0021-972X/92/7401-0198$03.00/0 .Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society
Vol. 74, No. 1 Printed
in
L’.S.A.
Pituitary-Adrenal Responses to Corticotropin-Releasing Hormone in Different Degrees of Adrenal 2 lHydroxylase Deficiency* AYRTON Endocrinology
C. MOREIRA Division,
AND LUCILA
Department
of
Medicine,
L. K. ELIAS Faculty of Medicine,
ABSTRACT. 21-Hydroxylase congenital adrenal hyperplasia (21-OHCAH) involves a orimarv defect of the adrenal gland and a secondary hrvolvement of ACTH secretion. The resbonses of the pituitary-adrenal axis to ovine CRH (oCRH, 1 fig/kg) were examined in subjects with different degrees of 21-OH deficiency. We studied 43 subjects: 7 classical and 6 nonclassical (NC) 21OHCAH patients,-15 heterozygotes (HT) and 15 control subjects. Baseline plasma ACTH levels were higher in classical CAH than in NC-CAH, HT, and control subjects (mean -t SEM, 66 + 14, 6 + 1.6, 4 f 0.5, and 5 + 0.5 pmol/L, respectively). The mean plasma ACTH resoonse to oCRH in NC-CAH (17 & 3 pmol/L) was higher than-in controls and HT (9 C 0.8 and 11 f 1.5 pmol/L). The highest ACTH responses to oCRH were obtained for classical CAH patients (126 f 29 pmol/L). Plasma
14049-RibeirCo
Preto, SP, Brazil
cortisol rose after oCRH in control, HT, and NC-CAH patients but did not change in classical CAH. After oCRH, plasma 17hydroxyprogesterone (17-OHP) were 4 * 0.5, 8 + 1.6, 93 f 28, and 359 + 110 nmol/L for controls, HT, NC-CAH, and classical patients, respectively. There was a significant correlation (r = 0.70) between 17-OHP and the ACTH resaonses to oCRH. The 17-OHP responses to oCRH were also correlated (r = 0.94) with the 17-OHP responses to the synthetic ACTH test. We conclude that the release of endogenous ACTH by oCRH result in graded 17-OHP responses on 21-OH deficiency. The present study also suggests that different degrees of adrenal biosynthetic defect may result in graded ACTH responses to oCRH. (J Clin Endocrinol Metab 74: 198-203, 1992)
I
N THEIR early studies, Wilkins et al. (1) and Bartter et al. (2) suggested that the secretory activity of pathological adrenal glands is dependent upon the tropic function of the pituitary in congenital adrenal hyperplasia (CAH). A partial or complete inability to synthesize cortisol in 21-hydroxylase (21-OH) deficiency CAH results in compensatory hypersecretion of ACTH. Later, elevated baseline plasma ACTH levels were demonstrated in classical 21-OH CAH patients (3, 4). In the last decade, improved biochemical assessment of adrenal function associated with HLA serotyping and molecular genetic evaluations have established graded severities of steroidogenic dysfunction in 21-OH CAH ranging from classical and nonclassical (NC) CAH to heterozygous carriers (5). Although the responses of ACTH to ovine CRH (oCRH) have been recently reported in two studies on NC-CAH patients (6, 7), detailed information on the pituitary-adrenal responses to oCRH in subjects with graded severities of 21-OH deficiency is lacking. The
purpose of the present investigation was to determine whether or not different degrees of adrenal biosynthetic defect would result in graded plasma ACTH and 17hydroxyprogesterone (17-OHP) responses to oCRH. We examined the responsiveness of the pituitary-adrenal axis to oCRH in patients and heterozygotes (HT) for 21OH deficiency. Materials
and Methods
Subjects
Forty-three subjects were divided into four groups as follows. 1) Seven classical 21-OH CAH patients (five females and two males ranging in age from 3-30 yr). Two patients had never been treated and five had stopped glucocorticoid therapy 10 days before the study. 2) Six NC 21-OH CAH patients (three females and three males, ranging in age from 7-29 yr). They had never been treated. Two patients were found to be compound HT by pedigree analysis, using HLA markers in conjunction with hormonal data. 3) Fifteen HT (six females and nine males, ranging in age from 4-44 yr). Twelve subjects were parents (obligate HT) and three asymptomatic subjects were brothers of classical CAH patients. The assignment of heterozygos-
Received December 14, 1990. Address correspondence and requests for reprints to: Ayrton C. Moreira, M.D., Department of Medicine, Faculty of Medicine, 14049, Ribeirb Preto, SP, Brazil. *This work was supported by Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq Grant 30.0834/82-91), Funda$50 de Amparo a Pesquisa do Estado de SHo Paulo and Hospital das Clinicas de RibeirGo Preto (HCFMRP-FAEPA). 198
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
CRH
TEST
IN 21-HYDROXYLASE
ity for the CAH gene was based on the presence of one HLA haplotype in common with the affected sib and plasma 17-OHP response to the ACTH test (5). 4) Fifteen control subjects (seven females and eight males, ranging in age from 9-28 yr). Twelve subjects were from the general population and three subjects were unaffected homozygous sibs who shared no HLA haplotypes with the affected CAH sib. All subjects were studied at the Endocrinology Division of the University Hospital (Ribeirao Preto, SP, Brazil) after informed consent was given by them or their parents. Postmenarchial females were all studied during the early follicular phase. CRH and ACTH testing
All tests were started at 0900 h after an overnight
fast.
CRH test
oCRH (1 pg/kg; Bachem, Torrance CA) was given as an iv bolus injection. Blood samples for hormone assays were collected 15 min before and 0, 15, 30, 45, 60, 90, and 120 min after the injection. Plasma ACTH, cortisol, and 17-OHP were measured at all sampling times. ACTH
test
ACTH (0.25 mg tetracosactrin; Organon, Sao Paulo SP) was administered iv over 60 s. Blood samples for 17OHP and cortisol measurement were obtained before and 60 min after the injection. Hormone assays
ACTH was determined by RIA after extraction from plasma using silicic acid (8). The assay sensitivity was 2 pmol/L. The intra- and interassay coefficients of variation (CVs) were 6% and 18%, respectively. Plasma cortisol and 17-OHP were determined by previously described RIA methods after ethanol extraction and extraction with ether and Sephadex LH-20 chromatography, respectively (9, 10). All steroid assays had intrassay CVs between 34% and interassay CVs between 7-16%. The assay sensitivity was 40 nmol/L for cortisol and 0.5 nmol/L for 17-OHP. All the samples obtained from each patient on both tests were analysed in duplicate in the same assay to avoid interassay variability. HLA typing was performed on peripheral blood lymphocytes using the standard microcytotoxicity test (11).
DEFICIENCY
199
concentrations. The integrated or mean ACTH, cortisol, and 17-OHP responses after oCRH administration were determined by calculating the sum or mean of all hormone values from 15 to 120 min. Statistical analysis of the results was performed using the Wilcoxon rank sum test and Spearman’s rank correlation procedure when appropriate. Differences of less than 0.05 were considered significant. Results Responsesof ACTH, cortisol, and 17-OHP to oCRH
The mean (~SEM) baseline plasma ACTH, cortisol, and 17-OHP values and their responses to oCRH in patients with 21-OH CAH deficiency, heterozygous carriers and control subjects are shown in Table 1 and Figs. 1 and 2. It can be seen that basal ACTH values were not different among controls, HT, and NC-CAH patients. However, the three groups had significant lower basal plasma ACTH than classical CAH patients. The mean plasma ACTH response in the HT after oCRH administration was 11 f. 1.5 pmol/L, slightly but not significantly higher than that in the control subjects (9 & 0.8 pmol/L). The ACTH responses to oCRH were higher (P < 0.05) in the NC-CAH patients (17 + 3 pmol/L) than in controls or heterozygous subjects. The highest ACTH responses to oCRH were obtained for classical CAH patients (126 + 29 pmol/L). The mean basal plasma cortisol levels were similar in the four groups. Plasma cortisol rose (P < 0.05) after oCRH compared to the basal levels in controls, HT, and NC-CAH patients but did not change in classical CAH patients. Baseline plasma 17-OHP levels were elevated (P < 0.001) in NC-CAH (12 + 1.8 nmol/L) and classical (303 + 99 nmol/L) patients when compared with HT and control subjects (3 f 0.4 nmol/L). The mean post-oCRH plasma 17-OHP level increased (P < 0.05) to 4.0 + 0.5, 8.0 & 1.6, and 93 k 28 nmol/L, in controls, HT, and NCCAH subjects, respectively. The high baseline plasma 17-OHP levels of classical CAH patients did not increase significantly after oCRH (359 + 110 nmol/L). The 17OHP responses to oCRH were higher (P c 0.001) in NCCAH patients than in HT. In addition, the mean 17OHP response of the latter group was significantly higher than those of the control group. However, some individual values overlapped those of the control subjects. Figure 3 shows a positive Spearman’s rank correlation between the time-integrated sum of the ACTH levels and 17-OHP levels after oCRH in all subjects as a group (r = 0.70, n = 41; P < 0.0001).
Statistical analysis
Comparison between adrenal hormone responsesto oCRH and ACTH stimulation test
Data are expressed as the mean + SEM. The baseline level was calculated as the mean of the -15 and 0-min
The mean (~SEM) plasma 17-OHP 60 min post-ACTH levels and peak post-oCRH 17-OHP levels were 4.5 f
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
200
MOREIRA
AND
ELIAS
J(‘E L M. 1%)” Vol 74. No 1
TABLE 1. Mean @EM) plasma ACTH, cortisol, and 17-OHP concentrations under baseline conditions and their mean responses after oCRH (1 pg/kg) in controls, heterozygous subjects, and NC and classical patients with 21-OH deficiency ACTH (pmol/L)
Variable or group
(n)
Controls
Basal
15
HT
5
15
NC patients
4
6
Classical patients
*
Mean response
0.5
+ 0.5
6-c
1.6
9 0.8 11 1.5 17 3o.h
f
126 2g0.h.’
+
66+ l.+’
7
Cortisol (nmol/L)
+
Basal
Mean response
300 -c 22
500 30 490 40 4x0
210
-e22 *
260
?I 25
50 220 r$jd’
240
f
60
17-OHP (nmol/L)
-
Basal
f
Mean response
3
f
f
9
+-
*
0.4 12
*
-c
303
r
4.0 f 0.5
0.4
8.0
f
1.6
1.8”.b
gg4.b.c
93
+
359 11(yLL’
”
‘2p.b
--.-_.
’ P < 0.05 us. controls. ’ P < 0.05 us. HT. ‘PC 0.05 us. NC.
250,~ 200-
IT
B
*
250
8
.-200
150
!” 0 0 . e
- 150 8
-L 11 u
-1 looi& 50 ’ 25 z E Lx 20? z
- 100
me: 0 _ -0. . _ .
-50 -35
0 0
0. . -L
-
0ON
-15
B
-‘O
t
15-
30
$’ 5-
II . iSCQ5 E :450-
450
I- l 0+3o -E-15
0
I 15304560 TIME
I
I (mln)
I 9C ’
lZO
0
I 15
111 30 45 TIME
a-
15&
0
8
-3
-5
0
t? I= g300v
I30 1
@
:g.
10-
.
MEAN RESPONSE
FIG. 1. A, Mean (+SEM) plasma ACTH (upper panel) and cortisol (lower panel) responses to oCRH (1 fig/kg) given at time zero in 7 patients with classical CAH (Cl), 6 patients with NC-CAH (m), and 15 HT for 21-OH deficiency (0). The hatched area represents the mean (*SD) for 15 controls (0). B, Individual values of mean response to oCRH for the 4 groups.
0.5 us. 4.0 It 0.5 nmoi/L (P = NS) for controls; 15 f 3.0 us. 11.5 & 2.2 nmol/L (NS) for HT; 142 + 21 us. 120 + 32 nmol/L (NS) for NC-CAH patients, and 626 + 191 us. 435 + 122 nmol/L (NS) for classical patients, respec-
60
(mln)
I
I
90
12
MEAN RESPONSE
FIG. 2. A, Mean (+SEM) plasma 17-OHP responses to oCRH (1 rg/ kg) given at time zero in 7 patients with classical CAH (Cl), 6 patients with NC-CAH (B), and 15 HT for 21-OH deficiency (0). The hatched area represents the mean (&SD) for 14 controls (0). B, Individual values of mean response to oCRH for the 4 groups.
tively. There was a positive Spearman’s rank correlation between 17-OHP responses to oCRH and ACTH administration in all subjects as a group (r = 0.94; n = 36; P < 0.0001; Fig. 4). The mean plasma cortisol levels obtained 60 min postACTH and the peak cortisol levels obtained post-oCRH were 660 + 30 us. 670 + 40 nmol/L for controls; 700 f 40 us. 610 + 50 nmol/L for HT; 410 + 20 us. 600 -C 75 nmol/L for NC-CAH patients, and 260 + 60 us. 290 f 65 nmol/L for classical CAH patients, respectively. In each group, plasma cortisol peak responses to oCRH were similar (P = NS) to those obtained after ACTH administration. In addition, peak cortisol responses of NC-CAH after exogenous ACTH were slightly but sig-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
CRH
TEST
IN
Zl-HYDROXYLASE
control tients.
0 Cl
201
DEFICIENCY
and heterozygous
subjects or of NC-CAH
pa-
0 0
Discussion
0
0
r= 070 pc 0.0001
I 20
.~.....I SUM
200
OF ACTH POST
..A
2000
( pmol / L )
oCRH
FIG. 3. Correlation between the sum of ACTH levels and the sum of plasma 17-OHP concentrations after oCRH in control (0) and heterozygous (0) subjects and patients with classical (Cl) and NC (m) 21-
0 no 0
0.3 t 0.3
I 3 IT-OHP
*
* * . . ., , , , . * . , .I , 30 300 ( n mol/L ) 60 min POST ACTH
. . . 4 3000
FIG. 4. Correlation between the 60-min ACTH stimulated plasma 17OHP level and the peak oCRH stimulated plasma 17-OHP level in control (0) and heterozygous (0) subjects and in patients with classical (Cl) and NC (W) 21-OH CAH.
nificantly lower than those of the control and heterozygous subjects. However, cortisol responses after oCRH did not differ among the three groups. The peak cortisol responses of classical CAH patients to oCRH or exogenous ACTH were significantly lower than those of the
The pathophysiology of 21-OH deficiency CAH involves a primary defect of the adrenal gland and a secondary increase of pituitary ACTH production. Although this dual gland interaction mechanism had been suggested as early as 40 yr ago (1, 2), the adrenal steroidogenesis defect has been more extensively studied than its pituitary counterpart (5, 12, 13). The present study demonstrates that the ACTH responses to oCRH taken as a whole, resulted in a spectrum ranging from lower responses of controls subjects through intermediate responses of HT and NC-CAH patients, to higher responses of classical CAH patients. The high baseline ACTH levels and the elevated ACTH levels attained after oCRH in the classical form of the disease confirm previous reports (3, 4, 14). A lack of cortisol negative feedback and possible hyperplasia of the corticotrophs may account for these findings (15, 16). However, mean baseline plasma cortisol levels in classical CAH patients were normal and indistinguishable from those in HT and NC-CAH patients. Only after adrenal stimulation with endogenous ACTH induced by oCRH or by the exogenous ACTH test, was decreased cortisol secretion detected in classical CAH patients. This feature was similar to patterns described for adrenal function in progressive autoimmune adrenalitis, in which increased plasma ACTH levels or a subnormal short ACTH test are more sensitive indices of early primary adrenal failure than resting plasma cortisol level (17). In addition, the NCCAH patients had normal basal plasma cortisol and normal endogenous ACTH-stimulated peak cortisol responses together with slightly lower 60 min post-ACTH plasma cortisol. These NC-CAH patients presented normal basal ACTH but an enhanced ACTH response to oCRH. These results partially agree with two studies recently reported for NC-CAH (6,7). These investigators found normal baseline ACTH and cortisol plasma levels in NC-CAH and decreased cortisol production detected by exogenous ACTH but not by the oCRH stimulus (7). However, normal ACTH responses to oCRH were found in both studies. The disagreement between these results and ours may probably reflect the genetic difference in the proportion of compound heterozygous and homozygous subjects for the mild allele among the three studies (13). The present study further indicates that in CAH the augmented ACTH response to oCRH associated or not with an increased baseline plasma ACTH may represent the early sign of compensated adrenal hypofunction. The pituitary gland may be very sensitive to a subtle reduction of cortisol production. This situation is
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
202
MOREIRA
analogous to that occurring in mild primary hypothyroidism (18). The mean plasma ACTH after oCRH in heterozygous subjects remained between those of controls and NCCAH but they were not significantly different from those of controls. Although, HT mean response was lower than the mean response of NC-CAH patients there was an overlap of values between these two groups. These data originally indicated that the ACTH-cortisol component of the pituitary-adrenal axis is in normal equilibrium in the heterozygous carriers for 21-OH CAH. However, based on the exaggerated 17-OHP response to oCRH of HT, the present study demonstrated that their ACTH17-OHP component of the pituitary adrenal axis is abnormal. These augmented 17-OHP responses to oCRH are comparable to responses classically described in HT after exogenous ACTH (12). However, 20-250 pg ACTH1-24 doses, commonly employed for adrenal function screening (19), were far in excess of that required to induce maximum steroidogenesis. Additionally, we have recently demonstrated that in normal subjects the increase of plasma ACTH after acute hCRH (1 pg/kg) was lower than obtained with hypoglycemic stress and that stimulation with synthetic CRH may mimic an endogenous ACTH pulse (20). Since there were an exaggerated 17-OHP response to oCRH in CAH HT, the present study indicates that the release of endogenous ACTH at levels equivalent to physiological ones by oCRH may be a sufficient stimulus to detect heterozygosis. We additionally demonstrated a good correlation between plasma 17-OHP and ACTH concentrations after oCRH in all subjects as a group. The ACTH responses to oCRH reflected the graded severities of adrenal 2lOH deficiency. These results suggest a parallelism between the primary defect of the adrenal glands and the secondary involvement of the pituitary in this disease. The present study further indicates an excellent correlation between adrenal hormone responses to oCRH and the exogenous ACTH stimulation test. These results confirm previous data comparing 17-OHP responses with oCRH and ACTH in NC-CAH (6, 7). We additionaly extended this comparison to different degrees of 21-OH deficiency. Although the 17-OHP responses to oCRH tended to be lower than those obtained after exogenous ACTH, the distribution of hormonal responses along a line in the following descending order: classical 21-OH CAH, NC 21-OH CAH, HT, and control subjects confirmed the spectrum of enzymatic deficiency in these groups that was established using the exogenous ACTH test (12). As far as the clinical applications of our findings are concerned, it seems that the use of exogenous ACTH as a diagnostic test is easier and cheaper than oCRH administration. In conclusion, the effects of oCRH on the pituitary-
AND
.I(‘E c hl* IYW
ELIAS
adrenal axis were documented in patients and HT for 21-OH deficiency. These findings suggest that different degrees of adrenal biosynthetic defect may result in graded ACTH and 17-OHP responses to oCRH. The variability of 21-OH deficiency probably is mirrored in the pituitary secretion of ACTH. Acknowledgments Reagents for ACTH RIA were supplied by the National Hormone and Pituitary Program, NIDDK (Bethesda, MD) and Dr. Toshihiro Suda (Japan). Tetracosactrin was the gift of Organon (Brazil). We are grateful to Mrs. Adriana Rossi, Mrs. Lucimara Bueno, and Mr. Jose Roberto Silva for technical assistance. We are greatly indebted to Dr. Julio C. Voltarelli and Mrs. Lucia H. Teixeira and Mr. Rubens Silveira for the HLA typing.
References 1. Wilkins L, Lewis RA, Klein R, et al. Treatment of congenital adrenal hyperplasia with cortisone. J Clin Endocrinol Metab. 1951;11:1-25. 2. Bartter FC, Albright F, Forbes AP, Leaf A, Dempsey E, Carrel E. The effects of adrenocorticotropic hormone and cortisone in the adrenogenital syndrome associated with congenital adrenal hyperplasia: an attempt to explain and correct its disordered hormonal pattern. J Clin Invest. 1951;30:23’7-51. 3. Sydnor KL, Kelley VC, Raile RB, Ely RS, Sayers R. Blood adrenocorticotrophin in children with congenital adrenal hyperplasia. Proc Sot Exp Biol Med. 1953;82:695-7. 4. Cacciari E, Cicognani A, Pirazzoli P, et al. GH, ACTH, TSH, LH, and FSH reserve in prepubertal girls with congenital adrenal hyperplasia. J Clin Endocrinol Metab. 1976;43:1146-52. 5
Vol. 74, No. 1 Printed
in
L’.S.A.
Pituitary-Adrenal Responses to Corticotropin-Releasing Hormone in Different Degrees of Adrenal 2 lHydroxylase Deficiency* AYRTON Endocrinology
C. MOREIRA Division,
AND LUCILA
Department
of
Medicine,
L. K. ELIAS Faculty of Medicine,
ABSTRACT. 21-Hydroxylase congenital adrenal hyperplasia (21-OHCAH) involves a orimarv defect of the adrenal gland and a secondary hrvolvement of ACTH secretion. The resbonses of the pituitary-adrenal axis to ovine CRH (oCRH, 1 fig/kg) were examined in subjects with different degrees of 21-OH deficiency. We studied 43 subjects: 7 classical and 6 nonclassical (NC) 21OHCAH patients,-15 heterozygotes (HT) and 15 control subjects. Baseline plasma ACTH levels were higher in classical CAH than in NC-CAH, HT, and control subjects (mean -t SEM, 66 + 14, 6 + 1.6, 4 f 0.5, and 5 + 0.5 pmol/L, respectively). The mean plasma ACTH resoonse to oCRH in NC-CAH (17 & 3 pmol/L) was higher than-in controls and HT (9 C 0.8 and 11 f 1.5 pmol/L). The highest ACTH responses to oCRH were obtained for classical CAH patients (126 f 29 pmol/L). Plasma
14049-RibeirCo
Preto, SP, Brazil
cortisol rose after oCRH in control, HT, and NC-CAH patients but did not change in classical CAH. After oCRH, plasma 17hydroxyprogesterone (17-OHP) were 4 * 0.5, 8 + 1.6, 93 f 28, and 359 + 110 nmol/L for controls, HT, NC-CAH, and classical patients, respectively. There was a significant correlation (r = 0.70) between 17-OHP and the ACTH resaonses to oCRH. The 17-OHP responses to oCRH were also correlated (r = 0.94) with the 17-OHP responses to the synthetic ACTH test. We conclude that the release of endogenous ACTH by oCRH result in graded 17-OHP responses on 21-OH deficiency. The present study also suggests that different degrees of adrenal biosynthetic defect may result in graded ACTH responses to oCRH. (J Clin Endocrinol Metab 74: 198-203, 1992)
I
N THEIR early studies, Wilkins et al. (1) and Bartter et al. (2) suggested that the secretory activity of pathological adrenal glands is dependent upon the tropic function of the pituitary in congenital adrenal hyperplasia (CAH). A partial or complete inability to synthesize cortisol in 21-hydroxylase (21-OH) deficiency CAH results in compensatory hypersecretion of ACTH. Later, elevated baseline plasma ACTH levels were demonstrated in classical 21-OH CAH patients (3, 4). In the last decade, improved biochemical assessment of adrenal function associated with HLA serotyping and molecular genetic evaluations have established graded severities of steroidogenic dysfunction in 21-OH CAH ranging from classical and nonclassical (NC) CAH to heterozygous carriers (5). Although the responses of ACTH to ovine CRH (oCRH) have been recently reported in two studies on NC-CAH patients (6, 7), detailed information on the pituitary-adrenal responses to oCRH in subjects with graded severities of 21-OH deficiency is lacking. The
purpose of the present investigation was to determine whether or not different degrees of adrenal biosynthetic defect would result in graded plasma ACTH and 17hydroxyprogesterone (17-OHP) responses to oCRH. We examined the responsiveness of the pituitary-adrenal axis to oCRH in patients and heterozygotes (HT) for 21OH deficiency. Materials
and Methods
Subjects
Forty-three subjects were divided into four groups as follows. 1) Seven classical 21-OH CAH patients (five females and two males ranging in age from 3-30 yr). Two patients had never been treated and five had stopped glucocorticoid therapy 10 days before the study. 2) Six NC 21-OH CAH patients (three females and three males, ranging in age from 7-29 yr). They had never been treated. Two patients were found to be compound HT by pedigree analysis, using HLA markers in conjunction with hormonal data. 3) Fifteen HT (six females and nine males, ranging in age from 4-44 yr). Twelve subjects were parents (obligate HT) and three asymptomatic subjects were brothers of classical CAH patients. The assignment of heterozygos-
Received December 14, 1990. Address correspondence and requests for reprints to: Ayrton C. Moreira, M.D., Department of Medicine, Faculty of Medicine, 14049, Ribeirb Preto, SP, Brazil. *This work was supported by Conselho National de Desenvolvimento Cientifico e Tecnologico (CNPq Grant 30.0834/82-91), Funda$50 de Amparo a Pesquisa do Estado de SHo Paulo and Hospital das Clinicas de RibeirGo Preto (HCFMRP-FAEPA). 198
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
CRH
TEST
IN 21-HYDROXYLASE
ity for the CAH gene was based on the presence of one HLA haplotype in common with the affected sib and plasma 17-OHP response to the ACTH test (5). 4) Fifteen control subjects (seven females and eight males, ranging in age from 9-28 yr). Twelve subjects were from the general population and three subjects were unaffected homozygous sibs who shared no HLA haplotypes with the affected CAH sib. All subjects were studied at the Endocrinology Division of the University Hospital (Ribeirao Preto, SP, Brazil) after informed consent was given by them or their parents. Postmenarchial females were all studied during the early follicular phase. CRH and ACTH testing
All tests were started at 0900 h after an overnight
fast.
CRH test
oCRH (1 pg/kg; Bachem, Torrance CA) was given as an iv bolus injection. Blood samples for hormone assays were collected 15 min before and 0, 15, 30, 45, 60, 90, and 120 min after the injection. Plasma ACTH, cortisol, and 17-OHP were measured at all sampling times. ACTH
test
ACTH (0.25 mg tetracosactrin; Organon, Sao Paulo SP) was administered iv over 60 s. Blood samples for 17OHP and cortisol measurement were obtained before and 60 min after the injection. Hormone assays
ACTH was determined by RIA after extraction from plasma using silicic acid (8). The assay sensitivity was 2 pmol/L. The intra- and interassay coefficients of variation (CVs) were 6% and 18%, respectively. Plasma cortisol and 17-OHP were determined by previously described RIA methods after ethanol extraction and extraction with ether and Sephadex LH-20 chromatography, respectively (9, 10). All steroid assays had intrassay CVs between 34% and interassay CVs between 7-16%. The assay sensitivity was 40 nmol/L for cortisol and 0.5 nmol/L for 17-OHP. All the samples obtained from each patient on both tests were analysed in duplicate in the same assay to avoid interassay variability. HLA typing was performed on peripheral blood lymphocytes using the standard microcytotoxicity test (11).
DEFICIENCY
199
concentrations. The integrated or mean ACTH, cortisol, and 17-OHP responses after oCRH administration were determined by calculating the sum or mean of all hormone values from 15 to 120 min. Statistical analysis of the results was performed using the Wilcoxon rank sum test and Spearman’s rank correlation procedure when appropriate. Differences of less than 0.05 were considered significant. Results Responsesof ACTH, cortisol, and 17-OHP to oCRH
The mean (~SEM) baseline plasma ACTH, cortisol, and 17-OHP values and their responses to oCRH in patients with 21-OH CAH deficiency, heterozygous carriers and control subjects are shown in Table 1 and Figs. 1 and 2. It can be seen that basal ACTH values were not different among controls, HT, and NC-CAH patients. However, the three groups had significant lower basal plasma ACTH than classical CAH patients. The mean plasma ACTH response in the HT after oCRH administration was 11 f. 1.5 pmol/L, slightly but not significantly higher than that in the control subjects (9 & 0.8 pmol/L). The ACTH responses to oCRH were higher (P < 0.05) in the NC-CAH patients (17 + 3 pmol/L) than in controls or heterozygous subjects. The highest ACTH responses to oCRH were obtained for classical CAH patients (126 + 29 pmol/L). The mean basal plasma cortisol levels were similar in the four groups. Plasma cortisol rose (P < 0.05) after oCRH compared to the basal levels in controls, HT, and NC-CAH patients but did not change in classical CAH patients. Baseline plasma 17-OHP levels were elevated (P < 0.001) in NC-CAH (12 + 1.8 nmol/L) and classical (303 + 99 nmol/L) patients when compared with HT and control subjects (3 f 0.4 nmol/L). The mean post-oCRH plasma 17-OHP level increased (P < 0.05) to 4.0 + 0.5, 8.0 & 1.6, and 93 k 28 nmol/L, in controls, HT, and NCCAH subjects, respectively. The high baseline plasma 17-OHP levels of classical CAH patients did not increase significantly after oCRH (359 + 110 nmol/L). The 17OHP responses to oCRH were higher (P c 0.001) in NCCAH patients than in HT. In addition, the mean 17OHP response of the latter group was significantly higher than those of the control group. However, some individual values overlapped those of the control subjects. Figure 3 shows a positive Spearman’s rank correlation between the time-integrated sum of the ACTH levels and 17-OHP levels after oCRH in all subjects as a group (r = 0.70, n = 41; P < 0.0001).
Statistical analysis
Comparison between adrenal hormone responsesto oCRH and ACTH stimulation test
Data are expressed as the mean + SEM. The baseline level was calculated as the mean of the -15 and 0-min
The mean (~SEM) plasma 17-OHP 60 min post-ACTH levels and peak post-oCRH 17-OHP levels were 4.5 f
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
200
MOREIRA
AND
ELIAS
J(‘E L M. 1%)” Vol 74. No 1
TABLE 1. Mean @EM) plasma ACTH, cortisol, and 17-OHP concentrations under baseline conditions and their mean responses after oCRH (1 pg/kg) in controls, heterozygous subjects, and NC and classical patients with 21-OH deficiency ACTH (pmol/L)
Variable or group
(n)
Controls
Basal
15
HT
5
15
NC patients
4
6
Classical patients
*
Mean response
0.5
+ 0.5
6-c
1.6
9 0.8 11 1.5 17 3o.h
f
126 2g0.h.’
+
66+ l.+’
7
Cortisol (nmol/L)
+
Basal
Mean response
300 -c 22
500 30 490 40 4x0
210
-e22 *
260
?I 25
50 220 r$jd’
240
f
60
17-OHP (nmol/L)
-
Basal
f
Mean response
3
f
f
9
+-
*
0.4 12
*
-c
303
r
4.0 f 0.5
0.4
8.0
f
1.6
1.8”.b
gg4.b.c
93
+
359 11(yLL’
”
‘2p.b
--.-_.
’ P < 0.05 us. controls. ’ P < 0.05 us. HT. ‘PC 0.05 us. NC.
250,~ 200-
IT
B
*
250
8
.-200
150
!” 0 0 . e
- 150 8
-L 11 u
-1 looi& 50 ’ 25 z E Lx 20? z
- 100
me: 0 _ -0. . _ .
-50 -35
0 0
0. . -L
-
0ON
-15
B
-‘O
t
15-
30
$’ 5-
II . iSCQ5 E :450-
450
I- l 0+3o -E-15
0
I 15304560 TIME
I
I (mln)
I 9C ’
lZO
0
I 15
111 30 45 TIME
a-
15&
0
8
-3
-5
0
t? I= g300v
I30 1
@
:g.
10-
.
MEAN RESPONSE
FIG. 1. A, Mean (+SEM) plasma ACTH (upper panel) and cortisol (lower panel) responses to oCRH (1 fig/kg) given at time zero in 7 patients with classical CAH (Cl), 6 patients with NC-CAH (m), and 15 HT for 21-OH deficiency (0). The hatched area represents the mean (*SD) for 15 controls (0). B, Individual values of mean response to oCRH for the 4 groups.
0.5 us. 4.0 It 0.5 nmoi/L (P = NS) for controls; 15 f 3.0 us. 11.5 & 2.2 nmol/L (NS) for HT; 142 + 21 us. 120 + 32 nmol/L (NS) for NC-CAH patients, and 626 + 191 us. 435 + 122 nmol/L (NS) for classical patients, respec-
60
(mln)
I
I
90
12
MEAN RESPONSE
FIG. 2. A, Mean (+SEM) plasma 17-OHP responses to oCRH (1 rg/ kg) given at time zero in 7 patients with classical CAH (Cl), 6 patients with NC-CAH (B), and 15 HT for 21-OH deficiency (0). The hatched area represents the mean (&SD) for 14 controls (0). B, Individual values of mean response to oCRH for the 4 groups.
tively. There was a positive Spearman’s rank correlation between 17-OHP responses to oCRH and ACTH administration in all subjects as a group (r = 0.94; n = 36; P < 0.0001; Fig. 4). The mean plasma cortisol levels obtained 60 min postACTH and the peak cortisol levels obtained post-oCRH were 660 + 30 us. 670 + 40 nmol/L for controls; 700 f 40 us. 610 + 50 nmol/L for HT; 410 + 20 us. 600 -C 75 nmol/L for NC-CAH patients, and 260 + 60 us. 290 f 65 nmol/L for classical CAH patients, respectively. In each group, plasma cortisol peak responses to oCRH were similar (P = NS) to those obtained after ACTH administration. In addition, peak cortisol responses of NC-CAH after exogenous ACTH were slightly but sig-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
CRH
TEST
IN
Zl-HYDROXYLASE
control tients.
0 Cl
201
DEFICIENCY
and heterozygous
subjects or of NC-CAH
pa-
0 0
Discussion
0
0
r= 070 pc 0.0001
I 20
.~.....I SUM
200
OF ACTH POST
..A
2000
( pmol / L )
oCRH
FIG. 3. Correlation between the sum of ACTH levels and the sum of plasma 17-OHP concentrations after oCRH in control (0) and heterozygous (0) subjects and patients with classical (Cl) and NC (m) 21-
0 no 0
0.3 t 0.3
I 3 IT-OHP
*
* * . . ., , , , . * . , .I , 30 300 ( n mol/L ) 60 min POST ACTH
. . . 4 3000
FIG. 4. Correlation between the 60-min ACTH stimulated plasma 17OHP level and the peak oCRH stimulated plasma 17-OHP level in control (0) and heterozygous (0) subjects and in patients with classical (Cl) and NC (W) 21-OH CAH.
nificantly lower than those of the control and heterozygous subjects. However, cortisol responses after oCRH did not differ among the three groups. The peak cortisol responses of classical CAH patients to oCRH or exogenous ACTH were significantly lower than those of the
The pathophysiology of 21-OH deficiency CAH involves a primary defect of the adrenal gland and a secondary increase of pituitary ACTH production. Although this dual gland interaction mechanism had been suggested as early as 40 yr ago (1, 2), the adrenal steroidogenesis defect has been more extensively studied than its pituitary counterpart (5, 12, 13). The present study demonstrates that the ACTH responses to oCRH taken as a whole, resulted in a spectrum ranging from lower responses of controls subjects through intermediate responses of HT and NC-CAH patients, to higher responses of classical CAH patients. The high baseline ACTH levels and the elevated ACTH levels attained after oCRH in the classical form of the disease confirm previous reports (3, 4, 14). A lack of cortisol negative feedback and possible hyperplasia of the corticotrophs may account for these findings (15, 16). However, mean baseline plasma cortisol levels in classical CAH patients were normal and indistinguishable from those in HT and NC-CAH patients. Only after adrenal stimulation with endogenous ACTH induced by oCRH or by the exogenous ACTH test, was decreased cortisol secretion detected in classical CAH patients. This feature was similar to patterns described for adrenal function in progressive autoimmune adrenalitis, in which increased plasma ACTH levels or a subnormal short ACTH test are more sensitive indices of early primary adrenal failure than resting plasma cortisol level (17). In addition, the NCCAH patients had normal basal plasma cortisol and normal endogenous ACTH-stimulated peak cortisol responses together with slightly lower 60 min post-ACTH plasma cortisol. These NC-CAH patients presented normal basal ACTH but an enhanced ACTH response to oCRH. These results partially agree with two studies recently reported for NC-CAH (6,7). These investigators found normal baseline ACTH and cortisol plasma levels in NC-CAH and decreased cortisol production detected by exogenous ACTH but not by the oCRH stimulus (7). However, normal ACTH responses to oCRH were found in both studies. The disagreement between these results and ours may probably reflect the genetic difference in the proportion of compound heterozygous and homozygous subjects for the mild allele among the three studies (13). The present study further indicates that in CAH the augmented ACTH response to oCRH associated or not with an increased baseline plasma ACTH may represent the early sign of compensated adrenal hypofunction. The pituitary gland may be very sensitive to a subtle reduction of cortisol production. This situation is
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 03:01 For personal use only. No other uses without permission. . All rights reserved.
202
MOREIRA
analogous to that occurring in mild primary hypothyroidism (18). The mean plasma ACTH after oCRH in heterozygous subjects remained between those of controls and NCCAH but they were not significantly different from those of controls. Although, HT mean response was lower than the mean response of NC-CAH patients there was an overlap of values between these two groups. These data originally indicated that the ACTH-cortisol component of the pituitary-adrenal axis is in normal equilibrium in the heterozygous carriers for 21-OH CAH. However, based on the exaggerated 17-OHP response to oCRH of HT, the present study demonstrated that their ACTH17-OHP component of the pituitary adrenal axis is abnormal. These augmented 17-OHP responses to oCRH are comparable to responses classically described in HT after exogenous ACTH (12). However, 20-250 pg ACTH1-24 doses, commonly employed for adrenal function screening (19), were far in excess of that required to induce maximum steroidogenesis. Additionally, we have recently demonstrated that in normal subjects the increase of plasma ACTH after acute hCRH (1 pg/kg) was lower than obtained with hypoglycemic stress and that stimulation with synthetic CRH may mimic an endogenous ACTH pulse (20). Since there were an exaggerated 17-OHP response to oCRH in CAH HT, the present study indicates that the release of endogenous ACTH at levels equivalent to physiological ones by oCRH may be a sufficient stimulus to detect heterozygosis. We additionally demonstrated a good correlation between plasma 17-OHP and ACTH concentrations after oCRH in all subjects as a group. The ACTH responses to oCRH reflected the graded severities of adrenal 2lOH deficiency. These results suggest a parallelism between the primary defect of the adrenal glands and the secondary involvement of the pituitary in this disease. The present study further indicates an excellent correlation between adrenal hormone responses to oCRH and the exogenous ACTH stimulation test. These results confirm previous data comparing 17-OHP responses with oCRH and ACTH in NC-CAH (6, 7). We additionaly extended this comparison to different degrees of 21-OH deficiency. Although the 17-OHP responses to oCRH tended to be lower than those obtained after exogenous ACTH, the distribution of hormonal responses along a line in the following descending order: classical 21-OH CAH, NC 21-OH CAH, HT, and control subjects confirmed the spectrum of enzymatic deficiency in these groups that was established using the exogenous ACTH test (12). As far as the clinical applications of our findings are concerned, it seems that the use of exogenous ACTH as a diagnostic test is easier and cheaper than oCRH administration. In conclusion, the effects of oCRH on the pituitary-
AND
.I(‘E c hl* IYW
ELIAS
adrenal axis were documented in patients and HT for 21-OH deficiency. These findings suggest that different degrees of adrenal biosynthetic defect may result in graded ACTH and 17-OHP responses to oCRH. The variability of 21-OH deficiency probably is mirrored in the pituitary secretion of ACTH. Acknowledgments Reagents for ACTH RIA were supplied by the National Hormone and Pituitary Program, NIDDK (Bethesda, MD) and Dr. Toshihiro Suda (Japan). Tetracosactrin was the gift of Organon (Brazil). We are grateful to Mrs. Adriana Rossi, Mrs. Lucimara Bueno, and Mr. Jose Roberto Silva for technical assistance. We are greatly indebted to Dr. Julio C. Voltarelli and Mrs. Lucia H. Teixeira and Mr. Rubens Silveira for the HLA typing.
References 1. Wilkins L, Lewis RA, Klein R, et al. Treatment of congenital adrenal hyperplasia with cortisone. J Clin Endocrinol Metab. 1951;11:1-25. 2. Bartter FC, Albright F, Forbes AP, Leaf A, Dempsey E, Carrel E. The effects of adrenocorticotropic hormone and cortisone in the adrenogenital syndrome associated with congenital adrenal hyperplasia: an attempt to explain and correct its disordered hormonal pattern. J Clin Invest. 1951;30:23’7-51. 3. Sydnor KL, Kelley VC, Raile RB, Ely RS, Sayers R. Blood adrenocorticotrophin in children with congenital adrenal hyperplasia. Proc Sot Exp Biol Med. 1953;82:695-7. 4. Cacciari E, Cicognani A, Pirazzoli P, et al. GH, ACTH, TSH, LH, and FSH reserve in prepubertal girls with congenital adrenal hyperplasia. J Clin Endocrinol Metab. 1976;43:1146-52. 5
