51
Figure 2: Quadratic calibration curves of PSOs in deproteined horse plasma on Day 1 and Day 2.
been approved by the US and EU drugs and medicines regulatory authorities (FDA and EMA respectively) [8]. However, with this powerful new technology comes the potential to misuse it [4].
Detection is essential to deterring gene doping
It is to counter of this potential abuse in sports, particularly in horse racing, that countries like the UK have already implemented research programs scheduled to span the next 5 years [2]. Genetic technologies could be used to enhance the performance of racehorses [4]. Genetic expression could be manipulated to improve muscle strength and sprinting ability, oxygen delivery or blood flow to muscles for enhanced endurance, energy metabolism in muscles to resist fatigue, pain alleviation or injury repair for post-race recovery [4,5]. Similarly, the performance of rival horses may be diminished by impairing any of these mechanisms through gene doping [4]. These genetic manipulations may also be applied to the germline of thoroughbreds, meaning
that not only the doped horse is affected but also potentially all of its offspring [4,5]. This would massively disrupt the selective breeding of thoroughbred horses with pedigrees and heritage traceable for generations, some all the way back to the 18th century when Arabian stallions were first introduced and bred with mares in the UK [4,13]. Moreover, as this technology is still relatively new, there is a lot we still do not know about its adverse effects, especially in the long term, which may prove to be highly detrimental and irreversible [4,5].
Although there has not yet been a gene doping incident detected in horseracing or breeding, the possibility of this form of doping is very real [2]. The rapid development of this technology may mean the eventual availability of a do-it-yourself gene-doping kit in the not so distant future [2]. In order to get out in front of this potentially deeply persistent form of doping, it is vital that robust and reliable methods of detection are developed and implemented [2,4,5,14]. An analytical method has been developed to detect oligonucleotides synthesised for gene doping. This method
relies on the detection of a key chemical moiety that has commonly been introduced to improve the structural integrity and stability of oligonucleotides, making them more resistant than native nucleic acids to degradation [15]. The moiety is that of phosphorothioate (PS) modification of nucleic acids, a modification that does not naturally occur in animals (see Figure 1) [14]. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyse horse plasma, an ideal matrix for gene doping tests, to identify and quantify any PS oligonucleotides (PSOs) present.
Experimental
Sample preparation To test the method developed to detect PSOs in equine serum, samples were prepared from equine plasma. One set of samples would be blank samples, serving as negative controls. Another set of samples would be spiked with PSOs, serving as the test samples. Horse plasma was obtained by centrifugal separation of whole blood collected with EDTA. The plasma was
Figure 3: Total ion chromatogram (TIC) of non-targeted analysis of PSOs in deproteinated horse plasma with 4.5 µg/mL of oligo dT (blank sample control; left graph), and with 100 ng/ml PSO (spiked sample containing Oligo 1; right graph).
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