Is Endotoxemia in Stable Hemodialysis Patients an Artefact? Limitations of the Limulus Amebocyte Lysate Assay and Role of (1→3)-β-D Glucan

Abstract

BackgroundElevated blood endotoxin levels are frequently reported in the dialysis population and are strongly linked with inflammation, a major predictor of mortality. Virtually all studies have employed the Limulus Amoebocyte Lysate (LAL) assay to detect endotoxin. However this assay is not endotoxin-specific and can be activated by (1→3)-β-glucan (BG), a component of fungal cell walls leading to false positive signals. Very few studies have taken account of this. We examined the influence of BG-based activation of the LAL assay on the detection of endotoxemia in this setting.MethodWe measured plasma endotoxin levels in 50 hemodialysis patients with and without the use of BG-blocking buffers. These buffers inhibit BG activation of the LAL assay to ensure that any signal detected is endotoxin-specific. Blood samples were measured for BG, interleukin-6 (IL-6), tumor necrosis factor-alfa (TNF-α) to examine the association between endotoxin signals, BG and inflammation.ResultsEndotoxin signals were detected in 50% of patients. On repeat measurement with a BG-blocking buffer, all detected endotoxin signals were extinguished. No patient had detectable endotoxemia. Plasma BG levels were significantly elevated in 58% of patients and were higher in those with detectable endotoxin signals using the LAL assay without BG-blocking buffers (78vs.54pg/mL;p<0.001). Endotoxin signal and BG levels did not correlate with levels of TNF-α or IL-6.ConclusionUse of the LAL assay for blood endotoxin detection in dialysis patients has its limitations due to high blood BG. Endotoxemia frequently reported in non-infected hemodialysis patients may be artefactual due to BG interference.

Lactobacillus rhamnosus L34 Attenuates Gut Translocation-Induced Bacterial Sepsis in Murine Models of Leaky Gut

“…Moreover, BG is also more stable than endotoxin due to the presence of endotoxin-degrading enzymes in blood (52). In addition, BG activation of unmodified Limulus amebocyte lysate (LAL) reagent can be a confounding factor in LAL-based endotoxin measurements (53).…”

Pro-resolving mediators promote resolution in a human skin model of UV-killed Escherichia coli–driven acute inflammation

“…Endotoxin was measured using the limulus amebocyte lysate (LAL) Pyrogent 5000 kit. Blister fluid was pretreated by diluting in Tween-20 buffer followed by incubating at 70°C for 15 minutes as described previously for biological matrices ( 58 ).…”

Intestinal Barrier Disturbances in Haemodialysis Patients: Mechanisms, Consequences, and Therapeutic Options

“…For example, beta D-glucan can be introduced to the circulation of patients through the use of cellulose membranes during HD [ 108 ]. It is possible that the LAL assay may not discriminate between endotoxin and beta D-glucan [ 27 ] resulting in false positive results, which has been supported by recent data [ 109 ]. Concerns relating to differing reporting units and possible interference of the LAL assay with contaminants (e.g., beta D-glucan) must be taken into account when interpreting and comparing data.…”