RESUMEN
Ammonia is a bi-product of protein metabolism in the body. It is able to cross the blood-brain barrier and elevated ammonia levels are toxic to the brain. Rats with hyperammonemia showed impaired learning ability and impaired function of the glutamate-nitric oxide-cyclic guanosine monophosphate (glutamate-NO-cGMP) pathway in the brain. Chronic treatment with sildenafil restored learning ability. We therefore tested the hypothesis that sildenafil has a protective effect on the brains of hyperammonemic rats. Hyperammonemia was induced in male rats by daily intraperitoneal (i.p.) injection of ammonium chloride (100 mg/kg body weight) for 8 weeks. Sildenafil citrate was administered intraperitoneally (10 mg/kg body weight/3 days) for 8 weeks. Treatment with sildenafil resulted in a significant reduction in plasma liver enzymes, lipid profile as well as brain lipid peroxidation and caspase-3 mRNA. Meanwhile, plasma NO as well as cGMP, antioxidants and endothelial nitric oxide synthase (eNOS) gene expression were significantly elevated in the brains of hyperammonemic rats. Our results showed that sildenafil exerts a protective effect on the brain by reversing oxidative stress during hyperammonemia and this could be due to (i) cytoprotective, antioxidant and anti-apoptotic effects (ii) increasing cGMP and enhancing the proper metabolism of fats which could suppress oxygen radical generation and thus preventing oxidative damage in the brain. The exact protective mechanism of sildenafil has to be still investigated and further studies are warranted. Consequently, therapeutic modulation of the NO/cGMP pathway might have important clinical applications to improve brain functions in patients with hyperammonemia or clinical hepatic encephalopathy.
Asunto(s)
Cloruro de Amonio/toxicidad , Antioxidantes/metabolismo , Encéfalo/efectos de los fármacos , Hiperamonemia/prevención & control , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Piperazinas/uso terapéutico , Sulfonas/uso terapéutico , Actinas/genética , Actinas/metabolismo , Cloruro de Amonio/farmacocinética , Animales , Biomarcadores/sangre , Encéfalo/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Hiperamonemia/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Estructura Molecular , Óxido Nítrico/sangre , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Inhibidores de Fosfodiesterasa 5/administración & dosificación , Inhibidores de Fosfodiesterasa 5/química , Piperazinas/administración & dosificación , Piperazinas/química , Purinas/administración & dosificación , Purinas/química , Purinas/uso terapéutico , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Citrato de Sildenafil , Sulfonas/administración & dosificación , Sulfonas/químicaRESUMEN
BACKGROUND: N-chlorotaurine, a long-lived oxidant produced by human leukocytes, can be applied in human medicine as an endogenous antiseptic. Its antimicrobial activity can be enhanced by ammonium chloride. This study was designed to evaluate the tolerability of inhaled N-chlorotaurine (NCT) in the pig model. METHODS: Anesthetized pigs inhaled test solutions of 1% (55 mM) NCT (n = 7), 5% NCT (n = 6), or 1% NCT plus 1% ammonium chloride (NH4Cl) (n = 6), and 0.9% saline solution as a control (n = 7), respectively. Applications with 5 ml each were performed hourly within four hours. Lung function, haemodynamics, and pharmacokinetics were monitored. Bronchial lavage samples for captive bubble surfactometry and lung samples for histology and electron microscopy were removed. RESULTS: Arterial pressure of oxygen (PaO2) decreased significantly over the observation period of 4 hours in all animals. Compared to saline, 1% NCT + 1% NH4Cl led to significantly lower PaO2 values at the endpoint after 4 hours (62 +/- 9.6 mmHg vs. 76 +/- 9.2 mmHg, p = 0.014) with a corresponding increase in alveolo-arterial difference of oxygen partial pressure (AaDO2) (p = 0.004). Interestingly, AaDO2 was lowest with 1% NCT, even lower than with saline (p = 0.016). The increase of pulmonary artery pressure (PAP) over the observation period was smallest with 1% NCT without difference to controls (p = 0.91), and higher with 5% NCT (p = 0.02), and NCT + NH4Cl (p = 0.05).Histological and ultrastructural investigations revealed no differences between the test and control groups. The surfactant function remained intact. There was no systemic resorption of NCT detectable, and its local inactivation took place within 30 min. The concentration of NCT tolerated by A549 lung epithelial cells in vitro was similar to that known from other body cells (0.25-0.5 mM). CONCLUSION: The endogenous antiseptic NCT was well tolerated at a concentration of 1% upon inhalation in the pig model. Addition of ammonium chloride in high concentration provokes a statistically significant impact on blood oxygenation.
Asunto(s)
Antiinfecciosos Locales/administración & dosificación , Antiinfecciosos Locales/efectos adversos , Mucosa Respiratoria/fisiología , Taurina/análogos & derivados , Administración por Inhalación , Cloruro de Amonio/administración & dosificación , Cloruro de Amonio/efectos adversos , Cloruro de Amonio/farmacocinética , Animales , Antiinfecciosos Locales/farmacocinética , Presión Sanguínea/fisiología , Espiración/fisiología , Modelos Animales , Mecánica Respiratoria/fisiología , Mucosa Respiratoria/efectos de los fármacos , Porcinos , Taurina/administración & dosificación , Taurina/efectos adversos , Taurina/farmacocinética , Volumen de Ventilación Pulmonar/fisiologíaRESUMEN
UNLABELLED: The intensity of the response to acid-base parameters in relation to the age after a defined acid load was studied in calves. 32 clinically healthy calves (age: 4-104 days) were infused with 5M NH4Cl solution (dose: 1.0 ml/kg) through a permanent intravenous catheter. Before (0 hrs) and after starting the infusion (2, 4, 6, 8 and 24 hrs) venous blood samples were collected for the determination of the various acid-base parameters. The intensity of the response of the acid-base parameters was estimated by using the "area under curve (AUC)" procedure. By 2-6 hrs after the infusion of the NH4Cl solution, the Henderson-Hasselbalch parameters decreased significantly (decrease pH, decrease [HCO3-]) as did Stewart's variables (decrease [Strong ion difference=SID3], decrease [Acid total = A(tot) or A-]). A transient moderate hyperchloraemic acidosis with a slight hypoproteinaemic alkalosis was observed in all calves in association with a respiratory compensation (decrease PCO2). The younger calves (1st-3rd week) showed a similar pattern of response to the same dose per kg 0.75 acid load with significantly greater acid-base parameters response (higher AUC values) than the older animals. The calculated pH was determined by using the three Stewart variables PvCO2, serum-[SID3] and serum-[A(tot)]. The mean difference was -0.03 to -0.09 compared with the measured pH (7.32-7.40). CONCLUSION: The Stewart model appears to be more successful in providing a comprehensive evaluation of acid-base status compared with the traditional Henderson-Hasselbalch model. The younger calves during the first week of life reacted more sensitively to an equal acidotic condition than the older animals.
Asunto(s)
Equilibrio Ácido-Base/fisiología , Acidosis/veterinaria , Cloruro de Amonio/farmacocinética , Enfermedades de los Bovinos/metabolismo , Infusiones Parenterales/veterinaria , Desequilibrio Ácido-Base/diagnóstico , Desequilibrio Ácido-Base/metabolismo , Acidosis/inducido químicamente , Acidosis/metabolismo , Factores de Edad , Envejecimiento/metabolismo , Animales , Área Bajo la Curva , Bovinos , Enfermedades de los Bovinos/diagnóstico , Infusiones Parenterales/métodos , Distribución AleatoriaRESUMEN
Influenza virosomes are reconstituted influenza virus envelopes that may be used as vaccines or as carrier systems for cellular delivery of therapeutic molecules. Here we present a procedure to generate influenza virosomes as a stable dry-powder formulation by freeze-drying (lyophilization) using an amorphous inulin matrix as a stabilizer. In the presence of inulin the structural integrity and fusogenic activity of virosomes were fully preserved during freeze-drying. For example, the immunological properties of the virosomes, i.e. the HA potency in vitro and the immunogenic potential in vivo, were maintained during lyophilization in the presence of inulin. In addition, compared to virosomes dispersed in buffer, inulin-formulated virosomes showed substantially prolonged preservation of the HA potency upon storage. Also the capacity of virosomes to mediate cellular delivery of macromolecules was maintained during lyophilization in the presence of inulin and upon subsequent storage. Specifically, when dispersed in buffer, virosomes with encapsulated plasmid DNA lost their transfection activity completely within 6 weeks, whereas their transfection activity was fully preserved for at least 12 weeks after incorporation in an inulin matrix. Thus, in the presence of inulin as a stabilizing agent, the shelf-life of influenza virosomes with and without encapsulated macromolecules was considerably prolonged. Formulation of influenza virosomes as a dry-powder is advantageous for storage and transport and offers the possibility to develop needle-free dosage forms, e.g. for oral, nasal, pulmonal, or dermal delivery.
Asunto(s)
Virus de la Influenza A/química , Inulina/química , Vacunas de Virosoma/inmunología , Virosomas/química , Cloruro de Amonio/farmacocinética , Animales , Formación de Anticuerpos/inmunología , Línea Celular , Centrifugación por Gradiente de Densidad , Cricetinae , Estabilidad de Medicamentos , Almacenaje de Medicamentos/métodos , Membrana Eritrocítica/inmunología , Membrana Eritrocítica/metabolismo , Femenino , Liofilización/métodos , Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Pruebas de Inhibición de Hemaglutinación , Virus de la Influenza A/inmunología , Virus de la Influenza A/ultraestructura , Fusión de Membrana , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Transmisión , Plásmidos/química , Plásmidos/genética , Transfección , Vacunas de Virosoma/administración & dosificación , Vacunas de Virosoma/química , Virosomas/genética , Virosomas/inmunología , Internalización del Virus/efectos de los fármacosRESUMEN
An analysis of arginine-vasopressin (AVP) V1a receptor-deficient (V1aR-/-) mice revealed that glucose homeostasis and lipid metabolism were altered in the mutant mice. Here, we used V1aR-/- mice to investigate whether the deficiency of the V1a receptor, which led to altered insulin sensitivity, affected protein metabolism. The serum 3-methylhistidine levels were increased in V1aR-/- mice under feeding conditions, indicating that proteolysis was enhanced in muscle tissue from V1aR-/- mice. Furthermore, serum amino acid profiling revealed that the amino acid levels, including glycogenic and branched-chain amino acids, were reduced in V1aR-/- mice. In addition, an alanine-loading test showed that gluconeogenesis was enhanced in V1aR-/- mice. Blood ammonia, which is a by-product of amino acid catabolism, was two times higher in V1aR-/- mice without hepatopathy under the feeding and fasting conditions than in wild-type mice. Amino acid profiling also revealed that the amino acid pattern was not typical of a urea-cycle enzymatic disorder. An ammonia tolerance test and an indocyanine green elimination test showed that V1aR-/- mice had lower ammonia clearance due to a decreased intrahepatic circulating blood volume. Metabolic acidosis, including lactic- and keto-acidosis, was not observed in V1aR-/- mice. These results provide evidence that proteolysis promotes the production of glucose in the muscles of V1aR-/- mice and that hyperammonaemia is caused by promoted protein catabolism and reduced intrahepatic blood volume. Thus, our study with V1aR-/- mice indicates that AVP plays a physiological role via the V1a receptor in regulating both protein catabolism and glucose homeostasis.
Asunto(s)
Volumen Sanguíneo , Glucosa/metabolismo , Hiperamonemia/metabolismo , Circulación Hepática , Hígado/irrigación sanguínea , Músculo Esquelético/metabolismo , Péptido Hidrolasas/metabolismo , Proteínas/metabolismo , Receptores de Vasopresinas/metabolismo , Aminoácidos/sangre , Amoníaco/sangre , Amoníaco/metabolismo , Cloruro de Amonio/sangre , Cloruro de Amonio/farmacocinética , Animales , Arginina Vasopresina/metabolismo , Glucemia/metabolismo , Dióxido de Carbono/sangre , Gluconeogénesis , Homeostasis , Concentración de Iones de Hidrógeno , Hiperamonemia/sangre , Hiperamonemia/enzimología , Hiperamonemia/genética , Hiperamonemia/fisiopatología , Cuerpos Cetónicos/sangre , Ácido Láctico/sangre , Metilhistidinas/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Vasopresinas/deficiencia , Receptores de Vasopresinas/genética , Factores de TiempoRESUMEN
In a patient with N-acetylglutamate synthase (NAGS) deficiency, incorporation of an isotopic label from ammonium chloride into urea was markedly reduced before treatment with N-carbamyl-L-glutamate (NCLG) and completely normalized following treatment. Blood ammonia rose following ammonium tracer ingestion before treatment but remained low following treatment. Serum urea concentration doubled following the treatment.
Asunto(s)
Acetiltransferasas/deficiencia , Glutamatos/uso terapéutico , Urea/metabolismo , Acetiltransferasas/genética , Adolescente , Adulto , N-Acetiltransferasa de Aminoácidos , Amoníaco/sangre , Cloruro de Amonio/farmacocinética , Nitrógeno de la Urea Sanguínea , Femenino , Glutamina/sangre , Humanos , Isótopos de Nitrógeno/farmacocinéticaRESUMEN
OBJECTIVE: In previous studies of the renal response to acute NH4Cl acidosis no correlation was found between systemic acid-base status and the traditionally used quantity, renal net acid excretion (NAE). If NAE is to be considered a physiologically meaningful quantity then this is surprising, as the extracellular acid-base status would be expected to be the key physiological trigger for renal NAE. The object of this study was to investigate the renal response to acute non-carbonic acid loading using a quantitative organ physiological approach. MATERIAL AND METHODS: Five-h NH4Cl loading studies were performed in 10 healthy men using a randomized, placebo-controlled, crossover design. Arterialized capillary blood, serum and urine were collected hourly during the loading studies for the measurement of electrolytes and acid-base status. Concentrations of non-metabolizable base (NB) and acid (NA) were calculated from measured concentrations of non-metabolizable ions according to Kildeberg. RESULTS: In the steady state (placebo) the rate of renal excretion of NA (=-NB) was close to zero, indicating that the net extrarenal input of NA (endogeneous production, gastrointestinal absorption. skeletal release, etc.) was likewise about zero. An inverse correlation was found between blood pH and the rate of renal excretion of NA. Only a small amount of the acid load (approximately 8%) was excreted during the 5-h study period and this was accompanied by massive calciuria, indicating that mobilization of NB from bone contributed substantially to the current net extrarenal NA input. CONCLUSION: From a physiological point of view, NB can be regarded as the actual substrate for renal acid-base control, and measurement of renal turnover of NB may give a more precise description of renal acid-base metabolism during acid loading than previously described methods.
Asunto(s)
Desequilibrio Ácido-Base/prevención & control , Cloruro de Amonio/administración & dosificación , Cloruro de Amonio/farmacocinética , Equilibrio Ácido-Base/fisiología , Desequilibrio Ácido-Base/diagnóstico , Adaptación Fisiológica , Estudios Cruzados , Historia del Siglo XVI , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Función Renal , Masculino , Valores de Referencia , Medición de Riesgo , Sensibilidad y Especificidad , UrinálisisRESUMEN
Children with hypoglycemia due to recessive loss of function mutations of the beta-cell ATP-sensitive potassium (K(ATP)) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K(ATP) channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1-/-) mice. Even though SUR1-/- mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1-/- islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1-/- islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1-/- islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-l-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid- and glucose-stimulated insulin secretion, and that beta-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Glutamina/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transducción de Señal/fisiología , Cloruro de Amonio/farmacocinética , Animales , Calcio/metabolismo , Glutamina/metabolismo , Gliburida/farmacología , Hipoglucemiantes/farmacología , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Ratones , Ratones Noqueados , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Isótopos de Nitrógeno , Canales de Potasio de Rectificación Interna , Receptores de Droga , Transducción de Señal/efectos de los fármacos , Receptores de SulfonilureasRESUMEN
This study was undertaken to determine whether gulf toadfish (Opsanus beta) could metabolize ammonia from their environment into other, less toxic products. To this end, gulf toadfish were exposed to 3.8 mM 15NH(4)Cl in seawater for 24 and 48 h. Liver, kidney, gill, brain and muscle samples were analyzed for distribution of 15N within the tissue and among various nitrogen-containing metabolites (ammonia, amino-N, glutamine-N, urea and protein). The data reported here show that the toadfish can indeed take up and metabolize ammonia. Analysis of individual metabolic products of ammonia indicates that the toadfish can convert this toxic chemical into other less toxic metabolites. Ammonia enrichment is significantly different over controls in the kidney, brain and muscle. Urea enrichment is most significant in the brain, with less significant enrichment occurring in the liver and muscle. While accumulation of ammonia into an amino acid pool was not a significant metabolic fate, protein synthesis was significantly enriched in all tissues (with the highest levels occurring in the gill) indicating that amino acid synthesis may be a pathway of ammonia detoxification en route to protein synthesis, and that environmental ammonia can be 'fixed' into protein. Finally, it was found that glutamine-N synthesis occurs at significant levels in the liver, brain and muscle.
Asunto(s)
Cloruro de Amonio/farmacocinética , Batrachoidiformes/metabolismo , Amoníaco/metabolismo , Animales , Compuestos de Nitrógeno/análisis , Isótopos de Nitrógeno , Distribución TisularRESUMEN
BACKGROUND: Low urinary pH is the commonest and by far the most important factor in uric acid nephrolithiasis but the reason(s) for this defect is (are) unknown. Patients with uric acid nephrolithaisis have normal acid-base parameters according conventional clinical tests. METHODS: We studied steady-state plasma and urinary parameters of acid-base balance in subjects with normouricosuric pure uric acid stones. We also tested the ability of these subjects to excrete ammonium in response to an acute acid load. We compared these parameters in patients with pure uric acid stones to patients with mixed uric acid/calcium oxalate stones, pure calcium stones, and normal volunteers. RESULTS: Pure uric acid stone formers have a much higher incidence of either diabetes or glucose intolerance. After equilibration to a control diet, patients with uric acid stones have lower urinary pH and they excrete less of their acid as ammonium. This is compensated by higher titratable acidity and hypocitraturia. Despite their low baseline urinary pH, uric acid stone formers further acidify their urine after an acid load because of a severely impaired ammonia excretory response. Their characteristics are significantly different from normal volunteers and pure calcium stone formers. Patients with mixed uric acid/calcium stones exhibit intermediate characteristics. CONCLUSION: We propose that certain patients with normouricosuric uric acid nephrolithiasis have a renal acidification disease. The primary defect lies in renal ammonium excretion, which may be linked to the insulin-resistant state. Although net acid excretion is maintained at the expense of increased titratable acidity and to some degree hypocitraturia, the compromise is acid urine pH and may result in uric acid nephrolithiasis.
Asunto(s)
Cálculos Renales/fisiopatología , Cálculos Renales/orina , Ácido Úrico/orina , Adulto , Anciano , Cloruro de Amonio/farmacocinética , Oxalato de Calcio/orina , Diuréticos/farmacocinética , Femenino , Humanos , Concentración de Iones de Hidrógeno , Cálculos Renales/etiología , Masculino , Persona de Mediana Edad , Triglicéridos/sangreRESUMEN
Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.
Asunto(s)
Líquido Intracelular/metabolismo , Riñón/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Cloruro de Amonio/metabolismo , Cloruro de Amonio/farmacocinética , Línea Celular , Cisteína Endopeptidasas , Inhibidores Enzimáticos/farmacología , Humanos , Cuerpos de Inclusión/metabolismo , Riñón/citología , Riñón/efectos de los fármacos , Lisosomas/metabolismo , Sustancias Macromoleculares , Complejos Multienzimáticos/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Enfermedades Neurodegenerativas/metabolismo , Nitratos/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico/farmacología , Oxidantes/farmacología , Complejo de la Endopetidasa Proteasomal , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Superóxidos/metabolismo , Superóxidos/farmacología , Sinucleínas , Transfección , Ubiquitinas/metabolismo , alfa-Sinucleína , Sinucleína betaRESUMEN
Hypocitraturia (HCit) is one of the most remarkable features of renal tubular acidosis, but an acidification defect is not seen in the majority of hypocitraturic patients, whose disease is denoted idiopathic hypocitraturia. In order to assess the integrity of urinary acidification mechanisms in hypocitraturic idiopathic calcium stone formers, we studied two groups of patients, hypocitraturic (HCit, N = 21, 39.5 + or - 11.5 years, 11 females and 10 males) and normocitraturic (NCit, N = 23, 40.2 + or - 11.7 years, 16 females and 7 males) subjects, during a short ammonium chloride loading test lasting 8 h. During the baseline period HCit patients showed significantly higher levels of titratable acid (TA). After the administration of ammonium chloride, mean urinary pH (3rd to 8th hour) and TA and ammonium excretion did not differ significantly between groups. Conversely, during the first hour mean urinary pH was lower and TA and ammonium excretion was higher in HCit. The enhanced TA excretion by HCit during the baseline period and during the first hour suggests that the phosphate buffer mechanism is activated. The earlier response in ammonium excretion by HCit further supports other evidence that acidification mechanisms react promptly. The present results suggest that in the course of lithiasic disease, hypocitraturia coexists with subtle changes in the excretion of hydrogen ions in basal situations
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Acidosis Tubular Renal/metabolismo , Ácido Cítrico/orina , Cálculos Urinarios/orina , Acidosis Tubular Renal/etiología , Acidosis Tubular Renal/fisiopatología , Cloruro de Amonio/farmacocinética , Cloruro de Amonio/orina , Concentración de Iones de Hidrógeno , Litiasis/complicaciones , Organofosfatos/orina , Factores de TiempoRESUMEN
BACKGROUND: The importance of urea nitrogen reutilization in the amino acid economy of the host remains to be clarified. OBJECTIVE: The objective was to explore the transfer of (15)N from orally administered [(15)N(2)]urea or (15)NH(4)Cl to plasma free and intestinal microbial amino acids. DESIGN: Six men received an L-amino acid diet (167 mg N*kg(-)(1)*d(-)(1); 186 kJ*kg(-)(1)*d(-)(1)) for 11 d each on 2 different occasions. For the last 6 d they ingested [(15)N(2)]urea or, in random order, (15)NH(4)Cl (3.45 mg (15)N*kg(-)(1)*d(-)(1)). On day 10, a 24-h tracer protocol (12 h fasted/12 h fed) was conducted with subjects receiving the (15)N tracer hourly. In a similar experiment, (15)NH(4)Cl (3.9 mg (15)N*kg(-)(1)*d(-)(1)) was given to 7 ileostomates. (15)N Enrichments of urinary urea and plasma free and fecal or ileal microbial protein amino acids were analyzed. RESULTS: (15)N Retention was significantly higher with (15)NH(4)Cl (47.7%; P < 0.01) than with [(15)N(2)]urea (29.6%). Plasma dispensable amino acids after the (15)NH(4)Cl tracer were enriched up to 20 times (0. 2-0.6 (15)N atom% excess) that achieved with [(15)N(2)]urea. The (15)N-labeling pattern of plasma, ileal, and fecal microbial amino acids (0.05-0.45 (15)N atom% excess) was similar. Appearance of microbial threonine in plasma was similar for normal subjects (0.14) and ileostomates (0.17). CONCLUSION: The fate of (15)N from urea and NH(4)Cl differs in terms of endogenous amino acid metabolism, but is similar in relation to microbial protein metabolism. Microbial threonine of normal and ileostomy subjects appears in the blood plasma but the net contribution to the body threonine economy cannot be estimated reliably from the present data.
Asunto(s)
Aminoácidos/metabolismo , Cloruro de Amonio/farmacocinética , Proteínas Bacterianas/metabolismo , Íleon/metabolismo , Nitrógeno/metabolismo , Urea/farmacocinética , Adulto , Aminoácidos/biosíntesis , Dieta , Heces/química , Heces/microbiología , Femenino , Humanos , Ileostomía , Masculino , Persona de Mediana EdadRESUMEN
We designed this study to determine whether the capacity of the liver to uptake ammonia and produce urea was affected by exercise (swimming at 24 degrees C with a 2.5% extra body-weight load). For this purpose, livers from sedentary rats at rest were perfused with a buffer containing increasing concentration of NH4Cl. The maximal hepatic capacity to produce urea was found at an NH4Cl concentration of 0.25 mM. Based on this finding all experiments with livers obtained from rats subject to swimming exercise were also carried out with a NH4Cl concentration of 0.25 mM. Thus, employing this concentration of ammonia, livers from sedentary and endurance trained rats, (for a period of 11 days ), that had either been resting or had been subjected to swimming exercise for 5 min or until exhaustion, were perfused in situ and ammonia uptake and urea production were measured. Clearly, both parameters were increased by exercise. However, these changes were not affected by swimming training. In addition, we demonstrate that the effect of an acute exercise on hepatic metabolism is not restricted to ammonia metabolism since livers from sedentary rats which had been subjected to swimming exercise for 5 min or until exhaustion showed higher urea production from L-glutamine. Our results also suggest that part of the changes in ureogenesis induced by exercise is mediated by cortisol (increased ammonia uptake) and part of the changes is mediated by glucagon (urea production).
Asunto(s)
Amoníaco/metabolismo , Glutamina/metabolismo , Hígado/metabolismo , Natación/fisiología , Urea/metabolismo , Amoníaco/farmacocinética , Cloruro de Amonio/administración & dosificación , Cloruro de Amonio/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Masculino , Perfusión , Ratas , Ratas WistarRESUMEN
In the rat terminal inner medullary collecting duct (tIMCD), Na+ pump inhibition reduces transepithelial net acid secretion (JtAMM) [JH = total CO2 absorption (JtCO2)+ total ammonia secretion] and increases resting intracellular pH (pHi). The increase in pHi and reduction in JH that follow ouabain addition do not occur in the absence of NH4+ nor when NH4+ is substituted with another weak base. The purpose of this study was to explore the mechanism of the NH4(+)-dependent reduction in JtCO2 and increase in pHi that follow ouabain addition. We hypothesized that NH4+ enters the tIMCD cell through the Na(+)-K(+)-ATPase with proton release in the cytosol. To test this hypothesis, tIMCDs were dissected from deoxycorticosterone-treated rats and perfused in vitro with symmetrical physiological saline solutions containing 6 mM NH4Cl. Since K+ and NH4+ compete for a common binding site on the Na+ pump, increasing extracellular K+ should limit NH4+ (and hence net H+) uptake by the Na+ pump. Upon increasing extracellular K+ concentration from 3 to 12 mM, the NH4(+)-dependent, ouabain-induced increase in pHi and reduction in JtCO2 were attenuated. In the presence but not in the absence of NH4+, reducing Na+ pump activity by limiting Na+ entry reduced JtCO2 and attenuated ouabain-induced alkalinization. Ouabain-induced alkalinization was not dependent on the presence of HCO3-/CO2 and was not reproduced with BaCl2 or bumetanide addition. Therefore, ouabain-induced alkalinization is not mediated by the Na(+)-K(+)-2Cl- cotransporter or a HCO3- transporter and is not mediated by changes in membrane potential. In conclusion, on the basolateral membrane of the tIMCD cell, NH4+ uptake is mediated by the Na(+)-K(+)-ATPase. These data provide an explanation for the reduction in net acid secretion in the tIMCD observed following administration of amiloride or with dietary K+ loading.
Asunto(s)
Cloruro de Amonio/farmacocinética , Concentración de Iones de Hidrógeno , Médula Renal/fisiología , Túbulos Renales Colectores/fisiología , Ouabaína/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Amilorida/farmacología , Animales , Bario/farmacología , Bumetanida/farmacología , Dióxido de Carbono/metabolismo , Epitelio/efectos de los fármacos , Epitelio/fisiología , Técnicas In Vitro , Médula Renal/efectos de los fármacos , Médula Renal/enzimología , Túbulos Renales Colectores/efectos de los fármacos , Túbulos Renales Colectores/enzimología , Masculino , Modelos Biológicos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Ratas , Ratas Sprague-Dawley , Sodio/metabolismoRESUMEN
An alternative method for the determination of [15N]ammonia enrichment in biological fluids was developed. It is based on the use of glutamate dehydrogenase of bovine liver (EC 1.4.1.2.) with 2-oxopentanoic acid as substrate, to convert the ammonia present in the sample into norvaline, the enrichment of which can be measured by gas chromatography/mass spectrometry as its tertiary butyldimethylsilyl (TBDMS) derivative under electron impact selective ion recording (SIR) conditions. The principal advantage of the present approach is that it is simpler and quicker than the previously described methods, because the synthetic product, norvaline, is not present in biological fluids and pre-processing of the sample is unnecessary. The procedure includes a pre-incubation stage which allows removal of contaminant ammonia present in the reagents used for the enzyme reaction. The contributions of other sources of nitrogen to norvaline production have been checked and quantified: these may provide limitations of the technique when samples for analysis are low in ammonia (e.g. arterial or hepatic venous blood). To reduce these contributions, short times of incubation are proposed. The results from two experiments in vivo in which two sheep were infused with [15N]ammonium chloride in the mesenteric vein are presented and the biological implications which arise from the results are discussed. The validity of the procedures was demonstrated by the quantitative recovery from the mesenteric and portal veins of [15N]ammonia infused.
Asunto(s)
Amoníaco/análisis , Amoníaco/sangre , Cloruro de Amonio/sangre , Cloruro de Amonio/farmacocinética , Animales , Bovinos , Cromatografía de Gases y Espectrometría de Masas , Glutamato Deshidrogenasa/metabolismo , Indicadores y Reactivos , Hígado/química , Hígado/enzimología , Ovinos , Valina/análogos & derivados , Valina/análisis , Valina/sangreRESUMEN
The effects of either low (25 mumol/min) or high (235 mumol/min) infusion of NH4Cl into the mesenteric vein for 5 d were determined on O2 consumption plus urea and amino acid transfers across the portal-drained viscera (PDV) and liver of young sheep. Kinetic transfers were followed by use of 15NH4Cl for 10 h on the fifth day with simultaneous infusion of [1-13C]leucine to monitor amino acid oxidation. Neither PDV nor liver blood flow were affected by the additional NH3 loading, although at the higher rate there was a trend for increased liver O2 consumption. NH3-N extraction by the liver accounted for 64-70% of urea-N synthesis and at the lower infusion rate the additional N required could be more than accounted for by hepatic removal of free amino acids. At the higher rate of NH3 administration additional sources of N were apparently required to account fully for urea synthesis. Protein synthesis rates in the PDV and liver were unaffected by NH3 infusion but both whole-body (P < 0.05) and splanchnic tissue leucine oxidation were elevated at the higher rate of administration. Substantial synthesis of [15N]glutamine occurred across the liver, particularly with the greater NH3 supply, and enrichments exceeded considerably those of glutamate. The [15N]urea synthesized was predominantly as the single labelled, i.e. [14N15N], species. These various kinetic data are compatible with the action of ovine hepatic glutamate dehydrogenase (EC 1.4.1.2) in periportal hepatocytes in the direction favouring glutamate deamination. Glutamate synthesis and uptake is probably confined to the perivenous cells which do not synthesize urea.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Aminoácidos/metabolismo , Cloruro de Amonio/farmacocinética , Hígado/metabolismo , Ovinos/metabolismo , Animales , Glutamina/biosíntesis , Inactivación Metabólica , Leucina/farmacología , Masculino , Nitrógeno/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Urea/metabolismoRESUMEN
Ruminants characteristically absorb a large proportion of dietary nitrogen across the portal-drained viscera as ammonia nitrogen which is detoxified by conversion to urea in the liver. In theory, ammonia can supply both nitrogen atoms of the urea molecule via mitochondrial (carbamoyl phosphate) and cytoplasmic (aspartate) precursor pathways of the ornithine cycle but the effect of amino acids on the flux of nitrogen from ammonia to each of the two urea nitrogen atoms has not been determined. We report a study designed to determine the distribution of [15N] ammonia between [15N1]urea and [15N2]urea in sheep hepatocytes in response to ammonia concentrations (0.33, 0.67 and 1.00 mM) in the presence or absence of amino acids. In the absence of amino acids, the enrichment of [15N2]urea rose more rapidly during incubations than [15N1]urea and attained enrichments of 66-88% within 5 min of incubation. At the end of 2.5 h of incubation, [15N2]urea represented 60% and 90% of the total urea molecules at low and high ammonia concentrations, respectively. The enrichments of glutamate and aspartate were similar to [15N1]urea in the cells at the end of the incubations, even in the presence of unlabelled amino acids, supporting the concept of mitochondrial ammonia being in equilibrium with cytosolic aspartate formation. In the presence of amino acids basal urea synthesis increased but ammonia uptake and 15NH4Cl conversion to urea was less than in the absence of amino acids. The rate of formation of [15N1]urea was greater in incubations containing amino acids but when ammonia concentration in the media was raised only [15N2]urea flux increased with no change in either [15N1]urea or the unlabelled species. Measurement of media amino acid concentrations after 2.5 h of incubation in the presence of amino acids revealed that arginine, glutamine, glycine and alanine were removed while there was net formation of aspartate, threonine, serine, glutamate, and the branched chain amino acids. However, less than 12% of the 15N transfer appeared in free amino acids. The increases in basal and unlabelled urea synthesis in the presence of amino acids could be numerically accounted as the sum of arginine and glutamine removal from incubations. It is concluded that in sheep hepatocytes 15NH4Cl removal leads to quantitative formation of [15N2]urea, even in the presence of a physiological mixture of amino acids. The increase in the formation of the [15N1]urea in the presence of amino acids can be explained by the preferential utilisation of the amide nitrogen of glutamine for urea synthesis.
Asunto(s)
Aminoácidos/fisiología , Cloruro de Amonio/farmacocinética , Hígado/metabolismo , Animales , Células Cultivadas , Hígado/citología , Masculino , Isótopos de Nitrógeno , Ovinos , Urea/metabolismoRESUMEN
Recent studies have suggested that defective medullary trapping of ammonia underlies the acidosis associated with renal failure and sets in motion maladaptive compensatory mechanisms that contribute to the progression of renal disease. Since a renal concentrating defect is an early functional abnormality in autosomal dominant polycystic kidney disease (ADPKD), defective medullary trapping and urinary excretion of ammonia may also occur early and have important pathophysiological consequences. The urinary pH and excretions of ammonia, titratable acid, and bicarbonate, were measured during a 24-hour baseline period and following the administration of ammonium chloride (100 mg/kg body wt) in ADPKD patients with normal glomerular filtration rate and in age- and gender-matched healthy control subjects. The distal nephron hydrogen ion secretory capacity was assessed during a bicarbonate infusion. Ammonia, sodium, pH, C3dg, and C5b-9 were measured in cyst fluid samples. The excretion rates of ammonia during the 24-hour baseline period and following the administration of ammonium chloride were significantly lower, and the relationship of ammonia excretion to urinary pH was significantly shifted downward in ADPKD. No difference in the increment of urinary pCO2 (delta pCO2) or the peripheral blood-urine pCO2 gradient (U-B pCO2) between ADPKD patients and control subjects was detected during a sodium bicarbonate infusion. Calculated concentrations of free-base ammonia in cyst fluid samples exceeded those calculated from reported concentrations of ammonia in renal venous blood of normal subjects. C3dg and C5b-9 were detected in some cyst fluids. The urinary excretion of ammonia is reduced in ADPKD patients with normal glomerular filtration rate.(ABSTRACT TRUNCATED AT 250 WORDS)