[Tetramine Contents in the Salivary Glands from 16 Species of Marine Carnivorous Gastropods Collected along Japanese Coasts].

Tetramine (tetramethyl ammonium ion), a neurotoxin, is present at high levels in the salivary glands of buccinid gastropods and is responsible for human intoxication due to consumption of the gastropods. We used LC-MS/MS to examine the tetramine contents of salivary glands from 16 species of carnivorous gastropods collected along Japanese coasts. Tetramine was detected in all specimens except for Babylonia japonica. High levels of tetramine were detected in whelks, Neptunea lamellosa (1,380-9,410 µg/g of salivary gland) and N. purpurea (1,190-7,400 µg/g of salivary gland). Although consumption of N. lamellosa is well-known cause of foodborne tetramine poisoning, it was newly discovered that N. purpurea has tetramine. In addition, we found 7 other species of gastropods containing tetramine: Siphonalia cassidariaeformis (117-135 µg/g), S. fusoides (204 µg/g), Buccinum inclytum (2.94-3.40 µg/g), and B. aniwanum (0.700 µg/g) of the family Buccinidae, and Fusinus perplexus (397 µg/g), F. ferrugineus (105 µg/g), and F. forceps salisburyi (67.5 µg/g) of the family Fasciolariidae. The present study, together with previous studies, shows that gastropods with salivary glands containing more than 1,000 µg tetramine/g of salivary gland, including the genus Neptunea as well as Fusitriton oregonesis and Hemifusus tuba, carry a high risk of tetramine poisoning, and their salivary glands should be removed before consumption to prevent food poisoning.

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.

Enzymatic properties and primary structures of hyaluronidases from two species of lionfish (Pterois antennata and Pterois volitans).

Lionfish are representative venomous fish, having venomous glandular tissues in dorsal, pelvic and anal spines. Some properties and primary structures of proteinaceous toxins from the venoms of three species of lionfish, Pterois antennata, Pterois lunulata and Pterois volitans, have so far been clarified. Our recent survey established the presence of hyaluronidase, presumably a toxin-spreading factor, in the venoms of P. antennata and P. volitans. This prompted us to examine enzymatic properties and primary structures of lionfish hyaluronidases. The hyaluronidases of P. antennata and P. volitans were shown to be optimally active at pH 6.6, 37°C and 0.1 M NaCl and specifically active against hyaluronan. These enzymatic properties are almost the same as those of stonefish hyaluronidases. The primary structures (483 amino acid residues) of the lionfish hyaluronidases were elucidated by a cDNA cloning strategy using degenerate primers designed from the reported amino acid sequences of the stonefish hyaluronidases. Both lionfish hyaluronidases share as high as 99.6% of sequence identity with each other and also considerably high identities (72-77%) with the stonefish hyaluronidases but rather low identities (25-40%) with other hyaluronidases from mammals and venomous animals. In consistent with this, phylogenetic tree analysis revealed that the lionfish hyaluronidases, together with the stonefish hyaluronidases, form a cluster independently of other hyaluronidases. Nevertheless, the lionfish hyaluronidases as well as the stonefish hyaluronidases almost maintain structural features (active site, glyco_hydro_56 domain and cysteine location) observed in other hyaluronidases.

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.

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.

Comparative Evaluation of Fatty Acid Composition, Tocopherols, and Volatile Compounds of Walnut Oil between Juglans mandshurica Maxim. var. sachalinensis (Komatsu) Kitam and J. regia L.

We investigated the fatty acid composition and regiospecific distribution of triacylglycerol in Juglans mandshurica Maxim. var. sachalinensis (Komatsu) Kitam and Juglans regia L. oils. Significant differences are observed in the fatty acid compositions and regiospecific distribution of triacylglycerol in both oils. In addition, we measured volatile compounds and tocopherol content in two walnut oils. In results of volatile compound analysis, vanillin is specifically detected from J. mandshurica var. sachalinensis oil, and was not detected in J. regia L. oil. Notably, γ-tocopherol content in the J. mandshurica var. sachalinensis oil was significantly higher than J. regia L. oil.

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.

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.

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.

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.

Evaluation of Hepatic Uptake of OATP1B Substrates by Short Term-Cultured Plated Human Hepatocytes: Comparison With Isolated Suspended Hepatocytes.

Hepatic uptake clearance has been measured in suspended human hepatocytes (SHH). Plated human hepatocytes (PHH) after short-term culturing are increasingly employed to study hepatic transport driven mainly by its higher throughput. To know pros/cons of both systems, the hepatic uptake clearances of several organic anion transporting polypeptide 1B substrates were compared between PHH and SHH by determining the initial uptake velocities or through dynamic model-based analyses. For cerivastatin, pitavastatin and rosuvastatin, initial uptake clearances (PSinf) obtained using PHH were comparable to those using SHH, while cell-to-medium concentration (C/M) ratios were 2.7- to 5.4-fold higher. For pravastatin and dehydropravastatin, hydrophilic compounds with low uptake/cellular binding, their PSinf and C/M ratio in PHH were 1.8- to 3.2-fold lower than those in SHH. These hydrophilic substrates are more prone to wash-off during the uptake study using PHH, which may explain the apparently lower uptake than SHH. The C/M ratios obtained using PHH were stable over an extended time, making PHH suitable to estimate the C/M ratios and hepatocyte-to-medium unbound concentration ratios (Kp,uu). In conclusion, PHH is useful in evaluating hepatic uptake/efflux clearances and Kp,uu of OATP1B substrates in a high-throughput manner, however, a caution is warranted for hydrophilic drugs with low uptake/cellular binding.

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.

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs.

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (Kp,uu) at the steady-state.

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.

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.

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".

Occurrence of a stonefish toxin-like toxin in the venom of the rabbitfish Siganus fuscescens.

Rabbitfish belonging to the order Perciformes are well-known venomous fish that are frequently involved in human accidents. However little research has been done into either the whole venom toxicities or the structures and properties of their venom toxins. In this study, we first examined biological activities of the crude venom extract prepared from dorsal spines of Siganus fuscescens, a rabbitfish most commonly found along the coasts of Japan. As a result, the crude venom extract was shown to have mouse-lethal activity, hemolytic activity against rabbit erythrocytes, edema-forming activity and nociceptive activity, similar to the known scorpaeniform fish toxins (stonefish toxins and their analogues). Then, the primary structure of the S. fuscescens toxin was successfully elucidated by the same cDNA cloning strategy as previously employed for the toxins of some scorpaeniform fish (lionfish, devil stinger and waspfish). The S. fuscescens toxin is obviously an analogue of stonefish toxins, being composed of two kinds of subunits, an α-subunit of 703 amino acid residues and a ß-subunit of 699 amino acid residues. Furthermore, the genes encoding both subunits were cloned from genomic DNA and shown to have an architecture of three exons and two introns, as reported for those of the scorpaeniform fish toxins. This study is the first to demonstrate the occurrence of stonefish toxin-like toxins in perciform fish.

A novel function of vitellogenin subdomain, vWF type D, as a toxin-binding protein in the pufferfish Takifugu pardalis ovary.

Marine pufferfish of the Tetraodontidae family contain high levels of tetrodotoxin (TTX) in the liver and ovary. TTX is suggested to transfer from the liver to the ovary in female pufferfish during maturation. TTX in pufferfish eggs may act as a repellent against predators and as a sexual pheromone to attract male pufferfish. The toxification mechanism of the pufferfish ovary is poorly understood. Here we evaluated the chemical form of TTX and its related substances in the ovary of the panther pufferfish Takifugu pardalis by LC-ESI/MS. TTX and its analogs 4-epi-TTX, 4, 9-anhydroTTX, deoxyTTX, dideoxyTTX, and trideoxyTTX were detected in a low molecular weight fraction by Sephacryl S-400 column chromatography. The finding of an unknown TTX-related substance in a high molecular weight fraction from the Sephacryl S-400 column suggested the occurrence of toxin-binding protein in the ovary. The toxin-binding protein in the ovary was purified by ion-exchange HPLC, gel filtration HPLC, and SDS-PAGE. Amino acid sequencing and cDNA cloning revealed that the toxin-binding protein, TPOBP-10 (Takifugu pardalis ovary toxin-binding protein with a molecular mass of 10 kDa) was homologous with the predicted vitellogenin-1-like protein [Takifugu rubripes] subdomain, a von Willebrand factor type D domain. TPOBP-10 mRNA was highly expressed in the ovary and liver and less in other organs of female individuals based on RT-PCR. These findings reveal a novel function of the vitellogenin subdomain as binding with TTX-related substances, and its involvement in the toxification of the pufferfish ovary.