Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.     

Product profiles in enzymic and non-enzymic oxidations of the lignin model compound erythro-1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol

The erythro form of the lignin model compound 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol (1) was oxidized with laccase/ABTS, lead(IV) tetraacetate (LTA), lignin peroxidase/H₂O₂, cerium(IV) ammonium nitrate (CAN) and Fenton's reagent. The product profiles obtained with the different oxidants were compared after separation, identification and quantification of the products using HPLC, UV-diode array detector and electrospray ionization mass spectrometry in positive ionization mode. The oxidants generated different product profiles that reflected their different properties. Oxidation with laccase/ABTS resulted almost exclusively in formation of 1-(3,4-dimethoxyphenyl)-3-hydroxy-2-(2-methoxyphenoxy)-1-propanone (2). Oxidation with LTA resulted in more 3,4-dimethoxybenzaldehyde (3) than ketone 2. Lignin peroxidase and CAN gave similar product profiles and aldehyde 3 was the predominant product (only small amounts of ketone 2 were formed). Oxidation with Fenton's reagent resulted in the formation of more aldehyde 3 than ketone 2 but the yields were very low. CAN served as an excellent model for the lignin peroxidase-catalyzed oxidation, while the laccase-mediator system, LTA and Fenton's reagent provided distinctly different product profiles. Erythro-1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol was present among the products obtained on oxidation with LTA, lignin peroxidase, CAN and Fenton's reagent. The differences in redox potential between the oxidants afford an explanation of the diverse product patterns but other factors may also be of importance. The reactions leading to cleavage of the β-ether bond with formation of 1-(3,4-dimethoxyphenyl)-1,2,3-propanetriol (veratrylglycerol) were found to proceed without affecting the configuration at the β-carbon atom.     

Fluorescence study of DNA-complexes formed after metabolic activation of benzo(a)pyrene derivatives

Benzo(a)pyrene derivatives (1-, 2-, 3-, 7-, and 9-hydroxy-benzo(a)pyrene and trans-9,10-dihydro-9,10-dihydroxy-, -4,5-dihydro-4,5-dihydroxy-, and -7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene) were metabolized by liver microsomes isolated from 3-methylcholanthrene-treated rats in the presence of calf thymus DNA. The isolated DNA was then assayed by fluorescence for bound metabolic products. Only 2-hydroxy-benzo(a)pyrene, 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene yielded detectable amounts of DNA-bound products. In contrast to the product(s) from 9-hydroxy-benzo(a)pyrene, the metabolites of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene, both strong carcinogens, had similar excitation spectra and gave considerably increased fluorescence intensities when the DNA was denatured. These data indicate structural similarities in the DNA complexes formed after metabolic activation of 2-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene.     

Heterocyclic polycyclic aromatic hydrocarbon carcinogenesis: 7H-dibenzo[c,g]carbazole metabolism by microsomal enzymes from mouse and rat liver

The metabolism of dibenzo[c,g]carbazole (DBC), was studied in vitro using microsomal fractions of mouse and rat liver from animals, which were treated with 3-methylcholanthrene (MC). The separation of extractable metabolites by high pressure liquid chromatography (HPLC) and thinlayer chromatography (TLC) as well as identification of most of them by nuclear magnetic resonance, mass spectrometry and comparison with synthetically obtained products are described. The microsomes of both species produced the same twelve compounds of which the following have been identified: five monohydroxylated derivatives (phenols), the product of further oxidation of one of them, and a dihydrodiol. The 5-OH-DBC (60% including its spontaneously-formed dimer) and the 3-OH-DBC (14%) are the main metabolites. Three minor metabolites cochromatographed with synthetically prepared 2-OH-DBC, 4-OH-DBC and 6-OH-DBC. The dihydrodiol detectable in small quantity (4–6%) was tentatively identified as 3,4-dihydroxy-3,4-dihydro-DBC by the sensitivity of its formation to very low concentrations of the inhibitor of microsomal epoxide hydrolase, 1,1,1-trichloropropene oxide, by its molecular ion and major fragment in mass spectrometry and by its dehydration product 3-OH-DBC. No other dihydrodiols were detected. The qualitative and quantitative effects of various modulators of metabolism (enzyme inhibitors, apparently homogeneous epoxide hydrolase, glutathione, supernatant fraction) were investigated. The results are discussed with respect to possible ultimate carcinogens.     

Diaryl dimers of estradiol and of estrone may be formed as major metabolites by mouse mammary glands

The formation of a novel estrogen metabolite by mammary tissues was investigated. Polar and nonpolar metabolites of endogenous estrogens are formed in liver and other tissues. Polar products such as the catechol estrogens are implicated in tumorigenesis in breast tissue, whereas a nonpolar metabolite, 2-methoxyestradiol, may be protective. Diaryl ether dimers, as a novel form, have been reported as nonpolar products from liver microsomes. We have noted major amounts of nonpolar metabolites in other tissues that were neither 2-methoxyestrogens nor estrogen fatty acid esters. The possible formation of such novel metabolites by breast tissues from adult nulliparous mice with [³H]-labeled estrogens as substrates was considered. Steroids were recovered from media by solid-phase extraction and profiles were obtained from HPLC (acetonitrile:water). Saponification was done with an internal standard of estradiol stearate. Major amounts of nonpolar metabolites were formed in all instances, with one or two principal peaks. Alkaline hydrolysis had no effect on the nonpolar product(s) but released estradiol from its stearate. Strong acid treatment also had no effect as shown by HPLC. Thus, it is suggested that diaryl dimers of estrogens may be formed as major metabolites by mouse mammary glands.     

Induction by 9-hydroxyellipticine of aryl hydrocarbon hydroxylase in perinatal rat liver and in primary fetal hepatocytes in culture

N-nitrosodiethanolamine is converted to N-(2-hydroxyethyl)-N-(formylmethyl)nitrosamine (EFMN) and N-(2-hydroxyethyl)-N-carboxymethyl) nitrosamine (ECMN) by rat S9 liver preparation as a result of beta-oxidation. The beta-oxidized metabolites were isolated and identified by gas chromatography-mass spectrometry (GC-MS) by comparison with authentic standards. An original gas chromatographic method with thermal energy detection was set up to measure both metabolites quantitatively. Under the experimental conditions described, when NAD+ was used as cofactor, about 1% of N-nitrosodiethanolamine (NDELA) was converted to EFMN and about half of the latter product was in turn converted to ECMN. The beta-nitrosamino aldehyde seems to transfer the nitroso moiety to other amino-compounds, even at physiological pH. The significance of the metabolic formation of EFMN in relation to the carcinogenicity of NDELA is discussed.     

Action of peptidases in brain synaptic membranes on the NH2-terminus of adrenocorticotropin using ACTH-(1-16)-NH2 as a model substrate

The action of brain peptidases on NH2-terminal sequences of adrenocorticotropin was studied by incubation of ACTH-(1-16)-NH2 under different pH conditions. Profiles of metabolites and time course of product formation were obtained by HPLC analysis of the digests. Fragments of ACTH-(1-16)-NH2 were isolated and characterized by their amino acid composition and NH2-terminal groups. Both at pH 7.4 and pH 8.5 the following fragments were found: ACTH-(3-16)-NH2, ACTH-(4-16)-NH2, ACTH-(5-16)-NH2, and ACTH-(7-16)-NH2. At pH 7.4 the major products were ACTH-(4-16)-NH2 and ACTH-(7-16)-NH2, while the peptide ACTH-(3-16)-NH2 was the main metabolite at pH 8.5. The nature of identified peptides and the time course of their formation demonstrates that aminopeptidase activities predominate in the conversion of the NH2-terminus of adreno-corticotropin and related peptides by brain synaptic membranes.     

Circulating and urinary thromboxane B2 metabolites in the rabbit: 11-dehydro-thromboxane B2 as parameter of thromboxane production

The metabolism of thromboxane B2 was studied in the rabbit. The aim of the study was to identify metabolites in blood and urine that might serve as parameters for monitoring thromboxane production in vivo. [5,6,8,9,11,12,14,15-3H8]-Thromboxane B2 was administered by i.v. injection to rabbits, and blood samples and urine were collected with brief intervals. The metabolic profiles were visualized by two-dimensional thin layer chromatography and autoradiography, and the structures of five major metabolites were determined using chromatographic and mass spectrometric methods. In urine the major metabolites were identified as 11-dehydro-TXB2 and 2,3,4,5-tetranor-TXB1, and other prominent products were 11-dehydro-2,3,4,5-tetranor-TXB1, 2,3-dinor-TXB1 and 2,3-dinor-TXB2. In the circulation, TXB2 was found to disappear rapidly. The first major metabolite to appear was 11-dehydro-TXB2, which also remained a prominent product in blood for the remainder of the experiment (90 min). With time, the profile of circulating products became closely similar to that in urine. TXB2 was not converted into 11-dehydro-TXB2 by blood cells or plasma. The dehydrogenase catalyzing its formation was tissue bound and was found to have a widespread occurrence: the highest conversion was found in lung, kidney, stomach and liver. The results of the present study suggest that 11-dehydro-TXB2 may be a suitable parameter for monitoring thromboxane production in vivo in the rabbit in blood as well as urinary samples, and possibly also several tissues. This was also demonstrated in comparative studies using radioimmunoassays for TXB2 and 11-dehydro-TXB2.     

Metabolic products and pathways of fluorotelomer alcohols in isolated rat hepatocytes

Fluorotelomer alcohols (FTOHs; CF(3)(CF(2))(x)C(2)H(4)OH; where x=3, 5, 7, 9) are a novel class of polyfluorinated contaminants, recently detected in the North American atmosphere, that are possible precursors to the series of perfluoroalkyl carboxylates (PFCAs) in human blood. An in vivo rat study validated earlier independent work that poly- and per-fluoroalkyl carboxylates were metabolites of FTOHs, but our detection of several novel metabolites prompted us to examine their pathways in greater detail using isolated rat hepatocytes. Using 8:2 FTOH (i.e. where x=7) as a model compound, the metabolic products formed by isolated rat hepatocytes were identified, and three synthesized intermediates were incubated separately to elucidate the metabolic pathways. For 8:2 FTOH, a major fate was direct conjugation to form the O-glucuronide and O-sulfate. Using 2,4-dinitrophenylhydrazine (DNPH) trapping, the immediate oxidation product of 8:2 FTOH was identified as 8:2 fluorotelomer aldehyde (8:2 FTAL; CF(3)(CF(2))(7)CH(2)C(H)O). 8:2 FTAL was transient and eliminated HF non-enzymatically to yield 8:2 fluorotelomer alpha,beta-unsaturated aldehyde (8:2 FTUAL; CF(3)(CF(2))(6)CFCHC(H)O) which was also short-lived and reacted GSH and perhaps other endogenous nucleophiles. Four polyfluorinated acid intermediates were also detected, including 8:2 fluorotelomer carboxylate (8:2 FTCA; CF(3)(CF(2))(7)CH(2)C(O)O(-)), 8:2 fluorotelomer alpha,beta-unsaturated carboxylate (8:2 FTUCA; CF(3)(CF(2))(6)CFCHC(O)O(-)), tetrahydroperfluorodecanoate (CF(3)(CF(2))(6)(CH(2))(2)CO(2)(-)), and dihydroperfluorodecenoate (CF(3)(CF(2))(6)CHCHCO(2)(-)). The pathways leading to 8:2 FTCA and FTUCA involve oxidation of 8:2 FTAL, however, the pathways leading to the latter two polyfluorinated acids remain inconclusive. The fate of the unsaturated metabolites, 8:2 FTUAL and FTUCA, included conjugation with GSH and dehydrofluorination to yield alpha,beta-unsaturated GSH conjugates, and GS-8:2 FTUAL which was subsequently reduced to the corresponding alcohol. Perfluorooctanoate (PFOA) and minor amounts of perfluorononanoate (PFNA) were confirmed as metabolites of 8:2 FTOH, and the respective roles of beta- and alpha-oxidation mechanisms are discussed. The analogous acids, aldehydes, and conjugated metabolites of 4:2, 6:2, and 10:2 FTOH (i.e. where x=3, 5, and 9, respectively) were also detected, and metabolite profiles among FTOHs generally differed only in the length of their perfluoroalkyl chains. Preincubation with aminobenzotriazole, but not pyrazole, inhibited the formation of metabolites from all FTOHs, suggesting that their oxidation was catalyzed by P450, not alcohol dehydrogenase.     

Characterisation of coupling products formed by biotransformation of biphenyl and diphenyl ether by the white rot fungus Pycnoporus cinnabarinus

Cells of the white rot fungus Pycnoporus cinnabarinus grown in glucose were able to hydroxylate biphenyl and diphenyl ether, although growth was inhibited by these substrates at concentrations above 250 microM. 2- and 4-Hydroxybiphenyl were detected as products of biphenyl metabolism and 2- and 4-hydroxydiphenyl ether as products of diphenyl ether metabolism in the culture media. After addition of 2-hydroxydiphenyl ether and 2-hydroxybiphenyl to cell-free supernatants containing laccase as the only ligninolytic enzyme, different coloured precipitates were formed. HPLC analysis revealed the formation of additional hydrophobic metabolites with one major product per transformation. Mass spectrometric analysis of the methyl derivatives of the polymer mixture indicated dimers and trimers with different binding types. The main products were identified as dimers with carbon-carbon bonds in para-position to the hydroxyl group of the monomers by mass spectroscopy and nuclear magnetic resonance spectroscopy.     

In vitro metabolism of 19-nor-1alpha, 25-(OH)2D2 in cultured cell lines: inducible synthesis of lipid- and water-soluble metabolites

The active vitamin D analog, 19-nor-1alpha,25-dihydroxyvitamin D2 (19-nor-1alpha,25-(OH)2D2), has a similar structure to the natural vitamin D hormone, 1a,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3), but lacks the C10-19 methylene group and possesses an ergosterol/ vitamin D2 rather than a cholesterol/vitamin D3 side chain. We have used this analog to investigate whether any of these structural features has any effect upon the type and rate of in vitro metabolism observed. Using a vitamin D-target cell, the human keratinocyte, HPK1A-ras, we observed formation of a number of metabolites, three of which were purified by extensive HPLC and conclusively identified by a combination of GC-MS and chemical derivatization as 19-nor-1alpha,24,25-(OH) 3D2, 19-nor-1alpha,24,25,26-(OH) 4D2, and 19-nor-1alpha,24,25,28-(OH)4,D2. The first metabolite is probably a product of the vitamin D-inducible cytochrome P450, P450cc24 (CYP24), while the latter two metabolites are likely to be further metabolic products of 19-nor-1alpha,24,25-(OH)3D2. These hydroxylated metabolites resemble those identified by other workers as products of the metabolism of 1alpha,25-(OH)2D2 in the perfused rat kidney. It therefore appears from the similar metabolic fate of 19-nor-1alpha,25-(OH)2D2 and 1alpha,25-(OH)2D2 that the lack of the C10-19 methylene group has little effect upon the nature of the lipid-soluble metabolic products and the rate of formation of these products seems to be comparable to that of products of 1alpha,25-(OH)2D3 in vitamin D-target cells. We also found extensive metabolism of 19-nor-1alpha,25(OH)2D2 to water-soluble metabolites in HPK1A-ras, metabolites which remain unidentified at this time. When we incubated 19-nor-1alpha,25-(OH)2D2 with the liver cell line HepG2, we obtained only 19-nor-1alpha,24,25-(OH)3D2. We conclude that 19-nor-1alpha,25-(OH)2D2 is efficiently metabolized by both vitamin D-target cells and liver cells.     

Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme.     

Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D3 to produce 20-hydroxyvitamin D3 and other poorly characterized hydroxylated products. The present study aimed to identify all the products of vitamin D3 metabolism by P450scc, as well as the pathways leading to their formation. Besides 20-hydroxyvitamin D3, other major metabolites of vitamin D3 were a dihydroxyvitamin D3 and a trihydroxyvitamin D3 product. The dihydroxyvitamin D3 was clearly identified as 20,23-dihydroxyvitamin D3 by NMR, in contrast to previous reports that postulated hydroxyl groups in positions 20 and 22. NMR of the trihydroxy product identified it as 17alpha,20,23-trihydroxyvitamin D3. This product could be directly produced by P450scc acting on 20,23-dihydroxyvitamin D3, confirming that hydroxyl groups are present at positions 20 and 23. Three minor products of D3 metabolism by P450scc were identified by MS and by examining their subsequent metabolism by P450scc. These products were 23-hydroxyvitamin D3, 17alpha-hydroxyvitamin D3 and 17alpha,20-dihydroxyvitamin D3 and arise from the three P450scc-catalysed hydroxylations occurring in a different order. We conclude that the major pathway of vitamin D3 metabolism by P450scc is: vitamin D3 --> 20-hydroxyvitamin D3 --> 20,23-dihydroxyvitamin D3 --> 17alpha,20,23-trihydroxyvitamin D3. The major products dissociate from the P450scc active site and accumulate at a concentration well above the P450scc concentration. Our new identification of the major dihydroxyvitamin D3 product as 20,23-dihydroxyvitamin D3, rather than 20,22-dihydroxyvitamin D3, explains why there is no cleavage of the vitamin D3 side chain, unlike the metabolism of cholesterol by P450scc.     

Synthesis and composition of vitamin D-3 metabolites in Solanum malacoxylon

The synthesis of vitamin D-3 hydroxylated metabolites in Solanum malacoxylon was investigated. When crude leaf homogenates and subcellular fractions were incubated with [³H]vitamin D-3 and [³H]25-hydroxy-vitamin D-3 under conditions described for animal vitamin D-3-25-hydroxylase and 25-hydroxy-vitamin D-3-1α-hydroxylase, respectively, labelled metabolites identified on the basis of their chromatographic properties as 25-hydroxy-vitamin D-3 and 1,25-dyhydroxy-vitamin D-3 were formed. Other unidentified product metabolites were also detected. Vitamin D-3-25-hydroxylase activity was localized in microsomes and 25-hydroxy-vitamin D-3-1α-hydroxylase in mitochondria and microsomes. Chromatography of sterols isolated from leaf extracts preincubated with β-glucosidase on Sephadex LH-20 columns permitted the isolation of three biologically active fractions with elution properties similar to vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3, respectively. Ultraviolet spectra characteristic of vitamin D-3 and its metabolites were obtained after purification of the fractions by TLC. Co-chromatography of individual fractions with authentic metabolites on TLC provided further evidence that the plant contains vitamin D-3, 25-hydroxy-vitamin D-3 and 1,25-dihydroxy-vitamin D-3 as glucoside derivatives. These results suggest that a similar pathway of vitamin D-3 hydroxylation as in animals may be operative in S. malacoxylon.     

Profiles of eicosanoid production from 14C-arachidonic acid and prostaglandin metabolism by rabbit esophageal mucosa and muscularis

In an attempt to elucidate the possible involvements of eicosanoids in esophageal functions and disorders, we have investigated the formation of both cyclooxygenase and lipoxygenase metabolites from 14C-arachidonic acid by rabbit esophageal tissues. Homogenates of rabbit esophageal mucosa and muscularis were incubated with 14C-arachidonic acid and after ether extraction eicosanoids were separated and quantified by reverse phase high performance liquid chromatography. The predominant cyclooxygenase products were 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 for mucosa and 6-keto-PGF1 alpha, and PGE2 for muscularis. The formation of these products was inhibited both by indomethacin and the dual pathway inhibitor, nordihydrogualaretic acid (NDGA). In mucosa the major eicosanoid was 12-HETE (12-hydroxyeicosatetraenoic acid) which was inhibited by NDGA but not by indomethacin which on the contrary enhanced its formation. Additionally four polar products were synthesized which appeared to be lipoxygenase-dependent as their formation was inhibited by NDGA but not by indomethacin. Muscularis produced as a minor lipoxygenase product only 12-HETE, which was inhibited by NDGA but unchanged in the presence of indomethacin. In addition, both tissues, but mucosa more than muscularis, possessed large prostaglandin catabolizing capacity. The present findings indicate that rabbit esophageal tissues can convert 14C-arachidonic acid into lipoxygenase as well cyclo-oxygenase products which may have a role in esophageal physiology and pathophysiology.     

Comparison of sister-chromatid exchange induction caused by nitrosoureas that alkylate or alkylate and crosslink DNA

We have investigated the induction of sister-chromatid exchanges (SCEs) in 9L rat brain tumor cells treated with the alkylating agent 1-ethyl-1-nitrosourea (ENU) and 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU), an agent that both alkylates and crosslinks DNA. Induction of SCEs by ACNU was found to be 143-fold greater than for ENU. However, on an equimolar basis, the alkylation of DNA by 14C-ACNU was approximately 3.2-fold higher than for 14C-ENU. After correction for this difference was made, the induction of SCEs by ACNU was calculated to be 45-fold greater than for ENU. While DNA alkylation products formed by ACNU and ENU are similar, the chloroethyl alkylation product(s) of ACNU can form DNA-interstrand crosslinks; the ethyl alkylation product(s) of ENU cannot. Based on these findings, we propose that the increased induction of SCEs caused by ACNU is a result of the formation of DNA interstrand crosslinks.     

Isolation and Characterization of a Dibenzofuran-Degrading Yeast: Identification of Oxidation and Ring Cleavage Products

We characterized the ability of a yeast to cleave the aromatic structure of the dioxin-like compound dibenzofuran. The yeast strain was isolated from a dioxin-contaminated soil sample and identified as Trichosporon mucoides. During incubation of glucose-pregrown cells with dibenzofuran, six major metabolites were detected by high-performance liquid chromatography. The formation of four different monohydroxylated dibenzofurans was proven by comparison of analytical data (gas chromatography-mass spectrometry) with that for authentic standards. Further oxidation produced 2,3-dihydroxydibenzofuran and its ring cleavage product 2-(1-carboxy methylidene)-2,3-dihydrobenzo[b]furanylidene glycolic acid, which were characterized by mass spectrometry and ¹H nuclear magnetic resonance spectroscopy. These two metabolites are derived from 2-hydroxydibenzofuran and 3-hydroxydibenzofuran, as shown by incubation experiments using these monohydroxylated dibenzofurans as substrates.     

Enzymatic synthesis of substituted epicatechins for bioactivity studies in neurological disorders

Glucuronidated and/or methylated metabolites of the proanthocyanidin (PA) monomer (-)-epicatechin are detected in both blood and brain following feeding of rodents with a monomeric grape seed PA extract shown to reduce symptoms in a mouse model of Alzheimer's disease. To generate metabolites for future mechanistic studies, we investigated the ability of recombinant human glucuronosyl transferases of the UGT1A and UGT2B families to glucuronidate epicatechin or 3'-O-methyl epicatechin in vitro. Of twelve enzymes tested, UGT1A9 was the most efficient, producing epicatechin 3'-O-glucuronide as the major product. Incubation of UGT1A9 with 3'-O-methyl-epicatechin resulted in two major products, one of which was identified as 3'-O-methyl-epicatechin 5-O-glucuronide, a major metabolite found in blood plasma and brain tissue of the rodents following feeding with a grape seed extract. We also investigated in vitro methylation of epicatechin and epicatechin glucuronides by human catechol O-methyltransferase. Enzymatic production of 3'-O-methyl-epicatechin 5-O-glucuronide was optimized to 50% overall yield. These studies form a basis for generation of mg quantities of pure epicatechin (methyl) glucuronides of biological significance, and provide clarification of structure of previously identified epicatechin metabolites.     

Synthesis and stereochemical determination of diunsaturated valproic acid analogs including its major diunsaturated metabolite

Valproic acid, an antiepileptic drug, is transformed into diunsaturated metabolites in humans. Synthesis of the geometric isomers of 2-(1'-propenyl)-2-pentenoic acid and 2-(1'-propenyl)-3-pentenoic acid was attempted using known procedures. The final product, a mixture of isomers, was converted into tert-butyldimethylsilyl or ethyl derivatives. Capillary gas-liquid chromatography-mass spectrometry analysis of the derivatives showed at least three isomeric dienoic acids from synthesized products. Argentation thin-layer chromatography was effective in resolving the isomeric mixture into a single isomer or mixture of two isomers. Thin-layer chromatography and gas-liquid chromatography retention data, photochemical isomerization studies, and nuclear magnetic resonance spectrometry were used to characterize the dienoic acids. By comparison of the retention times of the diunsaturated metabolites with synthesized reference compounds, the structure assigned to the major diunsaturated metabolite is 2-[(E)-1'-propenyl](E)-2-pentenoic acid.     

Metabolism of 25-hydroxyvitamin D3 by microsomal and mitochondrial vitamin D3 25-hydroxylases (CYP2D25 and CYP27A1): a novel reaction by CYP27A1

The metabolism of 25-hydroxyvitamin D(3) was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D(3) 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D(3) 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1alpha,25-dihydroxyvitamin D(3), 24,25-dihydroxyvitamin D(3) and 25,27-dihydroxyvitamin D(3). An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D(3) metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D(3) as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D(3) metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), 25,27-dihydroxyvitamin D(3) and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D(3) metabolites such as 1alpha,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D(3). The product had the mass spectrometic properties expected for 4beta,25-dihydroxyvitamin D(3). Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3) and 25,26-dihydroxyvitamin D(3). It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D(3).