Tiago B. Rodrigues

This is a tribute to Sebastian Cerdan, a brilliant and innovative NMR spectroscopist whose studies contributed greatly to the fundamental information to the understanding of brain metabolism, particularly in regard to multinuclear magnetic resonance spectroscopy (MRS) techniques. Sebastian Cerdan sadly passed away in May 2022. He was a wonderful mentor and colleague who will be greatly missed.

Imaging of the metabolism of hyperpolarized [1‐¹³C]pyruvate has shown considerable promise in preclinical studies in oncology, particularly for the assessment of early treatment response. The repeatability of measurements of ¹³C label exchange between pyruvate and lactate was determined in a murine lymphoma model in fasted and non‐fasted animals. The fasted state showed lower intra‐individual variability, although the [1‐¹³C]lactate/[1‐¹³C]pyruvate signal ratio was significantly greater in fasted than in non‐fasted mice, which may be explained by the higher tumor lactate concentrations in fasted animals. These results indicate that the fasted state may be preferable for the measurement of ¹³C label exchange between pyruvate and lactate, as it reduces the variability and therefore should make it easier to detect the effects of therapy. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

Purpose A resonance at ∼181 ppm in the ¹³C spectra of tumors injected with hyperpolarized [U-²H, U-¹³C]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 ¹³C-labeling using liquid chromatography tandem mass spectrometry (LC-MS/MS) Methods Tumor-bearing mice were injected with ¹³C₆ glucose and the ¹³C-labeled and total 6PG and 3PG concentrations measured. ¹³C MR spectra of glucose-6-phosphate dehydrogenase deficient (zwf1Δ) and wild-type yeast were acquired following addition of hyperpolarized [U-²H, U-¹³C]glucose and again ¹³C-labeled and total 6PG and 3PG were measured by LC-MS/MS Results Tumor ¹³C-6PG was more abundant than ¹³C-2PG/3PG and the resonance at ∼181 ppm matched more closely that of 6PG. ¹³C MR spectra of wild-type and zwf1Δ yeast cells showed a resonance at ∼181 ppm after labeling with hyperpolarized [U-²H, U-¹³C]glucose, however, there was no 6PG in zwf1Δ cells. In the wild-type cells 3PG was approximately four-fold more abundant than 6PG Conclusion The resonance at ∼181 ppm in ¹³C MR spectra following injection of hyperpolarized [U-²H, U-¹³C]glucose originates predominantly from 6PG in EL4 tumors and 3PG in yeast cells. Magn Reson Med 74:1543–1547, 2015.

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 [1-¹³C, U-²H₅] ethanol was hyperpolarized using dynamic nuclear polarization, injected into mice and its oxidation in the liver monitored using ¹³C MR spectroscopy and spectroscopic imaging. Results Oxidation of [1-¹³C, U-²H₅] ethanol to [1-¹³C] acetate was observed. Saturation of the acetaldehyde resonance, which was below the level of detection in vivo, demonstrated that acetate was produced via acetaldehyde. Irreversible inhibition of ALDH2 activity with disulfiram resulted in a proportional decrease in the amplitude of the acetate resonance. Conclusion ¹³C magnetic resonance spectroscopy measurements of hyperpolarized [1-¹³C, U-²H₅] ethanol oxidation allow real-time assessment of ALDH2 activity in liver in vivo. Magn Reson Med, 2014. © The Authors. Magnetic Resonance in Medicine Published by Wiley Periodicals, Inc. on behalf of International Society of Medicine in Resonance. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited. Magn Reson Med 73:1733–1740, 2015.

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. Procedures Luciferase-expressing cells were transfected with a lentivirus encoding the rat reno-hepatic organic anion transporter protein, Oatp1, which was identified as a potential transporter for d-luciferin. Light output was compared between cells expressing luciferase and those also expressing Oatp1. Results In two cell lines and in mouse xenografts, co-expression of Oatp1 with luciferase increased light output by several fold, following addition of luciferin. Conclusions The increase in light output thus obtained will allow more sensitive detection of luciferase-expressing cells in vivo.

Purpose To assess the potential of a gene reporter system, based on a urea transporter (UTB) and hyperpolarized [¹³C]urea. Methods Mice were implanted subcutaneously with either unmodified control cells or otherwise identical cells expressing UTB. After injection of hyperpolarized [¹³C]urea, a spin echo sequence was used to measure urea concentration, T₁, and diffusion in control and UTB-expressing tissue. Results The apparent diffusion coefficient of hyperpolarized urea was 21% lower in tissue expressing UTB, in comparison with control tissue (P < 0.05, 1-tailed t-test, n = 6 in each group). No difference in water apparent diffusion coefficient or cellularity between these tissues was found, indicating that they were otherwise similar in composition. Conclusion Expression of UTB, by mediating cell uptake of urea, lowers the apparent diffusion coefficient of hyperpolarized ¹³C urea in tissue and thus the transporter has the potential to be used as a magnetic resonance-based gene reporter in vivo. Magn Reson Med 73:1401–1406, 2015.

Astrocytic–Neuronal–Astrocytic Pathway Selection for Formation and Degradation of Glutamate/GABA

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, Gd³⁺-based, hepatotrophic contrast agent (gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid). Cells expressing the reporter showed readily reversible, intense, and positive contrast (up to 7.8-fold signal enhancement) in T₁-weighted magnetic resonance images acquired in vivo. The maximum signal enhancement obtained so far is more than double that produced by MRI gene reporters described previously. Exchanging the Gd³⁺ ion for the radionuclide, ¹¹¹In, also allowed detection by single-photon emission computed tomography, thus combining the spatial resolution of MRI with the sensitivity of radionuclide imaging. Significance Gene reporters can be used to track viable cells in vivo and their patterns of gene expression. There have been numerous attempts to develop gene reporters for magnetic resonance imaging (MRI), however these give only modest image contrast and often this is negative, which can be difficult to detect. We describe here a dual-imaging modality reporter that gives intense and positive contrast in magnetic resonance images (up to ∼8× increase in signal), which can also be used with radionuclide imaging, thus combining the sensitivity of radionuclide imaging with the spatial resolution of MRI. The contrast obtained is directly related to the degree of gene expression and is readily reversible, thus allowing longitudinal studies of changes in expression.

One of the most likely substrates for metabolic imaging of response to treatment in cancer is glucose, but until now, using hyperpolarized 13C-labelled glucose has been problematic because of the short lifetime of the hyperpolarization in this molecule. Using [U-13C, U-2H]glucose, Tiago Rodrigues et al. now show that they are able to image its glycolytic conversion to lactate in two mouse tumor models in vivo, and that in one model, flux is markedly reduced after treatment with the chemotherapeutic drug etoposide. 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. This technique could provide a new way of detecting early evidence of tumor treatment response in the clinic and of monitoring tumor pentose phosphate pathway activity.

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. In this work we have investigated cerebral metabolism in mice with Mct8 deficiency. Adult male mice were infused for 30 minutes with (1-¹³C) glucose and brain extracts prepared and analyzed by ¹³C nuclear magnetic resonance spectroscopy. Genetic inactivation of Mct8 resulted in increased oxidative metabolism as reflected by increased glutamate C4 enrichment, and of glutamatergic and GABAergic neurotransmissions as observed by the increases in glutamine C4 and GABA C2 enrichments, respectively. These changes were distinct to those produced by hypothyroidism or hyperthyroidism. Similar increments in glutamate C4 enrichment and GABAergic neurotransmission were observed in the combined inactivation of Mct8 and D2, indicating that the increased neurotransmission and metabolic activity were not due to increased production of cerebral T₃ by the D2-encoded type 2 deiodinase. In conclusion, Mct8 deficiency has important metabolic consequences in the brain that could not be correlated with deficiency or excess of thyroid hormone supply to the brain during adulthood.