Research article
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Abstract
A veterinary preparation known as TB-500 and containing a synthetic version of the naturally occurring peptide LKKTETQ has emerged. The peptide segment 17LKKTETQ23 is the active site within the protein thymosin β4 responsible for actin binding, cell migration and wound healing. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. TB-500 is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. In order to control the misuse of TB-500 in equine sports, a method to definitely identify its prior use in horses is required. This study describes a method for the simultaneous detection of N-acetylated LKKTETQ and its metabolites in equine urine and plasma samples. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction using ion-exchange cartridges, and analysed by liquid chromatography–mass spectrometry (LC/MS). These analytes were identified according to their LC retention times and relative abundances of the major product ions. The peptide N-acetylated LKKTETQ could be detected and confirmed at 0.02 ng/mL in equine plasma and 0.01 ng/mL in equine urine. This method was successful in confirming the presence of N-acetylated LKKTETQ and its metabolites in equine urine and plasma collected from horses administered with a single dose of TB-500 (containing 10 mg of N-acetylated LKKTETQ). To our knowledge, this is the first identification of TB-500 and its metabolites in post-administration samples from horses.
Highlights
► A veterinary preparation TB-500 (containing of N-acetylated LKKTETQ) has emerged. ► The amino acid sequence matching an active region (17LKKTETQ23) of the thymosin β4. ► Famous trainers experienced massive boosts in performance after its use on animals. ► First report of a method to control the misuse of TB-500 in horses. ► First identification of TB-500 and its metabolites in post-administration samples.
Keywords
TB-500
;
Thymosin β4
;
Horse urine
;
Horse plasma
;
Liquid chromatography–mass spectrometry
Introduction
Thymosin β4 is the most abundant member of a large family of highly conserved, small bioactive proteins [1], [2]. It consists of 43 amino acids and is present in different cells (except red blood cells) and body fluids like saliva, tears, blood and wound fluid. It has many structural functions via its actin-binding domain and biological effects on cells. Thymosin β4 has the potential of playing a significant role in tissue development, maintenance, repair and pathology [3]. It has been shown to promote cell migration [4], angiogenesis [5], [6], and to protect cells and tissues from oxidative damage [7]. It is critical for the repair and regeneration of the eye [8], heart [9], [10], skin [8], nerves and brain tissue [11], [12] following injury. Many of these activities are localised in distinct amino acid-specific peptide segments [3]. At least three different active sites have been identified, including peptide segment Ac-1SDKP4 for anti-inflammation and angiogenesis; 17LKKTETQ23 for actin binding, cell migration and wound healing; and Ac-1SDKPDMAEIEKFDKS15 for cytotoxicity protection. The activity and role of these three sites should be of great research interest for the development of targeted and specific therapeutics.
The peptide segment 17LKKTET22 was initially reported as a major actin-sequestering site on thymosin β4 [2]. With an additional amino acid, the segment LKKTETQ has demonstrated several biological functions beyond actin-binding, including exocytosis induction [13], dermal wound healing [14], angiogenesis [5] and hair growth [14]. In 2011, a veterinary preparation called TB-500 and containing the synthetic version of this naturally occurring peptide segment was marketed for equine or greyhound use. The key ingredient of TB-500 is the peptide LKKTETQ with artificial acetylation of the N-terminus. This product is claimed to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. It is said to help wound healing, muscle building and recovery of muscles fibres and cells. There have been rumours that some of the world's famous trainers had experienced massive boosts in performance after using of this preparation on their animals [15]. Although the preparation is declared ‘100% drug free’ and ‘does not swab’, TB-500 (N-acetylated LKKTETQ) is clearly prohibited in horse racing as implicit in Clause 10 of Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities) [16]. In order to control the misuse of TB-500 in equine sports, a definitive method to identify the prior use of TB-500 in horses is required.
The objective of this study was to develop a sensitive mass spectrometry based method for the identification of TB-500 (N-acetylated LKKTETQ) and its metabolites in biological samples from horses. Blood had long been considered a more suitable specimen for the doping control testing of protein- and peptide-based drugs. However, more and more reports on their detection in urine from horses [17] and humans [18] have recently been published. In view of this, the identification method for N-acetylated LKKTETQ was developed for equine urine as well as equine plasma. The metabolism of peptides occurs mainly in blood, liver and kidney in human [18]. The plasma half-life of peptide-based drugs is often short and limited to only a few minutes. It is thus important to try to identify their metabolites with a longer detection time. The possible metabolites of N-acetylated LKKTETQ were first identified from in vitro studies. The parent peptide and its metabolites were isolated from equine urine or plasma by solid-phase extraction (SPE) and analysed by liquid chromatography–mass spectrometry (LC/MS). In order to demonstrate the applicability of this method in doping control, plasma and urine samples collected from horses having been administered with TB-500 were successfully analysed by the developed method.
Section snippets
Materials
TB-500 was obtained from Medivet (NSW, Australia). Growth hormone releasing peptide-2 was from Prospectany Technogene (Rehovot, Israel). l-Ala2-dermorphin was from Genway (San Diego, CA, USA). l-Ala2-dermorphin was purchased from Genway as dermorphin and its identity (Tyr-(l-Ala)-Phe-Gly-Tyr-Pro-Ser-NH2) was confirmed in-house. Ammonia (25%, Emsure®), acetic acid (100%, Suprapur), formic acid (>98%, Suprapur), hydrochloric acid (30%, Suprapur) and methanol (LC gradient grade; LiChrosolv®) were
Identification of N-acetylated LKKTETQ
As mentioned previously, 17LKKTETQ23 is believed to be one of the active sites of thymosin β4. Thymosin β4 is naturally present in the body and its amino acid sequence is common amongst various species, like equine, human, bovine and rat. Thymosin β4 consists of 43 amino acids with average molecular mass of 4921.4 Da. The peptide fragment LKKTETQ is specific for thymosin hormone as indicated by the BLAST search results against the non-redundant protein database of The National Centre for
Conclusion
TB-500 is an emerging veterinary preparation for the racing industry, and claims to promote endothelial cell differentiation, angiogenesis in dermal tissues, keratinocyte migration, collagen deposition and decrease inflammation. The key ingredient in TB-500 is a synthetic peptide N-acetylated LKKTETQ, with an amino acid sequence matching an active region (17LKKTETQ23) of the thymosin β4 protein. Although peptide LKKTETQ could be a natural degradation product of thymosin β4, acetylation of the
Acknowledgements
The authors would like to thank Mr Chris Szeto and Mr Richard Chan for their technical assistance.
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