The urinary metabolite Ac-Tβ in huvec cells and rats treated with thymosin β4:A potential biomarker for doping tests

Abstract

Thymosin β4 (Tβ4) was reported to exert various beneficial bioactivites such as tissue repair and regeneration, anti-inflmmation, and reduced scar formation, and it is listed on the prohibited substnaces by the World Anti-Doping Agency. No metabolism studies of Tβ4 were not reported yet. Previously, our lab reported that total 13 metabolites were found by using multiple enzymes, and 6 metabolites (Ac-Tβ31-43, Ac-Tβ1-11, Ac-Tβ17-43, Ac-Tβ1-14, Ac-Tβ1-15, and β1-17) were confirmed by comparing with the synthetic standards. The study was aimed at identifying new metabolites of Tβ4 in biological systems such as leucine aminopeptidase, human kidney microsome, huvec cells and rats to develope biomarkers for detecting doping in sports. A method for detecting and quantifying Ac-Tβ1-14 was developed and validated using Q-Exactive orbitrap mass spectrometry. LOD and LOQ of the Ac-Tβ1-14 were 0.19 and 0.58 ng/ml and showed a good linearity (r2=0.9998). This work was conducted in in vitro pure enzyme and microsome systems, cultured huvec cells, and rats after administration of parent protein. Among 6 metabolites, Ac-Tβ1-14 was found in pure enzyme and microsome systems, in huvec cells exposed to 10 μg/ml, and in urine of rats intraperitoneally treated with 20 mg/kg Tβ4. As a result, Ac-Tβ1-14, the metabolite of Tβ4, was detected in all in vitro biological systems including enzyme systems, huvec cells, and urine samples of the rat. In addition, the metabolite Ac-Tβ1-14 was quantitatively determined to 48 hours in the rats, with the highest concentration occurred between 0-6 hrs. Ac-Tβ1-14 was not detected in non-treated control groups including human blank urine. This results strongly suggest that Ac-Tβ1-14 in urine is a potential biomarker for screening the parent Tβ4 in doping tests.

Keywords: Tβ4, Ac-Tβ1-14, urine, huvec cells, rats, biomarker, doping