Theses and Dissertations (Chemical Pathology)

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Design of a senolytic proteolysis targeting chimera against the B-cell lymphoma – extra large protein using in silico approaches
(University of Pretoria, 2020) Pillay, Tahir; Islam, Md Ataul; u14196973@tuks.co.za; Kotsovolos, Nikolaos
Senescent cells have been linked to age-related diseases and frailty. Their pathogenic effects are mediated by a group of cellular secretions called the senescence-associated secretory phenotype (SASP). Removal of senescent cells has been associated with the delay of the onset of age-related diseases as well as improved physical function. Senescent cells have upregulated pro-survival pathways which confer resistance to apoptosis. The anti-apoptotic BCLXL (B-cell lymphoma-extra large) protein is involved in the survival of senescent cells. Inhibition of BCLXL’s activity has been found to induce apoptosis in senescent human umbilical vein endothelial cells (HUVECs). The von Hippel-Lindau tumor suppressor protein (pVHL) is the substrate recognition subunit of an E3 ubiquitin ligase. The pVHL protein binds to substrate proteins, recruiting them to the E3 ligase for ubiquitination which will result in their degradation. A Proteolysis Targeting Chimera (PROTAC), is a bivalent molecule that promotes interaction between an arbitrary protein target and an E3 substrate recognition subunit. A PROTAC, therefore, forces the ubiquitination and subsequent degradation of a target protein. Computational methods were used to design PROTACs capable of recruiting BCLXL to the pVHL protein to trigger its degradation and therefore specifically induce apoptosis in senescent HUVECs. The techniques used included pharmacophore-based screening, structural screening, molecular docking and molecular dynamics (MD) simulation. A PROTAP (Proteolysis Targeting Peptide) targeting the same proteins was obtained from literature and recreated in silico to serve as a comparison and proof of concept. The structure of a PROTAC that interacted with pVHL and BRD4 was obtained from the PDB as an additional comparison. MD simulations were run on ligand-bound BCLXL and pVHL proteins, consequently relatively stable conformations were identified and five pharmacophore models for each protein were developed based on different structures. The BCLXL pharmacophores placed more importance on aromatic rings while the pVHL pharmacophores preferred hydrogen bond donors and acceptors. Pharmacophores were validated by decoy set validation, using BEDROC, accuracy, Güner-Henry score and area under curve as metrics, yielding barely passable results. A starting library of 2 million molecules from the Mcule database was screened, identifying 33005 and 221481 hits for BCLXL and pVHL respectively. Structural screening and molecular docking were used to search for molecules with good binding characteristics to BCLXL and pVHL, retaining 33 and 216 molecules respectively. Two potential BCLXL ligands and two potential pVHL ligand were joined in different combinations with different glycine linker lengths, yielding eight potential PROTACs. Ternary protein-PROTAC complexes were assembled and used in 100 ns long MD simulations. From the MD trajectories, the RMSD of the proteins and PROTACs, as well as the number of bonds/interactions between them, were examined. Comparisons were made to the RMSD and interactions of the reference PROTAC and PROTAP. Three attractive PROTACs were identified, with one having superior ADMET properties. The binding energy calculations did not find fault with any of the three candidate PROTACs. While there is currently no satisfactory way to evaluate the performance of a PROTAC, the procedures employed identified molecules with the potential to act as a PROTAC.
Effects of 25-hydroxyvitamin D3 on cell growth and cell death via regulation of the vitamin D metabolising system in a cervical adenocarcinoma cell line
(University of Pretoria, 2023) Punchoo, Rivak; Pillay, Tahir; u15016308@tuks.co.za; Zhou, Esther V.M.
Cervical cancer is the leading cause of cancer-related mortalities in South African women. Vitamin D and its metabolites exert anti-cancer actions in various cancers; however, few studies have explored the effects of vitamin D in cervical cancer. The vitamin D metabolising system (VDMS) is responsible for the activation and breakdown of the vitamin. Vitamin D is activated by two hydroxylation steps facilitated by 25-hydroxylase (CYP27A1/CYP2R1) and 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1). The resulting active hormone, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), binds vitamin D receptor (VDR) in target cells to exert its effects. Both 1,25(OH)2D3 and its precursor 25(OH)D3 are catabolised by 24-hydroxylase (CYP24A1). This study aimed to use the cervical cancer cell line, HeLa treated with 25(OH)D3, to investigate the cell health parameters (cell count, viability, and cell cycle), cell death (apoptosis, autophagy and necrosis) by brightfield and transmission electron microscopy, and flow cytometry, and regulation of expression of genes and proteins in the VDMS by qPCR and Western blot. Results indicated that treatment with 5000 nM 25(OH)D3 significantly decreased cell count and viability and increased the sub-G1 population without inducing cell cycle arrest. Treatment-induced intrinsic apoptosis through disruption of mitochondrial membrane potential, externalisation of phosphatidylserines and activation of executioner caspases -3 and -7. Classic morphological features of apoptosis were also observed with brightfield and transmission electron microscopy. In addition, high-dose treatment with vitamin D increased CYP27B1, CYP24A1 and VDR gene and protein expression. Interestingly, multinucleated cells were observed in all cultures, but the cells were significantly larger following high dose 25(OH)D3 treatment. Collectively, the findings indicate that the VDMS acts in an autocrine manner to mediate the effects of supraphysiological doses of 25(OH)D3 on the growth and death of HeLa cells. Based on the data, it can be inferred that 25(OH)D3 holds promise as a therapeutic agent for cervical cancer treatment, and further clinical studies are warranted to explore this potential.
Genetic analysis of Lamin A/C gene variants in cases of sudden cardiac death admitted to a medico-legal laboratory
(University of Pretoria, 2022) Van Niekerk, Chantal; Van Deventer, Barbara; nataliadasilver@gmail.com; Da Silva, Natalie
The sudden death (SD) of a young and healthy individual can be traumatic. Approximately 85% of all SD cases are of cardiac origin, a condition known as sudden cardiac death (SCD). Familial dilated cardiomyopathy (FDCM) accounts for a major portion of SCD cases with the pathogenesis of FDCM being associated with changes in the main components of the nuclear lamina, within cardiac myocytes. These components - the lamins- are proteins encoded by the Lamin A/C (LMNA) gene. Causative variants of the LMNA gene are a major risk factor for SD and can be highly predictive for SCD. This study aimed to identify variations in the LMNA gene among SCD cases, within a cohort representative of the South African population. Two synonymous single nucleotide variants (SNVs) were identified in both case and control samples- SNV c.861T>C; p.Ala287 (rs535089) and SNV c.1338T>C; p.Asp446 (rs505058). Although previously classified as benign, these variants have been associated with dilated cardiomyopathy (DCM) and other laminopathy phenotypes. Due to their unique diagnostic value, inclusion of these variants in genetic screening panels can aid in confirming diagnosis of DCM and heart failure (HF), preventing SCD in young, otherwise healthy, individuals as well as aiding in further investigating other LMNA-associated diseases, which may arise as our population grows and diversifies.
Analysis of the potassium voltage-gated channel subfamily Q member 1 gene in sudden unexplained deaths in a South African population
(University of Pretoria, 2022) Van Niekerk, Chantal; Van Deventer, Barbara; u15031153@tuks.co.za; Hawksley, Megan
This research aims to analyse the KCNQ1 gene in sudden unexpected deaths in a young South African population, to better understand the prevalence of variations within this gene. The KCNQ1 gene is a potassium ion channel protein, which is found in cardiac cells. Variations within this gene have been linked to cardiac channelopathies such as long and short QT syndromes. This research extracted DNA from blood samples of 66 deceased individuals received from the Pretoria Medico-Legal Laboratory, who were under the age of 50, and 19 control samples, received from volunteers who had signed informed consent. The exons of the KCNQ1 gene were optimised using primers and real-time PCR to be able to group similar cases together using HRM analysis. One case from each similarity group was sequenced. The respective chromatogram results, from the Sanger sequencing, were then used to determine if any variations were present. The variations found were then compared to previously published results to determine if they were novel. Intronic and exonic variants were found; all the variants were classified as benign. These findings showed that this gene may not be susceptible to pathogenic variations. However, an exon did have a prevalent variation, which could indicate the exon as a variable region, warranting further investigation.
Analysis of the role of nuclear factor-kappa B in insulin resistance caused by antiretroviral drugs
(University of Pretoria, 2020) Pillay, Tahir; charlesmatamela@gmail.com; Mabugana, Matamela Charles
Human immunodeficiency virus still remains the leading cause of death globally including women of child-bearing age. The rate of AIDS-related death has significantly declined since the introduction of antiretroviral treatment and other non-medical interventions such as the distribution and use of condoms. The introduction of antiretroviral treatment has however led to insulin resistance amongst users. Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated nuclease 9 (Cas) has been used to knockout NFκB to understand the pathway at which antiretroviral treatment causes insulin resistance. Heteroduplex mobility assay has shown that CRISPR-Cas9 knock out the gene of interest. These results have played a foundation in understanding how CRISPR-Cas9 can be integrated and utilized in medical research.
Potential anticancer actions of cholecalciferol on a cervical squamous carcinoma cell line
(University of Pretoria, 2020) Punchoo, Rivak; Pillay, Tahir; u15104533@up.ac.za; Bhoora, Sachin
Cervical cancer is the fourth most common female malignancy worldwide and is substantively higher in low-income and middle-income countries. In South Africa, cervical cancer is a leading cause of mortality amongst women. The anti-cancer actions of the vitamin D and its numerous metabolites are an active field of research. The family of vitamin D metabolites regulate numerous cellular pathways which are implicated in tumorigenesis. Pre-clinical studies and clinical studies have yielded promising, although conflicting results in various cancers. Some healthy and cancerous tissue express an autocrine vitamin D metabolising system (VDMS) which is capable of tightly regulating intracellular metabolism and growth. The VDMS expresses activating and inactivating enzymes and a vitamin D receptor (VDR). At the cellular level, the VDMS can activate and inactivate vitamin D precursors and transduce signals to the nucleus to regulate various cell health genes, including cell growth, metabolism and survival. Healthy and cancerous cervical tissue express a VDMS. The anti-cancer actions of cholecalciferol, an early precursor of activated vitamin D, is poorly studied in cervical cancer. This study aimed to characterise cholecalciferol’s action on cell growth, cell death and the VDMS in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Cell proliferation was enumerated by Ki67 nuclear antigen and the cell cycle profile analysed by flow cytometry. Apoptotic cell death was investigated by measuring mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and evaluation of DNA damage markers by flow cytometric analysis. The biochemical markers microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II) and lactate dehydrogenase (LDH) were also measured by flow cytometry and spectrophotometric analysis to identify autophagic cell death and necrosis, respectively. In addition, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis, autophagic cell death and necrosis. The VDMS in SiHa control and experimental cultures were characterised by the investigation of intracellular gene and protein expression of the cholecalciferol activating (CYP2R1 and CYP27A1) and inactivating (CYP24A1) enzymes, and the VDR. Qualitative microscopical analysis evaluated classical characteristics of cell death and semi-quantitative analysis of apoptosis was performed. Data were analysed using a one-way ANOVA and Bonferroni post-hoc test. p < 0.05 was considered statistically significant. A significant decrease in cell count and cell viability was identified in SiHa cell cultures treated with 2600 nM cholecalciferol. Furthermore, significant increase in biochemical markers of apoptosis were identified including, decreased ∆Ψm; PS exposure; terminal caspase activation; and nuclear damage at 2600 nM cholecalciferol treatment of SiHa cell cultures. Moreover, the biochemical findings were supported by brightfield microscopy and TEM, which observed classical apoptotic features viz. membrane blebbing, apoptotic bodies and nuclear fragmentation. Also, a significantly increased number of apoptotic cells were enumerated. There was no evidence of autophagic cell death and necrosis. Additionally, a significant increase in 25-hydroxylase (CYP2R1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Conversely, a significant decrease in 1α-hydroxylase (CYP27B1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, significant increase in both 24-hydroxylase (CYP24A1) and VDR expression at gene and protein levels were observed in 2600 nM experimental SiHa cultures. In conclusion, cholecalciferol exerts growth inhibition and apoptosis in SiHa cells at 2600 nM. This is accompanied by CYP2R1 and VDR upregulation which suggests autocrine activation to calcidiol and intracellular nuclear signalling, respectively. It is therefore hypothesised that calcidiol synthesised de novo binds to VDR and induces apoptosis in SiHa cell line.
Variational analysis of the Ryanodine Receptor 2 gene in cases of sudden unexpected death in the young and infants admitted to Pretoria Medico-Legal Laboratory
(University of Pretoria, 2019) Van Niekerk, Chantal; Van Deventer, Barbara; tristan.a.wallace@gmail.com; Wallace, Tristan A.
Sudden death can be defined as death from natural causes occurring instantaneous or within a few minutes after its cause (Merriam-Webster, 2019). It is globally considered as one of the leading causes of mortality and may also be referred to as sudden unexpected death (SUD) (Tester et al., 2012). This study was an investigation on this gene in the South African population using a number of molecular techniques such as HRM real time PCR, DNA sequencing analysis and variation analysis. Results included several DNA variations found in exons 2, 5, 7, 10, 12, 15, 16, 23 and 28. Most DNA variations were single nucleotide variations (SNVs), however some DNA variations were insertions. Variations found in exons 2, 5, 7, 10, 12, 23 and 28 were in the introns whereas variations found in exons 15, 16 and 28 were in the exons. Exon 15 particularly presented as a ‘hot-spot’ for several highly pathogenic DNA variations. Exon 16 also had one DNA variation in three cases that were found to be pathogenic.
The application of the N-terminal fragment of pro hormone brain natriuretic peptide (NT proBNP) in cardiac disease risk assessment
(University of Pretoria, 2012-10-11) Prof R Delport; Dr J Ubbink; Dr N Oosthuizen; yvette.hlophe@up.ac.za; Hlophe, Yvette Nkondo
NT proBNP is a precursor peptide that is derived from proBNP a cardiac natriuretic peptide (NP), which is measured in plasma to assist with management of cardiac related conditions. The first aim of this study was to examine the biological variation (BV) of NT proBNP and to calculate its reference change value (RCV) in healthy individuals. The second aim was to assess if the RCV calculated for NT proBNP could be used as prognostic marker in patients who presented with chest pain or acute coronary syndrome (ACS) symptoms at an emergency care unit. A second NT proBNP measurement was performed twenty four hours after admission. These patients were assessed for a likelihood of presenting with three outcomes; probability of mortality; angioplasty or bypass surgery after being monitored for a period of one year. The third aim of the study was to investigate if NT proBNP could be used to identify individuals with possible cardiac conditions for life insurance purpose. NT proBNP assays were measured using the Elecsys 2010 analyser from Roche Diagnostics which uses an electrochemilumeinesence immunoassay that functions on the basis of the sandwich technique. Statistical methods will include analysis of variance and Odd Ratio analysis to assess the risk of a particular outcome. The reference change value (RCV) was calculated as 67%. The odds of requiring angioplasty increased 3.6-fold if a RCV of >67% was observed. The RCV did not aid in predicting mortality, or in predicting need for bypass surgery. A significant association was observed between ECG findings and NT proBNP. The ECG and NT proBNP are not independent of each other in males, as is evident from the correlation between the two variables (r=0.6; p<0.005). This implies that the variables can be used interchangeably. The study concluded that the 67% RCV calculated allows for a more accurate prognostic analysis of each patient and it allows us to move away from the use of population based reference ranges. It was established that a 67% RCV can significantly indicate the likelihood of patients requiring angioplasty. The correlation that was assessed between NT proBNP and the ECG provides insurance companies with the option to choose the method that best accommodates them when assessing cardiac risk.
Applications of extractive-derivatization sample preparation in a clinical toxicology laboratory setting
(University of Pretoria, 2009-12-09) Laurens, Johannes B.; s20050781@tuks.co.za; Marais, A.A.S. (Adriaan Albertyn Scheepers)
The metabolism of absorbed xenobiotic compounds in humans results in a mixture of target compounds applicable for analysis, trapped in complex biological matrices. Gas chromatography-mass spectrometry (GC-MS) is a powerful analytical technique that has been successfully applied in the analysis of volatile and semi-volatile compounds from complex biological samples. This is due to the ability of GC-MS to separate different sample constituents at trace levels while providing accurate molecular structural information for the resolved compounds. The complexity of biological specimens and their largely aqueous nature, combined with the physicochemical properties of target analytes resulting from metabolism, greatly precludes direct analysis of biosamples by GC-MS. Traditionally, highly laborious and time consuming sample preparation procedures are performed to isolate and chemically alter target analytes to attain suitable amenity for the detection system. Furthermore, routine analytical procedures in clinical toxicology laboratories are signified by short specimen turn-around times. The commonplace use of GC-MS in modern-day laboratories still suffer from prolonged turn-around times that result from both sample preparation steps and lengthy instrumental analysis. Simplified and cost-effective analytical procedures capable of extracting multiple analytes, with divergent functional groups, from biological matrices in a timely manner are therefore required. To address this issue, this work describes the development of validated extractive-derivatization methods combined with fast GC-MS analysis for expedient and accurate quantitation of different analytes in occupational monitoring and workplace drug testing. Extractive alkylation of acidic analytes phenol, o-cresol, mandelic acid, hippuric acid, and (o-, m-, p-) methylhippuric acid for simultaneous urinary bio-monitoring of occupational exposure to benzene, toluene, ethylbenzene, and xylene, respectively, is performed. Extractive acylation for simultaneous urinary confirmation of basic analytes amphetamine, methamphetamine, norephedrine, methcathinone, ephedrine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), methylenedioxyethylamphetamine (MDEA) and N-methyl-1-(3,4 methylenedioxyphenyl)-2-butanamine (MBDB) in workplace drug testing is performed. The successful combination of abovementioned techniques alongside fast GC-MS allows increased sample throughput and decreased turn-around time for routine analysis while maintaining bioanalytical quantitative criteria, as required in a clinical toxicology laboratory setting.
Tryptophan and the kynurenine pathway in chronic renal failure patients on dialysis
(University of Pretoria, 2008-04-29) Viljoen, Margaretha; Laurens, Johannes B.; upetd@up.ac.za; Bipath, Priyesh
Tryptophan is metabolised along the kynurenine pathway under the influence of tryptophan 2,3 dioxygenase and indoleamine 2,3 dioxygenase. Quinolinic acid and kynurenine, two neuroactive metabolites of the kynurenine pathway are, in chronic renal failure patients, considered as uraemic toxins. Related research is generally hampered by the non-availability of relevant analytical techniques. The primary aim of this study was, therefore, to develop and validate suitable methods for the determination of tryptophan, kynurenine and quinolinic acid. The second aim was to quantify the levels of these substances in the blood of chronic renal failure patients on renal replacement therapies and to compare the levels of haemodialysis patients to those on peritoneal dialysis. Patients’ quality of life was investigated relative to disturbances in tryptophan metabolism. Gas chromatography coupled to mass spectrometry (GC-MS) gave the best results for the analysis of tryptophan, kynurenine and quinolinic acid. A Hewlett Packard HP GC 6890 series gas chromatographer was coupled to a MS 5973 series mass spectrometer. Analytes were separated on a DB-5MS column with a nominal length of 30 metres, a diameter of 250.0 µm and film thickness of 0.10 µm. Helium was used as carrier gas, and the chromatographic analysis run time 12.5 minutes. The validation results were within the acceptance criteria for newly developed methods. The linear calibration curves constructed for all of the analytes gave r2 correlation coefficients >0.99. Other validation data such as precision, bias, accuracy and stability all fell within acceptable validation limits. In the study on chronic renal failure patients significant differences were seen between patients and controls. Tryptophan levels were 5.34 SD 5.04 µM for the haemodialysis group, 6.73 SD 3.18 µM for the peritoneal dialysis group and 28.4 SD 4.31 µM for the control group. Kynurenine levels were 4.7 SD 1.9 µM for the haemodialysis group, 2.9 SD 2.0 µM for the peritoneal dialysis group and 2.1 SD 0.6 µM for the control group. Quinolinic acid levels were 4.9 SD 2.0 µM for the haemodialysis group, 2.8 SD 2.0 µM for the peritoneal dialysis group and 0.3 SD 0.1 µM for the control group. Tryptophan was lower for the total patient group than for controls with significantly lower levels for haemodialysis versus control (p<0.05) and peritoneal dialysis versus control (p<0.05). Kynurenine levels were higher in the total patient group with significantly higher levels for the haemodialysis versus control group (p=0.0001). The patient groups had higher quinolinic acid levels with significantly higher levels for the haemodialysis versus control (p<0.05) and peritoneal dialysis versus control (p<0.05) groups. This study was the first to determine the three substances simultaneously in both haemodialysis and peritoneal dialysis patients. The study showed significant tryptophan depletion, as well as kynurenine and quinolinic acid accumulation for both groups. No significant differences were found between the patient groups other than higher kynurenine levels in the haemodialysis group. The quality of life (SF-36) was largely similar in the two patient groups. This decrease in the quality of life strongly correlated with the degree of tryptophan depletion.
The impact of the cytochrome CYP2C9*2 and *3 polymorphisms in the South African Caucasian population on warfarin therapy protocols
(University of Pretoria, 2005-09-23) Bester, Megan J.; upetd@up.ac.za; Green, Pieter-Hendrik
Please read the abstract in the section 00front of this document
Increased osteoclastogenesis and bone resorption by peripheral blood mononuclear cells in chronic liver disease patients with osteopenia
(University of Pretoria, 2011-02-17) Mulder, Chris J.J.; De Vries, Teun J.; Van der Merwe, Schalk Willem; Vermaak, William J.H.; b.olivier@vumc.nl; Olivier, Brenda Jean
Please read the abstract on page 3 in the dissertation.
Diagnosis of helicobacter pylori infection with the 13C-urea breath test : analysis by means of gas chromatography with mass selective detection
(University of Pretoria, 2007) Laurens, Johannes B.; maraliese@yahoo.com; Jordaan, Maraliese
Please read the abstract in the section front of this document
Establishment of screening procedures for genetic disorders and risk factors in the South African Caucasian population
(University of Pretoria, 2005-08-02) Bester, Megan J.; upetd@up.ac.za; Adelekan, Adeboye Mutiu
Please read the abstract in the section 00front of this document
N-methylnicotinamide as marker for biological methylation in humans
(University of Pretoria, 2006-03-13) Ubbink, Johan B.; upetd@up.ac.za; Rosemann, G.M. (Gertruida Magdalena)
The purpose of this study was to determine whether the methylation of nicotinamide to N-methylnicotinamide could discriminate between differences in methionine nutritional status, and by implication methylation capacity, in healthy humans. As part of this thesis, a highly selective high performance liquid chromatography (HPLC) method for the determination of N-methylnicotinamide (NMN) in urine and plasma was developed and validated. Quantification was by fluorescence detection of the 1,6-naphthyridine derivatives, formed after incubation of NMN with acetophenone in alkaline conditions. Seven volunteers participated in a trial to evaluate the ability of a nicotinamide load test to discriminate between changes in the methylation status of the individual. The methylation status was measured as the time dependent changes in plasma NMN concentrations after a nicotinamide load. A basal nicotinamide load test was performed on each individual. The methylation status was then changed, by means of a methionine load, and the nicotinamide load test was repeated during the enhanced methylation state. The dynamic changes in N-methylnicotinamide levels indicated that the methionine load changed neither the plasma NMN concentrations, nor the rates of NMN formation. The conclusion of this study was that nicotinamide loading could not be used as a dynamic function test to assess biological methylation in healthy humans.
Quantitative analysis of catecholamines and their metabolites in human urine by gas chromatography - mass spectrometry as a screening method for sympatho-adrenal tumors
(University of Pretoria, 2009-04-15) Ubbink, Johan B.; Laurens, Johannes B.; brian@fdalab.co.za; Marais, Brian
The endogenous catecholamines and their metabolites play an integral role in establishing the presence or absence of a suspected sympatho-adrenal tumor. Highly elevated metabolites excreted in the urine are indicative of a tumor. For this reason numerous analytical methods has been developed to accurately quantify the levels of these compounds. However, current methods usually make use of conventional HPLC methods. Although effective, these methods require tedious sample preparation and are usually plagued by interferences. It was the aim of this work to develop a gas chromatographic – mass spectrometric (GC-MS) method that allow for the simultaneous analysis of the endogenous catecholamines, their basic and acidic metabolites using a single extraction procedure (which is easy to use and not tedious) with minimal derivatization steps. Furthermore, to develop GC-MS methods which do not require tedious sample preparation and yet be sensitive and accurate and allow for rapid analysis in the clinical pathology laboratory. Four different gas chromatographic - mass spectrometric methods were developed for the analysis of the catecholamines and their metabolites and are discussed in detail.
Kinetic studies of vitamin B6 metabolism in humans
(University of Pretoria, 2001) Ubbink, Johan B.; upetd@up.ac.za; Van der Westhuizen, Christian Abraham
The primal aim of this thesis was to establish whether kinetic aspects of vitamin B6 metabolism predispose to earlier observed racial differences found in plasma pyridoxal-5'-phosphate (PLP). The active forms of vitamin B6 namely plasma PLP and pyridoxal (PL) as well as the three enzymes expressed in the erythrocyte involved in B6 metabolism, PL kinase, PLP phosphatase and pyridoxamine -5'- phosphate (pyridoxine -5'- phosphate) [PMP(PNP) ] oxidase were measured by high performance liquid chromatography. Phase one supported earlier experimental evidence and lower plasma PLP concentrations were found in blacks in a group of200 male volunteers recruited from the South African National Defence Force (SANDF). The respective enzyme activities involved in vitamin B6 metabolism, from the same test subjects, suggested similar PLP production from PMP and PL as well as PLP dephosphorylation which result in the release of PL into the circulating fluid. Since applied exclusion criteria eliminated the majority of biochemical, physiological, genetical - and disease related factors that influence vit B6 status, dietary factors and individual preferences regarding food intake, were most likely to be responsible for the significantly lower circulating plasma PLP encountered in blacks. Phase two compared pharmacokinetic parameters between 7 black - and 9 white test subjects recruited from the South African Police Services after a single 10 mg oral supplement ofpyridoxine hydrochloride. Statistical analysis of the parameters elimination half-life, elimination rate constant, clearance, volume of distribution, mean residence time, maximum peak concentration and time to maximum peak concentration failed to demonstrate any significant differences between the two groups. These results suggest consistent appearance rate, distribution and metabolism for the metabolites PLP and PL in the study population. A tendency in slower appearance rate, for both the metabolites PLP and PL, were observed in blacks and needs to be investigated further. The end product of vitamin B6 metabolism, 4-pyridoxic acid, which was expressed in terms of 24 hour urine volume, again failed to illustrate any significant differences between blacks and whites. These results suggested similar excretion properties in my population study. Furthermore, the pharmacokinetic parameters calculated for plasma PLP and PL respectively, were found to display one-compartment - and two-compartment pharmacokinetic model characteristics. This mono- and bi exponential elimination characteristics displayed by PLP and PL respectively could be of value in future research efforts in terms of sampling time. The distribution half-life can be determined by the calculation of two-compartment macro-rate constants. Fasting blood-samples should be collected when true baseline values are needed in the case of PL. Following vit B6 supplementation, one should allow at least 5 times the distribution half-life (5-6 hr in the case of PL) before blood-sampling in order to achieve true pharmacological response. Phase three of this study was conducted to illustrate the metabolic interplay ofthe enzymes PL kinase and PMP (PNP) oxidase involved in PLP production. The kinetic parameters, Michaelis- Menten constant and maximum velocity rate, at varying substrate concentrations, for the enzymes PL kinase and PMP (PNP) oxidase, were compared in 14 white - and 14 black male test subjects recruited from the SANDF. Both the average Michaelis-Menten constant and maximum velocity rate were higher in whites, but these differences were not statistically significant. The high individual variability for both parameters calculated, can possibly be ruled out if a crystalline enzyme form is used and should be investigated further.

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