Identification of a novel monocyclic carotenoid and prediction of its biosynthetic genes in Algoriphagus sp. oki45.

Bacteria belonging to the genus Algoriphagus have been isolated from various sources, such as Antarctic sea ice, seawater, and sediment, and some strains are known to produce orange to red pigments. However, the pigment composition and biosynthetic genes have not been fully elucidated. A new red-pigmented Algoriphagus sp. strain, oki45, was isolated from the surface of seaweed collected from Senaga-Jima Island, Okinawa, Japan. Genome comparison revealed oki45's average nucleotide identity of less than 95% to its closely related species, Algoriphagus confluentis NBRC 111222 T and Algoriphagus taiwanensis JCM 19755 T. Comprehensive chemical analyses of oki45's pigments, including 1H and 13C nuclear magnetic resonance and circular dichroism spectroscopy, revealed that the pigments were mixtures of monocyclic carotenoids, (3S)-flexixanthin ((3S)-3,1'-dihydroxy-3',4'-didehydro-1',2'-dihydro-ß,ψ-caroten-4-one) and (2R,3S)-2-hydroxyflexixanthin ((2R,3S)-2,3,1'-trihydroxy-3',4'-didehydro-1',2'-dihydro-ß,ψ-caroten-4-one); in particular, the latter compound was new and not previously reported. Both monocyclic carotenoids were also found in A. confluentis NBRC 111222 T and A. taiwanensis JCM 19755 T. Further genome comparisons of carotenoid biosynthetic genes revealed the presence of eight genes (crtE, crtB, crtI, cruF, crtD, crtYcd, crtW, and crtZ) for flexixanthin biosynthesis. In addition, a crtG homolog gene encoding 2,2'-ß-hydroxylase was found in the genome of the strains oki45, A. confluentis NBRC 111222 T, and A. taiwanensis JCM 19755 T, suggesting that the gene is involved in 2-hydroxyflexixanthin synthesis via 2-hydroxylation of flexixanthin. These findings expand our knowledge of monocyclic carotenoid biosynthesis in Algoriphagus bacteria. KEY POINTS: • Algoriphagus sp. strain oki45 was isolated from seaweed collected in Okinawa, Japan. • A novel monocyclic carotenoid 2-hydroxyflexixanthin was identified from strain oki45. • Nine genes for 2-hydroxyflexixanthin biosynthesis were found in strain oki45 genome.

Identification and tissue distribution of fucoxanthinol and amarouciaxanthin A fatty acid esters in fucoxanthin-fed mice.

Administered carotenoid fatty acid esters are thought to be hydrolyzed to their free forms and absorbed into the body, and information on the tissue distribution of carotenoid fatty acid esters has been limited. Fucoxanthin, a marine carotenoid, exhibits various health benefits, including anti-diabetic and anti-obesity effects. However, fucoxanthin metabolism in mammals remains unclear. Herein, we investigated the fatty acid esters of fucoxanthin metabolites, fucoxanthinol and amarouciaxanthin A, in the tissues of male C57BL/6J mice fed a fucoxanthin-containing diet for one week. Fucoxanthinol and amarouciaxanthin A-3-esters accumulated abundantly in the liver and epididymal white adipose tissue, respectively. These esters were less detectable in the serum and other tissues. Therefore, it is suggested that fucoxanthinol and amarouciaxanthin A are partially acylated in the liver and epididymal white adipose tissue after being transported through the body as their free forms. This study presents a novel carotenoid metabolic pathway in mammals.

Preparation of Apoastaxanthinals and Evaluation of Their Anti-inflammatory Action against Lipopolysaccharide-Stimulated Macrophages and Adipocytes.

Apocarotenoids are carotenoid derivatives in which the polyene chain is cleaved via enzymatic or nonenzymatic action. They are found in animal tissues and carotenoid-containing foods. However, limited information on the biological functions of apocarotenoids is available. Here, we prepared apocarotenoids from astaxanthin via chemical oxidation and evaluated their anti-inflammatory action against macrophages and adipocytes. A series of astaxanthin-derived apoastaxanthinals, apo-11-, apo-15-, apo-14'-, apo-12'-, apo-10'-, and apo-8'-astaxanthinals, were successfully characterized by chromatography and spectroscopic analysis. The apoastaxanthinals inhibited inflammatory cytokine production and mRNA expression against lipopolysaccharide-stimulated RAW 264.7 macrophages. Apoastaxanthinals suppressed interleukin-6 overexpression in an in vitro model with macrophages and adipocytes in the following cultures: (1) contact coculture of 3T3-L1 adipocytes and RAW264.7 macrophages and (2) 3T3-L1 adipocytes in a RAW264.7-derived conditioned media. These results indicate that the apoastaxanthinals have the potential for regulation of adipose tissue inflammation observed in obesity.

Monocaprin Enhances Bioavailability of Fucoxanthin in Diabetic/Obese KK-Ay Mice.

Fucoxanthin is a marine carotenoid found in brown seaweeds and several microalgae. It has been reported that fucoxanthin has health benefits such as anti-obesity and anti-diabetic effects. To facilitate fucoxanthin applications in the food industry, it is important to improve its low bioavailability. We attempted the combined feeding of fucoxanthin-containing seaweed oil (SO) and monocaprin in a powder diet and analyzed the fucoxanthin metabolite contents in the liver, small intestine and serum of diabetic/obese KK-Ay mice. After 4 weeks of feeding with the experimental diets, the serum fucoxanthinol concentrations of the mice fed 0.2% SO and 0.5% monocaprin were higher than those of the 0.2% SO-fed mice. Furthermore, fucoxanthinol accumulation in the liver and small intestine tended to increase in a combination diet of 0.2% SO and 0.125-0.5% monocaprin compared with a diet of 0.2% SO alone, although amarouciaxanthin A accumulation was not different among the 0.2% SO-fed groups. These results suggest that a combination of monocaprin with fucoxanthin-containing SO is an effective treatment for improving the bioavailability of fucoxanthin.

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.

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.

Seco-type ß-Apocarotenoid Generated by ß-Carotene Oxidation Exerts Anti-inflammatory Effects against Activated Macrophages.

ß-Apocarotenoids are the cleavage products of ß-carotene. They are found in plants, carotenoid-containing foods, and animal tissues. However, limited information is available regarding the health benefits of ß-apocarotenoids. Here, we prepared seco-type ß-apocarotenoids through the chemical oxidation of ß-carotene and investigated their anti-inflammatory effects against activated macrophages. Oxidation of ß-carotene with potassium permanganate produced seco-ß-apo-8'-carotenal, in which one end-group formed an "open" ß-ring and the other was cleaved at the C-7',8' position. In lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells, seco-ß-apo-8'-carotenal inhibited the secretion and mRNA expression of inflammatory mediators such as nitric oxide, interleukin (IL)-6 and IL-1ß, and monocyte chemoattractant protein-1. Furthermore, seco-ß-apo-8'-carotenal suppressed phosphorylation of c-Jun N-terminal kinase and the inhibitor of nuclear factor (NF)-κB as well as the nuclear accumulation of NF-κB p65. Notably, since seco-ß-apo-8'-carotenal exhibited remarkable anti-inflammatory activity compared with ß-apo-8'-carotenal, its anti-inflammatory action could depend on the opened ß-ring structure. These results suggest that seco-ß-apo-8'-carotenal has high potential for the prevention of inflammation-related diseases.

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.

Identification of Paracentrone in Fucoxanthin-Fed Mice and Anti-Inflammatory Effect against Lipopolysaccharide-Stimulated Macrophages and Adipocytes.

SCOPE: Fucoxanthin is converted to fucoxanthinol and amarouciaxanthin A in the mouse body. However, further metabolism such as cleavage products (i.e., apocarotenoids) remains unclear. The fucoxanthin-derived apocarotenoid in vivo is investigated and the anti-inflammatory effect of apocarotenoids with fucoxanthin partial structure such as allenic bond and epoxide residue against activated macrophages and adipocytes in vitro is evaluated. METHODS AND RESULTS: LC-MS analysis indicates the presence of paracentrone, a C31 -allenic-apocarotenoid, in white adipose tissue of diabetic/obese KK-Ay and normal C57BL/6J mice fed 0.2% fucoxanthin diet for 1 week. In lipopolysaccharide-activated RAW264.7 macrophages, paracentrone as well as C26 - and C28 -allenic-apocarotenoids suppresses the overexpression of inflammatory factors. Further, apo-10'-fucoxanthinal, a fucoxanthin-derived apocarotenoid which retained epoxide residue, exhibits a most potent anti-inflammatory activity through regulating mitogen-activated protein kinases and nuclear factor-κB inflammatory signal pathways. In contrast, ß-apo-8'-carotenal without allenic bond and epoxide residue lacks suppressed inflammation. In 3T3-L1 adipocytes, paracentrone, and apo-10'-fucoxanthinal downregulate the mRNA expression of proinflammatory mediators and chemokines induced by co-culture with RAW264.7 cells. CONCLUSION: Dietary fucoxanthin accumulates as paracentrone as well as fucoxanthinol and amarouciaxanthin A in the mouse body. Allenic bond and epoxide residue of fucoxanthin-derived apocarotenoids have pivotal roles for anti-inflammatory action against activated macrophages and adipocytes.

Fucoxanthin inhibits hepatic oxidative stress, inflammation, and fibrosis in diet-induced nonalcoholic steatohepatitis model mice.

Nonalcoholic steatohepatitis (NASH) is associated with hepatocyte injury, excessive oxidative stress, and chronic inflammation in fatty liver, and can progress to more severe liver diseases, such as cirrhosis and hepatocellular carcinoma. However, currently there are no effective therapies for NASH. Marine carotenoid, fucoxanthin (Fx), abundant in brown seaweeds, has variable biological properties, such as anti-cancer, anti-inflammatory, anti-oxidative and anti-obesity. However, the effect of Fx on the development of NASH has not been explored. We investigated the protective effects of Fx in diet-induced NASH model mice fed choline-deficient L-amino acid-defined high fat diet (CDAHFD). Fx administration significantly attenuated liver weight gain and hepatic fat accumulation, resulting in the alleviation of hepatic injury. Furthermore, the Fx-fed mice, not only exhibited reduced hepatic lipid oxidation, but also decreased mRNA expression levels of inflammation and infiltration-related genes compared to that of the CDAHFD-fed mice. Moreover, fucoxanthinol and amarouciaxanthin A, two Fx metabolites exerted anti-inflammatory effects in the liver via inhibiting the chemokine production in hepatocytes. In case of fibrosis, one of the features of advanced NASH, the expression of fibrogenic factors including activated-hepatic stellate cell marker was significantly decreased in the liver of Fx-fed mice. Thus, the present study elucidated that dietary Fx not only inhibited hepatic oxidative stress and inflammation but also prevented early phase of fibrosis in the diet-induced NASH model mice.

Nutraceutical characteristics of the brown seaweed carotenoid fucoxanthin.

Fucoxanthin (Fx), a major carotenoid found in brown seaweed, is known to show a unique and wide variety of biological activities. Upon absorption, Fx is metabolized to fucoxanthinol and amarouciaxanthin, and these metabolites mainly accumulate in visceral white adipose tissue (WAT). As seen in other carotenoids, Fx can quench singlet oxygen and scavenge a wide range of free radicals. The antioxidant activity is related to the neuroprotective, photoprotective, and hepatoprotective effects of Fx. Fx is also reported to show anti-cancer activity through the regulation of several biomolecules and signaling pathways that are involved in either cell cycle arrest, apoptosis, or metastasis suppression. Among the biological activities of Fx, anti-obesity is the most well-studied and most promising effect. This effect is primarily based on the upregulation of thermogenesis by uncoupling protein 1 expression and the increase in the metabolic rate induced by mitochondrial activation. In addition, Fx shows anti-diabetic effects by improving insulin resistance and promoting glucose utilization in skeletal muscle.

The Effect of n-3 PUFA Binding Phosphatidylglycerol on Metabolic Syndrome-Related Parameters and n-3 PUFA Accretion in Diabetic/Obese KK-Ay Mice.

n-3 Polyunsaturated fatty acid binding phospholipids (n-3 PUFA-PLs) are known to be potent carriers of n-3 PUFAs and provide health benefits. We previously prepared n-3 PUFA binding phosphatidylglycerol (n-3 PUFA-PG) by phospholipase D-mediated transphosphatidylation. Because PG has excellent emulsifiability, n-3 PUFA-PG is expected to work as a functional molecule with properties of both PG and n-3 PUFAs. In the present study, the health benefits and tissue accretion of dietary n-3 PUFA-PG were examined in diabetic/obese KK-Ay mice. After a feeding duration over 30 days, n-3 PUFA-PG significantly reduced the total and non-HDL cholesterols in the serum of diabetic/obese KK-Ay mice. In the mice fed n-3 PUFA-PG, but not n-3 PUFA-TAG, hepatic lipid content was markedly alleviated depending on the neutral lipid reduction compared with the SoyPC-fed mice. Further, the n-3 PUFA-PG diet increased eicosapentaenoic acid and docosahexaenoic acid (DHA) and reduced arachidonic acid in the small intestine, liver, perirenal white adipose tissue, and brain, and the ratio of the n-6 PUFAs to n-3 PUFAs in those tissues became lower compared to the SoyPC-fed mice. Especially, the DHA level was more significantly elevated in the brains of n-3 PUFA-PG-fed mice compared to the SoyPC-fed mice, whereas n-3 PUFA-TAG did not significantly alter DHA in the brain. The present results indicate that n-3 PUFA-PG is a functional lipid for reducing serum and liver lipids and is able to supply n-3 PUFAs to KK-Ay mice.

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.

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.

Evaluating the Content and Distribution of Trans Fatty Acid Isomers in Foods Consumed in Japan.

Trans fatty acids (TFA) are considered risk factors for cardiovascular disease. However, detailed information on total content of TFA and TFA isomers and distribution of trans-octadecenoic acid positional isomers in foods consumed in Japan is not available till date. In this study, 250 foods, 169 processed foods and 81 foods derived from ruminant meat or milk, were analyzed. According to the results, most foods contained less than 1.0 g TFA / 100 g food. However, almost all foods containing butter had more than 1.0 g TFA / 100 g food. TFA isomers in foods were classified into two categories, monoene-rich type and polyenerich type. We hypothesized that these differences were attributed to diverse TFA formation mechanisms. Furthermore, we observed that trans-10-18:1 was also the dominant trans-18:1 positional isomer in foods consumed in Japan. These results are valuable for future analysis of the role of TFA in epidemiological studies in Japan.

Metabolism of Natural Highly Unsaturated Fatty Acid, Tetracosahexaenoic Acid (24:6n-3), in C57BL/KsJ-db/db Mice.

Tetracosahexaenoic acid (THA; 24:6n-3) is a natural, n-3 highly unsaturated fatty acid (n-3HUFA) that exists in fish, including Baltic herring (Clupea harengus) and the flathead flounder (Hippoglossoides dubius). In this study, natural n-3HUFAs, i.d. eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and THA were administrated to C57BL/KsJ-db/db mice for 4 weeks and the liver and serum lipid profiles, hepatic enzyme activity, expression of mRNA related to lipid metabolism, and adiponectin serum levels were then analyzed. The results showed that THA had the highest activity in suppressing hepatic triglyceride (TG) accumulation and increase in liver weight among the test groups. Furthermore, THA increased adiponectin levels in serum. These results indicate that THA is an excellent natural n-3HUFA that can suppress the development of metabolic syndromes and circulatory system diseases. The order of the n-3HUFA activity was THA > DHA > EPA in almost all the factors examined here. In a previous study of ours, the order was DHA > DPA > EPA, so the final order was summarized as THA > DHA > DPA > EPA. This order clearly translates to the rule that "the number of double bonds and carbon atoms in the n-3HUFA structure relates to their clinical functions".

Simple Quantification of Lactones in Milk Fat by Solvent Extraction Using Gas Chromatography-Mass Spectrometry.

The analysis of lactones as an indicator of milk quality is important in food manufacturing. However, the extraction of lactones requires sensitive conditions due to their volatility. In this study, the parameters for resolution of lactone standards were evaluated by gas chromatography-electron ionization/mass spectrometry (GC-EI/MS) to develop a rapid and simple method for the quantification and compositional analysis of lactones in edible fats, especially milk fat. Fourteen lactone standards consisting of 6-16 carbon atoms were analyzed and their correction factors (CFs) were obtained by using δ-undecalactone as an internal standard. The CFs of the lactone standards followed the same trend for δ-lactones and γ-lactones. Three volume equivalents of organic solvent per unit sample yielded the best recovery in the lactone analysis. Notably, 91-114% lactone recovery for the standards was achieved with methanol as the extractant. This method was also applicable to other fat samples, such as virgin coconut oil that is thought to contain large amounts of lactones. The recovery of lactones from virgin coconut oil was in the range of 87-104%, indicating that the developed method is also applicable to solid or semi-solid fat samples. The lactone content of butter oil, coconut oil, and butter samples was calculated by using the obtained CFs and the results were in good agreement with those of previous reports. Consequently, the GC-EI/MS method developed in this study is deemed applicable for the quantification of lactones in fat samples.

Quantitative Analysis of the Accumulation of Marine-derived Tocopherol in the Tissue of Mice Fed with Salmon Roe Oil Using HPLC-fluorescence.

In this study, we measured the quantity of marine-derived tocopherol (MDT), a monounsaturated vitamin E (VE), stored in the body tissue of mice fed with a diet containing a VE-rich fraction extracted from salmon roe. We first prepared the calibration curves for the MDT concentration using an HPLC-fluorescence system. Ranging from 0.016 to 50 µg/mL, the slope was expressed as first-order equations, with R2 values = 0.99. The mice were fed with an AIN-93 based diet containing MDT in doses of 21.4 mg/kg for 4 weeks, and the storage in the heart, lung, liver, stomach, small intestine, large intestine, kidney, pancreas, spleen, testis, skeletal muscle, visceral white adipose tissue (WAT), subcutaneous WAT and brain was quantified. MDT was widely distributed in tissues throughout the whole body, with higher accumulations observed in the adipose tissue, liver and kidney. These results demonstrate means to estimating the MDT concentration in natural products and in the bodies of animals and contribute to the understanding of the physiological functions of MDT in relation to human health.