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Gram-negative endotoxaemia

University of British Columbia

Endotoxins arelipopolysaccharide (LPS) complexes extractable from the outer membrane of gram negative bacilli. These complexes comprise the 0-antigen of gram-negative bacteria and are used in the serological typing of these organisms. The release of endotoxins from gram-negative bacteria is believed to be a contributing factor to the high mortality rate associated with gram-negative septic shock in humans. Indeed, the sequelae of gram-negative septic shock seen clinically can be reproduced in experimental animals by the parenteral administration of purified endotoxin. The clinical management of gram-negative septicaemia would, perhaps, be much more effective if agents capable of antagonizing the actions of endotoxin in vivo could be used in combination with appropriate antibiotic therapy. The work reported in this thesis has utilized both biochemical and physiological approaches to obtain more insight into the mechanism of action of E. coli endotoxin with the ultimate hope of finding an effective antagonist for this toxic substance. In vivo studies with rats and guinea pigs have indicated that elevations in plasma lysosomal enzyme activities can be used as a measure of the severity of endotoxic shock. The lysosomal enzymes examined in this study included acid phosphatase, N-acetyl-β-glucosaminidase and cathepsin D. Interestingly, when the investigation of these enzymes was extended to include patients in gram-negative septic shock, it was found that the plasma of the patients also contained lysosomal enzyme activities that were significantly elevated relative to controls. Furthermore, the average plasma activity of cathepsin D in gram-negative septic shock patients was found to be significantly higher than that seen in the plasma of patients in other forms of shock. Thus, these preliminary studies indicate that measurements of plasma cathepsin D activity may have diagnostic and possibly prognostic value in clinical gram-negative septic shock. Other in vivo investigations included tissue distribution studies with ⁵¹Cr-radiolabelled E. coli endotoxin in guinea pigs. A positive correlation (r = 0.964) was found to exist between the accumulation of endotoxin in lung tissue and toxicity (determined by plasma acid phosphatase activity). These results are consistent with what is known clinically regarding the lung being a primary organ involved in the pathophysiology of gram-negative septic shock. In vitro studies utilizing ⁵¹Cr-labelled E. coli endotoxin and human erythrocyte membranes revealed that endotoxin is capable of binding to membranes in a specific manner. The binding of endotoxin to membranes was associated with measureable changes in functional properties including inhibition of K⁺-p-nitrophenylphosphatase activity and protection of red blood cells from hypotonic lysis. Studies with chemically modified endotoxins and enzymatically modified human erythrocytes suggested that the stabilizing action of endotoxin involved lipid-lipid interactions between toxin and cell membrane. On the basis of these studies, certain membrane-active drugs including methylprednisolone, lidocaine and propranolol were selected for study as possible endotoxin antagonists. Of these agents, only propranolol effectively antagonized the binding of ⁵¹Cr-endotoxin to erythrocyte membranes in vitro. The effectiveness of propranolol as an endotoxin anta gonist was also demonstrated in vivo by its ability to significantly reduce the accumulation of endotoxin in lung tissue and to lower plasma acid phosphatase activity in endotoxin-treated animals. However, only the d-isomer was effective in vivo whereas the racemate was poorly tolerated by endotoxin-treated animals. These studies indicate that membrane-active drugs (such as propranolol) are capable of antagonizing certain actions of endotoxin in vivo and as such may prove to be valuable adjuncts to specific antibiotic therapy in the clinical management of gram-negative septicaemia.

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2010-06-13

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http://hdl.handle.net/2429/25667

Pharmacology

University of British Columbia

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