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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
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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


tube, without the need for technically sophisticated instru-
The salivary enzyme a-amylase has been proposed as a mentation. Despite the convenient assessment, some char-
marker for stress-induced activity of the sympathetic nervous acteristics of this salivary enzyme also entail some pitfalls
system (SNS) and recent studies have underscored the use- that might complicate study design and interpretation of
fulness of salivary a-amylase (sAA) in this regard. a-Amylase results. Since the literature reporting sAA findings is con-
is one of the major protein components of saliva. Its main tinually growing, guidelines for collection, analysis, and
function is the enzymatic digestion of carbohydrates (Baum, interpretation of this new sympathetic stress marker are
1993), but it is also important for mucosal immunity in the needed. The goal of this review is to provide recommenda-
oral cavity, as it inhibits the adherence and growth of bac- tions for the measurement of sAA in human studies in the field
teria (Scannapieco et al., 1993). of psychoneuroendocrinology (PNE). In the following, we will
Based on the early observation by Gilman et al. (1979) that therefore first provide a review of methodological issues,
sAA increases in response to exercise (Gilman et al., 1979), followed by a summary of determinants of basal and stress-
Chatterton et al. (1996) in their seminal paper reported a induced sAA activity in human saliva.
significant positive correlation between sAA and plasma
norepinephrine in response exercise (r = 0.64) and suggested 2. Methodological considerations for
the use of sAA as a marker for SNS activity (Chatterton et al.,
1996). Since then, several independent studies have con-
measuring salivary a-amylase in humans
firmed the responsivity of sAA to psychosocial stress and
physical exercise, and additional evidence for the association In this section, we will briefly explain the mechanism of saliva
of amylase responses with sympathetic activation has been production and protein secretion, describe the major saliva
reported (for a review see Nater and Rohleder (submitted)). collection techniques, laboratory assays, and derive recom-
As a substance that can be relatively easily and cost- mendations for the researcher. These recommendations are
effectively assessed in human saliva, it is likely that the summarized in Table 1.
number of studies using this parameter will increase in the
near future, especially given that evidence linking it to SNS 2.1. Mechanism of saliva secretion
function is accumulating, and as a consequence of the fact
that sAA as salivary parameter has some advantages over Saliva production and secretion is a complex process, and
classical electrophysiological measures such as skin conduc- knowledge of this process can help to avoid methodological
tance and heart rate measures. The major advantage of a errors during saliva collection, storage of samples, laboratory
saliva-based measure of SNS activity is the convenience of and statistical analysis. A detailed description can be found in
assessing activity of both major stress systems (i.e. SNS and excellent earlier reviews from the field of oral biology (see
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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 stimulated and unstimulated saliva. Specifically, under sti-
Rohleder (submitted). mulated conditions, the contribution of the parotid gland
It is of major importance that salivary a-amylase is not, like increases from 20% to more than 50% of total saliva (Hum-
salivary cortisol, a hormone that originates in an endocrine phrey and Williamson, 2001). This again has major implica-
gland, circulates in blood and diffuses into saliva. In contrast, tions for collection techniques, with regard to where the
amylase is produced by the highly differentiated epithelial saliva that is sampled comes from, and whether the collec-
acinar cells of the exocrine salivary glands (Baum, 1993). It is in tion technique stimulates salivary flow. We will therefore in
these same acinar cells that the primary fluid component of the following review the major collection methods for human
saliva is derived from the local vasculature, and enriched with saliva and discuss their relative advantages and disadvan-
salivary proteins, of which amylase is one of the most impor- tages.
tant. As Baum (1993) points out, there is no spontaneous
secretion of saliva or salivary proteins, but both processes, 2.2. Saliva collection methods
fluid and protein production, are stimulated by parasympa-
thetic and sympathetic nerves innervating the salivary glands. A wide array of different methods for collecting saliva has
The major implication of this mechanism and the fact that been described in the past years. Some of the methods have
amylase, as opposed to cortisol, is not passively transported by been summarized in earlier reviews (see for example White,
saliva, but produced together with saliva and other salivary 1977; Navazesh, 1993), but these works have not discussed
components, is that when collecting saliva, researchers need methods regarding their specific usefulness for the psycho-
to be aware of the fact that all factors influencing salivation neuroendocrinological researcher, e.g. without addressing
can also influence salivary a-amylase. issues of saliva sampling outside of the laboratory, when
Further, saliva is produced by three different types of study participants go after their regular daily lives, or in
major salivary glands, i.e. the parotid glands, the subman- repeated measures designs of acute stress studies.
dibular, and the sublingual glands, which are located pairwise In general, techniques for collecting human saliva can be
on the left and the right side of the oral cavity. These and a categorized into those sampling whole saliva vs. those sam-
number of smaller salivary glands secrete saliva through a pling saliva from specific salivary glands, and into those col-
network of salivary ducts, which enter the oral cavity at lecting stimulated vs. unstimulated saliva. Another distinction
different locations (Humphrey and Williamson, 2001). Par- that can be made is into collection techniques based on
otid saliva enters the oral cavity through Stensen’s ducts absorbent material vs. techniques based on passive drooling
located near the second molars in the upper jaw, while or spitting of saliva into collection tubes. The two techniques
submandibular and sublingual saliva enters the oral cavity most relevant for the PNE researcher are collection using the
through Wharton’s ducts located in the floor of the mouth cotton roll-based salivette (Sarstedt, Nümbrecht, Germany),
(Navazesh, 1993). which belongs to the former group, and the passive drooling
These specific glands differ in their relative contribution technique, which belongs to the latter group.
of protein and fluid saliva components, with about 80% of
amylase being produced by the parotid gland (Zakowski and 2.2.1. Saliva collection based on absorbent materials
Bruns, 1985). In addition, the relative contribution of each The most frequently used collection technique using absor-
specific gland to total salivary volume differs between bent material is probably the salivette. Salivettes consist of a
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472 N. Rohleder, U.M. Nater

cotton roll provided in a plastic container. Participants are researchers to keep their study protocols unchanged. Finally,
usually instructed to remove the cotton roll from the plastic the cotton serves as a filter so that after extraction of saliva
container and to place it in their mouth for intervals ranging by centrifugation, a clear fluid free of mucous components
from 1 to 5 min. Complete salivettes can be stored at con- and other contaminants is available for analysis. Other absor-
ditions described below until saliva is extracted by centrifu- bent techniques also have the advantage of being easy to
gation. This technique has been used by the majority of handle, for example in infants and other situations in which
studies sampling saliva for the determination of cortisol, salivary flow rate is low.
and it is therefore frequently used for amylase determination A disadvantage of salivettes or other absorbent-based
as well. In some experimental protocols, participants are collection techniques is that stimulation of salivary flow
instructed to chew on the cotton to stimulate salivary flow. cannot be completely excluded due to the presence of the
Researchers should be aware of the fact that while this does mechanical stimulus in the mouth, even if participants are
not have any effect on salivary cortisol, stimulated saliva instructed not to chew on the cotton roll (Navazesh, 1993). It
differs markedly from unstimulated saliva with regard to the has also been argued that low volumes cannot be recovered
source (i.e. the secreting gland) and therefore the relative from cotton (Zimmermann, 2008). However, this is a lesser
amount of amylase protein secreted. It is thus recommended problem since volume requirements of modern assays are
to keep this factor constant within each study. Related to extremely low (e.g. requiring only 20 ml of sample).
this, it has been suggested that the position of the cotton role
in the mouth influences from which specific gland the 2.2.2. Saliva collection using ‘‘drooling’’ or ‘‘spitting’’
sampled portion of saliva had been secreted. In fact, Harmon techniques
et al. have recently shown that saliva collected in different The ‘‘passive drooling’’ technique as described by Navazesh
areas of the mouth by placing absorbent cotton swabs in (1993) is probably the best method for collection of unsti-
locations to specifically collect saliva from the three major mulated whole saliva. Participants are instructed to empty
glands produced different results (Harmon et al., 2008). It is their mouths by swallowing all saliva, and then collect all
therefore further advisable to consistently instruct partici- subsequently secreted saliva in the oral cavity for an exact
pants within each study to either place the cotton roll in a period of time, usually between two and five minutes. In the
specific area of the mouth, or to move it around in a circular ‘‘draining method’’, saliva is allowed to drip off the lower lip
pattern to collect saliva from all glands. into the collection tube through a funnel. In the ‘‘spitting
method’’ the whole amount of collected saliva is spit into a
2.2.1.1. Other saliva collection techniques using absorbent collection tube, and the ‘‘suction method’’ implies aspirating
materials. Variations using other absorbent devices have the accumulated saliva from the bottom of the mouth. Of
been described for circumstances in which the use of saliv- these, only the spitting method appears to be feasible enough
ettes is not feasible, for example in infants or in adult to be performed by participants during most experimental
participants during sleep, when low sample volumes are to setups realized in the field of PNE.
be expected. Granger et al. (2007b) describe the use of
braided cotton dental rope (Richmond dental, Charlotte, 2.2.2.1. Variations. The major variation of spitting tech-
NC; Granger et al., 2007b). Briefly, cotton ropes can be niques described in the literature is to intentionally sti-
cut into appropriately sized lengths, usually longer than mulate saliva production either by using gustatory stimuli
needed, to enable the experimenter to hold onto the end such as citric acid (Froehlich et al., 1987) or by using
of the braid and prevent children from swallowing the mate- mechanical stimuli such as chewing on paraffin wax or
rial. The saliva-saturated end of the cotton rope is then parafilm (Mackie and Pangborn, 1990). The major advan-
placed into the barrel of a needleless syringe, and com- tage is that it enables the collection of adequate volumes
pressed using the plunger to express saliva into a collection of saliva. However, as discussed above, stimulating salivary
vial (Gunnar et al., 1989; Granger et al., 2007b). flow significantly changes the contribution of specific sali-
However, Harmon et al. (2007) showed that cotton-based vary glands to total saliva production, and also activates
collection devices are very effective in absorbing fluids, but secretion of salivary proteins. Hence, stimulation of saliva
that the extraction of small volumes by centrifugation or the production should only be used if saliva cannot be obtained
plunger of a syringe as described above can be problematic. otherwise, and should then be applied consistently
They have therefore suggested the use of hydrocellulose throughout each experiment.
microsponges. These are manufactured to absorb tear fluid
during eye surgery and are commercially available (Becton & 2.2.2.2. Advantages and disadvantages. The major advan-
Dickenson, Walton, MA). Tests have shown that these sponges tage of the passive drooling/spitting technique is that unsti-
absorb liquids up to 450 ml, which would make them parti- mulated whole saliva is collected, which yields a
cularly useful for collection of low volumes of saliva, for representative combination of saliva from all glands. Disad-
example in infant studies (Harmon et al., 2007). vantages are that passive drooling needs to be practiced, that
it requires the full attention of the participant, and that it is
2.2.1.2. Advantages and disadvantages. The major advan- not always perceived as clean, because participants are
tages of the salivette method are that saliva sampling is easy asked to spit in a relatively small tube, or use a straw to
to learn and easy to perform, even in public. This method is do so. Because of this, it is a technique that many partici-
usually perceived as clean. Therefore, this technique bears pants are not comfortable with practicing in public places, at
the highest probability of participants adhering to the study work, or generally in company of other people. This method
protocol. Furthermore, salivettes are already in use in many further yields ‘‘unfiltered’’ samples, which can be difficult to
psychoneuroendocrinological laboratories, which enables process for further analyses, because even ultracentrifugation
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Determinants of salivary a-amylase 473

is sometimes insufficient to completely remove mucous com- being collected in the field by participants going after their
ponents or contaminations. regular daily activities, they do not necessarily need to
freeze or refrigerate samples during the collection day,
2.2.3. Saliva collection from specific salivary glands and before and during shipping to the analyzing laboratory.
Specific suction devices have been designed to harvest saliva It is still recommended to store saliva samples in participants’
directly where the ducts enter the oral cavity (i.e. Stenson’s home refrigerators or freezers and to ship them in insulated
ducts for parotid saliva, Wharton’s ducts for sublingual and envelopes to avoid growth of bacteria and mold in samples.
submandibular saliva). These devices allow precise sampling Questions (2)—(4): For long-term storage, saliva samples
of saliva produced by specific glands. However, these meth- should be frozen at !20 8C or lower temperatures. Saliva can
ods are typically employed for very specific questions from be frozen in whole, un-centrifuged salivettes, or transferred
the field of oral biology, and will therefore not be described to space-saving containers such as deep-well plates or 1.7 ml
here (see White, 1977; Navazesh, 1993). vials. It has also been shown that amylase is not affected by
repeated freeze-thaw cycles (Granger et al., 2006). Because
2.2.4. Recommendations for collecting saliva freezing and thawing breaks down mucopolysaccharides in
Taken together both of the major collection techniques that saliva that could interfere with pipetting, it is recommended
have been in use for the measurement of salivary cortisol, to subject samples to at least one freeze-thaw-cycle, even if
i.e. salivettes and passive drooling, can generally be recom- time between sample collection and measurement is short
mended for sAA as well. From a theoretical standpoint, the (Shirtcliff et al., 2001).
passive drooling technique is superior to techniques using Question (5) There is no need for preservatives. It has
absorbent materials, because stimulation of saliva produc- been shown that sodium azide should not be used as a
tion is avoided and a representative combination of saliva preservative, because it increases amylase activity (Maurus
produced from the major salivary glands, i.e. whole saliva, is et al., 2008), thus leading to false high assay results (Decaro,
collected. However, passive drooling should only be used if 2008).
sample collection can be done under supervision, or if parti-
cipants are highly motivated and have been well trained in 2.4. Measurement of salivary a-amylase
the collection procedure. Therefore, from a practical point
of view, the use of salivettes is superior if circumstances do 2.4.1. Enzymatic measurement techniques
not allow thorough training and close monitoring of the The enzyme kinetic determination of pancreatic a-amylase
collection procedure and if saliva collection has to be done in human serum is an important everyday clinical diagnostic
by participants during their normal lives. If salivettes are test, which has led to the availability of a large number of
used, participants should be instructed to place the cotton different test methods. Since salivary and pancreatic a-
rolls in their mouths for an exactly defined period of time amylase share a homology of 97% (Lorentz, 1998), salivary
(usually, two minutes are sufficient), and they should be a-amylase can be measured using established methods with-
instructed not to chew on the cotton. out major modifications. Most of these commercially avail-
For all techniques, it is advisable to instruct participants able tests are produced for integrated biochemistry
to avoid eating or drinking (anything but water) at least an analyzers such as the Cobas systems (Roche, Basel, Switzer-
hour before saliva collection. Participants should also rinse land), which are typically used in hospital central labora-
their mouth with clear water to avoid contamination of saliva tories. While these might be available to some researchers,
samples with food components, and to avoid activation of those without access to one of those instruments or labora-
salivary flow or protein production by gustatory stimuli. tories can use the same reagents to build smaller scale
Furthermore, using citric acid or other acidic components assays.
is not recommended, as it can interfere with assay results Recommendations of the International Federation of Clin-
because it changes the pH of the sample. ical Chemistry and Laboratory Medicine (IFCC) for the mea-
surement of pancreatic and salivary a-amylase are provided
2.3. Handling and storage of samples by Lorentz (1998). Assays for enzyme kinetic measurement of
sAA can be performed using 96-well microtiter plates and
Several questions have to be answered to safely handle and standard laboratory absorbance readers as for example
store saliva samples that are going to be used for determina- described by Bosch et al. (1996, 2003).
tion of salivary a-amylase: (1) How long can samples be Briefly, to measure sAA using an enzyme kinetic assay,
stored at room temperature, or in regular household fridges saliva containing amylase has to be incubated with a specific
before frozen? (2) How long can samples be stored at !20 8C chromogenic substrate, for example 4,6-ethyliden-G7-PNP
or !80 8C? (3) How many freeze-thaw-cycles does amylase and the auxiliary enzyme a-glucosidase (both substances are
survive? (4) In which type of container should saliva be included in the substrate reagent ‘‘amylase EPS’’, Roche
stored? (5) Should preservatives be used? Diagnostics). a-Amylase cleaves the substrate into inter-
Question (1): A recent methodological study showed that mediate products, which are further broken down by the
amylase is stable at room temperature (22 8C), and even at auxiliary enzyme into p-nitrophenol (PNP) and glucose. PNP
higher temperatures (37 8C) for up to three weeks without absorbs light at a wavelength of 405 nm (yellow). The more
significant loss in activity (Decaro, 2008). These results are in a-amylase is present in the saliva sample, the more substrate
accordance with an earlier study, which revealed that amy- is broken down into PNP in a specific period of time, and the
lase is stable at room temperature, and at 4 8C for at least higher the optical density that can be measured at 405 nm.
four days (Granger et al., 2006). This has important implica- In a typical assay protocol, saliva is diluted between 1:200
tions for storage and shipping. Specifically, when saliva is and 1:1000 and 20 ml of diluted saliva are pipetted into the
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474 N. Rohleder, U.M. Nater

wells of a 96-well microtiter plate made out of clear plastic plication of amylase activity measured in U/ml with flow rate
(preferably polystyrene; ‘‘PS’’). Eighty microliters of com- measured in ml/min. Saliva flow rate is typically determined
mercially available substrate is added and the plate is incu- by collecting all saliva secreted during a specified time period
bated at 37 8C to start the enzymatic degradation of the of 2—5 min, and gravimetrically determining the volume
substrate. The color change is quantified by measuring the assuming that the density of saliva is 1.0 g/ml (Chicharro
microplate in a regular photometric plate reader twice. First et al., 1998). The advantage of the output measure is that it
after warming up to 37 8C and a second time after a defined accounts for changes in saliva flow rate. The disadvantage is
period of 2—5 min with a 405 nm filter. The resulting color that determining flow rate complicates sampling, making it
change is directly proportional to amylase activity. To trans- less suitable for field studies. A recent study comparing
form relative activity to standardized units, a standard curve activity and flow rate did not find differences in stress
made from commercially available standard solution (for responses between both measures (Rohleder et al., 2006).
example ‘‘Calibrator f.a.s.’’, Roche Diagnostics) should be
included on each plate. Alternatively, amylase activity can be 2.6. Statistical handling of salivary a-amylase
calculated using a formula that includes the volumes of data
sample and substrate, the dilution factor, the light path,
and the specific absorption of the p-nitrophenol. Reagents Salivary a-amylase data typically do not show normal dis-
can be obtained from several different vendors (e.g. Roche tributions, but are positively skewed. In addition, variability
Diagnostics), and prepackaged kits containing all reagents is frequently found to be considerably higher than that of for
are commercially available (for example Salimetrics, State example cortisol. This is most likely the result of the multi-
College, PA). tude of factors influencing acute amylase discussed in this
review. Amylase responds faster to acute stress than cortisol,
2.4.2. Other types of assays and its activation threshold in response to physical activity is
While the enzyme kinetic method is the standard technique lower. Furthermore, inconsistency in sampling techniques,
for measuring a-amylase, other assays have been employed. such as different duration of saliva accumulation and chew-
Yamaguchi et al. have described an automated analyzer for ing vs. not chewing, or in experimental procedures, such as
sAA in hand held format (Yamaguchi et al., 2006b). This not excluding sugar intake before saliva sampling, might be
instrument measures a-amylase activity in saliva loaded into further reasons for high variability. Variability might there-
the instrument using a test strip of absorbent paper. Saliva is fore be reduced by adhering to the recommendations given
transferred from the test strip to another strip of paper here. Furthermore, in most studies, distributions have been
saturated with a substrate, and a built-in optical sensor successfully normalized by using for example logarithmic
measures color change after 30 s. While it is unclear at (Rohleder et al., 2006; Nater et al., 2007), or square root
present how ambient temperature and pH of saliva influence transformations (Gordis et al., 2006).
assay results, and how these issues are dealt with, this might After normalization of distributions, further analysis stra-
be developed into a promising tool for field research. tegies depend on the type of data assessed. In studies
In addition, enzyme-linked immuno assays (ELISAs) and measuring acute sAA responses, it is advisable to collect
radio immuno assays (RIAs) have been employed for measure- saliva at baseline (i.e. before an intervention) and fre-
ment of sAA (Ito et al., 1985; Agarwal and Henkin, 1984). quently, e.g. every 10 min, thereafter until at least 30 min
However, ELISA techniques have not been used in any recent after the end of the intervention to capture increase and
studies. One reason might be that ELISA techniques are more recovery of the sAA response (see Fig. 2). Typically, research-
expensive and time-consuming than enzyme-kinetic meth- ers may want to calculate indices that describe the sAA
ods. Theoretically, they would have the advantage of speci- response. Here, simple delta scores between the post-inter-
fically measuring the concentration of the amylase protein, vention and the baseline level are the easiest approach. More
instead of indirectly deriving the concentration from its complex approaches such as the area-under-curve (AUC)
biological activity. have also been used. Given the acute nature of the response,
the AUC relative to increase might be better suited than the
2.5. Unit of measurement AUC relative to ground (Pruessner et al., 2003). Associations
between sAA and catecholamine increases, where detect-
Since most assay techniques employed to date make use of able, were found using simple delta scores (Rohleder et al.,
sAA’s enzymatic activity, the most frequently used unit of 2004).
measurement is enzyme units per milliliter (U/ml). An With respect to daily profiles, it is advisable to take
enzyme unit is defined as the amount of enzyme that cata- several samples throughout the day. As shown in Fig. 1,
lyzes the conversion of 1 mmol of substrate per minute, and is sAA concentrations are subject to strong diurnal variations,
directional proportional to the unit katal (kat), which was with lowest levels after awakening and highest levels in the
endorsed by the Nomenclature Committee of the Interna- late evening. To capture the full diurnal rhythm, samples
tional Union of Biochemistry (NC-IUB) in 1979 (NC-IUB, 1979). should be taken relative to awakening, i.e. at waking up, as
One U corresponds to 16.67 nkat. Although the unit katal is well as 30 and 60 min later (Nater et al., 2007). Further
recommended by the NC-IUB, most data are reported in U/ samples can be taken at specific times or a specific number of
ml. Thus, current assays do not provide concentrations in hours after waking up, but should cover the rest of the day. It
grams or mols per liter, as in other stress markers. is further advisable to assess diurnal amylase at two con-
An alternative way to report amylase data is to express secutive days at the minimum. Although Wolf et al. (2008)
amylase activity relative to saliva flow rate. This measure is have recently shown good stability of amylase measured
referred to as amylase output, and is obtained by multi- between 1 and 14 h after waking up (r’s between 0.48 and
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Determinants of salivary a-amylase 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
Figure 1 Diurnal rhythm of a-amylase and cortisol in saliva of words, specific factors might change amylase activity
n = 76 healthy men and women (mean age = 26.7 years, acutely, such as smoking one cigarette, but being a habi-
range = 18—58 years). (Reprinted from Nater et al., 2007, Copy- tual smoker might also influence basal a-amylase activity
right (2007) with permission from Elsevier.) 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
0.65, all p’s < 0.01), we cautiously assume that stability of
activation and baseline sAA activity, we will review the
diurnal sAA measurements is in the range determined for
effects of the major modulators of amylase activity in
cortisol, as shown for example by Hellhammer et al. (2007).
these three dimensions. A summary of determinants is
Analysis either using growth curve models/hierarchical
given in Table 2.
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 3.1. Basal activity of salivary a-amylase activity
production by using the area-under-curve with respect to
ground (Pruessner et al., 2003) and to calculate the slope of Many biological systems are subject to diurnal oscillations
the diurnal sAA profile. We have for example shown that daily (Mistlberger and Skene, 2004), and salivary flow as well as
amylase production measured with the AUC is associated with salivary protein production appear to be no exception from
shame and depression in young women (Rohleder et al., 2008). that. As early as 1972, Dawes et al. collected unstimulated
Finally, adherence to the protocol should be controlled. whole saliva and stimulated parotid saliva from a sample of
This can be done by either using electronic monitors that eight healthy men and women over several days at 07:00,
register whenever a participant opens a vial to use a salivette 11:00, 14:00, 17:00, and 22:00 h and measured salivary flow
(Nater et al., 2007), or by using palm pilots that signal and protein content, which both exhibited significant diurnal
participants to take a sample, and digitally store sampling rhythms, the latter only in parotid saliva (Dawes, 1972).
time (Rohleder et al., 2008). With regard to the wake-up
response, it has recently been shown that it is further 3.1.1. Earlier studies
advisable to objectively assess wake-up time of study parti- Ferguson et al. (1973) in their introduction provide an exten-
cipants by activity monitors (Dockray et al., 2008). 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
Figure 2 Acute responses of salivary a-amylase and plasma (Rantonen and Meurman, 2000) and in another study measur-
norepinephrine to the laboratory stress paradigm TSST in n = 12 ing amylase in unstimulated saliva sampled hourly between
healthy men and women. (Reprinted from Rohleder et al., 2004, 09:00 and 18:00 h in eight healthy male students (Li and
Copyright (2004) with permission from Wiley-Blackwell.) 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. nounced diurnal rhythm of sAA activity can be found. This
Yamaguchi et al. (2006a,b) measured amylase activity every rhythm is characterized by a strong decrease after waking-up
4 h between 07:00 and 22:00 h using a handheld electronic and gradually increasing levels with peaks in the late after-
measurement device (see above) in n = 15 healthy university noon (Rohleder et al., 2004). This pattern has later been
students and did not observe changes over time (Yamaguchi replicated in a larger sample of 76 healthy subjects of both
et al., 2006a). sexes (44 women and 32 men) again sampling saliva relative
to the individual diurnal rhythm (at wake-up, 30 and 60 min
3.1.2. Studies assessing salivary amylase activity after waking up) and hourly between 09:00 and 20:00 h using
relative to individual diurnal rhythm the cotton-based salivette (see Fig. 1; Nater et al., 2007).
Rohleder et al. have shown in a pilot study of 17 healthy Two more recent studies focusing on specific determinants of
subjects of both sexes using salivettes, that when samples daily sAA secretion (see below) were successful in further
are obtained relative to awakening, e.g. immediately, as well replicating this pattern in 56 healthy young women (Rohleder
as 30 and 60 min after waking up, and at different times in et al., 2008), and in children and adolescents of both sexes
the afternoon, such as 11:00, 15:00, and 20:00 h, a pro- between 8 and 18 years (Wolf et al., 2008).
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Determinants of salivary a-amylase 477

Michaud et al. investigated the impact of different sleep conditions. Again, no sex differences were found (Nater,
conditions on wake-up profiles of sAA in n = 12 healthy sub- 2004). In all studies, however, the number of participants
jects of both sexes (7 men and 5 women) using salivettes at was too low to exclude non-significant findings due to lack of
wake-up, 30 and 90 min later, at 12:00 and 17:00 h. a-Amy- power.
lase showed a pronounced rhythm with low levels in the
morning and higher levels in the afternoon. However, in 3.3.1.1. Effect of variation in sex hormones. The impact of
contrast to the studies above, there was no drop in amylase natural variation of gonadal steroids on sAA was investigated
activity in the first 30 min after waking up (Michaud et al., in some studies. Variations of salivary amylase activity over
2006). Diurnal rhythms of amylase were not influenced by the menstrual cycle have been investigated. Tenovuo col-
sleep disturbance (noise) or conditions (at home vs. sleep lected paraffin-stimulated whole saliva every second/third
laboratory). day of one menstrual cycle in 14 women not using oral
contraceptives. When analyses were restricted to the only
3.2. Acute salivary a-amylase responses three women in the sample displaying a regular menstrual
cycle, profound variation was observed. However, a-amylase
As described above saliva production and composition is levels did not differ between menstruation and ovulation in a
controlled by the autonomic nervous system. Since the SNS larger sample of 12 women. It is therefore unclear whether
is a fast-reacting stress system, and changes in autonomic the variation observed in the subsample of three women can
balance occur in response to for example physical activity be attributed to the menstrual cycle or is due to other factors
and psychological factors, it is not surprising that sAA shows (Tenovuo et al., 1981). Laine et al. (1991) found no impact of
high variability on top of the diurnal rhythm described above. menstrual cycle phase on salivary flow and amylase in par-
Factors that influence sAA activity, or modulate the sAA stress affin-stimulated whole saliva in 11 women not using contra-
response will be summarized in the following section. ceptives. There were also no differences between 11 women
using oral contraceptives and 10 men (Laine et al., 1991). In
3.3. Impact of sex n = 220 students aged 14—18 years, no variation of morning
and afternoon amylase over one menstrual cycle was found
Many hormonal systems show sex differences in basal activity (Bhoola et al., 1978).
or stress response patterns, as for example the hypothala- Laine et al. (1988) measured amylase and other salivary
mus—pituitary—adrenal-axis (Kirschbaum et al., 1999). The parameters in 16 women throughout pregnancy and lactation
SNS is subject to sex differences as well. Women are reported in paraffin-stimulated saliva and did not detect any signifi-
to show lower norepinephrine responses to activating stimuli cant changes (Laine et al., 1988). No significant differences
(Gustafson and Kalkhoff, 1982), and catecholamines vary in amylase were also measured in parotid saliva of 107
over the menstrual cycle (Goldstein et al., 1983). Therefore, pregnant women of all trimesters and seven controls (D’Ales-
it might be expected that basal sAA activity and stress sandro et al., 1989). In contrast, Salvolini et al. found some
responses are influenced by sex or sex hormones as well. variation of amylase measured in unstimulated whole saliva
of n = 45 pregnant women. Amylase activity was highest after
3.3.1. Sex differences in basal salivary a-amylase 10 weeks of pregnancy, decreased thereafter to reach similar
Women and men participated in most of the experiments levels as 15 non-pregnant control women at week 40 (Salvo-
assessing diurnal rhythms of sAA and acute responses to lini et al., 1998).
stress, exercise, or other conditions. Only some reported Taken together, the few available data do not support the
sex-specific analyses, but none of those yielded significant assumption that basal sAA differs between women and men,
differences. varies systematically with the menstrual cycle in women, or
Rantonen and Meurman (2000) did not find differences is affected by oral contraceptive use. Available data is
between 14 women and 16 men participating in their diurnal inconclusive with regard to pregnancy.
variation study (see above) (Rantonen and Meurman, 2000).
Of the 76 participants in our previous study assessing diurnal 3.3.2. Sex differences in acute responses
rhythms, 44 were premenopausal women. Growth curve Some acute stress studies investigated women and men, and
analyses revealed no sex differences in average sAA concen- reported results of sex-specific statistical analyses. Acute
trations nor in slopes of the diurnal rhythm (Nater et al., sAA responses to different stressors did not differ between
2007). Same results were also obtained in the pilot study in women and men. Unstimulated saliva was collected in n = 83
which twelve women and five men participated (Rohleder volunteers by passive drooling before and after viewing a
et al., 2004). video of an eye surgery procedure. a-Amylase responses did
Harm and Schlegel measured sAA before a parabolic flight not differ between men and women (Takai et al., 2007).
as a marker of motion sickness susceptibility in six women and Forty-two healthy adults (21 men and 21 women) partici-
10 men using salivettes, and no sex differences were found pated in a rowing ergometer competition. Unstimulated
(Harm and Schlegel, 2002). No sAA sex differences were whole saliva was collected before and after each ‘‘race’’
further found in a study comparing 18, 30, and 42 months of about 6—8 min duration. No sex differences in reactivity
old children (15 girls and 16 boys in each age group) (Dezan were found (Kivlighan and Granger, 2006). Finally, a-amylase
et al., 2002) and in a study comparing 31 men and 32 women was measured in saliva obtained using salivettes in 27 women
of different age groups (Ben-Aryeh et al., 1986). Finally, and 10 men before and after an oral academic examination of
Nater investigated resting amylase in saliva obtained with 30 min duration. Exams induced significant increases in sAA
salivettes from 27 women and 26 men, carefully controlling activity, but increases of men and women did not differ in
time of day, menstrual cycle phase in women, and ambient magnitude. Within the group of women, using oral contra-
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478 N. Rohleder, U.M. Nater

ceptives did not modulate sAA responses (Schoofs et al., an old group with 66 years average age. No age differences
2008). were found for stimulated and unstimulated whole saliva, but
In contrast, pregnancy had a profound effect on sAA the activity of amylase in resting and stimulated parotid
increases induced by the Trier social stress test (TSST). Stress saliva was significantly lower in the older group (Ben-Aryeh
responses were significantly reduced in 30 women in the et al., 1986). In contrast, no differences in amylase activity in
second trimester compared to 30 non-pregnant controls, stimulated parotid saliva was found comparing three age
and even further attenuated in 30 women in the third groups of 23—39 years, 40—59 years, and 60—84 years with
trimester (Nierop et al., 2006). a total of n = 128 participants (Aguirre et al., 1987). Similarly,
in a study comparing amylase activity in unstimulated whole
3.4. Impact of age saliva of n = 100 healthy subjects in four age groups (0—25
years, 26—40 years, 41—65 years, and 66—80 years) no
3.4.1. Impact of age on basal a-amylase differences were found (Salvolini et al., 1999). Pajukowski
3.4.1.1. Early development. Development of sAA produc- et al. investigated salivary parameters in n = 169 frail elderly
tion in the first months and years of the life of human persons between 69 and 96 years of age. Amylase activity in
newborns has been studied. In a cross-sectional design, stimulated saliva was not associated with age, dentate sta-
Bellavia et al. collected stimulated whole saliva from 25 tus, diseases and drug use (Pajukoski et al., 1997).
infants between 3 and 15 days after birth. Amylase activity Taken together, it appears that basal sAA activity does not
was measured and was found nine times lower in infants than change over the adult life span and remains constant even in
in saliva of four adults (Bellavia et al., 1979). Two long- older age. A general methodological problem with the studies
itudinal studies followed up newborns during the first weeks summarized above is that amylase was measured only once
and months of their lives. Amylase activity was repeatedly per subject at a single time-point. Since amylase is stress
measured in whole saliva of 13 premature infants between 26 responsive and underlies pronounced diurnal variations,
and 42 weeks gestational age and a positive relationship results from studies taking single saliva samples at more or
between age and amylase activity was observed. Unfortu- less standardized time points are of limited value. Unfortu-
nately, no statistical analysis of this association was reported nately, studies that assessed full diurnal profiles are rare, and
(Hodge et al., 1983). In another study, amylase was measured although many of them tested for age effects, their age
monthly from birth to the age of five months in unstimulated ranges are too limited to draw any conclusions (see for
saliva of n = 29 infants. Amylase increased significantly from example Nater et al., 2007; Rohleder et al., 2008; Wolf
undetectable concentrations in newborns to levels similar to et al., 2008).
adult concentrations at about 3 months (Sevenhuysen et al.,
1984). 3.4.2. Impact of age on acute responses
Another approach to investigate development of sAA Acute sAA responses have mainly been investigated in
activity was to compare amylase in infants of different age younger adult populations. However, some studies have sub-
groups. Amylase was measured in unstimulated saliva jected younger individuals to acute stress paradigms, and
obtained from n = 168 infants between 3 days and 12 months none have looked at older populations. Schaffer et al. (2008)
of age. A positive association of sAA with age was found in collected saliva using cotton swabs from n = 18 low birth
infants younger than 4 months; a plateau was reached in the weight and n = 36 normal weight neonates before and after
4—6 months old group; however, even the 12-month-old a heel prick test. Although sAA was in the detectable range of
group had significantly lower sAA concentrations than adults their assay used, no significant increases were found (Schaf-
(Ben-Aryeh et al., 1984). Looking at slightly older children, fer et al., 2008). Similarly, in a study subjecting n = 54 two-
Dezan et al. found that amylase activity in whole saliva of year olds to a range of toddler-specific stress tasks, no
male and female children was higher in 30-month-old com- significant responses were found. It remains to be investi-
pared to 18-month olds. No difference was observed between gated whether these non-responses are due to the nature of
the age groups 30 and 42 months (Dezan et al., 2002). Finally, the task, or if toddlers are too young to mount a sAA response
amylase in unstimulated saliva was found to be positively (Fortunato et al., 2008).
associated with age in five groups ranging from 25 infants (7— There is a larger body of literature in which acute sAA
11 months), over toddlers (2—3 years), children (6—8 years) responses are investigated in older children and adolescents.
and adolescents (12—14 years) to adults (25—63 years) (Ben- Results of these studies show that adolescents are capable of
Aryeh et al., 1990). mounting sAA responses in similar magnitudes as adults.
In summary, the emerging pattern is that sAA activity is However, no direct comparisons between youth and adults
very low to undetectable in the newborn and after then have been made within any of these studies. Gordis et al.
continually increases to reach adult levels within the first have subjected adolescents between 9 and 15 years of age to
3 years. Developmental stage should therefore be considered a modified version of the TSST in two separate studies with a
when interpreting sAA changes in subjects of varying ages. total n of 151 participants. a-Amylase measured in unstimu-
lated whole saliva increased significantly in both studies. In
3.4.1.2. Development over the adult life span and into one of the studies, association of sAA with age was statisti-
older age. Development of salivary a-amylase activity over cally tested, and came out non-significant (Gordis et al.,
the adult life span and into older age in healthy participants 2006, 2008). Stroud et al. compared n = 39 children aged 7—
has only been investigated cross-sectionally. Whole and par- 12 years with n = 43 adolescents aged 13—17 years on their
otid saliva composition was examined in 63 healthy volun- responses to a modified TSST for children and a peer rejection
teers, 39 of which were categorized into a younger group task. While the modified TSST failed to induce significant sAA
with an average age of 37 years, and 24 were categorized into increases, age differences emerged in responses to the peer
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Determinants of salivary a-amylase 479

rejection task, with significantly lower responses in children In summary, tobacco smoke does have a strong inhibitory
compared with adolescents (Stroud et al., in press). impact on salivary enzymes including a-amylase. Even if not
In summary, acute sAA stress responses appear to be all studies found generally lower concentrations in habitual
absent in newborns, are detectable in children who are at smokers, smoking status is emerging as an important variable
least 7 years old, and reach adult magnitude in adolescents. to control for, and acute smoking should be avoided before
taking saliva samples to avoid false negative measurements.
3.5. Smoking, drugs, and alcohol
3.5.2. Alcohol
3.5.1. Smoking 3.5.2.1. Acute response to alcohol consumption. An earlier
3.5.1.1. Acute response to tobacco smoke. Since nicotine animal study showed that acute intraperitoneal injection of
activates the SNS (Shinozaki et al., 2008), cigarette smoking ethanol into fasted rats reduced protein synthesis in major
could be expected to be associated with increased sAA activity. salivary glands (Proctor et al., 1993). In humans as well,
Instead, Zappacosta et al. report that smoking a single cigar- acute consumption of alcohol has been shown to impact
ette decreased sAA activity in n = 20 healthy smokers of both saliva secretion and composition. Enberg et al. report sig-
genders by 44% (Zappacosta et al., 2002). Similar results were nificant reductions of amylase activity in stimulated saliva of
obtained in studies in which whole human saliva was exposed to healthy volunteers who consumed 0.7 (men) or 0.6 (women)
cigarette smoke in vitro. Nagler et al. report 34% decreases of grams of alcohol per kg body weight (Enberg et al., 2001). In a
amylase activity after 3 h of incubation with intermittent more realistic experimental setup, 10 healthy participants
smoke exposure (Nagler et al., 2000), and another study drank 300 ml of alcoholic (5.2%) vs. non-alcoholic beer in a
documented a 85% decrease after 1 h incubation with cigarette cross-over design. Salivary flow rate and amylase activity was
smoke, and a 40% reduction after incubation with the parti- measured repeatedly before and after beer consumption in
culate part of cigarette smoke on amylase and other salivary stimulated saliva. Flow rate dropped slightly after alcoholic
enzymes (Greabu et al., 2007). This apparent contradiction can beer consumption, but no differences in amylase activity
be explained by direct noxious effects of tobacco smoke not were found (Brand et al., 2006).
only on oral tissues, but also on salivary proteins including a-
amylase. Specifically, aldehydes, which are a major component 3.5.2.2. Effect of habitual drinking on basal salivary a-
of tobacco smoke, react with and modify sulphydryl groups of amylase. Again, an important question is whether habitual
salivary enzymes, leading to functional impairment (Leuch- drinkers show differences in basal sAA activity. One animal
tenberger et al., 1974; Zappacosta et al., 2002). study showed that chronic alcohol treatment of rats over
three months decreased salivary flow rate and a-amylase
3.5.1.2. Effect of habitual smoking on basal salivary a- activity, along with structural changes and fat accumulation
amylase. An important question arising from these findings is in parotid glands (Maier et al., 1986). A later study, however,
whether sAA activity differs between habitual smokers and although treating rats for about the same time, did not
non-smokers. Callegari and Lami in 1984 reported lower a- detect changes in sAA, but in other proteins and in flow rate
amylase activity in saliva of n = 32 middle-aged habitual (Scott and Berry, 1989).
smokers who consumed more than 20 cigarettes per day, Two studies have investigated the impact of chronic alco-
in comparison with n = 40 non-smoking controls (Callegari hol consumption on sAA in humans. Nagaya and Okuno again
and Lami, 1984). In two studies, responses to different stress did not find differences between light drinkers (24 g alcohol
tests were compared between smokers and non-smokers, and per day in men, 5 g in women) (Nagaya and Okuno, 1993). In
children of mothers who were habitual smokers. Although contrast, Dutta et al. reported that salivary flow and amylase
stress tests in both studies did not significantly increase sAA, activity of stimulated parotid saliva were significantly
baseline levels were again lower in habitual smokers com- decreased in a group of n = 24 ‘‘alcoholic participants’’ com-
pared to non-smokers in both studies (Granger et al., 2007a; pared with a control group (Dutta et al., 1992).
Goi et al., 2007). Importantly, children of mothers who In summary, studies are unequivocal so far, but it might be
smoked also had lower amylase concentrations, which was cautiously concluded that acute as well as chronically high
probably mediated by smoke exposure, because these chil- alcohol consumption tends to lower sAA concentrations.
dren also had higher levels of cotinin (Granger et al., 2007a). Maier et al. speculate about the mechanism of altered
Some studies did not find altered basal sAA in habitual amylase output in chronic alcohol consumption (Maier
smokers. Nagaya and Okuno compared n = 106 smokers and et al., 1986). Alterations might be the consequence of
did not find differences although smokers consumed on aver- morphological alterations in salivary glands, which were
age 22 (men) or 10 (women) cigarettes per day (Nagaya and characterized by enlargement and fat accumulation
Okuno, 1993). In otherwise healthy periodontitis patients, a- observed in rats, or by alcohol-induced changes in autonomic
amylase in unstimulated whole saliva was not different innervation of the salivary glands, as reported by Perec et al.
between smokers and non-smokers (Zuabi et al., 1999). (1979). Future studies will have to replicate these findings
In the only study that assessed a diurnal profile with regard and determine thresholds. Until then it is advisable to control
to daily amylase activity, smokers’ sAA decreased more for chronic alcohol consumption and ask study participants
rapidly in the first hour after waking up, but levels did not not to drink any alcohol before experiments.
differ during the remainder of the day. Additionally, it was
found that having smoked one to three cigarettes in the hour 3.5.3. Medical drugs
before saliva collection, which happened in 7% of measure- Most attention has been given to agonist and antagonists of
ment occasions, was not associated with subsequent sAA adrenergic receptors. As outlined above and discussed in
levels (Nater et al., 2007). more detail in Nater and Rohleder (submitted), amylase
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480 N. Rohleder, U.M. Nater

release is stimulated by activation of b-adrenergic receptors Few studies have investigated shorter-term effects of
and reduced by blockade of these receptors. It is therefore specific diets or dietary changes on sAA. Patients with coeliac
advisable to carefully screen for, and control or exclude the disease (n = 128) who have to live on a gluten-free diet were
use of adrenergic agonists and antagonists that could be part not found to have different amylase activity in stimulated
of antihypertensive, asthma, or other medication. whole saliva than healthy controls (n = 55) (Lenander-Lumi-
kari et al., 2000). In a longitudinal study by Johansson and
3.5.4. Caffeine Birkhed, n = 20 participants (27—61 years, 16 women) shifted
Only two studies have investigated whether caffeine has an their diet from a mixed diet to a lactovegetarian diet, which
effect on sAA, but both show that caffeine appears to led to significant increases in carbohydrate consumption
stimulate sAA activity. In a study investigating stress and relative to fat and protein. No changes in amylase activity
noise in 11 nurses on a pediatric intensive care unit, caffeine in stimulated whole and parotid saliva were observed after 3,
intake was assessed as a control variable. Greater caffeine 6, 12 and 48 months (Johansson and Birkhed, 1994).
intake, but not stress and noise, predicted higher sAA con-
centrations (Morrison et al., 2003). Bishop et al. adminis- 3.6.2. Acute stimulation of amylase by food
tered 6 mg/kg body weight of caffeine vs. placebo in a cross- Several acute stimuli associated with food impact salivary
over design to eight endurance trained men. Caffeine inges- flow and composition. Gustatory or mechanical stimuli are
tion led to significant increases of amylase activity and frequently used to stimulate flow rate, for example applica-
output, which did not return to baseline at the last measure- tion of citric acid (Froehlich et al., 1987) or chewing on
ment occasion 3.5 h later (Bishop et al., 2006). These sti- paraffin wax (Mackie and Pangborn, 1990). Many of these
mulating effects of caffeine on sAA might be mediated by stimuli also change the protein content of saliva. In rats,
caffeine-induced activation of the SNS (Laurent et al., ascorbic acid increased salivary amylase activity and output
2000). It has not been investigated whether higher habitual (Ekstrom, 2001). In an experiment with rabbits, Gjörstrup
caffeine consumption is associated with generally higher sAA et al. report that different types of foods have differential
concentrations, and if acute responses of sAA differ in stimulatory effects of amylase in parotid saliva, with carrots
habitual caffeine consumers. inducing higher responses than food pellets (Gjorstrup,
1980). In humans, citric acid but not sucrose, sodium chloride
3.6. Food and starch stimulated a-amylase activity in parotid saliva of
10 healthy subjects (Froehlich et al., 1987). In contrast are
3.6.1. Association of carbohydrate contents of diet the findings of one study in which 10 human subjects of both
with basal a-amylase sexes provided parotid saliva after chewing parafilm, bread,
Wesley-Hadzija and Pigon compared a-amylase activity in or celery. Salivary flow and amylase output (expressed as
unstimulated saliva of three populations in Ghana living on units per min) but not amylase concentration was lowest
different diets. a-Amylase concentrations were signifi- after water, intermediate after parafilm and celery, and
cantly higher in a group of n = 18 Ghanaians who lived highest after chewing bread. Thus, amylase production
on a predominantly carbohydrate diet, as compared with was increased in accordance with higher fluid production,
Ghanaian students and Western Europeans living on a but this increased production did not translate to higher final
‘‘protein-balanced’’ mixed diet (Wesley-Hadzija and Pigon, amylase concentrations in saliva (Mackie and Pangborn,
1972). However, when Mazengo et al. compared sAA activ- 1990).
ity between individuals living in rural vs. urban areas of Effects of food on sAA seem to be more pronounced in
Tanzania, those in the rural areas with more carbohydrate response to real food intake. Two studies investigated the
consumption had lower amylase levels (Mazengo et al., effects of refeeding after a period of fasting. In rats, refeed-
1994). ing after overnight fasting doubled parotid and submandib-
More recently, Perry et al. investigated the association of ular protein secretion (Proctor et al., 1993), and similarly in
diet and sAA using a genetic approach. The number of humans, ingesting different types of carbohydrates induced
amylase gene copies, which is proportional to amylase pro- increases in amylase activity in parotid saliva of seven women
tein in saliva, was compared between different populations who had been fasting for 12 h (Behall et al., 1973).
characterized by high vs. low starch diets. Results show a Salivary a-amylase also increased within 15 min after
significantly higher number of amylase gene copies in agri- eating or ‘‘sham-eating’’ (chewing and expectorating) a
cultural high-starch populations (such as European-Ameri- standardized meal in n = 12 healthy men and women. How-
cans and Japanese) as compared with low-starch populations ever, these increases were significantly lower in the sham-
(such as Rainforest Hunter-Gatherers and the mainly fish- eating condition (Messenger et al., 2003). a-Amylase was
consuming Yakut). They conclude that these differences in shown to increase in response to a standardized lunch in
amylase gene copy number have developed through natural stimulated saliva of n = 18 healthy human subjects. Further-
selection (Perry et al., 2007). These associations between more, amylase was found positively associated with self-
historical diet and amylase gene copy number and baseline ratings of satiety and fullness, and inversely associated with
activity raises the question whether there should be ethnical hunger and desire to eat (Harthoorn and Dransfield, 2008).
or cultural differences in amylase activity. However, most of Taken together, there seems to be an association of long-
the studies summarized here have investigated sAA either in term diet, probably most pronounced on an evolutionary
European/North American or in Japanese populations, both time scale, with sAA production. Gustatory and mechanical
of which are characterized as high-starch populations by stimuli, but more effectively, ingestion of real food, induce
Perry et al. (2007). No further data are available on ethnical acute increases of sAA in most of the published studies. It is
or cultural differences in basal sAA activity. therefore highly advisable to assess and control for the time
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Determinants of salivary a-amylase 481

of food intake in studies measuring diurnal variation of sAA, The effects of minor physical activity on basal diurnal sAA
and to ask participants of acute stress studies not to have any were investigated in a recent study. No association of activ-
meals an hour before the experiment. Unfortunately, no data ities such as running for a bus, or climbing a set of stairs, in
are available to answer the questions whether acute stress the hour before saliva sampling was associated with
responses are influenced by nutritional status or recent food increased amylase concentrations. However, these activities
consumption. However, one study showed that having con- were of lower duration and intensity than any of those
sumed a glucose drink did not change the sAA response to summarized above (Nater et al., 2007).
exercise (Bishop et al., 2000).
3.7.2. Effect of training status on basal salivary a-
3.7. Physical exercise amylase
In contrast to the well-documented acute sAA response to
3.7.1. Acute response to exercise exercise, there are virtually no studies that investigated
Several earlier studies have investigated the effects of acute whether endurance-trained individuals differ from sedentary
physical exercise on sAA. Gilman et al. measured a signifi- people with regard to basal sAA activity.
cantly higher concentration of sAA during intense exercise in Taken together, it can be concluded that exercise is a
comparison to a control period (Gilman et al., 1979). Dawes reliable determinant of increases in sAA activity, even after
et al. reported significant increases in total protein contents short duration and with lower intensity. This implies that
of stimulated parotid saliva in response to running between 4 physical activity should be assessed and controlled for in
and 13 km (Dawes, 1981). Similarly, a cross-country run of 2 h experiments using sAA as an outcome measure. Diurnal
was found to increased activity of a-amylase in whole saliva profiles, however, seem to be relatively robust against minor
of n = 25 participants (Nexo et al., 1988). non-exercise activity.
Exercise was also one of the conditions that Chatterton
et al. (1996) used in their seminal paper for testing the 3.8. Somatic and psychiatric diseases
association of sAA and plasma catecholamine responses.
Here, significant increases of sAA were found after 20 min Salivary a-amylase has been, among other parameters of oral
of running as well as intermittent cycling on a stationary bike, health, investigated in oral and dental diseases. Periodontitis
and increases were associated with catecholamine changes. for example has been shown to be associated with changes in
In addition, they showed a dose—response effect with highest sAA concentrations (Henskens et al., 1996). Hence, oral
sAA increases after running as compared to light jogging and diseases should always be excluded or controlled for in
walking (Chatterton et al., 1996). Calvo et al. subjected experiments involving sAA. Additionally, amylase concentra-
n = 20 healthy young men to an incremental exercise test tions in saliva have also been found altered in other somatic
on a treadmill leading to exhaustion, and reported an initial diseases, while less data is available on sAA in psychiatric
decrease of sAA activity followed by a continuous increase. diseases.
Interestingly, this second, increasing phase paralleled that of Data on alterations of basal sAA concentrations in somatic
lactate concentrations in capillary blood, and the authors diseases are incomplete as well, except maybe a good num-
suggested using the sAA response to exercise as predictor for ber of studies on oral diseases, but there are for example no
aerobic fitness (Calvo et al., 1997). These results were later studies on sAA in cardiovascular disease. Nevertheless, some
replicated in n = 12 endurance trained men (Chicharro et al., diseases have been investigated. Lower sAA concentrations
1999). In n = 15 healthy men, 2 h of cycling at 60% maximal have been found in children suffering from asthma and atopic
oxygen consumption stimulated amylase in saliva collected dermatitis (Crespi et al., 1982; Ryberg et al., 1987; Wolf
with salivettes (Bishop et al., 2000). These findings were et al., 2008). Similarly, lower amylase has been found in
later replicated and it was further shown that caffeine saliva from children with juvenile idiopathic arthritis (Brik
potentiated the exercise stimulated sAA increase (Bishop et al., 2006) and in adolescents with cerebral palsy (Rodri-
et al., 2006). gues Santos et al., 2007). Some autoimmune diseases such as
Intermittent high-intensity exercise in n = 8 trained men Sjögren’s syndrome appear to be also associated with lower
induced fivefold increases in sAA, which returned to base- sAA activity. Since Sjögren’s syndrome can also be diagnosed
line after 2.5 h (Walsh et al., 1999). In a later study, the secondary to other autoimmune diseases such as rheumatoid
same group tested the effect of different times during the arthritis and systemic lupus erythematosus, autoimmune
day, this time employing 2-h cycling exercises, and found conditions should be excluded in study participants (Mathews
similar significant amylase increases in morning and after- et al., 2008).
noon exercises (Li and Gleeson, 2004). In the most recent Higher sAA concentrations have been reported in chronic
study from the same group, it was shown that sAA activity obstructive pulmonary disease (COPD) patients. Interest-
responded even to short-term exercise of 20 min at inten- ingly, within the group of COPD patients, those who were
sities of 50% maximal oxygen consumption (Allgrove et al., habitual smokers had lower sAA concentrations (Yigla et al.,
2008). Amylase increases were also found after more 2007), a finding that is consistent with the inhibitory effect of
intense and/or longer bouts of exercise, such as running cigarette smoke on sAA activity (see above). Higher sAA
a marathon, which led to 2-fold amylase increases in activity has been found in Parkinson’s disease patients (Tumi-
stimulated whole saliva of n = 21 runners (Ljungberg lasci et al., 2006).
et al., 1997), and competing in an Olympic Distance Finally, sAA activity has been investigated in individuals
triathlon, which led to 3.6-fold amylase increases in sti- with diabetes. A recent study reported increased a-amy-
mulated whole saliva of n = 42 healthy athletes (Steeren- lase in saliva of patients suffering from type-2 diabetes
berg et al., 1997). (Aydin, 2007). A series of earlier studies did not find sAA
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482 N. Rohleder, U.M. Nater

differences between patients with type-1 diabetes and Acknowledgements


controls (Tenovuo et al., 1986), or between type-1 and
type-2 diabetes and controls (Artino et al., 1998; Ben- The authors would like to thank Clemens Kirschbaum and
Aryeh et al., 1993). Ulrike Ehlert for their support and theoretical input. Further-
Taken together, while the literature on sAA alterations in more, NR acknowledges support by the German Research
somatic diseases is sparse at present, it can be safely con- Foundation (NR; DFG; Ro 2353/4-1) and the Michael Smith
cluded that somatic conditions which present with changes in Foundation for Health Research (MSFHR).
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 References
control for these conditions.
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salivary amylase: cross-reactivity with human and porcine pan-
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807.
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chemical quantitation of alpha-amylase and secretory Iga in
On the previous pages we have described the basic metho- parotid saliva from people of various ages. Arch. Oral Biol. 32,
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a-amylase, and we have derived some recommendations that Allgrove, J.E., Gomes, E., Hough, J., Gleeson, M., 2008. Effects of
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Artino, M., Dragomir, M., Ionescu, S., Badita, D., Nita, V., Chitoi, E.,
dations are summarized in Table 1.
1998. Diurnal behaviour of some salivary parameters in patients
We have further reviewed the recent literature on poten-
with diabetes mellitus (protein concentration, amylase activity,
tial factors influencing basal or stress-induced sAA measure- density)–—note I. Rom. J. Physiol. 35, 79—84.
ments. Although information is still scarce on some issues Aydin, S., 2007. A comparison of ghrelin, glucose, alpha-amylase and
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D., 1986. The salivary flow rate and composition of whole and
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to establish whether individual differences in acute stress- kallikrein and amylase in a population of schoolgirls, throughout
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Role of the funding source
2051.
Bishop, N.C., Walker, G.J., Scanlon, G.A., Richards, S., Rogers, E.,
NR was supported by personal (salary) fellowships by the 2006. Salivary Iga responses to prolonged intensive exercise
German Research Foundation (DFG; Ro 2353/4-1) and the following caffeine ingestion. Med. Sci. Sports Exerc. 38, 513—519.
Michael Smith Foundation for Health Research (MSFHR). UMN Bosch, J.A., de Geus, E.J., Veerman, E.C., Hoogstraten, J., Nieuw
has no funding source to report Amerongen, A.V., 2003. Innate secretory immunity in response to
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nomic activity. Psychosom. Med. 65, 245—258.
Conflict of interest Bosch, J.A., Brand, H.S., Ligtenberg, T.J., Bermond, B., Hoogstraten,
J., Nieuw Amerongen, A.V., 1996. Psychological stress as a
The authors do not have any conflicts of interest to report. determinant of protein levels and salivary-induced aggregation
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Determinants of salivary a-amylase 483

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