Metabolite

An HPLC system using solid-phase extraction and HPLC with UV detection has been validated in order to determine tramadol and o-desmethyltramadol (M1) concentrations in human plasma. The method developed was selective and linear for concentrations ranging from 50 to 3500 ng / ml (tramadol) and 50 to 500 ng / ml (M1) with mean recoveries of 94.36612.53% and 93.5267.88%, respectively. Limit of quantitation (LOQ) was 50 ng / ml. For tramadol, the intra-day accuracy ranged from 95.48 to 114.64% and the inter-day accuracy, 97.21 to 103.24%. Good precision (0.51 and 18.32% for intra-and inter-day, respectively) was obtained at LOQ. The system has been applied to determine tramadol concentrations in human plasma samples for a pharmacokinetic study.

A reversed-phase high performance liquid chromatography method has been developed and validated for determination and quantitation of the natural sesquiterpene (−)-α-bisabolol. Furthermore the application of the method was done by characterization of chitosan milispheres and liposomes entrapping Zanthoxylum tingoassuiba essential oil, which contains appreciable amount of (−)-α-bisabolol. A reversed-phase C18 column and gradient elution was used with the mobile phase composed of (A) acetonitrile–water–phosphoric acid (19:80:1) and (B) acetonitrile. The eluent was pumped at a flow rate of 0.8 mL/min with UV detection at 200 nm. In the range 0.02–0.64 mg/mL the assay showed good linearity (R2 = 0.9999) and specificity for successful identification and quantitation of (−)-α-bisabolol in the essential oil without interfering peaks. The method also showed good reproducibility, demonstrating inter-day and intra-day precision based on relative standard deviation values (up to 3.03%), accuracy (mean recovery of 100.69% ± 1.05%) and low values of detection and quantitation limits (0.0005 and 0.0016 mg/mL, respectively). The method was also robust for showing a recovery of 98.81% under a change of solvent in standard solutions. The suitability of the method was demonstrated by the successful determination of association efficiency of the (−)-α-bisabolol in chitosan milispheres and liposomes. Copyright © 2009 John Wiley & Sons, Ltd.

The phytochemical, antioxidant and antimicrobial in vitro assay of Pleurotus pulmonarius-LAU09 (JF736658) was evaluated. The metabolite obtained from Pleurotus pulmonarius was characterized by IR analysis. It revealed the absorption of O-H, C-H, C-O bonds and hydrated water peaks (1650.6cm -1 ), without no absorption at uronic acid peak (1730cm -1 ). 1 HNMR spectrum analysis of the metabolite has anomeric carbon peaks of 5.10 and 4.51ppm, characterized as α and β linkages of glucan compoumd. The phytochemical screening of the mushroom extract revealed the presence of alkaloids, saponins, steroids, phlobatannins, flavonoids and anthraquinones. The metabolite was active against all tested pathogens except Pseudomonas aeruginosa with percentage activity of 85.75%. The highest zone of inhibition was obtained against Staphylococcus aureus (30mm), while the lowest zone size obtained was against E. coli (7mm). The antioxidant activity of evaluated mushroom extracts gave positive results with free radical scavenging activity found to be higher in all used in vitro methods. The result obtained from this study has shown the potential of mushroom extract as a potent therapeutic agent and a food supplement.

"The rumen is a unique organ that serves as the primary site for microbial fermentation of ingested plant material for domestic livestock such as cattle, sheep and goats. The chemical composition of ruminal fluid is thought to closely reflect the healthy/unhealthy interaction between rumen microflora and diet. Just as diet and feed quality is important for livestock production, rumen health is also critical to the growth and production of high quality milk and meat. Therefore a detailed understanding of the chemical composition of ruminal fluid and the influence of diet on its composition could help improve the efficiency and effectiveness of farming and veterinary practices.
Consequently we have undertaken an effort to comprehensively characterize the bovine ruminal fluid metabolome.
In doing so, we combined NMR spectroscopy, inductively coupled plasma mass-spectroscopy (ICP-MS), gas chromatography-mass spectrometry (GC-MS), direct flow injection (DFI) mass spectrometry and lipidomics with computer-aided literature mining to identify and quantify essentially all of the metabolites in bovine ruminal fluid that can be routinely detected (with today’s technology).
The use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these techniques. Tables containing the set of 246 ruminal fluid metabolites or metabolite species, their concentrations, related literature reference and links to their known diet associations for the bovine rumen metabolome are freely available at http://www.rumendb.ca."

Saffron derived from  Crocus sativus is the most
expensive, flavored and colorful spice with worldwide
importance and utilities in many disease conditions. Many efforts
have been made to detect variability in this plant. We used
sequence specific amplified polymorphism (SSAP) and
retrotransposon microsatellite amplified polymorphism
(REMAP) markers for analyzing the genetic diversity among
thirty accessions of Crocus sativus. Besides, we also attempted
metabolite profiling in these accessions. This work aimed at
assessing the molecular and biochemical variability in saffron
since it has been reported to be highly stable in other parts of the
world.  Various primer combinations were used in SSAP and
REMAP for PCR amplification and the primers giving the best
pattern were used for further analysis. The retrotransposon based
molecular marker failed to find any variation at molecular level
inferring the inability of retrotransposon mobility to cause
polymorphism in geographically different cultivars of  Crocus
sativus. However, the HPLC profiling of samples collected from
17 selected locations among the  thirty samples, revealed
variations in metabolite concentrations of crocin and safranal.
The results obtained in this study supported the monomorphic
nature of Kashmir saffron at genetic level.

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca.

An industrial scale biomass production using batch or fed-batch fermentations usually optimized by selection of bacterial strains, tuning fermentation media, feeding strategy, and temperature. However, in-depth investigation of the biomass metabolome during the production may reveal new knowledge for better optimization. In this study, for the first time, the authors investigated seven fermentation batches performed on five Streptoccoccus thermophilus strains during the biomass production at Chr. Hansen (Denmark) in a real life large scale fermentation process. The study is designed to investigate effects of batch fermentation, fermentation time, production line, and yeast extract brands on the biomass metabolome using untargeted GC-MS metabolomics. Processing of the raw GC-MS data using PARAFAC2 revealed a total of 90 metabolites out of which 64 are identified. Partitioning of the data variance according to the experimental design was performed using ASCA and revealed that batch an...

The effect of high-pressure (HP) (0.1, 150 and 300 MPa, 15 min) on taste profiles of vinasse-cured ducks was investigated; the metabolite profiles were determined using 1 H NMR. HP at 150 MPa increased the taste intensity of products compared with the controls, while HP at 300 MPa did not further improve their taste compared with 150 MPa treated samples. The metabonome of vinasse-cured ducks was dominated by 27 metabolites. HP increased amino acids, glucose, alkaloids and organic acids, but decreased inosine monophosphate and its derivatives , compared with the controls. The increments of metabolites in vinasse-dry-cured duck were higher than those in vinasse-wet-cured duck. The change of metabolites could be related to the enzyme activity, the degradations of proteins, sugars and nucleotides, and the permeation from vinasse-curing agents to duck meat. These findings suggest that 150 MPa treatment was effective to improve the taste of vinasse-cured duck.

Artemisinin, a sesquiterpene lactone; is one of the most well–known secondary
metabolites with multi–functional therapeutic effects which is isolated from Artemisia annua. Due to
low levels of artemisinin in this plant, efforts are being made to optimize its production by various
strategies such as elicitation–infiltration. This is a procedure in which, elicitors are injected to the
plants by fine–needles directly. In this study, we identified the effect of bacterial and fungal elicitors
on artemisinin level and biomass accumulation in A. annua. Artemisinin content was measured by
high performance thin layer chromatography. The maximum level of artemisinin was accomplished
by injection of 0.4 mg total sugar/ml F. oxysporum and B. cereus decreased artemisinin content in
comparison to the controls. 1 McFarland S. aureus was the most successful elicitor, increasing the
fresh weight 1.34 fold over the controls.

A reversed-phase high performance liquid chromatography method has been developed and validated for determination and quantitation of the natural sesquiterpene (-)-a-bisabolol. Furthermore the application of the method was done by characterization of chitosan milispheres and liposomes entrapping Zanthoxylum tingoassuiba essential oil, which contains appreciable amount of ( )-a-bisabolol. A reversed-phase C 18 column and gradient elution was used with the mobile phase composed of (A) acetonitrile-water-phosphoric acid (19:80:1) and (B) acetonitrile. The eluent was pumped at a flow rate of 0.8 mL/min with UV detection at 200 nm. In the range 0.02-0.64 mg/mL the assay showed good linearity (R 2 = 0.9999) and specificity for successful identification and quantitation of ( )-a-bisabolol in the essential oil without interfering peaks. The method also showed good reproducibility, demonstrating inter-day and intra-day precision based on relative standard deviation values (up to 3.03%), accuracy (mean recovery of 100.69% ± 1.05%) and low values of detection and quantitation limits (0.0005 and 0.0016 mg/mL, respectively). The method was also robust for showing a recovery of 98.81% under a change of solvent in standard solutions. The suitability of the method was demonstrated by the successful determination of association efficiency of the (-)-a-bisabolol in chitosan milispheres and liposomes.

Recent in vitro data obtained in our laboratory revealed similarities between baboons and humans in the biotransformation of bupropion (BUP) by both hepatic and placental microsomes. These data supported the use of baboons to study BUP biotransformation during pregnancy. The aim of this investigation was to determine the pharmacokinetics of BUP in baboons during pregnancy and postpartum, as well as fetal exposure to the drug after intravenous administration. Pregnant baboons (n = 5) received a single intravenous bolus dose of bupropion hydrochloride (1 mg/kg) at gestational ages 94-108 days (midpregnancy), 142-156 days (late pregnancy), and 6 weeks postpartum. Blood and urine samples were collected for 12 and 24 hours, respectively. The concentrations of BUP, hydroxybupropion (OH-BUP), threohydrobupropion, and erythrohydrobupropion in plasma were determined by liquid chromatography-tandem mass spectrometry. Relative to the postpartum period, the average midpregnancy clearance of BUP...

Obesity rates among children are growing rapidly worldwide, placing massive pressure on healthcare systems. Untargeted metabolomics can expand our understanding of the pathogenesis of obesity and elucidate mechanisms related to its symptoms. However, the metabolic signatures of obesity in children have not been thoroughly investigated. Herein, we explored metabolites associated with obesity development in childhood. Untargeted metabolomic profiling was performed on fasting serum samples from 27 obese Caucasian children and adolescents and 15 sex- and age-matched normal-weight children. Three metabolomic assays were combined and yielded 726 unique identified metabolites: gas chromatography–mass spectrometry (GC–MS), hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC LC–MS/MS), and lipidomics. Univariate and multivariate analyses showed clear discrimination between the untargeted metabolomes of obese and normal-weight children, with 162 significantly dif...

Increased angiogenesis and vascular endothelial growth factor (VEGF) levels contribute to higher metastasis and mortality in uveal melanoma (UM), an aggressive malignancy of the eye in adults. (±)-MRJF22, a prodrug of the sigma (σ) ligand haloperidol metabolite II conjugated with the histone deacetylase (HDAC) inhibitor valproic acid, has previously demonstrated a promising antiangiogenic activity. Herein, the asymmetric synthesis of (R)-(+)-MRJF22 and (S)-(−)-MRJF22 was performed to investigate their contribution to (±)-MRJF22 antiangiogenic effects in human retinal endothelial cells (HREC) and to assess their therapeutic potential in primary human uveal melanoma (UM) 92-1 cell line. While both enantiomers displayed almost identical capabilities to reduce cell viability than the racemic mixture, (S)-(−)-MRJF22 exhibited the highest antimigratory effects in endothelial and tumor cells. Given the fundamental contribution of cell motility to cancer progression, (S)-(−)-MRJF22 may represent a promising candidate for novel antimetastatic therapy in patients with UM.

PLD-301, a phosphate prodrug of clopidogrel thiolactone discovered by Prelude Pharmaceuticals with the aim to overcome clopidogrel resistance, was evaluated for its in vivo inhibitory effect on ADP-induced platelet aggregation in rats. The potency of PLD-301 was similar to that of pra-sugrel, but much higher than that of clopidogrel. The results of pharmacokinetic analysis showed that the oral bioavailability of clopidogrel thiolactone converted from PLD-301 was 4-to 5-fold higher than that of the one converted from clopidogrel, suggesting that in comparison with clopidogrel, lower doses of PLD-301 could be used clinically. In summary, PLD-301 presents a potent and orally bioavailable antiplatelet agent that might have some advantages over clopidogrel, such as overcoming clopidogrel resistance for CYP2C19-allele loss-of-function carriers, and lowering dose-related toxicity due to a much lower effective dose.

High-performance liquid chromatography of bromophenacyl esters of mycolic acid was used as an aid to assign a particular organism to one of four mycolic acid-containing genera. A gradient elution system, with methanol and chloroform, was used to distinguish representative mycolic acid patterns for the genera Corynebacterium, Rhodococcus, Nocardia, and Mycobacterium.

A novel simple bioanalytical method of high performance liquid chromatography (HPLC) for simultaneous quantification of morinidazole and its carbonylation metabolite (M1) in human plasma was developed and validated. The calibration curves for morinidazole and M1 were linear over concentration ranges of 23.99-1.464 × 10 4 g L −1 and 5.407-3300 g L −1 , respectively. Intra-and inter-day precision and accuracy results satisfied the acceptable criteria for bioanalytical method validation. This method could be a useful method for clinical pharmacokinetic studies of morinidazole and its carbonylation metabolite, and it has been applied to a pharmacokinetic study involving 11 renal insufficiency patients and 11 healthy volunteers.

Purpose Individual differences in cytochrome P-450 efficiency partly explain their variations in resistance to tamoxifen and estrogen metabolism. Two polymorphisms of the CYP1A1 gene-A4889G and T6235C-are known to affect activation of estrone and estradiol and to deregulate concentration of highly active tamoxifen metabolites. However, the clinicopathologic implications of these findings have not yet been evaluated. Objective The objective of this study is to evaluate whether T6235C and A4889G gene polymorphisms are related to pathological presentations and clinical outcomes of ER?/PR? breast cancer (BC) in women using tamoxifen. Methods We included 405 women with ER?/PR? tumors, who used tamoxifen as their primary therapy, and for whom 5-year follow-up data were available. We evaluated associations within clinicopathologic features, including overall 5-year survival, with CYP1A1 gene status. Results Univariate analysis showed that a slightly higher proportion of women with AG/GG genotypes were of European descent (P = 0.05) and that TC/CC genotype was significantly associated with premenopausal status (P = 0.01); however, no significant association remained after multivariate adjustment. Women with CYP1A1 genotypes other than AA and TT were more prone to develop low-grade tumors; 85.9 % of tumors in AA and TT genotype groups were grade III, but only 76.1 % of tumors in carriers of the polymorphisms were grade III (adjusted P = 0.02; OR 0.51 for grade III disease; 95 % CI 0.28-0.93). After 60 months of follow-up, *75 % of the women were alive. There was no significant difference in survival related to the CYP1A1 gene status. Conclusions Breast cancer patients carrying CYP1A1 gene polymorphisms developed less aggressive tumors, but showed no evidence of better prognoses.

Background: In recent, various human health disorders including cancer, diabetes, neurodegenerative and metabolic diseases are noticed among human populations. Currently, genetic and proteomic approaches are highly reported to detect metabolic disorders that also include inborn error of metabolisms. These existing detection methods are faced with cost issue and time consuming factors. Therefore, metabolites as biomarkers are one of potential avenues to detect metabolic disorders. Further, exploitation of urine as potential source of metabolite biomarkers, there are limitation in this area of research due to abundance of non-metabolite components such as proteins and nucleic acids. Hence, methods and processes are required to precisely fractionate metabolites from urine of inborn error of metabolism patients and then identified by analytical tools such as LC-HRMS and GC-MS. Methods: Sterile filtered urine samples (750 microl) mixed with (250 microl) loading buffer were electrophorese...

Trimethylamine N-oxide (TMAO) exemplifies how Nature uses the solute effect as a simple chemical strategy to cope with hydrodynamic pressure or urea stress to maintain proteostasis. It is a gut-microbe-generated metabolite that strongly promotes the development of atherosclerosis. It remains unclear how TMAO exerts its effects. In this study, we experimentally characterized the profile of the preferential interaction potential of TMAO with proteins, a thermodynamic key to understanding the effects of TMAO on protein processes and the distinction of TMAO among osmolytes. TMAO is thus found to be highly preferentially excluded from most types of protein surface, which explains why TMAO is a strong globular protein stabilizer and identifies the dominant stabilizing factor as the unfavorable interaction of TMAO with the hydrophobic surface exposed upon unfolding. We dissected the mechanism of the counteracting effects of TMAO and urea: the contrary feature of the interaction profiles of the two solutes maximizes the possibility for them to offset each other's perturbing effect on protein processes. The interaction profile also predicts that TMAO promotes aggregation of amyloidogenic intrinsically disordered peptide, as demonstrated here in Aβ42, and that TMAO has a strong potential to impact protein processes in the absence of stressors. Our data suggest that although TMAO is an evolutionally selected chemical chaperone for some organisms or organs, its compatibility in vivo is conditional and determined by its interaction profile with biopolymers and the nature of the essential biopolymer processes. Our thermodynamic framework plus the TMAO-protein interaction profile provides a basis for exploring the broad biological significance of TMAO, including its pathological impact in the absence of stressors. We argue for the general importance of controlling in vivo background solutes and the pathological significance of a control failure.