1H magnetic resonance spectroscopy of 2H-to-1H exchange quantifies the dynamics of cellular metabolism in vivo

Abstract

Quantitative mapping of the in vivo dynamics of cellular metabolism via non-invasive imaging contributes to our understanding of the initiation and progression of diseases associated with dysregulated metabolic processes. Current methods for imaging cellular metabolism are limited by low sensitivities, costs or the use of specialized hardware. Here, we introduce a method that captures the turnover of cellular metabolites by quantifying signal reductions in proton magnetic resonance spectroscopy (MRS) resulting from the replacement of ¹H with ²H. The method, which we termed quantitative exchanged-label turnover MRS, only requires deuterium-labelled glucose and standard magnetic resonance imaging scanners, and with a single acquisition provides steady-state information and metabolic rates for several metabolites. We used the method to monitor glutamate, glutamine, γ-aminobutyric acid and lactate in the brains of unaffected and glioma-bearing rats following the administration of ²H₂-labelled glucose and ²H₃-labelled acetate. Quantitative exchanged-label turnover MRS should broaden the applications of routine ¹H MRS.

Figure 1.. Fundamentals of qMRS.

a, Schematic shows pathway, including important enzymes, for exchange of deuterium from [6,6′−²H₂]glucose and [2,2,2′−²H₃]acetate to downstream metabolites that can be detected with ¹H MRS. b, Displayed ¹H MRS spectra (10 min acquisition, 256 averages) acquired before and after 60 minutes of [6,6′−²H₂]glucose infusion shows decrease in Glu-H4 (Glu₄, blue line). Spectra were obtained from the midbrain region as shown in the representative anatomical image (yellow outline). qMRS difference spectra shows clear labelling of Glu₄, in addition to several Gln GABA, Glucose (Glc) and Asp resonances. Asp aspartate, ACS acetyl coA synthetase, GABA γ-aminobutyric acid, GAD glutamate decarboxylase, GDH glutamate dehydrogenase, Glc glucose, Glu glutamate, Gln glutamine, Glx Glu+Gln, GS glutamine synthetase, α-KG α-ketoglutarate, Lac lactate, LDH lactate dehydrogenase, NAA N-acetyl aspartate, PDH pyruvate dehydrogenase, tCr total creatine, tCho total choline

Figure 2.. Simultaneous qMRS and DMRS acquisition in normal rat brain.

a, Displayed ¹H MR spectra (10 min acquisition, 256 averages) show continuous reduction in 2.35 ppm Glu-H4 peak (Glu₄, red arrow) following infusion of [6,6′−²H₂]glucose. b, qMRS difference spectra show gradual accumulation of ²H labelled Glu-H4 beginning after 20 mins of infusion. c, Similarly, DMRS spectra (5 min acquisition, 1000 averages) also revealed a gradual increase in Glx peak observed beginning 20 min after infusion. Glu glutamate, Glx Glu+Gln, Lac lactate, NAA N-acetyl aspartate, tCr total creatine, tCho total choline

Figure 3.. Comparison of qMRS and DMRS metabolite quantification.

a, Graph shows changes in Glx concentration in normal rat brain (n=4) measured with qMRS (blue) and DMRS (red) made during [6,6′−²H₂]glucose infusion. For a visual aid, concentration estimates were fitted with the exponential plateau equation Y=Y_M −(Y_M-Y₀)*exp(−k*x), where Y₀ is the starting population, Y_M is the maximum population, and k is the rate constant. b, Correlation plot with Pearson’s correlation analysis shows relationship between these quantitative measurements at each time point post infusion. c, Bland-Altman plot and bias analysis showing the mean bias (solid line) and standard deviation (dashed lines) for Glx measurements made with qMRS and DMRS. Error bars represent the standard error of the mean.

Figure 4.. Kinetics of deuterium labelling of neural metabolites.

Graphs show fractional enrichment for (a) Glx, (b) Glu, (c) Gln, (d) GABA, and (e) NAA measured in normal rat brain (n=6) during [6,6′−²H₂]glucose infusion acquired over a 45 min period. For a visual aid, plots were fitted with the exponential plateau equation Y=Y_M −(Y_M-Y₀)*exp(−k*x), where Y₀ is the starting population, Y_M is the maximum population, and k is the rate constant. Error bars represent the standard error of the mean.

Figure 5.. Detection of glycolysis in rat glioblastoma.

Studies were performed in rats (n=3) bearing orthotopic F98 glioblastoma. a, Obtained spectrum (5 min acquisition, 128 averages) show a large lac/lipid peak observed at 1.33 ppm from a voxel placed within the tumour (inset). b, Spectrum acquired after 60 minutes of [6,6′−²H₂]glucose infusion shows a marked reduction in the 1.33 peak (green line). c, qMRS difference spectra (pre-post) obtained every 10 minutes post infusion show the increase in labelled lac at 1.33 ppm. Lac lactate, MM macromolecules, NAA N-acetyl aspartate, tCr total creatine, tCho total choline

Figure 6.. Metabolic imaging of neural metabolism.

a, Images show anatomical reference image (left) with sampled volume of interest (VOI, green box) for Chemical Shift Imaging and corresponding Glu and NAA metabolite maps (right) acquired in a normal rat brain at 9.4T before and after 60 min of [6,6′−²H₂]glucose infusion. b, Displayed spectra represent a single CSI voxel within the VOI (yellow box) acquired before and after infusion. A clear reduction in Glu-H4 resonance (Glu₄) can be observed post infusion (red arrow). Glu₄ peak was also observed in the corresponding difference spectrum. Glu glutamate, Glx glu-gln, Lac lactate, NAA N-acetyl aspartate, tCr total creatine, tCho total choline