Removing Masking Factors from Urinary Rhythm Data in Humans

We have previously developed simple models that enable the exogenous and endogenous components of the circadian rhythm of body temperature to be separated. The present paper extends the method to urinary data. First, we have shown that the basic superiority of the two-component model over the one-component model persists when temperature data are converted into a format that is appropriate for urine sampling (that is, a single overnight sample and two-hourly samples during waking). Second, we provide normative endogenous data for urinary sodium, potassium and urate, data obtained from about 80 constant routines. These data are required for the two-component model. Third, we have compared the rate of adjustment to a simulated eastward time-zone transition of 8hr in 8 subjects. This showed that the rate of adjustment assessed by the two-component model was significantly less than that assessed by the one-component model and much closer to that assessed in separate experiments (n=15 subjects) using constant routines. We conclude that the two-component model can be used upon urinary data to give a closer approximation to the shift of the endogenous component, as assessed by constant routines, than can estimates that do not take into account the problem of masking caused by exogenous factors.     

"Demasking" the temperature rhythm after simulated time zone transitions.

Simulated time zone transitions were performed in an isolation unit upon groups of one to four human subjects. In the first series of experiments, the adjustment of the circadian rhythm of body temperature, measured in the presence of sleep and other masking factors, was assessed by cosinor analysis and by cross-correlation methods. These methods modeled the circadian timing system either as a single component or as the sum of two components, those due to exogenous and endogenous influences. The one-component models described a more rapid adjustment of the temperature rhythm to the time zone transition than did the two-component models; we attribute this difference to the masking effects of the exogenous component. In a second series of experiments, we showed that the shift of the endogenous component, as assessed by the two-component models, was not significantly different from that measured during constant routines. The results also showed that, if the zeitgebers were phased in advance of the endogenous component, then advances of the endogenous component were produced only if this mismatch was less than about 10 hr. Mismatches greater than this, and cases where the zeitgebers were delayed with respect to the endogenous component, both produced delays of the endogenous component. We conclude that the two-component cross-correlation methods can be used to estimate shifts of the endogenous component of a circadian rhythm in the presence of masking factors. They are therefore an alternative to constant routines when these latter are impracticable to carry out.     

Masking in Humans: The Problem and Some Attempts to Solve IT

Different types of masking are discussed together with an account of the masking effect that the sleep-wake cycle exerts upon the circadian rhythms of body temperature and urinary excretion. The relative importance to masking of the several components of differences between sleeping and wakefulness are then assessed.

Means to deal with the problem of masking fall into two major categories. These attempt to minimise masking effects by protocols such as constant routines or control days, and mathematical models which separate results obtained in the presence of masking influences into endogenous and exogenous components. (The problem of the extent to which masking influences can render the endogenous component of a rhythm an impure reflection of the internal oscillator is considered also.) These different techniques are compared with respect to their usefulness and assumptions.

Finally, a brief speculation is given of the usefulness of masking.     

Measuring phase shifts in humans following a simulated time-zone transition: agreement between constant routine and purification methods

Twelve healthy participants were studied in an Isolation Unit. For the first 7 (control) days, subjects lived on UK time. Then the clock was advanced by 8 h, mimicking an eastward time-zone transition, and for days 8 to 12, participants lived on this new local time. Two constant routines (participants were not allowed to sleep, were restricted in movement, and ate regular, identical snacks) were undertaken, during the control days (days 3 to 4) and at the end of the experiment (days 11 to 12). Rectal temperature and activity were measured throughout, with activity used to correct the measured temperatures for the direct (masking) effects of the sleep-wake cycle. Phase changes of the temperature rhythm between the constant routines were assessed by cross-correlation and cosinor analysis. During days 8 to 10, the measured temperatures and those that had been corrected (purified) for masking were assessed by the same two methods, and the shifts were extrapolated to predict the values expected during the second constant routine. Individuals differed widely in the phase shifts of the temperature rhythm, but the correlations between the changes measured by constant routines and those estimated by the purification methods were high (r=0.771 to 0.903), and the differences between them were not significantly different from zero (p>0.24). Phase shifts of the measured (masked) temperature rhythm were poorer predictors of the shift obtained from the constant routines (r3+/-4.5 h). Limitations of the methods due to the variability of results are discussed, but we conclude that the mean phase shifts obtained from purified, but not raw, temperature data show acceptable agreement with those found using our version of the constant routine.     

Measuring Phase Shifts in Humans Following a Simulated Time‐Zone Transition: Agreement Between Constant Routine and Purification Methods

Twelve healthy participants were studied in an Isolation Unit. For the first 7 (control) days, subjects lived on UK time. Then the clock was advanced by 8 h, mimicking an eastward time‐zone transition, and for days 8 to 12, participants lived on this new local time. Two constant routines (participants were not allowed to sleep, were restricted in movement, and ate regular, identical snacks) were undertaken, during the control days (days 3 to 4) and at the end of the experiment (days 11 to 12). Rectal temperature and activity were measured throughout, with activity used to correct the measured temperatures for the direct (masking) effects of the sleep‐wake cycle. Phase changes of the temperature rhythm between the constant routines were assessed by cross‐correlation and cosinor analysis. During days 8 to 10, the measured temperatures and those that had been corrected (purified) for masking were assessed by the same two methods, and the shifts were extrapolated to predict the values expected during the second constant routine. Individuals differed widely in the phase shifts of the temperature rhythm, but the correlations between the changes measured by constant routines and those estimated by the purification methods were high (r=0.771 to 0.903), and the differences between them were not significantly different from zero (p>0.24). Phase shifts of the measured (masked) temperature rhythm were poorer predictors of the shift obtained from the constant routines (r≤0.605; mean±SD of differences >3±4.5 h). Limitations of the methods due to the variability of results are discussed, but we conclude that the mean phase shifts obtained from purified, but not raw, temperature data show acceptable agreement with those found using our version of the constant routine.     

The use of Constant Routines in Unmasking the Endogenous Component of Human Circadian Rhythms

A major problem in the study of the internal clock(s) that drives human circadian rhythms is that due to the effect produced by rhythmicity of habits and external influences (‘masking’). A particularly potent factor in this respect is the sleep-wake cycle. It is anomalous that, even though this masking influence is widely accepted, most studies of circadian rhythmicity have been performed in the presence of such interferences.

A protocol is described, the constant routine, by which these exogenous influences can be minimized, thereby enabling a closer scrutiny of the internal clock(s) to be made. An account is given of the different circumstances in which the constant routines have been used together with the results derived from such studies. Briefly, they indicate that nychthemeral studies can give misleading information about the rate of adjustment of the internal clock to various manipulations, e.g. time-zone transition, shift work.

In addition, future studies making use of constant routines are described, in particular those which might enable the presence of more than one internal clock to be established.     

The use of Constant Routines in Unmasking the Endogenous Component of Human Circadian Rhythms

A major problem in the study of the internal clock(s) that drives human circadian rhythms is that due to the effect produced by rhythmicity of habits and external influences ('masking'). A particularly potent factor in this respect is the sleep-wake cycle. It is anomalous that, even though this masking influence is widely accepted, most studies of circadian rhythmicity have been performed in the presence of such interferences.

A protocol is described, the constant routine, by which these exogenous influences can be minimized, thereby enabling a closer scrutiny of the internal clock(s) to be made. An account is given of the different circumstances in which the constant routines have been used together with the results derived from such studies. Briefly, they indicate that nychthemeral studies can give misleading information about the rate of adjustment of the internal clock to various manipulations, e.g. time-zone transition, shift work.

In addition, future studies making use of constant routines are described, in particular those which might enable the presence of more than one internal clock to be established.     

The Shape of the Endogenous Circadian Rhythm of Rectal Temperature in Humans

Fourteen healthy subjects have been studied in an isolation unit while living on a 30h “day” (20h awake, 10h asleep) for 14 (solar) days but while aware of real time. Waking activities were sedentary and included reading, watching television, and so forth. Throughout, regular recordings of rectal temperature were made, and in a subgroup of 6 subjects, activity was measured by a wrist accelerometer. Temperature data have been subjected to cosinor analysis after “purification,” a method that enables the endogenous (clock-driven) and exogenous (activity-driven) components of the circadian rhythm to be assessed. Moreover, the protocol enables effects due to the circadian rhythm and time-since-waking to be separated. Results showed that activity was slightly affected by the endogenous temperature rhythm. Also, the masking effects on body temperature exerted by the exogenous factors appeared to be less than average in the hours before and just after the peak of the endogenous temperature rhythm. This has the effect of producing a temperature plateau rather than a peak during the daytime. The implications of this for mental performance and sleep initiation are discussed. (Chronobiology International, 13(4), 261-271, 1996)     

A COMPARISON OF SOME DIFFERENT METHODS FOR PURIFYING CORE TEMPERATURE DATA FROM HUMANS

Nine healthy females were studied about the time of the spring equinox while living in student accommodations and aware of the passage of solar time. After 7 control days, during which a conventional lifestyle was lived under a 24h “constant routine,” the subjects lived 17 × 27h “days” (9h sleep in the dark and 18h wake using domestic lighting, if required). Throughout the experiment, recordings of wrist activity and rectal (core) temperature were taken. The raw temperature data were assessed for phase and amplitude by cosinor analysis and another method, “crossover times,” which does not assume that the data set is sinusoidal. Two different purification methods were used in attempts to remove the masking effects of sleep and activity from the core temperature record and so to measure more closely the endogenous component of this rhythm; these two methods were “purification by categories” and “purification by intercepts.” The former method assumes that the endogenous component is a sinusoid, and that the masking effects can be estimated by putting activity into a number of bands or categories. The latter method assumes that a temperature that would correspond to complete inactivity can be estimated from measured temperatures by linear regression of these on activity and extrapolation to a temperature at zero activity. Three indices were calculated to assess the extent to which exogenous effects had been removed from the temperature data by these purification methods. These indices were the daily variation of phase about its median value; the ratio of this variation to the daily deviation of phase about midactivity; and the relationship between amplitude and the square of the deviation of phase from midactivity. In all cases, the index would decrease in size as the contribution of the exogenous component to a data set fell. The purification by categories approach was successful in proportion to the number of activity categories that was used, and as few as four categories produced a data set with significantly less masking than raw data. The method purification by intercepts was less successful unless the raw data had been “corrected” to reflect the direct effects of sleep that were independent of activity (a method to achieve this being produced). Use of this purification method with the corrected data then gave results that showed least exogenous influences. Both this method and the purification by categories method with 16 categories of activity gave evidence that the exogenous component no longer made a significant contribution to the purified data set. The results were not significantly influenced by assessing amplitude and phase of the circadian rhythm from crossover times rather than cosinor analysis. The relative merits of the different methods, as well as of other published methods, are compared briefly; it is concluded that several purification methods, of differing degrees of sophistication and ease of application to raw data, are of value in field studies and other circumstances in which constant routines are not possible or are ethically undesirable. It is also concluded that such methods are often somewhat limited insofar as they are based on pragmatic or biological, rather than mathematical, considerations, and so it is desirable to attempt to develop models based equally on mathematics and biology. (Chronobiology International, 17(4), 539–566, 2000)     

A comparison of some different methods for purifying core temperature data from humans

Nine healthy females were studied about the time of the spring equinox while living in student accommodations and aware of the passage of solar time. After 7 control days, during which a conventional lifestyle was lived under a 24h “constant routine,” the subjects lived 17 × 27h “days” (9h sleep in the dark and 18h wake using domestic lighting, if required). Throughout the experiment, recordings of wrist activity and rectal (core) temperature were taken. The raw temperature data were assessed for phase and amplitude by cosinor analysis and another method, “crossover times,” which does not assume that the data set is sinusoidal. Two different purification methods were used in attempts to remove the masking effects of sleep and activity from the core temperature record and so to measure more closely the endogenous component of this rhythm; these two methods were “purification by categories” and “purification by intercepts.” The former method assumes that the endogenous component is a sinusoid, and that the masking effects can be estimated by putting activity into a number of bands or categories. The latter method assumes that a temperature that would correspond to complete inactivity can be estimated from measured temperatures by linear regression of these on activity and extrapolation to a temperature at zero activity. Three indices were calculated to assess the extent to which exogenous effects had been removed from the temperature data by these purification methods. These indices were the daily variation of phase about its median value; the ratio of this variation to the daily deviation of phase about midactivity; and the relationship between amplitude and the square of the deviation of phase from midactivity. In all cases, the index would decrease in size as the contribution of the exogenous component to a data set fell. The purification by categories approach was successful in proportion to the number of activity categories that was used, and as few as four categories produced a data set with significantly less masking than raw data. The method purification by intercepts was less successful unless the raw data had been “corrected” to reflect the direct effects of sleep that were independent of activity (a method to achieve this being produced). Use of this purification method with the corrected data then gave results that showed least exogenous influences. Both this method and the purification by categories method with 16 categories of activity gave evidence that the exogenous component no longer made a significant contribution to the purified data set. The results were not significantly influenced by assessing amplitude and phase of the circadian rhythm from crossover times rather than cosinor analysis. The relative merits of the different methods, as well as of other published methods, are compared briefly; it is concluded that several purification methods, of differing degrees of sophistication and ease of application to raw data, are of value in field studies and other circumstances in which constant routines are not possible or are ethically undesirable. It is also concluded that such methods are often somewhat limited insofar as they are based on pragmatic or biological, rather than mathematical, considerations, and so it is desirable to attempt to develop models based equally on mathematics and biology. (Chronobiology International, 17(4), 539-566, 2000)     

Rates of removal of exogenous and endogenous glucagon from the plasma of sheep in vivo

The removal of exogenous and endogenous glucagon from plasma was determined in vivo in sheep weighing 53 +/- 1 (mean +/- s.e.) kg. Porcine glucagon was infused intravenously for 90 min. The metabolic clearance rates (MCR) were determined from plateau immunoreactive glucagon (IRG) concentrations in plasma and infusion rates of glucagon. The mean clearance rate (+/0 s.e.) was 16.7 +/- 1.6 litres per hour (n = 20). Upon termination of the infusion, the decrease in IRG concentrations in plasma was determined. Least-squares regression analysis of non-linear functions indicated the data fit a two-component exponential function. The time constant for the rapid component of the plasma IRG disappearance function was -0.32 +/- 0.04 min-1 (mean +/- s.e.). The time constant for the slow component was -0.22 +/- 0.008 min-1. The rate of removal of endogenous glucagon was estimated during the infusion of somatostatin when glucagon secretion was inhibited. The time constants (mean +/. s.e., n = 8) for the decrease in IRG during somatostatin infusion were -0.42 +/- 0.08 and -0.003 +/- 0.002 min-1 for fast and slow components, respectively. The time constants for the rapid components of exogenous and endogenous glucagon were not significantly different. This suggests that endogenous and exogenous glucagon are similarly removed from plasma.     

A Mathematical Method for Studying the Endogenous Component of the Circadian Temperature Rhythm and the Effect of Aging

A mathematical model was developed in order to study the endogenous component of the circadian rhythm in body temperature. The model describes the fluctuations in body temperature as a function of a cosine-shaped endogenous rhythm plus an exogenous component which is linearly correlated with the time spent in active wakefulness. The model was evaluated in 4 young and 4 old rats. In 7 out of 8 rats there was a significant lack of fit when the traditional cosinor method was used, as compared with only 1 out of 8 when using our model. In all 8 rats the regression was highly significant and also useful as defined by the ? m criterion. The results from the model were in agreement with literature regarding constant routine studies in humans. The mean amplitude of the endogenous rhythm was 0.24°C in young rats and 0.19°C in old rats, whereas the amplitudes of the overt rhythm were 0.38 and 0.26°C, respectively. The age-related differences in the amplitude of the overt circadian temperature rhythm could to a large extent be attributed to age-related differences in activity-induced heat production. Finally, the acrophase of the endogenous rhythm occurred 18.7 minutes later than that of the overt rhythm. If applicable to human, the proposed method may form a valuable extension to existing constant routine protocols for studying the endogenous circadian rhythm in body temperature.     

Analysis of simulated and experimental fluorescence recovery after photobleaching. Data for two diffusing components.

Fluorescence recovery after photobleaching has been a popular technique to quantify the lateral mobility of membrane components. A variety of analysis methods have been used to determine the lateral diffusional mobility, D. However, many of these methods suffer from the drawbacks that they are not able to discern two-component diffusion (i.e., three-point fit), cannot solve for two components (linearization procedures), and do not perform well at low signal-to-noise. To overcome these limitations, we have adopted the approach of fitting fluorescence recovery after photobleaching curves by the full series solution using a Marquardt algorithm. Using simulated data of one or two diffusing components, determinations of the accuracy and reliability of the method with regard to extraction of diffusion parameters and the differentiation of one- versus two-component recovery curves were made under a variety of conditions comparable with those found in actual experimental situations. The performance of the method was also examined in experiments on artificial liposomes and fibroblast membranes labeled with fluorescent lipid and/or protein components. Our results indicate that: 1) the method was capable of extracting one- and two-component D values over a large range of conditions; 2) the D of a one-component recovery can be measured to within 10% with a small signal (100 prebleach photon counts per channel); 3) a two-component recovery requires more than 100-fold greater signal level than a one-component recovery for the same error; and 4) for two-component fits, multiple recovery curves may be needed to provide adequate signal to achieve the desired level of confidence in the fitted parameters and in the differentiation of one- and two-component diffusion.     

The effect of sleep upon human circadian rhythms

Subjects who slept for 4 h from 0000, and for a second 4 h variously distributed over the day, have provided values for rectal temperature and for urinary excretion of water, potassium, sodium, chloride, phosphate, creatinine, calcium and urate in the sleeping subject at all hours of the 24. These are compared with similar values in the wakeful subject. Temperature was lower during sleep at all hours except 1000 and 1200, and the difference was maximal shortly before 0000. At all hours potassium excretion was lower and phosphate excretion higher during sleep. Cosinor analysis of the different variables in the sleeping subject is compared with that in subjects following nycthemeral habits, and the interaction between endogenous rhythms and external influences such as sleep is discussed. The phasing of the temperature and urinary rhythms was essentially normal by the end of the observations. By contrast in a subject who slept at irregular hours mimicking the habits of an air pilot a free-running rhythm unrelated to the habits of sleep emerged. When he was finally living again on normal time his temperature and urinary acrophases had moved to the middle of the night. Phosphate excretion was largely exogenous, falling consistently when subjects rose after 8 h, but not after 4 h of sleep.     

The Adjustment of the Circadian Rhythm of Body Temperature to Simulated Time-Zone Transitions: A Comparison of the Effect of Using Raw Versus Unmasked Data

Deep body temperature and sleep/activity diaries data were recorded during control days and for 6 days after simulated time zone transitions of 8 h to the east (six subjects) or west (seven subjects). Circadian rhythms were assessed by cosinor analysis of both raw data (the conventional method) and purified data (corrected for the effects of sleep and activity). Analysis of raw data gives misleading information about the phase and amplitude of the rhythms due to the masking effects of the exogenous component. Use of purified data indicates that during the process of adjustment after an eastward shift (a) phase changes are more erratic than after a shift to the west; (b) no marked decrease in the amplitude of the rhythms is evident; and (c) no clear evidence exists that the circadian rhythm breaks up temporarily. The masking effect was less after the time zone transition if sleep maintenance was poor.     

The Adjustment of the Circadian Rhythm of Body Temperature to Simulated Time-Zone Transitions: A Comparison of the Effect of Using Raw Versus Unmasked Data

Deep body temperature and sleep/activity diaries data were recorded during control days and for 6 days after simulated time zone transitions of 8 h to the east (six subjects) or west (seven subjects). Circadian rhythms were assessed by cosinor analysis of both raw data (the conventional method) and purified data (corrected for the effects of sleep and activity). Analysis of raw data gives misleading information about the phase and amplitude of the rhythms due to the masking effects of the exogenous component. Use of purified data indicates that during the process of adjustment after an eastward shift (a) phase changes are more erratic than after a shift to the west; (b) no marked decrease in the amplitude of the rhythms is evident; and (c) no clear evidence exists that the circadian rhythm breaks up temporarily. The masking effect was less after the time zone transition if sleep maintenance was poor.     

Sleep and Circadian Rhythms of Temperature and Urinary Excretion on a 22.8 hr “Day”

Two groups of subjects (total N = 6) were studied in an isolation chamber for a period of 3 weeks whilst living on a 22.8 hr “day”. Regular samples of urine were taken when the subjects were awake, deep body temperature was recorded continuously and polygraphic EEG recordings were made of alternate sleeps. The excretion in the urine of potassium, sodium, phosphate, calcium and a metabolite of melatonin were estimated.

Measurements of the quantity and quality of sleep were made together with assessments of the temperature profiles associated with sleep. In addition, cosinor analysis of circadian rhythmicity in urinary variables and temperature was performed.

The 22.8 hr “days” affected variables and subjects differently. These differences were interpreted as indicating that the endogenous component of half the subjects adjusted to the 22.8 hr “days” but that, for the other three, adjustment did not occur. When the behaviour of different variables was considered then some (including urinary potassium and melatonin, sleep length and REM sleep) appeared to possess a larger endogenous component than others (for example, urinary sodium, phosphate and calcium), with rectal temperature behaving in an intermediate manner. In addition, a comparison between different rhythms in any subject enabled inferences to be drawn regarding any links (or lack of them) that might exist between the rhythms. In this respect also, there was a considerable range in the results and no links between any of the rhythms appeared to exist in the group of subjects as a whole.

Two further groups (total N=8) were treated similarly except that the chamber clock ran at the correct rate. In these subjects, circadian rhythms of urinary excretion and deep body temperature (sleep stages and urinary melatonin were not measured) gave no evidence for deterioration. We conclude, therefore, that the results on the 22.8 hr “day” were directly due to the abnormal “day” length rather than to a prolonged stay in the isolation chamber.     

Lack of evidence that feedback from lifestyle alters the amplitude of the circadian pacemaker in humans

Two groups of healthy subjects were studied indoors, first while living normally for 8 days (control section) and then for 18 × 27h “days” (experimental section). This schedule forces the endogenous (body clock-driven) and exogenous (lifestyle-driven) components of circadian rhythms to run independently. Rectal temperature and wrist movement were measured throughout and used as markers of the amplitude of the circadian rhythm, with the rectal temperature also “purified” by means of the activity record to give information about the endogenous oscillator. Results showed that, during the experimental days, there were changes in the amplitude of the overt temperature rhythm and in the relative amounts of out-of-bed and in-bed activity, both of which indicated an interaction between endogenous and exogenous components of the rhythm. However, the amplitude and the amount of overlap were not significantly different on the control days (when endogenous and exogenous components remained synchronized) and those experimental days when endogenous and exogenous components were only transiently synchronized; also, the amplitudes of purified temperature rhythms did not change significantly during the experimental days in spite of changes in the relationship between the endogenous and exogenous components. Neither result offers support for the view that the exogenous rhythm alters the amplitude of oscillation of the endogenous circadian oscillator in humans.     

The statistical analysis of circadian phase and amplitude in constant-routine core-temperature data.

Accurate estimation of the phases and amplitude of the endogenous circadian pacemaker from constant-routine core-temperature series is crucial for making inferences about the properties of the human biological clock from data collected under this protocol. This paper presents a set of statistical methods based on a harmonic-regression-plus-correlated-noise model for estimating the phases and the amplitude of the endogenous circadian pacemaker from constant-routine core-temperature data. The methods include a Bayesian Monte Carlo procedure for computing the uncertainty in these circadian functions. We illustrate the techniques with a detailed study of a single subject's core-temperature series and describe their relationship to other statistical methods for circadian data analysis. In our laboratory, these methods have been successfully used to analyze more than 300 constant routines and provide a highly reliable means of extracting phase and amplitude information from core-temperature data.