RESUMEN
This study investigates evidence of oxidative stress during bicarbonate hemodialysis by measuring total glutathione and lipid peroxidation products in plasma, and characterizes the free radicals produced by neutrophils from healthy volunteers when incubated in vitro with increasing concentrations of bicarbonate. Blood samples were taken from nine hemodialysis patients before and after two hemodialysis sessions. Plasma hydroperoxides and total glutathione were measured. A significant increase was found in total glutathione (1.04 +/- 0.4 versus 2.11 +/- 0.9 microM, P < 0.001) and hydroperoxides by ferrous oxidation in xylenol orange version 2 method (4.6 +/- 0.53 versus 6.4 +/- 0.63 microM, P < 0.001) after hemodialysis, which indicated increased oxidative injury during hemodialysis. Normal neutrophils, activated by contact adhesion, produced a dose-dependent increase in free radical production (measured by luminol-enhanced chemiluminescence) when incubated with increasing concentrations of bicarbonate (up to 35 mM). Bicarbonate had the same effect on the chemiluminescence of a cell-free hypoxanthine/acetaldehyde system generating superoxide, but not on a glucose oxidase/myeloperoxidase system generating hydrogen peroxide and hypochlorous acid. These findings are consistent with (1) the hypothesis that superoxide generated during hemodialysis reacts with bicarbonate to form the toxic carbonate and formate radicals and (2) our previous observation that some patients undergoing bicarbonate (but not lactate) dialysis have increased plasma concentrations of formate after hemodialysis. It is suggested that the increased plasma total glutathione and hydroperoxide concentrations are a result of lipid peroxidation by these species. These reactive radicals can initiate lipid peroxidation and contribute to the cardiovascular complications of hemodialysis patients.
Asunto(s)
Bicarbonatos/metabolismo , Diálisis Renal , Anciano , Bicarbonatos/administración & dosificación , Bicarbonatos/química , Sistema Libre de Células/química , Sistema Libre de Células/enzimología , Relación Dosis-Respuesta a Droga , Radicales Libres/química , Radicales Libres/metabolismo , Glutatión/sangre , Glutatión/efectos de los fármacos , Humanos , Peróxidos Lipídicos/sangre , Persona de Mediana Edad , Neutrófilos/química , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismoAsunto(s)
Nicotiana , Óxido Nítrico/farmacología , Estrés Oxidativo/efectos de los fármacos , Plantas Tóxicas , Plasma/efectos de los fármacos , Humo , Técnicas de Cultivo de Célula , Eritrocitos/metabolismo , Humanos , Peroxidación de Lípido/efectos de los fármacos , Mediciones Luminiscentes , Óxido Nítrico/análisis , Nitritos/sangre , Plasma/metabolismo , Humo/análisisRESUMEN
OBJECTIVE: The aim was to investigate whether the morphological changes previously described in endothelium exposed to cigarette smoke are linked with the oxidative burden imposed on the cells. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) were exposed to samples of plasma taken from volunteer smokers and to samples of plasma to which small doses of fresh cigarette smoke derived from a smoking machine had been added. Measurements of the pentose phosphate pathway and the extruded total glutathione (GSSG) were performed to assess the presence and degree of oxidative stress on cells. Angiotensin converting enzyme (ACE) release into the medium and the ATP content of the cells were used to assess early membrane damage and cytotoxicity. RESULTS: Treatment of endothelial cells with plasma exposed to cigarette smoke in vitro resulted in activation of the pentose phosphate pathway of glucose metabolism, increased extrusion of glutathione from the cells into the medium, a decrease in the ATP pool, and release of ACE from the cells into the medium. Plasma taken from volunteers immediately after smoking showed, as might be expected, similar but less marked changes in the release of glutathione and ACE. CONCLUSIONS: Plasma from human volunteer smokers or human plasma exposed to cigarette smoke in vitro produced injury in HUVEC as assessed by changes in the ATP pool and ACE release. The extrusion of glutathione from the cells and the activation of the hexose monophosphate shunt, which is necessary to keep glutathione in the reduced state, is indicative of oxidative stress. These findings support the view that cigarette smoke related endothelial injury is, at least in part, mediated by the oxidative burden imposed by the free radicals present in cigarette smoke.
Asunto(s)
Endotelio/metabolismo , Oxígeno/metabolismo , Fumar/metabolismo , Adenosina Trifosfato/metabolismo , Membrana Celular/efectos de los fármacos , Células Cultivadas , Radicales Libres/metabolismo , Glutatión/biosíntesis , Humanos , Técnicas In Vitro , Vía de Pentosa Fosfato/efectos de los fármacos , Peptidil-Dipeptidasa A/biosíntesis , Cordón Umbilical/citologíaRESUMEN
Nitric oxide, as well as being a major regulator of vascular reactivity, has been shown to be one of the mediators of cytotoxicity in macrophages. This cytotoxic effect seems to be due to the interaction between nitric oxide and oxygen-related free radicals. This study shows that, in vitro, nitric oxide reacts with hydrogen peroxide to release large amounts of chemiluminescence with the characteristics of the highly cytotoxic species, singlet oxygen. This is supported by the observation that when nitric oxide was added to a superoxide generating system, catalase inhibited the production of singlet oxygen while superoxide dismutase enhanced it.