The Relationship of Cortisol and Cortisone to Saturated Lecithin Concentration in Ovine Amniotic Fluid and Fetal Lung Liquid1 BARRY T. SMITH,2 D. WORTHINGTON, AND W. N. PIERCY Departments of Paediatrics and Obstetrics and Gynaecology, Queen's University, Ontario, Canada K7L 3N6 ABSTRACT. Cortisol and cortisone concentrations in ovine amniotic fluid and fetal lung liquid were analyzed with respect to gestational age and fetal lung maturation as reflected by saturated lecithin concentration. In amniotic fluid, the cortisol concentration rises as a function of gestational age while that of cortisone falls (P < 0.001). The ratio of cortisol/cortisone is significantly related to gestational age (r = 0.693, P < 0.001), and shows an even stronger correlation with saturated lecithin concentration (r = 0.832, P < 0.001). When examined
T
HE FUNCTIONAL maturation of the fetal lung is closely related to the emergence of its biochemical ability to produce and secrete saturated lecithin, the major component of the pulmonary surfactant (1). Since the original observations in the fetal lamb that the timetable of fetal lung development could be advanced by the administration of exogenous glucocorticosteroids (2,3), a similar phenomenon has been observed in a number of species (4,5) including man (6). Recently, several lines of evidence suggest that the normal course of intrauterine lung development may be dependent upon the interaction of the fetal lung with its corticosteroid milieu (7-12). Two recent studies have suggested a relationship between amniotic fluid cortisol levels and fetal lung development (13,14), although this relationship has been questioned (15). During an ongoing study of fetal respiratory-like activity utilizing a chronic fetal sheep preparation with indwelling amniotic and tracheal catheters, we thought it would be of interest to observe ovine Received November 16, 1976. 'Supported by grants (DG-147 and MA-5757) from the Medical Research Council of Canada. 2 Scholar, Medical Research Council of Canada.
Kingston,
independently of gestational age, the correlation of the cortisol/cortisone ratio to saturated lecithin concentration remains highly significant (r = 0.641, P < 0.001). Similar findings were observed in fetal lung liquid, except that in this fluid both the saturated lecithin concentration and the ratio of cortisol/ cortisone are significantly higher than simultaneously determined values in amniotic fluid. These observations provide further evidence that the process of fetal lung maturation is closely linked to the fetal hormonal milieu. (Endocrinology 101: 104, 1977)
amniotic and tracheal fluid cortisol levels and their relation to gestational age and fetal lung maturity, as reflected by the concentration of saturated lecithin in these fluids. Furthermore, since the fetal lung has recently been shown to possess the capacity to convert the biologically inactive, but readily available hormone, cortisone, to cortisol (16-18), cortisone levels have also been measured in the same context. Materials and Methods Fifteen gravid sheep of mixed breed with accurately dated pregnancies were used in this study. The length of gestation in this species is 147 days. Anesthesia was induced with iv sodium thiopental and, after tracheal intubation, maintained with methoxyfluorane-oxygen utilizing a rebreathing circuit. Under standard aseptic surgical conditions, the uterus was exposed through a 15 cm midline incision caudal to the umbilicus. The head and neck of the fetus were delivered through a 10 cm uterine incision and the head was placed in a surgical glove filled with warm physiologic saline. The edges of the uterine incision were held in close proximity to the fetal neck with Allis clamps to prevent escape of amniotic fluid. The fetal trachea was exposed through a midline incision in the neck and a non-occluding catheter was introduced in a caudal direction, its tip remaining above the carina, and sutured
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CORTISOL AND CORTISONE IN AMNIOTIC FLUID securely in place. A second catheter was secured in a fetal common carotid artery after direct exposure of this vessel. After closure of the fetal skin incisions, a third catheter was tied to the fetal neck posteriorly with its tip facing caudally. The fetus was replaced in the amniotic cavity along with a 25 cm loop of each catheter and the uterine incision closed. The abdomen was closed and the catheters were exteriorized through the maternal flank prior to closure of the abdominal skin incision. Finally, a catheter was placed in a maternal carotid artery under direct vision prior to the termination of anesthesia. Postoperatively, the ewe was given 1,000,000 units of penicillin G and 80 mg of gentamicin intramuscularly three times daily for two days. The catheters were flushed daily and kept filled with heparinized saline to maintain patency. The condition of the fetus was determined daily by arterial blood gas analysis. At the time of amniotic fluid and tiacheal fluid sampling, fetal blood gas analysis was carried out and samples were discarded if the values were not within normal limits. The mean arterial blood gas values at the time of sampling were as follows: pO2—18.4, pCO2—45.5, pH— 7.334, hematocrit—31.5. These determinations were carried out on one ml of fetal blood, and sampling was limited to this amount to minimize the risk of hypovolemia and/or anemia. Three to five ml of amniotic fluid and tiacheal fluid were extracted with 4 volumes of ethyl acetate after the addition of 3,000 dpm of repurified [4-14C]cortisol (New England Nuclear, SA 55 mCi/rnmol) and 3,000 dpm of repurified [4-14C]cortisone (New England Nuclear, SA 59.8 mCi/mmol). The organic phase was washed with 1/10 volume of 0.1N NaOH to remove chromogens and then with 1/10 volume of distilled water twice or until the ethyl acetate extracts reached neutrality. The extracts were then evaporated to dryness and applied to sbips (2 cm x 30 cm) of filter paper (Whatman, no. 2) in a few drops of methylene chloride. After equilibration, the strips were developed by descending paper chromatography in system B5 of Bush (19). The cortisol and cortisone fractions were located by radiochromatogram scanning and eluted with 10 ml of absolute ethanol. The eluates were evaporated to dryness and resuspended in 1.0 ml of ethanol. Duplicate 0.1 ml aliquots were taken for liquid scintillation counting to correct for experimental losses by the recovery of 14C and duplicate aliquots (usually 0.1 ml, depending upon steroid content) were dried in test tubes for
105
radiotransinassay as modified (20) from the procedure of Murphy (21). Briefly, this involves competition of the unknown steroid samples with 3 H-labelled steroid for binding sites on plasma corticosteroid binding globulin. For cortisol, 1 ml of a solution of 5% human plasma (Versatol-A, Warner Lambert) containing 20,000 dpm of repurified [l,2-3H]cortisol (New England Nuclear, SA 40 Ci/mmol) was added to each tube. In the case of cortisone, the source of transcortin was 5% dog plasma. The tubes were shaken and incubated at 45 C for 5 min then at 4 C for 30 min. After adding 40 mg of Florisil to each tube (70 mg in the case of cortisone), the tubes were vigorously shaken for 2 min and returned to the 4 C bath. Supernatant, 0.5 ml, was added to 10 ml of scintillation fluid and each sample counted in a Searle IsoCap 300 liquid scintillation counter for a period long enough to achieve a counting error of less than 5%. Each run of the assay was accompanied by a duplicate standard curve prepared with repurified cortisol or cortisone (Steraloids, Pawling, N.Y.). In the case of cortisol, the standard curve was linear between 2.5 and 25 ng per tube and for cortisone, 2.5 to 20 ng per tube. All unknowns were assayed within these ranges. Results were accepted when the differences between duplicate samples was less than 10%. [14C]Phosphatidylcholine, of pig liver origin (New England Nuclear, SA 50 mCi/mmol), was processed to isolate, the disaturated phosphatidylcholine component. The crude preparation was reacted with mercuric acetate (22) and the nonadducted, disaturated component was isolated by thin layer chromatography as previously described (22). The purity of this preparation was checked by osmium tetroxide oxidation followed by column chromatography over neutral alumina (23), and found to be greater than 97% disaturated. Three thousand dpm of this 14C-disaturated phosphatidylcholine was added to a second aliquot (1-4 ml) of amniotic fluid and tracheal fluid. The samples were then shaken with 1 volume of methanol and 2 volumes of chloroform. The lower phase was removed, filtered, and evaporated to dryness. The lipid extracts were reacted in 1 ml of carbon tetrachloride containing osmium tetroxide, 3.2 mg, for 15 min (23) and evaporated to dryness. Saturated lecithin was then recovered by column chromatography over neutral alumina (23). The column effluents were evaporated to dryness, resuspended in 1 ml of chloroform, and, after taking 0.1 ml for counting to correct for
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T
HE FUNCTIONAL maturation of the fetal lung is closely related to the emergence of its biochemical ability to produce and secrete saturated lecithin, the major component of the pulmonary surfactant (1). Since the original observations in the fetal lamb that the timetable of fetal lung development could be advanced by the administration of exogenous glucocorticosteroids (2,3), a similar phenomenon has been observed in a number of species (4,5) including man (6). Recently, several lines of evidence suggest that the normal course of intrauterine lung development may be dependent upon the interaction of the fetal lung with its corticosteroid milieu (7-12). Two recent studies have suggested a relationship between amniotic fluid cortisol levels and fetal lung development (13,14), although this relationship has been questioned (15). During an ongoing study of fetal respiratory-like activity utilizing a chronic fetal sheep preparation with indwelling amniotic and tracheal catheters, we thought it would be of interest to observe ovine Received November 16, 1976. 'Supported by grants (DG-147 and MA-5757) from the Medical Research Council of Canada. 2 Scholar, Medical Research Council of Canada.
Kingston,
independently of gestational age, the correlation of the cortisol/cortisone ratio to saturated lecithin concentration remains highly significant (r = 0.641, P < 0.001). Similar findings were observed in fetal lung liquid, except that in this fluid both the saturated lecithin concentration and the ratio of cortisol/ cortisone are significantly higher than simultaneously determined values in amniotic fluid. These observations provide further evidence that the process of fetal lung maturation is closely linked to the fetal hormonal milieu. (Endocrinology 101: 104, 1977)
amniotic and tracheal fluid cortisol levels and their relation to gestational age and fetal lung maturity, as reflected by the concentration of saturated lecithin in these fluids. Furthermore, since the fetal lung has recently been shown to possess the capacity to convert the biologically inactive, but readily available hormone, cortisone, to cortisol (16-18), cortisone levels have also been measured in the same context. Materials and Methods Fifteen gravid sheep of mixed breed with accurately dated pregnancies were used in this study. The length of gestation in this species is 147 days. Anesthesia was induced with iv sodium thiopental and, after tracheal intubation, maintained with methoxyfluorane-oxygen utilizing a rebreathing circuit. Under standard aseptic surgical conditions, the uterus was exposed through a 15 cm midline incision caudal to the umbilicus. The head and neck of the fetus were delivered through a 10 cm uterine incision and the head was placed in a surgical glove filled with warm physiologic saline. The edges of the uterine incision were held in close proximity to the fetal neck with Allis clamps to prevent escape of amniotic fluid. The fetal trachea was exposed through a midline incision in the neck and a non-occluding catheter was introduced in a caudal direction, its tip remaining above the carina, and sutured
104
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CORTISOL AND CORTISONE IN AMNIOTIC FLUID securely in place. A second catheter was secured in a fetal common carotid artery after direct exposure of this vessel. After closure of the fetal skin incisions, a third catheter was tied to the fetal neck posteriorly with its tip facing caudally. The fetus was replaced in the amniotic cavity along with a 25 cm loop of each catheter and the uterine incision closed. The abdomen was closed and the catheters were exteriorized through the maternal flank prior to closure of the abdominal skin incision. Finally, a catheter was placed in a maternal carotid artery under direct vision prior to the termination of anesthesia. Postoperatively, the ewe was given 1,000,000 units of penicillin G and 80 mg of gentamicin intramuscularly three times daily for two days. The catheters were flushed daily and kept filled with heparinized saline to maintain patency. The condition of the fetus was determined daily by arterial blood gas analysis. At the time of amniotic fluid and tiacheal fluid sampling, fetal blood gas analysis was carried out and samples were discarded if the values were not within normal limits. The mean arterial blood gas values at the time of sampling were as follows: pO2—18.4, pCO2—45.5, pH— 7.334, hematocrit—31.5. These determinations were carried out on one ml of fetal blood, and sampling was limited to this amount to minimize the risk of hypovolemia and/or anemia. Three to five ml of amniotic fluid and tiacheal fluid were extracted with 4 volumes of ethyl acetate after the addition of 3,000 dpm of repurified [4-14C]cortisol (New England Nuclear, SA 55 mCi/rnmol) and 3,000 dpm of repurified [4-14C]cortisone (New England Nuclear, SA 59.8 mCi/mmol). The organic phase was washed with 1/10 volume of 0.1N NaOH to remove chromogens and then with 1/10 volume of distilled water twice or until the ethyl acetate extracts reached neutrality. The extracts were then evaporated to dryness and applied to sbips (2 cm x 30 cm) of filter paper (Whatman, no. 2) in a few drops of methylene chloride. After equilibration, the strips were developed by descending paper chromatography in system B5 of Bush (19). The cortisol and cortisone fractions were located by radiochromatogram scanning and eluted with 10 ml of absolute ethanol. The eluates were evaporated to dryness and resuspended in 1.0 ml of ethanol. Duplicate 0.1 ml aliquots were taken for liquid scintillation counting to correct for experimental losses by the recovery of 14C and duplicate aliquots (usually 0.1 ml, depending upon steroid content) were dried in test tubes for
105
radiotransinassay as modified (20) from the procedure of Murphy (21). Briefly, this involves competition of the unknown steroid samples with 3 H-labelled steroid for binding sites on plasma corticosteroid binding globulin. For cortisol, 1 ml of a solution of 5% human plasma (Versatol-A, Warner Lambert) containing 20,000 dpm of repurified [l,2-3H]cortisol (New England Nuclear, SA 40 Ci/mmol) was added to each tube. In the case of cortisone, the source of transcortin was 5% dog plasma. The tubes were shaken and incubated at 45 C for 5 min then at 4 C for 30 min. After adding 40 mg of Florisil to each tube (70 mg in the case of cortisone), the tubes were vigorously shaken for 2 min and returned to the 4 C bath. Supernatant, 0.5 ml, was added to 10 ml of scintillation fluid and each sample counted in a Searle IsoCap 300 liquid scintillation counter for a period long enough to achieve a counting error of less than 5%. Each run of the assay was accompanied by a duplicate standard curve prepared with repurified cortisol or cortisone (Steraloids, Pawling, N.Y.). In the case of cortisol, the standard curve was linear between 2.5 and 25 ng per tube and for cortisone, 2.5 to 20 ng per tube. All unknowns were assayed within these ranges. Results were accepted when the differences between duplicate samples was less than 10%. [14C]Phosphatidylcholine, of pig liver origin (New England Nuclear, SA 50 mCi/mmol), was processed to isolate, the disaturated phosphatidylcholine component. The crude preparation was reacted with mercuric acetate (22) and the nonadducted, disaturated component was isolated by thin layer chromatography as previously described (22). The purity of this preparation was checked by osmium tetroxide oxidation followed by column chromatography over neutral alumina (23), and found to be greater than 97% disaturated. Three thousand dpm of this 14C-disaturated phosphatidylcholine was added to a second aliquot (1-4 ml) of amniotic fluid and tracheal fluid. The samples were then shaken with 1 volume of methanol and 2 volumes of chloroform. The lower phase was removed, filtered, and evaporated to dryness. The lipid extracts were reacted in 1 ml of carbon tetrachloride containing osmium tetroxide, 3.2 mg, for 15 min (23) and evaporated to dryness. Saturated lecithin was then recovered by column chromatography over neutral alumina (23). The column effluents were evaporated to dryness, resuspended in 1 ml of chloroform, and, after taking 0.1 ml for counting to correct for
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