Thesis (Ph. D.)--University of Wisconsin--Madison, 1956. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The generation of oxygen free radicals by the cytosolic enzyme, xanthine oxidase (XO), has been implicated in post-ischemic or reperfusion damage in several organs. XO catalyzes the conversion of hypoxanthine to urate with the concomitant production of superoxide anion free radical (0₂̅˙) and hydrogen peroxide (H₂O₂). Oxygen free radical-mediated injury has also been demonstrated in inflammatory lung disease. The possible involvement of XO in oxidative injury in the lung has not yet been studied. Therefore, this research project was designed to determine whether XO is present in the lung and to investigate its characteristics in porcine, bovine, rat and human lung and other tissues. Immunochemical analysis of xanthine oxidase in the tissues employed on polyclonal antibody raised to bovine milk XO. Proteins were separated by SDS-polyacrylamide gel electrophoresis of tissue homogenates. Proteins were transfered from the gels to nitrocellulose filters by Western blotting. After incubating the filters with a antisera containing the antibody to the purified bovine XO. XO on the filter was detected by its reaction with an enzyme-conjugated second antibody. XO was immunologically detectable in bovine lung and milk. Rat lung, kidney and liver all showed XO reactivity. XO was detectable in porcine liver but not detectable in porcine lung or kidney. Thus, the antibody to bovine XO was cross-reactive with porcine and rat XO. XO protein was not immunologically detectable in human lung possibly because the antibody was not cross reactive with the bovine antibody. In vivo, xanthine oxidase exists predominantly as a dehydrogenase rather than an oxidase. In this form as xanthine dehydrogenase (XDH) the enxyme does not produce either 0₂̅˙ or H₂O₂. The activity of both XDH and XO was measured in several tissues using a fluorometric assay which uses an artifical substrate, pterin which is catalytically converted to the fluorescent product isoxanthopterin (IXP). XO activity in porcine liver was of 1.1 x 10⁻³ µg IXP/mg protein/min although XO activity was not detectable in porcine lung and kidney, in rat lung of 1.7 x 10⁻² µg IXP/mg protein/min, rat kidney of 1.5 x 10⁻² µg IXP/mg protein/min, and rat liver of 2.2 x 10⁻² µg IXP/mg protein/min. Seven human lung biopsy samples were obtained after lung resection and initially tested for viability by determination of NADH oxidase activity and then assayed for XO-XDH. Three of these samples showed NADH oxidase activity indicating tissue viability, but only one of these three showed measurable XO activity of 5.35 x 10⁻⁶ µg IXP/mg protein/min. Irreversible conversion of XDH to XO is thought to be the result of limited proteolysis by a Ca²⁺/calmodulin activated protease, whereas reversible conversion of the enzyme occurs by oxidation of critical thiol groups. Studies on the rate and nature of fluorescence assay to detect catalytic activities of both enzyme forms. Incubation of lung homogenates with trypsin for 60 min caused irreverisble conversion of 90% of the XDH to XO. In contrast, incubation of homogenates at 15°C for 10 hours caused conversion of 100% of the XDH to XO. This conversion was reversible to the extent of 80% by reduction of thiol groups with dithiothreitol (DTT). The effects of free Ca²⁺ on the conversion of XDH to X0 was examined by using EDTA, a chelator of Ca²⁺ and other divalent cations; and EGTA, a more specific chelator of Ca²⁺. The presence of these chelating agents during homogenization of either normoxic or ischemic rat lung tissue did not inhibit reversible enzyme conversion. Increased XO activity was reversible by DTT. In the normoxic rat lung, homogenates prepared with EDTA and EGTA showed a similar conversion of 95% of XDH to XO which was reversible to 70% with DTT. In the ischemic rat lung, samples prepared with EDTA and EGTA showed a'conversion of 80% and 95% XDH to XO which was similar to control samples. The extent of reversibility to XDH was 75% with DTT incubation. In addition, perfusion of rat lungs with EDTA and DTT via a pulmonary artery cannula prior to 60 min of ischemia and homogenization did not affect the extent of XDH to XO conversion. These results indicate that irreversible Ca²⁺-mediated proteolytic conversion of XDH to XO does not occur to a great extent in the rat lung during either normoxia or ischemia. However, reversible conversion of XDH to XO does occur, suggesting that reversible thiol dependent conversion may play a role in the lung under both physiological and pathophysiological states. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No / Aromatic aldehydes are good substrates of aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. However, the oxidation of xenobiotic-derived aromatic aldehydes by thelatter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase and xanthine oxidase activities in the oxidation of isovanillin in separate preparations and also in freshly prepared and cryopreserved liver slices. The oxidation of isovanillin was also examined in the presence of specific inhibitors of each oxidizing enzyme. Minimal transformation of isovanillin to isovanillic acid was observed in partially purified aldehyde oxidase, which is thought to be due to residual xanthine oxidase activity. Isovanillin was rapidly metabolized to isovanillic acid by high amounts of purified xanthine oxidase, but only low amounts are present in guinea pig liver fraction. Thus the contribution of xanthine oxidase to isovanillin oxidation in guinea pig is very low. In contrast, isovanillin was rapidly catalyzed to isovanillic acid by guinea pig liver aldehyde dehydrogenase activity. The inhibitor studies revealed that isovanillin was predominantly metabolized by aldehyde dehydrogenase activity. The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared or cryopreserved liver slices has not been previously reported. In freshly prepared liver slices, isovanillin was rapidly converted to isovanillic acid, whereas the conversion was very slow in cryopreserved liver slices due to low aldehyde dehydrogenase activity. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. It is therefore concluded that isovanillin is predominantly metabolized by aldehyde dehydrogenase activity, with minimal contribution from either aldehyde oxidase or xanthine oxidase.

No description available.

Thesis (M.S.)--West Virginia University, 2010. / Title from document title page. Document formatted into pages; contains v, 88 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Chronic exercise has been recommended as a strategy for preventing several diseases associated with lifestyle such as heart disease, hypertension, osteoporosis and type II diabetes. Although regular physical exercise has benefits for health, all sports practitioners, and even sedentary people, have already feel delayed onset muscle soreness (DOMS) once, characterized by discomfort in skeletal muscle. As the most DOMS generator, acute eccentric exercise induce fatigue, strength reduction and performance impairment. Despite some researches demonstrating reactive oxygen species (ROS) in this context, there are few information about purine degradation as well as transient receptor potential vanilloid 1 (TRPV1) on DOMS development. In this context, animals performed a downhill running test (eccentric exercise) on treadmill until exhaustion for histologic evaluation, mechanical allodynea, strength force test and biochemical analysis. The results showed an increase in mechanical allodynea and ADP, AMP, uric acid and TRPV1 immunoreactivity levels. In conclusion, the results support the contribution of ROS and the participation of purine and TRPV1 on delayed onset muscle soreness. / O exercício físico crônico tem sido recomendado como estratégia para a prevenção de diversas doenças associadas ao estilo de vida, como doenças cardíacas, hipertensão, osteoporose e diabetes tipo 2. Embora o exercício físico regular traga benefícios para a saúde, todos os praticantes de atividade física e esporte e, até mesmo indivíduos sedentários, já experimentaram alguma vez na vida um episódio de dor muscular tardia (DMT), caracterizada pela sensação de desconforto na musculatura esquelética. Como grande gerador de DMT, destaca-se o exercício excêntrico agudo que induz fadiga, redução de força e perda de desempenho. Apesar de diversos estudos demonstrando a participação das espécies reativas de oxigênio neste quadro, pouco se sabe sobre a participação da degradação das purinas bem como a participação dos receptores de potencial transitório vaniloide 1 (TRPV1) no desenvolvimento da dor muscular tardia. Para tanto, os ratos wistar machos realizaram teste de downhill em esteira (exercício excêntrico) até a exaustão. Após foram analisados os danos histológicos nos músculos gastrocnêmio e sóleo, Outro set de animais após a exaustão foram avaliados nos testes de alodinea mecanica na pata traseira direita, teste funcional de força nas pastas dianteiras e análises bioquímicas no músculo gastrocnêmio. Os resultados demonstram aumento na alodinea, na carbonilação protéica, nos níves de ADP, AMP, ácido úrico, além de elevar os níveis de immureatividade do receptor TRPV1 e atividade da xantina oxidase. Esses dados apontam uma possível contribuição das espécies reativas de oxigênio, da degradação de purinas e dos receptores TRPV1 na dor muscular tardia.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22484号 / 農博第2388号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5264(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 小川 順, 教授 加納 健司, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No / Aliphatic aldehydes have a high affinity toward aldehyde dehydrogenase activity but are relatively poor substrates of aldehyde oxidase and xanthine oxidase. In addition, the oxidation of xenobiotic-derived aromatic aldehydes by the latter enzymes has not been studied to any great extent. The present investigation compares the relative contribution of aldehyde dehydrogenase, aldehyde oxidase, and xanthine oxidase activities in the oxidation of substituted benzaldehydes in separate preparations. The incubation of vanillin, isovanillin, and protocatechuic aldehyde with either guinea pig liver aldehyde oxidase, bovine milk xanthine oxidase, or guinea pig liver aldehyde dehydrogenase demonstrated that the three aldehyde oxidizing enzymes had a complementary substrate specificity. Incubations were also performed with specific inhibitors of each enzyme (isovanillin for aldehyde oxidase, allopurinol for xanthine oxidase, and disulfiram for aldehyde dehydrogenase) to determine the relative contribution of each enzyme in the oxidation of these aldehydes. Under these conditions, vanillin was rapidly oxidized by aldehyde oxidase, isovanillin was predominantly metabolized by aldehyde dehydrogenase activity, and protocatechuic aldehyde was slowly oxidized, possibly by all three enzymes. Thus, aldehyde oxidase activity may be a significant factor in the oxidation of aromatic aldehydes generated from amines and alkyl benzenes during drug metabolism. In addition, this enzyme may also have a role in the catabolism of biogenic amines such as dopamine and noradrenaline where 3-methoxyphenylacetic acids are major metabolites.

No / Background/Aims: The oxidation of xenobiotic-derived aromatic aldehydes with freshly prepared liver slices has not been previously reported. The present investigation compares the relative contribution of aldehyde oxidase, xanthine oxidase and aldehyde dehydrogenase activities in the oxidation of vanillin, isovanillin and protocatechuic aldehyde with freshly prepared liver slices. Methods: Vanillin, isovanillin or protocatechuic aldehyde was incubated with liver slices in the presence/absence of specific inhibitors of each enzyme, followed by HPLC. Results: Vanillin was rapidly converted to vanillic acid. Vanillic acid formation was completely inhibited by isovanillin (aldehyde oxidase inhibitor), whereas disulfiram (aldehyde dehydrogenase inhibitor) inhibited acid formation by 16% and allopurinol (xanthine oxidase inhibitor) had no effect. Isovanillin was rapidly converted to isovanillic acid. The formation of isovanillic acid was not altered by allopurinol, but considerably inhibited by disulfiram. Protocatechuic aldehyde was converted to protocatechuic acid at a lower rate than that of vanillin or isovanillin. Allopurinol only slightly inhibited protocatechuic aldehyde oxidation, isovanillin had little effect, whereas disulfiram inhibited protocatechuic acid formation by 50%. Conclusions: In freshly prepared liver slices, vanillin is rapidly oxidized by aldehyde oxidase with little contribution from xanthine oxidase or aldehyde dehydrogenase. Isovanillin is not a substrate for aldehyde oxidase and therefore it is metabolized to isovanillic acid predominantly by aldehyde dehydrogenase. All three enzymes contribute to the oxidation of protocatechuic aldehyde to its acid.

No description available.