# Identification of Metabolites of the Dietary Supplement Ingredient 5α‐Hydroxy‐Laxogenin

## TL;DR

This study identifies metabolites of 5α-hydroxy-laxogenin, a substance found in dietary supplements, using human cells, rats, and computer models.

## Contribution

The study identifies and characterizes specific metabolites of 5α-hydroxy-laxogenin in human and rat models.

## Key findings

- Five metabolites were predicted in silico and five were generated in human HepG2 cells.
- Three of these metabolites were also detected in rat serum samples.
- Hair analysis suggests contamination from saliva or urine, complicating interpretation.

## Abstract

Illegally added, undeclared dietary supplement ingredients represent one way of unintentional exposure to substances that are prohibited by the World Anti‐Doping Agency (WADA). In addition to the risk of anti‐doping rule violation, adulterated dietary supplements can also be a health risk for elite and recreational athletes. 5α‐hydroxy‐laxogenin is one of those illegal substances but is not prohibited yet. Though an in vitro bioassay showed androgen receptor activation, this androgenic effect could not be confirmed in the preclinical orchiectomized rat model, which might be due to a fast biotransformation into inactive metabolites. In the present study, we investigated the metabolism of 5α‐hydroxy‐laxogenin using three different approaches: in silico prediction using the GLORYx tool of NERRD, in vitro biotransformation using the human hepatocellular cell line HepG2 and the preclinical orchiectomized rat model, from which serum and hair samples were collected and investigated. Analyses were performed using high‐performance liquid chromatography‐high‐resolution mass spectrometry (HPLC‐HRMS). In silico metabolism predicted five different metabolites resulting from hydroxylation, reduction, or oxidation. The human HepG2 cells generated five metabolites resulting from reduction or hydroxylation and from a combination of both. Three metabolites generated in vitro and an additional mono‐hydroxylated metabolite were detected in the serum of the rats treated daily subcutaneously with 5α‐hydroxy‐laxogenin for 2 weeks. Hair analysis points to incorporation of 5α‐hydroxy‐laxogenin, though showing a rather high contamination most likely by saliva or urine. In conclusion, we identified and characterized metabolites of 5α‐hydroxy‐laxogenin, for which, however, the exact modification sites need to be investigated in future.

The biotransformation of the dietary supplement ingredient 5α‐hydroxy‐laxogenin was investigated using human HepG2 cells, the rat and in silico using the online software Next‐gen E‐Resource for Drug Discovery (NERDD). We identified five metabolites generated by the human HepG2 cells of which three and an additional mono‐hydroxylated metabolite were also found in the rat serum samples. The in silico prediction yielded five metabolites. Four of those metabolites most likely coincides with the in metabolites detected in vitro and in vivo respectively and might be suitable as targets for 5α‐hydroxy‐laxogenin screening in serum.