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Original Article 

Anaerobic Threshold and Salivary a-amylase 
during Incremental Exercise 



J. Phys. Ther. Sci. 
26: 1059-1063, 2014 



Kazunori Akizuki, RPT, MSc'' ^'*, Syouichirou Yazaki, RPT^\ Yuki Echizenya, RPT^, 
YuKARi Ohashi, RPT, PhD^) 

Department of Rehabilitation, Misato Central General Hospital: 745 Kobo Misato-city, Saitama 
341-8526, Japan 

Graduate School of Health Sciences, Ibaraki Prefectural University of Health Sciences, Japan 
^) Department of Rehabilitation, Nerima Hikarigaoka Hospital, Japan 
Department of Physical Therapy, Ibaraki Prefectural University of Health Sciences, Japan 



Abstract. [Purpose] The purpose of this study was to clarify the validity of salivary a-amylase as a method of 
quickly estimating anaerobic threshold and to establish the relationship between salivary a-amylase and double- 
product breakpoint in order to create a way to adjust exercise intensity to a safe and effective range. [Subjects and 
Methods] Eleven healthy young adults performed an incremental exercise test using a cycle ergometer. During 
the incremental exercise test, oxygen consumption, carbon dioxide production, and ventilatory equivalent were 
measured using a breath-by-breath gas analyzer. Systolic blood pressure and heart rate were measured to calculate 
the double product, from which double-product breakpoint was determined. Salivary a-amylase was measured to 
calculate the salivary threshold. [Results] One-way ANOVA revealed no significant differences among workloads 
at the anaerobic threshold, double-product breakpoint, and salivary threshold. Significant correlations were found 
between anaerobic threshold and salivary threshold and between anaerobic threshold and double -product break- 
point. [Conclusion] As a method for estimating anaerobic threshold, salivary threshold was as good as or better 
than determination of double-product breakpoint because the correlation between anaerobic threshold and salivary 
threshold was higher than the correlation between anaerobic threshold and double-product breakpoint. Therefore, 
salivary threshold is a useful index of anaerobic threshold during an incremental workload. 
Key words: Salivary a-amylase. Salivary threshold. Anaerobic threshold 

(This article -was submitted Dec. 20, 2013, and was accepted Jan. 13, 2014) 



INTRODUCTION 

With the rapid increase in the proportion of elderly in 
Japan, the proportion of adult diseases such as cancer, isch- 
emic heart disease, cerebrovascular disease, and diabetes 
mellitus has also increased, now accounting for about 60% 
of the causes of mortality'' Exercise plays a crucial role 
in the prevention of adult disease and is recommended to 
maintain or improve health^' 

It has been reported that a certain intensity of exer- 
cise is required for improvements in health and physical 
strength^"^'. At least a moderate exercise intensity is re- 
quired to improve the health of adults^', but comorbidities 
in the elderly may restrict their ability to exercise. There- 
fore, exercise should be prescribed at a suitable intensity 
for the elderly, i.e., the intensity at which the benefits of 
exercise can be gained but risks of exercise avoided. The 
balance between benefit and safety varies with the physical 



*Corresponding author. Kazunori Akizuki (E-mail: 
45030016@ipu.ac.jp) 

©2014 The Society of Physical Therapy Science. Published by IPEC Inc. 
This is an open-access article distributed under the terms of the Cre- 
ative Commons Attribution Non-Commercial No Derivatives (by-nc- 
nd) License <http://creativecommons.Org/licenses/by-nc-nd/3.0/>. 



condition of the individual but is considered to occur at a 

lower intensity in elderly people with existing disease than 
in healthy young adults. Therefore, exercise intensity in the 
elderly must be carefully adjusted. 

Anaerobic threshold (AT) has been proposed as an ob- 
jective criterion for precise adjustment of exercise inten- 
sity^'. AT is the exercise intensity just after which energy 
production by anaerobic metabolism is added to aerobic 
metabolic energy production during incremental exercise. 
It is estimated by the lactate threshold (LT), which is the 
workload at which the blood lactate concentration abruptly 
increases, and by the ventilatory threshold (VT), which is 
the workload at which the rate of pulmonary minute venti- 
lation abruptly increases nonlinearly during a progressive 
exercise test''- "'I Wasserman et al. contend that the ven- 
tilatory threshold is the same as the lactate threshold^'. It 
has been reported that both thresholds are safe and effective 
criteria"- However, both thresholds have disadvantages; 
specifically, the gas analyzer used to measure expired gas 
for determination of VT is expensive and is limited to use in 
a laboratory setting, and blood collection for determination 
of LT is invasive. 

Therefore, some alternatives for estimating AT simply 
have recently been developed. One is the double -product 
breakpoint (DPBP)'^', or the point at which double product 



1060 J. Phys. Ther. Sci. Vol. 26, No. 7, 2014 



(DP), the product of heart rate and systolic blood pressure, 
abruptly increases during a progressive exercise test. The 
DPBP can be calculated from the intersection of two re- 
gression lines, and its validity has already been established 
based on the high correlation between DPBP and AT (r = 
0.87, p < 0.001)i*-i6'. Another method for estimating AT is 
heart rate threshold (HRT), which uses only heart rate'^- 
HRT is obtained from the relationship between running 
speed and heart rate. The running speed-heart rate rela- 
tionship is linear from low to submaximal speeds and cur- 
vilinear from submaximal to maximal speeds. The speed of 
transition from the linear to the curvilinear phase coincides 
with the beginning of a sharp accumulation of blood lac- 
tate'^'. However, it has been reported that the DPBP method 
is inferior to expired gas analysis in detecting AT'"*^ and that 
the HRT occurs at a higher workload than AT'^\ For this 
reason, it is necessary to develop a more sensitive indicator 
or to create a method combining a number of indicators to 
improve the ability to detect AT and thus prescribe suitable 
exercise to subjects with reduced exercise tolerance. 

Both systolic blood pressure and heart rate, the com- 
ponents of DP, are controlled by the sympathetic nervous 
system. The plasma catecholamine concentration, which 
reflects sympathetic nervous system activity during incre- 
mental exercise, has been shown to start to rise abruptly 
at a workload similar to the LT. In addition, it has been 
reported that the plasma catecholamine concentration cor- 
relates with the blood lactate concentration^"- This sug- 
gests that DP may reflect changes in sympathetic nervous 
system activity and blood lactate concentration through the 
plasma catecholamine concentration. Therefore, it is pos- 
sible to detect the AT more precisely if an index is used 
that is a more sensitive reflection of sympathetic nervous 
system activity. The proposed index of sympathetic nervous 
system activity is salivary a-amylase, an enzyme in saliva 
that has been reported to be controlled by the sympathetic- 
adrenal-medullary system^^' -^^^ and to be a sensitive indi- 
cator of the activity of the sympathetic nervous system^"*'. 
Therefore, it is possible to detect changes in blood lactate 
concentration indirectly by measuring salivary a-amylase, 
a measurement that can be performed quickly under test 
conditions. Calvo et al. investigated the relationship be- 
tween the salivary threshold (Tsa), the workload at which 
salivary a-amylase abruptly increases, and the LT by mea- 
suring salivary a-amylase and blood lactate concentrations 
during a multistage incremental exercise^''. The authors 
concluded that Tsa had a high correlation with the LT (r = 
0.95, p < 0.001), and the detection rate was 80%. The results 
suggested that the high correlation between the Tsa and LT, 
as well as the ease of measuring salivary a-amylase, makes 
Tsa a valid and useful estimate of AT. However, there are 
few reports on the relationship between AT and Tsa. More- 
over, no report has investigated the relationship between 
Tsa and other methods of estimating AT. Determination of 
whether the detection rate is improved by combining Tsa 
and DPBP would provide meaningful information for exer- 
cise prescription, particularly for elderly people with preex- 
isting disease. 

The purpose of this study was to assess the validity of 



salivary a-amylase as a method for estimating AT quickly 
and to clarify the relationship between salivary a-amylase 
and DPBP in order to establish a way to adjust exercise in- 
tensity to a safe and effective range. 

SUBJECTS AND METHODS 

The subjects of this study were 11 healthy young adults 
(23.8 ± 1.8 years, height 173 ± 5.0 cm, body weight 64.6 ± 
6.3 kg, body mass index 21.7 ± 1.3 kg/m^). None of the sub- 
jects reported any neurological or vestibular disorders, or- 
thopedic conditions, or oral cavity diseases before partici- 
pating in this study. All subjects provided written informed 
consent before commencing the experiment. This study was 
conducted in accordance with the Declaration of Helsinki. 

We used an electrically braked cycle ergometer (AERO- 
BIKE 75XL, Combi Co. Ltd., Tokyo, Japan) for the exercise 
test. The timing of the experiment (6:00 p.m. to 8:00 p.m.) 
was chosen to minimize the influence of circadian varia- 
tion in salivary a-amylase^^l Room temperature was set to 
24°C. Since the activity of salivary a-amylase can be influ- 
enced by alcohol, medications, food, and caffeine, all sub- 
jects were instructed not to drink any alcohol the day before 
measurement and not to consume food or caffeine 2 hours 
before measurement. 

After a rest period of 3 min on the ergometer, subjects 
were instructed to try to maintain a cadence of 50 rpm while 
the workload level was increased by 20 watts per 3 min be- 
ginning with 10 watts. The exercise test was continued until 
subjects could no longer maintain the prescribed cadence 
due to fatigue or until completion of 2 stages following that 
at which AT was observed. 

Oxygen consumption (VO2), carbon dioxide production 
( VCO2), and ventilatory equivalent (VE) were measured us- 
ing a breath-by-breath gas analyzer (AE-300, Minato Medi- 
cal Science, Osaka, Japan). The V-slope method was used 
to determine AT-^l 

To calculate DP, systolic blood pressure and heart rate 
were measured during the last 30 seconds of each stage of 
the incremental exercise test. The same examiner measured 
systolic blood pressure using a stethoscope and mercury 
sphygmomanometer. Heart rate was measured using an 
electrocardiogram monitor (BSM-2401, Nihon Kohden, To- 
kyo, Japan). The DPBP was determined using a computer 
algorithm as follows: The linear regression lines of DP as a 
function of workload were calculated for all possible divi- 
sions of the data. DPBP was determined by choosing the 
intersection, or breakpoint, of the 2 lines representing the 
minimum residual sum of squares from among various in- 
tersections of the 2 lines. 

Salivary a-amylase was measured using a portable sali- 
vary amylase analyzer (Salivary amylase monitor, NIPRO, 
Osaka, Japan). It was verified that within the analyzer's 
linear range (10-230 kU/L), this handheld monitor's accu- 
racy (R^ = 0.989), precision (coefficient of variation < 9%), 
and measurement repeatability (range -3.1% to +3.1%) ap- 
proached those of a more elaborate laboratory-based auto- 
mated clinical chemistry analyzer (Olympus America Inc., 
Center Valley, PA, USA). Salivary a-amylase was measured 



1061 



from saliva samples collected during the last 30 seconds of 
each stage of an incremental exercise test by inserting the 
reagent test strip directly into the subject's oral cavity. The 
mask for expired gas analysis was briefly removed to obtain 
the saliva sample and then replaced after saliva collection. 
Tsa was determined in the same manner as DPBP. 

IBM SPSS for Windows Version 20.0 (IBM Corp., Ar- 
monk, NY, USA) was used to perform the statistical anal- 
ysis. Workload at the AT, Tsa, and DPBP were compared 
using one-way ANOVA to investigate the difference in 
workload between each index. Pearson's correlation coef- 
ficients were used to assess the relationship between work- 
load at the AT and Tsa and between the AT and DPBP. In 
addition, with Tsa-1 (or DPBP-1) representing 1 workload 
prior to that corresponding to Tsa (or DPBP), Tsa-2 repre- 
senting 2 workloads prior, Tsa-3 representing 3 workloads 
prior, and so on, Tsa, Tsa-1, Tsa-2, and Tsa-3 and workloads 
for DPBP, DPBP-1, DPBP-2, and DPBP-3 were compared 
by one-way ANOVA with repeated measures to identify the 
dynamics of salivary a-amylase and DPBP associated with 
an incrementally increasing workload. We chose an alpha 
level of 0.05 to indicate significant effects. 

RESULTS 

Subject characteristics are shown in Table 1 . In the pres- 
ent study, AT could be determined in all subjects (100%). 
However, Tsa and DPBP could not be detected in 2 of the 
subjects (detection rate of 82% for both methods) because 
the increase was not abrupt enough to identify 2 regression 
lines clearly. In the following analysis, therefore, data for 
the subjects whose Tsa and DPBP could not be detected 
were excluded. The results of one-way ANOVA revealed 
that there was no significant difference among workloads at 
the AT, DPBP, and Tsa (Table 2). 

There were significant correlations between AT and Tsa 
(r = 0.951, p < 0.01) and between AT and DPBP (r = 0.940, 
p < 0.01). The results of one-way ANOVA with repeated 
measures identified the significant main effects of salivary 
a-amylase (Fj 12 6 = 24.0, p < 0.01, partial = 0.75) and 
DP (Fi.34^ 10,3 = 44.7, p < 0.01, partial = 0.85), and post hoc 
analysis using Bonferroni's test was executed. The results 
showed that there were significant differences between Tsa 
and Tsa-1 (p < 0.05), between Tsa and Tsa-2 (p < 0.01), and 
between Tsa and Tsa-3 (p < 0.01). However, there were no 
significant differences between Tsa-1 and Tsa-2, between 
Tsa-1 and Tsa-3, or between Tsa-2 and Tsa-3. On the other 
hand, DP showed significant differences between all cor- 
responding workloads. 

DISCUSSION 

The purpose of this study was to assess the validity of 
Tsa as a workload index by clarifying the relationship be- 
tween AT, which was measured by analysis of expired gas, 
and Tsa, which represented the workload at which salivary 
a-amylase abruptly increased. We also investigated the re- 
lationship between DPBP, which has been established as 
a method for quickly estimating AT, and Tsa in order to 
assess the usability of Tsa as another method for quickly 



Table 1. SLibjccl characlcristics 





23.8 ± 1.8 




1.73 ± 0.05 


Wpitrht 


64.6 ± 6.3 


DJVli ^^Kg/m ) 


zi. / ± 1. J 


HR at rest (beats/min) 


70.0 ± 11.4 


HR at AT (beats/min) 


116.2 ± 16.4 


VO2 at AT (mL/min) 


1,170.2 ± 189.3 


VOj/W at AT (mL/min-kg) 


18.1 ±2.3 


Salivary a-amylase at rest (kU/L) 


22.2 ± 27.5 


Salivary a-amylase at Tsa (kU/L) 


40.0 ± 18.1 


DP at rest (mmHg • beats/min) 


8,740.4 ± 1,994.7 


DP at DPBP (mmHg • beats/min) 


14,868.2 ±3,153.1 



estimating AT. 

The results of this study indicate significant correlations 
between AT and Tsa (r = 0.951, p < 0.01). Furthermore, a 
significant difference was not observed in the workload at 
the AT and Tsa. This result suggests that Tsa has validity as 
a method for estimating AT quickly, which confirms results 
reported by Calvo et al-^^l Yamamoto et al. investigated the 
activity of the parasympathetic and sympathetic nervous 
systems during exercise using heart rate variability. The 
authors revealed that the indicator of parasympathetic ner- 
vous system activity decreased progressively from rest to 
a workload equivalent to 60% VT and that the indicator of 
sympathetic nervous system activity increased only when 
exercise intensity exceeded VT^^l These results suggest 
that the activity of the sympathetic nervous system changed 
at the VT, which was equivalent to the AT, and that AT 
might be estimated by monitoring the activity of the sym- 
pathetic nervous system. Moreover, it has been reported 
that the salivary a-amylase concentration increases with in- 
creased physical activity, such as treadmill exercise^'', run- 
ning-"*' and cycle exercise^"*' However, physical activ- 
ity per se does not necessarily increase salivary a-amylase. 
Chatterton et al. investigated salivary a-amylase during 
walking, jogging, and running and reported that jogging 
and running increased salivary a-amylase but that walk- 
ing did not affect salivary a-amylase^*'. This result con- 
firmed that the increase in salivary a-amylase was caused 
by physical activity exceeding a certain workload (i.e., the 
AT). Chatterton et al. also investigated the correlation be- 
tween salivary a-amylase and plasma catecholamines and 
showed significant correlations between them (r = 0.64 for 



Data presented as mean ± SD. BMI, body mass index; AT, an- 
aerobic threshold; DP, double product; DPBP, double -product 
breakpoint; HR, heart rate; Tsa, salivary threshold; W, Watt 



Table 2. Power output at AT, Tsa, and DPBP 

AT(W) 70.0 ±28.3 

Tsa (W) 61.1 ± 28.5 

DPBP (W) 65.6 ±21.9 

AT, anaerobic threshold; DPBP, double-prod- 
uct breakpoint; Tsa, salivary threshold; W, watt 



1062 J. Phys. Ther. Sci. Vol. 26, No. 7, 2014 



norepinephrine and r = 0.49 for epinephrine)^"^'. This result 
directly indicates that salivary a-amylase reflects the activi- 
ty of sympathetic nervous system. Since salivary a-amylase 
is an index that sensitively reflects the activity of the sym- 
pathetic nervous system and the activity of the sympathetic 
nervous system is caused by workloads above the AT, it is 
suggested that Tsa, which is the point of inflection of sali- 
vary a-amylase, is a good index by which to estimate AT. 
We noted significant strong correlation between AT and Tsa 
in the present study. 

The results of the present study also indicated that Tsa 
was at least as good as DPBP as a method for estimating 
AT quickly because the correlation between the AT and Tsa 
was 0.951 (p < 0.001), whereas the correlation between the 
AT and DPBP was 0.940 (p < 0.001). This result suggests 
that salivary a-amylase reflects the activity of the sympa- 
thetic nervous system more sensitively than DP, the com- 
ponents of which are controlled by the sympathetic nervous 
system. Spence et al. compared the systolic and diastolic 
blood pressure responses to exercise before the AT with 
those after the AT by using a cycle ergometry^-''. The au- 
thors concluded that the rate of increase of systolic blood 
pressure significantly increased after the AT. Tanaka et al. 
also demonstrated that the slope of a regression line of sys- 
tolic blood pressure and heart rate increased after the AT by 
comparing the slope of a regression line of systolic blood 
pressure and heart rate before and after the AT'^l However, 
other researchers reported that the slope of a regression line 
of systolic blood pressure and heart rate may not increase 
after the AT. Riley et al. investigated the relationship be- 
tween DPBP and AT, as determined from analysis of expi- 
ration gas, by measuring systolic blood pressure and heart 
rate during an exercise test'^^l Their results indicated that, 
although DPBP and AT are strongly correlated, the rates 
of increase of systolic blood pressure and heart rate do not 
always increase after the AT. In the study of Riley et al., the 
rate of increase of systolic blood pressure increased after 
the AT for some subjects but that for heart rate did not; other 
subjects indicated the opposite. Furthermore, Conconi et al. 
researched the relationship between HRT and LT by obtain- 
ing the HRT, which was calculated from running speed and 
heart rate'^'. The results indicated that there was a strong 
correlation between HRT and LT and that increased run- 
ning speed involved an increase in heart rate. However, 
the rate of increase of heart rate after the HRT decreased, 
which is in contrast to the results of Tanaka'^l Both Riley 
et al.'**' and Conconi et al.'**' demonstrated that changes in 
systolic blood pressure and heart rate before and after the 
AT differed among individuals. DP is highly correlated 
with AT because it consists of 2 variables, so that even if 
only I variable shows unique changes, the other variable 
is able to change in a complementary manner. However, 
using the product of variables that vary among individuals 
may lead inaccurate estimation of AT. Salivary a-amylase 
is therefore superior to DP in the degree of correlation to AT 
because salivary a-amylase can measure the activity of the 
sympathetic nervous system using only 1 variable. 

There are other ways in which Tsa is superior to DPBP 
for estimation of AT. To calculate DP, blood pressure and 
heart rate must be measured, but blood pressure measure- 



ment temporarily restricts subjects' physical activity. Ad- 
ditionally, measurement of heart rate requires equipment 
such as an electrocardiograph monitor, thus restricting the 
measurement environment. In contrast, measurement of 
salivary a-amylase requires only insertion of the reagent 
test strip directly into the subject's oral cavity to collect sa- 
liva samples without the need to interrupt activity, and it 
can be performed in various environments. Thus, salivary 
a-amylase estimates AT simply and has the advantages of 
easier measurement and more sensitive reflection of sym- 
pathetic nervous system activity than can be achieved with 
DP 

Interestingly, the results of the present study suggest that 
a more correct estimation of AT can be achieved by com- 
bining Tsa and DPBP. In this research, although AT was 
identified in all subjects using expired-gas analysis, Tsa and 
DPBP were unable to be determined in 2 subjects. However, 
because data allowing determination of DPBP were always 
collected, combining Tsa and DPBP allowed identification 
of AT in all subjects. This finding suggests that the vari- 
ables salivary a-amylase, systolic blood pressure, and heart 
rate are controlled by the sympathetic nervous system but 
that the expression of these variables in vivo differs among 
individuals. Therefore, inclusion of all the variables in the 
estimation of AT may allow a more precise estimation. AT 
estimation using salivary a-amylase is useful for subjects in 
whom Tsa can be identified even if this is the only method 
used because Tsa and AT are strongly correlated. However, 
the combination of DPBP and Tsa might be desirable for 
some subjects, since Tsa was unable to be identified in about 
18% of our study subjects. While it is better to measure sali- 
vary a-amylase to estimate AT because of its superiority 
to DPBP in the degree of correlation with AT, if Tsa is not 
able to be determined, DP can be calculated to complement 
the detection of AT. As a result, AT can be determined with 
a detection rate as high as that for expired-gas analysis. 
This result is in line with the study of Calvo et al.^"*', who 
mentioned that differences among subjects in their hydra- 
tion state before reporting to the laboratory or in the con- 
tribution of each salivary gland to the production of total 
saliva explained the inability to detect Tsa in 20% of their 
subjects. In this study, although we encouraged subjects to 
drink water before measurement and allocated a point un- 
der the tongue for saliva collection in order to minimize 
these influences, the detection rate was not improved. Fur- 
ther studies are needed to improve the precision of measure- 
ment of salivary a-amylase itself 

The results of this study clarified that Tsa is a useful in- 
dex for estimating AT during an incrementally increasing 
workload. Tsa was not able to be detected in 2 subjects, but 
in such cases, the rate of determination of AT can be im- 
proved by combining Tsa with DPBP. Determination of AT 
using salivary a-amylase is superior to other methods be- 
cause the measurement device is easily transportable, mak- 
ing measurement possible in various environments, and be- 
cause of the noninvasiveness of the technique. We believe 
that the ability to adjust exercise to the optimal intensity in 
various environments using salivary a-amylase can make 
exercise safer and more effective. 



1063 



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