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

Psychoneuroendocrinology 24 (1999) 567 – 579

Assessing dehydroepiandrosterone in saliva: a simple radioimmunoassay for use in studies of children, adolescents and adults Douglas A. Granger *, Eve B. Schwartz, Alan Booth, Mary Curran, Dena Zakaria Departments of Biobeha6ioral Health and Sociology, Beha6ioral Endocrinology Laboratory, The Pennsyl6ania State Uni6ersity, 315 E. Henderson Building, Uni6ersity Park, PA 16801 -6508, USA Received 23 December 1998; accepted 17 February 1999

Abstract 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. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; DHEA; Radioimmunoassay; Gender and developmental differences

1. Introduction There is an emerging consensus that monitoring circulating levels of dehydroepiandrosterone (DHEA) may afford developmental and health-oriented scientists a * Corresponding author. Tel.: +1-814-863-8402; fax: +1-814-863-7525. E-mail address: [email protected] (D.A. Granger) 0306-4530/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 3 0 ( 9 9 ) 0 0 0 1 3 - X

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unique view of the interacting effects of biological, environmental and behavioral processes. Despite technical advances that make the assay of some unconjugated hormones (e.g. cortisol, testosterone) in saliva possible, there remain wide gaps in information available to guide researchers in how to best collect and prepare samples, and assay this important hormone in saliva. This paper presents a rigorous evaluation of the internal and external validity of a salivary DHEA radioimmunoassay (RIA), makes specific recommendations regarding sample collection and preparation procedures, and characterizes individual differences in salivary DHEA levels by gender and time of day in children, adolescents and adults. Researchers studying the effects of biobehavioral processes on health and development have focused attention on the relationship between hormone products of the adrenal glands and a variety of psychological (e.g. Erb et al., 1981), developmental (e.g. McClintock and Herdt, 1996), and health constructs (e.g. Nafziger et al., 1991). DHEA, like cortisol, is one of the major hormones produced by the adrenal glands. In its native form or as a precursor of other steroids (e.g. androstenedione, testosterone, estradiol), DHEA affects a remarkable diversity of biologic actions including immune, cardiovascular, endocrine, central nervous system and metabolic effects (see Majewska, 1995). Circulating DHEA levels are known to be associated with individual differences in health risk behavior such as alcohol use and smoking (Field et al., 1994), and cognitive abilities, emotionality, and behavior (Leventhal and Brodie, 1981; Jacklin et al., 1988; Warren and Brooks-Gunn, 1989; Wolkowitz et al., 1995). More than 20 years ago, De Peretti and Forest (1976) described dramatic developmental differences in blood levels of DHEA in 442 children ranging in age from the first day of life to 15 years. During the first month, DHEA levels decreased significantly and then progressively declined throughout the first year of age. DHEA levels were very low in both sexes between the ages 1–6 years. At age 7, DHEA levels were higher than levels in any previous year (years 1–6) and increased in each subsequent year thereafter. For both boys and girls, DHEA levels increased substantially prior to the onset of puberty (Parker, 1991). These findings suggest that the biologic, behavioral and cognitive effects of DHEA may be age dependent. This may be particularly so during the peripubertal period, when in contrast to later in life, circulating levels of DHEA are several fold higher than its potent androgen metabolites. The concentration differential can facilitate rapid peripheral tissue conversion of DHEA to androstenedione and testosterone. It is noteworthy that in prepubertal children, and in females, peripheral conversion of DHEA is the major pathway for testosterone production. De Peretti and Forest (1976) also reported, based on studies in which ACTH1 – 24 was perfused, that children’s adrenal secretion of DHEA is under ACTH control. They described cases of acute clinical stress (e.g. respiratory distress) when children’s DHEA levels increased in response to activation of the HPA axis. Most interesting was the finding that: (1) ACTH up-regulation of DHEA was observed in children as young as 58 days old; and (2) DHEA levels after exogenous ACTH stimulation were highest at ages with elevated basal DHEA levels (early infancy and after the onset of adrenarche). These latter observations suggest possible

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developmental differences in the secretory products of the adrenal (e.g. ratio of DHEA to cortisol) during the stress response of the HPA axis. Monitoring DHEA in saliva has distinct advantages over doing so in other biological fluids (i.e. urine, serum or plasma). This is especially true when children are involved as participants in biobehavioral studies (e.g. Goodyer et al., 1996). Sampling saliva represents a less-invasive method for long-term or repeated sampling schedules; enables collection of samples in special populations and many circumstances in which blood or urine sampling is not viable. Lipid-soluble unconjugated steriods, such as DHEA, enter saliva predominantly via an intracellular route (Vining et al., 1983). Thus, the concentrations of these steroids in saliva are not dependent on saliva flow rate and accurately represent the unbound, biologically active, fraction in the general circulation (Vining et al., 1983). Also unless visibly contaminated with blood, human saliva is not considered a class II biohazard (Centers for Disease Control) affording researchers administrative and safety benefits. Surprisingly, with a few noted exceptions, scant information is available regarding the measurement of DHEA in saliva, especially in saliva of children and youth. Since the portion of DHEA in saliva represents only a fraction of the total in circulation (pg/ml in saliva versus ng/ml in serum) to capture the full range of individual differences across development it is possible that salivary assays may need to be designed to be ultra-sensitive (Boon et al., 1972; Hooper and Yen, 1975; De Peretti and Forest, 1976; New et al., 1981). We surveyed the literature to evaluate the characteristics of the assays that have been used to measure salivary DHEA. The description of the assay procedures was rarely presented in sufficient detail for us to reproduce the protocol or evaluate assay precision, validity and reliability. There was also little consistency between studies in the measurement procedures employed. As expected, some investigators were using ‘in house’ reagents; some employed extraction steps; and some used separation procedures. By contrast, the literature on sample collection and assay of other salivary biomarkers is substantial and is maturing at a relatively rapid rate. For instance, studies caution that saliva collection techniques must be carefully designed to maximize measurement validity when assaying testosterone and cortisol (Magnano et al., 1989; Dabbs, 1991; Schwartz et al., 1998; Granger et al., 1999). It is clear that, thus far, the depth of published information on the measurement and application of salivary DHEA in biobehavioral research is quite shallow. There is an apparent need for a sensitive, efficient, and reliable immunoassay with accessible reagents and materials for the determination of salivary DHEA. In this report, we fill that need with an immunoassay that health and developmental scientists can use to improve the next generation of their studies. The assay protocol is described so as to be reproducible from this report. Assay performance is demonstrated with respect to specificity, sensitivity, reliability of the standard curve, precision, accuracy, and linearity of dilution. DHEA levels are compared from matched saliva and serum samples and the relationship between salivary DHEA and serum DHEA is confirmed. The assay is shown to be sensitive to individual, gender, developmental and diurnal differences among samples from 208 normal male and female subjects ages 8 – 45 years. Alternative sample collection procedures

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are evaluated and specific recommendations about sample collection, storage, and preparation are presented.

2. Materials and methods

2.1. Reagents The protocol is a modification of the Diagnostic Systems Laboratories (Webster, TX) 125I double antibody test kit for the quantification of DHEA in serum. Our modifications of the kit are noted in the method section and overviewed in Table 1. The DHEA antiserum is rabbit anti-DHEA in a protein-based buffer with sodium azide as a preservative. The antibody cross-reacts 0.73% with isoandrosterone, 0.46% with androstenedione, and B0.05% with DHEA-sulfate, progesterone, androsterone, testosterone, and cortisol. Internal controls, supplied by DSL with the kit, contain DHEA in a human serum matrix. A PBS diluent (0.1% gel) is constructed by adding gelatin (Bovine Type B, Sigma) to 1× phosphate buffered saline. To 1 l of PBS solution, 1 g of gelatin is added and heated (not to boiling) until the gelatin is dissolved ( 15 min). Diluent must be returned to room

Table 1 Assay protocols for dehydroepiandrosterone (DHEA) determinations in saliva and seruma Saliva

Serum

Pipette 100 m1 of samples, controls or calibrators 100 m1 of samples, controls and/or calibrators + + 50 ml of DHEA antiserum diluted (1:4) 500 ml of DHEA [125I] reagent n + Vortex and incubate for 30 min @ 37°C 100 ml of DHEA antiserum + n Vortex and incubate @ 37°C 1 h 250 ml of DHEA [125I] reagent diluted (1:4) n + Vortex and incubate for 3 h 1 ml of precipitating reagent + n 500 ml precipitating reagent Vortex and incubate @ RT for 10–15 min n n Vortex and incubate for 20 min @ RT Centrifuge for 15–20 min @ 1500×g @ RT n n Centrifuge @ 1500×g for 20 min @ RT n Decant by simultaneous inversion with a sponge rack into a radioactive waste receptacle. Allow to drain, then blot tubes to remove excess droplets n Count all tubes in g counter for 2 min a

Note: RT = room temperature

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temperature prior to use and can be stored at 2–8°C. External controls, which contain multiple steroids in a human serum matrix, can be purchased from Bio-Rad (Anaheim, CA; Lypochek Immunoassay control serum). External controls can be constructed by diluting the Lypocheck level I control into the salivary range (pg/ml).

2.2. Methods 2.2.1. Radioimmunoassay The kit was modified as follows (see Table 1). Standards, provided in the kit, were diluted with PBS (0.1% gel) to give final concentrations of 20, 50, 100, 250, 500 and 1000 pg/ml. Calibrators are diluted as follows: for zero calibrator use 100 ml and 900 ml of diluent; for the 20, 100, 250 and 1000 pg/ml calibrator use 50 and 450 ml diluent; for the 50 pg/ml calibrator use 250 ml of the diluted zero calibrator and 250 ml of the 100 pg/ml calibrator; for 500 pg/ml calibrator use 250 of the diluted zero and 250 ml of the 1000 pg/ml calibrator. Antibody and tracer are diluted × 4 with diluent. The level I and II internal controls provided with the kit are diluted 15- and 4-fold, respectively, to obtain values in the low pg/ml and high pg/ml range. Polypropylene tubes are labeled and arranged in duplicate for total counts, NSB (nonspecific binding), standard tubes, controls, and unknowns. A total of 100 ml of standard, control, or saliva is pipetted into the appropriate tube. In place of antibody, 50 ml of PBS is added to each NSB tube. Then, 50 ml of diluted DHEA antiserum is added to all other tubes with the exception of the total count tubes. Vortex, cover the tubes with parafilm and incubate for 30 min in a 37°C waterbath with mixing (100 rpm). Thirty min later, 250 ml of diluted DHEA tracer [125I] is added to all tubes. The total count tubes are set aside. All other tubes are vortexed, covered with parafilm, and incubated in a 37°C waterbath with mixing for 3 h. Afterwards, 500 ml of precipitating reagent is added. The tubes are vortexed, and incubated at room temperature for 20 min. Finally, the tubes are centrifuged at 1500×g for 20 min at room temperature, aspirated or decanted, and counted for 2 min on a g counter (LKB Clinigamma). Results can be calculated in pg/ml using either log-linear or logit-log regression.

3. Results

3.1. Assay performance 3.1.1. Sensiti6ity The theoretical detection limit, defined as the minimal concentration of DHEA that can be distinguished from zero (Chard, 1990), was obtained by interpolating the mean minus 2 SD for ten replicates of the 0 pg/ml DHEA calibrator. By this method the limit of this assay’s sensitivity is 4.0 pg/ml.

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Table 2 Analytical recovery of dehydroepiandrosterone (pg/ml) added to saliva Endogenous

Added

Theoretical

Observed

Sample recovery (%)

I

303.5

II

160.2

III

23.1

50.0 100.0 300.0 50.0 100.0 300.0 50.0 100.0 300.0

353.3 403.5 603.5 210.2 260.2 460.2 73.1 123.1 323.1

339.1 399.4 677.4 209.8 278.5 493.8 61.5 129.1 321.8

95.9 99.0 112.0 99.8 93.4 107.3 84.1 104.9 99.6

3.1.2. Standard cur6e The standard curve was highly reproducible across 14 replicates, having an average correlation coefficient of −.995 (SEM= .001), an average ED 80 of 62.8 pg/ml (SEM= 4.4), ED 50 of 350 pg/ml (SEM= 18.1), and an average ED 20 of 1973.9 pg/ml (SEM=102.9). The readable range of the standard curve is from 4 to 1000 pg/ml, based on sensitivity and the highest standard. 3.1.3. Precision Intra-assay variation (CV) computed for the mean of 14 replicate tests of low (85.3 pg/ml) and high (335.6 pg/ml) concentration samples were 3.75 and 4.35%, respectively. Inter-assay variation was computed for the mean of average duplicates for samples representing 79.4 pg/ml (N= 10), 481.9 pg/ml (N= 14), and 658.1 pg/ml (N =14). CV% were 13.63, 10.27 and 6.32%, respectively. 3.1.4. Accuracy Method accuracy was determined from known amounts of unlabeled DHEA added to saliva samples containing various endogenous concentrations, as shown in Table 2. Recoveries ranged from 84.1 to 112% (M= 99.55%). 3.1.5. Linearity of dilution Parallelism was evaluated by measuring DHEA in 81.3, 64.9 and 34.4 pg/ml samples which were serially diluted incrementally to the low end of the assay’s range. Observed values were near those expected across the entire range of measurement. The average recovery was 92.03% (see Table 3). 3.2. External 6alidity 3.2.1. Comparison of matched serum and sali6a Simultaneous serum and saliva were collected from 39 adults (18 males, 21 females) between 0900 and 1000 h and were compared using the modified RIA for saliva and the manufacturer’s (DSL, Webster, TX) recommended protocol for

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DHEA in serum. The mean serum and saliva levels for males were 7.9 ng/ml (SEM= 0.8) and 277 pg/ml (SEM= 36.6) and for females 8.2 ng/ml (SEM= 0.8) and 264 pg/ml (SEM =32.8). Values obtained for saliva and serum DHEA levels showed a strong linear relationship, r(37)= .87, PB .0001, with, r(16)= .82, and, r(19) = .90, P B.001, for males and females, respectively.

3.2.2. De6elopmental, gender and diurnal differences As part of a larger study, saliva samples were collected from mothers, fathers and siblings from a cohort of 400 families of elementary school-aged or adolescent children. Following Dabbs (1991), saliva was collected by having subjects chew a stick of original flavor sugar-free Trident gum for at least 3 min to stimulate the flow of saliva and to deposit, over the course of 5–10 min, 3–6 ml of saliva into a plastic vial. Samples were collected by parents at home in the early morning (M= 0656 h) immediately after waking, and by interviewers in the late afternoonevening (M= 1831 h) at the beginning of the larger project’s assessment session. Samples collected at home were delivered by mail to The Pennsylvania State University Behavioral Endocrinology Laboratory where they were aliquoted, frozen (−40°C) and prepared for assay. A subset of 416 saliva samples from 64 children (eight boys and girls at each age 8, 9, 10 and 11 years), 96 adolescents (eight boys and girls at each age 12, 13, 14, 15, 16 and 17 years) and 48 adults (eight males and females in three age groups 30 – 34, 35–39, and 40–45 years) were assayed for DHEA as described above. Means (and SEMs) for salivary DHEA levels by gender, age, and time of day are presented in Table 4. The omnibus analytical design was a 2 (gender)× 2 (sampling time of day)× 13 (age group) GLM ANOVA. Age group and gender were included as blocking factors, with sampling time of day a repeated measure. The ANOVA revealed main effects of sampling time, F[1,180] =256.44, PB .0001, gender, F[1,180]= 4.08, P B .045, and age group, F[12,180] =8.33, PB .001. As expected, the most robust effect was for sampling time of day with DHEA levels higher in the AM (M= 328.5, SEM = 16.3) than in the PM (M= 132.9, SEM = 6.4). The gender main effect was qualified by a significant sampling time× gender interaction, F[1,180]= Table 3 Analytical recovery of dehydroepiandrosterone (pg/ml) in serially diluted samples Endogenous

Dilution

Theoretical

Observed

Sample recovery (%)

I

162.5

II

129.7

III

68.8

1:2 1:4 1:8 1:2 1:4 1:8 1:2 1:4 1:8

81.3 40.6 20.3 64.9 32.4 16.2 34.4 17.2 8.6

75.9 34.9 21.1 60.5 24.4 13.9 36.9 14.9 8.4

93.4 86.0 103.9 93.2 75.3 85.8 107.3 86.6 97.7

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Table 4 Means (and standard error of the mean) for salivary dehydroepiandrosterone (pg/ml) by gender, age and sample time of day Time of day

Morning (M = 0656 h)

Gender

Males

Adults (age in years) 40–45 351.3 (102.7) 35–39 464.0 (54.3) 30–34 467.4 (66.4) Adolescents (age in years) 17 355.2 (84.1) 16 479.4 (87.5) 15 303.3 (41.4) 14 204.6 (33.5) 13 220.3 (52.5) 12 330.4 (69.5) Children (age in years) 11 281.4 (57.9) 10 173.3 (42.0) 9 122.5 (44.0) 8 99.1 (19.9)

Evening (M=1831 h)

n

Females

n

Males

n

Females

n

8 8 8

223.5 (37.2) 370.4 (71.6) 608.6 (85.3)

8 8 8

134.2 (13.6) 198.1 (43.8) 204.0 (32.8)

8 8 8

121.3 (25.5) 165.1 (34.9) 215.9 (67.8)

8 8 8

8 8 8 8 8 8

615.1 421.4 412.5 534.7 395.4 242.2

(74.0) (89.9) (99.5) (132.4) (105.7) (61.1)

8 8 8 8 8 8

196.4 238.4 122.9 94.1 92.4 129.2

(51.5) (35.8) (14.6) (16.4) (16.5) (16.3)

8 8 8 8 8 8

212.2 157.8 122.1 176.2 138.1 104.1

(37.5) (35.5) (14.3) (22.4) (20.5) (16.6)

8 8 8 8 8 8

7 8 8 8

310.6 278.1 150.6 119.5

(42.9) (43.0) (39.7) (29.7)

8 8 8 8

115.0 72.7 59.3 29.6

(24.2) (17.6) (12.4) (6.8)

8 8 8 8

107.7 106.8 69.0 62.9

(20.3) (20.2) (12.8) (11.4)

8 8 8 7

5.68, PB .018. That is, females had higher DHEA levels (M= 360.2, SEM= 25.0) than males (M =296.5, SEM= 20.4) in the AM, but levels were comparable (M males=129.7, SEM=9.2; M females = 136.0, SEM= 9.1) in the PM. The relation-

Fig. 1. Developmental and diurnal differences in salivary dehydroepiandrosterone (pg/ml).

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ship between age and DHEA was quadratic, RSQ (203)= .31, PB .0001. As can be seen in Fig. 1, levels were higher in each subsequent year starting at age 8 (M =76.5, SEM=11.5), peaked at ages 30–34 (M= 374.0, SEM= 35.4), then sharply decreased at ages 35 – 39 (M = 299.4, SEM= 30.9), and then decreased again at ages 40 – 45 (M =207.6, SEM = 32.7). The relationship between age and DHEA was qualified by a significant sampling time× age group interaction, F[12,180]=3.05, P B.001, revealing that developmental differences in saliva DHEA levels were more pronounced in the AM than in the PM (see Fig. 1).

3.3. Sample collection As the number of studies assessing salivary hormones has increased, so too has our understanding that special circumstances present themselves when collecting saliva that have the potential to influence variability in the measured concentrations. In our previous studies (Schwartz et al., 1998; Granger et al., 1999) we have evaluated the effects of collection methods commonly used with younger (e.g. oral stimulants like powdered drink-mix crystals and cotton swabs) and older subjects (e.g. Salivette device; Sarstedt, Newton, NC) on salivary testosterone and cortisol. Here we report that the misuse of materials to absorb or stimulate saliva has the potential to artificially affect the assay of DHEA in saliva.

3.3.1. Cotton swabs and drink mix crystals Saliva from six individuals was collected by passive drool. Subjects rinsed their mouths with water, waited 5 min, then expectorated 6 ml of saliva through a short plastic straw into a plastic vial. Samples were left either untreated (clear), filtered through a cotton dental roll (Richmond, Charlotte, NC) by expressing saliva from the roll using a 10 cc syringe, filtered through the cotton swab used in the untreated Salivette device (Sarstedt, Newton, NC) by centrifugation, or spiked with 0.1 g/ml grape flavored sugar sweetened powdered crystals (a commonly used substance in a concentration used to stimulate saliva flow in studies with young children). All samples were stored at − 80°C until assay for DHEA. Compared to untreated samples, M= 173.6 pg/ml (SEM = 23.9), DHEA levels were higher, M =305.3 pg/ml (SEM =76.3), after filtering through dental cotton rolls, (t(5)= 1.53, P B .09), and higher, M= 1904.3 pg/ml (SEM= 88.5), after the use of the untreated-cotton Salivette device, (t(5)= 15.78, PB .001). Also when compared to untreated saliva, M =173.6 (SEM = 23.9), DHEA levels were higher, M =275.9 (SEM =70.4), after adding grape-flavored Kool-Aid, (t(5)= 1.81, PB .065). This increase was paralleled by a Kool-Aid-induced increase in sample acidity. The average pH of untreated saliva was 7.0 and decreased to 3.9 when Kool-Aid was added, (t(5) =15.36, P B.0001). By contrast, the pHs for cotton roll filtered samples (M =7.2) and for untreated-cotton Salivette filtered samples (M= 7.1), were not significantly different from untreated saliva (M= 7.0). 3.3.2. Sali6ettes: cotton, citric-acid and polyester Since a large number of laboratories are using the Salivette sampling devices and

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there are at least three different types available (untreated-cotton, cotton treated with citric acid to simulate saliva flow, and a polyester swab with a perforated plastic mantle) we evaluated the effects of each on DHEA levels relative to passive drool. As described above, saliva was collected from six individuals using the passive drool method. A 1 ml aliqout of each sample was left untreated, three other 1 ml aliqouts were filtered by centrifugation through each different Salivette device. All samples were stored at − 80°C until assay for DHEA. Paired t-tests compared samples collected by passive drool, (M= 94.0 pg/ml, SEM=20.1), to samples collected using the polyester swab, (M= 503.2 pg/ml, SEM = 81.1; t(5) =5.98, P B.002), the untreated-cotton swab, (M= 1211.6 pg/ ml, SEM =64.1; t(5) =17.75, P B .0001), and the citric acid-treated swab (M= 1526.76 pg/ml, SEM = 341.0; t(5) = 4.36, PB .007). The magnitude of the difference in DHEA levels between the passive drool and citric-acid Salivette method was associated with an increase in sample acidity. The average pHs were 7.3 for passive drool, 7.3 for the polyester swab, 6.5 for untreated-cotton, and 2.58 for citric-acid treated cotton.

3.3.3. Chewing gum The final technique evaluated involved participants chewing sugar-free original flavor Trident gum and expectorating directly into a plastic cup (Dabbs, 1991). Saliva was collected from six adults by passive drool immediately before and then at 1, 2, 3, 4 and 5 min time points while chewing the gum. Paired t-tests comparing baseline to each time point (1–5 min) while chewing the gum indicated DHEA levels were elevated over baseline at 1, 2 and 3 min while chewing gum with t(5) = 3.48, 2.09 and 2.77, PsB .05 (one-tailed) and means 141.7 (SEM=21.0), 185.6 (SEM = 28.2), 185.4 (SEM= 33.8), and 180.6 (SEM= 26.3), respectively. DHEA levels did not significantly differ from baseline after 4 min of chewing the gum.

3.4. Sample preparation Following Worthman et al. (1990), we recommend that no samples are processed through the assay fresh; instead, all should be frozen first and thawed before assay to break down mucopolysaccharides that can interfere with pipetting. We recommend that freeze – thaw cycles be kept to a minimum even though our preliminary data suggest that DHEA in saliva is robust to freeze–thaw. That is, seven samples representing DHEA levels from 90.1 to 497.6 pg/ml were thawed and refrozen at −80°C three times. Average DHEA levels did not significantly vary across these cycles. Means were 204.8 (SEM= 55.0), 195.8 (SEM=60.5), and 199.7 (SEM=57.2) for the first, second and third cycle. On the day of the assay, samples should be centrifuged (1500× g, 15 min) to remove particulate matter, then clear sample should be transferred into appropriate test tubes.

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4. Discussion This report presents an immunoassay protocol and validates its use in measuring DHEA in saliva. This immunoassay will enable researchers to capture 100% of the range of individual and diurnal differences in male and female salivary DHEA levels from middle childhood through adulthood. The protocol attains a high level of reliability and validity; it does not require sample separation or extraction. This assay represents a considerable savings in time and cost, and uses materials and reagents that are commercially manufactured and distributed. With respect to sample collection, we caution researchers not to use cotton swabs or either version (i.e. untreated-cotton, polyester or citric-acid treated swabs) of the Salivette device for samples to be assayed for DHEA using this protocol. The findings raise the possibility that multiple characteristics of the Salivette swabs interfere with antibody binding in this, and other, salivary immunoassays (Granger et al., 1999). Clearly, implicated here are changes in sample acidity induced by use of the citric-acid treated Salivette. But, plant hormones in the cotton swabs are also a potential source of interference (Dabbs, 1991). Since interference is present when using the polyester Salivette (i.e. in the absence of cotton or citric-acid) other factors yet to be identified also appear to contribute to measurement error. Researchers interested in applying this test should take precautions to minimize the impact of other sample collection procedures. In particular, if an oral stimulant is used, investigators might consider routinely performing checks to screen possible acid interference. This can be accomplished easily in the field by using pH paper. Collection procedures that lower sample pH below 5.0 should be ruled out. As a next step, confirmation is needed to verify that DHEA results from serially diluted samples decrease in proportion to the volume of the original sample analyzed (Chard, 1990). Ideally, some samples should be spiked with the stimulant directly, and a dose response curve generated to evaluate the effects on DHEA. Should interference be detected, the collection procedure should obviously be revised or abandoned. We have followed suggestions of Dabbs (1991) and have found that, when carefully applied, chewing Trident original flavor sugarless gum is a viable sample collection technique. Our findings suggest that there is a transient rise in measured DHEA in saliva during the first 3 min after chewing the gum. If this collection method is to be used, we recommend that researchers allow subjects to chew for at least 3 min before beginning to collect sample. Caution should be exercised by researchers studying young children as this collection method may be inappropriate to use with children under the age of 6 (American Academy of Pediatricians). Numerous studies have explored the relationship of salivary cortisol to developmental, behavioral, environmental, and health constructs (e.g. Kirschbaum et al., 1992). The present findings suggest that the inclusion of salivary DHEA into such analyses might contribute useful new information. Examining the ratio of the HPA axis production of DHEA to cortisol in response to stress may be particularly worthwhile. That DHEA is responsive to ACTH and the response shows developmental differences underscores this notion (e.g. Lashansky et al., 1991). For

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example, cortisol and DHEA are known to have opposite effects on the immune system. Indexing the ratio of these adrenal products would potentially contribute to our understanding of individual and developmental differences in the activation of the HPA axis by stress on immunity and health. An equally impressive literature has explored links between salivary testosterone, behavior and health constructs (e.g. Booth and Osgood, 1993; Booth et al., 1999). Given the role that DHEA plays in the production of testosterone in females and prepubertal males, its inclusion in biobehavioral studies might contribute substantially to an understanding of individual differences in testosterone–behavior relationships. This would seem especially likely when the primary source of testosterone is the adrenal gland rather than the gonads. In conclusion, it is now feasible for developmental and health scientists to incorporate salivary measurements of DHEA into their studies, but care must be taken to ensure accurate determinations before data collection starts. Employing this salivary DHEA protocol, and, following these recommendations for sample collection, storage and preparation, investigators can now easily and accurately monitor salivary DHEA levels from childhood to adulthood. We expect this effort will assist researchers to improve and expand their research activities.

Acknowledgements Thanks are due to Ruth Merritt, Jodi Heaton, Jyotika Mirchandani, Virginia Lucas, and the staff of the Penn State Behavioral Endocrinology Laboratory, as well as the staff of the Penn State Family Relations Project (Ann Crouter and Susan McHale, PIs) for their assistance with various aspects of this project. The studies were supported in part by the Pennsylvania State University Behavioral Endocrinology Laboratory, HYPERLINK http://bbh.hhdev.psu.edu/labs/bel/ bel.html, and the W.T. Grant Foundation. We gratefully acknowledge the contribution of reagents and materials by Diagnostic Systems Laboratories (Webster, TX).

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