Christine K. Fortunato

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.

Salivary cortisol mediates effects of poverty and parenting on executive functions in early childhood.

In a predominantly low-income population-based longitudinal sample of 1,292 children followed from birth, higher level of salivary cortisol assessed at ages 7, 15, and 24 months was uniquely associated with lower executive function ability and to a lesser extent IQ at age 3 years. Measures of positive and negative aspects of parenting and household risk were also uniquely related to both executive functions and IQ. The effect of positive parenting on executive functions was partially mediated through cortisol. Typical or resting level of cortisol was increased in African American relative to White participants. In combination with positive and negative parenting and household risk, cortisol mediated effects of income-to-need, maternal education, and African American ethnicity on child cognitive ability.

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.

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.

Focus on Methodology: Salivary bioscience and research on adolescence: An integrated perspective

The characterization of the salivary proteome and advances in biotechnology create an opportunity for developmental scientists to measure multi-level components of biological systems in oral fluids and identify relationships with developmental processes and behavioral and social forces. The implications for developmental science are profound because from a single oral fluid specimen, information can be obtained about a broad array of biological systems and the genetic polymorphisms related to their function. The purpose of this review is to provide a conceptual and tactical roadmap for investigators interested in integrating these measurement tools into research on adolescent health and development.

Salivary alpha-amylase and cortisol in toddlers: differential relations to affective behavior.

This study applies a minimally invasive and multi-system measurement approach (using salivary analytes) to examine associations between the psychobiology of the stress response and affective behavior in toddlers. Eighty-seven 2-year-olds (48 females) participated in laboratory tasks designed to elicit emotions and behavior ranging from pleasure/approach to fear/withdrawal. Saliva samples were collected pretask and immediately posttask, and assayed for markers of sympathetic nervous system (alpha-amylase or sAA) and hypothalamic-pituitary-adrenal axis (cortisol) activity. Individual differences in sAA were positively associated with approach behavior and positive affect; whereas, cortisol was positively associated with negative affect and withdrawal behavior. The findings suggest that individual differences in sAA may covary specifically with positive affect and approach behaviors or the predominant emotional state across a series of tasks. The results are discussed with respect to advancing biosocial models of the concomitants and correlates of young childrens affective behaviors.

Salivary flow and alpha-amylase: collection technique, duration, and oral fluid type.

There has been renewed interest in salivary alpha-amylase (sAA), a surrogate marker of autonomic/sympathetic activity, in biosocial research on stress vulnerability, reactivity, and recovery. This study explored the impact of saliva flow rate on sAA measurement by examining the influence of (1) the technique used to collect oral fluid-synthetic swab, cotton pledget, hydrocellulose microsponge, or passive drool; (2) collection point duration--the length of time the technique is employed (1-5min); and (3) oral fluid type--whole unstimulated saliva (not absorbed by any material) or oral fluid sampled from areas near the parotid, submandibular, or sublingual salivary glands. sAA activity (U/mL) was the highest in oral fluid collected from the parotid and submandibular gland areas. The volume (mL) of oral fluid collected increased, and the activity of sAA (U/mL) decreased, as collection point duration lengthened. The magnitude of these effects varied according to collection technique and oral fluid type. Across all conditions, there were positive correlations (range .70-.88) between sAA activity (U/mL) and sAA output (U/min). Management of these potential sources of measurement error will be essential to ensuring the success of future research on the correlates and concomitants of sAA activity, stress-related reactivity and recovery, and diurnal variation.

Associations between respiratory sinus arrhythmia reactivity and internalizing and externalizing symptoms are emotion specific

Internalizing and externalizing disorders are often, though inconsistently in studies of young children, associated with low baseline levels of respiratory sinus arrhythmia (RSA). RSA is thus considered to reflect the capacity for flexible and regulated affective reactivity and a general propensity for psychopathology. However, studies assessing RSA reactivity to emotional challenges tend to report more consistent associations with internalizing than with externalizing disorders, although it is unclear whether this is a function of the type of emotion challenges used. In the present study, we examined whether baseline RSA was associated with internalizing and/or externalizing severity in a sample of 273 young children (ages 5–6) with elevated symptoms of psychopathology. Following motivation-based models of emotion, we also tested whether RSA reactivity during withdrawal-based (fear, sadness) and approach-based (happiness, anger) emotion inductions was differentially associated with internalizing and externalizing symptoms, respectively. Baseline RSA was not associated with externalizing or internalizing symptom severity. However, RSA reactivity to specific emotional challenges was associated differentially with each symptom domain. As expected, internalizing symptom severity was associated with greater RSA withdrawal (increased arousal) during fearful and sad film segments. Conversely, externalizing symptom severity was related to blunted RSA withdrawal during a happy film segment. The use of theoretically derived stimuli may be important in characterizing the nature of the deficits in emotion processing that differentiate the internalizing and externalizing domains of psychopathology.

Aggression as an equifinal outcome of distinct neurocognitive and neuroaffective processes.

Early onset aggression precipitates a cascade of risk factors, increasing the probability of a range of externalizing and internalizing psychopathological outcomes. Unfortunately, decades of research on the etiological contributions to the manifestation of aggression have failed to yield identification of any risk factors determined to be either necessary or sufficient, likely attributable to etiological heterogeneity within the construct of aggression. Differential pathways of etiological risk are not easily discerned at the behavioral or self-report level, particularly in young children, requiring multilevel analysis of risk pathways. This study focuses on three domains of risk to examine the heterogeneity in 207 urban kindergarten children with high levels of aggression: cognitive processing, socioemotional competence and emotion processing, and family context. The results indicate that 90% of children in the high aggression group could be characterized as either low in verbal ability or high in physiological arousal (resting skin conductance). Children characterized as low verbal, high arousal, or both differed in social and emotional competence, physiological reactivity to emotion, and aspects of family-based contextual risk. The implications of this etiologic heterogeneity of aggression are discussed in terms of assessment and treatment.