Psychoneuroendocrinology (2009) 34, 486—496

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / p s y n e u e n

REVIEW

Salivary alpha-amylase as a non-invasive biomarker

for the sympathetic nervous system: Current state
of research
U.M. Nater a,*, N. Rohleder b

a
University of Zurich, Institute of Psychology, Department of Clinical Psychology and Psychotherapy, Zurich, Switzerland
b
Brandeis University, Dept. of Psychology, Waltham, MA, USA

Received 13 August 2008; received in revised form 23 January 2009; accepted 27 January 2009

KEYWORDS Summary Development of new biomarkers is a constantly evolving field of research endeavor
Alpha-amylase; in psychoneuroendocrinology. Salivary biomarkers have received special attention since they are
Stress; readily accessible and easily obtained. Salivary alpha-amylase (sAA) has been proposed as a
Biomarker; sensitive biomarker for stress-related changes in the body that reflect the activity of the
Sympathetic nervous sympathetic nervous system (SNS), and a growing body of research is accumulating to support
system the validity and reliability of this parameter. However, questions remain to be answered before
sAA can be accepted as an index of SNS activity. This review describes sAA as an emerging
biomarker for stress and provides an overview of the current literature on stress-related
alterations in sAA. It critically discusses how sAA might reflect changes in the autonomic nervous
system. Finally, current and future fields for the application of sAA measurement are outlined.
# 2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
2. What is salivary alpha-amylase?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
2.1. Anatomy and physiology of the salivary glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
2.1.1. Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
2.1.2. Secretory pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
2.2. Pathways of alpha-amylase secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
2.2.1. Rat studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488
2.2.2. Human studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
2.2.3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

* Corresponding author at: University of Zurich, Clinical Psychology & Psychotherapy, Binzmuehlestr. 14/Box 26, 8050 Zurich, Switzerland.
Tel.: +41 44 635 7382; fax: +41 44 635 7359.
E-mail address: u.nater@psychologie.uzh.ch (U.M. Nater).

0306-4530/$ — see front matter # 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.psyneuen.2009.01.014
Salivary alpha-amylase as a biomarker for the SNS 487

3. Stress-induced salivary alpha-amylase secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

3.1. Early findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489
3.2. Effects of psychological stress on salivary alpha-amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490
3.3. Effects of exercise on salivary alpha-amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
3.4. Is salivary alpha-amylase an indicator for the sympathetic nervous system? . . . . . . . . . . . . . . . . . . . . 491
3.5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
4. Applications of salivary alpha-amylase measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
4.1. Salivary alpha-amylase as an indicator of autonomic dysregulation . . . . . . . . . . . . . . . . . . . . . . . . . 492
4.2. Measurement of salivary alpha-amylase in treatment studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493

1. Introduction which one set is glycosylated and the other contains no

In recent years, salivary measures have become increasingly
important in psychoneuroendocrinological research. In the
early 1970s, Brown suggested that changes in saliva para-
meters could be regarded as an ‘‘index of specific states of
psychopathology’’ (Brown, 1970, p. 66). While research is not
yet quite at the point to use salivary measures in the way
Brown suggested, the scientific understanding of various
salivary parameters is progressing rapidly. However, apart
from the analysis of hormones such as cortisol and DHEA (see,
e.g. Vining and McGinley, 1987; see, e.g. Kirschbaum and
Hellhammer, 1994), few other salivary components have
been taken into consideration as meaningful physiological
markers in psychoneuroendocrinological research. A specific
focus of interest within this field of research is on stress. In
stress research, subjects might be examined either in the
field or in the laboratory. Whichever setting is chosen, valid
and reliable measures of changes associated with stress must
be applied. Moreover, the simple handling and easy sampling
of a stress measure is of utmost importance. A wide array of
possible parameters indicating stress-related changes has
been proposed over the years. Some have disappeared into
oblivion; others have endured for decades and are still being
used. Since stress is a multi-faceted phenomenon, it requires
a multidimensional measurement approach. As a conse-
quence, research can gain from additions to the canon of
psychobiological parameters. One parameter that has been
suggested to reflect stress-related changes in the body is the
salivary enzyme alpha-amylase (Chatterton et al., 1996;
Nater, 2004; Rohleder et al., 2004; Granger et al., 2007).
Salivary alpha-amylase (sAA) release is known to be elicited
by activation of the autonomic nervous system (ANS) which
controls the salivary glands.
The aim of this review is to assess whether sAA should be
incorporated in the canon of psychoneuroendocrinological
parameters measuring stress. The accompanying review by
Rohleder and Nater (2009) provides a detailed account of
methodological issues that arise when measuring sAA.
2. What is salivary alpha-amylase?
sAA (a-1,4-a-D-glucan 4-glucanohydrolase; EC 3.2.1.1) is one
of the most important enzymes in saliva. The enzyme was
first described in saliva by Leuchs in 1831 (Zakowski and
Bruns, 1985). It consists of two families of isoenzymes, of
carbohydrate. The molecular weight of the glycosylated form
is about 57,000; that of the non-glycosylated form is about
54,000. sAA accounts for 40% to 50% of the total salivary
gland-produced protein, most of the enzyme being synthe-
sized in the parotid gland (80% of the total) (Zakowski and
Bruns, 1985; Makinen, 1989). It is a calcium-containing
metalloenzyme that hydrolyzes the a-1,4 linkages of starch
to glucose and maltose. It is known to be mainly involved in
the initiation of the digestion of starch in the oral cavity.
However, sAA has also been shown to have an important
bacterial interactive function (Scannapieco et al., 1993).
2.1. Anatomy and physiology of the salivary
glands
In this section, we review the salivary glands and their major
product, saliva. A clear understanding of how and where
saliva is produced and how this process is controlled is
essential for gaining insight into the relationships between
salivary parameters and stress.
There are three major salivary glands on each side of the
face: the parotid, submandibular, and sublingual glands. In
addition, numerous minor glands contribute to salivary out-
flow. The salivary glands are part of the digestive tract.
Glands are comprised of different types of cells: acinar cells,
various duct system cells, and myoepithelial cells (Humphrey
and Williamson, 2001).
2.1.1. Saliva
The components of saliva are produced primarily by acinar
cells. Saliva is the main product of the salivary glands. It is a
clear, slightly acidic mucoserous exocrine fluid comprising a
complex mixture of secretions from major and minor salivary
glands and gingival crevicular fluid (Humphrey and Williamson,
2001; Kaufman and Lamster, 2002). This mixture of fluids
derived from different glands is called ‘‘whole saliva’’,
whereas the fluid secreted by single glands is called ‘‘duct
saliva’’ (Edgar, 1992). The constant flow of saliva from the
mouth into the gut has a protective function. This flushing
effect transfers, for example, food debris and exogenous and
possibly noxious agents into the gut (Tenovuo, 1998). Saliva is
routinely categorized as resting (unstimulated) or stimulated.
Under unstimulated conditions, 20% of saliva is derived from
the parotid, 65% from the submandibular, and 7—8% from the
sublingual glands. The minor glands contribute about 10% to
whole saliva. Under stimulation, the contributions of each
488
gland change, with the parotid contributing more than 50% of
total salivary secretions (Sreebny, 2000; Humphrey and Wil-
liamson, 2001).
2.1.2. Secretory pathways
For a detailed understanding of the secretory pathways of the
salivary glands, a distinction must be drawn between neural
and cellular mechanisms of secretion. This section provides a
detailed account of these pathways.
Acinar cells are innervated by both the sympathetic and
the parasympathetic branches of the ANS (Emmelin, 1987).
Autonomic nerves are adjacent to both acinar and ductal
cells. The afferent pathways for taste are via the facial and
glossopharyngeal nerves to a solitary nucleus in the medulla.
There is also input from higher centers in response to smell,
sight, etc. The parasympathetic efferent pathways for the
sublingual and submandibular glands are from the facial
nerve via the submandibular ganglion; for the parotid gland
they are from the glossopharyngeal nerve via the otic gang-
lion. The sympathetic postganglionic pathways are from the
cervical ganglion of the sympathetic chain. Neurotransmit-
ters are the first messengers in the communication pathway
between nerves and secretion. Neurotransmitters exert their
activity at the cell membrane; they communicate with intra-
cellular second messengers that have direct control of secre-
tory processes (Smith, 1996). Released in response to
secretory stimuli, they bind to specific receptor proteins
on the basolateral membrane, causing acute elevation of
intracellular Ca. This results in large-scale fluid and electro-
lyte transport, and modest exocytosis of stored protein.
Norepinephrine from sympathetic neurons binds to both
alpha- and beta-adrenergic receptors on the acinar cell.
Alpha-receptor activation is linked to elevation of intracel-
lular Ca, while beta-receptor activation causes elevation of
intracellular cyclic adenosine monophosphate (cAMP), which
is linked to the secretion of salivary proteins that are stored
in membrane-bound secretory granules (Baum, 1993; Castle
and Castle, 1998).
The secretory process of salivary proteins may be divided
into the three stages: (1) synthesis, (2) packaging and sto-
rage, and (3) release. Each of these stages is regulated by
phosphorylation of target proteins brought about by a protein
kinase such as cyclic AMP-dependent protein kinase (protein
kinase A) (Smith, 1996).
2.2. Pathways of alpha-amylase secretion
In acinar cells, release of salivary components is under
control of neuronal stimuli. Classic neurotransmitters and
specific bioactive peptides serve as the main stimuli for sAA
secretion. This section presents studies in rats and humans
that used blocking or stimulating pharmacological agents or
electrical stimuli to gain a better understanding of sAA
secretion.
2.2.1. Rat studies
The findings of Batzri and co-workers are among the first to
indicate the specific involvement of the ANS in the secretion
process of sAA. In studies on slices of the parotid gland in
rats, they found that beta-adrenergic receptors caused
secretion of sAA (Batzri and Selinger, 1973; Batzri et al.,
U.M. Nater, N. Rohleder
1973; Selinger et al., 1973). In a subsequent study, Anderson
et al. (1984) examined differential contributions of the two
branches of the ANS to sAA secretion. They found that
sympathetic stimulation in unconscious rats led to the secre-
tion of parotid saliva characterized by low salivary flow rate
and high total protein and amylase concentrations. In con-
trast, parasympathetic stimulation induced a rich flow of
saliva with low protein content, with mean concentrations
of sAA approximately 1% of those measured in sympatheti-
cally stimulated saliva. After performing adrenalectomy,
the authors observed significant reductions of sAA levels
upon parasympathetic stimulation of the parotid gland,
suggesting that circulating catecholamines (originating from
the adrenals) might play a specific role in sAA secretion.
Asking (1985) set out to compare sympathetic and parasym-
pathetic stimulation of the parotid gland in the rat both
separately and concomitantly. As in the Anderson et al.
study, sympathetic stimulation caused a low flow of saliva
containing amylase in very high concentration, whereas
parasympathetic stimulation produced saliva with a low
concentration of amylase. After combined sympathetic
and parasympathetic stimulation, however, sAA secretion
was much higher than the sum of the two separate stimula-
tion concentrations. Using the beta-1-antagonist pafenolol,
Asking (1985) showed that the higher sAA secretion due to
sympathetic stimulation superimposed on parasympathetic
background stimulation was elicited via beta-1-adrenocep-
tors. In a subsequent study (Asking and Gjorstrup, 1987),
these findings were replicated in that sympathetic and
parasympathetic stimulation led to equal sAA concentra-
tions and the combination of the two drastically increased
sAA concentrations. The authors found that sAA secreted on
sympathetic activation consisted of pre-formed amylase,
stored in granules, that was not replaced during secretory
activity under these conditions. Ongoing sympathetic sti-
mulation led to an attenuation of sAA concentration. Asking
and Proctor (1989) studied the effects of prolonged para-
sympathetic nerve stimulation in rat parotid glands on
alpha-amylase content in saliva and glands. The concentra-
tion and total output of sAA were measured throughout a
stimulatory period of 120 min, as were glandular concentra-
tions of sAA. The results suggest that amylase stores are
much more rapidly replenished by synthesis during para-
sympathetic than during sympathetic activity, whereas sym-
pathetic nerve excitation causes a pronounced loss of
amylase-containing acinar granules. No such loss could be
detected on parasympathetic stimulation, although the out-
put of amylase was about the same as during sympathetic
stimulation.
In a further examination of the adrenergic mechanisms
involved in sAA secretion, Skov Olsen et al. (1988) found that
stimulation of the beta-adrenergic receptors in rats
increased the concentration of sAA by a factor of 30, while
stimulation of the alpha-adrenergic receptors increased the
concentration of sAA by a factor of 10. Experiments with
several substances showed that sAA secretion was mainly
contributed by beta-1-adrenergic mechanisms. Following up
on these studies, Busch et al. set out to investigate the
differences in release of sAA by the parotid and the sub-
mandibular gland in rats. They found that submandibular sAA
did not respond with an increase to the administration of
isoproterenol, a beta-adrenergic agonist, whereas parotid
Salivary alpha-amylase as a biomarker for the SNS
sAA did. This effect was inhibited by the selective beta-1-
antagonist atenolol, but not by the beta-2-antagonist butox-
amine (Busch et al., 2002).
In sum, these findings demonstrate the involvement of the
ANS, and the sympathetic nervous system in particular, in the
release of sAA, with beta-adrenergic mechanisms as the main
contributing factor in sAA secretion. However, findings from
animal studies are not readily transferable to humans.
Furthermore, stimulation of the sympathetic nerves in iso-
lation, as used to elicit autonomic activation in the studies
described above, cannot be considered a physiological reflec-
tion of secretory processes in vivo. In humans, this approach
is not feasible. Use of pharmacological agents may be a more
appropriate methodological approach for determining the
role of the ANS in the secretion of sAA in humans, as the
following studies show.
2.2.2. Human studies
Speirs et al. (1974) provoked a sympathetic response either
by immersing subjects up to the waist in cold water (4—5 8C)
or by administering isoprenaline and propanolol (both are
beta-adrenergic blockers). Exposure to cold water and
isoprenaline raised sAA concentrations in the parotid gland,
whereas propranolol led to a reduction of sAA concentra-
tions. These results offered first evidence for the sympa-
thetic control of sAA secretion in humans (Speirs et al.,
1974). As shown in a number of the animal studies described
above, stimulation of beta-adrenergic receptors modulates
the synthesis and the release of sAA. Laurikainen et al.
(1988) studied the effects of timolol maleate, a widely used
beta-blocking agent, on the quantity and quality of saliva
secretion controlled by beta-adrenoceptors in healthy sub-
jects. Alpha-amylase concentrations in parotid saliva
decreased significantly after drug intake, thus corroborat-
ing similar findings from rat studies. Nederfors and co-
workers investigated the effects of therapeutic doses of
the selective beta-1-antagonist atenolol and the non-selec-
tive beta-antagonist propranolol on stimulated glandular
saliva. Atenolol, but not propranolol, resulted in a decrease
of parotid amylase in the morning, and in a decrease of
submandibular/sublingual amylase both in the morning and
at lunch time (Nederfors et al., 1994). The authors were
able to replicate their findings in a well-controlled study on
human hypertensive subjects with the selective beta-1-
antagonist metoprolol (Nederfors and Dahlof, 1996). Thus,
these studies demonstrate the importance of beta-adre-
nergic mechanisms of sAA secretion in humans, as already
shown in rats. Following up on these findings, van Stegeren
et al. (2006) conducted a placebo-controlled double-blind
study using propanolol in a stress—rest protocol. While the
placebo group showed a substantial increase in sAA subse-
quent to the stress test, the sAA response in the propanolol
group was attenuated. Finally, Ehlert et al. (2006) proposed
that sAA increases might reflect the interaction of stress-
dependent sympathetic and parasympathetic stimulation
via central nervous noradrenergic input. To examine this
hypothesis, they assessed the indirect effect of yohimbine
hydrochloride, an alpha-2-adrenergic receptor antagonist,
on sAA release in a randomized placebo-controlled study.
The results showed significant increases in sAA concentra-
tion in the yohimbine condition relative to the placebo
condition.
489
2.2.3. Summary
The results of studies on animals and humans indicate that
the ANS plays a powerful role in the secretion of sAA, with
contributions of both the alpha-adrenergic and the beta-
adrenergic mechanisms. These findings suggest that sAA
might be regarded as an indirect indicator of autonomic
activation.
3. Stress-induced salivary alpha-amylase
secretion
As outlined in the previous sections of this review, the
release of sAA is governed by activation of the ANS. Thus,
an increase in sAA may be expected during psychological
stress, when autonomic activation is high. The following
section reviews findings on sAA responses due to psycholo-
gical stress.
3.1. Early findings
The idea that sAA may serve as indicator of psychological
stress emerged in the late 1970s. To our knowledge, the
relationship between sAA and psychological stress was first
examined in a study by Gilman et al. (1979a), in which
subjects were exposed to eight days of hyperbaric pres-
sure. Findings showed increased concentrations of sAA.
The authors concluded that these increases were not only
due to the hyperbaric exposure and its effects on the ANS,
but also due to the psychological stress caused by the
procedure itself. In the 1980s, Donald Morse and his group
undertook a series of experiments with the goal of exam-
ining the impact of stress on various salivary parameters.
Their findings proved quite counterintuitive for research-
ers interested in salivary alpha-amylase: Morse et al. found
that relaxation, not stress, leads to increases in sAA (Morse
et al., 1983a,b). However, these studies were methodolo-
gically flawed in that there was no control condition or
random allocation to treatment groups. Furthermore, the
‘‘relaxation condition’’ always followed immediately after
the ‘‘stress condition,’’ suggesting that the increases in sAA
might be attributable to a stress response occurring after
the stressor. Since Morse concluded that salivary para-
meters were valid and reliable indicators for psychological
stress and relaxation, attempts were made to re-evaluate
this statement by comparing the usefulness of salivary
indicators with other known psychophysiological para-
meters (Borgeat et al., 1984). Borgeat et al. exposed
subjects to relaxation (Jacobson’s progressive muscle
relaxation) and to stress (written exercises, mental arith-
metic task). In addition to salivary parameters such as sAA,
they measured frontal muscle EMG, skin conductance,
heart rate, and skin temperature. The interventions
resulted in clear and distinct reaction patterns in the
‘‘classical’’ psychophysiological parameters, but no sali-
vary changes were observed. Thus, initial findings on
stress-related changes in sAA did not correspond with
the physiological literature outlined above. However,
further studies conducted under more rigorous conditions
and employing scientifically advanced designs, e.g. includ-
ing control groups etc., showed clear stress-related
increases in sAA, as summarized in the next section.
490
3.2. Effects of psychological stress on salivary
alpha-amylase
Whereas other stress-related factors in saliva, such as sali-
vary cortisol (Kirschbaum and Hellhammer, 1989, 1994),
received widespread scientific attention in the following
decade, interest in sAA as a stress marker was not sparked
again until Chatterton and colleagues published their findings
of increases in sAA in a variety of stressful conditions (Chat-
terton et al., 1996). Beyond this seminal study, a number of
studies applying psychological stress protocols have shown
that sAA is highly sensitive to stress-related changes. This
section briefly reviews these studies.
In a study of subjects exposed to an academic examina-
tion, Bosch et al. measured several salivary parameters
including sAA (Bosch et al., 1996). Whole unstimulated saliva
was taken 30 min before the examination, 2 weeks later, and
6 weeks later. Results indicated an increase in concentration
and output of sAA during the stress condition, while the
salivary flow rate did not change. In a separate analysis,
the authors found that the number and severity of critical life
events was related to sAA activity, thus suggesting that
everyday stress also contributes to stress-dependent changes
in salivary parameters (Bosch et al., 1998). Chatterton et al.
(1997) studied subjects preparing for skydiving and found
increased sAA prior to the jump than in control subjects who
did not jump. The highest levels were observed immediately
after landing. Using a stressful video game to induce labora-
tory stress, Skosnik et al. found a significant increase in sAA
after the 15-min stressor (Skosnik et al., 2000). In a further
study employing a laboratory task to induce acute stress
(Bosch et al., 2003), whole unstimulated saliva was measured
before, during, and after an active memory task, passive
watching of a gruesome video, and a control condition. sAA
output differed significantly between the three conditions,
with highest levels being measured during the passive video
task. A decrease in sAA output was found in the active
memory task, suggesting that the sAA response depends on
the nature of the stressor and the active or passive coping
capabilities of the subjects. Another research group used a
stressful and a relaxing video to induce stress and rest
conditions and directly compared the effects of both on
salivary cortisol and alpha-amylase responses in whole unsti-
mulated saliva (Takai et al., 2004, 2007). The stressful video
induced marked increases in both cortisol and sAA, whereas
the relaxing video produced no changes in salivary cortisol,
but significant decreases in sAA concentrations. Noto and
colleagues examined sAA levels during a mental arithmetic
task (Noto et al., 2005) and observed significant increases in
sAA.
sAA increases have also been reported in response to other
psychologically stressful conditions, such as experience of
medical procedures (Yamaguchi et al., 2006a), adverse musi-
cal stimuli in men (Nater et al., 2006a), mothers watching
their children being exposed to a stressful task (Granger
et al., 2006), the cold pressor test (West et al., 2006; van
Stegeren et al., 2008), achievement and interpersonal stress
(Stroud et al., 2006), a driving simulation (Yamaguchi et al.,
2006b), use of a noise burst and infant arm restraint in
depressive mothers (Shea et al., 2006), execution of neck/
face surgery by medical trainees (Yamakage et al., 2007), a
mental arithmetic task (Goi et al., 2007), oral academic
U.M. Nater, N. Rohleder
examination (Schoofs et al., 2007), a standardized test bat-
tery in toddlers (Fortunato et al., 2008), affective picture
viewing (van Stegeren et al., 2008), and in a peer rejection
paradigm in adolescents (Stroud et al., 2009). Interestingly,
only a few studies have found no changes in sAA in response to
stressful stimuli including noise (Morrison et al., 2003), the
heel prick test in neonates (Schaffer et al., 2008), or a
strange situation paradigm (Hill-Soderlund et al., 2008).
The scarcity of non-significant changes suggests that sAA is
indeed a highly sensitive parameter reflecting changes
caused by psychological stressors.
A number of studies have used a standardized psychosocial
stress test, i.e. the Trier Social Stress Test or TSST (Kirsch-
baum et al., 1993), to assess the usefulness of sAA as a
biomarker for psychologically induced stress. The TSST com-
prises a mock job interview and a mental arithmetic task,
both performed in front of an audience, leading to marked
increases in both psychological and physiological stress indi-
cators. In a pilot study, Rohleder et al. (2004) found marked
increases in sAA due to the TSST. These findings were corro-
borated in a subsequent study employing the TSST and a rest
condition (Nater et al., 2005) (see Fig. 1). The TSST led to
significant increases in sAA, while the rest condition did not
impact sAA levels.
A third study using the same study design again showed
marked increases in sAA due to the TSST (Nater et al., 2006b).
Other studies using the TSST and measuring sAA as a stress
marker have reported sAA increases in response to the TSST in
healthy children (Granger et al., 2006), healthy adults (Roh-
leder et al., 2006a,b; Grillon et al., 2007; Het and Wolf, 2007;
Payne et al., 2007; Schoofs et al., 2008), postpartum mothers
(Tu et al., 2006), and adolescents (Gordis et al., 2006, 2008).
Nierop et al. (2006) found that pregnant women showed
lower sAA responses than non-pregnant women when
exposed to the TSST.
In conclusion, the discrepancies between earlier and more
recent findings on the effects of stress on sAA may be
attributed to the different stressors and stress paradigms
used in the respective studies. Results have consistently
shown changes in sAA in response to psychological stress.
Figure 1 Salivary alpha-amylase activity in U/ml. Left: a
marked increase is observed in the stress condition. Right: no
significant changes occur in the rest condition (Nater et al.,
2005).
Salivary alpha-amylase as a biomarker for the SNS
Thus, sAA can be regarded as an excellent indicator of stress-
related body changes. Whereas the studies outlined above
measured changes in sAA in response to acute stress stimuli,
only a handful of studies have reported associations of
chronic stress and sAA. In one study, chronic stress was found
to be associated with diurnal profiles of sAA (Nater et al.,
2007). Subjects reporting relatively high chronic stress had
higher momentary sAA activity than subjects reporting low
chronic stress levels. Rohleder et al. recently showed that
daily sAA secretion is associated with self-reported shame
and depression in young women (Rohleder et al., 2008).
Another study reported higher basal values of sAA activity
in women exposed to Hurricane Katrina than in non-exposed
controls (Vigil et al., in press). Finally, Wolf and colleagues
found lower diurnal sAA in children with asthma than in
healthy controls (Wolf et al., 2008). The latter two studies
highlight the potential of measuring sAA as an index of
chronic stress in selected high-risk populations.
3.3. Effects of exercise on salivary alpha-
amylase
Another strong activator of the sympathetic nervous system
is physical activity. A variety of studies have examined the
effects of exercise on sAA. Gilman and co-workers observed a
significantly higher concentration of sAA during exercise than
in a control period. The authors discussed sAA as a valid
measure of sympathetic activity via adrenergic receptors
(Gilman et al., 1979b). Nexo and co-workers examined the
impact of a 2-h long cross-country race, collecting stimulated
whole saliva before and after the race. A marked increase in
sAA was found after the race; the median level increased
almost sevenfold compared to the beginning of the race
(Nexo et al., 1988). In two studies reported by Chatterton
et al. (1996), sAA was determined in subjects participating in
a running and a bicycle exercise task. Both protocols resulted
in increases of sAA. Another study investigated salivary
components before, during and 1 h after a 2-h marathon.
sAA activity increased significantly due to the strenuous
activity, and levels remained high 1 h after the marathon
(Ljungberg et al., 1997). In another study, sAA was used as an
indicator for the anaerobic threshold in subjects performing
a treadmill exercise test. Since the correlation between sAA
and the anaerobic threshold was almost 1 (r = 0.93), the
authors concluded that sAA was a good and valid measure
of the anaerobic threshold (Calvo et al., 1997). In a study of
triathletes, several salivary parameters in unstimulated
whole saliva were measured before and after a race consist-
ing of swimming, cycling, and running. Mean sAA activity
increased significantly during the triathlon (Steerenberg
et al., 1997). Another study examined eight well-trained
athletes during a high-performance 60-min cycle exercise
task. Unstimulated whole saliva was collected before exer-
cise, immediately after the task, and 1, 2.5, 5 and 24 h after
exercise. A significant increase in sAA activity was found after
exercise (Walsh et al., 1999). Similar results were obtained in
a more recent study (Bishop et al., 2006) examining the
influence of caffeine intake on sAA measures in an exercise
paradigm. The authors found that caffeine intake resulted in
an additional increase in sAA that was not observed in a
placebo condition. A study examining the impact of exercise
on a rowing ergometer on sAA levels showed increased levels
491
after exercise (Kivlighan and Granger, 2006). Interestingly,
sAA measures were positively associated with performance in
this study. Finally, in a study of exercise-induced sexual
arousal in women, exercise produced significant sAA
increases relative to a non-exercise condition (Hamilton
et al., 2008).
Taken together, a marked increase in sAA can be observed
during exercise. Exercise is known to increase sympathetic
activity, and the high protein level in saliva following exercise
may be due to increased adrenergic activity in the salivary
glands.
3.4. Is salivary alpha-amylase an indicator for
the sympathetic nervous system?
The evidence summarized above supports the view that sAA
may be a useful indicator for activity of the sympathetic
nervous system. Some studies have tested this hypothesis by
directly testing associations of sAA with various established
measures of sympathetic activity.
A series of studies with this specific aim was undertaken by
Chatterton and colleagues. In these studies, subjects were
exposed to physical (running, exercise, exposure to heat and
cold) and psychological (examination) stressors (Chatterton
et al., 1996). Increases observed in both sAA and plasma
catecholamines (norepinephrine and epinephrine) in
response to physical and (in some cases) psychological stres-
sors led Chatterton to conclude the two parameters might be
measured as substitutes for each other. Indeed, the authors
found significant correlations between sAA and both plasma
norepinephrine and epinephrine (r = 0.64 and r = 0.49,
respectively) in the exercise conditions of their study. The
authors therefore suggested that sAA might serve as an
indicator for plasma catecholamines (specifically, norepi-
nephrine). A close inspection of this study, however, shows
that only a small and non-significant correlation (r = 0.17)
was observed in the psychological stress condition (examina-
tion). The results therefore indicate that similar mechanisms
underlie increases in both catecholamines and sAA during
physiological stress experience. A pilot study using a stan-
dardized stress protocol reported correlations between
increases in sAA and plasma norepinephrine (r = 0.54) (Roh-
leder et al., 2004). In contrast, a study using the same stress
protocol in a bigger sample found no associations when the
plasma catecholamines norepinephrine and epinephrine
were measured concomitantly with sAA levels (Nater
et al., 2006b). Another study found increases in both plasma
norepinephrine and sAA after carbon dioxide inhalation, but
these two parameters did not correlate significantly (Wether-
ell et al., 2006). Thus, it seems doubtful that there is a 1:1
association between plasma catecholamines and sAA in the
body’s response to stressful conditions.
Further attempts have been made to relate sAA to other
indicators of sympathetic or parasympathetic activity. In a
study of subjects exposed to a stressful video showing a
surgical procedure or to a memory task, Bosch et al.
(2003) measured both sAA and various cardiovascular para-
meters. The authors found negative correlations between
left ventricular ejection time and sAA (in the video condition)
and between the square root of the mean squared differences
of normal-to-normal intervals (RMSSD, as an index of para-
sympathetic tone) and sAA during the memory task. These
492
findings suggest a predominant role of the sympathetic ner-
vous system in the secretion process of sAA, together with
vagal withdrawal, under psychological stress. They were
complemented by a study using a standardized psychosocial
stress protocol (Nater et al., 2006b), which reported a
positive correlation (r = 0.39) between sAA and the low
frequency/high frequency ratio thought to reflect sympa-
thetic tone. In another study, a positive association between
sAA and respiratory sinus arrhythmia (RSA) was observed
(r = 0.36), indicating an association of increased sAA with
vagal suppression (Granger et al., 2006). Finally, a recent
study revealed a moderate positive association (r = 0.26)
between sAA and basal skin conductance level (SCL), which
is regarded as a sympathetic activity marker (El-Sheikh et al.,
2008).
3.5. Summary
Taken together, it seems that sAA response patterns to both
physical and psychological stressors correspond to the
response patterns of the sympathetic nervous system.
Whereas no correspondence of plasma norepinephrine and
sAA could be established, associations between other mar-
kers of the ANS, such as cardiovascular parameters, have
been found. It should be noted, however, that the correla-
tions between sAA and sympathetic markers are relatively
small. Based on the pharmacological and electrophysiologi-
cal literature reviewed above, the pathways that lead to
secretion of sAA are clearly sympathetic/parasympathetic in
nature.
4. Applications of salivary alpha-amylase
measurement
The findings on the association of sAA and the sympathetic
nervous system summarized above indicate that sAA can
function as a useful biomarker in acute and chronic stress
studies. An overview of previous and potential applications in
stress research is given below.
4.1. Salivary alpha-amylase as an indicator of
autonomic dysregulation
Based on the knowledge that has been accumulated about
the role of stress and its underlying physiological mechanisms
in the secretion of sAA, it seems reasonable to conclude that
sAA concentration can serve as an index for pathological
dysregulation of the ANS in specific clinical and subclinical
conditions. Anxiety-related conditions are accompanied by
autonomic changes (Friedman et al., 1993; Shalev and Rogel-
Fuchs, 1993; Southwick et al., 1993; Lang et al., 2000;
Laederach-Hofmann et al., 2002; Coupland et al., 2003).
sAA measurements might provide additional information on
the autonomic changes occurring in anxiety patients. Some
preliminary findings indicate that sAA measurement may be
useful not only as an indicator of autonomic changes but also
as a marker for anxiety reports. Takai et al. (2004) used a
stressful video to induce stress and found sAA peak levels and
state anxiety measures to correlate highly (r = 0.535). A
similarly high correlation between state anxiety and
sAA was found in another study, in which sAA levels were
U.M. Nater, N. Rohleder
examined in healthy participants during a mental arithmetic
task (Noto et al., 2005). The task induced significant
increases in both state anxiety and sAA, with a correlation
of r = 0.589 between the two variables. These examples
demonstrate the potential of sAA measurement in a psychia-
tric context. To date, however, few studies with psychiatric
patients have employed sAA as an indicator for autonomic
changes. A notable exception is a recent study by Labudda
et al. (2007), who measured sAA in pathological gamblers and
found significant increases only in a subgroup of patients who
showed less disadvantageous decision-making patterns.
However, sAA might be also measured in the context of
somatic disorders in which autonomic dysregulation might be
present. Exaggerated autonomic responses to different sti-
muli can be observed in hypertensive patients (Fredrickson
et al., 1991; al’Absi and Wittmers, 2003) and patients with
HIV (Cole et al., 2001). It might be of interest to measure sAA
concentrations in these groups of patients. ANS dysregulation
can also be found in atopic diseases. In a study of patients
with dermatitis, Crespi and co-workers measured parotid
secretory response to intra-oral citric acid. Protein concen-
tration as well as sAA activity was significantly decreased in
patients with atopic dermatitis. The authors interpreted
these findings as a reflection of impaired beta-adrenergic
mediated responses in atopic dermatitis (Crespi et al., 1982).
In another study, Wolf et al. (2008) examined diurnal sAA in
patients with asthma and in controls. The found lower daily
sAA output in patients with asthma than in the control group.
4.2. Measurement of salivary alpha-amylase in
treatment studies
Given these findings of increased or attenuated sAA in clinical
populations, use of sAA to measure the effects of psychother-
apy, e.g. stress management training (Gaab et al., 2003) seems
warranted. A recent study has used sAA to assess stress levels in
healthy women administered Relora (a medication including
extracts of Magnolia officinalis and Phellodendron amurense)
(Kalman et al., 2008). Another study defined sAA as the end-
point of a treatment study using reflexology in nursing home
residents with dementia (Hodgson and Andersen, 2008).
Finally, a third study found decreased sAA levels in patients
undergoing surgery while exposed to natural sounds than in
patients who were not exposed to any sounds (Arai et al.,
2008). These examples show that sAA measurement is a pro-
mising approach for studies of treatment effects.
Taken together, sAA has already been used as a marker in a
variety of conditions. More recent developments suggest that
it may also be a useful marker in the context of pain (Shirasaki
et al., 2007) or sleep (Seugnet et al., 2006) research. It may
be particularly useful in ambulatory settings, such as field
studies, where assessment of sAA through saliva as an indi-
cator of autonomic functioning might present an easy, non-
invasive, and efficient sampling method.
5. Conclusion
The aim of this review was to evaluate the role of sAA activity
as a potential indicator for sympathetic activation in
response to stress. Although first findings on the role of
sAA in stress processes were published almost three decades
Salivary alpha-amylase as a biomarker for the SNS
ago, no attempts were made to scientifically elucidate this
relationship until the mid-1990s. Numerous studies have
since shown that changes in sAA are indeed dependent on
stressful stimuli, either physiological or psychological in
nature. The biological meaning of this phenomenon remains
to be clarified, however. In this context, it may be worthwhile
reconsidering the main property of alpha-amylase, namely its
digestive action. Alpha-amylase hydrolyzes starch to glucose
and maltose, initiating the digestion of starch in the oral
cavity; it has also been shown to have a bacterial interactive
function (Scannapieco et al., 1993). More studies are needed
to examine the cellular processes occurring after initiation of
stress-related responses in the oral environment. It is diffi-
cult to deduct the function of short-term increases in alpha-
amylase while the biological meaning of transient rises in the
anti-bacterial action of the enzyme remains unclear. How-
ever, such short-term increases may be useful to the body in
that energy is made available by increased digestive action in
response to stress. Physiological stress reactions comprise
orchestrated actions throughout the body, putting the organ-
ism in a state of overall preparedness to engage in fight or
flight. Thus, increases in amylase activity may be one of many
actions involved in activating the body’s resources to cope
with stressful events or threats to homeostasis. However, this
explanation applies only to reactions to short-term, acute
stressors. Further studies are needed to examine long-term
changes in sAA concentrations. If clinical or subclinical con-
ditions associated with an increase of activity in the ANS
result in chronically elevated concentrations of sAA, the
sustained higher sAA activity and therefore digestive action
may prove detrimental to oral health.
The studies summarized in this review clearly show that sAA
is increased in states of stress, i.e. when autonomic activation
is increased. We can therefore conclude that increases in sAA
may reflect changes in the ANS. Further studies are needed to
conclusively determine whether sAA can be included in the
canon of biological stress markers. In particular, the mechan-
isms underlying elevations in sAA in response to stress warrant
examination. Although a variety of studies have examined the
physiological mechanisms of sAA production and secretion in
animals, studies in humans are scarce. Use of pharmacological
agents that inhibit or activate the ANS may prove particularly
useful here, providing more detailed insights into the branches
of the ANS responsible for increases in sAA. Clearly, more
invasive techniques are available in animal studies. Experi-
mental damaging of nerve fibers or direct stimulation of nerve
cells is not feasible in a basic research context. It may be
interesting, however, to use electrical stimulation techniques
in awake or anaesthesized humans, e.g. in the clinical context
of a hospital. Measurement of direct sympathetic nerve activ-
ity via microneurography is considered to be the most accurate
technique for assessing sympathetic activation (Grassi and
Esler, 1999). Beyond peripheral measurements, the relation-
ship between central parameters and changes in sAA might
prove very interesting. Since cerebrospinal fluid concentra-
tions of norepinephrine reflect other mechanisms than do
peripheral catecholamines, it would be worthwhile comparing
sAA and central norepinephrine concentrations (Goldstein,
1998).
In summary, assessment of sAA as a non-invasive biomar-
ker for the sympathetic nervous system offers a multitude of
possibilities in different research areas. If further evaluated,
493
sAA may well become an important parameter in stress
research.
Role of the funding source
NR was supported by personal (salary) fellowships by the
German Research Association (DFG; Ro 2353/4-1) and the
Michael Smith Foundation for Health Research. UMN has no
funding source to report.
Conflicts of interest
The authors have no conflicts of interest and declare no
financial interests.
Acknowledgments
The authors would like to thank Ulrike Ehlert and Clemens
Kirschbaum for their support and theoretical input. Further-
more, NR acknowledges the support of the German Research
Foundation (DFG; grant no. Ro 2353/4-1) and the Michael
Smith Foundation for Health Research.
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