Endotoxin masking in human plasma: The role of protein interactions and lipopolysaccharide structure

Luisa Burgmaier; Johanna Lifka; Meltem Avci-Adali; Johannes Reich

doi:10.1016/j.ejpb.2025.114786

Endotoxin masking in human plasma: The role of protein interactions and lipopolysaccharide structure

Eur J Pharm Biopharm. 2025 Jun 13:114786. doi: 10.1016/j.ejpb.2025.114786. Online ahead of print.

Authors

Luisa Burgmaier¹, Johanna Lifka², Meltem Avci-Adali³, Johannes Reich⁴

Affiliations

¹ Microcoat Biotechnologie GmbH, Am Neuland 3, 82347 Bernried am Starnberger See, Germany; University Hospital Tuebingen, Department of Thoracic and Cardiovascular Surgery, Calwerstr. 7/1, 72076 Tuebingen, Germany. Electronic address: r.reich@microcoat.de.
² Management Center Innsbruck (MCI)-The Entrepreneurial School, Maximilianstrasse 2, 6020 Innsbruck, Austria.
³ University Hospital Tuebingen, Department of Thoracic and Cardiovascular Surgery, Calwerstr. 7/1, 72076 Tuebingen, Germany.
⁴ Microcoat Biotechnologie GmbH, Am Neuland 3, 82347 Bernried am Starnberger See, Germany.

PMID: 40518054
DOI: 10.1016/j.ejpb.2025.114786

Abstract

Reliable endotoxin detection in human plasma is compromised by masking effects, which interfere with Limulus Amebocyte Lysate (LAL)-based assays. While electrostatic interactions have been considered a major cause of masking, our study demonstrates that they alone cannot fully explain the phenomenon. We show that masking occurs rapidly in plasma, with endotoxin recoveries dropping below 50% within minutes. Although increasing the pH to 12 partially restores detection, high-salt treatments fail to disrupt endotoxin-protein complexes, indicating additional stabilizing forces. Plasma fractionation experiments revealed that specific proteins, particularly lysozyme, contribute significantly to masking, while human serum albumin plays only a minor role at physiological concentrations. Furthermore, structural differences between lipopolysaccharides (LPS) influence masking behavior: smooth LPS variants are masked more rapidly than rough mutants, suggesting that hydrophilic interactions and molecular conformation play a crucial role. Our findings highlight that endotoxin masking is not solely driven by electrostatic interactions but results from a complex interplay of structural and biochemical factors. Recognizing these mechanisms is essential for developing reliable detection strategies, ensuring the accuracy of endotoxin testing in clinical and pharmaceutical applications.

Keywords: Blood; Endotoxin; Human plasma; Limulus Amebocyte Lysate (LAL) Assay; Lipopolysaccharide (LPS); Masking.