Peroxidase-catalyzed oxidation of capsaicinoids: steady-state and transient-state kinetic studies

Capsaicinoids are the pungent compounds in Capsicum fruits (i.e., "hot" peppers). Peroxidases catalyze capsaicinoid oxidation and may play a central role in their metabolism. However, key kinetic aspects of peroxidase-catalyzed capsaicinoid oxidation remain unresolved. Using transient-state methods, we evaluated horseradish peroxidase compound I and II reduction by two prominent capsaicinoids (25 degrees C, pH 7.0). We determined rate constants approaching 2 x 10(7) and 5 x 10(5)M(-1)s(-1) for compound I and compound II reduction, respectively. We also determined k(app) values for steady-state capsaicinoid oxidation approaching 8 x 10(5)M(-1)s(-1) (25 degrees C, pH 7.0). Accounting for stoichiometry, these are in excellent agreement with constants for compound II reduction, suggesting that this reaction governs capsaicinoid-dependent peroxidase turnover. Ascorbate rapidly reduced capsaicinoid radicals, assisting in the determination of the kinetic constants reported. Because ascorbate accumulates in Capsicum fruits, it may also be an important determinant for capsaicinoid content and preservation in Capsicum fruits and related products.     

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.     

Characteristics of a plant deleterious rhizosphere pseudomonad and its inhibitory metabolite(s)

A cell-free culture filtrate of the plant growth inhibitory bacterial isolate Å313, identified as Pseudomonas fluorescens, was tested for its effect on wheat root elongation in vitro, with and without various pretreatments. The filtrate showed a strongly inhibitory effect on root elongation and could be heated to 100°C for 5 min or incubated at a pH within the range of 4–10 without losing its activity. Unlike other root growth-inhibitory bacterial metabolites the effect of the filtrate was not reversed by methionine, nor was any inhibitory activity present in the water phase after extraction with chloroform. The inhibitory agent(s) was formed when the bacterium was grown in an artificial medium as well as in root exudates from wheat. Two wheat cultivars differing in reaction to inoculation with living cells of Å313 showed the expected difference in growth reduction when exposed to culture filtrate, indicating a cultivar difference in sensitivity to the metabolite(s) formed by the bacterium. Isolate Å313 invaded intercellular spaces of the root cortex of gnotobiotically grown wheat plants, but did not produce pectolytic enzymes in vitro, nor induce a hypersensitive response in tobacco plants.     

Direct fluorometric analysis of benzo(a)pyrene metabolite formation by mouse liver microsomes

We have examined the metabolites produced by in vitro incubation of benzo(a)pyrene with 3-methylcholanthrene-induced mice liver microsomes. Our objective was to observe directly a possible difference in microsomal enzyme systems of animal models having different susceptibility to chemical carcinogens. The metabolites produced by the two animal models,$\text{C57BL}\text{6J}$ and $\text{DBA}\text{2}$ mice, were analyzed by a highly sensitive, “three-dimensional” fluorescence plotting technique. The fluorescence spectra of the total ethyl acetate-soluble metabolites clearly indicate that the metabolites produced by $\text{DBA}\text{2}$ enzymes were predominantly monohydroxylated benzo(a)pyrene while those produced by the liver microsomes of $\text{C57BL}\text{6J}$ were highly enriched with the 7,8-dihydrodihydroxybenzo(a)pyrene type.     

Amodiaquine as a prodrug: importance of metabolite(s) in the antimalarial effect of amodiaquine in humans

Existing analytical methods for assaying the 4-aminoquinoline antimalarial amodiaquine in body fluids are nonspecific and obscure the fact that little or no amodiaquine is present in the blood of dosed persons. We have isolated four metabolites of amodiaquine. The two major metabolites have been identified; one is desethylamodiaquine, and the other has been tentatively identified on the basis of proton nuclear magnetic resonance spectroscopy as 2-hydroxydesethylamodiaquine. We developed a reverse-phase high-performance liquid chromatographic (HPLC) method that separates the two major metabolites from each other and from amodiaquine, allowing separate quantification. The impact of these findings on in vitro sensitivity testing and blood analysis of persons dosed with amodiaquine is discussed.     

Feeding behaviour of Bactrocera cacuminata (Hering) (Diptera: Tephritidae) on methyl eugenol: a laboratory assay

Abstract Many dacine (Diptera: Tephritidae) species are attracted to one of two chemical substances, 4-allyl-1,2-dimethoxybenzene (methyl eugenol (ME)) or 4-( p -hydroxyphenyl)-2-butanone (cue lure). Despite the fact that these chemicals or analogs occur naturally, their significance in the biology/ecology of the Dacinae has seldom been examined. In this study, we examine the patterns in feeding behaviour of Bactrocera cacuminata (Hering), a monophagous non-pest fruit fly, on ME. Based on a laboratory assay, we found that males of this species fed on ME multiple times within a single day and on multiple days. The pattern of repeat feeding was not related to time since previous feeding or duration of prior feeding. Our results contrast those obtained on a similar study on a related polyphagous species ( Bactrocera dorsalis (Hendel)). We discuss the implications of our findings with a view to explaining the functional significance of dacine response to these plant-derived chemicals.     

Synthesis and absolute configuration of (-)-3-butyl-7-hydroxyphthalide, a cytotoxic metabolite of Penicillium vulpinum

Both the enantiomers as well as the racemate of 3-butyl-7-hydroxyphthalide (1) were synthesized, and the absolute configuration of the naturally occurring (-)-1 (a weakly cytotoxic metabolite of Penicillium vulpinum) was identified as S.     

Peroxidase-catalyzed formation of triplet acetone and chemiluminescence from isobutyraldehyde and molecular oxygen

It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.     

Isolation and characterization of a cytotoxic metabolite of Talaromyces bacillosporus.

A cytotoxic metabolite, talarotoxin, was isolated from a fungus, Talaromyces bacillosporus IFO 8397, cultured on rice. The structure of the toxin was elucidated and found to contain a pyrrolizidinedione connected with a trans delta 1-octalin through a conjugated triene.     

Recognition of human minor alloantigen(s) by cytotoxic lymphocytes in vitro

Lymphocytes from an extensively transfused patient with aplastic anemia were induced to cytotoxicity against target cells from several HLA-matched siblings by in vitro stimulation with allogeneic cells. Effective stimulating cells shared HLA-B7 with the patient, but not all B7 individuals were effective. An additional factor, which was found to segregate in both the patient's and an unrelated sibship, was also necessary. Segregation of this minor alloantigen, W, was also revealed among the patient's HLA-matched sibs by differential susceptibility to lysis by effectors from the patient. The ratio of six positive to four negative siblings suggests that the antigen difference might be coded by a single locus. Lymphocytes from a normal sib, who like the patient is lacking the minor antigen, could not be induced to cytotoxicity against positive targets. Thus in vivo sensitization of the donor of the responding cells appears to be necessary for the demonstration of the cytotoxic response to the minor antigen in vitro. No correlation was observed between the segregation pattern of W and of known blood group antigens, and no cytotoxic antibody to W was detected in the patient's serum in several trials.Abbreviations used in this paper MHC major histocompatibility complex - Tc thymus dependent cells capable of mediating cytotoxicity in the absence of Immoral antibody - GVHD graft versus host disease - CML cell mediated lympholysis - MLC unidirectional mixed lymphocyte culture - ADCC antibody-dependent cell mediated cytotoxicity     

Production of cytotoxic factor(s) in human T cell lines transformed by a human retrovirus

Human T cell lines, MT-2, TCL-Ter, TCL-Haz, and TCL-Kan which were transformed by a human retrovirus, constitutively produced cytotoxic factor(s) (CF) in the culture supernatants. In these cell lines, MT-2 produced the largest amount of CF. The amount of CF produced by MT-2 was 9-10 or 3-4 times larger than that produced by a human B cell line, RPMI 1788, or normal peripheral blood leukocytes stimulated with mitogens and phorbol ester. The kinetics of the production by MT-2 was similar in media with and without serum. The activity was stable at 56 degrees C for 30 min but was lost at 80 degrees C for 30 min and at pH 2 for 20 hr. On gel filtration, the molecular weight of the factor produced by MT-2 was approximately 90,000. On isoelectric focusing, the activity was recovered in the fraction at pH 6.5-7.0.     

Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (beta-hematin formation)

Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (beta-hematin formation) through its oxidation. Heme when added to HRP compound II (FeIV=O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, FeIII) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a1H=14.75 G, a2H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme.     

Vitamin K1 as a modulator of benzo(a)pyrene metabolism as measured by in vitro metabolite formation and in vivo DNA-adduct formation

Vitamin K1 (2-methyl-3-phytyl-1,4-napthoquinone) increases the microsomal metabolism of benzo(a)pyrene in rat liver microsomes in vitro. The increase is most marked in the 9,10 diol, 4,5 diol and 3-OH metabolites. The effect is seen at an in vitro concentration of 25 microM and disappears at higher concentrations of K1. The production of BP metabolite-DNA adducts in liver in vivo in ICR/Ha mice is reduced in dietary induced vitamin K deficient mice and this effect is reversed by vitamin K1. These findings indicate a role for vitamin K1 in the regulation of the microsomal mixed function oxidase system and suggest a reason for the low intracellular content and minimal body stores of this vitamin.     

S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

Radioimmunoassay of N-acetyl-N-formyl-5-methoxykynuramine (AFMK): a melatonin oxidative metabolite

N-acetyl-N-formyl-5-methoxykynuramine (AFMK) is a melatonin metabolite identified in rat brain by Hirata et al. (The Journal of Biological Chemistry 249 (1974) 1311). Since no assay has been described for its routine measurement, we have developed and validated such a radioimmunoassay. We synthesized AFMK and N-acetyl-5-methoxykynuramine (AMK), in order to produce anti-AFMK antibodies and to standardize the assay. The tracer [3H]-AFMK was obtained from [3H]-melatonin. The assay was preceded by a chromatographic step on Celite microcolumn in order to increase its specificity. The assay was suitable for the measurement of AFMK levels ranging from 59 to 1894 pmol/L. The detection limit of the assay was routinely set at 65 pmol/L. The intra- and inter-assay coefficients of variation were 3.5% and 11% respectively. Investigation of the 24 h plasma pattern in healthy volunteers did not reveal any AFMK levels in plasma samples. In rats, plasma AFMK showed a peak after melatonin injection, which confirmed the in vivo AFMK production as a melatonin metabolite. This AFMK assay is suitable for studies on melatonin metabolism.     

Branchpoint for heme alkylation and metabolite formation in the oxidation of arylacetylenes by cytochrome P-450

Phenylacetylene and biphenylacetylene are oxidized by cytochrome P-450 to the corresponding arylacetic acids. The acetylenic hydrogen shifts to the adjacent carbon and one atom of molecular oxygen is incorporated into the carboxylic acid group in these transformations, which are subject to a large kinetic isotope effect when the acetylenic hydrogen is replaced by deuterium. The same products and isotope effects are observed when the two arylacetylenes are oxidized by m-chloroperbenzoic acid rather than by the enzyme. In contrast, the inactivation of cytochrome P-450 that occurs during the oxidation of phenylacetylene is insensitive to deuterium substitution. The partition ratio between metabolite formation and enzyme inactivation consequently changes from 26 to 15 in going from phenylacetylene to the deuterated analogue. Metabolite formation therefore diverges from heme alkylation very early in the catalytic process.     

Benzimidazolone as potent chymase inhibitor: modulation of reactive metabolite formation in the hydrophobic (P1) region

A new class of chymase inhibitor featuring a benzimidazolone core with an acid side chain and a P₁ hydrophobic moiety is described. Incubation of the lead compound with GSH resulted in the formation of a GSH conjugate on the benzothiophene P₁ moiety. Replacement of the benzothiophene with different heterocyclic systems such as indoles and benzoisothiazole is feasible. Among the P₁ replacements, benzoisothiazole prevents the formation of GSH conjugate and an in silico analysis of oxidative potentials agreed with the experimental outcome.