Hemoglobin: a newly recognized binding protein for bacterial endotoxins (LPS).
Administration of purified hemoglobin (Hb) as a cell-free resuscitation fluid is associated with multiple organ toxicities. Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS). To better understand the potential role of LPS in the observed in vivo toxicities of Hb, we examined mixtures of Hb and LPS for evidence of LPS-Hb complex formation. LPS-Hb complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89% < 300 kDa) from LPS alone (90% > 300 kDa); density centrifugation through sucrose, which distinguished denser LPS alone from LPS-Hb complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of Hb in complexes compared to Hb alone. Interaction of LPS with Hb was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of human endothelial cell tissue factor. These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several of the physical characteristics of LPS and enhancement of the biological activities of LPS. These findings suggest that a mechanism for the toxicity of infused Hb in vivo may involve potentiation of the biological effects of LPS. In addition, these observations suggest a mechanism by which LPS-related morbidity during sepsis could be enhanced by erythrocyte hemolysis.
Department of Laboratory Medicine, University of California School of Medicine, San Francisco.
R I
RI
Roth
W
W
Kaca
J
J
Levin
D000209:Acute-Phase Proteins; D000818:Animals; D001802:Blood Substitutes / Q000009:adverse effects / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004340:Drug Contamination; D004731:Endotoxins / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008033:Limulus Test; D008070:Lipopolysaccharides / Q000009:adverse effects / Q000378:metabolism*; D008562:Membrane Glycoproteins; D011485:Protein Binding; D013925:Thromboplastin / Q000096:biosynthesis; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D001802:Blood Substitutes; D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D004731:Endotoxins; D006454:Hemoglobins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D013925:Thromboplastin; D000099231:Lipopolysaccharide-Binding Protein