Characterization of Ceramides and Glucosylceramides of the Satsuma Mandarin(Citrus unshiu) Fruit.

Ceramide (Cer) and glucosylceramide (GlcCer) were isolated from Satsuma mandarin (Citrus unshiu) fruits and characterized. 2,3-Dihydroxy fatty acids with C20 or longer acyl chains were found in Cer. GlcCers from the flesh of the fruit contained sphingosine (4-trans-sphingenine) as a major component. Notably, the Cer content was 1.5-fold higher than GlcCer content. The ratio of Cer plus GlcCer to the total lipid content in Satsuma mandarin was higher than that in the other citrus fruits analyzed in this study. Collectively, the pomace of the Satsuma mandarin fruit can be a good source of sphingolipids as functional components in foods.

Invariant Valpha14 natural killer T cell activation by edible mushroom acidic glycosphingolipids.

Invariant natural killer T (iNKT) cells regulate multi-immune response through Th1/Th2 cytokine release triggered by the recognition of CD1d-restricted glycosphingolipid antigens. Here we report that acidic glycosphingolipids (AGLs) of mushroom (Hypsizigus marmoreus and Pleurotus eryngii) presented by murine CD1d-transfected rat basophilic leukocytes induced interleukin-2 (IL-2) release from iNKT hybridoma cells. AGL-1, one of the AGLs, containing mannose at the non-reducing ends, induced CD1d-dependent IL-2 release. Al-though alpha-galactosylceramide (alpha-GalCer) presented by CD11c-positive cells induced both interferon-gamma (IFN-gamma) and IL-4 release, all of AGLs presented by CD11c-positive cells and AGL-1 presented by B cells induced IL-4 release from iNKT hybridoma cells. A single intravenous injection of AGLs into B6 mice induced only a little elevation of IL-4 in serum but repeated intravenous injection of AGLs induced prolonged retention of IL-4 in serum; therefore, these results suggested that edible mushroom AGLs might contribute to the retention of immunohomeostasis through the minimum induction of iNKT cell activation in vivo.

Content and fatty acid composition of sulfoquinovosyldiacylglycerol in conifer leaves grown in Hokkaido, Japan.

Sulfoquinovosyldiacylglycerol (SQDG) contents in conifer leaves and their fatty acid (FA) compositions were determined. The SQDG content was 16-36 mg/100 g, and was high in Picea glehnii. Palmitic and alpha-linolenic acid were the usually predominant FAs. In Picea, the proportion of alpha-linolenic acid was low, and those of oleic and linoleic acid were high. The essential oil residues of Abies sachalinensis leaves were found to be a potential source of SQDG material.

Simultaneous quantification of plant glyceroglycolipids including sulfoquinovosyldiacylglycerol by HPLC-ELSD with binary gradient elution.

Membrane lipids of photosynthetic organisms consist of glycerophospholipids and glyceroglycolipids. We investigated a method for the simultaneous quantitative analysis of neutral and acidic lipids using HPLC-ELSD, and quantified monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG). Ten complex lipid classes were separated with a binary gradient system consisting of chloroform and methanol-acetone-water-acetic acid (30:60:9:1, v/v/v/v) with 0.3% triethylamine (pH 4), and were eluted within 16 min. The contents of SQDG in ten edible plants ranged from 3 to 101 mg/100 g, and were positively correlated to the neutral glyceroglycolipids contents.

Prominent differences in leaf fatty acid composition in the F1 hybrid compared with parent trees Larix gmelinii var. japonica and L. kaempferi.

Fatty acid (FA) compositions in leaves were investigated for two families of F(1) hybrids of Larix gmelinii var. japonica x L. kaempferi (F(1)) and their parent clones. Twenty-one FAs, from C(12) to C(32), were found in the leaves of both adult trees and seedlings. The levels of 18:1/(18:2 + 18:3) increased in the order L. kaempferi, F(1), and L. gmelinii var. japonica, with significant differences between L. gmelinii var. japonica and F(1) in adult trees, but these differences were not found in the seedlings. Moreover, in the adult trees, the 18:1/(18:2 + 18:3) levels in the neutral phospholipid fraction and the SigmaC(18)/SigmaC(16), especially in the glycolipid fraction, showed significant differences among the three species. These characteristics are discussed from the viewpoint of lipid synthesis in the endoplasmic reticulum and chloroplasts, and of the activities and substrate specificities in sequential FA desaturation. Linear discriminant analysis suggested that the FA compositions are useable as an index in the identification of hybrid seedlings.

Analysis of sphingolipid classes and their contents in meals.

Sphingolipids have attracted attention as physiologically functional lipids. We determined their class and content in Japanese meals that had been prepared by a nutritionist, mainly by using HPLC-ELSD. In all 12 meals tested, cerebroside and/or sphingomyelin were generally detected as the major sphingolipids. The total amounts of sphingolipids in typical high- and low-calorie meal samples over 2 days were 292 and 128 mg/day, and 81 and 45 mg/day, respectively.

Anti-inflammatory effect of buckwheat sprouts in lipopolysaccharide-activated human colon cancer cells and mice.

In conducting an in vitro screening of ethanol extracts from various natural foods using a human colon cancer cell line (CoLoTC cells), an extract of buckwheat sprouts (ExtBS) was found to express significant anti-inflammatory activity. The anti-inflammatory activity of ExtBS was confirmed by oral administration of lipopolysaccharide (LPS) to mice. Inflammatory cytokines (interleukin 6 and tumor necrosis factor alpha) were markedly up-regulated in the spleen and liver from LPS-administrated mice, and combinatory treatment with LPS and ExtBS decreased up-regulation of them in both cytokines. Both serum cytokine levels corresponded to their gene expressions in tissues, but no anti-inflammatry effect in mice was observed when ExtBS was treated intraperitoneally. ExtBS oral administration also showed protective activity as to hepatic injury induced by galactosamine/LPS treatment. Based on these data, we suggest that ExtBS contains anti-inflammatory compounds.

Potato and soy peptide diets modulate lipid metabolism in rats.

Dietary plant and animal peptides have been shown to reduce serum lipids. However, the potential of food-derived peptides has yet to be fully elucidated. We investigated the physiological importance of potato peptides in rats fed on a cholesterol-free diet containing 20% potato peptides (PP), when compared with two diets containing either 20% casein (CN) or 20% soy peptides (SP). The high-density lipoprotein (HDL)-cholesterol (+13.8%) and serum triglyceride (-38%) concentrations in the PP-fed group, non-HDL-cholesterol level in the PP- (-22.5%) and SP- (-15.7%) fed groups, and serum total cholesterol concentration (-12%) in the SP-fed group, were significantly different from the control group at the end of the experiment. The fecal excretion of neutral and acidic sterols was higher in the PP- and SP-fed groups, respectively, relative to the control group. These results indicate that the observed changes in the serum cholesterol levels in rats fed on soy and potato peptide appear to have been due to different mechanisms.

Chemical properties of epidermal lipids, especially sphingolipids, of the Antarctic minke whale.

It is well known that sphingolipids specifically exist in the terrestrial mammal epidermis and correlate with skin barrier functions. However, the lipid properties of the marine mammal epidermis have not been examined in detail. We thus investigated the chemical composition of lipid components, especially sphingolipids, in the black epidermis (outer skin) of Antarctic minke whales (six mature and six immature specimens). Complex lipid fractions mainly contained cerebroside (CE), cholesteryl sulfate and sphingomyelin (SM), as well as two glycerophospholipids. Moreover, in the superficial layer of the black epidermis, CE was richly abundant but phospholipids were scarce. As component fatty acids, the non-hydroxy monounsaturated very long chain fatty acids (VLFA) within 34 carbons were generally present in CE and SM in the black epidermis. CE also consisted of alpha-hydroxy fatty acids with monounsaturation within C34 (17%) and a slight proportion of omega-hydroxy ones (32:1 and 34:1), the latter being probably derived from acyl-CE. Component sphingoid bases of both sphingolipids were predominantly 4-sphingenine (64%), followed by a C16 analogue (21%). When comparing these by different maturities, mature whales showed sphingolipid profiles with higher levels of unsaturated fatty acids and with shorter sphingoid base chains than those of immature ones. Component analysis revealed that CE sugars were 67% glucose and 33% galactose, and alpha-hydroxy fatty acids only bound to galactose.

Unusual fatty acid isomers of triacylglycerols and polar lipids in female limpet gonads of Cellana grata.

To investigate the occurrence of positional isomers of unsaturated fatty acids (FA) in triacylglycerols (TAG) and polar lipids (PL) in the female gonads of a dominant limpet, Cellana grata, fatty acid methyl esters (FAME) were fractionated according to the degree of their unsaturation by argentation thin-layer chromatography. Their structures were elucidated by gas chromatography-mass spectrometry of a combination of their FAME and picolinyl esters. A total of 125 different FA ranging from 12 to 24 carbon atoms were identified, and 105 unsaturated FA, including 34 nonmethylene-interrupted FA as minor components, were recognized. Although the major FA 16:0 and 18:1n-7, which accounted for more than 20 and 10%, respectively, of total FA, were present in TAG and PL, higher amounts of 20:4n-6 and 20:5n-3 were present in PL. Unsaturated FA consisting of more than five different positional isomers were 17:1, 19:1, 18:2, 19:2, 20:2, 21:2, 22:2, 20:3, and 22:3, most of which were present in TAG. Furthermore, a larger variety of nonmethylene-interrupted FA was found in TAG than in PL. This limpet gonad had a diverse assortment of unsaturated FA that comprised a wide range of unusual positional isomers with one to five double bonds.

Ethyl esterification of long-chain unsaturated fatty acids derived from grape must by yeast during alcoholic fermentation.

The composition of total fatty acid ethyl ester (FAEE) in yeast cells and the liquid phase separated from grape must during alcoholic fermentation at different temperatures was investigated by using the solid-phase extraction method. Thirteen FAEE from butyric to linolenic acids were detected during fermentation. Significant amounts of long-chain unsaturated FAEE, including linoleic and linolenic acids derived from grape material, had already accumulated in the yeast cells by day 3 during fermentation.

Novel Odd-Chain Fatty Acids with a Terminal Double Bond in Ovaries of the Limpet Cellana toreuma.

Our previous study characterized highly diverse dienoic fatty acids (FA), in particular an uncommon non-methylene-interrupted (NMI) FA, in the ovaries of the Japanese limpet Cellana toreuma belonging to the archaeogastropods, but many minor chemically unidentified FA remain. In this study, among previously unidentified FA (less than 0.1% of total FA), four novel NMI FA with a terminal double bond [7,18-nonadecadienoic (19:2Δ7,18), 11,18-nonadecadienoic (19:2Δ11,18), 7,20-heneicosadienoic (21:2Δ7,20), and 11,20-heneicosadienoic (21:2Δ11,20) acids] were found, along with known 14-pentadecenoic (15:1Δ14), 16-heptadecenoic (17:1Δ16), and 9,18-nonadecadienoic (19:2Δ9,18) acids, based on capillary GC-MS of their methyl esters, 3-pyridylcarbinol derivatives, and argentation thin-layer chromatography. From our findings, possible biosynthetic pathways for the novel FA are discussed.

(4Z,15Z)-Octadecadienoic Acid Inhibits Glycogen Synthase Kinase-3ß and Glucose Production in H4IIE Cells.

Many uncommon non-methylene-interrupted fatty acids (NMI FA) are present in limpet gonads, but their biological properties remain unknown. To investigate new biological effects of naturally occurring NMI FA in eukaryotic cells, the biological activities of structurally analogous (4Z,15Z)-octadecadienoic acid (1), (9Z,20Z)-tricosadienoic acid (2), and (12Z,23Z)-hexacosadienoic acid (3) were examined by using a yeast-based drug-screening system using the Ca2+-sensitive mutant strain, Saccharomyces cerevisiae (zds1Δ erg3Δ pdr1Δ pdr3Δ). Among 1-3, 1 showed restored growth activity at a dose of 80 µg/disc in the mutant yeast strain. This phenotype suggests that 1 suppresses Ca2+-signaling of the mutant yeast through inhibition of glycogen synthase kinase-3ß (GSK-3ß) or calcineurin pathways or both. From this result, the inhibitory activity of 1-3 against GSK-3ß was further determined. 1-3 showed potent inhibitory activity against GSK-3ß with IC50 values ranging from 8.7 to 21.9 µM. Inhibition of GSK-3ß reduces gene expression of the gluconeogenic key enzymes in liver, so we analyzed glucose production in rat hepatoma H4IIE cells to assess GSK-3ß inhibitory activity of 1-3. Acid 1 inhibited glucose production at 25 µM in H4IIE cells. Our results would open up new possibilities for an anti-diabetic effect of 1 and might provide important insights into understanding the biological properties of naturally occurring NMI FA.

An Unprecedented Occurrence of Δ5,9- and Δ9,15-Dienoic Fatty Acids in Ovaries of the Archaeogastropod Limpet Cellana toreuma.

A detailed structural diversity of dienoic fatty acids (FA), including non-methylene-interrupted dienoic FA, of triacylglycerols and polar lipids in ovaries of Cellana toreuma was clarified for the first time by using capillary gas chromatography-mass spectrometry of their 3-pyridylcarbinol esters and argentation thin-layer chromatography. Interestingly, in addition to 5,9-octadecadienoic (18:2Δ5,9), 5,9-eicosadienoic (20:2Δ5,9), 5,9-heneicosadienoic (21:2Δ5,9), 5,9-docosadienoic (22:2Δ5,9), 5,9-tricosadienoic (23:2Δ5,9), and 5,9-tetracosadienoic (24:2Δ5,9) acids, previously identified in ovaries of C. grata, rare FA 5,9-hexadecadienoic (16:2Δ5,9), 5,9-nonadecadienoic (19:2Δ5,9), and 21-methyl-5,9-docosadienoic (iso 23:2Δ5,9) were newly recognized in ovaries of C. toreuma. Detectable amounts of four Δ9,15-dienoic FA were present in the ovary lipids. The FA identified were one novel 9,15-heneicosadienoic (21:2Δ9,15) acid and known 9,15-docosadienoic (22:2Δ9,15), 9,15-tricosadienoic (23:2Δ9,15), and 9,15-tetracosadienoic (24:2Δ9,15) acids. The findings help to explain the broad evidence of the structural diversity in marine gastropods and suggest biomarkers to evaluate marine food web relations.

Fatty acids in must prepared from 11 grapes grown in Japan: comparison with wine and effect on fatty acid ethyl ester formation.

FA components of 11 musts made from grape cultivars grown for making red wine in various regions of Japan and of wines made from the musts were compared to elucidate variety-dependent characteristics and to clarify their effect on FA ethyl ester (FAEE) formation in wine. Sixteen FA with carbon chain lengths from 12 to 26 were detected in all musts, with palmitic, linoleic, and linolenic acids generally predominating. Higher levels of linoleic acid were found in musts from a cold region (Hokkaido), and higher levels of oleic acid occurred in musts from a warm region (Honshu). Moreover, the unsaturation indexes (1.31-1.56) of five grape musts from the cold region showed significant differences among varieties, corresponding to the grapevines' degree of cryotolerance. The FA levels (610-6,610 nmol/100 mL) of all wines were extremely low (1.2-12.8%) compared with those of must, but the FA compositions were similar to those of must. Additionally, significant amounts of FAEE, possibly derived from yeast activity, were found in wines by using a solid-phase extraction method. The amounts of FAEE in wine significantly differed among samples (245-904 nmol/100 mL) and were inversely correlated with the percentage of linoleic acid in musts (R = -0.883). Thus, higher linoleic acid levels in must may be related to lower FAEE formation by yeast.

Effects of Dietary Plant-Origin Glucosylceramide on Bowel Inflammation in DSS-Treated Mice.

The effects of dietary plant-origin glucosylceramide (GlcCer) on symptoms similar to those of inflammatory bowel diseasewere investigated in dextran sulfate sodium salt (DSS)-treated mice. Dietary GlcCer suppressed decreases in body weight due to DSS administration. To determine its effects on the colon, we examined its surface under a microscope following toluidine blue staining. Dietary GlcCer decreased DSS-induced chorionic crypt injury and elevated myeloperoxidase levels. Moreover, dietary GlcCer significantly suppressed the production of cytokines by the intestinal mucosa. These results provide evidence for the suppression of DSS-induced inflammation by dietary GlcCer.

Cytoprotective Effects of Lysophospholipids from Sea Cucumber Holothuria atra.

Lysophospholipids are important signaling molecules in animals and metazoan cells. They are widely distributed among marine invertebrates, where their physiological roles are unknown. Sea cucumbers produce unique lysophospholipids. In this study, two lysophospholipids were detected in Holothuria atra for the first time, lyso-platelet activating factor and lysophosphatidylcholine, with nuclear magnetic resonance and liquid chromatography-time-of-flight mass spectrometric analyses. The lipid fraction of H. atra contained lyso-platelet activating factor and lysophosphatidylcholine, and inhibited H2O2-induced apoptosis in the macrophage cell line J774A.1. The antioxidant activity of the lysophospholipid-containing lipid fraction of H. atra was confirmed with the oxygen radical absorbance capacity method. Our results suggest that the lysophospholipids from H. atra are potential therapeutic agents for the inflammation induced by oxidative stress.

Determination of carbon isotopic measurement conditions for ceramide in skin using gas chromatography-combustion-isotope ratio mass spectrometry.

The ceramide (Cer) content of skin and glucosylceramide (GlcCer) intake affect skin moisture conditions, but their mutual relation in skin remains unclear. For clarification of that mutual relation, carbon stable isotopes ((12)C and (13)C) are useful as a tracer. However, carbon isotopic measurement has not been applied to the study of clarifying their skin moisturizing effects. Therefore, we used gas chromatography / combustion / isotope ratio mass spectrometry (GC-C-IRMS) to ascertain the appropriate conditions for carbon isotopic measurements using synthesized Cer (SCer) in substitution for very low concentrations of Cer in skin. SCer was derivatized to trimethylsilylated SCer (TMS-SCer) quantitatively using N-trimethylsilylimidazole (TMSI) depending on the amount of SCer. The derivatization rates were 75-85%. Excess TMSI was removed using three cycles of hexane-water distribution. Under these conditions, carbon isotopic measurements of TMS-SCer conducted using GC-C-IRMS showed high repeatability and good inter-day variation (S.D. < 0.3‰). The carbon stable isotope ratio value (δ(13)C) of SCer calculated using a mass balance equation was compared with δ(13)C of underivatized SCer, which was regarded as the actual δ(13)C of SCer obtained using sealed tube combustion method. The difference between the calculated δ(13)C of SCer and δ(13)C of the underivatized SCer depended on the TMSI reagent supplier and on the number of hydroxyl groups to be derivatized in SCer. For accurate δ(13)C of Cer in skin using GC-C-IRMS, the measured δ(13)C of a target TMS-Cer must be calculated using a correction factor representing the difference in δ(13)C of underivatized standard SCer from that of TMS-standard SCer having a structure resembling that of the target Cer in skin. In addition, we show that the same lot of TMSI reagent from a specific supplier must be used throughout the experiments.

The Effect of Oral Intake of Low-Temperature-Processed Whey Protein Concentrate on Colitis and Gene Expression Profiles in Mice.

Inflammatory bowel disease (IBD) is an autoimmune disease of unknown etiology and can lead to inflammation and cancer. Whey proteins contain many bioactive peptides with potential health benefits against IBD. We investigated the effect of low-temperature-processed whey protein concentrate (LWPC) on the suppression of IBD by using a dextran sodium sulfate (DSS)-induced colitis model in BALB/c mice. Oral intake of LWPC resulted in improved recovery of body weight in mice. Histological analysis showed that the epithelium cells of LWPC-treated mice were healthier and that lymphocyte infiltration was reduced. The increase in mucin due to the LWPC also reflected reduced inflammation in the colon. Transcriptome analysis of the colon by DNA microarrays revealed marked downregulation of genes related to immune responses in LWPC-fed mice. In particular, the expression of interferon gamma receptor 2 (Ifngr2) and guanylate-binding proteins (GBPs) was increased by DSS treatment and decreased in LWPC-fed mice. These findings suggest that LWPCs suppress DSS-induced inflammation in the colon by suppressing the signaling of these cytokines. Our findings suggest that LWPCs would be an effective food resource for suppressing IBD symptoms.

Distribution of unusual cholesterol precursors, 4-methyl- and 4, 4-dimethylsterols with δ8 unsaturation, in gonads of marine archaeogastropods.

In the present study, we show that four unusual cholesterol precursors, 5α-cholesta-8,14-dien-3ß-ol, 4-methyl-5α-cholesta-8,24-dien-3ß-ol (4-methylzymosterol), 4,4,14-trimethyl-5α-cholesta-8,24-dien-3ß-ol (lanosterol), and 4,4-dimethyl-5α-cholesta-8,24-dien-3ß-ol (4,4-dimethylzymosterol) are present in testes of the limpet Cellana grata, which is part of one of the most primitive gastropod families (Nacelllidae Family). The distribution of these sterols in testes and ovaries of four dominant species of limpets, Cellana grata, Cellana toreuma, Nipponacmea concinna, and Nipponacmea fuscoviridis, was examined by capillary gas chromatography-mass spectrometry. Based on the data, we discuss about the biological roles and possible application as bio-resources of these sterols. This is the first identification of 5α-cholesta-8, 14-dien-3ß-ol, 4-methylzymosterol, and 4,4-dimethylzymosterol in marine invertebrate gonads.