OBJECTIVES:
The 24-hour circadian clock, located in the hypothalamus of the brain, controls the timing and pattern of sleep,
alertness, performance and mood, all of which has a major impact on safety when disrupted. The circadian clock
also controls many other aspects of human physiology, perturbation of which will affect longer-term health and
well-being. It is imperative that the timing of the circadian clock is measured routinely, as a standard measure of
health and behavior, during long-duration space missions to ensure that these multiple brain and body systems are
not disrupted and misaligned from either each other (internal desynchronization) or with the work schedule
(external desynchronization). Once measured, and if out of phase, circadian rhythms can be resynchronized to the
desired time using several countermeasures, including bright light, melatonin, and potentially exercise and meal
timing.
The effectiveness of these countermeasures depends critically on the circadian phase at which they are
administered. Mistimed countermeasures can exacerbate circadian disruption, leading to worsening of health and
performance outcomes rather than improvement. Circadian phase is highly individualized, however, varying by
up to five hours in healthy individuals under stable conditions, and is even more variable in microgravity. To
date, no feasible method exists to measure circadian phase accurately and reliably in real-time during space flight.
Existing methods currently being used inflight require collection of serial urine samples for up to 48 hours, from
which the concentration of the urinary metabolite 6-sulphatoxymelatonin is measured to determine the timing of
the circadian rhythm. Importantly, however, no technology exists to measure concentrations of urinary 6-
sulphatoxymelatonin directly inflight; therefore, knowledge about inflight circadian disruption can be determined
only during post-hoc analyses once the samples have been returned to Earth, which limits the ability to treat
circadian disruption in real time.
This study has the following specific aims:
- Validate the use of real-time measures of saliva cortisol and blood cholesterol to derive the circadian
phase.
- Develop a method to derive the circadian phase from as few as three salivary cortisol and/or blood
cholesterol samples.
APPROACH:
Twenty participants enrolled in this within-subject study to validate the stability of cortisol and cholesterol rhythms over two collection windows. Participants were generally free from acute and chronic illness, were similar to the astronaut population, and included equal numbers of males and females. Each participant was asked to collect three sample types over two approximately 48-hour periods: saliva, for the real-time measurement of salivary cortisol via the Soma Bioscience cortisol lateral flow device; blood, for the real-time measurement of cholesterol using a commercially available quantitative cholesterol meter (e.g., CardioChek ST Analyzer); and urine, for assay of urinary 6-sulphatoxymelatonin (aMT6s) completed by the Brigham Research Assay Core (BRAC).
Each participant met with the team’s research assistant for instructions on how to collect these samples and record measurements prior to the start of the first 48-hour collection period. Each participant was instructed to collect samples starting around wake time on day one and continuing every three hours (except during sleep) for the next two days. The final sample for the first collection window was collected at wake time on day three. Participants then repeated these procedures over another ~48-hour period approximately 1 week after the start of the first collection window. Due to COVID-19 restrictions, there were some changes to recruitment and data collection procedures. All research visits were conducted virtually, and sample materials and equipment were shipped to the participant and returned to us via return shipment.
Salivary cortisol collection: The Soma Bioscience point-of-care salivary cortisol kit includes oral fluid collectors (OFC), the cortisol lateral flow device (LFD-C), and the LFD reader. For each sample, the participant was instructed to collect a small amount of saliva into the OFC swab and place the swab into OFC buffer. Due to ongoing restrictions related to the COVID-19 pandemic, the LFD reader was not shipped to each participant. Instead, participants were instructed to freeze the sample according to the manufacturer’s instructions and record the time the sample was taken. Post-collection, one of the study investigators (MSH) thawed the samples, added two drops of each sample to each LFD-C test strip, and then inserted the test strip into the LFD reader at the 10-minute mark to obtain the quantitative cortisol value, per the manufacturer’s instructions.
Participants also collect saliva into a second tube at each time point. These samples were assayed by SolidPhase (Portland, ME) for saliva cortisol by radioimmunoassay to compare to the real-time samples.
Blood cholesterol collection: For each sample, the participant pricked their finger, collected a droplet of blood, placed the droplet onto a test strip, and inserted the strip into the meter. Participants recorded the quantitative cholesterol value along with the time the sample was collected.
Urinary aMT6s collection: At each urine void (approximately every three hours during wake), participants voided into a urine jug, recorded the volume of urine from the jug and the time of the void, aliquoted a 10 mL sample, and discarded the remaining urine. Participants immediately froze each sample. These samples were assayed by the Brigham Research Assay Core (Boston, MA) for 6-sulphatoxymelatonin and urinary free cortisol by LC/MS.
Assessment of circadian phase from each marker: The gold-standard method by which to estimate circadian phase from urinary aMT6s is via cosinor analysis and has also been applied to cortisol and cholesterol rhythms. Investigators applied the cosinor model to each 48-hour aMT6s, cortisol, and cholesterol profile to determine the presence of a significant circadian rhythm and the circadian phase.
Validation of cortisol and cholesterol circadian phase markers: To satisfy Aim 1, the investigators will determined whether circadian phase derived from real-time cortisol or cholesterol rhythms are stable across different 48-hour collection windows as compared to gold-standard marker urinary aMT6s. Derivation of circadian phase from less than 48 hours of samples: To satisfy Aim 2, the investigators applied their three-point circadian phase (3PCP) method to their real-time cortisol, real-time cholesterol, and aMT6s profiles.
RESULTS:
Validation of cortisol and cholesterol circadian phase markers (Aim 1): Urinary aMT6s exhibited significant and stable circadian rhythms in the majority of participants. Traditional salivary and urinary assays exhibited significant and stable circadian rhythms in the majority of participants. Real-time salivary cortisol did note correlate with assayed cortisol and did not exhibit significant circadian rhythms. Real-time cholesterol did not exhibit significant circadian rhythms in participants studied at home.
Derivation of circadian phase from less than 48 hours of samples (Aim 2): Circadian phase estimates based on three consecutive samples of urine aMT6s have high accuracy with circadian phase estimates taken from 48-hour urine aMT5s profiles. Real-time cholesterol and cortisol measures did not exhibit robust circadian rhythms as hypothesized, investigators were unable to develop an algorithm to estimate real-time circadian phase from these measures. Circadian phase estimates based on three non-consecutive samples of cholesterol and cortisol were made with the non-real-time measures and were found to not correlate strongly with the gold-standard marker.