Prostaglandin E2 directly protects isolated rat gastric surface cell membranes against bile salts.

Rat gastric surface cell membranes were prepared and the effect of taurocholic acid assessed by ESR spectroscopy using the 16-doxylstearic acid spin label. Taurocholic acid increased the polar part of the spectra, indicating an augmented amount of spin label molecules with a polar environment. Concomitantly, mobility of the spin label molecule was augmented. The effect of taurocholic acid was completely prevented by the previous addition of prostaglandin E2. This suggests a direct protective efficiency of prostaglandin E2 on rat gastric surface cell membranes without the metabolic participation of intact cells.

Ursodeoxycholate stabilizes phospholipid-rich membranes and mimics the effect of cholesterol: investigations on large unilamellar vesicles.

Ursodeoxycholate is used to treat primary biliary cirrhosis and is incorporated into hepatocyte plasma membranes. Its steroid nucleus binds to the apolar domain of the membrane, in a similar position to cholesterol. Therefore the question arises whether ursodeoxycholate has a similar effect on membrane structure and stability as cholesterol. Using differential scanning calorimetry the thermotropic behavior of egg phosphatidylcholine and dimyristoylphosphatidylcholine were studied after incubation with cholesterol or ursodeoxycholate. Large unilamellar vesicles were prepared with cholesterol contents of 0-50%. Following incubation of these vesicles with different amounts of ursodeoxycholate, vesicle stability in a gravitational field was investigated by measuring the phospholipid and cholesterol release. Vesicle size was studied by laser light scattering after incubation with cheno- and ursodeoxycholate, and the release of entrapped carboxyfluorescein was measured by means of fluorescence spectroscopy. Increasing cholesterol diminished the enthalpy of the phase transition in the membrane. Ursodeoxycholate decreased the enthalpy of the phase transition at even lower concentrations. Lipid release from vesicles in a high gravitational field diminished with increasing cholesterol content of the vesicles. Ursodeoxycholate had a comparable effect, which increased as the cholesterol content of the vesicles was decreased. Chenodeoxycholate damaged vesicles, whereas ursodeoxycholate did not. Cholesterol and ursodeoxycholate (below its critical micellar concentration) decreased the carboxyfluorescein release from vesicles induced by chenodeoxycholate. Thus like cholesterol, ursodeoxycholate is incorporated into phospholipid model membranes and reduces the change in enthalpy of the gel to liquid-crystalline phase transition. Like cholesterol ursodeoxycholate also maintains membrane stability and prevents membrane damage induced by mechanical and chemical stress.

The effect of bile salts and calcium on isolated rat liver mitochondria.

Intact mitochondria were incubated with and without calcium in solutions of chenodeoxycholate, ursodeoxycholate, or their conjugates. Glutamate dehydrogenase, protein and phospholipid release were measured. Alterations in membrane and organelle structure were investigated by electron paramagnetic resonance spectroscopy. Chenodeoxycholate enhanced enzyme liberation, solubilized protein and phospholipid, and increased protein spin label mobility and the polarity of the hydrophobic membrane interior, whereas ursodeoxycholate and its conjugates did not damage mitochondria. Preincubation with ursodeoxycholate or its conjugate tauroursodeoxycholate for 20 min partially prevented damage by chenodeoxycholate. Extended preincubation even with 1 mM ursodeoxycholate could no longer prevent structural damage. Calcium (from 0.01 mM upward) augmented the damaging effect of chenodeoxycholate (0.15-0.5 mM). The combined action of 0.01 mM calcium and 0.15 mM chenodeoxycholate was reversed by ursodeoxycholate only, not by its conjugates tauroursodeoxycholate and glycoursodeoxycholate. In conclusion, ursodeoxycholate partially prevents chenodeoxycholate-induced glutamate dehydrogenase release from liver cell mitochondria by membrane stabilization. This holds for shorter times and at concentrations below 0.5 mM only, indicating that the different constitution of protein-rich mitochondrial membranes does not allow optimal stabilization such as has been seen in phospholipid- and cholesterol-rich hepatocyte cell membranes, investigated previously.

High performance liquid chromatographic determination of free and conjugated bile acids in serum, liver biopsies, bile, gastric juice and feces by fluorescence labeling.

Separation and measurement of commonly occurring free and conjugated bile acids in serum, liver biopsies, bile, gastric juice and feces have been successfully accomplished using high performance liquid chromatography with fluorescence labeling. Free and conjugated bile acids were extracted from pretreated samples using Sep-Pak C18 cartridges and then fractionated on a piperidinohydroxypropyl Sephadex LH-20 column. Free and glycine conjugated bile acids were labelled with 4-bromomethyl-7-methoxycoumarin. Taurine conjugated bile acids were hydrolysed with cholylglycine hydrolase prior to derivatization with 4-bromomethyl-7-methoxycoumarin. Labelled bile acids were eluted using an acetonitrile/methanol/water gradient on an ultrasphere ODS column. The eluate was monitored by a fluorophotometer at 360 nm (excitation) and 410 nm (emission). Linearity was obtained between 50 and 400 nmol. Recoveries from serum, gastric juice and feces were greater than 87%. This is therefore a sensitive method for the precise quantification of bile acids in serum, liver biopsies, bile, gastric juice and feces.

LUV's lipid composition modulates diffusion of bile acids.

Large unilamellar vesicles were prepared from phosphatidylcholine (PC), sphingomyelin (SM), cholesterol (Chol) and cardiolipin (CL) by an extrusion technique (LUVETs). Diffusion of the more hydrophobic lithocholic acid (LCA) and the less hydrophobic chenodeoxycholic acid (CDCA) was investigated by using the pyranine fluorescence method. Membrane permeability was studied by measuring the inclusion of carboxyfluoresceine (CF) into the lipid vesicles, and membrane fluidity was determined with diphenylhexatriene (DPH) and trimethylammonium-diphenylhexatriene (TMA-DPH). All results indicate that, CDCA compared to LCA, exhibits a significantly better penetration into vesicles containing SM. LCA penetrates better into vesicles containing cholesterol. Small amounts of CL influenced the diffusional properties of CDCA more than those of LCA. Since Lamcharfi et al. (1997a) Euro. Biophys. 25, 285-291 have observed differences in the conformational forms of CDCA and LCA in solution, it is suggested that the diffusion rate of bile acids through (model-)membranes is not only dependent on hydrophobicity, but also on bile acid di-(poly-)meric associations and on membrane-lipid composition.

Peri-hepatic lymphadenopathy in primary biliary cirrhosis reflects progression of the disease.

OBJECTIVE: We prospectively investigated the peri-hepatic lymph node volume in patients with primary biliary cirrhosis (PBC) and healthy controls to evaluate the correlation with histology, biochemical and immunological features. MATERIALS AND METHODS: The total peri-hepatic lymph node volume in the liver hilus was evaluated by high-resolution ultrasound in 67 consecutive patients with PBC and in 43 healthy controls. Stages I-IV of PBC were biochemically, immunologically and histologically proven in all patients. RESULTS: Adequate visualization of the liver hilus was achieved in 59/67 patients (88%) with PBC and in 39/43 healthy controls (91%). Lymph nodes in the liver hilus were sonographically detected in all 59 patients with PBC and in 26/39 healthy controls (67%) with adequate visualization of the liver hilus. The mean peri-hepatic lymph node volumes were: stage I (n = 9): 0.8 +/- 0.5 ml; stage II (n = 28): 2.4 +/- 1.5 ml; stage III (n = 21): 4.2 +/- 2.3 ml; stage IV (n = 9): 3.2 +/- 1.0 ml. The peri-hepatic lymph node volume did not significantly correlate with cholestasis, liver function tests or the immunological status. CONCLUSIONS: Enlarged lymph nodes in the liver hilus are sonographically detectable in almost all patients with PBC. The total peri-hepatic lymph node volume in patients with PBC reflects histological stage, i.e. larger lymph nodes are observed in more advanced disease.

Detection of 6-acetylmorphine in vitreous humor and cerebrospinal fluid--comparison with urinary analysis for proving heroin administration in opiate fatalities.

The concentrations of morphine and 6-acetylmorphine (6-AM) in urine, cerebrospinal fluid (CSF), and vitreous humor (VH) and the morphine concentrations in blood were determined by gas chromatography-mass spectrometry for 29 fatalities after abuse of heroin either alone or in combination with alcohol and other drugs. 6-AM was found above a quantitation limit of 1 ng/mL in urine in 89% of the cases, in CSF in 68% of the cases, and in VH in 75% of the cases. The 6-AM concentrations in CSF (mean, 10 ng/mL) and VH (mean, 17 ng/mL) were in general much smaller than in urine (mean, 170 ng/mL); therefore, the different pharmacokinetic behavior of the fluids is discussed. There is no uniformity between the three fluids with respect to the presence or absence of 6-AM. Therefore, CSF or VH may be used as complementary or alternative materials to urine in order to prove heroin uptake in opiate fatalities.

Sex differences in rat liver bile acids.

Male Wistar rats show higher relative liver weights and higher weights of mitochondrial and microsomal fractions than females. Their relation of subcellular fraction weights tends more to the microsomal side. In liver homogenate and subcellular compartments, males contain significantly higher concentrations of beta-muricholic acid and other metabolites of chenodeoxycholic acid, females higher proportions of chenodeoxycholic acid. Higher concentrations of potentially toxic nonsulphated dihydroxy bile acids in the liver of female rats could be a sign of slower elimination, and the cause of higher susceptibility of females to liver injury induced by bile acid administration.

Dacarbazine (DTIC)-induced human liver damage light and electron-microscopic findings.

The first electron-microscopic description of DTIC-induced human liver injury is presented. A 61-year-old man developed signs of hepatic failure during the second treatment cycle with DTIC for malignant melanoma. Light-microscopic examination revealed extensive centrilobular liver necrosis. Terminal hepatic venules did not show any signs of vasculitis or thrombosis and there was a lack of inflammatory infiltration. At the ultrastructural level intracytoplasmic, membrane-bound, organelle-free vacuoles were found in the hepatocytes. Liver cells showed bleb formation. Bile canaliculi were dilated and their microvilli flattened. In the pericanalicular exoplasm electron-dense fibrillary material, thought to be of microfilamentous origin, accumulated. The patient received 250 mg methylprednisolone i.v. at the very onset of symptoms and was discharged 12 days after the peak rise of transaminases with normal liver parameters.

Radiological and ultrasonographic investigations with respect to patient selection and monitoring for chemical gallstone dissolution.

786 consecutive patients with suspected gallstones investigated radiologically and ultrasonographically, 231 (33.0%) were found to have stones. Radiological examinations showed that 59.3% of these were suitable for chemical dissolution with cheno- or ursodeoxycholic acid. The percentage of treatable patients fell from age 20 to age 65 from 71% to 48% (p less than 0.05) and then rose again to 61% (not significant). The benefit of litholysis is greater, the earlier it is begun. The results are in disagreement with those of two studies in operated patients. Here only 19-24% were suitable for stone dissolution. Comparative ultrasonographic and radiological studies in 23 patients have shown that monitoring of patients during litholysis can be carried out ultrasonographically. Stone size measured ultrasonographically corresponds sufficiently accurately with that obtained radiographically.

[Studies on the separation and photometric determination of antiepileptics (author's transl)]. / Untersuchungen zur Trennung und fotometrischen Bestimmung von Antiepileptica.

Well-known methods for the photometric determination of antiepileptics in body fluids were tested from the aspect of their utilization in a pharmacokinetic therapy service of clinical pharmacology. In the framework of a comparative synopsis of methods, specifications are given for the determination of phenytoin, phenobarbital, methylphenobarbital, and carbamazepine.

Bile acids in patients suffering from colorectal carcinoma--a pilot study.

Bile acids are increasingly discussed in the genesis of colorectal carcinoma. In a pilot study we analysed bile acids in tumor tissue, tumor-free mucosa, serum, and feces of 6 patients with rectal and 3 patients with colonic carcinoma. 5 patients dead from nongastroenterological diseases and two operated on for benign colonic stenosis served as controls for colorectal mucosa, 16 healthy persons as controls for feces and serum. Bile acids and their sulphates were determined after differential solvolysis by gas-liquid chromatography on QF-1 columns. Serum lithocholic acid was significantly (p less than 0, 05) elevated in all carcinoma patients. The feces showed a trend towards increased secondary bile acids. Tumor-free mucosa of patients with rectal carcinoma showed elevated unsulphated bile acids, sulphated lithocholic acid (p less than 0, 05) and sulphated chenodeoxycholic acid (p less than 0, 025) were significantly decreased. In rectal carcinoma tissue total bile acids were elevated, cholic acid (p less than 0, 01) and sulphated lithocholic acid decreased (p less than 0, 05). In nonaffected colonic mucosa of patients with colonic carcinoma total bile acids were decreased, in colonic carcinoma tissue only traces of bile acids were found. Whether these changes are causes or rather consequences of colorectal tumors--e.g. tumor-induced stasis in the gut--and whether they may be useful for diagnosis remains to be elucidated in further studies.

Differing effect of chenodeoxycholic acid and ursodeoxycholic acid on bile acids in rat colonic wall and contents.

Bile acids may promote experimental colonic cancer. Many studies correlate fecal bile acids and colorectal carcinomas. Little is known on bile acids in the colonic mucosa and their relation to luminal bile acids. We, therefore, studied bile acids in colonic wall and contents of normal female Wistar rats and after 14 days' administration of chenodeoxycholic acid or ursodeoxycholic acid (90 mg/kg daily), two bile acids used in medicamentous cholelitholysis. Both regimens increase total bile acids in colonic contents, ursodeoxycholic acid produces a higher rise in toxic lithocholic acid. In the colonic wall, only ursodeoxycholic acid causes an increase of most nonsulfated bile acids including lithocholic acid. Bile acid patterns do not correlate in colonic wall and contents. We conclude that increased colonic wall bile acids after ursodeoxycholic acid administration warrant control in man. In future colorectal carcinoma studies, not only fecal, but also mucosal bile acid concentrations should be correlated to carcinogenesis.

Antral mucosal bile acids in two types of chronic atrophic gastritis.

Bile acids may damage the gastric mucosa, and they are cocarcinogenic in experimental colonic and gastric cancer. Chronic atrophic gastritis (CAG) and chronic atrophic gastritis with intestinal metaplasia (CAGIM) are associated with gastric carcinoma. We, therefore, analysed bile acids in the antral mucosa in controls (n = 10), in patients with CAG (n = 12) and CAGIM (n = 20). In both forms of chronic antral gastritis, total mucosal bile acid concentrations drop, caused mainly by lower primary bile acids. The proportions of secondary bile acids rise, in particular of toxic lithocholic acid. This is probably caused by bacterial activity in the stomach. Whether secondary bile acids, especially lithocholic acid, alone or in combination with other bacterial degradation products, influence gastric carcinogenesis remains to be elucidated in further studies.

Ileal and colonic mucosal bile acids in Crohn's disease and right colonic carcinoma.

Bile acids are supposed to promote colonic cancer. In Crohn's disease, colonic carcinomas are relatively rare. We, therefore, compared ileal and right colonic mucosal bile acids analysed by gas-liquid chromatography in 8 patients with ileal Crohn's disease (14-48 yrs.) and 7 patients with right colonic carcinoma (28-77 yrs.) who underwent surgery. In both ileal and colonic mucosa, nonsulphated bile acid concentrations were somewhat higher in Crohn's disease (20.98 micrograms/g +/- 4.77 SEM; 12.09 micrograms/g +/- 2.55) than in colonic carcinoma (16.06 micrograms/g +/- 3.46; 7.75 micrograms/g +/- 4.28). In ileal mucosa, percentages of lithocholic and deoxycholic acids were slightly higher in colonic carcinoma (3.9%; 23.2%) than in Crohn's disease (1.1%; 14.9%). In colonic mucosa, carcinoma patients had more lithocholic (7.6%) and less deoxycholic acid (11.9%) than patients with Crohn's disease (1.7%; 20.3%). Bile acid sulphate esters were similar in both diseases (ca. 3.0 micrograms/g in ileal, 1.4 micrograms/g in colonic mucosa). Our results show that ileal and right colonic mucosal nonsulphated bile acids tend to be even lower in right colonic carcinoma than in Crohn's disease. This agrees well with our earlier findings of low mucosal bile acid concentrations in patients with left colonic carcinoma (Tokai J Exp Clin Med 8: 59-69, 1983) and does not support the assumption that bile acids are envolved in right colonic carcinogenesis.

Oral bile acid treatment of biliary cholesterol stones.

Cholesterol gallbladder stones can be dissolved with chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). Response rate is 60-90%, dissolution rate 60% in stones not exceeding 1.5 cm in diameter. Mean treatment time amounts to 18 months. To improve oral litholysis: 1) UDCA was combined with the amino acid taurine, 2) CDCA and UDCA were administered in a single bedtime dose, 3) they were combined, each bile acid in half dosage, and 4) they were mixed with terpenes. Although there was some improvement with the combination therapy, final outcome is still suboptimal. Many investigations have been performed concerning gallbladder function, mucus production and nucleating factors, showing both that cholesterol supersaturation of bile is the conditio sine qua non for gallstone formation and that other factors play an additional, important role for the development of the first nidus. These factors have to be considered when therapy shall be improved. As yet oral litholysis has shown neither drug-related side effects nor lethality. It is not more expensive than surgery. Direct contact litholysis with methyl tert-butyl ether could reduce the indication for oral treatment to floating stones or patients who refuse gallbladder puncture. But although oral litholysis does not provide us with optimal results and needs further improvements, it will always keep its place in gallstone therapy.

Ursodeoxycholic acid therapy in primary biliary cirrhosis.

In a total of 1004 patients in 11 controlled trials, treatment with ursodeoxycholic acid (UDCA) 8-15 mg/kg bodyweight per day led to a decrease of pruritus in 30-60% of cases, a decrease in aminotransferases and cholestasis-indicating enzymes in serum by 20-80%, and a decrease of serum bilirubin by 3-40%. A statistically significant improvement in liver histology was found in only two of these studies; in three others there was a positive trend. In three more trials histology was not examined, and in three studies there was no improvement. In the four studies investigating the time elapsed before liver transplantation and the number of deaths, only one definitely found that this was prolonged by UDCA, although in two of the other three there was a positive trend. During treatment, UDCA constitutes 30-50% of the total bile acids in bile and serum; however, its influence on the toxic bile acids is debatable. Cholic acid decreases, but deoxycholic acid and chenodeoxycholic acid are reduced to a lesser degree. UDCA therapy has now been practiced for 12 years and all authors consider the treatment to be safe, but the mode of action of UDCA is still unknown.

[Comparative study of superoxide production of monocytes in primary biliary cirrhosis]. / A monocyták szuperoxid termelésének összehasonlító vizsgálata primer biliaris cirrhosisban.

Monocytes appear to play a role in immunological abnormalities observed in primary biliary cirrhosis (PBC). Monocytes not only produce fibroproliferative factors, such as IL-1, TNF, and PDGF but also produce superoxide anion which can directly damage tissues, and thus may lead to fibrosis. The aim of this study was to compare the superoxide production in monocytes obtained from 12 control persons, 9 patients with non biliary cirrhosis, 6 untreated PBC patients, 6 patients with gallstones under urso- and chenodeoxycholicacid (Lithofalk) treatment and 32 PBC patients under ursodeoxycholicacid (UDCA) therapy. Monocytes were isolated and the production of superoxide anions with and without phorbol-myristate-acetate (PMA) stimulation was determined. In two occasion, the monocytes from control patients were preincubated with 10, 50, 100 microM UDCA. Unstimulated monocytes from PBC patients under UDCA therapy produce an average 43% more and the PMA stimulated monocytes an average 42% more superoxide than monocytes from the control or from the other cirrhotic patients. The UDCA preincubation did not influence the superoxide production of monocytes obtained from control patients. These findings suggest that the increased activity of monocytes may also play a role in liver damage and fibrosis in PBC.

[Drug therapy of cholestatic hepatopathies]. / Medikamentöse Therapie cholestatischer Hepatopathien.

Ursodeoxycholic acid is suitable for the treatment of primary biliary cirrhosis and primary sclerosing cholangitis in a dosage of 10 mg/kg bodyweight per day. In primary biliary cirrhosis not only laboratory parameters but the histological picture as well are markedly improved. Best results are obtained in the early stages of the disease (stage I, II), but there is an improvement in the late stages (stage III, IV) too. A long-term follow-up has shown that bile salt therapy is possible without any side effects and with excellent results during a period of 10 years. Excellent results have been obtained in newborns with primary bile duct atresia and in patients with cystic fibrosis.

[Drug therapy of gallstone disease]. / Medikamentöse Therapie des Gallensteinleidens.

Cholesterol stones in the gallbladder can be dissolved in 60 to 90% by oral administration of chenodeoxycholic acid, ursodeoxycholic acid or the combination of both. The effect depend on the number and size of stones. Treatment is without any side effects and lethality. A great number and large stones can be dissolved by direct instillation of methyl-tert-butyl-ether (MTBE) into the gallbladder using the percutaneous transhepatic method. The success rate amounts to 90%, the average therapy duration is 9 hours. Solvents for chemolitholysis of pigmented stones are being tested.