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Biomedical subjects

D Keppler

Publications and source records attributed to D Keppler.

At least 145 records · Page 8Linked to original sources
PubMed1986

Metabolism and analysis of cysteinyl leukotrienes in the monkey.

Predominant hepatobiliary elimination from blood and subsequent enterohepatic circulation of cysteinyl leukotrienes is demonstrated in the monkey Macaca fascicularis. From intravenous [3H]leukotriene C4, about 40% were recovered as metabolites in bile and about 20% in urine within 5 h. [3H]Leukotriene E4 was a predominant metabolite of defined structure in blood plasma, bile, and urine. From intraduodenal [3H]leukotriene C4, about 5% were recovered as metabolites in bile and about 8% in urine within 8 h. Endogenous cysteinyl leukotrienes generated in vivo were measured after implantation of a subcutaneously looped biliary bypass. Tapping of the loop allowed access to bile and prevented interference by leukotrienes produced by surgical trauma (Denzlinger, C., Rapp, S., Hagmann, W., and Keppler, D. (1985) Science 230, 330-332). Endogenous cysteinyl leukotrienes were analyzed in bile, urine, and blood plasma by the sequential use of high-performance liquid chromatography and a radioimmunoassay that was optimized for leukotriene E4 as a predominant metabolite detected in the tracer studies. Biliary leukotriene E4 rose from less than 0.2 to 9 nmol/liter, when leukotriene synthesis was elicited in anesthesized monkeys by staphylococcal enterotoxin B administered intragastrically. This study provides an approach to the analysis of cysteinyl leukotrienes in primates and serves to define the role of these mediators under pathophysiological as well as physiological conditions in vivo.

Animals↗
PubMed1986

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

Animals↗
PubMed1986

Requirement for lipopolysaccharide-responsive macrophages in galactosamine-induced sensitization to endotoxin.

Injection of D-galactosamine sensitizes mice many thousand-fold to the lethal action of endotoxin (lipopolysaccharide [LPS]). Comparable sensitization was practically absent in LPS-resistant C3H/HeJ mice, which after D-galactosamine treatment were about 500,000 times less sensitive to LPS lethality than histocompatible LPS-sensitive C3H/HeN mice. D-Galactosamine induces changes in the hepatocytes of treated animals, such as depletion of UTP and alterations in the pattern of UDP sugars. These early biochemical changes, which are necessary for development of sensitization, were similar in both mouse strains which we examined. High sensitivity to the lethal effects of LPS was achieved in C3H/HeJ mice after D-galactosamine treatment by transfer of C3H/HeN macrophages obtained in culture from bone marrow precursor cells.

Animals↗
PubMed1986

Inhibition of the cathepsin B like proteinase by a low molecular weight cysteine-proteinase inhibitor from ascitic fluid and plasma alpha 2 macroglobulin.

The cathepsin B like proteinase present in ascitic fluid of a patient with neoplasia has been purified and characterized after pepsin activation. From this fluid we have prepared the low molecular weight (LMW) cysteine-proteinase inhibitors. Three major inhibitor forms were found with isoelectric points of 7.4, 5.4, and 5.1, respectively. The interaction of the enzyme with the former inhibitor was studied because this inhibitor was the most abundant. The Ki value was found to be 4.3 X 10(-8) M. Two molecules of this proteinase were bound by one molecule of plasma alpha 2 macroglobulin (alpha 2M). The LMW inhibitor was able to bind to the enzyme entrapped in alpha 2M and reduced its endopeptidase activity on benzyloxycarbonyl-L-phenylalanyl-L-arginine-4-methyl-7-coumarylamide. These results may have a physiological significance in the regulation of the enzyme which, among other extracellular hydrolases, probably plays an important role in tumor invasion.

Ascites↗
PubMed1986

Effects of D-glucosamine and 6-azauridine on nucleotide contents, 5-fluorouridine uptake, and cytotoxicity in TA3 mammary tumor cells.

In TA3 mammary carcinoma cells in suspension culture, D-glucosamine X HCl (GlcN) induced a diversion of uridylate from UTP into UDP-N-acetylhexosamines, reducing the intracellular pool of UTP and eliciting an increased rate of de novo uridylate synthesis. This rise in de novo synthesis was completely suppressed by addition of 6-azauridine (6-AzaUrd) to the cell suspension in vitro or in the solid TA3 mammary tumor in NMRI mice in vivo. A synergistic depletion of UTP pools to less than 6% of the UTP in controls was observed in TA3 cell suspensions exposed to GlcN and 6-AzaUrd. In solid TA3 tumors in vivo, UTP was reduced by this combination to 19% of the control value. A high sensitivity of the solid tumor to inhibition of pyrimidine synthesis de novo was indicated by the reduction of the UTP content after administration of 6-AzaUrd alone. UTP deficiency in TA3 tumor cells was accompanied by CTP deficiency. In addition, 6-AzaUrd caused a lowering of GTP in the neoplastic tissue. Host liver was resistant to 6-AzaUrd but responded to treatment with GlcN with a decrease in UTP to 67%. Uridine-cytidine kinase was less inhibited in the presence of lowered UTP and CTP, which are potent feedback inhibitors of the enzyme, and enabled an enhanced formation of phosphorylated derivatives of 5-fluorouridine (FUrd). Aside from the formation of 5-fluoro-UTP, we have identified 5-fluoro-UDP-N-acetylhexosamines (FUDPHexNAc), which accumulated when FUrd and GlcN were sequentially administered. Treatment of TA3 cells with FUrd after a pretreatment with 6-AzaUrd and GlcN resulted in a 2.5-fold increase in [14C]FUrd uptake and a duplication of 5-fluorouridylate incorporation into the RNA. The proportion of FUDPHexNAc increased to 58% of the phosphorylated FUrd metabolites, as compared to 6% in TA3 cells exposed exclusively to FUrd. In vivo chemotherapy of mice bearing TA3 ascites tumors was most effective with respect to tumor growth inhibition and animal survival when GlcN and FUrd were combined.

Adenosine Triphosphate↗
PubMed1985

Leukotrienes as mediators in tissue trauma.

A significant increase in the production of cysteinyl leukotrienes was observed after mechanical or thermal trauma in the anesthesized rat. The amount of biliary N-acetyl-leukotriene E4, which represents a suitable indicator for blood plasma leukotrienes, was used as a measure of leukotriene generation. Cysteinyl leukotrienes were rapidly eliminated from blood plasma into bile where N-acetyl-leukotriene E4 was the major metabolite. Leukotrienes were at a much lower concentration in blood plasma than in bile and differed in the pattern of metabolites. The detected amounts of leukotrienes were sufficient to induce known phenomena associated with trauma, such as tissue edema and circulatory and respiratory dysfunction. Increased leukotriene generation appears to play an important role in the pathophysiology of tissue trauma.

Animals↗
PubMed1985

Production of peptide leukotrienes in endotoxin shock.

Arachidonate metabolites are potent mediators generated in endotoxin shock. Following endotoxin administration (15 mg/kg) 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.

Animals↗
PubMed1985

Uridylate-trapping sugar analogs in combination with inhibitors of uridylate synthesis de novo and 5-fluorouridine.

The combination of 3 types of antipyrimidines was studied in AS-30D hepatoma cells in suspension culture and in the rat in vivo. Cellular UTP and CTP pools can be depleted most effectively by combining an inhibitor of de novo UMP synthesis with sugar analogs diverting UMP to UDP-sugar analogs. The following UMP-trapping sugar analogs were employed: D-galactosamine, D-galactosone, D-glucosone, and D-glucosamine. These D-galactose and D-glucose analogs intensified the depletion of UTP and CTP pools induced by the following inhibitors of de novo UMP synthesis: acivicin, PALA, lapachol, pyrazofurin, and 6-azauridine. The sugar analogs, in the absence of inhibitors of the de novo pathway, enhanced the rate of de novo UMP synthesis several-fold, as indicated by incorporation of 14CO2 into intermediates and products of the pathway and by the expansion of the acid-soluble uracil nucleotide pool. Reduction of UTP and CTP contents to less than 5 and 15% of control, respectively, by D-galactosamine and PALA resulted in a decrease of the rate of RNA synthesis to 19% of control as calculated from the changes in specific activities of [14C]CTP and of [14C]cytidine in RNA after labeling with [14C]uridine. Hepatoma cells released uridine and cytidine into the extracellular fluid. This release was reduced to one third in UTP-deficient cells, indicating that pyrimidine nucleoside excretion is regulated by pyrimidine nucleotide levels, possibly by UTP and CTP regulation of uridine kinase. Determination of the rates of de novo pyrimidine synthesis, of the formation of RNA pyrimidines, and of pyrimidine nucleoside excretion indicates that de novo synthesis provides only about 67% of the pyrimidines required for the consuming processes. The difference, as well as the dilution of labeled pyrimidine nucleotide pools under conditions of a blocked de novo pathway, suggests a considerable salvage of pyrimidine nucleosides derived from RNA. This salvage of pyrimidines may be intracellular and/or by an excretion and re-uptake process. Depletion of UTP and CTP pools, induced in hepatoma cells by D-galactosamine and 6-azauridine, leads to growth inhibition in suspension culture; this inhibition becomes irreversible in an increasing percentage of cells, killing all cells after 20 hr of UTP deficiency. The enhanced uptake of 5-fluorouridine by UTP-deficient cells was associated with an increase of FUMP incorporation into RNA up to 4-fold and with stronger inhibition of cell growth.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Potentiation of antimetabolite action by uridylate trapping.

Uridylate-trapping analogs of D-galactose or D-glucose divert the uridylate moiety of UDPglucose and/or UTP to UDP-sugar analogs that accumulate while the pools of UTP and of related pyrimidine nucleotides are depleted. The uridylate-trapping action of sugar analogs is determined by the enzyme pattern of the target tissue. D-Galactose analogs are preferentially metabolized by hepatoma cells and hepatocytes. TA3-mammary tumor cells are susceptible to the action of D-glucosamine and other D-glucose analogs. A high rate of de novo pyrimidine synthesis and/or an active salvage of extracellular uridine compensate for the uridylate-trapping action of sugar analogs and prevent depletion of UTP pools. Accordingly, synergistic actions are induced by combining sugar analogs, such as D-galactosamine or D-glucosamine, with inhibitors of de novo pyrimidine synthesis, such as lapachol or 6-azauridine. Uridylate-trapping by D-galactosamine, acting on hepatocytes, shifts the balance between uridine consumption and uridine release by the liver and results in a fall of uridine and cytidine concentrations in blood plasma (20). Cultured hepatocytes produce uridine and cytidine. Combination of 5-fluorouridine with sugar analogs results in the formation of fluorinated UDP-sugar analogs in hepatoma cells or in mammary tumor cells. Formation of FUDP-sugar analogs transiently removes intracellular FUTP, but FUDP can be released subsequently in glycosyltransferase reactions (22). Pretreatment of hepatoma cells or of TA3-mammary tumor cells with an uridylate-trapping sugar analog in combination with an inhibitor of de novo pyrimidine synthesis enhances the uptake of 5-fluorouridine, its incorporation into RNA, and its growth inhibitory effect. The chemotherapeutic action of 5-fluorouridine in rats and mice, carrying the AS-30D and the TA3 ascites tumor, respectively, is significantly improved by pretreatment with an amino sugar together with 6-azauridine.

Amino Sugars↗
PubMed1985

First clinical experiences with a selective chemotherapy of hepatocellular carcinoma with 5-fluorouridine in combination with other antipyrimidines.

Sixteen unselected patients with non-resectable hepatocellular carcinoma were treated in a phase I study with 261 cycles of D-galactosamine and 6-azauridine prior to 5-fluorouridine. Thirty % of the patients survived for more than one year without signs of tumor progression and with an unchanged performance status. The compatibility of this chemotherapeutical method was quite satisfactory. The only extrahepatic side effect was a leucopenia and/or thrombocytopenia which was reversible upon reduction of the 5-fluorouridine dose. The heterogeneity of the 16 patients treated to date does not allow a definite statistical evaluation of the reported clinical observations and results. A final decision about the clinical applicability of this concept of a selective chemotherapy of hepatocellular carcinoma requires further experience.

Adult↗
PubMed1985

[Regulation of neoplasm-specific cathepsin B by cysteine-protease inhibitors present in cancerous exudates].

The Cathepsin B-like proteinase, a secretory form of lysosomal cathepsin B, is present in some cancerous exudates (i.e. pleural and ascitic fluids). It has been suggested that this enzyme may be involved in the invasive process, one of the most important aspects of cancer pathology. In the same fluids, two kinds of cysteine-proteinase inhibitors (C.P.I.'s) are found- HMr-CPI (90,000 daltons) and LMr-CPI's (11,000-13,000 daltons). The Cathepsin B-like enzyme is more strongly inhibited by the LMr-CPI's than by the HMr-CPI, the Ki values are 4.0 X 10(-9) M and 1.2 X 10(-7) M respectively. Theses results underline the similarity between this enzyme and the lysosomal Cathepsin B. On the other hand, the inhibitors could play a protective role against tumor invasion.

Ascitic Fluid↗
PubMed1984

D-glucosamine-induced changes in nucleotide metabolism and growth of colon-carcinoma cells in culture.

Human colon-carcinoma cells were exposed to D-glucosamine at 2.5, 5 and 10 mM, concentrations that were growth-inhibitory but not cytocidal in the presence of a physiological glucose concentration. Labelling of these HT-29 cells with D-[14C]-glucosamine, followed by nucleotide analyses, demonstrated that UDP-N-acetyl-hexosamines represented the major intracellular nucleotide pool and the predominant metabolite of the amino sugar. D-[14C]Glucosamine was not a precursor of UDP-glucosamine. After 4h exposure to D-glucosamine (2.5 mM), the pool of UDP-N-acetylhexosamines was increased more than 6-fold, whereas UTP and CTP were markedly decreased. UDP-glucuronate content increased by more than 2-fold, whereas purine nucleotide content was little altered. Uridine (0.1 mM) largely reversed the decrease in UTP, CTP, UDP-glucose and UDP-galactose, while intensifying the expansion of the UDP-N-acetylhexosamine pool. Uridine did not reverse the D-glucosamine-induced retardation of growth in culture. A 50% decrease in growth also persisted when uridine and cytidine, cytidine alone, or UDP, were added together with D-glucosamine. The growth-inhibitory effect of the amino sugar could therefore be best correlated with the quantitative change in the pattern of sugar nucleotides, and, in particular, with the many-fold increase in UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine.

Adenocarcinoma↗
PubMed1984

Role of peptide leukotrienes and their hepatobiliary elimination in endotoxin action.

The lethal action of endotoxin was studied in mice sensitized against the lipopolysaccharide by D-galactosamine. Protection was obtained by FPL 55712, a selective antagonist of sulfidopeptide leukotrienes, by diethylcarbamazine, an inhibitor of leukotriene biosynthesis, by BW 755C, a dual lipoxygenase and cyclooxygenase inhibitor, and by dexamethasone, which inhibits arachidonate release. Indomethacin incompletely antagonized endotoxin lethality; indoprofen did not protect at all. Leukotriene generation induced by endotoxin in vivo could be demonstrated in rats by employing a radioimmunoassay on bile extracts since intravascular sulfidopeptide leukotrienes were rapidly eliminated into bile. Additionally, however, endotoxin affected the hepatobiliary elimination of sulfidopeptide leukotrienes. From intravenously injected tracer [3H]leukotriene D4, 67% appeared only partially metabolized in bile within 30 min in control rats. Endotoxin and lipid A reduced the biliary [3H]leukotriene D4 secretion by up to 80% while enhancing the hepatic [3H]leukotriene D4 content up to twofold. This inhibition of hepatobiliary elimination was stronger than endotoxin-induced reductions of bile flow or of biliary [14C]taurocholate secretion. Endotoxin, as an activator of the arachidonate cascade, thus potentiates its action in vivo by interfering with the rapid hepatobiliary clearance of sulfidopeptide leukotrienes in addition to stimulating leukotriene and prostanoid formation.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1981

[Approaches to a selective chemotherapy of hepatocellular carcinoma (author's transl)].

1. An improvement of the chemotherapy of hepatocellular carcinoma with adriamycin or 5-fluorouracil and a reduction of side effects has been achieved by intra-arterial administration of the drugs. This treatment provides a somewhat extended survival but no cure. 2. The treatment of hepatocellular carcinoma in patients by reduction of an inactive precursor of a cytocidal alkylating agent by azoreductase of the tumor showed no therapeutic effect. 3. A selective hepatocellular uptake of drugs coupled to asialoglycoproteins has been described. An application of this concept for the chemotherapy of hepatocellular carcinoma seems doubtful since a loss of binding proteins for desialylated glycoproteins during experimental hepatocarcinogenesis has been demonstrated. 4. The increased uptake of 5-fluorouridine in hepatomas after induction of a tissue-specific depletion of uridine 5'-triphosphate and cytidine 5'-triphosphate provides an effective experimental chemotherapy with limited side effects. A clinical use of this new concept for the chemotherapy of hepatocellular carcinoma may serve as a useful approach.

Antineoplastic Agents↗