Renal metabolism of hyperpolarized [1-13C]aspartate

Aspartic acid is involved in several central metabolic pathways, including gluconeogenesis, the urea cycle, de novo nucleotide synthesis, the malate-aspartate shuttle, and via transmination the TCA cycle. The conversion in vivo of hyperpolarized [1-13C]pyruvate to aspartate, via pyruvate carboxylation to oxaloacetate, has been reported in rat kidney [1] and in the mouse liver [2]. Additionally, the metabolism of hyperpolarized [1-13C]aspartate to oxaloacetate has been noted in cultured cells supplemented with 2-oxoglutarate [3]. Aspartate is also of interest with its conversion to succinate in ischemic tissue implicated in subsequent reperfusion injury [4]. Here we report initial experiments with hyperpolarized [1-13C]aspartate in the rat kidney. [1-13C]Aspartic acid was formulated with OX063 trityl radical as in [3] as the tris salt, with the addition of glycerol (14% v/v) to improve glassing. The formulation is highly viscous and dissolves poorly, limiting the usable dose. Experiments were performed in a 9.4T animal scanner and a 7T polarizer operating at 1K, with automated rapid transfer and infusion. Rats (n=2) were isoflurane anesthesized, a femoral vein catheterized, and a quadrature 13C surface coil placed over the kidney. Respiration-triggered (TR ~3s) pulse-acquire (BIR4-30o) scans were started at the time of infusion of [1-13C]aspartate (~16 μmol/kg in 1.2 ml). Three metabolites were detected, with chemical shifts of 183.51 ppm (1.5%), 182.31 ppm (0.5%) and 181.26 ppm (0.6%), corresponding to [1-13C]malate, [4-13C]malate and an unidentified species provisionally assigned as N-acetyl[1-13C]aspartate. [4- 13C]aspartate was at natural abundance, but [4-13C]malate results from conversion to fumarate or succinate and label scrambling, and the greater prominence of [1-13C]malate indicates oxaloacetate as an intermediate. These results show the potential of hyperpolarized [1- 13C]aspartate as an in vivo metabolic probe.

Hyperpolarized Magnetic Resonance Methods to Investigate Metabolic Pathways in Liver and Immune Cells

Emine Can

Dysregulation of cellular metabolism is a key feature of major diseases such as diabetes and cancer. Monitoring cellular metabolic alterations is of critical importance for improved understanding of disease progression and the development of novel therapeutics. Carbon-13 magnetic resonance spectroscopy (13C MRS), which can detect and quantitate metabolites in a non-invasive manner, is well suited for clinical and pre-clinical metabolic studies. The low natural abundance of 13C allows one to trace exogenously administered 13C-labeled substrates by MRS and which enables the study of complex metabolic pathways in living subjects. However, the low sensitivity of 13C MRS limits the number of metabolites that can be detected with this technique. Hyperpolarized (HP) 13C MRS using dissolution dynamic nuclear polarization (d-DNP) is a novel metabolic imaging technique which allows real-time monitoring of the dynamics of in vivo metabolism by temporarily increasing the signal intensity more than 10,000-fold over conventional 13C MRS. This thesis is focused on the biomedical applications of HP 13C MRS, particularly to investigate in vivo hepatocellular energy metabolism in rodents and metabolic changes in active immune cells. Customized MRI/NMR probes and associated acquisition sequences were designed and implemented to improve the sensitivity and throughput of the HP MR experiments. A HP 13C MRS protocol for the sensitive in vivo detection of hepatic pyruvate metabolism was developed. HP [1-13C]pyruvate was infused at progressively decreasing doses to healthy rats under different nutritional conditions via a rapid automated infusion. It was shown that liver metabolism can be measured in vivo with HP [1-13C]pyruvate administered at near-physiological levels. While conversion to HP bicarbonate was detected in the liver of fasted rats, treatment with the phosphoenolpyruvate carboxykinase inhibitor 3-mercaptopicolinate resulted in significantly lower levels compared to fed rats, which supports the notion that hepatic gluconeogenic metabolism can be directly probed in vivo with HP pyruvate. Additionally, real-time in vivo hepatocellular glutamine and pyruvate metabolism were investigated in hepatocellular carcinoma (HCC) induced in liver receptor homolog-1 (LRH-1) knockout mice. The influence of LRH-1 on glutaminase activity was monitored through the conversion of HP [5-13C]glutamine to [5-13C]glutamate, and the impact of LRH-1 deletion on the citric acid cycle could be investigated by conversion of HP [1-13C]pyruvate to four-carbon mitochondrial metabolites. The results demonstrate the potential of HP 13C MRS as a non-invasive method for understanding the role of oncogenic mutations in nuclear receptors in the metabolic reprogramming of cancer cells. Several other 13C-labeled substrates were studied, including [1-13C]cysteine and Î³-glutamyl[1-13C]glycine, a novel probe to detect gamma-glutamyl transferase (GGT) activity. The metabolic changes in T cells upon activation was investigated using HP [1-13C]cysteine as a potential selective probe of active T cells and HP Î³-glutamyl[1-13C]glycine to detect increased GGT expression by T cells in the active state. This work includes the first report on applications of HP 13C MRS in T cells and demonstrates the feasiblity to study the immune response with this technique. In conclusion, this work shows the potential of HP 13C MRS to investigate in vivo liver metabolism with administered 13C-labeled

EPFL2017

[13C]bicarbonate labelled from hyperpolarized [1-13C]pyruvate is an in vivo marker of hepatic gluconeogenesis in fasted state

Josefina Adriana Maria Bastiaansen, Emine Can, Arnaud Comment, Rolf Gruetter, Hikari Ananda Infinity Yoshihara

Hyperpolarized [1-13C]pyruvate enables direct in vivo assessment of real-time liver enzymatic activities by 13C magnetic resonance. However, the technique usually requires the injection of a highly supraphysiological dose of pyruvate. We herein demonstrate that liver metabolism can be measured in vivo with hyperpolarized [1-13C]pyruvate administered at two- to three-fold the basal plasma concentration. The flux through pyruvate dehydrogenase, assessed by 13C-labeling of bicarbonate in the fed condition, was found to be saturated or partially inhibited by supraphysiological doses of hyperpolarized [1-13C]pyruvate. The [13C]bicarbonate signal detected in the liver of fasted rats nearly vanished after treatment with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, indicating that the signal originates from the flux through PEPCK. In addition, the normalized [13C]bicarbonate signal in fasted untreated animals is dose independent across a 10-fold range, highlighting that PEPCK and pyruvate carboxylase are not saturated and that hepatic gluconeogenesis can be directly probed in vivo with hyperpolarized [1-13C]pyruvate.

2022

Ultra high magnetic field for glial contribution into brain metabolism studied by MR spectroscopy and CEST methods for molecular imaging of glycogen

Elise Marie Catherine Vinckenbosch

Magnetic resonance at ultra-high field increases signal and spectral dispersion. In this thesis, I use those characteristics to investigate three different subjects in 13C spectroscopy, hyperpolarized methods and chemical exchange saturation transfer (CEST) used for molecular imaging. In the brain, metabolism is driven by glia-neuron interactions and is composed of at least two compartments with distinct TCA cycle kinetics. The first study examines cerebral metabolic adaptation after an induced glial TCA-cycle impairment using 1H and 13C spectroscopy. We treated rats with fluoroacetate, which specifically blocks glial aconitase and measured changes in neurochemical profile at euglycemic and hyperglycemic condi-tions by in vivo 1H MR spectroscopy. In addition, we evaluated the brain-compartmentalized metabolic adap-tation by following 13C-incorporation into brain amino acids by modern dynamic 13C MRS methods during infu-sion of [1,6-13C2]glucose. By following [1,6-13C2]glucose incorporation, we successfully observed an augmen-tation of pyruvate carboxylase and glutamine synthetase fluxes and a 20% decrease of glial TCA cycle flux. Neuronal metabolism was also affected. In the second study, we focused on hyperpolarized methods to increase acetate 13C polarization allowing us to follow the kinetics of its metabolic products in the TCA cycle. Indeed, infusion of hyperpolarized [1-13C]acetate, an astrocyte-specific precursor, and the in vivo detection of 2-oxo[5-13C]glutarate (2OG), a TCA intermediate, enables the direct analysis of glial Krebs cycle activity. We examined the effect of hyperpolar-ized acetate concentration on its cerebral metabolism and calculate the production rate of 2OG. A healthy group was compared to rodents treated with fluoroacetate. The conversion rates of acetate to 2OG were calculated and were found to depend on the substrate dose. We successfully estimated 2OG production rates. In partially inhibited TCA cycle conditions, the production rate of 2OG was reduced by a quarter. This level of reduction is agrees with the percentage of inhibition calculated in the first study. Finally, we investigated glycogen imaging in skeletal muscle using the CEST strategy. It has been demonstrat-ed in perfused liver that glycogen can be detected indirectly through the water signal using CEST. Glycogen detection is, due to its resonance close to water, strongly sensitive to spectral dispersion and improved by a higher static magnetic field. In contrast to liver, which contains ~300 mmol/g of glycogen and enjoys good glycogen specificity, myocellular content is also rich in creatine, which produces a spectral signal on the same order of amplitude as its glycogen content of ~80-30 mmol/g. In this study, we allowed glycogen to be de-tectable in vivo and distinguishable from the neighboring creatine at 14.1T. We propose to use creatine as an internal reference for muscular glycoCEST analysis. Scanning muscle after physical exercise, resulted in a 50% reduction of glycoCEST compared to muscle at resting state. This result supportis the specificity of the CEST method and is consistent with literature. This last study demonstrates for the first time that glycoCEST imag-ing is feasible in vivo.