The Metabolism of Serotonin (5-Hydroxytryptamine) *

WILLIAM M. MCISAAC AND IRVINE H. PAGE

From the Research Division of the Cleveland Clinic Foundation and The Frank E. Bunts Educational Institute, Cleveland, Ohio

(Received for publication, August 22, 1958)

Serotonin has assumed an important role as the focal point about which much current neurochemical investigation is cen- tered. The fact that its methylated derivatives and some com- pounds which antagonize its action can cause hallucinations has even led to the hypothesis that some defect in the body’s ability to metabolize serotonin might be the underlying cause of schizo- phrenia. Yet relatively little is known about its metabolic fate in the body and 5-hydroxyindoleacetic acid, the product of osidative deamination, is the only metabolite so far established (I). Although 5-hydrosyindoleacetic acid is allegedly the major metabolite, it represents only 33 per cent in the rat, 20 per cent in man, and 1.5 per cent in the rabbit, of administered serotonin (2). Experiments in vitro with rat liver and kidney preparations have also shown that less than 30 per cent of serotonin is con- verted to 5-hydrosyindoleacetic acid (1). Other pathways of metabolism have been suggested (3) and some unidentified in- dole derivatives in urines of patients with carcinoid syndrome and perfused rat livers have been reported (4-6). In addition, Chadwick and Wilkinson (7) have provisionally identified a metabolite of 5-hydroxytryptamine in rat liver homogenates as being the ethereal sulfate derivative and the osytocic activity of normal human urine has been attributed to the presence of unchanged 5-hydrosytryptamine and its methylated derivatives

(8).

EXPERIMENTAL

Materials and Afethods

CompozLnds-5-Hydrosytryptamine-P-C’ccreatininesulfate (see Fig. 5), with an activity of 184 PC. per gm., was prepared from material with a specific activity of 2 mc. per gm. by a lo-fold dilution with nonactive material and recrystallization to constant specific activity.

5 - Hydrozytryptamine-0, N - dibenzoate: 5 - Hydrosytrypta- mine creatinine sulfate, 0.5 gm., was benzoylated with 2.2 equiv- alents of benzoyl chloride and the resulting precipitate recrys- tallized from 75 per cent ethanol to yield 0.25 gm. of 5-hydrosytryptamine-0, N-dibenzoate as colorless needles, m.p. 172-3”. (Found C 74.92, H 5.25, N 7.29; CZ1H1903N2 requires C 75.16, H 5.0, N 7.31 per cent.)

.V-acetyl-5-hydrosytryptamine: Acetylation of 5-hydrosy- tryptamine by standard methods was found to give a mixture of N - acetyl- and 0, N - diacetyl- 5 - hydrosytryptamine. The former could be distinguished chromatographically by virtue of its free hydrosyl group and was found to have an RF of 0.75 in Solvent A (See Table I).

* The authors wish to thank Mrs. Gertrude H. Britton, whose generous financial support enabled this study to be undertaken.

Synthesis of the N-acetyl derivative was att.empted by acetyla- tion (glacial acetic: acetic anhydride, 1: 1) of 5-hydrosytryp- tamine benzyl ether, 500 mg., (RF 0.8 in Solvent A) to give 200 mg. of yellow crystals of 5-benzylosy-3&N-acetylaminoethyl- indole (RF 0.54 in Solvent A). Debenzylation in ethanolic solution was accomplished with 10 per cent palladium-charcoal as a catalyst and hydrogen at room temperature for 2 hours. After removal of the catalyst the filtrate was evaporated at re- duced pressure to leave a clear gum. Paper chromatography revealed the gum to be an indole with a free hydroxyl group, RF 0.71 and 0.81, in Solvents A and R respectively, presumably N-acetyl-5-hydrosytryptamine. Attempts to crystallize the gum, however, were unsuccessful.’

5-Hydrosyindoleaceturic acid: An attempt was made to syn- thesize 5-hydrosyindoleaceturic acid from 5-hydrosyindoleacetic acid, 250 mg., and glycine ethyl ester hydrochloride, 180 mg., by the method of Sheehan and Hess (9). Attempts to crystallize the glycine conjugate from the reaction misture failed but the presence of a phenolic indole derivative was demonstrated chro- matographically with RF 0.23 and 0.86 in Solvents A and B respectively.’ This was shown to be 5-hydrosyindoleaceturic acid since it gave an orange spot when sprayed with p-dimethyl- aminobenzaldehyde in acetic anhydride demonstrating azlactone formation which is characteristic of aceturic acids (10). This reaction was not given by 5-hydrosyindoleacetic acid or any of the other metabolites.

Animals-Female W7istar rats of 200 to 250 gm. and female rabbits of Dutch or albino strains weighing 2.5 to 3 kg. were used. Serotonin and 5-hydrosyindoleacetic acid in aqueous solution were administered by intraperitoneal injection unless otherwise stated. Animals were fed on a standard diet with water ad libitum and kept in metabolism cages while under experiment.

Analytical Xethods-Glucuronic acid and ethereal sulfate in urine were determined by the methods of Paul (11) and Sperber (12)) respectively. Results are given in Table VI. 5-Hydrosy- indoleacetic acid was measured calorimetrically (13) and serotonin fluorimetrically (14). For all fluorimetric determinations an Aminco-Bowman spectrophotofluorimeter was used.

Chromatographic Methods-For the detection of metabolites in urine and urine extracts descending chromatography with Whatman T\To. 1 or No. 4 paper was used. The solvents, RF values and color reactions of reference compounds employed are given in Table I. Radioactive chromatograms were used to produce radioautographs.

For partial fractionation of metabolites, an aluminum oside column 2 x 25 cm. was employed (8). Further fractionation

1 Similar difficulties in obtaining crystalline derivatives of serotonin have been encountered by other workers (29).

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TABLE I

RR values and color reactions of some compounds related to 5-hydroxytryptamine Solvent systems used were: A, propan-l-01 saturated with aqueous. NH, solution with Whatman No. 1 paper run for 14 hours; B,

n-butanol-acetic acid-water (4:1:5) with Whatman No. 1 paper run for 14 hours; C, ethyl methyl ketone-2-N-NH3 solution (2:l) with Whatman No. 4 paper run for 3 hours. For excitation of fluorescence an unfiltered ultraviolet lamp was used; Q means the background fluorescence of the paper was quenched. The sprays used for detecting compounds on paper were: Gibbs’ reagent, consisting of 2 per cent ethanolic solution of 2,6 dichloroquinonechloroimide followed by saturated aqueous NaHC03; Naphthanil diazo blue B

(tetrazotized di-o-anisidine) 3 per cent solution plus borate buffer pH 9. (3:2); Ehrlich’s reagent, p-dimethylaminobenzaldehyde 0.5 per cent solution in 1.5 N HCl.

Indole .............................................. Indole-3-acetic acid. ...................... .........

Indole-3-proprionic acid ............................. Indole-3-n-butyric acid. ............................ Indican .............................................

L-Tryptophan ....................................... 5-Hydroxytryptophan . .............................. Dihydroxy phenylalanine ............................

Tryptamine ......................................... 5.Hydroxytryptamine . .............................. N-methyl-5-hydroxytryptamine. ..................... N-dimethyl-5-hydroxytryptamine ....................

5-Hydroxyindole-3-acetic acid. ...................... N-acetyl-5.hydroxytryptamine. ...................... Bufotenidine ........................................

Bufothionine ........................................

Dehydrobufotenine. ................................. 5-Benzyloxy-3p-N-benzyl N-methylaminoethylindole. 5.Benzyloxy-3P-dimethylaminoethylindole. ...........

5Benzyloxyaminoethylindole sulfate .................

5.Benzyloxy-3p-N-acetylaminoethylindole . ........... 5.Hydroxyindoleaceturic acid ........................

RF values in solvent Color of spots on paper with

A B C Lhrlich’s reagent

0.94 0.97 0.35 0.95 0.42 0.95 0.50 0.95 0.40 0.43

0.26 0.62 0.11 0.42 0.06 0.33

0.72 0.70 0.48 0.54 0.73 0.49

0.92 0.62 0.15 0.80 0.75 0.81 0.20 0.59

0.24 0.51 0.46 0.76 0.90 0.90

0.89 0.76 0.80 0.81 0.54 0.74 0.23 0.84

0.97 0.12 0.19 0.26 0.56

0.08 0.03 0.00

0.94 0.86 0.83

0.91 0.03 0.86 0.15

0.34 0.09 0.97 0.97

0.96 0.64

Light blue Light blue Blue

Light. blue Light blue Light blue

Light blue

Q

Pink Purple

Purple Blue Brown

Pink Blue Pale green

Purple Blue Blue Blue Blue

Blue Blue Purple Purple

Blue Blue Blue

Blue Blue

was accomplished with a BW-200 (Brown Company) cellulose

column 2 x 25 cm. with the use of a one-phase solvent system

of n-propanol saturated with ammonia.

For final fractionation separation on Whatman No. 1 paper

with the solvents described in Table I was followed by ethanolic

elution of strips. After removal of the ethanol at reduced pres-

sure, aqueous solutions of the residues were employed for fluori-

metric and pharmacological tests.

Measurement of Radioactivity-Measurements were carried out on solid samples of “infinite thickness” on nickel planchetes with an end window counter tube, the background of which was

20 to 25 c.p.m. The specific activities were determined by com- parison with a stable polymer reference. A sample of 1 sq. cm.

containing 0.1 pc. of Cl4 per gm. of substance gave approsimately

270 c.p.m. Uririlze and Tissues-Since it has been shown by Parke (15)

that there is no significant difference in the results obtained by

wet combustion of urinary residue to BaC03 (16) and those ob- tained by counting the solid residue obtained by evaporation of the urine directly on the planchetes under infrared lamps, the latter method was employed.

The radioactivity of the tissues was measured in a similar way by drying tissue homogenates directly on the planchetes. The results for the various tissues are shown in Table II; Fig. 1

shows the average rate of elimination of radioactivity in the urine. Pharmacological J4ethods-Pharmacological characterization

Fluorescence Gibbs’ Naphthanil

reagent diazo blue

Red None

None None

None Blue Blue

Blue Blue Blue Blue

Blue Blue

Brown None None None None

None Blue None

Yellow Blue Blue

Blue Blue

Blue

None

None None ?;one

Blue

ru’one

None None Xone Blue

of serotonin and its derivatives was accomplished with the use of the osytocic response of the isolated estrus rat uterus and the antagonism of this response by lysergic acid diethylamide and

its bromo derivative. The tissue was suspended in an osygen- ated organ bath containing Tyrode solution at 30”.

RESULTS

Rate of Excretion and Distribution in Tissues of Administered 5-Hydroxytryptamine-/3-C14-creatinine Sulfate-The rate of es- cretion of metabolites in the urine and feces after administration of 5-hydrosytryptamine+U4-creatinine sulfate to rats and rab-

bits was measured by the activity present (Table II). More than 50 per cent of the activity appeared in the urine within 24 hours and the rate of elimination is shown graphically in Fig. 1.

Excretion of a small part of the close in the feces was established

not by the activity of the feces alone, which may have been sub- jected to urinary contamination, but also by the activity of the lower gut contents.

The percentage distribution of a dose of exogenous serotonin in the tissues of rats and rabbits is given in Table II.

Identijication of Metabolites-In a preliminary experiment one rat was dosed with 10 mg. of U4-5-hydrosytryptamine (200 PC.)

and three rats with 10 mg. each of nonactive material. The

24-hour urines were collected, combined, and acetone extracted

by the method of 13umpus and Page (8). Chromatography (Solvent A) of the concentrate revealed the presence of seven

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860 Metabolism of Xerotonin (5-Hydroxytryptamine) Vol. 234, No. 4

TABLE II peared to be separating into two components and an eluate of Distribution in tissues and excretion of (C14) this area was resolved in Solvent B into two spots, 5-hydroxy-

5-hydroxytryptamine indoleacetic acid, RF 0.8, and 5-hydroxyindoleaceturic acid (posi-

Rats Rabbits tive azlactone spot RF 0.84).

Animal No Further evidence for the formation of 5-hydrosyindoleaceturic

I- 1 2 3 4 5 acid was obtained by dosing three rats, each with 40 mg. of 5-hydroxyindoleacetic acid. The 24-hour urines were collected

Weight (gm.) : !70 260 180 2,500 2,500 Dose of (CP) 5-hydroxy-

and found to give a blue color with 2,6-dichloroquinonechloro-

tryptamine creatinine imide but did not give a positive naphthoresorcinol reaction.

sulfate (mg.). 10 / 10 ! 7 / 20 / 20 Chromatography of these urines disclosed two indole metabolites which proved to be 5-hydrosyindoleacetic acid, RF 0.8, and

I 1 I % dose excreted in urine

5-hydroxyindoleaceturic acid, RF 0.85 (Solvent B), the latter

4hr. 2.9 6.25 3.27 69.1 71.09 giving a positive azlactone reaction (10).

24hr. .._.. 44.8 18.8 66.3 13.6 16.12 The presence of two other metabolites was observed in the

48 hr.

72 hr.

8.1 * 1.37 4.65 4.15 urine of Rat 1 (Table II). The 24-hour urine was ether extracted

0.58 4.6 4.20 at pH 5 and 8. The extracts and aqueous residue were chro-

96hr. 0.57 3.35 matographed and radioautographs obtained (Fig. 2). These ___-

91.951 55.8 / 25.051 72.091

showed the presence of 5-hydroxyindoleacetic acid and N-acetyl- Total.............. 98.91 5-hydroxytryptamine in the acid extract, little or nothing in the

Feces . 3.5 0.41 5.33 0.46 0.15 basic extract, and four other metabolites in the aqueous residue.

Stomach contents 0.01 0.0 0.46 Removal of the 5-hydroxyindoleacetic acid by extraction left

Small gut. 0.10 the 5-hydrosyindoleaceturic acid, RF 0.23, in the aqueous layer.

Gut contents. 2.4 0.13 0.0 In addition there was some 5-hydrosytryptamine, RF 0.48, and

Blood-red cells 0.61 Blood-plasma 2.64 Heart. 0.07 0.32 0.05 0.0 Lung. 0.08 0.49 0.02 0.11 Liver. Kidney :

0.50 2.80 0.38 0.0 0.20 0.44 0.05 0.08

Spleen 0.15 0.19 0.03 0.27 Brain 0.01 0.03 0.07 0.34

l- _____ _____-

Total. 67.72 32.98 79.88 97.23

* This animal died 48 hours after dosing.

Percentage excretion

4 12 24 48 72

Time in hours

FIG. 1. Average urinary excretion of radioactivity following the administration of Cl4 serotonin to rats, X---X, and rabbits, o---e.

indole spots and radioautography showed three of these to be FIG. 2. Radioautographs of indole extracts from, left, rat

radioactive (Fig. 2). These were 5-hydroxyindoleacetic acid, urine, and right, rabbit urine. The extract from rat urine has

RF 0.15; 5-hydrosytryptamine, RF 0.48, and N-acetyl-5-hydroxy- been divided into an ether fraction, extreme left, containing 5-

tryptamine. RF 0.75. hydroxyindoleacetic acid and N-acetyl-5-hydroxytryptamine; and ̂

“̂ I The 5-hvdroxvindoleacetic acid snot, an- ” r an aqueous fraction contammg the other metabolites.

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two small spots at RF 0.35 and 0.60 which gave positive color TABLE IV reactions for indoles. The latter of these also gave a blue color Elimination of metabolites of serotonin in urine of animals

when sprayed with 2,6-dichloroquinonechloroimide or tetraz- receiving 5-hydroxytryptamine-a-C’4

otized o-dianisidine (Table I) and a red color with diazotized sulfanilic acid, showing it to be a hydroxy indole with an intact amino group in the side chain. Yet its chromatographic proper- ties and fluorescent spectra (X activation 305 rnp and X emission 360 rnp with maximal sensitivity at pH 7) showed that it was not unchanged serotonin (Table III). The most probable struc- ture to fit these observations would be that of an oxidative prod- uct of serotonin, either an oxindole or dihydroxy derivative. The results of color tests for dihydroxy indoles, however, proved inconclusive.

Quantitative estimations had shown some increase in the ex- cretion of glucuronic acid after administration of serotonin and the possibility that the former of these metabolites might be a glucuronide was confirmed in the following manner. Five rats were dosed with 20 mg. each of CW5-hydroxytryptamine, the 24-hour urines collected and subjected to the systematic lead acetate precipitation method (17), and a small amount of glu- curonide gum obtained from the basic lead acet,ate precipitate. A naphthoresorcinol reaction on the gum was masked by an intense red color which developed in the aqueous phase. The residual gum was chromatographed and showed the presence of two indoles, RF 0.35 and 0.5 (Solvent A). The latter proved to be serotonin but the former gave a negative Gibbs’ reaction (See Table I) showing that the hydroxyl group was not free. An eluate of this indole was found to be radioactive and to give a positive naphthoresorcinol reaction, indicating that it was the ether glucuronide of 5-hydroxytryptamine.

?F (sol ‘ent A)

0.15

0.23

0.35

0.48 0.60

Experiment No.

0

43.5 25 14.6

34.8

5.5

11

trace trace 7.3 9.4 3.5 2.5

5 25.4

60 55.4 72.1

- 4

-

5 78

0 2 5

2

92

-

5-Hydroxyindoleacetic acid.. 5-Hydroxyindoleaceturic acid.. 5-Hydroxytryptamine glucu-

ronide. 5.Hydroxytryptamine ? Oxidation product. N-acetyl&hydroxytryp-

tarnine......................

Sum of metabolites = total activity of urine.

TABLE V Quantitative aspects of conjugation of 5-hydroxytryptamine

and 5-hydroxyindoleacetic acid

amounts of serotonin and its N-acetyl derivative were also pres- ent (Fig. 2).

Quantitative Results-Since many of the possible metabolites were difficult to obtain in sufficient quantity for standard isotopic dilution techniques, quantitative estimation of metabolites had to be made by scanning radioactive chromatograms with a sensi-

The metabolic fate of serotonin in the rabbit was elucidated in a similar fashion with the urines obtained from Animals 4 and 5

tive end-window counter and plotting the activity against RF

(Table II). Autoradiography and chromatography disclosed

values.

the major metabolite to be 5-hydroxyindoleaceturic acid (pos-

The percentage excretion of different metabolites could

itive azlactone spot Rp 0.23 and 0.84 in solvents A and B, re- spectively) , with some free 5-hydroxyindoleacetic acid. Small

Compound Animals DOS.9

1 w./kg

B-Hydroxy- tm- tamine.

5-Hydroxy- twp- tamine.

5-Hydroxy- indoleace tic acid..

Rats 80

Rabbits 50

Rats 250

Conjugation ($ZO of dose) witht

Gly- cinef

Glucuronic acid ,thereal sulfatl E

.-

-

+

++

+

10 (0 to 30)

4 (0 to 12)

0 (0 to 0)

2.4(0 to 4.4;

0 (-3.6 to f6.6)

TABLE III Fluorescent spectra

t Act&- Fluores. ion max- cent

imum maximur n [ s Ultimate ensitivity

/

Indole. Indican. 5-Hydroxytryptophan. 5.Hydroxytryptamine. 5.Hydroxyindole-3.acetic acid. N-methyl-5-hydroxytryp-

280 355

300 400 310 350

295 540

300 355

310 355

310 355 310 370

Pfl

7

7 7 2

7

7

7 7

KS/ml. 0.002 0.001 0.005 0.003

0.002

had previously been estimated (Table II). This- method was satisfactory for the acetone extracts which were found to contain the total activity of the urine.

* This represented the maximal dose which could be well tol- erated.

However, in order to estimate the percentages of 5-hydroxyindoleacetic and -aceturic acids, which by this method were counted together, their RF values in

t Results are quoted as the average for three animals, with

Solvent A being very similar, use was made of the greater ether

range in parenthesis.

solubility of the unconjugated acid. In Experiments 1 and 4, therefore, known quantities of ether extracts and aqueous res- idues containing known proportions of the total activity were

$ Demonstrated chromatographically by colored azlactone

chromatographed and counted separately.

formation (10).

then be calculated (Table IV) since the total activity of the urine

Quantitative results of glucuronide and ethereal sulfate forma- tion were estimated with the use of rats and rabbits (Table V). 5-Hydroxyindoleacetic acid caused no increased excretion but 5-hydroxytryptamine gave rise to a slight increase in both species due to the formation of an ether glucuronide.

Metabolic Fate of Endogenous Serotonin in Patients with Car-

&&d, Syndrome-Three gallons of urine from two patients with

ta~ine.....................

N-dimethyl&hydroxytryp- tarnine.....................

N-acetyl-5-hydroxytryptamine -

0.004

0.002 0.001

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HEIGHT OF

CONTRACTION (cm.)

5

1

0.1 0.2 0.3 0.4 0.5 0,6 0.7 0.8 0.9

RF VALUE OF CHROMATOGRAM ELUATES

FIG. 3. Oxytocic activity of urinary metabolites of serotonin after separation by paper chromatography (Solvent A). When eluates according to Rp value were tested, maximal activity was obtained in the two fractions Rp 0.4 to 0.5 and 0.5 to 0.6 (hatched areas), When indole spots were eluted this activity (unhatched area) was found to be associated with one spot, RF 0.48 (0.44 to 0.53), which proved to be serotonin.

cnrcinoid syndrome containing 480 mg. of 5-hydroxyindoleacetic acid were acidified to pH 5 with glacial acetic acid and concen- trated at reduced pressure at 40” to 3 1. Urease was added and the concentrate incubated at 37” for 12 hours and then reduced to 500 ml. On the addition of 5 1. of acetone a precipitate formed and was filtered off. The filtrate was then finally reduced to a volume of 20 ml. Chromatography of this concentrate revealed the presence of at least six indole derivatives and urea, Rp 0.32 (Solvent A).

An aluminum oxide column was used to fractionate 3 ml. of this concentrate. The adsorbates were eluted with methanol diluted with increasing amounts of water as follows: 100 ml. of absolute methanol, 100 ml. of 75 per cent methanol, 100 ml. of 50 per cent methanol, 100 ml. of 25 per cent methanol; 50 ml. fractions were collected. Paper chromatography of the eight eluates showed a partial fractionation with most of the me- tabolites in the first three fractions, urea, and some hydroxy- indoleacetic acid in the remaining fractions. Use was made of this to purify further the rest of the crude urinary concentrate.

The purified mixture of metabolites was then fractionated on a cellulose column with the use of a one-phase n-propanol-am- monia solvent. Twenty fractions of 10 ml. were collected, evaporated to dryness under reduced pressure, and isolation of the metabolite attempted. Fractions 2 and 3 contained N- acetyl - 5 - hydroxytryptamine. Fractions 4 and 5 contained 5-hydroxytryptamine and indican. Fractions 6, 7, 8, and 9 con- tained 5-hydroxyindoleacetic acid. These were identified by paper chromatography in two solvents; when the fractions con- taining 5-hydroxyindoleacetic acid were chromatographed in Solvent B, in addition to the spot for 5-hydroxyindoleacetic acid, RF 0.80, there was another spot at RF 0.86, 5-hydroxyindole- aceturic acid.

Zsolation of Metabolites from Urine of Patients with Carcinoid

Syndrome-Serotonin was isolated from Fractions 4 and 5 which were pooled and benzoylated with excess benzoyl chloride, the precipitate filtered off and recrystallized from ethanol to give about 2 mg. of 5-hydroxytryptamine-0, N-dibenzoate, m.p. and mixed m.p. with authentic material, 171 to 173”.

Attempts to isolate N-acetyl-5-hydroxytryptamine and 5-hy-

droxyindoleacetic acid from the relevant fractions by forming benzoates or p-toluene sulfonates were unsuccessful.

Characterization of Metabolites-Some of the urinary concen- trate, 0.5 ml., was streaked on Whatman No. 1 paper and sep- aration of metabolites achieved with Solvent A. A control strip from the side of the chromatogram was sprayed and sec- tions of the paper corresponding to the metabolites were eluted with ethanol. The eluates were evaporated to dryness under reduced pressure and the residues dissolved in 1 ml. of water. These aqueous solutions were used for determination of the fluorescent spectra of the metabolites (Table IV), oxytocic ac- tivity (Fig. 3) and chromatographic identification in two solvents (Table I). In this way it was possible to identify the following five indole derivatives in order of their increasing Rp value in Solvent A: 5-hydroxyindoleacetic acid, RF 0.17; 5-hydroxy- indoleaceturic acid, RF 0.23; indoxyl potassium sulfate, RF 0.40; 5-hydroxytryptamine, RF 0.48; and N-acetyl-5-hydroxytryp- tamine, RF 0.72. A small amount of another unidentified indole derivative was present (RF 0.81 and 0.86 in Solvents A and B, respectively).

DISCUSSION

Exogenous serotonin is rapidly metabolized by rats and rabbits and excreted, 50 to 80 per cent being eliminated in the urine within 24 hours (Fig. 1). The only exception in Experiment 2, where excretion was 25 per cent in 24 hours, the animal developed paresis of the hind legs, became comatose, and died. Enlarged pale kidneys were revealed at necropsy.

Excretion of 0.5 to 5 per cent of a dose of serotonin via the gut was confirmed by the activity of the lower gut contents (Table II). Since these animals were dosed by intraperitoneal injection this must have been due to excretion. This excretion was ac- companied by increased gut motility.

Serotonin has been reported to cause hypernea in some animal species (18, 19) and a moderate tachypnea was observed during the course of these experiments which was of interest since the specific activity of the lung tissue was found to be fairly high, even 3 days after administration.

Platelets in vivo are not saturated with serotonin (20) and it has been shown (21) that they will take it up in vitro. This ap- pears to be true in zrivo since the activity of the blood following administration of CY-serotonin was largely associated with the platelet containing fraction of the plasma and the level was maintained for some time, being 3.25 per cent after 2 days and 3.3 =t 1.7 per cent after 3 days (Table II). The small amount of activity associated with the red cells might be due to their ability to absorb serotonin (22) though to a smaller extent than platelets. This evidence suggests that serotonin taken up by the platelets may be protected from metabolism and elimination for several days.

Much evidence has been presented for the inability of serotonin to cross the blood-brain barrier. The present radioactive studies, however, suggest that a small amount of it may do so since the brains of two of the rats were found to contain significant amounts of activity and in one of the rabbits, 4 days after dosing, the brain had a higher specific activity than the other organs (Table II). The fact that this activity might be due to the entry of metabolites rather than serotonin, however, cannot be discounted.

Species Difference in Metabolism-Several reports have been made of species differences in serotonin metabolism (2, 23). Sanyal and West (24) found such in anaphylactic shock and

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R= C6 Hg06 or S03H and XorY = H or OH

FIG. 4. Metabolism of 5-hydroxytryptamine-&CP. Major route arises from monoamine oxidase (MAO) activitv but acetvlation conjugation, and excretion unchanged are important alternatives. Oxidation is also a possibility. ~ ~’

Erspamer (2) noted that whereas carnivorous animals excreted 5-hydroxyindoleacetic acid as the major metabolite that herbiv- orous species excreted practically none. In the present work this species difference in the metabolism of serotonin has been confirmed. In both species oxidative deamination was the major pathway, but whereas all the rats excreted a mixture of 5-hy- droxyindoleacetic acid a,nd 5-hydroxyindoleaceturic acid, the rabbits excreted mainly the glycine conjugate (Table IV). This difference might account for the slightly more rapid excretion by rabbits (Fig. 1). Although there was no difference in glu- curonide formation (Table V) there were some minor quantitative differences in the excretion of the other metabolites.

ikfetubolic Fate of Serotonin-As can be seen from Table V the major metabolites of serotonin in the urine of rats and rabbits are those that result from amine oxidase action. Over 50 per cent of the dose excreted in the urine is accounted for by 5-hy- droxyindoleacetic and 5-hydroxyindoleaceturic acids. Subse- quent glycine conjugation occurs to a much greater extent in rabbits than in rats.

Acetylation plays an important part in the metabolism of serotonin, 5 to 25 per cent of the urinary metabolite being in the form of N-acetyl-5-hydroxytryptamine. This product has no oxytocic activity (see Fig. 3).

Excretion of unchanged serotonin has been found to account for 5 to 9 per cent of the dose, and serotonin is certainly the most potent oxytocic compound present in urine (Fig. 3).

Conjugation with glucuronic acid and ethereal sulfate has been found to occur, though only to a small extent; the ether glu- curonide of 5-hydroxytryptamine being formed. In comparison 5-hydroxyindoleacetic acid does not appear to form any ether conjugates, being excreted almost entirely as a mixture of the unchanged acid and its glycine conjugate.

One of the metabolites of norepinephrine has been shown to be 3-methoxy-4-hydroxy mandelic acid (25). This involves methyl- ation of the phenolic hydroxyl group. No evidence of O-methyl- ation of exogenous serotonin could be found in these experiments.

N-methylation of amino compounds in vivo has been shown e.g. N-methylation of histamine with subsequent excretion as N-methyl and N-dimethyl histamine in the urine (26). The possibility of serotonin being N-methylated is interesting since it has been shown that the N-methyl derivatives have hallucino- genic properties (27). However, in this study of the normal metabolic fate of exogenous serotonin no evidence has been found for the formation of methyl derivatives.

The possibility of further oxidation is of interest since it has been suggested by Dalgliesh (28) that serotonin might function through some more active derivative such as 5,6-dihydroxy- tryptamine. Although an exact characterization has not been possible, some evidence has been found in the present work that one of the minor metabolites of serotonin might be an oxida- tion product. Although this metabolite possessed little oxytocic activity, and other work in this laboratory has shown that 5,6-dihydroxytryptamine as a vasopressor is less potent than serotonin, it does not preclude the possibility that this derivative might be centrally active.

The metabolism of serotonin has proved to be complex and it may be possible to interpret the actions of some drugs, e.g. mono- amine oxidase inhibitors, by the way they interfere with the normal metabolic pattern. Since it is possible that some psy- choses may be due to abnormal metabolism of serotonin, elucida- tion of its normal metabolism ha.s been a necessary preliminary to the study of these other problems.

SUMMARY

1. A study has been made of the metabolism of exogenous CY- 5-hydroxytryptamine in rats and rabbits.

2. The activity of various tissues following administration of W-5-hydroxytryptamine has been estimated. Major activity was found in the platelet-containing fraction of the plasma and significant activity was found in lung and brain tissue.

3. The excretion of activity after administration of the radio- active compound to rats and rabbits has been found to be 50 to

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864 Metabolism of Serotonin (5-Hydroxytryptamine) Vol. 234, No. 4

98 per cent of the dose in 24 hours in the urine with a concomitant 6. A species difference was observed in the metabolism of excretion of 3 to 5 per cent in the feces, serotonin: rats excrete a mixture of 5-hydroxyindoleacetic acid

4. The following metabolites have been identified in the urine and 5-hydroxyindoleaceturic acid, however, rabbits excrete of rats and rabbits by chromatography, radioautography, fluo- mainly the glycine conjugate. rescent spectra, and biological activity: 5-hydroxyindoleacetic 7. Serotonin has been isolated from the urine of patients with acid; 5-hydroxyindoleaceturic acid; 5-hydroxytryptamine; N- acetyl- 5 - hydroxytryptamine, and 5- hydroxytryptamine glu-

carcinoid syndrome and in these subjects the metabolic fate ap-

curonide. A minor metabolite has been provisionally identified pears to be similar to that described in the rat.

as an oxidation product. 5. Quantitative estimation of these metabolites, by scanning Acknowledgments-The authors wish to thank Mrs. Pamela

radioactive chromatograms, has shown 35 to 83 per cent of the Taylor for her excellent technical assistance, Dr. K. E. Hamlin,

dose to be metabolized bv oxidative deamination and 5 to 25 of Abbott Laboratories, for supplying the radioactive serotonin,

per cent by N-acetylation. The other minor metabolites ac- and Dr. R. Bircher, of Sandoz, for generous supplies of serotonin

count for the remaining 5 to 10 per cent of the dose. and lysergic acid diethylamide.

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15.

REFERENCES

UDENFRIEND, S., TITUS, E., WEISSBACH, H., AND PETERSON, 16. CALVIN, M., HEIDELBERGER, C., REID, J. C., TOLBERT, B. H., R. E., J. Biol. Chem., 219, 335 (1956). AND YANKWICH, P. F., Isotopic carbon, John Wiley and Sons,

ERSPAMER, V., J. Physiol. London, 127, 118 (1955). Inc., New York, 1949. BLASCHKO, H., AND PHILPOT, F. J., J. Physiol. London, 122, 17. KAMIL, I. A., SMITH, J. N., AND WILLIAMS, R. T., Biochem. J.,

403 (1953). 50, 235 (1951). DALGLIESH, C. E., Biochem. J., 64, 481 (1955). 18. PAGE, I. H., J. Pharmacol. Exptl. Therap., 105, 58 (1952). JEPSON, J. B., Lancet, 1, 119, (1955). 19. MICHELSON, A. L., HOLLANDER, W., AND LOWELL, F. C., J. DALGLIESH, C. E., AND DUTTON, R. W., Biochem. J., 66, 21~ Lab. Clin. Med., 61, 57 (1958).

(1957). 20. HARDISTY, R. M., AND STACEY, R. S., J. Physiol. London, 130, CHADWICK, B. T., AND WILKINSON, J. H., Biochem. J., 68, 711 (1955).

lp (1958). 21. BRODIE, B. B., Proceedings of the symposium on B-hydroxy-

BUMPUS, F. M., AND PAGE, I. H., J. Biol. Chem., 212, 111 tryptamine, Pergamon Press, Ltd., New York, 1957, p. 71.

(1955). 22. STACEY, R. S., J. Physiol. London, 132, 39p (1956).

SHEEHAN, J., AND HESS, G., J. Am. Chem. Sot., 77,1067 (1955). 23. NAKAI, K., Nature, 181, 1734 (1958).

GAFFNEY, G. W., SCHREIER, K., DIFERRANTE, N., AND ALT- 24. SANYAL, R. K., AND WEST, G. B., Nature, 130, 1417 (1957).

MAN, K. I., J. Biol. Chem., 206, 695 (1954). 25. SHAW, K. N. F., MCMILLAN, A., AND ARMSTRONG, M. D.,

PAUL, J., Ph.D. Thesis: University of Glasgow, 1951. Federation Proc., 15, 353 (1956).

SPERBER, I., J. Biol. Chem., 172, 441 (1948). 26. KAPELLER-ADLER, R., AND IGGO, B., B&him. et Biophys.

UDENFRIEND, S., TITUS, E., AND WEISSBACH, H., J. Biol. Acta., 26, 394 (1957).

Chem., 216, 499 (1955). 27. FABING, H. B., AND HAWKINS, J. R., Science, 123, 886 (1956). 28. DALGLIESH, C. E., Proceedings of the symposium on ti-hydroxy-

BOGDANSKI, D. F., PLETSCHER, A., BRODIE, B. B., AND tryptamine, Pergamon Press, Ltd., New York, 1957, p. 60. UDENFRIEND, S. J. Pharmacol. Exptl. Therap., 117,82 (1956). 29. SPEETER, M. E., HEINZELMANN, R. F., AND WEISBLAT, D. I.,

PARKE, D. V., Biochem. J., 62, 339 (1956). J. Am. Chem. Sot., 73, 5516 (1951).

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