the hypothalamus is thought to play an important role in migraine pathogenesis [ 14 ] .
it is possible that periodic central disturbance of hypothalamic activity or labile threshold could account for the periodicity of the migraine attack .
it also provides a mechanism by which emotional disturbances could be mediated by pathways from the limbic system to the hypothalamus .
almost 60% of patients with migraine report symptoms of elation , irritability , depression , hunger , thirst , or drowsiness during the 24 h preceding headache , which also suggests a hypothalamic site of origin .
it has been suggested that if neuronal hyperexcitability and cortical spreading depression during migraine attack are part of the pathophysiology of the disorder , then aura is not the initiating event but rather is initiated by subcortical changes .
the suprachiasmatic nucleus ( scn ) is a possible subcortical origin of this changes , suggesting that a migraine attack might be an attempt at a drastic resetting of the biological clock in response to its previous decompensation .
compared with the environmental cycle of 24 h , the human biological pacemaker has an intrinsic periodicity of 25.7 hours . the conversion between the intrinsic rhythm and the environmental rhythm is known as 
the environmental agents that are able to synchronize the endogenous circadian clock are known as 
zeitgebers  , among them the most influential is the change in luminosity between day and night .
photic entrainment is mainly mediated by a direct retinal - hypothalamic pathway that conveys to the scn information about the level of ambient light .
photic information relayed from the scn to the pineal gland is closely reflected there in the secretion of melatonin , which is probably the strongest biological marker of the circadian rhythm in humans .
melatonin is absent during the day in human beings   and its nocturnal secretion is the main biological event signaling to the organism when it is night . once melatonin is synthesized in the pineal gland , it is quickly released , generating a blood melatonin rhythm reminiscent of that seen in the gland .
being an amphiphilic molecule , melatonin is capable of entering every cell in the organism ; additionally , it readily crosses all morphophysiological barriers , including the blood  brain barrier and the placenta .
melatonin is enzymatically - degraded in the liver to 6-hydroxymelatonin and finally excreted in the urine as 6-sulphatoxymelatonin ( amt6s ) .
urine analysis is widely used as a measure of melatonin secretion , since it is correlated with the nocturnal profile of plasma melatonin secretion .
the advantages of urinary metabolite measurement are substantial , particularly the non - invasive nature of fluid collection , the ability to perform very long - term studies and the feasibility of studies in circumstances where blood sampling is difficult .
substantial evidence points to melatonin as playing a role in the regulation of circadian rhythms , sleep , and headache .
an interictal decrease in the levels of nocturnal plasma melatonin and an alteration of the urinary amt6s excretion throughout the ovarian cycle have also been described in migraine patients [ 14 , 15 ] .
furthermore , seasonal variations in daily melatonin secretion have been reported , patients with migraine being more likely to have headache during the bright arctic summer season [ 16 , 17 ]
. these data prompted us to test the hypothesis of decreased levels of melatonin during migraine attacks , assessing amt6s levels in a large consecutive series of patients with migraine , comparing with controls .
the study was performed from february to december 2006 , at albert einstein hospital , so paulo , brazil .
the inclusion criteria were : patients with migraine and controls subjects of both sex , aged 1865 years , able to understand the consent form and explanations given by the research team , and with satisfactory diuresis .
migraine was defined according to international classification of headache disorders diagnostic criteria , second edition .
the exclusion criteria were : chronic diseases , instable medical condition , secondary headache , continuous usage of any kind of medication , drug addiction or alcohol abuse .
the patients who remained aroused with light exposure during the night of urine collection were also excluded .
all subjects provided written consent for the experimental procedure approved by the local ethics committee ( federal university of so paulo and albert einstein hospital , so paulo , brazil ) .
subjects were asked to collect urine into a plastic container from 8:00 p.m. to 8:00 a.m. of the next day ( 12-h period ) .
female patients were advised to collect the samples at least 5 days away from the beginning or the end of menses .
the patients were asked to provide information regarding the presentation or not of a migraine headache on the day of urine collection .
sample s volumes were recorded and 5-ml aliquots were stored frozen at 20c until analysis .
amt6s has been found to be extremely stable without preservative for at least 5 days at room temperature and for at least 2 years at 20c and no special precautions were necessary during sample collection .
amt6s was measured by quantitative elisa ( bhlmann laboratories , allschwil , switzerland ) . at a dilution of 1:200 ,
50 l of urine sample was assayed directly ; where necessary , samples were re - assayed at dilution of 1:400 . the chi - square test (  ) ( without yates correction ) was used for categorical data comparisons .
mean differences of continuous measurements were tested by the student s t test ( t ) followed by the mann 
whitney test , which without exception did not show any discrepant results ( only the parametric tests results will be reported ) .
the pearson s product - moment correlation coefficient ( r ) was used to assess the relationship between two continuous variables .
a p value of less than 0.05 was considered to indicate statistical significance ; all tests were two - tailed .
ninety - five percent confidence intervals ( ci ) were calculated for the difference between means .
all statistical analyses were performed on a personal computer with the statistical package spss 11.5.1 for windows .
the chi - square test (  ) ( without yates correction ) was used for categorical data comparisons .
mean differences of continuous measurements were tested by the student s t test ( t ) followed by the mann 
whitney test , which without exception did not show any discrepant results ( only the parametric tests results will be reported ) .
the pearson s product - moment correlation coefficient ( r ) was used to assess the relationship between two continuous variables .
a p value of less than 0.05 was considered to indicate statistical significance ; all tests were two - tailed .
ninety - five percent confidence intervals ( ci ) were calculated for the difference between means .
all statistical analyses were performed on a personal computer with the statistical package spss 11.5.1 for windows .
48 were excluded : 12 patients did not correctly provide their urine samples , 3 refused to take part in the study , 19 did not give reliable headache information and 17 had at least one exclusion criteria .
the remaining 220 subjects were in accordance with inclusion criteria . among them , 146 had migraine and 74 were control subjects .
there were no statistically significant differences between controls and patients with migraine regarding the mean age [ 38.4    9.0 vs. 38.5    11.2 , t(218 )  =  0.7 , 95% ci  =  3.0 to 2.8 , p  =  0.946 ] and sex [ 78 vs. 81% of women , (1 )  =  0.18 ; p  =  0.669 ] . among patients with migraine ,
their urinary amt6s concentration was significantly lower than in the urine of patients without pain [ 14.0  7.3 vs. 49.4  19.0 ; t(143 ) = 15.1 ; 95% ci  =  40.0 to 30.8 ; p < 0.001 ] .
however , there was no significant difference in the amt6s concentration of patients with migraine without pain on the day of their urine samples collection and controls [ 49.4    19.0 vs. 42.5    27.9 ; t(140 )  =  1.7 ; 95% ci  =  1.2 to 14.8 ; p  =  0.094 ] ( fig . 
1mean amt6 s concentration according to the diagnosis mean amt6 s concentration according to the diagnosis migraine with aura was observed in 24% of the patients .
there was no significant difference in the amt6s concentration of patients with migraine with and without aura [ 32.3    21.5 vs. 30.5    22.9 ; t(144 )  =  0.52 ; 95% ci  =  10.9 to 6.4 ; p  =  0.604 ] .
claustrat et al .   were the first to demonstrate lower plasma melatonin levels in samples from migraine patients compared with controls .
migraine patients without depression had lower levels than controls , but migraineurs with superimposed depression exhibited the greatest melatonin deficiency .
murialdo et al .   also found nocturnal urinary melatonin to be significantly decreased throughout the ovarian cycle of migraine patients without aura compared with controls . during the luteal phase ,
when melatonin levels should normally increase , migraine patients showed a less pronounced change when compared with controls .
brun et al .   studied urinary melatonin in women with migraine without aura attacks associated with menses and controls .
melatonin levels throughout the cycle were significantly lower in the migraine patients than in controls .
in the control group , melatonin excretion increased significantly from the follicular to the luteal phase , whereas no difference was observed in the migraine group .
lowered melatonin levels in patients with insomnia were observed compared with those without insomnia , and a phase delay in the melatonin peak in patients versus controls . to our knowledge , our study is the first to demonstrate reduction in melatonin levels during migraine attacks , not depending on the migraine type .
however , patients with migraine that did not present headache in the day of the urine sample collection did not have lower amt6s levels when compared with controls . in our opinion , two possible explanations could be made to this fact : first , the hypothalamic disturbance may occur only during attacks , mainly in the prodromal phase , so patients in the interictal period will not have reduction of melatonin levels .
the other possible explanation is that all patients with migraine could have lower levels of amt6s , but patients in the interictal phase could have a compensatory increase in melatonin levels .
 substantial evidence points to melatonin as playing a role in the regulation of circadian rhythms , sleep , and headache disorders . 
 the objective of the study was to assess 6-sulphatoxymelatonin ( amt6s ) levels in a large consecutive series of patients with migraine , comparing with controls . 
 a total of 220 subjects were evaluated146 had migraine and 74 were control subjects . 
 urinary samples were collected into the same plastic container since 8:00 p.m. to 8:00 a.m. of the next day ( 12-h period ) and amt6s was measured with quantitative elisa technique . among patients with migraine , 
 53% presented pain on the day of the urine samples collection . 
 their urinary amt6s concentration was significantly lower than in the urine of patients without pain [ 14.0    7.3 vs. 49.4    19.0 ; t(143 )  =  15.1 ; 95% ci  =  40.0 to 30.8 ; p  <  0.001 ] . 
 there was no significant difference in the amt6s concentration of patients with migraine without pain on the day of their urine samples collection and controls [ 49.4    19.0 vs. 42.5    27.9 ; t(140 )  =  1.7 ; 95% ci  =  1.2 to 14.8 ; p  =  0.094 ] . to our knowledge 
 , this is the first study to demonstrate reduction in melatonin levels during attacks in episodic and chronic migraine . 
Introduction
Materials and methods
Data analysis
Results
Discussion