Acidos biliares VIII: Los ácidos biliares mayoritarios presentes en bilis de cerdos cubanos / Major bile acids present in bile of Cuban pigs

Para establecer la composición de los ácidos biliares mayoritarios presentes en la bilis de cerdos cubanos, se emplearon métodos cromatográficos en capa delgada, cromatográficos por columna, así como procedimientos colorimétricos. Se estudiaron bilis de cerdos criados en empresas estatales y cerdos criados por pequeños agricultores. Se concluye que la bilis de cerdo doméstico presenta como componente fundamental el ácido hyodesoxicólico, mostrando en la mezcla de ácidos biliares crudos una distribución de ácidos mayoritarios similar a la de cerdos convencionales. En cambio, la bilis de cerdo estatal muestra como componente mayoritario el ácido quenodesoxicólico, coincidiendo la composición del crudo con la de cerdos genobióticos (animales libres de gérmenes). Se discuten las posibles causas de dichas diferencias y se evalúa de forma preliminar la posibilidad de explotación de las bilis

Acidos biliares séricos en la lesión hepática por giardia lamblia / Serum bile acids in the hepatic lesion by giardia lamblia

La determinación en suero de los ácidos biliares se ha convertido en la mejor prueba de significación clínica para detectar un "silencio bioquímico" en la enfermedad hepática, por lo tanto nos propusimos evaluar la utilidad de dichos ácidos y otras pruebas de funcionamiento hepático en pacientes con giardiasis. Se estudiaron 25 sujetos que presentaban trofozoitos de giardia lamblia en el drenaje biliar o frotis de la mucosa intestina. Fueron seguidos durante 3 meses. Se les normalizaron las pruebas de funcionamiento hepático, tales como: ácidos biliares, alanino-aminotransferasa, fosfatasa alcalina y timol, así como la histología hepática, en elperíodo de estudio. Después del tratamiento con distintas drogas antigiardiásicas, encontramos que la prueba más sensible en la lesión hepática por Giardia lamblia fue la de los ácidos séricos

Effects of ursodeoxycholic acid on gallbladder contraction and cholecystokinin release in gallstone patients and normal subjects.

It has been previously suggested that treatment with ursodeoxycholic acid leads to decreased gallbladder emptying. The proposed mechanism is decreased release of cholecystokinin through negative feedback control by an increased amount of intraduodenal bile acids. In the present study we examined cholecystokinin release and gallbladder contraction after oral administration of a commercial fatty meal (Sorbitract; Dagra, Diemen, The Netherlands) using ultrasonography in eight normal subjects and eight gallstone patients before and after 1 and 4 weeks of treatment with ursodeoxycholic acid (10 mg kg-1.day-1). Fasting gallbladder volume increased in 15 of 16 subjects during treatment (P less than 0.01). Minimal volume did not change. Therefore, both absolute and relative gallbladder emptying increased during therapy. Maximal decrement of gallbladder volume in milliliters and percentage as well as integrated gallbladder contraction during 90 minutes in milliliters and percentage were significantly increased after 1 and 4 weeks of treatment with ursodeoxycholic acid when compared with data before therapy. Gallstone patients tended to have larger fasting and residual gallbladder volumes than normal subjects, whereas parameters for the amount of bile expelled (maximal decrement of gallbladder volume and integrated gallbladder contraction in milliliters and percentage) did not differ. Release of cholecystokinin did not change during treatment and did not differ significantly between patients and normal subjects. Mean relative percentage of ursodeoxycholic acid in bile during treatment in 13 subjects consenting to have duodenal intubation was 47% (range 31%-60%). Changes of fasting gallbladder volume after institution of bile acid treatment correlated significantly (r = 0.74, P less than 0.01) with changes of cholesterol saturation index but not with relative percentage of ursodeoxycholic acid in bile. This study indicates that gallbladder emptying does not decrease during treatment with ursodeoxycholic acid. Moreover, there is no evidence of decreased cholecystokinin release.

Mutagenicity of bile and duodenal adenomas in familial adenomatous polyposis.

Duodenal adenomas occur almost inevitably in patients with familial adenomatous polyposis (FAP) whereas gastric adenomas are rare. FAP patients are also at high risk of duodenal cancer. Within the duodenum, adenomas cluster around the ampulla of Vater, as do the majority of duodenal cancers, suggesting that bile plays a role in tumour development. We therefore tested duodenal bile from 29 postcolectomy FAP patients (27 of whom had duodenal adenomas) and 24 non-FAP patients for mutagenicity, using techniques that detect point mutations in bacteria. Results which appeared to show that FAP bile was more mutagenic than control bile could be accounted for by a feeding effect, elimination of which also eliminated 'mutagenicity'. Under the conditions of our assays we conclude that if bile is an important factor in genesis of duodenal tumours, it does not act by inducing point mutation.

Mechanical property studies of human gallstones.

The recent development of gallstone fragmentation methods has increased the significance of the study of the mechanical properties of human gallstones. In the present work, fracture strength data and microhardness values of gallstones of various chemical compositions are presented as tested in both dry and simulated bile environments. Generally, both gallstone hardness and fracture strength values were significantly less than kidney stone values found in previous studies. However, a single calcium carbonate stone was found to have an outer shell hardness exceeding those values found for kidney stones. Diametral compression measurements in simulated bile conclusively demonstrated low gallstone fracture strength as well as brittle fracture in the stones tested. Based on the results of this study, one may conclude that the wide range of gallstone microhardnesses found may explain the reported difficulties previous investigators have experienced using various fragmentation techniques on specific gallstones. Moreover, gallstone mechanical properties may be relatively sensitive to bile-environment composition.

Caracterización morfológica al microscopio óptico y electrónico de "elementos inespecíficos" en el drenaje biliar / Morphologic characterization at the optic and electronic microscope of \"inespecific elements\" in the biliary drainage

Desde hace algunos años en nuestro país se informan "elementos inespecíficos" en drenajes biliares, denominando así a unas estructuras redondeadas de aproximadamente 12 *m que generalmente aparecen asociadas a trofozoitos de Giardia lamblia y desaparecen después de un tratamiento antigiardiásico. Muchos gastroenterólogos los interpretan como Giardia pero aún se desconoce su naturaleza. Nuestro objetivo fue estudiarlos al microscopio óptico y electrónico comparativamente con las formas biológicas de G. lamblia. Utilizamos muestras de drenaje biliar ricas en trofozoitos de G. lamblia y "elementos inespecíficos", trofozoitos de cultivo y heces ricas en quistes de G. lamblia. Las muestras fueron procesadas por la técnica habitual para microscopía electrónica de trasmisión con algunas modificaciones. Nuestros estudios demostraron que los caracteres morfológicos de los "elementos inespecíficos" corresponden a macrófagos

The effects of hypertonic glucose solutions on hepatic bile formation.

Bile secretion in the isolated guinea-pig liver was studied during perfusion with equi-osmolar hypertonic solutions containing either glucose, galactose, mannose, mannitol or sodium chloride. Perfusates made hypertonic with glucose, galactose or mannose decreased bile flow to the same extent and had similar effects on the ionic composition of bile: sodium, potassium and bicarbonate concentrations all increased. Mannitol had a smaller inhibitory effect and caused different changes in ionic composition: the increase in bile potassium concentration was proportionately greater; bicarbonate concentration did not change, but chloride was increased. Thus, glucose, galactose and mannose, can inhibit bile flow independently of extrinsic neural and hormonal mechanisms and exert a greater cholestatic effect than a non-metabolisable carbohydrate of similar molecular weight. The results also provide evidence for glucose reabsorption in the guinea-pig biliary tree, as shown in other species, and that galactose competes for this transport.

Gastrointestinal tract hemorrhage. The value of a nasogastric aspirate.

A bloody nasogastric aspirate is believed to imply active upper gastrointestinal tract bleeding, while a nonbloody yellow-green nasogastric aspirate that contains duodenal secretions suggests the absence of bleeding proximal to the ligament of Treitz. To validate these beliefs, physicians were asked to predict the presence of active gastrointestinal tract bleeding and whether bile was present in a nasogastric aspirate obtained immediately before endoscopy in 73 episodes of bleeding in 62 patients. A relationship was found between the physician's assessment of the presence of active bleeding demonstrated endoscopically and the appearance of the nasogastric aspirate. However, the sensitivity and specificity were low (79% and 55%, respectively). No association between the assessment of bile in the nasogastric aspirate and the presence of bile acids was demonstrated. These data do not support the placement of a nasogastric tube to determine whether or not a patient is bleeding, the location of the bleeding, and whether endoscopy should be performed.

Comparison of bile porphyrin concentrations in cattle and human beings with protoporphyria.

Blood and bile porphyrin concentrations were measured in cattle with protoporphyria and compared with those in human beings with the disease. Whereas the mean RBC porphyrin concentration in cattle was 18-fold greater than in human beings, the mean bile porphyrin concentration was only 78% greater. Sequential measurements over a 30-hour period in 1 animal with a bile fistula indicated that the ratio of total porphyrin to total bile acid in bile varied minimally. When the animal was given an IV infusion of taurocholate, the biliary excretion rate of porphyrin increased in parallel with that of bile acid, because of enhancement of bile flow. Thus, in cattle with protophorphyria, the concentration of porphyrin in bile is low compared with that of porphyrin in RBC, in contrast with findings in human beings, and adequate amounts of bile acids are secreted to maintain efficient protoporphyrin excretion. This explains, in part, why hepatobiliary disease has not been observed in cattle with protoporphyria, but has been seen in human beings with the disease.

Cholesterol absorption by the gall bladder.

Model and real biles were used to investigate factors influencing cholesterol and dextran (70,000 molecular weight) absorption by the gall bladder. Cholesterol absorption was proportional to cholesterol concentration when real bile was used, but model biles showed maximal absorption at cholesterol saturation. Reduction of temperature reduced cholesterol absorption and serosal secretion, but had little effect on dextran absorption. This indicates differences in uptake where cholesterol undergoes passive diffusion but dextran is taken up by fluid-phase endocytosis. Model bile prepared with a single bile salt showed lowest cholesterol uptake from cholate bile, but there was no difference in serosal secretion. Dextran uptake was also lowest from cholate bile, although serosal secretion was highest. These results show that an increase in the biliary content of dihydroxy bile salts increases gall bladder permeability to both hydrophobic and hydrophilic molecules and may lead to the accumulation of lipids in the mucosa, as seen in cholesterolosis.

Metabolism and effects on biliary lipid secretion of murocholic acid in the hamster.

The metabolism of murocholic acid (MC), a 6 beta-hydroxylated bile acid, was investigated after intravenous (i.v.), intraduodenal (i.d.) or intragastric (i.g.) administration to bile fistula hamsters. The effects on biliary cholesterol and phospholipid secretion were measured during intravenous infusions of increasing doses of [3H]MC. At an infusion rate of 0.1 or 1 mumol.min-1.kg-1, the hepatic uptake was effective. More than 90% of the dose was recovered in bile within 4 h. A bolus injection of 500 micrograms of [3H]MC in the duodenum led to a rapid and efficient biliary secretion of radioactivity. Increasing i.v. infused doses of MC had no effect on bile flow or biliary cholesterol output compared to the controls. Phospholipid secretion was significantly reduced (0.113 mumol.min-1.kg-1 versus 0.238 mumol.min-1.kg-1 in in controls per mumol.min-1.kg-1 of excreted bile acids) as MC progressively replaced the endogenous bile acid pool in bile. After i.v. and i.d. administration, MC was secreted in bile as glyco and tauro conjugates without additional hepatic hydroxylation, sulfation or glucuronidation. The i.g. ingestion of MC followed by the faecal analysis of metabolites showed the formation of hyodeoxycholic acid and 3 alpha-OH-6-oxo-5 beta-cholan-24-oic acid. An equivalent experiment with hyodeoxycholic acid gave MC and the same oxo bile acid. We concluded that MC is metabolized by the hamster liver as an endogenous bile acid, which undergoes intestinal bacterial transformation into a 6-oxo derivative and is then reduced into hyodeoxycholic acid. This process is completely reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Lovastatin added to ursodeoxycholic acid further reduces biliary cholesterol saturation.

The effects of lovastatin and ursodeoxycholic acid on cholesterol saturation of gallbladder bile were examined, alone and in combination. Nine volunteers were studied before any treatment and after each of three treatment periods: lovastatin, 40 mg, twice a day; ursodeoxycholic acid, 10 mg/kg per day; and the combination of both drugs. Treatment periods were randomly ordered, lasted 4-5 wk, and each was preceded by a 3-wk washout period. Mean cholesterol saturation index decreased from a baseline value of 1.40-0.92 on lovastatin (p less than 0.008). Mean cholesterol saturation index on ursodeoxycholic acid was 0.87 and decreased to 0.70 with the addition of lovastatin (p less than 0.030). There was a strong correlation (r = 0.87, p less than 0.003) between saturation index on ursodeoxycholic acid and the further incremental reduction in saturation index with addition of lovastatin. These findings raise the possibility that addition of lovastatin to ursodeoxycholic acid treatment might improve the efficacy of this bile salt for dissolution of cholesterol gallstones, especially in patients with a suboptimal response to ursodeoxycholic acid.

Biliary D-glucaric acid: its quantitation and preventive role in gallstone formation.

A method for quantitation of D-glucaric acid in bile has been developed involving extraction with tetrahexylammonium chloride, boiling for 40-60 min, and determination of the percentage inhibition of beta-glucuronidase activity at 56 degrees C and pH 4. D-glucaric acid, bilirubin, bile acid, and protein were determined in 106 human gallbladder biles obtained at autopsy, including 20 with gallstones. The mean D-glucaric acid content was 1125 +/- 159 microM (mean +/- SE). Biliary beta-glucuronidase activity was not affected by D-glucaric acid because of 1) no difference in biliary D-glucaric acid content, either absolute or corrected for per unit of bilirubin, bile acid, or protein, between those with and those without gallstones; 2) no negative correlation between D-glucaric acid content and beta-glucuronidase activity in the bile; and 3) minimal conversion of D-glucaric acid to D-glucaro-1,4-lactone at the usual pH of bile. We conclude that biliary D-glucaric acid plays no role in the prevention of gallstone formation.

The influence of barley fibre on bile composition, gallstone formation, serum cholesterol and intestinal morphology in hamsters.

Frequency of gallstones, concentration of bile acids and cholesterol in bile, concentration of cholesterol in serum, and structure of the small intestinal mucosa were analyzed in male Syrian Golden hamsters fed a stone provoking fibre-free diet with or without supplementation of brewer's spent grain (BSG), a concentrated barley fibre source from the by-product of brewing. A significantly lower frequency of gallstones was found in the animals with 10% BSG dietary supplementation. Addition of 30% BSG after an initial 6-week period with a fibre-free, stone provoking diet seemed to dissolve previously formed gallstones. Total bile acid concentration was higher in bile from animals given a diet supplemented with 10% BSG. In addition, the cholesterol concentration in both serum and bile was lower in the 30% BSG supplemented group. Structurally, a 10% BSG supplementation decreased ileal epithelium height whereas a high supplementation (30%) of BSG induced a decrease in epithelial height both of jejunal and ileal mucosa. The results show that BSG has significant effects on the metabolism of bile acids and cholesterol as well as on the morphology of the small intestinal mucosa.

An improved purification procedure for conjugated bile acids using octadecyl-bonded silica cartridges.

A rapid procedure for the extraction of conjugated bile acids from human fluids using pre-packed octadecyl-bonded silica cartridges is described. The method was compared with the other procedures reported in the literature and was found to produce a higher degree of sample purification and to achieve satisfactory accuracy and reproducibility. The present procedure is applicable to the high-performance liquid chromatographic assay of bile acid conjugates in human bile, gastric juice, serum and urine.

Sensitive and rapid colorimetric immunoenzymometric assay of ferritin in biological samples.

We describe a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for quantifying ferritin in human and rat biological fluids. We used chlorophenol red beta-D-galactopyranoside as the colorimetric substrate of beta-galactosidase (EC 3.2.1.23), which is coupled to specific antibodies to either human or rat liver ferritin. The assay is sensitive (detection limit for human assay = 0.58 micrograms/L and for rat assay = 0.37 micrograms/L), accurate (average recovery for human assay = 93% and for rat assay = 92%), and precise (total CVs for human assay = 2.3-12.2% and for rat assay = 5.6-11.3%). The results correlated well with those of an established immunoradiometric technique (r = 0.99691). This assay has a prolonged shelf-life, is inexpensive, and utilizes a stable colorimetric substrate that requires relatively short incubation.

High-performance liquid chromatographic separation of bilirubin conjugates: the effects of change in molarity and pH.

A fast, sensitive high-performance liquid chromatographic method has been developed for the separation and quantitation of biliary bile pigments; this utilizes a C18 reversed-phase column with two solvents, a buffer and an organic solvent, which were changed in a linear gradient from a polar to a less polar combination. Nine glycosidic conjugates of bilirubin as well as unconjugated bilirubin and a suitable internal standard, unconjugated mesobilirubin IX alpha, were all separated to baseline by gradient elution; the species eluted in a polar to less polar fashion. Increasing the molarity of the solvent decreased the binding of non-glucuronide pigments to the column, with a decrease in their retention times, whereas for bilirubin monoglucuronide they increased. Decrease in pH, similarly, preferentially increased bilirubin monoglucuronide retention times.

Altered bile composition during cholesterol gallstone formation: cause or effect?

Gallbladder stasis, increased gallbladder absorption, and elevated biliary levels of calcium, hydrogen ion, and bilirubin have been implicated as factors potentially critical to cholesterol crystal precipitation. Previous studies, however, have analyzed bile only when crystals or gallstones have already formed. Therefore, we tested the hypothesis that changes in bile composition are a late effect, occurring only after crystal formation. Adult male prairie dogs were fed a standard nonlithogenic control diet (n = 7) or a lithogenic 1.2% cholesterol diet for 5, 9, or 14 days to cause cholesterol saturation (n = 7), cholesterol monohydrate crystals (n = 7), or gallstones (n = 7). Gallbladder bile was examined microscopically for crystals, and analyzed for ionized calcium, bilirubin, pH, total protein, and biliary lipids. The ratio of gallbladder to hepatic bile radiolabeled cholic acid specific activity (Rsa) was calculated as an index of gallbladder stasis. Cholesterol saturation index was calculated. The results demonstrate that increased gallbladder bile cholesterol saturation and total protein concentration precede cholesterol monohydrate crystal precipitation. However, changes in gallbladder ionized calcium, unconjugated bilirubin, pH, stasis, and absorption were noted only after crystals and gallstones had already formed. These data indicate that alterations in gallbladder bile calcium, bilirubin, pH, stasis, and absorption are not early changes, but occur simultaneously with or after crystal formation. Increased biliary protein, however, which was elevated prior to nucleation, may be an important mediator of cholesterol precipitation in cholesterol-saturated bile.

Lack of effect of halofantrine on hepatic drug metabolism in the rat in vivo and in vitro.

The effect of the antimalarial drug halofantrine (Hf) on hepatic drug metabolism in the rat has been studied in vivo and in vitro using different model drug substrates. Hf in vitro produced no significant effect on the values of Km and Vmax for aminopyrine N-demethylation or 7-ethoxycoumarin O-dealkylation in microsomes incubated with Hf (0.01-0.1 mM) or on the rate of N-demethylation of aminopyrine or O-dealkylation of Ec in microsomes produced from rats dosed chronically with Hf (200 mg/kg) for 4 days. The disposition of antipyrine (Ap) was investigated in the isolated perfused rat liver preparation (IPRL). Following the administration of bolus doses of Hf (0.5, 2.5 and 5.0 mg) no significant changes were observed in the half-life (t1/2), clearance (Cl) or apparent volume of distribution (Vd) for Ap compared with controls. Pentobarbitone induced sleeping time was also assessed in mice. No significant difference was determined in time to recovery of the righting reflex for mice receiving Hf as single oral doses or chronically over 4 days when compared with appropriate controls. The potential for selective isoenzyme effects was studied in vivo. The three principal urinary metabolites of Ap, norantipyrine (Np), 3-OH and 4-OH Ap were measured in rat urine, with no significant change in urinary recovery of Ap or any of the metabolites in the presence of Hf (1.25 mg/kg i.p.) compared with controls. These results suggest that Hf is not, in contrast to many commonly used quinoline antimalarials, a potent or specific inhibitor of drug metabolism in vitro or in vivo.