McPherson, Mark MSc; Janssen, Ian PhD; Grundy, Anne MSc; Tranmer, Joan PhD; Richardson, Harriet PhD; Aronson, Kristan J. PhD
* Summarize the postulated contribution of altered melatonin secretion patterns to the increased health risks associated with shift work.
* Review the new findings on the relationship between physical activity and melatonin levels, including differences by intensity of energy expenditure and day versus night shift.
* Discuss the clinical significance of the study findings and the implications for future research.
Shift workers have a 40% higher risk of cardiovascular disease than the general population.1 With approximately 20% of the population working shift work,2 the population etiologic fraction of shift work on cardiovascular disease has been estimated to be nearly 7%.3 Long-term shift work involving night work has also been classified as a probable carcinogen by the International Agency for Research on Cancer.4 The increased risk of both cardiovascular disease and cancer in shift workers has been attributed to behavioral and social changes associated with disruption daily day–night routines and sleep–wake cycles. These may include altered eating and socializing schedules and added stresses through reduced family time.1,5 Although considerable research has examined the behavioral and social impacts of shift work on morbidity, biological mechanisms explaining these associations are not yet fully characterized.6 Some research has suggested that disturbances in the body's natural biological rhythms, or circadian disruption, could increase disease risk in the shift working population.7,8
Melatonin (N-acetyl-5-methoxytryptamine) is an important circadian indicator in humans and may play an important role on the biological pathway linking shift work with cardiovascular disease and cancer. Melatonin production is primarily influenced by light, and consequently shift workers may be susceptible to altered melatonin secretion patterns9 and lower circulating levels of melatonin10 because of their increased exposure to light at night. Melatonin can inhibit cardiovascular and carcinogenic pathological processes by acting directly as a free radical scavenger or indirectly as an antioxidant.11 Studies in humans suggest that exogenous melatonin administration reduces several cardiovascular risk factors including hypertension, dysrhythmias, and hypertrophic cardiomyopathy.12,13 Laboratory studies have also shown that breast cancer cells demonstrate decreased cell proliferation and invasiveness after the administration of physiological concentrations of melatonin.14
It is well-known that participation in moderate- to vigorous-intensity physical activity (MVPA) protects against the development of cardiovascular disease and colon, breast, and endometrial cancer.15,16 The proposed etiological mechanisms for this relationship include both the short- and long-term effects of physical activity including improved body composition, glucose management, and autonomic tone.17 Melatonin may also influence this pathological pathway13; however, this hypothesis remains largely untested. Few studies have investigated the role of physical activity in affecting melatonin levels in humans, and those that have are mainly experimental and have a wide range of results. Researchers have reported an increase,18 decrease,19 and no change20 in melatonin in response to a single bout of exercise. Both animal21 and human22 experimental studies have suggested that physical activity has a short-term effect (within minutes) on melatonin, with physical activity less than 12 hours prior to the commencement of sleep influencing circulating melatonin levels.
Two observational studies to date have aimed to characterize the relationship between physical activity and melatonin. Knight et al23 found that after stratifying by the timing of activity, there was a positive relationship between physical activity and melatonin in 213 female volunteers, and this relationship was strongest during the evening and night (4 PM to 4 AM), although physical activity explained only 5% of melatonin variation. In our previous pilot study, Grundy et al24 examined low-, moderate- and high-intensity acute physical activity among 61 rotating shift nurses and found no association with urinary 6-sulfatoxymelatonin among nurses working their day shift (P = 0.13).
The study of sedentary behavior, or inactivity, is of growing interest in physical activity epidemiology, although it has not been traditionally considered when classifying intensity. Sedentary behavior is at one extreme of the physical activity continuum and includes behaviors such as sitting and resting, and it has effects on health outcomes independent of low-, moderate-, and vigorous-intensity physical activity.25 The influence of sedentary behavior on melatonin has not been established in humans or animal models, and consequently this study will examine the independent roles of energy expended in both physical activity and sedentary behaviors on peak melatonin levels. If physical activity has a role in modulating the potential reduction in melatonin levels among shift workers, occupation-specific preventative recommendations can be developed to maximize health.
MATERIALS AND METHODS
Nurses working a rotating shift pattern of two 12-hour days (7 AM to 7 PM) after two 12-hour nights (7 PM to 7 AM) and then 5 days off at Kingston General Hospital were invited to volunteer for this study. Participants self-excluded if they were taking melatonin supplements or if they had been pregnant or lactating in the 6 months prior to enrollment. A total of 123 nurses enrolled, 5 were lost to follow-up or withdrew, and 12 completed only one of two data collection periods.
Nurses completed two 24-hour study periods, one during a day shift and one during a night shift. The two study periods took place 1 month apart to reduce melatonin variability due to menstrual stage. For the day shift study period, a morning void urine sample was collected preceding their second day shift (5 AM to 7 AM). For the night shift study period, a morning void urine sample was collected after completion of their second night shift (7 AM to 8 AM). This single urine sample was adequate for melatonin detection, as our previous research indicated that melatonin peaks at night during both the day and night shifts when working this rotating shift schedule,23 and consequently morning void samples were chosen to reflect the peak melatonin concentrations for both shifts. Participants were asked to record the time of urine collection in their 1-day diary to ensure that proper specimen protocol was followed. Urine samples were stored at −80°C prior to laboratory analyses.
Participants completed a diary recording the timing, duration, and intensity of physical activity and sedentary behaviors during each 24-hour study period. For each activity, participants were asked to list the length of time they were engaged in that activity, and the intensity recorded as low, moderate, or vigorous based on perceived perspiration and respiratory rate. Low-intensity activities were defined as activities requiring little to no physical effort, while moderate-intensity activities were described as slightly increasing heart rate and breathing and potentially causing light sweating. Finally, vigorous-intensity activities were explained as substantially increasing heart rate and breathing and causing heavy sweating. Although not validated, this scale was based on the diary utilized in our previous study on this topic.23 Sedentary behaviors were assessed through five questions regarding time spent watching television, using a computer, sitting, light chores, and standing. Physical activities and sedentary behaviors were reported separately for the morning/afternoon (7 AM to 3 PM), the afternoon/evening (3 PM to 11 PM), and the evening/morning (11 PM to 7 AM) time periods.
Information on potential confounders was collected at an initial interview, in a questionnaire completed at the commencement of the study, and in the diary, and included age, menopausal status, smoking history, ethnicity, alcohol and caffeine consumption, and medication usage (nonsteroidal anti-inflammatory drugs, sedatives, oral contraceptives, and migraine medication). Participants wore a StowAway Light Intensity Data Logger (Hoskin Scientific Ltd, Burnaby, BC, Canada) to measure light exposure. Height, weight, and waist circumference were measured by a study nurse at the time of study enrollment.
The type and intensity of each physical activity and sedentary behavior reported were converted to a metabolic equivalent (MET) using the Compendium of Metabolic Equivalents.26 A MET is a ratio of the metabolic rate of an activity compared with a standard resting metabolic rate (4.184 kJ kg−1 hr−1); thus, a MET of 3 indicates that the activity requires 3 times as much energy expenditure as is required at rest.23 Activities range from those of no intensity such as sitting (MET = 1) to activities of a very high intensity such as running (MET = 18). Activities with MET values less than 1.5 are defined as sedentary behaviors,27 activities with a MET between 1.5 and 3 are classified as being of low intensity, and activities with a MET value > 3 are classified as being of moderate or vigorous intensity.28
The duration of each physical activity and sedentary behavior reported in the diary was multiplied by its corresponding MET value to yield a MET minute variable (MET·min). MET·min values were summed for each intensity in each of the three time periods (7 AM to 3 PM, 3 PM to 11 PM, and 11 PM to 7 AM) and are a representation of the total energy expended.
Levels of 6-sulfatoxymelatonin, the main urinary melatonin metabolite, were measured using the Bühlmann 6-sulfatoxymelatonin enzyme-linked immunosorbent assay kit (ALPCO Diagnostics, Salem, NH). The Bühlmann 6-sulfatoxymelatonin enzyme-linked immunosorbent assay test kit is a competitive immunoassay that uses capture antibody principles to determine levels of 6-sulfatoxymelatonin in human urine. 6-Sulfatoxymelatonin concentrations have been shown to be representative of 70% of circulating melatonin.29 In contrast to blood and saliva melatonin measurements, morning void urine samples measure cumulative concentrations of melatonin, providing an indication of melatonin levels during the circadian peak. In adults with invariable sleeping schedules, nocturnal peak urinary melatonin levels remain fairly constant,30 whereas with disturbed sleeping patterns the amplitude of these peaks may fluctuate, so urinary 6-sulfatoxymelatonin provides a practical and noninvasive means of detecting such changes in circulating melatonin levels. Creatinine measurement was required to determine the volume of urine excreted in each sample, which could function to dilute or concentrate melatonin in the urine. The Parameter Creatinine Assay (R&D Systems, Inc, Minneapolis, MN) was utilized to assess levels of creatinine in diluted urine samples. Urinary 6-sulfatoxymelatonin concentrations were divided by concentrations of creatinine and log-transformed to yield a distribution that approximated the normal distribution.
To ensure high-quality laboratory data, samples were reanalyzed using appropriately adjusted dilutions if either duplicate was out of range of the standard curve, or if the standard curve fit the standards with an R2 of less than 0.95. Coefficients of variation between duplicates were calculated for each sample and retested if they exceeded 50. Two controls of known concentrations were also run in duplicate to confirm the accuracy of the results. Inter- and intra-assay precision was not calculated because of financial restraints.
Multivariable linear regression was used to determine the relationship between energy expenditure between 3 AM and 7 PM and melatonin. This time period was representative of the biologically relevant timeframe determined from previous literature19,24,31 and included both leisure-time and occupational activity. Additional analyses were carried out to confirm that this time frame was most predictive of melatonin levels. Separate linear regression models were used for each shift, to determine whether the relationship between physical activity, sedentary behavior, and peak melatonin levels was the same for both the day and night shift study periods. Moderate- and vigorous-intensity physical activities were combined and treated as one variable since very few subjects reported vigorous-intensity activity. Potential confounders were retained in the model if they changed the exposure parameter estimates by more than 10% upon deletion.32 All statistical analyses were completed using SAS (Version 9.1, SAS Institute, Cary, NC).
Nurses (n = 118) were mainly white (95%) with an average age of 41 years (Table 1). Sleep problems were commonly reported (59%). Characteristics that may have varied over time for the 106 participants who completed both study periods are presented in Table 2. In comparison to when the nurses were working on the day shift, participants reported an average of 1.7 hours less sleep after their night shift. The geometric mean of creatinine-adjusted 6-sulfatoxymelatonin concentrations was 27.1 mg/ng of creatinine after the day shift collection, and 25.3 mg/ng of creatinine after the night shift collection, and these values did not differ (P = 0.91).
The MET·min values for low-intensity physical activity and MVPA were not statistically different for the day-and-night 24-hour study periods (Table 3). During the biologically relevant timeframe (3 PM to 7 AM), the MET·min for low-intensity physical activity and sedentary behaviors were higher during the night shift study period than the day shift study period (P < 0.0001). During the day shift study period, 45% of the daily energy expenditure occurred during the biologically relevant timeframe, compared with 90% for the night shift study period.
Sedentary behaviors accounted for more than 45% of total MET·min during both study periods, although for the biologically relevant time period the MET·min for sedentary behavior were greater for the night shift than for the day shift (770 vs 423, P < 0.0001). The sedentary behaviors that contributed the most to the total MET·min during both 24-hour study periods were standing (76%), computer use (8%), and television watching (7%). Low-intensity activities comprised 37% and 35% of the total MET·min during the day and night shift periods, respectively. These mostly consisted of lifting and bathing patients (56%) and walking (16%). MVPA during the day and night shift periods contributed to 9.4% and 11.8%, respectively, of total MET·min. During the month prior to each study period, MVPA contributed to nearly 15% of total MET·min (data not shown).
Separate linear regressions were carried out for the day and night shift study periods for sedentary behavior, low intensity, and MVPA between 3 PM and 7 AM Age was the only covariate included in the linear regressions analyses, as other variables (ie, body mass index, waist circumference, menopausal status, smoking history, ethnicity, alcohol and caffeine consumption, and medication use) did not meet the modeling criterion for confounding.
Associations between the different intensities of physical activity with melatonin differed for the day and night shift periods (Table 4). For the day shift, MET·min accumulated during MVPA was negatively associated with melatonin levels (P = 0.024) while MET·min accumulated during low-intensity and sedentary behavior were not (P > 0.40). The partial regression coefficient was −0.21, indicating that for every 1 SD (298 MET·min) increase in MVPA, melatonin decreased by 0.21 SDs (0.60-mg melatonin/ng of creatinine; Fig. 1). For the night shift period, the MET·min accumulated during sedentary behavior was negatively associated with melatonin (P = 0.008), such that every 1 SD (190 MET·min) increase in sedentary behavior was associated with a decrease in melatonin of 0.28 SDs (0.91-mg melatonin/ng of creatinine; Fig. 2). The associations persisted after removal of the six most influential observations (data not shown).
Our analysis indicated that physical activity and sedentary behavior are associated with decreased melatonin levels during the biologically relevant timeframe. Specifically, MVPA during the day study period was negatively associated with 6-sulfatoxymelatonin levels, but was not associated with the outcome during the night shift study period. During the night shift period, sedentary behaviors were the only significant predictor of peak melatonin levels. Nevertheless, these exposures explained little variation in melatonin levels. Although these associations were statistically significant, they may not be clinically significant, and consequently more observational and experimental research is necessary to determine factors associated with melatonin downregulation among shift workers, if this potential mechanism proves important. Before substantial policy recommendations are made, the role of melatonin on the pathway linking shift work to both cardiovascular disease and cancer needs to be examined more thoroughly, as one of several potential pathways.
Our study attempted to address the issue of the relative importance of timing (biologically effective exposure) or intensity of physical activity exposures. In our study, energy expended in MVPA was associated with melatonin in the day shift, while energy expended in sedentary behavior was associated with melatonin in the night shift. The differences between shifts may indicate that the true biologically relevant timeframe by which physical activity and sedentary behavior affect melatonin may be later than that used in this study (3:00 PM to 7:00 AM). Since MVPA is largely unattainable during the 12-hour shift, most of the MVPA during the biologically relevant timeframe for the day shift study period would have occurred after the shift ended at 7:00 PM Conversely, most of the MVPA during the biologically relevant time frame for the night shift study period would have occurred before the 7:00 PM start time of the shift. For night shift, 90% of all daily sedentary energy expenditure occurred after 3:00 PM, because nurses were more likely to engage in sedentary behaviors on the job. Therefore, during the night shift study period, the primary source of energy expenditure during the true biologically relevant timeframe was sedentary behavior, hence its statistically significant association with melatonin. Thus, the timing of energy expenditure may play a more important role than the intensity.
Similar associations have been reported in both experimental and observational studies. Montelone et al33 found that after 20 minutes of high-intensity physical activity in human subjects, melatonin levels decreased for 3 hours, and similar results have also been shown in animal models.34 Reduced levels of melatonin may have been attributable to increased levels of cortisol that functions to reduce noradrenalin-stimulated melatonin release by the pineal gland. This relationship has been described in patients with hypercortisolemia, who show reduced levels of circulating melatonin.35
This study is the third epidemiological study on this topic. Knight et al23 found that exercise was positively associated with creatinine-adjusted melatonin levels in 214 healthy female volunteers, although it explained only 5.3% of melatonin variation.23 Our methods improved on this study by including sedentary behaviors and low-intensity physical activities. In addition, our study sample was rotating shift nurses who may be more susceptible to melatonin downregulation due to their altered sleep–wake schedules than healthy volunteers. In a sample from the same working population as the current research, Grundy et al24 found a nonsignificant positive association between physical activity and melatonin among those working the day shift, and a null association among those working the night shift.24 This was a small pilot study (n = 61), urine samples were taken at a different time for the day and the night shift, and melatonin measurements were not creatinine-adjusted.
A key strength of this study is that we assessed all intensities of physical activity and movement, not solely moderate- and vigorous-intensity exercise, the latter of which was uncommon in this working population. More than 45% of the activity-induced energy expenditure among these nurses was accrued through very low-intensity sedentary behaviors that were performed over a prolonged period, such as standing and sitting. Activities of this intensity would not have been captured using most existing physical activity questionnaires, which focus on MVPA. Both short- and long-term physical activity information was collected in this study, so the most biologically relevant timeframe could be established. In addition, the use of urine biomarkers in this study provided a validated method of assessing melatonin levels.
Limitations include errors in recall of physical activity, which would function to bias the results toward the null. Recall bias is not an issue because participants were unaware of their melatonin status. Overreporting and misclassification of physical activity and underreporting of sedentary behaviors are a common concern with physical activity diaries and questionnaires36 and may have influenced their reporting. General activity categories such as standing may introduce misclassification, as standing may include heavy lifting or other sources of energy expenditure not captured with this exposure assessment. Although collection periods were 1 month apart, which may have altered the day/night phase, light exposure was not determined to effect the relationships between physical activity, sedentary behavior, and melatonin. In terms of the external validity, the generalizability of the findings may be limited to female nurses working the 2-day, 2-night shift schedule.
Future studies should use more precise exposure assessment techniques such as accelerometers. These tools would eliminate any threat of self-report error and could provide a tool by which the biologically relevant timeframe linking physical activity and sedentary behaviors with melatonin could be further investigated. The validity of these results is dependent on the appropriate classification of the biologically relevant timeframe by which physical activity and sedentary behaviors affect melatonin production, which could have been misclassified by the broad timeframes used to classify the exposure in this study.
Cortisol measurements would provide a means by which the biological mechanisms could be further examined, and our research group has started a new study that will examine the relationship between melatonin and cortisol among nurses. Consideration should be given to the timing of the sample collection, to ensure that the cortisol measurements reflect the biologically relevant timeframe of physical activity.
The authors thank the nurses who participated in this study. We also thank Kathy Bowes, Karen Loller, Krista Smith, and Deborah Emerton for their assistance in data collection, and Annie Langley, Lindsay Kobayashi, Shannyn MacDonald-Goodfellow, and Dr Charles Graham for their assistance with the laboratory analysis. Financial support for this research was provided by the Workplace Safety and Insurance Board of Ontario. M. McPherson was supported by a graduate student award from the Heart and Stroke Foundation of Ontario, I. Janssen was supported by a New Investigator Award from the Canadian Institutes of Health Research and an Early Researcher Award from the Ontario Ministry of Research and Innovation, A. Grundy was supported by Terry Fox Foundation Training Program in Transdisciplinary Cancer Research, and J. Tranmer was supported by a Mid-Career Investigator Award from the Ontario Women's Health Council/Canadian Institutes of Health Research. We have no competing interests.
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