Technical aspects on middle molecules: separation, isolation, and identification

For the last few years we have attempted to separate and isolate from biological fluids the uremic Middle Molecules (MM). The first step in the treatment of plasma is an ultrafiltration through AN69 membrane. The samples are fractionated by Sephadex G-15 chromatography into 9 peaks "a" to "i' under precise conditions that have been selected in order to exhibit different patterns in the MM range (peak b and c) between uremic and uremic polyneuropathic patients. Further separation of peak b is performed by ion exchange chromatography with Sephadex DEAE A-25 into 7 sub-peaks b; sub-peak b 4-2 is the only one to correlate with neuropathy. Prior to b 4-2 identification studies, specific large scale isolation procedures are necessary. The purity of the product is monitored at each step by thin-layer chromatography on silica gel.