Using Ultra-high-performance Liquid Chromatography-mass Spectrometry Research Articles

ANTIOXIDANT CAPABILITIES OF Litsea garciae BARK EXTRACTS AND THEIR RELATION TO THE PHYTOCHEMICAL COMPOSITIONS

The plant species belonging to the Litsea genus are widely investigated due to their nutritional and medicinal purposes. In this regard, this study is another similar sincere effort in which the antioxidant property and phytochemical composition of Litsea garciae (L. garciae) bark’s hexane, chloroform, methanol, and aqueous extracts were evaluated to confirm its traditional benefits. The total flavonoid content (TFC) and total phenolic content (TPC) were determined first, followed by an assessment of in vitro antioxidant activity using the DPPH and FRAP assays. The composition of the secondary metabolites was determined using Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS). As a result, methanol extract was recorded to have the highest TPC value aligned with its positive appearance in phytochemical screening. Its antioxidant capacity indicated the least IC50. The results indicated that the significant free radical scavenging activity was due to the methanolic extract's high phenolic content. The secondary metabolites found in the methanol extract varied significantly according to UHPLC-MS analysis. The major phenolic compounds were found including N-trans-feruloyl-4-O-methyldopamine, N-cis-feruloyltyramine, epicatechin-(4beta->6)-epicatechin-(2beta->7,4beta->8)-epicatechin, 7-Hydroxy-3-(4-methoxyphenyl)-4-propyl-2H-1-benzopyran-2-one and 9-O-Methylneodunol. In general, the results indicate that L. garciae bark may be a promising source of novel natural compounds with antioxidative properties.

Liquid chromatography-mass spectrometry (LC-MS) based metabolomic fingerprinting in contrasting rice varieties for iron (Fe) excess.

Rice is an important cereal which is grown worldwide covering the requirement of 50% of the world population as the staple food. Diverse abiotic factors act as a limiting factor for its yield annually and can challenge sustainable productivity of the crop. Iron is an important micronutrient for plants participating in biochemical reactions including electron transport chain. Under the impact of excess iron in the rhizosphere, plants are posed with iron toxic condition. Most of the lowland rice is cultivated in submerged condition leading to accumulation of Fe2+ in the soil which is the toxic form for uptake and in turn can affect the yield of the crop. Present work is an extension of previously performed transcriptomic analysis of Northeast India lowland rice varieties under excess iron treatment using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS). In total 1105 peaks were detected for each root and shoot. Multivariate analysis based upon principal component analysis (PCA; PCA1 and PCA2) are accounted for 70.6% of the total variation in root and 77.2% in shoot. The metabolomics profiling has indicated significant alteration in primary and secondary metabolites. Majority of the secondary metabolites like quercetin, malvin, flavanones, delphinidin are upregulated indicating their role in anti-oxidative properties during iron toxicity. On the other hand, chelators of ferric ion like mugineic acid are down regulated to avoid excess iron uptake. This work is a comparative insight on the metabolic front in case of iron excess tolerance in contrasting lowland rice varieties.

Xiao-Luo-Wan treats propylthiouracil-induced goiter with hypothyroidism in rats through the PI3K-AKT/RAS pathways based on UPLC/MS and network pharmacology

Ethnopharmacological relevanceGoiter with hypothyroidism occurs in several thyroid diseases. Xiao-Luo-Wan (XLW), which contains Scrophularia ningpoensis Hemsl., Fritillaria thunbergii Miq. and Ostrea gigas Thunberg, has been used as an effective Chinese medicine for the treatment of goiters in China for hundreds of years. Based on clinical observations and experimental studies, XLW also exerts a certain effect on hypothyroidism. However, the therapeutic mechanism of XLW remains unclear. Aim of the studyThe present study aimed to investigate the therapeutic effect of XLW on propylthiouracil (PTU)-induced goiter with hypothyroidism in rats and to uncover the underlying molecular mechanism using ultra high-performance liquid chromatography-mass spectrometry (UPLC/MS), network pharmacology, and molecular docking simulations. Materials and methodsAfter successful modeling, the remaining rats were randomly divided into a model group, an Euthyrox group, an XLW group, and a control group. The corresponding drugs were given by gavage for four consecutive weeks. The growth status was monitored, the relative thyroid weight was calculated, and the total serum T3, T4, and TSH content were detected. Hematoxylin-eosin (H&E) staining was used to observe the pathological changes in the thyroid glands. The chemical components of the XLW were identified by UPLC/MS and the putative targets of XLW were predicted using multiple databases. We performed network pharmacology based on the intersection of goiter/hypothyroidism-related targets and XLW targets. Then, we performed KEGG pathway enrichment analysis, and key targets were further screened using protein-protein interaction (PPI) networks. Finally, molecular docking was used to predict the binding ability of XLW identified components and the key targets. ResultsXLW significantly increased the levels of T3 and T4, and reduced TSH, increased body weight, and decreased swollen thyroid glands in PTU-induced rats. XLW promoted the morphological recovery of thyroid follicles and epithelial cells. Twenty-one main chemical components of XLW were identified using UPLC/MS. 270 potential gene targets of XLW and 717 known targets of goiter/hypothyroidism disease were obtained by searching the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), Swiss Target Prediction, and UniProt databases. A total of 83 KEGG pathways were enriched with phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and RAS signaling pathways. PPI analysis revealed nine key targets of kinase-protein kinase B (AKT) 1, interleukin (IL) 6, vascular endothelial growth factor A (VEGFA), tumor necrosis factor (TNF), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), epidermal growth factor receptor (EGFR), GTPase HRas (HRAS), matrix metalloproteinase (MMP) 9, and heat shock protein 90 alpha family class A member 1 (HSP90AA1). Molecular docking verified which drug components had good binding ability to key targets (all ≤5 kcal/mol). ConclusionFor PTU-induced goiter with hypothyroidism in rats, XLW improves thyroid function, reduces goiter, increases body weight, and promotes the recovery of thyroid follicles and epithelial cells. The underlying molecular mechanism suggests that XLW may regulate thyroid hormone signaling by regulating the PI3K-AKT, RAS, and other signaling pathways. This study provides a pharmacological and biological basis for using XLW to treat goiter with hypothyroidism.

Metabolomics analysis of urine from rats given long-term high-protein diet using ultra-high-performance liquid chromatography-mass spectrometry

Previous studies have indicated high-protein diet (HPD) promotes weight loss and improves metabolic parameters, but most of these studies have focused on the impact of short-term, long-term effects remain unclear. In this study, male Wistar rats were fed two diets for 88 weeks: normal control diet (NCD, 20.5% of energy as protein) or HPD (30.5% of energy as protein). At 88 weeks intervention, compared to NCD rats, HPD rats had lower fat tissue and higher skeletal muscle to body weight ratio, but there were no significantly differences in body weight and food intake. To explore the mechanism underlying metabolism and diet, we further collected rat urine samples at 16, 40, 64 and 88 weeks diet treatment and analyzed metabolomics profiles using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). Partial least squares-discriminant analysis (PLS-DA) scores plots from ESI− or ESI+ model revealed a perfect separation between two diets at four time points. We identified 11 dramatically different metabolites (with VIP cut-off value > 1) in HPD, including 3 up-regulated and 8 down-regulated. And these 11 metabolites were identified as effective biomarkers, which were significantly related to HPD-induced metabolism related outcomes (fat tissue and skeletal muscle to body weight ratio). Our results provided vital information regarding metabolism in long-term HPD and more importantly, a few potentially promising metabolites were firstly identified which may related to metabolic responses.

Metabolomics analysis of the Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER)

AbstractBackgroundThe Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability (FINGER) is a randomized controlled trial that showed significant benefits on cognition for a 2‐year multidomain lifestyle intervention vs regular health advice in 1260 at‐risk older individuals from the general population. The aim of this study was to investigate the metabolic effects of the intervention and their association with cognitive outcomes.MethodWe acquired metabolomics data of blood plasma on two time points (baseline and 2 years) in a set of 1089 participants (547 intervention and 542 control). A multi‐technology platform consisting of using ultra‐high performance liquid chromatography ‐ mass spectrometry (UHPLC‐MS) and 1H nuclear magnetic resonance spectroscopy (NMR) was used to generate extensive measurement coverage of the metabolome. The data was acquired using both reversed‐phase chromatography (RPC) tailored for the retention and separation of both complex and neutral lipids and hydrophilic interaction liquid chromatography (HILIC) for the retention and separation of small polar metabolites. The UHPLC‐MS untargeted data contains a total of 10,825 metabolomic features, 1244 of them currently annotated. The NMR data contains measurements of both small and macromolecules, including 112 lipoprotein fractions (derived using Bruker lipoprotein subclass analysis: BI‐LISA). Data analysis was conducted using linear mixed models.ResultSeveral baseline metabolite levels had a statistically significant association with cognition. Results will also be presented from ongoing analyses to investigate intervention effects on change in metabolites.ConclusionComplex metabolomics analyses are new in the context of multidomain lifestyle dementia prevention trials. While causality is yet to be investigated further, some of the metabolite classes and pathways observed (e.g. sphingolipids) have been previously implicated in cognitive decline and neurological disorders, suggesting early interpretable leads in the rich dataset at hand.

The Algal Polysaccharide Ulvan Induces Resistance in Wheat Against Zymoseptoria tritici Without Major Alteration of Leaf Metabolome

This study aimed to examine the ability of ulvan, a water-soluble polysaccharide from the green seaweed Ulva fasciata, to provide protection and induce resistance in wheat against the hemibiotrophic fungus Zymoseptoria tritici. Matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) analysis indicated that ulvan is mainly composed of unsaturated monosaccharides (rhamnose, rhamnose-3-sulfate, and xylose) and numerous uronic acid residues. In the greenhouse, foliar application of ulvan at 10 mg.ml–1 2 days before fungal inoculation reduced disease severity and pycnidium density by 45 and 50%, respectively. Ulvan did not exhibit any direct antifungal activity toward Z. tritici, neither in vitro nor in planta. However, ulvan treatment significantly reduced substomatal colonization and pycnidium formation within the mesophyll of treated leaves. Molecular assays revealed that ulvan spraying elicits, but does not prime, the expression of genes involved in several wheat defense pathways, including pathogenesis-related proteins (β-1,3-endoglucanase and chitinase), reactive oxygen species metabolism (oxalate oxidase), and the octadecanoid pathway (lipoxygenase and allene oxide synthase), while no upregulation was recorded for gene markers of the phenylpropanoid pathway (phenylalanine ammonia-lyase and chalcone synthase). Interestingly, the quantification of 83 metabolites from major chemical families using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) in both non-infectious and infectious conditions showed no substantial changes in wheat metabolome upon ulvan treatment, suggesting a low metabolic cost associated with ulvan-induced resistance. Our findings provide evidence that ulvan confers protection and triggers defense mechanisms in wheat against Z. tritici without major modification of the plant physiology.

Disturbed microbial ecology in Alzheimer\u2019s disease: evidence from the gut microbiota and fecal metabolome

BackgroundGut microbiota (GMB) alteration has been reported to influence the Alzheimer’s disease (AD) pathogenesis through immune, endocrine, and metabolic pathways. This study aims to investigate metabolic output of the dysbiosis of GMB in AD pathogenesis. In this study, the fecal microbiota and metabolome from 21 AD participants and 44 cognitively normal control participants were measured. Untargeted GMB taxa was analyzed through 16S ribosomal RNA gene profiling based on next-generation sequencing and fecal metabolites were quantified by using ultrahigh performance liquid chromatography-mass spectrometry (UPLC-MS).ResultsOur analysis revealed that AD was characterized by 15 altered gut bacterial genera, of which 46.7% (7/15 general) was significantly associated with a series of metabolite markers. The predicted metabolic profile of altered gut microbial composition included steroid hormone biosynthesis, N-Acyl amino acid metabolism and piperidine metabolism. Moreover, a combination of 2 gut bacterial genera (Faecalibacterium and Pseudomonas) and 4 metabolites (N-Docosahexaenoyl GABA, 19-Oxoandrost-4-ene-3,17-dione, Trigofoenoside F and 22-Angeloylbarringtogenol C) was able to discriminate AD from NC with AUC of 0.955 in these 65 subjects.ConclusionsThese findings demonstrate that gut microbial alterations and related metabolic output changes may be associated with pathogenesis of AD, and suggest that fecal markers might be used as a non-invasive examination to assist screening and diagnosis of AD.

Comparative Metabolic Profiling of Grape Pulp during the Growth Process Reveals Systematic Influences under Root Restriction.

The compositions and contents of metabolites in the pulp tissue play critical roles in the fruit quality for table grape. In this study, the effects of root restriction (RR) on the primary and secondary metabolites of pulp tissue at five developmental stages were studied at the metabolomics level, using “Red Alexandria” grape berry (Vitis vinifera L.) as materials. The main results were as follows: 283 metabolites were annotated by using ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS); 28 and 16 primary metabolites contents were increased and decreased, and 11 and 19 secondary metabolites contents were increased and decreased, respectively, along the berry development; RR significantly decreased 12 metabolites (four amino acids and derivatives, three organic acids, four flavonoids and one other compound) contents, and improved 40 metabolites (22 amino acids and derivatives, six nucleotides, four carbohydrates, four cofactors, three cinnamic acids and one other compound) accumulation at the different developmental stages. Altogether, our study would be helpful to increase our understanding of grape berry’s responses to RR stress.

Cellular Metabolomics Profiles Associated With Drug Chemosensitivity in AML.

BackgroundAcute myeloid leukemia (AML) is a hematological malignancy with a dismal prognosis. For over four decades, AML has primarily been treated by cytarabine combined with an anthracycline. Although a significant proportion of patients achieve remission with this regimen, roughly 40% of children and 70% of adults relapse. Over 90% of patients with resistant or relapsed AML die within 3 years. Thus, relapsed and resistant disease following treatment with standard therapy are the most common clinical failures that occur in treating this disease. In this study, we evaluated the relationship between AML cell line global metabolomes and variation in chemosensitivity.MethodsWe performed global metabolomics on seven AML cell lines with varying chemosensitivity to cytarabine and the anthracycline doxorubicin (MV4.11, KG-1, HL-60, Kasumi-1, AML-193, ME1, THP-1) using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Univariate and multivariate analyses were performed on the metabolite peak intensity values from UHPLC-MS using MetaboAnalyst to identify cellular metabolites associated with drug chemosensitivity.ResultsA total of 1,624 metabolic features were detected across the leukemic cell lines. Of these, 187 were annotated to known metabolites. With respect to doxorubicin, we observed significantly greater abundance of a carboxylic acid (1-aminocyclopropane-1-carboxylate) and several amino acids in resistant cell lines. Pathway analysis found enrichment of several amino acid biosynthesis and metabolic pathways. For cytarabine resistance, nine annotated metabolites were significantly different in resistance vs. sensitive cell lines, including D-raffinose, guanosine, inosine, guanine, aldopentose, two xenobiotics (allopurinol and 4-hydroxy-L-phenylglycine) and glucosamine/mannosamine. Pathway analysis associated these metabolites with the purine metabolic pathway.ConclusionOverall, our results demonstrate that metabolomics differences contribute toward drug resistance. In addition, it could potentially identify predictive biomarkers for chemosensitivity to various anti-leukemic drugs. Our results provide opportunity to further explore these metabolites in patient samples for association with clinical response.

The Analyses of Chemical Components From Oldenlandia hedyotidea (DC.) Hand.-Mazz and Anticancer Effects in vitro.

Oldenlandia hedyotidea (DC.) Hand.-Mazz (OH), also known as sweet tea, is a valuable functional food with medicinal properties and is used for the treatment of cold, cough, gastroenteritis, heatstroke, herpes zoster, and rheumatoid arthritis. The phytochemicals in plant-based foods are responsible for the occurrence of these diseases to some extent. However, there is a scarcity of information on the chemical components of OH. We, therefore, aimed to investigate the phytochemical components of OH using ultra high-performance liquid chromatography–mass spectrometry (UHPLC-MS) and UHPLC triple time-of-flight mass spectrometry (UHPLC-Triple-TOF-MS). The main component of the OH extract, asperulosidic acid, was additionally quantified using UHPLC with ultraviolet detection (UHPLC-UV). The anticancer activity of the OH extract was assessed by a cell proliferation assay and a scratch assay using an esophageal cancer cell line. Ten compounds were tentatively identified in the aqueous extract of OH, including five iridoids, two anthraquinones, and one phenolic acid. The content of asperulosidic acid in the aqueous extract of OH was approximately 42 μg ml−1, and the extract exerted definite in vitro anticancer effects. The results can be used for quality control and assessment of the OH extract, which can serve as a promising source of functional ingredients for potential use in the food and drug industries.

Bioprospecting of rhamnolipids production and optimization by an oil-degrading Pseudomonas sp. S2WE isolated from freshwater lake

The present study revealed biosurfactants production by a novel oil-degrading Pseudomonas sp. S2WE isolated from hydrocarbon enriched water sample, where the genus Pseudomonas (48.65%) was dominated amongst several other genera. Biosurfactants produced by this strain showed the great potential for surface tension reduction (SFT) and emulsification. The extracted crude biosurfactants were characterized using ultra-high-performance liquid chromatography-Mass Spectrometry (UHPLC-MS) and identified various mono-and di-rhamnolipids homologs from the mixture. Moreover, the lowest SFT 33.05 ± 0.1 mN/m and highest emulsification of 60.65 ± 0.64% were achieved from rhamnolipids produced from glycerol with urea. Compared to initial screening, almost (>87%) higher emulsification was observed. In addition, the biosurfactants were found highly stable at different environmental factors i.e. temperature (4 °C-121 °C), pH (3–10) and NaCl conc. (1–9%). The high stable rhamnolipids produced by new Pseudomonas sp. S2WE in this study could widely be used in enormous industrial as well as environmental applications.

Resistance associated metabolite profiling of Aspergillus leaf spot in cotton through non-targeted metabolomics.

Aspergillus tubingensis is an important pathogen of economically important crops. Different biotic stresses strongly influence the balance of metabolites in plants. The aim of this study was to understand the function and response of resistance associated metabolites which, in turn are involved in many secondary metabolomics pathways to influence defense mechanism of cotton plant. Analysis of non-targeted metabolomics using ultra high performance liquid chromatography-mass spectrometry (UPLC-MS) revealed abundant accumulation of key metabolites including flavonoids, phenylpropanoids, terpenoids, fatty acids and carbohydrates, in response to leaf spot of cotton. The principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and partial least squares discriminant analysis (PLS-DA) score plots illustrated the evidences of variation between two varieties of cotton under mock and pathogen inoculated treatments. Primary metabolism was affected by the up regulation of pyruvate and malate and by the accumulation of carbohydrates like cellobiose and inulobiose. Among 241 resistance related (RR) metabolites, 18 were identified as resistance related constitutive (RRC) and 223 as resistance related induced (RRI) metabolites. Several RRI metabolites, identified in the present study were the precursors for many secondary metabolic pathways. These included phenylpropanoids (stilbenes and furanocoumarin), flavonoids (phlorizin and kaempferol), alkaloids (indolizine and acetylcorynoline) and terpenoids (azelaic acid and oleanolic acid). Our results demonstrated that secondary metabolism, primary metabolism and energy metabolism were more active in resistant cultivar, as compared to sensitive cultivar. Differential protein and fatty acid metabolism was also depicted in both cultivars. Accumulation of these defense related metabolites in resistant cotton cultivar and their suppression in susceptible cotton cultivar revealed the reason of their respective tolerance and susceptibility against A. tubingensis.

Towards a generic method for ion chromatography/mass spectrometry of low-molecular-weight amines in pharmaceutical drug discovery and development.

Low-molecular-weight amines are encountered in pharmaceutical analysis, e.g. as reactants in chemical syntheses, but are challenging to analyse using ultrahigh-performance liquid chromatography/mass spectrometry (UHPLC/MS) due to their high polarity causing poor retention. Ion chromatography/mass spectrometry (IC/MS) is an emerging technique for polar molecule analysis that offers better separation. A generic IC/MS method would overcome problems associated with using UHPLC/MS in drug discovery and development environments. Amine standards were analysed using IC/MS with gradient elution (variety of column temperatures evaluated). An electrospray ionisation (ESI) quadrupole mass spectrometer was operated in positive ion polarity in scanning mode. The make-up flow composition was evaluated by assessing the performance of a range of organic modifiers (acetonitrile, ethanol, methanol) and additives (acetic acid, formic acid, methanesulfonic acid). The ESI conditions were optimised to minimise adduct formation and promote generation of protonated molecules. The performance attributes were investigated and optimised for low-molecular-weight amine analysis. Organic solvents and acidic additives were evaluated as make-up flow components to promote ESI, with 0.05% acetic acid in ethanol optimal for producing protonated molecules. The hydrogen bonding capability of amines led to abundant protonated molecule-solvent complexes; optimisation of source conditions reduced these, with collision-induced dissociation voltage having a strong effect. The detection limit was ≤1.78 ng for the amines analysed, which is fit-for-purpose for an open-access chemistry environment. This study demonstrates the value of IC/MS for analysing low-molecular-weight amines. Good chromatographic separation of mixtures was possible without derivatisation. Ionisation efficiency was greatest using a make-up flow of 0.05% acetic acid in ethanol, and optimisation of ESI source conditions promoted protonated molecule generation for easy determination of molecular weight.

Effect of Juice and Extracts from Saposhnikovia divaricata Root on the Colon Cancer Cells Caco-2

Colorectal cancer ranks 3rd in terms of cancer incidence. Growth and development of colon cancer cells may be affected by juice and extracts from Saposhnikovia divaricata root. The objective of the research was to analyze the effect of S. divaricata juice and extracts on the viability, membrane integrity and types of cell death of Caco-2 cells. Juice and extracts were analyzed using Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and in respect of the presence of antioxidants, total carbohydrates, protein, fat and polyphenols. The contents of cimifugin β-D-glucopyranoside, cimifugin, 4′-O-glucopyranosyl-5-O-methylvisamminol, imperatorin and protein were the highest in juice. 50% Hydroethanolic extract had the greatest antioxidant potential, concentration of polyphenols and fat. Water extract was characterized by the highest content of glutathione. Juice and 75% hydroethanolic extract contained the most carbohydrates. After the application of juice, 50% extract and the juice fraction containing the molecules with molecular weights >50 kDa, a decrease of the cell viability was noted. Juice and this extract exhibited the protective properties in relation to the cell membranes and they induced apoptosis. The knowledge of further mechanisms of anticancer activity of the examined products will allow to consider their use as part of combination therapy.

Breaking the cycle: Reversal of flux in the tricarboxylic acid cycle by dimethyl fumarate.

ObjectiveTo infer molecular effectors of therapeutic effects and adverse events for dimethyl fumarate (DMF) in patients with relapsing-remitting MS (RRMS) using untargeted plasma metabolomics.MethodsPlasma from 27 patients with RRMS was collected at baseline and 6 weeks after initiating DMF. Patients were separated into discovery (n = 15) and validation cohorts (n = 12). Ten healthy controls were also recruited. Metabolomic profiling using ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS) was performed on the discovery cohort and healthy controls at Metabolon Inc (Durham, NC). UPLC-MS was performed on the validation cohort at the National Phenome Centre (London, UK). Plasma neurofilament concentration (pNfL) was assayed using the Simoa platform (Quanterix, Lexington, MA). Time course and cross-sectional analyses were performed to identify pharmacodynamic changes in the metabolome secondary to DMF and relate these to adverse events.ResultsIn the discovery cohort, tricarboxylic acid (TCA) cycle intermediates fumarate and succinate, and TCA cycle metabolites succinyl-carnitine and methyl succinyl-carnitine increased 6 weeks following treatment (q < 0.05). Methyl succinyl-carnitine increased in the validation cohort (q < 0.05). These changes were not observed in the control population. Increased succinyl-carnitine and methyl succinyl-carnitine were associated with adverse events from DMF (flushing and abdominal symptoms). pNfL concentration was higher in patients with RRMS than in controls and reduced over 15 months of treatment.ConclusionTCA cycle intermediates and metabolites are increased in patients with RRMS treated with DMF. The results suggest reversal of flux through the succinate dehydrogenase complex. The contribution of succinyl-carnitine ester agonism at hydroxycarboxylic acid receptor 2 to both therapeutic effects and adverse events requires investigation.

Abstract 2400: Complex gut microbiota modulate rat colon adenoma susceptibility, metabolites, and host gene expression

Abstract We used a multi-omics approach to identify signatures of differential susceptibility in the F344/NTac-Apc+/Pirc (Pirc) rat model of familial adenomatous polyposis. We assessed how different gut microbiomes (GMs) effect the metabolome and host transcriptome to reveal mechanisms of susceptibility. We previously found that differences in the GM are causative determinants of cancer susceptibility using complex microbiota targeted rederivation (CMTR), in which isogenic Pirc rat embryos are transferred into surrogate dams each harboring distinct specific pathogen free gut microbiota: GM-1 and GM-2. Fecal samples collected from CMTR Pirc rats at 1 month of age were analyzed using ultra-high performance liquid chromatography mass spectrometry (UHPLC/MS) to assess the impact of bacterial metabolites on disease susceptibility. At 6 months of age animals were sacrificed, tumor burden assessed, and normal colonic epithelium (NE) and tumor (T) tissues were collected for RNASeq analysis. Metabolomic analysis identified several features that were unique to either GM-1 or GM-2, and principal component analysis revealed a significant separation of the groups. Based on mass-charge ratios, 117 features were significantly different between the groups, of which 28 were putatively identifiable metabolites through the HMDB (Human Metabolite Database) and METLIN databases. Hierarchical cluster analysis of the 1 month metabolome data based on Euclidean distance measurements (Ward's algorithm) showed separation of samples based on tumor burden at sacrifice. Metabolites present prior to observable disease onset at 1 month were correlated with adenoma burden 5 months later. RNAseq between GM-1 and GM-2 tissues identified 2173 differentially expressed genes (DEGs) in the normal epithelium, and 3406 DEGs between tumor groups (FDR &amp;lt; 0.05). Ordinate analysis confirmed both NE and T samples were significantly distinct between rats harboring the two GM profiles. Integrated pathway (IP) analysis of metabolome and RNASeq data based on detected features showed that bile acid biosynthesis was enriched in GM-1, identifying 14 target genes affecting this pathway. Metabolome data from serum samples from an independent group of 1 month old Pirc and wildtype rats with GM-1 also identified metabolites from bile acid biosynthesis elevated in Pirc serum. Six out of the 14 target genes from the IP analysis showed significant differences (FDR &amp;lt; 0.05) between GM-1 and GM-2 in the NE and T tissues indicating a potential role of metabolites modulating both NE and T gene expression. DEG pathway analysis indicated increased fatty acid metabolism and mucin biosynthesis in the high susceptibility group, and apoptosis and calcium signaling pathways increased in the resistant group. These data show the utility of using metabolomics for early detection of adenomas and provide insights into the mechanism of how the GM modulates colon cancer. Citation Format: Susheel Bhanu Busi, Zhentian Lei, Lloyd Sumner, James Amos-Landgraf. Complex gut microbiota modulate rat colon adenoma susceptibility, metabolites, and host gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2400.

CCl₄-Enhanced Ultrasonic Irradiation for Ciprofloxacin Degradation and Antibiotic Activity.

In this study, an ultrasound/CCl4 system was used to degrade the fluoroquinolone antibiotic, ciprofloxacin, in aqueous solution. The effect of CCl4 concentration and initial solution pH on ciprofloxacin degradation were investigated. The results showed that ciprofloxacin degraded effectively under an ultrasound/CCl4 system, with degradation efficiency increasing from 0.51% to 50.92%, when the CCl4 concentration ranged from 0.0 to 41.4 mmol/L in 40 min. Radical scavenging experiments certified that both •OH and chlorine-containing radicals contributed to ciprofloxacin degradation. Eight intermediates were detected using ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS) method, including three chloro-intermediates. Based on these results, the possible degradation pathways of ciprofloxacin are proposed. Agar diffusion tests with E. coli and S. aureus showed that ciprofloxacin's antibacterial activity was completely removed in 40 min. This study indicates that an ultrasound/CCl4 system can degrade ciprofloxacin and remove its antibacterial activity, and thus is a promising process for treating fluoroquinolone antibiotics in wastewater.

Development and comparison of chromatographic methods for the analysis of long chain diols and alkenones in biological materials and sediment

We have compared and assessed the suitability of several chromatographic methods for the analysis of long chain alkenones and long chain diols and the associated paleotemperature proxies (UK’37 and LDI). We evaluated the traditional methods for the analysis of the UK’37 and the LDI, gas chromatography (GC) − flame ionization detection (FID) and GC mass spectrometry (MS) using selected ion monitoring (SIM), respectively, and developed a new method using GC–MS/MS in multiple reaction monitoring mode (MRM) for the analysis of long chain diols as well as a method for automatic silylation of diols using a robot autosampler. Finally, we evaluated liquid chromatography (LC) methods to simultaneously measure the UK’37 and the LDI, using ultra high performance LC (UHPLC) with low (nominal mass) resolution MS in SIM mode, and UHPLC with high resolution MS (HRMS). Detection and quantification limits and reproducibility were assessed by means of serial dilutions of culture extracts.Automated silylation by a robot autosampler showed similar reproducibility as off-line silylation while substantially decreasing sample preparation time. The novel MRM method had a slightly lower limit of quantification (LOQ; i.e. 0.3pgC28 1,13-diol injected on-column) than the traditional method (0.5pg) and improved reproducibility while allowing more unambiguous identification of LCDs in complex matrices. For diols, UHPLC–MS using SIM had the highest LOQ (i.e. 15pg) and a comparable reproducibility as GC–MS. UHPLC–HRMS had a LOQ of ca. 1.5pg, and an improved reproducibility for diol analysis. For alkenone analysis, both UHPLC–HRMS and UHPLC–MS using SIM were 2–3 orders of magnitude more sensitive (LOQ ca. 20 and 2pgC37:2 alkenone injected on-column, respectively) than GC-FID (LOD ca. 3ng), with a similar reproducibility of the UK’37 index. Hence, UHPLC–HRMS allows simultaneous analysis of the UK’37 and LDI at an increased sensitivity. In addition, it allows simultaneous measurement of TEX86, a temperature proxy based on the isoprenoid glycerol dialkyl glycerol tetraethers. This reduces the preparation time by excluding the need of derivatization and separation of the ketone (containing the long chain alkenones) and polar fractions (containing the long chain diols and GDGTs). However, synthetic standards are required to fully assess the accuracy of the new methods for determination of the LDI and UK’37.

Aqueous phase degradation of methyl paraben using UV-activated persulfate method

Methyl paraben (MP), a widely used preservative, exhibits endocrine-disrupting properties with estrogenic activities. The aqueous phase degradation of MP, using UV-activated persulfate method, was investigated in the present study. The combination of UV irradiation and persulfate anion successfully degraded MP showing 98.9% removal within 90min. A quenching experiment using ethanol (EtOH) and tert-butyl alcohol (TBA) showed the presence of both radicals (OH and SO4–) in the system; near neutral pH SO4– radical was the major species. The influence of various factors such as persulfate dose, initial MP concentration, solution pH, and water matrix components on the degradation kinetics was examined in view of the practical applications of the developed process. The degradation rate of MP was considerably increased as the amount of persulfate increased. The degradation of MP in the UV/persulfate system was pH dependent and more promising near neutral pH (6.5) conditions. The presence of anions such as Cl−, HPO42− and HCO3− showed inhibitory effect towards MP degradation. The presence of humic acid also suppressed the degradation efficiency of MP. The reaction rate followed pseudo-first-order kinetics for all of the degradations. The degradation of MP by UV/persulfate treatment led to the production of seven transformation byproducts, which were identified using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS). A degradation pathway for MP degradation was also proposed. The results of the present study reveal that the UV/persulfate process could be an effective approach to remove MP from aqueous solutions.

Polyphenol characterization, anti-oxidant, anti-proliferation and anti-tyrosinase activity of cranberry pomace

Background: Cranberry pomace (CP), an underutilized by-product from juice processing, contains a wide range of biologically active compounds that can be recovered and used in a variety of applications in functional foods and nutraceuticals. Methods: In this study, analytical chemical techniques such as solvent extractions and characterization of extracts in respect with their phenolic content were performed using ultra-high performance liquid chromatography mass spectrometry (UPLC-MS) and spectrophotometry. Crude CP extract and its phenolic acids, flavonols, anthocyanins and proanthocyanidins–rich fractions were then evaluated for their anti-oxidant capacity, tyrosinase inhibitory activity, and anti-proliferation activity against hepatocellular carcinoma HepG2 cells. Results: On a dry weight basis, the different CP fractions contained seven major anthocyanins (0.1-125 mg/g), six major phenolic acids (0.8-31 mg/g), seven flavonols (1-126 mg/g) and five flavan-3-ols (0.1-12 mg/g). Fractions rich in flavonols exhibited the most potent antioxidant capacities with ferric ion reducing antioxidant power values of 1.8-1.9 mmole/g and 2, 2-diphenyl-1-picrylhydrazyl radical scavenging IC50 values of 15.1-15.2 mg/L respectively. On the other hand, fractions rich in phenolic acids and flavan-3-ol monomers demonstrated the most potent anti-tyrosinase activity (IC50=6.1-6.2 mg/L) and anti-proliferative activity (IC50=7.8-15.8 mg/L). Generally, all the fractions exhibited a dose-response relationship in the selected biological activity assays.Conclusion: This study suggests an effective utilization of CP to obtain biologically active fractions with potential to be used in functional foods and nutraceuticals designed for the prevention of chronic diseases associated with oxidative stress.Keywords: cranberry pomace, polyphenol, anti-tyrosinase, anti-proliferation, nutraceuticals, functional foods