Identification of alexamorelin consumption biomarkers using human hepatocyte incubations and high-resolution mass spectrometry.
The study found that alexamorelin undergoes significant hepatic metabolism, with its signal decreasing approximately 150-fold after 3 hours, but the detected metabolite is not specific to alexamorelin consumption.
Where it sits
this study against the rest of the alexamorelin corpusSummary and findings
This study aimed to identify biomarkers associated with alexamorelin intake using human hepatocyte incubations. The researchers performed in silico metabolite predictions and analyzed samples via liquid chromatography-high-resolution tandem mass spectrometry. Significant hepatic metabolism of alexamorelin was observed, with a notable decrease in its signal after incubation.
Abstract
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.
Background
This paper addresses the need for effective doping monitoring related to alexamorelin, a synthetic peptide that mimics ghrelin and may enhance performance. Prior knowledge indicates that identifying biomarkers for such substances is essential for doping controls. This study is significant as it explores the metabolic pathways of alexamorelin and aims to provide insights into its detection.
Methods
The study utilized pooled human hepatocytes from 10 donors for in vitro incubations. In silico metabolite predictions were conducted using GLORYx freeware, and samples were analyzed using liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). The primary outcome was the identification of metabolites associated with alexamorelin intake.
Results
The primary endpoint indicated that the alexamorelin signal decreased approximately 150-fold after 3 h of incubation with hepatocytes. N-acetylation was the main predicted transformation with a probability score of 98%. Only one metabolite, examorelin, was detected, resulting from the C-terminal cleavage of the Ala amino acid.
Interpretation
The findings suggest that while significant hepatic metabolism of alexamorelin occurs, the detection of its metabolites, particularly examorelin, may not be specific to alexamorelin use. The effect size of the signal decrease is notable but does not imply clinical significance without further in vivo studies. Confounding factors include the use of pooled hepatocytes and the potential overlap with commercially available metabolites.
Key findings
- 21 single-reaction metabolites predicted using GLORYx freeware.
- N-acetylation identified as the primary transformation with a probability score of 98%.
- Alexamorelin signal decreased approximately 150-fold after 3 h of incubation.
- Only one metabolite, examorelin, was detected after 3 h, resulting from C-terminal cleavage of the Ala amino acid.
Limitations
- In vitro study may not reflect in vivo metabolism.
- Detected metabolite, examorelin, is commercially available and not specific.
- Small sample size of 10 donors for hepatocyte pooling.
- Short incubation duration of 3 hours may limit comprehensive metabolic profiling.