点击显示 收起
1 From the Nutrition Food and Health Research Centre, King's College London, and the Medical Research Council Epidemiology and Medical Care Unit, the Royal London School of Medicine and Dentistry, London.
2 Supported by a grant from Cultor Food Science. 3 Address reprint requests to TAB Sanders, Department of Nutrition and Dietetics, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, United Kingdom. E-mail: tom.sanders{at}kcl.ac.uk.
ABSTRACT
Background: An elevated postprandial lipid concentration is believed to be atherogenic and to increase the risk of thrombosis.
Objective: The objective was to test whether the consumption of a stearic acid–rich structured triacylglycerol has adverse effects on postprandial fibrinolytic activity and lipemia, factor VII coagulant (FVII:c) activity, and activated FVII (FVIIa) concentrations.
Design: A randomized crossover design was used to compare the effects on middle-aged healthy men (n = 17) and women (n = 18) of meals enriched with cocoa butter, high-oleate sunflower oil (oleate), or a structured triacylglycerol containing stearic acid.
Results: The mean increases from fasting in plasma triacylglycerol 3 h after the oleate, cocoa butter, and structured triacylglycerol meals were 1.36 (95% CI: 1.17, 1.56), 1.39 (1.17,1.63), and 0.65 (0.50, 0.82) mmol/L, respectively. Tissue plasminogen activator activity increased and plasminogen activator type 1 activity decreased after all 3 meals. Plasma FVII:c increased after the oleate and cocoa butter meals but not after the structured triacylglycerol meal. The values 6 h after the oleate and cocoa butter meals were 11.3% (7.0%, 15.6%) and 9.9% (4.7%, 15.2%), respectively, and were significantly different (P < 0.0001 and P = 0.001, respectively) from the value after the triacylglycerol meal [2.1% (-1.1%, 5.3%)]. Plasma FVIIa increased after all 3 meals, more so after the oleate and cocoa butter meals than after the structured triacylglycerol meal.
Conclusion: The consumption of stearic acid in the form of a structured triacylglycerol leads to less of an increase in plasma triacylglycerol and in FVII:c than does a meal enriched in cocoa butter or oleate.
Key Words: Factor VII • oleic acid • postprandial lipemia • saturated fatty acids • stearic acid • thrombosis • triacylglycerol • plasminogen activator
INTRODUCTION
An intake of >15 g triacylglycerols containing long-chain fatty acids (14 carbons) results in alimentary lipemia (1). Exaggerated postprandial lipemia is associated with accelerated atherosclerosis; chylomicron remnants can result in foam cell formation and become atherogenic (2). Alimentary lipemia may also increase the risk of coronary thrombosis by increasing factor VII coagulant (FVII:c) activity (3) and plasminogen activator inhibitor type 1 (PAI-1) activity (4). Besides the intake of fat, age, sex, and physical activity the extent of postprandial lipemia is influenced by (5). Some studies also suggested that saturated fatty acids increase postprandial lipemia (6). Several studies suggested that stearic acid (18:0) may have effects similar to those of oleic acid (18:1n-9) on fasting plasma LDL concentrations but may lower HDL cholesterol (7). It has been proposed that elevated plasma triacylglycerol concentrations promote cholesterol ester exchange reactions mediated by cholesteryl ester transfer protein (8). In addition, elevations in plasma triacylglycerol could result in HDL being transferred to triacylglycerol in exchange for cholesteryl ester with the consequence that the triacylglycerol-rich HDL particle becomes substrate for hepatic lipase, resulting in an increased clearance of HDL by the liver. Consequently, it can be hypothesized that a prolonged elevation in plasma triacylglycerol postprandially could lead to a decrease in HDL-cholesterol concentrations. A prospective study found that the intake of stearic acid is associated with an increased risk of coronary heart disease (9). It has been argued that stearic acid, unlike oleic acid, may promote thrombosis. Early studies in animals found that injection of stearate induced thrombosis (10) and subsequent studies (11) found that stearic acid increases FVII:c in vitro. Two studies reported an association between the proportion of stearic acid in plasma fatty acids and FVII:c activity (12, 13). It has been postulated that dietary stearate (18:0) activates factor XII and hence FVII by forming a negatively charged contact surface on triacylglycerol-rich lipoproteins (11).
FVII:c is a functional assay of factor VII activity that depends on the concentration of the zymogen FVII antigen (FVII:Ag) and the concentration of FVII circulating in the activated form (FVIIa). After a high-fat meal, FVIIa increases (14) but there is no change in FVII:Ag. Tholstrup et al (15) reported that a diet enriched in stearate results in lower fasting FVII:c than does a diet rich in palmitate or myristate. In that study, this change was accompanied by a lower plasma cholesterol concentration. In a later study, however, Tholstrup et al (16) suggested that a stearate-rich meal increases FVII:c postprandially but that the increase is not statistically significant. Mutanen et al (17) found no effect of a stearate-rich diet on FVII:c activity despite recording a decrease in LDL-cholesterol concentrations. Mennen et al (18) reported similar postprandial increases in FVIIa concentrations after meals enriched with stearate, palmitate, or linoleic acid. However, we observed previously that oleate results in a greater postprandial increase in FVII:c and FVIIa than does a meal rich in stearate (19). Synthetic structured triacylglycerols that contain stearic acid and short-chain fatty acids—mainly acetic, propionic, and butryic acids—are currently being used as a substitute in chocolate confectionery for cocoa butter, which is also rich in stearic acid. The aim of the present study was to compare the effects of such a structured triacylglycerol on postprandial changes in FVII:c and fibrinolytic activity with those of an oleate-rich triacylglycerol and cocoa butter.
SUBJECTS AND METHODS
Subjects
Volunteers were recruited from among staff and alumni of King's College London and from a general practice in north London. The subjects were healthy and exclusion criteria included the following: body mass index (in kg/m2) <22 or >35; plasma cholesterol <5.2 or >7.8 mmol/L; plasma triacylglycerol >3.0 mmol/L; self-reported weekly alcohol intake of >28 (for men) or 21 (for women) standard units of alcohol; a history of cholestatic liver disease, pancreatitis, diabetes mellitus, or myocardial infarction, use of antihypertensive or lipid-lowering medications; and current use of anticoagulant or antiplatelet agents (except for aspirin, which does not affect the postprandial increase in FVII:c induced by dietary fat). Fasting plasma lipoprotein and lipid concentrations, body weight, red and white blood cell counts, and liver function were confirmed to be within prescribed limits before entry into the study. The women were confirmed to be nonpregnant with the use of a pregnancy test. A total of 35 adults (18 women and 17 men) aged 40–60 y completed the study; 4 of the female subjects were current smokers. The subjects received a modest financial reimbursement for their participation in the study. The study protocol was reviewed and approved by the local human experimentation committee and all participants gave written, informed consent. Some characteristics of the subjects are shown in Table 1.
View this table:
TABLE 1. . Characteristics of the subjects1
Study design
A randomized crossover orthogonal Latin-square design was used. Each subject received 3 experimental meals [a structured triacylglycerol containing stearic acid (A), cocoa butter (B), or high–oleic acid sunflower oil (C)] in random order, 1 wk apart, over a 3-wk period. Subjects were randomly allocated to 1 of 6 treatment sequences (ABC, BCA, CAB, ACB, CBA, or BAC). Subjects were asked to avoid consuming foods high in fat the day preceding each test meal and to fast overnight beginning at 2200. Fasting venous blood samples were obtained between 0800 and 1000 the next morning. The test meal was consumed within 15 min and additional venous blood samples were obtained 3 and 6 h later. After the 3-h blood sample was taken, subjects consumed a standardized lunch (1.7 MJ) consisting of fresh fruit and low-fat yogurt (<1 g fat). To control for physical activity levels, subjects were asked to refrain from strenuous exercise, including cycling and sporting activities, and from the use of alcohol on the day before and on the day of the test meal.
Formulation of the test meals
The test meals consisted of a muffin and a freshly prepared strawberry milk shake and contained 50 g fat, 17 g protein, and 48 g carbohydrate. Each muffin contained 30 g test fat, 10 g wheat flour, 5 g cornstarch, 5 g cocoa powder, 15 g sugar, 20 mL skim milk, 2 g pasteurized egg white, 2 g vanilla essence, and 2 g baking powder. The test fats consisted of a high-oleate sunflower oil, cocoa butter, and a stearic acid–rich structured triacylglycerol (SALATRIM, type 23SO; Cultor Food Science, Ardsley, NY). The muffins were made in a single batch and stored at -20°C; their composition was confirmed by chemical analyses. The fatty acid composition of the test muffins was determined by gas-liquid chromatography of the fatty acid butyl esters. Because of the high volatility of short-chain fatty acids, it was necessary to prepare butyl esters as opposed to the methyl esters for analysis by gas-liquid chromatography. This was accomplished by reaction with 5 g HCl/L butanol. The fatty acid compositions of the high-oleate sunflower oil and safflower oil were determined by gas-liquid chromatography of the methyl esters, which were prepared by reaction with 5 g HCl/L methanol:toluene (4:1, by vol). The milk shake consisted of 150 g fresh strawberries, 150 g skim milk, 10 g pasteurized egg white powder, and 20 g vegetable oil. The milk shake accompanying the oleate test meal contained 20 g high-oleate sunflower oil and that accompanying the stearic acid–rich structured triacylglycerol and cocoa butter meals contained 14 g high-oleate sunflower oil and 6 g high-linoleate sunflower oil. This was done so that the linoleic acid content would be similar in the 3 meals. The analyzed fatty acid composition of the test meals is shown in Table 2.
View this table:
TABLE 2. . Analyzed fatty acid content of test meals enriched in oleate, stearic acid–rich triacylglycerol, or cocoa butter
Blood sample collection and handling
Venous blood samples were collected into evacuated tubes with the minimal compression necessary to display the vein. The first 4.5 mL blood was drawn into a tube containing no anticoagulant; the blood was allowed to clot and serum was separated by centrifugation at 1500 x g for 15 min at room temperature and stored at -20°C for measurement of serum triacylglycerol. For determination of plasma FVII:c activity and plasma FVIIa, FVII:Ag, PAI-1, and D-dimer concentrations, 4.5 mL blood was collected into 0.5 mL of 38 g trisodium citrate solution/L at room temperature and centrifuged at 1500 x g for 15 min at 20°C. To check the quality of the venipuncture sample, a 4.5-mL sample was collected into 0.5 mL of an anticoagulant mixture of Trasylol, EDTA, and a thrombin inhibitor (Byk-Sangtec, Dietzenbach, Germany) in a chilled tube and centrifuged at 1500 x g for 15 min at 4°C for determination of plasma fibrinopeptide A (FPA), which is a marker of thrombin activity on fibrinogen. Blood (4.5 mL) for the tissue-type plasminogen activator (tPA) assay was collected into precooled tubes containing 0.5 mL of 0.5 mol citrate buffer/L, pH 4.0, which results in a final pH of 5.5. Plasma was separated by centrifugation at 1500 x g for 15 min at 4°C. All plasma samples were snap frozen in liquid nitrogen and stored at -70°C until analyzed. Blood samples were processed within 1 h of blood collection.
Analytic methods
Serum triacylglycerol concentrations were measured by enzymatic assay (GPO-PAP; Boehringer-Mannheim, Lewes, United Kingdom). Plasma total and HDL cholesterol were measured by using reagents purchased from Wako Chemicals (Neuss, Germany) with a Cobas analyzer (Roche Diagnostics, Welwyn Garden City, United Kingdom). Plasma FVII:c activity was measured with a one-stage, semiautomated bioassay by using rabbit-brain thromboplastin (Diagen; Thame, Oxon, United Kingdom) and an FVII– deficient substrate plasma prepared as described elsewhere (20). Plasma FVIIa concentrations were measured according to the bioassay method described by Morrissey et al (21). Plasma FVII:Ag concentrations were determined with an enzyme-linked immunosorbent assay (Novo-Nordisk, Copenhagen). Plasma FPA was determined with a commercially available radioimmunoassay (Byk-Sangtec, Dietzenbach, Germany). Plasma D-dimer concentrations were measured by enzyme-linked immunosorbent assay (Chromogenix AB, Mölndal, Sweden); tPA and PAI-1 activities were determined by chromogenic assay (Chromogenix AB). All hemostatic assays were carried out at the coagulation laboratory at the Medical Research Council Epidemiology and Medical Care Unit. Samples for each subject were analyzed in the same run to avoid between-assay variation; the within-run CVs were 3.2% for FVII:c and 11.3% for FVIIa. The standard for FVII:c was a human, frozen, plasma pool calibrated in terms of the 1st International Standard for Blood Coagulation Factors II, VII, IX, and X in plasma (code no. 84/665). The standard for FVIIa was supplied by James Morrissey from the Oklahoma Medical Research Foundation (Oklahoma City, OK). This standard was calibrated against active site-titrated FVIIa to determine actual FVIIa concentrations and against the 1st British Standard for Blood Coagulation Factor VIIa concentrate.
Statistical analysis
Statistical analysis of the data was carried out by using two-factor repeated-measures analysis of variance models with SPSS (version 6.1.3; SPSS Inc, Chicago). When there were significant changes with time, values at 3 and 6 h were compared with fasting (0 h) values. The values after the different meals were compared by using the deviations from fasting values to allow for any differences from baseline, and diet x time interactions were included in these models. Data for plasma triacylglycerol were log-transformed before statistical analysis. When the F values were significant (P < 0.05), the statistical significance of specific contrasts was tested. All pairwise testing was adjusted for multiple comparisons by using a Bonferroni correction factor.
RESULTS
Serum triacylglycerol concentrations increased significantly after all 3 test meals (Table 3). There was no significant effect of sex on the response; therefore, the data for men and women were combined. There was a significant diet x time interaction (P = 0.002) for the analyses of the deviations of serum triacylglycerol from fasting values. The increase in serum triacylglycerol was significantly lower after the structured triacylglycerol meal than after the oleate and cocoa butter meals at 3 h (P < 0.0001 for both) and at 6 h (P = 0.04 and P = 0.001), respectively. The normalized integrated area under the curve for plasma triacylglycerol (Figure 1) was significantly lower (P < 0.001) after the structured triacylglycerol meal than after the oleate and cocoa butter meals (P < 0.001 for both). Plasma cholesterol concentrations fell after the test meals and remained low after the oleate meal. There was a significant diet x time interaction (P = 0.02) for the analysis of the deviations of plasma HDL from fasting values. Plasma HDL-cholesterol concentrations were greater 6 h after the oleate meal and were significantly different from the corresponding value after the cocoa butter meal.
View this table:
TABLE 3. . Serum triacylglycerol and plasma total and HDL cholesterol after the 3 test meals1
FIGURE 1. . Mean (±SE) normalized incremental areas under the curve for serum triacylglycerol concentrations after the high-oleate sunflower oil, stearic acid–rich triacylglycerol, and cocoa butter test meals. *Significantly different from the stearic acid–rich triacylglycerol meal, P < 0.001.
Plasma FPA values were generally low, indicating a satisfactory venipuncture technique. Plasma samples with FPA concentrations >10 µg/L were excluded from the statistical analysis and complete sets of data available for 22 subjects showed no significant differences in postprandial FPA between meals (data not shown). There were significant meal x time interactions for FVII:c and FVIIa (P < 0.0001 for both). Analysis of the deviations from fasting showed significant differences in FVII:c activity (P < 0.0001) and FVIIa concentrations (P < 0.001) between meals. Plasma FVII:c activity increased significantly after the oleate and cocoa butter meals but not significantly after the structured triacylglycerol meal (Table 4). The postprandial increase in FVII:c was significantly lower after the structured triacylglycerol meal than after the oleate and cocoa butter meals (P < 0.0001 and P = 0.001, respectively). There was a significant time effect for plasma FVII:Ag concentrations (P = 0.001); values at 3 and 6 h were lower than fasting values but values were not influenced by the type of meal consumed. However, plasma FVIIa concentrations increased significantly after all 3 meals. The increase in plasma FVIIa was significantly lower after the structured triacylglycerol meal than after the oleate and cocoa butter meals (P < 0.01 for both). The change in serum triacylglycerol was not significantly associated with the change in FVII:c or FVIIa. tPA activity increased postprandially (main time effect: P < 0.0001) and PAI-1 activity declined (main time effect: P = 0.0001) but there were no significant differences between test meals or significant diet x time interactions (Table 5). D-Dimer activity did not change significantly between meals or postprandially.
View this table:
TABLE 4. . Plasma factor VII coagulant (FVII:c) activity, activated factor VII (FVIIa) concentrations, and factor VII antigen (FVII:Ag) concentrations after the 3 test meals1
View this table:
TABLE 5. . Tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) activities and D-dimer concentrations after the 3 test meals1
DISCUSSION
The stearic acid–rich structured triacylglycerol meal resulted in a smaller postprandial increase in serum triacylglycerol than did the oleate and cocoa butter meals, which did not differ in this respect. This finding was not unexpected because the stearic acid–rich structured triacylglycerol contained fewer long-chain fatty acids than did the other fats, but the difference was much greater than would be expected on this basis alone and was probably a consequence of the poor digestibility of the triacylglycerol (22). A limitation of the present study was that blood samples were obtained only 3 and 6 h after the meals. However, in a previous study in which blood samples were obtained at hourly intervals, a stearate-rich oil derived from hydrogenated high-oleate sunflower oil resulted in less postprandial lipemia than did an oleate-rich sunflower oil (19). In the present study, the increase in serum triacylglycerol after the cocoa butter meal, which provided 11 g stearate and an intake of long-chain fatty acids similar to that from the oleate meal, was similar to that after the oleate meal. For cocoa butter, almost all of the stearate is present as 1,3-distearoyl-2-oleolyl glycerol, whereas for the stearic acid–rich structured triacylglycerol, stearate is present predominantly as randomly distributed monostearoyl triacylglycerol. The digestibility of cocoa butter is similar to that of corn oil (23). The results suggest that the postprandial increase in serum triacylglycerol is influenced by the position of stearate on the dietary triacylglycerol. Further research is needed to ascertain whether the stearate is excreted in feces.
We noted that the plasma cholesterol concentration fell postprandially after the oleate test meal and that the HDL-cholesterol concentration increased to a value that was greater than the fasting value 6 h postprandially. This change in HDL-cholesterol concentration was significantly different from the change after the cocoa butter meal. These preliminary results suggest that there may be subtle differences in postprandial lipoprotein metabolism between oleate and stearate that would be consistent with a modest HDL-cholesterol-lowering effect of stearate relative to oleate (24).
The type of fat in the test meals did not significantly influence markers of fibrinolytic activity. However, tPA activity increased and PAI-1 activity decreased postprandially. These findings agree with those of previous studies (25, 26) and probably reflect circadian variation in these variables. However, it was found that PAI-1 activity increases after a high-fat meal (4). In that study, the measurement of PAI-1 activity and PAI-1 antigen involved the addition of tPA to the anticoagulant. In the present study, the D-dimer concentration, measured as an indicator of global fibrinolytic activity, did not change significantly postprandially, in agreement with another study (25).
The increase in FVII:c activity and FVIIa concentrations after the oleate and cocoa butter meals was significantly different from the increase after the structured triacylglycerol meal. The increase in FVII:c after the structured triacylglycerol meal was not significantly different from the fasting value but the increase in FVIIa was. FVII:Ag concentrations tended to decrease postprandially but there were no significant differences between the test meals. The absence of an effect of the structured triacylglycerol meal on FVII was likely a consequence of its unusual triacylglycerol structure. In a previous study, stearate in the form of interesterified hydogenated sunflower oil was found to result in a lower postprandial increase in FVIIa than did oleate supplied as high–oleic acid sunflower oil (19). Larsen et al (27) found no significant differences in the postprandial response of plasma triacylglycerol and FVII:c activity after the consumption of 5 meals containing 70 g fat provided by rapeseed oil, olive oil, sunflower oil, palm oil, and butter. Mennen et al (18) reported increased FVIIa concentrations in elderly women after they consumed 50-g fat test meals enriched with 20 g palmitate, stearate, linoleate, or linoleate + linolenate. Hunter et al (28) found no significant differences in the effects of test meals enriched with 1,3-distearoyl-2-oleolyl glycerol or triolein on plasma triacylglycerol and FVIIa concentrations 3 h postprandially in 8 young adult male subjects. The results of the present study showed that stearate provided in the form of a stearic acid–rich structured triacylglycerol did not increase FVIIa concentrations or FVII:c activity but when provided in the form of cocoa butter, increased FVII:c and FVIIa to the same extent as did dietary oleate.
The results of the present study do not suggest that the consumption of a stearic acid–rich triacylglycerol increases FVII to a greater extent than does consumption of oleate in middle-aged men and women, but rather that it results in a smaller postprandial increase in serum triacylglycerol than do cocoa butter and high-oleate sunflower oil without an accompanying increase in plasma FVII:c activity.
ACKNOWLEDGMENTS
We thank David Howarth and Janet Attfield for conducting the coagulation assays and James Morrissey (Oklahoma Medical Research Foundation) for conducting the factor VII activated assay.
REFERENCES