ABSTRACT
Dim light melatonin onset (DLMO) is considered the most reliable marker of the circadian rhythm phase in humans. DLMO may moderately correlate with sleep onset and sleep offset time. There are no sufficient data about the correlations between DLMO and clinical scales assessing sleep quality and daytime symptoms of poor night sleep. The aim of the study was to determine the association between DLMO and basic sleep parameters from actigraphy and sleep diaries, as well as the association between DLMO and the following insomnia clinical scales: the Athens Insomnia Scale (AIS), Insomnia Severity Index (ISI), Epworth Sleepiness Scale (ESS), and chronotype questionnaires: Morningness-Eveningness Questionnaire (MEQ) and Composite Scale of Morningness (CSM). Participants of the study were healthy volunteers. Sleep parameters were measured by sleep diaries and actigraphy, and the following clinical scales: the AIS, ISI, and ESS, and chronotype questionnaires: MEQ and CSM. DLMO was calculated based on plasma melatonin concentration. The blood samples were collected hourly at five time points between 20:00 and 00:00 during the session in dim red light (<50 lux). Melatonin concertation was determined by LC-MS/MS. Twenty-one volunteers participated in the study. DLMO was calculated in 12 participants. There was a significant correlation between DLMO and ISI (r = 0.60, p = 0.038) and ESS (r = 0.61, p = 0.034). The correlation coefficient between the DLMO and the AIS was also high, however insignificant (r = 0.57, p = 0.054). There were no significant correlations between DLMO and chronotype scales MEQ and CSM. DLMO did not correlate with sleep onset and sleep offset; however, DLMO correlated with the Sleep Fragmentation Index (SFI) (r = 0.67, p = 0.017). DLMO is associated with poorer sleep maintenance, a stronger feeling of insomnia, and sleepiness during the day. Simultaneously, chronotype pattern and circadian rhythm parameters do not correlate with DLMO. Biological circadian rhythm does not reflect the real-life sleep-wake rhythm, indicating that the lifestyle is more often disconnected from the biological clock.
ABSTRACT
Melatonin (MELA) is a nocturnal hormone involved in the regulation of the circadian rhythm. MELA can be detected in plasma and saliva, and its salivary concentration strongly correlates with its plasma concentration. Dim light melatonin onset (DLMO) is considered to be the most accurate objective marker for assessing the circadian phase. The purpose of the study was to establish a method for the determination of MELA in plasma and saliva based on the liquid chromatography with tandem mass spectrometry (LC-MS/MS) and compare DLMO using both plasma and saliva matrices. The validation of the LC-MS/MS methods was performed in accordance with the European Medicines Agency (EMA) guideline. The study was conducted on a group of 21 volunteers, male and females, aged 26-54 years. Plasma and saliva were collected at five time points: between 20:00 and 00:00 hours. The MELA concentration was determined by the LC-MS/MS. The DLMO was considered as the point in time when MELA concentration exceeds 20 pg/mL in plasma and 7 pg/mL in saliva. The correlation coefficient between the plasma and salivary MELA concentration was r = 0.764 (p < .001). The ratio of the plasma/saliva MELA concentrations was 2.87. The mean time of the DLMO in the plasma was 21:30 ± 0:45 hours, and in the saliva was as follows: 21:34 ± 1:00 hours. The correlation between the DLMO, calculated based on the plasma and saliva MELA profiles, was r = 0.679 (p < .05). The determination of salivary MELA concentration using LC-MS/MS allows for the determination of the DLMO. Our method may be applied in clinical practice for the diagnosis and monitoring of circadian rhythm disorders.Abbreviations: CE: Collision Energy; CID: Collision-Induced Dissociation; DL: Desolvation Module; DLMO: Dim Light Melatonin Onset; EFSA: European Food Safety Authority; EMA: European Medicines Agency; ESI: electrospray ionization; HB: heat block; HPLC: high performance liquid chromatography; IS: internal standard; K3EDTA: ethylenediaminetetraacetic acid tripotassium salt; LC-MS/MS: liquid chromatography with tandem mass spectrometry; LLE: liquid-liquid extraction; LLOQ: lower limit of quantification; MELA: melatonin; MELA-D4: melatonin-d4; MRM: multiple reaction monitoring; Q1: quadrupole 1; Q3: quadrupole 3; RE: relative error; RIA: radioimmunoassay; RSD: relative standard deviation; SD: standard deviation; ULOQ: upper limit of quantification.
