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Jornal de Pediatria

Print version ISSN 0021-7557

J. Pediatr. (Rio J.) vol.83 no.2 Porto Alegre Mar./Apr. 2007 



Steroid analysis in saliva: a noninvasive tool for pediatric research and clinical practice



Wieland Kiess; Roland Pfaeffle

Hospital for Children and Adolescents, University of Leipzig, Leipzig, Germany



Saliva is a readily available, noninvasive fluid that can be used to monitor the presence and concentration of a wide variety of drugs, hormones, antibodies and other molecules. Saliva-based diagnostic tests are now frequently used in endocrine practice, pediatric research, in clinical studies in psychology and psychiatry, as well as in stress research. In particular, the use of saliva as a vehicle for the determination of plasma steroid hormone levels in children and adolescents has increased dramatically. In this issue of Jornal de Pediatria, Silva et al. report on their use of salivary cortisol to assess the hypothalamic-pituitary-adrenal axis in healthy children aged 45 days to 36 months.1 The authors established reference values for salivary cortisol using their in-house radioimmunoassay employing an antibody directed against cortisol-3-oxime conjugated to bovine albumin. These data are precious in that they can be used for reference in both pediatric day-to-day routine practice as well as in pediatric research. They also offer insight into the development of circadian rhythms and the physiology of the endocrine system in the human infant.

It is important to note that during recent years, saliva collection devices using for example cotton or plastic swabs have been developed (e.g.: Salivette®, Sarstedt, Germany). Each laboratory should test for cross-reacting substances in these premanufactured devices since in some instances cross-reactivity of the immunoassays with substances contained in the coating of the swabs were encountered (personal observation). In addition, a number of agents that can provoke increased saliva flow such as 1 or 5% citric acid are being increasingly used when saliva samples are collected. In newborns and very young children, aspiration with a tube and syringe after only gentle stimulation of saliva flow without using chemical irritants is the most practical and less invasive method to use. In older children and adolescents, saliva collecting tubes which provide swabs and ready to use two-chamber devices that can be centrifuged to remove detritus and also used to freeze-store samples prior to analysis are recommended. Highly sensitive immunoassays employed as radioimmunoassays, enzyme-based, or time-resolved fluorescent immunoassays are widely available. However, it is important to establish reference values over the life span with all such methods separately and at each center, since specificity, mainly determined by cross-reactivity of the first antibodies with different steroid metabolites, as well as sensitivity of the assays differ widely. For example, morning cortisol saliva concentrations reported by Silva et al. average 557.86 nmol/L and range between 76.88 and 1,620.08 nmol/L,1 while Tornhage & Alfven report on morning levels of 8.8 nmol/L in older children2 and our own reference values for morning cortisol levels in saliva range from less than 2 nmol/L to 100 nmol/L in infants, children and adolescents.3 In adults, morning salivary cortisol levels in healthy volunteers are above 12 nmol/L, while late-night concentrations were between 1.0 and 8.3 nmol/L.4

Silva et al. found in their recent paper that the difference between morning and afternoon cortisol levels in saliva become more apparent with age.1 Indeed, increased morning cortisol levels become apparent within the first three months of life2,3,5,6 while in neonates no circadian rhythm of cortisol saliva concentrations is present.5,7 Also, Custodio et al. showed that the mean age of emergence of salivary cortisol circadian rhythm was similar in monozygotic and dizygotic twins (7.8 vs. 7.4 weeks). Seven twin pairs showed coincidence of the emergence of cortisol rhythm, while 10 pairs were not coincident: these data suggest less genetic than environmental impact on the age of onset of cortisol circadian rhythm.8

Recent clinical studies using morning salivary cortisol measurements revealed that there is a weak negative association of cortisol levels with length of gestation, but no relation to birth weight or to systolic or diastolic blood pressure.9 In a study enrolling 68 boys and 72 girls aged 7-9 years, birth weight in boys was inversely related to salivary cortisol responses to stress, while in girls morning peak cortisol was inversely related to birth weight.10 Salivary cortisol measurements can be used in association with the overnight 1 mg dexamethasone suppression test: salivary cortisol was suppressed to less than 100 ng/dL after 1 mg dexamethasone in both control and obese patients in one study.6 A negative correlation of morning salivary cortisol concentrations and total cortisol concentration with body mass index was found in children with recurrent abdominal pain of psychosomatic origin in another study.2 These authors also found in their cohort of 159 healthy girls and 147 boys that salivary cortisol concentration was dependent on the time of sampling, age and menarche. In our sample of 138 children and 14 adults, cortisol levels were also age-dependent and also positively associated with weight and body mass index while we did not find any degree of sex difference (Kiess et al.). In a cohort of 119 healthy neonates, body mass index, arterial cord blood pH and time of saliva sampling after birth influenced salivary cortisol levels.7

At the present time saliva sampling and steroid hormone measurements are still competing with urinary collection and measurement and plasma and serum measurement for bound and free steroid hormones. This is surprising, since there are a number of studies that have already shown superiority of salivary hormone measurements.11,12 However, if salivary cortisol collection and analysis is to be used more widely, the following considerations should be taken into account:

1)The time of sampling and collection of saliva needs to be standardized.

2)Food intake, medications and basal conditions such as sleep, stress, fasting/feeding have to be standardized or documented.

3)Appropriate highly specific (immuno)assays have to be employed and reference values for these specific assays have to be made available by each laboratory.

4)Quality controls and standard operational procedures (SOPs) have to be established at each center.

While cortisol measurements in saliva are of particular interest for researchers studying stress responses and the hypothalamic-pituitary-adrenal axis, the determination of other steroid hormones in saliva can be used to monitor treatment in endocrine disorders: one of the prime examples of the use of steroid hormone measurements in saliva in this respect is the determination of salivary 17-hydroxy-progesterone, the use of which greatly facilitates treatment of children and adolescents with congenital adrenal hyperplasia.13 In summary, the paper by Silva et al. is timely, important and very useful for the practitioner as well as for the researcher in all pediatric subspecialties.



1. Silva ML, Mallozi MC, Ferrari GF. Salivary cortisol to assess the hypothalamic-pituitary-adrenal axis in healthy children under 3 years old. J Pediatr (Rio J). 2007;83:121-6.

2. Tornhage CJ, Alfven G. Diurnal salivary cortisol concentration in school-aged children: increased morning cortisol concentration and total cortisol concentration negatively correlated to body mass index in children with recurrent abdominal pain of psychosomatic origin. J Pediatr Endocrinol Metab. 2006;19:843-54.

3. Kiess W, Meidert A, Dressendörfer RA, Schriever K, Kessler U, König A, et al. Salivary cortisol levels throughout childhood and adolescence: relation with age, pubertal stage, and weight. Pediatr Res. 1995;37(4 Pt 1):502-6.

4. Vogeser M, Durner J, Seliger E, Auernhammer C. Measurement of late-night salivary cortisol with an automated immunoassay system. Clin Chem Lab Med. 2006;44:1441-5.

5. Grunau RE, Haley DW, Whitfield MF, Weinberg J, Yu W, Thiessen P. Altered basal cortisol levels at 3, 6, 8 and 18 months in infants born at extremely low gestational age. J Pediatr. 2007;150:151-6.

6. Castro M, Elias PC, Martinelli CE Jr., Antonini SR, Santiago L, Moreira AC. Salivary cortisol as a tool for psychological studies and diagnostic strategies. Braz J Med Biol Res. 2000;33:1171-5.

7. Klug I, Dressendörfer R, Strasburger C, Kuhl GP, Reiter HL, Reich A, et al. Cortisol and 17-hydroxyprogesterone levels in saliva of healthy neonates: normative data and relation to body mass index, arterial cord blood pH and time of sampling after birth. Biol Neonate. 2000;78:22-6.

8. Custodio RJ, Junior CE, Milani SL, Simoes AL, de Castro M, Moreira AC. The emergence of the cortisol circadian rhythm in monozygotic and dizygotic twin infants: the twin-pair synchrony. Clin Endocrinol (Oxf). 2007;66:192-7.

9. Koupil I, Mann V, Leon DA, Lundberg U, Byberg L, Vagero D. Morning cortisol does not mediate the association of size at birth with blood pressure in children born from full-term pregnancies. Clin Endocrinol (Oxf). 2005;62:661-6.

10. Jones A, Godfrey KM, Wood P, Osmond C, Goulden P, Phillips DI. Fetal growth and the adrenocortical response to psychological stress. J Clin Endocrinol Metab. 2006;91:1868-71.

11. Schmidt NA. Salivary cortisol testing in children. Issues Compr Pediatr Nurs. 1998;20:183-90.

12. Hanrahan K, McCarthy AM, Kleiber C, Lutgendorf S, Tsalikian E. Strategies for salivary cortisol collection and analysis in research with children. Appl Nurs Res. 2006;19:95-101.

13. Dressendörfer RA, Strasburger CJ, Bidlingmaier F, Klug I, Kistner A, Siebler T, et al. Development of a highly sensitive nonisotopic immunoassay for the determination of salivary 17-hydroxy-progesterone: reference ranges throughout childhood and adolescence. Pediatr Res. 1998;44:650-5.

2. Tornhage CJ, Alfven G. Diurnal salivary cortisol concentration in school-aged children: increased morning cortisol concentration and total cortisol concentration negatively correlated to body mass index in children with recurrent abdominal pain of psychosomatic origin. J Pediatr Endocrinol Metab. 2006;19:843-54.