CENTRAL ADRENAL INSUFFICIENCY COULD NOT BE CONFIRMED BY MEASUREMENT OF BASAL SERUM DHEAS LEVELS IN PUBERTAL CHILDREN

VAIANI E; MACEIRAS M,; CHALER E; LAZZATI J M,; CHIAVERO M; NOVELLE C; .RIVAROLA MA.,; BELGOROSKY A

HORMONE RESEARCH

KARGER

Lugar: Basel; Año: 2014

0301-0163

Abstract Background: Central adrenal insufficiency (CAI) is due to a decrease of CRH and/or ACTH secretion. ACTH-dependent dehydroepiandrosterone-sulphate (DHEAS) has been postulated as a possible marker of adrenal function in adult patients. Aims: To evaluate the usefulness of basal serum DHEAS determination to diagnose CAI in pubertal patients with a suspected diagnosis of CAI . Methods: Ninety-four pubertal patients suspected of having CAI were divided into two groups (Gr) according to sufficient (Gr1) or insufficient (Gr2) low-dose ACTH test (LDT) serum cortisol response. Concordance with low (<2.5th percentile) or normal (≥2.5th percentile) basal serum DHEAS levels for age and sex, respectively, were analyzed. Results: Fifty patients (56.2%) in Gr1 and and 44 (46.8%) in Gr2 were included. Median value of serum DHEAS levels in Gr2 (0.7 µmol/l, interquartile range: 0.44-1.49) was significantly lower than in Gr1, (2.13 µmol/l, interquartile range: 0.87-3.5) p<0.03. Nevertheless, serum basal DHEAS levels as a diagnostic marker of CAI showed 39% sensitivity and 80% specificity. Conclusion: In pubertal patients basal serum DHEAS levels do not seem to be a useful tool to diagnose either sufficiency or insufficiency of secondary adrenal function. Introduction Central adrenal insufficiency (CAI) is a clinical disorder that results from hypothalamic or pituitary disease or from prolonged administration of long-term supra physiological doses of glucocorticoids. CAI is characterized by impaired cortisol production by the adrenal glands due to a decrease of CRH and/or ACTH secretion. The deficiency is frequently partial, maintaining a reduced cortisol secretion, which leads to a potential risk of developing CAI in stressful situations. Once suspected, the definitive diagnosis can be confirmed by laboratory evaluation of adrenocortical function. A morning cortisol level lower than 83 to 100 nmol/l (3 to 3.62 µ/dl) is considered of diagnostic value for CAI, whereas a random cortisol level above 496 to 524 nmol/l (18-19 µ/dl ) rules it out [1-3]. The reference tests for establishing the integrity of the hypothalamic-pituitary-adrenal (HPA) axis require assessing the response to either a strong stimulus, such as insulin-induced hypoglycaemia (insulin tolerance test), or an interruption of the negative feedback from cortisol (metyrapone test). Nevertheless, these reference tests have major problems. The insulin tolerance test has raised safety concerns because of the possibility of inducing severe hypoglycaemia and the overnight metyrapone test carries a risk of an adrenal crisis. In addition, mistaken interpretation of the metyrapone test might occur in the presence of other drugs which affect metyrapone clearance [4-5]. The ACTH tests in its two versions, the high-dose 250µg ACTH test (HDT) and the low-dose 1µg ACTH test (LDT), are the most commonly used stimulation tests. Lack of continuous stimulation of the adrenal glands by endogenous ACTH leads to adrenal atrophy and hyporesponsiveness to exogenous ACTH, and hence, the ACTH test is an indirect measurement of pituitary function. These tests gained wide acceptance because of their safety compared to the metyrapone test and insulin tolerance test in paediatric and elderly patients. Although several authors demonstrated that both high- and low-dose ACTH tests provided similar reliability [6-8], currently, the LDT is preferred to the HDT, as the LDT has been demonstrated to have better sensitivity for the diagnosis of CAI, especially in mild forms of deficiency [2, 9, 10-14]. In addition, it has been postulated that the LDT provides serum ACTH levels that mimic physiological stress and more closely approximate responses to the insulin tolerance test than the standard dose of 250 µg corticotropin analogue [15-17]. Dehydroepiandrosterone-sulphate (DHEAS) is an ACTH-dependent adrenal androgen precursor and has been postulated as a possible marker of adrenal function. [18] Even though the regulation of DHEAS production by the zona reticularis of the adrenal cortex at the time of adrenarche is poorly understood, [19] it is well known that a trophic adrenal cortex is necessary for DHEAS synthesis. Therefore, in adult patients it has been proposed that serum DHEAS levels might be a good marker for the analysis of the HPA axis. [20-23]. To our knowledge, in paediatric patients data on the usefulness of baseline serum DHEAS to estimate central adrenal dysfunction are limited. This study was designed to evaluate basal serum DHEAS levels in the ACTH LDT in a group of pubertal patients referred to our paediatric endocrinology department with the suspicion of having CAI. Our findings suggest that, in pubertal patients with suspected insufficiency of the HPA axis, basal DHEAS serum levels do not seem to be a useful tool either to diagnose sufficiency or insufficiency in HPA function. Therefore, we suggest that the use of dynamic testing is necessary to assess the HPA axis in puberty. Subjects and Methods Ninety-four pubertal ambulatory patients suspected of CAI were studied. Based on the time that adrenarche takes place, all patients included in the study were older than 8 years. CAI was suspected in the presence of the following clinical risk factors: 1) withdrawal of chronic administration of exogenous glucocorticoids, 2) hypothalamic or pituitary conditions, such as central brain tumors, cranial irradiation, hypophysitis, traumatic brain injury, hystiocytosis, independently of the presence or absence of clinical signs or symptoms of adrenal insufficiency (fatigue, nausea, vomiting, anorexia, weight loss, abdominal pain), and 3) fasting morning (at 8 a.m.) basal serum cortisol levels ≤ 330.72 nmol/l (12 µ/dl) . In addition, in all patients included in the study basal serum ACTH levels were according to our normal reference values. Puberty was defined as testis volume ≥ 4 cc in males and breast Tanner stage 2 in females. Patients were divided into two groups according to pubertal development as follows: early or mid puberty: breast Tanner stage 2 and 3 in girls (n=22) and testis volume 4 to 15ml in boys (n=29); and late puberty: breast Tanner stage 4 and/or menarche in girls (n=17) and testis volume greater than 15ml in boys ( n=26 ). All patients with pituitary insufficiency other than ACTH deficiency were on hormone replacement therapy when the LDT was performed. Exclusion criteria were female patients on estrogen therapy, patients in whom chronic corticoid therapy was withdrawn less than 3 months before the LDT, and hypothalamus-pituitary tumor surgery less than 1 year previously. In addition patients on corticoids replacement therapy were also excluded from the study. LDT was performed in an outpatient setting, in the morning as it has previously been described.[16] In brief, one ampoule of 250µg /ml of tetracosactide acetate (Synacthen) was diluted in 0.9% Cl Na to reach the appropriate concentration performed by personnel with long-term experience in the dilution process. Each dose was stored at 4˚C for up to 12hs before administration. The LDT dose for children varies according to differents studies. Maghnie et al. [24] used a 1-µg dose, Gonc et al. [25] used 0.5µg /m2 and Rose et al. [26] used 1 µg/m2 body surface. Similarly to the latter study, we performed the LDT with a dose of 1µg per m2 of body surface but with a total maximal dose of 1.5µg, particularly in older adolescent patients. It was administered as an intravenous bolus. Serum cortisol was measured at baseline and at 30 and 60 minutes after Synacthen injection. According to previous reports [27-28], a serum cortisol cut-off limit equal to or higher than 18 µg/dl (496 nmol/l) at least once after the ACTH stimulation test was defined as a sufficient response [11]. Serum DHEAS levels were measured at LDT baseline. Serum values higher than or equal to the 2.5th percentile for chronological age (CA) and sex were considered normal according to a previous report. [29] For the analysis of the results patients were divided into two groups (Gr) according to sufficient (Gr1) or insufficient (Gr2) LDT serum cortisol response. Additionally, patients included in Gr1 and Gr2 were subdivided, depending on whether basal serum DHEAS levels were equal to or higher than (GR1a and GR2a), or lower than (GR1b and GR2b) the 2.5th percentile reference for age and sex. For group comparisons, Gr concordance was defined as follows: I. sufficient LDT and normal basal serum DHEAS levels, as well as, II. insufficient LDT response and low basal serum DHEAS levels (Gr1a and Gr2b, respectively); while Gr discordance was defined as follows: I. sufficient LDT response but low serum DHEAS levels, as well as, II. insufficient LDT response but normal basal serum DHEAS levels (Gr1b and Gr2a, respectively). This study was approved by the Ethics Committee of the Garrahan Pediatric Hospital. Methods DHEAS was measured using the Immulite 2000 Chemiluminescent Assay. Intraassay coefficients of varation (CV) were: level 1 (1.6 µmol/l) = 4.74%, level 2 (3.5µmol/l) = 13.7%. Interassay CV, level 1= 8.8%, level 2=6.3%. The 2.5th percentile of basal serum DHEAS levels for age and sex was established according to the percentile previously reported by Elmlinger M. et al. [29] Serum Cortisol (F) was measured by Immulite 2000 Chemiluminescent Assay. Intraassay CVs were: level 1(102 nmol/l) = 11.5% and level 2 (590 nmol/l): 3.6%, Interassay CVs level 1= 5.2% level 2 = 5.9%. Reference values, according to the manufacturer, are 138- 690 nmol/l (5-25 µg/dl) Statistical Analysis Statistical analysis was performed using the package Statistix 7 (Analytical Software, Tallahassee, FI). Categorical variables were presented as a percentage and continuous variables as a median and interquartile range. Comparison between groups was done using the Wilcoxon rank-sum test for non-parametric measurements. Categorical data were compared using chi-squared and Fisher?s exact test. Differences were considered significant when the two-sided values were less than 0.05. Sensitivity and specificity for serum DHEAS levels higher than or equal to the 2.5th percentile for age and sex to diagnose CAI were calculated using a contingency table. Results Clinical and laboratory features of the ninety-four pubertal patients with suspected CAI included in this study are shown in table 1. Median and (interquartile) chronological age (CA) were 13.9, (range:12-16) years; sex and pubertal status were equally distributed. The most frequent diagnoses leading to the indication of the LDT were: multiple pituitary insufficiencies secondary to a CNS tumor or their treatments (29.8%), chronic corticosteroid treatment (18%), and idiopathic hypopituitarism (13.8%). Supplemental table1 . Basal serum DHEA-S levels over the 2.5th percentile for age and sex, regardless of the type of LDT response, were found in 71.3% per cent of the patients studied. Fifty out of 94 patients (56.2%) had a sufficient LDT response and were included in Gr1 while 46 (43.8%) patients were included in Gr2. No significant differences in sex distribution, pubertal status, underlying diagnosis, Synacthen dose/body surface area, previous morning-basal serum cortisol or ACTH levels were found between Gr1 and Gr2. CA in Gr1 was significantly greater than in Gr2 (15.1 and 12.9 y, p< 0.001, respectively). Median value of basal serum DHEAS levels in Gr2 (0.7 µmol/l, interquartile range: 0.44-1.49 µmol/l) was significantly lower than in Gr1, p<0.03 (2.13 µmol/l , interquartile range: 0.87-3.5 µmol/l). A discrepancy between serum DHEAS levels (over the 2.5th percentile for age and sex in 61.4 % of patients in Gr2, and under the 2.5th percentile for age and sex in 20% of patients in Gr1) and LDT response was observed. In Gr1, this discrepancy remained in 25% of patients using a higher cortisol cut-off limit (22 microg/dl) [17, 22] in the LDT. Clinical characteristics and laboratory findings, in subjects with DHEAS levels above (Gr1a and Gr2a) or below (G1b and Gr2b, respectively) the 2.5th percentile for age and sex were analyzed. Median CA value was significantly lower in Gr1a (14.3, interquartile range: 12.6-15.9) than in Gr1b (17, interquartile range: 14.7-18.1y), p<0.02). No statistically significant difference in other clinical parameters or laboratory findings evaluated, such as sex distribution, Tanner pubertal status, underlying diagnosis, morning basal serum cortisol and ACTH levels, LDT response, and Synacthen doses, were found between Gr1a and Gr1b (Table 2, supplemental data). Similar to Gr1a and Gr1b, no statistical significant difference in clinical parameters was found between Gr2a and Gr2b, including CA and laboratory findings. Although no significant differences among diagnoses were found, when we analysed them as sub-groups, multiple pituitary insufficiencies found were significantly less in G2a vs Gr2b. The percentage of patients in each subgroup according to basal serum DHEA-S levels above (a) or below (b) the 2.5th percentile for age and sex are shown in Fig 1. As expected, a significantly greater percentage of basal serum DHEAS levels above the 2.5th percentile was found in Gr1a than in Gr2a (80 and 61.4% respectively, p< 0.03), and a significantly greater percentage of patients with serum DHEAS below the 2.5th percentile was observed in Gr2b than in Gr1b (38.6 and 20% respectively, p< 0.03) . Nevertheless, basal serum DHEAS levels above the 2.5th percentile for age and sex were found in a considerable percentage of patients from Gr2a (Table 3, Supplemental Data). Based on LDT response, basal serum DHEAS levels showed a 38.6 % sensitivity and a 80% specificity as a biomarker of CAI. Discussion Our findings suggest that the use of baseline serum DHEAS levels in pubertal patients with suspected insufficiency of the HPA axis does not seem to be a reliable tool to diagnose either sufficiency or insufficiency of secondary adrenal function. Previous studies, [20-21, 23, 30] particularly in adult patients, reported that basal serum DHEAS levels to be an excellent diagnostic tool to confirm the diagnosis of CAI, with a sensitivity of 100%. [20] Nevertheless, in the present study, even though median basal serum DHEAS levels were significantly lower in the insufficient compared to the sufficient LDT cortisol response groups, basal serum DHEAS levels within the normal range were found in a high percentage of patients included in the former group. In addition, a similar discordance between LDT cortisol response and serum basal DHEAS levels was observed in the sufficient LDT cortisol response group. Therefore, the sensitivity and specificity of basal serum DHEAS levels for CAI diagnosis were 39% and 80%, respectively. One potential explanation for the discrepancy between these results and those found in previous reports, [20-21] may be the fact that in the study by Nasrallah et al. [20], only adult patients were evaluated. Additionally, in the younger group of patients studied by Fischli et al [21], only 11 patients with an age range between 14 and 30 years were included. In our study, median (and interquartile range) age was clearly younger. DHEA and DHEAS are the main products of the adrenal zona reticularis (ZR). The rise of these C19 steroids occurs during a physiological event known as adrenarche .This process is biochemically evident at around 6 years of age in both sexes. Even though the regulatory mechanism involved in the onset of adrenarche remains unknown, [30] there is evidence that ACTH is not involved. Along this line, several studies have demonstrated that serum ACTH and cortisol levels remain constant when adrenarche is taking place. [30] Given that a high incidence of normal serum DHEAS levels was observed in the patients included in the insufficient LDT cortisol response group, we may speculate that adrenarche might take place even in the presence of CAI. On the other hand, as several reports [19] have shown that serum DHEAS levels significantly increase during pubertal development in both sexes, it may be proposed that a normal HPA axis function would be required for increasing adrenal androgen production. Nevertheless, the discrepancy found in 20% of patients in the sufficient cortisol LDT group remains unclear. In this regard, a cortisol/adrenal androgens dissociation in chronic or critical illness or in adults previously treated with glucocorticoids has been reported, suggesting that it may be a physiological advantage to switch the synthesis process towards cortisol synthesis [22, 32, 33]. Recently, Kassem et al [30] demonstrated that patients with impaired HPA function have a more severe loss of serum DHEA than of glucocorticoid secretion in the LDT. Since none of the responders were on chronic glucocorticoid treatment or had severe illness before the LDT was performed, the discrepancy between sufficient LDT serum cortisol response and low basal serum DHEAS levels cannot be explained by chronic exposure to exogenous glucocorticoids supplementation, as has been previously proposed [22]. Another potential explanation for the discrepancy between previous results and our study might be related to the accuracy of the LDT serum cortisol peak cut-off limit established. Giordano [6] reported that a cortisol cut-off limit of 500nmol/l yielded a sensitivity of 76.7% and a specificity of 73.3 %. In the meta-analysis reported by Kazlauskaite et al, [9] authors stated that a stimulated serum cortisol level <16.9 µg/dl (466 nmol/l ) confirms the diagnosis of ACTH deficiency , whereas a level of >24.4 µg/dl (673 nmol/l) rules it out. In agreement with the latter study in the insufficient LDT cortisol response group a median (interquartile range) LDT serum cortisol peak level of 400nmol/l (334.7- 441.4), 14.5 µg/dl (range; 12-16), was observed. Moreover, the prevalence of CAI in our study was of 46.8%, similar to that reported by other series ranging from 27% to 58%, with a mean of 33%. [17] Even though the diagnoses of the patients included in this study are heterogeneous, this sample is representative of the majority of pediatric patients in whom adrenal insufficiency diagnosis needs to be confirmed. Another issue to consider is that in the literature, ACTH testing is almost only reported in adults, data in children are limited. In summary, basal serum, DHEAS levels in late prepubertal or pubertal patients do not seem to be a useful tool to diagnose either sufficiency or insufficiency of secondary adrenal function. In our cohort of patients, differently from previously published cohorts, diseases that led to HPA axis insufficiency occurred during a critical period of great changes in androgen synthesis. We suggest that dynamic testing would be necessary to assess the HPA axis in late prepuperty as well as during pubertal development. Nevertheless, clinical judgment remains the most reliable way to define the initiation of supplementary corticosteroid therapy. 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[29] Elmlinger M, Kuhnel W & Ranke M: Reference ranges for serum concentrations of lutropin (LH), follitropin (FSH), estradiol (E2), prolactin, progesterone, sex hormone-binding globulin (SHBG), dehydroepiandrosterone sulfate (DHEAS), cortisol and ferritin in neonates, children and young adults Clin Chem Laborat Med 2002; 40(11),1151-1160. [30] Sayyed Kassem L, El Sibai K, Chaiban J, Abdelmannan D, Arafah BM: Measurements of Serum DHEA and DHEA Sulphate Levels Improve the Accuracy of the Low-Dose Cosyntropin Test in the Diagnosis of CentralAdrenal Insufficiency. J Clin Endocrinol Metabolism 2012; 97(10), 3655?3662. [31] Hornsby P. J. Adrenarche: a cell biological perspective J Endocrinol 2012; 214, 133?143. [32] Okonko DO, Crosato M, Kalra PR, Cicoira M, John M, Doehner W, Coats AJ, Poole-Wilson PA, Anker SD: Association of deranged adrenal steroid metabolism with anemia in chronic heart failure. Am J Cardiol. 2005; 1,96(1):101-3. [33] Parker CR Jr & Baxter CR:Divergence in adrenal steroid secretory pattern after thermal injury in adult patients. J Trauma.1985; 25(6):508-10. Table 1. Features of the 94 patients included in the study. Gr1: sufficient response, and Gr2: insufficient response. a Clinical features and diagnosis. Total Sufficient response G1 Insufficient response G2 N (%) 94 50 (53.2) 44 (46.8) Gender: Female n (%) Male n (%) 39 (41.5) 55 (58.5) 19 (38) 31 (62) 20 (45.5) 24 (54.5) Age, in years median, interquartile range 13.88, 12-16 15.1, 13-16.5 * 12.9 ,11.7- 14.3 Tanner pubertal status, n (%) Early or mid puberty Late puberty 51 (54.3) 43 (45.7) 24 (48) 26 (52) 27 (61.3) 17 (38.6) Underlying diagnosis, n (%): Chronic corticoid treatment Multiple pituitary insufficiencies: Autoimmune disease Other 17 (18.1) 54 (57.4) 6 (6.4) 17 (18.1) 10 (20) 29 (58.0) 1 (2) 10 (20) 7 (15.9) 25 (56.8) 5 (11.4) 7 (15.9) * G1 vs G2, p<0.01 b) Hormone laboratory findings: Maximal serum cortisol LDT response, basal serum DHEAS, and basal serum ACTH. Total Sufficient response G1 Insufficient response G2 N 94 50 44 Previous morning basal cortisol , median, interquartile range, in nmol/l (µg/dl) 171, 135.2 -245.5 (6.2, 4.9-8.9) 179.3, 146.2- 245.5 ( 6.5, 5.3-8.9) 165.5, 135.2-259.3 (6.0, 4.9-9.4) LDT cortisol peak, median, interquartile range in nmol/l (µg/dl) 502.1, 402.8-579.4 (18.2, 14.6-21.0) 579.4, 540.7- 620.7 * (20.8, 19.6-22.5) 400, 334.7- 441.4 (14.5, 12-16) Basal serum DHEAS, median, interquartile range in µmol/l (ng/ml) 1.13, 0.59-2.74 (418, 218-1013) 2.13, 0.87-3.5 * (785, 321-1292) 0.7, 0.44-1.49 (261, 163-550) Basal serum DHEAS, N ≥ 2.5th perc. (%) 67 (71.3) 40 (80) ** 27 (61.4) Basal serum DHEAS, N < 2.5th perc. (%) 27 (28.7) 10 (20) ** 17 (38.6) Basal serum ACTH (median; interquartile range, pg/ml) 17.6 ;13.-28.7 18; 14-30 17.4 ; 13.1-29.1 * G1 vs G2, p<0.01 ** G1 Vs G2, p < 0.05 Legend of figure Fig 1 Pubertal patients with a sufficient response to the low-dose ACTH test (LDT), Gr1, and serum DHEAS levels greater (Gr1a, concordant) or lower than (Gr1b, discordant) the 2.5th percentile for age and sex. Pubertal patients with an insufficient response (Gr2) and DHEA-S greater (Gr2a) and lower (Gr2b) than the 2.5th percentile for age and sex. n: number of patients, % percentage of patients * Gr1a vs Gr2a ** Gr2b Vs Gr1b, p <0.03