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,**1
*
Department for Thrombosis Research, University of Southern Denmark and Department of Clinical Biochemistry, Ribe County Hospital, Esbjerg, Denmark;
Department of Pharmacology and Pathobiology, and
**
Research Department of Human Nutrition, Royal Veterinary and Agricultural University, Frederiksberg, Denmark
1To whom correspondence should be addressed. E-mail: aako{at}ribeamt.dk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • animal models • diet • triglycerides • thrombosis • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Increased FVII coagulant activity (FVIIc) is believed to be associated with an increased risk of fatal ischemic heart disease (2
–5
), even though some studies have not confirmed this association (6
–8
). The postprandial elevation of FVIIc after consumption of high fat meals is due to activation of FVII protein (9
–15
). Contrary to what might be expected, postprandial FVII activation was not associated with increased concentrations of prothrombin fragment 1 + 2 (F1 + 2), a marker of thrombin generation in healthy volunteers (14
,16
–17
) and in patients with coronary atherosclerosis and stable angina pectoris (18
). This may be explained by the following: 1) absent or inadequate tissue factor (TF) expression; 2) tissue factor pathway inhibitor inhibition of activated FVII (FVIIa)-TF complexes; 3) increased hepatic clearance of F1 + 2 after a high fat meal; or 4) the possibility that local thrombin generation at sites of TF expression does not lead to systemic increase in F1 + 2. Therefore, it remains unknown whether the postprandial increase in FVIIa has any clinical importance.
Each of these possible explanations could be investigated in greater detail in an animal model. We recently rejected the minipig as a model for the study of thrombogenic effects of postprandial FVII activation because postprandial activation could not be achieved in this species (19
). In the present study, we tested the possibility of using the rat as a model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animal experiments were performed in accordance with the Danish Animal Experimentation Act on a license granted by the Ministry of Legal Affairs. All housing and procedures were performed according to the Convention ETS 123 of the Council of Europe.
The experiment was performed on 100 barrier-raised inbred female LEW/Mol rats weighing 144–183 g (median 166 g) (M&B, Ll. Skensved, Denmark). The rats had undergone health monitoring according to the Federation of European Laboratory Animal Science Association guidelines (20
). The rats were maintained in groups of 2–3 in U-1400 cages (Techniplast, Buguggiate, Italy) on aspen bedding (Finn Tapvei, Kaavi, Finland) at 22°C ± 1°C, at a relative humidity of 55–80%, and a light-dark cycle of 12 h (lights on, 0600–1800 h). The rats had free access to a standard laboratory pelleted diet (Altromin 1324, Chr. Petersen, Ringsted, Denmark) and tap water. Altromin 1324 contains 40 g crude fat/kg, 190 g crude protein/kg, 70 g fiber/kg and 11.9 MJ metabolizable energy/kg. The amount of food consumed during the study was recorded.
Lipid administration.
The study was performed in fed rats. Before the experiments, the rats were randomized into 1 group of 16 (A), and 6 groups of 14 (B–G). Recently, a method to obtain postprandial hyperlipidemia in rats was developed (Larsen, L. F., et al., Hansen, A. K., et al., unpublished observation). At 0800 h (t = 0), 1.4 g fat/rat (groups B–D) or 0.165 g glucose/rat (groups E–G) was administered through a gastric tube to unanaesthetized rats. Rats in group A were not intubated, and they were used to determine the baseline levels. A mixture of 2 mL Intralipid, 20% (Fresenius Kabi AB, Uppsala, Sweden) and 1 mL olive oil (Santagata Lurgi, Genoa, Italy) was used as fat source, and 3 mL isotonic glucose, 55g/L (Sygehusapotekerne, Denmark) was used as glucose source. The fatty acid composition of Intralipid/olive oil mixture was analyzed using gas chromatography.3
Blood sampling.
Immediately before blood sampling, rats were anesthetized by subcutaneous injection of a mixture containing 0.02 mg fentanyl, 0.75 mg fluanisone (Hypnorm, Janssen, Beerse, Belgium) and 0.38 mg midazolam (Dormicum, Roche AG, Basel, Schweiz) (total volume of 0.3 mL). Blood samples were obtained at baseline (group A), 2 h (groups B and E), 4 h (groups C and F) and 6 h (groups D and G) after fat/glucose administration. Heart punctures were performed with the rats in dorsal recumbency without awareness of the rat’s group designation. A 21-gauge, 25-mm long needle was inserted into the heart, and blood was sampled by use of a Vacutainer system (Terumen, Leuven, Belgium). The first 0.5 mL was collected in EDTA tubes and used for thrombin-antithrombin complex (TAT) analyses. Immediately after sampling, 10 µL D-phe-pro-arg chloromethylketone (2.63 g/L) (Calbiochem, Bad Soden, Germany) was added, and the tube was stored on crushed ice. The next 1.8 mL of blood was collected in 0.2 mL of 0.129 mol/L trisodium citrate at room temperature and used for FVII analyses. Finally, 1 mL blood was collected in plain tubes and used for triglyceride analyses. Blood sampling failed in one rat in group C. Immediately after blood sampling, the rats were killed by cervical dislocation. Within 2 h after blood sampling, citrated tubes were centrifuged at 20°C for 20 min at 2000 x g, and EDTA and plain tubes were centrifuged at 5°C for 20 min at 2000 x g. Serum and plasma were pipetted into plastic vials and stored at -70°C.
Blood analyses.
Serum and plasma samples were thawed rapidly in a water bath at 37°C and analyzed in one series for each variable. Serum triglycerides were analyzed by a commercial enzymatic method on a Vitros 950 (Johnson & Johnson Clinical Diagnostics, Rochester, NY). Plasma FVIIa was analyzed with the Staclot VIIa-rTF assay (Diagnostica Stago, Asnières, France), which contains human recombinant truncated TF specific for human FVIIa cofactor function. The analyses were performed on an ACL 7000 (Automatic Coagulation Laboratory, Instrumentation Laboratory, Milan, Italy). Concentrations of FVIIa (U/L) were estimated from a calibration curve prepared from human recombinant FVIIa. Plasma FVIIc was analyzed with a one-step clotting assay using human placenta thromboplastin (Thromborel S, Dade Behring, Marburg, Germany), and the measurements were performed on a BCT (Behring Coagulation Timer, Dade Behring). Levels of FVIIc were estimated from human plasma calibrators (Dade Behring) and expressed as percentages of these. It has previously been demonstrated that human TF reacts with rat FVII (21
). Plasma FVII amidolytic activity (FVIIam) was analyzed by a chromogenic method (Coa-set FVII, Kabi Diagnostica, Mölndal, Sweden) and expressed as a percentage of a human standard. FVIIam is an estimate of the FVII protein concentration (22
). TAT were analyzed with a commercial ELISA (Enzygnost TAT micro, Dade Behring). It has previously been demonstrated that this assay can detect TF-induced thrombin generation in rat plasma (23
).
Statistics.
The aim was to detect a postprandial increase in FVIIa concentration of at least 15 U/L at a significance level of 5% and a power of 85%. According to our pilot study, the population SD of FVIIa in rat plasma is 11 U/L. We decided on a group size of 14 rats, which was based on an ANOVA sample size calculation. The results were analyzed with nonparametric statistics due to the non-Gaussian distribution of residuals in some of the variables. Group differences within the fat-treated rats (Groups A–D) and glucose-treated rats (Group A, E–G) were analyzed with Kruskal-Wallis one-way ANOVA; if significant differences were found (P < 0.05), Dunn’s method was used to do paired comparisons. Differences between fat and glucose administration for each time point were analyzed with the Mann-Whitney rank sum test. The software Sigmastat 2.3 (Microsoft, Redmond, WA) was used for the calculations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Table 1
). Blood coagulation FVIIc, FVIIam and TAT were unaffected by fat administration. Rats fed glucose exhibited no postprandial changes in any variable (Fig. 1
, Table 1
). There were no differences in the ad libitum consumption of food between the groups (from 0700 h to the time of blood sampling: median, 1 g /rat). The baseline triglyceride and TAT concentrations were within the reference interval for rats (23
–24
), but FVIIc was higher than normally observed (25
). FVIIa and FVIIam have not previously been analyzed in plasma from rats. We found lower concentrations of FVIIa and higher concentrations of FVIIam in rats compared with humans.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Table 1
). No changes were observed in FVIIc, FVIIam and TAT. The fat-induced postprandial FVII activation observed indicates that the LEW/Mol rat is a relevant animal model for further studies of dietary FVII activation.
Consumption of 1–2 g fat/kg body leads to postprandial FVII activation in humans (10
–11
,14
). We used a dose of 1.4 g fat/rat (8.4 g fat/kg), metabolically corresponding to a dose of 1.8 g fat/kg in humans, as calculated from the formula: dose (g fat/kg) = 8.4 g/kg x 70-0.25/0.166-0.25 (assuming human body weight of 70 kg and rat body weight of 166 g) (26
). We observed that this amount of fat increases triglycerides and activates FVII also in rats.
No FVII activation was observed in the glucose-treated rats, indicating that the fat-induced postprandial FVII activation was not an artifact arising from gastric intubation or circadian variation, for example. This is also in agreement with human studies in which the postprandial FVII profiles seemed to be strongly influenced by the total fat content of the meal (1
). Only FVIIa concentrations were significantly raised after the fat load, whereas FVIIc and FVIIam were unchanged. This is consistent with human studies in which postprandial FVII activation is accompanied by only minor changes in FVIIc and no changes in FVIIam (10
,14
–16
). The magnitude of the postprandial FVII activation, and the timing of the FVIIa increase and its relation to the triglyceride peak are also comparable to what we have seen in humans (14
,18
). Blood coagulation FVIIc and FVIIam levels were higher than observed in humans. It has been shown by others (21
) that TF(human)-FVIIa(rat) complexes are hyperreactive compared with TF(rat)-FVII(rat) complexes. We used human TF in our clotting assays, which may explain the high FVIIc and FVIIam levels.
Because rats comprise different and heterogeneous stocks and strains, we conducted a pilot study in three strains of rats, i.e., Dark Agouti, Long Evans and LEW/Mol. Postprandial FVII activation was observed after a fat load in all three strains (results not presented), indicating that dietary FVII activation is a general phenomenon in rats. We selected the LEW/Mol rat for the present experiments due to the common use of this inbred strain in biochemical research and its easy handling. To obtain sufficient quantities of plasma and serum for analysis, blood was sampled only once from each rat (by heart puncture), increasing the number of animals needed (see power calculation in "Statistics").
Only one animal study of postprandial FVII activation has been published (19
). In that study, 4 g fat (Intralipid)/kg did not activate FVII in minipigs. The different postprandial FVII response in rats and minipigs is unexplained but may be related to differences in postprandial lipoprotein metabolism. Alternatively, the difference may arise from differences in the coagulation system in the two species. Pigs have considerably higher concentrations of factor X and the intrinsic factors IX, XI and XII than do rats and humans (24
); intrinsic coagulation may predominate in these animals, and it has been suggested that activation of intrinsic factors (e.g., factor IX) are intermediate steps in postprandial FVII activation (27
–31
). Because the concentrations of intrinsic factors are lower and the FVII activity higher in rats, the extrinsic coagulation system may play a more dominant role in blood coagulation in rats, like in humans (32
), and this may explain why FVII in rats is more sensitive to postprandial activation.
We used TAT as a marker of thrombin generation in the present study. We would have preferred to use F1 + 2 as a marker in line with most human studies, but it is not possible to detect F1 + 2 in rat plasma with available assays (23
). Most (84%) rats had TAT concentrations below the detection limit (2.0 µg/L). This may be explained by reduced affinity of anti-human-TAT antibodies against rat-TAT. However, in a previous study, four doses [(4.5, 45, 90, and 450 µL/(kg · h)] of thromboplastin were infused in rats, and dose-response effects of thromboplastin infusion on TAT concentrations were observed. The highest response (from <2 µg/L at baseline to 34 µg/L at the end of thromboplastin infusion after 1 h) was obtained with the highest dose of thromboplastin. Infusion of a thrombin inhibitor [Hirudin; 1 mg/(kg · h)] eliminated the TAT increase. We therefore believe that a real increase in TAT concentration can be detected with the assay used. Our observations indicate that postprandial FVII activation is not associated with increased thrombin generation in healthy rats, in agreement with observations in humans (14
,16
–18
). However, if the rat is used as a thrombosis model, and FVII is activated in the presence of high tissue factor concentrations, for example, TAT concentrations are expected to rise. Future studies will address this question.
In conclusion, our observations suggest that the LEW/Mol rat is a promising animal model for further studies of possible thrombogenic effects of postprandial FVII activation in humans (33
). Results of our present rat study and our previous minipig study (19
) indicate that the choice of animal species is crucial when establishing a model for the investigation of nutritional effects on blood coagulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 The fatty acid composition of the lipid mixture (g/100 g) was as follows: 16, 10%; 18, 3%; 18:1(n-9), 60%, 18:1(n-7), 2%; 18:2(n-6), 21%; 18:3(n-3), 2%; 22, 1%; and other fatty acids, 1%.
Manuscript received 6 September 2001. Revision accepted 14 December 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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