Delays of 5–15 min between awakening and the start of saliva sampling matter in assessment of the cortisol awakening response

Summary

Linking psychosocial measures to the cortisol awakening response (CAR) demands accurate saliva sampling times. Monitoring adherence to the saliva sampling protocol requires electronic monitoring of both awakening and sampling times since self-reported times are inaccurate. Delays greater than 15 min between awakening and commencement of saliva sampling reduce CAR magnitude. Less delay has been judged tolerable but remains unexplored for different magnitude measures, and for timing of the CAR peak.

Study 1: Fifty healthy females (21 ± 4 years) were instructed to collect saliva on four days at 0, 15, 30 and 45 min post-awakening (samples 1–4). Both self-reported awakening and sampling times were electronically monitored using actigraphy and track caps. Self-reported awakening was later than actigraph estimated awakening (median difference of 4 min). Estimates of CAR magnitude were significantly greater on non-adherent days (delay of 5–15 min) compared to adherent days (delay < 5 min). On non-adherent compared to adherent days cortisol on average peaked earlier, at sample 3 rather than at sample 4.

Study 2: Accurately timed cortisol values were obtained in an intensive investigation of 10 participants who collected saliva on 2 days every 5 min for 30 min post-awakening. Cortisol did not significantly increase until 10 min post-awakening, suggesting a time lag may be typical between awakening and observation of a cortisol increase.

We conclude that moderate delays between awakening and collection of saliva samples previously considered tolerable result in erroneous estimation of CAR magnitude and timing of the peak. These results are attributed to an approximate 10 min time lag between awakening and the start of the cortisol rise. The absence of this latent period in calculations leads to overestimation of the CAR magnitude on moderately non-adherent sampling days. These findings, if more universally generalizable, will further theoretical understanding of the physiology of the CAR, but are methodologically challenging for researchers since self-reported awakening times are not accurate enough to override the concerns raised. However accurate electronic measurement of adherence to protocol would enable sampling delays to be taken into account in computing CAR estimates.

Introduction

The cortisol awakening response (CAR) is a response to morning awakening (Wilhelm et al., 2007) and has become a much used biomarker of psychosocial variables and health (Clow et al., 2004, Fries et al., 2009). Determination of the CAR typically requires self-collection of saliva samples within the domestic setting at regular intervals post-awakening for between 30 and 60 min (e.g. 0, 15, 30 and 45 min post-awakening) and the peak of secretion characteristically occurs between 30 and 45 min post-awakening. CAR studies in the domestic setting usually rely upon unsupervised participant adherence to saliva sampling protocols during the immediate post-awakening period. This time period is associated with the presence of sleep inertia which is a state of reduced cognitive and motor performance (Tassi and Muzet, 2000) and is likely to increase the difficulty of adhering to protocol requested timings, even in well-intentioned participants. The dynamic and brief nature of the CAR makes it likely that errors in the timing of saliva sampling will impact upon its accurate measurement. Until recently, quantification of the CAR has almost exclusively focused upon its magnitude (e.g. mean increase [MnInc]) measures, or their near equivalent of areas under the curve with respect to increase [AUCi]. More economically, some have taken simple differences between awakening level and a single later measure (typically at 30 min). However the timing of the CAR peak is also an important variable. For example it has been shown to be related to gender, with males peaking earlier than females (Pruessner et al., 1997); menstrual cycle, with females in the ovulation phase peaking later (Wolfram et al., 2011); hormonal status, with pre-menarche females peaking earlier than menarche females (Oskis et al., 2009); and individuals with better cognitive function peaking earlier (Evans et al., 2012). This often overlooked CAR characteristic is likely to be increasingly studied and adds to the CAR's value as a biomarker. Although in recent years participant non-adherence to protocol has received increasing attention with respect to CAR magnitude, there has been no investigation of this issue with respect to the timing of the CAR peak, and even with respect to CAR magnitude there have been no comparative studies of delayed sampling on the different commonly used quantifications mentioned above.

Studies measuring the CAR in a domestic setting typically monitor participant adherence using self-report diary methods. However, sampling times can be electronically-monitored by track caps (e.g. medication event monitoring: MEM caps). Studies which have monitored sampling times using these devices have shown that real timings are later than participants’ self-reports (Kudielka et al., 2003, Broderick et al., 2004) with delays of more than about 10–15 min associated with a CAR of smaller magnitude (Kudielka et al., 2003, Broderick et al., 2004). True CAR estimates are also dependent upon accurate determination of awakening times which can be monitored electronically by electroencephalography, polysomnography and wrist-worn actigraphy. The latter can be used within the domestic setting and allows estimation of awakening time with reference to increased activity associated with awakening. Actigraphy has been validated against polysomnography (Lichstein et al., 2006) and is widely used in non-clinical and clinical studies (Lauderdale et al., 2006). Research using these devices suggests that participants are inaccurate in reporting awakening times resulting in a delay in collecting the ‘awakening’ sample (Kupper et al., 2005, Dockray et al., 2008, DeSantis et al., 2010) and this results in attenuated CAR magnitude. For instance, studies that have estimated awakening time by actigraph or polysomnography have reported that delays over 15 min lead to higher awakening samples and flatter CARs of smaller magnitude (Dockray et al., 2008, DeSantis et al., 2010, Okun et al., 2010). These studies did not electronically monitor sampling times, but more recently Griefahn and Robens (2011) electronically monitored awakening and sampling times and reported that delays of 11.5 min resulted in attenuated CAR magnitude.

Pooling the evidence, it seems to be broadly accepted that non-adherence of more than 15 min leads to under-estimation of CAR magnitude, i.e. apparently higher first samples and reduced CARs. By default these studies also imply that sampling delays of up to 10–15 min are tolerable and insignificant in this line of research, but studies have not directly tested this; no studies have examined the impact of virtually no delay (i.e. less than 5 min) compared to moderate delays (i.e. between 5 and 15 min). Delays of under 15 min being tolerable is a convenient assumption as it simplifies data collection processes which can be demanding in terms of resources; however it is also theoretically extremely puzzling. It is commonly assumed that the moment of awakening initiates the CAR (see Wilhelm et al., 2007, Clow et al., 2010) and that the rise in cortisol secretion is linear from awakening to 15–30 min later. If so, even delays of less than 15 min post-awakening should generate CARs of reduced magnitude as the first sample would be higher than it should be since a proportion of the CAR will have happened before sampling and thus be invisible to the researchers. The conundrum may be resolved, however, if awakening sets off a chain of events that normally takes between 5 and 15 min to manifest as increased cortisol concentrations in saliva (a latent period). This would mitigate any reduction in the CAR magnitude due to such moderate sampling delays. However an implication of this scenario may be that CAR magnitude is over-estimated in the presence of these more moderate delays. Such over-estimation would be a consequence of the real-time CAR-assessment period being shifted just sufficiently along the time axis to maximise the average level of cortisol measured in subsequent samples relative to the first sample but not being shifted sufficiently for that average to be influenced unduly by significantly lower post-peak values (unlike for delays > 15 min). Such a scenario would also impact on the apparent timing of the CAR peak, which would occur up to 15 min earlier than if the first sample was actually collected at the moment of awakening. Studies typically investigate the CAR in saliva samples collected at 15 min intervals with the assumption that cortisol rises linearly between sample points; however to our knowledge this assumption has not been investigated directly. In view of a burgeoning literature addressing associations between the CAR and psychosocial and health domains, what is or is not ‘tolerable’ measurement error is something in need of urgent investigation.

In summary, we monitored non-adherence to the saliva sampling protocol electronically and sought to investigate the impact of previously thought tolerable delays of up to 15 min on the CAR magnitude. We hypothesised that moderate non-adherence (delays of 5–15 min between awakening and commencement of saliva sampling) in comparison to adherence (defined as less than 5 min delay) would be associated with earlier timing of the CAR peak. Following the predictive speculation outlined above, we also explored effects of the same moderate delay interval on different measures of CAR magnitude. To extend the results of study one, study two explored directly whether there was a predicted time lag between awakening and the post-awakening rise in cortisol secretion in an intensive study utilizing 5 min intervals between post-awakening samples.