Human circadian rhythms: physiological and ...

Review Article Human circadian rhythms: physiological and therapeutic relevance of light and melatonin Debra J Skene and Josephine Arendt

Abstract Addresses Centre for Chronobiology, School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK Correspondence Professor Debra J Skene E-mail: [email protected] This article was prepared at the invitation of the Clinical Sciences Reviews Committee of the Association for Clinical Biochemistry.

Ocular light plays a key role in human physiology by transmitting time of day information. The production of the pineal gland hormone melatonin is under the control of the light–dark cycle. Its profile of secretion defines biological night and it has been called the ‘darkness hormone’. Light mediates a number of nonvisual responses, such as phase shifting the internal circadian clock, increasing alertness, heart rate and pupil constriction. Both exogenous melatonin and light, if appropriately timed, can phase shift the human circadian system. These ‘chronobiotic’ effects of light and melatonin have been used successfully to alleviate and correct circadian rhythm disorders, such as those experienced following travel across time zones, in night shift work and in circadian sleep disorders. The effectiveness of melatonin and light are currently being optimized in terms of time of administration, light intensity, duration and wavelength, and melatonin dose and formulation. The aim of this review is not to replicate information that has been reported in a number of reviews of the human circadian timing system and the role of melatonin and light, but rather to extract findings relevant to the field of clinical biochemistry. Ann Clin Biochem 2006; 43: 344–353

Introduction Most physiological (temperature, hormones, electrolytes) and behavioural (mood, alertness, sleep, performance) measures exhibit daily rhythms. Circadian rhythms are de¢ned as those rhythms that are endogenously generated, have a periodicity of approximately 24 h and whose rhythmicity persists when environmental conditions (light, temperature, posture, etc.) are kept constant. An endogenous circadian timing system allows an organism to anticipate and predict daily changes in the environment. Circadian rhythms are generated by a pacemaker localized in the hypothalamic suprachiasmatic nuclei (SCN); lesions of these nuclei produce arrhythmicity. In normal conditions, circadian rhythms are synchronized (entrained) to the 24 h day primarily by the light--dark cycle. Entrainment to the environmental light--dark cycle allows the timing of daily activities (e.g. sleeping and feeding) to be optimized. Circadian rhythm disorders can be caused by a mismatch between external and internal time (e.g. following £ight across time zones, 344

moving from a day shift to a night shift); can be a result of circadian ‘dysentrainment’ (e.g. circadian rhythm sleep disorders, advanced and delayed sleep phase insomnia [ASPS and DSPS]); or can relate to dysfunctional photic input to the SCN clock (e.g. in some types of blindness).

The human circadian system Human circadian rhythms Examples of human circadian rhythms are core body temperature, and secretion of cortisol, melatonin and thyroid-stimulating hormone (TSH). In normal conditions, these rhythms have a characteristic time of peak and nadir and there is a set time interval (phase relationship) between the di¡erent rhythms (Figure 1). In the absence of a light--dark cycle (such as experienced by totally blind individuals or in sighted individuals kept in very dim light conditions), circadian rhythms become desynchronized from the 24 h day and ‘freerun’at their intrinsic period length (t). Although there is some debate as to the average period length (t) in r 2006 The Association for Clinical Biochemistry

Human circadian rhythms

humans, most studies agree that t is highly individual. Studies in sighted people in dim light conditions and forced desychrony experiments have reported average t’s of approximately 24.2 h,1--3 whereas studies in totally blind people with free-running circadian rhythms have reported slightly longer t’s of 24.5 h.4--7 The reasons for this discrepancy in t are not yet known but may relate to methodological di¡erences or the

Plasma melatonin concentration (pg/mL)

(a)

60

40

20

0

Core body temperature (οC)

(b)

37.1

36.9

36.7

36.5

Subjective alertness (0 = not alert, 100 = very alert)

(c)

60

40

20

0

Task performance reaction time (ss)

(d)

1.8

1.6

1.4

345

di¡erences in light perception and previous light history between the study groups.

Measurement of circadian rhythms In order to establish whether a daily rhythm in a physiological parameter is endogenously generated (circadian) or a result of changes in the environment (exogenous), or both, ‘constant routine’ protocols are performed (this is currently considered the gold standard).8 In constant routine studies, all possible known confounding factors are kept ‘constant’ so that participants are required to remain awake; maintain a constant posture (e.g. semi-recumbent); in constant lighting conditions, usually dim light o10 lux (10 lux is approximately the light given by a candle 1ft away from an object; it is possible to read and see quite well in dim light once the eyes have adapted); and receive isocaloric meals hourly for at least 25 h. The contribution of the circadian system (endogenous) and the environment (exogenous) to an observed rhythm can also be quanti¢ed in ‘forced desynchrony’ experiments.3 In these experiments, participants live on a 20 or 28 h day for at least 12 days. Forced desynchrony protocols are designed to be able to separate the in£uence of the sleep--wake cycle from the in£uence of the circadian pacemaker. Although these experiments are extremely demanding on both participants and researchers, they reliably estimate the circadian component of an observed rhythm. There are also confounding factors even to forced desynchrony experiments (e.g. scheduling of sleep length), and because of ¢nancial and resource constraints (e.g. temporal isolation facilities are required), these experiments are not always possible. Fortunately, measurement of the endogenous melatonin rhythm provides a relatively reliable surrogate way of assessing the timing of the internal circadian clock in less-controlled conditions provided that certain precautions are taken (see later). Measurement of

1.2

Triacylglycerol concentration (mmol/L)

(e)

2.0

1.8

1.6

1.4

1.2 1000

1400

1800

2200 0200 Clock time (h)

0600

1000

1400

Figure 1 Diagrammatic representation of the circadian rhythms of plasma melatonin, core body temperature, subjective alertness, task performance (reaction time, in seconds) and triacylglycerol from human beings held in constant routine conditions (i.e. awake, controlled light, posture, activity and meals). The peak in the melatonin rhythm (panel a), shown by the dotted line, and the low point of the temperature rhythm (panel b) are within 1 h of each other. The low point of the alertness and performance rhythms (panels c and d, respectively) is shortly after the melatonin peak, and the peak in triacylglycerol (panel e) is close to the melatonin peak. Reproduced with permission from: Rajaratnam SM, Arendt J. Health in a 24-h society. Lancet 2001; 358: 999–100572 Ann Clin Biochem 2006; 43: 344–353

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melatonin in plasma or saliva -- or measurement of its major metabolite 6-sulphatoxymelatonin, usually in urine -- can enable long-term monitoring of the human circadian system in laboratory and real-life studies (see later section).

Molecular clockworks In recent years, major progress has been made in understanding the molecular mechanism underlying circadian oscillations within individual SCN cells. Clock genes and clock-controlled genes are rhythmically expressed primarily via an intracellular transcriptional/ translational negative feedback loop.9,10 In brief, during the day, transcription of a number of genes (Per1, Per2, Per3, Cry1, Cry2 and Reverba) is activated by heteromeric complexes of CLOCK and BMAL1. This transcription continues into the night until nuclear levels of PER and CRY proteins become su⁄ciently high to repress CLOCK/BMAL1 activation. Declining levels of PER/ CRY in the early morning then allows transcription of the genes again and the cycle continues. This constitutes the negative feedback limb of the oscillator core. There is an auxiliary loop driven by Reverba (and possibly Reverbb). The protein products, REVERBa and b, suppress Bmal1 transcription, disappearance of REVERB proteins allowing Bmal1 to peak at the end of the night (positive limb). Rhythms in clock gene expression have also been characterized in numerous peripheral tissues (e.g. liver, heart, lung), providing substantial evidence for the presence of peripheral clocks.11,12 The question of how these peripheral clocks interact with the central SCN clock (feedforward/feedback) and how they are synchronized (directly via hormonal and/or neural pathways and/or indirectly via behaviour [e.g. feeding]13) is presently a major research question.

Circadian photoreception Considerable advances have also been made regarding the neuroanatomical pathways involved in the transmission of light information from the retina to the SCN (circadian photoreception). Both animal14,15 and human16,17 studies have shown that, in addition to the classical rods and cones, a novel photoreceptor system is involved in mediating so-called ‘non-image forming’ visual functions. In addition to image formation, light entering the eyes is responsible for a number of nonimage-forming responses, such as circadian resetting and entrainment, suppression of nocturnal melatonin production, elevation of core body temperature and heart rate, increased alertness and performance, and pupil constriction. The discovery of melanopsin,18 a novel photopigment, and its localization in a network of intrinsically photosensitive retinal ganglion cells,19 has provided a basis for understanding how the light Ann Clin Biochem 2006; 43: 344–353

signal is captured and transmitted to the SCN clock, in addition to other brain structures. This melanopsin network, with some contribution from the rods and cones, is thought to mediate the non-visual e¡ects of light.20 Ocular light is the major time cue (zeitgeber) responsible for synchronizing the human circadian timing system. Although early work suggested that ‘non-photic’ time cues such as meals, exercise and the sleep--wake cycle were important synchronizers of the human circadian clock,21 today light is considered the primary time cue.22 Evidence to support this view has come from detailed studies of totally blind people, who exhibit free-running circadian rhythms in spite of living with strong social cues.5--7,23 The contribution of non-photic time cues to circadian entrainment, however, cannot be ruled out entirely. The demonstration of relative co-ordination in blind subjects with free-running circadian rhythms23,24 and non-photic entrainment in a few blind subjects4 suggests that non-photic zeitgebers may have a weak e¡ect on the circadian system. At present, however, the non-photic stimulus or stimuli responsible for these e¡ects have not been identi¢ed. The ability of appropriately timed exercise25 or exogenously administered melatonin26 to shift circadian rhythms represents the best evidence to date that indeed non-photic stimuli can a¡ect clock timing. Future studies investigating how photic and non-photic stimuli interact to determine circadian phase are required.

Melatonin Synthesis and metabolism The pineal hormone melatonin is intimately linked with the circadian timing system. The rhythm in melatonin synthesis is controlled by the SCN via a multisynaptic pathway (SCN -- paraventricular nuclei [PVN] -- superior cervical ganglia [SCG]) that terminates in the release of noradrenaline from postganglionic SCG ¢bres in the pineal gland.27 The SCN regulates the melatonin rhythm via GABAergic inhibition and glutaminergic stimulation of the PVN.28,29 The ratelimiting enzyme in the synthesis of melatonin is arylalkylamine N-acetyltransferase (AANAT). It is likely that the human AANAT enzyme is regulated primarily at a post-transcriptional level, whereas in rodents the key event appears to be cyclic adenosine monophosphate (cAMP)-dependent phosphorylation of a transcription factor that binds to the AANAT promoter. Rapid decline in activity with light treatment at night appears to depend on proteasomal proteolysis of AANAT following dephosphorylation and removal of a protective 14-3-3 protein.30,31 (Evidence from both structural studies and sequence analyses support the notion that the primary function of 14-3-3 proteins lies


in their preferential binding to phosphorylated substrates. In humans, there are seven 14-3-3 isoforms (b, g, e, z, Z, i, s).)30,31 Melatonin production peaks during the dark phase in all species studied to date. Melatonin is metabolized primarily in the liver via a cytochrome P450-mediated hydroxylation into 6-hydroxymelatonin. It is then conjugated, primarily with sulphate to form 6-sulphatoxymelatonin and, to a lesser extent, with glucuronic acid.32,33

Role of endogenous melatonin The role of endogenous melatonin in human physiology is not clearly de¢ned. Although the e¡ects of exogenous melatonin have been extensively studied (mainly at pharmacological doses),26,33,34 more extensive studies with pinealectomized patients and development of a speci¢c melatonin receptor antagonist for human studies may help to clarify its role further. Endogenous melatonin is a hormonal signal that gives time of day (and time of year) information to the body. Secreted at night in all species studied to date, it appears to reinforce darkness-related behaviours such as sleep initiation. An association between the rise in melatonin production at night and the increase in sleep propensity has been noted.35 In addition, abnormal timing of melatonin in totally blind individuals has been associated with increased napping.36 However, whether these e¡ects of endogenous melatonin are causal or merely correlational is not known.

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studies.33,46--48 The e¡ect of melatonin administration on gonadal hormones is more controversial. Most recent studies with near-physiological doses of melatonin show no short-term e¡ects on gonadotrophins and gonadal hormones.49,50 However, altered melatonin secretion has been reported in a number of reproductive pathologies (reviewed byArendt51,52).

Endogenous melatonin rhythm The circadian rhythm of melatonin has characteristics that can be used to measure the phase of the circadian system. In normal 24 h entrained conditions, melatonin peaks at 02.00--03.00 h, rising approximately 4 h earlier (22.00--23.00 h) and declining back to baseline levels in the morning (09.00--10.00 h), while sleep occurs 1--2 h after melatonin onset, ends 1--2 h before melatonin o¡set and usually lasts 7 h. The timing of the rhythm can be measured by estimating the melatonin onset, peak or o¡set (Figure 2). Although the time of melatonin onset in dim light conditions has been used frequently as a marker of circadian phase, it may be better to measure both melatonin onset and o¡set as there is evidence to suggest that these may be shifted di¡erentially.53 Ideally, however, the whole melatonin pro¢le should be measured. The amplitude of the rhythm and the amount of melatonin produced (e.g. area under the curve) is highly variable between individuals (thought to be related to the size of the pineal gland54). However, within an individual, melatonin production is fairly consistent.

Melatonin receptors

Importance of measuring melatonin

Melatonin receptors are high a⁄nity (picomolar KD) G-protein (mainly Gi) receptors. Three melatonin receptors have been cloned, namely mel1a (MT1), mel1b (MT2) and mel1c, by Reppert and colleagues.37--39 Although melatonin receptors have been found in the human SCN and pituitary stalk,40,41 relatively little work has been done in the area, most likely because of the lack of availability of human tissue. Further identi¢cation and localization of melatonin receptors may provide some clues as to melatonin’s physiological function in humans. In animals, melatonin receptors in the SCN have been linked to suppression (MT1) and phase shifting (not MT1, possibly MT2) SCN activity.42,43 In sheep, MT1 receptors in the pars tuberalis mediate the photoperiodic regulation of prolactin production.44 Melatonin receptors in the mediobasal hypothalamus have been proposed to mediate melatonin’s e¡ects on the gonadal axis of seasonal breeders.45 Whether endogenous melatonin has a similar role in prolactin production and gonadal development in humans has long been speculated but de¢nitive proof remains lacking. Exogenous melatonin has been reported to a¡ect prolactin production in some clinical

The timing of the melatonin rhythm is today considered the most reliable marker of the timing of the circadian clock. Control of the lighting environment and, to a lesser extent, posture, is required for accurate melatonin measurement but, apart from this, of all possible ‘rhythm markers’, melatonin is least a¡ected by activity, sleep, timing of meals, stress, menstrual cycle, etc. In contrast, other circadian rhythms such as core body temperature and secretion of cortisol have been shown to be signi¢cantly a¡ected by these factors. Measurement of melatonin and 6-sulphatoxymelatonin has been successfully used to monitor circadian desynchrony in blind people in their home environments5,55 as well as in DSPS,26 in people working night shifts56,57 and in people after crossing time zones.26 The timing of the melatonin rhythm in these studies has provided important information as to the individual’s circadian phase and has been used to optimize the timing of light and melatonin in the treatment of circadian rhythm disorders (discussed later on). In addition to being a marker of circadian phase, measurement of melatonin is also performed to provide an assessment of pineal gland function. Conditions Ann Clin Biochem 2006; 43: 344–353

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Duration

Plasma melatonin (pg/mL)

70 60 *

50

Acrophase (calculated peak time)

40 30

Mid-range crossing *

20

25% rise/fall

*

10 0

* *

Onset/offset *

*

1500 1700 1900 2100 2300 100 300 500 700 900 1100 1300 1500 1700

Clock time (h)

‘Biological night’

Figure 2 Normal profile of melatonin in plasma defining ‘biological night’ based on our laboratory normal values (n ¼ 133, healthy men and women, all ages). Parameters of this profile and that of salivary melatonin and urinary 6-sulphatoxymelatonin that are used to characterize the timing of the circadian clock are indicated, namely acrophase, duration, mid-range crossing, 25% rise and fall, onset and offset of secretion. 6-Sulphatoxymelatonin acrophase is approximately 2 h after the plasma melatonin acrophase. Redrawn from: Arendt J, Skene DJ. Melatonin as a chronobiotic. Sleep Med Rev 2005; 9: 25–39.26 in which pineal gland function might be compromised include uveitis, various pineal tumours, disruption of neural pathways to the pineal and/or the SCN (e.g. cervical cord lesion, diabetic autonomic neuropathy).33,52 Increased melatonin concentrations have been reported in conditions in which metabolism of melatonin is compromised (e.g. liver cirrhosis) and in some reproductive pathologies (e.g. amenorrhoea, hypogonadotrophic hypogonadism).33,52 Numerous studies have reported reduced melatonin production in other conditions, e.g. breast cancer, sleep disorders (inconsistent), depression, heart disease (reviewed byArendt51,52) and Alzheimer’s disease.58 Because of the well-reported inter-individual variation in melatonin concentrations as well as an age-related decline,33,58 a cautious approach to interpreting cross sectional studies should be adopted. Longitudinal, within-subject studies are preferable to determine whether a condition a¡ects pineal function or not.

Analytical measurement The gold standard for measuring melatonin is gas chromatography--mass spectrometry (GCMS).59 For routine measurement, radioimmunoassays (RIAs) have been developed and validated using GCMS and these serve as the most popular and widely used way of measuring melatonin and its metabolite, 6-sulphatoxymelatonin. The current assays and protocols have been recently reviewed.60 Assay reagents, antibodies and assay kits Ann Clin Biochem 2006; 43: 344–353

are available from a number of manufacturers, including Stockgrand Ltd (www.stockgrand.co.uk), ALPCO (www.alpco.com) and Buhlmann Laboratories (www.buhlmannlabs.ch). Development of enzymelinked immunosorbent assays (ELISAs) in this area has been slow but ELISAs for saliva melatonin and 6-sulphatoxymelatonin are currently available.

Factors affecting melatonin (reviewed by Arendt34) Factors known to a¡ect the timing and production of melatonin are several. The light--dark cycle has the most signi¢cant a¡ect on melatonin timing and production. Light is able to advance or delay the timing of the melatonin rhythm, depending on when it is administered. Light at night also has an acute suppressive e¡ect on melatonin synthesis.61,62 Light transmitted via the SCN--PVN--SCG pathway rapidly inhibits the activity of AANAT, melatonin’s rate-limiting biosynthetic enzyme, via proteasomal proteolysis (see previous section). Thus, it is essential that the light/dark environment is controlled or, if control is impossible, at least monitored, when measuring a person’s melatonin concentrations. Ideally samples should be collected from subjects in constant dim light conditions (o10 lux at eye level). Posture has also been shown to a¡ect the measurement of plasma melatonin,63 thus ideally this should also be controlled during the sampling period (routinely subjects are asked to remain seated or semirecumbent). Drugs reported to interfere with melatonin


measurements usually a¡ect the synthesis and/or metabolism of melatonin. Melatonin is synthesized from serotonin via a b1-adrenergic mechanism, and to a lesser extent an a-adrenergic mechanism. Drugs known to interfere with these processes (e.g. serotonin and noradrenaline reuptake inhibitors; a- and b1- agonists and antagonists) have been shown to a¡ect melatonin concentrations.32--34 Melatonin has been shown to be metabolized by CYP1A2 in both animals and humans.64,65 Thus, drugs known to a¡ect the activity of CYP1A2 (ca¡eine, nicotine) and drugs metabolized by CYP1A2 (e.g. £uvoxamine, 5-methoxypsoralen) are likely to interfere with the metabolism of melatonin. Early ¢ndings reporting high concentrations of melatonin following £uvoxamine66 and 5-methoxypsoralen67 administration can now be explained on the basis of a CYP1A2-mediated mechanism. There is also some preliminary evidence that the in£uence of oral contraceptives on melatonin concentrations68,69 may occur at the level of melatonin metabolism (unpublished data). Further studies investigating this mechanism are required.

Ideal sampling conditions The correct procedure to collect blood, saliva and urine for measurement of melatonin and 6-sulphatoxymelatonin is detailed in Table 1. Further details can also be found on the Stockgrand Ltd website (www.stockgrand.co.uk) and in a recent review.34

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Circadian rhythm disorders Circadian rhythm disorders are associated with sleep problems. As such these conditions have been classi¢ed in the International Classi¢cation of Sleep Disorders (ICSD)70 as Circadian Rhythm Sleep Disorders. Circadian rhythm sleep disorders may be the result of an abrupt shift in time (e.g. as experienced following transmeridian travel), or by an abrupt shift in sleep and work time (as in rotating shift workers). Sleep disturbance is caused by a mismatch between the internal circadian timing system and the environment. Following a period of re-adaptation, which depends on the number of time zones crossed, synchrony is achieved after time zone travel, but rarely in ‘normal’ night shift work. Thus, most shift workers are living in a state of intermittent, but chronic desynchrony.71,72 In unusual circumstances (social isolation on o¡shore oil rigs working certain schedules and during the polar winter), most people can completely adapt their internal clocks to night work.56,73 Circadian rhythm sleep disorders may also be a result of circadian misalignment such as advanced (ASPS) and delayed sleep phase insomnia (DSPS), in which su¡erers have abnormally early or late bedtimes, respectively. Apart from environmental causes such as lighting at inappropriate times, causes of ASPS and DSPS may be intrinsic and relate to abnormal light sensitivity or aberrant clock timing or both. Recent evidence suggests that polymorphisms in a number of human clock genes (which could

Table 1 Sampling details for measurement of melatonin and 6-sulphatoxymelatonin (aMT6s) Biological fluid

Analyte

Sampling procedure

Precautions

Blood

Melatonin

Collect blood into heparinized tubes. Centrifuge within 15 min. Store plasma at 201C.

Haemolysed plasma and plasma left in plastic pipettes for more than 2–3 min may give falsely elevated melatonin levels.

Saliva

Melatonin

The best method is to ask subjects to spit into polypropylene tubes. Do not stimulate saliva production. Store at 201C.

Do not eat within 30 min of sampling. Rinse mouth with tap water before spitting. Saliva left in plastic pipettes for more than 2–3 min may give falsely elevated melatonin levels. Use of salivettes may give falsely elevated melatonin levels, standards should be run through salivettes to correct for this.

Salivettes with an untreated cotton plug can be used. Centrifuge for 15 min at 3000 r.p.m. Store at 201C. Urine

aMT6s

Ask subjects to collect all urine passed over a pre-set period into a standard urine bottle. Measure and record the volume, store circa 5 mL at 201C. Urine should be collected at least every 3–4 h (longer during sleep period) for at least 24 h, preferably for 48 h or longer.

Do not wash urine bottles with bleach or another oxidant. No preservative is required as aMT6s is stable in urine for 1 day at room temperature, 2 days at 41C and for at least 2 years at 201C.

For further details, see www.stockgrand.co.uk Ann Clin Biochem 2006; 43: 344–353

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feasibly a¡ect t) may be associated with ASPS (per 2)74,75 and DSPS (per 3).76 Another less-common circadian rhythm disorder is desynchronization from the 24 h day as experienced most often in totally blind people.5,6 There are also some reports of this non-24 h sleep/wake disorder being observed in some sighted people77 and in some dim-light, long-winter environments,78 although it is rarely seen on British Antarctic Survey bases.79 Non-24 h sleep/wake disorder is cyclic, characterized by periods of good sleep (long duration sleep with no daytime napping) and periods of poor sleep (short duration sleep with daytime naps36). The periods of poor sleep have been shown to be associated with abnormal timing of the circadian clock.23,36

Consequences The physiological and behavioural consequences of circadian misalignment are known to anyone who has travelled across time zones or who has done night shift work: poor sleep of short duration with fatigue and reduced alertness, vigilance and performance during waking hours. These symptoms may predispose the individual to accidents, error and risk. The long-term consequences of repeated/continuous circadian desynchrony such as may be experienced by shift workers are just beginning to be studied. There is epidemiological evidence to show increased cardiovascular risk80,81 and cancer risk82 in night shift workers. Eating meals at night has been shown to produce increased blood triglycerides and evidence of insulin resistance,83,84 both risk factors for heart disease. Light exposure at night has been hypothesized to increase cancer risk.85 Although it is always di⁄cult to relate cause and e¡ect in chronic studies, these issues clearly require more study.

Treatment strategies The acute and possible long-term consequences of circadian rhythm disorders are driving the development of treatment strategies to alleviate or at least minimize some of the detrimental e¡ects of inappropriate circadian timing (such as tiredness, reduced performance). Currently, light and exogenous melatonins are the best candidate treatments as both have been shown to phase shift the timing of the human circadian clock. Although other drug strategies have been used (e.g. short acting benzodiazepines, moda¢nil, ca¡eine) to alleviate some of the consequences of circadian misalignment of jetlag and shift work, whether these drugs act directly on the circadian clock is not yet clear. However, by modifying sleep and wakefulness, these drugs indirectly a¡ect an individual’s light/dark exposure, which, in turn, would shift the timing of the clock. It is likely that the indirect e¡ect of these drugs on circadian timing will form part of future treatment strategies. Ann Clin Biochem 2006; 43: 344–353

However, side-e¡ects such as hangover and headache will need careful monitoring.

Light Light, appropriately timed, can advance or delay the circadian timing system. Optimization of light’s phaseshifting e¡ects is currently an intensive area of research. It is clear that the extent to which light can shift the clock is dependent upon the time of light administration86,87 as well as the intensity of light,88 its duration and wavelength.53,89,90 Broadly speaking, increasing the intensity and duration of the light stimulus increases its e¡ect. Recent data have shown that short wavelength light (420--480 nm) is most e¡ective at phase shifting the human circadian clock.53,89,90 In addition an individual’s light history (i.e. previous light exposure) may be an important modulator of light’s phase-shifting e¡ect. Direct proof of this is still outstanding but it is predicted on the basis of reports showing light history to a¡ect the ability of light to suppress melatonin.91--93

Exogenous melatonin Appropriately timed, exogenous melatonin has been shown to advance or, more controversially, delay the timing of the circadian clock.94,95 This phase-shifting e¡ect of melatonin has been employed successfully to entrain totally blind people with free-running circadian rhythms.96,97 Entrainment by melatonin in our studies has occurred by melatonin’s ability to phase advance the circadian clock.96,98 In addition to correcting the underlying desynchronized circadian disorder, melatonin has also been shown to improve sleep and reduce daytime napping in these subjects. However, melatonin also improved sleep in those subjects that did not entrain (but probably to a lesser extent). Melatonin’s phase-shifting e¡ect is presumably via receptors in the SCN, although this is yet to be de¢nitely proven. Current research is directed at determining the minimum e¡ective dose for entrainment. Melatonin at a daily dose of 0.5 mg has been shown to e¡ectively entrain free-running rhythms in blind people.98,99 At a single dose of 0.05 mg, phase shifts can also be seen100 and daily administration of this lower dose has recently been tried in a single blind subject.101 Melatonin has also been successful, when appropriately timed, in the alleviation of DSPS, jet lag and in helping shift workers to sleep during the day (reviewed by Arendt and Skene26 and Arendt34).

Future perspectives Although manipulation and optimization of the lighting environment remains key to minimizing the


detrimental e¡ect of circadian misalignment, the role of non-photic stimuli in combination with lighting requires further study. Timed exercise and meals may reinforce the e¡ect of light and help maintain entrainment. Melatonin is the treatment of choice for non-24 h sleep/wake disorder experienced by the blind, where light treatment is impossible. Further studies determining the minimum e¡ective dose and the ideal dosing regimen (daily/every second day) and formulation (fast/slow release) are required. How melatonin’s e¡ects are modulated by an individual’s t and pharmacokinetics is also not yet clear. Future lighting for industry, institutions and the home needs to incorporate the recent research developments into practical solutions. The e⁄cacy of lighting optimized on the basis of the current research ¢ndings (e.g. optimal spectral composition) now needs to be tested in the real-life situation. In addition, the long-term risk of these developments (e.g. blue light, daily melatonin) needs careful monitoring.

Acknowledgements The review was written during the tenure of an EU Marie Curie RTN grant (MCRTN-CT-2004-512362) to DJS, Health and Safety Executive/Energy Institute and AFI (Antarctic Funding Initiative CGS-6-15) grants to JA.

Competing interests: DJS and JA are directors of Stockgrand Ltd.

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