Analysis of an antioxidative defence system of hydrogen peroxide-treated pancreatic islet-derived 1.1B4 cells and siRNA targeting NR4A3-treated cells by microarray.

Pancreatic islet ß-cells weaken under oxidative stress. In this study, human pancreatic islet-derived 1.1B4 cells were exposed to H2O2 and analysed using a human microarray, which revealed that heme oxygenase 1 (HMOX1), glutamate-cysteine ligase, early growth response 1, nuclear receptor subfamily 4 group A member 3 (NR4A3) and jun B proto-oncogene were upregulated, whereas superoxide dismutase 1 and catalase were not. Expression of NR4A3 rapidly increased after H2O2 addition, and the 1.1B4 cells treated with siRNA targeting NR4A3 became sensitive to H2O2; further, HMOX1 expression was strongly inhibited, suggesting that NR4A3 is an oxidative stress-responsive transcription factor that functions through HMOX1 expression in pancreatic islet ß-cells. Expression of cyclin E1 and cyclin-dependent kinase 1 was also inhibited by siRNAs targeting NR4A3.

Effect of heat-moisture treated brown rice crackers on postprandial flow-mediated dilation in adults with mild endothelial dysfunction.

Background: Endothelial dysfunction is an early pathophysiological feature and independent predictor of a poor prognosis in most forms of cardiovascular disease. We evaluated the effect of brown rice crackers (BR-C) on endothelial function. Methods: Effect of heat-moisture treated (HMT) -BR-C on postprandial flow-mediated dilation (FMD) in adults with mild endothelial dysfunction was compared with that of BR-C and white rice crackers (WR-C) in 12 adults with mild endothelial dysfunction (less than 7.0% of FMD) by a randomized, single-blind, three-treatment three-period crossover trial (UMIN 000034898). Since we considered that the FMD increase was associated with the treatment of HMT-BR-C, we examined the effect of three possible factors: postprandial glucose levels, polyphenol content, and polyphenol release from the food matrix. Results: Mean pre-intake baseline FMD values of HMT-BR-C, BR-C, and WR-C were 4.9%, 5.1%, and 4.9%, respectively, and those values 1 h post-intake were 6.3%, 5.1%, and 4.8%, respectively. There was no difference in intergroup comparisons of FMD using Dunnett's multiple comparison test. There was a significant increase in FMD only in HMT-BR-C in intragroup comparisons (P = 0.042 by paired-t test). In comparison with BR-C, no significant difference was noted in the postprandial glucose level nor in the content of total polyphenols and ferulic acid derivatives in HMT-BR-C. However, the 70% ethanol extracted from HMT-BR-C contained a significantly larger amount of free and bound ferulic acids than from BR-C. Conclusion: HMT-BR-C intake increased the postprandial FMD response.

Evaluation of Fructo-, Inulin-, and Galacto-Oligosaccharides on the Maillard Reaction Products in Model Systems with Whey Protein.

The present study aimed to investigate the effects of fructo-, inulin-, and galacto-oligosaccharides (FOS, IOS, and GOS) on forming the Maillard reaction products such as browning, α-dicarbonyl compounds, and advanced glycation end products (AGEs). The model solutions at pH 6.8 containing each carbohydrate (mono-, di-, and oligosaccharides) and whey protein were incubated at 50 °C for 8 weeks. In the IOS model, sugars of DP3 or larger were significantly decreased at 4 weeks, whereas at 6 weeks in the FOS model. The residual amount of GOS after 8 weeks was higher than FOS and IOS; however, a large amount of 3-deoxyglucosone was formed compared to the other models. Nε-Carboxymethyllysine (CML) concentrations in oligosaccharide models were about half of those in monosaccharide and lactose models. The highest concentrations of glyoxal- and methylglyoxal-derived hydroimidazolones 3 (G-H3 and MG-H3) were observed in the IOS model, indicating the involvement of fructose units linked by ß-2 → 1 bonds. G-H3 and MG-H3 quantification could be a useful indicator to reflect the modification of an arginine residue by fructose if used acid-hydrolysis for AGE analysis. CML, G-H3, and MG-H3 were considerably formed even in the FOS model, which has no reducing terminal site, suggesting that degradation products of oligosaccharides probably participated in the formation of AGEs.

Evaluation of Blood-Brain Barrier Permeability of Polyphenols, Anthocyanins, and Their Metabolites.

Anthocyanins (ACs) are able to protect neurons against ß-amyloid-induced neurotoxicity. In this study, we evaluated blood-brain barrier (BBB) permeability of these compounds using a model kit to clarify the mechanism of AC on the brain. Black currant or strawberry AC extract was orally administrated to male Wistar rats. The urine and extirpated brain were collected before and after administration and analyzed quantitatively by liquid chromatography-tandem mass spectrometry. After administration of AC, several phenolic acids were detected in the urine samples. Further, AC and some AC metabolites were found in the brain tissue. BBB permeabilities of these compounds were much lower than the positive control. Epigallocatechin, daidzein, genistein, equol, and nobiletin presented high BBB permeability, whereas apigenin, luteolin, quercetin, and kaempferol showed medium permeability, and epicatechin, rutin, fisetin, resveratrol, and curcumin BBB permeation was neglected. These results suggested that ACs were difficult to cross BBB into the brain and ACs were not directly associated with the prevention of ß-amyloid-induced neurotoxicity.

Identification of dipyrrolone pigments and their precursors formed in the Maillard reaction of carnosine and pentose under weakly acidic conditions.

Colored compounds formed by the Maillard reaction of carnosine with xylose or glucose were investigated in this study. Yellow pigments showing an absorption maximum at 450 nm were found in a heated solution of carnosine with xylose at pH 5.0. These pigments were then isolated and identified as dicarnosyl-dipyrrolones A and B. The generation of dipyrrolones in the absence of lysine suggests that dipyrrolone pigments can be formed by pentose as well as every amino compound such as amino acids, peptides and proteins possessing a free amino group. Analysis of α-dicarbonyls using LC-MS/MS showed that pentosone, 1-deoxypentosone, 3-deoxypentosone (3-DP), and methylglyoxal were predominantly generated via degradation of Amadori compounds. Also, a potential formation pathway of dypyrrolones was established, indicating that an Amadori compound that could form 3-DP is likely to play a role as a main precursor for dipyrrolones.

Free advanced glycation end product distribution in blood components and the effect of genetic polymorphisms.

One hypothesis regarding the cause of diabetic complications is that advanced glycation end products (AGEs) bind to the AGE receptor and induce changes in gene expression. However, what AGEs exist in vivo and how individual AGEs are produced and impact body metabolic process to cause diabetes complications are not understood. We developed a new precise method to measure AGEs using LC-MS/MS with a new column and measured 7 free AGEs, including N(6)-carboxymethyllysine (CML), N(6)-(1-carboxyethyl)-l-lysine (CEL) and N5-(5-hydro-5-methyl-4-imidazolon-2-yl)L-ornithine (MG-H1), in human blood components. Blood was obtained from 9 people, and free AGEs were measured in individual blood components with LC-MS/MS before and after a meal. Free CML and CEL were abundant in erythrocytes, with 92% of free CML and 85% of free CEL localized in erythrocytes. In contrast, 60% of free MG-H1 was distributed in the serum. After the meal, free serum MG-H1 increased, but CML and CEL did not. CML and CEL are mainly distributed in erythrocytes and were not affected by the meal, indicating that they are produced in vivo. However, the main source of MG-H1 is the meal. The effect of genetic polymorphisms on AGEs was also investigated. Low activity type aldehyde dehydrogenase 2 (ALDH2) increased the CML concentration in the blood. This is the first observation that shows that the metabolic process of CML and CEL is different from that of MG-H1 and the effect of ALDH2 SNPs on CML.

High neutrophil incorporation rate of ascitic fluid cytology as an indicator of cancerous ascites.

The cell­in­cell phenomenon (CiCP) involves the incorporation of a viable cell by other cells (host cells) and includes two concepts: Emperipolesis and cell cannibalism. The former involves the incorporation of hematopoietic cells as the incorporated cells, while the latter involves cell incorporation by tumor cells as host cells. A total of 239 peritoneal cavity fluid cytology specimens were evaluated for CiCP and the number of singly detectable nuclei (SDN) were measured by examining virtual slide image files. The rates of CiCP­positive cases (RCPCs) and CiCP emergence rate (CER)/SDN were significantly higher in ascites samples than in peritoneal washing samples (P<0.0001 and P=0.0026, respectively), although the numbers of SDN were not significantly different between the groups (P=0.8063). Both the RCPCs and CER/SDN were significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0220 and P=0.0312, respectively), although the numbers of SDN were not significantly different between the samples (P=0.2471). Most of the incorporated cells were lymphocytes and the host cells were macrophages; however, the rate of neutrophil incorporation (NI) by host cells in the total CiCP cells in a sample was significantly higher in tumor­positive specimens than in tumor­negative specimens (P=0.0288). NI was mainly performed via emperipolesis by macrophages, with only six examples not by macrophages observed among all CiCP samples. The threshold NI rate/total CiCP (NI/CiCP) between tumor­positive and tumor­negative groups was 11.1% (P=0.0115). Using this threshold, the peripheral blood leukocyte count was significantly higher in the high­NI/CiCP group than in the low­NI/CiCP group (P=0.0022). The present findings revealed novel aspects of less frequently observed CiCP in ascitic fluid cytology by utilizing combined manual and computer assisted image analysis evaluation of samples. Notably, the present study indicated the importance of increased NI as an indicator of cancerous ascites.

Isolation, identification, and proposed formation mechanism of a novel hydrophilic compound formed by Maillard reaction between pyridoxamine and pentose.

Pyridoxamine (PM) could competitively protect amino groups in proteins from glycating agents. Although PM is expected to react with saccharides, available data therein are limited. In this study, a novel hydrophilic compound from a model reaction solution containing PM and xylose was isolated and identified as (6aR,9aR)-1,8,9-trihydroxy-2,6a-dimethyl-6a,9a-dihydrocyclopenta[5,6]pyrano[3,4-c]pyridin-7(5H)-one with a tricyclic structure. This compound appeared to be specifically formed from pentose via 1-deoxypentosone, and its formation was facilitated over a pH range of 7.0-8.0. After heating at 90 °C for 5 h in a reaction mixture containing 30 mM PM and pentose at pH 7.4, this compound was obtained at a yield of 6.95-8.53 mM.

Therapeutic Effects of Anthocyanins for Vision and Eye Health.

Anthocyanin (AC) is widely used as supplement of eye health in Europe and in East Asia. In this review, I describe AC effects to clarify the mechanism is important in order to understand the effects of AC on vision health. The bioavailability of AC is quite low but, reported as intact form and many kinds of metabolite. And AC passes through the blood-aqueous fluid barrier and blood-retinal barrier. In vitro study, AC had a relaxing effect on ciliary muscle which is important to treat both myopia and glaucoma. And AC stimulate the regeneration of rhodopsin in frog rod outer segment. Furthermore, AC could inhibit the axial length and ocular length elongation in a negative lens-induced chick myopia model. In addition, we summarized clinical studies of AC intake improved dark adaptation and transient myopic shift and the improvement on retinal blood circulation in normal tension glaucoma patients.

High Serum Advanced Glycation End Products Are Associated with Decreased Insulin Secretion in Patients with Type 2 Diabetes: A Brief Report.

OBJECTIVE: Advanced glycation end products (AGEs) are important in the pathophysiology of type 2 diabetes mellitus (T2DM). They directly cause insulin secretory defects in animal and cell culture models and may promote insulin resistance in nondiabetic subjects. We have developed a highly sensitive liquid chromatography-tandem mass spectrometry method for measuring AGEs in human serum. Here, we use this method to investigate the relationship between AGEs and insulin secretion and resistance in patients with T2DM. METHODS: Our study involved 15 participants with T2DM not on medication and 20 nondiabetic healthy participants. We measured the AGE carboxyethyllysine (CEL), carboxymethyllysine (CML), and methyl-glyoxal-hydro-imidazolone (MG-H1). Plasma glucose and insulin were measured in these participants during a meal tolerance test, and the glucose disposal rate was measured during a euglycemic-hyperinsulinemic clamp. RESULTS: CML and CEL levels were significantly higher in T2DM than non-DM participants. CML showed a significant negative correlation with insulin secretion, HOMA-%B, and a significant positive correlation with the insulin sensitivity index in T2DM participants. There was no correlation between any of the AGEs measured and glucose disposal rate. CONCLUSIONS: These results suggest that AGE might play a role in the development or prediction of insulin secretory defects in type 2 diabetes.

Simultaneous Quantitation of Advanced Glycation End Products in Soy Sauce and Beer by Liquid Chromatography-Tandem Mass Spectrometry without Ion-Pair Reagents and Derivatization.

The aim of this study was to develop a simple and sensitive method to analyze several advanced glycation end products (AGEs) simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to apply this method to the quantitation of AGEs in brown-colored foods. The developed method enabled to separate and quantitate simultaneously seven AGEs, and was applied to the determination of free AGEs contained in various kinds of soy sauce and beer. The major AGEs in soy sauce and beer were Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine (MG-H1). Using the developed LC-MS/MS method, recovery test on soy sauce and beer samples showed the recovery values of 85.3-103.9% for CML, 95.9-107.4% for CEL, and 69.5-123.2% for MG-H1. In particular, it is the first report that free CML, CEL, and MG-H1 were present in beer. Furthermore, long-term storage and heating process of soy sauce increased CML and MG-H1.

Characterisation of Maillard reaction products derived from LEKFD--a pentapeptide found in ß-lactoglobulin sequence, glycated with glucose--by tandem mass spectrometry, molecular orbital calculations and gel filtration chromatography coupled with continuous photodiode array.

Maillard reaction peptides (MRPs) contribute to taste, aroma, colour, texture and biological activity. However, peptide degradation or the cross-linking of MRPs in the Maillard reaction has not been investigated clearly. A peptide of LEKFD, a part of ß-lactoglobulin, was heated at 110 °C for 24h with glucose and the reaction products were analysed by HPLC with ODS, ESI-MS, ESI-MS/MS and HPLC with gel-filtration column and DAD detector. In the HPLC fractions, an imminium ion of LEK*FD, a pyrylium ion or a hydroxymethyl furylium ion of LEK*FD, and KFD and EK were detected by ESI-MS. Therefore, those products may be produced by the Maillard reaction. The molecular orbital of glycated LEKFD at the lysine epsilon-amino residue with Schiff base form was calculated by MOPAC. HPLC with gel-filtration column showed cross-linking and degradation of peptides.

Formation mechanism and characterization of dilysyl-dipyrrolones, the Maillard-type yellow pigments.

Our group has recently isolated and identified novel yellow compounds named dilysyl-dipyrrolones A (DPL A; 1) and B (DPL B; 2) in a heated aqueous solution containing xylose and lysine under weakly acidic conditions. In this study, we isolated and identified a novel DPL derivative (DPL C; 3), which has the same structure as DPL B, except for containing a hydroxymethyl group. To estimate the formation mechanism of DPL derivatives, (13)C-labeled DPLs were prepared and analyzed with LC/MS and NMR. (13)C-labeling experiments using [1-(13)C] ribose showed that the formation pathway of DPL C was different from those of DPLs A and B. In addition, (13)C-labeling experiments using [u-(13)C5] ribose and [1-(13)C] lysine showed that C-6 of a methine moiety in DPL C was derived from C-5 of ribose or acetic acid in buffer. Based on these results, we postulated the formation mechanism of DPLs. We then showed that DPLs A and B had potent antioxidant activities.

Formation and distribution of 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone, a pigment, an aroma and a biologically active compound formed by the Maillard reaction, in foods and beverages.

We recently identified 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone (DHDMT) from soy sauce as a low-molecular-weight pigment formed by the Maillard reaction. DHDMT has also been reported as an aroma compound in a model system and a biologically active compound of heated garlic. To utilize these functions efficiently, we here examined how DHDMT was formed during fermentation of soy sauce and in model systems. Although DHDMT was formed from cysteine and glucose, it was formed more from cystine and fructose in the model system. We also showed that this compound exists in various kinds of soy sauce and miso as well as in some brown foods and beverages such as roasted bread and beer.

Isolation and antioxidant activity of zeylaniin A, a new macrocyclic ellagitannin from Syzygium zeylanicum leaves.

Methanol extract obtained from Syzygium zeylanicum leaves exhibited potent antioxidant activity. The water extract obtained from this methanol extract by sequential extraction with hexane, chloroform, ethylacetate, and n-butanol also showed the strongest antioxidant activity among extracts. This water extract was further fractionated by column chromatography with various concentrations of methanol solutions. Among the 6 resultant fractions, the fraction developed with 20% methanol exhibited the most potent antioxidant activity. The one peak among the three major HPLC peaks in this fraction was isolated and purified using a preparative HPLC. The structure of a pure compound was elucidated as a novel macrocyclic ellagitannin using a (1)H/(13)C NMR and a high-resolution electrospray ionization mass spectrometer. This newly isolated compound, which was named zeylaniin A, exhibited potent antioxidant activities in the assays of DPPH, oxygen radical absorbance capacity, and malonadehyde/gas chromatography. S. zeylanicum leaves can be a possible source of natural antioxidants.

The identification of vitamin E homologues in medicinal plant samples using ESI(+)-LC-MS3.

The aim of this study was to elucidate the presence of vitamin E homologues in medicinal plants. To identify various homologues in the matrix of medicinal plant samples, a method for simultaneous determination was developed using ESI(+)-LC-MS3. A complete separation of each homologue was achieved within 20 min using a PFP column and an isocratic elution system of water/methanol (10:90, v/v) at a flow rate of 0.5 mL/min. The ESI-MS condition for each homologue was optimized, and the m/z value and the fragmentation pathway of each homologue were summarized. This LC-MS3 method made it possible to detect the homologues without the effect of matrix; therefore, high sensitive analysis was established, and then, the MS3 makes it possible to extract from plants with methanol only. The LC-MS3 method was applied to identify the eight vitamin E homologues in 11 medicinal plants.

Identification of 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone as a low-molecular-weight yellow pigment in soy sauce.

The color of soy sauce is considered to be mainly attributable to melanoidins formed by the Maillard reaction. However, the chemical structure of melanoidins cannot be clarified, because melanoidins are high-molecular-weight heterogeneous polymers. We isolated a low-molecular-weight pigment from soy sauce and identified 2,4-dihydroxy-2,5-dimethyl-3(2H)-thiophenone as this pigment formed by the Maillard reaction, although its contribution to the total color of soy sauce was very low.

Conditions for the formation of dilysyl-dipyrrolones A and B, and novel yellow dipyrrolone derivatives formed from xylose and amino acids in the presence of lysine.

Foods derived from plants contain pentose in addition to hexose. It is well known that pentose contributes more to browning by the Maillard reaction than hexose does. We have recently found novel yellow compounds formed from xylose and lysine under weakly acidic conditions, named dilysyldipyrrolones (dilysyl-DPLs) A and B. We indicate in this study that dilysyl-DPLs were specifically formed under weakly acidic conditions from pentose, but not hexose. Moreover, we found novel DPL derivatives which were formed from xylose and such amino acids as alanine, arginine, aspartic acid, glutamic acid, isoleucine, leucine, phenylalanine, serine, and valine in the presence of lysine.

Glutathione reacts with glyoxal at the N-terminal.

The elevation of such dicarbonyl compounds as glyoxal and the depletion of GSH occur simultaneously in diabetic patients. Enabling a nonenzymatic glycation reaction with GSH and glyoxal is therefore proposed. However, the reaction mechanism for GSH and glyoxal has not been precisely defined. We isolated in this study the major products obtained by the reaction of GSH and glyoxal under physiological conditions, and clarified the chemical structure of these compounds by MS and NMR analyses for the first time. We identified the major product after 24 h as N-[3-(2,5-dioxomorpholin-3-yl)propanoyl]cysteinylglycine, and the one after 30 min as N-glycoloyl-gamma-glutamylcysteinylglycine (the intermediate of the former compound). Our results suggest that GSH reacted with glyoxal at the alpha-NH(2) group of the glutamate residue, but not at the SH group of the cysteine residue.