Hyperpolarized ¹³C NMR Metabolomics of Urine Samples at Natural Abundance Applied to Chronic Kidney Disease

NMR is a central tool in the field of metabolomics, thanks to its ability to provide valuable structural and quantitative information with high precision. Most NMR-based metabolomics studies rely on 1D ¹H detection, which is heavily limited by strong peak overlap. ¹³C NMR benefits from a wider spectral dispersion and narrower signal line width but is barely used in metabolomics due to its low sensitivity. Dissolution dynamic nuclear polarization (d-DNP) offers an opportunity to improve ¹³C NMR sensitivity by several orders of magnitude. Here, we show that this emerging hyperpolarized metabolomics approach can provide meaningful information about clinical samples. Achieving sub-mM limits of detection with ¹³C at natural abundance in urine samples was made possible by a meticulous design of the experimental workflow. The analysis of human urine samples from patients with different stages of chronic kidney disease (CKD) was performed using ¹³C d-DNP NMR and benchmarked to conventional ¹H NMR metabolomics at a high magnetic field to explore the complementarity between the two methods. Multivariate analysis of the d-DNP ¹³C NMR dataset provided a statistical model able to distinguish patients with CKD from control patients. Moreover, ¹³C d-DNP NMR spectra highlighted several biomarkers known to be biologically relevant. Some of them were in agreement with those obtained with conventional ¹H NMR, and the results also highlighted the complementarity of biomarker coverage between hyperpolarized and conventional NMR metabolomics. In particular, ¹³C hyperpolarized NMR allowed the annotation of two biomarkers that could not be detected by ¹H NMR because of peak overlap (i.e., guanine and guanidoacetate).