Toward dynamic isotopomer analysis in the rat brain in vivo: automatic quantitation of ¹³C NMR spectra using LCModel

Abstract

The LCModel method was adapted to analyze localized in vivo13 C NMR spectra obtained from the rat brain in vivo at 9.4 T. Prior knowledge of chemical-shifts, J-coupling constants and J-evolution was included in the analysis. Up to 50 different isotopomer signals corresponding to 10 metabolites were quantified simultaneously in 400 ml volumes in the rat brain in vivo during infusion of [1,[6][7][8][9][10][11][12][13] C 2 ]glucose. The analysis remained accurate even at low signal-to-noise ratio of the order of 3:1. The relative distribution of isotopomers in glutamate, glutamine and aspartate determined in vivo in 22 min was in excellent agreement with that measured in brain extracts. Quantitation of time series of 13 C spectra yielded time courses of total 13 C label incorporation into up to 16 carbon positions, as well as time courses of individual isotopomer signals, with a temporal resolution as low as 5 min (dynamic isotopomer analysis). The possibility of measuring in vivo a wealth of information that was hitherto accessible only in extracts is likely to expand the scope of metabolic studies in the intact brain.

Modeling of Brain Metabolism and Pyruvate Compartmentation Using 13C NMR in Vivo: Caution Required

“…4 The resonances attributed to glutamate and glutamine C2, C3, and C4, aspartate C2 and C3, and smaller resonances from GABA, N-acetyl-aspartate, and lactate were identified. Multiplet resonances that are a consequence of 13 C spin-spin coupling were observed for the carbons of several intermediates, particularly glutamate C4.…”

“…Localized in vivo 13 C NMR spectra were acquired with a semiadiabatic distortion-less enhancement by polarization transfer sequence as previously described. 4 …”

“…4 Signals were converted to fractional enrichments using high-resolution 13 C NMR spectra of tissue extracts obtained via freeze-clamping at the end of the experiment.…”

Selective resonance suppression 1H‐[13C] NMR spectroscopy with asymmetric adiabatic RF pulses

“…The 13 C resonance frequency of Glu C3 was calculated from the frequency of 1 H (water) signal based on a previous in vivo calibration. To verify the precision of the frequency of Glu C3, the frequency of Glc C1␤ (96.81 ppm) was measured through an unlocalized distortionless enhancement by polarization transfer (DEPT) sequence (27) following the 99% enriched [U- 13 C 6 ] Glc bolus to derive the frequency of Glu C3 (26), and the discrepancy between the measured frequencies of Glu C3 and those calculated was below 15 Hz. All spectra were acquired in an interleaved fashion to minimize effects of instrumental drift.…”

Proton‐observed carbon‐edited NMR spectroscopy in strongly coupled second‐order spin systems

“…Although studies using direct detection have provided unique information, such as the resolved detection of C4, C3, and C2 carbon positions in glutamate and glutamine, and the detection of different isotopomers with distinct 13 C- 13 C coupling patterns (3)(4)(5), these studies generally required relatively large volumes. In contrast, studies using indirect detection have proved very useful for investigating small areas of the brain during functional activation (6,7) or distinct brain tissue types such as gray and white matter (8,9).…”