Preparation and Host-reactive Properties of Endotoxin with Low Content of Nitrogen and Lipid

Endotoxins of low lipid content prepared from S. enteritidis by the aqueous ether method have been further treated to remove bound lipid by non-hydrolytic procedures. Such endotoxins, containing as little as 2 per cent lipid A, were as potent in stimulating a variety of physiological responses as those prepared by the well known phenol-water or Boivin procedures which yield products containing as much as 30 per cent lipid A. To verify the difference in lipid content between the aqueous ether preparations and other types of endotoxins, three different methods of lipid analysis were employed: determination of chloroform-soluble material released by hydrolysis with hydrochloric acid (lipid A) or with acetic acid (lipid W), and estimation of total bound fatty acids. These methods were in accord in showing the magnitude of the difference. No more than one-half of the fatty acids present in endotoxin were associated with the fraction designated lipid A. Methods are described for the preparation of potent endotoxins with analytical values for nitrogen, phosphorus, hexosamine, carbohydrate, and fatty acid which do not differ appreciably from those of the classical, non-toxic, haptenic polysaccharides.