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Endocrinology, doi:10.1210/en.2003-0145
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Endocrinology Vol. 144, No. 7 3024-3030
Copyright © 2003 by The Endocrine Society

Leptin Distribution and Metabolism in the Pregnant Rat: Transplacental Leptin Passage Increases in Late Gestation but Is Reduced by Excess Glucocorticoids

Jeremy T. Smith and Brendan J. Waddell

School of Anatomy and Human Biology and the Western Australian Institute for Medical Research, The University of Western Australia, Crawley, Perth, Western Australia, 6009, Australia

Address all correspondence and requests for reprints to: Dr. Brendan Waddell, School of Anatomy and Human Biology, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. E-mail: bwaddell{at}anhb.uwa.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Materials and Methods
 Results
 Discussion
 References
 
Leptin is essential for the establishment of pregnancy and appears to promote fetal growth, but the mechanisms regulating fetal leptin exposure remain unclear. In rodents, indirect evidence suggests that fetal leptin is partly derived from the maternal circulation via transplacental passage. Indeed, the placenta expresses mRNA for Ob-Ra, one of the short forms of the leptin receptor (Ob-RS) important in leptin transport, and this expression increases markedly in late pregnancy. Therefore, we determined the transplacental passage of maternal leptin to the fetus in the rat and whether this transport increases near term in association with a rise in placental expression of Ob-RS protein. Because of the proposed role of leptin in promoting fetal growth, we also assessed the effect of glucocorticoid-induced fetal growth retardation on placental leptin transport. Anesthetized rats received a constant infusion of 125I-leptin via a jugular cannula before and at d 16 and 22 of pregnancy (term = d 23); plasma samples were obtained at 10, 20, 40, 60, 80, and 100 min, and fetuses and placentas were collected at the time of the final sample. The metabolic clearance rate of leptin fell (P < 0.01) from 3.08 ± 0.23 ml/min per kg in nonpregnant rats to 2.36 ± 0.13 ml/min per kg by d 22. Transplacental passage of 125I-leptin, estimated from its concentration in the whole fetus relative to maternal plasma, increased 10-fold (P < 0.005) between d 16 and d 22 of pregnancy. Over this same period, Ob-RS protein expression in the placental labyrinth zone increased by almost 2-fold. Transplacental leptin passage was reduced (P < 0.05) by 77% after maternal dexamethasone treatment, whereas suppression of endogenous glucocorticoid synthesis (by metyrapone) increased (P < 0.05) the transfer of maternal leptin to the fetus by 55%. These data show that transplacental passage of maternal leptin is a significant source of fetal leptin and increases markedly during late pregnancy. Consistent with the proposed role of leptin as a fetal growth factor, transplacental leptin passage is reduced in association with glucocorticoid-induced fetal growth retardation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE PEPTIDE product of the ob gene, is produced predominantly by adipocytes and acts to regulate food intake and energy expenditure via the hypothalamus (1, 2). Leptin has also been implicated as a key player in the hormonal regulation of several aspects of reproduction, including puberty onset (3), ovarian function (4), and fetal/placental growth (5, 6, 7, 8, 9). In rodents, the placenta synthesizes little, if any, leptin (10, 11), and the capacity of fetal adipocytes to synthesize leptin appears relatively low at least until late gestation (12, 13). Fetal plasma leptin levels increase near term (10), possibly due to stimulation of fetal adipocyte leptin synthesis by prolactin (13), but plasma leptin falls precipitously in the newborn (3). This suggests that fetal leptin is derived, in part, from the maternal circulation via transplacental passage. Such transport would likely involve interaction of leptin with one or more of the C-terminally truncated isoforms of the leptin receptor (Ob-RS) that are thought to facilitate transport of leptin across physiological barriers (14, 15). Indeed, we recently demonstrated placental expression of mRNA encoding one of these short isoforms (Ob-Ra) in the rat and showed that this expression increased severalfold late in pregnancy (8). Therefore, the present study assessed the ability of the rat placenta to transport maternal leptin to the fetus, and determined whether this transport increases near term and is accompanied by a rise in placental Ob-RS protein expression. The latter includes the Ob-Ra isoform and possibly other short isoforms (e.g. Ob-Rc) that are of comparable molecular weight and may be detected by the antibody used (16). In addition, we examined the effects of dexamethasone on transplacental leptin passage and placental Ob-RS protein expression, because glucocorticoids are known to potently stimulate maternal leptin levels yet reduce fetal leptin, suggestive of reduced transplacental leptin passage. The metabolic clearance rate (MCR) of leptin was also measured before and during pregnancy because previous reports had suggested that the progressive increase in maternal leptin during pregnancy (10, 17) may be due to reduced leptin metabolism associated with increased plasma leptin binding activity (17, 18). The MCR and transplacental passage of leptin were measured by constant infusion of 125I-leptin into the maternal circulation before and at d 16 and 22 of pregnancy (term = d 23) and on d 22 of pregnancy after altered fetal-placental glucocorticoid exposure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Materials and Methods
Results
 Discussion
 References
 
Animals and chemicals
Nullipararous albino Wistar rats aged between 9 and 12 wk were obtained from the Animal Resources Centre (Murdoch, Australia) and maintained under controlled conditions as previously described (19). Rats were mated overnight, and the day on which spermatozoa were present in vaginal smear was designated gestational d 1. Rats in this colony normally deliver on d 23. All procedures involving animals were conducted only after approval by the Animal Ethics Committee of The University of Western Australia. Dexamethasone, carbenoxolone, and metyrapone were purchased from Sigma (St. Louis, MO). Recombinant mouse leptin (Sigma) was radiolabeled with 125I by the chloramine-T method. The iodinated leptin (125I-leptin) was separated from free 125I on a column of Sephadex G-10 (Sigma). The specific activity of 125I-leptin was approximately 130 µCi/µg.

Glucocorticoid manipulations
Increased fetal-placental glucocorticoid exposure was achieved by maternal treatment either with the synthetic glucocorticoid dexamethasone or with carbenoxolone, an inhibitor of 11ß-hydroxysteroid dehydrogenase (11ß-HSD), from d 13–22 of pregnancy. Dexamethasone acetate was administered in drinking water (1 µg/ml), and carbenoxolone was administered twice daily (10 mg in 4% ethanol-saline, 0.1 ml sc injection at 0700 and 1730 h) over the same period. We have previously shown that these treatments reduce fetal weight at d 22 by 33% and 13%, respectively (8). To reduce glucocorticoid exposure, mothers were treated with the 11ß-hydroxylase inhibitor, metyrapone, administered in drinking water at a concentration of 500 µg/ml over d 13–22 of pregnancy. We have previously shown that this metyrapone treatment enhances both fetal and placental growth (8, 19).

Surgical procedures
Surgical preparation of rats for constant-infusion studies was performed as previously described by Waddell and Bruce (20). Briefly, each rat was anesthetized with sodium pentobarbitone (40 mg/kg, ip) and cannulas were positioned in the trachea to assist breathing, in the left common carotid artery for blood sampling, and in the jugular vein for administration of 125I-leptin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Materials and Methods
Results
 Discussion
 References
 
Following a stabilization period after surgery, the MCR and placental and fetal distributions of 125I-leptin were determined during constant infusion of 125I-leptin. Rats initially received a priming dose of 125I-leptin (1.0 x 106 cpm in 0.5 ml saline containing 0.1% BSA) via the jugular cannula, and this was followed immediately by a constant infusion of approximately 10,000 cpm/min 125I-leptin in the same vehicle (infusion rate, 50 µl/min) via an infusion pump (Batterie-Injectomat, Fresenius AG, Homburg, Germany) for 100 min. The exact infusion rate (counts per minute/minute) was determined for each rat at the end of the infusion by collecting three 1-min samples from the cannula tip. Six arterial blood samples (0.2 ml) were obtained via the carotid cannula into heparinized capillary tubes at 10, 20, 40, 60, 80, and 100 min of infusion. Samples were centrifuged, and plasma was stored at -20 C until subsequent analysis. Immediately after the final blood sample, placentas and fetuses were removed from pregnant rats, weighed, and then snap-frozen on liquid nitrogen. Fetal blood samples were also obtained from pregnant rats at d 22 by decapitation (blood from 4 fetuses from each mother was pooled to provide sufficient volume) and centrifuged, and plasma was stored at -20 C.

Analysis of blood and tissue samples
Estimates of intact 125I-leptin in blood and tissue samples were made as previously described by Banks et al. (21). Briefly, 450 µl of 10% trichloroacetic acid was added to 50 µl of plasma, and the mixture was vortexed and incubated for 30 min at 4 C. Precipitated proteins were pelleted by centrifugation (3600 x g, 4 C) for 20 min, and 125I radioactivity (indicative of intact 125I-leptin) was determined in a {gamma} counter (1282 CompuGamma, LKB-Wallac, Inc., Stockholm, Sweden). Frozen tissues were weighed and homogenized in an equal volume (wt/vol) of 0.1 M PBS, 10 mM HEPES, and 0.1% BSA. The homogenate was centrifuged at 3600 x g at 4 C for 20 min, and the supernatant was processed as described above for blood samples to determine intact 125I-leptin concentration.

Western blot analysis
Western blot analysis of placental Ob-RS was performed on additional groups of animals as described by Smith and Waddell (8). Portions of placental basal and labyrinth tissue were homogenized and centrifuged at 11,000 x g for 30 min. Supernatant protein concentration was determined (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories, Inc., Richmond, CA), and 100 µg was resolved by SDS-PAGE (7% separating) and transferred to nitrocellulose membrane (Hybond C-Super, Amersham Pharmacia Biotech, Sydney, Australia). Consistency of protein loading and transfer among samples was confirmed by Ponceau S dye staining (ICN Biomedicals, Inc., Aurora, OH). Membranes were incubated for 1 h in blocking solution containing 5% nonfat milk, then overnight at 4 C with the leptin receptor antibody (diluted 1:2000; Affinity BioReagents, Inc., Golden, CO). Preliminary analysis showed that this antibody detects both the long form (Ob-Rb, 120 kDa) and a shorter form (100 kDa) of the leptin receptor in the rat placenta, although the Ob-Rb signal is relatively weak. This 100-kDa form likely includes Ob-Ra because mRNA encoding Ob-Ra is expressed at relatively high levels in placenta (8); in addition, this band may also reflect placental expression of the other short Ob-R isoforms, Ob-Rc, Ob-Rd, and Ob-Rf (16). Immunoreactive bands were identified by incubation with horseradish peroxidase conjugate goat antirabbit secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; diluted 1:5000), and signals were visualized using a chemiluminescence detection kit (SuperSignal Substrate, Pierce Chemical Co., Rockford, IL). The resultant autoradiographs were quantified by densitometry using Scion Image analysis software (Release Beta 3b).

Data analysis
The MCR of leptin was calculated from the steady state plasma concentration of 125I-leptin during constant infusion as: MCR (milliliters/minute) = infusion rate (counts per minute/minute)/steady state plasma concentration (counts per minute/milliliter) (Ref. 22). The onset of steady state, a prerequisite for MCR calculation, was defined as the sample time after which no significant change in radiolabeled hormone concentration was observed, assessed within each group by repeated-measures ANOVA and subsequent least significant difference (LSD) test (20, 23). This occurred by the 80-min sample in the d 16 pregnant and d 22 carbenoxolone-treated rats, the 60-min sample in metyrapone-treated rats, and the 40-min sample in the remaining groups (Fig. 1Go). The concentration of 125I-leptin in placentas, whole fetuses, and fetal blood was expressed relative to the steady state concentration in maternal plasma. All data are expressed as the mean ± SEM, where one fetus/placenta was obtained from a minimum of three mothers per group. One-way ANOVAs were used to assess variation in MCR, tissue retention of 125I-leptin, and maternal, fetal, and placental weights. Two-way ANOVAs were used to determine variation in Ob-RS with gestational age and placental zone. Where the F test for the ANOVA reached statistical significance (P < 0.05), differences among specific means were assessed by LSD test (24). Differences in placental and fetal tissue retention of 125I-leptin between d 16 and 22 of pregnancy were assessed by unpaired t tests.



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  		Figure 1. Plasma concentration of 125I-leptin during its constant infusion for measurement of the MCR of leptin in nonpregnant rats. Values are the mean ± SEM (n = 4 per group). A priming dose of 125I-leptin was administered via a jugular cannula just before the infusion to reduce the time required to reach steady state. Repeated-measures ANOVA was used to determine the onset of steady state (i.e. the time after which no significant change in plasma 125I-leptin concentration occurred). Values without common notations (a–c) differ significantly (P < 0.05; LSD test), and so the onset of steady state in this group was 40 min.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Materials and Methods
 Results
Discussion
 References
 
Effect of pregnancy on the MCR of leptin
The MCR of leptin increased from 0.74 ± 0.02 ml/min in nonpregnant rats to 0.94 ± 0.04 ml/min by d 22 of pregnancy (22% increase; P < 0.05; Fig. 2AGo), but when account was taken of maternal weight, the MCR of leptin fell from 3.08 ± 0.23 ml/min per kg in nonpregnant rats to 2.36 ± 0.13 ml/min per kg at d 22 of pregnancy (23% decrease; P < 0.05; Fig. 2BGo).



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  		Figure 2. MCR of leptin in rats before (NP) and at d 16 and 22 of pregnancy. Values are expressed as milliliters/minute (A) and milliliters/minute per kilogram (B) and are the mean ± SEM (n = 3–5 per group). There was significant variation among groups (P < 0.05; one-way ANOVA), and values without common notations (a, b) differ significantly (P < 0.05; LSD test).

 
Effect of gestational age on the distribution of 125I-leptin to the placenta and fetus
The steady state concentration of 125I-leptin in the fetus (relative to that in maternal plasma) after 100 min of 125I-leptin constant infusion increased dramatically (10-fold; P < 0.005) from d 16 to d 22 of pregnancy (Fig. 3AGo). The placental concentration of 125I-leptin also increased over the same period (P < 0.005; Fig. 3BGo), but the magnitude of this increase (42%) was far less than that observed in the fetus.



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  		Figure 3. Steady state concentration of 125I-leptin in the fetus (A) and placenta (B) after its constant infusion for 100 min. Values are expressed relative to the concentration of 125I-leptin in maternal plasma at steady state and are the mean ± SEM (n = 3–5 per group). *, P < 0.05; **, P < 0.005 compared with value at d 16 (unpaired t test).

 
Effect of glucocorticoid manipulations on fetal and placental growth and transplacental passage of 125I-leptin
Litter size was similar among all experimental groups (P = 0.66; data not shown), but fetal and placental weights were significantly reduced by dexamethasone (33% and 36%, respectively; P < 0.001) and carbenoxolone treatments (9% and 15%, respectively; P < 0.01). In contrast, metyrapone treatment increased both fetal and placental weight (6% and 14%; P < 0.05; Table 1Go). Treatment of mothers with dexamethasone reduced the relative concentration of 125I-leptin in the fetus (77% decline; P < 0.01; Fig. 4AGo) and in fetal blood (53% decline; P < 0.05; Fig. 4BGo); although a similar trend was observed for 125I-leptin in the placenta (32% lower), this did not reach statistical significance (Fig. 4CGo). In contrast, an increase in fetal 125I-leptin was observed after maternal treatment with metyrapone (55% higher; P < 0.05; Fig. 4AGo). Carbenoxolone treatment had no effect on the relative concentration of 125I-leptin in the placenta, fetus, or fetal blood (Fig. 4Go). Maternal treatment with dexamethasone, carbenoxolone, or metyrapone had no effect on the MCR of leptin (Table 1Go).


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  		Table 1. Placental and fetal weights and MCR of leptin at d 22 of pregnancy in untreated rats and after maternal treatment with dexamethasone, carbenoxolone, or metyrapone from d 13

 


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  		Figure 4. Steady state concentration of 125I-leptin in the fetus (A), fetal blood (B), and placenta (C) after its constant infusion for 100 min at d 22 of pregnancy after no treatment (Con) and after maternal treatment with dexamethasone (Dex), carbenoxolone (CBX), or metyrapone (Met) from d 13–22. Values are expressed relative to the concentration of 125I-leptin in maternal plasma at steady state and are the mean ± SEM (n = 3–5 per group). The concentration of 125I-leptin in both fetus and fetal blood differed with treatment (P < 0.001 and P < 0.05, one-way ANOVAs, respectively), and values without common notations (a–c) differ significantly (P < 0.05; LSD test).

 
Placental expression of Ob-RS protein
Western analysis of Ob-RS in the basal and labyrinth zones of the placenta demonstrated a clear immunoreactive band at the expected size of approximately 100 kDa (Fig. 5AGo). The intensity of this band in the labyrinth zone increased (P < 0.01) by almost 2-fold between d 16 and 22 and was further enhanced by maternal treatment with metyrapone (2.6-fold higher than d 22 control; P < 0.05); dexamethasone and carbenoxolone treatments were without effect in the two placental zones (Fig. 5BGo).



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  		Figure 5. Western blot analysis of Ob-RS (100 kDa) protein expression in the basal (Bas) and labyrinth (Lab) zones of rat placenta at d 16 and d 22 of pregnancy (A) and at d 22 of pregnancy after no treatment (Con) and after maternal treatment with dexamethasone (Dex), carbenoxolone (CBX), or metyrapone (Met) (B). Values are expressed in arbitrary density units and are the mean ± SEM (n = 4–9 per group). A, Ob-RS protein varied with gestational age (P < 0.05) and placental zone (P < 0.001), and there was significant interaction between these sources of variation (P < 0.05; two-way ANOVA). Values without common notations (a and b) differ significantly (P < 0.05; LSD test). B, Ob-RS protein varied with treatment (P < 0.01) and placental zone (P < 0.01). All labyrinth zone values exceeded basal zone values in corresponding group; for labyrinth zone, values without common notations (a and b) differ significantly (P < 0.05; LSD test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Materials and Methods
 Results
 Discussion
References
 
This study investigated the distribution and metabolism of leptin in the pregnant rat, with particular emphasis on transplacental passage of leptin from mother to fetus. The major findings were that passage of maternal leptin to the fetus increased by around 10-fold over the final third of pregnancy, and this was associated with increased expression of Ob-RS protein in the placental labyrinth zone, the site of maternal-fetal exchange. The increase in transplacental leptin passage was almost abolished in association with glucocorticoid-induced fetal-placental growth retardation. In contrast, a reduction in endogenous glucocorticoid synthesis by metyrapone treatment enhanced both fetal-placental growth and transplacental passage of leptin from mother to fetus. These data show that the rise in fetal leptin over late gestation is due, in part, to increased transplacental passage of maternal leptin and that glucocorticoids suppress this transport of leptin.

The increased distribution of maternal 125I-leptin to the fetus (10-fold) between d 16 and 22 of pregnancy indicates that transplacental passage of leptin is greatly enhanced over this period. This change is consistent with our observation that expression of Ob-RS protein (100 kDa) in the placental labyrinth zone also increased markedly over this period. This increase in Ob-RS protein is comparable to our recent observation of a rise in mRNA expression for one of the short isoforms of the leptin receptor, Ob-Ra, in the labyrinth zone in late pregnancy (8). The Ob-Ra isoform has been shown to promote leptin transport across physiological barriers, including brain microvessels in vivo (14, 25) and Madin-Darby canine kidney epithelial cells in vitro (15). The Ob-RS detected by Western analysis may also include other short isoforms of the leptin receptor, most notably Ob-Rc, which has also been linked to leptin transport (25). In addition, it has been suggested that the soluble form of the leptin receptor, Ob-Re, may serve as a transport vehicle for leptin across the placenta (26), and we recently demonstrated that mRNA encoding this isoform also increases in the placenta from d 16–22 of pregnancy (8). The distribution of 125I-leptin also increased in the placenta over this period, albeit to a lesser extent than in the fetus, consistent with increased uptake via Ob-RS and/or the soluble receptor in the labyrinth zone. In addition to this greater capacity for specific leptin transport via placental expression of leptin receptor, it is possible that increases in placental blood flow (27) and surface area for maternal-fetal exchange (28) or a nonspecific reduction in the placental barrier (i.e. increased leakiness) also contribute to enhanced passage of maternal leptin to the fetus in late pregnancy.

The enhanced transport of maternal leptin to the late gestation fetus is consistent with the proposed role of leptin as an important fetal growth factor (5, 7). Thus, fetal leptin concentrations are positively correlated with fetal weight (5), and this effect appears to be independent of the IGFs (29). Leptin may promote fetal growth directly because leptin receptor expression has been identified in several fetal tissues, including brain, bone, cartilage, heart, liver, and testis (30, 31, 32, 33). Leptin may be particularly important in the central nervous system, because decreased brain weight and impaired expression of synaptic and glial proteins have been observed in both ob/ob and db/db mice (34). The specific growth-promoting actions of leptin within fetal tissues potentially involve both mitogenic (35) and angiogenic (36, 37) stimuli.

Consistent with a role for leptin in promoting fetal growth, dexamethasone-induced fetal growth retardation was associated with reduced passage of maternal leptin to the fetus. Such an effect of dexamethasone on placental leptin transport was suggested by previous reports showing opposite effects of dexamethasone on maternal and fetal plasma leptin concentrations (8, 38). Specifically, whereas maternal dexamethasone treatment (as used in the present study) increased maternal plasma leptin levels by almost 3-fold, fetal leptin was reduced by around 80% (8). Interestingly, the 11ß-HSD inhibitor carbenoxolone did not affect placental leptin transport at d 22 despite clear effects on fetal growth; this apparent inconsistency likely reflects the normal loss of 11ß-HSD2 expression in the placental labyrinth zone late in pregnancy (39, 40). Thus, it is possible that carbenoxolone did compromise transplacental leptin passage, but only before this loss of 11ß-HSD2 expression. In contrast to the effects of dexamethasone, suppression of endogenous glucocorticoid synthesis by maternal treatment with metyrapone increased fetal and placental growth and enhanced transplacental leptin passage. This effect of metyrapone was also associated with increased expression of Ob-Ra mRNA (8) and Ob-RS protein (present study) in the placental labyrinth zone, suggesting that placental Ob-Ra expression and fetal growth are normally restrained by physiological levels of glucocorticoids. Interestingly, although dexamethasone did not reduce placental Ob-Ra mRNA (8) or Ob-RS protein expression (present study), it did suppress placental expression of Ob-Rb mRNA and protein, the long form of the leptin receptor (8). This suggests that placental leptin transport may theoretically involve interaction between Ob-Rb and the shorter isoforms as has been demonstrated in vitro (41).

Although the absolute MCR of leptin (milliliters/minute) was around 20% higher in d 22 pregnant rats compared with nonpregnant rats, the MCR of leptin relative to maternal body weight (milliliters/minute per kilogram) fell significantly over pregnancy. Because cardiac output increases by around 50% during rat pregnancy (42), the associated increase in tissue perfusion would be expected to increase the MCR of leptin in parallel with body weight. The reason such an increase was not evident in the present study may reflect the rise in plasma leptin binding activity observed after midpregnancy (17). This plasma binding activity appears to be due to placental expression and secretion of the soluble form of the leptin receptor Ob-Re (8) which may restrict transport of plasma leptin to extravascular tissues, similar to the effects of binding proteins on transport of peptides such as CRH (43) and the IGFs (44). Although the relative MCR of leptin fell by late gestation, this cannot totally account for the approximately 2-fold increase in plasma leptin levels observed by midpregnancy (10, 17). Thus, our data support the suggestion that leptin production rate increases during pregnancy, consistent with a rise in leptin mRNA expression in adipose tissue observed by d 18 (10, 45).

In conclusion, this study shows that transplacental passage of maternal leptin to the fetus increases markedly over late gestation in association with increased expression of Ob-RS in the placental labyrinth zone. Changes in fetal growth induced by glucocorticoid manipulations were associated with parallel shifts in placental leptin transport, consistent with the proposed role of leptin as a fetal growth factor.


   Acknowledgments
 
We thank Mrs. Margaret Blackberry for valuable assistance with the iodination of leptin.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia (Project Grants 139104 and 254576). J.T.S. was the recipient of an Australian Postgraduate Research Award.

Abbreviations: 11ß-HSD, 11ß-Hydroxysteroid dehydrogenase; LSD, least significant difference; MCR, metabolic clearance rate.

Received January 29, 2003.

Accepted for publication March 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Materials and Methods
 Results
 Discussion
 References
 

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