No description available.

Thesis (MSc (Biochemistry))--University of Stellenbosch, 2009. / This study: 1. Describes the preparation of unfermented and fermented rooibos methanol and aqueous extracts. 2. Investigates the influence of unfermented and fermented rooibos methanol and aqueous extracts on the binding of natural steroid substrates to ovine adrenal microsomal cytochrome P450 enzymes, demonstrating that the binding of natural steroids is inhibited in the presence of rooibos extracts. 3. Describes an assay demonstrating the inhibitory effect of rooibos extracts on the catalytic activity of cytochrome 17α-hydroxylase (CYP17) and cytochrome 21-hydroxylase (CYP21) in ovine adrenal microsomes. 4. Investigates the influence of unfermented and fermented rooibos methanol extracts on the catalytic activity of individual cytochrome P450 enzymes – CYP17 and baboon CYP21, that are expressed in COS1 cells. 5. Demonstrates that fractions of the unfermented rooibos methanol extract inhibits the binding of natural steroid substrate to microsomal cytochrome P450 enzymes as well as the catalytic activity of baboon CYP21 expressed in COS1 cells. 6. Investigates the inhibitory influence of individual rooibos flavonoids on the catalytic activity of baboon CYP21 expressed in COS1 cells.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: This study describes: • the influence of a methanolic extract of unfermented Rooibos and five major Rooibos flavonoids, aspalathin, nothofagin, rutin, orientin and vitexin, on the activities of key adrenal steroidogenic enzymes - cytochrome P450 17β- hydroxylase/17,20-lyase (CYP17A1), 3β-hydroxysteroid dehydrogenase • the development of a novel UPLC-MS/MS method for the separation and quantification of 21 adrenal steroid metabolites; • the influence of Rooibos and aforementioned flavonoids on adrenal steroid hormone production in H295R cells - a human adrenal carcinoma cell line expressing the enzymes catalysing the production of mineralocorticoids, glucocorticoids and adrenal androgens, assayed under both basal (normal) and forskolin-stimulated (stressed) conditions; • the influence of Rooibos on the inter-conversion between cortisol and cortisone by 11βHSD1 and 11βHSD2 expressed in CHO-K1 cells; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male Wistar rats; • the influence of Rooibos consumption on circulating steroid hormone levels and ratios in male and female human test subjects at risk for developing cardiovascular disease. (3βHSD2), cytochrome P450 21-hydroxylase (CYP21A2) and cytochrome P450 11β-hydroxylase (CYP11B1), expressed in non-steroidogenic COS-1 cells; / AFRIKAANSE OPSOMMING: Hierdie studie beskryf: • die invloed van metanoliese ekstrakte van ongefermenteerde Rooibos en vyf van die hoof flavonoïedverbindings in Rooibos, aspalatien, notofagien, rutien, oriëntien en viteksien, op die aktiwiteite van ensieme wat steroïedbiosintese in die bynier kataliseer – sitochroom P450 17α-hidroksilase/17,20-liase (CYP17A1), 3β-hidroksisteroïed dehidrogenase (3βHSD2), sitochroom P450 21-hidroksilase (CYP21A2) en sitochroom P450 11β-hidroksilase (CYP11B1), uitgedruk in nie-steroïed produserende COS-1 selle; • die ontwikkeling van ‘n geskikte UPLC-MS/MS metode vir die skeiding en kwantifisering van 21 steroïedmetaboliete in die bynier; • die invloed van Rooibos en die bg. flavonoïede op steroïedproduksie in H295R selle – ‘n menslike bynier kanker sellyn gekenmerk deur die ekspressie van die steroidogeniese ensieme wat die produksie van mineralokortikoïede, glukokortikoïede en bynierandrogene kataliseer, geanaliseer onder beide basale (normale) en forskoliengestimuleerde (gestresde) kondisies; • die invloed van Rooibos op die omeenskakeling tussen kortisol en kortisoon deur 11βHSD1 and 11βHSD2 in CHO-K1 selle; • die invloed van Rooibosinname op vlakke van sirkulerende steroïed hormone en relatiewe verhoudings in die bloed van manlike Wistarrotte; • die invloed van Rooibosinname op sirkulerende steroïed hormoon vlakke en relatiewe verhoudings in die bloed van mans en vrouens met ‘n hoë risiko vir die ontwikkeling van kardiovaskulêre siektes.

The objectives were to investigate the effects of chromium (III) picolinate (CrPic; up to 2,000 ppb of Cr) on growing pigs subjected to a variety of stressors including thermal, immune, or dietary stress and to examine the effects of CrPic on steroidogenesis from adrenocortical cells. In the thermal stress study, high ambient temperature caused reduced weight gain and feed consumption (P andlt; 0.01), and low ambient temperature caused increased feed intake and feed:gain (P andlt; 0.01). However, these effects were not moderated by CrPic, and respiratory rate, plasma cortisol, or plasma glucose were unaffected by CrPic. In the immune stress study, pigs challenged with lipopolysaccharide (LPS) lost 951 g during 12 hours post injection, while the phosphate buffer saline (PBS) injected group gained 170 g (P andlt; 0.001). The LPS group showed higher rectal temperature (P andlt; 0.05), higher respiratory rate (P andlt; 0.05), greater plasma cortisol (P andlt; 0.001), and lower plasma glucose (P andlt; 0.05) than the PBS group. These effects were not ameliorated by CrPic. In the dietary stress study, pigs fed the high-fat diet (HFD) gained weight faster (P andlt; 0.05), consumed less feed (P andlt; 0.001), and had lower feed:gain (P andlt; 0.001). Plasma insulin concentration on d 14 decreased with CrPic (P andlt; 0.05) in a linear manner (P = 0.05). Consumption of the HFD resulted in increases of slaughter weight, perirenal fat, and back fat measurements (P andlt; 0.01). The CrPic resulted in linear reductions of carcass weight, last rib fat, last lumbar fat and average backfat (P andlt; 0.10). The effects of CrPic on carcass fat measurements were more significant in barrows than gilts. In the adrenocortical cell study, forskolin stimulated cortisol and DHEAs secretion from H295R cells. CrPic inhibited aspects of steroidogenesis in agonist-stimulated adrenocortical cells. Overall, dietary CrPic was unable to moderate the stress related effects due to high ambient temperature, low ambient temperature, or an endotoxin challenge. However, CrPic attenuated effects of HFD, mainly on body fat accretion of pigs, especially in barrows, and CrPic inhibited steroidogenesis in stimulated adrenocorticoid cells.

La majorité des hyperplasies macronodulaires bilatérales des surrénales avec syndrome de Cushing ACTH-indépendant (AIMAH) est due à l’expression aberrante de divers récepteurs hormonaux au niveau du cortex surrénalien. Les gènes responsables des AIMAH familiales avec récepteurs aberrants n’ont pas été identifiés. Le but de ce projet est de les identifier. Une étude de liaison, visant à identifier la ou les régions du génome comprenant le ou les gènes pouvant être en cause dans les AIMAH familiales, a été réalisée en utilisant l’ADN des membres d’une famille (10 malades et 7 sains) originaire du Québec, atteinte d’AIMAH et syndrome de Cushing et caractérisée par l’expression des récepteurs β-adrénergique et V1-vasopressine. Diverses régions chromosomiques entre les personnes atteintes et non-atteintes de la famille ont été soulignées. Un total de 707453 SNPs a été obtenu, et après analyse statistique, 159 SNPs significatifs, pouvant être associés au phénotype, ont été mis en évidence entre les deux groupes. Il a été constaté que la majorité de ces SNPs se situaient sur les régions chromosomiques 1q32.1 et 16q12.2. Une étude du transcriptome a aussi été réalisée en utilisant l’ADN des tumeurs de deux patients de la famille, ainsi que l’ADN d'autres tumeurs surrénaliennes. Les analyses statistiques ont permis d’identifier 15 gènes susceptibles d’être reliés à la maladie (11 surexprimés et 4 sous-exprimés). En utilisant les données de ces deux études, nous avons ciblé six gènes du chromosome 1 (ATP2B4, PPP1R12B, SOX13, CACNA1S, ADORA1et PHLDA3), un du chromosome 16 (CHD9) et un du chromosome 13 (SPRY2), afin de rechercher la présence de mutations. Le séquençage n’a révélé aucun changement de nucléotide dans les gènes PPP1R12B et SOX13. Dans les gènes ATP2B4, CACNA1S, ADORA1et PHLDA3, le séquençage a révélé des changements de nucléotides n’entrainant soit pas de changement d’acide aminé soit un changement d’acide aminé jugé « non pertinent », du fait qu’il ne permettait pas de différencier les sujets sains des sujets atteints. Pour ce qui est de CHD9 et SPRY2, le séquençage a permis d’identifier des changements de nucléotides entrainant des changements d’acides aminés de façon plus fréquente chez les sujets atteints par rapport aux sujets sains. En conclusion, nos travaux nous ont donc permis d’identifier, par étude de liaison et par analyse du transcriptome, des gènes candidats qui pourraient être responsables de cette pathologie. Le séquençage de ces gènes candidats a révélé des mutations de CHD9 et SPRY2. Ces résultats s’avèrent prometteurs puisque ces deux gènes produisent des protéines impliquées dans le remodelage de la chromatine et dans la régulation de la signalisation des protéines kinases. Le phénotypage et le génotypage des patients atteints doivent être poursuivis pour vérification. / The majority of ACTH-independent macronodular adrenal hyperplasia with Cushing's syndrome (AIMAH) is due to the aberrant expression of various receptors in the adrenal cortex. The genes responsible for familial AIMAH with aberrant receptors have not been identified. The aim of this project is to characterize them. A linkage study to identify the region or regions of the genome comprising the gene or genes that may be involved in familial AIMAH was performed using DNA of family members (10 affected and 7 non affected) born in Quebec and harboring AIMAH and Cushing's syndrome, under the aberrant regulation of B-adrenergic and V1-vasopressin receptors. Various chromosomal regions between patients and non-affected family were highlighted. A total of 707,453 SNPs were obtained, and after statistical analysis, 159 significant SNPs, possibly associated with phenotype, were found between the two groups. It was found that the majority of these SNPs were located on chromosomal regions 1q32.1 and 16q12.2. A transcriptome analysis was conducted using DNA from tumours of two patients of the family, as well as DNA from other adrenal tumours; Statistical analysis identified 15 genes that may be linked to disease (11 up-regulated and 4 under-expressed). Using data from these two studies, we identified six genes on chromosome 1 (ATP2B4, PPP1R12B, SOX13, ADORA1, CACNA1S and PHLDA3), one on chromosome 16 (CHD9) and one on chromosome 13 (SPRY2), to investigate the presence of mutations. The sequencing revealed no nucleotide changes in gene PPP1R12B and SOX13. In ATP2B4, CACNA1S, ADORA1 and PHLDA3, the sequencing not revealed nucleotides changes leading to either amino acid changes or an amino acid changes considered “not-relevant”, because they do not differentiate healthy individuals from affected. The sequencing of CHD9 and SPRY2 identified nucleotide changes causing amino acid changes more frequently in patients compared to healthy subjects. In conclusion, our work has therefore identified by linkage analysis and DNA microarray candidate genes that can be responsible to this disease, and mutations in two of these genes, CHD9 and SPRY2. These results are promising because these genes produce proteins involved in chromatin remodeling and regulation of signaling protein kinases. Phenotyping and genotyping of patients should be pursued further.

Este estudo avaliou a eficácia do propionato de clobetasol a 0,05% em pomada, com uso de moldeiras individuais de silicone, em 22 pacientes com lesões gengivais de doenças mucocutâneas auto-imunes e inflamatórias, por meio de um estudo duplo-cego, cruzado, placebo-controlado. Os pacientes foram distribuídos em dois grupos: grupo 1, que consistiu de 5 pacientes em uso de corticosteróide e/ou imunossupressor sistêmico para controle de lesões cutâneas e/ou gengivais associadas às doenças mucocutâneas; grupo 2, que consistiu de 17 pacientes sem utilização de corticosteróide e/ou imunossupressor sistêmico. Os pacientes de cada grupo receberam a bisnaga 1, e foram orientados a utilizá-la no preenchimento das moldeiras. Em seguida, foram instruídos a aplicar a pomada com a moldeira 3 vezes ao dia, durante 20 minutos, por um período de duas semanas. A freqüência de uso da pomada foi reduzida na 3 a semana para 1 vez ao dia (pela manhã), em dias alternados. Após esta fase, foi estabelecido um intervalo de 2 semanas sem tratamento após o qual houve a inversão das pomadas (bisnaga 2), e os pacientes passaram a utilizá-la da mesma maneira que a bisnaga 1. As consultas de avaliações foram realizadas na 2 a , 5 a , 7 a , e na 10 a semana após o início do teste, e a resposta terapêutica foi baseada no percentual da remissão dos sinais classificada como completa (100%), excelente (75% a 99%), boa (50% a 74%), regular (1% a 49%), inalterada e piorada; e da remissão dos sintomas classificada como completa, parcial, inalterada e piorada. Durante as consultas de retorno, os pacientes foram monitorados quanto à ocorrência de efeitos colaterais. Com relação à remissão dos sinais, nos pacientes do grupo 1, 4 pacientes (80%) mostraram resposta regular; e 1 paciente (20%) apresentou piora do quadro clínico após o uso do propionato de clobetasol. Nos pacientes do grupo 2, 13 pacientes (76,5%) apresentaram alguma melhora durante o uso do propionato de clobetasol, e 4 pacientes (23,5%) apresentaram piora do quadro clínico. Com relação aos sintomas, durante o uso do propionato de clobetasol, 3 (60%) pacientes do grupo 1, apresentaram melhora parcial, 1 paciente (20%) não verificou mudança na sintomatologia, e 1 paciente (20%) referiu piora dos sintomas. Nos pacientes do grupo 2, completa melhora dos sintomas foi verificado em 2 pacientes (11,8%) e resposta parcial em 9 pacientes (52,9%) durante o uso do propionato de clobetasol. A diferença dos resultados obtidos entre o período de uso do propionato de clobetasol e placebo, nos dois grupos de pacientes e para os parâmetros analisados, não foi estatisticamente significante (Teste exato de Fisher; p > 0,05). Apenas 2 pacientes (11,8 %) do grupo 2 desenvolveram candidose após o uso do propionato de clobetasol. Os resultados deste estudo demonstraram que a aplicação do propionato de clobetasol 0,05% em pomada, com o auxílio de moldeiras de silicone, apresenta eficácia boa a moderada no controle das lesões gengivais de doenças mucocutâneas, causando mínimo de efeitos colaterais. / This study evaluated the efficacy of 0.05% clobetasol propionate in ointment administered with trays in 22 patients with gingival lesions of autoimmune and inflammatory mucocutaneous diseases. The patients were subdivided into two groups: group 1, which was composed by 5 patients treated systemically with corticosteroid and/or other immunosuppressive drug for control of skin and/or gingival lesions associated to mucocutaneous diseases; and the group 2, which was composed by 17 patients not being under systemic corticotherapy. The patients of each group received the container number 1 and they were instructed to apply the ointment with the tray for 20 minutes, 3 times daily, for 2 weeks. The frequency of use of ointment was reduced in the third week for once a day on alternate days. After that, the patients were instructed to discontinue the treatment for 2 weeks (washout period), and then were given the container number 2, to be used in the same way as that of the container 1. Each patient was examined in the weeks 2, 5, 7, and 10 after the beginning of the study. The therapeutic response was determined according to remission of signs on percentage, and assessed as follow: complete (100%), excellent (75% to 99%), good (50% to 74%), poor (1% to 49%), failed and worsened; and on remission of symptoms assessed as complete, partial, failed and worsened. At every visit, the patients were also examined for the presence of side-effects. In group 1, 4 patients (80%) had a poor response, and 1 patient (20%) had a worse of clinical presentation after the use of clobetasol propionate. In patients of group 2, 13 (76.5%) presented some improvement after the use of clobetasol propionate, and 4 patients (23.5%) presented worse of signals. For symptoms, 3 patients (60%) of the group 1 showed partial improvement while 1 (20%) presented no response, and 1 (20%) had symptoms worsened after the use of corticosteroid. In the group 2, complete improvement of symptoms was observed in 2 (11.8%) and partial in 9 (52.9%) after the use of clobetasol. There was no statistical difference when compared the results obtained with clobetasol propionate and placebo in the two groups of patients for the parameters evaluated (Fisher test; P > 0.05). Only 2 patients (11.8%) of group 2 developed candidosis after the use of clobetasol propionate. This study showed that clobetasol propionate ointment present good to moderate efficacy, with minimal side-effects, in the treatment of gingival lesions of mucocutaneous diseases.

A mensuração da glândula adrenal nos cães ainda não está claramente estabelecida de acordo com o peso do animal, havendo uma ampla faixa de normalidade descrita e, além disso, esta não foi relacionada à dimensão da aorta. Este projeto objetivou estabelecer padrão de normalidade de mensuração ultrassonográfica para glândulas adrenais esquerda e direita de cadelas adultas jovens na faixa etária de 1 a 3 anos divididas por porte. A metodologia consiste em verificar se existe relação entre diâmetro da aorta mensurada logo cranialmente à sua trifurcação e a altura do polo caudal da glândula adrenal, na região de maior espessura, em três grupos de peso corporal (porte pequeno, médio e grande). As dimensões do polo caudal das glândulas adrenais direita e esquerda e o diâmetro da aorta, bem como, as relações aorta/adrenal direita e aorta/adrenal esquerda foram avaliadas por dois avaliadores experientes. Os animais foram divididos em grupo 1 (G1) até 10 kg (21 animais), grupo 2 (G2) de 10,1 a 25 kg (22 animais) e grupo 3 (G3) acima de 25,1 a 38 kg (20 animais). A média e o desvio padrão da mensuração do polo caudal da glândula adrenal direita foram de 0,37±0,072 cm (G1), 0,48±0,077 cm (G2) e 0,5±0,048 cm (G3) e da glândula adrenal esquerda de 0,38±0,082 cm (G1), 0,45±0,077 cm (G2) e 0,47±0,073 cm (G3). O diâmetro da aorta 0,52±0,1 cm (G1); 0,78±0,097 cm (G2); 0,94±0,071 cm (G3). Estatisticamente o grupo 2 e o grupo 3 foram semelhantes entre si e significativamente diferentes do grupo 1. Portanto, a mensuração do polo caudal das glândulas adrenais relacionadas ao peso corporal não foi considerada um parâmetro confiável. Os resultados indicaram que, nesse estudo, a relação entre aorta/glândulas adrenais de animais até 10 kg variou de 1,2 a 1,6 e de animais de 10,1 a 38 kg de 1,6 a 2,1. A relação aorta/glândulas adrenais provou ser um ótimo parâmetro para estabelecer mensuração normal das glândulas adrenais e, portanto, sugerido como novo parâmetro a ser utilizado na avaliação ultrassonográfica biométrica das glândulas adrenais. Ao relacionar estruturas do próprio animal, foi estabelecido de forma mais específica para o indivíduo uma relação entre a aorta e glândula adrenal e não mais um valor esperado em uma população normal. / Adrenal gland measurements remain unestablished in dogs since literature reports no consensus regarding to those values according to animal size. There can be seen a wide range of normality and no correlation between adrenal size and the dimension of aorta has yet been described. The present study aims to establish standard values for the ultrasonographic measurement of the right and left adrenal glands, considering 1 to 3-year-old healthy female dogs divided into size categories. Methods consist in verifying the relationship between the diameter of the aorta, measured immediately before its trifurcation, and height of the caudal extremity of the adrenal glands in three different groups according to body weight (small, medium and large dogs. The dimensions of the caudal pole right and left adrenal glands and the aorta, as well as, the aorta/ right adrenal and aorta/left adrenal relations were evaluated by two experienced professionals. Animals weighting up to 10 Kg (n=21) were classified in group 1 (G1); animals weighting from 10,1 to 25 Kg (n=22) were classified in group 2 (G2) and animals weighting 25,1 to 38 Kg (n=20) were classified in group 3 (G3). Mean values and standard deviation of the right adrenal gland were 0,37±0,072 cm (G1), 0,48±0,077 cm (G2) and 0,5±0,048 cm (G3) and for the left adrenal gland, the values were 0,38±0,082 cm (G1), 0,45±0,077 cm (G2) and 0,47±0,073 cm (G3). The values obtained for the aortic diameter were 0,52±0,1 cm (G1); 0,78±0,097 cm (G2) and 0,94±0,071 cm (G3). Groups 2 and 3 were statistically similar and differed from group 1. Therefore, the relationship between the measurement of the caudal extremity of the adrenal glands and the body weight was considered a non-reliable parameter. Results indicated that, in our study, the aorta/adrenal gland ratio ranged from 1,2cm to 1,6cm in dogs with up to 10 Kg and from 1,6cm up to 2,1cm for dogs weighting 10,1 Kg to 38 Kg. The aorta/adrenal gland ratio has proven to be an excellent parameter to establish normal adrenal values and, therefore, this parameter can be used for the biometric investigation of the adrenals, using ultrasound. The correlation between different structures from the same animal, based on the aorta/adrenal gland ratio in the present study, contributed to more accurate values when compared to the general values established for normal populations.

by Perpetua E. Tan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 149-157). / Abstract --- p.9 / Acknowledgments --- p.12 / List of abbreviations --- p.13 / List of Tables --- p.16 / List of Figures --- p.19 / Chapter CHAPTER 1 --- INTRODUCTION --- p.21 / Chapter CHAPTER 2 --- LITERATURE REVIEWS CATECHOLAMINES: NORADRENALINE AND ADRENALINE --- p.25 / Chapter 2.1 --- History --- p.25 / Chapter 2.2 --- Origin of plasma catecholamines --- p.25 / Chapter 2.3 --- Kinetics of entry and removal --- p.28 / Chapter 2.4 --- Levels present in plasma --- p.30 / Chapter 2.5 --- Some factors affecting plasma CA levels --- p.31 / Chapter 2.5.1 --- Effects of age --- p.31 / Chapter 2.5.2 --- Postural change --- p.32 / Chapter 2.5.3 --- Exercise --- p.32 / Chapter 2.5.4 --- Temperature change --- p.32 / Chapter 2.5.5 --- Stress --- p.33 / Chapter 2.5.6 --- Pregnancy --- p.34 / Chapter 2.5.7 --- Disease --- p.35 / Chapter 2.6 --- Actions in the body --- p.35 / Chapter 2.6.1 --- Plasma endogenous catecholamines --- p.35 / Chapter 2.6.2 --- Plasma exogenous catecholamines and medicine --- p.36 / Chapter 2.6.2.1 --- Clinical uses --- p.36 / Chapter 2.6.2.2 --- Effects --- p.37 / Chapter 2.6.2.3 --- Side effects --- p.38 / Chapter 2.7 --- Binding of catecholamines in plasma --- p.38 / Chapter 2.8 --- Measurement of catecholamines in plasma --- p.38 / Chapter 2.8.1 --- Chemistry --- p.38 / Chapter 2.8.2 --- Extraction and purification --- p.39 / Chapter 2.8.3 --- Biological methods --- p.40 / Chapter 2.8.4 --- Colorimetry --- p.41 / Chapter 2.8.5 --- Radioimmunoassay and radioenzymatic assay --- p.41 / Chapter 2.8.6 --- Enzyme-linked immunoassay --- p.42 / Chapter 2.8.7 --- Gas chromatography --- p.42 / Chapter 2.8.8 --- Liquid chromatography --- p.42 / Chapter 2.8.8.1 --- Fluorometry --- p.43 / Chapter 2.8.8.2 --- Electrochemical detection --- p.43 / Chapter 2.9 --- Plasma protein binding of basic drugs --- p.44 / Chapter 2.9.1 --- Binding to albumin --- p.45 / Chapter 2.9.2 --- Binding to alpha-1-acid-glycoprotein --- p.45 / Chapter 2.9.3 --- Binding to other proteins --- p.45 / Chapter 2.9.4 --- Factors affecting drug binding --- p.46 / Chapter 2.9.4.1 --- Pregnancy --- p.46 / Chapter 2.9.4.2 --- Age --- p.46 / Chapter 2.9.4.3 --- Disease states --- p.46 / Chapter 2.9.5 --- Separation procedures to reveal and follow drug protein binding --- p.47 / Chapter 2.9.5.1 --- Equilibrium dialysis --- p.47 / Chapter 2.9.5.2 --- Ultrafiltration --- p.48 / Chapter 2.9.5.3 --- Ultracentrifugation --- p.48 / Chapter 2.9.5.4 --- Gel Filtration --- p.48 / Chapter CHAPTER 3 --- ANALYTICAL TECHNIQUE : PLASMA CATECHOLAMINE ANALYSIS --- p.49 / Chapter 3.1 --- HPLC determination with coulometric detection of catecholamines --- p.49 / Chapter 3.1.1 --- Introduction --- p.49 / Chapter 3.1.2 --- Basic equipment --- p.49 / Chapter 3.1.3 --- Mobile phase preparation --- p.50 / Chapter 3.1.3.1 --- Reagent A (Citrate-acetate-EDTA buffer) --- p.50 / Chapter 3.1.3.2 --- Reagent B (ion pairing reagent) --- p.50 / Chapter 3.1.3.3 --- Mobile phase mixture --- p.50 / Chapter 3.1.4 --- Detector settings --- p.51 / Chapter 3.1.5 --- Sample collection and storage --- p.51 / Chapter 3.2 --- Reagents and solutions --- p.52 / Chapter 3.2.1 --- Acid-washed alumina --- p.52 / Chapter 3.2.2 --- Tris buffer solution --- p.53 / Chapter 3.2.3 --- Washing solution --- p.53 / Chapter 3.2.4 --- Acetic acid solution --- p.53 / Chapter 3.2.5 --- EDTA-HC1 solution --- p.53 / Chapter 3.2.6 --- Citric acid solution --- p.53 / Chapter 3.2.7 --- Stock solutions --- p.54 / Chapter 3.2.7.1 --- Catecholamine standards --- p.54 / Chapter 3.2.7.2 --- Dihydroxybenzylamine (Internal) standard --- p.54 / Chapter 3.2.8 --- Stripped fresh frozen plasma --- p.54 / Chapter 3.2.9 --- Sorensen's phosphate buffer containing 0.6% NaCl --- p.55 / Chapter 3.2.10 --- Control standards --- p.55 / Chapter 3.3 --- Voltammogram of catecholamines and internal standard used --- p.55 / Chapter 3.4 --- Maintenance of the HPLC-Coulometric detector system --- p.56 / Chapter 3.5 --- Optimization of the extraction method --- p.58 / Chapter 3.5.1 --- Amount of alumina for adsorption of CA --- p.58 / Chapter 3.5.2 --- pH of tris buffer for maximum uptake of CA onto alumina --- p.58 / Chapter 3.5.3 --- Optimum time for maximum uptake of CA onto alumina --- p.59 / Chapter 3.5.4 --- Optimum time for maximum desorption of CA into acid solution --- p.59 / Chapter 3.5.5 --- Optimum volume of acid solution for maximum desorption of CA --- p.60 / Chapter 3.6 --- Validation of the method --- p.60 / Chapter 3.6.1 --- Linearity --- p.60 / Chapter 3.6.2 --- Recovery --- p.61 / Chapter 3.6.3 --- Reproducibility --- p.62 / Chapter 3.6.4 --- Stability --- p.62 / Chapter 3.7 --- Results --- p.63 / Chapter 3.8 --- Discussion --- p.79 / Chapter CHAPTER 4 --- CLINICAL APPLICATIONS OF THE CATECHOLAMINE ASSAY --- p.84 / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Applications of catecholamines assay in clinical science --- p.84 / Chapter 4.2 --- : PLASMA CATECHOLAMINES AFTER INDUCTION OF ANAESTHESIA AT CAESARIAN SECTION --- p.84 / Chapter 4.2.1 --- Introduction --- p.84 / Chapter 4.2.2 --- Patients and methods --- p.86 / Chapter 4.2.3 --- Blood sampling and storage --- p.87 / Chapter 4.2.4 --- Statistics used --- p.87 / Chapter 4.2.5 --- Results --- p.88 / Chapter 4.2.6 --- Discussion --- p.99 / Chapter 4.3 --- EPINEPHRINE INFILTRATION IN SINUS SURGERY --- p.101 / Chapter 4.3.1 --- Introduction --- p.101 / Chapter 4.3.2 --- Patients and methods --- p.102 / Chapter 4.3.3 --- Blood sampling and storage --- p.103 / Chapter 4.3.4 --- Results --- p.104 / Chapter 4.3.5 --- Discussion --- p.108 / Chapter CHAPTER 5 --- ANALYTICAL TECHNIQUE: PLASMA PROTEIN BINDING OF CATECHOLAMINES --- p.110 / Chapter 5.1 --- Equilibrium dialysis for protein binding of drugs --- p.110 / Chapter 5.1.1 --- Introduction --- p.110 / Chapter 5.1.2 --- Dialyzing apparatus --- p.110 / Chapter 5.1.3 --- Sample collection and storage --- p.111 / Chapter 5.1.4 --- Reagents and solutions --- p.111 / Chapter 5.1.4.1 --- Ascorbic acid --- p.111 / Chapter 5.1.4.2 --- Glutathione --- p.111 / Chapter 5.1.4.3 --- Sodium metabisulfite --- p.111 / Chapter 5.1.4.4 --- Dialysis buffer --- p.111 / Chapter 5.1.5 --- Dialysis membrane --- p.112 / Chapter 5.1.6 --- Equilibrium dialysis --- p.112 / Chapter 5.2 --- Optimization of the binding parameters --- p.113 / Chapter 5.2.1 --- Types of preservatives for stability of catecholamines during dialysis --- p.113 / Chapter 5.2.2 --- Dialysis buffer --- p.114 / Chapter 5.2.3 --- Dialysis time and volume of sample --- p.114 / Chapter 5.2.4 --- Dialysis membrane --- p.115 / Chapter 5.2.5 --- Catecholamines concentration for dialysis --- p.114 / Chapter 5.3 --- Total protein analysis- Lowry Method --- p.115 / Chapter 5.3.1 --- Reagents and solutions --- p.116 / Chapter 5.3.1.1 --- Reagent A (Alkaline copper reagent) --- p.116 / Chapter 5.3.1.2 --- Reagent B (Folin-Ciocalteus phenol reagent with water) --- p.116 / Chapter 5.3.2 --- Stock standard and controls --- p.116 / Chapter 5.3.2.1 --- Human serum albumin standard --- p.116 / Chapter 5.3.2.2 --- Controls --- p.116 / Chapter 5.3.3 --- Procedure --- p.116 / Chapter 5.4 --- Results --- p.117 / Chapter 5.5 --- Discussion --- p.126 / Chapter CHAPTER 6 --- CONCLUSIONS --- p.130 / APPENDIX --- p.134 / CHEMICALS AND REAGENTS --- p.146 / REFERENCES --- p.149

Sam Sze Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 156-184). / Abstracts in English and Chinese. / Publications based on work in this thesis --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vii / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Corticosteroids --- p.2 / Chapter 1.1.1 --- Chemical Structure of Steroids --- p.3 / Chapter 1.1.2 --- Biosynthesis of Endogenous Corticosteroids --- p.3 / Chapter 1.1.2.1 --- Regulation of Cortisol synthesis and negative feedback system --- p.4 / Chapter 1.1.3 --- Biological Significance of Corticosteroids --- p.5 / Chapter 1.1.3.1 --- Involvement of corticosteroids as anti-inflammatory drugs --- p.6 / Chapter 1.1.3.2 --- Eicosanoid biosynthesis --- p.7 / Chapter 1.1.3.3 --- Lipoxygenase pathway --- p.9 / Chapter 1.1.3.4 --- Side-effects of prolonged use of corticosteroids --- p.9 / Chapter 1.2 --- Organisation of the Emetic Reflex --- p.11 / Chapter 1.2.1 --- Motor Pathway of Emetic Reflex --- p.12 / Chapter 1.2.1.1 --- Retching and vomiting --- p.12 / Chapter 1.2.1.2 --- Nausea --- p.13 / Chapter 1.2.2 --- Components of the Emetic Reflex --- p.14 / Chapter 1.2.2.1 --- The vomiting centre (VC) --- p.15 / Chapter 1.2.2.2 --- Area postrema (AP) / Chemoreceptor trigger zone (CTZ) --- p.15 / Chapter 1.2.2.3 --- The nucleus tractus solitarius (NTS) --- p.17 / Chapter 1.2.2.4 --- Gastrointestinal tract and vagus nerves --- p.17 / Chapter 1.2.2.5 --- Neurotransmitter receptors --- p.18 / Chapter 1.3 --- Chemotherapy-Induced Emesis --- p.19 / Chapter 1.3.1 --- Cancer as a cause of mortality in Man --- p.20 / Chapter 1.3.2 --- Chemotherapeutic Agents --- p.20 / Chapter 1.3.2.1 --- Different classes --- p.20 / Chapter 1.3.2.2 --- Emetogenic potential --- p.21 / Chapter 1.3.3 --- Cisplatin-Induced Emesis --- p.23 / Chapter 1.3.3.1 --- Unfavourable effects associated with chemotherapy-induced nausea and emesis --- p.24 / Chapter 1.3.3.2 --- Anticipatory nausea and vomiting --- p.24 / Chapter 1.3.3.3 --- Profile of cisplatin-induced emesis --- p.25 / Chapter 1.3.4 --- Animal Models of Cisplatin-Induced Acute and Delayed Emesis --- p.26 / Chapter 1.3.5 --- Mechanisms and Pathways Involves in Chemotherapy-Induced Emesis --- p.28 / Chapter 1.3.6 --- Anti-Emetic Drugs for the Treatment of Chemotherapy-Induced Emesis --- p.31 / Chapter 1.3.6.1 --- 5-HT3 receptor antagonists --- p.31 / Chapter 1.3.6.2 --- Dopamine receptor antagonists --- p.33 / Chapter 1.3.6.3 --- Benzodiazepines --- p.35 / Chapter 1.3.6.4 --- Cannabinoids --- p.35 / Chapter 1.3.6.5 --- Antihistamines and anticholinergics --- p.35 / Chapter 1.3.6.6 --- NK1 receptor antagonists --- p.37 / Chapter 1.3.6.7 --- Corticosteroids --- p.38 / Chapter 1.3.6.8 --- Multi-agent anti-emetic regimens --- p.39 / Chapter 1.4 --- Motion-Induced Emesis --- p.41 / Chapter 1.4.1 --- Incidence --- p.42 / Chapter 1.4.2 --- Mechanisms and Pathways Involved in Motion Sickness --- p.43 / Chapter 1.4.2.1 --- Importance of the vestibular apparatus --- p.44 / Chapter 1.4.2.2 --- Importance of the area postrema --- p.45 / Chapter 1.4.2.3 --- The nucleus tractus solitarius --- p.46 / Chapter 1.4.2.4 --- Hormone and neurotransmitters --- p.46 / Chapter 1.4.3 --- Animal models in Motion-Induced Emesis --- p.47 / Chapter 1.4.4 --- Anti-Emetic Drugs for the Treatment of Motion Sickness --- p.48 / Chapter 1.4.4.1 --- Anticholinergics --- p.49 / Chapter 1.4.4.2 --- Antihistamines --- p.49 / Chapter 1.4.4.3 --- Non-selective muscarinic and histamine receptor antagonists --- p.51 / Chapter 1.4.4.4 --- Sympathomimetics --- p.51 / Chapter 1.4.4.5 --- NK1i receptor antagonists --- p.51 / Chapter 1.4.4.6 --- 5-HT1A agonists --- p.52 / Chapter 1.4.4.7 --- 5-HT2 receptor agonist --- p.52 / Chapter 1.4.4.8 --- Arginine vasopressin (AVP) antagonists --- p.53 / Chapter 1.4.4.9 --- Opioid receptor agonists --- p.53 / Chapter 1.4.4.10 --- Dexamethasone and hormone levels --- p.54 / Chapter 1.4.4.11 --- Other anti-emetic drugs --- p.55 / Chapter 1.5 --- Aims of the Studies --- p.56 / Chapter 2 --- Methods --- p.59 / Chapter 2.1 --- Cisplatin-Induced Emesis Studies --- p.60 / Chapter 2.1.1 --- Animals --- p.60 / Chapter 2.1.2 --- Induction and Measurement of Emesis --- p.60 / Chapter 2.1.3 --- The Effects of Corticosteroids on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.63 / Chapter 2.1.4 --- "The Effects of Dexamethasone (1 mg/kg, i.p.) Administered as an Intervention Treatment on an Established Delayed Retching and Vomiting Response Induced by Cisplatin" --- p.63 / Chapter 2.1.5 --- The Effects of Cortrosyn Depot (Tetracosactrin) on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.63 / Chapter 2.1.6 --- The Effects of Metyrapone on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.64 / Chapter 2.1.7 --- The Effects of Indomethacin on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.64 / Chapter 2.1.8 --- "The Effects of DFU and L-745,337 Administered as an Intervention Treatments on an Established Delayed Retching and Vomiting Response Induced by Cisplatin" --- p.64 / Chapter 2.1.9 --- "The Effects of MK-886 (L-663,536) on Cisplatin-Induced Acute and Delayed Retching and Vomiting" --- p.65 / Chapter 2.1.10 --- The Effects of a Combination of Indomethacin and MK-886 on Cisplatin- Induced Acute and Delayed Retching and Vomiting --- p.65 / Chapter 2.1.11 --- Statistical Analysis --- p.66 / Chapter 2.2 --- Motion-Induced Emesis Studies --- p.67 / Chapter 2.2.1 --- Animals --- p.67 / Chapter 2.2.2 --- Measurement of Emesis --- p.67 / Chapter 2.2.3 --- Induction of Emesis in Motion-Naive Suncus murinus: Effects of Glucocorticoids --- p.68 / Chapter 2.2.4 --- Induction of Emesis in Motion-Sensitive Suncus murinus: Effects of Dexamethasone --- p.70 / Chapter 2.2.5 --- Preparation of Serum --- p.72 / Chapter 2.2.6 --- Measurement of Serum Cortisol by Enzyme-Linked Immunoassay (ELISA) --- p.72 / Chapter 2.2.6.1 --- Immunoassay kit --- p.72 / Chapter 2.2.6.2 --- Assay procedures --- p.73 / Chapter 2.2.7 --- Measurement of Serum Adrenocorticotrophin (ACTH) by Radioimmunoassay (RIA) --- p.75 / Chapter 2.2.7.1 --- Immunoassay kit --- p.75 / Chapter 2.2.7.2 --- Assay procedures --- p.76 / Chapter 2.2.8 --- Statistical Analysis --- p.79 / Chapter 3 --- Results --- p.81 / Chapter 3.1 --- Cisplatin-Induced Emesis --- p.82 / Chapter 3.1.1 --- General Profile of Emesis Induced by Cisplatin --- p.82 / Chapter 3.1.2 --- Antagonism of Cisplatin-Induced Emesis by Corticosteroids --- p.82 / Chapter 3.1.3 --- "The Effect of Dexamethasone (1 mg/kg, i.p.) Administered as an Intervention Treatment on an Established Delayed Retching and Vomiting Response Induced by Cisplatin" --- p.84 / Chapter 3.1.4 --- The Effect of Cortrosyn Depot (Tetracosactrin) on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.85 / Chapter 3.1.5 --- The Effect of Metyrapone on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.85 / Chapter 3.1.6 --- "The Effect of Indomethacin, DFU and L-745,337 on Cisplatin-Induced Acute and Delayed Retching and Vomiting" --- p.86 / Chapter 3.1.7 --- The Effect of MK-886 on Cisplatin-Induced Acute and Delayed Retching and Vomiting --- p.88 / Chapter 3.1.8 --- The Effect of Combination of Indomethacin and MK-886 on Cisplatin- Induced Acute and Delayed Retching and Vomiting --- p.89 / Chapter 3.2 --- Motion-Induced Emesis --- p.91 / Chapter 3.2.1 --- General Effect of Motion on Serum Cortisol and ACTH Levelsin Motion Naive Suncus murinus --- p.91 / Chapter 3.2.2 --- The Effect of Glucocorticoids on Motion-Induced Emesis and Cortisol and ACTH Levels in Motion-Naive Male Suncus murinus --- p.92 / Chapter 3.2.2.1 --- Effect of dexamethasone --- p.92 / Chapter 3.2.2.2 --- Effect of betamethasone --- p.93 / Chapter 3.2.2.3 --- Effect of methylprednisolone --- p.93 / Chapter 3.2.3 --- The Effect of Glucocorticoids on Motion-Induced Emesis and Cortisol and ACTH Levels in Motion Naive Female Suncus murinus --- p.94 / Chapter 3.2.3.1 --- Effect of dexamethasone --- p.94 / Chapter 3.2.3.2 --- Effect of betamethasone --- p.95 / Chapter 3.2.3.3 --- Effect of methylprednisolone --- p.95 / Chapter 3.2.4 --- The Effect of Dexamethasone on Motion-Induced Emesis and Cortisol and ACTH Levels in Motion-Sensitive Suncus murinus --- p.96 / Chapter 3.2.4.1 --- Effect of dexamethasone on male motion-sensitive animals --- p.97 / Chapter 3.2.4.2 --- Effect of dexamethasone on female motion-sensitive animals --- p.97 / Chapter 4 --- Discussion --- p.131 / Chapter 4.1 --- "Cisplatin (5 mg/kg, i.p.)-Induced Emesis in Control Animals" --- p.132 / Chapter 4.2 --- Anti-Emetic Action of Corticosteroids in the Ferret --- p.133 / Chapter 4.3 --- Metyrapone Study --- p.138 / Chapter 4.4 --- Cortrosyn Depot Study --- p.139 / Chapter 4.5 --- Role of Cycloxygenase --- p.141 / Chapter 4.6 --- Role of 5-Lipoxygenase --- p.143 / Chapter 4.7 --- Duel Inhibition of Cycloxygenase and 5-Lipoxygenase --- p.144 / Chapter 4.8 --- Anti-Emetic Potential of Glucocorticoids in Suncus murinus --- p.145 / Chapter 4.9 --- General Summary --- p.149 / Appendix I --- p.152 / Appendix II --- p.154 / References --- p.156

Prenatal stress exposure (PNS) has detrimental effects on the offspring’s brain and behaviour and has been identified as an etiological factor in inducing cognitive function deficits in rodents and humans. The neural mechanisms are unclear, however reprogramming of the neuroendocrine stress axis, the hypothalamo-pituitary- adrenal (HPA) axis is hypothesised. A psychosocial stressor (residentintruder paradigm) was used to generate PNS rat offspring, making these studies clinically compatible. The hippocampus and the medial prefrontal cortex (mPFC) are critical in regulating cognitive function and also contribute to the negative feedback control of the HPA axis via corticosteroid receptors, including the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Here the Barnes maze was used to assess spatial learning and memory in male and female PNS offspring during adulthood under different scenarios, including basal and acute and chronic stress conditions. Under basal conditions, PNS was associated with reduced GR and MR mRNA expression in the medial prefrontal cortex (mPFC) and the hippocampus, respectively; suggesting inhibitory feedback control of the HPA axis may be compromised in PNS rats. Moreover, impaired spatial learning was observed in male PNS rats following acute restraint stress. Bilateral lesions of the prelimbic cortex and central administration of an MR antagonist in control rats suggested acute stress-induced learning deficits in PNS males were a result of impaired hippocampus-mediated inhibitory feedback control of the HPA axis. Conversely, a one-week variable stress regimen facilitated spatial learning in PNS rats and this was associated with elevated MR mRNA expression in the dentate gyrus. Moreover, facilitated learning in the PNS rats exposed to chronic stress could be blocked by central administration of an MR antagonist, indicating a facilitatory role of hippocampal MR in spatial learning. In summary, opposite effects of PNS on spatial learning were observed under acute and chronic stress conditions, in which hippocampal MR played a key role in regulating behavioural performance. The effect of age was also examined in PNS rats, and the findings from middle-aged (10-11 months old) rats indicated PNS may accelerate cognitive decline. Sex differences were also studied, with control females’ out-performing males under basal conditions in terms of spatial learning and behavioural flexibility; however following prenatal or chronic stress these sex differences were no longer detected. Furthermore, acute stress impaired spatial learning to a greater extent in females, and this might be attributed to greater HPA axis responses to stress in females compared with males. In conclusion, prenatal stress alters later cognitive performance, in a sex- and stress context-dependent manner. Hippocampal MR plays a critical role in mediating spatial learning, particularly during stress conditions.