Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 56-61).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Abstracted in Dissertation abstracts, v. 18 (1958) no. 3, p. 1169. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Michigan, 1933. / "Reprinted from the Journal of Biological Chemistry vol. 103, no. 1 ... November, 1933." Bibliography: p. 150, 160.

Thesis (Ph. D.)--University of Michigan, 1933. / "Reprinted from the Journal of Biological Chemistry vol. 103, no. 1 ... November, 1933." Bibliography: p. 150, 160.

Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds. However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT) catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how, genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues, systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

O tolueno e o xileno são substâncias presentes, ao lado de outras, em vários produtos, entre eles as tintas, tíneres e colas, provocando exposição combinada dos trabalhadores a diversas substâncias químicas. Assim, foi objetivo deste trabalho desenvolver um método simples, rápido e confiável de cromatografia em fase gasosa que permitisse análise simultânea do ácido hipúrico (AH) e ácido metil-hipúrico (AMH) em urina, bioindicadores de dose interna utilizados na monitorização da exposição ocupacional ao tolueno e xileno respectivamente. Após extração líquido-líquido e derivação com trimetilfenilamôneo, foi realizada análise cromatográfica em coluna capilar 100% polidimetilsiloxano, usando-se o ácido heptadecanóico como padrão interno. Foram avaliados 69 trabalhadores que manipulavam tintas em atividades de repintura de veículos e 31 trabalhadores não-expostos (controle). O método mostrou-se linear entre 0,1 (limite de quantificação) e 5,0 g/L para o AH e 0,2 (limite de quantificação) e 5 g/L para o AMH. A recuperação média foi de 81 % e a inexatidão de aproximadamente - 8,0% (bias) para ambos os analitos. Os coeficientes de variação médios para as concentrações de 0,5; 2,0 e 4,0 g/L foram, para a precisão intra-ensaio, de 3,5% para o AH e de 3,7% para o AMH, e para a interensaio, de 10,5% para o AH e de 10,7% para o AMH. Os analitos permaneceram estáveis em até 3 ciclos de congelamento/descongelamento das amostras de urina. O valor médio obtido nas amostras de trabalhadores expostos foi de 0,55 g/g de creatinina (mediana = 0,36 g/g de creatinina). Apesar de diferença estatisticamente significativa (teste t Student, p ≤ 0,05) ter sido observada entre os valores médios de AH nos trabalhadores expostos e nos do grupo controle, esta diferença não foi detectada quando analisou-se as medianas. O hábito de fumar e de ingerir bebidas alcoólicas também não mostrou alterar significativamente a excreção dos bioindicadores no grupo avaliado. / Toluene and the xylene, among others substances, are solvents used in several products, such as inks, thiners and glues, and are responsible for mixed exposure in workers. Thus, the aim of this work was to develop a simple, fast and reliable gas chromatographic method to allow the simultaneous detection and quantitation of hippuric acid (HA) and methylhippuric acid (MHA) in urine, which are internai dose bioindicators in the monitoring of occupational exposure to toluene and the xylene, respectively. After liquid-liquid extraction and derivation with trimethylphenylammonium, the chromatographic analysis was accomplished using capillary column 100% methylsiloxane, and heptadecanoic acid as internai standard. 69 workers exposed to inks in activities such as vehicles painting and 31 nonexposed workers were evaluated. The method showed linearity range between 0,1 (quantitation limit) and 5,0 g/L for HA and 0,2 (quantitation limit) and 5 g/L for MHA. The recovery was 81 % and the inaccuracy of approximately - 8,0% (bias) for both analytes. The mean variation . coefficients for the concentrations of 0,5; 2,0 and 4,0 g/L were, for the intraassay precision, of 3,5% for HA and of 3,7% for MHA, and for the inter assay, 10,5% for HA and 10,7% for MHA. The analytes were stable for 3 freezing cycles of the urine samples. The medium value obtained in the exposed workers\' samples was of 0,55 creatinina g/g (medium = 0,36 creatinina g/g). In spite of the observed statistical significant differences (test t Student, p ≤ 0,05) among the medium values of HA in the exposed workers and the control groups, this difference was not detected when it was analyzed the respective median values. Smoking and drinking alcoholic beverages habits did not significantly alter the excretion of these bioindicators in the studied group.

Despite being the first biochemical reaction to be discovered, the glycine conjugation pathway remains poorly characterised. It has generally been assumed that glycine conjugation serves to increase the water solubility of organic acids, such as benzoic acid and isovaleric acid, in order to facilitate urinary excretion of these compounds. However, it was recently suggested that the conjugation of glycine to benzoate should be viewed as a neuroregulatory process that prevents the accumulation of glycine, a neurotransmitter, to toxic levels. The true importance of glycine conjugation in metabolism is therefore not well understood. However, no genetic defect of glycine conjugation has ever been reported. This seems to suggest that glycine conjugation is a fundamentally important metabolic process, whatever its function may be. Therefore, a major objective of this thesis was to develop a deeper understanding of glycine conjugation and its metabolic significance. A review of the literature on GLYAT and glycine conjugation suggested that the primary purpose of glycine conjugation is indeed to detoxify benzoate and other aromatic acids of dietary origin. However, the commonly held assumption, that glycine conjugation increases the water solubility of aromatic acids in order to facilitate urinary excretion, seems to be incorrect. A better explanation for the detoxification of benzoate by means of glycine conjugation is based on hydrophilicity, not water solubility. Because of its lipophilic nature, benzoic acid is capable of passively diffusing across the mitochondrial inner membrane into the matrix space, where it accumulates due to the pH gradient over the inner membrane. Although benzoate can be exported from the matrix by organic anion transporters, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. The conversion of benzoate to hippurate is a two-step process. First, benzoate is activated by an ATP-dependent acid:CoA ligase (ACSM2A) to form the more reactive benzoyl-CoA. Second, glycine N-acyltransferase (GLYAT) catalyses the formation of hippurate and CoASH from benzoyl-CoA and glycine. Another major objective of this thesis was to gain a better understanding of the structure and function of the GLYAT enzyme. While the substrate selectivity and enzyme kinetics of GLYAT have been investigated to some extent, almost nothing has been published on the structure, active site, or catalytic mechanism of GLYAT. Furthermore, while interindividual variation in the rate of glycine conjugation has been reported by several researchers, it is not known if, or how, genetic variation in the human GLYAT gene contributes to this interindividual variation. To address these issues, systems for the bacterial expression of recombinant bovine GLYAT and recombinant human GLYAT were developed. Because no crystal structure of GLYAT has been reported, homology modelling was used to generate a molecular model of bovine GLYAT. By comparing the molecular model to other acyltransferases for which the catalytic residues were known, Glu227 of bovine GLYAT was identified as a potential catalytic residue. Site directed mutagenesis was used to generate an E227Q mutant recombinant bovine GLYAT lacking the proposed catalytic residue. Characterisation of this mutant suggested that Glu227 was indeed the catalytic residue, and the GLYAT catalytic mechanism was elucidated. The molecular model was also used to identify Asn131 of bovine GLYAT as a potential active site residue. Site-directed mutagenesis was used to generate an N131C mutant, which was sensitive to inhibition by the sulfhydryl reagent DTNB. This suggests that the Asn131 residue of bovine GLYAT may be situated in the active site of bovine GLYAT, but more work is needed to confirm this result. Finally, site-directed mutagenesis was used to generate variants of recombinant human GLYAT corresponding to six of the known SNPs in the human GLYAT gene. Expression and characterisation of the recombinant human GLYAT variants revealed that the enzyme activity and KM (benzoyl-CoA) parameter of the recombinant human GLYAT were influenced by SNPs in the human GLYAT gene. This suggests that genetic variation in the human GLYAT gene could partly explain the interindividual variation in the rate of glycine conjugation observed in humans. Interestingly, the SNPs that negatively influenced enzyme activity also had low allele frequencies, suggesting that there may be some selective advantage to having high GLYAT activity. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2014

O tolueno e o xileno são substâncias presentes, ao lado de outras, em vários produtos, entre eles as tintas, tíneres e colas, provocando exposição combinada dos trabalhadores a diversas substâncias químicas. Assim, foi objetivo deste trabalho desenvolver um método simples, rápido e confiável de cromatografia em fase gasosa que permitisse análise simultânea do ácido hipúrico (AH) e ácido metil-hipúrico (AMH) em urina, bioindicadores de dose interna utilizados na monitorização da exposição ocupacional ao tolueno e xileno respectivamente. Após extração líquido-líquido e derivação com trimetilfenilamôneo, foi realizada análise cromatográfica em coluna capilar 100% polidimetilsiloxano, usando-se o ácido heptadecanóico como padrão interno. Foram avaliados 69 trabalhadores que manipulavam tintas em atividades de repintura de veículos e 31 trabalhadores não-expostos (controle). O método mostrou-se linear entre 0,1 (limite de quantificação) e 5,0 g/L para o AH e 0,2 (limite de quantificação) e 5 g/L para o AMH. A recuperação média foi de 81 % e a inexatidão de aproximadamente - 8,0% (bias) para ambos os analitos. Os coeficientes de variação médios para as concentrações de 0,5; 2,0 e 4,0 g/L foram, para a precisão intra-ensaio, de 3,5% para o AH e de 3,7% para o AMH, e para a interensaio, de 10,5% para o AH e de 10,7% para o AMH. Os analitos permaneceram estáveis em até 3 ciclos de congelamento/descongelamento das amostras de urina. O valor médio obtido nas amostras de trabalhadores expostos foi de 0,55 g/g de creatinina (mediana = 0,36 g/g de creatinina). Apesar de diferença estatisticamente significativa (teste t Student, p ≤ 0,05) ter sido observada entre os valores médios de AH nos trabalhadores expostos e nos do grupo controle, esta diferença não foi detectada quando analisou-se as medianas. O hábito de fumar e de ingerir bebidas alcoólicas também não mostrou alterar significativamente a excreção dos bioindicadores no grupo avaliado. / Toluene and the xylene, among others substances, are solvents used in several products, such as inks, thiners and glues, and are responsible for mixed exposure in workers. Thus, the aim of this work was to develop a simple, fast and reliable gas chromatographic method to allow the simultaneous detection and quantitation of hippuric acid (HA) and methylhippuric acid (MHA) in urine, which are internai dose bioindicators in the monitoring of occupational exposure to toluene and the xylene, respectively. After liquid-liquid extraction and derivation with trimethylphenylammonium, the chromatographic analysis was accomplished using capillary column 100% methylsiloxane, and heptadecanoic acid as internai standard. 69 workers exposed to inks in activities such as vehicles painting and 31 nonexposed workers were evaluated. The method showed linearity range between 0,1 (quantitation limit) and 5,0 g/L for HA and 0,2 (quantitation limit) and 5 g/L for MHA. The recovery was 81 % and the inaccuracy of approximately - 8,0% (bias) for both analytes. The mean variation . coefficients for the concentrations of 0,5; 2,0 and 4,0 g/L were, for the intraassay precision, of 3,5% for HA and of 3,7% for MHA, and for the inter assay, 10,5% for HA and 10,7% for MHA. The analytes were stable for 3 freezing cycles of the urine samples. The medium value obtained in the exposed workers\' samples was of 0,55 creatinina g/g (medium = 0,36 creatinina g/g). In spite of the observed statistical significant differences (test t Student, p ≤ 0,05) among the medium values of HA in the exposed workers and the control groups, this difference was not detected when it was analyzed the respective median values. Smoking and drinking alcoholic beverages habits did not significantly alter the excretion of these bioindicators in the studied group.

New Zealand’s 5.3 million strong dairy herd returns approximately 106 million litres of urine to pasture soils daily. The urea in that urine is rapidly hydrolysed to ammonium (NH₄⁺), which is then nitrified, with denitrification of nitrate (NO₃⁻) ensuing. Nitrous oxide (N₂O), a potent greenhouse gas (GHG), is produced via nitrification and denitrification, which are enzyme-catalysed processes mediated by soil microbes. Thus microbes are linked intrinsically to urine patch chemistry. However, few previous studies have investigated microbial dynamics in urine patches. Therefore the objective of these four experiments was to investigate the effects on soil microbial communities of cow urine deposition. Methods used included phospholipid fatty acid (PLFA) analyses of microbial community structure and microbial stress, dehydrogenase activity (DHA) assays measuring microbial activity, and headspace gas sampling of N₂O, ammonia (NH₃) and carbon dioxide (CO₂) fluxes. Experiment 1, a laboratory study, examined the influence of soil moisture and urinary salt content on the microbial community. Both urine application and high soil moisture increased microbial stress, as evidenced by significant changes in PLFA trans/cis and iso/anteiso ratios. Total PLFAs and DHA showed a short-term (< 1 week) stimulatory effect on microbes after urine application. Mean cumulative N₂O-N fluxes were 2.75% and 0.05% of the nitrogen (N) applied, from the wet (70% WFPS) and dry (35% WFPS) soils, respectively. Experiment 2, a field trial, investigated nutrient dynamics and microbial stress with plants present. Concentrations of the micronutrients, copper, iron and molybdenum, increased up to 20-fold after urine application, while soil phosphorus (P) concentrations decreased from 0.87 mg kg ⁻¹ to 0.48 mg kg⁻¹. Plant P was also lower in urine patches, but total PLFAs were higher, suggesting that microbes had utilised the available nutrients. Microbial stress again resulted from urine application but, in contrast to experiment 1, the fungal biomass recovered after its initial inhibition. Studies published during the course of this thesis reported that hippuric acid (HA) and its hydrolysis product benzoic acid (BA) significantly reduced N₂O-N emissions from synthetic cow urine, thus experiment 3 investigated this effect using real cow urine. Cumulative N₂O-N fluxes were 16.8, 5.9 and 4.7% of N applied for urine (U) alone, U+HA and U+BA, respectively. Since NH₃-N volatilisation remained unchanged, net gaseous N emissions were reduced. Trends in total PLFAs and microbial stress were comparable to experiment 1 results. Experiment 4 studied HA effects at different temperatures and found no inhibition of N₂O-N fluxes from HA-amended urine. However, mean cumulative N₂O-N fluxes were reduced from 7.6% of N applied at 15–20°C to 0.2% at 5–10°C. Total cumulative N emissions (N₂O-N + NH₃-N) were highest at 20°C (17.5% of N applied) and lowest at 10°C (9.8% of N applied). Microbial activity, measured as potential DHA, increased with increasing temperature. This work has clearly shown that the stimulation and inhibition of the soil microbial community by urine application are closely linked to soil chemistry and have significant impacts not only on soil nutrient dynamics but also on N₂O-N emissions and their possible mitigation.

Capillary zone electrophoresis is frequently used in various analyses. In this diploma thesis a hydrodynamic sample introduction method controlled by pressure pulse has been proposed for short-capillary electrophoresis. The base electrolyte flushes sample from the loop of a six-way sampling valve and is carried to the injection end of the capillary. At the time when the sample zone reached the capillary, a short pressure impulse is generated in the electrolyte stream, which provides injection of the sample into the capillary. Then the electrolyte flow is stopped and the separation voltage is turned on. The amount of sample introduced to the capillary is controlled by the duration of the pressure pulse. This new sample introduction method was tested in the determination of ammonia, histidine, creatinine, uric acid and hippuric acid in human urine and for rapid screening of the contents of the inorganic ions in cerebrospinal fluid and blood plasma. The determination was performed in a capillary with an overall length of 10,5 cm and two base electrolytes was tested - 50 mM MES + 5 mM NaOH (pH 5,10) and 1 M acetic acid + 1,5 mM crown ether 18-crown-6 (pH 2,40). Using dual detection techniques contactless conductivity and UV spectrometric detection, anorganic and organic substances in the sample could...