Unmasking metabolic endotoxemia: Proteinase K–enhanced detection of free and protein-associated endotoxin in human serum

Abstract

Metabolic endotoxemia is a low-grade endotoxin exposure that leaks from the gut into the bloodstream, contributing to low-grade chronic inflammation. The most used method to quantify endotoxins is the Limulus Amebocyte Lysate (LAL) assay; however, its accuracy is limited due to the strong affinity of endotoxins for plasma proteins like albumin and lipoproteins, which can mask them and interfere with detection by factor C. The aim of this study was to evaluate the use of proteinase K pretreatment to unmask protein-bound endotoxins in human serum, allowing quantification of total, free, and bound fractions using the LAL assay. Participants were classified as normal-weight or obese based on anthropometric and biochemical criteria. Blood samples were obtained under fasting conditions and 180 min after a balanced meal. Each sample was split into two aliquots: one untreated and the other digested with proteinase K. Protein degradation was confirmed by SDS-PAGE, and a formula was applied to estimate the percentage of protein-bound endotoxin. Results showed an approximately fivefold increase in detectable endotoxin levels after proteolysis under fasting conditions in both groups, and a smaller, though significant, postprandial increase. Obese participants showed a lower postprandial percentage of protein-bound endotoxin than normal-weight individuals, despite similar endotoxin after proteolysis levels in both groups. These findings highlight the need for proteolysis in accurately measuring endotoxin concentrations. Furthermore, the proportion of protein-bound endotoxin may serve as a marker of physiological detoxification capacity. The study suggests that inflammation risk is more closely tied to endotoxin bioavailability than total circulating levels.