Metabolic characteristics related to the hazardous effects of environmental arsenic on humans: A metabolomic review.

Arsenic (As) is a toxic metalloid exist ubiquitously in environment. Epidemiological studies and laboratory animal studies have verified that As damages multiple organs or tissues in the body and is associated with a variety of diseases. Changes in metabolites usually indicate disturbances in metabolic pathways and specific metabolites are considered as biomarkers of diseases or drugs/toxins or environmental effects. Metabolomics is the quantitative measurement of the dynamic multi-parameter metabolic responses of biological systems due to pathophysiological or genetic changes. Current years, some metabolomic studies on the hazardous effect of environmental As on humans have been reported. In this paper, we first overviewed the metabolomics studies of environmental As exposure in humans since 2011, emphasizing on the data mining process of metabolic characteristics related to the hazardous effects of environmental As on humans. Then, the relationship between metabolic characteristics and the toxic mechanism of environmental As exposure in humans were discussed, and finally, the prospects of metabolomics studies on populations exposed to environmental As were put forward. Our paper may shed light on the study of mechanisms, prevention and individualized treatment of As poisoning.

An Experimental Study Reveals the Protective Effect of Autophagy against Realgar-Induced Liver Injury via Suppressing ROS-Mediated NLRP3 Inflammasome Pathway.

Realgar, a poisonous traditional Chinese medicine, has been shown to cause liver injury when used for long periods or overdoses. However, the underlying molecular mechanisms and therapeutic targets have not been fully elucidated. The aim of this study is to explore the role of autophagy in sub-chronic realgar exposure-induced liver injury. Here, the liver injury model was established by continuously administrating mice with 1.35 g/kg realgar for 8 weeks. 3-methyladenine (3-MA) and rapamycin (RAPA) were used to regulate autophagy. The results showed that realgar induced abnormal changes in liver function, pathological morphology, expression of inflammatory cytokines, and upregulated NLRP3 inflammasome pathway in mouse livers. RAPA treatment (an inducer of autophagy) significantly improved realgar-induced liver injury and NLRP3 inflammasome activation, while 3-MA (an inhibitor of autophagy) aggravated the realgar-induced liver injury and NLRP3 inflammasome activation. Furthermore, we found that realgar-induced NLRP3 inflammasome activation in mouse livers is mediated by ROS. RAPA eliminates excessive ROS, inhibits NF-κB nuclear translocation and down-regulates the TXNIP/NLRP3 axis, consequently suppressing ROS-mediated NLRP3 inflammasome activation, which may be the underlying mechanism of the protective effect of autophagy on realgar-induced liver injury. In conclusion, the results of this study suggest that autophagy alleviates realgar-induced liver injury by inhibiting ROS-mediated NLRP3 inflammasome activation. Autophagy may represent a therapeutic target in modulating realgar-induced liver injury.

Triclosan-induced glycolysis drives inflammatory activation in microglia via the Akt/mTOR/HIF 1α signaling pathway.

Exposure to triclosan (TCS) has been implicated in neurotoxicity including autism spectrum disorders in vivo and oxidative stress and cell apoptosis in vitro. Thus, the molecular mechanisms underlying TCS-induced neurotoxicity warrants further research. In this study, we try to address the mode of action that TCS induced the expression of inflammatory cytokines by shifting metabolism to glycolysis. BV-2 cells were treated with 20 µM TCS for 24 h, and the conditional medium from TCS-induced activated microglia reduced the viability of the murine hippocampal neurons cell line HT22. Protein expression levels in the nuclear factor kappa B (NF-κB) signaling pathway were measured through Western blotting, and the expression levels of inflammatory cytokine were measured using quantitative real-time PCR. The results showed that exposure to TCS enhanced NF-κB activation, increased inflammatory cytokine expression including interleukin (IL) 1ß, IL-6, and tumor necrosis factor (TNF) α in the BV-2 cells. The glucose consumption and lactate production in BV2 cell increased sharply after exposure to TCS for 24 h. Based on our qPCR and Western blotting results, the expression of the key glycolysis enzymes-namely hexokinase 1, pyruvate kinase M2, and lactate dehydrogenase A-increased after treatment with 20 µM TCS. Furthermore, inhibiting glycolysis by 2-deoxy-D-glucose reduced the activation of NF-κB and the mRNA expression of the inflammatory cytokines in the TCS-activated BV-2 microglia. The expression of the proteins of the Akt/mTOR/HIF1α pathway examined through Western blotting, which regulates glycolysis, also increased in the BV2 cells exposed to TCS. Moreover, Akt and mTOR inhibition by using LY294002 and rapamycin, respectively, blocked inflammatory cytokine overexpression induced by TCS. In conclusion, TCS can induce glycolysis and directly drive inflammatory activation in microglia; with the mediation of the Akt/mTOR/HIF1α pathway.

Effects of realgar on GSH synthesis in the mouse hippocampus: Involvement of system XAG(-), system XC(-), MRP-1 and Nrf2.

Realgar is a type of mineral drug that contains arsenic and has neurotoxicity. Glutathione (GSH), which is the main antioxidant in the central nervous system, plays a key role in antioxidant defenses and the detoxification of arsenic. However, whether realgar interferes with the synthesis of GSH in the brain and the molecular mechanisms underlying its effects are largely unknown. Here, we used mouse models of exposure to realgar to show that realgar affects the synthesis of GSH in the hippocampus, leading to ultrastructural changes in hippocampal neurons and synapses and deficiencies in cognitive abilities, and that the mechanisms that cause this effect may be associated with alterations in the expression of system XAG(-), system XC(-), multidrug resistance-associated protein 1(MRP-1), nuclear factor E2-related factor 2 (Nrf2), γ-glutamylcysteine synthetase (γ-GCS), and the levels of glutamate (Glu) and cysteine (Cys) in the extracellular fluid. These findings provide a theoretical basis for preventing the drug-induced chronic arsenic poisoning in the nervous system that is triggered by realgar.

Metabonomic study of biochemical changes in urinary of type 2 diabetes mellitus patients after the treatment of sulfonylurea antidiabetic drugs based on ultra-performance liquid chromatography/mass spectrometry.

A metabonomic study on biochemical changes in the urine of type 2 diabetes mellitus (T2DM) patients after the treatment of sulfonylurea (SU) antidiabetic drugs was performed. An ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) method was used to generate metabolic fingerprints for the metabonomic analysis of urinary samples obtained from 20 T2DM patients without any drug treatment and 20 T2DM patients treated with SU antidiabetic drugs and 20 normal glucose tolerance subjects. The resulting data were subjected to chemometric analysis (principal component analysis and partial least squares discriminant analysis) to investigate the effect of SU antidiabetic drugs on urinary metabolite profiles of T2DM patients. Biomarkers such as xanthine, phenylalanine, tryptophan, hippurate, phenylacetylglutamine, carnitine C8:1, carnitine C10:3, uric acid and citrate were found to be responsible for the separation of T2DM and SU-treated groups, which indicates a potential effect of SU on energy metabolism, Tricarboxylic acid (TCA) cycle, gut microflora metabolism and oxidative stress. The study may be helpful to the understanding of the action of mechanism of SU antidiabetic drugs.

Effect of realgar on extracellular amino acid neurotransmitters in hippocampal CA1 region determined by online microdialysis­dansyl chloride derivatization­high-performance liquid chromatography and fluorescence detection.

An online microdialysis (MD)­dansyl chloride (Dns) derivatization­high-performance liquid chromatography (HPLC) and fluorescence detection (FD) system was developed for simultaneous determination of eight extracellular amino acid neurotransmitters in hippocampus. The MD probe was implanted in hippocampal CA1 region. Dialysate and Dns were online mixed and derivatized. The derivatives were separated on an ODS column and detected by FD. The developed online system showed good linearity, precision, accuracy and recovery. This online MD-HPLC system was applied to monitor amino acid neurotransmitters levels in rats exposed to realgar (0.3, 0.9 and 2.7 g/kg body weight). The result shows that glutamate concentrations were significantly increased (p<0.05) in hippocampal CA1 region of rats exposed to three doses of realgar. A decrease in γ-aminobutyric acid concentrations was found in rats exposed to medium and high doses of realgar (p<0.05). Elevation of excitotoxic index (EI) values in hippocampal CA1 region of realgar-exposed rats was observed (p<0.05). Positive correlation was found between EI values and arsenic contents in hippocampus of realgar-exposed rats, which indicates that the change in extracellular EI values is associated with arsenic accumulation in hippocampus. The developed online MD­Dns derivatization­HPLC­FD system provides a new experimental method for studying the effect of toxic Chinese medicines on amino acid neurotransmitters.

Excess iron intake induced liver injury: The role of gut-liver axis and therapeutic potential.

Excessive iron intake is detrimental to human health, especially to the liver, which is the main organ for iron storage. Excessive iron intake can lead to liver injury. The gut-liver axis (GLA) refers to the bidirectional relationship between the gut and its microbiota and the liver, which is a combination of signals generated by dietary, genetic and environmental factors. Excessive iron intake disrupts the GLA at multiple interconnected levels, including the gut microbiota, gut barrier function, and the liver's innate immune system. Excessive iron intake induces gut microbiota dysbiosis, destroys gut barriers, promotes liver exposure to gut microbiota and its derived metabolites, and increases the pro-inflammatory environment of the liver. There is increasing evidence that excess iron intake alters the levels of gut microbiota-derived metabolites such as secondary bile acids (BAs), short-chain fatty acids, indoles, and trimethylamine N-oxide, which play an important role in maintaining homeostasis of the GLA. In addition to iron chelators, antioxidants, and anti-inflammatory agents currently used in iron overload therapy, gut barrier intervention may be a potential target for iron overload therapy. In this paper, we review the relationship between excess iron intake and chronic liver diseases, the regulation of iron homeostasis by the GLA, and focus on the effects of excess iron intake on the GLA. It has been suggested that probiotics, fecal microbiota transfer, farnesoid X receptor agonists, and microRNA may be potential therapeutic targets for iron overload-induced liver injury by protecting gut barrier function.

Effects of chronic realgar exposure on liver lipidome in mice and identification sensitive lipid biomarker model for realgar-induced liver damage.

Chronic or excessive use of realgar induced liver damage. The biomarkers and exact mechanism have not been fully investigated. We performed an untargeted lipidomics study to investigate the effects of realgar on liver lipidome in mice and explore the sensitive biomarker model of realgar induced liver damage. It was found that realgar exposure induced arsenic accumulation in the liver, increased ROS generation, elevated MDA levels, decreased antioxidant enzymes levels, induced cell apoptosis, changed hepatocyte ultrastructure and morphology, and altered ALT, AST levels. Lipidomics study detected 30 classes and 1457 molecules in mice liver. The numbers of 49 and 103 lipid molecules were significantly altered (FDR<0.05) in the livers of 0.45 g/kg and 1.35 g/kg realgar-exposed mice. The glycerophospholipid and sphingomyelin were the most affected lipid class. We focused on the effect of chronic realgar exposure on the mutual transformation of lipid subclasses and the fatty acid chain composition of lipids in mouse liver, and found that realgar affected the mutual transformation of PE-PC, PC-LPC and SM-Cer. Notably, we found that realgar exposure increased PUFAs linked phospholipids in mouse liver tissues. We identified two sensitive lipid molecules [PE (44:2p) and PE (16:0/22:5)] in combination can accurately distinguish and predict realgar induced liver damage, they are associated with oxidative damage and mitochondrial respiration in liver tissue. Our study provides an experimental basis for the mechanism research and early detection of realgar-induced liver damage.

[Effect of subchronic realgar exposure on Glu and Gln in infant rat brain].

OBJECTIVE: To study the effect of realgar on Glu and Gln on rat brain tissues. METHODS: Forty-eight Wistar rats were divided into 4 groups randomly:control group,low dosage group, moderate dosage group and high dosage group. The treatment groups were treated with realgar by gastric perfusion at a dosage of 0.3 g/kg, 0.9 g/kg, 2.7 g/kg and the control group ones were orally given the same volume of 0.5% sodium carboxymethylcellulose (CMC-Na) for 6 weeks. The contents of inorganic arsenic, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) in brain tissues were measured by hydride generation-atomic absorption (HG-AAS) method. The contents of amino acid neurotransmitters in brain tissues of rats were determined by means of high performance liquid chromatography with precolumn derivatization. RESULTS: The levels of MMA and DMA in brain increased as the dosage of realgar increased, while the second methylation index declined. Compared with control group,the levels of Glu was significantly decreased in realgar treated group (P < 0.05); Gln also tended to decrease and that of low dosage group was obviously decreased compared with controls. CONCLUSION: Realgar exposure can cause accumulation of MMA and DMA,declination of second methylation index and the reduction of Glu and Gln in brain tissue.

Realgar facilitates the Nrf2-Keap1-p62 positive feedback signaling axis via MAPKs and AKT to interfere with autophagy-induced apoptosis and oxidative stress in the hippocampus.

Realgar, as a commonly used traditional Chinese medicine, exerts both pharmacological and biological effects. However, the mechanism by which it causes nervous system injury remains unclear. This study aimed to elucidate the specific mechanism underlying the hippocampal neurotoxicity caused by realgar. Nrf2 is an important receptor of exogenous toxic substances and oxidative stress. We utilized a p38-specific inhibitor (SB20358), ERK1/2-specific inhibitor (PD98059), JNK-specific inhibitor (SP600125) and AKT-specific inhibitor (LY249002) to establish the corresponding animal models and explore how realgar activates Nrf2. We established an Nrf2-shRNA gene silencing model in rats and an autophagy-specific inhibitor treatment model to further explore realgar-induced neurotoxicity and the role of Nrf2 in realgar-induced damage to the hippocampus. The results showed that realgar passed through the blood-brain barrier and accumulated in brain tissue to induce central nervous system toxicity. The specific mechanism was that realgar activated MAPKs and AKT signaling molecules to activate the Nrf2-Keap1-p62 positive feedback signaling axis, induced abnormal autophagy initiation and degradation, and promoted oxidative damage and apoptosis in neurons. Effective measures should be taken to prevent and control the arsenic poisoning caused by realgar in the early stage, and this study provides a theoretical and practical basis for the rational use of drugs in the clinic.

Insight into the regulation of NLRP3 inflammasome activation by mitochondria in liver injury and the protective role of natural products.

Due to high mortality rates and poor prognosis, liver injury remains one of the leading causes of mortality worldwide. Amounting evidence suggested that the activation of the nucleotide-binding oligomerization domain (NOD)-like receptor containing pyrin domain 3 (NLRP3) inflammasome, which promotes pro-interleukin-1ß (pro-IL-1ß) and pro-interleukin-18 (pro-IL-18) cleavage and maturation play a vital role in the occurrence and development of liver injury and liver disease. Mitochondrial dysfunction is a common co-occurring event in liver injury. Abnormal mitochondrial function has also been shown to be closely related to NLRP3 inflammasome activation. Currently, natural products have attracted the attention of researchers as potential therapeutic agents for liver injury and liver disease due to their less toxicity and multi-targeting advantages. A number of natural products have been discovered to prevent and treat liver injury by modulating the activation of NLRP3 inflammasome. In this review, we highlight the mechanisms involved in the regulation of NLRP3 inflammasome activation by mitochondria during liver injury and natural products that target mitochondrial function processes to prevent or treat liver injury. Our paper may shed insight into novel viewpoint and target for prevention and treatment of liver injury based on NLRP3 inflammasome.

Realgar Alleviated Neuroinflammation Induced by High Protein and High Calorie Diet in Rats via the Microbiota-Gut-Brain Axis.

PURPOSE: Gastrointestinal heat retention syndrome (GHRS) often occurs in adolescents, resulting into nervous system injury. Realgar, an arsenic mineral with neuroprotective effect, has been widely used to treat GHRS. However, its mechanism of action remains unknown. METHODS: A GHRS rat model was established using a high protein and high calorie diet. We performed macroscopic characterization by assessing bowel sounds, hot/cold preference, anal temperature, and fecal features. Atomic fluorescence spectroscopy was employed to evaluate brain arsenic level while hippocampal ultrastructural changes were analyzed using transmission electron microscopy. In addition, inflammatory cytokines and BBB breakdown were analyzed by western blotting, immunofluorescence assays, and immunohistochemistry staining. We also evaluated hippocampal metabolites by LC-MS while fecal microorganisms were assessed by 16S rDNA sequencing. RESULTS: Our data showed that the high protein and high calorie diet induced GHRS. The rat model depicted decreased bowel sounds, increased fecal characteristics score, preference for low temperature zone, and increased anal temperature. In addition, there was increase in inflammatory factors IL-6, Iba-1, and NF-κB p65 as well as reduced BBB structural protein Claudin-5 and Occludin. The data also showed appearance of hippocampus metabolites disorder and fecal microbial imbalance. Realgar treatment conferred a neuroprotective effect by inhibiting GHRS-specific characteristics, neuroinflammatory response, BBB impairment, metabolites disorder, and microbial imbalance in the GHRS rat model. CONCLUSION: Taken together, our analysis demonstrated that realgar confers a neuroprotective effect in GHRS rats through modulation of the microbiota-gut-brain axis.

Oxidative stress induced by realgar in neurons: p38 MAPK and ERK1/2 perturb autophagy and induce the p62-Keap1-Nrf2 feedback loop to activate the Nrf2 signalling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Due to the modernization of traditional Chinese medicine (TCM) and the influence of traditional medication habits (TCM has no toxicity or side effects), arsenic poisoning incidents caused by the abuse of realgar and realgar-containing Chinese patent medicines have occurred occasionally. However, the potential mechanism of central nervous system toxicity of realgar remains unclear. AIM OF THE STUDY: This study aimed to clarify the specific mechanism of realgar-induced neurotoxicity. MATERIALS AND METHODS: In this study, the roles of ERK1/2 and p38 MAPK in realgar-induced neuronal autophagy and overactivation of the nuclear factor erythroid-derived factor 2-related factor (Nrf2) signalling pathways was investigated in vivo and in vitro. RESULTS: The arsenic in realgar passed through the blood-brain barrier and accumulated in the brain, resulting in damage to neurons, synapses and myelin sheaths in the cerebral cortex and a decrease in the total antioxidant capacity. The specific mechanism is that the excessive activation of Nrf2 is regulated by the upstream signalling molecules ERK1/2 and p38MAPK. At the same time, p38 MAPK and ERK1/2 interfere with autophagy, thereby promoting autophagy initiation but causing subsequent dysfunctional autophagic degradation and inducing the p62-Keap1-Nrf2 feedback loop to promote Nrf2 signalling pathway activation and nerve cell apoptosis. CONCLUSIONS: This study confirmed the role of the signalling molecules p38 MAPK and ERK1/2 in perturbing autophagy and inducing the p62-Keap1-Nrf2 feedback loop to activate the Nrf2 signalling pathway in realgar-induced neurotoxicity.

Sub-chronic exposure to realgar induces liver injury via upregulating the TXNIP/NLRP3 pathway and disturbing bile acid homeostasis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Realgar is a traditional Chinese medicine used in China for a long history. Long-time or excessive use of realgar causes liver injury. However, its underlying mechanism is not fully clarified. AIM OF THE STUDY: In this study, we investigated the toxic effect of sub-chronic exposure to realgar on mice liver, and further revealed its underlying mechanism focused on the TXNIP/NLRP3 pathway and bile acid homeostasis. MATERIAL AND METHODS: Mice were divided into control and different doses of sub-chronic realgar exposed groups. Total arsenic levels in the blood and liver were determined by atomic fluorescence spectrometry. The effect of realgar on liver function was evaluated by biochemical analysis and histopathological examination. Assay kits were applied for the measurement of oxidative stress indexes, MPO and plasma inflammatory cytokines. The mRNA and proteins involved in the TXNIP/NLRP3 and NF-κB pathways were determined by RT-qPCR, western blot, Immunofluorescence and Immunohistochemistry. UHPLC/MS/MS was used for the quantitative analysis of bile acids (BAs) in mice plasma, liver and urine. The genes related to BAs metabolism were measured by RT-qPCR. RESULTS: Sub-chronic exposure to realgar led to arsenic accumulation and caused oxidative damage and inflammatory infiltration in mouse liver, finally resulting in liver injury. Realgar treatment activated the NF-κB pathway and significantly upregulated the TXNIP/NLRP3 pathway in mouse liver. Realgar altered the metabolic balance of BAs, which is related to the abnormal expression of BAs transporters and enzymes. CONCLUSION: Sub-chronic exposure to realgar caused liver injury in mouse, and the mechanism may involve the upregulation of the TXNIP/NLRP3 pathway and disordered BAs homeostasis.

Development and validation of UPLC-MS/MS method for simultaneous determination of gestodene and ethinyl estradiol in rat plasma.

A selective and sensitive ultra-performance liquid chromatography method with tandem mass spectrometric detection for simultaneous determination of gestodene (GES) and ethinyl estradiol (EE) in rat plasma was developed and validated. GES, EE and the internal standard, norgestrel, were extracted with ethyl acetate, derivatized (EE only) with dansyl chloride and then back-extracted into diethyl ether-hexane (2:1, v/v). The separation was performed on an ACQUITY UPLC BEH C(18) column with gradient elution using mobile phase consisting of acetonitrile and water (both containing 0.1% formic acid). The detection was carried out by means of electrospray ionization (ESI) mass spectrometry in positive ion mode with multiple-reaction monitoring. Calibration curves of GES and EE were linear (r(2) >or= 0.99) over the concentration ranges 1.59-159 and 0.196-78.4 ng/mL, respectively. The intra- and inter-day precisions were not more than 6.9 and 12.9% for GES and 10.6 and 9.0% for EE, and the accuracies were -2.5-8.0% for GES, and -7.2-0.19% for EE, respectively. The method herein described was superior to previous methods and was applicable to the pharmacokinetic study of GES and EE in rats.

Liver metabonomics study on the protective effect of glycyrrhetinic acid against realgar-induced liver injury.

Glycyrrhetinic acid (GA) is the bioactive ingredient in Glycyrrhizae Radix et Rhizoma. Our previous study has reported that GA has protective effect on realgar-induced hepatotoxicity. However, the details of the hepatoprotective mechanisms of GA on realgar-induced liver injury remain to be elucidated. In the study, mice were divided into control, GA-control, realgar, and co-treated groups. Their liver tissues were used for metabonomics study by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method. The results illustrate that GA significantly ameliorate the liver injury and metabolic perturbations caused by realgar. Some metabolites, such as phenylalanine, pyroglutamic acid (PGA), proline, carnitine, nicotinamide, choline, lysophosphatidylcholine (LPC) 16 : 0 and LPC 18 : 2 were found responsible for the hepatoprotective effect of GA. These metabolites are associated with the methylation metabolism of arsenic, cell membrane structure, energy metabolism and oxidative stress. From the results of this study, we infer that the potential hepatoprotective mechanism of GA on realgar-induced liver injury may be associated with reducing arsenic accumulation and its methylation metabolism in the liver, promoting the conjugation of arsenic and GSH to play detoxification effect, and ameliorating the liver metabolic perturbations caused by realgar.

Identification of neurotoxicity markers induced by realgar exposure in the mouse cerebral cortex using lipidomics.

Realgar is a traditional Chinese medicine containing arsenic and has neurotoxicity. This study used realgar exposure mice model, neurobehavioral tests, analytical chemistry, molecular biology and nontargeted lipidomics to explore the mechanism of realgar damages the nervous system. The arsenic contained in realgar passed through the BBB and accumulated in the brain. Neurons, synapses and myelin showed abnormal changes in the cerebral cortex. The number of autophagosomes were incresed as well as levels of MDA, Lp-PLA2, and cPLA2 but the CAT level was significant reduced. Finally, the cognition and memory of mice were decreased. Nontargeted lipidomics detected 34 lipid subclasses including 1603 lipid molecules. The levels of the LPC and LPE were significantly increased. Under the condition of variable importance for the projection (VIP)>1 and P < 0.05, only 28 lipid molecules satisfied the criteria. The lipid molecular markers SM (d36:2), PE (18:2/22:6) and PE (36:3) which were filtered by receiver operating characteristic (ROC) curve (AUC>0.8 or AUC<0.2) were used to identify the neurotoxicity induced by realgar. Therefore, realgar induces neurotoxicity through exacerbating oxidative damage and lipid dysfunction. Providing research basis for the clinical diagnosis and treatment of realgar-induced neurotoxicity.

Lysophosphatidylcholine--biomarker of Metformin action: studied using UPLC/MS/MS.

An UPLC/MS/MS based metabonomic method was developed and applied to the elucidation of biomarker of metformin action. The plasma metabolite profiling in healthy volunteers before and after per os metformin was determined with UPLC/MS/MS and analyzed by using multivariate statistics. Significant difference in endogenous metabolite profiles was revealed before and after administration of metformin. Four biomarkers found were lysophosphatidylcholines (LPCs), and their structures were tentatively identified to be 16:0 LPC, 18:0 LPC, 18:1 LPC and 18:2 LPC according to the molecular ions information and corresponding fragments of product ion scan. Lysophosphatidylcholine in blood may be involved in metformin treatment.

Quantitative determination of mitiglinide in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

A selective, rapid and sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantitative determination of mitiglinide in human plasma. With nateglinide as internal standard, sample pretreatment involved a one-step extraction with diethyl ether of 0.2 mL plasma. The separation was performed on an ACQUITY UPLCtrade mark BEH C(18) column (50 mm x 2.1 mm, i.d., 1.7 microm) with the mobile phase consisting of methanol and 10 mmol/L ammonium acetate (65:35, v/v) at a flow rate of 0.25 mL/min. The detection was carried out by means of electrospray ionization mass spectrometry in positive ion mode with multiple reaction monitoring (MRM). Linear calibration curves were obtained in the concentration range of 1.080-5400 ng/mL, with a lower limit of quantification of 1.080 ng/mL. The intra- and inter-day precision (RSD) values were below 15% and accuracy (RE) was from -3.5% to 7.3% at all QC levels. The method was fully validated and successfully applied to a clinical pharmacokinetic study of mitiglinide in 10 healthy volunteers following oral administration.

Determination and pharmacokinetics of orientin in rabbit plasma by liquid chromatography after intravenous administration of orientin and Trollius chinensis Bunge extract.

A high-performance liquid chromatography (HPLC) method was developed and validated for the determination of orientin in rabbit plasma using ultraviolet (UV) absorbance detection. Orientin is the active constituent of purified herbal extract (TRO PE) from the flower of Trollius chinensis Bunge. Protein precipitation was used as the sample preparation technique. A Diamonsil C18 column (150 mmx4.6 mm, 5 microm) was equilibrated with a mobile phase composed of 0.1% acetic acid/methanol/acetonitrile (80/5/15, v/v/v). The calibration curve of orientin in rabbit plasma was linear in the concentration range of 0.530-53.0 microg/mL. This validated method was successfully applied to a pharmacokinetic study in rabbits after the intravenous administrations of orientin and TRO PE at three different doses.