Evaluation of the use of the Endotoxin Activity Assay (EAA™) to Quantify Non-septic Exposure of Metabolic Endotoxemia

Abstract

Objective: Endotoxin, also known as lipopolysaccharide (LPS), is a potent immune stimulant. Low levels of endotoxin exposure (metabolic endotoxemia) play a pivotal role in metabolic disorders. However, there is no robust clinical assay to directly quantify metabolic endotoxemia. We aimed to validate the whole blood Endotoxin Activity Assay (EAA™) as a novel, rapid method to quantify low grade metabolic endotoxemia against the well-established lipopolysaccharide binging protein assay (LBP), a robust surrogate marker of endotoxaemia.Methods: 67 women and 47 men aged 21 to 47 years (35.4 ± 5.5 years, 34.5 ±7.2 years respectively) were assessed for adiposity (BMI, waist circumference and % body fat using bio-impedance), endotoxin levels (LBP, EAA™) and inflammatory status (serum CRP, IL-6, IL-8).Results: There was no direct relationship between EAA™ and LBP measures for quantitating metabolic endotoxemia for either women or men (R=0.146, p=0.284; R=0.283 p=0.09 respectively). In women, the traditional indirect marker of endotoxemia LBP correlated significantly with CRP and IL-6, measures of generalised immune activation and inflammation (R=0.664, p<0.001, R=0.296, p=0.028 respectively), but not with EAA™ assed endotoxemia. Supporting this relationship, LBP correlated with BMI and body fat percentage (R=0.306, p=0.022; R=0.301, p=0.024 respectively). However, the EAA™ only correlated with body fat percentage (R=0.382, p=0.014). In men, LBP was significantly related to CRP and IL-6 (R=0.345, p=0.046; R=0.421, p=0.009 respectively), but no relationship was observed between these inflammatory markers and EAA™ assed metabolic endotoxemia (R=0.206, p=0.243; R=0.280, p=0.093 respectively). There was no relationship between EAA ™ or LBP and any of the three measures of adiposity. Conclusion:In conclusion, the existing rapid whole blood EAA™ method of analysis was not suitable to detect low levels of endotoxemia known to be present in the obese state, while the results suggest LBP indirect analysis remains the superior tool for measuring low grade endotoxemia in this population.

Metabolic Endotoxemia, Feeding Studies and the Use of the Limulus Amebocyte (LAL) Assay; Is It Fit for Purpose?

“…Currently, there are three very different analytical techniques to assess human endotoxin levels; the Limulus amebocyte assay (LAL assay), the lipopolysaccharide binding protein (LBP) assay and the endotoxin activity assay (EAA™), which we have previously demonstrated is not suitable for use under conditions of metabolic endotoxemia (ME) [5]. The LBPenzyme-linked immunosorbent assay (ELISA) assay provides an indirect measurement of sub-acute endotoxin which has been designed specifically for biological samples and measures an acute-phase reactant protein, predominantly produced by the liver, in response to endotoxin exposure [6]. The biological role of LBP is to deliver endotoxin to a co-receptor CD14, facilitating an interaction between endotoxin and TLR4, triggering a signaling cascade.…”