Original Research As Short Communication

Obesity (2007) 15, 820–824; doi: 10.1038/oby.2007.592

Sex-specific Difference in the Interconversion of Cortisol and Cortisone in Men and Women*

Heinrich Vierhapper*, Georg Heinze† and Peter Nowotny*

  1. *Division of Endocrinology and Metabolism, Department of Internal Medicine, Medical University of Vienna, Vienna, Austria.
  2. †Section of Clinical Biometrics, Medical Statistics and Informatics, Medical University of Vienna, Vienna, Austria.

Correspondence: Heinrich Vierhapper Clinical Division of Endocrinology and Metabolism, Department of Internal Medicine III, Währinger Gürtel 18–20, A-1090 Wien, Austria. E-mail: heinrich.vierhapper@meduniwien.ac.at

*The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 3 November 2005; Revised  00; Accepted 29 September 2006.

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Abstract

Objective: Our objective was to demonstrate that the smaller oxoreductase activity of 11beta-HSD1 in women would shift the interconversion of cortisol and cortisone toward cortisone, resulting in a larger amount of generated labeled cortisone in healthy women than in healthy men.

Research Methods and Procedures: Using mass spectrometry, the amount of cortisone generated from a continuous infusion (8 AM to 6 PM) of stable-labeled cortisol (1alpha,2alpha-d-cortisol) was determined in non-obese and in obese (BMI >35 kg/m2) men and women during steady-state conditions (from 2 PM to 6 PM). In this setting, the amount of generated labeled cortisone (expressed as % of the achieved steady-state concentrations of labeled cortisol) reflects the sum of the bi-directional conversion of cortisol into cortisone (and vice versa) by 11beta-hydroxysteroid dehydrogenase.

Results: The amount of generated labeled cortisone was higher in men than in women (p < 0.0001). This sex difference was higher in obese than in non-obese patients (p = 0.0062).

Conclusions: The interconversion of cortisol and cortisone during steady-state conditions is shifted toward cortisol in men as compared with women. This suggests a higher overall oxoreductase activity of 11beta-hydroxysteroid dehydrogenase type 1 in men than in women. This sex-specific difference is maintained in obesity.

Keywords:

cortisol, cortisone, interconversion, production rates, metabolic clearance rates

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Introduction

Urinary free cortisol excretion rates, the metabolic clearance rates, and the endogenous production rates of cortisol are higher in men than in women (1, 2, 3). This sex-specific difference does not depend on the prevailing androgen concentrations (4). It could be due to differences in the mode of action and/or in the activity of the bidirectionally acting enzyme, 11-beta-hydroxy-steroid-dehydrogenase type 1 (11beta-HSD1),1 i.e., to a higher oxoreductase activity of 11beta-HSD1 in men as compared with women (5, 6). To verify this concept, we determined the amount of labeled cortisone generated during steady-state conditions from an infusion of stable-labeled cortisol in healthy men and women, which reflects the overall (possibly bidirectional) activity of 11beta-HSD1. We sought to demonstrate that the smaller oxoreductase activity of 11beta-HSD1 in women would shift the interconversion of cortisol and cortisone toward cortisone, resulting in a larger amount of generated labeled cortisone in healthy women than in healthy men.

Analogous experiments were subsequently performed in obese men and women. Despite some evidence to the contrary (7), obesity is thought to be associated with an increase in the activity of 11beta-HSD1 in adipose tissue (8, 9, 10, 11, 12, 13). However, hepatic 11beta-HSD1 activity is reduced in obesity (8), thus counterbalancing the enhanced formation of cortisol in adipose tissue. Hence, in our experimental setting, a decrease in the share of generated cortisone could be explained by an increased oxoreductase activity in the adipose tissues of obese individuals, whereas an increase in generated cortisone later would presumably correspond to the reduced (hepatic) reconstitution of cortisol.

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Research Methods and Procedures

Infusions of stable-labeled cortisol (1alpha,2alpha-d-cortisol) were given to 12 non-obese healthy individuals (7 men: age, 30 plusminus 5 years; BMI, 23.8 plusminus 2.5 kg/m2; and 5 women: age, 25 plusminus 3 years; BMI, 20.4 plusminus 1.7 kg/m2) and to 15 patients with obesity (8 men: age, 38 plusminus 12 years; BMI, 38.4 plusminus 3.6 kg/m2; and 7 women: age, 33 plusminus 8 years; BMI, 43.0 plusminus 6.9 kg/m2). Except for hypertension (n = 6), the obese individuals were clinically healthy at the time of the investigation and did not report any pertinent previous diseases. They had not been on a dietary regimen before the investigation and did not receive any medication other than antihypertensive drugs.

Thyroid function was normal in each case, and endogenous hypercortisolism was excluded by an overnight (1.0 mg) dexamethasone suppression test. The latter was done at least 2 weeks before the investigation. The protocol was approved by the local ethics committee and was carefully explained to each participant in terms of aims and the possible risks. On the day of the investigation, each patient provided a 24-hour urine sample for the determination of urinary steroid excretion rates. An indwelling catheter was inserted into an antecubital vein, and a constant (40 mL/h) intravenous infusion of 1alpha,2alpha-d-cortisol (2.0 mg in 500 mL 0.9% saline) was started at 8 AM. At the beginning and at the end of each infusion, a sample of the infusate from the end of the infusion line was obtained to determine losses by adsorption. After an equilibration period of 6 hours (at 2 PM), a second indwelling catheter was inserted into the contralateral arm, and blood samples were obtained at 20-minute intervals from 2 PM until 6 PM. These blood samples were subsequently pooled, and the pooled samples were used for analysis of the concentrations of labeled and unlabeled cortisol and cortisone by gas chromatography/mass spectrometry (GC/MS). To determine the amount of generated labeled urinary tetrahydrocortisol (THF), allotetrahydrocortisol (aTHF), and tetrahydrocortisone (THE), healthy volunteers collected a second 24-hour urine sample starting with the beginning of the infusion of labeled cortisol.

Materials

All organic solvents were of high-performance liquid chromatography or derivatization grade and were purchased from Pierce, Rockford, IL. Non-active cortisol (F; 11beta,17,21-trihydroxy-4-pregnene-3–20-dione), progesterone (4-pregnen-3,30-dione), and dihydrotestosterone (5alpha-androstan-17betaol-3-one) were obtained from Sigma, St. Louis, MO, or Steraloids, Wilton, NH. Radioactive [3H]1,2,6,7-cortisol (specific activity 60 Ci/mmol) and stable-labeled 1alpha,2alpha-d-cortisol (isotopic enrichment: 99.0%) were purchased from Amersham, Amersham, UK and from CIL, Andover, MA, respectively.

Urine Analysis by GC/MS

Urine samples were processed as reported previously (14, 15). Quantification of excreted steroids with individual standardization for each steroid was done by GC/MS in selective ion monitoring mode and after electric fragmentation using an HP 5973 MSD equipped with a 25-m CB5 fused-silica column. Dihydrotestosterone and progesterone were used as internal standards to assess the quality of the respective derivatization. M+ or M+-31 was used as a tracing ion (m/e 578 for THE, m/e 580 for d2-THE, me/652 for THF and a-THF and m/e 654 for d2-THF and for d2-aTHF). The estimated peak areas of the labeled compounds were corrected for the share of the isotopic peak of the respective natural compounds. Sensitivity was 1 pg per injection (THF, allo-THF, and THE) or 5 pg per injection (cortisol, cortisone).

Sample Preparation and Analysis by GC-MS

Plasma samples (2.5 mL) supplemented with 50,000 dpm of 3H-cortisol for later control of recovery were extracted with ethylacetate and separated by thin layer chromatography (cyclohexane-ethylacetate = 1:99). The zone containing cortisol and cortisone was eluted (2 times 2.5 mL methanol) and supplemented with tetrahydrocortisone (5beta-pregnan-3alpha,17,21-triol-11,20,dione) as an internal standard for GC/MS analysis. Subsequently, derivatization was performed using methoxyamine and trimethylsililation for the oxo- and the hydroxy-groups, respectively. Analysis by GC-MS (Hewlett-Packard HP5995; Hewlett-Packard, Palo Alto, CA) equipped with a 25-m CB5 fused silica column was made using selective ion monitoring mode and electric ionization (resolution 800). The sensitivity at a peak-to-noise ratio of 5:1 was 20 pg.

The tracer ions were m/e 609 for the internal standard tetrahydrocortisone (M+), m/e 605 for cortisol (M+ - 31), m/e 607 for 1,2alpha,-d2-cortisol (M+ - 31), m/e 531 for cortisone (M+ - 31) and m/e 533 for 1,2alpha,-d2-cortisone (M+ - 31). Since m/e 607 and m/e 533 also represent the natural isotopic peaks of native cortisol and cortisone, the detected peak areas of the labeled compounds (1,2alpha,-d2-cortisol, 1,2alpha,-d2-cortisone) had to be corrected for the share of the natural isotopic peak of the respective natural compounds. In addition, corrections were made for the losses during purification (using 3H-cortisol) and for the actual injected amount (internal standard). Finally, repeated (n > 5) injections of standard materials were used to establish the ratios of the detected peak areas of cortisol, cortisone, and the internal standard, tetrahydrocortisone.

Production rates of cortisol (PR[F]) were calculated from the product of the known infusion rate (Rt) and the ratio of tracer infusate enrichment (Et) to tracer dilution in the plasma (Es): (PR[F] = Rt x (Et/Es-1). Plasma concentrations of cortisone were calculated as (F times A[E] times f)/A[F]), where F is the concentration of cortisol as determined by GC/MS, A[E] is the detected peak area for cortisone (m/e 531), A[F] is the detected peak area of cortisol (m/e 605), and f is the previously established ratio between cortisone and cortisol.

The conversion rate (cortisol into cortisone) ratios were calculated as (A[E]corr times100 times fc)/A[F]corr), where A[E]corr is the detected peak area of cortisone (m/e 533) minus the area of the natural isotopic peak of natural cortisone (m/e 531), A[F] corr is the the peak area of cortisol (m/e 607) minus the area of the natural isotopic peak of natural cortisol (m/e 605), and fc is the ratio between the internal standard, tetrahydrocortisone, and cortisone.

Statistical Analysis

Continuous variables were described by mean plusminus standard deviation and compared between groups defined by sex and BMI using analysis of covariance (ANCOVA), adjusting for age. Because of skew distributions and heteroskedasticity, non-parametric ANCOVA analysis was performed by replacing the original data by their ranks. A potential interaction of sex and BMI group was accounted for by including a corresponding interaction term into the ANCOVA model. Group comparisons were corrected for multiple testing by adjusting p values by Tukey's method. Since the distribution of BMIs of obese males did not match exactly that of obese females, we included BMI also as a continuous variable instead of a categorical factor into the ANCOVA model to verify if that would change any statistical conclusions. p Values lower than 0.05 were considered as indicating statistical significance. The SAS System, version 9.1 (SAS Institute, Inc., Cary, NC) was used for statistical analysis.

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Results

The metabolic clearance rates of cortisol were 7.5 plusminus 2.3 L/h in non-obese men, 8.8 plusminus 2.8 L/h in obese men, 5.5 plusminus 1.8 L/h in non-obese women, and 8.8 plusminus 4.2 L/h in obese women. Production rates of cortisol were 0.42 plusminus 0.17 mg/h in non-obese men, 0.51 plusminus 0.20 in obese men, 0.38 plusminus 0.1 mg/h in non-obese women, and 0.53 plusminus 0.36 mg/h in obese women. There was no significant effect of BMI, sex, or age on metabolic clearance rates or on the production rates of cortisol.

Obese patients had higher (2.40 plusminus 0.60 mg/d) excretion rates of THE than non-obese patients (0.90 plusminus 0.12 mg/d; p = 0.0209). If BMI was entered as a continuous variable, these results remained unchanged (p = 0.0267 for the effect of BMI). There was no difference in the excretion rates of THE between males and females and no effect of age. Obese patients had higher (2.49 plusminus 0.66) ratios of THE/THF than non-obese (1.60 plusminus 0.35) individuals, irrespective of how BMI was entered into the analysis (p = 0.0001 for BMI categories, p = 0.0003 for continuous BMI). There was no difference between males and females and no effect of age. Obese patients had higher (1.45 plusminus 0.13) ratios of THE/THF + aTHF than non-obese (0.83 plusminus 0.07) individuals, irrespective of how BMI was entered into the analysis (p = 0.0002 for BMI categories, p = 0.048 for continuous BMI). There was no difference between males and females, and no effect of age could be ascertained.

The percentage of infused labeled cortisol excreted as (labeled) THE in the urine collected during the 24 hours after the beginning of the infusion of 1alpha,2alpha-d-cortisol was 0.6 plusminus 0.3% in both non-obese men and non-obese women.

The amount of generated labeled cortisone was 6.0 plusminus 1.8% in non-obese men, 3.9 plusminus 0.5% in obese men, 9.7 plusminus 2.4% in non-obese women, and 14.1 plusminus 5.8% in obese women. Males exhibited lower values than females (p < 0.0001; Figure 1). This sex-specific difference was significantly higher in obese than in non-obese patients (p = 0.0062). No significant difference between obese and non-obese patients could be detected, either in the total sample or if broken down by gender. Conclusions did not change if BMI or age was entered into the analysis as continuous variable.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Amount of generated labeled cortisone (%) during the infusion of stable-labeled cortisol in non-obese (open bars) and obese (striped bars) men and women (2 PM to 6 PM)

Full figure and legend (65K)

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Discussion

The 11beta-hydroxysteroid dehydrogenases are microsomal enzymes that catalyze the interconversion between the biologically active cortisol and the biologically inactive cortisone. 11beta-HSD type 2 serves to protect mineralocorticoid target organs (e.g., the kidneys) from an overabundance of cortisol by inactivating it to cortisone. 11beta-HSD type 1 acts predominantly as an oxoreductase restoring cortisol from cortisone in adipose tissues and in the liver but may also catalyze the reaction in the opposite direction (16).

Differences in the activity of 11beta-HSD1 between men and women were initially based on findings in men with hypopituitarism, i.e., in the absence of pituitary feedback mechanisms, and in elderly men (5, 6) who present, albeit with a substantial overlap between the members of the two sexes, a relative preponderance of urinary metabolites with an 11-hydroxy group (metabolites of cortisol) over their 11-keto homologues (metabolites of cortisone) as compared with women, suggesting a higher oxoreductase activity of 11beta-HSD1 in men. As demonstrated previously (17) and confirmed by the results of the present investigation, these differences are not apparent in young, non-obese men and women with intact pituitary function who show an identical relationship of the urinary metabolites of cortisol and of cortisone. Hence, the determination of glucocorticoid metabolites in 24-hour urine samples, which reflects the action of two different 11beta-hydroxysteroid dehydrogenases (11beta-HSD type 1 and type 2), and the variable activity of 11beta-HSD1 in different tissues are not precise enough to document sex-specific differences in healthy individuals.

The stable labeled isotope technique can be used as an alternative way to assess the interconversion of cortisol and cortisone in vivo. It has been used by others to investigate the renal activity of 11beta-HSD and the individual activities of the two isoenzymes in single individuals after the oral administration of deuterium-labeled cortisol (18, 19). Using a cortisol with a deuterium label in position 11, Andrew et al. (20) were able to differentiate between the activities of the two insoenzymes of 11beta-HSD1 in healthy men. The tracer used in the present study is labeled in positions 1 and 2, but not in position 11. While we, therefore, cannot separate the oxoreductase activity from its activity as a dehydrogenase, the amount of labeled cortisone generated during steady-state conditions is reflected in the overall activity of 11beta-HSD. Using this technique, we were indeed able to demonstrate that, in men, the amount of labeled cortisone generated from labeled cortisol is smaller than in women. This shift toward cortisol is in keeping with the postulated higher oxoreductase activity of 11beta-HSD1 in men than in women (5, 6). This sex-specific difference was higher in obese than in non-obese individuals. Our protocol investigated only the time period between 2 PM and 6 PM. Although there is no reason to believe that the interconversion of cortisol and cortisone changes throughout the day (i.e., as a function of the diurnal variation in cortisol secretion), we cannot formally exclude this possibility.

The urinary excretion rates of free cortisol and of its main metabolites are elevated in obese individuals (21, 22), and an increase in the metabolic clearance and in the endogenous production rates of cortisol is seen in obese women (17, 23) and in some obese men (17). An enhanced activity of 11beta-HSD1 in adipose tissue of obese individuals (8, 9, 10, 11, 12, 13, 24) should promote the conversion of cortisone into cortisol and, therefore, suggests a preponderance of urinary metabolites of cortisol over those of cortisone. However, as confirmed by the results of the present investigation, glucocorticoid metabolite excretion rates in obesity are, indeed, shifted toward the metabolites of cortisone. Since the amount of cortisone generated from infused labeled cortisol was similar in obese and in non-obese (male and female) individuals, the preponderance of urinary cortisone metabolites remains, at present, unexplained.

In summary, our results suggest that men have a higher (overall) oxoreductase activity of 11beta-hydroxysteroid dehydrogenase type 1 than women. This sex-specific difference in the activity of 11beta-HSD1 is maintained, indeed even more pronounced, in the presence of obesity.

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Notes

1 Nonstandard abbreviations: 11beta-HSD1, 11-beta-hydroxy-steroid-dehydrogenase type 1; GC/MS, gas chromatography/mass spectrometry; THF, tetrahydrocortisol; aTHF, allotetrahydrocortisol; THE, tetrahydrocortisone; ANCOVA, analysis of covariance.

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Acknowledgments

There was no funding/outside support for this study. The authors thank Annerose Fürst, Heidelinde Lendner, Astrid Hofer, and Erika Nowotny for technical assistance.

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