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Biomedical subjects

I M Rietjens

Publications and source records attributed to I M Rietjens.

At least 19 recordsLinked to original sources
PubMed2001

The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones.

The effect of the pH on antioxidant properties of a series of hydroxyflavones was investigated. The pKa of the individual hydroxyl moieties in the hydroxyflavones was compared to computer-calculated deprotonation energies. This resulted in a quantitative structure activity relationship (QSAR), which enables the estimation of pKa values of individual hydroxyl moieties, also in hydroxyflavones for which these pKa values are not available. Comparison of the pKa values to the pH-dependent antioxidant profiles, determined by the TEAC assay, reveals that for various hydroxyflavones the pH-dependent behavior is related to hydroxyl moiety deprotonation, resulting in an increase of the antioxidant potential upon formation of the deprotonated forms. Comparison of these experimental results to computer calculated O-H bond dissociation energies (BDE) and ionization potentials (IP) of the nondeprotonated and the deprotonated forms of the various hydroxyflavones indicates that especially the parameter reflecting the ease of electron donation, i.e., the IP, and not the BDE, is greatly influenced by the deprotonation. Based on these results it is concluded that upon deprotonation the TEAC value increases (radical scavenging capacity increases) because electron-, not H*-, donation becomes easier. Taking into account that the mechanism of radical scavenging antioxidant activity of the neutral form of the hydroxyflavones is generally considered to be hydrogen atom donation, this implies than not only the ease of radical scavenging, but also the mechanism of antioxidant action changes upon hydroxyflavone deprotonation.

Antioxidants↗
PubMed2001

Excretion and metabolism of desogestrel in healthy postmenopausal women.

The metabolism of desogestrel (13-ethyl-11-methylene-18,19-dinor-17alpha-pregn-4-en-20-yn-17-ol), a progestagen used in oral contraceptives and hormone replacement therapy, was studied in vivo after a single oral administration of 150 microg [14C]-labeled desogestrel and 30 microg ethinylestradiol under steady state conditions to healthy postmenopausal women. After this oral administration, desogestrel was extensively metabolized. The dosed radioactivity was predominantly ( approximately 60%) excreted via urine, while about 35% was excreted via the feces. Desogestrel was metabolized mainly at the C3-, C5-, C6- and C13-CH(2)CH(3) positions. At the C3-position, the 3-keto moiety was found and in addition, 3beta-hydroxy and 3alpha-hydroxy groups were observed in combination with a reduced Delta(4)-double bond (5alpha-H). Hydroxy groups were introduced at the C6- (6beta-OH), the C13-ethyl (C13-CH(2)CH(2)OH) and possibly the C15- (15alpha-OH) position of desogestrel. Conjugation of the 3alpha-hydroxy moiety with sulfonic acid and conjugation with glucuronic acid were also major metabolic routes found for desogestrel in postmenopausal women. The 3-keto metabolite of desogestrel (the biologically active metabolite) was the major compound present in plasma at least up to 24 h after administration of the radioactive dose. Species comparison of the metabolic routes of desogestrel after oral administration indicates that in rats and dogs desogestrel is also mainly metabolized at the C3-position, similar to what is now found for postmenopausal women. Most other metabolic routes of desogestrel were found to differ between species. Finally, major metabolic routes found in the present study in postmenopausal women are in line with outcome of previous in vitro metabolism studies with human liver tissue (microsomes and postmitochondrial liver fractions) and intestinal mucosa.

Administration, Oral↗
PubMed2001

Structure-activity study on the quinone/quinone methide chemistry of flavonoids.

A structure-activity study on the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids was performed. Using the glutathione trapping method followed by HPLC, (1)H NMR, MALDI-TOF, and LC/MS analysis to identify the glutathionyl adducts, the chemical behavior of the quinones/quinone methides of the different flavonoids could be deduced. The nature and type of mono- and diglutathionyl adducts formed from quercetin, taxifolin, luteolin, fisetin, and 3,3',4'-trihydroxyflavone show how several structural elements influence the quinone/quinone methide chemistry of flavonoids. In line with previous findings, glutathionyl adduct formation for quercetin occurs at positions C6 and C8 of the A ring, due to the involvement of quinone methide-type intermediates. Elimination of the possibilities for efficient quinone methide formation by (i) the absence of the C3-OH group (luteolin), (ii) the absence of the C2=C3 double bond (taxifolin), or (iii) the absence of the C5-OH group (3,3',4'-trihydroxyflavone) results in glutathionyl adduct formation at the B ring due to involvement of the o-quinone isomer of the oxidized flavonoid. The extent of di- versus monoglutathionyl adduct formation was shown to depend on the ease of oxidation of the monoadduct as compared to the parent flavonoid. Finally, unexpected results obtained with fisetin provide new insight into the quinone/quinone methide chemistry of flavonoids. The regioselectivity and nature of the quinone adducts that formed appear to be dependent on pH. At pH values above the pK(a) for quinone protonation, glutathionyl adduct formation proceeds at the A or B ring following expected quinone/quinone methide isomerization patterns. However, decreasing the pH below this pK(a) results in a competing pathway in which glutathionyl adduct formation occurs in the C ring of the flavonoid, which is preceded by protonation of the quinone and accompanied by H(2)O adduct formation, also in the C ring of the flavonoid. All together, the data presented in this study confirm that quinone/quinone methide chemistry can be far from straightforward, but the study provides significant new data revealing an important pH dependence for the chemical behavior of this important class of electrophiles.

Benzoquinones↗
PubMed2001

Transformation of the insecticide teflubenzuron by microorganisms.

Transformation of teflubenzuron, the active component in the insecticide commercialized as Nomolt, by soil microorganisms was studied. It was shown that microorganisms, belonging to Bacillus, Alcaligenes, Pseudomonas and Acinetobacter genera are capable to perform the hydrolytic cleavage of the phenylurea bridge of teflubenzuron in different positions, especially active was Bacillus brevis 625. The structure of the intermediates formed was established using TLC, HPLC, mass-spectrometry and 19F NMR techniques. It was shown that for a dose range of 53-132 microM and upon 12 days of fermentation about 30% of the teflubenzuron was modified. About 10-15% was transformed into 2,6-difluorobenzamide, 3-5% into 2,6-difluorobenzoic acid, 10-12% into 2,4-difluoro-3,5-dichloro-aniline. The late compound gave rise to formation of a condensed compound, identified as 1,2-bis(2,4-difluoro-3,5-dichlorophenyl)urea with molecular mass of 420. The results obtained indicate degradation of teflubenzuron by soil microorganisms to be a process to be mediated by microbial consortia, and starting with hydrolysis of the phenylurea bridge by several bacterial species. Subsequent further degradation of the aromatic degradation products has to be mediated by other strains known to be capable of degradation of halogenated aromatics.

Acinetobacter calcoaceticus↗
PubMed2000

Modelling flavin and substrate substituent effects on the activation barrier and rate of oxygen transfer by p-hydroxybenzoate hydroxylase.

The simulation of enzymatic reactions, using computer models, is becoming a powerful tool in the most fundamental challenge in biochemistry: to relate the catalytic activity of enzymes to their structure. In the present study, various computed parameters were correlated with the natural logarithm of experimental rate constants for the hydroxylation of various substrate derivatives catalysed by wild-type para-hydroxybenzoate hydroxylase (PHBH) as well as for the hydroxylation of the native substrate (p-hydroxybenzoate) by PHBH reconstituted with a series of 8-substituted flavins. The following relative parameters have been calculated and tested: (a) energy barriers from combined quantum mechanical/molecular mechanical (QM/MM) (AM1/CHARMM) reaction pathway calculations, (b) gas-phase reaction enthalpies (AM1) and (c) differences between the HOMO and LUMO energies of the isolated substrate and cofactor molecules (AM1 and B3LYP/6-31+G(d)). The gas-phase approaches yielded good correlations, as long as similarly charged species are involved. The QM/MM approach resulted in a good correlation, even including differently charged species. This indicates that the QM/MM model accounts quite well for the solvation effects of the active site surroundings, which vary for differently charged species. The correlations obtained demonstrate quantitative structure activity relationships for an enzyme-catalysed reaction including, for the first time, substitutions on both substrate and cofactor.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2000

Peroxidase-catalyzed formation of quercetin quinone methide-glutathione adducts.

The oxidation of quercetin by horseradish peroxidase/H(2)O(2) was studied in the absence but especially also in the presence of glutathione (GSH). HPLC analysis of the reaction products formed in the absence of GSH revealed formation of at least 20 different products, a result in line with other studies reporting the peroxidase-mediated oxidation of flavonoids. In the presence of GSH, however, these products were no longer observed and formation of two major new products was detected. (1)H NMR identified these two products as 6-glutathionylquercetin and 8-glutathionylquercetin, representing glutathione adducts originating from glutathione conjugation at the A ring instead of at the B ring of quercetin. Glutathione addition at positions 6 and 8 of the A ring can best be explained by taking into consideration a further oxidation of the quercetin semiquinone, initially formed by the HRP-mediated one-electron oxidation, to give the o-quinone, followed by the isomerization of the o-quinone to its p-quinone methide isomer. All together, the results of the present study provide evidence for a reaction chemistry of quercetin semiquinones with horseradish peroxidase/H(2)O(2) and GSH ultimately leading to adduct formation instead of to preferential GSH-mediated chemical reduction to regenerate the parent flavonoid.

Chromatography, High Pressure Liquid↗
PubMed2000

A mechanism for oxygen exchange between ligated oxometalloporphinates and bulk water.

Oxygen exchange between high-valent metal-oxo complexes and bulk water has been monitored for nonligated model porphyrins (hemin, FeTDCPPS, MnTMPyP) and the axially ligated microperoxidase-8 (MP-8). Exchange extents up to 90% were measured for MP-8 in spite of the presence of an axial histidine ligand and accompanied by the formation of nonlabelled H(2)O(2) from H(2)(18)O(2). These results point to the existence of a mechanism for oxygen exchange between the high-valent iron-oxo complex and the solvent different from the so-called "oxo-hydroxo tautomerism." Regeneration of the primary oxidant, H(2)O(2), and oxygen exchange by axially ligated porphyrins can be explained by a mechanism involving the reversibility of compound I formation.

Aminophenols↗
PubMed2000

Excretion balance and metabolism of the progestagen Org 30659 in healthy postmenopausal women.

Metabolism of Org 30659 ((17alpha)-17-hydroxy-11-methylene-19-norpregna-4, 15-dien-20-yn-3-one), a new potent progestagen currently under clinical development by NV Organon for use in oral contraception and hormone replacement therapy, was studied in vivo after oral administration to healthy postmenopausal women. After oral administration of [14C]-Org 30659 to postmenopausal women, the compound was extensively metabolized. The dosed radioactivity was predominantly excreted via urine. Org 30659 was to a large extent metabolized at the C3- and the C17-positions. Phase II metabolism, and in particular conjugation with glucuronic acid at the 17beta-hydroxy group, is the major metabolic route for Org 30659 in vivo. Not only phase II metabolism was observed for Org 30659 after oral administration to postmenopausal volunteers, but also metabolism in the A-ring occurred, especially reduction of the 3-keto-Delta(4) moiety to give 3alpha-hydroxy, 5alpha(beta)-dihydro and 3beta-hydroxy, 5alpha-dihydro derivatives. Oxidative metabolism (6beta-hydroxylation) observed in human liver preparations in vitro, was not observed to a significant extent in vivo. So, in vitro human metabolism is different from the in vivo metabolism, indicating that the in vitro-in vivo extrapolation is far from straightforward, at least when only liver preparations are used. The proper choice of the in vitro system (e.g., microsomes, hepatocytes, slices or individually expressed enzymes) and the substrate concentration can be very important determinative factors for the predictability of the in vitro system for the in vivo situation. Species comparison of the metabolic routes of Org 30659 after oral administration indicated that the monkey seems to be a better representative species than the rat for the metabolism of Org 30659 in humans.

Administration, Oral↗
PubMed2000

Characterization of different commercial soybean peroxidase preparations and use of the enzyme for N-demethylation of methyl N-methylanthranilate To produce the food flavor methylanthranilate.

The potential of different peroxidase preparations for the N-demethylation of methyl N-methylanthranilate to produce the food flavor methylanthranilate (MA) was investigated. All tested peroxidase preparations were able to catalyze the N-dealkylation. The tested soybean preparations vary widely with respect to their heme content. Furthermore, the operational stability of purified soybean peroxidase (SP) is at least 25-fold lower than that of horseradish peroxidase and only 5-fold higher than that of microperoxidase 8. Thus, the presence of a large protein chain around a porphyrin cofactor in a peroxidase is, by itself, insufficient to explain the observed differences in operational stability. Despite its relatively low operational stability, SP proved to be the most efficient biocatalyst for the production of MA with high yield and purity, especially observed at the high temperature and low pH values at which SP appeared to be optimally active.

Flavoring Agents↗
PubMed2000

Regioselectivity and reversibility of the glutathione conjugation of quercetin quinone methide.

The chemical reactivity, isomerization, and glutathione conjugation of quercetin o-quinone were investigated. Tyrosinase was used to generate the unstable quercetin o-quinone derivative which could be observed upon its subsequent scavenging by glutathione. Identification of the products revealed formation of 6-glutathionyl-quercetin and 8-glutathionyl-quercetin adducts. Thus, in particular, glutathione adducts in the A ring of quercetin were formed, a result which was not expected a priori. Quantum mechanical calculations support the possibility that the formation of these glutathione adducts can be explained by an isomerization of quercetin o-quinone to p-quinone methides. Surprisingly, additional experiments of this study reveal the adduct formation to be reversible, leading to interconversion between the two quercetin glutathione adducts and possibilities for release and further electrophilic reactions of the quercetin quinone methide at cellular sites different from those of its generation.

Chromatography, High Pressure Liquid↗
PubMed2000

Heme-(hydro)peroxide mediated O- and N-dealkylation. A study with microperoxidase.

The mechanism of microperoxidase-8 (MP-8) mediated O- and N-dealkylation was investigated. In the absence of ascorbate (peroxidase mode), many unidentified polymeric products are formed and the extent of substrate degradation correlates (r = 0.94) with the calculated substrate ionization potential, reflecting the formation of radical intermediates. In the presence of ascorbate (P450 mode) formation of polymeric products is largely prevented but, surprisingly, dealkylation is not affected. In addition, aromatic hydroxylation and oxidative dehalogenation is observed. The results exclude a radical mechanism and indicate the involvement of a (hydro)peroxo-iron heme intermediate in P450-type of heteroatom dealkylation.

Alkylation↗
PubMed2000

Identification of fluoropyrogallols as new intermediates in biotransformation of monofluorophenols in Rhodococcus opacus 1cp.

The transformation of monofluorophenols by whole cells of Rhodococcus opacus 1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and (19)F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The (19)F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2, 3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in R. opacus 1cp.

Biotransformation↗
PubMed1999

Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: description of quantitative structure-activity relationships.

Polyphenolic antioxidants exhibited a dose-dependent toxicity against human promyelocytic leukemia cells (HL-60). Their action was accompanied by malondialdehyde formation, and was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine. This points to a prooxidant character of their cytotoxicity. A quantitative structure-activity relationship (QSAR) has been obtained to describe the cytotoxicity of 13 polyphenolic antioxidants belonging to three different groups (flavonoids, derivatives of gallic and caffeic acid): log cL50 (microM) = (2.7829+/-0.2339)+(1.2734+/-0.4715) Ep/2 (V)-(0.3438+/-0.0582) log P (r2 = 0.8129), where cL50 represents the concentration for 50% cell survival, Ep/2 represents the voltammetric midpoint potential, and P represents the octanol/water partition coefficient. Analogous QSARs were obtained using enthalpies of single-electron oxidation of these compounds, obtained by quantum-mechanical calculations. These findings clearly point to two important characteristics determining polyphenol cytotoxicity, namely their ease of oxidation and their lipophilicity.

Antioxidants↗
PubMed1999

Preferential oxidative dehalogenation upon conversion of 2-halophenols by Rhodococcus opacus 1G.

The regiospecificity of hydroxylation of C2-halogenated phenols by Rhodococcus opacus 1G was investigated. Oxidative defluorination at the C2 position ortho with respect to the hydroxyl moiety was preferred over hydroxylation at the non-fluorinated C6 position for all 2-fluorophenol compounds studied. Initial hydroxylation of 2,3, 5-trichlorophenol resulted in the exclusive formation of 3, 5-dichlorocatechol. These results indicate that, in contrast to all other phenol ortho-hydroxylases studied so far, phenol hydroxylase from R. opacus 1G is capable of catalyzing preferential oxidative defluorination but also oxidative dechlorination.

Biodegradation, Environmental↗
PubMed1999

Differential substrate behaviour of phenol and aniline derivatives during conversion by horseradish peroxidase.

For the first time saturating overall k(cat) values for horseradish peroxidase (HRP) catalysed conversion of phenols and anilines are described. These k(cat) values correlate quantitatively with calculated ionisation potentials of the substrates. The correlations for the phenols are shifted to higher k(cat) values at similar ionisation potentials as compared to those for anilines. (1)H-NMR T(1) relaxation studies, using 3-methylphenol and 3-methylaniline as the model substrates, revealed smaller average distances of the phenol than of the aniline protons to the paramagnetic Fe(3+) centre in HRP. This observation, together with a possibly higher extent of deprotonation of the phenols than of the anilines upon binding to the active site of HRP, may contribute to the relatively higher HRP catalysed conversion rates of phenols than of anilines.

Aniline Compounds↗
PubMed1999

Quantitative structure activity relationships for the electron transfer reactions of Anabaena PCC 7119 ferredoxin-NADP+ oxidoreductase with nitrobenzene and nitrobenzimidazolone derivatives: mechanistic implications.

The steady state single electron reduction of polynitroaromatics by ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) from cyanobacterium Anabaena PCC 7119 has been studied and quantitative structure activity relationships are described. The solubility of the polynitroaromatics as well as their reactivity towards ferredoxin-NADP+ oxidoreductase are markedly higher than those for previously studied mononitroaromatics and this enabled the independent measurement of the kinetic parameters-k(cat) and Km. Interestingly, the natural logarithm of the bimolecular rate constant, k(cat)/Km, and also the natural logarithm of k(cat) correlate with the calculated energy of the lowest unoccupied molecular orbital of the polynitroaromatic substrates. The minimal kinetic model in line with these quantitative structure activity relationships is a ping-pong mechanism which includes substrate binding equilibria in the second half reaction.

Anabaena↗
PubMed1999

The effect of iron to manganese substitution on microperoxidase 8 catalysed peroxidase and cytochrome P450 type of catalysis.

This study describes the catalytic properties of manganese microperoxidase 8 [Mn(III)MP8] compared to iron microperoxidase 8 [Fe(III)MP8]. The mini-enzymes were tested for pH-dependent activity and operational stability in peroxidase-type conversions, using 2-methoxyphenol and 3,3'-dimethoxybenzidine, and in a cytochrome P450-like oxygen transfer reaction converting aniline to para-aminophenol. For the peroxidase type of conversions the Fe to Mn replacement resulted in a less than 10-fold decrease in the activity at optimal pH, whereas the aniline para-hydroxylation is reduced at least 30-fold. In addition it was observed that the peroxidase type of conversions are all fully blocked by ascorbate and that aniline para-hydroxylation by Fe(III)MP8 is increased by ascorbate whereas aniline para-hydroxylation by Mn(III)MP8 is inhibited by ascorbate. Altogether these results indicate that different types of reactive metal oxygen intermediates are involved in the various conversions. Compound I/II, scavenged by ascorbate, may be the reactive species responsible for the peroxidase reactions, the polymerization of aniline and (part of) the oxygen transfer to aniline in the absence of ascorbate. The para-hydroxylation of aniline by Fe(III)MP8, in the presence of ascorbate, must be mediated by another reactive iron-oxo species which could be the electrophilic metal(III) hydroperoxide anion of microperoxidase 8 [M(III)OOH MP8]. The lower oxidative potential of Mn, compared to Fe, may affect the reactivity of both compound I/II and the metal(III) hydroperoxide anion intermediate, explaining the differential effect of the Fe to Mn substitution on the pH-dependent behavior, the rate of catalysis and the operational stability of MP8.

Catalysis↗