An adapted isotope dilution 1H–13C heteronuclear single-quantum correlation (ID-HSQC) for rapid and accurate quantification of endogenous and exogenous plasma glucose
A wide variety of methods, such as enzymatic methods, LC-MS, and LC-MS/MS, are currently available for the concentration determination of plasma glucose in studies of diabetes, obesity, exercise, etc. However, these methods rarely discriminate endogenous and exogenous glucose in plasma. A novel NMR strategy for discriminative quantification of the endogenous and exogenous glucose in plasma has been developed using an adapted isotope dilution 1H–13C heteronuclear single-quantum correlation (ID-HSQC) with uniformly 13C-labeled glucose as a tracer of exogenous glucose. This method takes advantage of the distinct 1H–13C chemical shifts of the hemiacetal group of the α-D-glucopyranose and makes use of the 13C–13C one-bond J-coupling (1JCC) in uniformly 13C-labeled glucose to differentiate the 1H–13C HSQC signal of labeled glucose from that of its natural counterpart when data are acquired in high-resolution mode. The molar ratio between the endogenous and exogenous plasma glucose can then be calculated from the peak intensities of the natural and labeled glucose. The accuracy and precision of the method were evaluated using a series of standard mixtures of natural and uniformly 13C-labeled glucose with varied but known concentrations. Application of this method is demonstrated for the quantification of endogenous and exogenous glucose in plasma derived from healthy and diabetic cynomolgus monkeys. The results nicely agree with our previous LC-MS/MS results. Considering the natural abundance of 13C isotope at the level of 1.1% in endogenous glucose, comparable peak intensities of quantitatively measurable signals derived from natural and labeled glucose imply that the ID-HSQC can tolerate a significantly high ratio of isotope dilution, with labeled/natural glucose at ~ 1%. We expect that the ID-HSQC method can serve as an alternative approach to the biomedical or clinical studies of glucose metabolism.
KeywordsIsotope dilution HSQC Carbon-13 labeling Quantification Glucose
The authors are grateful for financial supports from the National Key R & D Program of China (2017YFC0906900), the National Natural Science Foundation of China (21505142), and Yunnan Provincial Science and Technology Department (2012HA015).
This study received financial supports from the National Key R & D Program of China (2017YFC0906900), the National Natural Science Foundation of China (21505142), and Yunnan Provincial Science and Technology Department (2012HA015).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Research involving animals
Animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (2011) and protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Kunming Biomed International.
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