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.

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.

Ursodeoxycholic acid and prednisolone versus ursodeoxycholic acid and placebo in the treatment of early stages of primary biliary cirrhosis.

BACKGROUND: Ursodeoxycholic acid probably is not able to cure primary biliary cirrhosis. Therefore in this study ursodeoxycholic acid was administered together with prednisolone, since monotherapy with glucocorticoids has been shown to have some positive effects. METHODS: Thirty patients with primary biliary cirrhosis (stages I-III) were entered into the study. Fifteen were treated with ursodeoxycholic acid 10 mg.kg-1.day-1 and placebo (group A), 15 with ursodeoxycholic acid and 10 mg prednisolone (group B) for 9 months. Apart from the usual laboratory examinations, liver biopsies were taken from 29 patients before and after therapy. RESULTS: Liver enzymes decreased significantly compared to the initial values in both groups (p < 0.001), but in group B cholestasis-indicating enzymes and the immunoglobulins G and A improved more rapidly. Between both groups the differences for AP, GGT, IgG, IgA and gamma-globulins were significant (p < 0.05), but only for short terms. In group B, liver histology improved significantly (p < 0.003), which correlated with the decrease of IgG. Ursodeoxycholic acid became the predominant bile acid in the serum. Toxic bile acids did not increase. Bone densitometry revealed a slight deterioration of preexisting osteoporosis in one patient. CONCLUSIONS: Although combination therapy with ursodeoxycholic acid and prednisolone was not superior to monotherapy with ursodeoxycholic acid with regard to liver function tests, it had a highly beneficial influence on liver histology. In our previous trials with monotherapy histology remained unchanged. An early decrease in IgG during combination therapy seems to be an indicator of an amelioration of liver histology.

[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.

TUDCA and UDCA are incorporated into hepatocyte membranes: different sites, but similar effects.

The aim of this paper is to point out that: 1) CDCA and DCA increase the polarity of cell membranes and cause the release of cholesterol and phospholipid from the membranes; 2) the extent of this damage is inversely correlated with the cholesterol content of the membrane investigated; 3) UDCA, TUDCA and GUDCA decrease membrane polarity; 4) they prevent membrane damage when added prior to CDCA or DCA; 5) UDCA appears to be incorporated into the apolar domain of the membrane, TUDCA, GUDCA into the interface; 6) UDCA decreases HLA class I expression on hepatocyte membranes; 7) CDCA induces GLDH-release from liver mitochondria and increases mitochondrial membrane polarity and mobility; and 8) UDCA reduces the release of GLDH from mitochondria caused by CDCA.

Crigler-Najjar syndrome type II. New observation of possible autosomal recessive inheritance.

The inheritance of Crigler-Najjar syndrome type II (CNS II) is still unclear. Both autosomal dominant transmission with variable penetrance and autosomal recessive transmission have been reported. We describe the diagnosis of CNS II in an adult patient with unconjugated serum bilirubin levels up to 19.6 mg/dl and no detectable activity of bilirubin UDP-glucuronosyltransferase in the liver biopsy. Serum bilirubin levels decreased markedly on phenobarbital treatment. The parents of our patient are first cousins. The mother and three of the patient's five sibs were jaundiced within a few days of birth. Our patient and her jaundiced siblings have 11 children, all healthy and anicteric. We conclude from these data that the inheritance of this very rare disease follows an autosomal recessive pattern, with pseudodominance in this family.

Effects of ursodeoxycholic acid after 4 to 12 years of therapy in early and late stages of primary biliary cirrhosis.

Twenty-two patients with primary biliary cirrhosis were treated with ursodeoxycholic acid, 10 mg/kg per day. Fourteen patients with stages I/II were treated for 4-12 years (mean 7.5), and eight patients with stages III/IV for 5-12 years (mean 6.5). Twelve of 13 patients with early stages became asymptomatic. Aminotransferases, cholestasis-indicating enzymes and IgM improved (p < 0.01) and remained low during the whole treatment period. Ursodeoxycholic acid was the predominant serum bile acid, and lithocholic acid did not increase in the serum but did increase in the stool. Of eight patients with stages III/IV, seven were symptomatic, and four became asymptomatic. In all eight patients, laboratory data improved. Of these eight patients three experienced haemorrhage from oesophageal varices, two had to be transplanted, and one of them died. In one patient splenic rupture occurred, and in three liver function tests deteriorated. Although the number of patients was small, this is the longest treatment period so far reported. Ursodeoxycholic acid had no side effects for up to 12 years, and in patients with early stages it seemed to have a beneficial effect on symptoms and the progression of the disease. However, even with up to 12 years of therapy, ursodeoxycholic acid did not cause antimitochondrial antibodies to disappear either in the early or in the late stages, it was unable to prevent rebound effects during therapy intermission even after more than 5 years of continuous therapy, there was no decisive influence on liver histology and it did not cure the disease. Finally, although ursodeoxycholic acid improved life quality and laboratory data in all patients with late stages of the disease, it did not prevent complications due to cirrhosis.

Manual and automatic gallstone dissolution with methyl tert-butyl ether.

The aim of the study was to establish the efficiency of cholesterol gallstone dissolution with methyl tert-butyl ether in a large group of patients and to compare the results of patients treated manually by a nurse or using an automatic pump. Gallbladder puncture was successful in 228 patients (99%). After 9 hr, 211 patients (91%) were stone-free; 144 (68%) of them left the hospital on the fourth day. In radiolucent stones not isodense with bile on a CT scan, dissolution rate decreased by 10%, treatment time was prolonged by 40%. Forty-two of the 228 patients were selected for the hand-syringed group, 42 patients, who matched these patients in stone size and number, were treated with an automatic pump (Baxter). Stone burden in matched pairs was comparable. Stones dissolved in 96% of the patients in both groups. Sludge remained in the gallbladder in 52% after manual treatment and 60% after automatic therapy. Side effects were identical in both groups. None of the side effects were pump-related. Automatic therapy reduced the time needed by the nurse to treat each patient by 70%.

Molecular aspects of membrane stabilization by ursodeoxycholate [see comment].

BACKGROUND: Ursodeoxycholate, used for therapy in biliary liver diseases, prevents bile salt damage in animal experiments. Using isolated red blood cell and both canalicular and basolateral hepatocyte membranes, the present study examined this protective effect. METHODS: Membranes were incubated with chenodeoxycholate, with chenodeoxycholate and ursodeoxycholate simultaneously, and first with ursodeoxycholate followed by chenodeoxycholate. Changes in membrane structure were investigated by electron paramagnetic resonance spectroscopy, using different spin labels. Data were confirmed by analysis of membrane lipids and studies with 14C-labeled bile acids. RESULTS: The increase of polarity in the apolar domain of the membrane caused by chenodeoxycholate corresponded to the amount of solubilized lipids. After preincubation with ursodeoxycholate or its conjugates, membrane damage by subsequent chenodeoxycholate incubation was prevented. This effect was caused by binding of ursodeoxycholate in the apolar domain, of its conjugates in the interface of the membrane. CONCLUSIONS: Chenodeoxycholate solubilizes membrane lipids and permits water to permeate into plasma membranes. The steroid nucleus of ursodeoxycholate is bound to the apolar domain and that of the conjugates to the interface of the membrane, thus stabilizing membrane structure.

[In vitro contractility of the musculature of human gallbladders with and without gallstones--relevance of the prostaglandin system for CCK regulated motoricity]. / In-vitro-Kontraktilitätsverhalten humaner Muskulatur von Gallenblasen mit und ohne Steinerkrankung--Relevanz des Prostaglandinsystems die CCK-regulierte Motorik.

This study describes the influence of endogenous and exogenous prostaglandins upon CCK-induced motility patterns of human gallbladders with and without stones (indomethacin and nocloprost; an exogenous PGE2-analogon). From 48 gallbladders with- and 22 gallbladders without stones (control group) longitudinal muscle stripes were dissected and transferred to an organ bath and CCK, indomethacin and nocloprost dose response curves were established. In another experimental protocol, the effect of CCK after indomethacin or nocloprost preincubation is demonstrated. Moreover, specimens of gallbladders were taken for histology and gallstones for analyse. The results demonstrate that gallbladders with stones have a significant higher basic tonus and phasic activities compared to the stone-free controls. Because of these different responses to CCK, gallbladders of the stone-diseased group were divided in two groups: 64% of the gallbladders show a sensitivity and tonic response to CCK like the controls (contractors), 36% demonstrate a reduced sensitivity to CCK and only a slight tonic response (non-contractors). Indomethacin causes a fall in tonus in both stone-diseased groups. It stops spontaneous activity in the contractor and non-contractor group. With indomethacin preincubation all three groups response to CCK with a significant reduced sensitivity. CCK-induced activity is reduced in the control and contractor group. In the non-contractor group, muscle strips do not contract after indomethacin preincubation. Nocloprost induces significant contractions in the control and contractor group. In both groups, the response to CCK after nocloprost preincubation is stronger than the reaction without preincubation. In the non-contractor group, a change in tonus after nocloprost application cannot be demonstrated, there also is no response to CCK after nocloprost preincubation. These results corroborate the notion of a significant contribution of the endogenous prostaglandin system to the regulation of gallbladder motility by CCK.

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.

Cholic acid and ursodeoxycholic acid therapy in primary biliary cirrhosis. Changes in bile acid patterns and their correlation with liver function.

We treated 6 patients with Stage II primary biliary cirrhosis with cholic acid (CA) 10 mg.kg-1 per day for 3 months and then with the same dose of ursodeoxycholic acid (UDCA). A matching group of 6 patients was observed for 3 months without any therapy. Liver function tests and serum and stool bile acids were investigated before, during and at the end of CA and UDCA therapy. The results of liver function tests deteriorated after 6-8 weeks of CA therapy and the changes were correlated (r = 0.92) with an increase in alpha-dihydroxy-bile acids (chenodeoxycholic acid and deoxycholic acid) in the serum. The 24 h excretion of DCA in 24 h faeces was markedly increased. Ursodeoxycholic acid treatment improved liver function tests; after 4 weeks glutamate dehydrogenase (GLDH) had decreased. After 8-12 weeks of therapy ursodeoxycholic acid had increased to 50-60% of the total serum bile acids whereas the more apolar bile acids were significantly decreased. No changes in liver function tests or bile acid metabolism were found in the untreated group. Since CA and UDCA are non-toxic in man, this trial indicates that the apolar bile acids chenodeoxycholic acid and deoxycholic acid may be responsible for the deterioration of liver function in primary biliary cirrhosis. However, the therapeutic effect of UDCA cannot be explained merely by the decrease in alpha-dihydroxy-bile acids in the serum, since the laboratory results had improved prior to the decrease in the serum apolar bile acids.

Treatment of chronic active hepatitis and primary biliary cirrhosis with ursodeoxycholic acid.

Ursodeoxycholic acid (UDCA) improves liver function tests in patients with chronic active hepatitis (CAH) and primary biliary cirrhosis (PBC). UDCA will reduce biochemical parameters of both cholestasis and hepatocellular damage. The effects may be less beneficial in patients with advanced stages of chronic liver disease: in PBC we found the improvement of laboratory parameters in stage I and II very impressive, in stage III and IV it was less marked. Data of two controlled trials in PBC showed an improvement in liver histology, in one study the improvement was statistically significant. UDCA can be administered for at least 10 years without any adverse effects, the treatment is safe and improves life quality. The mode of action of UDCA seems to be in its displacement of toxic hydrophobic bile salts from the bile acid pool and the hepatocellular membrane. In in-vitro investigations a direct protective effect of UDCA on isolated sinusoidal hepatocyte membranes against toxic bile salts has been shown. This protective effect of a more general nature may explain the efficacy of UDCA in different chronic, especially cholestatic liver diseases.

Experimental dissolution of pigment gallstone material using alkaline EDTA and adjuvant bile salts/non-bile salt detergents, thiols and urea, with respect to local chemolitholysis.

In order to enhance the dissolution capacity and the kinetics of topical solvents used in local pigment chemolitholysis, a series of dissolution experiments was performed with intact brown and black pigment stones and with standardized solutes such as pigment stone powder and compressed powder (static disc method). The basic dissolution medium was a 0.1 M boric acid/sodium carbonate buffer (pH 9.5), and the basic lytic agent was EDTA-4Na, working satisfactorily at 1-3 g/dl. It could be demonstrated that the dissolution efficiency of this basic solvent was enhanced significantly in the presence of a detergent (surfacant) and of urea. Among the detergents the zwitterionic (e.g., Sulfobetain-12) and the nonionic types (e.g., a polyoxyethylene ether like Lubrol PX) proved to be most effective. The adjuvant effect of the investigated thiols was disappointing. Only dithioerythritol/dithiothreitol and N-acetylcysteine showed any moderate, if consistent, lytic activity. The highest dissolution rates in dissolving compressed powder standards (disc method) were achieved with the ternary solvent (1% w/v EDTA/80 mM Lubrol PX/1 M urea, pH 9.5). Intact black pigment stones, well known as problematic candidates for chemolitholysis, could be largely dissolved up to approximately 70% of their initial weight. This was not merely a physical disintegration, but a chemical process.

[Posterior exenteration with plastic reconstruction in advanced vulvar cancer]. / Die hintere Exenteration mit plastischer Wiederherstellung beim fortgeschrittenen Vulvakarzinom.

Between 1984 and 1990, twenty-four patients suffering from advanced primary (7) or recurrent (17) valvular carcinoma were treated by posterior exenteration and myocutaneous reconstruction of the defect. During a follow-up of 34 months, the overall survival was 49%. Eight patients died. Tumour diameter and tumour-free margins of the resected specimens were the most important prognostic factors. In all cases, an improved quality of life could be achieved by the operation. It is concluded that, even in extremely difficult situations, posterior exenteration, followed by plastic reconstruction entails an essential benefit for the patient.

[The effect of pH and amount of antacids on bile acid binding in a quasi-natural reflux milieu]. / Einfluss von pH und Antazidummenge auf die Gallensäurenbindung in quasinatürlichem Refluxmilieu.

Bile acid adsorption may be one therapeutical mechanism of antacids. Little is known about the effect of pH and amount of antacid on bile acid adsorption. Therefore we carried out the following investigations using a lattice [correction of lettuce] layer antacid as a model substance. 5 ml of "quasi-natural reflux milieu" were mixed with 0.5, 1 or 2 ml of hydrotalcite and adjusted to pH 3, 5 or 7. The highest total bile acid adsorption was found at pH 3, the degree of bile acid adsorption correlated with bile acid lipophilicity, i.e. the most lipophilic and toxic bile acids are adsorbed best. High adsorption of lipophilic and particularly toxic bile acids even at low gastric pH may help to explain the good therapeutic effect of low-dose antacids in gastric ulcer.

Gallstone dissolution with methyl tert-butyl ether in 120 patients--efficacy and safety.

Of 612 patients with cholesterol gallbladder stones, 120 were eligible for percutaneous transhepatic litholysis with methyl tert-butyl ether (MTBE). Puncture of the gallbladder was successful in 117/120 (97.5%). In 113/117 (96.6%) the stones dissolved. With solitary stones, treatment lasted for an average of 4 hr, with multiple stones 10 hr. Mean hospitalization was 3.6 days. In 3/117 (2.6%) patients a bile leakage developed; 33% reported mild complaints. After the end of treatment 34% had some residue in the gallbladder; two of these patients developed recurrent stones. MTBE is exhaled, is distributed in fatty tissue, and is excreted renally together with its metabolite tert-butanol. Methanol was found only in traces. Gallbladder histology of six patients showed chronic cholecystitis. Since these findings were independent of treatment time and the interval between treatment end and operation, they are most consistent with stone-related changes rather than caused by MTBE.

[Endoscopy, shockwave lithotripsy and local lysis in complicated pigmentary calculi of extra- and intrahepatic bile ducts]. / Endoskopie, Stosswellenlithotripsie und lokale Lyse bei komplizierten Pigmentsteinen der extra- und intrahepatischen Gallengänge.

Endoscopy, extracorporeal shockwave lithotripsy (ESWL) and local lysis with alkaline solution of EDTA and bile salts in water were applied in combination in four patients with extra- and intrahepatic pigment stones as well as calcium bilirubinate covered concrements of the biliary tract. In the first patient (a man aged 80 years) a giant concrement of the bile duct was broken up after ESWL by three weeks of local chemical lysis and the fragments were removed by endoscopy. In the second case (man, aged 72), a nonextractable pigment stone was at first reduced in size by four-day local lysis and then removed endoscopically. Intrahepatic pigment stones were completely removed in the other two patients (boy of 12, man of 62) by local lysis only in 3 and 15 weeks, respectively. Even long-term use of the alkaline solution may not cause any serious side effects. Breaking up of stones after size reduction with ESWL of giant stones, size reduction of intact stones and contact lysis of intrahepatic stones are three important indications for chemical dissolution of biliary tract stones, respectively.