diets and platelet phospholipids, and the index of thrombogenicity of Ulbricht and Southgate [1]. Four groups of 16 pigs were fed for 70 days on diets containing one of three test fats/oils; anhydrous milkfat (AMF), fish oil (MaxEPATM) or hydrogenated coconut oil (HCO) at a level of 100 g/kg, or a basal diet containing starch at the expense of the test fat/oil. Blood samples were collected from each pig at 56 and 70 days. ADP- and collagen-induced platelet aggregation were measured by the turbidometric method at 56 days, intra-platelet cGMP response to sodium nitroprusside activator was measured by radioimmunoassay at 56 days, and the fatty acid composition of platelet phospholipids was measured at 70 days. There was a significant effect of diet on collagen-induced platelet aggregation (%) (AMF 76.0, MaxEPA 70.8, HCO 81.3, basal 86.8) which was lower in the MaxEPA group compared to the hydrogenated coconut oil and basal groups, and on the proportions (% total fatty acids) of the major monounsaturated (18:1n-9, AMF 16.6, MaxEPA 13.1, HCO 14.6, basal 15.9; 18:1n-7, AMF 0.9, MaxEPA 3.3, HCO 1.1, basal

* Corresponding author. Tel.: ⫹64-6-356-8300; fax: ⫹64-6-351-7050. E-mail address: [email protected] (K.A.C. James). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 4 2 5 - 6

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1.2) and polyunsaturated (18:2n-6, AMF 7.6, MaxEPA 7.4, HCO 8.7, basal 7.6; 20:4n-6, AMF 13.3, MaxEPA 4.8, HCO 16.2, basal 15.6; 20:5n-3, AMF 0.9, MaxEPA 10.3) fatty acids in platelet phospholipids. There were no significant effects of diet on ADP-induced platelet aggregation or platelet cGMP. The relative pattern of platelet aggregation across dietary treatments was consistent with empirical indices based on the ratios of the n-6/n-3 fatty acids in the diets and platelet phospholipids, but did not match the pattern based on the index of thrombogenicity [1]. The data suggest that useful factors for predicting platelet aggregation are the proportions of the fatty acids 18:2n-6 ⫹ 20:4n-6/18:3n-3 ⫹ 20:5n-3 ⫹ 22:6n-3 in the diets and platelet phospholipids. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Porcine; Platelet aggregation; Platelet fatty acid composition; Dietary fats/oils; Indices of thrombogenicity

1. Introduction Over recent years, the public perception of milkfat has been adversely affected by the contention that its relatively high saturated fatty acid content raises blood cholesterol level and increases the risk of coronary heart disease. In spite of this there have been few systematic studies investigating directly the influence of dietary milkfat in relation to coronary heart disease. Most of the adverse medical and public opinion has arisen from extrapolation of the physiological effects of dietary saturated fatty acids in general. Death from coronary heart disease usually results from the formation of an occlusive thrombus following rupture of an atheromatous plaque in a coronary artery [2]. Among the behavioural risk factors implicated in the development of coronary heart disease are dietary fatty acids, and the saturated fatty acids in particular [3]. However, the issue remains complex and the research focus is now predominantly on physiological responses to individual fatty acids since many of these may be considered atherogenic, thrombogenic or both [1]. It has been recognised recently that these two processes, atherogenesis and thrombosis, are closely linked [4,5]. The purpose of this study was to measure aspects of platelet composition and function for pigs fed diets containing anhydrous milkfat, a fish oil concentrate of the fatty acids 20:5n-3 and 22:6n-3 (MaxEPA™) or hydrogenated coconut oil. ADP- and collagen-induced platelet aggregation and intra-platelet cGMP response to the guanylate cyclase activator, sodium nitroprusside, were measured as potential predictors of platelet hyperaggregability and arterial platelet mediated thrombogenicity [6,7]. The pattern of experimentally determined platelet aggregation across dietary treatments, and associated empirical indices calculated from the fatty acid compositions of the diets and platelet phospholipids, were compared with the index of thrombogenicity calculated from the fatty acid composition of the diets by the formula of Ulbricht and Southgate [1]. The results presented here are part of a wider study developing a porcine thrombosis model for evaluating milkfat in human food products as a risk factor in coronary heart disease. The pig was selected because it is widely accepted as a model in thrombosis research [8 –10].

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2. Materials and methods 2.1. Experimental animals Sixty four male pigs, comprising 16 sets of four Large White ⫻ Landrace littermates, sourced from the Pig Research Unit, Massey University, were used in the trial. The pigs comprised three genotypes which included seven sets of 3⁄4 Large White ⫻ 1⁄4 Landrace, seven sets of 1⁄2 Large White ⫻ 1⁄2 Landrace, and two sets of 1⁄4 Large White ⫻ 3⁄4 Landrace. All pigs were weaned at approximately 28 days of age, and then fed a commercial weaner diet until they were started on the experimental diets at approximately 56 days of age (range 53–58 days, 11.2–19.6 kg live weight). All procedures involving animals in this study were conducted under guidelines established by the Massey University Animal Ethics Committee and with their prior approval. 2.2. Experimental diets There were four dietary treatments with 16 pigs per treatment. Three of the diets contained ‘high fat’ levels (12.0 –12.3%, w/w) with ca. 80% of the total fat supplied by anhydrous milkfat, fish oil (MaxEPA™) or hydrogenated coconut oil as the test fats/oils. One diet (basal) contained a ‘normal fat’ level (2.6%, w/w) for the domestic pig and contained starch at the expense of the test fat/oil. The basal diet served as a reference point for the other three diets. The main comparisons were between the three ‘high fat’ diets. Barley was the main source of digestible energy, and lactic casein and soy protein isolate were the main sources of crude protein. Sunflower seed oil was included in all diets to ensure that the level of the fatty acid 18:2n-6 was adequate to meet essential fatty acid requirements. The diets were formulated, as far as practicable, to contain purified ingredients so that most of the dietary fatty acids were provided by the test fats/oils. All fats/oils were analysed for ␣-tocopherol, and MaxEPA also for ␣-tocopheryl acetate. After all the values had been transformed to IU of vitamin E, dl-␣-tocopherol was added to anhydrous milkfat and hydrogenated coconut oil to balance the levels of antioxidant. After supplementation, anhydrous milkfat and hydrogenated coconut oil were stored at ⫺20°C in 3 kg batches. MaxEPA and sunflower seed oil were stored at 5°C, with MaxEPA under N2. Formulations for the protein sources were based on the total calculated lysine level of the ingredients, with all other essential amino acids balanced with respect to lysine. Diets were formulated using linear programming to meet or exceed certain nutrient specifications and nutritional adequacy was determined by comparison with the requirements for growing pigs [11]. The test and basal diets contained (g/kg): 248 crude protein, 15 lysine, 11.9 Ca, 8.3 P, 2.8 Na, 3.5 K, 2.0 Cl. The test diets contained 13.3 MJ DE/kg and the basal diet contained 11.2 MJ DE/kg. The diets containing test fats/oils, which were relatively high in total lipid content for pigs, were formulated to contain 100 g/kg of test fat/oil and 10 g/kg of sunflower seed oil. The total lipid contents of the diets were (g/kg): 122.8 anhydrous milkfat, 122.6 MaxEPA, 119.6 hydrogenated coconut oil, 26.0 basal. The ingredient compositions of the experimental diets are given in Table 1. A base mix (112.5 kg) was made, divided into four portions (27 kg) and 3 kg of test fat/oil,

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Table 1 Ingredient composition of experimental diets9 Ingredient

Dietary treatment 1

Barley Lactic casein2 Soy protein isolate3 Anhydrous milkfat4 MaxEPA5 Hydrogenated coconut oil6 Sunflower oil7 Wheat bran Corn starch Salt Limestone Dicalcium phosphate Disodium hydrogen orthophosphate Vitamin and trace element premix8 Ethoxyquin

HCO1

AMF

MaxEPA

540 100 100 100 0 0 10 100 3.553 1.95 17.45 17.43 6.68 2.75 0.187

g/kg, as fed 540 540 100 100 100 100 0 0 100 0 0 100 10 10 100 100 3.553 3.553 1.95 1.95 17.45 17.45 17.43 17.43 6.68 6.68 2.75 2.75 0.187 0.187

Basal 540 100 100 0 0 0 10 100 103.553 1.95 17.45 17.43 6.68 2.75 0.187

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Spec 100, Tui Milk Products, Palmerston North, New Zealand. 3 Supro 590, Columbit (New Zealand) Ltd, Auckland, New Zealand. 4 New Zealand Dairy Board, Wellington, New Zealand, including added dl-␣-tocopherol (1.307 g/kg, Roche Products New Zealand Ltd, Auckland, New Zealand). 5 Seven Seas Ltd, Hull, England, including d-␣-tocopheryl acetate. 6 Confectionary fat 92, Abels Ltd, Auckland, New Zealand, including added dl-␣-tocopherol (1.353 g/kg). 7 Abels Ltd, Auckland, New Zealand. 8 Vitamin and trace element premix was prepared specifically for the trial by NRM New Zealand Ltd, Auckland, New Zealand, and supplied in the finished diets (per kg): 8000 IU vitamin A; 1500 IU vitamin D3; 100 mg vitamin E; 1 mg vitamin K; 10 mg pantothenic acid; 3 mg riboflavin; 15 mg niacin; 1 mg pyridoxine; 1 mg thiamine; 15 ␮g vitamin B12; 50 ␮g biotin; 2 mg folic acid; 200 mg choline; 0.5 mg cobalt; 1 mg iodine; 10 mg copper; 80 mg iron; 60 mg manganese; 100 mg zinc; 0.2 mg selenium; 125 mg ethoxyquin; 100 mg vitamin C; 8 mg sodium fluoride; 195 mg inositol; 20 mg p-aminobenzoic acid. 9 Initially, 112.5 kg of base mix (excluding test fat/oil) was prepared in a wet mash mixer (ca. 200 kg capacity). Subsequently, the base mix was divided into 4 portions of 27 kg and each portion was transferred to the bowl of a dough mixer (ca. 50 kg capacity) and 3 kg of test fat/oil poured in with constant stirring and mixed for 8 min. 1 2

or starch, was added with mixing. Experimental diets were made up freshly to ensure that each batch was used within 4 days. Diets were stored in plastic drums at ambient temperature while being used. Samples of all experimental diets were taken on three separate occasions at the time of preparation, and stored at ⫺70°C for determination of fatty acid composition (Table 2). 2.3. Experimental procedure Eight pigs (two sets of littermates) were housed in each of eight pens. Each pen was comprised of a yard adjoined to a fully enclosed, insulated and heated sleeping area and a

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row of individual feeding stalls with restricted access. Overhead infrared lamps were used in the sleeping areas to maintain a temperature of 24°C. Natural lighting was used in the test house except when artificial lighting was required at feeding and bleeding times. One pig from each set of littermates was allocated randomly to each treatment. The start of the trial was staggered over 12 weeks, with each set of littermates being introduced to the experimental diets on 16 different days. Experimental diets were offered to pigs at a level of 0.10 of the metabolic body weight (kg0.75) per day, adjusted weekly. Pigs were fed twice daily in approximately equal portions, at 08:30 and 16:00, for a period of 70 days. Diet refusals were recorded. Water was continuously available from drinking nozzles situated in the yards and, in addition, from water added to the diets during feeding. To collect blood samples, pigs were sedated with Zoletil (an equal mixture of zolazepam hydrochloride 25 mg/mL and tiletamine hydrochloride 25 mg/mL, Virbac Laboratories, France) at a dose rate of 5 mg/kg live weight by intramuscular injection in the neck. The pigs were placed in dorsal recumbency and blood was taken from the anterior vena cava or aorta/common carotid artery using an 18 gauge ⫻ 38 mm needle (⬍50 kg live weight) or 18 gauge ⫻ 64 mm spinal needle (⬎50 kg live weight) (Terumo Corporation). A staggered bleeding schedule for each set of littermates was used to allow for the completion of platelet aggregation measurements within 2 h of blood collection. After 56 days on the experimental diets, following overnight starvation, 9 volumes of blood (18 mL) were withdrawn into a 20 mL disposable syringe containing 1 volume (2 mL) of 0.1 mol/L trisodium citrate for platelet aggregation and cGMP studies. These end-points were determined 14 days before the end of the feeding period because of other experimental commitments on day 70. At the completion of the trial, at 70 days, following overnight starvation, 9 volumes of blood (45 mL) were collected into a 50 mL syringe containing 1 volume (5 mL) of 0.1 mol/L trisodium citrate for determination of platelet phospholipid fatty acid composition. 2.4. Analytical methods 2.4.1. Platelet aggregation in vitro. Blood was transferred to 12 mL plastic tubes and centrifuged at 200 ⫻ gmax for 10 min at ambient temperature (3360 rotor, Omnifuge 2.0 RS, Heraeus Sepatech, Kalkberg, Germany) and the supernatant removed as platelet-rich plasma (PRP). The remaining contents were centrifuged at 5000 ⫻ gmax for 15 min at ambient temperature and the supernatant aspirated as platelet-poor plasma (PPP). When necessary, any remaining red blood cells were removed from the PRP by additional centrifugation at 500 ⫻ gmax for 2 min. Platelet concentration in PRP was determined manually using a haemocytometer. The PRP was then diluted with PPP to give a final platelet concentration of 0.36 ⫻ 106/␮L. Platelet aggregation in PRP was measured at 37°C by the turbidometric method [12] using a 4 channel aggregometer (Monitor IV, Helena Laboratories, Beaumont, TX, USA). After preincubation of 250 ␮L of PRP in the aggregometer for 5 min at 37°C, aggregation was initiated by adding 50 ␮L of either ADP or collagen agonist. ADP (Sigma Chemical Co, St Louis, MO, USA) was used at final concentrations of 2, 4, 6 and 10 ␮mol/L and collagen

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(Equine collagen, Paton Scientific Pty Ltd, Victor Harbour, SA, Australia) at final concentrations of 2, 4, 6 and 8 mg/L. There were 4 runs on the aggregometer for each blood sample, with the 4 concentrations of either agonist run simultaneously. Duplicate runs using ADP were followed by duplicate runs using collagen. Aggregation was recorded for 5 min and results were expressed as a percentage of maximum aggregation. Fresh working solutions of ADP were prepared daily from stock solution (60 ␮mol/L) stored in aliquots at ⫺85°C. Stock collagen solution (160 mg/L) was stored at 2°C and diluted daily to make up working solutions. 2.4.2. Intra-platelet cGMP assay. The volume of PRP required for the preparation of each cGMP extract was calculated using the empirical formula: volumePRP (mL) ⫽ 300/(platelets/nL). To meet the requirement of the concentration range for cGMP in the radioimmunoassay, PRP was diluted with PPP to a platelet count of 600/nL. Approximately 2.2 mL of adjusted PRP was preincubated at 38°C for 5 min, and then 2 ⫻ 0.5 mL aliquots were incubated for a further 3 min with 20 ␮L of sodium nitroprusside to give a final concentration of 10 ␮mol/L, and another 2 ⫻ 0.5 mL aliquots were incubated with 20 ␮L of 9 g/L NaCl as zero sodium nitroprusside controls. After incubation, the 4 samples were centrifuged at 4,000 ⫻ gmax for 2 min to separate plasma from platelets. The supernatant was removed, and 0.5 mL of 4 mmol/L Na2EDTA was added to the platelets before samples were placed in a boiling water bath for 5 min for cGMP extraction [6]. The platelet extracts were centrifuged at 5,000 ⫻ gmax for 10 min and then stored at ⫺70°C until assayed for cGMP using the cGMP [125I] radioimmunoassay system (Amersham, England). 2.4.3. Fatty acid composition of platelet phospholipids. PRP was prepared and, after further centrifugation at 2,000 ⫻ gmax for 10 min at ambient temperature, the sedimented platelets were washed 3 times with 20 mmol/L tris buffer pH 7.4 containing 138 mmol/L NaCl and 1 mmol/L Na2EDTA (5 mL) [13], resuspended in 0.1 mol/L KCl (1 mL), transferred to 15 mL tubes (Kimax), and total lipids were extracted using chloroform/methanol (2/1, v/v) (8 mL) [14]. Thin layer chromatography (TLC), using hexane/diethyl ether/acetic acid (85/15/1, v/v/v) as solvent (8 mL) [15], was used to identify the components of the extracts. A preliminary purification of the samples was carried out by passing the sample, dissolved in chloroform, through the column of BioSilA used for separation of fatty acid methyl esters. After passing 10 mL of chloroform through the column, cholesterol and phospholipids were eluted with 25 mL of methanol. Phospholipids in the lipid extracts were transesterified by incubation with 14% boron triflouride in methanol (1 mL) for 60 min at 100°C [16]. After cooling, water (2 mL) and hexane (2 mL) were added and the fatty acid methyl esters and cholesterol extracted into hexane. TLC was used to check transesterification of fatty acids. Fatty acid methyl esters were separated from cholesterol by silicic acid column chromatography, using a glass column of BioSilA 100-200 mesh (140 mm ⫻ 10 mm id) activated at 80°C for 4 h [17]. The sample was loaded in chloroform (0.5 mL), and the fatty acid methyl esters were eluted with the first 10 mL of chloroform passed through the column.

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Fatty acid methyl esters were fractionated by gas-liquid chromatography (GLC) in a Hewlett Packard Gas Chromatograph (Model 5890 A) using a stainless steel column (25 m ⫻ 0.22 mm id) packed with BPX70 and helium carrier gas, programmed over 140-210°C at 2%/min. There was evidence of residual contamination in the early part of the GLC trace and as a consequence the fatty acid peaks of chain length 聿12 were excluded from the calculation of area %. 2.4.4. Fatty acid composition of experimental diets and lipid content of faeces. Total lipid was extracted from subsamples of diets (ca. 2 g) and faeces (ca. 5 g) using chloroform/methanol (2/1, v/v) (40 mL) and 0.1 mol/L KCl (8 mL) [14]. With the faeces extraction, 3 drops of conc. HCl were added with KCl to ensure any free fatty acids were in the acid form and extractable into chloroform. Triacylglycerols in the lipid extracts of the diets (ca. 20 mg) were transesterified with 14% boron trifluoride and the fatty acid methyl esters fractionated by GLC as for the platelet phospholipids. 2.5. Statistical analyses The experiment was a split plot (or nested) design, with eight pens (blocks), two litters per pen (main plots) and four pigs per litter (sub-plots). Differences between the ages of the pigs at the start of the trial were accounted for by differences between litters. The genotypes were not distributed in any balanced way among pens, so analysis of variance (ANOVA) was not fully efficient at separating genotype effects from pen and litter differences. In addition to ANOVA, the data were also examined using the Residual Maximum Likelihood (REML) method of Patterson and Thompson [18]. Diet differences and interactions between diets and genotypes could be properly assessed ignoring pen effects, so for simplicity, the results of the ANOVA are presented unless indicated otherwise. REML analysis indicated that in most instances there were no important differences between pens and that ANOVA was not giving seriously biased conclusions. Where there were missing values, the split-plot ANOVA was used with missing values estimation. The platelet phospholipid fatty acid and cGMP data were log transformed before analysis to stabilise the variance. All analyses were carried out using Genstat 5 [19]. With the platelet aggregation data there was a higher number of missing values. Data for one pig were missing and there were several cases where data for one of the duplicates at all or some of the agonist concentrations were missing. To reduce the number of missing values, mean data across duplicates were used in the statistical analysis. The data were analysed using the split-plot analysis described with adjustment for remaining missing values. The different agonist levels were considered as split split-plot treatments within ANOVA, but as they were each applied to the same blood sample from each individual pig, they were not independent and so an unadjusted ANOVA was likely to be biased. Adjustments were therefore made using the method of Greenhouse and Geisser [20] which provided a correction factor for the degrees of freedom used in F-tests within ANOVA that involved agonist concentration.

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Table 2 Fatty acid composition of experimental diets Fatty acid

Dietary treatment 1

AMF

MaxEPA

HCO1

Basal

2

g/100 g total fatty acids n-saturated 12:0 14:0 16:0 18:0 20:0 n-unsaturated 14:1 16:1 18:1 trans 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3

3.1 9.6 27.6 12.4 0.8 1.1 3.7 21.6 14.0 1.8

0.2 8.2 21.9 3.2 3.4

38.0 15.0 11.5 3.2

0.4 0.5 17.5 2.9

10.3

19.7

13.9 0.8

55.3 3.7

43.2 17.0 13.1 3.6

0.1 0.1 4.3 0.7

11.7

4.9

15.8 0.9

13.7 0.9

9.5 11.3 3.2 13.6 1.4 0.2 11.8 5.3 g/kg diet, as fed2

n-saturated 12:0 14:0 16:0 18:0 20:0 n-unsaturated 14:1 16:1 18:1 trans 18:1n-9 18:1n-7 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 1 2

3.6 11.2 32.2 14.5

0.9 1.3 4.3 25.2 16.3 2.1

0.2 9.6 25.5 3.7 4.0

11.1 13.2 3.7 15.8 1.6 0.2 13.7 6.2

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Values are arithmetic means, n ⫽ 3.

3. Results The mean live weights of the pigs in the four dietary groups at the start of the feeding trial were within a 1 kg range (Table 3). After 70 days on the dietary treatments, there was a highly significant effect of diet on live weight, but this was due to differences between pigs fed the ‘high fat’ test diets compared to the ‘low fat’ basal diet. Further statistical analysis

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Table 3 Live weight and faecal lipid content for pigs fed diets containing different fats/oils Parameter

Time (days) 2

Live weight (kg)

0 70

Faecal lipid (g/kg, dry)2

70

Dietary treatment 1

AMF

15.7 72.5 168

MaxEPA 15.9 71.8 154

1

HCO

Basal

15.2 71.8

14.9 64.9

141

107

SED

df

Level of significance3

0.50 1.88

39 39

ns ***

9.04 5.75

39

*** *

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Values are means adjusted for any missing values. 3 Overall F test of treatment differences, ns ⫽ not significant at the 5% level,* P ⬍0.05,***P ⬍0.001. 4 To compare the basal treatment with any experimental treatment. 5 To compare between experimental treatments. 1 2

of the live weights at 70 days, using the zero time live weights as a covariate, gave similar results but with an increased level of significance between the ‘high fat’ diets and the basal diet. There were no differences in mean pig live weight between any groups of pigs fed the ‘high fat’ diets at the end of the feeding trial (Table 3). All pigs were healthy throughout the trial and, despite the relatively high dietary fat levels for pigs, their faeces were generally firm. The mean faecal lipid content of pigs fed the basal diet was significantly lower than the values for pigs fed the ‘high fat’ diets, which were all significantly different from each other (Table 3). The fatty acid compositions of the platelet phospholipids are given in Table 4. In the platelet phospholipids, there were no significant effects of diet on the levels of the main Table 4 Fatty acid composition of platelet phospholipids for pigs fed diets containing different fats/oils Fatty acid

Dietary treatment AMF1

14:0 16:0 18:0 18:1n-9 18:1n-74 18:2n-6 20:4n-6 20:5n-34,5 22:6n-3

1.37 (3.9) 3.32 (27.5) 2.86 (17.4) 2.81 (16.6) ⫺0.08 (0.9) 2.03 (7.6) 2.59 (13.3) ⫺0.11 (0.9) 0.2

MaxEPA

HCO1

g/100 g total fatty acids2 1.03 (2.8) 1.51 (4.5) 3.33 (27.8) 3.34 (28.2) 2.89 (18.0) 2.94 (18.9) 2.57 (13.1) 2.68 (14.6) 1.21 (3.3) 0.11 (1.1) 2.00 (7.4) 2.16 (8.7) 1.56 (4.8) 2.79 (16.2) 2.33 (10.3) tr

SED

df

Level of significance3

0.153 0.047 0.047 0.055 0.223 0.057 0.076 0.186

30 30 30 30 30 30 30 10

* ns ns *** *** * *** ***

Basal 1.19 (3.3) 3.41 (30.3) 2.89 (18.0) 2.77 (15.9) 0.18 (1.2) 2.03 (7.6) 2.75 (15.6) tr

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Values are means of log transformed data, and values in parentheses are these means back transformed. Value for 22:6n⫺3 is an arithmetic mean. Data from litters 1–3 inclusive were excluded. 3 Overall F test of diet differences, ns ⫽ not significant at the 5% level, * P ⬍0.05, ***P ⬍0.001. 4 Trace values were replaced by 0.4. 5 HCO and basal treatments were not included in statistical analysis. 1 2

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Table 5 Platelet aggregation within dietary treatment, across agonist concentrations, for pigs fed diets containing different fats/oils Agonist

Dietary treatment AMF1

3

ADP Collagen4

42.1 76.0

HCO1

MaxEPA

SED

df

Level of significance5

3.64 4.24

38 38

ns ***

Basal

% maximum aggregation2 47.0 42.6 70.8 81.3

49.3 86.8

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Values are means adjusted for any missing values. 3 2, 4, 6, 8 ␮mol/L. 4 2, 4, 6, 10 mg/L. 5 Overall F test for effect of diet, ns ⫽ not significant at the 5% level,***P ⬍0.001. 1 2

saturated fatty acids, 16:0 and 18:0, despite markedly higher levels in the anhydrous milkfat diet compared with the other three diets. However, there were significant effects of diet on all of the main unsaturated fatty acids in platelet phospholipids. The level of 20:4n-6 in the MaxEPA group was significantly lower than in all other groups. In contrast, the level of 20:5n-3 was significantly higher in the MaxEPA group, reflecting the relatively high content of this fatty acid in the diet. There was a low, but measurable, level of 20:5n-3 in the platelet phospholipids of pigs fed the anhydrous milkfat diet. The levels of 18:1n-9 and 18:2n-6 in platelet phospholipids were similar for all dietary groups despite the relatively higher levels of 18:1n-9 in the anhydrous milkfat and basal diets, and of 18:2n-6 in the basal diet. The in vitro platelet aggregation data using ADP and collagen agonists are presented in Tables 5 and 6, and in Figs. 1 and 2. With ADP, there was only one wave of aggregation after the addition of agonist, irrespective of the concentration used. There were statistically significant interactions between genotype and diet, and between genotype and agonist concentration. The genotype by diet interaction indicated that the differences between diets for platelet aggregation varied with genotype and were due to the lower level of aggregation

Table 6 Platelet aggregation within agonist concentration, across dietary treatments, for pigs fed diets containing different fats/oils Agonist concentration1

Agonist 1

2

3

SED

df

Level of significance3

0.93 1.83

134 113

*** ***

4 2

ADP Collagen

21.9 52.0

% maximum aggregation 44.5 57.5 82.1 94.9

57.2 83.9

ADP; 1 ⫽ 2, 2 ⫽ 4, 3 ⫽ 6, 4 ⫽ 8 ␮mol/L. Collagen; 1 ⫽ 2, 2 ⫽ 4, 3 ⫽ 6, 4 ⫽ 10 mg/L. Values are means adjusted for any missing values. 3 Overall F test for effect of agonist concentration,***P ⬍0.001. 1 2

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Fig. 1. ADP-induced platelet aggregation in vitro in platelet-rich plasma from pigs fed ‘high fat’ diets containing anhydrous milkfat ■, MaxEPA Œ or hydrogenated coconut oil ⴙ as the test fat/oils, or a ‘low fat’ basal diet containing carbohydrate in place of the test fat/oil }. The error bars represent the least significant differences at the 5% level of significance for comparison of means between ADP concentrations, within dietary treatment (i), and between dietary treatments, within ADP concentration (ii).

in the 1⁄4 Large White pigs fed the MaxEPA diet. The genotype by agonist concentration interaction indicated that the shapes of the dose response curves were different between genotypes, and this was due to the steeper increase in aggregation between 6 and 10 ␮mol/L for the 3⁄4 Large White pigs. Although there was no significant interaction between diet and agonist concentration, there was a significant effect of agonist concentration (Fig. 1). There was no significant overall effect of diet on ADP-induced platelet aggregation (Table 5). In contrast, with collagen there were no significant interactions between genotype and diet, genotype and agonist concentration, or diet and agonist concentration. However, there was a highly significant overall effect of diet (Table 5), in which maximum aggregation in platelets from pigs fed the MaxEPA diet was significantly lower than in platelets from pigs fed the hydrogenated coconut oil and basal diets (Fig. 2). Furthermore, platelet aggregation in pigs fed the anhydrous milkfat diet was significantly lower than in those fed the basal diet. There was also a highly significant overall effect of agonist concentration (Fig. 2), in which aggregation increased with increasing collagen concentration up to 6 mg/L, but decreased between 6 and 8 mg/L. The intra-platelet cGMP data are shown in Table 7 and, although there were no strongly significant differences overall between the dietary treatments or genotypes, there was a significant difference between the cGMP response to sodium nitroprusside in vitro; the response was higher in pigs fed the hydrogenated coconut oil diet than those fed the MaxEPA diet.

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Fig. 2. Collagen-induced platelet aggregation in vitro in platelet-rich plasma from pigs fed ‘high fat’ diets containing anhydrous milkfat ■, Max EPA Œ or hydrogenated coconut oil ⴙ as the test fat/oil, or a ‘low fat’ basal diet containing carbohydrate in place of the test fat/oil }. The error bars represent the least significant differences at the 5% level of significance for comparison of means between collagen concentrations, within dietary treatment (i), and between dietary treatments, within collagen concentration (ii).

4. Discussion 4.1. Platelet function versus calculated indices of thrombogenicity The most important outcome from this study was that the relative pattern of platelet aggregation across dietary treatments was consistent with empirical indices based on the fatty Table 7 Platelet guanylate cyclase activity for pigs fed diets containing different fats/oils Parameter

2,3

cGMP

Dietary treatment AMF1

MaxEPA

HCO1

0.93 (2.5)

pmol/109 platelets 0.70 (2.0) 1.16 (3.2)

SED

df

Level of significance4

0.169

42

ns (0.05 ⬍ P ⬍ 0.10)

Basal 1.07 (2.9)

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Values are means adjusted for any missing values. 3 Values are means of log transformed data, and values in parentheses are these means back transformed. 4 Overall F test of diet differences, ns ⫽ not significant at the 5% level. 1 2

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Table 8 Summary of indices of thrombogenicity, other empirical indices and collagen-induced platelet aggregation for pigs fed diets containing different fats/oils Indices

Dietary treatment 1

Thrombogenicity Diet n-6/n-32 Platelet phospholipid n-6/n-33 Platelet aggregation4

AMF

MaxEPA

HCO

Basal

1.91 7.8 23.2 76.0

0.45 0.7 1.2 70.8

2.05 17.4 62.3 81.3

0.43 14.9 58.0 86.8

1

Calculated according to Ulbricht and Southgate [1] from the fatty acid compositions of the diets (Table 2). The formula is: mS/(nM ⫹ oM' ⫹ p(n-6) ⫹ q(n-3) ⫹ (n-3/n-6)) where S ⫽ sum of 14:0, 16:0 and 18:0, M ⫽ 18:1, M' ⫽ sum of other MUFA, n-6 ⫽ sum of n-6 PUFA, n-3 ⫽ sum of n-3 PUFA, and m, n, o, p and q are empirical constants where m ⫽ 1, n ⫽ 0.5, o ⫽ 0.5, p ⫽ 0.5 and q ⫽ 3. 2 Ratio of fatty acids 18:2n-6 ⫹ 20:4n-6/18:3n-3 ⫹ 20:5n-3 ⫹ 22:6n-3 (Table 2). 3 Ratio of fatty acids 18:2n-6 ⫹ 20:4n-6/18:3n-3 ⫹ 20:5n-3 ⫹ 22:6n-3 (Table 4). 4 Collagen data (Table 5).

acid compositions of the diets and platelet phospholipids, but did not match the pattern based on the index of thrombogenicity (Table 8). In this study, MaxEPA and hydrogenated coconut oil were included as experimental treatments to maximise the opportunity of demonstrating statistically significant effects of diet on platelet function. This expectation was based on the fatty acid composition of the triacylglycerols in the pure fats/oils and, consequently, in the experimental diets (Table 2). Indices of thrombogenicity for the diets were calculated according to the empirical formula of Ulbricht and Southgate [1]. The formula is defined in the legend to Table 8. The values demonstrated a wide range in predicted thrombogenicity. Indices for the anhydrous milkfat diet (1.91) and hydrogenated coconut oil diet (2.05) were markedly higher than either the MaxEPA diet (0.45) or the basal diet (0.43) (Table 8). Milkfat ranked poorly, and the index supports the public perception that dairy products containing milkfat are relatively high risk foods in terms of cardiovascular disease. We have found a highly significant effect of diet on collagen-induced platelet aggregation in PRP from pigs. Platelet aggregation was lowest in pigs fed the MaxEPA diet, and highest in pigs fed the hydrogenated coconut oil and basal diets (Tables 6, 8). The relative pattern of platelet aggregation across dietary treatments was not consistent with the pattern obtained from the index of thrombogenicity. The highest collagen-induced platelet aggregation was found in pigs fed the basal diet, yet this finding was consistent with our scores calculated from the fatty acid compositions of the diets and platelet phospholipids (Table 8). This finding suggests that a critical factor in predicting the level of platelet aggregation is the pattern of fatty acids in the diet and not the total amounts, and highlights a difficulty in accepting the Ulbricht and Southgate formula as an index of thrombogenicity, given that platelet aggregation is intimately involved in thrombogenesis.

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4.2. Platelet aggregation and platelet phospholipid fatty acid composition There were significant effects of diet on all the major monounsaturated and polyunsaturated fatty acid components of the platelet phospholipids (Table 2). The most pronounced differences were in pigs fed the MaxEPA diet, where there was a significantly higher level of 20:5n-3, and a lower level of 20:4n-6, than in the platelet phospholipids of pigs fed any of the other diets. The substitution of 20:5n-3 for 20:4n-6 in the phospholipids of platelets has been consistently demonstrated in other dietary studies where animals or humans have been fed diets containing fish or fish oil. Our finding is consistent with studies in the rat [21], rabbit [22], pig [23–27] and human [28 –30]. There are few reports in the literature of studies in the pig investigating the link between platelet aggregation and the type of dietary fat/oil. Our finding, that the source of fat/oil did not influence ADP-induced platelet aggregation, is in contrast to those of Verdouw and colleagues. A reduction in platelet aggregation was found in the whole blood of pigs fed a combined lard and fish oil diet compared to lard alone, following either induced stenosis of a coronary artery or a preliminary period on a hypercholesterolaemic diet [25,31–33]. We were unable to demonstrate a lower ADP-induced platelet aggregation in pigs fed the MaxEPA diet compared with the anhydrous milkfat, hydrogenated coconut oil or basal diets. The difference in the ADP results may be due to the pretreatments used prior to feeding the fish oil diets to the pigs by the Dutch group, or the inherent variability of the turbidometric aggregation assay. There are reports in other species, particularly the rat, where there was no effect of dietary fish oil [34] or butter [35] on ADP-induced platelet aggregation. 4.3. cGMP and platelet aggregation Recent studies indicate that cGMP plays an important role in the regulation of platelet aggregation [36]. The cGMP system operates as a negative feedback mechanism, mediated by endogenous nitric oxide, where excessive aggregation results in the stimulation of guanylate cyclase and, consequently, in the synthesis of cGMP which leads to disaggregation. In in vitro experiments, guanylate cyclase can be activated by exogenous nitric oxide, or nitric oxide donors such as sodium nitroprusside [6,36]. The measurement of cGMP production in response to sodium nitroprusside formed the basis of the assay used in this study for assessing the sensitivity of platelet guanylate cyclase. Guanylate cyclase activity was measured in relation to dietary treatment and ADP- and collagen-induced platelet aggregation in order to determine whether the type of dietary fat/oil influenced enzyme activity and whether the cGMP system could be part of a primary control mechanism mediating differences in platelet aggregation. With this scenario, increased guanylate cyclase activity would be associated with decreased platelet aggregation. There was no evidence that dietary treatment influenced guanylate cyclase activity, or that the observed differences in dietary dependent collagen-induced platelet aggregation were associated with a complementary pattern in guanylate cyclase activity.

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4.4. Platelet function and omega-3 fatty acids The principal omega-3 fatty acids in MaxEPA, 20:5n-3 and 22:6n-6, inhibit platelet aggregation [37] by regulation of the biosynthesis of thromboxanes, TXA2 and TXA3, from fatty acid precursors in the platelet membrane [38]. The reduced level of platelet aggregation in pigs fed the MaxEPA diet is consistent with the contention that it is regulated by the ratio of 20:5n-3 to 20:4n-6 in the phospholipids of the platelet membranes. The level of 20:4n-6 in the platelet phospholipids of pigs fed the anhydrous milkfat diet was 2.9% and 2.3% lower than in the phospholipids of pigs fed the hydrogenated coconut oil and basal diets respectively. This reduction was partly compensated for by an increased level of 20:5n-3, where 0.9% was found in the phospholipids of pigs fed the anhydrous milkfat diet but only traces in the phospholipids of the hydrogenated coconut oil or basal groups of pigs. These changes in the fatty acid composition of the platelet phospholipids in the anhydrous milkfat-fed pigs probably result from the relatively higher level of 18:3n-3 in the diet, as demonstrated by the lower ratio of 18:2n-6 to 18:3n-3 (anhydrous milkfat ⫽ 8, hydrogenated coconut oil ⫽ 17, basal ⫽ 15). The increase in 20:5n-3 in platelet phospholipids of pigs fed the anhydrous milkfat diet is consistent with findings in rats fed a butter diet [39], although the smaller increase found in our study may be due to the lower level of dietary milkfat (10% anhydrous milkfat v. 29.7% butter) and the inclusion of 1% sunflower oil in the diets. Furthermore, the 20:5n-3/20:4n-6 ratio in rat platelet phospholipids was found to increase when the dietary ratio of 18:3n-3/ 18:2n-6 increased [40]. 4.5. Use of the pig as a model The pig was selected as an animal model because of its extensive use by Fuster and colleagues in the study of pathophysiological responses to injury and in evaluation of antithrombotic therapies [2,9,41], and its acceptability as an animal model for studies of platelet function in humans [10]. An advantage of the pig model is that it allows the long term feeding of controlled and relatively high amounts of single sources of fatty acids included in a mixed cereal-based diet. Over the course of this 70-day trial the live weight of pigs increased almost five-fold, representing a considerable proportion of the growth phase. The study demonstrated the successful formulation and use of diets containing 10% by weight of a single test fat/oil, and of feeding to pigs long term without health problems. The experimental diets were prepared from purified ingredients, with fats/oils and protein ingredients of human food grade quality, in order to be able to attribute any differences between treatment groups to the lipid composition of the test fat/oil. All diets were fed at 10% of metabolic body weight per day and, in most cases, all rations were eaten. The feeding level was a balance between maximum live weight gain and avoidance of feed refusals so that the fatty acid intakes per unit of metabolic body weight for all pigs fed the test fat/oil diets were similar. There were significant differences in pig live weight at the end of the trial but this reflected the expected differences between pigs fed the basal diet as opposed to those fed the diets containing the test fats/oils. Although there were no differences between pigs fed the test diets, the lower live weight for pigs fed the basal diet was due to the lower caloric density

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of the diet and the design principle adopted that the intake of all dietary components, except the test fats/oils, would be the same across all treatments. 4.6. General perspective In this study, several physiological indicators implicated in the thrombogenic process were measured in a porcine model in relation to the source of dietary fat/oil. A statistically significant effect of diet was only found for collagen-induced platelet aggregation, yet when the numbers for ADP-induced platelet aggregation were taken into account, there was a similar trend with respect to diet. Collagen-induced platelet aggregation was considered to be a highly relevant end-point to measure as an indicator of thrombosis because of the exposure of collagen in the arterial wall in vivo following rupture of atherosclerotic plaque. A summary of the index of thrombogenicity, empirical indices based on the fatty acid compositions of the diets and platelet phospholipids, and collagen-induced platelet aggregation, for all dietary treatments, is given in Table 8. The two indices based on the ratios of n-6/n-3 fatty acids in the diets and platelet phospholipids do not adequately rank the relative effects of platelet aggregation in a continuous sense, but would appear to be quite adequate in ranking this physiological end-point, and hence the diets, into groups of high, medium and low thrombogenicity. In contrast, the Ulbricht and Southgate [1] index of thrombogenicity did not rank the diets into the same broad groups as the n-6/n-3 indices. Although the MaxEPA diet was ranked low by all indices, relativity between the anhydrous milkfat and basal diets was markedly different depending on the index. The anhydrous milkfat diet was ranked highly thrombogenic by the Ulbricht and Southgate index but was in the mid range according to the other indices. Furthermore, the basal diet ranked low by the Ulbricht and Southgate index but high by the other indices and also led to the highest collagen-induced platelet aggregation. In summary, the data demonstrated a dietary dependent effect on collagen-induced platelet aggregation for the pig, and that the rankings into broad groups describing this propensity to aggregation were reflected by the ratios of the proportions of n-6/n-3 fatty acids in both the diets and platelet phospholipids, but were not reflected by the Ulbricht and Southgate index of thrombogenicity.

Acknowledgments The roles of the authors in this study were as follows: KACJ, experimental design, conduct of trial, data collation, manuscript preparation; KGT, experimental design, conduct of trial, manuscript preparation; AJM, experimental design, conduct of trial, manuscript preparation; CLB, platelet aggregation; DRB, fatty acid analyses, YYC, cGMP analyses; RCB, statistical analyses; PJM, experimental design, manuscript preparation; WFL, experimental design, manuscript preparation. We thank Mrs J.S. Shoemark, New Zealand Institute for Crop & Food Research Limited, Palmerston North, and Mr E.A.C. James, for technical assistance in carrying out the pig trial; Mr D.V. Thomas, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, for supply of diet ingredients; and Mr

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