RESUMEN
Ursodeoxycholic acid (UDCA) is currently used for the treatment of cholestatic liver disease and for cholesterol gallstone dissolution. Various formulations have been designed to enhance its intestinal absorption or to improve patient compliance through once-a-day administration. The pharmacokinetics and bioavailability of four commercially available modified-release UDCA formulations (450 mg) were studied in 12 healthy subjects randomly receiving the four drugs under study. Serum samples were collected hourly for a 12-h period after administration and UDCA concentrations were measured using a specific enzyme immunoassay. For each formulation, Cmax, tmax, and the area under the curve (AUC) were determined and compared. Although all formulations were designed to provide sustained release, we observed different pharmacokinetics among the studied formulations. One of the formulations (sustained-release ursodeoxycholic acid Ratiopharm 450 mg tablets) showed high bioavailability, but failed to produce sustained release. In contrast, two other formulations (modified-release ursodeoxycholic acid Dorom 450 mg capsules and controlled-release Ursobil HT 450 mg capsules) provided sustained release, but did not offer efficient bioavailability. A fourth formulation (Ursilon retard 450 mg) exhibited gradual UDCA release lasting over 10 h, with efficient bioavailability, similar to that of conventional prompt-release formulations administered at the same dose. These data highlight the variability of commercially available sustained-release formulations. Manufacturers should be encouraged to provide drug kinetics and bioavailability data to further support the claimed pharmacokinetics.
Asunto(s)
Ácido Ursodesoxicólico/administración & dosificación , Ácido Ursodesoxicólico/farmacocinética , Adulto , Análisis de Varianza , Disponibilidad Biológica , Química Farmacéutica , Estudios Cruzados , Preparaciones de Acción Retardada/administración & dosificación , Preparaciones de Acción Retardada/farmacocinética , Esquema de Medicación , Femenino , Humanos , Masculino , Ácido Ursodesoxicólico/sangreRESUMEN
A new sustained-release formulation (sustained release Ibifen) that gradually releases ketoprofen within 24 h and ensures therapeutic plasma concentration for the entire period has been developed. It consists of tableted pH-dependent barrier film-coated ketoprofen granules and was administered at a single dose of 200 mg to 12 volunteers. Ketoprofen plasma profiles were compared with: (1) administration of Orudis retard 200 capsule (200 mg); (2) two 12-h doses of prompt release Ibifen capsules (100 mg). In vitro dissolution kinetics and ketoprofen plasma levels were measured by HPLC. Sustained release Ibifen dissolution rate was constant for 10 h, whereas Orudis retard 200 dissolution profile presented one higher slope (0-6 h) and a lower one (6-12 h). Both formulations showed a delayed kinetics with respect to prompt release Ibifen. After sustained release Ibifen administration, ketoprofen plasma peak, reached within 2 h, remained practically constant for at least 12 h (average 4 microg/ml), which is higher than therapeutic levels. Differently, Orudis retard 200 produced a delayed, higher C(max) (5.91+/-0.66 vs. 4.51+/-0.65 microg/ml; P<0.01) and disappeared more quickly. In conclusion, sustained release Ibifen can ensure therapeutic ketoprofen plasma levels for the entire 24 h period, avoiding plasma concentration spikes, with bioavailability similar to other ketoprofen preparations.
Asunto(s)
Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/farmacocinética , Cetoprofeno/administración & dosificación , Cetoprofeno/farmacocinética , Adulto , Área Bajo la Curva , Disponibilidad Biológica , Cromatografía Líquida de Alta Presión , Preparaciones de Acción Retardada , Humanos , Masculino , SolubilidadRESUMEN
A chemiluminescent enzyme-immunoassay for urinary 1-hydroxypyrene has been developed and optimized. The enzymatic activity of horseradish peroxidase-labeled tracer was measured with an enhanced chemiluminescent system and the results were compared with those from conventional colorimetric detection. The method fulfilled all the requirements of accuracy and precision and the detection limit was 0.001 pmol/well, which enabled analysis in less than 1 microL urine. Subjects working in the center of Bologna who were exposed daily to vehicular exhaust gas were studied. Their urinary 1-hydroxypyrene concentrations were compared with the levels of benzo( a)pyrene in air particulate matter. Urinary 1-hydroxypyrene, which ranged from 0.5 to 10 nmol L(-1), correlated poorly with the concentration of benzo( a)pyrene in air particulate matter, which ranged from 5 to 140 ng m(-3). No significant effect of vehicle exhaust gas exposure was observed among the different groups of subjects working in different areas of the town. Thus, at a relatively low level of exposure 1-hydroxypyrene does not seem to be a sensitive biomarker of exposure to polycyclic aromatic hydrocarbons.
Asunto(s)
Técnicas para Inmunoenzimas/métodos , Mutágenos/análisis , Pirenos/análisis , Urinálisis/métodos , Adulto , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Femenino , Peroxidasa de Rábano Silvestre/metabolismo , Humanos , Italia , Mediciones Luminiscentes , Masculino , Persona de Mediana Edad , Mutágenos/química , Hidrocarburos Policíclicos Aromáticos , Pirenos/química , Sensibilidad y Especificidad , Emisiones de VehículosRESUMEN
Many recent bioanalytical systems based on immunologic and hybridization reactions in a mono- or bidimensional microarray format require technology that can produce arrays of spots containing biospecific molecules. Some microarray deposition instruments are commercially available, and other devices have been described in recent papers. We describe a system obtained by adapting a commercial ink-jet printer and used to produce mono- and bidimensional arrays of spots containing horseradish peroxidase on cellulose paper. In a few minutes, it was possible to obtain bidimensional arrays containing several thousands of spots with a diameter as low as 0.2 mm, with each of which requiring only a few nanoliters of the enzyme deposition solution. The quantity of enzyme in each spot was evaluated with a chemiluminescent reaction and a charge-coupled device-based, low-light imaging luminograph. The chemiluminescence measurements revealed that the reproducibility of the enzyme deposition was satisfactory for analytical purposes, with the variation coefficients being lower than 10% in almost all cases.
Asunto(s)
Proteínas/análisis , Peroxidasa de Rábano Silvestre/metabolismo , Mediciones LuminiscentesRESUMEN
Analytical chemiluminescence and bioluminescence represent a versatile, ultrasensitive tool with a wide range of applications in diverse fields such as biotechnology, pharmacology, molecular biology, clinical and environmental chemistry. Enzyme activities and enzyme substrates and inhibitors can be efficiently determined when directly involved in luminescent reactions, and also when they take part in a reaction suitable for coupling to a final light-emitting reaction. Chemiluminescence detection has been exploited in the fields of flow-injection analysis and column-liquid chromatographic and capillary-electrophoretic separative systems, due to its high sensitivity when compared with colorimetric detection. It has widely been used as an indicator of reactive oxygen species formation in cells and whole organs, thus allowing the study of a number of pathophysiological conditions related to oxidative stress. Chemiluminescence represents a sensitive and rapid alternative to radioactivity as a detection principle in immunoassays for the determination of a wide range of molecules (hormones, food additives, environmental pollutants) and in filter membrane biospecific reactions (Southern, Northern, Western, dot blot) for the determination of nucleic acids and proteins. Chemiluminescence has also been used for the sensitive and specific localization and quantitation of target analytes in tissue sections and single cells by immunohistochemistry and in situ hybridization techniques. A relatively recent application regards the use of luminescent reporter genes for the development of bioassays based on genetically engineered microorganisms or mammalian cells able to emit visible light in response to specific inorganic and organic compounds. Finally, the high detectability and rapidity of bio- and chemiluminescent detection make it suitable for the development of microarray-based high throughput screening assays, in which simultaneous, multianalyte detection is performed on multiple samples.
Asunto(s)
Técnicas de Química Analítica/instrumentación , Técnicas de Química Analítica/métodos , Mediciones Luminiscentes , Oxidación-Reducción , Animales , Enzimas/química , Enzimas/metabolismo , Genes Reporteros , Humanos , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The analysis of bile salts in biological samples has remained a difficult task, due to the complex nature of the salts and also to their low concentration in common sample fluids such as plasma and urine. Given their importance, the development of accurate and sensitive methods of instrumental analysis has been the subject of intensive research, and recent advances have eliminated or lessened some of the difficulties. Currently available techniques are the following: thin-layer chromatography, gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, gas chromatography-mass spectrometry and capillary electrophoresis. Liquid chromatography coupled with mass spectrometry (thermospray, fast atom bombardment, electrospray and ionspray), a method undergoing continuous improvement, is also being applied to bile salts analysis. In this paper, these various techniques, which differ greatly in specificity, accuracy and simplicity, are reviewed and discussed, in terms of analytical performance, applicability to a given sample fluid, major limitations, ability to identify uncommon bile salts, including unsaturated oxo derivatives, glucuronides, sulfates, glycosides and bile alcohols.
Asunto(s)
Ácidos y Sales Biliares/aislamiento & purificación , Cromatografía de Gases/métodos , Cromatografía Líquida de Alta Presión/métodos , Cromatografía en Capa Delgada/métodos , Electroforesis Capilar/métodos , Ácidos y Sales Biliares/análisis , HumanosRESUMEN
A chemiluminescent urease activity assay has been developed and optimized using the chemiluminescent pH indicator phthalhydrazidylazoacetylacetone. This compound is stable at pH = 7 and decomposes at higher pH values, emitting light in the presence of H2O2. Urease catalyzes hydrolysis of urea to form NH3 and CO2 which increase the pH of the reaction medium, thus allowing the chemiluminescent indicator to decompose and produce photons. The emitted light is proportional to the urease activity when urea is in excess. Urease tests based on colorimetric pH indicators like phenol red are commercially available and commonly used for the rapid diagnosis of Helicobacter pylori infection in gastric mucosa biopsy specimens, since this bacterium produces high amounts of urease. Such colorimetric tests often lack sensitivity, giving false-negative results. The developed chemiluminescent test proved to be at least 50-fold more sensitive than the colorimetric tests, permitting early diagnosis of infection, and it is more rapid, giving results in 1-10 min compared to 30 min. Further applications of this assay could be the in situ localization of urease activity, corresponding to the presence of H. pylori, in gastric mucosa cryosections and the development of high-throughput screening assays of antimicrobial drugs able to inactivate the bacterium.
Asunto(s)
Mucosa Gástrica , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori , Hidrazinas , Indicadores y Reactivos , Ftalazinas , Ureasa/metabolismo , Adulto , Anciano , Biopsia , Colorimetría , Estudios de Evaluación como Asunto , Femenino , Infecciones por Helicobacter/patología , Humanos , Hidrazinas/química , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/química , Mediciones Luminiscentes , Masculino , Persona de Mediana Edad , Estructura Molecular , Ftalazinas/química , Sensibilidad y EspecificidadRESUMEN
A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as visualization of the spatial distribution of biomolecules such as antigens, enzymes, antibodies, DNA probes in tissue, or cells. The system consists of a low-light imaging Vidicon videocamera connected to an optical microscope, able to measure light at the single photon level and perform 3D image analysis of the subcellular distribution of the analyte. The concentration and the spatial distribution of enzymes, or enzyme-labeled biospecific reagents can be determined using appropriate chemiluminescent substrates. Analytes are also determined with coupled enzymatic reactions terminating in light emission. Oxirane acrylic beads (250-micron-diameter macroporous particles) with immobilized horseradish peroxidase have been used as a model system to optimize the experimental conditions in terms of signal intensity and spatial resolution as a function of different chemiluminescent substrates such as luminol/enhancer/H2O2 and acridancarboxylate ester/H2O2. Localization of oxirane beads immobilized acetylcholinesterase has been also used to optimize a system in which the detection and localization of the primary enzyme involves two secondary enzymes in solution, choline oxidase and horseradish peroxidase, leading to a final light emission. Immunoenzymatic reactions for the detection of viral antigens and in situ hybridization assays for the detection of viral DNAs (cytomegalovirus, herpes simplex virus) have been performed in cells using peroxidase-labeled antibodies or cDNA probes and the analytical performance of different chemiluminescent substrates for the enzyme has been evaluated. The results obtained showed the possibility to sharply image the bioprobes in single cells and peroxidase is a suitable label when luminol/H2O2 system is used in conjunction with enhancer as in the ECL and SuperSignal Ultra reagents; other substrates such as Lumigen PS-3, despite adequate detectability, showed problems of localization of the signal as a result of the relatively long half-life of the excited emitting species and its diffusion in the chemiluminescent cocktail. The system has proven to be highly sensitive, able to perform quantitative analysis, and relatively simple.
Asunto(s)
Acetilcolinesterasa/química , Oxidorreductasas de Alcohol/química , Sondas de ADN , Enzimas Inmovilizadas/química , Peroxidasa de Rábano Silvestre/química , Microscopía/métodos , Línea Celular , ADN Viral/análisis , Humanos , Inmunohistoquímica , Hibridación in Situ , Cinética , Mediciones LuminiscentesRESUMEN
The prevention of the hepatotoxic effects produced by intravenous infusion of taurochenodeoxycholic acid (TCDCA) by coinfusion with taurohyodeoxycholic acid (THDCA) was evaluated in bile fistula rats; the hepatoprotective effects of the latter were also compared with those of tauroursodeoxycholic acid (TUDCA). Rats infused with TCDCA at a dose of 8 micromol/min/kg showed reduced bile flow and calcium secretion, as well as increased biliary release of alkaline phosphatase (AP) and lactate dehydrogenase (LDH). This was associated with a very low biliary secretion rate of TCDCA (approximately 1 micromol/min/kg). Simultaneous infusion of THDCA or TUDCA at the same dose preserved bile flow and almost totally abolished the pathological leakage of the two enzymes into bile. The effect was slightly more potent for THDCA. The maximum secretion rate of TCDCA increased to the highest value (8 micromol/min/kg) when coinfused with either of the two hepatoprotective bile acids (BA), which were efficiently and completely secreted in the bile, without metabolism. Calcium output was also restored and phospholipid (PL) secretion increased with respect to the control saline infusion. This increase was higher in the THDCA study. These data show that THDCA is highly effective in the prevention of hepatotoxicity induced by intravenous infusion of TCDCA by facilitating its biliary secretion and reducing its hepatic residence time; this was associated with selective stimulation of PL biliary secretion.
Asunto(s)
Colagogos y Coleréticos/farmacología , Colestasis/prevención & control , Ácido Tauroquenodesoxicólico/antagonistas & inhibidores , Ácido Taurodesoxicólico/análogos & derivados , Fosfatasa Alcalina/análisis , Animales , Calcio/análisis , Colagogos y Coleréticos/administración & dosificación , Colagogos y Coleréticos/análisis , Colagogos y Coleréticos/química , Colestasis/inducido químicamente , Inyecciones Intravenosas , L-Lactato Deshidrogenasa/análisis , Hígado/efectos de los fármacos , Hígado/metabolismo , Fosfolípidos/análisis , Ratas , Ácido Tauroquenodesoxicólico/análisis , Ácido Tauroquenodesoxicólico/química , Ácido Tauroquenodesoxicólico/farmacología , Ácido Taurocólico/química , Ácido Taurodesoxicólico/administración & dosificación , Ácido Taurodesoxicólico/análisis , Ácido Taurodesoxicólico/química , Ácido Taurodesoxicólico/farmacologíaRESUMEN
The development, analytical performance and applications of chemiluminescence imaging as a tool for quantitative analyte localization in target biological specimens are described. The detection of acetylcholinesterase activity both in array format and on a target surface are described. A proposed application of the method is a 384 well microtiter format assay for high throughput screening of acetylcholinesterase inhibitors such as tacrine, a drug widely used in the treatment of Alzheimer's disease, and two recently developed analogues. The chemiluminescent system in conjunction with optical microscopy allowed localization of acetylcholinesterase in brain tissue sections. We also describe the chemiluminescent immunohistochemical localization of interleukin 8 in Helicobacter pylori infected gastric mucosa cryosections and an in situ hybridization assay for the detection of herpes simplex virus DNA in single cells.
Asunto(s)
Bioensayo/métodos , Mediciones Luminiscentes , Acetilcolinesterasa/análisis , Animales , Encéfalo/enzimología , ADN Viral/análisis , Mucosa Gástrica/química , Infecciones por Helicobacter/patología , Helicobacter pylori , Infecciones por Herpesviridae/patología , Hibridación in Situ , Interleucina-8/análisis , Fotometría , Ratas , Ratas Sprague-Dawley , Simplexvirus/genéticaRESUMEN
The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe(2+)-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production.
Asunto(s)
Antioxidantes , Ácidos y Sales Biliares/química , Ácidos y Sales Biliares/fisiología , Glicina , Peroxidasa de Rábano Silvestre , Hipoxantina , Indicadores y Reactivos , Cinética , Mediciones Luminiscentes , Luminol , Micelas , Taurina , Xantina OxidasaRESUMEN
A double-chemiluminescence in situ hybridization has been developed that combines the advantages of chemiluminescence with the detection of two different viral DNAs, i.e., herpes simplex virus (HSV) DNA and cytomegalovirus (CMV) DNA, in infected cells in the same specimen. For the simultaneous detection of these two different viral DNAs, we used a biotinylated HSV DNA probe, which can be visualized by a streptavidin-horseradish peroxidase (HRP) complex amplified with biotinyl tyramide. This probe was followed by the use of a luminol-based chemiluminescent substrate for HRP and a digoxigenin-labeled CMV DNA probe visualized by antidigoxigenin Fab fragments conjugated with alkaline phosphatase (AP). This is followed by the detection with a dioxetane phosphate derivate as chemiluminescent substrate for AP. Since the final product of both chemiluminescent reactions was light emission, sequential images for the two hybridizations were taken and analyzed using a high-performance luminograph connected to an optical microscope and to a personal computer for image analysis. Positive signals for the presence of both HSV DNA and CMV DNA were noticed in infected cells in the same specimen with a sharp localization, absence of cross reactions and absence of background.
Asunto(s)
Citomegalovirus/genética , Genoma Viral , Hibridación in Situ/métodos , Simplexvirus/genética , Línea Celular , Sondas de ADN , ADN Viral/análisis , Fibroblastos , Células HeLa , Humanos , Mediciones Luminiscentes , Sensibilidad y EspecificidadRESUMEN
The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 µm. Other applications, such as the alkaline phosphatase localization in 8 µm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigenin-labeled probes and alkaline phosphatase-labeled anti-digoxigenin antibody, using chemiluminescent substrate for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 µm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.
RESUMEN
The critical micellar concentration (cmc) values of some mixed systems containing two bile salts were determined by a maximum pressure bubble method and compared with those derived from a theoretical model developed for nonionic surfactants to assess the applicability of this model to such systems. Some assumptions on which the presumed validity of this model was based are discussed. The following binary mixtures were investigated: sodium chenodeoxycholate with cholate, ursocholate and ursodeoxycholate, either unconjugated or conjugated with taurine and glycine at different mole fractions (0, 0.25, 0.5, 0.75, 1) in 0.15 M NaCl. For these mixtures, experimentally determined data were in good agreement with values predicted by the theoretical model: both the cmc and the surface tension at this concentration of the mixtures were intermediate between those of the two pure bile salts; also, as the total bile salt concentration increased, the mixed micelles became enriched with the bile salt having the highest cmc, whereas the total monomer activity, determined by a potentiometric method employing a bile salt-selective electrode, increased only slightly. To test this model in an in vitro system, surface tension was also measured in ox bile samples that were enriched by 50% with sodium ursodeoxycholate, chenodeoxycholate, or their taurine amidates. The cmc and the surface tension at this concentration of the artificial bile increased when enriched with a bile salt with a cmc higher than that of endogenous salts (e.g. ursodeoxycholate versus taurocholate), whereas the reverse occurred for mixtures enriched with a bile salt with a lower cmc, such as chenodeoxycholate.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácidos y Sales Biliares/química , Ácido Quenodesoxicólico/química , Colesterol/metabolismo , Ácido Cólico , Ácidos Cólicos/química , Matemática , Micelas , Modelos Químicos , Modelos Teóricos , Tensión Superficial , Tensoactivos/químicaRESUMEN
Little is known about the effect of posture on the circulatory abnormalities of advanced cirrhosis. We evaluated the systemic hemodynamics, measured by Doppler-echocardiography, atrial natriuretic factor, plasma renin activity and plasma norepinephrine, in 10 patients with cirrhosis and ascites and 10 healthy controls, after 2 h of standing and during lying down for a further 2 h. Standing hemodynamic patterns of controls and patients with cirrhosis did not differ significantly. The latter, however, showed higher plasma renin activity, norepinephrine and atrial natriuretic factor. The assumption of the supine position led to greater increases in cardiac index and atrial natriuretic factor, and reduction in systemic vascular resistance in patients with cirrhosis. Norepinephrine and plasma renin activity declined in both groups to a similar extent, while heart rate only slowed in controls. Thus, after 2 h in the supine position, patients with cirrhosis showed hyperdynamic circulation with increased cardiac index and heart rate and reduced systemic vascular resistance. Norepinephrine, plasma renin activity and atrial natriuretic factor were also elevated. The hyperdynamic circulation in advanced cirrhosis appears during or is enhanced by lying down. This finding suggests that this syndrome is, at least in part, attributable to excessive blood volume translocation towards the central area. However, the persistent activation of renin-angiotensin and sympathoadrenergic systems suggests that a concomitant reduced vascular sensitivity to vasoconstrictors concurs in its development.
Asunto(s)
Ascitis/fisiopatología , Hemodinámica/fisiología , Cirrosis Hepática/fisiopatología , Postura/fisiología , Adulto , Anciano , Ascitis/sangre , Ascitis/etiología , Factor Natriurético Atrial/sangre , Enfermedad Crónica , Ecocardiografía Doppler , Humanos , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Masculino , Persona de Mediana Edad , Norepinefrina/sangre , Renina/sangreRESUMEN
The present work describes the development of HPLC-mass spectrometric systems equipped with an electrospray interface for the quantitative analysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C18 reversed-phase column (3 microns particle size, 70 x 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradient mode, at a flow-rate of 0.3 ml/min. This system permits post-column splitting of the eluate for analysis by two different detectors: (1) electrospray-mass spectrometer with a flow-rate of 18 microliters/min; and (2) a complementary evaporative light scattering mass detector. When bile salts were ionized in the electrospray interface operating in the negative-ion mode, only [M-H]- molecular ions were generated; the detection limit was 15 pg injected for all bile acids studied. In the second system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC pump was reduced to 1.4 microliters/min and bile acids were separated with a micro-bore C18 column (3 microns particle size, 150 x 0.30 I.D.), using the same mobile phase as above. With this latter system, a head-column enrichment technique can be used: the amount injected can be increased from 60 to 200 nl, permitting an improvement in the detection limit to 5 pg injected. Application of the HPLC-electrospray-mass spectrometric method to bile and serum bile acid analysis is described; preliminary data on the ability of the first system to determine the 13C/12C isotope ratio in 13C-labeled bile acid enriched serum is also critically discussed.
Asunto(s)
Ácidos y Sales Biliares/análisis , Líquidos Corporales/química , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Animales , Bilis/química , Ácidos y Sales Biliares/sangre , Isótopos de Carbono , Cricetinae , HumanosRESUMEN
We recently showed that patients with compensated cirrhosis can dispose of their fluid overload while reclining. In contrast, patients with ascites fail to develop supine-induced natriuresis. To assess the effect of reclining on renal sodium handling in patients with advanced cirrhosis and the mechanisms blunting natriuresis in this situation, renal function and plasma concentrations of atrial natriuretic factor, aldosterone and norepinephrine were evaluated in 10 nonazotemic patients with cirrhosis and ascites and 10 healthy controls standing for 2 h and reclining for 2 h. While standing, all patients showed marked sodium retention and significantly elevated plasma atrial natriuretic factor levels, aldosterone and norepinephrine. Glomerular filtration rate did not differ from healthy controls. The reclining increased renal sodium excretion in both groups, but this change was far less marked in patients; natriuresis only rose to the control range in two of them. An increase in atrial natriuretic factor and a depression of plasma aldosterone and norepinephrine was seen in both controls and patients. In the latter, despite the greater change in atrial natriuretic factor and aldosterone, the aldosterone to atrial natriuretic factor ratio, which was inversely correlated with natriuresis during both standing and reclining remained significantly elevated. In the two patients who achieved normal natriuresis during reclining, reclining was associated with both the normalization of the aldosterone/atrial natriuretic factor ratio, and with an increase in glomerular filtration rate. The supine-induced increase in atrial natriuretic factor was not only preserved but was even enhanced in cirrhosis with ascites.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Riñón/metabolismo , Cirrosis Hepática/metabolismo , Natriuresis/fisiología , Posición Prona/fisiología , Sodio/metabolismo , Adulto , Anciano , Aldosterona/sangre , Ascitis , Factor Natriurético Atrial/sangre , Humanos , Riñón/fisiopatología , Cirrosis Hepática/fisiopatología , Masculino , Persona de Mediana Edad , Norepinefrina/sangre , Posición Supina/fisiologíaRESUMEN
To evaluate the effects of a single oral dose of erythromycin on gastric and gallbladder emptying, 10 volunteers, without a known history of gastrointestinal disease, were investigated. Erythromycin stearate (500 mg) or placebo was given on separate mornings 30 min before a standard solid meal in a randomized, double-blind, crossover study. Gastric and gallbladder emptying rates were simultaneously evaluated by means of real-time ultrasonography. Gastric antral area and gallbladder volume were determined before the meal and 30, 60, 120, 180, 240, and 300 min after commencing eating. Erythromycin, compared to placebo, significantly accelerates and increases the degree of both gastric and gallbladder emptying. As previously reported for intravenous and chronic oral assumption, also a single dose of oral erythromycin is able to accelerate gastric and gallbladder emptying in normal human subjects.