Preparative Isolation of Middle Molecular Weight Fractions from the Hemofiltrate of Patients with Chronic Uremia

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Abstract
Fractions in the [molecular weight] range of the so-called middle molecules (500-3000 daltons) were isolated on a preparative scale from hemofiltrate of patients. Hemofiltration was performed with an RP-6 dialyzer using a predilution technique. The hemofiltrate was concentrated and desalinated by reverse osmosis with membranes with a nominal cut-off of 500 daltons. The retentate was freeze-dried, redissolved in volatile buffer and fractionated on Sephadex G-15 macrocolumns. The middle molecule MW range in the elution profiles (detected at 206 and 280 nm simultaneously) was marked with peptides. Isolated fractions in the middle molecule MW range showed strong inhibitory activity upon 3H-thymidine incorporation into rat bone-marrow cells in vitro. The described combination of hemofiltration, reverse osmosis and gel chromatography is suggested as a useful approach to the isolation of these ninhydrin-positive substances which, according to their proved heterogeneity, are to be subfractionated by further procedures.