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ARCHIVAL REPORT Intrinsic Circadian Period of Sighted Patients with Circadian Rhythm Sleep Disorder, Free-Running Type Shingo Kitamura, Akiko Hida, Minori Enomoto, Makiko Watanabe, Yasuko Katayose, Kentaro Nozaki, Sayaka Aritake, Shigekazu Higuchi, Yoshiya Moriguchi, Yuichi Kamei, and Kazuo Mishima Background: Circadian rhythm sleep disorder, free-running type (FRT), is an intractable sleep disorder in which sleep and wake times progressively delay each day even in normal living environments. This disorder severely affects the social functioning of patients because of periodic nighttime insomnia, excessive daytime sleepiness, and a high rate of comorbid psychiatric disorders. Although abnormal regula- tion of the biological clock is suspected, the pathophysiology of FRT has yet to be elucidated. In this study, the endogenous circadian period, , of FRT patients with normal vision was compared with that of healthy individuals whose circadian rhythms are entrained to a 24-hour cycle. Methods: Six FRT patients and 17 healthy individuals (9 intermediate chronotypes and 8 evening chronotypes) were subjected to a 7-day, 28-hour sleep–wake schedule according to the forced desynchrony protocol. Phase shifts in melatonin rhythm were measured under constant routine conditions to calculate . Results: In FRT patients, was significantly longer than in intermediate chronotypes, whereas in evening chronotypes, it ranged widely and was not significantly different from that in FRT patients. Moreover, of melatonin rhythm in FRT patients showed no significant correlation with of sleep–wake cycles measured before the study. Conclusions: The findings suggest that although a prolongation of may be involved in the onset mechanism of FRT, a prolonged is not the only factor involved. It appears that several factors including abnormal entrainment of circadian rhythms are involved in the onset of FRT in a multilayered manner. Key Words: Biological clock, circadian period, circadian rhythm sleep disorder, sleep–wake cycle, forced desynchrony, chronotype M echanisms involved in the alternating appearance of sleep and wakefulness are thought to be regulated by the mu- tually inhibitory flip-flop circuit (1,2) present between the sleep-promoting nuclei represented by the ventrolateral preoptic area and the wakefulness-promoting nuclei represented by the tuberomammillary nucleus and the basal forebrain. Moreover, cir- cadian signals from the suprachiasmatic nucleus (SCN), the biolog- ical clock situated in the hypothalamus in the central nervous sys- tem, play a role in determining the timing of the sleep–wake switch (3). However, because the circadian rhythm cycle () generated in the SCN is slightly deviated from a 24-hour day, it is necessary to compensate for the daily shift to synchronize with the 24-hour day–night cycle. Circadian rhythm sleep disorder, free-running type (FRT), is one type of sleep disorder (4). FRT patients cannot synchronize their circadian rhythms to a 24-hour day because of an abnormality in the circadian regulatory mechanism, and this results in progressive delay in sleep and wake times. These patients experience periodic nighttime insomnia and strong daytime sleepiness, and their social activities are severely disturbed because of their inability to stay awake during socially acceptable hours. In addition, FRT patients frequently have comorbid psychiatric disorders. Hayakawa et al. reported that 30 (52.6%) of 57 FRT patients had psychiatric prob- lems before and after the time of disease onset (5). Although the pathophysiology of FRT is mostly unknown, im- pairment in the circadian rhythm entrainment system and/or ab- normal free-running cycles () are thought to be the cause of the disorder (6–8). Because the environmental light– dark cycle is the most powerful entrainment factor for mammalian circadian rhythms, reduced ambient light signals or decreased light sensitiv- ity of the biological clock can increase the risk of developing FRT (9). In fact, the prevalence of FRT is high in individuals with visual impairment (10 –14). However, there have been few reports of FRT in healthy, sighted individuals. Excessively long can be a cause of FRT in the absence of abnormalities in visual or photic entrainment functions (6,15). The average period measured using the strict forced desynchrony protocol (FD protocol) (16,17), which is free of the masking factors of mammalian circadian rhythms, is 24.18 hours and thus is fairly close to the environmental light– dark period (16). However, a large individual variation exists in (18,19), and the longer gets beyond 24 hours, the larger the phase difference that needs to be adjusted daily. In fact, healthy individuals with longer exhibit a strong tendency to be an evening type (20 –22). Therefore, when is significantly longer, typical exposure to environmental and indoor light may not compensate a beyond 24-hour shift in , resulting in a continuous phase delay as is seen in patients with FRT. No previous studies have reported on abnormal entrainment of circadian rhythms or abnormal among sighted patients with FRT. Therefore, we investigated whether any abnormality is associated with of sighted patients with long-term FRT using an isolation laboratory and using the FD protocol (16,17). Methods and Materials Participants Twenty-three participants, 6 FRT patients and 17 healthy indi- viduals, participated in this study. None of the participants exhib- ited clinical signs of visual impairment, and fundus examination detected no morphologic abnormalities in the retina. FRT patients were recruited from individuals treated at the sleep clinic of the National Center Hospital, National Institute of Neurology and Psy- From the Department of Psychophysiology, National Institute of Mental Health, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan. Address correspondence to Kazuo Mishima, M.D., Ph.D., Department of Psychophysiology, National Institute of Mental Health, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187- 8553, Japan; E-mail: [email protected]. Received Apr 11, 2012; revised and accepted Jun 26, 2012. BIOL PSYCHIATRY 2013;73:63– 69 0006-3223/$36.00 http://dx.doi.org/10.1016/j.biopsych.2012.06.027 © 2013 Society of Biological Psychiatry

Intrinsic Circadian Period of Sighted Patients with Circadian Rhythm Sleep Disorder, Free-Running Type

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Page 1: Intrinsic Circadian Period of Sighted Patients with Circadian Rhythm Sleep Disorder, Free-Running Type

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ARCHIVAL REPORT

Intrinsic Circadian Period of Sighted Patients withCircadian Rhythm Sleep Disorder, Free-Running TypeShingo Kitamura, Akiko Hida, Minori Enomoto, Makiko Watanabe, Yasuko Katayose, Kentaro Nozaki,Sayaka Aritake, Shigekazu Higuchi, Yoshiya Moriguchi, Yuichi Kamei, and Kazuo Mishima

Background: Circadian rhythm sleep disorder, free-running type (FRT), is an intractable sleep disorder in which sleep and wake timesprogressively delay each day even in normal living environments. This disorder severely affects the social functioning of patients because ofperiodic nighttime insomnia, excessive daytime sleepiness, and a high rate of comorbid psychiatric disorders. Although abnormal regula-tion of the biological clock is suspected, the pathophysiology of FRT has yet to be elucidated. In this study, the endogenous circadian period,�, of FRT patients with normal vision was compared with that of healthy individuals whose circadian rhythms are entrained to a 24-hour cycle.

Methods: Six FRT patients and 17 healthy individuals (9 intermediate chronotypes and 8 evening chronotypes) were subjected to a 7-day,28-hour sleep–wake schedule according to the forced desynchrony protocol. Phase shifts in melatonin rhythm were measured underconstant routine conditions to calculate �.

Results: In FRT patients, � was significantly longer than in intermediate chronotypes, whereas in evening chronotypes, it ranged widely andwas not significantly different from that in FRT patients. Moreover, � of melatonin rhythm in FRT patients showed no significant correlationwith � of sleep–wake cycles measured before the study.

Conclusions: The findings suggest that although a prolongation of � may be involved in the onset mechanism of FRT, a prolonged � is nothe only factor involved. It appears that several factors including abnormal entrainment of circadian rhythms are involved in the onset of FRT

n a multilayered manner.

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Key Words: Biological clock, circadian period, circadian rhythmsleep disorder, sleep–wake cycle, forced desynchrony, chronotype

M echanisms involved in the alternating appearance of sleepand wakefulness are thought to be regulated by the mu-tually inhibitory flip-flop circuit (1,2) present between the

sleep-promoting nuclei represented by the ventrolateral preopticarea and the wakefulness-promoting nuclei represented by thetuberomammillary nucleus and the basal forebrain. Moreover, cir-cadian signals from the suprachiasmatic nucleus (SCN), the biolog-ical clock situated in the hypothalamus in the central nervous sys-tem, play a role in determining the timing of the sleep–wake switch(3). However, because the circadian rhythm cycle (�) generated inthe SCN is slightly deviated from a 24-hour day, it is necessary tocompensate for the daily shift to synchronize with the 24-hourday–night cycle.

Circadian rhythm sleep disorder, free-running type (FRT), is onetype of sleep disorder (4). FRT patients cannot synchronize theircircadian rhythms to a 24-hour day because of an abnormality inthe circadian regulatory mechanism, and this results in progressivedelay in sleep and wake times. These patients experience periodicnighttime insomnia and strong daytime sleepiness, and their socialactivities are severely disturbed because of their inability to stayawake during socially acceptable hours. In addition, FRT patientsfrequently have comorbid psychiatric disorders. Hayakawa et al.reported that 30 (52.6%) of 57 FRT patients had psychiatric prob-lems before and after the time of disease onset (5).

From the Department of Psychophysiology, National Institute of MentalHealth, National Center of Neurology and Psychiatry, Kodaira, Tokyo,Japan.

Address correspondence to Kazuo Mishima, M.D., Ph.D., Department ofPsychophysiology, National Institute of Mental Health, National Centerof Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8553, Japan; E-mail: [email protected].

NReceived Apr 11, 2012; revised and accepted Jun 26, 2012.

0006-3223/$36.00http://dx.doi.org/10.1016/j.biopsych.2012.06.027

Although the pathophysiology of FRT is mostly unknown, im-airment in the circadian rhythm entrainment system and/or ab-ormal free-running cycles (�) are thought to be the cause of theisorder (6 – 8). Because the environmental light– dark cycle is theost powerful entrainment factor for mammalian circadian

hythms, reduced ambient light signals or decreased light sensitiv-ty of the biological clock can increase the risk of developing FRT (9).n fact, the prevalence of FRT is high in individuals with visualmpairment (10 –14). However, there have been few reports of FRTn healthy, sighted individuals. Excessively long � can be a cause ofRT in the absence of abnormalities in visual or photic entrainmentunctions (6,15). The average � period measured using the strictorced desynchrony protocol (FD protocol) (16,17), which is free ofhe masking factors of mammalian circadian rhythms, is 24.18 hoursnd thus is fairly close to the environmental light– dark period (16).owever, a large individual variation exists in � (18,19), and the

onger � gets beyond 24 hours, the larger the phase difference thateeds to be adjusted daily. In fact, healthy individuals with longer �xhibit a strong tendency to be an evening type (20 –22). Therefore,hen � is significantly longer, typical exposure to environmental

nd indoor light may not compensate a beyond 24-hour shift in �,esulting in a continuous phase delay as is seen in patients with FRT.

No previous studies have reported on abnormal entrainment ofircadian rhythms or abnormal � among sighted patients with FRT.herefore, we investigated whether any abnormality is associatedith � of sighted patients with long-term FRT using an isolation

aboratory and using the FD protocol (16,17).

ethods and Materials

articipantsTwenty-three participants, 6 FRT patients and 17 healthy indi-

iduals, participated in this study. None of the participants exhib-ted clinical signs of visual impairment, and fundus examinationetected no morphologic abnormalities in the retina. FRT patientsere recruited from individuals treated at the sleep clinic of the

ational Center Hospital, National Institute of Neurology and Psy-

BIOL PSYCHIATRY 2013;73:63–69© 2013 Society of Biological Psychiatry

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64 BIOL PSYCHIATRY 2013;73:63–69 S. Kitamura et al.

chiatry and who had been diagnosed with FRT in accordance withthe International Classification of Sleep Disorders, 2nd edition(ICSD-2) criteria. There were two men (mean age � SD � 27.5 �10.6 years; age range, 20 –35 years) and four women (mean age �SD � 33.8 � 16.2 years; age range, 17–56 years; Table 1). All patientshad a more than 3-year history of FRT, with the longest durationbeing 24 years (mean � SD � 10.83 � 8.4 years). A history of FRTwas confirmed based on the self-administered sleep diary that pa-tients had continued for at least 6 months (Figure 2). Although fourpatients had a history of mood disorder (two major depression, onedysthymic disorder, and one seasonal affective disorder), they werein remission during the study period. Because two of the patients(Subjects F4 and F6 in Table 1) had been undergoing maintenancedrug therapy (triazolam, flunitrazepam, zolpidem, lorazepam, bro-mazepam, and fluvoxamine), we fixed the amount of drugs andcontinued the therapy throughout the study period. At the time ofproviding informed consent, four patients (Subjects F2, F3, F4, andF6 in Table 1) were undergoing chronotherapy (bright light ther-apy, melatonin, and/or ramelteon), and the therapy was discontin-ued at least 2 weeks before the study.

The healthy individuals (control group) consisted of 17 healthymale, paid volunteers, aged 20 to 39 years (mean age � SD � 22.6 �4.4 years) without any known sleep, physical, or psychiatric disor-ders or any history of using psychoactive drugs. None had workednight shifts or traveled across time zones in the previous 6 months.These data were obtained by a semistructured interview with apsychiatrist, all-night clinical polysomnography, blood chemistrytests, and several screening questionnaires (Minnesota MultiphasicPersonality Inventory-2, Center for Epidemiology Study Depres-sion, and Pittsburgh Sleep Quality Index). On the basis of the resultsof the Morningness-Eveningness Questionnaire (23), nine of thehealthy individuals were classed as intermediate type (I-type; scorerange, 42–58 points, mean � SD � 52.8 � 4.1 points), and eightwere classed as evening type (E-type; score range, 19 – 41 points,mean � SD � 34.5 � 7.1 points). None of the participants exhibitedclinical signs of visual impairment, and fundus examination de-tected no morphologic abnormalities of the retina.

ProtocolAll subjects participated in a 13-day FD protocol in a temporal

isolation laboratory free from external time cues (Figure 1). Subjectsentered the laboratory (5.9 m2 polysomnographic bedroom, 67.9m2 living room, restroom, and bathroom) at 5 PM on Day 1, and after

aving a meal and taking a bath, they turned the lights off and wento bed at 12 AM. The FD protocol used in this study was conductedntirely in the laboratory and comprised the following three stud-

es: 1) measurement of melatonin rhythm under a constant routineCR) condition (24) (first CR) followed by 2) a 28-hour sleep–wakechedule for 7 days, and 3) a second measurement of melatoninhythm under CR (second CR). The 28-hour sleep–wake schedule

Table 1. Clinical Features in Patients of Circadian Rhyth

Subject Sex Age at Study Age at Onset

F1 F 17 14F2 F 31 15F3 M 20 17F4 F 31 25F5 F 56 32F6 M 35 22

F, female; M, male.

ncluded 9.33 hours of sleep (promoting sleep/bedrest with lightsaf

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ff) and 18.67 hours of wakefulness (prohibiting sleep). Because a8-hour life cycle is beyond the scope of the entrainment functionf the human biological clock, the biological clock continues en-ogenous oscillations of an approximately 24-hour cycle withoutntrainment (16,25).

During the FD protocol, subjects stayed in the laboratory insolation from external time cues such as clocks, radios, newspa-ers, cell phones, and the Internet. They were asked to maintainakefulness under a low-intensity light condition (�15 lux) during

he scheduled wake period and to sleep in the bedroom with theights off (0 lux) during the sleep period. Throughout the study,ubjects were under constant surveillance by a researcher and wereerbally awakened when they unintentionally took a nap duringhe wake period. During the wake period, they were allowed to

ove freely around in the laboratory, read and write, enjoy musicnd videos, play videogames, and engage in conversation with theesearcher. Ambient temperature and humidity in the laboratoryere maintained at 25°C � .5°C and 50% � 5% RH, respectively.

easurement of � of Melatonin RhythmTo calculate �, the difference in the melatonin rhythm phases

easured during the first CR and second CR conducted at the

ep Disorder, Free-Running Type

leep–Wake Period (�)Before Treatment Comorbid Disorders

25.17 Major depression25.02 —25.71 Orthostatic disturbance25.87 Dysthymic disorder24.66 Seasonal affective disorder24.69 Major depression

igure 1. Forced desynchrony (FD) protocol. Filled bars, scheduled sleep (0ux); open bars, wakefulness in dim light (�15 lx); hatched bars, constantoutine (CR). The FD protocol used in this study was a 28-hour sleep–wakeycle consisting of 9.33 hours of sleep and 18.67 hours of wakefulness. Theycle of biological rhythms was determined based on the phase of melato-in rhythm measured during the CRs conducted before and after applying

he FD protocol. CR conditions included low-intensity lighting (�15 lux),emirecumbent (45’) posture, distributed calorie intake (200 kCal/2 hours),

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nd 38.67 hours of continuous wakefulness, thus excluding the maskingactors of melatonin rhythm.

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beginning and end of the study, respectively, was divided by thenumber of experimental days. Dim light melatonin onset time(DLMO) and acrophase (ACR) of the cosine-fitted curve were usedto calculate the indices of the circadian rhythms, �DLMO and �ACR.

During CR, to exclude masking factors of the melatonin rhythm,subjects were asked to maintain a semirecumbent (45’) postureunder low-intensity light conditions (�15 lux) and consume smallmeals (approximately 200 kcal) separated by a 2-hour interval. Inaddition, during 38.67 hours of sustained wakefulness, blood sam-ples collected every 60 min via a stopcock attached directly tointravenous catheter were centrifuged, and the plasma collectedwas frozen at �80°C for radioimmunoassay of melatonin. Concen-trations of plasma melatonin were assayed by an radioimmunoas-say (RIA) technique (SRL, Tokyo, Japan). The assay sensitivity was 2.8pg/mL for plasma melatonin. In two patients (Subjects F1 and F5),saliva samples were collected instead of blood using a Salivette tube(Sarstedt AG & Co., Nümbrecht, Germany) and assayed by an RIA tech-nique.

DLMO was defined as the time when plasma melatonin concen-trations rose from a low background level to above 10 pg/mL (26).The same 10-pg/mL threshold value was applied to the salivarymelatonin level because the level of melatonin in saliva is as abun-dant as that in plasma.

ACR was defined as time of peak in the 24/12-hour compositecosine model fitted to the z score standardized data using theChronoLab 3.0 (27). Cosinor p value for 24 hours. A p value of lesshan .05 was considered statistically significant.

etermination of � in the Sleep–Wake CycleTo assess � of the sleep–wake rhythm, times of the midpoint

leep were plotted against day number and subjected to linearegression. The slope of this line indicates the average daily shift inhe midpoint sleep. Hence, � is equal to 24 plus (or minus) the slopef the regression. We calculated � of the sleep–wake rhythm beforetarting the chronotherapy (pretreatment stage).

hronotherapy Procedure and Treatment Response AnalysisAll six FRT patients had undergone chronotherapy several times

t some point during the observation period before and after ap-lying the FD protocol. Chronotherapy consisted of high-intensity

ight therapy and the administration of melatonin or a melatonineceptor agonist (ramelteon) (Figure 2). According to the phaseesponse curves (28,29), light exposure and melatonin administra-ion were performed during the time periods (as described later)hen a phase shift can be expected. Ramelteon was administered

n accordance with melatonin, and these were all performed as anpen trial.

Less than 6 hours after waking up, patients were exposed toigh-intensity light (5000 – 8000 lux) for 2–3 hours at home or hos-ital. Patients took 1, .5, and .5 mg of melatonin 7, 5.5, and 4 hoursnd took 4 mg of ramelteon 7 hours before going to bed on therevious day. Drug trials were considered successful if the 95%onfidence interval of the regression coefficient included 0 (i.e., 24ours), and patients were considered to have responded to the

herapy if 50% of the drug trials were successful.Written consent was obtained from all participants following

xplanations about the study and its purposes. The study was ap-roved by the Ethics Committee of the National Center of Neurol-gy and Psychiatry.

tatistical AnalysisThe unpaired t test was used for comparisons of � between

atients and control subjects. One-way analysis of variance was (

sed for comparisons of � among the FRT patients, the I-type con-rols, and the E-type controls. Multiple comparisons using the Bon-erroni method were then conducted on those with observed mainffects, and significance probabilities were determined with appro-riately adjusted p values (30). Results are reported as mean � SEM.PSS version 11.5J (SPSS Japan Inc., Tokyo, Japan) was used fortatistical analysis, setting 5% for all critical probability levels.

esults

of Melatonin Rhythm in FRT PatientsThe DLMO, ACR, �DLMO, and �ACR in FRT patients and healthy

ndividuals during the first and second CR are shown in Table 2Figure S1 in Supplement 1, individual triple plots of melatoninhythm in the first CR and second CR; Figure S2 in Supplement 2,ndividual melatonin onset time in the first CR and second CR).

The mean DLMO and ACR (range) during the first CR were2.67 � 1.28 hours (20.05–.38 hours) and 3.68 � 1.08 hours (1.87–.27 hours) in I-type healthy individuals and .51 � 2.03 hours

21.22–3.20 hours) and 5.37 � 1.83 hours (2.67– 8.53 hours) in E-ype healthy individuals, respectively. The mean DLMO and ACR of-type individuals showed significant phase delays compared withhose of I-type individuals [DLMO: t (15) � 2.364, p � .032; ACR:(15) � 2.347, p � .033]. The DLMO and ACR of FRT patients duringhe first CR were distributed widely throughout the day and nightTable 2, Figure S2 in Supplement 2) because they had been free-unning before the FD protocol.

The �DLMO in FRT patients was 24.48 � .05 hours, which wasignificantly longer than the corresponding period, 24.17 � .05ours, in the 17 healthy individuals [t (21) � 3.535, p � .002]. Simi-

arly, the �ACR in FRT patients was 24.43 � .05 hours and thusignificantly longer than 24.18 � .05 hours in the healthy individu-ls [t (21) � 2.729, p � .013].

The � of melatonin rhythm showed distinct patterns in I- and-type individuals (Figure 3A; Figure S1 in Supplement 1). The �DLMO

nd �ACR in FRT patients ranged from 24.30 to 24.69 hours and 24.28o 24.64 hours, and their ranges seldom overlapped with those of-type individuals (23.95–24.31 hours and 23.90 –24.37 hours). Onhe other hand, the �DLMO and �ACR of E-type individuals rangedidely from 23.89 to 24.68 hours and from 23.90 –24.67 hours,

overing the � values of I-type individuals and FRT patients. There-ore, whereas the �DLMO and �ACR of FRT patients were significantlyonger than the �DLMO (24.12 � .04 hours) and �ACR (24.14 � .05ours) of I-type individuals (�DLMO: p � .004, �ACR: p � .025, Bonfer-

oni test, Figure 2B), the �DLMO and �ACR values of E-type individualsere 24.22 � .10 hours and 24.24 � .09 hours and were not signif-

cantly different from those of FRT patients (�DLMO: p � .051, �ACR:� .214, Bonferroni test).

haracteristics of Sleep-Wake Rhythms in FRT Patients andheir Treatment Response to Chronotherapy

Mean � of the sleep–wake rhythm before therapy was 25.18 �21 hours, ranging widely from 24.66 hours (Subject F5) to 25.87ours (Subject F4; Table 1, Figure 2). Before chronotherapy, � wasignificantly longer than � of melatonin rhythm [�DLMO: t (5) � 3.687,� .014; �ACR: t (5) � 3.909, p � .011], although there was no

ignificant correlation between the two [�DLMO: r � .435, p � .389;

ACR: r � .415, p � .413].The number of chronotherapy trials in FRT patients ranged from

(Subject F2) to 6 times (Subjects F3 and F4), and the number ofleep episodes per chronotherapy was 55.2 � 8.2. Three patients

Subjects F3, F4, and F6) responded to the therapy, and another

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66 BIOL PSYCHIATRY 2013;73:63–69 S. Kitamura et al.

Figure 2. Sleep logs of circadian rhythm sleep disorder, free-running type (FRT) patients (double plot). A part of the self-administered sleep diary kept by FRTpatients is shown (periods without recording are marked by a compressed horizontal line). The horizontal axis indicates a double 24-hour plot over 48 hours.

Black bars, sleep period; gray bars, incidence of drowsiness; hatched bars, period of melatonin or melatonin agonist (ramelteon) administration; open bars,period of light therapy. Patients F1 through F6 are presented in the order of longer to shorter � in melatonin rhythm as determined during the FD protocol.

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S. Kitamura et al. BIOL PSYCHIATRY 2013;73:63–69 67

three patients failed to achieve entrainment to the 24-hour cycleand maintained a free-running condition during the therapy.

Discussion

To our knowledge, this is the first study to use isolation experi-ments to assess accurately the presence of abnormal � in sightedpatients with FRT. The results of this study clearly indicate that � ofFRT patients was significantly longer than that of healthy individu-

Table 2. Circadian Phases and Circadian Periods in Case

Subject Sex Age at Study CR

FRTF1 F 17 21F2 F 31 6F3 M 20 21F4 F 31 10F5 F 56 19F6 M 35 7

Evening TypeE1 M 22 2E2 M 22 1E3 M 20 2E4 M 22 3E5 M 20 22E6 M 23 23E7 M 21E8 M 22 21

Intermediate TypeI1 M 19 21I2 M 23 23I3 M 21 23I4 M 22 22I5 M 22I6 M 20 22I7 M 24 23I8 M 22 23I9 M 39 20

ACR, acrophase; CR, constant routine; DLMO, dim ligh

Figure 3. Comparison of � of melatonin rhythm between free-running typeshift (Day 0 –Day 1) in �DLMO in FRT patients. Filled circles, FRT patients; gray tr

healthy control subjects with intermediate chronotype (I-type). (B) Mean � of mepatients showed longer � than that the I-type controls (p � .004, Bonferroni test)

ls who were in synchrony with the 24-hour cycle. This is in line withhe pathophysiology-based hypothesis (6,15) that long � is a riskactor for the FRT phenotype associated with a daily delay in theleep phase.

Species-specific � is known to determine free-running periodength and phase angle differences between circadian rhythm andnvironmental light– dark cycle (31). Klerman et al. have shown thatlonger � could be a promoting factor for phase delay in circadian

Controls

latonin Onset Melatonin Peak

CR 2 �DLMO CR 1 CR 2 �ACR

4.10 24.69 3.40 9.13 24.6411.25 24.52 12.80 16.80 24.44

1.70 24.51 2.20 5.93 24.4115.05 24.49 15.93 20.13 24.4723.09 24.39 1.07 4.20 24.3510.55 24.30 13.73 16.27 24.28

5.50 24.36 6.87 10.07 24.363.08 24.23 5.40 7.67 24.251.14 23.89 6.13 6.07 23.999.36 24.68 8.53 14.60 24.672.64 24.44 3.73 8.33 24.51

22.78 23.93 4.87 4.00 23.90.88 24.08 4.73 5.27 24.06

22.51 24.14 2.67 3.93 24.14

21.00 23.95 1.87 1.00 23.9023.63 24.03 4.47 5.13 24.07

.71 24.17 3.53 5.47 24.2123.28 24.10 3.67 4.60 24.10

.40 24.00 5.27 5.60 24.0423.43 24.10 3.67 4.80 24.13

1.71 24.26 4.13 6.93 24.312.20 24.31 4.33 7.67 24.37

21.36 24.14 2.20 3.13 24.10

latonin onset; F, female; FRT, free-running type; M, male.

patients and healthy control subjects. (A) Horizontal axis represents a dailys, healthy control subjects with evening chronotype (E-type); open squares,

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latonin rhythm in I-type and E-type healthy controls and FRT patients. FRT. DLMO, dim light melatonin onset.

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rhythms under the entrained condition by computer simulatedquantitative model (32), which was verified by the finding thatE-type individuals often have longer � in humans (20 –22). Deduc-ively, more severely elongated � is thought to be a risk factor foresynchrony as the further � drift away from 24 hours, the larger theagnitude of a phase shift required for the entrainment to the

xternal environment.A previous study showed that although 6 of 7 blind subjects

hose � was shorter than 24.6 hours could achieve entrainment byrally administered melatonin, the last subject who had the longest(24.9 hours) could not (33). Another study also demonstrated that

he incidence of free-running sleep–wake rhythms was higher inlind individuals with longer � (�24.5 hours) than those with thehorter � (88% vs. 38%, respectively) (12). The results of our studyuggest that even in individuals with no visual impairment, longer �s a risk factor for the onset of FRT. The sighted patients with FRT

ho participated in our study were apparently unable to compen-ate for the daily phase shift even under the 24-hour cycle of envi-onmental light exposure.

In this study, we obtained preliminary data on the relationshipetween � of melatonin rhythm and patient response to chrono-

herapy. Although a linear relationship was not observed betweenhe two because of the small sample size, this study revealed thatwo patients (Subjects F1 and F2) with longer � did not respond tohronotherapy and one patient (Subject F6) with the shortest � did.nother patient (Subject F5) with the second shortest period was

egarded as nonresponsive; however, this patient did achieve en-rainment to a 24-hour cycle in 1 of 3 trials, and in the other 2 trials,he cycle of the sleep–wake rhythm became shorter (Figure 2). If theelationship between � and chronotherapy response is verified inuture studies, individuals’ � could hold promise as a physiologic

arker for the outcome of FRT.This study also indicates that long � in FRT patients is not the

nly factor that influences the free-running of sleep–wake cycles inreal-life setting. The lengths of � in both E-type individuals and FRTatients were greatly overlapped, although these E-type individu-ls were in synchrony with the 24-hour cycle. This suggests that inddition to long �, other factors that make entrainment to the4-hour cycle difficult might be related to the onset of FRT as well ashe modification of sleep–wake rhythm in a real-life setting. Oneossible factor is a functional decline in entrainment mechanisms.he prevalence of FRT is high in individuals with visual impairment,ypically with blindness (11). Although our FRT patients had nolinical signs of visual impairment or morphologic abnormalities,his does not necessarily guarantee the intactness of retinohypo-halamic tract projecting from the melanopsin-containing ganglionells in the retina to the hypothalamus, primary pathway for photicntrainment (34). Further systematic studies are needed to clarifyhether any functional decline in light sensitivity is present in

ighted FRT patients.It also appears that the temporal relationship (phase angle)

etween the external environment and biological rhythms is al-ered in FRT patients. The real-life � of the sleep–wake cycles of FRTatients was significantly longer and varied with time than � of theelatonin rhythm measured under the FD experimental condition

Figure 2). Previous studies have shown that sleep onsets under thesolated condition frequently occur around falling limb to nadirime of core body temperature rhythm (35,36), which is coincidentith the transition from the phase delay to advance portion of the

ight phase response curve (37). Theoretically, there is more oppor-unity for sighted FRT patients to be exposed to environmental lightn the phase delay portion and less opportunity on the phase

dvance portion (eye close in sleep time) in self-selected light– dark

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chedules. Klerman et al. reported that decreased light exposureuring the 6 hours after the nadir time of core body temperature

hythm lead to prolonged � observed in self-selected light– darkchedules under a 150-lux light intensity, compared with that mea-ured in the FD experimental condition by computer simulation32). If this is the case for sighted patients with FRT, a prolongedeal-life � of their sleep–wake cycles may be caused by nonuniformxposure to daily environmental light across the whole circadianhases. In support of this, Uchiyama et al. (38) found that the rise inleep propensity was delayed relative to the melatonin phase inighted FRT patients. This functional desynchrony may promote ahase delay of biological rhythms, which in turn results in a dailyelay in the sleep phase. Similar results have been reported ineveral case studies (15,39,40). Taken together, these findings sug-est that, in addition to long �, declined sensitivity (entrainmentbility) toward the ambient light or inappropriate timing (or insuf-cient amount) of light exposure may play a multifactorial andecondary role in the onset of FRT.

There are some limitations to this study. First, sex and age wereot matched between patients and healthy control subjects. FRTatients who participated in this study were significantly older than

he control subjects [31.7 � 13.8 years vs. 22.6 � 4.4 years, t (21) �.461, p � .023] and included four women (66.7%). Although noffect of age difference (23.7 vs. 67.4 years) on � was observed in arevious study using the FD protocol (16), Duffy et al. (18) reported

hat � in women was slightly shorter (.1 hours) than that in men.ven if the latter finding is correct, it would not have played a role inhe prolonged � seen in the FRT patients in the present study. Wehus speculate that the average age difference of 10 years and sexifference would not have resulted in the significantly longer �btained in the present study. Second, the menstrual cycle of the

emale participants was not controlled. It is difficult to conduct a-week experiment while female participants are either in the fol-

icular or luteal phase. One subject had reached menopause, butenstruation started in the three other female subjects between

xperimental Days 4 and 7 after the first CR. The potential role ofstrogen in shortening � was previously shown in a diurnal animal,he hamster (41). Even if estrogen plays a similar role in humans, itould not have played any role in the prolonged � seen in the

emale FRT patients in our study.In conclusion, this is the first study to have accurately assessed �

f sighted FRT patients using the FD protocol. It was found that � inighted FRT patients was significantly longer than that in healthyndividuals. However, no significant difference in � was observedetween FRT patients and healthy individuals with an E-type diur-al preference. These findings suggest that not only a prolongationf � but also entrainment mechanisms and patterns of light expo-ure are potentially involved in the multifactorial structure of theathogenesis of FRT.

This study was supported by a Grant-in-Aid for the Strategic Re-earch Program for Brain Sciences from the Ministry of Education, Cul-ure, Sports, Science and Technology of Japan (Understanding of Mo-ecular and Environmental Bases for Brain Health), an Intramuralesearch Grant (No. 23-3) for Neurological and Psychiatric Disorders ofational Center of Neurology and Psychiatry, and a Grant-in-Aid forcientific Research (Grant No. 21390335) from Japan Society for theromotion of Science.

The authors report no biomedical financial interests or potentialonflicts of interest.

Supplementary material cited in this article is available online.

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