[Extrahepatic manifestations of liver diseases: skeleton]. / Extrahepatische Manifesta- tionen von Leberkrankheiten: Skelett.

Since nowadays liver diseases are being diagnosed more frequently than in former years, an increasing number of patients with skeletal alterations is detected. Predominant is osteoporosis with the feared complications of bone fractures. The measurement of bone mineral density enables us to differentiate between normal, osteopenic and osteoporotic conditions. Despite the great number of data available on bone disease we are still incapable to predict which patients with liver disease will develop osteoporosis or bone fractures and when. Treatment should focus on the prevention of development of osteoporosis and fractures and rather not on the treatment of complications. We are still lacking convincing long-term treatment studies with a sufficient number of patients who are well stratified.

Ursodeoxycholic acid in the therapy for primary biliary cirrhosis: effects on progression and prognosis.

The effects in clinical studies of UDCA on the endpoints "death" or "pre-transplantation survival" can only be shown when UDCA therapy is started in an early disease phase, preferably in stage I but no later than stage II, and is then continued into stages III/IV, or preferably stage IV. The reasons for this lie in the observation that, in stages I/II, no patient suffers from progressive disease that irrevocably leads to death or transplantation, while a measurable effect of UDCA, as is true for other drugs and other hepatic diseases, continues to dwindle and finally disappears as patients progress through the fibrotic and cirrhotic stages III and IV. Hence, administration of UDCA must begin in the phase of progressive inflammation (stages I and II) and the outcome documented after many years of long-term therapy. This requires very large, probably unattainable, patient collectives. Whether it is justified to administer placebo to one-half of these patients over such an extended period of time represents a profound ethical dilemma. Because these arguments were not considered in the two meta-analyses cited above or in any other study, they do not allow a definitive statement on the life expectancy of patients on UDCA therapy. On the other hand, it is possible using generally accepted, independent prognostic variables and mathematical models, whose limitations are well-known and must be considered, to predict with a high degree of accuracy the disease course of treated and untreated patients and calculate their life expectancy and/or pre-transplantation survival. Because UDCA exerts a significant positive effect on the most important prognostic markers for PBC, such as serum bilirubin, piecemeal necroses, histological disease progression, ascites and edema, and apparently the scores for pruritus and fatigue, this permits us to demonstrate not only a decrease in the incidence of transplantation but also to calculate a prolongation in life expectancy.

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.

Medical treatment of primary biliary cirrhosis and primary sclerosing cholangitis.

Treatment of primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) with ursodeoxycholic acid (UDCA) has been in common use since 1985. In PBC, treatment with UDCA improves laboratory data, liver histology, enables a longer transplantation-free interval and prolongs disease survival. Because UDCA is unable to cure the disease newer drugs or combination therapies are still needed. Studies with UDCA and immunosuppressants such as prednisone, budesonide and azathioprine have shown that in selected patients combination therapy may be superior to UDCA monotherapy. PSC is treated successfully with UDCA and endoscopic dilatation of the bile duct strictures. Treatment of extrahepatic manifestations of cholestatic liver disease such as pruritus, fatigue, osteoporosis and steatorrhea can be problematic and time-consuming.

[Cholestatic hepatopathy: primary biliary cirrhosis]. / Cholestatische Hepatopathie: Primär biliäre Zirrhose.

Primary biliary cirrhosis is a cholestatic autoimmune disease of the liver. Its prevalence has significantly increased over the last seven years, therefore it can no longer be considered to be a rare liver disease. It is seen in 94/100,000 women older than 40 years. The treatment consists of administration of ursodeoxycholic acid, better therapy results seem to be achieved by combining UDCA with immunosuppressants. While UDCA-monotherapy proved to extend the time until liver transplantation and thus leads to life prolongation, this could not yet be shown for the combination therapies. Therapy of the often agonizing pruritus and of osteoporosis remains problematic. Frequently this can only be managed by the combination of several treatment measures.

Characterisation of patients with primary biliary cirrhosis responding to long term ursodeoxycholic acid treatment.

BACKGROUND: In some patients with primary biliary cirrhosis, ursodeoxycholic acid causes full biochemical normalisation of laboratory data; in others, indexes improve but do not become normal. AIMS: To characterise complete and incomplete responders. METHODS: Seventy patients with primary biliary cirrhosis were treated with ursodeoxycholic acid 10-15 mg/kg/day and followed up for 6-13 years. RESULTS: In 23 patients (33%) with mainly stage I or II disease, cholestasis indexes and aminotransferases normalised within 1-5 years, except for antimitochondrial antibodies. Histological findings improved. Indexes were not normalised in 47 patients (67%) although the improvement of their biochemical functions parallelled the trend in the first group. In these incomplete responders histological findings improved to a lesser extent. The only difference between the two groups before treatment was higher levels of alkaline phosphatase and gamma glutamyl transpeptidase in the incomplete responders. At onset of treatment the discriminant value separating responders from incomplete responders was 660 U/l for alkaline phosphatase and 131 U/l for gamma glutamyl transpeptidase. One year later it was 239 and 27 U/l (overall predictive value for responders 92%, for incomplete responders 81%). There were no differences between the two groups concerning immune status, antimitochondrial antibody subtypes, liver histology, or any other data. HLA-B39, DRB1*08, DQB1*04 dominated in both groups. CONCLUSIONS: In patients with mainly early stages of primary biliary cirrhosis, higher values of alkaline phosphatase and gamma glutamyl transpeptidase are the only biochemical indexes which allow discrimination between patients who will completely or incompletely respond to ursodeoxycholic acid treatment.

Oral budesonide and ursodeoxycholic acid for treatment of primary biliary cirrhosis: results of a prospective double-blind trial.

BACKGROUND & AIMS: Ursodeoxycholic acid (UDCA) is used for treatment of primary biliary cirrhosis. Previous studies showed that, compared with UDCA monotherapy, bile salts plus prednisolone had no further effect on laboratory data but improved liver histology. Thirty percent of these patients had prednisolone-related side effects. Budesonide is a glucocorticoid with a high receptor affinity and a high first-pass metabolism. In this study we investigated whether budesonide and UDCA are superior to UDCA monotherapy. METHODS: A 2-year prospective, controlled double-blind trial was performed. Twenty patients (mainly with early-stage disease) were treated with UDCA at a dose of 10-15 mg/kg daily in addition to 3 mg budesonide 3 times daily (group A), and 19 patients (1 dropped out for personal reasons) were treated with UDCA plus placebo (group B). Liver biopsy specimens were taken before, after 12 months, and at the end of study. Glucose tolerance tests, serum cortisol levels, and adrenocorticotropin-stimulated cortisol secretion were assessed at regular intervals. Bone mass density was measured by dual-energy photon absorptiometry. RESULTS: Compared with pretreatment values, liver enzyme and immunoglobulin M and G levels decreased significantly in both groups. Improvement in group A was significantly more pronounced (P < 0.05) than in group B. Titers of antimitochondrial antibodies did not change. In group A, the point score of liver histology improved by 30.3%; in group B, it deteriorated by 3.5% (P < 0.001). Changes in bone mineral density after 2 years were -1.747% in group A and -0.983% in group B (P = 0.43). Budesonide had little influence on the hypothalamic-pituitary-adrenal axis. One patient in group A had budesonide-related side effects; in 3 patients in group B, complications of liver disease developed. CONCLUSIONS: Combination therapy with UDCA and budesonide is superior to UDCA and placebo.

Exon-intron organization of the human multidrug-resistance protein 2 (MRP2) gene mutated in Dubin-Johnson syndrome.

BACKGROUND & AIMS: The Dubin-Johnson syndrome is characterized by conjugated hyperbilirubinemia and by impaired secretion of anionic conjugates from hepatocytes into bile. Absence of the multidrug-resistance protein 2 (MRP2; symbol ABCC2), an adenosine triphosphate-dependent conjugate export pump, from the hepatocyte canalicular membrane is the molecular basis of this syndrome. The aim of this study was the elucidation of all exon-intron boundaries of the MRP2 gene as a prerequisite for the analysis of mutations in patients with Dubin-Johnson syndrome. METHODS: Exon-intron boundaries of MRP2 were determined, and the amplified exons were screened for mutations. Immunofluorescence microscopy served to localize the MRP2 protein in human liver. RESULTS: The human MRP2 gene is approximately 45 kilobases long; it contains 32 exons and a high proportion of class 0 introns. In 2 patients with Dubin-Johnson syndrome, we detected a nonsense mutation at codon 1066 and a 6-nucleotide deletion mutation affecting codons 1392-1394. The MRP2 protein was absent from the canalicular membrane of both patients. CONCLUSIONS: The mutations detected so far show that various mutations in the MRP2 gene can lead to the Dubin-Johnson syndrome. The exon-intron boundaries established in this article will facilitate the analysis of additional mutations in the MRP2 gene.

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.

Small and large unilamellar vesicle membranes as model system for bile acid diffusion in hepatocytes.

Uptake of bile acids into the liver cell occurs via active transport or passive diffusion. In a model system, passive diffusion was studied in liposomes using pyranine fluorescence. Rate constants for the diffusion of diverse more polar or more apolar bile acids were examined. Hydrophobic lithocholic acid (LCA) revealed a maximal rate constant of 0.057 s(-1); with the polar ursodeoxycholic acid (UDCA), the value was 0.019 s(-1). UDCA (3 mol%) effectively decreased the rate constant of 0.1 mM chenodeoxycholic acid (CDCA), whereas cholesterol reached a similar decrease only between 5 and 10 mol%. At higher concentrations of CDCA (above 1 mM) or LCA (0.3-0.4 mM), breaking up of liposomal structure was confirmed by light-scattering decrease and increase of carboxyfluorescein fluorescence. Changes in lipid composition of phosphatidylcholine (PC)- small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) also caused decreasing rate constants. For a cardiolipin (CL):PC ratio of 1:20 the CDCA (0.1 mM) rate constant was 71% lower (0.015 s(-1)) and for a sphingomyelin (SM):PC ratio of 2:1 the rate constant was 50% lower (0.026 s(-1)). Changes in membrane fluidity were detected using membrane anisotropy measurements with the 1,6-diphenyl-1,3, 5-hexatriene (DPH) method. Membrane fluidity was reduced with cholesterol- but not with CL- or SM-containing SUVs (ratio: cholesterol, CL, SM:PC of 1:5). This model system is currently used for the analysis of more complex lipid vesicles resembling the plasma/hepatocyte membrane, which is either stabilized or destabilized by appropriate conditions. The results should become clinically relevant.

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.

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.

[Cholestasis: drug therapy]. / Cholestase: Medikamentöse Therapie.

For the medical treatment of cholestasis plant alkaloids, phenobarbital and S-adenosyl-L-methionine (SAMe) have been used. Although the mode of action of these substances is understood in part, the treatment of patients was unsuccessful. In contrast, ursodeoxycholic acid, a physiologically occurring bile acid in man, was successful. The daily dosage of ursodeoxycholic acid is 10-15 mg/kg bodyweight. Best results have been obtained in primary biliary cirrhosis where symptoms improved markedly, the cholestasis-indicating enzymes and immunoglobulin M decreased significantly. After long-term therapy even liver histology improved. Recently it has been shown that ursodeoxycholic acid prolongs the interval to liver transplantation. Ursodeoxycholic acid has to be taken lifelong, because interruption of therapy, even after long periods of continued treatment will induce a rebound of cholestasis. Ursodeoxycholic acid therapy is without side effects. In patients with primary sclerosing cholangitis ursodeoxycholic acid prolongs life expectancy only in combination with endoscopic bile duct dilatation.

Dissolution of gallbladder stones with methyl tert-butyl ether and stone recurrence: a European survey.

Since there are now several ways to treat symptomatic gallstone disease, one is able to select treatment on the basis of the patient's comfort, the practicability, effectiveness, and side effects of the technique, and the relative costs. In order to assess the present status of contact dissolution with methyl tert-butyl ether with regard to these aspects, the present enquiry reports the data of 21 European hospitals. Eight hundred three patients were selected for contact litholysis of cholesterol gallbladder stones using methyl tert-butyl ether. Percutaneous transhepatic puncture of the gallbladder was performed under x-ray or ultrasound guidance. Dissolution rate, side effects, and treatment times of 268 patients from one single center were compared to those of 535 patients from the other 20 centers. Two hundred sixty-four patients were followed for five years to assess stone recurrence. Physicians were asked how they assessed the expenditure of the method, the discomfort to the patients, and the staffing situation. Patients were asked to indicate their acceptance on an analog scale. Puncture was successful in 761 (94.8%) patients. Prophylactic administration of antibiotics was not necessary. Stones were dissolved in 724 (95.1%) patients. In 315 (43.5%) sludge remained in the gallbladder. The most severe complication was bile leakage, which led 12 (1.6%) patients to have elective cholecystectomy. Toxic injuries due to the ether were not reported. Method-related lethality amounted to 0%, 30-day-lethality to 0.4%. Stone recurrence rate was about 40% in solitary stones and about 70% in multiple stones over five years. Patients with multiple stones developed recurrent stones almost twice as often as those with solitary stones. The probability of stone recurrence in patients with sludge in the gallbladder after catheter removal was not statistically significantly different from those without sludge. Seventy to 90% of the centers found the puncture to be simple and not distressing for patients and the relation between expenditure and therapeutic success to be acceptable. The acceptance of contact litholysis by the patients was excellent. Contact litholysis when applied by an experienced team provides real advantages in the treatment of gallstone disease. The method is technically simple, well accepted by the patients, and can be easily applied in community hospitals. Contact litholysis may be of particular value in patients who are not suitable for anesthesia or surgery.

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.

Chemolysis of gallbladder debris left over after contact litholysis with methyl tert-butyl ether.

Rapid and safe gallbladder clearance from residual, post-MTBE stone debris is believed to be absolutely necessary to reduce stone recurrence after contact litholysis. Because the clearing effect of prokinetic agents is considered an uncertain postdissolution trial, we investigated by in vitro experiments whether and to what extent debris from various cholesterol and "mixed stones" could be removed by direct (topical) chemolysis. Debris from radiolucent cholesterol stones could be dissolved very easily, using the aqueous solvent S-01, composed of EDTA-2Na (1-2%), lauryl sulfobetaine-12 (0.1 M), and 0.1 M sodium carbonate/boric acid buffer, pH 9,5. Its dissolution capacity (DC) was 8.06 +/- 2.3 mg debris/ml and its dissolution efficacy (DE) was 16.2 +/- 4.6 mg debris/ml/hr. Debris from mixed, slightly to moderately calcified stones needed another treatment with S-05, composed of sodium citrate (0.25 M), lauryl sulfobetaine-12 (0.01 M), and citric acid. The initial pH was 5.2. The DC of S-05 ranged from 1.61 +/- 1.1 (debris enriched with Ca-phosphate) to 3.94 +/- 1.3 mg/ml (debris enriched with Ca-carbonate). Stones which did not respond immediately to MTBE because of a thin rim of inorganic or/and organic Ca salts could be made ready for MTBE litholysis by pretreatment with S-01 or S-05 or with a combination of both solvents. Debris containing large portions of biliary mucus could be dissolved most effectively by successive application of S-01 and S-06 (2 M urea).