Separation of an inhibitor of erythropoiesis in "middle molecules" from hemodialysate from patients with chronic renal failure.

A "middle molecule" inhibitor of erythropoiesis in patients with chronic renal failure was separated from a large amount of hemodialysate. Hemodialysate was passed through Amberlite XAD-4 resin, Sephadex G-50 gel, and DEAE-Sephadex A-25, followed by reversed-phase liquid chromatography. The erythroid colony assay (CFU-E) was used to detect the inhibitory effect of the sample solution. Certain fractions from the DEAE-Sephadex A-25 column showed a dose-related inhibition of CFU-E formation as great as that of standard spermine. The inhibitory effect of these fractions decreased to the control value after proteolytic digestion. The inhibitor was eluted from the liquid-chromatographic column by a solvent gradient containing 390 to 425 mL of methanol per liter. These results suggest there is an inhibitor of CFU-E with a relative molecular mass of 1000 to 10 000 and an active site composed of peptide. This technique may prove useful for separation of the inhibitor of erythropoiesis in uremic body fluid.