Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate.

Hyperpolarized metabolites offer a tremendous sensitivity advantage (>10(4) fold) when measuring flux and enzyme activity in living tissues by magnetic resonance methods. These sensitivity gains can also be applied to mechanistic studies that impose time and metabolite concentration limitations. Here we explore the use of hyperpolarization by dissolution dynamic nuclear polarization (DNP) in mechanistic studies of alanine transaminase (ALT), a well-established biomarker of liver disease and cancer that converts pyruvate to alanine using glutamate as a nitrogen donor. A specific deuterated, (13)C-enriched analog of pyruvic acid, (13)C3D(3)-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct (13)C observation and indirect observation through methyl protons introduced by ALT-catalyzed H-D exchange. Exchange on injecting hyperpolarized (13)C3D(3)-pyruvate into ALT dissolved in buffered (1)H(2)O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5s timescale. ALT introduced, on average, 0.8 new protons into the methyl group of the alanine produced, indicating the presence of an off-pathway enamine intermediate. The opportunities for exploiting mechanism-dependent molecular signatures as well as indirect detection of hyperpolarized (13)C3-pyruvate and products in imaging applications are discussed.

Dynamic changes in oxygenation of intracranial tumor and contralateral brain during tumor growth and carbogen breathing: a multisite EPR oximetry with implantable resonators.

INTRODUCTION: Several techniques currently exist for measuring tissue oxygen; however technical difficulties have limited their usefulness and general application. We report a recently developed electron paramagnetic resonance (EPR) oximetry approach with multiple probe implantable resonators (IRs) that allow repeated measurements of oxygen in tissue at depths of greater than 10mm. METHODS: The EPR signal to noise (S/N) ratio of two probe IRs was compared with that of LiPc deposits. The feasibility of intracranial tissue pO(2) measurements by EPR oximetry using IRs was tested in normal rats and rats bearing intracerebral F98 tumors. The dynamic changes in the tissue pO(2) were assessed during repeated hyperoxia with carbogen breathing. RESULTS: A 6-10 times increase in the S/N ratio was observed with IRs as compared to LiPc deposits. The mean brain pO(2) of normal rats was stable and increased significantly during carbogen inhalation in experiments repeated for 3months. The pO(2) of F98 glioma declined gradually, while the pO(2) of contralateral brain essentially remained the same. Although a significant increase in the glioma pO(2) was observed during carbogen inhalation, this effect declined in experiments repeated over days. CONCLUSION: EPR oximetry with IRs provides a significant increase in S/N ratio. The ability to repeatedly assess orthotopic glioma pO(2) is likely to play a vital role in understanding the dynamics of tissue pO(2) during tumor growth and therapies designed to modulate tumor hypoxia. This information could then be used to optimize chemoradiation by scheduling treatments at times of increased glioma oxygenation.

Exchange facilitated indirect detection of hyperpolarized 15ND2-amido-glutamine.

Hyperpolarization greatly enhances opportunities to observe in vivo metabolic processes in real time. Accessible timescales are, however, limited by nuclear spin relaxation times, and sensitivity is limited by magnetogyric ratios of observed nuclei. The majority of applications to date have involved direct (13)C observation of metabolites with non-protonated carbons at sites of interest ((13)C enriched carbonyls, for example), a choice that extends relaxation times and yields moderate sensitivity. Interest in (15)N containing metabolites is equally high but non-protonated sites are rare and direct (15)N observation insensitive. Here an approach is demonstrated that extends applications to protonated (15)N sites with high sensitivity. The normally short relaxation times are lengthened by initially replacing protons (H) with deuterons (D) and low sensitivity detection of (15)N is avoided by indirect detection through protons reintroduced by H/D exchange. A pulse sequence is presented that periodically samples (15)N polarization at newly protonated sites by INEPT transfer to protons while returning (15)N magnetization of deuterated sites to the +Z axis to preserve polarization for subsequent samplings. Applications to (15)ND(2)-amido-glutamine are chosen for illustration. Glutamine is an important regulator and a direct donor of nitrogen in cellular metabolism. Potential application to in vivo observation is discussed.

¹H nuclear magnetic resonance spectroscopy characterisation of metabolic phenotypes in the medulloblastoma of the SMO transgenic mice.

BACKGROUND: Human medulloblastomas exhibit diverse molecular pathology. Aberrant hedgehog signalling is found in 20-30% of human medulloblastomas with largely unknown metabolic consequences. METHODS: Transgenic mice over-expressing smoothened (SMO) receptor in granule cell precursors with high incidence of exophytic medulloblastomas were sequentially followed up by magnetic resonance imaging (MRI) and characterised for metabolite phenotypes by ¹H MR spectroscopy (MRS) in vivo and ex vivo using high-resolution magic angle spinning (HR-MAS) ¹H MRS. RESULTS: Medulloblastomas in the SMO mice presented as T2 hyperintense tumours in MRI. These tumours showed low concentrations of N-acetyl aspartate and high concentrations of choline-containing metabolites (CCMs), glycine, and taurine relative to the cerebellar parenchyma in the wild-type (WT) C57BL/6 mice. In contrast, ¹H MRS metabolite concentrations in normal appearing cerebellum of the SMO mice were not different from those in the WT mice. Macromolecule and lipid ¹H MRS signals in SMO medulloblastomas were not different from those detected in the cerebellum of WT mice. The HR-MAS analysis of SMO medulloblastomas confirmed the in vivo ¹H MRS metabolite profiles, and additionally revealed that phosphocholine was strongly elevated in medulloblastomas accounting for the high in vivo CCM. CONCLUSIONS: These metabolite profiles closely mirror those reported from human medulloblastomas confirming that SMO mice provide a realistic model for investigating metabolic aspects of this disease. Taurine, glycine, and CCM are potential metabolite biomarkers for the SMO medulloblastomas. The MRS data from the medulloblastomas with defined molecular pathology is discussed in the light of metabolite profiles reported from human tumours.

Targeting mitogen-activated protein kinase kinase with the inhibitor PD0325901 decreases hepatocellular carcinoma growth in vitro and in mouse model systems.

Hepatocellular carcinoma (HCC) is a common cause of death from solid organ malignancy worldwide. Extracellular signal-regulated/mitogen-activated protein kinase kinase (MEK) signaling is a critical growth regulatory pathway in HCC. Targeting MEK with a novel small molecule inhibitor, PD0325901, may inhibit HCC tumorigenesis. PD0325901 (0.01-100 nM) inhibited growth and MEK activity in vitro in immortalized murine transforming growth factor alpha (TGF-alpha) transgenic hepatocyte (TAMH) cells, derived from the livers of TGF-alpha transgenic mice. Treatment of athymic mice bearing TAMH flank tumors with vehicle or PD0325901 (20 mg/kg) revealed a significant reduction of MEK activity ex vivo 24 hours after a single PD0325901 dose. The growth rate of TAMH flank tumors over 16 days was reduced threefold in the treatment arm (1113 +/- 269% versus 3077 +/- 483%, P < 0.01). PD0325901 exhibited similar inhibitory effects in HepG2 and Hep3B human HCC cells in vitro and in Hep3B flank tumors in vivo. To confirm this in a developmental model, MT-42 (CD-1) TGF-alpha mice were treated with vehicle or PD0325901 (20 mg/kg) for 5 weeks. Gross HCC was detected in 47% and 13.3% of the control and treatment mice, respectively. Tumor growth suppression by PD0325901 relative to vehicle was also shown by magnetic resonance imaging. These studies provide compelling preclinical evidence that targeting MEK in human clinical trials may be promising for the treatment of HCC.

Intravenous polyethylene glycol successfully treats severe acceleration-induced brain injury in rats as assessed by magnetic resonance imaging.

OBJECTIVE: Polyethylene glycol (PEG) is a nontoxic molecule with known efficacy as a cell membrane sealant, improving histological and behavioral outcomes in trauma models. Diffusion-weighted (DW) magnetic resonance imaging (MRI) is the most sensitive method of detecting in vivo diffuse axonal injury (DAI), where a decreased apparent diffusion coefficient (ADC) of water reflects cytotoxic edema. We use DW-MRI to assess severe DAI in rats treated with a single acute postinjury injection of PEG. METHODS: Rats were divided into uninjured, injured saline-treated, and injured PEG-treated groups. Injury groups received a severe brain injury using an impact-acceleration weight-drop model. Saline or PEG was administered acutely as a single intravenous dose to injured saline-treated and injured PEG-treated groups, respectively. DW-MRI analysis was performed at postinjury day 7 with a 9.4-T magnet. ADC was calculated for cortex, corpus callosum/hippocampus, and thalamus in each group. RESULTS: An expected decrease in ADC, representing cytotoxic edema, was observed in the injured saline-treated group. The injured PEG-treated group demonstrated no decrease in ADC relative to the uninjured rats, and the difference between ADC in saline and PEG-treated groups reached significance for all 3 zones of assessed brain. Differences were seen grossly between injured saline-treated and injured PEG-treated groups on representative color-mapped ADC images. CONCLUSION: A single intravenous dose of PEG dramatically limits sequelae of severe acceleration-induced brain injury--in this case, assessed by cytotoxic edema on DW-MRI--by intervening at the primary injury level of neuronal membrane disruption. This outcome is unprecedented, as no prior treatments for DAI have demonstrated similar efficacy. DAI treatment with intravenous PEG may have future clinical relevance and warrants further investigation.

Fat and water 1H MRI to investigate effects of leptin in obese mice.

Leptin is known to be associated with regulation of body weight and fat content. The effects of exogenous leptin on abdominal visceral (VS) and subcutaneous (SC) fat volume and hepatic fat-to-water ratio in leptin-deficient obese mice were investigated by (1)H magnetic resonance imaging (MRI). Chemical shift-selected fat and water (1)H MRI of control and leptin-treated mice were obtained 1 day before treatment and after 7 days of treatment (0.3 mg/kg/day). Hepatic fat-to-water ratio and VS fat volume decreased significantly with treatment, whereas SC fat volume did not change. Noninvasive measurement of fat and water content in different body regions using MRI should prove useful for evaluating new drugs for the treatment of obesity and other metabolic disorders.

Predicting and monitoring response to chemotherapy by 1,3-bis(2-chloroethyl)-1-nitrosourea in subcutaneously implanted 9L glioma using the apparent diffusion coefficient of water and 23Na MRI.

PURPOSE: To examine the effects of the alkylating anticancer drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on (23)Na MRI and the water apparent diffusion coefficient (ADC) in subcutaneously- (sc-) implanted 9L glioma in rats. MATERIALS AND METHODS: (23)Na MRI and (1)H water ADC measurements were performed on sham-treated control (N = 6) and BCNU-treated (N = 15) Fisher rats one day before BCNU injection and then one, three, and five days after BCNU injection. RESULTS: The BCNU-treated tumors were divided into BCNU-responsive (R(BCNU)) and BCNU-nonresponsive (NR(BCNU)) groups depending on the tumor volume changes that occurred after therapy. The pretreatment (23)Na MRI signal intensity (SI) and water ADC values were higher in R(BCNU) tumors compared to NR(BCNU) tumors. (23)Na MRI SI and water ADC increased with tumor growth in control and NR(BCNU) groups, but these changes were interrupted by BCNU therapy in R(BCNU) group. CONCLUSION: (23)Na MRI and water ADC measurements may be useful for predicting and monitoring response to chemotherapy in some tumors. However, the changes that occurred in (23)Na MRI SI and water ADC in sc-implanted 9L tumors are in contrast to previously published results for BCNU therapy of orthotopic 9L tumors. This may have important implications for monitoring therapy response in tumors.

Non-invasive temperature imaging with thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetramethyl-1,4,7,10-tetraacetic acid (TmDOTMA-).

Non-invasive thermometry using hyperfine-shifted MR signals from paramagnetic lanthanide complexes has attracted attention recently because the chemical shifts of these complexes are many times more sensitive to temperature than the water 1H signal. Among all the lanthanide complexes examined thus far, thulium tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTMA-) appears to be the most suitable for MR thermometry. In this paper, the feasibility of imaging the methyl 1H signal from TmDOTMA- using a frequency-selective radiofrequency excitation pulse and chemical shift-selective (CHESS) water suppression is demonstrated. A temperature imaging method using a phase-sensitive spin-echo imaging sequence was validated in phantom experiments. A comparison of regional temperature changes measured with fiber-optic probes and the temperatures calculated from the phase shift near each probe showed that the accuracy of imaging the temperature with TmDOTMA- is at least 0.1-0.2 degrees C. The feasibility of imaging temperature changes in an intact rat at 0.5-0.6 mmol/kg dose in only a few minutes is demonstrated. Similar to commonly used MRI contrast agents, the lanthanide complex does not cross the blood-brain barrier. TmDOTMA- may prove useful for temperature imaging in many biomedical applications but further studies relating to acceptable dose and signal-to-noise ratio are necessary before clinical applications.

Noninvasive thermometry using hyperfine-shifted MR signals from paramagnetic lanthanide complexes.

MR thermometry techniques based on the strong water 1H signal provide high spatial and temporal resolution and have shown promise for applications such as laser surgery and RF ablation. However, these techniques have low temperature sensitivity for hyperthermia applications and are greatly influenced by local motion and susceptibility variations. 1H NMR signals from paramagnetic lanthanide complexes of Pr3+, Yb3+ and Tm3+ show up to 300-fold stronger temperature dependence compared to the water 1H signal. In addition, 1H chemical shifts of many of these complexes are insensitive to other factors such as the concentration of the paramagnetic complex, pH, [Ca2+], and the presence of plasma macro-molecules and ions. Applications of lanthanide complexes for temperature measurement in intact animals and the feasibility of mapping temperatures in phantoms have been demonstrated. Among all the lanthanide complexes examined so far, thulium 1, 4, 7, 10-tetramethyl-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetate (TmDOTMA-) appears to be the most attractive for in vivo MR thermometry. The 1H signal from the methyl groups on this complex is relatively intense because of 12 equivalent protons and provides high temperature sensitivity because of the large paramagnetic shifts induced by thulium. The possibility of imaging TmDOTMA2--in intact animals at physiologically safe concentrations has recently been demonstrated. Overall, MR thermometry methods based on hyperfine-shifted MR signals from paramagnetic lanthanide complexes appear promising for animal applications, but further studies relating to acceptable dose and signal-to-noise ratio are necessary before clinical use.

Heat-induced changes in intracellular Na+, pH and bioenergetic status in superfused RIF-1 tumour cells determined by 23Na and 31P magnetic resonance spectroscopy.

The acute effects of hyperthermia on intracellular Na+ (Nai+), bioenergetic status and intracellular pH (pHi) were investigated in superfused Radiation Induced Fibrosarcoma-1 (RIF-1) tumour cells using shift-reagent-aided 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy. Hyperthermia at 45 degrees C for 30 min produced a 50% increase in Na, a 0.42 unit decrease in pHi and a 40-45% decrease in NTP/P(i). During post-hyperthermia superfusion at 37 degrees C, pHi and NTP/P(i) recovered to the baseline value, but Na initially decreased and then increased to the hyperthermic level 60 min after heating. Hyperthermia at 42 degrees C caused only a 15-20% increase in Nai+. In the presence of 3 microM 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na+/H+ exchanger, the increase in Nai+ during 45 degrees C hyperthermia was attenuated, suggesting that the heat-induced increase in Nai+ was mainly due to an increase in Na+/H+ anti-porter activity. EIPA did not prevent hyperthermia-induced acidification. This suggests that pHi is controlled by other ion exchange mechanisms in addition to the Na+/H+ exchanger. EIPA increased the thermo-sensitivity of the RIF-1 tumour cells only slightly as measured by cell viability and clonogenic assays. The hyperthermia-induced irreversible increase in Nai+ suggests that changes in transmembrane ion gradients play an important role in cell damage induced by hyperthermia.

Noninvasive MR thermometry using paramagnetic lanthanide complexes of 1,4,7,10-tetraazacyclodoecane-alpha,alpha',alpha'',alpha'''-tetramethyl-1,4,7,10-tetraacetic acid (DOTMA4-).

Noninvasive techniques to monitor temperature have numerous useful biomedical applications. However, MR thermometry techniques based on the chemical shift, relaxation rates, and molecular diffusion rate of the water 1H signal suffer from poor thermal resolution. The feasibility of MR thermometry based on the strong temperature dependence of the hyperfine-shifted 1H signal from the paramagnetic lanthanide complex thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA-) was recently demonstrated. The use of paramagnetic lanthanide complexes for MR thermometry can be further enhanced by improving the signal-to-noise ratio (SNR) of the observed signal. In this study, the use of lanthanide complexes of a methyl-substituted analog of DOTA4-, 1,4,7,10-tetramethyl 1,4,7,10-tetra azacyclodoecane-1,4,7,10-tetraacetic acetate (DOTMA4-) was evaluated. DOTMA4- complexes have 12 magnetically equivalent methyl protons, which provide an intense and sharper resonance compared to the corresponding DOTA- complexes. Experiments with paramagnetic Pr3+, Yb3+, Tb3+, Dy3+, and Tm3+ complexes of DOTMA4- showed that the Tm3+ complex is most favorable for MR thermometery because of the high temperature dependence of its chemical shift and its relatively narrow linewidth. The chemical shift of the methyl 1H signal from TmDOTMA- was approximately 60 times more sensitive to temperature than the water 1H shift and was insensitive to changes in concentration, pH, [Ca2+], or the presence of other ions and macromolecules. The application of TmDOTMA- for measuring temperature in a subcutaneously implanted tumor model was demonstrated. Lastly, the feasibility of obtaining 3D images from the methyl 1H resonance of TmDOTMA- was demonstrated in phantom and live animal experiments. Overall, TmDOTMA- appears to be a promising probe for MR thermometry in vivo.

The role of mitochondrial uncoupling in 3,4-methylenedioxymethamphetamine-mediated skeletal muscle hyperthermia and rhabdomyolysis.

Use of the popular club drug ecstasy (3,4-methylenedioxymethamphetamine, MDMA) can result in life-threatening hyperthermia and rhabdomyolysis. Recent studies show a link between skeletal muscle uncoupling proteins in MDMA-mediated hyperthermia. The mechanisms by which MDMA interacts with skeletal muscle mitochondria are largely unknown. The present study was designed to comprehensively evaluate the effects of MDMA on bioenergetics and toxicity of skeletal muscle. Using (31)P nuclear magnetic resonance (NMR) and serum creatine kinase levels, we demonstrate evidence for uncoupling of oxidative phosphorylation in the skeletal muscle of MDMA (40 mg/kg)-treated rats. In vivo, rats treated with MDMA had significantly elevated serum creatine kinase levels, a marker of rhabdomyolysis, 4 h post-MDMA treatment (955 +/- 132 IU/l) compared with saline-treated controls (373.2 +/- 59 IU/l). beta-ATP signal areas after MDMA treatment showed significant reductions (15%) from the baseline values with corresponding increases in inorganic phosphate (88% increases) and decreases in intracellular pH. Clark electrode experiments on isolated skeletal muscle mitochondria in vitro (1-5 mM MDMA) and ex vivo in MDMA-treated animals demonstrated no evidence of uncoupling of oxidative phosphorylation. In vitro experiments using L6 myotubules cocultured with primary hepatocytes demonstrated the presence of uncoupling protein-3 in the L6 myotubules, but no evidence of a direct effect of MDMA or its potential metabolites on cellular creatine kinase concentrations. These findings suggest that MDMA uncouples skeletal muscle mitochondria in vivo but that this uncoupling is the result of indirect mechanisms.

Effects of temperature on intracellular sodium, pH and cellular energy status in RIF-1 tumor cells.

Most perfused tumor cell experiments are performed at 37 degrees C, the normal healthy body temperature. However, the temperature of subcutaneously implanted tumors in small animals is generally 29-33 degrees C when the rectal temperature of the animal is maintained at 37 degrees C. We have investigated the acute effects of increasing the temperature of perfused radiation-induced-fibrosarcoma (RIF-1) tumor cells from 33 to 37 degrees C (30 min) on intracellular sodium (Na(i)+) , intracellular pH (pH(i)), and bioenergetic status. Heating the cells by 4 degrees C produced a reversible increase in Na(i)+, slight acidification and no change in nucleotide triphosphate to inorganic phosphate ratio (NTP/P(i)) as measured by shift-reagent-aided (23)Na and (31)P NMR spectroscopy. In the presence of 3 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a potent and specific inhibitor of Na(+)/H(+) antiporter, the increase in Na(i)+ during the heating was completely abolished suggesting that the heat induced increase in Na(i)+ was caused by an increase in Na(+)/H(+) antiporter activity. However, the changes in pH(i) with the heating were identical with or without EIPA, indicating that pH(i) is controlled by other ion exchange mechanisms in addition to Na(+)/H(+) antiporter. NTP/P(i) was significantly higher in presence of EIPA for some time points during the heating suggesting that both NTP production and consumption rates may be altered during the heating. These results indicate that a slight increase in temperature from 33 to 37 degrees C induces significant changes in Na(+) physiology largely because of activation of Na(+)/H(+) antiporter but other ion exchange mechanisms are also involved in maintaining pH(i) in the RIF-1 tumor cells. Thus, care must be taken in choosing the temperature for perfused cell studies.

Non-invasive magnetic resonance thermometry using thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA(-)).

Non-invasive thermometry is pivotal to the future advances of regional hyperthermia as a cancer treatment modality. Current magnetic resonance (MR) thermometry methods suffer from poor thermal resolution due to relatively weak dependence of chemical shift of the (1)H water signal on temperature. This study evaluated the feasibility of using thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA(-)) for MR thermometry. TmDOTA(-) is non-toxic and the gadolinium complex of DOTA(4-) is widely used as a MR contrast agent. The results demonstrate that the temperature dependence of the TmDOTA(-) proton shifts are about two orders of magnitudes higher than the water proton and, thus, provide excellent accuracy and resolution. In addition, TmDOTA(-) proton shifts are insensitive to the paramagnetic complex concentration, pH, Ca(2+) or presence of plasma macromolecules and ions. Because hyperthermia is known to produce changes in tissue pH and other physiological parameters, these properties of TmDOTA(-) greatly simplify the procedures for using the lanthanide complex for MR thermometry. Application of TmDOTA(-) for measurement of temperature in a subcutaneously implanted human melanoma xenograft is demonstrated. Finally, the feasibility of imaging one of the (1)H resonances of the lanthanide complex is demonstrated in phantom experiments. Overall, TmDOTA(-) appears to be a promising probe for MR thermometry in vivo.