Isolation of 10 cyclosporine metabolites from human bile

Ten metabolites of cyclosporine were isolated from the ethyl ether extract of bile from four liver transplant patients receiving cyclosporine. Two of the metabolites were unique and previously unidentified. Liquid-liquid partitioning into diethyl ether with subsequent defatting with n-hexane was used for the initial extraction form bile. Separation of the individual metabolites (A-J) was performed using a Sephadex LH-20 column and a gradient high performance liquid chromatographic method. The molecular weights of the isolated metabolites were determined by fast atom bombardment/mass spectrometry. Gas chromatography with mass spectrometic amino acid analysis was also used to identify the amino acid composition and the hydroxylation position of metabolites A, B, C, D, and G. Proton nuclear magnetic resonance spectra were utilized to disinguish the chemical shifts of N-CH3 singlets and NH doublets of metabolites A, B, C, and D. Metabolites A, E, F, H, I, and J were reported previously in human urine and animal bile. Metabolites C and D are dihydroxylated compounds which cannot be clearly described as previously isolated compounds. Metabolites B and G are novel metabolites with a mass fragment which corresponded to a loss of 131 Da from the protonated molecular ion (MH+) in the fast atom bombardment/mass spectrometry, suggesting that the double bond in amino acid 1 has been modified. Metabolites B and G were primarily isolated from the bile of one of the liver transplant patients which contained abnormally high concentrations of these two metabolites. The method described is an efficient procedure for isolating milligram quantities of the major metabolites with greater than 95% purity

Genetic regulation of mouse liver metabolite levels.

We profiled and analyzed 283 metabolites representing eight major classes of molecules including Lipids, Carbohydrates, Amino Acids, Peptides, Xenobiotics, Vitamins and Cofactors, Energy Metabolism, and Nucleotides in mouse liver of 104 inbred and recombinant inbred strains. We find that metabolites exhibit a wide range of variation, as has been previously observed with metabolites in blood serum. Using genome-wide association analysis, we mapped 40% of the quantified metabolites to at least one locus in the genome and for 75% of the loci mapped we identified at least one candidate gene by local expression QTL analysis of the transcripts. Moreover, we validated 2 of 3 of the significant loci examined by adenoviral overexpression of the genes in mice. In our GWAS results, we find that at significant loci the peak markers explained on average between 20 and 40% of variation in the metabolites. Moreover, 39% of loci found to be regulating liver metabolites in mice were also found in human GWAS results for serum metabolites, providing support for similarity in genetic regulation of metabolites between mice and human. We also integrated the metabolomic data with transcriptomic and clinical phenotypic data to evaluate the extent of co-variation across various biological scales

Phenolic metabolites of anthocyanins following a dietary intervention study in post-menopausal women

Scope Numerous studies feeding anthocyanin-rich foods report limited bioavailability of the parent anthocyanins. The present study explores the identity and concentration of the phenolic metabolites of anthocyanins in humans. Methods and results Anthocyanin metabolites were quantified in samples collected from a previously conducted 12-wk elderberry intervention study in healthy post-menopausal women. Individual 1-, 2- and 3-h post-bolus urine samples and pooled plasma samples following acute (single bolus) and chronic (12-wk supplementation) anthocyanin consumption (500 mg/day) were analysed using HPLC-ESI-MS/MS. Twenty-eight anthocyanin metabolites were identified in urine and 21 in plasma (including sulfates of vanillic, protocatechuic and benzoic acid). Phenolic metabolites reached peak concentrations of 1237 nM in plasma, while anthocyanin conjugates only reached concentrations of 34 nM. Similarly, in urine, phenolic metabolites were detected at concentrations of 33 185 ± 2549 nM/mM creatinine, while anthocyanin conjugates reached concentrations of 548 ± 219 nM/mM creatinine. There was no evidence that chronic exposure had any impact on either the profile or quantity of metabolites recovered relative to acute exposure. Conclusion An extensive range of phenolic metabolites of anthocyanin was identified following elderberry consumption in humans, including 11 novel metabolites, which were identified at much higher concentrations than their parent compounds

Intracellular metabolites in marine microorganisms during an experiment evaluating microbial mortality

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Longnecker, K., & Kujawinski, E. B. Intracellular metabolites in marine microorganisms during an experiment evaluating microbial mortality. Metabolites, 10(3), (2020): 105, doi: 10.3390/metabo10030105.Metabolomics is a tool with immense potential for providing insight into the impact of biological processes on the environment. Here, we used metabolomics methods to characterize intracellular metabolites within marine microorganisms during a manipulation experiment that was designed to test the impact of two sources of microbial mortality, protozoan grazing and viral lysis. Intracellular metabolites were analyzed with targeted and untargeted mass spectrometry methods. The treatment with reduced viral mortality showed the largest changes in metabolite concentrations, although there were organic compounds that shifted when the impact of protozoan grazers was reduced. Intracellular concentrations of guanine, phenylalanine, glutamic acid, and ectoine presented significant responses to changes in the source of mortality. Unexpectedly, variability in metabolite concentrations were not accompanied by increases in microbial abundance which indicates that marine microorganisms altered their internal organic carbon stores without changes in biomass or microbial growth. We used Weighted Correlation Network Analysis (WGCNA) to identify correlations between the targeted and untargeted mass spectrometry data. This analysis revealed multiple unknown organic compounds were correlated with compatible solutes, also called osmolytes or chemical chaperones, which emphasizes the dominant role of compatible solutes in marine microorganisms.This research was funded by the US National Science Foundation (OCE-1154320 to EBK and KL, OCE-1634016 to EBK) and WHOI’s Ocean Life Institute (to EBK and KL). The mass spectrometry samples were analyzed at the WHOI FT-MS Users’ Facility with instrumentation funded by the National Science Foundation (OCE-0619608 and OCE-1058448)

Toward a Standardized Strategy of Clinical Metabolomics for the Advancement of Precision Medicine

Despite the tremendous success, pitfalls have been observed in every step of a clinical metabolomics workflow, which impedes the internal validity of the study. Furthermore, the demand for logistics, instrumentations, and computational resources for metabolic phenotyping studies has far exceeded our expectations. In this conceptual review, we will cover inclusive barriers of a metabolomics-based clinical study and suggest potential solutions in the hope of enhancing study robustness, usability, and transferability. The importance of quality assurance and quality control procedures is discussed, followed by a practical rule containing five phases, including two additional "pre-pre-" and "post-post-" analytical steps. Besides, we will elucidate the potential involvement of machine learning and demonstrate that the need for automated data mining algorithms to improve the quality of future research is undeniable. Consequently, we propose a comprehensive metabolomics framework, along with an appropriate checklist refined from current guidelines and our previously published assessment, in the attempt to accurately translate achievements in metabolomics into clinical and epidemiological research. Furthermore, the integration of multifaceted multi-omics approaches with metabolomics as the pillar member is in urgent need. When combining with other social or nutritional factors, we can gather complete omics profiles for a particular disease. Our discussion reflects the current obstacles and potential solutions toward the progressing trend of utilizing metabolomics in clinical research to create the next-generation healthcare system.11Ysciescopu

In vitro metabolic studies of REV-ERB agonists SR9009 and SR9011

SR9009 and SR9011 are attractive as performance-enhancing substances due to their REV-ERB agonist effects and thus circadian rhythm modulation activity. Although no pharmaceutical preparations are available yet, illicit use of SR9009 and SR9011 for doping purposes can be anticipated, especially since SR9009 is marketed in illicit products. Therefore, the aim was to identify potential diagnostic metabolites via in vitro metabolic studies to ensure effective (doping) control. The presence of SR9009 could be demonstrated in a black market product purchased over the Internet. Via human liver microsomal metabolic assays, eight metabolites were detected for SR9009 and fourteen metabolites for SR9011 by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Structure elucidation was performed for all metabolites by LC-HRMS product ion scans in both positive and negative ionization mode. Retrospective data analysis was applied to 1511 doping control samples previously analyzed by a full-scan LC-HRMS screening method to verify the presence of SR9009, SR9011 and their metabolites. So far, the presence of neither the parent compound nor the metabolites could be detected in routine urine samples. However, to further discourage use of these potentially harmful compounds, incorporation of SR9009 and SR9011 into screening methods is highly recommended

Structure-based selection of human metabolite binding P4 pocket of DRB1*15:01 and DRB1*15:03, with implications for multiple sclerosis.

Binding of small molecules in the human leukocyte antigen (HLA) peptide-binding groove may result in conformational changes of bound peptide and an altered immune response, but previous studies have not considered a potential role for endogenous metabolites. We performed virtual screening of the complete Human Metabolite Database (HMDB) for docking to the multiple sclerosis (MS) susceptible DRB1*15:01 allele and compared the results to the closely related yet non-susceptible DRB1*15:03 allele; and assessed the potential impact on binding of human myelin basic peptide (MBP). We observed higher energy scores for metabolite binding to DRB1*15:01 than DRB1*15:03. Structural comparison of docked metabolites with DRB1*15:01 and DRB1*15:03 complexed with MBP revealed that PhenylalanineMBP92 allows binding of metabolites in the P4 pocket of DRB1*15:01 but ValineMBP89 abrogates metabolite binding in the P1 pocket. We observed differences in the energy scores for binding of metabolites in the P4 pockets of DRB1*15:01 vs. DRB1*15:03 suggesting stronger binding to DRB1*15:01. Our study confirmed that specific, disease-associated human metabolites bind effectively with the most polymorphic P4 pocket of DRB1*15:01, the primary MS susceptible allele in most populations. Our results suggest that endogenous human metabolites bound in specific pockets of HLA may be immunomodulatory and implicated in autoimmune disease

Circulating Pro- and Anti-Inflammatory Metabolites and Its Potential Role in Rheumatoid Arthritis Pathogenesis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that affects synovial joints, leading to inflammation, joint destruction, loss of function, and disability. Although recent pharmaceutical advances have improved the treatment of RA, patients often inquire about dietary interventions to improve RA symptoms, as they perceive pain and/or swelling after the consumption or avoidance of certain foods. There is evidence that some foods have pro- or anti-inflammatory effects mediated by diet-related metabolites. In addition, recent literature has shown a link between diet-related metabolites and microbiome changes, since the gut microbiome is involved in the metabolism of some dietary ingredients. But diet and the gut microbiome are not the only factors linked to circulating pro- and anti-inflammatory metabolites. Other factors including smoking, associated comorbidities, and therapeutic drugs might also modify the circulating metabolomic profile and play a role in RA pathogenesis. This article summarizes what is known about circulating pro- and anti-inflammatory metabolites in RA. It also emphasizes factors that might be involved in their circulating concentrations and diet-related metabolites with a beneficial effect in RA

Determination of fluphenazine, related phenothiazine drugs and metabolites by combined high-performance liquid chromatography and radioimmunoassay.

Antibodies have been produced in rabbits immunized with a fluphenazine succinate-human serum albumin conjugate. By radioimmunoassay it is possible to quantify fluphenazine (FPZ), related phenothiazine drugs and several of their metabolites at the femtomole level. As little as 370 fmol (160 pg) of FPZ can be detected and up to 0.4 ml of plasma can be added to the incubation mixture (final volume = 1.1 ml). The phenothiazine heterocyclic nucleus is immunodominant and determines the specificity of the antiserum. When a parent drug cross-reacts significantly with antibody, its 7-hydroxide, N-oxide and N-10 side chain altered metabolites can also be determined by the assay. The 8-hydroxide, sulfoxide and 7-hydroxyglucuronide metabolites are not detectable unless present in large amounts. High-performance liquid chromatography was used to separate phenothiazine drugs and metabolites. Since the antiserum has broad specificity, a combined high-performance liquid chromatography and radioimmunoassay procedure permits the identification and quantification of a phenothiazine drug and its serologically reactive metabolites. Patterns of high-performance liquid chromatographic elution and extent of immunologic cross-reaction are characteristic for metabolites relative to the parent drug. This procedure offers distinct advantages in the analysis of this complex family of compounds. FPZ was quantitatively extracted from plasma samples obtained from patients receiving FPZ per os. Although large amounts of serological activity were present in the samples 2 to 6 hr after FPZ ingestion, only 2 to 23% was extractable. The major contributors to the serological activity at times greater than 6 hr were FPZ metabolites. In a preliminary application of the combined techniques, FPZ and a metabolite identified as N-[alpha-(trifluoromethylphenothiazinyl-10)propyl]perazine were quantified in the organic extract of one plasma sample