Boosting annotation in nutrimetabolomics by Feature-Based Molecular Networking: analytical and computational strategies applied to human urine samples from an untargeted LC-MS/MS based bilberry-blueberry intervention study.

Urine represents a challenging metabolite mixture to decipher. Yet, it contains valuable information on dietary intake patterns as typically investigated using randomized, single-blinded, intervention studies. This research demonstrates how the use of Feature-Based Molecular Networking in combination with public spectral libraries, further expanded with an 'In-house' library of metabolite spectra, improved the non-trivial annotation of metabolites occurring in human urine samples following bilberry and blueberry intake. Following this approach, 65 berry-related and human endogenous metabolites were annotated, increasing the annotation coverage by 72% compared to conventional annotation approaches. Furthermore, the structures of 15 additional metabolites were hypothesized by spectral analysis. Then, by leveraging the MzMine quantitative information, several molecular families of phase II (e.g., glucuronidated phenolics) and phase I (e.g., phenylpropionic acid and hydroxybenzoic acid molecular scaffolds) metabolism were identified by correlation analysis of postprandial kinetics, and the dietary impact of endogenous and exogenous metabolites following bilberry-blueberry intake was estimated.

Metabolite G-Protein Coupled Receptors in Cardio-Metabolic Diseases

G protein-coupled receptors (GPCRs) have originally been described as a family of receptors activated by hormones, neurotransmitters, and other mediators. However, in recent years GPCRs have shown to bind endogenous metabolites, which serve functions other than as signaling mediators. These receptors respond to fatty acids, mono- and disaccharides, amino acids, or various intermediates and products of metabolism, including ketone bodies, lactate, succinate, or bile acids. Given that many of these metabolic processes are dysregulated under pathological conditions, including diabetes, dyslipidemia, and obesity, receptors of endogenous metabolites have also been recognized as potential drug targets to prevent and/or treat metabolic and cardiovascular diseases. This review describes G protein-coupled receptors activated by endogenous metabolites and summarizes their physiological, pathophysiological, and potential pharmacological roles.

Combined Metabolomics and Network Toxicology to Explore the Molecular Mechanism of Phytolacca acinose Roxb-Induced Hepatotoxicity in Zebrafish Larvae in Vivo

Phytolacca acinosa Roxb (PAR), a traditional Chinese medicine, has been widely used as a diuretic drug for a long period of time for the treatment edema, swelling, and sores. However, it has been reported that PAR might induce hepatotoxicity, while the mechanisms of its toxic effect are still unclear. In this study, network toxicology and metabolomic technique were applied to explore PAR-induced hepatotoxicity on zebrafish larvae. We evaluated the effect of PAR on the ultrastructure and the function of the liver, predictive targets, and pathways in network toxicology, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS. PAR causes liver injury, abnormal liver function, and apoptosis in zebrafish. The level of arachidonic acid in endogenous metabolites treated with PAR was significantly increased, leading to oxidative stress in vivo. Excessive ROS further activated the p53 signal pathway and caspase family, which were obtained from KEGG enrichment analysis of network toxicology. The gene levels of caspase-3, caspase-8, and caspase-9 were significantly increased by RT-PCR, and the level of Caps3 protein was also significantly up-regulated through western blot. PAR exposure results in the liver function abnormal amino acid metabolism disturbance and motivates hepatocyte apoptosis, furthermore leading to liver injury.

Uncovering Xenobiotics in the Dark Metabolome using Ion Mobility Spectrometry, Mass Defect Analysis and Machine Learning

The identification of xenobiotics in nontargeted metabolomic analyses is a vital step in understanding human exposure. Xenobiotic metabolism, excretion, and co-existence with other endogenous molecules however greatly complicate nontargeted studies. While mass spectrometry (MS)-based platforms are commonly used in metabolomic measurements, deconvoluting endogenous metabolites and xenobiotics is often challenged by the lack of xenobiotic parent and metabolite standards as well as the numerous isomers possible for each small molecule m/z feature. Here, we evaluate the use of ion mobility spectrometry coupled with MS (IMS-MS) and mass defect filtering in a xenobiotic structural annotation workflow to reduce large metabolomic feature lists and uncover potential xenobiotic classes and species detected in the metabolomic studies. To evaluate the workflow, xenobiotics having known high toxicities including per- and polyfluoroalkyl substances (PFAS), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) were examined. Initially, to address the lack of available IMS collision cross section (CCS) values for per- and polyfluoroalkyl substances (PFAS), 88 PFAS standards were evaluated with IMS-MS to both develop a targeted PFAS CCS library and for use in machine learning predictions. The CCS values for biomolecules and xenobiotics were then plotted versus m/z, clearly distinguishing the biomolecules and halogenated xenobiotics. The xenobiotic structural annotation workflow was then used to annotate potential PFAS features in NIST human serum. The workflow reduced the 2,423 detected LC-IMS-MS features to 80 possible PFAS with 17 confidently identified through targeted analyses and 48 additional features correlating with possible CompTox entries.

Serum Predose Metabolic Profiling for Prediction of Rosuvastatin Pharmacokinetic Parameters in Healthy Volunteers

Rosuvastatin is a well-known lipid-lowering agent generally used for hypercholesterolemia treatment and coronary artery disease prevention. There is a substantial inter-individual variability in the absorption of statins usually caused by genetic polymorphisms leading to a variation in the corresponding pharmacokinetic parameters, which may affect drug therapy safety and efficacy. Therefore, the investigation of metabolic markers associated with rosuvastatin inter-individual variability is exceedingly relevant for drug therapy optimization and minimizing side effects. This work describes the application of pharmacometabolomic strategies using liquid chromatography coupled to mass spectrometry to investigate endogenous plasma metabolites capable of predicting pharmacokinetic parameters in predose samples. First, a targeted method for the determination of plasma concentration levels of rosuvastatin was validated and applied to obtain the pharmacokinetic parameters from 40 enrolled individuals; then, predose samples were analyzed using a metabolomic approach to search for associations between endogenous metabolites and the corresponding pharmacokinetic parameters. Data processing using machine learning revealed some candidates including sterols and bile acids, carboxylated metabolites, and lipids, suggesting the approach herein described as promising for personalized drug therapy.

Pharmaco-Metabolomics of Inhaled Corticosteroid Response in Individuals with Asthma

Metabolomic indicators of asthma treatment responses have yet to be identified. In this study, we aimed to uncover plasma metabolomic profiles associated with asthma exacerbations while on inhaled corticosteroid (ICS) treatment. We determined whether these profiles change with age from adolescence to adulthood. We utilized data from 170 individuals with asthma on ICS from the Mass General Brigham Biobank to identify plasma metabolites associated with asthma exacerbations while on ICS and examined potential effect modification of metabolite-exacerbation associations by age. We used liquid chromatography–high-resolution mass spectrometry-based metabolomic profiling. Sex-stratified analyses were also performed for the significant associations. The age range of the participating individuals was 13–43 years with a mean age of 33.5 years. Of the 783 endogenous metabolites tested, eight demonstrated significant associations with exacerbation after correction for multiple comparisons and adjusting for potential confounders (Bonferroni p value < 6.2 × 10−4). Potential effect modification by sex was detected for fatty acid metabolites, with males showing a greater reduction in their metabolite levels with ICS exacerbation. Thirty-eight metabolites showed suggestive interactions with age on exacerbation (nominal p-value < 0.05). Our findings demonstrate that plasma metabolomic profiles differ for individuals who experience asthma exacerbations while on ICS. The differentiating metabolites may serve as biomarkers of ICS response and may highlight metabolic pathways underlying ICS response variability.

A GC-MS-based Metabolomic Strategy to Investigate the Protective Effects of Mulberry Polysaccharide on CCl4-induced Acute Liver Injury in Mice

BackgroundMulberry (Morus alba) fruits of the woody mulberry tree (family: Moraceae Morus) is a type of mulberry fruit grown in the southern Xinjiang region,which polysaccharides have antioxidant and liver protective effects.This article further preliminary study on the protective effects of mulberry polysaccharide (MP) on liver.MethodsA detection kit was used to assess serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver malondialdehyde (MDA), superoxide dismutase (SOD) and other indicators. Liver tissue sections were stained with hematoxylin and eosin (H&E) and observed under a microscope. The entire endogenous metabolite profiling was acquired via metabolomics strategy using gas chromatography-mass spectrometry (GC-MS) to assess the underlying protective mechanisms of MPs. ResultsResults indicated that MPs exerted a hepatoprotective effect on acute liver injury by decreasing serum ALT and AST levels, hepatic MDA, and restored hepatic SOD glutathione peroxidase (GSH-Px) activities. A total of 33 possible endogenous metabolites associated with lipid, glucose, and energy metabolism including amino acids, sugars, and fatty acids, were found.ConclusionsThe results of the present study provide a reference for elucidating the protective mechanisms of MPs against acute liver injury.

Rat Endogenous Intoxication Level under Experimental Nephropathy and Reduced Glutathione Application

The purpose of the study, therefore, was to investigate the content of substances of low and medium molecular weight, total and tyrosine-containing peptides and to assess the level of endogenous intoxication of the rat organism under conditions of experimental nephropathy and the use of reduced glutathione. Materials and methods. The experiment was performed on nonlinear white rats weighing 160-180 g. The animals were in a vivarium with a constant temperature (18-22°C), relative humidity (50-55%) in individual exchange cells in the formed groups, on a constant balanced mode of a diet with free access to water and food. Nephropathy was modeled by a single intraperitoneal injection of folic acid at a dose of 250 mg/kg. Glutathione was administered intragastrically (100 mg/kg) for 7 days after folic acid intoxication. Results and discussion. Under conditions of nephropathy in the blood serum of rats, an increase in the level of molecules of average weight was observed by 2 times, as well as low-molecular substances by 1.6 times compared with the control. Daily administration of reduced glutathione to animals with nephropathy for 7 days led to a decrease in the level of substances of medium and low molecular weight by 21% and 27% in comparison with the indicators of animals with nephropathy, but still significantly differed from the control indicators. Against the background of an increase in the content of total oligopeptides by 2.2 times in the blood serum of experimental rats, the level of tyrosine-containing peptides increased by 1.8 times in comparison with the control. The introduction of glutathione to rats with nephropathy led to a decrease in these parameters by 40 and 35%, respectively, compared with the experimental control. As a result of the established changes in the level of albumin and average weight molecules in the blood serum of animals, under conditions of nephropathy, an increase in the coefficient of endogenous intoxication was observed by 2.3 times. This imbalance indicates a violation of the processes of detoxification of endogenous metabolites in the liver of animals under conditions of nephropathy. In the groups of animals with nephropathy, which were injected with an antioxidant, the coefficient of endogenous intoxication decreased by 41% compared with animals that did not receive glutathione, but this indicator did not return to the level of control values. Conclusion. Thus, under the conditions of experimental nephropathy in the blood serum of rats, an increase in the content of total and tyrosine-containing peptides, substances of low and medium molecular weight, as well as an increase in the values of the intoxication coefficient was observed, which indirectly indicates a violation of the processes of detoxification of endogenous metabolites in the liver of experimental animals with kidney pathology. In the group of animals with nephropathy that received reduced glutathione for 7 days, only a tendency towards normalization of the studied parameters characterizing the level of endogenous intoxication under conditions of nephropathy was noted

The Changes in Endogenous Metabolites in Hyperlipidemic Rats Treated with Herbal Mixture Containing Lemon, Apple Cider, Garlic, Ginger, and Honey

An herbal mixture composed of lemon, apple cider, garlic, ginger and honey as a polyphenol-rich mixture (PRM) has been reported to contain hypolipidemic activity on human subjects and hyperlipidemic rats. However, the therapeutic effects of PRM on metabolites are not clearly understood. Therefore, this study aimed to provide new information on the causal impact of PRM on the endogenous metabolites, pathways and serum biochemistry. Serum samples of hyperlipidemic rats treated with PRM were subjected to biochemistry (lipid and liver profile) and hydroxymethylglutaryl-CoA enzyme reductase (HMG-CoA reductase) analyses. In contrast, the urine samples were subjected to urine metabolomics using 1H NMR. The serum biochemistry revealed that PRM at 500 mg/kg (PRM-H) managed to lower the total cholesterol level and low-density lipoprotein (LDL-C) (p < 0.05) and reduce the HMG-CoA reductase activity. The pathway analysis from urine metabolomics reveals that PRM-H altered 17 pathways, with the TCA cycle having the highest impact (0.26). Results also showed the relationship between the serum biochemistry of LDL-C and HMG-CoA reductase and urine metabolites (trimethylamine-N-oxide, dimethylglycine, allantoin and succinate). The study’s findings demonstrated the potential of PRM at 500 mg/kg as an anti-hyperlipidemic by altering the TCA cycle, inhibiting HMG-CoA reductase and lowering the LDL-C in high cholesterol rats.