A uremic toxic compound with molecular weight 1007.94 was determined to be an octapeptide by mass spectrometry.Its amino-acid sequence was given as follos: Val-Val-Arg-Gly-Cys-Thr-Trp-Trp.Spin systems for amino acid residues in the octapeptide were identified through analysis of 2D NMR 1H-1H DQF-COSY,TOCSY and ROESY spectra acquired in H2O and D2O.Moreover,the complete assignment of proton resonances for the backbone and side chain was achieved.Based on the secondary chemical shift（Δδ） of the residues,the secondary structure of octapeptide was surveyed.Conformational analyses according to Chemical Shift Index（CSI） showed that the secondary structure of the octapeptide was principally α-helix.The CD spectra of the peptide in aqueous solution gave the same result.Additions of linear polymers made the conformations of octapeptide stretch.These experimental results provide a basis for further comprehension of interaction regulation between biomacromolecule and polymer absorbing materials.
Sera and urine from patients with severe uremia and healthy subjects were seperated by means of gel permeation chromatography on Sephadex G15 column with N(C 2H 5) 3 H 2CO 3 buffer as eluent. Two middle molecular peaks(A and B) were detected at 206 nm in normal urine, uremic serum and uremic urine, but these two peaks were hardly observed in the profile of normal sera. In contrast, 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. Fractions A from normal urine, uremic serum and urine were collected and resolved into 8 to 9 subpeaks at 230 nm by anion exchange chromatography. One of these subpeaks, A 3, was detected in uremic serum and normal urine but undetectable in uremic urine. After a gel permeation chromatography with bidistilled water as eluent for desalting, subfraction A 3 was seperated into two parts designated A 3 Ⅰ and A 3 Ⅱ in order. The results of MALDI TOF MS revealed that the two peaks from both samples were identical respectively, fraction A 3 Ⅰ contained three kinds of components with molecular weight 839.69, 1 007.94 and 2 015.16 and fraction A 3 Ⅱ consisted of other three kinds of components with molecular weight 873.69, 1 106.67 and 1 680.28.
Isolation and comparison of uremic sera and urine and normal sera and urine were performed by gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography. Two uremic middle molecular fractions (A and B) were obtained from uremic sera and urine and normal urine by gel permeation chromatography, but not from normal sera. The anion exchange chromatographic results of fraction A from different origins demonstrate that subfraction A-3 could be excreted in urine by healthy subject, but accumulated in uremic serum for renal failure of patient with uremia. After desalinization subfraction A-3 was analyzed by MALDI-TOF-MS. The results show that subfraction A-3 consists of six compounds with molecular weight 839, 873, 1007.94, 1106, 1680 and 2015 respectively. Finally, by reversed-phase high performance liquid chromatography, subfraction A-3 was further resolved into six independent fractions. Thus, the isolation and purification of six middle molecular compounds in subfraction A-3 came true by our method.
Chitosan resins, which clinically served as adsorbents in hemoperfusion therapy, were prepared with reversed-phase suspension methodology using three differently structured crosslinking agents, methanal, glyoxal and glutaraldehyde. And the glyoxal and glutaraldehyde crosslinked chitosan resins were reduced with NaBH4 afterwards. By analyzing the results from FTIR and SEM, it was found that the reduction treatment to the adsorbents efficiently improved the chemical stability of these chitosan resins, and the shifts in crosslinking agents exerted influences over the morphologies of the adsorbents obviously. After being put to use in the adsorption tests upon some model uremic middle molecular toxins and BSA in vitro, all three adsorbents demonstrated a fairly realistic adsorption capability to the model toxins but little to BSA. And the adsorption process reached the equilibrium in a clinically qualified short time. But the adsorption capacities of these adsorbents to the model toxins were quite different. It had been found that with the growing of fatty chain length of crosslinking agent, these adsorbents showed a gradually increased adsorption capacity to the model toxins, and the glutaraldehyde crosslinked chitosan resin behaved best.
Glutaraldehyde crosslinked chitosan used in the adsorption upon model uremic middle molecular toxins was studied. In comparison with untreated glutaraldehyde crosslinked chitosan resin beads, the hydrogen reduced ones showed a quite realistic behavior in the adsorption upon those model toxins, and the amount of adsorption was fairly high while the equilibrium time was obviously shortened.
