A study of the pyrogenic lipopolysaccharide of Proteus vulgaris

Boyle, William (1966) A study of the pyrogenic lipopolysaccharide of Proteus vulgaris. PhD thesis, University of Glasgow.

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Abstract

The major aim of this research was the isolation and characterisation of the heat-stable pyrogenic factor from the cells of Protous vulgaris RCST 53, in order to compare it with the fever-producing factor, obtained by a colleague, from culture filtrates of the same organism. This was undertaken because a report on the fever induced by this bacterium in rabbits, claimed that there existed qualitatively different cell-associated and filtrate pyrogens. Recent studies had shown that the pyrogenic activity and Gram-negative bacteria was associated with the endotoxic complex and could most conveniently be obtained as the lipopolysaccharide component. However, no definitive study of the lipopolysaccharide of P. vulgaris had been reported her was information available on a comparison of lipopolysaccharide obtained from cells and culture filtrates of any micro-organism. The culture filtrate pyrogen had to be obtained from synthetic medium cultures but the quantity of cells from this source was small. Therefore, initially Crude pyrogenic lipopolysaccharide preparations were extracted with phenol from cells grown in synthetic medium and in nutrient broth cultures. Both cell extracts were shown to contain the same lipopolysaccharide on the basis of their similar elemental analysis, monosaccharide constitution, equivalent pyrogenicity and the serological identity of their major heat-stable component. Therefore, methods of purification and analysis were investigated using extracts from cells grown in nutrient broth cultures since they could be obtained in greater quantity. The extract from nutrient broth culture cells was purified by differential ultracentrifugation. The purified lipopolysaccharide (LPS) had a reducing sugar value of 32%, and contained 16% hexosamine and 30-32% bound lipid. Monosaccharide constituents were glucose, galactose and an aldoheptose (probably D-glycero-L-mannoheptose), glucosamine and galactosamine. Protein could not be detected and nucleic acid contamination did not exceed 2%. Physical and chemical methods did not reveal heterogeneity in excess of 4% and immunological homogeneity was established. The lipid component contained all the saturated fatty acids of chain length C10 to C20 plus two long chain unsaturated acids and beta-hdroxymyristic acid. The lipopolysaccharide was highly pyrogenic in rabbits; the Minimum pyrogenic Dose was 0.002 mug/kg. body weight. The LD50 in mice was 1 mg. total dose, and in rabbits dosage in excess of 20 mug/kg. was toxic. From synthetic medium cluture cells a small quantity of lipopolysaccharide (LPI) was extracted, purified and analysed by the methods which had been used for the lipopolysaccharide (LIS) from nutrient broth culture cells. These lipopolysaccharides (LPI and LPS) were compared with a lipopolysaccharide (FPS) prepared by a colleague from synthetic medium culture filtrates. Their elemental analysis, carbohydrate and lipid contents were similar. Common constituents of all three preparations were the monosaccharides and fatty acids listed above. They gave serological reactions of complete identity and their pyrogenic activities were qualitatively and quantitatively alike. Minor dissimilarities in the relative content of two fatty acids was the only significant disparity among the products. In summary the same lipopolysaccharide was apparently being obtained from cells and from culture filtrates. Since the lipopolysaccharide (FPS) accounted for all the pyrogenic activity of the culture filtrate, there seems no justification for belief in the hypothesis of the existence of distinct cell-associated and culture filtrate pyrogens. This hypothesis was based on the appearance of single or biphasic fever responses in rabbits, but it was found that all three lipopolysaccharide preparations could produce either type of fever dependent purely on the dosage administered. The secondary aim of this research project was the pharmaceutical formulation of a standard reference pyrogen. A preparation was made by freeze-drying the lipopolysaccharide LPS with a mannitol carrier and its properties investigated. The preparation obtained appears to be a suitable reference standard because it is readily dispersable with full recovery of activity, can be terminally sterilised, is unaffected by the presence of a bacteriostatic agent and is stable on storage.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: James P Todd
Keywords: Chemical engineering
Date of Award: 1966
Depositing User: Enlighten Team
Unique ID: glathesis:1966-72175
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:11
Last Modified: 24 May 2019 15:11
URI: http://theses.gla.ac.uk/id/eprint/72175

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