Alan S. Gelbard, Richard S. Bonus, Robert E. Reiman, Joseph M.McDonald,John J. Vomero, and John S. Laughlin

Memorial Sloan-Kettering Cancer Center, New York, New York

In normal volunteersand cancer patients,studiesusingL-(N-13)glutamate asan Imagingagent showedlocalizationof N-13 activity in the heart. Other organsthat were well visualizedincludethe liver, pancreas,and salivaryglands. In tensubjects the average myocardial uptake after Intravenous injection of labeled glutamate was (5.7 ±O.39)% (s.e.m.) of Injecteddose,as determinedby a quantitative scanningsystem.The concentrationof N-13 activity in the humanheart couldnotbe predictedfrompreviousstudiesinvolvingmyocardlaluptakelfldogsandrodentsafter administrationof L-(N-13)glutamate.

J NucIMed21: 988—991,1980

We have been using L-(N- 13)glutamate as an imaging agent for bone tumors in patients (1 ) and have beenevaluating its uptake as an indicator of the early responseof these tumors to chemotherapy (2). In these patientsas well as in normal volunteers, we noted that the heart

was well visualized after i.v. administration of L-(N13)glutamate. This finding differed from its failure toconcentrate in the dog heart (3) and its lower uptake inrodent myocardial tissue (4), compared with N- 13 ammonia. The present report quantitates the uptake ofN-I3 in thehumanheartafterinjectionof labeledLglutamate.

MATERIALS AND METHODS

The cyclotron production of N-i 3 ammonia and itssubsequent use in the enzymatic conversion to L-(NI3)glutamate have been described (3). To make the labeled amino acid suitable for patient studies, this synthesis has been modified by immobilizing glutamate

dehydrogenase (L-glutamate: NAD(P) oxidoreductase(deaminating) EC I .4.1.3) on an activated Sepharosesupport (5). The immobilization procedure prevents theentry of potential pyrogenic or antigenic enzyme proteininto the labeled product. To immobilize the enzyme, 10mg of bovine-liver glutamate dehydrogenase* is dialyzed

Received Dec. 10, 1979; revision accepted May 21, 1980.

For reprints contact: Alan S. Gelbard, BiophysicsLaboratory,Memorial Sloan-Kettering Cancer Ctr., I275 York Ave., New York,NY 10021.

overnight against 0.05 M sodium phosphate buffer (pH8.0) and is then mixed with 15 zmoles a-ketogiutarate,15 @molesadenosine diphosphate, 2 zmoles reducednicotinamide adenosine dinucleotide, and 0.50 gCNBr-activated Sepharose. The mixture is brought to15 ml by the addition of 0.05 M sodium phosphate buffer(pH 8.0) and is shaken overnight at 4°Cwith a wristaction shaker.t The suspension is poured into a glasscolumn and unbound enzyme is removed by passage of500 ml of the phosphate buffer through the column.

A reaction mixture containing 100—200mCi of N-13ammonia, S @smolesof a-ketoglutarate, and 3 zmolësofreduced nicotinamide adenine dinucleotide in 3 ml of0.05 M sodium phosphatà(pH 8.0) is passed through thecolumn containing the bound enzyme. The column isthen washed with 3 ml of the phosphate buffer: Toseparate the labeled L-glutamate from unreacted N-i 3ammonia, the combined effluents are then passedthrough an AG-SOion-exchange column equilibrated atpH 4.0. The eluate of the ion-exchange column is passedthrough a Millex filter with 0.22 zm pore sizeinto asterile vial, is made isotonic with NaCl, and is ready forintravenous injection. In a typical preparation, 80 mCiof L-(N- I3)glutamate is synthesized, in a volume of 6ml, 4 mm after collection of 150 mCi of N-l3 ammonia.

The radiopurity of L-(N-l3)glutamate was determined by analyzing thóproduct with high-pressute liquidchromatography. A 20-s1 sample was injected onto aWhatman SAX anion column (10 .tm diameter, 25-cm

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Imagingof the HumanHeartafter Administrationof L-(N-13)Glutamate

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PRELIMINARY NOTES

length). Elution with a 5 mM potassium phosphate-HCIbuffer (pH 3.5), at 22°Cand 750 psi resulted in a singleradioactive peak with a retention time the same as thatof a known sample of glutamate (6).

Samples of L-(N-l 3)glutamate were routinely testedand found to be sterile and free of pyrogens. No adversereactions have been observed in patients. The procedureswere reviewed and approved by the Center's ClinicalInvestigations Committee and the Committee on Radiation.

The myocardial uptake of activity from intravenouslyadministered L-(N- 13)glutamate was studied in tensubjects, each receiving up to 10 mCi. These includedtwo normal subjects (ages 57 and 70) and eight patients(ages 10-23) with osteogenic sarcoma, Ewing's sarcoma,or benign ganglioneuroma. Normal subject WM (age70) was imaged with L-(N-l 3)glutamate and N-i 3ammonia on succeeding weeks. In some subjects, bloodclearance data for the first 20 mm after injection wereobtained from venous blood samples counted in a gammascintillation spectrometer. In a normal subject, the dynamic uptake of N-i 3 activity in the thoracic and abdominal regions was measured for the first 3 mm afterinjection of glutamate by means of our Total OrganKinetic Imaging Monitor (TOKIM) gamma camera(7).Thissystemenablesonetointegrateregionsof interest and thus to yield time-dependent organ uptakedata.

Quantitative whole-body scans were begun 5 mm afterinjection with the High-Energy Gamma (HEG) dualdetector rectilinear scanner. This instrument is equippedwith digital data recorders and constant-response collimators (8). The procedure for calculating in vivo organuptake from the I-lEG scan data has been described (9).Briefly, the calculation of myocardial uptake is performed as follows: the scan data are obtained in digitalform on magnetic tape and are then read by computerand corrected for the physical decay of the N-i 3 labelduring the scanning period. A boundary-detecting algorithm is applied to the image to obtain the silhouetteof the heart, and a second algorithm is applied to obtainthe total count rate within that silhouette. By means ofdata points in regions adjacent to the heart, the contribution of activity in overlying muscle and skin is estimated and subtracted from the heart data. Finally, thenet count rate is normalized to the subject's bodythickness and compared with the count rate derived froma phantom of similar size containing a known amountof activity. The results are expressed as percent of administered activity. No correction was made for activitycontained in the cardiac blood pool; for L-(N-i3)glutamate this quantity is estimated to be less than 0.5% of theadministered activity.

RESULTS

Figure i shows the clearance of N-13 activity from the

a

)3.Jcnz

)LLJC')S

SECONDSPOSTINJECTON

FIG. 1. Venousbloodclearanceanddynamicuptakecurvesforheart, pancreas, and liver in human subjects. Data for blood werenot obtained before 50 sec. Organ curves are intendedto demonstrate time course of distribution and not relative amount of N-13uptake, since only portions of liver and pancreas were monitored.

blood of three subjects after i.v. injection of L-(N- 13)-glutamate. About 95% of the injected activity clears fromthe blood with a half-time of 0.4- 1.6 mm and ‘@-5%remains in the vascular compartment for a time that is longcompared with the period of observation. The count ratesin the heart, liver, and pancreas, measured with the

TOKIM system as a function of time, are shown in thesame figure. Figure 2 shows anterior images of normalsubject WM, taken with the HEG rectilinear scanner.

FIG. 2. Organdistributionstudieswith(A) L-(N-13)glutamateand(B) N-13 ammonia. Both studies were performed with digital rectilinear scanning system designed for quantitative studies. Imagingwasbegun5mmafteri.v.injectionandrequired25mmtocomplete.The L-(N-13)glutamate scan visualizes the heart, liver, pancreas,and salivaryglands. N-13ammonia scan shows localizationof activity in brain, heart, lIver, kidneys, and urinary bladder.

.@,@

@ . . ji@

A B

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GELBARD. RENIJA. REIMAN. MCDONALD.VOMERO,AND LAUGHLIN

(A) is a scan after administration of 10 mCi of L-(Nl3)glutamate. High uptake is seen in the heart, liver,pancreas, and salivary glands. Little N- 13 activity isconcentrated in the brain. A different organ distributionis seen in Fig. 2B, which is a scan made after administration of 10 mCi of N-l3 ammonia. Areas of high radioactive concentration appear in the brain, heart, liver,kidneys, and bladder. There was 7.7% of the total injected dose in the heart region of this subject after administration of L-(N-l 3)glutamate. In ten subjects, the

@ average myocardial uptake of N-l3 activity after injection of labeled glutamate was 5.7 ±0.39% (s.c.m.) ofdose.

DISCUSSION

The present study demonstrates that L-(N- 13)glutamate is taken up at a high concentration in the humanheart. These results differ from that found in the dog (3)where myocardial uptake was less than 0.5% of the totaldose, compared with 3.6% ofdose after administrationof N-l3 ammonia. Ten minutes after i.v. injection ofL-(N- I 3)glutamate, @@-‘O.5%of the dose concentrates inthe myocardium of mice and rats. This is one-half (calculated from Ref. 4) and one-third (B. R. Freed and A.S. Gelbard, unpublished data) of the percent dose inthese species, respectively, after administration of N-l 3ammonia. Species-related variation in myocardial uptake of labeled amino acids has been observed previously.Nitrogen-13 activity from asparagine, labeled in theamide position, localized in the dog heart at a muchhigher concentration than did N- 13 activity from ammonia (10), but did not localize more effectively thanN-I 3 from ammonia in the myocardium of the rabbit orofman (II). The N-l3 ofL-glutamate and L-glutaminewas found in the myocardium of a monkey at a higherlevel than in that ofa dog (12). Similar species differences have been noted when L-(H-3)arginine localizedin the mouse myocardium, whereas L-(C- I4)asparagineconcentrated in the dog heart (/3). Whether speciesspecificities in myocardial uptake of amino acids reflectdifferences in enzyme levels, in intracellular amino acidtransport systems, or in cardiac amino acid pools, areareas for further study.

The use of amino acids as myocardial imaging agentshas not been extensively studied. Their role as nutritionalor energy sources in myocardial metabolism is considered to be limited in comparison with that of fatty acidsor of carbohydrates. Amino acids account for only 5.6%of the nutritional supply for the oxidative metabolismin the heart of a fasting human being (14). The intramolecular transfer of the amino group of amino acids inthe myocardium is high, however, because of the presence of high levels of glutamate a-keto acid transaminases. Thus, amino acids may be taken up in the myocardium and their carbon skeletons metabolized tocomponents of the citric acid cycle to supply energy

through subsequent oxidation. Indeed, when Mudge etal. (15) measured arteriovenous differences of all naturally occurring amino acids in normal subjects and inpatients with coronary artery disease, they found thatglutamate was the only amino acid extracted by theheart, and that more glutamate was taken up by themyocardium of patients with coronary artery diseasethan by normal myocardium.

Two case studies have indicated uptake of N- 13 activity in the human heart after the injection of L-(NI 3)amino acids. Lathrop et al. (4) imaged the heart afterthe injection of L-(N-l3)glutamate, and Cohen (16)demonstrated myocardial localization of N-l 3 afteradministration of L-(N- 13)alanine. In neither study wasthe quantity of N-l 3 label in the myocardium reported.The metabolic fate in the myocardium of the N-l 3 labelwas not determined in these studies, nor in our work.Thus it is not yet known whether the labeled amino groupof alanine must be transaminated to glutamate to betaken up by the heart, as suggested by the results ofMudge et al. (15).

The present study shows that N- 13 of glutamate isconcentrated in the human heart. L-(N-l 3)glutamatemay thus prove useful for imaging the heart and forstudying the higher extraction by patients with coronaryartery disease (15). Nitrogen-l3-labeled amino acidsmay also be useful for studying the observed increase andsubsequent decrease in uptake of amino acids in ischemicand failing hearts in animal model systems (17).

FOOTNOTES

* Boeringer-Mannheim, Indianapolis, IN.t Burreli Corp., Pittsburgh, PA.

ACKNOWLEDGMENTS

The authors express their appreciation to Dr. William G. Myers forhis valuable discussions and advice. We are indebted to Dr. GeraldRosen and his patients for their cooperation. We thank Ms. DorothyBorek for her secretarial assistance.

The work was supported in part by D.O.E. Contract No. EE-77-S-4268 and by Grant No. CA-I8153-03 and Core Grant No. CA08748-14 awarded by the National Cancer Institute, DHEW.

This paper was presented in part at the June 1979 Annual Meeting

of the Society of Nuclear Medicine in Atlanta, Georgia.

REFERENCES

I. GELBARD AS, BENUA RS, LAUGHLIN iS, et a):Quantitative scanning of osteogenic sarcoma with nitrogen-I 3 labeledL-glutamate. J Nucl Med 20:782-784, 1979

2. ROsEN G, GELBARD AS, BENUA RS, et al: N-13 Glutamatescanning to detect the early response of primary bone tumorsto chemotherapy. Proc Am Assoc Cancer Res 20:189, 1979(abst)

3. GELBARDAS, CLARKE LP, MCDONALDJM, et al: Enzymatic synthesis and organ distribution studies with ‘3N-Iabeled L-glutamine and L-glutamic acid. Radiology I 16:127-132,1975

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PRELIMINARY NOTES

4. LATHROPKA,HARPERPV,RICHBH,etal:Rapidincorporation of short-lived cyclotron-produced radionuclides intoradiopharmaceuticals. In Radiopharmaceuticals and LabelIedCompounds.Vol 1,Vienna,IAEA, 1973,pp 471-483

5. HAVEKESL, BUCKMANNF, VISSERJ: Immobilizedglutamate dehydrogenase: some catalytic and structural aspects.BiochimBiophysActa334:272-286,1974

6. COOPERAJL,McDoNALDJM,GELBARDAS,Ctal:Themetabolic fate of ‘3N-Iabeledammonia in rat brain. J BiolChem 254:4982-4992, 1979

7. MONAHAN WG, BEATFIE JW, POWELL MD, Ct al: Totalorgan kinetic imaging monitor: System design and applications. In Medical Radioisotopes Scintigraphy. Vol I, Vienna,IAEA, 1973, pp 285-298

8. CLARKE LP, LAUGHLIN iS, MAYER K: Quantitativeorgan-uptake measurement. Radiology 102:375—382,1972

9. CLARKELP, MAUGHAMEZ, LAUGHLINiS, et al:Calibration methods for measuring splenic sequestration by cxternal scanning. Med Phys 3: 324—327,1976

10. GELBARD AS, CLARKE LP, LAUGHLIN iS: Enzymaticsynthesis and use of ‘3N-Iabeled L-asparagine for myocardialimaging.J NuclMed 15:1223-1225,1974

II. MAJUMDAR C, STARK V. LATHROP K, et al: Species differences in the myocardial localization of N-13 L-asparagine.JNuclMed 19:701,1978(abst)

12. GELBARD AS, MCDONALD JM, REIMAN RE, et a1:Speciesdifferences in myocardial localization of N-13 labeled aminoacids. J NucI Med 16:529, 1975 (abst)

13. COHEN MB, MASUOKA DT, ALCARAZ AF, et al: Acutedistribution ofvarious C-14 compounds in mice as determinedby whole-body autoradiography: Adjunct for radiopharmaceutical synthesis. mt J AppI Radial 1501 28:491-496,1977

14. BING R: The metabolism of the heart. Harvey Lectures. NewYork, Academic Press Inc, 1956, pp 27—70

15. MUDGE GH, MILLS RM, TAEGTMEYER H, et al: Alterations of myocardial amino acid metabolism in chronic ischemic heart disease. J Clin Invest 58:1 185—1192, 1976

16. COHENMB: Synthesis and utilization of ‘3Ncompounds forpositron scanning. Int I NucI Med Biol 5:201 , I978 (abst)

17. PETERSON MB, MEAD Ri, WELTY iD: Protein and freeamino acid metabolism in the failing canine heart. In RecentAdvances in Studies on Cardiac Structure and Metabolism.vol. 3, N. 5. Dhalla, ed. Baltimore, University Park Press,1972, pp 615—623

The5thAnnualWesternRegionalMeetingoftheSocietyofNuclear MedicinewillbeheldOctober9-12,l980atthe MarriottHotel.

Invited Guest Lecturers are David Rollo, M.D., Ph.D., Director of Nuclear Medicine at Vanderbilt University and H.WilliamStrauss, M.D.,Director of Nuclear Medicine at Massachusetts General Hospital.

The Special Program willbe “Instrumentatonfor the 80's―Panelists: John Verba, Ph.D., Michael Phelps. Ph.D., LeonKaufman,Ph.D.,DavidWilliams,Ph.D.,DavidRollo,M.D.,H.WilliamStrauss,M.D.,withL.StephenGraham,Ph.D.serving as the moderator.

The 5th AnnualWesternRegionalMeetingwillhave commercialexhibitsand all interested companies are invited.Pleasecontact the WesternRegionalofficeat the address listedbelow.

Seven refresher courses are scheduled asfollows: 1. Nuclear Medicine Instrumentation , Ernest Garcia, Ph. D. 2. Updateon Assessmentof CardiacFunction,WilliamAshburn,M.D.3.Updateon AssessmentofMyocardialProfusion,HeinzSchelbert, M.D. 4. Imaging Studies of the Gastrointestinal Tract, Robert Stadalnik, M.D. 5. Diagnosis and Therapy ofThyroid Disease, Lawrence Greenfield, M.D.6. Update on Bone Imaging, Frederick Mishkin, M.D.7.CurrentConceptsof Radioimmunoassay, Albert Nichols, M.D.

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1980;21:988-991.J Nucl Med.   Alan S. Gelbard, Richard S. Benua, Robert E. Reiman, Joseph M. McDonald, John J. Vomero and John S. Laughlin  Imaging of the Human Heart after Administration of L-(N-13)Glutamate

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