Karl Decker

Experimental hepatitis induced by d-galactosamine

Abstract Repeated intraperitoneal injections of d -galactosamine produce within 24–48 hours in rat livers alterations that closely resemble human viral hepatitis. Histologic changes include necroses and inflammatory infiltration of periportal areas, mitoses and cell proliferation, the appearance of Councilman bodies, and an increased number of Kupffer cells. Serum levels of transaminases, glutamate-dehydrogenase, and other enzymes are greatly increased while total serum protein, including prothrombin, is reduced. No fatty infiltration of the liver occurs. Liver glycogen is rapidly depleted after galactosamine administration. The levels of several metabolites in liver, including adenine nucleotides and UDP-glucose, are significantly reduced. The ATP ADP- and lactate/pyruvate ratios remain in the normal range. These effects of galactosamine are dose dependent and can also be produced in other animals, though differences in quantitative response seem to exist. Several other chemically related compounds do not produce similar effects indicating a high specificity of d -galactosamine. Galactosamine hepatitis may serve as a biochemical and pathologic model.

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

Abstract The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum . The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H 2 O → 1 butyrate − + 2 HCO 3 − + 3 H + + 2 H 2 (1) Glucose + 4 H 2 O → 2 acetate − + 2 HCO 3 − + 4 H + + 4 H 2 (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases. The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations. The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP + .

Rat hepatic sinusoidal endothelial cells in monolayer culture: Biochemical and ultrastructural characteristics

Sinusoidal endothelial cells were isolated by collagenase-pronase digestion of rat livers followed by centrifugal elutriation. The main endothelial cell fraction consisted of more than 85% endothelial cells as shown by electron microscopy and enzyme histochemistry. Contamination by Kupffer cells was less than 5%. The endothelial cells formed a coherent stable monolayer on dishes coated with collagen type IV in the presence of an RPMI 1640 medium supplemented with 4% Ultroser. Fc receptors were undetectable immediately after elutriation but reappeared after 12 h in culture. Von Willebrand factor (formerly factor VIII-related antigen) could not be detected unequivocally by immunofluorescence. Unchallenged endothelial cells did not produce eicosanoids. In the presence of free arachidonate, however, prostaglandins D2 and E2 as well as thromboxane B2 and 6-keto-prostaglandin F1 alpha were detected by radioimmunoassay and by high-performance liquid chromatography analysis of [3H]arachidonate-exposed cells. Cells treated with the Ca2+ ionophore A23187 produced the same spectrum of immunologically measured prostanoids. In contrast to Kupffer cells in primary culture, eicosanoid formation by endothelial cells was neither triggered by phagocytotic stimuli nor suppressed by pretreatment with dexamethasone.

The release of tumor necrosis factor from endotoxin-stimulated rat Kupffer cells is regulated by prostaglandin E2 and dexamethasone

Evidence is presented that upon stimulation with endotoxin (lipopolysaccharide, LPS), Kupffer cells, the bodys largest pool of sessile macrophages, synthesize and liberate a factor whose immunological, cytotoxic and chemical properties are those described for tumor necrosis factor (TNF)-alpha. Hepatocytes and sinusoidal endothelial cells do not produce detectable amounts of this protein. Ten nanograms of LPS per ml medium are sufficient to stimulate a substantial release of this mediator. Recombinant interferon-gamma (rIFN gamma) per se is a poor inducer of TNF release. Costimulation with endotoxin and rIFN gamma shows only a slight increment in the release of this cytotoxic factor, relative to LPS alone. Exposure of Kupffer cells to the Ca2+ ionophore A23187 or to elicitors of the oxidative burst and superoxide production, e.g. zymosan or phorbol 12-myristate 13-acetate, stimulates only a fraction (20%) of the TNF release seen after endotoxin challenge. Prostaglandin E2, the synthesis of which is strongly enhanced after challenge of rat Kupffer cells with LPS, suppresses the release of TNF by these cells. This autoregulatory mechanism may explain the kinetics of TNF production by stimulated Kupffer cells. Dexamethasone is another important mediator capable of reducing the LPS-elicited TNF formation. An effect of the glucocorticoid hormone can still be provoked if it is added simultaneously with or shortly after LPS. This rapid action requires a mechanism that is different from the time-consuming one leading to the inhibition of prostaglandin synthesis in Kupffer cells.

Involvement of tumor necrosis factor in endotoxin-triggered neutrophil adherence to sinusoidal endothelial cells of mouse liver and its modulation in acute phase.

Tumor necrosis factor (TNF) has been shown to mediate lipopolysaccharide-induced neutrophil adhesion to liver sinusoidal endothelium in vivo. Female NMRI mice received either 5 micrograms lipopolysaccharide (R595) per animal alone (model A) or together with 116 mumol D-galactosamine (model B). One hour after injection, TNF activity in the serum was detectable to an equal extent in both models. Neutrophils in the liver, which had been identified by chloroacetate esterase staining of liver sections and quantitated by light microscopy, started to increase at 1 h and were elevated 10-fold above baseline at 6 h after application in (A) and (B). If 0.5 micrograms TNF instead of lipopolysaccharide was injected alone (model C) or together with D-galactosamine (model D), neutrophil influx into the liver was comparable to that observed in (A) or (B). Alanine aminotransferase activity in the serum was nearly normal in (A) and (C) 6 h after injection, while it reached levels up to 50-fold above baseline in models (B) and (D). This reflects the well-known D-galactosamine sensitization against lipopolysaccharide or TNF. Furthermore, degranulation of a large number of intrasinusoidal neutrophils could be observed 9 h after lipopolysaccharide-galactosamine injection. The administration of 116 mumol D-galactosamine per animal alone led neither to a measurable TNF activity in the serum nor to an increase in alanine aminotransferase activity or number of liver neutrophils. If the animals had received 50 microliter turpentine subcutaneously 24 h prior to lipopolysaccharide, TNF or D-galactosamine injection, the induced acute-phase reaction suppressed the increase of liver neutrophils in all models. Acute-phase reaction also prevented neutrophil degranulation and the rise of alanine aminotransferase in (B) to a great extent, while serum TNF activity was only minimally affected. It is concluded that TNF mediates neutrophil adhesion to the sinusoidal endothelium in vivo and that acute-phase reactants prevent lipopolysaccharide- or TNF-induced neutrophil influx into the liver.

Comparative study of cytotoxicity, tumor necrosis factor, and prostaglandin release after stimulation of rat Kupffer cells, murine Kupffer cells, and murine inflammatory liver macrophages.

Macrophages (MØ) and MØ‐depleted (nonadherent) nonparenchymal cells (NPC) of the liver were examined for their cytotoxic potential against tumor cells, production of tumor necrosis factor (TNF), and release of prostaglandins (PG) following stimulation by lipopolysaccharide (LPS), interferon‐γ (IFNγ), and zymosan.

Enzymic determination of uracil nucleotides in tissues

Abstract An enzymic assay for the sequential determination of UDP-glucose, UDP-galactose, UTP, UDP, and 5′-UMP in tissues is described. Two moles of NAD are reduced per mole of uracil nucleotide by use of UDPG dehydrogenase as indicator enzyme. The procedure is highly specific for the measurement of uridine 5′-phosphates. Snake venom phosphodiesterase is used for complete hydrolysis of nucleoside diphosphate sugars and nucleoside di- and tri-phosphates. Quantitative formation of 5′-UMP from uracil 5′-nucleotides and its enzymic measurement provide a specific determination of total uracil nucleotides. This principle can be applied for the quantitative enzymic estimation of total adenine 5′-nucleotides and of various UDP-sugars. The procedure uses commercially available enzymes and is highly reproducible, with standard deviations of less than 2%. Uracil nucleotides in normal and orotate-treated rat liver, in kidney, brain, and skeletal muscle of the rat, and in mouse liver, have been measured after freeze-stop of the tissues.

Regulation of endothelin-1 action on the perfused rat liver

Endothelin‐1 (ET‐1) was found to be a very potent stimulus for contraction and glycogenolysis in the perfused rat liver. At 1 nM it caused a dramatic increase in portal pressure of 22.1 ± 2.7 cm water and enhanced the glucose output up to 3‐fold. Extracellular Ca2+ and protein kinase C were involved in the signal transduction of ET‐1. ET‐1 action does not seem to be mediated by endogenous eicosanoids. The effects of ET‐1 were significantly reduced in the presence of 1 μM Iloprost, a prostaglandin I2 analogue, or by 100 μM sin‐1, a nitric oxide donor. In cultured hepatocytes, glycogenolysis was also stimulated by ET‐1 although to an extent too small to explain the high glucose output found in the perfused liver.

Liver restitution after acute galactosamine hepatitis: autoradiographic and biochemical studies in rats.

Abstract Autoradiographic and biochemical studies were performed to elucidate the restitution of rat liver after galactosamine hepatitis. The maxima of proliferation of Kupffer cells were seen after 25.5 hours, of periportal mesenchymal cells and of bile duct epithelia after 48 hours, and of hepatocytes after 72 hours, when galactosamine was given six times within 24 hours. A single injection produced a corresponding labelling pattern of the different liver cell types. The normalization of serum enzyme levels (GOT, SDH) and of the liver glycogen store was paralleled to the histologic signs of regeneration.

Toxicity of d-galactosamine for rat hepatocytes in monolayer culture

Hepatocellular injury was induced by exposure of primary cultures of rat hepatocytes to 4 mM D-galactosamine. The cell damage was very similar to that seen in vivo and in the isolated perfused rat liver, both in biochemical and in structural terms. The severity of the lesions caused by D-galactosamine was dependent on the age of the culture being treated. Less severe damage was found with older cultures. Since the primary metabolic effects of D-galactosamine were age-independent, the reduction in cell damage seems to be due to progressive cell dedifferentiation. Dexamethasone (1 microM) suppressed the full development of the injury, while 1 microM triiodo-L-thyronine enhanced it. A protection of hepatocytes by alpha 2-macroglobulin against the effects of D-galactosamine could be observed neither in vivo nor in vitro. Direct cytotoxic effects of endotoxin from Salmonella minnesota R 595 could be demonstrated only on hepatocytes in the early phases of primary culture using rather high doses of the purified lipopolysaccharide. It is unlikely that they play a major role in the hepatocellular injury seen following endotoxinemia in vivo. Lowering of extracellular Ca2+ concentration and additions of calcium/calmodulin inhibitors did not prevent cell injury after treatment with D-galactosamine. The results suggest that cell death is not due to an increased influx of Ca2+ into the cells.