吉田 優

We developed a practical analytical system for high-throughput and comprehensive lipid profiling using a supercritical fluid chromatography (SFC) system coupled to an Orbitrap Fourier transform mass spectrometer (Orbitrap FT-MS). Using our SFC method, polar lipid molecular species were separated based on not only their fatty acyl moieties but also their polar head groups, using a single octadecylsilyl (ODS) column. In addition, because automatic data processing software was used for the identification of lipid molecular species, the analysis time including data processing was about a half an hour per sample. A variety of lipid molecular species were detected in mouse plasma, and isomers which often co-elute in reverse phase separation were identified accurately and quantified individually. To the best of our knowledge, this is the first report describing the chromatographic separation of lipids based on both fatty acyl moieties and polar head groups, using a single ODS column. Our results demonstrate that SFC/MS is a powerful tool for the simultaneous analysis of diverse lipid molecular species.

BACKGROUND: Claudins have been demonstrated to be associated with inflammatory bowel disease (IBD), but the specific role of claudin-2 in colorectal inflammation remains undefined. AIMS: We aimed to determine the role of claudin-2 in TNFα-induced colorectal inflammation. METHODS: We used claudin-2 (-/-) mice to assess the role of claudin-2 in colon. The mice were intraperitoneally injected with 3 μg of recombinant murine TNFα, and the NF-κB signaling and mRNA expression levels of proinflammatory cytokines and myosin light chain kinase (MLCK) were evaluated. Moreover, in claudin-2 (-/-) mice, colitis was induced by the administration of dextran sodium sulfate (DSS). The involvement of claudin-2 in colorectal inflammation was also investigated using the Caco-2 human colon adenocarcinoma cell line, and the expression of claudin-2 was downregulated using claudin-2 siRNA. RESULTS: TNFα-induced colorectal inflammation via NF-κB signaling activation was enhanced in claudin-2 (-/-) mice compared with that in claudin-2 (+/+) mice. MLCK expression level in the colon tissue of claudin-2 (-/-) mice treated with TNFα was enhanced in comparison to that of the claudin-2 (+/+) mice. DSS-induced colitis was more severe in the claudin-2 (-/-) mice than in the claudin-2 (+/-) mice. In in vitro experiments, the decreased expression of claudin-2 enhanced the expressions of IL-6, IL-1β and MLCK. CONCLUSIONS: Our findings concerning the role of claudin-2 in epithelial inflammatory responses enrich our collective understanding of mucosal homeostasis and intestinal diseases such as IBD. Furthermore, the results of this study indicate that claudin-2 and MLCK are potential therapeutic targets for treatments against intestinal disease.

Identifying population structure forms an important basis for genetic and evolutionary studies. Most current methods to identify population structure have limitations in analyzing haplotypes and recombination across the genome. Recently, a method of chromosome painting in silico has been developed to overcome these shortcomings and has been applied to multiple human genome sequences. This method detects the genome-wide transfer of DNA sequence chunks through homologous recombination. Here, we apply it to the frequently recombining bacterial species Helicobacter pylori that has infected Homo sapiens since their birth in Africa and shows wide phylogeographic divergence. Multiple complete genome sequences were analyzed including sequences from Okinawa, Japan, that we recently sequenced. The newer method revealed a finer population structure than revealed by a previous method that examines only MLST housekeeping genes or a phylogenetic network analysis of the core genome. Novel subgroups were found in Europe, Amerind, and East Asia groups. Examination of genetic flux showed some singleton strains to be hybrids of subgroups and revealed evident signs of population admixture in Africa, Europe, and parts of Asia. We expect this approach to further our understanding of intraspecific bacterial evolution by revealing population structure at a finer scale.

AIM: To study gastric mucosal interleukine-8 (IL-8) mRNA expression, the cytotoxin-associated gene A (cagA) mutation, and serum pepsinogen (PG) I/II ratio related risk in Thai gastric cancer. METHODS: There were consent 134 Thai non-cancer volunteers who underwent endoscopic narrow band imaging examination, and 86 Thais advance gastric cancer patients who underwent endoscopic mucosal biopsies and gastric surgery. Tissue samples were taken by endoscopy with 3 points biopsies. The serum PG I, II, and Helicobacter pylori (H. pylori) immunoglobulin G (IgG) antibody for H. pylori were tested by enzyme-linked immunosorbent assay technique. The histopathology description of gastric cancer and non-cancer with H. pylori detection was defined with modified Sydney Score System. Gastric mucosal tissue H. pylori DNA was extracted and genotyped for cagA mutation. Tissue IL-8 and cyclooxygenase-2 (COX-2) mRNA expression were conducted by real time relative quantitation polymerase chain reaction. From 17 Japanese advance gastric cancer and 12 benign gastric tissue samples, all were tested for genetic expression with same methods as well as Thai gastric mucosal tissue samples. The multivariate analysis was used for the risk study. Correlation and standardized t-test were done for quantitative data, P value < 0.05 was considered as a statistically significant. RESULTS: There is a high non cagA gene of 86.8 per cent in Thai gastric cancer although there are high yields of the East Asian type in the positive cagA. The H. pylori infection prevalence in this study is reported by combined histopathology and H. pylori IgG antibody test with 77.1% and 97.4% of sensitivity and specificity, respectively. The serum PG I/II ratio in gastric cancer is significantly lower than in the non-cancer group, P = 0.045. The serum PG I/II ratio of less than 3.0 and IL-8 mRNA expression ≥ 100 or log10 ≥ 2 are significant cut off risk differences between Thai cancer and non-cancer, P = 0.03 and P < 0.001, respectively. There is a significantly lower PGI/II ratio in Japanese than that in Thai gastric cancer, P = 0.026. Serum PG I/II ratio at cut off less than 3.0 and IL-8 mRNA expression Raw RQ > 100 or log10 > 2 are significantly difference between Thai cancer group when compared to non-cancer group, P = 0.013 and P < 0.001, respectively. In the correlation study, low PG I/II ratio does not associate with chronic atrophic gastritis severity score in Thais non-cancer cases. However, there is a trend, but not significant convert correlation between IL-8 mRNA expression level and low PG I/II ratio in Thai positive H. pylori infection. The high expression of IL-8 gene demonstrates a poorer prognosis by stage and histology. CONCLUSION: Predominant gastric mucosal IL-8 mRNA expression level, H. pylori infection, and low PG I/II ratio are relative risks for Thai gastric cancer without correlation with cagA mutation.

BACKGROUND: A metabolomic approach using umbilical cord blood from infants at birth has not been studied widely yet. AIM: We examined changes in metabolite levels in umbilical cord blood at birth via gas chromatography/mass spectrometry (GC/MS)-based metabolomics, with the aim of achieving a detailed understanding of fetal stress during labor. STUDY DESIGN: All procedures were reviewed and approved by the Institutional Review Board of Kobe University School of Medicine. This was a cohort study of pregnant women based in Palmore Hospital, which is located in an urban area of Japan, and was carried out between December 2010 and May 2011. SUBJECT: Umbilical cord arterial blood samples were obtained from 41 infants immediately after delivery. OUTCOME MEASURES: Metabolites in the blood samples were measured using GC/MS to investigate whether the delivery method (spontaneous onset of labor, induction of labor or elective cesarean section) affected the metabolite profile in umbilical cord blood. RESULTS: Elective cesarean section without labor led to lower levels of isoleucine, fructose, mannose, glucose, allose, glucuronic acid, inositol and cysteine in comparison with vaginal delivery following spontaneous labor and without medication. CONCLUSION: It is proposed that the stress associated with labor be involved in alterations in the levels of metabolites, particularly saccharides such as glucose, in umbilical cord blood.

Metabolomics has recently undergone rapid development; however, metabolomic analysis in cerebrospinal fluid (CSF) is not a common practice. We analyzed the metabolite profiles of preoperative CSF samples from 32 patients with histologically confirmed glioma using gas chromatography/mass spectrometry (GC/MS). We assessed how alterations in the metabolite levels were related to the World Health Organization (WHO) tumor grades, tumor location, gadolinium enhancement on magnetic resonance imaging (MRI), and the isocitrate dehydrogenase (IDH) mutation status. Sixty-one metabolites were identified in the CSF from glioma patients using targeted, quantitative and non-targeted, semi-quantitative analysis. The citric and isocitric acid levels were significantly higher in the glioblastoma (GBM) samples than in the grades I-II and grade III glioma samples. In addition, the lactic and 2-aminopimelic acid levels were relatively higher in the GBM samples than in the grades I-II glioma samples. The CSF levels of the citric, isocitric, and lactic acids were significantly higher in grade I-III gliomas with mutant IDH than in those with wild-type IDH. The tumor location and enhancement obtained using MRI did not significantly affect the metabolite profiles. Higher CSF levels of lactic acid were statistically associated with a poorer prognosis in grades III-IV malignant gliomas. Our study suggests that the metabolomic analysis of CSF from glioma patients may be useful for predicting the glioma grade, metabolic state, and prognosis of gliomas.

Lipid peroxidation products are known to cause toxicity by reacting with biologically significant proteins, but the inducing role of peroxidation products has been not noted to produce degenerative disease-related eicosanoids. Here, 9-oxononanoic acid (9-ONA), one of the major products of peroxidized fatty acids, was found to stimulate the activity of phospholipase A2 (PLA2), the key enzyme to initiate arachidonate cascade and eicosanoid production. An exposure of fresh human blood to the atmosphere at 37°C accumulated 9-ONA, increasing peroxide value and thiobarbituric acid reactive substances in the blood. The lipid peroxidation was accompanied by significant increases of PLA2 activity and thromboxane B2 (TxB2) production, which is a stable metabolite of thromboxane A2 (TxA2) and a potent agonist of platelet aggregation. These events were abolished by standing the blood under nitrogen. The addition of organically synthesized 9-ONA resumed the activity of PLA2 and the production of TxB2. Also, 9-ONA induced platelet aggregation dose-dependently. These results indicated that 9-ONA is the primary inducer of PLA2 activity and TxA2 production, and is probably followed by the development of diseases such as thrombus formation. This is the first report to find that a lipid peroxidation product, 9-ONA, stimulates the activity of PLA2.

Supercritical fluid chromatography/tandem mass spectrometry (SFC/MS/MS) with methylation was used for the simultaneous profiling of diverse polar lipids in a mixture. A high throughput, high resolution analysis of nineteen classes of polar lipids including phospholipids, lysophospholipids, and sphingolipids was performed in 6 min. Methylation by trimethylsilyl-diazomethane suppressed peak tailing and improved detection sensitivity of phosphatidylserine (PS), phosphatidic acid (PA), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), lysophosphatidic acid (LPA), ceramide-1-phosphate (Cer1P), sphingosine-1-phosphate (So1P), and sphinganine-1-phosphate (Sa1P). The limits of detection for PS, PA, LPS, LPI, LPA, Cer1P, So1P, and Sa1P were enhanced 7.5-, 26.7-, 600-, 116.7-, 500-, 75-, 3000-, and 4500-fold, respectively. Global qualitative and quantitative analysis of not only the high-abundance species but also the low-abundance species in the polar lipids was achieved. When the method was applied to mouse liver, 4 PSs, 24 PAs, 3 lysophosphatidylethanolamines, 11 LPSs, 6 lysophosphatidylglycerols, 4 LPIs, 13 LPAs, 7 sphingomyelins, 11 Cer1Ps, So1P, and Sa1P were additionally analyzed. Furthermore, the quantification of various molecular species in each polar lipid was carried out.

BACKGROUND: We aimed to clarify the lifestyle factors associated with erosive esophagitis and non-erosive reflux disease (NERD) in a Japanese population. METHODS: Among 886 subjects who underwent health screening, we selected, according to their scores on the FSSG (frequency scale for symptoms of gastroesophageal reflux disease; GERD) questionnaire and the findings of upper gastrointestinal endoscopy, 138 subjects with erosive esophagitis (EE), 148 subjects with NERD (absence of esophagitis, FSSG score ≥8, and acid reflux-related symptoms score ≥4), and 565 control subjects (absence of esophagitis and FSSG score ≤7). We compared clinical characteristics and various lifestyle factors in these three groups. RESULTS: The lifestyle factors significantly associated with NERD compared with findings in the control group were intake of egg (odds ratio [OR] 1.89, 95% confidence interval [CI] 1.01-3.50), sleep shortage (OR 2.44, 95% CI 1.54-3.88), and strong psychological stress (OR 1.77, 95% CI 1.18-2.62). In male subjects, current smoking (OR 2.06, 95% CI 1.13-3.74; OR 1.87, 95% CI 1.09-3.20) was a significant risk factor for both NERD and EE. Moreover, alcohol >200 kcal/day (OR 3.99, 95% CI 1.03-15.55) and intake of a large quantity of food at supper (OR 7.85, 95% CI 1.66-37.05) were significant risk factors for EE in subjects with hiatal hernia. Intake of a large quantity of food at supper (OR 2.09, 95% CI 1.06-4.13) was more common in the NERD group than in the EE group. CONCLUSIONS: There were differences in the associated lifestyle factors between patients with NERD and those with EE, and there was also a gender-related difference between these groups.

In our previous study, it was found that linoleoyl ethanolamide (LE) is present in sake lees, which are produced as a byproduct during the making of Japanese sake. LE is a fatty acid ethanolamide, which have been demonstrated to exert a variety of biological functions, and in this study, the anti-inflammatory effects of LE were examined using in vitro cell culture and in vivo animal experiments. In mouse RAW264.7 macrophages, LE suppressed the lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. In addition, LE inhibited LPS-induced increases in the levels of cyclooxygenase enzyme-2 and prostaglandin E(2), which are indicators of inflammation. The inhibitory effect of LE on the release of TNF-α was stronger than that of dipotassium glycyrrhizinate, which is widely used in external human skin care treatments. LE also suppressed the LPS-induced activation of Toll-like receptor 4 signaling and nuclear translocation of nuclear factor-κB (NF-κB) p65. In a contact dermatitis animal model, applying LE to affected ear skin ameliorated 2,4-dinitrofluorobenzene-induced contact dermatitis and pro-inflammatory cytokine expression at inflamed sites. These results indicate that LE exerts anti-inflammatory effects by inhibiting NF-κB signaling, and LE is proposed to be a useful therapeutic agent against contact dermatitis.

Oral food intake influences the morphology and function of intestinal epithelial cells and maintains gastrointestinal cell turnover. However, how exactly these processes are regulated, particularly in the large intestine, remains unclear. Here we identify microbiota-derived lactate as a major factor inducing enterocyte hyperproliferation in starvation-refed mice. Using bromodeoxyuridine staining, we show that colonic epithelial cell turnover arrests during a 12- to 36-h period of starvation and increases 12-24 h after refeeding. Enhanced epithelial cell proliferation depends on the increase in live Lactobacillus murinus, lactate production and dietary fibre content. In the model of colon tumorigenesis, mice exposed to a carcinogen during refeeding develop more aberrant crypt foci than mice fed ad libitum. Furthermore, starvation after carcinogen exposure greatly reduced the incidence of aberrant crypt foci. Our results indicate that the content of food used for refeeding as well as the timing of carcinogen exposure influence the incidence of colon tumorigenesis in mice.

The patient was a 60-year-old man with encephalopathy without liver cirrhosis. CT angiography revealed a patent ductus venosus between the anterior segmental branch of the portal vein and the middle hepatic vein. Coils were framed in the patent ductus venosus and then used to fill in the frame. After treatment, transarterial portography showed that the shunt flow of the ductus venosus had decreased significantly. After one day, the patient's disturbance of consciousness disappeared. Our case involved the adult-onset of a patent ductus venosus, which is extremely rare. This case is the first in which coil embolization was successfully achieved in a noncirrhotic elderly patient with a patent ductus venosus.

Once lipids are oxidized, various volatiles are produced by cleavage of the fatty acid side chain. Considering the variety of lipids present in the body, a large number of possible volatiles might originate from oxidized lipids. However, only specific volatiles such as aldehydes are exclusively examined in current studies, and there is no reported method for the exhaustive analysis of all volatiles. We developed a sensitive analytical method for the detection of all possible volatiles for multimarker profiling, applying a new extraction method called in-tube extraction. Oxidized phosphatidyl choline standards were prepared in vitro and analyzed in order to determine the potential variety of volatiles. Over 40 compounds, including alcohols, ketones, and furanones, were identified in addition to the aldehydes reported previously. Based on this result, we applied our analytical method to mouse plasma and identified 12 volatiles, including 1-octen-3-ol, which is correlated to disease states. To determine the volatile profile after oxidation, we oxidized plasma in vitro under various conditions and identified 27 volatiles, including 1-octen-3-ol and benzaldehyde. The generation capacity of each volatile was different. This method allows sensitive and exhaustive analysis of various volatiles in addition to aldehydes.

BACKGROUND: Endoscopic submucosal dissection (ESD) has come to be widely performed for reduced invasiveness; however, its safety in patients with co-morbidities is not fully examined. We aimed to evaluate the safety and efficacy of gastric ESD with co-morbidities categorized according to ASA Physical Status Classification. METHODS: Two hundred and forty patients of ASA 1 (no co-morbidities), 268 of ASA 2 (mild), and 19 of ASA 3 (severe) were treated by ESD for gastric neoplasms. We retrospectively compared clinicopathological features and treatment results of these three groups. RESULTS: Cases (by percent) treated with anticoagulant/platelet agents were more common in the higher ASA grades (ASA 1, 5.8%; ASA 2, 29.1%; ASA 3, 31.6%; P < 0.0001). There were no significant differences in case numbers treated under guideline criteria, curative resection (ASA 1, 79.6%; ASA 2, 79.9%; ASA 3, 78.9%), or complications related to the ESD procedure (e.g., postoperative bleeding, perforation, thermal injury). By a patient risk prediction model on surgery, i.e., P-POSSUM, morbidity was halved, and no patients died compared to a predicted death rate of 0.5-2%; however, total and complications unrelated to ESD procedure (e.g., aspiration pneumonia, ischemic heat attack) were more common in higher ASA grades (ASA 1, ASA 2, ASA 3: 15.4, 23.9, 26.3%, respectively, P = 0.014; 0.4, 7.1, 0%, respectively, P = 0.00087). Deviation rates from clinical pathway were more frequent and hospital stay (days) longer in higher ASA grades (ASA 1, ASA 2, ASA 3: 11.3, 17.9, 26.3%, respectively, P = 0.014; 8, 8, 9%, respectively, P = 0.0053). CONCLUSIONS: ESD is an efficient treatment for gastric neoplasms with co-morbidities. However, additional caution is required because co-morbidity is a risk factor for both total complications and complications unrelated to the ESD procedure, and may cause deviations in the clinical course and prolonged hospital stay.

BACKGROUND: Transcatheter arterial chemoembolization (TACE) is an effective treatment for hepatocellular carcinoma (HCC) that can occasionally lead to the shortening of life expectancy. We aimed to make a new and more accurate prognostic model taking into account the course of disease after TACE. METHODOLOGY/PRINCIPAL FINDINGS: We performed a prospective cohort study involving 100 HCC patients who underwent TACE at Kobe University Hospital. Indirect calorimetry and blood biochemical examinations were performed before and 7 days after TACE. Time-dependent and time-fixed factors associated with 1-year mortality after TACE were assessed by multivariate analyses. A predictive model of 1-year mortality was established by the combination of odds ratios of these factors. Multivariate analyses showed that the ratio of non-protein respiratory quotient (npRQ) (7 days after/before TACE) and Cancer of Liver Italian Program (CLIP) score were independent factors of 1-year mortality after TACE (p = 0.014 and 0.013, respectively). Patient-specific 1-year mortality risk scores can be calculated by summarizing the individual risk scores and looking up the patient-specific risk on the graph. CONCLUSIONS: The short-term reduction of npRQ was a time-dependent prognostic factor associated with overall survival in HCC patients undergoing TACE. CLIP score was a time-fixed prognostic factor associated with overall survival. Using the prediction model, which consists of the combination of time-dependent (npRQ ratio) and time-fixed (CLIP score) prognostic factors, 1-year mortality risk after TACE would be better estimated by taking into account changes during the course of disease.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation of the gastrointestinal tract. It is unknown whether β-1,3;1,6-glucan can induce immune suppressive effects. Here, we study intestinal anti-inflammatory activity of Lentinula edodes-derived β-1,3;1,6-glucan, which is known as lentinan. Dextran sulfate sodium (DSS)-induced colitis mice were used to elucidate effects of lentinan in vivo. In the cellular level assessment, lentinan was added into a co-culture model consisting of intestinal epithelial Caco-2 cells and LPS-stimulated macrophage RAW264.7 cells. Ligated intestinal loop assay was performed for assessing effects of lentinan on intestinal epithelial cells (IECs) in vivo. Oral administration of lentinan (100 µg/mouse) significantly ameliorated DSS-induced colitis in body weight loss, shortening of colon lengths, histological score, and inflammatory cytokine mRNA expression in inflamed tissues. Lentinan reduced interleukin (IL)-8 mRNA expression and nuclear factor (NF)-κB activation in Caco-2 cells without decreasing of tumor necrosis factor (TNF)-α production from RAW264.7 cells. Flow cytometric analysis revealed that surface levels of TNF receptor (TNFR) 1 were decreased by lentinan treatment. A clathrin-mediated endocytosis inhibitor, monodansylcadaverine, canceled lentinan inhibition of IL-8 mRNA expression. Moreover, lentinan inhibited TNFR1 expression in Caco-2 cells in both protein and mRNA level. Lentinan also inhibited TNFR1 mRNA expression in mouse IECs. These results suggest that lentinan exhibits intestinal anti-inflammatory activity through inhibition of IL-8 mRNA expression associated with the inhibition of NF-κB activation which is triggered by TNFR1 endocytosis and lowering of their expression in IECs. Lentinan may be effective for the treatment of gut inflammation including IBD.

OBJECTIVE: Helicobacter pylori is known to affect the host's nutritional status. This study was performed to elucidate the relationship between H. pylori status and the dynamics of the ghrelin system, in the context of ghrelin O-acyltransferase (GOAT) expression. METHODS: We conducted a clinical study of 30 subjects focusing on the following points: 1) the effects of H. pylori infection on the concentrations of circulating ghrelin isoforms and on ghrelin and GOAT mRNA expression in the gastric mucosa, and 2) the effects of H. pylori eradication on the same parameters. RESULTS: The plasma acyl-ghrelin and desacyl-ghrelin concentrations of 16 H. pylori positive participants were significantly lower than those of 14 H. pylori negative controls. The acyl-ghrelin/desacyl-ghrelin ratio was not significantly different between the H. pylori positive and H. pylori negative participants. The levels of ghrelin and GOAT mRNA in the gastric mucosa were significantly lower in the H. pylori positive participants than in the H. pylori negative controls. In 11 subjects in whom H. pylori eradication was successful, their plasma acyl-ghrelin levels tended to increase after H. pylori eradication, but the difference was not significant; however, their plasma desacyl-ghrelin levels were significantly reduced. Although gastric ghrelin mRNA expression increased significantly after H. pylori eradication, gastric GOAT mRNA expression tended to increase but was not significantly altered. CONCLUSION: H. pylori status might affect the host's nutritional status through changes in the plasma levels of ghrelin isoforms and the gastric expression levels of ghrelin and GOAT mRNA.

Phospholipids that contain polyunsaturated fatty acid are easily oxidized by free radicals or oxidants and can yield numerous oxidation species, including positional and structural isomers. However, it is difficult to separate these oxidation products for structural analysis. In this study, a high-resolution separation analytical system based on supercritical fluid chromatography with tandem mass spectrometry (SFC/MS/MS) was established for the separation and identification of oxidized phosphatidylcholine (PC) isomers derived from esterified linoleic acid or arachidonic acid. Separation of oxidatively modified PC containing hydroxy, epoxy and hydroperoxy groups was achieved by SFC. Positional isomers of hydroxides and epoxides were identified based on MS/MS fragment information. To investigate whether this method is applicable to biological samples, we then analyzed oxidized PC isomers from mouse liver. Oxidized isomers, such as hydroxides, hydroperoxides and epoxides, were simultaneously observed. This method may be a powerful tool for providing further insight into how oxidized phospholipids are produced and are correlated with various diseases.

Novel and effective drugs against acute pancreatitis are required. Therefore, we examined the changes in the metabolite levels in the serum and pancreatic tissue of mice with cerulein- and arginine-induced pancreatitis using gas-chromatography/mass-spectrometry (GC/MS) and investigated whether these alterations affected the severity of acute pancreatitis. In the cerulein-induced pancreatitis model, 93 and 129 metabolites were detected in the serum and pancreatic tissue, respectively. In the L-arginine-induced acute pancreatitis model, 120 and 133 metabolites were detected in the serum and pancreatic tissue, respectively. Among the metabolites, the concentrations of tricarboxylic acid (TCA) cycle intermediates and amino acids were altered in pancreatitis, and in pancreatic tissue, the levels of the intermediates involved in the initial part of the TCA cycle were increased and those of the intermediates involved in the latter part of the TCA cycle were decreased. Some metabolites exhibited similar changes in both pancreatitis mouse models, e.g., the levels of glutamic acid and O-phosphoethanolamine were significantly decreased in the pancreatic tissue. Supplementation with glutamic acid and O-phosphoethanolamine attenuated the severity of cerulein-induced acute pancreatitis. Our results suggest that GC/MS-based metabolomics is capable of accurately representing the status of acute pancreatitis, leading to the discovery of therapeutic agents for pancreatitis.

We investigated viscoelastic and photoresponsive properties of the microparticle/liquid-crystal (LC) composite gels. The mechanical strength of the colloidal gels can be widely tuned by varying particle concentrations. With increasing particle concentration, a storage modulus of the particle/LC composite gels increased and reached over 10(4) Pa, showing good self-supporting ability. We demonstrated for the first time that the particle/LC composite gels exhibited rapid and repetitive recovery of the mechanical strength after large-amplitude oscillatory breakdown. In addition, photoresponsive properties of the composite gels were investigated by the cis-trans photoisomerization of the azobenzene compound doped into the host LCs. The photochemical gel-sol transition could be repeatedly induced by changing the phase structure of the host LCs between nematic and isotropic, using the photoisomerization. The particle/LC composite gels can be applied to optically healable materials by the site-selective gel-sol transition based on the photochemical modulation of the phase structures of the host LCs.

BACKGROUND: Transcatheter arterial chemoembolization (TACE) is an effective treatment for hepatocellular carcinoma (HCC) that can cause deterioration of liver function. We aimed to make an early predictive model of long-term liver dysfunction after TACE. METHODS: We performed a prospective cohort study involving 109 HCC patients who underwent TACE at Kobe University Hospital. Indirect calorimetry and blood biochemical examinations were performed before and 7 days after TACE. As an indicator of liver function, the Child's score was evaluated before and 3 months after TACE. Patients with and without Child's score deterioration were compared, and the independent risk factors for Child's score deterioration were statistically examined. An early predictive model of Child's score deterioration after TACE was developed using multivariate logistic regression. RESULTS: Multivariate analyses showed that the non-protein respiratory quotient (npRQ) and prealbumin (preAlb) ratios (7 days after/before TACE) were independent determinants of Child's score deterioration (p = 0.039 and 0.020, respectively). Decreases of the npRQ and preAlb ratios were significantly related to increases of Child's score 3 months after TACE (p = 0.007 and 0.002, respectively). The following predictive model of Child's score deterioration was developed: exp(-6.383 × npRQ ratio - 3.038 × preAlb ratio + 7.755)/(1 + exp(-6.383 × npRQ ratio - 3.038 × preAlb ratio + 7.755)). The model discriminated well between patients with and without Child's score deterioration (area under the receiver operating curve [ROC]; AUC 0.713; 95% confidence interval [CI] 0.613-0.812). The optimal cut-off point for the Child's score was 0.449, and the sensitivity and specificity of the model were 57.1 and 79.1%, respectively. CONCLUSIONS: Reductions in npRQ and preAlb 7 days after TACE were associated with the long-term deterioration of liver function. With our model, we were able to identify high-risk patients.

Dietary fucoxanthin has been reported to exert several physiological functions, and fucoxanthinol is considered to be the primary active metabolite of fucoxanthin. However, there is no information about the pharmacokinetics of fucoxanthinol in human subjects. In the present study, eighteen human volunteers were orally administered kombu extract containing 31 mg fucoxanthin, and their peripheral blood was collected 5 min before and 0·5, 1, 2, 4, 8 and 24 h after the treatment. Plasma fucoxanthinol concentrations were measured by HPLC, and the pharmacokinetics of fucoxanthinol were as follows: maximum concentration, 44·2 nmol/l; time at maximum concentration, 4 h; terminal half-time, 7·0 h; area under the curve (AUC) for 1-24 h, 578·7 nmol/l × h; AUC(∞), 663·7 nmol/l × h. In addition to fucoxanthinol, we also attempted to detect amarouciaxanthin A, a hepatic metabolite of fucoxanthinol, using HPLC, but it was not present in the volunteers' plasma. On the other hand, a peak that was suspected to represent the cis-isomer of fucoxanthinol was found in the HPLC chromatogram. By comparing the present results with those of a previous study using mice, we found that the bioavailability and metabolism of fucoxanthinol differ between human subjects and mice.

Epoxy carotenoids, which are products of carotenoid oxidation, are potential oxidative stress markers. However, it is difficult to profile epoxy carotenoids owing to their small amount and difficulty in their separation from hydroxy carotenoids. In this study, a high-performance analytical system based on supercritical fluid chromatography (SFC) coupled with tandem mass spectrometry (MS/MS) was developed for the simultaneous analysis of carotenoids and epoxy carotenoids. SFC is an effective separation technique for hydrophobic compounds, by which major carotenoids in human serum and their epoxidation products can be analyzed within 20 min. The use of MS/MS increased the sensitivity; the detection limit for each carotenoid was of the sub-fmol order. When the constructed method was applied to biological samples such as human serum and low-density lipoprotein (LDL), the precise detection of the target carotenoids was disturbed by several isomers. However, highly selective detection of epoxy carotenoids was performed by targeting product ions that were generated with a structure-specific neutral loss of 80Da. Furthermore, the sample volume needed for the analysis was only 0.1ml for the serum, indicating the efficiency of this system in performing small-scale analyses. Using the analytical system developed in this study, highly sensitive and selective analysis of epoxy carotenoids could be performed in a short time. These features show the usefulness of this system in application to screening analysis of carotenoid profiles that are easily modified by oxidative stress.

Autophagy has been demonstrated to be associated with the pathogenesis of cancer, but no consensus has been reached about its precise role. Therefore, we investigated whether autophagy in the intestinal epithelium is involved in the pathogenesis of intestinal tumors. To evaluate the relationship between autophagy and intestinal tumors, GFP-LC3-APC(min/+) mice were generated by mating GFP-LC3 transgenic mice with APC(min/+) mice. Autophagy was weakly induced in the intestinal polyp regions of the mice in comparison to their non-polyp regions. Under starved conditions, autophagy was not induced in the polyp regions, whereas it was observed in the non-polyp regions. Then, to examine whether a lack of autophagy in the intestinal epithelium enhances the induction of intestinal tumor, Atg7flox/flox:vil-cre-APC(min/+) mice, in which Atg7 had been conditionally deleted in the intestinal epithelium, were generated by mating Atg7flox/flox:vil-cre mice with APC(min/+) mice. However, there was no significant difference in the number of intestinal polyps between the Atg7flox/flox:vil-cre-APC(min/+) and the corresponding control Atg7flox/flox-APC(min/+) mice. These results indicate that autophagy in the intestinal epithelium is not involved in the pathogenesis of intestinal tumors, and future research should focus on regulating autophagy as a form of cancer therapy.

Sugar alcohol derivatives with multi azobenzene arms are photochemically and isothermally liquefied from a powdered solid upon irradiation with ultraviolet light at room temperature, and then solidified on irradiation with visible light, where the transition between solid and liquid are reversible. These compounds possess similar chemical structures to comb-like liquid crystalline oligomers.

Conventional tumor markers are unsuitable for detecting carcinoma at an early stage and lack clinical efficacy and utility. In this study, we attempted to investigate the differences in serum metabolite profiles of gastrointestinal cancers and healthy volunteers using a metabolomic approach and searched for sensitive and specific metabolomic biomarker candidates. Human serum samples were obtained esophageal (n = 15), gastric (n = 11), and colorectal (n = 12) cancer patients and healthy volunteers (n = 12). A model for evaluating metabolomic biomarker candidates was constructed using multiple classification analysis, and the results were assessed with receiver operating characteristic curves. Among the 58 metabolites, the levels of nine, five and 12 metabolites were significantly changed in the esophageal, gastric and colorectal cancer patients, respectively, compared with the healthy volunteers. Multiple classification analysis revealed that the variations in the levels of malonic acid and L-serine largely contributed to the separation of esophageal cancer; gastric cancer was characterized by changes in the levels of 3-hydroxypropionic acid and pyruvic acid; and L-alanine, glucuronoic lactone and L-glutamine contributed to the separation of colorectal cancer. Our approach revealed that some metabolites are more sensitive for detecting gastrointestinal cancer than conventional biomarkers. Our study supports the potential of metabolomics as an early diagnostic tool for cancer.

Autophagy, a ubiquitous degradation pathway, is important for the survival and homeostasis of cells. Previous studies have demonstrated the role of autophagy in host defense against bacterial infection, but the importance of autophagy in the intestinal epithelium for the regulation of bacterial infection has not been fully elucidated. In this study, we showed that the essential autophagy protein Atg7 is required for resistance to Citrobacter rodentium infection in the intestinal epithelium. Infected mice in which Atg7 had been conditionally deleted from the intestinal epithelium exhibited greater clinical evidence of disease and higher expression levels of pro-inflammatory cytokine mRNA in the large intestine. Moreover, C. rodentium clearance was reduced in the Atg7 conditional knockout mice. These results demonstrate that autophagy in intestinal epithelial cells plays an important role in host defense against C. rodentium infection and the regulation of C. rodentium infectious colitis.

We report on a chronic hepatitis C patient who developed hepatocellular carcinoma (HCC) 18 years after achieving a sustained virological response (SVR) to interferon therapy. We also review other reports of patients who developed HCC a long time after interferon therapy. The patient was a 67-year-old man with chronic hepatitis C who achieved an SVR to interferon therapy at the age of 49 years in 1992. Eighteen years later, however, a tumor measuring 19 mm in diameter in segment 7 of the liver was found by abdominal ultrasound. The tumor had a typical HCC enhancement pattern by dynamic computed tomography. A moderately to poorly differentiated HCC was confirmed by fine-needle aspiration biopsy. Fibrosis and inflammation in the liver parenchyma improved pathologically from F3A2 to F2A1 according to the New Inuyama Classification. Liver steatosis remained after achieving an SVR and the serum alanine aminotransferase level was persistently slightly elevated. The HCC was treated by transcatheter arterial chemoembolization combined with radiofrequency ablation. Patients with chronic hepatitis C who have achieved an SVR to interferon therapy, and those who have risk factors for the development of HCC, such as being male, of advanced age (<50 years is rare), or with progressive liver fibrosis and steatosis as a hepatic manifestation of metabolic syndrome, should undergo careful long-term follow-up.