A simple, accurate, and sensitive assay method of dehydroepiandrosterone sulfate: application for quantitative...

Radioimmunoassay (RIA) is the most prevalent method for measuring small amounts of hormones, peptides, and other compounds in human body fluids. The method, however, has several problems, such as cross reactions or non-specific reactions of the antibody used. In order to establish an improved method for assaying dehydroepiandrosterone sulfate (DHEAS) and cholesterol, which are the largest components of human breast cyst and duct fluids, we describe a simple, accurate, and sensitive method using high-performance liquid chromatography (HPLC). The samples were treated with cholesterol oxidase for quantitation of dehydroepiandrosterone (DHEA) and free cholesterol, and the respective oxidized substances, 4-androstene-3,17-dione and 4-cholesten-3-one, were extracted with n-hexane. The extracts were analyzed by straight phase HPLC. Effluents were monitored by measuring absorption at 240 nm, where a newly introduced chromophoric group, an alpha,beta-unsaturated ketone, showed intense absorption (epsilon = 16,000). When the total amount of DHEA (DHEAS plus DHEA) was measured, the sample had been solvolyzed by sulfatase beforehand. The amounts of DHEAS were quantified by comparing the amounts of DHEA before and after solvolysis. Levels of free cholesterol, DHEAS, and DHEA in human breast cyst fluids (n = 30) were 1.77 +/- 1.12 mmol/dl, 8.27 +/- 10.24 micromol/dl, and 0.02 +/- 0.02 micromol/dl (means +/- SD), respectively. The levels of sterol and steroid measured in breast duct fluids that were turbid, brown, dark green, or milky in color (n = 9) (mean levels, 3.20 +/- 2.97 mmol/dl for free cholesterol and 14.77 +/- 13.75 micromol/dl for DHEAS) were significantly (P < 0.01) higher than the levels in clear or serous breast fluids (n = 21) (mean levels, 0.14 +/- 0.13 mmol/dl for free cholesterol and 0.04 +/- 0.07 micromol/dl for DHEAS).