Effects of cooking methods and levels on formation of heterocyclic aromatic amines in chicken and fish with Oasis extraction method

Heterocyclic aromatic amines (HCAs) are potent mutagenic and carcinogenic compounds formed during heat processing of proteinaceous food such as beef, poultry, and fish. The objective of this study was to measure nine HCAs in chicken chops and fish fillets cooked by various methods (microwave, oven, hot plate, pan-frying, and barbecuing) to different degrees of doneness (rare, medium, well done, and very well done). Total HCA amount in chicken changed between 0.24 and 8.21 ng/g, and not only the highest total amount but also the lowest total HCA amount was found in microwave cooked chicken samples. The highest total HCA amount found in fish for microwave, oven, hot plate, pan-frying, and barbecuing were 18.09, 4.28, 3.12, 6.98, and 5.22 ng/g, respectively. The results show that microwave cooking alone is found to possess the highest total HCA amount, followed by pan-frying, and barbecuing of meat samples, and the total HCA amount in cooked samples is low if cooked to rare and medium degrees of doneness. 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), 2-amino-9H-pyrido[2,3-b]indole (AαC), and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) were not detected in any samples.     

Inhibitory effects of pomegranate seed extract on the formation of heterocyclic aromatic amines in beef and chicken meatballs after cooking by four different methods

Beef and chicken meatballs with a 0.5% (w/w) pomegranate seed extract were cooked using four different cooking methods (oven roasting, pan cooking, charcoal-barbecue, and deep-fat frying) and six heterocyclic aromatic amines; IQ, MeIQx, 4,8-DiMeIQx, PhIP, norharman, and harman were observed. In the beef meatballs, the highest inhibitory effects of pomegranate seed extract on heterocyclic aromatic amines formation were 68% for PhIP, 24% for norharman, 18% for harman, 45% for IQ, and 57% for MeIQx. Total heterocyclic aromatic amine formation was reduced by 39% and 46% in beef meatballs cooked by charcoal-barbecue and deep-fat frying, respectively. In the chicken meatballs, the highest inhibitory effects were 75% for PhIP, 57% for norharman, 28% for harman, 46% for IQ, and 49% for MeIQx. When the pomegranate seed extract was added to the chicken meatballs cooked by deep-fat frying, the total heterocyclic aromatic amine formation was inhibited by 49%, in contrast the total heterocyclic aromatic amine contents after oven roasting increased by 70%.     

Effect of cooking methods on the formation of heterocyclic aromatic amines in chicken and duck breast

Heterocyclic aromatic amines (HAAs), potent mutagens/carcinogens, are pyrolysis formed during the cooking of meat and fish. In the present study, the effects of various cooking methods, pan-frying, deep-frying, charcoal grilling and roasting on the formation of HAAs in chicken breast and duck breast were studied. The various HAAs formed during cooking were isolated by solid-phase extraction and analyzed by high-performance liquid chromatography (HPLC). Results showed that chicken breast cooked by charcoal grilling contained the highest content of total HAAs, as high as 112 ng/g, followed by pan-fried duck breast (53.3 ng/g), charcoal grilled duck breast (32 ng/g), pan-fried chicken breast (27.4 ng/g), deep-fried chicken breast (21.3 ng/g), deep-fried duck breast (14 ng/g), roasted duck breast (7 ng/g) and roasted chicken breast (4 ng/g). For individual HAA, the most abundant HAA was 9H-pyrido-[4,3-b]indole (Norharman), which was detected in charcoal grilled chicken breast at content as high as 32.2 ng/g, followed by 1-methyl-9H-pyrido[4,3-b] indole (Harman) and 2-amino-1-methyl-6-phenylimidazo[4,5-f]pyridine(PhIP) at 32 and 31.1 ng/g in charcoal grilled chicken breast, respectively. The content of PhIP in pan-fried duck and chicken breast were 22 and 18.3 ng/g, respectively. Generally, the type and content of HAAs in cooked poultry meat varies with cooking method and cooking conditions.     

Effect of green tea marinades on the formation of heterocyclic aromatic amines and sensory quality of pan-fried beef

The effect of a green tea marinade on the formation of heterocyclic aromatic amines (HAs) was examined in pan-fried beef cooked at 180–200 °C for 4 min each side. Different marinating times were assayed and unmarinated samples cooked in similar conditions provided reference HAs levels. A green tea marinade catechin rich was prepared taking 1 g of green tea and infusing with 125 ml of boiling tap water during 10 min. Four HAs were quantified in unmarinated and marinated meat samples during 1, 2, 4 and 6 h at 5 °C. Compared with the unmarinated samples, marinating in green tea resulted in a significant decrease (p < 0.05) of levels of PhIP and AαC. Person correlation indicated that the reduction of the levels of these HAs in meat marinated with the increase of marinating time was significant (respectively, r = −0.799, p < 0.0001; r = −0.631, p < 0.05). No reduction was observed for 4,8-DiMeIQx and MeIQx.     

Effects of breeds on the formation of heterocyclic aromatic amines in smoked lamb

Heterocyclic aromatic amines (HAAs) are mutagenic and carcinogenic compounds, which are commonly detectable in cooked meat products. The objective of this study was to evaluate the effect of sheep breeds on the formation of HAAs in smoking cooked lamb. The results showed that HAAs in smoked lamb meat products were generally low (2.74–5.42 ng g^?1), with most being Harman and Norharman. IQ, MeIQx, 4,8‐DiMeIQx, Trp‐p‐2, PhIP and MeAaC were not detected in smoked lamb meat products in the present study. The total content of HAAs differed between meat products of different sheep breeds, but no difference in the order of magnitude was determined. Smoking altered the content of protein, fat, moisture and free amino acids in lamb meat products, which was probably mostly contributed by the reduction in meat moisture. Free tryptophan decreased in all breeds after smoking, which was probably used to synthesise HAAs. In summary, HAAs were low in smoked lamb meat products of all sheep breeds; thus, consumption of smoked lamb meat products should contribute very limited intake of HAAs.     

A new quick solid-phase extraction method for the quantification of heterocyclic aromatic amines

A new solid-phase extraction (SPE) method using only one SPE cartridge is described as a clean-up procedure for the determination of heterocyclic aromatic amines (HAAs). In particular, the polar HAAs imidazoquinolines and imidazoquinoxalines, which are well-known toxic compounds in thermally treated food, can be determined by this quick and simple method. For validation of the method meat extracts were analyzed. For determination of the percentage recovery and standard deviation the beef extract was spiked (40 ng/g of each HAA) and analyzed 10 times. The polar HAAs were determined in this meat extract with a recoveries of 62 % to 95% and standard deviations of 3% to 5%. The recovery rates of the less polar HAAs Harman and Norharman were much lower (25±5% to 32±5%). The limit of detection for polar and less polar HAAs was between 3 ng/g and 9 ng/g in the meat extract matrix. The method comprises extraction with methanolic NaOH, centrifugation and SPE using a commercially available polystyrene copolymer cartridge. After different washing steps the eluate was analyzed by high-performance liquid chromatography with diode-array detection. The advantages of this new method are the reduced amounts of time and organic solvents required. Received: 29 November 1999 / Revised version: 21 February 2000     

Exposure to heterocyclic aromatic amines from the consumption of cooked red meat and its effect on human cancer risk: A review

This review covers the bibliographic data from the last 10 years on the possible carcinogenicity of heterocyclic aromatic amines (HAAs) in humans. Aspects such as red meat intake, cooking methods applied to red meat, and doneness of cooking are discussed from an epidemiological point of view. The role in the carcinogenicity of the HAAs has been assigned to two main factors: first, the very high frequency of consumption of red meat; and, second, very darkly browned meats from cooking. However, there are some uncertainties associated with epidemiological results such as the presence of other carcinogens, co-carcinogens and anti-carcinogens in the diet, analytical results on the content of HAAs in foods, food frequency questionnaires, and mainly genetic susceptibility to HAAs. It is concluded that there is not sufficient scientific evidence to support the hypothesis that human cancer risk is due specifically to the intake of HAAs in the diet.     

Mitigation strategies to reduce the impact of heterocyclic aromatic amines in proteinaceous foods

BackgroundMeat and fish are currently the main sources of proteins necessary for a healthy diet. Cooking proteinaceous food helps reduce biological risks and produce odor-active compounds, but it also generates heat-induced toxicants, of which heterocyclic aromatic amines (HAAs) are probably the most problematic as they are strongly mutagenic and carcinogenic.Scope and approachThis review highlights the most promising strategies for mitigating the impacts of HAAs on human health. These strategies revolve around reducing HAA formation by impacting HAA precursors, controlling the process, adjusting formulations or adapting diets to limit HAA assimilation and metabolism.Key findings and conclusionsIdentifying the different mechanisms of HAA formation and metabolism has made it possible to propose mitigation strategies to limit the risks related to HAA consumption. Various kinds of levers exist. While cooking methods for industrial processed foods can be regulated, it is far more difficult to influence household practices. Mitigation strategies involving other food ingredients are probably more promising from a consumer point of view if pushed by health education campaigns. Efforts to reduce the health risk from HAA consumption should now turn to ingredients like carvacrol that present different concomitant modes of action.     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 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-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 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-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998     

Production of heterocyclic aromatic amines in meat: Chemistry,health risks and inhibition. A review

Heterocyclic aromatic amines are compounds produced in meat subjected to high temperature cooking. Formation of these substances is highly influenced by cooking method, cooking time, cooking temperature, and type of meat. Heterocyclic amines contain heterocyclic rings and nitrogen containing groups within their structure. Risks of colon, pancreas, gastrointestinal tract, lung, liver, prostate, skin and breast cancers are decidedly associated with the consumption of heterocyclic amines. These mutagenic compounds can be attenuated by addition of different fruits and vegetable extracts. Several spices and antioxidants can also be used for the reduction of these heterocyclic amines. The level of heterocyclic amines can be reduced by cooking at low temperature and by decreasing the cooking time. Formation of heterocyclic amines can also be prevented by marinating the meat before frying or grilling and by microwave pre-treatments. Objectives of this review are to create awareness about health risks, and to stimulate further research on other suitable ways to reduce the cancer risks associated with the consumption of cooked meat products.     

Inhibitory effect of marinades with hibiscus extract on formation of heterocyclic aromatic amines and sensory quality of fried beef patties

Heterocyclic aromatic amines (HAA) are carcinogenic compounds found in the crust of fried meat. The objective was to examine the possibility of inhibiting HAA formation in fried beef patties by using marinades with different concentrations of hibiscus extract (Hibiscus sabdariffa) (0.2, 0.4, 0.6, 0.8 g/100 g). After frying, patties were analyzed for 15 different HAA by HPLC-analysis. Four HAA MeIQx (0.3–0.6 ng/g), PhIP (0.02–0.06 ng/g), co-mutagenic norharmane (0.4–0.7 ng/g), and harmane (0.8–1.1 ng/g) were found at low levels. The concentration of MeIQx was reduced by about 50% and 40% by applying marinades containing the highest amount of extract compared to sunflower oil and control marinade, respectively. The antioxidant capacity (TEAC-Assay/Folin–Ciocalteu-Assay) was determined as 0.9, 1.7, 2.6 and 3.5 μmol Trolox antioxidant equivalents and total phenolic compounds were 49, 97, 146 and 195 μg/g marinade. In sensory ranking tests, marinated and fried patties were not significantly different (p > 0.05) to control samples.     

Effects of different types of soy sauce on the formation of heterocyclic amines in roasted chicken

The objective of the study was to determine the effect of different types of soy sauce and marinating time on the formation of heterocyclic amines (HCAs) in roasted chicken. Chicken breast samples were marinated with sweet, salty, light and dark soy sauce at 0, 3, 6 and 12 h (control treatment was the chicken without marinade). The concentrations of free amino acids, sugars and creatinine were determined before roasting while HCA concentrations were determined after roasting. All types of soy sauce significantly increased (p ≤ 0.05) the concentration of HCAs in roasted chicken with increasing marinating time. The highest increment of total concentration of HCAs was found in samples marinated with light soy sauce (887%) followed by dark (375%), salty (193%) and sweet (169%) at 12 h. PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) showed a substantial reduction in samples only momentarily marinated with sweet, salty and dark soy sauce (0 h). Free amino acids were found to be more strongly correlated with the formation of HCAs than reducing sugars or creatinine.     

加工肉制品中杂环胺的形成与控制

煎炸熏烤肉制品容易导致杂环胺的产生。杂环胺大多具有致癌致突变性,特别是2-氨基-3-甲基咪唑并[4,5-f]喹啉(IQ),已被国际癌症研究中心列为"对人类高可疑致癌物(2A级)",对人体的健康存在极大的危害。主要对杂环胺形成的影响因素进行了分析,并提出了具体的抑制措施,以增加公众对杂环胺的认识,为健康饮食提供科学依据。     

Effects of four cooking methods on the heavy metal concentrations of sea bass fillets (Dicentrarchus labrax Linne, 1785)

Heavy metals (Pb, Cd, Cr, Co, As, Ni) were analysed in cooked sea bass (Dicentrachus labrax). Different cooking treatments were used (baking, grilling, microwaving and frying). The results obtained were statistically compared with those of raw fish. The Co concentrations were below limits of detection in all samples (<0.05). Furthermore, Cr in fried samples and Ni in raw fish were not detected. Cd was only detected in microwaved samples. The Pb concentrations of microwaved and baked fish were significantly decreased. The As concentrations of fried and microwaved samples were significantly increased. The Cd and As concentrations of the fillets subjected to microwave cooking were 0.741 and 1.41 mg/kg, respectively. However, the As concentration was also significantly increased in fried samples. This value was 2.66 mg/kg. Microwaving and frying are not suitable for sea bass.     

Effects of skin and grilling method on formation of heterocyclic amines in chicken pectoralis superficialis muscle

Mutagenic heterocyclic amines (HAs) originate in processed proteinaceous foods. The effects of the presence of skin (with vs. without) and of grilling method (two plate vs. infrared) on the content of HAs in grilled chicken pectoralis superficialis muscle (temperature, 220 °C) were investigated. HA precursors (creatine, creatinine, free amino acids and carbohydrates) and HAs of these raw and grilled breast muscles were determined. The muscles originated from 24 birds of either sex (provenance Ross; aged 40-45 days). The HA content was determined in homogenates of the upper and lower surface slices of the grilled muscles (T_i = 82 °C). A higher content of total free amino acids was seen for the muscle (27.1 mmol kg⁻¹ raw meat) than for the skin (21.7 mmol kg⁻¹ raw meat). The creatine, creatinine and carbohydrate levels in the skin were below the limits of detection. The contents of creatine (31.8-38.7 mmol kg⁻¹) and creatinine (0.24-0.33 mmol kg⁻¹) in the breast muscle were determined. Relatively high levels were seen for glucose (23 mmol kg⁻¹ raw meat) and fructose (10 mmol kg⁻¹ raw meat) in the muscle, with other sugars present at low levels (<2 mmol kg⁻¹ raw meat). For the chicken muscle grilled on a two-plate grill, the contents of total HAs (PhIP, MeIQx, DiMeIQx, Harman and Norharman) were lower with the skin in place than in the muscle grilled without the skin (3.5 μg kg⁻¹vs. 4.8 μg kg⁻¹). Also, during infrared grilling with the skin, lower amounts of HAs were formed than with grilling on the two-plate grill (2.4 μg kg⁻¹vs. 3.5 μg kg⁻¹). On average, the infrared-grilled samples with skin contained 3-fold more total HAs than similar samples without the skin (2.4 μg kg⁻¹vs. 0.8 μg kg⁻¹), with the highest levels seen for PhIP and MeIQx.     

Effects of traditional chinese cooking methods on formation of heterocyclic aromatic amines in lamb patties

Different amounts of the potent mutagenic and/or carcinogenic heterocyclic aromatic amines (HAAs) are formed in muscle-based foods under different cooking methods. HAAs (9 varieties) in lamb patties cooked using traditional Chinese cooking methods (roasting, frying, panfrying, and stewing in seasonings) were investigated. The total HAAs contents in roasted, fried, pan-fried, and stewed patties were 4.39-123.15 ng/g, 3.59-43.24 ng/g, 0.71-10.05 ng/g, and 51.07-120.32 ng/g, respectively. Amounts of HAAs increased as cooking time increased. 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) was the dominant HAAs in deep roasted and fried samples, while 1-methyl- 9H-pyrido [3,4-b] indole (Harman) and 9H-pyrido [3,4-b] indole (Norharman) were the main HAAs in pan-fried and stewed samples. Types and contents of HAAs formed at different cooking times using different methods are unique. Stewing in seasoning generated a higher HAAs content than the high-temperature cooking methods roasting, frying, and pan-frying.     

Fatty acids of neutral and phospholipids of three endangered trout: Salmo trutta caspius Kessler, Salmo trutta labrax Pallas and Salmo trutta macrostigma Dumeril

Total (TL), neutral (NL) and phospholipid (PL) amounts and fatty acid (FA) composition of female Salmo trutta caspius, Salmo trutta labrax and Salmo trutta macrostigma were investigated during one year. Twenty-three FAs were identified in both NLs and PLs. The principal FAs of both fractions were palmitic acid in saturated fatty acid, oleic acid in monounsaturated fatty acid, docosahexaenoic acid (DHA) in n-3 polyunsaturated fatty acids (n-3 PUFAs) and linoleic acid in n-6 PUFAs. The highest values for TLs, NLs and PLs were found in winter. As a general trend, the highest n-3/n-6 ratios and eicosapentaenoic acid (EPA) + DHA amounts were found in the winter and this coincided with the lowest gonado-somatic index.     

Metabolism and toxicology of heterocyclic aromatic amines when consumed in diet: Influence of the genetic susceptibility to develop human cancer. A review

There is not sufficient scientific evidence to support the hypothesis that human cancer risk is specifically due to the intake of heterocyclic aromatic amines (HAAs) in diet. Epidemiological evidence appears to imply two main factors in the HAAs carcinogenicity. These factors are the very high frequency of consumption of red meats, and very darkly browned meats from cooking, which are HAAs-containing foods. The present review focuses on the fact that the cancer risk is notably enhanced when certain genotypes related to HAAs metabolism are present. Thus, genetic predisposition seems to be the main factor in cancer development related to HAAs, and possibly the co-presence of other mutagenic compounds in diet.