Balzan Riccardo, Fernandes Laetitia, Comment Arnaud, Pidial Laetitia, Tavitian Bertrand, Vasos Paul R
LCBPT - UMR8601, Institut de Chimie, Université Paris Descartes;
LCBPT - UMR8601, Institut de Chimie, Université Paris Descartes.
J Vis Exp. 2016 Feb 23(108):53548. doi: 10.3791/53548.
The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.
基于核磁共振（NMR）的研究的主要局限性在于灵敏度较低。这就需要较长的采集时间，从而阻碍了对代谢转化进行实时NMR测量。通过溶解动态核极化（dissolution DNP）实现的超极化克服了部分灵敏度问题，这得益于电子到原子核的自旋极化转移所产生的大量非平衡核磁化。所获得的高NMR信号可用于实时监测化学反应。超极化NMR的缺点在于信号采集的可用时间窗口有限，通常约为核自旋纵向弛豫时间常数T1的量级，或者在有利情况下，约为与耦合核单重态相关的弛豫时间常数TLLS的量级。内源性分子的细胞摄取和代谢率可为肿瘤发展和药物反应提供重要信息。此前众多超极化NMR研究已证明丙酮酸作为代谢底物用于监测体内酶活性的相关性。这项工作详细描述了通过超极化NMR研究酶促反应，特别是在存在乳酸脱氢酶（LDH）的情况下丙酮酸向乳酸的转化率所需的实验装置和方法。
