Endocrine Abstracts

Background: Steroid hormones such as the estrogen 17β-estradiol and the androgen testosterone play an important role in the etiology of hormone dependent diseases. To investigate factors influencing endogenous steroid hormone synthesis also biologically less active glucuronides and sulfates should be considered since they can serve as circulating reservoir for bioactive hormones. As the sensitivity of the available mass spectrometry (MS)-based method is lower for glucuronides than for unconjugated steroids and sulfates, the specific hydrolysis of glucuronides can extend the accessible metabolic profile. However, there is no published method available which allows the simultaneous analysis of unconjugated steroids, hydrolyzed glucuronides and intact sulfates from the same sample.

Objectives: Thus, the aim of the present study was to extend the accessible steroid hormone profile in human plasma by inclusion of detection of steroid glucuronides after hydrolysis in addition to the detection of conjugated and unconjugated steroids.

Methods: After extraction of unconjugated steroids and quantitation thereof by gas chromatography–MS/MS, the remaining aqueous phase was divided for a) the direct quantification of steroid conjugates by liquid chromatography–MS/MS and b) the indirect quantification of steroid glucuronides after hydrolysis with Escherichia coli-β-glucuronidase. Putative formation of artifacts during sample cleanup was investigated by spiking plasma with (isotope-labelled) reference compounds.

Results: The enzymatic hydrolysis of steroid glucuronides was successfully implemented and no changes in the steroid profile due to putative artifact formation by oxidation, reduction or unintended hydrolysis during sample cleanup were observed. Out of 42 steroids included in the method, 27 were quantifiable in most plasma samples derived from 10 pre- and 10 postmenopausal women as well as 5 men.

Conclusions: The extended method will contribute to a comprehensive insight in the steroid hormone profile in human plasma.