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Endocrinology, doi:10.1210/en.2005-1161
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Endocrinology Vol. 147, No. 9 4281-4291
Copyright © 2006 by The Endocrine Society

The Adrenocorticotropin Stimulation Test: Contribution of a Physiologically Based Model Developed in Horse for Its Interpretation in Different Pathophysiological Situations Encountered in Man

Alain Bousquet-Mélou, Enrique Formentini, Nicole Picard-Hagen, Laure Delage, Valerie Laroute and Pierre-Louis Toutain

Unité Mixte de Recherche 181 de Physiopathologie et Toxicologie Expérimentales, Institut National de la Recherche Agronomique et Ecole Nationale Vétérinaire de Toulouse, Ecole Nationale Vétérinaire de Toulouse, 31076 Toulouse cedex 03, France

Address all correspondence and requests for reprints to: Alain Bousquet-Mélou, Unité Mixte de Recherche 181 de Physiopathologie et Toxicologie Expérimentales, Institut National de la Recherche Agronomique et Ecole Nationale Vétérinaire de Toulouse, Ecole Nationale Vétérinaire de Toulouse, 23 Chemin des Capelles, 31076 Toulouse cedex 03, France. E-mail: a.bousquet-melou{at}envt.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study aimed to characterize the adrenal response to ACTH. A model was developed that coupled the nonlinear disposition of cortisol with a physiologically based model for cortisol secretion by the adrenals. It was assumed that the response to ACTH resulted from two mechanisms: a stimulation of the cortisol secretion rate and control of the duration of the secretion. Seven dose levels of ACTH were tested in horses, a species similar to man as regards adrenal function. The main result was that the secretion rate of the adrenal gland can be modelized by a zero order process that is maximal for a relatively low dose of ACTH (0.1 µg/kg). Beyond this dose, the increasing adrenal gland response is only due to the prolongation of the time of its secretion. The consequences of these different features were explored by simulation to reproduce classical pathophysiological situations encountered in man. Our model was able to reproduce and simply explain many adrenal gland responses that are dimmed by the different nonlinearities of the system.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ACTH STIMULATION TESTS are among the most commonly used methods for clinical assessment of the hypothalamic-pituitary-adrenal (HPA) axis. The iv injection of high doses (250 µg) of ACTH is a standard test for HPA evaluation (1, 2), but during the past decade, a low-dose (1 µg or 0.5 µg/m2) ACTH test has been advocated as a more sensitive method for the detection of certain impairments of adrenal function (3, 4, 5). Since then, a debate has arisen as to whether or not the low-dose ACTH test is more sensitive than the high-dose test in detecting mild degrees of adrenal insufficiency and on the place of rapid ACTH tests in comparison with insulin and metyrapone tests (6, 7, 8).

The interpretation of rapid ACTH stimulation tests is currently based on the measurement of serum or plasma concentrations of total cortisol before and 30 and 60 min after iv injection of ACTH (2). In fact, the measurement of total plasma cortisol concentration is only a surrogate biomarker that imperfectly represents the function of interest, namely the time profile of adrenal gland response in terms of cortisol secretion rate. Using the total plasma cortisol concentration as an index of adrenal gland function may in fact lead to difficulties due to the nonlinear relationship existing between the measured total plasma cortisol concentration and the actual adrenal gland secretion rate.

The total cortisol concentration in blood is controlled not only by cortisol production (CP) rate (the parameter of interest) but also by processes of cortisol elimination and transport, the saturable binding to the corticosteroid-binding globulin (CBG), and a given total plasma cortisol concentration may correspond to different combinations of its three determinants. This is the case when the CBG level is altered in response to severe stress (9, 10).

To limit this first difficulty, we have developed a modeling approach to directly assess in vivo the cortisol binding parameters and the disposition of circulating cortisol not bound to CBG from the total plasma cortisol concentrations (11).

The second difficulty in interpreting rapid ACTH tests is that no more than two or three snapshot measurements of plasma cortisol are generally used as indicators of an overall adrenal response (AR). Little information is available to justify the choice of the standard sampling times used in these tests. More importantly, the factors governing the dose-response curve of the stimulatory effect of ACTH on circulating cortisol are poorly understood, and it will become apparent that peak cortisol concentration or area under the curve of cortisol vs. time profiles cannot be used indiscriminately as indicators of the adrenal gland response (12, 13) because they are not exploring the same underlying mechanisms.

A rational choice of cortisol sampling times may be documented from a mechanism-based model of the time development of cortisol secretion in response to iv ACTH. Because intraadrenal cortisol storage is minimal, the acute action of ACTH on the adrenal cortex is mainly to increase cortisol synthesis (14) and only secondarily to increase cortisol secretion. It has been shown that the EC50 of ACTH(1–24) is in the picomolar range for CP in human adrenocortical cells (15) or the stimulation of cAMP production with cloned mouse ACTH receptors (16). On the other hand, peak plasma ACTH concentrations in humans have been reported to be around 1,900 and 66,000 ng/ml after low- and high-dose ACTH tests, respectively (6), corresponding to 633 and 22,000 pM concentrations. Comparison of these in vitro and in vivo data suggests that the ACTH concentrations obtained just after ACTH administration are able to maximally stimulate the ACTH receptors in the adrenals, thereby ensuring a maximal response in terms of cortisol secretion. In this context, the duration of maximal stimulation of the adrenal gland will depend mainly on the decay of the plasma ACTH concentrations, i.e. on the plasma ACTH half-life and not on the adrenal gland responsiveness.

In the present study, we aimed to characterize the time development of the AR to increased doses of iv administered ACTH, using horses as an experimental model because of the similarities of their HPA axis function to that of humans, in terms of cortisol plasma levels, circadian rhythm, and production rate (17, 18). Simultaneous recourse to a model for the disposition of cortisol not bound to CBG and a physiologically based model for cortisol secretion by the adrenals enabled us to explore the overall plasma cortisol vs. time profile in response to iv ACTH and to simulate different what-if pathophysiological scenarios.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Eight healthy saddle-bred horses (~9 yr old) weighing 512 ± 29 kg were used. The horses were weighed three times during the course of the experiments with a 2-month interval between each weighing; the coefficients of variation were less than 4%. The horses were exercised 1–2 h/d, housed in individual boxes, and fed an appropriate commercial diet of two meals per day, with straw and water given ad libitum. They were accustomed to the sampling procedures and did not show any sign of agitation or stress.

All procedures involving animals were performed in accordance with the French legal requirements regarding the protection of laboratory animals and with the Authorization for Animal Experimentation no. 001889 of the French Ministry of Agriculture.

Experimental design, administrations, and blood sampling
Total and CBG-free plasma cortisol concentration vs. time profiles were obtained after administration of seven doses of synthetic peptide ACTH(1–24) (Synacthène immédiat, Novartis, Basel, Switzerland; 0.25 mg/ml). The experiment was carried out with an eight x eight crossover design, each horse receiving a placebo (0.9% NaCl) and seven ACTH doses (0.005, 0.01, 0.1, 0.5, 1, 2, and 10 µg/kg). The time between consecutive ACTH administrations was about 4–7 d. Two horses were withdrawn during the study for intercurrent diseases not related to ACTH administration (colic).

Twelve and 2 h before the administration of ACTH, dexamethasone (Cortamethasone, Vetoquinol S.A., Magny-Vernois, France) was administered im at 0.1 mg/kg (50 ml for a horse of 500 kg) to suppress the basal secretion of cortisol.

ACTH was injected into the right jugular vein at 0900 h via an indwelling catheter inserted 5 min before administration. The catheter was rinsed with 2 ml of 0.9% NaCl after the ACTH injection. Blood (10 ml) was drawn by direct venipuncture (22-gauge needle) from the left jugular vein at –40 –20, 5, 10, 20, 30, 40, 50, 60, 90, and 120 min and each hour until 4 h (0.005 µg/kg ACTH dose) or 8 h (0.01, 0.1, 0.5, 1, and 2 µg/kg ACTH doses), and at –40, –20, 5, 15, 30, 60, 90, and 120 min and each hour until 8 h after the 10 µg/kg ACTH dose.

Blood samples were collected in heparinized tubes and centrifuged at 1400 x g at 4 C for 10 min within 2 h of collection. The plasma was separated and aliquoted in three fractions and stored at –20 C until assay.

Total cortisol assay
Cortisol was assayed by RIA (cortisol assay kit, Beckman Coulter, Roissy, France) in duplicate using 100-µl aliquots of plasma. The calibration curve (10–300 ng/ml) was obtained with horse plasma stripped of steroids by charcoal. The level of quantification of the assay was 10 ng/ml. Within- and between-day coefficients of variation were lower than 15%.

Data analysis
Cortisol kinetic and binding parameters.
The pharmacokinetic part of the pharmacokinetic/pharmacodynamic model developed in this study is described by the set of equations presented in the supplemental data (published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Briefly, this pharmacokinetic model describes the behavior of the total and free fractions of an analyte exhibiting nonlinear binding to plasma proteins. Such a model was first developed by Toutain et al. (19) for angiotensin-converting enzyme inhibitors and adapted for cortisol disposition by Picard-Hagen et al. (11).

This model incorporates the main kinetic and binding parameters for cortisol not bound to CBG (CBG-free cortisol) controlling circulating cortisol profiles: plasma clearance of CBG-free cortisol (ClCBG-free), CBG maximal binding capacity (Bmax), and Kd CBG-free, equilibrium dissociation constant for CBG-free cortisol binding on CBG. The numerical values of these parameters had been determined during a previous experiment performed on the same horses. Briefly, this experiment consisted of the iv administration of cortisol at four dose levels (0.125, 0.25, 0.5, and 1 mg/kg). The four individual plasma cortisol concentration profiles obtained for each horse were simultaneously fitted using the set of equations presented in the supplemental data.

The mean values of ClCBG-free, Bmax, and Kd CBG-free are presented in Table 1Go. The binding parameters were used to calculate the plasma CBG-free cortisol concentrations (CBGfree) from the total plasma cortisol concentrations (CORtot) using the following quadratic equation derived by Tait and Burnstein (20):

Formula 1(1)


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  		TABLE 1. Mean pharmacokinetic and CBG binding parameters of CBG-free cortisol in eight horses

 
AR to ACTH stimulation.
Different indices were used to describe the AR to increasing doses of ACTH: plasma peak (Cmax) of total or CBG-free cortisol, area under the plasma concentration vs. time curve of total or CBG-free cortisol from time zero to the last concentration measured (AUC0-clast), and CP (micrograms per kilogram). The Cmax values of total and CBG-free cortisol were obtained by visual inspection of the plasma profiles of both fractions. The AUC0-clast of total and CBG-free cortisol were calculated using the arithmetic trapezoidal rule. The CP over a specific time interval was calculated using the following equation:

Formula 2(2)
The ACTH-cortisol dose-response curves were fitted using a sigmoid Emax model, the so-called Hill equation, which is the Michaelis-Menten equation extended by a power coefficient and is traditionally used for sigmoid curve fitting (21):

Formula 3(3)
where AR is as previously defined, D is the dose of ACTH, Rmax is the maximal AR, ED50 is the dose of ACTH producing the half-maximum response, a parameter expressing ACTH potency, and n is the power coefficient, which describes the slope of the dose-effect curves and is a measure of sensitivity of the dose-effect relationship.

For each AR, the Rmax, ED50, and n values were estimated by simultaneous fitting of individual dose-response curves using nonlinear least squares regression (Scientist, MicroMath Software, Salt Lake City, UT).

Cortisol secretion process.
The cortisol secretion by adrenals after iv injection of ACTH was modeled using a physiologically based model taking into account the nonlinear disposition of cortisol (equations 1Go and 2Go in the supplemental data) and for which the cortisol input rate (cortisol secretion) was modeled as a zero order event (k0; micrograms per kilogram per minute) over releasing time (RT; minutes). These parameters (k0, RT) are linked to CP by the following equation:

Formula 4(4)
Estimates of the secretion rate, k0, and duration of secretion, RT, were obtained at each ACTH dose level by simultaneous fitting of the total plasma cortisol concentration profiles obtained for all the horses.

The increasing response of the adrenals when the ACTH doses increased was modeled by assuming that k0 and RT were dependent on the ACTH dose. The curves resulting from the relationships between k0 and RT estimates (dependent variables) and ACTH doses (independent variable) were fitted using the following equations:

Formula 5(5)

Formula 6(6)
where k0, RT, D, Rmax, ED50, and n are as previously defined, and I is a factor with the dimension of a dose. Equation 5Go was empirically selected to take into account the tendency toward a decrease in adrenal responsiveness to higher ACTH doses; the term:

Formula 6
is classically used in modified Michaelis-Menten equations describing enzymatic kinetics that exhibit inhibition by substrate excess (22).

Finally, plasma cortisol concentration profiles corresponding to different ACTH doses were simulated with a pharmacokinetic-pharmacodynamic model combining the pharmacokinetic disposition model for circulating CBG-free cortisol (see supplemental data) and the pharmacodynamic model describing the relations between ACTH doses and the cortisol secretion process (equations 5Go and 6Go).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma cortisol profiles in response to ACTH stimulation
In all experiments, dexamethasone administration was followed by suppression of spontaneous cortisol secretion (total cortisol concentrations were below the quantification limits of the assay). The placebo (0.9% NaCl) did not elicit any quantifiable increase in total cortisol levels, indicating that the experimental procedures (e.g. placing catheters, giving injections, collecting samples, etc.) were without measurable stimulatory effect on the HPA axis.

The plasma concentration vs. time profiles of total cortisol after iv administration of increasing doses of ACTH indicated a clear dose-response relationship (Fig. 1Go, A and B). The profiles corresponding to CBG-free cortisol (Fig. 1Go, C and D) were obtained from equation 1Go.


Figure 1
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  		FIG. 1. Mean plasma cortisol concentrations after iv administration of ACTH in six to eight horses. The doses tested were: 0.005 ({blacktriangleup}), 0.01 ({circ}), 0.1 ({blacksquare}), 0.5 ({diamond}), 1 (bullet), 2 ({square}), and 10 ({Delta}) µg/kg. Arithmetic (A and C) and semilogarithmic (B and D) plots represent total (A and B) and CBG-free (C and D) plasma cortisol concentration profiles. Visual inspection indicates for the ACTH dose of 0.1 µg/kg and beyond that the secretion rate of the adrenal gland plateaued and that higher ACTH doses only increased the duration of the adrenal gland secretion.

 
ARs to ACTH stimulation
The relations between the ACTH doses and the five indices used to describe AR, namely total and CBG-free Cmax, total and CBG-free AUC0-clast, and CP, are presented in Fig. 2Go. These dose-response curves were fitted using a sigmoid model (equation 3Go). Fitted curves and the corresponding estimated parameters are shown in Fig. 2Go.


Figure 2
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  		FIG. 2. Dose-response relationship between ACTH doses (0.005–10 mg/kg) and AR in terms of CP (A), AUC of plasma cortisol profiles (B), and peak plasma cortisol (C). bullet, Total cortisol; {circ}, CBG-free cortisol. Experimental data (mean ± SD) and fitted curves (—). Inspection of the figure indicates a lack of parallelism between total and CBG-free cortisol when the AUC is selected as an endpoint (B), whereas the responses are roughly parallel for total cortisol and CBG-free cortisol when the maximal plasma concentration is considered (C). Parameters of the dose-response relationship (equation 3Go) are presented: Rmax, maximal response; ED50, ACTH dose producing half of the maximal response; n, Hill coefficient. Values shown are mean estimates (coefficients of variation, percentage).

 
The values of ED50 (ACTH potency) exhibited considerable differences when the adrenal gland response was assessed either in terms of Cmax or overall plasma cortisol exposure (AUC0-clast), namely 0.012 and 0.054 µg/kg for total and CBG-free Cmax compared with 0.14 and 0.30 for total and CBG-free AUC0-clast. In practice, this means that the Cmax dose-response curve is shifted to the left compared with the AUC0-clast dose response and that Cmax reaches a maximal value at ACTH doses much lower than those required to obtain a maximal value for AUC0-clast (Fig. 2Go).

AUC0-clast CBG-free and CP responses exhibited similar values for ED50 and n, as expected inasmuch as they are linked by a constant proportionality factor (equation 2Go).

Modeling the cortisol secretion process
Two models, assuming either a first or zero order process for cortisol release by the adrenals, were investigated to analyze simultaneously the different profiles of total cortisol obtained with each dose of ACTH. Visual inspection of fitting revealed obvious misfits with the model that assumed a first order secretion process (data not shown) and goodness-of-fit criteria confirmed a better fitting of the model that assumed a zero order adrenal secretion process.

The fitted total plasma cortisol profiles of all the horses for the seven ACTH doses are presented in Fig. 3Go. Estimated values of k0 (secretion rate) and RT (duration of secretion), and the corresponding CP (equation 4Go) were obtained from these fittings and are presented in Table 2Go. Both k0 and RT exhibited an obvious tendency to increase with increasing ACTH doses (Table 2Go), with k0 tending to plateau at ACTH doses higher than 0.1 µg/kg, whereas RT continued to increase over the range of ACTH doses tested.


Figure 3
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  		FIG. 3. Plasma concentration vs. time curves of total cortisol after iv administration of ACTH in six to eight horses. ACTH doses were 0.005 (A), 0.01 (B), 0.1 (C), 0.5 (D), 1 (E), 2 (F), and 10 µg/kg (G). Experimental data (mean ± SD) and fitted curves (—) are presented. Data were analyzed with a compartmental model taking into account the nonlinear disposition of cortisol and assuming that cortisol is released by an ACTH-stimulated adrenal gland according to an on/off zero order process over a given duration of time.

 

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  		TABLE 2. Parameters of CP by adrenals after iv ACTH in six to eight horses assuming a zero-order secretion process

 
The highest CP value calculated from equation 4Go, assuming a zero order secretion process for cortisol (136 µg/kg, Table 2Go), was close to the maximal CP value estimated with equation 3Go (123 µg/kg, Fig. 2Go).

Relations between cortisol secretion parameters and ACTH doses
Both parameters of cortisol secretion (k0 and RT) were clearly related to the ACTH doses (Fig. 4Go). The k0 and RT vs. ACTH dose curves were best described by equations 5Go and 6Go, respectively. Figure 4Go shows the fitted curves and the corresponding parameters.


Figure 4
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  		FIG. 4. Dose-response relationship between ACTH doses (0.005–10 mg/kg) and parameters of cortisol secretion by adrenals. A, k0, zero order rate constant of cortisol secretion; B, RT, duration of cortisol secretion by adrenals. Experimental data ({circ}) were obtained from the fitting of the post-ACTH cortisol profiles presented in Fig. 3Go. —, Fitted curves using equations 5Go (A) and 6Go (B). Inspection of A indicates that k0 reached a near-maximal value (– – –) over the (0.1–10 µg/kg) ACTH dose range, whereas RT (B) increased log-linearly (– – –) over the (0.1–10 µg/kg) ACTH dose range. Parameters of the dose-response relationship (equations 5Go and 6Go) are presented: Rmax, maximal response; ED50, ACTH dose producing half of the maximal response; n, Hill coefficient; I, dimension-less parameter. Values shown are mean estimates (coefficients of variation, percentage).

 
Inspection of Fig. 4Go clearly indicated that k0 reached a plateau for low ACTH doses, whereas RT continued to increase over the range of ACTH doses tested. Considering the 0.1–10 µg/kg range of ACTH doses, the relations between k0 or RT and the ACTH dose can be summarized as an average maximal value of 0.91 µg/(kg·min) for k0 (Fig. 4AGo, dashed line) and a log-linear relationship for RT (Fig. 4BGo, dashed line).

Pharmacokinetic/pharmacodynamic model linking ACTH doses to plasma cortisol profiles
By coupling the pharmacodynamic model connecting ACTH doses to the cortisol secretion process (k0, RT) with the pharmacokinetic disposition model involving cortisol binding to CBG, it was possible to simulate total and CBG-free plasma cortisol concentration vs. time profiles for any ACTH dose from 0.005–10 µg/kg.

Figure 5Go shows the agreement between simulated CBG-free cortisol profiles and the corresponding average profiles obtained from experimental data over the ACTH range tested.


Figure 5
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  		FIG. 5. Plasma concentration vs. time curves of CBG-free cortisol after iv administration of ACTH in six to eight horses. ACTH doses were 0.005 (A), 0.01 (B), 0.1 (C), 0.5 (D), 1 (E), 2 (F), and 10 µg/kg (G). Experimental data (mean ± SD) and simulated curves (—) are presented. Experimental data were obtained by calculation of CBGfree from measured total cortisol and estimated binding parameters. Simulated curves were obtained with the general pharmacokinetic/pharmacodynamic model taking into account both the nonlinear disposition of cortisol and the ACTH-dependent cortisol secretion by adrenals.

 
Simulations
By modifying some parameters from the cortisol secretion process, the disposition of circulating cortisol or both factors, we were able to simulate the AR to ACTH under several pathophysiological situations. Simulations were performed using three ACTH doses: 0.014, 0.05, and 5 µg/kg. We selected these ACTH doses to be relevant for comparisons with the doses used in humans for low- and high-dose ACTH tests. Indeed, in humans weighing 50–70 kg, the standard high ACTH dose (250 µg) corresponds to 3–5 µg/kg and the standard low dose (1 µg) to 0.01–0.02 µg/kg. So, in horses, the 0.014 µg/kg dose is in the range of the low-dose ACTH test in humans, and the 5 µg/kg dose corresponds to the high-dose ACTH test (the 0.05 µg/kg dose is intermediate).

We first performed simulations corresponding to HPA axis stimulation with the three doses of ACTH (Fig. 6Go). Although differences in total CP were obvious between doses, the total cortisol concentration at 30 min post-ACTH was similar for the two highest doses.


Figure 6
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  		FIG. 6. Influence of the ACTH dose on the shape of the plasma concentration profiles of total cortisol. ACTH doses: 0.014 (thin line), 0.05 (dashed line), and 5 (thick line) µg/kg. {circ}, Simulated total cortisol concentrations at 30 and 60 min post-ACTH. Inspection of the figure shows that the total plasma cortisol has the same value at 30 min post-ACTH after the two highest doses, whereas a later sampling (60 min) may be able to distinguish between these two dose levels.

 
Secondly, we performed simulations mimicking an adrenal insufficiency, corresponding to the halving of CP in response to iv ACTH. Figure 7Go compares the total cortisol plasma profiles obtained by either halving the RT or k0 of the control condition, using ACTH doses 0.05 and 5 µg/kg. Adrenal insufficiency simulated by decreased k0 led to diminished plasma cortisol responses at 30 and 60 min with both ACTH doses. The same was true for adrenal insufficiency due to decreased RT but only for the lower ACTH dose. In the simulation corresponding to reduced RT, plasma cortisol concentrations 30 and 60 min after the higher ACTH dose did not differ from normal, although sampling at 120 min allowed a clear-cut discrimination between normal and deficient states.


Figure 7
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  		FIG. 7. Simulations of plasma concentration profiles of total cortisol after iv ACTH at 5 (A) and 0.05 (B) µg/kg. Three different conditions were simulated: plasma cortisol profile obtained with the cortisol secretion parameters (k0, RT) estimated from experimental data (thick line), plasma cortisol profile obtained by halving k0 (thin line), and plasma cortisol profile obtained by halving RT (dashed line). {circ}, Simulated total cortisol concentrations at 30 and 60 min post-ACTH; bullet, simulated total cortisol concentrations at 120 min post-ACTH. Inspection of the figure indicates that an ACTH dose of 5 µg/kg is appropriate to diagnose a condition characterized by a reduction of the secretion rate when samples are obtained at 30 and 60 min but not for a condition for which only the duration of the secretion rate is reduced except if a 120-min sample is collected. In contrast, for the small ACTH dose (B), the two pathological conditions are acknowledged with samples at 30 and 60 min, but their etiology (level or duration or secretion rate) remains indistinguishable.

 
In a final set of simulations, we investigated the effects on plasma cortisol profiles after ACTH administration (5 µg/kg) of modifications of plasma cortisol binding, i.e. a modification of Bmax, resulting from variations in CBG concentrations. Firstly, changing the binding capacities induced obvious alterations in the concentration profiles of total cortisol (Fig. 8AGo), whereas there was no effect on the cortisol not bound to CBG (Fig. 8BGo). Secondly, we simulated simultaneous modifications of adrenal function (2-fold increase of the secretion rate, k0) and plasma cortisol binding (halving of CBG level Bmax), a situation corresponding to chronic stress in horses and in humans (see Discussion). Comparison of the simulated cortisol profiles in normal and impaired situations showed that the decreased CBG concentrations compensated for the increased CP rate, such that the resulting total cortisol profiles did not differ sufficiently to permit discrimination at 30 and 60 min after ACTH (Fig. 8CGo). However, the CBG-free cortisol profiles after 5 µg/kg ACTH differed in a manner that permitted discrimination between the two situations at the 30- and 60-min sampling times (Fig. 8DGo).


Figure 8
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  		FIG. 8. Simulations of plasma concentration profiles of total (A and C) and CBG-free (B and D) cortisol after an iv dose of ACTH at 5 µg/kg. A and B, Plasma cortisol profiles obtained with the cortisol secretion parameters (k0, RT) and the CBG-binding capacity (Bmax) estimated from experimental data (thick line), plasma cortisol profiles obtained by halving (thin line) or doubling (dashed line) Bmax. C and D, Plasma cortisol profiles obtained with the cortisol secretion parameters (k0, RT) and the CBG-binding capacity (Bmax) estimated from experimental data (thick line), plasma cortisol profiles obtained by simultaneously halving Bmax and doubling k0 (thin line). {circ}, Simulated total cortisol concentrations at 30 and 60 min post-ACTH. Visual inspection of A and B indicates that CBG concentration has a major impact on the total plasma cortisol concentration but no effect on CBG-free cortisol. C and D, Conditions characterized by both an increase of cortisol secretion rate and a decrease of CBG concentration are not distinguishable from the measurement of the total plasma cortisol concentration but easily detected from the CBG-free plasma cortisol.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main result of the present experiment was the 200-fold difference between the potency of ACTH to increase the two proposed mechanisms of the overall adrenal gland response to an ACTH treatment, namely k0 and RT of the cortisol secretion rate. ACTH potency as measured by the ED50 was 0.00347 and 0.72 µg/kg for k0 and RT, respectively. Thus, the increased CP in response to increasing ACTH doses within the tested ACTH dose range (0.005–10 µg/kg) was mainly due to the increase of RT, whereas k0 reached a maximal value at very low ACTH doses (0.1 µg/kg). In other words, the ACTH tests are only able to explore concentration-dependent mechanisms of the adrenal gland response at very low ACTH doses but then reflect different time-dependent mechanisms as soon as the ACTH dose exceeds 0.1 µg/kg.

In the present study, we aimed to build a physiologically based model describing the relation between an iv bolus of ACTH and AR in terms of plasma cortisol concentration vs. time profiles. The genuine feature of our model was to integrate in a single set of equations the quantitative relationship existing between adrenal cortisol secretion and dose of iv ACTH and the in vivo nonlinear disposition of cortisol. The ultimate goal was to use this model to simulate the ARs to ACTH and explore various possible pathophysiological conditions known in man to modify the secretion and/or disposition of cortisol. This model offers a framework for interpretation of cortisol responses to rapid ACTH tests and should provide valuable information of clinical relevance, especially to justify the choice of ACTH dose and select the most appropriate blood sampling time schedule for assessing adrenal gland responsiveness. For the present investigation, we used parameters actually obtained in horses by applying a nonradiolabeled method to estimate in vivo the adrenal gland secretion rate and corresponding cortisol disposition parameters (11). However, the same hypothesis regarding the validity or nonvalidity of free vs. total cortisol as a surrogate of adrenal gland response could be tested with our model but including sets of possible guest values obtained from the literature to extend our conclusions to other species, including man.

There is extensive literature describing the relationship between exogenous ACTH and cortisol concentrations, and various studies have provided an interpretation of the dose (concentration)-response curves in animals (23) or humans (12, 13, 24, 25). At the same time the importance of measuring free, not total, cortisol in ACTH stimulation tests is also gaining recognition (10, 26, 27). In our modeling approach, we combine a pharmacodynamic model and a pharmacokinetic model, both based on previous knowledge of cortisol secretion and disposition, that can reproduce and explain the AR to ACTH in terms of circulating free and total cortisol.

Several endpoints related to circulating cortisol concentrations have been used to describe the HPA axis response to various stimulations. These include snapshot cortisol concentrations, area under the total cortisol concentration vs. time curve, CP, peak cortisol concentration, or time to peak cortisol. Our model indicates that none of these endpoints alone is able to describe univocally the time course of the HPA axis response to ACTH stimulation. This is due to the conjunction of two unrelated properties of the investigated system, namely the nonlinear disposition of cortisol and the existence of two different mechanisms governing the adrenal gland response when stimulated by a low or high ACTH dose.

The nonlinearity of cortisol disposition is due to its saturable binding to CBG, the maximal binding capacity of CBG being exceeded during an ACTH test. This explains the lack of parallelism between the dose-effect curves expressed in terms of total vs. CBG-free cortisol. This is particularly evident when the selected endpoint is an AUC; in contrast, the distortion due to the nonlinearity is minimal when considering the peak cortisol concentration because, whatever the tested ACTH dose, the Cmax is observed within a range of cortisol concentrations for which the CBG is saturated meaning that all maximal plasma cortisol concentrations were obtained in conditions where the cortisol disposition had become linear (see Fig. 2Go). The best approach to circumvent the confounding factor represented by the nonlinearity of total cortisol disposition is to take into account the nonlinear binding of cortisol to plasma CBG because more than 90% of the circulating cortisol in humans is bound to CBG (28), and the proportion of cortisol bound to CBG is from 67–87% in several domestic species including horses (17).

The part of the present model describing the nonlinear disposition of circulating cortisol due to the binding to CBG (see supplemental data) has been presented in detail elsewhere (11). This model was used to fit the total plasma cortisol vs. time profiles observed after different doses of ACTH and enabled us to perform simulations to investigate the influence of alterations in cortisol binding to CBG on circulating cortisol profiles during rapid ACTH tests. We showed that modification of the CBG concentrations (i.e. the binding parameter, Bmax) induced changes in the simulated total cortisol concentration vs. time profiles (Fig. 8AGo). Considering their amplitude, such modifications seem highly relevant for the interpretation of rapid ACTH tests and confirm recent work suggesting that normal interindividual variations of CBG levels in healthy subjects can affect the results of rapid ACTH tests (10). As expected, the corresponding plasma concentration profiles of cortisol not bound to CBG showed little or no modification, rightly indicating the lack of alteration of the cortisol secretion process (Fig. 8CGo).

An increasing number of studies report situations characterized by alterations of both HPA axis activity and plasma CBG levels. In particular, HPA axis activation and a decrease in CBG levels are observed during severe stress in patients with inflammation and burn injury (29), septic shock (9), or undergoing cardiac surgery (30). The same pattern has been described in other species, during acute stress in rats (31) or social stress in horses (32). Endocrinometabolic disorders such as obesity (33, 34) and insulin resistance syndrome (35) were also associated with both these alterations in HPA axis activity and plasma CBG levels. Our simulations to compare the physiological situation with one in which there was both an increase in adrenal cortisol secretion (increased k0) and a decreased CBG (decrease in Bmax) indicated that, when considering only the plasma concentration profiles of total cortisol, these changes tended to cancel each other, so that the HPA axis alteration was not detected by the ACTH test (Fig. 8BGo). Conversely, because the plasma concentration profiles of cortisol not bound to CBG were only controlled by the cortisol secretion process, the ACTH test was able to detect the HPA axis modification (Fig. 8DGo). Thus, as recently stated by Dhillo et al. (10), circulating cortisol levels should always be interpreted in reference to CBG concentrations, and the modeling approach proposed here should be very useful for investigating this point. An alternative is to select an analytical technique that specifically measures the plasma free cortisol concentration.

Whatever the analytical technique used for the cortisol measurement (free or total), the shape of the dose (concentration)-response curve linking the ACTH dose and the cortisol (free or total) response need to be understood to properly interpret the results of an ACTH test or/and select an optimal sampling time to discriminate between different adrenal gland conditions.

A major result of our modeling of adrenal gland secretion was to propose that cortisol secretion by the adrenals following ACTH stimulation is governed by both a dose-dependent k0 and a dose-dependent duration of adrenal gland secretion (RT).

These modeling results are consistent with the fact that the adrenal glands do not accumulate a pool of releasable cortisol (for which a first order rate constant would be expected to describe the response to an ACTH administration) but instead stimulate a preestablished enzymatic pool, i.e. only increase cortisol synthesis (14). The zero order release of cortisol by the adrenals is also in agreement with previous studies indicating that plasma cortisol concentrations attained a plateau when high ACTH doses were administered as a bolus (13, 36, 37).

On the basis of the observed k0 variations in response to the range of tested ACTH doses (Table 2Go and Fig. 4AGo), a constant value for k0 can be considered without loss of generality in the range of 0.1–10 µg/kg ACTH. This indicates that the ACTH-induced increase in cortisol secretion rate rapidly attained a maximum and that beyond this, the increased CP in response to exogenous ACTH is only due to the increase in RT (Table 2Go). Consequently, when blood samples are taken before the end of adrenal secretion, our model predicts that the measured cortisol concentrations will be the same whatever the administered ACTH dose within this range. In particular, the simulated plasma cortisol concentrations at 30 min after ACTH were identical for the 5 and 0.05 µg/kg ACTH doses, which are equivalent to the high and low doses used for ACTH stimulation tests in humans (Fig. 6Go). These findings are in full agreement with reports indicating that cortisol concentrations 30 (or 20) min after ACTH did not differ after standard low (0.5 µg/m2)- and high (250 µg)-dose ACTH tests in healthy humans (3, 4, 6, 13). In the same way, the ability of the low-dose ACTH test in humans to stimulate maximal adrenal secretion up to 30 min postinjection (13, 38) can be interpreted in the framework of our model as the capacity of a low dose of ACTH to induce maximal k0, a higher ACTH dose only being able to increase RT (and thereby the overall CP).

Moreover, it has been described in normal subjects that cortisol levels at 60 min were lower than those at 30 min after a low dose of ACTH, whereas the inverse was observed after the high-dose ACTH test (4, 6, 38, 39). These observations can be reproduced in our model by simply assuming an identical k0, and an RT of 30–60 min for the low ACTH dose and more than 60 min for the high ACTH dose (Fig. 6Go; 0.05 µg/kg for the low ACTH doses and 5 µg/kg for the high ACTH doses).

Another matter of debate concerning standard ACTH tests is their ability to discriminate between patients with a normal or insufficient HPA axis. Several reports have concluded that both low- and high-dose ACTH tests failed to effectively detect mild degrees of secondary adrenal insufficiency (6, 7, 8). The simulations performed here lead to similar conclusions if adrenal insufficiency corresponds to an unchanged k0 associated solely with a decrease in RT, so that the cortisol levels at 30 or 60 min are not modified (Fig. 7AGo). The framework permitting interpretation of some adrenal insufficiencies in terms of decreased RT is consistent with clinical reports of patients showing an insufficient overall cortisol response to sustained stress but a normal response to rapid ACTH tests (5, 7, 40).

The main factor able to control the duration of maximal secretion is the kinetic disposition of ACTH and the fact that RT increased log-linearly with the ACTH dose (Fig. 4Go) would be expected if the duration of the adrenal gland secretion is an on/off process relying on some critical threshold plasma ACTH concentration. One consequence of this is that the selection of sampling time is of particular importance for the screening performance of both low- and high-dose ACTH tests. The choice of the sampling times used in standard ACTH tests has been justified by the correlations between cortisol concentrations at 30 min after high ACTH doses and peak cortisol concentrations during insulin hypoglycemia (1, 41), but this continues to be debated (2, 6). Our simulations strongly suggest that information about the shape of the cortisol concentration vs. time profiles in response to ACTH, and particularly about the physiological value of RT, are essential to select appropriate sampling times for the detection of adrenal insufficiencies characterized by blunted capacities to maintain prolonged cortisol secretion. Moreover, direct investigation of the ACTH kinetic disposition could be helpful in discriminating between an altered ACTH disposition and a decrease in adrenal gland responsiveness, leading to an alteration of the threshold ACTH concentration at which cortisol secretion is initiated.

In conclusion, this study demonstrated the value of a pharmacokinetic/pharmacodynamic model describing the relation between iv ACTH and AR reflected by plasma cortisol concentration vs. time profiles. Although further experiments will be needed to investigate this modeling approach in humans, our simulations of the conditions encountered in evaluating the human HPA axis suggest that such an approach should increase the understanding of AR to ACTH tests and thus help to improve their clinical relevance.


   Footnotes
 
Present address for E.F.: Catedra de Farmacologia, Departamento de Salud Animal, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, Kreder 2805, Esperanza, Argentina.

A.B.M., E.F., N.H.P., L.D., V.L., and P.L.T. have nothing to declare.

First Published Online June 8, 2006

Abbreviations: AR, Adrenal response; AUC0-clast, area under the plasma concentration vs. time curve of total or CBG-free cortisol from time zero to the last concentration measured; Bmax, CBG maximal binding capacity; CBG, corticosteroid-binding globulin; ClCBG-free, plasma clearance of CBG-free cortisol; CBGfree, plasma CBG-free cortisol concentration; Cmax, plasma peak; CP, cortisol production; HPA, hypothalamic-pituitary-adrenal; RT, releasing time.

Received September 9, 2005.

Accepted for publication May 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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