Cortisol metabolism and its inhibition by glycyrrhetinic acid in the isolated perfused human placental lobule

.7. Steroid Biochem. Molec. BioL Vol. 62, No. 4, pp. 337-343, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0960-0760(97)00039-3 o96o-076o/97 $17.oo + o,oo

Pergamon

Cortisol M e t a b o l i s m and Its Inhibition by G l y c y r r h e t i n i c A c i d in the Isolated P e r f u s e d H u m a n P l a c e n t a l Lobule H . M . D o d d s , 1'2. P . J. T a y l o r , 2 L. P . J o h n s o n , 3 R . H . M o r t i m e r , 4,s S . M . P o n d 1'~ a n d G . R . C a n n e U 3 1University of Queensland, Department of Medicine, Pn'ncess Alexandra Hospital, Brisbane, Queensland, Australia; 2Department of Clinical Pharmacology, Princess Alexandra Hospital, Brisbane, Queensland, Australia; 3Department of Pathology, Royal Brisbane Hospital, Brisbane, Queensland, Australia; 4Conjoint Endocrine Laboratory, Royal Brisbane Hospital, Brisbane, Queensland, Australia and 5University of Queensland Department of Obstetrics and Crynaecology, Royal Brisbane Hospital, Brisbane, Queensland, Australia

We have previously reported the placental m e t a b o l i s m of prednisolone to prednisone, 20~- and pdihydroprednisone and 20~-dihydroprednisolone. In this study, the disposition o f cortisol was investigated in v i t r o in the dual perfused, isolated h u m a n placental lobule after the addition o f cortlsol (1.2/anol, n = 3 and 12 pmol, n = 4) to the maternal compartment. Analysis o f 5 h maternal and fetal perfusate samples by high performance liquid chromatography-electrospray-tandem m a s s spectrometry (HPLC-ESI--MS/MS) revealed that cortisol was mainly metabolized to cortisone, but a significant production of 20~-dihydrocortisone, 20~-dihydrocortisone, 20~-dihydrocortisol and 20~dihydrocortisol was also detected. Saturability o f m e t a b o l i s m but not transfer was demonstrated. Metabolism was eliminated by co-perfusion with the potent l l~-hydroxysteroid dehydrogenase ( l l ~ H S D ) enzyme inhibitor 18~-glycyrrhetinic acid (GA). The disposition o f GA was analysed using H P L C - a t m o s p h e r i c pressure chemical ionisation-MS/MS ( H P L C - A P C I - M S / M S ) . GA was found to transfer f r o m the maternal to the fetal circulations without detectable m e t a b o l i s m during 6 h o f perfusion. © 1997 Elsevier Science Ltd.

57. Steroid Biochem. Mole,:. Biol., Vol. 62, No. 4, pp. 337-343, 1997

INTRODUCTION T h e placenta is rich in 1 lfl-hydroxysteroid dehydrogenase type 2 (llI3-HSD2) which inactivates glucocorticoids to their l l-keto metabolites, and plays a key role in modulating exposure of the fetus to maternal glucocorticoids [l]. Other steroid metabolising enzymes such as the 3~-, 3fl-, 20~- and 20/~-hydroxysteroid dehydrogenases are also present [2, 3] in placenta but their role in glucocorticoid metabolism is less clear. Although considerable information about h u m a n placental corticosteroid transfer and metabolism is available, only one study [4] has investigated the metabolism of cortisol utilizing the perfused h u m a n *Correspondence to H. M. Dodds. Tel: +61 7 3240 2596; Fax: +61 7 3240 5031: e-mail: [email protected]. Received 12 Nov. 1996; accepted 26 Mar. 1997. 337

placental lobule. T h a t study suggested that the only metabolic modification to cortisol was oxidation at C11 to form cortisone, but used relatively insensitive analytical techniques. We have previously, using a recirculating placental perfusion model, reported the metabolism of prednisolone to prednisone, 20/3-dihydroprednisolone and 20~- and fl-dihydroprednisone [5], but the formation of 20~- and 20/3- metabolites of cortisol has not until now been investigated. Glucocorticoids are administered to pregnant women with a wide variety of medical disorders, such as autoimmune diseases or asthma, where fetal exposure to steroids is undesirable. In some circumstances, such as the induction of fetal lung surfactant, glucocorticoids are, however, given to the pregnant woman with the express intention of treating the fetus. Glycyrrhetinic acid (GA), one of the principal components in the root of the licorice plant, Glycyrrhiza glabra, is a potent inhibitor of 1 I]3-HSD2

H . M . Dodds et al.

338

[6]. Maternal administration of GA offers a possible mechanism for increasing fetal exposure to endogenous or administered maternal glucocorticoid. In this study we have, with new sensitive techniques, examined placental cortisol metabolism and its interaction with GA, and studied the placental metabolism and transfer of GA.

MATERIALS AND METHODS

Materials Cortisol, 18/~-GA and dextran (approximate molecular weight 40 500) were obtained from the Sigma Chemical Co. (St Louis, U.S.A.). Sep-Pak ClS cartridges were obtained from Waters (Milford, U.S.A.). All solvents were of analytical grade and were glass distilled before use. High performance liquid chromatography (HPLC) grade methanol (EM Science, Gibbstown, U.S.A.) and glacial acetic acid (Mallinckrodt, Clayton, Australia) were obtained from their respective suppliers. Reagent grade deionised water (Milli-Q, Millipore Co, U.S.A.) was used throughout the investigation. Methods Placental perfusion. These studies were approved by the research ethics committees of the Royal Women's and Princess Alexandra Hospitals and the University of Queensland. Placentas were obtained from normal women, without a history of drug ingestion, delivered at or near term by repeat cesarian section. T h e placental perfusion techniques, materials, conditions, and the viability of the preparation have been described previously [5, 7]. Maternal circuit perfusate was circulated at a rate of 25 ml/min and fetal circuit perfusate circulated at a rate of 3 ml/min. Perfusion experiments were performed using cortisol in concentrations of 1.2 pmol/1 (n = 3) or 12 #mol/l (n = 4). Additional experiments were performed using cortisol 12pmol/1 in the presence of GA 16.67mmol/1 (n " 2). Maternal and fetal artery samples (3 ml) were collected at 0, 15, 30, 45 min and 1, 1.5 and 2 h and then hourly to the end of the experiment for the measurement of cortisol and its metabolites. GA concentrations were measured in samples taken at 0, 3 and 6 h from perfusions in the presence of GA. Analysis of cortisol and its metabolites. Perfusate samples were analysed for cortisol, cortisone, their 20~- and 20fl-dihydro metabolites and their tetrahydro metabolites by high performance liquid chromatography-electrospray-tandem mass spectrometry ( H P L C - E S I - M S / M S ) [8]. Briefly, the internal standard (IS) (6ct-methylprednisolone) was added to perfusate samples (3 ml), which were extracted using Cls solid phase extraction cartridges as described previously [5]. T w o different standard curves were used to analyse samples for the low- and high-dose per-

fusions, 10-200 gg/1 and 100-2000 pg/1, respectively. T h e H P L C separation was achieved using a Novapak Cls column (100 x 2.1 ram) at ambient temperature with a mobile phase containing 53% methanol and 47% buffer (10 m M ammonium formate, p H 4.0). A P E - S C I E X API III triple quadrupole instrument (PE-SCIEX, Thornhill, Canada) was used for mass spectrometric detection. Protonated precursor ions [M + H] + of each compound of interest were analysed using multiple reaction-ion monitoring (MRM). Analysis of 183-GA Standards. Standards were prepared in perfusion medium at concentrations of 100, 500, 1000 and 2500 #g/1. 6~-Methylprednisolone was used as the IS. Preparation of samples. T h e IS was added to standards, control and experimental samples which were then extracted with Sep-Pak Cxs (Waters) cartridges. T h e analytes were eluted with methanol, evaporated to dryness under air (40°C), and reconstituted in mobile phase before injecting an aliquot onto the column. For quality control purposes a standard curve was prepared and assayed daily along with perfusate samples and blank perfusate. T h e imprecision of the m e t h o d was determined by the method of Krouwer and Rabinowitz [9], by replicate analysis (n = 3) of spiked perfusate samples (200 and 2000/~g/1), over 5 days. T h e accuracy of the assay was determined by dividing the mean assayed concentration by the weighed-in concentration.

HPLC-atmospkeric pressure chemical ionisation-tandem mass spectrometry analysis of 18fl-GA T h e H P L C system consisted of a solvent delivery system (LKB, Bromma, Sweden) with an ISS 200 autoinjector (Perkin Elmer, Danbury, U.S.A.). Chromatographic separation was achieved on a Cls Activon column (30 x 3 m m i.d.) at 65°C, using methanol/isopropanol/10 m M ammonium formate buffer (pH 4.5, 55/5/40, v/v/v) as the mobile phase. A flow rate of 500 ml/min was used, giving a chromatographic run time of 12 min. A P E - S C I E X API III triple quadrupole instrument (PE-SCIEX) was used for quantitative mass spectrometric detection. The atmospheric pressure chemical ionisation (APCI) interface was at 500°C and in positive ion mode. T h e system was optimized for the production of the protonated molecular ion of GA [M + H] +. T h e orifice voltage and the interface heater were set to 70 V and 60°C, respectively. Quantitative analysis was achieved with the internal standard method using 6~-methylprednisolone. Analytes were measured by M R M (GA m/z 471.2 ~ 188.9, IS m/z 375.2--*357.2). Standard curves were constructed using weighted (1/Z2) linear least squares regression. Data were collected and manipulated on a Macintosh computer operating RAD and M A C Q U A N software (PE-SCIEX).

339

Glycyrrhetinic Acid and Placental Cortisol Metabolism

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Disposition of cortisol in low-dose, high-dose and GA perfusions Low-dose perfusions. Five cortisol metabolites were identified in the placenral perfusate, namely cortisone, 20~-dihydrocortisone, 20/~-dihydrocortisone, 20~dihydrocortisol and 20fl-dihydrocortisol. Metabolites formed by 6/%hydroxylation or cleavage of the C17Czo bond were not detected. Cortisol concentrations in the maternal circuit perfusate decreased rapidly in the first 30 rain of the perfusion to 7.0 _+ 1.2% of initial concentrations, and then decreased more slowly to be undetectable at 3 h. At 30 min, similar (4.4 :k 0.4%) concentrations of cortisol were detectable in the fetal circuit, and these also decreased to undetectable concentrations by 3 h . Large amounts of cortisone were found in the maternal and fetal circuits at 3 0 r a i n (45.4-t-7.7 and 26.4 ___12.5%, respectively). Cortisone concentrations then increased slowly 1:o reach 49.6 ± 16.8% in the maternal and 31.6_+ 10.4% in the fetal perfusate at 5 h [Fig. I(A)]. Low concentrations of 20~- and 20/%dihydrocortisone were detectable at 30 min in maternal and fetal perfusate and rose ower the time of the perfusion (Table 1). Similar concentrations of 20~- and 20/% dihydrocortisol could be demonstrated in maternal perfusate at 30 min, renaaining relatively constant over the time of the perfusion. Although 20/%dihydrocortisol was measurable in maternal and fetal perfusate at 30 rain, and 20~-dihydrocortisol was found at 30 min in maternal perfusate, no 20~-dihydrocortisol was demonstrable in fetal perfusate until 3 h (Table 1).

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Fig. 1. Comparison of the disposition of cortisol (omaternal, Ofetal) and its major metabolite cortisone (Bmaternal, IS]fetal) at (k) low-dose, 0.18/~ (n=3) and (B) high-dose, 1 . 8 / ~ (n = 4) substrate concentrations. (C) A typical concentration-time profile of tort±sol (omaternal and Ofetal) for perfusions in the presence o f GA.

High-dose perfusions. T o test the possibility of saturation of the steroid metabolizing enzymes further 6 h perfusions (n = 3) were performed using an initial unlabelled cortisol concentration of 12 mmol/1 (10 times the concentration used in the low-dose experiments). T h e disposition of cortisol was significantly different in these experiments. At 3 0 m i n cortisol concentrations in maternal and fetal perfusates (47.5 __ 5.8% and 12.7 __ 3.3%, respectively) were significantly greater than concentrations at 30 min in low-dose perfusion experiments ( P < 0.001 and P < 0.008, respectively). Conversely, at the same time cortisone concentrations were significantly higher (45.4 + 7.7% in maternal and 2 6 . 4 + 12.5% in fetal perfusate) ( P < 0 . 0 0 1 and P < 0 . 0 1 , respectively) [Fig. I(B)]. Figure 2 illustrates chromatograms for cortisol in

Table 1. Hourly production (%) (mean + SD) of the 20-dihydro reduced cortisol metabolites in maternal (M) and fetal (F) arteries during 5 h in the low-dose pe~fusions Product

20a-Dihydrocol"6sone 20fl-DihydrocoJxisone 20a-DihydrocoJxisol 20fl-Dihydroco~rtisol M, maternal; F, fetal.

Perfusion time (h)

M F M F M F M F

0.5

1.0

3.0

5.0

2.64 ± 0.99 2.95 + 1.24 2.61±0.99 2.77 ± 0.88 2.98 ± 0.90 ND 2.79 ± 0.87 2.29 ± 0.97

2.50 ± 0.97 2.76 ± 0.94 2.50 ± 0 . 9 6 2.78 ± 0.88 2.49 ± 1.03 2.77 ± 1.03 2.36 ± 0.98 2.57 ± 0.99

2.49 ± 0.98 2.78 ± 0.94 2.72±1.0 2.89 ± 0.75 2.80 ± 0.99 2.77 ± 1.04 2.39 ± 0.98 2.44 ± 0.90

2.84 ± 1.26 2.45 ± 0.96 3.53±1.1 3.51 ± 0.64 2.89 ± 1.28 2.45 ± 1.02 2.77 ± 1.21 2.36 ± 0.96

H . M . Dodds et al.

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tisol. Indeed, almost all of the cortisol remained intact and was evenly distributed between maternal and fetal compartments throughout the course of perfusion. l l(C) illustrates the complete inhibition of cortisol metabolism and free transfer of cortisol from the maternal to the fetal compartment when administered with GA. G A did not interfere with the H P L C - E S I MS/MS analysis of cortisol.

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Time (min) Fig. 2. Chromatograms for cortisol (rrdz 364.0 --* 121.0) in the high-dose perfusions of (A) blank perfusate, (B) maternal artery 0 It, (C) fetal artery 0 h, (D) maternal artery 5 h, and (E) fetal artery 5 h. blank perfusate, maternal and fetal circuits at 0 and 5 h, and effectively demonstrates that the metabolism of cortisol is completely exhausted after 5 h. Concentrations of 20a- and fl-dihydrocortisols and dihydrocortisones were similar to those seen in low-dose perfusions (Table 2). Effect of G A on the disposition of cortisol. The H P L C E S I - M S / M S analysis of perfusions in the presence of G A revealed no appreciable biotransformation of cor-

18fl-glycyrrhe-

Full-scan positive-ion spectra for both GA and the IS produced [M + H] + precursor ions of m/z 471.2 and m / z 375.2, respectively. By using argon as the collision gas, fragmentation of these precursor ions was observed, and parameters were adjusted to give maximum intensity to a major product ion. Both precursor and product ions were selected to optimize both sensitivity and specificity for M R M quantitative analysis (GA m/z 471.2--* 188.9 and IS m/z 375.2--* 357.2). Collision-assisted dissociation spectra for G A and IS are shown in Fig. 3. The assay was linear from 100 to 2500 pg/l, with a correlation coefficient of 0.999 (n = 5). Imprecision studies at 200 and 2000 #g/1 showed that the assay was reproducible with total coefficient of variations <4.0%. T h e validation data in terms of accuracy and imprecision (total, intra- and inter-day) are presented in Table 3. Chromatograms of the blank and spiked perfusate for GA are shown in Fig. 4. One unknown peak was observed in the spiked perfusate and experimental extracts. T h e mobile phase composition and flow rate were optimized to achieve baseline separation of the impurity and GA. T h e impurity peak is believed to be a contaminant of the standard and is most likely the 18~- isomer of GA, but this was not confirmed. Figure 5 illustrates G A samples from maternal and fetal arteries at 0 and 6 h, respectively. Although it was postulated that GA would be metabolized, perfusion over 6 h failed to reveal any loss of GA in the system.

Table 2. Hourly production (%) (mean + SD) of the 20-dihydro reduced cortisol metabolites in maternal (M) and fetal (F) arteries during 5 h in the high-dose perfusions Product

20a-Dihydrocortisone 20fl-Dihydrocorfisone 20~-Dihydrocortisol 20fl-Dihydrocortisol M, matemal; F, fetal.

Perfusion time (h)

M F M F M F M F

0.5

1.0

2.75 ± 1,91 2.76 ± 2.56 4.56 ± 3.38 3.33 ± 2.58 3.32±3.11 ND 2.87 ± 2.24 2.89 _+2.38

3.56 ± 2,97 2.85 ± 2.07 5.46 ± 4.59 3.90±2.47 3.17±2.59 ND 3.30 ± 2.45 2.93 ± 2.33

3.0

5.0

3.37 ± 2,69 4.38 _+3.77 3,18 ± 2.44 4,08 _+2.64 9.20 _+6.50 14.70_+12.53 8.10± 5,11 11.8±7.90 3.24_+2.84 3.02_+2.67 3.17±2.66 2.99±2.65 3.26 ± 2.59 4.51 ± 2.59 3.46 _+2.89 3.62 _+2.56

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DISCUSSION We have previously reported production of 20~and fl-dihydroprednisolae and 20fl-dihydroprednisolone from prednisolone in the perfused h u m a n placenta [5]. In the present study we have applied H P L C - E S I - M S / M S methodology to measure concentrations of cortisol ~Lnd its metabolites in the perfused placental model to confirm rapid metabolism of cortisol to cortisone and the production of 20~- and 20fl-dihydrocortisones zLnd dihydrocortisols. Our findings differ from earlier studies of cortisol metabolism, using the perfused placenta or placental minces and relatively insensitive analytical methods, which found cortisone to be the only placental metabolite of cortisol. In the placental perfusion study [4] the conversion rates of cortisol to cortisone in maternal and fetal vein perfusate samples were 37 and 73%, respectively, in a Table 3. Imprecision and ~ccuracy of H P L C - A P C I - M S / M S for 18fl-glycyrrhetinic acid Coefficient of variation

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i

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Fig. 4. C h r o m a t o g r a m s for 18fl-glycyrrhetinic a c i d (mlz 471.2 --* 188.9) o f (A) b l a n k p e r f u s a t e a n d (B) a 1000 pg/l s t a n dard.

non-recirculating (i.e. both maternal and fetal circuits open) perfusion dosed with 100 m M cortisol. These results were, however, from one analysis only and were taken after 1 h of perfusion. Incubation of [3H]cortisol with minces of mid-gestation and term placenta revealed cortisol conversion rates of 67% [10] and 81% [4]. T h e present study confirms that cortisone is the major placental metabolite of cortisol. Our finding, that the conversion of cortisol to cortisone is significantly lower with perfusate cortisol concentrations of 12/~mol/l than 1.2 #mold, is consistent with partial saturation of l lfl-HSD2 by higher cortisol concentrations. This enzyme has recently been cloned, and when transfected into mammalian C H O cells has an apparent Km for cortisol of 43.9 n M [11], which may approximate intracellular cortisol concentrations in our studies. T h e fact that equilibrium between compartments was established rapidly in both sets of perfusions indicates saturation of metabolism, but not transfer. In addition to the 1 l fl-HSD2 conversion of cortisol to cortisone, observed in this series of experiments, the 20-hydroxysteroid dehydrogenase enzyme system was also active. T h e r e is little information on the role played by the 20-dihydroisomers of cortisol and cortisone in human physiology. Urinary metabolites of cortisol identified in man include cortisone, the 6-

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hydroxy isomers of cortisol and cortisone, the cortols and cortolones, tetrahydrocortisol and the 20fl-isomers of cortisol and cortisone. T h e r e is one early report [12], using large quantities of urine, of the detection of 20~-dihydrocortisol in h u m a n urine. More recently, however, Schoneshofer and colleagues [13, 14] reported that both the ~- and fl-isomers of cortisol and cortisone are present in h u m a n urine, and that the a-isomers are predominant. It has long been recognized that 20~-hydroxysteroid dehydrogenase is present in the placenta. T h e activity of this enzyme in the placenta was first evident when 20~dihydroprogesterone was isolated [15], and more direct evidence was subsequently presented [16]. There has been extensive investigation into its activity in the placenta throughout pregnancy, its necessary cofactor requirements and kinetics. In microsomal preparations, the 20~-hydroxysteroid dehydrogenase activity for progesterone in h u m a n placenta is 5-fold greater in term placenta compared at 12 to 20 weeks of gestation [17]. T h e activity of 20fl-hydroxysteroid dehydrogenase in the placenta has not, however, been elucidated. 20fl-Dihydroprogesterone is formed, although in smaller quantities compared to the 20~isomer [18]. T h e placental rates of formation of the 20-dihydro reduced metabolites of progesterone are in contrast to

that observed in our study on cortisol. T h e 20fl-isomers of cortisol and cortisone were in greater abundance than the 20e-isomers, indicating that 20flhydroxysteroid dehydrogenase may have a higher affinity for cortisol. Second, it is also likely that the rates of formation of these metabolites are slower than that of 1 lfl-HSD2, indicating that a longer perfusion time is required to detect these metabolites. T h e formation of 20fl-dihydro metabolites for cortisol and cortisone, along with evidence with prednisolone in a parallel study [5], have provided direct evidence for the presence of the 20fl-hydroxysteroid dehydrogenase enzyme system in placental tissue. T h e aims of including GA, as a substrate, in the cortisol perfusate medium were 2-fold. T h e first was to confirm our previous finding [19] that in the presence of GA, the activity of 1 lfi-HSD2, measured by the conversion of cortisol to cortisone, would be effectively blocked. This was demonstrated to be the case. T h e second was to determine whether significant inhibition of l lfi-HSD2 would direct the metabolism of cortisol towards a different pathway. T h e observed results did not reveal an ahemative pathway. We have previously demonstrated the almost complete inhibition of placental prednisolone metabolism by coperfusion with GA [19]. We proposed a metabolic pathway consisting of conversion of prednisolone to prednisone, formation of 20c~- and 20fi-dihydroprednisone from prednisone and 20fl-dihydroprednisolone from 20fl-dihydroprednisone. T h e results of the present study suggest that, after the conversion of cortisol to cortisone, 20e- and 20fi-dihydrocortisone are produced from cortisone and subsequently converted to 20e- and 20fl-dihydrocortisol. Inhibition of l lflH S D 2 therefore blocks production of cortisone and the 20e- and 20fl-dihydro metabolites of cortisone. Investigating the scope of GA usage and its mechanism of action as a therapeutic agent warrants further study. T o initiate this, the disposition of GA in the human placenta was investigated. A H P L C A P C I - M S / M S assay was developed for GA because analysis by H P L C with UV detection is subject to interference from endogenous compounds, and necessitates considerable clean-up of the samples [2023]. We have combined two specific techniques ( H P L C and MS) and have further increased the specificity of the assay by using tandem MS to quantitare the disposition of GA in the perfused human placental lobule in vitro. T o produce intense molecular ions for the mass spectrometric monitoring of GA, it was necessary to buffer the mobile phase to p H 4.5. T h e method developed is simple, fast and reproducible. For studies with biological fluids, a more appropriate IS may be selected. T h e use of 6~methylprednisolone was necessary due to the determination in the concurrent study of cortisol, cortisone, their 20-dihydro reduced and tetrahydro metabolites, where the same sample extracts were re-assayed.

Glycyrrhetinic Acid and Placental Cortisol Metabolism

T h e observed G A profiles in the perfusate from each side of the placenta were identical. T h e transfer of G A from the maternal to the fetal c o m p a r t m e n t was slow, probably due to its carboxy group and the probable high protein binding. G A metabolites have been reported in patients with licorice-induced pseudoaldosteronism [24], and in healthy volunteers administered different oral doses of the c o m p o u n d [25]. A review of current literature has not revealed a safe dosage regimen for GA, nor a quantitative risk assessm e n t of a single G A close administered to mothers presenting with potentizl p r e m a t u r e delivery. Because the effect of G A on I l f l - H S D 2 is reversible, single doses of the acid yielding concentrations sufficient to inhibit metabolism may not pose a problem, as long as the enzyme is inhibited only for a short period. A single dose m a y present no adverse effect to the fetus due to its i m m a t u r e liver and enzyme activation and release of the acid directly back to the maternal comp a r t m e n t for elimination. T o date, a comprehensive comparative investigation of high- and low-dose glucocorticoid treatment during h u m a n pregnancy has not been initiated, and it is unclear if elevated maternal cortisol concentrations cross the placenta sufficiently to increase fetal exposure. This study has demonstrated that, at higher doses, saturation of metabolism but not transfer is observed. T h e simple and specific assay ( H P L C - A P C I - M S / MS) described in this investigation m a y be useful for quantitative risk assessments of G A usage, and to further studies of the value of G A in enhancing the availability of maternal cortisol and other glucocorticoids to the fetus. Acknowledgements--We thank the nursing staff of the Royal Women's Hospital for the collection of placental tissue and Drs Russell Addison and David Maguire for useful discussions. This work was supported by the N H M R C and the Princess Alexandra Hospital Research and Development Foundation.

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