PRODUCTION OF CORTISOL PROM CORTISONE By THE ISOLATED, PERPUSRD PEPAL RABBITLUNG J. S. Torday, E. B. Olson, Jr. and N. L. First The Endocrinology-ReproductivePhysiology Program and The Department of Preventive Medicine University of Wisconsin Madison, Wisconsin 53706 Received:

3/29/76 ABSTRACT

Perfusion of the isolated 26 day fetal rabbit lung with 3R-cortizone resulted in its converzlon to 3R-cortisol and rele e into the occurred. perfuzate. Little conversion of 14C-cortisol to l&ortisone Quantitative study of homogenized fetal rabbit lung revealed the development of both the cofactor and the enzyme for 118-hydroxy steroid dehydrogenase activity between 21 and 29 days gestation. These results suggest increasing production of cortizol from cortisone by the fetal rabbit lung as a function of gestational age. This conversion may be of significance with respect to both lung development and parturition, both events being accelerated by cortisol treatment. INTRODUCTION Exogenous glucocorticoidshave been shown to have a marked effect on fetal development in several species. They accelerate not only the rate of organ growth and maturation (l-4), but the time of parturition itself (5). Circulating glucocorticoids exist in either an 11-hydroxylated or 11-ketonic form, Lhe latter having no biological activity (6). In the adult these two forms are freely Interconvertedby peripheral tissues (7) and the ll-hydroxylated form predominates (8). In the fetus the 11-keto form prevails (9-14) and it has been shown that 11-reductase activity is low or absent in the fetal compartment (15). However, it

has recently been reported that both human

(16) and rabbit (17) fetal lungs in culture do, indeed, convert cortisone to cortisol. Since such reactivation of glucocorticoid by

VoZwne 27, Nwnber 6







fetal tissue is of potential physiological significance because of its implication in organogenesis and parturition, confirmation of this observation was sought using the isolated, perfused fetal rabbit lung.

Furthermore, in order to quantitatively assess llg-hydroxy

steroid dehydrogenase activity as a function of gestational age, this enzyme was assayed using a homogenized fetal rabbit lung preparation. MATERIALS AND MRTRODS Experiment 1 Perfusion. A total of 12 pairs of lungs from the fetuses of three pregnant white New Zealand rabbits at 26 days gestation were perfused. Four airs of lungs were assigned to each of the following treatments: 1) 5H-cortisone was passed through the perfusion apparatus without occluding the ductus arteriosus, in effect bypassing the lungs and only reflecting non-pulmonary conversion of cortisone to cortisol; 2) 3R-cortisone was perfused through the fetal lungs by ligating the ductus arteriosus to assess the pulmonary conversion of cortisone to cortisol; 3) 3H-cortisone and 14C-cortisol were simultaneously passed through the fetal lungs with the ductus arteriosus occluded in order to determine the interconversion of cortisone and cortisol. The does were anaesthetized with sodium pentobarbital (intravenous, 30 mgfkg), tracheostomized and artifically respirated with 5% CO2 95% 02. Four fetuses were removed from the uterus by laparotomy, and their thoraces were opened. The ductus arteriosus was exposed and ligated unless indicated otherwise. Then the pulmonary artery was cannulated via the right ventricle. The aorta was severed at the level of the diaphragm to allow free drainage of perfusate from the lungs. At this point the lower extremities were severed and the four sets of lungs were perfused simultaneously in a closed chamber maintained at 37'C, saturated with water vapor. The perfusion solution consisted of: Krebs-Ringer bicarbonate buffer, 2% bovine serum albumin, 3% Dextran (70,000 molecular weight) and 0.1% glucose. The perfusion medium was equilibrated with 5% CO2 - 95% 02 to maintain a pH of 7.4. The flow rate was kept at 0.67 ml/minute[lung. Changes in lung fluid equilibrfum were monitored with a torsfon balance to detect edema. The lnngs were perfused for an initial equilibration period of lo-20 minutes without added steroids, following which radiolabeled steroid was injected to each of the four individual lung preparations in equal quantities through injection ports. To determine the metabolic fate of cortisone after passage through the lungs, aliquots of the perfusate were retrieved from a tray above the main collecting reservoir, the remainder being readministered equally to the four Lung preparations. Following these samplings of the perfusate, the perfusion was allowed to continue for a maximum of one hour, at the end of which time the individual lung preparations were perfused with

methylene blue to determine the status of the vasculature. In the instance where the ductus arteriosus was not occluded, tne dye was not taken up, In all other cases, the pulmonary vaaculature filled with dye. At this point the lungs were removed and all samples were stored at -20°C until further analysis. In experiment 1, treatments 1 and 2, all procedural losses were corrected for by the addition of 14C-eortisol prior to processing (18). Perfusates were extracted with 5 volumes of methylene chloride and the organic phase was evaporated under nitrogen; lung tissue was homogevized in a Potter-Elvehjem tissue grinder in 10 ml distilled water. The homogenized tissue was precipitated with acetone:methanol::3:1,the supernatant was filtered through Nhatman 82 paper and the deproteinized extract was evaporated under a stream of nitrogen. The dried extracts were redissolved in 10 ml of distilled water and processed by the same method as were the perfusates. Aliquots of total organic extracts were taken for counting. Identification and Quantification of Metabolites. The methylene chloride extracts were separated by paper chromatography in the system benzene:methanol:water::l0:5:5(19) in parallel with standards of cortisol and cortisone. The zones corresponding to reference cortisol and cortisone were eluted with ethanol, evaporated under nitrogen and redissolved in a known amount of ethanol. Appropriate aliquots were counted in a Chicago Nuclear liquid scintillation spectrometer (machine efficiency 44.3% and 66.6% for tritium and carbon-14, respectively) in a toluene-Gmnifluor (New England Nuclear, 3oston, Mass.) mixture. Steroids. Cortisone-1,2-3H (48.4 Ci/mmol), cortisone-4-14C (59.8 mCi/mmol) and cortisol-4-14C (58.0 mCi/mmol) were purchased from New England Nuclear Corporation, Boston, Mass., and were at least 98% pure on paper chromatography. Unlabeled steroids were purchased from Steraloids, Inc., Pawling, New York. In treatments 1 and 2 only tracer 3H-cortisone was used; in treatment 3 equimolar amounts of 3H-cortisone and 14C-cortisol were perfused simultaneously; in order to render their specific activities equivalent, labeled cortisone was diluted with an appropriate amount of non-radioactive cortisone. Double Isotope Derivative Assay. The identity of the cortisol and cortisone fractions was further established by acetylation and chromatography of the acetates and of their respective chromium trioxide oxidation products, as described by Drayer and Giroud (20). Hadiochemical purity was established when at least two sequential isotope ratios proved to be constant within an experimental error of less than 10% (18). Experiment 2 Homogenate Preparation and Incubation. For this study 4 of a total of 16 pregnant does of known gestation were sacrificed on each of the following days of pregnancy - 21, 23, 25, 29. The fetuses from any given doe were removed from the uterus and the fetal rabbit lungs were excised and rinsed with chilled 0.9% saline. The tissue was blotted on filter paper, weighed, and a 10% (w/v) homogenate in 0.25M sucrose was prepared in a Virtis homogenizer. Each incubation flask consisted of 0.2 ml of the homogenate (20 mg wet weight equivalent), 0.3 ml NADPH-generating system (where indicated) and made up to 2.0 ml with




0.1 M phosphate buffer, pH 7.4, and labelled steroid substrate which had previously been pipetted in ethanol and dried under nitrogen. The mixture was incubated in a Dubnoff metabolic shaker at 37°C under air for 2 hrs. At the end of the incubation the flasks were frozen and stored at -2O'C until analyzed. Statistical Analysis of Experiment 2. Following transformation to loglO(x+l) the data from days 23, 26 and 29 were compared by analysis of variance for a factorial design (2x3~4) with rabbits nested within day. Sums of squares for single degree of freedom comparisons were calculated by the method of orthogonal polynomials (21). RESULTS Experiment 1 Treatments 1 and 2 - Perfusion with 3H-Cortisone. The results of perfusion of the 26 day fetal rabbit lung with cortisone without occlusion of the ductus arteriosus (treatment 1) are illustrated in figure 1A.

There was little conversion of cortisone to cortisol as

indicated by the percentage of cortisol in the perfusate and tissue. In contrast to the above, ligation of the ductus arteriosus resulted in significant conversion of cortisone to cortisol (figure 1B).


1 cycle (5 minutes) through the lungs 28% of the radioactivity in the perfusate was identifiable as cortisol. During this exposure the concentration of cortisone was 1 X lo-'M. A subsequent one hour perfusion through the same set of lungs at a diluted concentration of 1 x lo-loM resulted in an additional 22% conversion of cortisone to cortisol, yielding a total of 50% cortisol from cortisone in the pulmonary effluent. The intrapulmonary ratio of cortisol to cortisone was nine-fold higher than that in the perfusate, although this represented less than 10% of the total radioactivity. Nearly 80% of the initial radioactivity perfused was accounted for, 75% appearing as either cortisone or cortisol.




Figure 1. The distribution of cortisone and cortisol as reflected by the percentage of these steroids in the perfuaate and tissue sampled after various times during recirculation through the isolated, perfused Figure IA represents the metabolism of 3?l-cortisone fetal rabbit lun (1 x 10-g to lo-f' OM) in the absence of ductus arteriosus closure; figure 1B represents the metabolism of 3H-cortisone (1 x low9 to 1O-1oM) when the ductus arteriosus is occluded; figure 1C represents the simultsneous metabolism of 3H-cortisone and 14C-cortisol (each at 1.25 x 10S6 to 10%) expressed as the net conversion of cortisone to cortisol by subtracting the simultaneous conversion of cortisol to cortisone at any given time.




Treatment 3 - Perrusion of the Fetal Rabbit Lung with Equimolar Quantities of 3H-Cortisone and

14 C-Cortisol. This procedure (figure

1C) again reflected significant reduction of cortisone. The net conversion of cortisone to cortisol (conversion of 3H-cortisone to cortisol minus the conversion of

3 H-

14 14 C-cortisol to C-cortisone)

following one pass (5 minutes) through the lungs was 7.1%; after a second pass (10 minutes) this conversion had increased to 13.7%. At the end of an hour of recirculation the accrued conversion amounted to 27.0%. The concentration of both cortisone and cortisol was 1.25 X 10B6M during the first and second passages and 1 X 10B8M during the remainder of the one hour perfusion period. The percentage conversion, when expressed as the mass of substrate converted, represented 119, 294, and 412 ng of cumulative cortisol produced during the three sampling times. As in treatment 2 the conversion rate in the tissue was considerably higher than that reflected by the perfusate (49.6 versus 27.0%). Experiment 2 Homogenized Lung Incubation. Fetal rabbit lung homogenates were studied at various gestational ages to quantitatively assess the enzymatic conversion of cortisone to cortisol. Figure 2 represents both the mean conversion and rate of this reaction on days 21, 23, 25 and 29 of gestation. No US-hydroxysteroid dehydrogenase activity could be detected on day 21, whether cofactor was added or not.


conversion of cortisone to cortisol was apparent on day 23, and was markedly enhanced by the addition of cofactor. Statistical analysis revealed a significant (pc.01) increase in activity between days 23 and 29.

Between days 23 and 25 there appeared to be an increase in both


Figure 2. Conversion of cortisone to cortisol by fetal rabbit lung homogenate. The bars represent the meen conversions; the closedcircles and triangles represent the mean slopes (b values) of the dose-response curves. Standard deviations are not shown since they were all lesa than 1%. The incubation condi ions were: cortisone (1 X 10m6 to 1 X lO_qMI)in the presence of 1,2-&l-cortisone (3.8 X 10' dpm), 0.2 ml (20 mg wet weight equivalent) of fetal rabbit lung homogenate in 0.25 M sucrose, 0.3 4. of an NADPX-generating system* - in a total volume of 2.0 ml (adjusted to volume with 0.1 M phosphate buffer, pli7.4). The mixture was incubated in a Dubnoff metabolic shaker for 2 hours at 37*C under air. *NADPH-generating system: 0.1 ml each of 0.1 M glucose-d-phosphate; 2 ug/ml glucose-ii-phosphate dehydrogenase; 6 mH NABP. Each solution was diluted with 0.1 M phosphate buffer, pH 7.4.




the basal and cofactor-stimulated conversions. There was a marked increase in the basal dose-response, while the magnitude of the increase in the stimulated dose-response did not appear to be as great. The change from day 25 to 29 was apparently brought about by increases in both basal conversion and dose-response accompanied by a decrease in cofactor-stimulated conversion with no change in the dose-response. DISCUSSION The results of the present experiments support the previous claim of US-hydroxysteroid dehydrogenase activity in the fetal rabbit lung in culture (17). Such activity was first reported in cultured human fetal lung at midgestation (16) and was in sharp contrast with reports that the human fetus is devoid of such activity (15). The confirmation of llS-hydroxysteroid dehydrogenase activity in fetal tissue is of particular significance since the ability of the fetal lung to convert cortisone to cortisol enables it to utilize circulating cortisone, the predominant unconjugated glucocorticoid in fetal circulation in a number of species (9-14). Inhibition of this conversion in culture inactivates cortisone, thus rendering it unable to induce differentiated lung function (17). This loss of activity is solely due to inhibition of conversion of cortisone to cortisol since cortisone does not bind to the receptor in this tissue (18,19). Furthermore, results obtained in tissue culture were indicative not only of intracellular conversion of cortisone to cortisol, but of release of cortisol into the extracellular milieu (16,17). The present results, derived from the isolated, perfused lung, clearly demonstrate such release since cortisol formed from exogenous cortisone was identified in the perfusate. This capacity of the fetal lung to extract

cortisone from circulation, convert it to cortisol and release it may prove of physiological importance since it represents a site of reactivation of otherwise inactive hormone (6), resulting in a net increase in cortisol in fetal circulation. Study of the origins of cortisol in the fetal rhesus monkey supports the importance of cortisol derived from maternal cortisone, this reute accounting for more than 50% of the cortisol in fetal circulation (22). Furthermore, no difference could be found when data from intact fetuses was compared with that from decapitated fetuses (23), corroborating the metabolic origin of the cortisol in fetal circulation. During the course of the perfusion experiments it was noted that there was a consistently higher ratio of cortisol to cortisone in the tissue than in the perfusate, suggesting intracellular retention of cortisol. This


in agreement with previous reports in both human (24)

and rabbit (25) models of fetal lung receptors for glucocorticoids. The presence of such receptors is an important component of steroid hormone target tissue activity (26). Quantitation of ll$-hydroxysteroiddehydrogenase activity between days 21 and 29 was indicative of a correlation between cortisol production and lung maturation. This conversion was totally absent on day 21 and significantly increased with gestational age. crease in activity in the presence of


The marked in-

indicated that the

cofactor was rate limiting throughout this period. The proportional increase in both basal and cofactor-stimulatedactivities between days 23-25 implies that enzyme activity had increased, although these results do not exclude the possibility that endogenous cofactor




concentration had also increased. However, by day 29 the increase in maximal activity, accompanied by a moderate increase in endogenous activity, suggests that cofactor concentration alone had increased. These results indicate the ontogeny of both the enzyme and cofactor components of llE+hydroxysteroid dehydrogenase activity in the maturing fetal rabbit lung. The decrease in maximal activity by day 29 in all probability reflects dilution of enzyme since the dose-response did not change. This may have been due to proliferation of other cell-types or hypertrophy of the responsible cells in this rapidly growing organ. The shunting of total cardiac output through the perfused fetal lung, resulting from the occlusion of the ductus arteriosus, should be noted as an artificial condition in which 100% of the circulation passes through the fetal lung, whereas this organ is unly exposed to about 10% of the cardiac output -in utero (27) when the ductus arteriosus is patent. These differences in the pattern of flow are exemplified by contrasting the results of the two perfusion experiments in which the ductus arteriosus was either left open or ligated. In the former case only small quantities of radioactivity were present in the tissue and perfusate, although the radioactivity in the tissue did reflect high rates of conversion. The difference between these results is mainly due to the bypassing of the lungs by the ductus arteriosus, and the altering of this pattern might lead to spurious results. However, quantitative considerations reinstate the claim that the lung may be an important site of cortisol production. The lungs were able to convert cortisone to cortisol even at a concentration of 1 X 10W6M, which is some 40-fold greater than that found in circulation (31) *

In this light even a 10%

perfusion rate would be adequate to metabolize such quantities of cortisone. Recent reports indicate that elevation of fetal cortieol levels is intimately related to both normal lung function (28,29) and to parturition (30). If

a causal relationship exists between peri-

pheral conversion of cortisone to cortisol by the lung and the birth process, it may represent a mech~sm

for coordination of respiration

and the process of parturition. ACKN(%UDGMENTS This investigation was supported in part by NIH training grant number 5-TOl-BDO0104-10 from NICRRD and by Ford Foundation grant number 630-05058. The advice of Dr. E. G. Coggins on statistical analysis is gratefully acknowledged. RBFBRENCES 1.

2. 3. 4. 5, 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.


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19. Bush, I. E., BIOCHEM. J. 50, 370 (1952). 20. Drayer, N. M. and C. J. P. Giroud, STEROIDS 5, 289 (1965). 21. Steel, R. G. D. and J. H. Torrie,in PRINCIPLES AND PROCEDURES OF STATISTICS, pp. 341-343, McGraw-Hill, New York (1960). 22. Kittinger, G. W., STEROIDS, 23, 229 (1974). 23. McNulty, W., F. Hagemenas, N. Beamer and G. Kittinger,in PROGRAM OF THE ENDOCRINE SOCIETY, Chicago (1973). 24. Ballard, P. L. and R. A. Ballard, J. CLIN. INVEST. 53, 477 (1974). 25. Giannopoulos, G., S. Mulay and S. Solomon, BIOCHEM. BIOPHYS. RES. COMM. 47, 411 (1972). 26. Jensen, E. V., M. Numata, P. I. Brecher and E. R. DeSombre in THE BIOCHEMISTRY OF STEROID HORMONE ACTION, (R. M. S. Smellie, ea.), p. 133, Academic Press (1971). 27. Dawes, G. S., J. C. Mott and J. G. Widdicombe, J. PHYSIOL. 126, 563 (1954). 28. Murphy, B. E. P., AM. J. OBST. GYNEC. 119, 112 (1974). 29. Fencl, M. and D. Tulchinsky, NEW ENGLAND J. MED. 292, 133 (1975). 30. Challis, J. R. G. and G. D. Thorburn, BRIT. MED. BULL. 3& 57 (1975). 31. Torday, J. S., unpublished observation.

Production of cortisol from cortisone by the isolated, perfused fetal rabbit lung.

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