Pyruvic and lactic acids, both normal metabolites inthe myocardium under physiological and pathophysiological conditions (i.e., ischemia and myocardial infarction), have been studied in animals using C-14-labeled substrates (1 ,2). The availability of efficientmethods for the synthesis of [1-' ‘C]pyruvicacid and

1C]lactic acid would permit in vivo metabolic in

vestigations using positron emission tomography

(PET).DL-[l- !@ C]Lactic acid was first synthesized in 1941

by Cramer and Kistiokowsky (3), and later by Winsteadet al. (4). Physiologically active C-I I-labeled L-lacticacid, L-alanine, and pyruvic acid were first prepared by

Received Mar. I, I984; revision accepted Apr. 27, 1984.Forreprintscontact:J. R. Barrio,PhD, UCLA Schoolof Medicine,

Laboratoryof NuclearMedicine,Divisionof Biophysics,LosAngeles,CA90024.

Cohen et al. (5,6) in 1979. These methods are not suitable for routine, metabolic studies with PET in man,since either the radiochemical yields were too low (5,6)or the procedure used did not isolate the physiologicallyactive form and required excessive synthesis time (3,4).Recent work involving the carboxylation of a maskedanion has been directed toward developing a more selective method for synthesis of 11Cjpyruvic acid, although the formation of undesirable by-products couldnot be avoided (7).

We report here that DL-[l-' ‘C]alanine,easily prepared from hydrogen [I ‘C]cyanide,can be used for theefficient immobilized-enzyme production of multimillicurie amounts of [1-' 1Cjpyruvic acid, L-[l-' ‘C]lacticacid, and L-[1-' ‘C]alanine. We have developed conditions under which the enzymatic transformations becomeroutine procedures.

Volume 25, Number 8 887

EnzymaticSynthesisof [1@@11C]PyruvicAcid,L-[1-11C]LacticAcidand

L—[1- 11C]Alanine via ii-[1 - 11C]Alanine

Jim A. Ropchanand Jorge R. Barrio

Universityof California,andLaboratoryof BiomedicalandEnvironmentalSciences,LosAngeles,California

L-[1-@CJLaCtlCacid was prepared enzymatlcally from [1-@C]pyruvIcacid byway of @i-[1-@C]alanIne,usingremote, semlautomatedprocedures.The Di. isomersof alanlnewerepreparedbya modificationof theBucherer-Streckerreactionfromno-carrier-added(NCA) hydrogen[“Cjcyanlde.The enantlomermixturewastransformed to [1-@C]pyruvlc acid by successive elutlon through columns of (a)ImmobilizedD-amlnoacid oxidase(D-AAO)/catalase and (b) ImmobilizedL-alanine dehydrogenase(L-AID) or L-amlnoacid oxidase(L-AAO/catalase). [1-@CJPyruvic acid was subsequentlyconverted to L@[1@HC]lacticacid by passagethrougha L-laCtiCdehydrogenase(L-LDH) column.L-[1-―C]Alanineand [1-―C]-pyruvic acid were separated chromatographicallyby way of a cation-exchangecolumn(AG5OW-X2,H@form). Typicallythe synthesistime was 35-40 mm aftercyclotron productionof hydrogen[@C]cyanide (400 mCi), wfth radiochemicalyieldsof 25mCi(25%) forL-[1-1'Cjlacticacid,35mCi(29%) for [1-@C]pyruvicacid, and 20 mCi (20%) for L-[1-1'C]alanine.The use of immobilizedenzymeseliminatesthe possibilityof proteincontaminationand assuresthe productionofsterile, pyrogen-freeproducts,allowingfor rapidandeffectiveregio-andstereospecifictransformations.

J NucI Med 25: 887—892,1984

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ROPCHAN AND BARRIO

ACETALDEHYDE.B ISUIFI1@EADDUCT

\@ a)(@0@42C03.H11CN\@Na0H

DL-(1.11C]ALANIN(

[1.llC]PYRUVA1( (1.11C]PYRUVAT

I L.LDH 7

L.(1.llC]LACTAT(

MATERIALS AND METHODS

I. Enzyme immobilization. Enzymes were immobilizedon CNBr-activated Sepharose in the presence of cofactors and substrates as previously described for D-aminoacid oxidase (D-AAO) (8) and glutamate dehydrogenase(GDH) (9). A brief description of the conditions forimmobilization of each enzyme is given below.

D-Amino acid oxidase (E. C. 1 .4.3.3)/catalase(E.C.1.11.1.6). Porcine-kidney D@AAO* (340 units) anddog-liver catalase* (500 units) were immobilized on 570mg of CNBr-activated Sepharose in the presence of sodium pyrophosphate (30 mM, pH 8.3)/NaC1 (0.5zM)/flavin adenine dinucleotide (FAD, 1.0 @tM)aspreviously described (8).

L-Amino acid oxidase (E.C. 1.4.3.2. , L-AAO)/catalose. Crotalus atrox (Type VI) L@AAOt (330 units), anddog-liver catalase (500 units) were immobilized in thesame manner as D-AAO above, but using sodiumphosphate buffer pH 7.5, optimum for enzyme activity(10). In addition, a larger quantity of Sepharose (5.0 g)

was necessary due to the low specific activity of thecommercially available L-AAO.

L-Alanine dehydrogenase (E.C. I .4. 1. 1. , L-AID).Bacillus subtilis L-AID (300 units) was immobilizedin the presence of sodium phosphate (30 mM, pH 8.5),nicotinamide adenine dinucleotide (NAD, 200 SM),and L-alanine (1 mM).

L-Lactic dehydrogenase (E.C. 1.1.1.27, L-LDH).Rabbit-muscle (Type II) L@LDHt(500 units) was immobilized utilizing sodium phosphate (30 mM, pH 7.3)and NADH (1 1.0 @.tM).

All enzymes were stored at 4°Cin either a potassiumchloride solution (i.e., D-AAO-3OmM sodium pyrophosphate, pH 7.5, 1.0 @MFAD, 2M KCI; L-AAO30mM sodium phosphate, pH 7.5, 1.0 j.tM FAD, 2MKCI; L-LDH-3OmM sodium phosphate, pH 7.2, 11.01tiM NADH, 2M KC1), or in an ethylenediaminotetraacetic acid (EDTA) solution (i.e., L-AID-3OmM sodiumphosphate, pH 8.5, 5mM EDTA, 200 @MNAD).Before each run the enzymes were warmed to roomtemperature and washed for 1 hr with the appropriatebuffers. Periodic checks of the enzyme columns with coldsubstrate standards ensured their efficient performance.Under the storage conditions described, the enzymes arestable and columns can be reused for more than 6 mo.

II. Synthesis of DL-I1-―C]alanine.This preparationfrom hydrogen [I 1Clcyanide followed the same procedure as that previously reported for DL-[l-' ‘C]leucine(8), with the exception that sodium bisulfite/acetalde

hyde adduct was used instead of the free aldehyde. Hydrogen [I 1C]cyanide (“@-‘400mCi) prepared by the14N(p,n)1 ‘Creaction on nitrogen was bubbled into a

I .5-ml solution containing (NH4)2C03 (0.75 mmol),NH4C1 (0. 125 mmol), and NaOH (1 smole). The solution was transferred to a stainless steel reaction vessel

D.u0

1) 1—AID

L@A0

L.(1.llCJALANIpst

FIG. 1. Scheme outlining preparation of oL-[1-@C]alanine, L-[1-1‘C]alanine, [1-@C]pyruvic acid, and L-[1-'1C]lactic acid.

containing the sodium bisulfite/acetaldehyde adduct(0.5 mmol) and heated under pressure at 125°Cfor 5mm. After cooling, NaOH (6.25N, 1.0 ml) was added,the vessel resealed and heated at 125°Cfor 5 mm to formthe radiolabeled DLamino acid. An additional 5 mm wasnecessary to hydrolyze the alkylhydantoin when the openvessel was used. All times are optimum and were predetermined by C-14 experiments.

III. Enzymatic synthesis and purification of [1-NC]-pyruvic acid (path A, Fig. 1).

A. Via D-amino acid oxidase and L-alanine dehydrogenase. To the reaction vessel from the DL-[1-I ‘Cjalanine reaction (see above, II) was added glacial

acetic acid (0.61 ml, final pH 6.0—6.5),and the mixturewas transferred to a column (1 .5 by 17 cm) containingion-retardation resin (AG-i iA8). The column waseluted with deionized water, and the radioactive fraction(8.0 ml) that contained DL-[1-' ‘C]alaninewas collected,and H202 (50 mM, 0.1 ml) and a buffer (i.I ml) contaming sodium pyrophosphate (225 mM, pH 8.3)/FAD(75 zM) was added. The solution was passed througha light-protected D-AAO enzyme column (8). The enzyme column was washed with sodium pyrophosphatebuffer (30 mM, pH 8.3)/FAD (10 tiM), and the radioactive fraction containing pyruvic acid and L-alaninewas collected, treated with 1.5 ml of sodium carbonatebuffer (1 00 mM, pH i 1.0)/NAD@ (0.4 mM), andNaOH (6.25 N, 0.05 ml)—which adjusts the pH of thesolution to about 10.0-10.5—-and passed through theL-AID column (1.0 by 5.0 cm). The radioactive fractioncontaining [1-' ‘C]pyruvicacid was neutralized (iNHC1), made isotonic, then sterilized by passage througha 0.22-@zm-pore filter into a sterile vial (Table i).

B. Via D- and L-amino acid oxidases. The solution

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TABLEITypical

Compound Syntheticprocedtre amounts@mCiRadiochemicalyieldsb,%Radiochemicalp@gfty,%Totalreaction

timesc mm

BASIC SCIENCESRADIOCHEMISTRY AND RADIOPHARMACEUTICALS

oi-[1-11C]AlanmneL-[1-11CjAlaflmfle[1-―C]PyruvicAcidL-[1-'1C]LacticAcid

403540

Bucherer-StreckerD-AAO

D-AAO/L-AID1L-LDH@

100 45202925

98>98

98>97

203525

a Starting from 400 mCi of hydrogen [‘1C]cyanlde.

b Decay-corrected.

C For radlopharmaceutlcal preparation after cyclotron production of hydrogen [‘1C]cyanide.

d For closed-vessel reaction. Open-vessel reaction time in parenthesis.

. D-AAO: Immobilized D-amino acid oxidase co-immobilized with catalase.

f L-AID: Immobilized alanine dehydrogenase. Total reaction time was 40 mm when L-AAO (immobilized L-amiriO acid oxidase

co-immobilIzedwithcatalase)wasusedinplaceofL-AID.Lowerradlochemicalyieldsof [1-11C]pyruvicacid(25%)wereobtainedwithL-AAO.

aL-LDH:ImmobilizedL-lacticaciddehydrogenase.

containing [1-' ‘C]pyruvicacid and L-[l-' ‘C]alanine,after elution from the D-AAO column (Section lilA,above), was treated with H202 (50 mM, 0.1 ml) and abuffer (1.2 ml) containing sodium phosphate (225 mM,pH 7.5)/FAD (75 zM), and the initial 10 ml (void volume) eluted to waste. The solution was allowed to remainin contact with the enzyme for 8-iO mm, then elutedthrough the light-protected (<550 nm) L-AAO/catalasecolumn (1.5 by 7.0 cm). The enzyme column was washedwith 10 ml (void volume) plus an additional 4.0 ml of 30mM sodium phosphate buffer to ensure that all the[1-' ‘C]pyruvicacidwascollected(Table i).

Iv. Enzymaticconversionof[I-―C]pyruvicacidintoi41-@C]Iactic acid. If L-[l-' 1C]lactic acid was desired,to the neutralized eluate from the L-AID or L-AAOcolumns (see above, III) was added i.2 ml of sodiumphosphate buffer (100 mM, pH 7.5)/NADH (I 1 @zM).The buffered substrates were allowed to pass through theL-LDH column; the column was then washed with 30mM sodium phosphate buffer, pH 7.5, and the fractioncontaining L-[i-' ‘C]lacticacid collected. The solutionwas made isotonic and then sterile as described earlierfor [i-1 ‘C]pyruvicacid. The enzymatic conversion ofpyruvic acid to L-lactic acid was essentially quantitative(Table 1).

V. Isolation of L41-@C@aIanine(path B, Fig. 1). Theradioactive fraction collected from the D-AAO columncontaining pyruvic acid and L-alanine (Section lilA,above)- was acidified to pH 1-2 (iN HC1, 1 ml) andeluted through a i.0 X 15 cm AG5OW-X2 cation-exchange resin column (hydrogen form) equilibrated withdeionized water. The column was washed with 10 ml ofdeionized water, and the eluate, containing [i-' ‘C]-pyruvic acid, was collected for further processing (neutralization, sterilization, etc.) or discarded if not needed.The positively charged L-[i-' ‘C]alanineretained in the

column was released by the passage of 10 ml of 100 mMsodium phosphate, pH 12.0. Due to the pH of the columnafter acid washing, the concentration, and the pH of thesodium phosphate, the fraction containing L-[l-' ‘C]alanine (iO ml) was isotonic. The pH was adjusted (7.4)before sterilization through a 0.22-jzm-pore filter intoa sterile, pyrogen-free vial (Table 1).

VI. Verification of radiochemical properties.The radiochemical purities of [i-' ‘C]pyruvicacid and L-[1-I ‘C]lactic acid were verified by reversed-phase HPLC

(Organic Acid Aminex Column HPX-87H, 300 X 7.8mm, H@resin; mobile phase 0.013 N H2S04; flow rate0.6 mI/mm; room temperature; radioactivity detector;retention time 10.5 mm for [1-' ‘C]pyruvicacid, and 14mm for L-[I-―C]lacticacid). DL-and L-[1-@C]AIaninepurities were verified using the o-phthaldialdehyde(OPT) precolumn fluorescence derivatization procedure,as previously described for 13N-and ‘‘C-labeledL-aminoacids (8,9,11), using reversed-phase HPLC (UltrasphereODS, 5 @m,4.6 X 150 mm column; 55% 100 mM potassium phosphate buffer, pH 7.0/45% methanol; flowrate I .0 ml/min; fluorescence detector; radioactivitydetector; retention time 10 mm for L-[I-' ‘C]alanine).In addition, the enantiomeric purity of L-alanine wasverified by elution of a small portion of the radiotracersolution through the L-AID enzyme column, followedby HPLC analysis as indicated above for DL-alanine.The OPT derivatization procedurewas used for the determination of the specific activity of DL-[i-' ‘C]alanine(115—165Ci/mmol).Thismethodallowedforthecalculation of the specific activities of [1-' 1C]pyruvic acid(70—95Ci/mmole), L-[1-‘‘C]lacticacid and L-[ 1-I ‘C]alanine (60—80 Ci/mmol) at the end of synthesis.

Pyrogenicity testing verified all samples to be pyrogenfree.

VII. Remote, semiautomated synthesis systems. The

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ROPCHANAND BARRIO

synthesis, isolation and purification of [1-' 1C]pyruvicacid, L-[l-' ‘C]lacticacid, and L-[1-' ‘C]alaninewereperformed using a remote, semiautomated synthesissystem that ensures minimum radiation exposure to the

chemist (< 1 mR). The system consists of five units: (a)DL-amino acid synthesis, (b) deionization (c) and (d)enzymatic resolution and/or purification, and (e) sterilization. The basic components and operation of the

system are similar to those described elsewhere(8,9,11,12).

RESULTS AND DISCUSSION

The general reaction pathway outlined in Fig. 1 hasbeen used to synthesize L-[I-' ‘C]alanine,[1-' ‘C]pyruvicacid, and 1@ C]lactic acid from hydrogen [1‘C]-cyanide.

The rates and optimum reaction times for the synthesis of D,L-amino acids using the Bucherer-Streckerreaction vary widely according to the carbonyl component employed. The volatility of acetaldehyde and theobvious problems associated with its use (particularlypolymerization during the reaction) precluded its efficient transformation into DL-alanine. Accordingly, thepossibility of using the sodium bisulfite/acetaldehydeadduct for the synthesis of DL-[1-' ‘C]alaninewas investigated.

Initial experiments with C- 14-labeled hydrogen cyanide showed that satisfactory rates and yields of radiolabeled DL-alanine were obtained. Although theabove C- 14 reactions were analyzed by HPLC, whichallowed for only approximate reaction rates, they permitted the selection of satisfactory reaction conditionseasily applicable to carbon-i 1 (Table 1). Thus, after

desalting (ion-retardation resin) (8), 100 mCi of pureDL-[1-' ‘C]alanine were isolated from 400 mCi ofstarting hydrogen [1 ‘C]cyanide, with total synthesistimes of 17 mm (closed vessel) or 22 mm (open vessel).Since much larger amounts of hydrogen [l ‘C]cyanide(several curies) are easily produced, higher yields ofDL-[I-' ‘C]alanineare attainable.

Most recently a great deal of renewed interest in immobilized enzymes has emerged due to their versatilityand their potential for new applications in syntheticchemistry, biochemistry, and pharmacology (13). Thisincreasing use of immobilized enzymes is not suprising

and, in fact, was predictable. In the radiopharmaceuticalfield, we and others (8,9,11,12,14—17)have been usingimmobilized-enzyme techniques for a variety of purposes, and their advantages over many synthetic procedures are clear—particularly when rapid chemicaltransformations are necessary.

Close scrutiny of the salient features of the enzymatictransformation of D,L-alanine into pyruvic acid revealsits inherent efficiency. Firstly, both the D and L isomersof alanine are utilized in the production of radiolabeled

pyruvic acid, which permits its preparation in multimillicurie amounts suitable for metabolic studies usingPET. Secondly, since D- and L-alanine are the bestsubstrates for D-AAO (18—21)and L-AID (22—25),respectively, rapid and efficient transformations to[1-' ‘C]pyruvicacid are obtained. Thirdly, these enzymesare commercially available, ready for immobilizationwithout any further purification steps, and are reusablebeyond 6 mo. The fourth advantage is the compatibilityof immobilized-enzyme columns with remote processingsystems (8,9,1 1 ,12), which assures only minimum radiation exposure (< 1 mR) to the operator during thesynthesis. Lastly, enzyme immobilization eliminates thepossibility of protein contamination, and assures sterile,

pyrogen-free products.D-AAO, a flavoprotein oxidase (18—20),selectively

deaminates D-amino acids and gives a maximum oxidation rate with D-alanine (18—21). These features, andits commercial availability in a purified form ready forimmobilization (8), make it a good choice for the enzymatic transformation to [1-' ‘C]pyruvicacid. Similarly,the commercially available L-AID was selected for theoxidative deamination of L-alanine to form pyruvic acid,since its substrate specificity is highest for L-aianineunlike L-AAO (10,26,27) and glutamate dehydrogenase

‘(GDH,28).Reversed-phase HPLC analysis of the radioactive

U

0@0a

FIG. 2. Chromatographlc profile (OPT precolumn fluorescencederivatizatlon)of reactionof Dt-[1-11C]alaninewith immobilizedD-aminoacidoxidase(experimentalcondftionsdescribedin text).Radioactivityis expressedin arbitraryunits,andpeakintensftiesare not decay-corrected.

0 5 10Time (mm)

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BASIC SCIENCESRADIOCHEMISTRY AND RADIOPHARMACEUTICALS

0 5 10 15 0 5 10 15

Ti@•(asia)

Ua0

a

FIG. 3. Final-product chromato@aphicanalyses of [1-1'C]pyruvic acid (PanelA)andL-[1-11C]Iactlcacid(PanelB)usinganorganicacidaminexcolumn(experimentalconditionsdescribedin text). Retentiontimes of 0-14 markers are indicated.

fractions after elution of DL-[i-' ‘C]alaninethroughD-AAO showed only peaks of [1-' ‘C]pyruvicacid andL-[l-' ‘C]alanine(Fig. 2). If complete transformationto [I-' ‘C]pyruvicacid was desired for PET studies, themixture was eluted through the L-AID column withcarbonate buffer (100 mM, pH 10.5) containing NAD@(0.4 mM) as the required cofactor (22,23) (Fig. 3, panelA). In this manner all the DL-[i-' ‘C]alaninewas specifically and rapidly (5—10mm) converted to [1-' ‘C]-pyruvic acid in excellent yield (Table 1).

Alternatively, and as outlined in the experimentalsection, L-alanine can be converted to pyruvic acid byway of L-AAO. We note, however, that L-alanine is notamong the best substrates for L-AAO (10,26). This wasclear when the substrate was completely oxidized onlyafter allowing it to remain in contact with the immobilized enzyme for 8—10mm. It should be stressed, however,that the low enzymatic activity in the commercialpreparation demands a large amount of activatedSepharose (5.0 g) for immobilization, all of which mayhave been a contributing factor for the limited efficiencyof the procedure. Aside from the obvious limitations ofimmobilized L-AAO, satisfactory yields of [1-‘‘C]pyruvic acid were obtained. Further purification of L-AAOwould afford an improved procedure for oxidation ofL-[I-' ‘C]alanineto [1-' ‘C]pyruvicacid.

L-Lactic dehydrogenases (L-LDH) from differentsources have been purified, and their physical, chemical,and catalytic properties investigated (29—31). SolubleL-LDH, in combination with glutamate-pyruvatetransaminase (GOT), was used in the transformation of[3-―C]alanine (present as DL mixture) to L-[3-―C]-lactic acid (32); Immobilized L-LDH, however, had not

been previously utilized to produce L-lactic acid labeledwith a positron emitter. We have found this immobilized

enzyme to work very effectively (both quantitatively and

rapidly) and specifically in the transformation of [1-â€â€˜C]pyruvic acid to L-[I-' ‘C]lactic acid, producing ra

diopharmaceutical preparations ready for injection. Ineffect, when the [1-' ‘C]pyruvicacid collected from theL-AID column was preconditioned (see ExperimentalSection, IV) and eluted through the L-LDH column, arapid and quantitative conversion to L-[1-' ‘C]lacticacidwas observed (Fig. 3, Panel B). These enzymatic transformations are clear examples of the usefulness of enzymes for rapid synthesis of radiopharmaceuticals withchiral centers. Due to their great structural and geometric binding specificity, their remarkable catalyticpower, substrate specificity, and mild operating conditions, enzymes offer clear advantages over man-madecatalysts in a variety of situatipns.

Finally, the enzymatic procedures described in thiswork (Fig. I) allow for the preparation and isolation ofL-[1-' ‘C]alaninefrom DL-[i-' ‘C]alanineby enzymatictransformation of the D isomer to pyruvic acid, followedby chromatographic (cation-exchange resin) separationof the pyruvic acid from the L-isomer. The enantiomericpurities of L-[1-' ‘C]aminoacids, produced by D-AAOtreatment of DL-amino acids, can be analyzed using reversed-phase HPLC and Cu2@/L-proline buffer as themobile chiral phase (8,33). This procedure, efficient withhydrophobic amino acids, is unfortunately inadequatefor the analysis of L-[l-' ‘C]alanine(33). We havetherefore routinely verified the enantiomeric purity ofradiolabeled L-alanine using L-AID, an enzyme withstrict stereospecific requirements (23). After enzymatictreatment, no amino acid remained, indicating the purityof the@@ C]alanine, since only the L-isomer can betransformed to pyruvic acid by the enzyme L-AID. Inall cases L-[1-' ‘C]alaninewas obtained with >98% radiochemical purity. Since the best possible syntheticscheme is one in which all the product(s) formed can be

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ROPCHAN AND BARRIO

used, this method is unusual in accomplishing this goal.By this procedure, two different studies can be conductedusing either [I-―C]pyruvic or L-[l-' ‘C]lacticacid andL-[l-' ‘C]alanine.

In conclusion, [1@IIC]pyruvic acid, 1C] lacticacid, and L-[l-' ‘C]alaninewere prepared in amountssuitable for PET studies and with excellent radiochemical purities, using either a closed-vesselor an open-vesselreaction. The use of immobilized enzymes—along withthe versatility of our remote, semiautomated systems—allowedus the flexibility of producing any one orcombination of radiolabeled compounds, rapidly andeffectively, with minimum radiation exposure (<1 mR)to the operator. The immobilized enzymes are reusablebeyond 6 mo, eliminate the possibilty of protein contamination, and assure sterile, pyrogen-free products.

FOOTNOTES

C Sigma Chemical Co., St. Louis, Mo.

t Boehringer Mannheim Biochemicals, Indianapolis, IN.

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892 THE JOURNAL OF NUCLEAR MEDICINE

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1984;25:887-892.J Nucl Med.   Jim R. Ropchan and Jorge R. Barrio 

C]Alanine11via dl-[1-C]Alanine11C]Lactic Acid and l-[1-11C]Pyruvic Acid, l-[1-11Enzymatic Synthesis of [1-

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