6 a-Amylase as a Reliable and Convenient Measure of Sympathetic Activity

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alpha-Amylase as a Reliable and Convenient Measure of Sympathetic Activity
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  INVITED VIEWPOINT a -Amylase as a reliable and convenient measure of sympathetic activity: don’t start salivating just yet! Jos A. Bosch a,b, *, Enno C.I. Veerman c , Eco J. de Geus d , Gordon B. Proctor e a College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK  b Mannheim Institute of Public Health and Preventive Medicine (MIPH), University of Heidelberg, Germany  c Department of Oral Biology, Academic Centre for Dentistry Amsterdam (ACTA), Amsterdam, The Netherlands  d Department of Biological Psychology, Vrije Universiteit, Amsterdam, The Netherlands  e King’s College London Dental Institute, Guys’ Hospital, London, UK  Received 6 November 2010; received in revised form 23 December 2010; accepted 23 December 2010 Recent years have shown a burgeoning interest in salivary  a -amylase (sAA) as a non-invasive marker for sympatheticnervous system (SNS) activity. sAA is a digestive enzyme thatbreaks down insoluble starch into soluble maltose and dex-trin. sAA can be measured quickly and reproducibly withcommercially available kits based on substrates that utilizeits enzymatic activity. This activity, expressed as units per Psychoneuroendocrinology (2011)  36 , 449—453 KEYWORDS a -Amylase;Stress;Saliva;Oral biology;Sympathetic nervoussystem;Psychophysiology;Endocrinology;Adrenergic Summary  Recentyearshaveseenagrowinginterestinsalivary a -amylase(sAA)asanon-invasivemarkerforsympatheticnervoussystem(SNS)activity.Salivaoffersmanyadvantagesasabiomarkerfluid and sAA is one of its most plentiful components. sAA is a digestive enzyme that breaks downstarch, which provides a simple means of quantification by measuring its enzymatic activity. Thiscommentary will address a number of common misconceptions and methodological issues thatsurround the use of sAA as a marker of SNS activity and limit its utility in biobehavioral research.The usefulness of sAA as an SNS marker is undermined by the fact that the parasympatheticnerves also play a significant role in sAA release. Local parasympathetic nerves regulate sAAactivity via: (1)  a -amylase release from glands that are solely or mainly parasympatheticallyinnervated; (2) via synergistic sympathetic—parasympathetic effects on protein secretion (knownas ‘augmented secretion’); and (3) via effects on salivary flow rate. Regarding methodology, wediscuss why it is problematic: (1) to ignore the contribution of salivary flow rate; (2) to useabsorbent materials for saliva collection, and; (3) to stimulate saliva secretion by chewing. Whilethese methodological problems can be addressed by using standardized and timed collection of unstimulated saliva, the physiological regulation of sAA secretion presents less resolvable issues.We conclude that at present there is insufficient support for the use and interpretation of sAAactivity as a valid and reliable measure of SNS activity. # 2011 Elsevier Ltd. All rights reserved. * Corresponding author at: College of Life and EnvironmentalSciences, University of Birmingham, Edgbaston, Birmingham B152TT, UK. Tel.: +44 121 414 8105; fax: +44 121 414 4121. E-mail address:  j.a.bosch@bham.ac.uk (J.A. Bosch).available at www.sciencedirect.comjournal homepage: www.elsevier.com/locate/psyneuen0306-4530/$ — see front matter # 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.psyneuen.2010.12.019  milliliter (U/ml), is often taken as a proxy for sAA concen-tration although the two only modestly correlate ( r   = .60)(Mandel et al., 2010).In early studies sAA activity emerged as a measure of parasympathetic activity, whereby sAA levels were found toincrease during relaxation (Morse et al., 1983). The mid-nine-ties,however,sawthefirststudiesshowingthatsAAactivityisalso increased during stress and correlates with norepinephr-ine release during exercise (Bosch et al., 1996; Chattertonet al., 1996). Although stress studies failed to replicate thelatterobservation(NaterandRohleder,2009),sAAactivitywasquickly adopted as a measure of SNS activity. If true, thancollecting saliva would allow simultaneous assessment of thetwo major stress systems, the HPA-axis and the SNS, and it istherefore difficult to overstate its potential.At a first glance the supporting data looks compelling.Numerous studies in humans and animals have revealed thatsympathetic activation induced by stress, by exercise, bypharmacological means, or via local nerve stimulation, uni-formly increases sAA release or its activity in whole mouthsaliva(Boschetal.,2002;NaterandRohleder,2009).Moreover,administration of the beta-adrenergic antagonist propanololreduces amylase activity in unstimulated whole mouth saliva(Nederfors and Dahlo¨f , 1992) and abrogates stress induced increases in sAA activity (Nater and Rohleder, 2009). Thehypothesis also seems consistent with the extensively charac-terizedglandularbiologyinmanandinanimalmodels,wherebythe parasympathetic nerves mainly (but not exclusively) con-trolfluidsecretion,andthesympatheticnervesmainly(butnotexclusively) regulate salivary protein secretion, including thesecretion of sAA (Proctor and Carpenter, 2007).Notwithstanding, we will argue here that there is cur-rently no strong scientific basis for the use of sAA activity as areliable measure of SNS activity. This commentary willaddress a number of common misconceptions and methodo-logicalissues that, wehope, willclarify the limitations of sAAin biobehavioral research. 1. Is sAA activity determined by sympatheticactivity? Most psychophysiologists are aware that measuring sympa-theticactivityisaknottyissue:activationoftheSNSdoesnotoccur in the generalized manner that is sometimes assumedand the various measures of SNS activation do not correlatewell (Grassi and Esler, 1999; Folkow, 2000). The latter alsoapplies to sAA: stress studies show that changes in sAAactivity do not, or only modestly, correlate with changesin other SNS markers, such as cardiac pre-ejection period,skin conductance, and plasma norepinephrine. Even withinthe salivary glands the SNS does not act in a concertedfashion: the secreto-motor sympathetic nerve fibers, respon-sible for the glandular secretion of sAA, are activated inde-pendently of the vasoactive sympathetic nerve fibers thatregulate vasoconstriction in glandular tissue (Proctor andCarpenter, 2007).WhilesuchissuescomplicatetheinterpretationofsAAasameasure of SNS activity, more problematic is the evidencethat parasympathetic activity likewise plays an importantrole in sAA and protein secretion, thereby invalidating theuse of sAA as an exclusive read-out of sympathetic activity.There are three main pathways whereby parasympatheticactivity can influence sAA concentrations: (1) via  a -amylaserelease from glands that are solely or mainly parasympathe-tically innervated, e.g., the palate and sublingual glands; (2)via synergistic sympathetic—parasympathetic interactionswhereby parasympathetic activity amplifies sympatheticeffects; and (3) via the effects of (parasympathetically-mediated) salivary flow rate. These aspects of salivary glandbiology and physiology are further discussed below. Sincecollection of saliva is the key to reliable and interpretableresults (c.f., Rohleder and Nater, 2009), we will discuss alsothe methodological pitfalls which are frequently encoun-tered in saliva-based psychobiological research. 2. Basic concepts 2.1. Not all salivary glands respond the same Most sAA literature refers to ‘‘saliva’’ without acknowledge-ment that this fluid is a complex mixture derived from manydifferent glands and different cell types within glands. Inshort, saliva is produced by three pairs of major glands; theparotid glands, the submandibular glands and the sublingualglands. In addition there are numerous minor glands in thesubmucosa underlying the lip, cheeks and palate with asubstantial contribution to salivary protein content (Hum-phrey and Williamson, 2001). Individually these glands differgreatly in the amount of sAA they produce (Veerman et al.,1996), in the autonomic innervations they receive, and thetype of transmitter and neuropeptide receptor they express(Proctor and Carpenter, 2007). For example, the parotid andminor palatine glands contain the highest amounts of amy-lase (Veerman et al., 1996), and the parenchyma of theseminor glands is mainly, if not entirely, innervated by para-sympathetic nerves (Proctor and Carpenter, 2007). Thus,amylase release is most likely also elicited by parasympa-thetic stimulation of sAA-rich glands.The latter point is relevant to psychophysiology: Boschet al. (2003) showed that a passive-coping stressor thatevoked parasympathetic activation (viewing a surgicalvideo), as measured by increases in salivary flow and cardiacvagal tone, also strongly (2.5-fold) increased the release of sulfated-MUC5B;aproteinthatisalmostexclusivelysecretedby the parasympathetically innervated palatine and sublin-gual glands. Significantly, this stressor also evoked an sAArelease that was much larger than the release during astressor that elicited a dominant (cardiac-) sympatheticactivation in conjunction with a vagal withdrawal and areduced flow rate (i.e., a time-pace memory task) (Boschet al., 2003). 2.2. Sympathetic effects on sAA secretion areaugmented by concurrent parasympatheticactivity Although heart rate has been successfully used as an index of psychological stress for more than one hundred years, andlikewise has been erroneously labelled as a measure of sympathetic activity, it is now clear that heart rate responsesreflect a mixture of additive and interactive changes in localparasympathetic and sympathetic activity (Berntson et al.,450 J.A. Bosch et al.  2008).Thisprinciplealsoappliestothesalivaryglands,andinparticulartothesubmandibularandtheparotidglandswherethe two autonomic branches collaboratively evoke salivarysecretion of both fluid and protein (there is no autonomicantagonism in the salivary glands) (Proctor and Carpenter,2007). For example, an extensive experimental literatureshows that the sympathetic effects on secretion of sAA, andother salivary proteins, are amplified by concurrent para-sympathetic activity (Asking, 1985; Carpenter et al., 1998).This is termed ‘augmented secretion’ and refers to the factthatsAA secretionduringconcomitantsympatheticandpara-sympathetic nerve stimulation is much greater than the sumof the sAA secreted during individual nerve stimulation(Proctor and Carpenter, 2007). 3. Faulty methods 3.1. Ignoring the contribution of salivary flowrate Probablyoneofthemajorcauses ofconfusion insAAresearchis that researchers do not consider secretion rate. Indeed, of the sAA studies published in Psychoneuroendocrinology onlyone attempted to quantify the possible confounding effectsof flow rate. This omission is most likely inherited fromcortisol research, in which, due to the nature of this analyte,flow rate does not play a significant role. In contrast tocortisol, sAA is synthesized in the acinar cells (i.e., the mainsecretory cells) of the salivary glands, where it is stored ingranules before secretion. Upon neuronal activation, thecontent of these granules (containing amylase and otherproteins) is secreted into saliva: i.e., the amount of amylasethat is secreted per unit of time is directly related to theextentofsympatheticactivity(ProctorandCarpenter,2007).Therefore, the amylase output per unit of time, rather thanits concentration, would appear the better proxy for neuro-nal activity. That is, sAA concentration reflects the combinedeffectofsalivaryflowrate(whichislargelyparasympathetic)and protein secretion (which, in sympathetically innervatedglands, is largely sympathetic). If sAA is to be regarded as avalid measure of sympathetic activity, than the parasympa-theticeffect onsalivary flowrateisaconfounding factorthatneeds adjustment.The reciprocity between concentration and flow rate isalso evident from stress studies. These show that when acondition has little or no effect on flow rate, then theconfounding effects of flow rate are small (Bosch et al.,1996; Rohleder et al., 2006). However, this picture changeswhen there is a larger effect on flow rate. For example, wecompared the effects of different laboratory stress tasks onsaliva flow and sAA secretion and found that changes in sAAactivity were for 25—40% due to changes in salivary fluidsecretion (Bosch et al., 2003). 3.2. Use of cotton rolls for saliva collection Researchers whousecotton spongessuch asthe Salivette willhave noticed that saliva, a slimy and turbid fluid, comes outof the collection device watery and clear: what goes inevidently does not come out. Indeed, studies have shownthatthe salivette introduces measurement error, sometimes quitesubstantial, to a number of salivary analytes, including sAA(Strazdins et al., 2005; DeCaro, 2008; Harmon et al., 2008;Beltzeretal.,2010).ThespongedoesnotfullyreleaseitssAA,and this retention shows a strong inverse relation with theamount of fluid absorbed. Nearly complete sAA retention wasobserved when the cotton absorbed 0.25 ml of saliva, whichapproximates the normal amount of unstimulated saliva pro-ducedover1 min(DeCaro,2008).Thisimpliesthattheamountof saliva, which is related to flow rate and/or duration of collection,willindirectlyinfluencesAAvalues.Thedurationof saliva collection and the stimulus determining flow rate(see discussion below) are rarely standardized. A furtherlimitation is that salivary flow rate is difficult to assessreliably using Salivettes, because the absorbent capacitydecreases when more fluid is taken up and because valueshave a ceiling effect due to saturation of the material(see discussion by Beltzer et al., 2010). While recently newerabsorbent materials have been marketed, there is little datato suggest that these will do a much better job with sAA.Why use those absorbent materials in the first place? Theymay of course have an application in research where salivacollection is complicated, such as with newborns or duringconditions like strenuous exercise. But otherwise it wouldseem sensible to adhere to what oral biologists have longestablished as the standard procedures for unstimulated‘whole saliva’ collection (Navazesh, 1993). The suggestionthat study participants are uneasy with spitting into a tubeappearsoverstatedandiscontradictedbyevidence(Strazdinset al., 2005). Admittedly, the ‘‘drooling method’’ may soundunappealing, but drooling is avoided with the equally reliable‘‘spitting method’’ (Navazesh, 1993; Navazesh and Kumar,2008). There is also no strong reason to assert that spittingin a tube is impractical: most laboratory suppliers provide alarge variety of tubes and vials likely to fit every researchcondition. These collection vials come with another conve-nience: most cost only a fraction of absorbent products. 3.3. Stimulated and unstandardized collection of saliva Stimulation of mechano-receptors in the mouth during chew-ing induces local autonomic reflex activity which is wellknown to enhance glandular secretion independent of cen-tral regulation, i.e., independent of the ‘higher’ neuraleffects of stress (Garrett, 1987). Indeed, sAA is principallya digestive enzyme and its secretion is heavily influenced bylocal reflex stimuli in order to deliver large amounts duringeating. The fact that the majority of sAA studies use salivacollection methods which involve mechanical stimulationthuscomplicatesinterpretation ofdata:e.g.,towhatextenddoes activation of local reflexes modify or over-rule thecentral SNS effects on sAA release? This issue can be com-pared to the effects of movement and changing posture onheart rate, which similarly induce autonomic reflexes thatwilldistorttheheartrateeffectsofpsychologicalstress(c.f.,Bosch et al., 2009).Complicating matters even further, in most studies theglandular stimulation is unstandardized. For example, parti-cipants are instructed ‘‘to gently move the Salivette aroundin the mouth’’ or ‘‘to chew on the Salivette’’. If not stan-dardized, how can a researcher be certain that a relaxedIs salivary  a -amylase a reliable measure of SNS activity? 451  participant will chew with the same vigor as a distressedparticipant? Or, how will we know whether the instruction to‘gently move the Salivette around’ will be acted upon withthe same gentleness by female and male participants (orchildren vs. adults, etc.)? What we  do  know is that thestrength of mechanical stimulation corresponds to theamount of saliva produced (Hector and Linden, 1987). Thisincreased flow rate will likely affect sAA concentration sinceprotein concentration is the combined result of protein out-put and flow rate, as discussed above. We may add thatwithin the first several minutes of chewing-induced secretionthere are decreases in sAA output and concentration fromindividual glands, even with standardization of the stimulus(Proctor and Carpenter, 2001). Regretfully, the duration of saliva collection is typically also not standardized.Athird,andperhapsthemostsignificantconcernassociatedwith mechanically stimulating flow rate, is that it drasticallychanges salivary protein composition, owing to: (1) the differ-ent responsivity of the parotid and submandibular glands tostimulation; and (2) the differing amounts of sAA and otherproteins that these glandular salivas contain. Under passiveconditions(i.e.,withoutmechanicalorgustatorystimulation),most saliva is secreted by the submandibular glands and onlyabout20%derivesfromtheparotidgland,whichhappenstobeveryrichinsAA(Schenkelsetal.,1995;HumphreyandWilliam-son, 2001). However, in response to chewing the contributionof individual glands changes, whereby now about half of allsaliva is from the parotid glands. This is highly significant asparotid saliva contains a 4—10-fold higher AA concentrationthan submandibular saliva (Veerman et al., 1996).In sum, the commonly used method of unstandardizedmechanical stimulation invokes local autonomic reflexactivity, shifting the balance from submandibular secretion(modest sAA concentration) to parotid secretion (high flowrate, high  a -amylase content)  independent  of central SNSregulation. Overall therefore, it is extremely difficult toobtain a value from stimulated ‘whole mouth’ sAA thatcan confidently be attributed to central effects on localSNS activity. 4. Conclusion While biopsychology boasts a strong tradition of endocrine,immune, and cardiovascular research, scholars of this fieldrarely had training in oral biology. It is likely that with suchtraining many would have made different choices withregard to methods and the interpretation of sAA data.Currently most researchers adhere to a methodology thatwas validatedfor cortisol research.For example, among thesAA studies published in Psychoneuroendocrinology to date,virtually none controlled for the potentially confoundingeffects of salivary flow rate, most do not standardize salivacollectionintermsofstimulationorcollectionduration,andthe majority used absorbent materials that are known todistort sAA values. We found that a significant number of studiesdidnotprovideanydetailonhowsalivawascollectedor how participants were instructed (apart from the simplestatement that ‘Salivettes were used’). Also, many authorsconfuse the distinctions between sAA activity, sAA concen-trationandsAAsecretion,inoneexamplepresentingdatainimplausible concentration units while citing the use of anassaythatdoesnotexist.Wehopetohaveclarifiedhowsuchmethodology makes the bulk of sAA findings difficult tointerpret.But even with more rigorous methodology one shouldperhaps anticipate disappointment. Experimental evidencefrom oral biology research indicates that the idea of sAAactivity as a valid and reliable measure of SNS activity is toosimplistic. The salivary glands are a sophisticated and het-erogeneous group of organs, capable of responding with ahigh level of specificity to stimuli relevant to digestion,speech, and immune function. Whole mouth sAA can beviewed as the sum of a large number of contributing factors;glandular sympathetic—adrenergic stimulation during stressis only one of such factors. Role of the funding sources The authors received no funding in support of this work. Conflict of interest The authors have no conflicts of interests to declare. Acknowledgements We are grateful to Prof. Robert Dantzer for his invitation tosubmit this commentary, and thank Dr. Rose-Marie Bluthe´ forher editorial support. References Asking, B., 1985. Sympathetic stimulation of amylase secretionduring a parasympathetic background activity in the rat parotidgland. Acta Physiol. Scand. 124 (4), 535—542.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—296.Berntson, G.G., Norman, G.J., Hawkley, L.C., Cacioppo, J.T., 2008.Cardiac autonomic balance versus cardiac regulatory capacity.Psychophysiology 45 (4), 643—652.Bosch, J.A., Brand, H.S., Ligtenberg, T.J.M., Bermond, B., Hoogstra-ten, J., Nieuw Amerongen, A.V., 1996. Psychological stress as adeterminantofproteinlevelsandsalivary-inducedaggregationof  Streptococcus gordonii  in human whole saliva. Psychosom. Med.58 (4), 374—382.Bosch, J.A., de Geus, E.J., Carroll, D., Goedhart, A.D., Anane, L.A.,van Zanten, J.J., Helmerhorst, E.J., Edwards, K.M., 2009. Ageneral enhancement of autonomic and cortisol responses duringsocial evaluative threat. Psychosom. Med. 71 (8), 877—885.Bosch, J.A., de Geus, E.J.C., Veerman, E.C.I., Hoogstraten, J.,Nieuw Amerongen, A.V., 2003. Innate secretory immunity inresponse to laboratory stressors that evoke distinct patterns of cardiac autonomic activity. Psychosom. Med. 65 (2), 245—258.Bosch,J.A.,Ring,C.,deGeus,E.J.,Veerman,E.C.,Amerongen,A.V.,2002. Stress and secretory immunity. Int. Rev. Neurobiol. 52,213—253.Carpenter, G.H., Garrett, J.R., Hartley, R.H., Proctor, G.B., 1998.Theinfluenceofnerveson the secretionofimmunoglobulinA intosubmandibular saliva in rats. J. Physiol. 512 (Pt 2), 567—573.Chatterton Jr., R.T., Vogelsong, K.M., Lu, Y.C., Ellman, A.B., Hud-gens, G.A., 1996. Salivary alpha-amylase as a measure of endog-enous adrenergic activity. Clin. Physiol. 16 (4), 433—448. 452 J.A. Bosch et al.
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