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Randomised controlled trial of the effects of L-ornithine on stress markers and
sleep quality in healthy workers
Nutrition Journal 2014, 13:53 doi:10.1186/1475-2891-13-53
Mika Miyake (m-miyake@kirin.co.jp)
Takayoshi Kirisako (Takayoshi_Kirisako@kirin.co.jp)
Takeshi Kokubo (Takeshi_Kokubo@kirin.co.jp)
Yutaka Miura (yu-miura@kirin.co.jp)
Koji Morishita (koji.morishita@kyowa-kirin.co.jp)
Hisayoshi Okamura (okamura_hisayoshi@med.kurume-u.ac.jp)
Akira Tsuda (tsuda_akira@kurume-u.ac.jp)
ISSN 1475-2891
Article type Research
Submission date 8 January 2014
Acceptance date 14 May 2014
Publication date 3 June 2014
Article URL http://www.nutritionj.com/content/13/1/53
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Randomised controlled trial of the effects of L-
ornithine on stress markers and sleep quality in
healthy workers
Mika Miyake1*
* Corresponding author
Email: m-miyake@kirin.co.jp
Takayoshi Kirisako1
Email: Takayoshi_Kirisako@kirin.co.jp
Takeshi Kokubo1
Email: Takeshi_Kokubo@kirin.co.jp
Yutaka Miura1
Email: yu-miura@kirin.co.jp
Koji Morishita2
Email: koji.morishita@kyowa-kirin.co.jp
Hisayoshi Okamura3
Email: okamura_hisayoshi@med.kurume-u.ac.jp
Akira Tsuda4
Email: tsuda_akira@kurume-u.ac.jp
1 Research Laboratories for Health Science & Food technologies, Research &
Development Division, Kirin Co., Ltd., 1-13-5, Fukuura, Kanazawa-ku,
Yokohama, Kanagawa 236-0004, Japan
2 Healthcare Products Development Center, Kyowa Hakko Bio Co., Ltd., 2,
Miyukigaoka, Tsukuba, Ibaraki, 305–0841, Japan
3 Cognitive and molecular of Brain disease, Kurume University, 67, Asahi-machi,
Kurume, Fukuoka 830-0011, Japan
4 Department of Psychology, Kurume University, 1635, Miimachi, Kurume,
Fukuoka 839-8502, Japan
Abstract
Background
L-ornithine is a non-essential, non-protein amino acid. Although L-ornithine is contained in
various foods, the amount is usually small.
Recently, studies have shown that orally administered L-ornithine reduced the stress response
in animals.
From these findings, we speculated that L-ornithine may play a role in the relieve of stress
and improve sleep and fatigue symptoms in humans. Through a randomised, double-blind,
placebo-controlled clinical study, we asked if L-ornithine could be beneficial to stress and
sleep in healthy workers.
Method
Fifty-two apparently healthy Japanese adults who had previously felt slight stress as well as
fatigue were recruited to be study participants and were randomly divided into either the L-
ornithine (400 mg/day) or placebo group. They orally consumed the respective test substance
every day for 8 weeks. Serum was collected for the assessment of cortisol and
dehydroepiandrosterone-sulphate (DHEA-S). Perceived mood and quality of sleep were
measured by the Profile of Mood States (POMS), Athens Insomnia Scale (AIS), and Ogri-
Shirakawa-Azumi sleep inventory MA version (OSA-MA).
Results
Serum cortisol levels and the cortisol/DHEA-S ratio were significantly decreased in the L-
ornithine group in comparison with the placebo group. Also, anger was reduced and
perceived sleep quality was improved in the L-ornithine group.
Conclusion
L-ornithine supplementation has the potential to relieve stress and improve sleep quality
related to fatigue, both objectively and subjectively.
Keywords
L-ornithine supplementation, Stress and sleep, Clinical trial
Background
Sleep is both necessary and universal. Like eating and drinking, without it we will eventually
die. Sleep is affected by various psychosocial factors, stress, and the daily routine, and it
similarly affects all areas of life. Sleep is reported to be an important mediator of health
through the autonomic nervous and immune systems and endocrine function [1-5].
Sleep and fatigue often correlate highly in cross-sectional studies, and reduced sleep duration
involves the gradual accumulation of sleepiness/fatigue [6]. Fatigue is generally considered to
be important in daily life as it is closely related to quality of life and well being. Chronic or
accumulated fatigue plays a role in an individual’s performance of various functions. At
worst, long-term accumulated fatigue can lead to karoshi (death from overwork) [7].
Nevertheless, fatigue is a complex multidimensional concept that involves physical and
psychosocial aspects. Psychological fatigue is closely associated with stress [8,9].
Stress has been shown to induce a physiological response that is mediated by the
hypothalamic-pituitary-adrenal (HPA) axis leading to the release of cortisol in humans and
corticosterone in mice [10]. Cortisol has long been used as a marker of stress [11].
Dehydroepiandrosterone (DHEA) and its sulfate ester (DHEA-S) are the most abundant
adrenal androgens. They are produced as precursors to the sex hormones estradiol and
testosterone from adrenal glands and their serum levels decrease with age [12-14]. DHEA-S
has cortisol-lowering effects [15] and may attenuate the adverse health effects of
hypercortisolism [16]. DHEA-to-cortisol ratios in serum and saliva are likely to be more
reliable than concentrations of either hormone alone, with lower morning ratios seen in
depression [17-19].
Recently it was reported that orally administered L-ornithine reduced restraint stress-induced
activation of the HPA axis in mice accompanied by a reduction in the serum corticosterone
concentration [20]. It was shown that an intracerebroventricular (i.c.v.) injection of L-
ornithine attenuated the stress response in neonatal chicks. These actions were suggested to
be mediated by the gamma-aminobutyric acid (GABA) receptor.
From these findings, we speculated that L-ornithine may play a direct role in the central
nervous system, relieve stress and improve sleep and fatigue symptoms in humans. L-
ornithine is a non-essential, non-protein amino acid. Although L-ornithine is contained in
various foods, the amount is usually small. Since ancient times, corbicula, a genus of the
clam, has been considered to be good for the liver. They were found to contain 159.9 mg L-
ornithine per 100 g of the extract [21], which is high compared with other foods [22] but still
a relatively small amount.
Orally administered L-ornithine is transferred to the portal vein from the intestines and
delivered to various tissues, such as the liver, kidney and muscle [23]. In liver, L-ornithine
plays a central role in the urea cycle which converts ammonia to urea [24]. L-ornithine
administration has been known to enhance detoxification of ammonia in the liver [25].
Using a randomised, double-blind, placebo-controlled clinical trial, we evaluated the effect of
long-term ingestion of L-ornithine on stress-related markers in serum and subjective feelings
associated with stress and sleep in study subjects who indicated feelings of slight fatigue.
Methods
Study design
This study was a randomised, double-blind and placebo-controlled trial. Subjects were
randomly allocated to either the L-ornithine group or placebo group.
Most previous studies have evaluated the short term effect of L-ornithine supplementation on
healthy volunteers [26,27]; we investigated the long term effect of L-ornithine
supplementation on healthy volunteers. Eight weeks was chosen as the study period after
consideration of the seasonal effect and the volunteer's burden.
Subjects ingested either L-ornithine or placebo capsules before going to bed every day for 8
weeks. Blood was collected four times during this clinical trial: before supplementation (0w),
corresponding to the screening evaluation; 2 weeks (2w) and 4 weeks (4w) after taking the
supplement; and at the end of the trial (8w).
We used the following three questionnaires to evaluate perceived stress, sleep quality and
mood state: POMS [28], Athens Insomnia Scale (AIS) [29] and Ogri-Shirakawa-Azumi sleep
inventory MA version (OSA-MA) [30]. The POMS questionnaire is a well established,
factor-analytically derived measure of psychological distress, such as mood, for which high
levels of reliability and validity have been documented. We used the short-form POMS,
which consists of thirty adjectives rated on a 0–4 scale that can be consolidated into six mood
scales: “tension-anxiety”, “depression-dejection”, “anger-hostility”, “vigor”, “fatigue” and
“confusion”. The T score of the POMS questionnaire was calculated using the standard
method [28], showing that higher T scores represent high levels of distress, with the
exception of “vigor”. This questionnaire was completed by each study participant at 0, 2, 4, 6
and 8 weeks.
The AIS is a useful tool to assess the existence of insomnia. This self-administered
psychometric instrument consists of eight items: difficulty with sleep induction, awakening
during the night, early morning awakening, total sleep time, overall quality of sleep,
problems with sense of well-being, and functioning, and sleepiness during the day. Each item
was rated on a scale of 0 to 3, with 0 corresponding to “no problem at all” and 3 indicating “a
very serious problem”. Thus, the total AIS score ranges from 0 (denoting absence of any
sleep-related problem) to 24 (representing the most severe degree of insomnia). Volunteers
completed this questionnaire at 0, 4, and 8 weeks.
The OSA sleep inventory is popularly used for evaluation of sleep quality in Japan. The MA
version is more useful for middle-aged and old-aged people and consists of sixteen adjectives
with responses rated on a 0–4 scale that can be consolidated into five factors: “sleepiness on
rising”, “initiation and maintenance of sleep”, “frequent dreaming”, “refreshing” and “sleep
length”. The OSA-MA score was calculated using an MS-Excel sheet [30], with higher
scores indicating a good quality of sleep: this questionnaire was completed weekly for 8
weeks.
The present study was conducted according to the guidelines laid down in the Declaration of
Helsinki and all procedures involving human subjects were approved by the local ethics
committee of Medical Corporation, Akihabara Medical Clinic. Written informed consent was
obtained from all participants.
Study population
Fifty-two apparently healthy Japanese individuals participated in this study. The volunteers
ranged in age from 30 to 60 years (male : female ratio, 2:3), and had full-time jobs, excluding
those who engaged in shift work or physical work, such as carpenter or delivery person, and
irregular work schedules. Subjects were selected based on their POMS questionnaire score,
which should be above 50 in T scores of “fatigue” and below 50 in T scores of “vigor”.
Smokers, pregnant or lactating women, or persons who habitually took L-ornithine,
medication or supplements to improve stress, fatigue or sleep were excluded from this study.
Also excluded were those with a past history of diabetes, hepatic disease, renal disease,
hypertension, ischemic heart disease or abnormal glucose tolerance.
Test substance
L-Ornithine monohydrochloride and microcrystalline cellulose (FD-301) were purchased
from Kyowa Hakko Bio (Tokyo, Japan) and Asahi Kasei Chemicals (Tokyo, Japan),
respectively. Two kinds of small hard capsules were prepared, with one type of capsule
containing 500 mg of L-ornithine monohydrochloride (400 mg L-ornithine) and 160 mg of
microcrystalline cellulose per 2 capsules and the other (placebo) containing 560 mg of
microcrystalline cellulose per 2 capsules.
Measurement of serum cortisol and DHEA-S
Participants were instructed not to eat breakfast after 8:00 am and abstain from smoking and
caffeinated drinks prior to blood sampling. Blood samples were collected by venipuncture
from all participants between 11:00 and 13:00. Serum cortisol and DHEAS levels were
analyzed by commercial laboratories (BML Inc., Tokyo, Japan).
Statistical analysis
Values are presented as the mean ± standard error (SEM). Two-way analysis of variance was
used to evaluate the significance of differences between the placebo and L-ornithine groups
followed by pairwise comparisons and unpaired t tests. P values less than 0.05 were
considered to be statistically significant.
Results
Background information or demographic data
There were no statistical differences between the two groups at baseline (Table 1). During the
8 study weeks of the study, no adverse events were observed.
Table 1 Characteristics of subjects at 0 week
Placebo group
Ornithine group
men (n) 11 10
women (n) 15 16
Average SE Average SE
Age (years) 43.38
1.56
43.31
1.46
POMS tension-anxiety 63.35
1.66
65.23
1.99
depression-dejection 65.69
2.32
64.19
2.26
anger-hostility 61.85
2.29
63.31
2.28
vigor 35.62
1.05
36.88
1.12
fatigue 66.96
1.26
68.77
1.28
confusion 68.31
1.85
69.77
1.92
AIS 7.96
0.61
8.54
0.54
OSA sleepiness on rising 9.37
0.87
7.98
1.10
initiation and maintenance of sleep 15.21
1.34
12.12
1.17
frequent dreaming 21.73
1.62
23.04
1.40
refreshing 10.92
1.09
10.27
0.88
sleep length 13.31
1.11
14.71
1.40
cortisol (µM) 0.21
0.02
0.23
0.01
DHEA-S (µM) 4.25
0.32
4.72
0.44
cortisol/DHEA-S x 100 5.22
0.40
5.67
0.56
Most of the study subjects were general office workers with 24 out of 26 participants in the
placebo group, and 23 out of 26 participants in the L-ornithine group.
L-Ornithine supplementation improved mood state in study participants
Changes in POMS scores are shown in Figure 1. There was a trend toward an improved
mood indicated by each score compared to 0 weeks but there was no significant difference
between the two groups. There was a significant improvement in self-reported “anger-
hostility” at 2 weeks and 6 weeks in the L-ornithine group compared to the placebo group
(Figure 1C).
Figure 1 Effect of L-ornithine supplementation on POMS. Means of the change from 0
weeks of each POMS score (A, tension-anxiety; B, depression-dejection; C, anger-hostility;
D, vigor; E, fatigue; F, confusion) to 2, 4, 6 and 8 weeks: mean ± SE. White circles (○)
indicate the placebo and black circles (●) indicate L-ornithine.
L-Ornithine supplementation improved sleep quality in study participants
As shown in Figure 2, the AIS score revealed a trend towards improved insomnia in both
groups from 0 weeks to 4 weeks, with no significant difference between groups. However,
the AIS score indicated significant improvement in the L-ornithine group at 4 weeks in
comparison with the placebo group.
Figure 2 Effect of L-ornithine supplementation on AIS. Means of the change from 0
weeks of each AIS score to 4 and 8 weeks: mean ± SE. White circles (○) indicate the placebo
and black circles (●) indicate L-ornithine.
Scores for the OSA-MA for each group over an 8-week period are shown in Figure 3. Scores
of three OSA-MA items, “sleepiness on rising” (Figure 3A), “initiation and maintenance of
sleep” (Figure 3B) and “refreshing” (Figure 3D), tended towards improved sleep quality in
both groups without significant between-group differences. Scores for “frequent dreaming”
(Figure 3C) and “sleep length” (Figure 3E) were unchanged in the placebo group, however,
the score for the L-ornithine group significantly improved for self-reported “initiation and
maintenance of sleep” (Figure 3B) at 4 weeks and “sleep length” (Figure 3E) at 5 to 7 weeks
in comparison with the placebo group.
Figure 3 Effect of L-ornithine supplementation on OSA. Means of the change from 0
weeks of each OSA score (A, sleepiness on rising; B, initiation and maintenance of sleep; C,
frequent dreaming; D, refreshing; E, sleep length) to 1, 2, 3, 4, 5, 6, 7 and 8 weeks: mean ±
SE. White circles (○) indicate the placebo and black circles (●) indicate L-ornithine.
L-Ornithine supplementation attenuated the serum cortisol/ DHEA-S molar
ratio in study participants
While the concentration of serum DHEA-S was not increased by L-ornithine intake, serum
cortisol concentration decreased in the L-ornithine group. Although the change in either level
did not differ significantly between the two groups at any examination point (Figure 4A, 4B),
the change in cortisol/DHEA-S ratio significantly decreased in the L-ornithine group after 4
weeks (Figure 4C). The cortisol/DHEA-S (x100) ratio was calculated based on serum cortisol
and DHEA-S concentrations.
Figure 4 Effect of L-ornithine supplementation on serum stress markers. Means of the
change from 0 weeks of each stress marker level (A, DHEA-S; B, cortisol; C,
cortisol/DHEA-S) to 2, 4, and 8 weeks: mean ± SE. White circles (○) indicate the placebo
and black circles (●) indicate L-ornithine.
Discussion
Our results showed that L-ornithine supplementation had a favourable effect on the cortisol
response as an objective stress marker and improved perceived mood and sleep quality
related to fatigue as well as subjective feelings derived from stress.
Previous studies reported that i.c.v. injections of L-ornithine had sedative and hypnotic
effects on neonatal chicks exposed to acute stressful conditions [31,32]. That action was
mediated by GABAA receptors. It was also confirmed that orally administered L-ornithine
can be transported into the brain of mice [20], and as a result, reduced the plasma
corticosterone concentration induced by restraint stress in mice. L-ornithine levels in the
brain increased after oral administration of L-ornithine in mice [20]. Moreover, it was shown
that i.c.v. injected L-arginine, the precursor of L-ornithine, increased both L-arginine and L-
ornithine concentrations in the telencephalon and diencephalon in chicks 10 min post-
injection [33], however, the GABA content was not changed. This suggests that the sedative
and hypnotic effects of L-ornithine were not due to changes in GABA synthesis [32].
The role of GABA in HPA axis regulation has been well established, indicating that
corticotropin-releasing hormone (CHR) neurons receive robust GABAergic inhibition [34].
In addition, micro infusion of GABA agonists, such as the stress-derived neurosteroid
tetrahydrodeoxycorticosterone (THDOC), into the paraventricular nucleus (PVN) decreased
circulating levels of stress hormones [35]. Our result showing that orally administered L-
ornithine decreased the serum cortisol level in human subjects was in agreement with
previous animal studies on the effectiveness of stress reduction through the alleviation of
HPA axis hyperactivity [20,31-34].
We observed that L-ornithine supplementation for 8 weeks reduced the serum cortisol level
and cortisol/DHEA-S ratio, mainly due to reduction in the cortisol level. An imbalance
between cortisol and DHEA-S may be a key factor in physical and psychiatric disease
[36,37]. The molar DHEA-S/cortisol ratio was shown to be significantly lower in non-
medicated depressed patients than in control subjects, and evening salivary DHEA/cortisol
ratios were inversely correlated with the length of the current depressive episode [38].
Elevated cortisol/DHEA-S ratios in schizophrenia patients were positively associated with
higher scores for anxiety and anger, depression and hostility, age, age at onset of illness, and
duration of illness [39].
These reports provide support for our results showing that the decrease in cortisol/DHEA-S
ratios through the administration of L-ornithine corresponded to the improvement in mood
related to “anger-hostility” as well as sleep quality.
The initial decreasing tendency of the cortisol/DHEA-S ratios in the placebo group may be
due to a non-specific, natural change which occurs after formal ornithine and placebo intake
treatments. It is widely accepted that on average, brief initial interventions yield outcomes
similar to those with prolonged treatments, suggesting that changes could be triggered after a
brief phase of treatment [40,41].
“Anger-hostility”, an item on the POMS, is one of the phenotypes resulting from activation of
sympathetic nerves. A physiological change accompanying stress was shown to be the
increased excretion of cortisol and adrenaline [42]. Adrenalin promotes activation of the
sympathetic nerve system suggesting L-ornithine supplementation might affect, not only the
HPA axis, but also the autonomic nervous system. Unfortunately, we did not examine
whether L-ornithine supplementation could affect either the autonomic nervous system or
adrenaline levels.
Our results also suggested that sleep quality was improved by L-ornithine, as revealed by
both AIS and OSA-MA questionnaires, along with alleviation of stress (cortisol/DHEA-S).
The present finding is in accordance with a previous report that L-ornithine supplementation
after alcohol consumption improved sleep quality as perceived upon awakening in flushers
[43] and that ornithine increased the amount of non-rapid eye movement (NREM) sleep
without reducing the power spectrum density of NREM sleep in mice [44]. This result
replicated findings of our previous study, that ornithine could improve sleep in an animal
model and suggested the effectiveness of orally administered L-ornithine on stress reduction
through improvement of sleep quality in human subjects.
Furthermore, it was shown that L-ornithine administration stimulated release of growth
hormone [45,46], which is secreted as the largest pulse after the onset of sleep, and that there
is a correlation between night-time growth hormone release and sleep satisfaction [47-49].
A serotonin metabolite (5-hydroxyindole acetic acid, 5-HIAA) was induced in the striatum
after L-ornithine supplementation [20]. Day-time serotonin levels stimulate production of
melatonin during the night [50-52], therefore, L-ornithine might be considered an important
nutrient to maintain the circadian rhythm and to allow individuals to sleep well.
Job stress is one of the most important social problems for workers today. This study has
provided further objective evidence of the usefulness and effectiveness of L-ornithine for
managing stress and sleep quality related to fatigue. Future studies must address how L-
ornithine affects regulation of blood glucose and the autonomic nervous system.
Limitations exist in this study: first, we did not evaluate plasma ornithine levels, so we were
unable to assess any direct correlation between ornithine and suppressing stress markers or
improved subjective feelings. Second, we used three tests to assess fatigue and mood states,
however, many tests, using differing strategies, are required to properly evaluate
psychological status. Third, we determined the sample size of this study on the basis of a
previous report [26], however, this sample size (n = 26) was not sufficient to lead to draw
firm conclusions and more extensive studies are needed to confirm our findings.
Conclusion
L-ornithine plays a central role in the urea cycle which converts ammonia to urea in the liver.
L-ornithine administration has been known to enhance detoxification of ammonia in the liver.
Recent reports have shown that L-ornithine has a positive effect on animal stress models. Our
study suggested that L-ornithine has a positive effect on stress and sleep in healthy workers.
L-ornithine might be beneficial for people who live a stressful life.
Abbreviations
DHEA-S, Dehydroepiandrosterone-sulphate; DHEA, Dehydroepiandrosterone; POMS,
Profile of Mood States; AIS, Athens Insomnia Scale; OSA-MA, Ogri-Shirakawa-Azumi
sleep inventory MA version; HPA, Hypothalamic-pituitary-adrenal; GABA, Gamma-
aminobutyric acid; CHR, Corticotropin-releasing hormone; THDOC,
Tetrahydrodeoxycorticosterone; PVN, Paraventricular nucleus; NREM, Non-rapid eye
movement; 5-HIAA, 5-hydroxyindole acetic acid
Competing interests
In this study, we used L-ornithine monohydrochloride, a product of Kyowa Hakko Bio
Company, Limited. This company is an affiliate of Kirin Company, Limited, to which the
authors belong. None of the authors had a personal or financial conflict of interest.
Authors’ contributions
MM was involved in designing the trial, writing the trial protocol, calculating the sample
size, analysing data, drafting and finalising the manuscript. TKo, TKi and YM were involved
in designing the trial, writing the trial protocol, supervising analysis of data, and drafting and
revising the manuscript. MK was involved in designing the trial. AT and HO supervised the
trial as the principal investigator and participated in drafting and revising the manuscript. All
authors contributed to the data interpretation and approved the final version of the
manuscript.
Acknowledgements
Financial support
This study was provided by Kirin Co., Ltd and Kyowa Hakko Bio Co., Ltd.
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