The freshwater prawn, Macrobrachium rosenbergii (de Man, 1879) is an important aquaculture species but one that has the disadvantage of heterogeneous individual growth (HIG) according to different morphotypes. Chemical cues, especially, pheromones, are one of the most important communication types between individual prawns, along with visual and tactile methods. Testing pheromones, whilst restricting other cues, may therefore lead to a better understanding of the influence of these communicatory compounds on the prawn reproductive process. The three principle objectives of this study were therefore: 1) To examine the effect of moult stage and morphotype on pheromone-induced sexual behaviour 2) To examine the role of pheromone / urine concentrations on sexual attraction behaviour 3) To describe the functional morphology of the antennal gland and examine its possible role in pheromone production and release. Identical bioassay tanks were designed and constructed to study the reproductive behaviour of prawns. Experiments were set up to examine responses to pheromone release by live prawns over 30 minutes and behavioural response observations were made with the aid of a Closed-Circuit Videotape System (CCVS). Results were statistically analysed using a repeated measures general linear model (GLM). Three trials were designed to test the effect of moult stage of both males and females and male morphotypes on sexual attraction behavioural responses. Twelve prawns were used for each trial and each prawn was used five times (1 no-pheromone control and 4 for experimental tests). The first trial studied the effect of female moult stages (pre-, inter and newly-moulted) on sexual attraction behaviour of blue claw (BC) male. Results of this trial showed that newly-moulted females spent significantly (p<0.05) less time approaching the BC male than the pre- and inter-moult females. The second trial studied the effect of male moult stage (pre-, inter and newly-moulted) on sexual attraction to receptive females. Results showed that the time taken by the inter-moult males was (p<0.05) less than the pre- and newly-moulted males in approaching the newly-moulted female. The third trial tested the effect of male morphotypes (small male, SM, orange claw, OC and dominant blue claw, BC) on sexual attraction behaviour towards newly-moulted females. Results showed that the BC male was significantly more attractive (p<0.05) than other morphotypes to newly-moulted females and that the OC male was the least attractive. The role of moulting stage for both male and female prawns on reproductive response behaviour was investigated. Because BC males responded significantly faster towards newly-moulted female more than to either pre-or inter-moult females, results of the first trial suggest that BC males are able to use different chemical cues to gather information about a conspecific’s gender and can differentiate female’s moult stages. Since BC males responded significantly faster towards newly-moulted females more than to either pre-or inter-moult females, this suggests that females at this particular stage released a distinct sexual pheromone or concentration of pheromone that differed from those pheromones released by both pre- and inter-moult females. In contrast, newly-moulted females prefer the inter-moult BC males which indicate that females have an ability to distinguish the moult status of BC males. Furthermore, it indicates that pheromone characteristics change with the moult status of BC males. Also, newly-moulted females are most likely to be avoiding the potential costs of mate guarding with soft shell BC males. Results obtained from the third trial suggested that a newly-moulted female can discriminate male morphotypes (SM, OC and BC) from their pheromone cues. This indicates that male morphotypes release pheromones which differ from each other in some way. Newly-moulted females responded positively to both SM and BC males with different levels of attraction with the greatest attraction to BC males to BC males suggesting that pheromone released from the BC male may carry information relating to dominance status. Urine is believed to be one of the main carriers of pheromone and is usually released from the antennal gland. Different urine concentrations (0.1, 1.0, 2.0, 3.0, 5.0 and 10µl l-1) of collected urine from BC males were used to test the sexual attraction behaviour of receptive newly-moulted females. Also, the attractant capability of fresh urine following exposure to different temperature regimes (cooled at 4ºC, frozen at -70ºC and heated at 70ºC) was tested. Since newly-moulted female M. rosenbergii were attracted to BC male urine, this indicates the existence of sex pheromone in the fresh urine. Also, it was found that the sexual response of females to fresh urine of BC males was directly proportional to urine concentration with faster responses observed with increasing urine concentrations. At the three fresh urine concentrations 0.1 µl l-1, 1.0 µl l-1 and 2.0 µl l-1, statistical analysis indicated no significant difference (p>0.05) between these three concentrations while a significant (P<0.05) response was to concentrations more than 3.0 µl l-1. This may indicate that these three concentrations were not sufficient to elicit attraction behaviour in newly-moulted females. A concentration of 3.0 µl l-1 of fresh urine is suggested to be a sufficient concentration to elicit a significant sexual attraction under laboratory conditions. Response of newly-moulted female prawns to the various temperature treatments tested declined in response to nominally increasingly degradative treatments. Also, statistical analysis showed that temperature treatment and concentration added both had a significant effect on the response of females. The greatest degradation of urine attractiveness was found with the 70ºC heat treatment. It can be concluded that the pheromone components of prawn urine are friable when exposed to high temperatures. Using light and transmission electron microscopes, ultrastructural observation of the antennal gland (AG) of M. rosenbergii suggests that it has four distinct regions, the coelomosac, the nephridial tubules, the labyrinth and the bladder. Morphological and functional descriptions of each of these regions were compared with those of other aquatic Crustacea.

Le cancer du rein est un problème majeur de santé publique. Un diagnostic précoce améliore les chances de survie. Le diagnostic repose essentiellement sur les examens d'imagerie comme l'échographie, la tomodensitométrie et l'IRM. Ces examens sont parfois associés à la biopsie et sont couteux et parfois invasifs. De plus, l'imagerie n'est pas capable de faire la distinction entre les tumeurs bénignes et les tumeurs malignes et entre les sous-types histologiques de carcinome à cellules claires qui est le plus fréquent. Il n'existe pas dans le cancer rénal de marqueur comme la PSA dans le cancer de la prostate ou la Foetoprotéine et l'HCG dans le cancer du testicule. Le but de cette étude est concentré sur la recherche des marqueurs mARNs ou miARNs dans les liquides biologiques (sérum, plasma, urine) pour le cancer du rein à cellules claires. Nous avons montré que les petits ARNs dans le sérum et les urines et l'intégrité des ARNs dans les urines étaient des outils diagnostiques dans le cancer du rein à cellules claires. Si ces petits ARNs circulants sont validés, on peut éventuellement imaginer l'intérêt pratique en clinique comme la détection des petits ARNs circulants dans l'urine pour prédire le cancer, la classification de la tumeur pour aider le clinicien, la prédiction d'une récidive ou d'une progression soit après néphrectomie soit au cours d'un traitement médical

The aims of this thesis were to: 1) develop a new method for the determination of interstitial pH at rest and during exercise in vivo, 2) systematically explore the effects of different ingestion regimes of 300 mg.kg-1 sodium citrate on blood and urine pH at rest, and 3) to combine the new interstitial pH technique with the findings of the second investigation in an attempt to provide a greater understanding of H+ movement between the extracellular compartments. The purpose of the first study was to develop a method for the continuous measurement of interstitial pH in vastus lateralis was successfully developed using microdialysis and 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). To avoid the presence of an artificial alkalosis during exercise, it was necessary to add 25 mM HCO3- to the perfusate. The outlet of the probe was cut less than 10 mm from the skin and connected to a stainless steel tube completing the circuit to a microflow-through cuvette (8 fÝl) within a fluorescence spectrophotometer. This prevented the loss of carbon dioxide from the dialysate and any subsequent pH artefact. Interstitial pH was collected from six subjects before, during and after five minutes of knee-extensor exercise at three intensities 30, 50, and 70 W. Mean,,bSEM interstitial pH at rest was 7.38,,b0.02. Exercise reduced interstitial pH in an almost linear fashion. The nadir value for interstitial pH at 30, 50 and 70 W exercise was 7.27, 7.16 and 7.04, respectively. The lowest pH was obtained 1 min after exercise, irrespective of workload, after which the interstitial pH recovered in a nearly exponential manner. The mean half time of interstitial recovery was 5.2 min. The changes in interstitial pH exceeded the changes in venous blood pH. This study demonstrated that interstitial pH can be measured using microdialysis and that it is continuously decreased during muscle activity. The purpose of the second study was to establish an optimal ingestion regime for the ingestion of 300 mg.kg-1 of sodium citrate and maximise the alkalotic effect while minimising any side effects. Increasing the effectiveness of alkali ingestion may lead to further increases in muscle performance. Ingesting 300 mg.kg-1 sodium citrate at a rate of 300 mg.min-1 was identified as the optimal ingestion regime to maximise alkalosis at rest, which occurred 3.5 h post-ingestion. This was determined by monitoring eight human subjects ingesting 300 mg.kg-1 sodium citrate at five different rates, control (no ingestant), bolus, 300, 600 and 900 mg.kg.min-1 on five days separated by at least 48 hours. Sodium citrate was ingested in capsule form with water ad libitum, with the exception of bolus, which was combined with 400 ml less than 25 percent orange juice and consumed in less than 1 min. Arterialised blood (mean 71.3,,b3.5 mmHg) acid-base and electrolyte status was assessed via the withdrawal of ~5 ml of blood every 30 min across an eight hour duration, placed on ice and analysed within five minutes. No alkalotic difference was found between ingestion rates (mean 7.445,,b0.004, 7.438,,b0.004 and 7.442,,b0.004 for 300, 600 and 900 mg.min-1, respectively). All experimental ingestion regimes were associated with elevations in [HCO3-] (29.6, 29.7, 29.8, 29.9 and 26.3 mmol.l-1 for bolus, 300, 600, 900 and control, respectively). The 300 ingestion regime had the greatest impact on [H+], a 0.66 meq.l-1,,e10-8 change. Bolus ingestion (3.93,,b0.08 mmol.l-1) of sodium citrate had no effect on control (4.06,,b0.08 mmol.l-1) blood [K+], however, 300 mg.min-1 decreased blood [K+] (p less than 0.05). There was no effect of sodium citrate on blood [Cl-], but after 2.5 h blood [Cl-] was lower than pre-ingestion values (p less than0.05). All ingestion rates of sodium citrate increased (p less than 0.05) urine pH above control. This is the first study to investigate the effect of varying ingestion rates on acid-base status at rest in humans. The results suggest that ingesting sodium citrate in small doses in quick succession induce a greater blood alkalosis than the commonly practised bolus protocol. Using the interstitial pH technique described above and the optimal ingestion regime (300 mg.min-1) identified above, the final experiment was designed to assess the influence of sodium citrate ingestion on interstitial pH at both rest and during exercise. Five subjects ingested 300 mg.kg-1 sodium citrate at 300 mg.min-1 again in capsule form with water ad libitum. Prior to ingestion, each subject had a cannula placed into their cephalic vein and one microdialysis probe (CMA-60) inserted into their left thigh, orientated along the fibres of vastus lateralus. This probe was used for the measurement of pH as described above. At the end of this period, an exercise protocol required five subjects to perform light exercise (10 W) for 10 min, before starting an intense exercise period (~90-95% leg VO2peak) to exhaustion followed by a 15 min recovery period. Dialysate and blood samples were collected across all periods. Mean,,bSEM interstitial pH for placebo and alkalosis were 7.38,,b0.12 and 7.24,,b0.16, respectively. Sodium citrate ingestion was not associated with an interstitial alkalosis. An exercise induced acidosis was observed in the interstitium during placebo but not during alkalosis (p less than 0.05). Mean,,bSEM venous pH were 7.362,,b0.003 and 7.398,,b0.003 for placebo and alkalosis, respectively. Sodium citrate ingestion was not associated with a venous alkalosis. Sodium citrate ingestion was associated with an increase in mean,,bSEM venous [HCO3-] (placebo 25.5,,b0.2, alkalosis 28.1,,b0.2). This increase in the blood bicarbonate buffer system was not associated with an increase in time to exhaustion (placebo 352,,b71, alkalosis 415,,b171). This was the first study to investigate the effects of sodium citrate ingestion on interstitial pH. The results of this study demonstrated that an interstitial alkalosis does not ensue after alkali ingestion, however, it was associated with the lack of an exercise induced acidosis suggesting an improved pH regulation during exercise.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.

A doença da urina do xarope do bordo (MSUD) é causada pela deficiência na atividade do complexo da desidrogenase dos U-cetoácidos de cadeia ramificada (BCKAD), promovendo o acúmulo dos aminoácidos de cadeia ramificada (BCAA) leucina (Leu), isoleucina (Ile) e valina (Val) e seus U-cetoácidos correspondentes (BCKA). A MSUD caracteriza-se por cetoacidose, ataxia, coma, retardo mental e psicomotor. Estudos em animais demonstraram que BCAA e BCKA estimulam a lipoperoxidação e reduzem capacidade antioxidante cerebral em ratos. Também há evidências de que o estresse oxidativo ocorra em pacientes com MSUD no diagnóstico e durante o tratamento e que devido à terapia com dieta restrita e hipoproteica eles possuam deficiência de L-carnitina (L-car), um importante composto para o metabolismo energético. Recentemente, estudos demonstraram o papel antioxidante e anti-inflamatório da L-car, através de sua ação antiperoxidativa, sequestradora de espécies reativas e efeito estabilizador de danos às membranas celulares. Considerando que a fisiopatologia da MSUD ainda é pouco compreendida e que existe um crescente número de estudos enfatizando o envolvimento do estresse oxidativo na doença, neste trabalho foi investigado o efeito in vitro e in vivo da L-car sobre o estresse oxidativo e o dano inflamatório na MSUD tendo como objetivos: A) estudar a indução ao dano oxidativo pelos metabólitos acumulados na MSUD, verificando o possível papel antioxidante da L-car sobre o dano ao DNA in vitro; B) avaliar o efeito in vivo da suplementação de 50 mg/kg/dia de L-car sobre: b.1) a indução do dano ao DNA em leucócitos de pacientes com a MSUD tratados com dieta de restrição proteica, correlacionando as concentrações dos principais metabólitos acumulados nesta doença e verificando o possível papel antioxidante da suplementação da Lcar; b.2) a concentração de citocinas pró-inflamatórias em plasma de pacientes com MSUD tratados com dieta de restrição proteica e a correlação com o estresse oxidativo; b.3) os parâmetros de dano oxidativo à biomoléculas em urina de pacientes com MSUD sob dieta de restrição proteica; C) avaliar o efeito da L-car sobre o estresse oxidativo causado pelos metabólitos acumulados na MSUD em córtex cerebral e cerebelo de ratos Wistar, através de um modelo crônico de indução química da doença. Verificou-se que a Leu e o seu - cetoácido correspondente, o ácido -cetoisocapróico (KIC), causaram danos ao DNA in vitro e L-car foi capaz de diminuir significativamente essas alterações, principalmente as causadas pelo KIC. Quando testado o efeito da suplementação de L-car sobre o dano ao DNA em pacientes MSUD, observou-se um aumento significativo de lesões ao DNA em pacientes com dieta de restrição proteica quando comparados aos controles e a terapia com L-car foi capaz de diminuir significativamente os níveis desses danos. Também foram verificadas correlações do tipo negativa entre as concentrações de L-car e os índices de dano ao DNA e do tipo positiva entre as lesões ao DNA e níveis de MDA, marcador de lipoperoxidação, explicitando uma relação entre o dano ao DNA observado nos pacientes com MSUD, estresse oxidativo e o benefício da suplementação de L-car. Também averiguou-se o efeito da terapia de L-car sobre as citocinas pró-inflamatórias interleucina 1Y (IL-1Y), interleucina 6 (IL-6) e interferon gama (INF- Z). Constatou-se aumentos significativos de IL-1Y, IL-6 e INF- Z no plasma de pacientes com MSUD antes da suplementação de L-car e uma reversão completa desses valores aos níveis dos controles para IL-1Y e INF- Z após a administração de L-car. Ainda, verificou-se que a L-car pode auxiliar na defesa celular contra a inflamação e o estresse oxidativo, observando-se uma correlação negativa entre todas citocinas testadas e as concentrações de L-car, e uma correlação positiva entre o conteúdo de MDA e níveis de IL-1Y e IL-6. Constatou-se também que as medidas de di-tirosina (dano oxidativo a proteínas) e isoprostanos (dano de lipoperoxidação) estavam aumentadas e a capacidade antioxidante total diminuída na urina de pacientes com MSUD sem terapia com L-car e a suplementação deste composto induziu efeitos benéficos sobre estes parâmetros, reduzindo os níveis de di-tirosina e isoprostanos e aumentando a capacidade antioxidante medida em urina. Foi também observado um aumento de KIC urinário após dois meses de tratamento com L-car, quando comparado com o grupo controle, demonstrando um incremento da excreção deste metabolito tóxico. Desta forma, esses resultados sugerem um efeito de reversão de dano oxidativo pela L-car e que a urina pode ser utilizada para monitorar este tipo de lesão em pacientes afetados pela MSUD. Por fim, foram analisados em córtex cerebral e cerebelo de ratos Wistar submetidos ao modelo crônico de MSUD: espécies reativas ao ácido tiobarbitúrico (TBARS), para avaliar lipoperoxidação, conteúdo de carbonilas (dano oxidativo proteico), oxidação de diclorofluoresceína (DCF), para quantificar produção de espécies reativas teciduais, conteúdo de glutationa reduzida (GSH) que é um importante antioxidante não enzimático e a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx) e glicose-6-fosfato-desidrogenase (G6PD). Os resultados mostraram que a administração crônica de BCAA estimulou a lipoperoxidação, o dano oxidativo proteico, aumento de espécies reativas e diminuição das defesas antioxidantes enzimáticas e não enzimáticas, especialmente em córtex cerebral e o tratamento com L-car foi capaz de prevenir estes efeitos, exceto o dano oxidativo a proteínas. Em conjunto, estes resultados demonstram que os metabólitos acumulados na MSUD induzem dano oxidativo a biomoléculas (lipídios, proteínas e DNA), diminuem o status antioxidante e promovem aumento de processos inflamatórios. Ainda, estes dados podem contribuir para a compreensão dos mecanismos de ação dos efeitos citotóxicos dos metabólitos acumulados na MSUD e evidenciar o papel do estresse oxidativo e da inflamação na neuropatofiosiologia desta doença, além do efeito protetor da L-car sobre este processo. O estudo de antioxidantes, como a L-car, pode propor uma abordagem terapêutica adicional ao que é empregado atualmente para pacientes com MSUD, que é essencialmente dietética e, portanto, de difícil manejo. / Maple syrup urine disease (MSUD) is caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain U-ketoacid dehydrogenase (BCKAD). The metabolic defect leads to accumulation of the branched chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val) and the corresponding branched-chain U-keto acids. The clinical features of MSUD include ketoacidosis, seizures, coma, psychomotor delay and mental retardation. Treatment consists in Leu, Val and Ile restricted diet. Studies in animals have demonstrated that lipid peroxidation is stimulated by BCAA and BCKA in brain of rats and these metabolites reduce in vitro and in vivo the cerebral capacity to modulate the damage associated to increased free radical production. Also, there is evidence that oxidative stress occurs in MSUD patients at diagnosis and during treatment and that due to terapy with protein restricted diet they present L-carnitine (L-car) deficiency, an important compound for energy metabolism. Recent studies have demonstrated the antioxidant and anti-inflammatory role of L-carnitine (L-car), through its action against peroxidation in different tissues by various mechanisms, a scavenger of reactive oxygen species and the stabilizing effect of damage to cell membranes. Considering that the pathophysiology of MSUD is still poorly understood, and that there is an increasing number of studies emphasizing the oxidative stress involvement in the disease, this study investigated the in vitro and in vivo effect of L-car on oxidative stress and inflammatory damage in MSUD with the following purposes: A) to study the induction of damage by accumulated metabolites in MSUD, analyzing the possible antioxidant role of L-car on DNA damage in vitro; B) to evaluate the in vivo effect of 50 mg/kg/day of L-car supplementation about: b.1) the induction of DNA damage in leukocytes of MSUD patients treated with protein-restricted diet, correlating this damage with the concentrations of the major metabolites accumulated in this disorder and checking the possible antioxidant role of L-car supplementation; b.2) plasma inflammatory cytokines in treated MSUD patients with protein-restricted diet and the correlation with oxidative stress; b.3) oxidative damage parameters in urine of MSUD patients with protein-restricted diet supplemented with L-car; C) to investigate the BCAA effect on some oxidative stress parameters and evaluate the L-car efficacy against these possible pro-oxidant effects in cerebral cortex and cerebellum of rats submitted to a chronic chemically-induced model of MSUD. DNA damage index (DI) showed that Leu and -ketoisocaproic acid (KIC) groups was significantly higher than that of the control group, and that L-car was able to significantly prevent this damage, especially that due to KIC. Accordingly, DNA DI in MSUD patients under BCAA-restricted diet was significantly increased as compared to controls and L-car supplementation was able to significantly decrease this parameter. It was also verified a significant positive correlation between DNA DI and MDA content, a marker of lipid peroxidation. Furthermore, we found an inverse significant correlation between DI and L-car levels. These results strengthen a relationship between DNA damage observed in MSUD patients, oxidative stress and the L-car supplementation benefit. The role of L-car on plasma inflammatory cytokines interleukin-1Y (IL-1Y), interleukin-6 (IL-6) and interferon-gamma (INF- Z) was also evaluated in these patients. Significant increases of IL-1Y, IL-6, and INF- Z were observed before the treatment with L-car. Moreover, there is a negative correlation between all cytokines tested and L-car concentrations and a positive correlation among the MDA content and IL-1Y and IL-6 values after L-car supplementation. It was also demonstrated that the oxidative stress parameters di-tyrosine (oxidative protein damage) and isoprostanes (lipid peroxidation assay) were increased and the antioxidant capacity was reduced in urine of MSUD patients without L-car therapy and that the supplementation of this compound induced beneficial effects on these parameters, so reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary KIC after 2 months of L-car treatment compared to control group, demonstrating an increased excretion of this toxic metabolite. In conclusion, these results suggest a reversion effect of the oxidative damage by L-car and that urine can be used to monitorize oxidative damage in patients affected by this disease. The following parameters were analysed in cerebral cortex and cerebellum of Wistar rats submitted to MSUD chemically-induced chronic model: thiobarbituric acid reactive species (TBA-RS), to evaluate lipid peroxidation, carbonyl content to evaluate protein oxidative damage, DCF oxidation to quantify reactive species production, reduced glutathione (GSH), an important non-enzymatic antioxidant and the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD). The results showed that the chronic administration of BCAA was able to promote both lipid and protein oxidation, increase of reactive species production and decreased brain antioxidant defenses, especially in cerebral cortex and that L-car was able to prevent these effects, except for oxidative damage to proteins. Taken together, these results demonstrate that the metabolites accumulated in MSUD cause oxidative damage to biomolecules (lipids, proteins and DNA), decrease antioxidant status and promote increased inflammatory processes. These results may contribute to the understanding of the mechanism of action of the cytotoxic effect of the metabolites accumulated in MSUD and the role of oxidative stress and inflammation in the MSUD neuropathophysiology besides the protective effect of L-car on this process. The study of antioxidants like L-car can opens an additional therapeutic approach to that currently employed for MSUD patients, which is primarily dietary and therefore difficult to handle.

Measurement of urine absorbance which has been irradiate by a dose of ionising radiation with addition of Mn2+ should serve to find the dose of radiation. This method could work quickly and reliably for homogeneous irradiation of person or as a rough estimate of the dose which the person received during a radiation accident. This method should serve for quick classification of the person. The target of this thesis is to find out if the irradiated urine with the addition of a solution of manganese chloride will change absorbance according to radiation dose. And if urine can be used as a biological dosimeter. In the theoretical part I describe the basic areas related to the topic and target of my thesis. This part is divided to seven subchapters: ionising radiation, radiation protection, radiotherapy, particle accelerators, spectrophotometry, excretion and urine, and dosimetry and its methods. Methods of this thesis are not clear. I tried different procedures during experiments with different results. First, I always prepared samples of urine in tubes and irradiated it in a linear accelerator Clinac 2100C/D in České Budějovice, a.s. hospital with doses from 1 to 25 Gy. Before each measurement I had two sets of tubes with these doses: 0, 1, 2, 3, 5, 7, 10, 15, 20 and 25 Gy. The following procedure was different in each experiment. Sometimes I tried adding a solution of manganese chloride to all tubes at once. Sometimes I tried to adding a solution of manganese chloride to each tube separately. I added the solution to irradiated urine at various concentrations of solution (from 1 do 5 mols) and different amounts (from 1 to 3 ml). Another difference was the use of centrifuges. A centrifuge was use in about half of experiments. Other measurements were made without centrifugation.In discussion I propose recommended methods. I recommend measuring only without centrifuges. Each cell should be measured as soon as possible after irradiation and simultaneously as soon as possible after adding the solution of manganese chloride. Results of the thesis are not clear. Only some experiments which were measuring with centrifuge were clear. I can say that this method does not work when a centrifuge is used at any tested concentration of solution of manganese chloride. The absorbance of single doses of ionising radiation does not change and the values were the same when using 1M, 3M and 5M solutions of MnCl2. The resulting graphs from all experiments have a constant absorbance value of all measured doses. (subchapter 3.1).In the remaining experiments measured without the centrifuge the results were much more interesting. In some experiments the measured absorbance really changed with the dose of ionising radiation so the hypothesis of this study was confirmed. But the differences were too small for this method to be used for measuring radiation doses (subchapter 3.2).The results were compared with the results of the thesis ?Measurement of urine extinction in depending on ionising radiation? from author Š. Radová. She performed a similar experiment, but with a different indicator - FeSO4. 7 H20. It was found that the indicator FeSO4. 7 H20 is preferable to measuring doses of ionising radiation in urine. In conclusion I can say that the hypothesis of this study was confirmed, but the method could not be used in practice and irradiated urine with added MnCl2 indicator does not function as a biological dosimeter.

[pt] Neste trabalho foram desenvolvidas metodologias eletroanalíticas baseadas na voltametria adsortiva de redissolução catódica com varredura de potencial de onda quadrada e de pulso diferencial para a determinação do ciclofenil em urina e em formulações farmacêuticas, e na voltametria anódica com varredura de potencial de onda quadrada e de pulso diferencial para a determinação da primaquina em formulações farmacêuticas. Comparando-se as duas técnicas de aquisição, a voltametria de onda quadrada foi escolhida para realizar as determinações do ciclofenil em urina e em formulação farmacêutica por se mostrar, além de uma técnica mais rápida, maior sensíbilidade. Para a determinação da primaquina a melhor técnica foi à voltametria de pulso diferencial. As condições experimentais que possibilitaram um melhor desempenho analítico em termos da obtenção do menor limite de detecção e maior reprodutibilidade das leituras foram otimizados. No caso do ciclofenil, o composto mostrou ser instável no meio aquoso e orgânico para determinação voltamétrica, com sistemática diminuição da corrente faradaica. Assim, fez-se uso das propriedades eletroquímicas de um derivado estável obtido pela derivatização fotoquímica do composto (o composto foi irradiado com irradiação UV por 45 minutos em um reator fotoquímico), a corrente máxima obtida apresentou um potencial a -1,28 V. As condições experimentais que possibilitaram este sinal foram obtidas com 30 s de tempo de deposição do analito sobre o eletrodo de mercúrio, solução Britton-Robbinson pH 9,0 como eletrólito de suporte, potencial de acumulação de -0,9 V, amplitude de 250 mV, incremento de potencial de 2,0 mV no modo de onda quadrada. Desta forma foi obtido limite de detecção da ordem de 10-8 mol L-1, faixa linear dinâmica de 2 ordens de grandeza, condições estas que possibilitaram a determinação do composto tanto em formulações farmacêuticas (Menopax) como em amostra de urina enriquecida com o analito de interesse. Foram feitos testes com subtâncias concomitantes da formulação farmacêutica e constatou-se que não existia problema com interferência na análise nesse tipo de amostra. Nas determinações em urina não houve a necessidade de se fazer tratamento prévio da amostra, apenas a irradiação UV para estabilizar o composto, as análises foram realizadas com o método de adição de analito, de forma a se corrigir a interferência de matriz. Em ambos os casos os resultados obtidos se encontram na faixa de 93,6 a 106,5 por cento, dentro da faixa de recuperação aceitável para este tipo de problema analítico segundo estabelecido pela Farmacopéia dos Estados Unidos da América. Usou-se ainda os resultados provenientes das voltametrias de pulso diferencial, de voltametria de onda quadrada e da voltametria cíclica para se obter informações sobre o processo redox que ocorria no eletrodo de trabalho. Constatou-se que a reação é reversível, com um processo de controle adsortivo, sendo que apenas o reagente adsorve no eletrodo de trabalho, não ocorrendo adsorção do produto da reação de redução. Constatou-se também que o processo envolve a transferência de apenas 1 elétron, e que a reação não possuía contribuição cinética. No caso da primaquina o sinal de redução ocorre em região anódica (0,592 V), foi portanto necessário utilizar um eletrodo de carbono para se poder fazer a determinação da primaquina. Visando a possibilidade do uso do eletrodo de mercúrio, tentou-se fazer uso da formação de derivados complexos da primaquina com vanádio, com cobre ou com íon iodeto (complexo de tranferência de carga), porém não foram observados sinal da reação redox na janela de potencial procurada. Nas condições experimentais otimizadas não se / [en] In this work, square-wave and differential pulse voltammetric methods were developed for the determination of cyclofenil and primaquine in pharmaceutical formulations and in urine samples. The use of the square- wave acquisition technique was found to enable better sensitivity and faster analysis time compared to the differential pulse technique. Experimental and instrumental conditions were optimized to allow the best analytical performance in terms of limit of detection and repeatability of the readings. In the case of cyclofenil, its unstable behavior in aqueous and organic solvents, with systematic decreasing of analyte current signal, makes impossible any voltammetric determination. As an alternative way, the electrochemical properties of a stable photochemical derivative of cyclofenil was used (the compound was irradiated with UV radiation for 45 min) with maximum current at -1,28 V. This analyte photoderivative could also be accumulated in the working electrode. The experimental conditions that allowed the maximum current was a 30 s of deposition time at the mercury electrode, Britton- Robbinson (pH 9,0) supporting electrolyte, accumulation potential of -0,9 V, amplitude of pulse of 250 mV, scan increment 2,0 mV. These optimized conditions allowed a limit of detection of 10-8 mol L-1 and dynamic linear range of 2 orders of magnitude to be achieved. These analytical figures of merit made possible the determination of cyclofenil either in a pharmaceutical formulation (Menopax) and in urine samples spiked with the analyte of interest. The potential interferences from concomitant substances used in the pharmaceutical formulation were also evaluated. For the analyte determination in urine, only UV irradiation of sample was necessary, in order to obtain stable cyclofenil derivative. The analyte addition method was used to analyze urine in order to minimize matrix interferences. Recovery results for the analysis of Menopax and for the analysis of urine were between 96,5 and 107,6 percent, within the acceptable recovery range established by the United States Pharmacopoeia. Information concerning the analyte redox reaction and electrode processes was also obtained from differential pulse voltammetry, square-wave voltammetry and cyclic voltammetry. It was verified that the cyclofenil photoderivative eletrochemical reaction is reversible with adsorption of only the reagent on the surface of the electrode. The adsorption of the electrochemical reduction product does not occur. It was also verified that the process involves the transference of only one electron, and there is no kinetics contribution in the reaction. In the case of the primaquine the analyte reduction occurs in anodic region (0,592 V), therefore, it was necessary the use the carbon glass electrode to allow the determination of this analyte. The pre-concentration of the analyte in the working carbon glass electrode was also not attained with the experimental conditions used. Several attempts were made to make possible the use of the mercury electrode, including the formation of charge transfer complexes with iodine and complexation with vanadium. However no success was obtained. Using the carbon glassy electrode and DPV technique the determination of primaquine in pharmaceutical formulations and urine was performed with average recovery of 101.7 percent. Limit of detention of 1,0 x 10-6 mol L- 1 was obtained.

Made available in DSpace on 2015-03-26T13:46:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 246842 bytes, checksum: f7b7371c8cb299837b636e055567a1b8 (MD5) Previous issue date: 2005-03-02 / This trial was carried out to evaluate the effect of periods of urine and feces collection on urinary excretion of creatinine, urea and purine derivatives, absorbed purine, microbial nitrogen compounds production (Nmic), apparent digestibility of dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), neutral detergent fiber (NDF) and nonfiber carbohydrates (CNF) and total digestible nutrients contents (TDN) of Nelore bovines four categories (heifers, steers, bulls and lactating cows) fed 25 or 50% concentrate, total dry matter basis. The plasma N-urea concentrations (NUP) were evaluated and the Nmic obtained in urine spot samples was compared with that of total collection. The experiment was divided in two experimental periods of 28 days each, when the feces and urine total collection were performed at 22nd and 28th day of each experimental period. Feces were colleted directly from the floor after excretion and the urine was obtained using catheters in females and funnels in males. A 25% concentrate-based diet was fed to the animals in the first period and a 50% concentrate-based diet in the second one, all of them with 12% CP. Sixteen Nelore bovines, under feedlot, housed in individual pens, were assigned to a completely randomized design, in a split plot scheme, where the treatments were represented by the plots (2 x 4 factorial scheme), with two levels of concentrate (25 or 50%) and four Nelore categories, and the split plots were represented by the urine collections. The comparisons for digestibility evaluation were made by regression analyses, that were performed considering 4 days of feces collection of each period. The equations were obtained by comparing the nutrients digestibility referent to one (24h), two (48h) or three days (72h) compared to four days (96h of collection). Comparison with six days of total feces collection, using equations (second experimental period [50% concentrate]) was considered reference (144h total feces collection). No interaction (P>0.05) among concentrate levels, Nelore categories and collection days for the urinary volume, the creatinine excretion and Nmic production was observed. Urinary volume was not affected (P>0.05) by the concentrate levels and collection days, but significant effect (P<0.05) was observed for cows. Creatinine excretion was not affected (P>0.05) by treatments and collection days, considering average of 27.1 mg/kg0.75. Absorbed purines and microbial nitrogen compounds production were also not influenced (P>005) by the treatments and collection days. Nmic production estimated by the urinary spot collection differed (P>0.05) neither from that obtained by total collection total, nor among the concentrate levels and Nelore categories. No significant difference (P>0.05) was observed for any evaluated digestibility and TDN contents during the total feces collection period. The results suggest that the coefficients of variation decresed as the period of collection days increased. Considering the results of creatinine excretion and of microbial protein production, it was concluded that a 24-h period is enough for researchs with Nelore, independently of the category, and that urinary spot sample collection can be used to estimate microbial protein production in all Nelore bovines (heifers, steers, bulls and lactating cows). Total feces collection from 1 to 6 days to evaluate nutrients apparent digestibility are precise, but better results could be obtained by increasing the collection period. / O presente trabalho foi conduzido com o objetivo de avaliar o efeito da duração do período de coletas de urina e de fezes sobre a excreção urinária de creatinina, de uréia e de derivados de purinas, as purinas absorvidas, a produção de compostos nitrogenados microbianos (Nmic), as digestibilidades aparentes da matéria seca (MS), matéria orgânica (MO), proteína bruta (PB), extrato etéreo (EE), fibra em detergente neutro (FDN) e de carboidratos não-fibrosos (CNF) e os teores de nutrientes digestíveis totais (NDT) em Nelores de quatro categorias (novilhas, machos castrados, machos inteiros e vacas em lactação) alimentados com 25 ou 50% de concentrado na base da matéria seca total das dietas. Avaliou-se ainda as concentrações de N-uréia plasmática (NUP) e comparou-se também a produção de Nmic obtida em amostras spot de urina com aquela da coleta total. O experimento foi conduzido em dois períodos experimentais com duração de 28 dias cada, sendo as coletas totais de urina e de fezes efetuadas do 22o ao 28o dias de cada período experimental. As fezes foram retiradas do piso imediatamente após excreção e a urina obtida com sondas em fêmeas e funis nos machos. Utilizou-se dieta com 25% de concentrado no primeiro período e com 50% no segundo experimental. Todas as dietas continham aproximadamente 12% de PB. Utilizaram-se 16 animais da raça Nelore, mantidos em confinamento, alojados em baias individuais, distribuídos em delineamento inteiramente casualizado, em esquema de parcelas subdivididas, tendo nas parcelas os tratamentos em esquema fatorial 2 x 4, sendo dois níveis de concentrado (25 ou 50%) e quatro categorias de Nelores e nas subparcelas os seis dias de coletas de urina. Já para a avaliação das digestibilidades, as comparações foram feitas através de análises de regressão, que foram efetuadas considerando quatro dias de coletas de fezes em cada período, sendo as equações obtidas sempre comparando as digestibilidades dos nutrientes referentes a um dia (24 h), 2 dias (48 h) ou 3 dias (72 h) em relação aos 4 dias (96 h de coleta). Foram feitas também comparações através de equações, utilizando os seis dias de coleta total de fezes, referentes ao segundo período experimental (50% de concentrado), sendo nesse caso utilizado como referência os seis dias de coleta total (144 h de coleta total de fezes). Não houve interação (P>0,05) entre níveis de concentrado, categorias de Nelore e dias de coleta para o volume urinário, a excreção de creatinina e a produção de Nmic. O volume urinário não foi influenciado (P>0,05) pelos níveis de concentrado e dias de coleta, contudo foi significativamente maior (P<0,05) para as vacas. A excreção de creatinina não foi afetada (P>0,05) pelos tratamentos e dias de coletas, observando-se média de 27,1 mg/kg0,75. As purinas absorvidas e a produção de compostos nitrogenados microbianos também não foram influenciadas (P>0,05) pelos tratamentos e dias de coleta. A produção de Nmic estimada através de amostra spot de urina não diferiu (P>0,05) daquela obtida pela coleta total, nem entre os níveis de concentrados e categorias de Nelore. Não houve diferença significativa (P>0,05) para nenhuma das digestibilidades avaliadas e também para os teores de NDT entre os dias de coleta total de fezes, contudo, observou-se que os coeficientes de variação diminuíram à medida que se aumentou o número de dias de coleta. Concluiu-se considerando as respostas obtidas para excreção de creatinina e a produção de proteína microbiana, que um período de coletas de urina de 24 horas é suficiente para trabalhos com Nelores, independente de serem novilhas, machos castrados ou inteiros e vacas em lactação e que a coleta de amostra spot de urina também pode ser utilizada para estimar a produção de proteína microbiana em novilhas, machos inteiros ou castrados e vacas lactantes da raça Nelore. Concluiu-se também que para avaliar a digestibilidade aparente dos nutrientes, coletas totais de fezes feitas durante um a seis dias são exatas. Contudo, a precisão é melhorada com o aumento dos dias de coleta.

A doença da urina do xarope do bordo (MSUD) é causada pela deficiência na atividade do complexo da desidrogenase dos U-cetoácidos de cadeia ramificada (BCKAD), promovendo o acúmulo dos aminoácidos de cadeia ramificada (BCAA) leucina (Leu), isoleucina (Ile) e valina (Val) e seus U-cetoácidos correspondentes (BCKA). A MSUD caracteriza-se por cetoacidose, ataxia, coma, retardo mental e psicomotor. Estudos em animais demonstraram que BCAA e BCKA estimulam a lipoperoxidação e reduzem capacidade antioxidante cerebral em ratos. Também há evidências de que o estresse oxidativo ocorra em pacientes com MSUD no diagnóstico e durante o tratamento e que devido à terapia com dieta restrita e hipoproteica eles possuam deficiência de L-carnitina (L-car), um importante composto para o metabolismo energético. Recentemente, estudos demonstraram o papel antioxidante e anti-inflamatório da L-car, através de sua ação antiperoxidativa, sequestradora de espécies reativas e efeito estabilizador de danos às membranas celulares. Considerando que a fisiopatologia da MSUD ainda é pouco compreendida e que existe um crescente número de estudos enfatizando o envolvimento do estresse oxidativo na doença, neste trabalho foi investigado o efeito in vitro e in vivo da L-car sobre o estresse oxidativo e o dano inflamatório na MSUD tendo como objetivos: A) estudar a indução ao dano oxidativo pelos metabólitos acumulados na MSUD, verificando o possível papel antioxidante da L-car sobre o dano ao DNA in vitro; B) avaliar o efeito in vivo da suplementação de 50 mg/kg/dia de L-car sobre: b.1) a indução do dano ao DNA em leucócitos de pacientes com a MSUD tratados com dieta de restrição proteica, correlacionando as concentrações dos principais metabólitos acumulados nesta doença e verificando o possível papel antioxidante da suplementação da Lcar; b.2) a concentração de citocinas pró-inflamatórias em plasma de pacientes com MSUD tratados com dieta de restrição proteica e a correlação com o estresse oxidativo; b.3) os parâmetros de dano oxidativo à biomoléculas em urina de pacientes com MSUD sob dieta de restrição proteica; C) avaliar o efeito da L-car sobre o estresse oxidativo causado pelos metabólitos acumulados na MSUD em córtex cerebral e cerebelo de ratos Wistar, através de um modelo crônico de indução química da doença. Verificou-se que a Leu e o seu - cetoácido correspondente, o ácido -cetoisocapróico (KIC), causaram danos ao DNA in vitro e L-car foi capaz de diminuir significativamente essas alterações, principalmente as causadas pelo KIC. Quando testado o efeito da suplementação de L-car sobre o dano ao DNA em pacientes MSUD, observou-se um aumento significativo de lesões ao DNA em pacientes com dieta de restrição proteica quando comparados aos controles e a terapia com L-car foi capaz de diminuir significativamente os níveis desses danos. Também foram verificadas correlações do tipo negativa entre as concentrações de L-car e os índices de dano ao DNA e do tipo positiva entre as lesões ao DNA e níveis de MDA, marcador de lipoperoxidação, explicitando uma relação entre o dano ao DNA observado nos pacientes com MSUD, estresse oxidativo e o benefício da suplementação de L-car. Também averiguou-se o efeito da terapia de L-car sobre as citocinas pró-inflamatórias interleucina 1Y (IL-1Y), interleucina 6 (IL-6) e interferon gama (INF- Z). Constatou-se aumentos significativos de IL-1Y, IL-6 e INF- Z no plasma de pacientes com MSUD antes da suplementação de L-car e uma reversão completa desses valores aos níveis dos controles para IL-1Y e INF- Z após a administração de L-car. Ainda, verificou-se que a L-car pode auxiliar na defesa celular contra a inflamação e o estresse oxidativo, observando-se uma correlação negativa entre todas citocinas testadas e as concentrações de L-car, e uma correlação positiva entre o conteúdo de MDA e níveis de IL-1Y e IL-6. Constatou-se também que as medidas de di-tirosina (dano oxidativo a proteínas) e isoprostanos (dano de lipoperoxidação) estavam aumentadas e a capacidade antioxidante total diminuída na urina de pacientes com MSUD sem terapia com L-car e a suplementação deste composto induziu efeitos benéficos sobre estes parâmetros, reduzindo os níveis de di-tirosina e isoprostanos e aumentando a capacidade antioxidante medida em urina. Foi também observado um aumento de KIC urinário após dois meses de tratamento com L-car, quando comparado com o grupo controle, demonstrando um incremento da excreção deste metabolito tóxico. Desta forma, esses resultados sugerem um efeito de reversão de dano oxidativo pela L-car e que a urina pode ser utilizada para monitorar este tipo de lesão em pacientes afetados pela MSUD. Por fim, foram analisados em córtex cerebral e cerebelo de ratos Wistar submetidos ao modelo crônico de MSUD: espécies reativas ao ácido tiobarbitúrico (TBARS), para avaliar lipoperoxidação, conteúdo de carbonilas (dano oxidativo proteico), oxidação de diclorofluoresceína (DCF), para quantificar produção de espécies reativas teciduais, conteúdo de glutationa reduzida (GSH) que é um importante antioxidante não enzimático e a atividade das enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx) e glicose-6-fosfato-desidrogenase (G6PD). Os resultados mostraram que a administração crônica de BCAA estimulou a lipoperoxidação, o dano oxidativo proteico, aumento de espécies reativas e diminuição das defesas antioxidantes enzimáticas e não enzimáticas, especialmente em córtex cerebral e o tratamento com L-car foi capaz de prevenir estes efeitos, exceto o dano oxidativo a proteínas. Em conjunto, estes resultados demonstram que os metabólitos acumulados na MSUD induzem dano oxidativo a biomoléculas (lipídios, proteínas e DNA), diminuem o status antioxidante e promovem aumento de processos inflamatórios. Ainda, estes dados podem contribuir para a compreensão dos mecanismos de ação dos efeitos citotóxicos dos metabólitos acumulados na MSUD e evidenciar o papel do estresse oxidativo e da inflamação na neuropatofiosiologia desta doença, além do efeito protetor da L-car sobre este processo. O estudo de antioxidantes, como a L-car, pode propor uma abordagem terapêutica adicional ao que é empregado atualmente para pacientes com MSUD, que é essencialmente dietética e, portanto, de difícil manejo. / Maple syrup urine disease (MSUD) is caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain U-ketoacid dehydrogenase (BCKAD). The metabolic defect leads to accumulation of the branched chain amino acids (BCAA) leucine (Leu), isoleucine (Ile) and valine (Val) and the corresponding branched-chain U-keto acids. The clinical features of MSUD include ketoacidosis, seizures, coma, psychomotor delay and mental retardation. Treatment consists in Leu, Val and Ile restricted diet. Studies in animals have demonstrated that lipid peroxidation is stimulated by BCAA and BCKA in brain of rats and these metabolites reduce in vitro and in vivo the cerebral capacity to modulate the damage associated to increased free radical production. Also, there is evidence that oxidative stress occurs in MSUD patients at diagnosis and during treatment and that due to terapy with protein restricted diet they present L-carnitine (L-car) deficiency, an important compound for energy metabolism. Recent studies have demonstrated the antioxidant and anti-inflammatory role of L-carnitine (L-car), through its action against peroxidation in different tissues by various mechanisms, a scavenger of reactive oxygen species and the stabilizing effect of damage to cell membranes. Considering that the pathophysiology of MSUD is still poorly understood, and that there is an increasing number of studies emphasizing the oxidative stress involvement in the disease, this study investigated the in vitro and in vivo effect of L-car on oxidative stress and inflammatory damage in MSUD with the following purposes: A) to study the induction of damage by accumulated metabolites in MSUD, analyzing the possible antioxidant role of L-car on DNA damage in vitro; B) to evaluate the in vivo effect of 50 mg/kg/day of L-car supplementation about: b.1) the induction of DNA damage in leukocytes of MSUD patients treated with protein-restricted diet, correlating this damage with the concentrations of the major metabolites accumulated in this disorder and checking the possible antioxidant role of L-car supplementation; b.2) plasma inflammatory cytokines in treated MSUD patients with protein-restricted diet and the correlation with oxidative stress; b.3) oxidative damage parameters in urine of MSUD patients with protein-restricted diet supplemented with L-car; C) to investigate the BCAA effect on some oxidative stress parameters and evaluate the L-car efficacy against these possible pro-oxidant effects in cerebral cortex and cerebellum of rats submitted to a chronic chemically-induced model of MSUD. DNA damage index (DI) showed that Leu and -ketoisocaproic acid (KIC) groups was significantly higher than that of the control group, and that L-car was able to significantly prevent this damage, especially that due to KIC. Accordingly, DNA DI in MSUD patients under BCAA-restricted diet was significantly increased as compared to controls and L-car supplementation was able to significantly decrease this parameter. It was also verified a significant positive correlation between DNA DI and MDA content, a marker of lipid peroxidation. Furthermore, we found an inverse significant correlation between DI and L-car levels. These results strengthen a relationship between DNA damage observed in MSUD patients, oxidative stress and the L-car supplementation benefit. The role of L-car on plasma inflammatory cytokines interleukin-1Y (IL-1Y), interleukin-6 (IL-6) and interferon-gamma (INF- Z) was also evaluated in these patients. Significant increases of IL-1Y, IL-6, and INF- Z were observed before the treatment with L-car. Moreover, there is a negative correlation between all cytokines tested and L-car concentrations and a positive correlation among the MDA content and IL-1Y and IL-6 values after L-car supplementation. It was also demonstrated that the oxidative stress parameters di-tyrosine (oxidative protein damage) and isoprostanes (lipid peroxidation assay) were increased and the antioxidant capacity was reduced in urine of MSUD patients without L-car therapy and that the supplementation of this compound induced beneficial effects on these parameters, so reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary KIC after 2 months of L-car treatment compared to control group, demonstrating an increased excretion of this toxic metabolite. In conclusion, these results suggest a reversion effect of the oxidative damage by L-car and that urine can be used to monitorize oxidative damage in patients affected by this disease. The following parameters were analysed in cerebral cortex and cerebellum of Wistar rats submitted to MSUD chemically-induced chronic model: thiobarbituric acid reactive species (TBA-RS), to evaluate lipid peroxidation, carbonyl content to evaluate protein oxidative damage, DCF oxidation to quantify reactive species production, reduced glutathione (GSH), an important non-enzymatic antioxidant and the activities of antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD). The results showed that the chronic administration of BCAA was able to promote both lipid and protein oxidation, increase of reactive species production and decreased brain antioxidant defenses, especially in cerebral cortex and that L-car was able to prevent these effects, except for oxidative damage to proteins. Taken together, these results demonstrate that the metabolites accumulated in MSUD cause oxidative damage to biomolecules (lipids, proteins and DNA), decrease antioxidant status and promote increased inflammatory processes. These results may contribute to the understanding of the mechanism of action of the cytotoxic effect of the metabolites accumulated in MSUD and the role of oxidative stress and inflammation in the MSUD neuropathophysiology besides the protective effect of L-car on this process. The study of antioxidants like L-car can opens an additional therapeutic approach to that currently employed for MSUD patients, which is primarily dietary and therefore difficult to handle.

En metod för detektering och kvantifiering av gamma-hydroxysmörsyra (GHB) i urin med gaskromatografi (GC) är framtagen på Sahlgrenska universitetssjukhuset. Metoden är relativt unik då den inte kräver upparbetning i form av derivatisering, indunstning eller extraktion. Urinen surgörs med koncentrerad saltsyra och internstandard, gamma-valerolakton, tillsätts. GHB övergår då till laktonformen, gamma-butyrolakton (GBL). Därefter injiceras provet direkt på en GC-FID med en kapillärkolonn för glykoler och alkoholer. Detektion ner till 100 μmol/L är möjligt med en variationskoefficient mellan 6 och 12 %. Provsvar erhålls efter 6,5 minuter. Metoden är dock inte fullständig då en del frågetecken kvarstår. Bland annat bör det undersökas om andra föreningar, som kan förekomma i urin, kan eluera samtidigt som GHB. Om ja så bör vidare analyser genomföras för att separera GHB och den andra föreningen. Metoden kan däremot användas i nuläget som en screeninganalys för att snabbt få ett svar på om GHB finns närvarande eller inte. Verifiering kan sedan ske med GC-MS. / A method for determination and quantification of gamma-hydroxyburyric acid (GHB) in urine samples is developed at Sahlgrenska universitetssjukhus. No time consuming procedures as derivatization and exctration is required, which makes the method fairly unique. Hydrochloric acid and internal standard, gamma-valerolakton, is added to the urine sample before the sample is injected to a gas chromatograph with a flame ionization detector and a column for glycols and alcohols. The hydrochloric acid makes the GHB convert into gamma-butyrolactone (GBL) which is easier to separate in the gas chromatograph. Limit of detection was found to be 100 μmol/L and test result is received after 6,5 minutes. There are still some question marks around the method, for example, there is a possibility that another substance elute at the same time as GHB. More tests are required to determine whether or not it is so. For now the method can be used as a screening analysis to hastily detect GHB presence. Verification can be done with GC-MS.