A Simplified Physico-chemical Method for Determination of Human Urinary ÂŒstrogens

Journal/Book: Reprinted from Nature Vol. 183 p. 1607 only June 6 1959. 1959;

Abstract: Donner Laboratory of Biophysics and Medical Physics University of California Berkeley. Feb. 23. * Donner Postdoctorate Fellow in Biophysics. Present address ; Worcester Foundation for Experimental Biology Shrewsbury Mass. THE methods for estimating urinary Âœstrogens are open to criticism and are not easily applied as a routine in the laboratory because of their complexity. Our chief aim in the present investigation is to develop a reliable practical and simplified method for studying the excretion of urinary Âœstrogens by patients with cancer of the breast. 100 ml. urine of 24-hr. sample was adjusted to pH 5 with concentrated sulphuric acid. 10 per cent by volume of acetate buffer was added followed by 300 Fishman units of -glucuronidase/ml. urine. This was incubated at 37° C. for five days1. After completion of hydrolysis the urine was extracted four times with 100 ml. peroxide-free ethyl ether. The ether extract was washed with (a) saturated sodium carbonate solution pH 10 5 (b) 2 N sodium hydroxide solution which was neutralized to pH 10 with 8 per cent sodium bicarbonate solution and shaken again with the ether extracts (c) 8 per cent solution of sodium bicarbonate (d) water2. The ether extract was evaporated to dryness and the residue dissolved in about 80 ml. toluene ; this was extracted four times with 025 volume N sodium hydroxide and washed twice with 0 05 volume water. Neutral steroids remain in toluene layer. Alkaline extracts and washings were acidified to pH 9 with sulphuric acid (6 N) and extracted four times with 0 25 volume ether. This was evaporated and the residue was transferred to small storage bottles with methyl alcohol and evaporated to dryness at 60° C. Counter-current distribution in 50 per cent methanol 50 per cent water (upper layer)/carbon tetrachloride (lower layer)3 was then carried out for 24 transfers. ÂŒstrogen extract was dissolved in 0 2 ml. ethanol and heated in a water-bath with 1 ml. 90 per cent sulphuric acid at 80° C. for 10 min. This was diluted with 4 ml. 65 per cent sulphuric acid and cooled in crushed ice for 30 min. Fluorescence was measured in a Farrand photofluorometer modal A using Corning glass No. 3389 and No. 5113 as the lamp filter (436 mµ) with Nos. 3387 and 4308 and a 483 mµ interference filter as the photocell filter4-6. ... ___MH

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