As alterations in tissue pH underlie many pathological processes, the capability to image tissue pH in the clinic could offer new ways of detecting disease and response to treatment. Dynamic nuclear polarization is an emerging technique for substantially increasing the sensitivity of magnetic resonance imaging experiments. Here we show that tissue pH can be imaged in vivo from the ratio of the signal intensities of hyperpolarized bicarbonate (H(13)CO(3)(-)) and (13)CO(2) following intravenous injection of hyperpolarized H(13)CO(3)(-). The technique was demonstrated in a mouse tumour model, which showed that the average tumour interstitial pH was significantly lower than the surrounding tissue. Given that bicarbonate is an endogenous molecule that can be infused in relatively high concentrations into patients, we propose that this technique could be used clinically to image pathological processes that are associated with alterations in tissue pH, such as cancer, ischaemia and inflammation.

Dynamic nuclear polarization of 13 C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-13 C2]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-13 C2]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized 13 C-labeled malate from [1,4-13 C2]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically. magnetic resonance imaging ͉ spectroscopy ͉ metabolism ͉ cell death ͉ lymphoma

Mechanistic details of mammalian metabolism in vivo and dynamic metabolic changes in intact organisms are difficult to monitor because of the lack of spatial, chemical, or temporal resolution when applying traditional analytical tools. These limitations can be addressed by sensitivity enhancement technology for fast in vivo NMR assays of enzymatic fluxes in tissues of interest. We apply this methodology to characterize organ-specific short chain fatty acid metabolism and the changes of carnitine and coenzyme A pools in ischemia reperfusion. This is achieved by assaying acetyl-CoA synthetase and acetyl-carnitine transferase catalyzed transformations in vivo. The fast and predominant flux of acetate and propionate signal into acyl-carnitine pools shows the efficient buffering of free CoA levels. Sizeable acetyl-carnitine formation from exogenous acetate is even found in liver, where acetyl-CoA synthetase and acetylcarnitine transferase activities have been assumed sequestered in different compartments. In vivo assays of altered acetate metabolism were applied to characterize pathological changes of acetate metabolism upon ischemia. Coenzyme pools in ischemic skeletal muscle are reduced in vivo even 1 h after disturbing muscle perfusion. Impaired mitochondrial metabolism and slow restoration of free CoA are corroborated by assays employing fumarate to show persistently reduced tricarboxylic acid (TCA) cycle activity upon ischemia. In the same animal model, anaerobic metabolism of pyruvate and tissue perfusion normalize faster than mitochondrial bioenergetics.Acetyl coenzyme A (acetyl-CoA) 2 is a central metabolite that connects metabolic paths such as fatty acid degradation and synthesis, cholesterol biosynthesis, glycolysis, and the TCA cycle (Fig. 1). Thus, acetyl-CoA is among the key molecules of energy and intermediary metabolism. Acetyl-CoA is generated by a number of enzymes in mammals including pyruvate dehydrogenase, ␤-ketothiolase, and ATP citrate-lyase (1). A less well-explored metabolic pathway forming acetyl-CoA in mammals is the acetyl-CoA synthetase (AceCS)-catalyzed catabolism of acetate (Fig. 1), which accounts for up to 10% of the energy expenditure in humans (2).Esterification of carboxylic acids is a common means of generating activated molecules for entrance into metabolic pathways and renders the CoA-activated metabolite largely membrane impermeable. Pools of acyl-CoA esters in different cellular compartments require tight regulation because of their metabolic activity and because of the signaling function of some CoA esters (3). Under normal conditions, free CoA is regenerated by the metabolism esters, whereas large amounts of CoA esters may accumulate under stress conditions (4). Sufficient pools of free CoA are ensured under these conditions by buffering acyl-CoA:CoA ratios through transesterifications of acylCoA with carnitine. Carnitine also provides a shuttle for the flux of CoA-activated metabolites between intracellular compartments (5). Transesterification between CoA and carnitine est...

Central carbon metabolism of living Saccharomyces cerevisiae is visualized by DNP-NMR. Experiments are conducted as real time assays that detect metabolic bottlenecks, pathway use, reversibility of reactions and reaction mechanisms in vivo with subsecond time resolution.

ratio maintains cellular redox homeostasis and is a cellular metabolic

Dissolution dynamic nuclear polarization (DNP) provides a broadly applicable and rather simple means of developing probes for the real-time molecular imaging of cellular functions in vivo. The development of novel dissolution DNP substrate formulations is only rewarding for substrates that yield detectable metabolism within few minutes. In addition, in vivo preparations usually require amorphous samples at molar substrate concentrations for an efficient and reproducible DNP step with sufficient material. The composition ranges of novel substrate preparations need to be established experimentally owing to the solute's impact on vitrification behavior. Here, we describe simple rationales employed in the development of novel substrate preparations for dissolution DNP-magnetic resonance. Solution state substrate polarizations between 10 and 40 % have been obtained for *40 metabolic substrates in highly concentrated preparations that yield physiologically tolerable solutions with sufficient T 1 for in vivo nuclear magnetic resonance. Substrate metabolism is observed for novel in vivo substrates such as 3-hydroxybutyrate and aspartate.

Edited by Christian Griesinger Keywords:In vivo NMR Spectroscopy Metabolism Model organism Escherichia coli a b s t r a c tThe direct tracking of cellular reactions in vivo has been facilitated with recent technologies that strongly enhance NMR signals in substrates of interest. This methodology can be used to assay intracellular reactions that occur within seconds to few minutes, as the NMR signal enhancement typically fades on this time scale. Here, we show that the enhancement of 13 C nuclear spin polarization in deuterated glucose allows to directly follow the flux of glucose signal through rather extended reaction networks of central carbon metabolism in living Escherichia coli. Alterations in central carbon metabolism depending on the growth phase or upon chemical perturbations are visualized with minimal data processing by instantaneous observation of cellular reactions.

Powerful analytical tools are vital for characterizing the complex molecular changes underlying oncogenesis and cancer treatment. This is particularly true, if information is to be collected in vivo by noninvasive approaches. In the recent past, hyperpolarized 13 C magnetic resonance (MR) spectroscopy has been employed to quickly collect detailed spectral information on the chemical fate of tracer molecules in different tissues at high sensitivity. Here, we report a preclinical study showing that a-ketoisocaproic acid (KIC) can be used to assess molecular signatures of tumors with hyperpolarized MR spectroscopy. KIC is metabolized to leucine by the enzyme branched chain amino acid transferase (BCAT), which is found upregulated in some tumors. BCAT is a putative marker for metastasis and a target of the proto-oncogene c-myc. Very different fluxes through the BCAT-catalyzed reaction can be detected for murine lymphoma (EL4) and rat mammary adenocarcinoma (R3230AC) tumors in vivo. EL4 tumors show a more than 7-fold higher hyperpolarized 13 C leucine signal relative to the surrounding healthy tissue. In R3230AC tumor on the other hand branched chain amino acid metabolism is not enhanced relative to surrounding tissues. The distinct molecular signatures of branched chain amino acid metabolism in EL4 and R3230AC tumors correlate well with ex vivo assays of BCAT activity.Modern genetic tools have markedly improved the understanding of cancer as a genetic disease by linking the development, progression and remission of cancer to underlying genetic changes. 1,2 These approaches have also revealed the genetic heterogeneity of tumors. Thus, an understanding of the molecular signatures of the disease with noninvasive techniques would be highly desirable in order to define molecular targets for a tumor-specific or even personalized diagnosis and treatment.3 This can be achieved by hyperpolarized chemical shift imaging (CSI), which is a recently devised imaging modality for the visualization of molecular processes in vivo. [4][5][6] The method relies on a signal enhancement of the inherently weak nuclear magnetic resonance (NMR) signal by several orders of magnitude in a process termed dynamic nuclear polarization (DNP). This process increases the magnetization of nuclear spins ex situ to generate a 'hyperpolarized' molecule, which is injected as a nonradionuclide imaging marker. 7 The enhancement of detectable NMR signal by several orders of magnitude renders imaging experiments with a variety of substrates possible. As NMR is a high-resolution spectral technique, the method bears a unique potential to monitor chemical modifications of the hyperpolarized molecule in vivo. Previous studies using hyperpolarized pyruvate have detected cancer tissues by their increased anaerobic metabolism. 4,5,8 Notably, the method has also shown the potential to measure early tumor responses to therapy. 9 Cellular amino acid metabolism is regulated by the activity, organ distribution and cellular compartmentalization of metabolic enzymes incl...