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Research on food and nutrition characteristics of conjugated fatty acids a
Tsuyoshi Tsuduki a
Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan Published online: 30 Mar 2015.
Click for updates To cite this article: Tsuyoshi Tsuduki (2015): Research on food and nutrition characteristics of conjugated fatty acids, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1027656 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1027656
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Bioscience, Biotechnology, and Biochemistry, 2015
Award Review
Research on food and nutrition characteristics of conjugated fatty acids Tsuyoshi Tsuduki* Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan Received February 4, 2015; accepted March 4, 2015
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http://dx.doi.org/10.1080/09168451.2015.1027656
In this study, the physiological effects of fatty acids with conjugated double bonds were widely examined in vitro and in vivo. Initially, a method for determination of conjugated fatty acids in food and biological samples was established. I then clarified that the oxidative stability of conjugated fatty acids was improved by the form of triacylglycerol and addition of an antioxidant, and the influence of this effect on the metabolism and pharmacokinetics of conjugated fatty acids was clarified in vivo. In addition, antitumor, anti-angiogenesis, and antiobesity effects of conjugated fatty acids were found for the first time, thus demonstrating the usefulness of conjugated fatty acids. This communication mainly outlines the data obtained for conjugated linolenic acid. In addition, this review summarizes my research on conjugated fatty acid. Key words:
conjugated fatty acid; conjugated linolenic acid; eleostearic acid; conjugated EPA; tumor
Fatty acids with conjugated double bonds, i.e. conjugated fatty acids, occur naturally in addition to the more common fatty acids with double bonds. Beef and dairy products contain conjugated linoleic acid (CLA, 18:2), a positional and cis/trans isomer of linoleic acid (LA, 9Z12Z-18:2) (Fig. 1). CLA has anticarcinogenic and antiarteriosclerotic effects, and also improves fat metabolism, enhances immune function, and improves bone metabolism, among other benefits. Because of these effects, CLA is sold as a dietary supplement.1–4) Other naturally occurring conjugated fatty acids are not as well studied as CLA. Seeds of certain plant species such as Paulownia and balsam pear (Momordica balsamina) contain α-eleostearic acid (ESA, 9Z11E13E18:3), which has a conjugated triene structure (Fig. 1).5,6) Red and green algae contain conjugated eicosapentaenoic acid (EPA) and conjugated docosahexaenoic acid (DHA), which are conjugated fatty acids with aliphatic chains longer than those of ESA and CLA.7,8) While CLA has been intensively studied, few studies have reported on the physiological
activities and nutritional properties of other conjugated fatty acids. Studies of conjugated fatty acids other than CLA are important, because humans likely consume numerous conjugated fatty acids as food constituents. To investigate the safety and usefulness of conjugated fatty acids in terms of their application to human consumption, I first developed a method for creating methyl derivatives from conjugated fatty acids, which prevents their oxidization and isomerization, as well as a safe method for using conjugated fatty acids while minimizing oxidation. I also investigated the disposition of conjugated fatty acids and discovered a unique conjugated fatty acid metabolic pathway that has not been previously reported, in which animals metabolize ESA into CLA. I also discovered that highly unsaturated conjugated fatty acids (CLA, conjugated EPA, conjugated DHA, etc.) specifically induced the apoptosis of cancer cells through lipid peroxidation, suppressed the formation of tumor vessels, and reduced lipid accumulation by activating fatty acid β-oxidation. Conjugated linolenic acid used in this study was the ESA from tung oil, and conjugated EPA and conjugated DHA were prepared by alkaline treatment of EPA and DHA because conjugated EPA and conjugated DHA were difficult extraction from seaweed. In this article, I discuss findings from studies conducted using ESA, a conjugated linolenic acid.
I. Optimization of the methyl derivatization method To study conjugated fatty acids, it is essential that they can be accurately quantified. For conjugated fatty acids such as ESA, optimal methylation conditions have not been established. Because conjugated triene structures are highly reactive, it was necessary to review conventional analysis methods. I therefore attempted to determine optimal methylation conditions for conjugated fatty acids for use in gas chromatography while minimizing their isomerization. Methylation methods are classified into acid catalysis methods and base catalysis methods. Acid catalysis methods can be used to methylate both esterified and free fatty acids,
*Email: [email protected] This review was written in response to the author’s receipt of the JSBBA Award for Young Scientists in 2014 © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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COOH 12
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Linoleic acid (9Z12Z-18:2)
Conjugated linoleic acid (9Z11E-18:2)
α -Eleostearic acid (9Z11E13E-18:3)
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Fig. 1. Chemical structures of conjugated fatty acids. Note: Chemical structures of fatty acids.
but have some disadvantages, such as stringent reaction conditions and a long reaction time. Base catalysis methods require less stringent methylation conditions and can be accomplished in a shorter time, but they are more selective in terms of the lipids they are able to methylate. It should be possible to stably methylate ESA using base catalysis methods by optimizing methylation conditions. However, because base catalysis methods have lipid selectivity that limits the samples that can be used, it is necessary to combine the trimethylsilyldiazomethane method and the sodium methoxide method to stably methylate ESA. I found that the condition optimized 14% BF3/MeOH method achieved ESA methylation while minimizing its isomerization. Therefore, samples containing ESA were treated with weak alkali, after which I applied the 14% BF3/MeOH method or a method combining the trimethylsilyldiazomethane method and the sodium methoxide method.9)
II. Assessment of oxidation stability When conducting a physiological activity test for conjugated fatty acids that can be applied to food, information on oxidation stability is essential; therefore, the reactivity of ESA with oxygen was assessed. LA, CLA, α-linoleic acid (LnA, 9Z12Z15Z-18:3), and ESA were placed in a test tube, in which they formed a film. The tube with the fatty acids was left at 37 °C to allow autoxidation. The amount of oxygen absorbed was measured at 0, 6, 12, and 24 h. Prior to each measurement, the reaction was stopped by the addition of butylated hydroxytoluene. The amounts of fatty acid hydroperoxide (primary oxidation product), thiobarbituric acid reactive substance (TBARS), and residual fatty acids (secondary oxidation products) were measured. In addition, with the aim of improving oxidation stability, tests were performed using either triacylglycerol (TG) fatty acids or an antioxidant. The oxidation stability of free fatty acids (LA, CLA, LnA, and ESA) was assessed; based on the amounts of residual fatty acids and absorbed oxygen measured, I found that conjugated fatty acids (CLA and ESA) were more easily oxidized than nonconjugated fatty acids (LA and LnA). However, little fatty acid hydroperoxide and TBARS were generated from conjugated fatty acids. It was therefore suggested that there were differences in
the oxidation mechanisms that operate on conjugated and nonconjugated fatty acids. The oxidation rate of ESA was much faster than that of CLA, and conjugated fatty acids in the esterified form were more easily oxidized than nonconjugated fatty acids. However, esterified conjugated fatty acids had oxidation stability 10 times higher than that of free conjugated fatty acids. Conjugated TG began to oxidize earlier than did nonconjugated TG, but its oxidation rate was lower. This result may be due to differences in oxidation radical reactions between the conjugated and nonconjugated fatty acids. The oxidation stability of conjugated fatty acids was increased by nearly 2-fold by the addition of the antioxidant tocopherol. Furthermore, conjugated fatty acids were influenced more strongly by the addition of an antioxidant than were nonconjugated fatty acids. Based on these results, it was concluded that the oxidation stability of conjugated fatty acids was dramatically increased to a level equivalent to that of nonconjugated fatty acids by esterification and the addition of an antioxidant, enabling their application to food.10)
III. Effects on lipid metabolism It was shown in the aforementioned experiments that conjugated fatty acids were more easily oxidized than were nonconjugated fatty acids.10) It has been suggested that ingestion of conjugated fatty acids might promote oxidative stress in the body, but no studies have investigated the disposition (e.g. absorption) of ingested ESA in the body. Because this information is necessary for the application of ESA in food, I studied the absorption and metabolism mechanisms of ESA and its effects on lipid peroxidation in rats. Four rat groups, each consisting of 10 animals, were reared for 4 weeks. The rats were given feed containing 10% fat, 10% of which (1% of the weight of the feed) was 1 of the 4 test fatty acids: LA, CLA, LnA, and ESA. After the experiment, lipid composition, fatty acid composition, and the amounts of peroxidized phospholipids and TBARS (oxidative stress indicators) were determined for various tissues. There were no significant differences among the groups in the lipid composition and oxidative stress indicators for the plasma and liver, indicating that conjugated fatty acids did not have negative effects on the animals. Interestingly, an unidentified fatty acid with retention time equal to that of CLA was detected in the fatty acids comprising the total lipids in the plasma and the liver in the ESA group. Gas chromatography/electron impact mass spectrometry (GC-EI/MS) determined the unidentified fatty acid to be 9,11-CLA. It was therefore shown that the conjugated triene portion of ESA was converted to CLA, a conjugated diene, by the Δ13 saturation reaction when it was orally ingested by rats.11) However, the cis/trans isomerism of CLA and the method by which ESA was metabolized into CLA were still unknown. Therefore, using normal and germfree rats, I determined the structure of the CLA generated from ESA to determine where and how the Δ13 saturation reaction occurred. Using Ag+-HPLC, CLA was isolated from the fatty acid methyl esters of the total liver lipids in the ESA group. Based on the positional
isomerism determined by GC-EI/MS and the cis/trans isomerism determined by 13C NMR, the isolated CLA was determined to be 9Z11E-CLA.12) Next, rats were forcefully administered ESA orally, and ESA and CLA concentrations were measured in the plasma, liver, kidney, intestinal mucosa, and contents of the cecum from 0 to 24 h after administration. In addition, to investigate the involvement of intestinal bacteria, germfree rats were administered ESA in the same manner, and ESA and CLA concentrations were measured 6 h after administration. Because 9Z11ECLA was detected in the germfree rats and the cecal contents of the normal rats, but not in the cecal contents of the germfree rats, it was suggested that the Δ13 saturation reaction occurred in both intestinal bacteria and the body tissues of rats. However, considering that 9Z11E-CLA was detected in large amounts in the bodies of the germfree rats and in minute amounts in the cecal contents of the normal rats, it was suggested that much of the CLA was metabolized in the small intestine and liver of the rats. Furthermore, because CLA was detected in various tissues (plasma, liver, kidney, intestinal mucosa, and cecal content) only 3 h after oral administration, and the CLA generated as a result of ESA ingestion was 9Z11E-CLA, the reaction was considered to be an enzymatic reaction.12) The plasma, liver, intestinal mucosa, and kidneys of rats were ground into homogenates, and for each homogenate a coenzyme (NADH, NAD, NADPH, or NADP) and ESA were reacted at 37 °C. Then, for each homogenate, 9Z11E-CLA content was determined and enzyme activity was calculated with a correction for protein abundance. When the liver homogenate was reacted with NADPH, 9Z11E-CLA generation was promoted (Fig. 2). It was therefore shown that position Δ13 of ESA was saturated by an NADPH-dependent enzyme and metabolized into 9Z11E-CLA. Because the Δ13 saturation reaction was confirmed in the homogenates of the liver, kidney, and intestinal mucosa regardless of rat type (SD and Wistar), as well as in intestinal bacteria, it was concluded that the reaction occurred in the tissues and cells of various species.12)
CLA [μ mol/(min·kg protein)]
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With the confirmation of the occurrence of the Δ13 saturation reaction in the intestinal mucosa, I investigated the metabolism of ESA in the small intestine. I also investigated the metabolism of punic acid (PA; 9Z11E13Z-18:3) in the small intestine. PA is a cis isomer of ESA at position Δ13, which is subject to a saturation reaction. A cannula was inserted into the thoracic ducts of the rats and an emulsion of sample oil (ESA, PA, CLA, or LnA) was injected into the stomach 24 h later. Lymph was then continually collected for 24 h and the amount of fatty acids was quantified by GC. CLA converted from PA was confirmed in the lymph of the rats given PA, and the chemical structure of the CLA was determined as 9Z11E-18:2 using 13 C-NMR and GC-EI/MS. Because ESA and PA are metabolized into CLA in the small intestine, the CLA recovered in the lymph was considered to represent the total amount of recovered ESA, PA, and CLA. Twentyfour hours after ESA, PA, CLA, and LnA were administered each drug was recovered at a rate greater than 90%. The recovered amounts of CLA, ESA, and PA were smaller than that of LnA in the early hours after administration, but there were no significant differences in the final recovered amounts of the 4 compounds. CLA converted from ESA to PA accounted for approximately 20 and 10%, respectively, of the recovered drug. It was therefore suggested that large proportions of ESA and PA were absorbed by the body without being metabolized. Moreover, it was also shown that compounds that were trans isomers at the metabolism site (Δ13) were metabolized twice as efficiently as those that were cis isomers.13) In addition, there were no significant differences in gastrointestinal absorption rates among the CLA isomers.14) However, in an experiment in humans, when 9Z11E-CLA and 10E12Z-CLA were ingested in equal amounts, more 9Z11E-CLA was detected in the body, indicating that the metabolism rate was dependent on the structure of the conjugated double bonds of the compounds.15) These results showed that the portion of ESA with a conjugated triene structure was converted to 9Z11E-CLA with a conjugated diene structure by the Δ13 saturation reaction when it was administered
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Fig. 2. The Δ13 saturation reaction in rat liver homogenate. Notes: 9Z11E-CLA concentration in liver homogenate (HG) of male SD rats aged 5 weeks. ESA and co-enzyme (CE) were added to liver homogenate. The reaction was stopped after 10 min, and the 9Z11ECLA concentration was analyzed by GC. *Not detected. Values are mean ± SEM, n = 6. Values not sharing the same superscript letter (* is excluded) in each tissues are significantly different, p < 0.05.
COOH
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CLA Conjugated diene
In vivo Fig. 3. The Δ13 saturation reaction. Notes: The Δ13 saturation reaction in the formation of CLA (9Z11E-18:2) in rats fed α-ESA (9Z11E13E-18:3) diet.
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orally. The Δ13 saturation reaction is an NADPH-dependent enzymatic reaction that occurs in the small intestine and the liver, but it is particularly common in the liver (Fig. 3). ESA is found at high concentrations in the seeds of some plant species, and future utilization of vegetable fat and oil containing ESA as a source of CLA is expected.
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IV. Anticancer and other physiological effects It was shown in the aforementioned experiments that ESA is converted to CLA in the body, which suggested that ESA, like CLA, might have beneficial physiological effects against cancer and obesity. Therefore, I compared the anticancer effects of ESA and CLA. DLD-1 human colon cancer cells were subcutaneously transplanted in the backs of nude mice, which were divided into 4 groups, each consisting of 10 animals, and orally administered a test lipid (LA, 9Z11E-CLA, 10E12ZCLA, or ESA) forcefully for 32 days. After the experimental period, the livers and tumors were collected for measurements of peroxidized phospholipids and TBARS (oxidative stress indicators), in addition to the concentrations of conjugated fatty acids. The DNA fragmentation rate, which is an apoptosis indicator, was measured in the tumors. It was found that the cancer growth suppression effects of ESA were stronger than those of CLA (Fig. 4) and that ESA did not affect the liver. The aforementioned experiments did not find significant differences in lipid composition or oxidative
Control
CLA
ESA
Fig. 4. The antitumor effect of ESA. Notes: The back of nude mice transplanted with DLD-1 cells that received forcible fatty acid medication for 32 days. Control, Transplanted mice fed the control (safflower) diet; CLA, Transplanted mice fed the CLA diet; ESA, Transplanted mice fed the tung oil diet.
stress indicators in plasma and liver samples from ESA-exposed subjects.11) Therefore, it was suggested that ESA selectively induced the death of cancer cells. 9Z11E-CLA was detected in mice that ingested ESA, and because it was shown in the aforementioned studies that ESA ingested by rats was metabolized into 9Z11E-CLA,12) it was suggested that ESA might also be metabolized into 9Z11E-CLA in mice. The amounts of membrane phospholipid hydroperoxide and TBARS were increased in the tumors from mice that ingested ESA, indicating that lipid peroxidation might be involved in the cancer suppression effects of ESA. Therefore, I investigated the cancer suppression mechanism of ESA in a cell culture experiment. First, a cell growth experiment was conducted using the WST-1 assay, which showed that ESA had strong tumoricidal effects. Next, DNA was extracted from cells, the DNA fragmentation rate was calculated, and the activity of apoptosis effectors caspase-3, caspase-8, and caspase-9, as well as the expression levels of their respective mRNAs, were measured. The results of the DNA fragmentation and caspase assays confirmed that ESA induced apoptosis. In addition, oxidative stress caused by ESA was assessed by measuring the amounts of peroxidized phospholipids and TBARS in the exposed cells, and suppression of apoptosis was confirmed by the addition of the antioxidant tocopherol. The amounts of membrane phospholipid hydroperoxide and TBARS increased in the cancer cells to which ESA was added, and oxidative stress and cell death were suppressed by the addition of tocopherol. Thus, it was concluded that ESA suppressed cancer growth more strongly by lipid peroxidation than did CLA.16) Moreover, conjugated EPA and conjugated DHA have also been shown to have strong antitumor effects.17–19) The aforementioned experiments showed that the cancer suppression effect of ESA was stronger than that of CLA. In addition, it was found that the cancer tissue collected from the ESA-administered mice showed internal discoloration and necrosis, indicating that nutrition did not reach the tissue interior because vessels that would have transported nutrients were never formed. Therefore, it was suggested that ESA might suppress angiogenesis. The circulatory system is closely involved in the development and growth of cancers. Currently, substances that suppress angiogenesis are receiving intense research focus as potential cancer treatments. The antiangiogenesis effect of ESA has been studied both in vivo and in vitro, and the mechanism through which it produces this effect has been investigated at the gene expression level. In an in vivo experiment in which ESA was administered to mice subcutaneously in the back, ESA strongly suppressed angiogenesis. Furthermore, in human umbilical vein endothelial cells (HUVECs) ESA suppressed cell growth, migration, and tube formation, which are important steps in the process of angiogenesis (Fig. 5). Mechanistic studies showed that the antiangiogenic mechanism of ESA was mediated by its suppression of the expression of vascular endothelial growth factor receptors through activation of peroxisome proliferatoractivated receptor gamma, which is a nuclear receptor.20) Strong antiangiogenic effects of ESA have thus been confirmed as a new physiological action for the
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ESA, which have not been reported previously, as well as developing methods that are easier to use.
Acknowledgments Control
VEGF
VEGF +ESA
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Fig.5. The anti-angiogenic effect of ESA. Notes: Effects of ESA on tube formation by HUVEC. HUVEC cocultured with fibroblasts were incubated with medium only or with medium containing VEGF and ESA. After 11 days, the cells were visualized with anti-CD31 antibody and photographed. Control cells were cultured in medium without VEGF.
compound. The antiangiogenic effects of conjugated EPA and conjugated DHA have also been confirmed.21,22) Based on these results, it was suggested that ESA could be used as a food additive to prevent angiogenic diseases such as cancer, arteriosclerosis, and diabetes, which account for 60% of deaths among the Japanese population. As with other physiological effects, antiobesity effects of conjugated EPA and conjugated DHA have also been confirmed.23–25) Because ESA can be prepared more easily than CLA and has stronger physiological activities, it is expected that ESA will supplant CLA as a dietary supplement.
V. Conclusion This report discusses studies conducted to evaluate the safety and effectiveness of ESA, a CLA found in the seeds of easily available vegetables such as Paulownia and balsam pear (Momordica balsamina), which suggest that it might replace CLA due to its superior characteristics as a bioactive lipid. The 14% BF3/MeOH method, which involves pretreatment with a weak alkali, and the combined method using the trimethylsilyldiazomethane method and the sodium methoxide method were found to be optimal methylation methods for ESA. While ESA is very easily oxidized, the oxidative stability of ESA is dramatically increased by modification of its carboxyl group and the addition of an antioxidant. It has been confirmed that ESA orally administered to rats was metabolized into 9Z11E-CLA by the Δ13 saturation reaction, which was particularly active in the liver of rats and was dependent on NADPH. Because ESA does not alter lipid composition or promote oxidative stress in normal animals, ESA is considered to be safe and could be consumed by humans with the aim of producing its beneficial physiological activities. The strong cancer suppression effects of ESA in comparison with CLA have been confirmed in vitro and in vivo, where ESA specifically induced apoptosis in cancer cells by lipid peroxidation. Based on these results, ESA seems to be a promising dietary supplement that could be administered to humans, although further studies to evaluate the effects of long-term ESA administration are necessary. I am currently studying the metabolic aspects of
The author thank the members of the selection committee for awarding me the prestigious JSBBA Award for Young Scientists. The series of studies mentioned in this paper was conducted at the Graduate School of Agricultural Science, Tohoku University, and I offer my heartfelt thanks to Professor Teruo Miyazawa of the aforementioned school, who has provided invaluable advice that has supported my research since I was a student there. I would also like to offer my deepest gratitude to other faculty members and joint researchers for their support and advice. Lastly, I would like to thank the graduates and current students of the graduate and undergraduate schools of Tohoku University.
Disclosure statement No potential conflict of interest was reported by the author.
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[13] Tsuzuki T, Kawakami Y, Abe R, Nakagawa K, Koba K, Imamura J, Iwata T, Ikeda I, Miyazawa T. Conjugated linolenic acid is slowly absorbed in rat intestine, but quickly converted to conjugated linoleic acid. J. Nutr. 2006;136:2153–2159. [14] Tsuzuki T, Ikeda I. Slow absorption of conjugated linoleic acid in rat intestines, and similar absorption rates of 9c,11t-conjugated linoleic acid and 10t,12c-conjugated linoleic acid. Biosci. Biotechnol. Biochem. 2007;71:2034–2040. [15] Sato K, Shinohara N, Honma T, Ito J, Arai T, Nosaka N, Aoyama T, Tsuduki T, Ikeda I. The change in conjugated linoleic acid concentration in blood of the Japanese fed a conjugated linoleic acid diet. J. Nutr. Sci. Vitaminol. (Tokyo). 2011;57:364–371. [16] Tsuzuki T, Tokuyama Y, Igarashi M, Miyazawa T. Tumor growth suppression by alpha-eleostearic acid, a linolenic acid isomer with a conjugated triene system, via lipid peroxidation. Carcinogenesis. 2004;25:1417–1425. [17] Tsuzuki T, Tanaka K, Kuwahara S, Miyazawa T. Synthesis of the conjugated trienes 5E,7E,9E,14Z,17Z-eicosapentaenoic acid and 5Z,7E,9E,14Z,17Z-eicosapentaenoic acid, and their induction of apoptosis in DLD-1 colorectal adenocarcinoma cells. Lipids. 2005;40:147–154. [18] Tsuzuki T, Kambe T, Shibata A, Kawakami Y, Nakagawa K, Miyazawa T. Conjugated EPA activates mutant p53 via lipid peroxidation and induces p53-dependent apoptosis in DLD-1 colorectal adenocarcinoma human cells. Biochim. Biophys. Acta. 2007;1771:20–30. [19] Shinohara N, Tsuduki T, Ito J, Honma T, Kijima R, Sugawara S, Arai T, Yamasaki M, Ikezaki A, Yokoyama M, Nishiyama K,
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Research on food and nutrition characteristics of conjugated fatty acids a
Tsuyoshi Tsuduki a
Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan Published online: 30 Mar 2015.
Click for updates To cite this article: Tsuyoshi Tsuduki (2015): Research on food and nutrition characteristics of conjugated fatty acids, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2015.1027656 To link to this article: http://dx.doi.org/10.1080/09168451.2015.1027656
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Bioscience, Biotechnology, and Biochemistry, 2015
Award Review
Research on food and nutrition characteristics of conjugated fatty acids Tsuyoshi Tsuduki* Laboratory of Food and Biomolecular Science, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan Received February 4, 2015; accepted March 4, 2015
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http://dx.doi.org/10.1080/09168451.2015.1027656
In this study, the physiological effects of fatty acids with conjugated double bonds were widely examined in vitro and in vivo. Initially, a method for determination of conjugated fatty acids in food and biological samples was established. I then clarified that the oxidative stability of conjugated fatty acids was improved by the form of triacylglycerol and addition of an antioxidant, and the influence of this effect on the metabolism and pharmacokinetics of conjugated fatty acids was clarified in vivo. In addition, antitumor, anti-angiogenesis, and antiobesity effects of conjugated fatty acids were found for the first time, thus demonstrating the usefulness of conjugated fatty acids. This communication mainly outlines the data obtained for conjugated linolenic acid. In addition, this review summarizes my research on conjugated fatty acid. Key words:
conjugated fatty acid; conjugated linolenic acid; eleostearic acid; conjugated EPA; tumor
Fatty acids with conjugated double bonds, i.e. conjugated fatty acids, occur naturally in addition to the more common fatty acids with double bonds. Beef and dairy products contain conjugated linoleic acid (CLA, 18:2), a positional and cis/trans isomer of linoleic acid (LA, 9Z12Z-18:2) (Fig. 1). CLA has anticarcinogenic and antiarteriosclerotic effects, and also improves fat metabolism, enhances immune function, and improves bone metabolism, among other benefits. Because of these effects, CLA is sold as a dietary supplement.1–4) Other naturally occurring conjugated fatty acids are not as well studied as CLA. Seeds of certain plant species such as Paulownia and balsam pear (Momordica balsamina) contain α-eleostearic acid (ESA, 9Z11E13E18:3), which has a conjugated triene structure (Fig. 1).5,6) Red and green algae contain conjugated eicosapentaenoic acid (EPA) and conjugated docosahexaenoic acid (DHA), which are conjugated fatty acids with aliphatic chains longer than those of ESA and CLA.7,8) While CLA has been intensively studied, few studies have reported on the physiological
activities and nutritional properties of other conjugated fatty acids. Studies of conjugated fatty acids other than CLA are important, because humans likely consume numerous conjugated fatty acids as food constituents. To investigate the safety and usefulness of conjugated fatty acids in terms of their application to human consumption, I first developed a method for creating methyl derivatives from conjugated fatty acids, which prevents their oxidization and isomerization, as well as a safe method for using conjugated fatty acids while minimizing oxidation. I also investigated the disposition of conjugated fatty acids and discovered a unique conjugated fatty acid metabolic pathway that has not been previously reported, in which animals metabolize ESA into CLA. I also discovered that highly unsaturated conjugated fatty acids (CLA, conjugated EPA, conjugated DHA, etc.) specifically induced the apoptosis of cancer cells through lipid peroxidation, suppressed the formation of tumor vessels, and reduced lipid accumulation by activating fatty acid β-oxidation. Conjugated linolenic acid used in this study was the ESA from tung oil, and conjugated EPA and conjugated DHA were prepared by alkaline treatment of EPA and DHA because conjugated EPA and conjugated DHA were difficult extraction from seaweed. In this article, I discuss findings from studies conducted using ESA, a conjugated linolenic acid.
I. Optimization of the methyl derivatization method To study conjugated fatty acids, it is essential that they can be accurately quantified. For conjugated fatty acids such as ESA, optimal methylation conditions have not been established. Because conjugated triene structures are highly reactive, it was necessary to review conventional analysis methods. I therefore attempted to determine optimal methylation conditions for conjugated fatty acids for use in gas chromatography while minimizing their isomerization. Methylation methods are classified into acid catalysis methods and base catalysis methods. Acid catalysis methods can be used to methylate both esterified and free fatty acids,
*Email: [email protected] This review was written in response to the author’s receipt of the JSBBA Award for Young Scientists in 2014 © 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry
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COOH 12
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Conjugated linoleic acid (9Z11E-18:2)
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Fig. 1. Chemical structures of conjugated fatty acids. Note: Chemical structures of fatty acids.
but have some disadvantages, such as stringent reaction conditions and a long reaction time. Base catalysis methods require less stringent methylation conditions and can be accomplished in a shorter time, but they are more selective in terms of the lipids they are able to methylate. It should be possible to stably methylate ESA using base catalysis methods by optimizing methylation conditions. However, because base catalysis methods have lipid selectivity that limits the samples that can be used, it is necessary to combine the trimethylsilyldiazomethane method and the sodium methoxide method to stably methylate ESA. I found that the condition optimized 14% BF3/MeOH method achieved ESA methylation while minimizing its isomerization. Therefore, samples containing ESA were treated with weak alkali, after which I applied the 14% BF3/MeOH method or a method combining the trimethylsilyldiazomethane method and the sodium methoxide method.9)
II. Assessment of oxidation stability When conducting a physiological activity test for conjugated fatty acids that can be applied to food, information on oxidation stability is essential; therefore, the reactivity of ESA with oxygen was assessed. LA, CLA, α-linoleic acid (LnA, 9Z12Z15Z-18:3), and ESA were placed in a test tube, in which they formed a film. The tube with the fatty acids was left at 37 °C to allow autoxidation. The amount of oxygen absorbed was measured at 0, 6, 12, and 24 h. Prior to each measurement, the reaction was stopped by the addition of butylated hydroxytoluene. The amounts of fatty acid hydroperoxide (primary oxidation product), thiobarbituric acid reactive substance (TBARS), and residual fatty acids (secondary oxidation products) were measured. In addition, with the aim of improving oxidation stability, tests were performed using either triacylglycerol (TG) fatty acids or an antioxidant. The oxidation stability of free fatty acids (LA, CLA, LnA, and ESA) was assessed; based on the amounts of residual fatty acids and absorbed oxygen measured, I found that conjugated fatty acids (CLA and ESA) were more easily oxidized than nonconjugated fatty acids (LA and LnA). However, little fatty acid hydroperoxide and TBARS were generated from conjugated fatty acids. It was therefore suggested that there were differences in
the oxidation mechanisms that operate on conjugated and nonconjugated fatty acids. The oxidation rate of ESA was much faster than that of CLA, and conjugated fatty acids in the esterified form were more easily oxidized than nonconjugated fatty acids. However, esterified conjugated fatty acids had oxidation stability 10 times higher than that of free conjugated fatty acids. Conjugated TG began to oxidize earlier than did nonconjugated TG, but its oxidation rate was lower. This result may be due to differences in oxidation radical reactions between the conjugated and nonconjugated fatty acids. The oxidation stability of conjugated fatty acids was increased by nearly 2-fold by the addition of the antioxidant tocopherol. Furthermore, conjugated fatty acids were influenced more strongly by the addition of an antioxidant than were nonconjugated fatty acids. Based on these results, it was concluded that the oxidation stability of conjugated fatty acids was dramatically increased to a level equivalent to that of nonconjugated fatty acids by esterification and the addition of an antioxidant, enabling their application to food.10)
III. Effects on lipid metabolism It was shown in the aforementioned experiments that conjugated fatty acids were more easily oxidized than were nonconjugated fatty acids.10) It has been suggested that ingestion of conjugated fatty acids might promote oxidative stress in the body, but no studies have investigated the disposition (e.g. absorption) of ingested ESA in the body. Because this information is necessary for the application of ESA in food, I studied the absorption and metabolism mechanisms of ESA and its effects on lipid peroxidation in rats. Four rat groups, each consisting of 10 animals, were reared for 4 weeks. The rats were given feed containing 10% fat, 10% of which (1% of the weight of the feed) was 1 of the 4 test fatty acids: LA, CLA, LnA, and ESA. After the experiment, lipid composition, fatty acid composition, and the amounts of peroxidized phospholipids and TBARS (oxidative stress indicators) were determined for various tissues. There were no significant differences among the groups in the lipid composition and oxidative stress indicators for the plasma and liver, indicating that conjugated fatty acids did not have negative effects on the animals. Interestingly, an unidentified fatty acid with retention time equal to that of CLA was detected in the fatty acids comprising the total lipids in the plasma and the liver in the ESA group. Gas chromatography/electron impact mass spectrometry (GC-EI/MS) determined the unidentified fatty acid to be 9,11-CLA. It was therefore shown that the conjugated triene portion of ESA was converted to CLA, a conjugated diene, by the Δ13 saturation reaction when it was orally ingested by rats.11) However, the cis/trans isomerism of CLA and the method by which ESA was metabolized into CLA were still unknown. Therefore, using normal and germfree rats, I determined the structure of the CLA generated from ESA to determine where and how the Δ13 saturation reaction occurred. Using Ag+-HPLC, CLA was isolated from the fatty acid methyl esters of the total liver lipids in the ESA group. Based on the positional
isomerism determined by GC-EI/MS and the cis/trans isomerism determined by 13C NMR, the isolated CLA was determined to be 9Z11E-CLA.12) Next, rats were forcefully administered ESA orally, and ESA and CLA concentrations were measured in the plasma, liver, kidney, intestinal mucosa, and contents of the cecum from 0 to 24 h after administration. In addition, to investigate the involvement of intestinal bacteria, germfree rats were administered ESA in the same manner, and ESA and CLA concentrations were measured 6 h after administration. Because 9Z11ECLA was detected in the germfree rats and the cecal contents of the normal rats, but not in the cecal contents of the germfree rats, it was suggested that the Δ13 saturation reaction occurred in both intestinal bacteria and the body tissues of rats. However, considering that 9Z11E-CLA was detected in large amounts in the bodies of the germfree rats and in minute amounts in the cecal contents of the normal rats, it was suggested that much of the CLA was metabolized in the small intestine and liver of the rats. Furthermore, because CLA was detected in various tissues (plasma, liver, kidney, intestinal mucosa, and cecal content) only 3 h after oral administration, and the CLA generated as a result of ESA ingestion was 9Z11E-CLA, the reaction was considered to be an enzymatic reaction.12) The plasma, liver, intestinal mucosa, and kidneys of rats were ground into homogenates, and for each homogenate a coenzyme (NADH, NAD, NADPH, or NADP) and ESA were reacted at 37 °C. Then, for each homogenate, 9Z11E-CLA content was determined and enzyme activity was calculated with a correction for protein abundance. When the liver homogenate was reacted with NADPH, 9Z11E-CLA generation was promoted (Fig. 2). It was therefore shown that position Δ13 of ESA was saturated by an NADPH-dependent enzyme and metabolized into 9Z11E-CLA. Because the Δ13 saturation reaction was confirmed in the homogenates of the liver, kidney, and intestinal mucosa regardless of rat type (SD and Wistar), as well as in intestinal bacteria, it was concluded that the reaction occurred in the tissues and cells of various species.12)
CLA [μ mol/(min·kg protein)]
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With the confirmation of the occurrence of the Δ13 saturation reaction in the intestinal mucosa, I investigated the metabolism of ESA in the small intestine. I also investigated the metabolism of punic acid (PA; 9Z11E13Z-18:3) in the small intestine. PA is a cis isomer of ESA at position Δ13, which is subject to a saturation reaction. A cannula was inserted into the thoracic ducts of the rats and an emulsion of sample oil (ESA, PA, CLA, or LnA) was injected into the stomach 24 h later. Lymph was then continually collected for 24 h and the amount of fatty acids was quantified by GC. CLA converted from PA was confirmed in the lymph of the rats given PA, and the chemical structure of the CLA was determined as 9Z11E-18:2 using 13 C-NMR and GC-EI/MS. Because ESA and PA are metabolized into CLA in the small intestine, the CLA recovered in the lymph was considered to represent the total amount of recovered ESA, PA, and CLA. Twentyfour hours after ESA, PA, CLA, and LnA were administered each drug was recovered at a rate greater than 90%. The recovered amounts of CLA, ESA, and PA were smaller than that of LnA in the early hours after administration, but there were no significant differences in the final recovered amounts of the 4 compounds. CLA converted from ESA to PA accounted for approximately 20 and 10%, respectively, of the recovered drug. It was therefore suggested that large proportions of ESA and PA were absorbed by the body without being metabolized. Moreover, it was also shown that compounds that were trans isomers at the metabolism site (Δ13) were metabolized twice as efficiently as those that were cis isomers.13) In addition, there were no significant differences in gastrointestinal absorption rates among the CLA isomers.14) However, in an experiment in humans, when 9Z11E-CLA and 10E12Z-CLA were ingested in equal amounts, more 9Z11E-CLA was detected in the body, indicating that the metabolism rate was dependent on the structure of the conjugated double bonds of the compounds.15) These results showed that the portion of ESA with a conjugated triene structure was converted to 9Z11E-CLA with a conjugated diene structure by the Δ13 saturation reaction when it was administered
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Fig. 2. The Δ13 saturation reaction in rat liver homogenate. Notes: 9Z11E-CLA concentration in liver homogenate (HG) of male SD rats aged 5 weeks. ESA and co-enzyme (CE) were added to liver homogenate. The reaction was stopped after 10 min, and the 9Z11ECLA concentration was analyzed by GC. *Not detected. Values are mean ± SEM, n = 6. Values not sharing the same superscript letter (* is excluded) in each tissues are significantly different, p < 0.05.
COOH
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In vivo Fig. 3. The Δ13 saturation reaction. Notes: The Δ13 saturation reaction in the formation of CLA (9Z11E-18:2) in rats fed α-ESA (9Z11E13E-18:3) diet.
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orally. The Δ13 saturation reaction is an NADPH-dependent enzymatic reaction that occurs in the small intestine and the liver, but it is particularly common in the liver (Fig. 3). ESA is found at high concentrations in the seeds of some plant species, and future utilization of vegetable fat and oil containing ESA as a source of CLA is expected.
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IV. Anticancer and other physiological effects It was shown in the aforementioned experiments that ESA is converted to CLA in the body, which suggested that ESA, like CLA, might have beneficial physiological effects against cancer and obesity. Therefore, I compared the anticancer effects of ESA and CLA. DLD-1 human colon cancer cells were subcutaneously transplanted in the backs of nude mice, which were divided into 4 groups, each consisting of 10 animals, and orally administered a test lipid (LA, 9Z11E-CLA, 10E12ZCLA, or ESA) forcefully for 32 days. After the experimental period, the livers and tumors were collected for measurements of peroxidized phospholipids and TBARS (oxidative stress indicators), in addition to the concentrations of conjugated fatty acids. The DNA fragmentation rate, which is an apoptosis indicator, was measured in the tumors. It was found that the cancer growth suppression effects of ESA were stronger than those of CLA (Fig. 4) and that ESA did not affect the liver. The aforementioned experiments did not find significant differences in lipid composition or oxidative
Control
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Fig. 4. The antitumor effect of ESA. Notes: The back of nude mice transplanted with DLD-1 cells that received forcible fatty acid medication for 32 days. Control, Transplanted mice fed the control (safflower) diet; CLA, Transplanted mice fed the CLA diet; ESA, Transplanted mice fed the tung oil diet.
stress indicators in plasma and liver samples from ESA-exposed subjects.11) Therefore, it was suggested that ESA selectively induced the death of cancer cells. 9Z11E-CLA was detected in mice that ingested ESA, and because it was shown in the aforementioned studies that ESA ingested by rats was metabolized into 9Z11E-CLA,12) it was suggested that ESA might also be metabolized into 9Z11E-CLA in mice. The amounts of membrane phospholipid hydroperoxide and TBARS were increased in the tumors from mice that ingested ESA, indicating that lipid peroxidation might be involved in the cancer suppression effects of ESA. Therefore, I investigated the cancer suppression mechanism of ESA in a cell culture experiment. First, a cell growth experiment was conducted using the WST-1 assay, which showed that ESA had strong tumoricidal effects. Next, DNA was extracted from cells, the DNA fragmentation rate was calculated, and the activity of apoptosis effectors caspase-3, caspase-8, and caspase-9, as well as the expression levels of their respective mRNAs, were measured. The results of the DNA fragmentation and caspase assays confirmed that ESA induced apoptosis. In addition, oxidative stress caused by ESA was assessed by measuring the amounts of peroxidized phospholipids and TBARS in the exposed cells, and suppression of apoptosis was confirmed by the addition of the antioxidant tocopherol. The amounts of membrane phospholipid hydroperoxide and TBARS increased in the cancer cells to which ESA was added, and oxidative stress and cell death were suppressed by the addition of tocopherol. Thus, it was concluded that ESA suppressed cancer growth more strongly by lipid peroxidation than did CLA.16) Moreover, conjugated EPA and conjugated DHA have also been shown to have strong antitumor effects.17–19) The aforementioned experiments showed that the cancer suppression effect of ESA was stronger than that of CLA. In addition, it was found that the cancer tissue collected from the ESA-administered mice showed internal discoloration and necrosis, indicating that nutrition did not reach the tissue interior because vessels that would have transported nutrients were never formed. Therefore, it was suggested that ESA might suppress angiogenesis. The circulatory system is closely involved in the development and growth of cancers. Currently, substances that suppress angiogenesis are receiving intense research focus as potential cancer treatments. The antiangiogenesis effect of ESA has been studied both in vivo and in vitro, and the mechanism through which it produces this effect has been investigated at the gene expression level. In an in vivo experiment in which ESA was administered to mice subcutaneously in the back, ESA strongly suppressed angiogenesis. Furthermore, in human umbilical vein endothelial cells (HUVECs) ESA suppressed cell growth, migration, and tube formation, which are important steps in the process of angiogenesis (Fig. 5). Mechanistic studies showed that the antiangiogenic mechanism of ESA was mediated by its suppression of the expression of vascular endothelial growth factor receptors through activation of peroxisome proliferatoractivated receptor gamma, which is a nuclear receptor.20) Strong antiangiogenic effects of ESA have thus been confirmed as a new physiological action for the
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ESA, which have not been reported previously, as well as developing methods that are easier to use.
Acknowledgments Control
VEGF
VEGF +ESA
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Fig.5. The anti-angiogenic effect of ESA. Notes: Effects of ESA on tube formation by HUVEC. HUVEC cocultured with fibroblasts were incubated with medium only or with medium containing VEGF and ESA. After 11 days, the cells were visualized with anti-CD31 antibody and photographed. Control cells were cultured in medium without VEGF.
compound. The antiangiogenic effects of conjugated EPA and conjugated DHA have also been confirmed.21,22) Based on these results, it was suggested that ESA could be used as a food additive to prevent angiogenic diseases such as cancer, arteriosclerosis, and diabetes, which account for 60% of deaths among the Japanese population. As with other physiological effects, antiobesity effects of conjugated EPA and conjugated DHA have also been confirmed.23–25) Because ESA can be prepared more easily than CLA and has stronger physiological activities, it is expected that ESA will supplant CLA as a dietary supplement.
V. Conclusion This report discusses studies conducted to evaluate the safety and effectiveness of ESA, a CLA found in the seeds of easily available vegetables such as Paulownia and balsam pear (Momordica balsamina), which suggest that it might replace CLA due to its superior characteristics as a bioactive lipid. The 14% BF3/MeOH method, which involves pretreatment with a weak alkali, and the combined method using the trimethylsilyldiazomethane method and the sodium methoxide method were found to be optimal methylation methods for ESA. While ESA is very easily oxidized, the oxidative stability of ESA is dramatically increased by modification of its carboxyl group and the addition of an antioxidant. It has been confirmed that ESA orally administered to rats was metabolized into 9Z11E-CLA by the Δ13 saturation reaction, which was particularly active in the liver of rats and was dependent on NADPH. Because ESA does not alter lipid composition or promote oxidative stress in normal animals, ESA is considered to be safe and could be consumed by humans with the aim of producing its beneficial physiological activities. The strong cancer suppression effects of ESA in comparison with CLA have been confirmed in vitro and in vivo, where ESA specifically induced apoptosis in cancer cells by lipid peroxidation. Based on these results, ESA seems to be a promising dietary supplement that could be administered to humans, although further studies to evaluate the effects of long-term ESA administration are necessary. I am currently studying the metabolic aspects of
The author thank the members of the selection committee for awarding me the prestigious JSBBA Award for Young Scientists. The series of studies mentioned in this paper was conducted at the Graduate School of Agricultural Science, Tohoku University, and I offer my heartfelt thanks to Professor Teruo Miyazawa of the aforementioned school, who has provided invaluable advice that has supported my research since I was a student there. I would also like to offer my deepest gratitude to other faculty members and joint researchers for their support and advice. Lastly, I would like to thank the graduates and current students of the graduate and undergraduate schools of Tohoku University.
Disclosure statement No potential conflict of interest was reported by the author.
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[13] Tsuzuki T, Kawakami Y, Abe R, Nakagawa K, Koba K, Imamura J, Iwata T, Ikeda I, Miyazawa T. Conjugated linolenic acid is slowly absorbed in rat intestine, but quickly converted to conjugated linoleic acid. J. Nutr. 2006;136:2153–2159. [14] Tsuzuki T, Ikeda I. Slow absorption of conjugated linoleic acid in rat intestines, and similar absorption rates of 9c,11t-conjugated linoleic acid and 10t,12c-conjugated linoleic acid. Biosci. Biotechnol. Biochem. 2007;71:2034–2040. [15] Sato K, Shinohara N, Honma T, Ito J, Arai T, Nosaka N, Aoyama T, Tsuduki T, Ikeda I. The change in conjugated linoleic acid concentration in blood of the Japanese fed a conjugated linoleic acid diet. J. Nutr. Sci. Vitaminol. (Tokyo). 2011;57:364–371. [16] Tsuzuki T, Tokuyama Y, Igarashi M, Miyazawa T. Tumor growth suppression by alpha-eleostearic acid, a linolenic acid isomer with a conjugated triene system, via lipid peroxidation. Carcinogenesis. 2004;25:1417–1425. [17] Tsuzuki T, Tanaka K, Kuwahara S, Miyazawa T. Synthesis of the conjugated trienes 5E,7E,9E,14Z,17Z-eicosapentaenoic acid and 5Z,7E,9E,14Z,17Z-eicosapentaenoic acid, and their induction of apoptosis in DLD-1 colorectal adenocarcinoma cells. Lipids. 2005;40:147–154. [18] Tsuzuki T, Kambe T, Shibata A, Kawakami Y, Nakagawa K, Miyazawa T. Conjugated EPA activates mutant p53 via lipid peroxidation and induces p53-dependent apoptosis in DLD-1 colorectal adenocarcinoma human cells. Biochim. Biophys. Acta. 2007;1771:20–30. [19] Shinohara N, Tsuduki T, Ito J, Honma T, Kijima R, Sugawara S, Arai T, Yamasaki M, Ikezaki A, Yokoyama M, Nishiyama K,
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