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Biomedical subjects

A Roda

Publications and source records attributed to A Roda.

162 records · Page 9Linked to original sources
PubMed1975

[Therapy of cholesterol lithiasis with chenodeoxycholic acid (Chenoxyl)].

Chenodesoxycholic acid (500-750 mg/day Chenoxyl) was employed over a period of 9 months in the treatment of 40 patients with radiotransparent calculi in a functioning gallbladder and 8 with choledochal lithiasis. Radiological examination and complete hepatological exploration were carried out before and after the treatment in all cases. Complete dissolution was obtained in 6 and 2 cases respectively, with reduction of size in 13 and 2, and no change in 21 and 4. No significant variations were notes in the blood lipid picture or in liver function. There were no cases of diarrhoea.

Adult↗
PubMedDate not supplied

HPLC-fluorescence determination of individual free and conjugated bile acids in human serum.

A method for the quantitative analysis of unconjugated and conjugated bile acids (BA) in serum of patients with primary biliary cirrhosis (PBC) before and after therapy with antibiotic or ursodeoxycholic acid (UDCA) is described. After separation of the free, glycine and taurine conjugated (F, G and T conjugated) fractions by solid-phase extraction, the isolated T conjugates were hydrolysed enzymatically using cholyglycine hydrolase. The BA fractions were derivatized using 2-bromoacetyl-6-methoxynaphthalene (Br-AMN) and detected fluorimetrically (lambda exc = 300 nm, lambda em = 460 nm). The derivatization reaction was performed under mild conditions (10 min at 40 degrees C) in an aqueous medium in the presence of tetrakis (decyl) ammonium bromide (TDeABr). The HPLC separation was achieved using an ODS column and with a mobile phase gradient mixture of A-B, where A is water and B is acetonitrile:methanol (60:40 v/v) for elution at a flow-rate of 1.2 mL/min. The reproducibility, recovery and separation of individual BA under gradient elution conditions were satisfactory, allowing a sensitive detection of each BA in serum samples with a detection limit of about 1-2 pmol.

Amidohydrolases↗
PubMedDate not supplied

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role.

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.

Antioxidants↗
PubMedDate not supplied

Continuous-flow bioluminescent determination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate.

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25 degrees C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The ATP content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.

Adenosine Triphosphate↗
PubMedDate not supplied

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme.

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H2O2/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H2O2 and o-phenylendiamine as substrates. Recovery of the assay was 97-112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.

Animals↗
PubMedDate not supplied

Ursodeoxycholic acid vs. chenodeoxycholic acid as cholesterol gallstone-dissolving agents: a comparative randomized study.

Cholesterol gallstones are dissolved in man by chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA). To test the comparative efficacy of these two cholelitholytic bile acids, 223 gallstones patients were randomly treated with either UDCA or CDCA at two different doses: 7 to 8 mg per kg per day and 14 to 15 mg per kg per day. Efficacy and factors influencing dissolution (dose, size of the stones, and time) were evaluated after 3, 6, and 12 months of treatment. UDCA was significantly more efficacious than was CDCA after 3 and 6 months of treatment, whereas after 12 months, no significant differences were observed. UDCA was equally effective at high and low doses, both on small and large stones. CDCA was significantly more effective at high doses and on small stones. Seventy-four per cent of the total dissolutions with UDCA and 42% with CDCA occurred within the first 6 months of treatment. Diarrhea and hypertransaminasemia occurred only in the CDCA-treated patients. We conclude that UDCA seems to be the bile acid of choice in dissolving cholesterol gallstones.

Adolescent↗
PubMedDate not supplied

Diagnostic effectiveness of serum bile acids in liver diseases as evaluated by multivariate statistical methods.

The aims of this study were to determine the diagnostic effectiveness of fasting and postprandial serum bile acid determinations in liver diseases, and to compare results with those of conventional liver function tests. In 322 patients with biopsy-proved liver disease and 93 healthy subjects, fasting and postprandial (2 hr) serum levels of cholic, chenodeoxycholic, and lithocholic acid conjugates and conventional liver function tests were evaluated. Data were subjected to variance and discriminant and factor analyses. Fasting serum bile acids were higher in patients when compared to controls and were significantly higher in severe than in mild liver diseases. Determination of cholic plus lithocholic acid provided the highest discrimination capacity. The percent of correct allocation was 75.4% for conventional liver function tests, 70.1% for fasting serum bile acids and increased to 79.6% when liver function tests plus serum bile acids were considered. Postprandial percentages were always lower than fasting. Factor analysis identified two factors possibly related to cytolysis and protein synthesis. The serum bile acid concentrations highly correlated with both factors. We conclude that serum bile acid determinations increase the diagnostic and discriminant capacities of liver function tests and are more sensitive and discriminant when obtained in fasting than postprandially.

Adult↗
PubMedDate not supplied

Effect of nuclear hydroxy substituents on aqueous solubility and acidic strength of bile acids.

The aqueous solubility and thermodynamic dissociation constants of a representative series of bile acids with varying numbers and configuration of nuclear hydroxyl substituents were determined. The pKa values were calculated by extrapolating pKa' values determined in solutions of aqueous methanol of different mole fractions at 25 degrees C. All bile acids had the same acidic strength in water, indicating that the pattern of hydroxyl nuclear substituents does not affect the acidic strength probably because of the distance between the hydroxyl and ionizing groups. In contrast, aqueous solubility was dependent on the number, position and orientation of the hydroxyl groups. The solubility values ranged from 5 X 10(-8) M (0.05 microM) for 3 alpha-hydroxy-5 beta-cholanoic acid (lithocholic acid) to 1.67 X 10(-3) M (1,670 microM) for 3 alpha,7 beta,12 alpha-trihydroxy-5 beta-cholanoic acid (ursocholic acid). The hydroxy groups notably affect solubility by forming hydrogen bonds with water but also by reducing, according to their orientation, hydrophobicity of the steroid nucleus.

Bile Acids and Salts↗
PubMedDate not supplied

23-Methyl-3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic acid: dose-response study of biliary secretion in rat.

A side chain derivative of ursodeoxycholic acid, 23-methylursodeoxycholic acid, was synthesized and the effect of i.v. infusion of the acid at different doses (0.75, 1.5, 3.0 and 6.0 mumoles per min per kg body weight over 1 hr) on bile flow, on its hepatic biotransformations and on biliary lipid secretion has been studied in bile fistula rats. The results were compared with those of ursodeoxycholic and cholic acid administered under similar conditions. 23-Methylursodeoxycholic acid is poorly secreted into bile and poorly taurine and glycine conjugated, at all infusion doses. Ursodeoxycholic acid is quantitatively recovered at low doses and recovered less at high infusion rates. Cholic acid is almost entirely recovered at all infusion doses. Ursodeoxycholic acid conjugation pattern is dependent on the dose, and glucuronidation and sulfation operate at high doses. Cholic acid is taurine conjugated at low doses; at high doses, large amounts of unconjugated bile acids are observed. Methylursodeoxycholic acid presents a delayed secretion and hypercholeresis. Ursodeoxycholic acid presents similar results at high infusion rates, possibly by reaching a high intrahepatic concentration of free form. The octanol/water partition coefficients of ursodeoxycholic acid and 23-methylursodeoxycholic acid are similar and higher than that of cholic acid. A chole-hepatic shunting of 23-methylursodeoxycholic acid may explain both the low recovery in bile and hypercholeresis and is consistent with its hydrophilicity of cholic acid, on the contrary, makes possible its high recovery in bile. The effect on biliary lipid secretion is unpredictable and affected by the dose and, in consequence, by the conjugation pattern of the bile acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Bio- and chemiluminescence in bioanalysis.

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.

Animals↗
PubMedDate not supplied

A radioimmunoassay for lithocholic acid conjugates in human serum and liver tissue.

A sensitive and specific radioimmunoassay for glycine and taurine conjugates of lithocholic acid (CLCA) has been developed. 3H-glycolithocholic acid (S.A. = 17Ci/mmol) was used as tracer. Separation of free from antibody-bound bile acid was carried out using ammonium sulphate (saturated solution). The antiserum showed high specificity for both glyco and tauro conjugated lithocholate (100% cross reaction) and lithocholic acid (25% cross reaction). The sensitivity of the assay (1 pmole/tube), was adequate for measuring CLCA in peripheral blood and hepatic tissue in man.

Antibody Specificity↗
PubMedDate not supplied

Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate.

Enzymatic and molecular cytochemistry was used to detect and follow the hepatotoxic effects caused in overnight-fasted Sprague-Dawley rats by a 1-h continuous intrafemoral infusion of taurochenodeoxycholate at 0.4 and 0.8 mumol-1 min-1 100 g-1 body weight dose levels. Rats were killed at 0, 1 and 24 h from the end of perfusion. Their livers were examined for morphology, DNA fragmentation (by a TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-nick end-labelling assay), cell regeneration (by in vivo bromodeoxydurine incorporation), reduced glutathione, calcium and several enzyme cytochemical activities. Isolated injured hepatocytes randomly scattered throughout the liver were already evident at the end of perfusion. DNA fragmentation and cytoplasm shrinkage were prominent and early features of injured hepatocytes, which later showed calcium loading and chromatin clumping. Preserved cytochemical enzymatic activities indicated that plasma and mitochondria membranes were not severely damaged. Inflammatory response was absent. These observations indicate that an acute exposure to taurochenodeoxycholate induces a cell death process with apoptotic features.

Animals↗
PubMedDate not supplied

A radioimmunoassay of primary bile acid conjugates in human serum.

The fasting levels of chenodeoxycholic and cholic acid conjugates in various groups of patients [25 healthy subjects, 20 patients with chronic active liver disease (CALD), 21 patients with primary biliary cirrhosis (PBC) and 5 patients with cholesterol gallstones before and after chenodeoxycholic acid therapy] were determined by using a sensitive, accurate and precise radioimmunoassay. The results obtained with this method were compared with those of a gas-chromatographic method with methylation and acetylation of the bile acids using a 2 m x 0.4 cm glass column packed with 3% of AN 600 on Anakrom Q 110-120 mesh. The results obtained with both methods are in agreement; therefore the method used can be applied for quantitative analysis. In diagnosis, the measurement of serum bile acid levels, fasting or after meals, proved to be a very sensitive method, although further correlations are necessary with standard liver function tests.

Animals↗