Protein hormones
Currently, in human sports drug testing, methods are accepted for the analysis of hCG and for EPO only. Tests for hCG are based on immunoprocedures (Cowan et al. 1991; Laidler et al. 1994). A method to identify the different isoform pattern of recombinant EPO from that of endogenous material using isoelectric focusing (Figure 1) has been developed recently (Lasne and Ceaurriz 2000; Lasne 2001), whereas a method based on the perturbation of a number of blood parameters (haematocrit, reticulocyte haematocrit, percentage macrocytes, serum EPO and soluble transferrin receptor; Parisotto et al. 2001) was used at the Sydney Olympics in 2000. New forms of EPOs are now reaching the marketplace. Catlin et al. (2002) have published the detection of the administration of darbepoietin, a novel erythrocyte stimulating protein (with a half-life about three times that of endogenous human EPO), using the isoelectric focusing method of Lasne and Ceaurriz (2000). Interestingly, this method has been used to confirm the presence of recombin ant EPO in the canine as well as detecting human urine substituted for canine urine (Bartlett et al. 2006). A test to prove growth hormone (GH) admin istration was first adopted at the Athens Olympics in 2004. Since recombinant GH does not contain the 20 kDa isoform, the absence of this isoform is the basis of this test (Wu et al. 1999). An alternative approach is based on using a discriminant function with the GH-sensitive substance insulin-like growth factor 1 (IGF-1) produced by the liver and procollagen type III (P-III-P) (Sonksen 2001; Powrie et al. 2007). This latter approach has the benefit of a longer detection time than the isoform method. Rumour concerning the abuse of GH in horseracing has been circulating for a number of years, but interest in developing methods for its control intensified in 1998 with the approval, in Australia, of recombinant equine GH for veterinary use. IGF-1 has been confirmed as a universal marker to detect abuse of the three commercially available recombinant GH variants (equine, porcine and bovine GH) which were shown to be active in the horse (De Kock et al. 2001b). However, as IGF-1 is endogenous to the horse, the current international rules of racing would require its control through a threshold value.
In a collaborative study, IGF-1 levels in normal serum samples of horses in South Africa, Australia and the UK were determined by an immunoradiometric assay (IRMA) and were shown to have a distribution close to normal (De Kock et al. 2001c). Also, significant increases in IGF-1 levels in serum have been demonstrated after GH administration (De Kock et al. 2001b; Faustino-Kemp et al. 2001; Noble and Sillence 2001; Popot et al. 2001a, 2001b). LC-MS methods for IGF-1 determination have been developed (Bobin et al. 2001; De Kock et al. 2001a) to support screening on the basis of immunochemical methods. In addition, LC-MS has been shown to provide a viable approach for the quantification of IGF-1 (Popot et al. 2001c; Guan et al. 2007).
Misuse of recombinant human erythropoietin (rhEPO) in horses, supposedly to improve per formance, has received wide publicity. However, the horse is a natural blood doper in that, under exercise, contraction of the spleen can increase haematocrit by 33%. Thus, misuse of rhEPO presents a serious threat to the welfare of the horse, as its administration coupled with splenic contraction could result in a marked increase in blood viscosity. This increase in an animal undergoing severe exercise could have serious health implications. Some physiological implications of rhEPO administration to horses were reviewed by McKeever (1996). ELISA tests to detect rhEPO administration to horses and greyhounds have been evaluated and shown to be effective in serum (Tay et al. 1996). The double Western blotting technique developed by Lasne and Ceaurriz (2000) for EPO analysis in urine has also been evaluated in the horse (Y. Bonnaire, LAB, France, personal communication) and shown to be effective. As the natural equine EPO does not cross-react with the antibody used in the test, the test provides a better distinction between positive and negative samples when applied to equine urine as opposed to human urine.
Figure 1 Autoradiograph of isoelectric patterns of exogenous and endogenous erythropoietin (EPO). Images were obtained by chemiluminescent immunodetection of blotted EPO after isoelectric focusing. Lanes: α, Purified commercial human urinary natural EPO (Sigma); b, recombinant EPO-β (Neorecormon, France); c, recombinant EPO- α (Eprex, France); d, urine from a control subject; e, f, urine from two patients treated with Neorecormon EPO for post-haemorrhagic anaemia; g,h, urine from two cyclists from Tour de France 1998 (samples concentrated by ultrafiltration). Note the ‘mixed’ appearance of the pattern in lane e. The cathode is at the top; pH values are indicated on the left.
