Insights into Neuronal Cell Metabolism Using NMR Spectroscopy: Uridyl Diphosphate N‐Acetyl‐Glucosamine as a Unique Metabolic Marker

Abstract

Making the switch: Compounds 1 and 2 are used as metabolic markers for NMR detection. When neuronal cells switch to a glycolytic state, an uneven distribution of 13C in the N‐acetyl group results, thus giving a mixture of the metabolites 1 and 2. It is therefore possible to monitor flux through different metabolic pathways, such as glycolysis, the tricarboxylic acid cycle, and the hexosamine biosynthetic pathway, using a single molecule.

NMR Spectroscopy for Metabolomics and Metabolic Profiling

“…To reduce the spectral complexity arising from 13 C enrichment, Cahoreau et al used 2D heteronuclear J-resolved spectroscopy (2D-JRES), which records the scalar couplings in the second dimension instead of chemical shifts, decreasing experiment time compared with other 2D experiments. 75 Other 2D experiments (such as 1 H− 13 C HSQC) can also be used to monitor isotopomer enrichment by taking advantage of the dispersion of a second dimension and the enhanced detection sensitivity of the 1 H nucleus coupled to 13 C. 76 A recent publication by Fan and Lane applied a variety of 2D and 2D-edited experiments on either [U− 13 C]-glucose or [U− 13 C, 15 N]-glutamine enriched cancer cells. 77 The 2D experiments described include 1 The advent of ultrafast 2D NMR experiments, which allow the acquisition of a multidimensional NMR experiment in a single scan, has greatly reduced experiment times.…”

Metabolic Measurements of Nonpermeating Compounds in Live Cells Using Hyperpolarized NMR

“…Nuclear magnetic resonance (NMR), a spectroscopic technique capable of recognizing chemical identities even in complex mixtures, has long been applied to the determination of metabolic products and kinetics. 1–3 More recently, hyperpolarization of nuclear spins has been introduced as a method of enhancing signals and extending the reach of NMR to the characterization of metabolic pathways at an overall metabolite concentration that is reduced by several orders of magnitude compared to conventional non-hyperpolarized experiments. 4–6 Dissolution dynamic nuclear polarization (D-DNP) is a hyperpolarization technique that utilizes the larger electron Zeeman splitting, “transferring” the higher electron spin polarization to nuclear spins.…”

“…Nuclear magnetic resonance (NMR), a spectroscopic technique capable of recognizing chemical identities even in complex mixtures, has long been applied to the determination of metabolic products and kinetics. − More recently, hyperpolarization of nuclear spins has been introduced as a method of enhancing signals and extending the reach of NMR to the characterization of metabolic pathways at an overall metabolite concentration that is reduced by several orders of magnitude compared to conventional nonhyperpolarized experiments. − Dissolution dynamic nuclear polarization (D-DNP) is a hyperpolarization technique that utilizes the larger electron Zeeman splitting, “transferring” the higher electron spin polarization to nuclear spins. This polarization process occurs at a low temperature in a magnetic field, after which hyperpolarized aliquots are dissolved and can be transferred to the NMR detector at room temperature. − In combination with magnetic resonance imaging (MRI), metabolic turnover of substrates such as pyruvate can readily be monitored in vivo. , D-DNP has also emerged as a tool capable of characterizing metabolic flux in vitro, either in perfused organs or in cell cultures. , These in vitro experiments have been used for basic metabolic characterization of cell lines as well as for the development of methods for later use in in vivo MRI …”

“…Hyperpolarization. The hyperpolarized samples consisted of 0.89 M [1-13 C]-pyruvate (Cambridge Isotope Laboratories, Andover, MA), 15 mM tris[8-carboxy-2,2,6,6-tetrakis(2hydroxyethyl)benzo[1,2-d:4,5-d′]bis [1,3]-dithiol-4-yl]methyl free radical sodium salt (OX063; Oxford Instruments, Abingdon, U.K.), and 0.5 mM gadolinium diethylenetriaminepentaacetic acid in a solvent consisting of 3:2 v/v ethylene glycol/D 2 O. Sample aliquots of 5 μL volume were hyperpolarized on 13 C by irradiation of microwaves with 60 mW power and 93.974 GHz frequency, at a temperature of 1.4 K in a HyperSense DNP polarizer (Oxford Instruments).…”