31 research outputs found
Mass Spectrometric Based Approaches in Urine Metabolomics and Biomarker Discovery
Urine metabolomics has recently emerged as a prominent field for the discovery of non-invasive biomarkers that can detect subtle metabolic discrepancies in response to a specific disease or therapeutic intervention. Urine, compared to other biofluids, is characterized by its ease of collection, richness in metabolites and its ability to reflect imbalances of all biochemical pathways within the body. Following urine collection for metabolomic analysis, samples must be immediately frozen to quench any biogenic and/or non-biogenic chemical reactions. According to the aim of the experiment; sample preparation can vary from simple procedures such as filtration to more specific extraction protocols such as liquid-liquid extraction. Due to the lack of comprehensive studies on urine metabolome stability, higher storage temperatures (i.e. 4 °C) and repetitive freeze-thaw cycles should be avoided. To date, among all analytical techniques, mass spectrometry (MS) provides the best sensitivity, selectivity and identification capabilities to analyze the majority of the metabolite composition in the urine. Combined with the qualitative and quantitative capabilities of MS, and due to the continuous improvements in its related technologies (i.e. ultra high-performance liquid chromatography [UPLC] and hydrophilic interaction liquid chromatography [HILIC]), liquid chromatography (LC)-MS is unequivocally the most utilized and the most informative analytical tool employed in urine metabolomics. Furthermore, differential isotope tagging techniques has provided a solution to ion suppression from urine matrix thus allowing for quantitative analysis. In addition to LC-MS, other MS-based technologies have been utilized in urine metabolomics. These include direct injection (infusion)-MS, capillary electrophoresis-MS and gas chromatography-MS. In this article, the current progresses of different MS-based techniques in exploring the urine metabolome as well as the recent findings in providing potentially diagnostic urinary biomarkers are discussed
Development of a validated LC- MS/MS method for the quantification of 19 endogenous asthma/COPD potential urinary biomarkers
Saskatchewan Health Research Foundation; AllerGen NCE Inc; Canada Foundation for Innovation; College of Pharmacy and Nutrition, University of SaskatchewanPeer ReviewedObstructive airways inflammatory diseases sometimes show overlapping symptoms that hinder their early and correct diagnosis. Current clinical tests are tedious and are of inadequate specificity in special population such as the elderly and children. Therefore, we are developing tandem mass spectrometric (MS/MS) methods for targeted analysis of urine biomarkers. Recently, proton-nuclear magnetic resonance (1H-NMR) analysis proposed 50 urinary metabolites as potential diagnostic biomarkers among asthma and chronic obstructive pulmonary disease (COPD) patients. Metabolites are divided into 3 groups based on chemical nature. For group 1 (amines and phenols, 19 urinary metabolites), we developed and validated a high performance liquid chromatographic (HPLC)-MS/MS method using differential isotope labeling with dansyl chloride. Method development included the optimization of the derivatization reaction, the MS/MS conditions, and the chromatographic separation. Linearity varied from 2 to 4800 ng/mL and the use of 13C2-labeled derivatives allowed for the correction of matrix effects as well as the unambiguous confirmation of the identity of each metabolite in the presence of interfering isomers in urine. Despite the challenges associated with method validation, the method was fully validated as per the food and drug administration (FDA) and the European medicines agency (EMA) recommendations. Validation criteria included linearity, precision, accuracy, dilution integrity, selectivity, carryover, and stability. Challenges in selectivity experiments included the isotopic contributions of the analyte towards its internal standard (IS), that was addressed via the optimization of the IS concentration. In addition, incurred sample analysis was performed to ensure that results from patient samples are accurate and reliable. The method was robust and reproducible and is currently being applied in a cohort of asthma and COPD patient urine samples for biomarker discovery purposes
Comparison of accuracy and precision between multipoint calibration, single point calibration and relative quantification for targeted metabolomic analysis
Natural Sciences and Engineering Research Council of Canada (NSERC); Western Economic Diversification CanadaPeer ReviewedTargeted metabolomics requires accurate and precise quantification of candidate biomarkers, often through tandem mass spectrometric (MS/MS) analysis. Differential isotope labeling (DIL) improves mass spectrometric (MS) analysis in metabolomics by derivatizing metabolites with two isotopic forms of the same reagent. Despite its advantages, DIL-liquid chromatographic (LC)-MS/MS can result in substantial increase in workload when fully validated quantitative methods are required. To decrease the workload, we hypothesized that single point calibration or relative quantification could be used as alternative methods. Either approach will result in significant saving in resources and time. To test our hypothesis, six urinary metabolites were selected as model compounds. Urine samples were analyzed using a fully-validated multipoint dansyl chloride-DIL-LC-MS/MS method. Samples were reprocessed using single point calibration and relative quantification modes. Our results demonstrated that the performance of single point calibration or relative quantification was inferior, for some metabolites, to multipoint calibration. The lower limit of quantification failed in the quantification of ethanolamine in most of participant samples using single point calibration. In addition, its precision was not acceptable in one participant during serine and ethanolamine quantification. On the other hand, relative quantification resulted in the least accurate data. In fact, none of the data generated from relative quantification for serine was comparable to that obtained from multipoint calibration. Finally, while single point calibration showed an overall acceptable performance for the majority of the model compounds, we cannot extrapolate the findings to other metabolites within the same analytical run. Analysts are advised to assess accuracy and precision for each metabolite in which single point calibration is the intended quantification mean
Respiratory syncytial virus replication induces Indoleamine 2,3-dioxygenase (IDO) activation in human dendritic cells
Urine metabolomic profiling of children with respiratory tract infections in the emergency department: a pilot study
Comparative analysis of creatinine and osmolality as urine normalization strategies in targeted metabolomics for the differential diagnosis of asthma and COPD
Peer ReviewedIntroduction Urine is an ideal matrix for metabolomics investigation due to its non-invasive nature of collection and its rich metabolite content. Despite the advancements in mass spectrometry and 1H-NMR platforms in urine metabolomics, the statistical analysis of the generated data is challenged with the need to adjust for the hydration status of the person. Normalization to creatinine or osmolality values are the most adopted strategies, however, each technique has its challenges that can hinder its wide application.
Objective Assessment of whether the statistical model established using a targeted urine metabolomics dataset for the differential diagnosis of asthma and chronic obstructive pulmonary disease (COPD) would be improved by normalization to osmolality instead of creatinine.
Methods A metabolomics dataset from 51 patient urine samples previously analyzed using two liquid chromatography-tandem mass spectrometry methods was used. The data was normalized to creatinine and osmolality. The statistical analysis was achieved using partial least square discriminant analysis and models of separation were generated and compared.
Results Creatinine and osmolality values in asthma and COPD patients were strongly correlated. Using the same metabolites, we found that normalization to osmolality did not significantly change the results. The metabolites of importance in separation remained the same for both methods. The statistical strength of the creatinine model was somewhat better than the osmolality normalized model (R2Q2=0.919, 0.705 vs R2Q2 =0.929, 0.671).
Conclusion Our findings suggest that targeted urine metabolomics data can be normalized to creatinine or osmolality with no significant impact on the diagnostic accuracy of the model
Identifying hypoxia in a newborn piglet model using urinary NMR metabolomic profiling.
Establishing the severity of hypoxic insult during the delivery of a neonate is key step in the determining the type of therapy administered. While successful therapy is present, current methods for assessing hypoxic injuries in the neonate are limited. Urine Nuclear Magnetic Resonance (NMR) metabolomics allows for the rapid non-invasive assessment of a multitude breakdown products of physiological processes. In a newborn piglet model of hypoxia, we used NMR spectroscopy to determine the levels of metabolites in urine samples, which were correlated with physiological measurements. Using PLS-DA analysis, we identified 13 urinary metabolites that differentiated hypoxic versus nonhypoxic animals (1-methylnicotinamide, 2-oxoglutarate, alanine, asparagine, betaine, citrate, creatine, fumarate, hippurate, lactate, N-acetylglycine, N-carbamoyl-β-alanine, and valine). Using this metabolomic profile, we then were able to blindly identify hypoxic animals correctly 84% of the time compared to nonhypoxic controls. This was better than using physiologic measures alone. Metabolomic profiling of urine has potential for identifying neonates that have undergone episodes of hypoxia
A Pediatric Case of Diffuse Alveolar Hemorrhage Secondary to Poststreptococcal Glomerulonephritis
This report summarizes a case of a 4-year-old girl with poststreptococcal glomerulonephritis and diffuse alveolar hemorrhage, an atypical presentation in this age group and type of vasculitic disease. We propose that her rapid improvement in clinical status was due to her treatment, continuous renal replacement therapy (CRRT). This mechanism would have impacted recovery by removing factors such as endothelial microparticles, superantigens, and immune complexes that have been postulated as the pulmonary-renal link. This may be an interesting avenue of exploration going forward given the lack of evidence in treating such conditions and emergence of CRRT