Factors influencing the adrenocorticotropin test:: Role of contemporary cortisol assays, body composition, and oral contraceptive agents

Context: The normal cortisol response to an ACTH test remains inconsistently defined, possibly caused by various subject- and test-condition-related factors. Objective: Our objective was to evaluate the impact of newer automated immunoassays; gender, age, body composition, and endogenous sex-hormone levels; corticosteroid-binding globulin levels; and test conditions (fasting/nonfasting, rest/intermittent exercise). Methods: A 250-mu g ACTH test (0800-1000 h) was performed in 100 unmedicated subjects, 13 women taking oral contraception (OC), and six men with nephrotic syndrome. Tests were performed fasting supine (n = 119), nonfasting supine (n = 38), and fasting with intermittent exercise (n = 45). Serum cortisol was analyzed by three immunoassays. Results: Even with a negligible between-assay mean bias, individual samples from unmedicated subjects differed by as much as 110 nmol/liter. The normative 2.5th percentile for total cortisol ranged from 475-523 nmol/liter when analyzed by the three assays. In multivariate analyses, 30-min total cortisol was predicted by baseline cortisol (men plus women) and central adiposity (men) but not by gender, age, and endogenous sex hormones, corticosteroid-binding globulin, fasting/nonfasting, and exercise. Compared with unmedicated subjects, OC women had 2-fold elevated 30-min cortisol (P < 0.001) but lowered calculated free cortisol (P < 0.001), whereas nephrotic syndrome patients had lowered 30-min cortisol (P < 0.01) in two of three assays, but similar calculated free cortisol (P < 0.1). Conclusion: The normal response to an ACTH test is assay specific, even with newer methods, and this also applies to calculated free cortisol. Both total cortisol and calculated free cortisol were severely affected by OC, and the test is therefore only reliable if OC has been discontinued. The ACTH test is, however, robust for most of the other evaluated subject- and test-condition-related factors.