Determination of 15N/14N of Ammonium, Nitrite, Nitrate, Hydroxylamine, and Hydrazine Using Colorimetric Reagents and Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS).

In the nitrogen (N) cycle, nitrogenous compounds are chemically and biologically converted to various aqueous and gaseous N species. The 15N-labeling approach is a powerful culture-dependent technique to obtain insights into the complex nitrogen transformation reactions that occur in cultures. In the 15N-labeling approach, the fates of supplemented 15N- and/or unlabeled gaseous and aqueous compounds are tracked by mass spectrometry (MS) analysis, whereas MS analysis of aqueous N species requires laborious sample preparation steps and is performed using isotope-ratio mass spectrometry, which requires an expensive mass spectrometer. We developed a simple and high-throughput MS method for determining the 15N atoms percent of NH4+, NO2-, NO3-, NH2OH, and N2H4, where liquid samples (<0.5 mL) were mixed with colorimetric reagents (naphthylethylenediamine for NO2-, indophenol for NH4+, and p-aminobenzaldehyde for N2H4), and the mass spectra of the formed N complex dyes were obtained by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS. NH2OH and NO3- were chemically converted to NO2- by iodine oxidation and copper/hydrazine reduction reaction, respectively, prior to the above colorimetric reaction. The intensity of the isotope peak (M + 1 or M + 2) increased when the N complex dye was formed by coupling with a 15N-labeled compound, and a linear relationship was found between the determined 15N/14N peak ratio and 15N atom% for the tested N species. The developed method was applied to bacterial cultures to examine their N-transformation reactions, enabling us to observe the occurrence of NO2- oxidation and NO3- reduction in a hypoxic Nitrobacter winogradskyi culture. IMPORTANCE15N/14N analysis for aqueous N species is a powerful tool for obtaining insights into the global N cycle, but the procedure is cumbersome and laborious. The combined use of colorimetric reagents and MALDI-TOF MS, designated color MALDI-TOF MS, enabled us to determine the 15N atom% of common aqueous N species without laborious sample preparation and chromatographic separation steps; for instance, the 15N atom% of NO2- can be determined from >1,000 liquid samples daily at <$1 (U.S.) per 384 samples for routine analysis. This convenient MS method is a powerful tool that will advance our ability to explore the N-transformation reactions that occur in various environments and biological samples.

Effect of Yokukansan on Nitric Oxide Production and Hydroxyl Radical Metabolism During Cerebral Ischemia and Reperfusion in Mice.

BACKGROUND: The purpose of this study was to investigate the effects of yokukansan on forebrain ischemia. Because we can measure nitric oxide production and hydroxyl radical metabolism continuously, we investigated the effect of yokukansan on nitric oxide production and hydroxyl radical metabolism in cerebral ischemia and reperfusion. METHODS: Yokukansan (300 mg per kg per day) was mixed into feed and given to 16 mice for 10days. Sixteen additional mice received normal feed (control). Nitric oxide production and hydroxyl radical metabolism were continuously monitored using the salicylate trapping method. Forebrain ischemia was producedin all mice by occluding the common carotid artery bilaterally for 10minutes. Levels of the nitric oxide metabolites nitrite and nitrate were determined using the Griess reaction. Survival rates of hippocampal CA1 neurons were calculated and 8-hydroxydeoxyguanosine-immunopositive cells were counted to evaluate the oxidative stress in hippocampal CA1 neurons 72hours after the start of reperfusion. RESULTS: Arterial blood pressure and regional cerebral blood flow were not significantly different between the 2 groups. The level of nitrate was significantly higher in the yokukansan group than in the control group during ischemia and reperfusion. Levels of 2,3- and 2,5-dihydroxybenzoic acid were significantly lower in the yokukansan group than in the control group during ischemia and reperfusion. Although survival rates in the CA1 did not differ significantly, there were fewer 8-hydroxydeoxyguanosine-immunopositive cells in animals that had received yokukansan than in control animals. CONCLUSIONS: These data suggest that yokukansan exerts reducing hydroxyl radicals in cerebral ischemic injury.

Randomized double-blind placebo-controlled multicenter trial of Yokukansan for neuropsychiatric symptoms in Alzheimer's disease.

AIM: Yokukansan (YKS), a traditional herbal medicine, has been used to treat behavioral and psychological symptoms of dementia (BPSD). The present study is the first double-blind, randomized, placebo-controlled trial to determine the efficacy and safety of YKS for the treatment of BPSD in Alzheimer's disease (AD). METHODS: A total of 22 sites consisting of clinics, hospitals and nursing homes participated. A total of 145 patients with AD were randomized. Active YKS (7.5 g/day) and placebo were supplied to 75 and 70 participants, respectively. The primary outcome measure was the 4-week change in total score of the Neuropsychiatric Inventory Brief Questionnaire Form (NPI-Q), an instrument that evaluates BPSD. Secondary outcome measures included 12-week changes in NPI-Q scores, changes in NPI-Q subcategory scores and total scores of the Mini-Mental-State Examination. RESULTS: Four-week changes in NPI-Q total scores did not differ significantly between the treatment and placebo groups. There were also no significant differences between groups in 12-week changes in total NPI-Q scores, NPI-Q subcategory scores or total Mini-Mental-State Examination scores. However, a subgroup with fewer than 20 points on the Mini-Mental-State Examination at baseline showed a greater decrease in "agitation/aggression" score in the YKS group than in the placebo group (P = 0.007). No serious adverse effects were observed during the study. CONCLUSIONS: Our data did not reach statistical significance regarding the efficacy of YKS against BPSD; however, YKS improves some symptoms including "agitation/aggression" and "hallucinations" with low frequencies of adverse events. Geriatr Gerontol Int 2017; 17: 211-218.

Open-label study to evaluate the pharmacodynamics, clinical efficacy, and safety of meropenem for adult bacterial meningitis in Japan.

The aim of this study was to assess the efficacy, safety, and concentration of meropenem in cerebrospinal fluid when meropenem (2 g every 8 h) was administered to Japanese adult patients with bacterial meningitis. Five Japanese patients (mean age 60.6 years [range 35-71]) were enrolled. Infection with Streptococcus pneumoniae (three patients), Streptococcus salivarius (one patient), and Staphylococcus aureus (one patient) was confirmed by cerebrospinal fluid culture. Meropenem (2 g every 8 h) was administered to all five patients. Treatment duration ranged from 14 to 28 days (mean 22.6 days). All the patients were successfully treated. The concentration of meropenem in cerebrospinal fluid ranged from 0.27 to 6.40 µg/ml up to 8.47 h and was over 1 µg/ml 3 h after starting meropenem infusion. In each patient, the present study confirmed for the first time that the concentration of meropenem in cerebrospinal fluid exceeded the minimal inhibitory concentration for these pathogens. Eleven clinical and laboratory adverse events considered to be related to meropenem were observed in all patients, but no serious adverse event and no discontinuance of treatment due to adverse events occurred. Thus meropenem appeared to be a well-tolerated and effective agent for Japanese adult patients with bacterial meningitis. 2 g every 8 h of meropenem was delivered to CSF and its concentration was exceed in MICs for the detected pathogens.

[The practical use of acupuncture and moxibustion treatment cooperated with neurological practice].

The greatest number of patients in our department are those referred from the Department of Neurology. These patients usually present symptoms or conditions such as peripheral facial paralysis, primary headache, or cerebrovascular disorders; acupuncture and moxibustion treatment have resulted in an overall favorable effect. Regarding the effect of acupuncture and moxibustion treatment on the prevention of migraine attacks, a two-month course of acupuncture has been shown to decrease the number of days with a slight or moderate headache. Furthermore, we have seen a relation between the alleviation of headache and that of muscular tenderness in the neck-shoulder region and masticatory muscles. The results of Arterial Spin-Labeling MRI, by which we determine changes in cerebral blood flow before and after acupuncture stimulation in patients with migraine, have revealed that acupuncture stimulation induces an increase of blood flow in the thalamus and hypothalamus, opercular part, cingulate gyrus, and islet. This differs from the response obtained in healthy individuals. It has been shown that acupuncture and moxibustion for tension headache is highly effective and the action mechanism of acupuncture and moxibustion is associated with relaxation of masticatory muscles hypertonicity in the neck-shoulder region and normalization of circulation dynamics, contributing thereby to the alleviation of headache. Moreover, the automatic nervous system has been found to be related to such analgesic mechanism. Based on our experience, we consider that traditional medicine based on acupuncture and moxibustion is highly effective in patients with neurological symptoms.

Arterial spin-labeled MRI study of migraine attacks treated with rizatriptan.

Spin-tag perfusion imaging is an MRI method that quantitatively measures cerebral blood flow. Compared with conventional perfusion techniques, advantages of this arterial spin-labeling (ASL) include repeatability and the avoidance of intravenous contrast administration. In the present study, we performed an analysis of 3T high-field MRI examinations utilizing ASL perfusion during migraine attacks. A 32-year-old male patient was studied in three situations: during migraine attack within 1 h post-onset, 30 min after oral administration of rizatriptan 10 mg, and attack-free period. Normalized ASL images acquired during migraine attack showed significant relative hypoperfusion in the bilateral median thalamic areas including hypothalamus and significant relative hyperperfusion in the frontal cortex compared to images acquired during the migraine-free state. When normalized ASL images acquired 30 min after treatment were compared with those acquired during the attack, relative improvement of perfusion in the bilateral median thalamic areas including hypothalamus was observed. Hypothalamus and its surrounding areas may participate in the pathogenesis in migraine attack.

Effect of nilvadipine on regional cerebral blood flow in a patient with early Alzheimer disease.

Hypertension in an 83-year-old woman with early Alzheimer disease was treated with a calcium channel blocker, nilvadipine, for 3 months. Before and after nilvadipine treatment, global and regional cerebral blood flow (gCBF and rCBF) were measured using Tc-99m ethyl cysteinate dimer SPECT. This treatment elevated both the Mini-Mental State Examination score from 23 to 27 and gCBF from 37.6 to 42.0 mL/100 g/min. This treatment also elevated rCBF prominently in the bilateral frontal cortex, right parietal cortex, and posterior cingulate cortex. These areas with rCBF increase generated by subtraction of pretreatment SPECT from post-treatment SPECT were demonstrated on a coregistered MRI.