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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0492
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 10 3794-3803
Copyright © 2008 by The Endocrine Society

Role of Corticotropin-Releasing Hormone Testing in Assessment of Hypothalamic-Pituitary-Adrenal Axis Function in Infants with Congenital Central Hypothyroidism

David A. van Tijn, Jan J. M. de Vijlder and Thomas Vulsma

Department of Pediatric Endocrinology, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: David A. van Tijn, M.D., Ph.D., Department of Pediatric Endocrinology, Emma Children’s Hospital, G8-205, Academic Medical Center, University of Amsterdam, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. E-mail: tijn1{at}planet.nl.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: The Dutch neonatal congenital hypothyroidism (CH) screening program detects infants with CH of central origin (CH-C). These infants have a high likelihood of multiple pituitary hormone deficiencies. ACTH deficiency especially poses an additional risk for brain damage and may be fatal.

Objective: Our objective was to evaluate different tools for assessment of the integrity of the hypothalamus-pituitary-adrenocortex (HPA) axis in young infants, aiming for a strategy for reliable and timely diagnosis.

Design, Setting: This is a Dutch nationwide prospective study (enrollment 1994–1996). Patients were included if neonatal CH screening results were indicative of CH-C and HPA axis function could be tested within 6 months of birth.

Patients: Nine male and three female infants with CH-C and four infants with false-positive screening results or transient hypothyroidism were included in the study.

Main Outcome Measures: CRH test results, multiple cortisol plasma concentrations, and cortisol excretion in 24-h urine were measured.

Results: Six (50%) of the CH-C patients had abnormal CRH test results. Three of them had discordant test results: impaired increase of plasma cortisol in response to CRH, despite substantial increase of plasma ACTH. The other three infants, with concordant impaired responses of both ACTH and cortisol to CRH, had a very low urinary cortisol excretion in comparison with the subjects with normal CRH test results.

Conclusions: The CRH test proves to be a fast and reliable tool in the assessment of HPA axis (dys)function. It enables timely diagnosis in (asymptomatic) neonates at risk for serious morbidity and mortality. The discordant response type, which has not been described before, may be an early phase of HPA axis dysfunction. Alternatively, patients with this response type may constitute a separate pathogenetic subset of HPA axis-deficient patients.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
In neonates with multiple pituitary hormone deficiencies (MPHDs), ACTH deficiency, resulting in sustained hypoglycemia and neuroglycopenia, is a major cause of morbidity and mortality (1, 2, 3, 4). Under basal conditions, clinical signs of hypothalamus-pituitary-adrenocortex (HPA) axis dysfunction are usually absent, and even under stress they are seldom clearly indicative of hypocortisolism. In a former retrospective study (5, 6) on congenital hypothyroidism (CH) of central origin (CH-C), performed before the adaptation of the Dutch CH screening procedure (7, 8, 9), HPA axis dysfunction proved to be the major determinant of morbidity and mortality. Retrospectively, a mortality rate as high as 14% was estimated in all CH-C patients, of whom approximately 50% were diagnosed with HPA axis dysfunction. Unfortunately, of the different hypothalamus-pituitary-target organ axes, the HPA axis is probably the most difficult to assess in the neonate. Because during the first 6 months of life the circadian rhythm of cortisol excretion has not yet been consolidated (10), basal measurements of morning vs. evening plasma cortisol concentrations cannot be conclusive. Likewise, reference values for the salivary and urine cortisol excretion are not reliably available for neonates. The insulin-induced hypoglycemia test (11) and metyrapone test (12, 13), considered the standard investigations in adults and older children, are contraindicated in young children, due to the risk of provoking an acute adrenal crisis, resulting in severe and hazardous hypoglycemia (14). The short and long ACTH stimulation tests can be of some help, but as provisional screening tests only. A normal cortisol response to synthetic ACTH excludes complete adrenocortical insufficiency, but not partial insufficiency, that still hampers sufficient ACTH production to respond adequately to stress or hypoglycemia (14, 15). Probably the most complete test in the assessment of HPA axis function in the neonate is the CRH test, in which both the ACTH secretion by the pituitary gland and the subsequent cortisol secretion by the adrenal cortex can be assessed (16). Where in the performance of function tests using hypothalamic hormones TRH (17) or GnRH (18) the responses of the peripheral hormones (T4, T3, and 17β-estradiol/testosterone, respectively) do not enhance the diagnostic power of the test, the cortisol peak response to CRH is considered a valuable marker of HPA axis integrity (19). The relation between the ACTH and cortisol responses to CRH and the relative importance of both responses are among the main topics addressed in this paper. The overall aim of the study was to develop a diagnostic workup for fast and reliable assessment of HPA axis function in neonates with CH-C, detected by neonatal screening.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Study patients

The subjects, participants in a Dutch national prospective study, were enrolled from April 1, 1994, to April 1, 1996. All had neonatal CH-screening results indicative of CH-C, and subsequent plasma free T4 (FT4) concentrations below the predefined cutoff of 0.93 ng/dl (12 pmol/liter) (8) and plasma TSH concentrations less than 15 µU/ml (15 mU/liter). Anterior pituitary function was primarily assessed by stimulation tests. TRH and CRH tests took place on consecutive days as soon as the suspected CH-C patient was admitted to our department. Gonadotropic and somatotropic function was assessed around the age of 3 months, when euthyroid status had been accomplished by T4 supplementation. In addition, in most patients, magnetic resonance imaging (MRI) of the cerebrum was performed around the age of 3 months, to assess the anatomical integrity of hypothalamus and pituitary. See Ref. 8 for detailed study design and an overview of function test results. In 2001 and 2006/2007, all cases were reevaluated for revised diagnoses, additional morbidity, growth, and treatment data. The study protocol was approved by the Dutch Pediatric Endocrine Society and by the Medical Ethics Committees of the participating academic centers. Parental informed consent was obtained in all cases.

Patients excluded from the overall analysis (Fig. 1Go and Table 1Go)

The original study population consisted of 26 subjects suspected of CH-C. Consequently, these patients had a high likelihood of MPHDs, including HPA axis dysfunction (5, 6, 8).


Figure 1
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  		FIG. 1. Tree diagram of the enrollment of subjects suspected of CH-C on the basis of neonatal CH screening result in the CH-C study (8 ) and subsequent inclusion or exclusion from the current analysis of HPA axis function.

 

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  		TABLE 1. Perinatal data of all patients

 
Subjects 22–26 were considered too small or too ill to undergo a CRH test in the neonatal period and, therefore, had to be excluded from the overall analysis.

Subjects 17–21 were excluded from the overall analysis for the following reasons. Subject 17 was not referred and tested before the age of 2 yr, despite neonatal screening results indicative of CH-C. Because age differences may influence the test results (20), it was considered inappropriate to analyze his test results together with the results of the other subjects who underwent a CRH test within 6 months after birth. Subjects 18–20 were given cortisol supplementation as a safety first measure. After discontinuation of the cortisol treatment at age 1–5 yr, they had no signs of HPA axis dysfunction. Subject 21 developed HPA axis deficiency around 8 yr of age, as confirmed by repeated testing.

Patients included in the overall analysis (Fig. 1Go and Table 1Go)

From the remaining 16 subjects (12 boys), the age at inclusion in the study ranged from 14–137 d (median 38).

Subjects 1–12 had abnormal (type 2 or 3) TRH test results (8, 17), in addition to plasma FT4 less than 0.93 ng/dl (<12 pmol/liter) without elevated basal TSH concentration. Consequently, they were diagnosed with CH-C. Two subjects need some additional remarks. Subject 7, born in India, was screened and tested at the age of 4 months after the family had moved to The Netherlands. Subject 12, born to a mother with previously unnoticed Graves’ disease, had abnormal TRH test results (type 2 response) at the age of 17 d. By then, his FT4 concentration had spontaneously increased to 0.91 ng/dl (11.7 pmol/liter), close to the cutoff level of 0.93 ng/dl (12 pmol/liter). Therefore, further restoration of thyroid function could safely be awaited (21). As from the next determination, TSH and FT4 plasma concentrations remained within the normal range. After the CRH test, we refrained from further endocrine function testing and from MRI. Because a repeated TRH test at the age of 13 months demonstrated normal TSH secretion (type 0 response), this patient was diagnosed with transient CH-C, due to maternal gestational hyperthyroidism (8, 22).

Subjects 13–16 had normal (type 0) TRH test results (8, 17) and normal basal FT4 plasma concentrations, i.e. 0.93 ng/dl or more (≥12 pmol/liter) at the day of testing. After 5 yr follow-up, none of these four children had developed signs or symptoms of pituitary hormone deficiencies. Subject 13, a girl, had initially unrecognized deficiency of T4-binding globulin, a harmless variation that causes false-positive screening results. Subject 14, a boy with diminished FT4 plasma concentration at referral [0.83 ng/dl (10.7 pmol/liter)], without elevated plasma TSH, had normal TSH and FT4 concentrations at long-term follow-up. Subject 15, a boy with persistently low to low-normal FT4 values [0.75–1.00 ng/dl (9.7–12.9 pmol/liter)] and normal basal IGF-I and testosterone plasma concentrations, showed normal growth and psychomotor development. Subject 16, a boy with a FT4 plasma concentration close to the cutoff level at referral [0.89 ng/dl (11.5 pmol/liter)] and normal basal IGF-I and testosterone plasma concentrations, had normal TSH and FT4 concentrations, and showed normal growth and psychomotor development at long-term follow-up.

Hormone assays

All hormone assays were performed in the Academic Medical Center, Amsterdam. The two-site Nichols human 1–39 ACTH immunoluminometric assay kit (Nichols Institute Diagnostics Inc., San Juan Capistrano, CA) is specific for intact 1–39 ACTH and does not recognize ACTH-cleavage products. The lower limit of detection was 1 pg/ml. The intraassay coefficients of variation (CVs) were 3.7% at 31.8 pg/ml and 4.3% at 319 pg/ml; the interassay CVs were 5.1% at 31.8 pg/ml and 5.4% at 319 pg/ml. Because ACTH is very susceptible to proteolysis, all samples were collected in fresh EDTA containing tubes on ice, immediately centrifuged at 3000 rpm at 4 C and stored at –70 C until use. The laboratory of Endocrinology of the Academic Medical Center participated in an External Quality Assurance Scheme and scored with the ACTH assay in the middle of all participants. Cortisol was measured by fluorescence polarization immunoassay (TDx; Abbott Laboratories, North Chicago, IL). The lower limit of detection was 1.8 µg/dl (50 nmol/liter). The intraassay CVs were 6.4% at 7.2 µg/dl (200 nmol/liter), 5.8% at 13.4 µg/dl (370 nmol/liter), and 3.6% at 30.8 µg/dl (850 nmol/liter). The interassay CVs were 9.0% at 7.2 µg/dl (200 nmol/liter), 7.0% at 13.4 µg/dl (370 nmol/liter), and 4.7% at 30.8 µg/dl (850 nmol/liter). TSH and thyroid hormone assays were performed as previously described (8, 17).

Investigation of the HPA axis

All tests were conducted by the same investigator (D.A.v.T.). CRH tests were performed at 18–172 d (median 49) of age in the CH-C patients and 15–94 d (median 54) of age in the infants with false-positive screening results. Plasma ACTH and cortisol were measured before, and 5, 10, 15, 30, 45, 60, 120, and 150 min after iv administration of CRH [1–41 CRH (CRH-Ferring, Ferring BV, Hoofddorp, The Netherlands)] in a dosage of 1 µg/kg body mass. An adequate response was defined by an ACTH peak concentration of 80 pg/ml or more (≥17.6 pmol/liter) or four times baseline level and a cortisol peak concentration of 18 µg/dl or more (≥500 nmol/liter) (23, 24). In selected cases [children with adequate ACTH response but cortisol peak < 18 µg/dl (<500 nmol/liter)], a short, 60-min, ACTH test was performed using 1–24 ACTH (Synacthen; Ciba-Geigy, Basel, Switzerland) in a dosage of 20 µg/kg body mass. An adequate response to ACTH was defined by a cortisol peak concentration of 18 µg/dl or more (≥500 nmol/liter) (14). In all patients multiple random plasma cortisol samples were taken in the 24-h period between TRH and CRH tests; measurement of at least one random concentration of 18 µg/dl or more (≥500 nmol/liter) was considered safe (14). In addition, in 16 subjects the cortisol excretion in urine, collected in the same 24-h period, was determined. Because for the age group of our study patients, reference values are lacking in the literature, we cautiously focused on those for 2- to 6-yr-old children, in whom a urine cortisol content of 2.2 µg or more (≥6 nmol) per 24 h is considered the lower limit (14).

MRI of the hypothalamus-pituitary region

To examine pituitary morphology, MRI studies were performed under general anesthesia using a 1.5 tesla Siemens Magnetom (Siemens AG, Munich, Germany). Transversal (5 mm), sagittal (3 mm), and coronal (3 mm) T1-weighted spin-echo images were obtained. In the transversal plane, proton density as well as T2-weighted turbo-spin echo sequences were used. In addition, a T1-weighted three-dimensional series (Magnetization Prepared RApid Gradient Echo) was taken.

Statistical analysis

SPSS 10.1 (SPSS, Inc., Chicago, IL) was used for statistical computations. All reported P values are two sided. For all analyses, a two-tailed P value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Assessment of HPA axis function: profiling (Table 2Go)

The assessment of HPA axis function was based on CRH and ACTH tests, multiple random plasma cortisol samples taken in the 24-h period between TRH and CRH tests, determination of cortisol excretion in 24-h urine samples collected during this same interval, and long-term follow-up. For each patient the results of all endocrine examinations, including the other hypothalamic-pituitary axes, in combination with the results of cerebral MRI, added up to profiles on which overall diagnoses of HPA function were based. Diagnoses were reevaluated after 5 and 10 yr follow-up (false positives, 3–5 yr follow-up). In this way, six of the 12 patients (50%) included in the overall analysis were diagnosed as HPA axis deficient. All four subjects with false-positive screening results included in the overall analysis were diagnosed as having sufficient HPA axis function.


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  		TABLE 2. Assessment of HPA axis function of patients 1–21

 
Of the 12 CH-C patients included in the overall analysis, three showed diminished peak responses to CRH of both ACTH and cortisol (subjects 1–3). In addition, their highest measured random plasma cortisol concentrations and 24-h urine cortisol excretions were below the predefined cutoffs of 18 µg/dl (500 nmol/liter) and 2.2 µg/24 h (6 nmol/24 h), respectively. HPA axis function was considered deficient in these patients, and cortisol supplementation was initiated.

Another four infants (subjects 4–6 and 12) showed adequate ACTH peak response, but diminished cortisol peak response. This discordant response was considered abnormal (see Results). In two of these infants (subjects 4 and 5), a short ACTH test was also performed, producing diminished cortisol peak response. Although subject 4 had a 24-h urine cortisol excretion above the predefined cutoff, HPA axis function of both infants was considered deficient, and cortisol supplementation was initiated. In addition to the discordant CRH test result and a highest measured random plasma cortisol concentration below detection level [<1.8 µg/dl (<50 nmol/liter)], subject 6 was found to be deficient in GH, had abnormal GnRH test results, and an underdeveloped penis, whereas cerebral MRI showed posterior pituitary ectopia (PPE). This abnormal profile created a strong likelihood of HPA axis dysfunction, and cortisol supplementation was initiated. Subject 12 had transient CH-C, due to untreated maternal gestational Graves’ hyperthyroidism. In response to CRH, he showed an adequate ACTH peak concentration in combination with a near-normal cortisol peak concentration [17.4 µg/dl (480 nmol/liter)]. His highest measured random plasma cortisol concentration of 18.5 µg/dl (510 nmol/liter) was considered safe. His 24-h urine cortisol excretion was below the predefined cutoff. Although these results were not unequivocal, they created a strong likelihood of sufficient HPA axis function, and we refrained from cortisol supplementation. At long-term follow-up, he never showed clinical signs of HPA axis dysfunction.

Subjects 7–11 showed adequate peak response of both ACTH and cortisol to CRH. In addition, their 24-h urine cortisol excretion was above the predefined cutoff. Consequently, their HPA axis function was considered sufficient, and we refrained from cortisol supplementation. None of the latter patients had PPE on cerebral MRI, and so far, none has shown signs of pituitary dysfunction besides CH-C.

Of the four infants with false-positive CH screening results (subjects 13–16), three showed peak responses of both ACTH and cortisol to CRH above predefined cutoff as described in the Patients and Methods section. One infant (subject 15) showed an adequate ACTH peak response and a borderline cortisol response to CRH. The subsequent short ACTH test produced a safe cortisol peak response. His 24-h urine cortisol excretion was far above 2.2 µg (6 nmol) per 24 h, the lower limit in 2- to 6-yr-old children (14) that we used as a reference for our study population. The overall conclusion was sufficient HPA axis function, and we, therefore, refrained from cortisol supplementation.

Analysis of interrelations between the different investigations of HPA axis function

To evaluate the different determinants in the assessment of HPA axis function, both separately and combined, the interrelations between the different investigations were studied. CRH and ACTH test results were related to the highest measured random plasma cortisol concentrations and the 24-h urine cortisol excretion. Of 13 subjects for whom sufficient random cortisol samples were available, including the four subjects with false-positive screening results (Table 2Go), only six had one or more samples with a cortisol concentration of 18 µg/dl or more (≥500 nmol/liter), whereas 10 of 13 subjects for whom a 24-h urine analysis was available, including the four subjects with false-positive screening results, had a 24-h urine cortisol excretion more than or equal to 2.2 µg/24 h (≥6 nmol/24 h). The CRH-induced cortisol peaks were significantly correlated with the highest measured random plasma cortisol concentrations (Pearson; P = 0.012), but not with the 24-h urine cortisol excretion (P = 0.082). The CRH-induced ACTH peaks were not significantly correlated with the CRH-induced cortisol peaks (P = 0.141), or with the highest measured random plasma cortisol concentrations (P = 0.127) or 24-h urine cortisol excretion (P = 0.742).

Three infants (subjects 4–6) had adequate ACTH response but inadequate cortisol response to CRH. This response type was termed a discordant response, in contrast to a concordant response, in which the ACTH and cortisol responses are both normal or both impaired (concordant normal and concordant impaired responses, respectively). Subjects 1–3 had concordant impaired responses.

Finally, to evaluate the separate tests in the assessment of HPA axis function, we performed an analysis driven by the predefined hypothesis that CRH-induced ACTH and cortisol peaks, the highest measured random cortisol plasma concentrations, and the 24-h urine cortisol excretion would differ between HPA axis sufficient vs. deficient subjects. This hypothesis was tested using Mann-Whitney U tests. Significant differences were observed in the CRH-induced cortisol peaks (P = 0.001) and highest measured random cortisol plasma concentrations (P = 0.005), but not in the CRH-induced ACTH peaks (P = 0.415) or the 24-h urine cortisol excretion (P = 0.063). There were no significant differences between HPA axis sufficient vs. deficient subjects in age (n = 16; P = 0.301) or FT4 plasma concentration (n = 16; P = 0.745) on the day of testing.

MRI of the brain

Five of the 11 CH-C patients included and three of the five patients excluded from the overall analysis who underwent cerebral MRI (46 and 60%, respectively) had PPE (Table 3Go). Many had additional cerebral developmental disorders, such as dysgenesis of the corpus callosum, gray matter heterotopia, internal hydrocephalus, or extensive extracerebral hygromas. All patients with PPE had MPHDs. In all but one (subject 21), multiple deficiencies were already present at the age of 3 months. In subject 21 GH deficiency (and, thus, MPHDs) manifested at the age of 2.5 yr. Of the two patients with MPHDs without PPE, one (subject 7) was found to have anterior pituitary hypoplasia. Genetic investigations of this patient revealed compound heterozygosity for two mutations in the POU1F1 gene (25). The other (subject 4) had no manifest dysmorphia in the cerebrum.


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  		TABLE 3. Pituitary function evaluation, auxological, dysmorphic, genetic, and MRI abnormalities of subjects 1–21

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
An adaptation of the Dutch neonatal CH screening program in the mid-1990s resulted in enhanced detection of CH-C (7, 8, 9). Dutch pediatricians have not only become more aware of the occurrence of CH-C, but also of the accompanying pituitary hormone deficiencies in the majority of CH-C patients (8, 18). The early detection of congenital TSH deficiency enables early correction of the deficient thyroid hormone state. Thus, potential damage to the central nervous system can be significantly reduced (26, 27, 28). Congenital ACTH deficiency and consequent hypocortisolism, resulting in sustained hypoglycemia and neuroglycopenia, pose an even more acute and severe threat to the central nervous system and are potentially life threatening, especially in combination with GH deficiency (1, 2, 3, 4, 5). Therefore, embedded in the ongoing prospective study, we performed the current analysis to assess the fastest and most reliable way to test the integrity of the HPA axis in young infants.

During 24 months (in the yr 1994–1996), all Dutch children with CH screening results indicative of CH-C were enrolled in a prospective study. Because for the majority of the CH-C patients (and for all those included in the current analysis) the abnormal CH-screening result was the first sign of disturbance of the hypothalamus-pituitary system, the study design provided us not only with an early detected, but also with an unbiased study population.

An anticipated disadvantage of the study design was the fact that many of the patients had not yet reached euthyroidism at the moment of HPA axis testing. Theoretically, this might have influenced the functional integrity of the HPA axis and, thus, the test results. However, there were no significant differences between HPA axis sufficient vs. deficient subjects in FT4 plasma concentration on the day of testing.

Six of the 12 CH-C patients (and none of the four subjects with false-positive CH screening results) included in the analysis had abnormal CRH test results. Three of the six patients with abnormal CRH test results had discordant CRH test responses: impaired increase of plasma cortisol in response to an iv bolus injection of CRH, despite an ACTH peak plasma concentration above the predefined reference range (23, 24) (Table 2Go). We presume that this phenomenon (the discordant response type), which has not been described before, is a consequence of adrenocortex hypotrophia due to a lack of stimulation by ACTH, whereas apparently the adrenocorticotropic cell population in the pituitary gland is (still) able to respond substantially to exogenous CRH, at least at this early age (median 1.6 months). The discordant responses in these patients, of whom two (subjects 5 and 6) were found to have PPE on MRI, may indicate a predominant disturbance at the hypothalamic level or hindrance of the communication between hypothalamus and pituitary due to the insufficient portal venous connection between those two organs, as in PPE. We hypothesize that the insufficient production of cortisol might result in significant stimulation of the pituitary adrenocorticotropic cell population (via negative feedback control) in the latter infants, enough to enable a substantial increase in ACTH production in response to exogenous CRH. An alternative hypothesis, release of biologically inactive ACTH in response to exogenous CRH by these patients, is unlikely because ACTH is a small, unglycosylated polypeptide.

Is there any indication that the distinction between the two types of impaired CRH test responses is clinically relevant? Remarkably, among the patients with concordant impaired CRH test responses (both ACTH and cortisol response impaired), the 24-h urine cortisol excretion was very low in comparison with the subjects with normal CRH test results, whereas the only patient with a discordant CRH test response whose urine cortisol excretion was determined had an excretion in the normal range. Obviously, this set of observations is too small to support the hypothesis that the discordant response type indicates a less severe or variant type of HPA axis deficiency. As we are becoming more and more aware of gradually evolving pituitary hormone deficiencies (29), the discordant response as measured in these young infants may precede a concordant impaired type of response. Hopefully, future reports of similar investigations in young infants will further refine and validate the novel features reported here.

Although in comparison with the TRH and GnRH test responses (17, 18) the individual CRH test responses have a less uniform course (a graphical representation of the individual test results can be found on the JCEM web site at http://jcem.endojournals.org), the early data and 10-yr follow-up show that the CRH test is as valuable as the TRH and GnRH test in the diagnostic workup of pituitary hormone deficiencies in infants detected by neonatal CH screening. Especially in view of the existence of the discordant response type, the cortisol peak in response to CRH is the most informative marker of HPA axis integrity at this early age. This finding is in line with the statement made by Dickstein (19), that the cortisol rather than the ACTH response is the most clinically useful measure in the insulin-induced hypoglycemia test. Further fine-tuning is needed, especially regarding the influence age and maturation may have on the test results. Several authors [e.g. Karlsson et al. (16)] have already drawn attention to the fact that for infants, a cutoff of 18 µg/dl (500 nmol/liter) might be too high. In our study, two of the subjects with convincing evidence of sufficient HPA function (subjects 12 and 15) had CRH-induced cortisol peak values just below the predefined cutoff [17.0 µg/dl (470 nmol/liter) and 17.4 µg/dl (480 nmol/liter), respectively]. In addition, like Dickstein (19), who pointed out that the reproducibility of cortisol responses (to insulin induced hypoglycemia) is limited (30), we would like to underscore the importance of the establishment of a local set of reference values. Most important, in patients with a very high likelihood of HPA axis dysfunction, like in our study, the cutoff should be chosen very cautiously because unrecognized HPA axis dysfunction may be fatal. Cortisol peak levels around the cutoff should be interpreted with great care, and with regard to the clinical condition, before decisions are made. Regarding the clinical condition, we would like to point out that patients with HPA axis dysfunction (subjects 1–6) showed considerably more, and more severe, perinatal morbidity (especially persistent vomiting, pathological jaundice, and hypoglycemia), compared with those with TSH deficiency with (subjects 7 and 8) or without (subjects 9–12) GH deficiency, but without HPA axis dysfunction.

Using cutoff values of 18 µg/dl (500 nmol/liter) for maximum random or ACTH-induced cortisol concentrations and 2.2 µg/24 h (6 nmol/24 h) for urine cortisol excretion, we found concordance rates of 0.77 (10/13) between CRH test results and maximum random cortisol concentrations and 0.85 (11/13) between CRH tests and urine cortisol excretion. However, only six of 13 subjects in whom at least four random plasma samples were taken, including all four subjects with false-positive screening results, had one or more samples with a cortisol concentration of 18 µg/dl or more (≥500 nmol/liter), whereas in 10 of 13 subjects, including all four subjects with false-positive screening results, the urine cortisol excretion was found to be more than or equal to 6 nmol/24 h. Therefore, these determinations are not appropriate as sole or principal determinants of HPA axis integrity. Especially the 24-h cortisol excretion in urine does not serve the primary goal, which is prediction of HPA axis (dys)function under stress. An ACTH test cannot be used as the sole diagnostic criterion either because a normal test result does not exclude a partial HPA axis insufficiency that hampers sufficient ACTH production to respond adequately to stress or hypoglycemia (14, 15, 16).

In summary, the cortisol peak response to CRH is the most valuable marker of HPA axis function. Ten years of follow-up have taught us that it has the highest predictive value of all criteria evaluated in our study (Fig. 2Go). When clinicians become accustomed to the performance and interpretation of CRH tests, including the possibility of discordant ACTH and cortisol responses, the short ACTH test will remain of use only in newborns too ill or too small to undergo a CRH test. Possibly, the CRH test may be simplified regarding timing and number of samples; this will be the subject of future research. Multiple plasma cortisol sampling and determination of the 24-h urine cortisol excretion do further substantiate the diagnosis of HPA axis deficiency, but one cannot rely on these investigations as the sole or principal criterion in the diagnosis of congenital HPA axis dysfunction. Furthermore, the finding of PPE on cerebral MRI in a patient with CH-C urges for the assessment of HPA axis function because these patients invariably exhibit or develop MPHDs, including a very high likelihood of HPA axis deficiency (≥75% in the current study). Finally, neonates with CH-C or other pituitary hormone deficiencies and apparent normal HPA axis function according to test results in early infancy should be closely monitored throughout childhood and beyond because HPA axis dysfunction may develop later in life.


Figure 2
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  		FIG. 2. Flow diagram of the assessment of HPA axis function and therapeutic consequences: central role for CRH test. CRH test: ACTH and cortisol peak values, as measured in plasma in response to iv bolus of CRH. Random cortisol plasma measurements: four to six samples taken in the 24-h period between TRH and CRH tests. Urine cortisol excretion: as measured in the 24-h period between TRH and CRH tests. ACTH test: highest of cortisol plasma concentrations 30 and 60 min after iv bolus of ACTH. a, FT4 plasma concentration less than 0.93 ng/dl (<12 pmol/liter) and TSH plasma concentration less than 15 µU/ml (<15 mU/liter) (8 ). b, Perinatal signs such as hypoglycemia, persistent vomiting, and hyperbilirubinemia and/or elevated transaminases could indicate HPA axis dysfunction; central hypothyroidism, hypogenitalism/maldescensus testis, hyponatremia, small for gestational age/poor growth and midline defects, septooptic dysplasia, and (other) facial dysmorphias may provide circumstantial evidence. c, An adequate response to CRH was defined by an ACTH peak concentration of 80 pg/ml or more (17.6 pmol/liter) or four times baseline level (23 24 ). d, An adequate response to CRH was defined by a cortisol peak concentration of 18 µg/dl or more (≥500 nmol/liter) (23 24 ). e, The cortisol response may be considered the best bioassay for ACTH; a normal cortisol response proves normality of ACTH (19 ). f, Under basal conditions at least one random plasma concentration of 18 µg/dl or more (≥500 nmol/liter) (14 ) was considered safe. Because for the age group of our study patients reference values are lacking in the literature, we cautiously focused on those for 2- to 6-yr-old children, in whom a urine cortisol content more than or equal to 2.2 µg (≥6 nmol) per 24 h is considered the lower limit (14 ).

 


   Acknowledgments
 
We are indebted to the patients and their parents; to the referring pediatricians and pediatric endocrinologists; to Brenda Wiedijk, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, for her indispensable assistance in collection of the data from all over the country; to Erik Endert, for sharing his expertise in clinical-chemical matters concerning the adrenal gland; to Heather Houlihan for review of the English syntax.


   Footnotes
 
This work was supported by Grant 28-1060-2 (to J.J.M.d.V. and T.V.) from The Netherlands Organization for Health Research and Development (ZONMW, The Hague, The Netherlands).

Disclosure Summary: The authors have nothing to disclose.

First Published Online July 22, 2008

Abbreviations: CH, Congenital hypothyroidism; CH-C, congenital hypothyroidism of central origin; CV, coefficient of variation; FT4, free T4; HPA, hypothalamus-pituitary-adrenocortex; MPHD, multiple pituitary hormone deficiency; MRI, magnetic resonance imaging; PPE, posterior pituitary ectopia.

Received March 3, 2008.

Accepted July 10, 2008.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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