The extraction of tritiated testosterone metabolites from pooled fecal samples using pentane:ethyl acetate (80:20) and ethyl ether resulted in 82% and 86% recovery, respectively. Decreasing the sample size to less than 30 mg and increasing the solvent volume to 4 ml increased the efficiency to 90%. buy birth control online
Ether extraction of pooled fecal samples wetted with 10% methanol gave the highest recovery of free (ether soluble) testosterone, 94.9% (Table 1). Ether extraction without wetting recovered 74.1%. Solubilization with 10% methanol recovered only 35.7%. All fecal samples were wetted for the second extraction with the 250 pl H2O that had been added during the first extraction.
The addition of (3-glucuronidase to the fecal sample with subsequent ether extraction did not increase the amount of testosterone measured with the ELISA in comparison to that in nonhydrolyzed controls. The extraction efficiency of the polar and nonpolar radiolabeled testosterone metabolites excreted into feces was measured using the 10% methanol solubilization method and recovered 87 ± 2% (n = 3, mean ± SD).
Radiolabeled Testosterone Metabolism
Averages of 5.61 ± 0.62 g and 10.43 ± 1.74 g feces total weight and of 10.74 ± 5.38 ml and 9.78 ± 0.73 ml total volume were collected per animal for Mus and Pero-myscus, respectively (n = 3, mean ± SD). The proportion of the injected testosterone excreted in feces was 62 ± 14% for Mus and 58 ± 13% for Peromyscus.
TABLE 1. Amount and percentage of uncogugated testosterone extracted.
|Parameter||10% Methanol solubilization||Etherextraction||Ether extraction with wetting|
|Primary extraction*||+ 1||114 ± 8||150 ± 6|
|Additional ether extrac||115 ± 11||40 ± 5||8 ± 1|
|% Extraction of ether-soluble testosterone of primary extraction**||36||74||95|
* Values for the amount of unconjugated testosterone extracted represent nanograms testosterone per gram of feces.
** Percentage recovery is based on the maximum recovery of a second ether extraction.