Johanna Bergman PROJEKTARBETEAvalon Ernstson 2011-2012NV09

Changes in salivary α-amylase activity caused by the possible stressor of a 100 meter sprint

Handledare: Håkan Klarén

AbstractIt 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

Table of Contents

2

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

Appendix 3 – Uncertainty calculations...................................................................................................26

Starch solution - concentration...........................................................................................................26

α-amylase activity...............................................................................................................................26

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Appendix 4 – Survey..............................................................................................................................27

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IntroductionStress 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.312 Rolf Ekman och Bengt Arnetz (red), Stress: Individen, samhället, organisationen, molekylerna (Stockholm, Liber AB, 2005) p.763 http://www.1177.se/Halland/Fakta-och-rad/Sjukdomar/Stress/ (Hämtad 15.37 2012-01-13 senast ändrad 2010-05-17)4 Rolf Ekman och Bengt Arnetz (red), Stress: Individen, samhället, organisationen, molekylerna (Stockholm, Liber AB, 2005) p.372

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Background

Stress reactions in the sympathetic nervous systemDuring 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.

SalivaSaliva 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

StarchStarch 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.5546 Karlsson, Molander, Wickman, Biologi 2 för gymnasieskolan (Stockholm: Liber AB, 1997), p. 142-1437 Cambell, Reece, Biology sixth edition (San Francisco: Pearson Education Inc., 2002) p.860-8618 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. 189 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. 1910 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. 2011 Cambell, Reece, Biology sixth edition (San Francisco: Pearson Education Inc., 2002) p. 66-67

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α-amylase and exerciseChanges 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 stressorA 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 subjectsFor 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. 21413 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, 2114 Karlsson, Molander, Wickman, Biologi 2 för gymnasieskolan (Stockholm: Liber AB, 1997), p. 2915 Cambell, Reece, Biology sixth edition (San Francisco: Pearson Education Inc., 2002) p. 17016 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

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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 activityTo 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 activityExperiment 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 solution450 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 dataThe 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.

17 Maria Svensson, A study of the individual differences in α-amylase activity (Extended essay IB Sannarpsgymnasiet 2005)

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ResultsTable 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)Amount of

training(training/week)

Estimated stress level today

(scale 1-5)*

Estimated stress level of week

(scale 1-5)*

Food intake today

A Female 17 166 67 4-5 2 2 2B Female 18 171 68 5-6 1 1 1C Male 16 184 72 2-3 3 4 3D Male 18 175 72 3 1 1 0E Male 19 191 98 5 1 1 2

*1 is not stressed at all and 5 is very stressed.

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 1Sample

(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 Directly after warm-up 105 (±15) 9523.3 Before 100m sprint 135 (±15) 7413.4 Directly after 100m sprint 90 (±15) 11103.5 10 min after 100m sprint 150 (±15) 6673.6 20 min after 100m sprint 300 (±15) 3333.7 30 min after 100m sprint 120 (±15) 8333.8 40 min after 100m sprint 90 (±15) 1110

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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) 4173.3 Before 100m sprint 360 (±15) 2783.4 Directly after 100m sprint 375 (±15) 2673.5 10 min after 100m sprint 720 (±15) 1393.6 20 min after 100m sprint 1170 (±15) 863.7 30 min after 100m sprint 1170 (±15) 863.8 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 1Sample

(subject C)Description Time to break

down starch(s)

α-amylase activity(µg starch/s)

3.1 Base value (before warm-up) 105 (±15) 9523.2 Directly after warm-up 135 (±15) 7413.3 Before 100m sprint 165 (±15) 6063.4 Directly after 100m sprint 225 (±15) 4443.5 10 min after 100m sprint 285 (±15) 3513.6 20 min after 100m sprint 420 (±15) 2383.7 30 min after 100m sprint 315 (±15) 3173.8 40 min after 100m sprint 240 (±15) 417

Table 5 – Experiment 1Sample

(subject D)Description Time to break

down starch(s)

α-amylase activity(µg starch/s)

3.1 Base value (before warm-up) 90,0 (±15) 11103.2 Directly after warm-up 150 (±15) 6673.3 Before 100m sprint 90,0 (±15) 11103.4 Directly after 100m sprint 210 (±15) 4763.5 10 min after 100m sprint 255 (±15) 3923.6 20 min after 100m sprint 225 (±15) 4443.7 30 min after 100m sprint 285 (±15) 3513.8 40 min after 100m sprint 240 (±15) 417

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Table 6 –Experiment 1Sample

(subject E)Description Time to break

down starch(s)

α-amylase activity(µg starch/s)

3.1 Base value (before warm-up) 300 (±15) 3333.2 Directly after warm-up 165 (±15) 6063.3 Before 100m sprint 615 (±15) 1633.4 Directly after 100m sprint 225 (±15) 4443.5 10 min after 100m sprint 330 (±15) 3033.6 20 min after 100m sprint 540 (±15) 1853.7 30 min after 100m sprint 450 (±15) 2223.8 40 min after 100m sprint 405 (±15) 247

3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.80

200

400

600

800

1000

1200

1400

Experiment 1 - 100 meter sprint and α-amylase activity

ABCDE

Sample

α-am

ylas

e ac

tivity

(µg

star

ch/s

)

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.

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3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.80

200

400

600

800

1000

1200

1400

Experiment 1 - 100 meter lap and α-amylase activity

Sample

α-am

ylas

e ac

tivity

(µg

star

ch/s

)

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 activitySaliva 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 2Sample

(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 Value after 10 min 447 (±15) 2244.3 Value after 20 min 369 (±15) 2714.4 Value after 30 min 207 (±15) 4834.5 Value after 40 min 258 (±15) 3864.6 Value after 50 min 318 (±15) 3144.1* Control of method 240 (±15) 417

*Sample 4.1 was analyzed twice to control the reliability in the method.

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Table 8 – Experiment 2Sample

(subject B)Description Time to break

down starch(s)

α-amylase activity(µg starch/s)

4.1 Base value 420 (±15) 2384.2 Value after 10 min 927 (±15) 1084.3 Value after 20 min 858 (±15) 1174.4 Value after 30 min 807 (±15) 1244.5 Value after 40 min 540 (±15) 1584.6 Value after 50 min 507 (±15) 197

Table 9 – Experiment 2Sample

(subject D)Description Time to break

down starch(s)

α-amylase activity(µg starch/s)

4.1 Base value 189 (±15) 5294.2 Value after 10 min 267 (±15) 3754.3 Value after 20 min 207 (±15) 4834.4 Value after 30 min 180 (±15) 5564.5 Value after 40 min 120 (±15) 8334.6 Value after 50 min 180 (±15) 556

Table 10 – Experiment 2Sample

(subject E)Description Time to break

down starch(s)

α-amylase activity(µg starch/s)

4.1 Base value 207 (±15) 4834.2 Value after 10 min 867 (±15) 1154.3 Value after 20 min 327 (±15) 3064.4 Value after 30 min 360 (±15) 2784.5 Value after 40 min 249 (±15) 4024.6 Value after 50 min 309 (±15) 324

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4.1 4.2 4.3 4.4 4.5 4.60

200

400

600

800

1000

1200

1400Experiment 2 - rest and α-amylase activity

A

B

D

E

Sample

α-am

ylas

e ac

tivity

(µg

star

ch/s

)

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.

4.1 4.2 4.3 4.4 4.5 4.60

200

400

600

800

1000

1200

1400

Experiment 2 - rest and α-amylase activity

Sample

α-am

ylas

e ac

tivity

(µg

star

ch/s

)

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.

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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 α-amylase 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, 21719 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. 2221 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. 44122 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

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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 α-amylase 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 α-amylase 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 α-amylase makes the uncertainty of ±15 s percentage higher compared with those with lower α-amylase 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.

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ReferencesFelipe 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)

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Appendix 1 – Initial experiments

Initial experiment 1The 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 2Initial 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 1The 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

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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 1100 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 1Sample(A.E)

Description Time to break down starch

(s)

α-amylase activity (µg starch/s)

1.1 Base value (before warm-up) 360 (±30) 50 000 µg starch/360 s = 139 µg starch/s

1.2 Before 100m sprint 150 (±30) 3331.3 Directly after 100m sprint 630 (±30) 79,41.4 10 min after 100m sprint 360 (±30) 1391.5 20 min after 100m sprint 480 (±30) 1041.6 30 min after 100 m run 510 (±30) 98,01.7 40 min after 100m sprint 420 (±30) 119

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Sample(J.B)

Description Time to break down starch

(s)

α-amylase activity (µg starch/s)

1.1 Base value (before warm-up) 1110 (±30) 45,01.2 Before 100m sprint 1260 (±30) 39,71.3 Directly after 100m sprint 1770 (±30) 28,21.4 10 min after 100m sprint 1740 (±30) 28,71.5 20 min after 100m sprint 840 (±30) 59,51.6 30 min after 100m sprint 540 (±30) 92,61.7 40 min after 100m sprint 660 (±30) 75,8

Table 12 – Initial experiment 1

1.1 1.2 1.3 1.4 1.5 1.6 1.70

200

400

600

800

1000

1200

1400

Initial experiment 1

A.EJ.B

Sample

α-am

ylas

e ac

tivity

(µg

star

ch/s

)

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.

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Initial experiment 2A 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 2Sample(A.E)

Description Time to break down starch

(s)

α-amylase activity(µg starch/s)

2.1 Base value (before warm-up) 180 (±30) 100000 µg starch/180 s = 556 µg starch/s

2.2 After 400 m jog 210 (±30) 4762.3 After additionally 400 m jog 210 (±30) 4762.4 After 4x20 m running exercises 210 (±30) 4762.5 After 60 m soraway running 780 (±30) 1282.6 Before 100m sprint 180 (±30) 5562.7 Directly after 100m sprint 480 (±30) 2082.8 15 min after 100m sprint 210 (±30) 4762.9 30 min after 100m sprint 360 (±30) 278

Table 14 – Initial experiment 2Sample

(J.B)Description Time to break

down starch(s)

α-amylase activity(µg starch/s)

2.1 Base value (before warm-up) 750 (±30) 1332.2 After 400 m jog 360 (±30) 2782.3 After additionally 400 m jog 930 (±30) 1082.4 After 4x20 m running exercises 2400 (±30) 41,72.5 After 60 m soraway running 1170 (±30) 85,52.6 Before 100m sprint 2100 (±30) 47,62.7 Directly after 100m sprint 660 (±30) 151,52.8 15 min after 100m sprint >2100 (±30) < 47,62.9 30 min after 100m sprint 1170 (±30) 85,5

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2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.90

200

400

600

800

1000

1200

1400

Intial experiment 2

A.EJ.B

Sample

α-am

ylas

e ac

tivity

(µg

star

ch/s

)

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.

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Appendix 2 – Tests of method

Test 1Four 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 2During 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 3Though 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.

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Appendix 3 – Uncertainty calculations

Starch solution - concentrationInitial 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 / vcstarch solution = 1,25 g / 500 ml = 0,00250 g/ml Uncertainty cstarch solution = 0,00250 · 0,03 = 0,000075 ≈ 0,00008 g/mlcstarch solution = 0,00250 ± 0,00008 g/ml

Initial experiment 2 and experiments 1 and 2

c = m / vcstarch solution = 2,50 g / 500 ml = 0,0050 g/ml Uncertainty cstarch solution = 0,0050 · 0,03 = 0,00015 ≈ 0,0002 g/mlcstarch 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

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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.

_____________________________________________________________

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