Separation and analyses of uremic middle molecules by chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Sera and urine from patients with severe uremia and healthy subjects were s eparated by means of gel permeation chromatography on Sephadex G15 column w ith N(C2H5)(3)-H2CO3 buffer as eluent. Two middle molecular peaks (A and B) were detected at 206 nm in normal urine, uremic serum and uremic urine, bu t these two peaks were hardly observed in the profile of normal sera. In co ntrast, the absorption at 206 nm of fractions A and B from uremic serum and urine were less than that of fractions A and B from normal urine. Fraction s A from normal urine, uremic serum and urine were collected and resolved i nto 8 to 9 subpeaks at 230 nm by anion exchange chromatography. One of thes e subpeaks, A-3, was detected in uremic serum and normal urine but undetect able in uremic urine. After a gel permeation chromatography with bidistille d water as eluent for desalting, subfraction A-3 was separated into two par ts designated A-3-I and A-3-I in order. The results of MALDI-TOF-MS reveale d that the two peaks from both samples were identical respectively, fractio n A-3-I contained three kinds of components with molecular weight 839.69, 1 007.94 and 2 015.16 and fraction A-3-I consisted of other three kinds of c omponents with molecular weight 873.69, 1 106.67 and 1 680.28.