Very few reference intervals for salivary steroids in children have been established to date (1 ). Even the manufacturers of salivary steroid assays do not provide sufficient reference data for their products. This lack of information is surprising because the measurement of salivary steroids has been accepted as being noninvasive and stress-free (2, 3 ). In particular, psychiatric and neuroendocrinologic experiments are frequently designed with saliva as the medium of choice for steroid analysis (4 -6 ).A large variety of stressors can rapidly affect the adrenal cortex, causing increased adrenal steroid concentrations. For example, hypoglycemia (7 ) or physical exercise (8 ) are potent physiologic stressors, whereas fear (9 ), feelings of inferiority (10, 11 ), or experiences at school (12, 13 ) can affect the adrenal cortex activity as psychologic stressors. The taking of blood can also influence adrenal steroid concentrations in children; saliva collection, however, is almost stress-free (14 ). The use of saliva for steroid analysis in children is therefore an excellent alternative to blood.The aim of our study was to establish age-dependent reference values for salivary cortisol, 17␣-hydroxyprogesterone (17OHP), and progesterone in a large cohort of healthy children. The availability of such reference intervals will improve the applicability of saliva analysis as a diagnostic tool in pediatric endocrinology.We collected 252 saliva profiles from healthy children and adolescents (125 boys; age range, 4 days to 15 years; 127 girls; age range, 6 days to 13 years) with normal body length/height and weight. None of the girls had developed a regular menstrual cycle. The parents of the children gave informed consent.Saliva was collected either with the Salivette ® , using polyester swabs (Sarstedt), from children Ͼ1 year of age or with modified medical pacifiers (Bü ttner-Frank; see Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem. org/content/vol49/issue10/) from infants Ͻ1 year. The teats of the pacifiers were perforated, and strips of filter paper, as used for blood-spot collection, were inserted into the teats. The absorbent material had no effect on the steroid analysis in the RIAs, in contrast to the cotton Salivette, particularly for 17OHP (15 ) and progesterone (16 ).Samples were taken three times a day (0700, 1300, and 1900) at home. After sample collection, the saliva was immediately frozen and sent to the laboratory in a cooler. We tried to exclude any potential stressful events; thus the samples were taken only on so-called "quiet" days at home, e. g., days with no school-related stress or sports, or on weekends. All of the children participating in the study lived in an intact familial environment.We used adapted commercial RIAs (DSL), as described in detail elsewhere (17,18 ), that require 50 L of saliva per tube. The sensitivities and intra-and interassay CVs were 0.2 nmol/L (6% and 9.6%) for cortisol, 9 pmol/L (6% and 8.6%) for 17OHP, ...
