Pergamon Press

Life Sciences Vol . 22, PP . 473-478 Printed in the U .S .A .

INCORPORATION OF 18 02 INTO BRAIN SEROTONIN IN VIVO AS A PROCEDURE FOR ESTIMATING TURNOVER : A FEASIBILITYSTUDY IN ANIMALS C . L . Galli, J . W . Commissiong, E . Costa and N . H . Neff Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D .C . 20032 (Received in final form December 5, 1977)

SUMMARY Serotonin (5HT) containing 18 0 instead of 16 0 was found in brain after exposing rats to an atmosphere of 1802 . The 18 0 incorporation appeared linear with time and 228 of the 5HT in striatum contained 18 0 after one hour of exposure . 18 0 entered 5HT by enzymatic reaction rather than by isotope exchange as treatment with p-chlorophenylalanine, to block tryptophan hydroxylase, prevented incorporation, while more 1 8 0-5HT than normal was found in brain after treatment with pargyline, to block monoamine oxidase . Our studies suggest that the incorporation of 18 02 into brain 5HT might be a feasible approach for evaluating 5HT turnover in animals and with some modification might be applied in man . Abnormal serotonin (5HT) metabolism has been implicated in several psychiatric disorders (1) . Procedures for estimating the rate of formation of 5HT in human and animal brain are available (2-4), however, they involve administering radioactive materials or drugs that block either the formation of 5HT or the elimination from brain of its major metabolite, 5-hydroxyindoleacetic acid (5HIAA) . These procedures are unsatisfactory for human studies and in animal studies generate data that sometimes can not be readily interpreted . The initial biosynthetic event for the formation of 5HT involves the utilization of 02 during the enzymatic hydroxylation of tryptophan by try tophan hydroxylase . Presumably, 18 0 2 could be substituted for 1 02 in the inspired air resulting in the in vivo labelling of endogenous 5HT with 18 0, a stable isotope (5,6) . A gas chromatograph-mass spectrometer (GC-MS) could then be used to isolate 5HT and distinguish molecules containing 160 from those containing 180 . Such an experimental approach might be tried in man as equipment already exists for administering gases to man and 18 02 is now available at a relatively reasonable cost . Turnover rate estimates might be made from the relative abundance of 18 0 incorporated into 5HT and 5HIAA in blood, urine and cerebrospinal fluid .

Our objective for this study was to determine if significant quantities of 180 were incorporated into brain 5HT in vivo after exposing rats to an atmosphere containing 18 02 . Theexperimental model could then be tested by evaluating the relative abundance of 473

47 4

Incorporation of 1802 into 5HT

Vol .

22, No . 6, 1978

18 0-5HT in brain after blocking of tryptophan hydroxylase or monoamine oxidase . METHODS Male Sprague-Dawley rats, 150-180 g, obtained from ZivicMiller Labs . (Allison Park, Pa .) were placed in a closed chamber and exposed to room air or air containing 18 02 . The exposure chamber and the utilization of 1802 by the test animals have been described in detail elsewhere (7) . In brief, initially the chamber which holds four rats contains room air and is connected to a gas bottle of 180 2 . Access of 1802 to the chamber is regulated by the gas pressure in the system . As the animal utilizes the 02 the carbon dioxide formed is trapped on Ascarite (A . H . Thomas, Philadelphia, Pa .), thus, the pressure in the chamber is reduced until it triggers an opening of a valve which admits 18 02 into the chamber . As the experiment proceeds, the percentage of I80 in the chamber increases and approaches the concentration of 102 in the gas bottle which in our studies was 958 1802 : 5% 1602 . The 1802 was prepared by the electrolysis of water-180 (95 atom % 180) . Rats were placed in the chamber for 30 or 60 min and then decapitated . Brains were dissected and the striata removed . In one series of studies, animals were treated with p-chlorophenylalanine methyl ester (PCPA), 300 mg/kg, i .p ., to block tryptophan hydroxylase, or pargyline, 75 mg/kg, i .p ., to block monoamine oxidase, 24 hr prior to exposure to 1802 . Treatment with the drugs 24 hr before testing had no apparent effect on the utilization of 02 as measured by the total volume of 1 802 consumed by the animals in the exposure chamber . Brain tissues were prepared for GC-MS analysis of indoles as In brief, samples were homodescribed by Cattabeni et al . (8) . genized in 0 .1 M ZnS04, 200 pl . After neutralization with 0 .1 M Ba(OH)2, 200 pl, and centrifugation at 20,000 x g for 15 min, a portion of supernatant, 200 ul, was placed in a 1 ml Microflex tube (Kontes, Evanston, I1 .) . Alpha-methyl serotonin (a-M5HT), 100 pmol, was added to each sample to serve as an internal stanPerfluoropropiodard . The samples were then dried under vacuum . nic anhydride (PFPA), 100 ul and ethyl acetate, 20 ul, were added Before analysis, they and the samples reacted for 16 hr at 65 0 . were dried under nitrogen and the residue dissolved in ethyl acetate for injection into the GC-MS . A Finnigan gas chromatograph-quadrupole mass spectrometer, Model 3200, with programmed multiple ion monitoring was used for the analysis . Assays were performed employing a 3 m silanized glass column, 3 mm i .d ., packed with 38 OV 17 on Gas Chrom Q, 100-200 mesh (Supelco, Bellefonte, Pa .) . The column parameters were : injector,250 0 ; oven,195 0 and helium flow rate,20 ml/min . The agments (m/e) monitored were : 160-5HT, 438, 451 ; a-M5HT, 465 and 0-5HT, 453 .

fi

RESULTS

Placing rats in the exposure chamber had no apparent effect on the steady-state level of 5HT in the striatum (Table 1) . Molecular oxygen (1802), however, was rapidly incorporated into 5HT, with 180-5HT reaching about 22 percent of the total 5HT in the striatum after 60 min in the exposure chamber (Fig . 1) .

Incorporation of 1802 into 5HT

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TABLE 1 RELATIVE ABUNDANCE OF 18 0-5HT IN STRIATUM FROM COI OR PCPA- TREATED RATS AFTER EXPOSURE TOE 8 0 2

PARGYLINE-

SEROTONIN CONCENTRATION (nmol/g +_ SEM (N = 4) ) 160-5HT 180-5HT

TREATMENT 16 0I

1 .6

+

0 .3

-

18 0I

1 .5

+

0 .1

0 .19 + 0 .01

0 .43

+

0 .08*

N .D .

3 .9

+

0 .5*

0 .31 + 0 .05*

18 02 + PCPA

18 0I + Pargyline

Rats were exposed to an atmosphere of 18 02 or 16 02 as described in METHODS for 30 min . One group of animals was treated with PCPA, 300 mg/kg, i .p ., and one with pargyline, 75 mg/kg, i .p ., 24 hours prior to exposure to 18 02 . N .D . indicates not detectable above background . * P c 0 .05 when compared to 1802 exposed rats . After blockade of tryptophan hydroxylase by treatment with PCPA, 5HT levels decreased by about 70 percent and there was no detectable 18 0-5HT found in the striatal tissue after 30 min of exposure to 1802 (Table 1) . Pargyline treatment results in a blockade of the oxidative deamination of 5HT in brain and 5HT levels increased (Table 1) . Moreover, there was significantly more 180-5HT in striatum after treatment with pargyline when compared with rats exposed to 18 02 without pargyline treatment. DISCUSSION There are numerous methods for studying 5HT formation in brain (2-4), none of which is entirely satisfactory for evaluating human brain 5HT formation . Our study is an attempt to provide the basis for a more useful approach for studying 5HT in human brain . Similar studies for the catecholamines have already been described along with some of the problems encountered (6 ,7 9) . Our studies with rats demonstrate that the incorporation of ' 8 02 can be used for studying the formation of 5HT in vivo . The incorporation is apparently linear during the initial phase, and proceeds by enzymatic reaction rather than by isotope exchange . The latter notion is supported by the finding that incorporation is blocked by treatment with PCPA and metabolism is inhibited by treatment with pargyline . From studies with radioisotopes, it has been calculated that between 50-70 percent of the rat brain 5HT pool is turned over per hr (3) . We found that about 22 percent of the 5HT pool in rat striatum consisted of 180-5HT after 1 hr of exposure to 1802 . This is only a minimal value as no correction has been made for metabolism of 180-5HT during the 1 hr of exposure . Moreover, as des-

Incorporation of 1802 into 5HT

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Vol . 22, No . 6, 1978

h IWZ

vz

ô

m Q

The relative abundance of 18 0-5HT in exposure to an atmosphere containing represents the percentage of 180-5HT in the tissue and is reported as the 4 rats .

rat striatum after 18 02 . Each value of the total 5HT mean _+ S .E .M . for

cribed in METHODS, the concentration of 18 02 in the exposure chamber gradually increases with time, and therefore, the rates would appear to be less than maximal . Our studies show that rather large quantities of 18 02 are incorporated into brain 5HT during a relatively short period . The rate of incorporation is consistant with the rate found using radioactive methods (3) . The experimental design is simple and therefore might easily be adapted for studies in man . Such studies would require that a method be developed for assaying 18 0-5HIAA, the major metabolite of 18 0-5HT, in biological fluids .

Vol . 22, No . 6, 1978

Incorporation of 18 02 into 5HT

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Studies with 18 02 offer the following advantages over existing methods for studying 5HT formation in human brain : (a) 180 2 is not radioactive, has no known toxicity and can be easily administered to humans, (b) it is not necessary to administer drugs or tryptophan to make turnover estimates, so studies can be conducted under steady-state conditions, and (c) 18 0 is incorporated into 5HT at the rate-limiting enzymatic step, the hydroxylation of tryptophan (10), and therefore may give a more meaningful estimate of turnover than existing pharmacological procedures . In conclusion, animal studies suggest that the incorporation of 1 802 into brain 5HT might be a feasible approach for evaluating 5HT formation in human brain . REFERENCES 1.

A. J . Mandell, in The Impact of Biology on Modern Psychiatry , pp . 165-178 (edited E . S . Gérshon, R . Belmaker, S . S . Kety and M . Rosenbaum), Plenum Press, New York (1977) . 2 . N. H . Neff and T . N . Tozer, in Advances in Pharmacology 6A, pp . 97-109 (edited by E . Costa, S . Garattini, M . Sandlerand P . Shore), Academic Press, New York (1968) . 3 . N. H . Neff, P . F . Spano, A . Groppetti, C . T . Wang and E . Costa, J . Pharmacol . E . Ther . 17 6 701-710 (1971) . 4 . H . M. Van Praag, J . Korf, L . C . W .Dols and T . Schmitt, Psychopharmacologia 25 14-21 (1972) . 5 . E . Costa J . Psychiat. Res . 1 1 295-300 (1974) . 6 . C . L. Gail i, J . W . Commissiong and N . H . Neff, Biochem . Pharmacol . 26 1271-1273 (1977) . 7 . N . H . Neff, C . L . Galli and E . Costa, in Advances in Biochemical Psychopharmacology 16, pp . 187-192 (edited by E . Costa and G . L . Gessa) RavenPress, New York (1977) . 8 . F . Cattabeni, S . H . Koslow and E. Costa, Science 178 166-168 (1972) . 9 . A. Mayevsky, B . Sjoquist, C . G . Fri, D . Samuel and G . Sedvall, Biochem. Biophys . Res . Commun . 5 1 746-755 (1973) . 10 . W. Lovenberg, E . Jequier and A. Sjoerdsma, Science 155 217-219 (1967) .

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