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ORIGINAL RESEARCH COMMUNICATION |
1 From Cellular and Molecular Nutrition, Instituto de la Grasa, Seville, Spain (YMP, BB, SL, RA, and FJGM), and the Service of Internal Medicine, Hospitales Universitarios Virgen del Rocío, Seville, Spain (JV)
2 YMP and BB contributed equally to this work. 3 Supported by grants from the Ministerio de Ciencia y Tecnología (MCYT) and Ministerio de Educación y Ciencia (MEC) (AGL2001-0584 and AGL2004-04958); fellowships to YMP, BB, and SL; and in part by grants from Puleva Biotech SA (2002C00413) and the Spanish G03/181 Network. 4 Reprints not available. Address correspondence to FJG Muriana, Cellular and Molecular Nutrition, Instituto de la Grasa (CSIC), 41012 Seville, Spain. E-mail: muriana{at}ig.csic.es.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: We investigated whether the ratio of oleic to palmitic acid [and that of monounsaturated to saturated fatty acids (MUFA:SFA)] in the diet affects postprandial concentrations of triacylglycerols, tissue factor (TF), fibrinogen, tissue-type plasminogen activator (t-PA), and plasminogen activator inhibitor 1 (PAI-1).
Design: We studied the effects of diets enriched in olive oil (ROO), high-palmitic sunflower oil (HPSO), butter, or a mixture of vegetable and fish oils (VEFO) on circulating concentrations of the aforementioned factors in 14 healthy men. The fats had ratios of oleic to palmitic acid (MUFA:SFA) of 6.83 (5.43), 2.36 (2.42), 0.82 (0.48), and 13.81 (7.08).
Results: The largest and longest-lasting postprandial changes in plasma triacylglycerol concentrations were found with the butter-based diet (all P < 0.05). No correlation was observed between the net incremental area under the curve (netAUC) for triacylglycerol and the ratio of oleic to palmitic acid (or MUFA:SFA) in the dietary fats. The netAUCs for TF and PAI-1, however, were inversely related to the ratio of oleic to palmitic acid (and MUFA:SFA) in ROO, HPSO, butter, and VEFO. Similar results were found for the fibrinogen netAUC when VEFO was omitted from the analysis. The netAUC for t-PA was inversely correlated with postprandial concentrations of triacylglycerol.
Conclusions: Postprandial concentrations of TF, fibrinogen, and PAI-1 are associated with the ratio of oleic to palmitic acid (MUFA:SFA) in dietary fats. The postprandial t-PA response is related to postprandial concentrations of triacylglycerol.
Key Words: Oleic acid • palmitic acid • postprandial responses • diet • monounsaturated fatty acids • saturated fatty acids • fatty acid ratio
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most persons who consume a Western-style diet are postprandial most of the time. Postprandial triacylglycerol-rich lipoproteins may affect endothelial function and hemostasis in a prothrombotic or a profibrinolytic manner (7, 8). The magnitude of the postprandial lipidic response is determined by several factors, such as the amount and composition of the fat ingested, fasting triacylglycerol concentrations, age, lifestyle, and habitual dietary fat composition (9). It is generally agreed that consumption of butter fat [which contains short-chain and medium-chain saturated fatty acids (SFAs)] but not fats containing long-chain SFAs produces higher postprandial lipemic responses than does the consumption of monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids (10, 11). This suggests that the nature of dietary lipids may have an important regulatory role in the activation of the hemostatic system during the postprandial period (12). It is generally recommended that the dietary intake of fats rich in SFAs (mainly palmitic acid, 16:0) be replaced with those rich in MUFAs (mainly oleic acid, 18:1n–9), which highlights the importance of investigating the effects of high-oleic fats on postprandial events. Thus, we set out to study the effects of natural fats containing different relative amounts of oleic and palmitic acid on postprandial concentrations of TF, fibrinogen, t-PA, and PAI-1 in healthy subjects. We hypothesized that a meal rich in oleic acid (typical of a MUFA-rich Mediterranean diet) and with high ratio of oleic to palmitic acids might be more favorable for the postprandial hemostatic variables than meals with less oleic acid (MUFA) and more palmitic acid (SFA).
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was carried out with 14 healthy, male nonsmokers with no evidence of established coronary artery disease who were recruited by advertising (Table 1
). Subjects were excluded if they displayed biochemical evidence of renal impairment, hypothyroidism, or liver dysfunction.
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). The evening before the postprandial study, the subjects ate a meal relatively high in carbohydrates (60% of energy) made up of mainly plain pasta. Fasting blood samples were taken 12 h after the evening meal (baseline values).
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. The subjects were asked to consume the meal in <15 min, after which time blood was collected from a cubital vein catheterized with a small-bore extension set and a Smartsite needleless valve port (Alaris, San Diego, CA). A blood sample was drawn every hour for a total of 8 h into precooled tubes containing sodium citrate (final concentration, 0.129 mmol/L). The plasma was separated immediately by centrifugation (2000 x g, 4 °C, 20 min), and the aliquots were transferred into sterile, 1-mL cryovials and stored at –70 °C until analyzed further. For a blank test, the subjects who followed the NCEP diet during the second week consumed a test meal containing no fat, to determine the diurnal fluctuations in the thrombogenic and fibrinolytic variables (15, 16). The test meals were well tolerated by the subjects and had no unpleasant side effects. The subjects rested and read during the postprandial period. Only water was available on request.
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Analysis of dietary fats
Fatty acid composition was determined according to the method described in EC/796/2002 (17) with a gas chromatography system (HP-5890; Hewlett-Packard, Palo Alto, CA) with a flame ionization detector and an SP-2380 (Supelco, Bellefonte, PA) fused silica capillary column (50 m x 0.25 mm internal diameter) coated with cyanopropylpolysiloxane (0.25 µm film thickness). The oven temperature program was isothermal at 165 °C for 10 min before rising to 200 °C at 1.5 °C/min. The injector and detector temperatures were 220 °C and 250 °C, respectively. Hydrogen was used as the carrier gas at a column head pressure of 130 kPa. Sample injections were performed in the split mode.
Laboratory measurements
Triacylglycerols and other plasma lipids were quantified by using commercially available reagents on a Hitachi Modular Analytics D-2400 analyzer (Roche Diagnostics, Basel, Switzerland). Hemostatically active fibrinogen was measured according to the method of Clauss by using fibrinogen reagent (Stago Diagnostica, Asnieres-sur-Seine, France). An STA Unicalibrator was used as a reference standard and Preciclot I/II were used as quality controls for accuracy and reproducibility (Stago Diagnostica). TF was assayed by enzyme-linked immunosorbent assay (matched-pair set for human TF; Kordia, Leiden, Netherlands). As reference standards, several dilutions of a recombinant human tissue factor (not lipidated) were used (American Diagnostica, Stamford, CT).
t-PA and PAI-1 were measured with commercial kits (Imulyse t-PA and Imulyse PAI-1, Biopool; Kordia). In addition to active and latent PAI-1, the PAI-1 kit measured the t-PA:PAI-1 and urokinase-PA:PAI-1 complexes, albeit less efficiently. The assay included the use of quenching and normal antibodies to exclude false positives (immunologic specificity and accuracy control, or ISAC). The t-PA kit enabled single-chain and two-chain t-PA antigen to be quantified and used the same double-antibody method (ISAC).
Statistical analysis
Individual data from each subject were plotted and evaluated qualitatively. Statistical analyses were carried out to compare the effects of each fat on the fasting and postprandial values and to analyze the values from each fat at different time intervals. The net incremental area under the curve (netAUC), including the entire incremental area below the curve and the area below the fasting concentration, was analyzed by a one-factor repeated-measures analysis of variance (ANOVA). A Bonferroni correction was used for the post hoc detection of significant pairwise differences. The netAUC was calculated by the trapezoidal method by using Microsoft EXCEL 2000 v.9 (Microsoft Corp, Redmond, WA). Univariate correlation analysis between variables was performed with Pearson’s product-moment correlations. The data were analyzed by using STATVIEW v.5 for WINDOWS (SAS Institute, Cary, NC). The designated level of significance was P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As expected, no plasma triacylglycerol was apparent when the diet was not supplemented with fat. Conversely, there was a marked increase in plasma triacylglycerol when supplementary fat was consumed, which peaked at different times postprandially depending on the fat tested (P < 0.05). Overall, the HPSO supplement was associated with a lower plasma triacylglycerol response (netAUC was 3.75 ± 0.51 mmol · h/L; P < 0.05) than were ROO (4.45 ± 0.67 mmol · h/L) and VEFO (4.42 ± 0.45 mmol · h/L). The largest and longest-lasting changes in plasma triacylglycerol concentrations were found with the butter supplement, which produced the highest postprandial response (6.02 ± 0.82 mmol · h/L). No correlation was observed between postprandial triacylglycerol responses (netAUC) and the ratio of oleic to palmitic acid (MUFA:SFA) with ROO, HPSO, butter, and VEFO (Table 4).
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). There was then a sudden clearance of plasma TF to baseline values, except when butter was used for fat enrichment. Indeed, the butter supplement induced the highest TF postprandial response (netAUC was 33.03 ± 4.52 ng · h/L; P < 0.05; Figure 3). Moreover, the netAUC for TF differed significantly (P < 0.05) after the ingestion of the different fat supplements analyzed in the following descending order: HPSO (23.57 ± 5.11 ng · h/L), ROO (11.09 ± 1.65 ng · h/L), and VEFO (–13.75 ± 2.47 ng · h/L). No correlation was observed between the triacylglycerol and TF postprandial responses (netAUC) after the ingestion of any of the fat supplements. Nevertheless, a significant inverse correlation was observed between the netAUC for TF and the ratio of oleic to palmitic acid (MUFA:SFA) with ROO, HPSO, butter, and VEFO (P < 0.05; Table 4
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and 3; P < 0.05). When butter or no fat was added to the meal, there was a similar slight decrease in plasma fibrinogen with respect to the baseline value (–0.47 ± 0.07 g · h/L for butter, –0.38 ± 0.08 g · h/L in the absence of the fat supplement). However, VEFO enrichment produced an even greater decrease (–2.29 ± 0.35 g · h/L; P < 0.05). No correlation was observed between the postprandial triacylglycerol and fibrinogen responses (netAUC) after the ingestion of any test fat, but there was a significant positive correlation between the netAUC for fibrinogen and the ratio of oleic to palmitic acid (MUFA:SFA) after ROO, HPSO, and butter enrichment (Table 4; P < 0.05).
Postprandial fibrinolytic factors
Normal diurnal variations in t-PA and PAI-1 were observed in all subjects (Figure 2). However, when the meal was enriched with butter or when no fat was supplemented, there was a comparable drop in the plasma t-PA response from the baseline value (the netAUC was –15.66 ± 2.01 µg · h/L for butter and –15.41 ± 2.46 µg · h/L in the absence of the fat supplement; Figure 3). HPSO enrichment induced a significantly smaller change in the netAUC value for t-PA (–9.13 ± 1.24 µg · h/L; P < 0.05), whereas ROO and VEFO enrichment had a similar effect (–12.39 ± 1.63 µg · h/L for the ROO meal and –11.79 ± 1.37 µg · h/L for the VEFO meal). No correlation was observed between the t-PA postprandial responses (netAUC) and the ratio of oleic to palmitic acid (MUFA:SFA) after any fat supplement, but there was a significant inverse correlation between the netAUC for t-PA and triacylglycerol after the ingestion of ROO, HPSO, butter, and VEFO (Table 4; P < 0.05).
Diurnal postprandial PAI-1 concentrations were significantly higher after the ingestion of butter than after the other fat supplements (Figure 2; P < 0.05). Moreover, butter and HPSO enrichment significantly reduced the changes in the netAUC value for PAI-1 (–55.17 ± 8.35 µg · h/L for the butter meal and –64.95 ± 9.16 µg · h/L for HPSO; P < 0.05; Figure 3). When ROO or VEFO was added to the meal, there was a comparable decrease in plasma PAI-1 with respect to the baseline value (–76.13 ± 8.83 µg · h/L for ROO and –75.42 ± 7.46 µg · h/L for VEFO). No correlation was observed between the triacylglycerol and PAI-1 postprandial responses (netAUC) after the ingestion of any test fat. However, there was a significant inverse correlation between the netAUC for PAI-1 and the ratio of oleic to palmitic acid (MUFA:SFA) after ROO, HPSO, butter, and VEFO enrichment (Table 4; P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Postprandial triacylglycerol responses were similar for dietary enrichment with ROO and VEFO. This agrees with a recent study in which no difference was found in postprandial concentrations of triacylglycerol after MUFA enrichment in the presence or absence of n–3 long-chain polyunsaturated fatty acids (LCPUFAs; 19). Our results also showed that the postprandial triacylglycerol response was significantly higher after the butter supplement, whereas it was lowest after HPSO enrichment. Indeed, butter as a fat source was previously shown to cause an increase in postprandial triacylglycerol concentrations when compared with ROO (11). In contrast, no differences were observed in other studies involving diets enriched in high-palmitic or high-oleic fats (20), nor were differences identified between butter fats and ROO in terms of lower postprandial triacylglycerol concentrations (21). These discrepancies are probably due to differences in the amount and nature of the fats administered, the composition of the test meals, the standardization of the diet before the postprandial study, and the subjects involved. In accordance with a recent study with interesterified fats in healthy young males, we did not observe any association between the postprandial triacylglycerol response and the oleic to palmitic acid ratio (MUFA:SFA) in dietary fats (22).
The present study focused on the influence of the oleic to palmitic acid ratio (MUFA:SFA) in dietary fats on the postprandial responses of TF, fibrinogen, t-PA, and PAI-1. Despite observations indicating that thrombotic complications might be mediated by high circulating concentrations of TF (23) and might be accelerated during postprandial lipemia (24), few data are available regarding the postprandial effects of dietary fats on TF. In contrast, much more information exists regarding the effects of dietary fat composition on postprandial concentrations of activated factor VII (FVIIa; 12). Activation of FVII by TF represents a critical event in thrombogenesis. Indeed, the transient rise in FVIIa after a fat-rich meal is generally detectable 2–3 h postprandially and persists for 
8 h, thus displaying dose-response characteristics similar to those reported here for TF. The increase in FVIIa is correlated with fasting triacylglycerol concentrations, but not with postprandial triacylglycerol concentrations (25). Interestingly, the main dietary determinants of postprandial changes in FVIIa are oleic and palmitic acid (26), which agrees with the correlation found between the netAUC for TF and the ratio of oleic to palmitic acid (MUFA:SFA) in the ROO-, HPSO-, butter-, and VEFO-enriched meals. Nevertheless, the link between TF and FVIIa and their equivalence as procoagulant postprandial markers given a controlled dietary background should be studied further.
Elevated fibrinogen appears to be a risk factor for arterial thrombosis (27), which is relevant in the postprandial period because fibrinogen can bind to newly created triacylglycerol-rich lipoproteins (28). The coating of fibrinogen with triacylglycerol-rich lipoproteins involves hydrophobic patches on the surface of the lipoproteins. It is therefore likely that the high postprandial fibrinogen response observed after the intake of the ROO supplement was a consequence of an increase in surface lipoproteins due to the small number and larger size of nascent postprandial triacylglycerol-rich lipoproteins from ROO (29, 30). In our study, the netAUCs for fibrinogen after the ROO, HPSO, and butter supplements were significantly correlated with the ratio of oleic to palmitic acid (MUFA:SFA). The lower netAUC for fibrinogen after VEFO enrichment and the absence of such a correlation when the VEFO meal was included in the analysis suggest that ingestion of a low amount of n–3 LCPUFAs could reverse this effect on postprandial fibrinogen concentrations. Thus, our data stress importance of n–3 LCPUFAs as a determinant of the direction and magnitude of the postprandial response of fibrinogen to dietary fats. Similar observations were made regarding postprandial vascular reactivity to high-MUFA meals (19), with the presence of n–3 LCPUFAs reverting the pattern of fibrinogen while exerting no effect on postprandial triacylglycerol concentrations. The present study confirms that n–3 LCPUFAs may partially reduce the thrombotic potential of fat-rich meals (31, 32).
In prospective clinical (33) and in multicenter (34) studies of angina pectoris and postinfarction patients, the t-PA antigen can serve to predict subsequent acute coronary syndromes. Our data showed a marked decline in postprandial t-PA and PAI-1 concentrations from 0 to 8 h after all meals, which agrees with the strong diurnal (characteristic U-shaped) variations in net fibrinolytic activity (15, 16). We found that butter enrichment induced the lowest postprandial t-PA response, as is commonly observed for SFA-rich diets (35). This suggests that butter enrichment does not decrease postprandial clot lysis mediated by t-PA when compared with ROO, HPSO, and VEFO. We also observed a significant correlation between the netAUC for t-PA and the netAUC for triacylglycerol, but not with the ratio of oleic to palmitic acid (MUFA:SFA) after ROO, HPSO, butter, and VEFO enrichment. Notably, fasting t-PA antigen is linked to the metabolic syndrome, and it is positively related to fasting triacylglycerol and insulin, as well as to the waist-to-hip ratio in familial combined hyperlipidemia (36). However, our data show that the postprandial triacylglycerol response and not dietary fatty acid composition influences postprandial concentrations of t-PA antigen in healthy subjects.
Circulating PAI-1 is derived from a variety of sources, including the vascular endothelium, adipose tissue, and liver, and it is involved in the onset of obesity, diabetes, and cardiovascular disease (37). Notable ROO, HPSO, and butter enrichment induced a peak in PAI-1 2 h postprandially. This novel finding of postprandial PAI-1 peaks had remained undetected in other studies, possibly because of the failure to take blood samples under fasting conditions and hourly over 8 h. In addition, the netAUC for PAI-1 was significantly correlated with the ratio of oleic to palmitic acid (MUFA:SFA) in the ROO-, HPSO-, butter-, and VEFO-enriched meals. Butter enrichment produced the lowest ratio of oleic to palmitic acid (MUFA:SFA) and induced the highest PAI-1 postprandial response. A similar temporary increase in postprandial concentrations of PAI-1 antigen and impaired fibrinolytic activity after a butter-enriched meal was reported previously (38). Moreover, improved fibrinolytic activity has been associated with a decline in postprandial PAI-1 after high-MUFA (oleic acid) meals (39, 40). Together, these observations could have important clinical consequences, because the morning peak in PAI-1 antigen corresponds with the circadian peak in the incidence of acute myocardial infarction (41).
One limitation of the current study was that we evaluated the effects of meals containing large amounts of specific dietary fats that did not correspond in fatty acid content to the background diets to which the subjects had become accustomed. To date, the effect of varying the fatty acid composition in individuals who habitually consume comparable background diets is uncertain.
In conclusion, our novel data are consistent with the view that the ratio of oleic to palmitic acid (MUFA:SFA) in dietary fats has a regulatory influence on certain thrombogenic and fibrinolytic markers during the postprandial state in healthy subjects. Increases in postprandial concentrations of TF (prothrombotic effect) and PAI-1 (antifibrinolytic effect) were observed when the ratio of oleic to palmitic acid decreased (MUFA:SFA). Postprandial concentrations of fibrinogen are also sensitive to n–3 LCPUFAs, whereas postprandial concentrations of t-PA are dependent on those of triacylglycerol. We suggest that a Mediterranean diet can reduce cardiovascular events in part due to the high oleic acid content of olive oil (and thus the high oleic-to-palmitic acid and MUFA-to-SFA ratios), the moderate consumption of fish rich in n–3 LCPUFAs, and the combined effect of these factors on the postprandial hemostatic system.
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ACKNOWLEDGMENTS |
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YMP, RA, and FJGM participated in the study design and performed the data analysis, and FJGM wrote the manuscript. YMP and RA assisted with the editing of the manuscript. YMP, BB, SL, and JV were involved in the lipid analysis, and JV was responsible for recruiting patients and collecting samples. YMP participated in the development of the study menus and was responsible for implementing the dietary protocol. All authors participated in the discussion of the results and approved the final version of the manuscript. None of the authors had any personal or financial conflicts of interest.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or a meal supplemented with olive oil (ROO; 
), high-palmitic sunflower oil (HPSO; 
), butter (
), or a mixture of vegetable and fish oils (VEFO; 
). n = 14. There was a significant effect of the type of fat, a significant time effect, and a significant meal x time interaction (P < 0.05). Bars not sharing a common letter are significantly different, P < 0.05 (repeated-measures ANOVA with Bonferroni correction).
