The use of a balloon angioplasty model of arterial injury to compare the thrombogenicity of dietary anhydrous milkfat, fish oil and hydrogenated coconut oil in pigs

Nutrition Research 23 (2003) 761–773 www.elsevier.com/locate/nutres

The use of a balloon angioplasty model of arterial injury to compare the thrombogenicity of dietary anhydrous milkfat, fish oil and hydrogenated coconut oil in pigs Keith G. Thompsona,*, Kerry A.C. Jamesb, Donald G. Arthura, Alison J. Maccollc, Richard B. Broadhurstb, Christine L. Boothc, Ruth C. Butlerb, Paul J. Moughand, Wilhelm F. Lubbee a

Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand b New Zealand Institute for Crop & Food Research Limited, Palmerston North, New Zealand c Fonterra Research Centre, Palmerston North, New Zealand d Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand e Department of Medicine, School of Medicine, University of Auckland, Auckland, New Zealand Received 23 September 2002; received in revised form 20 February 2003; accepted 28 February 2003

Abstract Three groups each of 16 pigs were fed for 70 days on a diet containing one of three test fats/oils (anhydrous milkfat, fish oil (MaxEPATM) or hydrogenated coconut oil) at a level of 100 g/kg. On day 70, a balloon catheter was inserted under anaesthesia into each femoral artery and inflated to 620.5 kPa for five 30 sec intervals. One h after angioplasty, while still anaesthetised, the pigs were killed by exsanguination. The injured segments of femoral artery were perfused in vivo with 0.9% saline and 10% neutral buffered formalin before being harvested. Thrombus size was determined by the counts of technetium-99m-labelled platelets in the injured arteries and by morphometry of the thrombus area in microscopic sections. Of 91 arteries subjected to angioplasty, mural thrombi were seen macroscopically in all nine arteries that had deep injury with exposure of the tunica media, as well as in 19 of the remaining 82 arteries (23%) in which there was no evidence of a medial tear. Many small platelet-rich thrombi, attached to the denuded endothelium, were also detected microscopically. There was no statistically significant effect of diet on either thrombus area or platelet deposition at sites of arterial injury. Failure to show significant differences may have been due to the large within-group variation in thrombus size, a factor which may limit the use of this model in studies aimed at detecting

* Corresponding author. Tel.: ⫹64-6-356-9099; fax: ⫹64-6-350-2270. E-mail address: [email protected] (K Thompson). 0271-5317/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0271-5317(03)00032-0

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differences in thrombogenicity between different dietary fats and oils. © 2003 Elsevier Inc. All rights reserved. Keywords: Balloon angioplasty; Arterial thrombosis; Milkfat; Fish oil; Coconut oil; Pig; Porcine model

1. Introduction Thrombosis is not only a key event in acute occlusive vascular syndromes, such as myocardial infarction, but is considered to play an important role in the progression of atherosclerosis [1–3]. As a result, there has been increasing interest in identifying risk factors for thrombogenicity, both in epidemiological and experimental studies. Much of this interest has focused on the effect of dietary lipids, particularly individual fatty acids, on the promotion or reduction of thrombosis [4]. However, the role of dietary factors on thrombogenicity remains poorly defined. Several studies have shown that certain long-chain saturated fatty acids such as myristic (14:0), palmitic (16:0) and stearic (18:0) are thrombogenic [5,6] while monounsaturated and some polyunsaturated fatty acids, particularly those of the n-3 series from fish oils, are antithrombogenic [4,5,7–9]. Such information has encouraged estimation of the thrombogenicity of various foods and diets based on their relative content of particular saturated and unsaturated fatty acids [4]. Dairy products, with their relatively high concentration of long chain saturated fatty acids, rank poorly when estimated in this manner, however, this presumption is not supported by objective data from experimental studies. In fact, one major epidemiological study has suggested that the consumption of milk and butter might even be protective [10]. In spite of the benefits of epidemiological studies they are unable to determine causal relationships. For this reason, and because of the speculative nature of assessing the thrombogenicity or atherogenicity of diets on the basis of their fatty acid composition, there is a need for animal models in which specific diets can be tested and compared. A wide range of inhibitors and promoters of both coagulation and fibrinolysis, some of which are used clinically to provide an indication of thrombotic tendency in humans, have been identified in blood [11,12]. Such regulators can also be measured in animals but because of the complexity of the haemostatic process, a thrombosis end-point that reflects the interaction between primary and secondary haemostatic systems, the fibrinolytic system, and the naturally occurring anticoagulants, is highly desirable. The purpose of this study was to evaluate a porcine arterial thrombosis model based on balloon angioplasy of femoral arteries by comparing the thrombogenicity of dietary anhydrous milkfat, a fish oil concentrate (MaxEPATM) and hydrogenated coconut oil. In addition to objective measurement of the thrombosis end-point, as described in this article, other measures of thrombogenicity and putative risk factors for coronary heart disease were measured during this study and have been reported separately [13].

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2. Materials and methods 2.1. Experimental design Four groups of 16 large White x Landrace male domestic pigs, which had been weaned at 28 days of age and fed a commercial weaner diet until introduction to the test diet at 53-58 days of age, were fed for 70 days on ‘high fat’ diets containing anhydrous milkfat, fish oil (MaxEPATM) or hydrogenated coconut oil as the test fat/oil at a level of 100 g/kg, or a ‘low fat’ basal diet which contained 100 g/kg cornstarch in place of the test fat. The experimental diets were offered to pigs at 10% by weight of their metabolic body weight (kg [0.75] per day, adjusted after weekly weighings. The size of treatment groups was limited to 16 because of restrictions on available space in the trial shed, and because of the complex nature of the procedures that were to be performed at the end of the feeding period. Furthermore, because of the expense of the trial, a larger group size would have made future use of the model uneconomic for our purposes. Through practical necessity, the pigs were of three different genotypes, varying from 3⁄4 Large White x 1⁄4 Landrace to 1⁄4 Large White x 3⁄4 Landrace. Two sets of four littermates were placed in each of eight pens with littermates each receiving a different diet. Although pigs on all four diets were tested for indicators of thrombogenicity, the results of which have been published previously [13], only those on diets containing one of the three test fats/oils were included in the present study. On day 70 each pig was anaesthetised and subjected to balloon angioplasty of both femoral arteries. Anaesthesia was maintained for 1 h after angioplasty. At this stage the abdominal aorta was catheterised with a 10 mm diameter polyethylene tube and the hind limbs were perfused with 0.9% saline followed by 10% neutral buffered formalin. The perfusion was carried out using a standardised gravity feed system that mimicked arterial systolic pressure. The pigs were killed by exsanguination at the commencement of saline perfusion while still under general anaesthesia. 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 The composition of the experimental diets is presented in Table 1. Barley was the principal source of digestible energy while 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 concentration of linoleic acid was adequate to meet the essential fatty acid requirements. All fats/oils were analysed for ␣-tocopherol, and MaxEPA also for ␣-tocopheryl acetate. After all the values had been transformed into IU of vitamin E, dl-␣-tocopheryl acetate was added to anhydrous milkfat and hydrogenated coconut oil to balance the antioxidant contents of the diets. The diets were made up freshly to ensure that each batch was used within four days.

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Table 1 Ingredient composition of experimental dietsa Ingredient

Dietary treatment AMFa

MaxEPA

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

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

HCOa

Basal

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

540 100 100 0 0 0 10 100 103.553 1.95 17.45 17.43 6.68

g/kg, as fed Barley Lactic caseinb Soy protein isolatec Anhydrous milkfatd MaxEPAe Hydrogenated coconut oilf Sunflower oilg Wheat bran Corn starch Salt Limestone Dicalcium phosphate Disodium hydrogen orthophosphate Vitamin and trace element premixh Ethoxyquin

0.187

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Spec 100, Tui Milk Products, Palmerson North, New Zealand. c Supro 590, Columbit (New Zealand) Ltd, Auckland, New Zealand. d New Zealand Dairy Board, Wellington, New Zealand, including added dl-␣-tocopherol (1.307 g/kg, Roche Products New Zealand Ltd, Auckland, New Zealand). e Seven Seas Ltd, Hull, England, including added d-␣-tocopheryl acetate. f Confectionary fat 92, Abels Ltd, Auckland, New Zealand, including added dl-␣-tocopherol (1.353 g/kg). g Abels Ltd, Auckland, New Zealand. h 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. i 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 fast/oil poured in with constant stirring and mixed for 8 min. a

b

2.3. In vivo thrombus preparation and measurement 2.3.1. Anaesthesia and femoral angioplasty Following overnight starvation, anaesthesia was induced by intramuscular injection of a tiletamine hydrochloride/zolazepam hydrochloride combination, 25 mg/mL of each (Zoletil, Virbac Laboratories, France), at a dose rate of 5 mg/kg body weight. Anaesthesia was maintained with 3% halothane/oxygen administered by endotracheal tube. The medial saphenous artery was exposed through a 2-3 cm parallel cut-down at a distance of 15 cm from the angioplasty site. Approximately 1 ml of 2% lignocaine was dripped on to the exposed artery to reduce the risk of vasospasm. An ultra thin wall 19- gauge percutaneous arterial needle (William A. Cook Ltd, Australia) was inserted into the arterial

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lumen and a 0.035 inch diameter wire guide advanced through the lumen of the needle to a distance of approximately 15 cm from the tip of the needle. After withdrawing the needle a 5 F (8 x 30 mm) balloon angioplasty catheter (Meditech Inc., USA) was advanced over the guide into the femoral artery. Once the catheters were in position, in both femoral arteries, the balloons were inflated concurrently to 620.5 kPa 5 times for 30 sec with 30 sec between inflations. The catheters were withdrawn immediately after the final inflation and cotton swabs applied under pressure to the entry sites until bleeding ceased. 2.3.2. Platelet labeling Autologous platelets were labeled with technetium-99m using procedures similar to those described for human patients. Approximately 4 h before femoral angioplasty, pigs were anaesthetised with the tiletamine hydrochloride/zolazepam hydrochloride combination (5 mg/mL) and 43 mL of blood were collected from the anterior vena cava into 7 mL of acid-citrate-dextrose anticoagulant. Blood samples were centrifuged at 200 x g for 10 min at room temperature in 60 mL polypropylene conical tubes (Falcon Labware). The supernatant, consisting of platelet rich plasma (PRP) was aspirated and centrifuged at 1000 x g for 10 min to form a platelet pellet. The resulting platelet-poor plasma (PPP) was removed and saved for subsequent resuspension of labeled platelets. The pellet was initially resuspended in 5 mL of acid-citratedextrose-saline and approximately 200 Mbq of Tc-99m-hexamethyl propylene amine oxime (HMPAO) (Amersham) added to the platelet suspension. This mixture was incubated at room temperature for 60 min with constant mixing before centrifugation at 1000 x g for 10 min in a 12 mL polypropylene conical tube (Falcon Labware). The supernatant was discarded after determination of its radioactivity and the pellet resuspended in 5.5 mL of PPP. Any platelet aggregates and erythrocytes were removed by centrifugation of the mixture at 100 x g for 5 min. Five mL of this solution of labelled platelets were injected into an ear vein of the original donor pig 15-30 min before angioplasty was performed. The activity of Tc-99m-HMPAO was measured in a dose calibrator (Mediac Dose Calibrator, ‘Nuclear Chicago’) at each step of the labelling procedure. The platelet labelling efficiency was calculated by dividing the radioactivity in the platelet pellet by the total radioactivity in the pellet and supernatant. 2.3.3. Quantitation of platelet deposition Immediately prior to perfusion with saline and formalin, a 10 mL blood sample was collected into an evacuated tube containing ethylene diamine tetraacetic acid anticoagulant. This sample was used to determine the total platelet count and ␥-radioactivity in whole blood. After perfusion, both femoral arteries were exposed and dissected free of adventitial tissues. The length of femoral artery between the branches of the lateral circumflex femoral and medial saphenous arteries, containing the 3 cm segment subjected to angioplasty, was removed, its length measured and placed in 10% neutral buffered formalin. A similar length of carotid artery was removed from each pig and handled similarly. Radioactivity was measured in an LKB gamma counter approximately 20 h after angioplasty and was determined for each femoral and carotid arterial segment, and for the blood samples. The number

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of platelets at the angioplasty site in each femoral artery was calculated by the following formula, using radioactivity in carotid artery segments to correct for any background levels. Number of platelets at angioplasty site (x 106 c/segment) ⫽ [blood platelet concentration (x 109/L) x [femoral a. radioactivity (cpm/segment) – [[carotid a. radioactivity (cpm/segment) x femoral a. length (mm)]/carotid a. length (mm)]]]/blood radioactivity concentration (cpm/mL)

2.3.4. Morphometrical analysis The 3 cm lengths of femoral artery subjected to balloon angioplasty were cut transversely into 10 segments of equal thickness and embedded in paraffin. Sections of 5 ␮m thickness were then cut from each paraffin block and stained with haematoxylin and eosin and in some cases by Verhoffs/Van Gieson. All sections were examined microscopically to confirm endothelial denudation (mild injury) and the presence or absence of medial tears. Arteries with medial tears were classified as having deep injury. The total cross-sectional area of thrombi in 10 sections from each artery (one section from each segment) was measured by image analysis using a Sigma Scan scientific measurement system (Jandel Scientific, San Rafael, CA, USA). 2.4. Statistical analysis The analysis of data was complicated because angioplasty was not performed on two pigs and in one pig was only performed on one leg. In addition, nine arteries had deep injury, and these were not distributed equally between the three diets. Also, there were possible differences between pigs of different genotypes, between litters, and between pigs from different pens, with litters nested within pens, and individual pigs nested within litters. The platelet deposition and thrombus area data were therefore examined using the residual maximum likelihood (REML) [14] method, which enables relatively unbiased estimates of treatment effects to be made for unbalanced and nested data. Differences between treatments were tested with a Wald test [14], using the results of this method. As well as differences between the diets, the independent difference in thrombus size between arteries with deep versus superficial injury was examined, but there was insufficient data available to test whether this varied with diet. Adjustments were also included for any differences between genotypes. The data were transformed using natural logarithms before analysis in order to stabilise the variance. There were some zero thrombus areas, so a value of 0.0005 was added to areas before taking logs. Because of the unbalanced and nested nature of the data, each individual treatment comparison can have a unique standard error. Therefore for simplicity, for tables with more than two means, the mean of the standard errors of the difference (SED) for all possible comparisons is given. Actual SEDs varied by up to 1.5% of the mean SED given. Numbers of arteries with deep or superficial injury found with each diet were compared with a chi-squared test for contingency tables. The types of thrombi found for arteries with superficial injury were compared between diets using the same method. All analyses were carried out using Genstat 5 [15].

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Table 2 Number of femoral arteries with deep or superficial injury at angioplasty sites in growing pigs fed for 70 days on experimental diets containing one of three different dietary fats/oils Injury typeb

Deep Superficial

Dietary treatment

Total arteries

AMFa

MaxEPA

HCOa

4 26

1 28

4 28

9 82

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Number of femoral arteries with each form of injury, including all arteries in which successful angioplasty was performed. a

b

3. Results The pigs remained in good health throughout the 70-day study and the mean live weight was 72.0 kg. There were no significant differences in mean live weight between the treatment groups [13]. Results were not available from two pigs that died under anaesthesia and spasm of the saphenous artery prevented successful angioplasty of one femoral artery in another pig. Therefore, from the 48 pigs that had received the test diets, a total of 91 injured vessels were available for analysis. 3.1. Prevalence of thrombosis and deep injury A dilated segment corresponding to the site of angioplasty was usually visible in the femoral arteries at the time of their removal from the pigs. A summary of the number of arterial segments showing deep or mild injury from pigs in each dietary group is presented in Table 2, and a summary of the number of arterial segments with superficial injury and associated type of thrombus from pigs in each dietary group is presented in Table 3. Deep injury at the site of angioplasty, characterised by disruption of the internal elastic lamina and tearing of medial smooth muscle, occurred in 9 of the 91 arteries (9.9%). Macroscopic mural thrombi were invariably present at these sites. Histologically, they consisted predominantly of platelets but were usually intersected by thin bands of fibrin and contained variable Table 3 Number of femoral arteries with superficial injury and the type of associated thrombus at angioplasty sites in growing pigs fed for 70 days on experimental diets containing one of three different fats/oils Thrombus typeb

Dietary treatment a

Macroscopic Microscopic None a b

Total arteries a

AMF

MaxEPA

HCO

7 16 3

4 22 2

8 19 1

19 57 6

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Number of femoral arteries with superficial injury and associated macroscopic or microscopic thrombi.

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Fig. 1. Photomicrograph of a mural thrombus at a site of superficial injury in the femoral artery of a growing pig fed for 70 days on a diet containing 10% anhydrous milkfat. Although this thrombus was large enough to be visible macroscopically the underlying internal elastic lamina (arrows) remains intact. Verhoff’s/Van Gieson. Bar ⫽ 200␮m.

numbers of trapped erythrocytes and polymorph neutrophils. Fibrin deposition was greatest in deep layers of the thrombus immediately adjacent to the damaged tunica media. The remaining 82 arteries suffered superficial injury with denudation of the endothelium being confirmed histologically. Mural thrombi were seen macroscopically in 19 of the 82 (23.2%) arteries without deep injury. In these arteries, the presence of an intact internal elastic lamina beneath the thrombi was confirmed histologically (Fig. 1). Numerous scattered small platelet-rich mural thrombi were also detected microscopically in these vessels. A further 57 (69.5%) arteries with superficial injury contained microscopic mural thrombi, while only 6 (7.3%) arteries with mild injury had no thrombi detectable histologically. The proportion of arteries with deep or superficial injury did not vary significantly between diets (P ⫽ 0.37) and neither did the proportions of arteries with each thrombus type for the superficially injured arteries (P ⫽ 0.52). 3.2. Platelet deposition and thrombus morphometry Mean values for platelet deposition and thrombus area at sites of angioplasty are given in Table 4. Results for arteries with deep injury were excluded from these analyses to ensure that any differences between treatment groups were due to diet, rather than to the severity of vascular injury.

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Table 4 Platelet deposition and thrombus area at femoral angioplasty sites with superficial injury in growing pigs fed for 70 days on diets containing one of three different dietary fats/oils Parameter

Dietary treatment a

AMF Platelet deposition (⫻106 c/segment)b Thrombus area (mm2)b

a

MaxEPA

HCO

Mean SEDc (df ⬇ 36)

8.77 (2.171)

10.47 (2.349)

9.92 (2.294)

(0.363)

0.0215 (⫺3.834)

0.0076 (⫺4.878)

0.0246 (⫺3.704)

(1.119)

AMF ⫽ anhydrous milkfat, HCO ⫽ hydrogenated coconut oil. Values shown are back-transformed means calculated from the results of the statistical analysis of log transformed data which are shown in parentheses. Sample size (n) ⫽ 26 for AMF, 28 for Max EPA and HCO. c Mean SED ⫽ Mean standard error of the difference. Mean SED is the mean over all comparison of the SED between two means on the transformed scale (see text); df is the approximate associated degrees of freedom. a

b

Although the mean thrombus area measured in pigs fed the fish oil diet was substantially less than that for pigs fed either the anhydrous milkfat or hydrogenated coconut oil diets, the data variability was large and the differences between dietary treatments were not statistically significant (P ⫽ 0.28). Similarly, there were no statistically significant differences between dietary treatments for mean platelet deposition (P ⫽ 0.86). The correlation between platelet deposition and thrombus area across all three diets for all arteries with mild injury was 0.45. The correlation increased to 0.65 when arteries with deep injury were included in the analysis, however, this larger correlation was primarily because of the much larger values recorded when deep injury was present. 3.3. Comparison of deep and mild injury Thrombus area and platelet deposition were both significantly greater in arteries with deep injury when compared with mild injury (P ⬍ 0.01) when averaged across all treatment groups (Table 5). Table 5 Platelet deposition and thrombus area at femoral angioplasty site in association with deep or superficial injury in growing pigs averaged over all three dietary treatments Parameter

Deep injury Platelet deposition (⫻106 c/segment)a Thrombus area (mm2)a a

SEDb (df ⬇ 36)

Injury type Superficial injury

27.06 (3.298)

9.92 (2.294)

(0.331)

0.3322 (⫺1.133)

0.0165 (⫺4.101)

(1.053)

Values shown are back-transformed means calculated from the results of the statistical analysis of log transformed data which are shown in parentheses. Sample size (n) ⫽ 9 for deep injury. 82 for superficial injury. b SED ⫽ standard error of the difference. df is the approximate degrees of freedom.

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4. Discussion Balloon angioplasty of femoral arteries in pigs fed diets containing one of three different dietary fats/oils for 70 days led to a threefold increase in mean thrombus area in the pigs fed either the anhydrous milkfat or hydrogenated coconut oil diets compared with the pigs fed the MaxEPA diet. However, because of the large variability in the data this difference was not statistically significant. The model used in this study developed from descriptions of a carotid angioplasty model previously used in pigs for studying the pathophysiological responses to injury [16,17] and assessing the efficacy of various antithrombotic therapies [18 –23]. In those studies, mural thrombosis was only achieved in arteries subjected to deep injury with exposure of the tunica media [16,17,19,20]. Lam et al. [17] observed macroscopic mural thrombi within an hour of angioplasty in 29 (91%) of 32 carotid arteries that had a medial tear but in none of 24 arteries without deep injury. This is in contrast to the results reported in the present study where macroscopic mural thrombi were detected in all nine arteries with deep injury, and also in 19 (23%) of the remaining 82 arteries that had no evidence of a medial tear, even after microscopic examination of serial sections. Many small platelet-rich thrombi were also detected microscopically, in addition to the expected platelet monolayer that replaced the denuded endothelium. In our study, the aim was to use balloon angioplasty to induce superficial rather than deep injury in femoral arteries. This was based on a preliminary study (unpublished) in which mural thrombosis was demonstrated in femoral arteries with only superficial injury and a concern that deep injury would be more difficult to standardize, thus resulting in greater within-group variability in thrombus size. The marked variation in thrombus size within treatment groups raises the possibility that some thrombi, which formed during the one-hour period after angioplasty were dislodged before the injured segment was harvested. This would be consistent with previous observations in a model system involving exposure of de-endothelialised vessel wall (simulating mild injury) to porcine blood at high shear rate in a perfusion chamber [24,25]. In this system, platelet deposition reached a maximum within 5-10 minutes of exposure and, although thrombus formation occurred, such thrombi could easily be dislodged by flowing blood. Simulation of deep injury by exposure of fibrillar collagen in the vessel wall, not only resulted in greater platelet deposition but also produced thrombi that were not dislodged [26]. In vivo, expression of thromboplastin and tissue factor activity would be expected to further enhance thrombogenicity in vessels with deep injury [27,28]. This is consistent with the increased quantities of fibrin adjacent to the exposed media in thrombi that had formed at sites of deep injury in our study and with the observation of Maihac et al. [29]. The relatively low correlation between thrombus area and platelet deposition was surprising since both would be expected to reflect the total volume of thrombus in each injured arterial segment. The presence of fibrin and trapped erythrocytes in some large thrombi would have contributed to measurements of thrombus area without contributing to the number of radiolabelled platelets and may have partly accounted for the discrepancy, but the variability of both estimates of thrombus size used in this model needs further investigation. In theory, platelet deposition would be expected to provide a more reliable measure of

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thrombus volume since it is based on the number of radiolabelled platelets throughout the entire segment rather than a morphological ‘snap-shot’ at specific sites. In this study, the balloon angioplasty model of arterial thrombosis was used to compare the thrombogenicity of dietary anhydrous milkfat with that of hydrogenated coconut oil, which is considered to be thrombogenic [5], and fish oil, which has antithrombogenic properties [4,30,31]. It was decided to include dietary fats/oils at either end of the ‘thrombogenicity spectrum’ in order to provide the best chance of determining whether or not the model is capable of detecting differences in thrombogenicity between diets. Although the variation within treatment groups in this study was too great for any statistically significant differences to be detected it is premature to suggest that the model is inappropriate for this purpose since the differences in thrombus area did follow the predicted pattern. Deep injury of arteries by angioplasty would be expected to generate larger mural thrombi than those observed in this study and may reduce the variability encountered with superficial injury by increasing the deposition of radiolabelled platelets at the angioplasty site and minimizing the risk of thrombi becoming dislodged prior to harvest. It may also be possible to reduce the variability by using a different artery. In another study, larger thrombi and a smaller standard deviation were obtained in coronary arteries subjected to deep injury than in carotid arteries, presumably because a more consistent degree of injury can be achieved in coronary arteries (JJ Badimon, personal communication). Continuous recording of radioactivity immediately after angioplasty until the injured vascular segment is harvested may also improve the model. This would allow active monitoring of radiolabelled platelets accumulating at the site of injury and provide evidence of the dislodgement of any thrombi during the recording period. Ideally, the artery would need to be exposed surgically so that a gamma probe with collimator could be directed towards the angioplasty site. The femoral artery would be more appropriate than either the carotid or coronary arteries for this purpose. The feeding period of 70 days in this trial allowed sufficient time for changes in platelet membrane fatty acid composition to reflect the fatty acid composition of the diets since platelets in most animal species, including pigs, have an average life-span of less than 10 days [32]. This was confirmed by analyses carried out in our study [13] but alterations in platelet function and thrombogenicity of blood do not necessarily coincide with alterations in platelet membrane composition. Thorngren et al. [33], in a study of healthy men, maintained for 6 weeks on a fish diet, found that although the fatty acid composition of platelet membrane phospholipids changed within one week on the diet, bleeding time did not increase until the sixth week. Similarly, the effect of diet on the aggregability of platelets also failed to mirror changes in their fatty acid composition. Despite these reservations, the variability in the thrombosis end-point encountered in our porcine model would not have allowed detection of differences in thrombogenicity between diets even if they did exist at the end of the 70-day feeding period. Within-group variation in serum lipid profiles in response to dietary manipulation might be expected to contribute to variation in the response of individual animals to balloon angioplasty. However, this is unlikely to have been a contributing factor in the present study where data variability was low enough to allow detection of significant differences between dietary treatment groups in both serum total cholesterol and triacylglycerol concentrations (unpublished data).

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In conclusion, we have shown that mural thrombosis induced by balloon angioplasty can occur in femoral arteries of pigs at sites of superficial injury, but that a model based on this system is too variable to allow detection of differences in thrombogenicity between treatment groups. Deep injury and continuous monitoring of platelet deposition during the 60-minute period following angioplasty may assist in reducing the variability of the thrombosis end-point and provide a suitable model for measuring the comparative thrombogenicity of different dietary fats/oils.

Acknowledgments The roles of the authors in this study were as follows: KGT, experimental design, conduct of trial, surgery, data collation, manuscript preparation; KACJ, experimental design, conduct of trial, experimental diets, manuscript preparation; DGA, experimental design, conduct of trial, surgery, data collection; AJM, experimental design, conduct of trial; RBB, radiolabelled platelet preparation; CLB, conduct of trial; RCB, statistical analyses; PJM, experimental design; WFL, experimental design, manuscript preparation. We thank Mrs JS Shoemark, New Zealand Institute for Crop & Food Research Limited, Palmerston North, and Mr EAC James, for technical assistance in carrying out the pig trial; Mr DV Thomas, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, for supply of diet ingredients, and Mr PM Weber, Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, for supply of pigs. This work was partly supported by a grant from the Foundation for Research, Science and Technology, New Zealand.

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