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Integrating the measurement of salivary α-amylase into studies of child health, development, and social relationships Douglas A. Granger, Katie T. Kivlighan, Clancy Blair, Mona El-Sheikh, Jacquelyn Mize, Jared A. Lisonbee, Joseph A. Buckhalt, Laura R. Stroud, Kathryn Handwerger and Eve B. Schwartz Journal of Social and Personal Relationships 2006 23: 267 DOI: 10.1177/0265407506062479 The online version of this article can be found at: http://spr.sagepub.com/content/23/2/267

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Integrating the measurement of salivary -amylase into studies of child health, development, and social relationships Douglas A. Granger, Katie T. Kivlighan, & Clancy Blair Pennsylvania State University

Mona El-Sheikh, Jacquelyn Mize, Jared A. Lisonbee, & Joseph A. Buckhalt Auburn University

Laura R. Stroud Brown Medical School

Kathryn Handwerger Tufts University

Eve B. Schwartz Salimetrics LLC, State College PA

ABSTRACT

To advance our understanding of how biological and behavioral processes interact to determine risk or resilience, theorists suggest that social developmental models will need to include multiple measurements of stress-related biological processes. Identified in the early 1990s as a surrogate marker This research was supported in part by the Behavioral Endocrinology Laboratory and the Child Youth and Families Consortium at The Pennsylvania State University as well as the National Institute of Child Health and Development (PO1HD39667–01A1), National Science Foundation (0126584), Alabama Agricultural Experiment Station (ALA010–008), and a Lindsey Foundation Grant. Thanks are due to Vincent Nelson and Mary Curran for biotechnical support with assays. Reagents and materials were contributed in part by Salimetrics LLC (State College, PA). All correspondence concerning this article should be addressed to Douglas A. Granger, Behavioral Endocrinology Laboratory, Department of Biobehavioral Health, 315 Health and Human Development East, The Pennsylvania State University, University Park, PA 16802, USA [e-mail: [email protected]]. Journal of Social and Personal Relationships Copyright © 2006 SAGE Publications (www.sagepublications.com), Vol. 23(2): 267–290. DOI: 10.1177/0265407506062479


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Journal of Social and Personal Relationships 23(2)

of the sympathetic nervous system component of the stress response, salivary -amylase has not been employed to test biosocial models of stress vulnerability in the context of child development until now. In this report, we describe a standard assay that behavioral scientists can use to improve the next generation of studies and specific recommendations about sample collection, preparation, and storage are presented. More importantly, four studies are presented with mother– infant dyads (N = 86), preschoolers (N = 54), children (N = 54), and adolescents (N = 29) to illustrate individual differences in stress-related change in -amylase levels, that patterns of -amylase stress reactivity distinctly differ from those measured by salivary cortisol, and associations between individual differences in -amylase and social relationships, health, negative affectivity, cognitive/academic/behavior problems, and cardiovascular reactivity. We conclude that the integration of measurements of the adrenergic component of the locus ceruleus/autonomic (sympathetic) nervous system, as indexed by salivary -amylase, into the study of biosocial relationships may extend our understanding of child health and development to new limits. KEY WORDS:

biosocial relationships • child development • cortisol • salivary alpha-amylase • salivary biomarkers

Technical advances that make the assessment of biomarkers in saliva possible have enabled researchers to study a variety of biosocial processes related to stress vulnerability, social behavior, and health in naturalistic and developmental contexts. Much of the recent attention has focused on the activity of the limbic hypothalamic-pituitary-adrenal (LHPA) axis as indexed by individual differences and intra-individual change in salivary cortisol (Gunnar & Donzella, 2002; Kirschbaum, Read, & Hellhammer, 1992; Shirtcliff, Granger, Booth, & Johnson, 2005). Yet, behavioral endocrinologists have known for decades that the psychobiology of the stress response has at least two principal components. The first involves corticotropin-releasing hormone, activation of the LHPA axis, and the secretion of glucocorticoids (e.g., cortisol) into circulation. The second, and fasteracting component, involves activation of the locus ceruleus/autonomic (sympathetic) nervous system and the release of catecholamines (e.g., norepinephrine) into the blood stream (Chrousos & Gold, 1992). To advance our understanding of how biological, social, and behavioral processes interact to determine risk or resilience, several investigative teams have argued that developmental models of behavioral phenomenon need to include multiple measurements of stress-related biological processes (Bauer, Quas, & Boyce, 2002; Donzella, Gunnar, Krueger, & Alwin, 2000; Granger & Kivlighan, 2003). Unfortunately, our ability to do so using noninvasive salivary assessments has been restricted. That is, in contrast to the highly sensitive, accurate, and


Granger et al.: Salivary -amylase

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valid measurement of LHPA products (salivary cortisol and DHEA; Granger, Schwartz, Booth, Curron, & Zakaria, 1999; Schwartz, Granger, Susman, Gunnar, & Laird, 1998), the measurement of catecholamines has proven technically challenging in saliva (Kirschbaum et al., 1992). To address this gap we present a rigorous evaluation of the internal and external validity of a salivary -amylase assay; illustrate findings from four preliminary studies involving infants, children, adolescents, and adults; and provide guidelines to assist social and behavioral scientists through the process of integrating salivary -amylase into their behaviorally-oriented research. Salivary -amylase: A primer Given concerns raised about the meaning of directly measured catecholamines in saliva (Schwab, Heubel, & Bartels, 1994), we conducted a search for potential surrogate markers of sympathetic nervous system activity in saliva. A small literature, largely conducted with adult participants, revealed salivary -amylase as a viable candidate. Assuming most readers to be unfamiliar with -amylase, we digress to introduce its basic features, functions, and developmental significance, and then briefly review evidence causally linking -amylase to stress-related adrenergic reactivity, and its associative relationship with cortisol and measures of cardiovascular psychophysiology. Enzyme with antibacterial properties In contrast to the majority of salivary biomarkers employed on a regular basis in biobehavioral research (e.g., cortisol, testosterone, dehydroepiandrosterone), -amylase is an enzyme. Also, it is not actively transported, nor does it passively diffuse, into saliva from plasma. Rather, it is produced locally in the oral mucosa by the salivary glands. Thus, under normal conditions, -amylase is present in saliva in relatively high concentrations. Its primary function is the digestion of macromolecules such as carbohydrates and starch. But, the literature on oral biology also assigns -amylase a secondary role in the prevention of bacterial attachment to oral surfaces, and bacterial clearance from the mouth (Marcotte & Lavoie, 1998). Thus, individual differences in -amylase levels have been associated with nutritional benefits derived from the diet (i.e., caloric intake from the digestion of carbohydrates) and a variety of processes related to oral health (bacteria load, caries, and periodontal disease). Much of what is known about the biobehavioral implications of individual differences in salivary -amylase levels is derived from clinically-oriented literature on oral biology and disease that is largely tangential to students of developmental and social science. Developmental significance The literature on oral biology does reveal a marked developmental difference in salivary -amylase levels (O’Donnell & Miller, 1980). That is,


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-amylase is not present in the saliva of newborns. Correspondingly, young infants do not have the same capacity as do adults to digest complex macromolecules. This transitional physiological state is considered adaptive because macromolecules are highly immunogenic and represent a significant source of threat to the neonatal immune system. Moreover, during this period of immune immaturity, newborns are primarily protected against foreign antigens by passive immunity received from their mothers (i.e., maternal antibodies, IgA) colostrum or breast milk. Given the absence of salivary -amylase, maternal antibodies can be ingested without being destroyed (essentially digested) by the infant. Salivary levels of -amylase show a sharp rise in the 0.9–1.9 year period reaching maximum levels by 5–6 years of age (O’Donnell & Miller, 1980). The age of onset in the rise of -amylase levels parallels the timing of the introduction of solid foods in the diet and the emergence of teeth needed to chew those solids. Salivary marker of the adrenergic component of the stress response More than a decade ago a series of elegant studies by Chatterton and colleagues linked levels of salivary -amylase to the sympathetic nervous system (SNS) component of the stress response (e.g., Chatterton, Vogelsong, Lu, Ellman, & Hudgens, 1996; Chatterton, Vogelsong, Lu, & Hudgens, 1997; Skosnik, Chatterton, Swisher, & Park, 2000). Most importantly for present purposes, the studies show that levels of salivary -amylase increase under both physically (i.e., exercise, heat and cold stress) and psychologically (i.e., written examinations) stressful conditions known to increase plasma catecholamines. That is, salivary -amylase concentrations are associated with baseline plasma catecholamine levels (r = .64, p < .01), particularly norepinephrine (NE), and are also highly correlated with NE change in response to stress (Chatterton et al., 1997). The linkage between NE and -amylase is underscored by the finding that stress-related increases in salivary -amylase can be inhibited by the adrenergic blocker propranolol (Speirs, Herring, Cooper, Hardy, & Hind, 1974). Also, betaadrenergic agonists are capable of stimulating -amylase release without increasing salivary flow (Gallacher & Petersen, 1983). Therefore, the same stimuli that increase concentrations of catecholamines in the blood activate sympathetic input to the salivary glands. In sum, these early studies suggested that blood levels of NE, associated with the stress response of the locus ceruleus/autonomic (sympathetic) nervous system, can be estimated by the concentrations of -amylase in whole saliva specimens, and that salivary -amylase measurements may be employed as a noninvasive measure of plasma NE concentrations in human participants. More recent studies with adult participants corroborate that salivary -amylase is sensitive to psychosocial stress (e.g., Trier Social Stress Test) and sympathetic tone generally, but question its specific association with stress-related changes in catecholamines (Nater et al., 2005, 2006; Rohleder, Nater, Wolf, Ehlert, & Kirschbaum, 2004). What has yet to be determined is whether salivary -amylase is a useful and valid marker of individual differences in children’s stress responsivity, and correspondingly



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how intra-individual change in salivary -amylase may be related to social factors and children’s behavior. Relation to hypothalamic-pituitary-adrenal stress reactivity Noteworthy is the fact that stress-related changes in cortisol levels do not appear to correlate with changes in salivary -amylase levels during stress (Chatterton et al., 1996; Nater et al., 2006). This observation is key because it suggests that individual differences in -amylase reactivity to stress represent a reaction to a signal that is not redundant with measures of change in the LHPA axis. This raises the possibility that the inclusion of both salivary cortisol and -amylase would enable more complete measurement of the psychobiology of the stress response than by measuring either component separately (Nater et al., 2006). While it is clear that the LHPA and SNS systems work in co-ordination to generate the physiologic changes associated with the stress response, the exact nature of the co-ordination (e.g., additive or interactive; opposing or complementary) is a subject of debate. These subsystems are activated in response to different situational demands (i.e., defense vs. defeat; Henry, 1992) and are differentially activated depending on a confluence of experiential, person, and contextual variables. Therefore, examining the associations and dissociations between concurrent actions across these systems in relation to social behavior and relationships may allow a more complete understanding of the physiological processes involved as intervening variables than examining either single system. More generally, understanding how social forces influence the co-ordination of stressresponse systems may provide insight into how disruption of physiological processes contribute to social, behavioral, and cognitive problems. For instance, Bauer et al. (2002) suggest that optimal functioning is most likely when the SNS and LHPA responses are balanced. Concurrent activation or deactivation would be the most adaptive and activational asymmetries would be maladaptive. Studies aimed to determine factors intrinsic to the individual as well as objective features of social environmental events that predict correlated versus dissociated rates of change between cortisol and salivary -amylase seem worthwhile. Relation to cardiovascular stress reactivity Chatterton and colleagues (1996) reported a positive relationship between salivary -amylase and heart rate that strengthened with the intensity of physical stress. To date only two studies report relationships between salivary -amylase and measures of neural regulation of heart rate. Bosch, de Geus, Veerman, Hoogstraten, and Nieuw Amerongen (2003) observed increases in sympathetic activity (shortened pre-ejection period) and amylase secretion and decreases in parasympathetic activity (decrease in heart rate variability) in response to a laboratory stressor. Nater et al. (2006) report a positive relationship between amylase and sympathetic tone during stress. Taken together, these observations support the status of salivary -amylase as a marker of SNS activation, but clearly the depth of


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our knowledge is shallow here. No studies (to our knowledge) have explored these relationships in children. Salivary biomarkers: Prospects and pitfalls The ability to measure biological variables, like -amylase, noninvasively in saliva has created many opportunities for behavioral and social scientists to test biosocial models of individual differences and intra-individual change in mood, cognition, social behavior, and psychopathology. Monitoring salivary biomarkers has several obvious advantages over doing so in other diagnostic fluids such as urine, blood, or dried blood spots. Saliva sampling represents a less-invasive method for long-term or repeated sampling schedules and enables collection in everyday circumstances. Saliva sampling also enables researchers’ access to youth and special populations who otherwise would be unlikely to participate willingly in studies requiring traditional biological samplings (Kirschbaum et al., 1992; Malamud & Tabak, 1993). Despite the obvious advantages, the literature cautions researchers that sample collection techniques and assay protocols must be carefully designed to maximize measurement validity. Studies show that salivary measurements can be substantially influenced by the process of sample collection and storage (e.g., Granger, Shirtcliff, Booth, Kivlighan, & Schwartz, 2004; Kivlighan, Granger, Schwartz, Nelson, & Curran, 2004; Schwartz et al., 1998; Whembolua, Granger, Kivlighan, Marguin & Singer, in press). Additionally, there are environmental sources (bovine products in food stuffs, hormone-like substances in breast milk and formula, medications) capable of interfering with the assay of some salivary biomarkers (Magnano, Diamond, & Gardner, 1989; Masharani et al., 2005). To the best of our knowledge, little information is available to steer researchers around these potential problems or to minimize the influence of extraneous factors on the measurement of salivary -amylase (but see Enberg, Alho, Loimaranta, & Lenander-Lumikari, 2001). The present study addresses some of these knowledge gaps. Purpose of the present study Although there has been a renewed interest in -amylase as a surrogate marker of stress in studies with adults (e.g., Nater et al., 2005, 2006; Rohleder et al., 2004), salivary -amylase has yet to be integrated into the mainstream study of the psychobiology of stress, health, and social behavior, and has never, to our knowledge, been employed to test biosocial models of stress vulnerability in the context of child development. To date, the majority of our knowledge comes from specialized clinical literatures on oral biology and laboratory stressors with adult participants. There is precious little available information on biobehavioral relationships involving salivary -amylase



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in infants, children, and adolescents. In this report we describe an assay that social and developmental science can use to improve the next generation of studies as we address these basic questions. The assay is described and its performance demonstrated with respect to sensitivity, reliability, precision, accuracy, and linearity of dilution. Analyses are conducted to evaluate alternative sample collection procedures and specific recommendations about sample collection, preparation, and storage are presented. Next, four studies are reviewed involving mother–infant dyads, preschoolers, children, and adolescents. Collectively the findings reveal age- and stress-related differences in salivary -amylase levels, patterns of intra-individual -amylase change in response to challenge that distinctly differ from those measured by salivary cortisol, as well as associations between salivary -amylase levels and social behavior, health, negative affectivity, cognitive/ academic problems, and cardiovascular activity. Study 1: Measuring salivary -amylase by kinetic reaction assay Before substantive research questions testing biosocial links involving -amylase can be addressed there are fundamental gaps in knowledge that must be filled. One of these gaps is an immediate need for a highly sensitive, standardized, and reliable assay with accessible reagents and materials for the determination of salivary -amylase that can be employed consistently within and across studies. This study addressed this need. Kinetic reaction assay background and protocol Given that -amylase is an enzyme, a kinetic reaction assay was designed to quantitatively measure it in saliva. The assay employs a chromagenic substrate, 2-chloro-p-nitrophenol, linked to maltotriose. The enzymatic action of -amylase on this substrate yields 2-chloro-p-nitrophenol, which can be spectrophotometrically measured at 405 nm using a standard laboratory plate reader. The amount of -amylase activity present in the sample is directly proportional to the increase (over a 2-minute period) in absorbance at 405 nm. Given that this assay protocol recently became commercially available (Salimetrics, State College, PA) we present only a brief overview here. Saliva samples (10 ul) are diluted 1:200 in assay diluent and well mixed. 8 ul of diluted sample or control are then pipetted into individual wells of a 96-well microtiter plate. 320 ul of preheated (37C) -amylase substrate solution (2-chloro-p-nitrophenol, linked to maltotriose) is added to each well and the plate is rotated at 500–600 RPM at 37C for 3 minutes. Optical density (read at 405 nm) is determined exactly at the 1-minute mark and again at the 3-minute mark. Results are computed in U/mL of amylase using the formula: [Absorbance difference per minute total assay volume (328 ml) dilution factor (200)]/[millimolar absorptivity of 2-chloro-pnitrophenol (12.9) sample volume (.008 ml) light path (.97)].


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Assay performance characteristics Intrassay variation (CV) computed for the mean of 10 replicate tests of low (17.7 U/mL), medium (108.8 U/mL), and high (474.6 U/mL) concentration samples were 7.2%, 6.7%, and 2.5%, respectively. Interassay variation computed for the mean of average duplicates for eight separate runs for lower (10.6 U/mL) and higher (166.0 U/mL) concentration samples were 5.8% and 3.6%. Method accuracy was determined from known amounts of -amylase (range 3.14 to 77.09 U/mL) added to five saliva samples containing various endogenous concentrations (range 29.99 to 123.28 U/mL). Recoveries ranged from 87.4% to 111.6% (M = 101.04%). Parallelism was evaluated by measuring salivary -amylase in lower (40.96 U/mL) and higher concentration samples (1943.16 U/mL) which were then serially diluted (1:2 to 1:16; and 1:3 to 1:27) to the low end of the assay’s range. Observed values were as expected across the entire range of measurement. The average recovery was 96.43% (range 83.2 to 108.5%). Sample collection, handling, and storage The literature cautions that sample collection techniques must be carefully designed to maximize measurement validity of salivary biomarkers (e.g., Granger et al., 2004). To the best of our knowledge, no information is available to steer researchers around these potential problems or to minimize the influence of extraneous factors on the measurement of salivary -amylase. The present study addressed some of these knowledge gaps. Sample collection. A series of studies has evaluated the effects of different sample collection techniques on the measurement of a variety of salivary biomarkers (Granger et al., 2004; Schwartz et al., 1998; Shirtcliff, Granger, Schwartz, & Curran, 2001). The most common saliva collection methods typically involve absorbing sample using cotton-based products. While this method seems appropriate for some markers (i.e., cotinine, cortisol) it causes substantial interference in the assay of many others including sIgA, DHEA, testosterone, estradiol, and progesterone (Shirtcliff et al., 2001). Therefore, we experimented to determine if products that absorb saliva during sample collection would artificially influence -amylase results. First, saliva samples were collected by passive drool (N = 8) and then either left untreated, or absorbed using the Salivette device. The Salivette device (Sarstedt, NC) absorbs saliva using a (.5 inch diameter, 2 inch length) cotton pledget. Individual differences in salivary -amylase levels were highly conserved, r (6) = .99, p < .0001, and averaged levels were not significantly different between the passive drool (M = 317.81 U/mL, SEM = 120.29) and Salivette (M = 315.35 U/mL, SEM = 132.72) conditions. Next, samples were collected by passive drool (N = 10) and then either left untreated or absorbed using a microsponge (BD Opthalamic Systems, Walton, MA). The microsponge device employs a very small hydrocellulose absorbent ‘sponge’ fixed to the end of a short-shafted plastic applicator. As with the Salivette, individual differences in salivary -amylase levels were



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again highly conserved when the microsponge was used, r(8) = .99, p < .0001, and averaged levels were not significantly different between the passive drool (M = 151.07 U/mL, SEM = 34.22) and microsponge (M = 110.78 U/mL, SEM = 35.03) conditions. Sample handling and storage. To evaluate the influence of freeze–thaw cycles on -amylase levels two samples were assayed fresh (M = 1176.71 U/mL), then frozen at –40C, and then tested again after being subject to one (M = 1058.80 U/mL), two (M = 1014.56 U/mL), or three (M = 1101.28 U/mL) freeze–thaw cycles. Multiple freeze–thaw cycles did not have significant effects on the assay of salivary -amylase. To determine effects of temporary storage and transit conditions on -amylase, we collected ten saliva samples by passive drool. Aliquots were then stored for 96 hours at room (RT) temperature (as if samples were mailed unrefrigerated or left on the bench in the lab inadvertently), 4C (to show effects as if samples were kept on ice in the mail or left in the lab refrigerated), or –80C (as if samples were stored under more ideal laboratory conditions). -amylase levels were not significantly influenced by these storage temperature conditions over the 96-hour period. Means were 109.82 U/mL (SEM = 18.19), 110.55 U/mL (SEM = 18.35) and 107.15 U/mL (SEM = 21.52) for the RT, 4C, and –80C conditions respectively. Summary The quantitative measurement of -amylase in saliva was accomplished using a kinetic assay approach. The protocol proved internally and externally valid according to standard assay performance criterion (Chard, 1990). The assay has a low limit of sensitivity and very small test volume requirement (10 ul). Saliva samples to be assayed for -amylase may be conveniently collected using the most commonly employed methods – passive drool, cotton, or microsponge. At least in the short run (96 hours), the effects of storage and transport temperatures on salivary -amylase levels seem negligible. Study 2: Salivary -amylase and cortisol stress reactivity in 6-month-old infants and their mothers To our knowledge, no studies that have explored biosocial relationships during infancy have included salivary -amylase. In this study, Kivlighan and colleagues (Kivlighan, Granger, Blair, & The Family Life Project Investigators, 2005) did so in mother–infant dyads as 6-month-old infants participated in a series of tasks designed to elicit emotional distress. While the infants actively participated in the task series, mothers were asked to watch without intervening. We expected the task to be a mild social challenge for mothers and a moderate stressor for most of the infants, and therefore were interested in stress reactivity of each individual but also the degree of attunement in stress responsivity between members of the dyad.


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Method Participants and procedures Participants were selected from the first 100 mother–infant dyads to complete the 6-month wave of assessment in a large (N = 1200) multisite (Central Pennsylvania and Piedmont of North Carolina) prospective longitudinal study of infant development under conditions of rural poverty (NICHD, PO1HD39667). Complete data were available for 86 mother–infant dyads. There were 45 boys and 41 girls ranging in age from 6.12 to 10.20 months (M = 7.22). Sixty mothers were Caucasian and 26 were African American, the majority (51.2%) were single and most had never married; 76.2% had incometo-needs ratios less than 200% of the poverty line. As part of the larger project’s home interview, infants were presented with four ‘challenge tasks’ designed to elicit emotional reactivity. The task procedures had been previously validated (e.g., Buss & Goldsmith, 1998; Kochanska, Coy, Tjebkes, & Husarek, 1998; Stifter & Braungart, 1995) and three (masks, barrier, and arm restraint) were from the Laboratory Temperament Assessment Battery (Goldsmith & Rothbart, 1996; Kochanska et al., 1998). For the first tasks, a toy reach and then mask presentation, mothers were seated beside their child, and for the last tasks, barrier and then arm restraint, mothers were out of the child’s sight. Tasks were presented in the following order: reach, masks, barrier, and arm restraint. For the arm restraint task, an experimenter crouched behind the infant and gently restrained the child’s arms for 2 minutes or until 20-seconds of hard crying ensued. Mothers were asked not to intervene but were told they could stop the tasks at any time. Three saliva samples were collected from the dyad; a baseline collected prior to administration of the challenge tasks, 20 minutes following the infants’ peak arousal to the arm-restraint task, and 40 minutes after peak arousal. Assays for -amylase were conducted as described above. Samples were assayed for salivary cortisol using a highly sensitive enzyme immunoassay (Salimetrics, PA). The test used 25 µl of saliva, had a lower limit of sensitivity of .007 µg/dL, range of sensitivity from .007 to 1.8 µg/dL, and average intra- and interassay coefficients of variation of less than 5% and 10% respectively.

Results Analyses used salivary -amylase (and cortisol for comparative purposes) as the dependent variable in a 2 3 2 mixed-model ANOVA. The design was dyad member (infant vs. mother) by sampling occasion (pre task vs. 20 and 40 minutes post task) by infant gender (male v. female). For -amylase there was a main effect for dyad, F(1, 78) = 49.20, p < .001, and a sampling occasion for dyad interaction, F (2, 156) = 5.26, p < .01 (see Figure 1). As expected, on average, -amylase levels were higher for mothers. (M = 272.15 U/ml, SD = 141.65) than for infants (M = 161.35 U/mL, SD = 99.05). On average, the mothers’ -amylase levels were higher 20 minutes post task, t(83) = 3.58, p < .01, compared to pre task, and levels 40 minutes post task remained higher than pre task, t(82) = 2.07, p < .05. Surprisingly, on average, -amylase levels for the infants did not change in response to the challenge tasks. Using a 10% difference from pre task as a criterion for change, 44.4% of the infants showed -amylase, and 48.1% a decrease from pre to 20 minutes post task. Downloaded from spr.sagepub.com by Douglas Maple on September 15, 2010


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-Amylase U/mL

Cortisol ug/dl

FIGURE 1 Infants demonstrate LHPA response to challenging tasks as indexed by salivary cortisol (ug/dl), while mothers demonstrate an SNS response to infant distress as indexed by salivary -Amylase (U/mL).

Time Note. On average, infants have higher salivary cortisol levels than their mothers whereas mothers have higher salivary -amylase levels than their infants (Kivlighan et al., 2005).

By comparison, the pattern for cortisol was different from -amylase in important ways. There were main effects for dyad, F(1, 82) = 6.04, p < .05, and sampling occasion, F(2,164) = 7.96, p < .01, and a dyad sampling occasion interaction, F(2, 164) = 37.21, p < .001. Overall, cortisol levels were higher for infants (M = .63 g/dl, SD = .42) than for mothers (M = .48 g/dl, SD = .26). The interaction (see Figure 1) revealed that consistent with the diurnal decline across the day in cortisol levels, mothers’ levels were progressively lower across all three sampling occasions. Infants’ cortisol levels, on the other hand, were elevated over pre task baseline at 20 minutes post task, t(84) = 3.86, p < .001. Downloaded from spr.sagepub.com by Douglas Maple on September 15, 2010

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Infants’ cortisol levels 40 minutes post task were lower than the peak level reached 20 minutes post task, t(83) = 3.44, p < .01, but still remained higher than pre task, t(83) = 2.17, p < .05. Using a 10% difference from pre task as a criterion for change, 61.2% of the infants showed a cortisol increase, and 25.9% a decrease from pre to 20 minutes post task. To more directly compare the -amylase and cortisol levels, bivariate correlations were computed using baseline and reactivity scores. Baseline levels were simply pre task measures and reactivity scores were percent change scores computed across the pre to 20-minute post task period. Within individuals, neither salivary cortisol or -amylase baseline levels or reactivity scores were significantly associated for either infants or their mothers. Finally, we evaluated whether -amylase or cortisol levels were associated between mothers and their infants. Overall, mothers’ and infants’ baseline levels of -amylase, r = .29, p < .01, and cortisol, r = .31, p < .01, were positively associated. However, a more careful evaluation revealed this relationship was true for mother–son dyads (-amylase: r = .33, p < .05, cortisol: r = .44, p < .01) but not for mother–daughter dyads.

Summary This exploratory study revealed several novel findings. First, mothers had higher -amylase and lower cortisol levels than did their 6-month-old infants. Second, on average, mothers showed -amylase but not cortisol reactivity to observing their infants participate in the challenge tasks. By contrast, infants showed cortisol but not -amylase reactivity to the tasks. While this was true on average, an evaluation of individual differences revealed that approximately 45% of the infants had an -amylase rise greater than 10% in response to the tasks. Third, there was very little evidence that -amylase or cortisol levels or reactivity were associated within individuals, yet within each dyad mothers with higher baseline levels of -amylase or cortisol had infant sons with similarly high levels. An especially unique observation is the apparent gender-linked difference in the ‘attunement’ in the LHPA and SNS between mothers and their infants. Study 3: Stress in child care – cortisol and -amylase may reflect different components of the stress response in preschoolers In addition to the scant information available regarding salivary -amylase in infants, our literature search revealed a similar gap in knowledge about -amylase levels, stress reactivity, and biobehavioral relationships during the preschool period. Recently, Mize and colleagues (Mize, Lisonbee, & Granger, 2005) addressed this gap and studied these relationships in a subset of preschoolers who were participating in a larger prospective study of how childcare experiences relate to kindergarten adjustment (NSF 6048857). Salivary -amylase and cortisol were examined as a function of



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stress experience, teacher–child relationship quality (teacher report of closeness), and health (parent report). Method Participants and procedures The sample included 52 4-year olds (29 boys; 23 girls; M age 52.7 months) enrolled in full-day child care in a small city in the southeastern United States. Each child’s primary teacher completed the Student–Teacher Relationship Scale (Pianta, Steinberg, & Rollins, 1995), from which a measure of teacher– child closeness was derived. Parents completed a shortened version of the Rand Health Survey about the child (Eisen, Donald, & Ware, 1980). Children participated in a series of five developmentally appropriate challenge tasks or games intended to provoke mild frustration or disappointment. The tasks were: A coordination game (based on the dart toss game; Kochanska, 1997), a disappointment experience (Cole, Zahn-Waxler, & Smith, 1994), an impossible puzzle task (Smiley & Dweck, 1994), a delay of gratification task (Kochanska & Aksan, 1995), and an inhibitory motor activity task (Kochanska, Murray, & Harlen, 2000). The challenge task series lasted approximately 30 minutes. Children reported self-perceived skill at puzzles before and after the failure experience (Smiley & Dweck, 1994). Following the challenge tasks, the child’s primary teacher helped the child to make a block structure and read a book to the child (Teacher Interaction). Saliva samples were collected by passive drool from children before (pre) and after (post) the challenge tasks, and after the teacher interaction (follow-up) and stored frozen until shipped on dry-ice overnight to be assayed for -amylase and cortisol at Salimetrics Laboratories as described earlier. Results Both salivary biomarkers were stable across the three assessment intervals (r = .66 to .87), but cortisol and -amylase were not correlated at any assessment, nor were changes in cortisol correlated with changes in -amylase. There were no differences in either cortisol or -amylase as a function of sex or child age. For most children, cortisol fell slightly from pre to post challenge (80%) and from post challenge to follow-up (70%). However, a few children experienced cortisol increases of greater than 10% from pre to post challenge, and from post challenge to follow-up (14% and 10% of children, respectively). Interestingly, more children experienced increases in -amylase than cortisol over the same assessments (45% and 37%). -Amylase was associated with both children’s health and teacher–child relationships. Children with higher -amylase were more susceptible to illness, r = .31 to .45 at three assessments, and had less close relationships with teachers, r = –.30 to –.45. Children with greater -amylase increases from pre challenge to follow-up had more illness, r = .40 and less close relationships with teachers, r = –.44. The associations between -amylase and illness were somewhat stronger for girls than for boys, for example, rs = .55 vs. .14 at pre challenge; the sex-by--amylase increase from pre to follow-up challenge approached significance, = .61, p = .06.


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Summary Consistent with Kivlighan, Granger, Blair, and The Family Life project Investigators (2005), approximately 40% of the children showed an increase in -amylase greater than 10% in response to this series of ageappropriate challenging tasks, and there was no evidence that -amylase and cortisol levels or reactivity were associated. To the best of our knowledge, the robust pattern of positive associations between -amylase levels and illness susceptibility is novel. Although unique, the finding is not surprising, given the volumes of research suggesting linkages between norepinephrine, immune function, and health (e.g., Ader, Cohen, & Felten, 1995). Also noteworthy is the observation that higher levels of -amylase were associated with less close relationships with teachers. Taken together with the tentative link between mother and infant -amylase levels noted earlier, this finding hints that individual differences in -amylase levels during early childhood may be regulated by social relationships (with mothers and teachers) central in young children’s immediate social worlds.

Study 4: -Amylase responsivity to stress predicts behavioral adjustment, physical health, and cognitive functioning during middle childhood El-Sheikh, Mize, and Granger (2005) explored biosocial relationships involving -amylase during middle childhood. In a preliminary report they examined relationships between parasympathetic nervous system activity (vagal regulation), LHPA (salivary cortisol), and -amylase reactivity to stress in elementary school-aged children. Associations between these physiological markers of stress vulnerability and children’s psychological adjustment, physical health, and cognitive functioning were also explored. Method Participants and procedures Fifty-four children (24 boys, 30 girls) participated. Children ranged in age between 8 and 9 years (M = 8.86 years; SD = .28), and were recruited in a small city in the southeastern United States. Children were healthy (they did not have acute or chronic illnesses), and attended third grade at state schools in regular classes (no special education). To minimize confounds, exclusion criteria were based on parent report and included chronic or acute physical illness, mental retardation, learning disabilities, and ADHD. The majority of children lived with both of their parents (74%), and the rest lived in stepfamilies mostly composed of the child’s biological mother and stepfather. Socioeconomic status (SES) of the sample represented the complete spectrum of possible economic backgrounds (Hollingshead, 1975) with 21% in Levels 1–2 (e.g., unskilled and semiskilled workers), 34% in Level 3 (e.g., skilled workers), 32% in Level 4 (e.g., minor professionals), and only 13% in Level 5 (e.g., professionals). Sixtyseven per cent were European American and 33% African American. Downloaded from spr.sagepub.com by Douglas Maple on September 15, 2010


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Children came to a university laboratory for one session during which their vagal regulation and cognitive functioning were examined. Children were administered six tests of the Woodcock-Johnson III Tests of Cognitive Abilities (WJ-III; Woodcock, McGrew, & Mather, 2001). The WJ-III is a well-normed, highly reliable and valid comprehensive system for measuring general intellectual ability (g) and specific cognitive abilities in nine factorial dimensions of intelligence, or broad stratum II abilities, and several narrow stratum I abilities within each broad dimension. During the lab session, children’s respiratory sinus arrhythmia (RSA) was examined as an index of vagal regulation through procedures similar to those employed in El-Sheikh (2005) and El-Sheikh, Harger, and Whitson (2001). Electrodes and a respirometer belt were attached to the child while the parent was seated in the same lab room as the child. The research assistant conversed with both the parent and the child while attaching the electrodes to help the child relax (around 10 minutes). Then the child was told that the parent would go next door. Following the parent’s departure, a 2-minute adaptation period followed to allow the child to further acclimate to the laboratory environment. Baseline assessments (3 minutes) were then obtained for RSA, and children listened to an audiotaped argument through speakers, which supposedly occurred between a man and a woman next door (3 minutes). To increase generalizability of findings, two themes were used for the arguments at either T1 or T2: in-laws and leisure activities issues, and a similar number of boys and girls were exposed to each theme (note that potential theme-related effects were examined and no significant effects were found). The arguments were characterized by verbal expressions of anger. Similar scripts have been used in other studies, and were effective in inducing RSA suppression in children (El-Sheikh et al., 2001; 2005). Following the argument, a recovery period followed, then each child completed a star-tracing task (3 minutes). A board was put across the child’s chair, and the child was given a sheet of paper with a picture of a star. The star was blocked from direct view but visible through a mirror. Children were asked to trace the star using only the mirror image as a visual guide. Saliva was collected from the children before and 20 minutes after the stressful laboratory procedures. Samples were stored frozen until shipped overnight on dry ice to be assayed for cortisol and -amylase at Salimetrics Laboratories as described earlier. Parents reported on children’s adjustment using the Personality Inventory for Children (PIC-2; Lachar & Gruber, 2001), which is a comprehensive inventory of child functioning, including externalizing and internalizing problems. Teachers reported on children’s adjustment and achievement problems using the Student Behavior Survey (Lachar, 1999). Physical health problems were examined via mothers’ reports on the Cornell Medical Index (Brodman, Erdmann, & Wolff, 1960), and the child version of the Rand Corporation Health Insurance Scale (Eisen et al., 1980).

Results Salivary cortisol, -amylase, and RSA were stable across assessment times, r = .33 to .87, p < .05. There was no association between salivary cortisol and amylase levels or reactivity. -Amylase was associated with deficits in vagal suppression, r = .36, p < .05. Whereas vagal suppression (RSA stressor < RSA baseline) is the typical response to environmental challenges, and is associated Downloaded from spr.sagepub.com by Douglas Maple on September 15, 2010

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with positive child outcomes, children with higher levels of -amylase exhibited vagal augmentation (RSA > RSA baseline). Higher levels of salivary cortisol and -amylase were associated with lower SES and African American ethnicity. Girls with higher poststress -amylase had more health problems, social problems, and aggression, r = .36 to .63, p < .05, as reported by both mothers and teachers. Boys with higher baseline -amylase had more cognitive/ academic problems as assessed by the Woodcock-Johnson III and teachers’ reports, and more aggression, r = .42 to .66, p < .05. Controlling for the effects of ethnicity and SES, -amylase continued to predict many of the aforementioned outcomes.

Summary The findings confirm the preliminary observations noted earlier of the lack of correspondence between salivary cortisol and -amylase stress responsivity (Kivlighan et al., 2005; Mize et al., 2005), that higher levels of amylase are associated with children’s health problems (Mize et al., 2005), and that individual differences in -amylase may be related to social behavior and relationships (Kivlighan et al., 2005; Mize et al., 2005). With respect to the latter, the pattern of positive associations between -amylase and social problems, aggressive behavior, and cognitive/academic problems is especially noteworthy. It is tempting to speculate that this pattern is responsible for the observation that children with higher -amylase had less close relationships with teachers (Mize et al., 2005). This study also revealed that in middle childhood, individual differences in -amylase during stress conditions may be associated with deficits in vagal suppression to challenging situations. That is, children who showed the highest amylase levels were less effective in removing parasympathetic inhibition over their heart rate and increased sympathetic activation to successfully deal with the current challenge.

Study 5: Saliva -amylase stress reactivity in children/adolescents – developmental differences in relation to cortisol, cardiovascular, and affective responses Similar to infancy, early, and middle childhood, little is known about the effects of stress on -amylase over adolescence or links between -amylase reactivity and reactivity of other stress response systems during this period. Stroud, Handwerger, Kivlighan, Granger, and Niaura (2005) tested the validity of individual differences in saliva -amylase levels as a marker for physiological stress reactivity in adolescents. More specifically, Stroud and colleagues conducted a detailed analysis of the time course of the -amylase response to a modified Trier Social Stress Test for Children (TSST-C; Buske-Kirschbaum, Jobst, Wustmans, & Kirschbaum, 1997), tested the possibility of age-related differences in -amylase reactivity, and examined associations between -amylase and salivary cortisol and cardiovascular Downloaded from spr.sagepub.com by Douglas Maple on September 15, 2010


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responses to the modified TSST-C. Finally, given prior research in adults showing specific emotional correlates of cortisol versus sympathetic responses to stress (e.g., Frankenhaeuser, 1982), Stroud et al. (2005) examined associations between cortisol and -amylase reactivity and particular emotional responses to the stressors. Method Participants and procedures Participants were 29 healthy children/adolescents (12 girls; 17 boys) aged 7 to 16 (M = 12.1, SD = 2.4) recruited through community postings and direct mailings as part of a larger study of physiological responses to stress over the transition from childhood to adolescence. Exclusion criteria were based on factors known to influence cortisol and cardiovascular reactivity, including the use of oral contraceptives, thyroid medications, steroids, and psychotropic medications. Participants with a history of psychological or behavioral problems, or current physical illnesses were also excluded from the study. The study involved two sessions conducted on separate days in a psychophysiological stress laboratory. In the first ‘rest’ session, participants became accustomed to the laboratory and physiological monitors while completing a battery of questionnaires. The second (stress) session lasted approximately 2 hours, and included a baseline period (reading easy (grade K-2) books and watching G-rated movies and television shows), three stressors, and a recovery period (watching G-rated movies and television shows). Stressors included a speech task and a mental arithmetic task modified from the TSST-C as well as a mirror-tracing task. The speech task involved a preparation period and an academic speech in front of a two-person audience who rated the speaker for quality and accuracy. Speeches focused on academic topics (e.g. history, science, and book report) and were adjusted to the age of the participant. Participants were given 5 minutes to prepare and 5 minutes to deliver the speech, which was subsequently ‘judged’ by the experimenter and a second research assistant. The mental arithmetic task involved serial subtraction under time pressure in front of the same two-person audience, with difficulty adjusted by age. Finally, the mirror-tracing task (adapted from Allen & Matthews, 1997) involved tracing the figure of a six-sided star while viewing only its mirror image using a mirror star tracing apparatus (Layfayette Instruments, 1987). Mistakes were counted and marked by a sound and a white light. Seven to nine saliva samples were collected over the session and stored frozen at –70C until shipped overnight on dry ice to be assayed for cortisol and -amylase at Salimetrics Laboratories as described earlier. Cardiovascular measures (systolic and diastolic blood pressure and heart rate; SBP, DBP, HR) were assessed continuously and self-reported affect was assessed at six or seven time points throughout the sessions.

Results Data from two participants were excluded due to amylase values greater than 3 standard deviations from the mean, leaving 27 participants for most analyses. As shown in Figure 2, -amylase levels increased significantly over the course Downloaded from spr.sagepub.com by Douglas Maple on September 15, 2010

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of the stress session, F(5, 120) = 4.37, p < .01; there were also significant increases from baseline to maximum poststress -amylase levels, t(26) = 6.49, p < .001. Increasing age was also associated with increased -amylase reactivity to stress (defined as the difference between baseline and maximum poststress -amylase levels), r = .44, p < .05. To further examine the influence of age on -amylase responses over the stress session, participants were divided into younger (