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Quantitative Analysis of Blood Plasma Metabolites Using Isotope Enhanced NMR Methods

机译:同位素增强核磁共振法定量分析血浆血浆

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NMR spectroscopy is a powerful analytical tool for both qualitative and quantitative analysis. However, accurate quantitative analysis in complex fluids such as human blood plasma is challenging, and analysis using one-dimensional NMR is limited by signal overlap. It is impractical to use heteronuclear experiments involving natural abundance ~(13)C on a routine basis due to low sensitivity, despite their improved resolution. Focusing on circumventing such bottlenecks, this study demonstrates the utility of a combination of isotope enhanced NMR experiments to analyze metabolites in human blood plasma. ~(1)H-~(15)N HSQC and ~(1)H-~(13)C HSQC experiments on the isotope tagged samples combined with the conventional ~(1)H one-dimensional and ~(1)H-~(1)H TOCSY experiments provide quantitative information on a large number of metabolites in plasma. The methods were first tested on a mixture of 28 synthetic analogues of metabolites commonly present in human blood; 27 metabolites in a standard NIST (National Institute of Standards and Technology) human blood plasma were then identified and quantified with an average coefficient of variation of 2.4percent for 17 metabolites and 5.6percent when all the metabolites were considered. Carboxylic acids and amines represent a majority of the metabolites in body fluids, and their analysis by isotope tagging enables a significant enhancement of the metabolic pool for biomarker discovery applications. Improved sensitivity and resolution of NMR experiments imparted by ~(15)N and ~(13)C isotope tagging are attractive for both the enhancement of the detectable metabolic pool and accurate analysis of plasma metabolites. The approach can be easily extended to many additional metabolites in almost any biological mixture.
机译:NMR光谱是用于定性和定量分析的强大分析工具。但是,在诸如人血浆等复杂流体中进行准确的定量分析具有挑战性,并且使用一维NMR进行的分析受到信号重叠的限制。尽管灵敏度提高,但由于灵敏度低,因此常规使用涉及自然丰度〜(13)C的异核实验是不切实际的。专注于规避此类瓶颈,这项研究证明了结合同位素增强NMR实验分析人类血浆中代谢物的实用性。 〜(1)H-〜(15)N HSQC和〜(1)H-〜(13)C HSQC实验与传统的一维〜(1)H和〜(1)H- 〜(1)H TOCSY实验提供了血浆中大量代谢物的定量信息。首先对人类血液中常见的28种代谢产物的合成类似物的混合物进行了测试。然后,在标准NIST(美国国家标准技术研究院)人血浆中鉴定和定量了27种代谢物,其中17种代谢物的平均变异系数为2.4%,考虑所有代谢物时的平均变异系数为5.6%。羧酸和胺代表了体液中的大部分代谢产物,通过同位素标记对其进行分析,可以显着增强代谢池,以供生物标记物发现应用。 〜(15)N和〜(13)C同位素标记赋予的NMR实验更高的灵敏度和分辨率对于增强可检测的代谢库和精确分析血浆代谢物均具有吸引力。该方法可以轻松扩展到几乎任何生物混合物中的许多其他代谢产物。

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