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EnzymaticSynthesisof [1@@11C]PyruvicAcid,L-[1-11C]LacticAcidand L—[1- 11C] 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) [“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 (L-AID) or L-amlnoacid oxidase(L-AAO/catalase). [1-@CJ 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] 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- 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 .B ISUIFI1@EADDUCT \@ a)(@[email protected] I. Enzyme immobilization. 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 D.u0 (GDH) (9). A brief description of the conditions for immobilization of each enzyme is given below. D- 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 /acetaldehyde adduct L- (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 (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 -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] 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

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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. The pH was adjusted (7.4) ume) eluted to waste. The solution was allowed to remain before sterilization through a 0.22-jzm-pore filter into in contact with the enzyme for 8-iO mm, then eluted a sterile, pyrogen-free vial (Table 1). through the light-protected (<550 nm) L-AAO/catalase VI. Verification of radiochemicalproperties.The ra column (1.5 by 7.0 cm). The enzyme column was washed diochemical purities of [i-' ‘C]pyruvicacid and L-[1- with 10 ml (void volume) plus an additional 4.0 ml of 30 I ‘C]lactic acid were verified by reversed-phase HPLC mM sodium phosphate buffer to ensure that all the (Organic Acid Aminex Column HPX-87H, 300 X 7.8 [1-' ‘C]pyruvicacidwascollected(Table i). mm, H@resin; mobile phase 0.013 N H2S04; flow rate Iv. Enzymaticconversionof[I-―C]pyruvicacidinto 0.6 mI/mm; room temperature; radioactivity detector; i41-@C]Iactic acid. If L-[l-' 1C]lactic acid was desired, retention time 10.5 mm for [1-' ‘C]pyruvicacid, and 14 to the neutralized eluate from the L-AID or L-AAO mm for L-[I-―C]lacticacid). DL-and L-[1-@C]AIanine columns (see above, III) was added i.2 ml of sodium purities were verified using the o-phthaldialdehyde phosphate buffer (100 mM, pH 7.5)/NADH (I 1 @zM). (OPT) precolumn fluorescence derivatization procedure, The buffered substrates were allowed to pass through the as previously described for 13N-and ‘‘C-labeledL-amino L-LDH column; the column was then washed with 30 acids (8,9,11), using reversed-phase HPLC (Ultrasphere mM sodium phosphate buffer, pH 7.5, and the fraction ODS, 5 @m,4.6 X 150 mm column; 55% 100 mM po containing L-[i-' ‘C]lacticacid collected. The solution tassium phosphate buffer, pH 7.0/45% methanol; flow was made isotonic and then sterile as described earlier rate I.0 ml/min; fluorescence detector; radioactivity for [i-1 ‘C]pyruvicacid. The enzymatic conversion of detector; retention time 10 mm for L-[I-' ‘C]alanine). pyruvic acid to L-lactic acid was essentially quantitative In addition, the enantiomeric purity of L-alanine was (Table 1). verified by elution of a small portion of the radiotracer V. Isolation of L41-@C@aIanine(path B, Fig. 1). The solution through the L-AID enzyme column, followed radioactive fraction collected from the D-AAO column by HPLC analysis as indicated above for DL-alanine. containing pyruvic acid and L-alanine (Section lilA, The OPT derivatization procedurewas used for the de above)- was acidified to pH 1-2 (iN HC1, 1 ml) and termination of the specific activity of DL-[i-' ‘C]alanine eluted through a i.0 X 15 cm AG5OW-X2 cation-ex (115—165Ci/mmol).Thismethodallowedforthecal change resin column (hydrogen form) equilibrated with culation of the specific activities of [1-' 1C]pyruvic acid deionized water. The column was washed with 10 ml of (70—95Ci/mmole), L-[1-‘‘C]lacticacid and L-[ 1- deionized water, and the eluate, containing [i-' ‘C]- I ‘C]alanine (60—80 Ci/mmol) at the end of synthesis. pyruvic acid, was collected for further processing (neu Pyrogenicity testing verified all samples to be pyrogen tralization, sterilization, etc.) or discarded if not needed. free. The positively charged L-[i-' ‘C]alanineretained in the VII. Remote, semiautomated synthesis systems. The

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synthesis, isolation and purification of [1-' 1C]pyruvic pyruvic acid, which permits its preparation in multi acid, L-[l-' ‘C]lacticacid, and L-[1-' ‘C]alaninewere millicurie amounts suitable for metabolic studies using performed using a remote, semiautomated synthesis PET. Secondly, since D- and L-alanine are the best system that ensures minimum radiation exposure to the substrates for D-AAO (18—21)and L-AID (22—25), chemist (< 1 mR). The system consists of five units: (a) respectively, rapid and efficient transformations to DL-, (b) deionization (c) and (d) [1-' ‘C]pyruvicacid are obtained. Thirdly, these enzymes enzymatic resolution and/or purification, and (e) ster are commercially available, ready for immobilization ilization. The basic components and operation of the without any further purification steps, and are reusable system are similar to those described elsewhere beyond 6 mo. The fourth advantage is the compatibility (8,9,11,12). of immobilized-enzyme columns with remote processing systems (8,9,1 1 ,12), which assures only minimum ra RESULTS AND DISCUSSION diation exposure (< 1 mR) to the operator during the synthesis. Lastly, enzyme immobilization eliminates the The general reaction pathway outlined in Fig. 1 has possibility of protein contamination, and assures sterile, been used to synthesize L-[I-' ‘C]alanine,[1-' ‘C]pyruvic pyrogen-free products. @ acid, and 1 C]lactic acid from hydrogen [1‘C]- D-AAO, a flavoprotein oxidase (18—20),selectively cyanide. deaminates D-amino acids and gives a maximum oxi The rates and optimum reaction times for the syn dation rate with D-alanine (18—21). These features, and thesis of D,L-amino acids using the Bucherer-Strecker its commercial availability in a purified form ready for reaction vary widely according to the carbonyl compo immobilization (8), make it a good choice for the enzy nent employed. The volatility of acetaldehyde and the matic transformation to [1-' ‘C]pyruvicacid. Similarly, obvious problems associated with its use (particularly the commercially available L-AID was selected for the polymerization during the reaction) precluded its effi oxidative deamination of L-alanine to form pyruvic acid, cient transformation into DL-alanine. Accordingly, the since its substrate specificity is highest for L-aianine possibility of using the sodium bisulfite/acetaldehyde unlike L-AAO (10,26,27) and glutamate dehydrogenase adduct for the synthesis of DL-[1-' ‘C]alaninewas in ‘(GDH,28). vestigated. Reversed-phase HPLC analysis of the radioactive Initial experiments with C- 14-labeled hydrogen cy anide showed that satisfactory rates and yields of ra diolabeled DL-alanine were obtained. Although the above C- 14 reactions were analyzed by HPLC, which allowed for only approximate reaction rates, they per mitted the selection of satisfactory reaction conditions easily applicable to carbon-i 1 (Table 1). Thus, after desalting (ion-retardation resin) (8), 100 mCi of pure DL-[1-' ‘C]alanine were isolated from 400 mCi of starting hydrogen [1‘C]cyanide, with total synthesis times 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 of DL-[I-' ‘C]alanineare attainable. Most recently a great deal of renewed interest in im mobilized enzymes has emerged due to their versatility U 0 and their potential for new applications in synthetic @0 a chemistry, , and pharmacology (13). This increasing use of immobilized enzymes is not suprising and, in fact, was predictable. In the radiopharmaceutical field, we and others (8,9,11,12,14—17)have been using immobilized-enzyme techniques for a variety of pur poses, and their advantages over many synthetic proce dures are clear—particularly when rapid chemical 0 5 10 Time (mm) transformations are necessary. Close scrutiny of the salient features of the enzymatic FIG. 2. Chromatographlc profile (OPT precolumn fluorescence derivatizatlon)of reactionof Dt-[1-11C]alaninewith immobilized transformation of D,L-alanine into pyruvic acid reveals D-aminoacidoxidase(experimentalcondftionsdescribedintext). its inherent efficiency. Firstly, both the Dand L isomers Radioactivityis expressedin arbitraryunits,andpeakintensfties of alanine are utilized in the production of radiolabeled are not decay-corrected.

890 THE JOURNAL OF NUCLEAR MEDICINE BASIC SCIENCES RADIOCHEMISTRY AND RADIOPHARMACEUTICALS

U a 0 a

FIG. 3. Final-product chromato@aphic analyses of [1-1'C]pyruvic acid (PanelA) andL-[1-11C]Iactlcacid(PanelB)usingan organicacidaminexcolumn(experimental conditionsdescribedin text). Retention 0 5 10 15 0 5 10 15 times of 0-14 markers are indicated. Ti@•(asia)

fractions after elution of DL-[i-' ‘C]alaninethrough rapidly) and specifically in the transformation of [1- D-AAO showed only peaks of [1-' ‘C]pyruvicacid and ‘‘C]pyruvic acid to L-[I-' ‘C]lactic acid, producing ra L-[l-' ‘C]alanine(Fig. 2). If complete transformation diopharmaceutical preparations ready for injection. In to [I-' ‘C]pyruvicacid was desired for PET studies, the effect, when the [1-' ‘C]pyruvicacid collected from the mixture was eluted through the L-AID column with L-AID column was preconditioned (see Experimental carbonate buffer (100 mM, pH 10.5) containing NAD@ Section, IV) and eluted through the L-LDH column, a (0.4 mM) as the required (22,23) (Fig. 3, panel rapid and quantitative conversion to L-[1-' ‘C]lacticacid A). In this manner all the DL-[i-' ‘C]alaninewas spe was observed (Fig. 3, Panel B). These enzymatic trans cifically and rapidly (5—10mm) converted to [1-' ‘C]- formations are clear examples of the usefulness of en pyruvic acid in excellent yield (Table 1). zymes for rapid synthesis of radiopharmaceuticals with Alternatively, and as outlined in the experimental chiral centers. Due to their great structural and geo section, L-alanine can be converted to pyruvic acid by metric binding specificity, their remarkable catalytic way of L-AAO. We note, however, that L-alanine is not power, substrate specificity, and mild operating condi among the best substrates for L-AAO (10,26). This was tions, enzymes offer clear advantages over man-made clear when the substrate was completely oxidized only catalysts in a variety of situatipns. after allowing it to remain in contact with the immobil Finally, the enzymatic procedures described in this ized enzyme for 8—10mm. It should be stressed, however, work (Fig. I) allow for the preparation and isolation of that the low enzymatic activity in the commercial L-[1-' ‘C]alaninefrom DL-[i-' ‘C]alanineby enzymatic preparation demands a large amount of activated transformation of the D isomer to pyruvic acid, followed Sepharose (5.0 g) for immobilization, all of which may by chromatographic (cation-exchange resin) separation have been a contributing factor for the limited efficiency of the pyruvic acid from the L-isomer. The enantiomeric of the procedure. Aside from the obvious limitations of purities of L-[1-' ‘C]aminoacids, produced by D-AAO immobilized L-AAO, satisfactory yields of [1-‘‘C]py treatment of DL-amino acids, can be analyzed using re ruvic acid were obtained. Further purification of L-AAO versed-phase HPLC and Cu2@/L- buffer as the would afford an improved procedure for oxidation of mobile chiral phase (8,33). This procedure, efficient with L-[I-' ‘C]alanineto [1-' ‘C]pyruvicacid. hydrophobic amino acids, is unfortunately inadequate L-Lactic (L-LDH) from different for the analysis of L-[l-' ‘C]alanine(33). We have sources have been purified, and their physical, chemical, therefore routinely verified the enantiomeric purity of and catalytic properties investigated (29—31). Soluble radiolabeled L-alanine using L-AID, an enzyme with L-LDH, in combination with glutamate-pyruvate strict stereospecific requirements (23). After enzymatic (GOT), was used in the transformation of treatment, no amino acid remained, indicating the purity @ [3-―C]alanine (present as DL mixture) to L-[3-―C]- of the C]alanine, since only the L-isomer can be lactic acid (32); Immobilized L-LDH, however, had not transformed to pyruvic acid by the enzyme L-AID. In been previously utilized to produce L-lactic acid labeled all cases L-[1-' ‘C]alaninewas obtained with >98% ra with a positron emitter. We have found this immobilized diochemical purity. Since the best possible synthetic enzyme to work very effectively (both quantitatively and scheme is one in which all the product(s) formed can be

Volume 25, Number 8 891 ROPCHAN AND BARRIO

used, this method is unusual in accomplishing this goal. 13. MAUGH TH: A renewedinterestin immobilizedenzymes. By this procedure, two different studies can be conducted Science 223:474—476,1984 using either [I-―C]pyruvic or L-[l-' ‘C]lacticacid and 14. GELBARDAS, BENUA RS, LAUGHLIN iS, et a!: Quanti tative scanning of osterogenic sarcoma with nitrogen- 13- L-[l-' ‘C]alanine. labeled L-glutamate. J Nuc! Med 20:782—784,1979 In conclusion, [1@IIC]pyruvic acid, 1C] lactic 15. WASHBURN LC, SUN IT, BYRD BL, et al:High level pro acid, and L-[l-' ‘C]alaninewere prepared in amounts ductionof C-I1-carboxyl-labeledaminoacids.Radiophar suitable for PET studies and with excellent radiochem maceuticals II: Proceedings of the 2nd International Sym ical purities, using either a closed-vesselor an open-vessel posium on Radiopharmaceuticals, Seattle, Washington, 767—777,1979 reaction. The use of immobilized enzymes—along with 16. CASEY DL, DIGENISGA, WESNER DA, et al: Preparation @ the versatility of our remote, semiautomated sys and preliminary tissue studies of optically active ‘C-D- and tems—allowedus the flexibility of producing any one or L-phenylalaninc. In: I App! Rad iso! 32:325—330,1981 combination of radiolabeled compounds, rapidly and 17. COHEN MB, SPOLTER L, CHANG CC, Ct al: Immobilized effectively, with minimum radiation exposure (<1 mR) enzymesin the productionof radiopharmaccuticallypure amino acids labeled with ‘3N.J Nuc! Med 15:1192—1195, to the operator. The immobilized enzymes are reusable 1974 beyond 6 mo, eliminate the possibilty of protein con 18. DixoN M, KLEPPE K: D-Amino acid oxidase: I. Dissociation tamination, and assure sterile, pyrogen-free products. and recombinationsofthe holoenzyme.BiochimBiophysAda 96:357—367,1965 19. DIxoN M, KLEPPEK: D-Amino acid oxidase: II. Specificity, FOOTNOTES competitive inhibition and reaction sequence. Biochim Bio phys Ada 96:368—382,1965 20. DIxON M, KLEPPEK: D-Amino acidoxidase. III. Effect of C Sigma Chemical Co., St. Louis, Mo. pH.BiochemBiophysActa96:383-389,1965 t Boehringer Mannheim Biochemicals, Indianapolis, IN. 21. Tu S-C, MCCORMICK DB: Photoinactivation of porcine D-amino acid oxidase with flavin adenine dinucleotide. JBio! Chem 248:6339—6347,i973 REFERENCES 22. YO5HIDA A, FREESEE: Enzymatic properties of alanine dehydrogenase of Bacillus Subtilis. Biochim Biophys Ada I. ISSEKUTZ B, SHAW WAS, ISSEKUTZ A: Lactate metabo 96:248-262,1965 lism in resting and exercising dogs. J App! Physiol 40:312— 23. O'CoNNoR J, HALVORSON H: The substrate specificity of 319, 1976 L-alanine dehydrogenase. Biochim Biophys Ada 48:47—55, 2. GOLDSTEIN RA, KLEIN MS, SOBEL BE: Detection of I961 myocardial ischemia before infarction, based on accumulation 24. GOLDMANDS:Enzymesystemsinthemycobacteria.VII. oflabcledpyruvate.JNuclMed2l:lIOl—1104, 1980 Purification, properties and mechanism of action of the ala 3. 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J Academic Press, NY, 1963, pp 609-648 Labe! Compd Radiopharm XVI:63-65, 1979 28. STRUCKJ, SIZER 1W:The substrate specificity of glutamic 7. KILBOURNMR. WELCHMJ: No-carrier-addedsynthesis acid dehydrogenase. Arch Biochem Biophys 86:260-266, @ of [1-' ‘C]pyruvicacid. ml App! Radiat Iso: 33:359-36 1, i960 1982 29. EvERsE J, KAPLAN NO: Lactate dehydrogcnascs: Structure 8. BARRIO JR, KEEN RE, ROPCHAN JR, Ct al: L-[l-―C] and function. in Adv Enzymo!. Vo!. 37, A. Meister, ed. New Leucine: Routine synthesis by enzymatic resolution. I Nuc! York,AcademicPress,1973,pp61-133 Med24:515—521,1983 30. SCHWERTOW, WINERAD: Lactatedehydrogenase.In The 9. BARRIO JR. BAUMGARThER FJ, HENZE E, Ct al: Synthesis Enzymes, Vo!.7. Boycr PD, Lardy H, Myrback K, eds. New and myocardial kinetics of N-i 3 and C- I I labeled York,AcademicPress,1963,pp 127-146 branched-chain L-amino acids. J Nuc! Med 24:937—944, 31. HOLBROOK ii, LIUAS A, STEINDEL J, Ct al: Lactate Do 1983 . In the Enzymes, Vol. 2, Boyer PD, Lardy H, 10. GUILBAULT GO: Handbook of Enzymatic Methods of Myrback K, eds. New York, Academic Press, 1970, pp Ana!ysis. New York, Marcel Dekker, Inc., Vol. 4, pp 68-71, 191—292 I31-I36,208-222 and 294—298,1976 32. KLOSTER 0, LAUFERP: Enzymatic synthesis and chroma II. BAUMGARTNER FJ, BARRIO JR. HENZE E, et al: ‘3N- tographicpurificationof L-3-['‘C]-lacticacid via D,L-3- Labeled L-amino acids for in vivo assessment of local myo [I ‘C]-alanine. J Label CompdRadiopharm XVII:889-894, cardial . J Med Chem 24:764-766, 1981 i979 12. BARRIOJR.EGBERTJE,HENZEE,etal: L-[4-' ‘CjAspartic33. HARE PE, GIL.Av E: Separation of D and L amino acids by Acid:Enzymaticsynthesis,myocardialuptakeandmetabo liquid chromatography. Usc of chiral eluants. Science 204: lism. I Med Chem 25:93-96, 1982 1226—1228,1979

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