Association between Brain and Plasma Glutamine Levels in Healthy Young Subjects Investigated by MRS and LC/MS

Both glutamine (Gln) and glutamate (Glu) are known to exist in plasma and brain. However, despite the assumed relationship between brain and plasma, no studies have clarified the association between them. Proton magnetic resonance spectroscopy (MRS) was sequentially performed twice, with a 60-min interval, on 10 males and 10 females using a 3T scanner. Blood samples for liquid chromatography-mass spectrometry (LC/MS) to measure Gln and Glu concentrations in plasma were collected during the time interval between the two MRS sessions. MRS voxels of interest were localized at the posterior cingulate cortex (PCC) and cerebellum (Cbll) and measured by the SPECIAL sequence. Spearman's correlation coefficient was used to examine the association between brain and plasma metabolites. The Gln concentrations in PCC (mean of two measurements) were positively correlated with Gln concentrations in plasma (p < 0.01, r = 0.72). However, the Glu concentrations in the two regions were not correlated with those in plasma. Consideration of the different dynamics of Gln and Glu between plasma and brain is crucial when addressing the pathomechanism and therapeutic strategies for brain disorders such as Alzheimer's disease and hepatic encephalopathy.

Improved ¹H-[¹³C] and ¹H NMR methods for in vivo measurement of cerebral metabolism

Lijing Xin

Nuclear magnetic resonance (NMR) spectroscopy can be applied in vivo to measure static or dynamic biochemical information, e.g., concentrations of metabolites and metabolic fluxes, using various nuclei such as 1H, 13C, 31P and 15N. The work of this thesis involves both 1H and 13C nuclei and focuses on improving 1H NMR detection methods for measuring metabolites in vivo in rat brain. 13C NMR spectroscopy can be used to monitor the flow of 13C label from a 13C enriched substrate, such as glucose or acetate, into different NMR detectable metabolites, e.g., glutamate (Glu) and glutamine (Gln), for the quantitative study of cerebral metabolism in vivo. 1H-observed 13C-edited (1H-[13C]) NMR spectroscopy is an alternative to direct 13C NMR detection of 13C labeled metabolites, allowing a higher spatial and temporal resolution, albeit at a lower spectral resolution. In this context, a hybrid full signal intensity 1H-[13C] NMR sequence, combining a 13C editing block based on an inversion B1 insensitive spectral editing pulse (BISEP) with a spin-echo based localization (SPECIAL), was developed and implemented at ultra-high magnetic field (14.1 T) to benefit from increased sensitivity and spectral resolution at high B0. As a result, high quality 1H-[13C] NMR spectra were obtained, leading to an improved quantification of 13C labeled metabolites, which allowed the measurement of time courses of Glu C4, Gln C4, as well as, for the first time, of Glu C3 and of Gln C3, with high temporal resolution from a small acquisition volume. Although at high magnetic field spectral resolution and sensitivity are improved, spectral overlap is still present, e.g., the N-acetylaspartate (NAA) C6 is easily obscured by the intensive labeling of Glu C3 and Gln C3 (Glx C3), due to their complex coupling patterns. To improve the detection of unresolved 13C labeled metabolites, we developed an alternative 1H-[13C] NMR editing sequence termed RACED-STEAM (selective Resonances suppression by Adiabatic Carbon Editing and Decoupling single-voxel STimulated Echo Acquisition Mode), which can selectively suppress 1H resonances bound to a specific 13C to uncover resonances that are difficult to resolve in the 1H-[13C] NMR spectrum. Results showed the efficient suppression of Glx C3 and Glu C4, allowing the detection of NAA C6 and Gln C4 at 9.4 T. The application of this method to measure the time course of NAA C6 demonstrated that NAA C6 turnover can be measured at very low levels of isotopic enrichment in a small volume, within a time frame of a few hours. The proposed scheme could also be extended to lower magnetic fields provided that the 13C chemical shifts remain sufficiently resolved. Similarly, due to limited spectral resolution and sensitivity, editing techniques for the in vivo detection of metabolites using 1H NMR spectroscopy are still in development. Many in vivo 1H NMR editing sequences are usually performed at moderate to long echo times (TE). Furthermore, several 1H NMR spectroscopy studies are performed at long TE to avoid the confounding effect of macromolecular signals on metabolite quantification. In such cases, proton T2 relaxation times of metabolites have to be taken into account for proper quantification of metabolite concentrations. While T2 relaxation times of singlets have been characterized in several studies, due to the relative experimental simplicity, similar information is lacking from coupled spin resonances of cerebral metabolites. In this thesis, spectral simulations based on the density matrix formalism were initially performed to predict the response of spin systems to the pulse sequence used. T2 relaxation times of coupled spin resonances and singlet resonances of cerebral metabolites were then measured in rat brain in vivo at 9.4 T. Data analysis was performed using LCModel combined with simulated TE-specific spectra. The aforementioned spectral simulations were further used to optimize pulse sequence parameters for the detection of metabolites, such as glycine, whose resonance signal is overlapped with the more intense myo-inositol resonances. A favorable short TE (i.e., 20 ms) was sufficient to reduce the signal intensity of myo-inositol, allowing the detection of glycine in vivo in rat brain at 9.4 T. This work suggests that at high magnetic fields, glycine can be measured at a relatively short TE without additional editing efforts. Moreover, glycine is present at a particularly high concentration in the medulla oblongata (MO). Therefore, we further measured regional distribution of glycine in the hippocampus, cortex, striatum and MO of the rat brain, as well as the highly specific neurochemical profile of the MO. In conclusion, dedicated approaches for 1H NMR detection developed and validated in this thesis lead to improved dynamic measurement of 13C labeling time courses such as the time courses of 13C labeling of Glu C3 and Gln C3, as well as the spectral resolved NAA C6, the direct in vivo detection of glycine in rat brain at 9.4 T and the measurement of T2 relaxation times of numerous J-coupled metabolites for the first time.

EPFL2009

Quantitative proton spectroscopic imaging of the neurochemical profile in rat brain with microliter resolution at ultra-short echo times

Giulio Gambarota, Rolf Gruetter

Proton spectroscopy allows the simultaneous quantification of a high number of metabolite concentrations termed the neurochemical profile. The spin echo full intensity acquired localization (SPECIAL) scheme with an echo time of 2.7 ms was used at 9.4T for excitation of a slab parallel to a home-built quadrature surface coil in conjunction with phase encoding in the two remaining spatial dimensions to yield an effective spatial resolution of 1.7 microL. The absolute concentrations of at least 10 metabolites were calculated from the spectra of individual voxels using LCModel analysis. The calculated concentrations were used for constructing quantitative metabolic maps of the neurochemical profile in normal and pathological rat brain. Summation of individual spectra was used to assess the neurochemical profile of unique brain regions, such as corpus callosum, in rat for the first time. Following focal ischemia in rat pups, imaging the neurochemical profile indicated increased choline groups in the ischemic core and increased glutamine in the penumbra, which is proposed to reflect glutamate excitotoxicity. We conclude that it is feasible to achieve a sensitivity that is sufficient for quantitative mapping of the neurochemical profile at microliter spatial resolution.

Wiley-Blackwell2008

Mathematical Modeling of Brain Energy Metabolism, Measured with PET and MRS in Rodents

Bernard Lanz

Positron emission tomography (PET) and nuclear magnetic resonance spectroscopy (MRS) are two biomedical measurement techniques developed in the end of the XXth century, which drastically improved the amount of accessible information available in vivo. PET became popular through the most widely used tracer, fluorodeoxyglucose (FDG), which enables the measurement of the local glucose utilization and is nowadays routinely applied in clinical practice. Nuclear magnetic resonance is used in a large array of applications such as in analytical chemistry or chemical structure determination and is essentially known for its versatile medical imaging capabilities, grouped under the name of magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS). In vivo MRS measures concentrations of metabolites by interacting with various nuclei such as 1H, 13C or 15N, with the major asset that MRS enables the identification of the chemical position at which the detected nuclei is located in the measured molecule. When coupled with labeled substrate infusions, dynamic MRS gives the opportunity to probe specific biochemical reactions directly in vivo, which opens the way to a wide range of metabolic studies. However, the correct interpretation of the elaborate dynamic labeling data acquired either with MRS or PET tracer experiments requires the application of adapted metabolic models to derive quantitative metabolic rates characterizing the biochemical processes under study. The work of this thesis involves both PET and MRS studies of brain energy metabolism in rodents and focuses on the development of adapted metabolic models to derive reliable metabolic fluxes characterizing various brain metabolic processes, such as glucose consumption, glial and neuronal oxidative metabolism and neurotransmission. FDG-PET enables the measurement of the cerebral metabolic rate of glucose (CMRGlc) using a three-compartment metabolic model for FDG transport across the blood-brain barrier and FDG phosphorylation to FDG-6P. Although this method is well established, a main drawback of CMRGlc measurement with FDG-PET is the necessity to characterize the available FDG concentration in the plasma over the experiment duration, the so-called arterial input function. This constraint is a strong limitation for preclinical studies in rodents, due to their low blood volume. We developed therefore a new method to extract the FDG input function directly from the PET images in rats and mice, using the time-activity curve of a voxel located in the inferior vena cava. The method was validated by comparison with a dedicated external blood counter and manual blood sampling. Using this method, a CMRGlc of ~0.22 μmol/g/min was determined in the mouse cortex. Glial oxidative metabolism can be specifically assessed using glial specific substrates, in particular [1-11C] acetate. However, no existing metabolic model described the 11C tissue activity curve in terms of neuroglial energy metabolism. In order to extract quantitative metabolic fluxes characterizing the glial TCA cycle rate and glutamate/glutamine cycling, we adapted the neuroglial two-compartment modeling approach previously used in 13C MRS cerebral metabolic studies to interpret positron emission data following 11C-acetate injection in the rat. The precision and accuracy of the estimated metabolic parameters was tested and a composite glial metabolic flux Vgtg=0.136 ± 0.042 μmol/g/min and a neurotransmission flux Vnt=0.170 ± 0.103 μmol/g/min were obtained. This approach enabled a direct comparison of the metabolic fluxes measured using 11C PET with the values determined with 13C MRS. In the field of 13C MRS, we took advantage of the higher sensitivity and spectral resolution available at high magnetic field (14.1T) to measure glial and neuronal oxidative metabolism, glutamatergic neurotransmission and pyruvate carboxylation in more details and with higher precision using both [1,6-13C2] glucose and [2-13C] acetate infusions in rats. In the acetate study, the glial specific uptake of acetate enabled a quantification of glial oxidative metabolism with unrivalled precision. We could estimate for the first time separately the transmitochondrial flux Vx in the glial and neuronal compartment, a biochemical pathway which has been subject of strong controversy. The glial and neuronal Vx were determined with high precision, as calculated through Monte Carlo simulation, and their value was on the same order of magnitude than the respective glial and neuronal TCA cycle flux. The model was extended to estimate the fraction of glutamate located in the glial compartment. A glial glutamate concentration of 0.6 ± 0.1 μmol/g was found. The values of the different metabolic fluxes characterizing the neuroglial system (Vtcag=0.27, Vxg=0.17, VPC=0.087, VNT=0.15, Vtcan=0.37 and Vxn=0.46 μmol/g/min) were in very good agreement with the values found using [1,6-13C2] glucose infusion. In addition, the effect of the MRS temporal resolution, experiment duration and signal- to-noise ratio on the precision of the derived metabolic fluxes was analyzed. Previous simulation studies showed that two-compartment modeling of neuroglial metabolism using 13C glucose infusion could lead to unreliable parameter estimations. However, using polarization transfer 13C MRS at 14.1T, the carbon positions C4, C3 and C2 of glutamate and glutamine as well as the positions C3 and C2 of aspartate could be measured with high SNR. The C2 positions were used in two-compartment modeling for the first time, enabling a precise measurement of the apparent glutamatergic neurotransmission and glial metabolism, including pyruvate carboxylation, as confirmed by Monte Carlo simulation. The glial dilution at the level of acetyl-CoA, which is related to the metabolism of other substrates than glucose, could also be characterized. Brain metabolism under hyperammonemic conditions was probed with 15N MRS under labeled ammonia infusion and modeled using a modified neuroglial two- compartment model describing the dynamics of [5-15N] glutamine, [2-15N] glutamate and [2-15N] glutamine. Due to detoxification processes, the total glutamine concentration, measured with 1H MRS, was steadily increasing. This non-steady- state metabolic condition resulting from the hyperammonemic stress was taken into account in the model, resulting in the determination of the apparent neurotransmission (0.26 ± 0.030 μmol/g/min), the glutamate dehydrogenase (0.029 ± 0.002 μmol/g/min) and net glutamine accumulation (0.033 ± 0.001 μmol/g/min). Finally, compartmental modeling was applied to 13C labeling studies in the awake rat, fed during 5, 24 or 48 h with a [1-13C]-labeled glucose solution, to determine brain glycogen and NAA turnover times (τGlyc=5.3 ± 3.2 h and τNAA=15.6 ± 6.5 h), using 13C spectra of brain extracts. A group of rats followed a mild brain activation protocol over 5 hours, which resulted in a decreased glycogen turnover time (τGlyc =2.9 ± 1.2 h).