Lipopolysaccharide solution

Kav is the availability constant of a substance with molecular weight M, Ve is the elution volume, Vt is the total volume of the column, V0 is the void volume or the interstitial space between the beads in the bed. The void volume could not be determined by the elution volume of Blue Dextran 2000 (Pharmacia Fine Chemicals AB, Uppsala, Sweden, Mw about two million) since it would have separated in the column. We used a 0.5% lipopolysaccharide solution (Bacto Lipopolysaccharide W, Salmonella typhosa 0901 ref. nr. 3124-25, Difco Laboratories, Detroit, MI, USA) with a molecular weight of above 150,000,000, which is excluded by the gel. 

Fig. 6.21. Principle of detection of lipopolysaccharide (LPS) with the CD14-derived probe. It relies on the formation of a ground state complex between fluorescein and rhodamine in aqueous solution with quenching of donor and acceptor fluorescence. Spectrum A shows hypothetical fluorescence emission spectra of this complex. After LPS binding, the peptide sequence gets straightened prohibiting the close contact between the two fluorophores and leading to the recovery of red fluorescence (Spectra B).

Work at the outset focused on physical and chemical characterization of the whole lipopolysaccharide antigens of each of the seven Fisher lmmunotypes. All give opalescent solutions in water and have very high apparent molecular weights. 

Adsorbents are used in medicine mainly for the treatment of acute poisoning, whereas other extracorporeal techniques based on physico-chemical principles, such as dialysis and ultrafiltration, currently have much wider clinical applications [1]. Nevertheless, there are medical conditions, such as acute inflammation, hepatic and multi-organ failure and sepsis, for which mortality rates have not improved in the last forty years. These conditions are usually associated with the presence of endotoxin - lipopolysaccharide (LPS) or inflammatory cytokines - molecules of peptide/protein nature [2]. Advantages of adsorption over other extracorporeal techniques include ability to adsorb high molecular mass (HMM) metabolites and toxins. Conventional adsorbents, however, have poor biocompatibility. They are used coated with a semipermeable membrane of a more biocompatible material to allow for a direct contact with blood. Respectively, ability of coated adsorbents to remove HMM solutes is dramatically reduced. In this paper, preliminary results on adsorption of LPS and one of the most common inflammatory cytokines, TNF-a, on uncoated porous polymers and activated carbons, are presented. The aim of this work is to estimate the potential of extracorporeal adsorption technique to remove these substances and to relate it to the porous structure of adsorbents. 

Since LPS varies in chemical composition from one bacterial species to another, different methods are used for its extraction. LPS is generally extracted from smooth strains of bacteria in a mixture of phenol and water (PW) at 68°C as described by Westphal and Jann (19). After extraction, the solution is allowed to cool and partition. Nearly all proteins remain in the phenol phase or interphase, while polysaccharides, LPS, and nucleic acids remain in the aqueous phase. In some instances, the LPS from certain organisms (i.e. Brucella abortus) remain in the phenol phase. Lipopolysaccharide is extracted from rough strains of bacteria in a mixture of phenol, chloroform, and petroleum ether (PCP) at room temperature as described by Galanos, et (20). After extraction, the chloroform and petroleum ether are removed by rotary evaporation and the LPS is then precipitated from the phenol with water. With some bacteria, PW or PCP will not extract sufficient quantities of LPS (21) and EDTA (22), chloroform-methanol (23), or butanol (11) must be used. 

The 5-0-a-L-rhamnopyranosyl derivative of KDO and KDO 7-(2-aminoethylphosphate) have both been isolated from the inner core region of the lipopolysaccharide (LPS) of E.coli K12. The syntheses of KDO-7-phosphate and 7-(2-acetamidoethyl phosphate) have been carried out by phosphorylation of an appropriately-protected derivative. It has been shown that in solution KDO 8-phosphate exists as a mixture of a-pyranose (66%), P-pyranose (3%), a-furanose (19%) and P-furanose (12%) forms. By use of the two isomeric 2-deoxy analogues of KDO 8-phosphate it was shown that KDO 8-phosphate phosphatase is speciflc for the a-pyranose form of the substrate, so that a mutarotation must be involved before the next stage in the formation of LPS, since CMP-KDO synthetase is specific for the P-pyranose form of KDO. 0 Other references to 2-deoxy-KDO and 2-deoxy-NeuNAc can be found in Section 1 of this Chapter. 

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