Quantitative determination of endotoxin in plasma by means of a new chromogenic peptide substrate. A methodological study.

Endotoxins are highly biologically active compounds found in the cell wall of Gram-negative bacteria. A method has been developed to determine endotoxins quantitatively in plasma, using Limulus lysate and a new chromogenic peptide substrate (S-2423, KabiVitrum, AB, Stockholm, Sweden). Blood samples were collected in pyrogen-free tubes (Mallinckrodt Inc., St. Louis, USA) containing heparin as anticoagulance. Platelet poor plasma was prepared by centrifugation at 1500 x g at 4 degrees C for 10 min. Standard solutions were prepared by adding E-coli endotoxin (Mallinckrodt Inc., St. Louis, USA) to plasma from normals. Limulus lysate (Pyrogent, Mallinckrodt Inc., St. Louis, USA) was incubated with samples and standards for 30 min. at 37 degrees C. The chromogenic substrate /S-2423) was then added and the mixture incubated for 3 1/2 min. The reaction was stopped with 50% acetic acid. Absorbance read against a separate plasma blank was recorded photometrically at 405 nm. The standard curve was linear in the range 0.025 to 0.200 ng/ml.