NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

J Biol Inorg Chem (2007) 12:777–787 DOI 10.1007/s00775-007-0229-7To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

J Biol Inorg Chem (2007) 12:777–787 DOI 10.1007/s00775-007-0229-7To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme

Molecular mechanisms of anti-inflammatory activity mediated by flavonoids

Flavonoids (or bioflavonoids) are naturally occurring compounds, ubiquitous in all vascular plants. These compounds have been considered to possess anti-inflammatory properties, both in vitro and in vivo. Although not fully understood, these health-promoting effects have been mainly related to their interactions with several key enzymes, signaling cascades involving cytokines and regulatory transcription factors, and antioxidant systems. The biological effects of flavonoids will depend not only on these pharmacodynamic features but also on their pharmacokinetics, which are dependent on their chemical structure, administered dose schedule and route of administration. Thus, the therapeutic outcome mediated by flavonoids will result from a complex and interactive network of effects, whose prediction require a deep and integrated knowledge of those pharmacokinetic and pharmacodynamic factors. The aim of the present review is thus to provide an integrated update on the bioavailability and biotransformation of flavonoids and the mechanisms of activity at the molecular, cellular, organ and organism levels that may contribute to their anti-inflammatory effects

Iron and copper complexation by angiotensin-converting enzyme inhibitors. A study by ultraviolet spectroscopy and electrospray mass spectrometry

Captopril, enalaprilat and lisinopril, angiotensin-converting enzyme (ACE) inhibitors, are effective HO. radical scavengers and seem to have some degree of metal binding capabilities. By metal chelation iron and copper may become inactive in radical generation or the antioxidant effect can be operative by site-specific scavenging. In order to clarify the ability of the ACE inhibitors to bind iron and copper, their interactions were investigated by spectrophotometry and electrospray ionisation mass spectrometry. The spectrophotometric results showed that when iron or copper was added to the ACE inhibitors a decrease of the absorbance at 211 nm was observed. Significant modifications of the spectra in the range 250-400 nm were also observed when copper was added. An association of the ACE inhibitors with iron or copper may be inferred from these studies. The electrospray mass spectra of captopril, enalaprilat and lisinopril, when copper was added, exhibited peaks attributed to the chelation of copper by the three ACE inhibitors, with a preference for 1:1 metal : ligand stoichiometry. The electrospray data also suggest that iron is not as effective as copper for the formation of metal : ligand complexes for captopril, enalaprilat and lisinopril

Nitrite Reductase Activity of Rat and Human Xanthine Oxidase, Xanthine Dehydrogenase, and Aldehyde Oxidase: Evaluation of Their Contribution to NO Formation <i>in Vivo</i>

Nitrite is presently considered a NO “storage form” that can be made available, through its one-electron reduction, to maintain NO formation under hypoxia/anoxia. The molybdoenzymes xanthine oxidase/dehydrogenase (XO/XD) and aldehyde oxidase (AO) are two of the most promising mammalian nitrite reductases, and in this work, we characterized NO formation by rat and human XO/XD and AO. This is the first characterization of human enzymes, and our results support the employment of rat liver enzymes as suitable models of the human counterparts. A comprehensive kinetic characterization of the effect of pH on XO and AO-catalyzed nitrite reduction showed that the enzyme’s specificity constant for nitrite increase 8-fold, while the <i>K</i>_m^{NO₂^–} decrease 6-fold, when the pH decreases from 7.4 to 6.3. These results demonstrate that the ability of XO/AO to trigger NO formation would be greatly enhanced under the acidic conditions characteristic of ischemia. The dioxygen inhibition was quantified, and the <i>K</i>_i^O₂ values found (24.3–48.8 μM) suggest that <i>in vivo</i> NO formation would be fine-tuned by dioxygen availability. The potential <i>in vivo</i> relative physiological relevance of XO/XD/AO-dependent pathways of NO formation was evaluated using HepG2 and HMEC cell lines subjected to hypoxia. NO formation by the cells was found to be pH-, nitrite-, and dioxygen-dependent, and the relative contribution of XO/XD plus AO was found to be as high as 50%. Collectively, our results supported the possibility that XO/XD and AO can contribute to NO generation under hypoxia inside a living human cell. Furthermore, the molecular mechanism of XO/AO-catalyzed nitrite reduction was revised

Surto de hepatite A. Repercussões e intervenção no Distrito de Évora

Introdução: Actualmente Portugal é considerado um país de baixa endemicidade de hepatite A. Neste padrão epidemiológico são mais frequentes os surtos, habitualmente em grupos etários mais velhos. Em 2004 e 2005 verificou-se um aumento temporário do número de casos declarados de hepatite A em Portugal.Objectivo: Analisar os casos de hepatite A declarados no Distrito de Évora durante o surto de 2004-2005, descrever a investigação epidemiológica dos mesmos e referir as medidas de saúde pública implementadas na comunidade.Metodologia: Análise retrospectiva dos impressos de Doenças de Declaração Obrigatória, das fichas de urgência e dos processos de internamento (Serviço de Pediatria do Hospital do Espírito Santo de Évora) dos casos declarados de hepatite A, durante o surto de 2004-2005, no Distrito de Évora. Pesquisa dos dados de investigação epidemiológica e medidas de contenção do surto.Resultados: Dos 24 casos declarados de hepatite A(o primeiro em Junho e o último em Dezembro de 2004), a maioria (67%) era de etnia cigana, 20 (83%) tinham idade inferior a catorze anos e treze (54%) ficaram internados. Todos tiveram evolução clínica favorável à excepção de um (4%) que necessitou de transplante hepático por insuficiência hepática fulminante. Ainvestigação epidemiológica revelou um tipo de transmissão pessoa a pessoa e, na maioria dos casos, foi possível identificar uma ligação epidemiológica entre os casos confirmados. Foi administrada a primeira dose da vacina contra o VHA a todos os contactos próximos dos casos.Conclusões: Salienta-se a importância da declaração sistemática e rápida dos casos de hepatite A e do conhecimento dos dados epidemiológicos na implementação de medidas de prevenção e de controlo de surtos da doença

Noninvasive ventilation in COVID-19 patients aged ≥ 70 years : a prospective multicentre cohort study

Background: Noninvasive ventilation (NIV) is a promising alternative to invasive mechanical ventilation (IMV) with a particular importance amidst the shortage of intensive care unit (ICU) beds during the COVID-19 pandemic. We aimed to evaluate the use of NIV in Europe and factors associated with outcomes of patients treated with NIV. Methods: This is a substudy of COVIP study-an international prospective observational study enrolling patients aged >= 70 years with confirmed COVID-19 treated in ICU. We enrolled patients in 156 ICUs across 15 European countries between March 2020 and April 2021.The primary endpoint was 30-day mortality. Results: Cohort included 3074 patients, most of whom were male (2197/3074, 71.4%) at the mean age of 75.7 years (SD 4.6). NIV frequency was 25.7% and varied from 1.1 to 62.0% between participating countries. Primary NIV failure, defined as need for endotracheal intubation or death within 30 days since ICU admission, occurred in 470/629 (74.7%) of patients. Factors associated with increased NIV failure risk were higher Sequential Organ Failure Assessment (SOFA) score (OR 3.73, 95% CI 2.36-5.90) and Clinical Frailty Scale (CFS) on admission (OR 1.46, 95% CI 1.06-2.00). Patients initially treated with NIV (n = 630) lived for 1.36 fewer days (95% CI - 2.27 to - 0.46 days) compared to primary IMV group (n = 1876). Conclusions: Frequency of NIV use varies across European countries. Higher severity of illness and more severe frailty were associated with a risk of NIV failure among critically ill older adults with COVID-19. Primary IMV was associated with better outcomes than primary NIV