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PubMed2003

HPLC and LC-MS studies of hydroxylation of phenylalanine as an assay for hydroxyl radicals generated from Udenfriend's reagent.

An HPLC assay method and an LC-MS method were used to study the Udenfriend reaction and its variations by using phenylalanine as the hydroxylation substrate. The results indicate that (1). citric acid can replace EDTA as the promoter for the production of hydroxyl radicals in the Undenfriend reaction, albeit in a somewhat less efficient way, (2). dihydroxylation of the hydroxylation substrate, phenylalanine, readily occurs with the Udenfriend systems (with either EDTA or citric acid), and (3). a novel oxidative degradation pathway may exist for o-tyrosine. It is cautioned that dihydroxylation needs to be accounted for when interpreting hydroxylation results in HPLC-based HO(z.rad;) assay systems with phenylalanine as the substrate.

Chromatography, High Pressure Liquid↗
PubMed1998

Salicylate hydroxylation as an indicator of hydroxyl radical generation in dextran sulfate-induced colitis.

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease. This study investigated hydroxyl radical (.OH) generation in the inflamed colon of dextran sulfate sodium (DSS)-induced colitis by measuring the .OH-specific product of salicylate hydroxylation, 2,3-dihydroxybenzoic acid (DHB). Colitis was induced in 6-7 week old CBA/H male mice by supplementing the drinking water with 5% DSS for 7 days. On the last day of dextran exposure, mice were injected with salicylate (SAL) (100 mg/kg i.p.) 60 min before sacrifice, and mucosal homogenates were assayed for SAL and 2,3-DHB by HPLC with fluorescence and electrochemical detection. Mucosal 2,3-DHB levels in mice exposed to 5% DSS were increased by 83% (p < .005); however, SAL levels were also elevated by 182% (p < .001). This translated to a 34% decrease in the ratio 2,3-DHB:SAL in inflamed mucosa, possibly indicating greater catabolism or decreased production of 2,3-DHB. In vitro investigation of the stability of DHBs and SAL in the presence of oxidants of inflammatory lesions revealed that 2,3-DHB and 2,5-DHB were rapidly degraded by hypochlorous acid (HOCl), with initial decomposition rates of 190 and 281 nmol/min, respectively (100microM DHB with 200microM HOCl). Methionine prevented decomposition of DHBs in vitro; however, in mice with 5% DSS-induced colitis, where mucosal myeloperoxidase activity was ten-fold control levels (p < .001), administration of methionine (up to 200 mg/kg i.p.) with SAL was ineffective at increasing the ratio 2,3-DHB:SAL. SAL was also degraded in vitro by HOCl (4.7 nmol/min) resulting in the formation of new fluorescent species which may be useful as indicators of HOCl-mediated injury. Salicylate hydroxylation was unable to provide conclusive evidence supporting a role for .OH in the tissue injury of DSS-induced colitis, as metabolic disturbances in the diseased animals other than changes in .OH generation may have altered 2,3-DHB levels. This problem is relevant to any study involving the in vivo use of trapping molecules. In particular, the susceptibility of 2,3-DHB to degradation by HOCl brings into question the usefulness of salicylate hydroxylation for measurement of .OH-generation in any neutrophilic inflammatory lesion.

Animals↗
PubMed1986

An aromatic hydroxylation assay for hydroxyl radicals utilizing high-performance liquid chromatography (HPLC). Use to investigate the effect of EDTA on the Fenton reaction.

A highly sensitive HPLC method for the separation of hydroxylation products derived from the attack of hydroxyl radical upon phenol is described. Catechol and hydroquinone are the major hydroxylation products formed, with little resorcinol. The effect of EDTA upon hydroxyl radical generation from an iron (II)-H2O2 system is shown to depend upon the order of addition of chelator and metal ion to the reaction mixture, the ratio [iron salt]/[chelator] and the presence or absence of a phosphate buffer. Reasons for these different effects are discussed.

Chromatography, High Pressure Liquid↗
PubMed1997

Hydroxylation of salicylate and phenylalanine as assays for hydroxyl radicals: a cautionary note visited for the third time.

Hydroxylation of salicylate to 2,3- and 2,5-dihydroxy-benzoates (DHBs) is widely used as an index of hydroxyl radical (OH.) formation in vivo and in vitro. Several recent studies indicate that peroxynitrite can lead to generation of DHBs from salicylate and it is uncertain as to whether or not OH. is involved. A similar problem may occur in the use of phenylalanine as an OH. detector. Hence formation of hydroxylation products from salicylate (or phenylalanine) may not in itself be a definitive index of OH. generation, especially in cases where such generation in physiological systems is decreased by inhibitors of nitric oxide synthase. Determination of salicylate (or phenylalanine) nitration products can allow distinction between peroxynitrite-dependent aromatic hydroxylation and that involving "real" OH..

Animals↗
PubMed1997

A para-site-specific hydroxylation of aromatic compounds by Mycobacterium sp. strain 12523: stabilization of the hydroxylation activity.

Mycobacterium sp. strain 12523 has a para-site-specific hydroxylation activity, which produce para-substituted phenols from various aromatic compounds. However, the activity is unstable and the reactions are inactivated within 24 h. In order to extend the reaction period, the factors that affected reaction stability were examined. The hydroxylation activity of the cells incubated in buffer was significantly stabilized by the inclusion of an inducer such as methyl ethyl ketone. It is suggested that a regulatory mechanism is involved in controlling the activity. This study resulted in the development of a convenient method to stabilize the hydroxylation activity, involving the addition of an inducer, such as acetone, to the reaction system. This method permitted the hydroxylation reaction to continue for more than 67 h.

Butanones↗
PubMed2000

Fatty acid-specific, regiospecific, and stereospecific hydroxylation by cytochrome P450 (CYP152B1) from Sphingomonas paucimobilis: substrate structure required for alpha-hydroxylation.

Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is an unusual cytochrome P450 enzyme that hydroxylates the alpha-carbon of fatty acids in the presence of H2O2. Herein, we describe our investigation concerning the utilization of various substrates and the optical configuration of the alpha-hydroxyl product using a recombinant form of this enzyme. This enzyme can metabolize saturated fatty acids with carbon chain lengths of more than 10. The Km value for pentadecanoic acid (C15) was the smallest among the saturated fatty acids tested (C10-C18) and that for myristic acid (C14) showed similar enzyme kinetics to those seen for C15. As shorter or longer carbon chain lengths were used, Km values increased. The turnover numbers for fatty acids with carbon chain lengths of more than 11 were of the same order of magnitude (10(3) min(-1)), but the turnover number for undecanoic acid (C11) was less. Dicarboxylic fatty acids and methyl myristate were not metabolized, but monomethyl hexadecanedioate and omega-hydroxypalmitic acid were metabolized, though with lower turnover values. Arachidonic acid was a good substrate, comparable to C14 or C15. The metabolite of arachidonic acid was only alpha-hydroxyarachidonic acid. Alkanes, fatty alcohols, and fatty aldehydes were not utilized as substrates. Analysis of the optical configurations of the alpha-hydroxylated products demonstrated that the products were S-enantiomers (more than 98% enantiomerically pure). These results suggested that this P450 enzyme is strictly responsible for fatty acids and catalyzes highly stereo- and regioselective hydroxylation, where structure of omega-carbon and carboxyl carbon as well as carbon chain length of fatty acids are important for substrate-enzyme interaction.

Arachidonic Acid↗
PubMed1983

Generation of hydroxyl radicals during the enzymatic reductions of the Fe3+-ADP-phosphate-adriamycin and Fe3+-ADP-EDTA systems. Less involvement of hydroxyl radical and a great importance of proposed perferryl ion complexes in lipid peroxidation.

A system which contains NADPH, purified cytochrome P-450 reductase (enzyme) and Fe3+-ADP-adriamycin complex in Tris-HCl buffer does not produce hydroxyl radical, but possesses a strong lipid peroxidation activity on exogenously added phospholipid micelles. Fe3+-ADP-adriamycin complex, a tightly coordinated complex in Tris-HCl buffer, could be dissociated to Fe3+-ADP-phosphate complex and adriamycin in phosphate buffer. Hydroxyl radical, which can be detected by a spin trapping method using N-tert-butyl-alpha-phenylnitrone, is produced during the enzymatic reduction of a mixture of Fe3+-ADP-phosphate complex and adriamycin or of Fe3+-ADP-EDTA complex while it is not involved in phospholipid peroxidation under the conditions used. With hydroxyl radical-generating systems, little or no quenching of hydroxyl radical in Tris-HCl buffer could be demonstrated. The oxidative cleavage of phospholipid is initiated by the proposed perferryl ion complex, which may be generated by the interaction of Fe2+-ADP-adriamycin complex with O2. A similar perferryl ion complex is also produced during the enzymatic reduction of Fe3+-ADP-EDTA complex with a molar ratio of 2 for [Fe3+]/[EDTA] in the presence of air. This is also able to catalyze lipid peroxidation.

Adenosine Diphosphate↗
PubMed1990

Hydroxylation of the thiophene ring by hepatic monooxygenases. Evidence for 5-hydroxylation of 2-aroylthiophenes as a general metabolic pathway using a simple UV-visible assay.

The 5-hydroxylation of tienilic acid by rat liver microsomes was measured by a new, simple method involving the detection of 5-hydroxytienilic acid by UV-visible spectroscopy. This assay allowed continuous detection of this metabolite and could be easily used to determine the kinetic parameters of the reaction (Vmax and Km being respectively 1 +/- 0.2 nmol product formed/mg protein/min and 14 +/- 2 microM for liver microsomes from phenobarbital-treated rats). This activity was found to be dependent on NADPH and to be inhibited by CO, SKF 525A and metyrapone, indicating that it is dependent on cytochromes P-450. This UV-visible assay is based on intrinsic properties of 5-hydroxy 2-aroylthiophenes which exist as highly conjugated anions at physiological pH and exhibit large epsilon values around 390 nm. Its application to other 2-aroylthiophenes like suprofen, 2-parachlorobenzoylthiophene and a series of 2-aroylthiophenes with various substituents on the aroyl group showed that, in general, thiophene compounds bearing a 2-arylketo substituent appear to be hydroxylated at position 5 by rat liver microsomes. The kinetic parameters of the 5-hydroxylation of suprofen and 2-parachlorobenzoylthiophene by liver microsomes from phenobarbital-treated rats were determined and found to be similar to those for tienilic acid hydroxylation.

Animals↗
PubMed1995

Hydroxylation of the antimalarial drug 58C80 by CYP2C9 in human liver microsomes: comparison with mephenytoin and tolbutamide hydroxylations.

58C80 [2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone] is an experimental naphthoquinone antimalarial drug which undergoes extensive alkyl hydroxylation to a single t-butylhydroxy metabolite in man in vivo and also in human liver microsomes, where this is catalysed primarily by a 54 kDa CYP2C9 form of cytochrome P450, P450hB20-27. Microsomal 58C80 hydroxylation (58OH) activity showed a marked inter-individual variation in a bank of 39 individual human livers but did not correlate with the immunoquantified levels of either of two microsomal proteins (54 and 50 kDa, respectively) recognised by a polyclonal antibody against CYP2C9 (P450hB20-27). Neither 58OH activity nor the concentrations of the CYP2C9-immunorelated proteins showed any relationship with the individuals' age, sex, cigarette smoking habit, alcohol consumption or clinical drug treatment, including long term antiepileptic therapy with phenobarbitone or phenytoin. 58OH activity did not correlate with either TBOH (tolbutamide hydroxylation) or MPOH (S-mephenytoin 4'-hydroxylation) activities, while 58C80 inhibited both TBOH and MPOH in human liver microsomes non-competitively (Ki = 30 and 175 microM for TBOH and MPOH, respectively). 58C80 could be a useful model substrate for measuring human CYP2C activity in vitro.

Antimalarials↗
PubMed1984

Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography with electrochemical detection of phenol and salicylate hydroxylation products.

Evidence is presented for a sensitive method useful for the detection of hydroxyl free radical generation in various systems. The methodology employs high pressure liquid chromatography with electrochemical detection (LCED) for the quantification and identification of the hydroxylation products from the reaction of OH with both phenol and salicylate. A detection limit of less than 1 pmol for the hydroxylation products has been achieved with electrochemical detector responses linear over at least three orders of magnitude. Detection and quantitation of the hydroxylation products obtained and formed during OH generation from biologically meaningful systems have been demonstrated. The three systems utilized were ADP/FE(II)/H2O/, hypoxanthine/xanthine oxidase plus chelated iron, and UV photolysis of H2O2.

Chromatography, High Pressure Liquid↗
PubMed2000

Intracranial self-stimulation increases differentially in vivo hydroxylation of tyrosine but similarly in vivo hydroxylation of tryptophan in rat medial prefrontal cortex, nucleus accumbens and striatum.

We have examined using microdialysis the effect of intracranial self-stimulation (ICSS) on the in vivo hydroxylation rate of tyrosine and tryptophan in the medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and striatum (STR). A decarboxylase inhibitor NSD-1015 was included in the perfusate, which enabled the simultaneous measurement of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) as an index of the in vivo hydroxylation level of tyrosine and tryptophan. When rats were exposed to 1 h of ICSS at the medial forebrain bundle (MFB), their extracellular levels of DOPA significantly increased in the mPFC, NAC and STR, but with a different magnitude and time course. The same stimulation produced a delayed increase in extracellular 5-HTP, compared to DOPA, in these brain regions. The profile of 5-HTP response demonstrated no apparent difference among the regions. These findings indicate that ICSS of the MFB can increase differentially the in vivo hydroxylation of tyrosine but similarly the in vivo hydroxylation of tryptophan in the mPFC, NAC and STR.

5-Hydroxytryptophan↗
PubMed1997

A single amino acid substitution (Leu160His) in cytochrome P450 CYP2A6 causes switching from 7-hydroxylation to 3-hydroxylation of coumarin.

Human populations are thought to metabolize coumarin almost exclusively by 7-hydroxylation. We have identified an individual who is homozygous for a single amino acid substitution (Leu160His) in the cytochrome P450 CYP2A6 arising from the variant CYP2A6*2 allele. On administration of coumarin (2 mg orally) no detectable 7-hydroxycoumarin was excreted in the 0-8-hr urine, rather, approximately 50% of the dose was eliminated as 2-hydroxyphenylacetic acid, the end-product of coumarin 3-hydroxylation. His immediate family members, who were heterozygous for the CYP2A6*2 allele, excreted little 2-hydroxyphenylacetic acid and mainly 7-hydroxycoumarin, when similarly tested. These findings raise a question regarding human risk evaluations for environmental coumarin exposures, since 7-hydroxylation is regarded as a detoxication pathway, but 3-hydroxylation as the process required to lead to macromolecular covalent binding of coumarin. Persons homozygous for the CYP2A6*2 allele may constitute 1-25% of various populations.

Adolescent↗
PubMed2000

Peptidylglycine-alpha-hydroxylating monooxygenase generates two hydroxylated products from its mechanism-based suicide substrate, 4-phenyl-3-butenoic acid.

The bifunctional enzyme peptidylglycine-alpha-amidating monooxygenase mediates the conversion of C-terminal glycine-extended peptides to their active alpha-amidated products. Peptidylglycine-alpha-hydroxylating monooxygenase (PHM, EC 1.14.17. 3) catalyzes the first reaction in this two-step process. The olefinic compound 4-phenyl-3-butenoic acid (PBA) is the most potent irreversible, mechanism-based PHM inactivator known. While the details of the inhibitory action of PBA on PHM remain undefined, covalent modification of the protein has been proposed as the underlying mechanism. We report here that, in the process of inactivating PHM, PBA itself serves as a substrate without covalently labeling the enzyme. Approximately 100 molecules of PBA are metabolized per molecule of PHM inactivated, under saturating conditions. The metabolism of PBA by PHM generates two hydroxylated products, 2-hydroxy-4-phenyl-3-butenoic acid and its allylic isomer, 4-hydroxy-4-phenyl-2-butenoic acid. While one enantiomer for each product is significantly favored in the reaction, both are produced. From these observations, we conclude that hydroxylated PBA products are formed by a delocalized free radical mechanism and that the lack of absolute stereospecificity indicates significant freedom of movement within the catalytic site. The ability of PHM to metabolize PBA suggests that the physiological functions of PHM may include the hydroxylation of substrates other than those containing terminal glycines.

Catalytic Domain↗
PubMed1998

Protective effects of nicaraven, a new hydroxyl radical scavenger, on the endothelial dysfunction after exposure of pig coronary artery to hydroxyl radicals.

Recently, we have reported that a new synthetic compound, 1,2bis(nicotinamido)-propane (nicaraven), improved cardiac function following preservation and reperfusion. In this study, we investigated the efficacy of nicaraven as a radical scavenger by using an in vitro model of oxidative stress, to clarify mechanisms of the protective effect of this new compound on reperfusion injury in rat heart. Ring segments of epicardial right coronary arteries (RCA) of pig were suspended in organ chambers and exposed to hydroxyl radicals (.OH), generated (by two different systems) by 0.28 mM FeSO4/0.28 mM H2O2 and DHF/Fe3+-ADP (2.4 mM, 43 nM, and 1.56 uM, respectively) to the bathing solution for 60 min. Prior exposure of the coronary arteries to .OH significantly produced right-ward shift of the dose-response curves of the bradykinin-induced endothelium-dependent relaxations (an increase in the ED50 value for bradykinin by 4.37 and 1.98 times than control in two different .OH generating systems, respectively), but did not affect the maximum relaxation responses. The presence of nicaraven (10(-4) and 10(-5) M) in the .OH generating system, shifted the dose-response curves to bradykinin to the control level, suggesting a significant hydroxyl radical scavenging effect of the drug. These results indicate that nicaraven, a new hydroxyl radical scavenger, exhibits a protective effect on hydroxyl radical-induced endothelial dysfunctions of pig coronary artery.

Animals↗
PubMed1977

Hydroxylation of aromatic compounds by reduced nicotinamide-adenine dinucleotide and phenazine methosulphate requires hydrogen peroxide and hydroxyl radicals, but not superoxide.

1. A mixture of NADH and phenazine methosulphate hydroxylates aromatic compounds at acidic pH values. 2. Hydroxylation is inhibited by catalase and by scavengers of the hydroxyl radical (-OH) but not by superoxide dismutase. 3. It is concluded that neither O2 leads to nor HO2- is sufficiently reactive to hydroxylate aromatic rings.

Hydrogen Peroxide↗
PubMed2006

The production of hydroxyl radicals in the fetal lamb brain resulting from occlusion of the umbilical circulation and the transplacental effect of MCI-186 to inhibit hydroxyl radical production.

The present study evaluated hydroxyl radical production in fetal lamb brain during and after umbilical cord occlusion and examined the effects of injecting MCI-186 (3-metyl-1-phenyl-2-pyrazolin-5-one; Edaravone), a hydroxyl radical scavenger, into the maternal circulation. In 11 chronically instrumented lambs, intermittent total umbilical cord occlusions 1 min out of every 3 min for 1 h and 10-min persistent total umbilical cord occlusion were performed with brain microdialysis using 5 mM of sodium salicylate. In the remaining four lambs, 60 mg of MCI-186 was administered into the maternal circulation from shortly before the end of 10-min persistent total umbilical cord occlusion. Concentrations of 2,3-dihydroxy-benzoic acid (2,3-DHBA), produced by hydroxyl radical reactions with sodium salicylate, were measured in perfusate by HPLC. Concentration of 2,3-DHBA in perfusate was 23.05 +/- 10.95 nM before umbilical cord occlusion. Levels of 2,3-DHBA tended to increase slightly during and after intermittent umbilical cord occlusion, and were significantly increased by the end of 10-min occlusion (40.06 +/- 21.36 nM) and after occlusion (93.74 +/- 29.17 nM). Infusion of MCI-186 suppressed 2,3-DHBA concentration to 29.35 +/- 14.95 nM after occlusion. Administration of MCI-186 into the maternal circulation reduces hydroxyl radical production induced by umbilical cord occlusion in the fetal lamb brain.

Animals↗
PubMed1994

Cytochrome P450-dependent N-hydroxylation of an aminoguanidine (amidinohydrazone) and microsomal retroreduction of the N-hydroxylated product.

1. The first example of a P450-dependent N-hydroxylation of an aminoguanidine (amidinohydrazone) is reported for 2-amino-5-chlorobenzophenone amidinohydrazone 1 (G 256) as substrate. 2. The N-hydroxylated metabolite 2 (2-amino-5-chlorobenzophenone N-hydroxyamidinohydrazone NOH-G256) and a further metabolite of 1, the phenol 3, were identified by tlc and ms analysis. 3. The microsomal reduction of an N-hydroxyaminoguanidine (N-hydroxy-amidino-hydrazone) was also demonstrated for the transformation of 2 to 1. 4. Both the N-hydroxylation of the aminoguanidine and the retroreduction of the N-hydroxyaminoguanidine were characterized by quantitative hplc analysis. 5. The conversion of the aminoguanidine 1 to N-hydroxyaminoguanidine 2 may be considered as an analogue of the physiological N-hydroxylation of arginine to N-hydroxyarginine by NO synthases.

Animals↗
PubMed1984

Hydroxylations in biosynthesis of bile acids. Isolation of a cytochrome P-450 from rabbit liver mitochondria catalyzing 26-hydroxylation of C27-steroids.

A cytochrome P-450 catalyzing 26-hydroxylation of C27-steroids was purified from liver mitochondria of untreated rabbits. The enzyme fraction contained 10 nmol of cytochrome P-450/mg of protein and showed only one protein band with a minimum Mr = 53,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified mitochondrial cytochrome P-450 showed apparent molecular weight similar to microsomal cytochromes P-450LM4 but differed in spectral and catalytic properties from these microsomal isozymes. The purified cytochrome P-450 catalyzed 26-hydroxylation of cholesterol, 5-cholestene-3 beta,7 alpha-diol, 7 alpha-hydroxy-4-cholesten-3-one, 5 beta-cholestane-3 alpha,7 alpha-diol, and 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol up to 1000 times more efficiently than the mitochondria. The cytochrome P-450 required both ferredoxin and ferredoxin reductase for catalytic activity. Microsomal NADPH-cytochrome P-450 reductase could not replace ferredoxin and ferredoxin reductase. The cytochrome P-450 was inactive in 7 alpha-, 12 alpha- and 25-hydroxylations of C27-steroids. The results suggest that mitochondrial 26-hydroxylation of various C27-steroids is catalyzed by the same species of cytochrome P-450.

Adrenal Cortex↗