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Original Research Communication |
1 From the Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, Reading, United Kingdom (KGJ and CMW), and the Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, United Kingdom (MDR, BAF, and KNF).
2 Previously published in abstract form (Proc Nutr Soc 2001;60:94A). 3 Supported by the Biotechnology and Biological Research Council (BBSRC) UK. 4 Reprints not available. Address correspondence to KG Jackson, Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, University of Reading, Reading RG6 6AP, United Kingdom. E-mail: k.jackson{at}afnovell.reading.ac.uk.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: Our goal was to examine whether different dietary oils have effects on the storage of triacylglycerol as a result of differences in their digestion, absorption, and incorporation into chylomicrons.
Design: A single-blind, randomized, within-subject crossover design was used to study the effects of palm oil, safflower oil, a mixture of fish and safflower oil, and olive oil on postprandial apolipoprotein (apo) B-48, retinyl ester, and triacylglycerol in the Sf > 400 fraction with the use of a sequential meal protocol.
Results: For triacylglycerol, retinyl ester, and apo B-48, the time to reach peak concentration was significantly earlier after the second meal than after the first meal (P < 0.005). This was apparent with each of the dietary oils. The pattern of the apo B-48 response differed significantly among the dietary oils, with olive oil resulting in higher concentrations after both meals (P = 0.003). The ratio of triacylglycerol to apo B-48 was significantly lower after olive oil feeding than after feeding with the other oils (P = 0.02).
Conclusions: The rapid entry of chylomicrons after the ingestion of a second meal 5 h after a first meal was seen with all of the oils investigated. The short-term ingestion of olive oil produced more chylomicrons than did the other dietary oils, which may have been due to differences in the metabolic handling of olive oil within the gut.
Key Words: Apolipoprotein B-48 • retinyl ester • postmenopausal women • postprandial lipemia • second meal effect • dietary fatty acids • chylomicrons • triacylglycerol
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1 h after the second meal; these chylomicrons have been shown to carry fat ingested in the first meal (3). These data suggest that a proportion of fat from the first meal remains in the gut lumen or in the enterocyte and enters with subsequent meal ingestion. The location of the triacylglycerol storage pool and the effects that different fats have on this storage are currently unknown. The results of studies in humans examining the short-term effects of dietary fatty acids on postprandial lipemia suggest that the fatty acid composition of the test meal may influence the absorption, synthesis, and secretion of dietary triacylglycerol, as well as the size of the chylomicron particle (5, 6). A review of the data shows lipemic responses to vary in the order of saturated fatty acids (SFAs) = n-9 monounsaturated fatty acids (MUFAs) > n-6 polyunsaturated fatty acids (PUFAs) > n-3 PUFAs (7, 8). However, little is known about the effects of different fatty acids on postprandial lipemia after sequential meals. In the studies conducted, safflower oil or maize oil (both n-6 PUFAs) were used as the fat source in the first test meal (3, 4, 9). The effect of the rapid entry of chylomicrons into the circulation after the second meal on the postprandial lipemia occurring as a result of the first meal is important in view of the potentially adverse effects associated with elevated concentrations of chylomicrons and their remnants in the circulation (10).
The aim of the present study was to examine whether the type of fat given in the first meal influences the storage of triacylglycerol as a result of differences in the digestion, absorption, and incorporation of the fat into chylomicron particles. A chylomicron-enriched fraction of plasma (Sf > 400) was isolated to determine the effects of palm oil, safflower oil, a mixture of fish and safflower oil, and olive oil on the metabolism of large triacylglycerol-rich chylomicrons after sequential meals. The vitamin A loading technique was used to label chylomicrons with retinyl ester in the enterocyte and to provide a marker for the entry of fat from the first meal into the circulation. Another more specific marker of chylomicrons, the structural protein apolipoprotein (apo) B-48, was used to determine the number of chylomicron particles present in the circulation after each of the test meals and to estimate their particle size. The results of this study were published previously in abstract form (11).
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study had a single-blind, within-subject, crossover design in which subjects attended an investigation unit at either the University of Reading or the Radcliffe Infirmary, Oxford, on 4 occasions separated by 
1 mo. Four test meals of different fatty acid composition were given to the subjects in the form of a breakfast (milkshake containing the test oil, bowl of cereal, and a banana) given at 0800. This meal contained 41 g fat. The fatty acid composition of the test meals was modified by substituting 40 of the 41 g fat in the breakfast meal with different dietary oils. These were 1) palm oil, rich in SFAs (Anglia Oils Ltd, Hull, United Kingdom); 2) safflower oil, rich in n-6 PUFAs (Anglia Oils Ltd); 3) a 50:50 mixture of deodorized, highly purified fish oil (EPAX 3000 TG; Pronova Biocare, Lysaker, Norway) and safflower oil, rich in long chain PUFAs; and 4) olive oil, rich in n-9 MUFAs. At 1300, a second test meal (low fat, 6 g) was consumed by the subjects and the same meal was given on each of the 4 visits. Retinyl palmitate (aqueous form; 200000 IU; Roche, Welwyn Garden City, United Kingdom) was added to the breakfast test meals only. The nutrient composition of the 2 test meals and the fatty acid composition of the breakfast meals are shown in Tables 1 and 2
, respectively.
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Plasma separation and analytic methods
Blood samples were collected into heparin-containing tubes or syringes for the analysis of plasma triacylglycerol and apo B-48, retinyl ester, and triacylglycerol in the chylomicron-enriched Sf > 400 fraction. Plasma was separated by centrifugation at 1700 x g for 15 min in a bench-top centrifuge at 4°C. Samples for the analysis of plasma triacylglycerol were stored at –20°C. EDTA (0.5 mol/L), phenylmethylsulfonyl fluoride (10 mmol/L dissolved in isopropanol), and aprotinin [10000 kallikrein inactivator units (KIU)/mL; Bayer PLC, Newbury, United Kingdom] were added immediately to the isolated plasma to prevent proteolytic degradation of apo B. The plasma was stored overnight at 4°C until isolation of the triacylglycerol-rich lipoproteins.
Density-gradient ultracentrifugation according to the method of Karpe and Hamsten (13) was used to prepare the chylomicron-enriched fraction (Sf > 400) from plasma. Briefly, the density (d) of plasma was adjusted to d = 1.10 kg/L with the addition of a KBr solution with d = 1.42 kg/L. Four milliliters of density-adjusted plasma was placed into a 13.4-mL Ultra-Clear centrifuge tube (Beckman Instruments UK Ltd, High Wycombe, United Kingdom) and carefully overlayered with 3.0 mL d = 1.063 kg/L, 3.0 mL d = 1.020 kg/L, and 2.8 mL d = 1.006 kg/L NaCl solutions. Ultracentrifugation was performed at 40000 rpm (202000 x g) for 32 min in an SW 40 Ti swinging bucket rotor (Beckman) at 15 °C. The top 1 mL of the gradient (Sf > 400 fraction) was carefully isolated, divided into portions, and stored at -20°C for future analysis. The interassay CV for the isolation of the Sf > 400 fraction by ultracentrifugation has been shown to be 8.3% (13). Samples for the analysis of retinyl ester were covered with aluminum foil to protect from degradation by light. To further protect apo B-48 from proteolytic cleavage, another preservative cocktail was added to the appropriate tubes before the addition of the triacylglycerol-rich lipoproteins to give a final concentration of 5% (by volume) (14).
Triacylglycerol concentrations in both plasma and the Sf > 400 fraction were measured by using an IL Monarch centrifugal analyzer (Instrumentation Laboratory, Warrington, United Kingdom) and an enzyme-based colorimetric kit supplied by Instrumentation Laboratory. All samples for each subject were analyzed within a single batch, and the intraassay CV for triacylglycerol was 1.7%. Normal-phase HPLC was used to measure total retinyl ester in the Sf > 400 fraction (15). The intraassay CV was 1.9% at 10 µmol/L. Apo B-48 was measured by a specific competitive enzyme-linked immunosorbent assay (ELISA) (16) with a few modifications. Briefly, a heptapeptide-thyroglobulin conjugate consisting of the terminal residues of the apo B-48 molecule was used as the coating material in the ELISA format. Samples were incubated with a specific polyclonal anti–apo B-48 antibody that recognizes the carboxyl-terminal region of the protein on the surface of lipoprotein particles and does not show cross-reactivity for apo B-100 (17). A standard curve was prepared by serial dilution of the apo B-48 heptapeptide in phosphate-buffered saline containing 10 g human serum albumin/L. The intraassay CV was 4.5% at 92 pmol/L.
Calculation of triacylglycerol and retinyl ester contents of the chylomicron particles
The Sf > 400 fraction is composed of large triacylglycerol-rich lipoproteins with a particle size >75 nm that are predominantly chylomicrons (18). The ratio of triacylglycerol to apo B-48 in this fraction was calculated by dividing individual triacylglycerol concentrations by those for apo B-48 for each time point of the postprandial period. This calculation is subject to the relative number of triacylglycerol-rich particles containing apo B-48 and apo B-100 in the Sf > 400 fraction at various sampling times. The ratio of retinyl ester to apo B-48 was also determined as above. These calculations together provide an approximation of the relative amounts of triacylglycerol and retinyl ester in the chylomicrons released after each of the dietary oils.
Statistical analysis
Data were analyzed by using SPSS version 9 (SPSS Inc, Chicago). Results presented in time courses are mean values. To improve the clarity of the triacylglycerol, apo B-48, and retinyl ester responses after the 4 breakfast meals, SE bars were added to the mean values of 2 of the dietary oils only in each figure to indicate the degree of variability in the data. In the tables, results are presented as means ± SEMs. The incremental area under the curve (IAUC) for the entire time period (ie, 0–480 min) was calculated by using the trapezoidal rule. The postprandial time courses for the lipemic responses after the different test oils and the maximum concentrations reached after the breakfast and second meal (peak concentration) were analyzed by using two-factor repeated-measures analysis of variance (ANOVA) with interaction. A Holm’s sequential Bonferroni correction was used for the post hoc detection of significant pairwise comparisons. All of the data were log transformed to render their distribution normal before statistical analysis, with the exception of the time to reach peak concentration, which could not be normalized. The time to reach peak concentration after the breakfast and second meal was analyzed by using a nonparametric, one-way repeated-measures ANOVA (Friedman test). To compare the time to reach peak concentration after the breakfast and second meal for each dietary oil, nonparametric paired t tests were used (Wilcoxon’s test). As before, a Bonferroni correction was used for the post hoc detection of significant pairwise differences. To assess concordance of the time courses of the markers for chylomicrons, triacylglycerol, retinyl ester, and apo B-48 after the different dietary oils, time-series analysis with cross-correlation was used (19). Values of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Fasting retinyl ester concentrations were below the limit of detection of the HPLC system.
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). There were no significant differences in the patterns of response for either triacylglycerol or retinyl ester (Figure 1), although there was a tendency for higher retinyl ester responses after the olive oil meal.
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). As expected, the 300-min triacylglycerol (P = 0.005), retinyl ester (P < 0.005), and apo B-48 (P < 0.02) concentrations were significantly higher than their corresponding fasting concentrations for each visit.
Postprandial responses after the second meal (300–480 min)
Repeated-measures ANOVA showed a significant meal effect for the apo B-48 responses after the second meal. In this instance, the olive oil meal showed a different pattern of response from the safflower oil meal (P = 0.0001). Significant differences were not observed in the patterns of the triacylglycerol or retinyl ester responses after the second meal, although there was a tendency for higher triacylglycerol concentrations after the olive oil meal (Figure 1).
The time to reach peak concentration after the second meal was not significantly different after the 4 test oils for triacylglycerol, retinyl ester, or apo B-48. Comparison of the time to reach peak concentration after the breakfast and the second meal showed a significantly earlier time to peak after the second meal. This was consistently shown for the triacylglycerol, retinyl ester, and apo B-48 responses after each of the different test oils (P = 0.005; Table 4).
Contribution of the Sf > 400 triacylglycerol-rich lipoprotein response to the total plasma lipemic response
The IAUC (0–480 min) for the Sf > 400 triacylglycerol responses for the palm, safflower, fish+safflower, and olive oils were 42.9 ± 6, 51.8 ± 16, 53.5 ± 10, and 54.7 ± 12 mmol/L x 480 min respectively. The IAUC (0–480 min) for the plasma triacylglycerol responses after the palm, safflower, fish+safflower, and olive oils were 145.9 ± 29, 135.0 ± 40, 193.2 ± 49, and 158.7 ± 35 mmol/L x 480 min, respectively. The IAUC for the Sf > 400 triacylglycerol responses were significantly lower than the corresponding plasma triacylglycerol IAUC after each of the dietary oils (P < 0.01). On average, the lipemic response in the Sf > 400 fraction represented 31% (range: 29–38%) of the total plasma response. The postprandial plasma triacylglycerol responses after the palm, safflower, fish+safflower, and olive oils are included for reference (Figure 2).
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. Repeated-measures ANOVA showed a significant meal effect over the whole time course (0–480 min) in the ratio of triacylglycerol to apo B-48. The ratio of triacylglycerol to apo B-48 showed a significantly lower response and a less marked postprandial change with time after olive oil than after the palm, safflower, and fish+safflower oils (P = 0.02). The ratio of retinyl ester to apo B-48 also showed a significant meal effect over the 0–480 min postprandial period, with the palm oil ratio being lower than the safflower oil ratio (P = 0.007; Figure 3).
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for each of the dietary oils used in the breakfast test meal. Concordant time response curves were observed between each of the markers compared, which were shown to be highly significant by cross correlation (rs 0.700, P < 0.001) with no time lag.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A surprising observation was the significant effect of meal fatty acid composition on the apo B-48 response, with olive oil showing higher concentrations over the postprandial time course than did the other dietary oils. Olive oil also showed a tendency for higher retinyl ester concentrations after both meals, and after the second meal for triacylglycerol. These data suggest that olive oil caused the production of more chylomicrons over the postprandial period than did the palm, safflower, and fish+safflower oils. Similar findings were observed in single test meal studies comparing the postprandial responses of olive oil with those of other dietary oils (21, 22). In these studies, liquid drinks (22) and emulsions (21) were used to provide test meals enriched in SFAs, n-6 PUFAs, and MUFAs. Results from the study by Higashi et al (22) showed significantly higher plasma triacylglycerol and retinyl ester concentrations after olive oil than after high-linoleic safflower oil and cow milk fat. De Bruin et al (21) also showed significantly higher retinyl palmitate and apo B-48 concentrations after olive oil than after cream and soybean oil, but no significant differences in triacylglycerol responses. These data along with those from the present study suggest that olive oil may influence one or several steps involved in the formation or metabolism of the nascent chylomicrons in the circulation.
Hussain (23) proposed the sequential assembly model for the formation of chylomicrons in the enterocyte after fat ingestion. In this model, 3 steps are thought to occur: 1) formation of the primordial particle, 2) formation of triacylglycerol-rich droplets, and 3) core expansion resulting from the fusion of the primordial particle with lipid droplets enabling synthesis of nascent chylomicrons. Studies in Caco-2 cells, a human colon carcinoma cell line, have shown an increase in the number of particles secreted after incubation with oleic acid as opposed to linoleic and palmitic acid (24). The increase in apo B–containing lipoproteins released from the cells was attributed to the specific increase in apo B-48, suggesting that olive oil may increase the expression of the apo B messenger RNA editing enzyme.
Van Greevenbroek et al (24) also showed unsaturated fatty acids, especially oleic acid, to be more efficiently incorporated into lipoproteins by the microsomal triacylglycerol transfer protein (MTP). It had been thought that MTP was involved only in the co-translational lipidation of apo B, protecting the protein from early intracellular degradation (step 1 of model). However, MTP was shown in Caco-2 cells to be involved in the further lipidation of the primordial particle along the secretory pathway (25). These data suggest that the short-term ingestion of olive oil may increase the number of apo B-48–containing particles by modulating the activity or expression of MTP or the messenger RNA editing enzyme within the enterocyte. Further work is needed to determine the effect of dietary fatty acids on the expression of genes involved in lipoprotein assembly in the enterocyte.
The fatty acid composition of a chylomicron particle has been shown to be an important determinant of its metabolism in the circulation. Unsaturated fatty acids, especially PUFAs, were shown to increase the size of chylomicron particles compared with those from a test meal containing SFAs (5, 26). After their release into the circulation, large triacylglycerol-rich chylomicrons are preferentially hydrolyzed by lipoprotein lipase (EC 3.1.1.34), and removal of 70–90% of the triacylglycerol produces a cholesterol ester–rich particle termed a chylomicron remnant. Before their uptake by a receptor-mediated process in the liver, the particles interact with hepatic lipase, which further hydrolyzes triacylglycerol and phospholipid (27).
In vivo and in vitro studies in rats have shown chylomicrons enriched with n-6 PUFAs to be hydrolyzed more rapidly by lipoprotein lipase than are particles enriched with SFAs, MUFAs, and n-3 PUFAs (28). This may explain the significantly different apo B-48 responses after the different breakfast test meals. The safflower oil meal, which was rich in n-6 PUFAs, showed the lowest apo B-48 response over the entire postprandial period but the greatest ratios of triacylglycerol to apo B-48 and retinyl ester to apo B-48, especially compared with olive oil. Therefore, the larger triacylglycerol-rich, safflower oil–enriched chylomicrons may have been rapidly hydrolyzed by lipoprotein lipase before uptake by the liver. In contrast, olive oil produced the lowest ratio of triacylglycerol to apo B-48, suggesting that although these particles are present in the Sf > 400 fraction of plasma, they are less enriched with triacylglycerol than are the safflower oil chylomicrons. Therefore, olive oil–enriched chylomicrons may be poorly hydrolyzed by lipoprotein lipase as a result of their triacylglycerol content or greater particle number and so may rely on an alternative route for their removal from the circulation. Findings by de Bruin et al (21) and Brouwer et al (29) suggest that hepatic lipase plays a significant role in the removal of olive oil–enriched particles. The apo B-48 concentrations at the end of the postprandial period (480 min) in the present study were not significantly different between the 4 dietary oils, suggesting that olive oil–enriched chylomicrons are rapidly removed from the circulation, possibly as a result of hydrolysis by hepatic lipase.
Analysis of apo B-48, retinyl ester, and triacylglycerol in the Sf > 400 fraction provided an opportunity to compare the markers commonly used for chylomicron particles. Surprisingly, the triacylglycerol, retinyl ester, and apo B-48 responses observed after each of the dietary oils given in the breakfast meal were concordant. The time to reach peak concentration was also not significantly different, with the tendency for fish+safflower oil to peak earlier than the other oils after the first meal being mirrored in the triacylglycerol, retinyl ester, and apo B-48 data. However, this is not a consistent finding in the literature. The type of vitamin A (aqueous, tablet, or oil) and predominant fatty acid in the test meal have been shown to cause a delay in the retinyl palmitate–retinyl ester response compared with that of apo B-48 and triacylglycerol (30, 31). Karpe et al (32) suggested that n-6 PUFAs (soybean oil) delay the absorption of vitamin A. However, this was not observed in the present study, in which the apo B-48, retinyl ester, and triacylglycerol responses were concordant after each of the dietary oils, including the n-6 PUFA safflower oil. Despite criticism of the vitamin A loading method, its use in the present study provided valuable information in verifying the origin of the triacylglycerol released rapidly after consumption of the second test meal.
In conclusion, the rapid entry of triacylglycerol-rich lipoproteins after the second meal was a phenomenon of all the dietary oils examined. The magnitude of the response, however, was dependent on the type of fatty acids contained in the first test meal. The ingestion of olive oil in this group of postmenopausal women caused a significantly higher postprandial apo B-48 response and a tendency for higher retinyl ester concentrations than did the ingestion of palm, safflower, and fish+safflower oils. Therefore, olive oil may influence the formation and composition of nascent chylomicrons by modifying the activity or expression of the proteins and enzymes involved in lipoprotein assembly (eg, MTP), which may enable stabilization of the nascent chylomicron particles with smaller lipid droplets. Further work is required to determine the precise locus for this difference in the metabolic handling of olive oil, which is only observed with short-term ingestion and not in subjects habituated to high-MUFA diets (33).
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-6 and -3 series reduce postprandial lipoprotein levels. Chronic and acute effects of fat saturation on postprandial lipoprotein metabolism. J Clin Invest 1988;82:1884–93.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), safflower oil (•), fish+safflower oil (
), and olive oil (
). n = 10 women. To improve the clarity of the patterns of the response, SE bars were added to the means of 2 of the dietary oils only in each panel to indicate the degree of variability in the data. For apo B-48, there was a significant time effect (P < 0.001), a main effect of the fatty acid composition of the first meal (P = 0.003), and a meal x time interaction (P = 0.003) by repeated-measures ANOVA.
