Lane Daniel, Skinner Thomas E, Gershenzon Naum I, Bermel Wolfgang, Soong Ronald, Dutta Majumdar Rudraksha, Liaghati Mobarhan Yalda, Schmidt Sebastian, Heumann Hermann, Monette Martine, Simpson Myrna J, Simpson André J
Environmental NMR Centre, University of Toronto Scarborough, 1265 Military Trail, Toronto, ON, M1C 1A4, Canada.
Department of Physics, Wright State University, Dayton, OH, 45735, USA.
J Biomol NMR. 2019 Feb;73(1-2):31-42. doi: 10.1007/s10858-018-0221-2. Epub 2019 Jan 2.
In vivo Nuclear Magnetic Resonance (NMR) spectroscopy has great potential to interpret the biochemical response of organisms to their environment, thus making it an essential tool in understanding toxic mechanisms. However, magnetic susceptibility distortions lead to 1D NMR spectra of living organisms with lines that are too broad to identify and quantify metabolites, necessitating the use of 2D H-C Heteronuclear Single Quantum Coherence (HSQC) as a primary tool. While quantitative 2D HSQC is well established, to our knowledge it has yet to be applied in vivo. This study represents a simple pilot study that compares two of the most popular quantitative 2D HSQC approaches to determine if quantitative results can be directly obtained in vivo in isotopically enriched Daphnia magna (water flea). The results show the perfect-HSQC experiment performs very well in vivo, but the decoupling scheme used is critical for accurate quantitation. An improved decoupling approach derived using optimal control theory is presented here that improves the accuracy of metabolite concentrations that can be extracted in vivo down to micromolar concentrations. When combined with 2D Electronic Reference To access In vivo Concentrations (ERETIC) protocols, the protocol allows for the direct extraction of in vivo metabolite concentrations without the use of internal standards that can be detrimental to living organisms. Extracting absolute metabolic concentrations in vivo is an important first step and should, for example, be important for the parameterization as well as the validation of metabolic flux models in the future.
体内核磁共振（NMR）光谱在解释生物体对其环境的生化反应方面具有巨大潜力，因此使其成为理解毒性机制的重要工具。然而，磁化率畸变导致活生物体的一维NMR光谱线过宽，无法识别和定量代谢物，因此需要使用二维H-C异核单量子相干（HSQC）作为主要工具。虽然定量二维HSQC已经成熟，但据我们所知，它尚未应用于体内。本研究是一项简单的初步研究，比较了两种最流行的定量二维HSQC方法，以确定是否可以在同位素富集的大型溞（水蚤）体内直接获得定量结果。结果表明，完美HSQC实验在体内表现良好，但所使用的去耦方案对于准确定量至关重要。本文提出了一种使用最优控制理论推导的改进去耦方法，该方法提高了体内可提取的代谢物浓度的准确性，低至微摩尔浓度。当与二维电子参考获取体内浓度（ERETIC）方案相结合时，该方案允许直接提取体内代谢物浓度，而无需使用可能对活生物体有害的内标。在体内提取绝对代谢浓度是重要的第一步，例如，这对于未来代谢通量模型的参数化和验证应该很重要。
