Basic neurochemistry pp. 638 (1999). Acknowledgments Supported by NIH (P41RR08079, R21DK58004), the Graduate School, and the Whitaker Foundation.
In vivo 13C NMR measurement of total brain glycogen concentrations in the conscious rat In-Young Choi1, Rolf Gruetter1
1University of Minnesota, Medical School, Center for Magnetic Resonance Research, Minneapolis, MN USA; Introduction Glycogen is an endogenous store of carbohydrates that can serve as an energy reserve. Compared to other organs, glycogen concentration in the brain is relatively low (2 - 5µmol/g in rodents) (1). To date, brain glycogen concentration was mainly measured by invasive methods such as biochemical analysis and enzymatic analysis, which can introduce errors in measurements due to rapid postmortem degradation of brain glycogen (2). Brain glycogen can be measured non-invasively by 3D-localized 13C NMR (3, 4). Slow turnover of NAA (0.6 µmol/g/h) has been shown previously using 13C-labeled glucose infusion (5). The purpose of this study was (a) to estimate total concentration of brain glycogen in conscious rats and (b) to estimate the turnover rate of brain glycogen relative to NAA and glutamate metabolism. Methods After a 24 hour fast, six male Sprague-Dawley rats were fed ad libitum only with 99%-enriched [1-13C] glucose solution (~5% wt/vol) for over two days (13C-prelabeled rat). The animals were allowed to move freely without any surgery or restraint. To measure 13C-label incorporation by NMR, animals were intubated and mechanically ventilated. Right after preparation, α-chloralose (24 mg/kg/h) was applied and 13C NMR spectra were acquired from a 3D-localized volume (~500 µl) of the brain (3, 4). [1-13C] glycogen and [6-13C] NAA concentrations were measured using the external reference method (6). Results and Discussion Label incorporation into NAA was very slow (0.6 µmol/g/h), but measurable (Fig. 1), with a turnover time of T1/2 ~14 h (n = 6). The NAA C6 was initially labeled ~90 min faster than the NAA C3. This delayed 13C-label incorporation into aspartyl NAA C3 was consistent with the slower labeling of its precursor, neuronal aspartate, compared to the precursor of NAA C6, neuronal acetyl CoA. NAA turnover was estimated to be complete and implied one compartment for NAA synthesis. Therefore, from the observation of a slow turnover rate and 13C-label incorporation into NAA similar to that of brain glycogen (3), we assumed that the fractional enrichment (FE) of NAA was half of that of brain glycogen after over two days of [1-13C] glucose administration. The 13C NMR spectrum of the 13C-prelabeled rat brain in Fig. 2 shows labeling of NAA, glycogen and glucose, glutamate (Glu) and glutamine (Gln), aspartate (Asp), and glutathione (GSH). An excellent correlation between brain glycogen C1 and NAA C6 concentrations was observed in Fig. 3 (r = 0.93, p < 0.01 for correlation, solid line). Total concentration of brain glycogen (open squares in Fig. 3) was estimated based on the estimated FE of glycogen assumed to be twice that of NAA (Fig. 3) and the stable concentration of total NAA (8.5 ± 0.1 µmol/g by 1H MRS). Estimated total concentration of brain glycogen was 3.3 ± 0.3 µmol/g (mean ± SE), in excellent agreement with the literature (7). In contrast to the tight correlation between glycogen C1 and NAA C6, 13C-labeled concentrations of brain glycogen and glutamate C4 showed no statistically significant correlation (r = - 0.1, p > 0.86), indicating that glutamate turnover was faster than brain glycogen turnover. This result is consistent with slow metabolism of brain glycogen in the conscious rat brain. Conclusions Total concentration of brain glycogen in vivo can be reliably estimated during chronic ingestion of 13C-labeled substrates. The correlation between label incorporations into NAA and into brain glycogen suggests that brain glycogen metabolism is slower than glutamate metabolism and comparable to NAA metabolism in conscious animals. References 1. Strang and Bachelard J Neurochem 18: 1067 (1971) 2. Sagar, et al. Brain Res 417: 172 (1987) 3. IY Choi, et al. J Neurochem 73: 1300 (1999) 4. IY Choi, et al. Magn Reson Med 44: 387 (2000) 5. IY Choi and R Gruetter J Neurochem S86D (2000)
6. R Gruetter, et al. Magn Reson Med 20: 327 (1991) 7. DD Clarke, et al. Circulation and energy metabolism of the brain in Basic neurochemistry pp. 638 (1999) Acknowledgments Supported by NIH (P41RR08079, R21DK58004), the Graduate School, and the Whitaker Foundation.
Figure 1. Time-resolved observation of NAA Metabolism. The detection of 13C-label incorporation into NAA C3 and C6 during [1-13C] glucose infusion.
Figure 2. 13C NMR spectrum of the 13C-prelabeled rat brain. Spectrum acquired from the rat brain after over 48 h administration of 99% enriched [1-13C] glucose ad libitum.
Figure 3. Total brain glycogen concentration (open squares) and the relationship between 13C label in glycogen C1 and in NAA C6.
Proc. Intl. Soc. Mag. Reson. Med 9 (2001)
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Proc. Intl. Soc. Mag. Reson. Med 9 (2001)
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