Hyperammonaemia is not an infrequent presentation in the newborn or neonatal period. While the majority are transitory in nature and due to infective processes or liver pathology/immaturity, a significant number are due to defects in enzymes of the urea cycle. This cycle has evolved to cope with waste nitrogen disposal and the de novo synthesis of arginine. There are five distinct enzymatic steps in the urea cycle, and defects in each, result in a biochemically distinct disease. Four of these diseases, deficiencies of carbamyl phosphate synthetase (CPS), ornithine transcarbamylase (OTC), argininosuccmic acid synthetase (ASS), and argininosuccinate lyase (ASL) can present dramatically within the first 24 to 48 hrs of life with progressive lethargy, hypothermia and apnea, all related to very high plasma ammonia levels. These diseases may also present later in infancy, childhood and adulthood with hyperammonemia and episodic mental status changes. The fifth defect, arginase deficiency presents as progressive spastic quadriplegia and mental retardation but with milder elevation of blood ammonia levels. The molecular genetics of these disorders in South Africans has not been explored and there is thus very little information on phenotype/genotype relationships, specific for citizens of this country. This study aims to correct this imbalance and has concentrated initially on OTC deficiency, which is X-linked and therefore the most common defect encountered. Initial work on this project has concentrated on subjects with a classical X-linked OTC phenotype.

Hyperhomocysteinaemia is a recently established risk factor for the development of vascular disease and is caused by a variety of defects in the metabolism of methionine as well as dietary deficiencies of the vitamin cofactors (B6, B12 and folate) of the enzymes involved in methionine metabolism. Cystathionine β-synthase (CBS) is the most common genetic cause of homocystinuria, the severe form of the disease. The incidence of CBS deficiency in a group of 12 young patients of varied ethnic origin, who had peripheral vascular disease (PVD) that could not be ascribed to any of the conventional risk factors and were selected for having hyperhomocysteinaemia, either in the fasting state or after methionine load, was investigated. Nine out of the ten patients tested, showed abnormally elevated plasma homocysteine levels after methionine load, indicating a high incidence of deficient transsulfuration, which may have been caused by defects in CBS. Very wide variation in the CBS assay has hampered efforts to establish the contribution of CBS deficiency to the hyperhomocysteinaemia observed in this population. Therefore, a major part of this work has focussed on the source of this variation and the data suggests that between experiment variation as a result of changes in enzyme activity during the culture of the fibroblasts makes the biggest contribution. The most appropriate criterion to identify heterozygotes for CBS deficiency under these circumstances is to measure reduced CBS activity on several separate occasions compared to a control group. Only one of the group of 12 PVD patients (patient 1000) was identified as a heterozygote for CBS deficiency using this standard. Heterozygosity for CBS deficiency therefore seems to make only a minor contribution to the observed hyperhomocysteinaemia in this group of patients. Molecular genetic investigations were performed on selected individuals. Patient 1000 was confirmed to be a heterozygote for CBS deficiency. An A to G transition at nucleotide 695 leading to histidine to arginine substitution at amino acid 232 was found in one allele of this patient. A young homocystinuric female (patient 960) was confirmed to be compound heterozygote for CBS deficiency, with the common Celtic G₉₁₉A transition on the one allele and a novel duplication of the 7 bases between position 1553 and 1559 on the other allele. This 7bp insertion was identified as coming from the mother (patient 961). In an attempt to find an alternative or perhaps more sensitive method for the detection of defects in methionine metabolism, dual metabolic labelling of cultured fibroblasts with L-[methyl-³H]-methionine and L-[³⁵S]-methionine was developed to investigate these pathways in homozygotes and heterozygotes for CBS deficiency compared to controls. Although, no differences in the ratio of ³H/³⁵S were found that could be used to identify the zygosity of the patient for CBS deficiency, changes in the ratio of ³H/³⁵S over time in certain cellular compartments suggest that further development of this approach may prove to be useful.

The SR Ca²⁺-ATPase is an ATP driven pump that removes calcium from the sarcoplasm and myofibrils to allow muscle relaxation. The sulfhydryl crosslinker, 1,4- phenylenebismaleimide, reacts with Ca²⁺-ATPase (110 kD) to form a species with an apparent molecular weight of 125 kD, as well as dimers and high order oligomers, on SDS-P AGE. During the course of this study we have optimised and characterised the reaction of 1,4-phenylenebismaleimide with SR Ca²⁺-ATPase to produce the 125 kD species that is reminiscent of an E 125 species formed by intramolecular crosslink with glutaraldehyde. The glutaraldehyde crosslink involves the active site Lys 492 and Arg 678, in a zero distance link that overlaps with the ATP binding pocket, since it can be inhibited by nucleotides. It has been previously shown that the putative intramolecular crosslink with 1,4-phenylenebismaleimide is also sensitive to nucleotide binding. We show that the formation of the putative intramolecular crosslink of SR vesicles ( approximately 20 % of ATPase) with 1,4-phenylenebismaleimide is optimum at alkaline pH with micromolar concentrations of the crosslinker. The formation of ATPase dimers and high order oligomers, which were prominent in the reaction with SR vesicles, were eliminated by solubilising in Triton X-100. Under these conditions and in the presence of calcium, two intramolecular crosslinks are formed as seen in the formation of 125 and 130 kD species. The former seems to be in proximity of the y-phosphate and the latter in the β-phosphate region of the ATP binding site according to nucleotide protection studies. In the presence of detergent (Triton X-100) and absence of calcium, only the 125 kD species is formed and requires stabilisation by thapsigargin, a sesquiterpene lactone that binds the transmembrane α-helices. These conditions yield up to 60 % intramolecularly crosslinked ATPase. Trypsin digestion altered the apparent molecular weight of the 125 kD species to 135 kD, suggesting, in accordance with the results of glutaraldehyde crosslink, that the putative intramolecular crosslink 1s between tryptic fragments A and B. [¹⁴C]1,4-phenylenebismaleimide was synthesised to further characterise the reaction and to elucidate crosslinked amino acid residue following protein digestion, radioactive peptide purification, and sequencing. From filtration studies it was evident that a number of sulfhydryl residues were derivatized in both SR vesicles and solubilised Ca²⁺-ATPase. The results suggests that there is very fast reacting set of sulfhydryl groups, which could comprise of sulthydryls from Ca²⁺-ATPase and/or a minor contaminant protein as previous studies have indicated. Only this fast set was reduced by nucleotide binding. In Triton X-100, the total reactive residues increased two-fold and the biphasic nature of the curve showed that the intramolecular crosslink possibly involves a fast reacting sulfhydryl residue and a slow reacting one. Derivatization with [¹⁴C]1,4-phenylenebismaleimide followed by digestion and HPLC analysis revealed radio labelled peaks. Purification and sequencing of the adducts identified 8 reactive cysteines, namely Cys 12, Cys 344, Cys 364, Cys 471, Cys 498, Cys 636, Cys 670 and Cys 674. The cysteines involved in the putative intramolecular crosslink could not be identified but it is proposed that either Cys 471 or Cys 498 crosslink with Cys 670 or Cys 674.

Includes bibliographical references (leaves 67-72). / Liver dysfunction is common, often unrecognised and likely to increase in incidence in the population in parallel with the obesity and attendant type 2 diabetes mellitus epidemics. Liver fibrosis is a significant finding in liver pathology as it imparts important clinical staging and prognostic information, is a risk marker of adverse clinical outcome yet, even if advanced, is capable of reversal. Histological examination of liver biopsy material is the reference standard in the assessment of liver fibrosis, but it is impractical to biopsy all patients suspected of having liver disease. Serumprolidase is among novel biomarkers that have been described in diagnosing and/or staging liver fibrosis. This study evaluated the measurement and the diagnostic accuracy of serumprolidase in determining the potential presence and degree of liver fibrosis compared with liver biopsy.

Gonadotropin-releasing hormone (GnRH), a central regulator of reproductive function in all vertebrates, exerts its effects via binding to the GnRH receptor (GnRHR) in the pituitary gonadotrophs. The GnRHR is a member of the G-protein coupled receptor (GPCR) superfarnily. A second form of the GnRHR (type II), other than the pituitary gonadotrope GnRHR (type I) has been proposed to exist and to play a role other than the classical endocrine role of the pituitary GnRHR. Elucidation of amino acid residues of the GnRHR that are crucial for ligand binding, activation of the receptor, and coupling to the G-protein, is important in understanding structure-function relationships towards the design of drugs for therapeutic intervention. Such information can often be deduced by a comparison between conserved and non-conserved amino acid residues of GnRHRs from different species. At the start of this project no non-mammalian or invertebrate, and only some of the eutherian mammalian type I GnRHRs had been cloned. The aim of this project was to clone novel GnRHRs, i.e. type I and type II GnRHRs from redbait and mole and type II mouse and human GnRHRs using polymerase chain reaction (PCR) strategies. PCR was performed with degenerate primers designed to human type I GnRHR to areas that are not conserved between GPCRs in general, but are conserved between mammalian GnRHRs.

The Dalmatian coach hound, when compared to other dog breeds, exhibits three characteristic abnormalities of uric acid metabolism, namely hyperuricaemia, hyperuricosuria and increased renal uric acid clearance. These properties are associated with hypoallantoinaemia and hypoallantoinuria. A result of these abnormalities is a high incidence of urate urolithiasis in this breed. Other diseases such as recurrent dermatitis, chronic cystitis and deafness are also found in the Dalmatian and whether there is any causal relationship with the uric acid disorder is unknown. In terms of the quantity of uric acid excreted, the Dalmatian dog resembles man more closely than the non-Dalmatian. On the other hand, in its high renal urate clearance, this breed of dogs differs from man, whose renal clearance values are lower and therefore closer to those of the non-Dalmatian. In this respect the Dalmatian resembles man affected with the inborn renal urate transport defect of renal hypouricaemia. The significance of uric acid metabolism in the Dalmatian has attracted many investigators in search of the underlying metabolic defect(s). Study of the Dalmatian may also be relevant to the understanding of disorders of purine metabolism in man for example hyperuricosuria, uric acid urolithiasis and hereditary renal hypouricaemia.

Includes bibliographical references (leaves 105-111). / Microsatellite markers were used to measure genetic diversity and population differentiation within and between domestic dog breeds. The German Shepherd Dog was compared with typical outbred mongrel dogs, Dachshunds, Staffordshire Bull Terriers and a cohort of other pedigreed dogs representing 30 recognised breeds. Although archaeological records report that grey wolves (Canis lupus) were domesticated approximately 14 000 years ago, mtDNA analysis suggests that domestic dogs (Canis familiaris) and grey wolves diverged in multiple events over 100 000 years ago. Subsequently, the movement of humans and their dogs resulted in extensive gene flow between dog populations for thousands of years. Breeding practices to obtain distinctive pnenotypic uniformity were recently introduced, resulting in pure-bred dogs becoming essentially closed gene pools. However, further mtDNA analyses have reported unexpectedly high levels of variability, supported by microsatellite loci with heterogeneities of between 36% and 55% being reported for some dog breeds. Microsatellite analyses of 15 polymorphic canine loci are reported. German Shepherd Dogs and outbred mongrel dogs expressed diversity values of 4.0 alleles per locus in the former and 6.4 in the later (corrected for population size by jack-knifing with 1 000 pseudoreplications), with expected heterozygosities of 62% and 83%, respectively. German Shepherd Dogs showed a moderate loss of genetic diversity relative to outbred dogs, but not sufficient to describe the breed as highly inbred. However, in comparison with other pure-bred dogs examined, they expressed the least genetic diversity, with Dachshunds having 5.2, Staffordshire Bull Terriers 4.8 and the composite group of pedigreed dogs 6.0 alleles per locus, with expected heterozygosities of 72%, 67% and 80%, respectively. Significant population differentiation (GST = 0.103; RST = 0.058) between German Shepherd Dogs and the outbred dogs illustrates the effect of genetic drift since the breed was established just over 100 years ago. This study would benefit future breeding programs, as management should be facilitated by knowledge of relative measures of inbreeding and differentiation, especially between various separate breeding stocks within the breed.

This dissertation validates a Beckman-Coulter DxC(R) assay for total bicarbonate in urine and then proceeds to design, prototype and cost an inter-laboratory comparison (ILC) program for the above urine bicarbonate based on the validation. Furthermore, this work serves as a case study for how to establish proficiency testing - and thereby achieve accreditation - for tests without external quality assurance because of analyte instability.

This project aims to investigate the role of multimeric (high molecular weight) adiponectin in the development of metabolic disease resulting from anti-retroviral therapy. Specifically, the aim is to quantify the circulating levels of both total and high molecular weight (HMW) adiponectin and to establish whether a link exists between HMW adiponectin levels and susceptibility to HIV-induced lipodystrophy. Although total adiponectin levels have been shown to be significantly reduced in patients with HIVinduced lipodystrophy, there is no information on whether HMW adiponectin, which appears to be the most biologically active form of adiponectin, is altered in HAARTinduced lipodystrophy, and whether patients with low levels of the HMW form are more susceptible to lipodystrophy.

Captive black rhinoceroses in the United States are threatened by three major clinical disorders. Among these, haemolytic anaemia ranks as the number one cause of death. The object of this study therefore was to determine the metabolic lesion responsible for the haemolytic anaemia. Since black rhinoceros red blood cells were shown to be susceptible to oxidative stress, the first stage of the work focused on the characterisation of the hexose monophosphate shunt in these cells. Results were compared to human red blood cells. To determine how these cells responded to oxidative stress and to stimulation of this pathway, rhinoceros red blood cells were incubated with the known shunt stimulants ascorbate and methylene blue. Red blood cells were incubated with 14C-Iabelled glucose and then acid extracted. The rate of flux through the shunt was measured by counting 14C trapped on a filter placed inside the incubation vessel and saturated with NaOH. Red cell extracts were used for lactate and reduced glutathione determinations. These experiments showed that black rhinoceros red blood cells were capable of increasing flux through the shunt in response to oxidative stress although the magnitude of this response was significantly lower than that observed in human red blood cells. To determine whether the cells were capable of recycling metabolites through the shunt in response to prolonged oxidative stress, the red cells were incubated with 2-14C-Iabelled glucose. Recycling through the shunt was observed to function efficiently in these cells. Since no particular metabolic lesion could be defined with respect to the functioning of the lIMP, we shifted our attention to an unusual peak with cytidine-like absorbance properties which dominated the rhinoceros HPLC profile. A range of cytidine nucleotides and other nucleotides were analysed by reverse phase HPLC in an attempt to match the elution position of the unknown peak. This search yielded a surprising candidate, the amino acid tyrosine. Co-elution of this peak with standard tyrosine strengthened this identification. Diode array analysis of the HPLC peak yielded an identical wavelength maximum to standard tyrosine. Amino acid analysis of rhinoceros red blood cell extracts showed that rhinoceros red blood cells did indeed have elevated levels of tyrosine relative to human red blood cells. These levels were in the range of 20 to 50 times that measured in human red blood cells. The peak was then fractionated, concentrated by freeze drying and analysed by mass spectroscopy, which showed that it had the molecular weight expected for the amino acid tyrosine. Red blood cells of wild black rhinoceroses were found to contain 0.78 ± O.llmM (n=8) tyrosine. The finding of such a high concentration of a free amino acid in red blood cells appears to be unprecedented. In an attempt to elucidate the function of tyrosine in rhinoceros red blood cells we drew on the analogy of taurine which is present at very high concentrations in heart epithelial cells and neutrophils. Taurine protects these cells against the respiratory burst oxidants during periods of acute inflammation. Exposure of rhinoceros red blood cells to hydrogen peroxide resulted in the accumulation of dityrosine, a highly fluorescent tyrosine dimer. The formation of dityrosine has previously been shown to occur between protein tyrosyl residues. In our system we describe the crosslinking of free tyrosine in response to hydrogen peroxide in rhinoceros red blood cells. The formation of dityrosine was followed by fluorimetry. Human red blood cells showed no significant production of dityrosine under the same conditions. The accumulation of dityrosine in rhinoceros red blood cells was found to be reciprocally related to GSH concentration. A series of cell-free experiments showed that dityrosine only accumulated in the absence of sufficient GSH and that its production was catalysed by haemoglobin. This study therefore describes the identification of tyrosine present in rhinoceros red blood cells at levels approaching 1 mM. Haemoglobin catalyses the production of dityrosine in response to hydrogen peroxide in a manner that is inversely proportional to the GSH concentration. It is our hypothesis that this tyrosine response to hydrogen peroxide may form a backup antioxidant mechanism for the glutathione peroxidase / reductase system in these cells which are relatively deficient in catalase, a major enzyme for the protection from hydrogen peroxide in other mammals. Analysis of red blood cell extracts of 30 captive black rhinoceroses was found to have significantly lower tyrosine levels (0.37 ± 0.14mM) than the wild rhinoceroses (0.78 ± O.llmM) analysed. These low levels of tyrosine combined with an iron overload syndrome recently described by D. Paglia at UCLA, suggests that black rhinoceroses in captivity may face a higher level of oxidative challenge. The unusual mechanisms in the rhinoceros red cell described here for handling oxidative stress, in which catalase has been replaced by reliance on the glutathione peroxidase system together with a tyrosine buffer, seem to be inadequate under the environmental and dietary circumstances of the captive state. These findings however may give new insight into therapeutic or preventative measures based on dietary modification which will address iron absorption and antioxidants.