Lethal but Non-Pyrogenic Products of Endotoxin Obtained after Hydrolysis with Lithium Hydroxide

Abstract

An endotoxin was extracted from Salmonella typhosa, strain O-901, with trichloroacetic acid and phenol-water (TCA-PW ET). TCA-PW ET was treated with 0.5N lithium hydroxide at 37C for 30min. The hydrolysate was chromatographed in a Sephadex G-150 column. The absorbancy at 485mμ showed five successive peaks (Frs. 1 through 5). Analytical centrifugation indicated that these fractions were composed of diverse sizes. The sedimentation coefficient of Fr. 5 was the smallest being approximately 6.5S. The composition of each fraction was shown to be different by chemical analysis. Fraction 5 consisted mostly of hexosamine and fatty acids and some neutral sugars. With gas chromatography Fr. 5 showed a distinct difference when compared with either TCA-PW ET or Frs. 1 to 3 in its reduced lauric acid as well as increased β-hydroxymyristic acid contents. Rabbit pyrogenicity of Fr. 5 as compared with TCA-PW ET was approximately one thousandth, while chick embryo lethality was almost the same. Biological activities of the other fractions were intermediate in relative potencies. Fr. 5 was further chromatographed in Sephadex G-75 and G-50 columns and six fractions were obtained. All of these fractions were highly lethal with slight differences. Phosphorus, heptose, 2-keto-3-deoxyoctonate and glucosamine contents of the endotoxin products were relatively high, while the fatty acid contents were partly decreased. Relationship between lethality and chemical constituents of the endotoxin products is discussed.