Quantification of rate constants for successive enzymatic reactions with DNP hyperpolarized MR

Abstract

A kinetic model is provided to obtain reaction rate constants in successive enzymatic reactions that are monitored using NMR spectroscopy and hyperpolarized substrates. The model was applied for simulation and analysis of the successive oxidation of choline to betaine aldehyde, and further to betaine, by the enzyme choline oxidase. This enzymatic reaction was investigated under two different sets of conditions: two different choline molecular probes were used, [1,1,2,2-D4 , 1-(13) C]choline chloride and [1,1,2,2-D4 , 2-(13) C]choline chloride, in different MR systems (clinical scanner and high-resolution spectrometer), as well as in different reactors and reaction volumes (4.8 and 0.7 mL). The kinetic analysis according to the model yielded similar results in both set-ups, supporting the robustness of the model. This was achieved despite the complex and negating influences of reaction kinetics and polarization decay, and in the presence of uncontrolled mixing characteristics, which may introduce uncertainties in both effective timing and effective pulses. The ability to quantify rate constants using hyperpolarized MR in the first seconds of consecutive enzyme activity is important for further development of the utilization of dynamic nuclear polarization-MR for biological determinations.

The Sensitivity of Phosphocholine 13C Chemical Shifts to pH

“…We have previously shown that the longitudinal relaxation time of sp3 carbons in Cho can be prolonged by deuteration and that this enables visualization of Cho and its metabolites using hyperpolarized MR [11]. Hyperpolarized 13 C nuclei in Cho metabolites, namely acetylcholine, betaine aldehyde hydrate, and betaine, have been visualized and enabled the quantification of the rates of these metabolic conversions [11][12][13][14][15]. The 13 C label at Cho position 1 (Fig.…”

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

“…The dissolution dynamic nuclear polarization (dDNP) technology, that can enhance the liquid-state nuclear magnetic resonance (NMR) signal of 13 C nuclei by 3-4 orders of magnitude compared to ambient conditions, has improved the abilities of magnetic resonance in applications from analytical NMR, [1,2] reaction monitoring, [3,4] ligand screening [5,6] to real-time measurements of metabolism ex vivo and in vivo in preclinical models [7] and in vivo in human subjects. [8][9][10] In this method, the compound of interest is mixed with a free radical in a solution that vitrifies upon rapid freezing to cryogenic temperatures.…”

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

“…[7] Herein, the signal of the b anomero fG 6P vanishes for t ! [7] Herein, the signal of the b anomero fG 6P vanishes for t !…”

“…[4] To investigate this physiologicallyi mportant sequence of events that takes place in the context of PPP deficiency,a nd without the direct intervention of any enzyme, we have used dissolution dynamic nuclear polarisation (D-DNP). [5][6][7][8] Such as ensitivity enhancement renders the study of chemical reactionso nt ime scaleso fafew seconds possible by making signal averaging unnecessary.T his provides au nique access to potentially rich information,b ya llowingt he simultaneous monitoring of transformation kinetics of severalspecies. D-DNP allows to overcome the low sensitivity of 13 CNMR by providings ignal enhancements up to five orderso fm agnitude for aw ide range of hyperpolarised substrateso rm etabolites.…”

“…15 mmol s À1 to ensure complete conversion of the injected hyperpolarised Glc within af ew seconds;i .e.,w hile the hyperpolarisation is still high enough.O ne unit of enzyme activity converts 1.0 mmol of substrate per minute, sot hat the relevant enzyme masses could be readily calculated from their specific activities (i.e.,u nits mg À1 of enzyme,a sg iven by the supplier). [13] Alternatively, 13 C, 2 H doubly labelled compounds may be used to extend the 13 CT 1 relaxation times of methylene [7,14] or methine groups (as in the case of glucose) [15][16][17][18][19][20][21] Note that deuterationm ay in principle affect kinetics:af act that should be considered for virtually all experiments based on such substrates. The initial goal was to obtain, after af ew seconds, as olution containing only d-PGL, and its two reaction products g-PGL and 6PGA.…”