RESUMEN
Drug of abuse (DOA) screening in urine is often performed in the clinical emergency setting. However, there is considerable evidence that questions the usefulness of this screening in the acute management of patients with suspected intoxications. The used method is an immunoassay, in which cross reactivity with false positive results may occur. A positive result does not always indicate current toxicity, a negative result does not exclude drug use or a current intoxication. Therefore, DOA screening has limited value in the acute clinical management of patients with intoxications.
Asunto(s)
Inmunoensayo/métodos , Detección de Abuso de Sustancias/métodos , Trastornos Relacionados con Sustancias/diagnóstico , Urinálisis/métodos , Urgencias Médicas , Servicio de Urgencia en Hospital , Reacciones Falso Positivas , Humanos , Reproducibilidad de los Resultados , Trastornos Relacionados con Sustancias/orinaRESUMEN
BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Enfermedad de Leigh/enzimología , Enfermedad de Leigh/genética , Metiltransferasas/genética , Proteínas Mitocondriales/genética , Mutación/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Secuencia de Bases , Preescolar , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/genética , Familia , Femenino , Homocigoto , Humanos , Enfermedad de Leigh/diagnóstico por imagen , Enfermedad de Leigh/metabolismo , Leucocitos Mononucleares/enzimología , Imagen por Resonancia Magnética , Masculino , Metiltransferasas/química , Proteínas Mitocondriales/química , Datos de Secuencia Molecular , Marruecos , Linaje , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
BACKGROUND: The m.3243A>G mutation in the mitochondrial tRNA(Leu(UUR)) gene is an example of a mutation causing a very heterogeneous phenotype. It is the most frequent cause (80%) of the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), but it can also lead in addition or separately to type 2 diabetes, deafness, renal tubulopathy and/or cardiomyopathy. METHODS: To identify pathogenic processes induced by this mutation, we compared global gene expression levels of muscle biopsies from affected and unaffected mutation carriers with controls. RESULTS AND CONCLUSIONS: Gene expression changes were relatively subtle. In the asymptomatic group 200 transcripts were upregulated and 12 were downregulated, whereas in the symptomatic group 15 transcripts were upregulated and 52 were downregulated. In the asymptomatic group, oxidative phosphorylation (OXPHOS) complex I and IV genes were induced. Protein turnover and apoptosis were elevated, most likely due to the formation of dysfunctional and reactive oxygen species (ROS) damaged proteins. These processes returned to normal in symptomatic patients. Components of the complement system were upregulated in both groups, but the strongest in the symptomatic group, which might indicate muscle regeneration--most likely, protein damage and OXPHOS dysfunction stimulate repair (protein regeneration) and metabolic adaptation (OXPHOS). In asymptomatic individuals these processes suffice to prevent the occurrence of symptoms. However, in affected individuals the repair process terminates, presumably because of excessive damage, and switches to muscle regeneration, as indicated by a stronger complement activation. This switch leaves increasingly damaged tissue in place and muscle pathology becomes manifest. Therefore, the expression of complement components might be a marker for the severity and progression of MELAS clinical course.
Asunto(s)
Síndrome MELAS/genética , Mutación Puntual , ARN de Transferencia de Leucina/genética , Adolescente , Adulto , Anciano , Apoptosis , Niño , Preescolar , Activación de Complemento , Femenino , Perfilación de la Expresión Génica , Heterocigoto , Humanos , Síndrome MELAS/fisiopatología , Masculino , Persona de Mediana Edad , Músculo Esquelético/fisiopatología , Fosforilación Oxidativa , Proteínas/metabolismo , ARN de Transferencia de Leucina/metabolismoRESUMEN
Complex I activity of the mitochondrial respiratory chain is difficult to measure in blood lymphocytes because of the limited access of substrates to the enzyme complex in these cells. The results of the present study show that permeabilization of human blood lymphocytes in the presence of protease inhibitors by three cycles of freeze-thawing enables reproducible detection of the rotenone-sensitive complex I activity. To that end, the water-soluble coenzyme Q(10) analogue CoQ(1) and a relatively high concentration of blood lymphocytes were combined in small quartz cuvettes so that the amount of blood needed for this assay remained low. The relationship between the initial rate of NADH oxidation by complex I and the protein concentration was quasi-linear. The fractional inhibition of the total NADH:CoQ(1) oxidoreductase by a saturating concentration of rotenone decreased sharply at CoQ(1) concentrations higher than 20 muM, which is indicative, but does not prove the involvement of a second CoQ(1) binding site at complex I. Since the present complex I assay requires only a small amount of blood, the functionality of this important respiratory chain complex can be assessed in an easy and reliable manner not only in adult patients but also in children suspected to have a mitochondrial disease.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Linfocitos/enzimología , Proteínas Mitocondriales/metabolismo , Adulto , Niño , Complejo I de Transporte de Electrón/sangre , Humanos , Cinética , Mitocondrias/enzimología , NAD/metabolismo , Oxidación-Reducción , Ubiquinona/metabolismoRESUMEN
AIM: Leukocytes have been implicated in restenosis following percutaneous transluminal coronary angioplasty. We investigated the link between the activated status of circulating neutrophils and restenosis after angioplasty. METHODS AND RESULTS: The population of 108 patients with single, de novo lesions located in native coronary arteries were treated with elective balloon angioplasty (n=44) or stenting (n=64). Pre-, post-procedure and 6-month follow-up, angiograms were analysed by an independent core laboratory. Blood samples were collected immediately before treatment and the antigen CD66, which is specifically expressed by activated neutrophils, was measured. Overall, the average expression of CD66 was 6.4+/-3.6 of mean fluorescence intensity. In the stepwise linear regression model, which included biological, clinical and angiographic variables, absolute gain showed a direct association (P<0.001) with relative late loss (relative late loss=absolute late loss/pre-procedure reference diameter), whereas CD66 expression was inversely associated with relative late loss (P=0.004). CD66 expression also showed an inverse association with relative late loss in the balloon angioplasty treated patients (P=0.002, beta=-0.49). In the stent subgroup, only reference vessel diameter and acute gain were independent predictors of relative late loss. CONCLUSION: Our results confirm the beneficial role of activated neutrophils pre-procedure in the restenotic process after balloon angioplasty. The lack of a relationship between CD66 expression by neutrophils and relative late loss after stenting suggests that this leukocyte may be involved in the remodelling process.
Asunto(s)
Angioplastia Coronaria con Balón , Oclusión de Injerto Vascular/fisiopatología , Activación Neutrófila/fisiología , Antígenos CD/fisiología , Antígenos de Diferenciación/fisiología , Moléculas de Adhesión Celular , Angiografía Coronaria , Femenino , Citometría de Flujo , Humanos , Modelos Lineales , Masculino , Persona de Mediana Edad , Monocitos/fisiología , Valor Predictivo de las PruebasRESUMEN
The present study was undertaken to investigate the hypothesis that multiple oxygen radical generating systems contribute to the tumor necrosis factor (TNF) alpha-stimulated transcriptional activation of the vascular cell adhesion molecule (VCAM)-1 in endothelial cells. Experimental evidence has implicated the cytochrome P450 monooxygenase and a phagocyte type NADPH-oxidase as a source of oxygen radicals in these cells. We show here that endothelial cells exhibit cytochrome P450 activity by measuring the O-dealkylation of the exogenous substrate 7-ethoxyresorufin, but components of the phagocyte-type NADPH oxidase could not be demonstrated in endothelial cells. In that latter respect it was surprising that the NADPH oxidase inhibitor apocynin completely prevented the accumulation of VCAM-1 mRNA. However, we found that apocynin also acts as an inhibitor of cytochrome P450 activity in endothelial cells. Therefore the inhibitory effect of apocynin on the induction of VCAM-1 may no longer be used to demonstrate a role for the NADPH oxidase in this process. Furthermore, different cytochrome P450 inhibitors Co2+, metyrapone, SKF525a decreased the endothelial VCAM-1 expression stimulated by TNFalpha. Also under hypoxic conditions the expression of VCAM-1 was reduced. On this basis we assume that the oxygen dependent step in the intracellular signalling cascade underlying the TNFalpha stimulated transcriptional activation of VCAM-1 resides in the activity of a cytochrome P450 dependent monooxygenase. The finding that the phospholipase A2 inhibitor bromophenacylbromide inhibited the expression of VCAM-1 may indicate that arachidonic acid serves as a substrate for the cytochrome P450 monooxygenase reaction, but further research is needed to elucidate the particular cytochrome P450 family member mediating the expression of VCAM-1.
Asunto(s)
Acetofenonas/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , NADPH Oxidasas/metabolismo , Compuestos Onio/farmacología , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Alquilación/efectos de los fármacos , Northern Blotting , Hipoxia de la Célula , Células Cultivadas , Cobalto/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Combinación de Medicamentos , Medicamentos Herbarios Chinos/farmacología , Transporte de Electrón/efectos de los fármacos , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Radicales Libres , Glycyrrhiza , Humanos , Metirapona/farmacología , NADPH Oxidasas/antagonistas & inhibidores , Neutrófilos/enzimología , Oxazinas/metabolismo , Oxidación-Reducción , Paeonia , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A/farmacología , Fosfolipasas A2 , Reacción en Cadena de la Polimerasa , Transducción de Señal/efectos de los fármacos , Venas Umbilicales , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
The present studies were aimed at investigating the possible roles of dopamine (DA) and iron in production of hydroxyl radicals (OH) in rat striatum after Mn2+ intoxication. For this purpose, DA depletions were assessed concomitant with in vivo 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formation from the reaction of salicylate with OH, of which 2,3-DHBA is a nonenzymatic adduct. Following intrastriatal Mn2+ injection, marked 2,3-DHBA increases were observed in a time- and dose-dependent fashion reaching maximum levels at 6-18 h and a plateau beyond 0.4 micromol (fourfold increase). The delayed increase of 2,3-DHBA levels suggestS that Mn2+ induces OH formation in the living brain by an indirect process. The early DA depletion (2 h) and relatively late OH formation (6 h) indicate independent processes by Mn2+. In addition, depletion of DA (about 90%) by reserpine pretreatment not significantly alter Mn2+-induced 2,3-DHBA formation or the extent of DA depletion, suggesting that DA or DA autoxidation are not participating in Mn2+-induced OH formation in vivo. Furthermore, Mn2+ injection did not significantly alter the low molecular weight weight iron pool in striatum, and co-injections of the iron-chelator deferoxamine with Mn(2+) into striatum did not significantly attenuate Mn(2+)-induced 2,3-DHBA formation. These findings suggest no role of chelatable iron in generation of Mn(2+)-induced OH, but do not exclude a role for mitochondrial heme-iron or peroxynitrite (Fe-indepeNdent) in Mn2+-induced OH formation.
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Cuerpo Estriado/metabolismo , Dopamina/deficiencia , Radical Hidroxilo/metabolismo , Quelantes del Hierro/farmacología , Manganeso/farmacología , Animales , Cuerpo Estriado/efectos de los fármacos , Deferoxamina/farmacología , Relación Dosis-Respuesta a Droga , Hierro/química , Hierro/metabolismo , Masculino , Peso Molecular , Neurotoxinas/farmacología , Concentración Osmolar , Oxidopamina/metabolismo , Ratas , Ratas Wistar , Reserpina/farmacología , Factores de TiempoRESUMEN
BACKGROUND: The purpose of this pilot study was to identify biological risk factors for restenosis after percutaneous transluminal coronary angioplasty (PTCA) to predict the long-term outcome of PTCA before treatment. METHODS AND RESULTS: To investigate whether blood granulocytes and monocytes could determine luminal renarrowing after PTCA, several characteristics of these phagocytes were assessed before angioplasty in 32 patients who underwent PTCA of one coronary artery and who had repeat angiograms at 6-month follow-up. The plasma levels of interleukin (IL)-1 beta, tumor necrosis factor-alpha, IL-6, fibrinogen, C-reactive protein, and lipoprotein(a) before angioplasty were assessed as well. We found that the expression of the membrane antigens CD64, CD66, and CD67 by granulocytes was inversely associated with the luminal renarrowing normalized for vessel size (relative loss) at 6 months after PTCA, while the production of IL-1 beta by stimulated monocytes was positively associated with the relative loss. Next, these univariate predictors were corrected for the established clinical risk factors of dilation of the left anterior descending coronary artery and current smoking, which were statistically significant classic predictors in our patient group. Only the expression of CD67 did not predict late lumen loss independent of these established clinical risk factors. Multiple linear regression analysis showed that luminal renarrowing could be predicted reliably (R2 = .65; P < .0001) in this patient group on the basis of the vessel dilated and only two biological risk factors that reflect the activation status of blood phagocytes, ie, the expression of CD66 by granulocytes and the production of IL-1 beta by stimulated monocytes. CONCLUSIONS: The results of the present study indicate that activated blood granulocytes prevent luminal renarrowing after PTCA, while activated blood monocytes promote late lumen loss. To validate this new finding, further study in an independent patient group is required.
Asunto(s)
Angina de Pecho/terapia , Angioplastia Coronaria con Balón , Antígenos CD/análisis , Antígenos de Diferenciación/análisis , Enfermedad Coronaria/etiología , Interleucina-1/metabolismo , Fagocitos/inmunología , Angina de Pecho/inmunología , Moléculas de Adhesión Celular , Enfermedad Coronaria/inmunología , Femenino , Humanos , Modelos Lineales , Activación de Linfocitos/fisiología , Masculino , Persona de Mediana Edad , Fagocitos/metabolismo , Proyectos Piloto , Recurrencia , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
The early vascular effects of photodynamic therapy (PDT) include transient vasoconstriction and platelet aggregation. Since endothelium-derived relaxing factor (EDRF) is a potent vasodilator and inhibitor of platelet aggregation, we questioned whether PDT impairs the production of EDRF. To study this possible effect of PDT, endothelium-dependent relaxations of thoracic aortas obtained from male Wistar rats were determined. The aortic rings were connected to a isometric force transducer, exposed to various doses of Photofrin porfimer sodium (Photofrin) (0.1-1.0 microgram/ml), and illuminated with red light (wavelength > 610 nm, 14.6 +/- 1.5 mW/cm2) for different time periods (5-25 min). Endothelium-dependent relaxation was induced by acetylcholine in precontracted aortic rings. This EDRF-mediated relaxation was decreased after PDT in a light dose- and drug dose-dependent manner. Light microscopic examination did not show loss of endothelial cells. Similar results were obtained with rat aortas exposed to Photofrin in vivo and illuminated in vitro. Direct smooth muscle relaxation induced with sodium nitroprusside was not impaired, showing that PDT did not reduce the ability of smooth muscles to relax. No effect on the contractile responses was found either. We conclude that PDT impairs the production or release of EDRF by the endothelium. This could play an important role in the initial events occurring in vivo during and after PDT.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Derivado de la Hematoporfirina/farmacología , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico/biosíntesis , Fotoquimioterapia , Acetilcolina/farmacología , Animales , Aorta Torácica/química , Aorta Torácica/fisiología , Relación Dosis-Respuesta a Droga , Derivado de la Hematoporfirina/análisis , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso Vascular/fisiología , Ratas , Ratas WistarRESUMEN
The importance of the glucose/fatty acid cycle in the control of cardiac lipolysis is emphasized by the following observations. Addition of the glycogen debranching inhibitor deoxynojirimycin or an O2-vehicle, fluorocarbon F-43, to media perfusing paced, lipid-enriched, Langendorff hearts lower cardiac lactate and glycerol 3-phosphate levels together with inhibition of glucagon-stimulated glycerol (and lactate) release. The absence of fluorocarbon during perfusion of 5 Hz paced langendorff hearts probably results in limited tissue oxygenation, resulting in glycogenolysis and lipolysis. The results indicate hormonal control of cardiac lipolysis by glyco(geno)lysis.
Asunto(s)
Glucógeno/metabolismo , Glucólisis , Lipólisis , Miocardio/metabolismo , 1-Desoxinojirimicina , Animales , Glucagón/farmacología , Glucosamina/análogos & derivados , Glucosamina/farmacología , Glicerol/metabolismo , Glucólisis/efectos de los fármacos , Hipoxia , Técnicas In Vitro , Lactatos/metabolismo , Lipólisis/efectos de los fármacos , RatasRESUMEN
When during aerobic perfusion of the 5 Hz paced rat Langendorff heart, under constant aortic pressure of 8.3 kPa, the pH of the medium is changed from 7.5 to 7.0 a short period of positive inotropy is followed by a dramatic loss of contractility. The hearts, rapidly frozen after 10 min pH 7.0 perfusion, show moderate loss of high-energy phosphates and accumulation of lactate and glycerol-3-phosphate, indicative of tissue anaerobiosis. This can be overcome by including fluorocarbon, an O2 vehicle, in the media. The transient positive inotropy is interpreted as H(+)-induced release of plasmalemma-bound Ca2+ into the cytosol. The accompanying morphologic alterations are as described in this issue by Vandeplassche and Borgers (1990) and by Verkleij et al. (1990).
Asunto(s)
Acidosis/prevención & control , Corazón/fisiología , Oxígeno/farmacología , Animales , Calcio/metabolismo , Estimulación Cardíaca Artificial , Membrana Celular/metabolismo , Fluorocarburos/farmacología , Corazón/efectos de los fármacos , Concentración de Iones de Hidrógeno , Miocardio/citología , Miocardio/metabolismo , RatasRESUMEN
When the pH of the perfusion medium of rat Langendorff heart, paced at a rate of 300 beats/min, is abruptly lowered from pH 7.5 to 7.0, the hearts stop beating within 6 min in more than half of the cases. Reperfusion with pH 7.5 medium after 10 min pH 7.0 perfusion does not cause contractility to resume within 5 min. The causative factor is intracellular acidosis, resulting in severe morphological alterations of plasma membrane and mitochondria. It is probably initiated by the loss of membrane-bound calcium. Oleate, complexed with albumin included in the perfusion media, protects the hearts. This may be explained by maintenance of capillary flow and limitation of cellular acidosis.
Asunto(s)
Ácidos Grasos/farmacología , Corazón/fisiología , Contracción Miocárdica , Animales , Corazón/efectos de los fármacos , Concentración de Iones de Hidrógeno , Miocardio/ultraestructura , Perfusión , RatasRESUMEN
Lipopolysaccharide (LPS), the active principle of certain endotoxins, protein-free perfused in rat hearts leads in 3 h to a considerable loss of lipoprotein lipase (LPL) activity. In the presence of albumin LPS has virtually no effect. Tumor necrosis factor (TNF) added instead of LPS had no effects on LPL activity during 3 h in vitro perfusion. LPS injected into rats intravenously leads within 3 h to severe toxic phenomena amongst which increased capillary permeability. This was visualized as increased rate of interstitial fluid formation in Langendorff hearts mounted 3 h after rats had been treated with LPS. LPL activity did not decline in 3 h lasting endotoxemia. Six hours after LPS injection, however, cardiac LPL activity was considerably lowered, although immunoblotting and immunohistochemistry still showed LPL protein to be present. These date indicate the presence of a considerable pool of inactive LPL protein in addition to active LPL, that can be released in the presence of heparin. The LPL activity is lowered by LPS injection after a lag phase of at least 3 h, while capillary endothelial cells are influenced more rapidly. The relatively late expression of TNF toxicity in cardiomyocytes of the intact heart is discussed.
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Lipopolisacáridos/farmacología , Lipoproteína Lipasa/metabolismo , Miocardio/enzimología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Corazón/efectos de los fármacos , Lipopolisacáridos/administración & dosificación , Lipopolisacáridos/toxicidad , Masculino , Perfusión , Ratas , Ratas EndogámicasRESUMEN
Evidence is presented that, as in cardiomyocytes, vascular endothelial cells use fatty acids, in addition to glucose, as a respiratory fuel. Attention is focused on the cardiac interstitium, lined by vascular cells and cardiomyocytes, which may be enriched with metabolic products from these cells. Also, certain proteins are present in the interstitial fluid (Qi) such as plasma proteins and fatty acid binding protein (FABP). However, the concentration of FABP is so low in Qi that albumin is more important to shuttle long chain fatty acids in the interstitial fluid between cardiomyocytes and the vascular compartment. Under hypoxic conditions (hypo)xanthine, lactate and fatty acids may be expected to accumulate in the interstitium, as well as proteins from adjacent cells, such as xanthine oxidase from endothelial cells. This enzyme, acting upon the elevated level of (hypo)xanthine, giving rise to O2-., may be involved in the damage of the ischaemic heart. The significance of the interstitium in ischaemia and in fibrosis following long standing cardiac lipidosis is briefly discussed, as well as the possible mechanisms involved in fatty acid transport in the heart.