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2 Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan; 3 Department of Food Management, School of Food, Agricultural and Environment Sciences, Miyagi University, Sendai 982-0215, Japan; 4 Faculty of Nursing and Nutrition, Siebold University of Nagasaki, Nagasaki 851-2195, Japan; 5 Plantech Research Institute, Yokohama, Japan; 6 Department of Bioresource Science, Faculty of Agriculture, Tamagawa University, Tokyo, Japan; 7 The Nisshin OilliO Group, Shinkawa 1-chome, Chuo-ku, Tokyo 104-8285, Japan; and 8 Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
* To whom correspondence should be addressed. E-mail: tsuduki{at}myu.ac.jp.
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ABSTRACT |
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-eleostearic acid (-ESA; 9Z11E13E-18:3) is converted to 9Z11E-conjugated linoleic acid (CLA) in rats through a 
13-saturation reaction. To investigate this further, we examined the absorption and metabolism of -ESA in rat intestine using a lipid absorption assay in lymph from the thoracic duct. In this study, we used 4 test oils [tung oil, perilla oil, CLA-triacylglycerol (TG), and pomegranate seed oil, containing -ESA, -linolenic acid (LnA; 9Z12Z15Z-18:3), CLA, and punicic acid (PA; 9Z11E13Z-18:3), respectively]. Emulsions containing the test oils were administered to rats, and lymph from the thoracic duct was collected over 24 h. The positional and geometrical isomerism of CLA produced by PA metabolism was determined using GC-electron impact (EI)-MS and 13C-NMR, respectively; the product was confirmed to be 9Z11E-CLA. A part of -ESA and PA was converted to 9Z11E-CLA 1 h after administration; therefore the lymphatic recoveries of -ESA and PA were modified by the amount of recovered CLA. Cumulative recovery of CLA, -ESA, and PA was lower than that of LnA only during h 1 (P < 0.05), and cumulative recovery of -ESA and PA was significantly lower than that of LnA and CLA for 8 h (P < 0.05). Therefore, the absorption rate was LnA > CLA > -ESA = PA. The conversion ratio of -ESA to 9Z11E-CLA was higher than that of PA to 9Z11E-CLA over 24 h (P < 0.05). These results indicated that -ESA and PA are slowly absorbed in rat intestine, and a portion of these fatty acids is quickly converted to 9Z11E-CLA.
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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1% (wt:wt), thus preventing natural fats from being used as health-promoting foods containing CLA. Therefore, at present, oils that include CLA are prepared from alkali-isomerization of vegetable oils such as safflower oil, and these products are marketed as health supplements (6,7).
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-eleostearic acid (-ESA; 9Z11E13E-18:3) and punicic acid (PA; 9Z11E13Z-18:3) at levels of 60–80% (wt:wt) (Fig.1) (8–11). In Okinawa, a region inhabited by the longest-living people in Japan, which is in itself one of the leading countries in the world in terms of life expectancy, people often eat bitter gourds (Momordica charantia). The seed oil of such gourds contains 60% -ESA (wt:wt) and the flesh contains a small amount of -ESA. We are particularly interested in seed oils that contain conjugated linolenic acids (CLnA), which are the only conjugated fatty acids that can be prepared from natural sources in bulk. We showed previously that CLnA has a stronger antitumor effect than CLA in vitro and in vivo (9). -ESA (which is a CLnA) also has useful physiological effects. We showed that -ESA, a conjugated triene, is partially saturated at the 13 position and converted to 9Z11E-CLA, a conjugated diene, after administration in rats; the 13-saturation reaction is an NADPH-dependent enzymatic reaction that occurs most rapidly in rat liver and small intestine (Fig. 1) (8,12). However, the characteristics of absorption and metabolism of -ESA have yet to be clearly determined in vivo, and the absorption and metabolism of -ESA in the small intestine are particularly unclear. For example, the following issues remain unresolved: the ratio of -ESA conversion to CLA in the small intestine; whether absorption of -ESA is higher than that of unconjugated fatty acids and CLA; whether structural differences in conjugated fatty acids affect absorption in the small intestine; and whether the saturation reaction occurs regardless of the structure of the conjugated double bond. CLA and -ESA have useful physiological functions; therefore it is also important to examine the conversion of -ESA to CLA in vivo from this perspective. In addition, we were interested in this reaction because it is a rare example of fatty acid metabolism involving saturation of a double bond; hence, we investigated the absorption and metabolism of -ESA in the rat intestine using a lipid absorption assay in lymph from the thoracic duct. This assay was performed as described previously (13,14).
To clarify the absorption and metabolism of -ESA in rat intestine, we used 4 test oils (tung oil, perilla oil, CLA- triacylglycerol (TG) and pomegranate seed oil): tung oil contains aTG form of -ESA, and was used as the test oil (Fig.1); perilla oil contains a TG form of -linolenic acid (LnA), which is an isomer of -ESA, and was used as a control oil; CLA-TG contains a TG form of CLA, and was also used as a control oil (Fig. 1); and pomegranate seed oil contains a TG form of punicic acid (PA), and was used as an isomeric comparison for the 13 saturation reaction. In punicic acid, the double bond that potentially undergoes the saturation reaction in rats has a Z configuration, whereas in -ESA this double bond has an E configuration (Fig. 1). To confirm that PA was converted to CLA, the structure of the CLA product was determined using GC-electron impact (EI)-MS and 13C-NMR, as previously reported (12,15).
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Materials and Methods |
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Fatty acid composition of test oils. Perilla oil, CLA-TG, tung oil, or pomegranate seed oil and a known amount of heptadecanoic acid (17:0, Sigma Chemical) as an internal standard were methylated by the addition of sodium methoxide:methanol for 5 min at room temperature, as described previously (15,16). Each of the 4 test oils was dissolved in 1 mol/L sodium methoxide:methanol solution. After 5 min of incubation at room temperature, the reaction was stopped by the addition of saturated NaCl solution, and FAME were then extracted with n-hexane and subjected to GC (GC 353B gas chromatograph, GL Sciences) using a flame ionization detector and a Supelcowax-10 fused silica capillary column (60 m x 0.32 mm i.d., Supelco). Helium was used as the carrier gas. The injector and detector temperatures were 200 and 250°C, respectively, and the column oven temperature was increased by 20°C/min from 50 to 220°C and then held constant for 31.5 min. Peak components were identified by comparing their retention times with those of commercial FAME (Funakoshi). The fatty acid compositions of the 4 test oils are shown in Table 1.
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Measurement of fatty acids in lymph. Total lipids from the lymph were extracted by Folch's procedure (17,18), and methylated (along with a known amount of heptadecanoic acid as an internal standard) by the addition of trimethylsilyldiazomethane for 30 min at room temperature and sodium methoxide/methanol for 5 min at room temperature, as previously reported (15,16). Total lipids in 9 mL of MeOH:benzene (2:7, v:v) were added to 1.3 mmol/L trimethylsilyldiazomethane in n-hexane. After standing for 30 min at room temperature, the reaction mixture was dried under a stream of nitrogen gas, and the dried residue was dissolved in 1 mol/L sodium methoxide:methanol solution. After a 5-min incubation at room temperature, the reaction was stopped by adding saturated NaCl solution. FAME were extracted by n-hexane, and subjected to GC under the same conditions as those described above. Peak components were identified by comparing their retention times with those of commercial FAME, and also by analyzing the corresponding fatty acid dimethyloxazoline derivatives by GC-EI-MS (GCMS-QP5050A, Shimadzu ), as previously described (12). For the GC-EI-MS analysis, 4,4-dimethyloxazoline (DMOX) derivatives were prepared from the corresponding fatty acids as follows. Total lipids in the lymph were processed to free fatty acids, and the free fatty acids were mixed with 2-amino-2-methyl-1-propanol. The test tube was purged with nitrogen gas, screw-capped, and heated at 170°C. After 30 min, the tube was cooled, and a saturated NaCl solution and n-hexane were added, followed by vigorous shaking. After centrifugation, the hexane phase was dried under a stream of nitrogen gas, and the dried residue was then redissolved in an adequate amount of n-hexane. GC-EI-MS was performed using a GC (GC-17A, Shimadzu) equipped with a Supelcowax-10 fused silica capillary column coupled to an EI-MS (GCMS-QP5050A, Shimadzu). Helium was used as the carrier gas. The injector temperature was 200°C. The oven temperature was programmed to match the temperature used in the corresponding GC analysis described above. The temperature of the ion source was 250°C. Electron impact mass spectra were recorded at 70 eV and analyzed using the CLASS 5000 data system (Shimadzu).
Determination of CLA structure. To determine the positional and geometrical isomerism of the CLA produced by PA metabolism, total lipids in the lymph of pomegranate seed oil-treated rats 1–2 h after administration were converted into methyl ester derivatives (using the method described above), and the compounds were isolated using HPLC, as previously described (12). The lymph lipid methyl esters were dissolved in hexane, and this solution was fractionated by normal-phase HPLC using an 880-PU pump (Jasco) with an SPD-M10AVP detector (Shimadzu). A ChromSpher Lipids column impregnated with silver (5-µm particle size, 4.6 mm x 250 mm, Chrompack,) was used. In the first separation, a hexane:acetonitrile (100:0.5, v:v) mobile phase was used, with a flow rate of 1.0 mL/min and detection at 210 nm (all FAME) and 235 nm (fatty acid methyl esters with a conjugated diene). A substance showing a large peak at 235 nm was fractionated, and the collected fraction was purified in a second separation, using a mobile phase with a different composition. In the second separation, a hexane:acetonitrile (100:0.15, v:v) mobile phase was used, with a flow rate of 1.0 mL/min and detection at 210 and 235 nm. The substance showing a large peak at 235 nm was again fractionated. The fraction was collected and dried completely under nitrogen gas, and confirmed to be a single compound using GC. The isolated compound was converted to its 4,4-dimethyloxazoline derivative before being subjected to GC-EI-MS analysis, as described above. The geometrical isomerism of the conjugated double bonds was distinguished easily using 13C NMR, under conditions similar to those reported previously (12). The isolated compound was dissolved in CDCl3 (Wako Pure Chemicals Industries) at a concentration of 1 g/L, and the 13C NMR spectra were recorded on a Varian Unity 600 spectrometer operating at 150 MHz.
Statistical analysis. Statistical analysis was performed using 1-way ANOVA, followed by a Newman-Keuls test for multiple comparisons among several groups. Differences were considered to be significant at P < 0.05.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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-ESA (9Z11E13E-18:3), a conjugated LnA, in the rat small intestine. Tung oil containing the TG form of -ESA at a level of 76% was used as the test oil (Fig.1, Table 1). Perilla oil containing 62.8% of the TG form of LnA, an isomer of -ESA, and CLA-TG containing 77.6% of the TG form of CLA were used as controls (Fig. 1, Table 1). In addition, pomegranate seed oil containing 74.5% the TG form of PA was used as a comparison sample for the 13 saturation reaction (Fig. 1, Table 1): in PA, the double bond undergoing the saturation reaction is in the Z configuration, whereas the same double bond in -ESA is in the E configuration. CLA was not detected in the tung oil or pomegranate seed oil (Table 1). The total amounts of conjugated fatty acids in the test oils were 0% in perilla oil, 77.6% in CLA-TG, 79.1% in tung oil, and 75.9% in pomegranate seed oil (Table 1). The lymph from the thoracic duct of rats infused with emulsions containing each oil had normal flow properties (Supplemental Figure 1), confirming that the surgery and maintenance of rats was carried out appropriately. Lymph flow rates did not differ and were 95 ± 12, 107 ± 17, 99 ± 13, and 104 ± 24 mL/24 h in the perilla oil, CLA-TG, tung oil, and pomegranate seed oil groups, respectively (Supplemental Figure 1).
There were no conjugated fatty acids in rat lymph immediately before administration of the oils, based on GC analysis (Supplemental Figure 2A). A peak due to the administered oils appeared in the GC of the lymph 1 h after administration (Supplemental Figure 2B–E). A peak for LnA was apparent in the GC of the lymph of rats administered perilla oil, compared with the lymph before administration (Supplemental Figure 2B), and peaks for 9Z11E-CLA and 10E12Z-CLA appeared in the GC of the lymph of rats administered CLA-TG (Supplemental Figure 2C). Similarly, peaks for -ESA and 9Z11E-CLA appeared in the GC of the lymph of rats administered tung oil (Supplemental Figure 2D), showing that -ESA was converted to 9Z11E-CLA in the rat intestine. Peaks for PA and 9Z11E-CLA appeared in the GC of the lymph of rats administered pomegranate seed oil (Supplemental Figure 2E), suggesting that PA may have been converted to 9Z11E-CLA. To explore this further, the structure of the CLA formed by PA metabolism was determined using GC-EI-MS and 13C-NMR.
To determine the geometrical isomerism of the CLA formed from PA, the CLA was first isolated and purified using HPLC. Total lipids were extracted from the lymph of rats 1 h after administration of pomegranate seed oil, converted to methyl ester derivatives, and analyzed using HPLC. In the first separation, a chromatogram at 235 nm showed a clear peak with a retention time of 6 –7 min due to methyl esters of fatty acids with a conjugated diene, and this peak was isolated. In the second separation, a chromatogram at 235 nm showed a large peak with a retention time of 16–17 min, and this peak was also isolated. In subsequent GC analysis, the contents of the isolated HPLC peak appeared as a single peak, confirming that a pure compound had been isolated. Based on the retention time, the compound was determined to be 9Z11E-CLA or 9E11Z-CLA.
The methyl ester of the isolated CLA was converted to its DMOX derivative and analyzed using GC-EI/MS, in which a fragment due to the presence of a conjugated double bond was apparent. The GC-EI-MS spectrum of the DMOX derivative of this CLA had a molecular ion with an m/z ratio of 333, and fragments of m/z 182 and 262, due to allylic cleavage of the fatty acid chain. Furthermore, the mass spectrum exhibited a mass difference of 12 units between fragments of m/z 196 and 208, and between fragments of m/z 222 and 234, indicating the presence of conjugated double bonds at carbon atoms 9 and 11. In the 13C-NMR spectrum, the methyl ester of the isolated CLA gave signals of 
c 174.32 (C-1), 34.08 (C-2), 24.86 (C-3), 29.05 (C-4), 29.12–29.67 (C-5/C-6/C-7), 27.66 (C-8), 129.87 (C-9), 128.75 (C-10), 125.60 (C-11), 134.76 (C-12), 32.91 (C-13), 29.41 (C-14), 28.95 (C-15), 31.77 (C-16), 22.65 (C-17), 14.12 (C-18), and 51.42 ppm (COOCH3). The chemical shifts were compared with those for methyl esters of 9Z11E-CLA, which we reported previously(12); the chemical shifts for C-8 to C-13 were consistent with data for the 9Z11E-CLA methyl ester, confirming that the CLA isolated from rats administered pomegranate seed oil was indeed 9Z11E-CLA.
LnA was present in lymph collected from –2 h to 0 h (Supplemental Figure 2). Therefore, the recovery of LnA in lymph 0–24 h after administration was corrected by the amount of LnA in the lymph collected from –2 h to 0 h. With this correction, the lymphatic recovery of LnA in perilla oil–treated rats was 78% 8 h after administration and 91% after 24 h (Fig. 2A); therefore, almost all administered LnA was recovered. Similarly, the lymphatic recovery of CLAs in CLA-TG-treated rats was 71% 8 h after administration and 93% after 24 h (Fig. 2B). The lymphatic recovery curve for 9Z11E-CLA and 10E12Z-CLA in CLA-TG–treated rats was almost the same, showing that the absorption rates for these CLAs did not differ. Because -ESA is converted to 9Z11E-CLA, the lymphatic recovery of -ESA in tung oil–treated rats was taken as the total of recovered -ESA and 9Z11E-CLA; based on this assumption, the lymphatic recovery of -ESA in tung oil–treated rats was 54% 8 h after administration and 91% after 24 h (Fig. 2C): 72% of the -ESA administered to rats was recovered unchanged, and 19% was collected as 9Z11E-CLA due to metabolism of -ESA; therefore, 21% of the -ESA absorbed in rats was converted to 9Z11E-CLA. PA was also converted to 9Z11E-CLA in vivo, and therefore the lymphatic recovery of PA in pomegranate seed oil–treated rats was taken as the total of recovered PA and 9Z11E-CLA. The lymphatic recovery of PA in pomegranate seed oil–treated rats was 53% 8 h after administration and 90% after 24 h (Fig. 2D): 79% of the PA administered to rats was recovered unchanged, and 11% was collected as 9Z11E-CLA converted from PA; therefore, 12% of PA absorbed in rats was converted to 9Z11E-CLA. Overall, the results show that most of the LnA, CLA, -ESA and PA administered was absorbed within 24 h.
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-ESA, and PA in the lymph was compared at each time point (Fig. 3A). Recovery of CLA in the thoracic duct lymph of rats infused with CLA-TG was less than that of LnA in the thoracic duct lymph of rats infused with perilla oil for h 1 only (P < 0.05); recovery of CLA was 73% that of LnA in h 1, but cumulative recoveries of CLA and LnA were similar for all other times. Because -ESA and PA were partly converted to 9Z11E-CLA, the cumulative recovery of -ESA or PA in tung oil or pomegranate seed oil–treated rats was taken to be the total of 9Z11E-CLA and -ESA or PA. The recovery of -ESA+CLA and PA+CLA did not differ at any time point. The recovery of -ESA and PA was lower than that of LnA and CLA after 8 h (P < 0.05): recovery of -ESA was 69% that of LnA and 76% that of CLA, and recovery of PA was 68% that of LnA and 75% that of CLA. The cumulative recovery after 24 h did not differ among the four groups.
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-ESA, and PA were calculated again to compare for each interval (Fig. 3). There was a significant difference among recoveries in the 4 groups for the 0–1 h interval, as described above (Fig. 3A). Recovery of -ESA and PA was significantly greater than that of LnA and CLA in the 8- to 24-h interval (P < 0.05, Fig. 3B); in this interval, lymphatic recovery of -ESA was 293% that of LnA and 170% that of CLA, and the lymphatic recovery of PA was 294% that of LnA and 170% that of CLA. The 4 groups did not differ for any other intervals. Overall, these results show that the absorption rates of CLA, -ESA, and PA were lower than that of LnA: the final order of absorption rates was determined to be LnA > CLA > -ESA = PA. Next, the lymphatic recovery of -ESA and PA were calculated again for a comparison at each time point (Fig. 3C). The cumulative recoveries of -ESA and PA did not differ until 8 h, but the cumulative recovery of PA was higher than that of -ESA after 24 h (P < 0.05): 72 and 78% for -ESA and PA, respectively. The ratios of conversion of -ESA and PA to 9Z11E-CLA were also compared (Fig. 3D). The conversion ratio did not differ until 6 h, but the conversion ratio of -ESA was higher than that of PA after 8 h (P < 0.05), and these ratios were 19 and 11% for -ESA and PA, respectively, after 24 h. Therefore, -ESA is converted to 9Z11E-CLA 1.6 times higher than the conversion of PA, and unchanged PA remains at a higher concentration than unchanged -ESA after administration to rats. |
Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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-ESA and PA), which occur naturally in large amounts in rat intestine. It was first confirmed that the PA contained in pomegranate seed oil was metabolized to 9E11Z-CLA in rat intestine. In our previous study, CLA was detected in rats administered -ESA, and the molecular structure of this CLA was determined to be 9Z11E-18:2 using GC-EI-MS and 13C-NMR (12). Hence, it is likely that PA, which also contains a conjugated triene system (9Z11E13Z), might be converted to 9Z11E-CLA, similarly to -ESA (which contains a 9Z11E13E conjugated triene system). Using GC, was isolatedthe peak due to CLA in lymph from rats administered pomegranate seed oil (Supplemental Figure 2); this peak reflected CLA formed by the metabolism of PA present in the oil. The CLA was purified using HPLC and its structure was determined using GC-EI-MS and 13C NMR (12). This analysis showed that the CLA from the metabolism of PA was of the 9E11Z-18:2 form, suggesting that PA can be used as a source of 9E11Z-CLA in vivo, similarly to -ESA.
In our previous study, very little -ESA was detected in the plasma and liver of rats fed a diet including 1% -ESA for 1 mo, but CLA was detected (8). In addition, 9E11Z-CLA was detected in rat tissues (plasma, liver, kidney, and small intestine mucosa) a short time (3 h) after oral administration of -ESA, and 9E11Z-CLA was detected in rat tissues (plasma, liver, kidney, and small intestine mucosa) of germ-free rats 6 h after -ESA administration, suggesting that most of the -ESA was converted to 9E11Z-CLA through a 13-saturation reaction in the small intestine (12). However, in the current study of thoracic duct lymph from rats, 70% of the -ESA administered was absorbed as unchanged -ESA without conversion to 9E11Z-CLA (Figs. 2 and 3). In our previous study, we showed that the 13-saturation reaction occurs not only in the small intestine but also in the liver and the kidney (12). Therefore, our results suggest that the majority of -ESA administered to rats is absorbed as -ESA, and then converted to 9E11Z-CLA in organs such as liver and kidney, although some -ESA is converted to 9E11Z-CLA in the small intestine. Using nude mice with transplanted tumor cells (9), we also showed previously that -ESA suppresses the growth of cancer cells more strongly than does 9E11Z-CLA,. The difference in bioactivation of -ESA and 9E11Z-CLA was difficult to explain after this observation because we thought that the majority of -ESA would be converted to 9E11Z-CLA after oral administration. However, the current results suggest that intact -ESA may reach the cancer cells because the majority of -ESA is absorbed in an unchanged state, which leads to stronger suppression of cancer growth upon administration of -ESA, compared with 9E11Z-CLA. Moreover, because the majority of PA administered to rats was absorbed as unchanged PA without conversion to 9E11Z-CLA, PA may also exert a physiological action different from that of CLA (Figs. 2 and 3). PA was reported to have useful physiological functions, but a comparison with CLA has not been made (10,11); however, our results suggest that the potential utility of PA should not be disregarded.
The conversion of -ESA to 9E11Z-CLA was greater than that of PA (Figure 3). Using rat tissue homogenates, we showed previously that this conversion is due to an NADPH-dependent enzyme reaction (12); therefore, it is possible that the difference in conversion rates of -ESA and PA was due to substrate-specific effects of this enzyme. The double bond at the 13 position of -ESA is an E-isomer, whereas that in PA is a Z-isomer (Fig. 1), and the double bond may more easily undergo enzymatic 13-saturation when in an E configuration. The enzyme responsible for the 13-saturation reaction is probably a type of oxidoreductase because it is NADPH dependent and able to saturate (reduce) double bonds. With -ESA, this reaction proceeded rapidly until completion, i.e., until the -ESA substrate is almost completely absent and a large amount of CLA product has accumulated, suggesting that the enzyme should be classified as part of the drug metabolism system, and not as part of the ß-oxidation enzyme group, in the fatty acid metabolic pathway (8,9). Leukotriene (LT) B4) a known activator of leukocytes, contains a conjugated triene structure, and although 
-oxidation is the major deactivation pathway of LTB4 in leukocytes, another pathway proceeding by reduction of conjugated double bonds has been reported (19–21). In the reductive pathway, LTB4 is converted to 12-oxo-LTB4 by LTB4 12-hydroxydehydrogenase, which is distributed in various porcine tissues and leukocytes (19,20). 12-oxo-LTB4 is converted further into 10,11,14,15-tetrahydro-12-oxo-LTB4 (conjugated dienoic acid) by another enzyme, which has yet to be identified (19–21). Therefore, we speculate that either a novel enzyme recognizing conjugated trienoic acids or the enzyme active in the LTB4 reductive pathway may convert -ESA (a conjugated triene) to CLA (a conjugated diene) through 13-saturation (reduction).
Our previous study and other studies showed that the 9Z11E-CLA concentration is always significantly higher than the 10E12Z-CLA concentration after administration of these compounds to mice and rats (8,9,22). We thought initially that the structural difference in the conjugated double bond in these isomers affected absorption in the small intestine; however, in the current study, there was no difference in lymphatic recovery of 9Z11E-CLA and 10E12Z-CLA after CLA-TG was administered to rats (Fig. 2), suggesting that geometrical and positional isomerism of the conjugated double bond did not influence absorption. An alternative explanation is that 10E12Z-CLA is more easily metabolized than 9Z11E-CLA after administration of equal amounts of these compounds to rats, and it was reported that 10E12Z-CLA more strongly activates the ß-oxidation system, compared with 9Z11E-CLA (23–26). Lymphatic recovery of the conjugated fatty acids (CLA and CLnA) was significantly lower than that of the unconjugated fatty acid (LnA) 1 h after administration, and this difference showed a tendency to persist for 8 h after administration (Fig. 3). However, the lymphatic recovery of the 4 fatty acids did not differ 24 h after administration (Fig. 3); the appearance of this difference in lymphatic recovery only at an early time point (Fig. 3) suggests that conjugated fatty acids remain in small intestinal cells for a longer time than unconjugated fatty acids. The times for which 50% of LnA, CLA, -ESA, and PA in each test oil-treated rats were secreted in the lymph are: 3.4, 4.0, 6.9, and 7.3 h after administration, respectively (Fig. 2). Thus, it was shown that the time for -ESA and PA to pass through the small intestine was double that of LnA. It is unlikely that there is a large difference among samples in the absorption rate in the small intestine because the test oils were administered in the triglyceride form. Conjugated fatty acids obstruct the movmen of apolipoproteins, making them move more slowly to chylomicrons compared with unconjugated fatty acids, before secretion into the lymph. Furthermore, lymphatic recovery of conjugated triene fatty acids (-ESA and PA) was significantly lower than that of CLA 1 h after administration, and this difference tended to persist for 8 h after administration (Fig. 3). The difference in absorption of CLA and conjugated triene fatty acids may be due to greater conversion of conjugated triene fatty acids to CLA; because conjugated triene fatty acids are slowly secreted from the small intestine, they have sufficient time to undergo the 13-saturation enzyme reaction. The lymphatic recovery of LnA, CLA, -ESA, and PA in rats treated with each test oil was 
90% 24 h after administration (Fig. 2). This results show that most of the LnA, CLA, -ESA, and PA administered was absorbed in 24 h.
A high-energy, high-fat diet is a risk factors for colon carcinoma, and mortality due to this disease is increasing annually. In contrast, development of colon carcinoma can be prevented and progress of the disease can be slowed by altering the composition of the diet; therefore, effectual measures for cancer prevention can be taken through diet modification. Compared with other CLA isomers, 9Z11E-CLA has few side effects (23–26), and because -ESA and PA are quickly converted to 9Z11E-CLA in vivo, it is likely that side reactions of -ESA and PA will also be minor. Therefore, the addition of -ESA and PA to foods as cancer preventative agents may be a useful strategy in the future. However, CLA is present in natural sources in only minute amounts, and it is extremely difficult to purify this compound from such sources. Furthermore, it is difficult to separate CLA isomers prepared by alkali isomerization in bulk. On the contrary, -ESA can be purified relatively easily from tung oil and bitter gourd seed oil because these oils contain CLnA as almost their only component; similarly, PA can be purified relatively easily from pomegranate seed oil because this oil essentially contains CLnA as a single component. Therefore, safety tests and functional evaluation can be performed using these oils, and because -ESA and PA are present in plant seeds in such large amounts, they can act as a source of 9Z11E-CLA after metabolism, providing the physiological activities of conjugated unsaturated fatty acids. Therefore, once the safety of -ESA and PA is confirmed, these fatty acids may have value as health food supplements.
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FOOTNOTES |
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9 Abbreviations used: -ESA, -eleostearic acid (9Z11E13E-18:3); CFA, conjugated fatty acids; CLA, conjugated linoleic acid; CLnA, conjugated linolenic acid; DMOX, 4,4-dimethyloxazoline; GC-electron impact- (EI-)MS, LnA, -linolenic acid (9Z12Z15Z-18:3); LT, leukotriene; PA, punicic acid (9Z11E13Z-18:3); TG, triacylglycerol.
Manuscript received 1 April 2006. Initial review completed 10 May 2006. Revision accepted 22 May 2006.
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LITERATURE CITED |
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1. Ha YL, Grimm NK, Pariza MW. Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis. 1987;8:1881–7.
2. Lee KN, Kritchevsky D, Pariza MW. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis. 1994;108:19–25.[Medline]
3. Park Y, Albright KJ, Storkson JM, Liu W, Cook ME, Pariza MW. Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid. Lipids. 1999;34:243–8.[Medline]
4. Ip C, Chin SF, Scimeca JA, Pariza MW. Mammary tumor prevention by conjugated dienoic derivative of linoleic acid. Cancer Res. 1991;51:6118–24.
5. Igarashi M, Miyazawa T. The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells. Biochim Biophys Acta. 2001;1530:162–71.[Medline]
6. Larsen TM, Toubro S, Astrup A. Efficacy and safety of dietary supplements containing CLA for the treatment of obesity: evidence from animal and human studies. J Lipid Res. 2003;44:2234–41.
7. Gaullier JM, Halse J, Hoye K, Kristiansen K, Fagertun H, Vik H, Gudmundsen O. Supplementation with conjugated linoleic acid for 24 months is well tolerated by and reduces body fat mass in healthy, overweight humans. J Nutr. 2005;135:778–84.
8. Tsuzuki T, Igarashi M, Komai M, Miyazawa T. A metabolic conversion of 9, 11, 13-eleostearic acid (18:3) to 9, 11-conjugated linoleic acid (18:2) in the rat. J Nutr Sci Vitaminol (Tokyo). 2003;49:195–200.[Medline]
9. Tsuzuki T, Tokuyama Y, Igarashi M. Miyazawa T. Tumor growth suppression by -eleostearic acid, a linolenic acid isomer with a conjugated triene system, via lipid peroxidation. Carcinogenesis. 2004;25:1417–25.
10. Arao K, Yotsumoto H, Han SY, Nagao K, Yanagita T. The 9cis,11trans,13cis isomer of conjugated linolenic acid reduces apolipoprotein B100 secretion and triacylglycerol synthesis in HepG2 cells. Biosci Biotechnol Biochem. 2004;68:2643–5.[Medline]
11. Yamasaki M, Kitagawa T, Koyanagi N, Chujo H, Maeda H, Kohno-Murase J, Imamura J, Tachibana H, Yamada K. Dietary effect of pomegranate seed oil on immune function and lipid metabolism in mice. Nutrition. 2006;22:54–9.[Medline]
12. Tsuzuki T, Tokuyama Y, Igarashi M, Nakagawa K, Ohsaki Y, Komai M, Miyazawa T. -Eleostearic acid (9Z11E13E–18:3) is quickly converted to conjugated linoleic acid (9Z11E–18:2) in rats. J Nutr. 2004;134:2634–9.
13. Tomoyori H, Kawata Y, Higuchi T, Ichi I, Sato H, Sato M, Ikeda I, Imaizumi K. Phytosterol oxidation products are absorbed in the intestinal lymphatics in rats but do not accelerate atherosclerosis in apolipoprotein E–deficient mice. J Nutr. 2004;134:1690–6.
14. Ikeda I, Tsuda K, Suzuki Y, Kobayashi M, Unno T, Tomoyori H, Goto H, Kawata Y, Imaizumi K, et al. Tea catechins with a galloyl moiety suppress postprandial hypertriacylglycerolemia by delaying lymphatic transport of dietary fat in rats. J Nutr. 2005;135:155–9.
15. Igarashi M, Tsuzuki T, Kambe T, Miyazawa T. Recommended methods of fatty acid methylester preparation for conjugated dienes and trienes in food and biological samples. J Nutr Sci Vitaminol (Tokyo). 2004;50:121–8.[Medline]
16. Tsuzuki T, Igarashi M, Iwata T, Yamauchi-Sato Y, Yamamoto T, Ogita K, Suzuki T, Miyazawa T. Oxidation rate of conjugated linoleic acid and conjugated linolenic acid is slowed by triacylglycerol esterification and -tocopherol. Lipids. 2004;39:475–80.[Medline]
17. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497–509.
18. Tsuzuki T, Igarashi M, Miyazawa T. Conjugated eicosapentanoic acid inhibits transplanted tumor growth via membrane lipid peroxidation in nude mice. J Nutr. 2004;134:1162–6.
19. Yokomizo T, Uozumi N, Takahashi T, Kume K, Izumi T, Shimizu T. Leukotriene A4 hydroxylase and leukotriene B4 metabolism. J Lipid Mediat Cell Signal. 1995;12:321–32.[Medline]
20. Yokomizo T, Izumi T, Takahashi T, Kasama T, Kobayashi Y, Sato F, Takekuni Y, Shimizu T. Enzymatic inactivation of leukotriene B4 by a novel enzyme found in the porcine kidney. J Biol Chem. 1993;268:18128–35.
21. Wainwright SL, Powell WS. Mechanism for the formation of dihydro metabolites of 12-hydroxyeicosanoids. Conversion of leukotriene B4 and 12-hydroxy-5, 8, 10, 14-eicosatetraenoic acid to 12-oxo intermediates. J Biol Chem. 1991;266:20899–906.
22. Hargrave KM, Azain MJ, Miner JL. Dietary coconut oil increases conjugated linoleic acid-induced body fat loss in mice independent of essential fatty acid deficiency. Biochim Biophys Acta. 2005;1737:52–60.[Medline]
23. Pariza MW, Park Y, Cook ME. The biologically active isomers of conjugated linoleic acid. Prog Lipid Res. 2001;40:283–98.[Medline]
24. Evans M, Brown JM. McIntosh MK. Isomer-specific effects of conjugated linoleic acid (CLA) on adiposity and lipid metabolism. J Nutr Biochem. 2002;13:508–16.[Medline]
25. Nagao K, Wang YM, Inoue N, Han SY, Buang Y, Noda T, Kouda N, Okamatsu H, Yanagita T. The 10 trans, 12 cis isomer of conjugated linoleic acid promotes energy metabolism in OLETF rats. Nutrition. 2003;19:652–6.[Medline]
26. Masso-Welch PA, Zangani D, Ip C, Vaughan MM, Shoemaker SF, McGee SO, Ip MM. Isomers of conjugated linoleic acid differ in their effects on angiogenesis and survival of mouse mammary adipose vasculature. J Nutr. 2004;134:299–307.
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