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).