The identification of corticosterone in human plasma and its assay by isotope dilution.

Corticosterone has been demonstrated in human adrenal perfusates (l), and in adrenal venous blood obtained by cannulation of the adrenal vein immediately prior to adrenalectomy (2). Several investigators (Morris and Williams (3), Bush and Sandberg (4), Weichselbaum and Margraf (5), and Sweat (1)) have reported the presence of corticosterone or “corticosterone-like” substances in the peripheral blood in man. Morris and Williams (3) and Weichselbaum and Margraf (5) used relatively non-selective methods of measurement. The former used a polarographic measurement, and the latter a col&imetric assay. Bush and Sandberg (4) carried out an extensive purification of the plasma extracts; however, because of the small quantities of corticosterone present, they were not able to make more than an approximate estimate of the quantity of corticosterone, or to give rigorous proof of the presence of corticosterone. Sweat (1, 6) used relatively small quantities of plasma, a column chromatographic purification, and a fluorescence assay (7) that afforded great sensitivity and considerable selectivity. Among these groups of investigators, widely variable concentrations of plasma corticosterone have been reported. These variable results indicate either a lack of specificity of the methods or inadequate recoveries of corticosterone. None of these reports contains sufficient data regarding proof of specificity of the methods for determining corticosterone in plasma extracts. In this paper a rigorous proof of both the presence and the identity of corticosterone in human peripheral plasma is presented. Also, a method has been developed for the quantitative estimation of corticosterone based on the principle of isotope dilution (8). The method offers a special advantage in that it is possible to carry out an extensive purification of the plasma extract without concern about minor losses of steroid. Because of this purification and, in addition, the use of a fluorometric method of assay on a relatively pure product, a considerable degree of specificity and sensitivity is obtained.