THE ENZYMATIC DEAMINATION OF AMPHETAMINE (BENZEDRINE) By J&LIUS AXELROD (From the Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Public Health Service, United States Department of Health, @dueation, and Welfare, Bethesda, Maryland) Raptinted from Tb Jomar, or B~oxmrclu, Cammmr Vol. 214, No. 9, June, 19115 &ads' in -United &ate8 af Amdca Reprinted from THE JOURNAL OF BIOLOGICAL CHEMIBTRY Vol. 214, No. 2, June, 1955 Made an united state8 0f AWW~C~ THE ENZYMATIC DEAMINATION OF AMPHETAMINE (BENZEDRINE)* BY JULIUS AXELROD (From the Laboratory of Chemical Pharmacology, National Heart Institute, Nation.al Institutes of Health, Public Health Service, U&ted States Department of Health, Education, and Weljare, Bethesda, Maryland) (Received for publication, November 23, 1954) A previous study on the metabolism of amphetamine (I-phenyl-2-amino- propane), widely used as a stimulant of the central nervous system, showed t,hat its major route of biotransformat.ion in the rat and dog involved hy- droxylation of the aromatic ring. In t.he rabbit, however, the compound was not hydroxylated, but was found to be transformed in another man- ner (2). The present report describes an enzyme system in the rabbit which deaminates amphetamine to yield phenylacetone. It will be shown that the enzyme system is localized in the microsomal fraction of the cell and requires oxygen and reduced triphosphopyridine nucleotide. Furthermore, these studies indicate that there are inhibitory factors present in t,he liver of the dog, rat, and guinea pig which may explain, in part, the inabilit,y of these species to deaminate amphetamine. Materials-l- and d-amphetamine sulfate was obtained through the cour- tesy of the Smith, Kline and French Laboratories. I-Norephedrine hydro- chloride was kindly supplied by K. H. Beyer of Sharpe and Dohme. Tri- phosphopyridine nucleotide (TPN) and diphosphopyridine nucleotide (DPN) 80 per cent purity, glucose-6-phosphate dehydrogenase, and glu- cose-6-phosphate were obtained from the Sigma Chemical Company. Re- duced triphosphopyridine nucleotide (TPNH) was prepared by the proce- dure of Kaplan et al. (3). Methods-Amphetamine, ephedrine, norephedrine, and met,hylampheta- mine were determined by the methyl orange reaction of Brodie and Uden- friend (4) with benzene as the extractant, as previously described (2, 5). An essentially similar procedure was used for the estimation of l-phenyl-l- aminopropane, 1-phenyl-3-aminobutane, phenylethylamine, isoamylamine, 2-aminoheptane, and benzylamine with chloroform as the extractant. Tyramine was estimated according to Udenfriend and Cooper (6) and p- * A preliminary report of this work has appeared (1). The material in this in- vestigation will appear in a thesis in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmarology, Graduate Council, George Washington Iiniversity. 753 754 ENZYMATIC DEAMINATION OF AMPHETAMINE hydroxyamphetamine according to Axelrod (2). Ammonia was estimat,ed by the Conway microdiffusion procedure (7). Phenylacetone was determined by its reaction with 2,4-dinitrophenyl- hydrazine as follows: An aliquot of biological material was transferred to a 40 ml. glass-stoppered centrifuge tube containing 0.5 ml. of 1 N NaOH and 20 ml. of carbon tetrachloride (reagent grade) and shaken for 20 min- utes. The tube was cent,rifuged and the aqueous phase removed by as- piration. The carbon tetrachloride extract, was washed with 5 ml. of water to remove "blank" material which reacted with dinitrophenylhy- drazine. 15 ml. of t.he carbon tet,rachloride phase were transferred to a 40 ml. glass-stoppered centrifuge tube cont.aining 5 ml. of 0.1 per cent 2,4-dinitrophenylhydrazine in 2 N HCl and shaken for 2 hours. The aqueous phase was removed by aspiration and the organic phase washed three times with 5 ml. portions of 2 N HCl to remove unchanged dinitro- phenylhydrazine. 6 ml. of t,he carbon tetrachloride extract containing the dinitrophenylhydrazone derivative of phenylacetone were t,ransferred to a cuvette containing 1 ml. of 10 per cent KOH in ethanol, and the opt(ica1 density was read in a spectrophotometer at 430 rnk. A blank consisting of biological material carried through the procedure was set at 100 per cent transmission. Standards mere prepared by adding a known amount of phenylacetone to biological material and proceeding as above. Phenyl- acetone added t'o biological materials in amounts from 0.5 to 5 pmoles are recovered with adequate precision 90 f 6 per cent. Preparation of Tissue Xamples-The preparation of all tissue samples was carried out at O-3". Male albino rabbits were stunned and exsanguinated. The livers were immediately removed and homogenized with 2 volumes of 0.1 M phosphate buffer, pH 7, with a Potter-Elvehjem type of homogenizer. The homog- enates were centrifuged at 9000 X g for 10 minutes to remove unbroken cells, nuclei, and mit,ochondria, and the resulting supernatant fraction was dialyzed against 0.01 M phosphate buffer, pH 7.0, for 20 hours. The super- natant fraction could be stored for &t least 2 weeks at -10" without loss of act'ivity. The particulat,e fractions of liver were prepared by differential cent@fu- gation of 12 per cent homogenates in 0.25 M sucrose (8). The nuclear fraction was sedimented by centrifugation at 600 X g for 10 minut'es; the mitochondria at 9000 X g for 10 minutes; microsomes were separated from t,he soluble supernatant fract,ion by centrifugation at 78,000 X g for -2.5 minutes. The microsomes were washed once with 0.25 M sucrose and re- centrifuged. ParGculate fractions were resuspended in a 0.1 M phosphat,e buffer, pH 7.0, t,o a concent,ration 3 t,imes that of t'he original homogenat,e. The particulate and soluble supernatant fractions were dialyzed against 0.01 M phosphate buffer, pH 7.0, for 20 hours. J. AXELROD 755 Measurement of Enzyme Activity-A typical incubation mixture was pre- pared as follows: In a 20 ml. beaker were placed 0.3 ml. of enzyme prep- aration (equivalent to 100 mg. of liver), 5 rmoles of nicotinamide, 0.1 pmole of TPN, 5 pmoles of MgC12, 0.6 pmole of Z-amphetamine, 0.5 ml. of phosphate buffer pH 7.4 (0.2 M), and water to make a final volume of 4 ml. The mixture was incubated in a Dubnoff metabolic shaking apparatus for 2 hours at 37" in air. At the end of the incubation period an aliquot of the reaction mixture was immediately transferred to a 60 ml. glass-stop- pered bottle containing 0.5 ml. of 1 N NaOH and assayed for amphetamine. Enzyme act'ivity was expressed as micromoles of amphetamine metab- olized. After the incubation the residual amphetamine was identified by com- paring its distribution ratios between buffers of various pH values and benzene with those of authentic amphetamine. The results indicated that the apparent amphetamine measured after incubation had the same solu- bility characteristics as authentic amphetamine and that the compounds were presumably the same. Results Tissue Distribution of Enzyme Activity-l-Amphetamine (0.6 pmole) was incubated with 100 mg. of minced liver, lung, diaphragm, muscle, kidney, and brain of the rabbit under the conditions described under "Methods." The liver metabolized 0.35 pmole of Z-amphetamine in 2 hours, while the other tissues were unable to metabolize the drug. Properties of Enzyme System-An undialyzed supernatant fraction of rabbit liver failed to metabolize Z-amphetamine in appreciable amounts without the addition of nicotinamide and either TPN or DPN. Nicotin- amide presumably served to protect the pyridine nucleotides against enzymatic destruction (9). The effect of the two pyridine nucleotides was then examined in a supernatant fraction of rabbit liver dialyzed for 20 hours. It was found that the activity of the dialyzed supernatant fraction was restored after the addition of TPN, while DPN was inactive (Fig. 1). Mg++ stimulated activity about 10 per cent. Negligible activity was observed under anaerobic conditions, but incubation in air was as effective as incubation in oxygen. In phosphate buffers the maximal en- zyme activity occurred at pH 7.4, and the activity was sharply reduced below and above this pH. Rate of Metabolism-The rate of metabolism of Z-amphetamine by the rabbit supernatant fraction is shown in Fig. 2. About 50 per cent of the drug was metabolized in 3 hours, with no further metabolism after this time. To examine the lability of the enzyme, the rabbit liver supernatant fraction was preincubated at 37" for 3 hours. On the addition of Z-am- phetamine, no metabolism of the drug took place. 756 ENZYMATIC DEAMINATION OF AMPHETAMINE The relationship between substrate concenkation and substrate dis- appearance is shown in Table I. At concent,rations of amphetamine vary- ing from 0.6 to 2.4 pmoles a constant amount of the drug was metabolized. Intracellular Localization of Enzyme System-Nuclear, mkochondrial, microsomal, and soluble supcrnatant fractions of t'he rabbit liver were sep- arated by differeutial cerkifugation (8) and assayed for their capacity to metabolize Z-amphetamine. Negligible enzyme activity was observed in each cellular fraction (Table II). However, when t,he microsomal and soluble supernatant fractions were combined, the drug was met,abolized o DPN I I I I I ,uM TPN OR DPN ADDED FIG. 1 5 Y I 1 I I 3. 0 I 2 3 4 5 TIME IN HOURS FIG. 2 FIG. 1. Requirement for TPN. 0.3 ml. of dialyzed rabbit liver supernatant frsc- tion was incubated for 2 hours at 37" with 0.6 pmole of Z-amphetamine, varying amounts of TPN or DPN, and cofactors described under "Methods." FIG. 2. Rate of metabolism of Z-amphetamine. Six beakers, each containing 0.3 ml. of rabbit liver supcrnatant fraction, were incubated at 37" with 0.6 pmole of l-amphetamine and cofactors described under "Methods." almost as effectively as by t#he whole homogenate. From these observa- tions it was evident that factors present in both the microsomes and t'he soluble supernatant fraction were required to carry out the metabolism of amphetamine. Requirement for TPNH-The possible r81e of the soluble supernatant fraction in the metabolism of amphetamine was examined by add&g a number of substrat,es normally present in this fraction to the microsomes. It was found that by replacing t#he soluble supernatant fluid with TPY and either glucose-6-phosphate or isocitric acid, unwashed microsomes metab- olized amphetamine; washed microsomes in t,he presence of these factors were unable to metabolize t'he drug. These observations suggested that TPN-dependent dehydrogenases associated with the unwashed micro- somes were involved in the enzymatic conversion of amphetamine. In J. AXELROD 757 the presence of glucose-Bphosphate, glucose-6-phosphate dehydrogenase, and TPN, washed microsomes metabolized amphetamine (Table III). These results suggested that reduced TPN was the actual cofactor and that TABLE I Relationship between Substrate Concentration and Substrate Disappearance 0.3 ml. of rabbit liver supernatant fraction was incubated at 37" for 2 hours with various amounts of I-amphetamine and cofactors described under "Methods." I-Amphetamine added l-Amphetamine metabolized ptt!OlL?S rmole 0.12 0.12 0.30 0.20 0.60 0.25 1.20 0.24 2.40 0.22 TABLE II Intracellular Distribution of Amphetamine Deamination Activity Various intracellular fractions were prepared from rabbit liver by differential centrifugation as described under "Methods." Aliquots of the various fractions equivalent to that contained in 100 mg. of whole rabbit liver were incubated at 37" for 2 hours with 0.6 rmole of Z-amphetamine and cofactors described under "Meth- ods." Intracellular fraction l-Amphetamine metabolized Whole homogenate ...................................... Nuclei* ................................................. Mitochondria ........................................... Microsomes ............................................. Soluble supernatant ..................................... Nuclei* and soluble supernatant ......................... Mitochondria and soluble supernatant. .................. Microsomes and soluble supernatant ..................... pmole 0.27 0.06 0.00 0.03 0.00 0.08 0.02 0.20 * This fraction contained unbroken cells and red blood cells as well as nuclei. TPN-dependent dehydrogenases in the soluble fraction of the cell served bo generate this cofactor. To confirm this, the effect of chemically pre- pared TPNH on the metabolism of amphetamine by microsomes was ex- amined. Reduced TPN was found to be as effective as the supernatant fraction in promoting the metabolism of amphetamine by the washed microsomal fraction of rabbit liver (Table III). DPNH could not replace TPNH. From these results, it appears that the supernatant fraction 758 ENZYMATIC DEAMINATION OF AMPHETAMINE served to maintain a reservoir of reduced TPN by catalyzing the oxidation of glucoseB-phosphate and other substrates and that the deamination of amphetamine was mediated by a TPNH-dependent enzyme located in the microsomal fraction of the rabbit liver. Amphetamine Disappearance and Phenylacetone Formation-Supernatant fluid obtained from 20 gm. of rabbit liver was incubated at 37" for 1 hour with 100 pmoles of nicotinamide, 10 pmoles of TPN, 100 pmoles of MgC12, 120 rmoles of l-amphetamine, 10 ml. of phosphate buffer, pH 7 (0.2 M), and examined for the presence of phenylacetone, the deaminated metabo- lite of amphetamine. Apparent phenylacetone formed in the enzymatic TABLE III Requirement for Redwed TPN 0.3 ml. of liver microsomal fraction, 0.6 pmole of l-amphetamine, 5 rmoles of MgC12, 5 pmoles of nicotinamide, 0.5 ml. of 0.2 M phosphate buffer, pH 7.4, added cofactors, and water to make a final volume of 4 ml. were incubated at 37" for 2 hours. Cofactors added I-Amphetamine metabolized pmole TPN 1 Nmole*. " 0.1 rmole, glucose-6-phosphate 5 Hmoles.. " 0.1 " `I 5 `I glucose-6- phosphate dehydrogenase 1 mg.. . . . TPNH 1 pmole*. .: DPNHl " Soluble supernatant from 100 mg. of rabbit liver + TPN 0.1 pmole 0.01 0.03 0.25 0.26 0.03 0.26 * These cofactors were added in five divided portions over a period of 90 minutes. deamination of amphetamine was extracted from the incubation mixture with carbon tetrachloride and treated with 2,4-dinitrophenylhydrazine as described under "Methods." The absorption spectra of the hydrazone of the enzymatically formed phenylacetone and that of the hydrazone of an authentic sample of phenylacetone were identical. Further evidence for the identity of `the hydrazone of phenylacetone was obtained by ascending paper chromatography with water, t-butanol, and n-butanol (6:6:5$ as the solvent system. The apparent and aut,hentic hydrazones of phenyl- acetone were found to have the same RF (0.70). The quantity of phenylacetone in the enzymatic deamination of Z-am- phetamine was equivalent to only about one-half that of the Z-ampheta- mine metabolized (Table IV). However, phenylacetone incubated wit,h rabbit microsomes and TPNH was also metabolized. On incubation of 0.5, 1, and 2 rmoles of phenylacetone with rabbit liver microsomes, about J. AXELROD 759 the same perc'entage of the phenylacetone disappeared. It was possible, therefore, to make an approximate correction for the metabolism of phenyl- acetone formed in the enzymatic deamination of Z-amphetamine. The re- TABLE IV Enzymatic Deamination of l-Amphetamine to Phenylacetone Each beaker containing 1.5 ml. of rabbit liver microsomal fraction, 25 /*moles of MgC12, 25 pmoles of nicotinamide, 2 ml. of phosphate buffer, pH 7.4 (0.2 M), added cofactors, substrates, and water to make a final volume of 10 ml. was incubated at 37" for 2 hours. Experiments 1 and IA contained 3 pmoles of TPNH added in five divided portions over a period of 90 minutes. Experiments 2 and 2A contained 10 pmoles of glucose&phosphate, 1 mg. of glucose-6-phosphate dehydrogenase, and 0.3 Mmole of TPN. Experi- ment No. Substrate added 1 Z-Amphetamine 4 rmoles 1A Phenylacetone 1 .O pmole 2 Z-Amphetamine 4 pmoles 2A Phenylacetone 1 .O pmole Lmphetamim metabolized ,umolcs 1.26 1.10 Calculated Phenylace- Phenyl- anmunt of tone found acetone metabolized phenyl- afz% /mole 1 *er cent 1 #moles 0.63 1.21 48 0.66 1.02 35 M ICROSOMES - 0 2 4 6 8 IO RATIO OF RAT MIGROSOMES TO RABBIT MIGROSOMES FIQ. 3. Inhibition and activation of amphetamine-deaminating activity of rab- bit liver microsomes by rzt liver microsomes. A, each beaker contained 0.6 pmole of Z-amphetamine, 0.1 pmole of TPN, 5 pmoles of MgCL, 5 pmoles of nicotinamide, 0.5 ml. of phosphate buffer, pH 7.4 (0.2 M), microsomal fraction and soluble super- natant fraction obtained from 100 mg. of rabbit liver, and varying amounts of micro- somes obtained from rat liver. The reaction mixture was incubated at 37" for 2 hours. B, same as A, but rat microsomes were preheated for 2 minutes in a boiling water bath. 760 ENZYMATIC DEhMINATION OF AMPHETAMINE suits shown in Table IV indicate that about 1 mole of phenylacetone was formed for each mole of amphetamine metabolized. From 50 to 100 per cent of the theoretical amount of ammonia was found to be liberated in the enzymatic deaminat'ion of amphetamine, but, TABLE V Substrate Specificity 0.3 ml. of rabbit liver supernatant fraction was incubated at 37" for 2 hours with 0.6 pmole of substrate and cofactors as described under "Methods." The reaction mixture was examined for the amount of substrate remaining. In the absence of TPN none of the substrates were metabolized. Substrate Relative activity l-Amphetamine ....................................... d-Amphetamine .................................... Z-p-Hydroxyamphetamine. .......................... Z-Ephedrine ..................................... d-Ephedrine .......................................... l-Norephedrine., ................................... dl-Methylamphetamine .............................. dl-1 -Phenyl-1 -aminopropane. ....................... dl-1-Phenyl-3-aminobutane. ..................... Phenylethylamine*. .............................. dl-a-Phenylethylamine*. .............................. Tyramine* ........................................... Beneylamine* .................. ....................... 2-Aminoheptane ...................................... Isoamylamine* .................................... per cent 100 27 14 150 11 70 77 40 70 30 27 0 0 20 0 * The supernatant fluid was preincubated at 37" for 10 minutes with 1OV M iso- propylisonicotinylhydraaine before the addition of substrates metabolized by mono- amine oxidase. At this concentration isopropylisonicotinylhydrazine completely inhibits monoamine oxidase activity (10) without effecting amphetamine-deaminat- ing activity. owing to the relatively large amounts of endogenous ammonia also formed by the enzyme preparations, an accurate balance study could not be made. Enzyme Activity in Various Species-The activity of the amphetafiine- deaminating enzyme was examined in dog, guinea pig, and rat liver super- natant fraction. Only small amounts of enzyme activity were found in these species compared to t.he rabbit. The possibility was entertained that the low degree of deaminat,ion activity resulted from inhibitory factors in microsomes. This was examined by measuring the effect of microsomes of dog, guinea pig, and rat, liver on the enzyme activity of rabbit micro- somes. The activity of bhe rabbih liver microsomes was markedly de- J. AXELROD 761 pressed in the presence of the microsomes of these species. On the other hand, the rabbit microsomal enzyme was not inhibited by the soluble supernatant fractions of dog, guinea pig, or rat liver. These observations indicated that an inhibitory factor was present in the microsomes of dog, guinea pig, and rat liver. Fig. 3, A shows the inhibitory action of rat microsomes on the amphetamine-deaminating activity of rabbit' liver microsomes. When the microsomal fraction of rat liver was heated at 100" for 2 minutes prior to its add&ion to the rabbit enzyme, a marked stimulation of enzyme activity was observed (Fig. 3, B). These results indicate the presence of a heatstable activating factor which is masked by a heat-labile inhibitory factor. Preheated microsomes of dog, guinea pig, and rabbit liver also possessed stimulatory activity. Substrate Speci,f&-The metabolism of a number of amines by rabbit liver supernatant fraction is recorded in Table V. Substrates having a phenylpropylamine or phenylbutylamine structure were ext'ensively me- tabolized and the enzyme preparation showed relative specificity for the levo isomers. None of the amines were metabolized in the absence of TPN. It appeared that phenolic-substituted amines, phenylethylamines, and aliphatic amines were metabolized slightly or not at all. It was pos- sible that the arylamines could also be metabolized by hydroxylation of the aromatic nucleus. However, it seemed unlikely that the phenyliso- propylamines were hydroxylated in the rabbit, since it was demonstrated that this species could not hydroxylate these amines in vivo (2, 5). It seemed likely that the rabbit liver microsomal enzyme system metabolized the amphetamine analogues by deamination. DISCUSSION On the basis of the studies described in this paper, the over-all reaction for the enzymatic deamination of amphetamine is represented in the ac- companying scheme. Amphetamine is deaminated to phenylacetone and ammonia in the presence of reduced TPN and oxygen by an enzyme sys- tem which is localized in the microsomal fraction of t,he rabbit liver. A reservoir of reduced coenzyme is maintained by TPN-dependent dehydro- genases and their oxidizable substrates present in the soluble supernatant fraction. Hz H H2 L-CH I IjI a 02, C-C-CH, + NH, I II NH, TI'KH 0 762 ENZYMATIC DEAMINATION OF AMPHETAMINE The enzyme which deaminates amphetamine differs from other deami- nating enzymes such as monoamine oxidase, u-amino acid oxidase, L-amino acid oxidase, and glutamic a,cid dehydrogenase with respect to its sub- strate specificity, cellular localization, and cofactor requirements. The r81e of reduced TPN in an enzyme system catalyzing the oxidative deamination of an amine is not understood. Reduced TPN could con- ceivably act by the generation of hydrogen peroxide through the transfer of it,s hydrogen by an intermediate electron transport system to molecular oxygen. Preliminary studies indicate that hydrogen peroxide generated from n-amino acid oxidase was unable to replace TPNH in t'he deamina- tion of amphetamine by rabbit liver microsomes. The activating action of heated rat microsomes suggests the requirement for other factors be- sides TPNH. The nature of the additional cofactor is unknown. Previous studies have shown considerable species differences in the me- tabolism of amphet'amine in Co (2). The dog and the rat transformed amphetamine mainly by hydroxylat8ion, while t'he rabbit metabolized the drug, presumably by deamination. The species differences in the metab- olism of amphetamine may be explained in part by the presence of inhibi- tory factors in the dog and rat. Inhibitory factors in these species may act by blocking the deamination of amphetamine so that the drug is excreted unchanged or metabolized by alternat'ive pathways involving hydroxylation. It is becoming increasingly evident that enzymes in liver microsomes which have a specific requirement for reduced TPN and oxygen are of major importance in the detoxification of many drugs and foreign organic compounds. Such enzyme systems have been found to carry out a variety of react,ions such as dealkylation of alkylamines (11, 12), side chain oxida- tion of barbiturates,' cleavage of aromatic ethers2 and hydroxylation of aromatic compounds (13). Another property common to these enzyme systems is their inhibition by P-diethylaminoethyl diphenylpropylacetate (l-1, 15). SUMMARY An enzyme system in rabbit liver microsomes catalyzes the deamigation of amphetamine to yield phenylacetone and ammonia. The enzyme sys- tem requires reduced triphosphopyridine nucleotide and oxygen. The TPN-dependent dehydrogenases in the soluble supernatant fraction of the liver serve to maintain a reservoir of reduced triphosphopyridine nucleo- tide. Species differences in the metabolism of amphetamine may be explained, in part, by the presence of inhibitory fact,ors in the microsomes of the dog 1 Cooper, J. R., and Brodie, B. B., unpublished findings. 2 Axelrod, J., unpublished work. J. AXELROD 763 and rat liver. A heat-stable factor which can stimulate the enzymatic deamination of amphetamine is also present in the microsomes. BIBLIOGRAPHY 1. Axelrod, J., J. Pharmacol. and Exp. Therap., 110, 2 (1954). 2. Axelrod, J., J. Pharmacol. and Ezp. Therap., 110, 315 (1954). 3. Kaplan, N. O., Colowick, S. P., and Neufeld, E. F., J. Biol. Chem., 196, 107 (1952). 4. Brodie, B. B., and Udenfriend, S., J. Biol. Chem., 168, 705 (1945). 5. Axelrod, J., J. Pharmaeol. and Exp. Therap., 199, 62 (1953). 6. Udenfriend, S., and Cooper, J. R., J. Biol. Chem., 196, 227 (1952). 7. Conway, E. J., Micro-diffusion analysis and volumetric error, London, 2nd edi- tion (1947). 8. Schneider, W. C., J. Biol. Chem., 176, 259 (1948). 9. Mann, J. J. G., and Quastel, J. H., Biochem. J., 36, 502 (1941). 10. Zeller, E. A., Barsky, J., Fouts, J. R., Kircheimer, W. F., and Van Orden, L. S., Ezperientia, 8, 349 (1952). 11. Axelrod, J., Federation Proc., 13, 332 (1954). 12. La Du, B. N., Gaudette, L., Trousof, N., and Brodie, B. B., J. Biol. Chem., 214, 741 (1955). 13. Mitoma, C., and Udenfriend, S., J. Pharmacol. and Exp. Therap., in press. 14. Axelrod, J., Reichenthal, J., and Brodie, B. B., J. Pharmacol. and Exp. Therap., 112, 49 (1954). 15. Cooper, J. R., Axelrod, J., and Brodie, B. B., J. Pharmacol. and Exp. Therap.. 112, 55 (1954).