The efficacy of the standard high dose
ACTH stimulation test (HDT), using a pharmacological 250-microg dose of synthetic ACTH-(1-24), in the diagnosis of central
hypoadrenalism is controversial. The
insulin hypoglycemia test is widely regarded as the gold standard dynamic stimulation test of the hypothalamo-pituitary-adrenal (HPA) axis that provides the most reliable assessment of HPA axis integrity and reserve. Alternatively, a prolonged infusion of
ACTH causes a continuing rise in plasma
cortisol levels that may predict the adrenals' capacity to respond to severe ongoing stress. In nine normal subjects, we compared plasma
ACTH and
cortisol levels produced by three i.v. bolus low doses of ACTH-(1-24) (0.1, 0.5, and 1.0 microg/1.73 m2; LDTs) with those stimulated by
hypoglycemia (0.15 U/kg
insulin) and with the
cortisol response to a standard 250-microg dose of ACTH-(1-24). The normal
cortisol response to an 8-h ACTH-(1-24) infusion (250 microg at a constant rate over 8 h) was determined using three modern
cortisol assays: a high pressure liquid chromatography method (HPLC), a fluorescence polarization immunoassay (FPIA), and a standard RIA. In the LDTs, stepwise increases in mean peak plasma
ACTH were observed (12.4 +/- 2.0, 48.2 +/- 7.2, 120.2 +/- 15.5 pmol/L for the 0.1-, 0.5-, and 1.0-microg LDTs, respectively; P values all <0.0022 when comparing peak values between tests). The peak plasma
ACTH level after
insulin-induced
hypoglycemia was significantly lower than that produced in the 1.0-microg LDT (69.6 +/- 9.3 vs. 120.2 +/- 15.5 pmol/L; P < 0.0002), but was higher than that obtained during the 0.5-microg LDT (69.6 +/- 9.3 vs. 48.2 +/- 7.2 pmol/L; P < 0.02). In the LDTs, statistically different, dose-dependent increases in peak
cortisol concentration occurred (355 +/- 16, 432 +/- 13, and 482 +/- 23 nmol/L; greatest P value is 0.0283 for comparisons between all tests). The peak
cortisol levels achieved during the LDTs were very different from those during the HDT (mean peak
cortisol, 580 +/- 27 nmol/L; all P values <0.00009. However, the mean 30 min response in the 1.0-microg LDT did not differ from that in the HDT (471 +/- 22 vs. 492 +/- 22 nmol/L; P = 0.2). In the 8-h
ACTH infusion test, plasma
cortisol concentrations progressively increased, reaching peak levels much higher than those in the HDT [995 +/- 50 vs. 580 +/- 27 nmol/L (HPLC) and 1326 +/- 100 vs 759 +/- 31 nmol/L (FPIA)]. Significant differences in the basal, 1 h, and peak
cortisol levels as determined by the three different assay methods (HPLC, FPIA, and RIA) were observed in the 8-h infusion tests. Similarly, in the HDTs there were significant differences in the mean 30 and 60 min
cortisol levels as measured by HPLC compared with those determined by FPIA. We conclude that up to 30 min postinjection, 1.0 microg/1.73 m2
ACTH-(1-24) stimulates maximal adrenocortical secretion. Similar lower normal limits at 30 min may be applied in the 1.0-microg LDT and the HDT, but not when lower doses of
ACTH-(1-24) are administered. The peak plasma
ACTH level produced in the 1.0-microg LDT is higher than in the
insulin hypoglycemia test, but is of the same order of magnitude. The peak
cortisol concentration obtained during an 8-h
synthetic ACTH-(1-24) infusion is considerably higher than that stimulated by a standard bolus 250-microg dose, potentially providing a means of evaluating the adrenocortical capacity to maintain maximal
cortisol secretion. Appropriate interpretation of any of these tests of HPA axis function relies on the accurate determination of normal response ranges, which may vary significantly depending on the
cortisol assay used.