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 Author's personal copy

Psychoneuroendocrinology (2009) 34, 469—485

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

Determinants of salivary a-amylase in humans and

methodological considerations
Nicolas Rohleder a,*, Urs M. Nater b

a
Department of Psychology, Brandeis University, MS 062, PO Box 549110, 415 South Street, Waltham, MA 02454, USA
b
Institute of Psychology, Dept. of Clinical and Psychotherapy, University of Zurich, Switzerland

Received 12 August 2008; received in revised form 6 December 2008; accepted 8 December 2008

KEYWORDS Summary Salivary a-amylase (sAA) has been proposed as a marker for activity of the sympa-
Psychological stress; thetic nervous system (SNS). Recent studies in support of this hypothesis have led to an increased
Human; number of researchers integrating amylase measurements into their study designs. Salivary a-
Diurnal rhythm; amylase is produced locally in the salivary glands, controlled by the autonomic nervous system.
Sympathetic nervous This entails some methodological consequences and potential pitfalls that might lead to
system; increased error variance and thus prevent successful testing of hypotheses. The goal of this
Salivary biomarker; review is to summarize basic and recent findings on methodological issues and potential factors
Salivary a-amylase; influencing sAA measurement, and to derive a set of recommendations enabling researchers to
Salivary a-amylase output; successfully using sAA in psychoneuroendocrinological experiments.
Salivary flow rate # 2008 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
2. Methodological considerations for measuring salivary a-amylase in humans . . . . . . . . . . . . . . . . . . . . . . . . 470
2.1. Mechanism of saliva secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
2.2. Saliva collection methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
2.2.1. Saliva collection based on absorbent materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
2.2.2. Saliva collection using ‘‘drooling’’ or ‘‘spitting’’ techniques . . . . . . . . . . . . . . . . . . . . . . . . . 472
2.2.3. Saliva collection from specific salivary glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
2.2.4. Recommendations for collecting saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
2.3. Handling and storage of samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
2.4. Measurement of salivary a-amylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
2.4.1. Enzymatic measurement techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
2.4.2. Other types of assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
2.5. Unit of measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
2.6. Statistical handling of salivary a-amylase data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474
3. Determinants of salivary a-amylase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475

* Corresponding author. Tel.: +1 781 736 3319; fax: +1 781 736 3291.
E-mail address: rohleder@brandeis.edu (N. Rohleder).

0306-4530/$ — see front matter # 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.psyneuen.2008.12.004
 Author's personal copy

470 N. Rohleder, U.M. Nater

3.1. Basal activity of salivary a-amylase activity . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 475

3.1.1. Earlier studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 475
3.1.2. Studies assessing salivary amylase activity relative to individual diurnal rhythm . . . . . . . . . . . . 476
3.2. Acute salivary a-amylase responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 477
3.3. Impact of sex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 477
3.3.1. Sex differences in basal salivary a-amylase . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 477
3.3.2. Sex differences in acute responses. . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 477
3.4. Impact of age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 478
3.4.1. Impact of age on basal a-amylase . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 478
3.4.2. Impact of age on acute responses . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 478
3.5. Smoking, drugs, and alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 479
3.5.1. Smoking. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 479
3.5.2. Alcohol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 479
3.5.3. Medical drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 479
3.5.4. Caffeine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 480
3.6. Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 480
3.6.1. Association of carbohydrate contents of diet with basal a-amylase . . . ..... . . . . . . . . . . . . 480
3.6.2. Acute stimulation of amylase by food . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 480
3.7. Physical exercise . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 481
3.7.1. Acute response to exercise. . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 481
3.7.2. Effect of training status on basal salivary a-amylase . . . . . . . . . . . . ..... . . . . . . . . . . . . 481
3.8. Somatic and psychiatric diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 481
4. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 482
4.1. Summary of recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 482
4.2. Conclusions and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 482
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 482
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 482

1. Introduction hypothalamus—pituitary—adrenal [HPA]-axis) in a single test

The salivary enzyme a-amylase has been proposed as a
marker for stress-induced activity of the sympathetic nervous
system (SNS) and recent studies have underscored the use-
fulness of salivary a-amylase (sAA) in this regard. a-Amylase
is one of the major protein components of saliva. Its main
function is the enzymatic digestion of carbohydrates (Baum,
1993), but it is also important for mucosal immunity in the
oral cavity, as it inhibits the adherence and growth of bac-
teria (Scannapieco et al., 1993).
Based on the early observation by Gilman et al. (1979) that
sAA increases in response to exercise (Gilman et al., 1979),
Chatterton et al. (1996) in their seminal paper reported a
significant positive correlation between sAA and plasma
norepinephrine in response exercise (r = 0.64) and suggested
the use of sAA as a marker for SNS activity (Chatterton et al.,
1996). Since then, several independent studies have con-
firmed the responsivity of sAA to psychosocial stress and
physical exercise, and additional evidence for the association
of amylase responses with sympathetic activation has been
reported (for a review see Nater and Rohleder (submitted)).
As a substance that can be relatively easily and cost-
effectively assessed in human saliva, it is likely that the
number of studies using this parameter will increase in the
near future, especially given that evidence linking it to SNS
function is accumulating, and as a consequence of the fact
that sAA as salivary parameter has some advantages over
classical electrophysiological measures such as skin conduc-
tance and heart rate measures. The major advantage of a
saliva-based measure of SNS activity is the convenience of
assessing activity of both major stress systems (i.e. SNS and
tube, without the need for technically sophisticated instru-
mentation. Despite the convenient assessment, some char-
acteristics of this salivary enzyme also entail some pitfalls
that might complicate study design and interpretation of
results. Since the literature reporting sAA findings is con-
tinually growing, guidelines for collection, analysis, and
interpretation of this new sympathetic stress marker are
needed. The goal of this review is to provide recommenda-
tions for the measurement of sAA in human studies in the field
of psychoneuroendocrinology (PNE). In the following, we will
therefore first provide a review of methodological issues,
followed by a summary of determinants of basal and stress-
induced sAA activity in human saliva.
2. Methodological considerations for
measuring salivary a-amylase in humans
In this section, we will briefly explain the mechanism of saliva
production and protein secretion, describe the major saliva
collection techniques, laboratory assays, and derive recom-
mendations for the researcher. These recommendations are
summarized in Table 1.
2.1. Mechanism of saliva secretion
Saliva production and secretion is a complex process, and
knowledge of this process can help to avoid methodological
errors during saliva collection, storage of samples, laboratory
and statistical analysis. A detailed description can be found in
excellent earlier reviews from the field of oral biology (see
 Author's personal copy

Determinants of salivary a-amylase 471

Table 1 Recommendations for saliva sampling and storage, amylase measurement, and data analysis.

Recommendations for sampling, storage, measurement, and data analysis

Sampling Salivettes and passive drooling are the methods of choice. It depends on practical considerations which one to
favor. Salivettes are usually preferable in field studies and whenever instruction and close monitoring of the
sampling procedure is problematic.
Samples for assessing acute responses should be obtained at baseline and frequently (every 10 min)
after that until 20—30 min after the end of the intervention.
Samples for assessing the diurnal rhythm should be taken relative to waking up, ideally immediately
after waking up, 30 and 60 min later, and equally distributed over the rest of the day.
Storage Saliva can be stored at room temperature for up to three weeks. To prevent mold and bacterial growth, it is
recommended to freeze, or at least refrigerate samples as quickly as possible.
Long-term storage in the laboratory should be done at !20 8C or colder.
Measurement Enzyme kinetic assays can be commercially obtained as pre-packaged kits, or reagents and materials can
be bought separately.
Measurements can also be done at most hospital laboratories.
Data analysis Amylase data will typically deviate from normal distribution and require log- or square root transformation.
The best index for acute responses is a simple delta score between baseline and post-intervention maximum.
Diurnal variation should either be analyzed using growth curve models. Alternatively, the area-under-curve
with respect to ground (AUC ground) and linear slope can be calculated as indices for daily amylase output
and integrity of the diurnal rhythm, respectively.

for example White, 1977; Baum, 1993), and in Nater and
Rohleder (submitted).
It is of major importance that salivary a-amylase is not, like
salivary cortisol, a hormone that originates in an endocrine
gland, circulates in blood and diffuses into saliva. In contrast,
amylase is produced by the highly differentiated epithelial
acinar cells of the exocrine salivary glands (Baum, 1993). It is in
these same acinar cells that the primary fluid component of
saliva is derived from the local vasculature, and enriched with
salivary proteins, of which amylase is one of the most impor-
tant. As Baum (1993) points out, there is no spontaneous
secretion of saliva or salivary proteins, but both processes,
fluid and protein production, are stimulated by parasympa-
thetic and sympathetic nerves innervating the salivary glands.
The major implication of this mechanism and the fact that
amylase, as opposed to cortisol, is not passively transported by
saliva, but produced together with saliva and other salivary
components, is that when collecting saliva, researchers need
to be aware of the fact that all factors influencing salivation
can also influence salivary a-amylase.
Further, saliva is produced by three different types of
major salivary glands, i.e. the parotid glands, the subman-
dibular, and the sublingual glands, which are located pairwise
on the left and the right side of the oral cavity. These and a
number of smaller salivary glands secrete saliva through a
network of salivary ducts, which enter the oral cavity at
different locations (Humphrey and Williamson, 2001). Par-
otid saliva enters the oral cavity through Stensen’s ducts
located near the second molars in the upper jaw, while
submandibular and sublingual saliva enters the oral cavity
through Wharton’s ducts located in the floor of the mouth
(Navazesh, 1993).
These specific glands differ in their relative contribution
of protein and fluid saliva components, with about 80% of
amylase being produced by the parotid gland (Zakowski and
Bruns, 1985). In addition, the relative contribution of each
specific gland to total salivary volume differs between
stimulated and unstimulated saliva. Specifically, under sti-
mulated conditions, the contribution of the parotid gland
increases from 20% to more than 50% of total saliva (Hum-
phrey and Williamson, 2001). This again has major implica-
tions for collection techniques, with regard to where the
saliva that is sampled comes from, and whether the collec-
tion technique stimulates salivary flow. We will therefore in
the following review the major collection methods for human
saliva and discuss their relative advantages and disadvan-
tages.
2.2. Saliva collection methods
A wide array of different methods for collecting saliva has
been described in the past years. Some of the methods have
been summarized in earlier reviews (see for example White,
1977; Navazesh, 1993), but these works have not discussed
methods regarding their specific usefulness for the psycho-
neuroendocrinological researcher, e.g. without addressing
issues of saliva sampling outside of the laboratory, when
study participants go after their regular daily lives, or in
repeated measures designs of acute stress studies.
In general, techniques for collecting human saliva can be
categorized into those sampling whole saliva vs. those sam-
pling saliva from specific salivary glands, and into those col-
lecting stimulated vs. unstimulated saliva. Another distinction
that can be made is into collection techniques based on
absorbent material vs. techniques based on passive drooling
or spitting of saliva into collection tubes. The two techniques
most relevant for the PNE researcher are collection using the
cotton roll-based salivette (Sarstedt, Nümbrecht, Germany),
which belongs to the former group, and the passive drooling
technique, which belongs to the latter group.
2.2.1. Saliva collection based on absorbent materials
The most frequently used collection technique using absor-
bent material is probably the salivette. Salivettes consist of a
 Author's personal copy
472
cotton roll provided in a plastic container. Participants are
usually instructed to remove the cotton roll from the plastic
container and to place it in their mouth for intervals ranging
from 1 to 5 min. Complete salivettes can be stored at con-
ditions described below until saliva is extracted by centrifu-
gation. This technique has been used by the majority of
studies sampling saliva for the determination of cortisol,
and it is therefore frequently used for amylase determination
as well. In some experimental protocols, participants are
instructed to chew on the cotton to stimulate salivary flow.
Researchers should be aware of the fact that while this does
not have any effect on salivary cortisol, stimulated saliva
differs markedly from unstimulated saliva with regard to the
source (i.e. the secreting gland) and therefore the relative
amount of amylase protein secreted. It is thus recommended
to keep this factor constant within each study. Related to
this, it has been suggested that the position of the cotton role
in the mouth influences from which specific gland the
sampled portion of saliva had been secreted. In fact, Harmon
et al. have recently shown that saliva collected in different
areas of the mouth by placing absorbent cotton swabs in
locations to specifically collect saliva from the three major
glands produced different results (Harmon et al., 2008). It is
therefore further advisable to consistently instruct partici-
pants within each study to either place the cotton roll in a
specific area of the mouth, or to move it around in a circular
pattern to collect saliva from all glands.
2.2.1.1. Other saliva collection techniques using absorbent
materials. Variations using other absorbent devices have
been described for circumstances in which the use of saliv-
ettes is not feasible, for example in infants or in adult
participants during sleep, when low sample volumes are to
be expected. Granger et al. (2007b) describe the use of
braided cotton dental rope (Richmond dental, Charlotte,
NC; Granger et al., 2007b). Briefly, cotton ropes can be
cut into appropriately sized lengths, usually longer than
needed, to enable the experimenter to hold onto the end
of the braid and prevent children from swallowing the mate-
rial. The saliva-saturated end of the cotton rope is then
placed into the barrel of a needleless syringe, and com-
pressed using the plunger to express saliva into a collection
vial (Gunnar et al., 1989; Granger et al., 2007b).
However, Harmon et al. (2007) showed that cotton-based
collection devices are very effective in absorbing fluids, but
that the extraction of small volumes by centrifugation or the
plunger of a syringe as described above can be problematic.
They have therefore suggested the use of hydrocellulose
microsponges. These are manufactured to absorb tear fluid
during eye surgery and are commercially available (Becton &
Dickenson, Walton, MA). Tests have shown that these sponges
absorb liquids up to 450 ml, which would make them parti-
cularly useful for collection of low volumes of saliva, for
example in infant studies (Harmon et al., 2007).
2.2.1.2. Advantages and disadvantages. The major advan-
tages of the salivette method are that saliva sampling is easy
to learn and easy to perform, even in public. This method is
usually perceived as clean. Therefore, this technique bears
the highest probability of participants adhering to the study
protocol. Furthermore, salivettes are already in use in many
psychoneuroendocrinological laboratories, which enables
N. Rohleder, U.M. Nater
researchers to keep their study protocols unchanged. Finally,
the cotton serves as a filter so that after extraction of saliva
by centrifugation, a clear fluid free of mucous components
and other contaminants is available for analysis. Other absor-
bent techniques also have the advantage of being easy to
handle, for example in infants and other situations in which
salivary flow rate is low.
A disadvantage of salivettes or other absorbent-based
collection techniques is that stimulation of salivary flow
cannot be completely excluded due to the presence of the
mechanical stimulus in the mouth, even if participants are
instructed not to chew on the cotton roll (Navazesh, 1993). It
has also been argued that low volumes cannot be recovered
from cotton (Zimmermann, 2008). However, this is a lesser
problem since volume requirements of modern assays are
extremely low (e.g. requiring only 20 ml of sample).
2.2.2. Saliva collection using ‘‘drooling’’ or ‘‘spitting’’
techniques
The ‘‘passive drooling’’ technique as described by Navazesh
(1993) is probably the best method for collection of unsti-
mulated whole saliva. Participants are instructed to empty
their mouths by swallowing all saliva, and then collect all
subsequently secreted saliva in the oral cavity for an exact
period of time, usually between two and five minutes. In the
‘‘draining method’’, saliva is allowed to drip off the lower lip
into the collection tube through a funnel. In the ‘‘spitting
method’’ the whole amount of collected saliva is spit into a
collection tube, and the ‘‘suction method’’ implies aspirating
the accumulated saliva from the bottom of the mouth. Of
these, only the spitting method appears to be feasible enough
to be performed by participants during most experimental
setups realized in the field of PNE.
2.2.2.1. Variations. The major variation of spitting tech-
niques described in the literature is to intentionally sti-
mulate saliva production either by using gustatory stimuli
such as citric acid (Froehlich et al., 1987) or by using
mechanical stimuli such as chewing on paraffin wax or
parafilm (Mackie and Pangborn, 1990). The major advan-
tage is that it enables the collection of adequate volumes
of saliva. However, as discussed above, stimulating salivary
flow significantly changes the contribution of specific sali-
vary glands to total saliva production, and also activates
secretion of salivary proteins. Hence, stimulation of saliva
production should only be used if saliva cannot be obtained
otherwise, and should then be applied consistently
throughout each experiment.
2.2.2.2. Advantages and disadvantages. The major advan-
tage of the passive drooling/spitting technique is that unsti-
mulated whole saliva is collected, which yields a
representative combination of saliva from all glands. Disad-
vantages are that passive drooling needs to be practiced, that
it requires the full attention of the participant, and that it is
not always perceived as clean, because participants are
asked to spit in a relatively small tube, or use a straw to
do so. Because of this, it is a technique that many partici-
pants are not comfortable with practicing in public places, at
work, or generally in company of other people. This method
further yields ‘‘unfiltered’’ samples, which can be difficult to
process for further analyses, because even ultracentrifugation
 Author's personal copy
Determinants of salivary a-amylase
is sometimes insufficient to completely remove mucous com-
ponents or contaminations.
2.2.3. Saliva collection from specific salivary glands
Specific suction devices have been designed to harvest saliva
directly where the ducts enter the oral cavity (i.e. Stenson’s
ducts for parotid saliva, Wharton’s ducts for sublingual and
submandibular saliva). These devices allow precise sampling
of saliva produced by specific glands. However, these meth-
ods are typically employed for very specific questions from
the field of oral biology, and will therefore not be described
here (see White, 1977; Navazesh, 1993).
2.2.4. Recommendations for collecting saliva
Taken together both of the major collection techniques that
have been in use for the measurement of salivary cortisol,
i.e. salivettes and passive drooling, can generally be recom-
mended for sAA as well. From a theoretical standpoint, the
passive drooling technique is superior to techniques using
absorbent materials, because stimulation of saliva produc-
tion is avoided and a representative combination of saliva
produced from the major salivary glands, i.e. whole saliva, is
collected. However, passive drooling should only be used if
sample collection can be done under supervision, or if parti-
cipants are highly motivated and have been well trained in
the collection procedure. Therefore, from a practical point
of view, the use of salivettes is superior if circumstances do
not allow thorough training and close monitoring of the
collection procedure and if saliva collection has to be done
by participants during their normal lives. If salivettes are
used, participants should be instructed to place the cotton
rolls in their mouths for an exactly defined period of time
(usually, two minutes are sufficient), and they should be
instructed not to chew on the cotton.
For all techniques, it is advisable to instruct participants
to avoid eating or drinking (anything but water) at least an
hour before saliva collection. Participants should also rinse
their mouth with clear water to avoid contamination of saliva
samples with food components, and to avoid activation of
salivary flow or protein production by gustatory stimuli.
Furthermore, using citric acid or other acidic components
is not recommended, as it can interfere with assay results
because it changes the pH of the sample.
2.3. Handling and storage of samples
Several questions have to be answered to safely handle and
store saliva samples that are going to be used for determina-
tion of salivary a-amylase: (1) How long can samples be
stored at room temperature, or in regular household fridges
before frozen? (2) How long can samples be stored at !20 8C
or !80 8C? (3) How many freeze-thaw-cycles does amylase
survive? (4) In which type of container should saliva be
stored? (5) Should preservatives be used?
Question (1): A recent methodological study showed that
amylase is stable at room temperature (22 8C), and even at
higher temperatures (37 8C) for up to three weeks without
significant loss in activity (Decaro, 2008). These results are in
accordance with an earlier study, which revealed that amy-
lase is stable at room temperature, and at 4 8C for at least
four days (Granger et al., 2006). This has important implica-
tions for storage and shipping. Specifically, when saliva is
473
being collected in the field by participants going after their
regular daily activities, they do not necessarily need to
freeze or refrigerate samples during the collection day,
and before and during shipping to the analyzing laboratory.
It is still recommended to store saliva samples in participants’
home refrigerators or freezers and to ship them in insulated
envelopes to avoid growth of bacteria and mold in samples.
Questions (2)—(4): For long-term storage, saliva samples
should be frozen at !20 8C or lower temperatures. Saliva can
be frozen in whole, un-centrifuged salivettes, or transferred
to space-saving containers such as deep-well plates or 1.7 ml
vials. It has also been shown that amylase is not affected by
repeated freeze-thaw cycles (Granger et al., 2006). Because
freezing and thawing breaks down mucopolysaccharides in
saliva that could interfere with pipetting, it is recommended
to subject samples to at least one freeze-thaw-cycle, even if
time between sample collection and measurement is short
(Shirtcliff et al., 2001).
Question (5) There is no need for preservatives. It has
been shown that sodium azide should not be used as a
preservative, because it increases amylase activity (Maurus
et al., 2008), thus leading to false high assay results (Decaro,
2008).
2.4. Measurement of salivary a-amylase
2.4.1. Enzymatic measurement techniques
The enzyme kinetic determination of pancreatic a-amylase
in human serum is an important everyday clinical diagnostic
test, which has led to the availability of a large number of
different test methods. Since salivary and pancreatic a-
amylase share a homology of 97% (Lorentz, 1998), salivary
a-amylase can be measured using established methods with-
out major modifications. Most of these commercially avail-
able tests are produced for integrated biochemistry
analyzers such as the Cobas systems (Roche, Basel, Switzer-
land), which are typically used in hospital central labora-
tories. While these might be available to some researchers,
those without access to one of those instruments or labora-
tories can use the same reagents to build smaller scale
assays.
Recommendations of the International Federation of Clin-
ical Chemistry and Laboratory Medicine (IFCC) for the mea-
surement of pancreatic and salivary a-amylase are provided
by Lorentz (1998). Assays for enzyme kinetic measurement of
sAA can be performed using 96-well microtiter plates and
standard laboratory absorbance readers as for example
described by Bosch et al. (1996, 2003).
Briefly, to measure sAA using an enzyme kinetic assay,
saliva containing amylase has to be incubated with a specific
chromogenic substrate, for example 4,6-ethyliden-G7-PNP
and the auxiliary enzyme a-glucosidase (both substances are
included in the substrate reagent ‘‘amylase EPS’’, Roche
Diagnostics). a-Amylase cleaves the substrate into inter-
mediate products, which are further broken down by the
auxiliary enzyme into p-nitrophenol (PNP) and glucose. PNP
absorbs light at a wavelength of 405 nm (yellow). The more
a-amylase is present in the saliva sample, the more substrate
is broken down into PNP in a specific period of time, and the
higher the optical density that can be measured at 405 nm.
In a typical assay protocol, saliva is diluted between 1:200
and 1:1000 and 20 ml of diluted saliva are pipetted into the
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474
wells of a 96-well microtiter plate made out of clear plastic
(preferably polystyrene; ‘‘PS’’). Eighty microliters of com-
mercially available substrate is added and the plate is incu-
bated at 37 8C to start the enzymatic degradation of the
substrate. The color change is quantified by measuring the
microplate in a regular photometric plate reader twice. First
after warming up to 37 8C and a second time after a defined
period of 2—5 min with a 405 nm filter. The resulting color
change is directly proportional to amylase activity. To trans-
form relative activity to standardized units, a standard curve
made from commercially available standard solution (for
example ‘‘Calibrator f.a.s.’’, Roche Diagnostics) should be
included on each plate. Alternatively, amylase activity can be
calculated using a formula that includes the volumes of
sample and substrate, the dilution factor, the light path,
and the specific absorption of the p-nitrophenol. Reagents
can be obtained from several different vendors (e.g. Roche
Diagnostics), and prepackaged kits containing all reagents
are commercially available (for example Salimetrics, State
College, PA).
2.4.2. Other types of assays
While the enzyme kinetic method is the standard technique
for measuring a-amylase, other assays have been employed.
Yamaguchi et al. have described an automated analyzer for
sAA in hand held format (Yamaguchi et al., 2006b). This
instrument measures a-amylase activity in saliva loaded into
the instrument using a test strip of absorbent paper. Saliva is
transferred from the test strip to another strip of paper
saturated with a substrate, and a built-in optical sensor
measures color change after 30 s. While it is unclear at
present how ambient temperature and pH of saliva influence
assay results, and how these issues are dealt with, this might
be developed into a promising tool for field research.
In addition, enzyme-linked immuno assays (ELISAs) and
radio immuno assays (RIAs) have been employed for measure-
ment of sAA (Ito et al., 1985; Agarwal and Henkin, 1984).
However, ELISA techniques have not been used in any recent
studies. One reason might be that ELISA techniques are more
expensive and time-consuming than enzyme-kinetic meth-
ods. Theoretically, they would have the advantage of speci-
fically measuring the concentration of the amylase protein,
instead of indirectly deriving the concentration from its
biological activity.
2.5. Unit of measurement
Since most assay techniques employed to date make use of
sAA’s enzymatic activity, the most frequently used unit of
measurement is enzyme units per milliliter (U/ml). An
enzyme unit is defined as the amount of enzyme that cata-
lyzes the conversion of 1 mmol of substrate per minute, and is
directional proportional to the unit katal (kat), which was
endorsed by the Nomenclature Committee of the Interna-
tional Union of Biochemistry (NC-IUB) in 1979 (NC-IUB, 1979).
One U corresponds to 16.67 nkat. Although the unit katal is
recommended by the NC-IUB, most data are reported in U/
ml. Thus, current assays do not provide concentrations in
grams or mols per liter, as in other stress markers.
An alternative way to report amylase data is to express
amylase activity relative to saliva flow rate. This measure is
referred to as amylase output, and is obtained by multi-
N. Rohleder, U.M. Nater
plication of amylase activity measured in U/ml with flow rate
measured in ml/min. Saliva flow rate is typically determined
by collecting all saliva secreted during a specified time period
of 2—5 min, and gravimetrically determining the volume
assuming that the density of saliva is 1.0 g/ml (Chicharro
et al., 1998). The advantage of the output measure is that it
accounts for changes in saliva flow rate. The disadvantage is
that determining flow rate complicates sampling, making it
less suitable for field studies. A recent study comparing
activity and flow rate did not find differences in stress
responses between both measures (Rohleder et al., 2006).
2.6. Statistical handling of salivary a-amylase
data
Salivary a-amylase data typically do not show normal dis-
tributions, but are positively skewed. In addition, variability
is frequently found to be considerably higher than that of for
example cortisol. This is most likely the result of the multi-
tude of factors influencing acute amylase discussed in this
review. Amylase responds faster to acute stress than cortisol,
and its activation threshold in response to physical activity is
lower. Furthermore, inconsistency in sampling techniques,
such as different duration of saliva accumulation and chew-
ing vs. not chewing, or in experimental procedures, such as
not excluding sugar intake before saliva sampling, might be
further reasons for high variability. Variability might there-
fore be reduced by adhering to the recommendations given
here. Furthermore, in most studies, distributions have been
successfully normalized by using for example logarithmic
(Rohleder et al., 2006; Nater et al., 2007), or square root
transformations (Gordis et al., 2006).
After normalization of distributions, further analysis stra-
tegies depend on the type of data assessed. In studies
measuring acute sAA responses, it is advisable to collect
saliva at baseline (i.e. before an intervention) and fre-
quently, e.g. every 10 min, thereafter until at least 30 min
after the end of the intervention to capture increase and
recovery of the sAA response (see Fig. 2). Typically, research-
ers may want to calculate indices that describe the sAA
response. Here, simple delta scores between the post-inter-
vention and the baseline level are the easiest approach. More
complex approaches such as the area-under-curve (AUC)
have also been used. Given the acute nature of the response,
the AUC relative to increase might be better suited than the
AUC relative to ground (Pruessner et al., 2003). Associations
between sAA and catecholamine increases, where detect-
able, were found using simple delta scores (Rohleder et al.,
2004).
With respect to daily profiles, it is advisable to take
several samples throughout the day. As shown in Fig. 1,
sAA concentrations are subject to strong diurnal variations,
with lowest levels after awakening and highest levels in the
late evening. To capture the full diurnal rhythm, samples
should be taken relative to awakening, i.e. at waking up, as
well as 30 and 60 min later (Nater et al., 2007). Further
samples can be taken at specific times or a specific number of
hours after waking up, but should cover the rest of the day. It
is further advisable to assess diurnal amylase at two con-
secutive days at the minimum. Although Wolf et al. (2008)
have recently shown good stability of amylase measured
between 1 and 14 h after waking up (r’s between 0.48 and
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Determinants of salivary a-amylase
Figure 1 Diurnal rhythm of a-amylase and cortisol in saliva of
n = 76 healthy men and women (mean age = 26.7 years,
range = 18—58 years). (Reprinted from Nater et al., 2007, Copy-
right (2007) with permission from Elsevier.)
0.65, all p’s < 0.01), we cautiously assume that stability of
diurnal sAA measurements is in the range determined for
cortisol, as shown for example by Hellhammer et al. (2007).
Analysis either using growth curve models/hierarchical
linear modeling is considered gold standard when time series
data are available, see for example (Nater et al., 2007). An
alternative analysis strategy is to calculate the total daily sAA
production by using the area-under-curve with respect to
ground (Pruessner et al., 2003) and to calculate the slope of
the diurnal sAA profile. We have for example shown that daily
amylase production measured with the AUC is associated with
shame and depression in young women (Rohleder et al., 2008).
Finally, adherence to the protocol should be controlled.
This can be done by either using electronic monitors that
register whenever a participant opens a vial to use a salivette
(Nater et al., 2007), or by using palm pilots that signal
participants to take a sample, and digitally store sampling
time (Rohleder et al., 2008). With regard to the wake-up
response, it has recently been shown that it is further
advisable to objectively assess wake-up time of study parti-
cipants by activity monitors (Dockray et al., 2008).
Figure 2 Acute responses of salivary a-amylase and plasma
norepinephrine to the laboratory stress paradigm TSST in n = 12
healthy men and women. (Reprinted from Rohleder et al., 2004,
Copyright (2004) with permission from Wiley-Blackwell.)
475
3. Determinants of salivary a-amylase
activity
As noted above, variability and skewed distributions are
characteristic features of sAA values. This can have several
reasons. First, sAA concentrations have been described as
fast-reacting biological responses. Hence there is a large
array of factors with potential to stimulate or inhibit acute
amylase secretion. Second, there might be long-term influ-
ences on sAA secretion, such as age, sex, habitual smoking,
personal fitness, and psychological factors such as person-
ality, chronic stress, or psychopathology.
When looking at factors that potentially modulate amy-
lase activity, we have to differentiate between influences
on basal levels, and influences on acute responses. In other
words, specific factors might change amylase activity
acutely, such as smoking one cigarette, but being a habi-
tual smoker might also influence basal a-amylase activity
in general, and it might modulate the ability of acute
stress to increase sAA activity. In this section, after briefly
describing acute responses to psychosocial stress / physical
activation and baseline sAA activity, we will review the
effects of the major modulators of amylase activity in
these three dimensions. A summary of determinants is
given in Table 2.
3.1. Basal activity of salivary a-amylase activity
Many biological systems are subject to diurnal oscillations
(Mistlberger and Skene, 2004), and salivary flow as well as
salivary protein production appear to be no exception from
that. As early as 1972, Dawes et al. collected unstimulated
whole saliva and stimulated parotid saliva from a sample of
eight healthy men and women over several days at 07:00,
11:00, 14:00, 17:00, and 22:00 h and measured salivary flow
and protein content, which both exhibited significant diurnal
rhythms, the latter only in parotid saliva (Dawes, 1972).
3.1.1. Earlier studies
Ferguson et al. (1973) in their introduction provide an exten-
sive review of previous studies assessing diurnal variation of
several different salivary parameters. Salivary amylase was
found lower in the morning in one of the studies reviewed,
and higher in the afternoon in four others (out of seven that
measured sAA) (Ferguson et al., 1973). Jenzano et al. mea-
sured sAA at different times of the day in a sample of 14 male
and female healthy dental students. Amylase in stimulated
whole saliva was lowest at 08:00 h and highest between 14:00
and 17:00 h on two separate test days (Jenzano et al., 1987).
Artino et al. (1998) compared morning (07:30—08:00 h) sAA
concentrations in unstimulated saliva and late afternoon
concentrations (17:30—18:00 h) between diabetes patients
and controls. While concluding that patients and controls did
not differ, it was reported that afternoon values were sig-
nificantly higher (Artino et al., 1998). Similar findings of
lower morning sAA values were also found in saliva obtained
by passive drooling in a mixed sex group of 30 students
(Rantonen and Meurman, 2000) and in another study measur-
ing amylase in unstimulated saliva sampled hourly between
09:00 and 18:00 h in eight healthy male students (Li and
Gleeson, 2004).
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476 N. Rohleder, U.M. Nater

Table 2 Summary of determinants of basal and stress-induced salivary a-amylase activity and recommendations.

Determinants of basal and stress-induced salivary a-amylase activity

Sex Current data do not support sex differences in basal amylase activity.
At present, no sex differences in acute amylase responses have been described.
Pregnancy appears to attenuate stress responses.
Age Basal amylase activity is very low to undetectable in the newborn and after then
continually increases to reach adult levels within the first 3 years.
Basal amylase activity does not change over the life span and remains stable in older age.
Acute stress responses are absent in the newborn; develop through childhood to reach
adult magnitude in adolescence.
No data are available on acute stress responses in older age.
Smoking Tobacco smoke acutely inhibits amylase activity.
Habitual smokers showed lower basal amylase in some but not all studies.
No data is available on acute stress responses in habitual smokers.
Recommendation: smoking should be controlled for or smokers excluded.
Alcohol Data are inconclusive so far, but some studies found lower amylase in chronic drinkers.
Recommendation: current findings should caution us to control or exclude at least
excessive alcohol consumption.
Medical drugs Adrenergic agonists and antagonists have a strong impact on salivary a-amylase.
Data on other drugs are scarce.
Recommendation: anti-hypertensive drugs, asthma medication, and similar medication
using adrenergic agonists or antagonists needs to be controlled for or excluded.
Caffeine Acute administration can stimulate amylase activity.
No data is available on differences between individuals with high vs. low habitual
caffeine consumption.
Recommendation: acute caffeine consumption should be avoided an hour before
participation in experiments.
Food Amylase responds acutely to gustatory and mechanical stimuli.
There is evidence in support of the hypothesis that basal amylase is higher in populations
with higher carbohydrate consumption.
Recommendation: Participants should be instructed not to eat and drink
(anything except water) an hour before sampling.
Exercise Physical exercise acutely elevates salivary a-amylase.
No data is available on basal or response differences between well-trained vs.
sedentary individuals.
Recommendation: exercise should be avoided prior to participation in experiments.
Physical activity of lower intensity appears to have a lesser impact.
Somatic and psychiatric diseases Somatic diseases have been shown linked to lower and higher amylase concentrations.
Recommendation: somatic and psychiatric diseases should therefore be carefully
controlled or excluded.

One study did not find signs of a diurnal rhythm of sAA.
Yamaguchi et al. (2006a,b) measured amylase activity every
4 h between 07:00 and 22:00 h using a handheld electronic
measurement device (see above) in n = 15 healthy university
students and did not observe changes over time (Yamaguchi
et al., 2006a).
3.1.2. Studies assessing salivary amylase activity
relative to individual diurnal rhythm
Rohleder et al. have shown in a pilot study of 17 healthy
subjects of both sexes using salivettes, that when samples
are obtained relative to awakening, e.g. immediately, as well
as 30 and 60 min after waking up, and at different times in
the afternoon, such as 11:00, 15:00, and 20:00 h, a pro-
nounced diurnal rhythm of sAA activity can be found. This
rhythm is characterized by a strong decrease after waking-up
and gradually increasing levels with peaks in the late after-
noon (Rohleder et al., 2004). This pattern has later been
replicated in a larger sample of 76 healthy subjects of both
sexes (44 women and 32 men) again sampling saliva relative
to the individual diurnal rhythm (at wake-up, 30 and 60 min
after waking up) and hourly between 09:00 and 20:00 h using
the cotton-based salivette (see Fig. 1; Nater et al., 2007).
Two more recent studies focusing on specific determinants of
daily sAA secretion (see below) were successful in further
replicating this pattern in 56 healthy young women (Rohleder
et al., 2008), and in children and adolescents of both sexes
between 8 and 18 years (Wolf et al., 2008).
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Determinants of salivary a-amylase
Michaud et al. investigated the impact of different sleep
conditions on wake-up profiles of sAA in n = 12 healthy sub-
jects of both sexes (7 men and 5 women) using salivettes at
wake-up, 30 and 90 min later, at 12:00 and 17:00 h. a-Amy-
lase showed a pronounced rhythm with low levels in the
morning and higher levels in the afternoon. However, in
contrast to the studies above, there was no drop in amylase
activity in the first 30 min after waking up (Michaud et al.,
2006). Diurnal rhythms of amylase were not influenced by
sleep disturbance (noise) or conditions (at home vs. sleep
laboratory).
3.2. Acute salivary a-amylase responses
As described above saliva production and composition is
controlled by the autonomic nervous system. Since the SNS
is a fast-reacting stress system, and changes in autonomic
balance occur in response to for example physical activity
and psychological factors, it is not surprising that sAA shows
high variability on top of the diurnal rhythm described above.
Factors that influence sAA activity, or modulate the sAA stress
response will be summarized in the following section.
3.3. Impact of sex
Many hormonal systems show sex differences in basal activity
or stress response patterns, as for example the hypothala-
mus—pituitary—adrenal-axis (Kirschbaum et al., 1999). The
SNS is subject to sex differences as well. Women are reported
to show lower norepinephrine responses to activating stimuli
(Gustafson and Kalkhoff, 1982), and catecholamines vary
over the menstrual cycle (Goldstein et al., 1983). Therefore,
it might be expected that basal sAA activity and stress
responses are influenced by sex or sex hormones as well.
3.3.1. Sex differences in basal salivary a-amylase
Women and men participated in most of the experiments
assessing diurnal rhythms of sAA and acute responses to
stress, exercise, or other conditions. Only some reported
sex-specific analyses, but none of those yielded significant
differences.
Rantonen and Meurman (2000) did not find differences
between 14 women and 16 men participating in their diurnal
variation study (see above) (Rantonen and Meurman, 2000).
Of the 76 participants in our previous study assessing diurnal
rhythms, 44 were premenopausal women. Growth curve
analyses revealed no sex differences in average sAA concen-
trations nor in slopes of the diurnal rhythm (Nater et al.,
2007). Same results were also obtained in the pilot study in
which twelve women and five men participated (Rohleder
et al., 2004).
Harm and Schlegel measured sAA before a parabolic flight
as a marker of motion sickness susceptibility in six women and
10 men using salivettes, and no sex differences were found
(Harm and Schlegel, 2002). No sAA sex differences were
further found in a study comparing 18, 30, and 42 months
old children (15 girls and 16 boys in each age group) (Dezan
et al., 2002) and in a study comparing 31 men and 32 women
of different age groups (Ben-Aryeh et al., 1986). Finally,
Nater investigated resting amylase in saliva obtained with
salivettes from 27 women and 26 men, carefully controlling
time of day, menstrual cycle phase in women, and ambient
477
conditions. Again, no sex differences were found (Nater,
2004). In all studies, however, the number of participants
was too low to exclude non-significant findings due to lack of
power.
3.3.1.1. Effect of variation in sex hormones. The impact of
natural variation of gonadal steroids on sAA was investigated
in some studies. Variations of salivary amylase activity over
the menstrual cycle have been investigated. Tenovuo col-
lected paraffin-stimulated whole saliva every second/third
day of one menstrual cycle in 14 women not using oral
contraceptives. When analyses were restricted to the only
three women in the sample displaying a regular menstrual
cycle, profound variation was observed. However, a-amylase
levels did not differ between menstruation and ovulation in a
larger sample of 12 women. It is therefore unclear whether
the variation observed in the subsample of three women can
be attributed to the menstrual cycle or is due to other factors
(Tenovuo et al., 1981). Laine et al. (1991) found no impact of
menstrual cycle phase on salivary flow and amylase in par-
affin-stimulated whole saliva in 11 women not using contra-
ceptives. There were also no differences between 11 women
using oral contraceptives and 10 men (Laine et al., 1991). In
n = 220 students aged 14—18 years, no variation of morning
and afternoon amylase over one menstrual cycle was found
(Bhoola et al., 1978).
Laine et al. (1988) measured amylase and other salivary
parameters in 16 women throughout pregnancy and lactation
in paraffin-stimulated saliva and did not detect any signifi-
cant changes (Laine et al., 1988). No significant differences
in amylase were also measured in parotid saliva of 107
pregnant women of all trimesters and seven controls (D’Ales-
sandro et al., 1989). In contrast, Salvolini et al. found some
variation of amylase measured in unstimulated whole saliva
of n = 45 pregnant women. Amylase activity was highest after
10 weeks of pregnancy, decreased thereafter to reach similar
levels as 15 non-pregnant control women at week 40 (Salvo-
lini et al., 1998).
Taken together, the few available data do not support the
assumption that basal sAA differs between women and men,
varies systematically with the menstrual cycle in women, or
is affected by oral contraceptive use. Available data is
inconclusive with regard to pregnancy.
3.3.2. Sex differences in acute responses
Some acute stress studies investigated women and men, and
reported results of sex-specific statistical analyses. Acute
sAA responses to different stressors did not differ between
women and men. Unstimulated saliva was collected in n = 83
volunteers by passive drooling before and after viewing a
video of an eye surgery procedure. a-Amylase responses did
not differ between men and women (Takai et al., 2007).
Forty-two healthy adults (21 men and 21 women) partici-
pated in a rowing ergometer competition. Unstimulated
whole saliva was collected before and after each ‘‘race’’
of about 6—8 min duration. No sex differences in reactivity
were found (Kivlighan and Granger, 2006). Finally, a-amylase
was measured in saliva obtained using salivettes in 27 women
and 10 men before and after an oral academic examination of
30 min duration. Exams induced significant increases in sAA
activity, but increases of men and women did not differ in
magnitude. Within the group of women, using oral contra-
 Author's personal copy
478
ceptives did not modulate sAA responses (Schoofs et al.,
2008).
In contrast, pregnancy had a profound effect on sAA
increases induced by the Trier social stress test (TSST). Stress
responses were significantly reduced in 30 women in the
second trimester compared to 30 non-pregnant controls,
and even further attenuated in 30 women in the third
trimester (Nierop et al., 2006).
3.4. Impact of age
3.4.1. Impact of age on basal a-amylase
3.4.1.1. Early development. Development of sAA produc-
tion in the first months and years of the life of human
newborns has been studied. In a cross-sectional design,
Bellavia et al. collected stimulated whole saliva from 25
infants between 3 and 15 days after birth. Amylase activity
was measured and was found nine times lower in infants than
in saliva of four adults (Bellavia et al., 1979). Two long-
itudinal studies followed up newborns during the first weeks
and months of their lives. Amylase activity was repeatedly
measured in whole saliva of 13 premature infants between 26
and 42 weeks gestational age and a positive relationship
between age and amylase activity was observed. Unfortu-
nately, no statistical analysis of this association was reported
(Hodge et al., 1983). In another study, amylase was measured
monthly from birth to the age of five months in unstimulated
saliva of n = 29 infants. Amylase increased significantly from
undetectable concentrations in newborns to levels similar to
adult concentrations at about 3 months (Sevenhuysen et al.,
1984).
Another approach to investigate development of sAA
activity was to compare amylase in infants of different age
groups. Amylase was measured in unstimulated saliva
obtained from n = 168 infants between 3 days and 12 months
of age. A positive association of sAA with age was found in
infants younger than 4 months; a plateau was reached in the
4—6 months old group; however, even the 12-month-old
group had significantly lower sAA concentrations than adults
(Ben-Aryeh et al., 1984). Looking at slightly older children,
Dezan et al. found that amylase activity in whole saliva of
male and female children was higher in 30-month-old com-
pared to 18-month olds. No difference was observed between
the age groups 30 and 42 months (Dezan et al., 2002). Finally,
amylase in unstimulated saliva was found to be positively
associated with age in five groups ranging from 25 infants (7—
11 months), over toddlers (2—3 years), children (6—8 years)
and adolescents (12—14 years) to adults (25—63 years) (Ben-
Aryeh et al., 1990).
In summary, the emerging pattern is that sAA activity is
very low to undetectable in the newborn and after then
continually increases to reach adult levels within the first
3 years. Developmental stage should therefore be considered
when interpreting sAA changes in subjects of varying ages.
3.4.1.2. Development over the adult life span and into
older age. Development of salivary a-amylase activity over
the adult life span and into older age in healthy participants
has only been investigated cross-sectionally. Whole and par-
otid saliva composition was examined in 63 healthy volun-
teers, 39 of which were categorized into a younger group
with an average age of 37 years, and 24 were categorized into
N. Rohleder, U.M. Nater
an old group with 66 years average age. No age differences
were found for stimulated and unstimulated whole saliva, but
the activity of amylase in resting and stimulated parotid
saliva was significantly lower in the older group (Ben-Aryeh
et al., 1986). In contrast, no differences in amylase activity in
stimulated parotid saliva was found comparing three age
groups of 23—39 years, 40—59 years, and 60—84 years with
a total of n = 128 participants (Aguirre et al., 1987). Similarly,
in a study comparing amylase activity in unstimulated whole
saliva of n = 100 healthy subjects in four age groups (0—25
years, 26—40 years, 41—65 years, and 66—80 years) no
differences were found (Salvolini et al., 1999). Pajukowski
et al. investigated salivary parameters in n = 169 frail elderly
persons between 69 and 96 years of age. Amylase activity in
stimulated saliva was not associated with age, dentate sta-
tus, diseases and drug use (Pajukoski et al., 1997).
Taken together, it appears that basal sAA activity does not
change over the adult life span and remains constant even in
older age. A general methodological problem with the studies
summarized above is that amylase was measured only once
per subject at a single time-point. Since amylase is stress
responsive and underlies pronounced diurnal variations,
results from studies taking single saliva samples at more or
less standardized time points are of limited value. Unfortu-
nately, studies that assessed full diurnal profiles are rare, and
although many of them tested for age effects, their age
ranges are too limited to draw any conclusions (see for
example Nater et al., 2007; Rohleder et al., 2008; Wolf
et al., 2008).
3.4.2. Impact of age on acute responses
Acute sAA responses have mainly been investigated in
younger adult populations. However, some studies have sub-
jected younger individuals to acute stress paradigms, and
none have looked at older populations. Schaffer et al. (2008)
collected saliva using cotton swabs from n = 18 low birth
weight and n = 36 normal weight neonates before and after
a heel prick test. Although sAA was in the detectable range of
their assay used, no significant increases were found (Schaf-
fer et al., 2008). Similarly, in a study subjecting n = 54 two-
year olds to a range of toddler-specific stress tasks, no
significant responses were found. It remains to be investi-
gated whether these non-responses are due to the nature of
the task, or if toddlers are too young to mount a sAA response
(Fortunato et al., 2008).
There is a larger body of literature in which acute sAA
responses are investigated in older children and adolescents.
Results of these studies show that adolescents are capable of
mounting sAA responses in similar magnitudes as adults.
However, no direct comparisons between youth and adults
have been made within any of these studies. Gordis et al.
have subjected adolescents between 9 and 15 years of age to
a modified version of the TSST in two separate studies with a
total n of 151 participants. a-Amylase measured in unstimu-
lated whole saliva increased significantly in both studies. In
one of the studies, association of sAA with age was statisti-
cally tested, and came out non-significant (Gordis et al.,
2006, 2008). Stroud et al. compared n = 39 children aged 7—
12 years with n = 43 adolescents aged 13—17 years on their
responses to a modified TSST for children and a peer rejection
task. While the modified TSST failed to induce significant sAA
increases, age differences emerged in responses to the peer
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Determinants of salivary a-amylase
rejection task, with significantly lower responses in children
compared with adolescents (Stroud et al., in press).
In summary, acute sAA stress responses appear to be
absent in newborns, are detectable in children who are at
least 7 years old, and reach adult magnitude in adolescents.
3.5. Smoking, drugs, and alcohol
3.5.1. Smoking
3.5.1.1. Acute response to tobacco smoke. Since nicotine
activates the SNS (Shinozaki et al., 2008), cigarette smoking
could be expected to be associated with increased sAA activity.
Instead, Zappacosta et al. report that smoking a single cigar-
ette decreased sAA activity in n = 20 healthy smokers of both
genders by 44% (Zappacosta et al., 2002). Similar results were
obtained in studies in which whole human saliva was exposed to
cigarette smoke in vitro. Nagler et al. report 34% decreases of
amylase activity after 3 h of incubation with intermittent
smoke exposure (Nagler et al., 2000), and another study
documented a 85% decrease after 1 h incubation with cigarette
smoke, and a 40% reduction after incubation with the parti-
culate part of cigarette smoke on amylase and other salivary
enzymes (Greabu et al., 2007). This apparent contradiction can
be explained by direct noxious effects of tobacco smoke not
only on oral tissues, but also on salivary proteins including a-
amylase. Specifically, aldehydes, which are a major component
of tobacco smoke, react with and modify sulphydryl groups of
salivary enzymes, leading to functional impairment (Leuch-
tenberger et al., 1974; Zappacosta et al., 2002).
3.5.1.2. Effect of habitual smoking on basal salivary a-
amylase. An important question arising from these findings is
whether sAA activity differs between habitual smokers and
non-smokers. Callegari and Lami in 1984 reported lower a-
amylase activity in saliva of n = 32 middle-aged habitual
smokers who consumed more than 20 cigarettes per day,
in comparison with n = 40 non-smoking controls (Callegari
and Lami, 1984). In two studies, responses to different stress
tests were compared between smokers and non-smokers, and
children of mothers who were habitual smokers. Although
stress tests in both studies did not significantly increase sAA,
baseline levels were again lower in habitual smokers com-
pared to non-smokers in both studies (Granger et al., 2007a;
Goi et al., 2007). Importantly, children of mothers who
smoked also had lower amylase concentrations, which was
probably mediated by smoke exposure, because these chil-
dren also had higher levels of cotinin (Granger et al., 2007a).
Some studies did not find altered basal sAA in habitual
smokers. Nagaya and Okuno compared n = 106 smokers and
did not find differences although smokers consumed on aver-
age 22 (men) or 10 (women) cigarettes per day (Nagaya and
Okuno, 1993). In otherwise healthy periodontitis patients, a-
amylase in unstimulated whole saliva was not different
between smokers and non-smokers (Zuabi et al., 1999).
In the only study that assessed a diurnal profile with regard
to daily amylase activity, smokers’ sAA decreased more
rapidly in the first hour after waking up, but levels did not
differ during the remainder of the day. Additionally, it was
found that having smoked one to three cigarettes in the hour
before saliva collection, which happened in 7% of measure-
ment occasions, was not associated with subsequent sAA
levels (Nater et al., 2007).
479
In summary, tobacco smoke does have a strong inhibitory
impact on salivary enzymes including a-amylase. Even if not
all studies found generally lower concentrations in habitual
smokers, smoking status is emerging as an important variable
to control for, and acute smoking should be avoided before
taking saliva samples to avoid false negative measurements.
3.5.2. Alcohol
3.5.2.1. Acute response to alcohol consumption. An earlier
animal study showed that acute intraperitoneal injection of
ethanol into fasted rats reduced protein synthesis in major
salivary glands (Proctor et al., 1993). In humans as well,
acute consumption of alcohol has been shown to impact
saliva secretion and composition. Enberg et al. report sig-
nificant reductions of amylase activity in stimulated saliva of
healthy volunteers who consumed 0.7 (men) or 0.6 (women)
grams of alcohol per kg body weight (Enberg et al., 2001). In a
more realistic experimental setup, 10 healthy participants
drank 300 ml of alcoholic (5.2%) vs. non-alcoholic beer in a
cross-over design. Salivary flow rate and amylase activity was
measured repeatedly before and after beer consumption in
stimulated saliva. Flow rate dropped slightly after alcoholic
beer consumption, but no differences in amylase activity
were found (Brand et al., 2006).
3.5.2.2. Effect of habitual drinking on basal salivary a-
amylase. Again, an important question is whether habitual
drinkers show differences in basal sAA activity. One animal
study showed that chronic alcohol treatment of rats over
three months decreased salivary flow rate and a-amylase
activity, along with structural changes and fat accumulation
in parotid glands (Maier et al., 1986). A later study, however,
although treating rats for about the same time, did not
detect changes in sAA, but in other proteins and in flow rate
(Scott and Berry, 1989).
Two studies have investigated the impact of chronic alco-
hol consumption on sAA in humans. Nagaya and Okuno again
did not find differences between light drinkers (24 g alcohol
per day in men, 5 g in women) (Nagaya and Okuno, 1993). In
contrast, Dutta et al. reported that salivary flow and amylase
activity of stimulated parotid saliva were significantly
decreased in a group of n = 24 ‘‘alcoholic participants’’ com-
pared with a control group (Dutta et al., 1992).
In summary, studies are unequivocal so far, but it might be
cautiously concluded that acute as well as chronically high
alcohol consumption tends to lower sAA concentrations.
Maier et al. speculate about the mechanism of altered
amylase output in chronic alcohol consumption (Maier
et al., 1986). Alterations might be the consequence of
morphological alterations in salivary glands, which were
characterized by enlargement and fat accumulation
observed in rats, or by alcohol-induced changes in autonomic
innervation of the salivary glands, as reported by Perec et al.
(1979). Future studies will have to replicate these findings
and determine thresholds. Until then it is advisable to control
for chronic alcohol consumption and ask study participants
not to drink any alcohol before experiments.
3.5.3. Medical drugs
Most attention has been given to agonist and antagonists of
adrenergic receptors. As outlined above and discussed in
more detail in Nater and Rohleder (submitted), amylase
 Author's personal copy
480
release is stimulated by activation of b-adrenergic receptors
and reduced by blockade of these receptors. It is therefore
advisable to carefully screen for, and control or exclude the
use of adrenergic agonists and antagonists that could be part
of antihypertensive, asthma, or other medication.
3.5.4. Caffeine
Only two studies have investigated whether caffeine has an
effect on sAA, but both show that caffeine appears to
stimulate sAA activity. In a study investigating stress and
noise in 11 nurses on a pediatric intensive care unit, caffeine
intake was assessed as a control variable. Greater caffeine
intake, but not stress and noise, predicted higher sAA con-
centrations (Morrison et al., 2003). Bishop et al. adminis-
tered 6 mg/kg body weight of caffeine vs. placebo in a cross-
over design to eight endurance trained men. Caffeine inges-
tion led to significant increases of amylase activity and
output, which did not return to baseline at the last measure-
ment occasion 3.5 h later (Bishop et al., 2006). These sti-
mulating effects of caffeine on sAA might be mediated by
caffeine-induced activation of the SNS (Laurent et al.,
2000). It has not been investigated whether higher habitual
caffeine consumption is associated with generally higher sAA
concentrations, and if acute responses of sAA differ in
habitual caffeine consumers.
3.6. Food
3.6.1. Association of carbohydrate contents of diet
with basal a-amylase
Wesley-Hadzija and Pigon compared a-amylase activity in
unstimulated saliva of three populations in Ghana living on
different diets. a-Amylase concentrations were signifi-
cantly higher in a group of n = 18 Ghanaians who lived
on a predominantly carbohydrate diet, as compared with
Ghanaian students and Western Europeans living on a
‘‘protein-balanced’’ mixed diet (Wesley-Hadzija and Pigon,
1972). However, when Mazengo et al. compared sAA activ-
ity between individuals living in rural vs. urban areas of
Tanzania, those in the rural areas with more carbohydrate
consumption had lower amylase levels (Mazengo et al.,
1994).
More recently, Perry et al. investigated the association of
diet and sAA using a genetic approach. The number of
amylase gene copies, which is proportional to amylase pro-
tein in saliva, was compared between different populations
characterized by high vs. low starch diets. Results show a
significantly higher number of amylase gene copies in agri-
cultural high-starch populations (such as European-Ameri-
cans and Japanese) as compared with low-starch populations
(such as Rainforest Hunter-Gatherers and the mainly fish-
consuming Yakut). They conclude that these differences in
amylase gene copy number have developed through natural
selection (Perry et al., 2007). These associations between
historical diet and amylase gene copy number and baseline
activity raises the question whether there should be ethnical
or cultural differences in amylase activity. However, most of
the studies summarized here have investigated sAA either in
European/North American or in Japanese populations, both
of which are characterized as high-starch populations by
Perry et al. (2007). No further data are available on ethnical
or cultural differences in basal sAA activity.
N. Rohleder, U.M. Nater
Few studies have investigated shorter-term effects of
specific diets or dietary changes on sAA. Patients with coeliac
disease (n = 128) who have to live on a gluten-free diet were
not found to have different amylase activity in stimulated
whole saliva than healthy controls (n = 55) (Lenander-Lumi-
kari et al., 2000). In a longitudinal study by Johansson and
Birkhed, n = 20 participants (27—61 years, 16 women) shifted
their diet from a mixed diet to a lactovegetarian diet, which
led to significant increases in carbohydrate consumption
relative to fat and protein. No changes in amylase activity
in stimulated whole and parotid saliva were observed after 3,
6, 12 and 48 months (Johansson and Birkhed, 1994).
3.6.2. Acute stimulation of amylase by food
Several acute stimuli associated with food impact salivary
flow and composition. Gustatory or mechanical stimuli are
frequently used to stimulate flow rate, for example applica-
tion of citric acid (Froehlich et al., 1987) or chewing on
paraffin wax (Mackie and Pangborn, 1990). Many of these
stimuli also change the protein content of saliva. In rats,
ascorbic acid increased salivary amylase activity and output
(Ekstrom, 2001). In an experiment with rabbits, Gjörstrup
et al. report that different types of foods have differential
stimulatory effects of amylase in parotid saliva, with carrots
inducing higher responses than food pellets (Gjorstrup,
1980). In humans, citric acid but not sucrose, sodium chloride
and starch stimulated a-amylase activity in parotid saliva of
10 healthy subjects (Froehlich et al., 1987). In contrast are
the findings of one study in which 10 human subjects of both
sexes provided parotid saliva after chewing parafilm, bread,
or celery. Salivary flow and amylase output (expressed as
units per min) but not amylase concentration was lowest
after water, intermediate after parafilm and celery, and
highest after chewing bread. Thus, amylase production
was increased in accordance with higher fluid production,
but this increased production did not translate to higher final
amylase concentrations in saliva (Mackie and Pangborn,
1990).
Effects of food on sAA seem to be more pronounced in
response to real food intake. Two studies investigated the
effects of refeeding after a period of fasting. In rats, refeed-
ing after overnight fasting doubled parotid and submandib-
ular protein secretion (Proctor et al., 1993), and similarly in
humans, ingesting different types of carbohydrates induced
increases in amylase activity in parotid saliva of seven women
who had been fasting for 12 h (Behall et al., 1973).
Salivary a-amylase also increased within 15 min after
eating or ‘‘sham-eating’’ (chewing and expectorating) a
standardized meal in n = 12 healthy men and women. How-
ever, these increases were significantly lower in the sham-
eating condition (Messenger et al., 2003). a-Amylase was
shown to increase in response to a standardized lunch in
stimulated saliva of n = 18 healthy human subjects. Further-
more, amylase was found positively associated with self-
ratings of satiety and fullness, and inversely associated with
hunger and desire to eat (Harthoorn and Dransfield, 2008).
Taken together, there seems to be an association of long-
term diet, probably most pronounced on an evolutionary
time scale, with sAA production. Gustatory and mechanical
stimuli, but more effectively, ingestion of real food, induce
acute increases of sAA in most of the published studies. It is
therefore highly advisable to assess and control for the time
 Author's personal copy
Determinants of salivary a-amylase
of food intake in studies measuring diurnal variation of sAA,
and to ask participants of acute stress studies not to have any
meals an hour before the experiment. Unfortunately, no data
are available to answer the questions whether acute stress
responses are influenced by nutritional status or recent food
consumption. However, one study showed that having con-
sumed a glucose drink did not change the sAA response to
exercise (Bishop et al., 2000).
3.7. Physical exercise
3.7.1. Acute response to exercise
Several earlier studies have investigated the effects of acute
physical exercise on sAA. Gilman et al. measured a signifi-
cantly higher concentration of sAA during intense exercise in
comparison to a control period (Gilman et al., 1979). Dawes
et al. reported significant increases in total protein contents
of stimulated parotid saliva in response to running between 4
and 13 km (Dawes, 1981). Similarly, a cross-country run of 2 h
was found to increased activity of a-amylase in whole saliva
of n = 25 participants (Nexo et al., 1988).
Exercise was also one of the conditions that Chatterton
et al. (1996) used in their seminal paper for testing the
association of sAA and plasma catecholamine responses.
Here, significant increases of sAA were found after 20 min
of running as well as intermittent cycling on a stationary bike,
and increases were associated with catecholamine changes.
In addition, they showed a dose—response effect with highest
sAA increases after running as compared to light jogging and
walking (Chatterton et al., 1996). Calvo et al. subjected
n = 20 healthy young men to an incremental exercise test
on a treadmill leading to exhaustion, and reported an initial
decrease of sAA activity followed by a continuous increase.
Interestingly, this second, increasing phase paralleled that of
lactate concentrations in capillary blood, and the authors
suggested using the sAA response to exercise as predictor for
aerobic fitness (Calvo et al., 1997). These results were later
replicated in n = 12 endurance trained men (Chicharro et al.,
1999). In n = 15 healthy men, 2 h of cycling at 60% maximal
oxygen consumption stimulated amylase in saliva collected
with salivettes (Bishop et al., 2000). These findings were
later replicated and it was further shown that caffeine
potentiated the exercise stimulated sAA increase (Bishop
et al., 2006).
Intermittent high-intensity exercise in n = 8 trained men
induced fivefold increases in sAA, which returned to base-
line after 2.5 h (Walsh et al., 1999). In a later study, the
same group tested the effect of different times during the
day, this time employing 2-h cycling exercises, and found
similar significant amylase increases in morning and after-
noon exercises (Li and Gleeson, 2004). In the most recent
study from the same group, it was shown that sAA activity
responded even to short-term exercise of 20 min at inten-
sities of 50% maximal oxygen consumption (Allgrove et al.,
2008). Amylase increases were also found after more
intense and/or longer bouts of exercise, such as running
a marathon, which led to 2-fold amylase increases in
stimulated whole saliva of n = 21 runners (Ljungberg
et al., 1997), and competing in an Olympic Distance
triathlon, which led to 3.6-fold amylase increases in sti-
mulated whole saliva of n = 42 healthy athletes (Steeren-
berg et al., 1997).
481
The effects of minor physical activity on basal diurnal sAA
were investigated in a recent study. No association of activ-
ities such as running for a bus, or climbing a set of stairs, in
the hour before saliva sampling was associated with
increased amylase concentrations. However, these activities
were of lower duration and intensity than any of those
summarized above (Nater et al., 2007).
3.7.2. Effect of training status on basal salivary a-
amylase
In contrast to the well-documented acute sAA response to
exercise, there are virtually no studies that investigated
whether endurance-trained individuals differ from sedentary
people with regard to basal sAA activity.
Taken together, it can be concluded that exercise is a
reliable determinant of increases in sAA activity, even after
short duration and with lower intensity. This implies that
physical activity should be assessed and controlled for in
experiments using sAA as an outcome measure. Diurnal
profiles, however, seem to be relatively robust against minor
non-exercise activity.
3.8. Somatic and psychiatric diseases
Salivary a-amylase has been, among other parameters of oral
health, investigated in oral and dental diseases. Periodontitis
for example has been shown to be associated with changes in
sAA concentrations (Henskens et al., 1996). Hence, oral
diseases should always be excluded or controlled for in
experiments involving sAA. Additionally, amylase concentra-
tions in saliva have also been found altered in other somatic
diseases, while less data is available on sAA in psychiatric
diseases.
Data on alterations of basal sAA concentrations in somatic
diseases are incomplete as well, except maybe a good num-
ber of studies on oral diseases, but there are for example no
studies on sAA in cardiovascular disease. Nevertheless, some
diseases have been investigated. Lower sAA concentrations
have been found in children suffering from asthma and atopic
dermatitis (Crespi et al., 1982; Ryberg et al., 1987; Wolf
et al., 2008). Similarly, lower amylase has been found in
saliva from children with juvenile idiopathic arthritis (Brik
et al., 2006) and in adolescents with cerebral palsy (Rodri-
gues Santos et al., 2007). Some autoimmune diseases such as
Sjögren’s syndrome appear to be also associated with lower
sAA activity. Since Sjögren’s syndrome can also be diagnosed
secondary to other autoimmune diseases such as rheumatoid
arthritis and systemic lupus erythematosus, autoimmune
conditions should be excluded in study participants (Mathews
et al., 2008).
Higher sAA concentrations have been reported in chronic
obstructive pulmonary disease (COPD) patients. Interest-
ingly, within the group of COPD patients, those who were
habitual smokers had lower sAA concentrations (Yigla et al.,
2007), a finding that is consistent with the inhibitory effect of
cigarette smoke on sAA activity (see above). Higher sAA
activity has been found in Parkinson’s disease patients (Tumi-
lasci et al., 2006).
Finally, sAA activity has been investigated in individuals
with diabetes. A recent study reported increased a-amy-
lase in saliva of patients suffering from type-2 diabetes
(Aydin, 2007). A series of earlier studies did not find sAA
 Author's personal copy
482
differences between patients with type-1 diabetes and
controls (Tenovuo et al., 1986), or between type-1 and
type-2 diabetes and controls (Artino et al., 1998; Ben-
Aryeh et al., 1993).
Taken together, while the literature on sAA alterations in
somatic diseases is sparse at present, it can be safely con-
cluded that somatic conditions which present with changes in
SNS activity have the potential to alter sAA concentrations.
We therefore recommend to carefully screen participants for
the presence of any disease process and either exclude or
control for these conditions.
4. Summary and conclusions
4.1. Summary of recommendations
On the previous pages we have described the basic metho-
dological requirements for measuring and analyzing salivary
a-amylase, and we have derived some recommendations that
should help researchers in the field of PNE to successfully
design and analyze studies employing sAA. These recommen-
dations are summarized in Table 1.
We have further reviewed the recent literature on poten-
tial factors influencing basal or stress-induced sAA measure-
ments. Although information is still scarce on some issues
such as the impact of psychiatric diseases on sAA, in other
areas, such as age- and sex-differences, more data is avail-
able. Nevertheless, data are sufficient in many areas to
derive recommendations for excluding or controlling for
specific determinants. A summary of determinants of basal
and stress-induced sAA is given in Table 2, along with recom-
mendations on how to deal with each.
4.2. Conclusions and future directions
While the current state of the literature enabled us to give
some specific recommendations on practical issues of sAA
measurements, such as sampling method, storage and
analysis, as well as on the impact of some standard impact
factors, the review of the determinants also shows that
many important questions have not been explored. Future
studies will have to find answers to such basic questions as
the impact of training status or body mass index, and the
impact of age on sAA stress responses. It will also be
important to explore if basal sAA activity is increased in
psychiatric conditions that are characterized by heigh-
tened sympathetic activity, and further research will need
to establish whether individual differences in acute stress-
responses or basal sAA activity are useful as predictors of
future diseases.
Role of the funding source
NR was supported by personal (salary) fellowships by the
German Research Foundation (DFG; Ro 2353/4-1) and the
Michael Smith Foundation for Health Research (MSFHR). UMN
has no funding source to report
Conflict of interest
The authors do not have any conflicts of interest to report.
N. Rohleder, U.M. Nater
Acknowledgements
The authors would like to thank Clemens Kirschbaum and
Ulrike Ehlert for their support and theoretical input. Further-
more, NR acknowledges support by the German Research
Foundation (NR; DFG; Ro 2353/4-1) and the Michael Smith
Foundation for Health Research (MSFHR).
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