production of Steroids byin VitroSuperfusion of Endocrine Tissue. I. Apparatus and a Suitable Analytical Method for Adrenal Steroid Output

Abstract

An apparatus has been developed suitable for superfusion of rat adrenal glands with continuous collection of the incubation medium. A rapid and simple procedure for the simultaneous estimation of 18-hydroxy B [18-hydroxycorticosterone], aldosterone, 18-hydroxy DOC [18-hydroxy-deoxycorticosterone] and corticosterone is described. The extracted steroids are oxidized with periodic acid and the resultant etiolactones formed from the 18-oxygenated steroids are separated from corticosterone etioacid by partitioning from aqueous alkali and chromatographed separately on Bush-type paper systems. Quantitation is by continuous automatic scanning of the developed soda fluorescence directly on paper using a modified Turner fluorometer connected to a Texas Instrument recorder. The accuracy and precision of the fluorometric assay was determined by repeated estimations of known amounts of standard aldosterone etiolactone over a range of 0.05-2.5 [mu]g. A linear response was obtained with a calculated regression line of y=(1.02[plus or minus]0.04)x-0.004 [plus or minus] 0.016. The slope was not significantly different from 1, indicating no systematic error in the method. The error of the estimate was 0.016, 0.025, 0.076, 0.132 ug over the range of 0.05-0.15, 0.1-0.25, 0.25-1.0 and 1.0-2.5 ug. respectively. The maximum sensitivity was therefore 0.05, with a 30% error. The precision of the method for the 4 steroids over a range 0-5 ug calculated from duplicate and triplicate analyses for samples of rat adrenal gland incubations gave a mean coefficient of variation of 10, 14, 12 and 14% for 18-hydroxy B, aldosterone, 18-hydroxy DOC and B, respectively. The mean recovery for all 4 steroids was of the order of 70%. The specificity of the method has been shown to be satisfactory for the measurement of the 4 steroids from incubations of rat adrenals of intact and hypophy-sectomized rats, but saponification of the etiolactones, removal of the neutral steroids and relactonization may be necessary when inert or radioactive precursors are used. The accuracy of the method is similar with this extra procedure.