Mechanisms of TGFß in prostaglandin synthesis and sperm guidance in Caenorhabditis elegans.

BACKGROUND: The transparent epidermis of Caenorhabditis elegans makes it an attractive model to study sperm motility and migration within an intact reproductive tract. C elegans synthesize specific F-series prostaglandins (PGFs) that are important for guiding sperm toward the spermatheca. These PGFs are synthesized from polyunsaturated fatty acid (PUFA) precursors, such as arachidonic acid (AA), via a novel pathway, independent of the classical cyclooxygenases (Cox) responsible for most PG synthesis. While the enzyme(s) responsible for PG synthesis has yet to be identified, the DAF-7 TGFß pathway has been implicated in modulating PG levels and sperm guidance. RESULTS: We find that the reduced PGF levels in daf-1 type I receptor mutants are responsible for the sperm guidance defect. The lower level of PGs in daf-1 mutants is due in part to the inaccessibility of AA. Finally, lipid analysis and assessment of sperm guidance in daf-1;daf-3 double mutants suggest DAF-3 suppresses PG production and sperm accumulation at the spermatheca. Our data suggest that DAF-3 functions in the nervous system, and possibly the germline, to affect sperm guidance. CONCLUSION: The C elegans TGFß pathway regulates many pathways to modulate PG metabolism and sperm guidance. These pathways likely function in the nervous system and possibly the germline.

Comprehensive profiling of prostaglandins in human ovarian follicular fluid using mass spectrometry.

Prostaglandins are formed by enzymatic and non-enzymatic mechanisms. They have been detected in human ovarian follicular fluid (HFF), a medium rich in growth factors and nutrients important for oocyte growth and fertility. However, the comprehensive identification of HFF prostaglandins has not been addressed. Here we use hybrid triple quadrupole time-of-flight and triple quadrupole mass spectrometers to comprehensively analyze prostaglandins in HFF. We identified PGE1, PGE2, PGF2α, and other prostaglandins synthesized via prostaglandin-endoperoxide synthase (i.e. Cox) cascades. We also identified specific PGF2α isomers (F2-isoprostanes) and PGF3α analogs whose structures are inconsistent with Cox-dependent formation. A prospective cohort pilot study of infertility patient subtypes revealed two potential associations. F2-isoprostanes are decreased in the diminished ovarian reserve subtype and elevated PGF2α may be associated with decreased live birth. Other than PGF2α, only body mass index >25kg/m2 correlated with poor in vitro fertilization outcome. Our studies suggest that HFF contains prostaglandins formed from at least two mechanisms, which may correlate with distinct clinical parameters.

A heterogeneous mixture of F-series prostaglandins promotes sperm guidance in the Caenorhabditis elegans reproductive tract.

The mechanisms that guide motile sperm through the female reproductive tract to oocytes are not well understood. We have shown that Caenorhabditis elegans oocytes synthesize sperm guiding F-series prostaglandins from polyunsaturated fatty acid (PUFA) precursors provided in yolk lipoprotein complexes. Here we use genetics and electrospray ionization tandem mass spectrometry to partially delineate F-series prostaglandin metabolism pathways. We show that omega-6 and omega-3 PUFAs, including arachidonic and eicosapentaenoic acids, are converted into more than 10 structurally related F-series prostaglandins, which function collectively and largely redundantly to promote sperm guidance. Disruption of omega-3 PUFA synthesis triggers compensatory up-regulation of prostaglandins derived from omega-6 PUFAs. C. elegans F-series prostaglandin synthesis involves biochemical mechanisms distinct from those in mammalian cyclooxygenase-dependent pathways, yet PGF(2α) stereoisomers are still synthesized. A comparison of F-series prostaglandins in C. elegans and mouse tissues reveals shared features. Finally, we show that a conserved cytochrome P450 enzyme, whose human homolog is implicated in Bietti's Crystalline Dystrophy, negatively regulates prostaglandin synthesis. These results support the model that multiple cyclooxygenase-independent prostaglandins function together to promote sperm motility important for fertilization. This cyclooxygenase-independent pathway for F-series synthesis may be conserved.

VAPB/ALS8 MSP ligands regulate striated muscle energy metabolism critical for adult survival in caenorhabditis elegans.

Mutations in VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), two motor neuron diseases that often include alterations in energy metabolism. We have shown that C. elegans and Drosophila neurons secrete a cleavage product of VAPB, the N-terminal major sperm protein domain (vMSP). Secreted vMSPs signal through Roundabout and Lar-like receptors expressed on striated muscle. The muscle signaling pathway localizes mitochondria to myofilaments, alters their fission/fusion balance, and promotes energy production. Here, we show that neuronal loss of the C. elegans VAPB homolog triggers metabolic alterations that appear to compensate for muscle mitochondrial dysfunction. When vMSP levels drop, cytoskeletal or mitochondrial abnormalities in muscle induce elevated DAF-16, the Forkhead Box O (FoxO) homolog, transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels in adults, and extends lifespan, despite reduced muscle mitochondria electron transport chain activity. Finally, Vapb knock-out mice exhibit abnormal muscular triacylglycerol levels and FoxO target gene transcriptional responses to fasting and refeeding. Our data indicate that impaired vMSP signaling to striated muscle alters FoxO activity, which affects energy metabolism. Abnormalities in energy metabolism of ALS patients may thus constitute a compensatory mechanism counterbalancing skeletal muscle mitochondrial dysfunction.

Improved high sensitivity analysis of polyphenols and their metabolites by nano-liquid chromatography-mass spectrometry.

This study was conducted to assess the value of a high resolution, high mass accuracy time-of-flight analyzer in combination with nanoliquid chromatography for the analysis of polyphenols and their metabolites. The goal was to create a method that utilizes small volumes of biological fluids and provides a significant improvement in sensitivity compared with existing methods. Accordingly, nanoLC-MS and nanoLC-pseudo-multiple reaction monitoring (MRM) methods were developed that had a lower limit of quantification of 0.5 nM for several polyphenols and were linear over 2-3 orders of magnitude (R(2)>0.999). Using urine samples, the ability to observe and quantify polyphenols in such a complex biological fluid depended on much narrower mass windows (0.050 amu or less) on a TOF analyzer than those used on a quadrupole analyzer (0.7 amu). Although a greater selectivity was possible with the low mass resolution of a triple quadrupole instrument using the MRM approach, for the daidzein metabolite O-DMA, a chromatographically resolvable second peak could only be substantially reduced by using a 0.01 amu mass window. The advantage of a TOF analyzer for product ion data is that the whole MSMS spectrum is collected at high mass accuracy and MRM experiments are conducted in silico after the analysis.

LC-MS/MS method for proline-glycine-proline and acetylated proline-glycine-proline in human plasma.

The extracellular cellular matrix (ECM) maintains tissue structure and regulates signaling functions by continuous degradation and remodeling. Inflammation or other disease conditions activate proteases including matrix metalloproteinases (MMPs) that degrade ECM proteins and in particular generate fragments of collagen and elastin, some of which are biologically active ECM peptides or matrikines. Stepwise degradation of collagen by MMP 8, 9 and prolyl endopeptidase release the matrikine proline-glycine-proline (PGP) and its product acetyl-PGP (AcPGP). These peptides are considered as potential biomarkers and therapeutic targets for many disease conditions such as chronic lung disease, heart disease, and cancer. However, there is no published, validated method for the measurement of PGP and AcPGP in plasma and therefore, we developed a sensitive, selective and reliable, isotope dilution LC-multiple reaction monitoring MS method for their determination in human plasma. The chromatographic separation of PGP and AcPGP was achieved in 3 min using Jupiter column with a gradient consisting of acidified acetonitrile and water at a flow rate of 0.5 ml/min. The limit of detection (LOD) for PGP and AcPGP was 0.01 ng/ml and the limit of quantification (LOQ) was 0.05 ng/ml and 0.1 ng/ml, respectively. Precision and accuracy values for all analytes were within 20 % except for the lowest QC of 0.01 ng/ml. The mean extraction recoveries of these analytes were > 90 % using a Phenomenex Phree cartridge and the matrix effect was < 15 % for all the QCs for PGP and AcPGP except the lowest QC. The stability of PGP and AcPGP was > 90 % in several tested conditions including autosampler use, storage at -80 °C, and after 6 times freeze-thaw cycles. Using this method, we successfully extracted and determined PGP levels in human plasma from healthy and COPD subjects. Therefore, this method is suitable for quantification of these peptides in the clinical setting.

The gap junctional protein INX-14 functions in oocyte precursors to promote C. elegans sperm guidance.

Innexins are the subunits of invertebrate gap junctions. Here we show that the innexin INX-14 promotes sperm guidance to the fertilization site in the Caenorhabditis elegans hermaphrodite reproductive tract. inx-14 loss causes cell nonautonomous defects in sperm migration velocity and directional velocity. Results from genetic and immunocytochemical analyses provide strong evidence that INX-14 acts in transcriptionally active oocyte precursors in the distal gonad, not in transcriptionally inactive oocytes that synthesize prostaglandin sperm-attracting cues. Somatic gonadal sheath cell interaction is necessary for INX-14 function, likely via INX-8 and INX-9 expressed in sheath cells. However, electron microscopy has not identified gap junctions in oocyte precursors, suggesting that INX-14 acts in a channel-independent manner or INX-14 channels are difficult to document. INX-14 promotes prostaglandin signaling to sperm at a step after F-series prostaglandin synthesis in oocytes. Taken together, our results support the model that INX-14 functions in a somatic gonad/germ cell signaling mechanism essential for sperm function. We propose that this mechanism regulates the transcription of a factor(s) that modulates prostaglandin metabolism, transport, or activity in the reproductive tract.

Gamma-ray Irradiation of Rodent Diets Alters the Urinary Metabolome in Rats with Chemically Induced Mammary Cancer.

In this study, a comparative, untargeted metabolomics approach was applied to compare urinary metabolite profiles of rats fed irradiated and non-irradiated diets. γ-Irradiated and non-irradiated NIH 7001 diet was given orally to animals beginning 5 days after exposure to the carcinogen N-methyl-N-nitrosourea and continued for 120 days. There was a 36% reduction in mammary tumor incidence in rats consuming the γ-irradiated diet, compared to rats receiving the non-irradiated form of the same diet. Urine samples from rats fed with γ-irradiated and non-irradiated diets were analyzed using nanoLC-MS/MS on a Q-TOF mass spectrometer, collecting positive and negative ion data. Data processing involved feature detection and alignment with MS-DIAL, normalization, mean-centering and Pareto scaling, and univariate and multivariate statistical analysis using MetaboAnalyst, and pathway analysis with Mummichog. Unsupervised Principal Component Analysis and supervised Partial Least Squares-Discriminant Analysis of both negative and positive ions revealed separation of the two groups. The top 25 metabolites from variable importance in projection scores >1 showed their contributions in discriminating urines the γ-irradiated diet fed group from non-irradiated control diet group. Consumption of the γ-irradiated diet led to alteration of several gut microbial metabolites such as phenylacetylglycine, indoxyl sulfate, kynurenic acid, hippurate and betaine in the urine. This study provides insights into metabolic changes in rat urine in response to a γ-irradiated diet which may be associated with mammary cancer prevention.

Dietary isoflavone absorption, excretion, and metabolism in captive cheetahs (Acinonyx jubatus).

Dietary isoflavones, capable of influencing reproductive parameters in domestic cats (Felis catus), have been detected in commercial diets fed to captive cheetahs (Acinonyx jubatus). However, the absorptive and metabolic capacity of cheetahs towards isoflavones has not yet been studied. Experiments were designed to describe the plasma concentration-time curve, metabolite profile, and urinary and fecal excretion of genistein and daidzein in cheetahs following consumption of isoflavones. Four adult cheetahs were administered a single oral bolus of genistein and daidzein, and five juvenile cheetahs consuming a milk replacer formula found to contain isoflavones were also included. Urine was collected from all animals, and blood and feces were also collected from adult cheetahs following isoflavone exposure. Samples were analyzed for isoflavone metabolite concentration by liquid chromatography-electrospray ionization-multiple reaction ion monitoring mass spectrometry and high-performance liquid chromatography. Sulfate conjugates were the primary metabolites detected of both genistein and daidzein (60-80% of total isoflavones present) in the plasma and urine of cheetahs. A smaller proportion of daidzein was detected as conjugates in the urine of juvenile cheetahs, compared to adult cheetahs. Other metabolites included unconjugated genistein and daidzein, O-desmethylangolensin, and dihydrodaidzein, but not equol. Only 33% of the ingested genistein dose, and 9% of daidzein, was detected in plasma from adult cheetahs. However, of the ingested dose, 67% of genistein and 45% of daidzein were detected in the feces of adults. This study revealed that cheetahs appear efficient in their conjugation of absorbed dietary isoflavones and only a small fraction of ingested dose is absorbed. However, the capacity of the cheetah to conjugate genistein and daidzein with sulfate moieties appears lower than reported in the domestic cat. This may confer greater opportunity for biologic activity of isoflavones in the cheetah than would be predicted from findings in the domestic cat. However, further investigation is required.

Sperm-Guiding Unconventional Prostaglandins in C. elegans: Synthesis and Signaling.

Prostaglandins comprise a family of lipid signaling molecules derived from polyunsaturated fatty acids and are involved in a wide array of biological processes, including fertilization. Prostaglandin-endoperoxide synthase (a.k.a. cyclooxygenase or Cox) initiates prostaglandin synthesis from 20-carbon polyunsaturated fatty acids, such as arachidonic acid. Oocytes of Caenorhabditis elegans (C. elegans) have been shown to secrete sperm-guidance cues prostaglandins, independent of Cox enzymes. Both prostaglandin synthesis and signal transduction in C. elegans are environmentally modulated pathways that regulate sperm guidance to the fertilization site. Environmental factors such as food triggers insulin and TGF-ß secretion and their levels regulate tissue-specific prostaglandin synthesis in C. elegans. This novel PG pathway is abundant in mouse and human ovarian follicular fluid, where their functions, mechanism of synthesis and pathways remain to be established. Given the importance of prostaglandins in reproductive processes, a better understanding of how diets and other environmental factors influence their synthesis and function may lead to new strategies towards improving fertility in mammals.

Transcriptomic Profiling of DAF-7/TGFß Pathway Mutants in C. elegans.

The transforming growth factor beta superfamily encompasses a large family of ligands that are well conserved across many organisms. They are regulators of a number of physiological and pathological processes. The model nematode Caenorhabditis elegans has been instrumental in identifying key components of the transforming growth factor beta (TGFß) pathway. In C. elegans, the TGFß homolog DAF-7 signals through the DAF-1 Type I and DAF-4 Type II receptors to phosphorylate downstream R-SMADs DAF-8 and DAF-14. These R-SMADs translocate into the nucleus to inhibit Co-SMAD DAF-3. Many of the roles of the canonical DAF-7 pathway, involving both DAF-1 and DAF-3, have been identified using targeted genetic studies. Few have assessed the global transcriptomic changes in response to these genes, especially in adult animals. In this study, we performed RNA sequencing on wild type, daf-1, and daf-1; daf-3 adult hermaphrodites. To assess the overall trends of the data, we identified differentially expressed genes (DEGs) and performed gene ontology analysis to identify the types of downstream genes that are differentially expressed. Hierarchical clustering showed that the daf-1; daf-3 double mutants are transcriptionally more similar to wild type than daf-1 mutants. Analysis of the DEGs showed a disproportionally high number of genes whose expression is increased in daf-1 mutants, suggesting that DAF-1 acts as a general repressor of gene expression in wild type animals. Gene ontology analysis of the DEGs produced many significantly enriched terms, including Molting Cycle, Response to Topologically Incorrect Protein, and Response to Biotic Stimulus. Understanding the direct and indirect targets of the DAF-7 TGFß pathway through this RNA-seq dataset can provide insight into novel roles of the multifunctional signaling pathway, as well as identify novel genes that may participate in previously reported functions of TGFß signaling.

Signature profile of cyclooxygenase-independent F2 series prostaglandins in C. elegans and their role in sperm motility.

We previously discovered that Caenorhabditis elegans synthesizes Cox-independent F-series prostaglandins (PGs). To delineate the Cox-independent prostaglandin pathways and evaluate their role in sperm motility in C. elegans, we developed a novel biochemical method for the rapid production of F-series PGs using arachidonic acid as the substrate and worm lysate as source of enzyme(s). Among the four F2-series PGs produced in the reaction, three of them were identified as 8-isoPGF2α, 5iPF2 VI, and PGF2α based on their retention times and MS/MS spectral comparison with standards using LC-MS/MS. PG production was not markedly affected by specific antioxidants, or Cox, Lox, and Cyp inhibitors, suggesting that these PGs are formed through a novel, biologically regulated mechanism in C. elegans. This study also assessed the ability of 8-isoPGF2α, 5iPF2 VI, PGF2α, and a mixture containing these PGs in a 0.5/0.08/1 ratio that reflects their synthetic composition to modulate sperm motility in fat-2 mutants. PGF2α and the PG mixture at 25 µM concentration significantly stimulated sperm velocity by 28% and 38%, whereas 8-isoPGF2α and 5iPF2 VI reduced the velocity by 21% and 30%, respectively, compared to vehicle control. These results indicate that the sperm motility effects of PGs are structure- and composition-dependent in C. elegans.

Effect of cranberry juice concentrate on chemically-induced urinary bladder cancers.

The chemopreventive efficacy of cranberry juice concentrate in an experimental model of urinary bladder cancer was evaluated using female Fischer-344 rats. The animals received N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) for a period of eight weeks. Cranberry juice concentrate was administered at doses of 1.0 or 0.5 ml/rat/day beginning one week after the final OH-BBN treatment and continuing until the end of the study. The urinary bladders of all the rats were weighed and examined grossly for lesions, and all masses were submitted for pathological evaluation. A dose-dependent preventive effect of cranberry treatment was observed, with a reduced number of urinary bladder cancers (38%) in the 1.0 ml/rat/day group versus the control group. The cranberry extract neither affected body weight gain nor caused other signs of toxicity. For the metabolic studies, serum and urine were collected at 4 and 12 h after the administration of the cranberry juice concentrate and were analyzed by LC-MS/MS. Quercetin and its methylated derivative were detected in the urine samples. However, no quercetin was detected in the serum samples, indicating its poor bioavailability. These data suggest that components of cranberries may be effective in preventing urinary bladder carcinogenesis.

A liquid chromatography mass spectrometry assay for determination of PD168393, a specific and irreversible inhibitor of erbB membrane tyrosine kinases, in rat serum.

For the first time, a rapid, sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method using an atmospheric pressure chemical ionization (APCI) source for the quantification of PD168393 in rat serum was developed and validated. Serum samples were pretreated with methanol for protein precipitation. The chromatographic separation was performed on a Jupiter-C5 column (250 mm x 2.0 mm i.d.) pre-equilibrated with 0.1% formic acid. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 369/313 for PD168393 and m/z 343/308 for the internal standard triazolam, using positive ion mode. The MS/MS response was linear over the concentration range from 2 ng/mL to 5000 ng/mL, with a lower limit of quantification (LLQ) of 2 ng/mL. At the lowest quality control (4 ng/mL), the intra- and inter-day precisions (CV%) for PD168393 were less than 10% and the accuracies were between 92% and 111%. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies.

Effects of botanical dietary supplements on cardiovascular, cognitive, and metabolic function in males and females.

BACKGROUND: The onset of menopause marks a pivotal time in which the incidence of hypertension and of cardiovascular disease (CVD) begins to increase dramatically in women. Before menopause, the incidences of these diseases are significantly lower in women than in age-matched men. After menopause, the rates of these diseases in women eventually approximate those in men. The loss of endogenous estrogen at menopause has been traditionally believed to be the primary factor involved in these changes. OBJECTIVE: This review summarizes recent findings regarding the effectiveness of botanicals in the treatment of some menopausal symptoms and other symptoms of aging (eg, rise in arterial pressure, cognitive decline, insulin resistance, and hyperlipidemia). METHODS: Articles were selected for inclusion in this review based on the significance of the research and contribution to the current understanding of how each botanical elicits cardioprotective effects. To this end, PubMed and MEDLINE databases were searched, using terms that included the name of the specific botanical along with the relevant aspects of its action(s), such as blood pressure, glycemic control, and lipids. Most of the articles used were published within the past 5 years, although some older articles that were seminal in advancing the current understanding of botanicals were also included. RESULTS: Soy has been found to lower plasma lipid concentrations and arterial pressure in postmenopausal women and age-matched men, and to have protective effects in heart disease and atherosclerosis of the carotid and coronary circulation. Soy was also found to lower fasting insulin concentrations and glycosylated hemoglobin concentrations. Grape seed extract, another frequently used botanical, contains polyphenols that have been found to reduce arterial pressure and salt-sensitive hypertension in estrogendepleted animal models. CONCLUSION: Several botanical compounds have been found to have beneficial effects in the treatment of the symptoms of menopause and other symptoms of aging, including CVD, cognitive decline, and metabolic diseases.

Unique Lipid Signatures of Extracellular Vesicles from the Airways of Asthmatics.

Asthma is a chronic inflammatory disease process involving the conductive airways of the human lung. The dysregulated inflammatory response in this disease process may involve multiple cell-cell interactions mediated by signaling molecules, including lipid mediators. Extracellular vesicles (EVs) are lipid membrane particles that are now recognized as critical mediators of cell-cell communication. Here, we compared the lipid composition and presence of specific lipid mediators in airway EVs purified from the bronchoalveolar lavage (BAL) fluid of healthy controls and asthmatic subjects with and without second-hand smoke (SHS) exposure. Airway exosome concentrations were increased in asthmatics, and correlated with blood eosinophilia and serum IgE levels. Frequencies of HLA-DR+ and CD54+ exosomes were also significantly higher in asthmatics. Lipidomics analysis revealed that phosphatidylglycerol, ceramide-phosphates, and ceramides were significantly reduced in exosomes from asthmatics compared to the non-exposed control groups. Sphingomyelin 34:1 was more abundant in exosomes of SHS-exposed asthmatics compared to healthy controls. Our results suggest that chronic airway inflammation may be driven by alterations in the composition of lipid mediators within airway EVs of human subjects with asthma.

Loss of Merlin induces metabolomic adaptation that engages dependence on Hedgehog signaling.

The tumor suppressor protein Merlin is proteasomally degraded in breast cancer. We undertook an untargeted metabolomics approach to discern the global metabolomics profile impacted by Merlin in breast cancer cells. We discerned specific changes in glutathione metabolites that uncovered novel facets of Merlin in impacting the cancer cell metabolome. Concordantly, Merlin loss increased oxidative stress causing aberrant activation of Hedgehog signaling. Abrogation of GLI-mediated transcription activity compromised the aggressive phenotype of Merlin-deficient cells indicating a clear dependence of cells on Hedgehog signaling. In breast tumor tissues, GLI1 expression enhanced tissue identification and discriminatory power of Merlin, cumulatively presenting a powerful substantiation of the relationship between these two proteins. We have uncovered, for the first time, details of the tumor cell metabolomic portrait modulated by Merlin, leading to activation of Hedgehog signaling. Importantly, inhibition of Hedgehog signaling offers an avenue to target the vulnerability of tumor cells with loss of Merlin.

Applications of LC-MS in the study of the uptake, distribution, metabolism and excretion of bioactive polyphenols from dietary supplements.

Specific and quantitative analyses of the bioactive components and their metabolites in body fluids are essential to assess the interaction between groups of compounds in dietary supplements and preparations of psychoactives. Reverse-phase LC separations combined with tandem mass spectrometry provide the necessary specificity and sensitivity. In this paper, applications of these methods are described for the analysis of isoflavones, salvinorin A, synephrine isomers and their metabolites in serum, urine and aqueous humor.

Metabolism and growth inhibitory activity of cranberry derived flavonoids in bladder cancer cells.

In the present study, anti-proliferative activities of cranberry derived flavonoids and some of their in vivo metabolites were evaluated using a panel of human bladder tumor cell lines (RT4, SCABER, and SW-780) and non-tumorigenic immortalized human uroepithelial cells (SV-HUC). Among the compounds tested, quercetin 3-O-glucoside, isorhamnetin (3'-O-methylquercetin), myricetin and quercetin showed strong concentration-dependent cell growth inhibitory activities in bladder cancer cells with IC50 values in a range of 8-92 µM. Furthermore, isorhamnetin and myricetin had very low inhibitory activity against SV-HUC even at very high concentrations (>200 µM) compared to bladder cancer cells, indicating that their cytotoxicity is selective for cancer cells. To determine whether the differential cell growth inhibitory effects of isomeric flavonoids quercetin 3-O-glucoside (active) and hyperoside (quercetin 3-O-galactoside) (inactive) are related to their metabolism by the cancer cells, SW-780 cells were incubated with these compounds and their metabolism was examined by LC-MS/MS. Compared to quercetin 3-O-glucoside, hyperoside undergoes relatively less metabolic biotransformation (methylation, glucuronidation and quinone formation). These data suggest that isorhamnetin and quercetin 3-O-glucoside may be the active forms of quercetin in prevention of bladder cancer in vivo and emphasize the importance of metabolism for the prevention of bladder cancer by diets rich in cranberries.

Training in metabolomics research. I. Designing the experiment, collecting and extracting samples and generating metabolomics data.

The study of metabolism has had a long history. Metabolomics, a systems biology discipline representing analysis of known and unknown pathways of metabolism, has grown tremendously over the past 20 years. Because of its comprehensive nature, metabolomics requires careful consideration of the question(s) being asked, the scale needed to answer the question(s), collection and storage of the sample specimens, methods for extraction of the metabolites from biological matrices, the analytical method(s) to be employed and the quality control of the analyses, how collected data are correlated, the statistical methods to determine metabolites undergoing significant change, putative identification of metabolites and the use of stable isotopes to aid in verifying metabolite identity and establishing pathway connections and fluxes. The National Institutes of Health Common Fund Metabolomics Program was established in 2012 to stimulate interest in the approaches and technologies of metabolomics. To deliver one of the program's goals, the University of Alabama at Birmingham has hosted an annual 4-day short course in metabolomics for faculty, postdoctoral fellows and graduate students from national and international institutions. This paper is the first part of a summary of the training materials presented in the course to be used as a resource for all those embarking on metabolomics research. The complete set of training materials including slide sets and videos can be viewed at http://www.uab.edu/proteomics/metabolomics/workshop/workshop_june_2015.php. Copyright © 2016 John Wiley & Sons, Ltd.