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Quantification of high-resolution ¹H-[¹³C] NMR spectra from rat brain extracts. | LitMetric
NMR spectroscopy in combination with (13)C-labeled substrate infusion is a unique technique to obtain information about dynamic metabolic fluxes noninvasively in vivo. In many cases, the in vivo information content obtained during dynamic (13)C studies in rodents can be enhanced by high-resolution (1)H-[(13)C] NMR spectroscopy on brain extracts. Previously, it has been shown that (1)H NMR spectra from rat brain extracts can be accurately quantified with a spectral fitting routine utilizing simulated basis sets using complete prior knowledge of chemical shifts and scalar couplings. The introduction of (13)C label into the various metabolites presents complications that demand modifications of the spectral fitting routine. As different multiplets within a given molecule accumulate various amounts of (13)C label, the fixed amplitude relationship between multiplets typical for (1)H NMR spectra must be abandoned. In addition, (13)C isotope effects lead to spectral multiplet patterns that become dependent on the amount of (13)C label accumulation, thereby preventing the use of a common basis set. Here a modified spectral fitting routine is presented that accommodates variable (13)C label accumulation and (13)C isotope effects. Spectral fitting results are quantitatively compared to manual integration on column-separated samples in which spectral overlap is minimized.
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