The frequent Marburg I polymorphism impairs the pro-urokinase activating potency of the factor VII activating protease (FSAP).

The recently reported plasmatic, Factor Seven Activating Protease (FSAP), has also been found to be a potent activator of pro-urokinase [single-chain plasminogen activator, urinary type (scuPA)]. An initial epidemiological study surprisingly showed that plasmas of 5-10% of healthy blood donors had an impaired potential to activate scuPA. Analysis of the respective genomic DNAs revealed one particular single nucleotide polymorphism of FSAP resulting in an identical amino acid exchange (G511E), which correlates with the reduced activities. The corresponding mutation was named FSAP Marburg I. Thrombelastographies of wild-type and mutant plasmas were performed, facilitating the auto-activation of the intrinsic FSAP pro-enzymes by addition of dextran sulfate (DXS) and accelerated clot lysis by addition of scuPA. On these conditions, tissue-factor-induced coagulation revealed that clot lysis was significantly delayed in the Marburg I mutant plasmas as compared with wild-type plasmas. Furthermore, in the presence of DXS and scuPA, a FSAP-deficient plasma revealed significantly prolonged plasma clot lysis times, whereas the addition of purified FSAP pro-enzyme plus scuPA reversed this effect. These results support the hypothesis that FSAP contributes to the scuPA-dependent plasma fibrinolytic potential, which can be impaired in plasmas containing the FSAP Marburg I polymorphism, for instance.

[Biotransformation of the lipoxygenase inhibitor 2-hydroxy-5-methyl-laurophenone-oxime (FLM 5011)]. / Untersuchungen zur Biotransformation des Lipoxygenasehemmers 2-Hydroxy-5-methyl-laurophenon-oxim (FLM 5011).

2-Hydroxy-5-methyl-laurophenone-oxime (FLM 5011, 1) is an inhibitor of the lipoxygenase with antiinflammatory and antiallergic actions. The studies on the biotransformation using in vivo investigations and in vitro test systems resulted in finding of at least eight metabolites. Four of these compounds have been detected and identified in urine and faeces after p.o. administration in male Wistar rats. By means of cultures of hepatocytes, lymphocytes and myeloma cells additional metabolites were found and the main pathways of metabolism could be suggested. Furthermore it was possible to confirm the sequence of the metabolic reactions. First of all, 1 is hydroxylated in the omega-position of the lauryl side chain by the cytochrome P-450 system. The further oxidation to the carboxylated compound is followed by the stepwise degradation of the side chain by beta-oxidation similarly to the pathways of fatty acid metabolism. Simultaneously the oxime group is converted to the keto group. The metabolites and 1 partly occur as sulfate or glucuronide conjugates. Additionally all compounds produced by beta-oxidation are conjugated with other partners, probably amino acids. By omega-oxidation, compounds with higher inhibitory potency on the lipoxygenase than the parent compound are formed. These results suggest that the activity of 1 is partly caused by the initial metabolites.

[The biotransformation of the immunostimulant 3-(2-mercaptoethyl)quinazoline-2,4(1H,3H)-dione (AWD 100-041)]. / Untersuchungen zur Biotransformation des Immunstimulans 3-(2-Mercaptoethyl)chinazolin-2,4(1 H,3 H)-dion (AWD 100-041).

3-(2-Mercaptoethyl)quinazoline-2,4(1 H,3 H)-dione (1; AWD 100-041) is a substance with immunomodulating and immunorestorative activity. After p.o. administration in male Wistar rats at least 7 metabolites are formed and excreted in urine and faeces. The compounds were isolated and identified on the basis of UV and mass spectra. They are S-methylated structures in which sulfoxidation and ring-hydroxylation have been taken place. Four metabolites are also present as sulfate or glucuronide conjugates. The quantity ratio of the phase I to phase II metabolites amounts to 4:1. In the isolated perfused rat liver and rat hepatocyte culture 6 and 5 of the in vivo identified compounds are formed. The sequence of the metabolic pathways could be confirmed by in vitro experiments in which the incubation of synthetically prepared metabolites and the identification of generated biotransformation products were performed. In the lymphocyte and myeloma cell culture solely the disulfide of 1 is formed. After incubation of the S-methyl compound metabolites originate detectable also in vivo. Regarding the main ways of metabolism firstly 1 is attacked by methyltransferases forming the initial metabolite. After that oxidative processes take place leading to the formation of sulfoxides, sulfones as well as ring-hydroxylated compounds. A part of the ring-hydroxylated metabolites are conjugated.

[The biotransformation of the anticonvulsant 4-p-chlorophenylpyrrol-3-morpholino-2-carboxylic acid methyl ester (AWD 140-076)]. / Untersuchungen zur Biotransformation des Antikonvulsivums 4-p-Chlorophenylpyrrol-3-morpholino-2-carbonsäuremethylester (AWD 140-076).

4-p-Chlorphenylpyrrole-3-morpholino-2-carboxylic acid methylester (1; AWD 140-076) is a substance with anticonvulsive properties. After p.o. administration in male wistar rats many metabolites in relatively small concentration are excreted in urine and faeces, six of them could be isolated and identified as quantitative dominating compounds. Compound 1 is attacked in different sites of the molecule by the cytochrome P-450 system. At the morphine ring N- and O-dealkylation reactions take place leading to the cleavage of the ring. After that a stepwise degradation by oxidative and reductive processes occurs. Further reactions concern the N-oxidation of the morpholine nitrogen as well as the hydroxylation of the pyrrole skeleton forming the main metabolites. 5 metabolites are also present as sulfate or glucuronide conjugates. The quantity ratio of the phase I to phase II metabolites amounts to 9:1. In the in vitro test system isolated perfused rat liver and rat hepatocytes culture solely the two main metabolites are formed. Compound 1 is characterized by enzyme inducing activities. The oxidative demethylation of p-nitroanisole is increased 4-fold after pretreatment.

[Comparative studies on the cultivability and morphology of rat hepatocytes isolated by trypsin or collagenase]. / Vergleichende Untersuchungen zur Kultivierbarkeit und Morphologie von Rattenhepatozyten, die mittels Trypsin oder Collagenase isoliert wurden.

In the present study rat hepatocytes isolated either by trypsin or by collagenase were investigated concerning its cultivability and its morphological properties. The cultivation was carried out as monolayer for 24 h. The yields of cells prepared by trypsin or collagenase amounted to 8 x 10(7) cells per liver and 15-30 x 10(7) cells per liver, respectively, with viabilities measured by trypan blue exclusion test of 70-80 and 90-95%, respectively. Nonhepatocytes were not taken into consideration. Using the electron microscopy it could be established that both freshly isolated and 24 h cultured hepatocytes were intact. There were no morphological differences between cells isolated by trypsin and cells isolated by collagenase. After 24 h cultivation hepatocytes prepared by trypsin showed a little tendency in forming a slightly flattened appearance and in forming intercellular contacts.