Simultaneous analysis of degradation products of Novichok agents and conventional nerve agents in human urine by ion chromatography-tandem mass spectrometry using ammonium regeneration solution.

An ion chromatography (IC)-tandem mass spectrometry (MS/MS) method to analyze nerve agent degradation products in human urine was developed. Six degradation products of conventional nerve agents and six Novichok agent degradation products were analyzed simultaneously despite their differences in hydrophilicity and acidity. Using ammonium regeneration solution improved the peak shapes greatly compared with the results obtained with the ordinary IC-MS/MS configuration. For urine samples, a simple pretreatment method of dilution with water and ultrafiltration was used. The detection limits of the nerve agent degradation products were sufficiently low (10-250 ng/mL) and the calibration curves showed acceptable linearity. Due to the absence of a derivatization step, throughput was higher than for our previous derivatization-liquid chromatography-MS/MS method.

Risk of non-hypoglycemic agents for hypoglycemia-related hospitalization in patients with type 2 diabetes: a large-scale medical receipt database analysis.

INTRODUCTION: Hypoglycemia is listed as an adverse effect in the package inserts of not only hypoglycemic agents but also many other drugs. We aimed to clarify real-world factors related to an increased risk of hypoglycemia-related hospitalization (HRH) in Japanese patients with type 2 diabetes (T2D) on non-hypoglycemic agents that have been associated with hypoglycemia. RESEARCH DESIGN AND METHODS: This cross-sectional study was performed using data from the Medical Data Vision administrative claims database. We identified patients with T2D who were enrolled in the database between April 2014 and October 2019. Logistic regression analyses were performed to identify clinical factors associated with HRH due to non-hypoglycemic agents. RESULTS: Among 703 745 patients with T2D, 10 376 patients (1.47%) experienced HRH. The use of 332 non-hypoglycemic agents was associated with hypoglycemia. Multivariate analysis was performed to calculate OR for HRH. Seventy-five drugs had an OR greater than 1, and the values were significant. The OR was the highest for diazoxide (OR 15.5, 95% CI 4.87 to 49.3). The OR was higher than 2.0 for methylphenidate (OR 5.15, 95% CI 1.53 to 17.3), disulfiram (OR 4.21, 95% CI 2.05 to 8.62) and hydrocortisone (OR 2.89, 95% CI 1.11 to 7.51). CONCLUSION: This large retrospective analysis revealed that the risk of HRH from some non-hypoglycemic agents in patients with T2D may be increased. The results of this study are expected to support treatment planning by physicians and healthcare professionals involved in diabetes care.

Analysis of degradation products of nerve agents in biological fluids by ion chromatography-tandem mass spectrometry.

PURPOSE: The detection of hydrolysis products of nerve agents (alkyl methylphosphonic acids; RMPAs) in biological samples from victims is important to confirm exposure to nerve agents. However, analysis of RMPAs is difficult due to their high hydrophilicity. The aim of this study was to develop ion chromatography-tandem mass spectrometry (IC-MS/MS) methods using commercially available equipment and columns to analyze RMPAs in human urine and serum with high sensitivity and without using complicate techniques. METHODS: A Dionex IonPac AS11-HC anion-exchange column was used to analyze six RMPAs (MPA, EMPA, IMPA, iBuMPA, CHMPA, and PMPA). For pretreatments of biological fluids, we developed two pretreatment methods (Method 1: dilution and ultrafiltration; Method 2: removal of chloride ions with Ag cartridges). RESULTS: Six RMPAs including highly hydrophilic methylphosphonic acid and ethyl methylphosphonic acid could be analyzed with sufficient retention times and peak shape. The detection limits of RMPAs were improved using Dionex OnGuard II Ba/Ag/H cartridges and MetaSEP IC-Ag cartridges (urine: 0.5-5 ng/mL; serum: 1-5 ng/mL). These methods were also applied to the test samples for the Organisation for the Prohibition of Chemical Weapons Biomedical Proficiency Tests. CONCLUSIONS: RMPAs could be sufficiently analyzed by IC-MS/MS. In addition, the limits of detection were superior to those obtained in our previous study involving LC-MS/MS or derivatization-LC-MS/MS method. For analysis of biological samples, an appropriate pretreatment method can be chosen according to the amount of sample available for analysis and expected RMPA concentrations.

Analysis of degradation products of Novichok agents in human urine by hydrophilic interaction liquid chromatography-tandem mass spectrometry.

PURPOSE: The detection of hydrolysis products of Novichok agents in biological samples from victims is important for confirming exposure to these agents. However, Novichok agents are new class of nerve agent and there have been only few reports on analyses of Novichok agent degradation products. Here, we developed hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry (MS/MS) methods to detect Novichok agent degradation products in human urine with simple pretreatment and high sensitivity. METHODS: A Poroshell 120 HILIC-Z column was used to analyze six Novichok agent degradation products. For urine samples, we used a simple pretreatment method, which consisted of deproteinization with acetonitrile and microfiltration. We calculated the pKa values of the OH groups, the log P values, and the molecular weights to investigate the difference in chromatographic behaviors of the Novichok agent degradation products and the degradation products of conventional nerve agents. RESULTS: Six Novichok agent degradation products, including N-(bis-(diethylamino)methylidene)-methylphosphonamidic acid (MPGA), which could not be detected by our previous method, could be analyzed with sufficient peak shape and mutual separation. The detection limits of six Novichok agent degradation products were sufficiently low (1-50 ng/mL) and the calibration curves showed sufficient linearity. The physicochemical parameters of Novichok agent degradation products were different from those of conventional nerve agent degradation products, and this explains the difference in chromatographic behaviors. CONCLUSION: Six Novichok agent degradation products were successfully analyzed by HILIC-MS/MS. Due to the absence of a derivatization step, throughput performance was higher than our previous derivatization-liquid chromatography-MS/MS method.

Optimized Mouse-on-mouse Immunohistochemical Detection of Mouse ESR2 Proteins with PPZ0506 Monoclonal Antibody.

Despite the physiological significance of ESR2, a lack of well-validated detection systems for ESR2 proteins has hindered progress in ESR2 research. Thus, recent identification of a specific anti-human ESR2 monoclonal antibody (PPZ0506) and its specific cross-reactivity against mouse and rat ESR2 proteins heightened momenta toward development of appropriate immunohistochemical detection systems for rodent ESR2 proteins. Building upon our previous optimization of ESR2 immunohistochemical detection in rats using PPZ0506, in this study, we further aimed to optimize mouse-on-mouse immunohistochemical detection using PPZ0506. Our assessment of several staining conditions using paraffin-embedded ovary sections revealed that intense heat-induced antigen retrieval, appropriate blocking, and appropriate antibody dilutions were necessary for optimization of mouse-on-mouse immunohistochemistry. Subsequently, we applied the optimized immunostaining method to determine expression profiles of mouse ESR2 proteins in peripheral tissues and brain subregions. Our analyses revealed more localized distribution of mouse ESR2 proteins than previously assumed. Moreover, comparison of these results with those obtained in humans and rats using PPZ0506 revealed interspecies differences in ESR2 expression. We expect that our optimized methodology for immunohistochemical staining of mouse ESR2 proteins will help researchers to solve multiple lines of controversial evidence concerning ESR2 expression.

Neuromedin U-deficient rats do not lose body weight or food intake.

Studies in genetically modified mice establish that essential roles of endogenous neuromedin U (NMU) are anorexigenic function and metabolic regulation, indicating that NMU is expected to be a potential target for anti-obesity agents. However, in central administration experiments in rats, inconsistent results have been obtained, and the essential role of NMU energy metabolism in rats remain unclear. This study aims to elucidate the role of endogenous NMU in rats. We generated NMU knockout (KO) rats that unexpectedly showed no difference in body weight, adiposity, circulating metabolic markers, body temperature, locomotor activity, and food consumption in both normal and high fat chow feeding. Furthermore, unlike reported in mice, expressions of Nmu and NMU receptor type 2 (Nmur2) mRNA were hardly detectable in the rat hypothalamic nuclei regulating feeding and energy metabolism, including the arcuate nucleus and paraventricular nucleus, while Nmu was expressed in pars tuberalis and Nmur2 was expressed in the ependymal cell layer of the third ventricle. These results indicate that the species-specific expression pattern of Nmu and Nmur2 may allow NMU to have distinct functions across species, and that endogenous NMU does not function as an anorexigenic hormone in rats.

Hypothalamic KNDy neuron expression in streptozotocin-induced diabetic female rats.

Kisspeptin neurons, i.e. KNDy neurons, in the arcuate nucleus (ARC) coexpress neurokinin B and dynorphin and regulate gonadotropin-releasing hormone/luteinizing hormone (LH) pulses. Because it remains unclear whether these neurons are associated with reproductive dysfunction in diabetic females, we examined the expression of KNDy neurons detected by histochemistry in streptozotocin (STZ)-induced diabetic female rats 8 weeks after STZ injection. We also evaluated relevant metabolic parameters - glucose, 3-hydroxybutyrate, and non-esterified fatty acids - as indicators of diabetes progression. Severe diabetes with hyperglycemia and severe ketosis suppressed the mRNA expression of KNDy neurons, resulting in low plasma LH levels and persistent diestrus. In moderate diabetes with hyperglycemia and moderate ketosis, kisspeptin-immunoreactive cells and plasma LH levels were decreased, while the mRNA expression of KNDy neurons remained unchanged. Mild diabetes with hyperglycemia and slight ketosis did not affect KNDy neurons and plasma LH levels. The number of KNDy cells was strongly and negatively correlated with plasma 3-hydroxybutyrate levels. The vaginal smear analysis showed unclear proestrus in diabetic rats 3-5 days after STZ injection, and the mRNA expression of kisspeptin in the ARC was decreased 2 weeks after STZ injection in severely diabetic rats. Kisspeptin neurons in the anteroventral periventricular nucleus (AVPV), which induce an LH surge, were unaffected at 2 and 8 weeks after STZ injection regardless of the diabetes severity. These results suggest that diabetes mellitus progression in females may negatively affect ARC kisspeptin neurons but not AVPV kisspeptin neurons, implicating a potential role of ARC kisspeptin neurons in menstrual disorder and infertility.

A self-degradable hydrogel sensor for a nerve agent tabun surrogate through a self-propagating cascade.

Nerve agents that irreversibly deactivate the enzyme acetylcholinesterase are extremely toxic weapons of mass destruction. Thus, developing methods to detect these lethal agents is important. To create an optical sensor for a surrogate of the nerve agent tabun, as well as a physical barrier that dissolves in response to this analyte, we devise a network hydrogel that decomposes via a self-propagating cascade. A Meldrums acid-derived linker is incorporated into a hydrogel that undergoes a declick reaction in response to thiols, thereby breaking network connections, which releases more thiols, propagating the response throughout the gel. A combination of chemical reactions triggered by the addition of the tabun mimic initiates the cascade. The dissolving barrier is used to release dyes, as well as nanocrystals that undergo a spontaneous aggregation. Thus, this sensing system for tabun generates a physical response and the delivery of chemical agents in response to an initial trigger.

Optimization of immunohistochemical detection of rat ESR2 proteins with well-validated monoclonal antibody PPZ0506.

Although there are few well-validated antibodies against ESR2 proteins, a recent validation assessment identified a specific monoclonal antibody against human ESR2 proteins (PPZ0506). Furthermore, our previous study confirmed its cross-reactivity and specificity against rodent ESR2 proteins, enabling the determination of true ESR2 distribution profiles in rodents. Therefore, we aimed to determine optimal conditions for ESR2 detection by PPZ0506 immunostaining and analyze ESR2 distribution in rats. We evaluated several staining conditions using paraffin-embedded and frozen ovary sections. Immunohistochemical staining with PPZ0506 antibody required strong antigen retrieval and appropriate antibody dilution. Subsequent immunohistochemical analysis in multiple tissues under optimized conditions revealed that rat ESR2 proteins are expressed in a more localized manner than previously assumed. Our results suggest that previous immunohistochemical studies using inadequately validated antibodies against ESR2 proteins overestimated their distribution profiles. We expect that optimized immunohistochemical detection with PPZ0506 antibody can help researchers solve several conflicting problems in ESR2 research.

Analysis of nitrogen mustard degradation products via post-pentafluorobenzoylation liquid chromatography-tandem mass spectrometry.

A pentafluorobenzoylation (PFBz)-liquid chromatography-tandem mass spectrometry method was developed for qualitative and quantitative analysis of ethanolamines (EAs, nitrogen mustard degradation products). With this method, highly hydrophilic EAs can be sufficiently analyzed with a commonly used reversed phase column (retention times: (PFBz)2-methyl diethanolamine, 9.1 min; (PFBz)2-ethyl diethanolamine, 9.8 min; and (PFBz)3-triethanolamine, 17.6 min). The applicability of the method for real samples was investigated via recovery tests. Methyl diethanolamine and ethyl diethanolamine were detected at concentrations as low as 1 ng/mL in serum and 10 ng/mL in urine, and quantified within the range of 1-1000 ng/mL and 10-1000 ng/mL, respectively.

Search for Substances That Inhibit Histamine Production Using Used Tea Leaves.

ABSTRACT: As food waste has become a major problem in recent years, measures against food loss have become an urgent issue. When manufacturing or making green tea beverages, large quantities of tea leaves are subsequently disposed of, which results in potential food loss. Moreover, because many of the tea components remain in the used tea leaves, these continue to have value, as these leaves exhibit antibacterial action. Furthermore, histamine is produced from histidine via histidine decarboxylase that is produced by microorganisms, with histamine accumulation potentially causing histamine food poisoning. Although we have been trying to develop a simple method for detecting histamine, there has yet to be a quick detection method established. We examined whether a method using a low concentration of bromocresol indicator in the culture medium was capable of rapidly detecting histamine. Our results demonstrated that when using lower indicator concentrations, there was a faster detection of histamine production, within 4 h. Using this method, we also investigated whether used tea leaf components, which have antibacterial effects, could suppress histamine production. In this study, used leaves from green, oolong, and black teas were analyzed according to different extraction processes. Compared with green tea, oolong and black teas were able to suppress histamine production using lower concentrations, 25 and 12.5% extracts, respectively. In contrast, the inhibitory effect on histamine production by used green tea leaves required a high concentration of 50% used tea leaf extracts. Furthermore, our results suggested that used tea leaves suppress histamine production and that the inhibitory effects vary according to different extracts. Based on these findings, we propose that (i) a more rapid detection method for histamine should be established and (ii) used tea leaf extracts may have applications in the storage and processing of foods associated with an undesirable production of histamine.

Applicability of Anti-Human Estrogen Receptor ß Antibody PPZ0506 for the Immunodetection of Rodent Estrogen Receptor ß Proteins.

Several lines of controversial evidence concerning estrogen receptor ß (ERß) remain to be solved because of the unavailability of specific antibodies against ERß. The recent validation analysis identified a monoclonal antibody (PPZ0506) with sufficient specificity against human ERß. However, the specificity and cross-reactivity of PPZ0506 antibody against ERß proteins from laboratory animals have not been confirmed. In the present study, we aimed to validate the applicability of PPZ0506 to rodent studies. The antibody exhibited specific cross-reactivity against mouse and rat ERß proteins in immunoblot and immunocytochemical experiments using transfected cells. In immunohistochemistry for rat tissue sections, PPZ0506 showed immunoreactive signals in the ovary, prostate, and brain. These immunohistochemical profiles of rat ERß proteins in rat tissues accord well with its mRNA expression patterns. Although the antibody was reported to show the moderate signals in human testis, no immunoreactive signals were observed in rat testis. Subsequent RT-PCR analysis revealed that this species difference in ERß expression resulted from different expression profiles related to the alternative promoter usage between humans and rats. In conclusion, we confirmed applicability of PPZ0506 for rodent ERß studies, and our results provide a fundamental basis for further examination of ERß functions.

Analysis of degradation products of nitrogen mustards via hydrophilic interaction liquid chromatography-tandem mass spectrometry.

A hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for qualitative and quantitative analysis of ethanolamines (EAs), which are nitrogen mustard degradation products. With this method, the retention times of the highly hydrophilic EAs on the HILIC column were sufficient (retention times: methyl diethanolamine, 12.2 min; ethyl diethanolamine, 11.2 min; and triethanolamine, 9.5 min) and the EAs were analyzed more efficiently than with reported HILIC-MS/MS methods. The detection limits of methyl diethanolamine and ethyl diethanolamine in serum and urine using this approach were 15-20 ng/mL. The suitability of the method for real samples was evaluated via recovery tests involving urine and serum, and the method was validated. The MS/MS fragmentation of EAs was discussed based on density functional theory calculations.

Analysis of degradation products of nerve agents via post-pentafluorobenzylation liquid chromatography-tandem mass spectrometry.

In the work reported here, a screening procedure was developed for the detection and identification of RMPAs (nerve agent degradation products) after pentafluorobenzylation using liquid chromatography-tandem mass spectrometry (LC-MS/MS). With this method, all RMPAs, including highly hydrophilic types such as methylphosphonic acid (MPA) and ethyl methylphosphonic acid (EMPA), were sufficiently retained in commonly used reversed-phase columns (retention times: 15.7 and 11.0 min.), and the presence of RMPAs was determined more efficiently than with the conventional direct LC-MS/MS method. The detection limits of RMPAs using this approach (<33 ng) were mostly superior to those observed with direct LC-MS/MS (<74 ng) and gas chromatography-mass spectrometry (GC-MS) after pentafluorobenzylation (<1.1 µg). The applicability of newly developed method toward real samples was evaluated via recovery tests involving urine/serum and wipe tests on various surfaces.

Deprotonative Metalation of Methoxy-Substituted Arenes Using Lithium 2,2,6,6-Tetramethylpiperidide: Experimental and Computational Study.

The reaction pathways of lithium 2,2,6,6-tetramethylpiperidide (LiTMP)-mediated deprotonative metalation of methoxy-substituted arenes were investigated. Importantly, it was experimentally observed that, whereas TMEDA has no effect on the course of the reactions, the presence of more than the stoichiometric amount of LiCl is deleterious, in particular without an in situ trap. These effects were corroborated by the DFT calculations. The reaction mechanisms, such as the structure of the active species in the deprotonation event, the reaction pathways by each postulated LiTMP complex, the stabilization effects by in situ trapping using zinc species, and some kinetic interpretation, are discussed herein.

One-pot Annulation for Biaryl-fused Monocarba-closo-dodecaborate through Aromatic B-H Bond Disconnection.

We have developed a one-pot annulation reaction of monocarba-closo-dodecaborate with cyclic diaryliodonium salts to afford biaryl-fused derivatives. Aryl functionalities are introduced at both the 1-carbon and unreactive ortho-boron vertices of the "σ-aromatic" carborane cage without the need for pre-functionalization. DFT calculations revealed that the palladium-catalyzed C-B bond-formation step in this process proceeds through a concerted metalation-deprotonation (CMD)-type pathway for the B-H bond disconnection on the aromatic cage, though such bonds are generally regarded as hydridic.

"Dumbbell"- and "Clackers"-Shaped Dimeric Derivatives of Monocarba-closo-dodecaborate.

We designed, synthesized, and characterized two types of dimeric forms of monocarba-closo-dodecaborate, namely, a "dumbbell"-shaped dianion having a C-C bond and a "clackers"-shaped monoanion having an iodonium linker. The unique architectures of these anionic molecules were established by X-ray analysis. Spectroscopic analysis, DFT calculations, and reactivity experiments revealed high anionic and chemical stability of both anions, which are crucial properties for weakly coordinating anions.

Ameloblastin Upregulates Inflammatory Response Through Induction of IL-1ß in Human Macrophages.

Ameloblastin (AMBN) is an enamel matrix protein that has various biological functions such as healing dental pulp and repairing bone fractures. In the present study, we clarified the effect of AMBN on the expression of an inflammatory cytokine, interleukin-1ß (IL-1ß) in lipopolysaccharide (LPS)-treated human macrophages. Real-time RT-PCR analysis showed that LPS treatment upregulated expression of the IL-1ß gene in U937 cells. Interestingly, AMBN significantly enhanced IL-1ß gene expression in LPS-treated U937 cells as well as the secretion of mature IL-1ß into culture supernatants by these cells. AMBN also activated caspase-1 p10 expression in LPS-treated U937 cells. Pretreatment with a caspase-1 inhibitor, Z-YVAD-FMK, downregulated the mature IL-1ß expression enhanced by AMBN treatment in LPS-treated U937 cells. A co-immunoprecipitation assay showed that treatment with LPS and AMBN upregulated toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88) interactions, but there was no significant difference compared with LPS treatment alone in U937 cells. In contrast, western blot analysis revealed that AMBN remarkably prolonged the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), a member of the mitogen-activated protein kinase (MAPK) family. An ERK1/2-selective inhibitor, U0126, suppressed expression of the IL-1ß gene as well as its protein expression in U937 cells treated with LPS and AMBN. Taken together, these results indicate that AMBN enhances IL-1ß production in LPS-treated U937 cells through ERK1/2 phosphorylation and caspase-1 activation, suggesting that AMBN upregulates the inflammatory response in human macrophages and plays an important role in innate immunity. J. Cell. Biochem. 118: 3308-3317, 2017. © 2017 Wiley Periodicals, Inc.

Conjugation between σ- and π-Aromaticity in 1-C-Arylated Monocarba-closo-dodecaborate Anions.

Conjugation between σ- and π-aromatic moieties in 1-C-arylated monocarba-closo-dodecaborate anion derivatives 2 has been identified by means of kinetic experimental studies combined with theoretical calculations. We found that the reaction rate of iodination at the 12-B vertex of the carborane anion cage was affected by distal substituents on the benzene ring connected at the antipodal carbon vertex. Hammett and Yukawa-Tsuno plots indicated that substantial resonance effects are involved. Density functional theory calculations enabled detailed interpretation of the electronic interaction.