Nuclear magnetic resonance metabolic labeling

Eakin, R.T., Morgan, L.O., Gregg, C.T., Matwiyoff, N.A. (1972). Carbon-13 nuclear magnetic resonance spectroscopy of living cells and their metabolism of a specifically labeled 13C substrate. FEBS Lett. 28,259-264. 

As the glucose is metabolized, the metabolic derivatives become enriched with the isotopic carbon label and are detected using advanced technologies such as mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Importantly, incorporation of isotopes into molecules can alter reaction mechanisms including the rate of a reaction-this is termed isotopic substitution and forms the basis of the kinetic isotope effect. 

The newer MS experiments in a data-dependent acquisition mode provide the MS and MS" data from a single injection. Accurate mass measurements, software-assisted data acquisition, and processing methods have been very useful for metabolite detection and identification. In addition, when MS is combined with other analytical techniques such as derivatization, H/D exchange, and stable isotope labeling have been proven very useful for structural characterization of unusual, uncommon, and difficult metabolites. Further, the flexibility and broad applications of mass spectrometry have allowed for the creation of hybrid instruments and coupling to other powerful analytical techniques, most notably nuclear magnetic resonance (NMR), to further enhance the utility in the field of drug metabolism. 

Fast sampling has especially been of interest for metabolic flux analysis. This method is based on the analysis of a large number of cellular compounds. Mostly this method includes radioactive labeling, following fast sampling and analysis of the medium and cell fractions by nuclear magnetic resonance (NMR). 

The -based metabolic flux analysis is a more advanced technique that calculates the metabolic flux vector r by additionally using the -labelled pattern of the stable and abundant protein-bound amino acids determined by either gas chromatography coupled with mass spectroscopy (GC/MS) and/or nuclear magnetic resonance (NMR) (Christensen et al. 2002 Sauer 2006 Wiechert et al. 2(X)1 Zamboni et al. 2005, 2009). The most sophisticated technique known as kinetic flux profiling has recently been developed to calculate the metabolic flux vector r by measuring the dynamic incorporation of labeled substrates (e.g. C, N) into downstream intermediate metabolites. This measurement can be subsequently used to calculate metabolic fluxes (rates) directly without relying on the simplified metabolic network like the traditional MFA approach (Yuan et al. 2006, 2008,2010). 

The measurement of internal metabolic fluxes is more difficult. The direct measurement of intracellular fluxes is possible with in vivo nuclear magnetic resonance (NMR) spectroscopy. However, the inherent insensitivity of NMR limits its applicability. An improvement over this approach can be found with isotopic tracer techniques [8]. In isotope tracer methods the cells to be studied are provided with a substrate specifically labeled with a detectable isotope (usually or C). The incorporation of label into cellular material and by-products is governed by the fluxes through the biochemical pathways. The quantity and distribution of label is measured and combined with knowledge of the metabolic network to estimate some of the intracellular fluxes. The choices of substrate labeling patterns, as well as which by-products to measure, are guided by careful analysis of the assumed biochemical network. These experiments are usually performed at isotopic steady state so that the flow of isotope into each atom of a metaboHte equals the flux out. For the nth atom of the fcth metabolite the flux balance is [9] ... 

Recently, C-labeling experiments have been applied for MFA, known as C-MFA. C-labeled carbon source(s) are incorporated into the cells, and the abundances of in intracellular metabolites are measured by nuclear magnetic resonance spectroscopy and/or mass spectrometry. The abundance of in each metabolite reflects how the carbon sources are metabolized. Therefore, the metabolic flux distribution can be estimated from the abundance of in the metabolites. 

London RE (1988) C Labeling in studies on metabolic regulation. Progress In Nuclear Magnetic Resonance Spectroscopy 20 337-383. 

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