基于氘的同位素标记研究中动力学同位素效应和标记丢失的表征,ACS Chemical Neuroscience

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基于氘的同位素标记研究中动力学同位素效应和标记丢失的表征
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2020-12-15 , DOI: 10.1021/acschemneuro.0c00711
Robin A de Graaf ₁ , Monique A Thomas ₁ , Kevin L Behar ₂ , Henk M De Feyter ₁

Affiliation

氘代谢成像 (DMI) 是一种基于磁共振 (MR) 的新型 3D 方法，用于绘制体内氘化底物的代谢图。用氘核替换质子可能会导致动力学同位素效应 (KIE)，其中由于存在较重的同位素，氘化底物的代谢率会降低。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² H 标记损失。此处使用双底物/双标记策略和¹ H-去偶联¹³研究葡萄糖和乙酸的氘 KIE 和体内标记丢失大鼠神经胶质瘤细胞和大鼠脑组织代谢物提取物中的 C NMR。由于广泛的² H– ¹³ C 和¹³ C– ¹³ C 标量耦合而产生的独特光谱模式允许识别所有可能的代谢产物。^{当使用 [6,6- 2} H ₂ ]-葡萄糖作为代谢底物时，在大鼠脑的乳酸、谷氨酸和谷氨酰胺中观察到的² H 标记损失分别为 15.7 ± 2.6、37.9 ± 1.1 和 41.5 ± 5.2% 。对于 [2- ² H ₃ ]-乙酸盐，谷氨酸和谷氨酰胺的² H 标记损失分别为 14.4 ± 3.4 和 13.6 ± 2.2%，与预测值非常吻合。稳态²H 标签在谷氨酸和谷氨酰胺 C4 位置的积累与 C2 或 C3 位置没有标签积累形成鲜明对比，这表明在三羧酸循环的整个循环过程中，所有² H 标签都丢失了。对于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。

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Characterization of Kinetic Isotope Effects and Label Loss in Deuterium-Based Isotopic Labeling Studies

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 loss due to exchange reactions is required for DMI-based measurements of absolute metabolic rates. Here the deuterium KIE and label loss in vivo are 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 couplings allow the identification of all possible metabolic products. The ²H label loss observed in lactate, glutamate, and glutamine of rat brain was 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 loss in glutamate and glutamine was 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.

更新日期：2021-01-06

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