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Salivary Alpha-Amylase Enzymatic Kit

Technical Summary

Assay Protocol
Rev. 04.08.16
Specifications
Catalog#: 1-1902
Format: 96-well plate
Assay Time: ~ 30 mins
Sample Volume/Test: 10 µL of saliva then 8 µL of X200 dilution
Sensitivity: 0.4 U/mL
Assay Range: 5.1 pg/mL - 500pg/mL
Storage Requirements: 2-8°C
Tests Per Kit
Singlet: 94
Duplicate: 46
Target Analyte
Technical Documentation
Technical Bulletin - Running Multiple Amylase Strips
SDS Available on Request

Salivary Alpha-Amylase Assay Kit Overview

Intended Use

The Salimetrics Alpha-Amylase Kinetic/Enzymatic Assay Kit is specifically designed and validated for the kinetic measurement of salivary α-amylase activity. It is not intended for diagnostic use. It is intended only for research use in humans and some animals. Salimetrics has not validated this kit for serum or plasma samples.

Introduction

Technical advances that make the assessment of biomarkers in saliva possible have enabled researchers to non-invasively study biosocial processes related to stress in naturalistic contexts. Secretion of alpha-amylase from the salivary glands is controlled by autonomic nervous signals, and substantial literature reveals that salivary alpha-amylase is a correlate of sympathetic activity under conditions of stress. Studies show that levels of salivary alpha-amylase increase under a variety of physically (i.e., exercise, heat and cold) and psychologically (i.e., written examinations) stressful conditions in human subjects. Interestingly, studies show that cortisol levels often do not correlate with α-amylase during stress, suggesting that individual differences in alpha-amylase represent a response to a stress signal independent of the LHPA axis.

Early studies on salivary alpha-amylase showed that its concentrations are predictive of plasma catecholamine levels, particularly norepinephrine (NE), and are highly correlated with NE changes in response to stress. However, more recent studies call this relationship into question. The literature does show that stress-related increases in salivary alpha-amylase can be inhibited by the adrenergic blocker propranolol and also that beta-adrenergic agonists are capable of stimulating alpha-amylase release without increasing salivary flow. This link suggests that the same stimuli that increase autonomic (sympathetic) arousal may activate sympathetic input to the salivary glands. The salivary alpha-amylase response to stress is complex, however, and it appears also to involve the parasympathetic system to a lesser degree. A recent article has emphasized the contribution of the parasympathetic system to salivary alpha-amylase secretion, pointing out in particular that autonomic reflex activity from the oral cavity, which can increase the parasympathetic signaling to the salivary glands, may have the potential to obscure the effects of central SNS activity. However, a subsequent study has found that salivary alpha-amylase responses significantly predict responses to the TSST for norepinephrine (NE) but not for epinephrine (E). The relationship between salivary alpha-amylase and NE was stronger than the relationship between NE and E responses, indicating the predictive power of salivary alpha-amylase is well within the expected range for different SNS markers.

Although further work is necessary to understand better the underlying physiological factors that influence salivary alpha-amylase secretion, studies have already shown that salivary α-amylase measurements may be employed as a non-invasive measure of autonomic nervous system activation and are related to a variety of behavioral, social, health, and cognitive phenomena in human subjects.

Enzymatic Alpha-Amylase Test Principle

This method for alpha amylase activity determination utilizes a chromagenic substrate, 2-chloro-p-nitrophenol linked with maltotriose. (17)  The enzymatic action of α-amylase on this substrate yields 2-chloro-p-nitrophenol, which can be spectrophotometrically measured at 405 nm.  The amount of α-amylase activity present in the sample is directly proportional to the increase in absorbance at 405 nm. For ease of use, the reaction is read in a 96-well microtiter plate with controls provided.

References & Salivary Alpha-Amylase Research

    1. Granger, D.A., Kivlighan, K.T., El-Sheikh, M., Gordis, E., & Stroud, L.R. (2007). Salivary alpha-amylase in biobehavioral research: Recent developments and applications. Ann N Y Acad Sci, 1098, 122-44.
    2. Chrousos, G.P. & Gold, P.W. (1992). The concepts of stress and stress system disorders: Overview of physical and behavioral homeostasis. JAMA, 267(9), 1244-52. Erratum in JAMA (1992), 268(2), 200.
    3. Kirschbaum, C., Read, G.F., & Hellhammer, D.H. (1994). Assessment of hormones and drugs in saliva in biobehavioral research. Göttingen: Hogrefe & Huber.
    4. Scannapieco, F.A., Torres, G., & Levine, M.J. (1993). Salivary α-amylase: Role in dental plaque and caries formation. Crit Rev Oral Biol Med, 4(3/4), 301-7.
    5. Rogers, J.D., Palmer, R.J., Jr., Kolenbrander, P.E., & Scannapieco, F.A. (2001). Role of Streptococcus gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm formation. Infect Immun, 69(11), 7046-56.
    6. Chatterton, R.T., Jr., Vogelsong, K.M., Lu, Y.C., Ellman, A.B., & Hudgens, G.A. (1996). Salivary alpha-amylase as a measure of endogenous adrenergic activity. Clin Physiol, 16(4), 433-48.
    7. Nater, U.M., & Rohleder, N. (2009). Salivary alpha-amylase as a non-invasive biomarker for the sympathetic nervous system: Current state of research. Psychoneuroendocrinology, 34(4), 486-96.
    8. Speirs, R.L., Herring, J., Cooper, W.D., Hardy, C.C., & Hind, C.R. (1974). The influence of sympathetic activity and isoprenaline on the secretion of amylase from the human parotid gland. Arch Oral Biol, 19(9), 747-52.
    9. van Stegeren, A., Rohleder, N., Everaerd, W., & Wolf, O.T. (2006). Salivary alpha-amylase as a marker for adrenergic activity during stress: Effect of betablockade. Psychoneuroendocrinology, 31(1), 137-41.
    10. Gallacher, D.V. & Petersen, O.H. (1983). Stimulus-secretion coupling in mammalian salivary glands. Int Rev Physiol, 28, 1-52.
    11. Rohleder, N., Wolf, J.M., Maldonado, E.F., & Kirschbaum, C. (2006). The psychosocial stress-induced increase in salivary alpha-amylase is independent of saliva flow rate. Psychophysiology, 43(6), 645-52.
    12. Bosch, J.A., Veerman, E.C., de Geus, E.J., & Proctor, G.B. (2011). α-Amylase as a reliable and convenient measure of sympathetic activity: Don’t start salivating just yet! Psychoneuroendocrinology, 36(4), 449-53.
    13. Thoma, M.V., Kirschbaum, C., Wolf, J.M., & Rohleder, N. (2012). Acute stress responses in salivary alpha-amylase predict increases of plasma norepinephrine. Biol Psychol, 91(3), 342–48.
    14. Granger, D. A., Kivlighan, K. T., Blair, C., El-Sheikh, M., Mize, J., Lisonbee, J.A., Buckhalt, J. A., et al. (2006). Integrating the measurement of salivary alpha-amylase into studies of child health, development, and social relationships. J Soc Pers Relat, 23(2), 267-90.
    15. Segal, S.K., & Cahill, L. (2009). Endogenous noradrenergic activation and memory for emotional material in men and women. Psychoneuroendocrinology, 34(9), 1263-71.
    16. Susman, E.J., Dockray, S., Granger, D.A., Blades, K.T., Randazzo, W., Heaton, J.A., & Dorn, L.D. (2010). Cortisol and alpha amylase reactivity and timing of puberty: Vulnerabilites for antisocial behaviour in young adolescents. Psychoneuroendocrinology, 35(4), 557-69.
    17. Wallenfels, K., Foldi, P., Niermann, H., Bender, H., Linder, D. (1978). The enzymic synthesis, by transglucosylation of a homologous series of glycosidically substituted malto-oligosaccharides, and their use as amylase substrates. Carbohyd Res, 61(1), 359-68.
    18. Weiner, D., Levy, Y., Khankin, E.V., Reznick, A.Z. (2008). Inhibition of salivary amylase activity by cigarette smoke aldehydes. J Physiol Pharmacol, 59(Suppl 6), 727-37.
    19. Klein, L.C., Bennett, J.M., Whetzel, C.A., Granger, D.A., & Ritter, F.E. (2010). Caffeine and stress alter salivary α-amylase activity in young men. Human Psychopharmacol, 25(5), 359-67.
    20. Nater, U.M., Rohleder, N., Scholtz, W., Ehlert, U., & Kirschbaum, C. (2007). Determinants of the diurnal course of salivary alpha-amylase. Psychoneuroendocrinology, 32(4), 392-401.
    21. Mackie, D.A. & Pangborn, R.M. (1990). Mastication and its influence on human salivary flow and alpha-amylase secretion. Physiol Behav, 47(3), 593-95.
    22. Lo Piparo, E., Scheib, H., Frei, N., Williamson, G., Grigorov, M., & Chou, C.J. (2008). Flavonoids for controlling starch digestion: Structural requirements for inhibiting human α-amylase. J Med Chem, 51(12), 3555-61.
    23. Hara, K., Ohara, M., Hayashi, I., Hino, T., Nishimura, R., Iwasaki, Y., Ogawa, T., et al. (2012). The green tea polyphenol epigallocatechin gallate precipitates salivary proteins including alpha-amylase: Biochemical implications for oral health. Eur J Oral Sci, 120(2), 132-39.
    24. Beltzer, E.K., Fortunato, C.K., Guaderrama, M.M., Peckins, M.K., Garramone, B.M., & Granger, D.A. ( 2010). Salivary flow and alpha-amylase: Collection technique, duration, and oral fluid type. Physiol Behav, 101(2), 289-96.
    25. Harmon, A.G., Towe-Goodman, N.R., Fortunato, C.K., & Granger, D.A. (2008). Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: A preliminary note of caution. Horm Behav, 54(5), 592-96.

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