Biclustering analysis on tree-shaped time-series single cell gene expression data of Caenorhabditis elegans.

BACKGROUND: In recent years, gene clustering analysis has become a widely used tool for studying gene functions, efficiently categorizing genes with similar expression patterns to aid in identifying gene functions. Caenorhabditis elegans is commonly used in embryonic research due to its consistent cell lineage from fertilized egg to adulthood. Biologists use 4D confocal imaging to observe gene expression dynamics at the single-cell level. However, on one hand, the observed tree-shaped time-series datasets have characteristics such as non-pairwise data points between different individuals. On the other hand, the influence of cell type heterogeneity should also be considered during clustering, aiming to obtain more biologically significant clustering results. RESULTS: A biclustering model is proposed for tree-shaped single-cell gene expression data of Caenorhabditis elegans. Detailedly, a tree-shaped piecewise polynomial function is first employed to fit non-pairwise gene expression time series data. Then, four factors are considered in the objective function, including Pearson correlation coefficients capturing gene correlations, p-values from the Kolmogorov-Smirnov test measuring the similarity between cells, as well as gene expression size and bicluster overlapping size. After that, Genetic Algorithm is utilized to optimize the function. CONCLUSION: The results on the small-scale dataset analysis validate the feasibility and effectiveness of our model and are superior to existing classical biclustering models. Besides, gene enrichment analysis is employed to assess the results on the complete real dataset analysis, confirming that the discovered biclustering results hold significant biological relevance.

Untargeted and stable isotope-assisted metabolomic analysis of MDA-MB-231 cells under hypoxia.

INTRODUCTION: Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics. OBJECTIVES: Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia. METHODS: Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC-MS based untargeted and stable isotope assisted metabolomic techniques. RESULTS: Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells. CONCLUSION: The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.

Establishment and optimization of NMR-based cell metabonomics study protocols for neonatal Sprague-Dawley rat cardiomyocytes.

A quenching, harvesting, and extraction protocol was optimized for cardiomyocytes NMR metabonomics analysis in this study. Trypsin treatment and direct scraping cells in acetonitrile were compared for sample harvesting. The results showed trypsin treatment cause normalized concentration increasing of phosphocholine and metabolites leakage, since the trypsin-induced membrane broken and long term harvesting procedures. Then the intracellular metabolite extraction efficiency of methanol and acetonitrile were compared. As a result, washing twice with phosphate buffer, direct scraping cells and extracting with acetonitrile were chosen to prepare cardiomyocytes extracts samples for metabonomics studies. This optimized protocol is rapid, effective, and exhibits greater metabolite retention.

An integrated metabonomic and proteomic study on Kidney-Yin Deficiency Syndrome patients with diabetes mellitus in China.

AIM: To investigate specific changes in metabolites and proteins of Kidney-Yin Deficiency Syndrome (KYDS) patients with diabetes mellitus (DM) in China. METHODS: KYDS (n=29) and non-KYDS (n=23) patients with DM were recruited for this study. The KYDS was diagnosed by two senior TCM clinicians separately. The metabonomic and proteomic profiles of the patients were assessed using a metabonomic strategy based on NMR with multivariate analysis and a proteomic strategy based on MALDI-TOF-MS, respectively. RESULTS: Eighteen upregulated peptides and thirty downregulated peptides were observed in the plasma of the KYDS patients. Comparing the proteomic profiles of the KYDS and non-KYDS groups, however, no significantly differentially expressed peptides were found. At the same time, major metabolic alterations were found to distinguish the two groups, including eight significantly changed metabolites (creatinine, citrate, TMAO, phenylalanine, tyrosine, alanine, glycine and taurine). The levels of creatinine, citrate, TMAO, phenylalanine and tyrosine were decreased, whereas the levels of alanine, glycine and taurine were increased in the KYDS patients. These biochemical changes were found to be associated with alterations in amino acid metabolism, energy metabolism and gut microflora. CONCLUSION: The identification of distinct expression profiles of metabolites and signaling pathways in KYDS patients with DM suggests that there are indeed molecular signatures underlying the principles of 'Syndrome Differentiation' in traditional Chinese medicine.

[Investigation of Evolution Rules of Phlegm and Blood Stasis Syndrome in Hyperlipidemia and Ath- erosclerosis by NMR-based Metabolic Profiling and Metabonomic Approaches].

OBJECTIVE: To explore evolution rules of phlegm and blood stasis syndrome ( PBSS) in hyperlipidemia and atherosclerosis (AS) using NMR-based metabolic profiling and metabonomic approaches based on formulas corresponding to syndrome. METHODS: Totally 150 SD rats were divided into the normal group, the model group, the Erchen Decoction (ED) group, the Xuefu Zhuyu Decoction (XZD) group, the Lipitor group, 30 in each group. The hyperlipidemia and AS rat model was duplicated by suturing carotid artery, injecting vitamin D3, and feeding with high fat diet. ED and XZD were used as drug probes. Blood samples were withdrawn at week 2, 4, and 8 after modeling. Blood lipids, blood rheology, histopathology and metabolomics were detected and analyzed. Results Results of blood lipids and pathology showed hyperlipidemia and early AS rat models were successfully established. At week 2 after modeling, levels of total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) significantly increased, which reached the peak at week 4 and maintained at higher levels at week 8. ED exerted obvious effect in improving TC and LDL-C levels of early models, while XZD could greatly improve levels of TC and LDL-C of late models. Rheological results showed at week 2, there was no significant difference in whole blood viscosity, plasma viscosity, or hematocrit between the model group and the normal group (P > 0.05). At week 4 partial hemorheological indicators (such as plasma viscosity) were abnormal. Till week 8 whole blood viscosity, plasma viscosity, and hematocrit were significantly abnormal (P <0. 05, P < 0.01). As time went by, whole blood viscosity, plasma viscosity, and hematocrit showed gradual increasing tendency in the ED group, while they showed gradual decreasing tendency in the XZD group. Results of metabonomics showed significant difference in spectra of metabolites between the normal group and the model group. As modeling time was prolonged, contents of acetyl glucoprotein and glucose in the model group increased in late stage, which was in. line with results of blood lipids and hemorheology. ED showed more obvious effect in early and mid-term modeling (at week 2 and 4), and increased contents of partial metabolites (such as choline, phosphatidyl choline, glycerophosphocholine), but these changes in the XZD group were consistent with those of the model group. In late modeling (at week 8) XZD showed more obvious effect in improving contents of lactic acid, acetyl glycoprotein, LDL, creatine, choline, and glucose. CONCLUSIONS: ED and XZD not only showed regulatory effects on lipid disorders, but also could improve dysbolism of Chos. In formulas corresponding to syndrome, damp-phlegm was main pathogenesis of hyperlipidema and AS in early and mid stages. Blood stasis syndrome began to occur along with it progressed. Phlegm can result in blood stasis and intermingles with stasis. Phlegm turbidity runs through the whole process.

Metabolomic Profiling of Autoimmune Hepatitis: The Diagnostic Utility of Nuclear Magnetic Resonance Spectroscopy.

Autoimmune hepatitis (AIH) is often confused with other liver diseases because of their shared nonspecific symptoms and serological and histological overlap. This study compared the plasma metabolomic profiles of patients with AIH, primary biliary cirrhosis (PBC), PBC/AIH overlap syndrome (OS), and drug-induced liver injury (DILI) with those of healthy subjects to identify potential biomarkers of AIH. Metabolomic profiling and biomarker screening were performed using proton nuclear magnetic resonance spectroscopy (1H NMR) coupled with a partial least-squares discriminant analysis. Compared with the levels in healthy volunteers and other liver disease patients, AIH patients exhibited relatively high levels of plasma pyruvate, lactate, acetate, acetoacetate, and glucose. Such metabolites are typically related to energy metabolism alterations and may be a sign of metabolic conversion to the aerobic glycolysis phenotype of excessive immune activation. Increased aromatic amino acids and decreased branched-chain amino acids were found in the plasma of AIH patients. The whole NMR profiles were stepwise-reduced, and nine metabolomic biomarkers having the greatest significance in the discriminant analysis were obtained. The diagnostic utility of the selected metabolites was assessed, and these biomarkers achieved good sensitivity, specificity, and accuracy (all above 93%) in distinguishing AIH from PBC, DILI, and OS. This report is the first to present the metabolic phenotype of AIH and the potential utility of 1H NMR metabolomics in the diagnosis of AIH.

Extraction, derivatization, and determination of metabolome in human macrophages.

A GC/TOF-MS was applied to the determination of metabolites in human macrophages. The extraction conditions and quenching conditions were investigated and optimized. The results indicated that 0.9% w/v sodium chloride at 4°C was the most favorable condition to quench macrophage, 1 mL 50% ACN for 2 min in ice bath was the optimal condition to extract 5 × 10(6) cells. Two hundred six peaks could be detectable with peak area over 50 using this method. Among these peaks, 45 peaks with the similarity over 700 were identified using standard compounds for endogenous metabolites. Thirty-seven out of 45 metabolites could be quantified directly by this method. Twenty metabolites were selected randomly, and 15 amino acids were used for method validation. The correlation coefficients (r) ranging from 0.9902 to 0.9977 were obtained for 15 amino acids in the range of 2.35-150.20 µg/mL. The intraday and interday precisions were lower than 19.90% for the randomly selected 20 endogenous metabolites. Using this development method and multivariate statistical technique, several potential biomarkers were found from human macrophages infected by different Mycobacterium tuberculosis (M. tuberculosis) strains. The results suggest that the method could be applied to the investigation of the pathogenicity of tuberculosis.

Metabonomics reveals plasma metabolic changes and inflammatory marker in polycystic ovary syndrome patients.

Polycystic ovary syndrome (PCOS) is a common, clinically heterogeneous endocrine disorder affecting women of reproductive age, associated with endocrinopathy and metabolic abnormalities. Although some metabolic parameters have been investigated, very little information has been reported on the changes of small metabolites in biofluids. The aim of this study was to establish the metabolic profile of PCOS and compare it with that of controls. In this cross-sectional study of 34 women with PCOS and 36 controls, contents of small metabolites and lipids in plasma samples were measured using nuclear magnetic resonance (NMR)-based techniques and analyzed using multivariate statistical methods. Significant decrease (P < 0.05) in the levels of amino acids (leucine, isoleucine, methionine, glutamine, and arginine), citrate, choline, and glycerophosphocholine/phosphocholine (GPC/PC), and increase (P < 0.05) in the levels of lactate, dimethylamine (DMA), creatine, and N-acetyl glycoproteins were observed in PCOS patients compared with the controls. Subgroups of patients with obesity, metabolic syndrome, or hyperandrogenism exhibited greater metabolic deviations than their corresponding subgroups without these factors. PCOS patients have perturbations in amino acid metabolism, the tricarboxylic acid (TCA) cycle, and gut microflora, as well as mild disturbances in glucose and lipid metabolism. The elevated level of N-acetyl glycoproteins demonstrates the existence of low-grade chronic inflammation in PCOS patients.

Metabolomic analysis of biochemical changes in the plasma and urine of first-episode neuroleptic-naïve schizophrenia patients after treatment with risperidone.

Early findings propose that impaired neurotransmission in the brain plays a key role in the pathophysiology of schizophrenia. Recent advances in understanding its multiple etiologies and pathogenetic mechanisms provide more speculative hypotheses focused on even broader somatic systems. Using a targeted tandem mass spectrometry (MS/MS)-based metabolomic platform, we compared metabolic signatures consisting of monoamine and amino acid neurotransmitter (NT) metabolites in plasma/urine simultaneously between first-episode neuroleptic-naïve schizophrenia patients (FENNS) and healthy controls before and after a 6-week risperidone monotherapy, which suggest that the patient NT profiles are restoring during treatment. To detect and identify potential biomarkers associated with schizophrenia and risperidone treatment, we also performed a combined ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) and 1H nuclear magnetic resonance (NMR)-based metabolomic profiling of the same samples, indicating a further deviation of the patients' global metabolic profile from that of controls. The NTs and their metabolites together with the 32 identified biomarkers underpin that metabolic pathways including NT metabolism, amino acid metabolism, glucose metabolism, lipid metabolism, energy metabolism, antioxidant defense system, bowel microflora and endocrine system are disturbed in FENNS. Among them, pregnanediol, citrate and α-ketoglutarate (α-KG) were significantly associated with symptomatology of schizophrenia after Bonferroni correction and may be useful biomarkers for monitoring therapeutic efficacy. These findings promise to yield valuable insights into the pathophysiology of schizophrenia and may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes.

Interaction with polyglutamine-expanded huntingtin alters cellular distribution and RNA processing of huntingtin yeast two-hybrid protein A (HYPA).

Huntington disease (HD) is an autosomal inherited disorder that causes the deterioration of brain cells. The polyglutamine (polyQ) expansion of huntingtin (Htt) is implicated in the pathogenesis of HD via interaction with an RNA splicing factor, Htt yeast two-hybrid protein A/forming-binding protein 11 (HYPA/FBP11). Besides the pathogenic polyQ expansion, Htt also contains a proline-rich region (PRR) located exactly in the C terminus to the polyQ tract. However, how the polyQ expansion influences the PRR-mediated protein interaction and how this abnormal interaction leads to the biological consequence remain elusive. Our NMR structural analysis indicates that the PRR motif of Htt cooperatively interacts with the tandem WW domains of HYPA through domain chaperoning effect of WW1 on WW2. The polyQ-expanded Htt sequesters HYPA to the cytosolic location and then significantly reduces the efficiency of pre-mRNA splicing. We propose that the toxic gain-of-function of the polyQ-expanded Htt that causes dysfunction of cellular RNA processing contributes to the pathogenesis of HD.

[Efficacy of lamivudine on acute-on-chronic liver failure in patients with chronic hepatitis B].

To observe the therapeutic effects of lamivudine treatment in patients with early- to mid-stage hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF). Clinical data of 73 hospitalized patients with HBV-ACLF were retrospectively analyzed. Prothrombin time (PT, active coagulation), HBV DNA, and model for end-stage liver disease (MELD) score data from treatment weeks 4, 8, 24, and 48 were collected and analyzed using the statistical t-test. During the treatment duration, the complete virologic response rates were 57.5% (42/73) at 4 weeks, 71.0% (44/62) at 8 weeks, 83.1% (49/59) at 24 weeks, and 86.5% (45/52) at 48 weeks. The partial virologic response rates were 30.1% (22/73) at 4 weeks, 25.8% (16/62) at 8 weeks, 17.0% (10/59) at 24 weeks, and 13.5% (7/52) at 48 weeks. At week 48, the survival rate was 71.2% (52/73) and the probability of survival was higher in the complete virological response rate (VRR) group than in the partial VRR group [45/73 (61.6%) vs. 7/73 (30.1%), respectively; P = 0.000]. In addition, there were significant improvements in the serum normalization rate of HBV DNA, alanine aminotransferase, aspartate aminotransferase, albumin, total bilirubin, PT and MELD score in surviving patients compared to baseline (P less than 0.05) and in the complete VRR group compared to the partial VRR group (P less than 0.05). Antiviral therapy using lamivudine may be an effective therapeutic option for patients with HBV-ACLF.

A proton nuclear magnetic resonance metabonomics approach for biomarker discovery in nonalcoholic fatty liver disease.

This study was undertaken to discover novel biomarkers for the noninvasive early diagnosis of nonalcoholic fatty liver disease (NAFLD). A methionine and choline deficient (MCD) diet was used to represent different stages of NAFLD in male C57BL/6 mice. (1)H NMR spectroscopy and principal components analysis (PCA) were used to investigate the time-related biochemical changes in mice sera induced by the MCD diet. Many serum metabolites' concentrations changed between control and MCD-fed mice. Hierarchical cluster analysis (HCA) and artificial neural networks (ANNs) were used to select the least number of metabolites to be used for the noninvasive diagnosis of various stages of NAFLD; four potential biomarkers, serum glucose, lactate, glutamate/glutamine, and taurine were selected. To verify the diagnostic accuracy of these selected metabolites, their serum concentrations were measured in healthy controls (n = 28), NAFLD patients with steatosis (n = 15), steatosis patients with necro-inflammatory disease (n = 11), and NASH patients (n = 6). On the basis of results from MCD-fed mice model, clinical tests, and previous reports, we propose using the levels of the four metabolites for diagnosing NAFLD at various stages. Furthermore, the probability of developing NAFLD at a particular stage was assessed by multinomial logistic regression (MLR) based on the clinical results of the four serum metabolites.

Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. To date, the molecular mechanisms of DN remain largely unclear. The present study aimed to identify and characterize novel proteins involved in the development of DN by a proteomic approach. Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice. Consistently, the activity of HMGCS2 in kidneys and 24-h urinary excretion of the ketone body ß-hydroxybutyrate (ß-HB) were significantly increased in db/db mice. Immunohistochemistry, immunofluorescence, and real-time PCR studies further demonstrated that HMGCS2 was highly expressed in renal glomeruli of db/db mice, with weak expression in the kidneys of control mice. Because filtered ketone bodies are mainly reabsorbed in the proximal tubules, we used RPTC cells, a rat proximal tubule cell line, to examine the effect of the increased level of ketone bodies. Treating cultured RPTC cells with 1 mM ß-HB significantly induced transforming growth factor-ß1 expression, with a marked increase in collagen I expression. ß-HB treatment also resulted in a marked increase in vimentin protein expression and a significant reduction in E-cadherin protein levels, suggesting an enhanced epithelial-to-mesenchymal transition in RPTCs. Collectively, these findings demonstrate that diabetic kidneys exhibit excess ketogenic activity resulting from increased HMGCS2 expression. Enhanced ketone body production in the diabetic kidney may represent a novel mechanism involved in the pathogenesis of DN.

NMR-based metabonomic analysis of the hepatotoxicity induced by combined exposure to PCBs and TCDD in rats.

A metabonomic approach using (1)H NMR spectroscopy was adopted to investigate the metabonomic pattern of rat urine after oral administration of environmental endocrine disruptors (EDs) polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) alone or in combination and to explore the possible hepatotoxic mechanisms of combined exposure to PCBs and TCDD. (1)H NMR spectra of urines collected 24h before and after exposure were analyzed via pattern recognition by using principal component analysis (PCA). Serum biochemistry and liver histopathology indicated significant hepatotoxicity in the rats of the combined group. The PCA scores plots of urinary (1)H NMR data showed that all the treatment groups could be easily distinguished from the control group, so could the PCBs or TCDD group and the combined group. The loadings plots of the PCA revealed remarkable increases in the levels of lactate, glucose, taurine, creatine, and 2-hydroxy-isovaleric acid and reductions in the levels of 2-oxoglutarate, citrate, succinate, hippurate, and trimethylamine-N-oxide in rat urine after exposure. These changes were more striking in the combined group. The changed metabolites may be considered possible biomarker for the hepatotoxicity. The present study demonstrates that combined exposure to PCBs and TCDD induced significant hepatotoxicity in rats, and mitochondrial dysfunction and fatty acid metabolism perturbations might contribute to the hepatotoxicity. There was good conformity between changes in the urine metabonomic pattern and those in serum biochemistry and liver histopathology. These results showed that the NMR-based metabonomic approach may provide a promising technique for the evaluation of the combined toxicity of EDs.

Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatography-electrospray tandem mass spectrometry.

Salinomycin is a polyether ionophore antibiotic that is widely used in poultry and livestock. Exposure of humans to salinomycin via inhalation or ingestion can cause severe toxicity. The aim of the present work was to develop a simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid identification and quantification of salinomycin in human plasma. After removing protein using methanol, plasma samples were eluted from a Waters Xterra(®) MS C18 column with an isocratic mobile phase. Detection and quantification of the drug were performed with a triple-quadruple mass spectrometer by monitoring for two specific transitions in the electrospray, positive-ion, multiple-reaction monitoring mode. Assay validation showed good linearity (r(2) = 0.998). The detection and quantification limits of the method were 0.6 and 16 pg/mL, respectively. The inter- and intraday coefficients of variation for the assay were both <15%. Twelve authentic plasma samples from intoxicated patients were analyzed using this method. Salinomycin was detected in six samples, at concentrations of between 0.6 and 46.5 pg/mL. The described assay method allows the sensitive and rapid identification and quantification of salinomycin in human plasma, and thus provides a valuable tool for the specific diagnosis of salinomycin intoxication in clinical and emergency rescue practice.

[NMR-spectroscopy-based metabonomic study on mouse model of blood deficiency syndrome induced by compound method of bleeding, starved feeding and exhausting and effect of siwutang].

OBJECTIVE: To explore the essence of blood deficiency syndrome and the mechanism of the effects of siwutang based on the characteristic changes in metabolic profiles of C57 mice with blood deficiency induced by compound method of bleeding, starved feeding and exhausting before and after the treatment of Siwutang. METHOD: Thirty C57 mice were randomly divided into three groups of 10 animals: the control group and two blood deficiency model groups without or with siwutang treatment. The animals were sacrificed after induction by compound method of bleeding,starved feeding and exhausting for 10 days and the blood, spleen, thymus and bone marrow were obtained. The metabolic profiles of serum, aqueous and lipidic extracts of thymus, spleen and bone marrow were investigated using 1H nuclear magnetic resonance spectroscopy (NMR). The NMR spectra were integrated in segments of 0.01, normalized and then analyzed using SIMCA-P software to visualize the similarities and differences in metabolic profiles between these groups. RESULT: Compared with the control group, the model group contained different levels of lactate, choline, glucose, taurine, alanine, LDL, glycerin, UFA, creatinine, acetoacetate, glutamate and beta-hydroxybutyrate (beta-HB). In the Siwutang treated group, the above-mentioned changes were reversed. CONCLUSION: The disorder of energy metabolism, damage of lymphocyte, and disorder of immune function were observed in blood deficiency model by NMR-based-metabonomics method, and the Siwutang can improve these effects. Metabonomics may be a valuable tool in the pharmacological evaluation of siwutang.

[Comparative study on metabonomics and on liver and kidney toxicity of Aristolochia fangchi and Stephania tetrandra].

OBJECTIVE: Combined the blood biochemical markers, the renal histopathological changes and the metabonomics profile were investigated to study the toxicity differences between Aristolochia fangchi and Stephania tetrandra. METHOD: Ten rats were randomly selected from 70 male Wistar rats as blank control group. The remaining 60 rats were divided into three groups. The two treated groups were orally administrated by 8.1 g x kg(-1) of A. fangchi and S. tetrandra respectively and the control group by equal volume of distilled water for 4weeks. Before the administrated and every 2 weeks, urine and plasma were collected and their 1H-NMR spectra were acquired, and then subjected to data process and PCA. Blood biochemical analysis and histopathological examination were carried out. RESULT: On the 2nd weekend, the BUN of the two treated groups, the AST of A. fangchi group were all markedly higher than that of the control group (P < 0.05). Compared with the A. fangchi group, the SCr higher in the S. tetrandra group (P < 0.05). The kidney pathological changes were apparently in the two treated groups and the pathological changes in the liver apparently in the S. tetrandra group. Along with the lasting of administration to the 4th week, the BUN, ALT and AST of the two treated groups, the SCr of A. fangchi group were all significantly higher than that of the control group (P < 0.01). The renal and liver injuries in the two treated groups were all become more seriously. Comparing the A. fangchi group, the BUN, SCr and AST were all higher in the S. tetrandra group (P < 0.05). Compared with control group, the urinary concentrations of citrate, 2-oxo-glutarate, taurine, hippurate, TMAO, creatine and the plasma concentrations of 3-D-hydroxybutyrate, acetone, NAC, OAC, creatinine were all changed. CONCLUSION: The A. fangchi and S. tetrandra all can induce the renal and liver lesion and its seriousness is correspondent to the lasting of administration. The liver and kidney toxicity of S. tetrandra are all more serious than the A. fangchi.

[NMR-spectroscopy-based metabonomic approach to analysis of Siwutang, a novel prescription, treated blood deficiency in mice].

OBJECTIVE: To discover the characteristic changes of metabolic profiles in C57 mice with cyclophosphamide induced blood deficiency and the effect of Siwutang. METHOD: An integrated metabonomic study using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy has been applied to investigate the metabolic profiles of serum, aqueous and lipidic extracts of thoracic gland, spleen, bone marrow obtained from control, model group (intraperitoneal injection of cyclophosphamide at a dose of 250 mg x kg(-1)) and Siwutang treated model group. The NMR spectra were integrated in segments of 0.04 ppm and then analyzed by principal component analysis (PCA) using SIMCA-P software to visualize the similarities and differences in metabolic profiles between these groups. RESULT: PCA result showed conspicuous difference in the metabolic profiles between groups. Compared with the control group, the model group contained lower concentration of lactate, 3-hydroxybutyrate, choline, glucose, and higher concentration of VLDL/LDL, leucine/isoleucine in serum. Lower concentration of taurine choline, Fbeta:RCH2CH2CO, Epi-coprostanol and lactate were found in both in thoracic gland extracts and spleen extracts. And in spleen extracts, we also found the lower concentration of 3-HB. In the extracts of bone marrow, the lower concentration of lactate, choline, glucosee were observed. When they were dosed with Siwutang 10 g x kg(-1) x d(-1) for 7 days, the effects above-mentioned were reversed. CONCLUSION: The injury established by injecting CTX is a kind of proper model to develop further metabonomics researches. The damage of mitochondria, disorder of energy metabolism and osmoregulation are observed in cyclophosphamide caused blood deficiency model by NMR-based-metabonomics method, and the Siwutang can improve these effects.

Differential metabolic responses in breast cancer cell lines to acidosis and lactic acidosis revealed by stable isotope assisted metabolomics.

Extracellular acidosis is considered as a hallmark of most human tumors, which plays an important role in promoting tumor malignant and aggressive phenotype in tumorigenesis. Acidosis and lactic acidosis can induce different responses in tumors. Previous studies have associated the response to lactic acidosis of tumors with good survival outcomes. In this study, we investigated the metabolomic changes in triple negative and luminal subtype breast cancer cell lines in response to acidosis and lactic acidosis. Our results showed that acidosis results in the reduction of cell viability and glycolysis in breast cancer cells, which is reversely correlated with the malignancy of cell lines. Under lactic acidosis, this reduction is reversed slightly. Untargeted metabolomic profiling revealed that glutaminolysis and fatty acid synthesis in cancer cells under acidosis are increased, while TCA cycle and glycolysis are decreased. Under lactic acidosis, the pentose phosphate pathway and acetate release are increased in MDA-MB-231 cells. The current results uncovered the different metabolic responses of breast cancer cells to acidosis and lactic acidosis, demonstrating the power of combined untargeted and stable isotope assisted metabolomics in comprehensive metabolomic analysis.

Metabolomic analysis of biofluids from rats treated with Aconitum alkaloids using nuclear magnetic resonance and gas chromatography/time-of-flight mass spectrometry.

The Aconitum alkaloids aconitine, mesaconitine, and hypaconitine are the main toxic components in a commonly used traditional Chinese herbal medicine Fu Zi. To provide guidelines for the safe use of this medicine, metabolic changes in Wistar rats caused by these compounds were investigated by means of integrated analysis of two metabonomic approaches: (1)H nuclear magnetic resonance (NMR) and gas chromatography/time-of-flight mass spectrometry (GC/TOF-MS). Rats were given a single dose of aconitine, mesaconitine, hypaconitine, or vehicle. The largest metabolic changes were observed 6 h after treatment. Every group receiving a dose had higher urine concentrations of glucose, acetate, dimethylglycine, succinate, and alanine and had lower concentrations of creatinine, citrate, 2-oxoglutarate, N-acetylated metabolites, and trimethylamine-N-oxide (TMAO) than did the control group. These results may reflect the perturbation of renal tubular function within the first 24 h after treatment. The results also revealed a larger perturbation of metabolic profiles in the aconitine group than in the mesaconitine and hypaconitine groups, illustrating how these alkaloids exhibit different toxicities. An analysis of plasma samples collected 7 days postdose showed that there were higher levels of lactate, alanine, and lipids along with lower levels of glucose, beta-hydroxybutyrate, and creatine in the plasma of the aconitine and mesaconitine groups than there were in the control and hypaconitine groups. The GC/TOF-MS data from the plasma samples showed that the number of metabolites, with significant changes or with a tendency to change, in the aconitine and mesaconitine groups were dissimilar, suggesting a possible difference in the acute toxicity mechanisms of these alkaloids.