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ADRENALINE RELEASES LEVEL ON SKIN-TO SKIN
TOUCHES
Version (2)
Bachelor Thesis Project in Biomedicine 30 ECTS Spring term 2020
Maryan George A13marge@student.his.se Supervisor:Linda Handlin Linda.handlin@his.se Examiner: Johan Haux johan.haux@vgregion.se School of Health and Education,
University of Skövde, Box 408, 541 28 Skövde
Abstract
Human pleasant touches promote feelings of security, supportiveness, and wellbeing. Conversely, human unpleasant touches promote the body for either “fight or flight” or “short term acute stress”
during emergencies, feeling of stress or danger. The promoted stress response is released from the hypothalamus by the sympathetic nerve system further to the spinal cord to reach the signals to the adrenal medulla, where stress hormones adrenaline is released. Adrenaline, which is characterized by a mimic sympathetic nerve system, interacts with α and β receptors on different organs. The aim for this study was to investigate whether the stroker (partner/stranger) touch effects on adrenaline hormone releases. The null hypothesis for this study entails a significant adrenaline reduction in partners’ touches compared with strangers’ touches. Indirect competitive ELISA method was used, and concentration data of a total of sixteen participants was obtained. Whitney-U test was carried out to compare group differences within stroker (stranger/partner) touches and adrenaline releasing level. In addition, correlation in adrenaline with noradrenaline and oxytocin hormones was obtained using Spearman’s correlation test. The significant p-value 0.05 was conducted. The result of this study showed no differences between stroker (partner/stranger) associated with adrenaline
hormone release. Correlation between partner maximum (max) concentration data for both oxytocin and adrenaline had significant differences. However, max variables for adrenaline and noradrenaline within stroker did not show significant differences. The conclusion of this study is that the gentle touch stimulus used in this study was not enough to detect stress hormone in adrenaline.
Keyword: Touches, fight or flight, adrenaline, indirect competitive ELISA, non-parametric test, human animal interaction, noradrenaline, and oxytocin.
Popular scientific summary
Skin-to-skin touching is one of the most powerful forces in human- development, connection, communication, and survival. Gentle touch has a huge influence on social bonding to express gratitude, emotion, and interaction. However, touch plays a critical role in generating emotions that are either pleasant or unpleasant. In an unpleasant touch, the body will release stress hormones such as adrenaline from the kidney; this is known as “flight or fight”. Common adverse effects of adrenaline release are anxiety, fear, palpitations, pale skin, shortness of breath, sweating and tremors.
The adrenaline study was performed at Linköping University and included 31 healthy female participants. The females who participated in this study were touched by their partner and by a stranger in two randomized runs. The stroker movement was taken on the right dorsal arm and palm of the female’s hand. During touches, blood samples were taken at seven timepoints. The aim of this study was to observe whether the female participant's adrenaline hormone level differs between their partner and stranger by gentle touch. The suggested theory of this study was that adrenaline level was higher for stranger touches than for partner touches.
The method that has been used in this experiment is called: “3 CAT ELISA” which is a fast and easy method to detect and observe the level of adrenaline hormone in blood plasma. The Kit included two major parts; one was the extraction part and one was adrenaline ELISA. The extraction part included extracting adrenaline from plasma and adrenaline ELISA was to detect the level of adrenaline substance within the samples. To determine the adrenaline level in the participants’ blood plasma, a statistical program named SPSS was used to observe the differences and similarities between female participants with the (partner/stranger) group.
Besides adrenaline, two other hormones were included in the statistical part. One adrenaline hormone was investigated by a classmate which is also known as fight or flight stress hormone and another hormone known as the love hormone, known as oxytocin which was done previously by researchers.
The result of this study did not show any differences in stress hormone adrenaline release by stroker (partner/stranger) touch. In addition, the connection between adrenaline and noradrenaline did not show any connection with each other in both strokes. However, there was a connection between the female’s partner touch in oxytocin and adrenaline, but not in stranger touch.
The finding of this research is a fundamental start to understanding how stress hormone release in adrenaline can be affected by touch. That would be one of the non-medical treatments to add to prevent mental health problems like memory impact, anxiety, and dementia.
Content
Abstract ...
Popular scientific summary ...
Content ...
1. Introduction ... 1
1.1. Meaning of touches ... 1
1.2. Sensory neuron somatosensory system of human touch ... 1
1.3. Pleasant and unpleasant touches in nerves related ... 1
1.3.1. Pleasant touch ... 1
1.3.2. Unpleasant touch in Adrenaline releasing ... 2
1.4. Endocrine system in Catecholamines releases ... 2
1.4.1. Catecholamines in general ... 2
1.4.2. Synthesis and pathways of adrenergic neuron ... 2
1.5. Adrenaline stimulations pathway ... 2
1.5.1. Adrenaline releasing in adrenal medulla. ... 2
1.5.2. Adrenaline adrenergic receptor in cAMP amplification ... 3
1.6. Adrenaline effectiveness on the body ... 3
1.6.1. Cardiovascular ... 3
1.6.2. Adrenaline influences on glucose metabolism ... 3
1.6.3. Leukocytes and adrenaline. ... 3
1.7. Adrenaline and oxytocin, inverse relationship ... 4
1.8. Adrenaline and noradrenaline relationship ... 4
1.9. Indirect competitive ELISA ... 5
Aim of the study ... 5
2. Material and Method ... 6
2.1. Study design ... 6
2.1.1. Participants ... 6
2.2. 3-CAT ELISA-kit in Adrenaline analyses ... 6
2.3. Instruments ... 7
2.4. Spike recovery ... 7
2.5. Statistical uses by SPSS-statistical program ... 7
3. Results ... 8
3.1. Participants ... 8
3.2. Distribution analyses ... 8
3.2.1. Q-Q plot result. ... 8
3.3. Croupwise comparison by Mann-Witney U-test and histogram. ... 9
3.4. Confidience interval (CI) 99%. ... 10
3.5. Spearman’s test ... 10
3.6. Spike recovery ... 11
4 Discussion ... 12
4.1. Indirect competitive ELISA result analyses. ... 12
4.2. Spike recovery ... Fel! Bokmärket är inte definierat. 4.3. Adrenaline hormone in this study ... 13
4.4. Adrenaline and noradrenaline related to animal experiments ... 14
4.5. Rolls of hormone adrenaline and oxytocin in skin touch. ... 15
5. Ethical aspect and impact of the research on the society ... 17
6. Conclusion/future perspective ... 18
7. Acknowledgement ... 19
8. References ... 20
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1. Introduction
1.1. Meaning of touches
Touches by hand to hand or skin to skin are evolutionary and are known as the language of compassion which have a unique impact on social bonds to express gratitude, emotion, and interaction. In addition, touch consider as one of the most powerful forces in human development, connection, communication, and survival. However, touch plays a critical role in generating emotions that are either pleasant or unpleasant emotional expression (Von Mohr et al., 2017). In unpleasant touches, stress stimulations promotes the body for “flight or fight” or “acute stress response” which activates the sympathetic nerve system that will trigger adrenal medulla catecholamine (adrenaline, noradrenaline and dopamine) secretion into the bloodstream. While in human pleasant touches, parasympathetic nerve system will be activating to promote releases of oxytocin hormone to obtain feelings of calmness and appreciated (Keltner, 2017).
1.2. Sensory neuron somatosensory system of human touch
Skin touches are a complex process that has been developed over millions of years, which are made up of billions of cells that send neurochemical signals to the region of the somatosensory cortex. The somatosensory cortex in the brain shows a remarkable capacity of object sensing, recognition, sensory motor feedback, temperature, sensing pressure and pain (Abraira and Ginty, 2014). Senses of touch arise from multiple touch receptors that innervate our skin, which can be qualitatively different. The somatosensory system is exteroceptive and interoceptive which both serves our body to detect any sense and reaction outside and inside the body; the last one is proprioceptive which is responsible for body position and balance. In general, the somatosensory pathway begins with activation of primary sensory neurons within dorsal root ganglia (DRG), which are pseudo-unipolar that spread to the periphery associated target and cranial sensory ganglia (CSG); this penetrates the spinal cord and in some cases, carries on to the nuclei in the brainstem. Therefore, the exteroceptive somatosensory and DRG neurons are crucial to the uptake of different sensory organs to determine whether the sense of touch is pleasant or unpleasant stimuli (Abraira and Ginty, 2014).
1.3. Pleasant and unpleasant touches in nerves related
1.3.1. Pleasant touch
Mechanoreceptive afferents fibres that include fast-conducting, myelinated A- beta or slow- conducting and unmyelinated C-tactile have a certain property that affect the brain differently. To begin with, myelinated A-beta fibres respond to major touch stimulation, while with unmyelinated C- tactile, it is enough to have a low touching in contact in order to feel pleasantness of all other type of afferents (Ellingsen et al., 2016).
To sense pleasant or unpleasant touches, there are different neural networks involved that are connected to the brain. For the pleasant touch, medial orbitofrontal is commonly activated, while unpleasant touch is activated in the right frontal-insular cortex (Lamm et al., 2015). Brain mechanism and touching are modified in an integrated fashion with top-down information (Ellingsen et al.,
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2016). Top-down information is driven by cognitive processes that include learning, attention, and locomotion which have an important factor on modulate sensory processing (Samaha et al.,2015).
1.3.2. Unpleasant touch in Adrenaline releasing
The foundation of the integumentary system which contains nerves that work as primary sensing for external stressors such as (adrenaline) stimulation. These nerves are responsible for sending stimulation signals through the spinal cord and on into the brain which will respond to the signals.
Epidermal keratinocytes and melanocytes will secrete adrenaline which activates β2-adrenoreceptor by being touched in an unpleasant way. This leads to an increase in cAMP in keratinocytes, which in turn increases calcium concentration Ca2+ through protein kinase C (PKC) activation. It results in high calcium concentration levels which may influence epidermal health. At the same time, adrenaline will be surrounded by melanocytes which can promote melanogenesis (Chen & Lyga, 2014).
1.4. Endocrine system in Catecholamines releases
1.4.1. Catecholamines in general
From adrenal medulla, catecholamine hormones (Adrenaline, Noradrenaline and Dopamine) are secreted, which are considered as neurotransmitter hormones and sympathomimetic amines that contain the 3,4 -dihydroxybenzene group. Catecholamines have an extraordinary property that includes the following: high potency, rapid inactivation, and poor penetration into the central nerve system (CSN). The first category of catecholamines is adrenaline, which is characterized by a mimic sympathetic nerve system. Second category is noradrenaline hormone which is stimulating all types of adrenergic receptors. Third category is dopamine which is a metabolic precursor to noradrenaline further to adrenaline in adrenal medulla. Dopamine hormones occur naturally in the CNS in basal ganglia, where it functions as a neurotransmitter (Harvey, 2015a).
1.4.2. Synthesis and pathways of adrenergic neuron
All three hormones adrenaline, noradrenaline and dopamine synthesized in adrenal medulla because of the sympathetic nervous system effect. Hormones synthesis stimulation in adrenal medulla begins with tyrosine hydroxylated converted to dihydroxyphenylalanine L-dopa by tyrosine hydroxylase. L-dopa is converted to dopamine by dopa decarboxylase. At this stage, two important pathways will determine what kind of receptor is targeted. The two pathways could either target the dopaminergic receptor or target catecholamines synthesis. When dopaminergic receptor is the target, the pathway will be stopped then; however, when adrenergic receptor is the target, then conversion of dopamine will synthesize to noradrenaline and may be converted to adrenaline (Simmons and Wohl, 2019).
1.5. Adrenaline stimulations pathway
1.5.1. Adrenaline releasing in adrenal medulla.
In mammals, adrenaline production is produced in the centre medulla of the adrenal gland, whose secretion is required for maintenance of life (Romero, 2019). Adrenal medulla is composed of pheochromoblasts, also known as chromaffin cells which have a major function to synthesize and store adrenaline. Synthesis and storage of adrenaline are obtained by the response of cholinergic stimulation by preganglionic sympathetic fibres. (Hinson and Chew, 2010). On stimulation, the adrenal medulla releases about 80% of adrenaline further into the blood circulation.
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1.5.2. Adrenaline adrenergic receptor in cAMP amplification
Adrenaline is a hydrophilic hormone that interacts effectively to the water which cannot cross the hydrophobic plasma membrane of the target cells. Therefore, adrenergic agonist adrenaline needs to bind to the adrenoreceptor either to α or β receptors in the plasma membrane to respond to short acute body stress (Harvey, 2015 and Lodish et al., 2000). When adrenaline signals bind to adrenergic receptors on the cells, the G-protein-coupled receptors (GPCRs) first messenger will be activated.
This activation will result in GTP binding to adenylyl cyclase enzyme, which catalyses the synthesis of many molecules of ATP to second messenger cAMP. The constant of increasing cAMP level leads to, activate of serine/threonine kinase called protein kinase in certain cell types (Reece et al., 2014). The feedback inhibition of adrenaline hormone is due to antagonist hormone, which acts as an inhibitor by competing for binding sites to block the physiology activity of hormone (Lodish et al., 2000).
The amount of adrenaline being released interacts in different strengths on both α and β receptors, at low amounts β effects (vasodilation) on the vascular system predominate and high amounts, α effects (vasoconstriction) are the greatest (Harvey, 2015b).
1.6. Adrenaline effectiveness on the body
1.6.1. Cardiovascular
Adrenaline release has a major action on the cardiovascular system. A high level of adrenaline release will target contractility of the myocardium (positive inotrope: β1 action) and elevate contraction rates (positive chronotropic: β1 action) which increase the cardiac output. As a result, the need of oxygen will increase on the myocardium. Adrenaline hormone will later activate β1 receptors on kidney, in order to release renin enzyme, a potent vasoconstrictor, viscera (α effects), constrict arterioles in the skin and dilate vessels going to the liver and skeletal muscles (β1 effects).
The action of adrenaline will decrease renal blood flow, increase cumulative effect on systolic blood pressure and decrease the diastolic pressure due to β2 receptor vasodilation in the skeletal muscle vascular bed (Harvey, 2015a).
1.6.2. Adrenaline influences on glucose metabolism
In acute stress, adrenaline hormone is released, which in turn increases adenylyl cyclase activation in muscle cells, resulting in increases of glycogenolysis. Elevation of glycogenolysis will not convert the glucose-6phospate into free glucose, which goes directly into the glycolysis process. This would relieve muscle cells to eliminate glucose from the blood and save blood glucose during nervous system activation (Norris and James, 2013). In mammals, stimulation of glycogen breakdown starts when adrenaline binds to β- adrenergic receptors on the surface of hepatic (liver) cells or muscle cells. Thus, these adrenergic receptors will activate G-protein coupled receptors to activate formation of adenylyl cyclase. In each adenyl cyclase molecule catalyses 100 cAMP while each molecule of protein kinase A phosphorylates 10 molecules. These signals amplifications allow the adrenaline hormone substance to bind to liver cells or muscle cells which release hundreds of millions of glucose molecules converted from glycogen (Reece et al., 2014 and Lodish et al., 2000).
1.6.3. Leukocytes and adrenaline.
Leukocytes are part of the immune system, which consist of inter alia (neutrophils, monocytes, or lymphocyte). Adrenaline releasing hormone affect, leads to a rapid significant redistribution and mobilization of immune cells into the bloodstream. However, induces leukocyte redistribution may
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be a fundamental survival response to specific target organs and significantly enhances the speed, efficacy, and regulation of immune response (Dhabhar et al., 2012).
1.6.4. Memory and learning
Adrenaline and memory have an inverted-U shape relationship; high or low presence of adrenaline concentration in the blood seems to affect memory and learning. Improvement of memory is possible only when adrenaline is present following training. However, if the adrenaline is administered before or after training, learning is not affected (Pravosudov, 2010).
1.7. Adrenaline and oxytocin, inverse relationship
The direct connection between affective touch and adrenaline hormone release has been poorly investigated within the research area. However, studies and theories have improved the stress adrenaline hormone stabilization thanks to the oxytocin hormone by skin to skin touches. Oxytocin hormone and its receptor have a major ability to decrease the sympathetic nerve system from taking over and further increasing the parasympathetic nerve system to retain stress hormone level adrenaline in the body (Magon and Kalra, 2011). The First theory is based on when a new mother welcomes her child by putting the baby on her chest. The skin to skin contacts will reduce the adrenaline levels steeply after the birth thanks to calm hormone Oxytocin. This may limit maternal bleeding and uterine contraction in the mother and reduce energy consumption for the baby (Buckley, 2015). Other theories have been shown and provided evidence that massage promotes relaxation and relieves stress (Kim et al., 2016). Having massage by skin to skin for one hour seems to influence the activation of the parasympathetic system by oxytocin hormone, which promotes long sedation, and inhibition of adrenaline releasing hormone in circulation. As a result, blood levels of stress-related adrenal hormone adrenaline will be reduced, heart rate and blood pressure will optimally also be reduced (Beck, 2016). Also, much research has shown that having a dog in human life will increase oxytocin hormone and decrease anxiety and stress in people who have cognitive health problems (Feldman, 2018).
1.8. Adrenaline and noradrenaline relationship
Adrenaline and noradrenaline hormones are considered as neurotransmitters which both play an important role in the body’s fight or flight response. In result, hormone releases will increase into the bloodstream which in turns increase blood sugar, heart rate and blood pressure. These symptoms occur and react differently depending on which cell is targeted. Each cell has a different receptor that both hormones can react to on a cell membrane surface to react differently. During adrenaline releasing, the hormone will bind to α and β-adrenoreceptor where it is located on muscles, lungs, heart, and blood vessels. While noradrenaline releasing hormone will primarily act on the α- adrenoreceptor in the blood vessels (Eske, 2019). The field of human animal interaction research has shown the benefits and positive changes in physiological stress such as fear and anxiety and improvement in people’s long-term mental health condition (Feldman, 2018). Pets' interaction with humans has shown an improvement in the decline of adrenaline and noradrenaline in patients hospitalized with heart failure (Cole et al., 2007).
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1.9. Indirect competitive ELISA
Indirect competitive Enzyme-Linked Immunosorbent Assay (indirect c-ELISA) is a microplate layout used to immobilize solution antigens to target antigen specific antibodies. The aim of using (indirect c-ELISA) is to obtain antibody-antigen complexes where it is added to microliter plates whose wells are coated with purified antigen. The reason for choosing indirect c-ELISA was to be able to give fast and accurate results by obtaining a standard curve after reading absorbance. Thus, the higher antigen concentration within samples, the weaker the resultant signal will be. The common use of (Indirect c-ELISA) is to be able to obtain the detection and quantification of antibodies against viral diseases in biological samples (Kohl and Ascoli, 2017).
Aim of the study
This project is a sub-study of a major study that has been performed at Linköping University. The aim of the major study was to investigate what kind of hormones were releasing beside oxytocin in pleasant touch.
For the sub-study, the new aim of this present study was to analyse how effective touches of stroker (partner/stranger) influence stress adrenaline hormone release. Hypothesis of this thesis work will be a significant reduction of adrenaline levels during partner touches compared to stranger’s touches. In addition, the result of this study will also be compared to other catecholamines (noradrenaline and dopamine) that two of my student peers have conducted and oxytocin from the major study.
3 CAT ELISA was used in this study due accuracy and specificity to determine adrenaline hormone release in plasma. This kind of kit indicates the adaption of the body stress hormone in response to acute and chronic stress.
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2. Material and Method
2.1. Study design
2.1.1. Participants
At Linköping University students of 31 participated in this study. Females were chosen to take the blood samples which left the touching task for men. Chosen females of age were selected between 19-40 years, were not pregnant, not taken hormone treatment and free from contraceptive medication (Handlin et al., 2020).
2.1.2. Stranger/partner stroking stimulation
Touches stimulus was obtained by male partner and male stranger. All males’ participants were asked to touch their female partner on the right dorsal arm and palm, the way they used to touch each other without having any force or direction pattern of stroking movement. Stranger and partner Stroking touch stimulation was therefore obtained by two random runs in a total of seven minutes/run.
2.1.3. Blood samples
Blood samples were taken by a magnet-safe catheter, inserted into the cubital vein of the female participants’ left arm. That would minimize the short-term effect of needle insertion on plasma hormone level during the experiment. In addition, fMRI-analysing was conducted only when blood samples were taken at each timepoint (Handlin et al., 2020). The Timepoints were collected at seven different timepoints including baseline, run 1 at (1 and 6.5 minute), rest, run 2 at (1 and 6.5 minutes) and final samples were taken at the end of the experiment. The choice of stroker (partner or stranger) was pseudorandomized within two runs. Samples were collected in EDTA-tubes, centrifuged at 4 °C at 10000g for 10 min and stored at -20°C (Handlin et al., 2020).
2.2. 3-CAT ELISA-kit in Adrenaline analyses
Investigation of whether the adrenaline releasing hormone level is released or not, by touching, 3- CAT ELISA Kit (ImmuSmol. REF: BA E-6600, Bordeaux, France) manual was therefore used. All steps were followed according to the 3-CAT ELISA kit. However, there were two steps that went differently during ELISA part, one was Enzyme solution of 45 µl was added instead of 25 µl and 80 µl of extracted plasma was added instead of 100 µl. This change was taken because the final volume of extracted plasma ended with 175 µl/timepoints which did not have enough extracted plasma to fulfil duplicate ELISA of total volume 100 µl/well. In this experiment each ELISA- plate contained 96 wells, 12 wells in horizontal and 8 wells in vertical. Therefore, 5 ELISA-plates were used, and each plate had a different layout, depended on how many participants were included in each plate. Hence, for all ELISA-plates included in this experiment were only taken in duplicate in each timepoint for all 26 participants. The OD-values from ELISA were obtained by plotting both absorbance 1 (450 nm) and absorbance 2 (650 nm) to estimate a non-linear standard curve. Since the lab work is based on competitive indirect ELISA assay, OD-value is decreasing with increased concentration of the analyte and opposite.
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2.3. Instruments
In the extraction part, a microplate shaker TM (Thermo Scientific, USA) was used. While during ELISA assay performance, two different instruments were included. A Multiskan TM FC Microplate photometer (ThermoFisher Scientific, Finland), provided with a 450 nm and 650 nm, was used for absorbance measurements. A Wellwash Microplate Washer TM (ThermoFisher Scientific, Vantaa, Finland), provided by washing the plate in triplet time (3x). Both instruments were mainly suitable for using enzyme-linked immunosorbent assay (ELISA) microplates in 96-well plate format.
2.4. Spike recovery
Spike recovery method was used to validate the accuracy of the ELISA kit and observe whether there are some factors within the sample that could affect the kit (Boden, 2016). During spike recovery, 5 blood samples were taken from the participants who did not have complete timepoints. Spike recovery is obtained by following extraction and ELISA steps according to the kit. Spike recovery is performed by taking a known amount of extracted plasma, buffer and one specific standard (D) which was collected from the kit. Three Eppendorf’s tubes were taken from each participant and each tube had different content as shown in number 1,2 and 3. Each content of all the numbers had a total volume of 110 µl. Percentage level of spike recovery was calculated by using number 4.
Percentage values 80-110% was the interval to determine whether ELISA kit-spike recovery was within accuracy level or not.
Extracted plasma + Known concentration of standards (1) Buffer + Known concentration of Standard (2)
Extracted plasma + Buffer (3)
(4)
2.5. Statistical uses by SPSS-statistical program
To determine whether the ELISA variables' concentration values were distributed or not, an analyzing distribution for Q-Q-plot was gained and showed a non-normality distribution. Also, Confidence Interval (CI) 99%, was run instead of the normal range of statistical use 95%, due to being more confident in their differences or not within selective data. The non-parametric test, Mann-Whitney U test was run to compare if the nominal (partner/stranger) adrenaline level would differ for both maximum and mean values. In addition, Spearman’s correlation has also been used to conduct the direction (positive/negative) and correlation between hormones (Noradrenaline and Oxytocin) with
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adrenaline hormone in gentle touch. Spearman’s test significant (2-tailed) p-value was determined at p<0,05.
3. Results
3.1. Participants
Q-Q- plot test was obtained for all 26 participants of adrenaline data. To determine the null hypothesis for this current study, maximum and the mean data were analyzed in total 16 of 26 participants. The reason for taking 16 participants instead of 26, was because some of the concentration was not obtained. Therefore, a data set was collected from participants which had complete adrenaline concentration values to be accurate as much as possible.
3.2. Distribution analyses
3.2.1. Q-Q plot result.
By plotting all seven points' concentration for a total 26 participants, the Q-Q plot data was obtained, see (Figure 1). As seen in the (Figure 1), the data plotting points are not scattered around the line which indicates non-normality distribution (Figure 1).
Figure 1. Normal Q-Q plot concentration data showed non-normality distributed to a total of 26 participants. As it is seen in the figure, the scattered plots are not within line which indicates a non- normally distributed data.
Figure 1. Normal Q-Q plot concentration data showed non-normality distributed of total 26 participants. As it seen in the figure, the scattered plots are not within line which indicated a non-normally distributed data.
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3.3. Croupwise comparison by Mann-Whitney U-test and histogram.
The null hypothesis of this study, there are no differences between stroker touches in order to release adrenaline hormone. To compare the critical significance p-value (0,05) and p-values in (Table 1), Mann-Whitney- U test showed that all the exact significant values are greater than 0,05, which showed no differences between stroker (partner/stranger) and adrenaline hormone releasing.
Further, a bar chart of histogram was also obtained to visualize the differences between variables and groups (Figure 1). Histogram graph in (Figure 1), showed no differences between stroker (partner/stranger) and (max and mean) variables. To generate both Mann Whitney U-test p-value values in (Table 1) and Histogram graph in (Figure 1), showed a non differences between stroker (Partner/stranger) and touches in this research.
Figure2. Stroker (partner/stranger) values for Mean and Maximum are represented in total of sixteen participants. On x-axis presented the variables of stroker (Maximum and Mean) Values. On Y-axis shows the adrenaline concentration in (ng/ml) subunit. Error Bars of 99% (CI) represented reliability of the mean population samples. Mean Inferential Errors Bars represented total amount of four variables: Max partner n=14, max stranger n=15, mean partner n=14, and mean stranger n=15.
Table 1. Shows exact significances of p-value for Mean and Max for stroker (Partner/stranger) Mean Partner Mean Stranger Max Partner Max Stranger
Exact Sig (2sided test) 0,699 0,328 0,797 0,529
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3.4. Confidence Interval (CI) 99%.
Confidence interval (CI) 99% was also obtained in both graph (Figure 2) and by destructive statistical test which showed range of likely mean differences in values followed by (lower and upper) ranges;
max partner [-0,55221 4,923], max stranger [-0,453 4,426], mean partner [-0,720 4,774] and mean stranger [-0,58090 4,311]. Significant or non-significant difference statement in (CI) depends whether the values are included zero or not. In a significant statement the zero number is not included, while no significant statement the zero number is included. Since the statistical result of confidence interval 99% (CI) was all included value zero in ranges, that determined a non-significant difference between stroker and variables.
3.5. Spearman’s test
Spearman’s correlation test was also included with this study, which determined correlation between adrenaline with dopamine and oxytocin. However, dopamine did not include this test because the concentration data set were not enough to be compared with adrenaline hormone. Spearman’s correlation in max partner between adrenaline and noradrenaline had no correlation with each other with p-value of 0,132 (table 2). In max, a stranger between adrenaline and noradrenaline spearman’s correlation showed a p-value of 0,069 with positive correlation (r=0,596). A negative correlation coefficient for adrenaline-oxytocin max partner was (r=-0,825) and p<0,002 which was greater than a significant correlation of p= 0,05. While max strangers between adrenaline and oxytocin had no correlation with each gathering with p-value of 0,974, see (table 2).
Table 2. Spearman’s correlation values of adrenaline with noradrenaline and oxytocin. Stroker respective p- value and coefficient correlation (R) data is generated. Correlation significant P-value is 0,05.
Stroker P-value Coefficient correlation (R) Max Partner (Adrenaline-Noradrenaline) 0,132 Adrenaline (1,000)
Noradrenaline (+0,440) Max Partner (Adrenaline-Oxytocin) 0,002** Adrenaline (1,000) Oxytocin (-0,825) Max Stranger (Adrenaline-Noradrenaline) 0,069 Adrenaline (1,000) Noradrenaline (+0,596)
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Max Stranger (Adrenaline-Oxytocin) 0,974 Adrenaline (1,000) Oxytocin (+0,011)
** Shows a strong correlation between max partner in (adrenaline and oxytocin) with p value of lower than 0,05.
3.6. Spike recovery
By spike recovery, a non-linearity of the standard curve was obtained and showed of R2 (0,974) which was close to a perfect fit of 1. However, the controls had different ranges to compare, control 1 had normal mean concentration (5,5425 ng/ml) while control 2 had a high mean concentration (50,145 ng/ml). According to the kit, control 1 has a normal range between (8-8,4) ng/ml and control 2 has normal range between (30-34) ng/ml. Spike recovery validation did not obtain any concentration data in order to calculate the percentage for ELISA- kit accuracy. Unlucky, Spike recovery was insufficient to be detected and showed a poor accuracy to compare with ELISA assay.
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4 Discussion
4.1. Indirect competitive ELISA result analyses.
Detection of adrenaline hormones in 10 of 26 participants' samples did not obtain any concentration values to be included in this study. The analytical performance of the indirect competitive Enzyme- Linked Immunosorbent Assay (ic-ELISA) was conducted according to 3-CAT ELISA Kit (ImmuSmol, Bordeaux, France). Series of concentrations were carried out by Multiskan TM FC Microplate photometer, by running a duplicate in each timepoint, which provide strength of data to validate the result (Mathieu, 2020). The competitive ELISA kit process works when antigen in samples competes for a limited antibody binding site with enzymes conjugated that will end by adding substrate to the samples. This result has an inverse relationship between antigen concentration and strength of substrate color. In other words, a dark substrate of yellow substrate color will detect high absorbance measurement which generates low antigen concentration. However, light substrates of yellow substrate color will detect low absorbance measurement which generate high antigen concentration in samples (Mathieu, 2020).
Since some of ELISA- Microplate photometers did not retain any concentration, the conclusion for this result is that adrenaline hormone peptide antigen had low molecules to be detected by the Multiskan TM FC Microplate photometer. Other reasons may have a role of not obtaining any concentration could be possible due adrenaline hormones were degraded during steps. Also, the pipette technique error would be one of the reasons the adrenaline antigen did not uptake as it should be during steps. Finally, the result it did not obtain was by delay in time and low speed of pipetting between steps.
4.2. Spike recovery
Validation of ELISA kit by spike recovery did not present any concentration for all 5 participants. To adjust the insufficient result between sample matrix and spike recovery experiment several things could be added. As an option BSA addition to the samples or purified protein as a stabilizer or carrier to obtain optimum results (Thermo scientific, 2007). Furthermore, the standard curve had adequate fit with R value (0,974). The reasons for not having spike recovery perhaps the lack of experience to work with the ELISA method. Also, the speed of moving step to step was too slow which leaves the matrix content on the bottom and pipetting non-rich content on the top. In fact, the Spike recovery process would be repeated once again, but due to circumstances with Covid-19 epidemic outbreak, spike recovery validation for the second time was not obtained.
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4.3. Adrenaline hormone in this study
In this study, adrenaline hormone releasing depends on skin -to- skin touches between (partner/stranger) in a similar way have not been well known in previous studies and research’s area.
Therefore, the result of this current study cannot be compared with earlier published articles.
However, the understanding of how adrenaline’s mechanism during a different concept of touches than experimental touches (partner/stranger) allows us to understand the connection between adrenaline releasing hormone and skin-to-skin touches.
The hypothesis of this quantitative research is to predict differences between groups (Partner/stranger) and variables to maintain a significant or non-significant statement. The hypothesis of this study has a significant reduction of adrenaline levels during partner touches compared to stranger’s touches. Based upon the finding in this research, showed that there are no significant differences between groups whether it started with the partner touch or stranger touch to elicit the release of adrenaline hormone. This finding was detected by a non-parametric statistical test, Mann-Whitney U-test, which included stroker (max and mean) variables in a total of sixteen participants. Significant P-values were greater than 0,05 which retained the null hypothesis for this study.
Adrenaline itself is known as fight-or-flight as a response to threat, stress, and tension, but stress is mostly common of causing a more frequent release of adrenaline into the blood circulation (Sissons, 2018). According to the “3-CAT ELISA”, the physiological range for adrenaline plasma is between (0,018-6,667) ng/m, and all the concentrations that were included in the experiment were within range. In addition, the reference value for adrenaline plasma is <0,1 ng/ml. By including stroker Mean and Max concentrations for this study, the highest concentration was 2,987 ng/ml and the lowest was 0,1 ng/ml. This indicated a normal range of hormone peptides released within a total of sixteen participants.
Assumption of this result from groupwise comparison showed no differences between groups (partner/stranger) to release adrenaline hormone. This may be due either the participants were calm during the experiment or needed additional emotional stress in order to analyze whether partner or stranger touches would be affected on validation of ELISA kit by spike recovery did not present any concentration for all 5 participants. To adjust the insufficient result between sample matrix and spike recovery experiment several things could be added. As an option BSA addition to the samples or purified protein as a stabilizer or carrier to obtain optimum results (Thermo scientific, 2007).
Furthermore, the standard curve had adequate fit with R value (0,974). The reasons for not having spike recovery perhaps the lack of experience to work with the ELISA method. Also, the speed of moving step to step was too slow which leaves the matrix content on the bottom and pipetting non- rich content on the top. In fact, the Spike recovery process would be repeated once again, but due to circumstances with Covid-19 epidemic outbreak, spike recovery validation for the second time was not obtained. releasing hormones. Normally, if there are any signs of high levels of adrenaline hormone it would target the major muscle groups, including heart and lungs and air passage dilution to provide the muscles with oxygen in need (Hormone Health network, 2020). Since none of the participants experienced any symptoms, the adrenaline hormone level either was too low to be detected or some of the female’s participants did not feel any stress during the experiment. The
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correlation between adrenaline and noradrenaline in max partner showed no correlation between (partner/stranger) touches. While adrenaline and oxytocin hormones showed two different correlations in max (partner/stranger) touches. One had a strong correlation between max partners, and one had no correlation between max strangers.
4.4. Adrenaline and noradrenaline related to animal experiments
Studies which are based on animal experiments, provide adrenaline and noradrenaline hormones released level on both rat and dogs by touches. One study was conducted on anesthetized rats to measure the neuron changes by non-noxious and noxious stroking stimulation. Noxious pinching stimulation was taken on rat lower chest skin for 3 min which resulted in a high level of adrenaline and noradrenaline hormones. However, non-noxious brushing stimulation of the lower chest for 3 min on anesthetized rats, showed a reduction in nerve activity which decreased adrenaline and noradrenaline during stimulation (Araki et al., 1948). Another animal study conducted the optimal time for measuring stress in dog presence. The study provides strong evidence in dog interaction that has a positive effect on reduction and changes of endocrine response of adrenaline and noradrenaline hormone releases (Beetz et al., 2012). Another study which also was related to animal- assisted was carried out by a dog visited, heart failure patient in hospital in 12 minutes. The study design was obtained in three randomized groups in a total of 76 patients’ adults, divided as group 1, group 2 and control group. Compared with the group, the therapy dog experiments showed a significantly greater decreases in adrenaline level before (-15,86 pg/ml, p=0,04) and after (-17,54 pg/ml, p= 0,04), and noradrenaline level before (-232,36 pg/ml, p= 0,02) and after (-240,14 pg/ml , p=0,02). This result improved a therapy dog-visited at 12 minutes decreases anxiety and neurohormone releases in patients hospitalized with heart failure (Cole et al., 2007).
Pleasant or unpleasant stimulation on rats and dog interaction with healthy and unhealthy individuals showed a difference of hormones releasing in both cases. These studies do not agree and support the current study that showed no correlation between adrenaline and noradrenaline depends on human gentle touch in (partner/stranger). In “power of touch” provides, which class of tactile fibers it innervates the hair skin which represent the neurobiological substrate properties of touch (McGlone et al., 2014). The differences between animal touch/interaction and human gentle touch experiments possibly depends on how powerful stroking was to release those two stress hormones. Despite the comparison between two different species (human and rats), myelinated A beta fibers and unmyelinated C-tactile had a major role of reflexing effect on adrenal sympathetic efferent activation to secrete adrenal medullary hormones. Study that was conducted on euthanized rats, showed an optimum significant observation in adrenaline and noradrenaline hormone releases in anxious and non-anxious stroking stimulation. In study was conducted that rat’s pleasant tactile stimulation promotes the afferent C-fibres by substantia gelatinosa (lamina II) in spinal cord (Andrew, 2010). Other study suggested that unpleasant stimulation in rats with electrical stimulation of 5 Hz showed an observant rats’ synaptic response from C-fibers to myelinated fibres afferents in higher electrical response (Pitcher and Henry, 2004). While in human, mechanoreceptive afferent response in pleasant or unpleasant skin touch, A-beta fibres respond to unmyelinated C-fibres in pleasant touch while unpleasant touch elicit sympathetic nervous system and promote myelinated A- beta
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fibres (Ellingsen et al., 2016). This research improves and strengthens the possibilities of why some of the participants did not obtain any detection of both hormones releasing, while some of the samples showed a low level of adrenaline and noradrenaline hormones released by ELISA method detection.
Adrenaline and noradrenaline stress hormones belong to the catecholamine group which contains both nucleus catechol and amine groups releasing in the adrenal medulla. Each of the groups has a different role to affect the target cells in different ways. Catechol group has two adjacent hydroxyl group which acts for ligand-increasing of cAMP level, while amine single side chain roll is to estimate the affinity of the ligand for the receptor (Lodish et al., 2000; Gnegy and Siegel 2012). Adrenaline and noradrenaline correlation were conducted in this study to elevate up the influences of catecholamines releasing depending on gentle touch. Adrenaline and noradrenaline are neurotransmitter hormones which both act on α and β adrenergic receptors. However, adrenaline has a strong affinity for both adrenergic receptors while noradrenaline has a higher affinity of α- adrenergic receptors than β-adrenergic receptors (Lodish et al., 2000; Nguyen and Gerstein 2019).
Therefore, both hormones released and regulated by preganglionic sympathetic fibers activation act on nicotinic cholinergic receptors during stress which have a major part of the fight or flight response (Melmed et al., 2015).
In this experiment adrenaline and noradrenaline hormones released showed a non-correlation between partners with p-value of 0,132. However, stranger touch between hormones showed a value of 0,069 which is remarkably close to the significant p value 0.05 with positive correlation of R=
0,440. The correlation between adrenaline and noradrenaline would perhaps be significant if the whole participants of 26 data were determined in this study. Since p-value of stranger was much closer than p-value of partner in both hormones, this may be a hint or weak evidence that both have a weak correlation in unpleasant touch despite non-correlation where obtained by Spearman’s test.
To compare this study with animal experiments provides that both hormones are increasing or decreasing parallel depending on what kind of touch stimulation is received and what kind of fibers are in interaction. However, the conclusion for this study is that female participants who did not detect any hormone concentration, felt calm during the experiment. Hence to animal studies and hence to current study, the conclusion of this non correlation was due to enhancing low threshold unmyelinated C-tactile stimulation to take over instead of myelinated A-beta fibers in both partner and stranger touches. However, female participants whose concentration levels of both stress hormones were determined were possibly due to some of the female participants feeling pressure and stress during an experiment which promoted slight velocity of sympathetic nervous system activation and therefore myelinated A-tactile was taken over.
4.5. Rolls of hormone adrenaline and oxytocin in skin touch.
In this current experiment the correlation between oxytocin and adrenaline hormone releases level by (partner/stranger) touches were carried out by Spearman’s correlation test. The result showed female participants touched with a partner had a strong negative correlation between oxytocin and adrenaline. While a non-correlation was conducted between oxytocin and adrenaline in stranger touches. This result corresponds and supports other numerous studies which examined the
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importance of oxytocin hormone to enhance anti-stress effect on human relationship specially between partners (Uvnäs-Moberg et al., 2015). Another study which also assessing the involvement of oxytocin in married heterosexual partner may have a crucial impact on stress reduction on physical touch. A regular warm touch between married couple partners allows oxytocin hormone levels to elevate and lower stress hormones in both men and women, particularly lower blood pressure in husbands (Holt-Lundstedt et al., 2008). Oxytocin hormone is a warm and calm hormone released by low density, non-anxious stroking by either unmyelinated mechanoreceptor C-tactile afferent presence or not. These tactile are found in hairy skin which responses to low velocity of light touch (walker, 2017).
Oxytocin is a particularly important reproductive neuropeptide hormone in women (Neumann, 2000). Therefore, another study finds that innate hormonal physiology childbearing and breastfeeding with skin to skin contact has significant benefits for mothers and infants. Therefore, separation of mother and new-born child leads to elevations of adrenaline hormone and decline oxytocin hormone (Buckley, 2015). There is another study which carried out oxytocin releasing level investigation on heterosexual par in response with their dog after being separated from the dog during a normal stress day work. 10 men and 10 women were included in this study which showed a significant elevation of oxytocin in women and decreased stress burden after being touched with a dog. While the male partner did not show any significant differences (Miller et al., 2009).
Correlation between max concentrations values in adrenaline and oxytocin hormones were conducted in this study for two reasons, one oxytocin considered as essential reproductive neuropeptide in women, and one that oxytocin is anti-stress response which has effect on adrenaline hormone reduction. To compare this study with previous studies in human bonding relationships in partners has a significant reduction on adrenaline releasing. These findings may be an additional strong evidence of a healthy relationship in partners enhancing oxytocin hormone releasing which decreases further adrenaline hormone releasing. This supported the current study, where it showed a strong positive correlation between adrenaline and oxytocin in partner touches unlike stranger touches.
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5. Ethical aspect and impact of the research on the society
Ethical aspect of this project was obtained by the Regional Ethical Review Board in Linköping, Sweden. The research obtained high ethical standards which protected and ensured participants from potential adverse consequences that would be obtained by research tasks. All participants were informed of their consent according to the Declaration of Helsinki. The Declaration of Helsinki was developed by the World Medical Association which is known as the best policy and guidance to protect health/sick human project material and data above science and society. The main foundation in any medical practice or medical research is to maintain high standard and full protection of individuals involving risk and burden. In other words, medical research related to human biological subjects must be taken on individuals with appropriate scientific training and qualifications.
Individuals who are conducted in research investigations whether on healthy or sick individuals, specialists or health care professionals are required to maintain full protection and to avoid high potential risks.
The overall impact of the research on the society of using human material is to improve prophylactic, diagnostic and therapeutic procedures for human wellbeing. In addition, human material used from healthy or sick individuals provides a research area for understanding aetiology and pathogenesis of some diseases which improve new medication and clinical picture. The finding for this search is to add a new hypothesis of how adrenaline could be involved in human related health problems by stress or treating life situations.
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6. Conclusion/future perspective
In this thesis work, adrenaline releasing level depends on skin-to-skin is the first report and new concept of human subject in the research area and in medical research. This elevated study of hormone releasing behaviour was obtained by young and healthy female’s participants who were not taking any hormone therapy, were not pregnant and were free from estrogen-based contraceptive use. Each female has been touched with their male partner and with male stranger in two runs. The aim of this study was therefore to detect whether adrenaline hormone would differ between partner and stranger touches.
The conclusion of this result showed that no differences between female (partner/stranger) gentle touch to obtain adrenaline releasing levels between groups. The result of this study determined that gentle touches were weak to observe a significant result in adrenaline hormone releases within groups in different variables. In biological response related to nerve signal, some of the females maybe did not felt any discomfort of being touched by a stranger and some of the females felt a weak pressure during the experiment which was not enough to observe a significant observation on adrenaline releasing hormone. This result maintained that female participants were calm during the experiment which promoted unmyelinated C-tactile in gentle touch. A strong evidence to draw from this conclusion is when adrenaline and oxytocin correlation was obtained by the same female’s plasma. It showed that there is a correlation between adrenaline and oxytocin in max partner with (R= -0,825, p= 0,002) unlike max stranger partner (R= 0,596, p= 0,069). That would be one of the important findings of how important and strong of gentle touches between partner bonding is compared with stranger touches.
In future perspective, the same idea and concept of experiment could be followed but, to receive unpleasant touches such as (needles or pinches) stimulation instead of gentle touches to determine whether adrenaline hormone is released in humans or not. The experiment design in this study could change into many suggestions to obtain the high quality of research that involves how much adrenaline produces due to human touching. Another option could also be that the experiment includes males and females at the same study concept to understand how much adrenaline releases differ in both genders. Understanding the influences of touches related to adrenaline stimulation, gives an additional knowledge of how important different touches promote a different type of symptoms. It is well known that unpleasant touching enhances adrenaline releasing to the bloodstream which results in high blood pressure, increases heart rate, and increases vascular resistance. But in normal life major stresses, adrenaline also affect other sides in human beings that occur unrelated to fight or flight response. In other words, adrenaline hormone releasing causes human anxiety, depression, damaging the blood vessels, headache, insomnia, and weight gain. These symptoms have a big part in human life which enhance and promote several serious conditions in the future. Therefore, to understand how much of adrenaline hormone releases affect our normal life stress it could be one of the reasons why people develop for example, heart attack risks, memory impact, anxiety, and dementia.
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7. Acknowledgement
I would like to express my deepest gratitude to the special person who has guided me and supported me in all the best ways to present this written thesis work. I am deeply honoured and privileged to work with my supervisor Linda Handlin and to be part of her work to develop the meaning of touch influences on human beings and touch benefits on sociality. I would like to thank Lisa Svedbom who helped me out in the lab to maintain the best lab work as professional by accessing additional lab tools it needed to maintain this practical project.
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