Determination of normal human serum erythropoietin levels, using mouse bone marrow

A simple, rapid, and economic method for measuring epo bioactivity in small samples of normal human serum has been developed. The method requires an initial fractionation of the test sample on WG-Sepharose beads. The material eluted by 1 mM N,N'-diacetylchitobiose is then assayed for its ability to stimulate the uptake of 59Fe into heme by mouse marrow cells under conditions previously optimized for the stimulation of epo-dependent hemoglobin synthesis. Potential secondary effects on 59Fe uptake, due to variations in the iron-transferrin content of different serum samples, were eliminated by the introduction of a centrifugation and cell-wash step prior to the addition of 59Fe. Preliminary chromatography of test samples on WG was necessary because different samples of untreated human serum were still found to give variable secondary effects on 59Fe uptake when assayed in the presence of increasing amounts of an epo standard. These were more clearly revealed when human serum was chromatographed on Sephadex G150, and the presence of a number of "inhibitors" and "stimulators" (ranging from 8000 to more than 150,000 daltons) were demonstrated. In contrast, only material that was additive with epo was demonstrable in the WG column eluate, and this activity coeluted with epo on Sephadex G150. With the WG fractionation procedure, sera from 20 normal volunteers between the ages of 20 and 40 were assayed for epo bioactivity. Epo levels ranging from 0.3 to 1.3 U/ml (average 0.8) were obtained. Values for men and women were not significantly different. Comparison of the extent of 59Fe incorporation obtained before and after WG fractionation showed no correlation, indicating significant natural variation in the relative amounts of non-epo stimulators and inhibitors in the sera of different normal individuals.