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An extract prepared from species of Paris is the most widely consumed herbal product in China. The genus Paris includes a variety of genotypes with different medicinal component contents but only two are defined as official sources. Closely related species have different medicinal properties because of differential expression of proteins and metabolites. To better understand the molecular basis of these differences, we examined proteomic and metabolomic changes in rhizomes of P. polyphylla var. chinensis, P. polyphylla var. yunnanensis, and P. fargesii var. fargesii using a technique known as sequential window acquisition of all theoretical mass spectra as well as gas chromatography-time-of-flight mass spectrometry. In total, 419 proteins showed significant abundance changes, and 33 metabolites could be used to discriminate Paris species. A complex analysis of proteomic and metabolomic data revealed a higher efficiency of sucrose utilization and an elevated protein abundance in the sugar metabolic pathway of P. polyphylla var. chinensis. The pyruvate content and efficiency of acetyl-CoA-utilization in saponin biosynthesis were also higher in P. polyphylla var. chinensis than in the other two species. The results expand our understanding of the proteome and metabolome of Paris and offer new insights into the species-specific traits of these herbaceous plants. SIGNIFICANCE: The traditional Chinese medicine Paris is the most widely consumed herbal product for the treatment of joint pain, rheumatoid arthritis and antineoplastic. All Paris species have roughly the same morphological characteristics; however, different members have different medicinal compound contents. Efficient exploitation of genetic diversity is a key factor in the development of rare medicinal plants with improved agronomic traits and malleability to challenging environmental conditions. Nevertheless, only a partial understanding of physiological and molecular mechanisms of different plants of Paris can be achieved without proteomics. To better understand the molecular basis of these differences and facilitate the use of other Paris species, we examine proteomic metabolomic changes in rhizomes of Paris using the technique known as SWATH-MS and GC/TOF-MS. Our research has provided information that can be used in other studies to compare metabolic traits in different Paris species. Our findings can also serve as a theoretical basis for the selection and cultivation of other Paris species with a higher medicinal value.

Two-phase flow is important in both science and engineering. In this paper, a D2Q5 multi-relaxation-time lattice Boltzmann model is proposed for solving Allen–Cahn equation, which is a convection–diffusion equation for the order parameter. Through Chapmann–Enskog analysis, the macroscopic equations can be recovered from the D2Q5 MRT LB model. Then, a detailed numerical study on several classical problems is performed to give a comparison between the present model for the AC equation and the previous study. The results show that the present model gets better accuracy and has better stability than that of single-relaxation-time LB model, and has higher computational efficiency than that of D2Q9 MRT LB model. Furthermore, as an important application, the model is also used to study the deformation field problem because of its excellent ability to cope with complex and changeable boundaries.

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. 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. 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. 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. 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.

Food spoilage is one of the most common factors of food poisoning. In this research, we characterized the metabolic features of three spoiled foods using a nontargeted metabolic profiling strategy based on two dimensional gas chromatography time-of-flight mass spectrometry profiling method. Five time points (0, 1, 3, 5 and 7 days) during the spoilage processes of three different foods (bread, chicken egg and cucumber) were evaluated. More than 2000 chromatographic peaks were obtained for subsequent data analysis. Thirteen metabolites, including amino acids, biogenic amines and organic acids, were mined and identified as differential metabolites. The major metabolic alternations in the spoilage processes of different foods were closely related to some metabolic pathways, such as amino acid metabolism, tricarboxylic acid cycle and ammonia recycling. It showed that these potential metabolites screened using this method had good predication performance and could reflect the changing trend of the spoilage process of those three foods. This method could be used to further develop chemical sensors for food safety, and be helpful to gain further understanding on the spoilage.

Ethnopharmacological relevance Aconitum carmichaelii Debx. (Fuzi), a commonly use traditional Chinese medicine (TCM), has often been used in combination with Rhizoma Glycyrrhizae (Gancao) to reduce its toxicity due to diester diterpenoid alkaloids aconitine, mesaconitine, and hypaconitine. However, the mechanism of detoxication is still unclear. Glycyrrhetinic acid (GA) is the metabolite of glycyrrhizinic acid (GL), the major component of Gancao. In present study, the effect of GA on the changes of metabolic profiles induced by mesaconitine was investigated using NMR-based metabolomic approaches. Materials and methods Fifteen male Wistar rats were divided into a control group, a group administered mesaconitine alone, and a group administered mesaconitine with one pretreatment with GA. Their urine samples were used for NMR spectroscopic metabolic profiling. Statistical analyses such as orthogonal projections to latent structures-discriminant analysis (OPLS-DA), t-test, hierarchical cluster, and pathway analysis were used to detect the effects of pretreatment with GA on mesaconitine-induced toxicity. Results The OPLS-DA score plots showed the metabolic profiles of GA-pretreated rats apparently approach to those of normal rats compared to mesaconitine-induced rats. From the t-test and boxplot results, the concentrations of leucine/isoleucine, lactate, acetate, succinate, trimethylamine (TMA), dimethylglycine (DMG), 2-oxo-glutarate, creatinine/creatine, glycine, hippurate, tyrosine and benzoate were significantly changed in metabolic profiles of mesaconitine-induced rats. The disturbed metabolic pathways include amino acid biosynthesis and metabolism. Conclusions GA-pretreatment can mitigate the metabolic changes caused by mesaconitine-treatment on rats, indicating that prophylaxis with GA could reduce the toxicity of mesaconitine at the metabolic level.

Tuberculosis is a common infectious disease caused by a bacterium called M. tuberculosis. Invasion of human macrophages is a critical step for establishing a mycobacterial infection. Many protocols, such as M. tuberculosis genomics and proteomics, have been used to discuss the pathogenesis of M. tuberculosis. This work describes a GC/MS metabolomics approach to investigate the metabolome variation of human macrophages infected by different M. tuberculosis strains. The pathogenic mechanism of M. tuberculosis was discussed from the metabolic terminal of macrophages. It was quite remarkable that most of the differential metabolites had the very similar regularity and variation tendency after infection by different virulent strains. The results suggested that M. tuberculosis invaded and infected macrophages largely through three different pathways, including glutathione anabolic metabolism, lipid biosynthesis and metabolism, and carbohydrate biosynthesis and metabolism. These pathways were affected after the ...

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.

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.

Ethnopharmacological relevance Fuzi, the processed lateral root of Aconitum carmichaelii Debeaux, is a traditional Chinese medicine used for its analgesic, antipyretic, anti-rheumatoid arthritis and anti-inflammation effects; however, it is also well known for its toxicity. Gancao, the root of Glycyrrhiza uralensis Fisch., is often used concurrently with Fuzi to alleviate its toxicity. However, the mechanism of detoxication is still not well clear. Aim of the study In this study, the effect of Gancao on the metabolic changes induced by Fuzi was investigated by NMR-based metabonomic approaches. Materials and methods Fifty male Wistar rats were randomly divided into five groups (group A: control, group B: Fuzi decoction alone, group C: Gancao decoction alone, group D: Fuzi decoction and Gancao decoction simultaneously, group E: Fuzi decoction 5 h after Gancao decoction) and urine samples were collected for NMR-based metabolic profiling analysis. Statistical analyses such as unsupervised PCA, t-test, hierarchical cluster, and pathway analysis were used to detect the effects of Gancao on the metabolic changes induced by Fuzi. Results The behavioral and biochemical characteristics showed that Fuzi exhibited toxic effects on treated rats (group B) and statistical analyses showed that their metabolic profiles were in contrast to those in groups A and C. However, when Fuzi was administered with Gancao, the metabolic profiles became similar to controls, whereby Gancao reduced the levels of trimethylamine N-oxide, betaine, dimethylglycine, valine, acetoacetate, citrate, fumarate, 2-ketoglutarate and hippurate, and regulated the concentrations of taurine and 3-hydroxybutyrate, resulting in a decrease in toxicity. Furthermore, important pathways that are known to be involved in the effect of Gancao on Fuzi, including phenylalanine, tyrosine and tryptophan biosynthesis, the synthesis and degradation of ketone bodies, and the TCA cycle, were altered in co-treated rats. Conclusions Gancao treatment mitigated the metabolic changes altered by Fuzi administration in rats, demonstrating that dosing with Gancao could reduce the toxicity of Fuzi at the metabolic level. Fuzi and Gancao administered simultaneously resulted in improved toxicity reduction than when Gancao was administrated 5 h prior to Fuzi. In summary, co-administration of Gancao with Fuzi reduces toxicity at the metabolic level.

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.

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.

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.

Functional dyspepsia (FD) is a common gastrointestinal disorder with multiple pathogenic mechanisms seen in clinical practice, and acupuncture may potentially be an alternative therapy for it. In order to investigate the biological effects of FD and the effect of acupuncture on metabolism, 1H nuclear magnetic resonance (NMR)-based metabonomic techniques have been used to compare the plasma metabolic profiles of six female FD patients with those of six female healthy control subjects. Plasma metabolic profiles of FD patients treated by acupuncture at the Foot-Yangming Meridian were also collected and compared. Data obtained from NMR spectroscopy were subjected to principal components analysis (PCA). The results show that there are relatively higher levels of glucose, acetate, high-density lipoprotein (HDL), and phosphatidylcholine (PtdCho), and lower levels of lactate, leucine/isoleucine, N-acetyl glycoprotein (NAc), and low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) in FD patients than in healthy controls. Acupuncture treatment of FD patients significantly changed the levels of leucine/isoleucine, lactate and glucose, and slightly changed lipids level towards those of the healthy controls, demonstrating its therapeutic effects on the relief of FD symptoms. Due to the limited number of subjects, the present work is just a proof-of-principle study and further researches with larger number of subjects are needed. Our work shows the potential of an NMR-based metabonomic approach in the study of biological effects of acupuncture.

A metabolomic investigation of intoxication with Aconitum sp. alkaloids was carried out. Plasma obtained from Wistar rats administered these alkaloids was analyzed using gas chromatography/time-of-flight mass spectrometry. Samples were analyzed following protein precipitation, liquid-liquid extraction, and derivatization. Thirty-six metabolites were identified among the detected compounds. Subsequent data analysis using the t test and principal component analysis revealed metabolic differences between the control rats and treated groups as well as between the groups of rats administered different alkaloids. Twenty-seven metabolites were significantly different between plasma samples from the controls and treated groups. The significant decreased plasma levels of glutamine and creatinine in all treated groups suggested impaired heart and muscle function caused by alkaloids. The plasma levels of 22 metabolites in the hypaconitine group were significantly decreased. In contrast, only 8 and 13 metabolites were observed with significantly decreased levels in the aconitine and mesaconitine groups, respectively.These results indicated that Aconitum sp. alkaloids can cause metabolic disorders in rats. The toxicity and corresponding mechanism of hypaconitine was different from those of aconitine and mesaconitine, based on the differences of perturbed metabolic patterns between groups.

Fulminant hepatic failure (FHF) is one of the most challenging gastrointestinal emergencies encountered in clinical practice. Early identification of patients with FHF who need liver transplantation is very important. To construct a prediction model for the early diagnosis and prognosis of FHF, we studied the dynamics of metabolic intermediates and metabolic profiles with a D-galactosamine (GalN)/lipopolysaccharide (LPS)-treated BALB/c mouse model of FHF. Levels of plasma metabolites were quantified with gas chromatography/time-of-flight mass spectrometry, and data were processed with partial least squares discriminant analysis (PLS-DA). Distinct clustering differences were observed 5 and 6 hours after GalN/LPS treatment between mice that survived and those that died, but there were no differences between these groups 4 hours after treatment. Five hours after treatment, plasma levels of some metabolites differed significantly between the survival, dead, and control groups. Ketogenesis and the tricarboxylic acid cycle were inhibited in both the survival and dead groups, but in the dead group, the urea cycle was also inhibited, and glycolysis was elevated. PLS-DA indicated that principal component weighting was greatest for plasma levels of phosphate, beta-hydroxybutyrate, urea, glucose, and lactate. The Y-predicted scatter plot in the partial least squares (PLS) model assigned samples to the survival or dead groups with an a priori cutoff of 0.10 with 100% sensitivity and specificity. Similar results were observed in 11 FHF patients with different outcomes. In conclusion, the PLS model based on metabonomic analysis can be used to predict outcomes well, and plasma levels of phosphate, beta-hydroxybutyrate, urea, glucose, and lactate may constitute a set of markers for the early diagnosis and prognosis of FHF.

Metabonomics methods have gradually become important auxiliary tools for screening disease biomarkers. However, recognition of metabolites or potential biomarkers closely related to either particular clinical symptoms or prognosis has been difficult. The current study aims to identify potential biomarkers of functional dyspepsia (FD) by a new strategy that combined hydrogen nuclear magnetic resonance (1H NMR)-based metabonomics techniques and an integrative multi-objective optimization (LPIMO) method. First, clinical symptoms of FD were evaluated using the Nepean Dyspepsia Index (NDI) and plasma metabolic profiles were measured by 1H NMR. Correlations between the key metabolites and the NDI scores were calculated. Then, LPIMO was developed to identify a multi-biomarker panel by maximizing diagnostic ability and correlation with the NDI score. Finally, a KEGG database search elicited the metabolic pathways in which the potential biomarkers are involved. The results showed that glutamine, alanine, proline, HDL, β-glucose, α-glucose and LDL/VLDL levels were significantly altered in FD patients. Among them, phosphatidycholine (PtdCho) and leucine/isoleucine (Leu/Ile) were positively and negatively correlated with the NDI Symptom Index (NDSI) respectively. Our procedure not only significantly improved the credibility of the biomarkers, but also demonstrated the potential of further explorations and applications to diagnosis and treatment of complex disease.

An integrated metabonomics study using high-resolution (1)H nuclear magnetic resonance (NMR) spectroscopy has been applied to investigate the biochemical composition of urine, serum, liver tissue aqueous extracts (acetonitrile/water) and liver tissue lipidic extracts (chloroform/methanol) obtained from control and Bay41-4109 treated rats (10, 50, 400mg.kg(-1).d(-1) for 5 days, i.g.). Principal components analysis was used to visualize similarities and differences in biochemical profiles. The results showed the effects induced by Bay41-4109 at 400mg.kg(-1).d(-1) are different from those induced at 10, 50mg.kg(-1).d(-1). The biochemical profiles of 400mg.kg(-1).d(-1) group might reflect the hepatotoxicity of Bay41-4109 more exactly. The elevation in the level of 3-HB, lactate, 2-hydroxy-acetol and d-glucose was found in the urine, and the levels of VLDL/LDL(CH(2))(n), VLDL/LDL-CH(3), 2-oxo-3-methyl-n-valerate, 3-HB, lactate, acetate, taurine, 2-hydroxy-isovalerate in serum were increased significantly in 400mg.kg(-1).d(-1) group. The predominant changes identified in liver tissue aqueous extracts included an increase in the signal intensities of lactate, 3-amino-isovalerate, pyruvate, choline, trimethylamine-N-oxide (TMAO) and a reduction in the intensities of taurine, hippurate and d-glucose. In liver tissue chloroform/methanol extracts, there was a remarkably increase in many of the lipid signals including the triglyceride terminal methyl, methylene groups, and CH(2)CO, N(+)(CH(3))(3), CH(2)OPO(2), CH(2)OCOR. These observations all provide evidence that fatty acid metabolism disorder and mitochondrial inability might contribute to the hepatotoxicity of Bay41-4109. The application of (1)H NMR spectroscopy to an array of biological samples comprising urine, serum and liver tissue extracts yields new insight into the hepatotoxicity of xenobiotics.

Hepatocellular carcinoma (HCC) is a common malignancy that has region specific etiologies. Unfortunately, 85% of cases of HCC are diagnosed at an advanced stage. Reliable biomarkers for the early diagnosis of HCC are urgently required to reduced mortality and therapeutic expenditure. We established a non-targeted gas chromatography–time of flight–mass spectrometry (GC-TOFMS) metabolomics method in conjunction with Random Forests (RF) analysis based on 201 serum samples from healthy controls (NC), hepatitis B virus (HBV), liver cirrhosis (LC) and HCC patients to explore the metabolic characteristics in the progression of hepatocellular carcinogenesis. Ultimately, 15 metabolites were identified intimately associated with the process. Phenylalanine, malic acid and 5-methoxytryptamine for HBV vs. NC, palmitic acid for LC vs. HBV and asparagine and β-glutamate for HCC vs. LC were screened as the liver disease-specific potential biomarkers with an excellent discriminant performance. All the metabolic perturbations in these liver diseases are associated with pathways for energy metabolism, macromolecular synthesis and maintaining the redox balance to protect tumor cells from oxidative stress.

Antiangiogenic compound has been believed to be an ideal drug in the current cancer biological therapy, but the angiogenesis inhibitors suffer setback for unknown toxicity now. A novel synthetic indolin-s-ketone small molecular compound, 3Z-3-[({sup 1} H-pyrrol-2-yl)-methylidene]-1-(1-piperidinylmethyl)-1,3-2H-indol-2-one (Z24) can inhibit angiogenesis in new blood vessels. The hepatotoxicity effects of Z24 oral administration (dosed at 60, 130 and 200 mg/kg) have been investigated in female Wistar rats by using metabonomic analysis of {sup 1}H NMR spectra of urine, plasma and liver extracts, as well as by clinical chemistry analysis, liver histopathology and electron micrographs examination. The {sup 1}H NMR spectra of the biofluids were analyzed visually and via pattern recognition by using principal component analysis. The metabonomic trajectory analysis on the time-related hepatotoxicity of Z24 was carried out based on the {sup 1}H NMR spectra of urine samples, which were collected daily predose and postdose over an 8-day period. Urinary excretion of citrate, lactate, 2-oxo-glutarate and succinate increased following Z24 dosing. Increased plasma levels of lactate, TMAO and lipid were observed, with concomitant decrease in the level of glucose and phosphatidylcholine. Metabolic profiling on aqueous soluble extracts of liver tissues with the high dose level of Z24 showed an increase in lactatemore » and glutamine, together with a decrease in glucose, glycogen and choline. On the other hand, studies on lipid soluble extracts of liver tissues with the high dose level of Z24 showed increased level in lipid triglycerides and decreased level in unsaturated fatty acids and phosphatidylcholine. Moreover, the most notable effect of Z24 on the metabolism was the reduction in the urinary levels of creatinine and TMAO and the increase in acetate, citrate, succinate and 2-oxo-glutamate with time dependence. The results indicate that in rats Z24 inhibits mitochondrial function through altering the energy and lipid metabolism, which results in the accumulation of free fatty acids and lactate because of the lack of aerobic respiration. These data show that the metabonomic approach represents a promising new technology for the toxicological mechanism study.« less

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J(NN)-correlated [(15)N, (1)H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147-14151, 1998) can be applied to measure (h)J(HN) in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA(Trp), since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J(NN)-correlated [(15)N, (1)H] TROSY experiment which enables direct measurement of (h)J(HN) in large nucleic acids.

15N-Labeled Bacillus subtilis tRNATrp wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques. These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNATrp molecules. Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs whose peaks were not clearly observed. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. These studies also revealed two components of tRNATrp, which could not be separated by high pressure liquid chromatography, corresponding to s4U and U at position 8 of the tRNATrp, as indicated by two different sets of peaks for the TψC and D arms. The modification at position 8 altered the local conformation of the core region of the tRNA. Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to the s4U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.

Background. The objective of this study was to identify potential biomarkers of electroacupuncture (EA) on relieving acute migraine through metabolomic study.Methods. EA treatments were performed on both acupoints and nonacupoints on the nitroglycerin (NTG)-induced migraine rat model. NMR experiments and multivariate analysis were used for metabolomic analysis.Results. The number of head-scratching, the main ethology index of migraine rat model, was significantly increasedP<0.01after NTG injection. The plasma metabolic profile of model group was distinct from that of the control group. Glutamate was significantly increasedP<0.01, whereas lipids were significantly decreasedP<0.01in model rats. After EA at acupoints, the metabolic profile of model rats was normalized, with decreased glutamateP<0.05and increased lipidsP<0.01. In contrast, EA at nonacupoints did not restore the metabolic profile, but with six metabolites significantly different from acupoints group. Interestingly, the number of head-scratching and glutamate level were significantly decreasedP<0.05after receiving EA at both acupoints and nonacupoints.Conclusions. EA at acupoints may relieve acute migraine by restoring the plasma metabolic profile and plasma glutamate, while EA at nonacupoints may modestly relieve acute migraine by decreasing plasma glutamate.

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.

Identifying biomarkers for acupuncture treatment is crucial to understand the mechanism of acupuncture effect at molecular level. In this study, we investigate the metabolic profiles of acupuncture treatment on several meridian points in human. To identify the subsets of metabolites that best characterize the acupuncture effect for each meridian point, a linear programming based model is proposed to identify biomarkers from the high-dimensional metabolic data. Specifically, we use nearest centroid as prototype to simultaneously minimize the number of selected features and leave-one-out cross validation error of the classifier. As a result, we reveal novel metabolite biomarkers for acupuncture treatment. Our result demonstrates that metabolic profiling might be a promising method to investigating the molecular mechanism of acupuncture. Comparison with other existing methods shows the efficiency and effectiveness of our new method. In addition, the method proposed in this paper is general and can be used in other high-dimensional applications, such as cancer genomics.

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.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.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.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.

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.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.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 (P0.05). Compared with the A. fangchi group, the SCr higher in the S. tetrandra group (P0.05). 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 (P0.01). 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 (P0.05). 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.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.

The metabonomics changes of plasma and brain tissue after dietary supplementation with blueberry extracts (BBE) and cyanidin-3-O-galactoside from blueberry (BBM) in aged mice were investigated by (1)H NMR technique. The mice received intragastric administration of BBE (200 mg/kg/day), BBM (50 mg/kg/day), and saline water (0.9%) for 6 weeks, respectively, in the BBE, BBM, and control groups. At the end of the experiment, plasma and brain samples were collected for NMR analysis. The results demonstrated that the level of choline in plasma from BBE and BBM groups were obviously elevated relative to the control group, whereas the levels of lactate and phosphocholine in plasma were remarkably reduced. Compared with those in the control group, the levels of choline and GABA in the brain from the BBE group were obviously increased, whereas glutamate and phosphocholine in the BBE group were significantly decreased. The level of taurine in the brain from the BBM group was particularly higher than that in the control group. These results indicated supplementation with BBE or BBM might induce similar changes of endogenous plasma and brain metabolic profiles in aged mice.

Background Perfluorododecanoic acid (PFDoA) is a perfluorinated carboxylic chemical (PFC) that has broad applications and distribution in the environment. While many studies have focused on hepatotoxicity, immunotoxicity, and reproductive toxicity of PFCAs, few have investigated renal toxicity. Methodology/Principal Findings Here, we used comparative proteomic and metabonomic technologies to provide a global perspective on renal response to PFDoA. Male rats were exposed to 0, 0.05, 0.2, and 0.5 mg/kg/day of PFDoA for 110 days. After 2-D DIGE and MALDI TOF/TOF analysis, 79 differentially expressed proteins between the control and the PFDoA treated rats (0.2 and 0.5 mg-dosed groups) were successfully identified. These proteins were mainly involved in amino acid metabolism, the tricarboxylic acid cycle, gluconeogenesis, glycolysis, electron transport, and stress response. Nuclear magnetic resonance-based metabonomic analysis showed an increase in pyruvate, lactate, acetate, choline, and a variety of amino acids in the highest dose group. Furthermore, the profiles of free amino acids in the PFDoA treated groups were investigated quantitatively by high-coverage quantitative iTRAQ-LC MS/MS, which showed levels of sarcosine, asparagine, histidine, 1-methylhistidine, Ile, Leu, Val, Trp, Tyr, Phe, Cys, and Met increased markedly in the 0.5 mg dosed group, while homocitrulline, α-aminoadipic acid, β-alanine, and cystathionine decreased. Conclusion/Significance These observations provide evidence that disorders in glucose and amino acid metabolism may contribute to PFDoA nephrotoxicity. Additionally, α2u globulin may play an important role in protecting the kidneys from PFDoA toxicity.

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.

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 β-hydroxybutyrate (β-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 β-HB significantly induced transforming growth factor-β1 expression, with a marked increase in collagen I expression. β-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.

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

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.

Two oleanane-type oligoglycosides, cernuosides A (1) and B (2), were isolated from the roots of Pulsatilla cernua (Ranunculaceae). Structure elucidation was accomplished by 1D and 2D NMR (DQF-COSY, TOCSY, HMQC, HMBC, and ROESY) methods, FABMS, and hydrolysis. Both compounds showed moderate activity against sucrase.

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.

A metabolomic investigation of intoxication with Aconitum sp. alkaloids was carried out. Plasma obtained from Wistar rats administered these alkaloids was analyzed using gas chromatography/time-of-flight mass spectrometry. Samples were analyzed following protein precipitation, liquid-liquid extraction, and derivatization. Thirty-six metabolites were identified among the detected compounds. Subsequent data analysis using the t test and principal component analysis revealed metabolic differences between the control rats and treated groups as well as between the groups of rats administered different alkaloids. Twenty-seven metabolites were significantly different between plasma samples from the controls and treated groups. The significant decreased plasma levels of glutamine and creatinine in all treated groups suggested impaired heart and muscle function caused by alkaloids. The plasma levels of 22 metabolites in the hypaconitine group were significantly decreased. In contrast, only 8 and 13 metabolites were observed with significantly decreased levels in the aconitine and mesaconitine groups, respectively.These results indicated that Aconitum sp. alkaloids can cause metabolic disorders in rats. The toxicity and corresponding mechanism of hypaconitine was different from those of aconitine and mesaconitine, based on the differences of perturbed metabolic patterns between groups.

The putative translation initiation factor eIF5A is essential for cell viability and is highly conserved from archaebacteria to mammals. This factor is the only cellular protein that undergoes an essential posttranslational modification dependent on the polyamine spermidine, called hypusination. Although this protein may be involved in many important physiological functions, the precise molecular functions of eIF-5A remain to be clarified. To determine the solution structure and the protein interactions of eIF5A with its potential substrates, we performed NMR studies. Here, we report the nearly complete assignment of the eIF5A.

In this work, metabonomic methods utilizing (1)H NMR spectroscopy and multivariate statistical technique have been applied to investigate the metabolic profiles of SAM. The serum metabolome of senescence-prone 8 (SAMP8), a murine model of age-related learning and memory deficits and Alzheimer's disease (AD), was compared with that of control, senescence-resistant 1 (SAMR1), which shows normal aging process. Serum samples were collected for study from both male and female 12-month-old SAMP8 and age matched SAMR1 ( n = 5). (1)H NMR spectra of serum were analyzed by pattern recognition using principal components analysis. The results showed that the serum metabolic patterns of SAMP8 and SAMR1 were significantly different due to strains and genders. Subtle differences in the endogenous metabolite profiles in serum between SAMP8 and SAMR1 were observed. The most important metabolite responsible for the strain separation was lack of inosine, which meant the protective function of anti-inflammation, immunomodulation and neuroprotection might be attenuated in SAMP8. Other differential metabolites observed between strains included decreased glucose, PUFA, choline, phosphocholine, HDL, LDL, D-3-hydoxybutyrate, citrate and pyruvate and increased lactate, SFA, alanine, methionine, glutamine and VLDL in serum of SAMP8 compared with those of SAMR1, suggesting perturbed glucose and lipid metabolisms in SAMP8. Besides the differences observed between the strains, an impact of gender on metabolism was also found. The females exhibited larger metabolic deviations than males and these gender differences in SAMP8 were much larger than in SAMR1. Higher levels of VLDL, lactate and amino acids and lower levels of HDL, LDL and unsaturated lipids were detected in female than in male SAMP8. These facts indicated that the metabolism disequilibrium in female and male SAMP8 was different and this may partly explain that females were more prone to AD than males. The results of this work may provide valuable clues to the understanding of the mechanisms of the senile impairment and the pathological changes of AD, as well as show the potential power of the combination of the NMR technique and the pattern recognition method for the analysis of the biochemical changes of certain pathophysiologic conditions.

Metabonomic approaches are believed to be one of the powerful methods for disease diagnosis and assessment of patients' conditions. However, many publications have been providing the evidences of the biases caused by many factors such as diet, physical exercises, environment and individual differences in the clinical researches. For the purpose of ensuring the comparability of metabonomic 1H NMR data from different studies, it is necessary to control those nonexperimental factors in the study design and the data analysis.

The purpose of this paper was to study the effects of Hei-Shun-Pian, the processed lateral root of Aconitum carmichaelii Debx. (Ranunculaceae), on the metabolic profile of rats, to discuss the mechanism of toxicology and to find out the potential biomarkers of the toxic effects. Twenty male Wistar rats were divided into four groups (n=5) and each group were administered orally with the decoction of Hei-Shun-Pian (88.1g/kg per day, 35.6g/kg per day, 17.6g/kg per day) or equal volume of drinking water respectively for 14 days. Urine of every 24-h and the plasma of the last day were collected for NMR experiments, and then analyzed by multivariate analysis methods. Decreases in urinary excretion of taurine and trimethylamine-N-oxide (TMAO) and increases in urinary levels of citrate, 2-oxoglutarate (2-OG), succinate and hippurate were observed in the high and medium dosed groups at the early stage of the dosing period. Taurine level increased at the later stage of the dosing period to the normal value, and then even to a value higher than that of the control group at the end of the experiment. No metabolic differences were observed between low dosed and control groups until the later stage of the dosing period when a slight increase in urinary taurine level was observed, suggesting a cumulative effect. These results suggest the toxic effect of Hei-Shun-Pian on rat heart in a dose dependent manner.

The purpose of this study was to characterize the changes in metabolic intermediates and to investigate the metabolic profile of a mouse model of fulminant hepatic failure (FHF), induced by D-galactosamine/lipopolysaccharide (GalN/LPS). Plasma metabolite levels were detected using gas chromatography/time-of-flight mass spectrometry, and the acquired data were transferred into Simca-P and processed using principal components analysis (PCA). In total, 45 metabolites were identified from the 267 distinct compounds found in the study. Whereas significant differences were noted in the plasma levels of the control and FHF groups, no differences in gluconeogenesis or glycolysis were noted following GalN/LPS treatment. Our data also suggest that the production of ketone bodies, and the tricarboxylic acid and urea cycles, was inhibited. PCA data suggest that 5-hydroxyindoleacetic acid, glucose, beta-hydroxybutyrate, and phosphate parameters had the highest weights on each of the principal components, and that they were the most important metabolites contributing to the separation of groups. In conclusion, this metabonomic approach can be used as a powerful tool to characterize changes in metabolic intermediates and to search for metabolic markers under certain pathophysiological conditions, such as FHF. Our data also demonstrate that a combination of 5-hydroxyindoleacetic acid, glucose, beta-hydroxybutyrate, and phosphate concentrations in the plasma is a potential marker for FHF, as well as for the early prognosis of FHF.

Drastisch verkurzt wird die transversale Relaxation bei der Transversal-Relaxations-optimierten Spektroskopie (TROSY) infolge der Interferenz zwischen Dipol-Dipol-Wechselwirkungen und der Anisotropie der chemischen Verschiebung. Damit wird die NMR-Spektroskopie zu einer geeigneten Methode fur die Untersuchung sehr groser Biomolekule, die optimal genutzt werden kann, sobald 1-GHz-Spektrometer zur Verfugung stehen werden. Durch Verwendung von neuen Phasencyclen und Datenverarbeitungsprogrammen konnte die Empfindlichkeit des TROSY-Experiments um den Faktor √2 erhoht werden, was fur Anwendungen in Hochfeld-NMR-Untersuchungen an grosen Proteinen von erheblicher Bedeutung ist.

Dramatically shortened transverse relaxation times in transverse relaxation optimized spectroscopy (TROSY) result from interference between dipole-dipole interactions and the anisotropy of the chemical shift. Thus NMR spectroscopy becomes a suitable method for studying large biomolecules, with optimal performance when 1-GHz spectrometers become available. By using new phase cycles and data-processing methods, the sensitivity of the TROSY experiment was increased by a factor of √2, which is of considerable importance for applications in high-field NMR studies on large proteins.