Targeted Neurotransmitters Profiling Identifies Metabolic Signatures in Rat Brain by LC-MS/MS: Application in Insomnia, Depression and Alzheimer's Disease.

Epidemiological, cross-sectional, and prospective studies have suggested that insomnia, Alzheimer's disease (AD) and depression are mutually interacting conditions and frequently co-occur. The monoamine and amino acid neurotransmitter systems in central nervous system were involved in the examination of neurobiological processes of this symptom complex. However, few studies have reported systematic and contrastive discussion of different neurotransmitters (NTs) changing in these neurological diseases. Thus, it is necessary to establish a reliable analytical method to monitoring NTs and their metabolite levels in rat brain tissues for elucidating the differences in pathophysiology of these neurological diseases. A rapid, sensitive and reliable LC-MS/MS method was established for simultaneous determination of the NTs and their metabolites, including tryptophan (Trp), tyrosine (Tyr), serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA), dopamine (DA), acetylcholine (ACh), norepinephrine (NE), glutamic acid (Glu), and γ-aminobutyric acid (GABA) in rat brain tissues. The mobile phase consisting of methanol and 0.01% formic acid in water was performed on an Inertsil EP C18 column, and the developed method was validated well. Results demonstrated that there were significant differences for 5-HT, DA, NE, Trp, Tyr and ACh between model and control group in all three models, and a Bayes linear discriminant function was established to distinguish these three kinds of nervous system diseases by DA, Tyr and ACh for their significant differences among control and three model groups. It could be an excellent strategy to provide perceptions into the similarity and differentia of mechanisms from the point of NTs' changing in brain directly and a new method to distinguish insomnia, depression and AD from view of essence.

The investigation of immunoprotective and sedative hypnotic effect of total polysaccharide from Suanzaoren decoction by serum metabonomics approach.

Suanzaoren decoction, as one of the traditional Chinese medicine prescriptions, has been most commonly used in Asian countries and reported to inhibit the process of immunodeficiency insomnia. Polysaccharide is important component which also contributes to the role of immunoprotective and sedative hypnotic effects. This study was aimed to explore the immunoprotective and sedative hypnotic mechanisms of polysaccharide from Suanzaoren decoction by serum metabonomics approach. With this purpose, complex physical and chemical immunodeficiency insomnia models were firstly established according to its multi-target property. Serum samples were analyzed using UHPLC/Q-TOF-MS spectrometry approach to determine endogenous metabolites. Then, principal component analysis was used to distinguish the groups, and partial least squares discriminate analysis was carried out to confirm the important variables. The serum metabolic profiling was identified and pathway analysis was performed after the total polysaccharide administration. The twenty-one potential biomarkers were screened, and the levels were all reversed to different degrees in the total polysaccharide treated groups. These potential biomarkers were mainly related to vitamin, sphingolipid, bile acid, phospholipid and acylcarnitine metabolisms. The result has indicated that total polysaccharide could inhibit insomnia triggered by immunodeficiency stimulation through regulating those metabolic pathways. This study provides a useful approach for exploring the mechanism and evaluating the efficacy of total polysaccharide from Suanzaoren decoction.

Metabolomics Strategy Using High Resolution Mass Spectrometry Reveals Novel Biomarkers and Pain-Relief Effect of Traditional Chinese Medicine Prescription Wu-Zhu-Yu Decoction Acting on Headache Modelling Rats.

Headache is a common episodic or chronic neurologic disorder. Treatment options and diagnosis are restricted by an incomplete understanding of disease pathology and the lack of diagnostic markers. Wu-Zhu-Yu decoction (WZYD), a traditional Chinese medicine (TCM) formula containing four TCM herbs, is commonly used in the treatment of headache in China. To deeply understand more about headache and investigate the pain-relief mechanism of WZYD, a comprehensive metabolomics study combined with multivariate data processing strategy was carried out. An LC-high resolution mass spectrometry-based metabolomics approach was applied to characterize metabolic biomarker candidates. Multiple pattern recognition including principal component analysis-discriminant analysis, partial least squares-discriminant analysis and hierarchical cluster analysis were used to determine groups and confirm important variables. A total of 17 potential biomarkers were characterized and related metabolic pathways were identified. The study demonstrated that the established metabolomics strategy is a powerful approach for investigating the mechanism of headache attack and WZYD. In addition, the approach may highlight biomarkers and metabolic pathways and can capture subtle metabolite changes from headache, which may lead to an improved mechanism understanding of central nervous system diseases and TCM treatment.

An integrative investigation of the toxicity of Aconiti kusnezoffii radix and the attenuation effect of its processed drug using a UHPLC-Q-TOF based rat serum and urine metabolomics strategy.

Aconiti kusnezoffii radix (AKR), the root of Aconitum kusnezoffii Reichb., is commonly used in the treatment of the rheumatoid arthritis. However, the clinical application is limited due to its potential toxicity. Therefore, to investigate the mechanism of its potential neurotoxicity and nephrotoxicity, a comprehensive metabolomics study combined with serum biochemistry and histopathology measurements was carried out. A UHPLC-Q-TOF mass spectrometry based metabolomics approach was applied to characterize the AKR toxicity, while the toxicity attenuation effects of Aconiti kusnezoffii radix cocta (AKRC) on Wistar rats were also investigated. Two chromatographic techniques involving reversed-phase chromatography and hydrophilic interaction chromatography were combined for the serum and urine detection, which balanced the integrity and selectivity of the two matrices. Principal component analysis was used to determine the groups, and principal component analysis discriminant analysis was carried out to confirm the important variables. Then, the developed integrative toxicity evaluation method was applied to assess the toxicity of AKR and the attenuation effect of AKRC. The highly sensitive and specific toxic biomarkers, which can provide practical bases were identified for the diagnosis of the neurotoxicity and nephrotoxicity induced by AKR. In all, a total of 19 putative biomarkers were characterized, and related metabolic pathways were identified. The study demonstrated that the established metabolomics strategy is a powerful approach for investigating the mechanisms of herbal toxicity and the attenuation effect of a processing method and would provide medical solutions for other toxic herbal medications and further clinical evidence on how AKR improves symptoms of rheumatoid arthritis patients.

Simultaneous quantitative determination of 13 active components in the traditional Chinese medicinal preparation Suanzaoren oral liquid by HPLC coupled with diode array detection and evaporative light scattering detection.

To control the quality of different forms of Suanzaoren decoction, an effective and reliable method for the simultaneous determination of 13 major components (neomangiferin, mangiferin, spinosin, liquiritin apioside, liquiritin, 6'''-feruloylspinosin, senkyunolide I, timosaponin BII, isoliquiritoside, timosaponin C, jujuboside A, jujuboside B, and timosaponin AIII) was developed and validated for the first time in this study using high-performance liquid chromatography with diode array detection and evaporative light scattering detection. The chromatographic separation was performed on a Venusil MP C18 column (250 mm × 4.6 mm, 5 µm) at 30°C with a gradient of acetonitrile/redistilled water as the mobile phase. Diode array detection was carried out at a wavelength of 275 nm. The drift tube temperature and the nitrogen gas flow rate of the evaporative light scattering detection were set at 50°C and 1.6 L/min, respectively. The newly developed method was successfully applied to the determination of 13 components in lab-prepared Suanzaoren oral liquid, Suanzaoren mixture, and clinical Suanzaoren granules, and this study showed that this was a useful way to comprehensively evaluate the quality of Suanzaoren decoction in different forms of the preparation.

An Investigation on the Quantitative Structure-Activity Relationships of the Anti-Inflammatory Activity of Diterpenoid Alkaloids.

Diterpenoid alkaloids are extracted from plants. These compounds have broad biological activities, including effects on the cardiovascular system, anti-inflammatory and analgesic actions, and anti-tumor activity. The anti-inflammatory activity was determined by carrageenan-induced rat paw edema and experimental trauma in rats. The number of studies focused on the determination, quantitation and pharmacological properties of these alkaloids has increased dramatically during the past few years. In this work we built a dataset composed of 15 diterpenoid alkaloid compounds with diverse structures, of which 11 compounds were included in the training set and the remaining compounds were included in the test set. The quantitative chemistry parameters of the 15 diterpenoid alkaloids compound were calculated using the HyperChem software, and the quantitative structure-activity relationship (QSAR) of these diterpenoid alkaloid compounds were assessed in an anti-inflammation model based on half maximal effective concentration (EC50) measurements obtained from rat paw edema data. The QSAR prediction model is as follows: log ( E C 50 ) = - 0.0260 × SAA + 0.0086 × SAG + 0.0011 × VOL - 0.0641 × HE - 0.2628 × LogP - 0.5594 × REF - 0.2211 × POL - 0.1964 × MASS + 0.088 × BE + 0.1398 × HF (R² = 0.981, Q² = 0.92). The validated consensus EC50 for the QSAR model, developed from the rat paw edema anti-inflammation model used in this study, indicate that this model was capable of effective prediction and can be used as a reliable computational predictor of diterpenoid alkaloid activity.

Halitosis and helicobacter pylori infection: A meta-analysis.

BACKGROUND: Halitosis is used to describe any disagreeable odor of expired air regardless of its origin. Numerous trials published have investigated the relation between Helicobacter pylori (H pylori) infection and halitosis, and even some regimes of H pylori eradication have been prescribed to those patients with halitosis in the clinic. We conducted a meta-analysis to define the correlation between H pylori infection and halitosis. OBJECTIVES: To evaluate whether there is a real correlation between H pylori infection and halitosis, and whether H pylori eradication therapy will help relieve halitosis. METHODS: We searched several electronic databases (The Cochrane Library, MEDLINE, EMBASE, PubMed, Web of Science, and Wanfangdata) up to December 2015. Studies published in English and Chinese were considered in this review. After a final set of studies was identified, the list of references reported in the included reports was reviewed to identify additional studies. Screening of titles and abstracts, data extraction and quality assessment was undertaken independently and in duplicate. All analyses were done using Review Manager 5.2 software. RESULTS: A total of 115 articles were identified, 21 of which met the inclusion criteria and presented data that could be used in the analysis. The results showed that the OR of H pylori infection in the stomach between halitosis-positive patients and halitosis-negative patients was 4.03 (95% CI: 1.41-11.50; P = 0.009). The OR of halitosis between H pylori-positive patients and H pylori-negative patients was 2.85 (95% CI: 1.40-5.83; P = 0.004); The RR of halitosis after successful H pylori eradication in those H pylori-infected halitosis-positive patients was 0.17 (95% CI: 0.08-0.39; P <0.0001), compared with those patients without successful H pylori eradication. And the RR of halitosis before successful H pylori eradication therapy was 4.78 (95% CI: 1.45-15.80; P = 0.01), compared with after successful H pylori eradication therapy. CONCLUSIONS: There is clear evidence that H pylori infection correlates with halitosis. H pylori infection might be important in the pathophysiological mechanism of halitosis, and H pylori eradication therapy may be helpful in those patients with refractory halitosis.

Identification and determination of the major constituents in Kai-Xin-San by UPLC-Q/TOF MS and UFLC-MS/MS method.

In order to have overall chemical material information of Kai-Xin-San (KXS), the reliable ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometer (UHPLC-Q-TOF-MS) and ultra-fast liquid chromatography mass spectrometer (UFLC-MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC-Q-TOF-MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC-Q-TOF-MS method was achieved on Agilent 6520 Q-TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple-reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.

Simple risk factors to predict urgent endoscopy in nonvariceal upper gastrointestinal bleeding pre-endoscopically.

The goal of this study is to evaluate how to predict high-risk nonvariceal upper gastrointestinal bleeding (NVUGIB) pre-endoscopically. A total of 569 NVUGIB patients between Match 2011 and January 2015 were retrospectively studied. The clinical characteristics and laboratory data were statistically analyzed. The severity of NVUGIB was based on high-risk NVUGIB (Forrest I-IIb), and low-risk NVUGIB (Forrest IIc and III). By logistic regression and receiver-operating characteristic curve, simple risk score systems were derived which predicted patients' risks of potentially needing endoscopic intervention to control bleeding. Risk score systems combined of patients' serum hemoglobin (Hb) ≤75 g/L, red hematemesis, red stool, shock, and blood urine nitrogen ≥8.5 mmol/L within 24 hours after admission were derived. As for each one of these clinical signs, the relatively high specificity was 97.9% for shock, 96.4% for red stool, 85.5% for red hematemesis, 76.7% for Hb ≤75 g/L, and the sensitivity was 50.8% for red hematemesis, 47.5% for Hb ≤75 g/L, 14.2% for red stool, and 10.9% for shock. When these 5 clinical signs were presented as a risk score system, the highest area of receiver-operating characteristic curve was 0.746, with sensitivity 0.675 and specificity 0.733, which discriminated well with high-risk NVUGIB. These simple risk factors identified patients with high-risk NVUGIB of needing treatment to manage their bleeding pre-endoscopically. Further validation in the clinic was required.

Characterization of multiple constituents in Kai-Xin-San prescription and rat plasma after oral administration by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry.

A sensitive and reliable ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method was established to separate and identify the chemical constituents of Kai-Xin-San prescription, a classic traditional Chinese medicine formula that plays an important role in the treatment of Alzheimer's disease. The detection was performed on an Agilent 6520 Accurate-Mass quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source in negative modes. With the optimized conditions, a total of 54 compounds were identified or tentatively characterized. Out of the 54 compounds, six compounds were identified by comparing the retention time and mass spectrometry data with reference standards, the rest were characterized by analyzing mass spectrometry data and retrieving the literature data. Results indicated ginsenosides, polygala saponins, terpenoids, and oligosaccharide esters were the major effective constituents in Kai-Xin-San prescription. There were 26 prototype ingredients that were assigned for identification in rat plasma. It is also concluded that the developed ultra high performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry method with high sensitivity and resolution is suitable for identifying and characterizing the chemical constituents of Kai-Xin-San prescription, and the analysis provides a helpful chemical basis for further research on Kai-Xin-San prescription and the clinical diagnostics of Alzheimer's disease.

Determination of catecholamines and their metabolites in rat urine by ultra-performance liquid chromatography-tandem mass spectrometry for the study of identifying potential markers for Alzheimer's disease.

In order to investigate the potential links between catecholamines (CAs) and Alzheimer's disease (AD), rapid and sensitive ultra-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) methods in different ionization modes for the quantification of 14 CAs and their metabolites in rat urine without derivatization or complex sample pre-treatments were developed. After addition of the internal standard, isoproterenol, the urine samples were extracted by protein precipitation and separated on an Inertsil ODS-EP column (Shimadzu, Japan) at a flow of 1.0 ml min(-1). Tandem mass spectrometric detection was performed on a 4000Q UPLC-MS/MS in the multiple reaction monitoring mode with turbo ion spray source. Tyrosine, dopamine, noradrenaline, epinephrine, 3-methoxytyramine, normetanephrine and metanephrine were determined in positive mode, while 3,4-dihyroxy-L-phenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid, DL-3,4-dihydroxymandelic acid, DL-3,4-dihydroxyphenyl glycol, homovanillic acid, DL-4-hydroxy-3-methoxymandelic acid and 4-hydroxy-3-methoxy-phenylglycol were determined in negative mode. The methods were examined and were found to be precise and accurate within the linearity range of the assays. The intra-day and inter-day precision and accuracy of the analytes were well within acceptance criteria (±15%). The mean extraction recoveries of analytes and internal standard were all more than 60%. The validated methods have been successfully applied to compare CAs profiles in normal and AD rats. The results indicated the urine levels of DL-3,4-dihydroxyphenyl glycol and 4-hydroxy-3-methoxy-phenylglycol in AD rats were significantly higher than those in the normal group, and the other CAs have an opposite performance. These may attribute to the difference of some enzyme activity between rats with AD and normal. Furthermore, this may be helpful in clinical diagnostics and monitor the efficacy of AD treatment.

A UFLC-MS/MS method with a switching ionization mode for simultaneous quantitation of polygalaxanthone III, four ginsenosides and tumulosic acid in rat plasma: application to a comparative pharmacokinetic study in normal and Alzheimer's disease rats.

A fast, sensitive and reliable ultra fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) method has been developed and validated for simultaneous quantitation of polygalaxanthone III (POL), ginsenoside Rb1 (GRb1), ginsenoside Rd (GRd), ginsenoside Re (GRe), ginsenoside Rg1 (GRg1) and tumulosic acid (TUM) in rat plasma after oral administration of Kai-Xin-San, which plays an important role for the treatment of Alzheimer's disease (AD). The plasma samples were extracted by liquid-liquid extraction using ethyl acetate-isopropanol (1:1, v/v) with salidrdoside as internal standard (IS). Good chromatographic separation was achieved using gradient elution with the mobile phase consisting of methanol and 0.01% acetic acid in water. The tandem mass spectrometric detection was performed in multiple reaction monitoring mode on 4000Q UFLC-MS/MS system with turbo ion spray source in a negative and positive switching ionization mode. The lower limits of quantification were 0.2-1.5 ng/ml for all the analytes. Both intra-day and inter-day precision and accuracy of analytes were well within acceptance criteria (±15%). The mean absolute extraction recoveries of analytes and IS from rat plasma were all more than 60.0%. The validated method has been successfully applied to comparing pharmacokinetic profiles of analytes in normal and AD rat plasma. The results indicated that no significant differences in pharmacokinetic parameters of GRe, GRg1 and TUM were observed between the two groups, while the absorption of POL and GRd in AD group were significantly higher than those in normal group; moreover, the GRb1 absorbed more rapidly in model group. The different characters of pharmacokinetics might be caused by pharmacological effects of the analytes.