Corrected amplitudes of fitted water and metabolite peaks were converted to concentration (in millimolar) using the preinfusion, natural abundance water peak as an internal reference equal to 10.12 mM. The latter was determined assuming a 55.5 M water concentration, 80% water content in brain tissue and 70% water content in the liver, and a deuterium natural abundance of 0.0115 % (15, 44). Concentrations of substrates and metabolites were reported as concentration of the labeled molecule by normalizing the 2H NMR signal to the average number of deuterons per molecule. The 2H NMR signal amplitudes of water and [6,6′-2H2]glucose were normalized by dividing the quantified signal by two, whereas the observed 2H-labeled lactate signal was normalized by three and corrected for 2H label loss assumed to be 10% based on our in vitro data. The peak annotated as Glx contains signals from [4,4′-2H2]glutamate, [4-2H]glutamate, [4′-2H]glutamate, [4,4′-2H2]glutamine, [4-2H]glutamine, and [4′-2H]glutamine. The average number of deuterons at the C4 position of Glx is always lower than two because of 2H label loss in the TCA cycle at the conversion of acetyl-CoA to citrate. On the basis of the theoretical chance that both deuterons or only a single 2H atom is transferred from acetyl-CoA to Glx, the average number of deuterons of Glx will be 1.33. In case of [2H3]acetate metabolism, Glx contains a theoretical average of 2.00 deuterons on the C4 position.

The deuterium-labeled metabolite levels calculated on the nominal MRSI grid (for example, 11 × 11 × 11 on rat brain) were interpolated on a per-slice basis to a grid that is fivefold larger per dimension (that is, 55 × 55 × 11 on rat brain). 2D interpolation was achieved by convolving the nominal DMI data with a Gaussian kernel, whereby the convolution also provided an inherent Gaussian smoothing equal to 1.2- to 1.8-pixel widths. Interpolated DMI maps were overlaid with anatomical MR images as amplitude color maps.

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