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Biomedical subjects

V Ullrich

Publications and source records attributed to V Ullrich.

At least 127 records · Page 7Linked to original sources
PubMed1985

A new monooxygenase product from 7-ethoxycoumarin and its relation to the O-dealkylation reaction.

The widely used fluorometric microsomal monooxygenase test for 7-ethoxycoumarin O-dealkylation was reinvestigated with regard to other possible hydroxylation products. By HPLC-analysis no beta-hydroxylation of the ethyl group and no 8-hydroxylation could be detected. Only a small percentage of 6-hydroxylation occurred, but as a new major metabolite 7-ethoxy-3-hydroxycoumarin was found in quantities depending on the microsomal preparation used. The ratio of O-dealkylation to 3-hydroxylation varied according to species, induction, buffer and pH, suggesting that different isozymes of cytochrome P450 were involved. The isozyme mainly responsible for 3-hydroxylation exhibited a great dependence on cytochrome b5 as the donor for the second electron. The fluorometric test does not include 3-hydroxylation due to the virtual absence of an emission spectrum above 450 nm.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1984

Characterization of the benzene monooxygenase system in rabbit bone marrow.

The microsomal fraction of bone marrow contains cytochrome P-450 (39 +/- 11 pmoles/mg microsomal protein) and monooxygenase activity could be demonstrated by the O-dealkylation of 7-ethoxycoumarin (114 +/- 65 pmoles/(min X mg microsomal protein] and the hydroxylation of benzene to phenol (51 +/- 8.6 pmol/45 min X mg microsomal protein). This monooxygenase system differs from that in liver in various aspects. The conversion of benzene to phenol calculated as molecular activity was about 4 times higher than in liver and no induction by phenobarbital could be observed. Aroclor 1254 induced the cytochrome P-450 content about twofold but lowered the O-dealkylation activity of 7-ethoxycoumarin in contrast to liver. Pretreatment with benzene did not change the O-dealkylation in bone marrow, but had a stimulating effect on benzene monooxygenation and covalent binding of 14C-benzene metabolites. From these results we conclude that the bone marrow monooxygenase system develops its own pattern of cytochrome P-450 isoenzymes. Especially after chronic exposure to benzene this system can convert this chemical to phenol and secondary metabolites. The similar behaviour of phenol formation and covalent binding strengthens the hypothesis of a common pathway for metabolism and toxicity but the active intermediate still remains unknown.

Animals↗
PubMed1984

Spectral intermediates of prostaglandin hydroperoxidase.

Microsomes from ram seminal vesicles or purified prostaglandin H synthase supplemented with either arachidonic acid or prostaglandin G2 formed an unstable spectral intermediate with maxima at 430 nm, 525 nm and 555 nm and minima at 410 nm, 490 nm and 630 nm. At -15 degrees C the band at 430 nm disappeared within 4 min whereas the trough at 410 nm increased three fold. At higher temperatures (10-37 degrees C) spectral complex formation and decay were observed in less than 1 s. An apparent KS-value of about 3 microM was determined for the titration of purified prostaglandin synthase with prostaglandin G2 at -20 degrees C. Substrates for cooxidation reactions of prostaglandin synthase such as phenol, hydroquinone and reduced glutathione as well as the peroxidase inhibitors cyanide and azide inhibited the prostaglandin G2-induced spectral complex formation. The oxene donor iodosobenzene and hydrogen peroxide formed a spectral intermediate analogous to the complex observed with prostaglandin G2 or arachidonic acid in ram seminal vesicle microsomes as well as with the purified prostaglandin synthase. These results are interpreted as the formation of a ferryl-oxo complex (FeO)3+ of the heme of prostaglandin synthase with prostaglandin G2 analogous to the formation of compound I of horseradish peroxidase.

Animals↗
PubMed1983

Conjugation of 1-naphthol and transport of 1-naphthol-conjugates in the vascularly perfused small intestine of the mouse.

A method is described which allows the simultaneous vascular and luminal perfusion of the murine small intestine. This preparation was used for the investigation of 1-naphthol conjugation in the gut and the sidedness of conjugate release. The viability of this preparation can be maintained for more than 1 hr as indicated by morphological controls, measurement of tissue metabolism and the transport of 3-O-methyl-glucose against a concentration gradient. When 100 microM 1-naphthol was administered on the luminal side, it was conjugated at a constant rate, yielding 1-naphthyl-glucuronide and 1-naphthyl-sulfate in a molar ratio of 1:2. Both metabolites were excreted into the blood at the contraluminal side of the epithelium. The results are discussed with respect to the sidedness of intestinal transport systems for anionic conjugates of xenobiotics and drugs.

3-O-Methylglucose↗
PubMed1983

Singlet oxygen formation detected by low-level chemiluminescence during enzymatic reduction of prostaglandin G2 to H2.

Addition of arachidonic acid to a suspension of ram vesicular gland microsomes or purified prostaglandin synthase, causes a rapid burst of light emission in the range 600-750 nm, as detected by single-photon counting. Maximal light emission intensity is obtained within 15-30 s after the addition of arachidonic acid and is followed by a rapid decay to the background level. The intensity of chemiluminescence is dependent on the amount of ram vesicular gland microsomes or isolated prostaglandin synthase and arachidonic acid concentration (Km about 6 microM). Spectral analysis of arachidonic acid-induced photoemission of isolated prostaglandin synthase in the range 600-750 nm showed two distinctive peaks at about 634 and 703 nm. The similar relative intensities of these peaks, along with the lower intensity at about 668 nm is indicative of singlet oxygen dimol emission. Chemiluminescence with arachidonate is enhanced by 1,4-diazabicyclo[2,2,2]octane and inhibited by azide, indomethacin, acetylsalicylic acid and beta-carotene. Cooxygenation substrates such as phenol, hydroquinone and reduced glutathione, inhibited the arachidonic acid-induced chemiluminescence. Dioxygen is a requirement for the observation of singlet oxygen dimol emission with arachidonic acid as a substrate for ram vesicular gland microsomes or purified prostaglandin synthase. However, when prostaglandin G2 is substituted for arachidonic acid, light emission is not dependent on oxygen. Thus, singlet oxygen can be formed in the dismutation reaction, 2 PGG2 leads to 2 PGH2 + 1O2, catalysed by prostaglandin hydroperoxidase.

Animals↗
PubMed1982

Oxidase and oxygenase function of the microsomal cytochrome P450 monooxygenase system.

The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of O2(-) radicals by microsomes, a stoichiometry of O2(-) to H2O2 close to 2:1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of H2O2. The rates of O2(-) as well as H2O2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%. It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 microM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.

Animals↗
PubMed1982

A quantitative test for superoxide radicals produced in biological systems.

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.

Animals↗
PubMed1982

The mechanism of reductive dehalogenation of halothane by liver cytochrome P450.

The reductive dehalogenation of halothane leading to 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE) has been investigated in vitro using at liver microsomes under anaerobic conditions. The stimulation of NADPH oxidation by halothane as well as the formation of the products were dependent upon cytochrome P450 as indicated by their CO and metyrapone inhibition. After replacement of NADPH by sodium dithionite as a reducing agent CDE was the only product of the enzymatic reaction. The product pattern was influenced by pretreatment with 3-methylcholanthrene, benzo(a)pyrene, phenobarbitone and Arochlor 1254 and by addition of anti-cytochrome P450-PB immunoglobulin. The CTE:CDE ratio was shifted by addition or inhibition of cytochrome b5 and by pH variation indicating a crucial role of the second electron donation to cytochrome P450 in determining the product pattern. The intermediate complex of cytochrome P450 with a Soret band at 470 nm formed with halothane in reduced liver microsomes was shown to decompose spontaneously to give CDE. Therefore we propose the 470 nm peak to represent a cytochrome P450 Fe3+----CHCl-CF3 carbanion complex. From these results a reaction pathway could be derived which includes radical and carbanion intermediates as reactive precursors of CTE and CDE, respectively.

Animals↗
PubMed1982

The reductive metabolism of halogenated alkanes by liver microsomal cytochrome P450.

Under anaerobic conditions various polyhalogenated alkanes (CCl3-CCl3, HCl2C-CCl3, CF3-CCl3, CCl4, CF-CHCIBr) stimulate the oxidation of NADPH by liver microsomal fractions. The participation of cytochrome P450 in the NADPH oxidation was shown by inducers and inhibitors of the monooxygenase system. The products of the reductive pathway of hexachloroethane were tetrachloroethene (99.5%) and pentachloroethane (0.5%). From pentachloroethane as substrate trichloroethene (96%) and tetrachloroethane (4%) were produced. The stoichiometry of NADPH oxidation and product formation was close to 1:1. There was a synergistic effect in the presence of NADPH and NADH for both hexa- and pentachloroethane. The influence of dioxygen and radical traps (RSH) on the formation of products from hexachloroethane with reduced cytochrome P450 has been investigated. The results indicate the possibility of a reductive in vivo metabolism of polyhalogenated alkanes even at physiological dioxygen concentrations. For the reductive dehalogenation of polyhalogenated alkanes by microsomal cytochrome P450 a reaction scheme is proposed: the reduction proceeds by two subsequent one electron reductions forming first a radical and then a carbanion. The carbanion can form an alkene via beta-elimination of chloride.

Animals↗
PubMed1982

[Prevention by metyrapone of halothane induced liver necrosis in rats (author's transl)].

Liver necrosis and an increase of serum sorbitol dehydrogenase activity can be produced in rats by halothane anesthesia (1% v/v in oxygen) following pretreatment with polychlorinated biphenyls. Using this model, it was shown that administration of metyrapone (2-methyl-1,2-bis(3-pyridyl)-1-propanone) (100 mg/kg b.w.) 1 h prior to anesthesia prevents liver necrosis and the concomitant increase in serum sorbitol dehydrogenase activity. No significant differences were observed when halothane was applied in air instead of oxygen, but the protective effect of metyrapone was abolished.

Animals↗
PubMed1981

Cytochrome b5 as electron donor for oxy-cytochrome P-450.

The steady-state concentration of the oxycomplex of microsomal cytochrome P-450 in the presence of NADPH could be elevated by increasing pH and ionic strength or by adding anti-(cytochrome b5) immunoglobulin. Low pH and low ionic strength as well as incorporation of cytochrome b5 into microsomal membranes decreased the steady-state level. Parallel to these effects, the b5 antibody monooxygenase activity at low pH and low ionic strength, but was not effective at high pH and high ionic strength. This establishes a role for cytochrome b5 in donating electrons for the reduction of oxy-cytochrome P-450 to the active oxygen complex of cytochrome P-450 but also points to large variations in the importance of this role depending on the experimental conditions, the species of P-450 involved and the substrates employed.

Animals↗
PubMed1980

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes.

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.

Animals↗
PubMed1980

Separation and purification of liver microsomal monooxygenases from induced and untreated pigs.

From liver microsomes of phenobarbital-, beta-naphthoflavone- and ethanol-pretreated male pigs and of controls NADPH-cytochrome P-450 reductase and four cytochrome P-450 fractions were isolated according to a modified procedure of Imai and Sato. Electrophoretic, immunological, spectral and catalytic properties suggested that at least five different forms of cytochrome P-450 could be separated, two of which were obtained in a pure state. Using selective inhibitors and antisera it was concluded that microsomes from phenobarbital-pretreated pigs contain at least three forms which all were metyrapone-sensitive. beta-Naphthoflavone induced probably two 7,8-benzoflavone-sensitive forms. After ethanol administration the main form of cytochrome P-450 was identical to that of controls.

Animals↗