Predominant Metabolite Research Articles

Regulator Of G Protein Signaling 14 Disruption Affects The Gut Microbiota And Metabolome In Mice

The gut microbiota is linked to brown adipose tissue (BAT), but the mechanisms and microbes facilitating BAT production or function are unknown. A novel mouse model containing a gene knock-out of regulator of G protein signaling 14 (RGS14 KO) has increased BAT. Our early studies found distinct microbes in RGS14 KO mice and BAT-specific metabolites. PURPOSE: To identify key gut microbial species and uncover BAT-specific metabolites in RGS14 KO mice. Further, we aim to identify and associate metabolites produced in specific tissue samples with these key gut microbes. METHODS: Twenty-two mice (N = 13 RGS14 KO, N = 9 Wild type (WT)) were used to identify predominant microbes and metabolites. Gut microbiota profiles were obtained by sequencing bacterial ribosomal operons. Metabolomics used ultra-high performance liquid chromatography to evaluate polar and nonpolar untargeted metabolites in fecal, cecal, brain, and BAT samples. Microbiome analysis used Kulczynski distance to compare WT to RGS14 KO reads in two-dimensional non-metric multidimensional scaling (NMDS) plots. Two-tailed t-tests were used to compare WT and RGS14 KO metabolite means (p < 0.05). MetaboAnalyst 5.0 was used to identify significant metabolite pathways in tissue samples and generate pathway plots. RESULTS: Approximately 500 k rRNA reads post QA/QC ((84% identify; >1000 bp alignment) were obtained from all samples by MegaBlast. NMDS plots showed significant bacterial community differences (Genus: p = 0.035; species: p = 0.028; strain: p = 0.037) between WT and RGS14 KO mice. Specifically, RGS14 KO mice housed two unique strains of Akkermansia muciniphilia (A. muciniphilia BIOML-A22 and A. muciniphilia AN78) while WT animals contained A. muciniphilia EB-AMDK-1. Untargeted metabolomics identified 82 significantly different (p < 0.05) unique metabolites were between RGS14 KO and WT mice in one of the four samples. Specifically, RGS14 KO animals had significantly higher levels of G6P, glycyl-l-proline, glyercophosphocholine, isoleucine, pipecolic acid, thymidine, UDP-D-glucose, malate, leucic acid, NADP+, and guanosine in BAT compared to WT animals. CONCLUSION: The gut microbiome differs between RGS14 KO and WT mice from genus to strain level. Unique metabolites found in RGS14 KO BAT may be linked to BAT function. Supported by ONR Grant 826640

Untargeted Metabolomics Analysis of Crocus cancellatus subsp. damascenus (Herb.) B. Mathew Stigmas and Their Anticarcinogenic Effect on Breast Cancer Cells.

Safranal, crocin, crocetin, and picrocrocin are major known compounds in the stigma extract of Crocus sativus with various medicinal properties. Crocus cancellatus is another Crocus species that grows extensively in Iran's various regions, such as the Kurdistan province. The predominant metabolites and biological properties of C. cancellatus have not yet been investigated. The ingredients of the stigma ethanol extract of C. cancellatus were investigated using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem mass spectrometry (LC-MS). The ROIMCR approach was performed to analyze the LC-MS full scan data sets. This method searches the MS regions of interest (ROI) data in the m/z domain and analyses the results using the multivariate curve-resolution alternating least squares (MCR-ALS) chemometrics technique for simultaneous resolution of two extracts. Also, the antiproliferative properties of C. cancellatus against MDA-MB-231 and MCF-7 cancer cells were examined by MTT, dual acridine orange/ethidium bromide test, Annexin V-FITC/PI, and zymography. The GC-MS and LC-MS untargeted metabolomics data analysis of the extract indicated the presence of cytotoxic agents including safranal, crocin, picrocrocin, and crocetin in the stigma ethanol extract of C. cancellatus. Biological tests showed that the viability of MDA-MB-231 and MCF-7 cancer cells is decreased following C. cancellatus treatment in a time- and dose-dependent way in both monolayer and 3D cell cultures. The MCF-7 cell spheroids had greater resistance to the cytotoxic activity of the extract in 3D cell culture than the MDA-MB-231 cell spheroids. The morphological changes of the cells treated with C. cancellatus stigmas extract were indicative of apoptosis. Zymography analysis revealed a similar trend of matrix metallopeptidase-2 (MMP-2) and matrix metallopeptidase-9 (MMP-9) activity in the treated cells with C. cancellatus extract in comparison with doxorubicin treatment as a positive control. The findings of this research indicate that the ethanolic extract of C. cancellatus stigmas was a good source of bioactive metabolites with anticancer activity.

Identification of etazene metabolites in human urine by LC-HRMS

Etazene (or etodesnitazene) is a novel and highly active synthetic opioid belonging to the rapidly evolving and emerging group of ‘nitazenes’. We examined the in-vivo metabolism of etazene through analysis of a human (overdose) urine sample. The sample was obtained from a 25-year-old man who attempted suicide by taking an NPS cocktail purchased online. Next to etazene, N-ethylpentedrone, deschloroketamine and flubromazepam were also taken by the patient. The urine sample was analyzed on a Q-Exactive high resolution mass spectrometer (HRMS) operating in full scan mode (range 70-700 m/z) with data-dependent acquisition based on an inclusion list. Etazene metabolites were predicted with the BioTransformer 3.0 software and the exact masses were added into the inclusion list. The urine sample was analyzed both with simple dilution of the sample (addition of internal standard and dilution with aqueous mobile phase) and after β-glucuronidase treatment (addition of internal standard and Abalonase® to the sample with subsequent incubation at 37 °C for 30 min). Six possible metabolites were identified in the urine sample of the patient. The parent compound was less abundant than some metabolites. In line with previous findings for other nitazene opioids, N- and O-deethylation were identified as the predominant metabolism routes, resulting in M1 (O-deethylated etazene; most abundant metabolite based on the peak area), M2 (N-deethylated etazene) and M3 (N,O-dideethylated etazene) metabolites. Less abundant hydroxylated products of these deethylated metabolites (M5: hydroxylated M2 and M6: hydroxylated M1) and etazene (M4) were also found. Hydroxylation is presumed at the benzimidazole ring. For M5 and M6, different chromatographic peaks with the same MS 2 spectra were detected, suggesting the presence of positional isomers of the hydroxyl-group at the benzimidazole ring. Additionally, in the analysis without β-glucuronidase treatment, M1- and M3-glucuronide phase II metabolites were found. A rapid and straightforward HRMS method was developed to study the in-vivo metabolites of etazene. N- and O-deethylated products were the predominant urinary metabolites. Detection of these metabolites in urine can be useful to demonstrate etazene exposure. Our findings are comparable to results obtained in a metabolism study performed in rats.

Morphological and chemical characterization of Alternaria populations from apple fruit

Alternaria is a frequent contaminant of apple fruit, causing severe economic losses. It can produce external lesions and mouldy core, characterised by a rotten area in the apple core.In the present study, morphological and chemical characterization of Alternaria from apples was performed, evaluating differences related to agricultural practices and type of disease.A low morphological diversity was observed; most of the isolates were identified as A. tenuissima sp.-grp. (95 %). A. arborescens sp.-grp. and A. gaisen sp.-grp. were present in a proportion of 1 %, and 3 % of the isolates showed intermediate characteristics between these sp.-grps. and were identified as Alternaria sp. The chemical diversity was greater; 27 secondary metabolites were produced by the apple isolates. The most frequents were altertoxin-I (85 %), altechromone A (76 %), tentoxin (69 %), and tenuazonic acid (68 %). The alternariols were produced in a lower frequency when comparing with isolates from other crops; alternariol, 58 % and alternariol monomethyl ether, 57 %. The predominant secondary metabolite profile included compounds from different chemical families, such as dibenzopyrones, tetramic acids, perylene quinones, and cyclic tetrapeptides.A wider metabolomic capacity was observed in isolates from conventional apples when compared to those from organic fruit, with the predominance of strong producers of altertoxins and alternariols. The isolates from mouldy core showed higher ability to produce metabolites from different chemical families than those from external lesions. The wide chemical diversity of the Alternaria apple population should be considered to assess the health risk associated with apple by-products.

Levels of organochlorine pesticides in five species of fish from Lake Ziway, Ethiopia

The Ethiopian Rift Valley (ERV) lakes region is an agricultural area in Ethiopia with intense year-round irrigated framing where widespread legal and illegal pesticide use was documented. The aim of the study is to assess tissue concentration of organochlorine pesticides (OCPs) in fillets of fives species of fish from Lake Ziway, an ERV lake. The influence of trophic position and fish size on accumulation of OCPs was investigated. Sample preparation for the analysis of OCPs was done following the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method and OCPs were analyzed using gas chromatography and compound specification was made using mass spectrometry. Dichloro-diphenyl-trichloroethanes (DDTs) were dominant among investigated OCPs. 4,4’-dichloro-diphenyl-dichloro-ethylene (p,p’-DDE) was the predominant DDT metabolite constituting about 99.2% of total DDTs. Historic DDT input was suggested as the source of current DDT contamination dominated by p,p’-DDE. Geometric mean value of p,p’-DDE varied from 1.07 to 3.64 ng g–1ww. Species variation in accumulation of p,p’-DDE was found that could result from differences in trophic level, lipid content, age, specific habitat, and feeding habit. Carassius carassius occupying a higher trophic position and with relative high lipid content had the highest p,p’-DDE levels. Positive associations between log-transformed p,p’-DDE, and total length were found for C. carassius and Cyprinus carpio. p,p’-DDE was biomagnified through the local food web. Generally, the levels of DDT in the present study are lower than levels reported from lake Ziway in earlier studies. The present findings may contribute contemporary OCP contamination data in investigated fish species.

Biotransformation and transplacental transfer of the anti-viral remdesivir and predominant metabolite, GS-441524 in pregnant rats.

SummaryBackgroundRemdesivir was the first prodrug approved to treat coronavirus disease 2019 (COVID-19) and has the potential to be used during pregnancy. However, it is not known whether remdesivir and its main metabolite, GS-441524 have the potential to cross the blood-placental barrier. We hypothesize that remdesivir and predominant metabolite GS-441524may cross the blood-placental barrier to reach the embryo tissues.MethodsTo test this hypothesis, ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) coupled with multisite microdialysis was used to monitor the levels of remdesivir and the nucleoside analogue GS-441524 in the maternal blood, fetus, placenta, and amniotic fluid of pregnant Sprague-Dawley rats. The transplacental transfer was evaluated using the pharmacokinetic parameters of AUC and mother-to-fetus transfer ratio (AUCfetus/AUCmother).FindingsOur in-vivo results show that remdesivir is rapidly biotransformed into GS-441524 in the maternal blood, which then readily crossed the placenta with a mother-to-fetus transfer ratio of 0.51 ± 0.18. The Cmax and AUClast values of GS-441524 followed the order: maternal blood > amniotic fluid > fetus > placenta in rats.InterpretationWhile remdesivir does not directly cross into the fetus, however, its main metabolite, GS-441524 readily crosses the placenta and can reside there for at least 4 hours as shown in the pregnant Sprague-Dawley rat model. These findings suggest that careful consideration should be taken for the use of remdesivir in the treatment of COVID-19 in pregnancy.FundingMinistry of Science and Technology of Taiwan.

Comparative In Vitro and In Vivo Hydroxylation Metabolization of Polychlorinated Biphenyl 101 in Laying Hens: A Pilot Study.

Polychlorinated biphenyls (PCBs) can be metabolized into hydroxylated PCBs (OH-PCBs) that exhibit greater toxicity than their parent compounds. In particular, 2,2',4,5,5'-pentachlorobiphenyl (PCB 101) is commonly found in chicken feeds and breeding environments, although information on the biotransformation of this PCB in chickens is lacking. In this study, the hydroxylation metabolization of PCB 101 was assessed based on in vitro trials with Sanhuang chicken liver microsomes and in vivo experiments with Hy-Line Brown hens. The para-substituted metabolite 4'-OH-PCB 101 is the predominant metabolite of PCB 101. 4'-OH-PCB 101 is preferentially retained in the chicken bloodstream and partly distributed into different tissues of laying hens. The blood-brain barrier can effectively prevent the OH-PCB from entering the brain, and the adipose tissue contains a relatively low residue concentration of the OH-PCB. The laying hen can deplete the OH-PCB via laying eggs and excrement. The ratio of 4'-OH-PCB 101/PCB 101 in egg yolk is about 1:2. These results first provide definite evidence for the previous hypothesis of the PCB 101 metabolism by chickens. They could assist in predicting the environmental fate of PCBs, and in the risk assessment of PCBs and OH-PCBs in chicken-based foodstuffs.

Lentilactobacillus laojiaonis sp. nov., isolated from the mud in a fermentation cellar for the production of Chinese liquor.

A novel Gram-stain-positive, rod-shaped, non-motile bacterial strain, designated IM3328T, was isolated from a mud cellar which has been continuously used over hundreds of years for the fermentative production of Chinese strong-flavour baijiu. It is asporogenous, facultative anaerobic and does not exhibit catalase activity. Strain IM3328T can grow at pH 4.5-8.5 (optimum, pH 7.0), 15-45 °C (optimum, 37 °C), with 0-75% (w/v) ethanol with and 0-6% (w/v) NaCl. The API 50CH assay revealed that strain IM3328T can metabolize l-arabinose, d-ribose, d-xylose, d-glucose, d-fructose, d-mannose, N-acetylglucosamine, gluconate, methyl β-d-pyranoside, methyl α-d-glucopyranoside, methyl α-d-glucopyranoside and raffinose among the 49 studied carbon sources. Lactic acid, acetic acid, ethanol, isopentanol and butyl acetate are he predominant metabolites in the fermentation broth of strain IM3328T when cultured in liquid de Man, Rogosa and Sharpe medium under micro-aerobic or anaerobic conditions. The polar lipids of strain IM3328T consist of diphosphatidylglycerol, phosphatidylglycerol, one unidentified phospholipid, two unidentified glycolipids and two unidentified lipids. The major cellular fatty acids (≥10%) consist of C16 : 0, C18:1 ω9c and summed feature 7. The cell wall contains ribose, glucose, galactose, lysine, alanine, glutamic acid and aspartic acid. The complete genome of strain IM3328T contains a circular chromosome of 1242019 bp with 1242 genes and 33 mol% G+C content. On the basis of the 16S rRNA gene phylogenetic tree, Lentilactobacillus senioris DSM 24302T (95.9% similarity), Lentilactobacillus rapi DSM 19907T (95.7% similarity) and Lentilactobacillus parabuchneri DSM 5707T (95.1% similarity) were chosen to compare with strain IM3328T to reveal the physiological differences. The low average nucleotide identity values (69.7-71.2%) between strain IM3328T and phylogenetically related reference strains demonstrated that this strain represents a novel species of the genus Lentilactobacillus, and the name Lentilactobacillus laojiaonis sp. nov. (type strain IM3328T=CGMCC 1.18832T=JCM 34630T) is proposed.

Morphine-3-Glucuronide, Physiology and Behavior.

Morphine remains the gold standard painkiller available to date to relieve severe pain. Morphine metabolism leads to the production of two predominant metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). This metabolism involves uridine 5′-diphospho-glucuronosyltransferases (UGTs), which catalyze the addition of a glucuronide moiety onto the C3 or C6 position of morphine. Interestingly, M3G and M6G have been shown to be biologically active. On the one hand, M6G produces potent analgesia in rodents and humans. On the other hand, M3G provokes a state of strong excitation in rodents, characterized by thermal hyperalgesia and tactile allodynia. Its coadministration with morphine or M6G also reduces the resulting analgesia. Although these behavioral effects show quite consistency in rodents, M3G effects are much more debated in humans and the identity of the receptor(s) on which M3G acts remains unclear. Indeed, M3G has little affinity for mu opioid receptor (MOR) (on which morphine binds) and its effects are retained in the presence of naloxone or naltrexone, two non-selective MOR antagonists. Paradoxically, MOR seems to be essential to M3G effects. In contrast, several studies proposed that TLR4 could mediate M3G effects since this receptor also appears to be essential to M3G-induced hyperalgesia. This review summarizes M3G’s behavioral effects and potential targets in the central nervous system, as well as the mechanisms by which it might oppose analgesia.

Identification of the metabolites of methylophiopogonanone A by ultra-high-performance liquid chromatography combined with high-resolution mass spectrometry.

Methylophiopogonanone A (MOA) is a naturally occurring homoisoflavonoid from the Chinese herb Ophiopogon japonicus, which has been demonstrated to attenuate myocardial apoptosis. However, the metabolism of MOA remains unknown. The goal of the present work was to investigate the in vitro metabolism of MOA using liver microsomes and hepatocytes. The metabolites were generated by incubating MOA with rat, monkey and human liver microsomes or hepatocytes. The resulting samples were analyzed by using a quadrupole-orbitrap high-resolution mass spectrometer. The metabolites were identified through the measurements of the exact mass, elemental composition and product ions. A total of 15 metabolites were detected and identified. Among these metabolites, M7 (demethylenation) was the most abundant metabolite in liver microsomes, while M6 (hydroxylation) was the predominant metabolite in hepatocytes, and glucuronidation metabolites (M9 and M10) were also the main metabolites in hepatocytes. The metabolic pathways of MOA included hydroxylation, demethylenation, glucuronidation, methylation, sulfation and glutathione conjugation. This study for the first time provides valuable data on the metabolites of MOA, which will be of great importance for a better understanding of its disposition and to predict human pharmacokinetics.

Exploring Vaccinium vitis-idaea L. as a potential source of therapeutic agents: antimicrobial, antioxidant, and anti-inflammatory activities of extracts and fractions

Ethnopharmacological relevanceVaccinium vitis-idaea L. (lingonberry) leaves and fruits have traditionally been used in Asian and European countries as a natural solution for urinary tract infections, gastrointestinal distress, neurodegenerative diseases, and related inflammatory disorders, which are overall associated with free radical damage and presence of triggering pathogenic strains in the human body. Considering growing attention to natural products, there are not enough scientific data to confirm predominant specialized metabolites, responsible for the traditional therapeutic use of lingonberries. Aim of the studyThe present study aimed at an in-depth study of specialized metabolite profiling and biological activity evaluation of lingonberry crude extracts and isolated fractions. Materials and methodsCrude dry extracts and fractions from lingonberry leaves and fruits were analyzed by the UPLC-MS method. Potential inhibiting properties against different bacterial strains and hyaluronidase, ability to scavenge hydrogen peroxide, and effect on its production in a macrophage culture J774 were examined. ResultsFindings suggested the tentative presence of 59 compounds, mainly phenolics, displayed higher bioactivities of particular fractions than that of crude extracts and elucidated particular compounds as candidates in pharmaceuticals. Trimeric and dimeric proanthocyanidins from lingonberry leaves and fruits were shown to have the strongest antimicrobial, antioxidant, and anti-inflammatory potential. ConclusionsThis study revealed specialized metabolites responsible for the traditional medicinal properties of lingonberries and pointed out demand for further purification and new research directions of proanthocyanidins in the frame of their multipharmacological perspectives.

English

Aflatoxin occurrence in poultry feed causes aflatoxicosis in birds and poses health hazards to the consumers. Aflatoxin B1 (AfB1) is the most predominant and toxic metabolite that is controlled through addition of argillaceous clays as non-nutritive additives. Non-smectitic indigenous clay reserves need testing as AfB1 adsorbent. With detailed mineral and adsorption characteristics, three indigenous non-smectitic clays: (i) palygorskite, (ii) palygorskite-smectite mix, and (iii) interstratified smectite with hydroxy interlayered smectite were tested against 250 µg kg-1 AfB1 contamination in a poultry feeding trial with three replications at 1% and 2% (w/w) with positive and negative controls. A total of 330 Ross-308 male broiler chicks, initially reared on clean feed under controlled conditions of light and humidity, were distributed into eleven treatment combinations in a completely randomized design with 30 birds in each treatment on an equal weight basis with three replications at day 14 and fed on experimental feeds for three weeks. Three birds from each pen were randomly slaughtered at day 35 and for each replicated treatment, body weight gain, feed intake, and internal organs weight and morphology were recorded. AfB1 contamination reduced weight gain (p 0.0001), feed conversion ratio (p 0.0001) and feed intake (p 0.0001) while clays addition in the toxin feed effectively controlled AfB1 toxicity as suggested by improved body weight, weight gain rate and feed consumption compared to the toxin fed birds. The liver morphology was comparatively better in palygorskite-smectite mix treatment when applied at 1% and the darkish colour was also improved with addition of the clay in the toxin feed. AfB1 feeding caused a 75% reduction in weight gain compared to the clean feed. Palygorskite and palygorskite-smectite mix clays were better than interstratified clay in increasing weight gain and caused a 60% and 65% increase over toxin feed treatment when applied at 1% and 2%, respectively. In conclusion, the indigenous clay sources overall and palygorskite-smectite mix in particular has the potential for use as a mycotoxin binder for controlling AfB1 incidence in poultry

Clostridium thailandense sp. nov., a novel CO2-reducing acetogenic bacterium isolated from peatland soil.

Some species of the genus Clostridium are efficient acetate producers and have been deemed useful for upgrading industrial biogas. An acetogenic, strictly anaerobic, Gram-stain-positive, subterminal endospore-forming bacterium designated strain PL3T was isolated from peatland soil enrichments with H2 and CO2. Cells of strain PL3T were 0.8-1.0×4.0-10.0 µm in size and rod-shaped. Growth of strain PL3T occurred at pH 6.0-7.5 (optimum, pH 7.0), at 20-40 °C (optimum, 30 °C) and with 0-1.5 % (w/v) NaCl (optimum, 0.5%). Biochemical analyses revealed that strain PL3T metabolized lactose, maltose, raffinose, rhamnose, lactic acid, sorbitol, arabinose and glycerol. Acetic acid was the predominant metabolite under anaerobic respiration with H2/CO2. The major cellular fatty acids were C16 : 0, C16 : 1 cis 9 and C17 : 0 cyc. The main polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, aminolipid and aminophospholipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PL3T belongs to the genus Clostridium with the highest sequence similarity to Clostridium aciditolerans DSM 17425T (98.6 %) followed by Clostridium nitrophenolicum (97.8 %). The genomic DNA G+C content of strain PL3T was 31.1 mol%.The genomic in silico DNA-DNA hybridization value between strain PL3T and C. aciditolerans DSM 17425T was 25.1 %, with an average nucleotide identity of 80.2 %. Based on phenotypic, chemotaxonomic and phylogenetic differences, strain PL3T was suggested to represent a novel species of the genus Clostridium, for which the name Clostridium thailandense sp. nov. is proposed. The type strain is PL3T (=DSM 111812T=TISTR 2984T).

The Effect of Breed and Age on the Growth Performance, Carcass Traits and Metabolic Profile in Breast Muscle of Chinese Indigenous Chickens.

Indigenous chickens possess desirable characteristics and account for considerable proportions of the total chicken production in China. The current study examined the growth performance, carcass characteristics and muscle metabolites among a crossbred broiler and two indigenous, yellow-feathered chickens (Mahuang and Tuer) with different ages (60 and 75 days). Results indicated that the crossbred broiler had better feed efficiency, higher breast and thigh muscle yield, as well as a lower abdominal fat percentage than Mahuang and Tuer chickens (p < 0.05). Gas chromatography–mass spectrometry-based metabolomics and multivariate analysis revealed sugars, amino acids and organic acids were the predominant metabolites that differed among the three chicken breeds. Growth performance and carcass traits of yellow-feathered chickens exhibited significant differences with the extension of the feeding period (p < 0.05). Moreover, differential metabolites reflected altered aminoacyl-tRNA biosynthesis, ATP-binding cassette transporters, pantothenate and CoA biosynthesis, as well as glutathione metabolism in yellow-feathered chickens affected by age. Collectively, this study contributes to a deeper understanding of the production efficiency and chemical composition of precursor flavor in Chinese indigenous, yellow-feathered chicken.

Detection of bazedoxifene, a selective estrogen receptor modulator, in human urine by liquid chromatography-tandem mass spectrometry.

Bazedoxifene, a selective estrogen receptor modulator, has been explicitly included in the prohibited list issued by the World Anti-Doping Agency (WADA) since January 2020. A high-resolution liquid chromatography-tandem mass spectrometric detection method was developed to identify bazedoxifene and its metabolites in human urine and to quantify bazedoxifene (free plus glucuronide) for doping control purposes. Bazedoxifene acetate (20 mg) was orally administered to seven male volunteers, and the urine samples collected were analyzed using the developed method. The linearity ranged from 0.5 to 200 ng/ml, and the limit of detection was <0.2 ng/ml. The interday precision (2.2% to 3.6%) and the interday accuracy (-10.0% to 1.9%) were adequate. Bazedoxifene, bazedoxifene-N-oxide, and bazedoxifene glucoconjugates were identified in the urine samples. The profiles of the urinary excretion indicated the presence of small amounts of free bazedoxifene and bazedoxifene-N-oxide, whereas bazedoxifene glucuronide was the predominant metabolite. The cumulative excretion amount of bazedoxifene (free form plus glucuronide conjugate) within 78 h after the administration was 0.7% to 1.3% of the total dose. In all subjects, bazedoxifene (free plus glucuronide) could be detected in urine up to 78 h after administration.

Cancer Chemopreventive Potential and Chemical Profiling of Euphorbia abyssinica Endowed with Docking Studies.

Chemical profiling of both fruit and aerial part extracts of Euphorbia abyssinica via ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) showed them to be a rich source of diverse compounds. A total of 39 compounds in both extracts including flavonoids and phenolic compounds were identified as predominant metabolites. The antioxidant activity of both extracts was evaluated using three different in vitro assays (DPPH, ABTS, and FRAP assays). The E. abyssinica fruit extract demonstrated more potent activity compared to the aerial part extract (IC50 of 85.1 ± 1.07 and 562.3 ± 1.01 μg/mL, respectively) in the DPPH assay. Furthermore, using ABTS and FRAP assays, the antioxidant capacities of the fruit extract were 1063.03 ± 37.8 and 1476.5 ± 95.6, respectively, calculated as μM Trolox equivalent/mg extract. One of the existing markers for cancer chemoprevention is the induction of phase II detoxifying enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1), which plays a vital role in cytoprotection against oxidative damage. The extracts were assessed to test their chemopreventive potential via NQO1 enzyme induction. The methanolic extract of fruits demonstrated a concentration-dependent increase in the cancer chemopreventive marker enzyme NQO1 at the protein expression level in a murine hepatoma cell line (Hepa1c1c7). The interaction with Kelch-like ECH-associated protein 1 (KEAP1) is an essential transcription factor that controls the expression of the NQO1 enzyme. The demonstrated induction of NQO1 by the fruit extract is consistent with a molecular docking study of the effect of dereplicated compounds on the KEAP1 target. Among the dereplicated compounds, hesperidin, naringin, and rutin have been established as promising inducer compounds for the chemopreventive marker NQO1. Our results highlight the E. abyssinica fruit extract as a future chemopreventive lead.

1H NMR-Based Chemometrics to Gain Insights Into the Bran of Radiation-Induced Colored Wheat Mutant.

Recently, wheat has attracted attention as a functional food, rather than a simple dietary energy source. Accordingly, whole-grain intake increases with an understanding of bioactive phytochemicals in bran. The development of colored wheat has drawn more attention to the value of bran owing to its nutritional quality, as well as the antioxidant properties of the colorant. The present 1H NMR-based chemometric study evaluated the compositional improvement of radiation-induced mutants in purple wheat by focusing on the predominant metabolites with high polarity. A total of 33 metabolites, including three choline derivatives, three sugar alcohols, four sugars, 13 amino acids, eight organic acids, and two nucleosides, were identified throughout the 1H NMR spectra, and quantification data were obtained for the identified metabolites via peak shape-based quantification. Principal component and hierarchical cluster analyses were conducted for performing multivariate analyses. The colored original wheat was found to exhibit improvements compared to yellow wheat in terms of the contents of primary metabolites, thus highlighting the importance of conducting investigations of polar metabolites. The chemometrics studies further revealed mutant lines with a compositional enhancement for metabolites, including lysine, proline, acetate, and glycerol.

The Multiomics Analyses of Gut Microbiota, Urine Metabolome and Plasma Proteome Revealed Significant Changes in Allergy Featured with Indole Derivatives of Tryptophan.

ObjectiveTo explore changes in the gut microbiota (GM), urine metabolome and plasma proteome in individuals with allergies using multiomics analyses, and identify the key components and mechanism.MethodsThis was a cross-sectional study. All subjects were recruited to collect fecal, urine and blood samples. 16S rDNA sequencing was used to analyze the structure and function of the GM, liquid chromatography mass spectrometry was used to quantify metabolites in the urine, and data-independent acquisition quantitative proteome analysis was used to detect proteins in the plasma. Differences in GM, urine metabolites and plasma proteins between allergic and healthy individuals were displayed using principal component analysis (PCoA) and heatmap, and the co-occurrence network was visualized in Cytoscape using Spearman correlation among differential predominant genera, metabolites and proteins. The functional analysis was performed according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) dataset. The allergy-related cytokines, IL-4, IL-6 and IL-13, were measured to evaluate the effect of indole derivatives on LPS-induced macrophage activation.ResultsGM α indexes, β distances and the relative abundance of the core differential genera in the allergic group were different from those of healthy individuals, which resulted in a separate distribution in the PCoA and enterotypes. Similarly, the concentrations of 393 metabolites and 144 proteins were different between allergic and healthy individuals. Then, 634 significant correlations were identified among 6 predominant differential genera, 24 differential metabolites and 104 differential proteins, 301 of which were negative and 333 of which were positive. Notably, a core network centered on tryptophan metabolites, indole-3-butyric acid (IBA) and indole-3-lactic acid (ILA), displayed high consistency with the results of KEGG pathway analysis. In the LPS-stimulated macrophages, IBA reduced the expression of IL-4 and IL-6, and ILA inhibited the upregulation of IL-6.ConclusionThe GM, urine metabolome and plasma proteome underwent systematic change in allergic individuals compared to healthy individuals, among which indole derivatives from tryptophan metabolism might play key roles in the progression of allergies and could serve as therapeutic targets of allergy.

Bioactive Phenolic and Fatty Acid Composition of Canadian Wild Gooseberry and Blackcurrant

Wild berry varieties are widely distributed in Canada and mainly used by the indigenous population as food. Gooseberries and blackcurrant are the two tart berries with strong taste that are used to make preserves such as jams, jellies, and wine. Phenolic compounds and fatty acids are the predominant bioactive metabolites present in these berries. Recently, researchers are more interested in exploring the fruits such as berries for functional properties that benefit human health. Although wild berries are known for their health benefits, research evidence related to profiling of bioactive molecules in these wild berries such as Canadian gooseberry and blackcurrant is limited. In this study we assessed and compared the bioactive phenolic and fatty acid composition in these two berries using liquid and gas chromatography and mass spectrometry. The cyanidine glucoside, which is a polyphenolic anthocyanin present in blackcurrant is significantly higher (832 μg/g Dry wt.) compared to that in Canadian gooseberry (150 μg/g Dry wt.). Linolenic acid (20.3 %) and linoleic acid (18.2 %) were the most abundant fatty acids in blackcurrant while linoleic acid (15.0 %) and palmitic acid (11.2 %) were abundant in gooseberry. Other fatty acids such as oleic acid, linoleic, and alpha linolenic acid, which are important in cardiovascular health, were present in both blackcurrant and gooseberry.

CYP2C8-Mediated Formation of a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Cytochrome P450 and Aldehyde Oxidase Substrate.

Predicting human disproportionate metabolites is difficult, especially when drugs undergo species-specific metabolism mediated by cytochrome P450s (P450s) and/or non-P450 enzymes. This study assessed human metabolites of DS-1971a, a potent Nav1.7-selective blocker, by performing human mass balance studies and characterizing DS-1971a metabolites, in accordance with the Metabolites in Safety Testing guidance. In addition, we investigated the mechanism by which the major human disproportionate metabolite (M1) was formed. After oral administration of radiolabeled DS-1971a, the major metabolites in human plasma were P450-mediated monoxidized metabolites M1 and M2 with area under the curve ratios of 27% and 10% of total drug-related exposure, respectively; the minor metabolites were dioxidized metabolites produced by aldehyde oxidase and P450s. By comparing exposure levels of M1 and M2 between humans and safety assessment animals, M1 but not M2 was found to be a human disproportionate metabolite, requiring further characterization under the Metabolites in Safety Testing guidance. Incubation studies with human liver microsomes indicated that CYP2C8 was responsible for the formation of M1. Docking simulation indicated that, in the formation of M1 and M2, there would be hydrogen bonding and/or electrostatic interactions between the pyrimidine and sulfonamide moieties of DS-1971a and amino acid residues Ser100, Ile102, Ile106, Thr107, and Asn217 in CYP2C8, and that the cyclohexane ring of DS-1971a would be located near the heme iron of CYP2C8. These results clearly indicate that M1 is the predominant metabolite in humans and a human disproportionate metabolite due to species-specific differences in metabolism. SIGNIFICANCE STATEMENT: This report is the first to show a human disproportionate metabolite generated by CYP2C8-mediated primary metabolism. We clearly demonstrate that DS-1971a, a mixed aldehyde oxidase and cytochrome P450 substrate, was predominantly metabolized by CYP2C8 to form M1, a human disproportionate metabolite. Species differences in the formation of M1 highlight the regio- and stereoselective metabolism by CYP2C8, and the proposed interaction between DS-1971a and CYP2C8 provides new knowledge of CYP2C8-mediated metabolism of cyclohexane-containing substrates.