Analysis of clenbuterol in human plasma using liquid chromatography/atmospheric-pressure chemical-ionization mass spectrometry.

Clenbuterol is a beta-agonist drug used illegally as a growth stimulant in meat-producing animals and human athletes. The analysis of clenbuterol in spiked human plasma was performed using on-line liquid chromatography/atmospheric-pressure chemical-ionization mass spectrometry (LC/APCI-MS) using a conventional bore LC column (flow rate = 1.0 mL/min).

Chemical and enzymatic oxidation of benzimidazoline-2-thiones: a dichotomy in the mechanism of peroxidase inhibition.

Derivatives of imidazole-2-thiones block reactions catalyzed by thyroid peroxidase (TPX) and the closely related lactoperoxidase (LPX), and this property is used therapeutically to treat hyperthyroidism. The reactions of a series of benzimidazoline-2-thiones with chemical and enzymatic oxidants were investigated to probe systematically the mechanism of inhibition. Oxidation of benzimidazoline-2-thione (I) and 1-methylbenzimidazoline-2-thione (II) with 3-chloroperbenzoic acid (PBA) yielded reaction products and stoichiometry consistent with benzimidazole-2-sulfenic acids as reactive intermediates.

Metabolism of benzimidazoline-2-thiones by rat hepatic microsomes and hog liver flavin-containing monooxygenase.

The metabolism of benzimidazoline-2-thione (I) and the 1-methyl (II) and 1,3-dimethyl (III) derivatives was studied to elucidate the mechanisms of hepatic oxidation for this class of thionosulfur-containing xenobiotics. NADPH-dependent metabolism of I, II, and III to the corresponding benzimidazoles Ia, IIa, and IIIa, respectively, was observed in dexamethasone-pretreated rat hepatic microsomes. III was the only thiocarbamide converted to an amide metabolite (IIIb).

Covalent binding of 14C- and 35S-labeled thiocarbamides in rat hepatic microsomes.

The covalent binding of a series of 14C- or 35S-labeled benzimidazole-2-thione (MBI) derivatives to rat liver microsomal proteins was studied to determine the mechanisms of hepatic monooxygenase oxidation of model anti-hyperthyroid compounds. All thiocarbamides tested (including methimazole) produced an NADPH-dependent loss of cytochrome P450 (P450) chromophore which could be prevented by the addition of glutathione (GSH). The covalent binding of MBI to liver microsomal proteins from dexamethasone (DEX)-pretreated rats was enhanced 10-fold with NADPH, unaffected by P450 inactivation with 1-aminobenzotriazole (ABT) and attenuated by GSH addition.

Liquid chromatographic determination of ethylenethiourea using pulsed amperometric detection.

A liquid chromatographic method was developed using pulsed amperometric detection at a gold working electrode to measure residue levels of ethylenethiourea (ETU) in crops and groundwater. Use of the sequential pulsing program eliminates electrode fouling while preserving the sensitive and selective detection of ETU. Minimum detection limits in crops were 5-10 ppb (1.

Peroxidase-catalyzed S-oxygenation: mechanism of oxygen transfer for lactoperoxidase.

The mechanism of organosulfur oxygenation by peroxidases [lactoperoxidase (LPX), chloroperoxidase, thyroid peroxidase, and horseradish peroxidase] and hydrogen peroxide was investigated by use of para-substituted thiobenzamides and thioanisoles. The rate constants for thiobenzamide oxygenation by LPX/H2O2 were found to correlate with calculated vertical ionization potentials, suggesting rate-limiting single-electron transfer between LPX compound I and the organosulfur substrate. The incorporation of oxygen from 18O-labeled hydrogen peroxide, water, and molecular oxygen into sulfoxides during peroxidase-catalyzed S-oxygenation reactions was determined by LC- and GC-MS.

Determination of ethylenethiourea in crops using particle beam liquid chromatography/mass spectrometry.

Several food crops were analyzed for residues of ethylenethiourea (ETU), a suspect thyroid and liver carcinogen present in EBDC fungicides, using a commercial particle beam (PB) LC/MS method. The PB/LC/MS detection limits for ETU in crops (5 ppb, 1.25 ng) are comparable to those obtained by LC with electrochemical detection.

Peroxygenation mechanism for chloroperoxidase-catalyzed N-oxidation of arylamines.

The metabolism of three arylamine substrates by H2O2 in the presence of each of the peroxidative enzymes chloroperoxidase (CPX) and pea seed peroxygenase (PSM) was conducted with normal H2O2 and with 18O-labeled H2O2. The resulting C-nitroso aromatic metabolites were examined by GC-MS methods to determine the extent of 18O incorporation. The arylamine substrates were p-toluidine, 4-chloroaniline, and 3,4-dichloroaniline.

Rat hepatic microsomal metabolism of ethylenethiourea. Contributions of the flavin-containing monooxygenase and cytochrome P-450 isozymes.

The contributions of the rat hepatic flavin-containing monooxygenase (FMO) and cytochrome P-450 isozymes (P-450) in the ethylenethiourea (ETU) mediated inactivation of P-450 isozymes and covalent binding of the compound to microsomal proteins were investigated. In vitro, ETU was found to inhibit P-450 marker activities in microsomes obtained from untreated (UT) and phenobarbital (PB), beta-naphthoflavone (BNF), and dexamethasone (DEX) pretreated rats. This inhibition was dependent on the presence of NADPH and was completely abolished by coincubation with glutathione (GSH).

Mechanism of thyroid peroxidase inhibition by ethylenethiourea.

Ethylenethiourea (ETU) is a thyroid carcinogen present in foods formed by degradation and metabolism of ethylenebis[dithiocarbamate] fungicides. ETU inhibits thyroid peroxidase (TPX), the enzyme that catalyzes synthesis of thyroid hormones. Inhibition of TPX-catalyzed reactions by ETU occurs only in the presence of iodide ion with concomitant oxidative metabolism to imidazoline and bisulfite ion.

Oxygenation of organosulfur compounds by peroxidases: evidence of an electron transfer mechanism for lactoperoxidase.

The oxygenation of benzyl methylsulfide, thioanisole, and thiobenzamide to the respective sulfoxides was found to be catalyzed by chloroperoxidase, lactoperoxidase, and horseradish peroxidase. The activities of lactoperoxidase and horseradish peroxidase were similarly low toward benzyl methylsulfide and thioanisole but lactoperoxidase efficiently catalyzed the oxygenation of thiobenzamide while horseradish peroxidase showed low activity. Chloroperoxidase had high reactivity toward all three substrates tested in halide-independent reactions and only small differences in the rates of enzymatic sulfoxidation were observed.

The action of alpha-ketoglutarate dehydrogenase on 4-chloronitrosobenzene: evidence for species-dependent differences in active site properties.

The reaction of bovine (Bos taurus) and porcine (Sus scrufa) cardiac alpha-ketoglutarate dehydrogenase complex (alpha-KGD) with 4-chloronitrosobenzene (I) was shown to produce a hydroxamic acid (IV) and a product due to a Bamberger rearrangement as previously shown for Escherichia coli alpha-KGD. The conversion of I into an active site-bound electrophile was general among the three alpha-KGD enzymes tested, but quantitative differences in products and kinetics were shown. The reaction of I was specific for the resolved alpha-ketoglutarate decarboxylase subunit.

Primary arylamine oxidation by a flavinhydroperoxide. A study of the basis for the substrate specificity of the flavoprotein monooxygenase.

4a-Hydroperoxy-5-ethyl-3,8,10-trimethylisoalloxazine (FlEtOOH) was prepared as a chemical model for the flavin-containing monooxygenase enzyme of mammalian liver. FlEtOOH was found to oxidize a series of para-substituted primary arylamines to the corresponding arylnitroso compound. The rates of arylamine oxidation were found to correlate with the Hammett substituent constant, sigma, as well as with amine basicity.

Hydroperoxyflavin-mediated oxidations of organosulfur compounds. Model studies for the flavin monooxygenase.

Kinetic and product studies were carried out for the reaction of a synthetic hydroperoxyflavin with a series of organosulfur compounds as a model for the flavin-containing monooxygenase of mammalian liver (FMO). S-Oxidized products were identified, and the kinetics of the oxidation reactions were consistent with a mechanism involving attack of a sulfur nucleophile on the terminal oxygen atom of the hydroperoxyflavin. These results provide information about the substrate oxygenation step of FMO not available from steady-state enzyme kinetics.

Oxidation of organosulfur compounds by the microsomal fraction of germinating pea seeds (Pisum sativum).

The oxidation of organosulfur functional groups by the microsomal fraction of germinating pea seeds has been investigated. Arylsulfides , but not thioamides , were converted to sulfoxides by this hemoprotein in the presence of hydrogen peroxide.

The synthesis and mutagenicity of the N-formyl analog of N-hydroxyphenacetin.

The synthesis and purification of N-hydroxy-N-formyl-p-phenetidine (N-OH-FP) is described. This new compound was subjected to mutagenicity testing using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 both in the presence and absence of the post-mitochondrial fraction of rat liver homogenate. Simultaneous mutagenicity testing of the known phenacetin metabolite, N-hydroxyphenacetin (N-OH-AP), was conducted with the same tester strains.

Unsaturated fatty acid-dependent oxidation of epinephrine in homogenates of the gorgonian, Pseudoplexaura porosa.

A novel epinephrine oxidation system in homogenates of the gorgonian Pseudoplexaura porosa was discovered. The enzymatic reaction required an unsaturated fatty acid and molecular oxygen or hydrogen peroxide. Diphenylisobenzofuran was also oxidized by Ps.