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-Eleostearic Acid (9Z11E13E-18:3) Is Quickly Converted to Conjugated Linoleic Acid (9Z11E-18:2) in Rats
Food & Biodynamic Chemistry Laboratory and * Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
1To whom correspondence should be addressed. E-mail: miyazawa{at}biochem.tohoku.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-eleostearic acid (-ESA; 9Z11E13E-18:3) is converted to conjugated linoleic acid (CLA; 9,11–18:2) in the liver and plasma of rats that were given diets including 1% -ESA for 4 wk. In this study, we investigated this phenomenon in detail. First, the chemical structure of CLA produced by -ESA administration was determined. After -ESA was orally administered to rats, CLA in rat liver was isolated by HPLC. The positional and geometric isomerism was determined using GC-EI/MS and 13C-NMR, respectively, and the CLA generated in rats after -ESA feeding was confirmed to be 9Z11E-CLA. Next, the concentrations of -ESA and CLA were determined 0, 3, 6, and 24 h after oral administration of -ESA to rats. Moreover, we also investigated whether enteric bacteria are involved in the conversion of -ESA to CLA using germ-free rats. -ESA was orally administered to germ-free and normal rats and -ESA and CLA were detected in the organs of both groups. In addition, to confirm that this reaction was enzyme-mediated, -ESA was reacted with tissue homogenates (liver, kidney, and small intestine mucous) and coenzymes (NADH, NAD+, NADPH, and NADP+), and the enzyme activities were estimated from the amount of CLA produced. CLA was detected when -ESA was reacted with liver, kidney, and small intestine mucous homogenates and a coenzyme (NADPH). These results indicated that -ESA is converted to 9Z11E-CLA in rats by a 
13-saturation reaction carried out by an NADPH-dependent enzyme.
KEY WORDS: • conjugated linoleic acid • conjugated linolenic acid • eleostearic acid • germ-free rat • GC-EI/MS
Conjugated fatty acid is a generic term used for fatty acids with conjugated double-bond systems, as exemplified by conjugated linoleic acid (CLA)2 (1). Several CLA isomers exist due to positional and geometrical isomerism of the conjugated double bonds, and the major naturally occurring CLA isomer is referred to as 9Z11E-18:2 (1). CLA was first reported to have an anticarcinogenic effect and subsequently various physiological effects were also shown, including an antiarteriosclerotic effect and a role in regulation of lipid metabolism (1–5). These CLA activities are associated with the conjugated double-bond system in the molecules.
CLA is found naturally and is especially present in ruminant fats such as beef tallow and milk fat (1). However, the CLA level in these foodstuffs is around 1% (w:w), and this does not allow natural fats to be used as a health-promoting food 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.
Conjugated fatty acids other than CLA exist in nature. Seed oils of certain plants include conjugated triene fatty acids such as -eleostearic acid (-ESA; 9Z11E13E-18:3) and calendic acid (8E10E12Z-18:3) at levels of 30 to 80% (w:w) (6,7). 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 (w:w) and the flesh contains a small amount of -ESA. However, there have only been a few studies on the physiological function of these fatty acids.
We are particularly interested in seed oils including conjugated linolenic acids (CLnA), which are the only conjugated fatty acids that can be prepared from natural sources in bulk. We previously showed that CLnA has a stronger antitumor effect than CLA in vitro and in vivo (8,9). -ESA (a CLnA) also has useful physiological effects, and we previously showed that -ESA is converted to CLA in rats that were fed a diet including 1% -ESA (w:w) for 4 wk (10) (Fig. 1). However, the in vivo metabolic pathway of -ESA has yet to be clearly determined. CLA has various physiological roles, and therefore it is of importance to examine the conversion of -ESA to CLA in vivo. 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 mechanism of the reaction and the organ in which it occurs in rats.
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-ESA administration was determined. In our earlier study, both GC and GC-electron impact (EI)/MS analyses showed that the CLA formed from -ESA was either 9Z11ECLA or 9E11Z-CLA (10). In the present study, the CLA was isolated by HPLC and the positional and geometric isomerism was determined using GC-EI/MS and 13C-NMR, respectively. 13C-NMR is an excellent method for determination of the geometric isomerism of a conjugated fatty acid (11). In our previous study, the presence of CLA was confirmed after 4 wk, but measurement of CLA levels at earlier time points was not performed (10). Hence, after -ESA was orally administered to rats, the concentrations of -ESA and CLA were determined 0, 3, 6, and 24 h after administration. Moreover, we also investigated whether enteric bacteria are involved in conversion of -ESA to CLA using germ-free rats. To confirm that this reaction was enzyme-mediated, -ESA was reacted with tissue homogenates (liver, kidney, and small intestine mucous) and coenzymes (NADH, NAD+, NADPH, and NADP+), and the enzyme activities were estimated from the amount of CLA produced. |
MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fatty acid composition of tung oil. Tung oil fatty acids were prepared from tung oil, as previously reported (8,9). Tung oil fatty acids, with a known amount of heptadecanoic acid (17:0, Sigma) as an internal standard, were methylated by the addition of 14% BF3/MeOH for 30 min at room temperature (BF3/MeOH method), also as previously reported (12).
Animals and treatments. This study was conducted in conformity with the policies and procedures detailed in the Animal Experiment Guidelines of Tohoku University. Four-week-old male Sprague-Dawley rats were obtained from Japan SLC, and housed in stainless-steel wire-mesh cages in a room kept at 23 ± 1°C with a 12-h light:dark cycle. After acclimatization with MF3 Standard Rodent Chow (Oriental Yeast) and distilled water (free access) for 1 wk, each rat was administered 1 g of tung oil via a stomach tube. Before and 3–24 h after administration, rats were killed by decapitation, and blood was collected into an EDTA-treated blood collection tube. Plasma was prepared from the blood by centrifugation at 1000 x g for 15 min at 4°C and was then stored at –80°C until analysis. Immediately after blood collection, the liver, kidney, small bowel mucosa, and cecal contents were removed and stored at –80°C until analysis.
Structure determination of CLA.
To determine the positional and geometric isomerism of CLA produced by -ESA administration, all lipids in the liver of tung oil-fed rats 6 h after administration were converted into methyl ester derivatives (using the above method, see Fatty acid composition of tung oil), and the compounds were isolated using HPLC, based on the method reported by Juanéda and Sébédio (14). The rat liver 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 x 250 mm, Chrompack) was used. In the first separation, a hexane:acetonitrile (100:0.5, v:v) mobile phase was used, and the flow rate was 1.0 mL/min. Detection was at 210 nm (all FAME) and 235 nm (FAME with a conjugated diene). A substance showing a large peak at 235 nm was fractionated, and the collected fraction (referred to as Fraction A) 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, and the flow rate was 1.0 mL/min. Detection was at 210 nm (all FAME) and 235 nm (FAME with a conjugated diene). A substance showing a large peak at 235 nm was again fractionated. The collected fraction (referred to as Fraction B) was collected and dried completely under nitrogen gas and confirmed as a single compound using GC (see Fatty acid composition of tung oil).
The isolated compound (B) was converted to its 4,4-dimethyloxazoline (DMOX) derivative prior to GC-EI/MS analysis, as previously reported (10). Geometric isomerism of the conjugated double bonds was distinguished easily using 13C-NMR (11), using conditions similar to those previously reported (15). Compound B was dissolved in CDCl3 (Wako Pure Chemicals Industries) at a concentration of 1 g/L. The 13C-NMR spectra were recorded on a Varian Unity 600 spectrometer (Varian) operating at 150 MHz.
Determination of -ESA and CLA concentrations.
The liver, kidney, small bowel mucosa, and cecal contents were homogenized with 4 vol of ice-cold saline. Total lipids from all homogenates and plasma were extracted by Folch’s procedure (13). Total lipids were similarly determined using GC (see fatty acid composition of tung oil).
Germ-free rat assay.
To investigate whether the conversion of -ESA to CLA is caused by enteric bacteria, tung oil was orally administered to germ-free rats (Wistar rats, male, 20–24 wk old), as described above, and the concentrations of -ESA and CLA in the plasma, liver, and cecal contents were determined 6 h after administration (see Animals and treatments and Determination of -ESA and CLA concentrations). Germ-free rats were obtained from Komai et al. (Laboratory of Nutrition, Graduate School of Life Science and Agriculture, Tohoku University) and bred according to a previously published procedure (16,17).
Enzyme assay.
To investigate whether the conversion of -ESA to CLA is enzyme-mediated, male SD rats age 5 wk were bred and killed using the above procedure, and the liver, plasma, kidney, and small bowel mucosa were removed and homogenized with Tris-acetate-sucrose buffer (0.01 mol/L Tris-acetate, 0.25 mol/L sucrose, pH 7.4, in 1 mmol/L DTT and 1 mmol/L EDTA, 0.7 mg/L pepstatin A, 1 mmol/L benzylsulfonyl fluoride; PMSF) to prepare 20% homogenates (w:v). Following this, 0.25 mL of homogenate was mixed with 0.73 mL of Tris-acetate-sucrose buffer and 0.02 mL of 0.1 mol/L coenzymes (NADH, NAD, NADPH, and NADP) and incubated at 37°C for 2 min, followed by the addition of 0.01 mL of tung oil fatty acid (10 g/L EtOH) to the solution and further incubation at 37°C for 10 min. Subsequently, lipids were extracted using Folch’s method, as described above, and the -ESA and CLA concentrations were determined using GC analysis (see Determination of -ESA and CLA concentrations). Enzyme activity was corrected for the amount of protein, and protein content was determined by the method of Lowry et al. (18).
Statistical analysis. Statistical analysis was performed using a one-way ANOVA, followed by a Newman-Keuls test for multiple comparisons among several groups. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-eleostearic acid (9Z11E13E-18:3), 3.9% ß-eleostearic acid (9E11E13E-18:3), and 3.0% others (w:w). CLA was not detected in the tung oil. Immediately after forced oral tung oil administration (0 h), neither CLA nor -ESA was detected, but both were detected in the liver 6 h after administration, showing clear peaks in the GC chromatogram (Fig. 2). To determine the geometric isomerism of the CLA, the compound was isolated using HPLC and its structure was analyzed using GC-EI/MS and 13C-NMR. Total lipids were extracted from the liver of rats 6 h after tung oil administration, and these were converted to methyl ester derivatives and analyzed using HPLC. Hexane:acetonitrile (100:0.5, v:v) was used as the mobile phase in the first separation (Fig. 3a), in which wavelengths of 210 and 235 nm were used for detection of normal FAME and methyl esters of fatty acids with a conjugated diene, respectively. A chromatogram at 235 nm showed a clear peak with a retention time of 6–7 min, and the compound responsible for this peak (Fraction A) was isolated. Subsequently, the mobile phase was changed to hexane:acetonitrile (100:0.15, v:v), and Fraction A was analyzed using HPLC, using the same conditions except for the altered mobile phase (Fig. 3b). A chromatogram at 235 nm showed a large peak with a retention time of 16–17 min, and the compound responsible for this peak (Fraction B) was isolated. In subsequent GC analysis, Fraction B appeared as a single peak, confirming that it has been isolated as a pure compound (Fig. 3c). Based on the retention time, Fraction B was concluded to be 9Z11E-CLA or 9E11Z-CLA. Fraction B was converted to its DMOX derivative and analyzed using GC-EI/MS, in which a fragment that was clearly due to the presence of conjugated double bonds was apparent (Fig. 4). The GC-EI/MS spectrum of the DMOX derivative of Fraction B 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 mass 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. Table 1 shows the results of 13C-NMR analysis of compound B, with a comparison to previously reported chemical shifts for methyl esters of 9Z11E-CLA and 9E11Z-CLA (11). The chemical shifts for C-8 to C-13 were consistent with those for 9Z11E-CLA methyl ester but differed from those of 9E11Z-CLA methyl ester, confirming that fraction B was indeed 9Z11E-CLA methyl ester.
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-ESA and 9E11Z-CLA were not detected 0 h after tung oil administration, but were detected 3 h after tung oil administration (Fig. 5). Fatty acid concentrations differed between organs. The plasma 9E11Z-CLA concentration increased with time (P < 0.05), but the plasma -ESA concentration decreased from 6 h after administration (P < 0.05). The liver 9E11Z-CLA concentration increased with time (P < 0.05), and the liver -ESA concentration increased until 6 h after administration (P < 0.05), but had decreased by 24 h after administration (P < 0.05). The kidney 9E11Z-CLA concentration also increased with time (P < 0.05), and, similar to the liver, the kidney -ESA concentration increased until 6 h after administration (P < 0.05), but had decreased by 24 h after administration (P < 0.05). A similar phenomenon occurred in the small bowel mucosa, in which the 9E11Z-CLA concentration increased with time (P < 0.05), and the -ESA concentration increased for 6 h (P < 0.05) and then decreased 24 h after administration (P < 0.05). In the cecal contents, trace amounts of 9E11Z-CLA were detected and the concentration increased with time (P < 0.05). The amount of -ESA in the cecal contents also increased with time (P < 0.05), indicating that not all the -ESA was absorbed. The 9E11Z-CLA detected in the cecal contents suggests that -ESA might be metabolized to 9E11Z-CLA by enteric bacteria.
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-ESA was detected in the liver, plasma, and cecal contents within 6 h (Fig. 6), which suggested that -ESA was absorbed in germ-free rats and metabolized to 9E11Z-CLA. 9E11Z-CLA was not detected in the cecal contents of germ-free rats. These results suggest that -ESA is metabolized by rat tissues.
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-ESA enhanced production of 9E11Z-CLA to a greater extent than addition of other coenzymes (P < 0.001) (Fig. 7). NADH addition also increased production of 9E11Z-CLA compared with NAD+ and NADP+ addition (P < 0.001). These results suggest that metabolism of -ESA to 9E11Z-CLA occurs through a NADPH-dependent enzymatic reaction. A comparison of enzyme activity between the plasma, liver, kidney, and small intestine mucosa suggested that the liver had a higher enzyme activity than the other organs (P < 0.001) (Fig. 8). The activity level was liver > kidney > small intestine mucous > plasma, each different from each other (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-ESA, a CLnA that occurs naturally in large amounts, was metabolized to 9E11Z-CLA in rats. In our previous study, 9,11-CLA was detected in rats that were fed a diet including 1% -ESA for 4 wk, and the site of the double bonds was determined using GC-EI/MS (10). Here, we successfully isolated 9E11Z-CLA using HPLC (Fig. 3) and determined its structure using GC-EI/MS and 13C-NMR (Fig. 4 and Table 1).
CLA was detected in rat tissues (plasma, liver, kidney, and small intestine mucosa) a short time (3 h) after oral administration (Fig. 5), and the CLA produced from -ESA administration was present as the 9Z11E-CLA isomer only, suggesting that it was synthesized through an enzymatic reaction. Addition of NADPH to a mixture of rat liver homogenate and -ESA enhanced production of 9Z11E-CLA (Fig. 7), and we therefore concluded that -ESA was saturated at the 13 position by a NADPH-dependent enzyme and converted to 9Z11E-CLA. 9E11Z-CLA was not detected in the cecal contents of germ-free rats 6 h after tung oil administration and was not detected in the presence of -ESA alone (Figs. 6and 7), suggesting that -ESA cannot be converted into CLA in the absence of a particular enzyme. 9E11Z-CLA was detected in the cecal contents of normal rats 6 h after tung oil administration, but was not detected in the cecal contents of germ-free rats (Figs. 5and 6), which suggests that enteric bacteria might be involved in -ESA metabolism. However, we concluded that only a small amount of -ESA metabolism is associated with enteric bacteria. The 13-saturation reaction was most active in the liver (Fig. 7), but in rats most -ESA was converted to CLA in the small intestine (Fig. 5). This may have occurred because -ESA was supplied continuously and the small intestine was the first organ to encounter -ESA and may have absorbed it in large amounts, due to the large intestinal surface area.
The presence of the 13-saturation reaction was also confirmed in the kidney and small intestine mucosa (Fig. 8) in various strains of rats (SD and Wistar) (Figs. 5and 6), which suggests that this reaction may be general across strains. In addition, we previously reported that -ESA administration to transplanted tumor cells in mice inhibited tumor growth more strongly than CLA administration, and CLA was detected in the liver and tumors of mice fed -ESA (9).
The enzyme that carries out the 13-saturation reaction is probably a kind of oxidoreductase, because it was NADPH-dependent and able to saturate (reduce) double bonds. This reaction proceeded rapidly until the -ESA substrate was almost completely absent and the CLA product had accumulated (Fig. 5) (9,10), which suggests 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. Leukotriene B4 (LTB4), a known activator of leukocytes, contains a conjugated triene structure. Although 
-oxidation is the major deactivation pathway of LTB4 in leukocytes, another pathway that proceeds by the reduction of conjugated double bonds was 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 known to be 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). Accordingly, in our rat model, we speculate that either a novel enzyme recognizing conjugated trienoic acid or the enzyme active in the LTB4 reductive pathway may convert ESA (a conjugated triene) to CLA (a conjugated diene) through 13-saturation (reduction).
CLA is present in natural sources in only minute amounts, which makes it extremely difficult to purify from such sources. Furthermore, it is difficult to separate CLA isomers prepared by alkali isomerization in bulk, and therefore CLA mixtures are currently on the market only as health supplements. On the contrary, -ESA (CLnA) can be purified relatively easily from tung oil and bitter gourd seed oil. Furthermore, these seed oils contain CLnA as almost a single component, and therefore it is easy to conduct studies using these oils. Because -ESA is converted to 9Z11E-CLA in rats, -ESA has the potential to provide the same physiological effects as 9Z11E-CLA although the physiological function of 9Z11E-CLA is only anticarcinogenic and antiangiogenic effects (22–24). The colon carcinoma is promoted with the high-energy diet and the high-fat diet, etc., and the mortality due to colon carcinoma increases every year. The accrual of colon carcinoma can be prevented and the progress of the colon carcinoma can be lagged by changing food composition. Therefore, an effectual measure is to add a safe cancer prevention material to food. Compared with other CLA isomers, the side effect of 9Z11E-CLA small (22). -ESA is quickly converted to 9Z11E-CLA inside the body so the side reaction of -ESA is thought to be small, too. Therefore, it might be effective to add -ESA to foods as a cancer prevention material in the future.
In conclusion, orally administered -ESA, a conjugated triene, was partially saturated at the 13 position and converted to 9Z11E-CLA, a conjugated diene, in rats. The 13-saturation reaction is an NADPH-dependent enzymatic reaction that occurs at the highest rate in rat liver and small intestine mucosa. The use of -ESA based on the physiological activities of conjugated unsaturated fatty acids may be plausible, because -ESA is present in some plant seeds in large amounts and might act as a source of 9Z11E-CLA, following -ESA metabolism. Useful physiological effects of conjugated fatty acids other than CLA and -ESA were reported (25), and further studies on both CLA and other conjugated fatty acids will hence be of importance.
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FOOTNOTES |
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-ESA, -eleostearic acid (9Z11E13E-18:3); EI, electron impact. 
3 Approximate composition of MF (g/kg diet): carbohydrate, 540; protein, 238; fat, 51; fiber, 32; moisture, 78; ash, 61.
Manuscript received 12 March 2004. Initial review completed 27 April 2004. Revision accepted 6 August 2004.
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LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Ha, Y. L., Grimm, N. K. & Pariza, M. W. (1987) Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis 8:1881-1887.
2. Lee, K. N., Kritchevsky, D. & Pariza, M. W. (1994) Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108:19-25.[Medline]
3. Park, Y., Albright, K. J., Storkson, J. M., Liu, W., Cook, M. E. & Pariza, M. W. (1999) Changes in body composition in mice during feeding and withdrawal of conjugated linoleic acid. Lipids 34:243-248.[Medline]
4. Ip, C., Chin, S. F., Scimeca, J. A. & Pariza, M. W. (1991) Mammary tumor prevention by conjugated dienoic derivative of linoleic acid. Cancer Res. 51:6118-6124.
5. Igarashi, M. & Miyazawa, T. (2001) 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 1530:162-171.[Medline]
6. Hopkins, Y. (1972) Fatty acids with conjugated unsaturation. Gunstone, F. D. eds. Topics in Lipid Chemistry 1972:37-87 ELEK Science London. .
7. Badami, R. C. & Patil, K. B. (1981) Structure and occurrence of unusual fatty acids in minor seed oils. Prog. Lipid Res. 19:119-153.
8. Igarashi, M. & Miyazawa, T. (2000) Newly recognized cytotoxic effect of conjugated trienoic fatty acids on cultured human tumor cells. Cancer Lett. 148:173-179.[Medline]
9. Tsuzuki, T., Tokuyama, Y., Igarashi, M. & Miyazawa, T. (2004) Tumor growth suppression by -eleostearic acid, a linolenic acid isomer with a conjugated triene system, via lipid peroxidation. Carcinogenesis 25:1419-1425.
10. Tsuzuki, T., Igarashi, M., Komai, M. & Miyazawa, T. (2003) 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. 49:195-200.
11. Marcel, S. F., Jie, L. K., Pasha, M. K. & Alam, M. S. (1997) Synthesis and nuclear magnetic resonance properties of all geometrical isomers of conjugated linoleic acids. Lipids 32:1041-1044.[Medline]
12. Igarashi, M., Tsuzuki, T., Kambe, T. & Miyazawa, T. (2004) Recommended methods of fatty acid methylester preparation for conjugated dienes and trienes in food and biological samples. J. Nutr. Sci. Vitaminol. 50:121-128.
13. Folch, J., Lees, M. & Sloane-Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.
14. Juanéda, P. & Sébédio, J.-L. (1999) Combined silver-ion and reversed-phase high-performance liquid chromatography for the separation and identification of C20 metabolites of conjugated linoleic acid isomers in rat liver lipids. J. Chromatogr. B 724:213-219.
15. Tagiri-Endo, M., Ono, K., Nakagawa, K., Yotsu-Yamashita, M. & Miyazawa, T (2002) Ozonation of PC in ethanol: separation and identification of a novel ethoxyhydroperoxide. Lipids 37:1007-1012.[Medline]
16. Shirakawa, H. & Kimura, S. (1990) Antibiotic-induced vitamin K deficiency and role of the presence of intestinal flora. Int. J. Vitamin Nutr. Res. 60:245-251.
17. Komai, M. & Kimura, S. (1987) Effect of dietary fiber on fecal steroid profiles in germfree and conventional mice. Nutr. Rep. Int. 36:365-375.
18. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265-275.
19. Yokomizo, T., Uozumi, N., Takahashi, T., Kume, K., Izumi, T. & Shimizu, T. (1995) Leukotriene A4 hydroxylase and leukotriene B4 metabolism. J. Lipid Mediat. Cell Signal. 12:321-332.[Medline]
20. Yokomizo, T., Izumi, T., Takahashi, T., Kasama, T., Kobayashi, Y., Sato, F., Takekuni, Y. & Shimizu, T. (1993) Enzymatic inactivation of leukotriene B4 by a novel enzyme found in the porcine kidney. J. Biol. Chem. 268:18128-18135.
21. Wainwright, S. L. & Powell, W. S. (1991) 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. 266:20899-20906.
22. Masso-Welch, P. A., Zangani, D., Ip, C., Vaughan, M. M., Shoemaker, S. F., McGee, S. O. & Ip, M. M. (2004) Isomers of conjugated linoleic acid differ in their effects on angiogenesis and survival of mouse mammary adipose vasculature. J. Nutr. 134:299-307.
23. Ip, C., Dong, Y., Ip, M. M., Banni, S., Carta, G., Angioni, E., Murro, E., Spada, S., Melis, M. P. & Saebo, A. (2002) Conjugated linoleic acid isomers and mammary cancer prevention. Nutr. Cancer 43:52-58.[Medline]
24. Masso-Welch, P. A., Zangani, D., Ip, C., Vaughan, M. M., Shoemaker, S. F., Ramirez, R. A. & Ip, M. M. (2002) Inhibition of angiogenesis by the cancer chemopreventive agent conjugated linoleic acid. Cancer Res. 62:4383-4389.
25. Tsuzuki, T., Igarashi, M. & Miyazawa, T. (2004) Conjugated eicosapentanoic acid inhibits transplanted tumor growth via membrane lipid peroxidation in nude mice. J. Nutr. 134:1162-1166.
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