Discrimination of recombinant and endogenous urinary erythropoietin by calculating relative mobility values from SDS gels.

Erythropoietin (EPO) promotes the production of red blood cells, the key factor in the regulation of the oxygen transport, and has been abused by athletes for performance enhancement in endurance sports. Current methods to detect EPO misuse are based on isoelectric focussing (IEF), double blotting, and chemiluminescence detection. A new approach utilizing SDS-PAGE mobilities of target analytes is presented. Employing two internal standards (novel erythropoiesis stimulating protein and recombinant rat EPO), the assay provides a tool which allows the calculation of relative mobility values for endogenous urinary EPO and recombinant epoetins (e.g., Dynepo) and, thus, the distinction of these analytes in doping control samples. A reference group of 53 healthy volunteers and samples originating from a Dynepo (epoetin delta) excretion study conducted with a single person were analyzed and led to a significant discrimination of endogenous urinary and recombinant EPO. A clear differentiation was accomplished over a period of four days post-administration of a single injection of 50 IU/kg body weight. Hence, the method may be useful as a screening procedure in doping control or as complementary confirmation tool to the established IEF assay.

Apoptotic volume decrease, pH acidification and chloride channel activation during apoptosis requires CD45 expression in HPB-ALL T cells.

Mitochondrial-perturbating agents such as toxic coumponds induce apoptosis. We note that the loss of CD45 expression in the lymphoblastic leukemia cell line HPB-ALL (HPB45.0) leads to an inhibition of nuclear apoptosis. Our hypothesis is that the absence of CD45 disturbs protein function regulated by a proto-oncogene of the Src family playing a significant role in nuclear apoptosis. In this work we explore the importance of a chloride efflux on DNA fragmentation. The role of tyrosine kinase in the function and regulation of the chloride channels was determined. Our results showed a disturbance of ionic homeostasis in CD45 deficient lymphocytes (CD45-) in contrast to normal lymphocytes (CD45+). The phosphorylation levels of the chloride channels are considerably inhibited in CD45-, while the expression levels of these channels are similar in the two types of cells. A hypertonic medium inhibits DNA fragmentation in CD45+ while a hypotonic medium increases DNA fragmentation in CD45-. Thus CD45 plays a significant role in nuclear apoptosis by the regulation of the chloride channels responsible for ionic homeostasis of the cell essential for the DFF40 activation.