title: Highly purified lipid X is devoid of immunostimulatory activity. Isolation and characterization of immunostimulating contaminants in a batch of synthetic lipid X.
abstract: Lipid X, an early precursor in the biosynthesis of lipid A has been reported to directly induce cytokine release in macrophages but also to inhibit endotoxin-induced tumor necrosis factor (TNF) induction. In this report we provide evidence that these conflicting results could be due to contaminants present in different batches of lipid X used. Thus, in an apparently pure batch of crystalline lipid X as obtained by a published procedure (Macher, I. (1987) Carbohydr. Res. 262, 79-84) small amounts of N,O-acylated disaccharide-1-phosphates could be identified. Their isolation was achieved by gel filtration on Sephadex LH-20 and further analysis of fractions showing elevated limulus amebocyte lysate values by thin layer chromatography and reverse-phase high performance liquid chromatography (HPLC) in combination with bioassays. Identification of immunostimulatory by-products was possible by testing HPLC-fractions for TNF-induction in bone marrow-derived mouse macrophages. Applying these procedures a disaccharide-1-phosphate, containing four 3(R)-hydroxymyristic acids at positions 2, 3, 2', 3', was identified as the main immunostimulatory side product. Two isomeric hydrolysis products of this compound with only three 3(R)-hydroxymyristic acid moieties attached to the disaccharide-1-phosphate were also identified. Surprisingly, these compounds behave quite differently in the TNF induction test. The disaccharide-1-phosphate, acylated at positions 2, 2', 3', is a very potent inducer of TNF-release whereas the corresponding isomer containing the 3(R)-hydroxymyristic acids in positions 2, 3, 2', does not induce TNF release, but strongly inhibits TNF release as induced by the former compound. Thus, contamination of "pure" lipid X with immunostimulatory or immunoinhibitory impurities may explain the divergent pharmacological profiles which were attributed to synthetic lipid X.
authors.affiliation: Sandoz Forschungsinstitut, Vienna, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Aschauer
authors.forename: A
authors.initials: A
authors.lastname: Grob
authors.forename: J
authors.initials: J
authors.lastname: Hildebrandt
authors.forename: E
authors.initials: E
authors.lastname: Schuetze
authors.forename: P
authors.initials: P
authors.lastname: Stuetz
mesh_terms: D000215:Acylation; D000276:Adjuvants, Immunologic / Q000302:isolation & purification*; D000818:Animals; D001681:Biological Assay; D001854:Bone Marrow Cells; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D004340:Drug Contamination; D005260:Female; D006017:Glycolipids / Q000138:chemical synthesis / Q000276:immunology* / Q000302:isolation & purification; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D015394:Molecular Structure; D019814:Myristic Acid; D009227:Myristic Acids / Q000032:analysis; D013403:Sugar Phosphates / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D000276:Adjuvants, Immunologic; D004187:Disaccharides; D006017:Glycolipids; D009227:Myristic Acids; D013403:Sugar Phosphates; D014409:Tumor Necrosis Factor-alpha; D019814:Myristic Acid; C044283:lipid X