Determinants of the diurnal course of salivary alpha-amylase

Summary

Objective

Previous data from our group and others have shown that salivary alpha-amylase activity increases in response to stress. It has been suggested that salivary alpha-amylase may be a marker for adrenergic activity. Less is known about other determinants of salivary alpha-amylase activation. The objective of the current study was to describe the diurnal pattern of salivary amylase and its determinants.

Methods

Saliva samples were collected immediately after waking-up, 30 and 60 min later, and each full hour between 0900 and 2000 h by 76 healthy volunteers (44 women, 32 men). Compliance was controlled by electronic monitors. In order to control factors which might influence the diurnal profile of salivary alpha-amylase (such as momentary stress, mood, food, or body activity), at each sampling time point the subjects filled out a diary examining the activities they had carried out during the previous hour.

Results

Salivary alpha-amylase activity shows a distinct diurnal profile pattern with a pronounced decrease within 60 min after awakening and a steady increase of activity during the course of the day. Mixed models showed a relative independence of diurnal salivary alpha-amylase from momentary stress and other factors, but significant associations with chronic stress and mood.

Conclusions

Our results suggest that diurnal profiles of salivary alpha-amylase are relatively robust against momentary influences and therefore may prove useful in the assessment of sympathetic nervous system activity. The findings underscore the need to control for time of day in studies using salivary alpha-amylase as a dependent variable.

Introduction

Daily oscillations characterize the secretion of almost all endogenous substances (Mistlberger and Skene, 2004). These oscillations are driven by a circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus. However, this biological clock is not the only factor responsible for regular fluctuations. The orchestrated action of different zeitgebers leads to specific release patterns of endogenous substances during the sleep–wake cycle. Among the most important zeitgebers are exposure to light, changing of time zones, and shift work. In addition, factors like age, sex, emotions, and stress further influence biological rhythms significantly. The knowledge of not only the time course of the naturally occurring release of a given substance but also of the factors that may alter this course is of outmost importance.

In recent years, components of saliva have gained increasing interest as indicators of bodily changes following stress. Alpha-amylase is one of the principal salivary proteins. It is a calcium-containing metalloenzyme that hydrolyzes the α1,4 linkages of starch to glucose and maltose (Zakowski and Bruns, 1985). Since salivary alpha-amylase secretion is regulated by the sympathetic nervous system which stimulates acinar cells of the salivary glands via beta-adrenergic receptors (Baum, 1987), the measurement of salivary alpha-amylase activity has been proposed to reflect stress-related changes in the autonomic nervous system (Chatterton et al., 1996; Ehlert et al., 2006; van Stegeren et al., 2006). Studies using physiological (Nexo et al., 1988; Walsh et al., 1999; Kivlighan and Granger, 2006) and psychological stressors (Chatterton et al., 1997; Bosch et al., 2003; Kivlighan and Granger, 2006; Tu et al., 2006) have found increased activity of salivary alpha-amylase due to stress. Studies from our laboratories show that salivary alpha-amylase activity might be considered a readily accessible non-invasive parameter to evaluate psychological stress responses (Rohleder et al., 2004; Nater et al., 2005; Nater et al., 2006a; Nater et al., 2006b; Rohleder et al., 2006). Besides other salivary parameters such as cortisol (Kirschbaum and Hellhammer, 1994), concomitant measurement of salivary alpha-amylase activity seems to be a unique possibility to complement a more comprehensive evaluation of stress responses. However, whereas cortisol is known to show a distinct circadian release pattern (Kirschbaum and Hellhammer, 1989), not much is known about the circadian oscillations of salivary alpha-amylase activity. There are a number of studies reporting that both salivary flow rate and saliva composition vary rhythmically over a 24-h period (Dawes, 1974). In a review of previous studies Ferguson et al. reported low values of salivary alpha-amylase in the morning and high values in the afternoon (Ferguson et al., 1973). In a more recent study, stimulated whole saliva was collected by healthy volunteers. Samples were obtained at 0800, 1100, 1400, and 1700 h on two different days. For salivary alpha-amylase activity, significant changes were observed over the day, with a peak in the early evening and a trough in the morning at 0800 h (Jenzano et al., 1987). Another study investigated unstimulated and stimulated whole saliva collected 5 times during a 12-h period in the laboratory. Salivary alpha-amylase activity varied significantly within the subjects during the collection period, with lowest values found in the morning (Rantonen and Meurman, 2000). In yet another study, two groups (patients with Diabetes mellitus and healthy controls) were examined with respect to diurnal rhythm of salivary alpha-amylase activity. Unstimulated whole saliva was collected in the morning (0730–0800h), and in the early evening (1730–1800h). Peaks of salivary alpha-amylase activity were found at the latter time point. However, the two groups did not differ from each other (Artino et al., 1998). A recent study, however, found that there were no distinct changes in salivary alpha-amylase activity over the course of the day at all (Yamaguchi et al., 2006). These discrepant findings might be explained by the fact that most of these studies took place in a laboratory environment. Furthermore, salivary samplings were either irregular or too spaced throughout the day. While these studies were predominantly focused on determining the diurnal course of salivary alpha-amylase, confounding factors have not been taken into account. Potential confounding factors are: (1) those that activate the sympathetic nervous system, such as exercise and stress; (2) those that are generally associated with saliva production, such as eating and drinking; and (3) those that are known to influence other stress systems, such as gender, age, sleep, or time of awakening.

In order to use salivary alpha-amylase as a biological measure in stress research, it is crucial to establish its diurnal pattern, as well as the determinants of this pattern. This information will enable researchers to choose the correct time of day for acute stress studies, and to choose an appropriate sampling strategy in studies investigating chronic psychosocial conditions.

We therefore carried out a field study in which the endogenous rhythm of salivary alpha-amylase release was unaffected by a laboratory or hospital setting. Thus, our healthy volunteers were asked not to change their habits during the day of the study. In order to get a precise diurnal profile, samples of saliva were collected hourly. Moreover, we took various external and internal factors into account that could influence salivary alpha-amylase activity (e.g., emotion, food, physical activity or personality). We additionally measured possible confounding variables, such as sex, smoking, and menstrual cycle phase. Salivary cortisol levels were obtained from the same samples for a comparison of the diurnal course of both salivary alpha-amylase and cortisol.