Rates of Chemical Reactions Embedded in a Metabolic Network by Dissolution Dynamic Nuclear Polarisation NMR

Abstract

The isomerisation of 6-phosphogluconolactones and their hydrolyses into 6-phosphogluconic acid form a non enzymatic side cycle of the pentose-phosphate pathway (PPP) in cells. Dissolution dynamic nuclear polarisation can be used for determining the kinetic rates of the involved transformations in real time. It is found that the hydrolysis of both lactones is significantly slower than the isomerisation process, thereby shedding new light onto this subtle chemical process.

The Effect of Gadolinium Doping in [13C6,2H7]Glucose Formulations on 13C Dynamic Nuclear Polarization at 3.35 T

“…Chemicals [1][2][3][4][5][6][7][8][9][10][11][12][13] For the [ 13 C 6 , 2 H 7 ]glucose formulations, three stock solutions were prepared by dissolving OX063 to a concentration of 20 mM in D 2 O with and without addition of 0.5 M solution of Gd-DOTA to the following Gd-DOTA concentrations: 0 mM, 2 mM and 6 mM.…”

“…dDNP studies with hyperpolarized [ 13 C 6 , 2 H 7 ]glucose have given insight into enzyme kinetics, [11,12] the glycolytic pathways in living cells of E. coli, [13,14] yeast, [15][16][17][18][19] and cancer, [19][20][21][22] body distribution, [23] as well as reporting on glycolysis in vivo in tumors [7] and in the mouse brain. dDNP studies with hyperpolarized [ 13 C 6 , 2 H 7 ]glucose have given insight into enzyme kinetics, [11,12] the glycolytic pathways in living cells of E. coli, [13,14] yeast, [15][16][17][18][19] and cancer, [19][20][21][22] body distribution, [23] as well as reporting on glycolysis in vivo in tumors [7] and in the mouse brain.…”

“…Glucose is an important compound for metabolic studies. dDNP studies with hyperpolarized [ 13 C 6 , 2 H 7 ]glucose have given insight into enzyme kinetics, [11,12] the glycolytic pathways in living cells of E. coli, [13,14] yeast, [15][16][17][18][19] and cancer, [19][20][21][22] body distribution, [23] as well as reporting on glycolysis in vivo in tumors [7] and in the mouse brain. [24] Although the hyperpolarized lifetime of [ 13 C 6 , 2 H 7 ]glucose (T 1 up to 20 s) is much shorter than the gold standard of dDNP metabolism studies, namely [1-13 C]pyruvate (T 1 � 60 s), glucose can be injected in vivo at higher concentrations and can be labeled with more 13 C sites per molecule, such that it could potentially represent a more versatile probe than [1-13 C]pyruvate.…”

Transport of hyperpolarized samples in dissolution-DNP experiments

“…Dissolution dynamic nuclear polarization (D-DNP) is a method to enhance signal amplitudes in solution-state nuclear magnetic resonance (NMR) experiments. 1 Spectacular signal enhancements above four orders of magnitude 2 can be attained, rendering novel applications possible in fields ranging from the monitoring of chemical reactions, 3,4 metabolomics, 5,6 studies of interactions 7 or real-time biomolecular NMR. 8 The D-DNP method is based on spin hyperpolarization of a sample at temperatures close to 1 K by employing slightly off-resonance microwave saturation of the Electron Paramagnetic Resonance (EPR) spectra of paramagnetic polarization agents, such as stable paramagnetic molecules, [9][10][11] radical mixtures, 12 UV-induced radical centers 13 or paramagnetic microporous matrices.…”

Observation of glucose-6-phosphate anomeric exchange in real-time using dDNP hyperpolarised NMR

“…The anomerization rate of glucose (Glc) intrigued researchers back at the turn of the 20 th century. 1 Numerous studies into both Glc and glucose-6-phosphate (G6P) anomerization have been performed since, [2][3][4][5][6][7][8][9][10][11][12][13][14][15] due to their importance for the activities of anomer specic enzymes such as G6P dehydrogenase (which converts D-G6P to 6-phospho-D-glucono-1,5-lactone and is specic to b-G6P 2,16 ), phosphoglucomutase (which converts G6P to G1P, and vice versa, and is specic to a-G6P 2 ), and G6P isomerase (which interconverts G6P and fructose-6phosphate (F6P) and is specic to a-G6P 2 and a-F6P 17 ). G6P is the product of Glc phosphorylation which is carried out by hexokinases including glucokinase.…”