The Response of C 19 Δ 5-steroids to ACTH Stimulation and Hypoglycemia in Insulin Tolerance Test for Adrenal Insufficiency

Studies on the time course of ACTHor insulin-induced hypoglycemia stimulating adrenal androgens are usually limited to dehydroepiandrosterone and/or its sulphate. Our data on dehydroepiandrosterone (DHEA) and its hydroxylated metabolites clearly show that measurements of DHEA and its sulphate (DHEAS) are valuable markers of the integrity of the HPA (hypothalamus-pituitary-adrenal) axis. Assessments of HPA function should rely on measurements of baseline and/or stimulated serum cortisol concentrations, and C19 Δ -steroids may provide additional information. The art of stimulation of 7and 16-hydroxylated metabolites of DHEA can help our understanding of the formation sequence of these compounds. http://dx.doi.org/10.14712/23362936.2016.10 © Charles University – Karolinum Press, 2016 This study was supported by grant no. NT 11277-6 IGA of the Ministry of Health of the Czech Republic. Mailing Address: Prof. RNDr. Luboslav Stárka, MD., DSc., Institute of Endocrinology, Národní 8, 116 94 Prague 1, Czech Republic; e-mail: lstarka@endo.cz


Introduction
The adrenal gland is a key provider of androgens in women (Short, 1960).The adrenal androgens dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstenedione, 5-andostenediol and 11β-hydroxyandrostenedione exert only little androgenic activity; however, they provide a pool of circulating precursors for more active androgens or steroids, with a wide spectrum of important physiological effects.The role of DHEA and DHEAS as neurosteroids has been intensively studied (Baulieu and Robel, 1998;Morfin and Stárka, 2001;Labrie, 2010;Li and Bigelow, 2010;Hill et al., 2015;Stárka et al., 2015).Its 7-hydroxylated metabolites modulate our immunity (Pélissier et al., 2004(Pélissier et al., , 2006;;Stickney et al., 2011) and take part in the control of local cortisol levels through competition in the 11β-hydroxysteroid dehydrogenase system (Hennebert et al., 2007;Sedláčková et al., 2012).The 16-hydroxylated derivatives may play some role in hormonal homeostasis (Hampl and Stárka, 2000).In spite of the importance of C 19 Δ 5 -steroids, their responsiveness to corticotrophin or insulin-induced hypoglycemia has not been well studied (Rege et al., 2013).In the present study, we quantified ten C 19 Δ 5 -steroids plasma samples from women before and after ACTH stimulation, and compared the results with stimulation of the hypothalamus-pituitary-adrenal (HPA) system by insulin-induced hypoglycemia.Dynamic testing of adrenal function is a standard procedure for the diagnosis of adrenal insufficiency, and we tried to find differences in the ACTH stimulation test in terms of C 19 Δ 5 -steroids between healthy women and patients with adrenal insufficiency.

Material and Methods
Our study involved six premenopausal females (with mean/median 43.4/42 age (SD ±6.2) years, and mean/median 25.4/24.6BMI (body mass index) (SD ±3.9) kg/m 2 ) with primary adrenal insufficiency verified by clinical symptoms and biochemical markers.The control group consisted of seven healthy BMI and age matched women.The women used no medications and had no history of using corticosteroids.All signed informed consent before initiating the study.The study was approved by the Ethical Commission of the Institute of Endocrinology.
All patients underwent the 250 μg "high dose" ACTH test.The control group was given two tests: the 250 μg "high dose" ACTH test and the insulin tolerance test (ITT).The minimum time between tests was one week.All tests were performed after an overnight fast, and started in the morning between 7 and 9 a.m.Synacthen and insulin were administered through a cannula inserted into the cubital vein, 15 minutes after insertion of the cannula.
Insulin-induced hypoglycemia stimulation -ITT: 0.1 IU per 1 kg Actrapid insulin was given intravenously.During the test, blood glucose was regularly checked with a glucometer (Accu-Chek Perform), and blood pressure and pulse rate were measured every five minutes during the first hour and every ten minutes thereafter.There was a decrease in blood glucose below 2.2 mmol/l in all of the tests, and all controls had a spontaneous blood glucose response during the first hour followed by normalization.Blood samples were taken prior to the administration of insulin at time = 0, and then after 20, 30, 40, 60, 90, and 120 minutes.The ITT was only carried out in the control group since it is unpleasant and may introduce risks in patients with adrenal insufficiency.

Analytical measurements Steroid hormones measured by the GC/MS method
The levels of C 19 Δ 5 -steroids and of additional 27 unconjugated steroids and their polar conjugates were measured in cubital vein blood using our original GC/MS method (Hill et al., 2010).In brief, free steroids were extracted from plasma by diethyl-ether; steroid conjugates were then hydrolyzed and extracted.The resulting residues were derivatized by methoxyamine hydrochloride and analysed by GC/MS as described below.

Instruments
Measurements of steroid levels were performed on a GCMS-QP2010 Plus system by Shimadzu (Kyoto, Japan) consisting of a gas chromatograph equipped with automatic flow control, an AOC-20s autosampler, and a single quadrupole detector with an adjustable electron voltage of 10-195 V.A capillary column with a medium polarity RESTEK Rxi phase (diameter 0.25 mm, length 15 m, film thickness 0.1 μm) was used for analyses.Electron impact ionization with electron voltage fixed at 70 V and emission current set to 160 μA was used.The temperatures of the injection port, ion source and interface were maintained at 220 °C, 300 °C, and 310 °C, respectively.Analyses were carried out in the splitless mode with a constant linear velocity of the carrier gas (He), which was maintained at 60 cm/s.The septum purge flow was set at 3 ml/min.The samples were injected using the high pressure mode (200 kPa), which was maintained for 1 min.The detector voltage was set to 1.4 kV.

Statistical analysis
Data were transformed by Box-Cox transformation before further processing due to non-Gaussian distribution and non-constant variance (heteroscedasticity) in all variables.Repeated-measures analysis of variance (ANOVA) was used for monitoring levels of steroids during the tests.The ANOVA model was followed by least significant difference (LSD) multiple comparisons.The statistical software Statgraphics Centurion, version XVI from Statpoint Inc. (Warrenton, VA, USA) was used for data transformations and ANOVA analyses.

Results
Basal plasma levels of C 19 Δ 5 -steroids and their levels at 60 min after ACTH stimulation are given in Table 1, and for insulin-induced hypoglycemia in Table 2.The trends of the steroid levels after the stimulation of adrenal secretion by ACTH and by insulin-induced hypoglycemia are shown in Figures 1-5.7α,7β,

105)
Prague Medical Report / Vol.117 (2016) No. 2-3, p. 98-107 1) For all steroids, both free and conjugated, levels during the ACTH tests in patients did not show significant increases, in contrast to the levels in healthy controls.2) Except for free DHEA and 7β-hydroxylated derivatives of DHEA, even the starting levels of the steroids significantly differed between the patients with adrenal insufficiency and healthy controls.3) In contrast to free steroids, changes of the levels of their conjugates during the ACTH test were insignificant.4) During the ITT, the increase of steroid conjugates was also either insignificant, or in some rare cases, as for conjugated 16α-hydroxy-DHEA, the increase was delayed.5) All free steroids (with the exception of 16α-hydroxy-DHEA) increased after both stimuli; however, the increase during the ITT had a latent time of about 20-40 min.6) The differences between the baseline and peak concentration at 60 min of C 19 Δ 5 -steroids in healthy controls was higher during the ACTH test than during the ITT.

Discussion
Dynamic testing of adrenal function is a standard procedure for the diagnosis of proper adrenal function.Testing with various doses of ACTH-24 or with insulininduced hypoglycemia (insulin tolerance test -ITT) is commonly used, and the concentration of circulating cortisol, either in plasma or in saliva, generally serves as the marker measured.In establishing the diagnosis of adrenal insufficiency, several authors have recommended measurements of baseline serum cortisol and DHEAS levels (Abdu et al., 1999;Al-Aridi et al., 2011).Our results indicated that it may be useful to additionally follow the stimulation of C 19 Δ 5 -steroids during the course of stimulation by either ACTH or ITT.Increases of DHEA or DHEAS after stimulation are observed in plasma but not in saliva (Dušková et al., 2016), due to the insufficient ultrafiltration of these compounds to saliva.
Our present data also show that the levels of C 19 Δ 5 -steroids are reliable markers of adrenal insufficiency.Even the basal levels of conjugated DHEA, free or conjugated 5-androstenediol, free 7α-hydroxy-and 16α-hydroxymetabolites of DHEA differed significantly between healthy subjects and patients with impaired adrenal function.In contrast to healthy individuals, there was no increase of C 19 Δ 5 -steroids during the ACTH test in the group of patients with adrenal insufficiency.
The lack of an increase of conjugated C 19 Δ 5 -steroids both during the ACTH and ITT tests before 60 min is in agreement with other findings of the postponed increase of conjugated DHEA (Sayyed Kassem et al., 2012), and with the observation that there is a stimulation of DHEA but no response in DHEAS production by ACTH in normal adrenocortical cell suspensions (Fehér et al., 1985).This is compatible with the differences in the half-times of DHEA (2-3 h) and DHEAS (20 h) (Kroboth et al., 1999) and with the concept that the biosynthesis of DHEA is under the control of ACTH, while other factors may contribute to the regulation of the sulphate pathway of DHEA secretion under normal conditions.
The effective and rapid stimulation of adrenal C 19 Δ 5 -steroids by ACTH is an argument against the hypothesis of the presence of a special unknown hypothalamic or pituitary adrenal androgen-stimulating hormone (Parker and Odell, 1979;Odell andParker, 1984-1985), a hypothesis that until now has neither been confirmed nor refuted.
Whereas changes of DHEA or DHEAS levels in the course of dynamic testing have been reported in many studies (Griffing et al., 1985;Abdu et al., 1999;Al-Aridi et al., 2011;Sayyed Kassem et al., 2012), the response of hydroxylated derivatives of DHEA had not yet been studied.7α-hydroxymetabolites are rapidly stimulated by both ACTH and ITT in coordination with DHEA levels, while an increase of 7β-hydroxymetabolites is either delayed or absent.This is in agreement the concept that 7α-hydroxylation is the primary reaction and that 7β-epimers are a later product of either direct isomerization or conversion through a 7-oxo-derivative.
A quite exceptional reaction to stimulation was observed for 16α-hydroxy-DHEA.We found that the levels of free 16α-hydroxy-DHEA did not change significantly during either the ACTH or ITT tests; however, conjugated 16α-hydroxy-DHEA increased during the ACTH test in the group of adrenal insufficient women as well as in controls during the ITT.
In conclusion, our data clearly show that DHEA and DHEAS measurements are valuable markers of the integrity of the HPA axis.Assessments of HPA function should rely on the measurement of baseline and/or ACTH-stimulated serum cortisol concentrations, and C 19 Δ 5 -steroids may provide additional information.The art of stimulation of hydroxylated metabolites of DHEA can help improve our understanding of the sequence of the formation of these compounds.

Figure 1 -
Figure 1 -Plasma dehydroepiandrosterone (DHEA) and conjugated DHEA in high dose ACTH stimulation test (HDST) for adrenal insufficient women and HDST and insulin-induced hypoglycemia stimulation test (ITT) for controls.
Figure7α,7β, in high dose ACTH stimulation test (HDST) for adrenal insufficient women and HDST and insulin-induced hypoglycemia stimulation test (ITT) for controls.

Table 1 -Baselines and values at 60 th min of ACTH test for patients and controls
Median and quartiles are given; *significance at p<0.05

Table 2 -Baselines and values at 60 th min of ITT test for controls
Median and quartiles are given; *significance at p<0.05