Bound amino acids in uremic sera: search for peptidic middle molecules by preparative polyacrylamide gel...

The samples of normal and uremic sera were ultrafiltered, separated on SEP-PAK C18 cartridges and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The serum material extracted from the slab gel was purified from SDS and further fractionated by reverse-phase high performance liquid chromatography (HPLC). The obtained fractions were examined by amino acid analysis and mass spectrometry. An increased number of moderately polar fractions containing bound amino acids was found in sera of uremic patients on hemodialysis. Two most prominent uremic fractions corresponded to N-benzoylglycine (hippuric acid) (fraction I, k' = 9,4) and phenylacetylglutamine (fraction II, k' = 9,7). Increased amounts of bound glutamine, glycine, serine, leucine, asparagine, alanine, valine, phenylalanine were found in other moderately polar uremic fractions. These fractions (k' range from 12.1 to 13.2) contained no free amino acids, nor any other known small uremic serum compounds; they were considered as peptide 'middle molecules' (MM) of a molecular mass smaller than 1700.