Development of an Analytical Method for Unsaturated Disaccharides from Heparan Sulfate Using Dansylhydrazine Derivatization and High-Performance Liquid Chromatography with Fluorescence Detection.

Glycosaminoglycans (GAGs), such as heparan sulfate (HS), play essential roles in living organisms. Understanding the functionality of HS and its involvement in disease progression necessitates the sensitive and quantitative detection of HS-derived unsaturated disaccharides. Conventionally, fluorescence derivatization precedes the HPLC analysis of these disaccharides. However, the presence of excess unreacted derivatization reagents can inhibit rapid and sensitive analysis in chromatographic determinations. In this study, we describe analytical methods that use dansylhydrazine as a derivatization agent for the detection and determination of HS-derived unsaturated disaccharides using HPLC. In addition, we have developed a straightforward method for removing excess unreacted reagent using a MonoSpin NH2 column. This method may be employed to remove excess pre-labeling reagents, thereby facilitating the analysis of HS-derived unsaturated disaccharides with satisfactory reproducibility.

Elucidation of Degradation Behavior of Barbiturates in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (V).

The gastric stability of eight barbiturates (BARs) (barbital, primidone, allobarbital, phenobarbital, cyclobarbital, pentobarbital, secobarbital, and thiobutabarbital (TBB)) was examined in artificial gastric juice using LC/UV detection. Among the eight BARs, only TBB was degraded at higher temperatures. Furthermore, the degradation product of TBB was isolated, structurally analyzed, and finally identified as 5-butan-2-yl-5-ethyl-1,3-diazinane-2,4,6-trione, also known as butabarbital. The study elucidated that butabarbital was formed by substituting the sulfur atom of the carbonyl group at the 2-position of TBB with an oxygen atom under acidic condition.

Elucidation of Degradation Behavior of Nitrazepam, a Benzodiazepine Drug, under Basic Conditions: Study on Degradability of Drugs in Stomach (IV).

This study investigates the stability of nitrazepam (NZP), a benzodiazepine drug, under basic conditions, since alkaline putrefactive amines and ammonia are produced once bodies are left to decompose for a long period postmortem after a murder involving NZP or an accidental overdose of NZP. The degradation of NZP in an aqueous alkaline solution was investigated by LC/photodiode array detector (PDA) where the NZP degradation product was isolated and purified by solid-phase extraction using Oasis® MCX, and its chemical structure was determined by LC/time-of-flight (TOF)-MS, NMR spectroscopy, and single-crystal X-ray crystallography. The results revealed that NZP was immediately degraded under basic conditions with 2-amino-5-nitrobenzophenone being an intermediate which further degraded to provide 2-hydroxy-5-nitrobenzophenone as the final degradation product. These results are expected to be useful in clinical chemistry and forensic science, such as the detection of drugs during postmortem examination and suspected addiction.

[Implementation and Evaluation of Risk Communication Regarding Residual Pesticides].

In this study, a public seminar on risk communication methods was conducted to raise awareness and disseminate accurate knowledge about residual pesticides to consumers. Additionally, surveys on consumer awareness were conducted on the attendees before and after the seminar to evaluate its effectiveness. Responses were obtained from 84 participants. The paired t-test was used to analyze the changes in awareness before and after the seminar. The results showed significant improvements in "trust in the government" and "understanding of residual pesticides." Furthermore, step-wise multiple regression analysis was performed to explore the factors influencing satisfaction with the risk communication seminar, and the item "understanding of the safety of residual pesticides in food" was extracted. Understanding food safety is a crucial concern in daily life for consumers. To enable consumers to have an accurate understanding of food risks and make appropriate judgments, it is essential to continue implementing risk communication and conveying information about food safety and security in the future.

Determination of Cyanide and Cyanoglycosides in Sweetened Bean Paste by HPLC with Fluorescence Detection.

It is necessary to evaluate the efficiency of reduction for cyanide and cyanoglycosides during the manufacturing process from raw material beans to sweetened bean paste in a food hygiene control system from the viewpoint of food safety. Analytical methods for cyanide and cyanoglycoside determination in sweetened bean paste by HPLC with fluorescence detection were developed. In analysis of collection time of free cyanide in the free cyanide assay, the recovery was improved by extending the collection time, the recovery rate was >80% by 2 h. The accuracy, repeatability and intra-laboratory precision of the free cyanide assay were 82.3, 2.0, and 2.4%, respectively. The method for cyanoglycoside analysis was evaluated by 5 repeated spiked recovery experiments at a concentration of 10 ppm. The accuracy, repeatability and intra-laboratory precision of the cyanoglycoside method were 82.2, 1.9, and 3.4%, respectively. These analytical methods will enable the analysis of cyanide and cyanoglycosides in sweetened bean paste without using steam distillation method in the pretreatment.

Analysis of Methamphetamine in Urine by HPLC with Solid-Phase Dispersive Extraction and Solid-Phase Fluorescence Derivatization.

We have developed a fluorescence detection-liquid chromatography (HPLC-FL) method that involves sample pretreatment by solid-phase dispersive extraction (SPDE) and solid-phase fluorescence derivatization for the simple and rapid analysis of methamphetamine (MA) in urine. This method uses a reversed-phase polymeric solid-phase gel to clean up analytes in SPDE, followed by fluorescence derivatization with 9-fluorenylmethyl chloroformate (FMOC) in the solid-phase. The optimal conditions for SPDE and solid-phase fluorescence derivatization were obtained when J-SPEC PEP was used as the solid-phase gel and 0.5 mmol/L FMOC in 50 mmol/L borate buffer solution (pH 10) was used as the fluorescence derivatization reagent. The recovery experiment of MA in urine yielded a clean chromatogram with no interfering peaks, demonstrating the validity of our method; the recoveries were 83.6% when spiked at a low concentration level (100 ng/mL) and 80.7% when spiked at a high concentration level (1000 ng/mL). Compared with the conventional liquid-phase method, the reaction product (FMOC-MA) of solid-phase fluorescence derivatization had higher stability. Reaction rate constants were calculated by changing the temperature conditions, and physicochemical parameters, including activation energy and activation entropy involved in the degradation reaction, were obtained from the Arrhenius plot and analyzed thermodynamically. Taken together, our results suggest that the HPLC-FL method with SPDE and solid-phase fluorescence derivatization for sample pretreatment provides a simple and rapid means of analyzing MA in urine samples.

Elucidation of Degradation Behavior of Hydroxy Group-Containing Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (III).

The degradation behavior of three benzodiazepines (BZPs)-lormetazepam (LMZ), lorazepam, and oxazepam-with hydroxy groups on the diazepine ring in artificial gastric juice and the effect of storage pH conditions on drug degradability were monitored using an LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. Although the three BZPs degraded in artificial gastric juice, none could be restored, despite increasing the storage pH, implying that the degradation reaction was irreversible. As for LMZ, we discussed the physicochemical parameters, such as the activation energy and activation entropy involved in the degradation reaction as well as the reaction kinetics; one of the degradation products was isolated and purified for structural analysis. In the LMZ degradation experiment, peaks corresponding to degradation products, (A) and (B), were detected through the LC/PDA measurements. Regarding the degradation behavior, we hypothesized that LMZ was degraded into (B) via (A), where (A) was an intermediate and (B) was the final product. Although the isolation of degradation product (A) was challenging, degradation product (B) could be isolated and was confirmed to be "methanone, [5-chloro-2-(methylamino)phenyl](2-chlorophenyl)-" based on structure determination using various instrumental analyses. The compound exhibited axis asymmetry as determined using single-crystal X-ray structure analysis. Because the formation of degradation product (B) was irreversible, it would be prudent to target the final degradation product (B) and LMZ for identification when detecting LMZ in human stomach contents, such as during forensic dissection.

[Severe neuropsychiatric disorder developing during busulfan-containing regimen for stem cell transplantation].

Here we describe two patients that required interruption of a busulfan (BU) containing conditioning regimen due to severe mental disorder before stem cell transplantation. The first patient was a 66-year-old man scheduled for unrelated peripheral blood stem cell transplantation with fludarabine/BU conditioning for myelodysplastic syndrome. He received 9.6 mg/kg BU and developed hallucinations that worsened the next day. BU was stopped on the final day, but the patient became comatose (grade 4). He recovered the next day. The second patient was a 69-year-old man scheduled for autologous peripheral blood stem cell transplantation with thiotepa (TT)/BU conditioning for cerebral nervous system relapse of mantle cell lymphoma. He received 12.8 mg/kg BU and developed hallucinations. His mental symptoms worsened on the next day, and thus administration was stopped on the second day of TT. His symptoms improved the next day. Both patients were over 65 years old, and their psychiatric symptoms worsened 1-2 days after the final dose of BU. Our findings suggest that BU may cause psychiatric disorders in elderly patients. When performing BU conditioning, it may be necessary to avoid azole antifungal medication and acetaminophen and to reduce the dose or perform therapeutic dose monitoring for elderly patients.

Serum-Induced Expression of Brain Natriuretic Peptide Contributes to Its Increase in Patients with HFpEF.

Brain natriuretic peptide (BNP) levels are increased in both patients with heart failure with preserved (HFpEF) and reduced ejection fraction (HFrEF), but the reasons for this remain unclear. Our purpose was to examine whether serum-induced BNP (iBNP) expression partly contributes to increased BNP in patients with HFpEF. BNP reporter cardiomyocytes from pBNP-luc-KI mice were stimulated with serum from patients with HFpEF or HFrEF (n = 114 and n = 82, respectively). Luciferase activity was examined as iBNP and the iBNP-to-BNP ratio was evaluated. Patient characteristics and clinical parameters were compared, and multivariate regression analysis was performed to determine independent predictors of the iBNP-to-BNP ratio. Female sex and frequencies of atrial fibrillation, hypertension and the use of a calcium channel blocker (CCB) were higher in HFpEF. The iBNP-to-BNP ratio was significantly higher in HFpEF (26.9) than in HFrEF (16.1, p < 0.001). Multivariate regression analysis identified the existence of HFpEF as an independent predictor of the iBNP-to-BNP ratio after adjusting for all other measurements (ß = 0.154, p = 0.032). Age, hemoglobin, CCB usage and deceleration time were also independent predictors (ß = 0.167, p = 0.025; ß = 0.203, p = 0.006; ß = 0.138, p = 0.049; and ß = 0.143, p = 0.049, respectively). These results indicate that the elevated BNP in patients with HFpEF is partly due to iBNP from the heart.

Elucidation of Degradation Behavior of Nitrazepam and Other Benzodiazepines in Artificial Gastric Juice: Study on Degradability of Drugs in Stomach (II).

The degradation behavior of eight benzodiazepines (BZPs): alprazolam, etizolam, diazepam, triazolam, nitrazepam (NZP), flunitrazepam (FNZ), bromazepam, and lorazepam, in artificial gastric juice was monitored by a LC/photodiode array detector (PDA) to estimate their pharmacokinetics in the stomach. For drugs that were degradable, such physicochemical parameters as reaction rate constant were measured to evaluate the effect of storage conditions on drug degradability, such as whether the degradation proceeds faster by increasing storage temperature, or whether the degradation reaction is reversible by adjusting pH. As a result, it was confirmed that although the eight BZPs degraded in artificial gastric juice, most of them could be restored when pH was increased, and the restoration rates differed depending on the pH and the type of BZP. As for NZP, an Arrhenius plot was drawn to obtain the physicochemical parameters, such as activation energy and activation entropy involved in the degradation reaction, and the reaction kinetics was discussed. In addition, two substances were confirmed as the degradation products of NZP in artificial gastric juice: one was a reversible degradation product (A) (intermediate) and the other was an irreversible degradation product (B) (final degradation product). The intermediate was identified as 2-amino-N-(2-benzoyl-4-nitrophenyl)-acetamide, and the final degradation product was 2-amino-5-nitrobenzophenone. Therefore, when detecting NZP in human stomach contents, such as during judicial dissection, it would be prudent to target NZP as well as the intermediate (A) and the final degradation product (B).

[Evaluation of a Risk Communication Program for Pesticide Residues].

To promote and raise the awareness of accurate knowledge on pesticide residues, the symposium program on risk communication on pesticide residues was held by the broadcasted online style. The risk communication program was statistically evaluated using a pre- and post-program online questionnaire survey. We had the questionnaire answers of the 105 valid participants. The analysis of post-program questionnaires shows that the risk communication program was effective in terms of levels of understanding and interest. Pre-program risk perception or awareness of safety assessments was significantly and positively correlated with awareness for establishing standard values of pesticide residues by the analysis of pre-program questionnaires. Risk perception after the program was significantly higher than before the program, suggesting that risk perception increased due to the program by analysis the same question between pre- and post-program questionnaires. Multiple regression analysis suggests that the participants with higher pre-program awareness of safety assessments or pre-program awareness for establishing standard values appeared to have higher levels of understanding and post-program risk perception.

Sodium-powered stators of the bacterial flagellar motor can generate torque in the presence of phenamil with mutations near the peptidoglycan-binding region.

The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error-prone PCR to introduce single point mutations into the sodium-powered Vibrio alginolyticus/Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodium-channel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C-terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion-channel blockers.

Elucidation of Degradation Behavior of Tricyclic Antidepressant Amoxapine in Artificial Gastric Juice.

The degradation behavior of eight tricyclic antidepressants (TCAs; amitriptyline, amoxapine (AMX), imipramine, clomipramine, desipramine, doxepin, dothiepin, and nortriptyline) in artificial gastric juice was investigated to estimate their pharmacokinetics in the stomach. As a result, among the eight TCAs, only AMX was degraded in artificial gastric juice. The degradation was a pseudo first-order reaction; activation energy (Ea) was 88.70 kJ/mol and activation entropy (ΔS) was -80.73 J/K·mol. On the other hand, the recovery experiment revealed that the degradation product did not revert to AMX and accordingly, this reaction was considered to be irreversible. In the AMX degradation experiment, peaks considered to be degradation products A (I) and B (II) were detected at retention times of around 3 min and 30 min in LC/UV measurements, respectively. Structural analysis revealed that compound (I) was [2-(2-aminophenoxy)-5-chlorophenyl]-piperazin-1-yl-methanone, a new compound, and compound (II) was 2-chlorodibenzo[b,f][1,4]oxazepin-11(10H)-one. As for the degradation behavior, it was estimated that AMX was degraded into (II) via (I), i.e., (II) was the final product. The results are expected to be useful in clinical chemistry and forensic science, including the estimation of drugs to be used at the time of judicial dissection and suspected drug addiction.

[Lupus anticoagulant-hypoprothrombinemia syndrome associated with follicular lymphoma].

A 56-year-old woman was referred to our hospital with symptoms of swelling, purpura, and pain in her limbs. Prior to referral, bleeding in her limbs had spontaneously appeared and disappeared several times. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged, and the factor II level was 17%. The plasma-mixing test indicated lupus anticoagulant (LA), which was confirmed using aPTT-LA and dilute Russell's viper venom time (dRVVT). Therefore, she was diagnosed with lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS). During screening for underlying disorders, chest computed tomography (CT) revealed a retrosternal mass. Biopsy was not performed because the administration of freshly frozen plasma failed to correct her coagulopathy. Prednisolone (PSL) treatment (1 mg/kg) was initiated, which normalized the coagulation tests. The retrosternal mass also disappeared. PSL was tapered without LAHPS recurrence; however, the follow-up CT revealed systemic lymphadenopathy. Follicular lymphoma was diagnosed using lymph-node biopsy. Considering the subsequent LAHPS recurrence, six cycles of bendamustine + rituximab were administered. Complete response with no LAHPS recurrence was observed at the time of drafting this report. LAHPS is rare and distinct from antiphospholipid syndrome because it can cause severe bleeding. Underlying disorders should be evaluated in cases of LAHPS.

Clinical utility of chlormadinone acetate (Lutoral™) in frozen-thawed embryo transfer with hormone replacement.

PURPOSE: The clinical utility of chlormadinone acetate tablets (Lutoral™), an orally active progestin which has been available since June 2007, was compared to an in-house vaginal suppository formulation of progesterone used between 2006 and 2007 for assisted reproductive technology (ART). METHODS: We retrospectively evaluated the efficacy and safety of chlormadinone acetate by comparing the pregnancy rates and the incidences of birth defects and hypospadias in frozen-thawed embryo transfer cycles using the in-house vaginal progesterone and those using chlormadinone acetate for luteal phase support. RESULTS: The pregnancy rates in the frozen-thawed embryo transfer cycles were 31.2% (259/831) with vaginal progesterone for luteal phase support and 31.6% (4228/13 381) with chlormadinone acetate (no significant difference). In the cycles resulting in live birth following administration of chlormadinone acetate between July 2007 and December 2015, the incidence of birth defects was 2.8% (80/2893), and the incidence of hypospadias was 0.03% (1/2893). CONCLUSIONS: These results indicate that the pregnancy rate following frozen-thawed embryo transfer using chlormadinone acetate for luteal phase support was comparable with that using vaginal progesterone, with no increased risk of birth defects, including hypospadias, which has been a concern following the use of progestins.

Performance of anti-Müllerian hormone (AMH) levels measured by Beckman Coulter Access AMH assay to predict oocyte yield following controlled ovarian stimulation for in vitro fertilization.

PURPOSE: We evaluated the performance of anti-Müllerian hormone (AMH) measured by the Beckman Coulter fully automated Access assay to predict oocyte yield following controlled ovarian stimulation (COS) for in vitro fertilization (IVF). METHODS: The correlation between the Access assay and the pre-mixing method with Generation II ELISA assay (Gen II pre-mix assay) was assessed using 230 blood samples. The relationship of AMH level measured by the Access assay and the actual number of oocytes retrieved following COS was assessed using 3296 IVF cycles. The performances of AMH, follicle stimulating hormone (FSH), and estradiol (E2) in predicting the responses to COS were also evaluated by constructing receiver operating characteristic (ROC) curves. RESULTS: The AMH levels measured just before oocyte retrieval by the Access assay and the number of oocytes retrieved following COS showed a good correlation with R = 0.655. The ROC analysis revealed that the sensitivity of AMH was comparable with or lower than that of E2 but higher than that of FSH. CONCLUSIONS: With the improved Access AMH assays, AMH was as sensitive as E2 and could become an accurate marker of ovarian response to COS in more than 3000 Japanese IVF patients.

The first nationwide surveillance of antibacterial susceptibility patterns of pathogens isolated from skin and soft-tissue infections in dermatology departments in Japan.

To investigate the trends of antimicrobial resistance in pathogens isolated from skin and soft-tissue infections (SSTI) at dermatology departments in Japan, a Japanese surveillance committee conducted the first nationwide survey in 2013. Three main organisms were collected from SSTI at 30 dermatology departments in medical centers and 10 dermatology clinics. A total of 860 strains - 579 of Staphylococcus aureus, 240 of coagulase-negative Staphylococci, and 41 of Streptococcus pyogenes - were collected and shipped to a central laboratory for antimicrobial susceptibility testing. The patient profiles were also studied. Among all 579 strains of S. aureus, 141 (24.4%) were methicillin-resistant (MRSA). Among 97 Staphylococcus epidermidis strains, 54 (55.7%) were methicillin-resistant (MRSE). MRSA and MRSE were more frequently isolated from inpatients than from outpatients. Furthermore, these methicillin-resistant strains were also isolated more frequently from patients with histories of taking antibiotics within 4 weeks and hospitalization within 1 year compared to those without. However, there were no significant differences in MIC values and susceptibility patterns of the MRSA strains between patients with a history of hospitalization within 1 year and those without. Therefore, most of the isolated MRSA cases at dermatology departments are not healthcare-acquired, but community-acquired MRSA. S. pyogenes strains were susceptible to most antibiotics except macrolides. The information in this study is not only important in terms of local public health but will also contribute to an understanding of epidemic clones of pathogens from SSTI.

Important role of calcium chloride in preventing carbon monoxide generation during desflurane degradation with alkali hydroxide-free carbon dioxide absorbents.

We investigated whether calcium chloride (CaCl2), a supplementary additive in carbon dioxide (CO2) absorbents, could affect carbon monoxide (CO) production caused by desflurane degradation, using a Japanese alkali-free CO2 absorbent Yabashi Lime®-f (YL-f), its CaCl2-free and 1% CaCl2-added derivatives, and other commercially available alkali-free absorbents with or without CaCl2. The reaction between 1 L of desflurane gas (3-10%) and 20 g of desiccated specimen was performed in an artificial closed-circuit anesthesia system for 3 min at 20 or 40 °C. The CO concentration was measured using a gas chromatograph equipped with a semiconductor sensor detector. The systems were validated by detecting dose-dependent CO production with an alkali hydroxide-containing CO2 absorbent, Sodasorb®. Compared with YL-f, the CaCl2-free derivative caused the production of significantly more CO, while the 1% CaCl2-added derivative caused the production of a comparable amount of CO. These phenomena were confirmed using commercially available absorbents AMSORB® PLUS, an alkali-free absorbent with CaCl2, and LoFloSorb™, an alkali-free absorbent without CaCl2. These results suggest that CaCl2 plays an important role in preventing CO generation caused by desflurane degradation with alkali hydroxide-free CO2 absorbents like YL-f.

Evidence of exposure to chemicals and heavy metals during pregnancy in Japanese women.

Purpose: Prenatal exposure to environmental chemicals is a growing concern, because such exposures have been shown to be associated with various diseases. The levels of chemicals and heavy metals in maternal blood, cord blood, maternal urine and amniotic fluid in Japanese pregnant women were investigated. Methods: A total of 145 women, including 14 fetal growth restriction cases, were included in the present study. The levels of phthalates (di[2-ethylhexyl]phthalate and mono[2-ethylhexyl]phthalate), perfluorinated compounds (perfluorooctane sulfonate, perfluorohexanoic acid, perfluorooctanoic acid, and perfluorononanoic acid), pesticides (dimethylphosphate, dimethylthiophosphate, diethylphosphate, diethylthiophosphate, 3-phenoxybenzoic acid, and octachlorodipropyl ether), bisphenol A, nicotine (nicotine, nornicotine, cotinine, norcotinine, and trans-3'-hydroxycotinine), polybrominated diphenyl ethers, and heavy metals were measured. The relationship between fetal growth and the levels of chemicals and heavy metals were investigated. Results: Phthalates, perfluorinated compounds, pesticides, polybrominated diphenyl ethers, and heavy metals were detected in high frequency, whereas nicotine and bisphenol A were almost negative. Phthalates, perfluorinated compounds, and several heavy metals were transferred to the fetus. High perfluorononanoic acid levels in the maternal blood and cord blood, and low perfluorooctanoic acid level in the cord blood were significantly and negatively associated with fetal growth. Conclusions: The present study showed that pregnant women in Japan and their fetuses are exposed to a variety of chemicals and heavy metals.

[Collaboration of Hospitals and Home Care Providers - Lessons to be Learned from an Actual Case to Enable Patients to Keep Living at Home by Their Own Values].

The aging society has arrived, and home care options are being promoted. To date, because of an increase in the number of patients with higher disability levels in greater need of medical help and senior people living alone or in senior-person households, more patients are in need of discharge support. Many medical institutions cannot consider the option of staying at home and receiving home care services for such patients, and such patients often give up on going home. In the case of service recipients suffering from incurable diseases or disabilities and undergoing age-related changes, it is necessary that they decide on their own"where and how they want to live."For their decision to be respected, a key is collaboration with those who support patient discharge from the hospital and those who support patient home care. This paper explores a patient appropriate for level-3 care with brain hemorrhage and paralysis of the left half of the body. This patient required supportive and adaptive devices, a cane for walking, and constant observation. In consideration of the wishes of the patient and his family, he was allowed to go home.