Real-time metabolic imagingKlaes Golman*, Rene in ‘t Zandt†, and Mikkel Thaning

Amersham Health R&D AB, GE Healthcare, Medeon, SE-205 12 Malmo, Sweden

Communicated by Alexander Pines, University of California, Berkeley, CA, March 7, 2006 (received for review November 22, 2005)

The endogenous substance pyruvate is of major importance tomaintain energy homeostasis in the cells and provides a windowto several important metabolic processes essential to cell survival.Cell viability is therefore reflected in the metabolism of pyruvate.NMR spectroscopy has until now been the only noninvasivemethod to gain insight into the fate of pyruvate in the body, butthe low NMR sensitivity even at high field strength has onlyallowed information about steady-state conditions. The medicallyrelevant information about the distribution, localization, and met-abolic rate of the substance during the first minute after theinjection has not been obtainable. Use of a hyperpolarizationtechnique has enabled 10–15% polarization of 13C1 in up to a 0.3M pyruvate solution. i.v. injection of the solution into rats and pigsallows imaging of the distribution of pyruvate and mapping of itsmajor metabolites lactate and alanine within a time frame of �10s. Real-time molecular imaging with MRI has become a reality.

13C � dynamic nuclear polarization � hyperpolarized � MRI � spectroscopy

The technique for increase in signal-to-noise ratio of �10,000times in liquid-state NMR and the use of this technique for

molecular imaging with endogenous substances generatedthrough the process of dynamic nuclear polarization (DNP) hasbeen reported (1, 2). In both these studies, 13C-enriched urea wasused as an example of an endogenous substance that could bepolarized to a high degree (37% for 13C and 8% for 15N) and usedfor high-resolution imaging of the cardiovascular system in rats.

It was suggested that the signal enhancement could be usednot only for visualizing the cardiovascular system but also forimproved perfusion measurements and that it may allow real-time metabolic mapping of other endogenous substances such asalanine, glutamine, and acetate. Such studies should be possibleif the relaxation time of the 13C-labeled site in the hyperpolarizedmolecule is long enough and the metabolic products retainsufficient fraction of the nonequilibrium polarization. The pos-sibilities for doing perfusion studies using 13C labeled hyperpo-larized substances have recently been reviewed by Månsson et al.(3), but the application of visualizing metabolic processes byusing hyperpolarized substances have not yet been described.

To reveal information about the metabolic status of the tissue,magnetic resonance (MR) spectroscopy has been used, employ-ing nuclei like 1H, 13C, 31P, and 19F (4, 5). The main applicationareas have been brain, muscle, and prostate tissue. Informationon fluxes through metabolic pathways is less straightforward toobtain though. Traditionally, 13C NMR spectroscopy in combi-nation with 13C-labeled (enriched) substrates has been used tovisualize the label applied, its metabolic intermediates, and�orits end products during steady-state conditions. In certain casesthe metabolic rates can be indirectly estimated by using math-ematical modeling (6), for example, in determining the fluxthrough the tricarboxylic acid cycle in vivo (7–10).

As reviewed by Ross et al. (11) injection of 13C-labeled glucoseand acetate now can uncover hitherto unknown disorders ofN-acetyl aspartate (NAA) synthesis, tricarboxylic acid cycle, andglycolysis by using clinical 13C NMR (1.5 T) spectroscopy in man.The inherent low intrinsic sensitivity of 13C NMR in combinationwith low in vivo concentrations for relevant metabolites resultedin a need to sample for several minutes, i.e., 35 min (12). If

information about flux rates is wanted, one needs to follow thefate of 13C-labeled substrate up to hours after injection (9, 10).

The increased signal amplitude available by the hyperpolar-ization method should allow the sampling times to be reduced toseconds. This reduction would open up a completely differentperspective on metabolic mapping using NMR: detection ofnewly formed 13C compounds within minutes after injection ofthe 13C-hyperpolarized label with high image quality and nobackground interference. To examine the potential of the hy-perpolarization technique for visualization of in vivo metabolicprocesses, we have chosen the substance pyruvate-13C enrichedin the C1 position. Pyruvate is an intermediate common to threemajor metabolic and catabolic pathways in the mammalian cells(Fig. 1). Depending on the intracellular energy status of thetissue, pyruvate will be converted to a different degree intoalanine, lactate, or carbon dioxide.

After an i.v. injection, pyruvate is rapidly distributed in thebody and absorbed by the cells of most organs. It is known thatonly insignificant amounts of the injected pyruvate leaves thebody via the normal excretory pathways, the bile and urine (13,14). Consequently, pyruvate is completely metabolized within ashort time after its injection.

The relative amount of metabolites produced from the in-jected pyruvate will depend on the actual condition of the cellsand a number of basic cell viability parameters such as pO2, pH,and need for protein synthesis. If one therefore could determinenot only pyruvate metabolite levels but also metabolic rate invivo, it would be of great medical diagnostic importance fororgan function and disease quantification. It is the aim of thiswork to evaluate whether the metabolic fate of injected pyruvatecan be mapped and followed within the medically relevant timeframe of seconds.

It is important to realize that hyperpolarized samples arecharacterized by a strong nonthermal polarization and that thehyperpolarization is created ex situ to the examined object. Thenonthermal polarization condition cannot be regenerated afterthe NMR investigation. Characteristic for hyperpolarized sam-ples is that during and after dissolution of the sample into theliquid space, the nuclear polarization will decrease according tothe longitudinal relaxation time T1 (3). In addition, the distri-bution in the body will lead to a decrease of concentration of theagent depending on body size. Therefore, we conducted theexperiments in both rats and pigs where the pharmacokineticparameters widely differ. It will be shown that in both species itis possible to map pyruvate and some of its metabolic productsin resting skeletal muscle within a clinically useful time frame of�10 s.

Results and DiscussionMost applications in in vivo NMR spectroscopy are performedfrom NMR signals of carbon-bound ‘‘nonexchangeable’’ protons

Conflict of interest statement: K.G., R.i.t.Z., and M.T. are employees of Amersham HealthR&D AB Malmo, which is now part of GE Healthcare.

Abbreviations: CSI, chemical shift imaging; MR, magnetic resonance.

*To whom correspondence should be sent at the present address: Mellemvang 3B, DK-2970Hoersholm, Denmark. E-mail: golman@mail.tele.dk.

†Present address: Imagnia AB, S-20502 Malmo, Sweden.

© 2006 by The National Academy of Sciences of the USA

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in small molecules (Mw � 500). In general, several minutes areused for signal averaging to obtain sufficient signal-to-noise ratioto obtain an interpretable NMR spectrum. The spectra thereforeinforms in most applications about the steady-state situationonly. Endogenous pyruvate concentrations in plasma are be-tween 0.1 and 0.2 mM and thus currently challenging to examinewith conventional 1H NMR spectroscopic techniques. Especiallybecause of the high H-background signal, it is impossible to studythe metabolism of pyruvate and its metabolic rate within aclinically interesting time frame using 1H NMR spectroscopy.The 13C spectral region is �10 times that of proton, but becauseof the low gyromagnetic ratio (1�4 of 1H) the intrinsic 13C NMRsensitivity is nearly 2 orders of magnitude lower than that of 1H.

To examine the capability of monitoring the metabolic fate ofpyruvate in vivo in a noninvasive manner, 13C1-enriched hyper-polarized pyruvate (0.79 mmol�kg) was injected, and the 13CNMR spectra were acquired from the time of injection for 50 s.By injecting the hyperpolarized 13C1-enriched pyruvate in a rat,we obtained a NMR spectrum (Fig. 2) showing the fate ofpyruvate in the lower half of the rat during the first 50 s after theinjection. The dynamic of the production of its main metabolicproducts lactate and alanine can be followed as well as the rateof the 13C NMR signal disappearance. When working withhyperpolarized substances, the signal decay caused by the exci-

tation pulses and the respective T1 of the formed products mustbe considered. An estimation of the in vivo T1 value of the 13C1of pyruvate, lactate, and alanine shows that they are all within15 � 5 s in the rat and 20 � 5 s in the pig. In addition to themetabolites, the pyruvate hydrate [CH3C(OH)2

13COO�] also canbe seen in the NMR spectra. Pyruvate hydrate, which is notmetabolically active, is formed (�8%) under the conditions ofpH 7.5–8.2 in the injection solution.

The third main metabolite formed from 13C1-pyruvate is13CO2, which is in rapid equilibrium with H13CO3

�. We coulddetect the H13CO3

� signal only after all of the spectra acquiredduring the 50-s time window (Fig. 2) were added. The relativelow amounts of detectable H13CO3

� could be because of a shortT1 of this compound, although experiments indicate that the T1of bicarbonate should be close to the T1 of pyruvate (K.G. andR.i.t.Z., unpublished results). It is more likely that the skeletalmuscle tissue that constitutes most of the volume covered by theradiofrequency (rf)-coil does not metabolize pyruvate to a largeextent through the tricarboxylic acid cycle in the resting musculartissue of the anesthetized animal under the experimental con-ditions (15, 16). Rates for pyruvate oxidation and formation oflactate and alanine have been calculated by using 13C-glucose assubstrate (17). This kind of data has been obtained understeady-state conditions by using physiological levels of substrate.It is possible that a different metabolic pattern can be foundwhen using high levels of 13C-pyruvate to visualize metabolismunder non-steady-state conditions with a short time window. Theratio between CO2 and HCO3

� in tissue is directly related to thepH, and a low concentration of HCO3

� therefore also may beexplained by a low pH in the examined area.

The spectra presented in Fig. 2 are affected by the fact thatthey cover most of the rat and do not inform about the relativecontribution to the metabolism from the different organs in thebody.

Spatial information can be obtained by using, for example, achemical shift imaging (CSI) sequence (18, 19). Every excitationrf-pulse irreversibly destroys a certain fraction of the longitudi-nal magnetization; a 90° pulse, for example, uses all of themagnetization available within the excited slice in one single

Fig. 1. Simplified overview of the main metabolic pathways of pyruvate.

Fig. 2. Metabolic production of lactate and alanine after the injection of 13C1-pyruvate. (A and B) The spectra (B) are acquired with a time interval of 3 s fromthe lower part of the animal as indicated by the proton MR image (A). (C) The formation of bicarbonate can be seen when adding all spectra.

Golman et al. PNAS � July 25, 2006 � vol. 103 � no. 30 � 11271

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rf-pulse. For this reason a compromise should be made betweenthe rf-pulse angle and the matrix size, e.g., the amount ofrf-pulses needed to acquire a single CSI. It is feasible to acquirefive chemical shift images with a modest matrix size of 12 � 12and at the same time increase the rf-pulse angle with everysubsequent measurement to compensate for the loss of signalover time.

CSI experiments in rats injected with hyperpolarized 13C1-enriched pyruvate were carried out to examine the differences inthe metabolic transformation of pyruvate to the various oxida-tive, reductive, or transaminated products. The result of such anapproach is shown in Fig. 3 where the CSI allows us to follow thefate of the injected pyruvate. (i) About 13 s after the injection,most of the signal originates from pyruvate located in the centralvascular structures. (ii) At 21 s pyruvate has distributed into thewhole body and already been metabolized in significant amountsto alanine and lactate. (iii) At 37 s even more has beenmetabolized, and it can be seen that the lumbar muscles are themost active areas for metabolism.

The strength of the NMR modality using the CSI sequence isthat the chemical shift allows us to identify the metabolic productand at the same time localize them within the body organs. Theresults in Fig. 3B where the metabolite concentration changeswith time indicate that metabolic rate measurements are possi-ble, provided that the relaxation rates of all metabolites involved

are known. It also shows that it is possible to simultaneouslylocalize 13C1-pyruvate and the production of its metabolites inthe rat with a spatial resolution of 7.5 � 7.5 � 32 mm3 and a timeresolution of 8 s at a 1.5 T clinical NMR machine. The color scaleused in Fig. 3B might suggest the lack of pyruvate in the animalin the 37-s image. To illustrate the presence of pyruvate in thewhole image (t � 37 s), the pyruvate intensity has been rescaledwith a factor of 13 20 in Fig. 3C. Even after 37 s the amplitudeof pyruvate is higher than that of the metabolites in any voxel.

If one acquires the entire signal during the period 30–43 s, ahigher spatial resolution (5 � 5 � 10 mm3) can be obtained (Fig.4) at the expense of the metabolic rate information. These resultsdemonstrate the previously undescribed feasibility to monitorthe metabolic transformation of pyruvate, a key species involvedin cellular energetics, noninvasively, in the region of interest,within a clinically interesting time frame, and coregistration ofsuch maps with the anatomy.

To show that the method is also applicable in a larger animal,we decided to perform a nearly similar study as performed in ratsin pigs. Such a large animal would be better suited in a clinicalNMR system. It would provide important information as towhether the 13C-pyruvate dynamics also could be visualized in amore clinically relevant setting. The examination area chosen inthe pig covers mainly muscular tissue in the legs as depicted fromthe standard proton-based image (Fig. 5A). If one measures the

Fig. 3. The time course of the build up of lactate and alanine in the imaged slice of the rat. (A) The location of the image slab in the rat and the correspondingtransversal 1H NMR image are illustrated. (B) The NMR signal obtained simultaneously from pyruvate, lactate, and alanine is shown. The evolution of themetabolic maps as a function of time visualizes the production of lactate and alanine in the skeletal muscle. To facilitate the interpretation, the 13C-metabolicimages have been superimposed on the anatomical 1H NMR image from A. The total amplitude of pyruvate varies with a factor of 5 between the bolus passage(t � 5 s) and the final image (t � 37 s). (C) To illustrate the presence of pyruvate in the whole image (t � 37 s), the pyruvate intensity was rescaled with factorsof 1, 4, and 20. The image in C Right shows the pyruvate amplitude being higher than the alanine and lactate amplitudes over the whole image.

11272 � www.pnas.org�cgi�doi�10.1073�pnas.0601319103 Golman et al.

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NMR signal amplitude generated at the different frequenciescharacteristic for the 13C1 in pyruvate, alanine, lactate, andpyruvate hydrate, it can be seen (Fig. 5B) that the time windowof 30–45 s after the injection is an optimum time for imaging themetabolites. Consequently, a single CSI was collected during thistime window. Fig. 5C shows the localization and indicates therelative concentration of pyruvate, alanine, and lactate in the

chosen part of the legs of the pig. We thus can noninvasively getlocalized information about the metabolic status of the musculartissue of interest. The low or missing signal in the upper part ofthe right leg is due to the fact that a femoral arterial bloodsampling catheter limits the delivery of pyruvate in this area.

Pyruvate is a molecule central to delivering energy to the bodycells, and the metabolism is under control of a range of differentphysiological conditions (20–22). Despite this control, it ispossible to create metabolic images of 13C-lacate and 13C-alaninewithin a minute after injection of supraphysiological levels of13C-pyruvate. General changes in organ function will affect theenergy consumption (viability) of the cells and consequentlythe metabolism of pyruvate. These changes will be reflected inthe capacity of the cells to label the existing lactate and alaninelevels with 13C and the production of these metabolites. Abilityto localize quantity and determine metabolic rate of pyruvatemay be of importance for diagnosis and treatment of medicaldiseases. In this study, we did not pay attention to quantificationof metabolite concentration but focused on the aim to prove thatreal molecular imaging with MRI within a clinically interestingtime frame has become a reality.

Materials and MethodsEndogenous Substance. The 13C1-labeled pyruvate was polarizedin its acid form with the electron paramagnetic agent Tris(8-carboxy-2,2,6,6,-tetra(methoxyethyl)benzo[1,2-d:4,5d�]bis(1,3)dithiole-4-yl)methyl sodium salt present in a 15 mM concen-tration in the neat acid. The polarization and subsequent disso-lution of the substance were performed in a manner similar tothe process described in ref. 2. After the polarization process (60min of microwave irradiation at 1.2 K), the polarized sample of40 mg (31.5 �l) labeled pyruvic acid for rat and 500 mg labeledpyruvic acid for pig was thawed, dissolved, and neutralizedwithin 2 s by using 5.7 ml of a heated aqueous buffer fordissolving the rat sample and 18.7 ml of a heated aqueous bufferfor dissolving the pig sample. The final injection solution for ratcontained 78.8 mM pyruvate, 68.8 mM sodium ion, 20 mM Trisbuffer, 0.27 mM Na2EDTA, and 83 �M paramagnetic agent. Forpig the final injection solution contained 300 mM pyruvate, 100mM Tris buffer, 0.27 mM Na2EDTA, 250 mM sodium ion, and0.32 mM paramagnetic agent. The injectant temperature was�30°C, and pH was 7.5–8.2. The polarization measured imme-diately after the dissolution was 15–20% and was measured in ahomebuilt polarimeter. The T1 of the 13C pyruvate was 55 s, andthe transfer time to the imaging magnet was 15–20 s, resulting ina polarization of 10–15% at the moment of injection.

Fig. 4. Acquisition of a single CSI image at a higher matrix size of 16 � 16 and a field of view of 80 � 80 � 10 mm. The location of the image slab in the ratand the corresponding transversal 1H NMR image are illustrated. The NMR signal obtained simultaneously from pyruvate, lactate, and alanine also is shown. Timeof acquisition is between 30 and 43 s after the start of the 12-s-long injection. The alanine production is mainly localized in the skeletal muscle.

Fig. 5. Pyruvate and its metabolism in the hind leg of a pig. (A) Unlocalized13C NMR spectra were acquired from the lower legs of the pig as illustrated inthe proton projection image. (B) The signal amplitudes of pyruvate, pyruvatehydrate, alanine, and lactate are plotted as a function of time. (C) A separateexperiment in which a chemical shift image obtained from 30 to 43 s after startof the injection maps the production of lactate and alanine in the skeletalmuscle of the lower legs. To facilitate the interpretation, the 13C-metabolicimages have been superimposed on the anatomical proton NMR image.

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Dosing. Hyperpolarized 13C-pyruvate was injected at a dose of0.79 mmol�kg for rats, whereas for pigs the dose was 0.2mmol�kg. The injection volume was 3 ml for the rat, and theinjection rate was 0.25 ml�s, whereas in the pig 16 ml of thesolution was injected at an injection rate of 1.25 ml�s. Thistechnique results in a total injection time of 12 s in both species.

Animal Handling. Male Wistar rats (300–350 g) were anesthetizedby using isoflurane (2–3%) in 97% oxygen. A catheter wasintroduced into the tail vein, and another catheter was insertedinto A. carotis communis sinistra. They were placed on a home-built pad that was heated to �37°C by means of circulatingFC-104 Fluorinert to avoid background signals in the MRexperiments. Body temperature was constantly kept at 37°C.Anesthesia was continued by means of the isoflurane mixture.The arterial catheter was connected via a T-tube to a pressurerecorder and a pump delivering saline (0.15 ml�min�1) to preventcatheter clotting. After the examination, the animals were killedby a lethal injection of pentobarbital.

Pig Experiment. A Swedish land pig (25 kg) was premedicated with10 ml of Ketalar (50 mg�ml; Warner–Lambert, Ann Arbor, MI)and 1.5 ml of Midazolan (5 mg�ml; Pharma Hameln, Hameln,Germany) intramuscularly. The pig was tracheally intubated; twocatheters were inserted i.v., one in the hind leg for administrationof anesthesia and one in the front leg for administration ofRinger-acetate solution (150 ml�h) for hydration and injection ofthe 13C-pyruvate. Full anesthesia was induced by using an injectionof 0.5 ml�kg Pentothal natrium (25 mg�ml; Abbott). The pig wasconnected to a volume-controlled respirator (PV 301A; BreasMedical AB, Molnlycke, Sweden; 6–8 ml�kg; 20 breaths per min).Blood pressure and heart rate were continuously recorded througha catheter in A. carotis, and body temperature was measuredthrough a rectal probe. Anesthesia during the examination wasmaintained by using a mixture containing isotonic NaCl (26 vol%),Ketalar (50 mg�ml; 42 vol%) (Pfizer AB, Sollentuna, Sweden),Norcuron (10 mg 5 ml sterile water; 21 vol%) (Organon), andMidazolam (5 mg�ml; 11% vol) (Pharma Hameln) administeredusing an infusion pump at a rate of 0.6 ml�min. Injection of thehyperpolarized 13C-pyruvate was performed by a manual injection.After the examination, the animals were killed by a lethal injectionof pentobarbital.

All animal experiments were approved by the local ethicalcommittee.

MRI Equipment. The spectroscopic and imaging experiments wereperformed on a 1.5-T clinical MRI (Sonata; Siemens) by usinga 1H-13C Tx�Rx birdcage coil for the rat experiments (diameter8.3 cm, length 10 cm; Rapid Biomedical, Rimpor, Germany)operating at 63.67 and 16.00 MHz, respectively. Pigs werepositioned in a pig 13C-Tx�Rx NMR coil (diameter 26 cm, length35 cm; Rapid Biomedical) and imaged with the proton body coilby using a standard proton NMR imaging sequence to ensurereproducible positioning of the hind legs of the pig.

NMR Protocol. All automatic in line adjustments of the MRscanner were disabled to avoid the use of unwanted rf-pulses thatwould destroy the hyperpolarization signal. The 90° reference rfpulse was calibrated by using the natural abundance 13C-lipidsignal. Based on the proton frequency as determined by theNMR system, the NMR frequency for 13C1-alanine was calcu-lated according to frequency 13C1-alanine � 0.25144 � [(systemfrequency proton � 1.00021) � 0.000397708]. The frequencycalculated will position the NMR signal arising from 13C1-alanine in the middle of the 13C-NMR spectrum with 13C1-lactate on the left and 13C1-pyruvate resonating on the right of13C1-alanine. An unlocalized NMR spectroscopy sequence wasrun to check that the 13C-NMR coil and the system NMR

frequency is setup correctly (bandwidth 10 KHz; 2K complexpoints; 90° rf-pulse; TR � 800 ms; 128 averages).

Unlocalized Free Induction Decay Experiments. Pigs and rats wereinjected with 13C-pyruvate, and a series of 25 low-flip-angle (10°)unlocalized NMR spectra were acquired with an interval of 3 s,where the first NMR spectrum was acquired just before theinjection of the 13C-pyruvate started. This finding gives insight in themetabolic rate of the 13C-labeled pyruvate and relative concentra-tion of the metabolites in the whole animal. The 13C NMR signalobtained with the rat coil used covers the area from the kidneysdown to the tail, whereas in case of experiments with pigs the 13Ccoil covers the lower legs of the animal. The 13C NMR spectraobtained therefore should be considered as a reflection of theaverage metabolism of pyruvate over the field of view.

Chemical-Shift Imaging Experiments. For the chemical-shift imag-ing a standard sequence was used (Siemens V21B) except thatcentric K-space acquisition was added, and the possibility wascreated to be able to change the repetition time to be as short aspossible as the timing of the sequence would allow.Rat. Chemical shift images were acquired with the 13C-imagelocation positioned to cover the region of interest (80 � 80 � 32mm3) with a matrix containing 12 � 12 elements. In thereconstruction phase, the matrix was zero-filled to 32 � 32.The injection of the pyruvate was started simultaneously with thestart of the CSI sequence. In one version of the experiment, atotal of five chemical shift images were acquired with a rf pulseangle of 2, 3, 4, 5, and 10°, respectively, with a time resolution ofone chemical shift image of 8 s (Tr � 90 ms). In a secondexperiment, a single chemical shift image was acquired starting30 s after start of the injection with a matrix size of 16 � 16 anda field of view of 80 � 80 � 10 mm3. The rf pulse angle used was10°, and the total scan time 13.9 s (Tr � 90 ms).Pig. Chemical shift images were acquired with the same field ofview as for the results of the unlocalized 13C NMR spectraexperiments (250 � 250 � 80 mm3). NMR imaging resulted ina matrix containing 16 � 16 elements in which each element orvoxel�pixel contains a 13C NMR spectrum. In the reconstructionphase, the matrix was zero-filled to 32 � 32. Thirty seconds afterthe start of the injection, i.e., 18 s after finishing the injection, thechemical shift 13C-NMR sequence was started (Tr � 90 ms; totalacquisition time 13.9 s).

Analysis. The series of 25 unlocalized 13C NMR spectra wereanalyzed by using the algorithm AMARES (23) as implementedin JMRUI 2.2 (24, 25) with the following prior knowledge: Lorent-zian line shape fitting; assumption of equal linewidth for alanine,lactate, pyruvate, and pyruvate hydrate; relative phase zero withrespect to main phase; zero-order phase fixed, e.g., �30.1 for pigand �49.6 for rat free induction decay experiments; first-orderphase fixed at 0.5 ms (delay between rf pulse and acquisition),256 points in analysis; and to neglect the first 2 points of freeinduction decay in analysis. All graphs were made with GRAPH-PAD PRISM 4.03 (GraphPad, San Diego).

The metabolic images were calculated from the CSI data setby using a home written program coded in MATLAB 6.5.1 (Math-Works, Natick, MA). This program incorporates time domainfitting algorithms (23). After manual phasing of the spectra, theamplitudes were estimated assuming constant phase; identicallinewidth for alanine, lactate and pyruvate; and a fixed fre-quency shift between lactate and alanine (106 Hz) and pyruvateand alanine (�92 Hz). The amplitudes estimated for the me-tabolites pyruvate, alanine, and lactate are shown as color maps.

We thank Fredrik Ellner, Andreas Gram, Britt-Marie Lilja, BirgitPersson, and Kerstin Thyberg for excellent technical assistance andMathilde Lerche for helpful discussions.

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on

Apr

il 13

, 202

0