INTRODUCTION

Noninvasive in vivo detection of ¹³C labeling of glutamate (Glu) and glutamine (Gln) is a powerful tool for investigating Glu and Gln metabolism and neurotransmission in the brain ¹. Attempts have been made to circumvent the hardware limitation of ¹³C MRS by measuring the changes in short TE ¹H MRS spectra caused by incorporation of ¹³C labels into brain amino acids ². However, it has been difficult to reliably separate Glu and Gln in the crowded ¹H MRS spectra, especially after the incorporation of ¹³C labels. Quantification of ¹³C labeling of both Glu and Gln using ¹H MRS in the human brain has not been reported. In this work, we demonstrate the feasibility of measuring ¹³C fractional enrichments of Glu and Gln at 7 T using a recently published single-step spectral editing ¹H MRS pulse sequence, which induces intense Glu and Gln H4 singlets at TE = 56 ms using an always-on editing pulse at 2.12 ppm ³.

METHODS

Five healthy volunteers were scanned on a Siemens Magnetom 7 T scanner equipped with a 32-channel receiver head coil. In each scan session, the subject was first scanned to acquire the pre-¹³C MRS data from a 3.5 × 1.8 × 2 cm³ voxel in the dorsal anterior cingulate cortex (dACC) of the brain. Subsequently, the subject exited from the scanner and was orally administered 20% w/w 99% enriched [U-¹³C]glucose solution at a dosage of 0.75 g [U-¹³C]glucose per kg of body weight. After a rest period, the subject reentered the scanner. Post-¹³C MRS data were repeatedly acquired from the same voxel as the pre-¹³C scan. The pre-¹³C MRS data were processed first and the process was similar to that described previously ³. Using an in-house developed fitting program, the reconstructed pre-¹³C spectrum was fitted in the range of 1.8 – 3.4 ppm by linear combination of numerically computed basis spectra of acetate (Ace), N-acetyl-aspartate (NAA), N-acetylaspartylglutamate (NAAG), γ-aminobutyric acid (GABA), Glu, Gln, glutathione (GSH), aspartate (Asp), total creatine (tCr), total choline (tCho), taurine (Tau), myo-inositol (mI), and scyllo-inositol (sI), as well as a cubic spline baseline with 13 knots. After the metabolite concentrations in arbitrary unit were obtained from the fitting, we computed the metabolite ratios, which were defined as the concentration of a metabolite divided by the sum of concentration of tCr and three times the concentration of tCho ([tCr]+3[tCho]), which weighs approximately equally the intensities of the tCr and tCho singlet peaks.

The post-¹³C spectra were reconstructed in the same way as the pre-¹³C spectrum. Additional basis spectra of ¹³C satellites of Glu H4, Gln H4 and Asp H3 were simulated for the subsequent fitting process. When fitting each post-¹³C spectrum, the metabolites ratios (/ [tCr]+3[tCho]) of Ace, NAA, NAAG, GABA, GSH, Asp, tCr, tCho, Tau, mI, and sI were fixed to the pre-¹³C values. The sum of metabolite ratios of Glu and its ¹³C satellites was constrained to be the same as the metabolite ratio of Glu obtained from the pre-¹³C spectrum. Similar constraints were also applied to Gln and Asp. After obtaining the metabolite concentrations from the fitting process, the ¹³C fractional enrichment of Glu H4 for a post-¹³C spectrum was computed as the ratio of the concentration of its ¹³C satellites to the total concentration of Glu. The fractional enrichment of Gln H4 was similarly computed.

RESULTS

Figure 1 shows the basis spectra of Glu, Gln, Asp, and their ¹³C satellites. The spectrum of [4,5-¹³C]Glu is highly similar to that of [4-¹³C]Glu because the spectral pattern is dominated by the large ¹J_HC. As both carbons of the acetyl CoA are ¹³C-labeled after administration of uniformly labeled glucose ⁴, [4,5-¹³C]Glu was chosen to represent the ¹³C satellite signals of Glu H4 in the fitting process. Similarly, [4,5-¹³C]Gln and [3,4-¹³C]Asp were chosen to represent the ¹³C satellite signals of Gln H4 and Asp H3, respectively. Figure 2 displays the time-course spectra of subject 1 and corresponding fitted spectra of Glu, Gln and their ¹³C satellites. The in vivo spectra and corresponding fits for the pre-¹³C MRS scan and the last post-¹³C scan of subject 1 are displayed in Figure 3. The plots of fractional enrichments of Glu H4 and Gln H4 vs. time after oral administration of [U-¹³C]glucose for all five subjects are displayed in Figure 4. Table 1 lists the metabolite ratios (/[tCr]) and end point fractional enrichments of Glu H4 and Gln H4 in the dACC of all five subjects.

DISCUSSION AND CONCLUSION

We demonstrated the feasibility of using a proton-only single-step spectral editing sequence with TE = 56 ms to measure fractional enrichments of Glu and Gln in the dACC of healthy volunteers after oral administration of [U-¹³C]glucose. Compared to the existing indirect ¹H-[¹³C] MRS techniques that use ¹H and ¹³C surface coils, this ¹H MRS technique can acquire MRS data from a voxel away from the neocortex using a standard ¹H head coil. This technique offers the ability to measure glutamate neurotransmission in the human brain with the high sensitivity and spatial resolution of ¹H MRS using standard commercial equipment. Brain regions inaccessible to surface coils can now be investigated using the technique described in this work.

Acknowledgements

This work was supported by the intramural program of the NIH and NIMH.