Chromatographic Characterization of Adrenocorticotrophin in Human Plasma

Abstract

A chromatographic procedure has been developed for the characterization of ACTH- and lipotrophin- (LPH) related peptides in human plasma under acid-dissociating conditions to minimize artifacts of protein binding. The recovery and sensitivity of this method permits identification of ACTH at normal physiological levels in the circulation. Plasma profiles obtained from normal subjects and patients with pituitary dependent Cushing's disease, Addison's disease and Nelson's syndsrome showed only one significant peak of ACTH activity eluting in the position of purified native human 1--39 ACTH. However, the plasma profiles obtained from all the patients with the ectopic ACTH syndrome demonstrated a second peak of immunoreactive larger-molecular-weight ACTH and in some plasma samples this was the only form of ACTH observed. This larger-molecular-weight ACTH eluted midway between the void volume and 1--39 ACTH and co-eluted with a protein marker of molecular weight 22 000.

Gel chromatographic characterization of immunoreactive adrenocorticotropin in patients with ACTH hypersecretion

“…In patients with Addison's disease, Nelson's syndrome and Cushing's disease we found only a single peak of immunoreactive ACTH eluting at the same position as labeled 1-39 ACTH. These results are in accordance to the investigation of Ratter et al [11]. In contrast, Thoren et al [14] observed a high molecular weight form of ACTH in normal subjects and patients with Nelson's syndrome by gel filtration which eluted in the void volume.…”

“…Incomplete or aberrant processing of POMC with secretion of'big' ACTH appeared to be a characteristic feature of the ectopic Cushing's syndrome. Ratter et al [11] demonstrated at least one other peak of ACTH immunoreactivity suggestive of a high molecular weight form of ACTH in 20 patients with the ectopic ACTH syndrome.…”

“…In normal subjects the only circulating form of ACTH immunoreactivity is 1-39 ACTH [11]. In patients with disorders of the hypothalamic-pituitary-adrenal axis abnormalities of the processing of POMC have been observed resulting in the secretion of various large molecular weight forms of ACTH [5,6,7,11,12,15,16]. In patients with Addison's disease, Nelson's syndrome and Cushing's disease we found only a single peak of immunoreactive ACTH eluting at the same position as labeled 1-39 ACTH.…”

“…Analysis of the molecular size of circulating immunoreactive ACTH was performed by gel chromatography, as described by Ratter et al [11] in detail. Briefly, a column of Sephadex G-75 (superfine; 100 x 1.5 cm) equilibrated with 1 % formic acid was used.…”

“…Abnormalities in the processing ofPOMC have been reported in the ectopic ACTH syndrome resulting in the secretion of various high molecular weight forms of ACTH [11,15]. Since differentiation of patients with ectopic Cushing's syndrome from pituitary dependent Cushing's syndrome (Cushing's disease) may be difficult in some cases it has been suggested that variations in the secretion pattern of ACTH may have diagnostic significance [5].…”