Effect of reducing isoflurane level on glucosamine uptake in the mouse brain during magnetic resonance imaging studies.

Anesthesia is often required during magnetic resonance imaging (MRI) examinations in animal studies. Anesthetic drugs differ in their capacity to interfere with homeostatic mechanisms responsible for glucose metabolism in the brain, which may create a constraint in the study design. Recent studies suggest that the chemical exchange saturation transfer (CEST) MRI scanning technique can detect localized metabolic changes in rodent brains induced by the uptake of glucose or its analogs; however, most of these studies do not account for the impact of anesthesia type on the brain metabolism. Herein, we aimed to evaluate the effect of reduced isoflurane levels on the preclinical imaging of glucosamine (GlcN) uptake in healthy mouse brains to establish optimal conditions for future brain imaging studies using the CEST MRI technique. The commonly used anesthesia protocol for longitudinal MRI examinations using 1.5% isoflurane level was compared to that using a mixture of low isoflurane (0.8%) level combined with midazolam (2 mg/kg, SC). Magnetization transfer ratio asymmetry (MTRasym) and area under the curve (AUC) analyses were used to characterize GlcN signals in the brain. The results indicated that mice injected with GlcN and anesthetized with 1.5% isoflurane exhibited low and insignificant changes in the MTRasym and AUC signals in the frontal cortex, whereas mice administered with 0.8% isoflurane combined with midazolam demonstrated a significant increase in these signals in the frontal cortex. This study highlights the diverse GlcN metabolic changes observed in mouse brains under variable levels of isoflurane anesthesia using the CEST MRI method. The results suggest that it is feasible to maintain anesthesia with low-dose isoflurane by integrating midazolam, which may enable the investigation of GlcN uptake in the brain. Thus, reducing isoflurane levels may support studies into mouse brain metabolism using the CEST MRI method and should be considered in future studies.

Metabolic brain imaging with glucosamine CEST MRI: in vivo characterization and first insights.

The utility of chemical exchange saturation transfer (CEST) MRI for monitoring the uptake of glucosamine (GlcN), a safe dietary supplement, has been previously demonstrated in detecting breast cancer in both murine and human subjects. Here, we studied and characterized the detectability of GlcN uptake and metabolism in the brain. Following intravenous GlcN administration in mice, CEST brain signals calculated by magnetization transfer ratio asymmetry (MTRasym) analysis, were significantly elevated, mainly in the cortex, hippocampus, and thalamus. The in vivo contrast remained stable during 40 min of examination, which can be attributed to GlcN uptake and its metabolic products accumulation as confirmed using 13C NMR spectroscopic studies of brain extracts. A Lorentzian multi-pool fitting analysis revealed an increase in the hydroxyl, amide, and relayed nuclear Overhauser effect (rNOE) signal components after GlcN treatment. With its ability to cross the blood-brain barrier (BBB), the GlcN CEST technique has the potential to serve as a metabolic biomarker for the diagnosis and monitoring various brain disorders.

Phosphate buffer-catalyzed kinetics of mutarotation of glucosamine investigated by NMR spectroscopy.

Glucosamine (2-amino-2-deoxy-d-glucose, GlcN) is a naturally occurring amino monosaccharide that is essential for a variety of biological functions, it is mainly involved in the formation of polysaccharide structures. It was recently reported to enable the imaging of cancerous tumors as an exogenous contrast agent using the MRI technique of chemical exchange saturation transfer (CEST). In preparation for the clinical use of GlcN, its anomeric equilibrium and mutarotation rate constants were directly investigated in this study utilizing high resolution 1H and 13C NMR spectroscopy. The effects of GlcN concentration, temperature, pH and buffer on the mutarotation rate constant and mutarotation equilibrium were measured. The mutarotation rate constant increased markedly with increasing GlcN concentrations. The rate constant of mutarotation of GlcN at room temperature was 2.2 × 10-4 - 5.0 × 10-4 s-1 at concentrations of 0.02-0.5 M, corresponding to a time of 3.8-1.7 h to reach 95% equilibrium. The anomeric ratio was strongly pH-dependent. The influence of phosphate buffer on the apparent rate constant of GlcN mutarotation was investigated. For phosphate buffer saline values between 0 and 50 mM, there was a six-fold increase in rate at pH 7.0. The mutarotation rate constant rose rapidly with pH at a phosphate concentration of 50 mM: from 0.4 × 10-3 s-1 at pH 5.0 to 7.8 × 10-3 s-1 at pH 9.4, suggesting that the catalysis is due to the HPO42- and PO43- ions. These findings might help researchers design the experimental setting for employing GlcN for cancer detection using GlcN-CEST MRI.

Breast cancer imaging with glucosamine CEST (chemical exchange saturation transfer) MRI: first human experience.

OBJECTIVES: This study aims to evaluate the feasibility of imaging breast cancer with glucosamine (GlcN) chemical exchange saturation transfer (CEST) MRI technique to distinguish between tumor and surrounding tissue, compared to the conventional MRI method. METHODS: Twelve patients with newly diagnosed breast tumors (median age, 53 years) were recruited in this prospective IRB-approved study, between August 2019 and March 2020. Informed consent was obtained from all patients. All MRI measurements were performed on a 3-T clinical MRI scanner. For CEST imaging, a fat-suppressed 3D RF-spoiled gradient echo sequence with saturation pulse train was applied. CEST signals were quantified in the tumor and in the surrounding tissue based on magnetization transfer ratio asymmetry (MTRasym) and a multi-Gaussian fitting. RESULTS: GlcN CEST MRI revealed higher signal intensities in the tumor tissue compared to the surrounding breast tissue (MTRasym effect of 8.12 ± 4.09%, N = 12, p = 2.2 E-03) with the incremental increase due to GlcN uptake of 3.41 ± 0.79% (N = 12, p = 2.2 E-03), which is in line with tumor location as demonstrated by T1W and T2W MRI. GlcN CEST spectra comprise distinct peaks corresponding to proton exchange between free water and hydroxyl and amide/amine groups, and relayed nuclear Overhauser enhancement (NOE) from aliphatic groups, all yielded larger CEST integrals in the tumor tissue after GlcN uptake by an averaged factor of 2.2 ± 1.2 (p = 3.38 E-03), 1.4 ± 0.4 (p =9.88 E-03), and 1.6 ± 0.6 (p = 2.09 E-02), respectively. CONCLUSION: The results of this initial feasibility study indicate the potential of GlcN CEST MRI to diagnose breast cancer in a clinical setup. KEY POINTS: • GlcN CEST MRI method is demonstrated for its the ability to differentiate between breast tumor lesions and the surrounding tissue, based on the differential accumulation of the GlcN in the tumors. • GlcN CEST imaging may be used to identify metabolic active malignant breast tumors without using a Gd contrast agent. • The GlcN CEST MRI method may be considered for use in a clinical setup for breast cancer detection and should be tested as a complementary method to conventional clinical MRI methods.

What do we know about dynamic glucose-enhanced (DGE) MRI and how close is it to the clinics? Horizon 2020 GLINT consortium report.

Cancer is one of the most devastating diseases that the world is currently facing, accounting for 10 million deaths in 2020 (WHO). In the last two decades, advanced medical imaging has played an ever more important role in the early detection of the disease, as it increases the chances of survival and the potential for full recovery. To date, dynamic glucose-enhanced (DGE) MRI using glucose-based chemical exchange saturation transfer (glucoCEST) has demonstrated the sensitivity to detect both D-glucose and glucose analogs, such as 3-oxy-methyl-D-glucose (3OMG) uptake in tumors. As one of the recent international efforts aiming at pushing the boundaries of translation of the DGE MRI technique into clinical practice, a multidisciplinary team of eight partners came together to form the "glucoCEST Imaging of Neoplastic Tumors (GLINT)" consortium, funded by the Horizon 2020 European Commission. This paper summarizes the progress made to date both by these groups and others in increasing our knowledge of the underlying mechanisms related to this technique as well as translating it into clinical practice.

Molecular imaging of cancer by glucosamine chemical exchange saturation transfer MRI: A preclinical study.

Glucosamine (GlcN) was recently proposed as an agent with an excellent safety profile to detect cancer with the chemical exchange saturation transfer (CEST) MRI technique. Translation of the GlcN CEST method to the clinical application requires evaluation of its sensitivity to the different frequency regions of irradiation. Hence, imaging of the GlcN signal was established for the full Z spectra recorded following GlcN administration to mice bearing implanted 4T1 breast tumors. Significant CEST effects were observed at around 1.5, 3.6 and -3.4 ppm, corresponding to the hydroxyl, amine/amide exchangeable protons and for the Nuclear Overhauser Enhancement (NOE), respectively. The sources of the observed CEST effects were investigated by identifying the GlcN metabolic products as observed by 13 C NMR spectroscopy studies of extracts from the same tumor model following treatment with [UL-13 C] -GlcN·HCl. The CEST contribution can be attributed to several phosphorylated products of GlcN, including uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is a substrate for the O-linked and N-linked glycosylated proteins that may be associated with the increase of the NOE signal. The observation of a significant amount of lactate among the metabolic products hints at acidification as one of the sources of the enhanced CEST effect of GlcN. The proposed method may offer a new approach for clinical molecular imaging that enables the detection of metabolically active tumors and may play a role in other diseases.

Molecular imaging of tumors by chemical exchange saturation transfer MRI of glucose analogs.

Early detection of the cancerous process would benefit greatly from imaging at the cellular and molecular level. Increased glucose demand has been recognized as one of the hallmarks of cancerous cells (the "Warburg effect"), hence glucose and its analogs are commonly used for cancer imaging. One example is FDG-PET technique, that led to the use of chemical exchange saturation transfer (CEST) MRI of glucose ("glucoCEST") for tumor imaging. This technique combines high-resolution MRI obtained by conventional imaging with simultaneous molecular information obtained from the exploitation of agents with exchangeable protons from amine, amide or hydroxyl residues with the water signal. In the case of glucoCEST, these agents are based on glucose or its analogs. Recently, preclinical glucoCEST studies demonstrated the ability to increase the sensitivity of MRI to the level of metabolic activity, enabling identification of tumor staging, biologic potential, treatment planning, therapy response and local recurrence, in addition to guiding target biopsy for clinically suspected cancer. However, natural glucose limits this method because of its rapid conversion to lactic acid, leading to reduced CEST effect and short signal duration. For that reason, a variety of glucose analogs have been tested as alternatives to the original glucoCEST. This review discusses the merits of these analogs, including new data on glucose analogs heretofore not reported in the literature. This summarized preclinical data may help strengthen the translation of CEST MRI of glucose analogs into the clinic, improving cancer imaging to enable early intervention without the need for invasive techniques. The data should also broaden our knowledge of fundamental biological processes.

Quantification of hydroxyl exchange of D-Glucose at physiological conditions for optimization of glucoCEST MRI at 3, 7 and 9.4 Tesla.

AIMS: To determine individual glucose hydroxyl exchange rates at physiological conditions and use this information for numerical optimization of glucoCEST/CESL preparation. To give guidelines for in vivo glucoCEST/CESL measurement parameters at clinical and ultra-high field strengths. METHODS: Five glucose solution samples at different pH values were measured at 14.1 T at various B1 power levels. Multi-B1 -Z-spectra Bloch-McConnell fits at physiological pH were further improved by the fitting of Z-spectra of five pH values simultaneously. The obtained exchange rates were used in a six-pool Bloch-McConnell simulation including a tissue-like water pool and semi-solid MT pool with different CEST and CESL presaturation pulse trains. In vivo glucose injection experiments were performed in a tumor mouse model at 7 T. RESULTS AND DISCUSSION: Glucose Z-spectra could be fitted with four exchanging pools at 0.66, 1.28, 2.08 and 2.88 ppm. Corresponding hydroxyl exchange rates could be determined at pH = 7.2, T = 37°C and 1X PBS. Simulation of saturation transfer for this glucose system in a gray matter-like and a tumor-like system revealed optimal pulses at different field strengths of 9.4, 7 and 3 T. Different existing sequences and approaches are simulated and discussed. The optima found could be experimentally verified in an animal model at 7 T. CONCLUSION: For the determined fast exchange regime, presaturation pulses in the spin-lock regime (long recover time, short yet strong saturation) were found to be optimal. This study gives an estimation for optimization of the glucoCEST signal in vivo on the basis of glucose exchange rate at physiological conditions.

3-O-Methyl-D-glucose mutarotation and proton exchange rates assessed by 13C, 1H NMR and by chemical exchange saturation transfer and spin lock measurements.

3-O-Methyl-D-glucose (3OMG) was recently suggested as an agent to image tumors using chemical exchange saturation transfer (CEST) MRI. To characterize the properties of 3OMG in solution, the anomeric equilibrium and the mutarotation rates of 3OMG were studied by 1H and 13C NMR. This information is essential in designing the in vivo CEST experiments. At room temperature, the ratio of α and ß 3OMG anomers at equilibrium was 1:1.4, and the time to reach 95% equilibrium was 6 h. The chemical exchange rates between the hydroxyl protons of 3OMG and water, measured by CEST and spin lock at pH 6.14 and a temperature of 4 °C, were in the range of 360-670 s-1.

CEST MRI of 3-O-methyl-D-glucose on different breast cancer models.

PURPOSE: To test the ability of chemical exchange saturation transfer (CEST) MRI of 3-O-methyl-D-glucose (3OMG) to detect tumors in several breast cancer models of murine and human origin, for different routes of administration of the agent and to compare the method with glucoCEST and with 18 FDG-PET on the same animals. METHODS: In vivo CEST MRI experiments were performed with a 7T Biospec animal MRI scanner on implanted orthotopic mammary tumors of mice before and after administration of 3OMG. RESULTS: A marked 3OMG-CEST MRI contrast that was correlated with the administrated dose was obtained in different breast cancer models and by intravenous, intraperitoneal, and per os methods of administration. The most aggressive breast cancer model yielded the highest CEST contrast. 3OMG-CEST contrast reached its maximum at 20 min after administration and lasted for more than an hour, while that of glucose was lower and diminished after 20 min. 3OMG-CEST showed comparable results to that of FDG PET. CONCLUSION: The sensitivity of the 3OMG-CEST MRI method indicates its potential for the detection of tumors in the clinic. Magn Reson Med 79:1061-1069, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Glucosamine and N-acetyl glucosamine as new CEST MRI agents for molecular imaging of tumors.

The efficacy of glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) as agents for chemical exchange saturation transfer (CEST) magnetic resonance molecular imaging of tumors is demonstrated. Both agents reflect the metabolic activity and malignancy of the tumors. The method was tested in two types of tumors implanted orthotopically in mice: 4T1 (mouse mammary cancer cells) and MCF7 (human mammary cancer cells). 4T1 is a more aggressive type of tumor than MCF7 and exhibited a larger CEST effect. Two methods of administration of the agents, intravenous (IV) and oral (PO), gave similar results. The CEST MRI observation of lung metastasis was confirmed by histology. The potential of the clinical application of CEST MRI with these agents for cancer diagnosis is strengthened by their lack of toxicity as can be indicated from their wide use as food supplements.

NMR studies of the equilibria and reaction rates in aqueous solutions of formaldehyde.

Formaldehyde has an important role in the chemical industry and in biological sciences. In dilute aqueous solutions of formaldehyde only traces of the molecular formaldehyde are present and the predominant species are methylene glycol and in lower concentrations, dimethylene glycol. The chemical equilibria and reaction rates of the hydration of formaldehyde in H2O and D2O solutions at low concentrations were studied by (1)H and (13)C NMR at various conditions of pH (1.8-7.8) and temperature (278-333 K). These measurements became possible by direct detection of formaldehyde (13)C and (1)H peaks. The equilibrium and rate constants of the dimerization reaction of methylene glycol were also measured. The rate constants for both the hydration and the dimerization reactions were measured by a new version of the conventional selective inversion transfer method. This study, together with previous published work, completes the description of dynamics and equilibria of all the processes occurring in dilute aqueous formaldehyde solutions.

Functional molecular imaging of tumors by chemical exchange saturation transfer MRI of 3-O-Methyl-D-glucose.

PURPOSE: To evaluate the feasibility to detect tumors and metastases by the chemical exchange saturation transfer (CEST) MRI technique using 3-O-Methyl-D-glucose (3OMG), a nonmetabolizable derivative of glucose that is taken up rapidly and preferentially by tumors and is entirely excreted by the kidneys. METHODS: In vivo CEST MRI experiments were performed on a Bruker 7 Tesla Biospec on implanted orthotopic mammary tumors of mice before and following i.p. injection of 3OMG. The CEST images were generated by a series of gradient-echo images collected from a single 1 mm coronal slice after a 1.2 s presaturation pulse, applied at offsets of ±1.2 ppm from the water and at B(1) power of 2.5 µT. RESULTS: Following 3OMG (1.5 g/kg) i.p. injection, an enhanced CEST effect of approximately 20% was visualized at the tumor within a few minutes. The signal slowly declined reaching half of its maximum at approximately 80 min. CONCLUSION: Due to the large CEST effect of 3OMG and its low toxicity 3OMG-CEST may serve for the detection of tumors and metastases in the clinic.

NMR studies of proton exchange kinetics in aqueous formaldehyde solutions.

Aqueous solutions of formaldehyde, formalin, are commonly used for tissue fixation and preservation. Treatment with formalin is known to shorten the tissue transverse relaxation time T2. Part of this shortening is due to the effect of formalin on the water T2. In the present work we show that the shortening of water T2 is a result of proton exchange between water and the major constituent of aqueous solutions of formaldehyde, methylene glycol. We report the observation of the signal of the hydroxyl protons of methylene glycol at 2ppm to high frequency of the water signal that can be seen at low temperatures and at pH range of 6.0±1.5 and, at conditions where it cannot be observed by the single pulse experiment, it can be detected indirectly through the water signal by the chemical exchange saturation transfer (CEST) experiment. The above finding made it possible to obtain the exchange rate between the hydroxyl protons of the methylene glycol and water in aqueous formaldehyde solutions, either using the dispersion of the spin-lattice relaxation rate in the rotating frame (1/T1ρ) or, at the slow exchange regime, from the line width hydroxyl protons of methylene glycol. The exchange rate was ∼10(4)s(-1) at pH 7.4 and 37°C, the activation energy, 50.2kJ/mol and its pH dependence at 1.1°C was fitted to: k (s(-1))=520+6.5×10(7)[H(+)]+3.0×10(9)[OH(-)].

Molecular imaging of tumors and metastases using chemical exchange saturation transfer (CEST) MRI.

The two glucose analogs 2-deoxy-D-glucose (2-DG) and 2-fluoro-2-deoxy-D-glucose (FDG) are preferentially taken up by cancer cells, undergo phosphorylation and accumulate in the cells. Owing to their exchangeable protons on their hydroxyl residues they exhibit significant chemical exchange saturation transfer (CEST) effect in MRI. Here we report CEST-MRI on mice bearing orthotopic mammary tumors injected with 2-DG or FDG. The tumor exhibited an enhanced CEST effect of up to 30% that persisted for over one hour. Thus 2-DG/FDG CEST MRI can replace PET/CT or PET/MRI for cancer research in laboratory animals, but also has the potential to be used in the clinic for the detection of tumors and metastases, distinguishing between malignant and benign tumors and monitoring tumor response to therapy as well as tumors metabolism noninvasively by using MRI, without the need for radio-labeled isotopes.