Combination of conventional and high-performance liquid chromatographic techniques for the isolation of so-called "uraemic toxins"

Abstract

Using fluids from the artificial kidney as an example, a generally useful combination of separation techniques is described for the preparative isolation of biologically active subfractions from extremely heterogeneous and diluted biological fluids. Haemofiltrate (20 l) and dialysate (100 l), respectively, are desalted and concentrated in one step by reverse osmosis using membranes with a nominal cut-off of 500 Daltons. The retentate with high concentrations of "uraemic toxins" is fractionated by preparative ion-exchange chromatography (double column technique with detection at 206 nm) and size exclusion chromatography yielding large amounts of ninhydrin-positive subfractions which inhibit DNA synthesis of rat bone marrow and HeLa cells in vitro, respectively. These fractions were analyzed by reversed-phase and size exclusion high-performance liquid chromatography. Many of the isolated fractions were shown to contain peptides.