Development and Validation of a Fecal Testosterone Biomarker: MATERIALS AND METHODS(8)


Separation of Free Steroid

Samples initially extracted with 10% methanol were reextracted using ethyl ether to obtain primarily unconjugated testosterone and nonpolar metabolites of testosterone. Extracts were concentrated into 2-ml vials, dried under nitrogen, and reconstituted in ethanol. HPLC separations were obtained using a Waters 5 X 100-mm Nova-Pac C18 column contained in an RCM Radial Compression Cartridge Holder (Waters, Milford, MA). A linear gradient of 20-75% acetonitrile (Fisher) in water at a 1 ml/min flow rate over 30 min, followed by 75% acetonitrile for 5 min, was used to separate testosterone metabolites. After separation on the C18 column, samples were passed through a UV detector to determine retention time of cold steroid samples or spiked 14C samples. HPLC elution fractions containing 3H-labeled and nonlabeled steroid standards were run to determine separation efficiency and retention times of the metabolite peaks. proventil inhaler

Samples were collected using a fraction collector. Half of each sample was measured on the LSC and the other half with the testosterone ELISA. Some samples were also measured using a Packard Flo-One Beta Series A-500 ra-dio-chromography in-line detector. The percentage unconjugated testosterone excreted in feces and the number of major metabolites were determined.

This entry was posted in Testosterone and tagged Biomarker, Mus musculus, Peromyscus maniculatus, Testosterone.