Hyperpolarized ¹³C NMR Spectroscopy of Urine Samples at Natural Abundance by Quantitative Dissolution Dynamic Nuclear Polarization

Abstract

Hyperpolarized nuclear magnetic resonance (NMR) offers an ensemble of methods that remarkably address the sensitivity issues of conventional NMR. Dissolution Dynamic Nuclear Polarization (d-DNP) provides a unique and general way to detect 13 C NMR signals with a sensitivity enhanced by several orders of magnitude. The expanding application scope of d-DNP now encompasses the analysis of complex mixtures at natural 13 C abundance. However, the application of d-DNP in this area has been limited to metabolite extracts. Here, we report the first d-DNP-enhanced 13 C NMR analysis of a biofluid -urine-at natural abundance, offering unprecedented resolution and sensitivity for this challenging type of sample. We also show that accurate quantitative information on multiple targeted metabolites can be retrieved through a standard addition procedure.

Optimization of heteronuclear ultrafast 2D NMR for the study of complex mixtures hyperpolarized by dynamic nuclear polarization

“…Ribay et al showed that the absolute quantification of hyperpolarized natural abundance metabolites in freeze-dried urine samples was possible relying on a standard addition protocol, while retaining a repeatability of the concentration determination below 10%. 49 In any case, relative quantification of each metabolite across a set of samples remains possible thanks to the good linearity and repeatability of the method, as required for untargeted metabolomics.…”

“…44 It was applied to the analysis of metabolic fluxes on labelled cell and tissue extracts 45–47 and also at natural 13 C abundance on plant extracts 48 or freeze-dried urine. 49 Despite very encouraging results, these studies also pointed out that direct 13 C detection with d-DNP lacked structural information, since peak assignment can be very challenging. Peak overlap is also an issue, due to the potentially very rich quaternary 13 C fingerprint in the 160–220 ppm range.…”

Hyperpolarization-Enhanced NMR Spectroscopy of Unaltered Biofluids Using Photo-CIDNP

“…7 In another study, Dey et al presented an untargeted NMR-based metabolomic workflow based on dissolution DNP thereby enabling hyperpolarized 13 C metabolomics of plant extracts at natural abundance. 8 Despite these and several other extremely encouraging results, 9,10 ments, e.g., long polarization times in combination with the occasional need for expensive additional instrumentation, which render biocompatible and automated high-throughput NMR measurements challenging. Photochemically induced dynamic nuclear polarization (photo-CIDNP), another nuclear spin-selective technique, uses somewhat milder experimental conditions and involves the instantaneous (low-power) LED-or laser light-induced generation of transient radical pairs in the presence of a small amount of a photosensitizer yielding spin-polarized molecular species.…”

Hyperpolarized 1H and 13C NMR Spectroscopy in a Single Experiment for Metabolomics

“…22 This optimization enabled the acquisition of hyperpolarized 13 C spectra of freeze-dried urine at natural abundance, allowing the quantification of key metabolites that were difficult to access with conventional NMR. 23 While hyperpolarized 13 C NMR spectra are highly valuable, hyperpolarized 1 H NMR spectra would also be of high interest for complex mixture analysis since (i) they can provide improved limits of detection and (ii) extensive workflows and databases are available for their analysis. Hyperpolarized 13 C and 1 H NMR spectra are thus expected to be highly complementary.…”

“…Recently, a fine optimization of the d-DNP setting provided a significant sensitivity improvement (about 5 times improvement on quaternary 13 C and 50 times improvement on protonated 13 C) with excellent repeatability (in the range of 3%) . This optimization enabled the acquisition of hyperpolarized 13 C spectra of freeze-dried urine at natural abundance, allowing the quantification of key metabolites that were difficult to access with conventional NMR …”