Glucocorticoid Bioactivity Research Articles

GR CALUX assay detects synthetic glucocorticoids in calf urine: a validation study

ABSTRACTMember States of the EU are required to monitor the use of pharmacologically active substances in food-producing animals. There is evidence, however, that the target-based approach currently applied in official monitoring plans might under-estimate the real incidence of growth promoter abuse in livestock. As demonstrated for sex hormones, the association of effect-oriented biological screening with chemical confirmatory techniques could be the best strategy in revealing the abuse of veterinary drugs. Here we demonstrate the reliability of a cell-based assay to screen calf urine samples for synthetic glucocorticoids. The validation included the most widely used synthetic drugs (flumethasone, dexamethasone, betamethasone, methylprednisolone and prednisolone) and was developed according to the Commission Decision 2002/657/EC, thus including the verification of cut-off level, the β error, the specificity, ruggedness and stability. The study was carried out using prednisolone as representative substance at 5 ng mL−1 concentration. All blank and spiked urine fulfilled the EU criteria, moreover the method resulted in being specific and sound, and the analytes in urine were stable for at least 30 days. The assay results indicated its suitability for a qualitative analysis of calf urine samples. This method enabled the detection of low doses of synthetic glucocorticoids (GCs) in matrix (<2 ng mL−1 for flumethasone, dexamethasone, betamethasone; < 4 ng mL−1 for methylprednisolone; 5 ng mL−1 for prednisolone), with the possibility of detecting new or unknown molecules and cumulative effects of low-level mixtures with glucocorticoid bioactivity.

Balancing the Budget: Accounting for Glucocorticoid Bioactivity and Fate during Water Treatment.

Numerous studies have identified the presence and bioactivity of glucocorticoid receptor (GR) active substances in water; however, the identification and activity-balance of GR compounds remained elusive. This study determined the occurrence and attenuation of GR bioactivity and closed the balance by determining those substances responsible. The observed in vitro GR activity ranged from 39 to 155 ng dexamethasone-equivalent/L (ng Dex-EQ/L) in the secondary effluents of four wastewater treatment plants. Monochromatic ultraviolet light of 80 mJ/cm(2) disinfection dose was efficient for GR activity photolysis, whereas chlorination could not appreciably attenuate the observed GR activity. Ozonation was effective only at relatively high dose (ozone/TOC 1:1). Microfiltration membranes were not efficient for GR activity attenuation; however, reverse osmosis removed GR activity to levels below the limits of detection. A high-sensitivity liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was then developed to screen 27 GR agonists. Twelve were identified and quantified in effluents at summed concentrations of 9.6-21.2 ng/L. The summed Dex-EQ of individual compounds based on their measured concentrations was in excellent agreement with the Dex-EQ obtained from bioassay, which demonstrated that the detected glucocorticoids can entirely explain the observed GR bioactivity. Four synthetic glucocorticoids (triamcinolone acetonide, fluocinolone acetonide, clobetasol propionate, and fluticasone propionate) predominantly accounted for GR activity. These data represent the first known publication where a complete activity balance has been determined for GR agonists in an aquatic environment.

Adrenocortical Hormonal Activity in 20-Year-Old Subjects Born Small or Appropriate for Gestational Age

Background: Altered adrenocortical activity is one suggested mechanism relating small birth size with the metabolic syndrome in adulthood. Adrenal androgen concentrations are higher in children born small (SGA) than appropriate for gestational age (AGA). Aim: To compare adrenocortical hormonal activity between 20-year-old subjects born SGA or AGA. Methods: Seventy 20-year-old subjects (35 SGA and 35 age- and gender-matched AGA controls) were studied. Serum cortisol, cortisone, corticosteroid-binding globulin (CBG), glucocorticoid bioactivity (GBA), aldosterone, dehydroepiandrosterone sulfate (DHEAS) and androstenedione were measured, and the free cortisol index (FCI) was calculated. Results: The mean levels of glucocorticoid parameters, aldosterone, DHEAS or androstenedione did not differ between the SGA and AGA groups. In both groups, the males had lower cortisol (p < 0.05) and CBG levels (p < 0.01) and higher DHEAS (p < 0.01) concentrations than the females. Females who used hormonal contraceptives had higher cortisol and CBG levels (p < 0.01) but similar FCI, GBA and DHEAS levels than females who did not use contraceptives. Conclusion: No differences in adrenocortical activity were found between 20-year-old SGA and AGA subjects. Enhanced peripubertal adrenal androgen secretion seems to disappear by early adulthood in full-term born SGA subjects. FCI and GBA are useful parameters in the evaluation of the glucocorticoid milieu during hormonal contraceptive use.

Abdominal Fat Accumulation in Adults Born Preterm Exposed Antenatally to Maternal Glucocorticoid Treatment Is Dependent on Glucocorticoid Receptor Gene Variation

Preterm birth is associated with short stature, abdominal adiposity, insulin resistance, and hypertension, resembling effects of increased glucocorticoid bioactivity. Although antenatal glucocorticoid treatment does not substantially contribute to these associations, it is unknown whether genetic variants in the glucocorticoid receptor gene could modulate the effects of antenatal glucocorticoid treatment on the above phenotype. Our objective was to test the effects of the R23K and N363S variants, associated with decreased and increased sensitivity to cortisol, respectively, on the metabolic profile in adults born preterm of whom some had been treated with glucocorticoids antenatally and/or in the early postnatal phase. This was a prospective follow-up study that included 263 19-year-olds born at a gestational age under 32 wk from the Dutch Project on Preterm and Small-for-Gestational-Age Infants cohort. This was a nationwide multicenter follow-up study. Adult height and body composition, fasting serum glucose, insulin and cholesterol levels, and blood pressure were evaluated. At 19 yr of age, waist circumference was 1.67 ± 0.90 sd score in 363S carriers who had been treated antenatally with glucocorticoids (n = 4), which was much higher than that of the other groups (P for interaction = 0.03). A similar association was found for the waist-to-hip ratio sd score (P = 0.03). Similar associations were absent with the R23K polymorphism. There was no interaction between these genotypes and postnatal glucocorticoid treatment on serum levels of glucose, insulin, and cholesterol or blood pressure. In prematurely born individuals carrying the 363S variant, antenatal glucocorticoid treatment predisposes to abdominal adiposity at age 19 yr.

Serum regulates cortisol bioactivity by corticosteroid‐binding globulin‐dependent and independent mechanisms, as revealed by combined bioassay and physicochemical assay approaches

Corticosteroid-binding globulin (CBG) is the principal carrier of natural glucocorticoids in the circulation, and we hypothesized that it modulates glucocorticoid bioactivity (GBA). Alterations in CBG, the presence of noncortisol, naturally occurring glucocorticoids and the use of potent, synthetic glucocorticoids, all make it difficult to assess adrenal activity in-vivo; these problems can be addressed by a glucocorticoid bioassay. A bioassay was developed for serum GBA and a physicochemical ultrafiltration-liquid chromatography-tandem mass spectrometry assay for free serum cortisol (FreeF). We studied individuals homozygous and heterozygous for a nonfunctioning CBG variant (CBG G237V) and healthy controls. FreeF concentrations were similar in healthy controls, and those with absent functional CBG, but surprisingly we found low GBA in CBG null individuals. This may suggest that CBG delivers cortisol to target cells. However, further experiments revealed that dilution of serum in the bioassay caused release of cortisol from CBG, resulting in elevated GBA measurements in all but the CBG G237V homozygotes. Furthermore, we identified a specific and potent inhibitory effect of high concentration serum on glucocorticoid sensitivity of the recipient cells used in the bioassay. Analysis of inflammatory synovial fluid, a filtrate of serum with lower CBG concentration, revealed elevated free cortisol compared to noninflammatory synovial fluid, a change not attributable to interconversion between cortisol and cortisone. Our findings reveal that dilution of CBG enhances cortisol release, and so bioactivity, and also that serum potently induces glucocorticoid resistance in target cells.

Intra-articular corticoid injection induces circulating glucocorticoid bioactivity and systemic immune activation in juvenile idiopathic arthritis

Objective: To study the systemic effects of intra-articular (IA) glucocorticoid (GC) injections in juvenile idiopathic arthritis (JIA).Methods: The study group comprised 21 JIA patients being treated with IA methylprednisolone [MP (n = 15) or MP plus triamcinolone hexacetonide (THA) (n = 6)] prescribed on clinical indications. The systemic effect of MP was assessed by measuring circulating glucocorticoid bioactivity (GBA) with a recombinant cell transactivation assay 7 and 24 h after the IA injections, and after 2 months. The systemic immunological responses were studied with a novel assay for testing patient serum-induced changes in the secretion of interferon (IFN)-γ and interleukin (IL)-5 from target cells.Results: Administration of IA GC induced serum GBA (p = 0.001) and suppressed circulating cortisol levels (p = 0.002) 7 h after the injection. Serum withdrawn 24 h after the IA injection induced less IL-5 secretion from mitogen-activated target cells when compared with pre-treatment sera (p = 0.036). This decrease in target cell T helper (Th)2 response (IL-5) was MP dose related (r = −0.550, p = 0.018). High IL-5 secretion from target cells prior to the IA injections was associated with good clinical outcome at 2 months, seen as a low number of active (p = 0.044) and restricted joints (p = 0.049).Conclusion: IA GC injections have systemic effects that are reflected in the serum as an immediate elevation of GBA, a decrease of endogenous cortisol as well as a suppressive effect of patient serum on target cell IL-5 secretion. These systemic effects may play a role in the attenuation of disease activity.

Establishing a sensitive and specific assay for determination of glucocorticoid bioactivity

Glucocorticoids are hormones that play a major role in energy homeostasis and stress response of the body. As drugs they are most frequently used for immunosuppressive and anti-inflammatory purposes. Glucocorticoids are exploited successfully in the treatment of a wide variety of diseases; however, some patients develop side-effects, while others fail to respond to this form of therapy. Alterations in pharmacodynamic and pharmacokinetic actions might contribute to individual differences in glucocorticoid sensitivity. Antibody-based methods such as RIA (Radioimmunoassay) and ELISA (Enzyme-linked immunosorbent assay) are routinely used to determine glucocorticoid serum levels. However, as these techniques measure the total amount of a specific glucocorticoid and do not discriminate between protein-bound and freely available (i.e. biologically active) glucocorticoids, the results do not necessarily reflect the active levels of glucocorticoid, i.e. the "glucocorticoid milieu" in a patient. Being able to determine glucocorticoid bioactivity in serum or other body fluids could help identifying glucocorticoid-sensitive or -resistant patients and help finding explanations for different responses in individual patients. For this reason, we established a glucocorticoid bioactivity assay that is based on the measurement of glucocorticoid-dependent reporter gene activity. Making use of a human T-cell leukemia line, equipped with the glucocorticoid receptor and the fluorescence protein Venus as the assay's reporter (Jurkat(GR)-MMTV-VNP), glucocorticoid bioactivity can be determined from small amounts of serum or other biologic fluids. The developed glucocorticoid bioassay is both sensitive and reproducible, without any relevant cross-reactivity with steroid hormones other than glucocorticoids and can be practically applied in daily laboratory routine.

Serum glucocorticoids and adiponectin associate with insulin resistance in children born small for gestational age

Altered glucocorticoid activity is one possible mechanism linking fetal growth restriction with later insulin resistance (IR) and type 2 diabetes. We aimed to investigate whether serum glucocorticoid parameters are related to IR in children born small for gestational age (SGA). A total of 110 children (55 age- and gender-matched pairs born SGA or appropriate for gestational age (AGA) in a case-control setting) were studied at the mean age of 12.2 (s.d. 0.2) years. Serum cortisol, corticosteroid-binding globulin (CBG), free cortisol index (FCI=cortisol/CBG), and glucocorticoid bioactivity (GBA, transactivation assay) were analyzed and related to serum adiponectin and insulin-like growth factor-binding protein 1 (IGFBP1) concentrations and homeostasis model assessment for IR (HOMA-IR) and QUICKI indices. In the pooled study population, GBA correlated well with cortisol and FCI (r=0.681 and 0.586 respectively; P<0.001 for both). Serum cortisol, CBG, FCI, GBA, HOMA-IR, or QUICKI did not differ between the SGA and AGA subjects, but the SGA children had lower body mass index (P=0.005) and waist circumference (WC) (P=0.001). The mean GBA in the highest GBA quartile was higher among the SGA subjects than among the AGA subjects (138.6 vs 96.4 nmol/l cortisol equivalents, P<0.001). In the SGA children, GBA correlated positively with HOMA-IR (r=0.522, P<0.001) and inversely with adiponectin (r=-0.278, P=0.042) (WC/height ratio adjustments), and in logistic regression analysis, higher GBA (odds ratio (OR) 1.3; P=0.013), lower adiponectin (OR 1.4; P=0.038), and lower IGFBP1 (OR 1.9; P=0.010) associated independently with higher HOMA-IR. These findings suggest that increased glucocorticoid activity and low serum adiponectin concentrations associate with IR in SGA children.

199: Glucocorticoid bioactivity in syncytiotrophoblast in relation to maternal obesity

199: Glucocorticoid bioactivity in syncytiotrophoblast in relation to maternal obesity

Glucocorticoid bioactivity does not predict response to steroid therapy in severe pediatric ulcerative colitis

The pathophysiological basis for corticosteroid (CS) failure in ulcerative colitis (UC) is unknown. A transactivation glucocorticoid bioassay (GBA) was developed to measure the biological activity of CS by quantifying glucocorticoid response elements. This approach eliminates differences in bioavailability, chemistry, affinity, and other potential differences between the various steroids regarding their ability to activate the glucocorticoid receptor. In this multicenter prospective study, we aimed to evaluate whether CS bioavailability plays a role in CS refractoriness in severe pediatric UC. GBA (using COS-1 transfected cells) was measured in the serum of 50 children (52% males, age 13.4 +/- 3.5 years) admitted for acute severe UC on the third day of CS treatment. Demographic, clinical, and laboratory data were prospectively recorded. Of the children enrolled, 16 (32%) failed CS therapy and required infliximab (n = 14) or colectomy (n = 2) within a median of 10 days (interquartile range [IQR] 6.5-14.5). Reflecting internal validity of the assay, GBA was highly correlated with the last CS dose and the time interval to bloodletting (r = -0.41 and r = -0.54, respectively; P < 0.001). There was no statistically significant difference in the GBA levels between responders and nonresponders (249 nM versus 200 nM cortisol equivalent, P = 0.18). In a multivariate regression model adjusted for time elapsed from CS and the administered dose, GBA did not predict response to CS (P = 0.34). The lack of correlation of GBA level and treatment outcome lends support to the hypothesis that the bioavailability, type, and dosing of intravenous CS are not associated with response or failure to the drug.

Circulating Adiponectin as a Marker for Glucocorticoid‐related Side Effects in Children and Adolescents With Inflammatory Bowel Disease

Glucocorticoid therapy is widely used in clinical practice. However, treatment with steroids carries the risk of side effects. We investigated changes in serum adiponectin and leptin in response to systemic glucocorticoid treatment in 18 pediatric patients with inflammatory bowel disease and contrasted these findings to circulating glucocorticoid bioactivity measured with a mammalian cell bioassay. Interestingly, serum adiponectin levels at 2 to 4 weeks of therapy were significantly higher in patients who experienced acute glucocorticoid-related side effects (22.9 +/- 2.6 microg/mL, n = 7) than in those who did not (16.0 +/- 2.1 microg/mL, n = 11, P < 0.05). Serum leptin was not associated with side effects. Circulating adiponectin may serve as an early and readily available endogenous biomarker for acute glucocorticoid-related side effects in pediatric patients.

Circulating Glucocorticoid Bioactivity During Peroral Glucocorticoid Treatment in Children and Adolescents With Inflammatory Bowel Disease

Our objective was to investigate the changes in circulating glucocorticoid bioactivity (GBA) at the onset of systemic glucocorticoid therapy in pediatric patients with inflammatory bowel disease. Prednisolone (1 mg/kg/d) or budesonide (9 mg/d) was introduced as a single daily dose, and the patients (n=22) were subsequently followed up at 2 to 4 week intervals. The limit for a raised value of serum GBA was defined in pediatric patients (mean+2 SD; 118 nM cortisol equivalents; n=142). Two weeks of prednisolone brought about an increase in serum GBA from 84+/-14 to 336+/-38 nM cortisol equivalents (mean+/-SE; P<0.001). Young patients (<10 y) had similar GBA values to older patients, even though their prednisolone dose was higher (1.3 vs. 0.79 mg/kg; P<0.05). Patients treated with budesonide displayed a minor increase in GBA (151+/-20 vs. 267+/-21 nM cortisol equivalents after 4 wk of treatment; P<0.05; n=3), and when switched to prednisolone (n=2), their GBA level increased 3-fold. GBA levels did not predict the development of glucocorticoid-related side effects. Prednisolone doses used in the treatment of pediatric inflammatory bowel disease patients elicit a 4-fold increase in serum GBA that is significantly higher than the increase induced by budesonide. The GBA measurement is an additional tool for assessing steroid therapy at an individual level during systemic glucocorticoid treatment.

Detection of glucocorticoid bioactivity in bovine urine samples using a reporter gene assay

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of low-level cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems. We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC 50 of 0.79 ng mL −1 for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay. Cross-reactivity data for a range of 11β-hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques.

Circulating glucocorticoid bioactivity and serum cortisol concentrations in premature infants: the influence of exogenous glucocorticoids and clinical factors

Glucocorticoids are widely used before preterm delivery and in preterm infants may bear serious adverse effects. Better knowledge about the circulating glucocorticoid milieu after glucocorticoid treatment could improve treatment modalities. Therefore, we investigated the influence of exogenous glucocorticoids and clinical factors on serum cortisol (F) levels and circulating glucocorticoid bioactivity (GBA) in preterm infants. Eighty-nine infants (gestational age (GA) 23.6-33.1 weeks at birth) were enrolled in a prospective cohort study in two tertiary neonatal centres. Cord, day of birth (D0), fourth day (D4) and 36 weeks postmenstrual age serum F and GBA levels were measured. The cord GBA was 5.8-fold and D0 GBA 2.3-fold higher in the infants exposed to antenatal steroids within 12 h before birth when compared with those unexposed or exposed >7 days before birth (95% CI 3.8-8.6; P<0.0001, and 1.8-3.0; P<0.0001 respectively). In the infants treated with early postnatal dexamethasone, D4 GBA was 1.7-fold (1.3-2.2; P<0.0005) higher when compared with levels in the infants without this treatment. Clinical factors indicating perinatal distress, such as Apgar scores <7 and low GA, were associated with higher cord, D0 and D4 serum F levels. Both ante- and postnatally administered glucocorticoids increase circulating GBA not attributable to endogenous F. Perinatal distress and preceding glucocorticoid treatment need to be taken into account when circulating glucocorticoid milieu is evaluated in preterm infants. The GBA assay may prove to be a useful instrument in the development of new glucocorticoid treatment strategies.

High Circulating Glucocorticoid Bioactivity Predicts a Steroid‐dependent Disease in Children with IBD

High Circulating Glucocorticoid Bioactivity Predicts a Steroid‐dependent Disease in Children with IBD

Methylprednisolone Exposure in Pediatric Renal Transplant Patients

Glucocorticoid (GC) dosing is commonly based on body mass or surface area in children, although the drug effects appear to correlate with steroid exposure, rather than dose. We compared the area under the serum concentration-time curve (AUC) of methylprednisolone (MP) with a recombinant cell bioassay measuring serum glucocorticoid bioactivity (GBA), in prediction of side effects in 16 pediatric patients (5.4-18.4 years of age) 2.0-14.9 years after renal transplantation (TX). They received 0.3 mg/kg of MP orally and timed blood samples were drawn up to 8 h postdose. Serum MP concentrations correlated moderately with GBA (r= 0.65, p < 0.0001) with best linear fit at 6 and 8 h (r= 0.72, 0.79, respectively, p < 0.001). MP-AUC(t = 0-8) and GBA(t = 6) were significantly greater in patients who gained excessive weight soon after TX. Change in growth after TX was inversely correlated with MP-AUC (r= 0.73, p < 0.05) and GBA(t = 6) (r= 0.62, p < 0.05). No correlation of MP-AUC or GBA was found with blood glucose or serum lipid concentrations, glomerular filtration rate, bone mineral density or graft histology. In conclusion, GC exposure varies individually and dosing should be adjusted accordingly to control the adverse effects. GBA might provide a complementary tool for monitoring GC exposure but further studies are needed.

Transgenic over-expression of macrophage migration inhibitory factor renders mice markedly more susceptible to experimental colitis

Enhanced production of macrophage migration inhibitory factor (MIF) is recognized in patients with inflammatory bowel disease (IBD) and mice with experimental colitis; however, the precise molecular function of MIF in colitis is not fully understood. To further investigate this matter, we examined the pathological features of MIF transgenic mice with dextran sulphate sodium (DSS)-induced colitis. We generated transgenic mice carrying a murine MIF cDNA driven by a cytomegalovirus enhancer and a beta-actin/beta-globin promoter. Mice were orally administered 1-4% DSS in drinking water for 7 days. Clinical disease activity, survival and histological features were evaluated. The level of myeloperoxidase (MPO) activity in the colon tissue was measured to assess neutrophil infiltration. The level of corticosterone in the serum was measured by enzyme linked-immunosorbent assay (ELISA). MIF mRNA and protein were markedly up-regulated in the colon and serum obtained from MIF transgenic mice. The severity of the colitis induced by 1% DSS treatment was markedly higher in MIF transgenic mice than in wild-type mice. We also found that MPO activity was significantly higher in MIF transgenic mice than wild-type mice in response to DSS stimulation. Interestingly, the corticosterone level remained unchanged in MIF transgenic mice. MIF enhances DSS-induced colitis, in part via neutrophil accumulation and inhibition of glucocorticoid bioactivity.

Late follicular phase administration of mifepristone suppresses circulating leptin and FSH – mechanism(s) of action in emergency contraception?

Low dose mifepristone (RU486) is highly effective in emergency post-coital contraception (EC), although the mechanism(s) of action remains unclear. We studied the endocrine actions of 10 mg mifepristone administered orally as a single dose to eight healthy volunteers (aged 20-45 years) during the late follicular phase. Serum levels of LH, FSH, oestradiol, progesterone, leptin, mifepristone, cortisol, and gluco-corticoid bioactivity (GBA) were measured before and 1, 2, 4 and 8 h after ingestion of mifepristone on cycle day 10 or 11 (study day 1), and follow-up was continued for 10 days. Ovarian ultrasonography was performed on study days 1 and 7. Similar measurements were carried out during a control cycle. Mifepristone postponed ovulation, as evidenced by a 3.4+/-1.1 day (means+/-s.d.) delay (P < 0.005) in the LH surge and 3.6+/-4.0 day prolongation of the treatment cycle (P = 0.08). During the mifepristone cycle, an LH surge was displayed by five subjects when serum mifepristone levels had declined to 9.5+/-7.1 nmol/l. During the day of mifepristone administration, circulating GBA (P < 0.001) and leptin (P < 0.001) levels declined. On the day after mifepristone administration, mean serum FSH and leptin levels were lower than pretreatment values (3.8+/-1.8 IU/l vs 5.2+/-1.1 IU/l, n = 7, P < 0.05; 28.9+/-6.7 microg/l vs 33.2+/-9.0 microg/l, n = 7, P < 0.05 respectively), and the corresponding difference in the mean serum oestradiol concentration was borderline (452+/-252 pmol/l vs 647+/-406 pmol/l, n = 7, P = 0.056). In contrast to the control cycle, individual leptin levels declined during the follow-up after ingestion of mifepristone (n = 8, P < 0.01). These data showed that the commonly employed dose of mifepristone for EC delays ovulation and prolongs the menstrual cycle, when given during the late follicular phase. The mechanism of action of mifepristone may include a reduction of FSH secretion via a decrease in circulating leptin.

Circulating glucocorticoid bioactivity in the preterm newborn after antenatal betamethasone treatment.

Antenatal glucocorticoid treatment of mothers at risk of premature delivery is highly cost-effective in reducing neonatal mortality and morbidity. However, there is only limited information on the actual glucocorticoid bioactivity (GBA) reaching the fetus. By employing a recently developed recombinant cell bioassay, we studied circulating GBA in preterm newborns exposed to the standard antenatal betamethasone regimen (12 mg betamethasone twice, 24-h interval, for the mother; repeated in 7-10 d if required). Plasma GBA and cortisol concentrations were measured in cord blood of 71 infants (mean gestational age, 28.9 wk; range, 24.6-32 wk; mean birth weight, 1208 g; range, 480-2010 g). The median time between the last administered betamethasone dose and birth was 2.0 d. Cord GBA ranged from less than 15.6 to 170 nmol/liter cortisol equivalents. The level was highly dependent on the time between the last betamethasone dose and birth, i.e. infants born shortly (<12 h) after the last steroid dose displayed on average 4-fold higher GBA than that in the reference group (infants with >7 d since the last betamethasone dose before birth or without treatment; 74 vs. 21 nmol/liter cortisol equivalents; P < 0.0001). By contrast, if more than 72 h had elapsed between the last steroid dose and birth, circulating GBA was strongly dependent on cord cortisol (r = 0.85; P < 0.0001; n = 30). In multiple regression analysis adjusted for cord cortisol concentration and the time since the last steroid dose, increased umbilical artery resistance, a sign of severe fetal distress, was associated with lower cord GBA (P = 0.01). In conclusion, antenatal exposure of preterm fetuses to betamethasone causes a sizeable, but brief, peak of supraphysiological GBA, and approximately 3 d after the last betamethasone dose, circulating GBA derives from cord cortisol concentration.

Termination of pregnancy with mifepristone and prostaglandin suppresses transiently circulating glucocorticoid bioactivity.

Mifepristone is a potent antiglucocorticoid, the administration of which results in a dose-dependent activation of the hypothalamic-pituitary-adrenal axis. However, the net effect of this compound on circulating glucocorticoid activity is not known. We have used a recombinant cell bioassay to study glucocorticoid bioactivity (GBA), measured directly from serum, in 18 women undergoing medical termination of an early pregnancy with 200 mg mifepristone, followed by 0.8 mg misoprostol, a prostaglandin. Increased serum mifepristone was accompanied by an increase in serum cortisol that was insufficient to maintain circulating GBA within the normal (pre-mifepristone) range (34.7-93.8 nM cortisol equivalents); after approximately 43, 46, and 68 h of mifepristone ingestion, the mean serum GBA levels were much lower than the mean pre-mifepristone level (P < 0.0001). At the corresponding times, 16, 13, and 12 women displayed subnormal serum GBA levels (ranges, <15.6-23, <15.6-25.6, and <15.6-32.5 nM cortisol equivalents, respectively). Altogether 11 subjects displayed subnormal serum GBA (range, <15.6-32.5 nM cortisol equivalents) continuously in the presence of high concentrations of mifepristone. Two weeks after mifepristone administration, circulating GBA had returned to normal levels in all subjects. We conclude that 200 mg mifepristone elicits a significant suppression of serum GBA, to one third of the pretreatment value, despite the compensatory increase in the serum cortisol concentration.