PubMed · 8199437
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A Fleck. Monitoring nutritional support.. https://pubmed.ncbi.nlm.nih.gov/8199437/
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The adsorption of human serum proteins to mono-, di- and tri-cyanocarbon ligands was studied in the presence of the water-structuring salt Na2SO4. All of the ligands adsorbed proteins to varying degrees when coupled to agarose via a divinylsulfone-derived spacer arm (DVS), whereas an insignificant or very low protein adsorption capacity occurred when the ligands were coupled through the bisoxirane-derived spacer arm. Studies of the DVS-coupled cyanocarbons showed that adsorption capacity increases with the number of cyano substituents carried by the ligand. The selectivity toward human serum proteins appeared to be similar to that of other electron acceptor ligands, but different from that of hydrophobic ligands. Tricyanoaminopropene-DVS-agarose was found to be the most potent protein adsorbent.
A binding protein for the globular head domains of complement component C1q, designated gC1qR, recently described to be present on vascular and blood cells (Ghebrehiwet, B., Lim, B.-L., Peerschke, E. I. B., Willis, A. C., and Reid, K. B. M. (1994) J. Exp. Med. 179, 1809-1821 was expressed in recombinant form in bacteria to investigate its functional and structural properties. The recombinant gC1qR was found to be functional because tetramerization of the 24.3-kDa polypeptide occurred as described for the native protein, and the binding of the ligand C1q by recombinant gC1qR was indistinguishable from binding shown by gC1qR isolated from Raji cells. Recombinant gC1qR immobilized to microspheres was used to search for additional binding proteins unrelated to C1q. Surprisingly, it was found that vitronectin or complexes containing vitronectin were retained from plasma or serum, and subsequent analysis revealed the specific binding of the ternary vitronectin-thrombin-antithrombin complex to gC1qR. Because the thrombin-antithrombin complex was unable to interact with gC1qR, direct binding with vitronectin was investigated in a purified system. The heparin binding multimeric form of vitronectin but not the plasma form of vitronectin was found to bind specifically to gC1qR isolated from Raji cell membrane as well as to recombinant gC1qR. This interaction was saturable (KD approximately 20 nM) and inhibitable by glycosaminoglycans such as heparin but not by chondroitin sulfate. C1q and vitronectin did not compete with each other for binding to gC1qR, and both ligands seem to interact with different parts of the gC1qR because a truncated version of recombinant gC1qR lacking the N-terminal 22-amino acid portion hardly interacted with vitronectin but bound C1q as well as the intact gC1qR. These findings establish gC1qR as a novel vitronectin-binding protein that may participate in the clearance of vitronectin-containing complexes or opsonized particles or cooperate with vitronectin in the inhibition of complement-mediated cytolysis.
Many clinical laboratories employ gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) to detect abnormal compounds occurring in urine and serum due to disease. The methods, particularly GC-MS, often require laborious sample pre-treatment, and separation times may exceed an hour. We describe the use of capillary electrophoresis (CE) equipped a with a diode-array detector in an attempt to improve the efficiency of an analytical system routinely used for diagnosis of human metabolic disease. It was found that urine samples could be injected directly onto the CE instrument without any pre-treatment, and over 50 metabolites were separated in 15 min. Identification of abnormal metabolites was based on migration times and characteristic diode-array spectra. The method readily diagnosed adenolysuccinase deficiency, 5-oxoprolinuria, propionic acidemia and disorders have orotic acid as diagnostic metabolite (e.g. the HHH-syndrome). The results show that CE may become a useful additional tool for diagnosis of metabolic disease. In a different project CE was used to study sera from the Janus-bank. This large serum bank comprises samples collected at intervals from nearly 300,000 blood donors. As the sera are stored at -25 degrees C and not at a lower temperature, a major concern has been the stability of the specimens. GC-MS, 2D-protein electrophoresis, certain immunological assays and enzyme measurements have previously been used to evaluate the stability of the sera. We can now also show that the protein profile, as determined by CE, is remarkably stable even after 22 years of storage. The results moreover confirmed that the CE-method and traditional gel electrophoresis gave almost identical results, except for small amounts of fibrinogen which did not show up on the CE-pattern.