Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2009) 19 P327

SFEBES2009 Poster Presentations Steroids (36 abstracts)

Measuring cortisone production in man using a new stable isotope tracer

K A Hughes , R M Reynolds , R Andrew & B R Walker

University of Edinburgh, Edinburgh, UK.

Background: 11β-hydroxysteroid dehydrogenases (11β-HSD1&2) interconvert cortisol (F) and cortisone (E). Although 11β-HSD1 reductase activity has been measured in vivo, E production (dehydrogenase activity) has not been quantified using a Gold Standard technique, steady state tracer infusion.

Aim: To develop a method to measure E production in vivo using the stable isotope tracer d2-cortisone (d2E).

Methods: LCMS conditions were optimised to allow steroid measurement. To establish if the deuteriums exerted a primary isotope effect on enzymatic kinetics, h11β-HSD1 transfected HEK293 cells were incubated with E or d2E (2 μM; 5 h) and production rates of F or d2F compared. HEK293/h11β-HSD1 cells were incubated (6/24 h) with a fixed concentration of [3H]2E (5 nM) and increasing concentrations of E or d2E(95–4995 nM). In healthy men, pharmacokinetics for d2E were derived in volunteers (with Ethical Approval) by measuring plasma d2E following bolus injection (141 μg), and cortisone production rate was calculated from enrichment of cortisone with d2E-tracer steady state d2E infusion (105 μg/h, loading dose 76 μg).

Results: Incubation of HEK293/h11β-HSD1 cells with d2E or E resulted in a time-dependent production of d2F or F respectively (0.4±0.2 vs 1.0±0.8 pmol/min per 105 cells). No significant isotope effect on competition with [3H]2E was observed (d2EvsE:Vmax1.8±0.6 vs 2.2±0.8 pmol/min per 105 cells; apparent Km 2.3±1.0 vs 2.8±1.3 μM). In vivo d2E t1/2 was 48.9 min and Vd was 39.5 l, compared with t1/2 28 min1 previously reported for E. Whole body E production was 21.5±5.1 nmol/min at a prevailing circulating concentration of F of 246.7±30.0 nM. This is comparable with results previously calculated from non-steady state kinetics using a d4F tracer (24.1 nmol/min, adjusted for substrate concentration and Vd2). Clearance of d2E was 0.27 l/min, similar to that of cortisol (0.28 l/min)2.

Conclusion: We have developed a method of measuring cortisone production in steady state. Tissue-specific cortisone production can now be measured using d2E allowing further understanding of the contribution of the 11β-HSDs to glucocorticoid homeostasis in obesity and hypertension.

1. Peterson. JCI 1957 36 (9) 1301.

2. Andrew. JCEM 2002 87 277.

Article tools

My recent searches

No recent searches.