Addition of perchloric acid to blood samples for colorimetric limulus test using chromogenic substrate: comparison with conventional procedures and clinical applications

Abstract

Preexposure of blood samples to perchloric acid permitted an accurate, quantitative measurement of endotoxin levels as low as 1 pg/ml using a colorimetric limulus test. Conventional chloroform and dilution-heating methods were unsatisfactory because of high residual nonspecific amidolytic activity and poor recovery. The normal peripheral plasma endotoxin level was less than 10 pg/ml when Escherichia coli 0111:B4 endotoxin was used as a reference. One nanogram in this assay was equivalent to 2.9 endotoxin units of USP reference standard endotoxin (E. coli 0113). High values were noted in portal venous blood and in cases of acute hepatitis, liver cirrhosis, strangulation ileus, pyothorax, lung abscess, diffuse panbronchiolitis, and pneumonia. Normal human plasma and serum exhibited a high capacity to inactivate added endotoxin. E. coli 0111:B4 and Salmonella minnesota 9700 were more susceptible to inactivation than Pseudomonas aeruginosa endotoxin. This inactivating activity was temperature dependent, was maximal between 37 degrees and 45 degrees C, and disappeared completely after heating plasma or serum to 56 degrees C for 30 minutes prior to the addition of endotoxin. The E. coli 0111:B4 endotoxin-inactivating activity of normal platelet-rich plasma, platelet-poor plasma, and serum, all at 37 degrees C, was 8.1 +/- 3.1, 11.7 +/- 4.5, and 15.2 +/- 4.9 micrograms/min/ml (mean +/- SD; n = 4), respectively. Endotoxin-inactivating activity was markedly decreased in plasma from patients with endotoxemia, but returned to normal with recovery from the underlying illness.