Urinary Metabolite Quantification Employing 2D NMR Spectroscopy

Abstract

Two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy is a fairly novel method for the quantification of metabolites in biological fluids and tissue extracts. We show in this contribution that, compared to 1D 1H spectra, superior quantification of human urinary metabolites is obtained from 2D 1H-13C heteronuclear single-quantum correlation (HSQC) spectra measured at natural abundance. This was accomplished by the generation of separate calibration curves for the different 2D HSQC signals of each metabolite. Lower limits of detection were in the low to mid micromolar range and were generally the lower the greater the number of methyl groups contained in an analyte. The quantitative 2D NMR data obtained for a selected set of 17 urinary metabolites were compared to those obtained independently by means of gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry of amino acids and hippurate as well as enzymatic and colorimetric measurements of creatinine. As a typical application, 2D-NMR was used for the investigation of urine from patients with inborn errors of metabolism.

Discrimination of steatosis and NASH in mice using nuclear magnetic resonance spectroscopy

“…Using established protocols (Gronwald et al 2008), selected compounds from aqueous and lipophilic extracts were quantified using 2D and 1D spectra, respectively. In the lipophilic extracts, concentrations were calculated relative to the signal of the CHCl 3 fraction contained in CDCl 3 .…”

“…Glucose showed high values only in IC 2 and separated the steatosis group from the other two groups. Quantitative data for the metabolites identified, except for betaine that could not be measured above the lower limit of quantification (LLOQ) (Gronwald et al 2008), and the P-values for the ANOVA tests and all t-statistics are given in Table 2. For taurine significantly increased values were found in the steatosis group compared to both the control and the NASH group.…”

Hyperpolarized NMR of plant and cancer cell extracts at natural abundance

“…Heteronuclear ( 1 H→ 13 C) correlation spectroscopy can in principle offer a satisfactory dispersion. 5,6 So far, 13 C NMR is not routinely used in metabolomic studies, but hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) combined with cross polarization (CP) could boost its popularity in this field.Hyperpolarization techniques are capable of generating spin polarization levels that are about 50 000 times above Boltzmann equilibrium at room temperature. 7-9 Once prepared and transferred to a solution-state NMR spectrometer, the resulting magnetization can be exploited within a limited time-span that depends on the longitudinal relaxation time T 1 of the hyperpolarized nuclei.…”

2D-Cosy NMR Spectroscopy as a Quantitative Tool in Biological Matrix: Application to Cyclodextrins

“…However, the direct quantitative properties observed in 1 H-NMR cannot be extrapolated to 2D-NMR and especially in 2D-COSY. Indeed, cross-peak intensities are influenced by other factors than concentration such as relaxation times, mixing times, evolution times, uneven magnetization transfers, coupling constants (J)… (10)(11)(12). Moreover, the influence of the biological matrix has to be taken into account and the presence of a huge amount of water requires the use of adapted pulse sequences including a pre-saturation step in order to drastically reduce the water signal which can greatly impact the quality of the spectra.…”