Abstract D-allulose, D-sorbose and D-tagatose are D-fructose isomers that are called rare sugars. These rare sugars have been studied intensively in terms of biological production and food application as well as physiological effects. There are limited papers with regard to the transporters mediating the intestinal absorption of these rare sugars. We examined whether these rare sugars are absorbed via sodium-dependent glucose cotransporter 1 (SGLT1) as well as via GLUT type 5 (GLUT5) using rats. High-fructose diet fed rats, which express more intestinal GLUT5, exhibited significantly higher peripheral concentrations, Cmax and AUC_{0–180 min} when D-allulose, D-sorbose and D-tagatose were orally administrated. KGA-2727, a selective SGLT1 inhibitor, did not affect the peripheral and portal vein concentrations and pharmacokinetic parameters of these rare sugars. The results suggest that D-allulose, D-sorbose and D-tagatose are likely transported via GLUT5 but not SGLT1 in rat small intestine.

The oral administration of pure monosaccharides is effective for improving intestinal function such as nutrient digestion and absorption. However, day-to-day diets tend not to include high purity monosaccharides for intestinal health. Honey possesses large amounts of monosaccharides including glucose and fructose in the same ratio. In this study, we have evaluated the nutritional properties of honey and examined the effects of its oral ingestion on the recovery of intestinal function in the total parenteral nutrition (TPN) rat model. It was observed that honey remarkably recovered the function of the small intestine including the villous morphology, nutrient digestion, and absorption capabilities. In particular, the expression of disaccharidase was significantly enhanced by the ingestion of honey after TPN treatment. Therefore, oral intake of honey is effective in recovering and maintaining small intestinal functions and can potentially be used as a supplement for promoting small intestinal function recovery.

d-Allose is the C3 epimer of d-glucose and has been reported to have beneficial health effects. The transporter mediating intestinal transport of d-allose is unknown. We examined whether d-allose is absorbed via sodium-dependent glucose cotransporter 1 (SGLT1) as well as via glucose transporter type 5 (GLUT5) using rats. For examination of absorption via SGLT1, KGA-2727, an SGLT1-specific inhibitor, and d-allose were orally administered. KGA-2727 blocked the increase of plasma d-allose levels and suppressed them throughout the experiment (0-180 min), whereas without KGA-2727, the plasma d-allose levels peaked at around 60-90 min. For examination of absorption via GLUT5, rats were fed a high-fructose diet for 3weeks to increase the abundance and activity of GLUT5 in the small intestine. High-fructose diet-fed rats did not exhibit significant changes in the plasma d-allose levels compared to control rats fed a high-glucose diet. These results indicate that SGLT1 but not GLUT5 mediates the intestinal absorption of d-allose.

OBJECTIVE: To evaluate blood pressure (BP)-lowering effects of Umezu polyphenols, polyphenols contained in Japanese plums, in a community-based sample by double-masked and placebo-controlled design. METHODS: Seventy-two Japanese community-dwellers who were interested in prevention or control of their BP (preferably high-normal BP or grade I hypertension) but without antihypertensive medication were randomized into Umezu polyphenols or placebo groups. Each subject took 800 mg/day of Umezu polyphenols or placebo for 12 weeks, followed by a 2-week washout period. Their home and office BP were monitored for 14 weeks in a double-masked manner. We analyzed 56 subjects who met the inclusion criteria. RESULTS: Home BP increased gradually in both the groups during the intervention period, while diastolic office BP insignificantly decreased in the intervention group. During the washout period, home systolic BP in the morning elevated only in the intervention group. CONCLUSIONS: The study failed to collect consistent evidence of a clear persistent hypotensive effect of Umezu polyphenols.

High intakes of fructose are associated with metabolic diseases, including hypertriglyceridemia and intestinal tumor growth. Although small intestinal epithelia consist of many different cell types, express lipogenic genes, and convert dietary fructose to fatty acids, there is no information on the identity of the cell type(s) mediating this conversion and on the effects of fructose on lipogenic gene expression. We hypothesized that fructose regulates the intestinal expression of genes involved in lipid and apolipoprotein synthesis, that regulation depends on the fructose transporter solute carrier family 2 member a5 [Slc2a5 (glucose transporter 5)] and on ketohexokinase (Khk), and that regulation occurs only in enterocytes. We compared lipogenic gene expression among different organs from wild-type adult male C57BL mice consuming a standard vivarium nonpurified diet. We then gavaged twice daily for 2.5 d fructose or glucose solutions (15%, 0.3 mL per mouse) into wild-type, Slc2a5-knockout (KO), and Khk-KO mice with free access to the nonpurified diet and determined expression of representative lipogenic genes. Finally, from mice fed the nonpurified diet, we made organoids highly enriched in enterocyte, goblet, Paneth, or stem cells and then incubated them overnight in 10 mM fructose or glucose. Most lipogenic genes were significantly expressed in the intestine relative to the kidney, liver, lung, and skeletal muscle. In vivo expression of Srebf1, Acaca, Fasn, Scd1, Dgat1, Gk, Apoa4, and Apob mRNA and of Scd1 protein increased (P < 0.05) by 3- to 20-fold in wild-type, but not in Slc2a5-KO and Khk-KO, mice gavaged with fructose. In vitro, Slc2a5- and Khk-dependent, fructose-induced increases, which ranged from 1.5- to 4-fold (P < 0.05), in mRNA concentrations of all these genes were observed only in organoids enriched in enterocytes. Fructose specifically stimulates expression of mouse small intestinal genes for lipid and apolipoprotein synthesis. Secretory and stem cells seem incapable of transport- and metabolism-dependent lipogenesis, occurring only in absorptive enterocytes.

Fructose is metabolized in the cytoplasm by the enzyme ketohexokinase (KHK), and excessive consumption may affect bone health. Previous work in calcium-restricted, growing mice demonstrated that fructose disrupted intestinal calcium transport. Thus, we hypothesized that the observed effects on bone were dependent on fructose metabolism and took advantage of a KHK knockout (KO) model to assess direct effects of high plasma fructose on the long bones of growing mice. Four groups (n = 12) of 4-week-old, male, C57Bl/6 background, congenic mice with intact KHK (wild-type, WT) or global knockout of both isoforms of KHK-A/C (KHK-KO), were fed 20% glucose (control diet) or fructose for 8 weeks. Dietary fructose increased by 40-fold plasma fructose in KHK-KO compared to the other three groups (p < 0.05). Obesity (no differences in epididymal fat or body weight) or altered insulin was not observed in either genotype. The femurs of KHK-KO mice with the highest levels of plasma fructose were shorter (2%). Surprisingly, despite the long-term blockade of KHK, fructose feeding resulted in greater bone mineral density, percent volume, and number of trabeculae as measured by µCT in the distal femur of KHK-KO. Moreover, higher plasma fructose concentrations correlated with greater trabecular bone volume, greater work-to-fracture in three-point bending of the femur mid-shaft, and greater plasma sclerostin. Since the metabolism of fructose is severely inhibited in the KHK-KO condition, our data suggest mechanism(s) that alter bone growth may be related to the plasma concentration of fructose.

d-Allulose has been reported to have beneficial health effects. However, the transport system(s) mediating intestinal d-allulose transport has not yet been clearly identified. The aim of this study was to investigate whether intestinal d-allulose transport is mediated by glucose transporter type 5 (GLUT5). When d-allulose alone was gavaged, plasma d-allulose levels were dramatically higher in rats previously fed fructose. This suggests enhanced intestinal d-allulose absorption paralleled increases in GLUT5 expression observed only in fructose-fed rats. When d-allulose was gavaged with d-fructose, previously observed increases in plasma d-allulose levels were dampened and delayed, indicating d-fructose inhibited transepithelial d-allulose transport into plasma. Tracer D-[14C]-fructose uptake rate was reduced to 54.8% in 50 mM d-allulose and to 16.4% in 50 mM d-fructose, suggesting d-allulose competed with D-[14C]-fructose and the affinity of d-allulose for GLUT5 was lower than that of d-fructose. GLUT5 clearly mediates, likely at lower affinity relative to d-fructose, intestinal d-allulose transport.

Background: Mammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. Small intestinal epithelial permeability is ascribed primarily to the properties of TJs between adjoining enterocytes (ENTs), because there is almost no information on TJ composition and the paracellular permeability of nonenterocyte cell types that constitute a small but significant fraction of the intestinal epithelia. Results: Here we directed murine intestinal crypts to form specialized organoids highly enriched in intestinal stem cells (ISCs), absorptive ENTs, secretory goblet cells, or Paneth cells. The morphological and morphometric characteristics of these cells in organoids were similar to those in vivo. The expression of certain TJ proteins varied with cell type: occludin and tricellulin levels were high in both ISCs and Paneth cells, while claudin-1, -2, and -7 expression was greatest in Paneth cells, ISCs, and ENTs, respectively. In contrast, the distribution of claudin-15, zonula occludens 1 (ZO-1), and E-cadherin was relatively homogeneous. E-cadherin and claudin-7 marked mainly the basolateral membrane, while claudin-2, ZO-1, and occludin resided in the apical membrane. Remarkably, organoids enriched in ENTs or goblet cells were over threefold more permeable to 4 and 10 kDa dextran compared to those containing stem and Paneth cells. The TJ-regulator larazotide prevented the approximately tenfold increases in dextran flux induced by the TJ-disrupter AT1002 into organoids of different cell types, indicating that this ZO toxin nonselectively increases permeability. Forced dedifferentiation of mature ENTs results in the reacquisition of ISC-like characteristics in TJ composition and dextran permeability, suggesting that the post-differentiation properties of TJs are not hardwired. Conclusions: Differentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellular pathway of epithelia.

Nutrient sensing triggers responses by the gut-brain axis modulating hormone release, feeding behavior and metabolism that become dysregulated in metabolic syndrome and some cancers. Except for absorptive enterocytes and secretory enteroendocrine cells, the ability of many intestinal cell types to sense nutrients is still unknown; hence we hypothesized that progenitor stem cells (intestinal stem cells, ISC) possess nutrient sensing ability inherited by progenies during differentiation. We directed via modulators of Wnt and Notch signaling differentiation of precursor mouse intestinal crypts into specialized organoids each containing ISC, enterocyte, goblet, or Paneth cells at relative proportions much higher than in situ as determined by mRNA expression and immunocytochemistry of cell type biomarkers. We identified nutrient sensing cell type(s) by increased expression of fructolytic genes in response to a fructose challenge. Organoids comprised primarily of enterocytes, Paneth, or goblet, but not ISC, cells responded specifically to fructose without affecting nonfructolytic genes. Sensing was independent of Wnt and Notch modulators and of glucose concentrations in the medium but required fructose absorption and metabolism. More mature enterocyte- and gobletenriched organoids exhibited stronger fructose responses. Remarkably, enterocyte organoids, upon forced dedifferentiation to reacquire ISC characteristics, exhibited a markedly extended lifespan and retained fructose sensing ability, mimicking responses of some dedifferentiated cancer cells. Using an innovative approach, we discovered that nutrient sensing is likely repressed in progenitor ISCs then irreversibly derepressed during specification into sensing-competent absorptive or secretory lineages, the surprising capacity of Paneth and goblet cells to detect fructose, and the important role of differentiation in modulating nutrient sensing. NEW & NOTEWORTHY Small intestinal stem cells differentiate into several cell types transiently populating the villi. We used specialized organoid cultures each comprised of a single cell type to demonstrate that 1) differentiation seems required for nutrient sensing, 2) secretory goblet and Paneth cells along with enterocytes sense fructose, suggesting that sensing is acquired after differentiation is triggered but before divergence between absorptive and secretory lineages, and 3) forcibly dedifferentiated enterocytes exhibit fructose sensing and lifespan extension.

カンキツリモノイドであるリモニン，ノミリンの脂質代謝に与える影響を検討した。Sprague-Dawley系雄性ラットに高脂肪・高コレステロール食を給餌し，リモニンまたはノミリンを50mg/kgBW/day経口投与し，5週間飼育した。摂餌量，体重，肝臓および内臓脂肪重量，血糖値，血中脂質，肝臓中脂質について，ノミリン群でのLDL-Chol上昇を除いて，有意な差は認められなかった。ノミリン投与によりChrebp，Pparα，Fxr，Lxrαの発現量は有意に低下し，Srebflc，Abcalの発現量も低下傾向が示された。脂肪酸生合成（Acaca，Fasn，Scd1，Me1)，β酸化（Cpt1a），コレステロール代謝（Hmgcr，Ldlr，Srebf2，Acat2），胆汁酸代謝（Shp，Lrh-1，Hnf4a，Cyp7a1，Cyp27a1)，トリグリセリド合成（Dgat1，Dgat2）,リポタンパク会合（Mttp），脂質輸送（Abcg5，Abcg8，Abcb11，Cd36）に関連する有意な差は認められなかった。リモニン投与では，いずれの遺伝子発現にも差は認められなかった。

Medications or lifestyle changes to prevent or improve hypertension often press considerable efforts on patients suffering from mild hypertension. Capsules including Umezu polyphenols (UP), polyphenols in Japanese plums, may help them to control their blood pressure (BP). The aim of this study is to evaluate the effectiveness of UP on BP and its safety. A total of 15 healthy workers without antihypertensive medication who had some concerns about their BP, preferably normal-high BP or hypertension level 1, were randomized in a double-blind manner into UP ingesting and placebo groups. Each subject was instructed to take four capsules daily for 12 weeks (daily UP dose, 800 mg for the UP ingesting group; and 0 mg for the placebo group). These subjects were followed for 12 weeks, and their BP both at home and at the examination site, as well as self-perceived quality-of-life outcomes and possible side effects, was monitored during that period. Group x time interactions on BP changes were examined. All of the 15 subjects completed the 12-week intervention trial. The BP changes did not significantly differ between the UP ingesting and placebo groups, neither at the examination site nor at home. But during the study period, no adverse effects were observed. No remarkable effect of UP on BP was observed. However, a higher dose of UP was confirmed safe and high in adherence in this 12-week randomized controlled trial. Its effect on BP and other outcomes shall be confirmed in a larger sample.

ウメ由来ポリフェノール抽出物(JAPE)を用いて，ラット小腸二糖類水解酵素への阻害活性ならびに食後血糖値上昇抑制作用を検討した。JAPEは，マルターゼ，グルコアミラーゼ，ラクターゼに対して阻害作用を示し，阻害様式はそれぞれ混合型非拮抗阻害，不拮抗阻害，拮抗阻害であった。スクラーゼ，イソマルターゼ，トレハラーゼに対しては阻害作用を示さなかった。ラットへの経口糖負荷試験では，マルトース，スクロース，デンプン投与時にJAPEによる有意な血糖値上昇抑制作用が認められ，グルコース投与時には，有意な影響は認められなかったことから，JAPEはグルコース吸収には影響せず，血糖値上昇抑制作用は二糖類水解酵素阻害によることが示唆された。AUC 0-120minは変化がなかったことから，JAPEはグルコース吸収量には影響しなかった。JAPEに含まれるヒドロキシ桂皮酸誘導体が二糖類水解酵素阻害にある程度関与していると考えられる。これらの知見より，JAPEは2型糖尿病や肥満などの生活習慣病のリスクを減らす食品素材として有用である可能性が示された。

The fruit of mume, Japanese apricot (Prunus mume Sieb. et Zucc.), was evaluated for its phenolics content, high performance liquid chromatography (HPLC) pro- file and antioxidative activities. The phenolics content of mume fruit was relatively high, the flesh of fully matured fruit containing up to 1% of phenolics on a dry weight basis. Reflecting such a high content of phenolics, the ORAC (oxygen radical absorbance capacity) value for mume fruit flesh showed high values, ranging from 150 to 320 μmol/g Trolox equivalent, depending upon the stage of maturation. 5-O-Caffeoylqunic acid (chlorogenic acid), 3-O-caffeoylquinic acid and tetra-O-acylated sucrose-related compounds were isolated from the flesh of mume fruit, although many unknown peaks were also apparent in the HPLC chromatogram. An alkali hydrolysate comprised four main phenolic acids, caffeic acid, cis/trans-p-coumaric acid and ferulic acid. No flavonoids were observed in the analysis. These results suggest that the majority of phenolics in mume fruit were hydroxycinnamic acid derivatives.

A simplified method was developed for the simultaneous determination of oxytetracycline, tetracycline, chlortetracycline, and doxycycline in milk. Isolation of the target compounds was performed using an Ultrafree-MC/PL centrifugal ultrafiltration device without prior sample preparation. Analyses were carried out via high-performance liquid chromatography using a Discovery HS F5 column with a gradient mobile phase which consisted of 30 mM citric acid solution (pH 3, in water) and acetonitrile (60:40 -> 40:60, v/v). Recovery of the target compounds from spiked samples at four levels (0.05, 0.1, 0.2, and 0.5 mu g/mL) was higher than 87%, with relative standard deviations of less than 6%. Limits of quantification ranged from 0.01 to 0.04 mu g/mL. (C) 2010 Elsevier Ltd. All rights reserved.

The mitochondrial (mt) DNA C5178A and A10398G polymorphisms have been reported to be associated with mental disorders such as bipolar disorder. However, the effects of these polymorphisms on temperament in healthy people are poorly understood. Evaluating healthy subjects can have the advantage of providing new strategies for maintaining psychological health and preventing mental illness. We examined the association between mtDNA polymorphisms and temperament in Japanese students. There was no significant difference in examined temperament when analysed by genotypes, 5178-10398 haplotypes, or sex. The subgroup analysis based on sex indicated that there was an interactive effect of the mtDNA A10398G polymorphism and sex on anxiety and obsession. This finding is preliminary and cannot exclude the possibility of false-positive due to small sample size (144 subjects) and multiple statistical testing. Further studies involving a larger sample size or other ethnic groups are necessary to confirm that mtDNA A10398G polymorphism can be a genetic factor for temperament.

Multiresidue analysis of six sulphonamides (SAs) (sulphadiazine, sulphadimidine, sulphamonomethoxine, sulphamethoxazole, sulphadimethoxine, and sulphaquinoxaline) in meat (beef, pork, and chicken) using liquid chromatography (LC)-mass spectrometry (MS) with photodiode array (PDA) detection is presented. The sample preparation is carried out by normal-phase matrix solid-phase dispersion (MSPD) with an ethanol solution. The LC-MS determination is performed using a Mightysil RP-4 GP column and an isocratic mobile phase of 0.3% (v/v) acetic acid solution (pH 3.4, in water)-ethanol (83:17, v/v) with an atmospheric pressure chemical ionization (APCI) MS on positive-ion mode. Average recoveries spiked at 0.05-0.5 ppm for each drug were higher than 90% with relative standard deviations between 1% and 6%. In all the processes, no toxic solvents were used at all. (C) 2005 Elsevier Ltd. All rights reserved.

Simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their N-4-acetyl metabolites in eggs using restricted-access media high performance liquid chromatography (RAM-HPLC) with a photo-diode array detector is developed. The target compounds are extracted by a handheld ultrasonic homogenizer with saturated ammonium sulfate solution followed by centrifugation. The separation is performed by a Hisep shielded hydrophobic phase column, isocratic elution with 0.3% acetic acid solution (pH 2.9, in water)-ethanol (75:25, v/v). Average recoveries from samples spiked at 0.1-1.0 ppm for each drug were > 91% with relative standard deviations within 4%. The limits of quantitation were <= 0.08 ppm. No hazardous-chemicals were used in all the processes. (c) 2006 Elsevier Ltd. All rights reserved.

The authors have developed a method of determining zeranol residues in bovine tissues (muscle, kidney, liver, and fat) without using toxic chemicals, organic solvents, and reagents in sample preparation and reversed-phase high performance liquid chromatography (HPLC) separation. Isolation is achieved by homogenization using a handheld ultrasonic homogenizer, which is easy to use and portable, followed by solid-phase extraction with an anion exchanger. For determination and identification of zeranol, the HPLC method uses an isocratic mobile phase and a photodiode-array detector. The authors obtained average recoveries of zeranol > 84% with relative standard deviations around 3.0%-4.8% in spiked samples. The total analytical time and quantitation limit were < 40 min/sample and 0.04 mu g/g, respectively.

The authors have developed a method of determining zeranol residues in bovine tissues (muscle, kidney, liver, and fat) without using toxic chemicals, organic solvents, and reagents in sample preparation and reversed-phase high performance liquid chromatography (HPLC) separation. Isolation is achieved by homogenization using a handheld ultrasonic homogenizer, which is easy to use and portable, followed by solid-phase extraction with an anion exchanger. For determination and identification of zeranol, the HPLC method uses an isocratic mobile phase and a photodiode-array detector. The authors obtained average recoveries of zeranol > 84% with relative standard deviations around 3.0%-4.8% in spiked samples. The total analytical time and quantitation limit were < 40 min/sample and 0.04 mu g/g, respectively.

Presented is a sample preparation-high performance liquid chromatography-diode array detection method for the simultaneous determination of five sulphonamides in milk. The procedure, performed under 100% aqueous conditions, does not use organic solvents or reagents and is, therefore, harmless to both humans and the environment.

A simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N-4-acetyl metabolites in chicken plasma, muscle, liver, and eggs using gradient high-performance liquid chromatography (HPLC) with a photo-diode array detector is developed. All the compounds are extracted by a handheld ultrasonic homogenizer with ethanol followed by centrifugation. The separation is performed by a reversed-phase C4 column with a gradient elution (ethanol: 1% (v/v) acetic acid, v/v; 10: 90 --> 20:80). Average recoveries from samples spiked at 0.1-1.0 mu g g(-1) or mu g ml(-1) for each drug were >90% with relative standard deviations within 4%. The limits of quantitation were <30 ng g(-1) or ng ml(-1). (C) 2005 Elsevier B.V. All rights reserved.

An "environmentally-friendly" method has been established for simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their N-4-acetyl metabolites in chicken plasma. The sample is prepared by mixing with 4 mol L-1 ammonium sulfate solution then centrifugation, and analysis is performed by high-performance liquid chromatography (HPLC) on a polyethylene glycol reversed-phase column with 0.001 mol L-1 sodium acetate solution as mobile phase and photodiode-array detection. Average recoveries from samples spiked with 0.1, 0.5, and 1.0 mug mL(-1) of each drug were >78% and relative standard deviations were within 4%. The practical quantitation limits were less than or equal to0.09 mug mL(-1). No organic solvents or hazardous reagents were used at any stage of the analysis.

The authors have developed a method for determining sulfamethazine residue in pork using 100% water in sample preparation and high performance liquid chromatography (HPLC) separation. Sample preparation comprises homogenization with 100% water by means of a handheld ultrasonic homogenizer followed by centrifugal ultrafiltration. HPLC is performed on a reversed-phase C1 column with a 100% water mobile phase and a photodiode-array detector. The average recoveries, total analytical time, and limit of quantitation were less than or equal to 81 % (relative standard deviations were < 5.8%), < 30 min/sample, and 0.09 mug/g, respectively.

A hazardous-chemical free method for simultaneous determination of sulfamonomethoxine (SMM), sulfadimethoxine (SDM), and their N-4-acetyl metabolites in raw milk using shielded column liquid chromatography is developed. The target analytes are extracted by mixing with ethanol-acetic acid (97:3, v/v) followed by centrifugation. The procedure uses a Hisep shielded hydrophobic phase (SUP) column, isocratic elution with 0.1% acetic acid solution (pH 3.1, in water)-ethanol (75:25, v/v), and a photo-diode array detector. Average recoveries from samples spiked at 25-500 ng/ml for each drug were >81% with relative standard deviations within 5%. The limits of quantitation were <25 ng/ml. (C) 2004 Elsevier B.V. All rights reserved.

A simultaneous HPLC determination of six sulfonamides (SAs) [sulfadiazine (SDA), sulfadimidine (SDD), sulfamonomethoxine (SMM), sulfamethoxazole (SMX), sulfadimethoxine (SDM), and sulfaquinoxaline (SQ)] in meat (chicken, beef, and pork) is presented. The sample preparation is carried out by normal-phase matrix solid-phase dispersion (MPSD) with an aqueous ethanol solution. The HPLC determination is performed using a Mightysil RP-4 GP column and an isocratic mobile phase of 2% (v/v) acetic acid solution (pH 2.7, in water)-ethanol (75: 25, v/v) with a photodiode array detector. Average recoveries spiked at 0.05-0.5 ppm for each drug are higher than 85% with standard deviations within 10%. In all the processes, no toxic/harmful solvents are used at all.

The five sulphonamides, sulphadiazine (SDZ), sulphadimidine (SDD), sulphamethoxazole (SMX), sulphamonomethoxine (SMM) and sulphaquinoxaline (SQ), were fed to laying hens at a dietary concentration of 100 mg kg(-1), respectively. On the 7th day after the start of feeding, the drug concentrations in the plasma, muscle and the main tissues involved in egg formation, the liver, and ovary and oviducts (magnum and isthmus plus shell grand) were determined by high-performance liquid chromatography. Dietary sulphonamides were distributed throughout the above tissues. SQ was found at the highest concentration in all tissues, while the reverse was true for SDD. The ratio of SDD concentrations in the main tissues involved in egg formation to that in the plasma were greater than those for the other drugs.

The five sulphonamides, sulphadiazine (SDZ), sulphadimidine (SDD), sulphamethoxazole (SMX), sulphamonomethoxine (SMM) and sulphaquinoxaline (SQ), were fed to laying hens at a dietary concentration of 100 mg kg-1, respectively. On the 7th day after the start of feeding, the drug concentrations in the plasma, muscle and the main tissues involved in egg formation, the liver, and ovary and oviducts (magnum and isthmus plus shell grand) were determined by high-performance liquid chromatography. Dietary sulphonamides were distributed throughout the above tissues. SQ was found at the highest concentration in all tissues, while the reverse was true for SDD. The ratio of SDD concentrations in the main tissues involved in egg formation to that in the plasma were greater than those for the other drugs.

A simplified method for routine monitoring of 7 residual sulfonamides (SAs) (sulfadiazine (SDZ), sulfamerazine (SMR), sulfadimidine (SDD), sulfamonomethoxine (SMM), sulfamethoxazole (SMX), sulfadimethoxine (SDM), and sulfaquinoxaline (SQ)) in milk using high-performance liquid chromatography (HPLC) with a photodiode array detector is described. The spiked and blank samples were cleaned up by using an Ultrafree (R) -MC/PL centrifugal ultrafiltration unit. For determination/identification, a Mightysil (R) RP-4 GP column and a mobile phase of 25% (v/v) ethanol in water with a photodiode array detector were used. Average recoveries from milk samples spiked with 0.05. 0.1, 0.2, and 0.5 mug mL(-1) of each drug were > 82%. The inter- and intra-assay variabilities were 2.0-3.1%. The practical detection limits for 7 SAs were 0.005-0.02 mug mL(-1). The total time and amount of solvent required for the analysis of one sample were < 40 min and <6 mL of ethanol, respectively. No toxic solvents were used.

Simultaneous determination of the six sulfonamides (SAs) sulfadiazine, sulfadimidine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine and sulfaquinoxaline in chicken using matrix solid-phase dispersion (MSPD) with neutral aluminium oxide as an MSPD sorbent and high-performance liquid chromatography (HPLC) is presented. In the present MSPD, six SAs could be isolated by only one step, elution with a 70% (v/v) aqueous ethanol solution, without the sorbent conditioning and the sorbent-tissue matrix washing. For the HPLC determination, a LiChrospher 100 RP-8 and a mixture of 1% acetic acid solution (pH 3.0, in water)-acetonitrile-N,N-dimethylformamide (78:22:5, v/v/v) as the mobile phase with a photodiode array detector were used. Average recoveries were greater than 87.6% with relative standard deviations between 0.5 and 8.6%. The total time and amount of solvent required for the analysis of one sample were <1.5 h and < 12 ml, respectively. (C) 2001 Elsevier Science B.V. All rights reserved.

To examine whether 1,5-anhydroglucitol (AG) is derived from starch degradation in plant tissues, we colorimetrically measured AG contents of germinating amaranth seeds and ripening banana pulp. In both cases, as starch degradation proceeded, AG levels were significantly increased, but were 1,700-5,000 times lower than those of total soluble carbohydrates. alpha -1,4-Glucan lyase activity, which is measured by the 1,5-anhydrofructose (AF) liberated from non-reducing glucose residues of starch or glycogen, was too low to be detected in amaranth or banana by the 3,5-dinitrosali-cylic acid method. On the other hand, AF reductase, which reduces AF to AG, was detected in germinating amaranth seeds and banana pulp. Thus, the increases in AG levels are conceived to be derived from starch breakdown, although further investigation is needed to answer whether the starch degradation pathway via alpha -1,4-glucan lyase/AF reductase exists in plant tissues.

以下の2つの実験を実施した。 実験1 SD系雄ラットに①Glu＋大豆油、②Fru＋大豆油、③Glu＋魚油、④Fru＋魚油を含む飼料を4週間給餌し、各種パラメーターを解析した。肝臓重量はFru群で有意に高く、副睾丸脂肪および腎周囲脂肪重量は魚油群で有意に低かった。血漿トリグリセリド濃度は交互作用がある傾向が見られ、他の3群と比較してFru＋大豆油群で有意に高くなった。肝臓ではFasn、Acacaの発現がFru＋大豆油群で有意に高く、Acox1の発現は魚油群で有意に高かった。腸間膜脂肪ではAcox1の発現は魚油群で有意に低かった。また、Lpl発現はFru＋魚油群が他の3群と比較して有意に低かった。以上の結果より、魚油の作用としてよく知られている脂質代謝改善作用は高Fru食給餌下でも観察されるが、腸間膜脂肪では脂肪酸酸化はむしろ低下していることが分かった。 実験2 SD系雄ラットに①Glu＋ラード、②Fru＋ラード、③Glu＋中鎖脂肪、④Fru＋中鎖脂肪を含む飼料を4週間給餌し、各種パラメーターを解析した。最終体重は、ラード群に高い傾向が見られた。血漿中Glu濃度はMCT群、Fru群で有意に高く、血漿中トリグリセリド(TG)濃度はラード群、Fru群で有意に高かった。肝臓重量はFru群が有意に高く、Acaca，Fasn等の脂肪酸合成系関連遺伝子発現はMCT群で有意に高かった。腸間膜脂肪重量に差は見られなかったが、Fasn, Scd1, Acaca, Me1, Chrebp等の脂肪酸合成系関連遺伝子発現がMCT群、Glu群で有意に高かった。これらの結果から、腸間膜脂肪では、Fruによる脂肪合成促進作用、MCTによる脂肪酸酸化作用は見られず、むしろGluまたはMCTで脂肪酸合成関連遺伝子発現が高まり、一方で腸間膜脂肪重量には影響しないことが分かった。

令和2年度は脂肪と糖質の割合のみが異なる普通食（ND）と高脂肪食（HFD）をC57BL/6Jマウスに5週間摂取させて初期のメタボリックシンドロームの状態に誘導し、16時間の絶食の後に採血と肝臓、脂肪組織、小腸組織の採取を行った。このうち、肝臓の定量プロテオミクス解析を実施し、絶食時には、脂肪組織から流入した脂肪の分解、アセチルCoAからのケトン体の合成、TCA回路の抑制と解糖系の更新などが起こることが知られているが、HFD群ではND群に比べて、①脂肪酸のβ酸化、②ケトン体やコレステロールの合成に関わる酵素の量が増加した。ここから、極初期のメタボリックシンドロームでは糖新生の進行が抑制されており、脂肪分解で生じたグリセロールからピルビン酸に至る経路上の代謝物が蓄積している可能性が考えられた。 令和3年度は、NDとHFDをC57BL/6Jマウスに同じく5週間摂取させた後、最終日に絶食を行わずに、両群共にND食を摂取させることで条件をそろえ、昨年度と同様に肝臓の定量プロテオミクス解析を行った。その結果、本条件では、ND群とHFD群間では肝臓プロテオームプロファイルに大きな違いは存在せず、脂肪酸分解や合成反応に関する酵素など脂肪の蓄積量を反映した変化しか見いだせなかった。ここから、メタボリックシンドロームにおける代謝異常は空腹時における異常が引き金となっている可能性が示唆された。 また、令和3年度は、他に高脂肪食の摂取期間を10週に伸ばして同様の実験を行い、肝臓プロテオームの測定を実施しており、現在解析を進めている。他に令和2年度3年度に採取した脂肪組織や小腸組織のプロテオーム測定も進めデータの蓄積を行っている。

前年度に引き続き、生体発光イメージング法によるサンショウの抗肥満成分の同定を行なった。前年度の実験結果でも見られていた様に、睾丸でのNon-specificなシグナルが認められ、投与効果によるのかそれとも単なるアーチファクト化の判断が難しく、生体発光イメージング法による有効成分の同定は、ペンディングすることとした。一方、褐色脂肪組織やベージュ化した鼠径部皮下脂肪への18FDGの取り込みが増加することが知られている。サンショウ成分の投与によって、ベージュ化が起こっていれば、白色脂肪組織に18FDGが取り込まれるはずである。今回は、高脂肪食とそれにサンショウ成分を添加したものでの比較と、標準食とそれにサンショウ成分を添加したものでの比較のPETイメージング法による解析を行った。高脂肪食とともにサンショウ成分を４週間投与したマウスの比較では、褐色脂肪組織においては取り込みに変化はなく、鼠径部皮下脂肪組織では、有意差は得られないもののサンショウ投与群で取り込みの上昇傾向が見られた。さらに８週間投与したマウスの比較では、褐色脂肪組織では取り込みが減少し、反対に鼠径部皮下脂肪組織では、取り込みの上昇傾向が見られた。標準食にサンショウ成分の添加の有無による比較では、褐色脂肪組織においては、サンショウを同時添加した群では４週間・８週間投与したマウスは共に有意に取り込みが低下していた。一方、鼠径部皮下組織においては、４週間投与群では有意ではないが増加傾向を示し、８週間投与群では有意に18FDGの取り込みが増加した。以上の結果から、元々UCP-1遺伝子発現が高い褐色脂肪組織では、サンショウ成分の摂取により18FDGの取り込みが減少し、鼠径部白色脂肪組織においては、サンショウ成分の摂取により18FDGの取り込みが増加することが明らかとなった。これはベージュ化していることを示唆している。

フルクトースと各種脂肪酸の相互作用による代謝変動を明らかにするため、フルクトースを53%、各種油脂（ラード、魚油、大豆油、中鎖脂肪）を15%含む飼料を6週齢の雄性SDラットに4週間給餌した。飼育期間中の摂餌量には有意な差は認められなかった。飼育終了時の体重は、魚油群が大豆油群に対して有意に低い値を示した。副睾丸周囲脂肪、腎周囲脂肪は魚油群が有意に低かった。血中および肝臓中のトリグリセリド濃度は、魚油群が他群より著しく低く、フルクトース誘導性の脂質代謝異常が魚油の摂取により抑制されていることが示唆された。中鎖脂肪摂取によるトリグリセリド低下作用は認められなかった。血中コレステロール濃度は、魚油群が有意に低い、あるいは低い傾向を示したが、肝臓中コレステロール濃度は中鎖脂肪群が有意に低い、あるいは低い傾向を示した。中鎖脂肪の摂取により肝臓でのコレステロール合成が抑制される報告があることから、今後、肝臓での遺伝子およびタンパク発現解析により、生体内での代謝変動を明らかにしていく。小腸での二糖類水解酵素活性は、有意な差は認められず、摂取する油脂（脂肪酸）の違いによる影響は観察されなかった。血中フルクトース濃度も有意な差は認められず、各群にフルクトース吸収および代謝に差がないことが示唆された。血中ASTおよびALT濃度は、各群ともに正常値の範囲内であり、4週間の投与試験では、肝障害等の影響は引き起こされなかったものと考えられた。

黒酵母Aureobasidium pullulans由来のβ-グルカンを用いて、p-クマル酸とその類縁体を難水溶性化合物モデルとして、その可溶化とその安定性を検討した。さらに動物試験でのその吸収評価法を検討した。また、β-グルカンの生産性向上についても検討した。その結果、変異株の取得とβ-グルカンの高生産かつ高純度回収法を確立した。アルカリ変性させた変性β-グルカンを用いた場合にｐ-クマル酸の溶解度は約1.5倍に、特にカフェ酸を用いた場合は3倍となった。一方、変性β-グルカンを用いたp-クマル酸分散液を用いた動物経口試験において、その吸収性への阻害影響は見られないことを確認した。

1. 梅酢に含まれる各種ヒドロキシ桂皮酸の内、カフェ酸がインスリン抵抗性惹起因子レジスチン遺伝子発現を有意に、かつ濃度依存的に低下させることを明らかにした。またカフェ酸の濃度依存的にレジスチン・レポーター遺伝子発現が抑制された。これをヌードマウス皮下に移植し、カフェ酸投与後数日間でレジスチン遺伝子発現抑制傾向が見られた。 2. 県特産物由来機能性成分（特許出願中につき成分は未開示）について、高脂肪食に混餌しC57BL/6マウスを用いて検討を行った。3％混餌群では、摂取後1週間で高脂肪食群に比し有意に体重増加抑制効果が見られ、1％、3％混餌群と濃度依存的にUCP-1遺伝子発現が上昇した。

ありふれた遺伝子多型と食習慣および生理学的パラメータとの関連について検討した。日本人学生を対象とした研究では、FABP2のAla54Thr多型において、 Thrキャリアが三大栄養素の摂取量が有意に高いこと、ミトコンドリアA10398多型と性別がメンタルヘルスに対して交互作用を示すことが認められた。また、中国系マレーシア人を対象とした研究では、肉と炭水化物に偏った食事パターンとVEGFR-2多型(rs1870377)がHbAlc濃度および」血中相対総コレステロール濃度(TC/HDLC)に対して交互作用を示すことを見出した。

食の安全がおびやかされている昨今、消費者にとって安全で安心できる食糧の供給体制の確立は急務である。本研究は、養殖魚の残留医薬品問題を解決するための一方策として、生産者が残留濃度を経時的に把握できるような簡便・迅速な分析方法の検討を行った。また、分析に用いる溶媒使用量を減らすなど、環境負荷の軽減も重要なテーマである。テトラサイクリン系抗生物質4種(オキシテトラサイクリン、テトラサイクリン、クロルテトラサイクリン、ドキシサイクリン)を対象として養殖魚(ブリ)からの分析法を検討した。微量分析が可能なビーズ式細胞破砕装置を用い、従来のロータ・ステータ方式ホモジナイザーとの抽出効率の比較を行った結果、同程度の回収率・精度が得られサンプル量、溶媒量及び分析時間の大幅な削減が達成できた。測定条件の検討として、LC/MSでの分析及び毒性を考慮して酢酸・エタノール・水系での分離を試みたが達成できなかった。一般的な逆相系カラムでは4種化合物の分離が困難であったため、Pentafluorophenylpropyl基を導入したDiscovery HS F5カラムを用いてクエン酸・アセトニトリル・水系移動相によるHPLC分析を行った。平均回収率約70%、変動係数10%未満の結果が得られたが、本法は従来法とは異なり抽出液を蒸発乾固させたのち一定量にメスアップして定量する操作を省いているため、定量における誤差が生じることも考えられることから、現段階ではスクリーニング法としては簡便で有効な分析法であると思われる。