Methylation Hypothesis Ross J. Baldessarini, MD;
Giorgio Stramentinoli, PhD; Joseph
\s=b\ L-Methionine had no behavioral effects in normal humans and failed to increase concentrations of S-adenosylmethionine (methyl donor) in human or rat blood, while increasing rat liver levels more than fivefold. Methionine or S-adenosylmethionine in very high doses had almost no effect on methylation of tritiated levodopa in rodent tissues; various "methyl acceptor" molecules, including nicotinamide, guanidineacetic acid, and estradiol similarly had little effect. In rabbit lung, methionine and S-adenosylmethionine not only failed to increase production of dimethyltryptamine, but actually decreased it, possibly due to end-product inhibition by S-adenosylhomocysteine, which also strongly inhibited methylation of dopa in rat. These results fail to support several predictions of the "methylation hypothesis" concerning the pathophysiology and potential treatment of idiopathic psychotic disorders and leave the consistent clinical worsening effects of methionine in schizophrenia unexplained. (Arch Gen Psychiatry 36:303-307, 1979)
many years, there has been considerable interest in chemistry and pharmacology of biological transmethylation in the field of neuropsychiatry.1- This interest was largely stimulated by the fact that many natural or synthetic substances that produce a variety of psychotropic effects, including hallucinations or other reactions that also occur in psychotic illnesses, are methylated amines. As early as 1952, Harley-Mason suggested that abnormal transmethylation of an endogenous amine, possibly
For
the
dopamine, might produce a psychotomimetic compound like mescaline. Such products were proposed to be formed by the action of the methyl group (CH,) donor S-adenosyl¬ methionine—a metabolite of methionine and adenosine
triphosphate (ATP) synthesized by a specific adenosyltransferase (see Baldessarini1). Evidence apparently consistent with this hypothesis was the observation that methionine, uniquely among several amino acids, and especially when combined with an inhibitor of monoamine oxidase, led to striking and reversible exacerbations of the psychotic symptoms of chronic schizophrenic patients with¬ out inducing obvious signs of delirium.'4 It has also been reported that S-adenosylmethionine levels in the blood of acutely psychotic,"' but not chronically psychotic,' " schizo¬ phrenic subjects are low, possibly indicating its excessive utilization or deficient production."' Moreover, it has been reported recently that intravenously injected S-adenosylAccepted
for publication March 27, 1978. From the Mailman Laboratories for Psychiatric Research, McLean Division of Massachusetts General Hospital, Belmont, Mass, and the Department of Psychiatry, Harvard Medical School, Boston. Dr Stramentinoli is currently with the Department of Biochemistry, BioResearch Co, Liscate
(Milan), Italy.
Portions of this work were discussed at an International Symposium on Methylation in the Central Nervous System, Naples, Italy, May 2, 1978. Reprints not available.
Downloaded From: by a UNIVERSITY OF SYDNEY LIBRARY User on 03/26/2018
F.
Lipinski,
MD
methionine can exert clinically useful psychotropic effects in depression.7 There have also been repeated suggestions that there -'·8 or indomay be unusual methylated phenethylamines' leamines12S11 in the urine or blood of psychotic patients. On the other hand, the significance of these findings has been questioned or not supported by several studies reported over the past decade. Many criticisms have been made of this search for unusual patterns of production or excretion of methylated metabolites on methodological grounds, particularly the use of ambiguous analytical techniques and inadequate control of dietary and other extraneous variables, as well as uncertainties regarding clinical diagnosis.1 L"" Clinical studies of this kind have continued to the present time, and recent work is charac¬ terized by the use of increasingly specific biochemical methods and greater attention to clinical variables. These results have been both positive"""1 and negative1'17-0 regarding relationships between blood levels or excretion of methylated indoleamines or phenethylamines and schiz¬ ophrenia or other severe psychiatric disorders. The hypoth¬ esis that unusual methylated amine compounds might contribute to the pathophysiology of schizophrenia or manic-depressive illness has been given further encourage¬ ment by reports of lower activity of monoamine oxidase in blood platelets of patients with these disorders.2' While the possible contribution of psychoactive methy¬ lated amines to the major idiopathic mental illnesses remains obscure, the observation that 10- to 20-g loading doses of methionine, of several amino acids, uniquely led to exacerbation of psychosis in schizophrenic patients is highly unusual among biological findings in severe psychiatric illness in that it has been independently confirmed repeatedly in the past decade.1412 The metabolic basis for this action of methionine in schizophrenic patients, as well as the more recently reported antidepressant action of S-adenosylmethionine, are still not under¬ stood. While interest in the transmethylation hypothesis has waned in recent years, the fact remains that the effect of methionine is one of the few viable clinical clues to a metabolic abnormality in psychotic illness. The initial suggestion that methionine might act by increasing the production of potentially psychotomimetic methylated amines by way of increased availability of S-adenosyl¬ methionine1'2 is only partially supported by the available evidence. Thus, there is excellent metabolic evidence in animals, and very little in man, that the availability of S-adenosylmethionine is dependent on the availability of its precursor, methionine, and on the rate of utilization of the methyl donor, which can be increased by giving large doses of certain "methyl acceptors."2224 On the other hand, the evidence that methionine loading increases blood or
tissue levels of S-adenosylmethionine in patients remains untested even though the means of doing so have been available for more than ten years. Furthermore, it remains unproven that increased availability of S-adenosyl¬ methionine can increase the production of methylated biogenic amines. These considerations have led us to reopen the study of transmethylation in animals and man. We were particular¬ ly interested to learn whether increased availability of exogenous methionine can increase levels of the methyl donor S-adenosylmethionine in human subjects. Further¬ more, we have attempted to test the prediction offered by the methylation hypothesis that increased levels of methionine and S-adenosylmethionine in animals might increase the production of methylated índoles or catechols. METHODS
With the approval of the US Food and Drug Administra¬ tion and our Hospital Human Studies Review Committee, normal adult human subjects consented to ingesting lmethionine and having antecubital blood samples taken for assay of S-adenosylmethionine by a sensitive and specific
radioenzymatic method,6'23 using [methyl-,4C]-S-adenosylL-methionine. Parallel experiments were also conducted in laboratory rats to assess the influence of methionine on blood S-adenosylmethionine levels, using liver as a control for the effect of methionine on tissue levels of S-adenosyl¬ methionine.23-24 In other experiments,
evaluated the effects of lmethionine, or a chemically stable salt of S-adenosyl¬ methionine (Samyr), and of several other substances obtained from commercial sources in the highest available purity. In some of the experiments we studied the methy¬ lation of chromatographically pure [G-3H]-L-dihydroxyphenylalanine (tritium-levodopa, 10 Ci/mmole), given systemically in a tracer dose (25 µ0 /200 g male SpragueDawley rat, intraperitoneally). This was done by pretreating the rats with the powerful and irreversible monoamine oxidase-inhibitor pargyline hydrochloride the evening prior to giving test substances followed by tritium-levodo¬ pa. Five to thirty minutes later, tissues were removed and the radioactive metabolites were extracted: catechols and methylated metabolites were separated by aluminum oxide column chromatography and counted by scintillation spectrometry. To compensate for potential artifactual changes in uptake of the tracer into some tissues (notably, the known competition of methionine and dopa for entry into the brain1), metabolic data were computed as ratios of radioactivity in the methylated to the free-catechol frac¬ tions, rather than as absolute quantities of the metabolites. These methods have been described in detail previously.25 In another laboratory experiment we evaluated the effect of L-methionine or S-adenosylmethionine on the accumulation of ['4C]-A7,Ar-dimethyltryptamine in lung tissue of New Zealand rabbits. The animals were pretreated with the monoamine oxidase inhibitor pheniprazine (Catron) hydrochloride to prevent breakdown of the labeled indoleamines. Later, they were given [14C]-
combination of silica gel thin layer chromatography followed by cocrystallization with authentic -2V,A/"-dimethyltryptamine and oxalic acid to constant specific radioactiv¬ ity. This method is sensitive and highly selective for isolating iV^iV-dimethyltryptamine as its oxalate, as has been described in detail elsewhere.26-27 RESULTS When methionine was given to rats by orogastric tube in a high dose (300 mg/kg), the levels of S-adenosylmethio¬ nine in liver increased more than fivefold, and remained elevated for more than two hours (Table 1). When parallel experiments were done with rat (Table 1) and human subjects (Table 2), blood levels of S-adenosylmethionine were not altered by large doses of methionine. In the human subjects, no change was detected in whole blood, plasma, or in the cellular fractions (erythrocytes plus buffy
coat) (Table 2). Nevertheless,
decided to test the possible conse¬ levels of S-adenosylmethionine by methionine, as this effect had been well documented in laboratory animals even at lower doses of methionine than used in the human subjects in the present work (Table 1; see also Baldessarini121). We were particularly interested in testing the prediction that increased S-adenosylmethioquences of
we
increasing
Table 1— Blood and Liver S-adenosylmethionine Levels in Rat After Oral L-Methionine*
S-adenosylmethionine
we
N-methyltryptamine (50 Ci/mole,
9.2
µCi/rabbit) by
ear
vein. Five minutes later the rabbits were killed and radioactive material was extracted from the whole lung and ["CJ-Aysf-dimethyltryptamine was separated by a
Downloaded From: by a UNIVERSITY OF SYDNEY LIBRARY User on 03/26/2018
Levels
*_
Time, min
Blood, /ig/ml 1.99 ± 0.25
30
2.20 ± 0.21 2.38 ± 0.14t 2.49 ± 0.13t
60 120
Liver, ;
Giorgio Stramentinoli, PhD; Joseph
\s=b\ L-Methionine had no behavioral effects in normal humans and failed to increase concentrations of S-adenosylmethionine (methyl donor) in human or rat blood, while increasing rat liver levels more than fivefold. Methionine or S-adenosylmethionine in very high doses had almost no effect on methylation of tritiated levodopa in rodent tissues; various "methyl acceptor" molecules, including nicotinamide, guanidineacetic acid, and estradiol similarly had little effect. In rabbit lung, methionine and S-adenosylmethionine not only failed to increase production of dimethyltryptamine, but actually decreased it, possibly due to end-product inhibition by S-adenosylhomocysteine, which also strongly inhibited methylation of dopa in rat. These results fail to support several predictions of the "methylation hypothesis" concerning the pathophysiology and potential treatment of idiopathic psychotic disorders and leave the consistent clinical worsening effects of methionine in schizophrenia unexplained. (Arch Gen Psychiatry 36:303-307, 1979)
many years, there has been considerable interest in chemistry and pharmacology of biological transmethylation in the field of neuropsychiatry.1- This interest was largely stimulated by the fact that many natural or synthetic substances that produce a variety of psychotropic effects, including hallucinations or other reactions that also occur in psychotic illnesses, are methylated amines. As early as 1952, Harley-Mason suggested that abnormal transmethylation of an endogenous amine, possibly
For
the
dopamine, might produce a psychotomimetic compound like mescaline. Such products were proposed to be formed by the action of the methyl group (CH,) donor S-adenosyl¬ methionine—a metabolite of methionine and adenosine
triphosphate (ATP) synthesized by a specific adenosyltransferase (see Baldessarini1). Evidence apparently consistent with this hypothesis was the observation that methionine, uniquely among several amino acids, and especially when combined with an inhibitor of monoamine oxidase, led to striking and reversible exacerbations of the psychotic symptoms of chronic schizophrenic patients with¬ out inducing obvious signs of delirium.'4 It has also been reported that S-adenosylmethionine levels in the blood of acutely psychotic,"' but not chronically psychotic,' " schizo¬ phrenic subjects are low, possibly indicating its excessive utilization or deficient production."' Moreover, it has been reported recently that intravenously injected S-adenosylAccepted
for publication March 27, 1978. From the Mailman Laboratories for Psychiatric Research, McLean Division of Massachusetts General Hospital, Belmont, Mass, and the Department of Psychiatry, Harvard Medical School, Boston. Dr Stramentinoli is currently with the Department of Biochemistry, BioResearch Co, Liscate
(Milan), Italy.
Portions of this work were discussed at an International Symposium on Methylation in the Central Nervous System, Naples, Italy, May 2, 1978. Reprints not available.
Downloaded From: by a UNIVERSITY OF SYDNEY LIBRARY User on 03/26/2018
F.
Lipinski,
MD
methionine can exert clinically useful psychotropic effects in depression.7 There have also been repeated suggestions that there -'·8 or indomay be unusual methylated phenethylamines' leamines12S11 in the urine or blood of psychotic patients. On the other hand, the significance of these findings has been questioned or not supported by several studies reported over the past decade. Many criticisms have been made of this search for unusual patterns of production or excretion of methylated metabolites on methodological grounds, particularly the use of ambiguous analytical techniques and inadequate control of dietary and other extraneous variables, as well as uncertainties regarding clinical diagnosis.1 L"" Clinical studies of this kind have continued to the present time, and recent work is charac¬ terized by the use of increasingly specific biochemical methods and greater attention to clinical variables. These results have been both positive"""1 and negative1'17-0 regarding relationships between blood levels or excretion of methylated indoleamines or phenethylamines and schiz¬ ophrenia or other severe psychiatric disorders. The hypoth¬ esis that unusual methylated amine compounds might contribute to the pathophysiology of schizophrenia or manic-depressive illness has been given further encourage¬ ment by reports of lower activity of monoamine oxidase in blood platelets of patients with these disorders.2' While the possible contribution of psychoactive methy¬ lated amines to the major idiopathic mental illnesses remains obscure, the observation that 10- to 20-g loading doses of methionine, of several amino acids, uniquely led to exacerbation of psychosis in schizophrenic patients is highly unusual among biological findings in severe psychiatric illness in that it has been independently confirmed repeatedly in the past decade.1412 The metabolic basis for this action of methionine in schizophrenic patients, as well as the more recently reported antidepressant action of S-adenosylmethionine, are still not under¬ stood. While interest in the transmethylation hypothesis has waned in recent years, the fact remains that the effect of methionine is one of the few viable clinical clues to a metabolic abnormality in psychotic illness. The initial suggestion that methionine might act by increasing the production of potentially psychotomimetic methylated amines by way of increased availability of S-adenosyl¬ methionine1'2 is only partially supported by the available evidence. Thus, there is excellent metabolic evidence in animals, and very little in man, that the availability of S-adenosylmethionine is dependent on the availability of its precursor, methionine, and on the rate of utilization of the methyl donor, which can be increased by giving large doses of certain "methyl acceptors."2224 On the other hand, the evidence that methionine loading increases blood or
tissue levels of S-adenosylmethionine in patients remains untested even though the means of doing so have been available for more than ten years. Furthermore, it remains unproven that increased availability of S-adenosyl¬ methionine can increase the production of methylated biogenic amines. These considerations have led us to reopen the study of transmethylation in animals and man. We were particular¬ ly interested to learn whether increased availability of exogenous methionine can increase levels of the methyl donor S-adenosylmethionine in human subjects. Further¬ more, we have attempted to test the prediction offered by the methylation hypothesis that increased levels of methionine and S-adenosylmethionine in animals might increase the production of methylated índoles or catechols. METHODS
With the approval of the US Food and Drug Administra¬ tion and our Hospital Human Studies Review Committee, normal adult human subjects consented to ingesting lmethionine and having antecubital blood samples taken for assay of S-adenosylmethionine by a sensitive and specific
radioenzymatic method,6'23 using [methyl-,4C]-S-adenosylL-methionine. Parallel experiments were also conducted in laboratory rats to assess the influence of methionine on blood S-adenosylmethionine levels, using liver as a control for the effect of methionine on tissue levels of S-adenosyl¬ methionine.23-24 In other experiments,
evaluated the effects of lmethionine, or a chemically stable salt of S-adenosyl¬ methionine (Samyr), and of several other substances obtained from commercial sources in the highest available purity. In some of the experiments we studied the methy¬ lation of chromatographically pure [G-3H]-L-dihydroxyphenylalanine (tritium-levodopa, 10 Ci/mmole), given systemically in a tracer dose (25 µ0 /200 g male SpragueDawley rat, intraperitoneally). This was done by pretreating the rats with the powerful and irreversible monoamine oxidase-inhibitor pargyline hydrochloride the evening prior to giving test substances followed by tritium-levodo¬ pa. Five to thirty minutes later, tissues were removed and the radioactive metabolites were extracted: catechols and methylated metabolites were separated by aluminum oxide column chromatography and counted by scintillation spectrometry. To compensate for potential artifactual changes in uptake of the tracer into some tissues (notably, the known competition of methionine and dopa for entry into the brain1), metabolic data were computed as ratios of radioactivity in the methylated to the free-catechol frac¬ tions, rather than as absolute quantities of the metabolites. These methods have been described in detail previously.25 In another laboratory experiment we evaluated the effect of L-methionine or S-adenosylmethionine on the accumulation of ['4C]-A7,Ar-dimethyltryptamine in lung tissue of New Zealand rabbits. The animals were pretreated with the monoamine oxidase inhibitor pheniprazine (Catron) hydrochloride to prevent breakdown of the labeled indoleamines. Later, they were given [14C]-
combination of silica gel thin layer chromatography followed by cocrystallization with authentic -2V,A/"-dimethyltryptamine and oxalic acid to constant specific radioactiv¬ ity. This method is sensitive and highly selective for isolating iV^iV-dimethyltryptamine as its oxalate, as has been described in detail elsewhere.26-27 RESULTS When methionine was given to rats by orogastric tube in a high dose (300 mg/kg), the levels of S-adenosylmethio¬ nine in liver increased more than fivefold, and remained elevated for more than two hours (Table 1). When parallel experiments were done with rat (Table 1) and human subjects (Table 2), blood levels of S-adenosylmethionine were not altered by large doses of methionine. In the human subjects, no change was detected in whole blood, plasma, or in the cellular fractions (erythrocytes plus buffy
coat) (Table 2). Nevertheless,
decided to test the possible conse¬ levels of S-adenosylmethionine by methionine, as this effect had been well documented in laboratory animals even at lower doses of methionine than used in the human subjects in the present work (Table 1; see also Baldessarini121). We were particularly interested in testing the prediction that increased S-adenosylmethioquences of
we
increasing
Table 1— Blood and Liver S-adenosylmethionine Levels in Rat After Oral L-Methionine*
S-adenosylmethionine
we
N-methyltryptamine (50 Ci/mole,
9.2
µCi/rabbit) by
ear
vein. Five minutes later the rabbits were killed and radioactive material was extracted from the whole lung and ["CJ-Aysf-dimethyltryptamine was separated by a
Downloaded From: by a UNIVERSITY OF SYDNEY LIBRARY User on 03/26/2018
Levels
*_
Time, min
Blood, /ig/ml 1.99 ± 0.25
30
2.20 ± 0.21 2.38 ± 0.14t 2.49 ± 0.13t
60 120
Liver, ;
