Introduction

In our previous study, circadian rhythm sleep disorders have been reported in pediatric and adolescent populations (Tomoda, Miike, Uezono, & Kawasaki, 1994). Pediatric practitioners now commonly encounter sleep disturbance in previously healthy children and adolescents (Boergers, Hart, Owens, Streisand, & Spirito, 2007; Giannotti, Cortesi, Sebastiani, & Ottaviano, 2002; Stein, Mendelsohn, Obermeyer, Amromin, & Benca, 2001). The characteristic clinical features are well known, but the specific causes remain unknown. New types of circadian rhythm sleep disorders, such as familial advanced sleep phase syndrome (ASPS) and delayed sleep phase syndrome (DSPS), non-24-h sleep-wake syndrome (non-24), and morningness-eveningness have been described during the last decade. Such disorders are probably caused by various disturbances of circadian expression of the clock gene (Archer et al., 2003; Ebisawa et al., 2001; Iwase et al., 2002; Pirovano et al., 2005; Takimoto et al., 2005; Toh et al., 2001; Wijnen, Boothroyd, Young, & Claridge-Chang, 2002). Polymorphisms in clock genes are known to induce circadian rhythm sleep disorders. For example, mutations in the period2 (Per2) gene (S662G) or casein kinase1 d (CK16) gene (T44A) cause familial ASPS; furthermore, missense polymorphisms in the Per3 (V647G) and CK1e (S408N) genes increase or decrease the risk of developing DSPS.

In our clinical practices, we recognized that the majority of our patients have a circadian rhythm disorder even though they usually do not mention or recognize this problem at the first interview. We hypothesized that there could be certain relationship between biological rhythm disorders in these patients and their indefinite symptoms as well as their sleep disturbances. This chapter introduces sleep patterns, circadian rhythms of core body temperature (CBT), glucose metabolism, and human clock gene profile in children and adolescents with sleep disturbance.

2. Methods 2.1 Protocol

This study included 22 unmedicated patients with sleep disturbances (Table 1). All patients satisfied diagnostic criteria for circadian rhythm sleep disorders of the Diagnostic and Statistical Manual of Mental Disorders, 4th Edition, Text Revision (DSM-IV-TR®). The diagnosis was made by three raters using the Structured Clinical Interview. The severity of those symptoms was measured using self-reported ratings (performance status scores), as described previously {Kuratsune, 2002 #890;Tomoda, 2007 #925}. Their performance status scores on admission were higher than 5 (mean, 5.6; SD, 0.8).

For at least one month prior to the initial assessment, prophylactic drugs (e.g. tranquilizers) were not given. Patients who had just recently started treatment with antidepressants or hypotension drugs, or who were diagnosed as having neurological illness, migraine, obstructive sleep apnea, below average intelligence, or serious psychopathology were excluded from the study. Serious psychopathology was evaluated by referral to at least one psychiatrist if the patient presented with some indicative symptoms. No patient had a history of drug abuse. Table 1 presents physical characteristics of the present subjects. The protocol was approved by the Committee of Life Ethics, Graduate School of Medicine, Kumamoto University. All participants gave written informed consent.

p-value: significant difference in ANOVA.

Table 1. Circadian rhythm of core body temperature: Results of a cosinor analysis.

2.2 Recording of the sleep-wake rhythm

Each subject kept daily recordings (logs) of their time of sleeping and awaking for 4 or longer weeks. These logs were used to analyze their sleep pattern during a 24-hour period. According to the International Classification of Sleep Disorders (ICSD) revised by the Association of Sleep Disorders Center in North America in 1990 (Diagnostic Classification Committee, 1990), our patients were diagnosed as either delayed sleep phase syndrome (DSPS), non-24-hour sleep-wake syndrome (non-24), irregular sleep, or long sleeper. DSPS is characterized by difficulty in falling asleep at night and an inability to be easily aroused in the morning, and this diagnosis corresponds to DSM-III-R: Sleep-Wake Schedule Disorder. Non-24 presents sleep-wake cycles longer than 24 hours, and this corresponds also to DSM-III-R: 307.45. Irregular sleep is characterized with no recognizable circadian patterns of sleep onset or waking time, and this does not correspond a sleep disorder diagnosis in DSM-III-R. Long sleeper have sleep times longer than 9 hours although they do not have any organic abnormalities, and this correspond to DSM-III-R: 780.54.

2.3 Circadian rhythm of core body temperature

Continuous monitoring of CBT for 3 days and at every one minute was carried out by using a deep body temperature monitor (Terumo Corp., Tokyo, Japan).

Mean values of the 3 measurements at each time point during the 3 consecutive days were used in the examination. A chronograph was used to determine the circadian rhythm, and the single cosinor method, to analyze the CBT circadian variation for both groups (Halberg et al. 1977). A cosine curve with a period of 24 hours was fitted to the data by using the least squares method, and the following parameters were obtained: mesor (°C, rhythm-adjusted average), amplitude (difference between the highest and lowest temperature), and acrophase (time of the highest point in the rhythm defined by a fitted cosine curve). To obtain data in normal age-matched persons, we recruited 9 healthy school children as volunteers. They were 6 males and 3 females, aged 10-21 years (mean age, 17.3 years), and who had no mental retardation, physical problems, or psychiatric psychopathology. In statistical analysis, ANOVA was used, and when the p-value was less than 0.05, the group difference was considered to be statistically significant.

2.4 Hormonal secretion profiles

Melatonin, cortisol, fi-endorphm and temperature circadian rhythms. 24-hour blood sampling was performed through an indwelling catheter in a forearm vein at 4-hour intervals. Each blood sample was immediately centrifuged at 4°C and stored at -80°C until melatonin, cortisol and 6-endorphin were assayed by radioimmuno assay (RIA). The lower limit of melatonin sensitivity was determined to be 3 pg/ml.

Comparative data concerning the timing of hormonal production were obtained for a group of six normally-sighted healthy male volunteers aged 20-22 years (mean age, 20.6 years) who had no mental retardation or serious psychopathology.

The recordings of the deep body temperature were carried out with a deep body temperature monitor (Terumo Co., Tokyo, Japan) below Lanz's point every 1 minute for three consecutive days for the patient and the control group.

Both a chronograph and the single cosinor method were used to examine the rhythmicity and to analyze the circadian variation.

A cosine curve with a period of 24 hours was fitted to the data using the least squares method, and the following parameters were established; mesor (rhythm-adjusted mean), amplitude (difference between mesor and nadir) and acrophase (lag of the crest time in the best fitted cosine curve in relation to a given reference time). When the p-value was less than 0.05, the rhythm was considered to be statistically significant.

2.5 Evaluation of carbohydrate metabolism

A 3-h oral glucose tolerance test was performed the morning after a subject had fasted overnight. After the fasting blood sample was drawn, a subject was given a solution containing a predetermined amount of glucose based on body weight (1.75 g/kg to a maximum of 75 g). After glucose ingestion, blood samples were drawn at 30, 60, 90, 120, 150, and 180 min to measure blood glucose (BG) levels and immunoreactive insulin (IRI) response. Serum BG level was determined using the glucose oxidase reaction method. Serum IRI response was measured using radioimmunoassay (Eiken Chemical Co. Ltd., Tokyo, Japan). The BG levels, IRI response, cumulative BG (sigma BG), cumulative IRI (sigma IRI), insulin/ glucose ratio (delta IRI/delta BG), and insulinogenic index (sigma IRI/sigma BG)

were then compared to normal control data that had been reported previously for 8 subjects aged 12-16 years without a personal or family history of diabetes mellitus or any factor affecting glucose metabolism (Iwatani et al., 1997). The control subjects were within ±2.0 SD of standard height, and within ±20% of ideal body weight. All indices were calculated using the same methods as those reported previously (Iwatani et al., 1997).

2.6 Experimental procedure for human clock gene measurement

Subjects were exposed to natural and fluorescent lighting of the institution during the awake period. Lights were turned off during the sleeping period. An indwelling catheter was placed in the antecubital vein for a 24-h period. Blood samples were taken at 4-h intervals beginning at 10:00 a.m. on the second day of hospitalization and continued until 6:00 a.m. of the following day. Samples were obtained under dim light (less than 30 Lux) without waking the patients during the sleeping period. We previously reported that subjects 12 years of age and older show similar metabolic characteristics to those of an adult (Iwatani et al., 1997). Therefore, we recruited 10 men aged 20-41 years (mean age, 27.4 years; SD, 6.1 years) as normal subjects from whom data were obtained (Reppert & Weaver, 2002; Takimoto et al., 2005): none had below-average intelligence, physical problems, psychiatric psychopathology, or irregular sleep or meal schedules.

Blood was collected in blood RNA kit tubes (PAXgene; Qiagen K.K., Tokyo, Japan). The tubes were incubated at room temperature for 24 h; then the total ribonucleic acid (RNA) was isolated according to the manufacturer's instructions. For quality assessment of total RNA during protocol development, deoxyribonucleic acid (DNA) digestion of the samples was performed with the RNase-Free DNase Set (Qiagen K.K.). Synthesis of complementary DNA was conducted (ReverTra Ace-a-®; Toyobo Co. Ltd., Osaka, Japan) for use with the reverse-transcription polymerase chain reaction (RT-PCR) kit. Quantitative real-time RT-PCR (TaqMan®) was performed using a sequence detection system (ABI PRISM® 7900; Applied Biosystems, Foster City, CA) to determine the expression levels of hPer1, hPer2, hPer3, hBmal1, hClock, and housekeeping gene hp-actin expression relative to hpactin, with the standard protocol described by the manufacturer. Relative expression of the clock gene was determined as the ratio of expression of the clock gene to that of the P-actin gene for each sample. Values were normalized so that the peak value equaled 100%. The TaqMan® hpactin control reagents and primer sets, Assays-on-DemandTM Gene Expression Product for hPer1, hPer2, hPer3, hBmal1, and hClock were purchased from Applied Biosystems for the following: hPer1, Hs00242988_m1; hPer2, Hs00256144_m1; hPer3, Hs00213466_m1; hBmal1, Hs00154147_m1; hClock, Hs00231857_m1. In addition, hPer2 was selected as the daily expression of the clock gene for determination of the circadian profile (Takimoto et al., 2005).

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