Search

Melatonin is produced in the pineal gland under very tight control through the influences of light and the suprachiasmatic nucleus. As such, melatonin circulates in the blood at levels <3 pg/ml during the day and is only actively secreted at night reaching levels of approximately 100 pg/ml. As a consequence of binding to plasma proteins, free melatonin appears in saliva at approximately one third the plasma concentration. Measurement of melatonin is technically challenging because of these very low concentrations and while a number of commercial immunoassay kits are available and mass spectrometry assay methods have been published, not all are fit for purpose and can lead to unreliable conclusions. In this review I discuss the aspects of pineal melatonin production that saliva melatonin reflects, the factors influencing melatonin production or metabolism, saliva collection and analysis methods. Examples are provided of the appropriate use of saliva melatonin measurements; Dim Light Melatonin Onset (DLMO) assessment, impact of light on melatonin and the monitoring of rhythms prior to specific treatments. Examples of inappropriate use of saliva melatonin measurements are also provided including the use of poorly validated assays, morning saliva collections, attempts to stimulate melatonin, and linking specific illnesses to saliva melatonin levels.

Circadian rhythm disruption is a core symptom of bipolar disorder (BD), also reflected in altered patterns of melatonin release. Reductions of grey matter (GM) volumes are well documented in BD. We hypothesized that levels and timing of melatonin secretion in bipolar depression could be associated with depressive psychopathology and brain GM integrity. The onset of melatonin secretion under dim light conditions (DLMO) and the amount of time between DLMO and midsleep (i.e. phase angle difference; PAD) were used as circadian rhythm markers. To study the time course of melatonin secretion, an exponential curve fitting the melatonin values was calculated, and the slope coefficients (SLP) were obtained for each participant. Significant differences were found between HC and BD in PAD measures and melatonin profiles. Correlations between PAD and depressive psychopathology were identified. Melatonin secretion patterns were found to be associated with GM volumes in the Striatum and Supramarginal Gyrus in BD. Our findings emphasized the role of melatonin secretion role as a biological marker of circadian synchronization in bipolar depression and provided a novel insight for a link between melatonin release and brain structure.

Dim light melatonin onset (DLMO) is considered the most reliable circadian phase marker in humans. However, the methods to calculate it are diverse, which limits the comparability between studies. Given the key role of DLMO to diagnose circadian rhythm sleep-wake disorders and determine the optimal timing of chronotherapies, the establishment of clear and validated guidelines on the methodology to assess DLMO is very important. We performed a repeatability study (n = 31) and an agreement study (n = 62) in healthy young adults with hourly blood samples collected under dim light conditions (<8 lux) during a chronobiological protocol. We assessed the repeatability of DLMO with three different methods (fixed threshold, dynamic threshold and hockey stick) across two nights and assessed agreement of each method with the mean visual estimation made by four chronobiologists. Analyses included Bland-Altman diagrams, intraclass correlation coefficients and equivalence tests. The repeatability of the four methods across two nights ranged from good to perfect. The agreement study highlighted that the hockey stick showed equivalent or superior performance (ICC: 0.95, mean difference with visual estimation: 5 min) in healthy subjects compared to the dynamic and fixed thresholds. Thanks to its objective nature, the hockey stick method may provide better estimates than the mean of the visual estimations of several raters. These findings suggest that the hockey stick method provides the most reliable estimate of DLMO within the tested methods and should be considered for use in future studies.

Circadian misalignment remains a distinct challenge for night shift workers. Variability in individual sleep-wake/light-dark patterns might contribute to individual differences in circadian alignment in night shift workers. In this simulation study, we compared the predicted phase shift from a mathematical model of the effect of light on the human circadian pacemaker to the observed melatonin phase shift among individuals who completed one of four interventions during simulated night shift work. Two inputs to the model were used to simulate circadian phase: sleep-wake/light-dark patterns measured from a wrist monitor (Simulation 1) and sleep-wake/light-dark patterns measured from a wrist monitor enhanced by known light levels measured at the level of the eye during simulated night shifts (Simulation 2). The estimated phase shift from the model was within 2 hours of the observed phase shift in ~80% of night shift workers for both simulations; none of the model-predicted phase shifts was more than ~3 hours from the observed phase shift. Overall, the root-mean-square error between observed and predicted phase shifts was better for Simulation 1. The light input from the wrist monitor informed by actual light level measured at the eye performed better in the sub-group exposed to bright light during their night shifts. The findings from this simulation study suggest that using a mathematical model combined with sleep-wake and light exposure data from a wrist monitor can facilitate the design of shift work schedules to enhance circadian alignment, which is expected to improve sleep, alertness, and performance.

Determination of circadian phase is required to diagnose and treat circadian abnormalities, but the measurement of dim light melatonin onset (DLMO), the most common phase marker, is laborious. As sleep timing reflects circadian phase, measurement of sleep markers (e.g., sleep onset, sleep midpoint, sleep offset) provides a simple way to estimate DLMO. The study aim was to compare methods to estimate DLMO from markers derived from the Pittsburgh Sleep Quality Index (PSQI), Munich Chronotype Questionnaire (MCTQ), sleep diaries, and actigraphy. PSQI, MCTQ, and 1 week of diary and actigraphy data were collected from 72 (36 f, 36 m) healthy adults aged 23.1 (± 3.6) y prior to a laboratory sleep study. Saliva samples were collected hourly in dim light during the second evening of the study. The sleep markers most strongly associated with DLMO from each source were PSQI onset, MCTQ average midpoint, 7-d diary midpoint, and 7-d actigraphy midpoint. Estimates of DLMO as a fixed interval before the sleep marker exhibited proportional bias. DLMO estimated from regression models based on sleep midpoint from 7 d of diary or 7 d of actigraphy showed the narrowest limits of agreement with measured DLMO without proportional bias (±1.8 h and ±1.9 h, respectively). Our findings indicate none of the methods provided precise estimates of DLMO from sleep markers. The best estimates were from linear regressions on sleep midpoints from 7 d of diary or actigraphy, and these estimates of DLMO may be suitable for limited research purposes.

Studies on circadian timing in depression have produced variable results, with some investigations suggesting phase advances and others phase delays. This variability may be attributable to differences in participant diagnosis, medication use, and methodology between studies. This study examined circadian timing in a sample of unmedicated women with and without unipolar major depressive disorder. Participants were aged 18-28 years, had no comorbid medical conditions, and were not taking medications. Eight women were experiencing a major depressive episode, nine had previously experienced an episode, and 31 were control participants with no history of mental illness. Following at least one week of actigraphic sleep monitoring, timing of salivary dim light melatonin onset (DLMO) was assessed in light of <1 lux. In currently depressed participants, melatonin onset occurred significantly earlier relative to sleep than in controls, with a large effect size. Earlier melatonin onset relative to sleep was also correlated with poorer mood for all participants. Our results indicate that during a unipolar major depressive episode, endogenous circadian phase is advanced relative to sleep time. This is consistent with the early-morning awakenings often seen in depression. Circadian misalignment may represent a precipitating or perpetuating factor that could be targeted for personalized treatment of major depression.

We examined phase shifts to bright morning light when sleep was restricted by delaying bedtimes. Adolescents (n = 6) had 10-h sleep/dark opportunities for 6 days. For the next 2 days, half were put to bed 4.5 h later and then allowed to sleep for 5.5 h (evening room light + sleep restriction). The others continued the 10-h sleep opportunities (sleep satiation). Then, sleep schedules were gradually shifted earlier and participants received bright light (90 min, ~6000 lux) after waking for 3 days. As expected, sleep satiation participants advanced (~2 h). Evening room light + sleep restriction participants did not shift or delayed by 2-4 h. Abbreviations: DLMO: dim light melatonin onset.

In mammals, daily physiological events are regulated by the circadian rhythm, which comprises two types of internal clocks: the central clock and peripheral clocks. Circadian rhythm plays an important role in maintaining physiological functions including the sleep-wake cycle, body temperature, metabolism and organ functions. Circadian rhythm disorder, which is caused, for example, by an irregular lifestyle or long-haul travel, increases the risk of developing disease; therefore, it is important to properly maintain the rhythm of the circadian clock. Food and the circadian clock system are known to be closely linked. Studies on rodents suggest that ingesting specific food ingredients, such as the flavonoid nobiletin, fish oil, the polyphenol resveratrol and the amino acid L-ornithine affects the circadian clock. However, there are few reports on the foods that affect these circadian clocks in humans. In this study, therefore, we examined whether L-ornithine affects the human central clock in a crossover design placebo-controlled human trial. In total, 28 healthy adults (i.e. ≥20 years) were randomly divided into two groups and completed the study protocol. In the 1st intake period, participants were asked to take either L-ornithine (400 mg) capsules or placebo capsules for 7 days. After 7 days' interval, they then took the alternative test capsules for 7 days in the 2nd intake period. On the final day of each intake period, saliva was sampled at various time points in the dim light condition, and the concentration of melatonin was quantified to evaluate the phase of the central clock. The results revealed that dim light melatonin onset, a recognized marker of central circadian phase, was delayed by 15 min after ingestion of L-ornithine. Not only is this finding an indication that L-ornithine affects the human central clock, but it also demonstrates that the human central clock can be regulated by food ingredients.

Since there is less movement during sleep than during wake, the recording of body movements by actigraphy has been used to indirectly evaluate the sleep-wake cycle. In general, most actigraphic devices are placed on the wrist and their measures are based on acceleration detection. Here, we propose an alternative way of measuring actigraphy at the level of the arm for joint evaluation of activity and body position. This method analyzes the tilt of three axes, scoring activity as the cumulative change of degrees per minute with respect to the previous sampling, and measuring arm tilt for the body position inference. In this study, subjects (N = 13) went about their daily routine for 7 days, kept daily sleep logs, wore three ambulatory monitoring devices and collected sequential saliva samples during evenings for the measurement of dim light melatonin onset (DLMO). These devices measured motor activity (arm activity, AA) and body position (P) using the tilt sensing of the arm, with acceleration (wrist acceleration, WA) and skin temperature at wrist level (WT). Cosinor, Fourier and non-parametric rhythmic analyses were performed for the different variables, and the results were compared by the ANOVA test. Linear correlations were also performed between actimetry methods (AA and WA) and WT. The AA and WA suitability for circadian phase prediction and for evaluating the sleep-wake cycle was assessed by comparison with the DLMO and sleep logs, respectively. All correlations between rhythmic parameters obtained from AA and WA were highly significant. Only parameters related to activity levels, such as mesor, RA (relative amplitude), VL5 and VM10 (value for the 5 and 10 consecutive hours of minimum and maximum activity, respectively) showed significant differences between AA and WA records. However, when a correlation analysis was performed on the phase markers acrophase, mid-time for the 10 consecutive hours of highest (M10) and mid-time for the five consecutive hours of lowest activity (L5) with DLMO, all of them showed a significant correlation for AA (R = 0.607, p = 0.028; R = 0.582, p = 0.037; R = 0.620, p = 0.031, respectively), while for WA, only acrophase did (R = 0.621, p = 0.031). Regarding sleep detection, WA showed higher specificity than AA (0.95 ± 0.01 versus 0.86 ± 0.02), while the agreement rate and sensitivity were higher for AA (0.76 ± 0.02 versus 0.66 ± 0.02 and 0.71 ± 0.03 versus 0.53 ± 0.03, respectively). Cohen's kappa coefficient also presented the highest values for AA (0.49 ± 0.04) and AP (0.64 ± 0.04), followed by WT (0.45 ± 0.06) and WA (0.37 ± 0.04). The findings demonstrate that this alternative actigraphy method (AA), based on tilt sensing of the arm, can be used to reliably evaluate the activity and sleep-wake rhythm, since it presents a higher agreement rate and sensitivity for detecting sleep, at the same time allows the detection of body position and improves circadian phase assessment compared to the classical actigraphic method based on wrist acceleration.