A dissertation submitted to the Faculty of Science, University of the Witwatersrand in fulfilment of the requirements for the degree of Master of Science. University of the Witwatersrand, Johannesburg, March 2016 / A liquid phase microextraction based on single hollow fibre followed by liquid chromatographic determination was developed for the extraction and quantification of the hallucinogenic muscimol and its two precursors, tryptophan and tryptamine from urine samples. A multivariate design of experiment was used in which a half fractional factorial approach was applied to screen six potential factors (donor phase pH, acceptor phase concentration, supported liquid membrane composition, stirring rate, extraction time and salt content) for their extent of vitality on the extraction of muscimol, tryptophan and tryptamine using the developed method. Four factors were identified as essential for an enhanced enrichment of each of the three research analytes from diluted urine samples. The paired vital factors were then optimized using central composite designs where empirical quadratic response models were used to visualize the response surface through contour plots, surface plots and optimization plots of response output. When the muscimol-based optimum factor levels were applied for the simultaneous extraction of the three research analytes, a composite desirability of 0.687 was obtained implying that the set conditions were ideal for a combined extraction of the analytes from the donor phase into the acceptor phase across a supported liquid membrane impregnated with a carrier molecule. This was an acceptable result considering that only the optimized muscimol factor levels were set as universal factor values. Muscimol was the analyte of interest in this research. The composite desirability value was predicted by setting the extraction conditions to 20% (w/w) di-(2-ethylhexyl) phosphoric acid (DEHPA) in dihexyl ether (DHE) supported on the walls of a hollow fibre into a 200 mM HCl acceptor phase inside the hollow fibre from a 20% (v/v) diluted urine donor phase spiked in the 0.1 – 10 μg mL-1 analyte concentration range maintained at pH 4 and stirred at 800 rpm for 60 mins. Experimentally, average enrichments of 4.1, 19.7 and 24.1 were obtained for muscimol, tryptophan and tryptamine, respectively. iv The complexity of urine and the anionic nature of the carrier molecule embedded on the supported liquid membrane resulted in interfering peaks that could not be completely resolved from the analyte peaks. Thus matrix-based calibration curves were used to address matrix effects. Various statistical approaches were used to validate suitability of the developed method for its potential use in quantifying muscimol and its precursors from urine samples. These validation measures were used as a way of determining the method’s ability to maintain the extraction process at equilibrium over a specific range of analyte concentrations over a period of analyte existence in a urine sample. The r² values of the matrix-based linear regression prediction models ranged from 0.9933 to 0.9986. The linearity of the regression line of the matrixbased calibration for each analyte was directly linked to the analyte enrichment repeatability. Simultaneous analyte enrichment repeatability over a 0.1 – 10 μg mL-1 analyte spiking concentration ranged from an RSD value of 8.3% to 13.1%. Limits of detection were 0.021 μg mLˉ¹, 0.061 μg mL-1 and 0.005 μg mL-1 for muscimol, tryptophan and tryptamine, respectively. Other validation parameters that were considered included specificity (and selectivity), accuracy, robustness, extraction range and system suitability. The accuracy of the developed method was reported as the reproducibility of enrichment factor values over six spiking concentrations used in constructing matrix-based calibration curves. System suitability was limited to an HPLC-UV approach. Method suitability was addressed through a comparative summary in which the LOD, LOQ and r² values for the developed method were compared to other methods that have been used to extract muscimol from urine samples. The relevance or acceptability of the enrichment factor values obtained for the extraction of the three analytes was achieved by comparison with enrichment factor values of several compounds with similar polarity that have been extracted from urine samples using carrier-mediated hollow fibre liquid phase microextraction. / GR2016

The effect that organic urinary macromolecules have on the crystallization of calcium oxalate from a synthetic urine-like solution was studied in a mixed suspension-mixed product removal (MSMPR) continuous crystallizer. Precipitation of calcium oxalate crystals occurs during the continuous passage of urine through the renal system (kidney, bladder and tubules). While in normal circumstances these crystals remain small in size and exit the system unimpeded, in the pathologic condition calcium oxalate crystals are observed to aggregate and grow beyond a critical size where there is a significant probability of being trapped inside the renal system, e.g., on the kidney wall or in the tubules. Once trapped, the crystals become a nidus for further solute deposition and aggregation, giving origin to a renal calculus or stone. It is shown that this process is significantly affected by the presence or absence of organic macromolecules that act as modifiers of crystal growth, nucleation, and aggregation. An ultrafiltration technique was used to fractionate urine specimens from normal (N) and stone-forming (SF) persons into organic compounds of different molecular weight. These compounds were then added to the MSMPR system to test their effect on calcium oxalate crystallization. Significant differences were found to exist between N and SF urines in the composition, molecular weight distribution, and total quantity of these organic macromolecular compounds. The fraction of macromolecules responsible for the major effects on calcium oxalate crystallization was isolated, and its effect on crystal growth and nucleation rates was quantified. The steady state driving force (supersaturation) in the MSMPR system was measured. Striking differences in supersaturation versus residence time behavior between N and SF macromolecules were observed. The experimental conditions under which calcium oxalate crystals agglomerate were identified. Evidence which supports agglomeration as a key mechanism in urinary stone formation is presented.

[Truncated abstract] Urinary desmosines have been proposed as a biomarker for inflammatory lung damage. Desmosine, a breakdown product of elastin, is an effective marker of the degradation of elastin and has been studied in many disease scenarios where there is acute and chronic lung inflammation. Lung matrix degradation has been proven in vitro and in vivo with many experiments showing that the excess proteases degrades lung matrix. The secretion of proteases by neutrophils is an innate response of the body to the invasion by micro organisms and when secreted in excess, the protective anti-protease mechanism is swamped. Chronic inflammation and persistent infection eventually leads to bronchiectasis and respiratory failure. Urinary desmosine has been shown to be elevated in respiratory conditions with acute and chronic inflammation . . . Urinary desmosine levels in a large cohort of healthy children have been established using this method and predictive Z-score formulae have been developed to use in children with lung disease. Exploration of these scores in children with CF have shown that the levels of urinary desmosine appear to be sensitive to the clinical setting, where high urinary desmosine levels were present during exacerbation and significantly reduced when treated for infection with antibiotic therapy and physiotherapy. The study of young children under the age of seven was undertaken to determine if the urinary desmosine levels could indicate when lung damage was occurring and to determine what mechanisms might be involved. Since there appeared to be no apparent relationship between elevated desmosines and proteases in the lung in young children with CF, further studies are required to define the mechanisms behind increased elastin metabolism in those children.

Diabetic nephropathy (DN) is one of the leading causes of end stage renal disease (ESRD) in western world and has a trend to spread in developing countries. Pathogenesis of DN is not fully elucidated. Studies of recent years showed that podocyte loss and activation of the rennin-angiotensin system (RAS), especially intra-renal RAS, played important roles in this process. Although renal biopsy is currently the most common way used to determine the expression pattern of podocyte and RAS associated molecules in DN, this invasive procedure has its own risk and is not practical for serial monitoring. We hypothesized that measurement of messenger ribonucleic acid (mRNA) expression of related genes in the urinary sediment might be a useful way to assess the severity of DN. / Firstly, we found that urinary mRNA expressions of podocyte-associated molecules nephrin, podocin, synaptopodin, Wilm's tumor-1 (WT-1) and alpha-actinin-4 were higher in patients with DN than in healthy controls, and urinary nephrin, podocin and synaptopodin expression was related to proteinuria and baseline renal function. In addition, there was a close relationship between urinary mRNA expression of type 2 angiotensin converting enzyme (ACE2), a key element of RAS, and the degrees of proteinuria, renal function and rate of decline of glomerular filtration rate (GFR). Urinary mRNA expression of ACE also inversely correlated with the rate of renal function decline. / In the next step, we studied the change in urinary mRNA expression of nephrin, podocin, synaptopodin, ACE and ACE2 in patients with DN treated with angiotensin converting enzyme inhibitor (ACEI) and addition of angiotensin receptor blocker (ARB). We found that urinary mRNA expression of podocin, synaptopodin and propably nephrin increased with disease progression, and percentage change in urinary podocin expression negatively correlated with rate of decline of GFR. Furthermore, serial measurement of urinary expression of nephrin and possibly synaptopodin may reflect therapeutic response to ARB in these patients. Urinary mRNA expression of ACE and ACE2, however, remained unchanged during the study duration and did not correlate with therapeutic response. / In this series of work, we investigated (i) the relation between the gene expression profile of podocyte-associated molecules and RAS related molecules in the urinary sediment and the severity of DN, including clinically defined parameter of disease severity, histological scarring, and the degree of intra-renal podocyte loss, (ii) the relation between urinary and intra-renal gene expression of patients with DN, (iii) the application of urinary gene expression on the monitoring of disease progression and therapy response of DN. The urinary mRNA expression of related genes was quantified by real-time quantitative polymerase chain reaction (RT Q-PCR). The intra-renal mRNA expression of related genes was studied from the histologic specimens of kidney biopsy by laser catapult microdissection (LCM) and RT Q-PCR. The degree of renal scarring was determined by morphometric analysis. Glomerular podocyte number was determined by stereological study on serial sections of renal biopsy specimen. / Taken together, our results suggest that although urinary mRNA expression of podocyte and RAS associated molecules is not related to intra-renal expression, urinary expression has the potential to be used as a non-invasive tool to assess the severity and progression of DN, and serial measurements of urinary gene expression of podocyte associated molecules may be used to reflect therapy response for patients with DN. Our findings also indicate that the information from urinary gene expression is supplementary to, but not a surrogate of, the data obtained from renal biopsy. / We then examined the relation between urinary gene expression and histological changes in the kidney. We found that urinary WT-1 expression correlated with the degree of kidney fibrosis. Unlike intra-renal expression, urinary mRNA expression of podocyte associated molecules did not correlate with glomerular podocyte number. There was also no association between urinary and intra-renal mRNA expression. / Wang, Gang. / Adviser: Cheuk Chen Szeto. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3423. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.