We have for the first time, illustrated the dynamic evolution of multiplet isotopomers in the awake mouse brain by ex vivo 13
C NMR spectroscopy. Because of the superior resolution of the 13
C spectra, the signals arising from glutamate, glutamine, aspartate and GABA were not only detected separately, but were also discerned as multiplets due to the coupling of adjacent 13
C carbons within each molecule, which were precisely resolved using conventional NMR equipment. This method overcomes the common technical challenges posed by the mouse brain, namely the low sensitivity of 13
C-NMR techniques to resolve 13
C isotopomers and the notable effect of anesthesia on brain metabolism, while also allowing for the determination of isotopomer steady state commonly assumed in intraperitoneal injection approaches (Walls et al., 2011
). We have shown that an accurate resolution of 13
C multiplets in the awake mouse brain is feasible and potentially valuable to determine rates of substrate uptake and of major brain metabolic pathways with further assistance from metabolic modeling. Importantly, in contrast with other studies performed in rats (Patel et al., 2004
; Sibson et al., 1998
; Sibson et al., 2001
), the concentration of 13
C label infused avoided or minimized hyperglycemia (the maximum blood glucose level reached was 11 mM), preventing potential systemic metabolic confounders when excessive, supraphysiological glucose concentrations are reached.
Kinetic studies designed to measure rates of biochemical reactions within a metabolic network rely upon the analysis of multiple 13
C spectra. Steady-state isotopomer analysis can also allow for the determination of relative flux and labeling patterns of metabolic pathways, such as anaplerosis (Malloy, 1998
). For instance, in the heart (mostly considered as a single-type cell organ for the purposes of NMR analysis), a comprehensive isotopomer analysis of glutamate C4 under steady-state 13
C enrichment conditions allowed the determination of carbon flux through the oxidative and anaplerotic pathways (Malloy et al., 1987
). Moreover, a similar analysis of glutamate C4 – in this case not restricted to isotopic steady-state conditions- can be applied to estimate the enrichment of 13
C-acetyl-CoA in the intact tissue, a valuable parameter that provides information about substrate oxidation and competition in cells (Malloy et al., 1990
). In the brain, the same analysis can be developed to compute relative rates of anaplerosis and 13
C enrichment of acetyl-CoA, with the caveats imposed by a multi-compartment system in which the rate of anaplerosis may differ between compartments, and in which the pool of 13
Cacetyl-CoA may substantially diverge, depending of the availability and of substrate preference in each cell type considered.
As illustrated in , multiplets were well resolved in all isotopomers, with glutamate exhibiting the highest resolvability of multiplets in all carbon positions relative to other isotopomers. Two potential reasons explain this finding: 1) glutamate is highly abundant in the brain (~ 12 µmol/g of wet tissue) relative to other 13
C-labeled amino acids (Chang et al., 1981
), 2) and glutamate and α-ketoglutarate are in rapid exchange relative to neuronal and glial TCA cycle flux, resulting in the early appearance of glutamate in the 13
C spectrum relative to other isotopomers (Oz et al., 2004
; Yang et al., 2009
). On the other hand, the 13
C label incorporation (detected as 13
C multiplets) was found to be roughly proportional for interrelated molecules such as glutamate, glutamine and GABA. Overall, brain metabolic steady state was reached approximately at 150 min of infusion, which was significantly earlier than that previously observed in anesthetized mice (~250 min after infusion of larger amount of [1,6-13
]glucose than the present protocol (bolus: 0.130 mg/g, infusion: 0.016 mg/g/min; (Nabuurs et al., 2008
)). As noted in earlier studies of the rat brain, a likely explanation for this phenomenon is that anesthesia can limit the interpretation of metabolic fluxes as it significantly impacts the rate of certain key fluxes, such as the rate of the glutamate-glutamine cycle (Vnt
), which is notably higher in conscious rats (0.57 ± 0.21 µmol/g/min) (Oz et al., 2004
) relative to rats anesthetized with α-chloralose (0.2 ± 0.08 µmol/g/min) (Sibson et al., 2001
) or pentobarbital (0.04 ± 0.01 µmol/g/min) (Choi et al., 2002
Previous in vivo
NMR approaches to mouse brain metabolism were limited because of the low 13
C spectral resolution leading to a substantial overlap between adjacent isotopomers, i.e. glutamate and glutamine C3, and because animals were subjected to anesthesia that interferes, in all likelihood, with brain metabolism (Nabuurs et al., 2008
; Peled-Kamar et al., 1998
). The studies cited have analyzed the appearance of 13
C in TCA cycle-related metabolites (for example, glutamate and glutamine) by measuring the 13
C contents of individual carbons identified in the NMR spectra. However, we have shown that multiplets can provide additional information (superior to 13
C contents alone) for the computation of metabolic flux rates and the 13
C enrichment of TCA cycle substrates (Jeffrey et al., 1999
). For instance, the estimation of the oxygen consumption in the rat heart is significantly improved when multiplets are added to the kinetic analysis of NMR data, detecting 60% changes in oxygen utilization depending upon experimental conditions (Jeffrey et al., 1999
). This feature of the method can simplify the characterization of different physiological and pathological conditions associated with abnormal oxygen consumption and metabolic rate. On the other hand, the presented approach will potentially benefit from innovative 13
C-enhancing techniques such as cryogenically cooled probes and hyperpolarization (Kovacs et al., 2005
). Cryogenically cooled probes typically enable a 3–4-fold enhancement of the detection sensitivity in high-resolution NMR compared to conventional probes (Kovacs et al., 2005
). Additionally, methods that strengthen the polarization of nuclear spins (termed hyperpolarization methods) such as dynamic nuclear polarization (DNP) can increase the 13
C sensitivity by a factor of up to 10,000 (Ardenkjaer-Larsen et al., 2003
). We anticipate that these techniques may potentially improve even more the quality of the ex vivo 13
C spectra and will enhance the resolution of in vivo 13
C multiplets in the mouse brain.
In summary, the detection and analysis of time-dependent ex vivo 13C NMR spectroscopy data arising from awake mouse brain metabolism can be enhanced to include information-rich multiple resonances. In this work, we have presented a method for the generation of high-quality 13C spectra from the mouse brain that permitted accurate resolution of multiplets in glutamate, glutamine, GABA and aspartate. The results demonstrate that, overall, all detected TCA cycle-derived isotopomers manifest a similar evolution of multiplets reaching steady state at 150 min with a quasi-physiological [1,6-13C2]glucose infusion. We anticipate that this method will be useful for the estimation of the rates of major metabolic pathways in the normal awake mouse brain and also in transgenic mouse models of human neurological diseases.
We have developed a method for the ex vivo resolution of NMR multiplets arising from the awake mouse brain after the infusion of [1,6-13C2]glucose. (Most NMR studies have been conducted in large animals (often under anesthesia) because the weight of the target organ is a limiting factor for NMR).
NMR spectra obtained by this method display favorable signal-to-noise ratios that lend themselves to precise analysis.
The method enables the accurate resolution of multiplets over time in the awake mouse brain.
We anticipate that this method will be broadly applicable to compute brain fluxes in normal and transgenic mouse models of neurological disorders.