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Metabolites of labelled 5-hydroxytryptamine in urine and bile of rats after various routes of administration
Inc. .I. Ncurophnrmwl..
1964. 3, 149-156
Pergamon
Press.
Printed
in Cit. Britain.
[I table,
4 figs..
16 reis.]
METABOLITES OF LABELLED 5-HYDROXYTRYPTAMINE IN URINE AND BILE OF RATS AFTER VARIOUS ROUTES OF ADMINISTRATION* MAUKO M. AIRAKSINEN Department
of Pharmacology,
University of Helsinki, Finland
(5HT) were studied in Summary-Urinary metabolites of “C-labelled 5-hydroxytryptamine hydrated rats after subcutaneous (s.c.), rapid and slow intravenous (i.v.). and oral administration. Total excretion after an oral dose was about half of those after parenteral doses. Chromatography of an 8 hr urine sample showed that O-conjugates of SHT (mainly Oglucuronide, less O-sulphate) were the main metabolites after oral administration and that smaller quantities of the conjugates were excreted after S.C.than after i.v. administration. Conversely small amounts of 5-hydroxyindoleacetic acid (SHIAA) were excreted after oral administration and also less after i.v. than after S.C. injection. O-conjugates of SHIAA. also were excreted in smaller amounts after oral than after parenteral administrations. In anaesthetized rats the bile also was collected after S.C. and oral doses of ‘“C-SHT. It contained a small amount of radioactivity, which seemed to be due to O-conjugates of 5HT and SHIAA. INTRODUCTION
SOMEeffects of 5-hydroxytryptamine (serotonin, 5HT) are more pronounced after subcutaneous than after intravenous or intraperitoneal administration as, for example, in its antidiuretic action (ERSPAMER 1954, ERSPAMER and OTTOLENGH~ 1953) and also the potentiation of the barbiturate sleeping time (WESTERMANN, 1962). Preliminary studies from this laboratory (AIRAKSINENet al. 1960) showed that 5-hydroxyindoleacetic acid (SHIAA) is excreted to a greater extent after subcutaneous than after intravenous administration in rats, as shown in the Fig. 1. In contrast, chromatograms of rat urine showed that after 5HT administration the SHT-0-glucuronide spot was more intense after intravenous injection as compared with subcutaneous administration. In the present work the phenomenon were further studied by using llC labelled 5HT. The biliary metabolites were also measured in some experiments. METHODS
Plastic cannula filled with heparainized saline were placed in the jugular vein of adult male Sprague-Dawley rats (average weight 180 g) under light chloralose anaesthesia and 5HT was administered orally, subcutaneously or by rapid or slow injection into the jugular vein two days after inserting the cannula. 5-Hydroxytryptamine-3’J4C creatinine sulphate? was diluted with carrier: so that 1 PC contained 1 mg 5HT base. All doses of 5HT refer to the base and were given in saline. * Presented at the Second International t Radiochemical Centre, Amersham. : F. Hoffman-La Roche & Co.
Pharmacological
149
Meeting, Prague, August 1963.
150
MAUNO M. AIRAKSINEN T
SO ,’
1’ :i :: ’ : : :, : I .i
LO
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o-2 HOURS
AFTER OF
L-6
6-8
ADMINISTRATION 5HT
FIG. 1. Urinary excretion of 5-hydroxyindoleacecic acid (SHIAA) before and after the administration of SHT (1 mg/kg) intravenously (i.v.) or subcutaneously (SC.) in rats given water (3.5 per cent of body weight) every 2 hr. Values are means of five experiments. Vertical lines (UDENindicate standard error of mean. Determinations of SHIAA spectrophotometrically FRIEND, TITUS and WEISSBACH, 1955).
The rats were given tap water through a stomach tube (equivalent to 3.5 per cent of body weight) twice in the first hour and repeated after 4 hr. The radioactive 5HT or saline were also given after 4 hr and the urine collected thereafter. Water was administered also 4 and 8 hr after the 5HT administration. Later they were allowed to drink ad libitum and to eat once a day. Bile was collected through a plastic cannula with a thin glass point inserted into the hepatic duct through the abdominal wall. The bladder was also cannulated
Metabolites of labelled 5-hydroxytryptamine
151
through the same wound in the abdominal wall. All operative procedures were carried out under chloralose anaesthesia (1.2 g/kg intraperitoneally, and increased when needed). Urine samples were collected between O-8 hr and 8-24 hr after the 14C-5HT administration. For measuring the total radioactivity of the urine and bile, an aliquot usually l/10 or l/20, of the samples, or about O-5 ml, was dried in an aluminium plancetes under an infrared lamp and counted with a thin-window counter tube FHZ 15b (Frieseke-Hoepfner) in a lead chamber. Control samples were made by mixing a small volume of the injected 14C-5HT solution with a similar amount of non-radioactive urine or bile. Selecta paper 2043 bM of Sleicher & Schiill was used for ascending paper chromatography using the following solvents: n-butanol-acetic acid-water (4: 1 :5, organic layer), isopropanol-ammonia-water (10: 1: I), n-butanol-pyridine-water (1: 1: I), and 20 per cent aqueous KCl. All reagents were E.Merck’s p.s./for chromatography. The chromatograms were counted in an automatic radiochromatograph apparatus using two thin-window Geiger tubes (FHZ 15a) and thin protective membranes on each side of the paper. The background of two tubes was about 40 c.p.m. All the bile chromatograms and the least active urine chromatograms were counted with one tube without protective membrane. Two dimensional chromatograms were exposed to X-ray film (Kodak Blue Rand) and were also sprayed with p-dimethylaminobenzyaldehyde reagent (BLOCK et al., 1958). In addition to some commercial 5-hydroxyindoles, SHT-0-glucuronide purified from rabbit urine (AJRAKSINEN, 1961) and SHT-O-sulphate synthetized according to KISHIMOTO, et al. (1961) as well as SHIAA-O-sulphate containing material synthetized analogously to 5-HTO-sulphate were used as control substances. The spectral and biological properties of these substances as well as further details of the chromatography and other methods are published elsewhere (AIRAKSINEN, 1963).
RESULTS The urinary excretion of radioactivity after a 10 mg/kg dose of labelled 5HT in normal rats is shown in Fig. 2. About 50-60 per cent of the parenteral doses was found in the urine during the first 8 hr, 2-3 per cent during the following 16 hr, and nearly similar amounts during the following 3 days. After oral administration (through a stomach tube) only about half of these quantities were excreted in the first 8 hr; however, the excretion was as high or higher than after parenteral administration after 8 hr. The total faeces collected over 4 days after 5HT administration to three rats, extracted with acetone and water, was found to contain less than 10 per cent of the orally administered 14C-5HT. Subcutaneous 5HT caused anuria of long duration and tle urinary excretion of 5HT metabolites was slower in rats with the biliary+urinary fistulas than in normal nonanaesthetized rats. The 8-24 hr excretion seemed also to be higher in the anaesthetized animals after oral administration, as compared with that in non-anaesthetized animals though no complete anuria was found. A small but significant portion of radioactivity was found in the bile after subcutaneous and after oral administration. One dimensional chromatograms of a urine sample collected over the first 8 hr run in butanol-acetic acid, showed four main peaks of radioactivity. Comparison with twodimensional chromatograms of urine and control substances sprayed with different reagents (AIRAKSINEN 1961, 1963) indicated that the peak having the lowest Rf value was due to 5HT0-glucuronide and -O-sulphate. Two-dimensional autoradiograms of the same urine
MAUNO M. AIRAKSINLN
152
samples showed that StiT-O-sulphate, though always present, was the minor component of the peak; less than one-tenth of the radioactivity of the glucuronide conjugate.
FIG. 2. Urinary excretion of radioactive products after administration of 10 pg/kg (= 10 mg/ kg) of r4C-5HT by various methods of administration in non-anaesthetized rats. Means of five or six experiments in per cent of the radioactive dose. The slow intravenous (Lv.) administration lasted 60 min and the rapid injection less than 1 min. A plastic cannula was inserted in the jugular vein at least 2 days before the beginning of the experiment; both the rapid and slow i.v. injections were made through this cannula.
:
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urine
L
FIG. 3. Biliary and urinary excretion of radioactive products:after subcutaneous (s.c.) and oral dose of 10 pg/kg (= 10 mg/kg) of labelled 5HT to (three with SC., two with oral dose) rats under urethane anaesthesia. The rats had cannulas in the urinary bladder and in the bile duct (ductus hepaticus), inserted just before the beginning of the experiment.
Metabolites of labelled 5hydroxytryptamine
153
The second radioactive peak (Rf 0 *20-O*45) included the glucuronide and sulphate conjugates of SHIAA and a small amount of an unidentified substance (possibly oxidation product). The two corresponding to the glucuronide and sulphate conjugates of 5HAA were not completely resolved in most chromatograms. However, the SHIAA-O-sulphate seemed to contain a greater proportion of radioactivity than that of SHT-O-sulphate, though SHT-0-glucuronide often was present in higher amount than the glucuronide of SHIAA. A third small radioactive peak contained free 5HT and some other unidentified material found particularly after intravenous administration. The peak of highest Rf value (65-85) was indentified as SHIAA. After parenteral administration the peak included an additional weak spot (less than one-tenth of the activity of this area in the autoradiograms) of higher Rf value than that of SHIAA in the isopropanol-ammonia system. This spot did not react with p-dimethylaminobenzaldehyde; unlike the other spots which yielded a violet or blue colour. N-acetyl-5HT and the 0- or N-methyl conjugates of 5HT were not detected on the chromatograms. The amounts of different metabolites after various routes of 5HT administration are expressed as per cent of the total radioactivity excreted as shown in Table 1 or as per cent of dose in Fig. 4. After oral administration SHT-0-glucuronide was the major metabolite. The O-conjugates of 5HT constitutes 17.1 f 1.5 per cent of the administered 5HT and over 70 per cent of the total urinary metabolites excreted. TABLE 1. PROPORTIONS OF THE RADIOACTIVITY IN PER CENT OF THE TOTAL URINARY RADIOACTIVITY IN DIFFERENT AREAS OF PAPER CHROMATOGRAMS PREPARED FROM SAMPLES THE FIRST 8 HR URINE OF RATS GIVEN W-5HT (10 &KG). SOLVENT BUTANOL-ACETIC ACID WATER. MEANS & S.E. EACH GROUP FIVE OR SIX EXPERIMENTS
Total urinary radioactivity ‘A of dose
Rf O-G.20 (O-conjugates of 5HT)
Rf 0.21-0.42 (O-conjugates of SHIAA)*
Rf 0.43-055 (5HT, etc.)
Rf 0.7&0+?5 (SHIAA)
Subcutaneous
55.713.7
10.4&0.4
30.1%1.0
1.6hO.7
57.9k1.6
Slow intravenous
53.Oh8.7
16.3k3.2
35.3zL1.9
0.710.4
47.7A4.9
Rapid intravenous
56.4A3.8
24.112.3
30.8hl.9
2.2hO.7
42.3% 1.8
Oral
23.6& 1.7
72.2*3.4
8.5h1.2
1.4+ 1.4
17.832.4
Method of administration
* This peak was later found to contain also the O-conjugates of 5-hydroxytryptophol.
After rapid intravenous injection 13 *9f 1 a9 per cent of the labelled 5HT was excreted as 5HT conjugates in contrast with 9.6h2.3 per cent after slow intravenous and 5 *Sk l-3 per cent after subcutaneous administration. The SHIAA spot accounted for only 4.3f0.7 per cent of the oral 5HT dose and 17.8 per cent of the activity excreted. After slow and rapid intravenous administration the SHIAA accounted for 24*0&3*2 and 24*3f2-1 per cent of the dose, and 32.2f2.2 per cent after subcutaneous injection.
154
MAUNOM. AIRAKSINEN
100 [--1 5HIAA ~
O.conjugatesof 5HIA.~
5HT etc. I ~ l O-conjugatesof 5HT I.Z W U tY W 13_
50
S.C.
I.V. SLOW
I.V. PER OS RAPID
FIG. 4. Percentage of various metabolites of labelled 5HT in the first 8 hr urine of the anaesthetized rats as measured from the paper chromatograms. Means of five or six experiments in per cent of the administered 5HT. Significant differences (p<0'05 according to Student's t-test) were found between all the administration routes in 5HIAA and in O-conjugates of 5HT. Differences between rapid and slow intravenous administrations were not statistically significant (p>0"05). The O-conjugates of 5HIAA as well as 5HT, etc., were also significantly lower after oral than after parenteral administration Only small amouts of O-conjugates of 5 H I A A were detected after oral administration. The urinary excretion of the O-conjugates of 5 H I A A were not significantly different after intravenous or subcutaneous injection. M o r e free 5HT as well as the unknown substances were excreted after rapid intravenous administration compared with the other routes of administration. The O-conjugates of 5 H I A A and 5HT appeared to be the only metabolites of 5HT in the bile. 5HIAA-O-sulphate possibly was the main biliary metabolite after both subcutaneous and oral administration of 5HT, but 5HT-O-glucuronide also was present. DISCUSSION The amounts of radioactivity found in bile were comparable to those in rat faeces after parenteral administration of labelled 5 H T reported by results of MCISAAC and PAGE (1959) and KEGLEVIC et al. (1960). The extent of reabsorpfion of the metabolites of 5HT is unknown. The metabolic products could presumably enter into the gut contents by other routes. So far we have studied only the excretion into the gastric juice, which was very small (less than 1 per cent of the 5HT dose) (KEYR!LMNEN and AIRAKSINEN, 1963). After oral administration the glucuronide may be formed in the intestinal wall and liver before entering the general circulation. Oral administration of 5HT m a y have little general actions for this reason. In addition the gut and liver have high monoamine oxidase activity (BLASCHKO, 1952; TlSSARI, 1963), but at least in the Sprague-Dawley rat in vivo this enzyme seems to be of less importance in the metabolism of 5HT administered orally than glucuronide formation. Also in vitro 5HT, but not 5-hydroxytryptophan and 5HIAA, was a
Metabolites
of labelled 5-hydroxytryptamine
155
good substrate of uridine diphosphate transglucuronylase in the duodenal mucosa, kidney and liver of Sprague-Dawley rats; Wistar rats showed lower enzyme activities in the duodenum and kidney (AIRAKSINEN and MIETTINEN,1963). The reasons for the different metabolic patterns after intravenous and subcutaneous administration are not fully understood. It may be due in part to the monoamine oxidase activity found in fat tissue (STOCK and WESTERMAN, 1963) and in the walls of blood vessels (THOMPSONand TICKNER, 1951). Part of the subcutaneously given 5HT may therefore become deaminated before entering circulation. There was a greater tendency for the 5HT to be metabolized by conjugation, after rapid intravenous injection than after slow injection. The higher initial concentration of 5HT in the circulation after rapid intravenous administration than after subcutaneous or slow intravenous administration may cause a difference of uptake of the amine into the platelets and other tissues. Of interest, reserpine pretreatment almost eliminates the differences in amounts of 5HIAA excreted between the subcutaneous and intravenous administrations of 5HT (AIRAKSINENet al, 1960); also the effect of 5HT on the circulation itself could affect its distribution and subsequent mode of metabolism and may differ with different doses of 5HT. The differences in the fate of 5HT according to the method of administration seem to be connected with the differences in the antidiuretic action of 5HT in rats and rabbits (AJRAKSINEN,1963).
Rbum&Les mCtabolites urinaires de la 5-hydroxytryptamine (5HT) marquQ au C” ont Btt Ctudits chez la rat hydrate apr&s administration sous-cutan& (s.c.) intraveineuse (iv.) lente et rapide et per OS. Lors de l’administration orale, l’excr&ion totale est r6duite d’environ de moitiC par rapport aux injections parent&ales. La chromatographie d’un 6chantillon d’urine de 8h. montre que les 0-conjug6.s de 5HT (principalement l’O-glucuronide, moins I’O-sulphate) sont excr&s. Apr&s administration s.c., de plus petites quantites de conjuguCs sont excr&% qu’aprts injection i.v. Ce sont less m&abolites principaux aprbs administration orale. Inverstment, de petites quantities d’acide 5-hydroxyindoleacetique (5HIAA) sont excrCtCes par administration orale et egalement moins apr& l’i.v. qu’aprts la S.C. Les O-conjuguts de SHIAA sont egalement excr&s en quantitds moindres aprks le per OS qu’aprb administrations parent&ales. Chez le rat anesthbi6, la bile a kgalement BtBrecueillie apres administrations s.c.et orales de P-5HT. Elle contient une petite quantit6 de radioactivk semblant provenir des O-conjugub de 5HT et de SHIAA. (5HT) Zussammenfassung-Die Harnmetaboliten von ‘“C-markierten 5-Hydroxytryptamin wurden nach der subcutanen (s.c.) nach der schnellen und langsamen intravenbsen (i.v.) und nach der oralen Verabreichung in hydrieren Ratten untersucht. Die totale Exkretion war nach der oralen Dose etwa die Hllfte der Exkretion nach parenteralen Dosen. Die Chromatographie de3 Harns in den emsten 8 Stunden ziegte dass die 0-Konjugate von 5HT (haupstlchlich 0-Glukurunide, weinger 0-Sulfat) nach den S.C. Dosen in geringerem Masse ausgeschiedon wurden als nach den i.v. Dosen und das es die Hauptmetaboliten nach der oralen Verabreichung waren. Umgekehrt war die Exkretion als 5-Oxyindolessigslure (SHIAA) nach der oralen Verabreichung sehr gering; ebenso war sie kleiner nach den i.v. als nach den S.C. Injektionen. Auch die 0-Konjugate von SHIAA waren nach der oralen Verabreichung in kleineren Mengen vorhanden als nach den parenteralen. In narkotisierten Ratten wurde nach den S.C. und oralen Dosen von 14C-5HT such die Galle gesammelt. Sie enthielt enie geringe Menge RadioaktivitLt, die aus den 0-Konjugaten von 5HT und SHIAA zu stammen schien. Aufhor’s address-M. M. AIRAKSINEN, Department of Pharmacology, 10, Helsinki, Finland
University of Helsinki, Siltavuorenpenger
156
MAUNO M. AIRAKSINEN REFERENCES
AIRAKSINEN,M. M. (1961). The effect of the route of administration on the metabolism of S-hydroxytryptamine. Biochem. Pharmacol. 8: 245-246. AIRAKSINEN,M. M. (1963). Studies of the metabolism and antidiuretic action of 5-hydroxytryptamine and the effect of mode of administration. Ann. Med. exp. Fenn. 41: suppl. 4. AIRAKSINEN,M. M. and MIETTINEN,T. A. (1963). In preparation. AIRKSINEN,M. M., PAASONEN,M. K. and PELTOLA, P. (1960). New aspects in the 5-hydroxyindoleacetic acid excretion after administration of 5-hydroxytryptamine. Ann. Med. exp. Fenn. 38: 237-241. BLASCHKO,H. (1952). Amine oxidase and amine metabolism. PharmacoL Rev. 4: 415458. BLOCK. R. J.. DURRUM. E. L. and ZWEIG. G. (1958). A manual of uaDer chromato~ra~hv andmwer _-_ __ electrophiresis,.Academic’Press, New York,‘2nd Edit&n, p.710. ”_ _ ERSPAMER,V. (1954). Pharmacology of indolealkylamines. Plzarmacol. Rev. 6: 425-487. ERSPAMER,V. and OTTOLENGHI,(1953). Pharmacological studies on enteramine-VI11. Action of enteramine on the diuresis and the renal circulation of the rat. Arch. int. Pharmacodyn. 93: 177-201. KEGLEVIC,D., SUPEK,Z., KVEDER, S.,ISKRIC, S., KECKES,S. and KISIC, A. (1960). The metabolism of exogenous 14C-labelled 5-hydroxytryptamine in rats. Biochem. J. 73: 53-60. KEYRIL~INEN,0. and AIRAKSINEN,M. M. (1963). Unpublished results. MCISAAC, W. M. and PAGE, I. H. (1959). The metabolism of serotonin (5-hydroxytryptamine). J. biol. C’hem. 235: 858-864. STOCK, K. und WESTERMANN, E. (1963). Biogene Amine im Fettgewebe. Naynun-Schmiedeberg’s Arch. exp. Path. Pharmak. 246: 15-16. THOMPSON,R. H. S. and TICKNER, A. (1951) The occurrence and distribution of monoamine oxidase in blood.vessels. J. PhysioQLond.) 115:34-40. TISSARI,A. (1963). The distribution of 5-hydroxytryptophan decarboxylase and monoamine oxidase during foetal development of the guinea-pig. Second Znt. Pharmacol. Meeting, Prague, 20-23 August, 1963; abstract no. 129, Biochem. Pharmacol. 12: suppl. 39. UDENFRIED,S., TITUS, E. and WEISSBACH,H. (1955). The identification of 5-hydroxy-3-indoleacetic acid in normal urine and a method for its assay. J. biol. Chem. 216:499-505. WESTERMANN,E. 0. (1962). Discussion to T. C. Butler’s paper: Duration of action of drugs as affected by tissue distribution. Proc. First Znt. Pharmacol. Meeting, August 22-25, 1961. Vol. 6. (Edited by B. B. BRODIEand E. G. ERD&), Pergamon Press, Oxford, p. 205-211.
1964. 3, 149-156
Pergamon
Press.
Printed
in Cit. Britain.
[I table,
4 figs..
16 reis.]
METABOLITES OF LABELLED 5-HYDROXYTRYPTAMINE IN URINE AND BILE OF RATS AFTER VARIOUS ROUTES OF ADMINISTRATION* MAUKO M. AIRAKSINEN Department
of Pharmacology,
University of Helsinki, Finland
(5HT) were studied in Summary-Urinary metabolites of “C-labelled 5-hydroxytryptamine hydrated rats after subcutaneous (s.c.), rapid and slow intravenous (i.v.). and oral administration. Total excretion after an oral dose was about half of those after parenteral doses. Chromatography of an 8 hr urine sample showed that O-conjugates of SHT (mainly Oglucuronide, less O-sulphate) were the main metabolites after oral administration and that smaller quantities of the conjugates were excreted after S.C.than after i.v. administration. Conversely small amounts of 5-hydroxyindoleacetic acid (SHIAA) were excreted after oral administration and also less after i.v. than after S.C. injection. O-conjugates of SHIAA. also were excreted in smaller amounts after oral than after parenteral administrations. In anaesthetized rats the bile also was collected after S.C. and oral doses of ‘“C-SHT. It contained a small amount of radioactivity, which seemed to be due to O-conjugates of 5HT and SHIAA. INTRODUCTION
SOMEeffects of 5-hydroxytryptamine (serotonin, 5HT) are more pronounced after subcutaneous than after intravenous or intraperitoneal administration as, for example, in its antidiuretic action (ERSPAMER 1954, ERSPAMER and OTTOLENGH~ 1953) and also the potentiation of the barbiturate sleeping time (WESTERMANN, 1962). Preliminary studies from this laboratory (AIRAKSINENet al. 1960) showed that 5-hydroxyindoleacetic acid (SHIAA) is excreted to a greater extent after subcutaneous than after intravenous administration in rats, as shown in the Fig. 1. In contrast, chromatograms of rat urine showed that after 5HT administration the SHT-0-glucuronide spot was more intense after intravenous injection as compared with subcutaneous administration. In the present work the phenomenon were further studied by using llC labelled 5HT. The biliary metabolites were also measured in some experiments. METHODS
Plastic cannula filled with heparainized saline were placed in the jugular vein of adult male Sprague-Dawley rats (average weight 180 g) under light chloralose anaesthesia and 5HT was administered orally, subcutaneously or by rapid or slow injection into the jugular vein two days after inserting the cannula. 5-Hydroxytryptamine-3’J4C creatinine sulphate? was diluted with carrier: so that 1 PC contained 1 mg 5HT base. All doses of 5HT refer to the base and were given in saline. * Presented at the Second International t Radiochemical Centre, Amersham. : F. Hoffman-La Roche & Co.
Pharmacological
149
Meeting, Prague, August 1963.
150
MAUNO M. AIRAKSINEN T
SO ,’
1’ :i :: ’ : : :, : I .i
LO
: I
: .
30
VI :: . !z = N
20
2 z&
10
2-L
o-2 HOURS
AFTER OF
L-6
6-8
ADMINISTRATION 5HT
FIG. 1. Urinary excretion of 5-hydroxyindoleacecic acid (SHIAA) before and after the administration of SHT (1 mg/kg) intravenously (i.v.) or subcutaneously (SC.) in rats given water (3.5 per cent of body weight) every 2 hr. Values are means of five experiments. Vertical lines (UDENindicate standard error of mean. Determinations of SHIAA spectrophotometrically FRIEND, TITUS and WEISSBACH, 1955).
The rats were given tap water through a stomach tube (equivalent to 3.5 per cent of body weight) twice in the first hour and repeated after 4 hr. The radioactive 5HT or saline were also given after 4 hr and the urine collected thereafter. Water was administered also 4 and 8 hr after the 5HT administration. Later they were allowed to drink ad libitum and to eat once a day. Bile was collected through a plastic cannula with a thin glass point inserted into the hepatic duct through the abdominal wall. The bladder was also cannulated
Metabolites of labelled 5-hydroxytryptamine
151
through the same wound in the abdominal wall. All operative procedures were carried out under chloralose anaesthesia (1.2 g/kg intraperitoneally, and increased when needed). Urine samples were collected between O-8 hr and 8-24 hr after the 14C-5HT administration. For measuring the total radioactivity of the urine and bile, an aliquot usually l/10 or l/20, of the samples, or about O-5 ml, was dried in an aluminium plancetes under an infrared lamp and counted with a thin-window counter tube FHZ 15b (Frieseke-Hoepfner) in a lead chamber. Control samples were made by mixing a small volume of the injected 14C-5HT solution with a similar amount of non-radioactive urine or bile. Selecta paper 2043 bM of Sleicher & Schiill was used for ascending paper chromatography using the following solvents: n-butanol-acetic acid-water (4: 1 :5, organic layer), isopropanol-ammonia-water (10: 1: I), n-butanol-pyridine-water (1: 1: I), and 20 per cent aqueous KCl. All reagents were E.Merck’s p.s./for chromatography. The chromatograms were counted in an automatic radiochromatograph apparatus using two thin-window Geiger tubes (FHZ 15a) and thin protective membranes on each side of the paper. The background of two tubes was about 40 c.p.m. All the bile chromatograms and the least active urine chromatograms were counted with one tube without protective membrane. Two dimensional chromatograms were exposed to X-ray film (Kodak Blue Rand) and were also sprayed with p-dimethylaminobenzyaldehyde reagent (BLOCK et al., 1958). In addition to some commercial 5-hydroxyindoles, SHT-0-glucuronide purified from rabbit urine (AJRAKSINEN, 1961) and SHT-O-sulphate synthetized according to KISHIMOTO, et al. (1961) as well as SHIAA-O-sulphate containing material synthetized analogously to 5-HTO-sulphate were used as control substances. The spectral and biological properties of these substances as well as further details of the chromatography and other methods are published elsewhere (AIRAKSINEN, 1963).
RESULTS The urinary excretion of radioactivity after a 10 mg/kg dose of labelled 5HT in normal rats is shown in Fig. 2. About 50-60 per cent of the parenteral doses was found in the urine during the first 8 hr, 2-3 per cent during the following 16 hr, and nearly similar amounts during the following 3 days. After oral administration (through a stomach tube) only about half of these quantities were excreted in the first 8 hr; however, the excretion was as high or higher than after parenteral administration after 8 hr. The total faeces collected over 4 days after 5HT administration to three rats, extracted with acetone and water, was found to contain less than 10 per cent of the orally administered 14C-5HT. Subcutaneous 5HT caused anuria of long duration and tle urinary excretion of 5HT metabolites was slower in rats with the biliary+urinary fistulas than in normal nonanaesthetized rats. The 8-24 hr excretion seemed also to be higher in the anaesthetized animals after oral administration, as compared with that in non-anaesthetized animals though no complete anuria was found. A small but significant portion of radioactivity was found in the bile after subcutaneous and after oral administration. One dimensional chromatograms of a urine sample collected over the first 8 hr run in butanol-acetic acid, showed four main peaks of radioactivity. Comparison with twodimensional chromatograms of urine and control substances sprayed with different reagents (AIRAKSINEN 1961, 1963) indicated that the peak having the lowest Rf value was due to 5HT0-glucuronide and -O-sulphate. Two-dimensional autoradiograms of the same urine
MAUNO M. AIRAKSINLN
152
samples showed that StiT-O-sulphate, though always present, was the minor component of the peak; less than one-tenth of the radioactivity of the glucuronide conjugate.
FIG. 2. Urinary excretion of radioactive products after administration of 10 pg/kg (= 10 mg/ kg) of r4C-5HT by various methods of administration in non-anaesthetized rats. Means of five or six experiments in per cent of the radioactive dose. The slow intravenous (Lv.) administration lasted 60 min and the rapid injection less than 1 min. A plastic cannula was inserted in the jugular vein at least 2 days before the beginning of the experiment; both the rapid and slow i.v. injections were made through this cannula.
:
k!
4
urine
urine
L
FIG. 3. Biliary and urinary excretion of radioactive products:after subcutaneous (s.c.) and oral dose of 10 pg/kg (= 10 mg/kg) of labelled 5HT to (three with SC., two with oral dose) rats under urethane anaesthesia. The rats had cannulas in the urinary bladder and in the bile duct (ductus hepaticus), inserted just before the beginning of the experiment.
Metabolites of labelled 5hydroxytryptamine
153
The second radioactive peak (Rf 0 *20-O*45) included the glucuronide and sulphate conjugates of SHIAA and a small amount of an unidentified substance (possibly oxidation product). The two corresponding to the glucuronide and sulphate conjugates of 5HAA were not completely resolved in most chromatograms. However, the SHIAA-O-sulphate seemed to contain a greater proportion of radioactivity than that of SHT-O-sulphate, though SHT-0-glucuronide often was present in higher amount than the glucuronide of SHIAA. A third small radioactive peak contained free 5HT and some other unidentified material found particularly after intravenous administration. The peak of highest Rf value (65-85) was indentified as SHIAA. After parenteral administration the peak included an additional weak spot (less than one-tenth of the activity of this area in the autoradiograms) of higher Rf value than that of SHIAA in the isopropanol-ammonia system. This spot did not react with p-dimethylaminobenzaldehyde; unlike the other spots which yielded a violet or blue colour. N-acetyl-5HT and the 0- or N-methyl conjugates of 5HT were not detected on the chromatograms. The amounts of different metabolites after various routes of 5HT administration are expressed as per cent of the total radioactivity excreted as shown in Table 1 or as per cent of dose in Fig. 4. After oral administration SHT-0-glucuronide was the major metabolite. The O-conjugates of 5HT constitutes 17.1 f 1.5 per cent of the administered 5HT and over 70 per cent of the total urinary metabolites excreted. TABLE 1. PROPORTIONS OF THE RADIOACTIVITY IN PER CENT OF THE TOTAL URINARY RADIOACTIVITY IN DIFFERENT AREAS OF PAPER CHROMATOGRAMS PREPARED FROM SAMPLES THE FIRST 8 HR URINE OF RATS GIVEN W-5HT (10 &KG). SOLVENT BUTANOL-ACETIC ACID WATER. MEANS & S.E. EACH GROUP FIVE OR SIX EXPERIMENTS
Total urinary radioactivity ‘A of dose
Rf O-G.20 (O-conjugates of 5HT)
Rf 0.21-0.42 (O-conjugates of SHIAA)*
Rf 0.43-055 (5HT, etc.)
Rf 0.7&0+?5 (SHIAA)
Subcutaneous
55.713.7
10.4&0.4
30.1%1.0
1.6hO.7
57.9k1.6
Slow intravenous
53.Oh8.7
16.3k3.2
35.3zL1.9
0.710.4
47.7A4.9
Rapid intravenous
56.4A3.8
24.112.3
30.8hl.9
2.2hO.7
42.3% 1.8
Oral
23.6& 1.7
72.2*3.4
8.5h1.2
1.4+ 1.4
17.832.4
Method of administration
* This peak was later found to contain also the O-conjugates of 5-hydroxytryptophol.
After rapid intravenous injection 13 *9f 1 a9 per cent of the labelled 5HT was excreted as 5HT conjugates in contrast with 9.6h2.3 per cent after slow intravenous and 5 *Sk l-3 per cent after subcutaneous administration. The SHIAA spot accounted for only 4.3f0.7 per cent of the oral 5HT dose and 17.8 per cent of the activity excreted. After slow and rapid intravenous administration the SHIAA accounted for 24*0&3*2 and 24*3f2-1 per cent of the dose, and 32.2f2.2 per cent after subcutaneous injection.
154
MAUNOM. AIRAKSINEN
100 [--1 5HIAA ~
O.conjugatesof 5HIA.~
5HT etc. I ~ l O-conjugatesof 5HT I.Z W U tY W 13_
50
S.C.
I.V. SLOW
I.V. PER OS RAPID
FIG. 4. Percentage of various metabolites of labelled 5HT in the first 8 hr urine of the anaesthetized rats as measured from the paper chromatograms. Means of five or six experiments in per cent of the administered 5HT. Significant differences (p<0'05 according to Student's t-test) were found between all the administration routes in 5HIAA and in O-conjugates of 5HT. Differences between rapid and slow intravenous administrations were not statistically significant (p>0"05). The O-conjugates of 5HIAA as well as 5HT, etc., were also significantly lower after oral than after parenteral administration Only small amouts of O-conjugates of 5 H I A A were detected after oral administration. The urinary excretion of the O-conjugates of 5 H I A A were not significantly different after intravenous or subcutaneous injection. M o r e free 5HT as well as the unknown substances were excreted after rapid intravenous administration compared with the other routes of administration. The O-conjugates of 5 H I A A and 5HT appeared to be the only metabolites of 5HT in the bile. 5HIAA-O-sulphate possibly was the main biliary metabolite after both subcutaneous and oral administration of 5HT, but 5HT-O-glucuronide also was present. DISCUSSION The amounts of radioactivity found in bile were comparable to those in rat faeces after parenteral administration of labelled 5 H T reported by results of MCISAAC and PAGE (1959) and KEGLEVIC et al. (1960). The extent of reabsorpfion of the metabolites of 5HT is unknown. The metabolic products could presumably enter into the gut contents by other routes. So far we have studied only the excretion into the gastric juice, which was very small (less than 1 per cent of the 5HT dose) (KEYR!LMNEN and AIRAKSINEN, 1963). After oral administration the glucuronide may be formed in the intestinal wall and liver before entering the general circulation. Oral administration of 5HT m a y have little general actions for this reason. In addition the gut and liver have high monoamine oxidase activity (BLASCHKO, 1952; TlSSARI, 1963), but at least in the Sprague-Dawley rat in vivo this enzyme seems to be of less importance in the metabolism of 5HT administered orally than glucuronide formation. Also in vitro 5HT, but not 5-hydroxytryptophan and 5HIAA, was a
Metabolites
of labelled 5-hydroxytryptamine
155
good substrate of uridine diphosphate transglucuronylase in the duodenal mucosa, kidney and liver of Sprague-Dawley rats; Wistar rats showed lower enzyme activities in the duodenum and kidney (AIRAKSINEN and MIETTINEN,1963). The reasons for the different metabolic patterns after intravenous and subcutaneous administration are not fully understood. It may be due in part to the monoamine oxidase activity found in fat tissue (STOCK and WESTERMAN, 1963) and in the walls of blood vessels (THOMPSONand TICKNER, 1951). Part of the subcutaneously given 5HT may therefore become deaminated before entering circulation. There was a greater tendency for the 5HT to be metabolized by conjugation, after rapid intravenous injection than after slow injection. The higher initial concentration of 5HT in the circulation after rapid intravenous administration than after subcutaneous or slow intravenous administration may cause a difference of uptake of the amine into the platelets and other tissues. Of interest, reserpine pretreatment almost eliminates the differences in amounts of 5HIAA excreted between the subcutaneous and intravenous administrations of 5HT (AIRAKSINENet al, 1960); also the effect of 5HT on the circulation itself could affect its distribution and subsequent mode of metabolism and may differ with different doses of 5HT. The differences in the fate of 5HT according to the method of administration seem to be connected with the differences in the antidiuretic action of 5HT in rats and rabbits (AJRAKSINEN,1963).
Rbum&Les mCtabolites urinaires de la 5-hydroxytryptamine (5HT) marquQ au C” ont Btt Ctudits chez la rat hydrate apr&s administration sous-cutan& (s.c.) intraveineuse (iv.) lente et rapide et per OS. Lors de l’administration orale, l’excr&ion totale est r6duite d’environ de moitiC par rapport aux injections parent&ales. La chromatographie d’un 6chantillon d’urine de 8h. montre que les 0-conjug6.s de 5HT (principalement l’O-glucuronide, moins I’O-sulphate) sont excr&s. Apr&s administration s.c., de plus petites quantites de conjuguCs sont excr&% qu’aprts injection i.v. Ce sont less m&abolites principaux aprbs administration orale. Inverstment, de petites quantities d’acide 5-hydroxyindoleacetique (5HIAA) sont excrCtCes par administration orale et egalement moins apr& l’i.v. qu’aprts la S.C. Les O-conjuguts de SHIAA sont egalement excr&s en quantitds moindres aprks le per OS qu’aprb administrations parent&ales. Chez le rat anesthbi6, la bile a kgalement BtBrecueillie apres administrations s.c.et orales de P-5HT. Elle contient une petite quantit6 de radioactivk semblant provenir des O-conjugub de 5HT et de SHIAA. (5HT) Zussammenfassung-Die Harnmetaboliten von ‘“C-markierten 5-Hydroxytryptamin wurden nach der subcutanen (s.c.) nach der schnellen und langsamen intravenbsen (i.v.) und nach der oralen Verabreichung in hydrieren Ratten untersucht. Die totale Exkretion war nach der oralen Dose etwa die Hllfte der Exkretion nach parenteralen Dosen. Die Chromatographie de3 Harns in den emsten 8 Stunden ziegte dass die 0-Konjugate von 5HT (haupstlchlich 0-Glukurunide, weinger 0-Sulfat) nach den S.C. Dosen in geringerem Masse ausgeschiedon wurden als nach den i.v. Dosen und das es die Hauptmetaboliten nach der oralen Verabreichung waren. Umgekehrt war die Exkretion als 5-Oxyindolessigslure (SHIAA) nach der oralen Verabreichung sehr gering; ebenso war sie kleiner nach den i.v. als nach den S.C. Injektionen. Auch die 0-Konjugate von SHIAA waren nach der oralen Verabreichung in kleineren Mengen vorhanden als nach den parenteralen. In narkotisierten Ratten wurde nach den S.C. und oralen Dosen von 14C-5HT such die Galle gesammelt. Sie enthielt enie geringe Menge RadioaktivitLt, die aus den 0-Konjugaten von 5HT und SHIAA zu stammen schien. Aufhor’s address-M. M. AIRAKSINEN, Department of Pharmacology, 10, Helsinki, Finland
University of Helsinki, Siltavuorenpenger
156
MAUNO M. AIRAKSINEN REFERENCES
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