ATP stimulates human placental 11beta-hydroxysteroid dehydrogenase type 2 activity by a novel mechanism independent of phosphorylation.

The human placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is believed to play a key role in fetal development since this enzyme protects the fetus from exposure to high levels of maternal cortisol by virtue of converting maternal cortisol to its inert metabolite cortisone. The present study was undertaken to examine the effect of ATP on 11beta-HSD2 activity in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was stimulated more than six-fold by 0.5 mM ATP (EC(50) = 0.2 mM). Such stimulation appears to be mediated through a novel mechanism independent of ATP-induced phosphorylation of the reaction components since AMP-PNP, a non-hydrolyzable analogue of ATP, was equally effective. The ATP-induced stimulation of 11beta-HSD2 activity is adenine nucleotide specific in that a similar stimulation was observed with ADP and AMP but not with CTP, GTP, or UTP. Furthermore, ATP increased the maximal velocity (V(max)) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the apparent K(m) of 11beta-HSD2 for cortisol, suggesting that ATP may stimulate enzyme activity by interacting with the enzyme at a site other than that involved in substrate binding. In conclusion, the present study has identified ATP as a novel regulator of human placental 11beta-HSD2 in vitro. It is conceivable that intracellular ATP may have a profound effect on 11beta-HSD2 function in vivo.     

The 11 beta-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide

Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11 beta-hydroxysteroid dehydrogenase type 2 enzyme (11 beta-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11 beta-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC(50)=10 microM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC(50)=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11 beta-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11 beta-HSD2 may be critical for enzyme activity. Zn(2+) also inactivated enzyme activity (IC(50)=2.5 microM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD(+) (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC(50)=8 microM) in 11 beta-HSD2 activity, indicating that binding of NAD(+) to the microsomal 11 beta-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11 beta-HSD2, with NAD(+) binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn(2+) may play an important role in regulation of placental 11 beta-HSD2 activity.     

Increased production of 11beta-hydroxysteroid dehydrogenase type 2 in the kidney microsomes of squirrel monkeys (Saimiri spp.)

In squirrel monkeys (Saimiri spp.), cortisol circulates at levels much higher than those seen in man and other Old World primates, but squirrel monkeys exhibit no physiologic signs of the mineralocorticoid effects of cortisol. These observations suggest that squirrel monkeys have mechanisms for protection of the mineralocorticoid receptor (MR) from these high levels of cortisol. We previously showed that the serum cortisol to cortisone ratio in these animals is low relative to that in human serum, suggesting that production of the MR protective enzyme, 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), is increased in squirrel monkeys. Here, we directly evaluate whether increased production of 11beta-HSD2, which inactivates cortisol to cortisone, is a mechanism for protection of MR. In vitro assays showed that 11beta-HSD2 activity in squirrel monkey kidney microsomes was 3 to 7 times higher than that seen in kidney microsomes from pig or rabbit. 11beta-HSD2 protein detected by Western blot analysis was 4 to 9 times greater in squirrel monkey microsomes than in pig or rabbit microsomes. Comparison of the effect of expression of either human or squirrel monkey 11beta-HSD2 on MR transactivation activity showed similar inhibition of MR response to cortisol by both enzymes, indicating that the intrinsic activities of the human and squirrel monkey enzymes are similar. These findings suggest that one mechanism by which squirrel monkeys protect the MR from activation by high cortisol levels in the kidney is by upregulation of 11beta-HSD2 activity through increased production of the enzyme.     

11 Beta-hydroxysteroid dehydrogenase type 1 is induced in human monocytes upon differentiation to macrophages.

11beta-hydroxysteroid dehydrogenases (11beta-HSD) perform prereceptor metabolism of glucocorticoids through interconversion of the active glucocorticoid, cortisol, with inactive cortisone. Although the immunosuppressive and anti-inflammatory activities of glucocorticoids are well documented, the expression of 11beta-HSD enzymes in immune cells is not well understood. Here we demonstrate that 11beta-HSD1, which converts cortisone to cortisol, is expressed only upon differentiation of human monocytes to macrophages. 11beta-HSD1 expression is concomitant with the emergence of peroxisome proliferator activating receptor gamma, which was used as a surrogate marker of monocyte differentiation. The type 2 enzyme, 11beta-HSD2, which converts cortisol to cortisone, was not detectable in either monocytes or cultured macrophages. Incubation of monocytes with IL-4 or IL-13 induced 11beta-HSD1 activity by up to 10-fold. IFN-gamma, a known functional antagonist of IL-4 and IL-13, suppressed the induction of 11beta-HSD1 by these cytokines. THP-1 cells, a human macrophage-like cell line, expressed 11beta-HSD1 and low levels of 11beta-HSD2. The expression of 11beta-HSD1 in these cells is up-regulated 4-fold by LPS. In summary, we have shown strong expression of 11beta-HSD1 in cultured human macrophages and THP-1 cells. The presence of the enzyme in these cells suggests that it may play a role in regulating the immune function of these cells.     

The use of deuterium-labeled cortisol for in vivo evaluation of renal 11beta-HSD activity in man: urinary excretion of cortisol, cortisone and their A-ring reduced metabolites

This study describes a new approach using stable isotope methodology in evaluating 11beta-HSD activities in vivo based on urinary excretion of cortisol, cortisone, and their A-ring reduced metabolites. The method involved the measurement of deuterium-labeled cortisol and its deuterium-labeled metabolites by GC/MS simultaneously with endogenous cortisol, cortisone, and their A-ring reduced metabolites after oral administration of deuterium-labeled cortisol to normal human subjects. This stable isotope approach offered unique advantages in assessing the appropriateness of measuring unconjugated and total (unconjugated + conjugated) cortisol, cortisone, and their A-ring reduced metabolites in urine as indices of renal 11beta-HSD2 activity in man. Our results strongly support that the measurement of urinary unconjugated cortisol and cortisone is a significant advance in assessing 11beta-HSD2 activity.     

11 Beta-hydroxysteroid dehydrogenase type 1 from human liver: dimerization and enzyme cooperativity support its postulated role as glucocorticoid reductase

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme that catalyzes the reversible interconversion of receptor-active 11-hydroxy glucocorticoids (cortisol) to their receptor-inactive 11-oxo metabolites (cortisone). However, the physiological role of 11beta-HSD 1 as prereceptor control device in regulating access of glucocorticoid hormones to the glucocorticoid receptor remains obscure in light of its low substrate affinities, which is in contrast to low glucocorticoid plasma levels and low Kd values of the receptors to cortisol. To solve this enigma, we performed detailed kinetic analyses with a homogeneously purified 11beta-HSD 1 from human liver. The membrane-bound enzyme was successfully obtained in an active state by a purification procedure that took advantage of a gentle solubilization method as well as providing a favorable detergent surrounding during the various chromatographic steps. The identity of purified 11beta-HSD 1 was proven by determination of enzymatic activity, N-terminal amino acid sequencing, and immunoblot analysis. By gel-permeation chromatography we could demonstrate that 11beta-HSD 1 is active as a dimeric enzyme. The cDNA for the enzyme was cloned from a human liver cDNA library and shown to be homologous to that previously characterized in human testis. Interestingly, 11beta-HSD 1 exhibits Michaelis-Menten kinetics with cortisol and corticosterone (11beta-dehydrogenation activity) but cooperative kinetics with cortisone and dehydrocorticosterone (11-oxoreducing activity). Accordingly, this enzyme dynamically adapts to low (nanomolar) as well as to high (micromolar) substrate concentrations, thereby providing the fine-tuning required as a consequence of great variations in circadian plasma glucocorticoid levels.     

Evaluation of hepatic 11 beta-hydroxysteroid dehydrogenase activity by cortisone acetate test in young adults with diabetes mellitus type 1

Cortisone acetate test was performed in twelve young adult patients with diabetes mellitus type 1, after dexamethasone administration to suppress endogenous cortisol production. Previous screening revealed that all of the subjects had peak cortisol responses in the range from subnormal to normal, as determined by a low-dose Synacthen test. The aim was to find out whether these patients would exhibit different conversion of cortisone to cortisol by 11beta-hydroxysteroid dehydrogenase. Using multifactorial ANOVA the following significant relationships were obtained between cortisol or cortisol/cortisone ratio measured during the test and other parameters examined a) before dexamethasone suppression and b) during the test: a) Cortisol at 120(th) minute negatively correlated with daily insulin dose and positively with basal aldosterone. Cortisol/cortisone ratio at 60(th), 120(th), 180(th), and 240(th) minute negatively correlated with basal aldosterone/plasma renin activity ratio, urinary free cortisol/24 hours and positively with basal dehydroepindrosterone sulphate. b) Cortisol at 120(th) minute negatively correlated with suppressed basal serum glycemia; cortisol/cortisone ratio during the whole test negatively correlated with supressed basal ACTH. The examination of peripheral metabolism of cortisol using cortisone acetate test in patients with diabetes mellitus type 1 showed adaptive changes of 11beta-hydroxysteroid dehydrogenace activity associated with altered cortisol tissue supply.     

Gender-specific links between hepatic 11beta reduction of cortisone and adipokines

Objective: Reduction of cortisone to cortisol is mediated by 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1), a putative key enzyme in obesity‐related complications. Experimental studies suggest that adipokines, notably leptin and tumor necrosis factor‐α (TNF‐α), are of importance for 11βHSD1 activity. We hypothesized that the regulation of hepatic preceptor glucocorticoid metabolism is gender‐specific and associated with circulating levels of leptin and TNF‐α receptors and/or sex hormones. Research Methods and Procedures: A total of 34 males and 38 women (14 premenopausal and 22 postmenopausal) underwent physical examination and fasting blood sampling. Insulin sensitivity was tested by euglycemic hyperinsulinemic clamps, and hepatic 11βHSD1 enzyme activity was estimated by the conversion of orally‐ingested cortisone to cortisol. Results: Hepatic 11βHSD1 activity was negatively associated with leptin and soluble TNF (sTNF) r1 and sTNFr2 in males. These correlations remained significant after adjustment for age and insulin sensitivity, and for sTNF‐α receptors also after adjustment of BMI and waist circumference. In contrast, 11β reduction of cortisone was positively associated to leptin in females after adjustment for BMI and waist circumference. Discussion: Hepatic 11β reduction shows different links to circulating adipocyte‐derived hormones in males and females. This emphasizes the need for further studies on tissue‐specific regulation of 11βHSD1 in both genders.     

Calcium inhibits human placental 11beta-hydroxysteroid dehydrogenase type 2 activity

The effect of Ca2+ on the conversion of cortisol to its inert metabolite cortisone, the reaction catalyzed by the microsomal enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), was investigated in human placental microsomes. Placental microsomal 11beta-HSD2 activity, as determined by the rate of conversion of cortisol to cortisone, was inhibited up to 50% by increasing free Ca2+ concentrations from 22 to 268 nM. The Ca2+-induced inhibition was reversible since chelation of endogenous Ca2+ with EGTA increased 11beta-HSD2 activity up to 200%. Ca2+ decreased the maximal velocity (Vmax) of the 11beta-HSD2 catalyzed conversion of cortisol to cortisone without altering the Km of 11beta-HSD2 for cortisol, indicating that Ca2+ modulates the catalytic efficiency rather than the substrate binding of 11beta-HSD2. Moreover, the Ca2+-induced inhibition does not appear to involve altered cofactor (NAD+) binding since the inhibition of microsomal 11beta-HSD2 activity by a sub-maximal concentration of free Ca2+ was not overcome by increasing the concentration of NAD+. These findings in the microsomes were then extended to an intact cell system, JEG-3 cells, an established model for human placental trophoblasts. In these cells, an increase in cytosolic free Ca2+ concentration ([Ca2+]i) elicited by a known physiological stimulus, PGF(2alpha), was accompanied by a 40% decrease in the level of 11beta-HSD2 activity. Furthermore, the PGF(2alpha)-induced inhibition of 11beta-HSD2 activity was abrogated when increases in [Ca2+]i were blocked with the intracellular Ca2+ chelator, BAPTA. Collectively, these results demonstrate for the first time that Ca2+ inhibits human placental 11beta-HSD2 activity by a post-translational mechanism not involving substrate or cofactor binding.     

11beta-Hydroxysteroid dehydrogenase expression and glucocorticoid synthesis are directed by a molecular switch during osteoblast differentiation

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays an important role in the prereceptor regulation of corticosteroids by locally converting cortisone into active cortisol. To investigate the impact of this mechanism on osteoblast development, we have characterized 11beta-HSD1 activity and regulation in a differentiating human osteoblast cell line (SV-HFO). Continuous treatment with the synthetic glucocorticoid dexamethasone induces differentiation of SV-HFO cells during 21 d of culture. Using this cell system, we showed an inverse relationship between 11beta-HSD1 activity and osteoblast differentiation. 11beta-HSD1 mRNA expression and activity were low and constant in differentiating osteoblasts. However, in the absence of differentiation (no dexamethasone), 11beta-HSD1 mRNA and activity increased strongly from d 12 of culture onward, with a peak around d 19. Promoter reporter studies provided evidence that specific regions of the 11beta-HSD1 gene are involved in this differentiation controlled regulation of the enzyme. Functional implication of these changes in 11beta-HSD1 is shown by the induction of osteoblast differentiation in the presence of cortisone. The current study demonstrates the presence of an intrinsic differentiation-driven molecular switch that controls expression and activity of 11beta-HSD1 and thereby cortisol production by human osteoblasts. This efficient mechanism by which osteoblasts generate cortisol in an autocrine fashion to ensure proper differentiation will help to understand the complex effects of cortisol on bone metabolism.     

The effect of water loading on the urinary ratio of cortisone to cortisol in healthy subjects and a new approach to the evaluation of the ratio as an index for in vivo human 11β-hydroxysteroid dehydrogenase 2 activity

Factors that give rise to a large variation in the urinary ratio of free cortisone to cortisol (UFE/UFF) were investigated to accurately estimate 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) activity in humans in vivo. A water loading test was first carried out in two healthy subjects to examine the effect of water intake or urine volume on the urinary ratio of free cortisone to cortisol (UFE/UFF). The ratio was found to increase by water loading. We also examined urinary concentrations and amounts of cortisol, cortisone, creatinine, Na(+), K(+), and Cl(-), and urine volume, as possible factors affecting the urinary ratio (UFE/UFF), in 60 urine samples obtained from 15 healthy volunteers. Among these factors tested, the urinary concentration of cortisol was most highly correlated with the UFE/UFF ratio (r=-0.858), indicating that the in vivo activity of 11β-HSD2 (UFE/UFF) should fluctuate with the changes of the urinary concentration of cortisol. Based on the findings, we proposed a new estimation method of in vivo activity of 11β-HSD2 in humans, using the UFE/UFF ratio correlated with the urinary concentration of cortisol (UFE/UFF-cortisol concentration). Taking into consideration the intra-individual variabilities in the urinary concentration of cortisol, there were no significant within-day variations in 11β-HSD2 activity. The findings indicate that 11β-HSD2 activities can be accurately evaluated by simply measuring free cortisol and cortisone concentrations in spot urine samples. Furthermore, administrations of glycyrrhetinic acid in three healthy volunteers were performed to confirm the usefulness of the present assessment for the activity of 11β-HSD2.     

Antisense inhibition of 11betahydroxysteroid dehydrogenase type 1 improves diabetes in a novel cortisone-induced diabetic KK mouse model

The inhibition of 11βhydroxysteroid dehydrogenase 1 (11βHSD1), an enzyme that catalyzes the conversion of inactive cortisone to active cortisol, is an attractive target to treat diabetes by suppressing hepatic gluconeogenesis. To test this hypothesis, we developed a novel glucocorticoid-induced diabetic KK mouse model and used 11βHSD1 antisense oligonucleotide (ASO) as an inhibitory tool. KK mice were treated with 25 or 50 mg/kg/day of 11βHSD1 ASO for 28 days. On day 25, cortisone pellets were surgically implanted to induce diabetes. In the ASO-treated mice, plasma blood glucose levels were significantly reduced by up to 54%. In parallel, cortisol and other diabetes endpoints were also significantly reduced. Hepatic 11βHSD1 mRNA was suppressed by up to 84% with a concomitant respective decrease of up to 49% in the expression of PEPCK. The results suggest that inhibition of 11βHSD1 activity reduces the availability of cortisol to activate the glucocorticoid receptor, down regulates gluconeogenesis and thus reduces plasma glucose levels in cortisone-induced diabetic KK mice.     

Guinea pig 11beta-hydroxysteroid dehydrogenase type 1: primary structure and catalytic properties

The 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) enzyme is responsible for the interconversion of glucocorticoids and their inactive metabolites, and thus modulates the intracellular level of bioactive glucocorticoids. The present study was designed to clone and characterize 11beta-HSD1 in the guinea pig, a laboratory animal known for resistance to glucocorticoids. The cDNA encoding guinea pig 11beta-HSD1 was cloned by a modified 3'-RACE (rapid amplification of cDNA ends) protocol using the hepatic RNA as template. The cloned cDNA encodes a protein of 300 amino acids that shares 71 to 74% sequence identity with other known mammalian 11beta-HSD1 proteins. Sequence comparison analysis revealed that the deduced guinea pig 11beta-HSD1 was longer, by eight amino acids at the C terminus, than those of other mammals. Moreover, one of the two absolutely conserved consensus sites for N-glycosylation was absent. To examine the functional significance of these structural changes, we also characterized 11beta-HSD1 activity in the hepatic microsomes. Although the guinea pig hepatic enzyme was NADP(H)-dependent and reversible, it displayed equal affinity for cortisol and cortisone (apparent K(m) for both substrates was 3 microM). This is in marked contrast to 11beta-HSD1 in other mammals whose affinity for cortisone is approximately 10 times higher than that for cortisol (apparent K(m) of 0.3 vs. 3.0 microM). The apparent lower affinity of the guinea pig enzyme for cortisone would suggest that the intracellular bioformation of cortisol from circulating cortisone may be less efficient in this species. Northern blot analysis and RT-PCR revealed that the mRNA for 11beta-HSD1 was widely expressed in the adult guinea pig but at low amounts. In conclusion, the present study has identified distinct features in the deduced primary structure and catalytic function of 11beta-HSD1 in the guinea pig. Thus, the guinea pig provides a useful model in which the structural determinants of catalytic function of 11beta-HSD1 may be studied.     

Dehydrogenase and oxoreductase activities of porcine placental 11beta-hydroxysteroid dehydrogenase

Dehydrogenase (cortisol to cortisone) and oxoreductase (cortisone to cortisol) activities of porcine placental 11beta-hydroxysteroid dehydrogenase (11beta-HSD) were measured in tissue fragment cultures on day 75 of gestation. Dehydrogenase activity was over fivefold greater than oxoreductase activity (p < .001). There were positive linear associations (p < .01) between net dehydrogenase activity (dehydrogenase minus oxoreductase) and fetal weight, fetal length, and placental weight. These data indicate a predominance of placental 11beta-HSD dehydrogenase activity at this gestational stage that would insure a net conversion of cortisol to cortisone as it traverses the placenta. The data further suggest that 11beta-HSD activities may provide an optimal glucocorticoid environment that is supportive of enhanced fetal and placental growth.     

Investigation of factors influencing 11 beta-hydroxysteroid dehydrogenase (EC 1.1.1.146) activity in midgestational human fetal lung monolayer and explant cultures

We recently suggested that the 11 beta-hydroxysteroid dehydrogenase (11-HSD) activity in midgestational human fetal lung (HFL) cultures comprised at least two enzymes, one oxidative--associated with epithelial cells, the other reductive--related to fibroblast-like cells. In this study, the effects of various hormones on 11-HSD activity were studied by measuring the interconversion of [3H]cortisol and [14C]cortisone. Human chorionic gonadotrophin, placental medium, and low oxygen concentration increased the conversion of cortisone to cortisol while activity in the reverse direction remained unchanged. No effects were seen when adrenocorticotrophin, prolactin, placental lactogen, estrogens, triiodothyronine or oxytocin were added in physiological amounts.     

Sequence and expression of 11β-hydroxysteroid dehydrogenase type 1 cDNA cloned from pig testis

Pig 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11β-HSD type 1, and the sequence of 11β-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11β-HSD type 1.     

Validation and application of a highly specific and sensitive ELISA for the estimation of cortisone in saliva, urine and in vitro cell-culture media by using a novel antibody

It is generally acknowledged that local tissue concentrations of cortisol and cortisone are modulated by site-specific actions of 11β-hydroxysteroid dehydrogenase (11β-HSD) isoenzymes 1 and 2. Cortisone, the inactive metabolite of cortisol is produced by 11βHSD type 2. To assess 11β-HSD types 1 and 2 activities, the cortisol/cortisone ratio has to be accurately determined. Immunoassays to measure cortisone levels are not widely available and tend to lack specificity. The aim of this project was to develop a highly specific and sensitive ELISA method for the estimation of free cortisone levels in urine, saliva and in vitro media samples without chromatographic separation. Antibodies against cortisone were raised in rabbits using cortisone-3-CMO-KLH as immunogen. HRP-goat anti-rabbit IgG conjugate was used as enzyme tracer. Cross-reactivities of the untreated cortisone antiserum with major interfering steroids were minimal except for cortisol (3.15%). However, following an immune-affinity purification of the antibodies using CNBr-activated sepharose-cortisol-3-CMO-BSA, cross-reactivity of the purified cortisone antibody with cortisol was reduced to 0.27%. The minimum detection limit of cortisone ELISA was 28 pg/mL (77.7 pM). The validity of the cortisone ELISA was confirmed by the excellent correlation obtained before and after an HPLC fractionation step (Y=1.09X-0.21, R2=0.98). Intra-assay and inter-assay imprecision were 5.5-11.7% and 8.7-12.8% CV, respectively. Using this assay, salivary cortisone levels showed a circadian rhythm in men and women (11.2±7.3 nM at 08.00 h and 5.1±3.6 nM at 18.00 h), and the levels were reduced following liquorice ingestion. In media of adrenocortical H295 cell line incubations, basal cortisone levels were 4.24±0.22 nM that increased to 8.6±1.2 nM post forskolin treatment. Urinary free cortisone excretion levels in healthy subjects were 56.66±36.9 nmol/day. In human volunteers following ingestion of green coffee bean extract for 2 weeks, urinary free cortisol excretion reduced significantly from 66.67±22.3 to 42.66±17.5 nmol/day (p=0.02) and the cortisol/cortisone ratio from 2.04±1.33 to 1.49±1.13, p=0.05. In conclusion, a simple and highly specific and sensitive ELISA has been developed and applied to estimate cortisone levels in biological fluids and culture media.