Detection and quantification of the metabolite Ac-Tβin in vitro experiments and urine of rats treated with Ac-Tβ4: A potential biomarker of Ac-Tβ4 for doping tests.

Thymosin β4 (Tβ4) was reported to exert various beneficial bioactivities such as tissue repair, anti-inflammation, and reduced scar formation, and it is listed on the prohibited substances in sports by the World Anti-Doping Agency. However, no metabolism studies of Tβ4 were reported yet. Previously, our lab reported in in vitro experiment that a total of 13 metabolites were found by using multiple enzymes, and six metabolites (Ac-Tβ, Ac-Tβ, Ac-Tβ, Ac-Tβ, Ac-Tβ, and Ac-Tβ) were confirmed by comparing with the synthetic standards. This study was aimed at identifying new metabolites of Tβ4 leucine aminopeptidase (LAP), human kidney microsomes (HKM), cultured huvec cells, and rats after administration of Tβ4 protein to develop biomarkers for detecting doping drugs in sports. A method for detecting and quantifying Ac-Tβwas developed and validated using Q-Exactive orbitrap mass spectrometry. The limit of detection (LOD) and limit of quantification (LOQ) of the Ac-Tβwere 0.19 and 0.58 ng/mL, respectively, and showed a good linearity (r = 0.9998). As a result, among the six metabolites above, Ac-Tβ, as a common metabolite, was found in LAP, HKM, huvec cells exposed to Tβ4, and the urine of rats intraperitoneally treated with 20-mg/kg Tβ4. And the metabolite Ac-Tβwas quantitatively determined by 48 h in rats, with the highest concentration occurring between 0 and 6 h. Ac-Tβwas not detected in non-treated control groups, including human blank urine. These results suggest that Ac-Tβin urine is a potential biomarker for screening the parent Tβ4 in doping tests.