Hyperpolarized ketone body metabolism in the rat heart

The aim of this work was to investigate the use of ¹³C‐labelled acetoacetate and β‐hydroxybutyrate as novel hyperpolarized substrates in the study of cardiac metabolism. [1‐¹³C]Acetoacetate was synthesized by catalysed hydrolysis, and both it and [1‐¹³C]β‐hydroxybutyrate were hyperpolarized by dissolution dynamic nuclear polarization (DNP). Their metabolism was studied in isolated, perfused rat hearts. Hyperpolarized [1‐¹³C]acetoacetate metabolism was also studied in the in vivo rat heart in the fed and fasted states. Hyperpolarization of [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate provided liquid state polarizations of 8 ± 2% and 3 ± 1%, respectively. The hyperpolarized T₁ values for the two substrates were 28 ± 3 s (acetoacetate) and 20 ± 1 s (β‐hydroxybutyrate). Multiple downstream metabolites were observed within the perfused heart, including acetylcarnitine, citrate and glutamate. In the in vivo heart, an increase in acetylcarnitine production from acetoacetate was observed in the fed state, as well as a potential reduction in glutamate. In this work, methods for the generation of hyperpolarized [1‐¹³C]acetoacetate and [1‐¹³C]β‐hydroxybutyrate were investigated, and their metabolism was assessed in both isolated, perfused rat hearts and in the in vivo rat heart. These preliminary investigations show that DNP can be used as an effective in vivo probe of ketone body metabolism in the heart.     

Pyruvate‐lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1‐13C]pyruvate MRS and [18F]FDG‐PET

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1‐¹³C]pyruvate MRS and [¹⁸F]FDG‐PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1‐¹³C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1‐¹³C]pyruvate to lactate conversion was followed by ¹³C MRS. Subsequently [¹⁸F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [¹⁸F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant k_pl of ¹³C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [¹⁸F]FDG PET measurements and k_pl values for the xenografts of both tumor types. The k_pl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [¹⁸F]FDG‐PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1‐¹³C]pyruvate.     

Probing carbohydrate metabolism using hyperpolarized 13C‐labeled molecules

Glycolysis is a fundamental metabolic process in all organisms. Anomalies in glucose metabolism are linked to various pathological conditions. In particular, elevated aerobic glycolysis is a characteristic feature of rapidly growing cells. Glycolysis and the closely related pentose phosphate pathway can be monitored in real time by hyperpolarized ¹³C‐labeled metabolic substrates such as ¹³C‐enriched, deuterated D‐glucose derivatives, [2‐¹³C]‐D‐fructose, [2‐¹³C] dihydroxyacetone, [1‐¹³C]‐D‐glycerate, [1‐¹³C]‐D‐glucono‐δ‐lactone and [1‐¹³C] pyruvate in healthy and diseased tissues. Elevated glycolysis in tumors (the Warburg effect) was also successfully imaged using hyperpolarized [U‐¹³C₆, U‐²H₇]‐D‐glucose, while the size of the preexisting lactate pool can be measured by ¹³C MRS and/or MRI with hyperpolarized [1‐¹³C]pyruvate. This review summarizes the application of various hyperpolarized ¹³C‐labeled metabolites to the real‐time monitoring of glycolysis and related metabolic processes in normal and diseased tissues.     

A multisample 7 T dynamic nuclear polarization polarizer for preclinical hyperpolarized MR

Dynamic nuclear polarization (DNP) provides the opportunity to boost liquid state magnetic resonance (MR) signals from selected nuclear spins by several orders of magnitude. A cryostat running at a temperature of ~ 1 K and a superconducting magnet set to between 3 and 10 T are required to efficiently hyperpolarize nuclear spins. Several DNP polarizers have been implemented for the purpose of hyperpolarized MR and recent systems have been designed to avoid the need for user input of liquid cryogens. We herein present a zero boil‐off DNP polarizer that operates at 1.35 ± 0.01 K and 7 T, and which can polarize two samples in parallel. The samples are cooled by a static helium bath thermally connected to a 1 K closed‐cycle ⁴He refrigerator. Using a modified version of the commercial fluid path developed for the SPINlab polarizer, we demonstrate that, within a 12‐minute interval, the system can produce two separate hyperpolarized ¹³C solutions. The ¹³C liquid‐state polarization of [1‐¹³C]pyruvate measured 26 seconds after dissolution was 36%, which can be extrapolated to a 55% solid state polarization. The system is well adapted for in vitro and in vivo preclinical hyperpolarized MR experiments and it can be modified to polarize up to four samples in parallel.     

A comparison of quantitative methods for clinical imaging with hyperpolarized 13C‐pyruvate

Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized ¹³C‐labelled molecules, such as the conversion of [1‐¹³C]pyruvate to [1‐¹³C]lactate, to be dynamically and non‐invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model‐free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two‐way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time‐to‐peak and the lactate‐to‐pyruvate area under the curve ratio were simple model‐free approaches that accurately represented the full reaction, with the time‐to‐peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized ¹³C data. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.     

1H NMR and hyperpolarized 13C NMR assays of pyruvate–lactate: a comparative study

Pyruvate–lactate exchange is mediated by the enzyme lactate dehydrogenase (LDH) and is central to the altered energy metabolism in cancer cells. The measurement of exchange kinetics using hyperpolarized ¹³C NMR has provided a biomarker of response to novel therapeutics. However, the observable signal is restricted to the exchanging hyperpolarized ¹³C pools and the endogenous pools of ¹²C‐labelled metabolites are invisible in these measurements. In this study, we investigated an alternative in vitro ¹H NMR assay, using [3‐¹³C]pyruvate, and compared the measured kinetics with a hyperpolarized ¹³C NMR assay, using [1‐¹³C]pyruvate, under the same conditions in human colorectal carcinoma SW1222 cells. The apparent forward reaction rate constants (k_PL) derived from the two assays showed no significant difference, and both assays had similar reproducibility (k_PL = 0.506 ± 0.054 and k_PL = 0.441 ± 0.090 nmol/s/10⁶ cells; mean ± standard deviation; n = 3); ¹H, ¹³C assays, respectively). The apparent backward reaction rate constant (k_LP) could only be measured with good reproducibility using the ¹H NMR assay (k_LP = 0.376 ± 0.091 nmol/s/10⁶ cells; mean ± standard deviation; n = 3). The ¹H NMR assay has adequate sensitivity to measure real‐time pyruvate–lactate exchange kinetics in vitro, offering a complementary and accessible assay of apparent LDH activity. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous imaging of hyperpolarized [1,4‐13C2]fumarate, [1‐13C]pyruvate and 18F–FDG in a rat model of necrosis in a clinical PET/MR scanner

A co‐polarization scheme for [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate is presented to simultaneously assess necrosis and metabolism in rats with hyperpolarized ¹³C magnetic resonance (MR). The co‐polarization was performed in a SPINlab polarizer. In addition, the feasibility of simultaneous positron emission tomography (PET) and MR of small animals with a clinical PET/MR scanner is demonstrated. The hyperpolarized metabolic MR and PET was demonstrated in a rat model of necrosis. The polarization and T₁ of the co‐polarized [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate substrates were measured in vitro and compared with those obtained when the substrates were polarized individually. A polarization of 36 ± 4% for fumarate and 37 ± 6% for pyruvate was obtained. We found no significant difference in the polarization and T₁ values between the dual and single substrate polarization. Rats weighing about 400 g were injected intramuscularly in one of the hind legs with 200 μL of turpentine to induce necrosis. Two hours later, ¹³C metabolic maps were obtained with a chemical shift imaging sequence (16 × 16) with a resolution of 3.1 × 5.0 × 25.0 mm³. The ¹³C spectroscopic images were acquired in 12 s, followed by an 8‐min ¹⁸F‐2‐fluoro‐2‐deoxy‐d ‐glucose (¹⁸F–FDG) PET acquisition with a resolution of 3.5 mm. [1,4‐¹³C₂]Malate was observed from the tissue injected with turpentine indicating necrosis. Normal [1‐¹³C]pyruvate metabolism and ¹⁸F–FDG uptake were observed from the same tissue. The proposed co‐polarization scheme provides a means to utilize multiple imaging agents simultaneously, and thus to probe various metabolic pathways in a single examination. Moreover, it demonstrates the feasibility of small animal research on a clinical PET/MR scanner for combined PET and hyperpolarized metabolic MR.     

Application of hyperpolarized [1-(13) C]lactate for the in vivo investigation of cardiac metabolism

In addition to cancer imaging, ¹³C‐MRS of hyperpolarized pyruvate has also demonstrated utility for the investigation of cardiac metabolism and ischemic heart disease. Although no adverse effects have yet been reported for doses commonly used in vivo, high substrate concentrations have lead to supraphysiological pyruvate levels that can affect the underlying metabolism and should be considered when interpreting results. With lactate serving as an important energy source for the heart and physiological lactate levels one to two orders of magnitude higher than for pyruvate, hyperpolarized lactate could potentially be used as an alternative to pyruvate for probing cardiac metabolism. In this study, hyperpolarized [1‐¹³C]lactate was used to acquire time‐resolved spectra from the healthy rat heart in vivo and to measure dichloroacetate (DCA)‐modulated changes in flux through pyruvate dehydrogenase (PDH). Both primary oxidation of lactate to pyruvate and subsequent conversion of pyruvate to alanine and bicarbonate could reliably be detected. Since DCA stimulates the activity of PDH through inhibition of PDH kinase, a more than 2.5‐fold increase in bicarbonate‐to‐substrate ratio was found after administration of DCA, similar to the effect when using [1‐¹³C]pyruvate as the substrate. Copyright © 2012 John Wiley & Sons, Ltd.     

Glucose metabolism via the pentose phosphate pathway, glycolysis and Krebs cycle in an orthotopic mouse model of human brain tumors

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2‐¹³C₂]glucose. The [3‐¹³C]lactate/[2,3‐¹³C₂]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, ¹³C–¹³C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, ¹³C multiplets of γ‐aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67‐kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that ¹³C‐labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy. Copyright © 2012 John Wiley & Sons, Ltd.     

Hyperpolarized 13C‐lactate to 13C‐bicarbonate ratio as a biomarker for monitoring the acute response of anti‐vascular endothelial growth factor (anti‐VEGF) treatment

Hyperpolarized [1‐¹³C]pyruvate MRS provides a unique imaging opportunity to study the reaction kinetics and enzyme activities of in vivo metabolism because of its favorable imaging characteristics and critical position in the cellular metabolic pathway, where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl‐coenzyme A and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis relative to oxidative phosphorylation (i.e. Warburg effect). Although there is a strong theoretical basis for presuming that readjustment of the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized ¹³C‐pyruvate imaging studies had focused solely on [1‐¹³C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of ¹³C‐bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of ¹³C‐bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized ¹³C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of ¹³C‐lactate to ¹³C‐bicarbonate provides a more robust metric relative to ¹³C‐lactate for the assessment of the metabolic effects of anti‐angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti‐angiogenic treatment results in a rapid normalization in the relative tissue utilization of glycolytic and oxidative phosphorylation by tumor tissue. Copyright © 2016 John Wiley & Sons, Ltd.     

Increased hyperpolarized [1‐13C] lactate production in a model of joint inflammation is not accompanied by tissue acidosis as assessed using hyperpolarized 13C‐labelled bicarbonate

Arthritic conditions are a major source of chronic pain. Furthering our understanding of disease mechanisms creates the opportunity to develop more targeted therapeutics. In rheumatoid arthritis (RA), measurements of pH in human synovial fluid suggest that acidosis occurs, but that this is highly variable between individuals. Here we sought to determine if tissue acidosis occurs in a widely used rodent arthritis model: complete Freund's adjuvant (CFA)‐induced inflammation. CFA robustly evoked paw and ankle swelling, concomitant with worsening clinical scores over time. We used magnetic resonance spectroscopic imaging of hyperpolarized [1‐¹³C]pyruvate metabolism to demonstrate that CFA induces an increase in the lactate‐to‐pyruvate ratio. This increase is indicative of enhanced glycolysis and an increased lactate concentration, as has been observed in the synovial fluid from RA patients, and which was correlated with acidosis. We also measured the ¹³CO₂/H¹³CO₃^? ratio, in animals injected with hyperpolarized H¹³CO₃^?, to estimate extracellular tissue pH and showed that despite the apparent increase in glycolytic activity in CFA‐induced inflammation there was no accompanying decrease in extracellular pH. The pH was 7.23 ± 0.06 in control paws and 7.32 ± 0.09 in inflamed paws. These results could explain why mice lacking acid‐sensing ion channel subunits 1, 2 and 3 do not display any changes in mechanical or thermal hyperalgesia in CFA‐induced inflammation.     

Assessing the transport rate of hyperpolarized pyruvate and lactate from the intra‐ to the extracellular space

The use of [1‐ ¹³ C]pyruvate hyperpolarized by means of dynamic nuclear polarization provides a direct way to track the metabolic transformations of this metabolite in vivo and in cell cultures. The identification of the intra‐ and extracellular contributions to the ¹³ C NMR resonances is not straightforward. In order to obtain information about the rate of pyruvate and lactate transport through the cellular membrane, we set up a method that relies on the sudden ‘quenching’ of the extracellular metabolites' signal. The paramagnetic Gd–tetraazacyclododecane triacetic acid (Gd‐DO3A) complex was used to dramatically decrease the longitudinal relaxation time constants of the ¹³ C‐carboxylate resonances of both pyruvate and lactate. When Gd‐DO3A was added to an MCF‐7 cellular culture, which had previously received a dose of hyperpolarized [1‐ ¹³ C]pyruvate, the contributions of the extracellular pyruvate and lactate signals were deleted. From the analysis of the decay curves of the ¹³ C‐carboxylate resonances of pyruvate and lactate it was possible to extract information about the exchange rate of the two metabolites across the cellular membrane. In particular, it was found that, in the reported experimental conditions, the lactate transport from the intra‐ to the extracellular space is not much lower than the rate of lactate formation. The method reported herein is non‐destructive and it could be translated to in vivo studies. It opens a route for the use of hyperpolarized pyruvate to assess altered activity of carboxylate transporter proteins that may occur in pathological conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

Hyperpolarized δ‐[1‐13C]gluconolactone as a probe of the pentose phosphate pathway

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis), but simple methods for detecting changes in flux through this pathway are not available. MRI of hyperpolarized ¹³C–enriched metabolites offers considerable potential as a rapid, non‐invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ‐[1‐¹³C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPP_ox) in isolated perfused mouse livers. The appearance of hyperpolarized (HP) H¹³CO₃^? within seconds after exposure of livers to HP‐δ‐[1‐¹³C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPP_ox pathway to produce HP‐H¹³CO₃^? after three enzyme catalyzed steps (6P–gluconolactonase, 6‐phosphogluconate dehydrogenase, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H¹³CO₃^? after exposure to low levels of H₂O₂, while livers perfused with glucose and insulin showed a twofold increase in H¹³CO₃^? after exposure to peroxide. This indicates that flux through the PPP_ox is stimulated by H₂O₂ in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non‐polarized [1,2‐¹³C]glucose followed by ¹H NMR analysis of lactate in the perfusate verified that flux through the PPP_ox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized δ‐[1‐¹³C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.     

HDAC inhibition in glioblastoma monitored by hyperpolarized 13C MRSI

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized ¹³C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real‐time dynamic metabolic changes using a bioreactor system with live vorinostat‐treated or control cells. We found a significant 40% decrease in the ¹³C MRS‐detectable ratio of hyperpolarized [1‐¹³C]lactate to hyperpolarized [1‐¹³C]pyruvate, [1‐¹³C]Lac/Pyr, and a 37% decrease in the pseudo‐rate constant, k_PL, for hyperpolarized [1‐¹³C]lactate production, in vorinostat‐treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat‐treated cells. In vivo ¹³C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1‐¹³C]Lac/Pyr when comparing vorinostat‐treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC‐inhibited tumors. Thus, the ¹³C MRSI‐detectable decrease in hyperpolarized [1‐¹³C]lactate production could serve as a biomarker of response to HDAC inhibitors.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.     

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 ¹³C‐carbamazepine and its pharmacologically active metabolite ¹³C‐carbamazepine‐10,11‐epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single‐scan ¹³C 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. Copyright © 2010 John Wiley & Sons, Ltd.     

Thermoresponsive Properties of Sugar Sensitive Copolymer of N‐Isopropylacrylamide and 3‐(Acrylamido)phenylboronic Acid

Summary: The copolymer of N‐isopropylacrylamide and 3‐(acrylamido)phenylboronic acid (82:18, = 47 000 g · mol^?1) was prepared by free radical polymerization. The copolymer showed typical thermal precipitation behavior in aqueous solutions, its precipitation temperature (T_P) being increased from 23 to 32 °C by increasing the pH from 6.5 to 9.7, because of ionization of the phenylboronate units. The pK_a was evaluated as 8.9 ± 0.1 from the effect of pH on T_P. At pH > 9, i.e., in the anionic form of the copolymer, T_P was affected by a very low concentration of glucose (5.6 μM , ΔT_P = 1–1.5 °C), because of complex formation with a high binding constant. At a higher concentration of polyols (560 μM , pH > 8) the increase of T_P was maximal for the copolymer complexes with fructose (7–10 °C) and decreased in the order: fructose > glucose ≈ mannitol > pentaerythritol > galactose > Tris >glycerol. Di‐ and oligosaccharides (lactose, sucrose, and dextran) caused a slight increase of T_P at pH 7.5–8.7 while no effect was observed at pH > 9. Isothermal dissolution of the copolymer suspension in water (27 °C, pH 8.5) was possible in the presence of fructose or mannitol but required higher concentrations (1.4–3.6 × 10³ μM ) as compared to those which enabled the shift of T_P in the soluble copolymer. The dissolution rate increased with fructose concentrations.

Effect of pH on T_P of poly(NIPAAM‐co‐AAPBA) in the presence of various monosaccharides.     

In vivo measurement of aldehyde dehydrogenase‐2 activity in rat liver ethanol model using dynamic MRSI of hyperpolarized [1‐13C]pyruvate

To date, measurements of the activity of aldehyde dehydrogenase‐2 (ALDH2), a critical mitochondrial enzyme for the elimination of certain cytotoxic aldehydes in the body and a promising target for drug development, have been largely limited to in vitro methods. Recent advancements in MRS of hyperpolarized ¹³C‐labeled substrates have provided a method to detect and image in vivo metabolic pathways with signal‐to‐noise ratio gains greater than 10 000‐fold over conventional MRS techniques. However aldehydes, because of their toxicity and short T₁ relaxation times, are generally poor targets for such ¹³C‐labeled studies. In this work, we show that dynamic MRSI of hyperpolarized [1‐¹³C]pyruvate and its conversion to [1‐¹³C]lactate can provide an indirect in vivo measurement of ALDH2 activity via the concentration of NADH (nicotinamide adenine dinucleotide, reduced form), a co‐factor common to both the reduction of pyruvate to lactate and the oxidation of acetaldehyde to acetate. Results from a rat liver ethanol model (n = 9) show that changes in ¹³C‐lactate labeling following the bolus injection of hyperpolarized pyruvate are highly correlated with changes in ALDH2 activity (R² = 0.76). Copyright © 2012 John Wiley & Sons, Ltd.     

Automated transfer and injection of hyperpolarized molecules with polarization measurement prior to in vivo NMR

Hyperpolarized magnetic resonance via dissolution dynamic nuclear polarization necessitates the transfer of the hyperpolarized molecules from the polarizer to the imager prior to in vivo measurements. This process leads to unavoidable losses in nuclear polarization, which are difficult to evaluate once the solution has been injected into an animal. We propose a method to measure the polarization of the hyperpolarized molecules inside the imager bore, 3 s following dissolution, at the time of the injection, using a precise quantification of the infusate concentration. This in situ quantification allows for distinguishing between signal modulations related to variations in the nuclear polarization at the time of the injection and signal modulations related to physiological processes such as tissue perfusion. In addition, our method includes a radical scavenging process that leads to a minor reduction in sample concentration and takes place within a couple of seconds following the dissolution in order to minimize the losses due to the presence of paramagnetic polarizing agent in the infusate. We showed that proton exchange between vitamin C, the scavenging molecule and the deuterated solvent shortens the long carboxyl ¹³C longitudinal relaxation time in [1‐¹³C]acetate. This additional source of dipolar relaxation can be avoided by using deuterated ascorbate. Overall, the method allows for a substantial gain in polarization and also leads to an extension of the time window available for in vivo measurements. Copyright © 2013 John Wiley & Sons, Ltd.