Background
Japanese students suffering from psychosomatic disorders, such as those involving mood and sleep, may exhibit basic problems in their lifestyle, including deleterious changes in their living environment and dietary or lifestyle disturbances
A circadian pacemaker in the central nervous system regulates human sleep cycles, hormone secretion, subject alertness, objective performance levels and other physiologic functions over a 24-h period. Core body temperature, plasma cortisol, and plasma melatonin are three variables frequently used to estimate the phase of the human pacemaker
Carroll et al.
The purpose of the present study was to investigate individual variation in the levels of growth hormone in healthy subjects and to examine the relationship between an individual's hormone profile and his or her psychosomatic complaints.
Methods
The subjects and self-assessment questionnaire
Fifteen subjects (4 men and 11 women) without major medical disorders ranging in age from 21 to 22 years participated in this study. The study design was approved by the Ethics Committee of Chiba University, Japan, and informed consent was obtained from all subjects. A self-assessment questionnaire concerning psychosomatic symptoms was developed in accordance with data from the Health Behavior in School-Aged Children (HBSC) study of the WHO
Morning and evening psychosomatic condition scores collected from the self-assessment psychosomatic complaint questionnaire
High Self-Assessment Group (n = 7)
Low Self-Assessment Group (n = 8)
Morning Comparison
Morning
(08:00 h)
Evening
(20:00 h)
Morning
(08:00 h)
Evening
(20:00 h)
• Physical symptoms
1. Drowsiness
3.14
1.43
3.38
1.50
-0.67
2. Poor appetite
2.14
1.86
2.25
1.38
-0.30
3. Heaviness in the head
1.14
1.29
1.38
1.13
-1.00
4. Dizziness
1.00
1.00
1.75
1.25
-3.00*
5. Whole-body fatigue
2.00
1.57
2.63
1.75
-1.49
• Mental symptoms
6. Lack of motivation
2.00
2.00
2.75
2.13
-1.82
7. Easily irritated
1.00
1.14
1.50
1.50
-2.65*
8. Feelings of melancholy
1.00
1.14
1.88
2.13
-2.97*
9. Desire to rest
2.00
2.00
3.00
2.50
-2.45*
10. Anxiety
1.14
1.57
2.50
3.00
-3.80**
total
19.00
16.60
32.75
23.00
-3.96**
The scores shown are means. Each scale ranges from 1 to 4, with higher scores indicating a greater degree of psychosomatic symptoms (No, Somewhat No, Somewhat Yes, Yes).
**p < 0.01; *p < 0.05
Sample collection
Saliva was collected into Salivette tubes (Sarstedt, Germany) using polyester swabs from the subjects' mouths following 2 min of chewing. Samples were collected five times a day at home (20:00, 24:00, 04:00, 08:00, and 12:00 h). Both the day of sampling and the preceding day were required to be normal days (i.e., without special events or stressful circumstances). After sample collection, the saliva was stored at -20°C until being analysed.
Salivary growth hormone assay
On the day of testing, the samples were centrifuged at 3,000 rpm for 10 min to remove all mucin. A standard fluorescent determination immunoassay was used to assess the growth hormone concentrations in each sample. To avoid inter-assay variability, all determinations were performed in a single series. In the first step, 96-well fluoro-nunc plates (Nunc, Black MicroWell 137101, Denmark) were pre-coated with 100 μl of anti-growth hormone antibody (Quartett, 2071800210, Germany) and incubated for 1.5 h at room temperature. After incubation, the plate was washed three times with phosphate-buffered saline and blocked for 1 h. After washing, 100 μl of saliva or a standard solution was dispensed into each well and left for 1.5 h. After washing, primary antibody (Funakoshi, FU47500254, Japan) was added to the plates and incubated for 1.5 h. Next, incubation with a secondary antibody (Novus Biologicals, NB120-7112, USA) was performed for 1 h. After washing, rabbit anti-ovine immunoglobulin (Amersham Biosciences, ECF Western Blotting Reagent Pack, USA) was added. After 20 min of incubation, the plate was scanned using a Fluoromark Microplate Fluorometer (Bio-Rad, USA) with excitation at 485 nm and emission at 590 nm.
Data analysis
The significance of differences between group means was tested by analysis of variance (ANOVA), followed by protected
Results
Self-assessment psychosomatic complaints questionnaire
A factorial repeated measures ANOVA (high/low self-assessment versus morning/evening self-assessment scores) was conducted. ANOVA results showed significant differences (p < .05) between morning and evening self-assessment scores. Post hoc analyses revealed that, as shown in Table
Assessment of daily rhythmicity of salivary growth hormone secretion
We collected saliva profiles from 15 healthy students (4 men and 11 women). The amplitude of salivary growth hormone, defined as the difference between the highest and lowest salivary concentrations, was used to produce a standardisation, or Z, score. As shown in Figure
Daily variation in salivary level of growth hormone in the high and low self-assessment groups
Daily variation in salivary level of growth hormone in the high and low self-assessment groups. The results are presented as means ± S.E.M. * p < .05.
Discussion
Psychosocial factors have been previously shown to affect the psychosomatic symptoms reported by Japanese school children
Our assessment of salivary growth hormone secretion was not sensitive enough to detect significant daily rhythmicity, but the highest level measured in the subjects of our high self-assessment group occurred earlier in the day than the peak of the daily rhythm measured in a previous study
Our results indicate a relationship between the self-assessment scores and the levels of growth hormone. Subjects with high self-assessment scores in the morning showed significant variability in growth hormone secretion during the day, whereas subjects with low self-assessment scores did not. Thus, psychosomatic symptoms may be associated with hormonal rhythms related to basic lifestyle habits.
Nocturnal melatonin secretion can be suppressed by exposure to light on the order of several hundred lux, such as ordinary room light
Some limitations of our study must be emphasized. First, it is possible that the sleep-disrupting effect of waking at 00.00 and 04.00 to produce a saliva sample had a disruptive effect on hormonal secretion. Second, we observed a larger difference in terms of gender than has been previously described
Conclusion
Psychosomatic symptoms may be associated with circadian dysfunction, as inferred from blunted rhythmicity in growth hormone secretion.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MN designed the experiments, collected data and wrote the manuscript. KY managed the laboratory and adjusted the schedule of subjects. SW participated in the design of the study and performed statistical analysis. MN supervised the study. All authors read and approved the final version of the article.