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The renal erythropoietic factor (REF), in the absence of added normal serum, has been shown to have only slight erythropoietic activity (1,2 ) . Such activity is, however, easily demonstrable after incubation of REF with the serum of normal animals. The REF acts enzymatically on a serum substrate to produce active erythropoietin (ESF) . However, all assays of REF have been performed in vivo and the possibility exists that the serum of the assay mouse may provide the necessary substrate for REF to produce a small amount of ESF. I t is also conceivable that REF contains small amounts of ESF or an inactive precursor of ESF. Further REF studies were, therefore, carried out by use of a hemagglutination-inhibition (HAI) system using a gamma-globulin extract of anti-ESF serum. The results indicate that REF is immunochemically different from ESF, and that, after incubation of REF with normal serum, ESF is generated.Methods. The REF was extracted from the kidneys of five species of mammals ( 1). For rat REF, 110 ml of extract were obtained from 55 g of kidneys from Long-Evans rats ( 2 50-280 g) which had been rendered hypoxic by exposure to 0.42 atm of air for 19 hr. Part of the material was frozen and used immediately after thawing and the other part was lyophilized and kept in the freezer until used. The REF from other species was extracted from normal tissues using the same techniques. The incubation procedure in-1 Supported in part by PHS Research Grants 1 R01 H E 10567-04, 2 R01 H E 03357-11 and Contract No. ,4T-(40-1) -3547 from the Atomic Energy Commission. volved adding REF, dissolved in saline, to equal amounts of normal rabbit serum (NRS) that had previously been dialyzed first against 0.005 M Na2-EDTA and then against water at 4" (1). All test materials ( REF-saline, NRS-saline, REF-NRS ) were incubated for 30 min at 37" and assayed for ESF content by the in vivo and in vitro systems outlined below.The anti-ESF serum used in these studies was produced by immunizing a white New Zealand rabbit with human urinary ESF which was conjugated to methylated rabbit serum albumin by the method of Sueoka and Cheng ( 3 ) . The anti-ESF serum was absorbed with normal human serum and the titer was determined as previously outlined (4). The gamma-globulins were extracted by the method of Stanworth (5).In vivo assays for ESF activity were accomplished by injecting the test materials into ex-hypoxic polycythemic mice ( 6 ) . Five to eight mice were used to test each sample. Each mouse received either saline, ESF standard, serum, or REF-serum, as a single 2.0 ml ip injection on day 3 post-hypoxia. On day 5, 0.5 pCi 59Fe in 0.2 ml of saline was injected iv and on day 7 the mice were killed and the % RBC radioiron incorporation measured.For in vitro studies, a modification of the HA1 system outlined by Lange et al. (4) was used. A 0.075 M phosphate buffer (pH 7.4) was used as the diluent of the sensitized sheep red blood cdls in place of the 1% NRS diluted with 0.9% NaCl. Gamma-globulins were employed in place of whole antiserum. T...

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