we have demonstrated that , melatonin is a bioprecursor of hypnotic acetyl metabolites produced by enzymatic acetylation of melatonin and 2-oxomelatonin under the control of acetyltransferases , most probably the nat enzymes . in 1994 , in our laboratory , we developed a specific and highly sensitive gas chromatography - mass spectrometry ( gc - ms ) method to assay , simultaneously and distinct ! ) , plasma concentrations of endogenous melatonin ( d0melatonin ) and exogenous melatonin ( d7-melatonin ) , in which 7 atoms of h have been substituted by 7 atoms of deuterium . using the same human volunteers ( 12 young subjects in june 1994 and 12 elderly subjects in october 1994 ) , we determined the pharmacokinetics of exogenous d7-melatonin , when given orally and intravenously , and the kinetics of the pineal secretion of endogenous d0-melatonin . ,   the results shown in figure 2 led to the following conclusions : secretion of melatonin by the pineal gland occurs only during the night . pharmacokinetic analysis shows that the rate of melatonin secretion by the pineal gland is constant throughout the whole nocturnal pineal melatonin production , for the same subject . the beginning and end of melatonin secretion from the pineal gland are the same for each subject , whatever the season and night , length . duration of melatonin pineal secretion is between 7.5 and 8 h. therefore , melatonin secretion and sleep are contemporaneous . there is a large interindividual variability in the amount , of melatonin released in plasma by the pineal gland during the night in young and old subjects alike . results of previous related studies show that melatonin secretion , and therefore the presence of melatonin in the central nervous system ( cns ) , is necessary for the induction and maintenance of nocturnal sleep . however , the presence of melatonin in the cns is insufficient for the induction and maintenance of sleep . indeed , figure 3 and table i show results of observations in chicks in an alternate light ( l)-dark ( d ) program ( l / d , 12 h:12 h ) , in which the light phase lasted from 8.00 am until 8.00 pm . when melatonin was administered intramuscularly ( pectoralis major muscle ) during the light phase from 2.00 pm to 8.00 pm , the chicks did not . the absence of a hypnotic effect during the light phase correlated with the very low level of nat activity in the pineal glands of chicks measured at the same times . in contrast , when chicks were observed in a 7-day permanent light , program ( l / l , 12 h:12 h ) , during which nat activity level was constantly higher , the administration of melatonin induced a significant , hypnotic effect . the duration of sleep ( between 4 and 5 h ) was much greater than that , observed with diazepam ( between 1 and 2 h ) when it . was administered intramuscularly at the same dose ( 1 m per 100 g body weight , at 2.00 pm ) . these results lead to the following conclusions : the simultaneous presence of melatonin and nat in the cns ( pineal gland ) is a necessary and sufficient condition for the induction and maintenance of sleep . in contrast to the classic so - called  hypnotic drugs  ( eg , benzodiazepines , barbiturates , zopiclone , and zolpidem ) , melatonin does not have direct , hypnotic properties related to its chemical structure . during the development of the gc - ms method for the assay of melatonin in plasma , our attention was focussed on the chemical reactivity of melatonin at position 3 , which allows cyciization of the side chain after acylation . this proceeds by nucleophilic attack and leads to a fluoroacyl--carboline ( figure 4 ) . considering our previous observations , we assumed that melatonin undergoes enzymatic acetylation during the night , under the control of nat , and that this leads to an n - acetyl--carboline , which we call carbo2 . we conclude that melatonin is a bioprecursor of hypnotic acetyl metabolites , such as carbo2 . chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase , they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min . figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) . gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) . we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low . the results are presented in table iii , together with some reference compounds . the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect . in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced   sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep . chick pineal glands were observed during an alternate light - dark program at 37c for 7 days . in the middle of dark phase , they were treated with phjacetyl coenzyme a and melatonin ( or 2-oxomelatonin ) for 30 min . figure 5 and figure 6 show that melatonin ( or 2-oxomelatonin ) undergoes an aeetylation that is significantly higher ( p<0.002 , in the middle of dark phase ; p<0.0005 , 1 h before end of dark phase [ or p<0.00005 for 2oxomelatonin over the whole dark phase ] ) than that observed in controls ( nonsignificant when melatonin was replaced by phosphate buffer ) . gc - ms indicated the biosynthesis of [ h]carbo2 for five chick pineal glands collected in the middle of dark phase ( table ii ) . we have synthesized several acetyl derivatives , such as carbo2 ( figure 7 ) , which is an n-acetyl-p-carboline.we have found 20 to 40 pg carbo2 per gram of lamb pineal gland collected on the middle of the dark phase of an alternate light - dark program . the hypnotic activity of carbo2 has been observed and measured in chicks and beagles : in chicks , the tests were performed at 2.00 pm , in the middle of light phase , a time at which nat activity in the pineal gland is very low . the essential role of acetyl group is demonstrated by the fact that 10-mcthoxyharmalan ( as well as harmaline ) , which is the product of jv - deacetylation of compound carbo2 , does not exhibit any hypnotic effect . in contrast , it induces excitatory effects in chicks by increasing locomotor activity in beagles , polysomnographic studies showed that when carbo2 was administered intravenously , it induced   sleep of longer duration and shorter time latencies than the sleep induced by zolpidem and diazepam ( table iv ) . the most interesting feature , which provides more support for our assumption , is the eeg architecture of the sleep produced , which is similar to that of physiological sleep ( see results with placebo in table iv ) , characterized by the significant proportion of slow - wave deep sleep and rapid eye movement ( rem ) sleep , in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds , such as zolpidem or diazepam , which induce mainly drowsiness ( light sleep ) and little rem sleep . we have evidenced the role played by melatonin in both inducing and maintaining nocturnal sleep . melatonin is the bioprecursor of hypnotic acetyl metabolites , such as carbo2 , which result from the enzymatic acetylation of melatonin ( and 2-oxomelatonin ) by nat . since insomnia and sleep disorders may be due to a lack of nat enzymes in the pineal gland , a therapeutic approach to sleep disorders could be suggested . patients with insomnia may be treated by administering hypnotic acetyl metabolites of melatonin or their synthetic analogs .
pharmacokinetic studies of melatonin in young and elderly human volunteers , and the measurement of hypnotic effects in chicks under alternate light - dark or permanent light conditions , show that melatonin is a bioprecursor of hypnotic acetyl metabolites produced by the enzymatic acetylation of both melatonin and 2-oxomelatonin under the control of serotonin n - acetyltransferases ( nats ) , which are present in the pineal gland . 
 the acetyl metabolite of melatonin , which we call carbo2 , is an n - acetyl--carboline . 
 the electroencephalographs ( eeg ) architecture of the sleep produced by this compound is similar to thai of physiological sleep , and is characterized by the significant proportion of slow - wave deep sleep and rapid eye movement sleep . 
 this is in sharp contrast to the eeg sleep architecture observed with gabaergic ( gaba , -aminobutyric acid ) compounds . 
 since insomnia and sleep disorders are believed to be due to a lack of nat enzymes in the pineal gland , a new therapeutic approach of sleep disorders by administration of such hypnotic acetyl metabolites of melatonin , or synthetic analogs thereof , can be en visaged .