Review of studies aimed at such objectives using sAA and C

Based on this theoretical framework, a number of relevant studies have focused on the specific interaction between day-to-day and life stressors and the HPA axis stress responses they elicited to predict the development of the first MDE. In 1996, Goodyer and colleagues showed that high levels of evening cortisol (at 20:00), low levels of salivary dehydroepiandrosterone (DHEA) in the morning (at 8:00), or a high cortisol/DHEA ratio in the evening or at midnight (at 20:00 or at 24:00) were present in 50% of children or adolescents diagnosed with a first MDE. These associations were not affected by age or gender differences in this cohort of 147 participants. Moreover, about 50% of these children and adolescents remained depressed at follow-up, 36 and 52 weeks later (^{Goodyer et al., 1996}; ^{Herbert et al., 1996}) and, in a smaller selection of this cohort, an association between evening cortisol and co-morbid dysthymia emerged (^{Goodyer, Herbert, & Altham, 1998}). In line with these seminal works, a delayed recovery (flat diurnal rhythm of cortisol) and possibly a reduced cortisol awakening response (CAR) (multiple measurements of morning cortisol levels with strict respect to waking time; see ^{Nater & Rohleder, 2009} for a review) could be considered as reliable indicators of a more general risk status and may be part of a commonly-used method for determining psychopathology that is also used by other research groups (^{Merwin, Smith, Kushner, Lemay & Dougherty, 2017}; ^{Nederhof et al., 2015}; ^{Van den Bergh & Van Calster, 2009}).

In contrast with these results, another body of evidence that is currently emerging has pointed out that high morning levels of cortisol could be considered a risk factor for a MDE. For example, in one of these studies, ^{Halligan, Herbert, Goodyer, & Murray (2004}) reported that adolescents (13 year-olds; n = 87) whose mothers were diagnosed with postpartum depression exhibited a greater number and/or more severe levels of depressive symptoms and an elevated CAR, but not elevated evening levels of cortisol. These differences were accompanied by more variability in cortisol levels relative to controls during the observation period. In a later phase of the same study, these authors reported that children of mothers diagnosed with postpartum depression exhibited higher levels of morning cortisol at 13 years of age, which was the factor that best predicted the level of depressive symptomology at the age of 16. These associations were independent of confounders such as gender, BMI, and Tanner Stages (^{Halligan, Herbert, Goodyer, & Murray, 2007}). Thus, depressive symptoms observed during adolescence were statistically mediated by morning cortisol levels at the age of 13. A closely-related finding in female participants was recently reported by ^{Adam et al. in 2010}. ^{Dietrich et al. (2013}) confirmed this same association between morning cortisol levels and CAR with depressive problems in a cohort of 1604 adolescents. Nevertheless, the relationship was weaker than previously assumed and, in analyses of the clinical subsample, the association was found to be stronger among male participants. A similar conclusion was reached by ^{Vrshek-Schallhorn et al. (2013}) and ^{Nelemans et al. (2014}). Finally, ^{Keenan et al. (2013}) studied the specific association between the cortisol response to a laboratory stressor and depressive symptomatology in a cohort of 232 girls at 10 years and 12 years of age and their mothers. They reported that associations between HPA axis functioning and depression emerged around age 12.

Recent findings in this particular field of research have demonstrated that higher morning cortisol levels could also be associated with a greater risk of depression even in much younger children. For example, ^{Dougherty, Klein, Olino, Dyson, & Rose (2009}) reported that postpartum depression in mothers was associated with a higher level of cortisol and lower positive emotional temperament in 94 healthy boys and girls (aged 3-4 years). This result was independent of confounders such as age or gender. In a subsequent study, ^{Dougherty et al. (2013)} reported that higher negative emotional temperament at age three predicted high morning cortisol levels three years later. Interestingly, these authors proposed that these psychobiosocial characteristics could, as a whole, increase the risk of triggering a future MDE.

Although the studies reviewed in the previous paragraphs highlight the usefulness of cortisol in particular (especially when it is measured in form of the CAR) as an early predictor of a future triggering of a first MDE in adolescents (without gender differences in the majority of the reviewed studies), a recent and robust epidemiological study that examined a wide cohort showed that the predictive capacity of morning cortisol levels could be influenced by gender, thereby increasing misunderstanding of these initial results (^{Owens et al., 2014}). Specifically, these researchers observed that in the total sample of 1858 participants, morning cortisol was associated with a high risk of depression exclusively in male participants. Given the higher prevalence of depression in adolescent girls (^{Domènech-Llaberia et al., 2009}; ^{Fonseca-Pedrero, Paino, Lemos-Giráldez, & Muñiz, 2011}; ^{Mendes et al., 2012}; ^{Weissman et al., 2006}), these groups of results (^{Lewis, Jones, & Goodyer, 2016}; ^{Nelemans et al., 2014}; ^{Owens et al., 2014}) should alert us of the need to shift the focus of our attention towards the search for further sensitive biomarkers of early depression risk in girls. In line with this search for new, more precise biological markers of depression, the usefulness of sAA as a low-cost, biological marker of psychopathology has become a relevant new line of research, especially over the two last decades. Unfortunately, the results published on the utility of the association of sAA activity/output with depressive symptoms and/or symptoms of anxiety specifically are still scarce and inconclusive in adult and child populations (^{Bagley, Weaver, & Buchanan, 2011}; ^{Braithwaite, Ramchamandani, Lane, & Murphy, 2015}; ^{Ishitobi et al., 2010}; ^{Tanaka et al., 2012}; ^{Veen et al., 2013}). Interestingly, the only study on this issue to focus on a child and adolescent population showed an inverse relationship between baseline sAA activity and depressive symptoms in male adolescent survivors of Hurricane Katrina (^{Vigil, Geary, Granger, & Flinn, 2010}). As in the previously-reviewed studies that use C, the full meaning of Vigil et al.’s results will be made clear only by understanding the multiple interactions among genetic background, life stressors, and gender. Likewise, in line with the search for more sensitive biomarkers of depression risk, and according to principles of the interactive model of the function of the stress system, ^{Bauer, Quas, & Boyce (2002}) have suggested that the use of sAA/cortisol and/or cortisol/sAA ratios could be more sensitive biological measurements of anxious and depressive feelings associated with these internalizing disorders in comparison with a single measurement of any or both aspects of this biological response. (^{Ali & Pruessner, 2012}; ^{Bauer et al., 2002}; ^{Blair, Granger, & Razza, 2005}; ^{Vigil et al., 2010}).

Based on all the aforementioned results, the main aim for our study was to estimate the potential relative usefulness of morning cortisol, sAA activity, sAA output, and the ratios of sAA activity-output/cortisol in predicting levels of depressive symptoms in a community sample composed of 54 girls and 45 boys aged 8-11 years.

Participants

Participants in our cross-sectional study (n = 99) were selected from an initial pool of 116 children attending a public school in the metropolitan area of Malaga (Spain). According to parental reports, all of the children were in good general health with the exception of 17, who were excluded from this study because they were taking prescription medication with endocrine, immunological, or neurological effects or had a major illness. Thus, the final sample comprised 45 boys and 54 girls. The study was conducted according to the principles set forth in the Declaration of Helsinki. Informed consent was obtained in writing from the parents (fathers and/or mothers) of each participant in our study.

Procedure

Before data collection, we organized a briefing for the children’s parents and provided them all the necessary information about the aims of this research. Special attention was paid to the following four points: (a) instruction of parents and children on the correct timing of saliva sampling on awakening and +30 min later; (b) avoidance of food or liquids (except water) and refraining from brushing teeth during the time of saliva sampling; (c) appropriate conservation of saliva samples in the home freezer; and (d) a record sheet kept by the parents to note any violations of this protocol. Parents filled out a questionnaire which provided anthropometric and socio-demographic data on their child. Home saliva samples were collected over two typical school days (freely chosen by the parents, but always in the same week before psychological assessment) along with information about the sleeping habits of the participant.

Psychological measures

We employed the Achenbach System of Empirically Based Assessment (ASEBA) for school-age children and adolescents to assess the children’s emotional and/or behavioral problems and social competencies. In this study, only the results from the internalizing scales of the Teacher’s Report Form (TRF) were used. Only those teachers who had a minimum of one academic year of experience with the student provided information on the behavior of each participant. The name and composition of the three internalizing subscales of TRF are: anxious/depressed (Cronbach’s α = 0.97; indicated as TRF_anxiety in Tables 1, (2, and 3) with 8 items; withdrawn/depressed (Cronbach’s α = 0.79; shown as TRF_{depression-withdrawal} in Tables 1, (2, and 3) with 8 items, and somatic complaints (Cronbach’s α = 0.97; indicated as TRF_{psychosomatic} in Tables 1, 2, and 3) with 9 items. These three subscales were combined to calculate an overall score for internalizing symptoms (Cronbach’s α = 0.97; indicated as TRF_total in Tables 1, (2, and 3).

Physiological measurements and data reduction

Saliva samples were obtained using the passive method over a two-minute time period on two days (^{Navazesh, 1993}). On both days, two saliva samples were collected at home. The subjects were instructed on both mornings to avoid eating or brushing their teeth until the sampling had been completed. The first sample was taken immediately after awakening and the second was taken 30 minutes later. Parents supervised the children in the collection and conservation of samples, which were immediately frozen at home at -20º C and picked up later that day. For free cortisol measurement, 500 (l of saliva were taken for analysis using an automatized assay system (salivary cortisol on ROCHE Elecsys immunoassay system; see ^{Chiu, Collier, Clark, & Wynn-Edwards, 2003}, and ^{Vogeser, Durner, Seliger, & Auernhammer, 2006}, for more details). This system does not require duplicate analysis of each saliva sample. Cortisol levels were expressed in nmol/l. For the sAA activity and output assays, an enzymatic colorimetric assessment was used (see ^{Maldonado et al., 2008}, and ^{Sánchez-Navarro, Maldonado, Martínez-Selva, Enguix, & Ortiz, 2012}, for more details). sAA activity is considered as a measurement of the enzymatic activity of this enzyme in saliva (U/ml) whereas sAA output is a measurement of the secretion of this enzyme over time (U/min). Intra- and inter-CV were not calculated for cortisol or sAA in this study.

For statistical analyses, we reduced the raw salivary data. Only those participants who had collected all four samples were included in the statistical analyses. Salivary measurements of α-amylase activity and output, cortisol, delta cortisol (defined as the difference between the +30 min measurement of cortisol minus the awakening time cortisol measure), and the sAA/cortisol ratio were averaged over the two days of observation using the four data measurements from each participant. In case of violation of the normality assumption, the data were square-root transformed (in the case of psychological measurements) or natural-log transformed (ln(x-1) in the case of sAA activity, output, and sAA/cortisol ratio). The less-drastic data transformation was always employed, pursuant to the indications of ^{Tabachnick and Fidell, 2013}. The cortisol and delta cortisol data did not require any transformation.

Statistical analyses and data reduction

Our statistical analyses were conducted using a two-step strategy:

(1) First, descriptive statistics for age, anthropometric, socio-demographic (parental education and income), perceived socioeconomic status (SES) with respect to the country or city, psychological (TRF scores) and salivary measurements for the total sample and according to gender were calculated.

(2) For data reduction, we carried out the following steps:

(2.1.) Initially, we conducted an exploratory analysis to assess whether internalizing symptoms scores were linked to salivary markers through bivariate partial correlations (after controlling for age and BMI as indirect indicators of development) for the full sample and, separately, for both genders.

(2.2.) We then conducted various simple linear regression analyses (using the stepwise method) on the total sample to determine the relative predictive power of the analytes in question on TRF scores. After this step, and in accordance with our results, we specifically tested the moderating role of gender on the associations between sAA activity and the depression-withdrawal subscale of TRF.

Data were analyzed using the Statistical Package for Social Science version 22 (SPSS Institute, Chicago, IL). For all analyses, p-values < .05 were considered significant. Unless otherwise indicated, all results shown in figures are mean ± standard deviation (SD) (obtained from untransformed data) to facilitate comparison with other studies.

Association of internalizing psychopathology scores with salivary measures

Statistically significant partial Pearson product-moment correlation coefficients were observed among two subscales of TRF (the depression-withdrawal subscale and the total internalizing score in TRF) and mean levels of morning sAA activity (r = -.213, p < .05, n = 99; r = -.222; p < .05, n = 99, respectively). Moreover, various moderately-statistically significant partial Pearson product-moment correlation coefficients between TRF subscores and the salivary measures emerged when we repeated the same analysis exclusively on the subset of girls (Table 2). Thus, the depression-withdrawal subscore of TRF was inversely related to different salivary measurements (Mean sAA activity: r = -.343, p < .05, n = 54; Mean ratio sAA activity/cortisol: r = -.314, p < .05, n = 54). Nevertheless, these analyses yielded negative results in the subset of boys (Table 3).

Table 2: Partial correlations between Teacher’s Report Form scores and salivary markers after controlling for age and body mass index (BMI) in girls (n = 54). 

Table 3: Partial correlations between Teacher’s Report Form scores and salivary markers after controlling for age and body mass index (BMI) in boys (n = 45). 

Simple linear regression models of salivary measures on TRF scores

To determine the best salivary predictor of each TRF internalizing subscale in the sample as a whole, we performed simple linear regression analyses using the stepwise method for all salivary biomarkers on each of the TRF internalizing subscales. In the full sample, sAA activity fit best with the depression-withdrawal TRF subscore and the total TRF score. In Model 1 (β = -.213, t = -2.149, p < .05), sAA activity [F (1,97) = 4.620, p < .05] explained 4.5% of the total variance in the depression-withdrawal subscore of TRF (R ² = .045, Tolerance = 1, VIF = 1, Condition Index = .097; see Figure 1) whereas in Model 2 (β = -.221, t = -2.232, p < .05), sAA activity [F (1,97) = 4.980, p < .05] explained 4.9% of the total variance in the total score of TRF (R ² = .049, Tolerance = 1, VIF = 1, Condition Index = .097).

Figure 1 Square-root transformed values of morning salivary alpha-amylase activity on square-root transformed values of the TRF depression-withdrawal subscale (n = 99). 

According to the different results obtained in our exploratory analyses for boys and girls described in the previous section, we examined the possible moderating role of gender in regards to the association between sAA activity and depressive symptoms. The interaction analysis (Model 3) was not statistically significant for sAA activity (β = -.014, SE = .064, Beta = - .031, t = -.222, p = .825), gender (β = .687, SE = .663, Beta = .315, t = 1.036, p = .303), or for the interaction of sAA activity by gender (β = -0.150, SE = 0.093, Beta = -0.539, t = - 1.616, p = .109). In sum, low levels of sAA activity were associated with an increased score in depressive symptoms and total score on the TRF in the whole sample, and gender was not a moderator of the association between sAA activity and TRF scores despite the differences observed in our previous exploratory analyses.