There exists an inverse proportionality between number of heat-killed cells of Salmonella typhimurium injected intraperitoneally into mice and the quantity of urinary nitrogen the animals excrete during a 17 hour period following the subcutaneous administration of 2 units of ACTH. This relationship has been developed into an assay for bacterial endotoxin. Mice immunized against S. typhimurium require 10 to 20 times the number of cells needed by control animals to suppress urinary nitrogen excretion to the same extent. Intravenous saccharated iron oxide sensitizes animals so that fewer heat-killed salmonellae can be detected. Heat-killed cells of Staphylococcus aureus are without effect in the assay. Several lipopolysaccharides derived from Gram-negative bacteria are effective in preventing the rise of urinary nitrogen excreted in response to ACTH and the amount required, compared to the LD50, is in the same ratio for all of them. Citrated mouse serum partially inactivates the endotoxin during in vitro incubation for 1 hour at 37°C. while normal serum does not. Dichloroisoproterenol protects mice against the lethal effects of lipopolysaccharide and it lowers its effectiveness in the assay. The minimum amount of endotoxin reliably determined by the test is 0.25 µg. of an E. coli preparation that was given intravenously in mice in which the reticuloendothelial system had been "blocked" with saccharated iron oxide.

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