[Pathophysiology of chronic diarrhea]. / Pathophysiologie der chronischen Diarrhöe.

The symptom of diarrhoea is defined as an abnormally frequent discharge from the bowel (more than 3 times a day) and a semisolid or fluid consistency of the faecal matter. Diarrhoea is termed chronic when it lasts more than four weeks. Diarrhoea is the result of disturbances in enteral water and electrolyte balance. Increased intestinal motility is usually not the cause but the result of diarrhoea. Transport of water through the gut is dependent on the osmotic gradient between interstitium and gut lumen. The secretion of chloride ions by the cells of the intestinal glands plays a major role in water secretion into the gut lumen, while sodium and potassium absorption in the villous zone of the enterocytes is crucial for enteral water absorption. Enteral water and electrolyte balance is regulated by the autonomic and enteral nervous system, by gastrointestinal hormones and signal messengers of mesenchymal cells. Pathogenetically, one distinguishes between secretory and osmotic diarrhoea. Furthermore, mixed forms of both pathogenic types can occur. The various types can be differentiated clinically and by the "osmotic gap". Diarrhoea can be a symptom of various diseases. Its pathogenesis is illustrated using examples of diarrhoea in pathological bile acid absorption, bacterial infections, carbohydrate malabsorption or disaccharidase insufficiency and in chronic inflammatory bowel disease.

Survival benefit of patients with inoperable hepatocellular carcinoma treated by a combination of transarterial chemoembolization and percutaneous ethanol injection--a single-center analysis including 132 patients.

Hepatocellular carcinoma (HCC) is one of the most severe sequelae of chronic liver disease. The only potentially curative therapeutic options are surgical resection and orthotopic liver transplantation. In most HCC patients, however, at clinical presentation the tumors are unresectable because of multicentricity or poor hepatic functional reserve due to pre-existing cirrhosis or not transplantable because of too advanced tumor stage or severe co-morbidity. In clinical practice, therefore, percutaneous ethanol injection (PEI) and transarterial chemoembolization (TACE) are widely used non-surgical therapeutic strategies. We prospectively analyzed the clinical factors determining the prognosis of 132 inoperable HCC patients and assessed the feasibility, therapeutic efficacy and safety of PEI, TACE and a combination thereof. Mean age of patients was 64 years; 95% of patients had liver cirrhosis and 39% were Okuda stage I, 48% stage II and 13% stage III. Fifteen patients were treated by PEI (group 1), 33 by TACE (group 2), 39 by TACE and PEI (group 3) and 45 received best supportive care (group 4). Survival correlated with the Child-Pugh class of liver cirrhosis and the Okuda stage of HCC. Favorable prognostic parameters were alpha-fetoprotein (AFP) levels <100 ng/ml and absence of portal vein thrombosis. Median survival time was 18 months in group 1 [interquartile range (IQR) 10-19], 8 months in group 2 (IQR 5-15), 25 months in group 3 (IQR 13-36) and 2 months in group 4 (IQR 1-9). Multivariate analysis revealed that patients treated with a combination of TACE and PEI have a significantly better survival than patients receiving either PEI or TACE only (p = 0.001). Patients with inoperable HCCs treated by the combination of TACE and PEI have a clear survival benefit. A favorable outcome can be expected in patients with compensated cirrhosis, a low Okuda stage, a baseline AFP level <100 ng/ml and absence of portal vein thrombosis.

[New hepatitis viruses]. / Neue Hepatitisviren.

Until 1987 only the hepatitis viridae A, B and D were discovered. All other viral agencies of hepatitis were summarized under the term non-A-non-B virus. By modern methods of molecular biology and recombinant gene technology, several new hepatitis viridac could be identified and their structure partly characterized. The hepatitis virus E (HEV) is predominantly transferred through the fecal-oral path, the hepatitis viridae C and G predominantly parenteral. The structure and molecular biology of these new hepatitis viridae as well as the pathogenesis, epidemiology, diagnosis, clinical manifestation, prophylaxis and therapy of the various types of hepatitis are described and compared with the wellknown hepatitis viridae A and B.

Uptake of 3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate into isolated rat hepatocytes by three transport systems.

Uptake of norcholansulfonate (3 alpha, 7 alpha, 12 alpha-trihydroxy-24-nor-5 beta-cholan-23-sulfonate), an isogeometric analogue of cholate into isolated rat liver hepatocytes occurs only by saturable transport. In order to identify the transport systems involved, uptake of norcholansulfonate was studied using 7 beta-NBD-NCT ({N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl})-2'-aminoethanesulfonate) as a competing substrate. For transport of both bile salt derivatives, which mutually inhibit their mediated transport competitively, the existence of at least three transport systems must be assumed. Uptake studies using the cloned hepatic Na+/cholyltaurine cotransporting polypeptide stably expressed in CHO cells (Chinese hamster ovary cells) showed that both bile salt derivatives were transported and furnished the definite KT values of this single transport system and the ratio of the maximal uptake velocities. On the basis of these data, uptake of both bile salt derivatives into rat hepatocytes and their mutual competitive inhibition could be analyzed for three transport systems. The maximal flux rates J2 and the half-saturation constants KT2 in the presence of Na+ (143 mM) are for norcholansulfonate: J1(Na+ 143) = 1.0 +/- 0.2 nmol/(min . mg protein), KT1(Na+ 143) = 15 +/- 4 microM, J2(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT2(Na+ 143) = 15 +/- 2 microM, J3(Na+ 143) = 0.5 +/- 0.2 nmol/(min.mg protein), KT3(Na+ 143) = 60 +/- 15 microM, and for 7 beta-NBD-NCT J1(Na+ 143) = 0.14 +/- 0.04 nmol/(min.mg protein), KT1(Na+ 143) = 3.1 +/- 0.5 microM, J2(Na+ 143) = 0.014 +/- 0.005 nmol/(min.mg protein), KT2(Na+ 143) = 21 +/- 2 microM, J3(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein), KT3(Na+ 143) = 190 +/- 25 microM. The kinetic parameters are in accordance with the assumptions that the cloned Na+/cholyltaurine cotransporting polypeptide represents transport system 2 and that the kinetically identified additional transport system 1 is either strictly or partially Na(+)-dependent.

Irreversible inhibition of hepatic fatty acid salt uptake by photoaffinity labeling with 11, 11-azistearate.

In order to have a model compound for detection of proteins involved in transport and metabolism of long-chain fatty acid salts by photoaffinity labeling 11,11-azistearate and 11,11-azi[G-3H]stearate (specific radioactivity 2.78 TBq/mmol) were synthesized. The suitability of 11,11-azi[G-3H]stearate for photoaffinity labeling was demonstrated by incorporation into BSA (bovine serum albumin) and H-FABP (hepatic fatty acid salt-binding protein) of rat liver. Repeated photoaffinity labeling resulted in a clear decrease of the binding capacities of both proteins. Labeling of protein mixtures with 11,11-azi[G-3H]stearate showed that binding proteins for long-chain fatty acid salts interact specifically with this probe. Photoaffinity labeling of isolated hepatocytes using 300 microM 11,11-azistearate in the presence of 100 microM BSA resulted in the irreversible inhibition of the uptake of stearate and its analogue 2,2,3,3,18,18,18-heptafluorostearate nearly to the same extent of about 30%. Irreversible inhibition of the uptake of long-chain fatty acid salts by photoaffinity labeling did not alter the mediated transport of cholyltaurine and has no effect on the uptake of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, a compound that crosses the hepatocyte membrane by simple diffusion. The irreversible inhibition of membrane transport by photoaffinity labeling demonstrates the existence of a specific transport system for the uptake of long-chain fatty acid salts into hepatocytes.

Inhibition of duck hepatitis B virus replication by 2',3'-dideoxy-3'-fluoroguanosine in vitro and in vivo.

The antiviral activity of 2',3'-dideoxy-3'-fluoroguanosine (FdG) or its triphosphate was evaluated in the duck hepatitis B virus (DHBV) system in vitro and in vivo. In primary DHBV-infected hepatocytes FdG results in a dose-dependent inhibition of viral replication with a nearly complete inhibition at a concentration of 1 microM. Also in vivo, FdG treatment of DHBV-infected ducklings reduces DHBV DNA replication by more than 90%. These data demonstrate that FdG is a strong inhibitor of DHBV replication in vitro and in vivo.

Synthesis and applicability of a photolabile 7,7-azi analogue of 3-sulfated taurine-conjugated bile salts.

For the identification of proteins involved in hepatobiliary transport, the photolabile derivative 7,7-ASLCT ((7,7-azi-3 alpha-sulfato-5 beta-cholan-24-oyl)-2'-aminoethanesulfonate) was synthesized. 7,7-ASLCT is taken up into liver and excreted into bile completely unmetabolized at a rate between the excretion rate of SLCT ((3 alpha-sulfato-5 beta-cholan-24-oyl)-2'- aminoethanesulfonate) and SCCT ((7 alpha-hydroxy-3 alpha-sulfato-5 beta- cholan-24-oyl)-2'-aminoethanesulfonate). The dependency of flux rate of uptake into freshly isolated hepatocytes on the concentration of 7,7-ASLCT in presence of Na+ (143 mM) and with Na+ depletion (1 mM) is best described by the assumption of two simple transport systems, the kinetic parameters of which are similar to those of SLCT. As studied in the presence of Na+, 7,7-ASLCT and SLCT exhibit a clearly competitive cross-inhibition of uptake with inhibition constants that are similar to the corresponding half-saturation constants. Photoaffinity labeling of isolated hepatocytes using 7,7-ASLCT (400 microM) resulted in the irreversible inhibition of the uptake of 7,7-ASLCT and SLCT to similar extents, confirming the kinetic data that 7,7-ASLCT is a true competing substrate for the uptake of SLCT. Because in intact rat liver 7,7-ASLCT and SLCT mutually inhibit their biliary excretion, the photolabile derivative shares with SLCT the same pathways in uptake and in excretion. Thus, 7,7-ASLCT should be appropriate for the study of hepatobiliary transport of sulfated and taurine-conjugated bile salts by photoaffinity labeling.

Functional significance of interaction of H-FABP with sulfated and nonsulfated taurine-conjugated bile salts in rat liver.

To identify the cytosolic proteins of rat hepatocytes involved in transcellular transport of sulfated and taurine-conjugated bile salts in comparison with only taurine-conjugated bile salts, photoaffinity labeling studies were performed with [3H]-7,7-ASLCT (7,7-azi-3 alpha-sulfatolithocholyl[2'-3H(N)]taurine), [3H]-7,7-ACT ((7,7-azi-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)- 2'-[2'-3H(N)]aminoethanesulfonate), and [3H]-7,7-ALCT (7,7-azilithocholyl[2'-3H(N)]taurine) using isolated hepatocytes and intact liver tissue. Photoaffinity labeling of isolated hepatocytes with [3H]-7,7-ASLCT on the one hand and with [3H]-7,7-ACT and [3H]-7,7-ALCT on the other resulted in a different labeling pattern of cytosolic polypeptides, without a relevant incorporation of radioactivity into subunits of glutathione transferases. This suggests that glutathione transferases play no role in the transport of dianionic or of monoanionic bile salts. With [3H]-7,7-ACT and [3H]-7,7-ALCT a moderate incorporation of radioactivity was found in polypeptides with apparent M(r)s of 33,000, 38,000, and 54,000, whereas with [3H]-7,7-ASLCT, a polypeptide with an apparent M(r) of 14,000, identified as H-FABP, was markedly and almost exclusively labeled. Photoaffinity labeling of specimens of intact liver tissue resulted in a labeling pattern of cytosolic polypeptides comparable to that obtained from photolabeled isolated hepatocytes. All results suggest that transcellular transport of dianionic sulfated as well as taurine-conjugated bile salts and of monoanionic taurine-conjugated bile salts follows different pathways. In intracellular transport of taurine-conjugated bile salts, several cytosolic polypeptides may have a function, whereas, in transport of taurine-conjugated 3 alpha-sulfato bile salts, only H-FABP appears to be involved.

Inhibition of proteolysis by cell swelling in the liver requires intact microtubular structures.

In the perfused rat liver, proteolysis is inhibited by cell swelling in response to hypo-osmotic media, glutamine and insulin. Colchicine, an inhibitor of microtubules, did not affect cell swelling in response to these agonists. However, the antiproteolytic action of these effectors was largely blunted in the presence of colchicine or the microtubule inhibitors colcemid and taxol. On the other hand, inhibition of proteolysis by phenylalanine, asparagine or NH4Cl, i.e. compounds which exert their antiproteolytic effects by mechanisms distinct from cell swelling, was not sensitive to colchicine. Swelling-induced inhibition of proteolysis was not affected by cytochalasin B. The anti-proteolytic effect of hypo-osmotic cell swelling and insulin was largely abolished in freshly isolated rat hepatocytes; however, it reappeared upon cultivation of the hepatocytes for 6-10 h. The restoration of the sensitivity of proteolysis to cell volume changes was accompanied by a progressive reorganization of microtubule structures, as shown by immunohistochemical staining for tubulin. It is concluded that intact microtubules are required for the control of proteolysis by cell volume, but not for the control of proteolysis by phenylalanine, asparagine or NH4Cl. These findings may explain why others [Meijer, Gustafson, Luiken, Blommaart, Caro, Van Woerkom, Spronk and Boon (1993) Eur. J. Biochem. 215, 449-454] failed to detect an antiproteolytic effect of hypo-osmotic exposure of freshly isolated hepatocytes. This effect, however, which is consistently found in the intact perfused rat liver, also reappeared in isolated hepatocytes when they were allowed to reorganize their microtubular structures in culture.

Pattern of bile acid regurgitation and metabolism during perfusion of the bile duct obstructed rat liver.

Bile acid processing in the long-term, bile duct obstructed rat liver was studied ex vivo. Twenty four and 72 h, respectively, after bile duct obstruction the isolated liver was perfused with taurodeoxycholate (16 nmol/min per g liver) the bile duct still being closed. Uptake, metabolism and regurgitation profile were traced by bolus injection of tritium-labeled bile acid; in addition, concurrent histological changes were examined by light- and electron microscopy. Ligation caused dilatation of the intrahepatic ductular branches and increased the serum bile acid concentration to 740 +/- 75 microM (controls: 16 +/- 2.12), reaching its maximum within 24 h. At 16 nmol/min per g liver uptake rate was > 96% in controls and in bile duct obstructed rats. Maximal uptake rates (assessed separately) differed between controls and bile duct obstructed rats (700 nmol/min per g liver vs. 460). Controls excreted more than 80% of labeled bile acid in bile within 10 min after bolus injection. Biliary recovery of label was virtually completed after 30 min. In bile duct obstructed rats excretion of label back to the perfusate effluent (regurgitation) started quantitatively 5 min after bolus application and peaked between 10 and 40 min; after 80 min, effluent recovery was incomplete (about 60% of bolus injected). Biliary bile acids of controls consisted of about 20% taurodeoxycholate-metabolites; bile acids in the perfusate effluent of bile duct obstructed rats of about 55%. The major metabolite in all animal groups was taurocholate; minor metabolites were tauroursocholate, tauro-3 alpha,7 = 0,12 alpha-cholanoic acid and 3-sulfo-taurodeoxycholate. Histologically, inflammation and periportal edema were present after 1 day of bile duct obstruction. After 3 days, marked proliferation of bile ductules was the dominant histological feature. It is concluded that during initial bile duct obstruction, bile acid processing is not altered, although ultrastructural alterations occur early.

Hepatitis B virus infection without immunological markers after open-heart surgery.

Post-transfusion hepatitis is still an important problem, despite the screening of blood donors for hepatitis B (HBV) and C virus infections. We assessed whether HBV DNA might be detected by PCR in prospectively collected serum samples of patients with unexplained post-transfusion hepatitis but no immunological HBV markers. We found HBV DNA in 4 (20%) of 20 patients with unexplained post-transfusion hepatitis and in 5 patients with mildly increased aminotransferases. The clinical course of these HBV infections was usually mild and self-limiting. Thus we found that low-titre, immunologically negative HBV infections do exist and might represent a significant cause of post-transfusion hepatitis.

Therapy of hepadnavirus infection using antisense oligonucleotides.

Chronic infection with the hepatitis B virus (HBV) is a major health problem worldwide. The only established therapy is alpha-interferon with an efficacy of only 30-40% in highly selected patients. Major theoretical problems of therapeutical strategies against hepadnaviral infections are the limited immune response and the presence of covalently closed HBV DNA in the nucleus. Many nucleoside analogues and inhibitors of viral reverse transcriptases were tested in vitro and in vivo with transient effects and often severe side effects. Molecular therapeutic strategies include antisense DNA/RNA and ribozymes. In vitro antisense oligodeoxynucleotides could be shown to inhibit viral replication and gene expression in human hepatoma cell lines. In vivo an antisense oligodeoxynucleotide directed against the 5'-region of the preS gene of the duck hepatitis B virus inhibited the viral replication and gene expression in ducks. These results demonstrate the potential clinical use of antisense DNA/RNA as antiviral therapeutics.

Modulation of phosphoenolpyruvate carboxykinase mRNA levels by the hepatocellular hydration state.

Exposure of isolated perfused rat livers to hypo-osmotic (225 mosmol/l) perfusion media for 3 h led to a decrease of about 60% in mRNA levels for phosphoenolpyruvate carboxy-kinase (PEPCK) compared with normo-osmotic (305 mosmol/l) perfusions. Conversely, PEPCK mRNA levels increased about 3-fold during hyperosmotic (385 mosmol/l) perfusions. The anisotonicity effects were not explained by changes in the intracellular cyclic AMP (cAMP) concentration or by changes of the extracellular Na+ or Cl- activity. Similar effects of aniso-osmolarity on PEPCK mRNA levels were found in cultured rat hepatoma H4IIE.C3 cells, the experimental system used for further characterization of the effect. Whereas during the first hour of anisotonic exposure no effects on PEPCK mRNA levels were detectable, near-maximal aniso-osmolarity effects were observed within the next 2-3 h. PEPCK mRNA levels increased sigmoidally with the osmolarity of the medium, and the anisotonicity effects were most pronounced upon modulation of osmolarity between 250 and 350 mosmol/l. The aniso-osmolarity effects on PEPCK mRNA were not affected in presence of Gö 6850, protein kinase C inhibitor. cAMP increased the PEPCK mRNA levels about 2.3-fold in normo-osmotic media, whereas insulin lowered the PEPCK mRNA levels to about 8%. The effects of cAMP and insulin were also observed during hypo-osmotic and hyperosmotic exposure, respectively, but the anisotonicity effects were not abolished in presence of the hormones. The data suggest that hepatocellular hydration affects hepatic carbohydrate metabolism also over a longer term by modulating PEPCK mRNA levels. This is apparently unrelated to protein kinase C or alterations of cAMP levels. The data strengthen the view that cellular hydration is an important determinant for cell metabolic function by extending its regulatory role in carbohydrate metabolism to the level of mRNA.