title: Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones.
abstract: Erythropoietin (EPO) is normally present in urine at a low concentration (about 1IU/L, i.e., about 10ng/L) for a total protein concentration of at least 50mg/L. A method to study the isoelectric profile of this hormone from 20-ml urine aliquots without previous purification was developed. This method involves isoelectric focusing of the retentate from ultrafiltered urine. Both the ultrafiltration and the isoelectric focusing required precautionary measures to prevent EPO degradation by the proteases that are present in urine. Because classical immunoblotting gave rise to an unspecific detection of various urinary proteins in the focused retentate, it was essential to use the "double-blotting" process developed to solve this problem. Sufficient sensitivity was achieved using amplified chemiluminiscent detection after the blotting membrane was treated with dithiotreitol. The patterns that were revealed from various urinary samples proved to be highly heterogeneous as they were composed of more than 10 isoforms in a pI range of 3.7-4.7. Clear transformation of the patterns was observed in the case of treatment by the recombinant hormone, suggesting that this method can be regarded an efficient tool for indicating recombinant EPO misuse in sports. It may also open new investigations in the field of physiologic or pathologic exploration.
authors.affiliation: Laboratoire National de Dépistage du Dopage, 143 Avenue Roger Salengro, 92290 Châtenay-Malabry, France. f.lasne@lndd.com
authors.forename: Françoise
authors.initials: F
authors.lastname: Lasne
authors.forename: Laurent
authors.initials: L
authors.lastname: Martin
authors.forename: Nathalie
authors.initials: N
authors.lastname: Crepin
authors.forename: Jacques
authors.initials: J
authors.lastname: de Ceaurriz
mesh_terms: D015153:Blotting, Western / Q000379:methods; D004921:Erythropoietin / Q000008:administration & dosage / Q000302:isolation & purification / Q000652:urine*; D006728:Hormones / Q000302:isolation & purification / Q000652:urine; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods* / Q000592:standards; D020033:Protein Isoforms / Q000302:isolation & purification / Q000652:urine; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000302:isolation & purification / Q000652:urine; D012680:Sensitivity and Specificity; D014462:Ultrafiltration
chemical_list: D006728:Hormones; D020033:Protein Isoforms; D011994:Recombinant Proteins; D004921:Erythropoietin