Dietrich Keppler

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.

Production of peptide leukotrienes in endotoxin shock

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 ) into unanesthetized rats, we found a rapid biliary secretion of peptide leukotrienes. Analysis of bile for peptide leukotrienes included organic solvent extractions, reversed phase‐HPLC, radioimmunoassay (RIA), and spectrophotometry. The major immunoreactive endogenous leukotriene (LT) from bile was eluted between LTC4 and LTD4 in three chromatographic systems. It corresponded thereby to a biliary metabolite of injected LTC4 and LTD4 which in turn showed the ultraviolet spectrum of a peptide leukotriene. This demonstration of endotoxin‐induced generation of peptide LTs in vivo was possible by sequential HPLC and RIA analyses in bile into which peptide LTs are eliminated from blood.

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.

Identification of the major endogenous leukotriene metabolite in the bile of rats as N-acetyl leukotrience E4

Mercapturic acid formation, an established pathway in the detoxication of xenobiotics, is demonstrated for cysteinyl leukotrienes generated in rats in vivo after endotoxin treatment. The mercapturate N-acetyl-leukotriene E4 (N-acetyl-LTE4) represented a major metabolite eliminated into bile after injection of [3H]LTC4 as shown by cochromatography with synthetic N-acetyl-LTE4 in four different HPLC solvent systems. The identity of endogenous N-acetyl-LTE4 elicited by endotoxin in vivo was additionally verified by enzymatic deacetylation followed by chemical N-acetylation. The deacetylation was catalyzed by penicillin amidase. Endogenous cysteinyl leukotrienes were quantified by radioimmunoassay after HPLC separation. A N-acetyl-LTE4 concentration of 80 nmol/l was determined in bile collected between 30 and 60 min after endotoxin injection. Under this condition, other cysteinyl leukotrienes detected in bile by radioimmunoassay amounted to less than 5% of N-acetyl-LTE4. The mercapturic acid pathway, leading from the glutathione conjugate LTC4 to N-acetyl-LTE4, thus plays an important role in the deactivation and elimination of these potent endogenous mediators.

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.

The regulation of pyrimidine nucleotide level and its role in experimental Hepatitis

Abstract The GalN-induced liver injury is presented as an experimental tool for studies on the regulation of uridylate biosynthesis and on the metabolic consequences of changes in uracil nucleotide contents of the liver. The primary biochemical lesion consists of a deficiency of uridine phosphates and UDP-hexoses for a defined minimum period of time. Evidence is presented for a stimulation of uridylate de novo synthesis by release of feedback inhibition during the deficit period. Orotate administration results in a cycloheximide-sensitive increase of the activities of OMP pyrophosphorylase and OMP decarboxylase. The stimulated rate of uridylate biosynthesis is positively correlated to the activities of both enzymes under a variety of conditions. The intensity and the duration of the deficiency of uridine phosphates depend on the relative capacities of uridylate producing and consuming processes. The quantitation of the deficit period and its relation to changes of UMP biosynthesis and of UTP- and UDP-hexose-dependent processes enable an understanding of the effect of measures which protect against GalN hepatitis, of the existence of GalN-refractory conditions, and of the different susceptibilities of various species and organs to this amino sugar. If a critical value of the uridylate deficit period is surpassed, irreversible liver cell damage is consistently observed several hours later. Under these conditions, RNA synthesis is strongly depressed. The reduction of guanosine incorporation into RNA can be relieved within a short time by uridine administration. The concept of the deficit period appears to be a valuable approach to biochemical pathology.

w-Oxidation products of leukotriene E4 in bile and urine of the monkey

The intravenous administration of [3H]leukotriene C4 in the monkey Macaca fascicularis results in the biliary and urinary elimination of [3H]leukotriene D4 and [3H]leukotriene E4 in addition to more-polar metabolites. Separation of these polar metabolites and chromatographic comparison with synthetic w-oxidized leukotrienes indicated the in vivo formation of w-hydroxy-[3H]leukotriene E4 and w-carboxy-[3H]leukotriene E4. Time course studies of the [3H]leukotriene metabolite pattern in bile and urine showed that w-hydroxy-leukotriene E4 was decreasing as w-carboxy-leukotriene E4 and additional polar derivatives were increasing.

Changes in uridine nucleotides during liver perfusion with D‐galactosamine

The first steps of galactosamine metabolism in liver follow the pathway of D-galactose [ 1,2]. Galactosamine-l-phosphate is converted by UDPG: galactose-l-phosphate uridylyltransferase (EC 2.7.7.12) to UDP-galactosamine [2], whose epimerisation to UDPglucosamine was shown by Maley and Maley [3]. UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine were identified as further metabolites of galactosamine in rat liver [4] . In this communication we report that the perfusion of isolated rat livers with galactosamine leads to a strong decrease in several uridine nucleotides (UDPG, UTP, UDP). Evidently this is due to a trapping of uridine phosphates by formation of UDP-aminosugars. The investigation of these effects in the isolated perfused liver contributes to an understanding of galactosamine-induced hepatitis which was shown to be closely related to human viral hepatitis [5,6].

Increased formation of nucleotide derivatives of 5-fluorouridine in hepatoma cells treated with inhibitors of pyrimidine synthesis and D-galactosamine

Fluorinated pyrimidines and their nucleosides are widely used in the chemotherapy of tumors; their mode of action requires the formation of nucleotide derivatives (summa~ [I ] ). Uridine kinase (EC 2.7.1.48) plays a key role in the conversion of 5fluorouridine (5-FUrd) to its active metabolites. The feedback regulation of uridine kinase by UTP [2] can be used for an increase in the cell-specific uptake of some pyrimidine analogs after depletion of the cellular UTP pools [3] . Induction of selective UTP deficiency by D-galactosamine (GalN) and 6azauridine (6aza-Urd) in hepatoma cells [4] is associated with an increased formation of phosphorylated derivatives of 5-FUrd [3]. This approach [3] has the following implications: (i) Considerable selectivity for hepatoma cells and hepatocytes is based on the action of GalN [3,5] ; (ii) The increased activity of uridine kinase [6,7] and the high rate of de nova pyrimidine nucleotide synthesis in hepatoma cells relative to liver [7-IO] makes these malignant cells more susceptible to the action of 6aza-Urd. (iii) The formation and accumulation of 5-fluoxoUDP-amino sugars may serve as a metabolic store providing 5-FUrd phosphates; the latter become available in amino sugar transferase reactions or upon hydroly tic splitting of the fluorinated amino sugar nucieotide. In this communication we demonstrate the negative correlation between 5-FUrd uptake and intracellular UTP level. The replenishment of

Activity and distribution of the enzymes of uridylate synthesis from orotate in animal tissues

Abstract 1. 1.|Activity and distribution of orotidine 5′-monophosphate pyrophosphorylase (EC 2.4.2.10) and orotidine 5′-monophosphate decarboxylase (EC 4.1.1.23), were studied. The activities of both enzymes catalyzing uridylate biosynthesis from orotate were measured by the formation of 14 CO 2 from [ carboxy - 14 C]orotate and orotidine 5′-monophosphate (OMP), respectively. Improved conditions for their determination are given, including the use of excess EDTA, for the assay of OMP pyrophosphorylase with endogeneous OMP decarboxylase as indicator enzyme. 2. 2.|The subcellular distribution of both enzymes in rat liver indicates an exclusive localization in the cytosol. 3. 3.|The specific activity of OMP pyrophosphorylase in different rat tissues was found to decrease in the following order: regenerating liver > spleen > liver > gut > kidney > brain > skeletal muscle. The order of OMP decarboxylase activity was: spleen > regenerating liver > liver > kidney > gut > brain > skeletal muscle. 4. 4.|The hepatic activities of the two enzymes were decreasing during the post-natal development. 5. 5.|In the livers of different animal species the specific activities of both enzymes were highest in mice, lowest in guinea pigs, and intermediate in chicken and rats. 6. 6.|Under a variety of conditions, including fast growing tissues, OMP pyrophosphorylase and OMP decarboxylase showed closely parallel, possibly coordinate changes in activity.