Rapid probing of glucose influx into cancer cell metabolism: using adjuvant and a pH-dependent collection of central metabolites to improve in-cell D-DNP NMR

Abstract

Changes to metabolism are a hallmark of many diseases. Disease metabolism under physiological conditions can be probed in real time with in-cell NMR assays. Here, we pursued a systematic approach...

Visualizing Metabolism in Biotechnologically Important Yeasts with dDNP NMR Reveals Evolutionary Strategies and Glycolytic Logic

“…The samples for conventional NMR were rapidly transferred to an 800 MHz Bruker Avance III HD instrument equipped with a 5 mm TCI cryoprobe and thermally equilibrated to 30 °C. A time series of 256 13 C NMR spectra (30°flip angle and inverse gated decoupling of 1 H) was acquired by accumulating 16 transients with an interscan relaxation delay of 1.5 s by sampling the FID for 0.68 s (16384 complex data points). Time series of 13 C NMR spectra were implemented as pseudo-2D experiments acquiring the 256 time points with a time resolution of 0.58 min.…”

“…A time series of 256 13 C NMR spectra (30°flip angle and inverse gated decoupling of 1 H) was acquired by accumulating 16 transients with an interscan relaxation delay of 1.5 s by sampling the FID for 0.68 s (16384 complex data points). Time series of 13 C NMR spectra were implemented as pseudo-2D experiments acquiring the 256 time points with a time resolution of 0.58 min. All spectra were acquired using Topspin 3.5 pl6.…”

“…The hyperpolarization of [U-13 C, U-2 H]glucose, [1-13 C, 1-2 H]glucose, and [2- 13 C]fructose was conducted by solid state dynamic nuclear polarization as previously described. 7,8,11 Briefly, solid state dynamic nuclear polarization was conducted using trityl radical OX063 (27 mM; Oxford Instruments, Abingdon, U.K.) and gadoteridol (1.5 mM; Bracco Imaging, Italy) mixed with the labeled carbohydrate and ultrapure water.…”

“…These studies can be conducted on a time scale of seconds to minute, as the enhanced spin polarization fades with the nuclear T 1 relaxation constant, which is on the order of ∼13−14 s for deuterated 13 C sites in monosaccharides. Hence, hyperpolarized NMR using hexose substrates is most useful with glucose that is 13 C and 2 H enriched at the same sites or with fructose that is 13 C at the quaternary C2 site. Compound identifications from intracellular signals were conducted with the help of fully assigned reference compounds of the central metabolites.…”

“…Hyperpolarized NMR experiments provide pre-steady-state data that is useful for gaining insight into pathway usage, kinetic bottlenecks, and regulation. These experiments were subsequently compared to conventional NMR experiments probing the conversion of [U- 13 C]glucose on a time scale of minutes rather than seconds. Figure 4 tracks the conversion of [U- 13 C]glucose during a time scale of 120 min.…”