| Summary: | Thesis (Ph.D.)--Boston University === Introduction: On the basis of various in vitro studies, progesterone appeared to be an intermediate in the biosynthesis of some of the adrenal cortical hormones. In particular, perfusion of the isolated cow adrenal with blood media containing added progesterone had led to the isolation of 11β-hydroxyprogesterone, 17α-hydroxyprogesterone, corticosterone, 17α-hydroxycorticosterone, allopregnane-3,20-dione, allopregnane-17α-ol-3,20-dione, and allopregnane-3β,17α-diol-20-one. Since these studies demonstrated that progesterone did in fact lead to adrenal cortical hormones, it appeared possible that other hormones of known or unknown constitution might be present in the above perfusates. Therefore, in the present research, adrenal perfusions with progesterone were performed and the resultant mixtures investigated.
Thus, the purpose of this research was to perfuse bovine adrenals with blood containing added progesterone in order to obtain substances of adrenocortical or other biological activity, metabolites of these hormones or their intermediates, and in fact, any and all substances derived from progesterone.
To achieve the aims of the research, four basic stages were involved. These were: 1. perfusion, 2. isolation of the steroids from the perfusates, 3. fractionation of the resultant mixtures into pure crystalline components, and 4. identification of these components.
Perfusion: The first series of perfusions was performed at G. D. Searle and Co., Chicago. In total 600 cow adrenals were perfused with 600 liters of bovine blood - Tyrode's solution (1:1) containing 90 g. of added progesterone. The second series was performed by C. A. Fish at the Worcester Foundation. In this, 73 cow adrenals were perfused with 73 liters of citrated whole blood containing 5.1 g. of progesterone-4-C14 (1.47 millicuries/mmole).
Isolation: The steroids were isolated from the first perfusate by adsorbing them onto charcoal and subsequently eluting them with acetone. Control studies performed before this research had indicated the method to be sufficiently efficient to be used. During this work however, some disadvantages of the charcoal procedure such as destructive nature toward certain steroids were reported by other investigators. Further, several other procedures for isolating corticosteroids from blood were described. One of these involved the shaking of the blood with solvents such as isopropyl acetate. Since this latter method appeared to have merit, the present research included a quantitative comparison of it with the charcoal procedure. This aspect of the work consisted of perfusing bovine adrenals with progesterone-4-C14 and subjecting aliquots of the perfusate to both procedures. The respective extracts were then compared (among other factors) for total recovery of radioactivity, amount of formaldehydogenic substance (on oxidation with periodic acid), and number of zones detected by color tests on paper chromatography of the residues. In these experiments the charcoal was pretreated in various ways such as digestion with potassium cyanide and with Versene (a chelating agent) in an attempt to reduce destructive surfaces. Furthermore, an attempt was made to remove the steroids from the charcoal via countercurrent elution at room temperature in a column in order to minimize chance for destruction due to heat. The room temperature elution proved to be successful in that the bulk of the radioactivity was removed in this step. The recovery of radioactivity was the same for both methods but the solvent procedure did however appear to have an advantage in that the formaldehydogenic values obtained were somewhat higher than those with the charcoal procedure.
Finally, an attempt was made to compare the crystalline recovery of cortisone from water, using the treated charcoal and the isopropyl acetate procedures. This experiment was performed since it had been reported that using untreated charcoal to adsorb cortisone and subsequent, continuous extraction by hot solvents in a soxhlet apparatus, to elute the cortisone, a crystalline recovery of 25-44% was obtained. The present comparative experiment resulted in crystalline recoveries of 33 and 92% respectively using the above modified charcoal and solvent procedures. This final experiment on the crystalline recovery of cortisone clearly demonstrated the superiority of the solvent extraction procedure to the charcoal procedure. The former was therefore used in the subsequent progesterone-4-C14 perfusions.
Fractionation of the extracts: The methods used to fractionate the mixtures extracted from the perfusates included the following: adsorption chromatography on silica gel, partition chromatography on paper and a diatomaceous earth (Celite), and fractional crystallization. Components were generally considered sufficiently pure for structure determination when crystallized to constant melting point.
Identification of components: Since components were frequently isolated in quantities of the order of 1 mg., the identifications had to be made on a micro scale. The classical methods of organic chemistry such as degradations and microanalyses for the elements had to be used very judiciously. In many cases there was insufficient material for a microanalysis.
To overcome this difficulty and learn the nature of functional groups present, spectrophotometry was used either directly or in conjunction with various color tests and microreactions. Another property found to be very useful was rates of movement of substances on paper or column chromatography. A study of relative running rates frequently suggested the number of oxygen atoms present on the molecule. Thus, spectrophotometry (infrared, ultraviolet, and visible), and chromatographic mobility data, combined with analogy, tacit assumptions that components were steroidal, and intuition were most valuable tools used throughout this research.
Although supporting data were used, the infrared spectrum of either the isolated sample or a degradation product, obtained through an unambiguous reaction sequence, was compared with that of an authentic sample in order to establish identity.
Steroids identified from these perfusates:
1. progesterone
2. Δ4-pregnene-2β-ol-3-one
3. allopregnane-3,20-dione*
4. allopregnane-3β-ol-20-one
5. allopregnane-17α-ol-3,20-dione*
6. allopregnane-3β,17α-diol-20-one
7. 17α-hydroxyprogesterone*
8. 11β-hydroxyprogesterone*
9. Δ4-androstene-11β-ol-3,17-dione**
10. 6β-hydroxyprogesterone
ll. 17α-hydroxy-11-desoxycorticosterone
12. corticosterone*
13. 19-hydroxy-11-desoxycorticosterone
14. 17α-hydroxycorticosterone*
15. allopregnane-3α,17α,11β,21-tetrol-20-one
16. Δ4-pregnene-6β,17α,21-triol-3,20-dione
17. allopregnane-3β,17α,11β,21-tetrol-20-one
18. Δ4-pregnene-17α,19,21-triol-3,20-dione
*From progesterone and progesterone-4-C14
**From progesterone-4-C14
Test of purity experiments: Experiments were also performed on the purity of some of the steroids isolated from the progesterone-4-C14 perfusates. In essence the methods involved chromatographing an aliquot of the sample on paper and determining the radioactivity in the region corresponding to the desired component relative to the total radioactivity present. These experiments indicated that samples otherwise thought to be pure could contain considerable amounts of impurity which were not detected by other methods used. Furthermore, it was found that some destruction of steroid appeared to be occuring during paper chromatography; probably during the air drying in daylight. Evidence for this is that in several rechromatographies of the zone corresponding to 17α-hydroxycorticosterone, the purity was found to be considerably lower than in the first.
Discussion: The isolation of substances 10 and 16 represents the first demonstration of hydroxylation occuring at carbon 6 by adrenal perfusion. 6β-Hydroxylation has, however, been reported earlier following incubation of 11-desoxycorticosterone with hog adrenal brei. The isolation of substances 13 and 18 represents the first demonstration of hydroxylation at carbon 19 by adrenal perfusion and is the first demonstration of the formation of 21 carbon steroids hydroxylated at carbon l9 by any in vivo or in vitro system. Simultaneous with the identification of substance 13 in these perfusates, 19-hydroxy-Δ4-androstene-3,17-dione was identified as a component formed, following incubation of Δ4-androstene-3,17-dione with adrenal tissue. Oxidation of substance 18 with sodium bismuthate resulted in a mixture from which was crystallized and identified, 19-hydroxy-Δ4-androstene-3,17-dione. The structure of substance 18 (isolated in quantity of about 10 mg.) appears therefore to be established as Δ4-pregnene-17α,19,2l-triol-3,20-dione, a hitherto unknown substance. Although the latter compound originally appeared to be biologically active in sodium ion retention assays, subsequent assays have failed to reproduce the activity. It now appears that a trace impurity of extremely high activity was present in the sample originally tested. Although the two samples tested were purified differently, the melting point ranges were identical. The possibility of an impurity's causing the initial activity is now being investigated by assaying appropriate mother liquors.
The isolation of substance 9 is among the first direct indications of a C-21 steroid transformed into a C-19 steroid. It was reported earlier that trace amounts of Δ4-androstene-3,11,17-trione were isolated from adrenal perfusions of cortisone.
The isolation of substance 11 has added substantiation to the postulated scheme of corticosteroid biosynthesis in which it had been proposed as an intermediate but never isolated.
The isolation of substances 1,3,5,6,7,8,12 and 14 had been reported before this research and at present represents only confirmatory findings. The remaining substances identified are all reduction products in ring A or at C-20 of progesterone or progesterone derivatives.
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