Steroid Extraction: Get the best out of faecal samples

PALME R; TOUMA C; ARIAS N; DOMINCHIN MF; LEPSCHY M

WIENER TIERARZTLICHE MONATSSCHRIFT

B W K PUBLISHING SOLUTIONS & VERLAG

Año: 2013 p. 238 - 246

0043-535X

Measurements of steroid hormone metabolites in faecal samples as non-invasive parameters for reproductive functions and stress have become increasingly popular. The extraction of these steroids from the faecal matrix represents the initial step before quantification can be performed. The steroid metabolites present in the faecal matrix are of varying polarity and composition. Therefore the selection of a proper extraction procedure is essential. Some studies have already addressed this complex but rather unnoticed matter. Often radiolabelled steroids (e.g. cortisol or progesterone) were added to faecal samples to estimate the efficiency of the used extraction procedures. However, the added native, unmetabolized steroids are normally not present in the faeces and therefore the results are rather artificial and do not accurately reflect the actual recoveries of the substances of interest. In this respect, recovery experiments based on faecal samples from radiometabolism studies are more informative. In these samples, the metabolite content accurately reflects the mixture of metabolites actually present in the given species. Consequently, the evaluation of different extraction methods in these faecal samples, as demonstrated here for studies on sheep, horses, pigs, and dogs, utilized samples containing the naturally metabolised, 14C-labelled steroids. Based on our results, we recommend extracting faecal steroids by simply suspending the faeces in a high percentage of a primary alcohol (for glucocorticoid metabolites 80% aqueous methanol proved best suited for virtually all mammalian species tested so far). This procedure not only significantly increased the total radioactivity recovered, but also the relative portion of unconjugated metabolites, which are more likely to be recognized by the antibodies used in various immunoassays. Therefore the advantages of this extraction procedure are clear: it is very easy to use (no evaporation step is needed), it yields high recoveries and variation based on the extraction procedure is reduced to a minimum.