Iron sulfate inhibits Limulus activity by induction of structural and qualitative changes in lipid A

Abstract

Aims: The bacterial endotoxins test (BET) is a sensitive assay for measuring endotoxin levels in solution and uses the limulus amebocyte lysate (LAL) coagulation reaction. We sought to identify the mechanisms through which certain substances interfere with the interaction between LAL and bacterial lipopolysaccharide (LPS). Methods and Results: Endotoxin lipid A was inactivated by the addition of iron sulfate, which acted on endotoxin directly and strongly inhibited LAL coagulation activity. Size-exclusion, anion-exchange and reverse-phase liquid chromatography/mass spectrometry were used to examine changes in inactivated lipid A in terms of complex formation, negative charge status, hydrophobic interaction and structure. Furthermore, we verified the involvement of the lipid A phosphoryl group in the interference of iron sulfate with lipid A-factor C binding activity. Iron sulfate-inactivated lipid A was cleaved at its glycosidic bond, resulting in loss of hydrophobic interactions and disruption of lipid A complexes without alteration of negative charge status and lipid A-factor C interaction. Conclusions: Lipid A cleavage was a direct result of interfering factors, including iron sulfate, which acted on endotoxin directly to disrupt lipid A complexes rather than interfering with LAL coagulation. Significance and Impact of the Study: Our data provide new insights into the mechanisms of lipid A activity.

Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification

“…Moreover, LAL is based on an enzymatic cascade in which the proteins are subject to various physicochemical parameters. Temperature, pH, and ions can directly inhibit the LAL assay [95]. Some molecules, such as polysaccharides, are also known to interfere with the LAL test [96,97].…”

Nanosafety: An Evolving Concept to Bring the Safest Possible Nanomaterials to Society and Environment

“…The release of LPS, present on the outer membrane of Gram-negative bacteria [ 73 ], takes place after death and lysis of the cell; since microorganisms are ubiquitously present during nanomaterial preparation, unless precautionary measures are taken into consideration, the risk of inadvertent contamination is high. At the present, the Limulus Amebocyte Lysate (LAL) assay is considered the golden standard for assessing and quantifying endotoxin content in pharmaceutical products and medical devices; nevertheless, there are a few limitations for its application [ 74 ]. For that reason, it is important to use a second method to validate our results, such as the monocyte activation test [ 75 ], especially in the case of preparations containing nanomaterials, considering that interferences with toxicological assays are a concern [ 76 ], as discussed in the previous section.…”

Mechanism of Low Endotoxin Recovery Caused by a Solution Containing a Chelating Agent and a Detergent

“…Therefore, there is a possibility that the Takayama's monomeric LPS preparations were partially reaggregated and that the regenerated LPS aggregates reacted with the LAL. Certain metal ions decreased LPS activity [23][24][25][26][27]. Fujita et al [26] observed change in LPS aggregates by gel-filtration, and LPS solution with iron or cupper did not show the peaks of the original LPS solution probably because of increased particle sizes of LPS.…”