A continuous flow bioreactor system for high-throughput hyperpolarized metabolic flux analysis.

Hyperpolarized carbon-13 labeled compounds are increasingly being used in medical MR imaging (MRI) and MR imaging (MRI) and spectroscopy (MRS) research, due to its ability to monitor tissue and cell metabolism in real-time. Although radiological biomarkers are increasingly being considered as clinical indicators, biopsies are still considered the gold standard for a large variety of indications. Bioreactor systems can play an important role in biopsy examinations because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo. We demonstrate here a proof-of-concept bioreactor and microcoil receive array setup that allows for ex vivo preservation and metabolic NMR spectroscopy on up to three biopsy samples simultaneously, creating an easy-to-use and robust way to simultaneously run multisample carbon-13 hyperpolarization experiments. Experiments using hyperpolarized [1-13C]pyruvate on ML-1 leukemic cells in the bioreactor setup were performed and the kinetic pyruvate-to-lactate rate constants ( k PL ) extracted. The coefficient of variation of the experimentally found k PL s for five repeated experiments was C V = 35 % . With this statistical power, treatment effects of 30%-40% change in lactate production could be easily differentiable with only a few hyperpolarization dissolutions on this setup. Furthermore, longitudinal experiments showed preservation of ML-1 cells in the bioreactor setup for at least 6 h. Rat brain tissue slices were also seen to be preserved within the bioreactor for at least 1 h. This validation serves as the basis for further optimization and upscaling of the setup, which undoubtedly has huge potential in high-throughput studies with various biomarkers and tissue types.

Detection of myocardial medium-chain fatty acid oxidation and tricarboxylic acid cycle activity with hyperpolarized [1-13 C]octanoate.

Under normal conditions, the heart mainly relies on fatty acid oxidation to meet its energy needs. Changes in myocardial fuel preference are noted in the diseased and failing heart. The magnetic resonance signal enhancement provided by spin hyperpolarization allows the metabolism of substrates labeled with carbon-13 to be followed in real time in vivo. Although the low water solubility of long-chain fatty acids abrogates their hyperpolarization by dissolution dynamic nuclear polarization, medium-chain fatty acids have sufficient solubility to be efficiently polarized and dissolved. In this study, we investigated the applicability of hyperpolarized [1-13 C]octanoate to measure myocardial medium-chain fatty acid metabolism in vivo. Scanning rats infused with a bolus of hyperpolarized [1-13 C]octanoate, the primary metabolite observed in the heart was identified as [1-13 C]acetylcarnitine. Additionally, [5-13 C]glutamate and [5-13 C]citrate could be respectively resolved in seven and five of 31 experiments, demonstrating the incorporation of oxidation products of octanoate into the tricarboxylic acid cycle. A variable drop in blood pressure was observed immediately following the bolus injection, and this drop correlated with a decrease in normalized acetylcarnitine signal (acetylcarnitine/octanoate). Increasing the delay before infusion moderated the decrease in blood pressure, which was attributed to the presence of residual gas bubbles in the octanoate solution. No significant difference in normalized acetylcarnitine signal was apparent between fed and 12-hour fasted rats. Compared with a solution in buffer, the longitudinal relaxation of [1-13 C]octanoate was accelerated ~3-fold in blood and by the addition of serum albumin. These results demonstrate the potential of hyperpolarized [1-13 C]octanoate to probe myocardial medium-chain fatty acid metabolism as well as some of the limitations that may accompany its use.

Efficient Hyperpolarization of U-13 C-Glucose Using Narrow-Line UV-Generated Labile Free Radicals.

Free radicals generated by UV-light irradiation of a frozen solution containing a fraction of pyruvic acid (PA) have demonstrated their dissolution dynamic nuclear polarization (dDNP) potential, providing up to 30 % [1-13 C]PA liquid-state polarization. Moreover, their labile nature has proven to pave a way to nuclear polarization storage and transport. Herein, differently from the case of PA, the issue of providing dDNP UV-radical precursors (trimethylpyruvic acid and its methyl-deuterated form) not involved in any metabolic pathway was investigated. The 13 C dDNP performance was evaluated for hyperpolarization of [U-13 C6 ,1,2,3,4,5,6,6-d7 ]-d-glucose. The generated UV-radicals proved to be versatile and highly efficient polarizing agents, providing, after dissolution and transfer (10 s), a 13 C liquid-state polarization of up to 32 %.

Identification of Intracellular and Extracellular Metabolites in Cancer Cells Using 13C Hyperpolarized Ultrafast Laplace NMR.

Ultrafast Laplace NMR (UF-LNMR), which is based on the spatial encoding of multidimensional data, enables one to carry out 2D relaxation and diffusion measurements in a single scan. Besides reducing the experiment time to a fraction, it significantly facilitates the use of nuclear spin hyperpolarization to boost experimental sensitivity, because the time-consuming polarization step does not need to be repeated. Here we demonstrate the usability of hyperpolarized UF-LNMR in the context of cell metabolism, by investigating the conversion of pyruvate to lactate in the cultures of mouse 4T1 cancer cells. We show that 13C ultrafast diffusion- T2 relaxation correlation measurements, with the sensitivity enhanced by several orders of magnitude by dissolution dynamic nuclear polarization (D-DNP), allows the determination of the extra- vs intracellular location of metabolites because of their significantly different values of diffusion coefficients and T2 relaxation times. Under the current conditions, pyruvate was located predominantly in the extracellular pool, while lactate remained primarily intracellular. Contrary to the small flip angle diffusion methods reported in the literature, the UF-LNMR method does not require several scans with varying gradient strength, and it provides a combined diffusion and T2 contrast. Furthermore, the ultrafast concept can be extended to various other multidimensional LNMR experiments, which will provide detailed information about the dynamics and exchange processes of cell metabolites.

Stable Isotope-Resolved Analysis with Quantitative Dissolution Dynamic Nuclear Polarization.

Metabolite profiles and their isotopomer distributions can be studied noninvasively in complex mixtures with NMR. The advent of dissolution Dynamic Nuclear Polarization (dDNP) and isotope enrichment add sensitivity and resolution to such metabolic studies. Metabolic pathways and networks can be mapped and quantified if protocols that control and exploit the ex situ signal enhancement are created. We present a sample preparation method, including cell incubation, extraction and signal enhancement, to obtain reproducible and quantitative dDNP (qdDNP) NMR-based stable isotope-resolved analysis. We further illustrate how qdDNP was applied to gain metabolic insights into the phenotype of aggressive cancer cells.

High-field dissolution dynamic nuclear polarization of [1-(13)C]pyruvic acid.

[1-(13)C]pyruvate is the most widely used hyperpolarized metabolic magnetic resonance imaging agent. Using a custom-built 7.0 T polarizer operating at 1.0 K and trityl radical-doped [1-(13)C]pyruvic acid, unextrapolated solution-state (13)C polarization greater than 60% was measured after dissolution and rapid transfer to a spectrometer magnet, demonstrating the signal enhancement attainable using optimized hardware. Slower rates of polarization under these conditions can be largely overcome with higher radical concentrations.

Difference between Extra- and Intracellular T1 Values of Carboxylic Acids Affects the Quantitative Analysis of Cellular Kinetics by Hyperpolarized NMR.

Incomplete knowledge of the longitudinal relaxation time constant (T1 ) leads to incorrect assumptions in quantitative kinetic models of cellular systems, studied by hyperpolarized real-time NMR. Using an assay that measures the intracellular signal of small carboxylic acids in living cells, the intracellular T1 of the carboxylic acid moiety of acetate, keto-isocaproate, pyruvate, and butyrate was determined. The intracellular T1 is shown to be up to four-fold shorter than the extracellular T1 . Such a large difference in T1 values between the inside and the outside of the cell has significant influence on the quantification of intracellular metabolic activity. It is expected that the significantly shorter T1 value of the carboxylic moieties inside cells is a result of macromolecular crowding. An artificial cytosol has been prepared and applied to predict the T1 of other carboxylic acids. We demonstrate the value of this prediction tool.

Non-invasive in-cell determination of free cytosolic [NAD+]/[NADH] ratios using hyperpolarized glucose show large variations in metabolic phenotypes.

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.

Hyperpolarized [1,3-13C2 ]ethyl acetoacetate is a novel diagnostic metabolic marker of liver cancer.

An increased prevalence of liver diseases such as hepatitis C and nonalcoholic fatty liver results in an augmented incidence of the most common form of liver cancer, hepatocellular carcinoma (HCC). HCC is most often found in the cirrhotic liver and it can therefore be challenging to rely on anatomical information alone when diagnosing HCC. Valuable information on specific cellular metabolism can be obtained with high sensitivity thanks to an emerging magnetic resonance (MR) technique that uses 13C labeled hyperpolarized molecules. Our interest was to explore potential new high contrast metabolic markers of HCC using hyperpolarized 13C-MR. This work led to the identification of a class of substrates, low molecular weight ethyl-esters, which showed high specificity for carboxyl esterases and proved in many cases to possess good properties for signal enhancement. In particular, hyperpolarized [1,3-13C2 ]ethyl acetoacetate (EAA) was shown to provide a metabolic fingerprint of HCC. Using this substrate a liver cancer implanted in rats was diagnosed as a consequence of an ∼4 times higher metabolic substrate-to-product ratio than in the surrounding healthy tissue, (p=0.009). Unregulated cellular uptake as well as cosubstrate independent enzymatic conversion of EAA, made this substrate highly useful as a hyperpolarized 13C-MR marker. This could be appreciated by the signal-to-noise (SNR) obtained from EAA, which was comparable to the SNR reported in a literature liver cancer study with state-of-the-art hyperpolarized substrate, [1-13C]pyruvate. Also, the contrast-to-noise (CNR) in the EAA based metabolic ratio images was significantly improved compared with the CNR in equivalent images reported using [1-13C]pyruvate.

Magnetic resonance imaging of pH in vivo using hyperpolarized 13C-labelled bicarbonate.

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.

Hyperpolarized NMR probes for biological assays.

During the last decade, the development of nuclear spin polarization enhanced (hyperpolarized) molecular probes has opened up new opportunities for studying the inner workings of living cells in real time. The hyperpolarized probes are produced ex situ, introduced into biological systems and detected with high sensitivity and contrast against background signals using high resolution NMR spectroscopy. A variety of natural, derivatized and designed hyperpolarized probes has emerged for diverse biological studies including assays of intracellular reaction progression, pathway kinetics, probe uptake and export, pH, redox state, reactive oxygen species, ion concentrations, drug efficacy or oncogenic signaling. These probes are readily used directly under natural conditions in biofluids and are often directly developed and optimized for cellular assays, thus leaving little doubt about their specificity and utility under biologically relevant conditions. Hyperpolarized molecular probes for biological NMR spectroscopy enable the unbiased detection of complex processes by virtue of the high spectral resolution, structural specificity and quantifiability of NMR signals. Here, we provide a survey of strategies used for the selection, design and use of hyperpolarized NMR probes in biological assays, and describe current limitations and developments.

Real-time DNP NMR observations of acetic acid uptake, intracellular acidification, and of consequences for glycolysis and alcoholic fermentation in yeast.

Uptake and upshot in vivo: Straightforward methods that permit the real-time observation of organic acid influx, intracellular acidification, and concomitant effects on cellular-reaction networks are crucial for improved bioprocess monitoring and control. Herein, dynamic nuclear polarization (DNP) NMR is used to observe acetate influx, ensuing intracellular acidification and the metabolic consequences on alcoholic fermentation and glycolysis in living cells.

Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors.

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)C(2)]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)C(2)]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized (13)C-labeled malate from [1,4-(13)C(2)]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.

Detection of tumor glutamate metabolism in vivo using (13)C magnetic resonance spectroscopy and hyperpolarized [1-(13)C]glutamate.

Dynamic nuclear polarization can be used to increase the sensitivity of solution state (13)C magnetic resonance spectroscopy by four orders of magnitude. We show here that [1-(13)C]glutamate can be polarized to 28%, representing a 35,000-fold increase in its sensitivity to detection at 9.4 T and 37°C. The metabolism of hyperpolarized glutamate to α-ketoglutarate, catalyzed by the enzyme alanine transaminase, was detected in vitro in human hepatoma cells (HepG2). Incubation of the cells with sodium pyruvate increased the level of the hyperpolarized label in the α-ketoglutarate pool, with an associated increase in the apparent rate constant describing flux of hyperpolarized (13)C label between glutamate and α-ketoglutarate. The metabolism of hyperpolarized glutamate was observed in vivo following coadministration of pyruvate in a murine lymphoma model. This represents a new method to probe glutamate metabolism and citric acid cycle activity in vivo; as glutamate is an endogenous molecule, it has the potential to be used in the clinic.

Quantitative dynamic nuclear polarization-NMR on blood plasma for assays of drug metabolism.

Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring.

Metabolic contrast agents produced from transported solid 13C-glucose hyperpolarized via dynamic nuclear polarization.

Magnetic Resonance Imaging combined with hyperpolarized 13C-labelled metabolic contrast agents produced via dissolution Dynamic Nuclear Polarization can, non-invasively and in real-time, report on tissue specific aberrant metabolism. However, hyperpolarization equipment is expensive, technically demanding and needs to be installed on-site for the end-user. In this work, we provide a robust methodology that allows remote production of the hyperpolarized 13C-labelled metabolic contrast agents. The methodology, built on photo-induced thermally labile radicals, allows solid sample extraction from the hyperpolarization equipment and several hours' lifetime of the 13C-labelled metabolic contrast agents at appropriate storage/transport conditions. Exemplified with [U-13C, d7]-D-glucose, we remotely produce hyperpolarized 13C-labelled metabolic contrast agents and generate above 10,000-fold liquid-state Magnetic Resonance signal enhancement at 9.4 T, keeping on-site only a simple dissolution device.

Tissue-specific short chain fatty acid metabolism and slow metabolic recovery after ischemia from hyperpolarized NMR in vivo.

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 acetyl-carnitine 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.

Imaging of branched chain amino acid metabolism in tumors with hyperpolarized 13C ketoisocaproate.

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 alpha-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.