Project description:Oxidative stress plays a role in the onset and progression of a number of diseases, such as Alzheimer's disease, diabetes and cancer, as well as ageing. Oxidative stress is caused by an increased production of reactive oxygen species and reduced antioxidant activity, resulting in the oxidation of glutathione. The ratio of reduced to oxidised glutathione is often used as a marker of the redox state in the cell. Whereas a variety of methods have been developed to measure glutathione in blood samples, methods to measure glutathione in cultured cells are scarce. Here we present a protocol to measure glutathione levels in cultured human and yeast cells using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC⁻MS/MS).
Project description:The endocannabinoid system is expressed in cells throughout the body and controls a variety of physiological and pathophysiological functions. We describe robust and reproducible UPLC-MS/MS-based methods for analyzing metabolism of the endocannabinoids, 2-arachidonoyl-sn-glycerol and arachidonoyl ethanolamide, and related monoacylglycerols (MAGs) and fatty acid ethanolamides (FAEs), respectively, in mouse mucosal tissues (i.e., intestine and lung). These methods are optimized for analysis of activity of the MAG biosynthetic enzyme, diacylglycerol lipase (DGL), and MAG degradative enzymes, monoacylglycerol lipase (MGL) and alpha/beta hydrolase domain containing-6 (ABHD6). Moreover, we describe a novel UPLC-MS/MS-based method for analyzing activity of the FAE degradative enzyme, fatty acid amide hydrolase (FAAH), that does not require use of radioactive substrates. In addition, we describe in vivo pharmacological methods to inhibit MAG biosynthesis selectively in the mouse small-intestinal epithelium. These methods will be useful for profiling endocannabinoid metabolism in rodent mucosal tissues in health and disease.
Project description:A rapid and sensitive ultrafast performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was developed for the simultaneous determination of 11 compounds in Gualou Guizhi Granule (GLGZG), including liquiritin, isoliquiritin, liquirtin apioside, isoliquiritin apioside, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, paeoniflorin, albiflorin, and paeoniflorin sulfonate in rat plasma. UPLC-MS/MS assay with negative ion mode was performed on a Waters CORTECS C18 (2.1 × 100 mm, 1.6 μm) with the mobile phase consisting of 0.1% aqueous formic acid (A) and acetonitrile (B) in gradient elution at a flow rate of 0.25 mL·min-1. The method was linear for all analytes within the detection range (r ≥ 0.9597). The inter- and intraday precision (RSD) were 2.21-6.41% and 1.67-6.18%; the inter- and intraday accuracy (recover) were 92.48-114.03% and 90.23-112.04%. And the recovery rate ranged from 81.30% to 108.22%. The matrix effect values obtained for analytes ranged from 88.91% to 113.32%. This validated method was successfully applied to a pharmacokinetics study in rats after oral administration of GLGZG.
Project description:This West Coast Metabolomics Center pilot and feasibility project was granted to Ernst Lengyel (University of Chicago).<br>The biology of ovarian cancer (OvCa) is clearly distinct from that of most epithelial tumors, in that hematogenous metastases are rare, and ovarian tumors remain confined to the peritoneal cavity. The omentum, a large pad of fat tissue (20x13x3cm) covering the bowel, is the most common site of OvCa metastasis. It consists primarily of adipocytes, which become the principal microenvironment for the OvCa cells. The underlying hypothesis for this application is that, in the presence of adipocytes, the metabolism of OvCa cells is reprogramed and shifts towards lipid utilization, which provides energy that facilitates tumor growth and metastasis. Preliminary results suggest that primary human omental adipocytes secrete cytokines which promote the metastasis of OvCa cells to the omentum and their subsequent invasion. Once metastasis has occurred, OvCa cells induce lipolysis in omental adipocytes, and use the energy derived from these lipids to proliferate.<br>To study the metabolic changes in the tumor microenvironment we have established a 3D organotypic culture of the human omentum using primary human cells established from patient tissue. Metabolic studies will be performed on adipocytes and OvCa cells individually, on conditioned media and on adipocytes and OvCa cells co-cultured in our 3D model, with the goal of arriving at a comprehensive analysis of primary metabolites and lipids in the tumor microenvironment.<br>In the current investigation, untargeted analysis of primary metabolites and complex lipids were conducted on adipocytes and OvCa cells individually, on conditioned media and on adipocytes and OvCa cells co-cultured in our 3D model. Analysis of oxylipins was conducted on conditioned media. To gain better understanding of the dynamic regulation of metabolic pathways we will also perform metabolic flux analysis using labeled cells (13C-glucose, 13C-glutamine) in the 3D culture model.<br>The primary objective of this study is to gain insight into the dynamic interactions between OvCa cells and human adipocytes with the anticipation of elucidating targets of therapeutic intervention. <br><br>
Project description:The role of the gut microbiome in human health, and non-invasive measurement of gut dysbiosis are of increasing clinical interest. New high-throughput methods are required for the rapid measurement of gut microbial metabolites and to establish reference ranges in human populations. We used ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) -- positive and negative electrospray ionization modes, multiple reaction monitoring transitions -- to simultaneously measure three urinary metabolites (phenylacetylglutamine, 4-cresyl sulphate and hippurate) that are potential biomarkers of gut function, among multi-ethnic US men and women aged 40-59 from the INTERMAP epidemiologic study (n = 2000, two timed 24-hr urine collections/person). Metabolite concentrations were quantified via stable isotope labeled internal standards. The assay was linear in the ranges 1ng/mL (lower limit of quantification) to 1000ng/mL (phenylacetylglutamine and 4-cresyl sulfate) and 3ng/mL to 3000ng/mL (hippurate). These quantitative data provide new urinary reference ranges for population-based human samples: mean (standard deviation) 24-hr urinary excretion for phenylacetylglutamine was: 1283.0 (751.7) ?mol/24-hr (men), 1145.9 (635.5) ?mol/24-hr (women); for 4-cresyl sulphate, 1002.5 (737.1) ?mol/24-hr (men), 1031.8 (687.9) ?mol/24-hr (women); for hippurate, 6284.6 (4008.1) ?mol/24-hr (men), 4793.0 (3293.3) ?mol/24-hr (women). Metabolic profiling by UPLC-MS/MS in a large sample of free-living individuals has provided new data on urinary reference ranges for three urinary microbial co-metabolites, and demonstrates the applicability of this approach to epidemiological investigations.
Project description:The purpose of the present study was to investigate molecular compositions of lipid droplets changing in live hepatic cells stimulated with major fatty acids in the human body, i.e., palmitic, stearic, oleic, and linoleic acids. HepG2 cells were used as the model hepatic cells. Morphological changes of lipid droplets were observed by optical microscopy and transmission electron microscopy (TEM) during co-cultivation with fatty acids up to 5 days. The compositional changes in the fatty chains included in the lipid droplets were analyzed via Raman spectroscopy and chemometrics. The growth curves of the cells indicated that palmitic, stearic, and linoleic acids induced cell death in HepG2 cells, but oleic acid did not. Microscopic observations suggested that the rates of fat accumulation were high for oleic and linoleic acids, but low for palmitic and stearic acids. Raman analysis indicated that linoleic fatty chains taken into the cells are modified into oleic fatty chains. These results suggest that the signaling pathway of cell death is independent of fat stimulations. Moreover, these results suggest that hepatic cells have a high affinity for linoleic acid, but linoleic acid induces cell death in these cells. This may be one of the causes of inflammation in nonalcoholic fatty liver disease (NAFLD).
Project description:Analysis of whole cell lipid extracts of bacteria by means of ultra-performance (UP)LC-MS allows a comprehensive determination of the lipid molecular species present in the respective organism. The data allow conclusions on its metabolic potential as well as the creation of lipid profiles, which visualize the organism's response to changes in internal and external conditions. Herein, we describe: i) a fast reversed phase UPLC-ESI-MS method suitable for detection and determination of individual lipids from whole cell lipid extracts of all polarities ranging from monoacylglycerophosphoethanolamines to TGs; ii) the first overview of a wide range of lipid molecular species in vegetative Myxococcus xanthus DK1622 cells; iii) changes in their relative composition in selected mutants impaired in the biosynthesis of α-hydroxylated FAs, sphingolipids, and ether lipids; and iv) the first report of ceramide phosphoinositols in M. xanthus, a lipid species previously found only in eukaryotes.
Project description:Peanuts are a rich dietary source of lipids, which are essential for human health. In this study, the lipid contents of 13 peanut cultivars were analyzed using UPLC-Q-TOF-MS and GC-MS. The OXITEST reactor was used to test their lipid oxidation stabilities. A total of 27 subclasses, 229 individual lipids were detected. The combined analysis of lipid and oxidation stability showed that lipid unsaturation was inversely correlated with oxidation stability. Moreover, lipid profiles differed significantly among the different peanut cultivars. A total of 11 lipid molecules (TG 18:2/18:2/18:2, TG 24:0/18:2/18:3, TG 20:5/14:1/18:2, TG 18:2/14:1/18:2, PE 17:0/18:2, BisMePA 18:2/18:2, PG 38:5, PMe 18:1/18:1, PC 18:1/18:1, MGDG 18:1/18:1, TG 10:0/10:1/18:1) might be employed as possible indicators to identify high oleic acid (OA) and non-high OA peanut cultivars, based on the PLS-DA result of lipid molecules with a VIP value greater than 2. This comprehensive analysis will help in the rational selection and application of peanut cultivars.
Project description:Oat saponins are composed of triterpenoid and steroidal saponins, and their potential biological activities, such as antibacterial, antifungicidal, osteogenic, and anticancer activities, have been reported. In this study, qualitative and quantitative analyses of oat saponins were conducted by using UPLC-QToF-MS and UPLC-Triple Q-MS/MS. A total of 22 saponins were analyzed in seven Korean oat cultivars. Among them, 7 saponins were identified as new compounds in this source, which were tentatively confirmed as nuatigenin-type saponins with 26-O-diglucoside and 3-O-malonylglucoside forms and (25S)-furost-5-en-3β,22,26-triol-type saponins. In addition, the total content of these saponins ranged from 70.61 to 141.38 mg/100 g dry weight, and it was affected by the type of oat cultivar and the presence or absence of hulling. These detailed profiles will be suggested as fundamental data for breeding superior oat cultivars, evaluating of related products, and various industries.