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Gregory Pincus, A Diurnal Rhythm in the Excretion of Urinary Ketosteroids by Young Men, The Journal of Clinical Endocrinology & Metabolism, Volume 3, Issue 4, 1 April 1943, Pages 195–199, https://doi.org/10.1210/jcem-3-4-195
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Abstract
THE PRESENCE of a diurnal cycle in the urinary output of 17-ketosteroids might have some consequence in consideration of their functional significance. The marked quantitative changes in excretion accompanying various pathologic states (1, 2) may indicate that the processes involving ketosteroid secretion may be labile to ordinary daily stresses.
Seven young men ranging in age from 22 to 29 years undertook the collection of consecutive night and day urine specimens for periods ranging from 5 to 9 days. In most cases the attempt was made to collect 4 specimens for each 24-hour period, but this was not always possible.
Toluol was added as preservative and the urines hydrolyzed and extracted within 48 hours of collection. Our usual method of acid hydrolysis and ether extraction was practiced (3). The dried neutral fractions of the ether extract were separated into ketonic and nonketonic fractions by the micro-Girard reaction (3).