Endocrine Abstracts (2003) 5 P233

In vitro synthesis of 18-hydroxycortisol (18-OH-F) and 18-oxocortisol (18-oxo-F); clues to their origin in normal human subjects

LA Shakerdi1, EC Friel1, E Davies1, JMC Connell1, AM Wallace2 & R Fraser1

1MRC Blood Pressure Group, Western Infirmary, Glasgow, UK; 2Department of Clinical Chemistry, Macewen Building, Royal Infirmary, Glasgow, UK.

The plasma concentrations and urinary excretion rates of 18-OH-F and 18-oxo-F are high in Conn's syndrome and glucocorticoid suppressible hyperaldosteronism, reputedly because aldosterone synthase (AS) has abnormal access to cortisol (F). However, both compounds are also excreted in normal subjects. Two enzymes, 11beta-hydroxylase (11OH) and AS, located in the zona fasiculata (ZF) and zona glomerulosa (ZG) respectively, are capable of 18-hydroxylation but only AS catalyses 18-oxidation. To determine the origin of these compounds, we used V79 Chinese hamster lung cells transfected with their respective genes, CYP11B1 and CYP11B2 (donated by Dr. Rita Bernhardt, Saarbrucken) to compare their action on cortisol.
The V79 cells were grown to confluence in a culture flask. CYP11B1-transfected cells were incubated with cortisol (1-10 micromolar) or 11-deoxycortisol (S; 1-10 micromolar). CYP11B2-transfected cells were incubated with cortisol or 11-deoxycorticosterone (DOC)). Incubation was for 24h at 37 degC. Medium was removed and steroid content was analysed by gas chromatography-mass spectrometry.
Protein was measured by the Biorad procedure.
CYP11B1 transfected cells converted (S) to cortisol (c42%) in a dose-dependent manner. Small quantities of 18-OH-S were also found. Cortisol was converted to 18-OH-F (c0.5%) but no 18-oxo-F was found. CYP11B2-transfected cells converted (DOC) to corticosterone (c1.8%), 18-hydroxycorticosterone (c1.7%), and aldosterone (c3%), again in dose-dependent manner. Cortisol was converted to 18-OH-F (c4.3%) and 18-oxo-F (c1.1%).
Cortisol can be converted to 18-OH-F in both the ZF and ZG by 11beta-hydroxylase and AS respectively. Since, in vivo cortisol synthesis is restricted to the ZF, it is likely that most 18-OH-F in normal subjects is the product of 11beta-hydroxylase. 18-Oxo-F is a unique product of AS possibly resulting from the conversion of perfusing plasma cortisol or originating in the ill-defined transitional zone.

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