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Department of Biochemistry and Food Chemistry, University of Turku, 20014 Turku, Finland and * Department of Clinical Nutrition, University of Kuopio, 70211 Kuopio, Finland
2To whom correspondence should be addressed. E-mail: kaisa.yli-jokipii{at}utu.fi.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • triacylglycerol positional distribution • chylomicrons • postprandial lipemia • tandem mass spectrometry • humans
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The distribution of fatty acids (FA) varies greatly in dietary oils and fats. Melting qualities of fat depend on the isomeric structures of lipids, and FA released from the sn-3 and, to lesser extent, from the sn-1 position of TAG by the action of gastric lipase (1
) take part in the regulation of gastric motility and secretory functions (2
). The released FA also increase the activity of pancreatic lipase (3
), which has stereospecificity to ester bonds in the primary positions (1
). The release rates of FA in the stomach (4
,5
) and the potential of the released FA to activate pancreatic lipase differ (3
).
To be absorbed, lipids must be transferred from bile salt micelles to cell membranes and further to epithelial cells. At the brush border membrane, 2-monoacylglycerols are taken up by passive diffusion (6
), but long-chain FA mainly by a protein-mediated process. Fatty acid transport proteins may have different affinities for different FA (7
–9
).
Inside the epithelial cells, TAG are resynthesized through the 2-monoacylglycerol pathway. The positional distribution of the FA in the sn-2 position is thus largely retained from the fat ingested to chylomicron TAG. The enzymes of the 2-monoacylglycerol pathway may use some FA more efficiently than others (10
).
Lipoprotein lipase (LPL) hydrolyzes FA from the primary positions of lipoprotein TAG, and preferentially from the position sn-1. Thus, any individual FA that is preferentially located at position sn-2 or sn-3 is less susceptible to the initial attack of this lipase, and enriched in the diacylglycerols formed. LPL hydrolyzes FA at different rates depending on the FA themselves and the species studied (11
,12
).
The removal of TAG from chylomicrons is generally not saturated (13
,14
), but the removal of chylomicron remnants by liver appears to be saturable (14
–16
). Both chylomicron remnant size (17
,18
), and the number of chylomicron particles in blood (15
) have been associated with clearance rates. Imbalance between chylomicron production and clearance results in remnant accumulation. Delayed postprandial TAG clearance is an independent risk factor for cardiovascular disease (19
–21
).
TAG molecules may behave differently during absorption, chylomicron formation, TAG hydrolysis or chylomicron clearance. Differences may be most apparent in TAG with different positional distribution of long-chain saturated FA (22
–24
). In this study, chylomicron TAG molecular weight distribution and regioisomerism were followed in healthy women after two fat loads with identical FA composition but different positional distributions (palm oil and transesterified palm oil) to investigate whether some TAG were cleared in favor of others. An efficient tandem mass spectrometric analysis method was applied to the regioisomerism analysis.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two fats with identical FA composition but different positional distribution were served to 10 healthy female volunteers in a study with a randomized, double-blind, crossover design. Blood samples for investigation of the changes in chylomicron TAG molecular weight and regioisomerism were collected through a cannula in the antecubital vein 1.5 and 2 h postprandially, and hourly thereafter up to 6 h. The effects of the oral fat loads on clinical markers of postprandial lipemia, chylomicron TAG structures 3 h postprandially, and VLDL TAG structures 4 h postprandially were reported elsewhere (25
).
Ten of 11 healthy, normal weight premenopausal women recruited completed the study (25
). The mean body mass index (kg/m2) of the subjects was 20.5 ± 1.81 (mean ± SD) before the test with palm oil and 20.6 ± 1.88 before the test with transesterified palm oil. Similarly, fasting serum lipids (mmol/L) before the palm oil treatment and transesterified palm oil treatment were 3.96 ± 0.59 and 3.93 ± 0.46 for total cholesterol, 2.23 ± 0.39 and 2.24 ± 0.32 for LDL cholesterol, 1.48 ± 0.30 and 1.49 ± 0.24 for HDL cholesterol, and 0.67 ± 0.32 and 0.76 ± 0.35 for TAG, respectively. The subjects had normal fasting plasma glucose, blood pressure, hemoglobin, and liver, kidney and thyroid functions, and they were at the same stage of their menstruation cycle during both treatments. There were no differences in the fasting concentrations at the beginning of the oral fat loads. All subjects provided written consent for the study and they were free to discontinue their participation in the experiment at any point without explanation. The study plan was approved by the Ethics Committee of the University of Kuopio and Kuopio University Hospital.
The subjects were asked to fast overnight (14 h) and advised not to consume alcohol or engage in strenuous exercise for 5 d before the test. They kept food diaries from Wednesday to Saturday in the week preceding the test and were advised to eat as habitual. The energy percentages from fat, protein and carbohydrate calculated from the 4-d food records with Micronutrica software (Version 2.5) (26
) did not differ between the treatments, but the percentage of total energy from saturated fat was greater before the test with palm oil than before the test with transesterified palm oil (P = 0.031) (25
).
Due to a technical reason, the 6-h sample from one volunteer during the test with transesterified fat was missed; because of the nature of the statistical tests used, her results for the transesterified fat treatment were omitted.
A palm oil fraction was used as the experimental fat both as such and after transesterification. The two fats had identical FA compositions (Table 1
) but their positional distribution was different (Table 2
) (25
).
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Chylomicrons (Svedberg flotation > 400 kg/L) were isolated from plasma by ultracentrifugation (27
), lipids were extracted with chloroform/methanol (2:1, v/v) (28
) and TAG separated from the extracted lipid mixture with silica columns (29
).
The molecular weight distributions of the TAG extracted from chylomicrons were determined by ammonia-negative ion chemical ionization with a triple quadrupole tandem mass spectrometer (TSQ-700, Finnigan MAT, San Jose, CA) (30
). The samples were introduced into the ion source with a direct exposure probe. Chemical ionization with ammonia resulted in the formation of deprotonated TAG ions [M - H]-, which were analyzed by scanning the mass range from m/z 500 to 1000. The combined number of acyl carbons (ACN) and double bonds in the acyl chains (DB) of TAG were calculated according to the m/z values of the [M - H]- ions. The relative molar proportions of different molecular weight species were calculated using the [M - H]- ion abundances. The amount of naturally occurring 13C was taken into account when calculating the proportions of TAG. The analysis parameters were set according to the optimization carried out earlier in our laboratory (31
). Each sample was analyzed in quadruplicate.
TAG regioisomerism was determined with a tandem mass spectrometric (TSQ-700) analysis based on negative ion chemical ionization and collision-induced dissociation with argon gas (32
,33
). The results were calculated with TAG-100 and MSPECTRA programs (Nutrifen, Turku, Finland) (34
,35
). The regioisomers of the test fats and molecular weight species with ACN:DB 50:1, 52:2 and 52:3 in chylomicrons were analyzed in quadruplicate.
The statistical analyses were carried out with SPSS-PC+ statistical package (Version 10, SPSS, Chicago, IL). A two-way ANOVA for repeated measurements (general linear model; GLM) was used to test whether there were differences from linear trend. If the GLM was significant, a paired samples t test was used for comparison of individual time points. Relative abundances in chylomicrons were used. Differences of P < 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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TAG molecular weight affected TAG clearance from chylomicrons (Figs. 1
and 2
). TAG with ACN:DB 48:2, 50:3 and 50:2 decreased (P < 0.05) during postprandial lipemia after consumption of both oils. TAG 48:1 and 52:4 decreased (P < 0.05) after palm oil only, and TAG 46:1 after transesterified palm oil only. Proportions of 48:0, 52:3 and 54:4 remained constant in chylomicrons during postprandial lipemia.
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and 2
. The number of double bonds affected chylomicron TAG clearance. When TAG were compared within groups of equal number of acyl carbons (especially 48, 50, and 52), the TAG clearance was more rapid as the number of double bonds increased.
Chylomicron TAG positional distribution.
Regioisomerism of chylomicron TAG in molecular weight species with ACN:DB 50:1, 52:2 and 52:3 were analyzed. These chosen molecular weight species contained 49.5–55.4% of chylomicron TAG after palm oil load and 47.7–53.2% of TAG after transesterified palm oil load. No other ACN:DB species exceeded the 10% level. The fourth most abundant TAG in chylomicrons was ACN:DB 54:3, and because 46% of FA in the test fats were oleic acid, this molecular weight fraction contained mainly triolein. Similarly, ACN:DB fraction 48:0 contained mostly tripalmitin.
The regioisomers of chylomicron TAG after the two oral fat loads (Tables 3
and 4
) closely resembled the isomers of the appropriate fats ingested (Table 2)
. Palmitic acid was preferably located at the sn-1/3 positions in palm oil, and the FA of the transesterified palm oil were randomly distributed, which was reflected in the chylomicrons.
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and 4)
.
The ACN:DB fraction 52:2 contained TAG with two oleic acid residues and one palmitic acid residue. Significant differences from linear trend (P < 0.001 after palm oil, and P = 0.004 after transesterified palm oil) were seen in 1(3),2-dioleoyl-3(1)-palmitoyl-sn-glycerols (sn-18:1–18:1–16:0 + sn-16:0–18:1–18:1). After palm oil, the 3-h time point differed from the 1.5-, 2-, 4- and 5-h time points, and the 6-h time point from the 2- and 4-h time points. After transesterified palm oil, the 3- and 6-h time points differed from the 1.5- and 4-h time points and the 3-h time point from the 5-h time point. In both treatments, the 3- and 6-h time points were generally higher than the other time points, although the differences were not always significant (P = 0.002–0.074 and P = 0.001–0.224). The other regioisomer in ACN:DB 52:2, 1,3-dioleoyl-2-palmitoyl-sn-glycerol (sn-18:1–16:0–18:1), behaved linearly after both oils (Tables 3
and 4)
. No differences were seen in the relative proportions of the regioisomers from the ACN:DB 52:3 species.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–21
). However, in the most frequently applied analytical procedures for characterization of fats, the composition of individual TAG molecules is largely overlooked. In this study, the tandem mass spectrometric analysis method applied enabled us to observe the behavior of seven individual TAG regioisomers during postprandial lipemia after two fat loads with different positional distributions but identical FA composition in humans. The three most abundant molecular weight fractions were chosen for regioisomerism analysis. To our knowledge, such detailed information of human chylomicron TAG has not been published before, probably because the methods of analysis traditionally applied are laborious and require relatively large sample amounts.
The proportion of 1,3-palmitoyl-2-oleoyl-sn-glycerols (sn-16:0–18:1–16:0) was smaller at 1.5 h than at later time points in both treatments, which may be an indication of loss of palmitic acid released from the sn-1/3 positions in the gut. This was the only one of the regioisomers we studied that contained two palmitic acid residues at the primary positions. The proportion of 1(3),2-dioleoyl-3(1)-palmitoyl-sn-glycerols (sn-18:1–18:1–16:0 + sn-16:0–18:1–18:1) peaked at 3 h and tended to peak again at 6 h, but the physiologic explanation for this finding is unknown.
Despite the many ways in which FA positional distribution might affect chylomicron TAG composition, no clear trends indicating selective clearance of TAG regioisomers were found. However, in animal experiments, palmitic and stearic acids in the sn-2 position have been shown to slow down chylomicron clearance (22
,23
,36
). In some (36
,37
), but not all (22
,24
,38
) animal studies, TAG hydrolysis rates have also been affected by the positional distribution of saturated FA. In most of the animal experiments, a pure form or a mixture of two regioisomers was used. Our fats contained many different regioisomers, and it is possible that these would behave differently during the postprandial period if fed in pure form or as a mixture of only a few regioisomers. The total amount of fat given might also affect the results.
Despite differences in the positional distribution of FA in the two test fats, the clearance of TAG molecular weight species was similar after both treatments, further suggesting that the positional distribution of FA might not have a major effect on the clearance of TAG from chylomicrons.
It has been suggested that the physicochemical properties of TAG could cause selective mobilization of FA, at least from white fat cells (39
). Therefore, the molecular species of TAG might be one of the regulating factors in TAG mobilization. In this study, we found that the proportions of TAG containing palmitic and palmitoleic acid residues (ACN:DB 48:2) and palmitic, palmitoleic and oleic acid residues (ACN:DB 50:3 and 50:2) decreased during postprandial lipemia. At the same time, the proportion of triolein (ACN:DB 54:3) increased. TAG clearance seemed to be more rapid as the number of double bonds in TAG increased, but because the test fats did not contain unsaturated FA other than oleic and linoleic acids, research with more unsaturated and complex fats is warranted. Surprisingly, the amount of tripalmitin (ACN:DB 48:0) in chylomicron TAG remained fairly constant during lipemia, suggesting that the polarity of TAG might not be a major regulatory factor of TAG clearance.
Recently, it was found that postprandial TAG from dietary olive oil were cleared selectively in humans (40
). Triolein had the fastest rate of clearance, whereas the concentrations of TAG with polyunsaturated FA and saturated FA tended to increase. However, in that study, none of the stereospecific positions were separated. In contrast, we found that the concentration of triolein increased, and TAG with saturated FA either remained constant or decreased. It is possible that the FA composition of the fat ingested has considerable influence on the TAG composition of chylomicrons and VLDL, and therefore the results of different studies are difficult to compare.
The tandem mass spectrometric analysis method used in our study enables the study of TAG regioisomerism in individual molecular species. New information on selective clearance of chylomicron TAG during postprandial lipemia was obtained. Statistical differences were observed in the proportions of chylomicron TAG molecular weight species and some regioisomers during the course of postprandial lipemia, indicating that TAG molecular weight composition and to a lesser extent, positional distribution seem to affect the rates of chylomicron TAG clearance in humans. Molecular-level research with different fats and metabolic conditions is required to gain better understanding of the mechanisms controlling human postprandial triacylglycerol metabolism.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: ACN, acyl carbon number; DB, double bond; FA, fatty acid; LPL, lipoprotein lipase; TAG, triacylglycerol.
Manuscript received 6 November 2001. Initial review completed 3 December 2001. Revision accepted 4 February 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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