Separation of six uremic middle molecular compounds by high performance liquid chromatography and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Background: Since the Postulation of uremic middle molecule (UMM) hypothesi s made by Babb et al. [Trans-Am Soc Artif Intern Organs 18 (1972) 98], ther e has been great interest in the separation and identification of the role of UMM. However, few Of the Compounds isolated from UMM fractions were demo nstrated to play an important role in humans. Thus, the separation and iden tification of the real UMM is essential for UMM research. Methods Urine and serum samples from uremic patients and healthy subjects were separated by gel permeation chromatography. Two presumed UMM fractions, A and B, were ob tained from uremic sera and urine, normal urine, but not normal sera. Fract ion A was further isolated by anion exchange chromatography and a series of sub-peaks were obtained. The sub-fraction A-3 obtained in the second step was desalted on a Sephadex G-15 column, and characterized by IR, UV and mat rix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Further separation of sub-fraction A-3 was performed by hig h performance liquid chromatography (HPLC). Results: By gel permeation chro matography, two UMM peaks (A and B).,re detected at 206 nm in normal urine, uremic sera, but they were barely noticed in the profile of normal sera. I n contrast, the absorption at 206 nm of fractions A and B from uremic serum and urine were smaller than that of fractions A and B from normal urine, F ractions A from different origins were resolved into eight to nine sub-peak s at 230 nm by anion exchange chromatography. One of these sub-peaks. A-3, was detected in uremic serum and normal urine, but is undetectable in uremi c urine. After desalting, sub-fraction A-3 was separated into two parts des ignated as A-3-I and A-3-II. MALDI-TOF-MS revealed that fraction A-3-I and A-3-II from two origins were identical, respectively-fraction A-3-I contain ed three components with MW 839.69, 1007.94 and 2015.16 and fraction A-3-II consisted of another three components with MW 873.69, 1106.67 and 1680.28, Six middle molecular compounds in sub-fraction A-3 were thoroughly resolve d by HPLC. Conclusion: Our results demonstrated that the UMM sub-fraction A -3 contains the real UMM in the MW range of 800-2015 Da. By multi-step chro matographic isolation, six real middle molecular compounds were purified an d characterized with MALDI-TOF-MS. It is likely that three of these UMM com pounds are important. as they readily accumulated in sera of uremic patient s, but are normally excreted in healthy subjects. (C) 2001 Elsevier Science B.V. All rights reserved.