In vivo carbon-13 magnetization transfer effect. Detection of aspartate aminotransferase reaction.

One of the most remarkable achievements of in vivo NMR spectroscopy has been the detection of rapid enzyme-catalyzed exchange reactions using phosphorus-31 magnetic resonance spectroscopy-based magnetization transfer experiments. In this paper, we report, for the first time, the in vivo carbon magnetization transfer (CMT) effect and in vivo detection of the CMT effects of the alpha-ketoglutarate <--> glutamate and the oxaloacetate <--> aspartate reactions, both of which are catalyzed by aspartate aminotransferase. By saturating the carbonyl carbon of alpha-ketoglutarate at 206 ppm in alpha-chloralose anesthetized adult rat brain, the unidirectional glutamate --> alpha-ketoglutarate flux was determined to be 78 +/- 9 mumol/g/min (mean +/- SD, n = 11) following i.v. infusion of [1,6-(13)C(2)]D-glucose. Contribution from aspartate aminotransferase-catalyzed partial reactions to the observed CMT effects was emphasized. Because of the large chemical shift separation between the alpha-carbons of the amino acids and the carbonyl carbons of the corresponding cognate keto acids, the spillover of the saturation radiofrequency pulses to the alpha-carbon resonances was negligible. The results indicate that the magnetization transfer effects of aspartate aminotransferase-catalyzed reactions can be used as new biomarkers accessible to non-invasive in vivo magnetic resonance spectroscopy techniques.     

13C saturation transfer effect of carbon dioxide-bicarbonate exchange catalyzed by carbonic anhydrase in vivo.

Carbonic anhydrase catalyzes reversible hydration of carbon dioxide and dehydration of bicarbonate. In this article we report that the rapid exchange catalyzed by carbonic anhydrase causes a large magnetization (saturation) transfer effect on the 13C signal of bicarbonate at 160.7 ppm in vivo when the resonance of the undetectable carbon dioxide at 125.0 ppm is irradiated with RF pulses. In isoflurane-anesthetized adult rat brain the unidirectional, pseudo first-order rate constant of this exchange in the dehydration direction was determined to be 0.47 +/- 0.05 sec(-1) following intravenous infusion of uniformly 13C-labeled glucose for labeling bicarbonate. Intralateral ventricular administration of the highly specific carbonic anhydrase inhibitor acetazolamide, which is a drug used for treating glaucoma and epilepsy, was also shown to significantly attenuate the observed 13C magnetization transfer effect of the carbon dioxide-bicarbonate exchange in the rat brain.     

In vivo monitoring of hepatic glutathione in anesthetized rats by 13C NMR.

A method for in vivo (13)C NMR monitoring of hepatic glutathione (GSH) in intact, anesthetized rats has been developed. Studies were conducted using a triple-tuned, surgically implanted surface coil designed for this animal model. The coil permitted complete decoupling and sufficient resolution in the (13)C NMR spectrum to monitor the time course of hepatic (13)C-metabolites of intravenously administered 2-(13)C-glycine, particularly GSH at 44.2 ppm and serine signals at 61.1 and 57.2 ppm, respectively. It further allowed concomitant monitoring of high-energy phosphagens and intracellular pH by (31)P NMR. To confirm in vivo NMR peak assignments, we compared high-resolution 2D (1)H[(13)C] heteronuclear multiple quantum coherence and 1D (13)C spectra of hepatic perchloric acid extracts to those of authentic standards. The fractional isotopic enrichment of hepatic (13)C-glycine increased exponentially at a rate of 1.68 h(-1) and reached its plateau level of 81% in 2 h. The (13)C fractional isotopic enrichment of GSH increased exponentially at a rate of 0.316 h(-1) and reached 55% after 4 h of 2-(13)C-glycine infusion, but without achieving a plateau. To confirm that the resonance at 44.2 ppm resulted from GSH, a rat was given an intravenous dose of 2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor that increases intracellular GSH. As expected, with OTC administration the hepatic (13)C GSH-to-glycine peak area increased more than sevenfold.     

Pharmacokinetics of the 13C labeled anticancer agent temozolomide detected in vivo by selective cross-polarization transfer

The anticancer agent temozolomide labeled with ¹³C (8-Carbamoyl-3-13C-methylimidazo-[5,1-d]-1,2,3,5-tetrazin-4-(3H)-one), was noninvasively detected in subcutaneous RIF-1 tumors by a selective cross polarization ¹³C NMR method, at a field strength of 9.4T. Pharmacokinetics of the drug, at a dose of 150 mg/kg, were determined for intravenous and intraperitoneal modes of administration (three animals per mode). The half-life of the drug in the tumors was approximately 60 min. The uptake and clearance of the drug, however, varied significantly between individual hosts, for both modes of administration. These results demonstrate the feasibility of obtaining pharmacokinetics of anticancer agents for individual tumors without the need for a label that might modify drug activity (e.g., fluorine). The variability of the in vivo measurements, even within the same tumor model, demonstrates the necessity of directly monitoring the tumor to evaluate drug pharmacokinetics.     

Magnetization transfer measurements of exchange between hyperpolarized [1‐13C]pyruvate and [1‐13C]lactate in a murine lymphoma

Measurements of the conversion of hyperpolarized [1‐¹³C]pyruvate into lactate, in the reaction catalyzed by lactate dehydrogenase, have shown promise as a metabolic marker for the presence of disease and response to treatment. However, it is unclear whether this represents net flux of label from pyruvate to lactate or exchange of isotope between metabolites that are close to chemical equilibrium. Using saturation and inversion transfer experiments, we show that there is significant exchange of label between lactate and pyruvate in a murine lymphoma in vivo. The rate constants estimated from the magnetization transfer experiments, at specific points during the time course of label exchange, were similar to those obtained by fitting the changes in peak intensities during the entire exchange time course to a kinetic model for two‐site exchange. These magnetization transfer experiments may therefore provide an alternative and more rapid way of estimating flux between pyruvate and lactate to serial measurements of pyruvate and lactate ¹³C peak intensities following injection of hyperpolarized [1‐¹³C]pyruvate. Magn Reson Med 63:872–880, 2010. © 2010 Wiley‐Liss, Inc.     

Validation of 13C NMR measurements of liver glycogen in vivo

The natural abundance ¹³C NMR intensity of the glycogen C1 resonance was measured in the surgically exposed liver of rabbits in vivo (n = 17) by integration from 98 to 104 ppm and compared double blindedly to the subsequent biochemical measurement. Coil loading was measured each time from a reference sphere at the coil center and the NMR Intensity was normalized accordingly. For quantification, the normalized NMR intensity was calibrated using aqueous glycogen solutions ranging from 110 to 1100 μmol glucosyl units/g (n = 14). An in vivo range from 110 to 800 pmol glucosyl unit/g wet weight was measured with a highly linear correlation with concentration (r = 0.85, P < 0.001). The in vivo NMR concentration was 0.95 ± 0.05 (mean ± standard error, n = 17) of the concomitant enzymatic measurement of glycogen content. We conclude that the ¹³C NMR signal of liver glycogen C1 is essentially 100% visible in vivo and that natural abundance ¹³C NMR spectroscopy can provide reliable noninvasive estimates of in vivo glycogen content over the physiological range of liver glycogen concentrations when using adequate localization and Integration procedures.     

In vivo 13C magnetic resonance spectroscopy of human brain on a clinical 3 T scanner using [2‐13C]glucose infusion and low‐power stochastic decoupling

This study presents the detection of [2‐¹³C]glucose metabolism in the carboxylic/amide region in the human brain, and demonstrates that the cerebral metabolism of [2‐¹³C]glucose can be studied in human subjects in the presence of severe hardware constraints of widely available 3 T clinical scanners and with low‐power stochastic decoupling. In the carboxylic/amide region of human brain, the primary products of ¹³C label incorporation from [2‐¹³C]glucose into glutamate, glutamine, aspartate, γ‐aminobutyric acid, and N‐acetylaspartate were detected. Unlike the commonly used alkanyl region where lipid signals spread over a broad frequency range, the carboxylic carbon signal of lipids was found to be confined to a narrow range centered at 172.5 ppm and present no spectral interference in the absence of lipid suppression. Comparison using phantoms shows that stochastic decoupling is far superior to the commonly used WALTZ sequence at very low decoupling power at 3 T. It was found that glutamine C1 and C5 can be decoupled using stochastic decoupling at 2.2 W, although glutamine protons span a frequency range of ≈700 Hz. Detailed specific absorption rate analysis was also performed using finite difference time domain numerical simulation. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Proton magnetization transfer effect in rat liver lactate.

Off-resonance lactate magnetization transfer (MT) experiments were performed on the in situ rat liver under perfused and ischemic conditions. A significant MT effect for lactate methyl protons was observed. The effect was larger for the ischemic condition than for the perfused condition, and was largest in the blood-filled ischemic livers. The size of the motionally restricted lactate pool, determined using a two-pool model fit, was estimated to be about 1% in perfused livers and about 1.8-2.5% after more than 1 hr of onset of ischemia, suggesting that lactate in liver is almost fully NMR-visible. The MT data for both the perfused and the ischemic condition appeared to be better approximated when assuming a superLorentzian lineshape for the immobile pool rather than a Gaussian lineshape. Finally, the experiments demonstrated a coupling between the lactate methyl and water protons, which may be mediated by macromolecules.     

In vivo detection of cortical GABA turnover from intravenously infused [1-13C]D-glucose.

In this study [2-(13)C] gamma-aminobutyric acid (GABA) was spectrally resolved in vivo and detected simultaneously with [4-(13)C]glutamate (Glu) and [4-(13)C]glutamine (Gln) in the proton spectra obtained from a localized 40 microL voxel in rat neocortex with the use of an adiabatic (1)H-observed, (13)C-edited (POCE) spectroscopy method and an 89-mm-bore vertical 11.7 Tesla microimager. The time-resolved kinetics of (13)C label incorporation from intravenously infused [1-(13)C]glucose into [4-(13)C]Glu, [4-(13)C]Gln, and [2-(13)C]GABA were measured after acute administration of gabaculine, a potent and specific inhibitor of GABA-transaminase. In contrast to previous observations of a rapid turnover of [2-(13)C]GABA from [1-(13)C]glucose in intact rat brain, the rate of (13)C incorporation from [1-(13)C]glucose into [2-(13)C]GABA in the gabaculine-treated rats was found to be significantly reduced as a result of the blockade of the GABA shunt.     

In vivo 13 carbon metabolic imaging at 3T with hyperpolarized 13C-1-pyruvate.

We present for the first time dynamic spectra and spectroscopic images acquired in normal rats at 3T following the injection of (13)C-1-pyruvate that was hyperpolarized by the dynamic nuclear polarization (DNP) method. Spectroscopic sampling was optimized for signal-to-noise ratio (SNR) and for spectral resolution of (13)C-1-pyruvate and its metabolic products (13)C-1-alanine, (13)C-1-lactate, and (13)C-bicarbonate. Dynamic spectra in rats were collected with a temporal resolution of 3 s from a 90-mm axial slab using a dual (1)H-(13)C quadrature birdcage coil to observe the combined effects of metabolism, flow, and T(1) relaxation. In separate experiments, spectroscopic imaging data were obtained during a 17-s acquisition of a 20-mm axial slice centered on the rat kidney region to provide information on the spatial distribution of the metabolites. Conversion of pyruvate to lactate, alanine, and bicarbonate occurred within a minute of injection. Alanine was observed primarily in skeletal muscle and liver, while pyruvate, lactate, and bicarbonate concentrations were relatively high in the vasculature and kidneys. In contrast to earlier work at 1.5 T, bicarbonate was routinely observed in skeletal muscle as well as the kidney and vasculature.     

1H-localized broadband 13C NMR spectroscopy of the rat brain in vivo at 9.4 T.

Localized (13)C NMR spectra were obtained from the rat brain in vivo over a broad spectral range (15-100 ppm) with minimal chemical-shift displacement error (<10%) using semi-adiabatic distortionless enhancement by polarization transfer (DEPT) combined with (1)H localization. A new gradient dephasing scheme was employed to eliminate unwanted coherences generated by DEPT when using surface coils with highly inhomogeneous B(1) fields. Excellent sensitivity was evident from the simultaneous detection of natural abundance signals for N-acetylaspartate, myo-inositol, and glutamate in the rat brain in vivo at 9.4 T. After infusion of (13)C-labeled glucose, up to 18 (13)C resonances were simultaneously measured in the rat brain, including glutamate C2, C3, C4, glutamine C2, C3, C4, aspartate C2, C3, glucose C1, C6, N-acetyl-aspartate C2, C3, C6, as well as GABA C2, lactate C3, and alanine C3. (13)C-(13)C multiplets corresponding to multiply labeled compounds were clearly observed, suggesting that extensive isotopomer analysis is possible in vivo. This unprecedented amount of information will be useful for metabolic modeling studies aimed at understanding brain energy metabolism and neurotransmission in the rodent brain.     

Optimized detection of lactate at high fields using inner volume saturation.

In localized proton MR spectroscopy ((1)H-MRS) in vivo, the detection of lactate (Lac) is affected by modulation of its resonances due to homonuclear scalar couplings (J). A simple and convenient way to distinguish Lac from lipids is to set the TE to 1/J so that the Lac signal is inverted while other resonances (such as lipid) remain in-phase. However, at high field strengths, such as 3 Tesla or above, the modulation of the Lac signal is complicated by chemical shift effects that cause modulation patterns to vary within different subregions of the localized volume. Under some conditions the Lac signal may even disappear completely. In this note we introduce the concept of inner volume saturation (IVS), which makes use of high bandwidth spatial pulses to remove the signal corresponding to the regions of the localized volume that contribute unwanted modulation patterns. The method is described theoretically and demonstrated experimentally at 3 Tesla in a phantom and a patient with acute stroke. The phantom measurements indicate that virtually 100% of the Lac signal can be recovered using this method. The method should be feasible at magnetic fields above 3 Tesla, and may also be applied to other coupled spin systems in which modulation effects are important.     

Dynamic 13C−1H nuclear polarization of lipid methylene resonances applied to broadband proton-decoupled in vivo 13C MR spectroscopy of human breast and calf tissue

Dynamic nuclear polarization of the coupled ¹³C?^lH spin system was studied for optimizing the signal-to-noise ratio of in vivo ¹³C MR spectra. In particular, the truncated driven and transient nuclear Overhauser effect (NOE) of the proton-decoupled ¹³C resonances from methylene carbons in vegetable oil and in human calf tissue was observed. Maximum in vivo NOE enhancements n = 1.5 and 0.9 were found, respectively. Theoretical fits to the data yield ¹³C?¹ cross-relaxation times in the order of 0.6 s. Significant signal enhancement over the whole in vivo ¹³C chemical shift range is obtained with minimum expense utilizing the NOE of the dipolar coupled ¹³C?¹ spin system in addition to proton-decoupling. NOE-enhanced proton-decoupled in vivo ¹³C MR spectra were acquired within 17 min in volunteer examinations from the human breast and the calf. These spectra show well-resolved resonances of carbons in lipids and several other cellular compounds.     

13C editing of glutamate in human brain using J-refocused coherence transfer spectroscopy at 4.1 T

The method of single quantum ¹³C editing is analyzed and implemented with water suppressed J-refocused coherence transfer spectroscopy. Analysis of the ¹³C inversion pulse demonstrates that it is optimally placed into the second echo of the J-refocused sequence. We have used this method to acquire ¹³C-edited spectra of glutamate from phantoms and in vivo. The turnover of ¹³C4-labeled glutamate in human brain in vivo was observed in parasagittal gray matter using a volume head coil at 4.1 T with a time resolution of 5.3 min.     

Quantitation of erythrocyte pentose pathway flux with [2-13C]glucose and 1H NMR analysis of the lactate methyl signal.

A simple and sensitive NMR method for quantifying excess (13)C-enrichment in positions 2 and 3 of lactate by (1)H NMR spectroscopy of the lactate methyl signal is described. The measurement requires neither signal calibrations nor the addition of a standard and accounts for natural abundance (13)C-contributions. As a demonstration, the measurement was applied to approximately 3 micromol of lactate generated by erythrocyte preparations incubated with [2-(13)C]glucose to determine the fraction of glucose metabolized by the pentose phosphate pathway (PP). PP fluxes were estimated from the ratio of excess (13)C-enrichment in lactate carbon 3 relative to carbon 2 in accordance with established metabolic models. Under baseline conditions, PP flux accounted for 7 +/- 2% of glucose consumption while in the presence of methylene blue, a classical activator of PP activity, its contribution increased to 27 +/- 10% of total glucose consumption (P < 0.01).     

In Vivo Selective Measurement of {1−13C}-Glucose Metabolism in Tumors by Heteronuclear Cross Polarization

Selective detection of {1?¹³C}-glucose and its glycolytic product, {3?¹³C}-lactate, was achieved by selective ¹³C NMR spectroscopy with ¹H cross polarization. The total sensitivity of conventional broadband experiments was retained, and peak intensities were at least equivalent to those obtained with the inverse detection technique (i.e., ¹H{¹³C}) for single proton resonances. A key advantage of the method is that it maintains the specific absorption rate (SAR) within FDA limits of 5 W/kg by reducing power deposition during decoupling. In this study we have monitored the kinetics of metabolism of ¹³C-labeled glucose to lactate following intravenous infusion of 0.55 ml of 0.18 M labeled glucose. Physiological effects were minimized by a) maintaining total plasma glucose concentrations below 20 mM throughout the course of NMR experiment and b) by avoiding significant heating of the tumor.     

Four-angle saturation transfer (FAST) method for measuring creatine kinase reaction rates in vivo.

A new fast method of measuring kinetic reaction rates for two-site chemical exchange is described. The method employs saturation transfer magnetic resonance spectroscopy (MRS) and acquisition of only four spectra under partially saturated, high signal-to-noise ratio (SNR) conditions. In two acquisitions one of the exchanging species is saturated; the other two employ a control saturation. Each pair of acquisitions is applied with two different flip angles, and the equilibrium magnetization, relaxation times, and reaction rates are calculated therefrom. This four-angle saturation transfer (FAST) method is validated theoretically using the Bloch equations modified for two-state chemical exchange. Potential errors in the rate measurements due to the effects of exchange are evaluated for creatine kinase (CK) metabolism modeled for skeletal and heart muscle, and are found to be < 5% for forward CK flux rates of 0.05 < or = k(f) < or = 1.0 s(-1), and up to a 90% depletion of phosphocreatine (PCr). The effect of too much or too little saturating irradiation on FAST appears to be comparable to that of the conventional saturation transfer method, although the relative performance deteriorates when spillover irradiation cuts the PCr signal by 50% or more. "FASTer" and " FASTest" protocols are introduced for dynamic CK studies wherein [PCr] and/or k(f) changes. These protocols permit the omission of one or two of the four acquisitions in repeat experiments, and the missing information is recreated from initial data via a new iterative algorithm. The FAST method is validated empirically in phosphorus ((31)P) MRS studies of human calf muscle at 1.5 T. FAST measurements of 10 normal volunteers yielded the same CK reaction rates measured by the conventional method (0.29 +/- 0.06 s(-1)) in the same subjects, but an average of seven times faster. Application of the FASTer algorithm to these data correctly restored missing information within seven iterations. Finally, the FAST method was combined with 1D spatially localized (31)P MRS in a study of six volunteers, yielding the same k(f) values independent of depth, in total acquisition times of 17-39 min. These timesaving FAST methods are enabling because they permit localized measurements of metabolic flux, which were previously impractical due to intolerably long scan times.     

Localized sensitivity enhanced in vivo 13C MRS to detect glucose metabolism in the mouse brain.

The application of in vivo 13C MR spectroscopy to mouse brain models is potentially valuable for improving the understanding of cerebral carbohydrate metabolism and glutamatergic neurotransmission in various neuropathologies. However, the low sensitivity of 13C nuclei and contaminating signals of lipids in the relatively small mouse brain make this application rather challenging. To meet these technical challenges, localized semi-adiabatic distortionless enhanced polarization transfer (DEPT) MR spectroscopy in combination with a continuous intravenous [1,6-13C2] glucose infusion was implemented to detect glucose metabolism in isoflurane-anesthetized mice at 7T. The signal enhancement and high spectral resolution obtained in these experiments enabled the separate determination of 13C label incorporation into as much as 13 metabolites from a 175 microL volume. Signal increases of glucose (C6), glutamine (C3, C4), and glutamate (C3, C4) were determined with a time resolution of 8.6 min. This study demonstrates an optimized MR method for the application of in vivo 13C MRS in mouse brain.