Psycho pharmacology
Psychopharmacology54, 171-175 (1977)
9 by Springer-Verlag1977
Factors Affecting the Urinary Excretion of Endogenously Formed Dimethyltryptamine in Normal Human Subjects MICHAEL C. H. OON, ROBIN M. MURRAY*, RICHARD RODNIGHT**, MARION P. MURPHY, and JAMES L. T. BIRLEY Departments of Biochemistryand Psychiatry,Instituteof Psychiatry, De CrespignyPark, Denmark Hill, London,SE5 8AF, United Kingdom
Abstract. The hallucinogenic substance N',N'-dimethyltryptamine and its precursor N-methyltryptamine were found in 24-h specimens of urine from 19 normal human subjects; the mean excretion rates were 386 ng 24 h -1 and 856 ng 24 h - 1 respectively. The urinary excretion of both compounds was unrelated to age, sex, urinary volume, or creatinine, nor was any consistent diurnal pattern observed. Rates for the mono and dimethylated compounds were not correlated. Diet and the intestinal flora were excluded as a source of urinary dimethyltryptamine. Administration to 4 subjects of sufficient ammonium chloride to increase the H ion concentration of the urine caused a transient increase in dimethyltryptamine excretion but no consistent increase in the rate for N-methyltryptamine. Acidification of the urine did not appear to be the determining factor in this result since in one subject the same drop in urinary pH was achieved by feeding methionine without any increase in dimethyltryptamine excretion. Key words: Dimethyltryptamine -
Monomethyltryptamine - Urinary excretion of methylated indol e s - Mass fragmentography
It has often been suggested that some of the symptoms of schizophrenia could be due to the endogenous production of methylated compounds with hallucinogenic properties. Recently, attention has centred on the methylated indoleamines and in particular on N',N'-dimethyltryptamine (DMT), the psychotomimetic properties of which have been extensively studied (Domino, 1975). The major source of DMT and other Present address: Section on Clinical Neuropharmacology, NIMH, Bethesda,Maryland,USA ** To whomoffprintrequests should be sent *
methylated indoles in the body is dietary tryptophan, of which a very small proportion is decarboxylated to tryptamine by aromatic amino acid decarboxylase. The N-methylation of tryptamine to first monomethyltryptamine (NMT) and then DMT, using S-adenosylmethionine as methyl donor, was originally demonstrated in extracts of rabbit lung by Axelrod (1962); subsequently N-methylating activity towards tryptamine and serotonin was reported to occur in animal and human brain, albeit in much lower concentrations than in lung (Morgan and Mandell, 1969; Saavedra et al., 1973). However, the existence of N-methylating activity in brain towards indoleamines has been questioned on the grounds that inadequate procedures for product identification were used in much of the published work on the subject (Taylor and Hanna, ]975; Boarder and Rodnight, 1976; Gomes et al., 1976). Recent evidence derived from more rigorous identification procedures suggests that traces of indoleamine-N-methyltransferase activity do occur in mammalian brain, although the activity is so low that its significance is debatable (Taylor and Hanna, 1975; Boarder and Rodnight, 1976; Boarder et al., 1976; Guchhait, 1976). With regard to clinical observations, early work suggested that DMT occurs more frequently in the blood of subjects diagnosed as schizophrenic than in normal individuals (Narasimhachari et al., 1971), but a further study using more sensitive methods failed to confirm this conclusion (Angrist et al., 1976). The disadvantages of measuring DMT in blood are indicated by the work of Kaplan et al. (1974), who found that following an injection of DMT the hallucinogen disappeared very rapidly from the plasma. Other investigators have used urinary excretion as an index of the rate of formation of DMT in the body, but again the results have been contradictory, Rodnight et al. (1976) found DMT to occur significantly more often in the urine of schizophrenics and other psychotic
172
Psychopharmacology 54 (1977)
Table 1. subjects
Urinary excretion of DMT and NMT in 19 normal
Age/sex
Urinary volume (ml 24 h - 1)
Urinary pH
DMT (rig 24 h-1)
NMT (ng 24 h - 1)
32 5i 47 24 28 22 19 34 21 31 25 39 68 32 28 48 66 42 62
900 500 i100 1600 1000 2200 760 1600 2000 1050 2200 1900 1375 1680 1800 1700 1100 860 2000
6.5 6.0 5.8 6.4 6.4 6.2 5.8 6.8 6.5 6.0 6.2 6.0 6.3 6.1 6.4 5.4 6.0 5.7 6.2
250 425 287 100 142 299 175 450 400 350 250 100 250 20 280 2500 400 142 400
400 500 870 121 284 398 3000 500 250 400 624 800 3000 3000 222 250 337 714 600
M M M M F F F F F F E F F F F F M M M
individuals than in control subjects, whereas Carpenter et al. (1975) failed to differentiate a rather different schizophrenic population from normal controls. Very little is known about the source of the D M T occurring in the body fluids, and to elucidate the significance of observed differences in excretion between psychotic patients and normal individuals we need to know more about the factors affecting its excretion in normal subjects.
MATERIALS AND METHODS The subjects (7 males and i2 females, age range 19-68 years) had no previous history of any psychiatric illness and the majority were members of staff of either the Institute of Psychiatry or the Bethlem Royal and Maudsley Hospitals. None was receiving any medication during the experimental periods except that indicated. All the drugs were taken orally every 6 h unless otherwise specified. Enterically-coated tablets of ammonium chloride were obtained from either Macarthys Ltd., Romford, Essex, U.K. or from Eli Lilly & Co., Basingstoke, U.K.; d/-methionine, encapsulated in the Maudsley Hospital Pharmacy, from Haleswood Chemicals Ltd., Staines, Middlesex, U.K. ; neomycin from Glaxo Laboratories, Greenford, Middlesex, U.K. and 'Complan' from GlaxoFarley Foods Ltd., Plymouth, Devon, U.K. The psychoactive drugs were iproniazid (Marsilid, Roche Products Ltd.), thioridazine (Melleril, Sandoz Products Ltd.) and dithiepin HC1 (Prothiaden, Crookes Laboratories Ltd.). Urine was collected in plastic containers for 24 h (8.00 a.m. to 8.00 a.m.) without addition of preservative. Subjects were instructed to keep the sample cool during collection. Urinary pH and total creatinine were measured immediately on receipt of the sample which was.then frozen at 15~ and stored pending analysis.
Previous work has shown that DMT and NMT are stable for 3 months in urine frozen within 12 h of completing collection. The urinary methylated indoleamines were measured by a procedure with a limit of detection of 20 ng 24 h -1 for DMT and 50 ng 24 h -1 for NMT. The procedure will be described in detail elsewhere (Oon and Rodnight, 1977). Of the 24-h urine samples, 50~ was concentrated in a rotary evaporator to 70ml and extracted with toluene at alkaline pH. The resulting extract was purified by preparative thin layer chromatography, derivatised with trifluoroacetic anhydride and injected into a gas chromatograph (Perkin Elmer, F 17) fitted with a nitrogen-sensitive detector. An internal standard of 5-methyIdimethyltryptamine was added to the concentrated urine to account for variations in recovery. In the case of 10 urine extracts (4 from the present study and 6 from a patient population used for another study), combined gas chromatography and mass spectrometry (GC/MS) were used to examine the identity of peaks in the eluate from the gas chromatograph having the same retention time as the authentic DMT and NMT derivatives. Because of sensitivity problems it was impossible to obtain definitive mass spectra for the substance corresponding to the DMT peak. However, by using chemical ionisation and monitoring by the technique of mass fragmentography the pseudomolecular ion characteristic of DMT-trifluoracetate (m/e 285), the extracts were found to give a peak with precisely the same retention time as the pseudo-molecular ion of the authentic DMT derivative (for further details see Oon and Rodnight, 1977). This evidence still falls short of absolute identification, although the chance that the extracts contain a compound yielding an ion with identical mass and retention time as the pseudo-molecular ion of the DMT derivative is considered very unlikely. It is also significant that in these same extracts there was also close quantitative agreement between the results obtained by gas chromatography alone and by GC/MS.
RESULTS In 18 of the 19 samples analysed from drug-free subjects, the D M T excretion rate was below 500 ng 24 h - 1, and in 16 of the 19 samples the N M T rate was below 1000 ng 24 h - 1. There was no direct relationship between D M T and N M T excretion rates (Table 1). Neither were there any associations between D M T or N M T concentrations and age, sex, urinary volume, or creatinine. There was no general association between the indole contents and pH, but the subject with the lowest pH had a very high excretion of DMT. The mean pH for all the samples collected was 6.14 _+ 0.33 (SD) with a range 5 . 4 - 7 . 5 . Diurnal variation was studied in 5 individuals by collecting urine over three 8-h periods. The relationship between time of day and excretion of D M T and N M T varied between individuals and no consistent diurnal pattern emerged (Table 2). To examine whether any of the urinary D M T originates from gut-flora, two subjects took 8 g of neomycin daily for 2 days in an effort to diminish bacterial activity in the intestine. This treatment had no effect on the 24-h excretion of D M T and N M T over the 4 days studied. A preformed dietary source was excluded by an experiment in which a subject
173
M. C. H. Oon et al. : Urinary DMT in Normal Subjects
consumed only 'Complan' and water for 5 days; the daily excretion of both DMT and NMT was essentially unchanged throughout this period. Neither was there any observable change in the excretion of the two N-methylated compounds during a day of increased physical activity by two subjects. One subject collected 8 hourly specimens during a day of examinationinduced stress, but this apparently did not affect urinary DMT or NMT. The urinary excretion of amines is known to be affected by H ion balance in the kidney; for instance, acidification of the urine through administration of ammonium chloride greatIy enhances the rate of excretion of amphetamine by the kidney (Davis et al., 1971). In the present work 4 subjects had their urine acidified with ammonium chloride (8 g daily for 2 days), and in each case excretion of DMT increased considerably (Table 3); however, in no case did the peak of DMT excretion coincide with the lowest urinary pH. Surprisingly, urinary NMT appeared unaffected by acidification of the urine. In a further experiment one subject took 4 g of methionine over 3 days and although the urinary pH dropped to 5.6 there was no observable change in excretion of the two N-methylated compounds. Alkalinisation of the urine with sodium bicarbonate to a pH of 7.7 in one subject
Table 3 Urinary excretion of DMT and NMT after administration of ammonium chloride and methionine
Subject
Amine
Total excretion (ng) 0-8.00
1 1 2 2 3 3 4 4 5 5 6
DMT NMT DMT NMT DMT NMT DMT NMT DMT NMT DMT
216 433 100 38 154 300 200 600 86 150 20
6
NMT
2000
8.00-16.00 52 315
16.00-24.00
73 640 88 1352 18t 50
80 180 106 160 50 560 208 1150 20 670
not detectable
not detectable
1250
1000
-
and with potassium citrate to a pH of 7.6 in another subject did not alter to a significant extent the excretion of either DMT or NMT. In none of the above experiments on normal volunteers did any untoward psychological states coincide with high rates of DMT excretion following administration of ammonium chloride.
Subject (drug administered)
Day of collection
Urinary volume (ml 24 h -1)
Urinary pH
(ng 24 h--~)
NMT (rig 24 h 1)
1 (NH4C1,
1 2a 3 4 5
700 t850 1200 1850 1150
6.3 6.3 6.1 5.1 6.1
50 200 500 516 3000
400 620 1500 1500 500
1 2a 3a 4 5 6
900 1100 1100 1000 975 950
6.5 6.2 5.6 5.5 5.6 5.3
250 562 2500 50O 250 250
400 500 500 384 334 350
1 2~ 3~ 4
2200 2200 1400 1600
6.3 5.7 5.9 5.9
500 500 2500 100
625 300 500 300
1 2a
1600 2400 1850 80O 90O
7.0 7.0 5.8 6.3 6.7
350 300 300 1400 1500
400 400 300 400
1200 1650 1300 1350 1700
6.3 6.2 5.8 5.6 5.6
200 214 166 200 170
400 342 333 300 320
Macarthy)
2 (NH4CI,
Macarthy)
3 (NH,C1,
Macarthy) 4 (NH4CI,
Eli Lilly)
1 (Methionine) a The drug was administered on these days
Table 2. Urinary excretion of DMT and NMT over consecutive periods of 8 h
3~ 4 5
1 2a 3. 4" 5
DMT
3OO
174
Psychopharmacology54 (197'7) DISCUSSION
Iproniazid 2.8
I
q
f
'""',
2.0 T
.It'4
t
6'
~ Z
NMT
"',,, ~,/
\,
[
1.2
-.
rJ
I
I , A / / /
123 ;" 0.4
,,
,,
as ,,
/
L
i
5
i
38~
30
i
46
62~
5,*
70 90
Days
Fig. 1. Effectof iproniazid (Marsilid) on excretion of DMT and NMT in one subject. The dose of iproniazid was 75 mg daily over period indicated
3.0
NH4C I
N a HCO3
Neomyc in
Exercise
I
~t 2.0 DMT
>,
0
5 t
I
I
1
2
3
4
5
6
7
8
9
10 11
~2 13 14 15 16 17 18
Days
Fig.2. UrinaryDMT and NMT excretionin one subject;influence of NH4C], NaHCO3, neomycin,and exercise
The excretion of the two indoles was also studied in a patient with reactive depression given 75 mg daily of iproniazid, an inhibitor of both monoamine oxidase A and B. Figure I shows that the excretion of both D M T and N M T increased to double that of the previous drug-free period. Following cessation of the medication the D M T and N M T excretion rates decreased again. Another patient given a tricyclic antidepressant (150 mg of Prothiaden daily) over a 6-week period showed no change in the excretion of these methylated indoles. Likewise, 100 mg of thioridazine daily for 14 days did not modify D M T excretion. Figure 2 illustrates the effect of various experiments on the indole excretion of one subject.
The finding of an endogenous hallucinogen in the urine of normal individuals raises some interesting questions. D M T may arise as a by-product of some physiological process in the body unrelated to mental function, or alternatively it could have some physiological role in the nervous system in its own right. With present evidence the latter possibility seems unlikely in view of the apparent low activity of the N-methylating enzyme in brain. In a previous study (Rodnight et al., 1976) using a less sensitive method (limit 500 ng 24 h -1) than the present one we detected urinary DMT in only one out of 20 normal subjects. In the present study also only 1 out of 19 normal individuals excreted more than 500 ng 24 h-1. These findings go some way to explain the apparently contradictory results of studies of urinary D M T in schizophrenic and normal subjects (Carpenter et al., 1975; Rodnight et al., 1976). With a relatively insensitive method, the putative compounds may be detected in only occasional normal subjects, but with a very sensitive method they may be found in every case. That normal subjects excrete DMT is consistent with previous findings that DMT was present in the blood of some normal subjects (Mandel, 1974; Angrist et al., 1976). It may be that sequential studies using highly sensitive methods will demonstrate D M T in the blood of all normal subjects at some time. In fact, our study does not exclude the possibility that diurnal or other fluctuations do occur in the excretion of these two methylated indoles. Because of the sensitivity limits of our analysis, we could not measure the quantity excreted in urine collected over periods shorter than 8 h. We cannot, therefore, tell whether these compounds were excreted consistently over the periods we studied or only in short bursts. Sequential studies, preferably of blood, with a yet more sensitive analytical method will be required to answer this question. There are numerous pitfalls in the biochemical investigaton of mental illness (Rodnight, 1975). In the past researchers of schizophrenia have been misled by urinary substances that were later found to origb nate in the diet or were products of intestinal bacteria. This study suggests that neither of these two factors are relevant to the excretion of urinary DMT or NMT. Similarly, nonspecific stresses such as emotional tension or physical exertion do not appear to be major dererminants of the excretion of these indoles. Monoamine oxidase (MAO) is the major catabolic enzyme for D M T and N M T (Lu and Domino, 1976), as well as their precursor tryptamine. Thus, the inhibition of MAO might not only decrease catab-
M. C. H. Oon et al. : Urinary DMT in Normal Subjects
olism of DMT and NMT but also increase their biosynthesis by blocking the catabolism of tryptamine by MAO and increasing its N-methylation. [t is, therefore, hardly surprising that administration of a monoamine oxidase inhibitor resulted in the increased excretion of DMT and NMT in the subject studied. The increase in excretion of DMT following the administration of ammonium chloride merits further consideration. This 'ammonium chloride' effect did not coincide with the lowest urinary pH, and acidification with methionine had no effect (a finding of interest in view of the status of methionine as a methyl donor). It therefore seems unlikely that the increase in DMT excretion was a straightforward consequence of acidifying the body fluids. Nor is it likely that the ammonium ions were interfering with the catabolism of DMT since in that case a more prolonged increase in the concentration of urinary DMT would have been expected. It remains conceivable that ammonium chloride releases DMT from some store in peripheral tissues. These and other possibilities require further investigation. Acknowledgements. We are grateful to Judith Bastow and Imogen Dawes (The Pharmacy, Maudsley Hospital) and to John Garrod (Department of Pharmacy, Chelsea College) for advice. M.C.H.O. was supported by a grant from the Medical Research Council of the U.K.; M.P.M. by a grant from the Wellcome Trust; R.M.M. by a grant from the Research Fund of the Bethlem Royal and Maudsley Hospitals.
REFERENCES Axelrod, J.: The enzymatic N-methylation of serotonin and other amines. J. Pharnracol. Exp. Ther. 138, 28-33 (1962) Angrist, B., Gershon, S., Sathananthan, G., Walker, R. W., LopezRamos, B., Mandel, L. R., Vanden Heuvel, W. J. A. : Dimethyltryptamine levels in blood of schizophrenic patients and control subjects. Psycfiopharmacology 47, 29 - 32 (1976) Boarder, M. R., Oon, M. C. H., Rodnight, R. : Mass spectrometric identification of N-monomethyltryptamine following incubation of tryptamine with brain protein and S-adenosylmethionine or 5-methyltetrahydrofolic acid. Biochem. Pharmacol. 25, 21092112 (1976)
175 Boarder, M. R., Rodnight, R.: Tryptamine N-mcthyltransferase activity in brain tissue: a re-examination. Brain Res. 114, 359 - 364 (1976) Carpenter, W. T., Fink, E. B., Narasimhachari, N., Himwich, H. E.: A test of the transmethylation hypothesis in acute schizophrenic patients. Am. J. Psychiatry 132, 1067-1071 (1975) Davis, J. M., Kopin, I. J., Lemberger, L., Axelrod, J.: Effects of urinary pH on amphetamine metabolism. Ann. N.Y. Acad. Sci. 179, 493-501 (1971) Domino, E.F.: The indole hallucinogen model: is it worth pursuing? In: Predictability in psychopharmacology : preclinical and clinical correlations, A. Sudilovsky, S. Gershon, B. Beer, eds., pp. 247-268. New York: Raven Press 1975 Gomes, U. R., Neethling, A. C., Stanley, B. C.: Enzymatic Nmethylation of indoleamines by mammalian brain: fact ol- artefact? J. Neurochem. 27, 701 - 705 (1976) Guchhait, R. B.: Biogenesis of 5-methoxy-N,N,-dimethyltryptamine in human pineaI gland. J. Neurochem. 26, 187- 190 (1976) Kaplan, J., Mandel, L. R., Walker, R. W., Vanden Heuvel, W. J. A., Stillman, R., Gillin, J. C., Wyatt, R. J.: Blood and urine levels of N,N-dimethyltryptamine following administration of psychoactive dosages to human subjects. Psychopharmacologia (Bed.) 38, 239-245 (1974) Lu, L. J., Domino, E. F. : Effect of iproniazid and tranylcypromine on the half-life of N,N-dimethyltryptamine in rat brain and liver. Biochem. Pharmacol. 25, 1521-1527 (1976) Mandel, L. R.: Dimethyltryptamine. Psychopharmacol. Bull. 10, 55 - 56 (1974) Morgan, M., Mandell, A.J.: Indole(ethyl)amine N-methyltransferase in the brain. Science 165, 492-493 (1969) Narasimhachari, N., HelIer, B., Spaide, J., Haskovec, L., Meltzer, H., Strahilevitz, M., Himwich, H. E. : N,N,-dimethylated indoleamines in blood. Biol. Psychiatry 3, 21-23 (1971) Oon, M. C. H., Rodnight, R.: A gas chromatographic procedure for determining N,N-dimethyltryptamine and N-monomethyltryptamine in urine using a nitrogen detector. Biochemical Medicine (in press, 1977) Rodnight, R. : The significance of some biochemical abnormalities in schizophrenia. Acta Neurologica 30, 86-101 (1975) Rodnight, R., Murray, R. M., Oon, M. C. H., Brockington, I. F., Nicholls, P., Birley, J. L. T. : Urinary dimethyltryptamine and psychiatric symptomatology and classification. Psychol. Med. 6, 649-657 (1976) Saavedra, J. M., Coyle, J. R., Axelrod, J.: The distribution and properties of the non-specific N-methyltransferase in brain. J. Neurochem. 20, 743-752 (1973) Taylor, R. T., Hanna, M. L.: S-methyltetrahydrofolate aromatic alkylamine N-methyltransferase: an artefact of 5,10-methylene tetrahydrofolate reductase activity. Life Sci. 17, 111 - 120 (1975) Received April 4, 1977
Psychopharmacology54, 171-175 (1977)
9 by Springer-Verlag1977
Factors Affecting the Urinary Excretion of Endogenously Formed Dimethyltryptamine in Normal Human Subjects MICHAEL C. H. OON, ROBIN M. MURRAY*, RICHARD RODNIGHT**, MARION P. MURPHY, and JAMES L. T. BIRLEY Departments of Biochemistryand Psychiatry,Instituteof Psychiatry, De CrespignyPark, Denmark Hill, London,SE5 8AF, United Kingdom
Abstract. The hallucinogenic substance N',N'-dimethyltryptamine and its precursor N-methyltryptamine were found in 24-h specimens of urine from 19 normal human subjects; the mean excretion rates were 386 ng 24 h -1 and 856 ng 24 h - 1 respectively. The urinary excretion of both compounds was unrelated to age, sex, urinary volume, or creatinine, nor was any consistent diurnal pattern observed. Rates for the mono and dimethylated compounds were not correlated. Diet and the intestinal flora were excluded as a source of urinary dimethyltryptamine. Administration to 4 subjects of sufficient ammonium chloride to increase the H ion concentration of the urine caused a transient increase in dimethyltryptamine excretion but no consistent increase in the rate for N-methyltryptamine. Acidification of the urine did not appear to be the determining factor in this result since in one subject the same drop in urinary pH was achieved by feeding methionine without any increase in dimethyltryptamine excretion. Key words: Dimethyltryptamine -
Monomethyltryptamine - Urinary excretion of methylated indol e s - Mass fragmentography
It has often been suggested that some of the symptoms of schizophrenia could be due to the endogenous production of methylated compounds with hallucinogenic properties. Recently, attention has centred on the methylated indoleamines and in particular on N',N'-dimethyltryptamine (DMT), the psychotomimetic properties of which have been extensively studied (Domino, 1975). The major source of DMT and other Present address: Section on Clinical Neuropharmacology, NIMH, Bethesda,Maryland,USA ** To whomoffprintrequests should be sent *
methylated indoles in the body is dietary tryptophan, of which a very small proportion is decarboxylated to tryptamine by aromatic amino acid decarboxylase. The N-methylation of tryptamine to first monomethyltryptamine (NMT) and then DMT, using S-adenosylmethionine as methyl donor, was originally demonstrated in extracts of rabbit lung by Axelrod (1962); subsequently N-methylating activity towards tryptamine and serotonin was reported to occur in animal and human brain, albeit in much lower concentrations than in lung (Morgan and Mandell, 1969; Saavedra et al., 1973). However, the existence of N-methylating activity in brain towards indoleamines has been questioned on the grounds that inadequate procedures for product identification were used in much of the published work on the subject (Taylor and Hanna, ]975; Boarder and Rodnight, 1976; Gomes et al., 1976). Recent evidence derived from more rigorous identification procedures suggests that traces of indoleamine-N-methyltransferase activity do occur in mammalian brain, although the activity is so low that its significance is debatable (Taylor and Hanna, 1975; Boarder and Rodnight, 1976; Boarder et al., 1976; Guchhait, 1976). With regard to clinical observations, early work suggested that DMT occurs more frequently in the blood of subjects diagnosed as schizophrenic than in normal individuals (Narasimhachari et al., 1971), but a further study using more sensitive methods failed to confirm this conclusion (Angrist et al., 1976). The disadvantages of measuring DMT in blood are indicated by the work of Kaplan et al. (1974), who found that following an injection of DMT the hallucinogen disappeared very rapidly from the plasma. Other investigators have used urinary excretion as an index of the rate of formation of DMT in the body, but again the results have been contradictory, Rodnight et al. (1976) found DMT to occur significantly more often in the urine of schizophrenics and other psychotic
172
Psychopharmacology 54 (1977)
Table 1. subjects
Urinary excretion of DMT and NMT in 19 normal
Age/sex
Urinary volume (ml 24 h - 1)
Urinary pH
DMT (rig 24 h-1)
NMT (ng 24 h - 1)
32 5i 47 24 28 22 19 34 21 31 25 39 68 32 28 48 66 42 62
900 500 i100 1600 1000 2200 760 1600 2000 1050 2200 1900 1375 1680 1800 1700 1100 860 2000
6.5 6.0 5.8 6.4 6.4 6.2 5.8 6.8 6.5 6.0 6.2 6.0 6.3 6.1 6.4 5.4 6.0 5.7 6.2
250 425 287 100 142 299 175 450 400 350 250 100 250 20 280 2500 400 142 400
400 500 870 121 284 398 3000 500 250 400 624 800 3000 3000 222 250 337 714 600
M M M M F F F F F F E F F F F F M M M
individuals than in control subjects, whereas Carpenter et al. (1975) failed to differentiate a rather different schizophrenic population from normal controls. Very little is known about the source of the D M T occurring in the body fluids, and to elucidate the significance of observed differences in excretion between psychotic patients and normal individuals we need to know more about the factors affecting its excretion in normal subjects.
MATERIALS AND METHODS The subjects (7 males and i2 females, age range 19-68 years) had no previous history of any psychiatric illness and the majority were members of staff of either the Institute of Psychiatry or the Bethlem Royal and Maudsley Hospitals. None was receiving any medication during the experimental periods except that indicated. All the drugs were taken orally every 6 h unless otherwise specified. Enterically-coated tablets of ammonium chloride were obtained from either Macarthys Ltd., Romford, Essex, U.K. or from Eli Lilly & Co., Basingstoke, U.K.; d/-methionine, encapsulated in the Maudsley Hospital Pharmacy, from Haleswood Chemicals Ltd., Staines, Middlesex, U.K. ; neomycin from Glaxo Laboratories, Greenford, Middlesex, U.K. and 'Complan' from GlaxoFarley Foods Ltd., Plymouth, Devon, U.K. The psychoactive drugs were iproniazid (Marsilid, Roche Products Ltd.), thioridazine (Melleril, Sandoz Products Ltd.) and dithiepin HC1 (Prothiaden, Crookes Laboratories Ltd.). Urine was collected in plastic containers for 24 h (8.00 a.m. to 8.00 a.m.) without addition of preservative. Subjects were instructed to keep the sample cool during collection. Urinary pH and total creatinine were measured immediately on receipt of the sample which was.then frozen at 15~ and stored pending analysis.
Previous work has shown that DMT and NMT are stable for 3 months in urine frozen within 12 h of completing collection. The urinary methylated indoleamines were measured by a procedure with a limit of detection of 20 ng 24 h -1 for DMT and 50 ng 24 h -1 for NMT. The procedure will be described in detail elsewhere (Oon and Rodnight, 1977). Of the 24-h urine samples, 50~ was concentrated in a rotary evaporator to 70ml and extracted with toluene at alkaline pH. The resulting extract was purified by preparative thin layer chromatography, derivatised with trifluoroacetic anhydride and injected into a gas chromatograph (Perkin Elmer, F 17) fitted with a nitrogen-sensitive detector. An internal standard of 5-methyIdimethyltryptamine was added to the concentrated urine to account for variations in recovery. In the case of 10 urine extracts (4 from the present study and 6 from a patient population used for another study), combined gas chromatography and mass spectrometry (GC/MS) were used to examine the identity of peaks in the eluate from the gas chromatograph having the same retention time as the authentic DMT and NMT derivatives. Because of sensitivity problems it was impossible to obtain definitive mass spectra for the substance corresponding to the DMT peak. However, by using chemical ionisation and monitoring by the technique of mass fragmentography the pseudomolecular ion characteristic of DMT-trifluoracetate (m/e 285), the extracts were found to give a peak with precisely the same retention time as the pseudo-molecular ion of the authentic DMT derivative (for further details see Oon and Rodnight, 1977). This evidence still falls short of absolute identification, although the chance that the extracts contain a compound yielding an ion with identical mass and retention time as the pseudo-molecular ion of the DMT derivative is considered very unlikely. It is also significant that in these same extracts there was also close quantitative agreement between the results obtained by gas chromatography alone and by GC/MS.
RESULTS In 18 of the 19 samples analysed from drug-free subjects, the D M T excretion rate was below 500 ng 24 h - 1, and in 16 of the 19 samples the N M T rate was below 1000 ng 24 h - 1. There was no direct relationship between D M T and N M T excretion rates (Table 1). Neither were there any associations between D M T or N M T concentrations and age, sex, urinary volume, or creatinine. There was no general association between the indole contents and pH, but the subject with the lowest pH had a very high excretion of DMT. The mean pH for all the samples collected was 6.14 _+ 0.33 (SD) with a range 5 . 4 - 7 . 5 . Diurnal variation was studied in 5 individuals by collecting urine over three 8-h periods. The relationship between time of day and excretion of D M T and N M T varied between individuals and no consistent diurnal pattern emerged (Table 2). To examine whether any of the urinary D M T originates from gut-flora, two subjects took 8 g of neomycin daily for 2 days in an effort to diminish bacterial activity in the intestine. This treatment had no effect on the 24-h excretion of D M T and N M T over the 4 days studied. A preformed dietary source was excluded by an experiment in which a subject
173
M. C. H. Oon et al. : Urinary DMT in Normal Subjects
consumed only 'Complan' and water for 5 days; the daily excretion of both DMT and NMT was essentially unchanged throughout this period. Neither was there any observable change in the excretion of the two N-methylated compounds during a day of increased physical activity by two subjects. One subject collected 8 hourly specimens during a day of examinationinduced stress, but this apparently did not affect urinary DMT or NMT. The urinary excretion of amines is known to be affected by H ion balance in the kidney; for instance, acidification of the urine through administration of ammonium chloride greatIy enhances the rate of excretion of amphetamine by the kidney (Davis et al., 1971). In the present work 4 subjects had their urine acidified with ammonium chloride (8 g daily for 2 days), and in each case excretion of DMT increased considerably (Table 3); however, in no case did the peak of DMT excretion coincide with the lowest urinary pH. Surprisingly, urinary NMT appeared unaffected by acidification of the urine. In a further experiment one subject took 4 g of methionine over 3 days and although the urinary pH dropped to 5.6 there was no observable change in excretion of the two N-methylated compounds. Alkalinisation of the urine with sodium bicarbonate to a pH of 7.7 in one subject
Table 3 Urinary excretion of DMT and NMT after administration of ammonium chloride and methionine
Subject
Amine
Total excretion (ng) 0-8.00
1 1 2 2 3 3 4 4 5 5 6
DMT NMT DMT NMT DMT NMT DMT NMT DMT NMT DMT
216 433 100 38 154 300 200 600 86 150 20
6
NMT
2000
8.00-16.00 52 315
16.00-24.00
73 640 88 1352 18t 50
80 180 106 160 50 560 208 1150 20 670
not detectable
not detectable
1250
1000
-
and with potassium citrate to a pH of 7.6 in another subject did not alter to a significant extent the excretion of either DMT or NMT. In none of the above experiments on normal volunteers did any untoward psychological states coincide with high rates of DMT excretion following administration of ammonium chloride.
Subject (drug administered)
Day of collection
Urinary volume (ml 24 h -1)
Urinary pH
(ng 24 h--~)
NMT (rig 24 h 1)
1 (NH4C1,
1 2a 3 4 5
700 t850 1200 1850 1150
6.3 6.3 6.1 5.1 6.1
50 200 500 516 3000
400 620 1500 1500 500
1 2a 3a 4 5 6
900 1100 1100 1000 975 950
6.5 6.2 5.6 5.5 5.6 5.3
250 562 2500 50O 250 250
400 500 500 384 334 350
1 2~ 3~ 4
2200 2200 1400 1600
6.3 5.7 5.9 5.9
500 500 2500 100
625 300 500 300
1 2a
1600 2400 1850 80O 90O
7.0 7.0 5.8 6.3 6.7
350 300 300 1400 1500
400 400 300 400
1200 1650 1300 1350 1700
6.3 6.2 5.8 5.6 5.6
200 214 166 200 170
400 342 333 300 320
Macarthy)
2 (NH4CI,
Macarthy)
3 (NH,C1,
Macarthy) 4 (NH4CI,
Eli Lilly)
1 (Methionine) a The drug was administered on these days
Table 2. Urinary excretion of DMT and NMT over consecutive periods of 8 h
3~ 4 5
1 2a 3. 4" 5
DMT
3OO
174
Psychopharmacology54 (197'7) DISCUSSION
Iproniazid 2.8
I
q
f
'""',
2.0 T
.It'4
t
6'
~ Z
NMT
"',,, ~,/
\,
[
1.2
-.
rJ
I
I , A / / /
123 ;" 0.4
,,
,,
as ,,
/
L
i
5
i
38~
30
i
46
62~
5,*
70 90
Days
Fig. 1. Effectof iproniazid (Marsilid) on excretion of DMT and NMT in one subject. The dose of iproniazid was 75 mg daily over period indicated
3.0
NH4C I
N a HCO3
Neomyc in
Exercise
I
~t 2.0 DMT
>,
0
5 t
I
I
1
2
3
4
5
6
7
8
9
10 11
~2 13 14 15 16 17 18
Days
Fig.2. UrinaryDMT and NMT excretionin one subject;influence of NH4C], NaHCO3, neomycin,and exercise
The excretion of the two indoles was also studied in a patient with reactive depression given 75 mg daily of iproniazid, an inhibitor of both monoamine oxidase A and B. Figure I shows that the excretion of both D M T and N M T increased to double that of the previous drug-free period. Following cessation of the medication the D M T and N M T excretion rates decreased again. Another patient given a tricyclic antidepressant (150 mg of Prothiaden daily) over a 6-week period showed no change in the excretion of these methylated indoles. Likewise, 100 mg of thioridazine daily for 14 days did not modify D M T excretion. Figure 2 illustrates the effect of various experiments on the indole excretion of one subject.
The finding of an endogenous hallucinogen in the urine of normal individuals raises some interesting questions. D M T may arise as a by-product of some physiological process in the body unrelated to mental function, or alternatively it could have some physiological role in the nervous system in its own right. With present evidence the latter possibility seems unlikely in view of the apparent low activity of the N-methylating enzyme in brain. In a previous study (Rodnight et al., 1976) using a less sensitive method (limit 500 ng 24 h -1) than the present one we detected urinary DMT in only one out of 20 normal subjects. In the present study also only 1 out of 19 normal individuals excreted more than 500 ng 24 h-1. These findings go some way to explain the apparently contradictory results of studies of urinary D M T in schizophrenic and normal subjects (Carpenter et al., 1975; Rodnight et al., 1976). With a relatively insensitive method, the putative compounds may be detected in only occasional normal subjects, but with a very sensitive method they may be found in every case. That normal subjects excrete DMT is consistent with previous findings that DMT was present in the blood of some normal subjects (Mandel, 1974; Angrist et al., 1976). It may be that sequential studies using highly sensitive methods will demonstrate D M T in the blood of all normal subjects at some time. In fact, our study does not exclude the possibility that diurnal or other fluctuations do occur in the excretion of these two methylated indoles. Because of the sensitivity limits of our analysis, we could not measure the quantity excreted in urine collected over periods shorter than 8 h. We cannot, therefore, tell whether these compounds were excreted consistently over the periods we studied or only in short bursts. Sequential studies, preferably of blood, with a yet more sensitive analytical method will be required to answer this question. There are numerous pitfalls in the biochemical investigaton of mental illness (Rodnight, 1975). In the past researchers of schizophrenia have been misled by urinary substances that were later found to origb nate in the diet or were products of intestinal bacteria. This study suggests that neither of these two factors are relevant to the excretion of urinary DMT or NMT. Similarly, nonspecific stresses such as emotional tension or physical exertion do not appear to be major dererminants of the excretion of these indoles. Monoamine oxidase (MAO) is the major catabolic enzyme for D M T and N M T (Lu and Domino, 1976), as well as their precursor tryptamine. Thus, the inhibition of MAO might not only decrease catab-
M. C. H. Oon et al. : Urinary DMT in Normal Subjects
olism of DMT and NMT but also increase their biosynthesis by blocking the catabolism of tryptamine by MAO and increasing its N-methylation. [t is, therefore, hardly surprising that administration of a monoamine oxidase inhibitor resulted in the increased excretion of DMT and NMT in the subject studied. The increase in excretion of DMT following the administration of ammonium chloride merits further consideration. This 'ammonium chloride' effect did not coincide with the lowest urinary pH, and acidification with methionine had no effect (a finding of interest in view of the status of methionine as a methyl donor). It therefore seems unlikely that the increase in DMT excretion was a straightforward consequence of acidifying the body fluids. Nor is it likely that the ammonium ions were interfering with the catabolism of DMT since in that case a more prolonged increase in the concentration of urinary DMT would have been expected. It remains conceivable that ammonium chloride releases DMT from some store in peripheral tissues. These and other possibilities require further investigation. Acknowledgements. We are grateful to Judith Bastow and Imogen Dawes (The Pharmacy, Maudsley Hospital) and to John Garrod (Department of Pharmacy, Chelsea College) for advice. M.C.H.O. was supported by a grant from the Medical Research Council of the U.K.; M.P.M. by a grant from the Wellcome Trust; R.M.M. by a grant from the Research Fund of the Bethlem Royal and Maudsley Hospitals.
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175 Boarder, M. R., Rodnight, R.: Tryptamine N-mcthyltransferase activity in brain tissue: a re-examination. Brain Res. 114, 359 - 364 (1976) Carpenter, W. T., Fink, E. B., Narasimhachari, N., Himwich, H. E.: A test of the transmethylation hypothesis in acute schizophrenic patients. Am. J. Psychiatry 132, 1067-1071 (1975) Davis, J. M., Kopin, I. J., Lemberger, L., Axelrod, J.: Effects of urinary pH on amphetamine metabolism. Ann. N.Y. Acad. Sci. 179, 493-501 (1971) Domino, E.F.: The indole hallucinogen model: is it worth pursuing? In: Predictability in psychopharmacology : preclinical and clinical correlations, A. Sudilovsky, S. Gershon, B. Beer, eds., pp. 247-268. New York: Raven Press 1975 Gomes, U. R., Neethling, A. C., Stanley, B. C.: Enzymatic Nmethylation of indoleamines by mammalian brain: fact ol- artefact? J. Neurochem. 27, 701 - 705 (1976) Guchhait, R. B.: Biogenesis of 5-methoxy-N,N,-dimethyltryptamine in human pineaI gland. J. Neurochem. 26, 187- 190 (1976) Kaplan, J., Mandel, L. R., Walker, R. W., Vanden Heuvel, W. J. A., Stillman, R., Gillin, J. C., Wyatt, R. J.: Blood and urine levels of N,N-dimethyltryptamine following administration of psychoactive dosages to human subjects. Psychopharmacologia (Bed.) 38, 239-245 (1974) Lu, L. J., Domino, E. F. : Effect of iproniazid and tranylcypromine on the half-life of N,N-dimethyltryptamine in rat brain and liver. Biochem. Pharmacol. 25, 1521-1527 (1976) Mandel, L. R.: Dimethyltryptamine. Psychopharmacol. Bull. 10, 55 - 56 (1974) Morgan, M., Mandell, A.J.: Indole(ethyl)amine N-methyltransferase in the brain. Science 165, 492-493 (1969) Narasimhachari, N., HelIer, B., Spaide, J., Haskovec, L., Meltzer, H., Strahilevitz, M., Himwich, H. E. : N,N,-dimethylated indoleamines in blood. Biol. Psychiatry 3, 21-23 (1971) Oon, M. C. H., Rodnight, R.: A gas chromatographic procedure for determining N,N-dimethyltryptamine and N-monomethyltryptamine in urine using a nitrogen detector. Biochemical Medicine (in press, 1977) Rodnight, R. : The significance of some biochemical abnormalities in schizophrenia. Acta Neurologica 30, 86-101 (1975) Rodnight, R., Murray, R. M., Oon, M. C. H., Brockington, I. F., Nicholls, P., Birley, J. L. T. : Urinary dimethyltryptamine and psychiatric symptomatology and classification. Psychol. Med. 6, 649-657 (1976) Saavedra, J. M., Coyle, J. R., Axelrod, J.: The distribution and properties of the non-specific N-methyltransferase in brain. J. Neurochem. 20, 743-752 (1973) Taylor, R. T., Hanna, M. L.: S-methyltetrahydrofolate aromatic alkylamine N-methyltransferase: an artefact of 5,10-methylene tetrahydrofolate reductase activity. Life Sci. 17, 111 - 120 (1975) Received April 4, 1977
