DAMAGE OF TRACER ERYTHROPOIETIN RESULTS IN ERRONEOUS ESTIMATION OF CONCENTRATION IN MOUSE SUBMAXILLARY-GLAND

It has been previously reported that 1) plasma erythropoietin (Epo) ti ter during exposure to hypobaria is lower in nephrectomized rats and m ice whose submaxillary glands (SMG) were either ablated or atrophied t han in nephrectomized controls whose SMG were intact and 2) that the g land shows one of the highest levels of immunoreactive Epo (iEpo) in t he body. The latter observation, however, was questioned recently when it was observed that SMG extracts degrade labeled Epo used as tracer antigen in the radioimmunoassay (RIA), thus giving invalid estimates o f Epo. Since this interpretation was in turn questioned, the present s tudy was conducted to obtain more information on the subject and make these conflicting points clear. Investigation of the reported/possible degradation of Epo by SMG homogenates was conducted via polyacrylamid e gel electrophoresis followed by radioautography or by a RIA in solid phase in which there was no simultaneous incubation of the tracer ant igen with the SMG homogenates. It was observed that I-125-labeled rhEp o was degraded when incubated with SMG homogenates. Degradation was ra pid, being evident when incubation lasted 30 minutes, and occurred in the presence of a protease inhibitor. It showed a high degree of speci ficity since it did not occur when Epo was incubated with kidney homog enate or normal mouse serum. SMG homogenate did not degrade labeled th yrotrophic hormone and degraded alpha interferon (IFN-alpha) only part ially. When estimates of iEpo in SMG homogenate were performed in cond itions of simultaneous (SI-RIA) or nonsimultaneous (NSI-RIA) incubatio n of the homogenate with tracer Epo, it was observed that while estima tes of Epo in plasma were similar in both types of RIA and somewhat hi gher in kidney homogenate in the SI-RIA than in the NSI-RIA, estimates of Epo in SMG were about 60 times higher in the former than in the la tter. Therefore, it could be concluded that most of the Epo detected b y standard RIA in SMG homogenate does not represent true Epo because o f damage of tracer Epo which determines loss of the integrity of the R IA system.