In vitro simulation of the equine hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic-androgenic steroids in horses

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that beta-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including beta-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were setup, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC MS/MS method. The addition of beta-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or alpha T. Upon addition of ADD to the in vitro digestions, the transformation of ADD to beta-Bol was observed and this for all eight horses' inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% beta-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of beta-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1 ng/mL). As such, the digestive transformation of ADD could be linked to the detection of beta-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and beta-Bol. (C) 2015 Elsevier Ltd. All rights reserved.