Johanna Bergman PROJEKTARBETE
Avalon Ernstson 2011-2012
NV09
Changes in salivary α-amylase activity caused by the possible stressor of a 100 meter sprint
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
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
Appendix 3 – Uncertainty calculations 26
Starch solution - concentration 26
α-amylase activity 26
Appendix 4 – Survey 27
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?
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.
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.
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.
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 100000 µg starch.
Results
Table 1 – Results of survey (Appendix 4)
Answers from the survey handed out to the subjects after experiment 1.