Johanna Bergman
Avalon Ernstson
NV09
PROJEKTARBETE
2011-2012
Changes in salivary α-amylase activity caused
by the possible stressor of a 100 meter sprint
Handledare: Håkan Klarén
Abstract
It is suggested that changes in salivary α-amylase activity could be used to measure stress
levels. In this study, physical activity was used as a stressor. The aim was to see if there is a
change in salivary α-amylase activity following a 100 meter sprint. Two experiments were
performed. Firstly, five youths ran 100 meter sprint followed by collection of salivary
samples. Secondly, salivary samples from four of the subjects were collected during rest. The
results showed large individual variations. All though, directly following the 100 meter sprint
all subjects displayed a decrease in α-amylase activity. Increases and decreases in α-amylase
activity could be seen during rest. Even though the changes found during rest are less
significant, it cannot be concluded that the changes after the 100 meter sprint indicates a stress
reaction. This study does not support α-amylase activity as a single biomarker for measuring
and monitoring stress levels.
(150 words)
Key words: salivary α-amylase, stress, physical activity
Abstract in Swedish
Att mäta förändringar i α-amylasaktivitet i saliven kan vara ett sätt att mäta stressnivåer. I
denna studie användes fysisk aktivitet som stressor. Syftet var att se om α-amylasaktiviteten
påverkas av ett 100 meterslopp. Två försök utfördes. I det första sprang 5 ungdomar ett 100
meterslopp, salivprover samlades in under försöket. I det andra satt fyra av försökspersonerna
ner under vila medan salivprover samlades in. Resultaten visade en stor variation mellan
försökspersonerna. Direkt efter 100 metersloppet kunde dock en minskning i
α-amylasaktivitet ses hos alla försökspersoner. Ökningar och minskningar i α-amylasaktivitet
kunde ses även under vila. Trots att förändringarna var betydligt mindre under vila kan det
inte fastställas att förändringarna i α-amylasaktivitet efter 100 metersloppet visar på en
stressreaktion. Denna studie stödjer inte α-amylasaktivitet som en ensam biomarkör för att
mäta och följa stressnivåer.
(133 ord)
Nyckelord: saliv α-amylas, stress, fysisk aktivitet
2
Table of Contents
Introduction ............................................................................................................................................. 5
Background ............................................................................................................................................. 6
Stress reactions in the sympathetic nervous system ............................................................................ 6
Saliva ................................................................................................................................................... 6
Salivary α-amylase .............................................................................................................................. 6
Starch ................................................................................................................................................... 6
α-amylase and exercise........................................................................................................................ 7
Method .................................................................................................................................................... 7
Choice of subjects ............................................................................................................................... 7
Collecting of saliva samples ................................................................................................................ 8
Experiment 1 - 100 meter sprint and α-amylase activity ..................................................................... 8
Experiment 2 - rest and α-amylase activity ......................................................................................... 8
Preparation of starch solution .............................................................................................................. 8
Analysis of data ................................................................................................................................... 9
Results ................................................................................................................................................... 10
Table 1 – Results of survey ............................................................................................................... 10
Experiment 1 – 100 meter sprint and α-amylase activity .................................................................. 10
Experiment 2 - rest and α-amylase activity ....................................................................................... 13
Conclusion ............................................................................................................................................. 18
Further investigations ............................................................................................................................ 18
References ............................................................................................................................................. 19
Appendix 1 – Initial experiments .......................................................................................................... 20
Initial experiment 1 ........................................................................................................................... 20
Initial experiment 2 ........................................................................................................................... 20
Analysis of samples from initial experiment 1 .................................................................................. 20
Analysis of samples from initial experiment 2 .................................................................................. 21
Results ............................................................................................................................................... 21
Initial experiment 1 ....................................................................................................................... 21
Initial experiment 2 ....................................................................................................................... 23
Appendix 2 – Tests of method .............................................................................................................. 25
Test 1 ................................................................................................................................................. 25
Test 2 ................................................................................................................................................. 25
Test 3 ................................................................................................................................................. 25
3
Appendix 3 – Uncertainty calculations ................................................................................................. 26
Starch solution - concentration .......................................................................................................... 26
α-amylase activity ............................................................................................................................. 26
Appendix 4 – Survey ............................................................................................................................. 27
4
Introduction
Stress is the organism’s way of handling strain. Stress prepares the body for flight or fight and
functions unnecessary for the stress response are set aside. Stress is basically a positive
reaction necessary for the survival of the species.1 Despite that, stress is a growing problem in
today’s society. The problem is that the stressors have changed and are changing, but the
organism remains the same.2 There is a significant difference between running from a lion and
holding a work presentation, but the body will react the same way. When facing a lion flight
is the preeminent option, but in daily stress situations running away is not an alternative. The
body does not react in a natural way which can cause the unpleasant feeling of stress.
However, an occasional short-term stress reaction is not a problem. The problem comes when
there is long term strain and the body has no time to recover between the stress reactions, also
known as chronic stress.
Chronic stress is believed to cause several diseases, e.g. cardiovascular disease, fibromyalgia,
burnout and depression.3 To be able to state if some diseases are related to stress and to be
able to prevent stress related diseases, an objective way to measure stress levels are required.
Stress is a very complex reaction and all the consequences of stress are still not identified.4
Therefore most of the methods of measuring stress are uncertain today. The most common
ways of measuring stress is by levels of catecholamines and cortisol in the blood. This
requires a blood test. It has been found that salivary α-amylase activity might be another
method of measuring stress without requiring a blood test.
Physical activity and stress both affect the autonomic nervous system and give similar
reactions in the body, physical activity generate a stress response. A way to investigate if
salivary α-amylase activity could be a reliable way of measuring stress could be to use
physical activity as a stressor. In this study 100 meter sprint was used as stressor. 100 meter
sprint was expected to imitate a flight reaction and therefore cause an acute physical stress
reaction.
The aim of this study was to investigate if salivary α-amylase activity could be used as an
indicator of physical stress.
In what way is the salivary α-amylase activity affected by the possible stressor of a 100
meter sprint?
1
Rolf Ekman och Bengt Arnetz (red), Stress: Individen, samhället, organisationen, molekylerna (Stockholm,
Liber AB, 2005) p.31
2
Rolf Ekman och Bengt Arnetz (red), Stress: Individen, samhället, organisationen, molekylerna (Stockholm,
Liber AB, 2005) p.76
3
http://www.1177.se/Halland/Fakta-och-rad/Sjukdomar/Stress/ (Hämtad 15.37 2012-01-13 senast ändrad 201005-17)
4
Rolf Ekman och Bengt Arnetz (red), Stress: Individen, samhället, organisationen, molekylerna (Stockholm,
Liber AB, 2005) p.372
5
Background
Stress reactions in the sympathetic nervous system
During physical activity the sympathetic nervous system, which is a part of the autonomic
nervous system, is activated.5 The sympathetic nervous system prepares the body for flight or
fight. The heart rate increase, the bronchi dilates, release of glucose from the liver is
stimulated and the synthesis production of adrenaline and noradrenalin is stimulated. Functions
as salivary secretion, the activity in the pancreas and the food digestion is inhibited. Physical
activity and stress both activates the sympathetic nervous system.6 From this it can be
suggested that physical activity can be used as a stressor.
Saliva
Saliva is a colourless fluid found in the oral cavity of humans. Approximately one litre of
saliva is produced every day by three major and numerous minor salivary glands. 7, 8 Saliva is
secreted when eating but can also be secreted by external stimuli. The human saliva contains
of 99 percent water but also different types of proteins for example α-amylase, which
hydrolyzes starch, and mucin, a slippery glycoprotein.7, 9 The saliva also contains ions such as
Na+, Ca2+, Cl- and K+.10 In addition the human saliva has antibacterial effects and the ion
HCO3- which neutralizes acid and acts as a buffer.7
Salivary α-amylase
The enzyme α-amylase is one of the proteins in human saliva. The α-amylase is the first stage
in hydrolyzing starch and glycogen in food digestion. The starch and glycogen are digested to
smaller polysaccharides and the disaccharide maltose.7 α-amylase can be found in the cells of
the acinar region of the salivary glands packed into zymogen granules. When the α-amylase is
secreted it is released through exocytosis of the zymogen granules.5
Starch
Starch is an energy rich molecule that comes from plants. It is a polysaccharide, which is a
long chain of monosaccharides. Each chain can contain a few thousand monosaccharides.11
Iodine solution (KI/I2) can be used as an indicator for starch. If there is starch in a solution a
dark-blue complex will appear. As the starch amount decreases the blue colour will fade and
finally disappear.
5
Felipe Calvo, José L Chicharro, Fernando Bandrés, Alejandro Lucía, Margartia Pérez, Julián Álvarez, Luis L.
Mojares, Almudenaa F. Vaquero, Julio C. Legido, Anaerobic Threshold Determination With Analysis of Salivary
Amylase (Can. J. Appl. Physiol. 22(6): 553-561, 1997, Canadian Society for Exercise Physiology), p.554
6
Karlsson, Molander, Wickman, Biologi 2 för gymnasieskolan (Stockholm: Liber AB, 1997), p. 142-143
7
Cambell, Reece, Biology sixth edition (San Francisco: Pearson Education Inc., 2002) p.860-861
8
José L. Chicharro, Alejandro Lucía, Margarita Pérez, Almudena F. Vaquero, Rosario Ureña, Saliva
Composition and Exercise (Sports Med , 1998 Jul ; 26 (1) : 17-27), p. 18
9
José L. Chicharro, Alejandro Lucía, Margarita Pérez, Almudena F. Vaquero, Rosario Ureña, Saliva
Composition and Exercise (Sports Med , 1998 Jul ; 26 (1) : 17-27), p. 19
10
José L. Chicharro, Alejandro Lucía, Margarita Pérez, Almudena F. Vaquero, Rosario Ureña, Saliva
Composition and Exercise (Sports Med , 1998 Jul ; 26 (1) : 17-27), p. 20
11
Cambell, Reece, Biology sixth edition (San Francisco: Pearson Education Inc., 2002) p. 66-67
6
α-amylase and exercise
Changes in human salivary α-amylase are suggested to indicate stress. During stress and
exercise the sympathetic nervous system is activated which leads to a decreased salivary
secretion. Studies have shown that the concentration of proteins during stress or exercise is
higher than before the stress or the exercise. This may show that the decrease in salivary flow
might mainly consist of a decrease of water. That would leave the amount of proteins intact
and lead to a higher concentration of proteins in the saliva during stress or exercise. A higher
concentration of proteins will also result in a higher α-amylase activity since α-amylase is one
of the proteins in human saliva.12, 13
Another study has shown a correlation between α-amylase activity and lactate levels. The
cells in the body need oxygen to produce adenosine triphospate (ATP). ATP is a nucleotide
that has a central role in the energy usage in the cells, without ATP the cell cannot function.
During sufficient strenuous exercise the muscle cells do not get enough oxygen and starts to
work anaerobic, which means the cells produce ATP without oxygen. This can only happen
during short time and the waste product lactic acid is formed.14, 15 When the muscles go from
aerobic- to anaerobic work it is called the anaerobic threshold (AT). The study suggests that
the α-amylase activity decreases until the subjects reach exercise intensity were the anaerobic
threshold (AT) is reached. When AT is reached the α-amylase activity will start to rise
towards base levels again.16
Method
Choice of stressor
A 100 meter maximal sprint was chosen as a stressor. 100 meter sprint was expected to
imitate a flight reaction and therefore cause an acute physical stress reaction in the body. The
subjects did not run against each other to avoid the psychological stress a competition can
cause.
Choice of subjects
For the initial experiments, ourselves was used as subjects. For the main experiments five
healthy youths (three men and two women) who were used to physical activity were chosen.
Three of the subjects were track and field athletes and two were soccer players. The subjects
were all between the ages of 15-19 (mean 17,6 years). To avoid placebo effect none of the
subjects were aware of the aim of the study.
12
G. Ljungberg, T. Ericson, B. Ekblom, D. Birkhed, Saliva and marathon running (Scand J Med Sci Sports,
1997:7:214-219), p. 214
13
José L. Chicharro, Alejandro Lucía, Margarita Pérez, Almudena F. Vaquero, Rosario Ureña, Saliva
Composition and Exercise (Sports Med , 1998 Jul ; 26 (1) : 17-27), p. 19, 21
14
Karlsson, Molander, Wickman, Biologi 2 för gymnasieskolan (Stockholm: Liber AB, 1997), p. 29
15
Cambell, Reece, Biology sixth edition (San Francisco: Pearson Education Inc., 2002) p. 170
16
José L. Chicharro, Alejandro Lucía, Margarita Pérez, Almudena F. Vaquero, Rosario Ureña, Saliva
Composition and Exercise (Sports Med , 1998 Jul ; 26 (1) : 17-27), p. 22
7
Collecting of saliva samples
The subjects did not eat or chew anything within 1 hour before the experiments. To rinse
away food scraps that can contain starch, every experiment was started by gargling deionised
water for 60 seconds followed by 10 minutes of rest. Every saliva sample was collected by
gargling 20 ml of deionised water for 60 seconds. The salivary-water solution was spitted out
and collected in 10 ml plastic test tubes with lid. All samples were stored at -20ᵒ C.
Experiment 1 - 100 meter sprint and α-amylase activity
To design experiment 1 two initial experiments (Appendix 1) were performed.
Experiment 1 was to perform an individual maximal 100 meter sprint. Before the sprint the
subjects did a warm-up. Saliva samples were collected both before and after the sprint.
Before warm-up sample 3.1 was collected then the warm-up (800 meter jog on an outdoor
track, stretch, 4x20 meter running exercises, 60 meter soaraway running) was performed.
Directly after the warm-up sample 3.2 was collected. Sample 3.3 was collected after 15
minutes of rest. At an outdoor track a maximal individual sprint of 100 meter was performed.
Directly after finish, sample 3.4 was collected. Following samples (3.5-3.8) was collected
each 10 minutes for 40 minutes while resting. Each sample was collected in 10 ml plastic test
tubes with lid. The 100 meter race was performed individually without any impact from the
other participants, i.e. without competition or timing. After the experiment a survey
(Appendix 4) was handed out to all of the subjects.
Experiment 2 - rest and α-amylase activity
Experiment 2 was to collect saliva samples while the subjects were resting.
The saliva samples were collected while the subjects sat down without doing any kind of
physical activity. Six salivary samples was collected in a ten minutes interval (total time of
collection was 50 minutes). Each sample was collected in 10 ml plastic test tubes with lid.
Preparation of starch solution
450 ml of deionised water was boiled on a magnetic stirrer with heating device. 2,5 g starch
(starch soluble GR for analysis, Merck pro analysis, (C6H10O5)n) was mixed with 50 ml
deionised water. The 50 ml starch solution was poured into the boiling water. The solution
was boiled until clear. The 500 ml starch solution was cooled down to room temperature. The
solution was used within 36 h after it was made.
8
Analysis of data
The analyzing method was based on a method designed by a former student at
Sannarpsgymnasiet.17 The method was to analyze saliva samples by taking the time of how
long it takes for α-amylase to degrade a fixed amount of starch. Iodine solution (KI/I2) was
used as an indicator. When changed back to reference iodine colour all starch was considered
degraded. Long degrading time represents low α-amylase activity and short degrading time
represents high α-amylase activity.
Small test tubes (3 ml) were put into a test tube holder. 100 µl iodine solutions were pipetted
to every test tube. 20 ml starch solution was poured into a conical flask (250 ml) and covered
with parafilm to avoid evaporation. The saliva samples were thawed and warmed to 24,5 ᵒC in
a water bath. The conical flask with starch solution was also warmed to 24,5 ᵒC in the water
bath. A thermometer was used to control that the saliva samples and the conical flask with
starch solution was 24,5 ᵒC. 4 ml of the tempered saliva solution was pipetted into the
preheated starch solution. The solution was stirred and a stopwatch was directly started when
the saliva solution was pipetted into the starch solution. The saliva-starch solution was placed
in the water bath again to keep the temperature 24,5 ᵒC. 500 µl of the saliva-starch solution
was pipetted into a test tube every minute. When the colour shifted from dark blue to brown,
the interval was shorten to 15 seconds. The pipetting was continued until no further colour
change was noticed. The watch was stopped and the result written down. To ensure the
samples from the same subject had the same amount of hydrolyzed starch each finished
sample was compared with a chosen finished test tube from the same subject as a reference.
The α-amylase activity was determined to how long it took for the α-amylase in 4 ml saliva
solution to hydrolyze 100 000 µg starch.
Maria Svensson, A study of the individual differences in α-amylase activity (Extended essay IB
Sannarpsgymnasiet 2005)
17
9
Results
Table 1 – Results of survey (Appendix 4)
Answers from the survey handed out to the subjects after experiment 1.
Subject
Gender
Age
Height
(cm)
Weight
(kg)
A
Female
17
166
67
B
Female
18
171
68
C
Male
16
184
72
D
Male
18
175
72
E
Male
19
191
98
*1 is not stressed at all and 5 is very stressed.
Amount of
training
(training/week)
Estimated
stress
level
today
(scale 15)*
4-5
5-6
2-3
3
5
2
1
3
1
1
Estimated Food
stress
intake
level of
today
week
(scale 15)*
2
1
4
1
1
2
1
3
0
2
Experiment 1 – 100 meter sprint and α-amylase activity
A 100 meter individual maximal sprint was performed. Saliva samples (3.1 – 3.8) were
collected before and after the 100 meter maximal sprint. α-amylase activity was determined to
how long it took for the α-amylase in 4 ml saliva solution to hydrolyze 100 000 µg starch.
Table 2 – Experiment 1
Sample
(subject A)
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
3.1
Base value (before warm-up)
75 (±15)
100 000 µg starch/
75 s = 1330 µg/s
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Directly after warm-up
Before 100m sprint
Directly after 100m sprint
10 min after 100m sprint
20 min after 100m sprint
30 min after 100m sprint
40 min after 100m sprint
105 (±15)
135 (±15)
90 (±15)
150 (±15)
300 (±15)
120 (±15)
90 (±15)
952
741
1110
667
333
833
1110
10
Table 3 – Experiment 1
Sample
(subject B)
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
3.1
Base value (before warm-up)
675* (±15)
148*
3.2
Directly after warm-up
240 (±15)
417
3.3
Before 100m sprint
360 (±15)
278
3.4
Directly after 100m sprint
375 (±15)
267
3.5
10 min after 100m sprint
720 (±15)
139
3.6
20 min after 100m sprint
1170 (±15)
86
3.7
30 min after 100m sprint
1170 (±15)
86
3.8
40 min after 100m sprint
585 (±15)
179
*The first sample of 3.1 was spilled and another 3.1 sample was collected directly after the first one.
After a test (Appendix 2, test 3) the secondly taken sample 3.1 was stated as a non reliable value.
Table 4 – Experiment 1
Sample
(subject C)
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
Base value (before warm-up)
Directly after warm-up
Before 100m sprint
Directly after 100m sprint
10 min after 100m sprint
20 min after 100m sprint
30 min after 100m sprint
40 min after 100m sprint
105 (±15)
135 (±15)
165 (±15)
225 (±15)
285 (±15)
420 (±15)
315 (±15)
240 (±15)
952
741
606
444
351
238
317
417
Table 5 – Experiment 1
Sample
(subject D)
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Base value (before warm-up)
Directly after warm-up
Before 100m sprint
Directly after 100m sprint
10 min after 100m sprint
20 min after 100m sprint
30 min after 100m sprint
40 min after 100m sprint
90,0 (±15)
150 (±15)
90,0 (±15)
210 (±15)
255 (±15)
225 (±15)
285 (±15)
240 (±15)
1110
667
1110
476
392
444
351
417
11
Table 6 –Experiment 1
Sample
(subject E)
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Base value (before warm-up)
Directly after warm-up
Before 100m sprint
Directly after 100m sprint
10 min after 100m sprint
20 min after 100m sprint
30 min after 100m sprint
40 min after 100m sprint
300 (±15)
165 (±15)
615 (±15)
225 (±15)
330 (±15)
540 (±15)
450 (±15)
405 (±15)
333
606
163
444
303
185
222
247
Experiment 1 - 100 meter sprint and α-amylase
activity
1400
α-amylase activity
(µg starch/s)
1200
1000
A
800
B
600
C
400
D
E
200
0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Sample
Fig. 1. α-amylase activity from five subjects (A, B, C, D and E) who participated in warm-up and a 100 meter
maximal sprint. 3.1 was collected before warm-up. 3.2 was collected directly after warm-up. 3.3 was collected
before the 100 meter sprint. 3.4 was collected directly after the 100 meter sprint. 3.5 -3.8 were collected with a
10 minutes interval after the race while the participant were resting. Each line represents one subject.
12
Experiment 1 - 100 meter lap and α-amylase
activity
α-amylase activity
(µg starch/s)
1400
1200
1000
800
600
400
200
0
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
Sample
Fig. 2. α-amylase activity from five subjects (A, B, C, D and E) who participated in warm-up and a100 meter
sprint. 3.1 was collected before warm-up. 3.2 was collected directly after warm-up. 3.3 was collected before the
100 meter sprint. 3.4 was collected directly after the 100 meter sprint. 3.5 -3.8 were collected with a 10 minutes
interval after the race while the participant were resting. The horizontal line shows the mean value for all
subjects while the vertical line range between the lowest and the highest values.
Experiment 2 - rest and α-amylase activity
Saliva samples (4.1 – 4.6) was collected while the subjects sat down during total rest for 50
minutes. α-amylase activity was determined to how long it took for the α-amylase in 4 ml
saliva solution to hydrolyze 100 000 µg starch.
Table 7 – Experiment 2
Sample
(subject A)
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
4.1
Base value
258 (±15)
100 000 µg starch/
258 s = 388 µg/s
4.2
4.3
4.4
4.5
4.6
4.1*
Value after 10 min
Value after 20 min
Value after 30 min
Value after 40 min
Value after 50 min
Control of method
447 (±15)
369 (±15)
207 (±15)
258 (±15)
318 (±15)
240 (±15)
224
271
483
386
314
417
*Sample 4.1 was analyzed twice to control the reliability in the method.
13
Table 8 – Experiment 2
Sample
(subject B)
4.1
4.2
4.3
4.4
4.5
4.6
Table 9 – Experiment 2
Sample
(subject D)
4.1
4.2
4.3
4.4
4.5
4.6
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
Base value
Value after 10 min
Value after 20 min
Value after 30 min
Value after 40 min
Value after 50 min
420 (±15)
927 (±15)
858 (±15)
807 (±15)
540 (±15)
507 (±15)
238
108
117
124
158
197
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
Base value
Value after 10 min
Value after 20 min
Value after 30 min
Value after 40 min
Value after 50 min
189 (±15)
267 (±15)
207 (±15)
180 (±15)
120 (±15)
180 (±15)
529
375
483
556
833
556
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
207 (±15)
867 (±15)
327 (±15)
360 (±15)
249 (±15)
309 (±15)
483
115
306
278
402
324
Table 10 – Experiment 2
Sample
Description
(subject E)
4.1
4.2
4.3
4.4
4.5
4.6
Base value
Value after 10 min
Value after 20 min
Value after 30 min
Value after 40 min
Value after 50 min
14
1400
Experiment 2 - rest and α-amylase activity
1200
α-amylase activity
(µg starch/s)
1000
800
A
B
600
D
400
E
200
0
4.1
4.2
4.3
4.4
4.5
4.6
Sample
Fig. 3. α-amylase activity from four subjects (A, B, D and E) during total rest. Each sample was collected with
an interval of 10 minutes. Each line represents one subject.
Experiment 2 - rest and α-amylase activity
1400
α-amylase activity
(µg starch/s)
1200
1000
800
600
400
200
0
4.1
4.2
4.3
4.4
4.5
4.6
Sample
Fig. 4. α-amylase activity from four subjects (A, B, D and E) during total rest. Each sample was collected with
an interval of 10 minutes. The horizontal line shows the mean value for all subjects while the vertical line range
between the lowest and the highest values.
15
Discussion
The experiments show that the variations in α-amylase activity between the subjects are huge.
In experiment 1 the base value had a diversity from the fastest, 75 seconds (subject A), to the
slowest, 675 seconds (subject B). Subject A thus had a nine times higher α-amylase activity
than subject B. Because of this, the subjects are not going to be compared with each other.
What can be seen though is that between sample 3.4 (directly after the 100 meter sprint) and
sample 3.5 (10 minutes of rest after the 100 meter sprint) all of the subjects have a decrease in
α-amylase activity. Four out of five subject continues to have a decrease in α-amylase activity
until 3.6 (20 minutes of rest after the 100 meter sprint) were the α-amylase activity start to rise
towards baseline level again. This can be seen clearly in fig.2. The mean values of the αamylase activity are decreasing almost linearly until 3.6 were the lowest α-amylase activity
can be found. After 3.6 the α-amylase activity is starting to increase towards baseline levels
again. It seems like 100 meter sprint affects the α-amylase activity.
Increases and decreases in α-amylase activity can also be seen in experiment 2 were the
subjects were resting. The changes during rest are though less significant than the changes
found after the 100 meter sprint. It is possible though that the results found could be a result
of a random variation. The possible random variation in α-amylase activity could be because
of a deficiency in the method. The saliva was collected in a diluted form which makes the
amount of saliva in each sample unverifiable. Therefore the changes in α-amylase activity that
can be seen during the study could be a result of a different saliva concentration in each
sample. It is though noticeable that the changes in salivary α-amylase activity are larger
during physical activity than during rest. This indicates that it is not likely that the results
found are just a random variation in salivary α-amylase activity.
No differences between men and women can be seen in the results of the experiments. Neither
a correlation between α-amylase activity and weight, height, food intake or amount of training
each week can be seen. What can be seen though is that the only subject (subject C) which
claimed to be stressed at time of the experiment shows a more linear α-amylase activity than
the others. No further investigations have been made to be able to establish that the linear
α-amylase activity is a consequence of that.
Studies have found a change in α-amylase activity during physical activity. The results are
however a bit contradictive. Ljungberg et al. have found an increase in α-amylase activity
during physical activity. The increase in α-amylase activity is explained by the fact that the
salivary glands may not be given priority during conditions of strong exercise. The non
priority leads to a decreased level of water in the saliva which results in a higher
concentration of proteins in the saliva and therefore an increase in α-amylase activity.18
Chatterton et al. are on the same track, they have also found an increase in α-amylase activity
during physical activity. They suggest that there might be a correlation between concentration
of α-amylase and the plasma levels of catecholamines. Same stimuli that increase the levels of
plasma catecholamines may also activate sympathetic input to the salivary glands and
therefore lead to an increased α-amylase activity.19 On the other hand, Calvo et al. made a
study were no direct increase in α-amylase activity during physical activity could be seen.
Results from Calvo et al. suggests that the α-amylase activity decreases until the subjects
18
G. Ljungberg, T. Ericson, B. Ekblom, D. Birkhed, Saliva and marathon running (Scand J Med Sci Sports,
1997:7:214-219), p. 214, 217
19
Robert T. Chatterton Jr., Kirsten M. Vogelsong, Yu-cai Lu, Allsion B. Ellman, Gerald A. Hudgens, Salivary
α-amylase as a measure of endogenous adrenergic activity (Clinical Physiology, volume 16 number 4 july 1996,
433-448), p. 444-445
16
reach an exercise intensity were the anaerobic threshold (AT) is reached. When AT is reached
the study show that the α-amylase activity will start to rise towards base line levels again.20
This indicates that the intensity of the exercise might impact on the α-amylase activity. A
study by Chatterton et al. was designed to investigate the relationship between heart rate (and
thus exercise intensity) and α-amylase activity. The subjects walked, jogged and ran for
periods of 10 minutes with 35 minutes resting in between the activities. While performing the
activity heart rate and α-amylase data from the subjects were collected. The results showed an
increased heart rate in all three types of activities. The α-amylase activity on the other hand
only showed increased levels of activity while jogging and running.21
Running a 100 meter sprint is high intensity but short time exercise. The time it took for the
subject to run 100 meter was estimated to approximately 15 seconds (≈6,7 m/s.). None of the
studies above have performed running in such high speed and during such short time. Because
of the short time of running a 100 meter sprint it is possible that a change in α-amylase
activity can be seen firstly after the race. This could explain why a decrease in α-amylase
activity can be seen first 10 minutes after the race. The decrease in α-amylase activity
supports the results of Calvo et al. Calvo et al.’s study was based on twenty subjects that ran
on a treadmill with increasing speed intervals. α-amylase concentration and blood lactate
concentrations was measured during the test. The results showed a clear correlation between
the α-amylase concentrations and the blood lactate concentrations. The α-amylase activity
decreased until the subjects reached exercise intensity were the anaerobic threshold (AT) was
reached. When AT was reached the study showed that the α-amylase activity would start to
rise towards base line levels again.22 No measurements of blood lactate levels was though
made during the 100 meter sprint, therefore it cannot be established that the possible
correlation between α-amylase and lactate levels explains the changes in α-amylase activity
after the 100 meter sprint.
It is difficult to compare studies with each other, firstly because the saliva is collected
differently and secondly because there are different types of physical exercise. It also seems
like the α-amylase activity depends on both day of the week and time of the day. This can be
emphasized by comparing the base value in experiment 1 with the base value in experiment 2.
For all the subjects the base value differs a lot between the both experiments. A possible
explanation is that experiment 1 and 2 was not performed on the same day or on the same
time. This may be one of the main reasons why α-amylase activity is hard to use as a reliable
indicator for stress. Another reason is the large individual variations in α-amylase activity.
This makes it impossible to compare stress levels due to α-amylase activity between different
individuals. Stress is a complex reaction that affects different functions in the body. Because
of this, one single indicator is not enough to show a reliable result. α-amylase activity may
have potential application as a stress indicator but only in addition with other stress
biomarkers. The results of this study do not support α-amylase activity as a single biomarker
for measuring and monitoring stress levels.
20
José L. Chicharro, Alejandro Lucía, Margarita Pérez, Almudena F. Vaquero, Rosario Ureña, Saliva
Composition and Exercise (Sports Med , 1998 Jul ; 26 (1) : 17-27), p. 22
21
Robert T. Chatterton Jr., Kirsten M. Vogelsong, Yu-cai Lu, Allsion B. Ellman, Gerald A. Hudgens, Salivary
α-amylase as a measure of endogenous adrenergic activity(Clinical Physiology, volume 16 number 4 july 1996,
433-448), p. 441
22
Felipe Calvo, José L Chicharro, Fernando Bandrés, Alejandro Lucía, Margartia Pérez, Julián Álvarez, Luis L.
Mojares, Almudenaa F. Vaquero, Julio C. Legido, Anaerobic Threshold Determination With Analysis of Salivary
Amylase (Can. J. Appl. Physiol. 22(6): 553-561, 1997, Canadian Society for Exercise Physiology), p.555-559
17
Conclusion
The experiment seems to show that the α-amylase activity is affected by the possible stressor
of a 100 meter sprint. After the sprint the α-amylase activity is decreasing until a certain point
where it turns and starts to increase towards baseline levels again. Even if the changes in αamylase activity are less significant during rest, this study cannot clearly establish that the
changes seen after the 100 meter sprint indicate a stress reaction. This study does not support
α-amylase activity as a single biomarker for measuring and monitoring stress.
Further investigations
To be sure that the amount of saliva is the same in all samples, the saliva could be collected as
concentrated saliva. This can exclude that different saliva samples contain different amounts
of saliva and therefore exclude that the changes in α-amylase activity is caused by that.
Moreover the uncertainty in when all starch is degraded is large. Since the time of when the αamylase has hydrolyzed the starch to a fixed amount is depending on a subjective judgment of
colour. For those with high active α-amylase the uncertainty is even larger. A highly active αamylase makes the uncertainty of ±15 s percentage higher compared with those with lower αamylase activity. To overcome this problem the saliva can be diluted alternative the mass of
starch be increased to increase the degradation time. To overcome the shortcomings of this
method further investigation could be made with complementary forms of analysis.
A large source of uncertainty in this study is that the experiments were only performed once.
To get a more reliable result the experiments should be performed over again and with more
subjects. Moreover further studies should be made on whether a 100 meter sprint could be
used as a stressor or not. A further uncertainty is whether it is possible to form lactic acid after
a 100 meter sprint and therefore explain the result as a correlation between α-amylase activity
and lactate levels. To increase the chances that lactic acid is formed a 200 meter sprint could
be more appropriate to use. To determine a correlation between α-amylase and lactate levels
the lactate levels needs to be measured parallel to the α-amylase activity. Further
investigations should also evaluate the changes in α-amylase activity during rest.
As a single indicator for stress α-amylase activity is uncertain. To establish what the results of
this study tell about salivary α-amylase activity correlated to stress, further investigations
should be made on complementary measurements of other biological stress indicators.
18
References
Felipe Calvo, José L Chicharro, Fernando Bandrés, Alejandro Lucía, Margartia Pérez, Julián Álvarez,
Luis L. Mojares, Almudenaa F. Vaquero, Julio C. Legido, Anaerobic Threshold Determination With
Analysis of Salivary Amylase (Can. J. Appl. Physiol. 22(6): 553-561, 1997, Canadian Society for
Exercise Physiology)
José L. Chicharro, Alejandro Lucía, Margarita Pérez, Almudena F. Vaquero, Rosario Ureña, Saliva
Composition and Exercise (Sports Med , 1998 Jul ; 26 (1) : 17-27)
G. Ljungberg, T. Ericson, B. Ekblom, D. Birkhed, Saliva and marathon running (Scand J Med Sci
Sports, 1997:7:214-219)
Robert T. Chatterton Jr., Kirsten M. Vogelsong, Yu-cai Lu, Allsion B. Ellman, Gerald A. Hudgens,
Salivary α-amylase as a measure of endogenous adrenergic activity (Clinical Physiology, volume 16
number 4 july 1996, 433-448)
Maria Svensson, A study of the individual differences in α-amylase activity (Extended essay IB
Sannarpsgymnasiet 2005)
Rolf Ekman och Bengt Arnetz (red), Stress: Individen, samhället, organisationen, molekylerna
(Stockholm, Liber AB, 2005)
Karlsson, Molander, Wickman, Biologi 2 för gymnasieskolan (Stockholm: Liber AB, 1997)
Cambell, Reece, Biology sixth edition (San Francisco: Pearson Education Inc., 2002)
http://www.1177.se/Halland/Fakta-och-rad/Sjukdomar/Stress/ (Hämtad 15.37 2012-01-13 senast
ändrad 2010-05-17)
19
Appendix 1 – Initial experiments
Initial experiment 1
The initial experiments were performed as a foundation for the main experiments.
Initial experiment 1 was to perform an individual maximal 100 meter sprint. Before the sprint
a warm-up was performed. Saliva samples were collected before and after the sprint.
Before warm-up sample 1.1 was collected. Directly after the warm-up (800 meter jog on an
outdoor track, stretch, 4x20 meter running exercises, 60 meter soar away running) sample 1.2
was collected. At an outdoor track a maximal sprint of 100 meter was performed. Directly
after finish sample 1.3 was collected. Following samples (1.4-1.7) was collected each 10
minutes for 40 minutes while resting. Each sample was collected in 2 ml eppendorph tubes.
The 100 meter sprint was performed individually without any impact from the other
participants, i.e. without competition or timing.
Initial experiment 2
Initial experiment 2 was to perform an individual maximal 100 meter sprint. Before the sprint
a warm-up was performed. Saliva samples were collected before and during the warm-up and
before and after the sprint.
Before warm-up sample 2.1 was collected. The warm-up (800 meter jog on an outdoor track,
stretch, 4x20 meter running exercises, 60 meter soaraway running) was performed while
collecting samples. Sample 2.2 was collected after 400 meter of jog. Sample 2.3 was collected
after additionally 400 meter of jog. Sample 2.4 was collected after 4x20 meter running
exercises. Sample 2.5 was collected after 60 meter soaraway running. After a rest of 15
minutes sample 2.6 was collected. At an outdoor track a maximal sprint of 100 meter was
performed. Directly after finish sample 2.7 was collected. The following two samples (2.8 and
2.9) were collected each 15 minutes for 30 minutes while resting. Each sample was collected
in 10 ml sampling tubes. The 100 meter race was performed individually without any impact
from the other participants, i.e. without competition or timing.
Analysis of samples from initial experiment 1
The starch solution was prepared by boiling 450 ml of deionised water. 1,25 g starch (starch
soluble GR for analysis, Merck pro analysis, (C6H10O5)n) was mixed with 50 ml deionised
water. 50 ml starch solution was poured into the boiling water. The solution was boiled until
clear. The 500 ml starch solution was cooled down to room temperature. The solution was
used within 36 h after it was made.
Small test tubes (3 ml) were put into a tube rack. 100 µl iodine solution was pipetted to every
test tube. 20 ml starch solution was poured into a conical flask (250ml) and covered with
parafilm to avoid evaporation. The saliva samples was thawed and warmed to 24,5 ᵒC in a
water bath. The conical flask with starch solution was also warmed to 24,5 ᵒC in the water
bath. A thermometer was used to control that the saliva samples and the conical flask with
starch solution was 24,5 ᵒC. 1,5 ml of the tempered saliva solution was pipetted into the
preheated starch solution. The solution was stirred and a stopwatch was directly started when
the saliva solution was pipetted into the starch solution. 500 µl of the saliva-starch solution
20
was pipetted into a test tube every minute. When the colour shifted from dark blue to brown,
the interval was shortened to 30 seconds. The pipettering was continued until no further
colour changing was noticed. The watch was stopped and the result written down.
Analysis of samples from initial experiment 2
The starch solution was prepared by boiling 450 ml of deionised water. 2,5 g starch (starch
soluble GR for analysis, Merck pro analysis, (C6H10O5)n) was mixed with 50 ml deionised
water. 50 ml starch solution was poured into the boiling water. The solution was boiled until
clear. The 500 ml starch solution was cooled down to room temperature. The solution was
used within 36 h after it was made.
Small test tubes (3 ml) were put into a tube rack. 100 µl iodine solutions were pipetted to
every test tube. 20 ml starch solution was poured into a conical flask (250ml) and covered
with parafilm to avoid evaporation. The saliva samples was thawed and warmed to 24,5 ᵒC in
a water bath. The conical flask with starch solution was also warmed to 24,5 ᵒC in the water
bath. A thermometer was used to control that the saliva samples and the conical flask with
starch solution was 24,5 ᵒC. 4 ml of the tempered saliva solution was pipetted into the
preheated starch solution. The solution was stirred and a stopwatch was directly started when
the saliva solution was pipetted into the starch solution. The saliva-starch solution was placed
in the water bath again to keep the temperature 24,5 ᵒC. 500 µl of the saliva-starch solution
was pipette into a test tube every minute. When the colour shifted from dark blue to brown,
the interval was shorten to 30 seconds. The pipettering was continued until no further colour
changing was noticed. The watch was stopped and the result written down.
Results
Initial experiment 1
100 meter individual maximal sprint was performed. Saliva samples (1.1 – 1.7) were collected
before and after the 100 meter sprint. α-amylase activity was determined to how long it took
for the α-amylase in 1,5 ml saliva solution to hydrolyze 50 000 µg starch.
Table 11 – Initial experiment 1
Sample
Description
(A.E)
Time to break down
starch
(s)
α-amylase activity
(µg starch/s)
50 000 µg starch/360 s
= 139 µg starch/s
333
79,4
139
104
98,0
119
1.1
Base value (before warm-up)
360 (±30)
1.2
1.3
1.4
1.5
1.6
1.7
Before 100m sprint
Directly after 100m sprint
10 min after 100m sprint
20 min after 100m sprint
30 min after 100 m run
40 min after 100m sprint
150 (±30)
630 (±30)
360 (±30)
480 (±30)
510 (±30)
420 (±30)
21
Table 12 – Initial experiment 1
Sample
(J.B)
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
1.1
1.2
1.3
1.4
1.5
1.6
1.7
Base value (before warm-up)
Before 100m sprint
Directly after 100m sprint
10 min after 100m sprint
20 min after 100m sprint
30 min after 100m sprint
40 min after 100m sprint
1110 (±30)
1260 (±30)
1770 (±30)
1740 (±30)
840 (±30)
540 (±30)
660 (±30)
45,0
39,7
28,2
28,7
59,5
92,6
75,8
Initial experiment 1
α-amylase activity
(µg starch/s)
1400
1200
1000
800
600
A.E
400
J.B
200
0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
Sample
Fig. 5 α-amylase activity from two subjects (A.E and J.B) who participated in warm-up and 100 meter sprint. 1.1
was collected before warm-up. 1.2 was collected before the 100 meter sprint. 1.3 was collected directly after the
100 meter sprint. 1.4 -1.7 were collected with a 10 minutes interval after the race while the participant were
resting. Each line represents one subject.
22
Initial experiment 2
A warm-up and 100 meter individual maximal sprint were performed. Saliva samples 2.1 –
2.5 were collected before and during the warm-up. Saliva samples 2.6 – 2.9 were collected
before and after the 100 meter race. α-amylase activity was determined to how long it took for
the α-amylase in 4 ml saliva solution to hydrolyze 100 000 µg starch.
Table 13 – Initial experiment 2
Sample
Description
(A.E)
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
100000 µg starch/180 s
= 556 µg starch/s
476
476
476
128
556
208
476
278
2.1
Base value (before warm-up)
180 (±30)
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
After 400 m jog
After additionally 400 m jog
After 4x20 m running exercises
After 60 m soraway running
Before 100m sprint
Directly after 100m sprint
15 min after 100m sprint
30 min after 100m sprint
210 (±30)
210 (±30)
210 (±30)
780 (±30)
180 (±30)
480 (±30)
210 (±30)
360 (±30)
Table 14 – Initial experiment 2
Sample
(J.B)
Description
Time to break
down starch
(s)
α-amylase activity
(µg starch/s)
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
Base value (before warm-up)
After 400 m jog
After additionally 400 m jog
After 4x20 m running exercises
After 60 m soraway running
Before 100m sprint
Directly after 100m sprint
15 min after 100m sprint
30 min after 100m sprint
750 (±30)
360 (±30)
930 (±30)
2400 (±30)
1170 (±30)
2100 (±30)
660 (±30)
>2100 (±30)
1170 (±30)
133
278
108
41,7
85,5
47,6
151,5
< 47,6
85,5
23
α-amylase activity
(µg starch/s)
Intial experiment 2
1400
1200
1000
800
A.E
600
J.B
400
200
0
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
Sample
Fig. 6 α-amylase activity from two subjects (A.E and J.B) who participated in warm-up and 100 meter sprint. 2.1
was collected before warm-up. 2.2 was collected after 400 meter jog. 2.3 was collected after additionally 400
meter jog. 2.4 was collected after 4x20 meter running exercises. 2.5 was collected after 60 meter soraway
running. 2.6 was collected before the 100 meter sprint. 2.7 was collected directly after the 100 meter sprint. 2.8
and 2.9 was collected 15 minutes resp. 30 minutes after the race while the participant were resting. Each line
represents one subject.
24
Appendix 2 – Tests of method
Test 1
Four samples of the same salivary sample were analyzed to see if the results were reliable and
reproducible. The method for analyzing from experiment 1 and 2 was used. All of the four
samples had the same results (±30 s). This is regarded as valid since the estimated time
uncertainty is ±15 s due to the intervals of taking samples.
Test 2
During analyzing of samples from initial experiment 2 sample 2.1 (A.E) was analyzed twice,
firstly directly when the saliva reach 24,5 ᵒC and secondly after 1 h in the water bath of
24,5 ᵒC. The results differed by 30 sec. which is within the margin of error for the method.
This test was made a second time at experiment 2 with sample 4.1 from subject A. In this test
as well the results differed by 30 sec. Therefore the conclusion can be made that the samples
are not affected by being in a water bath of 24,5 ᵒC during 1 h.
Test 3
Though the first sample of subject B (sample 3.1) was spilled another 3.1 sample was taken directly
after the first one. One test was made to see how the α-amylase activity was affected when two
samples was taken directly after each other. The results show that the secondly taken sample
had a lower α-amylase activity than the firstly taken sample. This makes sample 3.1 from
subject B a non reliable value.
25
Appendix 3 – Uncertainty calculations
Starch solution - concentration
Initial experiment 1
Uncertainty balance= (0,001 g / 1,250 g) · 100 = 0,08 %
Uncertainty 500 ml measuring cylinder = (2,5 ml / 450 ml) · 100 ≈ 0,6 %
Uncertainty 50 ml measuring cylinder = (1 ml / 50 ml) · 100 = 2 %
Overall uncertainty of apparatus = 0,08 % + 0,6 % + 2 % = 2,7 % ≈ 3 %
c=m/v
cstarch solution = 1,25 g / 500 ml = 0,00250 g/ml
Uncertainty cstarch solution = 0,00250 · 0,03 = 0,000075 ≈ 0,00008 g/ml
cstarch solution = 0,00250 ± 0,00008 g/ml
Initial experiment 2 and experiments 1 and 2
c=m/v
cstarch solution = 2,50 g / 500 ml = 0,0050 g/ml
Uncertainty cstarch solution = 0,0050 · 0,03 = 0,00015 ≈ 0,0002 g/ml
cstarch solution = 0,0050 ± 0,0002 g/ml
α-amylase activity
Uncertainty starch solution = 3 %
Uncertainty volume starch solution = 0,5 ml / 20,0 ml = 0,025 = 2,5 %
Uncertainty pipette = ±1 %
Overall uncertainty of α-amylase activity except time = 3% + 2,5% + 1% = 6,5% ≈ 7 %
Estimated uncertainty of time = ±15 s
26
Appendix 4 – Survey
Namn:_____________________________________________________________________________
Ålder:_____________________________________________________________________________
Längd(ungerfärlig):__________________________________________________________________
Vikt
(ungefärlig):________________________________________________________________________
Träningsmängd (träningar/vecka):_______________________________________________________
Hur stressad känner du dig idag på en skala 1-5 (där 1 är ostressad och 5 är väldigt stressad):________
Hur stressad har du varit i veckan på en skala 1-5:__________________________________________
Hur många gånger har du ätit idag: _____________________________________________________
Jag godkänner att insamlad data används för eventuell publicering. All information kommer användas
anonymt.
_____________________________________________________________
Datum
Signatur
27