Maya R. Rashkova, Lora S. Ribagin, Nina G. Toneva Department of Pediatric Dentistry, Faculty of Dental Medicine, Medical University, Sofi a

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1 Copyright 2012 Medical University Plovdiv doi: /v CORRELATION BETWEEN SALIVARY α-amylase AND STRESS-RELATED ANXIETY Maya R. Rashkova, Lora S. Ribagin, Nina G. Toneva Department of Pediatric Dentistry, Faculty of Dental Medicine, Medical University, Sofi a ABSTRACT Salivary α-amylase is a useful biomarker that can be used in assessing human psychobiological and social behavioural processes. Studying it opens up possibilities for the creation of novel concepts concerning the interaction of biological and social processes and their impact on health and behaviour. MATERIALS AND METHODS: The levels of salivary α-amylase and situation anxiety self-assessment using Spielberger test were measured twice in 30 individuals aged ± 0.96 yrs (18 females and 12 males): once during stressful situation (prior to examination) and, again a month later, in stress-free environment (during a training session). Salivary α-amylase was measured using kinetic reaction kit Salimetrics LLC USA. RESULTS: The mean level of salivary α-amylase measured during the first measurement ± U/ml. During the second measurement in the absence of intense stress, the levels were two times lower ± U/ml and the differences were statistically significant (P < 0.001). We found a statistically significant correlation between the levels of salivary α-amylase in both measurements (P < 0.01). The correlation coefficient was r = (P < 0.01). CONCLUSIONS: The adapted version of the State-Trait Anxiety Inventory score (STAI) created by Spielberger is appropriate for assessment of stress-related anxiety in young individuals. Salivary α-amylase may be used as a biomarker for objective evaluation of the psychosomatic state of individuals in a stressful environment. The combination of psychological test and objective indicator such as salivary α-amylase is an excellent tool for objective evaluation of individual s state in stressful environment. Similar tests may be used in assessment of patients behaviours at dental treatment that may be considered a stressor in most patients. Key words: salivary α-amylase, salivary cortisol, biomarker, psychobiology, stress, anxiety self-assessment test, oral-based diagnostics INTRODUCTION The medium of oral fluids presents opportunities for studying biologically active substances related to various processes in the organism. When these substances have diagnostic or prognostic value they are referred to as biological markers (biomarkers) and their study in saliva is incorporated in a modern approach known as oral-based diagnostics. 1-3 Studying such biomarkers that can be used to assess human psychobiology and socio-behavioural processes makes it possible for researchers to introduce new concepts concerning the interaction of biological and social processes and their impact on health and behavior. 4 Test have been developed for psychological assessment of anxiety as a prerequisite for the psychological response to stress such as the social stress test (TSST), the Spielberger test of situational response to stress, etc. 5-9 These tests are subjective and difficult to apply in children. Modern science is looking for marker that are easy to apply and accurate enough to make an assessment of stress psychobiology. The main systems in human body basically associated with stress psychobiology are the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system There are two major markers that can be found in saliva: the salivary cortisol, associated with the former system and the salivary α-amylase that is associated with the latter. The serological mediators of the sympathetic activity are the catecholamines, which are very difficult Correspondence and reprint request to: M. Rashkova, Department of Pediatric Dentistry, Faculty of Dental Medicine, Medical University, Sofi a; mayarashkova@mail.bg; Mob.: (+359) Sv. G. Sofi iski blvd., Sofi a 1431, Bulgaria 46 Received 27 February 2012; Accepted for publication 17 May 2012

2 Correlation Between Salivary α-amylase and Stress-related Anxiety to study in blood samples. 13 Recent research has demonstrated that salivary α-amylase may be used as indirect marker of sympathetic nervous system activity. There is evidence of individual differences in the levels of salivary α-amylase associated with differences in individual behaviour in stress Unlike most diagnostically important salivary substances entering the saliva through the systemic circulation by active transport or by passive diffusion, salivary α-amylase is produced locally in the cells of the salivary glands and is directly influenced by sympathetic innervation of the gland. Alpha-amylases are included in the glycoside hydrolase family 13; the function of α-amylase is to break down the portions of carbohydrate taken by the mouth. 17 Evidence from comparative studies shows that changes in the levels of salivary α-amylase are a more accurate indicator of an individual s response to stress compared to those of cortisol, because of the higher sensitivity of the sympathetic nervous system compared to the complex hypothalamicpituitary-adrenal hormone axis. Correlation between both markers at the beginning and at the end of a stress producing experiment has been found by many researchers ; we have also found such correlation (unpublished results). The relationship between the salivary α-amylase level and psychological predisposition to certain diseases and conditions has been studied only recently. The conclusions that can be drawn at this stage is that individual differences in the levels of salivary α-amylase are associated with both the internalization (social alienation, anxiety, depression) and the externalization (aggression and symptoms of behavioural disorders) These studies prompted us to investigate the salivary α-amylase in stress situations so that we can suggest this enzyme to be used as a biomarker in stress psychobiology research. AIM To study the levels of salivary α-amylase and state anxiety in adolescents in stressful environment. 1. Assessment of the state anxiety in the study sample using the self-report anxiety assessment test introduced by Spielberger; 2. Measurement of the salivary α-amylase in young people in stressful environment; 3. Correlation between Spielberger test and salivary α-amylase during the experiment. MATERIALS AND METHODS STUDY PARTICIPANTS The present study recruited 30 third-year dental students (mean age ± 0.96 yrs, 18 females and 12 males) and exposed them to a stressful situation (immediately before an exam) and stress-free situation (during an exercise tutorial). The participants were given the self-assessment anxiety test suggested by Spielberger, the State- Trait Anxiety Inventory score (STAI), adapted in Bulgarian by D. Shtetinski and I. Paspalanov. 9,26,27 Salivary α-amylase in samples of unstimulated saliva was analyzed in parallel. The study was conducted in two stages: - First stage - before the exam (participants in a stressful situation) use of STAI-s test and analysis of salivary α-amylase; - Second stage - 30 days after the exam in a stressor-free situation use of STAI test (s-scale and t-scale) and analysis of salivary α-amylase. The inclusion criteria for participation in the study were: 1) no use of drugs that affect the sympathetic innervation (blood pressure controlling drugs), nicotine, and caffeine, and 2) no consumption of carbohydrate rich food 48 hours prior to test. These conditions are known to affect the α-amylase level in the mouth. Samples of saliva for analysis of salivary α-amylase. The enzyme may be detected in 10 μl of saliva. A sample of unstimulated saliva was collected in the morning on an empty stomach, at least ½ hour after brushing teeth. The saliva was collected in plastic containers for 10 minutes after rinsing the mouth with distilled water. A certain amount (2 ml) was taken from the container using pipette, placed in a micro tube and frozen in a refrigerator (at -20 C). Spielberger s State-Trait Anxiety Inventory test. The test consists of two subscales (1) the S- scale which evaluates anxiety as a transitory state and (2) the T-scale which evaluates anxiety as a personality trait, measuring relatively stable individual differences in anxiety proneness. Individual differences affect cognition, as well as the type and dynamics of human defence mechanisms. Both scales have 20 items. The S-scale evaluates how respondents feel at a particular time of perceived threat stress, while the second one (T-scale) - how they anticipate they would feel in general during hypothetical stressful situations. A 4-point scale is used to rate the intensity of feeling: in the S-scale - not at all, somewhat, a great deal, completely, 2012 Medical University Plovdiv 47

3 M. Rashkova, et al and in the T-scale - almost never, sometimes, often, almost always. The test may be completed in 10 minutes. We used the S-scale in 30 students before the examination in Pediatric Dental Medicine (in a stressful environment) and one month later, during a exercise tutorial in Pediatric Dental Medicine (stress-free environment), and the T-scale - only in the second assessment. The study is part of a project authorized by the Ethics Committee of Scientific Research at the Medical University - Sofia. Informed consent was obtained from every participant. Method for quantitative measurement of α-amylase in salivary samples. Salivary α-amylase was measured by kinetic reaction. The kinetic reaction kit Salimetrics LLC USA used includes chromatogenic substrate (2-chloro-p-nitrophenol) linked with maltotriose. The enzyme reaction with this substrate results in formation of 2-chloro-pnitrophenol and yellow colour measured by spectrophotometry, with a standard reader. Activeness of salivary α-amylase is proportional to the increasing absorption of light with a wavelength of 405 nm (optical density) for a period of 2 min. Individual levels are relatively stable and are sensitive to environmental changes in psychological stressors. Statistics. Statistical data was analysed using with SPSS v.16 (SPSS Inc.Chicago USA). We used analysis of variance (descriptive statistics); quantifiable results are presented as mean ± standard deviation (SD). Paired samples t-test was used to compare means. A value of P < 0.05 was considered statistically significant. RESULTS State anxiety assessment of the study sample usin g Spielberger s test STAI. The total score was calculated from the completed questionnaires: the S-scale (before examination) and the S- and T-scales 1 month after the exam as instructed in the test instruction manual. 3 The results are shown in Table 1. The mean score for state anxiety (S-score) in the first test (before examination) was significantly greater than the one (S-score) in the second stage in a relaxed environment (p < ). Anxiety as a character trait (T-score), measured in the second stage was significantly correlated with anxiety as a transitory state (S-score) from the same study (r = 0.394, P < 0.05). The relationships for all participants are presented in the Figs 1 and 2. Table 1. Mean scores of state anxiety as found during test n = 30 S-scale T-scale Mean ± SD Mean ± SD First stage of test ± 7.22 Not used in stress Second stage of test ± ± 7.80 t-test t = p = Figure 1. Level of state anxiety (S- score) in participants in stressful and stress-free environment S 1 - score S 2 - score T - score S 2 - score Figure 2. Anxiety level (S2-score and T-score) in participants in stress-free situation. Greater mean scores of S1 scale (in stressful environment) compared to S2 scores (in stressfree environment) were found in all participants indicating higher level of anxiety during a stressful situation. The statistically significant lower score in the second stage shows that anxiety decreases in a relaxed environment. In the second stage, conducted in a stress-free environment, the observed correlation (P < 0.05) between the mean scores for state anxiety (S-scale) and anxiety as a character trait (T-score) of the two tests supports the concept that state anxiety Medical University Plovdiv

4 Correlation Between Salivary α-amylase and Stress-related Anxiety as transient emotional state reflects susceptibility to anxiety as a relatively stable personality trait. Analysis of salivary α-amylase in subjects during stressful situation. Mean levels of salivary α-amylase in both stages of test are presented in Table 2. both measureshoweverprovides sufcient obective information about the psychosomatic state of participants in stress and in a relaxed environment. Table 2. Levels of salivary α-amylase observed in experiment n = 30 Stage 1 - measured before examination Stage 2 - measured a month after the examination α-amylase Mean S Mean S ml t p The mean level of salivary α-amylase in the rst stage of test was ml. n the second stagein the absence of strong stressors the level was two times lower ml and the difference was statistically signicant ( 0.001). There was also signicant correlation between the level of salivary α-amylase in both measurements ( 0.01). The correlation coefcient was r 0.42 ( 0.01). Figure shows clearly the relationship between the first and second measurements of salivary α-amylase for all participants with the lower mean scores of salivary α-amylase observed in the second assessment (in a relaxed environment). Relationship between Spielberger s STAI test and salivary α-amylase during the experiment. There was no correlation between mean anxiety scores and salivary α-amylase in both assessments ( 0.05). This can be easily accounted for by the fact that these two indicators are essential completely. Salivary α-amylase is an obective marer of the sympathetic nervous system functionwhile the mean score of state anxiety is a subective indicator obtained from self-reported answers to specic uestions. The combination of α-amylase-2 α-amylase-1 Figure 3. Salivary α-amylase level in participants in stressful and stress-free situation. DISCUSSION The present study found a two-fold increase of salivary α-amylase concentration in stress and its decrease while measured in a stress-free situation. Similar research has been nding similar results in recent years Salivary α-amylase increases in response to stressful conditions: physical exercise and exposure to heat and cold (sharp change in T)examination Trier Social Stress Test (watching very negative pictures (car crashes) and participating in exhaustive exercises t was as bac as 1990 in a series of studies conducted by Chatterton et al. that a correlation was found between the salivary α-amylase level and the components of the sympathetic nervous system in the body when it is involved in a stress response. 1 Shea Aet al. measured salivary α-amylase in mothers and their infants (6-10 months) subected to emotional distress and found that these are positively correlated. Similar results have been reported when separating infants from their mothers. 22 Correlation between behavioursalivary α-amylase levels and salivary cortisol was found in -9-yearold children. Correlation has been found between behaviourteaching staff and health in school children. The salivary α-amylase levels are associated with aggressive behaviour in boyswhile the poststress level of salivary α-amylase vary depending on the internaliation in girls A series of studies have found different interrelationships between salivary α-amylasebehaviour and cognitionleading to the obvious conclusion that salivary α-amylase may be a biomarer associated with physiological processes influenced by psychologicalbehavioural and social processes. This conclusion is consistent with the ndings in 2012 Medical University Plovdiv 49

5 M. Rashkova, et al our research which is the first in our country that studies this enzyme in unstimulated saliva samples. CONCLUSIONS The adapted Bulgarian version of STAI inventory by Spielberger for state anxiety assessment is a useful measure of anxiety in young individuals in stress; Salivary α-amylase may be used as a biomarker for objective assessment of individual psychosomatic state of in stressful environment; The combination of psychological test and objective indicator such as salivary α-amylase concentration is an excellent tool for objective evaluation of individual s state in stressful environment. Similar tests may be used in assessment of patients behaviors in dental treatment that may be considered a stressor in most patients. ACKNOWLEDGEMENTS The authors would like to thank Mrs. M. Karapeeva and Dr. Minkova who took part in the laboratory tests performed in Med-Dia Ltd. Clinical Laboratory in Sofia. The study received funding by Medical University Sofia under Project No 17 (contract No4/ closed 2010) REFERENCES 1. Malamud DL, Niedbala RS, eds. Oral-based Diagnostics. Ann N Y Acad Sci, Blackwell, 2007: Lawrence A, Tabak LA. Revolution in Biomedical Assessment: The Development of Salivary Diagnostics. J Dent Educ 2001;65(12): Thompson LA, Trevathan WR. Cortisol reactivity, maternal sensitivity, and learning in 3-month-old infants. Infant Behav Dev 2008;31(1): Chrousos, GP, Gold PW. The concepts of stress and stress system disorders. JAMA 1992;267(9): Lovallo WR, Thomas TL. Stress hormones in psychophysiological research: emotional, behavioural, and cognitive implications. In: Cacioppo JT, Tassinary LG, Bernston GG, eds. Handbook of Psychophysiology. 2nd ed. New York: Cambridge University Press; 2000: Kirschbaum C, Pirke KM, Hellhammer DH. The Trier Social Stress Test - a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology 1993;28: Raynolds C, Richmond B. What I think and feel: a revised measure of children s manifest anxiety. Journal of Abnormal Child Psychology 1978;21(6): Spielberger, C. Anxiety. Current trends in theory and research. New York: Academic Press; Taylor J, Deane FP. Development of a short form of the Test Anxiety Inventory (Tai). J Gen Psychol 2002;129(2): Kirschbaum C, Hellhammer DH. Salivary cortisol in psycho neuroendocrine research: recent developments and applications. Psychoneuroendocrinology 1994;19(4): Dawes C. Considerations in the development of diagnostic tests on saliva. In: Malamud D, Tabak LA, eds. Saliva as a diagnostic fluid. Ann NY Acad Sciences 1993;694: Gozansky WS, Lynn JS, Laudenslager ML, Kohrt WM. Salivary cortisol determined by enzyme immunoassay is preferable to serum total cortisol for assessment of dynamic hypothalamic-pituitary-adrenal axis activity. Clin Endocrinol (Oxf). 2005;63(3): Poggi ED, Granger GA. Developmental differences in infant salivary alpha-amylase and cortisol responses to stress. Psychoneuroendocrinology 2009;34,6: Stegeren AH, Wolf OT, Kindt Mvan Stegeren AH, Wolf OT, Kindt M. Salivary alpha amylase and cortisol responses to different stress tasks: impact of sex. Int J Psychophysiol 2008;69(1): Nater UM, et al. Human salivary alpha-amylase reactivity in a psychosocial stress paradigm. Int J Psychophysiol 2005;55(3): Nater UM, et al. Stress induced changes in human salivary alpha-amylase activity associations with adrenergic activity. Psychoneuroendocrinology 2006;31: Stroud LR, et al. Saliva alpha-amylase stress reactivity in children and adolescents: validity, associations with cortisol, and link behaviour. Presented at the Annual Meeting of the American Psychosomatic Society. Denver, CO, 2006; March Kivlighan KT, DA Granger. Salivary alpha-amylase response to competition: relation to gender, previous experiences, and attitudes. Psychoneuroendocrinology 2006;31: Allwood MA, Handwerger K, Kivlighan KT, Granger DA, Stroud LR. Direct and moderating links of salivary alpha-amylase and cortisol stress-reactivity to youth behavioral and emotional adjustment. Biol Psychol 2011;21(6): Skosnik PD, et al. Modulation of attentional inhibition by norephinephrine and cortisol after psychological stress. Int J Psychophysiol 2000;36: Granger DA, et al. Integrating the measurement of salivary alpha-amylase into studies of child health, development, and social relationships. J Pers Soc Relationships, Special Issue: Physiology and Human Relationships. 2006;23: Shea AK, et al. Maternal depression and salivary alpha-amylase response to stress in their infants. Presented at the Annual Meeting of the American Psychosomatic society. Denver, CO, 2006: Stoyanov D. A linkage of mind and brain: towards Medical University Plovdiv

6 Correlation Between Salivary α-amylase and Stress-related Anxiety translational validity between neurobiology and psychiatry. Biomed Rev 2011;22: Stoyanov DS. Translational cross-validation among neuroscience and psychiatry: Prospects for diagnostic assessment and psychopharmacology. European Psychiatry 2012;27: Mize J, Lisonbee J, Granger D. Stress in child care: cortisol and alpha-amylase may reflect different components of the stress response. Presented at the Biennial Meeting of Society for Research in Child Development. Atlanta, GA, March Shtetinski D, Paspalanov I. Manual for the Bulgarian version of questionnaire for anxiety assessment by Ch. Spielberger (STAI ), Institute of Psychology, Comprehensive study of human being and their brain. 1989: Salimetrics/Products and Services [Internet] Available from: КОРРЕЛЯЦИЯ МЕЖДУ α-амилазой СЛЮНЫ И ТРЕВОЖНОСТЬЮ ПРИ СТРЕС- СЕ М. Рашкова, Л. Рибагин, Н. Тонева РЕЗЮМЕ Слюнная α-амилаза представляет доказанный биомаркер, связанный с психобиологией и социальноповеденческими процессами у человека. Ее изучение создает возможности развития новых концепций относительно взаимодействия биологических и социальных процессов и их влияние на здоровье и поведение. МАТЕРИАЛ И МЕТОДЫ: 30 пробандов в возрасте ± 0.96 г. (18 девушек и 12 юношей), поставленных в стрессовую среду (перед экзаменом), а месяц после этого в спокойную среду (во время упражнения), подвергнуто обследованию уровней α-амилазы слюны и ситуативной тревожности с помощью теста для самооценки тревожности Spielberger. Слюнная α-амилаза измерена через кинетическую реакцию с помощью теста Salimetrics LLC, USA. РЕЗУЛЬТАТЫ: При первом исследовании средние количества α-амилазы слюны в границах ± U/ml. При втором исследовании при отсутствии сильного стресса они в два раза ниже ± U/ml, при чем разницы показывают значительную достоверность (Р < 0.001). Наблюдается значительная корреляция между уровнем α-амилазы слюны в обоих исследованиях (Р < 0.01). Коэффициент корреляции - r = (Р < 0.01). ВЫВОДЫ: Адаптированный у нас STAI - тест Spielberger относительно ситуативной тревожности является подходящим тестом оценки тревожности у молодых людей в состоянии стресса. Α-амилазу слюны можно использовать как биомаркер в целях объективной оценки психосоматического состояния человека в стрессовой среде. Сочетание между психологическим тестом с объективным показателем, каким является α-амилаза слюны, представляет отличное средство для объективизирования состояния человека, поставленного в стрессовую среду. Подобные тесты могут быть использованы и при оценке поведения пациентов в случаях дентального лечения, на которое можно смотреть как на стрессор для большинства пациентов Medical University Plovdiv 51