Hepatic zonation of [delta]-pyrroline-5-carboxylate metabolism

Pink, Desmond Barry Stephen (2002) Hepatic zonation of [delta]-pyrroline-5-carboxylate metabolism. Masters thesis, Memorial University of Newfoundland.

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Abstract

Metabolic zonation in the liver is characterized by a heterogeneous distribution of enzymes along the sinusoids of the liver acinus. Periportal hepatocytes are enriched in enzymes associated with gluconeogenesis, amino acid degradation and urea synthesis. Xenobiotic metabolism, lipogenesis and glutamine synthetase activity occur predominantly in the perivenous cells. The localization of proline oxidase activity has been examined to investigate the zonal implications of P5C metabolism. -- Administration of the hepatotoxins, carbon tetrachloride and bromobenzene, was used to induce damage (in vivo) to the perivenous zone. Allyl alcohol administration was used to damage the periportal zone of the acinus. Alternatively, controlled antegrade and retrograde perfusion of rat liver with digitonin was used to selectively permeabilize cells in the periportal or perivenous zone of the acinus. These techniques were combined with the collagenase method for hepatocyte isolation to provide enriched suspensions of specific hepatocyte populations. The activities of perivenous zone-specific marker enzymes, glutamine synthetase and ornithine aminotransferase, as well as the periportal zone-specific ornithine transcarbamylase were used in conjunction with histological analysis to ascertain the extent of damage. -- Administration of bromobenzene provided strong evidence to suggest a perivenous enrichment of proline oxidase. This result was further substantiated by retrograde digitonin-perfusion. In both instances, cells in the perivenous zone were destroyed and remaining cells demonstrated significantly decreased activities of perivenous zone markers and of proline oxidase. -- The results of the toxin and digitonin experiments have also demonstrated that the (perivenous) zone of proline oxidase enrichment is not as discrete as glutamine synthetase. The zone of positive proline oxidase activity was similar to the zone of positive ornithine aminotransferase activity. -- Proline production from P5C was used to assess the activity of P5C reductase in isolated hepatocytes following either toxin or digitonin treatment. The results indicated that as more of the perivenous region was damaged, proline production increased. Specifically, proline production was highest following bromobenzene intoxication, followed by carbon tetrachloride and allyl alcohol intoxication. These results suggest a periportal enrichment of P5C reductase. Retrograde digitonin-perfusion however, resulted in a significant decrease in proline production, indicative of a perivenous enrichment. -- The utilization of DL-P5C as a substrate for amino acid production was compared in hepatocytes from fed and fasted animals. Following a 48 hour fast, P5C was converted to proline and glutamate equally. In the fed state, P5C was preferentially converted to proline with marginal conversion to glutamate. The production of ornithine from P5C was not observed in either nutritional state.

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