Sub-minute kinetics of human red cell fumarase: H spin-echo NMR spectroscopy and C rapid-dissolution dynamic nuclear polarization.

Fumarate is an important probe of metabolism in hyperpolarized magnetic resonance imaging and spectroscopy. It is used to detect the release of fumarase in cancer tissues, which is associated with necrosis and drug treatment. Nevertheless, there are limited reports describing the detailed kinetic studies of this enzyme in various cells and tissues.

ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.

ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker.

Rapid Imaging of Tumor Cell Death In Vivo Using the C2A Domain of Synaptotagmin-I.

Cell death is an important target for imaging the early response of tumors to treatment. We describe here the validation of a phosphatidylserine-binding agent for detecting tumor cell death in vivo based on the C2A domain of synaptotagmin-I. The capability of near-infrared fluorophore-labeled and Tc- and In-labeled derivatives of C2Am for imaging tumor cell death, using planar near-infrared fluorescence imaging and SPECT, respectively, was evaluated in implanted and genetically engineered mouse models of lymphoma and in a human colorectal xenograft.

Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

Glycosylation is a ubiquitous post-translational modification, present in over 50 % of the proteins in the human genome,1 with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases,2 including cancer.3 We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe.

Imaging Glycosylation In Vivo by Metabolic Labeling and Magnetic Resonance Imaging.

Glycosylation is a ubiquitous post-translational modification, present in over 50% of the proteins in the human genome, with important roles in cell-cell communication and migration. Interest in glycome profiling has increased with the realization that glycans can be used as biomarkers of many diseases, including cancer. We report here the first tomographic imaging of glycosylated tissues in live mice by using metabolic labeling and a gadolinium-based bioorthogonal MRI probe.

(13) C magnetic resonance spectroscopy measurements with hyperpolarized [1-(13) C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo.

Purpose: Dissolution dynamic nuclear polarization can increase the sensitivity of the (13) C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize (13) C-labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.

MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model.

Objectives: Pancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select patients for surgery. We investigated here whether pancreatic preneoplasia could be detected prior to the development of invasive cancers in genetically engineered mouse models of PCa using metabolic imaging.

Development of Timd2 as a reporter gene for MRI.

Purpose: To assess the potential of an MRI gene reporter based on the ferritin receptor Timd2 (T-cell immunoglobulin and mucin domain containing protein 2), using T1- and T2-weighted imaging.

Methods: Pellets of cells that had been modified to express the Timd2 transgene, and incubated with either iron-loaded or manganese-loaded ferritin, were imaged using T1- and T2-weighted MRI. Mice were also implanted subcutaneously with Timd2-expressing cells and the resulting xenograft tissue imaged following intravenous injection of ferritin using T2-weighted imaging.

Gold nanoparticles functionalised with fast water exchanging Gd3+ chelates: linker effects on the relaxivity.

The relaxivity displayed by Gd(3+) chelates immobilized onto gold nanoparticles is the result of the complex interplay between the nanoparticle size, the water exchange rate and the chelate structure. In this work we study the effect of the length of ω-thioalkyl linkers, anchoring fast water exchanging Gd(3+) chelates onto gold nanoparticles, on the relaxivity of the immobilized chelates. Gold nanoparticles functionalized with Gd(3+) chelates of mercaptoundecanoyl and lipoyl amide conjugates of the DO3A-N-(α-amino)propionate chelator were prepared and studied as potential CA for MRI.

Hyperpolarized [U-(2) H, U-(13) C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells.

A resonance at ∼181 ppm in the (13) C spectra of tumors injected with hyperpolarized [U-(2) H, U-(13) C]glucose was assigned to 6-phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3-phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG (13) C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) METHODS: Tumor-bearing mice were injected with (13) C6 glucose and the (13) C-labeled and total 6PG and 3PG concentrations measured. (13) C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-(2) H, U-(13) C]glucose and again (13) C-labeled and total 6PG and 3PG were measured by LC-MS/MS RESULTS: Tumor (13) C-6PG was more abundant than (13) C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG.

(13) C magnetic resonance spectroscopic imaging of hyperpolarized [1-(13) C, U-(2) H5 ] ethanol oxidation can be used to assess aldehyde dehydrogenase activity in vivo.

Purpose: Aldehyde dehydrogenase (ALDH2) is an emerging drug target for the treatment of heart disease, cocaine and alcohol dependence, and conditions caused by genetic polymorphisms in ALDH2. Noninvasive measurement of ALDH2 activity in vivo could inform the development of these drugs and accelerate their translation to the clinic.

Methods: [1-(13) C, U-(2) H5 ] ethanol was hyperpolarized using dynamic nuclear polarization, injected into mice and its oxidation in the liver monitored using (13) C MR spectroscopy and spectroscopic imaging.

Oatp1 enhances bioluminescence by acting as a plasma membrane transporter for D-luciferin.

Purpose: Bioluminescence imaging is a powerful tool for studying gene expression and cell migration in intact living organisms. However, production of bioluminescence by cells transfected to express luciferase can be limited by the rate of plasma membrane transport of its substrate D-luciferin. We sought to identify a plasma membrane transporter for D-luciferin that could be expressed alongside luciferase to increase transmembrane flux of its substrate and thereby increase light output.

Detection of transgene expression using hyperpolarized 13C urea and diffusion-weighted magnetic resonance spectroscopy.

Purpose: To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [(13) C]urea.

Methods: Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [(13) C]urea, a spin echo sequence was used to measure urea concentration, T1 , and diffusion in control and UTB-expressing tissue.

(13)C NMR spectroscopy applications to brain energy metabolism.

(13)C nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying brain metabolism. Indeed, the most convincing data obtained to decipher metabolic exchanges between neurons and astrocytes have been obtained using this technique, thus illustrating its power. It may be difficult for non-specialists, however, to grasp thefull implication of data presented in articles written by spectroscopists.

Dual-modality gene reporter for in vivo imaging.

The ability to track cells and their patterns of gene expression in living organisms can increase our understanding of tissue development and disease. Gene reporters for bioluminescence, fluorescence, radionuclide, and magnetic resonance imaging (MRI) have been described but these suffer variously from limited depth penetration, spatial resolution, and sensitivity. We describe here a gene reporter, based on the organic anion transporting protein Oatp1a1, which mediates uptake of a clinically approved, Gd(3+)-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid).

Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose.

In this study, we monitored glycolysis in mouse lymphoma and lung tumors by measuring the conversion of hyperpolarized [U-2H, U-13C]glucose to lactate using 13C magnetic resonance spectroscopy and spectroscopic imaging. We observed labeled lactate only in tumors and not in surrounding normal tissue or other tissues in the body and found that it was markedly decreased at 24 h after treatment with a chemotherapeutic drug. We also detected an increase in a resonance assigned to 6-phosphogluconate in the pentose phosphate pathway.

Increased oxidative metabolism and neurotransmitter cycling in the brain of mice lacking the thyroid hormone transporter SLC16A2 (MCT8).

Mutations of the monocarboxylate transporter 8 (MCT8) cause a severe X-linked intellectual deficit and neurological impairment. MCT8 is a specific thyroid hormone (T4 and T3) transporter and the patients also present unusual abnormalities in the serum profile of thyroid hormone concentrations due to altered secretion and metabolism of T4 and T3. Given the role of thyroid hormones in brain development, it is thought that the neurological impairment is due to restricted transport of thyroid hormones to the target neurons.

Cerebral oedema is not responsible for motor or cognitive deficits in rats with hepatic encephalopathy.

Background & Aims: Low-grade cytotoxic oedema is considered a main contributor to the neurological (motor and cognitive) alterations in patients with hepatic encephalopathy (HE). This assumption is mainly based on studies with cultured astrocytes treated with very large ammonia concentrations or with animal models of acute liver failure with strong HE. However, the possible contribution of cerebral oedema (vasogenic or cytotoxic) to cognitive or motor alterations in chronic mild HE has not been demonstrated.

Amide conjugates of the DO3A-N-(α-amino)propionate ligand: leads for stable, high relaxivity contrast agents for MRI?

A novel synthetic methodology for preparing amide conjugates of the DO3A-N-(α-amino)propionate chelator is described, using the synthesis of the DO3A-N-(α-benzoylamido)propionate chelator as an illustrative example. The model Gd[DO3A-N-(α-benzoylamido)propionate] chelate displays accelerated water exchange, stability in a wide pH range and inertness towards transmetallation by Zn(2+). The Gd[DO3A-N-(α-benzoylamido)propionate] complex is mainly excreted via the kidneys, producing a significant increase in the kidney medulla/cortex enhancement ratio in MR images of Wistar rats, reflecting probably its higher lipophilicity compared with Gd(DTPA).

Brain glutamine synthesis requires neuronal-born aspartate as amino donor for glial glutamate formation.

The glutamate-glutamine cycle faces a drain of glutamate by oxidation, which is balanced by the anaplerotic synthesis of glutamate and glutamine in astrocytes. De novo synthesis of glutamate by astrocytes requires an amino group whose origin is unknown. The deficiency in Aralar/AGC1, the main mitochondrial carrier for aspartate-glutamate expressed in brain, results in a drastic fall in brain glutamine production but a modest decrease in brain glutamate levels, which is not due to decreases in neuronal or synaptosomal glutamate content.

Unambiguous assignment of the H3S and H3R deuterations of cerebral (2-13C) glutamate by 13C NMR at 18.8 Tesla.

We used high-field (13)C NMR (18.8 T) to assign unambiguously the isotopic shifts induced by the deuterium substitutions of the H3(proR) and H3(proS) hydrogens of (2-(13)C) glutamate in extracts of the brain from deuterated animals. Monodeuterated H3R or H3S glutamate diastereoisomers were produced stereospecifically either by chemical synthesis or by coupling the reactions of isocitrate dehydrogenase and aspartate aminotransferase in deuterated medium, respectively.