Volume 152, August 2015, Pages 180–192
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
Highlights
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- Static equine in vitro digestion simulations: proof of concept.
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- Supplemented ADD can be transformed to β-Bol by the intestinal microbiota.
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- In vitro digestions link phytosterol consumption to the detection of AED.
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- Both AED and ADD are steroids suspected to be β-Bol precursors.
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- Biotransformation ratios of ADD to β-Bol and phytosterols to AED are horse dependent.
Abstract
Traditionally,
steroids other than testosterone are considered to be synthetic,
anabolic steroids. Nevertheless, in stallions, it has been shown that
β-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 β-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
set up, 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 β-Bol gave rise to the formation of ADD
(androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro
digestions, the transformation of ADD to β-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% β-sitosterol) or
phytosterol-rich herbal supplements on the other hand, did not induce
the detection of β-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 β-Bol, and the
consumption of phytosterols to low concentrations of AED, but there is
no direct link between phytosterols and β-Bol.
Graphical abstract
Keywords
- In vitro digestion;
- Hindgut;
- Equine;
- Anabolic steroids;
- Endogenous;
- Phytosterols
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