Bastawrous Monica, Tabatabaei-Anaraki Maryam, Soong Ronald, Bermel Wolfgang, Gundy Marcel, Boenisch Holger, Heumann Hermann, Simpson Andre J
Environmental NMR Center, Department of Physical and Environmental Science, University of Toronto, 1265, Military Trail, Toronto, ON, M1C 1A4, Canada.
Bruker Biospin GmbH, Silberstreifen 4, 76287, Rheinstetten, Germany.
Anal Chim Acta. 2020 Nov 22;1138:168-180. doi: 10.1016/j.aca.2020.09.065. Epub 2020 Oct 4.
In-vivo Nuclear Magnetic Resonance (NMR) spectroscopy is a unique and powerful approach for understanding sublethal toxicity, recovery, and elucidating a contaminant's toxic mode of action. However, magnetic susceptibility distortions caused by the organisms, along with sample complexity, lead to broad and overlapping 1D NMR spectra. As such, 2D NMR in combination with C enrichment (to increase signal) is a requirement for metabolite assignment and monitoring using high field in-vivo flow based NMR. Despite this, it is not clear which NMR experiment and probe combinations are the most appropriate for such studies. In terms of experiments, H-C Heteronuclear Single Quantum Coherence (HSQC) and C-H Heteronuclear Correlation Spectroscopy (HETCOR) experiments are logical choices for molecular fingerprinting. HSQC uses H for detection and thus will be the most sensitive, while HETCOR uses C for detection, which benefits from improved spectral dispersion (i.e. a larger chemical shift range) and avoids detection of the huge in-vivo water signal which can be problematic in HSQC. NMR probes are available in two variations, inverse (inner coil H) which is best suited to H detection and observe (inner coil C) which is ideal for C detection. To further complicate matters, the low biomass in many aquatic organisms makes cryoprobes desirable, however, changing cryoprobes is time prohibitive, requiring at least a day to warmup and cool down, meaning only a single probe can be used to monitor "real-time" in-vivo responses. The key questions become: Is it best to use HSQC on an inverse cryoprobe and accept a compromised HETCOR? Or is it best to use HETCOR on an observe cryoprobe and accept a compromised HSQC? Here these questions are explored using living C enriched Daphnia as the test case. The number of metabolites identified across the different probe/experiment combinations are compared over a range of experiment times. Finally, the probes/experiments are compared to monitor an anoxic stress response. Both probes and experiments prove to be quite robust, albeit HSQC identified slightly more metabolites in most cases. HETCOR did nearly as-well and because of the lack of water complications would be the most accessible approach for researchers to apply in-vivo NMR to C enriched organisms, both in terms of experimental setup and flow system design. This said, when using an optimized flow system, HSQC did identify the most metabolites and an inverse probe design offers the most potential for H-only approaches which are continuously being developed and have the potential to eventually overcome the current limitation that requires C enriched organisms.
体内核磁共振（NMR）光谱法是一种独特且强大的方法，可用于了解亚致死毒性、恢复情况以及阐明污染物的毒性作用模式。然而，生物体引起的磁化率畸变以及样品的复杂性，导致一维NMR光谱宽泛且相互重叠。因此，二维NMR结合碳富集（以增加信号）是使用基于高场体内流动的NMR进行代谢物鉴定和监测的必要条件。尽管如此，尚不清楚哪种NMR实验和探头组合最适合此类研究。在实验方面，氢 - 碳异核单量子相干（HSQC）和碳 - 氢异核相关光谱（HETCOR）实验是进行分子指纹识别的合理选择。HSQC使用氢进行检测，因此将是最灵敏的，而HETCOR使用碳进行检测，这得益于改善的光谱分散性（即更大的化学位移范围），并且避免了检测在HSQC中可能存在问题的巨大体内水信号。NMR探头有两种变体，反向探头（内线圈为氢）最适合氢检测，观察探头（内线圈为碳）则最适合碳检测。更复杂的是，许多水生生物的生物量较低，这使得低温探头成为理想选择，然而，更换低温探头非常耗时，升温及降温至少需要一天时间，这意味着在“实时”监测体内反应时只能使用单个探头。关键问题变成：是最好在反向低温探头上使用HSQC并接受折衷的HETCOR？还是最好在观察低温探头上使用HETCOR并接受折衷的HSQC？在此，以富含碳的活体水蚤作为测试案例来探讨这些问题。在一系列实验时间内，比较不同探头/实验组合所鉴定出的代谢物数量。最后，比较探头/实验以监测缺氧应激反应。两种探头和实验都证明相当可靠，尽管在大多数情况下HSQC鉴定出的代谢物略多一些。HETCOR的表现也差不多，并且由于不存在水信号的干扰问题，从实验设置和流动系统设计两方面来看，对于研究人员将体内NMR应用于富含碳的生物体而言，HETCOR将是最容易采用的方法。话虽如此，当使用优化的流动系统时，HSQC确实鉴定出了最多的代谢物，并且反向探头设计对于仅基于氢的方法具有最大潜力，此类方法正在不断发展，并且最终有可能克服目前需要富含碳的生物体这一限制。
