Identification of alexamorelin consumption biomarkers using human hepatocyte incubations and high-resolution mass spectrometry.
This study investigated the metabolic fate of alexamorelin — a synthetic peptide growth hormone secretagogue (GHS) with potential performance-enhancing properties — to identify biomarkers useful for doping control. Researchers used in silico metabolite prediction software (GLORYx) alongside in vitro incubations with pooled human hepatocytes from 10 donors, analyzing samples via liquid chromatography–high-resolution tandem mass spectrometry (LC-HRMS/MS). GLORYx predicted 21 possible single-reaction metabolites, with N-acetylation ranked highest in probability (98%), and other transformations such as hydroxylation, N-oxidation, and glucuronidation predicted at lower probabilities. However, after 3 hours of hepatocyte incubation, only one metabolite was experimentally detected: examorelin (hexarelin), produced by carboxypeptidase-mediated cleavage of the C-terminal alanine residue. The parent compound decreased approximately 150-fold over the incubation period, indicating rapid and extensive hepatic metabolism. A key limitation is that examorelin is itself a commercially available GHS compound, meaning it cannot serve as a specific biomarker for alexamorelin use. The authors conclude that direct detection of alexamorelin itself remains the most reliable strategy for confirming its consumption in anti-doping contexts. This study was conducted entirely in vitro and does not involve human subjects or animal models.
Why this grade: All experimental work was performed in vitro using pooled human hepatocytes and computational modeling, with no human participants or animal subjects involved, limiting direct clinical extrapolation.
Alexamorelin is a synthetic peptide and growth hormone secretagogue (GHS) with potential performance-enhancing properties, making its use and abuse a topic of interest in clinical research and doping monitoring. Alexamorelin mimics the natural peptide hormone ghrelin by binding to the GHS type 1a receptor (GHS-R1a) in the pituitary gland, thereby promoting endogenous growth hormone release. Identifying alexamorelin and/or its metabolite biomarkers is crucial for effective doping controls. The purpose of this study was to determine and characterize biomarkers associated with alexamorelin intake. In silico metabolite predictions were performed using GLORYx freeware, and in vitro incubations were conducted with pooled human hepatocytes from 10 donors. Samples were analysed using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS), with data processed through Thermo Scientific's Compound Discoverer. GLORYx predicted 21 single-reaction metabolites. N-Acetylation was identified as the primary transformation, with the highest probability score (98%), and occurring either at the C-terminal Ala or the N-terminal Lys. Other predicted transformations included N-oxidation, hydroxylation, amide hydrolysis, oxidative deamination, and phase II N-glucuronidation, with probability scores below 40%. All these transformations were predicted to occur at the two C-terminal (Ala or His) or N-terminal (d-Phe or Lys) amino acids. After 3 h of incubation with hepatocytes, only one metabolite (known as examorelin or hexarelin) was detected, resulting from the C-terminal cleavage of the Ala amino acid; this metabolic reaction is mediated by a carboxypeptidase. The alexamorelin signal decreased approximately 150-fold after 3 h, indicating significant hepatic metabolism. However, examorelin itself is a commercially available GHS secretagogue, and thus, it is not specific to alexamorelin consumption. Detecting alexamorelin remains critical to documenting its use.
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