1 Introduction
Eicosapentaenoic acid (EPA) and linoleic acid are both polyunsaturated fatty acids (PUFAs) that are key structural components of lipids, which have been widely reported to confer health benefits, particularly in cardiovascular, neurological, and inflammation-related functions1) ,2) . Among these, EPA in particular has been associated with anti-inflammatory effects and reduced cardiovascular disease risk, and is therefore widely used in dietary supplements, functional foods, and cholesterol-lowering products1) . These biological effects are thought to arise from enzymatic oxidation of EPA in vivo by lipoxygenases (LOX) and cytochrome P450 (CYP450), leading to the formation of bioactive lipid mediators, such as resolvins, prostaglandins, and leukotrienes1) ,3) . During this conversion of EPA into physiologically active lipid mediators, several EPA hydroperoxide (EPA-OOH) have been identified as key intermediates, including 6E,8Z(E),11Z,14Z,17Z-EPA-5OOH, 5Z,8Z(E),10E,14Z,17Z-EPA-12OOH, 5Z,8Z,11Z(E),13E,17Z-EPA-15OOH, and 5Z,8Z,11Z,14Z(E),16E-EPA-18OOH3) ,4) . In addition, EPA-OOH can also be generated through auto-oxidation of EPA present in fish oil5) . In contrast to unoxidized EPA, EPA-OOH have been associated with concerns regarding possible adverse effects on human health. Studies using HeLa (human cervical carcinoma), L929 (mouse fibroblast), and RAW 264.7 (mouse macrophage-like) cell lines suggest that, when EPA accumulates extensively within mitochondria, EPA-OOH may trigger apoptotic cell death6) . Similarly, Iuchi et al. (2019) reported that oxidized EPA exhibits stronger antiproliferative and cytotoxic effects than unoxidized EPA in THP-1 (human monocytic leukemia) and DLD-1 (human colorectal ade-nocarcinoma) cells7) . In contrast, Sethi et al. (2002) demonstrated that EPA-OOH can exert anti-inflammatory effects via activation of peroxisome proliferator-activated receptor alpha (PPAR-α) in both in vivo and in vitro systems8) . EPA-OOH are therefore not necessarily detrimental and may also exert physiological functions. In this way, although several in vivo studies have examined EPA-OOH, it remains unclear how dietary EPA-OOH are handled in the body-specifically, whether they accumulate, are rapidly metabolized, or follow alternative metabolic pathways. Moreover, most previous investigations have relied on auto-oxidized EPA preparations, which contain not only EPA-OOH but also secondary oxidation products, such as aldehydes and alcohols. Consequently, the intrinsic biological effects of EPA-OOH themselves may not have been evaluated in isolation.
In our previous work, we employed stable isotope-labeled linoleic acid hydroperoxide (LA-OOH) and oleic acid hydroperoxide (OA-OOH), demonstrating that these lipid hydroperoxides undergo rapid β-oxidation and exhibit minimal accumulation in vivo9). 13C-labeled fatty acids provide a powerful tool for assessing the in vivo catabolic rates of administered fatty acids. Dietary fatty acids are degraded to acetyl-CoA and subsequently metabolized through the tricarboxylic acid cycle, ultimately yielding carbon dioxide (CO2). Accordingly, measurement of the 13C/12C ratio in expired breath enables quantitative evalua-tion of fatty acid catabolism. LA-OOH and OA-OOH have been reported to undergo gastric acid-mediated cleavage to form octanoic acid (C8:0) and 9-oxo-nonanoic acid, respectively, followed by rapid β-oxidation, suggesting limited potential for systemic accumulation9) . On this basis, EPA-OOH may likewise be susceptible to chemical degradation under gastric acidic conditions. Reports have shown that EPA-OOH generated via auto-oxidation of EPA can undergo further oxidative transformation to form hydroxy fatty acids and epoxy fatty acids10) . However, there is very limited information regarding the specific compounds produced when EPA-OOH decomposed under acidic conditions.
Accordingly, the present study aimed to elucidate the in vivo fate of dietary EPA-OOH, and, leveraging our established analytical approaches, we conducted a detailed evaluation of their catabolic rates. Stable isotope (13C)-labeled EPA-OOH were synthesized and orally administered to mice. Following administration, expired air was collected from the mice at defined time points, and the 13C/12C ratio was determined using isotope ratio mass spectrometry (IR-MS), allowing direct comparison of the catabolic kinetics of 13C-labeled EPA-OOH with those of unoxidized EPA. In addition, to mimic the human gastric environment, photo-oxidized EPA-OOH were treated with hydrochloric acid, and the resulting degradation products were structurally characterized using gas chromatography-mass spectrometry (GC-MS).
2 Experimental procedures
2.1 Chemicals and materials
EPA ethyl ester was purchased from Bizen Chemical Co., Ltd. (Okayama, Japan). 4-Dimethylaminopyridine (DMAP) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other chemicals were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
2.2 Preparation of lipid samples and emulsions
To estimate the catabolic rates of individual fatty acids, we synthesized 1-13C-labeled eicosapentaenoic acid ethyl esters (*EPA-EE) and its hydroperoxide (*EPA-EE-OOH) using a method modified from our previous study8) . Figure 1 shows the representative structures of *EPA-EE-OOH and *EPA-EE. Briefly, 10 g of EPA ethyl ester (EPA-EE) was mixed with ethanol, NaOH, and distilled water. The mixture was then saponified at 80°C for 1 h. After saponification, 10 % of sulfuric acid was added and purified by silica gel chromatography using hexane/ethyl acetate mixture (20/1, v/v). Then, 1-13C-labeled EPA (*EPA) was synthesized from purified EPA through a Barton decarboxylation, followed by a Grignard reaction with 13CO2. The resulting *EPA was esterified at 25 °C for 16 h under argon using ethanol, DMAP, and EDC in dichloromethane to obtain *EPA ethyl esters (*EPA-EE). Then, purities of *EPA-EE were determined to be >91 % by GC-flame ionization detection.
*EPA-EE-OOH was subsequently prepared by photo-oxidation as previously reported8) , with slight modifications. Briefly, *EPA-EE (1 g) was mixed with methylene blue (20 μM, dissolved in dichloromethane) and incubated in chloroform at 4°C for 24 h under illumination of 50,000 lx from a Suntech light (CN-304, SUNTECH Co., Ltd., Tokyo, Japan). The sample was loaded onto a Sep-Pak Silica cartridge (500 mg; Waters Co., Milford, MA, USA) pre-equilibrated with hexane, and methylene blue was removed by solution with chloroform. After evaporation, *EPA-EE-OOH was further purified by silica gel column chromatography using hexane/ethyl acetate (8:2, v/v) as the eluent to separate it from unoxidized *EPA-EE. Then, synthesis of *EPA-EE-OOH was confirmed by TLC using UV detection at 234 nm and a stain of 1.5 g of N,N'-dimethyl-p-phenylenediamine dihydrochloride/25 mL H2O/125 mL of MeOH/1 mL of acetic acid. The synthetic 13C-labeled fatty acid ethyl esters were stored at -80°C with an argon headspace until use.
Emulsion samples of *EPA-EE-OOH and *EPA-EE were prepared as previously described8) , with slight modifications. In brief, each 13C-labeled fatty acid ethyl ester (final concentration: 80 mM) was mixed with triolein, Triton X-100 (5 % of lipid), and 1 mL of distilled water in a vial and then mixed thoroughly via gentle inversion to prepare the emulsion samples. Emulsion samples were freshly prepared immediately prior to animal experiment.
2.3 Evaluation of the Catabolic Rates of Samples
Male Slc:ICR mice (3 weeks old; Japan SLC, Inc., Shizuoka, Japan) were housed in plastic cages with free access to tap water and CE-2 as a normal diet (CLEA Japan, Inc., Tokyo, Japan). These mice were maintained under controlled conditions with a 12:12-h light-dark cycle, a temperature of 22 ± 2 °C, and a relative humidity of 50 ± 5 %. A one-week acclimatization period was provided for all mice prior to the experiments. Following the acclimation period, mice (n = 5) received oral administration of emulsion samples containing *EPA-EE-OOH or *EPA-EE at a dose of 16 μmol per 20 g of body weight. At designated time points (0, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 240, 300, and 360 min post-administration), each animal was placed individually in a 450 mL plastic chamber for 5 min to collect expired air. The exhaled gas was then withdrawn using a 50 mL syringe and transferred into 12 mL evacuated vials (Exetainer, Labco Ltd., Lampeter, UK). During the entire experimental period (0-360 min), mice were maintained without access to food and water. Animal experiments in this study were approved by the Committee for the Care and Use of Laboratory Animals of Tokyo University of Marine Science and Technology (approval numbers: R2-6), and the animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals of Tokyo University of Marine Science and Technology.
The actual 13C/12C ratio in the 13CO2 gas exhaled by mice was analyzed using isotope ratio mass spectrometry (IRMS, ANCA-GSL, Sercon Ltd., Cheshire, UK), as described previously8) . The δ13C values were measured and converted to Δ13C (‰) relative to the baseline value at 0 min. The maximum Δ13C value (Cmax), the time to reach Cmax (Tmax), and the area under the Δ13C-time curve from 0 to 360 min (AUC0-360) were calculated. The Cmax and AUC reflect the absorption and β-oxidation of 13C-labeled fatty acids.
2.4 Analysis of EPA-EE-OOH Decomposition products
Non labeled EPA-EE-OOH were synthesized from EPA-EE by photo-oxidation as described above. EPA-EE-OOH (50 mg) was dissolved in a mixture of 2.5 mL of ethanol and 2.5 mL of 0.2 M HCl, and the mixture was incubated in a water bath at 37°C for 3 h with continuous shaking at 100 rpm. Following the reaction, 1 mL of hexane was added, and the mixture was vigorously mixed. The hexane layer was then collected and evaporated to dryness under reduced pressure. The resulting residue was reconstituted in hexane for subsequent GC-MS analysis. GC-MS analysis of the decomposition products was performed using a CP-Sil 88 column, following the method described previously 8) .
2.5 Statistical analyses
Data are presented as mean ± standard error. Differences between groups at each time point were evaluated using Student's t-test to assess time-specific variations in Δ13C values. Differences in AUC values were first evaluated using one-way analysis of variance (ANOVA), followed by the Tukey-Kramer multiple comparison test. A value of p < 0.05 was considered statistically significant.
3 Results and discussion
3.1 Analysis of the expired 13CO2 levels in mice
Using a photo-oxidation reaction, 170 mg of *EPA-EE-OOH was synthesized. The reaction yield was 15.2 %. As illustrated in Fig. 2, EPA-EE-OOH exists as multiple positional and geometric isomers. Auto-oxidation of EPA-EE yields 16 positional and geometric EPA-EE-OOH isomers: 6E,8Z,11Z,14Z,17Z-EPA-EE-5OOH, 6E,8E,11Z,14Z,17Z-EPA-EE-5OOH, 5Z,9E,11Z,14Z,17Z-EPA-EE-8OOH, 5Z,9E,11E,14Z,17Z-EPA-EE-8OOH, 5Z,7E,11Z,14Z,17Z-EPA-EE-9OOH, 5E,7E,11Z,14Z,17Z-EPA-EE-9OOH, 5Z,8Z,12E,14Z,17Z-EPA-EE-11OOH, 5Z,8Z,12E,14E,17Z-EPA-EE-11OOH, 5Z,8Z,10E,14Z,17Z-EPA-EE-12OOH, 5Z,8E,10E,14Z,17Z-EPA-EE-12OOH, 5Z,8Z,11Z,15E,17Z-EPA-EE-14OOH, 5Z,8Z,11Z,15E,17E-EPA-EE-14OOH, 5Z,8Z,11Z,13E,17Z-EPA-EE-15OOH, 5Z,8Z,11E,13E,17Z-EPA-EE-15OOH, 5Z,8Z,11Z,14Z,16E-EPA-EE-18OOH, and 5Z,8Z,11Z,14E,16E-EPA-EE-18OOH. In addition to these 16, photo-oxidation produces two additional isomers, 4E,8Z,11Z,14Z,17Z-EPA-EE-6OOH and 5Z,8Z,11Z,14Z,18E-EPA-EE-17OOH. Individual isolation of these isomers is technically challenging. Accordingly, in the present study, all experiments were conducted using the unresolved mixture of the 18 EPA-EE-OOH isomers. Since EPA contains five double bonds, oxidation could theoretically generate products bearing multiple hydroperoxide (-OOH) groups. However, previous studies indicate that the primary auto-oxidation products of EPA are predominantly mono-hydroperoxide EPA-OOH5) . However, as the report by Yin et al. analyzed EPA-OOH obtained by auto-oxidation, we cannot exclude the possibility that higher-order oxidation products may be present in the EPA-OOH prepared via photo-oxidation in this study. Nevertheless, in the present analysis, we focused on EPA-OOH species bearing a single hydroperoxide group as the major oxidation products. Fur-thermore, the present study is consistent with our previous report9) in that fatty acids were examined in their ethyl ester forms.
Fig. 2
Chemical structures of eighteen isomers of EPA-EE-OOH (6E,8Z,11Z,14Z,17Z-EPA-EE-5OOH, 6E,8E,11Z,14Z,17Z-EPA-EE-5OOH, 4E,8Z,11Z,14Z,17Z-EPA-EE-6OOH 5Z,9E,11Z,14Z,17Z-EPA-EE-8OOH, 5Z,9E,11E,14Z,17Z-EPA-EE-8OOH, 5Z,7E,11Z,14Z,17Z-EPA-EE-9OOH, 5E,7E,11Z,14Z,17Z-EPA-EE-9OOH, 5Z,8Z,12E,14Z,17Z-EPA-EE-11OOH, 5Z,8Z,12E,14E,17Z-EPA-EE-11OOH, 5Z,8Z,10E,14Z,17Z-EPA-EE-12OOH, 5Z,8E,10E,14Z,17Z-EPA-EE-12OOH, 5Z,8Z,11Z,15E,17Z-EPA-EE-14OOH, 5Z,8Z,11Z,15E,17E-EPA-EE-14OOH, 5Z,8Z,11Z,13E,17Z-EPA-EE-15OOH, 5Z,8Z,11E,13E,17Z-EPA-EE-15OOH, 5Z,8Z,11Z,14Z,18E-EPA-17OOH, 5Z,8Z,11Z,14Z,16E-EPA-EE-18OOH, and 5Z,8Z,11Z,14E,16E-EPA-EE-18OOH). These EPA-EE-OOH were oxidized by photo-oxidation (solid line) and auto-oxidation (dotted line). A (gray solid line) and B (gray dotted line) indicate the cleavage sites in the vicinity of the hydroperoxy group.
Synthesized *EPA-EE-OOH or *EPA-EE (control) was administered orally to mice, and the 13C/12C ratio in expired breath was subsequently compared between groups. The Δ13C values in exhaled CO2 were significantly higher in the *EPA-EE-OOH group than in the *EPA-EE control group during the 30-60 min period following administration (Fig. 3). The maximum Δ13C value (observed Cmax) in the *EPA-EE-OOH group reached 28.6 ‰, with a corresponding time to peak (observed Tmax) of 30 min. By contrast, the EPA-EE group exhibited an observed Cmax of 27.4 ‰ and an observed Tmax of 240 min. A similar time-dependent profile of exhaled Δ13C following administration of unoxidized EPA has also been reported in human studies. Specifically, Le'veille' et al. (2017) demonstrated that oral administration of 35 mg 13C-EPA methyl ester to healthy adult human males resulted in peak breath Δ13C levels approximately 4 h post-dose11) . The observation of a comparable temporal pattern in mice in the present study suggests that EPA metabolic kinetics may share conserved features across species. Taken together, these findings indicate that EPA-EE-OOH enters oxidative metabolic pathways at earlier time points than its unoxidized counterpart.
In contrast, no significant differences in exhaled Δ13C were detected between the *EPA-EE-OOH and *EPA-EE groups from 75 to 240 min after administration (Fig. 3). Moreover, the area under the curve (AUC) that integrated Δ13C (‰) values over the 6 h post-dose period revealed no significant difference in the amount of fatty acid-derived carbon eliminated as CO2 via β-oxidation between groups (Fig. 4). Thus, although the *EPA-EE-OOH group exhibited a more rapid initial rise in Δ13C, the absence of differences in AUC up to 6 h indicates that, despite divergent early metabolic kinetics, the overall extent of β-oxidation is comparable between EPA-EE-OOH and EPA-EE. Exhaled 13CO2 reflects whole-body β-oxidation rather than a specific organ; however, the liver is expected to be a major contributor during the early postprandial period. Therefore, EPA-OOH may undergo β-oxidation predominantly in the liver at early time points after administration.
Consistent with the AUC0-360 results, previous studies have shown that oral administration of 1-13C-labeled LA-OOH (*LA-EE-OOH) or OA-OOH (*OA-EE-OOH) results in substantial 13C expiration within 30-90 min, indicating rapid early β-oxidation of these hydroperoxides8) . In Fig. 4, AUC values obtained in the present study were compared with previously reported AUCs9) , which were generated by the same research group using the same experimental design and analytical procedures, calculated from breath Δ13C (‰) measurements of *LA-EE-OOH, 1-13C-labeled linoleic acid ethyl ester (*LA-EE), *OA-EE-OOH, 1-13C-labeled oleic acid ethyl ester (*OA-EE), and 1-13C-labeled octanoic acid ethyl ester (*C8-EE). These comparisons revealed that EPA and its oxidized derivatives produced lower amounts of expired 13CO2 than other fatty acids, with *EPA-EE and *EPA-EE-OOH exhibiting the lowest extent of β-oxidation among all compounds examined (Fig. 4). Although *LA-EE-OOH and *OA-EE-OOH tended to show larger AUC values than their respective unoxidized forms, this pattern was not observed for *EPA-EE and *EPA-EE-OOH. Notably, the reduced susceptibility of *EPA-EE to β-oxidation relative to *LA-EE and *OA-EE is consistent with earlier findings comparing 13CO2 production from triacylglycerol-bound EPA, LA, and OA12) . The observation that EPA undergoes β-oxidation less readily than other fatty acids aligns with reports that, following ingestion, EPA is preferentially incorporated into membrane phospholipids and utilized as a precursor for bioactive lipid mediators13) . This suggests that EPA exhibits metabolic characteristics favoring retention and functional utilization as a membrane constituent and signaling lipid, rather than rapid consumption as an energy substrate. Previous studies have also indicated that triacylglycerols containing *OA-OOH are not absorbed from the intestine in their peroxide form14) . In addition, LA-OOH and OA-OOH are reported to undergo gastric acid-mediated cleavage to yield medium-chain fatty acids and oxo-fatty acids with shorter carbon chains than the parent molecules, which are subsequently subject to rapid β-oxidation9) . Moreover, the medium-chain fatty acid C8:0 has been shown to be metabolized more rapidly via β-oxidation than the long-chain fatty acid palmitic acid (C16:0)15) . Collectively, these findings suggest that the significantly elevated Δ13C observed in expired breath during the 30-60 min period following *EPA-EE-OOH administration may reflect gastric degradation of EPA-OOH, followed by metabolism resembling that of medium-chain fatty acids. Although the absorbed molecular form remains unclear (intact dietary EPA-OOH or its degradation products), these findings suggest that EPA-OOH-derived carbon is absorbed from the small intestine and enters systemic metabolism. Accordingly, we conducted an acid-mediated decomposition assay of EPA-EE-OOH using hydrochloric acid to simulate gastric conditions.
3.2 Analysis of EPA-EE-OOH decomposition products
As shown in Fig. 2, EPA-EE-OOH exists as a mixture of 18 isomeric species. Cleavage of these EPA-EE-OOH isomers in the vicinity of the hydroperoxide group (at positions A or B indicated in Fig. 2) is predicted to generate hydrocarbons, oxo-fatty acids, aldehydes, and fatty acids, as summarized in Table 1. For example, cleavage of 6E,8Z,11Z,14Z,17Z-EPA-EE-5OOH on the left side of the hydroperoxy group (position A in Fig. 2) yields 1,3,6,9,12-pentadecapentaene and 5-oxo-pentanoic acid ethyl ester, whereas cleavage on the right side (position B in Fig. 2) produces butanoic acid ethyl ester and 2,4,7,10,13-hexadecapentaenal (Table 1).
Table 1
Predicted decomposition products of EPA-EE-OOH. Bold indicates decomposition products detected by GC/MS in this study
Following hydrochloric acid treatment of EPA-EE-OOH, GC-MS analysis based on mass fragmentation identified fatty acids, such as butanoic acid ethyl ester, decadienoic acid ethyl ester, and tridecatrienoic acid ethyl ester, together with aldehydes, including 2-butenal, 3,6-nonadienal, 2,4-heptadienal, and 2,4,7-decatrienal (Fig. 5). In the *EPA-EE-OOH used in the present study, the carbon on the carboxyl side is labeled with the stable isotope (13C). Accordingly, the aldehydes detected after hydrochloric acid treatment originate from carbon skeletons lacking the carboxyl group and therefore do not contain 13C. As a result, these aldehydes are not reflected in the δ13C values obtained in the breath analysis. Therefore, subsequent degradation analyses focused specifically on products retaining the carboxyl group.
Short-chain fatty acids are efficiently absorbed from the intestine; locally, they serve as a major energy source for colonic epithelial cells, while systemically they enter the circulation without accumulating and contribute rapidly to metabolic processes16) . Accordingly, butanoic acid ethyl ester is likely to be metabolized quickly and may have contributed to early expiration of labeled carbon. Moreover, since medium-chain fatty acids undergo rapid β-oxidation15) , decadienoic acid ethyl ester and tridecatrienoic acid ethyl ester detected in this study may exhibit medium-chain-like behavior and be metabolized rapidly. Taken together, these observations suggest that butanoic acid ethyl ester, decadienoic acid ethyl ester, and tridecatrienoic acid ethyl ester generated through gastric ac-id-mediated degradation of EPA-EE-OOH may behave in a manner similar to medium-chain fatty acids, potentially contributing to relatively rapid metabolism. Collectively, the conversion of EPA-EE-OOH into low-molecular-weight fatty acids under acidic gastric conditions may partly contribute to the elevated breath Δ13C observed during the 30-60 min period following *EPA-EE-OOH administration. However, as decadienoic acid ethyl ester and tridecatrienoic acid ethyl ester contain multiple unsaturated bonds, their metabolic rates may differ from those of typical medium-chain fatty acids. Future studies administering stable isotope-labeled forms of these degradation products to animals, followed by assessment of breath output and in vivo kinetics, would enable further validation of this hypothesis.
In the present study, no oxo-fatty acids or hydrocarbons were detected. In contrast, previous reports have shown that LA-OOH degradation under gastric acid conditions produces not only aldehydes, such as hexanal and 2,4-decadienal, but also medium-chain fatty acids including heptanoic and octanoic acids, as well as oxo-fatty acids, such as 8-oxo-octanoic acid and 9-oxo-nonanoic acid17) . Also, acid decomposition of LA-EE-OOH and OA-EE-OOH has been reported to yield 13-16 aldehydes and oxo-fatty acids ethyl ester, including 9-oxo-nonanoic acid ethyl ester, 10-oxo-8-decenoic acid ethyl ester, and, 12-oxo-9-dodecenoic acid ethyl ester9) . Among the degradation products predicted for EPA-OOH in the present study (Table 1), only seven compounds were detected, and no oxo-fatty acids were identified (Fig. 5). Since EPA-EE-OOH comprises multiple positional isomers (Fig. 2), degradation under gastric acid conditions may generate a wide variety of low-molecular-weight compounds. However, the individual abundances of these products are likely low, and some species may have fallen below the detection limits of GC-MS analysis. In addition, certain oxo-fatty acids predicted as potential degradation products, such as 11-oxo-5,8-undecadienoic acid ethyl ester, are not represented in the NIST library and corresponding analytical standards were unavailable, precluding their identification in this study. In addition, several minor peaks not assigned to identified compounds were observed in the GC-MS chromatograms. These peaks may represent additional degradation products present at low abundance; however, due to the lack of corresponding standards and library matches, they could not be reliably identified and are therefore not included in the present analysis. Accordingly, the absence of detectable oxo-fatty acids may be attributed to these analytical and compositional limitations. As suggested by the results of the simulated gastric degradation experiments, EPA-EE-OOH likely consists of multiple positional and geometrical isomers, with certain species preferentially formed during photo-oxidation. Notably, the detected gastric degradation products were consistent with cleavage reactions occurring adjacent to hydroperoxide groups in isomers such as 6E,8Z,11Z,14Z,17Z-EPA-EE-5OOH, 6E,8E,11Z,14Z,17Z-EPA-EE-5OOH, 5Z,8Z,12E,14Z,17Z-EPA-EE-11OOH, 5Z,8Z,12E,14E,17Z-EPA-EE-11OOH, 5Z,8Z,10E,14Z,17Z-EPA-EE-12OOH, 5Z,8E,10E,14Z,17Z-EPA-EE-12OOH, 5Z,8Z,11Z,15E,17Z-EPA-EE-14OOH, 5Z,8Z,11Z,15E,17E-EPA-EE-14OOH, and 5Z,8Z,11Z,14Z,18E-EPA-EE-17OOH. Under acidic conditions, these isomers may undergo cleavage at different positions, generating a range of structurally distinct low-molecular-weight compounds. Moreover, differences in the relative abundances of the parent isomers may have led to a limited number of decomposition products, which could explain why fewer products were observed compared with those listed in Table 1. Future studies employing LC-MS to analyze the isomeric composition of EPA-EE-OOH would help clarify the relative abundances of these species and further elucidate the mechanisms underlying their gastric degradation. Thus, owing to the presence of multiple positional isomers in EPA-EE-OOH, acid-mediated cleavage may yield small quantities of structurally diverse low-molecular-weight compounds, including fatty acids and aldehydes. Although these degradation products may be absorbed and metabolized shortly after administration, their low individual abundances and distribution across multiple metabolic pathways and time frames likely limit their overall contribution.
These findings suggest that while a fraction of EPA-EE-OOH-derived carbon undergoes rapid β-oxidation, another fraction is diverted to non-oxidative metabolic pathways or retained in tissue lipid pools, thereby limiting cumulative 13CO2 production. Consequently, in Fig. 4, the total expired 13CO2 (AUC) in the EPA-EE-OOH group was comparable to that with unoxidized EPA and did not increase to the same extent as with LA-OOH or OA-OOH. Future studies employing direct mass spectrometric analyses of degradation products generated from EPA-EE-OOH within the stomach or small intestine may clarify the mechanisms underlying the early rise in breath Δ13C and the absence of differences in AUC.
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