Is Current Practice Adhering to Guidelines Proposed for Metabolite Identification in LC-MS Untargeted Metabolomics? A Meta-Analysis of the Literature.

Metabolite identification remains a bottleneck and a still unregulated area in untargeted LC-MS metabolomics. The metabolomics research community and, in particular, the metabolomics standards initiative (MSI) proposed minimum reporting standards for metabolomics including those for reporting metabolite identification as long ago as 2007. Initially, four levels were proposed ranging from level 1 (unambiguously identified analyte) to level 4 (unidentified analyte). This scheme was expanded in 2014, by independent research groups, to give five levels of confidence. Both schemes provided guidance to the researcher and described the logical steps that had to be made to reach a confident reporting level. These guidelines have been presented and discussed extensively, becoming well-known to authors, editors, and reviewers for academic publications. Despite continuous promotion within the metabolomics community, the application of such guidelines is questionable. The scope of this meta-analysis was to systematically review the current LC-MS-based literature and effectively determine the proportion of papers following the proposed guidelines. Also, within the scope of this meta-analysis was the measurement of the actual identification levels reported in the literature, that is to find how many of the published papers really reached full metabolite identification (level 1) and how many papers did not reach this level.

Omega-6 oxylipins generated by soluble epoxide hydrolase are associated with knee osteoarthritis.

Omega-6 FAs are inflammatory mediators that are increased in joints with osteoarthritis (OA), but their association with OA progression is not yet well defined. To investigate the relationship between omega-6 FAs and knee OA, we measured with LC-MS the levels of 22 omega-6 lipids (arachidonic acid, linoleic acid, and 20 oxylipins) in synovial fluid (SF) from 112 knees of 102 individuals (58 with knee OA; 44 controls). We hypothesized that oxylipin metabolites would increase in OA knee SF and with radiographically progressive disease. We validated results by comparing samples from affected and unaffected knees in 10 individuals with unilateral OA. In adjusted analysis, SF levels of three omega-6 oxylipins [prostaglandin D2, 11,12-dihydroxyeicosatrienoic acid (DHET), and 14,15-DHET] were associated with OA. Of these, 11,12-DHET and 14,15-DHET were higher in affected versus unaffected knees of people with unilateral disease (P < 0.014 and P < 0.003, respectively). Levels of these and 8,9-DHET were also associated with radiographic progression over 3.3 years in 87 individuals. Circulating levels of all three were associated with gene variants at the soluble epoxide hydrolase enzyme. Lipidomic profiling in SF identified an additional inflammatory pathway associated with knee OA and radiographic progression.

Untargeted Metabolomics Pilot Study Using UHPLC-qTOF MS Profile in Sows' Urine Reveals Metabolites of Bladder Inflammation.

Urinary tract infections (UTI) of sows (characterized by ascending infections of the urinary bladder (cyst), ureters, and renal pelvis), are major health issues with a significant economic impact to the swine industry. The current detection of UTI incidents lacks sensitivity; thus, UTIs remain largely under-diagnosed. The value of metabolomics in unraveling the mechanisms of sow UTI has not yet been established. This study aims to investigate the urine metabolome of sows for UTI biomarkers. Urine samples were collected from 58 culled sows from a farrow-to-finish herd in Greece. Urine metabolomic profiles in 31 healthy controls and in 27 inflammatory ones were evaluated. UHPLC-qTOF MS/MS was applied for the analysis with a combination of multivariate and univariate statistical analysis. Eighteen potential markers were found. The changes in several urine metabolites classes (nucleosides, indoles, isoflavones, and dipeptides), as well as amino-acids allowed for an adequate discrimination between the study groups. Identified metabolites were involved in purine metabolism; phenylalanine; tyrosine and tryptophan biosynthesis; and phenylalanine metabolism. Through ROC analysis it was shown that the 18 identified metabolite biomarkers exhibited good predictive accuracy. In summary, our study provided new information on the potential targets for predicting early and accurate diagnosis of UTI. Further, this information also sheds light on how it could be applied in live animals.

Lipidomic UPLC-MS/MS Profiles of Normal-Appearing White Matter Differentiate Primary and Secondary Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a neurodegenerative inflammatory disease where an autoimmune response to components of the central nervous system leads to a loss of myelin and subsequent neurological deterioration. People with MS can develop primary or secondary progressive disease (PPMS, SPMS) and differentiation of the specific differences in the pathogenesis of these two courses, at the molecular level, is currently unclear. Recently, lipidomics studies using human biofluids, mainly plasma and cerebrospinal fluid, have highlighted a possible role for lipids in the initiation and progression of MS. However, there is a lack of lipidomics studies in MS on CNS tissues, such as normal-appearing white matter (NAWM), where local inflammation initially occurs. Herein, we developed an untargeted reverse phase ultra-performance liquid chromatography time of flight tandem mass spectrometry (RP-UPLC-TOF MSE)-based workflow, in combination with multivariate and univariate statistical analysis, to assess significant differences in lipid profiles in brain NAWM from post-mortem cases of PPMS, SPMS and controls. Groups of eight control, nine PPMS and seven SPMS NAWM samples were used. Correlation analysis of the identified lipids by RP-UPLC-TOF MSE was undertaken to remove those lipids that correlated with age, gender and post-mortem interval as confounding factors. We demonstrate that there is a significantly altered lipid profile of control cases compared with MS cases and that progressive disease, PPMS and SPMS, can be differentiated on the basis of the lipidome of NAWM with good sensitivity, specificity and prediction accuracy based on receiver operating characteristic (ROC) curve analysis. Metabolic pathway analysis revealed that the most altered lipid pathways between PPMS and SPMS were glycerophospholipid metabolism, glycerophosphatidyl inositol (GPI) anchor synthesis and linoleic acid metabolism. Further understanding of the impact of these lipid alterations described herein associated with progression will provide an increased understanding of the mechanisms underpinning progression and highlight possible new therapeutic targets.

Targeting the D Series Resolvin Receptor System for the Treatment of Osteoarthritis Pain.

OBJECTIVE: Pain is a major symptom of osteoarthritis (OA); currently available analgesics either do not provide adequate pain relief or are associated with serious side effects. The aim of this study was to investigate the therapeutic potential of targeting the resolvin receptor system to modify OA pain and pathology. METHODS: Gene expression of 2 resolvin receptors (ALX and ChemR23) was quantified in synovium and medial tibial plateau specimens obtained from patients with OA at the time of joint replacement surgery. Two models of OA joint pain were used for the mechanistic studies. Gene expression in the joint and central nervous system was quantified. The effects of exogenous administration of the D series resolvin precursor 17(R)-hydroxy-docosahexaenoic acid (17[R]-HDoHE) on pain behavior, joint pathology, spinal microglia, and astroglyosis were quantified. Plasma levels of relevant lipids, resolvin D2, 17(R)-HDoHE, and arachidonic acid, were determined in rats, using liquid chromatography tandem mass spectrometry. RESULTS: There was a positive correlation between resolvin receptor and interleukin-6 (IL-6) expression in human OA synovial and medial tibial plateau tissue. In rats, synovial expression of ALX was positively correlated with expression of IL-1ß, tumor necrosis factor, and cyclooxygenase 2. Treatment with 17(R)-HDoHE reversed established pain behavior (but not joint pathology) in 2 models of OA pain. This was associated with a significant elevation in the plasma levels of resolvin D2 and a significant reduction in astrogliosis in the spinal cord in the monosodium iodoacetate-induced OA rat model. CONCLUSION: Our preclinical data demonstrate the robust analgesic effects of activation of the D series resolvin pathways in 2 different animal models of OA. Our data support a predominant central mechanism of action in clinically relevant models of OA pain.

Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA:RNA interactions.

DNA/RNA chromatography presents a versatile platform for the analysis of nucleic acids. Although the mechanism of separation of double stranded (ds) DNA fragments is largely understood, the mechanism by which RNA is separated appears more complicated. To further understand the separation mechanisms of RNA using ion pair reverse phase liquid chromatography, we have analysed a number of dsRNA and single stranded (ss) RNA fragments. The high-resolution separation of dsRNA was observed, in a similar manner to dsDNA under non-denaturing conditions. Moreover, the high-resolution separation of ssRNA was observed at high temperatures (75 degrees C) in contrast to ssDNA. It is proposed that the presence of duplex regions/secondary structures within the RNA remain at such temperatures, resulting in high-resolution RNA separations. The retention time of the nucleic acids reflects the relative hydrophobicity, through contributions of the nucleic sequence and the degree of secondary structure present. In addition, the analysis of RNA using such approaches was extended to enable the discrimination of bacterial 16S rRNA fragments and as an aid to conformational analysis of RNA. RNA:RNA interactions of the human telomerase RNA component (hTR) were analysed in conjunction with the incorporation of Mg2+ during chromatography. This novel chromatographic procedure permits analysis of the temperature dependent formation of dimeric RNA species.