The UDP-glucuronosyltransferases (GT) represent a major family of drug-metabolising enzymes, but little is known about their multiplicity in man. The aims of this project were to purify and characterise a GT isozyme from human liver, and to investigate glucuronidation in rat and human hepatocytes, with the aim of using human hepatocytes in primary culture as an <i>in vitro</i> model for the study of human drug metabolism. Chromatofocusing of human liver microsomes produced separation of GT isozymes, providing evidence for heterogeneity. However, purification in an active form was not achieved, due to lability in the presence of detergent. Rat liver 17β-hydroxysteroid-GT was purified, and antibodies raised against this protein recognised a single protein in human liver microsomes. Rates of glucuronidation of 1-naphthol and phenolphthalein were significantly higher in rat hepatocyte homogenates than in hepatocytes, the magnitude of the difference being greater for 1-naphthol. This was attributed to the presence of excess UDPGA in homogenate assays and the limitation imposed by lipophilicity on substrate uptake into cells by passive diffusion. In contrast, the rate of bilirubin glucuronidation was greater in hepatocytes, possibly as a result of intact carrier-mediated uptake mechanisms, combined with a suitable environment for efficient delivery of bilirubin to the endoplasmic reticulum by membrane-membrane transfer. The same three substrates were glucuronidated at a reduced rate in human compared with rat hepatocytes, apparently due to a lower intracellular UDPGA level and isozyme-specific differences in intrinsic activity and latency. As a result, the rate of glucuronidation of all these substrates was greater in human hepatocyte homogenates than in hepatocytes. Isozyme-specific changes were observed in GT activities in human hepatocytes in primary culture, indicating the need to develop culture systems allowing stable expression of these enzymes before such a model could be used for predictive human drug metabolism.

The doubly labelled water technique (DLW) allows the estimation of the carbon dioxide production (r<SUB>co2</SUB>) of a free-living animal from which, with knowledge of the respiratory quotient (RQ), energy expenditure may be predicted. The aim of this project was to assess the performance of this technique in ruminant animals using sheep as a model. In a series of preliminary experiments physiological processes which may affect this technique were examined. It was found the administered isotopes equilibrated throughout the body water after 6 hours following either an oral or intravenous dosing. <SUP>2</SUP>H lost in the form of methane was estimated to lead to errors in r<SUB>co2</SUB> of between -3.27 and -6.54% and may be corrected for by use of a methane to urine enrichment ratio of 0.6536. Losses of <SUP>2</SUP>H in exchangeable sites in faeces were estimated to lead to errors in r<SUB>co2</SUB> of between -5.35 to -6.54%, again corrections factors were proposed. Sequestration of <SUP>2</SUP>H into carcase fat was also quantified, a maximum error of 0.67% was estimated on r<SUB>co2</SUB>, although more <SUP>2</SUP>H was detected in the fat free carcase this was assumed to be exchangeable <SUP>2</SUP>H. A maximum error of -2.39% on water turnover (r<SUB>H20</SUB>) was estimated to arise from the recycling of <SUP>2</SUP>H due to the catabolism of labelled body solids, although this was probably an overestimate. No convincing evidence was found to suggest the fractionation factors applied to human subjects are not equally applicable to ruminant animals. The baseline body water enrichment was altered by a change in diet from synthetic concentrates to fresh chopped grass. Isotopic estimates of r<SUB>co2</SUB> were compared with respiration chamber measurements of r<SUB>co2</SUB>. A number of curve fitting procedures were considered together with three multi-point, three ratio plot and three two-point estimations of r<SUB>co2</SUB>. Using a multi-point calculation and after making corrections for fractional water loss, loss of <SUP>2</SUP>H in methane and faecal solids, r<SUB>H20</SUB> was underestimated by 16.24%. The isotopic r<SUB>co2</SUB> estimates were on average 1.52% (SD 15.31) lower than the chamber measurements, although one animal was markedly different from the others, omitting this animal led to an average error of + 5.95% (SD 4.34). In applying this technique to animals in the field the influence of a free-living existence on the performance of the method was assessed. A number of r<SUB>co2</SUB> calculation procedures were again considered. Preliminary chamber measurements were used to estimate methane production and faecal dry matter output in the field. Estimates of r<SUB>H2O</SUB> in the field (mean 7.218.1. day<SUP>-1</SUP>) were lower than those measured in the chambers (mean 9.812 1.day<SUP>-1</SUP>). From predictions of dry matter intake in the field RQ was estimated (mean 1.067), using the isotopic r<SUB>co2</SUB> estimates (mean 590 1.day<SUP>-1</SUP>) energy expenditure was then predicted (mean 11586 kJ.day<SUP>-1</SUP>). In the light of this work it was concluded the DLW technique may be usefully applied to ruminant animals and a number of practical suggestions were made.

The work described in this Thesis was conducted to investigate aspects of copper metabolism in the plaice Pleuronectes platessa (L). Three major aspects were studied. The first was to investigate and partially characterize the distribution of copper and copper-dependent enzymes in the various tissues of the plaice. The second was to study the effect of cadmium on copper-dependent enzymes as a base line for understanding the competition between an essential metal ion and a non-essential toxic metal ion. This has established a foundation for the interpretation of the toxicity of pollutants to a marine organism. The third and the major part of the investigation was to purify and characterize the copper-dependent enzyme, ceruloplasmin, and to compare it with the isolated enzyme from the mammalian system. The results from the first part of the investigation showed that (i) Copper is relatively evenly distributed in the plaice. Apart from brain which has the highest concentration of copper, the concentrations of copper in the other tissues generally reflect the copper-dependent enzyme activities. (ii) The presence of the copper-dependent enzymes in the plaice are confirmed. The assay system used for the mammalian enzyme systems apply reasonably well to the plaice enzymes. (iii) The limited study on the relatively crude preparations of copper-dependent enzymes show that they are similarly distributed in mammals and that they have similar properties. Cytochrome oxidase, however, has a higher pH optimum than that of the mammalian enzymes. Exposure of the plaice to sublethal concentrations of cadmium as cadmium chloride results in initial increase in ceruloplasmin, cytochrome oxidase, monomaine oxidase and tryptophan oxygenase activities followed by a decrease after a prolonged exposure. Changes in the copper-dependent enzyme activities are accompanied by parallel changes in the concentrations of copper in the serum and liver. Thus cadmium alters copper metabolism in the plaice.

To study maternal glucose tolerance in pregnancy and its effects on the fetus, a rapid 25g intravenous glucose tolerance test was performed at about 32 weeks gestation in a group of randomly selected women. Full glucose tolerance data was available in 815 cases. The results were withheld from the patients and their obstetricians and paediatricians, and no treatment or advice was offered. Fasting plasma glucose and indices of glucose disposal were distributed unimodally with no evidence of a separate pathological group towards the diabetic end of the distributions. Glucose disposal rate was not, however, signficantly associated with the fasting plasma glucose, suggesting that glucose intolerance associated with elevation of the fasting plasma glucose might be a more clearly defined entity. New reference standards for fasting plasma glucose in pregnancy, which differ from those currently in use, are presented. The major determinants of relatively impaired maternal glucose tolerance in pregnancy were maternal age and obesity. Nonetheless, many cases of relative glucose intolerance occurred in the absence of any preexisting clinical indication. Significant association were found between maternal glucose metabolism and various measures of neonatal size and morbidity, including the incidence of congenital malformations and the occurrence of perinatal asphyxia in post-term infants. These effects were graded through much of the range of maternal glucose tolerance and not of predictive value in individual cases. The available evidence did not indicate that these relationships were mediated by fetal hyperinsulinism. It is concluded that the adverse consequences of impaired glucose disposal with normal fasting plasma glucose in pregnancy do not justify exhaustive measures to identify the condition. Screening for glucose intolerance during pregnancy should seek to identify those cases in which glucose intolerance is associated with elevation of fasting plasma glucose.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.