Spectroscopy for stable isotope measurements in metabolic physiology

稳定同位素是较重的元素，由于额外的中子（或中子）超过正常同位素，但它们是非放射性的。这些同位素用于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，已经使用稳定同位素标记的生物分子的引发输注的方法。输注的示踪剂是昂贵的，需要无菌和无热原的制备，并且仅产生短期（小时）结果，因为参与者在输注时被限制在床/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（包括脂质、RNA和DNA）D标记的方法。口服重水使我们能够对几种生物分子进行高灵敏度的通量测量，同时节省时间和金钱，并迅速推进我们的发现工作。与常规注入的示踪剂相比，重水的摄入具有显著的成本效益，由于其被摄入而不是注射，因此风险较低，并且具有更大的生物分子标记潜力，因此与常规注入的示踪剂相比具有若干方法学优势。我们还希望将重氧（18 O）分析，以增加更大的效用，这种稳定的同位素方法。我们寻求的仪器是一个腔衰荡光谱（CRDS）单元，它将测量生物样品中的D和18 O，以支持人体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CRDS单位将为HQP提供独特的培训机会。CRDS单元将通过缓解样品分析的重大瓶颈，大大降低分析成本，并扩大使用低成本同位素标记水来评估多种代谢途径，从而迅速推进计划的发现研究。CRDS可以分析18 O和D，并允许估计自由生活的能量消耗和全身含水量，这是每个申请人的关键研究应用。该设备将由三名高级和两名初级NSERC支持的研究人员使用，以促进他们在生理研究中使用稳定同位素。这些设备将由大约20名HQP在从本科到博士后研究员的各级培训中使用。由于它的易用性，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和我们建立新的国家和国际跨学科合作。

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.