Salivary alpha amylase not chromogranin A reflects sympathetic activity: exercise responses in elite male wheelchair athletes with or without cervical spinal cord injury
Leicht et al. Sports Medicine - Open
Salivary alpha amylase not chromogranin A reflects sympathetic activity: exercise responses in elite male wheelchair athletes with or without cervical spinal cord injury
Christof A. Leicht 0
Thomas A. W. Paulson 0
Victoria L. Goosey-Tolfrey 0
Nicolette C. Bishop 0
0 School of Sport, Exercise, and Health Sciences, The Peter Harrison Centre for Disability Sport, The National Centre for Sport and Exercise Medicine, Loughborough University , Loughborough LE11 3TU , UK
Background: Salivary alpha amylase (sAA) and chromogranin A (sCgA) have both been suggested as non-invasive markers for sympathetic nervous system (SNS) activity. A complete cervical spinal cord injury leading to tetraplegia is accompanied with sympathetic dysfunction; the aim of this study was to establish the exercise response of these markers in this in vivo model. Methods: Twenty-six elite male wheelchair athletes (C6-C7 tetraplegia: N = 8, T6-L1 paraplegia: N = 10 and non-spinal cord injured controls: N = 8) performed treadmill exercise to exhaustion. Saliva and blood samples were taken pre, post and 30 min post exercise and analysed for sAA, sCgA and plasma adrenaline concentration, respectively. Results: In all three subgroups, sAA and sCgA were elevated post exercise (P < 0.05). Whilst sCgA was not different between subgroups, a group × time interaction for sAA explained the reduced post-exercise sAA activity in tetraplegia (162 ± 127 vs 313 ± 99 (paraplegia) and 328 ± 131 U mL−1 (controls), P = 0.005). The post-exercise increase in adrenaline was not apparent in tetraplegia (P = 0.74). A significant correlation was found between adrenaline and sAA (r = 0.60, P = 0.01), but not between adrenaline and sCgA (r = 0.06, P = 0.79). Conclusions: The blunted post-exercise rise in sAA and adrenaline in tetraplegia implies that both reflect SNS activity to some degree. It is questionable whether sCgA should be used as a marker for SNS activity, both due to the exercise response which is not different between the subgroups and its non-significant relationship with adrenaline.
Adrenaline; Catecholamines; Cortisol; Sympathetic dysfunction; Testosterone; Wheelchair athlete; Wheelchair propulsion
This study shows a blunted alpha amylase but a
normal chromogranin A response to maximal
exercise in athletes with sympathetic dysfunction.
This study would favour the use of alpha amylase
over chromogranin A as a surrogate marker for
However, in contrast to the adrenaline response, the
alpha amylase response is not completely absent in
cervical spinal cord injury, implying it is also regulated
by mechanisms other than sympathetic activity.
Salivary secretions provide a non-invasive alternative to
blood-derived markers to quantify exercise stress in both
clinical and sports performance settings. Whilst salivary
catecholamines have been suggested to be poor markers
of acute sympathetic nervous system (SNS) activity ,
the salivary proteins α-amylase (sAA) and chromogranin
A (sCgA) have been proposed to serve this purpose [2, 3].
Indeed, sAA activity is responsive to exercise, particularly
that of a high-intensity nature, supporting the relationship
between sAA and SNS activity [4–7]. Further, adrenergic
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receptor blockade can inhibit the stress-induced secretion
of sAA . However, the correlations between sAA and
catecholamines in the circulation are relatively small [9, 10].
It has hence been questioned whether sAA truly reflects
SNS activity as the influence of parasympathetic activity on
sAA secretion may confound a stronger relationship [9, 11].
Chromogranin A is stored and co-released with
adrenaline and noradrenaline from secretory vesicles within the
adrenal medulla and post-ganglionic sympathetic axons
. It is also secreted from the submandibular gland
following stimulation by noradrenaline and acetylcholine
. In response to acute exercise stress, sCgA shows a
similar response to sAA [5, 14]. However, the correlation
to physiological responses during acute exercise has been
shown to be stronger for sCgA than for sAA . This
suggests a differential regulation of the two proteins and that
sCgA may provide a more accurate representation of SNS
activity. Conversely, non-exercise stress in the form of
noise  or delivering a lecture  has been shown to
increase sAA, whereas sCgA remained unaffected,
questioning to what extent sCgA is regulated by the SNS.
Cortisol and testosterone represent two further markers
of exercise stress and recovery that can be measured in
plasma as well as in saliva. In response to acute exercise,
both markers increase in a time and intensity-dependent
manner [17, 18]. Cortisol and testosterone, expressed as a
ratio, have also been put forward to describe the
anaboliccatabolic balance. In this context, they may help in the
diagnosis of the overtraining syndrome, even though it
has been made clear that supporting markers are needed
to define this condition .
The primary aim of this study was to investigate the
impact of exercise to exhaustion on established plasma
markers for SNS activity (adrenaline) and on proposed
salivary markers for SNS activity (sAA and sCgA) in a
human in vivo model of SNS dysfunction. The model of
spinal cord injury (SCI) was employed: A complete
injury to the cervical region of the spinal cord results in a
tetraplegia (TETRA) and the dysfunction of the SNS, as
evidenced by reduced cardiac acceleration  or a
reduced catecholamine concentration at rest or following
exercise . However, increases in sAA activity have
been found previously in both cervical- and thoracic-level
athletes with SCI and non-spinal injured controls
following strenuous exercise, which further questions sAA as a
true indicator of central sympathetic drive . We hence
hypothesise the exercise-induced changes in adrenaline to
be more closely related to sCgA than to sAA.
The secondary aim of this study was to investigate the
impact of exercise on plasma and salivary steroid
hormones in the SCI model. Whilst cortisol secretion is
governed by humoral mechanisms (the
hypothalamuspituitary-adrenal or HPA axis), testosterone secretion is
partly governed by neural pathways , explaining the
high proportion of testosterone deficiency in the SCI
population . We hence hypothesise a normal plasma
and salivary cortisol but a blunted testosterone response
to exercise in TETRA.
The present data have not been previously published,
but the samples were collected as part of a larger study
; as such, the participants studied and the exercise
protocol employed are identical to this publication.
Twentysix international-level male wheelchair athletes volunteered
to participate and were grouped according to their disability,
TETRA, (N = 8), paraplegia (PARA, N = 10) and disabilities
unrelated to SCI (non-spinal injured (NON-SCI), N = 8). All
participants with SCI had a motor and sensory complete
lesion in accordance with the American Spinal Injury
Association (ASIA) impairment scale . A summary of
the participants’ characteristics and their main peak
responses to exercise are presented in Table 1. All
procedures were approved by the Loughborough University
Ethical Advisory Committee and were in accordance with
the Declaration of Helsinki. Participants provided written
informed consent prior to the experiments, and they were
free from infectious symptoms and pressure sores.
Participants reported to the laboratory between 09:30
and 11:30 having been fasted for at least 2 h. They were
asked to refrain from strenuous physical activity and
caffeine intake 24 h prior to exercise. On arrival, their body
mass was obtained to the nearest 0.1 kg using
doublebeam seated scales (Marsden MPWS-300, Rotherham, UK).
All exercise tests were performed in the participants’
competition court sports wheelchair on a motorised treadmill
(HP Cosmos, Traunstein, Germany) as described previously
. First, a ~30-min warm-up at intensities covering a
range from 40 to 80% peak oxygen uptake V_ O2peak was
performed, followed by a 15-min passive recovery. A
graded exercise test to exhaustion (GXT) was then
completed at a constant speed. The gradient at the start
of the GXT was 1.0% for all subgroups, with subsequent
increases of 0.3% every minute for PARA and NON-SCI
and 0.1% every 40 s for TETRA to account for the
functional differences between subgroups and ensure a
minimum GXT duration of ~8 min. After the GXT,
participants recovered actively at a low intensity (1.2 m s−1
at a 1.0% gradient) for 5 min. Participants then performed
a verification test, designed as a test to exhaustion at
the same constant speed but 0.3 and 0.1% higher than
the maximal gradient achieved during the GXT for
NON-SCI/PARA and TETRA, respectively. The GXT and
the verification test were terminated when participants
Table 1 Participants’ characteristics and peak exercise responses
ASIA impairment scale
Training (h week−1)
V_ O2peak (L min−1)
HRpeak (b min−1)
BLapeak (mmol L−1)
SCI T6–L1/spina bifida
RPEpeak 20 (19–20) 19 (19–20) 19 (19–20)
Data are mean ± SD or median (interquartile range); P < 0.05
TETRA tetraplegia, PARA paraplegia, NON-SCI non-spinal injured, ASIA American Spinal Injury Association, HR heart rate, BLa blood lactate concentration, RPE rating
of perceived exertion, SCI spinal cord injury
aSignificantly different from NON-SCI
bSignificantly different from other subgroups
were unable to maintain the speed of the treadmill. Verbal
encouragement was given throughout the test, and
participants were allowed to consume water ad libitum during
Expired air was collected for at least the final three
consecutive minutes of the GXT and for 2 min during the
verification test and analysed using the Douglas bag technique,
using a gas analyser (Series 1400; Servomex Ltd, Sussex,
UK) and a dry gas meter (Harvard Apparatus, Kent, UK).
Blood lactate concentration (BLa) was determined using a
calibrated lactate analyser (YSI 1500 SPORT, YSI
Incorporated, Yellow Springs, OH, USA) from a capillary blood
sample obtained immediately after the GXT and
immediately after the verification test. At the same time
points, participants were asked to indicate an overall
rating of perceived exertion (RPE) using the 15-point
Borg scale according to previous instructions. Heart rate
was continuously recorded at 5-s intervals (Polar PE
4000, Polar, Kempele, Finland). The higher of the two
V_ O2peak; HRpeak and BLapeak values obtained in the GXT
and the verification test was taken as peak value. All
participants had prior experience of the physiological testing
procedure and were therefore familiar with the protocol.
Blood and saliva samples were collected before (pre),
immediately after the verification test (post) and 30 min
after exercise (post30). A 4.9-mL blood sample was drawn
from an antecubital vein into a K3EDTA vacutainer.
Timed, unstimulated saliva samples were collected as
described previously ; the participants’ head slightly
tilted forward with minimal orofacial movement during
collection after rinsing their mouth out with water
immediately before collection. Participants were allowed
to consume water ad libitum apart from 6 min before
each collection. Saliva flow rate was calculated by
dividing the obtained saliva volume by the collection time.
Plasma and saliva analysis
Blood samples were refrigerated until the final sample
from each participant was collected and then centrifuged
at 1500g for 10 min (at 4 °C). Whole saliva samples were
immediately centrifuged at 13,000 rpm for 2 min following
collection. The separated plasma was then immediately
stored at −80 °C and saliva at −20 °C. Plasma
concentrations of adrenaline, cortisol (pCort) and free testosterone
(pTest) were determined using quantitative
sandwichtype enzyme-linked immunosorbant assay (ELISA) kits
(adrenaline: IBL international, Hamburg, Germany; pCort
and pTest: DRG instruments, Marburg, Germany),
according to the manufacturers’ instructions. All samples
were analysed in duplicate. Saliva concentrations of
cortisol (sCort), testosterone (sTest) and sCgA were also
determined using ELISA (sCort and sTest: Salimetrics,
Newmarket, UK; sCgA: Demeditic Diagnostics GmbH,
Kiel, Germany) and sAA with an enzymatic activity assay
as described previously . All samples from one
participant were analysed on the same ELISA plate; the
withinassay coefficient of variation for the analyses performed
was as follows: adrenaline 2.7%, pCort 1.3%, pTest 4.5%,
sCort 2.8%, sTest 3.7%, sCgA 5.2% and sAA 4.9%.
Data were analysed using IBM SPSS for Windows version
19 (SPSS inc, Chicago, IL). Exercise responses were
analysed using a two-way mixed measures ANOVA with time
as within- and group as between-measures variable for
normally distributed variables, and significant main effects
were assessed using Sidak post hoc tests. Adrenaline and
sTest data were analysed using non-parametric Friedman
tests for each group separately; adrenaline differences
were assessed using Mann-Whitney U tests between
TETRA and the other two subgroups (with intact
sympathetic function) combined. Pearson-product moment
correlations for normally distributed data and Spearman’s
rho for non-normally distributed data were used to
determine the relationship between plasma and salivary
concentrations of cortisol and testosterone, as well as between
adrenaline and the salivary proteins sAA and sCgA.
Significance was set at P ≤ 0.05, and Bonferroni adjustments were
performed when performing multiple comparisons. Data
are presented as mean ± standard deviation.
A significant effect of time was found for plasma
adrenaline concentration for both PARA and NON-SCI with a
2.2-fold increase from pre (P < 0.05) but not for TETRA
(P = 0.74, Fig. 1, Table 2). Furthermore, the plasma
adrenaline concentration was significantly lower in TETRA
when compared with the other subgroups with intact
sympathetic function (P < 0.001). The concentrations of
sAA were highest at post exercise in all subgroups
(2.3fold increase from pre, P < 0.03), a main effect of group
was found in sAA with lower values in TETRA than in
PARA (P = 0.02) and a group × time interaction indicated
a blunted response in TETRA (P = 0.005). The same
pattern was found for the sAA secretion rate, with the
exception that a main effect of group indicated lowest
values for TETRA when compared with both other
subgroups (P < 0.05). Post-exercise concentrations of sCgA
were significantly elevated in all subgroups (threefold
from pre, P < 0.001) with no difference between subgroups
(P = 0.69). TETRA had a lower saliva flow rate than
NON-SCI (P = 0.02), the saliva flow rate of PARA was not
different from the other subgroups (P > 0.33). Saliva
flow rate did not change over time (P = 0.77).
Correlations between plasma adrenaline concentration
and salivary parameter post exercise were significant for
sAA (r = 0.60, P = 0.01), but not for sCgA (r = 0.06, P = 0.79,
Fig. 2). The correlation between sAA and sCgA was also
not significant (r = 0.25, P = 0.23).
Plasma and salivary steroid concentrations are shown in
Fig. 3 and in Table 2. Significant effects of time (P < 0.001)
demonstrate an increase in both plasma and salivary
cortisol concentrations following exercise, with the highest
concentrations found at post and post30 for plasma (both
1.4-fold elevated from pre), whilst salivary concentrations
were only elevated at post30 (1.9-fold from pre, P < 0.01).
No significant group (P > 0.89) or interaction (P > 0.68)
effects were found for plasma and salivary cortisol
concentrations. A main effect of time was found for pTest
(P = 0.02) which only showed a trend for post
concentrations to be elevated from the other time points (1.3-fold
Fig. 1 Plasma adrenaline, salivary α-amylase activity, chromogranin A and α-amylase secretion rate response to exhaustive exercise. Daggers: main
effect of time for PARA and NON-SCI; Asterisks: significant difference from pre for all subgroups (P < 0.05)
Table 2 Plasma and salivary responses to exhaustive exercise
Parameter (unit) Time
Plasma adrenaline concentration (ng/mL) Pre
Salivary chromogranin A concentration (pmol/mL)
Plasma cortisol concentration (ng/mL)
Salivary cortisol concentration (ng/mL)
Plasma testosterone concentration (ng/mL)
Salivary testosterone concentration (pg/mL)
Saliva flow rate (mL/min)
Data are mean ± SD; P < 0.05
aSignificantly different from pre
bSignificantly different from post
from pre, P = 0.06), whereas no group or time effects were
found for sTest (P > 0.05).
Significant correlations were found between plasma
and salivary concentrations of cortisol both at pre (r = 0.60,
P = 0.003) and post30 (r = 0.68, P < 0.001), whereas a trend
was evident at post (r = 0.43, P = 0.09). In contrast, no
significant correlation was found between corresponding
plasma and salivary testosterone concentrations (pre:
r = 0.08; post: r = 0.34; post30: r = 0.21; P > 0.05).
The main findings of the present study were that (1) both
sAA and sCgA concentrations increased following
exhaustive exercise in all spinal injury level subgroups;
(2) the post-exercise sAA response was blunted in
TETRA; (3) post-exercise plasma adrenaline
concentrations were significantly correlated with post-exercise sAA
concentrations, but not with sCgA concentrations; (4) in
contrast to the other subgroups, the plasma adrenaline
concentrations in TETRA did not alter in response to
exercise; and (5) salivary cortisol but not testosterone
reflected plasma activity, the responses not being
different between subgroups.
Are sAA and sCgA appropriate salivary markers for
The primary aim of this study was to investigate the
impact of strenuous exercise on established plasma
(adrenaline) and salivary (sAA and sCgA) markers for SNS
activity in a human in vivo model of SNS dysfunction. The
present results support the finding that sAA is partly
governed by SNS activity , as a blunted response
was observed in TETRA when compared with the other
subgroups. The correlation between adrenaline and sAA
Fig. 2 Suggested salivary markers for SNS activity and their relationship
with plasma adrenaline after exhaustive exercise. R2 coefficient
further imply that sAA could be used as a surrogate
marker for adrenaline. However, the variance explained
was only 36% (R2), leaving a considerable amount of
unexplained random variation, which is of a similar magnitude
as found previously . It is likely that some of this
random variation is explained by the contribution of the
parasympathetic nervous system , which contributes
to sAA, but not to adrenaline release. Therefore, whilst
plasma catecholamine concentrations represent a gold
standard with respect to SNS activity, sAA activity may be
used to help indicating SNS dysfunction in an exercise
context. These results are in contrast to earlier findings,
where no significant differences between the same
subgroups as investigated in the current study have been
found with respect to sAA activity following strenuous
interval exercise . However, closer inspection of these
previous data reveals a 22% lower sAA activity in TETRA
post exercise, even though the difference to the subgroups
with intact SNS was insignificant. It should further be
noted that the exercise performed during this previous
study was not performed to exhaustion as in the
current study. This could further explain the previous
non-significant difference, as the subgroups with intact
SNS activity were likely not to initiate the full potential
of their sAA response. Indeed, exercise of an
incremental nature with continuous data sampling shows a
continuous increase in sAA and sCgA concentration with
increasing exercise intensity which led to the suggestion
for their use as markers for exercise intensity .
Whilst sAA responds to stressors other than exercise,
such as emotional stressors , it is worth noting that
Bocanegra et al.  suggest sAA and sCgA as markers
for exercise intensity, not sympathetic activity in exercising
contexts. This is an aspect worth developing: correlation
(between salivary markers and exercise intensity) does not
necessarily imply causation (of their regulation by the
SNS), as demonstrated by comparing the present findings
with the data presented by Bocanegra et al. . Despite
the strong relationships between sAA, sCgA and exercise
intensity , factors other than SNS activity, such as
parasympathetic activity, reflex activity or, in the case of
SCI, receptor hypersensitivity , may also contribute
to the changes observed in these salivary markers in
response to exercise. Therefore, SNS activity cannot be
suggested as their main modulating component. This
thought may be further developed to question the use
of sCgA as a marker of SNS activity as previously
suggested , as the present data do not support sCgA as
a marker for SNS activity for two reasons: first, the
exercise response is not different between the wheelchair
athlete subgroups, and second, the relationship with
adrenaline is insignificant.
The steroid hormone and saliva flow rate response in SCI
The cortisol exercise response was not different between
subgroups. This is consistent with the humoral regulation
of cortisol release, which is independent of SNS function.
In contrast, testosterone is secreted partly through the
action of neuronal mechanisms , which again lends itself
to be studied with the SCI model. However, no group
or interaction effects were found for the testosterone
responses, implying that testosterone regulation is only
minimally, if at all, dependent on neural activity in the
context of an exercise intervention.
The present cortisol data corroborate earlier results: A
positive association between salivary and circulating plasma/
serum cortisol concentrations has been confirmed both
at rest [27–29] and following exercise [30–33]. Further,
we have shown that exercise induces a rise in plasma
cortisol concentration immediately following exercise, a
response which can be observed with a time lag in the
Fig. 3 Cortisol and testosterone response to exhaustive exercise. Asterisks: significant difference from pre for all subgroups; Dagger: significant
difference from pre and post for all subgroups (P < 0.05)
salivary concentrations. Similar effects have been found
previously in direct comparisons of plasma and salivary
cortisol concentrations , and exercise studies report
that the sCort peak trails the pCort peak, which is
usually found soon after cessation of exercise . As sCort
originates from the circulation, this lag is likely due to the
process of cortisol diffusion and ultrafiltration through
acinar cells , and the lag between pCort and sCort peak
values has been reported to be in the area of ~10–20 min
. This is consistent with our findings—the weakest
correlation between pCort and sCort was found post
exercise, where the sCort concentration had not reached its
maximum yet. It is therefore likely that the correlation
between these two markers could be improved when
comparing post-exercise pCort values with sCort sampled
with a delay of 10–20 min. Including a sampling lag for
sCort may also be relevant whenever the focus lies on
determining the true maximum cortisol value, for example
in the context of quantifying acute exercise stress  or
in the context of over-reaching .
A modest but significant increase was observed in
pTest, but not in sTest for all subgroups. The increases
in pTest are consistent with previous reports ,
therefore suggesting that pTest may be used in SCI subgroups
for the same diagnostic purposes as in the able-bodied
population. The poor relationships between salivary and
plasma testosterone also support previous research that
questions the diagnostic value of salivary testosterone
; even though significant relationships have been shown
between salivary and plasma/serum concentrations [27, 32],
this was not replicated by others . From a clinical
perspective, the present study does not provide any
evidence for testosterone deficiency in TETRA as previously
reported . However, previous research shows that
exercise training increases resting testosterone levels in
chronic SCI ; the highly trained nature of the
investigated participant group is hence a likely explanation of
Finally, the reductions in saliva flow rate in TETRA
underline the physiological difference of this subgroup—
saliva flow rate can be increased by adrenoreceptor
agonists but decreased by adrenoreceptor blocking drugs;
furthermore, it is affected by neural stimulation . The
chronically lower adrenaline concentrations and
interruption of neural pathways to the salivary glands in TETRA
are a likely reason for these observed reductions in saliva
In addition to adrenaline, a number of studies report
relationships between noradrenaline and suggested markers
for SNS activity [3, 13]. Whilst noradrenaline was not
measured in the present study, it is highly likely that
the noradrenaline response was similarly blunted as the
adrenaline response, as shown earlier for cervical SCI
[39, 40]. Despite this, we suggest assessing the
noradrenaline response in follow-up studies, which would allow
a distinction between secretion mechanisms governed
mainly by the adrenal glands (adrenaline) or secretion
by sympathetic nerve endings (noradrenaline).
The blunted post-exercise rise in sAA and adrenaline in
TETRA implies that both reflect SNS activity to some
degree. Even though the sAA response to exercise is not
absent, as is the case for adrenaline, sAA appears to reflect
some of the SNS dysfunction found in TETRA. Therefore,
rather than describing sAA as a marker of SNS activity in
its own right, it would be more accurate to refer to it as a
marker which partly reflects SNS activity but is also
regulated by other mechanisms. Despite this limitation, we
suggest that to date, sAA is the best surrogate salivary
marker for SNS activity, which is relevant in the absence
of available blood tests and if restricted to salivary
analyses. This is in contrast to earlier findings which have
proposed that sCgA may provide a more accurate
representation of SNS activity than sAA  but supports the
findings from non-exercise stress interventions that
failed to observe increases in sCgA in the presence of
sAA elevations [15, 16].
On a final note, plasma and salivary cortisol and
testosterone responses to exercise did not differ between
subgroups, implying a minimal involvement of sympathetic
innervation in the acute exercise response.
BLa: Blood lactate concentration; GXT: Graded exercise test to exhaustion;
HPA: Hypothalamus-pituitary-adrenal; HR: Heart rate; NON-SCI: Non-spinal
injured; PARA: Paraplegia; pCort: Plasma cortisol; pTest: Plasma testosterone;
RPE: Rating of perceived exertion; sAA: Salivary alpha amylase; sCgA: Salivary
chromogranin A; SCI: Spinal cord injury; sCort: Salivary cortisol;
SNS: Sympathetic nervous system; sTest: Salivary testosterone;
We thank the Peter Harrison Centre for Disability Sport for financial support.
Nicolette Bishop is supported by the National Institute for Health Research
(NIHR) Diet, Lifestyle and Physical Activity Biomedical Research Unit based at
the University Hospitals of Leicester and Loughborough University. The views
expressed are those of the authors and not necessarily those of the NHS, the
NIHR or the Department of Health.
All authors were involved in the conception and planning of the study. CL
was involved in the data analysis and manuscript preparation. TP was involved
in the data collection and manuscript review. VT and NB were involved in the
manuscript review. All authors read and approve the final manuscript.
Consent for publication
Ethics approval and consent to participate
This study was approved by the Loughborough University Ethical Advisory
Committee, and all participants gave written informed consent prior to the
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