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Hospital for Children and Adolescents, Friedrich Alexander University, 91054 Erlangen, Germany
Address all correspondence and requests for reprints to: Helmuth G. Dörr, M.D., Klinik mit Poliklinik für Kinder und Jugendliche, Loschgestrasse 15, 91054 Erlangen, Germany. E-mail: . hgdoerr{at}kinder.imeduni-erlangen.de
Abstract
We have analyzed the kinetics of salivary cortisol (F) and 17-hydoxyprogesterone (17OHP) after a single oral administration of hydrocortisone (HC; 10 mg; 0700 h) in healthy male volunteers (n = 10; 18–29 yr) and in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (males, n = 7; females, n = 3; 8.5–20.4 yr). The HC doses, related to body surface area, ranged from 6.3–9.2 mg/m2 in controls and from 4.2–10.7 mg/m2 in CAH patients. Saliva was collected over 5 h (at intervals of 15–30 min), and the steroids were measured with adapted RIAs. In healthy controls, maximal cortisol values (250.3 ± 35.9 nmol/liter) were reached after 30 min. Values showed a monophasic decrease. A t1/2 of 94.5 min was calculated. The proportion of the HC dose in the total area under the curve was 71.2 ± 3.2%. For 17OHP, a monophasic decrease was found, with a minimum level of 48 ± 27 pmol/liter after 300 min. In CAH patients the salivary steroid profiles showed individual kinetics (maximal cortisol values ranged from 107–726 nmol/liter). Here a monophasic decrease was found with a shorter t1/2 of 56.4 min. The HC dose proportion in the area under the curve was 88.3 ± 6%. 17OHP showed biphasic courses with a decrease to the minimum 17OHP level after 210 min at the latest and a subsequent gradual increase. Our findings of limited normalization of the adrenal cortex by oral HC administration underlines the necessity of optimizing therapy control and indicates the usefulness of kinetic studies for the judgement of therapy in CAH patients.
THE CORTISOL DEFICIT in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (CYP21) deficiency is substituted by physiological administration of oral glucocorticoids. The main target of the therapy is to enable normal growth and pubertal development. In practice, hydrocortisone (HC) is favored for the therapy of infants and children (1, 2, 3, 4) because it is well tolerated and is more convenient due to its shorter half-life compared with dexamethasone (5, 6, 7). Additionally, it has no influence on growth (8). Tablets of HC are administrated three times daily, with highest doses in the morning and lower doses at noon and in the evening. This procedure mimics the physiological diurnal rhythm and is used to normalize the activity of the hypothalamo-pituitary-adrenal axis (9, 10, 11). In this context the kinetics of cortisol (F) in plasma and serum after oral administration of HC have been studied (12, 13, 14), and it has been shown that the bioavailability of orally administered HC is high in the morning, with a peak at 1–2 h and a monoexponential decline thereafter (15).
In the therapeutic control of CYP21-deficient patients, the advantage of salivary steroid measurements has been accepted for many years. The main advantages of steroid measurements in saliva samples are the noninvasiveness of the sample collection (16, 17) and the fact that salivary steroids reflect the physiologically active fraction (18, 19, 20). In pediatric practice, saliva is collected at different hours during the day at home, and 17OHP is analyzed. To optimize CAH therapy, it would be advantageous to know how long a single oral HC dose can affect salivary 17OHP values. To answer this question, it is necessary to study hydrocortisone kinetics in saliva of CAH patients. However, to the best of our knowledge, this has not yet been done.
The aim of our study was to investigate the kinetics of F and 17OHP in saliva of CYP21-deficient patients and healthy controls after a single morning dose of HC to obtain information on the regulatory effects on the adrenal cortex and to establish salivary kinetic studies as an additional tool for the optimization of the therapeutic control of CAH patients.
Materials and Methods
Sample collection
Saliva was collected in 10 children and adolescents (males, n = 7; females, n = 3) with salt-wasting CAH due to 21-hydroxylase deficiency. Their chronological age was 8.5–20.4 yr, the body mass index varied from 18.7–26.0 kg/m2. The daily dose of HC (Hoechst, Darmstadt, Germany) was 10.8–17.9 mg/m2 body surface area, whereby the morning dose varied from 4.2–10.7 mg/m2. Additionally, the patients had morning doses of Astonin H (9
-fludrocortisone; Merck, Darmstadt, Germany) between 15 and 30 µg/m2. The adequacy of mineralocorticoid replacement was controlled by normal serum renin concentrations. All 3 female patients were premenarcheal. The clinical data of the patients are shown in Table 1
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Saliva was obtained at home using polyester Salivettes (Sarstedt, Nurnbrecht, Germany). Collection times were as follows: the first sample before the morning tablet (t0; 
0700 h), the next four samples after administration at 15-min intervals (t15–t60), and the last eight samples at intervals of 30 min (t90–t300). Saliva was stored frozen until measurement to avoid microbial decomposition of the steroids (23).
The patients and healthy volunteers gave informed consent to participate in this study, which was approved by the local ethics committee.
Measurements
The concentrations of F and 17OHP were determined with commercial RIAs (Diagnostics Systems Laboratories, Inc., Sinsheim, Germany) adapted for the use of saliva as sample matrix as previously described (24, 25). Intraassay variance of the F assay was 7.9% for 2.8 nmol/liter and 6.1% for 27.6 nmol/liter. Interassay variance was 11.2% for 2.8 nmol/liter and 9.6% for 27.6 nmol/liter, respectively. The lower determination limit was 0.22 nmol/liter. Coefficients of variation of the 17OHP assay were 6.2% for 90 pmol/liter and 5.9% for 30,000 pmol/liter (intraassay), and 9.5% for 90 pmol/liter and 8.6% for 30,000 pmol/liter (interassay), respectively. The determination limit was 12 pmol/liter.
Statistics
Statistical calculation was performed with PRISM software (GraphPad Software, Inc., San Diego, CA) using nonlinear regression analysis. Data are either presented as the mean ± SEM or as total ranges. A one-compartment model with first order absorption and elimination was fitted to the data. The fit was evaluated between observed and predicted data. The area under the curve (AUC) was calculated for F concentrations. To assess the proportion of the HC dose in the AUC, the physiological proportion was subtracted. This proportion was calculated by the linear trapezoidal method.
Results
Ranges of salivary F and 17OHP levels of healthy controls are given as the mean and SD (Fig. 1). Baseline F levels (33.1 ± 11 nmol/liter) increased significantly (P < 0.0001) to 251.2 ± 36 nmol/liter (Fmax) after 30 min. From 45 min on, salivary F levels showed a monophasic decrease and reached a minimum level of 13.2 ± 4.4 nmol/liter after 300 min (r2 = 0.96; P < 0.001). A t1/2 of 94.5 min was calculated for an average dose of 7.5 mg HC/m2 body surface. As F concentrations in the three volunteers without any tablet administration showed a linear decrease between t0 and t300, this physiological proportion was calculated for each volunteer in the same way. The mean AUC of total salivary F during the experiment was 27.13 µmol/liter with a proportion of the HC dose of 71.2 ± 3.2% (Fig. 2).
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). The basal level of 126 ± 33 pmol/liter declined after 300 min to 48 ± 27 pmol/liter. The correlation was the same as for F (r2 = 0.96; P < 0.001).
In contrast, F and 17OHP levels were different in CAH patients. According to the HC dose (4.2–10.7 mg/m2), Fmax ranged from 106.5–725.9 nmol/liter. Individual kinetic data are shown in Table 2. A monophasic decrease in F values was common to all patients (Fig. 3A). At the end of the study the lowest levels between 0.28–16.8 nmol/liter were measured. A mean t1/2 of 56.4 min was found (range, 10.6–139.4 min). In CAH patients, the AUC proportion of the HC dose in salivary F was 88.3 ± 6% (range, 75.2–94.1%). Salivary 17OHP levels also showed variable courses. Beginning with basal levels between 690-9150 pmol/liter, a biphasic course of 17OHP was found in all patients. With one exception (patient 3), the minimum concentration of 17OHP obtained after single HC application (17OHPmin) was observed after 180 min at the latest. Thereafter, the levels increased again (Fig. 3B).
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). The influence of HC on salivary 17OHP concentrations was different in the studied groups. Minimal 17OHP values were reached in healthy subjects toward the end of the study (270–300 min) with only one exception (17OHPmin after 210 min; Fig. 4B). In contrast, 17OHPmin was reached in CAH patients after 210 min (median, 180 min). In one patient (patient 7), 17OHPmin was reached after only 60 min. After 300 min, elevated 17OHP levels (up to 3690 pmol/liter) were found again (Table 2).
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The adequacy of glucocorticoid therapy in children with CAH due to 21-hydroxylase deficiency can be assessed by measuring different serum/plasma steroids, such as 17OHP, 21-deoxycortisol, androstenedione, and T (girls and prepubertal boys); 24-h urinary steroid excretion of pregnanetriol; and/or 17OHP in saliva (1, 25, 26). The advantage of salivary steroid measurements of 17OHP as an alternative to serum analysis is widely accepted in the treatment of CAH (27, 28, 29). The noninvasiveness of sample collection allows for large numbers and high densities of samples. However, studies of salivary kinetics of oral administrated HC and its effects on salivary 17OHP levels in CAH patients have not yet been published, whereas studies on the kinetics of HC in serum have already underlined the usefulness of performing individual profiles for optimizing the CAH therapy (14, 15). The results of these studies did not include changes in 17OHP levels in plasma. However, data on cortisol kinetics in plasma after oral HC administration described in these studies correspond well with our findings on salivary cortisol.
In our study we found clear differences between the kinetics of F and 17OHP in saliva of healthy volunteers and CAH patients. The rapid increase to Fmax and the shorter half-life of HC in CAH patients limit the suppression of the hypothalamo-pituitary-adrenal axis for longer than 3 h with a breakthrough of adrenal androgen secretion. Thus, 17OHP values again increased to pathological values in CAH, whereas in controls both F and 17OHP show normal physiological levels at the end of the study (23, 24, 30, 31). The CAH patients had different salivary steroid levels and kinetic statistics. For example, in patient 7, a turnaround of the 17OHP level was found after 60 min (30 min after Fmax), with nearly the same levels as at baseline. Our results suggest that the metabolism and clearance of HC is more rapid in CAH patients than in healthy controls. This could be caused by lower concentrations of corticosteroid-binding globulin, which is responsible for buffering endogenous and/or administrated cortisol. However, this discussion remains speculative, because to the best of our knowledge data on corticosteroid-binding globulin levels in CAH patients compared with those in healthy subjects are not yet available. Further investigations need to address this important point. The variability in 17OHP levels between different CAH patients suggests that other factors (e.g. genotype) may be involved in the feedback on 17OHP kinetics.
The finding of a restricted suppression of 17OHP levels in CAH patients suggests that HC might be inappropriate for the management of this disease. Accepting this conclusion, the strategy to use HC as the preferred drug for the treatment of CAH in children must be reevaluated. To normalize 17OHP levels, it might be useful to shorten the time intervals between the HC doses by inserting an additional dose after 3 h (e.g. splitting the morning dose). However, this strategy can cause compliance problems particularly in schoolchildren. Thus, dexamethasone with its longer half-life might be more appropriate. This is supported by a recent study showing that carefully adjusted doses of dexamethasone are at least as effective as HC in the treatment of CAH (32).
In summary, we have shown that the analysis of individual steroid kinetics can also be obtained from salivary profiles. It remains to be shown by long-term studies whether this might be useful for the optimization of CAH therapy. The differences in the salivary kinetics of F and 17OHP between CAH patients and healthy controls are related to the shorter half-life of HC in CAH patients, resulting in an early re-increase in 17OHP concentrations. The wide spectrum of the elevated salivary 17OHP levels among the CAH patients underlines the necessity for an individually tailored therapy.
Acknowledgments
We thank Mrs. Jutta Biskupek-Sigwart for her technical support, and Mrs. Patricia Schmid for linguistic editing.
Footnotes
Abbreviations: AUC, Area under the curve; CAH, congenital adrenal hyperplasia; F, cortisol; Fmax, maximal cortisol values; HC, hydrocortisone; 17OHP, 17-hydoxyprogesterone; OHPmin, minimum concentration of 17OHP obtained after single HC application.
Received May 31, 2001.
Accepted November 26, 2001.
References
-fluorhydrocortisone in the treatment for congenital adrenal hyperplasia. Eur J Pediatr 144:370–373[CrossRef][Medline]
-hydroxyprogesterone levels by filter paper blood spots in patients with 21-hydroxylase deficiency. Horm Res 44:247–252[Medline]
-hydroxyprogesterone in congenital adrenal hyperplasia. Clin Endocrinol (Oxf) 11:631–637[Medline]
uig A, Schwarz HP, Strasburger CJ 1995 Salivary cortisol levels throughout childhood and adolescence: relation with age, pubertal stage, and weight. Pediatr Res 37:502–506[Medline]
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: CAH-patients (n = 10); 
: healthy subjects (n = 10). Fmax: Maximal concentrations of cortisol reached after single application of hydrocortisone tablet; tmax: time to reach Fmax. B, Relationship between time and minimal 17OHP levels: