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Journal of Lipid Research, Vol. 44, 2065-2072, November 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology
* Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, OX3 7LJ, United Kingdom
Lipoprotein and Atherosclerosis Research Group, Department of Pathology, University of Ottawa Heart Institute, Ottawa, Ontario, Canada
Institute of Human Nutrition, University of Southampton, Southampton, SO16 6YD, United Kingdom
Published, JLR Papers in Press, August 16, 2003. DOI 10.1194/jlr.M300167-JLR200
1 To whom correspondence should be addressed. e-mail: fredrik.karpe{at}oxlip.ox.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In summary, the contribution of dietary fatty acids to early postprandial VLDL TG is substantial. The role of DHA in VLDL TG production will require further investigation.
Abbreviations: apoB, apolipoprotein B; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; RE, retinyl ester; TG, triacylglycerol
Supplementary key words chylomicron • apolipoprotein B • immunoaffinity chromatography • triacylglycerol • n-3 polyunsaturated fatty acids • palmitic acid • stable isotopes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VLDL and chylomicron remnants are similar in size and composition, and separation of the lipoprotein classes cannot be achieved by conventional methods such as ultracentrifugation. However, the structural protein, apolipoprotein B (apoB), differs between TRL derived from the intestine (apoB-48) and that derived from the liver (apoB-100) (6). ApoB-48 and apoB-100 are encoded by the same gene, but a post-transcriptional modification of apoB-100 occurs in the enterocyte, producing apoB-48 (7). ApoB-48 is homologous to the N-terminal 48% of apoB-100, but shares no homology with the C-terminal end of apoB-100. Monoclonal antibodies, which recognize epitopes in apoB-100 but not in apoB-48, have therefore been used to separate and examine the lipid and apolipoprotein composition of chylomicrons and VLDL, respectively (3, 4, 8–12). However, this technique has not been used to study the dynamic aspects of lipid metabolism, such as the incorporation of dietary fatty acids into TRL in the postprandial state. The first aim of this study was to investigate the appearance of dietary fatty acids in hepatic TRL in the postprandial state in normal healthy subjects.
Studies of postprandial lipid metabolism have shown that fatty acid uptake by adipose tissue is highly regulated and that significant amounts of hydrolysed fatty acids are not taken up by the tissue but are released as nonesterified fatty acids (NEFAs) (13, 14). In particular, postprandial uptake of both docosahexaenoic acid (DHA, C22:6 n-3) and eicosapentaenoic acid (EPA, C20:5 n-3) by adipose tissue is proportionally reduced, compared with saturated and monounsaturated fatty acids. In fact, accumulation of EPA and DHA in the NEFA pool has been observed in the postprandial period (15). Furthermore, it has also been reported that the EPA:DHA ratio is significantly decreased in the NEFA pool compared with that in the plasma TG pool (16–19). This may indicate selective metabolism between these two n-3 fatty acids. Few studies have looked at the EPA:DHA ratio in lipoprotein TG in the postprandial period (16, 20). Therefore, the second aim of this study was to investigate whether there is selective partitioning of dietary EPA and DHA in the postprandial state.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Study protocol
To standardize the subjects' nutritional state before the study, all subjects consumed a low-fat (<10 g fat) evening meal. All subjects were instructed to fast overnight and to refrain from alcohol and strenuous exercise. A cannula was inserted into an antecubital vein of the forearm, and blood samples were taken at -20, 0, 30, 60, 90, 120, 150, 180, 240, 270, 300, and 360 min. At 0 min, subjects were given a mixed meal consisting of 40 g Rice Krispies (Kelloggs, Warrington, UK), banana, and a warm chocolate milkshake containing 50 g of fat. The fat consisted of 30 g fish oil (EPAX3000TG; Pronova Biocare, Asslund, Norway), 10 g macadamia nut oil, and 10 g safflower oil (Anglia Oils Ltd., Hull, UK) to provide a range of fatty acids. Six hundred milligrams of [1-13C]palmitic acid (99 atom%; Cambridge Isotopes, Woburn, MA) were added to the test oil. The macronutrient composition and fatty acid composition of the meal are shown in Table 1 and Table 2, respectively. The breakfast was consumed within 10 min, and the subjects remained in the supine position throughout the study.
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Plasma glucose, TG, and lipoprotein TG concentrations were measured with kits from Instrumentation Laboratory (Warrington, UK). Cholesterol and NEFA concentrations were measured with kits from Randox Co. (Antrim, UK) and Alpha Laboratories (Eastleigh, UK), respectively. All of the metabolites were batch-analyzed and measured enzymatically with an IL Monarch automated analyzer (Instrumentation Laboratory), exhibiting an intra-assay coefficient of variation of <2.5%.
TRLs were separated by flotation in a density gradient (21). Ultracentrifugation was performed in a SW40Ti swinging bucket rotor at 40,000 rpm at 15°C (XL-70 Ultracentrifuge, Beckman Instruments, Palo Alto, CA). The gradients were run for 32 min to float Svedberg flotation rate (Sf) >400 lipoproteins and for a further 16 h to float Sf 20–400. The top 0.5–1 ml from each tube was aspirated, collected into another preweighed tube, and immediately put on ice. TRLs were separated from plasma taken at -20 and 0 min, and at 90, 180, 270 and 360 min after the mixed meal.
TRLs were further separated by immunoaffinity chromatography, using the specific monoclonal antibodies 3F5, 4G3, and 5E11 against apoB-100, which do not cross-react with apoB-48 (8, 22). Preparation of immunoabsorbants was performed essentially as described by Milne et al. (8). Typically, 4 mg of 3F5, 4 mg of 4G3, and 4 mg of 5E11 were coupled to 1 ml of cyanogen bromide-activated Sepharose. Separation of apoB-48 and B-100 was carried out in 1 ml columns containing the antibody-coupled Sepharose gel dissolved in phosphate-buffered saline (PBS, pH 7.4, 0.02% NaN3). The column was drained, and 0.5 ml TRL (<0.2 mg TRL protein) was added to the top of the column. The sample was passed through the column 10 times over a 2 h period to ensure optimum binding efficiency. The unbound fraction was collected by draining the column, and further unbound material was washed through with 1.5 ml of PBS. Any nonspecific binding was removed by eluting the column with sodium chloride (1 M NaCl, pH 7.4). The apoB-100-containing TRLs, which were bound to the column, were eluted by successive 1 ml washes with thiocyanate (3 M NaSCN, pH 7.4). Bound material was recovered in 10 ml sodium thiocyanate. Gels were washed with 10 ml PBS and stored at 4°C for later reutilization.
The bound material was concentrated by density gradient ultracentrifugation using a protocol similar to that used before. Briefly, 10 ml of bound sample was pipetted into polyvinyl alcohol-coated Ultra-Clear centrifuge tubes. Three milliliters PBS was added to the top of each tube to create a small gradient. Samples were centrifuged for 16 h as previously described. The bound samples were collected by aspiration into preweighed tubes and immediately put on ice.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (23) showed that the bound fraction was completely devoid of apoB-48. Ninety percent of apoB-100 was found in the bound fraction; this fraction will hereafter be called the VLDL fraction. ApoB in the unbound fraction consisted of 70% apoB-48 and 30% apoB-100 and will be called the "chylomicron remnant fraction," as it is chylomicron remnant rich. The recovery of lipoproteins using the immunoaffinity method was 73.4 ± 17.0% and 77.1 ± 9.3% as judged by recovery of apoB-100 and lipoprotein TG, respectively.
Gas chromatography and mass spectrometry
For analysis of specific fatty acids, lipids were extracted from plasma or from lipoprotein fractions by using chloroform-methanol (2:1; v/v) (24). After separation of the lipid classes by solid-phase extraction, and methylation of fatty acids with methanolic sulfuric acid, gas chromatography was used to analyze the fatty acid composition of plasma NEFA, Sf >400 TG, Sf 20–400 TG, chylomicron remnant TG, and VLDL TG (25, 26). The absolute concentrations of the individual fatty acids were calculated by reference to internal standards added to the plasma during lipid extraction—heptadecanoic acid for NEFA and triheptadecanoyl glycerol for lipoprotein TG. Isotope enrichment was analyzed by gas chromatography-isotope ratio mass spectrometry as described previously by Evans et al. (27). A test meal was homogenized and analyzed using gas chromatography to establish its specific fatty acid composition.
Statistics
Data were analyzed with SPSS software version 10.0 (SPSS UK Ltd., Chertsey, UK). Postprandial metabolite and lipoprotein responses were analyzed by repeated measures ANOVA, using ‘time’ as a within-subject factor. Differences between remnant and VLDL TG composition were also measured by repeated measures ANOVA, using ‘time’ and ‘lipoprotein’ as within-subject factors. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Incorporation of EPA and DHA into postprandial lipoprotein TG pools
The postprandial appearance of EPA and DHA in Sf >400 and Sf 20–400 TRL TG is shown in Fig. 4
. In Sf >400, there was a rapid rise in both EPA and DHA in lipoprotein TG (P < 0.001) and both remained at high relative concentrations 6 h after the meal. The relative increase of EPA to DHA in the Sf >400 mirrored the composition of the test meal (EPA:DHA ratio 1.73). Both EPA and DHA were rapidly incorporated into both VLDL and chylomicron remnant TG pools (P < 0.001) and appeared at the same time as labeled palmitic acid in both pools. However, there was a significant difference in the relative concentrations of n-3 PUFA in these two TG pools: in chylomicron remnant TG, EPA concentration peaked at 55.4 ± 12.0 µmol/l at 270 min, whereas DHA concentration peaked at 31.3 ± 7.6 µmol/l at 270 min; in VLDL TG, EPA concentration was 27.7 ± 8.1 µmol/l at 360 min, whereas DHA concentration was 31.4 ± 7.7 µmol/l at this time. The EPA:DHA ratio in remnant TG was 1.90 ± 0.16, yet the EPA:DHA ratio was significantly lower in the VLDL TG pool at 0.77 ± 0.16 (P < 0.003).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dietary fatty acids were rapidly incorporated into plasma TRL shortly after meal intake. Different incorporation was observed when Sf 20–400 lipoproteins were separated into chylomicron remnants and VLDL. Labeled palmitic acid was rapidly incorporated into the chylomicron remnant TG pool, yet after a 90 min delay, the incorporation into the VLDL TG pool was substantial. Perfused liver studies have shown a lag time of 45 min before incorporation of labeled oleic acid into newly secreted VLDL TG (29). In the current study, EPA and DHA were incorporated into the VLDL TG pool at the same time as labeled palmitic acid, a saturated fatty acid. Therefore, the consistent appearance of three different exogenous tracers reinforces the conclusion that dietary fatty acids are recirculated into newly synthesized VLDL from an early stage in the postprandial period.
Using several lines of reasoning, it may be possible to estimate the contribution of dietary fat to the postprandial VLDL TG pool under the present experimental conditions. First, the turnover rates of the VLDL and chylomicron remnants are similar (30), due to the similarities in composition of these two particles (10). Second, chylomicron turnover studies have demonstrated that 
20% of core lipid is transferred from Sf >400 chylomicrons to Sf 20–400 chylomicron remnants (31). Third, here the rate of appearance of labeled fatty acids in both the VLDL TG pool and the chylomicron remnant TG pool was similar, and therefore it can be assumed that the entry of dietary fat into both pools occurs at the same rate. Following this line of reasoning, it may be concluded that as much as 20% of dietary fat is recycled as VLDL TG within the postprandial period following a single meal.
It could be argued that the early appearance of dietary fat in the VLDL TG pool reflects the transfer of TG and cholesteryl ester between VLDL and chylomicrons in circulation by the cholesteryl ester transfer protein (CETP). Previous studies have examined the transfer of lipids between VLDL and chylomicrons by using retinyl palmitate-labeled chylomicrons and chylomicron remnants. Retinyl esters (REs) are produced in the intestine after ingestion of vitamin A, and are packaged into chylomicrons along with dietary fat. REs in chylomicron remnants are taken up by the liver, and either stored or released as unesterified retinol bound to retinyl binding protein. The results from these studies are mixed; in vitro studies show that very little RE is transferred from chylomicrons to VLDL (32, 33); however, in vivo studies suggest that this transfer is much greater. In vivo, and using techniques comparable to ours, Cohn et al. (3) separated apoB-100-containing lipoproteins from TRL collected following an oral fat load labeled with vitamin A, and found that 25% of RE was recovered in the apoB-100 TRL at 6 h after meal intake. The substantial appearance of RE in apoB-100 TRL in vivo may even suggest that RE can be secreted in VLDL particles. We believe that the high and early incorporation of dietary fatty acids into the VLDL pool cannot be explained to any substantial degree by CETP-mediated transfer. The examination of the TG fatty acid composition in the VLDL pool argues against CETP-mediated transfer. If the main entry of dietary fat into the VLDL TG pool were by transfer between VLDL and chylomicrons, it would be expected that the relative composition of dietary fatty acid tracers would be similar in these TG pools. We show that the n-3 PUFA compositions of VLDL TG and chylomicron TG pools were remarkably different: chylomicrons and their remnants contained TG enriched with DHA and EPA in concentrations corresponding to the meal, whereas VLDL TG was enriched more with DHA than with EPA. In fact, the EPA:DHA ratio in the VLDL TG pool was half that seen in the remnant TG pool, suggesting under-incorporation of EPA into VLDL TG. The different ratios support the idea that dietary fat is rapidly and substantially incorporated into the VLDL TG pool through hepatic secretion.
The relative incorporation of the three exogenous tracers, EPA, DHA, and [1-13C]palmitic acid into VLDL TG and chylomicron TG pools may also provide an insight into the hepatic partitioning of dietary fatty acid pools into VLDL TG production. By comparing the moles of either EPA or DHA per mole of labeled palmitic acid, it is clear that there is a difference between the different pools. Both the EPA:[1-13C]palmitic acid and DHA:[1-13C]palmitic acid ratios in the Sf >400 reflect those seen in the meal, yet the DHA:[1-13C]palmitic acid ratio increases by 3-fold in the VLDL TG pool, while the EPA:[1-13C]palmitic acid ratio remains constant. This demonstrates clearly that DHA is over-incorporated into VLDL TG, compared with both EPA and [1-13C]palmitic acid.
There are two pools of dietary fat being delivered to the liver in the postprandial period. The first pool contains dietary fatty acids that have been released by lipoprotein lipase but not taken up by skeletal muscle or adipose tissue. The second pool contains dietary fat contained in chylomicron remnants, which are taken up directly by the liver. It is estimated that as much as 50% of dietary fat is taken up as remnant TG by the liver, whereas a proportion of dietary fat escapes tissue uptake and is delivered to the liver as NEFA bound to albumin (34). In addition, it is well recognized that elevated NEFA concentration is associated with increased VLDL production, suggesting that NEFA is another major source of fatty acids for the pre-VLDL TG pool. We have found that the EPA:[1-13C]palmitic acid ratio in the NEFA pool was half that seen in the meal, yet the chylomicron remnants were enriched in EPA. If the chylomicron remnants were the main source of TG, it would be expected that VLDL TG would have a higher enrichment of EPA than observed. On the other hand, the DHA:[1-13C]palmitic acid ratio in the NEFA pool was twice that seen in the meal, and the VLDL TG pool showed signs of over-incorporation of DHA, suggesting that NEFA is a major precursor for VLDL TG. DHA enrichment in both the NEFA pool and fasting VLDL TG have been previously demonstrated (15, 16, 20). In particular, Summers et al. (15) noted that the EPA:DHA ratio in plasma NEFA was significantly less than that in the meal, despite similar composition of chylomicron TG and meal fatty acids. In a long-term dietary study, Sadou et al. (20) studied the incorporation of EPA and DHA into cholesteryl esters, phospholipids, and TGs of two lipoprotein fractions: a combined VLDL and LDL fraction and an HDL fraction. Meal EPA:DHA ratios were similar to those of Summers et al., yet the observed EPA:DHA ratios in (VLDL + LDL) TG were similar to the NEFA ratios observed by Summers et al., suggesting that the composition of the VLDL TG pool may be directly influenced by the NEFA pool, as demonstrated by the current study. The over-incorporation of DHA into both the VLDL TG and NEFA pools could be due to differential lipolysis of n-3 PUFA-containing TG (35, 36), as it has been shown that DHA-containing TGs are preferentially lipolysed, compared with EPA-containing TGs. In addition, it is possible that short-term differential tissue uptake of n-3 PUFA occurs in the postprandial state, further exaggerating the enrichment of DHA in the NEFA pool.
In conclusion, we have demonstrated a new method for tracing dietary fatty acids into TRL TG. This is the first study to show that dietary fatty acids are rapidly incorporated into the VLDL TG pool, enabling efficient recycling of dietary fatty acids for further uptake by extrahepatic tissues. There is also significant partitioning of fatty acids into different lipid pools within the liver before hepatic TG synthesis, as seen by differential incorporation of n-3 PUFA into the VLDL TG pool. In particular, dietary DHA appears to be preferentially recirculated into apoB-100 TRL particles.
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ACKNOWLEDGMENTS |
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Manuscript received April 23, 2003 and in revised form July 17, 2003.
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REFERENCES |
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