This thesis describes the development of separate assays to measure coenzyme Q₁₀ (CoQ₁₀) and vitamin K. Coenzyme Q is essential for the mitochondrial electron transport chain, and vitamin K for the blood coagulation cascade. Vitamin K deficiency is associated with haemorrhagic disease of the new-born, and CoQ₁₀ deficiency with HMG-CoA-reductase inhibitor (statin) therapy and heart failure. Coenzyme Q and vitamin K are usually measured by HPLC, using electrochemical and ultraviolet, and electrochemical and fluorescence detection, respectively. For vitamin K1, the limit of detection achieved using fluorescence and electrochemical detection was 0.28 and 0.12 nmol/L, respectively. Sensitivity of fluorescence detection is improved by using protic solvents in the mobile phase, and platinum-black catalysed alcohol reduction. The lipophilicity and low endogenous concentrations of vitamin K1 hinder its measurement, and further work is required to produce a rapid, reliable and robust assay for its measurement in human plasma. The limits of detection achieved using fluorescence, ultraviolet and electrochemical detection to measure CoQ₁₀ were 29, 4.8, and 0.34 nmol/L, respectively. Plasma CoQ₁₀ is not stable during long term storage at -13 ℃, but at -80 ℃ it is stable for at least 18 months. The reference interval for plasma total CoQ₁₀ in the New Zealand population is 0.47 - 1.80 µmol/L. There is no clinical requirement for stratification of the reference interval according to gender. Coenzyme Q₁₀ in human plasma is homeostatically controlled, varying little over a two month interval in healthy young males. Coenzyme Q₁₀ supplements have significantly different bioavailability, with the median increase in plasma CoQ₁₀ ranging from 0.14 to 0.59 µmol/L for seven different supplement brands. There is a large inter-individual variation in CoQ₁₀ absorption, and hence plasma concentrations should be monitored during supplementation. A plateau in CoQ₁₀ absorption, from a single dose, at approximately 200 mg suggests that the maximum dose ingested at one time should be 200 mg or less. Q-Gel capsules containing 30 mg of CoQ₁₀ are twice as effective at raising blood CoQ₁₀ as 100 mg capsules. Plasma CoQ₁₀ in patients with chronic heart failure are significantly lowered by approximately 33% when these patients receive Atorvastatin for six weeks. The absolute decrease in CoQ₁₀ showed a significant correlation with worsening endothelial function (r = + 0.548, p = 0.011). Coenzyme Q₉ was shown to be present in human plasma with a reference interval of 8.8 - 47.0 nmol/L.

Com o objetivo de se investigar o efeito da suplementação de coenzima Q10 sobre o desempenho de equinos em treinamento aeróbio, foram utilizados dez equinos do sexo masculino, castrados, da raça Puro Sangue Árabe, com idade de 48±8,15 meses e peso 473±34,75 kg, divididos em grupo controle (GC) e grupo suplementado (GS). O experimento foi conduzido no Laboratório de Pesquisa em Alimentação e Fisiologia do Exercício de Equinos (LABEQUI), pertencente à FMVZ-USP, no Campus Administrativo de Pirassununga, São Paulo, adotando-se o consumo diário individual de 2% do peso corpóreo, com base na matéria seca, sendo 50% de volumoso composto por feno de gramínea e 50% de concentrado comercial. Os animais inseridos no grupo suplementado (GS) receberam individualmente a inclusão diária de 800mg de coenzima Q10 adicionado à dieta de concentrado durante os 90 dias de experimento. Todos os animais foram exercitados cinco vezes por semana, durante sessenta minutos, na velocidade máxima de 15 km/h, em exercitador circular mecânico para cavalos, controlado eletronicamente. Durante o experimento foram realizados análise hematológica e bioquímica (AST, CK e LDH), mensuração da curva de glicose e de lactato, monitoramento da frequência cardíaca e sudorese dos equinos. Foi utilizado o delineamento inteiramente casualizado com medidas repetidas no tempo, com dados estatisticamente significantes sendo submetidos à análise de regressão. Através das análises hematológicas, bioquímicas, da curva glicose e de lactato, pode-se observar a higidez e a adaptação dos animais frente ao protocolo de treinamento. O grupo suplementado apresentou efeito de tratamento para a enzima creatinaquinase, e apresentou menor oscilação da frequência cardíaca e da taxa de sudação. Conclui-se que a suplementação de equinos atletas com coenzima Q10 submetidos a exercício aeróbio influenciou no desempenho atlético dos cavalos / With the goal of investigating the effect of Coenzyme Q10 supplementation on the performance of equines subject to aerobic exercise, ten pure Arabian geldings, aged 48±8,15 months and weighing 473±34,75 kg were divided into control group (GC) and supplemented group (GS). The experiment was performed at LABEQUI - Equine Nutrition and Exercise Physiology Research Laboratory, which belongs to FMVZ - USP, School of Veterinary Medicine and Animal Science of USP (São Paulo University), which belongs to Campus Pirassununga. Individual intake of food was considered 2% of body weight, of which 50% corresponded to grass hay and 50% to commercial pelleted concentrate. The horses in the supplemented group (GS) received a daily inclusion of 800 mg of coenzyme Q10, added to the concentrated food during the 80 days of the experiment. All animals were exercised five days per week, during sixty minutes, at a top speed of 15 km/h, in a electronically controlled circular mechanical walker. During the experiment, blood tests and biochemical analysis (AST, CK, and LDH) were conducted, as well as measurements of glucose and lactate curves and monitoring of heart frequency and perspiration. The method used was totally casual lineation with measures repeated in time, with statistically significant data being submitted to regression analysis. Animals’ healthiness and adaptation to the training protocol could be observed through hematological and biochemical analysis and glycemic and lactate curves. The supplemented group showed a treatment effect for the enzyme creatinekinase, and showed less fluctuation in heart rate and sweating rate.. It was concluded that supplementation with coenzyme Q10 of equine athletes submitted to aerobic exercise had a positive effect on the athletic performance of the horses

La mitochondrie joue un rôle majeur dans la mort cellulaire par nécrose et apoptose. Un des phénomènes hautement régulés conduisant à cette mort cellulaire est la transition de perméabilité mitochondriale qui est médiée par l'ouverture du pore de transition de perméabilité (PTP) localisé dans la membrane interne. Dans les mitochondries de foie de rat, il a été constaté que les analogues de l'ubiquinone ont trois types d'action sur l'ouverture du PTP : l'induction, l'inhibition ou l'interaction sans effet régulateur. Bien que l'inhibition du PTP soit habituellement protectrice de la mort cellulaire, il a été rapporté que des quinones inhibitrices provoquent une toxicité sur certaines lignées cellulaires.<br />Dans notre étude, nous nous sommes attachés à comprendre d'où peuvent provenir ces divergences, en travaillant sur des lignées hépatocytaires immortalisées et cancéreuses. Nous avons observé que la régulation du PTP par les analogues de l'ubiquinone peut être bouleversée par l'immortalisation et/ou la cancérisation, alors que l'effet de la ciclosporine A demeure inchangé. Tant en culture qu'en co-culture, cette caractéristique unique permet une toxicité ciblée, même entre deux lignées cellulaires très proches. Dans certaines lignées, certaines ubiquinones inhibitrices entraînent malgré tout une mort cellulaire. Nous avons pu montrer que cette toxicité est due à l'augmentation de la production radicalaire. Ce travail ouvre de nouvelles perspectives dans l'utilisation du PTP comme cible moléculaire de thérapie anticancéreuse sélective.

This thesis describes the development of separate assays to measure coenzyme Q₁₀ (CoQ₁₀) and vitamin K. Coenzyme Q is essential for the mitochondrial electron transport chain, and vitamin K for the blood coagulation cascade. Vitamin K deficiency is associated with haemorrhagic disease of the new-born, and CoQ₁₀ deficiency with HMG-CoA-reductase inhibitor (statin) therapy and heart failure. Coenzyme Q and vitamin K are usually measured by HPLC, using electrochemical and ultraviolet, and electrochemical and fluorescence detection, respectively. For vitamin K1, the limit of detection achieved using fluorescence and electrochemical detection was 0.28 and 0.12 nmol/L, respectively. Sensitivity of fluorescence detection is improved by using protic solvents in the mobile phase, and platinum-black catalysed alcohol reduction. The lipophilicity and low endogenous concentrations of vitamin K1 hinder its measurement, and further work is required to produce a rapid, reliable and robust assay for its measurement in human plasma. The limits of detection achieved using fluorescence, ultraviolet and electrochemical detection to measure CoQ₁₀ were 29, 4.8, and 0.34 nmol/L, respectively. Plasma CoQ₁₀ is not stable during long term storage at -13 ℃, but at -80 ℃ it is stable for at least 18 months. The reference interval for plasma total CoQ₁₀ in the New Zealand population is 0.47 - 1.80 µmol/L. There is no clinical requirement for stratification of the reference interval according to gender. Coenzyme Q₁₀ in human plasma is homeostatically controlled, varying little over a two month interval in healthy young males. Coenzyme Q₁₀ supplements have significantly different bioavailability, with the median increase in plasma CoQ₁₀ ranging from 0.14 to 0.59 µmol/L for seven different supplement brands. There is a large inter-individual variation in CoQ₁₀ absorption, and hence plasma concentrations should be monitored during supplementation. A plateau in CoQ₁₀ absorption, from a single dose, at approximately 200 mg suggests that the maximum dose ingested at one time should be 200 mg or less. Q-Gel capsules containing 30 mg of CoQ₁₀ are twice as effective at raising blood CoQ₁₀ as 100 mg capsules. Plasma CoQ₁₀ in patients with chronic heart failure are significantly lowered by approximately 33% when these patients receive Atorvastatin for six weeks. The absolute decrease in CoQ₁₀ showed a significant correlation with worsening endothelial function (r = + 0.548, p = 0.011). Coenzyme Q₉ was shown to be present in human plasma with a reference interval of 8.8 - 47.0 nmol/L.

Com o objetivo de se investigar o efeito da suplementação de coenzima Q10 sobre o desempenho de equinos em treinamento aeróbio, foram utilizados dez equinos do sexo masculino, castrados, da raça Puro Sangue Árabe, com idade de 48±8,15 meses e peso 473±34,75 kg, divididos em grupo controle (GC) e grupo suplementado (GS). O experimento foi conduzido no Laboratório de Pesquisa em Alimentação e Fisiologia do Exercício de Equinos (LABEQUI), pertencente à FMVZ-USP, no Campus Administrativo de Pirassununga, São Paulo, adotando-se o consumo diário individual de 2% do peso corpóreo, com base na matéria seca, sendo 50% de volumoso composto por feno de gramínea e 50% de concentrado comercial. Os animais inseridos no grupo suplementado (GS) receberam individualmente a inclusão diária de 800mg de coenzima Q10 adicionado à dieta de concentrado durante os 90 dias de experimento. Todos os animais foram exercitados cinco vezes por semana, durante sessenta minutos, na velocidade máxima de 15 km/h, em exercitador circular mecânico para cavalos, controlado eletronicamente. Durante o experimento foram realizados análise hematológica e bioquímica (AST, CK e LDH), mensuração da curva de glicose e de lactato, monitoramento da frequência cardíaca e sudorese dos equinos. Foi utilizado o delineamento inteiramente casualizado com medidas repetidas no tempo, com dados estatisticamente significantes sendo submetidos à análise de regressão. Através das análises hematológicas, bioquímicas, da curva glicose e de lactato, pode-se observar a higidez e a adaptação dos animais frente ao protocolo de treinamento. O grupo suplementado apresentou efeito de tratamento para a enzima creatinaquinase, e apresentou menor oscilação da frequência cardíaca e da taxa de sudação. Conclui-se que a suplementação de equinos atletas com coenzima Q10 submetidos a exercício aeróbio influenciou no desempenho atlético dos cavalos / With the goal of investigating the effect of Coenzyme Q10 supplementation on the performance of equines subject to aerobic exercise, ten pure Arabian geldings, aged 48±8,15 months and weighing 473±34,75 kg were divided into control group (GC) and supplemented group (GS). The experiment was performed at LABEQUI - Equine Nutrition and Exercise Physiology Research Laboratory, which belongs to FMVZ - USP, School of Veterinary Medicine and Animal Science of USP (São Paulo University), which belongs to Campus Pirassununga. Individual intake of food was considered 2% of body weight, of which 50% corresponded to grass hay and 50% to commercial pelleted concentrate. The horses in the supplemented group (GS) received a daily inclusion of 800 mg of coenzyme Q10, added to the concentrated food during the 80 days of the experiment. All animals were exercised five days per week, during sixty minutes, at a top speed of 15 km/h, in a electronically controlled circular mechanical walker. During the experiment, blood tests and biochemical analysis (AST, CK, and LDH) were conducted, as well as measurements of glucose and lactate curves and monitoring of heart frequency and perspiration. The method used was totally casual lineation with measures repeated in time, with statistically significant data being submitted to regression analysis. Animals&rsquo; healthiness and adaptation to the training protocol could be observed through hematological and biochemical analysis and glycemic and lactate curves. The supplemented group showed a treatment effect for the enzyme creatinekinase, and showed less fluctuation in heart rate and sweating rate.. It was concluded that supplementation with coenzyme Q10 of equine athletes submitted to aerobic exercise had a positive effect on the athletic performance of the horses

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23955号 / 農博第2504号 / 新制||農||1091(附属図書館) / 学位論文||R4||N5390(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 宮川 恒, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

京都大学 / 新制・課程博士 / 博士(農学) / 甲第25335号 / 農博第2601号 / 新制||農||1106(附属図書館) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三芳 秀人, 教授 白井 理, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

This dissertation presents a detailed computational investigation into the vibrational properties of quinones involved in solar energy conversion processes in photosynthetic reaction centers. In particular, we focus on the vibrational properties of the ubiquinone molecule that occupies the QA binding site in purple bacterial photosynthetic reaction centers. To provide a foundation upon which to base computational studies of pigments in protein binding sites density functional theory based calculations of the vibrational properties of neutral ubiquinone in the gas phase and in solvent were undertaken. From single point energy calculations it was shown that at least eight ubiquinone conformers, each with slightly different FTIR spectra, could be present in solvent at room temperature. The calculated and experimental spectra for neutral ubiquinone in solution are very different from the spectra associated with ubiquinone in the QA binding in purple bacterial reaction centers. For this reason an ONIOM method was undertaken in which the pigment was treated using density functional theory based methods while the protein was treated using molecular mechanics. The ONIOM calculations not only modeled the experimental QA FTIR difference spectra but also resolved the long standing issue of whether a very strong hydrogen bond exists between the bound ubiquinone and the imidazole nitrogen of a histidine residue (HisM219). To further validate the usefulness of the ONIOM approach experimental isotope edited FTIR spectra obtained using purple bacterial reaction centers with a range of chainless symmetrical quinones incorporated were modeled. Again, the agreement between the calculated and experimental spectra is outstanding. We also modeled the vibrational properties of the ubisemiquinone anion radical both in solvent and in the QA binding site. Vibrational modes of ubisemiquinone display a greater degree of mixing of the various molecular groups of the molecule. Nonetheless the calculated FTIR spectra for ubisemiquinone in solution and in the QA site agree very well with that found experimentally. Vibrational frequencies of ubisemiquinone obtained from ONIOM calculated Raman spectra also agree very well with that found in experimental resonance Raman spectra associated with the ubisemiquinone anion radical in the QA binding site.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.