Leah C. Hibel

Integration of salivary biomarkers into developmental and behaviorally-oriented research: Problems and solutions for collecting specimens

Saliva has been championed as a diagnostic fluid of the future. Much of the attention that saliva receives as a biological specimen is due to the perception that the nature of sample collection is quick, uncomplicated, and non-invasive. In most cases, this perception matches reality; however, in some special circumstances and populations collecting saliva can be unexpectedly difficult, time consuming, and may not yield sufficient sample volume for assay. In this report, we review the nature and circumstances surrounding some of these problems in the context of developmental science and then present alternatives that can be used by investigators to improve the next generation of studies. We expect our findings will ease the burden on research participants and assistants, reduce the rate of missing values in salivary data sets, and increase the probability that salivary biomarkers will continue to be successfully integrated into developmental and behaviorally-oriented research.

Medication effects on salivary cortisol: Tactics and strategy to minimize impact in behavioral and developmental science

The non-invasive measurement of cortisol in saliva has enabled behavioral scientists to explore the correlates and concomitants of the interaction between the activity of the hypothalamic-pituitary-adrenal (HPA) axis, intrinsic factors, and social forces as they occur naturally in everyday life. The widespread integration of salivary cortisol into behavioral science has also revealed that omnipresent features of everyday life such as, over-the-counter and prescription medications, have the capacity to influence measurement validity. We identify several pathways by which pharmacologic agents could influence salivary cortisol, including (a) direct agonistic and antagonistic effects on the HPA axis, (b) indirect effects on physiological systems networked with the HPA axis, (c) moderation or mediation effects on cortisol secretion via pharmacologically induced change in subjective experience, (d) iatrogenic effects on the availability or composition of saliva, or the diffusion of serum constituents into oral fluid, and (e) cross-reactivities with antibodies used to detect cortisol by immunoassay. Specific medications with the capacity to influence salivary cortisol via these pathways are documented in an effort to procedurally and statistically minimize this potential source of error variance in the next generation of studies.

Allostasis and allostatic load in the context of poverty in early childhood.

This paper examined the relation of early environmental adversity associated with poverty to child resting or basal level of cortisol in a prospective longitudinal sample of 1135 children seen at 7, 15, 24, 35, and 48 months of age. We found main effects for poor housing quality, African American ethnicity, and low positive caregiving behavior in which each was uniquely associated with an overall higher level of cortisol from age 7 to 48 months. We also found that two aspects of the early environment in the context of poverty, adult exits from the home and perceived economic insufficiency, were related to salivary cortisol in a time-dependent manner. The effect for the first of these, exits from the home, was consistent with the principle of allostatic load in which the effects of adversity on stress physiology accumulate over time. The effect for perceived economic insufficiency was one in which insufficiency was associated with higher levels of cortisol in infancy but with a typical but steeper decline in cortisol with age at subsequent time points.

Maternal and Child Contributions to Cortisol Response to Emotional Arousal in Young Children from Low-Income, Rural Communities.

Relations of maternal and child characteristics to child cortisol reactivity to and recovery from emotional arousal were examined prospectively at approximately 7 months of age (infancy) and then again at approximately 15 months of age (toddlerhood). The sample was diverse and population based (N = 1,292 mother-infant dyads) and included families from predominantly low-income, rural communities. Maternal behavior, family income-to-need ratio and social advantage, and child temperament, attention, and mental development were assessed, and childrens saliva was sampled before and after standardized procedures designed to elicit emotional arousal. Maternal engagement in infancy was associated with greater cortisol reactivity at the infancy assessment and with reduced overall cortisol level at the toddler assessment. Also at the toddler assessment, child attention, mental development, and temperamental distress to novelty were associated with increased cortisol reactivity and regulation, whereas temperamental distress to limitations and African American ethnicity were associated with reduced cortisol reactivity. Findings are consistent with prior work linking early caregiving to the development of the hypothalamic-pituitary-adrenal axis stress response system and with a conceptual model in which developing temperament is characterized by the interplay of emotional reactivity and the emergence of the ability to effortfully regulate this reactivity using attention.

Intimate Partner Violence Moderates the Association Between Mother-Infant Adrenocortical Activity Across an Emotional Challenge

This study examined the relationship between mother and infant adrenocortical levels and reactivity to an emotion eliciting task. The impact of intimate partner violence (IPV) on these relationships was assessed as a moderator. The sample (n = 702 mother-infant dyads) was racially diverse and from predominantly low-income, rural communities. During a home visit, the dyads saliva was sampled before, 20 min, and 40 min after standardized tasks designed to elicit the infants emotional arousal and later assayed for cortisol. Mothers completed self-report measures of their partners violence, and parenting behaviors were assessed via structured interview and mother-child interactions. In response to the task, infants had positive, and mothers had negative, cortisol slopes. Contrary to expectations, there were no IPV-related differences in mean pretask cortisol levels or reactivity in the mothers or infants. Mother-infant dyads from households characterized by either (1) violence or (2) restrictive and punitive parenting behaviors exhibited correlated cortisol reactivity measured in response to the infant challenge task. The findings suggest that social contextual features of the early caregiving environment may influence individual differences in the coordination between maternal and infant adrenocortical reactivity.

Blood contamination in children's saliva: Prevalence, stability, and impact on the measurement of salivary cortisol, testosterone, and dehydroepiandrosterone

The prevalence, stability, and impact of blood contamination in childrens saliva on the measurement of three of the most commonly assayed hormones were examined. Participants were 363 children (47% boys; ages 6-13 years) from economically disadvantaged families who donated saliva samples on 2 days in the morning, midday, and late afternoon. Samples (n=2178) were later assayed for cortisol (C), testosterone (T), and dehydroepiandrosterone (DHEA). To index the presence of blood (and its components) in saliva, samples were assayed for transferrin. Transferrin levels averaged 0.37 mg/dl (SD=0.46, range 0.0-5.5, Mode=0), and were: (1) highly associated within individuals across hours and days, (2) positively correlated with age, (3) higher for boys than girls, (4) higher in PM than AM samples, and (5) the highest (>1.0 mg/dl) levels were rarely observed in samples donated from the same individuals. Transferrin levels were associated with salivary DHEA and C, but less so for T. As expected, the relationships were positive, and explained only a small portion of the variance. Less than 1% of the statistical outliers (+2.5 SDs) in salivary hormone distributions had correspondingly high transferrin levels. We conclude that blood contamination in childrens saliva samples is rare, and its effects on the measurement of salivary hormones is small. Guidelines and recommendations are provided to steer investigators clear of this potential problem in special circumstances and populations.

Individual differences in salivary cortisol : Associations with common over-the-counter and prescription medication status in infants and their mothers

The purpose of the present study was to describe associations between the use of common over-the-counter (OTC) and prescription medications with individual differences in salivary cortisol in infants and their mothers. Participants were 1020 mothers and 852 infants (52.5% boys; ages 5.03-13.44 months) from economically disadvantaged and ethnically diverse families (38.4% African American) who donated saliva samples before, 20 and 40 min after infants participated in a series of challenging tasks. Samples (N=5616) were later assayed for cortisol. Medication information was content analyzed separately for infants (e.g., teething gels, nonsteroidal anti-inflammatory drugs, acetaminophen, decongestants) and mothers (e.g., narcotics, antidepressants, antipsychotics, contraceptives, glucocorticoids). A large percentage of infants (44%) and the majority of mothers (57.5%) had used at least one medication (range 0-4) in the previous 48 h. Most frequent were acetaminophen (e.g., Tylenol) and cold medications (e.g., decongestants) for infants and contraceptives and acetaminophen for mothers. Compared to infants not taking any medications, cortisol reactivity to the challenge tasks was less pronounced for infants taking acetaminophen. Cortisol levels were higher for mothers taking oral or transdermal contraceptives and acetylsalicylic acid (e.g., Aspirin) but lower for mothers taking pure agonist opioids (e.g., Oxycontin) compared to mothers not taking any medications. These medication-related differences remained significant after controlling for sampling time, fever, maternal anxiety and depression, infant temperament, ethnicity, SES, and health status. Recommendations are provided to steer investigators clear of these potential sources of unsystematic error variance in salivary cortisol.

Maternal sensitivity buffers the adrenocortical implications of intimate partner violence exposure during early childhood

This study prospectively examined the effect of intimate partner violence (IPV) on adrenocortical reactivity and recovery during early childhood. The sample (n = 1102 mother-infant dyads; 49.2% male) was racially diverse and from predominantly low-income, rural communities. To measure IPV exposure mothers completed the Conflicts Tactics Scale, and her caretaking behaviors were observed when her child was approximately 7, 15, and 24 months of age. Childrens saliva samples, later assayed for cortisol, were collected around challenge tasks designed to elicit emotional reactivity. IPV was related to a trajectory of increased cortisol reactivity from infancy to toddlerhood. By contrast, the trajectory for non-IPV-exposed children decreased in cortisol reactivity across 7 to 24 months of age. At the 24-month assessment, on average, toddlers did not exhibit a cortisol reaction; however, those exposed to high levels of violence continued to have reactivity. Accumulative levels of IPV across the first 2 years of life predicted cortisol reactivity at 24 months of age. Early (7-month) sensitive maternal behavior moderated this relationship, so that only children exposed to both early insensitivity and high accumulated IPV exhibited increased reactivity at the 24-month assessment. Findings are discussed in relation to the risky family framework.

Parenting stressors and morning cortisol in a sample of working mothers.

The cortisol awakening response (CAR) is a normative rise in cortisol levels across the 30 minutes post awakening. Both the levels and the degree of change in cortisol across this time period are sensitive to the perceived challenges of the day and are thought to prepare the individual to meet these tasks. However, working parents of young children may be under unique strains at this time as they attempt to simultaneously care for their children while also preparing themselves for the workday ahead. In these analyses we examined the contributions of both work and parenting stress on maternal cortisol levels and awakening responses, and how these relationships differed on workdays compared with nonworkdays. To do this, saliva samples were collected from 56 working mothers (25% single) with a child between the ages of 2 and 4 years old (mode = 2 children), at awakening and 30 min postawakening. Samples were collected on 4 consecutive days-2 nonworkdays followed by 2 workdays. Analyses revealed mothers reporting higher levels of parenting stress had higher average a.m. cortisol on workdays compared with nonworkdays. Further, mothers reporting a combination of high job strain and high parenting stress had significantly higher cortisol levels and steeper CAR increases on workdays compared with nonworkdays. Findings are discussed by integrating knowledge from the fields of parenting stress, work-family, and stress physiology.

Relationship of Salivary Alpha Amylase and Cortisol to Social Anxiety in Healthy Children Undergoing Laboratory Pain Tasks.

Objective Salivary alpha amylase (sAA) has been shown to be a sensitive and reliable marker of the autonomic nervous system (ANS) response to stress. A link between sAA, cortisol, and social/evaluative stress has been established in youth, but little is known about these relationships in response to other stressors in children, and how social anxiety might moderate these relationships. The current study explored the associations among sAA and salivary cortisol responses to laboratory pain tasks and self-reported social anxiety symptoms in a sample of healthy children. Method Two hundred thirty-one children (114 girls; 49.4%) with a mean age 12.68 years (SD=3.0; range 7–18) participated in the study. Participants completed self-report questionnaires prior to undergoing a series of laboratory pain tasks involving cold, pressure, and heat pain. Saliva samples were collected upon arrival to the laboratory (pre-task), following the completion of the pain tasks (post-task1), and 20 minutes after the completion of the pain tasks (post-task2). Results Demographic factors (age, sex, pubertal stage) did not predict either sAA or cortisol levels. However, children reporting higher levels of social anxiety demonstrated significantly higher sAA but not cortisol levels across three salivary collection times, compared to children reporting lower levels of social anxiety. Further, it does not appear that reduced state levels of anxiety before or during the tasks buffer this relationship. Conclusion These data highlight the possibility of identifying biomarkers of stress that are consistent across time and developmental stage. sAA appears to be a marker of stress response in children with self-reported social anxiety. There may also be a potentially unique relationship of sAA to stress in this population. In addition, sAA may reflect stable individual differences in levels of ANS arousal and may be a useful biomarker for identifying children at risk for stress.