**Clinical Approaches and Health State Modulation by Melatonin and Its Metabolites**

[32] Wurtman R, Cooper D. Physiology and available preparations of melatonin. Dostupno:

[33] Agil A, Navarro-Alarcón M, Ruiz R, Abuhamadah S, El-Mir MY, Vazquez GF. Beneficial effects of melatonin on obesity and lipid profile in young Zucker diabetic fatty rats.

[34] Sochor J, Ruttkay-Nedecky B, Babula P, Adam V, Hubalek J, Kizek R. Automation of Methods for Determination of Lipid Peroxidation. In: Catala A, editor. Lipid Peroxidation.

[35] Gutteridge JM. Lipid-peroxidation and antioxidants as biomarkers of tissue damage.

[36] Repetto M, Semprine J, Boveris A. Lipid Peroxidation: Chemical Mechanism, Biological Implications and Analytical Determination, Lipid Peroxidation Angel Catala. UK: IntechOpen; August 29th 2012. DOI: 10.5772/45943. Available from: https://www.intechopen.com/books/lipid-peroxidation/lipid-peroxidation-chemical-mechanism-biologi-

[37] Spirlandeli AL, Deminice R, Jordao AA. Plasma malondialdehyde as biomarker of lipid peroxidation: Effects of acute exercise. International Journal of Sports Medicine. 2014;**35**:

[38] Ayala A, Munoz MF, Arguelles S. Lipid peroxidation: Production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine

[39] Natalia Fagali AC. The antioxidant behaviour of melatonin and structural analogues during lipid peroxidation depends not only on their functional groups but also on the assay system. Biochemical and Biophysical Research Communications. 2012;**423**:873-877

[40] Mekhloufi J, Bonnefont-Rousselot D, Yous S, Lesieur D, Couturier M, Therond P, et al. Antioxidant activity of melatonin and a pinoline derivative on linoleate model system.

[41] Dillard CJ, Litov RE, Savin WM, Dumelin EE, Tappel AL. Effects of exercise, vitamin E, and ozone on pulmonary function and lipid peroxidation. Journal of Applied Physiology:

[42] Tutkun E. Melatonin administration in rats with acute swimming exercise prevents lipid peroxidation in muscle tissue. International Journal of Academic Research. 2013;**5**(3):1-5

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UK: InTech; 2012. p. 131-154

14-18

**Chapter 6**

Provisional chapter

**Melatonin in Childhood Epilepsy and in Child**

DOI: 10.5772/intechopen.80005

Melatonin (MLT) was isolated as a hormone by Lerner in 1958, and since then, intense studies have been under way with respect to its action and possibilities of application in various fields of medicine. Despite the existence of multiple antiepileptic medications and progress that has taken place in neurosurgical treatment of epilepsy, drug-resistant epilepsy continues to be a phenomenon that occurs in 30–35% children treated for epileptic seizures. Reports presented in the study have shown that children with epilepsy suffer from sleep disorders. Sleep deprivation may cause seizures, and on the other hand, an

Melatonin in Childhood Epilepsy and in Child

Justyna Paprocka, Marek Kijonka and Maria Sokół

increased frequency of seizures may lead to sleep disturbances.

Keywords: melatonin, children, epilepsy, autism, hypoxic-ischemic brain injury

The sleep/wake cycle, body temperature, and melatonin (MLT) rhythms have a stable internal phase relationship with maximum sleepiness coinciding with the melatonin excretion peak and the core body temperature nadir in humans and other diurnal species [1]. Several genes known as clock genes play a role of regulators of circadian rhythms generated by suprachiasmatic nucleus among them are PER, NPAS2, BMAL1, and CLOCK [2]. Also, Period genes (Per1, Per2, Per3) and Cryptochrome gene (Cry 1, Cry 2) are involved in auto-regulatory translation-

Melatonin, as a hormone, is secreted by the pineal gland, and its production is regulated by light and retino-hypothalamic tract. Melatonin secretion depends on the age—the highest values of its concentration are detected between 1 and 7 years of age. In healthy subjects, the

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Justyna Paprocka, Marek Kijonka and Maria Sokół

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80005

**Neurology**

Abstract

1. Melatonin

transcription feedback loops [3].

Neurology

#### **Melatonin in Childhood Epilepsy and in Child Neurology** Melatonin in Childhood Epilepsy and in Child Neurology

DOI: 10.5772/intechopen.80005

Justyna Paprocka, Marek Kijonka and Maria Sokół Justyna Paprocka, Marek Kijonka and Maria Sokół

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80005

#### Abstract

Melatonin (MLT) was isolated as a hormone by Lerner in 1958, and since then, intense studies have been under way with respect to its action and possibilities of application in various fields of medicine. Despite the existence of multiple antiepileptic medications and progress that has taken place in neurosurgical treatment of epilepsy, drug-resistant epilepsy continues to be a phenomenon that occurs in 30–35% children treated for epileptic seizures. Reports presented in the study have shown that children with epilepsy suffer from sleep disorders. Sleep deprivation may cause seizures, and on the other hand, an increased frequency of seizures may lead to sleep disturbances.

Keywords: melatonin, children, epilepsy, autism, hypoxic-ischemic brain injury

### 1. Melatonin

The sleep/wake cycle, body temperature, and melatonin (MLT) rhythms have a stable internal phase relationship with maximum sleepiness coinciding with the melatonin excretion peak and the core body temperature nadir in humans and other diurnal species [1]. Several genes known as clock genes play a role of regulators of circadian rhythms generated by suprachiasmatic nucleus among them are PER, NPAS2, BMAL1, and CLOCK [2]. Also, Period genes (Per1, Per2, Per3) and Cryptochrome gene (Cry 1, Cry 2) are involved in auto-regulatory translationtranscription feedback loops [3].

Melatonin, as a hormone, is secreted by the pineal gland, and its production is regulated by light and retino-hypothalamic tract. Melatonin secretion depends on the age—the highest values of its concentration are detected between 1 and 7 years of age. In healthy subjects, the

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

serum melatonin concentration peak occurs between 2 am and 4 am, and then it gradually declines; however, melatonin release may be shifted with time zone due to its day and night light dependence [3–5]. During the day, melatonin is not produced in measurable quantities.

and NO/L-arginine pathways and glutamate neurotransmission [11]. On the contrary, Steward and Leung suggested that proconvulsive action of melatonin is connected with the suppression of the GABA A receptors in pyramidal cells [12, 13]. Antioxidant properties of melatonin may also have a positive effect on children with epilepsy [14]. Our knowledge about possible melatonin role in epilepsy has increased in recent years but still remains controversial. For some time, melatonin has been recommended for children with epilepsy due to its ability to

Melatonin in Childhood Epilepsy and in Child Neurology http://dx.doi.org/10.5772/intechopen.80005 125

Sleep problems in children with developmental disabilities and epilepsy can be connected with an improper deranged circadian melatonin secretion, an insufficient melatonin production, or melatonin receptor insensitivity. Usually, falling asleep and maintain a sleep are the most frequently encountered problems of pediatric populations. The prevalence rates of sleep prob-

At the cellular level, sleep deprivation impairs synaptic plasticity, increases hippocampal oxidative stress, and facilitates neuronal loss, which can affect neurocognitive skills especially

In adults with epilepsy, 55% have insomnia, 34% have sleep-onset insomnia, and 52% have maintenance insomnia [15–20]. On nights with seizures, patients experience up to 50% reduction of REM sleep and an increased REM latency [15–20]. In specific childhood epilepsy syndromes, like juvenile myoclonic epilepsy, the sufficient amount of sleep may completely protect from seizures. In autosomal-dominant nocturnal frontal lobe epilepsy, the epileptic seizures dominate at night, while in juvenile myoclonic epilepsy after awakening in the

The stage of sleep also matters. Seizures are rare in REM sleep, and indeed the rate of REM seizures' onset is low (0–5%) [21]. According to the studies conducted by Minecam et al. and Herman et al., most related to sleep seizures appear in stage 2 NREM sleep (61–68%), and lower rates are evident in stages 3 and 4 as 9–14% [19, 21, 22]. Some authors believe that the area of REM discharges could be an indicator of epileptic zone [17–22]. Jan et al. postulated that the occurrence of seizures may show a 24-rhythmicity and circadian occurrence pattern

Sleep deprivation is one of the most frequent precipitating factors of seizures and interictal epileptiform discharges (IED). On the other hand, site epileptic seizures, epileptiform activity, or antiepileptic drugs (AEDs) may disrupt sleep pattern. The analysis of the questionnaires filled by the patients revealed that the most common sleep-related complaints are excessive daytime sleepiness (EDS), insomnia, and poor sleep quality. The other important remark is that more sleep abnormalities are concerned with focal than generalized epilepsies. Also, sudden unexpected death in epilepsy (SUDEP) is most frequent to appear between 6 am and noon [17–21]. Long-standing epilepsy can affect insula, anterior cingulated gyrus, ventromedial frontal cortex,

Antiepileptic drugs (AEDs) may affect sleep parameters. For example, a frequently administrated valproic acid, due to its interaction with GABA transmission at suprachiasmatic nucleus, may lower melatonin secretion [27]. However, Braam et al. [28, 29] compared the endogenous melatonin levels in children administered with valproate and those who did not use it, and

and through their influence on cardiac rhythm may provoke SUDEP [16–22].

promote sleep and to avoid sleep deprivation.

morning.

(Table 1) [23].

lems in childhood are estimated between 30 and 40% [15–20].

attention, behavioral, and emotional aspects of development.

Measurement of the whole 24-h rhythm of melatonin is considered to be the most robust sleep phase marker of various circadian rhythm sleep disorders [1–6]. Because melatonin secretion is suppressed by light, the melatonin levels should be measured in dim light conditions. Serial sampling of melatonin measured in the blood or saliva can be used to assess circadian timing by determining the dim light melatonin onset (DLMO), the parameter indicating the time point in which melatonin levels begin to rise in the evening above baseline [2]. Another useful circadian phase marker is dim light melatonin offset (DLMOff), the point in time when melatonin levels diminish in the morning. The melatonin secretion profile can also be analyzed in a more complex way—by approximation of the empirical or analytical models. The models of melatonin secretion can provide a set of parameters with biophysical and clinical significance that directly characterize melatonin cycle. Moreover, mathematical modeling facilitates statistical analysis of the patients' hormone levels, offering a set of parameters that enable the objectification of the secretion description.

Melatonin for many years remained the most mysterious and forgotten human hormone with not exactly understood role and suggested pleiotropic actions. It is well known that melatonin is released in a circadian pattern with a night peak. Endogenous melatonin production varies among individuals. Endogenous melatonin production varies among individuals. Melatonin is secreted mainly by pinealocytes from tryptophan through hydroxytryptophan and serotonin. Then, two enzymes, arylalkylamine-N-acetyltransferase (AA-NAT) and acetylserotonin-O-methyltransferase (ASMT), form melatonin from serotonin [1–6]. The organization of the sleep-wake rhythm is set around 6 months of age, but melatonin rhythm may be set earlier, from 3 months of age. At the age of 3, a stabilization of these rhythms is visible. Between 4 and 7 years, nocturnal melatonin secretion reaches the highest values [1–6].

Gender difference (a lower melatonin secretion in girls) and age-related decline have been described [7]. According to the latest Task Force of American Academy of Sleep Medicine, melatonin application is recommended in children with delayed sleep-wake phase disorders, children with neurological disorders, and with an irregular sleep cycle [5, 6].

Melatonin should be administered at a time related to DLMO—the onset of melatonin endogenous production [7–9]. Actually, prior to melatonin administration, DLMO should be measured to let for optimal treatment. In saliva, DLMO is defined as a melatonin range of 3–5 pg/ml [9, 10].

### 2. Sleep and epilepsy

A complex interaction between sleep and epilepsy is still a matter of debate. Sleep deprivation may activate epileptiform activity. Epilepsy per se and antiepileptic treatment may cause sleep deprivation or fragmentation causing the vicious circle.

Accumulating evidences suggest that melatonin modulates the electrical activity of neurons. Based on experimental studies, melatonin probably may mediate the GABA-ergic, 5HT-ergic, and NO/L-arginine pathways and glutamate neurotransmission [11]. On the contrary, Steward and Leung suggested that proconvulsive action of melatonin is connected with the suppression of the GABA A receptors in pyramidal cells [12, 13]. Antioxidant properties of melatonin may also have a positive effect on children with epilepsy [14]. Our knowledge about possible melatonin role in epilepsy has increased in recent years but still remains controversial. For some time, melatonin has been recommended for children with epilepsy due to its ability to promote sleep and to avoid sleep deprivation.

serum melatonin concentration peak occurs between 2 am and 4 am, and then it gradually declines; however, melatonin release may be shifted with time zone due to its day and night light dependence [3–5]. During the day, melatonin is not produced in measurable quantities. Measurement of the whole 24-h rhythm of melatonin is considered to be the most robust sleep phase marker of various circadian rhythm sleep disorders [1–6]. Because melatonin secretion is suppressed by light, the melatonin levels should be measured in dim light conditions. Serial sampling of melatonin measured in the blood or saliva can be used to assess circadian timing by determining the dim light melatonin onset (DLMO), the parameter indicating the time point in which melatonin levels begin to rise in the evening above baseline [2]. Another useful circadian phase marker is dim light melatonin offset (DLMOff), the point in time when melatonin levels diminish in the morning. The melatonin secretion profile can also be analyzed in a more complex way—by approximation of the empirical or analytical models. The models of melatonin secretion can provide a set of parameters with biophysical and clinical significance that directly characterize melatonin cycle. Moreover, mathematical modeling facilitates statistical analysis of the patients' hormone levels, offering a set of parameters that enable the

Melatonin for many years remained the most mysterious and forgotten human hormone with not exactly understood role and suggested pleiotropic actions. It is well known that melatonin is released in a circadian pattern with a night peak. Endogenous melatonin production varies among individuals. Endogenous melatonin production varies among individuals. Melatonin is secreted mainly by pinealocytes from tryptophan through hydroxytryptophan and serotonin. Then, two enzymes, arylalkylamine-N-acetyltransferase (AA-NAT) and acetylserotonin-O-methyltransferase (ASMT), form melatonin from serotonin [1–6]. The organization of the sleep-wake rhythm is set around 6 months of age, but melatonin rhythm may be set earlier, from 3 months of age. At the age of 3, a stabilization of these rhythms is visible. Between 4 and 7 years, nocturnal

Gender difference (a lower melatonin secretion in girls) and age-related decline have been described [7]. According to the latest Task Force of American Academy of Sleep Medicine, melatonin application is recommended in children with delayed sleep-wake phase disorders,

Melatonin should be administered at a time related to DLMO—the onset of melatonin endogenous production [7–9]. Actually, prior to melatonin administration, DLMO should be measured to let for optimal treatment. In saliva, DLMO is defined as a melatonin range of 3–5 pg/ml [9, 10].

A complex interaction between sleep and epilepsy is still a matter of debate. Sleep deprivation may activate epileptiform activity. Epilepsy per se and antiepileptic treatment may cause sleep

Accumulating evidences suggest that melatonin modulates the electrical activity of neurons. Based on experimental studies, melatonin probably may mediate the GABA-ergic, 5HT-ergic,

children with neurological disorders, and with an irregular sleep cycle [5, 6].

objectification of the secretion description.

124 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

melatonin secretion reaches the highest values [1–6].

deprivation or fragmentation causing the vicious circle.

2. Sleep and epilepsy

Sleep problems in children with developmental disabilities and epilepsy can be connected with an improper deranged circadian melatonin secretion, an insufficient melatonin production, or melatonin receptor insensitivity. Usually, falling asleep and maintain a sleep are the most frequently encountered problems of pediatric populations. The prevalence rates of sleep problems in childhood are estimated between 30 and 40% [15–20].

At the cellular level, sleep deprivation impairs synaptic plasticity, increases hippocampal oxidative stress, and facilitates neuronal loss, which can affect neurocognitive skills especially attention, behavioral, and emotional aspects of development.

In adults with epilepsy, 55% have insomnia, 34% have sleep-onset insomnia, and 52% have maintenance insomnia [15–20]. On nights with seizures, patients experience up to 50% reduction of REM sleep and an increased REM latency [15–20]. In specific childhood epilepsy syndromes, like juvenile myoclonic epilepsy, the sufficient amount of sleep may completely protect from seizures. In autosomal-dominant nocturnal frontal lobe epilepsy, the epileptic seizures dominate at night, while in juvenile myoclonic epilepsy after awakening in the morning.

The stage of sleep also matters. Seizures are rare in REM sleep, and indeed the rate of REM seizures' onset is low (0–5%) [21]. According to the studies conducted by Minecam et al. and Herman et al., most related to sleep seizures appear in stage 2 NREM sleep (61–68%), and lower rates are evident in stages 3 and 4 as 9–14% [19, 21, 22]. Some authors believe that the area of REM discharges could be an indicator of epileptic zone [17–22]. Jan et al. postulated that the occurrence of seizures may show a 24-rhythmicity and circadian occurrence pattern (Table 1) [23].

Sleep deprivation is one of the most frequent precipitating factors of seizures and interictal epileptiform discharges (IED). On the other hand, site epileptic seizures, epileptiform activity, or antiepileptic drugs (AEDs) may disrupt sleep pattern. The analysis of the questionnaires filled by the patients revealed that the most common sleep-related complaints are excessive daytime sleepiness (EDS), insomnia, and poor sleep quality. The other important remark is that more sleep abnormalities are concerned with focal than generalized epilepsies. Also, sudden unexpected death in epilepsy (SUDEP) is most frequent to appear between 6 am and noon [17–21]. Long-standing epilepsy can affect insula, anterior cingulated gyrus, ventromedial frontal cortex, and through their influence on cardiac rhythm may provoke SUDEP [16–22].

Antiepileptic drugs (AEDs) may affect sleep parameters. For example, a frequently administrated valproic acid, due to its interaction with GABA transmission at suprachiasmatic nucleus, may lower melatonin secretion [27]. However, Braam et al. [28, 29] compared the endogenous melatonin levels in children administered with valproate and those who did not use it, and


In epilepsy, melatonin secretion may be disturbed: higher nocturnal melatonin concentrations, a higher melatonin concentration after seizures, or loss/shift of the characteristic diurnal rhythm of secretion are reported by some authors [35–40], while other authors found low baseline levels [41, 42]. Melatonin concentration in patients with epilepsy is sometimes claimed

Melatonin in Childhood Epilepsy and in Child Neurology http://dx.doi.org/10.5772/intechopen.80005 127

However, we observed a statistical dependence between melatonin release amplitude and the number of seizures in different time intervals in the epilepsy of children. Moreover, the time since last seizure has a significant effect on the secretion of melatonin. It should be noted that antiepileptic treatment itself may affect melatonin secretion, which, in fact, was seen in our studies [35, 42]. On the contrary, Dabak et al. showed lower post-seizure melatonin levels in the patients with febrile and afebrile seizures [43]. On the other hand, a normal plasma melatonin curve in epilepsy patients under dim-lit conditions [25] as well as in the study

There is also no agreement between the animal models and the results obtained in the human studies. In animal studies, the data suggest anticonvulsant properties of melatonin, whereas in

Having in mind the heterogeneity of melatonin secretion and the mode of action in children with epilepsy, Praninskiene et al. postulated that probably not only peripheral melatonin levels but also measurements of melatonin receptors in the brain and melatonin level in central

Before the era of randomized trials with melatonin, we witnessed the add-on melatonin supplementation in a few described trials by Peled et al. (improvement in seizures' frequency in five of six children on 3-mg melatonin add-on therapy) [46], Ross et al. (clinical improvement in seizure control and sleep in 20 of 24 children treated with 2.5–7.5 mg of melatonin addon therapy) [47], and Molina-Carballo et al. (clinical improvement of one child with refractory

The first randomized, double-blind, placebo-controlled trial concerning melatonin in epilepsy was conducted in 2004 by Copolla et al. [49]. A total of 25 participants (with mental retardation and epilepsy) aged 3.6–26 years were randomized to oral synthetic fast release melatonin (an initial dose of 3 mg, possible tiltration to 9 mg). In 2 of 11 seizure-free patients, epileptic seizures appeared on melatonin supplementation [49]. Among seven patients with not adequately controlled epilepsy, the results were not promising (N = 1 seizure-free, N = 2 partial

In the same year, Gupta et al. assessed the effect of melatonin add-on supplementation in children with epilepsy aged 3–12 years on carbamazepine or valproic acid monotherapy using the parental questionnaire (Sleep Behavior Questionnaire) [50–54]. In these studies, the children were seizure-free for at least 6 months before the first visit; that is why the authors could

4. Melatonin supplementation in epilepsy in randomized trials

myoclonic epilepsy treated with melatonin add-on therapy of 200 mg daily) [48].

improvement, N = 2 unchanged, N = 2 increase of seizures) [49].

not report the influence of melatonin on seizures' frequency [50, 51].

to be slightly increased or unchanged as compared to normal values [38, 43].

involving epileptic children was found [4].

components may be of value [44, 45].

human studies, it is difficult to reach unambiguous conclusions.

Table 1. Seizures' circadian occurrence pattern [23–26].

Takaesu et al. [30] observed a minimal impact of sodium valproate on the low serum levels that do not have such supposition. Carbamazepine may increase slow-wave sleep, reduce REM, and reduce awakenings and arousals [31]. Similarly, lamotrigine and valproic acid appear to stabilize sleep (more REM and slow-wave sleep) [27–31]. Newer AEDs have little effect on sleep architecture (levetiracetam) or little is known about these effects (lacosamide, eliscarbazepine, and retigabine) [31]. The direct effect of AEDs on sleep is difficult to measure because of many confounding factors, with the leading one—polypharmacy. Antiepileptic treatment with more than one drug increases the risk of obstructive sleep apnea (OSA). The prevalence of OSA is significantly higher in the epilepsy group—35% versus healthy children—7.4%. In refractory epilepsy, 44% children have the diagnosis of OSA, in other form of epilepsy around 31% [32, 33]. It is especially important for clinicians, who frequently under-recognize and misinterpret sleep disorders in epilepsy patients.

### 3. Melatonin secretion in epilepsy

The dynamics of melatonin secretion in epileptic subjects is more complex compared to healthy subjects. Though it appears that human seizure occurrence may have 24-h rhythmicity (and such rhythmicity has also been shown in animals), but there is still no answer to the question on the relationship between the occurrence of seizures and the human circadian rhythm. Many studies on epilepsy have examined the processes that have circadian variation, like hormones secretion, body temperature changes, activity, sleep, and wakefulness, and it is obvious that circadian rhythm and epilepsy at least interact. Unfortunately, there are considerable gaps in the knowledge of such interaction, especially in humans [34].

In epilepsy, melatonin secretion may be disturbed: higher nocturnal melatonin concentrations, a higher melatonin concentration after seizures, or loss/shift of the characteristic diurnal rhythm of secretion are reported by some authors [35–40], while other authors found low baseline levels [41, 42]. Melatonin concentration in patients with epilepsy is sometimes claimed to be slightly increased or unchanged as compared to normal values [38, 43].

However, we observed a statistical dependence between melatonin release amplitude and the number of seizures in different time intervals in the epilepsy of children. Moreover, the time since last seizure has a significant effect on the secretion of melatonin. It should be noted that antiepileptic treatment itself may affect melatonin secretion, which, in fact, was seen in our studies [35, 42]. On the contrary, Dabak et al. showed lower post-seizure melatonin levels in the patients with febrile and afebrile seizures [43]. On the other hand, a normal plasma melatonin curve in epilepsy patients under dim-lit conditions [25] as well as in the study involving epileptic children was found [4].

There is also no agreement between the animal models and the results obtained in the human studies. In animal studies, the data suggest anticonvulsant properties of melatonin, whereas in human studies, it is difficult to reach unambiguous conclusions.

Having in mind the heterogeneity of melatonin secretion and the mode of action in children with epilepsy, Praninskiene et al. postulated that probably not only peripheral melatonin levels but also measurements of melatonin receptors in the brain and melatonin level in central components may be of value [44, 45].

### 4. Melatonin supplementation in epilepsy in randomized trials

Takaesu et al. [30] observed a minimal impact of sodium valproate on the low serum levels that do not have such supposition. Carbamazepine may increase slow-wave sleep, reduce REM, and reduce awakenings and arousals [31]. Similarly, lamotrigine and valproic acid appear to stabilize sleep (more REM and slow-wave sleep) [27–31]. Newer AEDs have little effect on sleep architecture (levetiracetam) or little is known about these effects (lacosamide, eliscarbazepine, and retigabine) [31]. The direct effect of AEDs on sleep is difficult to measure because of many confounding factors, with the leading one—polypharmacy. Antiepileptic treatment with more than one drug increases the risk of obstructive sleep apnea (OSA). The prevalence of OSA is significantly higher in the epilepsy group—35% versus healthy children—7.4%. In refractory epilepsy, 44% children have the diagnosis of OSA, in other form of epilepsy around 31% [32, 33]. It is especially important for clinicians, who frequently under-recognize and

Seizures' occurrence pattern Sleep Wakefulness

seizures, frontal lobe seizures

20% of temporal lobe seizures

• West syndrome: hypsarrhythmia most evident in early NREM sleep • Lennox-Gastaut syndrome: paroxysmal fast activity during sleep

EEG-stages 3 and 4 as effective facilitator of

Generalized epilepsy

activated by light NREM)

epileptiform discharges

• Time peak of occurrence

for frontal lobe seizures, NFLE:

Clonic, absence, atonic, myoclonic

Generalized epilepsy of unknown etiology: JME, GTCE (on EEG spike waves discharges most prominent in

• Time peak of occurrence

for temporal lobe seizures, TLE:

seizures

stage 2 sleep)

3–6 pm

11.00–17.00 (6 h before DLMO)

Seizures' types Tonic seizures, generalized tonic-clonic

126 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Epilepsy with focal seizures BECTS (interictal epileptiform discharges

6–9 am

23.00–5.00 (6–12 h after DLMO)

Relation with sleep 78% of frontal lobe seizures

Epilepsy with generalized

seizures

The dynamics of melatonin secretion in epileptic subjects is more complex compared to healthy subjects. Though it appears that human seizure occurrence may have 24-h rhythmicity (and such rhythmicity has also been shown in animals), but there is still no answer to the question on the relationship between the occurrence of seizures and the human circadian rhythm. Many studies on epilepsy have examined the processes that have circadian variation, like hormones secretion, body temperature changes, activity, sleep, and wakefulness, and it is obvious that circadian rhythm and epilepsy at least interact. Unfortunately, there are consider-

able gaps in the knowledge of such interaction, especially in humans [34].

misinterpret sleep disorders in epilepsy patients.

Table 1. Seizures' circadian occurrence pattern [23–26].

3. Melatonin secretion in epilepsy

Before the era of randomized trials with melatonin, we witnessed the add-on melatonin supplementation in a few described trials by Peled et al. (improvement in seizures' frequency in five of six children on 3-mg melatonin add-on therapy) [46], Ross et al. (clinical improvement in seizure control and sleep in 20 of 24 children treated with 2.5–7.5 mg of melatonin addon therapy) [47], and Molina-Carballo et al. (clinical improvement of one child with refractory myoclonic epilepsy treated with melatonin add-on therapy of 200 mg daily) [48].

The first randomized, double-blind, placebo-controlled trial concerning melatonin in epilepsy was conducted in 2004 by Copolla et al. [49]. A total of 25 participants (with mental retardation and epilepsy) aged 3.6–26 years were randomized to oral synthetic fast release melatonin (an initial dose of 3 mg, possible tiltration to 9 mg). In 2 of 11 seizure-free patients, epileptic seizures appeared on melatonin supplementation [49]. Among seven patients with not adequately controlled epilepsy, the results were not promising (N = 1 seizure-free, N = 2 partial improvement, N = 2 unchanged, N = 2 increase of seizures) [49].

In the same year, Gupta et al. assessed the effect of melatonin add-on supplementation in children with epilepsy aged 3–12 years on carbamazepine or valproic acid monotherapy using the parental questionnaire (Sleep Behavior Questionnaire) [50–54]. In these studies, the children were seizure-free for at least 6 months before the first visit; that is why the authors could not report the influence of melatonin on seizures' frequency [50, 51].

In 2005, Hancock et al. in the next randomized, double-blind, crossover trial evaluated melatonin supplementation (two dose regimen: 5 or 10 mg) in 8 patients aged 18 months to 31 years with epilepsy and tuberous sclerosis complex [55, 56]. During the study period of 6 months, no change in seizure frequency was noted at either dose [55, 56].

Miano et al. distinguished five sleep phenotypes in ADHD [61, 62]: 1. phenotype related to hypoarousal state, primary form of ADHD;

3. phenotype related to sleep-disordered breathing (SDB) from snoring to obstructive sleep

Melatonin in Childhood Epilepsy and in Child Neurology http://dx.doi.org/10.5772/intechopen.80005 129

The most common complaint is sleep-onset insomnia, and rarely, sleep problems are related to a delayed sleep phase syndrome. Also, SDB is highly associated with disturbed attention and

Based on trials conducted in this population, melatonin treatment in doses ranging between 3 and 6 mg/day may reduce sleep-onset delay and increased sleep duration time [61–63].

Autistic spectrum disorders are frequently connected with sleep disturbances (30–53% or up to 50–80%) [64, 65]. The most common medication used in sleep difficulties is melatonin, apart

In children with autistic spectrum disorders, melatonin levels are lower [65–67] or within normal values [64, 68, 69]. Based on parental questionnaires and clinician completed forms of 1518 ASD children aged 4–10 years, Braam et al. informed about a much higher percentage of sleep problems in ASD (71%) and a higher necessity of drug intake (>46% children on more than one drug promoting sleep) [29]. In the latest double-blind study conducted by Gringras et al., 125 ASD children (among them 3.2% children with the diagnosis of Smith-Magenis syndrome (SMS)) received prolonged-release melatonin or placebo for 13 weeks [70]. Melatonin treatment prolonged the total sleep time (melatonin 57.5 min vs. placebo 9.14 min) and decreased sleep latency (melatonin 39.6 min vs. placebo 12.5 min) [70]. Veatch et al. studied the possible genetic background of sleep problems in ASD by evaluation of two melatonin pathway genes: acetylserotonin O-methyltransferase (ASMT) and cytochrome P450 1A2 (CYP1A2) [68]. The authors found a higher prevalence of variants responsible for a decreased expression of ASMT and a lower CYP1A2 enzyme activity [68]. On the other hand, a lower CYP1A2 enzyme activity may be responsible for slow metabolism and the possibility of lack of efficacy of exogenous melatonin with time. That is why some children may benefit from low melatonin

Some authors speculate that melatonin as a hormone derived from serotonin may be of a special interest in autism neurobiology [73]. Another interesting finding is that melatonin levels may be negatively correlated with the severity of autistic features. This assumption was made by the examination of sulfatoxymelatonin level in urine of 60 mothers of a child with

4. phenotype related to restless leg syndrome and/or periodic limb movements; 5. phenotype related to sleep epilepsy and/or EEG interictal epileptic discharges.

hyperactivity, and children with SDB are more sensitive to oxidative stress [61–63].

2. phenotype related to delayed sleep phase syndrome;

6. Melatonin in autism spectrum disorders (ASDs)

dose like 0.5 mg rather than higher (exceeding 5–6 mg) [64, 65, 71, 72].

apnea;

from behavioral interventions.

ASD features and in control group.

Goldberg-Stern et al. conducted another trial investigating response to melatonin (10 mg) in 10 patients with a refractory epilepsy aged 9–32 years (N = generalized epilepsy, N = focal epilepsy) [57]. The mean seizure frequency was 7.75 per day on placebo and 4.6 on melatonin treatment [57]. The limitation of this study was the absence of dim light melatonin-onset measurement.

The randomized double-blind placebo-controlled trial performed by Jain et al. showed that a 9 mg sustained release melatonin formulation decreased sleep latency and wakefulness after sleep onset (WASO) as compared to placebo [58]. This group consisted of 10 children aged 6–11 years diagnosed with epilepsy (focal epilepsy N = 6, generalized epilepsy-childhood absence epilepsy N = 3, undetermined N = 1) with intelligence quotient (IQ) > 70 [58]. Melatonin was given for 30 min before bedtime for 9 weeks. Apart from melatonin, children received different antiepileptic drugs as monotherapy: zonisamide, lamotrigine, levetiracetam, oxcarbazepine, and carbamazepine. According to the authors of the study, no worsening in seizures frequency was observed. Eight participants remained seizure-free, and another two experienced 50% reduction in seizure frequency on melatonin treatment.

Elkhayat et al. in a group of 23 children with refractory epilepsy and in 14 children with controlled seizures (aged 2–15 years) measured melatonin level and assessed the sleep parameters before and after melatonin supplementation (melatonin dose of 1.5–3 mg daily) [59]. The most frequent antiepileptic drug was valproic acid (in intractable epilepsy in 78.2% of patients, in controlled seizures group—85.7%) [59]. After 3 months of melatonin therapy, children with intractable epilepsy experienced improvement in sleep continuity (bedtime resistance, sleep duration, sleep latency, frequent nocturnal arousals, and excessive daytime sleepiness), sleep apnea, nocturnal enuresis, sleep walking, forcible teeth grinding, and Epworth sleepiness score. Melatonin diurnal secretion and the frequency of seizures in controlled seizures group and refractory epilepsy did not differ significantly. Some children experience a decreased severity of seizures.

There are some significant limitation of the abovementioned studies like the small sample size and lack of the homogeneity of the sample: diversity of the epilepsy syndromes, different etiology of seizures, different seizure types, and short period of observation. But treating epilepsy with antiepileptic drugs may also improve sleep architecture and restore sleep cycle. The very limited number of randomized studies did not allow to draw definite conclusions about melatonin add-on therapy and influence of the treatment on epileptic seizures.

### 5. Melatonin in attention-deficit/hyperactivity disorder (ADHD)

About 25–50% of children with ADHD experience sleep problems [60]. The frequency of sleep problems is almost two-fold higher in a case of stimulant treatment. Sleep disturbances are included among diagnostic criteria for ADHD in the DSM third edition.

Miano et al. distinguished five sleep phenotypes in ADHD [61, 62]:


In 2005, Hancock et al. in the next randomized, double-blind, crossover trial evaluated melatonin supplementation (two dose regimen: 5 or 10 mg) in 8 patients aged 18 months to 31 years with epilepsy and tuberous sclerosis complex [55, 56]. During the study period of 6 months, no

Goldberg-Stern et al. conducted another trial investigating response to melatonin (10 mg) in 10 patients with a refractory epilepsy aged 9–32 years (N = generalized epilepsy, N = focal epilepsy) [57]. The mean seizure frequency was 7.75 per day on placebo and 4.6 on melatonin treatment [57]. The limitation of this study was the absence of dim light melatonin-onset measurement. The randomized double-blind placebo-controlled trial performed by Jain et al. showed that a 9 mg sustained release melatonin formulation decreased sleep latency and wakefulness after sleep onset (WASO) as compared to placebo [58]. This group consisted of 10 children aged 6–11 years diagnosed with epilepsy (focal epilepsy N = 6, generalized epilepsy-childhood absence epilepsy N = 3, undetermined N = 1) with intelligence quotient (IQ) > 70 [58]. Melatonin was given for 30 min before bedtime for 9 weeks. Apart from melatonin, children received different antiepileptic drugs as monotherapy: zonisamide, lamotrigine, levetiracetam, oxcarbazepine, and carbamazepine. According to the authors of the study, no worsening in seizures frequency was observed. Eight participants remained seizure-free, and another two experienced 50% reduction

Elkhayat et al. in a group of 23 children with refractory epilepsy and in 14 children with controlled seizures (aged 2–15 years) measured melatonin level and assessed the sleep parameters before and after melatonin supplementation (melatonin dose of 1.5–3 mg daily) [59]. The most frequent antiepileptic drug was valproic acid (in intractable epilepsy in 78.2% of patients, in controlled seizures group—85.7%) [59]. After 3 months of melatonin therapy, children with intractable epilepsy experienced improvement in sleep continuity (bedtime resistance, sleep duration, sleep latency, frequent nocturnal arousals, and excessive daytime sleepiness), sleep apnea, nocturnal enuresis, sleep walking, forcible teeth grinding, and Epworth sleepiness score. Melatonin diurnal secretion and the frequency of seizures in controlled seizures group and refractory epilepsy did not differ significantly. Some children experience a decreased

There are some significant limitation of the abovementioned studies like the small sample size and lack of the homogeneity of the sample: diversity of the epilepsy syndromes, different etiology of seizures, different seizure types, and short period of observation. But treating epilepsy with antiepileptic drugs may also improve sleep architecture and restore sleep cycle. The very limited number of randomized studies did not allow to draw definite conclusions

About 25–50% of children with ADHD experience sleep problems [60]. The frequency of sleep problems is almost two-fold higher in a case of stimulant treatment. Sleep disturbances are

about melatonin add-on therapy and influence of the treatment on epileptic seizures.

5. Melatonin in attention-deficit/hyperactivity disorder (ADHD)

included among diagnostic criteria for ADHD in the DSM third edition.

change in seizure frequency was noted at either dose [55, 56].

128 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

in seizure frequency on melatonin treatment.

severity of seizures.


The most common complaint is sleep-onset insomnia, and rarely, sleep problems are related to a delayed sleep phase syndrome. Also, SDB is highly associated with disturbed attention and hyperactivity, and children with SDB are more sensitive to oxidative stress [61–63].

Based on trials conducted in this population, melatonin treatment in doses ranging between 3 and 6 mg/day may reduce sleep-onset delay and increased sleep duration time [61–63].

### 6. Melatonin in autism spectrum disorders (ASDs)

Autistic spectrum disorders are frequently connected with sleep disturbances (30–53% or up to 50–80%) [64, 65]. The most common medication used in sleep difficulties is melatonin, apart from behavioral interventions.

In children with autistic spectrum disorders, melatonin levels are lower [65–67] or within normal values [64, 68, 69]. Based on parental questionnaires and clinician completed forms of 1518 ASD children aged 4–10 years, Braam et al. informed about a much higher percentage of sleep problems in ASD (71%) and a higher necessity of drug intake (>46% children on more than one drug promoting sleep) [29]. In the latest double-blind study conducted by Gringras et al., 125 ASD children (among them 3.2% children with the diagnosis of Smith-Magenis syndrome (SMS)) received prolonged-release melatonin or placebo for 13 weeks [70]. Melatonin treatment prolonged the total sleep time (melatonin 57.5 min vs. placebo 9.14 min) and decreased sleep latency (melatonin 39.6 min vs. placebo 12.5 min) [70]. Veatch et al. studied the possible genetic background of sleep problems in ASD by evaluation of two melatonin pathway genes: acetylserotonin O-methyltransferase (ASMT) and cytochrome P450 1A2 (CYP1A2) [68]. The authors found a higher prevalence of variants responsible for a decreased expression of ASMT and a lower CYP1A2 enzyme activity [68]. On the other hand, a lower CYP1A2 enzyme activity may be responsible for slow metabolism and the possibility of lack of efficacy of exogenous melatonin with time. That is why some children may benefit from low melatonin dose like 0.5 mg rather than higher (exceeding 5–6 mg) [64, 65, 71, 72].

Some authors speculate that melatonin as a hormone derived from serotonin may be of a special interest in autism neurobiology [73]. Another interesting finding is that melatonin levels may be negatively correlated with the severity of autistic features. This assumption was made by the examination of sulfatoxymelatonin level in urine of 60 mothers of a child with ASD features and in control group.

A few RDBPC trials showed that melatonin may improve communication [74] and anxiety in children with ASD [75]. Based on the knowledge from placebo-controlled studies, long-acting melatonin preparations at bedtime improve the sleep latency and the total sleep time [67, 69, 70, 74, 76–80].

Because of inverted melatonin circadian profile, a complex but promising treatment was found: a combination of acebutolol in the morning (10 mg/kg decrease melatonin level during

Melatonin in Childhood Epilepsy and in Child Neurology http://dx.doi.org/10.5772/intechopen.80005 131

During the conference in Rome in 2014, Bruni et al. postulated recommendation for melatonin treatment guidelines in children with neurodevelopmental disabilities and insomnia [15]:

2. if used as a chronobiotic 3–4 hs before bedtime (if used as a sleep inductor 30 min before sleep time) with starting dose 0.2–0.5 mg (tiltrated by 0.2–0.5 mg every week till maximum

3. treatment duration should not be <1 month, therapy adjusted to the patient; if normal sleep cycle is restored 1 week without melatonin treatment, once a year is recommended

During the last decade, melatonin has started to be considered as an attractive option in order to minimize the neurological sequelae from hypoxic-ischemic brain injury [93–95]. The brain itself is particularly sensitive to free radicals damage due to its high utilization of oxygen, its relatively poorly developed antioxidant defense, and its high amount of easily oxidizable fatty acids. Melatonin may serve as a potential therapeutic free radical scavenger (hydroxyl radicals, hydrogen peroxide, singlet oxygen) and broad-spectrum antioxidant (upregulation of antioxidant pathways: superoxide dismutase, glutathione, catalase, glutathione peroxidase, glutathione reductase) [96–98]. Based on experimental studies, melatonin may increase the number of neurons in the CA1, CA2–CA3 areas and dentate gyrus of the hippocampus and parietal cortex, reduce the expression of the glial fibrillary acidic protein, and regulate the expression of myelin basic protein and oligodendrocytes' function (regulation of myelination process)

Aly et al. examined the effect of melatonin on clinical, biochemical, neurophysiological, and radiological outcomes of neonates with hypoxic-ischemic encephalopathy (HIE) [99]. They performed a prospective trial involving 45 newborns randomized in the hypothermia alone and hypothermia and melatonin groups. All infants were studied with repeated EEG and brain MRI. In all patients, superoxide dismutase (SOD) and nitric oxide (NO) were measured. These examinations showed an increased melatonin and a decreased NO in the hypothermiamelatonin group [99]. Because of postulated unpredicted bioavailability of oral melatonin, Merchant et al. gave blood transfusion of 0.04–0.6 μg/kg melatonin to 18 preterm babies (less than 31 weeks gestation, less than 7 days old) for 2 h [100]. As a result they found melatonin concentration peak similar to adults. Another challenge might be the possibility to administer melatonin antenatally, in order to prevent or reduce brain hypoxic insult in preterm babies

the day) and melatonin in the evening [91].

dose of 3 (<40 kg) and 5 mg (>40 kg),

(especially during summer).

[96–98].

1. no age limit (safe administration >6 months of age),

8. Melatonin in hypoxic-ischemic brain injury

### 7. Melatonin in other neurodevelopmental disabilities (NDDs)

Reported prevalence of sleep disturbances in children with neurodevelopmental disabilities is up to 86% [81]. An interesting double-masked randomized placebo-controlled phase III trial was performed by Gringras et al. One hundred and forty-six children aged 3–15 years were treated with melatonin (0.5–12 mg) or placebo for 12 weeks [82]. In melatonin-treated group, the total sleep time increased by 23 min and sleep latency was reduced by around 38 min [82].

Melatonin may be affective in sleep problems in many genetic syndromes, especially in Angelman syndrome (AS), Smith-Magenis syndrome (SMS), Rett syndrome (RS), San Filippo syndrome, and tuberous sclerosis complex syndrome (TSCS) [83–90]. In these genetic conditions, sleep problems are one of the phenotype features. Sleep apnea is a frequent finding in children with Down syndrome and with Prader-Willi syndrome [83–91].

The results of Hancock et al. on the urinal 6-sulfatoxymelatonin excretion in seven TSCS patients revealed, however, no evidence of abnormal excretion of melatonin in patients with tuberous sclerosis complex and sleep disorder [55, 56, 83, 92]. All but one of the patients showed a normal circadian rhythm of melatonin secretion. However, the authors were aware that a small number of analyzed cases weakened their reasoning. Our investigations suggest that not only disordered sleep but also the shift of melatonin secretion may be expected in TSCS children with frequent seizures [88]. We also noticed that melatonin profiles are not homogeneous in TSCS patients [88]. Unfortunately, both researchers supposition are based on the results gathered from a small TSCS group.

Children with Angelman syndrome may present with sleep-onset insomnia as well as sleep maintenance problems, and low endogenous melatonin levels are often claimed to be an essential feature of melatonin secretion in their circadian rhythms [83–90]. Our studies with mathematical modeling of melatonin secretion showed that the phase parameters of melatonin cycle (DMLO parameters, phase or duration of melatonin amplitude) could be the key characteristic of AS children [87].

The recommended melatonin dose in Angelman syndrome is very small like 0.3–0.5 mg, because of the high prevalence of slow melatonin metabolizers [90]. In TSCS, a decreased sleep total time and multiple awakenings are evident; the recommended dose of melatonin is 5– 10 mg. Melatonin may reduce the sleep problems (the frequent awakenings) in Rett syndrome in the daily dose of 2.5–7.5 mg [83–85, 92]. Children with SMS have an early sleep onset (19.30– 20.30), repeated and prolonged walking at night, and an early sleep offset (04:00–05.00) [91]. Because of inverted melatonin circadian profile, a complex but promising treatment was found: a combination of acebutolol in the morning (10 mg/kg decrease melatonin level during the day) and melatonin in the evening [91].

During the conference in Rome in 2014, Bruni et al. postulated recommendation for melatonin treatment guidelines in children with neurodevelopmental disabilities and insomnia [15]:

1. no age limit (safe administration >6 months of age),

A few RDBPC trials showed that melatonin may improve communication [74] and anxiety in children with ASD [75]. Based on the knowledge from placebo-controlled studies, long-acting melatonin preparations at bedtime improve the sleep latency and the total sleep time [67, 69,

Reported prevalence of sleep disturbances in children with neurodevelopmental disabilities is up to 86% [81]. An interesting double-masked randomized placebo-controlled phase III trial was performed by Gringras et al. One hundred and forty-six children aged 3–15 years were treated with melatonin (0.5–12 mg) or placebo for 12 weeks [82]. In melatonin-treated group, the total sleep time increased by 23 min and sleep latency was reduced by around 38 min [82]. Melatonin may be affective in sleep problems in many genetic syndromes, especially in Angelman syndrome (AS), Smith-Magenis syndrome (SMS), Rett syndrome (RS), San Filippo syndrome, and tuberous sclerosis complex syndrome (TSCS) [83–90]. In these genetic conditions, sleep problems are one of the phenotype features. Sleep apnea is a frequent finding in

The results of Hancock et al. on the urinal 6-sulfatoxymelatonin excretion in seven TSCS patients revealed, however, no evidence of abnormal excretion of melatonin in patients with tuberous sclerosis complex and sleep disorder [55, 56, 83, 92]. All but one of the patients showed a normal circadian rhythm of melatonin secretion. However, the authors were aware that a small number of analyzed cases weakened their reasoning. Our investigations suggest that not only disordered sleep but also the shift of melatonin secretion may be expected in TSCS children with frequent seizures [88]. We also noticed that melatonin profiles are not homogeneous in TSCS patients [88]. Unfortunately, both researchers supposition are based on

Children with Angelman syndrome may present with sleep-onset insomnia as well as sleep maintenance problems, and low endogenous melatonin levels are often claimed to be an essential feature of melatonin secretion in their circadian rhythms [83–90]. Our studies with mathematical modeling of melatonin secretion showed that the phase parameters of melatonin cycle (DMLO parameters, phase or duration of melatonin amplitude) could be the key charac-

The recommended melatonin dose in Angelman syndrome is very small like 0.3–0.5 mg, because of the high prevalence of slow melatonin metabolizers [90]. In TSCS, a decreased sleep total time and multiple awakenings are evident; the recommended dose of melatonin is 5– 10 mg. Melatonin may reduce the sleep problems (the frequent awakenings) in Rett syndrome in the daily dose of 2.5–7.5 mg [83–85, 92]. Children with SMS have an early sleep onset (19.30– 20.30), repeated and prolonged walking at night, and an early sleep offset (04:00–05.00) [91].

7. Melatonin in other neurodevelopmental disabilities (NDDs)

130 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

children with Down syndrome and with Prader-Willi syndrome [83–91].

the results gathered from a small TSCS group.

teristic of AS children [87].

70, 74, 76–80].


### 8. Melatonin in hypoxic-ischemic brain injury

During the last decade, melatonin has started to be considered as an attractive option in order to minimize the neurological sequelae from hypoxic-ischemic brain injury [93–95]. The brain itself is particularly sensitive to free radicals damage due to its high utilization of oxygen, its relatively poorly developed antioxidant defense, and its high amount of easily oxidizable fatty acids. Melatonin may serve as a potential therapeutic free radical scavenger (hydroxyl radicals, hydrogen peroxide, singlet oxygen) and broad-spectrum antioxidant (upregulation of antioxidant pathways: superoxide dismutase, glutathione, catalase, glutathione peroxidase, glutathione reductase) [96–98]. Based on experimental studies, melatonin may increase the number of neurons in the CA1, CA2–CA3 areas and dentate gyrus of the hippocampus and parietal cortex, reduce the expression of the glial fibrillary acidic protein, and regulate the expression of myelin basic protein and oligodendrocytes' function (regulation of myelination process) [96–98].

Aly et al. examined the effect of melatonin on clinical, biochemical, neurophysiological, and radiological outcomes of neonates with hypoxic-ischemic encephalopathy (HIE) [99]. They performed a prospective trial involving 45 newborns randomized in the hypothermia alone and hypothermia and melatonin groups. All infants were studied with repeated EEG and brain MRI. In all patients, superoxide dismutase (SOD) and nitric oxide (NO) were measured. These examinations showed an increased melatonin and a decreased NO in the hypothermiamelatonin group [99]. Because of postulated unpredicted bioavailability of oral melatonin, Merchant et al. gave blood transfusion of 0.04–0.6 μg/kg melatonin to 18 preterm babies (less than 31 weeks gestation, less than 7 days old) for 2 h [100]. As a result they found melatonin concentration peak similar to adults. Another challenge might be the possibility to administer melatonin antenatally, in order to prevent or reduce brain hypoxic insult in preterm babies [94]. Denihan et al. employed untargeted metabolomics to identify metabolomic biomarkers of umbilical cord blood after hypoxic injury [101]. The analysis was performed using direct injection FT-ICR mass spectrometry. Some metabolites allowed for differentiation between children with perinatal asphyxia with recovery and children with perinatal asphyxia followed by hypoxic-ischemic encephalopathy like melatonin leucine, kynurenine, and 3-hydroxydodecanoic acid. HIE itself was associated with abnormalities in tryptophan and pyrimidine metabolism.

Author details

Justyna Paprocka<sup>1</sup>

References

Silesia, Katowice, Poland

ogy. 2017;15:434-443

186-195

\*, Marek Kijonka<sup>2</sup> and Maria Sokół

\*Address all correspondence to: justyna.paprocka@interia.pl

disrupted circadian timing. PLoS Genetics. 2008;30:1-8

Institute of Oncology, Gliwice Branch, Poland

2

Melatonin in Childhood Epilepsy and in Child Neurology http://dx.doi.org/10.5772/intechopen.80005 133

1 Department of Child Neurology, School of Medicine in Katowice, Medical University of

2 Department of Medical Physics, Maria Skłodowska-Curie Memorial Cancer Center and

[1] Lack L, Wright HR. Chronobiology of sleep in humans. CMLS. 2007;64:1205-1215

[2] Barnard AR, Nolan PM. When clocks go bad: Neurobehavioural consequences of

[3] Tordjman S, Chokron S, Delorme R, Charrier A, Bellissant E, Jaafari N, Fougerou C. Melatonin: Pharmacology, functions and therapeutic benefits. Current Neuropharmacol-

[4] Brzezinski A. Melatonin in humans. The New England Journal of Medicine. 1997;3363:

[5] Lewy AJ, Sack RL, Blood ML, Bauer VK, Cutler NL, Thomas KH. Melatonin marks circadian phase position and resets the endogenous circadian pacemaker in humans.

[6] Zisapel N. New perspectives on the role of melatonin in human sleep, circadian rhythms and their regulation. British Journal of Pharmacology. 2018. DOI: 10.1111/bph.14116

[7] Lewy AJ, Sack RL, Singer CM. Immediate and delayed effects of bright light on human melatonin production: Shifting "dawn" and "dusk" shifts the dim light melatonin onset

[8] Lewy AJ, Sack RL. The dim light melatonin onset as a marker for circadian phase position.

[9] Mandrell BN, Avent Y, Walker B, Loew M, Tynes BL, Crabtree VM. In home salivary melatonin collection: Methodology for children and adolescents. Developmental Psycho-

[10] Keijzer H, Smits MG, Peeters T, Looman CW, Endenburg SC, Gunnewiek JM. Evaluation of salivary melatonin measurements for dim Light Melatonin onset calculations in patients with possible sleep-wake rhythm disorders. Clinica Chimica Acta. 2011;17:417-418

(DLMO). Annals of the New York Academy of Sciences. 1985;453:253-259

Chronobiology International. 1989;6:93-102

biology. 2018;60:118-122

Ciba Foundation Symposium. 1995;183:303e17 [discussion 317e21]

Children after hypoxia-ischemia brain injury often develop circadian rhythm disorders. Yang et al. documented that in experimental studies, mRNA and protein expression of pineal arylalkylamine N-acetyltransferase (AANAT) and melatonin are impaired after hypoxic damage [102]. They postulated that miR-325-3p (micro RNA) may play a role of potential downregulator of AANAT-rate-limiting enzyme for melatonin synthesis [102].

### 9. Conclusions

Melatonin may be effective not only in primary sleep disorders but also in some abovementioned neurological disorders in children. In adults, postulated antioxidative potential of melatonin may be of value in neurodegenerative diseases like Parkinson, Alzheimer, and Huntington's disease [98].

Because disturbed circadian rhythms and poor sleep quality are associated with increased risks of cardiovascular, metabolic, and cognitive diseases, poor quality of life, and even with mortality, exogenously administered melatonin is often claimed to be a remedy for all these problems. However, many conflicting results obtained in various areas of research on the functions and roles of melatonin require caution and the extension of basic research [103– 108]. First of all further standardized studies of the human circadian rhythm and of its disturbances affecting melatonin rhythms by interfering with its production and secretion are necessary, as well as the studies of the interaction between circadian rhythm and seizures in animal models. Moreover, the melatonin role in epilepsy and the effects of antiepileptic drug treatment (in relation to the circadian rhythm phases) should be explored. As concerning exogenous melatonin application, larger study groups are required to identify proper therapeutic dosage regarding age, concrete disease, and to check the clinical efficacy of melatonin add-on therapy.

Filling up the gaps in the knowledge about the interactions of circadian rhythm, human epilepsy, and melatonin will improve our understanding of the undergoing processes and the patients' treatment quality.

### Conflict of interest

The authors declare no conflict of interest.

### Author details

[94]. Denihan et al. employed untargeted metabolomics to identify metabolomic biomarkers of umbilical cord blood after hypoxic injury [101]. The analysis was performed using direct injection FT-ICR mass spectrometry. Some metabolites allowed for differentiation between children with perinatal asphyxia with recovery and children with perinatal asphyxia followed by hypoxic-ischemic encephalopathy like melatonin leucine, kynurenine, and 3-hydroxydodecanoic acid. HIE itself was associated with abnormalities in tryptophan and pyrimidine

Children after hypoxia-ischemia brain injury often develop circadian rhythm disorders. Yang et al. documented that in experimental studies, mRNA and protein expression of pineal arylalkylamine N-acetyltransferase (AANAT) and melatonin are impaired after hypoxic damage [102]. They postulated that miR-325-3p (micro RNA) may play a role of potential down-

Melatonin may be effective not only in primary sleep disorders but also in some abovementioned neurological disorders in children. In adults, postulated antioxidative potential of melatonin may be of value in neurodegenerative diseases like Parkinson, Alzheimer, and

Because disturbed circadian rhythms and poor sleep quality are associated with increased risks of cardiovascular, metabolic, and cognitive diseases, poor quality of life, and even with mortality, exogenously administered melatonin is often claimed to be a remedy for all these problems. However, many conflicting results obtained in various areas of research on the functions and roles of melatonin require caution and the extension of basic research [103– 108]. First of all further standardized studies of the human circadian rhythm and of its disturbances affecting melatonin rhythms by interfering with its production and secretion are necessary, as well as the studies of the interaction between circadian rhythm and seizures in animal models. Moreover, the melatonin role in epilepsy and the effects of antiepileptic drug treatment (in relation to the circadian rhythm phases) should be explored. As concerning exogenous melatonin application, larger study groups are required to identify proper therapeutic dosage regarding age, concrete disease, and to check the clinical efficacy of melatonin

Filling up the gaps in the knowledge about the interactions of circadian rhythm, human epilepsy, and melatonin will improve our understanding of the undergoing processes and the

regulator of AANAT-rate-limiting enzyme for melatonin synthesis [102].

132 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

metabolism.

9. Conclusions

add-on therapy.

patients' treatment quality.

Conflict of interest

The authors declare no conflict of interest.

Huntington's disease [98].

Justyna Paprocka<sup>1</sup> \*, Marek Kijonka<sup>2</sup> and Maria Sokół 2

\*Address all correspondence to: justyna.paprocka@interia.pl

1 Department of Child Neurology, School of Medicine in Katowice, Medical University of Silesia, Katowice, Poland

2 Department of Medical Physics, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Poland

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**Chapter 7**

**Provisional chapter**

**Measurement of Plasma Tryptophan Metabolites:**

**Measurement of Plasma Tryptophan Metabolites:** 

**and Stress States Assessment**

**and Stress States Assessment**

Akikazu Takada, Fumiko Shimizu and

Akikazu Takada, Fumiko Shimizu and

http://dx.doi.org/10.5772/intechopen.78560

3-hydroxykynurenine, kynurenic acid

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Junichi Masuda

Junichi Masuda

**Abstract**

**1. Introduction**

important physiological roles.

**Clinical and Experimental Application for Depression**

**Clinical and Experimental Application for Depression** 

There are three pathways in tryptophan (TRP) metabolism. Serotonin (5-hydroxytryptamine; 5-HT) pathway is important in mood, anxiety, memory, and cognition and is impaired in depression. Kynurenine (KYN) pathways are involved in immunity, inflammation, muscles movement, and mental health. We investigated changes in TRP metabolites in plasmas of stressed rats and in depressive patients. TRP metabolite levels in 5-HT and KYN pathways in various brain areas and plasma were increased soon after electric foot shock given to rats but returned to normal 24 h later. Plasma levels of 5-HT were very low or undetectable in patients of monopolar depression. 5-hydroxyindole acetic acid (5-HIAA)/TRP ratios or KYN/TRP ratios were not different between healthy controls and depressive patients, indicating 5-HT quickly being degraded into 5-HIAA in patients of depression but KYN levels were not changed in depression. These results indicate that

TRP metabolism changes upon stress application and in patients of depression.

Tryptophan (TRP) is one of the essential amino acids, which must be taken as food. It is not only needed for protein synthesis but serves as a substrate for bioactive component with

**Keywords:** tryptophan, serotonin, 5-hydroxyindole acetic acid, kynurenine,

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.78560

#### **Measurement of Plasma Tryptophan Metabolites: Clinical and Experimental Application for Depression and Stress States Assessment Measurement of Plasma Tryptophan Metabolites: Clinical and Experimental Application for Depression and Stress States Assessment**

DOI: 10.5772/intechopen.78560

Akikazu Takada, Fumiko Shimizu and Junichi Masuda Akikazu Takada, Fumiko Shimizu and Junichi Masuda

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.78560

#### **Abstract**

There are three pathways in tryptophan (TRP) metabolism. Serotonin (5-hydroxytryptamine; 5-HT) pathway is important in mood, anxiety, memory, and cognition and is impaired in depression. Kynurenine (KYN) pathways are involved in immunity, inflammation, muscles movement, and mental health. We investigated changes in TRP metabolites in plasmas of stressed rats and in depressive patients. TRP metabolite levels in 5-HT and KYN pathways in various brain areas and plasma were increased soon after electric foot shock given to rats but returned to normal 24 h later. Plasma levels of 5-HT were very low or undetectable in patients of monopolar depression. 5-hydroxyindole acetic acid (5-HIAA)/TRP ratios or KYN/TRP ratios were not different between healthy controls and depressive patients, indicating 5-HT quickly being degraded into 5-HIAA in patients of depression but KYN levels were not changed in depression. These results indicate that TRP metabolism changes upon stress application and in patients of depression.

**Keywords:** tryptophan, serotonin, 5-hydroxyindole acetic acid, kynurenine, 3-hydroxykynurenine, kynurenic acid

#### **1. Introduction**

Tryptophan (TRP) is one of the essential amino acids, which must be taken as food. It is not only needed for protein synthesis but serves as a substrate for bioactive component with important physiological roles.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

There are three pathways in TRP metabolism: serotonin (5-HT), kynurenine (KYN), and indole-3-acetic acid pathways.

The targets are 17 of major metabolites, tryptophan (TRP), L-5-hydroxytryptophan (5-HTP), serotonin (5-HT), kynurenine (KYN), 5-hydroxy-tryptophol, tryptophol, 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid, anthranilic acid (AA), kynurenic acid (KYNA), quinaldic acid, 3-indolebutyric acid, 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), melatonin, and quinolinic acid (QA). Each compound is commercially available from major chemical regent manufacturers, such as Fujifilm-Wako chemical (Osaka,

Measurement of Plasma Tryptophan Metabolites: Clinical and Experimental Application…

http://dx.doi.org/10.5772/intechopen.78560

145

Analysis was performed by a liquid chromatograph tandem mass spectrometer, the LCMS-8060 mass spectrometer equipped with Nexera X2 liquid chromatograph system (Shimadzu

The targets are separated by reversed phase mode using ODS analytical column, L-Columns ODS (2.1 mm x 150 mm, CERI, Tokyo, Japan) with a gradient elution. Mobile phases were 0.1% formic acid solution and acetonitrile with 5% concentration of acetonitrile to 3 min, then 5–95% in 6 min followed by 5% in 3 min (12 min analytical cycle) at total flow rate of 0.4 mL/min. The

> **Monoisotopic mass**

DL-Trypotophan 204.225 204.090 205.10 188.10 205.10 146.10

Serotonin 176.215 176.095 177.10 160.10 177.10 115.05 L-Kynurenine 208.214 208.085 209.10 192.00 209.10 94.05 5-Hydroxytrypotophol 177.200 177.079 178.10 160.10 178.10 115.05 Trypotophol 161.200 161.084 162.10 144.05 162.10 117.10

Indole-3-acetic acid 175.184 175.063 176.10 130.05 176.10 77.05 Anthranilic acid 136.129 136.040 138.10 120.05 138.10 65.05 Kynurenic acid 189.167 189.043 190.10 144.05 190.10 89.10 Quinaldic acid 172.161 172.040 174.10 128.05 174.10 156.05 Indole-3-acetic acid 202.230 202.087 204.10 186.10 204.10 130.10 3-Hydroxykynurenine 224.213 224.080 225.15 208.20 225.15 162.15

Xanthurenic acid 205.167 205.038 206.15 160.20 206.15 132.20 Melatonin 232.279 232.121 232.20 174.10 232.20 130.05 Quinolinic acid 167.120 167.022 168.00 78.10 168.00 150.00

220.225 220.085 221.10 204.05 221.10 162.00

190.176 190.051 192.10 146.05 192.10 110.00

152.128 152.035 154.15 136.20 154.15 80.15

**Qualification (m/z) Qualification (m/z)**

**Precursorion Production Precursorion Production**

Japan) and Sigma-Aldrich (St. Louis, MO, USA).

**weight**

Corporation, Kyoto, Japan).

**Compound Molecular** 

L-5-

Hydroxytrypotophan

5-Hydroxyindole -3-acetic acid

Hydroxyanthranilic

**Table 1.** MRM transition.

acid

Since 5-HT is known as an important neurotransmitter and derived from TRP, many people know about TRP to some extent. 5-HT is really involved in the adaptive responses in the central nervous system and considered to be related to mood, anxiety, or cognition [1].

5-HT is further converted in the pineal body and the retina to N-acetyl serotonin (NAS) and melatonin which controls circadian rhythm [2].

In mammals, most of the free TRP is converted to KYN and generates metabolites involved in inflammatory, immune, responses, and neurotransmission [3].

We have recently succeeded in simultaneous measurements of almost all TRP metabolites including melatonin by using an ultrahigh speed liquid chrom`atography and mass spectrometry (LC-MS) [4], which is the first time in the world.

In this chapter, we report about the precise methodology of the simultaneous measurements and some results obtained from stressed rats and depressive patients.

#### **1.1. Measurements of TRP metabolites using LC-MS**

#### *1.1.1. Background*

Since 5-HT and its derivatives have the strong fluorescence, high-sensitive analyses of some TRP metabolites can be performed by liquid chromatography with fluorescence detection. However, it is very limited to analyze the total metabolites including kynurenine by photometric detection although kynurenic acid (KYNA) can be detected as zinc chelate compound under zinc acetate solution.

In the past years, liquid chromatograph mass spectrometry (LC-MS) has been widely spread in many areas including clinical and biological analyses. Since LC-MS with electrospray ionization (ESI) is suitable for determination of metabolites of TRP, LC-MS is expected as a powerful tool for this type of analysis.

In addition, the tandem mass spectrometer also has widely used in recent years with its high sensitivity and selectivity.

There are several researches for the simultaneous analyses of TRP metabolites using LC-MS or LC-MS/MS methods for clinical samples such as human serum and plasma [5, 6]. In general, isotope-labeled internal standards are used in LC-MS/MS analysis to improve the accuracy, although isotope-labeled regents are expensive and limited availability [7, 8]. Even though less accurate, acceptable results can be obtained without internal standards for the screening purposes. In this chapter, the simultaneous determination of TRP metabolites in human plasma by LC-MS/MS technique combined with simple pretreatment procedure is described.

#### *1.1.2. Regents and instrumentation*

The simultaneous analytical method was developed for major metabolites of TRP including melatonin.

The targets are 17 of major metabolites, tryptophan (TRP), L-5-hydroxytryptophan (5-HTP), serotonin (5-HT), kynurenine (KYN), 5-hydroxy-tryptophol, tryptophol, 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid, anthranilic acid (AA), kynurenic acid (KYNA), quinaldic acid, 3-indolebutyric acid, 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HAA), xanthurenic acid (XA), melatonin, and quinolinic acid (QA). Each compound is commercially available from major chemical regent manufacturers, such as Fujifilm-Wako chemical (Osaka, Japan) and Sigma-Aldrich (St. Louis, MO, USA).

Analysis was performed by a liquid chromatograph tandem mass spectrometer, the LCMS-8060 mass spectrometer equipped with Nexera X2 liquid chromatograph system (Shimadzu Corporation, Kyoto, Japan).

The targets are separated by reversed phase mode using ODS analytical column, L-Columns ODS (2.1 mm x 150 mm, CERI, Tokyo, Japan) with a gradient elution. Mobile phases were 0.1% formic acid solution and acetonitrile with 5% concentration of acetonitrile to 3 min, then 5–95% in 6 min followed by 5% in 3 min (12 min analytical cycle) at total flow rate of 0.4 mL/min. The


**Table 1.** MRM transition.

There are three pathways in TRP metabolism: serotonin (5-HT), kynurenine (KYN), and

Since 5-HT is known as an important neurotransmitter and derived from TRP, many people know about TRP to some extent. 5-HT is really involved in the adaptive responses in the central nervous system and considered to be related to mood, anxiety, or cognition [1].

5-HT is further converted in the pineal body and the retina to N-acetyl serotonin (NAS) and

In mammals, most of the free TRP is converted to KYN and generates metabolites involved in

We have recently succeeded in simultaneous measurements of almost all TRP metabolites including melatonin by using an ultrahigh speed liquid chrom`atography and mass spec-

In this chapter, we report about the precise methodology of the simultaneous measurements

Since 5-HT and its derivatives have the strong fluorescence, high-sensitive analyses of some TRP metabolites can be performed by liquid chromatography with fluorescence detection. However, it is very limited to analyze the total metabolites including kynurenine by photometric detection although kynurenic acid (KYNA) can be detected as zinc chelate compound

In the past years, liquid chromatograph mass spectrometry (LC-MS) has been widely spread in many areas including clinical and biological analyses. Since LC-MS with electrospray ionization (ESI) is suitable for determination of metabolites of TRP, LC-MS is expected as a

In addition, the tandem mass spectrometer also has widely used in recent years with its high

There are several researches for the simultaneous analyses of TRP metabolites using LC-MS or LC-MS/MS methods for clinical samples such as human serum and plasma [5, 6]. In general, isotope-labeled internal standards are used in LC-MS/MS analysis to improve the accuracy, although isotope-labeled regents are expensive and limited availability [7, 8]. Even though less accurate, acceptable results can be obtained without internal standards for the screening purposes. In this chapter, the simultaneous determination of TRP metabolites in human plasma by LC-MS/MS technique combined with simple pretreatment procedure is described.

The simultaneous analytical method was developed for major metabolites of TRP including

indole-3-acetic acid pathways.

*1.1.1. Background*

under zinc acetate solution.

sensitivity and selectivity.

*1.1.2. Regents and instrumentation*

melatonin.

powerful tool for this type of analysis.

melatonin which controls circadian rhythm [2].

144 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

inflammatory, immune, responses, and neurotransmission [3].

and some results obtained from stressed rats and depressive patients.

trometry (LC-MS) [4], which is the first time in the world.

**1.1. Measurements of TRP metabolites using LC-MS**

temperature of the column was 40°C. For LC-MS, electrospray ionization (ESI) was used with multi-reaction monitoring (MRM) mode.

Flow rate of the neutralizer and the drying gas were 2 L/min and 10 mL/min, respectively. The temperature of desolvation line (heated capitally tube) was 250°C. ESI interface was used at 400°C with 10 L/min of heating gas flow. Each MRM transition was optimized using each standard solution. Optimized results are shown in **Table 1**.

All mother solution of 1 mg/mL had been stocked under −80°C and standard samples for calibration curve were prepared before use as mixture solution by consideration of each range of measurement concentration.

#### *1.1.3. Analysis of human plasma*

Aliquot of 50 μL human plasma was used for each sample analysis. The procedure including deproteinization is shown in **Figure 1**. The typical chromatograms of 17 major metabolites are shown in **Figure 2** as standard solution and in **Figure 3** as human plasma sample. These chromatograms demonstrate the usefulness of the developed method for simultaneous analysis of TRP metabolites.

**1.2. Stress and TRP metabolism**

**Figure 2.** Chromatograms of 17 major metabolites of tryptophan.

noradrenaline and 5-HT [16, 17].

**Figure 3.** Chromatogram of human plasma sample.

Stress influences many functions related to mental and body health. We have shown that the application of electric shock increased plasma and brain TRP, 5-HT, and 5-HIAA levels [9, 10] and changed nicotine-induced release of 5-HT or dopamine in rats [11–14]. We also examined

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147

Stress induces a number of changes in the central system of neurotransmitters, particularly

A pathologic overabundance of endogenous excitotoxin, quinolinic acid, or hypofunction of KYNA has been hypothetically linked to the occurrence of seizures and nerve cell death [18, 19].

changes in serotonergic and kynurenic pathways in rats exposed to foot shock [15].

**Figure 1.** The procedure of deproteinization.

**Figure 2.** Chromatograms of 17 major metabolites of tryptophan.

#### **1.2. Stress and TRP metabolism**

Stress influences many functions related to mental and body health. We have shown that the application of electric shock increased plasma and brain TRP, 5-HT, and 5-HIAA levels [9, 10] and changed nicotine-induced release of 5-HT or dopamine in rats [11–14]. We also examined changes in serotonergic and kynurenic pathways in rats exposed to foot shock [15].

Stress induces a number of changes in the central system of neurotransmitters, particularly noradrenaline and 5-HT [16, 17].

A pathologic overabundance of endogenous excitotoxin, quinolinic acid, or hypofunction of KYNA has been hypothetically linked to the occurrence of seizures and nerve cell death [18, 19].

**Figure 3.** Chromatogram of human plasma sample.

**Figure 1.** The procedure of deproteinization.

temperature of the column was 40°C. For LC-MS, electrospray ionization (ESI) was used with

Flow rate of the neutralizer and the drying gas were 2 L/min and 10 mL/min, respectively. The temperature of desolvation line (heated capitally tube) was 250°C. ESI interface was used at 400°C with 10 L/min of heating gas flow. Each MRM transition was optimized using each

All mother solution of 1 mg/mL had been stocked under −80°C and standard samples for calibration curve were prepared before use as mixture solution by consideration of each range

Aliquot of 50 μL human plasma was used for each sample analysis. The procedure including deproteinization is shown in **Figure 1**. The typical chromatograms of 17 major metabolites are shown in **Figure 2** as standard solution and in **Figure 3** as human plasma sample. These chromatograms demonstrate the usefulness of the developed method for simultaneous analysis

multi-reaction monitoring (MRM) mode.

146 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

of measurement concentration.

*1.1.3. Analysis of human plasma*

of TRP metabolites.

standard solution. Optimized results are shown in **Table 1**.

#### *1.2.1. Stress and TRP metabolites in the brain*

Animals: Male Wistar rats (9 weeks old) were fed with standard laboratory foods and tap water *ad libitum*. Two weeks later, rats were randomly divided into two groups. One group received foot shock, which was given as a series of 10-s shock (o.19 mA) followed by 50 s intervals during a 60-min period. CT 110 cycle timer and NS-SG01 shock generator scrambler (Neuroscience Inc., Tokyo, Japan) were used. Samples of 10 rats were taken immediately after the foot shock and samples of another 10 rats were taken 24 h after the shock. Ten rats were used as controls.

Blood: Rats were anesthetized with pentobarbital, and blood was taken by heart puncture and put into a tube containing 3/13% sodium citrate. Plasma was obtained by centrifugation at 3000 rpm for 20 min.

Brain sampling: The brains were removed and chilled on ice. Eight regions (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus, cortex, and frontal cortex) were dissected and samples were immediately frozen. Frozen brain samples were homogenized in 0.15 N perchloric acid containing 0.025% EDTA (pH 3.0). The samples were centrifuged at 14,000 rpm for 20 min at 4°C. After centrifugation, the supernatant was filtered (0.45 μm Millipore filter) and stored at −80°C until assayed.

**Figure 4** shows TRP pathway and its metabolites.

**Figure 5** shows that 5-HT levels increased significantly at hypothalamus and midbrain soon after the shock but returned normal 24 h later.

5-HT levels were not increased in cerebellum, medulla, striatum, hippocampus, cortex, and frontal cortex.

**Figure 6** shows 5-HIAA levels of various brain areas after foot shock.

5-HIAA levels significantly increased in all the brain areas except striatum but returned normal 24 h later.

**Figure 7** shows KYN levels in various brain areas after foot shock.

KYN levels significantly increased in all the brain areas after foot shock but returned to normal 24 h later.

recruited them if there were no health problems such as diabetes, hypertension, or no serious diseases experienced in the past. They did not smoke in the past. We also excluded people who took drugs for dyslipidemia, hyperglycemia, or hypertension. We collected blood samples early morning. Participants were asked not to eat anything after 21.00 PM the previous evening. Plasma specimens were collected for assays of blood parameters. We obtained an informed consent prior to conducting the protocol which had been approved by the Ethical Committee of Showa Women's University and Yokohama North Hospital of

CO2H

CH2.CH.COOH

NH2

CO.CH2.CH.COOH

Tryptophan

NH2

Kynurenine

Measurement of Plasma Tryptophan Metabolites: Clinical and Experimental Application…

**5-hydroxytryptophan**

Serotonin

149

**5-hydroxyindole acetic acid**

http://dx.doi.org/10.5772/intechopen.78560

Melationin

CO-CH2.CH.COOH

NH2

NH2

N

OH

OH

OHC

COOH

COOH

*nonenzym atic*

HOOC

*3,4-dioxygenase*

N

*hydroxyanthraniate*

*3.*

COOH

OH

Kynurenic acid

N

*quinolinate ohosphoribosyl transferase*

*Kynurenine aminotransferases I.II.III*

**indole-3-acetic acid**

*Tryptophan dioxgenase or Indoleamine dioxygenase*

> *Kynurenine hydroxylase*

*Kynureninase*

NH2

COOH

CO2H

Quinolinic acid Picolinic acid

N

NH2

NH2

2-amino-3-carboxymuconatesemialdehyde *2-amino-3-carboxymuconatesemialdehyde decarboxylase*

3-Hydroxykynurenine

3-Hydroxyanthranilic acid

Patients were diagnosed to be monopolar depression at the psychiatry clinic of Yokohama North Hospital of Showa University. The Zung Self-Rating Depression Scale [20] was used, which is a short self-administered survey to quantify the depressed status of a patient. There are 20 items on the scale that rate the affective, psychological, and somatic symptoms associated with depression. We took blood from five male patients (26, 30, 45, 47, and 56 years old)

Showa University.

**Figure 4.** TRP pathways.

NAD

and four female patients (23, 25, 41, and 60 years old).

**Figure 8** shows plasma levels of TRP, 5-HT and 5-HIAA after foot shock.

Plasma levels of TRP, 5-HT, and 5-HIAA significantly increased after foot shock but returned to normal 24 h later.

**Figure 9** shows plasma levels of KYN,3-HKYN, and KYNA after foot shock.

Plasma levels of KYN, 3-HKYN, and KYNA increased significantly after foot shock but returned to normal 24 h later.

#### **1.3. TRP metabolites in plasma of patients of depression**

We asked male and female acquaintances older than 50 years old and male and female college students to participate in the experiments. We checked their health carefully and Measurement of Plasma Tryptophan Metabolites: Clinical and Experimental Application… http://dx.doi.org/10.5772/intechopen.78560 149

**Figure 4.** TRP pathways.

*1.2.1. Stress and TRP metabolites in the brain*

148 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Millipore filter) and stored at −80°C until assayed. **Figure 4** shows TRP pathway and its metabolites.

after the shock but returned normal 24 h later.

at 3000 rpm for 20 min.

frontal cortex.

mal 24 h later.

mal 24 h later.

to normal 24 h later.

returned to normal 24 h later.

Animals: Male Wistar rats (9 weeks old) were fed with standard laboratory foods and tap water *ad libitum*. Two weeks later, rats were randomly divided into two groups. One group received foot shock, which was given as a series of 10-s shock (o.19 mA) followed by 50 s intervals during a 60-min period. CT 110 cycle timer and NS-SG01 shock generator scrambler (Neuroscience Inc., Tokyo, Japan) were used. Samples of 10 rats were taken immediately after the foot shock and samples of another 10 rats were taken 24 h after the shock. Ten rats were used as controls. Blood: Rats were anesthetized with pentobarbital, and blood was taken by heart puncture and put into a tube containing 3/13% sodium citrate. Plasma was obtained by centrifugation

Brain sampling: The brains were removed and chilled on ice. Eight regions (cerebellum, medulla, hypothalamus, striatum, midbrain, hippocampus, cortex, and frontal cortex) were dissected and samples were immediately frozen. Frozen brain samples were homogenized in 0.15 N perchloric acid containing 0.025% EDTA (pH 3.0). The samples were centrifuged at 14,000 rpm for 20 min at 4°C. After centrifugation, the supernatant was filtered (0.45 μm

**Figure 5** shows that 5-HT levels increased significantly at hypothalamus and midbrain soon

5-HT levels were not increased in cerebellum, medulla, striatum, hippocampus, cortex, and

5-HIAA levels significantly increased in all the brain areas except striatum but returned nor-

KYN levels significantly increased in all the brain areas after foot shock but returned to nor-

Plasma levels of TRP, 5-HT, and 5-HIAA significantly increased after foot shock but returned

Plasma levels of KYN, 3-HKYN, and KYNA increased significantly after foot shock but

We asked male and female acquaintances older than 50 years old and male and female college students to participate in the experiments. We checked their health carefully and

**Figure 6** shows 5-HIAA levels of various brain areas after foot shock.

**Figure 7** shows KYN levels in various brain areas after foot shock.

**1.3. TRP metabolites in plasma of patients of depression**

**Figure 8** shows plasma levels of TRP, 5-HT and 5-HIAA after foot shock.

**Figure 9** shows plasma levels of KYN,3-HKYN, and KYNA after foot shock.

recruited them if there were no health problems such as diabetes, hypertension, or no serious diseases experienced in the past. They did not smoke in the past. We also excluded people who took drugs for dyslipidemia, hyperglycemia, or hypertension. We collected blood samples early morning. Participants were asked not to eat anything after 21.00 PM the previous evening. Plasma specimens were collected for assays of blood parameters. We obtained an informed consent prior to conducting the protocol which had been approved by the Ethical Committee of Showa Women's University and Yokohama North Hospital of Showa University.

Patients were diagnosed to be monopolar depression at the psychiatry clinic of Yokohama North Hospital of Showa University. The Zung Self-Rating Depression Scale [20] was used, which is a short self-administered survey to quantify the depressed status of a patient. There are 20 items on the scale that rate the affective, psychological, and somatic symptoms associated with depression. We took blood from five male patients (26, 30, 45, 47, and 56 years old) and four female patients (23, 25, 41, and 60 years old).

**Figure 5.** Serotonin (5-HT) levels in various brain areas after electric foot shock. con, control; E0, soon after the shock; E1, 24 h after the shock. \*p < 0.05 and \*\*p < 0.01.

5-HT was detected only in plasma of two patients. Both of them were young females. A 60-yearold woman took Jay Zoloft 25 mg and ethyl loflazepate 1 mg, and another 41-year-old woman took Cymbalta 20 mg and Luran 3 mg. 5-HT was not detected in plasmas of these women.

**Figure 7.** Kynurenine levels in various brain areas after foot shock. con, control; E0, soon after the shock; E1, 24 h after

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151

The number of healthy old men and young women were 20, and the number of depressive

**Figure 8.** Plasma levels of TRP, 5-HT, and 5-HIAA after electric foot shock. con, control; E0, soon after the shock; E1, 24 h

patients were 9.

the shock. \*\*p < 0.01 and \*\*\*p < 0.001.

after the shock. \*p < 0.05 and \*\*p < 0.01.

**Figure 6.** 5-HIAA levels in various brain areas after foot shock. con, control; E0, soon after the shock; E1, 24 h after the shock. \*p < 0.05.

Measurement of Plasma Tryptophan Metabolites: Clinical and Experimental Application… http://dx.doi.org/10.5772/intechopen.78560 151

**Figure 7.** Kynurenine levels in various brain areas after foot shock. con, control; E0, soon after the shock; E1, 24 h after the shock. \*\*p < 0.01 and \*\*\*p < 0.001.

**Figure 5.** Serotonin (5-HT) levels in various brain areas after electric foot shock. con, control; E0, soon after the shock; E1,

**Figure 6.** 5-HIAA levels in various brain areas after foot shock. con, control; E0, soon after the shock; E1, 24 h after the

24 h after the shock. \*p < 0.05 and \*\*p < 0.01.

150 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

shock. \*p < 0.05.

5-HT was detected only in plasma of two patients. Both of them were young females. A 60-yearold woman took Jay Zoloft 25 mg and ethyl loflazepate 1 mg, and another 41-year-old woman took Cymbalta 20 mg and Luran 3 mg. 5-HT was not detected in plasmas of these women.

The number of healthy old men and young women were 20, and the number of depressive patients were 9.

**Figure 8.** Plasma levels of TRP, 5-HT, and 5-HIAA after electric foot shock. con, control; E0, soon after the shock; E1, 24 h after the shock. \*p < 0.05 and \*\*p < 0.01.

**Figure 9.** Plasma levels of KYN (kynurenine), 3-HKYN (3-hydroxykynurenine), and KYNA (kynurenic acid). con, control; E0, soon after the shock; E1, 24 h after the shock. \*\*p < 0.01 and \*\*\*p < 0.001.


**Tables 2** and **3** show the background data of these patients.

**Figure 10.** 5-HT levels in plasmas of depressive patients and controls.

**Figure 11** shows 5-HT/TRP and 5-HIAA/TRP ratios.

**Figure 11.** 5-HT or 5-HIAA/TRP ratio.

young women. 5-HT was detected in plasmas of only two young females.

**Figure 10** shows plasma levels of 5-HT of healthy young women, old men and depressive patients. Plasma levels of 5-HT in depressive patients were very low compared to those of old men and

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Although 5-HT/TRP ratio of depressive patients was low compared to that of old men and young women, 5-HIAA/TRP ratio was almost the same as that of old men and young women.

**Table 2.** Basic backgrounds of healthy participants.


**Table 3.** Various parameters of patients.

**Figure 10.** 5-HT levels in plasmas of depressive patients and controls.

**Figure 9.** Plasma levels of KYN (kynurenine), 3-HKYN (3-hydroxykynurenine), and KYNA (kynurenic acid). con,

**Young men (n = 20) Young women (n = 20) Old men (n = 20)**

control; E0, soon after the shock; E1, 24 h after the shock. \*\*p < 0.01 and \*\*\*p < 0.001.

152 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**Table 2.** Basic backgrounds of healthy participants.

**Table 3.** Various parameters of patients.

Age 20.7 ± 1.5 21.2 ± 0.7 60.8 ± 9.9 Height (m) 1.72 ± 0.06 1.58 ± 0.05 1.69 ± 0.07 Weight (kg) 65.1 ± 8.9 51.4 ± 5.8 71.1 ± 13.1 BMI 22.1 ± 3.1 20.4 ± 1.6 24.9 ± 3.7

**Patients Sex Serotonin detection Visit (age) Medication** H1 Male — 45 None H2 Male — 47 None

H4 Female + 23 None H5 Female + 25 None H6 Male — 30 None

H8 Male — 56 None H9 Male — 26 None

H3 Female — 60 Jay Zoloft 25 mg, ethyl

H7 Female — 41 Cymbalta 20 mg, Luran 3 mg

loflazepate1 mg

**Tables 2** and **3** show the background data of these patients.

**Figure 10** shows plasma levels of 5-HT of healthy young women, old men and depressive patients.

Plasma levels of 5-HT in depressive patients were very low compared to those of old men and young women. 5-HT was detected in plasmas of only two young females.

**Figure 11** shows 5-HT/TRP and 5-HIAA/TRP ratios.

Although 5-HT/TRP ratio of depressive patients was low compared to that of old men and young women, 5-HIAA/TRP ratio was almost the same as that of old men and young women.

**Figure 11.** 5-HT or 5-HIAA/TRP ratio.

Some of TRP metabolites were shown to be neuroexcitatory and convulsive, thus toxic [29]. One of such neuroexcitatory factors in KYN pathway is 3-HKYN [24]. Its synthesis is catalyzed by kynurenine 3-hydroxylase. Although data of brain analyses were not shown here,

Measurement of Plasma Tryptophan Metabolites: Clinical and Experimental Application…

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3-HKYN is said to be the most toxic substance in TRP metabolism [30]. So stress induces disturbances in the central nervous system by increasing levels of 3-HKYN (**Figures 4**–**9**). KYN is usually hydroxylated to 3-HKYN and then further converted to 3-hydroxyanthranilic acid (3-HAA). 3-HAA is rapidly converted to QN by the non-enzymatic reaction and further to NAD<sup>+</sup>

The other pathway of KYN, the production of KYNA and xanthurenic acid (XA), is minor under normal conditions. KYNA is an endogenous antagonist of excitatory amino acid receptors and may serve as a modulator of excitatory nerve transmission. This study may suggest that stress induces the indirect modulation of excitatory amino acids in the central nervous

KYNA has an antagonist activity of the three ionotropic excitatory amino acid receptors [22, 31]. At low concentrations, KYNA blocks the glycine co-agonist site of N-methyl-D-aspartate receptor and may serve to prevent the overactivation of glutamic acid receptor. When brain kynurenic acid levels were increased experimentally, neuroprotection and seizure reduction

The roles of various metabolites of KYN pathway are reviewed [28], so we do not discuss

As to relationships between serotonin levels and depression, we analyzed plasma levels of

Although the concentration of 5-HT has been considered to be low in depressive patients [33], 5-HT concentration in the brains of suicide victims was not low [34]. Therefore, it is not known if 5-HT concentration is decreased in the brains of depressive patients (**Table 2**).

We decided to measure TRP metabolites in patients of monopolar depression. **Table 3** shows five male patients participated in the experiments. The age is 44–61 years old (41.2 ± 11.3). Plasma serotonin levels were detected only in two young female patients. We could not mea-

Although plasma serotonin levels and 5-HT/TRP ratio were low in depressive patients, the levels of 5-HIAA/TRP were not lower in depressive patients. This result indicates that 5-HT is degraded to 5-HIAA in depressive patients almost to the same extent to healthy old and

We measured the levels of KYN in healthy old men and young women and depressive patients.

These results suggest that in depressive patients 5-HT was quickly degraded to 5-HIAA, and

As to a relationship between serotonin pathway and KYN pathway, Lapin IP suggested that in depression tryptophan 2,3-dioxygenase in the liver shunted metabolism of serotonin away from 5-HT production to KYN production, resulting in serotonin deficiency [35]. KYN,

TRP seems to be degraded to KYN pathways to the same extent in these three groups.

sure 5-HT in plasma in seven persons, which is shown in **Figure 10**.

this seems to be a reason of low 5-HT levels in depressive patients.

.

155

3-HKYN levels increased in all the brain areas and plasma (**Figure 6**).

system by increasing KYNA.

have been reported [32].

TRP metabolites in patients of depression.

young women (**Figures 11** and **12**).

these roles in detail.

**Figure 12.** KYN/TRP ratio.

**Figure 12** shows that there were no differences in KYN/TRP ratios between that of old men, young women, and depressive patients.

### **2. Discussion**

As stated above, most of the metabolites of KYN pathway are found in the brain [1, 21–23]. Some metabolites of KYN pathway are neurotoxic and some are neuroprotective.

KYNA is an endogenous neuroprotective agent that is usually present in the brain at nanomolar concentration [24]. KYNA is an antagonist to quinolinic acid (QA) and acts on the glycine modulatory site of the NMDA receptor at low concentration [22] and at higher concentration at the glutamate site of the NMDA receptors and also on the a-amino3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors [25]. It also antagonizes the alpha 7 nicotinic acetylcholine receptors [26] and selectively activates a G-protein-coupled receptor, GPR35–48 [27]. Many neuroactive intermediates are shown in KYN pathways [28]. So, we decided to measure some of TRP metabolites in the brain and plasma in the stressed rats.

Our results show that 5-HT levels increased only in hypothalamus and midbrain, but its degradation product, 5-HIAA levels, increased in every part of the brain. These results may imply that 5-HT is quickly converted to 5-HIAA soon after shock, so that 5-HT levels apparently did not increase after shock. This finding is important in the explanation of plasma 5-HT and 5-HIAA levels in patients of depression.

Some of TRP metabolites were shown to be neuroexcitatory and convulsive, thus toxic [29]. One of such neuroexcitatory factors in KYN pathway is 3-HKYN [24]. Its synthesis is catalyzed by kynurenine 3-hydroxylase. Although data of brain analyses were not shown here, 3-HKYN levels increased in all the brain areas and plasma (**Figure 6**).

3-HKYN is said to be the most toxic substance in TRP metabolism [30]. So stress induces disturbances in the central nervous system by increasing levels of 3-HKYN (**Figures 4**–**9**).

KYN is usually hydroxylated to 3-HKYN and then further converted to 3-hydroxyanthranilic acid (3-HAA). 3-HAA is rapidly converted to QN by the non-enzymatic reaction and further to NAD<sup>+</sup> .

The other pathway of KYN, the production of KYNA and xanthurenic acid (XA), is minor under normal conditions. KYNA is an endogenous antagonist of excitatory amino acid receptors and may serve as a modulator of excitatory nerve transmission. This study may suggest that stress induces the indirect modulation of excitatory amino acids in the central nervous system by increasing KYNA.

KYNA has an antagonist activity of the three ionotropic excitatory amino acid receptors [22, 31]. At low concentrations, KYNA blocks the glycine co-agonist site of N-methyl-D-aspartate receptor and may serve to prevent the overactivation of glutamic acid receptor. When brain kynurenic acid levels were increased experimentally, neuroprotection and seizure reduction have been reported [32].

The roles of various metabolites of KYN pathway are reviewed [28], so we do not discuss these roles in detail.

As to relationships between serotonin levels and depression, we analyzed plasma levels of TRP metabolites in patients of depression.

**Figure 12** shows that there were no differences in KYN/TRP ratios between that of old men,

As stated above, most of the metabolites of KYN pathway are found in the brain [1, 21–23].

KYNA is an endogenous neuroprotective agent that is usually present in the brain at nanomolar concentration [24]. KYNA is an antagonist to quinolinic acid (QA) and acts on the glycine modulatory site of the NMDA receptor at low concentration [22] and at higher concentration at the glutamate site of the NMDA receptors and also on the a-amino3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors [25]. It also antagonizes the alpha 7 nicotinic acetylcholine receptors [26] and selectively activates a G-protein-coupled receptor, GPR35–48 [27]. Many neuroactive intermediates are shown in KYN pathways [28]. So, we decided to measure

Our results show that 5-HT levels increased only in hypothalamus and midbrain, but its degradation product, 5-HIAA levels, increased in every part of the brain. These results may imply that 5-HT is quickly converted to 5-HIAA soon after shock, so that 5-HT levels apparently did not increase after shock. This finding is important in the explanation of plasma 5-HT and

Some metabolites of KYN pathway are neurotoxic and some are neuroprotective.

some of TRP metabolites in the brain and plasma in the stressed rats.

young women, and depressive patients.

154 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

5-HIAA levels in patients of depression.

**2. Discussion**

**Figure 12.** KYN/TRP ratio.

Although the concentration of 5-HT has been considered to be low in depressive patients [33], 5-HT concentration in the brains of suicide victims was not low [34]. Therefore, it is not known if 5-HT concentration is decreased in the brains of depressive patients (**Table 2**).

We decided to measure TRP metabolites in patients of monopolar depression. **Table 3** shows five male patients participated in the experiments. The age is 44–61 years old (41.2 ± 11.3). Plasma serotonin levels were detected only in two young female patients. We could not measure 5-HT in plasma in seven persons, which is shown in **Figure 10**.

Although plasma serotonin levels and 5-HT/TRP ratio were low in depressive patients, the levels of 5-HIAA/TRP were not lower in depressive patients. This result indicates that 5-HT is degraded to 5-HIAA in depressive patients almost to the same extent to healthy old and young women (**Figures 11** and **12**).

We measured the levels of KYN in healthy old men and young women and depressive patients. TRP seems to be degraded to KYN pathways to the same extent in these three groups.

These results suggest that in depressive patients 5-HT was quickly degraded to 5-HIAA, and this seems to be a reason of low 5-HT levels in depressive patients.

As to a relationship between serotonin pathway and KYN pathway, Lapin IP suggested that in depression tryptophan 2,3-dioxygenase in the liver shunted metabolism of serotonin away from 5-HT production to KYN production, resulting in serotonin deficiency [35]. KYN, QN, and 3-HKYN were shown to be anxiogenic and KYNA were anxiolytic [36]. From these results, he tried to explain the effects of antidepressive drugs.

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[4] Matsuoka K, Kato K, Takao T, Ogawa M, Ishii Y, Shimizu F, Masuda J, Takada A. Concentrations of various tryptophan metabolites increase in patients of diabetes mellitus compared to healthy aged male adults. Diabetology International. 2016;**8**:69-72. DOI:

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Our results do not support this hypothesis. Metabolites of KYN pathways were not high in depressive patients.

There have not been enough studies as to 5-HT levels in the brain of patients of bipolar depression. Serotonin levels in cerebrospinal fluids of patients of bipolar depression were shown to be high [37] or normal [38]. So it seems to be very important to discriminate monopolar and bipolar depression to study roles of serotonin in the pathogenesis of disease.

Our results show that plasma 5-HT levels were low and metabolites of KYN pathway were not changed in patients of monopolar depression.

### **3. Statistics**

Standard ANOVA methodology was used and p < 0.05 was considered as significant difference. Results are expressed as mean ± SD. Bars of figures represent standard deviations.

### **4. Ethics**

This work has been approved by the Ethical committees of Showa Women's University, Showa University School of Medicine, and NPO "International projects on food and health" and has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments.

### **Acknowledgements**

Experiments were designed and performed by all of the authors. AT wrote the manuscript. Statistical analyses were done by FS. All authors read the manuscript and approve the final manuscript. All the authors had responsibilities for a final content. A part of the work was reported in Japanese (http://www5c.biglobe.ne.jp/~takada-a/protein%20and%20brain.pdf).

### **Author details**

Akikazu Takada1 \*, Fumiko Shimizu2 and Junichi Masuda3

\*Address all correspondence to: takadaa@mwd.biglobe.ne.jp

1 International Projects on Food and Health (NPO), Tokyo, Japan

2 Faculty of Life and Environmental Sciences, Showa Women's University, Tokyo, Japan

3 Global Application Development Center, Shimadzu Corporation, Kanagawa-ken, Japan

### **References**

QN, and 3-HKYN were shown to be anxiogenic and KYNA were anxiolytic [36]. From these

Our results do not support this hypothesis. Metabolites of KYN pathways were not high in

There have not been enough studies as to 5-HT levels in the brain of patients of bipolar depression. Serotonin levels in cerebrospinal fluids of patients of bipolar depression were shown to be high [37] or normal [38]. So it seems to be very important to discriminate monopolar and

Our results show that plasma 5-HT levels were low and metabolites of KYN pathway were

Standard ANOVA methodology was used and p < 0.05 was considered as significant difference. Results are expressed as mean ± SD. Bars of figures represent standard deviations.

This work has been approved by the Ethical committees of Showa Women's University, Showa University School of Medicine, and NPO "International projects on food and health" and has been carried out in accordance with The Code of Ethics of the World Medical Association

Experiments were designed and performed by all of the authors. AT wrote the manuscript. Statistical analyses were done by FS. All authors read the manuscript and approve the final manuscript. All the authors had responsibilities for a final content. A part of the work was reported in Japanese (http://www5c.biglobe.ne.jp/~takada-a/protein%20and%20brain.pdf).

and Junichi Masuda3

2 Faculty of Life and Environmental Sciences, Showa Women's University, Tokyo, Japan 3 Global Application Development Center, Shimadzu Corporation, Kanagawa-ken, Japan

bipolar depression to study roles of serotonin in the pathogenesis of disease.

results, he tried to explain the effects of antidepressive drugs.

156 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

not changed in patients of monopolar depression.

(Declaration of Helsinki) for experiments.

\*, Fumiko Shimizu2

\*Address all correspondence to: takadaa@mwd.biglobe.ne.jp

1 International Projects on Food and Health (NPO), Tokyo, Japan

**Acknowledgements**

**Author details**

Akikazu Takada1

depressive patients.

**3. Statistics**

**4. Ethics**


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**Chapter 8**

**Provisional chapter**

**Salivary Melatonin at Night: Responding to the Night**

**Salivary Melatonin at Night: Responding to the Night** 

DOI: 10.5772/intechopen.79816

**Lighting and Cow's Milk Consumption at Breakfast in**

**Lighting and Cow's Milk Consumption at Breakfast in** 

(1) In junior high school students aged 14–15 years old, the salivary melatonin level increased rapidly from 3.00 pg/ml at 21:45 to 9.18 pg/ml at 23:40 under orange light from light bulb, whereas it remained at less than 1.3 pg/ml under white light from fluorescent lamp. (2) In 3 weeks of intervention on university athlete students, the salivary melatonin concentration at 23:00 of G3 (protein-rich breakfast and following sunlight exposure and orange light from the light bulb at night) after intervention was significantly higher than that of G1 (protein-poor breakfast and not following exposure to sunlight and white light from the fluorescent lamp at night) and G2 (protein-rich breakfast and following exposure to sunlight and white lights from the fluorescent lamp at night). (3) This study evaluates the effects of cow's milk intake (Group 1: G1) for 20 days at breakfast on saliva melatonin concentration at 22:00 and 23:00 on 0, 10, and 21 days of the intervention period in Japanese university male athletes attending a university soccer club. In the intervention group (G1), salivary melatonin concentration increased at 22:00 in comparison with that before intervention, but there was no significant change in the control group (Group 2: G2). On the other hand, there were no significant differences in the melatonin at 23:00 between the both groups just after 21 days of intervention. Intake of cow's milk at breakfast might make the circadian phase in advance in the soccer athletes.

**Keywords:** junior high-school students, university soccer club students, salivary melatonin concentration in 22:00–23:00, orange or white lighting at night, cows' milk at

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Japanese Junior High and University Students**

**Japanese Junior High and University Students**

Hitomi Takeuchi and Tetsuo Harada

Hitomi Takeuchi and Tetsuo Harada

http://dx.doi.org/10.5772/intechopen.79816

**Abstract**

breakfast

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

#### **Salivary Melatonin at Night: Responding to the Night Lighting and Cow's Milk Consumption at Breakfast in Japanese Junior High and University Students Salivary Melatonin at Night: Responding to the Night Lighting and Cow's Milk Consumption at Breakfast in Japanese Junior High and University Students**

DOI: 10.5772/intechopen.79816

Hitomi Takeuchi and Tetsuo Harada Hitomi Takeuchi and Tetsuo Harada

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79816

#### **Abstract**

(1) In junior high school students aged 14–15 years old, the salivary melatonin level increased rapidly from 3.00 pg/ml at 21:45 to 9.18 pg/ml at 23:40 under orange light from light bulb, whereas it remained at less than 1.3 pg/ml under white light from fluorescent lamp. (2) In 3 weeks of intervention on university athlete students, the salivary melatonin concentration at 23:00 of G3 (protein-rich breakfast and following sunlight exposure and orange light from the light bulb at night) after intervention was significantly higher than that of G1 (protein-poor breakfast and not following exposure to sunlight and white light from the fluorescent lamp at night) and G2 (protein-rich breakfast and following exposure to sunlight and white lights from the fluorescent lamp at night). (3) This study evaluates the effects of cow's milk intake (Group 1: G1) for 20 days at breakfast on saliva melatonin concentration at 22:00 and 23:00 on 0, 10, and 21 days of the intervention period in Japanese university male athletes attending a university soccer club. In the intervention group (G1), salivary melatonin concentration increased at 22:00 in comparison with that before intervention, but there was no significant change in the control group (Group 2: G2). On the other hand, there were no significant differences in the melatonin at 23:00 between the both groups just after 21 days of intervention. Intake of cow's milk at breakfast might make the circadian phase in advance in the soccer athletes.

**Keywords:** junior high-school students, university soccer club students, salivary melatonin concentration in 22:00–23:00, orange or white lighting at night, cows' milk at breakfast

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **1. Background**

Night sleep duration of Japanese children aged 10–18 years has become shorter by 1 hour during 1970–2000 in Japan [1]. In 2016, sleep hours in Japanese infants were reported to be only 9 hours (ideal hours, 12–13 hours) on average [2], while it ranged mainly in 7–10 hours for 8–11 years old of Japanese children (ideal hours, 10–11 hours) [3]. The so-called 24-hour society, which is currently in progress in Japan, seems to change environmental conditions surrounding the children. For example, mobile phones are used by more than 90% in 2000 and more than 98% in 2017 of university students, and more than 30% in 2000 and 95% in 2017 of junior high school students living in the urban area of Kochi city (33°N) had their own mobile phone (currently, most ones have smart phones) [4, 5]. Students can communicate with their colleagues even in the middle of the night with mobile phones. Frequent or long-time (more than 30 min) usage of the mobile phone makes university and junior high school students more evening-typed [4, 5]. At least for Japanese students in junior and senior high schools and universities, convenience stores have critical items for usual life, and these provide many kinds of foods and other goods for usual life. The total number of convenience stores by the main eight Japanese companies is 55,395 in 24 April 2018 all over Japan (http:// mitok.info/?p=75099). Convenience stores are very bright places with luminance of 2000 Lux or more at the level of the eye height. Such bright light inside convenience stores may function as a merchandising technology which has been used all over the world for at least 75 years. Unconscious effects of using bright lights with higher color temperature in the evening or at night could be very serious for phase-delaying the circadian clock of students who had been exposed to bright lights [6].

was not seen under dim lights [19, 20]. Even room lights such as fluorescent lamps can attenuate melatonin excretion duration at night [21]. Especially, blue lights with 460 nm as peak wave length in the evening can be well absorbed by melanopsin in the neuro-ganglia cells in the retina [22, 23]. Evening lighting conditions are also said to affect circadian rhythms [24, 25] and mental health in mice [26]. Tryptophan intake at breakfast is effective for the onset and offset of sleep in young children [27]. Moreover, questionnaire surveys showed that young children exposed to sunlight for more than 30 min after having sources of protein at breakfast are more morning-typed than those exposed for less than 30 min [28] and that the more young children take in vitamin B6 at breakfast, the more they exhibit morning typology [29]. These findings can lead to appear the following hypothesis. It would be that morning tryptophan and vitamin B6 intake and following exposure to sunlight in the morning can induce serotonin synthesis in the daytime especially in the morning at pineal, and the following metabolism from serotonin to melatonin synthesis at night also at pineal can be promoted. However, it is impossible to test this hypothesis by a simple questionnaire study. Moreover, this melatonin synthesis could be inhibited by the night exposure to shortwave length (blue light, high color temperature) lights emitted from fluorescent lamps through melanopsin [30]. This hypothesis can be tested by an intervention field experiment which was done [31] and introduced here. In the case of adolescents and children, exposure to lights of 300 Lux or more during the first half of subjective night in the normal life might decrease their melatonin level and prevent the falling into sleep [32]. Japanese junior and senior students were known to study at home mainly at night and also in the private preparatory school (for upper schools). In such cases, they are exposed to bright lights in most cases from fluorescent light bulbs. Many blue or blue-green lights with 470–500 nm wave lengths which were included in the bright lights

Salivary Melatonin at Night: Responding to the Night Lighting and Cow's Milk Consumption…

http://dx.doi.org/10.5772/intechopen.79816

163

In the epidemiological studies made in 2003–2013 on university students in Kochi Prefecture (33°N), 21.4% of the students used frequently (more than 4–5 days or more per week) convenience stores [34]. Twenty-two percent of convenience store users went there after sunset. Among convenience store users, 30.2 and 6.5% of junior high school students stayed there for 15–30 min and more than 30 min per one use, respectively [8]. In Japan, many junior high school students were faced to the entrance examination for the upper senior high school and went to a private preliminary school after schools. About 62.4 and 18% of the students in preparation went and studied there for 2 or 3 hours till 21:00 and 22:00, respectively [8]. Generally, in Japanese education scene, some junior and senior students use convenience stores after sunset and were exposed to bright light with high color temperature with more than 2000 Lux from the fluorescent lamp. These exposures seem to suppress the plasma melatonin level [31, 35] and also are possible to make the circadian phase delayed in junior high school and

Tryptophan is one kind of amino-acid of 21 kinds and cannot be made up from another substance but only can be absorbed exclusively from meals in humans. After absorption of tryptophan, it was transported from alimentary canal through brain-vessel barrier into the pineal in the brain. There, it was metabolized first to 5-hydroxytryptamine (serotonin) in the morning time mainly, at first, by two kinds of enzymes. Serotonin was again metabolized into melatonin by another two kinds of enzymes again in the pineal at night [36, 37]. What is the

were powerful to suppress melatonin concentration [33].

university students.

Honma and Honma [6] reported an evidence that phase-delay effects were shown by light pulse exposure with higher color temperature from fluorescent lamp made only in the first half of the subjective night (about 19:00–24:00) in the circadian phase. On the other hand, phase advance of the circadian clock could be caused by the light exposure during 3 hours from the bottom point (about 4:00–5:00) of core body temperature rhythm. The effects of the night or evening use of convenience store was studied and showed that it was possible to make the diurnal rhythms of Japanese children mainly aged 12–15 years old become more evening-typed and take shorter sleep [7, 8]. Younger children attending kinder gardens and students attending elementary school were more sensitive to "light conditions" in normal life than university students, according to an epidemiological study [4].

Melatonin is synthesized in the pineal body of the hypothalamic area and secreted at night. It is well known as a key substance which may be effective in promoting the falling into night sleep by humans [9]. During daytime, the concentration of plasma melatonin was reported to be extremely low, whereas it increased rapidly during 22:00–23:00 as much as 10 or 20 times of daytime values [10, 11]. The melatonin intake caused higher EEG power density in the range of relatively low frequency of 5.25–9.0 Hz rather than that of placebo [12]. The melatonin level in the serum can be well and positively correlated with that in the saliva [13–16]. Secretion of melatonin exhibits circadian rhythms and is suppressed by bright light at night [17, 18]. Moreover, the plasma melatonin level at night was suppressed by the exposure of lights with 400 Lux for more than 4 min (or 300 Lux for more than 2 hours), whereas such suppression was not seen under dim lights [19, 20]. Even room lights such as fluorescent lamps can attenuate melatonin excretion duration at night [21]. Especially, blue lights with 460 nm as peak wave length in the evening can be well absorbed by melanopsin in the neuro-ganglia cells in the retina [22, 23]. Evening lighting conditions are also said to affect circadian rhythms [24, 25] and mental health in mice [26]. Tryptophan intake at breakfast is effective for the onset and offset of sleep in young children [27]. Moreover, questionnaire surveys showed that young children exposed to sunlight for more than 30 min after having sources of protein at breakfast are more morning-typed than those exposed for less than 30 min [28] and that the more young children take in vitamin B6 at breakfast, the more they exhibit morning typology [29]. These findings can lead to appear the following hypothesis. It would be that morning tryptophan and vitamin B6 intake and following exposure to sunlight in the morning can induce serotonin synthesis in the daytime especially in the morning at pineal, and the following metabolism from serotonin to melatonin synthesis at night also at pineal can be promoted. However, it is impossible to test this hypothesis by a simple questionnaire study. Moreover, this melatonin synthesis could be inhibited by the night exposure to shortwave length (blue light, high color temperature) lights emitted from fluorescent lamps through melanopsin [30]. This hypothesis can be tested by an intervention field experiment which was done [31] and introduced here.

**1. Background**

162 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

exposed to bright lights [6].

Night sleep duration of Japanese children aged 10–18 years has become shorter by 1 hour during 1970–2000 in Japan [1]. In 2016, sleep hours in Japanese infants were reported to be only 9 hours (ideal hours, 12–13 hours) on average [2], while it ranged mainly in 7–10 hours for 8–11 years old of Japanese children (ideal hours, 10–11 hours) [3]. The so-called 24-hour society, which is currently in progress in Japan, seems to change environmental conditions surrounding the children. For example, mobile phones are used by more than 90% in 2000 and more than 98% in 2017 of university students, and more than 30% in 2000 and 95% in 2017 of junior high school students living in the urban area of Kochi city (33°N) had their own mobile phone (currently, most ones have smart phones) [4, 5]. Students can communicate with their colleagues even in the middle of the night with mobile phones. Frequent or long-time (more than 30 min) usage of the mobile phone makes university and junior high school students more evening-typed [4, 5]. At least for Japanese students in junior and senior high schools and universities, convenience stores have critical items for usual life, and these provide many kinds of foods and other goods for usual life. The total number of convenience stores by the main eight Japanese companies is 55,395 in 24 April 2018 all over Japan (http:// mitok.info/?p=75099). Convenience stores are very bright places with luminance of 2000 Lux or more at the level of the eye height. Such bright light inside convenience stores may function as a merchandising technology which has been used all over the world for at least 75 years. Unconscious effects of using bright lights with higher color temperature in the evening or at night could be very serious for phase-delaying the circadian clock of students who had been

Honma and Honma [6] reported an evidence that phase-delay effects were shown by light pulse exposure with higher color temperature from fluorescent lamp made only in the first half of the subjective night (about 19:00–24:00) in the circadian phase. On the other hand, phase advance of the circadian clock could be caused by the light exposure during 3 hours from the bottom point (about 4:00–5:00) of core body temperature rhythm. The effects of the night or evening use of convenience store was studied and showed that it was possible to make the diurnal rhythms of Japanese children mainly aged 12–15 years old become more evening-typed and take shorter sleep [7, 8]. Younger children attending kinder gardens and students attending elementary school were more sensitive to "light conditions" in normal life

Melatonin is synthesized in the pineal body of the hypothalamic area and secreted at night. It is well known as a key substance which may be effective in promoting the falling into night sleep by humans [9]. During daytime, the concentration of plasma melatonin was reported to be extremely low, whereas it increased rapidly during 22:00–23:00 as much as 10 or 20 times of daytime values [10, 11]. The melatonin intake caused higher EEG power density in the range of relatively low frequency of 5.25–9.0 Hz rather than that of placebo [12]. The melatonin level in the serum can be well and positively correlated with that in the saliva [13–16]. Secretion of melatonin exhibits circadian rhythms and is suppressed by bright light at night [17, 18]. Moreover, the plasma melatonin level at night was suppressed by the exposure of lights with 400 Lux for more than 4 min (or 300 Lux for more than 2 hours), whereas such suppression

than university students, according to an epidemiological study [4].

In the case of adolescents and children, exposure to lights of 300 Lux or more during the first half of subjective night in the normal life might decrease their melatonin level and prevent the falling into sleep [32]. Japanese junior and senior students were known to study at home mainly at night and also in the private preparatory school (for upper schools). In such cases, they are exposed to bright lights in most cases from fluorescent light bulbs. Many blue or blue-green lights with 470–500 nm wave lengths which were included in the bright lights were powerful to suppress melatonin concentration [33].

In the epidemiological studies made in 2003–2013 on university students in Kochi Prefecture (33°N), 21.4% of the students used frequently (more than 4–5 days or more per week) convenience stores [34]. Twenty-two percent of convenience store users went there after sunset. Among convenience store users, 30.2 and 6.5% of junior high school students stayed there for 15–30 min and more than 30 min per one use, respectively [8]. In Japan, many junior high school students were faced to the entrance examination for the upper senior high school and went to a private preliminary school after schools. About 62.4 and 18% of the students in preparation went and studied there for 2 or 3 hours till 21:00 and 22:00, respectively [8]. Generally, in Japanese education scene, some junior and senior students use convenience stores after sunset and were exposed to bright light with high color temperature with more than 2000 Lux from the fluorescent lamp. These exposures seem to suppress the plasma melatonin level [31, 35] and also are possible to make the circadian phase delayed in junior high school and university students.

Tryptophan is one kind of amino-acid of 21 kinds and cannot be made up from another substance but only can be absorbed exclusively from meals in humans. After absorption of tryptophan, it was transported from alimentary canal through brain-vessel barrier into the pineal in the brain. There, it was metabolized first to 5-hydroxytryptamine (serotonin) in the morning time mainly, at first, by two kinds of enzymes. Serotonin was again metabolized into melatonin by another two kinds of enzymes again in the pineal at night [36, 37]. What is the function of serotonin as a precursor of melatonin? The shortage of serotonin in the human brain has been known to induce eating disorders, sleep disorders, obsessive compulsive disorder, panic disorder, and depression [38]. The lack of serotonin also seems to cause impulsive behavior and suicidal attempts, anxiety/aggression-driven depression, and aggression [39, 40]. Thus, serotonin is called the "key" substance in the psychiatry field. For example, serotonin reuptake inhibitors (SSRIs) were widely and commonly used for the treatment of affective disorders like as depression [41].

their soccer performances in advance [49]. Another intervention study was performed for 1 month on the Japanese university soccer team members using a leaflet entitled: "Three benefits: Go to bed early! Get up early! Eat a nutritionally rich breakfast!" [48]. As the result of this intervention, all of their soccer performance, sleep health, and mental health

Salivary Melatonin at Night: Responding to the Night Lighting and Cow's Milk Consumption…

http://dx.doi.org/10.5772/intechopen.79816

165

An increased intake of cow's milk at breakfast, as a source of tryptophan, is hypothesized to promote the amount of serotonin in the morning and the following synthesis to melatonin at night in the Japanese university soccer team athletes. However, this hypothesis has not been

Japanese junior high school students (four females and six males) as participants in this study were aged 14–15 years old [35]. They were Motoyama junior high school students in the third grade. This junior high school was located in the mountain area of Reihoku district (33.5°N) in Kochi Prefecture, Japan. Seven days were holidays for the participants before this study as intervention experiment. During the holidays before the experiment, they were recom-

The two groups "bright light experimental group (BLEG)" and "dim light control group (DLCG)" were set up for the intervention experiment for junior high school students. For the BLEG and DLCG, participants were selected to have similar circadian typology scores as the diurnal-type scale (DTS) of the morningness-eveningness (M-E) questionnaire of Torsvall and Åkerstedt [50] (mean ± SD: 15.00 ± 4.30 by BLEG and 14.80 ± 4.09 by DLCG). Participants of BLEG showed 23.0 ± 4.2 hours (mean ± SD, bedtime), 8.4 ± 1.9 hours (wake-up time), and 9.1 ± 1.4 hours (sleep hours) just before the experiment. On the other hand, the values were 23.8 ± 1.3 hours (bedtime), 8.9 ± 1.3 hours (wake-up time), and 9.5 ± 1.5 hours (sleep hours) for the DLCG participants. Each group has two females and three males. All participants collected their own saliva using "Salivette" by collecting tubes (SARSTEDT Aktiengesellschaft & Co., Numbrecht, Germany) at 22:30–23:00 under the 200–300 Lux light from fluorescent light

Most of the Japanese citizens are enjoying evening time under fluorescent light bulbs during the first half of the subjective night (about 19:00–24:00) based on a long-term epidemiological study [48] on 3700 small children aged 2–6 years and their mothers in 2003–2017 in Kochi.

Eighty-five percent or more of 3700 families of Japanese infants used fluorescent light bulbs as night lighting at home usually. When the illumination was measured at the level of 1 m above floor under a usual type of round-shaped fluorescent light bulb in a typical one-

**2. Effects of evening light conditions on salivary melatonin of** 

mended to keep usual diurnal rhythm (like as bedtime and wake-up times).

bulbs in their home on the day before the experimental day.

room apartment for university students, it was 340 Lux [31].

were improved.

**2.1. Methods**

*2.1.1. Participants*

tested. This study will try to test this hypothesis.

**Japanese junior high school students**

For keeping mental health, serotonin in the brain, one kind of catecholamine, would be a key substance. Medical doctors in the field of psychiatry have used serotonin reuptake inhibitors (SSRIs) widely in the past four decades for the treatment of affective disorders including depression [41]. However, mixed opinions have been expressed to whether SSRIs are effective for the treatment of depression in children and adolescents because there has been the shortage of coincident scientific evidence of SSRIs for young human beings. Synthesis of serotonin seems to be promoted by sunlight exposure after consuming protein-rich foods at breakfast [42]. This synthesis is hypothesized to occur mainly in the morning time. Another study on young Japanese children showed that the amount of tryptophan which was consumed at supper had neither related with morningness-eveningness (M-E) scores nor with sleep habits [43].

How do cow's milk proteins promote human health? Eight ways to promote human health were demonstrated by Nongonierma and FitzGerald [44]: (1) improving satiety and weight management, (2) reducing risk of heart disease, (3) having an antimicrobial role, (4) having anti-inflammatory effects, (5) showing anticancer effects, (6) exerting antioxidant effects, (7) affecting insulin secretion and serum glucose regulation, and (8) an action upon muscle protein synthesis.

Brezinova and Oswald [45] showed, using electroencephalography, that sleep was significantly improved (longer and uninterrupted night sleeps) in older people when they ate a combination of cow's milk and cereal before going to bed. Laird and Drexel [46] reported that a meal of cornflakes and milk strongly improved sleep quality (as judged from an uninterrupted night sleep), with regard to the relationship between sleep health and the intake of cow's milk. On the effects of morning-drinking cow's milk, only a few studies have been performed for any improvement in sleep health. The two results of a questionnaire study have been reported on Japanese infants aged 2–6 years old [47]. Infants who added protein-rich foods at breakfast to the usual breakfast by other infants were more morning-typed and slept with significantly better quality than the other infants. Moreover, infants who drank milk at breakfast were less frequently depressed than those who did not.

Protein intake at breakfast would mean the consumption of tryptophan from the alimentary canal to the blood. Such tryptophan could be transferred to the pineal where serotonin can be synthesized and promote mental health in the daytime as an antidepressive agent. Serotonin can be further synthesized into melatonin as a sleep-onset agent at night [27–29, 31, 48]. For Japanese children, drinking cow's milk at breakfast is an important source of tryptophan and could, on the basis of a questionnaire study [47], be supposed to promote mental and sleep health. Recently, an intervention of drinking cow's milk (200 ml) at breakfast was reported to make Japanese university soccer team athletes who were originally more evening-typed than evening-typed, and this intervention also made their soccer performances in advance [49]. Another intervention study was performed for 1 month on the Japanese university soccer team members using a leaflet entitled: "Three benefits: Go to bed early! Get up early! Eat a nutritionally rich breakfast!" [48]. As the result of this intervention, all of their soccer performance, sleep health, and mental health were improved.

An increased intake of cow's milk at breakfast, as a source of tryptophan, is hypothesized to promote the amount of serotonin in the morning and the following synthesis to melatonin at night in the Japanese university soccer team athletes. However, this hypothesis has not been tested. This study will try to test this hypothesis.

## **2. Effects of evening light conditions on salivary melatonin of Japanese junior high school students**

### **2.1. Methods**

function of serotonin as a precursor of melatonin? The shortage of serotonin in the human brain has been known to induce eating disorders, sleep disorders, obsessive compulsive disorder, panic disorder, and depression [38]. The lack of serotonin also seems to cause impulsive behavior and suicidal attempts, anxiety/aggression-driven depression, and aggression [39, 40]. Thus, serotonin is called the "key" substance in the psychiatry field. For example, serotonin reuptake inhibitors (SSRIs) were widely and commonly used for the treatment of

For keeping mental health, serotonin in the brain, one kind of catecholamine, would be a key substance. Medical doctors in the field of psychiatry have used serotonin reuptake inhibitors (SSRIs) widely in the past four decades for the treatment of affective disorders including depression [41]. However, mixed opinions have been expressed to whether SSRIs are effective for the treatment of depression in children and adolescents because there has been the shortage of coincident scientific evidence of SSRIs for young human beings. Synthesis of serotonin seems to be promoted by sunlight exposure after consuming protein-rich foods at breakfast [42]. This synthesis is hypothesized to occur mainly in the morning time. Another study on young Japanese children showed that the amount of tryptophan which was consumed at supper had neither related with morningness-eveningness (M-E) scores nor with sleep habits [43]. How do cow's milk proteins promote human health? Eight ways to promote human health were demonstrated by Nongonierma and FitzGerald [44]: (1) improving satiety and weight management, (2) reducing risk of heart disease, (3) having an antimicrobial role, (4) having anti-inflammatory effects, (5) showing anticancer effects, (6) exerting antioxidant effects, (7) affecting insulin secretion and serum glucose regulation, and (8) an action upon muscle pro-

Brezinova and Oswald [45] showed, using electroencephalography, that sleep was significantly improved (longer and uninterrupted night sleeps) in older people when they ate a combination of cow's milk and cereal before going to bed. Laird and Drexel [46] reported that a meal of cornflakes and milk strongly improved sleep quality (as judged from an uninterrupted night sleep), with regard to the relationship between sleep health and the intake of cow's milk. On the effects of morning-drinking cow's milk, only a few studies have been performed for any improvement in sleep health. The two results of a questionnaire study have been reported on Japanese infants aged 2–6 years old [47]. Infants who added protein-rich foods at breakfast to the usual breakfast by other infants were more morning-typed and slept with significantly better quality than the other infants. Moreover, infants who drank milk at

Protein intake at breakfast would mean the consumption of tryptophan from the alimentary canal to the blood. Such tryptophan could be transferred to the pineal where serotonin can be synthesized and promote mental health in the daytime as an antidepressive agent. Serotonin can be further synthesized into melatonin as a sleep-onset agent at night [27–29, 31, 48]. For Japanese children, drinking cow's milk at breakfast is an important source of tryptophan and could, on the basis of a questionnaire study [47], be supposed to promote mental and sleep health. Recently, an intervention of drinking cow's milk (200 ml) at breakfast was reported to make Japanese university soccer team athletes who were originally more evening-typed than evening-typed, and this intervention also made

breakfast were less frequently depressed than those who did not.

affective disorders like as depression [41].

164 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

tein synthesis.

#### *2.1.1. Participants*

Japanese junior high school students (four females and six males) as participants in this study were aged 14–15 years old [35]. They were Motoyama junior high school students in the third grade. This junior high school was located in the mountain area of Reihoku district (33.5°N) in Kochi Prefecture, Japan. Seven days were holidays for the participants before this study as intervention experiment. During the holidays before the experiment, they were recommended to keep usual diurnal rhythm (like as bedtime and wake-up times).

The two groups "bright light experimental group (BLEG)" and "dim light control group (DLCG)" were set up for the intervention experiment for junior high school students. For the BLEG and DLCG, participants were selected to have similar circadian typology scores as the diurnal-type scale (DTS) of the morningness-eveningness (M-E) questionnaire of Torsvall and Åkerstedt [50] (mean ± SD: 15.00 ± 4.30 by BLEG and 14.80 ± 4.09 by DLCG). Participants of BLEG showed 23.0 ± 4.2 hours (mean ± SD, bedtime), 8.4 ± 1.9 hours (wake-up time), and 9.1 ± 1.4 hours (sleep hours) just before the experiment. On the other hand, the values were 23.8 ± 1.3 hours (bedtime), 8.9 ± 1.3 hours (wake-up time), and 9.5 ± 1.5 hours (sleep hours) for the DLCG participants. Each group has two females and three males. All participants collected their own saliva using "Salivette" by collecting tubes (SARSTEDT Aktiengesellschaft & Co., Numbrecht, Germany) at 22:30–23:00 under the 200–300 Lux light from fluorescent light bulbs in their home on the day before the experimental day.

Most of the Japanese citizens are enjoying evening time under fluorescent light bulbs during the first half of the subjective night (about 19:00–24:00) based on a long-term epidemiological study [48] on 3700 small children aged 2–6 years and their mothers in 2003–2017 in Kochi.

Eighty-five percent or more of 3700 families of Japanese infants used fluorescent light bulbs as night lighting at home usually. When the illumination was measured at the level of 1 m above floor under a usual type of round-shaped fluorescent light bulb in a typical oneroom apartment for university students, it was 340 Lux [31].

#### *2.1.2. Procedure*

We collected all 10 participants in front of Motoyama junior high school at 8:00 in 4 January 2003. We moved the participants by a wagon car to the experimental place which was a Japanese-style hotel located at a mountain area, Yusuhara town in Kochi Prefecture, Japan. The Yusuhara town was located 126 km west from the Motoyama town. During driving, illumination inside the car was 350–500 Lux. The car arrived at the hotel around noon on 4 January 2004. It was heavily snowing through the day around the hotel. Behavior of all participants was controlled during the experimental day in the hotel till the next morning of the experimental day. In the experimental daytime, all participants played outside and were exposed to the lights with 6000–7500 Lux at the eye level during 12:30–13:30 and 14:50.

*2.1.3. Ethic treatment*

*2.1.4. Statistical analysis*

**2.2. Results**

SEM [35].

full and complete agreement from all of them.

Detailed explanation of the objectives and methods of the experiment was provided before the experimental performance to the participants and their parents. The research project received

Salivary Melatonin at Night: Responding to the Night Lighting and Cow's Milk Consumption…

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167

The software used for statistical analysis was SPSS 12.0 J for Windows (SPSS Inc., Chicago, IL, USA). The Wilcoxon test was used for the pair-wise test for melatonin concentration for temporal change of melatonin concentration before, through and after the intervention day.

Salivary melatonin concentration in the DLCG increased from 3.00 ± 3.34 (mean ± SD) pg/ml at 21:45 to 9.18 ± 7.66 pg/ml at 23:30 in the experimental day (t-test between values at 21:45 and 23:30; t = 3.60, df = 4, p < 0.05) (**Figure 1**). On the other hand, it remained at less than 1.3 pg/ ml till 23:30 in BLEG (t = 2.07, df = 4, p = 0.2). On the day before the experiment, significant difference was not shown in the melatonin concentration in saliva between BLEG and DLCG (Wilcoxon test: z = −1.163, p = 0.31). In comparison with the melatonin concentration at 22:30

**Figure 1.** Effects of light condition on salivary melatonin concentration. Values shown are means (n = 5 per group) and

All the participants were exposed to the sunlight with 6000–7500 Lux at the eye level during 12: 30–13:30 and 14:00–14:50 during they played in the yard of the hotel. After the playtime, they were allowed to have a rest in the living room of the hotel. The floor of the rest room was filled with 12 *tatami* mats. The illumination of the light at the eye level in the rest room was 250 Lux from fluorescent light bulbs during the resting time till 16:00. Bath was taken by the participants one by one between 16:30 and 18:00. After that, supper was together taken by all participants between 18:15 and 19:20 in the living room.

BLEG participants moved to a Japanese-style room with eight *tatami* mats at 19:25. They were exposed to the high color temperature (white-colored) light with 2000 Lux at the eye level from fluorescent light bulbs. On the other hand, the DLCG ones moved to another Japanese-style room with eight *tatami* mats at the same time. Instead, the DLCG ones were exposed to the low color temperature (orange-colored) light with 60 Lux from an electronic light bulb.

In each room for BLEG and DLCG, all the participants did homework (e.g., literatures and mathematics) or made a small wooden folk craft object which is typical in the Yusuhara district, under each light condition till 22:30. An oil heater in both rooms functioned to keep the room temperature 15 ± 2°C.

At 22:30, they moved back to the former living room (12 *tatami* mats) and stayed there under the light of 250 Lux till 23:40. After that, experimental staffs ordered female and male participants to move to separate rooms and go to bed just before 24:00. At 21:45, 22:30, and 23:40, the participants collected their own saliva using the "Salivette" as collecting tubes. Their salivary samples were preserved in a refrigerator at less than −20°C. Melatonin concentration in the samples was analyzed by a professional-analyzing company (MSL Co. Ltd.) which was a professional company for analyzing several chemicals and microbiological organisms.

All the participants of BLEG and DLCG were ordered to get up at 7:00 in the next morning by calling out. They were enforced to get up between 7:00 and 7:15. Then, they took breakfast and left the experimental place, the hotel, at 9:00 for Motoyama junior high school. Light exposure at the eye level was measured by using a digital illuminance meter through the study.

#### *2.1.3. Ethic treatment*

*2.1.2. Procedure*

14:50.

light bulb.

cal organisms.

room temperature 15 ± 2°C.

We collected all 10 participants in front of Motoyama junior high school at 8:00 in 4 January 2003. We moved the participants by a wagon car to the experimental place which was a Japanese-style hotel located at a mountain area, Yusuhara town in Kochi Prefecture, Japan. The Yusuhara town was located 126 km west from the Motoyama town. During driving, illumination inside the car was 350–500 Lux. The car arrived at the hotel around noon on 4 January 2004. It was heavily snowing through the day around the hotel. Behavior of all participants was controlled during the experimental day in the hotel till the next morning of the experimental day. In the experimental daytime, all participants played outside and were exposed to the lights with 6000–7500 Lux at the eye level during 12:30–13:30 and

All the participants were exposed to the sunlight with 6000–7500 Lux at the eye level during 12: 30–13:30 and 14:00–14:50 during they played in the yard of the hotel. After the playtime, they were allowed to have a rest in the living room of the hotel. The floor of the rest room was filled with 12 *tatami* mats. The illumination of the light at the eye level in the rest room was 250 Lux from fluorescent light bulbs during the resting time till 16:00. Bath was taken by the participants one by one between 16:30 and 18:00. After that, supper was together taken by all

BLEG participants moved to a Japanese-style room with eight *tatami* mats at 19:25. They were exposed to the high color temperature (white-colored) light with 2000 Lux at the eye level from fluorescent light bulbs. On the other hand, the DLCG ones moved to another Japanese-style room with eight *tatami* mats at the same time. Instead, the DLCG ones were exposed to the low color temperature (orange-colored) light with 60 Lux from an electronic

In each room for BLEG and DLCG, all the participants did homework (e.g., literatures and mathematics) or made a small wooden folk craft object which is typical in the Yusuhara district, under each light condition till 22:30. An oil heater in both rooms functioned to keep the

At 22:30, they moved back to the former living room (12 *tatami* mats) and stayed there under the light of 250 Lux till 23:40. After that, experimental staffs ordered female and male participants to move to separate rooms and go to bed just before 24:00. At 21:45, 22:30, and 23:40, the participants collected their own saliva using the "Salivette" as collecting tubes. Their salivary samples were preserved in a refrigerator at less than −20°C. Melatonin concentration in the samples was analyzed by a professional-analyzing company (MSL Co. Ltd.) which was a professional company for analyzing several chemicals and microbiologi-

All the participants of BLEG and DLCG were ordered to get up at 7:00 in the next morning by calling out. They were enforced to get up between 7:00 and 7:15. Then, they took breakfast and left the experimental place, the hotel, at 9:00 for Motoyama junior high school. Light exposure

at the eye level was measured by using a digital illuminance meter through the study.

participants between 18:15 and 19:20 in the living room.

166 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Detailed explanation of the objectives and methods of the experiment was provided before the experimental performance to the participants and their parents. The research project received full and complete agreement from all of them.

#### *2.1.4. Statistical analysis*

The software used for statistical analysis was SPSS 12.0 J for Windows (SPSS Inc., Chicago, IL, USA). The Wilcoxon test was used for the pair-wise test for melatonin concentration for temporal change of melatonin concentration before, through and after the intervention day.

#### **2.2. Results**

Salivary melatonin concentration in the DLCG increased from 3.00 ± 3.34 (mean ± SD) pg/ml at 21:45 to 9.18 ± 7.66 pg/ml at 23:30 in the experimental day (t-test between values at 21:45 and 23:30; t = 3.60, df = 4, p < 0.05) (**Figure 1**). On the other hand, it remained at less than 1.3 pg/ ml till 23:30 in BLEG (t = 2.07, df = 4, p = 0.2). On the day before the experiment, significant difference was not shown in the melatonin concentration in saliva between BLEG and DLCG (Wilcoxon test: z = −1.163, p = 0.31). In comparison with the melatonin concentration at 22:30

**Figure 1.** Effects of light condition on salivary melatonin concentration. Values shown are means (n = 5 per group) and SEM [35].

under fluorescent lamps emitting 250 Lux white lights, the value for BLEG at experimental 22:00 tended to be lower, whereas for DLEG, it was significantly higher. In the day before the experimental day, melatonin concentration under the fluorescent lamp with 200–400 Lux in each home of both BLEG and DLCG was similar and had no significant difference (Mann-Whitney U-test: z = −1.163, p = 0.31). At 22:30 of the experimental day, melatonin concentration of BLEG tended to be lower than that of BLEG on the day before the experiment (Wilcoxon test: z = −1.604, p = 0.109). On the other hand, the concentration of DLCG at 22:30 become higher than that of the previous day at home (z = −2.023, p = 0.043).

Group 1 (G1) consisted of 20 soccer players without intervention, Group 2 (G2) had 22 soccer club members who were asked to have protein-rich foods such as fermented soybeans and vitamin B6-rich foods such as bananas at breakfast and sunlight exposure after breakfast, and Group 3 (G3) consisted of 21 members who were asked the same breakfast contents and sunlight exposure after that and additionally were asked to use incandescent light as night

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For the night lighting, all the participants in the three groups have used fluorescent lamps (white light). Integrated questionnaires were administered to all participants three times to estimate the effects of the 1-month intervention. Questionnaire studies were performed just before the intervention period, soon after the end of the intervention, and 1 month after the intervention. The same questionnaire was used for study before the intervention and also 1 month after that. The contents of the questionnaire were the diurnal-type scale constructed by Torsvall and Åkerstedt [50], questions on sleep habits and meal habits [52], an Irritation Index, the General Health Questionnaire (GHQ), the Sense of Coherence (SOC) questionnaire, and FFQ (Food Frequency Questionnaire). After the intervention, the questionnaire which had been administered before it was again done. Moreover, other self-assessment questions were also administered to the members of the three groups. The self-assessment questions were on how many days during the month-long intervention period they followed the three recommendations. The first one was on protein-rich foods as breakfast contents, the second one was sunlight exposure after breakfast, and the third one was the usage of orange lightings

(like light bulbs which emit orange (lower color temperature)) lights at night.

the body height, body weight, and age, among the three groups.

Based on the two questionnaire scores of FFQ (good, mild, and bad: three groups) and the diurnal-type scale (DTS) (morning-typed, middle-typed, evening-typed), nine groups (3 × 3) were made. Members of each of nine groups were randomly divided into the three experimental groups. As the results of the treatment, no significant differences were made also in

Through the intervention of 30 days in October–November in 2010, we asked all participants to keep a sleep diary. The sleep diary included a question and a selection list of answer: "How was the depth of your last night's sleep?" "(1) Deep, (2) Relatively deep, (3) Relatively shallow, (4) Shallow." To the members of Group 3, incandescent light bulbs (emitting orange lights) were distributed one by one before the intervention period. After that, they were asked to install the light bulbs (distributed) in their bed room. The G1 and G2 members were asked to switch fluorescent lamps on as usual, whereas the G3 ones were asked to switch incandescent light bulbs (distributed) on instead, when they were asked on the lightings when they got back to their residences after sunset. Room lights for G1 and G2 (white: fluorescent lamp) and G3 (orange: incandescent lamp) were 100–400 Lux and <100 Lux, respectively. Sixty-seven percent of the participants (63 of 94) answered the first questionnaire before intervention and 81% of participants (51 of 63) wrote their own sleep diaries during the intervention of 1 month. On "high-protein content breakfast" and following "exposure to >30 min exposure to sunlight," the implementation scores were calculated as the sum of days when the "protein-rich

lighting. All participants were males.

*3.1.2. Procedure*

The bright light of 2000 Lux from fluorescent lamps with high color temperature seems to have suppressed the night increase of melatonin concentration of junior high school students. On the other hand, the relatively low color temperature light with 60 Lux did not. In Japan, the light condition with 2000 Lux and high color temperature can be seen usually inside all the convenience stores of Japan, which are now increasing and become common. Such bright light conditions are also common as room lightings in Japanese private schools, the so-called Juku, which are preliminary schools for preparation of going through the entrance examination, which is a very severe competition for upper schools. Therefore, exposure to bright lights in the evening "Juku" and convenience store would suppress the night increase in the plasma melatonin level as a direct effect also would make the circadian phase delayed and as a result phase of sleep wake cycle can be delayed.

#### **2.3. Discussion**

Traditional lightings by ancient Japanese citizens were mainly low color temperature lights which were emitted from a traditional Japanese hearth fire or an oil lamp or candle (20–30 Lux). Such "orange" lightings might be healthy for adolescent and children, because the ancient lightings included only a low amount of the light components with 460–480 nm wave lengths (blue or high color temperature lights), which are peaks of energy consumption by melanopsin (relative to conopsin and lodopsin). Melanopsin is included in neuro-segmental cells and key substance for melatonin suppression [30]. Such orange lights do not stimulate the melanopsin, and melatonin suppression also does not occur. Therefore, junior high school children can fall in night sleep very smoothly [51]. This part 2 was already published in [35].

### **3. A tryptophan-rich breakfast and exposure to light with low color temperature at night improve the sleep and salivary melatonin level in Japanese students**

#### **3.1. Methods**

#### *3.1.1. Participants*

Ninety-four participants were male university students aged 19–22 years old with averaged 20.33 who were belonging to a university soccer club [31]. They participated in an intervention study; 63 of them answered to the integrated questionnaire before and after the intervention period.

Group 1 (G1) consisted of 20 soccer players without intervention, Group 2 (G2) had 22 soccer club members who were asked to have protein-rich foods such as fermented soybeans and vitamin B6-rich foods such as bananas at breakfast and sunlight exposure after breakfast, and Group 3 (G3) consisted of 21 members who were asked the same breakfast contents and sunlight exposure after that and additionally were asked to use incandescent light as night lighting. All participants were males.

For the night lighting, all the participants in the three groups have used fluorescent lamps (white light). Integrated questionnaires were administered to all participants three times to estimate the effects of the 1-month intervention. Questionnaire studies were performed just before the intervention period, soon after the end of the intervention, and 1 month after the intervention. The same questionnaire was used for study before the intervention and also 1 month after that. The contents of the questionnaire were the diurnal-type scale constructed by Torsvall and Åkerstedt [50], questions on sleep habits and meal habits [52], an Irritation Index, the General Health Questionnaire (GHQ), the Sense of Coherence (SOC) questionnaire, and FFQ (Food Frequency Questionnaire). After the intervention, the questionnaire which had been administered before it was again done. Moreover, other self-assessment questions were also administered to the members of the three groups. The self-assessment questions were on how many days during the month-long intervention period they followed the three recommendations. The first one was on protein-rich foods as breakfast contents, the second one was sunlight exposure after breakfast, and the third one was the usage of orange lightings (like light bulbs which emit orange (lower color temperature)) lights at night.

#### *3.1.2. Procedure*

under fluorescent lamps emitting 250 Lux white lights, the value for BLEG at experimental 22:00 tended to be lower, whereas for DLEG, it was significantly higher. In the day before the experimental day, melatonin concentration under the fluorescent lamp with 200–400 Lux in each home of both BLEG and DLCG was similar and had no significant difference (Mann-Whitney U-test: z = −1.163, p = 0.31). At 22:30 of the experimental day, melatonin concentration of BLEG tended to be lower than that of BLEG on the day before the experiment (Wilcoxon test: z = −1.604, p = 0.109). On the other hand, the concentration of DLCG at 22:30

The bright light of 2000 Lux from fluorescent lamps with high color temperature seems to have suppressed the night increase of melatonin concentration of junior high school students. On the other hand, the relatively low color temperature light with 60 Lux did not. In Japan, the light condition with 2000 Lux and high color temperature can be seen usually inside all the convenience stores of Japan, which are now increasing and become common. Such bright light conditions are also common as room lightings in Japanese private schools, the so-called Juku, which are preliminary schools for preparation of going through the entrance examination, which is a very severe competition for upper schools. Therefore, exposure to bright lights in the evening "Juku" and convenience store would suppress the night increase in the plasma melatonin level as a direct effect also would make the circadian phase delayed and as a result

Traditional lightings by ancient Japanese citizens were mainly low color temperature lights which were emitted from a traditional Japanese hearth fire or an oil lamp or candle (20–30 Lux). Such "orange" lightings might be healthy for adolescent and children, because the ancient lightings included only a low amount of the light components with 460–480 nm wave lengths (blue or high color temperature lights), which are peaks of energy consumption by melanopsin (relative to conopsin and lodopsin). Melanopsin is included in neuro-segmental cells and key substance for melatonin suppression [30]. Such orange lights do not stimulate the melanopsin, and melatonin suppression also does not occur. Therefore, junior high school children can fall in night sleep very smoothly [51]. This part 2 was already published in [35].

**3. A tryptophan-rich breakfast and exposure to light with low color temperature at night improve the sleep and salivary melatonin level** 

Ninety-four participants were male university students aged 19–22 years old with averaged 20.33 who were belonging to a university soccer club [31]. They participated in an intervention study; 63 of them answered to the integrated questionnaire before and after the intervention period.

become higher than that of the previous day at home (z = −2.023, p = 0.043).

phase of sleep wake cycle can be delayed.

168 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**2.3. Discussion**

**in Japanese students**

**3.1. Methods**

*3.1.1. Participants*

Based on the two questionnaire scores of FFQ (good, mild, and bad: three groups) and the diurnal-type scale (DTS) (morning-typed, middle-typed, evening-typed), nine groups (3 × 3) were made. Members of each of nine groups were randomly divided into the three experimental groups. As the results of the treatment, no significant differences were made also in the body height, body weight, and age, among the three groups.

Through the intervention of 30 days in October–November in 2010, we asked all participants to keep a sleep diary. The sleep diary included a question and a selection list of answer: "How was the depth of your last night's sleep?" "(1) Deep, (2) Relatively deep, (3) Relatively shallow, (4) Shallow." To the members of Group 3, incandescent light bulbs (emitting orange lights) were distributed one by one before the intervention period. After that, they were asked to install the light bulbs (distributed) in their bed room. The G1 and G2 members were asked to switch fluorescent lamps on as usual, whereas the G3 ones were asked to switch incandescent light bulbs (distributed) on instead, when they were asked on the lightings when they got back to their residences after sunset. Room lights for G1 and G2 (white: fluorescent lamp) and G3 (orange: incandescent lamp) were 100–400 Lux and <100 Lux, respectively. Sixty-seven percent of the participants (63 of 94) answered the first questionnaire before intervention and 81% of participants (51 of 63) wrote their own sleep diaries during the intervention of 1 month.

On "high-protein content breakfast" and following "exposure to >30 min exposure to sunlight," the implementation scores were calculated as the sum of days when the "protein-rich breakfast and following light exposure" was implemented. On another intervention "night exposure to low color temperature light," it was defined as implementation score how many minutes per night they were exposed to the low color temperature light emitted from the incandescent bulb.

**3.2. Results**

period (Kruskal-Wallis test: χ<sup>2</sup>

Whitney U-test: z = −2.000, p = −0.071).

We could get significant positive correlation between the feeling of sleeping well in the last week period (LWP) of the intervention period and hours spent under incandescent light at night (Pearson's correlation test r<sup>2</sup> = 0.265, p = 0.034). Salivary melatonin concentration by G3 participants was significantly higher than that of G1 and G2 in the midpoint and the day before the last day of intervention period (Bonferroni multiple comparison test: G1 vs. G3, p = 0.018; G2 vs. G3, p = 0.011). On the other hand, we have got no significant differences in the salivary melatonin among the three groups on the day just before the start of the intervention

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(MP) of the intervention period of G3, the "high implementation period" tended to show a higher concentration of salivary melatonin than the "low implementation group" did (Mann-

Group 2 participants tended to follow the morning intervention recommendations (highprotein breakfast and sunlight exposure) on less days than G3 participants did (Mann-Whitney U-test: first week period (FWP), z = −1.952, p = 0.053; MP, z = −1.628, p = 0.105;

**Figure 3.** Salivary melatonin level just before intervention (A) (no significant difference: Kruskal-Wallis test: χ<sup>2</sup>

df = 2, p = 0.63). All three groups showed low salivary melatonin level of 2–5 pg/ml just before intervention. Melatonin in the saliva collected at the midpoint and on the day before the last day of the intervention (B) (Bonferroni multiple comparison test: G1 versus G3, p = 0.018; G2 versus G3, p = 0.011). Comparison of salivary melatonin concentration among the three groups. Group 1: no intervention; Group 2: recommendation of high-protein breakfast and exposure to sunlight; and Group 3: same as Group 2 plus the recommendation of exposure to orange lights from incandescent lamps.



We asked the participants of G2 and G3 to mark (marking as 0–100 points as satisfaction score) the scores on "To what extent do they satisfy on their carryout of the two (G2) or three (G3) intervention contents for 30 days in the total." We measured the salivary melatonin of the 10 participants of three groups. The 10 participants in each group were selected randomly because there was financial limitation for the chemical analysis. Ninety salivary samplings were needed for the experiment, because three groups prepared one sampling at 23:00 before, in the middle, and in the end of intervention (10 × 3 × 3). We asked participants to extract their own saliva at around 23:00 with cylindrical cotton (1 cm diameter, 3 cm long). If participants put them under their tongues for 3 min, full amount of saliva (1–3 ml) for analysis can be extracted outside and absorbed into the cotton. It was kept frozen at −25°C till analysis for 1–2 weeks. Melatonin concentrations in the saliva were determined using an ELISA kit (Direct Saliva Melatonin ELISA, Bulmann, Switzerland).

#### *3.1.3. Ethic treatment*

Participants received a full explanation with the code of the guideline for a study targeting humans before the study [53]. The explanation included that the results of the study would be used only for academic purposes and that all participants completely agreed to participate in this study.

#### *3.1.4. Statistical analysis*

We used a statistical analysis software as SPSS 12.0 J for Windows (SPSS Inc., Chicago, IL, USA). As nonparametric analyses, Kruskal-Wallis test and following Bonferroni multiple comparison were used among the three groups. Wilcoxon's signed-rank sum test was used for the pair-wise test in the same group and after the intervention (**Figure 2**).


**Figure 2.** Graphic schema of intervention study for university soccer club athletes.

#### **3.2. Results**

breakfast and following light exposure" was implemented. On another intervention "night exposure to low color temperature light," it was defined as implementation score how many minutes per night they were exposed to the low color temperature light emitted from the

We asked the participants of G2 and G3 to mark (marking as 0–100 points as satisfaction score) the scores on "To what extent do they satisfy on their carryout of the two (G2) or three (G3) intervention contents for 30 days in the total." We measured the salivary melatonin of the 10 participants of three groups. The 10 participants in each group were selected randomly because there was financial limitation for the chemical analysis. Ninety salivary samplings were needed for the experiment, because three groups prepared one sampling at 23:00 before, in the middle, and in the end of intervention (10 × 3 × 3). We asked participants to extract their own saliva at around 23:00 with cylindrical cotton (1 cm diameter, 3 cm long). If participants put them under their tongues for 3 min, full amount of saliva (1–3 ml) for analysis can be extracted outside and absorbed into the cotton. It was kept frozen at −25°C till analysis for 1–2 weeks. Melatonin concentrations in the saliva were determined using an ELISA kit (Direct

Participants received a full explanation with the code of the guideline for a study targeting humans before the study [53]. The explanation included that the results of the study would be used only for academic purposes and that all participants completely agreed to participate in this study.

We used a statistical analysis software as SPSS 12.0 J for Windows (SPSS Inc., Chicago, IL, USA). As nonparametric analyses, Kruskal-Wallis test and following Bonferroni multiple comparison were used among the three groups. Wilcoxon's signed-rank sum test was used

for the pair-wise test in the same group and after the intervention (**Figure 2**).

**Figure 2.** Graphic schema of intervention study for university soccer club athletes.

incandescent bulb.

*3.1.3. Ethic treatment*

*3.1.4. Statistical analysis*

Saliva Melatonin ELISA, Bulmann, Switzerland).

170 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

We could get significant positive correlation between the feeling of sleeping well in the last week period (LWP) of the intervention period and hours spent under incandescent light at night (Pearson's correlation test r<sup>2</sup> = 0.265, p = 0.034). Salivary melatonin concentration by G3 participants was significantly higher than that of G1 and G2 in the midpoint and the day before the last day of intervention period (Bonferroni multiple comparison test: G1 vs. G3, p = 0.018; G2 vs. G3, p = 0.011). On the other hand, we have got no significant differences in the salivary melatonin among the three groups on the day just before the start of the intervention period (Kruskal-Wallis test: χ<sup>2</sup> -value = 0.92, df = 2, p = 0.63) (**Figure 3**). In the middle period (MP) of the intervention period of G3, the "high implementation period" tended to show a higher concentration of salivary melatonin than the "low implementation group" did (Mann-Whitney U-test: z = −2.000, p = −0.071).

Group 2 participants tended to follow the morning intervention recommendations (highprotein breakfast and sunlight exposure) on less days than G3 participants did (Mann-Whitney U-test: first week period (FWP), z = −1.952, p = 0.053; MP, z = −1.628, p = 0.105;

**Figure 3.** Salivary melatonin level just before intervention (A) (no significant difference: Kruskal-Wallis test: χ<sup>2</sup> -value = 0.92, df = 2, p = 0.63). All three groups showed low salivary melatonin level of 2–5 pg/ml just before intervention. Melatonin in the saliva collected at the midpoint and on the day before the last day of the intervention (B) (Bonferroni multiple comparison test: G1 versus G3, p = 0.018; G2 versus G3, p = 0.011). Comparison of salivary melatonin concentration among the three groups. Group 1: no intervention; Group 2: recommendation of high-protein breakfast and exposure to sunlight; and Group 3: same as Group 2 plus the recommendation of exposure to orange lights from incandescent lamps.

LWP, z = 1.253, p = 0.221). The implementation rate in FWP tended to be higher than MP (Wilcoxon's signed-rank sum test: G2, z = −1.851, p = 0.064; G3, z = −1.914, p = 0.056) and LWP (G2, z = −2.298, p = 0.022, G3, z = −2.898, p = 0.004). The implementation rate in LWP in G2 and G3 tended to be lower than that in MP (G2, z = −1.681, p = 0.093; G3, z = −2.533, p = 0.011).

temperature lighting at night can facilitate achievement of high plasma melatonin at night in humans. Melatonin, a hormone secreted from the pineal gland, has been reported to cause the core body temperature to decrease and induces sleep [9, 12, 57]. An important role of the high plasma melatonin level at night was reported as a sleep-onset agent and sleep quality promoter [58]. There was a significant and positive correlation between the duration when participants spent under incandescent lights at night and the scores which

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High sleep quality would be promoted by high plasma melatonin in human beings. The principle theory of this study is promotion of serotonin synthesis in the morning and succeeding melatonin synthesis at night. The intervention of this study is composed of three issues: (1) having sources of tryptophan and vitamin B6 at breakfast, (2) following up breakfast with exposure to sunlight, and (3) the exposure to low color temperature lights as night lighting. Serotonin works as antidepression agent, and melatonin is a natural sleep-onset pill. Moreover, these two hormones would act as a circadian clock as inner-zeitgebers. As a result,

A limitation of this study of the intervention study is as follows: this intervention could not set a control group with low-tryptophan breakfast, sunlight exposure, and exposure to lowtemperature light at night for finding out the importance of the intake of tryptophan at breakfast in the physiological mechanism of serotonin-melatonin synthesis. This study was not the so-called physiological experiment to set up several experimental groups and control group under controlling their usual life like as meals, lightings, and social activity in some extents. In the future, another intervention study controlling more life habits would be possible. Another limitation of this study is that this intervention study was performed only with men. In the future, women participants from sports club could add important data for making gender differences in response to breakfast modulation and the change at night lighting into orange

**4. Effects of drinking cow's milk at breakfast on saliva melatonin** 

Sixty percent of 93 participants were born and grown in Shikoku island, whereas the others were from other parts of all over Japan. Participants in this study were 19–25 years old

Seventy-three participants of the experimental group were asked to drink 200 ml of cow's milk at breakfast each morning for a period of 21 days, from 13 November 2014 to 4 December

they marked to feel deep sleep, in this study.

shift to morning-typed would lead to promoted mental health.

light at night. This part 3 was already published in [31].

**concentration in Japanese university athletes**

**4.1. Methods**

*4.1.1. Participants*

*4.1.2. Procedure*

(Takeuchi et al., unpublished).

There was a significantly positive correlation between the regularity of time to take breakfast and supper and the implementation satisfaction index (maximum score, 100) (Kendall tau-b test: breakfast r2 = 0.058, p = 0.038; supper r2 = 0.057, p = 0.036). There was a significant positive correlation between the index on implementation days and the diurnal-type scale (DTS) at 1 month later after the intervention period (higher scores showing morning-typed) (Pearson's correlation test: r2 = 0.195, p = 0.006). The index on intervention days was how many days (among intervention of 30 days) the participants satisfied the implementation on the breakfast contents as high-protein foods.

A significant positive correlation was shown between the number of nights (among 30 days) when participants were exposed to orange-colored lights emitted from incandescent lamp and the index of meal time for three meals just after the intervention (Kendall tau b-test: breakfast r = −0.574, p = 0.007; lunch r2 = 0.146, p = 0.084; supper r2 = 0.215, p = 0.029). In comparison with those who ate breakfast less frequently for 1 month after the intervention, participants who took breakfast more frequently took late-night snacks with less frequency (Kendall tau-b test = −142, p = 0.003).

In G3, participants after the intervention showed a lower anger/irritation index than those before the intervention (Wilcoxon's signed-rank sum test: z = −3.072, p = 0.002). On the other hand, G1 showed only a tendency of reduced irritation (z = −1.786, p = 0.074), and G2 had no differences in mental health index after the intervention (z = −0.956, p = 0.340). Anger/irritation indices (the frequency to be irritated and the frequency to become angry due to small trigger) were also decreased after the intervention in G3 (Wilcoxon's signedrank sum test: irritation z = −2.496, p = 0.013; anger z = 2.714, p = 0.007).

#### **3.3. Discussion**

This study showed that a triple intervention concerning breakfast content, sunlight exposure after breakfast, and exposure to low temperature light emitted from incandescent bulbs are powerful methods for inducing secretion of high amounts of melatonin by the pineal gland inhuman adults. Underlying mechanisms can be hypothesized to consist of two components. The first component is that serotonin synthesis from tryptophan taken at breakfast may be enhanced by the exposure to sunlight just after taking breakfast. The second component is that the high potential of melatonin synthesis based on the high serotonin synthesis in the pineal during daytime might be available due to the night exposure to the "low-temperature light" emitted from incandescent bulbs. Many reports have shown that melatonin secretion is suppressed by evening or night light emitted from fluorescent lamps including shortwave length (with around 460 nm of wave length) components [10, 33, 35, 54–56]. Moreover, this study newly implies that the combined behaviors of (1) modifying breakfast content, (2) receiving sunlight exposure, and (3) receiving exposure to low color temperature lighting at night can facilitate achievement of high plasma melatonin at night in humans. Melatonin, a hormone secreted from the pineal gland, has been reported to cause the core body temperature to decrease and induces sleep [9, 12, 57]. An important role of the high plasma melatonin level at night was reported as a sleep-onset agent and sleep quality promoter [58]. There was a significant and positive correlation between the duration when participants spent under incandescent lights at night and the scores which they marked to feel deep sleep, in this study.

High sleep quality would be promoted by high plasma melatonin in human beings. The principle theory of this study is promotion of serotonin synthesis in the morning and succeeding melatonin synthesis at night. The intervention of this study is composed of three issues: (1) having sources of tryptophan and vitamin B6 at breakfast, (2) following up breakfast with exposure to sunlight, and (3) the exposure to low color temperature lights as night lighting. Serotonin works as antidepression agent, and melatonin is a natural sleep-onset pill. Moreover, these two hormones would act as a circadian clock as inner-zeitgebers. As a result, shift to morning-typed would lead to promoted mental health.

A limitation of this study of the intervention study is as follows: this intervention could not set a control group with low-tryptophan breakfast, sunlight exposure, and exposure to lowtemperature light at night for finding out the importance of the intake of tryptophan at breakfast in the physiological mechanism of serotonin-melatonin synthesis. This study was not the so-called physiological experiment to set up several experimental groups and control group under controlling their usual life like as meals, lightings, and social activity in some extents. In the future, another intervention study controlling more life habits would be possible. Another limitation of this study is that this intervention study was performed only with men. In the future, women participants from sports club could add important data for making gender differences in response to breakfast modulation and the change at night lighting into orange light at night. This part 3 was already published in [31].

## **4. Effects of drinking cow's milk at breakfast on saliva melatonin concentration in Japanese university athletes**

#### **4.1. Methods**

LWP, z = 1.253, p = 0.221). The implementation rate in FWP tended to be higher than MP (Wilcoxon's signed-rank sum test: G2, z = −1.851, p = 0.064; G3, z = −1.914, p = 0.056) and LWP (G2, z = −2.298, p = 0.022, G3, z = −2.898, p = 0.004). The implementation rate in LWP in G2 and G3 tended to be lower than that in MP (G2, z = −1.681, p = 0.093; G3, z = −2.533,

There was a significantly positive correlation between the regularity of time to take breakfast and supper and the implementation satisfaction index (maximum score, 100) (Kendall tau-b test: breakfast r2 = 0.058, p = 0.038; supper r2 = 0.057, p = 0.036). There was a significant positive correlation between the index on implementation days and the diurnal-type scale (DTS) at 1 month later after the intervention period (higher scores showing morning-typed) (Pearson's correlation test: r2 = 0.195, p = 0.006). The index on intervention days was how many days (among intervention of 30 days) the participants satisfied the implementation on the breakfast

A significant positive correlation was shown between the number of nights (among 30 days) when participants were exposed to orange-colored lights emitted from incandescent lamp and the index of meal time for three meals just after the intervention (Kendall tau b-test: breakfast r = −0.574, p = 0.007; lunch r2 = 0.146, p = 0.084; supper r2 = 0.215, p = 0.029). In comparison with those who ate breakfast less frequently for 1 month after the intervention, participants who took breakfast more frequently took late-night snacks with less frequency

In G3, participants after the intervention showed a lower anger/irritation index than those before the intervention (Wilcoxon's signed-rank sum test: z = −3.072, p = 0.002). On the other hand, G1 showed only a tendency of reduced irritation (z = −1.786, p = 0.074), and G2 had no differences in mental health index after the intervention (z = −0.956, p = 0.340). Anger/irritation indices (the frequency to be irritated and the frequency to become angry due to small trigger) were also decreased after the intervention in G3 (Wilcoxon's signed-

This study showed that a triple intervention concerning breakfast content, sunlight exposure after breakfast, and exposure to low temperature light emitted from incandescent bulbs are powerful methods for inducing secretion of high amounts of melatonin by the pineal gland inhuman adults. Underlying mechanisms can be hypothesized to consist of two components. The first component is that serotonin synthesis from tryptophan taken at breakfast may be enhanced by the exposure to sunlight just after taking breakfast. The second component is that the high potential of melatonin synthesis based on the high serotonin synthesis in the pineal during daytime might be available due to the night exposure to the "low-temperature light" emitted from incandescent bulbs. Many reports have shown that melatonin secretion is suppressed by evening or night light emitted from fluorescent lamps including shortwave length (with around 460 nm of wave length) components [10, 33, 35, 54–56]. Moreover, this study newly implies that the combined behaviors of (1) modifying breakfast content, (2) receiving sunlight exposure, and (3) receiving exposure to low color

rank sum test: irritation z = −2.496, p = 0.013; anger z = 2.714, p = 0.007).

p = 0.011).

contents as high-protein foods.

172 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

(Kendall tau-b test = −142, p = 0.003).

**3.3. Discussion**

#### *4.1.1. Participants*

Sixty percent of 93 participants were born and grown in Shikoku island, whereas the others were from other parts of all over Japan. Participants in this study were 19–25 years old (Takeuchi et al., unpublished).

#### *4.1.2. Procedure*

Seventy-three participants of the experimental group were asked to drink 200 ml of cow's milk at breakfast each morning for a period of 21 days, from 13 November 2014 to 4 December 2014. We provided the milk for them. Twenty participants in the control group did not drink the cow's milk in the morning at breakfast. Twenty men did not like milk originally. Between the two groups, no significant differences were shown in age, diurnal-type scale [50] scores, and sleep habits (wake-up time, bedtime, and sleep hours both in the weekdays and weekends) [52]. After the questionnaires and sleep diaries had been distributed, participants were allowed to answer them at home.

#### *4.1.3. Ethic treatment*

Before administrating the intervention study, all participants were given a written explanation that detailed the concepts and purposes of the study and also stated that we would use the saliva melatonin data only for academic purpose. All participants agreed completely with the proposal and gave written consents after the explanation. The explanation stated that they could withdraw at any time and the withdrawal (canceling) had negative consequences for them. However, there were no withdrawals at fact. The intervention research was performed in accordance with the guidelines which have been established by the *Chronobiology International* journal for the conduct of research on human beings [53]. The study was also permitted by the Kochi university soccer club committee and the committee in the Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences, Kochi University, which carried out ethical inspections regarding the contents of the methodology in this study.

#### *4.1.4. Statistical analysis*

The software used for statistical analysis was SPSS 12.0 J for Windows (SPSS Inc., Chicago, IL, USA). ANOVA and T-test were used for the tests on spacing ratio variables between ranked variables. Friedman test was used for paired variables. We were sometimes interested in changes *during* the intervention (Friedman).

**4.3. Discussion**

members.

The intake of cow's milk at breakfast made the evening-typed members more morning-typed, whereas the diurnal type of the morning-typed members shifted to more evening-typed [49]. Tryptophan included in the cow's milk was metabolized to serotonin in the morning and serotonin again synthesized to melatonin at night [28, 29, 31, 48, 49, 59]. Therefore, the sleep latency seems to become shorter during the intervention [49] due to the melatonin synthesis which has been known as the natural sleep inducer [60] based on cow's milk intake at breakfast. This shorter sleep latency leads to the longer sleep hours for the university soccer club

**Figure 4.** Comparisons of saliva melatonin concentration in saliva taken at 22:00 (A) and 23:00 (B) before intervention, 10 days later and 20 days later (just after the intervention). Black bars (cow's milk consumption group), white bars (no

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cow's milk group) (mean ± 95% confidence) (Takeuchi et al., unpublished).

The melatonin onset timing especially at dim light conditions can be used as a "sign" of the circadian phase [60]. For example, this melatonin-onset timing could be used for the signs of mood disorders and accompanied phase delay of the circadian phase shown by the monopolar and bipolar depression phenotypes [61]. The promotion of the melatonin concentration level at 22:00 due to morning cow's milk consumption might show the phase advance of university soccer team members. If the participants in this study would be senior high school students, the increase timing might be 1 or 2 hour(s) in advance, because the melatonin onset timing was around 21:00 to 22:00 for the senior high school students [62]. However, the number of samples was very small, and the significant differences in the saliva melatonin at 23:00 was not shown in this study. More number of samplings of saliva will be taken in the future study.

#### **4.2. Results**

In saliva collected at 22:00, melatonin concentration was increased during the intervention period in the cow's milk consumption group (ANOVA due to GLM repeating measurement analysis: df = 2, F-value = 8.038, p = 0.080; Friedman test: p = 0.044) (**Figure 4A**). Moreover, the differences in the individual levels in the saliva melatonin concentrations tended to be higher (more increased) in the cow's milk consumption group than the control group (T-test: t-value = 2.05, p = 0.061) (**Figure 4A**). On the other hand, at 23:00, there were no significant differences during the intervention in the melatonin level (ANOVA: df = 2, F-value = 1.999, p = 0.172; Friedman test: p = 0.867) in the experimental group (cow's milk consumption) (**Figure 4B**). On the other hand, there were no differences in the saliva melatonin concentrations through the intervention of 21 days both at 22:00 (**Figure 4A**) (ANOVA: df = 2, F-value = 0.794, p = 0.235; Friedman test: p = 0.867) and at 23:00 (**Figure 4B**) (ANOVA: df = 2, F-value = 0.176, p = 0.841; Friedman test: p = 0.867) in the control group (no cow's milk consumption).

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**Figure 4.** Comparisons of saliva melatonin concentration in saliva taken at 22:00 (A) and 23:00 (B) before intervention, 10 days later and 20 days later (just after the intervention). Black bars (cow's milk consumption group), white bars (no cow's milk group) (mean ± 95% confidence) (Takeuchi et al., unpublished).

#### **4.3. Discussion**

2014. We provided the milk for them. Twenty participants in the control group did not drink the cow's milk in the morning at breakfast. Twenty men did not like milk originally. Between the two groups, no significant differences were shown in age, diurnal-type scale [50] scores, and sleep habits (wake-up time, bedtime, and sleep hours both in the weekdays and weekends) [52]. After the questionnaires and sleep diaries had been distributed, participants were

Before administrating the intervention study, all participants were given a written explanation that detailed the concepts and purposes of the study and also stated that we would use the saliva melatonin data only for academic purpose. All participants agreed completely with the proposal and gave written consents after the explanation. The explanation stated that they could withdraw at any time and the withdrawal (canceling) had negative consequences for them. However, there were no withdrawals at fact. The intervention research was performed in accordance with the guidelines which have been established by the *Chronobiology International* journal for the conduct of research on human beings [53]. The study was also permitted by the Kochi university soccer club committee and the committee in the Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences, Kochi University, which carried out ethical inspections regarding the contents of the methodology

The software used for statistical analysis was SPSS 12.0 J for Windows (SPSS Inc., Chicago, IL, USA). ANOVA and T-test were used for the tests on spacing ratio variables between ranked variables. Friedman test was used for paired variables. We were sometimes interested in

In saliva collected at 22:00, melatonin concentration was increased during the intervention period in the cow's milk consumption group (ANOVA due to GLM repeating measurement analysis: df = 2, F-value = 8.038, p = 0.080; Friedman test: p = 0.044) (**Figure 4A**). Moreover, the differences in the individual levels in the saliva melatonin concentrations tended to be higher (more increased) in the cow's milk consumption group than the control group (T-test: t-value = 2.05, p = 0.061) (**Figure 4A**). On the other hand, at 23:00, there were no significant differences during the intervention in the melatonin level (ANOVA: df = 2, F-value = 1.999, p = 0.172; Friedman test: p = 0.867) in the experimental group (cow's milk consumption) (**Figure 4B**). On the other hand, there were no differences in the saliva melatonin concentrations through the intervention of 21 days both at 22:00 (**Figure 4A**) (ANOVA: df = 2, F-value = 0.794, p = 0.235; Friedman test: p = 0.867) and at 23:00 (**Figure 4B**) (ANOVA: df = 2, F-value = 0.176, p = 0.841; Friedman test: p = 0.867) in the control group (no cow's

allowed to answer them at home.

174 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

*4.1.3. Ethic treatment*

in this study.

**4.2. Results**

milk consumption).

*4.1.4. Statistical analysis*

changes *during* the intervention (Friedman).

The intake of cow's milk at breakfast made the evening-typed members more morning-typed, whereas the diurnal type of the morning-typed members shifted to more evening-typed [49]. Tryptophan included in the cow's milk was metabolized to serotonin in the morning and serotonin again synthesized to melatonin at night [28, 29, 31, 48, 49, 59]. Therefore, the sleep latency seems to become shorter during the intervention [49] due to the melatonin synthesis which has been known as the natural sleep inducer [60] based on cow's milk intake at breakfast. This shorter sleep latency leads to the longer sleep hours for the university soccer club members.

The melatonin onset timing especially at dim light conditions can be used as a "sign" of the circadian phase [60]. For example, this melatonin-onset timing could be used for the signs of mood disorders and accompanied phase delay of the circadian phase shown by the monopolar and bipolar depression phenotypes [61]. The promotion of the melatonin concentration level at 22:00 due to morning cow's milk consumption might show the phase advance of university soccer team members. If the participants in this study would be senior high school students, the increase timing might be 1 or 2 hour(s) in advance, because the melatonin onset timing was around 21:00 to 22:00 for the senior high school students [62]. However, the number of samples was very small, and the significant differences in the saliva melatonin at 23:00 was not shown in this study. More number of samplings of saliva will be taken in the future study. On the 10th day and 21st day of the intervention period, a questionnaire on performance/skill was administered to all participants [49]. The group who drank cow's milk showed higher frequency of improvement of soccer performance than the control group who did not drink [49]. The improved sleep quality which might be induced and enhanced at 22:00 in this study in the melatonin group could be related to such improvement of soccer performance.

2016–2019); (2) Fund from J-Milk "Meals and Education" (To T. Harada: 2013–2014); and (3) Fund from J-Mild "Research Study to Promote Health by Milk and Related Foods" (To T. Harada: 2016–2017). We would like to give many thanks also to Professor Emeritus Jim Waterhouse, Liverpool John Moores University, Liverpool, UK, for his valuable comments

Salivary Melatonin at Night: Responding to the Night Lighting and Cow's Milk Consumption…

http://dx.doi.org/10.5772/intechopen.79816

177

Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences,

[1] Fukuda K, Ishihara K. Research work on life habits and tiredness in Japanese elementary, junior high and senior high school students. In: Reports of Study Supported by Ministry of Sciences and Technologies for Science Promotion Japanese Ministry of Sciences and

[2] Takahashi M, Wang G, Adachi M, Jiang F, Saito M, Nakamura K. Differences in sleep problems between Japanese and Chinese preschoolers: A cross-cultural comparison within the Asian region. Sleep Medicine. 2018;**48**:42-48. DOI: 10. 1016/j.sleep.2017.11.1145

[3] Harada T, Takeuchi H. Scientific study on "Go to bed early! Get up early! Take nutritionally rich breakfast!". Chronobiology. 2017;**23**:9-16 (Jikanseibutugaku in Japanese)

[4] Harada T, Morikuni M, Yoshii S, Yamashita Y, Takeuchi H. Usage of mobile phone in the evening or at night makes Japanese students evening-typed and night sleep uncomfort-

[5] Kawada T, Kataoka T, Tsuji F, Nakade M, Krejci M, Noji T, et al. The relationship between a night usage of mobile phone and sleep habit and the circadian typology of Japanese

[6] Honma K, Honma S.A human phase-response curve for brightlight pulses. The Japanese

[7] Harada T, Takeuchi H. Epidemiological study on diurnal rhythm and sleep habits of Japanese students aged 9-22 years. Annals of Japanese Society for Chronobiology. 2001;

[8] Harada T, Takeuchi H. Do evening usages of convenience store, mobile phone and midnight programs of TV induce the sleep shortage of Japanese children? Japanese Journal

from an academic view point on this manuscript.

\*Address all correspondence to: haratets@kochi-u.ac.jp

Kochi University, Akebono-cho, Kochi, Japan

able. Sleep and Hypnosis. 2002;**4**:150-154

students aged 18-30 years. Psychology. 2017;**8**:892-902

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of Clinical Dentistry for Children. 2003;**8**:57-67 (in Japanese)

Hitomi Takeuchi and Tetsuo Harada\*

**Author details**

**References**

Technologies. 2000

**3**:37-46 (in Japanese)

### **5. Integrated discussion**

Melatonin secretion is very important for sleep induction at bedtime and also keeping sleep quality high [62]. Moreover, night melatonin for mothers can be related to brain development of their children. Braam et al. [63] showed a hypothesis that low parental melatonin levels could be one of the contributors to autism spectrum disorder (ASD) and possibly intellectual disability (ID) etiology. If this hypothesis is correct, this could lead to policies to detect future parents who are at risk and to treatment strategies to ASD and intellectual disability risk.

Pfeffer et al. [64] reviewed that the present contribution of melatonin confirmed the synchronizing effect of endogenous melatonin and the melatoninergic system in humans and rodents. However, they discussed that these effects would be subtle. These relatively subtle effects stand in contrast with their markable, overt therapeutic successes that have been achieved using melatonin as an externally applied chronobiotic in humans [65]. Thus, melatonin does not appear as the master of internal synchronization but as one component in an integrated system of synchronizing agents. Melatonin might be one indicator of a phase point of human circadian clock. And, it can be influenced by breakfast protein consumption and also evening lighting especially modern lighting which includes "blue lights (460–480 nm)" as peak wave lengths of melanopsin [66].

Several studies including Bouwmans et al. [67] that showed the possibilities of network mapping for dynamic person-specific psychological and biological data revealed that there were not only large heterogeneity between affect and fatigue in depression and melatonin secretion which can be related to fall in sleep and sleep quality, namely, negatively and positively correlation with wide personal variety in the relationship. Meaningful generalizations can be made on the interplay of melatonin with affect and fatigue in depression.

Anyway, melanopsin is a key photoreception substance to control circadian phase shift in human beings [68, 69] and moreover to relate to sleep and mood disorders in the parents [66]. Melanopsin function might be related to melatonin synthesis via circadian phase shift indirectly. Twenty-four hours of commercialization society might be dangerous for disruption of circadian phase and normal melatonin secretion through over usage of smartphone (e.g., line and game) and watching TV program at night for children.

### **Acknowledgements**

Thanks should be due to all the participants in this study. The following financial supports have been supplied to this study: (1) JSPS fund (Foundation No: JP 16 K01871; To H. Takeuchi: 2016–2019); (2) Fund from J-Milk "Meals and Education" (To T. Harada: 2013–2014); and (3) Fund from J-Mild "Research Study to Promote Health by Milk and Related Foods" (To T. Harada: 2016–2017). We would like to give many thanks also to Professor Emeritus Jim Waterhouse, Liverpool John Moores University, Liverpool, UK, for his valuable comments from an academic view point on this manuscript.

### **Author details**

On the 10th day and 21st day of the intervention period, a questionnaire on performance/skill was administered to all participants [49]. The group who drank cow's milk showed higher frequency of improvement of soccer performance than the control group who did not drink [49]. The improved sleep quality which might be induced and enhanced at 22:00 in this study

Melatonin secretion is very important for sleep induction at bedtime and also keeping sleep quality high [62]. Moreover, night melatonin for mothers can be related to brain development of their children. Braam et al. [63] showed a hypothesis that low parental melatonin levels could be one of the contributors to autism spectrum disorder (ASD) and possibly intellectual disability (ID) etiology. If this hypothesis is correct, this could lead to policies to detect future parents who are at risk and to treatment strategies to ASD and intellectual disability risk.

Pfeffer et al. [64] reviewed that the present contribution of melatonin confirmed the synchronizing effect of endogenous melatonin and the melatoninergic system in humans and rodents. However, they discussed that these effects would be subtle. These relatively subtle effects stand in contrast with their markable, overt therapeutic successes that have been achieved using melatonin as an externally applied chronobiotic in humans [65]. Thus, melatonin does not appear as the master of internal synchronization but as one component in an integrated system of synchronizing agents. Melatonin might be one indicator of a phase point of human circadian clock. And, it can be influenced by breakfast protein consumption and also evening lighting especially modern lighting which includes "blue lights (460–480 nm)" as peak wave

Several studies including Bouwmans et al. [67] that showed the possibilities of network mapping for dynamic person-specific psychological and biological data revealed that there were not only large heterogeneity between affect and fatigue in depression and melatonin secretion which can be related to fall in sleep and sleep quality, namely, negatively and positively correlation with wide personal variety in the relationship. Meaningful generalizations can be

Anyway, melanopsin is a key photoreception substance to control circadian phase shift in human beings [68, 69] and moreover to relate to sleep and mood disorders in the parents [66]. Melanopsin function might be related to melatonin synthesis via circadian phase shift indirectly. Twenty-four hours of commercialization society might be dangerous for disruption of circadian phase and normal melatonin secretion through over usage of smartphone (e.g., line

Thanks should be due to all the participants in this study. The following financial supports have been supplied to this study: (1) JSPS fund (Foundation No: JP 16 K01871; To H. Takeuchi:

made on the interplay of melatonin with affect and fatigue in depression.

and game) and watching TV program at night for children.

in the melatonin group could be related to such improvement of soccer performance.

**5. Integrated discussion**

176 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

lengths of melanopsin [66].

**Acknowledgements**

Hitomi Takeuchi and Tetsuo Harada\*

\*Address all correspondence to: haratets@kochi-u.ac.jp

Laboratory of Environmental Physiology, Graduate School of Integrated Arts and Sciences, Kochi University, Akebono-cho, Kochi, Japan

### **References**


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**Section 3**

**Pharmaceutical Formulations for Enhancing**

**Melatonin's Bioavailability**


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**Chapter 9**

**Provisional chapter**

**Melatonin Modified Release Formulations Designed**

**Melatonin Modified Release Formulations Designed** 

Melatonin (*N*-acetyl-5-methoxytryptamine, MLT), a hormone synthesized by the pineal gland and released at night, has a regulatory role on sleep in vertebrates, including humans. It has been shown to have a hypnotic action in animals and humans and it has been used as an agent for restoring circadian rhythms, disturbed by jet-lag, shift-work or aging. The physiological actions of melatonin in regulating seasonal and circadian rhythms are mediated through a family of specific, high affinity G protein-coupled membrane receptors. The beneficial effect of fast-release formulations on sleep initiation may come from the high amount of melatonin released immediately after administration, while the benefit of the sustained release systems comes from the release of melatonin in small dosages during the entire night period. This chapter covers the recent scientific

**Keywords:** melatonin, sleep onset, sleep maintenance, modified release, formulations

Melatonin (MLT), which was originally discovered in the bovine pineal gland in 1958, is a pleiotropic molecule with numerous cellular and physiological functions in vertebrates, including humans. MLT's production is regulated by a multisynaptic pathway from the biological clock in the suprachiasmatic nucleus (SCN), in the hypothalamus, to the pineal gland. The SCN is the primary site for both generation and integration of signals, which regulate melatonin's production by the pineal. Control at this central point ensures the high nocturnal

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.78337

**for Sleep Disorders**

**Abstract**

**1. Introduction**

**for Sleep Disorders**

Marilena Vlachou and Angeliki Siamidi

Marilena Vlachou and Angeliki Siamidi

http://dx.doi.org/10.5772/intechopen.78337

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

work on melatonin modified release formulations.

concentration and extremely low diurnal melatonin synthesis.

#### **Melatonin Modified Release Formulations Designed for Sleep Disorders Melatonin Modified Release Formulations Designed for Sleep Disorders**

DOI: 10.5772/intechopen.78337

Marilena Vlachou and Angeliki Siamidi Marilena Vlachou and Angeliki Siamidi

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.78337

#### **Abstract**

Melatonin (*N*-acetyl-5-methoxytryptamine, MLT), a hormone synthesized by the pineal gland and released at night, has a regulatory role on sleep in vertebrates, including humans. It has been shown to have a hypnotic action in animals and humans and it has been used as an agent for restoring circadian rhythms, disturbed by jet-lag, shift-work or aging. The physiological actions of melatonin in regulating seasonal and circadian rhythms are mediated through a family of specific, high affinity G protein-coupled membrane receptors. The beneficial effect of fast-release formulations on sleep initiation may come from the high amount of melatonin released immediately after administration, while the benefit of the sustained release systems comes from the release of melatonin in small dosages during the entire night period. This chapter covers the recent scientific work on melatonin modified release formulations.

**Keywords:** melatonin, sleep onset, sleep maintenance, modified release, formulations

### **1. Introduction**

Melatonin (MLT), which was originally discovered in the bovine pineal gland in 1958, is a pleiotropic molecule with numerous cellular and physiological functions in vertebrates, including humans. MLT's production is regulated by a multisynaptic pathway from the biological clock in the suprachiasmatic nucleus (SCN), in the hypothalamus, to the pineal gland. The SCN is the primary site for both generation and integration of signals, which regulate melatonin's production by the pineal. Control at this central point ensures the high nocturnal concentration and extremely low diurnal melatonin synthesis.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Melatonin has been shown to have a hypnotic action in animals and humans, and there has been considerable recent interest in the therapeutic potential of melatonin and its analogues as hypnotics and as agents for restoring circadian rhythms, disturbed by jet-lag, shiftwork, and aging. The physiological actions of melatonin in regulating seasonal and circadian rhythms are thought to be mediated through a family of specific, high affinity, and G-proteincoupled cell membrane receptors. One of its main uses is insomnia treatment, particularly among the elderly, with up to 50% of people over the age of 65 reporting trouble sleeping. Night time insomnia is associated with increased daytime sleepiness, reduced motor and cognitive performance, and reduced productivity in the workplace and is an important cause of industrial and road traffic accidents. Current hypnotic drugs are recommended only for short-term treatment of insomnia, but concerns about "hangover" effects and problems upon withdrawal persist. Many people with occasional sleep problems resort to self-medication and over-the-counter sales of medicines for sleep problems are increasing rapidly [1].

to pediatric patients that may have potential difficulties taking other oral dosage forms [3]. Dosages of 3 and 5 mg were intended for epileptic children, migraine prevention, neurodevelopmental disability, sleep disorders, and blindness, whereas dosages of 10 and 60 mg were used for Duchenne muscular dystrophy. Tablets have been produced with excipients for direct compression, having disintegration times of less than 25 s and with friability and hardness values that require no special storage or packaging conditions. The results indicated that these orodispersible tablets have been easily produced via direct compression, having low costs and optimal galenic assay results. To explore the therapeutic potential of melatonin, as an antioxidant agent, researchers have analyzed the red blood cell antioxidants and lipid peroxidation after 5 mg/daily immediate-release melatonin treatment of elderly with type 2 diabetes patients and healthy elderly subjects in comparison with 2 mg/daily sustained-release. The results suggest that both immediate and sustained release preparations may exert similar therapeutic effects related to

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Immediate release oral drug dosage forms are formulated in order to release the active substance immediately after oral administration. On the other hand, modified-release oral drug dosage forms are deliberately modified from those of an immediate release, to achieve a desired therapeutic objective or better patient compliance. The term modified release drug dosage forms is used to describe formulations that alter the time and/or the rate of release. With this in view, scientists have focused on modifying the release of melatonin especially to treat conditions that deal with circadian rhythmic disorders, like sleep syndrome, insomnia, jet lag, seasonal affective disease, shift work syndrome, etc. [5–7]. Modified-release melatonin treatment could be more useful to initiate and maintain sleep, compared with immediate-

Many scientists have focused on different ways to manufacture modified release tablets. One of the most common ways is the use of various excipients that facilitate the prolong release when tablet manufacturing [8–13]. These excipients usually involve hydroxypropylmethylcellulose, polyvinylpyrrolidone, and sodium alginate in various molecular weights or forms. Another way to achieve modified release is the production of different tablet formulations, like multilayer or bilayer, coated or uncoated tablets [14–16]. Other researchers have studied the use of liposomal formulations [17] or nanoparticles [18]. Recently, electrospun nanofibrous systems, incorporating melatonin, have been used for its modified release [19]. Another way is to employ a variety of techniques, like for example, the experimental design [20] in order to facilitate the modified release tablet production and therefore, to improve quality of sleep in patients, with minimal side effects. Both in the development of these systems and the immediate release formulations, the fact that melatonin displays both a circadian and circannual rhythm and is secreted only during the night has been taken into account. This physiological rhythm needs to be conserved or modulated (advanced, reversed, diminished, or amplified) according to the appropriate

melatonin's action as antioxidant [4].

release therapy.

therapeutic indications.

**2.2. Melatonin** *per os* **modified release formulations**

The administration of exogenous melatonin is effected via different formulations, but at present, the most studied is the oral route. Also, there are different dosages of melatonin. Because of its pharmacokinetic characteristics, it is necessary to maintain melatonin's concentration for a long time to imitate its physiological release, especially for insomnia treatment. To this end, prolonged-release formulations of melatonin have been developed covering the entire night cycle and improving sleep disorders. As the toxicology of melatonin's formulations is concerned, very little is known. However, the acute toxicity of melatonin, as seen in both animal and human studies, is extremely low. Nevertheless, further research needs to be undertaken, including regulatory studies.

### **2. Formulations of melatonin**

Melatonin is traditionally administered orally in immediate and modified release formulations, but has a poor and variable bioavailability. Apart from *per os* administration, melatonin is currently under research with respect to other routes of administration, such as sublingual, transbuccal/transmucosal, and intranasal for topical and systemic exposure, injectables (intravenous, i.v. bolus infusion, intramuscular, subcutaneous, and implant), topical preparations, and transdermal patches. This chapter aims at presenting an overview of all administration routes and different kinds of formulations of melatonin that are currently explored *in vitro* and *in vivo*, including experimental and clinical studies.

#### **2.1. Melatonin** *per os* **immediate release formulations**

*Per os* immediate release formulations imply that the full dose of the drug is absorbed into the bloodstream all at once. This pattern of fast released melatonin is most effective when against delayed sleep onset [2]. The immediate release dosage forms (fast released, orodispersible, sublingual tablets oral sprays, etc.) are usually administered to patients prior to sleep time in order to facilitate sleep onset. A research group has manufactured fast dissolving disintegrating tablets containing different dosages of melatonin for administration to pediatric patients that may have potential difficulties taking other oral dosage forms [3]. Dosages of 3 and 5 mg were intended for epileptic children, migraine prevention, neurodevelopmental disability, sleep disorders, and blindness, whereas dosages of 10 and 60 mg were used for Duchenne muscular dystrophy. Tablets have been produced with excipients for direct compression, having disintegration times of less than 25 s and with friability and hardness values that require no special storage or packaging conditions. The results indicated that these orodispersible tablets have been easily produced via direct compression, having low costs and optimal galenic assay results. To explore the therapeutic potential of melatonin, as an antioxidant agent, researchers have analyzed the red blood cell antioxidants and lipid peroxidation after 5 mg/daily immediate-release melatonin treatment of elderly with type 2 diabetes patients and healthy elderly subjects in comparison with 2 mg/daily sustained-release. The results suggest that both immediate and sustained release preparations may exert similar therapeutic effects related to melatonin's action as antioxidant [4].

#### **2.2. Melatonin** *per os* **modified release formulations**

Melatonin has been shown to have a hypnotic action in animals and humans, and there has been considerable recent interest in the therapeutic potential of melatonin and its analogues as hypnotics and as agents for restoring circadian rhythms, disturbed by jet-lag, shiftwork, and aging. The physiological actions of melatonin in regulating seasonal and circadian rhythms are thought to be mediated through a family of specific, high affinity, and G-proteincoupled cell membrane receptors. One of its main uses is insomnia treatment, particularly among the elderly, with up to 50% of people over the age of 65 reporting trouble sleeping. Night time insomnia is associated with increased daytime sleepiness, reduced motor and cognitive performance, and reduced productivity in the workplace and is an important cause of industrial and road traffic accidents. Current hypnotic drugs are recommended only for short-term treatment of insomnia, but concerns about "hangover" effects and problems upon withdrawal persist. Many people with occasional sleep problems resort to self-medication

and over-the-counter sales of medicines for sleep problems are increasing rapidly [1].

taken, including regulatory studies.

186 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**2. Formulations of melatonin**

and *in vivo*, including experimental and clinical studies.

**2.1. Melatonin** *per os* **immediate release formulations**

The administration of exogenous melatonin is effected via different formulations, but at present, the most studied is the oral route. Also, there are different dosages of melatonin. Because of its pharmacokinetic characteristics, it is necessary to maintain melatonin's concentration for a long time to imitate its physiological release, especially for insomnia treatment. To this end, prolonged-release formulations of melatonin have been developed covering the entire night cycle and improving sleep disorders. As the toxicology of melatonin's formulations is concerned, very little is known. However, the acute toxicity of melatonin, as seen in both animal and human studies, is extremely low. Nevertheless, further research needs to be under-

Melatonin is traditionally administered orally in immediate and modified release formulations, but has a poor and variable bioavailability. Apart from *per os* administration, melatonin is currently under research with respect to other routes of administration, such as sublingual, transbuccal/transmucosal, and intranasal for topical and systemic exposure, injectables (intravenous, i.v. bolus infusion, intramuscular, subcutaneous, and implant), topical preparations, and transdermal patches. This chapter aims at presenting an overview of all administration routes and different kinds of formulations of melatonin that are currently explored *in vitro*

*Per os* immediate release formulations imply that the full dose of the drug is absorbed into the bloodstream all at once. This pattern of fast released melatonin is most effective when against delayed sleep onset [2]. The immediate release dosage forms (fast released, orodispersible, sublingual tablets oral sprays, etc.) are usually administered to patients prior to sleep time in order to facilitate sleep onset. A research group has manufactured fast dissolving disintegrating tablets containing different dosages of melatonin for administration Immediate release oral drug dosage forms are formulated in order to release the active substance immediately after oral administration. On the other hand, modified-release oral drug dosage forms are deliberately modified from those of an immediate release, to achieve a desired therapeutic objective or better patient compliance. The term modified release drug dosage forms is used to describe formulations that alter the time and/or the rate of release. With this in view, scientists have focused on modifying the release of melatonin especially to treat conditions that deal with circadian rhythmic disorders, like sleep syndrome, insomnia, jet lag, seasonal affective disease, shift work syndrome, etc. [5–7]. Modified-release melatonin treatment could be more useful to initiate and maintain sleep, compared with immediaterelease therapy.

Many scientists have focused on different ways to manufacture modified release tablets. One of the most common ways is the use of various excipients that facilitate the prolong release when tablet manufacturing [8–13]. These excipients usually involve hydroxypropylmethylcellulose, polyvinylpyrrolidone, and sodium alginate in various molecular weights or forms. Another way to achieve modified release is the production of different tablet formulations, like multilayer or bilayer, coated or uncoated tablets [14–16]. Other researchers have studied the use of liposomal formulations [17] or nanoparticles [18]. Recently, electrospun nanofibrous systems, incorporating melatonin, have been used for its modified release [19]. Another way is to employ a variety of techniques, like for example, the experimental design [20] in order to facilitate the modified release tablet production and therefore, to improve quality of sleep in patients, with minimal side effects. Both in the development of these systems and the immediate release formulations, the fact that melatonin displays both a circadian and circannual rhythm and is secreted only during the night has been taken into account. This physiological rhythm needs to be conserved or modulated (advanced, reversed, diminished, or amplified) according to the appropriate therapeutic indications.

#### **2.3. Melatonin sublingual/transbuccal formulations**

The oral cavity is a perfect route of administration for both topical and systemic treatment. Considering melatonin, research has suggested that it is effective in treating pathologies like periodontitis, mucositis, cancers, and cytotoxicity from various drugs or biomaterials. Furthermore, melatonin has been observed to enhance osseointegration "functional ankylosis (bone adherence)" and bone regeneration, to promote the healing of tooth extraction sockets and may also impede the progression of oral cancer [21, 22]. On the other hand, sublingual and transbuccal/transmucosal administration of melatonin in the forms of sublingual tablets or oral sprays has shown comparable systemic results to other routes of administration.

on sedation, orientation, anxiety scores, and psychomotor performance, melatonin 5 mg, midazolam 15 mg or placebo was administered 90 min prior to anesthesia, sublingually to 66 patients undergoing laparoscopic cholecystectomy [27]. Sedation, orientation, and anxiety were quantified before 10, 30, 60, and 90 min after premedication and 15, 30, 60 and 90 min after admission to the recovery room. The results indicated that melatonin premedication was associated with preoperative anxiolysis and sedation without postoperative impairment of

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Intranasal administration is a route of administration for drugs used primarily for the treatment of conditions affecting the nasal cavity, but can also be used for cases requiring systemic exposure, since drugs can be absorbed into the circulation through the nasal mucosa. This kind of systemic administration offers lots of advantages, such as rapid onset of action and avoidance of first-pass metabolism [28]. Many researchers have investigated melatonin's intranasal administration and tested their formulations *in vitro* and *in vivo* in rats, rabbits, and

In another study, the role of inclusion complexes of melatonin with modified cyclodextrins (CDs) in order to improve melatonin's solubility and nasal absorption was investigated [29]. The formation of inclusion complex of melatonin with hydroxypropyl *β*-cyclodextrin (HP*β*CD) and randomly methylated *β*-cyclodextrin (RM*β*CD) was demonstrated in solution and solid state and both CD's at 1% w/v concentration were found to improve the nasal permeability (the *in vitro* permeability studies were carried out with EpiAirwayTM-100 cell cultures from

Intranasal melatonin encapsulated in nanosized niosomes has been preclinically evaluated in male Wistar rats [30]. It was found that intranasal melatonin niosomes that were bioequivalent to intravenous injection of melatonin could provide therapeutic level doses, deliver melatonin to the brain to induce sleep, and delay systemic circulation. The cross-over study, including eight rats, examined the intranasal administration of melatonin from a nasal formulation consisted of melatonin (2.0 mg/ml), *β*-cyclodextrin (7.5 mg/ml) dissolved in saline that also contained benzalkonium chloride (0.01% w/v) and EDTA (0.1% w/v) as preservatives, in comparison to the administration from an intravenous bolus injection. Tmax was recorded at 2.5 min in both routes of administration and an almost zero plasma concentration after

In another study, the research group [32] prepared melatonin gelatin microspheres (MLT-GMS) for intranasal administration and tested them in comparison to gelatin solution and i.v. injection. The *in vitro* release profile showed sustained effect, while the residence time of MLT-GMS in the rabbits' nasal cavity was longer than that of gelatin solution. After intranasal administration in rabbits, the bioavailability of MLT-GMS was 87.47%, while the bioavailability of melatonin solution was 69.72%, when compared to i.v. administration. The results showed that this formulation could meet the needs of intranasal administration, while increasing melatonin's bioavailability. Other scientists have studied the bioavailability of

MatTek Corporation) of melatonin from HPMC gel formulations.

psychomotor performance.

humans.

120 min [31].

**2.4. Melatonin intranasal formulations**

Many scientists have considered the oral cavity as a route of administering melatonin and compared it to other routes. The effect of transdermal, in comparison to oral controlled release and oral transmucosal drug delivery systems, on plasma concentrations of melatonin and its principal metabolite, 6-sulfatoxymelatonin, was evaluated in 12 human volunteers, using a crossover, single dose design. Oral transmucosal delivery provided prompt systemic drug levels with less variability than oral or transdermal delivery and no indication of mucosal deposition. The results indicated that oral transmucosal delivery was able to mimic the physiological plasma profiles of both melatonin and its principal metabolite [23]. Another study involved 60 patients, who were randomly assigned to receive either sublingual melatonin (3 mg) or placebo, 60 min before cataract surgery. The dose of 3 mg of melatonin sublingually, 60 min before surgery, was chosen, because the onset of melatonin-induced sedation is reported to begin approximately 20–30 min after sublingual administration. The results concluded that sublingual melatonin premedication for patients undergoing cataract surgery, under topical anesthesia, reduced the anxiety scores and provided excellent operating conditions [24]. A prospective, randomized, double-blinded, placebo-controlled study was designed to compare the perioperative effects of different doses of melatonin and midazolam [25]. Doses of 0.05, 0.1, or 0.2 mg/kg sublingual midazolam or melatonin or placebo were given to 84 women, approximately 100 min before a typical anesthetic. Sedation, anxiety, and orientation were quantified before, 10, 30, 60, and 90 min after premedication and 15, 30, 60, and 90 min after admission to the recovery room. Patients who received premedication with either midazolam or melatonin had a significant decrease in anxiety levels and increase in preoperative levels of sedation, compared with control subjects. Premedication with 0.05 mg/kg melatonin was associated with preoperative anxiolysis and sedation without the impairment of cognitive and psychomotor skills or affecting the quality of recovery. To evaluate the analgesic dose response of the effects of melatonin on pressure and heat pain threshold, tolerance, and its possible sedative effects, the research group of Stefani et al. [26] recruited 61 healthy subjects aged 19–47 years old and placed them randomly into one of four groups: placebo, 0.05 mg/kg sublingual melatonin, 0.15 mg/kg sublingual melatonin, or 0.25 mg/kg sublingual melatonin. Serum plasma melatonin levels were found to be directly proportional to the melatonin doses given to each subject indicating that the sublingual melatonin has a well-defined dose-dependent antinociceptive activity. These results provided a correlation between the plasma melatonin drug concentration and acute changes in the pain threshold. To compare the perioperative effects of melatonin and midazolam, given in premedication, on sedation, orientation, anxiety scores, and psychomotor performance, melatonin 5 mg, midazolam 15 mg or placebo was administered 90 min prior to anesthesia, sublingually to 66 patients undergoing laparoscopic cholecystectomy [27]. Sedation, orientation, and anxiety were quantified before 10, 30, 60, and 90 min after premedication and 15, 30, 60 and 90 min after admission to the recovery room. The results indicated that melatonin premedication was associated with preoperative anxiolysis and sedation without postoperative impairment of psychomotor performance.

#### **2.4. Melatonin intranasal formulations**

**2.3. Melatonin sublingual/transbuccal formulations**

188 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

The oral cavity is a perfect route of administration for both topical and systemic treatment. Considering melatonin, research has suggested that it is effective in treating pathologies like periodontitis, mucositis, cancers, and cytotoxicity from various drugs or biomaterials. Furthermore, melatonin has been observed to enhance osseointegration "functional ankylosis (bone adherence)" and bone regeneration, to promote the healing of tooth extraction sockets and may also impede the progression of oral cancer [21, 22]. On the other hand, sublingual and transbuccal/transmucosal administration of melatonin in the forms of sublingual tablets or oral sprays has shown comparable systemic results to other routes of administration.

Many scientists have considered the oral cavity as a route of administering melatonin and compared it to other routes. The effect of transdermal, in comparison to oral controlled release and oral transmucosal drug delivery systems, on plasma concentrations of melatonin and its principal metabolite, 6-sulfatoxymelatonin, was evaluated in 12 human volunteers, using a crossover, single dose design. Oral transmucosal delivery provided prompt systemic drug levels with less variability than oral or transdermal delivery and no indication of mucosal deposition. The results indicated that oral transmucosal delivery was able to mimic the physiological plasma profiles of both melatonin and its principal metabolite [23]. Another study involved 60 patients, who were randomly assigned to receive either sublingual melatonin (3 mg) or placebo, 60 min before cataract surgery. The dose of 3 mg of melatonin sublingually, 60 min before surgery, was chosen, because the onset of melatonin-induced sedation is reported to begin approximately 20–30 min after sublingual administration. The results concluded that sublingual melatonin premedication for patients undergoing cataract surgery, under topical anesthesia, reduced the anxiety scores and provided excellent operating conditions [24]. A prospective, randomized, double-blinded, placebo-controlled study was designed to compare the perioperative effects of different doses of melatonin and midazolam [25]. Doses of 0.05, 0.1, or 0.2 mg/kg sublingual midazolam or melatonin or placebo were given to 84 women, approximately 100 min before a typical anesthetic. Sedation, anxiety, and orientation were quantified before, 10, 30, 60, and 90 min after premedication and 15, 30, 60, and 90 min after admission to the recovery room. Patients who received premedication with either midazolam or melatonin had a significant decrease in anxiety levels and increase in preoperative levels of sedation, compared with control subjects. Premedication with 0.05 mg/kg melatonin was associated with preoperative anxiolysis and sedation without the impairment of cognitive and psychomotor skills or affecting the quality of recovery. To evaluate the analgesic dose response of the effects of melatonin on pressure and heat pain threshold, tolerance, and its possible sedative effects, the research group of Stefani et al. [26] recruited 61 healthy subjects aged 19–47 years old and placed them randomly into one of four groups: placebo, 0.05 mg/kg sublingual melatonin, 0.15 mg/kg sublingual melatonin, or 0.25 mg/kg sublingual melatonin. Serum plasma melatonin levels were found to be directly proportional to the melatonin doses given to each subject indicating that the sublingual melatonin has a well-defined dose-dependent antinociceptive activity. These results provided a correlation between the plasma melatonin drug concentration and acute changes in the pain threshold. To compare the perioperative effects of melatonin and midazolam, given in premedication, Intranasal administration is a route of administration for drugs used primarily for the treatment of conditions affecting the nasal cavity, but can also be used for cases requiring systemic exposure, since drugs can be absorbed into the circulation through the nasal mucosa. This kind of systemic administration offers lots of advantages, such as rapid onset of action and avoidance of first-pass metabolism [28]. Many researchers have investigated melatonin's intranasal administration and tested their formulations *in vitro* and *in vivo* in rats, rabbits, and humans.

In another study, the role of inclusion complexes of melatonin with modified cyclodextrins (CDs) in order to improve melatonin's solubility and nasal absorption was investigated [29]. The formation of inclusion complex of melatonin with hydroxypropyl *β*-cyclodextrin (HP*β*CD) and randomly methylated *β*-cyclodextrin (RM*β*CD) was demonstrated in solution and solid state and both CD's at 1% w/v concentration were found to improve the nasal permeability (the *in vitro* permeability studies were carried out with EpiAirwayTM-100 cell cultures from MatTek Corporation) of melatonin from HPMC gel formulations.

Intranasal melatonin encapsulated in nanosized niosomes has been preclinically evaluated in male Wistar rats [30]. It was found that intranasal melatonin niosomes that were bioequivalent to intravenous injection of melatonin could provide therapeutic level doses, deliver melatonin to the brain to induce sleep, and delay systemic circulation. The cross-over study, including eight rats, examined the intranasal administration of melatonin from a nasal formulation consisted of melatonin (2.0 mg/ml), *β*-cyclodextrin (7.5 mg/ml) dissolved in saline that also contained benzalkonium chloride (0.01% w/v) and EDTA (0.1% w/v) as preservatives, in comparison to the administration from an intravenous bolus injection. Tmax was recorded at 2.5 min in both routes of administration and an almost zero plasma concentration after 120 min [31].

In another study, the research group [32] prepared melatonin gelatin microspheres (MLT-GMS) for intranasal administration and tested them in comparison to gelatin solution and i.v. injection. The *in vitro* release profile showed sustained effect, while the residence time of MLT-GMS in the rabbits' nasal cavity was longer than that of gelatin solution. After intranasal administration in rabbits, the bioavailability of MLT-GMS was 87.47%, while the bioavailability of melatonin solution was 69.72%, when compared to i.v. administration. The results showed that this formulation could meet the needs of intranasal administration, while increasing melatonin's bioavailability. Other scientists have studied the bioavailability of melatonin in rabbits after nasal administration of two formulations (1.5 mg melatonin in 40% PEG 300 in the presence and absence of 1% sodium glycocholate) in comparison to the i.v. route [33]. The results documented that the bioavailabilities in rabbits correspond to much higher values, which indicates a potential advantage of using nasal delivery for melatonin and the possibility of producing a clinically relevant nasal formulation. In another study, starch microspheres of melatonin for intranasal administration were prepared by an emulsification crosslinking technique using a uniform design to optimize preparation conditions [34]. The *in vitro* release experiments showed that melatonin was released from the microspheres in a sustained manner. Nasal clearance studies in six healthy, male rabbits showed that >80% of the radioactivity from the starch microspheres was present in the nasal mucosa 2 h after administration, compared to only 30% radioactivity from the solution. The absorption rate after intranasal administration of the microspheres was rapid, and the absolute bioavailability was high, compared to the intranasal solution and a significant correlation between *in vivo* and *in vitro* data was recorded.

absolute bioavailability of melatonin was studied in 12 young healthy subjects (six males and six females) after administration at midday, on two separate occasions: 23 μg by intravenous infusion and 250 μg by oral solution of D7 melatonin (seven deuterium atoms replace seven hydrogen atoms in the melatonin molecule). Exogenous (D7) and endogenous (D0) melatonin were quantified simultaneously, but separately, by a highly specific assay, gas chromatography/negative ion chemical ionization mass spectrometry. After i.v. administration, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC) values were found to be significantly different in male and female subjects, but there was no significant gender difference in total body clearance when normalized to body weight. After oral administration, pharmacokinetic parameters used to quantify bioavailability were near 3 times greater in female subjects than in males, with large inter-individual variations [39]. In the review article of Gómez-Moreno et al. [40], the use of melatonin in implant dentistry has been proved to increase the new bone formation and bone-to-implant values, around dental implants, leading to a more stable bone area around the implants. In view of these findings, researchers are currently exploring further possibilities as to how

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Two i.v. formulations of melatonin (5 mg/ml) have been tested in Wistar rats: one formulation with hydroxypropyl-*β*-cyclodextrin and propylene glycol to increase solubility and stability and the other with an antioxidant and chelating agent to minimize oxidation and hydrolysis. The pharmacokinetic profiles and the plasma antioxidant activity results suggested a potential for use in the clinical study for these two i.v. melatonin formulations [41]. Another research group [42] studied the possibility of using high-dose intravenous melatonin as an anesthetic adjuvant and compared its effects with thiopental and propofol using Sprague Dawley rats. By administering to the rats bolus or cumulative i.v. doses of melatonin, thiopental, or propofol, they assessed the righting reflex, hind paw withdrawal to a noxious stimulus, response to tail clamping and hemodynamic effects. The results showed that melatonin caused a dose-dependent increase in paw withdrawal. Melatonin was comparable to thiopental and propofol in terms of its rapid onset of hypnosis and it was concluded that intravenous melatonin can exert hypnotic effects similar to those observed with thiopental and propofol. Other researchers have injected mice daily with 50 μg of melatonin, 12 h after lights on, for 18 weeks. The results indicated that mice underwent gonadal regression after 4–7 weeks and

The effect of differing doses and routes of administration of melatonin on plasma melatonin levels in sheep and goats has been examined by the researchers Kennaway and Seamark [44]. Melatonin injected subcutaneously in a saline or oil vehicle caused high transitory peaks in plasma melatonin, whereas oral administration, in either saline solution or adsorbed onto pelleted foodstuff, resulted in sustained elevated blood levels for periods exceeding 7 h. Oral dosages of about 2 mg proved adequate to raise the normal daytime plasma levels in both sheep and goats to levels within the normal night-time range. It was concluded that with ruminants the oral route of administration provides a facile and practical way of administering melatonin for physiological study. The review paper of Williams et al. [45] presents data from five research trials and 108 clinical trials conducted in three countries to validate the optimum use of melatonin to advance seasonal breeding patterns of a variety of breeds of

melatonin might benefit implant dentistry.

reproductive recrudescence after 15 weeks [43].

In humans, a cross-over study in three volunteers, receiving either intranasally 0.4 mg melatonin or intravenously 0.2 mg on two separate study days, was undertaken. The study reported a Tmax value of 5 min for intranasal administration and 10 min for intravenous administration [35]. Other researchers [36] have formulated melatonin, as a thermoreversible Pluronic gel for nasal administration for treating sleep disorders. The comparative electroencephalogram (EEG) pattern, derived from five healthy volunteers who participated in this crossover study after administration of melatonin tablet and nasal gel, revealed that nasal absorption of melatonin was faster and the sleep produced resembled to one during nocturnal chronobiological melatonin secretion. The optimized formulation has provided bimodal drug release extending over 5 h at significantly low dose (1 mg intranasal dose) as compared to 3 mg oral dose. The use of the novel thermoreversible Pluronic gel showed the desired bioadhesion to the nasal mucosa, with no sensitizing effect in subjects and reproducible sleep characteristics thus making this formulation an agent with an excellent commercial potential.

#### **2.5. Melatonin injectable formulations**

Injectable formulations also play an important role as melatonin modified release systems. Many researchers have focused on intravenous (i.v. bolus infusion), intramuscular, subcutaneous, and implant administration of melatonin formulations in human volunteers, small laboratory animals, as well as in larger animals like ewes, goats, and deer.

A research work showed that during melatonin infusion (n = 4 bolus intravenous injection of 5 or 10 μg/person and after a 5-h infusion of 20 μg per person in six healthy subjects), the plasma hormone level reached a steady-state after 60 and 120 min, which was equal to the nocturnal level. This particular infusion regime could be valuable in replacing blunted hormonal secretion in disease states [37]. A human positron emission tomography (PET) study, performed in a healthy volunteer with 11C-labeled melatonin, showed maximum activity in the brain 8.5 min following the injection, quite different from the curve observed for the plasma radioactivity (maximum at 3.5 min), confirming that melatonin crosses the blood-brain barrier and that 6-sulfatoxymelatonin is its main plasma metabolite [38]. The absolute bioavailability of melatonin was studied in 12 young healthy subjects (six males and six females) after administration at midday, on two separate occasions: 23 μg by intravenous infusion and 250 μg by oral solution of D7 melatonin (seven deuterium atoms replace seven hydrogen atoms in the melatonin molecule). Exogenous (D7) and endogenous (D0) melatonin were quantified simultaneously, but separately, by a highly specific assay, gas chromatography/negative ion chemical ionization mass spectrometry. After i.v. administration, the maximum plasma concentration (Cmax) and the area under the plasma concentration-time curve (AUC) values were found to be significantly different in male and female subjects, but there was no significant gender difference in total body clearance when normalized to body weight. After oral administration, pharmacokinetic parameters used to quantify bioavailability were near 3 times greater in female subjects than in males, with large inter-individual variations [39]. In the review article of Gómez-Moreno et al. [40], the use of melatonin in implant dentistry has been proved to increase the new bone formation and bone-to-implant values, around dental implants, leading to a more stable bone area around the implants. In view of these findings, researchers are currently exploring further possibilities as to how melatonin might benefit implant dentistry.

melatonin in rabbits after nasal administration of two formulations (1.5 mg melatonin in 40% PEG 300 in the presence and absence of 1% sodium glycocholate) in comparison to the i.v. route [33]. The results documented that the bioavailabilities in rabbits correspond to much higher values, which indicates a potential advantage of using nasal delivery for melatonin and the possibility of producing a clinically relevant nasal formulation. In another study, starch microspheres of melatonin for intranasal administration were prepared by an emulsification crosslinking technique using a uniform design to optimize preparation conditions [34]. The *in vitro* release experiments showed that melatonin was released from the microspheres in a sustained manner. Nasal clearance studies in six healthy, male rabbits showed that >80% of the radioactivity from the starch microspheres was present in the nasal mucosa 2 h after administration, compared to only 30% radioactivity from the solution. The absorption rate after intranasal administration of the microspheres was rapid, and the absolute bioavailability was high, compared to the intranasal solution and a significant correlation between *in vivo*

In humans, a cross-over study in three volunteers, receiving either intranasally 0.4 mg melatonin or intravenously 0.2 mg on two separate study days, was undertaken. The study reported a Tmax value of 5 min for intranasal administration and 10 min for intravenous administration [35]. Other researchers [36] have formulated melatonin, as a thermoreversible Pluronic gel for nasal administration for treating sleep disorders. The comparative electroencephalogram (EEG) pattern, derived from five healthy volunteers who participated in this crossover study after administration of melatonin tablet and nasal gel, revealed that nasal absorption of melatonin was faster and the sleep produced resembled to one during nocturnal chronobiological melatonin secretion. The optimized formulation has provided bimodal drug release extending over 5 h at significantly low dose (1 mg intranasal dose) as compared to 3 mg oral dose. The use of the novel thermoreversible Pluronic gel showed the desired bioadhesion to the nasal mucosa, with no sensitizing effect in subjects and reproducible sleep characteristics

thus making this formulation an agent with an excellent commercial potential.

laboratory animals, as well as in larger animals like ewes, goats, and deer.

Injectable formulations also play an important role as melatonin modified release systems. Many researchers have focused on intravenous (i.v. bolus infusion), intramuscular, subcutaneous, and implant administration of melatonin formulations in human volunteers, small

A research work showed that during melatonin infusion (n = 4 bolus intravenous injection of 5 or 10 μg/person and after a 5-h infusion of 20 μg per person in six healthy subjects), the plasma hormone level reached a steady-state after 60 and 120 min, which was equal to the nocturnal level. This particular infusion regime could be valuable in replacing blunted hormonal secretion in disease states [37]. A human positron emission tomography (PET) study, performed in a healthy volunteer with 11C-labeled melatonin, showed maximum activity in the brain 8.5 min following the injection, quite different from the curve observed for the plasma radioactivity (maximum at 3.5 min), confirming that melatonin crosses the blood-brain barrier and that 6-sulfatoxymelatonin is its main plasma metabolite [38]. The

and *in vitro* data was recorded.

190 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

**2.5. Melatonin injectable formulations**

Two i.v. formulations of melatonin (5 mg/ml) have been tested in Wistar rats: one formulation with hydroxypropyl-*β*-cyclodextrin and propylene glycol to increase solubility and stability and the other with an antioxidant and chelating agent to minimize oxidation and hydrolysis. The pharmacokinetic profiles and the plasma antioxidant activity results suggested a potential for use in the clinical study for these two i.v. melatonin formulations [41]. Another research group [42] studied the possibility of using high-dose intravenous melatonin as an anesthetic adjuvant and compared its effects with thiopental and propofol using Sprague Dawley rats. By administering to the rats bolus or cumulative i.v. doses of melatonin, thiopental, or propofol, they assessed the righting reflex, hind paw withdrawal to a noxious stimulus, response to tail clamping and hemodynamic effects. The results showed that melatonin caused a dose-dependent increase in paw withdrawal. Melatonin was comparable to thiopental and propofol in terms of its rapid onset of hypnosis and it was concluded that intravenous melatonin can exert hypnotic effects similar to those observed with thiopental and propofol. Other researchers have injected mice daily with 50 μg of melatonin, 12 h after lights on, for 18 weeks. The results indicated that mice underwent gonadal regression after 4–7 weeks and reproductive recrudescence after 15 weeks [43].

The effect of differing doses and routes of administration of melatonin on plasma melatonin levels in sheep and goats has been examined by the researchers Kennaway and Seamark [44]. Melatonin injected subcutaneously in a saline or oil vehicle caused high transitory peaks in plasma melatonin, whereas oral administration, in either saline solution or adsorbed onto pelleted foodstuff, resulted in sustained elevated blood levels for periods exceeding 7 h. Oral dosages of about 2 mg proved adequate to raise the normal daytime plasma levels in both sheep and goats to levels within the normal night-time range. It was concluded that with ruminants the oral route of administration provides a facile and practical way of administering melatonin for physiological study. The review paper of Williams et al. [45] presents data from five research trials and 108 clinical trials conducted in three countries to validate the optimum use of melatonin to advance seasonal breeding patterns of a variety of breeds of sheep. In order to define the optimum time for treatment in breeding flocks, ewes of three different breeds were treated with controlled-release 18-mg melatonin implants (Regulin®), with treatments commencing at various times ranging from 9 to 3 weeks, prior to joining with fertile rams. Overall, the studies presented in this paper showed that melatonin pretreatment of spring and early summer joined ewe flocks resulted in both a modest decrease in the number of barren ewes and an increase in the number of multiple births concluding that this treatment strategy maximizes the potential advantages expected from the melatonin treatment.

as topical application of melatonin along with vitamin E and C in human volunteers has been found to counteract ultraviolet induced erythema and the generation and adverse effects of free radical species [51]. Another research work [52] has dealt with the preclinical safety evaluation of the sunscreen formulation comprising of four US FDA approved UV filters; namely avobenzone, octinoxate, oxybenzone, titanium dioxide along with melatonin and pumpkin seed oil. The results obtained from this study indicated that the sunscreen formulation is nontoxic and safe in animal models and alongside with additional preclinical evaluations may serve as a basis for considering the formulation, as a potential candidate for further trials

Melatonin Modified Release Formulations Designed for Sleep Disorders

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193

The transdermal delivery of melatonin could be a good route for its administration, given the variability of absorption, short biological half-life, and extensive first-pass metabolism of melatonin when administered orally. The main obstacle is the barrier nature of the stratum corneum of the skin that requires the right choice of a suitable vehicle, where the drug can be

A research group [53] prepared and evaluated monolithic drug-in-adhesive type transdermal patches of melatonin using Eudragit E 100 as the adhesive polymer, containing penetration enhancers such as fatty alcohols, fatty acids, and terpenes. The results indicated that the addition of enhancers in the patch increased the permeation of melatonin through hairless rat skin. Decanol and undecanoic acid showed the maximum permeation of melatonin among the fatty alcohols and fatty acids, respectively, while menthol showed the maximum permeation of melatonin among all the enhancers studied. The release profile of melatonin from the patches followed first order kinetics. A lag time of 4–6 h was observed before a steady-state

The effect of transdermal in comparison to oral controlled-release and oral transmucosal drug delivery systems on plasma concentrations of melatonin and its principal metabolite (6-sulfatoxymelatonin (MT6s)) was evaluated in 12 human subjects using a crossover, single dose design [23]. The plasma concentrations of the parent drug and MT6s were measured by radioimmunoassay. Transdermal drug delivery resulted in a significant delay in systemic drug levels and a gradual decline in drug delivery after patch removal (patch dosage forms were removed after 10 h of application), possibly due to the deposition of melatonin

Other researchers suggested that transdermal melatonin may have advantages over fastrelease oral melatonin in improving sleep maintenance at adverse circadian cycles [54]. An experimental skin patch designed to deliver melatonin, such that plasma levels steadily increase for 6–8 h, and thus counteract the increasing circadian wake drive and improve daytime sleep was administered to 8 healthy subjects (2.1 mg melatonin or placebo, randomized, double-blind, crossover study) 1 h before an 8 h daytime sleep opportunity (09:00–17:00 h). The results indicated that transdermal melatonin delivery was effective in elevating plasma

melatonin levels for an extended duration during the daytime.

to establish its efficacy, tolerability, and applicability.

dissolved, then released, and finally penetrate the skin.

**2.7. Transdermal patches**

flux of melatonin was reached.

in the skin.

#### **2.6. Melatonin topical preparations**

As mentioned previously, melatonin has shown antioxidant and immunological properties and therefore, it may be beneficial as a topical drug for the use against oxidative damage in the skin or even as a potential sun protection element, against UV-induced oxidative damage. Melatonin skin penetration properties have been studied in alcoholic solutions, creams, in various vesicular approaches (liposomes and ethosomes), in undecanoic, lauric, and oleic acids.

The skin penetration properties of melatonin from three galenic preparations (0.01% in a cream and 0.01 and 0.03% in a solution) were investigated by the evaluation of the serum melatonin levels over a 24-h time period in a clinical study (15 healthy volunteers) conducted by a research group [46]. The cumulative melatonin serum values for each preparation were 7.1, 8.6, and 15.7 pg/ml, respectively, showing that the alcoholic solution was superior to the cream formulation for melatonin delivery. The strongly lipophilic substance melatonin is able to penetrate through the skin, leading to dose- and galenic-dependent melatonin levels in the blood.

Novel ethanolic liposomes (ethosomes) bearing melatonin were evaluated for transdermal administration potential [47]. Melatonin loaded ethosomes were prepared and characterized for vesicular shape and surface morphology, vesicular size, entrapment efficiency, stability, *in vitro* skin permeation, and *in vivo* skin tolerability. The results suggested that ethosomes may offer a suitable approach for transdermal delivery of melatonin.

The effects of vehicles and enhancers on skin permeation and lag time were evaluated for a more effective transdermal delivery of melatonin [48]. Skin permeation study was conducted in Franz diffusion cells using excised hairless mouse skins and samples were analyzed by HPLC. As vehicles, ethanol (EtOH), polyethylene glycol 400 (PEG), or propylene glycol (PG) were used alone or mixed with a phosphate buffer. The results indicated that the use of binary vehicles could effectively modulate the skin permeability of melatonin (to the limited extent) and the lag time observed. As enhancers, fatty acids were used and when compared with the binary vehicles, the use of oleic acid drastically enhanced the skin permeation of melatonin as well as shortened its lag time.

Apart from being an established radioprotectant and anticancer agent [49], melatonin has also been found to counteract UV-induced solar damage, which includes the generation of reactive oxygen species, skin aging, mitochondrial, and DNA damage [50]. The presence of melatonin along with the UV filters could provide a synergistic effect for optimum sun protection as topical application of melatonin along with vitamin E and C in human volunteers has been found to counteract ultraviolet induced erythema and the generation and adverse effects of free radical species [51]. Another research work [52] has dealt with the preclinical safety evaluation of the sunscreen formulation comprising of four US FDA approved UV filters; namely avobenzone, octinoxate, oxybenzone, titanium dioxide along with melatonin and pumpkin seed oil. The results obtained from this study indicated that the sunscreen formulation is nontoxic and safe in animal models and alongside with additional preclinical evaluations may serve as a basis for considering the formulation, as a potential candidate for further trials to establish its efficacy, tolerability, and applicability.

#### **2.7. Transdermal patches**

sheep. In order to define the optimum time for treatment in breeding flocks, ewes of three different breeds were treated with controlled-release 18-mg melatonin implants (Regulin®), with treatments commencing at various times ranging from 9 to 3 weeks, prior to joining with fertile rams. Overall, the studies presented in this paper showed that melatonin pretreatment of spring and early summer joined ewe flocks resulted in both a modest decrease in the number of barren ewes and an increase in the number of multiple births concluding that this treatment strategy maximizes the potential advantages expected from the melatonin treatment.

As mentioned previously, melatonin has shown antioxidant and immunological properties and therefore, it may be beneficial as a topical drug for the use against oxidative damage in the skin or even as a potential sun protection element, against UV-induced oxidative damage. Melatonin skin penetration properties have been studied in alcoholic solutions, creams, in various vesicular approaches (liposomes and ethosomes), in undecanoic, lauric, and oleic

The skin penetration properties of melatonin from three galenic preparations (0.01% in a cream and 0.01 and 0.03% in a solution) were investigated by the evaluation of the serum melatonin levels over a 24-h time period in a clinical study (15 healthy volunteers) conducted by a research group [46]. The cumulative melatonin serum values for each preparation were 7.1, 8.6, and 15.7 pg/ml, respectively, showing that the alcoholic solution was superior to the cream formulation for melatonin delivery. The strongly lipophilic substance melatonin is able to penetrate through the skin, leading to dose- and galenic-dependent melatonin levels in the

Novel ethanolic liposomes (ethosomes) bearing melatonin were evaluated for transdermal administration potential [47]. Melatonin loaded ethosomes were prepared and characterized for vesicular shape and surface morphology, vesicular size, entrapment efficiency, stability, *in vitro* skin permeation, and *in vivo* skin tolerability. The results suggested that ethosomes may

The effects of vehicles and enhancers on skin permeation and lag time were evaluated for a more effective transdermal delivery of melatonin [48]. Skin permeation study was conducted in Franz diffusion cells using excised hairless mouse skins and samples were analyzed by HPLC. As vehicles, ethanol (EtOH), polyethylene glycol 400 (PEG), or propylene glycol (PG) were used alone or mixed with a phosphate buffer. The results indicated that the use of binary vehicles could effectively modulate the skin permeability of melatonin (to the limited extent) and the lag time observed. As enhancers, fatty acids were used and when compared with the binary vehicles, the use of oleic acid drastically enhanced the skin permeation of melatonin as

Apart from being an established radioprotectant and anticancer agent [49], melatonin has also been found to counteract UV-induced solar damage, which includes the generation of reactive oxygen species, skin aging, mitochondrial, and DNA damage [50]. The presence of melatonin along with the UV filters could provide a synergistic effect for optimum sun protection

offer a suitable approach for transdermal delivery of melatonin.

**2.6. Melatonin topical preparations**

192 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

acids.

blood.

well as shortened its lag time.

The transdermal delivery of melatonin could be a good route for its administration, given the variability of absorption, short biological half-life, and extensive first-pass metabolism of melatonin when administered orally. The main obstacle is the barrier nature of the stratum corneum of the skin that requires the right choice of a suitable vehicle, where the drug can be dissolved, then released, and finally penetrate the skin.

A research group [53] prepared and evaluated monolithic drug-in-adhesive type transdermal patches of melatonin using Eudragit E 100 as the adhesive polymer, containing penetration enhancers such as fatty alcohols, fatty acids, and terpenes. The results indicated that the addition of enhancers in the patch increased the permeation of melatonin through hairless rat skin. Decanol and undecanoic acid showed the maximum permeation of melatonin among the fatty alcohols and fatty acids, respectively, while menthol showed the maximum permeation of melatonin among all the enhancers studied. The release profile of melatonin from the patches followed first order kinetics. A lag time of 4–6 h was observed before a steady-state flux of melatonin was reached.

The effect of transdermal in comparison to oral controlled-release and oral transmucosal drug delivery systems on plasma concentrations of melatonin and its principal metabolite (6-sulfatoxymelatonin (MT6s)) was evaluated in 12 human subjects using a crossover, single dose design [23]. The plasma concentrations of the parent drug and MT6s were measured by radioimmunoassay. Transdermal drug delivery resulted in a significant delay in systemic drug levels and a gradual decline in drug delivery after patch removal (patch dosage forms were removed after 10 h of application), possibly due to the deposition of melatonin in the skin.

Other researchers suggested that transdermal melatonin may have advantages over fastrelease oral melatonin in improving sleep maintenance at adverse circadian cycles [54]. An experimental skin patch designed to deliver melatonin, such that plasma levels steadily increase for 6–8 h, and thus counteract the increasing circadian wake drive and improve daytime sleep was administered to 8 healthy subjects (2.1 mg melatonin or placebo, randomized, double-blind, crossover study) 1 h before an 8 h daytime sleep opportunity (09:00–17:00 h). The results indicated that transdermal melatonin delivery was effective in elevating plasma melatonin levels for an extended duration during the daytime.

Another group of researchers examined the pharmacokinetics of melatonin incorporated in solid lipid nanoparticles, administered by oral or transdermal route [18]. Solid lipid nanoparticles were used as a reservoir system, permitting a constant and prolonged release of melatonin. In comparison to the standard formulation of orally administered melatonin, the absorption and elimination after administration of the solid lipid nanoparticle-melatonin complexes through the transdermal route demonstrated to be slow and melatonin plasma levels above 50 pg/ml were maintained for at least 24 h. Therefore, these systems disclose a potential for the sustained delivery of melatonin.

[3] Muñoz H, Castan H, Clares B, Ruiz MA. Obtaining fast dissolving disintegrating tablets with different doses of melatonin. International Journal of Pharmaceutics. 2014;**5**(1-2):

Melatonin Modified Release Formulations Designed for Sleep Disorders

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195

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[9] Vlachou M, Eikosipentaki A, Xenogiorgis V. Pineal hormone melatonin: Solubilization studies in model aqueous gastrointestinal environments. Current Drug Delivery.

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### **3. Conclusions**

In this chapter, a concise account of the different melatonin delivery routes is given. The choice of the most effective melatonin delivery system is circumstantial and depends on the dysfunction that needs to be treated. However, it seems that modified release formulations mimic closer the endogenous melatonin release pattern and thus a plethora of such systems are currently under thorough investigation.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Marilena Vlachou\* and Angeliki Siamidi

\*Address all correspondence to: vlachou@pharm.uoa.gr

School of Health Sciences, Department of Pharmacy, Division of Pharmaceutical Technology, National and Kapodistrian University of Athens, Athens, Greece

### **References**


[3] Muñoz H, Castan H, Clares B, Ruiz MA. Obtaining fast dissolving disintegrating tablets with different doses of melatonin. International Journal of Pharmaceutics. 2014;**5**(1-2): 84-89. DOI: 10.1016/j.ijpharm.2014.03.054

Another group of researchers examined the pharmacokinetics of melatonin incorporated in solid lipid nanoparticles, administered by oral or transdermal route [18]. Solid lipid nanoparticles were used as a reservoir system, permitting a constant and prolonged release of melatonin. In comparison to the standard formulation of orally administered melatonin, the absorption and elimination after administration of the solid lipid nanoparticle-melatonin complexes through the transdermal route demonstrated to be slow and melatonin plasma levels above 50 pg/ml were maintained for at least 24 h. Therefore, these systems disclose a

In this chapter, a concise account of the different melatonin delivery routes is given. The choice of the most effective melatonin delivery system is circumstantial and depends on the dysfunction that needs to be treated. However, it seems that modified release formulations mimic closer the endogenous melatonin release pattern and thus a plethora of such systems

potential for the sustained delivery of melatonin.

194 Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

are currently under thorough investigation.

The authors declare no conflict of interest.

Marilena Vlachou\* and Angeliki Siamidi

\*Address all correspondence to: vlachou@pharm.uoa.gr

School of Health Sciences, Department of Pharmacy, Division of Pharmaceutical Technology, National and Kapodistrian University of Athens, Athens, Greece

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**3. Conclusions**

**Conflict of interest**

**Author details**

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## *Edited by Cristina Manuela Drăgoi and Alina Crenguţa Nicolae*

Melatonin, the pineal neurohormone, is a pleiotropic molecule acting in the center of the integrative molecular mechanisms of the organism, based on interconnections of the regulatory systems: neural, endocrine, immune, and genetic, conveying into the uniqueness of human architecture.

This book provides a systematic and updated overview of melatonin biochemical mechanisms of action, pharmacological features, and clinical uses, clutching the subject with complete details of pharmaceutical formulations designed for different routes of administration and different health issues, aiming at optimal melatonin bioavailability when therapeutically delivered.

The book addresses a broad range of audiences, from healthcare professionals, medically and pharmaceutically based, to highly profiled medical specialists and biomedical researchers, helping them to expand their knowledge of the physiological and pathological implications of melatonin and its metabolites.

Published in London, UK © 2018 IntechOpen © enzart / iStock

Melatonin - Molecular Biology, Clinical and Pharmaceutical Approaches

Melatonin

Molecular Biology, Clinical

and Pharmaceutical Approaches

*Edited by Cristina Manuela Drăgoi* 

*and Alina Crenguţa Nicolae*