Relation of Structure to Function for the U. S. Reference Standard Endotoxin After Exposure to 60Co Radiation

Abstract

The structure and function of the highly purified U.S. reference standard endotoxin (RSE) were studied after exposure to ionizing radiation from a 60Co source. With increasing doses of radiation, the trilaminar ribbon-like structure of untreated endotoxin exhibited focal swelling, after which only spherical particles were seen by electron microscopy. These morphological changes were paralleled by the respective loss of O-side chain repeating units and pieces of the R-core from the lipopolysaccharide molecules, as demonstrated by electrophoresis. The biologic function of the irradiated endotoxin was assessed with a variety of tests. At higher doses of radiation, a direct relation was observed between the degradation of the molecular and supramolecular structure and the loss of biologic function. At lower doses of radiation, however, there was variability among the functional assays in their rate of change with progressive irradiation of the RSE. The results suggest that the carbohydrate moiety plays an important role both in determining the supramolecular structure and in modulating certain biologic activities of bacterial endotoxins.

Endotoxin-Induced Platelet Activation in Human Whole Blood In Vitro

“…Our finding that RSE exposed to low doses of y-radiation possesses enhanced platelet aggregating activity in human WB is in accord with our former observation in dog PRP in vitro (25). In both systems, we found that LPS molecules with short or lacking O-polysaccharide side chain (produced by low doses of ionizing radiation) are more effective in decreasing free platelet count than those with long O-side chains.…”

“…For comparison, platelet activation induced by microfibrillar collagen from equine tendon is shown in the same donor These doses of y-radiation result in "mutant" LPS molecules that consist of R-core and lipid A, sometimes in combination with a single O-side chain unit (25). The platelet activation by RSE was also evident in the change of the volume of free platelets by 25 min post-challenge (Fig.3").…”

“…We have shown previously that endotoxin exposed to high doses of ionizing radiation sustains a stepwise degradation (25).…”

“…Exposure of RSE to 4.32 Mrad completely eliminated its platelet aggregating activity in human WB (Fig.2). On the other hand, as a sensitive method for detection of platelet aggregation (25), determination of the free platelet count (at 25 min after challeng'e) revealed that RSE exposed to low doses of y-radiation (0.36 and 0.72 Mrad) was significantly more potent (p <0.05) for inducing platelet aggregation than unexposed RSE ( Fig.3d).…”

“…The RSE was prepared at a concentration of 50 pgmL in pyrogen-free sterile 0.9% NaCl. Some of the glass vials containing RSE in aqueous medium were exposed to y-radiation from a oCo-source as described previously (25).…”