Fred Hutchinson Cancer Center, Seattle, Washington 98109, United States.
Anal Chem. 2023 Aug 29;95(34):12923-12930. doi: 10.1021/acs.analchem.3c02478. Epub 2023 Aug 15.
Recent efforts in our laboratory have enabled access to an unprecedented number (∼90) of quantifiable metabolites in human blood by a simple nuclear magnetic resonance (NMR) spectroscopy method, which includes energy coenzymes, redox coenzymes, and antioxidants that are fundamental to cellular functions [ 2022, 12-13, 100082]. The coenzymes and antioxidants, however, are notoriously labile and are extremely sensitive to specimen harvesting, extraction, and measurement conditions. This problem is largely underappreciated and carries the risk of grossly inaccurate measurements and incorrect study outcomes. As a part of addressing this challenge, in this study, human blood specimens were comprehensively and quantitatively investigated using H NMR spectroscopy. Freshly drawn human blood specimens were treated or not treated with methanol, ethanol, or a mixture of methanol and chloroform, and stored on ice or on bench, at room temperature for different time periods from 0 to 24 h, prior to storing at -80 °C. Interestingly, the labile metabolite levels were stable in blood treated with an organic solvent. However, their levels in blood in untreated samples increased or decreased by factors of up to 5 or more within 3 h. Further, surprisingly, and contrary to the current knowledge about metabolite stability, the variation of coenzyme levels was more dramatic in blood stored on ice than on bench, at room temperature. In addition, unlike the generally observed phenomenon of oxidation of redox coenzymes, reduction was observed in untreated blood. Such preanalytical dynamics of the labile metabolites potentially arises from the active cellular metabolism. From the metabolomics perspective, the massive variation of the labile metabolite levels even in blood stored on ice is alarming and stresses the critical need to immediately quench the cellular metabolism for reliable analyses. Overall, the results provide compelling evidence that warrants a paradigm shift in the sample collection protocol for blood metabolomics involving labile metabolites.
最近，我们实验室的努力使得通过一种简单的核磁共振（NMR）光谱方法可以获得数量空前的（约 90 种）可量化的人类血液代谢物，其中包括对细胞功能至关重要的能量辅酶、氧化还原辅酶和抗氧化剂[2022,12-13,100082]。然而，这些辅酶和抗氧化剂极不稳定，对标本采集、提取和测量条件极其敏感。这个问题在很大程度上被低估了，存在测量结果严重不准确和研究结果不正确的风险。作为解决这一挑战的一部分，在这项研究中，我们使用 H NMR 光谱全面、定量地研究了人类血液标本。新鲜抽取的人类血液标本用甲醇、乙醇或甲醇和氯仿的混合物处理或不处理，并在冰上或室温下放置 0 至 24 小时不等，然后储存在-80°C 下。有趣的是，用有机溶剂处理的血液中不稳定代谢物的水平是稳定的。然而，在未处理的样本中，其水平在 3 小时内增加或减少了多达 5 倍或更多。此外，令人惊讶的是，与目前关于代谢物稳定性的知识相反，在冰上或室温下储存的血液中辅酶水平的变化更为剧烈。此外，与氧化还原辅酶通常观察到的氧化现象不同，未处理的血液中观察到还原。这种不稳定代谢物的预分析动力学可能源于细胞的活跃代谢。从代谢组学的角度来看，即使在冰上储存的血液中，不稳定代谢物水平的巨大变化也令人震惊，这强调了为了进行可靠的分析，需要立即抑制细胞代谢。总的来说，这些结果提供了令人信服的证据，证明需要对涉及不稳定代谢物的血液代谢组学样本采集方案进行范式转变。
