Abstract
Endotoxin neutralization, caused by plasma components, makes it difficult to detect endotoxins in human blood. In this study, we investigated which factors influence the recovery of endotoxins using limulus ameobocyte lysate (LAL)-based assays. The individual factors that were examined in more detail were lipoprotein content, type of blood anticoagulation, kinetics and serum levels of divalent cations. Furthermore, it was investigated whether there is a direct correlation between LAL activity and monocyte activation. We could show that polyanionic heparin increases endotoxin recovery in blood, while citrate anticoagulation promotes endotoxin neutralization. Furthermore, we could show that the endotoxin activity in human plasma and serum decreases strongly over time. Time-dependent endotoxin neutralization reaches its maximum after 4–6 h incubation. By means of filtration tests we could determine that endotoxins in the plasma bind to lipoproteins but do not influence their activity. Comparative measurements have shown that high LAL activity of endotoxins in plasma simultaneously possesses high monocyte activating properties in whole blood. For the maximum recovery of endotoxins in human blood the physiological calcium and magnesium concentrations are sufficient. In this study, it was shown that the endotoxin neutralizing plasma components have a molecular weight similar to β2-microglobulin (11.7 kDa). For the exact identification of the endotoxin neutralizing plasma components, which caused a modulation of the immunostimulating endotoxin activity, further investigations have to be carried out in the future.
Highlights
Endotoxin neutralization, caused by plasma components, makes it difficult to detect endotoxins in human blood
The Limulus Amebocyte Lysate (LAL) test is a common method for the detection of LPS
Both the endotoxin and the LAL reagent are subject to biological fluctuations
Summary
Endotoxin neutralization, caused by plasma components, makes it difficult to detect endotoxins in human blood. In contrast to the widespread use of the LAL test in industrial pharmaceutical settings, the application of the LAL test for the detection of LPS in blood has been significantly hindered by the presence of inhibitors[7] To overcome these inhibitions, several approaches of plasma treatment prior to the LAL test have been described. Studies on the role of the aggregate structure for biological activity have shown that cubic LPS aggregate structures have a high endotoxic activity, while lamellar aggregate structures have little or no endotoxic activity[16] This dependence has been demonstrated for a variety of endotoxin and non-endotoxin s tructures[17]. Ribi et al.[15] reported the effect of human plasma on the LAL activity of LPS in DOC They demonstrated that LPS potency in DOC was recovered after dilution. The potency was not recovered by dilution if human
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