In vivo 1H NMR spectroscopy and neurochemistry. Quantification matters

For a long time, in vivo H NMR spectroscopy of the brain has been limited to the evaluation of three or four resonances (e.g., NAA, Cr, Cho, and Lac). Comparisons of data (mostly signal ratios) have been difficult to achieve because of a number of methodological factors that had to be taken into account. Volume-localized short-echo-time spectroscopy, which was introduced in the late 1980s (1), has shown that the detection and quantification of multiple metabolites with J-coupled spin systems is possible. Since then, in vivo H NMR spectroscopy has evolved into a rather powerful tool for the assessment of neurochemistry. There is undoubtedly a consensus that in the ideal case, measured metabolite concentrations should be independent of the instrumentation and methodology used, including differences in field strength. The aim of our recent paper (2) was to demonstrate the feasibility of in vivo H NMR spectroscopy of the mouse brain at 9.4 T with high spatial and spectral resolution. We showed that with the use of a strong shim system, it is possible to compensate for macroscopic field gradients, thus enabling the quantification of many metabolites from small voxels located in different brain regions. This led us to compare quantified metabolite concentrations with the data reported by other groups, including Schwarcz et al. (3), who performed their studies at field strengths different from 9.4 T. In their letter, Michaelis and Frahm claim that we “failed to accomplish a fair comparison” with the study by Schwarcz et al. (3), and tried to create “the impression that proton MRS of mouse brain at low field strengths is of low value. ” These claims require a rebuttal. In our response we choose glucose quantification as an example to demonstrate and explain why some metabolite concentrations reported by Schwarcz et al. (3) deserve a critical assessment. A brain glucose concentration of 10 mM must be considered unphysiologically high for a healthy C57/BL6 mouse, independent of the B0 at which this value was measured. First, it implies a brain glucose concentration exceeding that in plasma under euglycemic conditions, which is contrary to known blood–brain barrier physiology (see Ref. 4 and references therein). Second, the explanation of the authors that a shift to anaerobic metabolism occurs would imply a further decrease of the brain glucose concentration due to increased CMRglc (see, e.g., Fig. 11 in Ref. 4). Third, the observation of a statistically significant, nonzero brain glucose concentration after cardiac arrest is contrary to established neurochemistry, because under hypoxic ischemia both brain glucose and glycogen are rapidly depleted (e.g., Ref. 5 and references therein). Lastly, it is clear that the mass balance was not preserved, when one considers that two molecules of lactate are generated from each molecule of glucose and glucosyl unit of glycogen under anoxic conditions. The postmortem increase in lactate by 12 mM reported by Schwarcz et al. (3) does not account for the decrease of glucose by 8.1 mM without including lactate generated from brain glycogen (6). Therefore, the high brain glucose concentration reported in Ref. 3 must be explained by methodological issues. The comment in the letter that “low-field spectra concentrate most of the intensity of the Glc protons in a well-defined apparent singlet at 3.43 ppm, which may be readily exploited for accurate quantification by LCModel” may indeed be true for well-resolved signals. However, at 2.35 T the apparent glucose singlet is significantly overlapped with other collapsed resonances, such as taurine (Tau). We gratefully acknowledge Michaelis and Frahm for supplementing the concentrations of Tau. It is interesting to note that with these new numbers, the sum of the Glc and Tau concentrations in C57BL/6 mice are in very good agreement with our data (2), which indicates that a correlation between Glc and Tau could have biased the glucose quantification in Ref. 3. We disagree with the statement that “the use of high field strengths introduces new problems, such as those caused by the pronounced splitting of strongly spin-coupled resonances. ” At 9.4 T most metabolites have a unique spectral pattern, which results in higher reliability (Cramer-Rao lower bounds) and less correlation in analyses of NMR spectra with overlapping resonances. The reduction in the signal-to-noise ratio (SNR) due to the multiplet structure is more than compensated for by the higher sensitivity at high fields, which allows to quantify about twice as many metabolites in fiveto 10fold smaller volumes at 9.4 T (2). Our paper (2) provides an excellent example of this: Two resonances of the same intensity at 3.25 ppm and 3.42 ppm, which are unique for Tau, are discernible in all spectra (see Fig. 1 in Ref. 2). In addition, the high concentration of Tau (8–13 mol/g) can be easily assessed by a visual comparison of the intensity of the well-resolved signal of the S-CH2 group of Tau (at 3.42 ppm) with the intensity of the methyl signal of creatine and phosphocreatine (at 3.03 ppm). Furthermore, the H signal of -glucose at 5.23 ppm is not overlapped by any other resonances. A glucose concentration of 10 mM should result in a clearly visible signal at 5.23 ppm well above the noise level (7), but was not observed in spectra from mice (2). Therefore, the ability to resolve and quantify different H signals critically depends on the linewidths achieved. Reported linewidths of 9 Hz may not be the lowest achiev*Correspondence to: Ivan Tkáč, 2021 6th Street SE, Minneapolis, MN 55455. E-mail: ivan@cmrr.umn. edu Received 16 February 2005; revised 10 June 2005; accepted 11 June 2005. DOI 10.1002/mrm.20658 Published online 13 September 2005 in Wiley InterScience (www.interscience. wiley.com).