Identification of 4-deoxythreonic acid present in human urine using HPLC and NMR techniques

Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
Journal of pharmaceutical and biomedical analysis (Impact Factor: 2.98). 07/2009; 50(5):878-85. DOI: 10.1016/j.jpba.2009.06.007
Source: PubMed


The 1H NMR spectrum of urine exhibits a large number of detectable and quantifiable metabolites and hence urine metabolite profiling is potentially useful for the study of systems biology and the discovery of biomarkers for drug development or clinical applications. While a number of metabolites (50-100) are readily detectable in urine by NMR, a much larger number is potentially available if lower concentration species can be detected unambiguously. Lower concentration metabolites are thought to be more specific to certain disease states and thus it is important to detect these metabolites with certainty. We report the identification of 4-deoxythreonic acid, a relatively low concentration endogenous metabolite that has not been previously identified in the 1H NMR spectrum of human urine. The use of HPLC and NMR spectroscopy facilitated the unequivocal and non-invasive identification of the molecule in urine which is complicated by extensive peak overlap and multiple, similar resonances from other metabolites such as 3-hydroxybutanoic acid. High-resolution detection and good sensitivity were achieved by the combination of multiple chromatographic fraction collection, sample pre-concentration, and the use of a cryogenically cooled NMR probe.

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Available from: Emmanuel Appiah-Amponsah, Feb 19, 2015
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    • "The use of chromatographic methods to simplify sample matrices by isolating metabolites of interest prior to NMR analysis has high utility for a variety of biological investigations [24,25,26,27,28,29]. This approach has also benefited from the use of sample pre-concentration techniques such as solid phase extraction (SPE) and column trapping to extend NMR detection limits significantly and thus circumvent the issue of sample dilution attributed to solvent mixing in the chromatographic step [26]. "
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    ABSTRACT: The complementary use of liquid chromatography (LC) and nuclear magnetic resonance (NMR) has shown high utility in a variety of fields. While the significant benefit of spectral simplification can be achieved for the analysis of complex samples, other limitations remain. For example, 1H LC-NMR suffers from pH dependent chemical shift variations, especially during urine analysis, owing to the high physiological variation of urine pH. Additionally, large solvent signals from the mobile phase in LC can obscure lower intensity signals and severely limit the number of metabolites detected. These limitations, along with sample dilution, hinder the ability to make reliable chemical shift assignments. Recently, stable isotopic labeling has been used to detect quantitatively specific classes of metabolites of interest in biofluids. Here we present a strategy that explores the combined use of two-dimensional hydrophilic interaction chromatography (HILIC) and isotope tagged NMR for the unambiguous identification of carboxyl containing metabolites present in human urine. The ability to separate structurally related compounds chromatographically, in off-line mode, followed by detection using 1H-15N 2D HSQC (two-dimensional heteronuclear single quantum coherence) spectroscopy, resulted in the assignment of low concentration carboxyl-containing metabolites from a library of isotope labeled compounds. The quantitative nature of this strategy is also demonstrated.
    09/2013; 3(3):575-591. DOI:10.3390/metabo3030575
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    • "A one-dimensional 1H selective TOCSY experiment (See additional file 1, Figure S1) observed three related 1H resonances in the same molecule. Further two-dimensional experiments [See additional file 1, Figure S2] allowed the full 1H and 13C characterization [See additional file 1, Table S1] of the two diastereoisomers of 2,3-dihydroxybutanoic acid [See additional file 1, Figure S3]: 4-deoxy-erythronic acid and 4-deoxy-threonic acid, both of which have previously been identified in human urine [41]. "
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    ABSTRACT: The 'exposome' represents the accumulation of all environmental exposures across a lifetime. Top-down strategies are required to assess something this comprehensive, and could transform our understanding of how environmental factors affect human health. Metabolic profiling (metabonomics/metabolomics) defines an individual's metabolic phenotype, which is influenced by genotype, diet, lifestyle, health and xenobiotic exposure, and could also reveal intermediate biomarkers for disease risk that reflect adaptive response to exposure. We investigated changes in metabolism in volunteers living near a point source of environmental pollution: a closed zinc smelter with associated elevated levels of environmental cadmium. High-resolution ¹H NMR spectroscopy (metabonomics) was used to acquire urinary metabolic profiles from 178 human volunteers. The spectral data were subjected to multivariate and univariate analysis to identify metabolites that were correlated with lifestyle or biological factors. Urinary levels of 8-oxo-deoxyguanosine were also measured, using mass spectrometry, as a marker of systemic oxidative stress. Six urinary metabolites, either associated with mitochondrial metabolism (citrate, 3-hydroxyisovalerate, 4-deoxy-erythronic acid) or one-carbon metabolism (dimethylglycine, creatinine, creatine), were associated with cadmium exposure. In particular, citrate levels retained a significant correlation to urinary cadmium and smoking status after controlling for age and sex. Oxidative stress (as determined by urinary 8-oxo-deoxyguanosine levels) was elevated in individuals with high cadmium exposure, supporting the hypothesis that heavy metal accumulation was causing mitochondrial dysfunction. This study shows evidence that an NMR-based metabolic profiling study in an uncontrolled human population is capable of identifying intermediate biomarkers of response to toxicants at true environmental concentrations, paving the way for exposome research.
    BMC Medicine 06/2012; 10(1):61. DOI:10.1186/1741-7015-10-61 · 7.25 Impact Factor
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    ABSTRACT: In this work, untargeted NMR metabonomics was employed to evaluate the effects of pregnancy on the metabolite composition of maternal urine, thus establishing a control excretory trajectory for healthy pregnancies. Urine was collected for independent groups of healthy non-pregnant and pregnant women (in 1(st), 2(nd), 3(rd) trimesters) and multivariate analysis performed on the corresponding NMR spectra. Models were validated through Monte Carlo Cross Validation and permutation tests and metabolite correlations measured through Statistical Total Correlation Spectroscopy. The levels of 21 metabolites were found to change significantly throughout pregnancy, with variations observed for the first time to our knowledge for choline, creatinine, 4-deoxyerythronic acid, 4-deoxythreonic acid, furoylglycine, guanidoacetate, 3-hydroxybutyrate and lactate. Results confirmed increased aminoaciduria across pregnancy and suggested a) a particular involvement of isoleucine and threonine in lipid oxidation/ketone body synthesis, b) a relation of excreted choline, taurine and guanidoacetate to methionine metabolism and urea cycle regulation and c) a possible relationship of furoylglycine and creatinine to pregnancy, based on a tandem study of non-fasting confounding effects. Results demonstrate the usefulness of untargeted metabonomics in finding biomarker metabolic signatures for healthy pregnancies, against which disease-related deviations may be confronted in future studies, as a base for improved diagnostics and prediction.
    Journal of Proteome Research 12/2012; 12(2). DOI:10.1021/pr301022e · 4.25 Impact Factor
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