Comparison of the Catabolic Rates of Linoleic and Oleic Acid Hydroperoxides Using 13CO2 Expired from Mice.

Unsaturated fatty acids, such as oleic and linoleic acids, are easily oxidized by exposure to temperature and light in the presence of air to form unsaturated fatty acid hydroperoxides as primary oxidation products. However, the catabolic rates of unsaturated fatty acid hydroperoxides in the human body remain unknown. In this study, ethyl esters of 13C-labeled linoleic acid (*C18:2-EE) and oleic acid (*C18:1-EE) and their hydroperoxides (*C18:2-EE-OOH and *C18:1-EE-OOH, respectively) prepared by the photo-oxidation of *C18:2-EE and *C18:1-EE, respectively, were administered to mice and their catabolic rates were determined by measuring the expired 13CO2 levels. *C18:2-EE-OOH and *C18:1-EE-OOH were ß-oxidized faster than *C18:2-EE and *C18:1-EE, respectively. Notably, rapid ß-oxidation of *C18:2-EE-OOH and *C18:1-EE-OOH was similar to that of medium-chain fatty acids, such as octanoic acid. Then, degradation products of C18:2-EE-OOH and C18:1-EE-OOH were analyzed under gastric conditions by gas chromatography/mass spectrometry. Major decomposition products of C18:2-EE-OOH and C18:1-EE-OOH were medium-chain compounds, such as octanoic acid ethyl ester, 9-oxo-nonanoic acid ethyl ester, and 10-oxo-8-decenoic acid ethyl esters, indicating that C18:2-EE-OOH and C18:1-EE-OOH isomers formed during photo-oxidation were decomposed under acidic conditions. These findings support previous reports that dietary lipid hydroperoxides are not absorbed into the intestine as lipid hydroperoxides but as degradation products. This is the first study to suggest that dietary lipid hydroperoxides decompose during gastric digestion to form medium-chain compounds that are directly absorbed into the liver via the portal vein and rapidly catabolized via ß-oxidation.

Formation of Epoxy Fatty Acids in Triacylglycerol Standards during Heating.

Epoxy fatty acid formation during heating was estimated using triolein (OOO) and trilinolein (LLL). Epoxy octadecanoic acids were found in heated OOO, while epoxy octadecenoic acids were found in heated LLL. The content of epoxy fatty acids increased with heating time, and trans-epoxy fatty acids were formed significantly more than cis-epoxy fatty acids. A comparison between OOO and LLL indicated that epoxy fatty acid formation was higher in the OOO than that in the LLL. Heating tests in the presence of α- tocopherol suggested that the formation of epoxy fatty acids could be suppressed by antioxidants.

Stable Isotope Tracer to Reveal the Interconversion between 3-Monochloro-1,2-propanediol Ester and Glycidyl Ester during the Deodorization Process.

In this study, the effects of the deodorization process on the interconversion between 3-monochloro-1,2-propanediol ester (3-MCPDE) and glycidyl ester (GE) using 3-MCPDE or GE standards containing deuterium-labeled palmitic acid (*P), oleic acid (*O), or linoleic acid (*L) were examined. Deuterium-labeled 3-MCPDE or GE was added to palm oil then deodorized at 250 °C for 20, 40, or 60 min. In the 3-MCPDE-spiked palm oil, the deuterium-labeled 3-MCPDE content decreased with deodorization time. Moreover, GE containing *P or *O was detected, but there was no GE containing *L in the 3-MCPDE-spiked palm oil. In the GE-spiked oil, GE containing *O or *L decreased with deodorization time, but the content of GE containing *P did not change over the time. Furthermore, deuterium-labeled 3-MCPDE was not detected in the GE-spiked oil. These results suggest that 3-MCPDE is converted into GE and that fatty acid species bound to 3-MCPDE or GE may affect their interconversion.

Antioxidative Self-Assembling Nanoparticles Attenuate the Development of Steatohepatitis and Inhibit Hepatocarcinogenesis in Mice.

Oxidative stress (OS) contributes to nonalcoholic steatohepatitis (NASH) and hepatocarcinogenesis. We investigated whether antioxidative self-assembling nanoparticles (SMAPoTN) could reduce the development of NASH and hepatocellular carcinoma (HCC) in p62/Sqstm1 and Nrf2 double knockout (DKO) mice and studied protective mechanisms. We measured disease development in male DKO mice fed a normal chow (NASH model) or a 60% high-fat diet (HFD; HCC model) with or without SMAPoTN administration for 26 weeks. SMAPoTN inhibited liver fibrosis in both groups and prevented HCC development (0% vs. 33%, p < 0.05) in the HFD group. SMAPoTN reduced OS, inflammatory cytokine signaling, and liver fibrosis. RNA-sequencing revealed that SMAPoTN decreased endoplasmic reticulum stress signaling genes in both groups, HCC driver genes, and cancer pathway genes, especially PI3K-AKT in the HFD groups. In the SMAPoTN treatment HFD group, serum lipopolysaccharide levels and liver lipopolysaccharide-binding protein expression were significantly lower compared with those in the nontreatment group. SMAPoTN improved the α-diversity of gut microbiota, and changed the microbiota composition. Oral SMAPoTN administration attenuated NASH development and suppressed hepatocarcinogenesis in DKO mice by improving endoplasmic reticulum stress in the liver and intestinal microbiota. SMAPoTN may be a new therapeutic option for NASH subjects and those with a high HCC risk.

Dietary triacylglycerol hydroperoxide is not absorbed, yet it induces the formation of other triacylglycerol hydroperoxides in the gastrointestinal tract.

The in vivo presence of triacylglycerol hydroperoxide (TGOOH), a primary oxidation product of triacylglycerol (TG), has been speculated to be involved in various diseases. Thus, considerable attention has been paid to whether dietary TGOOH is absorbed from the intestine. In this study, we performed the lymph duct-cannulation study in rats and analyzed the level of TGOOH in lymph following administration of a TG emulsion containing TGOOH. As we successfully detected TGOOH from the lymph, we hypothesized that this might be originated from the intestinal absorption of dietary TGOOH [hypothesis I] and/or the in situ formation of TGOOH [hypothesis II]. To determine the validity of these hypotheses, we then performed another cannulation study using a TG emulsion containing a deuterium-labeled TGOOH (D2-TGOOH) that is traceable in vivo. After administration of this emulsion to rats, we clearly detected unlabeled TGOOH instead of D2-TGOOH from the lymph, indicating that TGOOH is not absorbed from the intestine but is more likely to be produced in situ. By discriminating the isomeric structures of TGOOH present in lymph, we predicted the mechanism by which the intake of dietary TGOOH triggers oxidative stress (e.g., via generation of singlet oxygen) and induces in situ formation of TGOOH. The results of this study hereby provide a foothold to better understand the physiological significance of TGOOH on human health.

Quantitative Analysis of Lactone Enantiomers in Butter and Margarine through the Combination of Solvent Extraction and Enantioselective Gas Chromatography-Mass Spectrometry.

We quantified the enantiomeric distributions of δ- and γ-lactones in butter, fermented butter, and margarine through the combination of solvent extraction and enantioselective gas chromatography-mass spectrometry. The main lactones in butter and fermented butter comprised (R)-δ-decalactone, (R)-δ-dodecalactone, (R)-δ-tetradecalactone, (R)-δ-hexadecalactone, and (R)-γ-dodecalactone. In contrast, margarine samples consisted of only δ-decalactone and δ-dodecalactone in racemic forms, indicating that synthetic aroma chemicals were added to margarine. After heat treatment, 13 types of lactones were detected in butter and fermented butter. In heated butter and fermented butter, major δ-lactones in the (R)-form were abundant, but only δ-octalactone in the (S)-form was detected. In contrast, γ-dodecalactone (main γ-lactone in the heated samples) was abundant in the (R)-form, whereas other γ-lactones were detected in the racemic form. These results suggested that the major lactones in dairy products are in the (R)-form. Furthermore, the heat treatment affected the enantiomeric distribution of lactones in butter and fermented butter.

Comparison of Lipoprotein Cholesterol Levels in Golden Syrian Hamster Administrated trans-Octadecenoic Acid Positional Isomers.

We previously conducted a study using HepG2 cells to compare the effect on the secreted apolipoprotein B-100 and apolipoprotein A-1 ratio (B-100/A-1) corresponding to the ratio of low-density to high-density lipoprotein cholesterol (LDL/HDL) among 13 types of trans-octadecenoic acid (t-18:1) positional isomers. The results revealed that trans-5-18:1 (t5) significantly increased B-100/A-1. In this study, 1% of t5 in the diet, corresponding to 2.08 energy%, was administrated golden Syrian hamsters for 4 weeks to reveal the effects on lipid profiles, including LDL/HDL, by comparing cis-9-octadecenoic acid (OA, oleic acid), trans-9-octadecenoic acid (EA), trans-11-octadecenoic acid (VA), and trans-9,trans-12- octadecadienoic acid (TT). LDL/HDL was not significantly different among the groups. However, the cholesterol concentration of medium very low-density lipoprotein (VLDL) was significantly lower in the TT diet than in the OA and t5 diets. The cholesterol concentration of small VLDL was significantly lower in the TT diet than in the OA, t5, and EA diets. The cholesterol concentration of large LDL was significantly lower in the TT diet than in the t5 and EA diets. However, no significant difference was detected between the TT and OA diets. In contrast, the cholesterol concentration of very small HDL was significantly higher in the TT diet than in the t5 diet. These results would support that lipid metabolism is affected by the structure of TFA in animals. However, t5-18:1 did not significantly change any lipid profile compared to OA existing in nature, and the previous result from the cell experiment showing that t5 increased B-100/A-1 (LDL/HDL) was not confirmed in this animal experiment.

Visualization of dietary docosahexaenoic acid in whole-body zebrafish using matrix-assisted laser desorption/ionization mass spectrometry imaging.

Zebrafish models have been developed for several studies involving lipid metabolism and lipid-related diseases. In the present study, the migration of dietary docosahexaenoic acid (DHA) in whole-body zebrafish was estimated by stable-isotope tracer and matrix-assisted laser desorption/ionization mass spectrometry imaging. Administration of 1-13C-2,2-D2-labeled DHA ((+3)DHA) ethyl ester to male zebrafish was conducted to evaluate its accumulation, migration, and distribution in the body. The (+3)DHA content in the body of zebrafish after administering (+3)DHA for 10 and 15 d was significantly higher than that in the control group. (+3)DHA was observed as a constituent of phosphatidylcholine (PC) in the intestine of zebrafish that were administered (+3)DHA for 5 and 10 d. (+3)DHA-containing PC tended to accumulate in the intestines of zebrafish administered (+3)DHA for 1 d, indicating that recombination of (+3)DHA from ethyl ester to PC occurs quickly at intestine. After administration for 15 d, (+3)DHA-containing PC accumulated in the intestine, liver, and muscle of whole-body zebrafish. In contrast, (+3)DHA-containing PC was not detected in the brain. These results showed that dietary DHA is initially constructed into PC as a structural component of intestinal cell membranes and gradually migrates into peripheral tissues such as muscle.

Simultaneous Treatment of Long-chain Monounsaturated Fatty Acid and n-3 Polyunsaturated Fatty Acid Decreases Lipid and Cholesterol Levels in HepG2 Cell.

The n-3 type polyunsaturated fatty acids (n-3PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from fish oil exhibit health benefits such as triacylglycerol- and cholesterol-lowering effects. Some pelagic fishes contain long-chain monounsaturated fatty acids (LC-MUFAs) such as eicosenoic acid (C20:1), which exert health-promoting effects. However, no study has evaluated beneficial effects of n-3PUFA and LC-MUFA combination. Here, we investigated effects of simultaneous treatment with n-3PUFA (EPA and DHA) and LC-MUFA (cis-5-C20:1 and cis-7-C20:1) and found that n-3PUFA and LC-MUFA combination significantly decreased lipid accumulation and reduced total cholesterol in HepG2 cells. Cholesterol level was significantly lower in DHA + cis-7-C20:1 group than in DHA + EPA group. These results suggest the importance of LC-MUFA as a functional molecule in fish oil.

Incorporation of Dietary Arachidonic and Docosatetraenoic Acid into Mouse Brain.

It is essential to analyze the metabolism of dietary polyunsaturated fatty acids in the brain for the research and development of functional foods. In this study, a single dose of 2,2-dideuterium-labeled docosatetraenoic acid ((+2)DTA) or 2,2-dideuterium-labeled arachidonic acid ((+2)AA) was orally administered to Institute of Cancer Research (ICR) mice and its metabolism in the brain was investigated. In the (+2)DTA group, the (+2)DTA content in the brain was significantly increased at 4, 8, 24, and 96 h compared to 0 h after administration, while in the (+2)AA group, the (+2)AA content was significantly increased at 4, 8, 24, and 96 h compared to 0 h. However, there was no significant difference in the content of (+2)DTA, a metabolite of (+2)AA, among all the groups. These results suggest that dietary (+2)DTA and (+2)AA pass through the blood-brain barrier and dietary (+2)AA is rather stored in the brain than converted to (+2)DTA.

Application of Partial Hydrogenation for the Generation of Minor Tocochromanol Homologs and Functional Evaluation of Hydrogenated Tocotrienol-rich Vitamin E Oil in Diabetic Obese Mice.

Recent research has identified minor homologs of vitamin E with one or two double bonds in the side-chain, namely tocomonoenol (T1) and tocodienol (T2), in natural products. We first explored the effectiveness of partial hydrogenation for generating minor tocochromanols from tocotrienol (T3). During hydrogenation with pure α-T3 as a substrate, the side-chain was partially saturated in a time-dependent manner, and a large amount of α-T1 and α-T2 was obtained. To investigate the beneficial effects of the hydrogenated product, we fed diabetic obese KK-A y mice with a hydrogenated T3 mixture (HT3). Feeding HT3 revealed tissue-specific accumulation of tocochromanols, ameliorated hyperglycemia and improved ratio of high-density lipoprotein cholesterol to total cholesterol in serum, with invariant body weight and fat mass. Hence, we propose that hydrogenation is a useful method for generating T1 and T2 homologs, which can be applied to explore the structure-related function of tocochromanols.

Effect of trans-Octadecenoic Acid Positional Isomers on Tumor Necrosis Factor-α Secretion in RAW264.7 Cells.

We compared the cytotoxic effects and tumor necrosis factor-α (TNF-α) production induced by 13 trans-octadecenoic acid positional isomers (trans-4-C18:1 to trans-16-C18:1) in RAW264.7 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and enzyme-linked immunosorbent assay, respectively. No significant differences were observed in the cytotoxic effects among the 13 trans-C18:1 positional isomers and control on RAW264.7 cells. TNF-α production significantly decreased by treatment of trans-4-C18:1 as compared to control, but no significant differences in TNF-α production were observed among other trans-C18:1 positional isomers and control. These results suggest that the double bond position in trans-C18:1 may affect TNF-α production in cells.

Selective Visualization of Administrated Arachidonic and Docosahexaenoic Acids in Brain Using Combination of Simple Stable Isotope-Labeling Technique and Imaging Mass Spectrometry.

We developed a new method for monitoring the distribution of administrated fatty acids in the body by combination of a stable isotope-labeling technique and imaging mass spectrometry (IMS). The developed stable isotope-labeling technique is very simple and able to adapt to all the fatty acid species. In this study, we synthesized stable isotope-labeled arachidonic acid (AA) and docosahexaenoic acid (DHA), and they were simultaneously administrated to mice to examine their migrations and distributions in the brain. The administrated AA and DHA have two more molecular weights compared to the originals and apparently were distinguished from the originally accumulated AA and DHA in the brain using IMS. As a result, we reveal that the administered AA and DHA first accumulated in the hippocampus and cerebellar cortex in the brain. This technique does not use radio isotopes and would appear to elucidate the role of all kinds of fatty acid species in the body.

Effects of Heat Treatment on Lactone Content of Butter and Margarine.

The lactone content of butter, fermented butter, and margarine was compared using gas chromatography-mass spectrometry. The main lactones in butters and fermented butters consisted of δ-decalactone, δ-dodecalactone, δ-tetradecalactone, δ-hexadecalactone, and γ-dodecalactone. In contrast, the main lactones in margarines were δ-decalactone and δ-dodecalactone. The total lactone content in butters and fermented butters increased by approximately two-fold upon heat treatment, whereas, heat treatment did not affect the lactone content in margarine. The changes in lactone content caused by heat treatment were greater in fermented butters than in butters. These findings suggested that the fermentation process could increase lactone or lactone precursor content in butter.

Effect of Calcium Treatment on Catabolic Rates of 13C-Labeled Fatty Acids Bound to the α and ß Positions of Triacylglycerol.

The absorption efficacies and catabolic rates of fatty acids are affected by their binding position on triacylglycerol (TAG). However, the kind of effect calcium treatment has on the catabolism of fatty acids is unclear. In this study, the catabolic rates of 13C-labeled palmitic acid, oleic acid, and linoleic acid bound to sn-1, 3 (α) and sn-2 (ß) position of TAG in the presence of calcium were compared using isotope ratio mass spectrometry. The catabolic rates of 13C-labeled fatty acids were evaluated using the ratio of 13C to 12C in the carbon dioxide expired by mice. The catabolic rate of palmitic acid bound to the α position was significantly lower than that of palmitic acid bound to the ß position of TAG. The rates of 13CO2 formation from palmitic acid at the ß position remained higher for a long time. In contrast, oleic and linoleic acids at the α position were as well catabolized as those at the ß position. These results indicate that in the presence of calcium, the saturated fatty acid bound to the ß position is highly catabolized, whereas that bound to the α position is not well catabolized. Saturated fatty acid at the α position is hydrolyzed by pancreatic lipase to promptly form insoluble complexes with calcium, which are excreted from the body, and thereby reducing the catabolic rate of these fatty acids.

Simultaneous Separation of Triacylglycerol Enantiomers and Positional Isomers by Chiral High Performance Liquid Chromatography Coupled with Mass Spectrometry.

The rapid and simultaneous separation of triacylglycerol (TAG) enantiomers and positional isomers was achieved using chiral high performance liquid chromatography (HPLC). TAGs composed of two fatty acids, which were both saturated (P: palmitic acid or S: stearic acid) and unsaturated (O: oleic acid or L: linoleic acid; e.g., sn-PPO/sn-OPP/sn-POP: 1,2-dipalmitoyl-3-oleoyl-sn-glycerol/1-oleoyl-2,3-dipalmitoyl-sn-glycerol/1,3-dilpalmitoyl-2-oleoylglycerol), were resolved into three peaks using CHIRALPAK IF-3 without recycling on the HPLC system. For example, the mixture of sn-PPO/sn-OPP/sn-POP was resolved in 30 min, although it took 150 min to resolve sn-PPO/sn-OPP using CHIRALCEL OD-RH in a previous study using a recycling HPLC system. This novel chiral HPLC method was applicable for the separation of other TAG isomers, including sn-OOP/sn-POO/sn-OPO, sn-PPL/sn-LPP/sn-PLP, sn-LLP/sn-PLL/sn-LPL, sn-SSO/sn-OSS/sn-SOS, sn-OOS/sn-SOO/sn-OSO, sn-SSL/sn-LSS/sn-SLS, and sn-LLS/sn-SLL/sn-LSL. For TAGs composed of three fatty acids containing both saturated and unsaturated fatty acids, the POL isomers were not sufficiently separated but the PSO and SOL isomers were partially separated into several peaks. Their elution order could be estimated by the fragment ions generated in the ion source of the mass spectrometer. However, TAGs consisting of only saturated or unsaturated fatty acids (e.g., sn-PSP/sn-PPS/sn-SPP and sn-OLO/sn-OOL/sn-LOO) were not separated. This novel chiral HPLC method is especially applicable for the analysis of TAG composition of semi-solid fats such as palm oil.

Examination of the Catabolic Rates of 13C-Labeled Fatty Acids Bound to the α and ß Positions of Triacylglycerol Using 13CO2 Expired from Mice.

Fatty acids in triacylglycerol (TAG) are catabolized after digestion. However, the catabolic rates of several fatty acids bound to the α (sn-1, 3) or ß (sn-2) position of TAG have not been thoroughly compared. In this study, the catabolic rates of 13C-labeled palmitic acid, oleic acid, linoleic acid, α-linolenic acid, eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) bound to the α and ß position of TAG were compared using isotope ratio mass spectrometry. The catabolic rates of the studied fatty acids were evaluated using the ratio of 13C and 12C in carbon dioxide expired from mice. The results indicated that palmitic acid, oleic acid, or α-linolenic acid bound to the ß position was slowly catabolized for a long duration compared to that when bound to the α position. In contrast, EPA bound to the ß position was quickly catabolized, and EPA bound to the α position was slowly catabolized for a long time. For linoleic acid or DHA, no difference in the catabolic rates was detected between the binding positions in TAG. Furthermore, EPA and DHA were less catabolized than the other fatty acids. These results indicate that the catabolic rates of fatty acids are influenced by their binding positions in TAG and that this influence on the catabolic rate differed depending on the fatty acid species.

Comparison of the Effects of Long-chain Monounsaturated Fatty Acid Positional Isomers on Lipid Metabolism in 3T3-L1 Cells.

Long chain monounsaturated fatty acids (LC-MUFAs) have shown beneficial health effects in previous studies. They occur as mixtures of positional isomers (PIs) in food. The functionalities of LC-MUFA PIs have not been studied extensively. Common LC-MUFA PIs, namely cis-octadecenoic acid (c-18:1), cis-eicosenoic acid (c-20:1), and cis-docosenoic acid (c-22:1), were screened based on their effects on lipid accumulation. We selected nine fatty acids (FAs) to assess their effects on cellular lipid metabolism using 3T3-L1 preadipocytes. Lipid accumulation was found to be higher in cells treated with LC-MUFAs than in the non-treated cells. When comparing the influence of chain length of LC-MUFAs, TG levels tended to be higher in cells treated with c-22:1 group than that of the c18:1 and c-20:1 groups. Among the c-22:1 group, c9-22:1 treatment showed higher lipid accumulation, and was accompanied with elevated expression of transcription factors related to adipogenesis and lipogenesis, such as PPARγ and C/EBPα, and SREBP-1, respectively. In contrast, the effects of c-20:1 FAs were less pronounced than those of c-18:1 and c-22:1. Levels of accumulated lipid in cells treated with c15-20:1 were the same as in non-treated control. PPARγ, C/EBPα, and SREBP-1 were expressed at lower levels with c15-20:1 FA. Furthermore, mRNA levels of SCD-1 and FAS were lowered more by c15- and c11-20:1 than by other MUFAs. These results revealed that differences in the effects of LC-MUFAs on lipid metabolism depend on their chain lengths and on the position of the double bond.

Evidence for an alternative fatty acid desaturation pathway increasing cancer plasticity.

Most tumours have an aberrantly activated lipid metabolism1,2 that enables them to synthesize, elongate and desaturate fatty acids to support proliferation. However, only particular subsets of cancer cells are sensitive to approaches that target fatty acid metabolism and, in particular, fatty acid desaturation3. This suggests that many cancer cells contain an unexplored plasticity in their fatty acid metabolism. Here we show that some cancer cells can exploit an alternative fatty acid desaturation pathway. We identify various cancer cell lines, mouse hepatocellular carcinomas, and primary human liver and lung carcinomas that desaturate palmitate to the unusual fatty acid sapienate to support membrane biosynthesis during proliferation. Accordingly, we found that sapienate biosynthesis enables cancer cells to bypass the known fatty acid desaturation pathway that is dependent on stearoyl-CoA desaturase. Thus, only by targeting both desaturation pathways is the in vitro and in vivo proliferation of cancer cells that synthesize sapienate impaired. Our discovery explains metabolic plasticity in fatty acid desaturation and constitutes an unexplored metabolic rewiring in cancers.