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

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.