Spectroscopy for stable isotope measurements in metabolic physiology

代谢生理学中稳定同位素测量的光谱学

• 批准号: RTI-2022-00410
• 负责人: Phillips, Stuart
• 金额: $ 10.92万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743063
• 关键词: Spectroscopy; stable; isotope; measurements; metabolic

Stable isotopes are heavier elements due to an additional neutron (or neutrons) over the normal isotope, but they are non-radioactive. These isotopes are used to tag molecules that we wish to measure the metabolism of in experiments. There are very few laboratories that use stable isotope-labelled tracers to study human physiology. Traditionally, the method of a primed infusion of stable isotope-labelled biomolecules has been used. Infused tracers are expensive, require sterile and pyrogen-free preparation, and only yield short-term (hours) results as participants are confined to a bed/chair while being infused. Recently, we have begun to use oral ingestion of heavy water (heavy hydrogen - 2H- or deuterium D-labelled), which rapidly equilibrates and, via normal metabolism, labels biomolecules such as non-essential amino acids, which then become the tracer. We are now perfecting methods to measure the D-labelling of other molecules, including lipids, RNA, and DNA. The oral dosing of heavy water allows us to make highly sensitive flux measurements of several biomolecules, simultaneously saving time and money and rapidly advancing our discovery work. Compared to conventional infused tracers, ingestion of heavy water is remarkably cost-effective, lower risk as it is ingested rather than injected, and has greater biomolecule labelling potential, and thus has several methodological advantages over conventional infused tracers. We also wish to incorporate heavy oxygen (18O) analysis to add greater utility to this stable isotope methodology. The instrument we seek is a cavity ring-down spectroscopy (CRDS) unit that will measure both D and 18O in biological samples to support integrative and innovative studies of metabolic physiology and energy expenditure in humans. The CRDS unit is more affordable than, and yet as accurate as, the traditional (high-maintenance cost) isotope-ratio mass spectrometer. Acquisition of a CRDS unit would provide a unique training opportunity for HQP. The CRDS unit will result in a rapid advance of planned discovery research by alleviating a substantial bottleneck for sample analysis, greatly reducing analytical costs, and expanding the use of lower-cost isotope-labelled water for evaluating multiple metabolic pathways. The CRDS can analyze both 18O and D and allowing estimation of free-living energy expenditure and whole-body water content, which are key research applications for each of the applicants on this proposal. This equipment will be used by three senior and two junior NSERC-supported researchers to advance their use of stable isotopes in physiological research. The equipment will be used by ~20 HQP at various levels of training from undergraduate to postdoctoral fellows. Because of its ease of use, we will use the CRDS to run samples from various applications. Acquisition of the CRDS unit would elevate our research productivity and us to forge new national and international interdisciplinary collaborations.

稳定同位素是较重的元素，因为在正常同位素上多了一个中子（或中子），但它们是非放射性的。这些同位素被用来标记我们希望在实验中测量代谢的分子。很少有实验室使用稳定的同位素标记示踪剂来研究人体生理学。传统上，已使用稳定同位素标记的生物分子的引物输注方法。注射示踪剂价格昂贵，需要无菌和无热原制备，并且只能产生短期（小时）的结果，因为参与者在注射时被限制在床/椅子上。最近，我们已经开始使用口服重水（重氢- 2H-或氘- d标记），它迅速平衡，并通过正常代谢，标记生物分子，如非必需氨基酸，然后成为示踪剂。我们现在正在完善测量其他分子的d标记的方法，包括脂质、RNA和DNA。口服重水剂量使我们能够对几种生物分子进行高度敏感的通量测量，同时节省时间和金钱，并迅速推进我们的发现工作。与传统的输注示踪剂相比，摄入重水具有显著的成本效益，风险更低，因为它是摄入而不是注射的，并且具有更大的生物分子标记潜力，因此与传统输注示踪剂相比具有几个方法学优势。我们还希望纳入重氧（18O）分析，以增加这种稳定同位素方法的更大效用。我们寻找的仪器是一个空腔衰荡光谱（CRDS）单元，它将测量生物样品中的D和18O，以支持人类代谢生理学和能量消耗的综合和创新研究。CRDS装置比传统的（高维护成本）同位素比质谱仪更便宜，但同样准确。获得一个CRDS单元将为HQP提供一个独特的培训机会。CRDS装置将缓解样品分析的瓶颈，大大降低分析成本，并扩大使用成本较低的同位素标记水来评估多种代谢途径，从而促进计划中的发现研究的快速发展。CRDS可以分析18O和D，并允许估计自由生活的能量消耗和全身水分含量，这是本提案中每个申请人的关键研究应用。该设备将由nserc支持的三名高级和两名初级研究人员使用，以推进他们在生理研究中使用稳定同位素。该设备将用于约20名HQP在不同层次的培训，从本科生到博士后研究员。由于它易于使用，我们将使用CRDS来运行来自各种应用程序的示例。收购CRDS单位将提高我们的研究生产力，并使我们能够建立新的国内和国际跨学科合作。