In Chapter 1 a review of the literature concerning aspects of erythrocyte membrane transport and metabolism, and purine and pyrimidine metabolism is presented. The effects of pH, pO₂ and inorganic phosphate (Pi) on the uptake and metabolism of hypoxanthine by erythrocytes has been studied in Chapter 2. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low pO₂. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favouring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate (PRPP). Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3- bisphosphoglycerate (2,3-DPG). These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely PRPP synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue. In Chapter 3 the kinetics and mechanism of transport of orotate across the human erythrocyte membrane and the effect of pH and inorganic phosphate on its metabolism (in the erythrocyte) have been studied. It has been shown that orotate enters erythrocytes with non-saturable kinetics and with a capacity of 190 μmoles/1 packed cells/min at a concentration of 4-6 mmolar. The presence of competition for transport by a number of anions and the lack of competition by uridine is indicative of transport by a general anion transporter, with the ability for concentrative uptake in the absence of other external anions being compatible with transport via a ping-pong mechanism. Inhibition of transport by the specific band 3 inhibitors DIDS and CHCA confirm that transport is via the band 3 anion transporter. This explains the lack of significant uptake of orotate by most differentiated tissues which lack the intact band 3 protein. However, the demonstration of band 3 in rat hepatocytes (Cheng and Levy, 1980) provides a mechanism for the orotate transport which has been observed in liver (Handschumacher and Coleridge, 1979). Changes in pH and inorganic phosphate (Pi) concentrations have been shown to have marked effects on the relative quantities of metabolic products produced by the erythrocyte from orotate. There was an increase in orotate metabolised with increasing Pi, an effect augmented by lowering the pH, and most easily explained by the allosteric activation of PRPP synthetase by Pi. The increase in UTP levels with decreasing pH may be the consequence of both increased PRPP availability for the formation of uridine nucleotide from orotate, and decreased conversion of UMP to uridine by pyrimidine 5'-nucleotidase, which is known to be inhibited by phosphate. The accumulation of UDP sugars is optimal at a phosphate concentration of 10 mmolar, which is unexplained but would be compatible with an inhibitory effect of Pi on CTP synthetase. A PRPP wasting cycle at alkaline pH values is proposed to explain the apparent paradox where no PRPP was observed to accumulate in erythrocytes (Chapter 2) at pH values of 7.6 and above in the presence of 10 mmolar phosphate and no added hypoxanthine, yet the metabolism of orotate, which is a PRPP utilising reaction, at alkaline pH values was readily demonstrable here. This (apparent paradox) can be resolved if one assumes that even in the absence of added hypoxanthine and demonstrable intracellular IMP there are sufficient quantities of hypoxanthine and/or IMP to maintain a PRPP wasting cycle at alkaline pH values. The cycle is interrupted at acidic pH values as phosphate levels rise and inhibit 5'-nucleotidase, an effect augmented by the decreasing levels of 2,3-DPG which accompany decreasing pH. This wasting cycle has recently been confirmed by P. Berman (unpublished). The kinetics of orotate uptake by erythrocytes and its eventual release as uridine provides a role for the erythrocyte in the transport and distribution of pyrimidines to peripheral tissues. A model is proposed and involves the de novo production of orotate in the liver. In the next step erythrocytes take up the orotate secreted by the liver into the circulation, convert it into an intermediate buffer store of uridine nucleotides, whose distribution is a function of pH and phosphate concentration, and eventually release it as uridine, which is a readily available form of pyrimidine for utilisation by peripheral nucleated cells. The enhancement of uptake of labelled orotate into nucleic acids of cultured cells is demonstrated here. The degradative half of the cycle proposes that uracil and palanine are the predominant degradative forms of pyrimidines produced by peripheral cells, and their ultimate metabolic fate is complete catabolism in the liver to CO₂ and water. In the final chapter the possible role of the human erythrocyte in the prevention of reperfusion injury has been investigated. The development of a model of renal ischaemia in the rat is described. The ability of human erythrocytes, "primed" by preincubating in acid medium of high Pi concentration and low pO₂, to take up hypoxanthine in a concentrative manner when perfused through ischaemic rat kidney is demonstrated. Attempts to demonstrate improved survival and renal function in rats with "primed" human erythrocytes prior to reperfusion were, however, unsuccessful. It is further demonstrated that "unprimed" human erythrocytes, resident in ischaemic rat kidney for 3 hours, take up hypoxanthine and convert it to IMP. that erythrocytes could play a physiological prevention of reperfusion injury.

Medium-Chain Acyl-CoA Dehydrogenase Deficiency (MCADD) is the most common inborn error of metabolism affecting the fatty acid oxidation pathway. The deficiency is caused by a defect in the medium-chain acyl-CoA dehydrogenase enzyme which catalyzes the first step in the oxidation of medium-chain fatty acids. Long-chain fatty acids, after being transported into the mitochondria and activated into long-chain acyl-CoAs, are sequentially broken down until they become medium-chain acyl-CoAs. Medium-chain acyl-CoAs are then broken down until they become short-chain acyl-CoAs. Short-chain acyl-CoAs are broken down until only acetyl-CoA remains. The block in the oxidation of fatty acids in those with MCADD happens once the long-chain acyl-CoAs have been oxidized to medium-chain acyl-CoAs. The medium-chain acyl-CoAs cannot be further oxidized and build up. Without the breakdown of fatty acids, individuals with MCADD cannot produce enough energy during times of increased metabolic demand. Thus, prolonged exercise, fasting, or fever can precipitate clinical symptoms once the body enters a hypoketotic hypoglycemic state. Those with MCADD typically present in the early months of life with fasting intolerance, vomiting, lethargy, and, in more serious cases, seizures. Adult presentation is rare, but should not be ruled out of a differential diagnosis, because early detection and intervention can prevent permanent brain damage and death. Because early detection can prevent the serious effects of metabolic decompensation, MCADD was added to the Newborn Screen and is tested through measuring levels of medium-chain acylcarnitines in dried blood smears by tandem mass spectrometry. Metabolic decompensation is manifested clinically through dehydration, vomiting, and acidosis. In serious cases, metabolic decompensation can progress to seizures, coma, and death. Introduction of the Newborn Screen has reduced the morbidity of the deficiency, but has not eliminated it. Those with MCADD need to be closely monitored and emergency glucose needs to be available to them in case of a hypoglycemic emergency. The Newborn Screen has been effective in finding mutations in the ACADM gene that produce a mild phenotype of MCADD. Before the Newborn Screen, the most common variant, K329E, was detected in clinically diagnosed patients. However, the screen has shown that there are about 150 variants leading to MCADD. The most common variant of the MCAD protein, K329E, has been studied and characterized in order to further understand the pathogenesis of MCADD. This mutation substitutes a lysine for a glutamic acid, introducing hindrance and the inability of the protein to form its fully functional tetrameric form. The mutant protein also has an increased sensitivity to heat denaturation. Currently, there are no pharmacological treatments for MCADD. The idea of pharmacological chaperones is explored by using the example of tetrahydrobiopterin and phenylketonuria. Future studies will need be done to find a treatment for MCADD that is curative rather than treating the symptoms of the deficiency; however, curative therapies which target the mutant enzyme may be problematic since there is a wide array of mutations that result in a defective enzyme in affected individuals.

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix, thereby linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient c.C289T and c.T236A MPC1 alleles disrupt MPC function. MPC1 c.C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and full-length point mutant (R97W). MPC1 c.T236A encodes a full-length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from human patient MPC1 mutations and inform fundamental MPC structure-function relationships. These results also demonstrate an efficient molecular genetic system using the mouse C2C12 cell line to mechanistically investigate human inborn errors in pyruvate metabolism.

Genetics is concerned with inheritance, genomics with the study of genomes. Bioinformatics provides the tools to study the interface between the two. If a particular locus in the human genome could have 100 discrete alleles, then the genome (comprising an estimated 80,000 genes), could harbor 8 million different alleles. To record information about each of these alleles in a meaningful and systematic fashion is a task for the Mutation Database domain of bioinformatics. The HUGO Mutation Database Initiative is an international effort to capture, record and distribute information about variation in genomes. This initiative comprises a growing number of Locus-Specific Mutation databases, and a few large Federated Genomic databases [Cotton et al., 1998]. / Here I present work on a well recognized prototypical Locus-Specific database: PAHdb. PAHdb is a relatively large curated relational database. / This graduate project has had two major aims: to improve PAHdb , by careful analysis of version 1.0 and revision of its design, resulting in PAHdb version 2.0; to document the redesign process and share the experience by the conception of guidelines for content and structure of mutation databases in general. (Abstract shortened by UMI.)

Several of the inborn errors of vitamin B$ sb{12}$ (cobalamin, Cbl) metabolism (cblC, cblD, cblE, cblF, cblG) are associated with homocystinuria and hypomethioninemia due to a functional deficiency of the cytoplasmic enzyme methionine synthase which requires methylcobalamin (MeCbl) as a cofactor. We compared the growth of cultured fibroblasts from controls with those from patients with a selective deficiency of MeCbl (cblE and cblG) and with those from patients with a defect in both MeCbl and adenosylcobalamin (AdoCbl) (cblC, cblD and cblF). Cells were grown in methionine and folic acid free media and in fully supplemented medium. Control cells were able to grow in the deficient medium supplied with homocysteine, cobalamin and folate, while mutant cells were not, due to their inability to synthesize methionine from its immediate metabolic precursor, homocysteine. This differential growth is useful for screening for genetic defects of methionine biosynthesis. Moreover, by correcting methionine auxotrophy in these cells, it may be possible to isolate genes which code for the products that are deficient in these disorders.

Methionine synthase (MS) is a vitamin B12(cobalamin;cbl) dependent enzyme that catalyses the methylation of homocysteine to methionine. It uses methyl-cbl as coenzyme and in ethyl tetrahydrofolate as the methyl donor. Methionine sythase reductase (MSR) maintains MS in it active state using S-adenosyl methionine as the methyl donor. Functional MS deficiency may occur as a result of a defect in either enzyme. Patients with this disorder have been classified into two complemetation groups according to which protein is defective: cblG patients are deficient in MS and cblE patients in MSR. A subset of cblG, known as cblG variant, is unique in showing barely detectable MS activity and failure of cbl incorporation into MS in patient fibroblasts. I report the mutations responsible for three cblG variant patients, two of them siblings, and connect their phenotype to lack of protein expression. I also report the cloning of the MSR cDNA, aided by confirming the identity of the cDNA through the discovery of two deleterious mutations in three cblE patients. These findings contribute to the overall understanding of functional MS deficiency.

The first three studies in this thesis address the mechanism for the aberrant fall in serum 1,25-dihydroxyvitamin D$ sb3$ (1,25-(OH)$ sb2$D$ sb3 rbrack$ and increase in renal 1,25-(OH)$ sb2$D$ sb3$-24-hydroxylase(24-hydroxylase) activity in X-linked hypophosphatemic mice (Hyp). The 24-hydroxylase is the first enzyme in the C-24 oxidation pathway that degrades the vitamin D hormone to its final inactivation product, calcitroic acid. We demonstrated that: (i) the aberrant increase in 24-hydroxylase activity in Pi-deprived Hyp mice is specific to the kidney and is the result of an increase in enzyme Vmax, immunoreactive protein and mRNA abundance; (ii) the increase in 24-hydroxylase mRNA in both Pi-deprived Hyp mice and 1,25-(0H)$ sb2$D$ sb3$-treated normal littermates can be ascribed to an increase in the transcriptional activity of the 24-hydroxylase gene; (iii) 24-hydroxylase transcripts in normal mice, Pi-deprived Hyp and normal mice and 1,25-(OH)$ sb2$D$ sb3$-treated normal mice are localized to the proximal tubule by in situ hybridization; and (iv) recombinant human growth hormone administration normalizes the aberrant increase in 24-hydroxylase but that this response is not sufficient to correct serum 1,25-(OH)$ sb2$D$ sb3$ levels in Pi-deprived Hyp mice. / The fourth study addresses the mechanism whereby EB 1089, an analogue of 1,25-(OH)$ sb2$D$ sb3,$ is less calcemic than the vitamin D hormone, while being more potent in its antiproliferative action. We demonstrate that: (i) EB 1089 has a 50-fold lower affinity than 1,25-(OH)$ sb2$D$ sb3$ for the vitamin D catabolic enzyme, 24-hydroxylase; and (ii) EB 1089 and 1,25-(OH)$ sb2$D$ sb3$ exhibit tissue-specific differences in vitamin D receptor-mediated responses in vivo that may be ascribed, at least in part, to differences in binding affinities for the vitamin D receptor.

Proefschrift--Utrecht. / Title also in English. Summary in English. Vita. Includes bibliographical references.

Proefschrift--Utrecht. / Title also in English. Summary in English. Vita. Includes bibliographical references.

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