published_or_final_version / Biochemistry / Master / Master of Philosophy

Biochemical consequences of ascorbate deficiency were studied in the leaf tissue of Arabidopsis thaliana ascorbate-deficient vtc mutants with a view of understanding the relationship between ascorbate, stress response and metabolism. Ascorbate is an important antioxidant and is also a cofactor for 2-oxoglutarate-dependent dioxygenases, which are involved in the biosynthesis of a number of metabolites. The response of wild type (Col-0) and vtc1, vtc2-1, vtc2-2 and vtc3-1 mutants to high light intensity, wounding and salinity was investigated using a metabolomics and proteomics approach. Metabolite profiling and comparative proteomics were performed by liquid chromatography-quadrupole time of flight mass spectrometry (LC-QToF MS) and targeted analysis of plant hormones and flavonoids by liquid chromatography triple-quadrupole mass spectrometry (LC-QQQ-MS). These combined analyses revealed the effect of ascorbate deficiency and stress on metabolites and cell wall proteins. LC-QToF-MS based untargeted metabolite profiling methodologies were developed for analysis of metabolites on a large scale. Using this method about 3000-5000 metabolites (mass-retention time pairs) could be reproducibly detected in A. thaliana leaf extract and aligned between samples. Approximately 1000 metabolites were differentially expressed between WT and vtc mutants in different experiments. Of these, twenty eight compounds were confirmed to be differentially expressed by LC-QQQ-MS between WT and vtc mutants, and eight of these compounds were positively identified and validated with standards. The plant hormones abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) have all been implicated in plant stress responses and differences in their accumulation in some of the vtc mutants have been reported. A systematic study of the response to stress of these hormones in several vtc mutants was carried out using LC- QQQ- MS. While some of the mutants showed increased SA and SA-glycoside accumulation, stress-induced ABA and JA accumulation was generally unaffected. Methods for identifying the metabolites in a targeted manner by LC- QQQ-MS was developed and were shown that all vtc mutants were impaired in the accumulation of anthocyanin in response to HL treatment. In strong contrast to anthocyanin, flavonol glycosides were not affected by ascorbate deficiency. Therefore, ascorbate deficiency has a specific effect on the anthocyanin biosynthesis. Ascorbate occurs in the plant cell wall and isolation of apoplastic fluid showed that all vtc mutants have decreased apoplastic ascorbate compared to WT. Ionically-bound proteins were from the cell wall of A. thaliana leaves. Peroxidase specific activity in this fraction tended to be higher in vtc mutants than WT. High light intensity also increased peroxidase activity in WT and vtc mutants. To determine which peroxidase isoenzyme caused increased peroxidase activity, ionically-bound cell wall N-glycosylated proteins were isolated by Concanavalin A chromatography and analysed by LC-QToF-MS. Comparison of WT and vtc2-2 grown in low light and high light identified 937 peptides significantly different between WT and vtc2-2 and some are also affected by light intensity. Specifically, peroxidases 33 and 34 had increased abundance in vtc2-2. The results show that ascorbate deficiency causes a detectable change in the metabolome of A. thaliana leaves, with specific effects on anthocyanin accumulation being detected. Ascorbate deficiency also influences the expression of cell wall proteins. Peroxidase activity is increased, and this response could be related to the increased pathogen resistance reported in vtc mutants.

MMed (Paediatrics), Faculty of Health Sciences, University of the Witwatersrand / This prospective observational study analysed iron studies and vitamin C levels in patients with chronic kidney disease attending Johannesburg Hospital Paediatric Nephrology Clinic. The rationale behind this study was to determine the extent of iron deficiency among patients in chronic renal failure. Vitamin C deficiency is common among dialysis patients, it is easy to test for and easy to prevent. This study may assist in guiding future management with regards to vitamin C supplementation in patients with chronic renal insufficiency on dialysis. The study contained 45 patients of which 27 (60 %) were male and 18 (40 %) were female. The ages of the children varied from 2 years 1 month to 19 years and 7 months. The study included patients from all ethnic groups; 9 were Caucasian, 33 African, 2 Indian and 1 Coloured. Two male patients did not have Vitamin C levels analyzed. The patients were divided into 3 distinct groups; firstly those patients on haemodialysis (12 patients), those on peritoneal dialysis (22 patients) and those not yet dialysed (11 patients). In all patients who were not yet on dialysis the GFR ranged between 18.1 and 45 ml/min/1.73m2. There were no statistically significant differences between the three groups when the results of the iron studies were analysed. However, despite iron treatment 26.6 % of patients were iron deficient as indicated by their transferrin saturation which was less than 20 %. Vitamin C levels were also analysed in this study. Forty one percent of children in chronic renal failure were vitamin C deficient. There was no statistically significant variability among the three groups. Two patients (4.6%) were noted to be Vitamin C toxic. One of these patients was haemodialysed; the other was not yet on dialysis. Vitamin C deficiency in chronic renal insufficient patients on dialysis is easily correctable when identified. Vitamin C in specific well documented doses is safe to administer to this group of patients. It will also enhance the absorption of iron and thereby have an indirect effect on anaemia.

Orientador: Silvana Martinez Baraldi Artoni / Coorientador: Sarah Sgavioli / Banca: Ricardo de Albuquerque / Banca: Selma de Fátima Grossi / Banca: Lizandra Amoroso / Banca: Otto Mack Junqueira / Resumo: O objetivo deste estudo foi verificar quais os efeitos do aditivo com glicosaminoglicanos (sulfato de condroitina e de glucosamina) e vitamina C in ovo e na dieta em frangos de corte. Para isso realizaram-se dois experimentos de incubação e um de criação, respectivamente. Foram incubados 490 ovos férteis de frangos de corte, distribuídos em delineamento inteiramente casualizado com cinco tratamentos: ovos não injetados e ovos injetados com 0, 2, 4 e 6 μg de aditivo/100 μL de água no quarto dia da incubação, com 14 repetições de sete ovos por tratamento. Os dados foram submetidos a análise de variância pelo procedimento Modelo Linear Geral (GLM) do SAS. Avaliou-se o efeito dos tratamentos em relação aos parâmetros de incubação, qualidade do pinto pós-eclosão e características ósseas. A nutrição in ovo com glicosaminoglicanos (GAGs) e vitamina C não interferiu nos parâmetros de incubação e promoveu melhora do desenvolvimento ósseo nos pintos pós eclosão. No segundo experimento de incubação foram incubados 2.160 ovos férteis de frangos de corte, distribuídos em um delineamento casualizado com dois tratamentos: ovos não injetados e ovos injetados 4 μg de aditivo/100 μL de água no quarto dia da incubação, com 15 repetições de 72 ovos por tratamento. Após a eclosão, 760 aves foram alojadas e distribuídas em esquema fatorial 2x2. Sendo dois tratamentos na incubação (ovos não injetados e ovos injetados com 4 μg de aditivo) e dois tratamentos durante a criação (dieta sem a inclusão de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this study was to verify the additive effects with glycosaminoglycans (chondroitin sulfate and glucosamine) and vitamin C in ovo feeding and diet of broilers. Two incubation experiments and one rearing experiment were carried out, respectively. A total of 490 Cobb® fertile broiler eggs were incubated and distributed in completely randomized experimental design, with five treatments: un-injected egg, in injection egg of 0, 2, 4 and 6 μg additive/100 μL water on day four of incubation. Data were subjected to analysis of variance by the General Linear Model procedure (GLM) of SAS®. Were evaluated the treatments effect in relation the incubation parameters, chick quality post hatching and bone characteristics. In ovo feeding with glycosaminoglycans (GAGs) and vitamin C did not interfere on incubation parameters and promoted improvement of bone development in chicks after hatching. Two thousand hundred sixty (2,160) Cobb® fertile broiler eggs were distributed into five teen trays (repetitions) with seventy-two eggs/treatment/tray. After hatching, 760 birds were housed and distributed in a 2x2 factorial design. Were two incubation treatments (un injected eggs and eggs injected with 4 μg of additive) and two rearing treatments (with 0.74 g of additive/100 kg of feed). The purpose of this study was to determine if in ovo feeding or feed nutrition with glycosaminoglycans (chondroitin sulfate and glucosamine sulfate) and vitamin C would influence on bone and cartilage macroscopic, mineral composition, densitometry, breaking strength and histology of the femur and tibia, blood parameters, performance and carcass yield of broilers at 42 days of age.The effects of 4 μg additive in ovo feeding and 0.74 g/kg additive in feed nutrition were submitted to analysis of variance using the G... (Complete abstract click electronic access below) / Doutor

Reações de oxidação lipídica são responsáveis pelo desenvolvimento de sabores e odores desagradáveis tornando os alimentos impróprios para consumo, sendo necessário o uso de antioxidantes. O ácido ascórbico (AA) é um antioxidante muito eficaz, que exibe função vitamínica, no entanto é relativamente instável. Com o objetivo de aumentar a estabilidade do AA e, consequentemente, facilitar sua aplicação em produtos alimentícios, os métodos de encapsulação por coacervação complexa e por dispositivos microfluídicos foram testados. Primeiramente foi apresentada a Revisão Bibliográfica no Capítulo 1, e em seguida, a encapsulação por coacervação complexa, como será visto no Capítulo 2. Neste caso, nove tratamentos foram obtidos utilizando-se gelatina e goma arábica como materiais de parede e analisados com relação à morfologia, por microscopia ótica e eletrônica de varredura, umidade, atividade de água, higroscopicidade, solubilidade, potencial zeta, espectroscopia no infravermelho por transformada de Fourier (Ftir), tamanho e distribuição de tamanho de partículas, cor instrumental, eficiência de encapsulação e estabilidade do material encapsulado. No capítulo 3 serão apresentados resultados obtidos na encapsulação do AA pelo método de dispositivos microfluídicos. Cinco tratamentos foram obtidos, sendo analisados com relação à morfologia, por microscopia ótica, eletrônica de varredura e confocal, eficiência de encapsulação, tamanho e distribuição de tamanho de partícula e estabilidade do material encapsulado. A obtenção das microcápsulas de AA pelos dois métodos citados foi viável uma vez que apresentaram altos valores de eficiência de encapsulação e ótima atuação em relação à proteção do AA durante estocagem. Comparando-se os dois métodos, as cápsulas obtidas por dispositivos microfluídicos conferiram melhor estabilidade ao ácido ascórbico, no entanto amostras obtidas por coacervação complexa foram aplicadas em salsicha devido a maior quantidade de AA presente em sua constituição. O efeito da aplicação das microcápsulas nas salsichas foi avaliado durante 40 dias de armazenamento refrigerado como será visto no Capítulo 4. Cinco tratamentos foram elaborados e analisados de acordo com a estabilidade da emulsão cárnea durante o processamento, umidade, atividade de água, alteração do pH, determinação da cor instrumental, perfil de textura instrumental, estabilidade oxidativa pelo método de substâncias reativas ao ácido tiobarbitúrico (TBARS) e aceitação sensorial. A aplicação das microcápsulas de AA em salsicha foi possível sem comprometer a qualidade do produto final. Todos os dados obtidos foram analisados estatisticamente por análise de variância ANOVA e teste de Tukey, ao nível de 5% de significância com auxílio do programa SAS. Os experimentos relacionados à encapsulação por coacervação complexa e aplicação das microcápsulas em salsicha foram realizados no Laboratório de Produtos Funcionais, nas dependências do Departamento de Engenharia de Alimentos da FZEA/USP. Os experimentos relacionados à utilização de dispositivos microfluídicos foram realizados nos laboratórios do professor David A. Weitz, da Escola de Engenharia e Ciências Aplicadas de Harvard, da Universidade de Harvard, Cambridge, Estados Unidos. / Lipid oxidation reactions are responsible for the development of unpleasant tastes and odors making food unfit for consumption. For this reason, the use of antioxidant is necessary. Ascorbic acid (AA) is a very effective antioxidant with vitamin function, however it is relatively unstable. With the aim of increasing the stability of AA and thus improve its application in food products, the methods of encapsulation by complex coacervation and microfluidic devices were tested. First of all the Literature Review is presented in Chapter 1. The encapsulation by complex coacervation can be seen in Chapter 2. For this methodology, nine treatments were obtained using gelatin and gum Arabic as encapsulant agent and analyzed regarding to morphology by optical and scanning electron microscopy, moisture, water activity, hygroscopicity, solubility, Zeta Potential, Fourier transform infrared Spectroscopy (FTIR), particle size and particle size distribution, instrumental color, encapsulation efficiency and stability of the encapsulated material. The results obtained for AA encapsulation by microfluidic device will be presented in Chapter 3. Five treatments were obtained and analyzed regarding to morphology by optical, scanning electron and confocal microscopy, encapsulation efficiency, particle size and particle size distribution and stability of the encapsulated material. The production of AA microcapsules by the two methods mentioned was feasible once that showed high levels of encapsulation efficiency and optimal performance regarding to the protection of AA during storage. Comparing the two methods, the microcapsules obtained by microfluidic device conferred better stability to AA, however samples obtained by complex coacervation were applied in sausage due to the greater amount of AA in its constitution. The effect of the application of microcapsules in sausages was evaluated during 40 days at refrigerated storage as it will be seen in Chapter 4. Five treatments were prepared and analyzed according to the stability of the meat emulsion during processing, moisture, water activity, pH changes, determination of instrumental color, instrumental texture profile, oxidative stability by the method of thiobarbituric acid reactive substances (TBARS) and sensory acceptance. The application of AA microcapsules in sausage was possible without compromising the quality of the final product. All data were statistically analyzed by ANOVA and Tukey test, at 5% of significance with the use of SAS software. The experiments related to encapsulation by complex coacervation and application of microcapsules in sausage were carried out at Laboratory of Functional Products, at Department of Food Engineering, FZEA / USP. The experiments related to the use of microfluidic devices were performed in the laboratories of Professor David A. Weitz, at School of Engineering and Applied Sciences of Harvard, at Harvard University, Cambridge, USA.

Made available in DSpace on 2014-06-11T19:30:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-25Bitstream added on 2014-06-13T19:40:39Z : No. of bitstreams: 1 graciani_fs_dr_arafcf_parcial.pdf: 545160 bytes, checksum: d12c1e4909cabaf516ea500fd292c6e9 (MD5) Bitstreams deleted on 2014-12-19T18:32:47Z: graciani_fs_dr_arafcf_parcial.pdf,Bitstream added on 2014-12-19T18:33:34Z : No. of bitstreams: 1 000726391.pdf: 1117169 bytes, checksum: 0f0bc7cc07ba17b24618a33ee997eb8d (MD5) / câncer é considerado como uma das principais causas de morte no mundo. A compreensão das diferenças biológicas entre células cancerígenas e normais é fundamental para o desenvolvimento de novas estratégias terapêuticas, buscando sempre a maior seletividade em alvos que sejam críticos para células cancerígenas se comparado às células normais, sendo uma das características apresentadas em células cancerígenas o aumento de espécies reativas de oxigênio (EROs). O objetivo deste estudo consiste em estudar a geração de peróxido de hidrogênio (H2O2) por meio da associação ácido ascórbico/naftoquinona e sua aplicação em modelos de citotoxicidade, além de verificação de possível seletividade entre células normais e tumorais. Foram utilizados os métodos de avaliação do consumo de oxigênio, determinação da produção de H2O2, avaliação da ocorrência de cliclo redox por cromatografia líquida de alta eficiência (CLAE) e interação destas drogas com glutationa (GSH) ou nicotinamida adenina dinucleotídeo reduzida (NADH). O ensaio de citotoxicidade foi determinado pelo método da sulforodamina B em células câncer de mama (MCF-7 BUS) e células normais de ovário de hamster chinês (CHO). Foi descoberto que a presença de um grupo retirador de elétrons na molécula de naftoquinona teve um efeito direto sobre a eficiência da produção de H2O2. O composto 2- bromo-1,4-naftoquinona (BrQ), em que o átomo de bromo substituído no grupo metil na molécula 2-metil-1,4-naftoquinona (VK3), foi aproximadamente 10 a 19 vezes mais eficiente do que VK3 em termos de consumo de oxigênio e produção de H2O2, respectivamente. A proporção [H2O2] μM/minproduzido/[naftoquinona] μM/minconsumido foi 68 ± 11 and 5.8 ± 0.2 para BrQ e VK3, respectivamente, que revelou a maior eficácia de BrQ como um catalisador para a oxidação de ácido ascórbico (VC)... / Cancer is considered as one of the leading causes of death worldwide. Understanding the biological differences between normal cells and cancer cells is critical for the development of new therapeutic strategies, always seeking greater selectivity at targets that are critical for cancer cells compared to normal cells, one of the features shown in cancer cells is the increase reactive oxygen species (ROS). The aim of this study is to investigate the generation of hydrogen peroxide (H2O2) through association naphthoquinone/ascorbic acid and its application in models of cytotoxicity, and check possible selectivity between normal cells and tumor cells. Methods were used to evaluate the oxygen consumption, determination of H2O2 production, assessing the occurrence of redox cycle by HPLC and interaction of these drugs in combination with glutathione (GSH) or reduced nicotinamide adenine dinucleotide (NADH). The cytotoxicity assay was determined by the sulforhodamine B in breast cancer cell (MCF-7 BUS) and chinese hamster ovary (CHO). It has been discovered that the presence of an electron withdrawing group in the naphthoquinone had a direct effect on the efficiency of production of H2O2. The compound 2-bromo-1,4-naphthoquinone (BrQ) in which the bromine atom substituted the methyl group in the molecule 2-methyl -1,4- naphthoquinone (VK3) was approximately 10 to 19 times more efficient that VK3 in terms of oxygen consumption and the production of H2O2, respectively. The ratio [H2O2] μM/ minproduced/[naphthoquinone] μM/minconsumed was 68 ± 5.8 and 11 ± 0.2 for BrQ and VK3, respectively, which showed the greatest efficacy BrQ as a catalyst for the oxidation of ascorbic acid (VC). Both VK3 and BrQ reacted with GSH, but the substrate was BrQ more effective. Part of GSH was incorporated into naphthoquinone, giving rise to a nucleophilic substitution product (Q -SG)... (Complete abstract click electronic access below)

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-11-30Bitstream added on 2014-06-13T20:01:21Z : No. of bitstreams: 1 000751413.pdf: 2252740 bytes, checksum: 6e8ef15e5086c99f37df6243e30b0a16 (MD5) / Estresse calórico afeta o desempenho e as características morfofuncionais dos frangos. Manipulação das condições ambientais na fase fetal tem se mostrado efetiva na indução de características adaptativas ao calor na incubação. Manipulação nutricional da ave com anti-estressores também tem sido apontado como potencial indutor de adaptações fisiológicas ao calor na fase de criação. O presente estudo analisou os efeitos do estresse por calor na incubação associado ou não à injeção de ácido ascórbico (AA) intra ovo sobre características morfofuncionais dos pintos na eclosão e sobre seu desempenho pós-eclosão e características morfofuncionais à idade de abate quando criados sob temperatura fria, termoneutra ou quente. No Capítulo 1 apresentamos uma abordagem teórica sobre o tema. Nos Capítulos 2, 3 e 4 são abordados os efeitos da alta temperatura de incubação associada ou não com diferentes porcentagens de AA injetado intra ovo antes da incubação sobre parâmetros de incubação (temperatura da casca dos ovos, perda de massa dos ovos, eclodibilidade, mortalidade, condutância e qualidade dos pintos), hemodinâmica (eritrograma, leucograma e variáveis bioquímicas e gases) e desenvolvimento da bursa de Fabrícius (peso, área medular e cortical dos folículos, espessura de epitélio e proliferação celular). Para isso, ovos férteis de matrizes de frangos de corte (Cobb) foram utilizados em delineamento experimental inteiramente casualizado 2x5 (temperatura de incubação: 37,5ºC e 39ºC; sem injeção de AA e injeção de 0%, 2%, 4% e 6% de AA). Os dados do Capítulo 2 mostram que as porcentagens de AA utilizadas afetaram a perda de calor por condução e a taxa de eclosão dos ovos, e que não minimizaram os efeitos da alta temperatura de incubação sobre o desenvolvimento hepático e cardíaco... / Heat stress affects the performance and morphofunctional characteristics of broilers. Manipulation of environmental conditions during the fetal period has been effective in the induction of adaptive traits in heat incubation.Nutritional manipulation of the bird with anti-stressors has also been touted as a potential inducer of physiological adaptations to heat in the growing phase. The present study examined the effects of heat stress on incubation with and without intra-egg injection of ascorbic acid (AA) on the morphofunctional characteristics of chicks at hatching and their post-hatch performance and morphofunctional characteristics at slaughter age when raised under cold thermoneutral or hot temperatures. In Chapter 1 we present a theoretical approach to the topic. In Chapters 2, 3 and 4 the effects of high incubation temperature associated or not with different percentages of AA injected intra-egg before incubation on incubation parameters (temperature, eggshell, weight loss, hatchability, mortality, conductance and chick quality), hemodynamics (erythrocyte, leukocyte count and biochemical variables and gases) and development of the Fabrucius bursa (weight, medullary and cortical area of the follicles, epithelial thickness and cell proliferation) are addressed. For this fertile Cobb broiler eggs were used in a completely randomized design 2x5 (incubation temperature: 37.5º C and 39º C, not injection of AA, and injection of 0%, 2%, 4% and 6% of AA). The data in Chapter 2 show that the percentage of AA used affected the heat loss by conduction and hatching rate of the eggs and did not minimize the effects of high temperature incubation on the heart and liver development...

Made available in DSpace on 2014-06-11T19:31:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-03-19Bitstream added on 2014-06-13T18:41:30Z : No. of bitstreams: 1 jeronimo_m_dr_arafcf.pdf: 924549 bytes, checksum: deb5b1b247d16a3e3dd39dbb0f5987fb (MD5) / Universidade Estadual Paulista (UNESP) / Os laboratórios de análise de alimentos e fármacos necessitam demonstrar competência técnica para garantia da qualidade dos ensaios que realizam, assegurando a confiabilidade e comparabilidade dos resultados que emitem que subsidiam tomadas de decisões relativas a aspectos econômicos, de saúde pública e defesa do consumidor. O presente trabalho apresenta uma abordagem metrológica de métodos clássicos e instrumental para a determinação do ácido ascórbico (AA). Os parâmetros de validação de todos os métodos de análise abordados neste trabalho e o cálculo da incerteza atenderam as diretrizes da ISO-Guia-IEC-17025 e dos guias EURACHEM. Os métodos clássicos validados foram: N-Bromosuccinamida (NBS), Iodo, Iodato de Potássio e Método do 2,6 diclorofenol-indofenol (DCFI). O método instrumental validado foi o espectrofotométrico, utilizando o reagente 2,6 diclorofenol-indofenol. Os parâmetros avaliados foram: a faixa linear de trabalho, seletividade, linearidade, precisão pela norma da American Society for Testing and Materials (ASTM E 691-99) (sn), precisão por repetição (sr), limite de detecção (LD), limite de quantificação (LQ), robustez (teste de Younden-Steiner) e incerteza genérica. A linearidade avaliada pela análise de resíduo (Y-Yo) mostra que a curva é linear em toda a sua extensão ensaiada em todos os métodos validados e eles foram precisos, robustos e exatos. A importância da validação de métodos analíticos está na obtenção de resultados confiáveis e adequados ao uso pretendido / The laboratory analysis of food and drugs need to demonstrate technical competence for quality assurance tests to perform, ensuring the reliability and comparability of results that give off that support decision making on economic, public health and consumer protection. This paper presents a metrological approach of classical methods and instrument for the determination of ascorbic acid (AA). The validation parameters of all methods of analysis in this paper and the calculation of uncertainty within the guidelines of ISO-IEC-17025-Guide and EURACHEM guides. The classical methods have been validated: N-Bromosuccinamida (NBS), Iodine, Potassium iodate method and 2.6-dichlorophenol indophenol (DCPI). The instrumental method was validated spectrophotometric reagent using 2.6-dichlorophenol indophenol. The parameters evaluated were: the linear range of work, selectivity, linearity, accuracy in the standard American Society for Testing and Materials (ASTM E 691-99) (sn), by repetition accuracy (sr), limit of detection (LD), limit of quantification (LQ), robustness (test-Younden Steiner) and general uncertainty. Linearity was assessed by residual analysis (Y-Yo) shows that the curve is linear over its entire length in all tested methods validated and they were accurate and robust The importance of validation of analytical methods is to obtain reliable results and suitable for intended use

Die Bedeutung von Ascorbinsäure als „Krebsschutzfaktor“ wird auch weiterhin kontrovers diskutiert. Seit einiger Zeit wird vermutet, dass Ascorbinsäure oxidativen Stress auslöst. In der vorliegenden Untersuchung wurde die Wirkung von Ascorbinsäure auf 12 maligne und 3 benigne Zelllinien in vitro untersucht. Die Zellen wurden für 2 bzw. 14 Stunden mit unterschiedlichen Konzentrationen von Ascorbinsäure (5 bis 100 mmol/L) inkubiert und 24, 48 und 72 Stunden nach Versuchsbeginn der Anteil vitaler Zellen bestimmt. Die hierfür verwendeten Assays, WST-8 und Kristallviolett-Assay, ließen zudem Aussagen über die Stoffwechselaktivität (WST-8) und Zellvitalität (Kristallviolett) zu. Die schädigende Wirkung von Ascorbinsäure wurde als EC50-Wert angegeben, bei dieser Ascorbinsäure-Konzentration sind 50 % der Zellen zerstört. Ascorbinsäure wirkte nach 2 Stunden Inkubation kaum zelltoxisch, während nach 14 Stunden Inkubation eindeutige zelltoxische Effekte bei 6 der 12 malignen Zelllinien zu beobachten waren. So waren die drei getesteten Glioblastomzelllinien allesamt bereits bei einer Ascorbinsäure-Konzentrationen von 5 mmol/L nahezu vollkommen zerstört (EC50: 2,6-5,5 mmol/L). Die Mammakarzinomzelllinie BT-20 hingegen war am widerstandsfähigsten gegenüber dem zelltoxischen Effekt der Ascorbinsäure (EC50: 95 mmol/L). Als wesentliches Effektormolekül der zelltoxischen Wirkung der Ascorbinsäure wurde Wasserstoffperoxid identifiziert. Die Zugabe von Katalase schützt Ascorbinsäure- sensitive Zellen, in dem es Wasserstoffperoxid abbaut. Ein weiteres Indiz hierfür ist, dass Zelllinien, die gegenüber dem Ascorbinsäure-vermittelten Effekt unempfindlich waren, dies auch gegenüber Wasserstoffperoxid waren. Umgekehrt waren Zelllinien, die empfindlich gegenüber dem Ascorbinsäurevermittelten zelltoxischen Effekt reagierten, auch empfindlich gegenüber Wasserstoffperoxid. 45 Eine wesentliche sich aus den Daten dieser Arbeit ergebende Frage ist die, worin sich Ascorbinsäure-resistente Tumorzellen von Ascorbinsäure-empfindlichen Tumorzellen unterscheiden. Da Ascorbinsäure-empfindliche Zellen durch Zugabe von Katalase vor der zelltoxischen Wirkung der Ascorbinsäure geschützt werden, liegt die Vermutung nahe, dass eine wesentliche Ursache hierfür in der zelleigenen Katalase begründet liegt. Somit sollten Ascorbinsäureresistente Zellen mehr bzw. aktivere Katalase aufweisen, als Ascorbinsäureempfindliche Zellen. Diese Vermutung ist in weiteren Experimenten zu überprüfen. / Ascorbic acid (Asc) has a controversial history in supportive cancer treatment. The Asc-mediated cytotoxic effect is based on the local production of hydrogen peroxide (H2O2). Tumour cells lines were screened for their susceptibility or resistance to Asc-mediated cytotoxicity. The purpose of this study was to analyse the impact of ascorbic acid modifying tumour cell vitality. A panel of twelve tumour cell lines, carcinomas and glioblastoma, were exposed to serial dilutions of Asc (0-100 mmol/L) up to 2 and 14hrs in vitro. The Asc concentration that effectively decreased survival by 50% (EC50) was determined by wst-8 and crystal violet assay. The tested cell lines demonstrated obvious differences in their resistance to Asc. Fifty percent of the cancer cell lines had an EC50 >20mM and were resistant to Asc, whereas fifty percent had an EC50 <10mM; they were susceptible to Asc. In addition, Asc resistant cell lines were also cross-resistant to H2O2. Glioblastoma cell lines were found to be the most susceptible cell lines (EC50: 2.5-6mM). External catalase (250U) neutralizes the cytotoxic effect of Asc and protects against cell death.

Vitamin C occurs in relatively high concentrations in fresh and processed fruits and vegetables but is found to a lesser extent in animal tissues and animal-derived products. Nearly 90 % of vitamin C in the human diet is obtained from fruits and vegetables but this can be indirect by way of commercially prepared fruit juices. These juices are often enriched with vitamin C which has been synthetically prepared. There is a wide range of such juices on the New Zealand market, and they are a significant source of dietary vitamin C for many in the population. The focus of this research is on the Keri range of juice products.The present study monitors the fate of vitamin C during storage of Keri juices up to the best-before date, and under a range of other storage and consumption situations. Two methods were adopted for determining ascorbic acid (AA, the chemical identity of vitamin C). These were the titrimetric method, which is based upon the reduction of the dye 2,6-dichlorophenolindophenol by AA in acidic solution, and liquid chromatography, which is used to separate AA from its immediate oxidation product dehydroascorbic acid. In the latter method these two analytes can be measured independently. The liquid chromatography was less successful than the simpler titrimetric method, so most of the work was done by titration. However, the concentration of dehydroascorbic acid, which has vitamin C activity in vivo, remained uncertain. Moreover, the titrimetric method could not be applied to juices with high purple anthocyanin concentrations, like blackcurrant, because the colour change at the titration end point could not be detected. pH adjustment to change colour was ineffective, and decolourisation with charcoal led to the rapid and complete destruction of AA. The concentration of AA in Keri juices at the time of manufacture were always much higher than claimed on the labels. Storage for up to nine months at room temperature resulted in a loss in AA of between 37 and 68 %, depending on the juice and exposure to fluorescent light. However, the time of storage was a much more dominant factor than light exposure. The kinetics of loss, straight lines, were most easily explained by an aerobic model of AA degradation from oxygen diffusing across the polyethylene tetraphthalate bottle wall. Overall, the label claims made were defensible in terms of the best-before date, because it took at least 100 days of storage before the AA concentration in the most susceptible juices fell below the claimed value. This is because these drinks are fast moving consumer goods and storage beyond 100 days is unlikely. (Nonetheless, the supplier (Keri Juice Company) has since adopted its new unitised method of formulating juice. This has resulted in an initially higher concentration of vitamin C as compared to the juices under investigation.) In the nine months storage experiment there was some evidence for the presence of dehydroascorbic acid in blackcurrant drinks, but not in another three juices. Pasteurisation during preparation of these drinks resulted in up to 7 % loss of AA, probably due to oxygen dissolved in water, and accelerated by heat of pasteurisation. Higher temperatures in later storage also accelerated losses. Progressive exposure of juice to air during simulated consumption of 3 L bottles over a week also accelerated losses. Finally, exposure to sunlight in a diurnal temperature environment accelerated losses five-fold higher than in total darkness. Filtration of ultraviolet light approximately halved the loss due to sunlight. Overall however, it can be concluded that AA in the Keri range of juices is very resistant to degradation of AA.