Comparison and Optimization of Different Protein Nitrogen Quantitation and Residual Protein Characterization Methods in Dietary Fiber Preparations.

Proteins are plant cell wall components but they are not included in the definition of dietary fiber. Therefore, dietary fiber preparations have to be corrected for their residual protein contents. This is commonly done by calculating the residual protein concentrations from the nitrogen contents after Kjeldahl digestion. Here, three different methods to determine nitrogen in Kjeldahl digests were compared: conventional titration with hydrochloric acid after steam distillation, a colorimetric assay (24-well microplates and cuvettes), and the determination by using an ammonia electrode. All assays gave similar results but detection using the ammonia electrode was found to be the most time-efficient approach. Also, an amino-acid profiling method, which is not based on commercial kits and which is suitable for routine analysis of dietary fiber preparations, was established. For this purpose, an HPLC-FLD method following amino acid derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was optimized for fiber samples. Although all commonly used dietary fiber preparation methods involve the application of proteases the amino acid profiles of fiber samples from different sources were shown to be quite diverse. Considering the amino acid composition of the residual protein in various dietary fiber preparations, residual protein is probably not only based on structural proteins.

Development of a QuEChERS-Based Stable-Isotope Dilution LC-MS/MS Method To Quantitate Ferulic Acid and Its Main Microbial and Hepatic Metabolites in Milk.

Forage plants of the Poaceae family are grown as pasturage or used for the production of hay, straw, corn stover, etc. Although ferulic acid contents of grasses are generally high, the amount of ingested ferulic acid differs depending on the type of forage, resulting in varying contents of ferulic acid and its microbial and hepatic metabolites in milk. Concentrations and patterns of these metabolites may be used as markers to track different forages in livestock feeding. Therefore, we developed a stable isotope dilution assay to quantitate ferulic acid, 12 ferulic acid-based metabolites, p-coumaric acid, and cinnamic acid in milk. Because most analytes were not commercially available as stable isotope labeled standard compounds, they were synthesized as 13C- or deuterium-labeled standard compounds. A modification of the QuEChERS method, a Quick, Easy, Cheap, Effective, Rugged, and Safe approach usually applied to analyze pesticides in plant-based products, was used to extract the phenolic acids from milk. Determination was carried out by LC-ESI-MS/MS in scheduled multiple reaction monitoring modus. By using three different milk samples, the applicability of the validated approach was demonstrated.

Structural Transformation of 8-5-Coupled Dehydrodiferulates by Human Intestinal Microbiota.

Ingested dehydrodiferulates (DFAs) are partially released from cereal dietary fiber by human colonic microbiota, but little research has explored the further microbial metabolism of 8-5-coupled DFAs. This study investigated the in vitro microbial metabolism and elucidated major metabolites of free 8-5-DFAs (benzofuran and open forms) and an esterified analogue, 8-5-DFA diethyl ester (benzofuran). Synthesized standard compounds were incubated with fresh human fecal suspensions. Metabolites were isolated and structurally elucidated using high-resolution-LC-time-of-flight-(ToF)-MS, GC-MS, and NMR. Nine metabolite structures were unambiguously characterized with NMR, and four additional metabolites were tentatively identified to reveal structural conversion motifs: propenyl side chain hydrogenation (all substrates), O-demethylation and reductive ring-opening (8-5-DFA diethyl ester and free 8-5-DFA [benzofuran]), and de-esterification (8-5-DFA diethyl ester). A pathway of microbial 8-5-DFA metabolism was proposed based on metabolite formation kinetics. Importantly, de-esterification of the 8-5-DFA diethyl ester occurred primarily after and/or concurrently with other metabolism steps. Cleavage to monomers was not observed.

Conjugation of the mycotoxins alternariol and alternariol monomethyl ether in tobacco suspension cells.

The mycotoxins alternariol (AOH) and alternariol-9-O-methyl ether (AME) carry three and two phenolic hydroxyl groups, respectively, which makes them candidates for the formation of conjugated metabolites in plants. Such conjugates may escape routine methods of analysis and have therefore been termed masked or, more recently, modified mycotoxins. We report now that AOH and AME are extensively conjugated in suspension cultures of tobacco BY-2 cells. Five conjugates of AOH were identified by MS and NMR spectroscopy as ß-D-glucopyranosides (attached in AOH 3- or 9-position) as well as their 6'-malonyl derivatives, and as a gentiobiose conjugate. For AME, conjugation resulted in the d-glucopyranoside (mostly attached in the AME 3-position) and its 6'- and 4'-malonyl derivatives. Pronounced differences were noted for the quantitative pattern of AOH and AME conjugates as well as for their phytotoxicity. Our in vitro study demonstrates for the first time that masked mycotoxins of AOH and AME can be formed in plant cells.

Catechol metabolites of the mycotoxin zearalenone are poor substrates but potent inhibitors of catechol-O-methyltransferase.

The mycotoxin zearalenone (ZEN) elicits estrogenic effects and is biotransformed to two catechol metabolites, in analogy to the endogenous steroidal estrogen 17ß-estradiol (E2). Previous studies have shown that the catechol metabolites of ZEN have about the same potency to induce oxidative DNA damage as the catechol metabolites of E2, but are less efficiently converted to their methyl ethers by human hepatic catechol-O-methyltransferase (COMT). Here, we report that the two catechol metabolites of ZEN, i.e. 13-hydroxy-ZEN and 15-hydroxy-ZEN, are not only poor substrates of human COMT but are also able to strongly inhibit the O-methylation of 2-hydroxy-E2, the major catechol metabolite of E2. 15-Hydroxy-ZEN acts as a non-competitive inhibitor and is about ten times more potent than 13-hydroxy-ZEN, which is an uncompetitive inhibitor of COMT. The catechol metabolites of ZEN were also shown to inhibit the O-methylation of 2-hydroxy-E2 by hepatic COMT from mouse, rat, steer and piglet, although to a lesser extent than observed with human COMT. The powerful inhibitory effect of catechol metabolites of ZEN on COMT may have implications for the tumorigenic activity of E2, because catechol metabolites of E2 elicit genotoxic effects, and their impaired O-methylation may increase the tumorigenicity of steroidal estrogens.

Catechol metabolites of zeranol and 17ß-estradiol: a comparative in vitro study on the induction of oxidative DNA damage and methylation by catechol-O-methyltransferase.

α-Zearalanol (α-ZAL, zeranol) is a highly estrogenic macrocyclic ß-resorcylic acid lactone, which is used as a growth promotor for cattle in various countries. We have recently reported that α-ZAL and its major metabolite zearalanone (ZAN) are hydroxylated at the aromatic ring by microsomes from human liver in vitro, thereby forming two catechol metabolites each. Thus, the oxidative metabolism of α-ZAL and ZAN resembles that of the endogenous steroidal estrogens 17ß-estradiol (E2) and estrone (E1), which also give rise to two catechols each. As these catechol metabolites are believed to mediate the carcinogenicity of E2 and E1 by causing oxidative DNA damage and DNA adducts, their methylation by catechol-O-methyltransferase (COMT) is an important inactivation pathway. Here we report that hepatic microsomes from five species generate catechol metabolites of α-ZAL and ZAN, the highest amounts being formed by human liver microsomes, followed by rat, mouse, steer and swine. The microsomal extracts and the individual catechols of α-ZAL, ZAN, E2 and E1 were found to induce oxidative DNA damage, as measured by the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in a cell-free system. The ranking of pro-oxidant activity was 15-HO-ZAN>15-HO-α-ZAL≈4-HO-E2/E1≈2-HO-E2/E1>13-HO-ZAN>13-HO-α-ZAL. With respect to the rate of methylation by human hepatic COMT, the ranking was 2-HO-E2/E1>>4-HO-E2/E1>15-HO-α-ZAL/ZAN>>13-HO-α-ZAL/ZAN. Thus, some catechol metabolites of α-ZAL and ZAN are better pro-oxidants and poorer substrates of COMT than the catechols of E2 and E1. These findings warrant further investigations into the genotoxic potential of α-ZAL, which may constitute another biological activity in addition to its well-known estrogenicity.

Hydroxylation of the mycotoxin zearalenone at aliphatic positions: novel mammalian metabolites.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species and frequently found as a contaminant of food and feed. Earlier studies have disclosed that ZEN is biotransformed in microsomes from human and rat liver to multiple hydroxylated metabolites, two of which have recently been identified as products of aromatic hydroxylation. Here, we report for the first time on the structure elucidation of metabolites arising through hydroxylation of the aliphatic ring of ZEN at various positions. By using reference compounds and ZEN labeled with deuterium at specific positions, evidence was provided for the preferential hydroxylation of ZEN at C-8 and, to a lesser extent, at C-9, C-10, and C-5. In contrast, hydroxylation at C-6 could be ruled out, as could oxidation of the olefinic double bond. These results imply that the phase I metabolism of ZEN in the mammalian organism is more extensive than previously thought, and warrant further studies on the in vivo formation of the novel ZEN metabolites and their biological activities.

Genotoxicity and inactivation of catechol metabolites of the mycotoxin zearalenone.

Zearalenone (ZEN) is a highly estrogenic mycotoxin produced by Fusarium species. The adverse effects of ZEN and its reductive metabolite α-zearalenol (α-ZEL) are often compared to those of 17ß-estradiol (E2) and estrone (E1). These endogenous steroidal estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (1) hormonal activity and (2) genotoxic effects after cytochrome P450-catalyzed metabolic activation to catechols. Like E1 and E2, ZEN and α-ZEL exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites. The subsequent methylation of catechols by catechol-O-methyltransferase (COMT) is generally considered as a detoxifying pathway. Imbalances between the activation and inactivation reactions can lead to the formation of reactive semiquinones and quinones, which can alkylate DNA or produce reactive oxygen species by redox cycling. In the present study, the genotoxicity of the catechol metabolites of ZEN, α-ZEL, E1 and E2 was determined in a cell-free system by measuring 8-oxo-2'-deoxyguanosine using a LC-DAD-MS(2) method. Each of the individual catechols of ZEN, α-ZEL, E1 and E2 induced oxidative DNA damage in calf thymus DNA. The ranking order of the DNA damaging activity was 15-hydroxy-ZEN/α-ZEL ≈ 2/4-hydroxy-E1/E2 > 13-hydroxy-ZEN/α-ZEL. When hepatic microsomes from different species were incubated with ZEN, the rat had the highest activity for catechol formation, followed by human, mouse, pig and steer. The amount of catechol metabolites correlated directly with the amount of oxidative damage in calf thymus DNA. The ranking order for the rate of methylation by human hepatic COMT was 2-hydroxy-E1/E2 >> 4-hydroxy-E1/E2 >> 13/15-hydroxy-ZEN/α-ZEL. Thus, the catechol metabolites of the mycoestrogen ZEN and its reductive metabolite α-ZEL exhibit a DNA-damaging potential comparable to that of the catechol metabolites of E1 and E2, but are much poorer substrates for inactivation by human COMT.

Absorption and metabolism of the mycotoxin zearalenone and the growth promotor zeranol in Caco-2 cells in vitro.

SCOPE: Zearalenone (ZEN) and α-zearalanol (α-ZAL, zeranol) were studied in differentiated Caco-2 cells and in the Caco-2 Millicell® system in vitro to simulate their in vivo intestinal absorption and metabolism in humans. METHODS AND RESULTS: In addition to metabolic reduction/oxidation, extensive conjugation with glucuronic acid and sulfate of the parent compounds and their phase I metabolites was observed. The positional isomers of the glucuronides and sulfates were unambiguously identified: Sulfonation occurred specifically at the 14-hydroxyl group, whereas glucuronidation was less specific and, in addition to the preferred 14-hydroxyl group, involved the 16- and 7-hydroxyl groups. Using the Caco-2 Millicell® system, an efficient transfer of the glucuronides and sulfates of ZEN and α-ZAL and their phase I metabolites into both the basolateral and the apical compartment was observed after apical administration. The apparent permeability coefficients (P(app) values) of ZEN, α-ZAL and the ZEN metabolite α-zearalenol were determined, using an initial apical concentration of 20 µM and a permeation time of 1 h. CONCLUSION: According to the P(app) values, the three compounds are expected to be extensively and rapidly absorbed from the intestinal lumen in vivo and reach the portal blood both as aglycones and as glucuronide and sulfate conjugates in humans.

Identification of an aliphatic epoxide and the corresponding dihydrodiol as novel congeners of zearalenone in cultures of Fusarium graminearum.

The mycotoxin zearalenone (ZEN) is produced by various Fusarium fungi and frequently found as a contaminant in food and feed. There are reports in the literature that several closely related analogues of ZEN are also formed in cultures of Fusarium species. We have therefore analyzed the organic extract from a 40 day culture of Fusarium graminearum by LC-DAD-MS and detected 15 compounds, which could be congeners of ZEN because of their ultraviolet, mass spectroscopy, and tandem mass spectroscopy spectra. In addition to confirming the previously reported α- and ß-stereoisomers of 5-hydroxy-ZEN and 10-hydroxy-ZEN, we identified seven ZEN congeners for the first time. One of the major novel congeners was shown by nuclear magnetic resonance spectroscopy and chemical synthesis to have the structure of an aliphatic ZEN epoxide, whereas two minor products proved to be the corresponding dihydrodiols. In addition, three stereoisomers of a cyclization product of the dihydrodiols, carrying a spiro-acetal group, were identified as fungal products for the first time. The latter may be artifacts, because the ZEN epoxide and dihydrodiol are unstable under acidic conditions and rearrange easily to the spiro-acetal compounds.

Glucuronidation of zearalenone, zeranol and four metabolites in vitro: formation of glucuronides by various microsomes and human UDP-glucuronosyltransferase isoforms.

Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α-zearalanol (α-ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α-ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP-glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14-hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α-ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.

Aromatic hydroxylation and catechol formation: a novel metabolic pathway of the growth promotor zeranol.

Alpha-zearalanol (alpha-ZAL, zeranol) is a macrocyclic resorcylic acid lactone, which is highly estrogenic and used as a growth promotor for cattle in various countries. Little is known about the phase I metabolism of alpha-ZAL. We now report that alpha-ZAL and its major metabolite zearalanone (ZAN) are extensively monohydroxylated at the aromatic ring by microsomes from human liver in vitro. This novel pathway leads to catechols, the chemical structures of which were unambiguously established by the use of deuterium-labeled alpha-ZAL and ZAN, and by the synthesis of authentic standards. The aromatic hydroxylation of alpha-ZAL is almost exclusively mediated by the human cytochrome P450 (hCYP) 1A2 isoform. The catechol metabolites of alpha-ZAL and ZAN are unstable and readily oxidized to quinones, which could be detected among the metabolites of alpha-ZAL and ZAN generated by human hepatic microsomes and hCYP1A2. Furthermore, the quinone metabolites are able to form covalent adducts with N-acetylcysteine (NAC), as several of such adducts were found in microsomal incubations fortified with NAC. Aromatic hydroxylation of alpha-ZAL was also observed with bovine, porcine and rat hepatic microsomes. Further studies are needed to demonstrate the catechol pathway of alpha-ZAL in vivo and to assess its toxicological significance.

Aromatic hydroxylation is a major metabolic pathway of the mycotoxin zearalenone in vitro.

Zearalenone (ZEN) is a common mycotoxin, for which only reductive metabolites have been identified so far. We now report that ZEN is extensively monohydroxylated by microsomes from human liver in vitro. Two of the major oxidative metabolites arise through aromatic hydroxylation and are catechols. Their chemical structures have been unambiguously determined by using deuterium-labeled ZEN and by comparison with authentic reference compounds. Moreover, both catechol metabolites of ZEN were substrates of the enzyme catechol-O-methyl transferase. One of the monomethyl ethers represented the major metabolite when ZEN was incubated with rat liver slices, thus demonstrating that catechol formation also takes place under in vivo-like conditions. Out of ten major human cytochrome P450 (hCYP) isoforms only hCYP1A2 was able to hydroxylate ZEN to its catechols with high activity. Catechol formation represents a novel pathway in the metabolism of ZEN and may be of toxicological relevance.