Life Sciences, Vol . 23, pp . 1577-1582 Printed in the U .S .A .
Pergamon Press
LOW PLATELET MONOAMINE OXIDASE ACTIVITY AND CHRONIC MARIJUANA USE Richard C . Stilimam e-2 Richard Jed Wyatt, e Dennis L . Murphy,' and Frederick P . Rauscher e e Laboratory of Clinical Psychopharmacology, Division of Special Mental Health Research, Intramural Research Program, Saint Elizabeths Hospital Washington, D .C . 20032 Z Intramural Research Program, Division of Research National Institute ön Drug Abuse, Rockville, Maryland 20903 3 Clinical Neuropharmacology Branch, National Institute of Mental Health Bethesda, Maryland 20014 (Received in final form August 25, 1978)
Swnnary Mean platelet monoamine oxidase (MAO) activity in 26 consecutivelystudled male marijuana smokers was significantly lower than in a comparable group of non-marijuana smoking males . In addition, the level of current marijuana use reported by the subjects was significantly and inversely correlated with MAO activity . No acute reduction in MAO activity was found 1n response to smoking a marijuana cigarette containing 15 mg of delta-9-THC . Significant in vitro inhibition of MAO activity by THC was detected only at TH~concentrations above 10-5M, approximately 100 times the peak plasma concentrations seen in vivo following smoking . Reduced platelet monoamine oxidase activity has been found in several (but not all) studies of chronic schizophrenic patients (1-3) and in one study of identical twins discordant for schizophrenia (4) . Although platelet monoamine oxidase (MAO) activity was not reduced in acute schizophrenics (5) or in unipolar depressed patients, reduced MAO activity has been reported in bipolar patients (6) and 1n their first-degree relatives (7) . Thus, reduced platelet MAO activity has been implicated in at least two psychiatric disorders pf grave behavioral and personal consequence . Platelet MAO activity is highly replicable in most individuals over periods from 1 to 10 weeks, suggesting that platelet MAO activity may represent a relatively stable characteristic of individuals (B) . Studies of the effects of chronic treatment with delta-9-tetrahydrocannabinol (THC) on MAO activity in animals have found increases in enzyme activity in rat brain, uterus and other tissues including platelets (9-11) ; liver MAO activity, however, was unchanged (9,11) . One in vitro study reported inhibition of porci ne brain mi tochondri al MAO acts vi ty by~H~(1-2~ . In order to assess possible MAO activity differences in relation to marijuana use, we first measured MAO activity in a group of 26 known users . Fending that MAO activity was reduced in this group, we then studied the acute effects of THC on MAO activity both in yivo and in vitro , in order to assess whether any 0300-9653/78/1016-157702 .00/0
1578
Platelet hfAO Activity in hlarijuana Smokers
~'ol . ?.ï, ~o . 15, 19'R
traces of THC in the blood of habitual marijuana users might directly affect MAO activity . A.
Relation between MAO activity and current marijuana use Procedure Subjects :
Normal male paid volunteers, a e 20-34, were recruited for experiments on the effects of marijuana on memory 13,14) . All applicants for the experiments spent an initial half-day in a screening session, which included psychological and psychiatric examinations, a physical examination, routine blood analyses (CBC, chemistry panel) and a drug history taken personally by the psychiatrist in charge of the experiments . Subjects were assured that their answers would be confidential . During this interview on drug usage, the psychiatrist asked a number of questions about marijuana, which included the subjects' estimates of current use and how long they had smoked . (Other drugs included in the interview were alcohol, barbiturates, hypnotic/sedatives, stimulants, hallucinogens, cocaine and opiates .) No subjects reporting regular use of any of these other drugs except alcohol (under 1 ounce daily) were included in this study . Assa~ : Twenty-six consecutive male applicants for our marijuana experiments provided blood for MAO determinations and drug histories . Blood for MAO analysis was drawn concurrently with the hematology and clinical chemistry specimens ; it was collected in Becton-Dickinson Company Vacutainer (R) tubes with acidIt was kept in ice for no more than two hours and citrate-dextrose (ACD) . and separated into two samples, one for counting platelets and the other, obtained after centifugation, consisting of platelet-rich plasma . The assay for platelet MAO activity was then performed as described previously, using C-14-labelled benzylamine as the substrate (8) . MAO activity was expressed in units of nmoles per lOß platelets per hour . The non-marijuana smokers' bloods had been drawn previously from male volunteers and hospital personnel at two hospitals in the Washington, D .C . Area (NIH Clinical Center and Saint Elizabeths Hospital) . Results : First, the marijuana-user group had There were two significant findings : lower MAO activity than the non-user group . Second, within the marijuana-user group, MAO activity was inversely proportional to current use of marijuana . The group of 26 marijuana users (ages 25-35) had a mean MAO activity of 8 .56± .58 (S .E .M .) compared with a value for 348 normal males (ages 18-60) of 11 .041 .29 (8) . This difference is significant at p< .O1 (two-tailed t-test) . {Although the normal group included a wider range of subject ages, no relation was found in either group between age and MAO activity .) No statistically significant differences were found between the heavier marijuana users and the light marijuana users in their total use of other drugs during the past year (median split) . The amount of current marijuana use over the past two months reported by marijuana smokers was inversely correlated with platelet MAO activity (Pearson r=- .48) . Mean frequency of use was 4 .3 times weekly with a S .D . of 2 .8 times weekly . Total duration of marijuana use was uncorrelated with platelet MAO activity, r=- .14, NS ; however, the mean duration of use was fairly uniform (mean 5 .3 years with a S .D . of only .81 years) .
Vol . 23, No .
B.
15, 1978
Platelet MAO Activity in Marijuana Smokers
1579
Acute effects of THC on MAO activity Procedure :
Three marijuana-smoking subjects were selected randomly from the previously described group of users . Their current frequencies of use were once a day, twice a week, and a eight times a week, respectively . The subjects, tested individually on separate days, were given marijuana cigarettes to smoke which had been previously cut to a length to contain 15 mg of THC . They smoked over an 8-minute period, adjustin their smoking speed to consume an eighth of the cigarette each minute (13,14 . All three reported being quite "high" at the end of this period ; one said he would not want any more . Samples were collected prior to smoking, 20 minutes after beginning smoking, one hour after smoking, and four hours after smoking . Samples for THC analysis were drawn into citrated tubes which were spun at 50,000 RPM and the plasma decanted by pi petting . The plasma samples were analyzed for delta-9-THC by Dr . Roger Foltz at Batelle Columbus Laboratories in Ohio, using gas-chromatograph/mass spectroscopy (GS/MS) methods . Another portion of each sample was prepared and analyzed for MAO activity as described above . To evaluate the effect of THC on platelet MAO activity in vitro, aliquots of platelet sonicates from normal subjects which contained approx~mâtely 0 .3 mg protein were preincubated for 30 minutes at 25°C with 10 -9 to 10-3M THC prepared in 1% ethanol . The 14C-benzylamine substrate was then added and the entire assay procedure completed as described (8) . Controls preincubated similarly with 1% ethanol exhibited no MAO inhibition . Results : MAO activity in vivo over a four-hour period was unaffected by the inhalation of 15 mg THC in a marijuana cigarette, despite the appearance of significant concentrations of THC in the subjects' blood, the characteristic THC-in duced increase in heart rate and the marked subjective "high" reported by these experienced users (Figure 1) (13,14) . In vitro , THC at low concentrations had no inhibitory effect on platelet MAO activity (Figure 2) . At high concentrations (10-5 to 10-3M, approximately 100-10,000 fold higher than peak plasma THC levels measured in man after smoking a standard marijuana cigarette containing 15 mg THC), 20-35% inhibition of MAO activity was observed . DISCUSSION These findings suggest that marijuana users have lower MAO activity than non-users, with the heaviest users exhibiting the lowest enzyme activity of all . The negative intra-group correlation between current frequency of use and MAO activity might suggest that marijuana use directly affects this enzyme system . If this were so, it would seem related to chronic use, since in acute in vivo and in vitro studies, no significant inhibition of MAO by clinicallyatt~ble côncentrations of THC was observed . It is possible, though less likely, that the reduction of MAO activity might have resulted from either some component of marijuana other than THC, or from the past use of illicit drugs other than marijuana ; current users of other drugs have been screened from the study .
of An alternate explanation for the pattern of MAO activity in this sample marijuana users is that individuals with low MAO activity choose to use mari-
1580
Platelet MAO Activity in Marijuana Smokers
0 mln
20 n~ln
I h
2 Ir
Vol . 23, No .
15,
1978
3 4 6 hr Ir k
FIG . 1 Administration of 15 mg THC by inhalation (shaded bar) to three subjects . Ordinates are (1) change in heart rate (beats er minute over baseline), (2) change in high (subjective ratings 0-10), (3~ plasma concentration of THC (ng/ ml), and (4) platelet MAO activity (nmoles benzylamine/108 platelets/hour) .
50
0 m z
25
z
0
10_e
10~
10~`
THC (M)
FIG . 2 Percent inhibition of platelet MAO by THC . (p
Pergamon Press
LOW PLATELET MONOAMINE OXIDASE ACTIVITY AND CHRONIC MARIJUANA USE Richard C . Stilimam e-2 Richard Jed Wyatt, e Dennis L . Murphy,' and Frederick P . Rauscher e e Laboratory of Clinical Psychopharmacology, Division of Special Mental Health Research, Intramural Research Program, Saint Elizabeths Hospital Washington, D .C . 20032 Z Intramural Research Program, Division of Research National Institute ön Drug Abuse, Rockville, Maryland 20903 3 Clinical Neuropharmacology Branch, National Institute of Mental Health Bethesda, Maryland 20014 (Received in final form August 25, 1978)
Swnnary Mean platelet monoamine oxidase (MAO) activity in 26 consecutivelystudled male marijuana smokers was significantly lower than in a comparable group of non-marijuana smoking males . In addition, the level of current marijuana use reported by the subjects was significantly and inversely correlated with MAO activity . No acute reduction in MAO activity was found 1n response to smoking a marijuana cigarette containing 15 mg of delta-9-THC . Significant in vitro inhibition of MAO activity by THC was detected only at TH~concentrations above 10-5M, approximately 100 times the peak plasma concentrations seen in vivo following smoking . Reduced platelet monoamine oxidase activity has been found in several (but not all) studies of chronic schizophrenic patients (1-3) and in one study of identical twins discordant for schizophrenia (4) . Although platelet monoamine oxidase (MAO) activity was not reduced in acute schizophrenics (5) or in unipolar depressed patients, reduced MAO activity has been reported in bipolar patients (6) and 1n their first-degree relatives (7) . Thus, reduced platelet MAO activity has been implicated in at least two psychiatric disorders pf grave behavioral and personal consequence . Platelet MAO activity is highly replicable in most individuals over periods from 1 to 10 weeks, suggesting that platelet MAO activity may represent a relatively stable characteristic of individuals (B) . Studies of the effects of chronic treatment with delta-9-tetrahydrocannabinol (THC) on MAO activity in animals have found increases in enzyme activity in rat brain, uterus and other tissues including platelets (9-11) ; liver MAO activity, however, was unchanged (9,11) . One in vitro study reported inhibition of porci ne brain mi tochondri al MAO acts vi ty by~H~(1-2~ . In order to assess possible MAO activity differences in relation to marijuana use, we first measured MAO activity in a group of 26 known users . Fending that MAO activity was reduced in this group, we then studied the acute effects of THC on MAO activity both in yivo and in vitro , in order to assess whether any 0300-9653/78/1016-157702 .00/0
1578
Platelet hfAO Activity in hlarijuana Smokers
~'ol . ?.ï, ~o . 15, 19'R
traces of THC in the blood of habitual marijuana users might directly affect MAO activity . A.
Relation between MAO activity and current marijuana use Procedure Subjects :
Normal male paid volunteers, a e 20-34, were recruited for experiments on the effects of marijuana on memory 13,14) . All applicants for the experiments spent an initial half-day in a screening session, which included psychological and psychiatric examinations, a physical examination, routine blood analyses (CBC, chemistry panel) and a drug history taken personally by the psychiatrist in charge of the experiments . Subjects were assured that their answers would be confidential . During this interview on drug usage, the psychiatrist asked a number of questions about marijuana, which included the subjects' estimates of current use and how long they had smoked . (Other drugs included in the interview were alcohol, barbiturates, hypnotic/sedatives, stimulants, hallucinogens, cocaine and opiates .) No subjects reporting regular use of any of these other drugs except alcohol (under 1 ounce daily) were included in this study . Assa~ : Twenty-six consecutive male applicants for our marijuana experiments provided blood for MAO determinations and drug histories . Blood for MAO analysis was drawn concurrently with the hematology and clinical chemistry specimens ; it was collected in Becton-Dickinson Company Vacutainer (R) tubes with acidIt was kept in ice for no more than two hours and citrate-dextrose (ACD) . and separated into two samples, one for counting platelets and the other, obtained after centifugation, consisting of platelet-rich plasma . The assay for platelet MAO activity was then performed as described previously, using C-14-labelled benzylamine as the substrate (8) . MAO activity was expressed in units of nmoles per lOß platelets per hour . The non-marijuana smokers' bloods had been drawn previously from male volunteers and hospital personnel at two hospitals in the Washington, D .C . Area (NIH Clinical Center and Saint Elizabeths Hospital) . Results : First, the marijuana-user group had There were two significant findings : lower MAO activity than the non-user group . Second, within the marijuana-user group, MAO activity was inversely proportional to current use of marijuana . The group of 26 marijuana users (ages 25-35) had a mean MAO activity of 8 .56± .58 (S .E .M .) compared with a value for 348 normal males (ages 18-60) of 11 .041 .29 (8) . This difference is significant at p< .O1 (two-tailed t-test) . {Although the normal group included a wider range of subject ages, no relation was found in either group between age and MAO activity .) No statistically significant differences were found between the heavier marijuana users and the light marijuana users in their total use of other drugs during the past year (median split) . The amount of current marijuana use over the past two months reported by marijuana smokers was inversely correlated with platelet MAO activity (Pearson r=- .48) . Mean frequency of use was 4 .3 times weekly with a S .D . of 2 .8 times weekly . Total duration of marijuana use was uncorrelated with platelet MAO activity, r=- .14, NS ; however, the mean duration of use was fairly uniform (mean 5 .3 years with a S .D . of only .81 years) .
Vol . 23, No .
B.
15, 1978
Platelet MAO Activity in Marijuana Smokers
1579
Acute effects of THC on MAO activity Procedure :
Three marijuana-smoking subjects were selected randomly from the previously described group of users . Their current frequencies of use were once a day, twice a week, and a eight times a week, respectively . The subjects, tested individually on separate days, were given marijuana cigarettes to smoke which had been previously cut to a length to contain 15 mg of THC . They smoked over an 8-minute period, adjustin their smoking speed to consume an eighth of the cigarette each minute (13,14 . All three reported being quite "high" at the end of this period ; one said he would not want any more . Samples were collected prior to smoking, 20 minutes after beginning smoking, one hour after smoking, and four hours after smoking . Samples for THC analysis were drawn into citrated tubes which were spun at 50,000 RPM and the plasma decanted by pi petting . The plasma samples were analyzed for delta-9-THC by Dr . Roger Foltz at Batelle Columbus Laboratories in Ohio, using gas-chromatograph/mass spectroscopy (GS/MS) methods . Another portion of each sample was prepared and analyzed for MAO activity as described above . To evaluate the effect of THC on platelet MAO activity in vitro, aliquots of platelet sonicates from normal subjects which contained approx~mâtely 0 .3 mg protein were preincubated for 30 minutes at 25°C with 10 -9 to 10-3M THC prepared in 1% ethanol . The 14C-benzylamine substrate was then added and the entire assay procedure completed as described (8) . Controls preincubated similarly with 1% ethanol exhibited no MAO inhibition . Results : MAO activity in vivo over a four-hour period was unaffected by the inhalation of 15 mg THC in a marijuana cigarette, despite the appearance of significant concentrations of THC in the subjects' blood, the characteristic THC-in duced increase in heart rate and the marked subjective "high" reported by these experienced users (Figure 1) (13,14) . In vitro , THC at low concentrations had no inhibitory effect on platelet MAO activity (Figure 2) . At high concentrations (10-5 to 10-3M, approximately 100-10,000 fold higher than peak plasma THC levels measured in man after smoking a standard marijuana cigarette containing 15 mg THC), 20-35% inhibition of MAO activity was observed . DISCUSSION These findings suggest that marijuana users have lower MAO activity than non-users, with the heaviest users exhibiting the lowest enzyme activity of all . The negative intra-group correlation between current frequency of use and MAO activity might suggest that marijuana use directly affects this enzyme system . If this were so, it would seem related to chronic use, since in acute in vivo and in vitro studies, no significant inhibition of MAO by clinicallyatt~ble côncentrations of THC was observed . It is possible, though less likely, that the reduction of MAO activity might have resulted from either some component of marijuana other than THC, or from the past use of illicit drugs other than marijuana ; current users of other drugs have been screened from the study .
of An alternate explanation for the pattern of MAO activity in this sample marijuana users is that individuals with low MAO activity choose to use mari-
1580
Platelet MAO Activity in Marijuana Smokers
0 mln
20 n~ln
I h
2 Ir
Vol . 23, No .
15,
1978
3 4 6 hr Ir k
FIG . 1 Administration of 15 mg THC by inhalation (shaded bar) to three subjects . Ordinates are (1) change in heart rate (beats er minute over baseline), (2) change in high (subjective ratings 0-10), (3~ plasma concentration of THC (ng/ ml), and (4) platelet MAO activity (nmoles benzylamine/108 platelets/hour) .
50
0 m z
25
z
0
10_e
10~
10~`
THC (M)
FIG . 2 Percent inhibition of platelet MAO by THC . (p
