Psychoneuroendocrinology (2012) 37, 1990—1997

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Day and nighttime excretion of 6-

sulphatoxymelatonin in adolescents and young
adults with autistic disorder
Sylvie Tordjman a,b,*, George M. Anderson c, Eric Bellissant d,e, Michel
Botbol b, Henriette Charbuy f, Franc¸oise Camus f, Rozenn Graignic a, Solenn
Kermarrec a, Claire Fougerou d,e, David Cohen g, Yvan Touitou f

a
Hospital-University Department of Child and Adolescent Psychiatry, Guillaume Re´gnier Hospital, Rennes 1 University, Rennes,
France
b
Laboratory of Psychology of Perception, CNRS UMR 8158, Paris Descartes University, Paris, France
c
Child Study Center and Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA
d
Department of Clinical Pharmacology, University Hospital, Rennes 1 University, Rennes, France
e
Inserm CIC 0203 Clinical Investigation Centre, University Hospital, Rennes 1 University, Rennes, France
f
Medical Biochemistry and Molecular Biology, Pitie´-Salpe´trie`re School of Medicine, Paris and Chronobiology Unit,
Rothschild Foundation, Paris, France
g
Hospital-University Department of Child and Adolescent Psychiatry, Pitie´-Salpe´trie`reHospital, Paris 6 University, Paris, France

Received 27 December 2011; received in revised form 25 April 2012; accepted 26 April 2012

KEYWORDS
Melatonin;
6-Sulphatoxymelatonin;
Pineal;
Autistic disorder;
Autism severity;
Circadian rhythm;
Biological clocks
Summary
Background: Several reports indicate that nocturnal production of melatonin is reduced in
autism. Our objective was to examine whether melatonin production is decreased during the
whole 24-h cycle, whether the melatonin circadian rhythm is inverted, and whether the
reduction in melatonin production is related to the severity of autistic behavioral
impairments.
Latar belakang: Beberapa laporan menunjukkan bahwa produksi melatonin di malam hari
berkurang autis. Tujuan kami adalah untuk menguji apakah produksi melatonin menurun
selama seluruh siklus 24-jam, apakah ritme sirkadian melatonin terbalik, dan apakah
pengurangan produksi melatonin terkait dengan keparahan gangguan perilaku autistik

Method: Day and nighttime urinary excretion of 6-sulphatoxymelatonin (6-SM) was examined
during a 24-h period in post-pubertal individuals with autism (N = 43) and typically developing
controls (N = 26) matched for age, sex and pubertal stage.

Metode: Ekskresi urin siang dan malam hari 6-sulphatoxymelatonin (6-SM) diperiksa selama 24
jam pada individu pasca pubertas dengan autisme (N = 43) dan biasanya mengembangkan
kontrol (N = 26) yang disesuaikan dengan usia, jenis kelamin. dan tahap pubertas.

Results: Low 6-SM excretion (mean T SEM) was observed in autism, both at daytime (0.16 T
0.03 vs. 0.36 T 0.05 mg/h, p < 0.01), nighttime (0.52 T 0.07 vs. 1.14 T 0.23 mg/h, p <
0.05), and
during 24 h (8.26 T 1.27 vs. 18.00 T 3.43 mg/24-h collection, p < 0.001). Intra-individual
night-
time—daytime differences (delta values) in 6-SM excretion were smaller in individuals with
autism than in controls (0.36 T 0.07 vs. 0.79 T 0.23 mg/h, p < 0.05). Nocturnal excretion of
 6-SM was negatively correlated with autism severity in the overall level of verbal language
(Spearman r = —0.30, p < 0.05), imitative social play (Spearman r = —0.42, p < 0.05), and
repetitive use of objects (Spearman r = —0.36, p < 0.05).

Hasil: Ekskresi 6-SM rendah (rata-rata T SEM) diamati pada autisme, baik pada siang hari (0,16
T 0,03 vs 0,36 T 0,05 mg / jam, p <0,01), malam hari (0,52 T 0,07 vs 1,14 T 0,23 mg / h, p
<0,05), dan
selama 24 jam (8,26 T 1,27 vs 18,00 T 3,43 mg / 24 jam pengumpulan, p <0,001). Malam
intra-individu-
perbedaan waktu-siang hari (nilai delta) dalam ekskresi 6-SM lebih kecil pada individu dengan
autisme daripada pada kontrol (0,36 T 0,07 vs 0,79 T 0,23 mg / jam, p <0,05). Ekskresi
nokturnal 6-SM berkorelasi negatif dengan tingkat keparahan autisme pada keseluruhan
tingkat bahasa verbal (Spearman r = -0,30, p <0,05), permainan sosial imitatif (Spearman r =
-0,42, p <0,05), dan penggunaan berulang dari objek (Spearman r = —0,36, p <0,05).

Conclusion: A deficit in melatonin production is present both at daytime and at nighttime in

individuals with autism, particularly in the most severely affected individuals. These results
highlight interest in potential therapeutic uses of melatonin in autistic disorder, especially in
individuals with severe autistic impairment and/or low urinary 6-SM excretion.
Kesimpulan: Defisit dalam produksi melatonin hadir baik pada siang hari dan pada malam hari
pada individu dengan autisme, terutama pada individu yang paling parah terkena dampaknya.
Hasil ini menyoroti minat penggunaan melatonin dalam terapi potensial pada gangguan
autistik, terutama pada individu dengan gangguan autistik berat dan / atau ekskresi 6-SM
kemih yang rendah.

# 2012 Elsevier Ltd. All rights reserved.

1. Introduction nighttime values usually at least three times greater than

Melatonin is a neurohormone produced mainly by the
pineal gland and during the night. Pineal melatonin is
important for the regulation of human circadian rhythms
including the sleep—wake, neuroendocrine and body
temperature cycles (Axelrod, 1974; Zhdanova et al.,
1997). The measures of melatonin concentration in plasma
and saliva, or of the urinary excretion of its predominant
metabolite, 6-sulpha- toxymelatonin (6-SM), are
considered the best peripheral indices of human circadian
timing (Arendt, 2006). There is also increasing evidence
that melatonin is critically involved in early development
through its direct effects on placenta, developing neurons
and glia, and its role in the ontogenetic establishment of
diurnal rhythms (Niles et al., 2004; Iwasaki et al., 2005).
1. Perkenalan
Melatonin adalah neurohormon yang diproduksi terutama
oleh kelenjar pineal dan pada malam hari. Melatonin
pineal penting untuk pengaturan ritme sirkadian manusia
termasuk siklus tidur-bangun, neuroendokrin dan suhu
tubuh (Axelrod, 1974; Zhdanova et al., 1997). Ukuran
konsentrasi melatonin dalam plasma dan saliva, atau
ekskresi urin dari metabolit dominannya, 6-sulpha-
toxymelatonin (6-SM), dianggap sebagai indeks perifer
terbaik dari pengaturan waktu sirkadian manusia (Arendt,
2006). Ada juga semakin banyak bukti bahwa melatonin
secara kritis terlibat dalam perkembangan awal melalui
efek langsungnya pada plasenta, mengembangkan neuron
dan glia, dan perannya dalam pembentukan ontogenetik
dari ritme diurnal (Niles et al., 2004; Iwasaki et al.,
2005) .
The physiological increase in melatonin secretion during
the night is well established with a peak around 2 AM and
daytime values (Arendt, 1988). Pineal melatonin production
is powerfully suppressed by light acting through the retino-
hypothalamic tract (Revell and Skene, 2007). In addition to
light and consequently seasons (Lindblom et al., 2002), pineal
melatonin secretion can also be influenced by endo- genous
factors including sex, age and pubertal stage (Cavallo and
Ritschel, 1996; Touitou, 2001). At daytime, it has been
suggested that most of the melatonin production occurs
outside the pineal gland, in the wall of the gut (Bubenik,
2002).
Peningkatan fisiologis sekresi melatonin pada malam hari
mapan dengan puncak sekitar 2 pagi dan nilai-nilai malam
hari biasanya setidaknya tiga kali lebih besar dari nilai-nilai
siang hari (Arendt, 1988). Produksi melatonin pineal sangat
ditekan oleh cahaya yang bekerja melalui saluran retino-
hipotalamus (Revell dan Skene, 2007). Selain musim cahaya
dan konsekuensinya (Lindblom et al., 2002), sekresi
melatonin pineal juga dapat dipengaruhi oleh faktor endogen
termasuk jenis kelamin, usia dan tahap pubertas (Cavallo dan
Ritschel, 1996; Touitou, 2001). Pada siang hari, telah
disarankan bahwa sebagian besar produksi melatonin terjadi
di luar kelenjar pineal, di dinding usus (Bubenik, 2002).
Melatonin is of interest in autism due to its apparent role
in neurodevelopment (de Faria Poloni et al., 2011), reports of
sleep—wake rhythm disturbances in individuals with autism
(see Glickman, 2010 and Tordjman et al., 2005 for reviews),
as well as beneficial effects of melatonin when administered
to individuals with autism and sleep problems (Doyen et al.,
2011; Rossignol and Frye, 2011; Gue´nole´ et al., 2011). In
addition, central and peripheral alterations in serotonin in
autism have been widely reported and it is noteworthy that
melatonin is synthesized in only two steps from serotonin in
the pineal gland and the gut. (Richdale, 1999; Anderson,
2002; Nakamura et al., 2010). Prior studies of melatonin
production in autistic disorder were often limited by small
sample sizes and were not entirely consis- tent, but all
reported abnormalities in the melatonin pro- duction
(Ritvo et al., 1993; Nir et al., 1995; Kulman et al., 2000;
Tordjman et al., 2005; Melke et al., 2008; Mulder et al.,
2010). Our results (Tordjman et al., 2005), taken together
with the other studies (except Ritvo et al.’s study, 1993),
indicate that nocturnal secretion of melatonin is
frequently reduced in autism. However, given the limita-
tions of the available data, it has not been possible to
conclude if there is a general decrease in melatonin secre-
tion during the whole 24-h cycle, or if the melatonin circa-
dian rhythm is altered or inverted in autistic disorder. In
order to clarify these issues, we examined simultaneously
the diurnal and nocturnal excretion of urinary 6-SM in a
large, accessible and post-pubertal sample of adolescents
and young adults with autistic disorder, and in a group of
typically developing controls matched on age, sex and
stage of puberty. Post-pubertal participants were
recruited given the reported effect of puberty on
melatonin secretion (Cavallo and Ritschel, 1996; Touitou,
2001). We also exam- ined the relationship between 6-SM
excretion and the sever- ity of behavioral autistic
impairments.
Melatonin tertarik pada autisme karena perannya yang
jelas dalam pengembangan saraf (de Faria Poloni et al.,
2011), laporan gangguan tidur-bangun ritme pada individu
dengan autisme (lihat Glickman, 2010 dan Tordjman et
al., 2005 untuk ulasan) , serta efek menguntungkan dari
melatonin ketika diberikan kepada individu dengan
masalah autisme dan tidur (Doyen et al., 2011; Rossignol
dan Frye, 2011; Gue 'nole' et al., 2011). Selain itu,
perubahan sentral dan perifer dalam serotonin pada
autisme telah banyak dilaporkan dan perlu dicatat bahwa
melatonin disintesis hanya dalam dua langkah dari
serotonin di kelenjar pineal dan usus. (Richdale, 1999;
Anderson, 2002; Nakamura et al., 2010). Studi sebelumnya
tentang produksi melatonin pada gangguan autis sering
dibatasi oleh ukuran sampel kecil dan tidak sepenuhnya
konsisten, tetapi semua melaporkan kelainan pada
produksi melatonin (Ritvo et al., 1993; Nir et al., 1995;
Kulman et al., 2000; Tordjman et al., 2005; Melke et al.,
2008; Mulder et al., 2010). Hasil kami (Tordjman et al.,
2005), diambil bersama dengan penelitian lain (kecuali
studi Ritvo et al., 1993), menunjukkan bahwa sekresi
melatonin pada malam hari sering berkurang pada
autisme. Namun, mengingat keterbatasan data yang
tersedia, itu tidak mungkin untuk menyimpulkan apakah
ada penurunan umum dalam sekresi melatonin selama
seluruh siklus 24 jam, atau jika ritme sirkuler melatonin
diubah atau dibalikkan. pada gangguan autistik. Untuk
mengklarifikasi masalah ini, kami memeriksa secara
simultan ekskresi diurnal dan nokturnal urin 6-SM dalam
sampel remaja dan dewasa pubertas yang besar, dapat
diakses, dan dewasa muda dengan gangguan autistik, dan
dalam kelompok kontrol yang biasanya berkembang sesuai
usia. , jenis kelamin dan tahap pubertas. Partisipan paska
pubertas direkrut mengingat efek pubertas yang
dilaporkan pada sekresi melatonin (Cavallo dan Ritschel,
1996; Touitou, 2001). Kami juga meneliti hubungan antara
6-SM ekskresi dan parahnya gangguan autistik perilaku.
2. Methods
2.1. Participants
Adolescents and young adults with autistic disorder (N =
43), all post-pubertal, were matched with typically
developing controls (N = 26) on age, sex and Tanner stage
of puberty assessed by a pediatrician. Outpatients with
autistic disor- der were recruited from French day-care
facilities and included 31 males and 12 females [mean age
= 18.6 years, SEM (standard error of the mean) = 0.5].
Controls were recruited over a one-month period from a
preventive med- ical center where they went for a regular
check-up, and included 19 males and 7 females (mean age
= 19.8 years, SEM = 0.8). In addition, controls were
interviewed by a psychiatrist and had no sleep problems
and no psychopathol- ogy based on the Mini International
Neuropsychiatric Inter- view (MINI; Sheehan et al., 1998)
and determined to be free of any. There was no family
history of autistic disorder in the control group. All
patients and controls were Caucasian, non-obese, had no
history of encephalopathy or neuroendo- crinological
disease and were determined to be physically healthy
based on a pediatrician’s examination. All patients and
controls were unmedicated for at least one month before
urine collection. All subjects were sleeping in their
parents’ house and were attending high school or college
(controls) or day-care facilities (individuals with autism)
on a daily basis from 0900 h to 1600 h. The protocol was
approved by the ethics committee of Biceˆtre Hospital
(Krem- lin Biceˆtre, France) and written informed
consent was obtained from parents.
2. Metode
2.1. Peserta
Remaja dan dewasa muda dengan kelainan autistik (N = 43),
semuanya post-pubertas, dicocokkan dengan kontrol yang
berkembang secara khas (N = 26) pada usia, jenis kelamin dan
tahap pubertas Tanner yang dinilai oleh dokter anak. Pasien
rawat jalan dengan gangguan autistik direkrut dari fasilitas
penitipan siang hari Perancis dan termasuk 31 laki-laki dan 12
perempuan [usia rata-rata = 18,6 tahun, SEM (kesalahan
standar rata-rata) = 0,5]. Kontrol direkrut selama periode
satu bulan dari pusat medis preventif di mana mereka pergi
untuk pemeriksaan rutin, dan termasuk 19 laki-laki dan 7
perempuan (usia rata-rata = 19,8 tahun, SEM = 0,8). Selain
itu, kontrol diwawancarai oleh seorang psikiater dan tidak
memiliki masalah tidur dan tidak ada psikopatologi
berdasarkan Mini International Neuropsychiatric Wawancara
(MINI; Sheehan et al., 1998) dan bertekad untuk bebas dari
apa pun. Tidak ada riwayat keluarga dengan gangguan
autistik pada kelompok kontrol. Semua pasien dan kontrol
adalah Kaukasia, non-obesitas, tidak memiliki riwayat
ensefalopati atau penyakit neuroendrokrinologis dan bertekad
sehat secara fisik berdasarkan pemeriksaan dokter anak.
Semua pasien dan kontrol tidak diberi obat setidaknya selama
satu bulan sebelum pengumpulan urin. Semua subjek tidur di
rumah orang tua mereka dan menghadiri sekolah menengah
atau perguruan tinggi (kontrol) atau fasilitas penitipan anak
(individu dengan autisme) setiap hari dari pukul 09.00 hingga
1.600. Protokol telah disetujui oleh komite etika Rumah Sakit
Biceˆtre (Kremlin Biceˆtre, Prancis) dan persetujuan tertulis
diperoleh dari orang tua.
2.2. Cognitive and behavioral assessments
 Cognitive functioning of patients with autistic disorder
was assessed by two psychologists using French versions
of the WAIS-III (the age-appropriate Wechsler
intelligence scale; Wechsler, 2000) and the Kaufman K-
ABC (Kaufman and Kauf- man, 1993). All patients with
autistic disorder were cogni- tively impaired (mean full
scale IQ SEM: 42.1 0.5, with a range of 40—58; mean
verbal IQ SEM: 45.2 0.3, with a range of 45—57; mean
performance IQ SEM: 45.2 0.7, with a range of 45—
80).

2.2. Penilaian kognitif dan perilaku

Fungsi kognitif pasien dengan gangguan autistik

dinilai oleh dua psikolog menggunakan WAIS-III
versi Perancis (skala kecerdasan Wechsler yang
sesuai usia; Wechsler, 2000) dan Kaufman K-ABC
(Kaufman dan Kaufman, 1993). Semua pasien
dengan gangguan autistik secara kognitif
terganggu (rerata IQ skala penuh SEM: 42,1 0,5,
dengan kisaran 40-58; rerata IQ SEM: 45,2 0,3,
dengan kisaran 45-57; rerata kinerja IQ SEM: 45,2
0,7, dengan kisaran 45-80).
 Behavioral assessments were performed using the
French INSERM (Institut National de la Sante´ et de la
Recherche Me´dicale, Third edition, 1993) version of the
Autism Diag- nostic Interview-Revised (ADI-R; see Lord,
1997). The ADI-R, an extensive semi-structured parental
interview, was admi- nistered by two trained psychiatrists.
The ADI-R scale assessed the three major domains of
autistic impairments: reciprocal social interactions,
verbal/non-verbal communi- cation, and repetitive
behaviors/restricted interests. Based on the direct clinical
observation of the patient by two independent child
psychiatrists, the diagnosis of autistic disorder was made
according to the criteria of DSM-IV-TR, ICD-10 and CFTMEA
and was confirmed by the ADI-R ratings (Mise`s and
Quemada, 1993; American Psychiatric Associa- tion, 2000;
Tordjman et al., 2001).
Penilaian perilaku dilakukan dengan menggunakan
INSERM Prancis (Institut Nasional de la Sante et de
la Recherche Me´dicale, edisi Ketiga, 1993) versi
Autism Diagnostic Interview-Revisi (ADI-R; lihat
Lord, 1997). ADI-R, sebuah wawancara orang tua
semi-terstruktur yang luas, didaftarkan oleh dua
psikiater yang terlatih. Skala ADI-R menilai tiga
domain utama dari gangguan autistik: interaksi
sosial timbal balik, komunikasi verbal / non-
verbal, dan perilaku berulang / minat terbatas.
Berdasarkan pengamatan klinis langsung dari
pasien oleh dua psikiater anak independen,
diagnosis gangguan autistik dibuat sesuai dengan
kriteria DSM-IV-TR, ICD-10 dan CFTMEA dan
dikonfirmasi oleh peringkat ADI-R (Mise` s dan
Quemada, 1993; American Psychiatric Associa-
tion, 2000; Tordjman et al., 2001).
2.3. Urine collection
Urine samples were collected at home by parents during a
whole 24-h cycle from 8 PM to 8 AM (nighttime collection)
and from 0800 to 2000 h (daytime collection). Subjects
were instructed to empty their bladder between 1945 and
2000 h before starting urine collection. Because of possible
effects of season on the secretion of melatonin, urine was
collected for all subjects in spring (March—May). Parents
of autistic and control subjects completed a questionnaire
reporting on the following information for the night of
urine collection, and for what was typical over the month
before the collection: time to bed, time to sleep,
nighttime awakenings, if their child slept with the light on
or got up during the night and turned on the light, and
presence of other sleep distur- bances/problems. On the
night of urine collection, all sub- jects included in the
analyses went to bed with lights out between 9 PM and 10
PM, and none was exposed to light until their morning
wake-up between 0700 and 0745 h. Based on the brief
parental sleep questionnaire, none of the study
participants showed any significant sleep disturbance for
the night of the urine collection or for the month before
the collection. In addition, no bedwetting or accidental
loss of urine was reported for any of the subjects.
Collected urines were stored in a refrigerator until
delivered to the laboratory within 24 h of the urine
collection. The volume of the urine collection was measured
and a portion was frozen until analyzed for creatinine and
6-SM. It should be noted that urine was originally collected
from 41 controls; however, urine from 15 controls was
rejected prior to biochemical analyses due to either
parental report of late bedtimes or uncertainty regarding
whether the daytime collection was placed in the nighttime
collection jug (and vice versa).
2.3. Pengumpulan urin
Sampel urin dikumpulkan di rumah oleh orang tua
selama seluruh siklus 24 jam dari jam 8 malam
sampai jam 8 malam (pengumpulan malam hari)
dan dari jam 0800 hingga 2000 jam (pengumpulan
siang hari). Subjek diinstruksikan untuk
mengosongkan kandung kemih mereka antara 1945
dan 2000 jam sebelum memulai pengumpulan urin.
Karena kemungkinan efek musim pada sekresi
melatonin, urin dikumpulkan untuk semua subjek di
musim semi (Maret-Mei). Orang tua dari subjek
autistik dan kontrol menyelesaikan kuesioner yang
melaporkan informasi berikut untuk malam
pengumpulan urin, dan untuk apa yang khas selama
sebulan sebelum pengumpulan: waktu tidur, waktu
tidur, bangun malam, jika anak mereka tidur
dengan lampu menyala atau bangun di malam hari
dan menyalakan lampu, dan adanya gangguan /
masalah tidur lainnya. Pada malam pengumpulan
urin, semua subjek yang dimasukkan dalam analisis
pergi tidur dengan lampu mati antara pukul 21:00
dan 22:00, dan tidak ada yang terpapar cahaya
sampai bangun pagi mereka antara 0700 dan 0745
jam. Berdasarkan kuesioner tidur orang tua
singkat, tidak ada peserta penelitian yang
menunjukkan gangguan tidur yang signifikan untuk
malam pengumpulan urin atau untuk bulan sebelum
pengumpulan. Selain itu, tidak ada ngompol atau
kehilangan urin yang tidak disengaja dilaporkan
untuk semua subjek. Air seni yang terkumpul
disimpan dalam lemari es sampai dikirim ke
laboratorium dalam waktu 24 jam dari
pengumpulan urin. Volume pengumpulan urin
diukur dan sebagian dibekukan sampai dianalisis
untuk kreatinin dan 6-SM. Perlu dicatat bahwa urin
awalnya dikumpulkan dari 41 kontrol; Namun, urin
dari 15 kontrol ditolak sebelum analisis biokimia
karena laporan orang tua tentang waktu tidur yang
terlambat atau ketidakpastian mengenai apakah
pengumpulan siang hari ditempatkan dalam tabung
pengumpulan malam hari (dan sebaliknya).
2.4. Determination of urinary 6-SM
Melatonin production was assessed by measuring the urinary
excretion of 6-SM which has been well established to give
an accurate reflection of pineal melatonin secretion
(Bojkowski et al., 1987). Blinded analysis of urine 6-SM
levels was performed by radioimmunoassay using an assay
kit from Stockgrand Ltd (Guildford, UK). The urine samples
were diluted prior to assay (1/250). The intra-assay hour (mg/h) over the collection period: the urine 6-SM
coefficient of variation was 6% (N = 10) for a 0.030 mg/ml con- centration (mg/ml) was multiplied by the collection
control sample value. Excretion of 6-SM was expressed as volume in ml and divided by the 12 h of collection.
mg excreted per
2.4. Penentuan 6-SM kemih

Produksi melatonin dinilai dengan mengukur ekskresi urin 6-SM

yang telah mapan untuk memberikan refleksi akurat dari
sekresi melatonin pineal (Bojkowski et al., 1987). Analisis buta
kadar urin 6-SM dilakukan oleh radioimmunoassay menggunakan
alat uji dari Stockgrand Ltd (Guildford, Inggris). Sampel urin
diencerkan sebelum pengujian (1/250). Koefisien variasi intra-
assay adalah 6% (N = 10) untuk nilai sampel kontrol 0,030 mg /
ml. Ekskresi 6-SM dinyatakan sebagai mg diekskresikan per

jam (mg / jam) selama periode pengumpulan: konsentrasi urin

6-SM (mg / ml) dikalikan dengan volume pengumpulan dalam
ml dan dibagi dengan 12 jam pengumpulan.

2.5. Statistical analysis

Correlations between melatonin excretion rates and age,

as well as correlations between nighttime and daytime
mela- tonin excretion rates were assessed using Pearson
correlation analyses. Group comparisons of urine
collection volumes and of creatinine urinary excretions
were performed using two- tailed Student’s t-tests. Group
and subgroup comparisons of urine 6-SM levels were
performed using repeated-measures analyses of variance
and two-tailed Student’s t-tests. Rela- tionships between
melatonin excretion and autism severity within the major
behavioral domains of impairment were studied using
Spearman rank-order correlations. In order to balance type
I and type II errors in the statistical analysis of behavioral
domains, a hierarchical strategy was used (Cohen and
Cohen, 1983). First, total behavioral domains and sub-
domains were examined. If there was a significant result
for an overall domain or sub-domain, then a further level
of analysis was performed on the subscores included in the
domain. The severity of impairments in the behavioral
domains of autism were scored following a previously
described procedure that involved determining for each
domain the median value of the subset of ADI-R items
included in the ADI-R algorithm (Tordjman et al., 2001).
Alpha risk was set at 0.05 for all analyses.

2.5. Analisis statistik

Korelasi antara tingkat ekskresi melatonin dan usia, serta

korelasi antara tingkat ekskresi melatonin malam hari dan siang
hari dinilai menggunakan analisis korelasi Pearson.
Perbandingan kelompok volume pengumpulan urin dan ekskresi
urin kreatinin dilakukan dengan menggunakan uji-t Student dua
sisi. Perbandingan kelompok dan subkelompok kadar urin 6-SM
dilakukan dengan menggunakan analisis varians pengukuran
berulang dan uji-t Student dua sisi. Hubungan antara ekskresi
melatonin dan keparahan autisme dalam domain perilaku
utama dari penurunan dipelajari dengan menggunakan korelasi
rank-order Spearman. Untuk menyeimbangkan kesalahan tipe I
dan tipe II dalam analisis statistik domain perilaku, strategi
hierarkis digunakan (Cohen dan Cohen, 1983). Pertama, total
domain perilaku dan sub-domain diperiksa. Jika ada hasil yang
signifikan untuk keseluruhan domain atau sub-domain, maka
tingkat analisis lebih lanjut dilakukan pada subskala yang
termasuk dalam domain. Tingkat keparahan gangguan dalam
domain perilaku autisme dinilai setelah prosedur yang
dijelaskan sebelumnya yang melibatkan menentukan untuk
setiap domain nilai median dari subset item ADI-R yang
termasuk dalam algoritma ADI-R (Tordjman et al., 2001).
Risiko alfa ditetapkan 0,05 untuk semua analisis.

3. Results

3.1. Initial analyses

Mean ( SEM) urine collection volumes for nighttime (2000

— 0800 h) and daytime (0800—2000 h) periods were not
signifi- cantly different in the autism and control groups
(nighttime:
467 48 and 388 47 ml, respectively; daytime: 542 46
and 489 71 ml, respectively). Similarly, creatinine
urinary excretions were not significantly different in
autism and control groups (nighttime: 431 33 and 372 74
mg/collec- tion, respectively; daytime: 461 33 and 606
81 mg/col- lection, respectively). There were no
significant effects of gender or age on the nighttime or
daytime hourly 6-SM excretion rates in either the autism
or control group.

3. Hasil

3.1. Analisis awal

Volume pengumpulan urin rata-rata (SEM) untuk periode

malam hari (2000- 0800 jam) dan siang hari (0800-2000
jam) tidak berbeda secara signifikan dalam kelompok
autisme dan kontrol (malam hari:
467 48 dan 388 47 ml, masing-masing; siang hari: 542 46
dan 489 71 ml, masing-masing). Demikian pula, ekskresi
kreatinin urin tidak berbeda secara signifikan pada
autisme dan kelompok kontrol (malam hari: 431 33 dan
372 74 mg / koleksi, masing-masing; siang hari: 461 33 dan
606 81 mg / koleksi, masing-masing). Tidak ada efek
signifikan dari jenis kelamin atau usia pada tingkat
ekskresi 6-SM malam hari atau siang hari baik dalam
autisme atau kelompok kontrol.

3.2. Relationships between diagnosis and 6-

SM excretion rates

Repeated-measures analysis of variance conducted on

night- time and daytime hourly 6-SM excretions indicated
signifi- cant effects of group (F (1,65) = 12.24, p <
0.001), time (F (1,65) = 35.00, p < 0.0001), and group-
by-time interaction (F (1,65) = 4.88, p < 0.05). Post hoc
t-tests analyses showed significantly lower nighttime 6-
SM excretion rates in indivi- duals with autism compared
to controls (0.52 0.07 vs.
1.14 0.23 mg/h, p = 0.016); daytime 6-SM excretion rates
in individuals with autism were also lower compared to
controls (0.16 0.03 vs. 0.36 0.05 mg/h, p = 0.002) (see
Fig. 1). Furthermore, the total (nighttime + daytime) 6-
SM excretion was significantly lower in individuals with
autism

Figure 1 Daytime and nighttime urinary 6-sulphatoxymelato-
nin (6-SM) excretion rates (mg/h, mean T SEM) in individuals
with autism (N = 43) and typically developing controls (N =
26). Daytime and nighttime 6-SM excretion rates were
significantly lower in individuals with autism compared to
typically develop- ing controls (t = 3.15, df = 67, p = 0.002
and t = 2.53, df = 67,
p = 0.016, respectively). The total (nighttime + daytime) 6-SM
excretion was significantly lower in individuals with autism
compared to controls (t = 3.00, df = 67, p = 0.005). Finally,
the mean ( T SEM) intra-individual nighttime—daytime
differences (delta values) in 6-SM excretion rate were
significantly smaller in individuals with autism than in
controls (t = 2.21, df = 67,
p = 0.031).
compared to controls (8.26T 1.27 vs. 18.00 T3.43 mg/24 h
collection, p = 0.005).
3.2. Hubungan antara diagnosis dan tingkat ekskresi 6-SM
Analisis varians pengukuran berulang yang dilakukan pada
malam hari dan siang hari 6-SM ekskresi menunjukkan efek
signifikan kelompok (F (1,65) = 12,24, p <0,001), waktu (F
(1,65) = 35,00 , p <0,0001), dan interaksi kelompok-per-
waktu (F (1,65) = 4,88, p <0,05). Analisis post-hoc t-test
menunjukkan tingkat ekskresi 6-SM malam yang lebih
rendah secara signifikan pada individu dengan autisme
dibandingkan dengan kontrol (0,52 0,07 vs
1,14 0,23 mg / jam, p = 0,016); tingkat ekskresi 6-SM siang
hari pada individu dengan autisme juga lebih rendah
dibandingkan dengan kontrol (0,16 0,03 vs 0,36 0,05 mg /
jam, p = 0,002) (lihat Gambar. 1). Selain itu, total (malam
hari + siang hari) 6-SM ekskresi secara signifikan lebih
rendah pada individu dengan autisme dibandingkan dengan
kontrol (8,26 1,27 vs 18,00 pengumpulan 3,43 mg / 24
jam, p = 0,005).
Box and whisker plots for daytime, nighttime, and total
24 h 6-SM excretion, in individuals with autism and
controls are shown in Fig. 2A—C. The distributions of 6-SM
excretion rates appear shifted downward in the autism
group.
Box dan kumis plot untuk siang hari, malam hari, dan
total 24 jam 6-SM ekskresi, pada individu dengan autisme
dan kontrol ditunjukkan pada Gambar. 2A-C. Distribusi
tingkat ekskresi 6-SM tampak bergeser ke bawah pada
kelompok autisme.
Concerning the significant group-by-time interaction,
the meanT ( SEM) intra-individual nighttime—daytime
differences (delta values) in 6-SM excretion rate were
T T
significantly smaller in individuals with autism than in
controls (0.36 0.07 vs. 0.79 0.23 mg/h, p = 0.031) (see
Fig. 1).
Mengenai interaksi kelompok-oleh-waktu yang signifikan, rata-
rata (SEM) malam hari intra-individu - perbedaan siang hari (nilai
delta) dalam 6-SM tingkat ekskresi secara signifikan lebih kecil
pada individu dengan autisme daripada pada kontrol (0,36 0,07 vs
0,79 0,23 mg / h, p = 0,031) (lihat Gambar 1).
Among the 43 patients with autism, 30 (69.8%) displayed
greater 6-SM excretion at night compared to the daytime
(normal circadian rhythm), 10 (23.2%) displayed little or no
nighttime—daytime variation in 6-SM excretion rates (night-
time—daytime differences in 6-SM excretion rate less than
0.1 mg/h, with nighttime values less than 3 times greater
than daytime values), and 3 (7%) had higher daytime than
nighttime excretion (‘‘inverted’’ circadian rhythm). Of the 26
controls, 23 (88.5%) showed greater nighttime 6-SM excre-
tion, 1 (3.8%) displayed no nighttime—daytime variation in 6-
SM excretion rates, and 2 (7.7%) displayed higher daytime
excretion (see Fig. 3). The absence of circadian variation
(nighttime—daytime differences in 6-SM excretion rate) was
observed significantly more often than expected in
individuals with autism compared to controls ( p = 0.043,
Fisher Exact Test). Nighttime and daytime 6-SM excretion
rates were significantly correlated in both groups (individuals
with autism: r = 0.49, p < 0.001; controls: r = 0.44, p =
0.035).
Di antara 43 pasien autisme, 30 (69,8%) menunjukkan
ekskresi 6-SM lebih besar di malam hari dibandingkan
dengan siang hari (ritme sirkadian normal), 10 (23,2%)
menunjukkan sedikit atau tidak ada malam-variasi
siang hari dalam tingkat ekskresi 6-SM ( malam hari —
perbedaan siang hari dalam tingkat ekskresi 6-SM
kurang dari
0,1 mg / jam, dengan nilai malam hari kurang dari 3
kali lebih besar dari nilai siang hari), dan 3 (7%)
memiliki siang hari lebih tinggi daripada ekskresi
malam hari (‘‘ terbalik 'ritme sirkadian terbalik). Dari
26 kontrol, 23 (88,5%) menunjukkan ekskresi 6-SM
malam hari yang lebih besar, 1 (3,8%) tidak
menampilkan malam hari — variasi siang hari dalam
tingkat ekskresi 6- SM, dan 2 (7,7%) menampilkan
ekskresi siang hari lebih tinggi (lihat Gbr. 3). Tidak
adanya variasi sirkadian (perbedaan malam hari -
siang hari dalam tingkat ekskresi 6-SM) diamati secara
signifikan lebih sering daripada yang diperkirakan
pada individu dengan autisme dibandingkan dengan
kontrol (p = 0,043, Fisher Exact Test). Tingkat ekskresi
6-SM malam hari dan siang hari secara signifikan
berkorelasi pada kedua kelompok (individu dengan
autisme: r = 0,49, p <0,001; kontrol: r = 0,44, p =
0,035).
3.3. Relationships between autism severity and
6-SM excretion rates
Nighttime 6-SM excretion rates were significantly and nega-
tively correlated with severity of autistic impairment in the
 overall level of verbal language (verbal language is
defined in the ADI-R as the ‘‘daily, functional and
comprehensible use of spontaneous phrases of at least
three words, including at least sometimes a verb’’,
Spearman r = 0.30, p = 0.048), in the non-verbal
communication sub-domain C4 (‘‘Lack of varied
spontaneous make-believe or social imitative play’’,
Spearman r = 0.45, p = 0.040), and in the sub-domain
D4 (‘‘Preoccupations with part of objects or non-
functional elements of materials’’, Spearman r = 0.47,
p = 0.01). A further level of analysis on the subscores
indicated that the significant results in the C4 sub-
domain were mainly due to the subscore ‘‘Imitative
social play’’ (Spearman r = 0.42, p = 0.043), and in the
D4 sub-domain to the subscore ‘‘Repe- titive use of
objects’’ (Spearman r = 0.36, p = 0.045). There was no
significant correlation between nighttime 6-SM excre-
tion rates and IQ scores. Furthermore, there was no
signifi- cant correlation between daytime 6-SM
excretion rates and autism severity. However, daytime
6-SM excretion rates were significantly and positively
correlated with total IQ scores (Pearson r = 0.43, p =
0.014), with verbal IQ scores (Pearson r = 0.38, p =
0.033), and with performance IQ scores (Pearson r =
0.43, p = 0.014).
3.3. Hubungan antara tingkat keparahan autisme
dan tingkat ekskresi 6-SM
Tingkat ekskresi 6-SM malam hari secara
signifikan dan negatif berkorelasi dengan tingkat
keparahan gangguan autis di
keseluruhan tingkat bahasa verbal (bahasa verbal
didefinisikan dalam ADI-R sebagai '' penggunaan
harian, fungsional dan komprehensif dari frasa
spontan setidaknya tiga kata, termasuk
setidaknya kadang-kadang kata kerja '', Spearman
r = 0,30, p = 0,048), dalam sub-domain
komunikasi non-verbal C4 ('' Kurangnya beragam
keyakinan spontan atau permainan imitatif sosial
'', Spearman r = 0,45, p = 0,040), dan dalam sub-
domain D4 ('' Kesibukan dengan bagian benda
atau elemen non-fungsional material '', Spearman
r = 0,47, p = 0,01). Tingkat analisis lebih lanjut
pada subskala menunjukkan bahwa hasil yang
signifikan dalam sub-domain C4 terutama
disebabkan oleh subscore '' Permainan sosial
imitatif '' (Spearman r = 0,42, p = 0,043), dan
dalam sub-domain D4 ke subscore '' Penggunaan
berulang objek '' (Spearman r = 0,36, p = 0,045).
Tidak ada korelasi yang signifikan antara tingkat
ekskresi 6-SM malam hari dan skor IQ. Selain itu,
tidak ada korelasi yang signifikan antara tingkat
ekskresi 6-SM siang hari dan tingkat keparahan
autisme. Namun, tingkat ekskresi 6-SM siang hari
secara signifikan dan positif berkorelasi dengan
skor IQ total (Pearson r = 0,43, p = 0,014),
dengan skor IQ verbal (Pearson r = 0,38, p =
0,033), dan dengan skor IQ kinerja (Pearson r =
0,43, p = 0,014).
4. Discussion
A major finding of the study was that both daytime and
nocturnal 6-SM excretion rates, as well as the total (night-
time + daytime) 6-SM excretion, were significantly lower
in individuals with autism than in typically developing
controls. The finding of low nighttime urinary excretion of
6-SM in autism is consistent with our previous study
(Tordjman et al., 2005) and also with two other studies
(Nir et al., 1995; Kulman et al., 2000), but contradicts a
prior smaller study (Ritvo et al., 1993) that found
unaltered levels of nighttime urinary melatonin in autism.
However, in that study melatonin itself was measured,
melatonin levels were expressed as a urine concentration
(moles/liter), and a relatively small sample (N = 10) was
studied. Our finding of low daytime excretion of melatonin
in autism is consistent with Melke et al.’s study (2008), but
contrasts with previous smaller studies of urinary
melatonin (Ritvo et al., 1993) and plasma melatonin (Nir
et al., 1995) that have reported higher day- time levels in
autism.
4. Diskusi
Temuan utama dari penelitian ini adalah bahwa tingkat
ekskresi 6-SM siang hari dan nokturnal, serta ekskresi 6-SM
total (malam hari + siang hari), secara signifikan lebih
rendah pada individu dengan autisme daripada pada
kontrol yang biasanya berkembang. Temuan rendahnya
ekskresi urin malam hari 6-SM dalam autisme konsisten
dengan penelitian kami sebelumnya (Tordjman et al.,
2005) dan juga dengan dua penelitian lain (Nir et al.,
1995; Kulman et al., 2000), tetapi bertentangan dengan
penelitian sebelumnya yang lebih kecil (Ritvo et al., 1993)
yang menemukan kadar melatonin urin malam hari yang
tidak berubah pada autisme. Namun, dalam studi itu
melatonin itu sendiri diukur, kadar melatonin dinyatakan
sebagai konsentrasi urin (mol / liter), dan sampel yang
relatif kecil (N = 10) dipelajari. Temuan kami tentang
ekskresi melatonin siang hari yang rendah pada autisme
konsisten dengan penelitian Melke et al (2008), tetapi
berbeda dengan penelitian sebelumnya yang lebih kecil
tentang melatonin kemih (Ritvo et al., 1993) dan
melatonin plasma (Nir et al., 1995) yang melaporkan
tingkat autisme pada siang hari yang lebih tinggi.
Our results demonstrate that there is a deficit in
melatonin production in a substantial proportion of
individuals with autism and this deficit is present at night
and during the day, indicating that pineal and, possibly,
extra-pineal production of melatonin is lower in autism.
Furthermore, the small intra-individual 6-SM nighttime—
daytime differences and the significant absence of
melatonin variation found in autism, might be a reflection
of the lower day and nighttime levels, or an indication
that there exists a subgroup of individuals with autism that
have a dysregulation of their circadian rhythm, and more
precisely an absent circadian rhythm. The hypothesis of an
absent circadian rhythm in melatonin and other
neuroendocrine functions is supported by Kulman et al.’s
study (2000) in which 10 out of 14 children with autistic
disorder showed no melatonin circadian varia- tion, by
Zapella (1993) who found a blunted circadian rhythm of
melatonin secretion in a male adolescent with autism
and hypomelanosis of Ito, and by reports in autism of

Figure 2 (A) Box and whisker plot of daytime urinary 6-sul-
phatoxymelatonin (6-SM) excretion (mg/12 h collection) in
indi-
Figure 3 Distribution (%) of individuals with autism (N = 43)
and typically developing controls (N = 26) with normal
circadian rhythm (higher nighttime than daytime 6-SM
excretion rates), inverted circadian rhythm (higher daytime
than nighttime 6-SM excretion rates) and absent circadian
rhythm (no nighttime— daytime variation in 6-SM excretion
rates).
abnormalities in the circadian rhythm of cortisol secretion
including an absence of cortisol variation (Tordjman et al.,
1997). The possible existence and characteristics of a sub-
group of patients with autism showing a deficit in
melatonin production with no nighttime—daytime
variations may be fruitfully examined in future larger
studies. It is noteworthy that the circadian rhythm does
not appear in the present study to be inverted (day >
night) in more than a few of the individuals with autism
(3/43) and that a similar small fraction of the controls
(2/26) had inverted 6-SM excretion. This contrasts with
the Kulman et al. Study (2000) where 4 of 14 children with
autism were found to have inverted rhythms and is quite
distinct from Smith Magenis syndrome, a genetic syndrome
(del 17p11.2) displaying mental retardation, autistic
symptoms, and an inverted pattern of melatonin secretion
(Potocki et al., 2000).
Hasil kami menunjukkan bahwa ada defisit dalam produksi
melatonin di sebagian besar individu dengan autisme dan
defisit ini hadir di malam hari dan siang hari, menunjukkan
bahwa produksi melatonin pineal dan, mungkin, ekstra-
pineal lebih rendah autis. Lebih jauh lagi, malam-6-SM
kecil intra-individu - perbedaan siang hari dan tidak
adanya variasi melatonin yang ditemukan pada autisme,
mungkin merupakan cerminan dari tingkat hari dan malam
yang lebih rendah, atau indikasi bahwa ada subkelompok
individu dengan autisme yang memiliki disregulasi ritme
sirkadian mereka, dan lebih tepatnya ritme sirkadian
absen. Hipotesis tentang ritme sirkadian yang tidak ada
dalam melatonin dan fungsi neuroendokrin lainnya
didukung oleh penelitian Kulman et al. (2000) di mana 10
dari 14 anak dengan gangguan autistik tidak menunjukkan
variasi sirkadian melatonin, oleh Zapella (1993) yang
menemukan ritme sirkadian tumpul sekresi melatonin pada
remaja pria dengan autisme dan hipomelanosis Ito, dan
oleh laporan autisme kelainan pada ritme sirkadian sekresi
kortisol termasuk tidak adanya variasi kortisol (Tordjman
et al., 1997). Keberadaan dan karakteristik yang mungkin
dari sub-kelompok pasien dengan autisme menunjukkan
defisit dalam produksi melatonin tanpa malam hari —
variasi siang hari dapat diperiksa secara bermanfaat dalam
penelitian yang lebih besar di masa depan. Patut dicatat
bahwa ritme sirkadian tidak muncul dalam penelitian ini
untuk dibalikkan (siang> malam) di lebih dari beberapa
individu dengan autisme (3/43) dan bahwa sebagian kecil
yang sama dari kontrol (2/26) ) telah membalikkan
ekskresi 6-SM. Ini kontras dengan Kulman et al. Studi
(2000) di mana 4 dari 14 anak autisme ditemukan
memiliki irama terbalik dan sangat berbeda dari sindrom
Smith Magenis, sindrom genetik (del 17p11.2) yang
menunjukkan keterbelakangan mental, gejala autistik,
dan pola sekresi melatonin yang terbalik ( Potocki et al.,
2000).
It can be speculated that the present findings are
viduals with autism (N = 43) and typically developing controls
(N = 26). Means are indicated by the cross, medians by the
midline, second and third quartiles by the open box, and mini-
mums and maximums by the lower and upper whiskers. The
median values were 3.14 and 1.3 mg/12 h collection in the
control and autism groups, respectively. (B) Box and whisker
plot of nighttime urinary 6-sulphatoxymelatonin (6-SM) excre-
tion (mg/12 h collection) in individuals with autism (N = 43)
and typically developing controls (N = 26). Means are
indicated by the cross, medians by the midline, second and
third quartiles by the open box, and minimums by the lower
whiskers. The median values were 8.6 and 4.8 mg/12 h
collection in the control and autism groups, respectively.
Maximum nighttime 6-SM excretion
related to peripheral or central abnormalities in serotonin
physiology reported in autism possibly resulting from
genetic factors, given that melatonin is synthesized
directly from serotonin by N-acetylation and O-
methylation (Tordjman et al., 2001; Anderson et al., 2002;
Revell and Skene, 2007). The relationship of the melatonin
deficit to the well-replicated platelet hyperserotonemia of
autism is of special interest and there is some preliminary
evidence that lower melatonin production may be more
common in individuals with elevated platelet serotonin
(Melke et al., 2008; Mulder et al., 2010). Certainly,
values were 60.6 and 29.0 mg/12 h collection in the control
and autism groups, respectively (upper whiskers not
presented). (C) Box and whisker plot of combined night and
daytime urinary 6- sulphatoxymelatonin (6-SM) excretion
(mg/24 h collection) in individuals with autism (N = 43) and
typically developing controls (N = 26). Means are indicated by
the cross, medians by the midline, second and third quartiles
by the open box, and mini- mums by the lower whiskers. The
median values were 8.6 and
4.8 mg/24 h collection in the control and autism groups,
respec- tively. Maximum combined (night and daytime) 6-SM
excretion values were 69.7 and 42.7 mg/24 h collection in the
control and autism groups, respectively (upper whiskers not
presented).
the enzymes involved in the synthesis of melatonin from
serotonin should be examined as potential contributors to
the lower melatonin production. However, it appears that
the individual melatonin deficit is usually one of degree,
lessen- ing the likelihood that loss-of-function mutations
are fre- quently playing an important role. An initial report
(Melke et al., 2008) suggesting a primary deficit in the
enzyme catalyzing O-methylation (acetylserotonin O-
methyltrans- ferase, ASMT) and positing ASMT as a
susceptibility gene for autism, has not been borne out by
subsequent research (Toma et al., 2007; Jonsson et al.,
2010). The report of an increased occurrence of a partial
duplication in the ASMT gene in autism (6% vs. 2% in
controls) has also increased interest in melatonin synthesis
(Cai et al., 2008). The appar- ent lower production of both
nighttime and daytime mela- tonin observed in the present
study suggests that identification of aspects and regulatory
factors held in com- mon across the pineal and extrapineal
tissues might lead to underlying mechanisms. Thus, the
study of melatonin in other tissues, including gut,
placenta, and skin, should be considered (Bubenik, 2002;
Iwasaki et al., 2005; Slominski et al., 2008).
Dapat berspekulasi bahwa temuan ini terkait dengan
kelainan perifer atau sentral dalam fisiologi serotonin yang
dilaporkan dalam autisme mungkin dihasilkan dari faktor
genetik, mengingat melatonin disintesis langsung dari
serotonin oleh asetilasi-N dan metilasi-O (Tordjman et al.,
2001 ; Anderson et al., 2002; Revell dan Skene, 2007).
Hubungan defisit melatonin dengan hiperserotonemia
autisme trombosit yang direplikasi dengan baik merupakan
hal yang menarik dan ada beberapa bukti awal bahwa
produksi melatonin yang lebih rendah mungkin lebih umum
pada individu dengan serotonin trombosit tinggi (Melke et
al., 2008; Mulder et al., 2008). ., 2010). Tentu saja, enzim
yang terlibat dalam sintesis melatonin dari serotonin harus
diperiksa sebagai kontributor potensial terhadap produksi
melatonin yang lebih rendah. Namun, tampaknya defisit
melatonin individu biasanya adalah satu derajat,
mengurangi kemungkinan bahwa mutasi kehilangan fungsi
sering memainkan peran penting. Laporan awal (Melke et
al., 2008) menunjukkan defisit primer pada enzim yang
mengkatalisasi O-metilasi (asetilserotonin O-
metiltransferase, ASMT) dan menempatkan ASMT sebagai
gen kerentanan untuk autisme, belum ditanggapi oleh
penelitian selanjutnya. (Toma et al., 2007; Jonsson et al.,
2010). Laporan peningkatan kejadian duplikasi parsial
pada gen ASMT pada autisme (6% vs 2% pada kontrol) juga
meningkatkan minat dalam sintesis melatonin (Cai et al.,
2008). Produksi yang lebih rendah dari melatonin malam
hari dan siang hari yang diamati dalam penelitian ini
menunjukkan bahwa identifikasi aspek dan faktor regulasi
yang dimiliki bersama di seluruh jaringan pineal dan
ekstrapineal dapat menyebabkan mekanisme yang
mendasarinya. Dengan demikian, studi melatonin di
jaringan lain, termasuk usus, plasenta, dan kulit, harus
dipertimbangkan (Bubenik, 2002; Iwasaki et al., 2005;
Slominski et al., 2008).
In addition to characterizing the nature of the
alteration in melatonin production in autism, we aimed to
identify potential associations between melatonin and
behavioral expression of autism. We did observe that
nocturnal 6-SM excretion rate was significantly negatively
correlated with severity of impairment in the overall level of
verbal lan- guage, imitative social play and repetitive use of
objects. The observation of significant negative correlations
between nocturnal 6-SM excretion and severity of autistic
impairment in verbal communication and play replicates our
previous finding (Tordjman et al., 2005). It is noteworthy
that we replicated this result using the ADI-R (the ADI-R is
based on a parental interview) which differs from the ADOS
(the Autism Diagnostic Observation Schedule is based on a
direct observation of the patient; Lord, 1997) used in our
previous study. Nir et al. (1995) presented data suggestive of
reduced melatonin production in individuals with autism and
speech difficulties or with EEG abnormalities. More compel-
ling is the agreement between our finding of a negative
relationship of nocturnal 6-SM excretion with severity of
language impairment and the study by Hu et al. (2009) in
autism spectrum disorders (ASD) that reported substantially
reduced expression of the gene encoding arylalkylamine N-
acetyltransferase (AANAT, the rate limiting enzyme for mel-
atonin synthesis) in ASD individuals with severe language
impairment.
Selain mengkarakterisasi sifat dari perubahan dalam
produksi melatonin dalam autisme, kami bertujuan untuk
mengidentifikasi hubungan potensial antara melatonin dan
ekspresi perilaku autisme. Kami memang mengamati bahwa
tingkat ekskresi 6-SM nokturnal secara signifikan berkorelasi
negatif dengan tingkat keparahan penurunan tingkat
keseluruhan bahasa verbal, permainan sosial imitatif dan
penggunaan berulang objek. Pengamatan korelasi negatif
yang signifikan antara ekskresi 6-SM nokturnal dan keparahan
gangguan autistik dalam komunikasi verbal dan permainan
mereplikasi temuan kami sebelumnya (Tordjman et al.,
2005). Perlu dicatat bahwa kami mereplikasi hasil ini
menggunakan ADI-R (ADI-R didasarkan pada wawancara orang
tua) yang berbeda dari ADOS (Jadwal Pengamatan Diagnostik
Autisme didasarkan pada pengamatan langsung pasien; Lord,
1997) digunakan dalam penelitian kami sebelumnya. Nir et
al. (1995) menyajikan data yang menunjukkan penurunan
produksi melatonin pada individu dengan autisme dan
kesulitan bicara atau dengan kelainan EEG. Yang lebih
menarik adalah kesepakatan antara temuan kami tentang
hubungan negatif ekskresi 6-SM nokturnal dengan tingkat
keparahan gangguan bahasa dan penelitian oleh Hu et al.
(2009) dalam gangguan spektrum autisme (ASD) yang
melaporkan secara substansial mengurangi ekspresi gen yang
mengkode arylalkylamine N-acetyltransferase (AANAT, enzim
pembatas laju untuk sintesis melonin) pada individu-individu
ASD dengan gangguan bahasa yang parah.
We did not observe sleep problems in the subjects
studied, but the subjects were adolescents and young adults,
all post- pubertal individuals, and the assessment was brief
and not as detailed as a prior report linking lower melatonin
with less nighttime N3 sleep and increased daytime sleepiness
in children with autism (Leu et al., 2011).
Kami tidak mengamati masalah tidur pada subjek yang
diteliti, tetapi subjeknya adalah remaja dan dewasa muda,
semua individu post-pubertas, dan penilaiannya singkat dan
tidak sedetail laporan sebelumnya yang mengaitkan
melatonin yang lebih rendah dengan lebih sedikit tidur N3 di
malam hari dan meningkatkan siang hari kantuk pada anak
autis (Leu et al., 2011).
 We did not find significant correlations between
nocturnal 6-SM excretion rates and IQ scores. However,
the range of IQ scores in the patients was too narrow to
test thoroughly the relationship between IQ scores and 6-
SM levels. Previous studies of melatonin production in
autism have provided little or no information regarding
level of cognitive function- ing of study subjects, and it is
clear that IQ-matching would be desirable. Melatonin
production in non-syndromic mental retardation appears
unstudied, but it is of potential rele- vance that melatonin
production in Down syndrome has been reported to be
normal, while increased levels have been reported for
Fragile X subjects (Reiter et al., 1996; Gould et al., 2000).
Kami tidak menemukan korelasi yang signifikan antara
tingkat ekskresi 6-SM malam dan skor IQ. Namun,
kisaran skor IQ pada pasien terlalu sempit untuk
menguji secara menyeluruh hubungan antara skor IQ
dan level 6-SM. Studi sebelumnya tentang produksi
melatonin dalam autisme telah memberikan sedikit
atau tidak ada informasi mengenai tingkat fungsi
kognitif subjek penelitian, dan jelas bahwa pencocokan
IQ akan diinginkan. Produksi melatonin pada retardasi
mental non-sindrom tampaknya tidak dipelajari, tetapi
berpotensi penting bahwa produksi melatonin pada
sindrom Down telah dilaporkan normal, sementara
peningkatan level telah dilaporkan untuk subjek Fragile
X (Reiter et al., 1996; Gould et al., 2000).
Finally, given the apparent role of melatonin in
neural development (de Faria Poloni et al., 2011), it
might be possible that a lack of melatonin occuring in
utero or at birth could impair brain development.
Furthermore, it is quite speculative but thought
provoking to consider whether the lower mean
melatonin production, the significantly smaller day—
night differences and the significantly higher frequency
of absence of circadian variation observed in individuals
with autism compared to controls, might be playing a
role in, or be a reflection of, the hypothesized
alteration in time perception in autism (Boucher, 2001;
Wimpory et al., 2002). Indeed, melatonin signals can
drive daily rhythmicity and are also involved in the
synchroniza- tion of peripheral oscillators (i.e., in the
adjustment of the timing of existing oscillations) (Pevet
and Challet, 2011). Thus, Boucher’s model of autism
relates the very poor intuitive sense of time observed in
individuals with autism to abnormalities in circadian
physiological measures and sleep-wake cycle (Boucher,
2001). Boucher suggests that timing problems in
‘‘biological clocks’’ would have physio- logical and
psychological consequences that might be involved in
autistic impairments. Furthermore, Wimpory et al.
have theorized that anomalies in clock genes operating
as timing genes in high frequency oscillator systems
may underline timing deficits that could be important
in the development of autistic disorder (Wimpory et al.,
2002).
Akhirnya, mengingat peran nyata melatonin dalam
perkembangan saraf (de Faria Poloni et al., 2011),
dimungkinkan bahwa kurangnya melatonin yang terjadi
dalam rahim atau saat lahir dapat mengganggu
perkembangan otak. Lebih lanjut, ini cukup spekulatif
tetapi pemikiran yang memprovokasi untuk
mempertimbangkan apakah produksi melatonin rata-rata
yang lebih rendah, perbedaan siang-malam yang jauh lebih
kecil dan frekuensi signifikan lebih tinggi dari tidak adanya
variasi sirkadian yang diamati pada individu dengan
autisme dibandingkan dengan kontrol, mungkin
memainkan peran dalam , atau menjadi refleksi dari,
perubahan hipotesis dalam persepsi waktu dalam autisme
(Boucher, 2001; Wimpory et al., 2002). Memang, sinyal
melatonin dapat mendorong ritme harian dan juga terlibat
dalam sinkronisasi osilator periferal (mis., Dalam
penyesuaian waktu osilasi yang ada) (Pevet dan Challet,
2011). Dengan demikian, model autisme Boucher
menghubungkan rasa intuisi waktu yang sangat buruk yang
diamati pada individu dengan autisme terhadap kelainan
pada tindakan fisiologis sirkadian dan siklus tidur-bangun
(Boucher, 2001). Boucher menunjukkan bahwa masalah
waktu dalam ‘cl jam biologis’ akan memiliki konsekuensi
fisiologis dan psikologis yang mungkin terlibat dalam
gangguan autistik. Selanjutnya, Wimpory et al. telah
berteori bahwa anomali dalam gen jam beroperasi sebagai
gen waktu dalam sistem osilator frekuensi tinggi dapat
menggarisbawahi defisit waktu yang bisa menjadi penting
dalam pengembangan gangguan autistik (Wimpory et al.,
2002).
In summary, this is the first study showing clearly in a
large sample of individuals with autism that a deficit in
melatonin production is present both at night and during
the day, and that the deficit is greater in more severely
affected indivi- duals. The biochemical, neuronal and
genetic pathways gov- erning melatonin production are
well-characterized and offer promising avenues for
investigation. The present study was limited by the low
temporal resolution provided by the long 12-h urine
collection periods (a consequence of inherent difficulties
in obtaining urine collection at fixed time points in low
functioning individuals with autism), by the narrow range
of IQ scores in the autism group, and by the relatively
brief assessment of sleep problems. Thus, further research
is needed to determine whether the nocturnal melatonin
acro- phase is shifted, whether the melatonin deficit is
influenced by the level of intellectual functioning, and
whether the nocturnal deficit is important in the sleep
problems often reported to be associated with autism.
However, it is note- worthy that prior studies of the
prevalence of sleep problems in autism typically have not
used population-based samples and have rarely studied
post-pubertal individuals. Additional studies are warranted
to investigate the utility of urinary 6- SM in screening and
subtyping, to identify underlying genetic and biological
factors, and to elucidate the role of altered melatonin and
associated factors in the pathophysiology and behavioral
manifestations of autism. The results highlight interest in
potential therapeutic uses of melatonin in autism,
especially in individuals with severe behavioral autistic
impairment and/or low in urinary 6-SM excretion, and sug-
gest that urinary 6-SM could be examined as a potential
predictor of response to melatonin.
Singkatnya, ini adalah studi pertama yang
menunjukkan dengan jelas dalam sampel besar
individu dengan autisme bahwa defisit dalam
produksi melatonin hadir baik di malam hari dan
siang hari, dan bahwa defisit lebih besar di industri
yang terkena dampak lebih parah. Jalur biokimia,
neuron dan genetik yang mengatur produksi
melatonin dikarakterisasi dengan baik dan
menawarkan jalan yang menjanjikan untuk
diselidiki. Penelitian ini dibatasi oleh resolusi
temporal rendah yang diberikan oleh periode
pengumpulan urin 12 jam yang panjang
(konsekuensi dari kesulitan yang melekat dalam
memperoleh pengumpulan urin pada titik waktu
tetap pada individu yang berfungsi rendah dengan
autisme), oleh kisaran sempit skor IQ pada
kelompok autisme, dan dengan penilaian yang
relatif singkat tentang masalah tidur. Dengan
demikian, penelitian lebih lanjut diperlukan
untuk menentukan apakah fase melatonin
nokturnal digeser, apakah defisit melatonin
dipengaruhi oleh tingkat fungsi intelektual, dan
apakah defisit nokturnal penting dalam masalah
tidur yang sering dilaporkan dikaitkan dengan
autisme. Namun, perlu dicatat bahwa penelitian
sebelumnya tentang prevalensi masalah tidur
pada autisme biasanya tidak menggunakan
sampel berbasis populasi dan jarang mempelajari
individu pasca pubertas. Studi tambahan
diperlukan untuk menyelidiki kegunaan 6- SM
kemih dalam skrining dan subtipe, untuk
mengidentifikasi faktor genetik dan biologis yang
mendasarinya, dan untuk menjelaskan peran
melatonin yang berubah dan faktor terkait dalam
patofisiologi dan manifestasi perilaku autisme.
Hasil ini menyoroti minat dalam penggunaan
terapi melatonin potensial dalam autisme,
terutama pada individu dengan gangguan autistik
perilaku yang parah dan / atau rendah dalam
ekskresi 6-SM kemih, dan menunjukkan bahwa 6-
SM kemih dapat diperiksa sebagai prediktor
potensial respon untuk melatonin.
Role of funding sources
Funding sources had no impact on any aspect of the work
with the current manuscript (design, data collection,
analysis, interpretation, writing or submission).
Conflict of interest
There are no conflicts of interest for any of the authors.
Acknowledgments
Pr Tordjman gratefully acknowledges the support of the
Foundation de France (Paris). Dr. Anderson gratefully
acknowledges the support of the Alan B. Slifka Foundation
(New York City).
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