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INRA, Unité de Nutrition Lipidique, Dijon, France and * CEA-Saclay, Service des Molécules Marquées, Gif Sur Yvette, France
2To whom correspondence should be addressed. E-mail: chardign{at}dijon.inra.fr.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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KEY WORDS: • rumenic acid • triacylglycerols • bioavailability • rats • dairy fat
Rumenic acid (cis9, trans11–18:2) (1), is the major isomer of conjugated linoleic acid (CLA)2 naturally present in food, including dairy products and ruminant meat. Conjugated fatty acids result in part from the biohydrogenation of PUFA in the rumen, but also from the 
9 desaturation of trans vaccenic acid in various tissues, including the mammary gland of lactating cows (2). CLA isomers have been reported to have beneficial effects on health, including cancer (3), atherosclerosis (4), diabetes (5), immune function (6) and body composition (7). Most of these data were obtained using various synthetic CLA mixtures, but the trans10,cis12 isomers were reported to be the most effective on body composition in mice (8). On the other hand, rumenic acid was reported to have effects on cancer (9) and cytotoxicity (10). However, the mechanism(s) of its action is still not clearly understood. Moreover, the metabolic fate related to the position on the triacylglycerol molecule and its bioavailability have not been yet investigated.
Different mechanisms of action including effects on peroxisome proliferator- activated receptors (11) and eicosanoid production (12) have been proposed. However, no study has yet investigated the effect of the position of CLA on the triacylglycerol (TAG) on its metabolic fate and bioavailability. So far, the lymphatic absorption of CLA has been studied using CLA as free fatty acids (FFA) (13) or nonstructured TAG (14). However, the bioavailability may be modulated by the position of rumenic acid on the TAG. The present study was carried out to consider the metabolism of rumenic acid located at the external (sn-1 or sn-3) or internal (sn-2) position of the TAG. Additionally, the position of rumenic acid on butter and "Camembert" cheese TAG was also determined.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Two dairy products, "Camembert" cheese and butter, were purchased from local supermarkets and analyzed for their rumenic acid content on the TAG molecules. Lipids were extracted from lyophilized cheese and fresh butter according to Yurawecz et al. (15), with minor modifications. Briefly, lipids were extracted using a mixture of diethyl ether and hexane (1:1, v/v) after dispersion in ethanol. The upper phase was evaporated to dryness under a nitrogen stream.
The analysis of the triacylglycerol structure was conducted as proposed by Christie (16). The lipids (5–10 mg) were incubated for 5 min at 37°C in 2 mL of Tris buffer, pH 8, in the presence of 5000 IU of porcine pancreatic lipase (Sigma Chemicals, Saint Quentin Fallavier, France) and gum arabic (10 g/100 g). After extraction using hexane, the 2-monoacyl glycerols (Rf = 0.1) were separated from other fractions by TLC on silica gel plates using a mixture of hexane/diethyl ether/acetic acid (60:40:4, v/v/v). Finally, the fatty acids from the 2-monoacylglycerol fraction were transformed into methyl esters according to Christie et al. (17). Briefly, the 2-monoacylglycerols were dissolved in toluene and heated at 50°C for 5 min in the presence of sodium methanolate (0.5 mol/L). The resulting FAME were then extracted and analyzed by GC using a Hewlett-Packard series II (Hewlett-Packard, Palo Alto, CA) gas chromatograph packed with a fused silica CPSil88 (Chrompack, Middleburg, The Netherlands) capillary column (100 m x 0.32 mm i.d.). The injector and detector were maintained at 250°C, the oven temperature was programmed from 60°C to 190°C and H2 was used as carrier gas.
Total lipids were methylated and analyzed using the same procedure. The part of rumenic acid present in the sn-1 or sn-3 position was calculated using the equation proposed by Christie (16), [position 1 and 3 = 3 x (triacylglycerol–position 2)/2].
Rumenic acid and triacylglycerols.
[1-14C]-rumenic acid was synthesized as previously described (18). Briefly, sequential substitution of (E)-1,2-dichloro-ethene and stereoselective reduction were used as key reactions. A first metal-catalyzed cross-coupling reaction between (E)-1,2-dichloro-ethene and 2-non8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conjugated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative. Stereoselective reduction of the triple bond with disiamylborane after bromination with triphenylphosphine dibromide afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Formation of the Grignard reagent and carbonation with 14CO2 provided [1-14C]-(9Z,11E) CLA isomer (specify activity 1.9 GBq/mmol). Radiochemical and isomeric purities were >99% by reversed-phase (RP)-HPLC. The identification of [1-14C] rumenic acid was confirmed on its methyl ester derivative by comparison with an authentic sample using GC/MS (HP-23 column, Interchim, Montluçon, France) and HPLC (ChromSpher 5 Lipids column, Merck, Darmstadt, Germany).
Commercially available 1,3-dioleoyl-glycerol and 1,2-dileoyl-rac-glycerol (99 and 97% pure, respectively) were from Sigma. They were coupled with [1-14C] rumenic acid in the presence of dicyclohexyl-carbodiimide and 4-DMAP to provide labeled triacylglycerols (19). After purification by flash chromatography [silica gel; pentane/diethyl ether (90:10)], chemical and radiochemical purities (>99%) were determined by TLC and RP-HPLC.
Animals.
Male Wistar male rats (n = 9; Elevage Janvier, Le Genest Saint Isle, France), weighing 305–325 g were used in this experiment. The protocol was carried out according to the French regulation on animal experiments. They were housed in plastic cages in an animal house under controlled conditions (12-h light:dark cycle, temperature 26 ± 1°C, 55% humidity). They were fed a commercial diet (Harlan, Gannat, France). The day before the experiment, the rats were isolated in individual cages.
All of the experiments were started at the same time (1700 h) to avoid nyctemeral variations. Intragastric intubations of TAG containing labeled rumenic acid on the internal (sn-2) or external (sn-1 or sn-3) position were performed. The tracer was diluted in triolein to facilitate the transfer of labeled TAG (130 MBq) in 
270 mg of triolein to the rats.
Immediately after the force-feeding, the rats were placed in an air-tight plexiglas cage, as previously described (20). The expired CO2 was trapped in Carbosorb E (Packard, Groningen, The Netherlands) for further quantification. The rats were left in the cage for 16 h, with the light on from 1700 h until the end of the experiment.
Tissue analyses.
After the 16-h experiment, the rats were anesthetized by intraperitoneal injection of pentobarbital (Sanofi, Libourne, France). After laparotomy, blood was withdrawn into a heparinized syringe. Tissues (heart, liver, stomach, gastrocnemial muscle, kidneys, epididymal adipose tissue, brown adipose tissue, large and small intestine) were removed, blotted on filter paper and weighed. For the small intestine, the lumen and the mucosa were treated separately. The carcass of each rat was weighed before homogenization.
Three portions (30–80 mg) of each tissue were finely minced and five portions of the carcass (50–100 mg) as well as the two parts of the gastrointestinal tract were digested overnight at 50°C using 1 mL of Soluene (Packard, Groningen, The Netherlands). The radioactivity of the samples was then determined by liquid scintillation counting as described above, after addition of Hionic Fluor (Packard) scintillation cocktail.
Liver lipids were extracted according to Folch et al. (21). Lipid classes were separated by TLC on silica gel plates (Merck, Darmstadt, Germany), using a mixture of hexane/diethyl ether/acetic acid (80:20:1, v/v/v). The radioactivity incorporated into phospholipids (Rf = 0.1), TAG (Rf = 0.44) and cholesteryl esters (Rf = 0.73) was determined by scanning the plates using a linear TLC analyzer (Berthold).
Total liver fatty acids were esterified into methyl esters (FAME) as described above. FAME were analyzed using radio-GC analyses as previously described (20). Briefly, a Hewlett-Packard 5890 series II gas chromatograph (Palo Alto, CA) equipped with a splitless injector and a fused Stabilwax wide-bore silica column (60 m x 0.53 mm i.d. film thickness: 0.50 µm, Restek, Evry, France) was used. The output flow from the column was split between a flame-ionization detector (10%) and a copper oxide oven heated at 700°C to transform the labeled fatty acids into 14CO2 (90%). The radioactivity was determined with a radiodetector (GC-RAM, Lablogic, Sheffield, UK) by counting 14CO2 after mixing it with a mixture of argon and methane (9:1). The data were computed using Laura software (Lablogic).
Statistical analysis.
Results are expressed as means ± SEM. The means were compared using the SYStat software and the t test procedure. P-values of < 0.05 were considered to be significant.
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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In the sample of "Camembert" cheese and butter, rumenic acid was esterified mainly in the sn-1 and/or sn-3 position (74%) vs. only 26% esterified in the sn-2 position, clearly indicating that the major part of rumenic acid was released as FFA from the TAG in the intestinal lumen and was further absorbed as FFA.
Rumenic acid metabolism in rats.
At the time of killing, the recovery of the radioactivity was calculated as a percentage of the dose administered to the rats. These recoveries were 96.4 ± 2.7 and 97.4 ± 6.1% for the 1,3 TAG and 2-TAG, respectively, and they did not differ.
The total radioactivity was approximately the same between the absorbed and excreted radioactivity. The excreted radioactivity is represented by the sum of what was recovered in the feces, cecum as well as in the lumen (not mucosa) of the distal parts of the small intestine. The excretion was <15% of the radioactivity ingested, but the excretions of 1,3-TAG and 2-TAG (4.5 ± 0.9 and 13.5 ± 5.5%, respectively) differed (P < 0.05). Generally, the intestinal absorption of fatty acids is higher when they are present in the internal (sn-2) position of the TAG. Rumenic acid does not behave like a common fatty acid with a methylene-interrupted structure.
No previous study has reported the absorption of labeled rumenic acid. The intestinal absorption of CLA was reported only in comparison with linoleic acid absorption. Using a mixture of CLA isomers as FFA, Sugano et al. (13) reported that their intestinal absorption in rats was lower than that of linoleic acid. On the other hand, a CLA mixture administered to rats as TAG was absorbed as well as linoleic acid (14). Because TAG is the form in which fatty acids are consumed, it is possible that CLA would be absorbed as well as linoleic acid from the diet. However, in our study, the absorption of rumenic acid was affected by the position of the fatty acid in the glycerol moiety. Generally, fatty acids are better absorbed when present at the internal (sn-2) position, but this was not the case for rumenic acid in our study.
A major portion of the radioactivity administered to rats in this study was recovered in the labeled CO2 expired, and the percentage recovered was greater in rats force-fed 1,3-TAG than in those fed 2-TAG (P < 0.05) (Table 1). Incorporation of the radioactivity into other tissues was lower in the former group (43 vs. 56%, P < 0.05). To our knowledge, no data exist concerning the effect of the positional distribution of fatty acids on their metabolic oxidation. It would be of interest to compare our present data on rumenic acid with other nonconjugated fatty acids under similar conditions.
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The radioactivity in liver was primarily in the TAG (45.5 ± 5.3 and 50.6 ± 4.1%) in the 1,3-TAG and 2-TAG groups, respectively; P > 0.05), in agreement with previous reports (22,23). The incorporation into liver phospholipids did not differ between the groups (33–34%). In the cholesteryl esters, the incorporation into rats fed the 1,3-TAG (8.2%) was higher than in the 2-TAG group (4.9%, P < 0.05). We do not have an explanation for this difference.
Conversion into CLA metabolites (conjugated 18:3 or conjugated 20:3) did not occur according to radio-GC analyses, as was shown previously (22,24). This may be due to our shorter experiment or the use of a normal diet rather than an essential fatty acid–deficient diet as was used previously (24).
The present data suggest that rumenic acid from dairy fat may be well absorbed and used extensively for energy production, as already reported for FFA (25). Furthermore, the data suggest that dietary rumenic acid from cheese and butter is present mainly in the external position of the TAG, which enhances its metabolic fate in the oxidative pathway. It is not known whether a similar pathway exists in humans. This could be confirmed using 13C-labeled rumenyl-TAG, which would permit an exact determination of CLA metabolism and allow a comparison of the fate of rumenic acid in the nutritional context because differences exist between animal models and humans. For example, in previous work on the trans isomers of linoleic and linolenic acids, their was a similar trend in the two species, but the extent of ß-oxidation was lower in humans (20,26).
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FOOTNOTES |
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3 Abbreviations used: CLA, conjugated linoleic acid; FFA, free fatty acids; Rf, retention factor; RP, reversed-phase; TAG, triacylglycerol.
Manuscript received 3 April 2003. Initial review completed 22 May 2003. Revision accepted 30 September 2003.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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