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1.
Cell ; 185(15): 2678-2689, 2022 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-35839759

RESUMEN

Metabolic anomalies contribute to tissue dysfunction. Current metabolism research spans from organelles to populations, and new technologies can accommodate investigation across these scales. Here, we review recent advancements in metabolic analysis, including small-scale metabolomics techniques amenable to organelles and rare cell types, functional screening to explore how cells respond to metabolic stress, and imaging approaches to non-invasively assess metabolic perturbations in diseases. We discuss how metabolomics provides an informative phenotypic dimension that complements genomic analysis in Mendelian and non-Mendelian disorders. We also outline pressing challenges and how addressing them may further clarify the biochemical basis of human disease.


Asunto(s)
Genómica , Metabolómica , Diagnóstico por Imagen , Humanos , Metabolómica/métodos
2.
Proc Natl Acad Sci U S A ; 119(19): e2120595119, 2022 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-35512101

RESUMEN

Glutamine is consumed by rapidly proliferating cells and can provide the carbon and nitrogen required for growth through various metabolic pathways. However, delineating the metabolic fate of glutamine is challenging to interrogate in vivo. Hyperpolarized magnetic resonance, by providing high transient nuclear magnetic resonance signals, provides an approach to measure fast biochemical processes in vivo. Aminohydrolysis of glutamine at carbon-5 plays an important role in providing nitrogen and carbon for multiple pathways. Here, we provide a synthetic strategy for isotope-enriched forms of glutamine that prolongs glutamine-C5 relaxation times and thereby reveals in vivo reactions involving carbon-5. We investigate multiple enrichment states, finding [5-13C,4,4-2H2,5-15N]-L-glutamine to be optimal for hyperpolarized measurement of glutamine conversion to glutamate in vivo. Leveraging this compound, we explore pancreatic cancer glutamine metabolism in vivo. Taken together, this work provides a means for studying glutamine metabolic flux in vivo and demonstrates on-target effects of metabolic enzyme inhibitors.


Asunto(s)
Glutaminasa , Glutamina , Biomarcadores/metabolismo , Ciclo del Ácido Cítrico , Glutaminasa/metabolismo , Glutamina/metabolismo , Humanos , Metabolómica
3.
Anal Chem ; 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-39034921

RESUMEN

Metabolic flux analysis of live cells using NMR enables the study of cancer metabolism and response to treatment. However, conventional NMR platforms require often prohibitively high numbers of cells to achieve significant resolution. In this work, we present a double 1H/13C resonance NMR probe consisting of a solenoid coil with a less than 100 nL sensitive region. In-solution robustness is demonstrated through measurement of decaying hyperpolarized signals. A suspension of live cells and hyperpolarized (HP) [1-13C]pyruvate is loaded in the coil, and dynamic changes in pyruvate and lactate concentrations by fractions of femtomoles are detected from just 2000 live cells at a time, in seconds. Through an integrated microfluidic channel, the probe is used as high-throughput platform to perform nondestructive quantitative analysis of metabolic flux of different leukemia cell lines with sensitivity to detect on target treatment response. This approach platform provides an approach to study mass-limited samples and living cells with dramatically enhanced sensitivity in real time.

4.
Magn Reson Med ; 91(5): 2162-2171, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38230992

RESUMEN

PURPOSE: To test the hypothesis that lactate oxidation contributes to the 13 $$ {}^{13} $$ C-bicarbonate signal observed in the awake human brain using hyperpolarized 13 $$ {}^{13} $$ C MRI. METHODS: Healthy human volunteers (N = 6) were scanned twice using hyperpolarized 13 $$ {}^{13} $$ C-MRI, with increased radiofrequency saturation of 13 $$ {}^{13} $$ C-lactate on one set of scans. 13 $$ {}^{13} $$ C-lactate, 13 $$ {}^{13} $$ C-bicarbonate, and 13 $$ {}^{13} $$ C-pyruvate signals for 132 brain regions across each set of scans were compared using a clustered Wilcoxon signed-rank test. RESULTS: Increased 13 $$ {}^{13} $$ C-lactate radiofrequency saturation resulted in a significantly lower 13 $$ {}^{13} $$ C-bicarbonate signal (p = 0.04). These changes were observed across the majority of brain regions. CONCLUSION: Radiofrequency saturation of 13 $$ {}^{13} $$ C-lactate leads to a decrease in 13 $$ {}^{13} $$ C-bicarbonate signal, demonstrating that the 13 $$ {}^{13} $$ C-lactate generated from the injected 13 $$ {}^{13} $$ C-pyruvate is being converted back to 13 $$ {}^{13} $$ C-pyruvate and oxidized throughout the human brain.


Asunto(s)
Bicarbonatos , Imagen por Resonancia Magnética , Humanos , Imagen por Resonancia Magnética/métodos , Encéfalo/diagnóstico por imagen , Ácido Pirúvico , Ácido Láctico , Isótopos de Carbono
5.
Magn Reson Med ; 91(6): 2559-2567, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38205934

RESUMEN

PURPOSE: To investigate the safety and value of hyperpolarized (HP) MRI of [1-13C]pyruvate in healthy volunteers using deuterium oxide (D2O) as a solvent. METHODS: Healthy volunteers (n = 5), were injected with HP [1-13C]pyruvate dissolved in D2O and imaged with a metabolite-specific 3D dual-echo dynamic EPI sequence at 3T at one site (Site 1). Volunteers were monitored following the procedure to assess safety. Image characteristics, including SNR, were compared to data acquired in a separate cohort using water as a solvent (n = 5) at another site (Site 2). The apparent spin-lattice relaxation time (T1) of [1-13C]pyruvate was determined both in vitro and in vivo from a mono-exponential fit to the image intensity at each time point of our dynamic data. RESULTS: All volunteers completed the study safely and reported no adverse effects. The use of D2O increased the T1 of [1-13C]pyruvate from 66.5 ± 1.6 s to 92.1 ± 5.1 s in vitro, which resulted in an increase in signal by a factor of 1.46 ± 0.03 at the time of injection (90 s after dissolution). The use of D2O also increased the apparent relaxation time of [1-13C]pyruvate by a factor of 1.4 ± 0.2 in vivo. After adjusting for inter-site SNR differences, the use of D2O was shown to increase image SNR by a factor of 2.6 ± 0.2 in humans. CONCLUSIONS: HP [1-13C]pyruvate in D2O is safe for human imaging and provides an increase in T1 and SNR that may improve image quality.


Asunto(s)
Imagen por Resonancia Magnética , Ácido Pirúvico , Humanos , Estudios de Factibilidad , Imagen por Resonancia Magnética/métodos , Encéfalo/diagnóstico por imagen , Isótopos de Carbono , Solventes
6.
NMR Biomed ; 36(10): e4989, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37336778

RESUMEN

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths. Imaging plays a crucial role in the early detection of HCC, although current methods are limited in their ability to characterize liver lesions. Most recently, deuterium metabolic imaging (DMI) has been demonstrated as a powerful technique for the imaging of metabolism in vivo. Here, we assess the metabolic flux of [6,6'-2 H2 ] fructose in cell cultures and in subcutaneous mouse models at 9.4 T. We compare these rates with the most widely used DMI probe, [6,6'-2 H2 ] glucose, exploring the possibility of developing 2 H fructose to overcome the limitations of glucose as a novel DMI probe for detecting liver tumors. Comparison of the in vitro metabolic rates implies their similar glycolytic metabolism in the TCA cycle due to comparable production rates of 2 H glutamate/glutamine (glx) for the two precursors, but overall higher glycolytic metabolism from 2 H glucose because of a higher production rate of 2 H lactate. In vivo kinetic studies suggest that HDO can serve as a robust reporter for the consumption of the precursors in liver tumors. As fructose is predominantly metabolized in the liver, deuterated water (HDO) produced from 2 H fructose is probably less contaminated from whole-body metabolism in comparison with glucose. Moreover, in studies of the normal liver, 2 H fructose is readily converted to 2 H glx, enabling the characterization of 2 H fructose kinetics. This overcomes a major limitation of previous 2 H glucose studies in the liver, which were unable to confidently discern metabolic flux due to overlapped signals of 2 H glucose and its metabolic product, 2 H glycogen. This suggests a unique role for 2 H fructose metabolism in HCC and the normal liver, making it a useful approach for assessing liver-related diseases and the progression to oncogenesis.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ratones , Animales , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/metabolismo , Deuterio/metabolismo , Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/metabolismo , Cinética , Fructosa/metabolismo , Glucosa/metabolismo , Hígado/diagnóstico por imagen , Hígado/metabolismo , Ácido Láctico/metabolismo
7.
MAGMA ; 35(6): 895-901, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-35876917

RESUMEN

OBJECT: Spatial variation in the sensitivity profiles of receive coils in MRI leads to spatially dependent scaling of the signal amplitude across an image. In practice, total sensitivity of the coil array is either calibrated or corrected directly by comparison to a uniform sensitivity image, fitting of coil profiles, or indirectly by constraining the reconstructed image or coil profiles. In the absence of these corrections, popular coil summation strategies are often designed to maximize the signal-to-noise ratio or optimize under-sampled encoding but not necessarily estimate the value of the signal unscaled by the coil spatial sensitivity. MATERIALS AND METHODS: We use ratios of first-order statistics to approach the unscaled value of the signal at any position. Motivated by the assumption that the coil array is a sample from much larger number of possible coils, we present two approaches to scale the mean signal in all coils: (1) an argument for use of the mode of the normalized signals, and (2) using a one-dimensional analog derive an approximate expression for scaling with the ratio of the square-of-the-mean to the mean-of-the-squares. We test these approaches with simulation where idealized coil elements are arrayed around an object, and on directly acquired data with an 8-channel coil array on a uniform 13C phantom, and on Hyperpolarized 13C pyruvate brain MRI. RESULTS: We show improved image uniformity using the ratios of first order statistics compared to a simple homomorphic filter, noting that these approaches are more sensitive to noise. DISCUSSION: We present simple methods for correcting the spatial variation in sensitivity profiles in the context of a coil array. These methods can be used as an initial or adjunct step in data post-processing.


Asunto(s)
Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Relación Señal-Ruido , Fantasmas de Imagen , Piruvatos , Encéfalo/diagnóstico por imagen
8.
Magn Reson Med ; 85(2): 978-986, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32820566

RESUMEN

PURPOSE: To generate dynamic, volumetric maps of hyperpolarized [1-13 C]pyruvate and its metabolic products in vivo. METHODS: Maps of chemical species were generated with iterative least squares (IDEAL) reconstruction from multiecho echo-planar imaging (EPI) of phantoms of thermally polarized 13 C-labeled chemicals and mice injected with hyperpolarized [1-13 C]pyruvate on a preclinical 3T scanner. The quality of the IDEAL decomposition of single-shot and multishot phantom images was evaluated using quantitative results from a simple pulse-and-acquire sequence as the gold standard. Time course and area-under-the-curve plots were created to analyze the distribution of metabolites in vivo. RESULTS: Improved separation of chemical species by IDEAL, evaluated by the amount of residual signal measured for chemicals not present in the phantoms, was observed as the number of EPI shots was increased from one to four. Dynamic three-dimensional metabolite maps of [1-13 C]pyruvate,[1-13 C]pyruvatehydrate, [1-13 C]lactate, [1-13 C]bicarbonate, and [1-13 C]alanine generated by IDEAL from interleaved multishot multiecho EPI of live mice were used to construct time course and area-under-the-curve graphs for the heart, kidneys, and liver, which showed good agreement with previously published results. CONCLUSIONS: IDEAL decomposition of multishot multiecho 13C EPI images is a simple, yet robust method for generating high-quality dynamic volumetric maps of hyperpolarized [1-13 C]pyruvate and its products in vivo and has potential applications for the assessment of multiorgan metabolic phenomena.


Asunto(s)
Imagen Eco-Planar , Ácido Pirúvico , Animales , Isótopos de Carbono , Ácido Láctico , Análisis de los Mínimos Cuadrados , Imagen por Resonancia Magnética , Ratones , Fantasmas de Imagen
9.
NMR Biomed ; 34(3): e4447, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33314422

RESUMEN

Hyperpolarized [1-13 C] pyruvate can be used to examine the metabolic state of cancer cells, highlighting a key metabolic characteristic of cancer: the upregulated metabolic flux to lactate, even in the presence of oxygen (Warburg effect). Thus, the rate constant of 13 C exchange of pyruvate to lactate, kPL , can serve as a metabolic biomarker of cancer presence, aggressiveness and therapy response. Established in vitro hyperpolarized experiments dissolve the probe for each cell sample independently, an inefficient process that consumes excessive time and resources. Expanding on our previous development of a microcoil with greatly increased detection sensitivity (103 -fold) compared with traditional in vitro methods, we present a novel microcoil equipped with a 10-µL vertical reservoir and an experimental protocol utilizing deuterated dissolution buffer to measure metabolic flux in multiple mass-limited cell suspension samples using a single dissolution. This method increases efficiency and potentially reduces the methodological variability associated with hyperpolarized experiments. This technique was used to measure pyruvate-to-lactate flux in melanoma cells to assess BRAF-inhibition treatment response. There was a significant reduction of kPL in BRAFV600E cells following 24 and 48 hours of treatment with 2 µM vemurafenib (P ≤ .05). This agrees with significant changes observed in the pool sizes of extracellular lactate (P ≤ .05) and glucose (P ≤ .001) following 6 and 48 hours of treatment, respectively, and a significant reduction in cell proliferation following 72 hours of treatment (P ≤ .01). BRAF inhibition had no significant effect on the metabolic flux of BRAFWT cells. These data demonstrate a 6-8-fold increase in efficiency for the measurement of kPL in cell suspension samples compared with traditional hyperpolarized in vitro methods.


Asunto(s)
Ácido Láctico/metabolismo , Melanoma/metabolismo , Análisis de Flujos Metabólicos , Ácido Pirúvico/metabolismo , Línea Celular Tumoral , Humanos , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Resultado del Tratamiento
11.
Proc Natl Acad Sci U S A ; 111(27): 9983-8, 2014 Jul 08.
Artículo en Inglés | MEDLINE | ID: mdl-24961373

RESUMEN

Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.


Asunto(s)
Hígado Graso/fisiopatología , Hipoglucemiantes/farmacología , Metformina/farmacología , Factor 1 de Transcripción de Unión a Octámeros/fisiología , Tiamina/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Ratones , Ratones Noqueados , Factor 1 de Transcripción de Unión a Octámeros/efectos de los fármacos , Factor 1 de Transcripción de Unión a Octámeros/genética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Oxidación-Reducción
12.
Anal Chem ; 88(22): 11147-11153, 2016 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-27749041

RESUMEN

Nuclear magnetic resonance (NMR) spectroscopy is widely used in metabolomics to perform quantitative profiling of low-molecular weight compounds from biological specimens. The measurement of endogenous metabolites using NMR has proven to be a powerful tool to identify new metabolic biomarkers in physiological and pathological conditions, and to study and evaluate treatment efficiency. In this study we present a rapid approach to indirectly quantify 13C enriched molecules using one-dimensional (1D) 1H NMR. We demonstrate this approach using isotopically labeled [1,6-13C]glucose and in four different cell lines. We confirm the applicability of this approach for treatment follow-up, utilizing a renal cancer cell line with rapamycin as a tool compound to study changes in metabolic profiles. Finally, we validate the applicability of this method to study metabolic biomarkers from ex vivo tumor extracts, after infusion, using isotopically enriched glucose. Given the high throughput and increased sensitivity of direct-detect 1H NMR, this analytical approach provides an avenue for simple and rapid metabolic analysis of biological samples including blood, urine, and biopsies.


Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Metabolómica , Espectroscopía de Protones por Resonancia Magnética , Isótopos de Carbono , Línea Celular , Glucosa/química , Humanos , Estructura Molecular , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo
13.
Bioconjug Chem ; 27(1): 170-8, 2016 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-26649808

RESUMEN

Solid tumors are hypoxic with altered metabolism, resulting in secretion of acids into the extracellular matrix and lower relative pH, a feature associated with local invasion and metastasis. Therapeutic and diagnostic agents responsive to this microenvironment may improve tumor-specific delivery. Therefore, we pursued a general strategy whereby caged small-molecule drugs or imaging agents liberate their parent compounds in regions of low interstitial pH. In this manuscript, we present a new acid-labile prodrug method based on the glycosylamine linkage, and its application to a class of positron emission tomography (PET) imaging tracers, termed [(18)F]FDG amines. [(18)F]FDG amines operate via a proposed two-step mechanism, in which an acid-labile precursor decomposes to form the common radiotracer 2-deoxy-2-[(18)F]fluoro-d-glucose, which is subsequently accumulated by glucose avid cells. The rate of decomposition of [(18)F]FDG amines is tunable in a systematic fashion, tracking the pKa of the parent amine. In vivo, a 4-phenylbenzylamine [(18)F]FDG amine congener showed greater relative accumulation in tumors over benign tissue, which could be attenuated upon tumor alkalinization using previously validated models, including sodium bicarbonate treatment, or overexpression of carbonic anhydrase. This new class of PET tracer represents a viable approach for imaging acidic interstitial pH with potential for clinical translation.


Asunto(s)
Fluorodesoxiglucosa F18/química , Tomografía de Emisión de Positrones/métodos , Radiofármacos/química , Microambiente Tumoral , Aminas/química , Animales , Línea Celular Tumoral , Técnicas de Química Sintética , Humanos , Concentración de Iones de Hidrógeno , Masculino , Ratones Desnudos , Neoplasias Experimentales/diagnóstico por imagen , Oximas/química , Profármacos/química , Radioquímica/métodos , Radiofármacos/síntesis química , Ensayos Antitumor por Modelo de Xenoinjerto
16.
Prostate ; 75(14): 1601-9, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26177608

RESUMEN

BACKGROUND: Metabolic shifts in disease are of great interest for the development of novel therapeutics. In cancer treatment, these therapies exploit the metabolic phenotype associated with oncogenesis and cancer progression. One recent strategy involves the depletion of the cofactors needed to maintain the high rate of glycolysis seen with the Warburg effect. Specifically, blocking nicotinamide adenine dinucleotide (NAD) biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) inhibition depletes cancer cells of the NAD needed for glycolysis. To characterize this metabolic phenotype in vivo and describe changes in flux with treatment, non-invasive biomarkers are necessary. One such biomarker is hyperpolarized (HP) [1-(13) C] pyruvate, a clinically translatable probe that allows real-time assessment of metabolism. METHODS: We therefore developed a cell perfusion system compatible with HP magnetic resonance (MR) and positron emission tomography (PET) to develop translatable biomarkers of response to NAMPT inhibition in reduced volume cell cultures. RESULTS: Using this platform, we observed a reduction in pyruvate flux through lactate dehydrogenase with NAMPT inhibition in prostate cancer cells, and showed that both HP lactate and 2-[(18) F] fluoro-2-deoxy-D-glucose (FDG) can be used as biomarkers for treatment response of such targeted agents. Moreover, we observed dynamic flux changes whereby HP pyruvate was re-routed to alanine, providing both positive and negative indicators of treatment response. CONCLUSIONS: This study demonstrated the feasibility of a MR/PET compatible bioreactor approach to efficiently explore cell and tissue metabolism, the understanding of which is critical for developing clinically translatable biomarkers of disease states and responses to therapeutics.


Asunto(s)
Reactores Biológicos , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Nicotinamida Fosforribosiltransferasa/metabolismo , Tomografía de Emisión de Positrones/métodos , Neoplasias de la Próstata/metabolismo , Humanos , Masculino , Células Tumorales Cultivadas
17.
Magn Reson Med ; 74(3): 622-633, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25213126

RESUMEN

PURPOSE: Hyperpolarized (13) C magnetic resonance allows for the study of real-time metabolism in vivo, including significant hyperpolarized (13) C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized (13) C lactate but also understand its compartmentalization using diffusion-weighted MR will provide unique information for improved tumor characterization. METHODS: We used a bipolar, pulsed-gradient, double spin echo imaging sequence to rapidly generate diffusion-weighted images of hyperpolarized (13) C metabolites. Our methodology included a simultaneously acquired B1 map to improve apparent diffusion coefficient (ADC) accuracy and a diffusion-compensated variable flip angle scheme to improve ADC precision. RESULTS: We validated this sequence and methodology in hyperpolarized (13) C phantoms. Next, we generated ADC maps of several hyperpolarized (13) C metabolites in a normal rat, rat brain tumor, and prostate cancer mouse model using both preclinical and clinical trial-ready hardware. CONCLUSION: ADC maps of hyperpolarized (13) C metabolites provide information about the localization of these molecules in the tissue microenvironment. The methodology presented here allows for further studies to investigate ADC changes due to disease state that may provide unique information about cancer aggressiveness and metastatic potential.


Asunto(s)
Isótopos de Carbono/metabolismo , Imagen de Difusión por Resonancia Magnética/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Animales , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Isótopos de Carbono/análisis , Isótopos de Carbono/química , Línea Celular Tumoral , Humanos , Ácido Láctico/química , Ácido Láctico/metabolismo , Ratones , Fantasmas de Imagen , Ratas , Ratas Sprague-Dawley
18.
NMR Biomed ; 28(9): 1141-9, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26202449

RESUMEN

We have developed a 3D cell/tissue culture bioreactor compatible with hyperpolarized (HP) (13)C MR and interrogated HP [1-(13)C]lactate production and efflux in human renal cell carcinoma (RCC) cells. This platform is capable of resolving intracellular and extracellular HP lactate pools, allowing the kinetic measurement of lactate production and efflux in the context of cancer aggressiveness and response to therapy. HP (13)C MR studies were performed on three immortalized human renal cell lines: HK2, a normal renal proximal tubule cell line from which a majority of RCCs arise, UMRC6, a cell line derived from a localized RCC, and UOK262, an aggressive and metastatic RCC. The intra- (Lacin ) and extracellular (Lacex ) HP lactate signals were robustly resolved in dynamic (13)C spectra of the cell lines due to a very small but reproducible chemical shift difference (0.031 ± 0.0005 ppm). Following HP [1-(13)C]pyruvate delivery, the ratio of HP Lacin /Lacex was significantly lower for UOK262 cells compared with both UMRC6 and HK2 cells due to a significant (p < 0.05) increase in the Lacex pool size. Lacin /Lacex correlated with the MCT4 mRNA expression of the cell lines, and inhibition of MCT4 transport using DIDS resulted in a significant reduction in the HP Lacex pool size. The extension of these studies to living patient-derived RCC tissue slices using HP [1,2-(13)C2]pyruvate demonstrated a similarly split lactate doublet with a high Lacex pool fraction; in contrast, only a single NMR resonance is noted for HP [5-(13)C]glutamate, consistent with intracellular localization. These studies support the importance of lactate efflux as a biomarker of cancer aggressiveness and metastatic potential, and the utility of the MR compatible 3D cell/tissue culture bioreactor to study not only cellular metabolism but also transport. Additionally, this platform offers a sophisticated way to follow therapeutic interventions and screen novel therapies that target lactate export.


Asunto(s)
Reactores Biológicos , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Ácido Láctico/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Biomarcadores de Tumor , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Riñón/metabolismo , Neoplasias Renales/patología
19.
Chem Soc Rev ; 43(5): 1627-59, 2014 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-24363044

RESUMEN

The study of transient chemical phenomena by conventional NMR has proved elusive, particularly for non-(1)H nuclei. For (13)C, hyperpolarization using the dynamic nuclear polarization (DNP) technique has emerged as a powerful means to improve SNR. The recent development of rapid dissolution DNP methods has facilitated previously impossible in vitro and in vivo study of small molecules. This review presents the basics of the DNP technique, identification of appropriate DNP substrates, and approaches to increase hyperpolarized signal lifetimes. Also addressed are the biochemical events to which DNP-NMR has been applied, with descriptions of several probes that have met with in vivo success.


Asunto(s)
Imagen por Resonancia Magnética , Espectroscopía de Resonancia Magnética , Reactores Biológicos , Isótopos de Carbono/química , Deuterio/química , Enzimas/metabolismo , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Teoría Cuántica
20.
Proc Natl Acad Sci U S A ; 108(46): 18606-11, 2011 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-22042839

RESUMEN

Reduction and oxidation (redox) chemistry is involved in both normal and abnormal cellular function, in processes as diverse as circadian rhythms and neurotransmission. Intracellular redox is maintained by coupled reactions involving NADPH, glutathione (GSH), and vitamin C, as well as their corresponding oxidized counterparts. In addition to functioning as enzyme cofactors, these reducing agents have a critical role in dealing with reactive oxygen species (ROS), the toxic products of oxidative metabolism seen as culprits in aging, neurodegenerative disease, and ischemia/ reperfusion injury. Despite this strong relationship between redox and human disease, methods to interrogate a redox pair in vivo are limited. Here we report the development of [1-(13)C] dehydroascorbate [DHA], the oxidized form of Vitamin C, as an endogenous redox sensor for in vivo imaging using hyperpolarized (13)C spectroscopy. In murine models, hyperpolarized [1-(13)C] DHA was rapidly converted to [1-(13)C] vitamin C within the liver, kidneys, and brain, as well as within tumor in a transgenic prostate cancer mouse. This result is consistent with what has been previously described for the DHA/Vitamin C redox pair, and points to a role for hyperpolarized [1-(13)C] DHA in characterizing the concentrations of key intracellular reducing agents, including GSH. More broadly, these findings suggest a prognostic role for this new redox sensor in determining vulnerability of both normal and abnormal tissues to ROS.


Asunto(s)
Isótopos de Carbono/química , Ácido Deshidroascórbico/química , Oxidación-Reducción , Animales , Anisotropía , Ácido Ascórbico/química , Ácido Ascórbico/metabolismo , Encéfalo/metabolismo , Glutatión/metabolismo , Riñón/metabolismo , Cinética , Hígado/metabolismo , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Ratones , Neoplasias/metabolismo , Especies Reactivas de Oxígeno , Distribución Tisular
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