Salivary uric acid across child development and associations with weight, height, and body mass index.

Introduction: Obesity during childhood is a serious and growing chronic disease with consequences for lifelong health. In an effort to advance research into the preclinical indicators of pediatric obesity, we examined longitudinal assessments of uric acid concentrations in saliva among a cohort of healthy children from age 6-months to 12-years (n's per assessment range from 294 to 727). Methods: Using data from a subsample of participants from the Family Life Project (an Environmental influences on Child Health Outcomes Program cohort), we: (1) characterized salivary uric acid (sUA) concentrations from infancy to early adolescence by sex and race; (2) assessed changes in sUA levels across development; and (3) evaluated associations between sUA concentrations and measures of child weight, height, and body mass index (BMI). Across four assessments conducted at 6-, 24-, 90-, and 154-months of age, 2,000 saliva samples were assayed for UA from 781 participants (217 participants had sUA data at all assessments). Results: There were no significant differences in sUA concentrations by sex at any assessment, and differences in sUA concentrations between White and non-White children varied by age. At the 90- and 154-month assessments, sUA concentrations were positively correlated with measures of child weight, height, and BMI (90-month: weight- ρ(610) = 0.13, p < 0.01; height- ρ(607) = 0.10, p < 0.05; BMI- ρ(604) = 0.13, p < 0.01; 154-month: weight- ρ(723) = 0.18, p < 0.0001; height- ρ(721) = 0.10, p < 0.01; BMI- ρ(721) = 0.17, p < 0.0001). Group based trajectory modeling identified two groups of children in our sample with distinct patterns of sUA developmental change. The majority (72%) of participants showed no significant changes in sUA across time ("Stable" group), while 28% showed increases in sUA across childhood with steep increases from the 90- to 154-month assessments ("Increasing" group). Children in the Increasing group exhibited higher sUA concentrations at all assessments (6-month: t(215) = -5.71, p < 0.001; 24-month: t(215) = -2.89, p < 0.01; 90-month: t(215) = -3.89, p < 0.001; 154-month: t(215) = -19.28, p < 0.001) and higher weight at the 24- and 90-month assessments (24-month: t(214) = -2.37, p < 0.05; 90-month: t(214) = -2.73, p < 0.01). Discussion: Our findings support the potential utility of sUA as a novel, minimally-invasive biomarker that may help advance understanding of the mechanisms underlying obesity as well as further surveillance and monitoring efforts for pediatric obesity on a large-scale.

Sex-related Differences in Stress Reactivity and Cingulum White Matter.

The prefrontal cortex and limbic system are important components of the neural circuit that underlies stress and anxiety. These brain regions are connected by white matter tracts that support neural communication including the cingulum, uncinate fasciculus, and the fornix/stria-terminalis. Determining the relationship between stress reactivity and these white matter tracts may provide new insight into factors that underlie stress susceptibility and resilience. Therefore, the present study investigated sex differences in the relationship between stress reactivity and generalized fractional anisotropy (GFA) of the white matter tracts that link the prefrontal cortex and limbic system. Diffusion weighted images were collected and deterministic tractography was completed in 104 young adults (55 men, 49 women; mean age = 18.87 SEM = 0.08). Participants also completed self-report questionnaires (e.g., Trait Anxiety) and donated saliva (later assayed for cortisol) before, during, and after the Trier Social Stress Test. Results revealed that stress reactivity (area under the curve increase in cortisol) and GFA of the cingulum bundle varied by sex. Specifically, men demonstrated greater cortisol reactivity and greater GFA within the cingulum than women. Further, an interaction between sex, stress reactivity, and cingulum GFA was observed in which men demonstrated a positive relationship while women demonstrated a negative relationship between GFA and cortisol reactivity. Finally, trait anxiety was positively associated with the GFA of the fornix/stria terminalis - the white matter pathways that connect the hippocampus/amygdala to the hypothalamus. These findings advance our understanding of factors that underlie individual differences in stress reactivity.

Associations Between Secretory Immunoglobulin A and Social Network Structure.

PURPOSE: This study investigates the social determinants of health by examining how mucosal immunity is associated with the patterning of social connections in a network. Studies have suggested that social networks have biological underpinnings, but investigations at the scale of networks, rather than individuals, have remained elusive. We integrate salivary bioscience methods with advanced social network modeling to explore the association between salivary secretory immunoglobulin A (SIgA), a key component of mucosal immunity, and social network structure. METHOD: Friendship network data and saliva samples (later assayed for SIgA) were obtained from a large mixed-gender social organization (n = 155, 55% female, M age = 19.5 years). RESULTS: Exponential random graph modeling revealed that SIgA levels were positively associated with reporting more friendship ties with community members (i.e., social network activity), after controlling for other processes associated with network structure including preference to befriend others of the same age, gender, and extraversion, increased network popularity of agreeable individuals and those with lower levels of perceived stress, as well as network structural and organizational processes. CONCLUSION: By examining a wider range of associations between SIgA and network structure, we pinpoint that SIgA is positively associated with respondent's sociability. Our findings are consistent with social integration theories linking social relationships to health and highlight the role of humoral immunity as a possible mediator of these associations.

Adrenocortical regulation, eating in the absence of hunger and BMI in young children.

The purpose of this study was to examine relations among adrenocortical regulation, eating in the absence of hunger, and body mass index (BMI) in children ages 5-9years (N=43). Saliva was collected before and after the Trier Social Stress Test for Children (TSST-C), and was later assayed for cortisol. Area under the curve with respect to increase (AUCi) was used as a measure of changes in cortisol release from baseline to 60min post-TSST-C. Age- and sex-specific BMI scores were calculated from measured height and weight, and eating in the absence of hunger was assessed using weighed food intake during a behavioral procedure. We also included a measure of parents' report of child impulsivity, as well as family demographic information. Participants were stratified by age into younger (5-7years) and older (8-9years) groups. In younger children, parents' reports of child impulsivity were significantly and positively associated with BMI; cortisol AUCi was not associated with BMI or eating in the absence of hunger. In older children, however, greater stress-related cortisol AUCi was related to higher BMI scores and greater energy intake in the absence of hunger. The results suggest that cortisol AUCi in response to psychosocial stress may be linked to problems with energy balance in children, with some variation by age.

Defining MAP3 kinases required for MDA-MB-231 cell tumor growth and metastasis.

Analysis of patient tumors suggests that multiple MAP3 kinases (MAP3Ks) are critical for growth and metastasis of cancer cells. MAP3Ks selectively control the activation of extracellular signal-regulated kinase 1/2 (ERK1/2), Jun N-terminal kinase (JNK), p38 and ERK5 in response to receptor tyrosine kinases and GTPases. We used MDA-MB-231 cells because of their ability to metastasize from the breast fat pad to distant lymph nodes for an orthotopic xenograft model to screen the function of seven MAP3Ks in controlling tumor growth and metastasis. Stable short hairpin RNA (shRNA) knockdown was used to inhibit the expression of each of the seven MAP3Ks, which were selected for their differential regulation of the MAPK network. The screen identified two MAP3Ks, MEKK2 and MLK3, whose shRNA knockdown caused significant inhibition of both tumor growth and metastasis. Neither MEKK2 nor MLK3 have been previously shown to regulate tumor growth and metastasis in vivo. These results demonstrated that MAP3Ks, which differentially activate JNK, p38 and ERK5, are necessary for xenograft tumor growth and metastasis of MDA-MB-231 tumors. The requirement for MAP3Ks signaling through multiple MAPK pathways explains why several members of the MAPK network are activated in cancer. MEKK2 was required for epidermal growth factor receptor and Her2/Neu activation of ERK5, with ERK5 being required for metastasis. Loss of MLK3 expression increased mitotic infidelity and apoptosis in vitro. Knockdown of MEKK2 and MLK3 resulted in increased apoptosis in orthotopic xenografts relative to control tumors in mice, inhibiting both tumor growth and metastasis; MEKK2 and MLK3 represent untargeted kinases in tumor biology for potential therapeutic development.

Regulation of mitochondrial sn-glycerol-3-phosphate acyltransferase activity: response to feeding status is unique in various rat tissues and is discordant with protein expression.

Triacylglycerol plays a critical role in an organism's ability to withstand fuel deprivation, and dysregulation of triacylglycerol synthesis is important in the development of diseases such as obesity and diabetes. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step of glycerolipid synthesis and is therefore a potential site for regulation of triacylglycerol synthesis. Because several studies suggest that triacylglycerol synthesis is linked to the mitochondrial isoform, we studied mitochondrial GPAT expression and the effect of feeding status on the regulation of mitochondrial GPAT in various rat tissues. Liver, adipose, and soleus muscle have high levels of GPAT mRNA, but low protein expression, whereas heart and adrenal, tissues with low GPAT mRNA abundance, have the highest GPAT protein expression. In addition, heart, which has the highest expression of mitochondrial GPAT protein, has low mitochondrial GPAT specific activity (0.02 nmol/min/mg). Liver and adipose have the highest mitochondrial GPAT specific activity (0.17 nmol/min/mg), but very low protein expression. Discrepancies between GPAT protein expression and activity suggest that mitochondrial GPAT may be regulated acutely. In response to a 48-h fast, liver and adipose mitochondrial GPAT protein expression and activity decrease 30-50%. After 24-h refeeding of either chow or high-sucrose diet, mitochondrial GPAT protein expression and activity overshoot normal levels 30-60%. In kidney, mitochondrial GPAT protein and activity increase 65 and 30%, respectively, with refeeding, whereas in the heart, mitochondrial GPAT activity increases 2.3-fold after a fast, with no change in protein expression. We also found that hepatic mitochondrial GPAT activity in the neonatal rat constitutes a lower percentage of the total GPAT activity than in the adult. We postulate that GPAT expression is modulated uniquely in each tissue according to specific needs for triacylglycerol storage.

Mitochondrial glycerol phosphate acyltransferase contains two transmembrane domains with the active site in the N-terminal domain facing the cytosol.

The topography of mitochondrial glycerol-3-phosphate acyltransferase (GPAT) was determined using rat liver mitochondria and mutagenized recombinant rat GPAT (828 aa (amino acids)) expressed in CHO cells. Hydrophobicity analysis of GPAT predicts two transmembrane domains (TMDs), residues 472-493 and 576-592. Residues 224-323 correspond to the active site of the enzyme, which is believed to lie on the cytosolic face of the outer mitochondrial membrane. Protease treatment of rat liver mitochondria revealed that GPAT has a membrane-protected segment of 14 kDa that could correspond to the mass of the two predicted TMDs plus a loop between aa 494 and 575. Recombinant GPAT constructs containing tagged epitopes were transiently expressed in Chinese hamster ovary cells and immunolocalized. Both the C and N termini epitope tags could be detected after selective permeabilization of only the plasma membrane, indicating that both termini face the cytosol. A 6-8-fold increase in GPAT-specific activity in the transfected cells confirmed correct protein folding and orientation. When the C terminus and loop-tagged GPAT construct was immunoassayed, the epitope at the C terminus could be detected when the plasma membrane was permeabilized, but loop-epitope accessibility required disruption of the outer mitochondrial membrane. Similar results were observed when GPAT was truncated before the second TMD, again consistent with an orientation in which the loop faces the mitochondrial intermembrane space. Although protease digestion of the HA-tagged loop resulted in preservation of a 14-kDa fragment, consistent with a membrane protected loop domain, neither the truncated nor loop-tagged enzymes conferred GPAT activity when overexpressed, suggesting that the loop plays a critical structural or regulatory role for GPAT function. Based on these data, we propose a GPAT topography model with two transmembrane domains in which both the N (aa 1-471) and C (aa 593-end) termini face the cytosol and a single loop (aa 494-575) faces the intermembrane space.

Developmental effects of early immune stress on aggressive, socially reactive, and inhibited behaviors.

The origins of individual differences in social development are examined in relation to early stress (immune challenge) and social milieu (maternal behavior) in a genetic-developmental analysis using an animal model. Neonatal male mice (5 or 6 days of age) from two lines of mice selectively bred for high versus low levels of inter-male aggressive behavior received a standard immune challenge (i.p. injections of 0.05 mg/kg endotoxin or saline). Animals were reared by their line-specific biological dam or by a foster dam from a line bred without selection. Adult levels of social behaviors were assessed in a dyadic test (age 45-50 days). Mice from the high-aggressive line show more developmental sensitivity to immune challenge than mice from the low-aggressive line, and line differences persist regardless of the early maternal environment. As adults, endotoxin-treated mice from the high-aggressive line have lower levels of aggressive behavior, longer latency to attack, and higher rates of socially reactive and inhibited behaviors compared to saline controls. Developmental effects of endotoxin in the low-aggressive line are minimal: endotoxin increases socially reactive behaviors, compared to saline controls, but only for mice reared by their biological dams. Rearing by foster dams increases social exploration in the low-aggressive line. The findings raise novel questions regarding the openness of behavioral systems to effects of nonobvious but omnipresent features of the environment, such as antigenic load, how these effects are integrated to affect social development and psychopathology, and the nature of intrinsic factors that contribute to individual differences in sensitivity to early stressors.

Adrenocortical activity in at-risk and normally developing adolescents: individual differences in salivary cortisol basal levels, diurnal variation, and responses to social challenges.

The purpose of this study was to examine adrenocortical activity (basal, diurnal variation, and responses to social stressors) in adolescents at risk for psychopathology. Salivary cortisol levels were examined in normally developing and at-risk youth with internalizing and externalizing symptoms ranging from subclinical to clinical levels. Adolescents showed expected patterns of diurnal variation, with high early morning cortisol levels and a pattern of decline throughout the day. Females showed higher midday and late afternoon levels than males, and these patterns interacted with risk status. Internalizing problems sometimes were associated with gradual rather than steep declines in basal cortisol production. Both immediate and delayed cortisol reactivity to a social performance stressor were associated with internalizing symptoms. There was no evidence of relations between externalizing problems and underarousal of the hypothalamic-pituitary-adrenal (HPA) system. These and other results suggest that gender is an important moderating factor linking psychopathology. development, and context with HPA axis functioning in adolescence.

Measurement of gonadal hormones in dried blood spots versus serum: verification of menstrual cycle phase.

In the present study, we show that blood spot assays for estradiol, progesterone, and testosterone are a reliable, accurate, and sensitive means for measuring circulating gonadal hormones. The lower limit of sensitivity of each blood spot assay is sufficient to determine gonadal hormone levels in adult females. Correspondence of serum to blood spot measures is high, with blood spot hormone levels explaining an average of 88.60% of the variance in serum gonadal hormones in females, but only 46.20% in males. We provide formulas for converting hormone levels in blood to hormone levels in serum (which traditional endocrinology studies report). Finally, we show that careful attempts to estimate hormone status by day-count methods are unreliable when compared to hormone assay in blood spots.

Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently.

Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC ) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.

Use of salivary biomarkers in biobehavioral research: cotton-based sample collection methods can interfere with salivary immunoassay results.

In a series of studies, we evaluated the susceptibility of immunoassays for saliva biomarkers to interference effects caused by cotton materials used to absorb saliva during sample collection. Salivary assay results for testosterone, DHEA, progesterone, and estradiol are artificially high, and for sIgA artificially low, when samples are collected using cotton absorbent materials. In contrast, results for salivary cortisol, DHEA-S, and cotinine are not affected by the use of cotton collection methods. The order of individual results from samples collected using cotton versus no-cotton methods for certain markers is not conserved, suggesting that for some biomarkers this collection method can be a significant source of unsystematic error. It was shown, using DHEA as an example, that the cotton interference effect is of sufficient magnitude to attenuate the association between serum and saliva levels. Awareness of this issue is critical to ensure measurement validity in future studies and analyses of archived samples collected using cotton materials.

Human aggression and enumerative measures of immunity.

OBJECTIVE: A pattern of clinical, behavioral, and experimental findings suggests that individual differences in aggressive behavior may be related to immunologic processes. We evaluated two conflicting models of the relationship: 1) A positive association stems from an adaptive mechanism protecting aggressive individuals from increased exposure to immune stimuli and 2) a negative association is due to potential immunosuppressive effects of high testosterone levels. METHODS: We investigated the models using enumerative measures of cellular and humoral immunity in a sample of 4415 men aged 30 to 48 years who were interviewed and underwent a medical examination. RESULTS: Analysis revealed positive (and curvilinear) associations between aggressive behavior and enumerative measures of helper/inducer and suppressor/cytolytic T lymphocytes and B lymphocytes. The aggression-immunity relationship was independent of testosterone level, age, current health status, and negative health behaviors and was most pronounced for helper/inducer T cells. There was no evidence of a negative association between testosterone and any immune measure. CONCLUSIONS: In a large sample of men, individual differences in aggressive behavior were positively associated with enumerative measures of cellular immunity.

Assessing estradiol in biobehavioral studies using saliva and blood spots: simple radioimmunoassay protocols, reliability, and comparative validity.

We developed simple, reliable, and highly sensitive assay modifications of commercially available radioimmunoassay kits to measure estradiol in saliva and blood spot specimens. The saliva assay has average intra- and interassay coefficients of variation (CV) of 6.45 and 9.01%, with average analytical and serial dilution recoveries 100.65 and 89.25%. The blood spot assay has average intra- and interassay CVs of 7.57 and 8.22%, with analytical and serial dilution recoveries of 80.50 and 108.50%. The analytical sensitivity ranges of the saliva (0.25-7.50 pg/ml) and blood spot (2. 00-375 pg/ml) assays are sufficient to determine levels in the majority of pre- and postpubertal males and females. Blood spot assay results are correlated with serum estradiol levels for adult males, r (17) = 0.73, and females, r (18) = 0.96. In contrast, the serum-saliva correlation is only modest for adult females, r (14) = 0.60, and not significant for adult males. Substitution of blood spot assay results for serum values underestimates the known serum estradiol-behavior correlation by only 3.45%, whereas substitution of saliva assay results for serum values underestimates the association by 37.55%. The findings have important implications for the use and potential misuse of noninvasive measures of estradiol in studies of health and human development.

Testosterone and men's depression: the role of social behavior.

Medical research suggests that testosterone has positive effects on mood (thereby reducing the chances of depression), and social science research finds testosterone to be related to antisocial behavior, risk behavior, unemployment and low paying jobs, and being unmarried--factors known to be positively related to depression. Analysis of a sample of 4,393 men finds a parabolic model best fits the data. The relationship between testosterone and depression is inverse for men with below average testosterone and direct for those with above average testosterone. The relationship disappears for those with above average testosterone when controls for antisocial and risk behaviors and the absence of protective factors such as marriage and steady employment are in the equation. The relationship is unchanged for those with below average testosterone. The results help explain the difference between medical and social research findings. Mechanisms accounting for the findings are explored.

Maternal depression and risk for postpartum complications: role of prenatal corticotropin-releasing hormone and interleukin-1 receptor antagonist.

The pregnancies of 58 healthy adolescents (ages 13 to 19 years) were followed to examine links between symptoms of depression, corticotropin-releasing hormone (CRH), interleukin-1 beta, (IL-1 beta), and IL-1 receptor antagonist (IL-1ra) as possible predictors of maternal and infant outcomes. Maternal psychological adjustment and medical complications during gestation, labor, delivery, and the postpartum period were monitored. Plasma samples collected during gestation were assayed for CRH, IL-1 beta, and IL-1ra. During gestation, symptoms of maternal depression were found to be associated with lower levels of CRH; lower levels of CRH were associated with lower levels of IL-1ra. In addition, lower levels of IL-1ra predicted higher rates of maternal complications after childbirth. IL-1 beta, detected in only 4 mothers, was not associated with any predictor or outcome measures. During gestation, CRH may induce circulating cytokine inhibitors without significantly affecting cytokine production or synthesis. Maternal symptoms of depression during gestation may attenuate the association between CRH and IL-1ra.

Assessing dehydroepiandrosterone in saliva: a simple radioimmunoassay for use in studies of children, adolescents and adults.

While salivary assays for some hormones are widely used, the availability of assays for salivary DHEA is limited. By adapting a commercially available radioimmunoassay serum kit, we developed a reliable, efficient and sensitive measure of DHEA in saliva that does not require separation or extraction. The minimum detection limit was 4.0 pg/ml. Intra-assay coefficients of variation (CV%) were on average 4.05, and inter-assay CVs averaged 9.70. Method accuracy, determined by spike recovery, and linearity, determined by serial dilution, averaged 99.55 and 92.03%. Levels in matched serum and saliva samples showed strong linear relationships for adult males and females. Specific guidelines are developed for sample collection, storage, and preparation procedures. Reference ranges for salivary DHEA levels are provided for 64 children ages 8-11, 96 adolescents ages 12-17 and 48 adults ages 30-45. Salivary DHEA levels are shown to reflect developmental, gender and diurnal differences.

Corticotropin-releasing hormone and cortisol: longitudinal associations with depression and antisocial behavior in pregnant adolescents.

OBJECTIVE: To examine the concurrent and longitudinal associations between corticotropin-releasing hormone (CRH) and cortisol concentrations and depression and antisocial behavior (conduct disorder symptoms) in pregnant adolescents. METHOD: Fifty-nine adolescents were evaluated in early pregnancy (9-21 weeks' gestation), late pregnancy (32-34 weeks' gestation), and the postpartum period (4-5 weeks postpartum). Symptoms of depression and conduct disorder were obtained from the Diagnostic Interview Schedule for Children. RESULTS: Lower concentrations of CRH were related to a greater number of depression symptoms in early pregnancy (p < .05) and in late pregnancy (p < .05). Lower concentrations of CRH also were related to a greater number of conduct disorder symptoms in early pregnancy (p < .06) and in the postpartum period (p < .05). CONCLUSION: The findings support the long-standing hypothesis that stress-related products of the hypothalamic-pituitary-adrenal axis are associated with emotions and behavior during pregnancy.

Testosterone and men's health.

Medical and behavioral research depicts the influence of testosterone on health in opposite ways, the former finding beneficial effects and the latter potentially detrimental ones. We investigate the relationship between testosterone and health risk behavior, indicators of disease, and overall health in a sample of 4393 men who were interviewed and medically examined. Analysis revealed that having a high level of testosterone, compared to a low level, increased the odds of health risk behavior. With respect to disease, high testosterone increased the odds of some health problems but decreased the chances of others. At very high levels testosterone loses many of its beneficial effects. Overall, men with high testosterone would be healthier if they did not engage in health risk behavior.

Salivary testosterone determination in studies of child health and development.

Measurement of hormones in children's saliva has excited interest because of numerous potential applications in developmental studies. Although assays of children's saliva for some hormones (e.g., cortisol) are widely available and used, the availability and use of assays of children's saliva testosterone is restricted. By adapting a commercially available serum testosterone kit, our laboratory has developed a reliable, efficient, and highly sensitive procedure for measuring testosterone in children's saliva that does not require separation or extraction. The minimum detection limit was 0.8 pg/mL. Intraassay coefficients of variation (CV) were between 3.66 and 6. 78% at concentrations 9.25 to 86.41 pg/mL, and interassay CVs were between 5.70 and 6.61% at concentrations of 7.3 to 118.51 pg/mL. The standard curve was highly reproducible (M slope = -0.70 and Mr = 0. 99). Method accuracy, determined by spike recovery, and linearity, determined by serial dilution, were 99.20 and 92.80%, respectively. Values from matched serum and saliva samples showed strong linear relationships. The assay captured near 99.09% of the range of individual differences in boys' (N = 90) and girls' (N = 85), ages 8-12, am and pm salivary testosterone levels. This assay can be easily applied to the investigation of testosterone-behavior relations in the context of studies on child health and development. It may help many child development researchers improve or expand their research activities.