Interleukin-8 induces an elevation in the endotoxin activity assay (EAA) level: does the EAA truly measure the endotoxin level?

The endotoxin activity assay (EAA) is a FDA-approved blood endotoxin assay that is reported as a useful tool for the diagnosis of gram-negative bacterial infection. However, discrepancies between the results of the EAA and those of the limulus amebocyte lysate (LAL) assay have been reported. Thus, we verified these methods. Blood was incubated with anti-endotoxin antibody, the resultant polymorphonuclear activation to produce oxidants was measured and the EAA level calculated. As a reference endotoxin assay, we used an endotoxin-specific LAL assay. Significant increases in plasma LAL assay levels were observed only in patients with sepsis caused by gram-negative bacterial infections, whereas higher EAA levels were observed in almost all the sepsis cases and the SIRS cases, especially those with acute pancreatitis. Graded amounts of LPS (1-10,000 pg/ml) were spiked into normal blood to obtain dose-response curves: a good dose-response curve, from 1 to 1,000 pg/ml, was obtained for the LAL assay. A good dose-response curve was barely obtained for the EAA; the lowest detection limit seemed to be 1,000 pg/ml. Addition of methylprednisolone decreased the EAA levels. Interleukin-8 (IL-8) induced elevation in EAA levels when IL-8 was added to volunteers' blood samples. Overall, the EAA kit could not measure clinically relevant doses of endotoxin. Because IL-8 induced an increase in EAA level, it is suggested that the EAA level reflects the primed state of polymorphonuclear leukocytes.