Characterization of Kinetic Isotope Effects and Label Loss in Deuterium-Based Isotopic Labeling Studies.
Characterization of Kinetic Isotope Effects and Label Loss in Deuterium-Based Isotopic Labeling Studies.
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DOI：
10.1021/acschemneuro.0c00711
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发表时间：
2021-01-06
影响因子：
5
通讯作者：
De Feyter HM
中科院分区：
文献类型：
--
作者：
de Graaf RA;Thomas MA;Behar KL;De Feyter HM
氘代谢成像（DMI）是一种新颖的、3D的、基于磁共振（MR）的方法来绘制体内氘化底物的代谢。质子与氘核的替换可能潜在地导致动力学同位素效应（KIEs），其中氘化底物的代谢率由于较重的同位素的存在而降低。基于dmi的绝对代谢率测量需要了解体内KIE的程度和交换反应引起的2H标签损失。本研究采用双底物/双标记策略和1h -解耦13C核磁共振在大鼠胶质瘤细胞和大鼠脑组织代谢物提取物中研究了葡萄糖和醋酸盐在体内的氘KIE和标记丢失。由于广泛的2H-13C和13C-13C标量耦合，其独特的光谱模式允许识别所有可能的代谢产物。以[6,6- 2h2]-葡萄糖为代谢底物时，大鼠脑乳酸、谷氨酸和谷氨酰胺的2H标记损失分别为15.7±2.6、37.9±1.1和41.5±5.2%。对于[2-2H3]-乙酸，谷氨酸和谷氨酰胺的2H标签损失分别为14.4±3.4和13.6±2.2 %，与预测值非常吻合。谷氨酸和谷氨酰胺C4位置的稳态2H标签积累与C2或C3位置的无标签积累形成对比，表明在三羧酸循环的完整回合中，所有2H标签都丢失了。对于两种底物和所有测量的代谢产物，测量的KIE相对较小（4-6%）。这些结果为进一步发展定量DMI研究以生成体内代谢通量图铺平了道路。
Deuterium metabolic imaging (DMI) is a novel, 3D, magnetic resonance (MR)-based method to map metabolism of deuterated substrates in vivo. The replacement of protons with deuterons could potentially lead to kinetic isotope effects (KIEs) in which metabolic rates of deuterated substrates are reduced due to the presence of a heavier isotope. Knowledge of the extent of KIE in vivo, and 2H label losses due to exchange reactions is required for DMI-based measurements of absolute metabolic rates. Here the deuterium KIE and label loss in vivo is investigated for glucose and acetate using a double substrate/double labeling strategy and 1H-decoupled 13C NMR in rat glioma cells and rat brain tissue metabolite extracts. The unique spectral patterns due to extensive 2H-13C and 13C-13C scalar coupling allows the identification of all possible metabolic products. The 2H label loss observed in lactate, glutamate and glutamine of rat brain were 15.7 ± 2.6, 37.9 ± 1.1 and 41.5 ± 5.2 % when using [6,6-2H2]-glucose as the metabolic substrate. For [2-2H3]-acetate, the 2H label losses in glutamate and glutamine were 14.4 ± 3.4 and 13.6 ± 2.2 %, respectively, in excellent agreement with predicted values. Steady-state 2H label accumulation in the C4 position of glutamate and glutamine was contrasted by the absence of label accumulation in the C2 or C3 positions, indicating that during a full turn of the tricarboxylic acid cycle all 2H label is lost. The measured KIE was relatively small (4–6%) for both substrates, and all measured metabolic products. These results pave the way for further development of quantitative DMI studies to generate metabolic flux maps in vivo.
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影响因子：
158.5
作者：
SHULMAN, GI;ROTHMAN, DL;SHULMAN, RG
通讯作者：
SHULMAN, RG
影响因子：
2.2
作者：
Funk, Alexander M.;Anderson, Brian L.;Malloy, Craig R.
通讯作者：
Malloy, Craig R.
影响因子：
15.9
作者：
de Ven, Kim C. C. van;Tack, Cees J.;de Galan, Bastiaan E.
通讯作者：
de Galan, Bastiaan E.
影响因子：
6.3
作者：
Lu, Ming;Zhu, Xiao-Hong;Chen, Wei
通讯作者：
Chen, Wei
影响因子：
2.5
作者：
Wijnen, Jannie P.;Van der Graaf, Marinette;Heerschap, Arend
通讯作者：
Heerschap, Arend