Quantitation of carcinogenic heterocyclic aromatic amines and detection of novel heterocyclic aromatic amines in cooked meats and grill scrapings by HPLC/ESI-MS

A tandem solid-phase extraction method was used to isolate carcinogenic heterocyclic aromatic amines (HAAs) from cooked meats. The following 10 HAAs were identified by HPLC/ESI-MS/MS: 2-amino-9H-pyrido[2,3-b]indole (2-AalphaC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), and 2-amino-1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]); the latter HAA has not previously been reported in cooked meats. The concentrations of these HAAs ranged from <0.03 to 15 ppb in cooked meats and poultry, to 75 ppb in cooked beef extract, and to 85 ppb in grill scrapings. The product ion scan mode was used to confirm the identities of these HAAs. Six other compounds were detected that appear to contain the N-methylimidazoquinoxaline skeleton on the basis of their product ion spectra, and these compounds are probable isomers of IQx, 8-MeIQx, and DiMeIQx. A number of known HAAs and novel HAAs of unknown genotoxic potential are formed at appreciable levels in cooked meats.     

Identifying new lignin bioengineering targets: impact of epicatechin, quercetin glycoside, and gallate derivatives on the lignification and fermentation of maize cell walls

Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, primary maize cell walls were artificially lignified with normal monolignols plus various epicatechin, quercetin glycoside, and gallate derivatives added as 0 or 45% by weight of the precursor mixture. The flavonoids and gallates had variable effects on peroxidase activity, but all dropped lignification pH. Epigallocatechin gallate, epicatechin gallate, epicatechin vanillate, epigallocatechin, galloylhyperin, and pentagalloylglucose formed wall-bound lignin at moderate to high concentrations, and their incorporation increased 48 h in vitro ruminal fiber fermentability by 20-33% relative to lignified controls. By contrast, ethyl gallate and corilagin severely depressed lignification and increased 48 h fermentability by about 50%. The results suggest several flavonoid and gallate derivatives are promising lignin bioengineering targets for improving the inherent fermentability of nonpretreated cell walls.     

Inhibitory effect of antioxidant-rich marinades on the formation of heterocyclic aromatic amines in pan-fried beef

The inhibitory effect of antioxidant-rich marinades containing beer and white wine (with/without alcohol) alone or mixed with herbs commonly used as meat flavoring (garlic, ginger, thyme, rosemary, and red chili pepper) on the formation of heterocyclic aromatic amines (HAs) in pan-fried beef was studied. Radical-scavenging activity was evaluated by DPPH assay, before the addition of meat to the marinade (T0) and after 4 h of meat marinating (T4). At T0, wine with herbs possessed the highest scavenging activity (73.5%), followed by wine (72.5%), dealcoholized wine with herbs (53.4%), beer and herbs (41.7%), dealcoholized wine (39.6%), and beer (25.9%). At T4, a decrease in the radical-scavenging activity of all marinades was observed, although with a similar radical-scavenging profile. All of the six marinades under the study reduced the total amount of HAs, keeping meat with good overall sensory quality. Beer marinades were more efficient than white wine marinades, and the addition of herbs provided a superior inhibitory effect, reducing around 90% of HAs. No correlation was observed between radical-scavenging activity of marinades and total or individual HAs formation. Herbs explained around 30% of inhibition of PhIP formation, whereas alcohol increased PhIP formation.     

Model studies of ferulate-coniferyl alcohol cross-product formation in primary maize walls: implications for lignification in grasses

Ferulate and diferulates mediate cell wall cross-linking in grasses, but little is known about their cross-coupling reactions with monolignols and their role in lignin formation in primary cell walls. Feruloylated primary walls of maize were artificially lignified and then saponified to release ferulate and diferulates and their cross-products with coniferyl alcohol for analysis by GC-FID, GC-MS, and NMR spectroscopy. Ferulate and 5-5-coupled diferulate had a greater propensity than 8-coupled diferulates to copolymerize with coniferyl alcohol, forming mostly 4-O-beta' and 8-beta' and some 8-O-4' and 8-5' cross-coupled structures. Some 8-beta' structures de-esterified from xylans, but these cross-links were subsequently replaced as 8-coupled diferulates formed stable cross-coupled structures with lignin. Based on the incorporation kinetics of ferulate and diferulates and the predicted growth of lignin, cross-products formed at the onset of lignification acted as nucleation sites for lignin polymerization.     

Tissue-specific and developmental modifications of grape cell walls influence the adsorption of proanthocyanidins

Cell wall material from Vitis vinifera L. cv. Cabernet Sauvignon grape skin and flesh was isolated at different stages of grape maturity to determine whether developmental changes in cell wall composition in different tissue types influence the binding of proanthocyanidins (PAs). Trends in cell wall adsorption of, and selectivity for, PAs were determined using two skin PAs that differed in their average molecular masses. Flesh cell walls consistently bound a higher amount of PA than those from skin. Key structural differences that reduced PA adsorption in skin cell walls by comparison with flesh cell walls were endogenously higher concentrations of insoluble PA, Klason lignin, and lower cell wall-bound protein. These differences may confer reduced flexibility and porosity of skin cell walls relative to flesh cell walls. Analysis of skin and flesh cell wall properties revealed that the onset of ripening was associated with a loss of type I arabinogalactan and galacturonic acid, which indicated a degradation of pectin within the cell wall. Flesh cell walls consistently bound PAs of larger molecular mass, and changes in PA adsorption properties after the onset of ripening were minor. For skin cell walls, adsorption of PA was lowest immediately following solubilization of galacturonic acid, and high molecular mass PAs were poorly bound. As ripening progressed, PAs of higher molecular mass were selectively adsorbed by skin cell walls, which indicates that ongoing cell wall remodeling during ripening may confer an increased porosity within the skin cell wall matrix, resulting in a greater adsorption of PA within a permeable structure.     

Inhibitory effect of fruit extracts on the formation of heterocyclic amines

Natural extracts have attracted considerable attention for development into effective inhibitors against the formation of genotoxic heterocyclic amines (HAs) in processed foods. In this study, four fruit extracts (apple, elderberry, grape seed, and pineapple) were evaluated for their effects on HA formation in fried beef patties. Apple and grape seed extracts were found to be the most effective in both the degree of inhibition in the formation of individual HAs (2-amino-3,8-dimethylimidazo [4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), and 2-amino-1-methyl-6-henylimidazo [4,5-b]pyridine (PhIP)) and in the reduction of total HA content (approximately 70% relative to the control). Activity-guided analysis of apple extract using model systems (PhIP- and MeIQx-producing models) showed that the proanthocyanidins, phloridzin and chlorogenic acid were responsible for reducing the amount of HAs formed. Proanthocyanidins were identified as the dominant inhibitors because they were strongly active against HA formation in both the PhIP and MeIQx model systems. For phloridzin, the inhibitory effect was observed only on the formation of PhIP. In contrast, chlorogenic acid, although effective against the formation of MeIQx, significantly enhanced the formation of PhIP. This is the first report showing the inhibitory activities of apple phenolics on the formation of heterocyclic amines. The findings provide valuable information for the development of effective strategies to minimize HA content of cooked meats and to identify several new natural products that may have new applications in the food industry.     

Effect of oil marinades with garlic, onion, and lemon juice on the formation of heterocyclic aromatic amines in fried beef patties

Effects of the application of oil marinades with garlic, onion, and lemon juice on the formation of heterocyclic aromatic amines (HAAs) in fried beef patties were investigated. Two different statistical design models were used. In the screening experiment, a significant inhibition of formation of MeIQx was determined only by addition of garlic (p < 0.05). When the amount of garlic was changed from 2 to 20 g/100 g of marinade, the estimated MeIQx content in patties was reduced about 70%. MeIQx (0.38-1.22 ng/g), 4,8-DiMeIQx (n.d.-0.45 ng/g), PhIP (n.d.-0.09 ng/g), norharman (0.76-13.5 ng/g), and harman (2.9-21.5 ng/g) were found in fried patties. The results of two-level and three-level fractional factorial design experiments confirmed the first investigation. They showed a stronger reduction in MeIQx in patties with the addition of increasing amounts of garlic (p < 0.01) and onion (p < 0.05) in marinades. A higher content of lemon juice in marinades led to only a marginal reduction in MeIQx (p > 0.05). The optimum amounts of onion, garlic, and lemon juice that achieved a maximum reduction of HAAs were calculated as 31.2%, 28.6%, and 14.6% in marinade.     

大豆、玉米幼苗根细胞壁的制备与表面性质

根细胞壁对于植物养分吸收积累以及植物的环境抗性或耐性方面的作用和影响与细胞壁表面性质密切相关。本研究采用改进的匀浆洗涤法提取大豆（Glycine max L.,Type I细胞壁类型植物）、玉米（Zea mays L.,Type II细胞壁类型植物）幼苗的根细胞壁物质,获得了较好的细胞壁材料纯度。测定了根细胞壁材料以及整根的阳离子交换量（CEC）,并通过拟合解析根细胞壁的电位滴定曲线求解根细胞壁表面基团的解离常数pKa以及数量。结果表明,大豆的根细胞壁CEC显著（p0.05）高于玉米的根细胞壁CEC,分别为425±5、162±7 μmol/g,DW;大豆的根CEC也显著（p0.05）高于玉米根,与两种植物根细胞壁CEC的差异一致。从两种植物的根细胞壁上均识别出一种表观pKa值约为5.78的羧基基团。大豆根细胞壁的羧基含量也显著（p0.05）高于玉米根细胞壁,分别为604±15、252±4 μmol/g,DW,大豆根或根细胞壁的CEC较高与Type I细胞壁富含羧基有关。本文还进一步讨论了不同细胞壁类型植物的根细胞壁表面性质对于植物的营养过程以及环境胁迫抗性或耐性方面的影响与意义。     

Methyl esterification divergently affects the degradability of pectic uronosyls in nonlignified and lignified maize cell walls

Nonlignified cell walls from Zea mays (L.) cell suspensions were incubated with and without pectin methylesterase (PME) and a portion were artificially lignified to assess how methyl esters influence the release of pectic uronosyls and total sugars from cell walls by fungal enzymes. Treatment with PME reduced uronosyl concentrations from 97 to 92 mg/g, reduced uronosyl methylation from 57% to 21%, and increased Klason lignin concentrations in artificially lignified cell walls from 99 to 116 mg/g. Although PME treatment slightly enhanced uronosyl release from nonlignified cell walls, it reduced uronosyl release from artificially lignified cell walls by 55% after 4 h and by 7% after 72 h of enzymatic hydrolysis. Pectin hydrolysis in PME treated cell walls was probably impaired by enhanced benzyl ester cross-linking of uronosyls to lignin via quinone methide intermediates. Variations in uronosyl methylation had little effect on the overall release of total sugars from cell walls.     

Influence of different oligosaccharides and inulin on heterocyclic aromatic amine formation and overall mutagenicity in fried ground beef patties

The effects of different oligosaccharides [fructooligosaccharide (FOS), galactooligosaccharide (GOS), and isomaltooligosaccharide (MOS)] and inulin on heterocyclic aromatic amine (HAA) formation and overall mutagenicity in fried ground patties were evaluated. Different oligosaccharides and inulin was added directly to ground beef. Patties (100 g) were fried at 225 degrees C (surface temperature) for 10 min per side. FOS added at levels of 0.5, 1.0, 1.5, 2.0, and 2.5 g to 100 g of ground beef inhibited total HAA formation by 19, 32, 45, 51, and 58%, respectively. The addition of 1.5 g of FOS, GOS, MOS, and inulin to ground beef patties inhibited total HAA formation by 50, 47, 46, and 54%, respectively. They also reduced overall mutagenicity by 52, 51, 48, and 59%, respectively. These studies confirm that oligosaccharides and inulin have the potential to reduce HAA formation in cooked beef patties.     

Quantification of heterocyclic aromatic amines in fried meat by HPTLC/UV-FLD and HPLC/UV-FLD: a comparison of two methods

A recently developed HPTLC/UV-FLD method was compared to the routinely used HPLC/UV-FLD method for the quantification of heterocyclic aromatic amines (HAA) formed at trace levels during the heating process of meat. For formation of these process contaminants under normal cooking conditions, beef patties were fried in a double-contact grill at 230 degrees C for five different frying times and extracted by solid-phase extraction. The HAAs most frequently found, that is, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline (4,8-DiMeIQx), 9 H-pyrido[3,4- b]indole (norharman), and 1-methyl-9 H-pyrido[3,4- b]indole (harman), were quantified by two chromatographic methods, which were orthogonal to each other (normal versus reversed phase system). Both methods showed a similar performance and good correlation of the results ( R (2) between 0.8875 and 0.9751). The comparison of running costs and run time in routine analysis proved HPTLC/UV-FLD to be more economical (factor of 3) and faster (factor of 4) due to its capability of parallel chromatography. The HAA findings calculated by standard addition increased with the heating time from <1 to 33 microg/kg related to 3-6 min of frying time. The precision (RSD) was between 7 and 49% (HPTLC) and between 5 and 38% (HPLC) at these very low HAA levels formed.     

Effect of high water-holding capacity on the formation of heterocyclic amines in fried beefburgers

Mutagenic/carcinogenic heterocyclic amines (HAs) are formed in the crust during the cooking of meat. The influence of cooking loss, time, and temperature on the formation of HAs was investigated in fried beefburgers. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) were identified in all samples. The amounts of PhIP, MeIQx, and 4,8-DiMeIQx increased with increasing cooking time, and this effect was significant for all three HAs. The pan temperature had a significant effect on the formation of PhIP and 4,8-DiMeIQx. The addition of NaCl/sodium tripolyphosphate to the beefburgers reduced the cooking loss and decreased the formation of PhIP, MeIQx, and 4,8-DiMeIQx. This decrease was significant for MeIQx and 4,8-DiMeIQx. The results clearly show that it is possible to modify cooking practices to minimize the formation of HAs.     

Quantitation of 13 heterocyclic aromatic amines in cooked beef, pork, and chicken by liquid chromatography-electrospray ionization/tandem mass spectrometry

The concentrations of heterocyclic aromatic amines (HAAs) were determined, by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI-MS/MS), in 26 samples of beef, pork, and chicken cooked to various levels of doneness. The HAAs identified were 2-amino-3-methylimidazo[4,5- f]quinoline, 2-amino-1-methylimidazo[4,5- b]quinoline, 2-amino-1-methylimidazo[4,5- g]quinoxaline (I gQx), 2-amino-3-methylimidazo[4,5- f]quinoxaline, 2-amino-1,7-dimethylimidazo[4,5- g]quinoxaline (7-MeI gQx), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline, 2-amino-1,6-dimethyl-furo[3,2- e]imidazo[4,5- b]pyridine, 2-amino-1,6,7-trimethylimidazo[4,5- g]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline, 2-amino-1,7,9-trimethylimidazo[4,5- g]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-9 H-pyrido[2,3- b]indole, and 2-amino-3-methyl-9 H-pyrido[2,3- b]indole. The concentrations of these compounds ranged from <0.03 to 305 parts per billion (micrograms per kilogram). PhIP was the most abundant HAA formed in very well done barbecued chicken (up to 305 microg/kg), broiled bacon (16 microg/kg), and pan-fried bacon (4.9 microg/kg). 7-MeI gQx was the most abundant HAA formed in very well done pan-fried beef and steak, and in beef gravy, at concentrations up to 30 microg/kg. Several other linear tricyclic ring HAAs containing the I gQx skeleton are formed at concentrations in cooked meats that are relatively high in comparison to the concentrations of their angular tricyclic ring isomers, the latter of which are known experimental animal carcinogens and potential human carcinogens. The toxicological properties of these recently discovered I gQx derivatives warrant further investigation and assessment.     

Effect of red wine marinades on the formation of heterocyclic amines in fried chicken breast

Genotoxic heterocyclic amines (HAs) are formed via the Maillard reaction and free radical reaction mechanisms when meat or fish is cooked at usual cooking conditions. In this paper, the effect of the addition of red wine was tested to study if it interferes in HA formation. Fried chicken breast was the food item chosen, and three different red wines, characterized in terms of grape varieties, free amino acids, antioxidant properties, and metallic composition, were used to marinate meat prior to the heating process. Unmarinated samples and samples marinated with an ethanol-water mixture provided reference HA levels. The frying experiments were performed under well-controlled temperature and time conditions. The samples were analyzed for HA content using solid-phase extraction and LC-MS/MS. DMIP, PhIP, MeIQx, 4,8-DiMeIQx, IFP, TMIP, harman, and norharman were identified in fried chicken breast. Red wine marinades were found to reduce the formation of some of the HAs formed. PhIP, with a reduction of up to 88%, was the most minimized amine, although the formation of harman was enhanced.     

Cross-linking of maize walls by ferulate dimerization and incorporation into lignin

Cross-linking of xylans and lignin by ferulates was investigated with primary maize walls acylated with 2% ferulate and with ferulate ethyl esters. Peroxidase-mediated coupling of wall ferulate and ethyl ferulate yielded mostly 8-coupled products, including three new dehydrodimers. Significant quantities of 5-5-coupled diferulate formed only within walls, suggesting that matrix effects influence dimer formation. Over 60% of wall ferulate dimerized upon H2O2 addition, suggesting that xylan feruloylation is highly regulated during wall biosynthesis to permit extensive dimer formation at the onset of lignification. During lignification, ferulate and 5-5-coupled diferulate copolymerized more rapidly and formed fewer ether-linked structures with coniferyl alcohol than 8-5-, 8-O-4-, and 8-8-coupled diferulates. The potential incorporation of most ferulates and diferulates into lignin exceeded 90%. As a result, xylans become extensively cross-linked by ferulate dimerization and incorporation to lignin, but only a small and variable proportion of these cross-links is measurable by solvolysis of lignified walls.     

Addition of various carbohydrates to beef burgers affects the formation of heterocyclic amines during frying

The influence of the addition of carbohydrates with different physicochemical properties on weight loss and formation of heterocyclic amines (HAs) during the frying of beef burgers was examined. Furthermore, the capability of carbohydrates to bind HAs was tested. Beef burgers containing 1.5% NaCl and 0.3% tripolyphosphate (reference), with the addition of 1.5% carbohydrate, were fried for 5 min at 200 degrees C in a double-sided pan fryer. The beef burgers were analyzed for HAs with solid phase extraction and liquid chromatography/mass spectrometry. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), and 9H-pyrido[3,4-b]indole (Norharman) were detected in all of the beef burgers. The addition of carbohydrates affected both the weight loss and the formation of HAs during cooking. The formation of HAs could be correlated to depend on both the weight loss and the type of the added carbohydrate. Of the 11 different carbohydrates tested, raw potato starch was most capable of inhibiting the formation of HAs, while potato fiber gave the lowest weight loss and a comparably low amount of PhIP. Wheat bran and potato fiber were found to reversibly bind HAs. It is concluded that adding small amounts of certain carbohydrates may be a simple and effective way of reducing the amount of HAs and can easily be applied in households and commercial preparations of beef burgers.     

Antimutagenic activity of cacao: inhibitory effect of cacao liquor polyphenols on the mutagenic action of heterocyclic amines

We investigated the effect of polyphenols derived from cacao liquor on the mutagenic action of heterocyclic amines (HCAs) in vitro and ex vivo. In the Ames test, the cacao liquor polyphenols showed antimutagenic effects in bacteria treated with HCA in the presence of an S-9 mixture; however, they showed less efficacy than quercetin. On the other hand, the cacao liquor polyphenols showed potent antimutagenic activity in bacteria treated with activated forms of HCA, compared with quercetin. We also evaluated the effect of these compounds on enzymatic activation of HCA. They weakly suppressed the production of activated HCA. In the host-mediated assay in mice, a method used to estimate the potential carcinogenicity of chemicals ex vivo, oral administration of the cacao liquor polyphenols, reduced the number of colonies of revertant bacteria recovered from the liver. These data suggest that the cacao liquor polyphenols have an antimutagenic effect not only in vitro, but also ex vivo.