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Enzymaticsynthesisof [1@@11C]Pyruvicacid, L-[1-11C]Lacticacidand

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Enzymaticsynthesisof [1@@11C]Pyruvicacid, L-[1-11C]Lacticacidand 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 enzymatlcallyfrom [1-@C]pyruvIcacid by way of @i-[1-@C]alanIne,usingremote, semlautomatedprocedures.The Di. iso mersof alanlnewerepreparedbya modificationoftheBucherer-Streckerreaction fromno-carrier-added(NCA) hydrogen[“Cjcyanlde.The enantlomermixturewas transformed to [1-@C]pyruvlc acid by successive elutlon through columns of (a) ImmobilizedD-amlnoacid oxidase(D-AAO)/catalase and (b) ImmobilizedL-ala nine dehydrogenase(L-AID) or L-amlnoacid oxidase(L-AAO/catalase). [1-@CJ Pyruvic acid was subsequentlyconverted to L@[1@HC]lacticacid by passage througha L-laCtiCdehydrogenase(L-LDH) column.L-[1-―C]Alanineand [1-―C]- pyruvic acid were separated chromatographicallyby way of a cation-exchange column(AG5OW-X2,H@form). Typicallythe synthesistime was 35-40 mm after cyclotron productionof hydrogen[@C]cyanide(400 mCi), wfth radiochemical yieldsof 25mCi(25%) forL-[1-1'Cjlacticacid,35mCi(29%) for[1-@C]pyruvic acid, and 20 mCi (20%) for L-[1-1'C]alanine.The use of immobilizedenzymes eliminatesthe possibilityof proteincontaminationand assuresthe productionof sterile, pyrogen-freeproducts,allowingfor rapid andeffectiveregio-andstereo specifictransformations. J NucI Med 25: 887—892,1984 Pyruvic and lactic acids, both normal metabolites in Cohen et al. (5,6) in 1979. These methods are not suit the myocardium under physiological and pathophysio able for routine, metabolic studies with PET in man, logical conditions (i.e., ischemia and myocardial in since either the radiochemical yields were too low (5,6) farction), have been studied in animals using C-14-la or the procedure used did not isolate the physiologically beled substrates (1,2). The availability of efficient active form and required excessive synthesis time (3,4). methods for the synthesis of [1-' ‘C]pyruvicacid and Recent work involving the carboxylation of a masked 1C]lactic acid would permit in vivo metabolic in anion has been directed toward developing a more se vestigations using positron emission tomography lective method for synthesis of 11Cjpyruvic acid, al (PET). though the formation of undesirable by-products could @ DL-[l- ! C]Lactic acid was first synthesized in 1941 not be avoided (7). by Cramer and Kistiokowsky (3), and later by Winstead We report here that DL-[l-' ‘C]alanine,easily pre et al. (4). Physiologically active C-I I-labeled L-lactic pared from hydrogen [I‘C]cyanide,can be used for the acid, L-alanine, and pyruvic acid were first prepared by efficient immobilized-enzyme production of multimil licurie amounts of [1-' 1Cjpyruvic acid, L-[l-' ‘C]lactic Received Mar. I, I984; revision accepted Apr. 27, 1984. acid, and L-[1-' ‘C]alanine. We have developed condi Forreprintscontact:J. R. Barrio,PhD, UCLA Schoolof Medicine, Laboratoryof NuclearMedicine,Divisionof Biophysics,LosAngeles, tions under which the enzymatic transformations become CA90024. routine procedures. Volume 25, Number 8 887 ROPCHAN AND BARRIO MATERIALS AND METHODS ACETALDEHYDE.B ISUIFI1@EADDUCT \@ a)(@[email protected] I. Enzyme immobilization. Enzymes were immobilized \@Na0H on CNBr-activated Sepharose in the presence of cofac tors and substrates as previously described for D-amino DL-(1.11C]ALANIN( acid oxidase (D-AAO) (8) and glutamate dehydrogenase D.u0 (GDH) (9). A brief description of the conditions for immobilization 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) and dog-liver catalase* (500 units) were immobilized on 570 1) 1—AID mg of CNBr-activated Sepharose in the presence of so L@A0 dium pyrophosphate (30 mM, pH 8.3)/NaC1 (0.5 zM)/flavin adenine dinucleotide (FAD, 1.0 @tM)as previously described (8). [1.llC]PYRUVA1( (1.11C]PYRUVAT L.(1.llCJALANIpst L-Amino acid oxidase (E.C. 1.4.3.2. , L-AAO)/cata lose. Crotalus atrox (Type VI) L@AAOt (330 units), and I L.LDH 7 dog-liver catalase (500 units) were immobilized in the same manner as D-AAO above, but using sodium L.(1.llC]LACTAT( phosphate buffer pH 7.5, optimum for enzyme activity FIG. 1. Scheme outlining preparation of oL-[1-@C]alanine, L-[1- (10). In addition, a larger quantity of Sepharose (5.0 g) 1‘C]alanine, [1-@C]pyruvic acid, and L-[1-'1C]lactic acid. was necessary due to the low specific activity of the commercially available L-AAO. containing the sodium bisulfite/acetaldehyde adduct L-Alanine dehydrogenase (E.C. I .4. 1. 1., L-AID). (0.5 mmol) and heated under pressure at 125°Cfor 5 Bacillus subtilis L-AID (300 units) was immobilized mm. After cooling, NaOH (6.25N, 1.0 ml) was added, in the presence of sodium phosphate (30 mM, pH 8.5), the vesselresealed and heated at 125°Cfor 5 mm to form nicotinamide adenine dinucleotide (NAD, 200 SM), the radiolabeled DLamino acid. An additional 5 mm was and L-alanine (1 mM). necessary to hydrolyze the alkylhydantoin when the open L-Lactic dehydrogenase (E.C. 1.1.1.27, L-LDH). vessel was used. All times are optimum and were pre Rabbit-muscle (Type II) L@LDHt(500 units) was im determined by C-14 experiments. mobilized utilizing sodium phosphate (30 mM, pH 7.3) III. Enzymatic synthesis and purification of [1-NC]- and NADH (1 1.0 @.tM). pyruvic acid (path A, Fig. 1). All enzymes were stored at 4°Cin either a potassium A. Via D-amino acid oxidase and L-alanine dehy chloride solution (i.e., D-AAO-3OmM sodium pyro drogenase. To the reaction vessel from the DL-[1- phosphate, pH 7.5, 1.0 @MFAD, 2M KCI; L-AAO I ‘Cjalanine reaction (see above, II) was added glacial 30mM sodium phosphate, pH 7.5, 1.0 j.tM FAD, 2M acetic acid (0.61 ml, final pH 6.0—6.5),and the mixture KCI; L-LDH-3OmM sodium phosphate, pH 7.2, 11.0 was transferred to a column (1 .5 by 17 cm) containing 1tiM NADH, 2M KC1), or in an ethylenediaminotetra ion-retardation resin (AG-i iA8). The column was acetic acid (EDTA) solution (i.e., L-AID-3OmM sodium eluted with deionized water, and the radioactive fraction phosphate, pH 8.5, 5mM EDTA, 200 @MNAD). (8.0 ml) that contained DL-[1-' ‘C]alaninewas collected, Before each run the enzymes were warmed to room and H202 (50 mM, 0.1 ml) and a buffer (i.I ml) con temperature and washed for 1 hr with the appropriate taming sodium pyrophosphate (225 mM, pH 8.3)/FAD buffers. Periodic checks of the enzyme columns with cold (75 zM) was added. The solution was passed through substrate standards ensured their efficient performance. a light-protected D-AAO enzyme column (8). The en Under the storage conditions described, the enzymes are zyme column was washed with sodium pyrophosphate stable and columns can be reused for more than 6 mo. buffer (30 mM, pH 8.3)/FAD (10 tiM), and the ra II. Synthesis of DL-I1-―C]alanine.This preparation dioactive fraction containing pyruvic acid and L-alanine from hydrogen [I 1Clcyanide followed the same proce was collected, treated with 1.5 ml of sodium carbonate dure as that previously reported for DL-[l-' ‘C]leucine buffer (1 00 mM, pH i 1.0)/NAD@ (0.4 mM), and (8), with the exception that sodium bisulfite/acetalde NaOH (6.25 N, 0.05 ml)—which adjusts the pH of the hyde adduct was used instead of the free aldehyde. Hy solution to about 10.0-10.5—-and passed through the drogen [I 1C]cyanide (“@-‘400mCi) prepared by the L-AID column (1.0 by 5.0 cm). The radioactive fraction 14N(p,n)1 ‘Creaction on nitrogen was bubbled into a containing [1-' ‘C]pyruvicacid was neutralized (iN I .5-ml solution containing (NH4)2C03 (0.75 mmol), HC1), made isotonic, then sterilized by passage through NH4C1 (0. 125 mmol), and NaOH (1 smole). The so a 0.22-@zm-pore filter into a sterile vial (Table i). lution was transferred to a stainless steel reaction vessel B. Via D- and L-amino acid oxidases. The solution 888 THE JOURNAL OF NUCLEAR MEDICINE BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS ITypicalTABLE reaction Compound Syntheticprocedtre amounts@mCiRadiochemicalyieldsb,%Radiochemicalp@gfty,%Total timesc mm oi-[1-11C]Alanmne Bucherer-Strecker 100 45 98 L-[1-11CjAlaflmfle D-AAO 20 20 >98 40 [1-―C]PyruvicAcid D-AAO/L-AID1 35 29 98 35 L-[1-'1C]LacticAcid L-LDH@ 25 25 >97 40 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%)wereobtained with L-AAO. aL-LDH:ImmobilizedL-lacticaciddehydrogenase. containing [1-' ‘C]pyruvicacid and L-[l-' ‘C]alanine, column was released by the passage of 10 ml of 100 mM after elution from the D-AAO column (Section lilA, sodium phosphate, pH 12.0. Due to the pH of the column above), was treated with H202 (50 mM, 0.1 ml) and a after acid washing, the concentration, and the pH of the buffer (1.2 ml) containing sodium phosphate (225 mM, sodium phosphate, the fraction containing L-[l-' ‘C] pH 7.5)/FAD (75 zM), and the initial 10 ml (void vol alanine (iO ml) was isotonic.
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