Reprinted from PuysiococicaL Reviews Vol. 39, No. 4, 1959 Printed in U.S.A. Metabolism of Epinephrine and Other Sympathomimetic Amines JULIUS AXELROD Laboratory of Clinical Science, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland HE PHYSIOLOGICAL actions of epinephrine and other sympathomimetic | amines are well established, yet the metabolism of these compounds is poorly understood. Although several enzymes have been shown to transform these amines in vitro, the part that they play in the intact organism has been conjectural. In the past few years considerable information concerning the fate of sympatho- mimetic amines has become available. Many new enzymes have been found that are of importance in the transformation of these amines. This review will describe these newer developments in the metabolism and physiological disposition of catecholamine hormones and related compounds of therapeutic interest. METABOLISM OF CATECHOLAMINES When epinephrine or norepinephrine is administered to man (65, 66) and other species (131) only a small percentage of these compounds is excreted un- changed, indicating that they undergo almost complete transformation in the body. Many pathways have been proposed for the metabolism of catecholamines in vive, but until recently, no convincing evidence has been presented showing to what extent, if any, they are involved in the transormation of exogenous or endog- enous catecholamine hormones. In the past, the routes postulated for the metab- olism of these amines included deamination, oxidation and conjugation. Some of these pathways will be dealt with only briefly, since they have been extensively covered in past reviews (23, 27, 30, 84). Oxidation In 1938, Green and Richter (79) demonstrated that cytochrome oxidase can convert epinephrine to adrenochrome. Because of the widespread occurrence of this enzyme, it was assumed that it also metabolizes epinephrine in the body. However, Schayer has shown that the formation of adrenochrome from epi- nephrine in the body is unlikely (136). He observed that after the administration of C-adrenochrome to rats, the urine contained an intensely colored yellow pigment which showed a smear of radioactivity on paper chromatograms, with no distinct peaks (136). After the administration of C'-epinephrine, five radioactive peaks were found (135). This dissimilarity in chromatographic behavior suggested that adrenochrome was not a metabolite of epinephrine. The presence of adrenochrome in the plasma of man was reported by Hoffer (86). Using a specific and sensitive method, Szara et al. failed to confirm this (148). In man, over go per cent of ad- 751 752 JULIUS AXELROD Volume 39 ministered epinephrine can be accounted for by a number of metabolites, none of which have been identified as adrenochrome (102), The dangers of translating in vitro enzyme activity to metabolic events oc- curring in the intact animal might be pointed out at this time. Cytochrome oxi- dase metabolizes catecholamines in vitro more rapidly than any known enzyme (Axelrod, unpublished observations) but in the body it attacks catecholamines to a negligible extent. The question of how closely the results of controlled in vitro reactions parallel reactions in the body depends not only on enzyme activity, but on other competing reactions, localization and degree of binding of substrates, affinity of substrate for the enzyme, naturally occurring inhibitors, and passage of the compound across many membrane barriers to the intracellular site of metab- olism. Imaizumi and co-workers have described an enzyme, epinephrine dehydro- genase, in rabbit plasma that reversibly oxidizes epinephrine to adrenalone, diphosphopyridine nucleotide serving as the hydrogen acceptor (go, g1). These investigators have also reported that after the administration of epinephrine to guinea pigs, compounds were found in the urine that had the same Ry on chroma- tograms as adrenalone, protocatechuic aldehyde, protocatechuic acid and nor- epinephrine (gg). Treatment of guinea pigs with iproniazid resulted in an increased excretion of adrenalone. These findings indicated that the guinea pig not only oxidized epinephrine to adrenalone, but also demethylated it to norepinephrine. Weil-Malherbe and Bone did not succeed in finding the epinephrine dehydro- genase reported above (160). They have looked for this enzyme in rabbit and human plasma, and in extracts of rat liver, heart, spleen and brain. Using 6 H'- epinephrine, no evidence for the formation of adrenalone or norepinephrine has been found (13, 102). Conjugation Richter ingested large amounts of epinephrine, 3, 4-dihydroxyephedrine, and epinine and noted that these catecholamines were excreted as sulfo-conjugates (124, 125). These observations were confirmed in dogs (152). After the oral ad- ministration of epinephrine to rabbits, it was excreted mostly as epinephrine glucosiduronic acid. On the basis of these results, it was concluded that conju- gation constituted an important pathway for the metabolism of catecholamines. According to more recent studies, the intravenous administration of physiological doses of catecholamines led to the excretion of only small amounts of conjugated catecholamines (65, 66, 131). It is now generally believed that conjugation plays a minor role in the inactivation of physiological amounts of catecholamines. In the earlier experiments, the massive amounts of the catecholamines given may have overwhelmed the normal enzymatic mechanisms. Furthermore, after oral ingestion, the intestinal flora may have conjugated these amines before they were absorbed. Deamination by Monoamine Oxidase A number of investigators (31a, 116, 122) have demonstrated that epinephrine serves as a substrate for monoamine oxidase. Since this observation was made, October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 753 monoamine oxidase has been considered by many to be the enzyme chiefly con- cerned with the metabolism of epinephrine and other catecholamines in the intact organism (see refs. 30 and 53 for reviews on this subject). Burn (41) postulated that monoamine oxidase acts at the sympathetic nerve endings in the same manner as acetylcholine esterases in the parasympathetic system. This assumption rested mainly on the fact that monoamine oxidase is present in the autonomic nervous system and that after denervation of the nictitating membrane, iris and blood vessels in cats, a decrease in the enzyme was found in these tissues (43). A correla- tion between the fall in enzyme activity and increased sensitivity to norepi- nephrine was also observed. Armin et al. (3), however, showed that after sectioning of the sympathetic nerves amine oxidase persisted undiminished, while acetyl- choline esterase activity was markedly reduced. They attributed the hypersensi- tivity to epinephrine after denervation to a reduction of acetylcholine esterase rather than amine oxidase. Subsequent work of Burn eé al. (42) did not confirm the previous findings of Burn and Robinson (43) that degeneration of sympathetic nerves results in a fall in monoamine oxidase activity. In contrast to the high localization of acetylcholine esterase in cholinergic fibers there appears to be no selective association of monoamine oxidase with adrenergic nerves (101). The introduction of the potent monoamine oxidase inhibitors iproniazid (165) and choline-p-tolyl ether (40) made possible a reevaluation of the role of monoamine oxidase in the metabolism of catecholamine hormones. In an im- portant experiment Griesemer ef al. (80) found that inhibition of monoamine oxidase in vivo markedly prolonged the contraction of the cat’s nictitating membrane produced by phenylethylamine and tyramine, but no prolongation of the actions of administered epinephrine was observed. Following this report a number of investigators confirmed these findings and reported that the in vivo inhibition of monoamine oxidase had no effect on a yariety of actions of epi- nephrine and norepinephrine (24, 42, 52, 120). Moreover, Kamijo et al. (95) showed that any potentiating action of iproniazid on the effects of adrenergic nerve stimu- lation and of injected epinephrine were unrelated to its ability to inhibit mono- amine oxidase. Exposure of isolated strips of rabbit aorta to iproniazid until mono- amine oxidase was completely inhibited neither potentiated nor depressed the response of strips to epinephrine or norepinephrine, yet in these preparations the response to tyramine was potentiated (72). The administration of mohoamine oxidase inhibitors also increased the excretion of administered tyramine (132), tryptamine (137), and serotonin (52) but had little or no effect on the excretion (52, 71) or rate of disappearance (48, 71) of administered catecholamines. Stimulation of postganglionic sympathetic fibers to the spleen elevated the norepinephrine concentration of the venous blood leaving the spleen, but mono- amine oxidase inhibitors did not affect this increase (39). It was also noted that the adrenergic blocking agents Dibenamine and Dibenzyline increased the nor- epinephrine concentration in venous blood after sympathetic stimulation. As a result of these observations, it was suggested that the receptors inactivate nor- epinephrine, and that adrenergic blocking agents not only antagonize the actions of sympathomimetic amines, but also prevent the receptors from metabolizing 754 JULIUS AXELROD Volume 39 the amines. Von Euler and co-workers (63) found that the administration of monoamine oxidase inhibitors had no effect on the catecholamine content of cat tissues. All these observations strongly suggest that the metabolism of administered as well as endogenous catecholamines is unaffected by the inhibition of mono- amine oxidase. Evidence for the participation of monoamine oxidase in the metabolism of norepinephrine in the central nervous system has been presented by Brodie and co-workers (141, 145). After the administration of iproniazid to rabbits, a threefold increase in brain norepinephrine was noted. In addition, pretreatment with iproniazid completely blocked the metabolism of amines released by reserpine. From these observations, it was proposed that monoamine oxidase is the enzyme chiefly responsible for the transformation of norepinephrine in the nervous system. Carlsson et al. reported that iproniazid treatment elevated dopamine (46) but not norepinephrine (47) in the rabbit brain. Treatment with monoamine oxidase inhibitors did not increase the norepinephrine content in the brain of dogs and cats (63, 144, 156). These conflicting findings might be due to species differences in the metabolism of catecholamines in the central nervous system. Using radioactive epinephrine, Schayer and co-workers demonstrated that monoamine oxidase was involved at some step in the metabolism of epinephrine (136). After the administration to rats of C'-epinephrine labeled on the 8 carbon, almost all of the radioactivity was found in the urine (130), but when N-methyl C"*-epinephrine was injected, only 50 per cent of the radioactivity was excreted (136). However, rats receiving N-methyl C*-epinephrine after treatment with iproniazid excreted almost all of the radioactivity. Similar results were obtained with epinephrine in man (121) and with norepinephrine in rats (134). It was concluded that iproniazid blocks the cleavage between the 8 carbon and methyl carbon, presumably by inhibiting deamination, and that half of the administered catecholamines are metabolized by monoamine oxidase (136). Whether deami- nation occurred on the epinephrine molecule itself, or an amine-containing me- tabolite, was not established. When the urine of rats that received 8 C*-epinephrine was subjected to paper chromatography, five radioactive metabolites retaining the 8 carbon were found (130). Except for epinephrine, none of these compounds was identified. Paper chromatograms of urine of rats to which N-methy] C-epinephrine was given showed only three radioactive metabolites, one of which was epinephrine (135). From these experiments it appeared that epinephrine formed two major metab- olites that retained the methyl amino group and two in which this group was re- moved. METABOLISM OF Dora AND Dopamine IN Vivo The discovery of a heretofore unrecognized pathway for catecholamines stemmed from the observation of MacLaglan and Wilkinson (107), who found that phenolic compounds are O-methylated in the body. DeEds and co-workers also demonstrated that ingested catechol flavanoids were excreted as 3-methoxy- 4-hydroxyphenylacetic acid (homovanillic acid, ref. 33). Following this, Shaw October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 735 et al. (140) and DeEds ef al. (54) independently reported that homovanillic acid and 3,4-dihydroxyphenylacetic acid were metabolites of dihydroxyphenylaianine (dopa). Much smaller amounts of these compounds were formed from p- than from L-dopa (140). Armstrong et al. also identified homovanillic acid as a normal constituent of human and rat urine (5). The latter compound was proposed as a metabolite of endogenous L-dopa. Von Euler ef al. have also detected a compound chromatographically identical with dihydroxyphenylacetic acid in human urine (58). This compound has been reported to be present in plasma, vascular wall (64), splenic nerve (62) and brain (110). In rabbits and rats DeEds ¢¢ al. (54) found that L-dopa is also transformed to m-hydroxyphenylacetic acid. The latter reaction, which involves the removal of a hydroxy! group from an aromatic nucleus, is a new type of metabolic transformation. Shaw et al. reported that dehydrox- ylation of 3,4-dihydroxyphenylacetic acid did not occur to any significant extent in man or rat (140). Many years ago, Holtz et al. showed that dopa is converted to dopamine by the enzyme dopadecarboxylase (88). These investigators, using bioassay techniques, also found that man and animals excreted large amounts of dopamine after the administration of dopa (87), and that dopamine is a normal constituent of human urine. More recently, Pellerin and D’Iorio (114) identified free and conjugated dopamine in the urine when C" dopa was given to rats. In addition, 3, 4-dihydrox- yphenylpyruvic acid, as well as two unidentified metabolites, were also present. Since L-dopa can participate in transamination reactions (44), it appears likely that the pyruvic acid derivative arises from this type of transformation. "i iz 14 CmE=C—0H c- —t_teon HO’ HH ~ H oH a 3,4 ne PYRUVIC ACID a _ | HOW HO iE on ae i 48 — —C—NH “uw —C—oH . i ine a i 2 4 C—C=OH ——» HO HO 5 ° wm a 34 onvoron PHEN oH . y’ YL 3-METHOXYTYROSINE a OHH ACETIC ACID M-HYDROXYPHENYL- C—CNH, ACETIC. ACID C af 4 6 H ran HO tod 1 oi C—C—NH, C—C—OH it NOREPINEPHRINE i LS, HO to HO OCH, OCH; 3-METHOXYTYRAMINE HOMOVANILLIC ACID Fic. 1. Metabolism of dopa. 1; This reaction is presumably catalyzed by a transaminase. 3, 7: Dopadecarboxylase and pyridoxal are required for step 3 and possibly step 7. 2, 6, &: Catechol-O-methy] transferase catalyzes these reactions in the presence of S-adenosylmethionine and a divalent cation. Only reaction 8 has been shown to occur in vivo. 4: This step has never been demonstrated in vivo. 5: This interesting pathway is found in certain species. The enzyme catalyzing this step has not yet been described. 10, 11: These reactions are catalyzed by monoamine oxidase and aldehyde dehydrogenase. 9:The enzyme concerned with this reaction is not well characterized. 756 JULIUS AXELROD Volume 39 According to Shaw et al. (140), deamination precedes O-methylation in the formation of homovanillic acid from dopa. However, after the administration of dopamine to rats, Axelrod et al. found free and conjugated 3-methoxytyramine as well as homovanillic acid in the urine (18). Pretreating the animals with ipro- niazid caused a fivefold increase in the excretion of 3-methoxytyramine (18). These observations indicate that catecholamines are O-methylated prior to deamination, and that 3-methoxytyramine is deaminated to homovanillic acid by monoamine oxidase. Goldstein et al. (77) found that the administration of dopamine to rats results in the excreton of normetanephrine (3-O-methyl norepinephrine). Since dopa is O-methylated in vitro (20), the possibility of the formation of 3-methoxy- tyrosine should also be considered. The routes of metabolism of dopa are summarized in figure 1. O-METHYLATION OF EPINEPHRINE AND NorEPINEPHRINE IN Vivo In a recent study, Armstrong ¢ al. reported that 3-methoxy-4-hydroxy- mandelic acid is a normal constituent of human urine (4). The excretion of this compound was increased after the administration of norepinephrine or 3,4- dihydroxymandelic acid, and in subjects with pheochromocytomas. It was concluded by these investigators that 3-methoxy-4-hydroxymandelic acid is an important metabolic product of norepinephrine and that deamination of nor- epinephrine precedes O-methylation. Axelrod (11) found that metanephrine (3-O-methylepinephrine) and normetanephrine (3-O-methylnorepinephrine), in free and conjugated form, occur normally in rat urine, and that the excretion of these compounds was markedly elevated when epinephrine and norepinephrine were given to rats. These results demonstrated that the catecholamines undergo O-methylation before deamination. The extent to which O-methylation of epinephrine occurs was examined after intraperitoneal injection of physiological amounts of 6 H*-epinephrine to rats (13, 16). After collecting the urine for 24 hours, the O-methylated metabolites were extracted into organic solvents (18). About 87 per cent of the administered radioactivity appeared in the urine, 55 per cent of which was identified as free metanephrine and metanephrine glucosiduronic acid. Twelve per cent of the radioactivity was present as 3-methoxy-4-hydroxymandelic acid and only traces as 3,4 dihydroxymandelic acid. After the administration of largé@ amounts of epinephrine or metanephrine to rats, smaller percentages of metanephrine (free and conjugated) appeared in the urine (16). To study the sequential steps in the biotransformation of epinephrine, meta- nephrine was given to rats and its metabolic fate examined (13, 16). About the same amounts of metanephrine (free and conjugated) and 3-methoxy-4-hydroxy- mandelic acid were excreted after metanephrine as were found after epinephrine, indicating that 3-methoxy-4-hydroxymandelic acid is derived from metanephrine rather than 3,4-dihydroxymandelic acid. When rats were pretreated with ipro- niazid, virtually all the administered epinephrine or metanephrine was excreted as free and conjugated metanephrine. The metanephrine excretion increased at the expense of 3-methoxy-4-hydroxymandelic acid. A major metabolic product found October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 757 after the administration of norepinephrine to rats was normetanephrine (16). Pretreatment with iproniazid resulted in a twofold increase in the excretion of normetanephrine. These observations provided strong evidence that, in rodents, O-methylation is the first step in the metabolism of catecholamines in vivo, and that monoamine oxidase is concerned in the deamination of the O-methyl amines. Normetanephrine and metanephrine were found to occur normally in adrenal gland, spleen (18), pheochromocytoma tumors (142) and urine (18, 103). Normal occurrence of O-methylated amines in tissues rich in epinephrine and norepi- nephrine as well as in urine would indicate that the endogenous catecholamines are also O-methylated. However, the extent to which endogenous catecholamines are O-methylated remains to be established. The presence of large amounts of 3-methoxy-4-hydroxymandelic acid (4) and normetanephrine (103) in the urine of human subjects with pheochromocytomas suggests that O-methylation is an important route of metabolism of catecholamines. in man. After the intravenous infusion of physiological doses of 8 H®-epinephrine, about go per cent of the radioactivity appeared in the urine. Fifty-five per cent of the radioactivity was free and conjugated metanephrine and 30 per cent was 3- methoxy-4-hydroxymandelic acid (102). Kirshner et al. found essentially similar results in man (98). In rats, metanephrine is conjugated with glucuronic acid, while in man it is conjugated presumably with sulfuric acid. An additional me- tabolite, present in small quantities, was tentatively identified as 3 ,4-dihydroxy- mandelic acid (98, 121). When metanephrine was administered to man, the same fraction was excreted as metanephrine and 3-methoxy-4-hydroxymandelic acid as was observed after epinephrine (102), indicating that the acid arises mainly from the deamination of metanephrine. In iproniazid-treated subjects the excretion of metanephrine is increased, while that of 3-methoxy-4-hydroxymandelic acid is decreased (130). Normetanephrine and metanephrine possess relatively weak physiological and psychological activity (68, 102) suggesting that O-methylation of catechol- amines is an inactivation process. In the light of these findings, it can be concluded that the principal pathway for the metabolism of epinephrine and norepinephrine in man and rodents is O-methylation to metanephrine or normetanephrine, the latter amines being in turn either deaminated by monoamine oxidase or conjugated. Direct deamination of these catecholamines appears to be a minor route of transformation. The path- ways for the metabolism of epinephrine and norepinephrine are shown in figure 2. A new metabolic product of epinephrine and norepinephrine, 3-methyoxy- 4-hydroxyphenylglycol, has been identified in this laboratory (Axelrod, Kopin and Mann, unpublished observations). The metabolite arises from the deamination of metanephrine or normetanephrine followed by reduction of the resulting 3-methoxy- 4-hydroxyphenylglycol aldehyde. O-METHYLATION OF CATECHOLAMINES IN ViTRO An enzyme, catechol-O-methyl transferase, that O-methylates epinephrine and other catechols has been found in this laboratory (11, 20). The presence of 758 JULIUS AXELROD Volume 39 i ry mi \ C~E-NHCH / CTECNHCH, 7 C— C=NHCHs — ——____» —_—— HO HoH 9 HO HH RO HH 8 OCH, OCH, METANEPHRINE CONJUGATED META EPINEPHRINE NEPHRINE 2 s | OH O OH O { il { oo C-C—OH , ¢-C-OH 4 ad H HO HO OH OCH; 3,4 DIHYDROXYMANDELIC (3-METHOXY-4. HYDROXY- ACID MANDELIC ACID a mh rt ¢-C—NH, 6 S—E-NHe 8 Ly} —¢—NHa ——— CO Ho HH HO HOH RO HoH OH OCH; OCH, CONJUGATED NORMETA NOREPINEPHRINE NORMETANEPHRINE NEPHRINE Fic. 2. Metabolism of epinephrine and norepinephrine. 7, 6, 10: Catalyzed by catechol- O-methyl transferase, S-adenosylmethionine serving as the methyl donor. Divalent cations are required. 2, 3, 4, 5° These reactions take place in two steps, deamination by monoamine oxidase to form the corresponding aldehyde, followed by oxidation by aldehyde dehydrogenase and diphosphopyridine nucleotide. 7, 4: In may species, conjugation occurs by the transfer of glucuronic acid from uridine diphosphate glucuronic acid by a transferring enzyme in the liver microsomes. g- This reaction has been shown to occur in vitro in certain species. this enzyme has been confirmed by other investigators (75: 105, 115). Incubating the soluble supernatant fraction of rat liver with epinephrine, S-adenosylmethionine and Mg** resulted in the disappearance of one mole of the catecholamine and the appearance of one mole of metanephrine. In the absence of S-adenoslymethionine or Mg+* negligible metabolism occurred (11, 20). These observations demonstrated that the enzyme transfers the methyl group of S-adenosylmethionine to the 3- hydroxy group of epinephrine. Catechol-O-methyl transferase is widely distributed among A variety of species and tissues (table 1) and is localized in the soluble fraction of the cell (14, 20). Of all the tissues examined, enzyme activity is found to be consistently highest in the liver, while the activity in monkey liver is greater than in any other mamma- lian species examined. Particularly noteworthy is the high catechol-O-methyl transferase in such glandular tissues as salivary gland, pancreas and adenohy- pophysis (14). The magnitude of enzyme activity is the same in thyroid gland, spleen, blood vessels and brain in the monkey. Catechol-O-methyl transferase activity is also found in all peripheral nerves (14). The presence of this enzyme in those organs upon which norepinephrine and epinephrine exert their effects sug- gests that it might act locally in the transformation of these hormones. October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 759 Taste 1. DistrisuTION oF CATECHOL-O-METHYL TRANSFERASE CONC. conc, SPECIES TISSUE UNITS* SPECIES TISSUE UNITS* Monkey Liver 1700 Monkey = Aorta 120 Rat Liver goo Monkey Inf. vena cava 80 Cow Liver 430 Monkey Submax. gland 820 Pig Liver 380 Monkey Pancreas 680 Mouse Liver 300 Monkey Adenchypophysis 420 Guinea pig Liver 130 Monkey = Thyroid 115 Man Liver 100 Monkey Brain 105 Rabbit Liver 38 Rat Brain 26 Rat Kidney 260 Monkey Neurohypophysis 190 Rat Spleen 63 Monkey Sup. cerv. gangl. 130 Rat Sm. intestine 40 Monkey Vagus nerve 128 Rat Lung 40 Monkey Saphenous nerve 88 Rat Skel. muscle o Monkey = Splanchnic nerve 87 Rat Heart muscle 25 Monkey Sciatic nerve 80 Monkey — Sacral sympath. nerve 65 Monkey = Splenic nerve 60 * 1 unit = wg metanephrine formed from epinephrine in 1 hr. per gm tissue (values obtained from refs. 14, 20). Catechol transferase was purified about 30-fold from rat liver by procedures involving ammonium sulfate precipitation and adsorption and elution on calcium phosphate gel (20). The purified enzyme has an absolute requirement for S- adenosylmethionine and Mg. However, a number of divalent cations such as Cott, Mnt+, Cd, Fett and Nit could be substituted for Mgt. All catechols, regardless of the substituents on the aromatic nucleus, were O-methylated. The enzyme showed no stereospecificity toward the p or the L isomers of epinephrine, nor did it O-methylate monophenols. Among the catechols methylated were normally occurring compounds such as norepinephrine, dopamine, 3, 4-dihydrox- yphenylalanine, 3,4-dihydroxymandelic acid and dihydroxyphenylacetic acid, as well as many synthetic catechols (20). The optimal pH for enzyme activity ranged from 7.5 to 8.2. In the presence of p-chloromercuric benzoate and iodo- acetic acid, O-methylation was inhibited (20), pointing to a sulfhydryl group as the active site on the enzyme surface. Pyrogallol and other catechols were found to block the O-methylation of catecholamines in vitro (13a). Pretreating mice with a catechol-O-methyl transferase inhibitor (pyrogallol) markedly slowed the me- tabolism of epinephrine. A monoamine oxidase inhibitor (iproniazid), on the other hand, did not affect the rate of disappearance of epinephrine or norepinephrine. The specificity of the O-methyl transferase with respect to catechols and the requirement for divalent cations suggests a role for the cation (20). The metal could link the adjacent hydroxy groups of the catechol to the enzyme surface, O-methylation would then proceed by a nucleophilic attack of the hydroxy group by the electrophilic methyl carbon of S-adenosylmethionine. Since the 3-hydroxy group is strongly nucleophilic, the transfer takes place on the 3 position. Further support of such a mechanism is provided by the finding that catechols having a 4760 JULIUS AXELROD Volume 39 strongly nucleophilic group on position 4 are O-methylated on this position by the enzyme (139a). METABOLISM OF METANEPHRINE AND OTHER O-METHYLATED AMINES The O-methylated metabolites of catecholamines undergo further metabolic change in vivo and in vitro. The administration of metanephrine results in the excretion of 3-methoxy-4-hydroxymandelic acid (13, 102), When epinephrine, norepinephrine, dopamine, metanephrine or normetanephrine were given to. animals in which monoamine oxidase was inhibited, the excretion of the O-methy]- ated amines was increased at the expense of O-methylated acids (13, 16, 18). Consequently, deamination and oxidation of the O-methylated amines constitute a pathway in the metabolism of these compounds in vive, O-methylated amines, metanephrine, normetanephrine and 3-methoxytyramine also serve as substrates. for monoamine oxidase preparations (11, 12, 105). In addition, deaminated products can be oxidized to their corresponding acids by aldehyde dehydrogenase (11, 12, 105). Normetanephrine was found to be a better substrate for monoamine oxidase than either epinephrine or norepinephrine (105). An enzyme has been found in the microsomes of rabbit liver that can O- demethylate metanephrine to epinephrine (19). The enzyme requires reduced triphosphopyridine nucleotide and appears to be similar to other O-demethylating enzymes (10). A major route of metabolism of O-methylated amines in some animal species involves conjugation with glucuronic acid on the hydroxy group. The synthesis of glucuronides has been shown to be catalyzed by an enzyme system in the micro- somes of liver, and requires uridine diphosphate glucuronic acid as the glucuronide donor (55, 92). Incubation of metanephrine or normetanephrine with microsomes of rat liver and uridine diphosphate glucuronic acid resulted in the formation of the corresponding glucosiduronic acid of these amines (unpublished). FORMATION AND METABOLISM OF CATECHOLAMINES IN NERVOUS SYSTEM The presence and role of norepinephrine in the autonomic nervous system has been well established (61). Although norepinephrine has been shown to be highly localized in certain regions of the central nervous system (155), the functions of this catecholamine in the brain are not clear. In the sympathetic nervous system norepinephrine presumably arises from dopa via dopamine. Holtz and Wester- mark (89) have found dopa decarboxylase activity to be high in postganglionic sympathetic nerves, sympathetic ganglia and in the sympathetic trunk and brain stem. Furthermore, Schitimann has shown that sympathetic nerves contain do- pamine in relatively high concentrations (138). Both dopadecarboxylase and its metabolite, dopamine, are highly localized in the cytoplasm of the nerves, while norepinephrine is found in the particulate fraction of the nerve cell (139). Recently Goodall and Kirshner (78) reported that the incubation of C" dopa with sym- pathetic nerves and ganglia led to the formation of radioactive dopamine and norepinephrine. Negligible amounts of radioactive epinephrine were found. The October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 461 vagus nerve, on the other hand, formed no significant amounts of radioactive norepinephrine and only small amounts of radioactive hydroxytyramine. Using paper chromatographic techniques, Schitimann found norepinephrine and do- pamine, but no epinephrine, in the splenic nerve and spleen (139). On the basis of these observations, this investigator postulates that norepinephrine is the only transmitter substance at the sympathetic nerve endings. The concentration of H*-epinephrine was determined in various regions of the cat brain after its intravenous infusion (159). It did not exceed that expected from the blood concentration of the tissues, except in the hypothalamus, where small amounts of H*-epinephrine were found (159). It was concluded from these experiments that epinephrine is unable to cross the blood-brain barrier, except to a small extent in the hypothalamus (159). Since epinephrine is present in the brain (155), it must be formed from precursors which are able to cross the blood- brain barrier. It is probable that norepinephrine in the brain is formed from dopa, a compound that can cross the blood-brain barrier (46). Both dopa (110) and dopamine (46, 110) were found to occur normally in the brain of a number of mammalian species. Moreover, the intravenous injection of dopa to rabbits caused a considerable rise in the dopamine content of the brain (46). Pretreatment with iproniazid markedly enhanced the dopamine levels, but had little or no effect on norepinephrine levels in the brain (47). The presence of the deaminated product of dopamine, dihydroxyphenylacetic acid, in the splenic nerve (62) and brain (110) has also been reported. These observations would suggest that in the nervous system dopamine is readily attacked by monoamine oxidase. The manner in which the actions of the catecholamines in the nervous system are terminated is poorly understood. For many years, monoamine oxidase has been considered to be the principal enzyme involved in the inactivation of norepi- nephrine and epinephrine. However, since the introduction of monoamine oxidase inhibitors, considerable doubt has been cast upon the role of this enzyme in the metabolism of epinephrine and norepinephrine. (See above for a review of this subject.) Recently, O-methylation has been shown to be the principal pathway for the metabolism of administered epinephrine and norepinephrine (16, 102). Nor- metanephrine has been found to be present in organs rich in sympathetic fibers (18) and in the brain extract of rats treated with iproniazid (12). When ‘nor- metanephrine was incubated with brain mitochondria and aldehyde dehydro- genase, it was oxidatively deaminated to 3-methoxy-4-hydroxymandelic acid (12). This indicates that the endogenous norepinephrine could be O-methylated in the nervous system and then further metabolized. The enzyme, catechol-O-methyl transferase, which catalyzes the first reaction, has been found to be present through- out the nervous system, including the brain, sympathetic ganglia and postgan- glionic sympathetic fibers, as well as parasympathetic, motor and sensory nerves (table 1 and ref. 14). Within the central nervous system differences of enzyme activity ranging up to fourfold were found (14). The lowest values occurred in the cerebral cortex and amygdala, and the highest in the neurohypophysis. There 762 JULIUS AXELROD Volume 99 was no direct relationship between catechol-O-methy] transferase activity found in various regions of the monkey brain and norepinephrine levels reported in different areas in the dog brain. The Michaelis constant (Km) of catechol-O-methy] trans- ferase with respect to brain epinephrine is less than 10-5 m, which places it in the physiological range of tissue catecholamine concentrations (14). Although direct evidence of the main pathway of metabolism of the catecholamines in the nervous system is lacking, it appears likely that catechol-O-methy] transferase is an im- portant enzyme. S-adenosylmethionine, the donor of active methyl groups (45), is assuming considerable importance in the metabolism of biologically active amines. The cofactor is involved in the O-methylation of catecholamines (11), the N-methyl- ation of norepinephrine to epinephrine (97), and more recently it has been shown to be required for the conversion of histamine to methy] histamine (38). The latter reaction is of paramount importance in the inactivation of histamine (133). The ability of the nervous system to synthesize S-adenosylmethionine from adenosine triphosphate and methionine has been established (14). PHYSIOLOGICAL DisPosiITION OF EPINEPHRINE There is considerable information available concerning the localization of endogenous catecholamines (61). However, relatively little is known about the disposition of these hormones when they are released from the adrenal medulla and sympathetic nerve endings, or when administered intravenously. Raab and Gigee (117, 118) reported that epinephrine and norepinephrine were selectively taken up by heart muscle and other vascular tissues when massive doses of these compounds were administered to cats or dogs, but there was no demonstrable increase in the catecholamine content with doses below 2 milligrams per kilogram. Von Euler (60), on the other hand, did not find any significant changes in the catecholamine content of heart, spleen, liver, kidney or skeletal muscle of the cat when large or small amounts of catecholamines were given. These contradictory results might be due to the lack of precision and specificity of the methods em- ployed. More recently, the physiological disposition of epinephrine and its me- labolite, metanephrine, was examined in cats and mice after an intravenous in- fusion of physiological amounts of tritium-labeled 8 pt H*-epinephrine of high specific activity (22). The amines were separately determined by speeific procedures involving column chromatography and extraction in organic solvents. Immedi- ately after the end of a 30-minute infusion (3 yg/min/kg) the concentration of H*-epinephrine in heart, spleen, adrenal and pituitary gland exceeded that of the plasma several-fold. In kidney, liver, lung and intestines, its concentration was of the same magnitude as that of the plasma, while it was lower in skeletal muscle. After an intravenous infusion of radioactive epinephrine, Weil-Malherbe et ai. (159) found negligible amounts of radioactive epinephrine in the brain of the cat, while Schaepdryver (129) reported significant amounts of the radioactive cate- cholamine in the brain of the dog. Two hours after the administration of H- epinephrine, large quantities were found in heart and spleen, indicating that October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 763 these tissues not only accumulate the hormone, but also retain it for long periods of time. , Immediately after H*-epinephrine infusion, the concentration of meta- nephrine in plasma was about the same as that of epinephrine (22). The O-meth- ylated metabolite accumulated in heart, spleen, adrenal gland and liver, but in- significant amounts were found in the brain, indicating that metanephrine crosses the blood-brain barrier with difficulty. After the intravenous injection of epinephrine in rabbits and dogs, Pekkarinen (113) and Lund (106) reported that the amine was completely eliminated from the blood within 10 minutes. Norepinephrine disappeared from the plasma even more rapidly than epinephrine (106). An intravenous injection of H*-epinephrine was followed by a precipitous fall in the plasma levels of the amine within 5 minutes (22a). In contrast to the earlier findings, however, small but significant amounts of epinephrine were present in the plasma for more than 2 hours (22a, 129). Within 2 minutes after the intravenous injection of H*-epinephrine, its metabolite, metanephrine, reached a peak level in the plasma, with approximately the same or higher concentrations as that of epinephrine (25). Only small amounts of 3- methoxy-4-hydroxymandelic acid and 3,4-dihydroxymandelic acid (less than one-tenth that of metanephrine) were present in the plasma at that time (22a). These observations indicate that during the period of physiological inactivation of epinephrine, catechol-O-methyl transferase is the predominating enzyme. The disappearance of the metanephrine from the plasma then closely paralleled that of epinephrine (fig. 3). The initial drop in the plasma level was presumably due to passage into the tissues, as well as metabolic transformation. The dynamic factors involved in the elimination of epinephrine in the intact organism were examined in the mouse (22a). After the intravenous injection of 3 i) 200 @—* EPINEPHRINE 100 Onn =O METANEPHRINE 80 60 40 Fic. 3. Plasma levels of epinephrine and metanephrine after intravenous injection of 70 pg/kg epinephrine to a cat (22). i 20 Lit iit MICROGRAMS/LITER Nuh 8 WO ' MINUTES 4 0 30 60 90 120 764 JULIUS AXELROD Volume 39 micrograms of H?-epinephrine to mice, the catecholamine and its metabolite were determined in the whole animal at various time intervals. Within 10 minutes, more than half of the epinephrine disappeared. After 10 minutes, the amine disappeared much more slowly; more than ro per cent of the administered epinephrine was still found in the mouse after 3 hours. The rate of elimination of epinephrine in the whole mouse was independent of the amount of epinephrine administered over a range of 1-10 micrograms of injected epinephrine per mouse. In the initial phase, 1 mole of metanephrine appeared in the whole mouse for every mole of epineph- rine eliminated. These observations, together with those described above, suggest the following sequence of events in the elimination of circulating epinephrine: first phase (0-5 minutes), rapid dilution by redistribution into tissues, O-methylation by catechol-O-methyl transferase and binding onto certain tissues; second phase (5-180 minutes or longer), slow release of epinephrine from binding sites, followed by attack by catechol-O-methyl transferase. METABOLISM OF PHENYLETHYLAMINES Studies on the metabolism of amines with a phenylethylamine nucleus were initiated in 1910 by Ewins and Laidlow (69) who found that the administration of phenylethylamine and tyramine to dogs, cats and rabbits resulted in the ex- cretion of large amounts of phenylacetic acid and p-hydroxyphenylacetic acid, respectively. Guggenheim and Léffler confirmed these findings in rats and mice (81). More recently, Schayer reported that about go per cent of C tyramine was excreted as free and conjugated p-hydroxyphenylacetic acid and ro per cent as free and conjugated tyramine (132). These observations suggested the presence of an enzyme that could deaminate these amines in vivo. In 1928 Hare-Bernheim demonstrated an enzyme that catalyzes the oxidative deamination of tyramine to yield p-hydroxybenzaldehyde, peroxide and ammonia (82). The aldehyde is further oxidized to the corresponding carboxylic acid by aldehyde dehydrogenase and diphosphorpyidine nucleotide (161). It was subsequently observed that this enzyme, monoamine oxidase, deami- nates many alkyl and aromatic amines, including epinephrine and norepinephrine, to their corresponding aldehydes (31a, 116, 122). The substrate specificity of monoamine oxidase has been studied extensively, since so many amines have marked biological activity and are of therapeutic interest (see ref. $o for an ex- tensive review of the subject). Jn vitro, monoamine oxidase acts on amines which have an amine group attached to a terminal carbon atom, e.g. compounds of the type R—CH:—NH2. Aromatic amines such as aniline, in which the amine is attached to the aromatic ring, are not deaminated, but benzylamine and a- phenylethylamine are metabolized. Secondary amines are deaminated as rapidly as the corresponding primary amines. Introduction of N-methyl groups reduces the rate considerably, but does not abolish the reaction. Hordinine (HOC;H,CH,CH2N(CH$):) is a naturally occurring tertiary amine which is slowly oxidized by amine oxidase. Phenylamines in which the amino group is not attached to a terminal carbon atom are not deaminated by monoamine oxidase, October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 765 e. g. ephedrine and amphetamine. Not only are the phenylisopropylamines stable with respect to monoamine oxidase, but they also inhibit this enzyme in vitro (73). Gaddum and Kwiatkowski postulated that phenylisopropylamines delay the destruction of epinephrine in vive by inhibiting monoamine oxidase (74). However, the arguments put forward by these investigators have been challenged (23). Alles and Heegard (1) have determined the relative rates of deamination of a number of phenylamines. From the data obtained in these studies, it was suggested that more than one enzyme may be involved in the deamination of monoamines. This is also the view of Werle and Roewer (163), who believe that in ox liver, tyramine and n-butylamine are oxidized by two enzyme systems. Recently other amine oxidases have been identified, one isolated from ox plasma which shows marked activity towards spermine and spermidine (149), and another enzyme localized in liver microsomes which deaminates phenylisopropylamines (g). The further purification of monoamine oxidase (161) may make it possible to uncover other closely related amine oxidases. The evaluation of monoamine oxidase in the metabolism of phenylethylamine derivatives in vive was made possible by the introduction of potent monoamine oxidase inhibitors such as iproniazid (165) and choline-p-tolyether bromide (40). Pretreatment with iproniazid markedly prolonged the contraction of the nictitating membrane of the cat when phenylethylamine or tyramine was administered (80). The toxic action of phenylethylamine and tyramine, but not of ephedrine, was increased by pretreating guinea pigs with iproniazid (120). In mice, iproniazid or choline-p-tolylether bromide caused a pronounced increase in the excretion of C* tyramine after the administration of the latter substance (132). Several com- pounds, such as ephedrine, amphetamine and cocaine, which inhibit monaomine oxidase zn vitro, were found to have no effect in vivo (132). The metabolism of mescaline (3,4,5-trimethoxy phenylethylamine), the active psychotomimetic principle of ‘mescal buttons,’ has been the subject of many studies. After its administration to dogs and rabbits, Slotta and Muller (143) recovered large amounts of 3,4, 5-trimethoxy phenylacetic acid in the urine. These results could not be confirmed by Cochin ¢é a/. (49). In man and dog, a major fraction of mescaline is excreted unchanged (409, 123, 127). An interesting minor metabolite found in the urine of man after the ingestion of mescaline was identified as a conjugate of 3 ,4-dihydroxy-5-methoxyphenylacetic acid, indicating that this molecule can undergo O-demethylation. Despite the fact that mescaline contains a phenylethylamine nucleus, it is metabolized by diamine oxidase, rather than monoamine oxidase (166). Mescaline is also O-demethylated by an enzyme local- ized in the microsomes of the liver (10). After the administration of mescaline to the dog, the amine is widely distributed in all organ tissues, including the brain (49). Block et al. (32) have made the interesting observation that ingested mescaline is incorporated into liver proteins, and that the mescaline-containing protein may be involved in the production of hallucinations. Recently the incorporation of a variety of C-labeled amines in the soluble protein fraction of mammalian tissues has been reported (111, 128). This system 466 JULIUS AXELROD Volume 39 is activated by Catt and consists of a protein that accepts the amines as well as an enzyme catalyzing the incorporation. The participation of protein amide groups in this reaction was proposed by these investigators. METABOLISM AND PHYSIOLOGICAL DIsPosiTION OF PHENYLISOPROPYLAMINES Phenylisopropylamines constitute an important segment of the sympathomi- metic amines, yet until recently little was known about the disposition and metabo- lism of these compounds. Richter (123) reported that when the phenyliso- propylamines ephedrine and amphetamine were administered to man, they were excreted in the urine almost entirely unchanged. He ascribed the stability of the drugs zn vvo to the failure of monoamine oxidase to attack these compounds. Other investigators (6, 7, 29, 93, 96) did not corroborate these findings. They found that considerable amounts of administered phenylisopropylamines were metabolized in man, dog and rabbit. Moreover, when the liver function of dogs was impaired by the administration of carbon tetrachloride, the urinary excretion of ampheta- mine was increased (29). In the past, studies on the fate of phenylisopropylamines have been hampered by the lack of sensitive and specific methods for the estimation of these compounds in biological materials. The development of methods for the estimation of am- phetamine, p-hydroxyamphetamine (Paradrin), methamphetamine, ephedrine, norephedrine (Propadrine), p-hydroxyephedrine (Suprifen) and p-hydroxynor- ephedrine has made possible a study of the metabolism of these compounds in vivo and in vitro (6, 7). The accompanying scheme (fig. 4) describes the metabolic transformation of amphetamine in vive (7). The main route of metabolism of p-amphetamine in the dog involves hy- droxylation to p-hydroxyamphetamine, a potent pressor agent. After the adminis- tration of L-amphetamine, however, only small amounts of p-hydroxyamphetamine are excreted. The differences in the metabolism of the optical isomers of ampheta- mine are presumably due to a stereospecific enzyme (9) which can deaminate the levo isomer of amphetamine more readily than the dextro isomer. In the dog Hl NHCHs H NH2. — tot C—C—Ch, c-C—CH, I 4 re | ot H oH HOA METHAMPHETAMINE AMPHETAMINE Fic. 4. Metabolism of amphetamine. he ni Co C—CH, C—C—CH Ory —roir RO HH HO oH P-HYDROXYAMPHETAMINE P-HYDROXYAMPHE TAMINE _ CONJUGATE —_ October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 767 p-amphetamine disappears more slowly than its hydroxylated metabolite, sug- gesting that in this species at least, the major pressor effect is mediated by the parent compound. When hydroxyamphetamine is administered, it appears in the urine partly free and partly conjugated. After the administration of methampheta- mine to a dog, about half the compound is demethylated to amphetamine, part of which is hydroxylated to p-hydroxyamphetamine. From these observations, it appears that the pharmacological action of methamphetamine is mediated through its metabolic product, amphetamine. There are marked differences in the metabolism of p-amphetamine in various species. Dogs and rats hydroxylate considerable amounts of the amine, while rabbits almost completely metabolize amphetamine by deamination. Walkenstein et al. (157) have shown that congeners of amphetamine also undergo N-demethyla- tion and p-hydroxylation in the dog, rat and rabbit. At present, no studies on the metabolic fate of amphetamine have been made in man. Previous work has shown that a number of aromatic compounds, including acetanilid, aniline (34) and antipyrine (35) are hydroxylated in man; it seems likely that amphetamine would also hydroxylate in this species. Studies at a cellular level have uncovered an enzyme system in the microsomes of liver that can deaminate amphetamine to phenylacetone and ammonia in the presence of reduced triphosphopyridine nucleotide (TPNH) and oxygen (9). The enzymatic deamination of amphetamine is shown in the following equation: TPNH 2 C.H;CH,CH(NH,)CH; C;H;CH.COCH, + NH; The enzyme which deaminates amphetamine differs from other deaminating enzymes such as amine oxidase, D-amino acid oxidase, L-amino acid oxidase and glutamic acid dehydrogenase with respect to its substrate specificity, intracellular localization and cofactor requirements (9). The role of TPNH in an enzyme system catalyzing the oxidative deamination of an amine is not understood. TPNH could conceivably act by the generation of hydrogen peroxide through the transfer of hydrogen by an intermediate electron transport system to molecular oxygen. However, hydrogen peroxide generated from D-amino acid oxidase was unable to replace TPNH in the deamination of amphetamine. Gillette et al. (76) have found that TPNH is oxidized by an enzyme in liver microsomes in the absence of amine substrate to form a ‘peroxide.’ These investigators believe that the oxidation of TPNH is necessary to carry out other metabolic reactions in the microsomes. In addition to amphetamine, a number of other amines are deaminated by the TPNH-dependent enzymes in the microsomes (g). Amines having a phenyliso- propylamine or phenylbutylamine structure are extensively transformed by the enzyme, while phenolic-substituted amines, phenylethylamines and aliphatic amines are metabolized slightly or not at all. The enzyme also has some degree of stereospecificity, since the L-isomers of amphetamine are transformed more rapidly than the p-isomers. The deaminating activity of rabbit liver microsomes is 768 JULIUS AXELROD Volume 39 considerably depressed when rat microsomes are added. Heating of the rat liver microsomes unmasks the presence of a heat-stable activator for this reaction. The nature of this activating factor is unknown. Species differences in the metabolism of amphetamine may in part be explained by the presence of inhibitory and activ- ating factors in liver microsomes (9). Following the observation that an enzyme in the microsomes of liver re- quiring TPNH and oxygen can deaminate amphetamine, many TPNH-dependent enzyme systems have been found that carry out a wide variety of reactions. Meta- bolic transformations catalyzed by these enzyme systems include N-demethylation (8, 104), hydroxylation of aromatic compounds (109), side chain oxidation (51) and cleavage of ethers (10). A review of this subject has been published (37). The relative nonspecificity of the substrates atacked by the microsomal enzymes would suggest that they act as ‘scavenger’ enzymes and perform normal roles as well. Another property common to microsomal enzyme systems is their inhibition by 8-diethylaminoethyl diphenylpropylacetate (SKF 525A, refs. 17, 50). This com- pound has little pharmacological activity of its own, but prolongs the action of sympathomimetic amines as well as other pharmacological agents by inhibiting microsomal enzyme systems which are concerned with the inactivation of these compounds. Beyer (25) has observed that ascorbic acid can deaminate a number of phenyl- ethylamines and phenylisopropylamine derivatives nonenzymatically. This was substantiated in vive for amphetamine when it was reported that the excretion of the amine in dogs could be markedly diminished by the subcutaneous injections of large amounts of ascorbic acid (26). When the injections were discontinued, the excretion of amphetamine increased again. It has been reported that ascorbic acid in the presence of Fet*+, versene and oxygen hydroxylates aromatic compounds in the test tube (36, 151). Thus, in the model chemical system, tyramine is oxidized to dopamine (36) and amphetamine and ephedrine are hydroxylated to their corresponding para derivatives (36). In ascorbic acid-deficient guinea pigs the rate of the hydroxylation of aromatic compounds was reduced (21). The precise role that ascorbic acid plays in the metabolism of amines in the organism is not known. Figure 5 shows the route of metabolism of ephedrine (6). The principal pathway of biotransformation of ephedrine in the dog involves enc on yr ny t=—c— =—C— me Ce cor C=CH Cri ¢—CHy , C-C=CHy Ho HoH HH HH, ~HYDROXYEPHEORINE EPHEDRINE NOREPHEDRINE 2 | Fic. 5. Metabolism of _ Nie Aa ephedrine. ore HO H p ~HYDROXYNOREPHEORINE xr~0=-9 October 1959 METABOLISM OF SYMPATHOMIMETICG AMINES 4769 rapid N-demethylation to norephedrine, a relatively stable and potent pressor agent. It is probable that the activity of ephedrine is mediated to a considerable extent through its metabolite, norephedrine. Other minor routes of metabolism involve hydroxylation of both ephedrine and norephedrine to yield the corre- sponding p-hydroxy derivatives, both of which are potent pressor agents. No evi- dence has been found for the excretion of 3,4-dihydroxyephedrine. However, these catecholamines are readily O-methylated by catechol-O-methyltransferase, and if formed, they might be further metabolized to their corresponding O-methyl derivatives. Norephedrine is relatively stable, and is excreted in the urine mainly unchanged. Calculations of the renal excretion of norephedrine from plasma levels and urinary excretion yielded values which were considerably higher than the glomerular filtration rate, suggesting that a secretory transport mechanism is involved in its excretion (6). There are differences in the metabolism of ephedrine in mammalian species. In the dog and guinea pig, demethylation proceeds rapidly, constituting a major route of biotransformation. The rat, on the other hand, demethylates ephedrine slowly and considerable amounts of the drug are excreted both unchanged and as hydroxylated derivatives. Only very small quantities of norephedrine and ephed- rine are eliminated by the rabbit, indicating that in this species ephedrine is demethylated to norephedrine, which in turn is extensively metabolized by path- ways other than hydroxylation. After the subcutaneous administration of 3,4-dihydroxynorephedrine to man, only small amounts of the compound are excreted unchanged (67), indicating almost complete metabolic alteration of the molecule. Although the metabolic products of this catechol have not been identified, it is probable that it isO-methyl- ated and excreted as free or conjugated 3-methoxy-4-hydroxynorephedrine. Ephedrine is N-demethylated to norephedrine and formaldehyde by a TPNH-dependent enzyme system in the microsomes of the liver (8). The over-all reaction can be represented by the following equation: C,H,;CHOHCH,(NHCH,)CH,; ce C,H,CHOH(NH,)CH, + HCHO. 2 The microsomal preparation also metabolizes ephedrine by pathways other than demethylation (8). The ability of the microsomal enzyme system to demethylate other sympathomimetic amines is dependent on the structure of the amine. Sub- strates possessing a phenylisopropylamine nucleus are extensively demethylated, while those with a phenylethylamine nucleus are not. Amines with hydroxy groups on the aromatic nucleus are demethylated slightly or not at all. Species differences in the metabolism of ephedrine can be explained in part by the activity of the TPNH-dependent microsomal demethylating enzyme as compared to the deaminating enzyme. When ephedrine or norephedrine are administered to the rabbit, both compounds are extensively metabolized. The transformation of these amines is catalyzed by enzymes which demethylate ephedrine and deaminate norephedrine. In the dog the activity of the norephedrine-deaminating enzyme is 770 JULIUS AXELROD Volume 39 low, so that the demethylated ephedrine, once formed, is excreted unchanged. The activity of the enzymes which demethylate ephedrine and deaminate norephedrine in the rat is negligible, so that administered ephedrine in this species is excreted unchanged or as p-hydroxyephedrine. The tissue concentration of the phenylisopropylamines amphetamine, ephed- rine and: norephedrine, shows a similar pattern of distribution (6, 7). After their administration, these compounds are localized in organ tissues to a considerable extent, with small amounts in the bile and fat. The high concentration of the amines found in the brain and cerebrospinal fluid suggests that there is little hindrance to their passage across the blood-brain barrier. Catecholamines, on the other hand, cross the blood-brain barrier with difficulty or not at all (159). METABOLISM OF INDOLAMINES The metabolism of the indolamine, tryptamine, has been elucidated by Ewins and Laidlow many years ago (70). After its administration, it is deaminated to 3-indolacetic acid, which is then excreted as a glycine conjugate. Since trypta- mine is a substrate for monoamine oxidase (7, 31, 31a) it has been assumed that this enzyme deaminates the amine in the intact organism. More recently, Schayer ef al. (137) have shown that the inhibition of monoamine oxidase in vive increases the excretion of administered tryptamine. An indole base with an Ry» value corresponding to that of tryptamine has been found in normal urine (126). Tryptamine has also been found in tissues, including brain and liver, following the administration of a monoamine oxidase inhibitor and tryptophan (85). Although the decarboxylation of tryptophan has been demonstrated in vitro (158, 162) the origin and physiological significance of trypta- mine in the body have yet to be established. In the past 10 years, tryptamine derivatives have acquired a new significance when serotonin (5-hydroxytryptamine) was found to be a normal constituent in the body (119). The presence of serotonin in the brain (2, 150) and the provocative proposal of Wooley (164) and Gaddum (73) that this amine may be involved in the maintenance of mental health, stimulated great activity in this area of research. An exhaustive review of this subject has been published recently by Page (112), and the metabolism of serotonin will be covered only briefly here. Serotonin has been shown to arise from tryptophan in the body (154). Although tryptophan and 5-hydroxytryptophan form serotonin in man, tryptamine does not (152), indi- cating that hydroxylation of the indole nucleus precedes decarboxylation. A major pathway for the metabolism of serotonin is deamination by monoamine oxidase and aldehyde dehydrogenase to 5-hydroxyindolacetic acid (153) which is then conjugated with glycine and excreted in the urine. Other, though minor, routes for the metabolism of serotonin involve N-acetylation and conjugation with glucuronic acid (108). Szara has described the psychotomimetic actions of dimethyltryptamine (146). After its administration, this compound is excreted in the urine as 3-indolacctic acid in the rat (57) and man (146). Recently, new pathways for the metabolism of October 1959 METABOLISM OF SYMPATHOMIMETIC AMINES 771 | T NHCHy C= —— Vo N N7 4H N~METHYLTRYPTAMINE DIMETHYLTRYPTAMINE | . H | Cm x 7CHy —N N CHy ri-oer mi~o=-z m-0O—Ir H HH i it 1, TNH é-tane Fic. 6. Metabolism of ne how i wi boa ‘ce dimethyltryptamine. TRYPTAMINE 6-HYDROXYDIMETHYLTRYPTA MINE HO ee) i ou te —C—OH C—O Cust ST: n7 HO N°? WH 3-INDOLACETIC ACID 6-HYDROXYINDOLACETIC ACID indolamines have been described (147). The accompanying scheme (fig. 6) has been proposed for the biotransformation of dimethyltryptamine (147). Dimethyl- tryptamine is metabolized along two pathways involving N-demethylation and hydroxylation on the 6 position. The metabolites are further transformed by deamination. Jn vitro studies have shown that N-demethylation (147) and 6-hy- droxylation (94, 147) of the indolamine is catalyzed by TPNH-dependent enzymes in the liver microsomes. Although the indole-containing ergot alkaloids are widely used as pharmaco- logic agents, little is known about their biologic fate. One of these compounds, lysergic acid diethylamide (LSD), a potent hallucinogenic compound, is readily hydroxylated to 2-oxy-LSD by a microsomal enzyme in the liver of a number of mammalian species (15). Other ergot alkaloids may be hydroxylated in the same manner. When administered, LSD is almost completely altered in the body, and there are species differences in the rate of biotransformation of the compound (15). From the amount of LSD found in the brain of cats, it was calculated that the drug exerts its psychological effect at an exceedingly minute concentration. SUMMARY Routes for the metabolism of catecholamines in vivo have been established the past few years. The major pathway for the metabolism of epinephrine and norepinephrine involves O-methylation to yield metanephrine and normetaneph- rine. These physiologically inactive metabolites are then conjugated or deaminated to 3-methoxy-4-hydroxymandelic acid. A considerable body of evidence has accumulated indicating that monoamine oxidase is mainly concerned with the deamination of the O-methylated metabolites, rather than the catecholamines themselves. An enzyme, catechol-O-methyl transferase, catalyzes the O-methylation of epinephrine, norepinephrine and other normally occurring catechols. This enzyme is widely distributed in most organs and tissues, including the autonomic 772 JULIUS AXELROD Volume 39 and central nervous system. Catechol-O-methy]l transferase is chiefly concerned in the metabolism of circulating epinephrine and norepinephrine, but its role in the inactivation of endogenously released catecholamines remains to be established. Sympathomimetic phenylethylamines, phenylisopropylamines and indol- amines are metabolized by a variety of pathways, including deamination, N- and O-demethylation, hydroxylation and conjugation. © 13. REFERENCES . ALLES, G. A. anp E. V. HEEGAARD. Substrate specificity of amine oxidase. 7. Biol. Chem. 147: 487-513, 1943. . AMIN, A. H., T. B. B. CRAWFORD anp J. H. GADDUM. Distribution of substance P and 5-hy- droxytryptamine in central nervous system in dog. J. Physiol. 126: 596-618, 1954. . ARMIN, J., R. T. GRANT, R. H. S. THOMPSON anp A. TICKNER. An explanation for the height- ened vascular reactivity of the denervated rabbit’s ear. 7. Physiol. 121: 603-622, 1953. . ARMSTRONG, M. D., A. McMILLAN anv K. N. F. SHAW. 3-Methoxy-4-hydroxy-D-mandelic acid, a urinary metabolite of norepinephrine. Biochim. et biophys. acta 25: 442-423, 1957. . ARMSTRONG, M D., K. N. F. SHAW ann P. E. WALL. Phenolic acids of human urine. 7. Biol. Chem, 218: 293-303, 1958. . AXELROD, J. Studies on sympathomimetic amines. 1. Biotransformation and physiological disposition of “ephedrin and /-norephedrine. 7. Pharmacol. & Exper. Therap. 109: 62-73, 1953. . AXELROD, J. Studies on sympathomimetic amines. i, Biotransformation and physiological disposition of d-amphetamine, d-p-hydroxyamphetamine and d- methamphetamine. 7. Pharmacol. & Exper. Therap. 110: 915-326, 1954. . AXELROD, J. Enzymatic demethylation of ephed- rine. J. Pharmacol. @ Exper. Therap. 114: 430-438, 1955. . AXELROD, J. Enzymatic deamination of ampheta- mine. J. Biol. Chem. 214: 753-763, 1955. . AXELROD, J. Enzymic cleavage of aromatic ethers. Biochem. 7. 63: 634-639, 1956. . AXELROD, J. O-Methylation of catechol amines in vitro and in vivo. Science 126: 400-401, 1957- . AXELROD, J. Presence, formation and metabolism of normetanephrine in the brain. Science 127: 754- 755) 1958. AXELROD, J. Metabolism of catechol amines in vivo and in vitro. Pharmacol. Rev. 11: (Pt. 2): 402, 1959. tga. AXELROD, J. anp LAROCHE. Science. In press. 14. AXELROD, J., R. W. ALBERS anp C. D. CLE- MENTE. Distribution of catechol-O-methyl trans- ferase in the nervous system and certain tissues. 7. Neurochem. In press. . AXELROD, J., R. O. BRADY, B. WITKOP ano E. V. EVARTS. Distribution and metabolism of lysergic acid diethylamine. Ann. New York Acad. Sc. 66: 435-444, 1957- , AXELROD, J., J. K. INSCOE, S. SENOH anp B. WITKOP. O-Methylation, the principal pathway for the metabolism of epinephrine and norepineph- 20. 2. 22. rine in the rat. Biochim. et biophys. acta 27: 210-211, 1958. . AXELROD, J., J. REICHENTHAL anv B. B. BRODIE. Mechanism of potentiating action of 8-diethyl aminoethy] diphenylpropylacetate (SKF525A) 3. Pharmacol. @ Exper. Therap. 112: 49-54, 1954- . AXELROD, J., S. SENOH ann B. B. WITKOP. O-Methylation of catechol amines in vivo. 7. Biol. Chem. 233: 697-701, 1958. . AXELROD, J. ano S. SZARA. Enzymic conversion of metanephrine to epinephrine. Biochim. et biophys. acta 30: 188-189, 1958. AXELROD, J. anp R. TOMCHICK. Enzymatic O-methylation of epinephrine and other catechols. JF. Biol. Chem. 233: 702-705, 1958. AXELROD, J., S. UDENFRIEND anv B. B, BRODIE. Ascorbic acid in aromatic hydroxylation. mt, Effect of ascorbic acid on hydroxylation of acetanilide, aniline and antipyrine in vive. 7. Pharma- col. & Exper. Therap. 111: 176-181, 1954. AXELROD, J., H. WEIL-MALHERBE anv R. TOMCHICK. Physiological disposition of Hi adrenaline and its principal metabolite, metaneph- rine. Fed. Proc, 18: 364, 1959. 22a, AXELROD, J., H. WEIL-MAEHERBE anv R. 23. 24. 25. 26. 27. 28. 29. 30. 31. TOMCHICK. 7. Pharmacol. & Exper. Therap. In press. BACQ, Z. M. The metabolism of adrenaline. Pharma- col, Rev. 1: 1-26, 1949. BALZER, H. anv P. HOLTZ. Beeinflussung der Wirkung biogener Amine durch Hemmung der Aminoxydase. Arch. exper. Path. u. Pharmakol. 227: 547-558, 1956. BEYER, K. H. Action of vitamin C and phenol- oxidase in the inactivation of B-phenylpropylamines. JF. Pharmacol. @ Exper. Therap. 7%: 394-401, 1941. BEYER, K. H. Ascorbictacid in the inactivation of sympathomimetic amines. 3. Pharmacol. & Exper. Therap. 76: 149-155, 1942. BEYER, K. H. Sympathomimetic amines: Relation of structure to their action and inactivation. Physiol. Rev. 26: 169-197, 1946. BEYER, K. H. anv S. H. SHAPIRO. Excretion of conjugated epinephrine and related compounds. Am. J. Physiol. 144: 321-330, 1945. BEYER, K. H. anv J. T. SKINNER. Detoxication and excretion of 8-phenylisopropylamine (Benze- drine). 7. Pharmacol. & Exper. Therap. 68: 419-432, 1940. BLASCHKO, H. Amine oxidase and amine metabo- lism. Pharmacol. Rev. 4: 4157458, 1952. BLASCHKO, H. anv F. J. PHILPOT. Enzymic October 1959 oxidation of tryptamine derivatives. 7. Physiol. 122: 403-408, 1953. gia. BLASCHKO, H., D. RICHTER ann H. J. SCHLOSSMAN. Inactivation of adrenaline. 7. Physiol. go: 1-17, 1937. 32. BLOCK, W., K. BLOCK anv B. PATZIG. Zur wo 7 Physiologic des C'-radioaktiven Mescalins in Tier- versuch. 1. Mitteilung Verteilung der Radioaktivitat in den Organen in Abhangigkeit von der Zeit. Rischr. Physiol. Chem. 290: 230-243, 1952. . BOOTH, A. N., C. W. MURRAY, F. DEEDS ann F. T. JONES. Metabolic fate of rutin and quercitrin. Fed. Proc. 14: 321, 1958. . BRODIE, B. B. anp J. AXELROD. Fate of acetan- ilide in man. 7. Pharmacol. @ Exper. Therap. g4: 29-38, 1948. BRODIE, B. B. anp J. AXELROD. Fate of anti- pyrine in man. 7. Pharmacol. & Exper. Therap. 98: 97-104, 1950. . BRODIE, B. B., J. AXELROD, P. A. SHORE ano S. UDENFRIEND. Ascorbic acid in aromatic hydroxylation u. Products formed by reaction of substrate with ascorbic acid, ferrous ion and oxygen. J. Biol. Chem. 208: 741-750, 1954- . BRODIE, B. B., J. R. GILLETTE anv B. N. LA DU. Enzymatic metabolism of drugs and other foreign compounds. Ann. Rev. Biochem. 27: 427-454, 1958. . BROWN, D. D., J. AXELROD ann R. TOM- CHICK. Enzymatic N-methylation of histamine. Nature 183: 680, 1959. . BROWN, G. L. anv J. S. GILLESPIE. Output of sympathetic transmitter from the spleen of the cat. J. Physiol. 138: 81-102, 1957. . BROWN, B. G. anv P. HEY. Choline pheny! ethers as inhibitors of amine oxidase. Brit. 7. Pharmacol. rr: 58-65, 1956. 41. BURN, J. H. Mechanism of action of chemical sub- stances at nerve endings. Acta physiol. scandinav. 29: 40-49, 1953. . BURN, J. H., F. J. PHILPOT ann U. TRENDEL- ENBURG. Effect of denervation on enzymes in iris and blood vessels. Brit. F. Pharmacol. g: 423-429, 1954. 43. BURN, J. H. anp J. ROBINSON. Effect of denerva- 45 tion on amine oxidase in structures inervated by the sympathetic nerves. Brit, J. Pharmacol. 7: 304-318, 1952. , CAMMARATA, P. S. anp P, P. COHEN. Scope of transamination in tissues. 7. Biol. Chem. 187: 439- 452, 1950. . CANTONI, G. L. S-Adenosylmethionine; a new intermediate formed enzymatically from |-methionine and adenosinetriphosphate. J. Biol. Chem. 204: 403-416, 1953. . CARLSSON, A., M. LINDQVIST, T. MAGNUS- SON anp B. WALDECH. Presence of 3-hydroxy- tyramine in brain. Setence 127: 471, 1958. . CARLSSON, A., E. ROSENGREN, A. BERTLER anbD J. NILSSON. Effect of reserpine on the me- tabolism of catechol amines. In: Psychotropic Drugs, edited by S. Garattini and V. Ghetti. Amsterdam: Van Nostrand, 1958, pp. 363-372. . CELENDER, O. anp S. MELLANDER. Elimina- tion of adrenaline and noradrenaline from circulat- ing blood. Nature 176: 973, 1955- METABOLISM OF SYMPATHOMIMETIC AMINES 49. 50. 52. 53- 54- 55- 56. 57. 58. 59: 60. 62. 63. 64. 65. 66. 67. 68. 773 COCHIN, J., L. A. WOODS anp M. H. SEEVERS. Absorption, distribution and urinary excretion of mescaline in the dog. 7. Pharmacol & Exper. Therap. 101; 205-209, 1951. COOPER, J. R., J. AXELROD anp B. B. BRODIE. Inhibitory effects of f-diethylaminoethyl diphenyl- propylacetate-(SKF525A) 7. Pharmacol. @ Exper. Therap. 112: 49-54, 1954. . COOPER, J. R. ann B. B. BRODIE. Enzymatic metabolism of hexobarbital (Evipal). 7. Pharmacol @ Exper. Therap. 114: 409~417, 1955. CORNE, 8. J. anp J. D. P. GRAHAM. Effect of inhibition of monoamine oxidase in vivo on admin- istered adrenaline, noradrenaline, tyramine and serotonin. 7. Physiol. 135: 339-349, 1957. DAVISON, A. N. Physiological role of monoamine oxidase. Physiol. Reo. 38: 729-747, 1958. DEEDS, F., A. N. BOOTH ann F, T. JONES. Methylation and dehydroxylation of phenolic com- pounds by rats and rabbits. J. Biol. Chem. 225: 615- 6ar, 1957- DUTTON, G. J. anv I. D. E. STOREY. Uridine compounds in glucuronic acid metabolism. 1. Forma- tion of glucuronides in liver suspensions. Biochem. 7. 57: 275-283, 1954. EADE, N. R. Storage and release of catechol amines. Rev. Canad. Biol. 17: 299-311, 1958. ERSPAMER, V. Fate of indolalkylamines in the organism. 7. Physiol. 127: 118-133, 1955. von EULER, C., U.S. von EULER anp I, FLOD- ING. Biologically inactive catechol derivatives in urine. Acta physiol. scandinav. 33: Suppl. 118: 32~98, 1955- von EULER, U. S. Nature of adrenergic nerve mediators. Pharmacol. Rev. 3: 247-277, 1951. von EULER, U. S. Catechol amine content of various organs of the cat after injections and infu- sions of adrenaline and noradrenaline, Circulation Res. 4: 647-652, 1956. . von EULER, U. S. Noradrenaline. Springfield, IIL: Thomas, 1956. von EULER, U. S. Distribution and metabolism of catechol amines. Recent Progress Hormone Res. 14: 483-505, 1958. von EULER, U. S. anp S. HELLNER-BJORK- MAN. Effect of amine oxidase inhibitors on nor- adrenaline and adrenaline content of cat organs. Acta physiol. scandinav. 33, Suppl. 118: 21-25, 1955. von EULER, U. S. ann F. LISHAJKO. Catechol amines in the vascular wall. Acta physiol, scandinav. 42: 333-341, 1958. von EULER, U. S. anp R. LUFT. Noradrenaline output in urine after infusion in man. Brit. 7. Pkarma- col. 6: 286-288, 1951. von EULER, U. 8., R. LUFT anp T. SUNDEN. Excretion of urinary adrenaline in normals follow- ing intravenous infusion. Acta physiol. scandinav. 30: 249-257, 1953. von EULER, U.S. anp B. ZETTERSTROM, Role of amine oxidase in the inactivation of catechol amines injected in man. Acta physiol. scandinav. 33, Suppl. 118: 26-31, 1955. EVARTS, E. V., L. GILLESPIE, T. C. FLEMING anp A, SJOERDSMA. Relative lack of pharmaco- logical action of the 3-methoxy analogue of nor- 774 6g. 70. 71. 72. 73. 74: 75. 76. 77- 78. 79: 80. 8 82. 83. 84. 85. 86. 87. 88. JULIUS AXELROD epinephrine. Proc. Soc. Exper. Biol. @ Med. 98: 74-76, 1958. EWINS, A. J. ano P. P. LAIDLAW. Fate of p-hy- droxyphenylethylamine in the organism. 7. Physiol. 4: 78-87, Igto. EWINS,. A. J. anp P. P. LAIDLAW. Fate of indol- ethylamine in the organism. Biochem. J. 7: 18-25, 1913. FRIEND, G., M. S. ZILELI, J. R. HAMILIN anp F. W. REUTTER. Effect of iproniazid on the inactivation of norepinephrine in the human. 7. Clin. @ Exper. Psychotherap, 19: 61-68, 1958. FURCHTGOTT, R. F., P. WEINSTEIN, H. HUEBL, P. BOZORGMEHRI anv 8. R. MENSEN- DIEK. Effect of inhibition of monoamine oxidase on response of rabbit aortic strips to sympathomimetic amines. Fed. Proc. 14: 341-342, 1955. GADDUM, J. H. In: Ciba Foundation Symposium on Hypertension. Boston: Little, 1954. GADDUM, J. H. ann H. KWIATKOWSKI. Action of ephedrine. 7. Physiol. 94: 87-100, 1938. GAUDETTE, L. E., J. L. SCOTT anv P. KOSKY. Metabolism of DL-C-epinephrine in mammals. Fed. Proc. 17: 52, 1958. GILLETTE, J. R., B. B. BRODIE anp B. N. LA DU. Oxidation of drugs by liver microsomes: Role of TPNH and oxygen. 7. Pharmacol. & Exper. Therap. 119: 532-540, 1957. GOLDSTEIN, M., A, J. FRIEDHOFF, C. SIM- MONS anp N. PROCHOROFF. Conversion of g-hydroxytyramine /-C¥ to 3-methoxynorepinephrine in vivo. Fed. Proc. 18: 236, 1959. GOODALL, McC. anp N, KIRSHNER. Biosyn- thesis of epinephrine and norepinephrine by sympa- thetic nerves and ganglia. Circulation 17: 366-371, 1958. GREEN, D. E. anp D. E, RICHTER. Adrenaline and adrenochrome. Biochem. 7. 31: 596-616, 1937. GREISEMER, E. C., C. A. BARSKY, C. A. DRAG- STEDT, J. A. WELLS anv E. A. ZELLER. Poten- tiating effect of iproniazid on the pharmacological actions of sympathomimetic amines. Proc. Soc. Exper. Biol. @ Med. 84: 699-701, 1953. . GUGGENHEIM, M. anp W. LOFFLER. Das Schicksal protienogener Amine in Tierkérper: Bio- chem, Ktschr. 72: 325-350, 1915. HARE, M. L. C. Tyramine oxidase. 1. A new en- zyme system in liver. Biochem. 7. 22: 968-979, 1928. HARLEY-MASON, J., A. H. LAIRD anp J. R. SMYTHIES. Metabolism of mescaline in the human. Confinia neurol. 18: 152, 1958. HARTUNG, W. H. Inactivation and detoxication of pressor amines. Ann. Rev. Biochem. 15: 593-616, 1946. HESS, §., B. G. REDFIELD anp S. UDENFRIEND. Tryptamine in animal tissues following the adminis- tration of iproniazid, Fed. Proc. 18: 402, 1959. HOFFER, A. Adrenochrome in blood plasma. Am. J. Psychiat. 114: 752, 1958. HOLTZ, P., K. CREDNER ann W. KOEPP. Die enzymatische Entstehung von Oxytyramine in Organismus und die physiologische Bedeutung der Dopadecarboxylase. Arch. exper. Path. u. Pharmakol. 200: 356-388, 1942. HOLTZ, P.,.R. HEISE anp K. LUDTKE. Fermen- tativer Abbau von Dopa durch Niere. Arch. exper. Path. u. Pharmakol. 191 : 87-118, 1938. 8g. go. gl. 93- 94- 95: g6. 97. 98. 99. 100. 103. 104. 105. 106. 107. Volume 39 HOLTZ, P. anp E. WESTERMANN. Ueber die Dopadecarboxylase und Histidinedecarboxylase des Nevengewebes. Arch. exper. Path. u. Pharmakol, 227: 538~546, 1956. . : IMAIZUMIL, R. anp K. KAWAMOTO. Adrenaline dehydrogenase in blood. Med. 7. Osaka Univ. 3: 269-278, 1952. IMAIZUMI, R., K. KAWAMOTO, T. KITA anp H. SATO. Adrenaline dehydrogenase in blood. Med. F. Osaka Univ, 3: 279-283, 1952. - ISSELBACHER, K. ann J. AXELROD. Enzymatic formation of corticosteroid glucuronides. 3. Am. Chem. Soc. 77: 1070-1071, 1955. JACOBSEN, E. anp I. GAD. Das Verhalten des 8-Phenylisopropylamine in Kaninchenorganismus. Arch. exper. Path. u. Pharmakol, 196: 34-42, 1940. JEPSON, J. B., S. UDENFRIEND anv P. SALTZ- MANN. Enzymic conversion of tryptamine to 6-hy- droxytryptamine. Fed. Proc. 18: 254, 1959. KAMIJO, K., G. B. KOELLE ann H. H. WAG- NER. Modification of the effects of sympathomimetic amines and of adrenergic nerve stimulation by 1- isonicotinyl-2-isopropylhydrazine (IHH) and_ iso- nicotinic acid hydrazide (INH). 7, Pharmacol, & Exper. Therap. 117: 213-224, 1955. KELLER, R. E. anp W. C. ELLENBOGEN. De- termination of d-amphetamine in body fluids. 7. Pharmacol, & Exper. Therap. 106: 77-82, 1952. KIRSHNER, N. anp McC. GOODALL. Formation of adrenaline from noradrenaline. Biochim. et biophys. acta 24: 658-659, 1957. KIRSHNER, N., McC. GOODALL np L. ROSEN. Metabolism of dl-adrenaline-2-C™ in the human. Proc. Soc. Exper. Biol. & Med. 98: 627-630, 1958. KITA, T. Adrenaline metabolism, especially the formation of adrenalone, protocatechuic aldehyde and protocatechuic acid. Wippon Yakurigaku Zasshi, 53! 523-541, 1957. Chem. Abstr. 52: 10405, 1958. KOELLE, G. B. Histochemica! identification of acetylcholinesterase in cholinergic, adrenergic and sensory neurons. 7. Pharmacol. & Exper. Therap. 114: 167-184, 1955. - KOELLE, G. B. ann A. peT. VALK. Physiological implications of the histochemical localization of monoamine oxidase. 7. Physiol. 126: 434-447, 1954. . LaBROSSE, E. H., J. AXELROD anp S. S. KETY. O-Methylation, the principal route of metabolism of epinephrine in man. Science 128: 593-594, 1958. LaBROSSE, E. A., J. AXELROD anp A. SJOERDSMA. Urinary excretiod of normetaneph- rine by man. Fed. Proc. 17: 386, 1958. LA DU, B. N., L. GAUDETTE, N. TROUSOF anp B. B. BRODIE. Enzymatic dealkylation of aminopyrine and other alkylamines. 7. Biol. Chem. 214°7147752, 1955. LEEPER, L. C., H. WEISSBACH anv 8S. UDEN- FRIEND. Metabolism of norepinephrine, epineph- rine and their O-methyl analogoues by partially purified enzyme preparations. Arch. Biochem. @ Biophys. 77: 417-427, 1958. LUND, A. Elimination of adrenaline and nor- adrenaline from the organism. Acta pharmacol, et toxicol. 7: 297-308, 1951. MACLAGLAN, N. F. anp J. H. WILKINSON. Methylation of a phenolic hydroxyl group in the human body. Nature 168: 251, 1951. October 1959 108, 109. r10. Ini. 112. 113. 114. 115. 116. 117. 118, 119. 120, 122, 123, 124. 125. 126, 127 128, McISAAC, W. anp I. H. PAGE. New metabolites of serotonin in carcinoid urine. Science 128: 537, 1958. MITOMA, C., H. S. POSNER, H. C. REITZ and 8, UDENFRIEND. Enzymatic hydroxylation of aromatic compounds. Arch. Biochem. & Biophys. 61: 431-441, 1956. MONTAGU, K. A. Catechol compounds in rat tissues and brains of different animals. Nature 180: 244-245, 1957- MYCEK, M. J., D. D. CLARKE, A. NEIDLE anp H. WAELSCH. Enzyme-substrate relationship in amine incorporating system. Fed. Proc. 17: 280, 1958. PAGE, I. H. Serotonin (5-hydroxytryptamine): the last four years. Physiol. Rev. 38: 277-335, 1958. PEKKARINEN, A. Chemical determination, oc- currence and metabolism of adrenaline in blood and urine. Acta physiol. scandinav. 16: 54, 1-110, 1948 (uppl.) PELLERIN, J. anp A. D’IORIO. Metabolism of radioactive 3,4-dihydroxyphenylalanine a@-C'4 in the albino rat. Canad. 7. Biochem. & Physiol. 33: 1055- 1061, 1955. PELLERIN, J. ann A. DIORIO. Methylation of the 3-OH position of catechol acids by rat liver and kidney preparations. Canad. 7. Biochem. &@ Physiol. 26: 491-497, 1958. PUGH, C. E. M. anv J. H. QUASTEL. Oxidation of aliphatic amines by brain and other tissues. Bio- chem. J. 31: 286-2g1, 1937. RAAB, W. anv A. B, GIGEE. Specific avidity of the heart muscle to absorb and store epinephrine and norepinephrine. Circulation Res. 3: 553-560, 1955. RAAB, W. anv A. B. GIGEE, Uptake and break- down of catecholamines in vascular tissue. Angiology g: 283-289, 1958. RAPPORT, M. M., A. A. GREEN anp I. H. PAGE. Serum vasoconstrictor (serotonin). IV. Isolation and characterization, 7. Btol. Chem, 176: 1243-1251, 1948. RABHUN, J., S. M. FEINBERG anp E. A. ZEL- LER. Potentiating effects of iproniazid on the action of some sympathomimetic amines. Proc. Soc. Exper. Biol. @ Med, 87: 218-220, 1954. . RESNICK, O., J. M. WOLFE, H. FREEMAN ano F. ELMADJIAN. Iproniazid treatment and metabolism of labeled epinephrine in schizophrenics, Science 127: 1116-1117, 1958. RICHTER, D. Adrenaline and amine oxidase. Biochem, F. 31: 2022-2028, 1937. RICHTER, D. Elimination of amines in man. Biochem. 7. 32: 1763-1769, 1938. RICHTER, D. Inactivation of adrenaline in zivo in man. J. Physiol. 98: 361-374, 1940. RICHTER, D. anp F. C. MACINTOSH. Adrenal- ine ester. Am. 7. Physiol. 135: 1-5, 1941. RODNIGHT, R. Separation and characterization of urinary indoles resembling 5-hydroxytryptamine and tryptamine. Biochem. 7. 64: 621-626, 1956. . SALOMAN, K., B. W. GABRIO anp T. THALE. A study of mescaline in human subjects. 7. Pharmacol. @ Exper, Therap. 95: 455-459, 1949- SARKAR, N. K., D. D. CLARKE ann H. WAELSCH. An enzymatically catalyzed incorpora- METABOLISM OF SYMPATHOMIMETIC AMINES 129. 130. 131. 132. 133. 194. 135. 136. 137. 198. 139. 775 tion of amines into proteins. Biochim. et biophys. acta 25: 451-452, 1957. SCHAEPDRYVER, A. F. Secretion, Distribution and Excretion of Adrenaline and Noradrenaline. Bruges, Bel- gium: St. Catherine Press, 1959. SCHAYER, R. W. Metabolism of 8-C4 DL-adrenal- ine. 7. Biol. Chem. 189: 301-306,. 1951. SCHAYER, R. W. Metabolism of adrenaline con- taining isotopic carbon. 7. Biol. Chem, 192: 875-881, 1951. SCHAYER, R. W. In vive inhibition of monoamine oxidase studied with radioactive tyramine. Proc. Soc. Exper. Biol, @ Med. 84: 60-53, 1953. SCHAYER, R. W. Metabolism of histamine in various species. Brit. 7. Pharmacol. 11: 472-473, 1956. SCHAYER, R. W., R. L. SMILEY, K. J. DAVIS anD Y. KOBAYASHI. Role of monoamine oxidase in noradrenaline metabolism. Am. 7. Physiol. 182: 285-286, 1955. SCHAYER, R. W., R. L. SMILEY ann E. H. KAP- LAN. Metabolism of adrenaline containing isotopic carbon (a). J. Biol. Chem. 198: 545-551, 1952. SCHAYER, R. W., R. L. SMILEY anv J. KEN- NEDY. Metabolism of epinephrine containing iso- topic carbon ut. F. Biol. Chem. 202: 425-430, 1953. SCHAYER, R. W., K. Y, T. WU, R. L. SMILEY anp Y. KOBAYASHI. Monoamine oxidase in intact animals. 7. Biol. Chem. 210: 259-267, 1954. SCHUMANN, H. J. Nachweis von Oxytyramine (Dopamin) in sympathischen Nerven und Ganglien, Arch. exper. Path u. Pharmakol. 227: 566-573, 1956. SCHUMANN, H. J. Uber die Verteilung von Nor- adrenalin und Hydroxytyramin in sympathischen Nerven (Milznerven). Arch. exper. Path u. Pharmakol. 234: 17-25, 1958. 1g39a. SENOH, S., J. DALY, J. AXELROD anp B. 140. 141. 142. 143. 144. 145. 146. 147. 148. WITKOP. 7. Am. Chem. Soc. In press. SHAW, K. N. F., A. McMILLAN anp M. D. ARMSTRONG. Metabolism of 3,4-dihydroxy- phenylalanine, 7. Biol. Chem. 226: 255-266, 1957. SHORE, P. A., J. A. R. MEAD, R. G. KUNTZ- MAN, S. SPECTOR anv B. B. BRODIE. Physio- logical significance of monoamine oxidase in the brain. Science 126: 1063-1064, 1957. SJOERDSMA, A., W. M. KING, L. C. LEEPER. anp S, UDENFRIEND. Demonstration of the 3- methoxy analog of norepinephrine in man. Science 127: 876, 1958. SLOTTA, K. H. anp J. MULLER. Uber den Ab- bau des Mescalin und mescalinanlicher Stoffe in Organismus, tschr. Physiol. Chem, 238: 217-245, - 1936. SPECTOR, S., H. M. MALING ann P. A. SHORE. Effect of JB516, a monoamine oxidase inhibitor, on levels of serotonin and norepinephrine in brain and spinal cord. Fed. Proc. 18: 447, 1959. SPECTOR, S., D. PROCKOP, P. A. SHORE and B. B. BRODIE. Effect of iproniazid on brain levels of norepinephrine and serotonin. Science 127: 794, 1958. SZARA, S. Dimethyltryptamine: Its metabolism in man, the relation of its psychotic effects to serotonin metabolism. Experientia 12: 441, 1956. SZARA, S. anp J. AXELROD. Hydroxylation and demethylation of dimethyltryptamine in vitro and in vivo. Experientia 15: 216, 1959- SZARA, S., J. AXELROD anv S. PERLIN. Is 776 149. 150. 151. 152. 153. 154. 155. 156. 157. adrenochrome present in the blood? Am. 7. Psychiat. 115: 162-163, 1958. TABOR, C. W., H. TABOR anp 5. M. ROSEN- THAL. Purification of amine oxidase from beef plasma. 7. Biol. Chem. 208: 645-661, 1954. TWAROG, B. M. anv I. H. PAGE. Serotonin con- tent in some mammalian tissues and urine. dm. 7. Physiol. 175: 157-161, 1953. UDENFRIEND, S., C. T. CLARK, J. AXELROD anp B. B. BRODIE. Ascorbic acid in aromatic hydroxylation. 1. A model system for aromatic hydroxylation. 7. Biol. Chem. 208: 731-739, 1954. UDENFRIEND, S., C. 8. CREVELING, H. POS- NER, B. C. REDFIELD, J. DALY anv B. WITKOP. Inability of tryptamine to serve as a precursor of serotonin. Arch. Biochem. & Biophys. In press. UDENFRIEND, S., E. O. TITUS anv H. WEISS- BACH. Identification of 5-hydroxy-3-indoleacetic acid in normal urine and a method for its assay. 7. Biol. Chem. 216: 499-505, 1955. UDENFRIEND, S., E. O. TITUS, H. WEISSBACH AnD R. E. PETERSON. Biogenesis and metabolism of 5-hydroxyindole compounds. 7, Biel. Chem. 219: 335-344, 1956. VOGT, M. Concentration of sympathin in different parts of central nervous system under normal con- ditions and after administration of drugs. 7. Physiol. 123: 451-481, 1954. VOGT, M. Catechol amines in brain. Pharmacol. Rev. 11 (Pt. 2): 483, 1959. WALKENSTEIN, 8S. S., N. CHUMAKOW anp J. SHIFTER, Detoxication and excretion of mephen- JULIUS AXELROD 158. 159. 160. 162. 163. 164. 165. 166. Volume 39 termine. 7, Pharmacol. & Exper. Therap. 115: 16-20, 1955- WEISSBACH, H., W. KING, A. SJOERDSMA anv 8S, UDENFRIEND. Formation of indole-3 acetic acid and tryptamine in animals. 7. Biol. Chem. 234: 81-86, 1959. WEIL-MALHERBE, H., J. AXELROD anp R. TOMCHICK. The blood-brain barrier for adrenaline, Science 129: 1226, 1959. WEIL-MALHERBE, H. anp A. D. BONE. The fluorimetric estimation of adrenaline and nor- adrenaline in plasma. Biochem. 7. 67: 65-72, 1957. . WEISSBACH, H., B. G. REDFIELD anp S. UDEN- FRIEND. Soluble monoamine oxidase; its proper- ties and actions on serotonin. J. Biol. Chem. 229: 953, 1957- 7 WERLE, E. ann G. MENNICKEN. Uber die Bildung von Tryptamin aus Tryptophan und von Tyramin aus Tyrosin durch tierisches Gewebe. Biochem, Ztschr. 291: 325-327, 1937. WERLE, E. anv F. ROEWER. Uber tierische und Pflanzilche Monoaminoxydasen. Biochem. Ztschr. 322: 320-926, 1952. WOOLEY, D. W. anp E. SHAW. A biochemical and pharmacological suggestion about certain men- tal disorders. Proc. Natl, Acad. Sci. 40: 228-231, 1954. ZELLER, E. A. anv J. BARSKY. In vivo inhibition of liver and brain by 1-isonicotinyl-2-isopropylhy- drazine. Proc. Soc. Exper. Biol. & Med. 81: 459-461, 1952. ZELLER, E. A., J. BARSKY, E. R. BERMAN, M. S. CHERKAS anp J. R. FOUTS. Degradation of mescaline by amine oxidases. 7. Pharmacol. & Exper, Therap. 124: 282-289, 1958.