Translation Series No. 958

64/2 1964a). Within several days after hypophysectomy, this gland atrophies and the proportion of plasmatic cortisol diminishes: this proportion drops from 4.6 / g per 100 ml in the intact animal po less than 1 .)1g 7 days after hypophysectomy, however, a single injection of mammalian ACTH in the hypophysectomized animal, kept at 16° C., will bring about a return to normal glandular weight within an hour, and a considerable increase in the proportion of cortisol in circulation, to an amount of 20 to 30}ig per 100 ml of plasma. These rapid variations in the corticosteroid level thus indicate, in the interrenal of this Teleost, a considerable power of synthesis, leading to almost-immediate release of corticosteroids into the plasma. This considerable power of synthesis, also found in the experiments in vitro, makes interrenal of eel an excellent material for the study of corticosteroid production in Teleosts. MATERIAL AND TECHNIQUES From silver eels, weighing 220 to 700 g. and originating either in Moret (Seine et Marne), or the ponds of the Somme, the anterior cardinal veins were removed from the area indicated above. In the various experiments, from 60 to 200 mg of these veinous portions (mainly constituted of interrenals), taken from 3-10 kg of eels, were incubated in 5 ml of KrebsRinger bicarbonate medium containing 200 mg of glucose per 100 ml, 3 hours at 30°C, under 5% seltzogene powder in the presence of progesterone 4-C14 as a precursor (0.5-1 /11c of progesterone 4-014 provided by New England Nuclear Corporation, with specific activity: 145.821c per mg). In order to make the purification operations quantitative, we added to the incubation media, after elimination of the tissue used in the experiment,exactly 0.2 jpc of the following tritium-labelled corticosteroids: cortisol, aldosterone, and corticosterone (provided by the National Institute of Health, Bethesda l Maryland). They were extracted three times by a volume of an ether: chloroform mixture (3:1). The extracts Chicago, S s 725). Each sample is evaporated dry and dissolved ■ 65 obtained, dissolved in 70% methanol, were then delipiced in petrolic ether, after which the methanolic solution was fractioned by paper chromatography (Arches Paper no. 302 exhausted in ethanol and then in chloroform) by utilizing several systems of solvents in succession, as outlined below: Bush System B5 3. Polar zone Les olar zone (Corticosterone, progesterone) Bush System A. Corticosterone \.) ■1/ Progesterone Bush System B3 V Corticosterone (Cortisol, cortisone, aldosterone) Chloroform: Formamide L7 %, Cortisol Cortisone, aldosterone 11 / Bush System C Bue System C :1/ i,/ \ Cortisol Aldostârone ...,,‘ Cortisone After each chromatography, the distribution of carbon 14 (C14 ) on the chromatogram was determined by means of a narrowaperture Geiger-MUller counter (TGC 2 Tracerlab) connected to an integrator and an automatic recorder. Comparison of the recordings obtained and the reference corticosteroid chromatograms, produced simultaneously by tetrazolium blue, allows rapid identification of certain corticosteroids marked with C14 . The parts of the chromatograms corresponding with peaks of radioactivity 14. (C ) and those corresponding with the location of tritiumlabelled corticosteroids which cannot be located with the assistance of C14 (e.g. aldosterone) were removed and eluted by means of methanol. The methanolic elutates are then chromatographed in the following system. In the final eluates, the C 14 and the tritium (H3 ) are measured bÿ liquid scintillation (liquid scintillator, Nucaa in pure toluene (Nuclear Enterprises, Scintillation Grade) containing o.4% 2-5 diphenyloxazole and 0.01% POPOP. The radioactivity ratings of the eluates are calculated using the discrimination ratio method proposed by Okita et al. (1957) and modified by Stachenko et al. (1964). To deternine the quenching and correct it, if necessary, we used the Peng method (1964.) 65/2 The product of purification operations was assessed from the recuperated H3 which had been added to the incubation media. Taking the products into account, the percentage of C14 progesterone 4_ 1 by the interrenal during incubation was determined for each radioactive substance. The substances isolated were characterized by their R, in various solvent systems. They iere identified using these Rf but also, firstly, by their behavioupafter acetylation in 66 chromatography in B.Ash System B3 and, secondly, by their migration after chromic oxidation in chromatography on a thin layer of silica gel in the chloroform: ethanol system (90:10) (Duvivier, 1965). In the case of cortisol, aldosterone and corticosterone, the /„ stability of the H3/C14 ratio after acetylation and chromic oxidation served as the criterion of purity. Lastly, the specific activity of certain corticosteroids was calculated when a characteristic reaction revealed their dosage in the purified elilate (Porter & Silber reaction (1950) (J-Lj for cortisol and cortisone; sulph ric fluorescence (Sweat, 1954) fôr corticosterone). This was expressed bw_jac per mgAstable corticosteroid. RESULTS In a preliminary report (Leloup-Hâtey, 1965), we reviewed very briefly the results obtained by this incubation technique; therre sumarized in Table 1. From the incubation media of eel interrenal with progesterone 4-C14 , we isolated 8 radioactive fractions: Fraction 1(3-7%), very polar, remained in the origin. Fraction II (17.1-41.8%) was identical to cortisol: same t chromatographic behaviour, absorption at 240 m , positive Porter & Silber reaction, reduction\91 tetrazolium b_i ue. The H3 /C14 radioactivity ratio was 0.80 for the eluate, 0.84 after acetylation and 0.82 after chromic oxidation of the acetate. 66/2 Fraction III (4-5.5%) had the same Rf as cortisone in Bush Systems B5 and C was distinguished by a lower R, in the chloroform: formamide system (if the R. i2s calculated as a function of the mobility of the cortisol (1.00), Fraction III has an Rf of 1.44 and cortisone, 2.11). After acetylation, this compound migrated like the acetate of cortisone in Bush System B3). Fraction IV . (0.6 1.4%) migrated like tritium-labelled aldosterone. We shall discuss the significance of this phenomenon later. Fraction V. (4.3 9.3%) was identical to cortisone: same chromatographic behaviour, absorption at 240 ry, positive Porter & Silber reaction. Its acetate did not separate from that of pure cortisone and, after chromic oxidation, the products formed by acetates of Fraction V and cortisone were not seperated. Fraction VI (1.5 5.7%) was identical to corticosterone: same chromatographic behaviour, absorption at 240 74 sulphuric fluorescence. The 0/C 14 radioactivity ratio was 6.81 for the eluate, 7.25 for its acetate and 7.38 after chromic oxidation. Fraction VII (0.7 4.8%) was slightly less polar than corticosterone in Bush Systems A-and B3. The carbon 21 of the progesterone