Occurrence of heterocyclic amines in several home-cooked meat dishes of the Spanish diet

Heterocyclic amines (HAs) were determined in several of the most frequently eaten meat dishes in Spain such as fried beef hamburger, fried pork loin, fried chicken breast, fried pork sausages, griddled chicken breast, griddled lamb steak and griddled beef steak. All of the products tested were household cooked. The HAs were analysed in the selected meat dishes using an analytical method based on solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry. DMIP, MeIQx, 4,8-DiMeIQx, Norharman, Harman, PhIP, Trp-P-1, AalphaC and MeAalphaC were the amines most frequently found at concentrations of up to 47 ng g(-1) of cooked meat. Glu-P-2, IQ, MeIQ, Glu-P-1, 7,8-DiMeIQx and Trp-P-2 were only found in a few of the meat dishes and their concentrations were lower than 1 ng g(-1) of cooked meat. The highest amounts of HAs, especially PhIP and DMIP, were formed in fried chicken breast and the lowest were formed in fried beef hamburger and in fried pork sausages. Daily intake of HAs in Spain was estimated at 606 ng of mutagenic HAs per capita and day, DMIP and PhIP being the main contributors.     

Formation and stability of heterocyclic amines in a meat flavour model system. Effect of temperature, time and precursors

A model system based on a commercial meat flavour was used to evaluate the formation of heterocyclic amines, simulating the application of this seasoning in household cooking. The effects of different treatments in both dry and aqueous conditions were studied. The lyophilized meat flavour extract was heated at temperatures ranging between 100 and 200 degrees C for times ranging from 10 min to 2 h. Similarly, an aqueous suspension of the extract was heated at 175 degrees C for 1, 2 and 3 h. Precursors of HAs, such as creatinine, glucose, and the amino acids glycine, alanine and phenylalanine were added to the meat extract and their effect was tested by heating the mixture at 200 degrees C for 30 min, when dry conditions were used, and at 175 degrees C for 2 h in wet systems. All conditions led to the formation of HAs, PhIP being the amine that was detected at the highest level of concentration in most model systems (i.e. 173 ng g(-1) at 200 degrees C, 30 min). Moreover, the addition of creatinine and amino acids to the meat extract flavour produced an important increase in IQ and MeIQx content.     

Formation of heterocyclic aromatic amines in model systems

Heterocyclic aromatic amines (HAs) are mutagenic and carcinogenic substances that are formed in significant amounts during heating of meat or fish at temperatures of at least 150 degrees C. To investigate the chemistry lying behind the formation of these harmful substances model systems were established. The first aim was to identify the naturally occurring precursors, namely creatinine, amino acids and carbohydrates. Later these model systems were used to develop strategies for a reduction of the content of the heterocyclic aromatic amines and for the evaluation of the reaction mechanisms that lead to the formation of these substances. All these aspects are discussed in this review.     

Effects of Seasoning and Heating Device on Mutagenicity and Heterocyclic Amines in Cooked Beef

Pan-roasted beef showed a lower mutagenicity after various degrees of cooking than charcoaled one. The high mutagenicity of charcoaled beef was due to the formation of more heterocyclic amines, especially AαC (2-amino-9 H-pyrido- [2,3-b]indole) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) because of rapid and direct heating on the surface of the meat at a high temperature. Seasoning decreased mutagenicity of pan-roasted beef except the very well done sample with unchanged heterocyclic amine contents, but increased mutagenicity of charcoaled beef with decreased levels of AαC and PhIP, probably due to the change of heterocyclic amine precursors or alternatively to the occurrence of other mutagens.     

Estimates of heterocyclic amine intake in the US population

HA-specific meat concentration estimates using a method that combines laboratory data to predict HA concentrations from meat type, cooking method and meat doneness were used with national dietary data to estimate daily HA intake for segments of the US population. PhIP was found to comprise approximately 70% of US mean dietary intake of total HAs, with pan-frying and chicken being the single cooking method and meat type contributing the greatest to total estimated HA exposures. This analysis demonstrated significantly higher concentrations in grilled/barbecued meats than in other cooked meats. African-American males were estimated to consume nearly twofold and approximately 35 to 40% more PhIP (and total HAs) than white males at ages <16 and >30 years, respectively.     

Determination of less polar heterocyclic aromatic amines in standardised beef extracts and cooked meat consumed in Austria by liquid chromatography and fluorescence detection

An analysis method was developed for the determination of trace levels of less polar heterocyclic aromatic amines (HAs) in food samples. The development started from a frequently used sample pre-treatment scheme which was slightly improved to make it applicable with high-performance liquid chromatography (HPLC) with fluorescence detection. The method was applied for the analysis of a standardised beef extract containing 5-15 ng/g of HAs and the results are compared with those of the other participants in the same European project. In addition, the method was used for the analysis of less polar HAs in cooked meat consumed in Austria.     

Use of antioxidants to minimize the human health risk associated to mutagenic/carcinogenic heterocyclic amines in food

Heterocyclic amines (HAs) are mutagenic/carcinogenic compounds formed in meat during cooking. Several efforts have been made to minimize the risk associated to HA human exposure. Supplementation with antioxidants is considered a promising measure to reduce HA exposure because of their ability as inhibitors of HA formation or as blocking/suppressing agents on HA biotransformation/metabolism. The aim of this review is to present the current knowledge on the capability of synthetic and natural antioxidants to modulate HA-induced mutagenicity/carcinogenicity. Data show a general trend towards a reduction of HA formation both in model systems and in real foods as well as an effective modulation of biotransformation and metabolism. Phenolic compounds, particularly those from tea and olive oil, seem to be the most effective, although a great variability is observed because of the concentration-dependent pro- and antioxidant effects.     

Effects of heating time and antioxidants on the formation of heterocyclic amines in marinated foods

The effect of heating time and antioxidants on the heterocyclic amine (HAs) formation in marinated foods were studied. Food samples were cooked at 98 +/- 2 degrees C for 1, 2, 4, 8, 16 and 32 h in a closed pan in the presence of water, soy sauce and rock candy with or without antioxidants. The various HAs in marinated food samples and juice were analyzed by HPLC with photodiode-array detection. Results showed that the amount of HAs formed during heating followed an increased order for each increasing heating time. A larger variety and higher amount of HAs were generated in marinated pork when compared to marinated eggs and bean cake. In marinated juice, the levels of HAs were present in greater amount than in marinated foods. The incorporation of antioxidants Vitamin C, Vitamin E and BHT were found to be effective towards HAs inhibition, however, the effect was minor.     

Preparation of a beef-extract as a laboratory reference material for the determination of heterocyclic amines

The present paper describes the preparation of a suitable laboratory reference material (LRM) to validate analytical methods for the determination of heterocyclic amines (HAs) in foods. Three different lots of reference material were prepared using a beef extract which was contaminated with a well-known quantity of amines at different levels ranging from 10 to 75 ng/g. These materials were then lyophilised under determined conditions and, after grinding and sieving, homogenised and, finally, bottled and labelled. Homogeneity and stability studies were performed and no statistical differences were observed in the analysis of variances for within- and between-bottle results, thus demonstrating the homogeneity of the material. Stability at different storage temperatures (-18, +4, +25 and +40 degrees C) and times (1, 2, 3 and 6 months) was also tested. Therefore, the material can be considered homogeneous and stable and can be proposed for use in inter-comparison exercises for the determination of HAs.     

Determination of heterocyclic aromatic amines (HAs) content in samples of household-prepared meat dishes

Aminoazaarene content was investigated in 10 meat samples (including pork, beef, turey and chicken) thermally processed at home according to common recipes used by residents of Upper Silesia region in Poland. The clean-up procedure included tandem solid-phase extraction (SPE) using Extrelut-type columns filled with diatomaceous earth, propylsulphonic acid and chemically bounded phase-C18. Identification and quantitative analysis of HAs fraction was carried out using a HPLC system with DAD-type detector. Separation was achieved using TSK-gel ODS 80-TM column and a mixture of 5% acetonitrile and 95% triethylamine phosphate buffer (pH 3.3) as a mobile phase. The results of qualitative determinations were confirmed by GC-MS method. To achieve this, HAs fractions were derivatized to pentafluoropropionic acid (PFPA) amide derivatives. The summary content of five aminoazaarenes determined in investigated meat samples, i.e. 2-amino-3-methylimidazo [4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 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) falls within the range of 1.9-77.4 ng/g of sample. The calculated values of theoretically daily human exposure to five determined HAs were in the range of 0.2-7.7 microg per day per person.     

Evaluation of reversed-phase columns for the analysis of heterocyclic aromatic amines by liquid chromatography-electrospray mass spectrometry

Liquid chromatography coupled to mass spectrometry (LC-MS), especially by the use of electrospray ionisation source (ESI), is currently used for the analysis of heterocyclic aromatic amines (HAs) in complex samples. The present paper describes the study of the performance of different narrow-bore reversed-phase columns to achieve the best chromatographic separation for the determination of 16 HAs by LC-ESI-MS in food samples. Different parameters such as peak symmetry, resolution and number of theoretical plates have been evaluated for each column, using different chromatographic conditions. The column that provided the best results was TSK Gel Semi-Micro ODS-80TS of Tosohaas. Quality parameters have been established, obtaining good short-term precision in all cases (relative standard deviation (R.S.D.) lower than 7.7%) and low limits of detection (<13 pg injected in MS and <16 pg injected in MS/MS). The content of HAs in two beef extracts have been determined.     

Surface decontamination of beef inoculated with Salmonella Typhimurium DT104 or Escherichia coli O157:H7 using dry air in a novel heat treatment apparatus

AIMS: To determine the effectiveness of a novel dry air decontamination apparatus in the deactivation of Salmonella serotype Typhimurium DT104 or Escherichia coli O157:H7 on beef surfaces. METHODS AND RESULTS: A laboratory scale dry air decontamination apparatus, capable of producing repeatable and known heating time-temperature cycles on food surfaces was used in decontamination trials. Beef samples were surface inoculated with 7-8 log10CFU cm(-2) of S. Typhimurium DT104 or E. coli O157:H7 and heated at 60, 75, 90 and 100 degrees C using fast and slow heating rates and subsequently held at these temperatures for up to 600 s. A substantial reduction in pathogen numbers was achieved at higher temperatures (90 and 100 degrees C, 4.18-6.06 log10CFU cm(-2)) using both heating rates, but cell survival at these temperatures was also observed. At the lower temperatures, deactivation was small at 60 degrees C in particular it was less than one log unit after 3 min heating. No significant differences were observed when total reductions in pathogen counts were compared for all the temperature/heat up time combinations tested. During slow heating at 90 degrees C, and both heating rates at 100 degrees C, the pattern of deactivation of S. Typhimurium DT104 or E. coli O157:H7 was triphasic. CONCLUSIONS: This study has shown that heating meat surfaces with dry air can achieve substantial reductions in S. Typhimurium DT104 or E. coli O157:H7. As surface decontamination of beef surfaces with dry air had a negative effect on beef colour and appearance, such a decontamination apparatus would be unsuitable for producing meat for retail sale but it could be used to produce safer meat for use in the catering trade. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides researchers and food processors with data on the dynamic changes in S. Typhimurium DT104 and E. coli O157:H7 counts on intact beef surfaces during heating with dry air under realistic (time-varying) temperature conditions.     

Analysis of heterocyclic amines in food products: interlaboratory studies

A feasibility study and two interlaboratory exercises on the determination of selected heterocyclic amines (HAs) in beef extract, organised in the framework of a European project, are presented. The aim of these exercises was to improve the quality of the laboratories and to evaluate the performance of a standardised analytical method and also the methods currently used by each of the participants for the analysis of these compounds. Three lyophilised portions of a commercial beef material previously spiked with HAs at different concentration levels ranging from 10 to 75 ng g(-1) were used as laboratory reference materials (lot A, B and C). Firstly, a feasibility study was carried out using a test standard solution and the beef extract (lot A), which contained only five HAs. Then, two interlaboratory exercises were carried out using the laboratory reference materials lot B and lot C, containing 10 selected HAs at two different concentration levels, 75 and 10 ng/g, respectively. The results obtained by all participant laboratories using the proposed method showed satisfactory agreement and the CV(%) between-laboratories obtained were from 8.3 to 24.1% for lot B and from 8.7 to 44.5% for lot C. The standardised method evaluated in these collaborative studies is therefore proposed for the analysis of HAs in food material. Moreover, LC-MS is recommended as the most suitable technique for the analysis of a large number of HAs in food samples.     

Determination of heterocyclic aromatic amines in beef extract, cooked meat and rat urine by liquid chromatography with coulometric electrode array detection

This paper describes a method for the determination of heterocyclic aromatic amines (HAs; DMIP, IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, AalphaC, PhIP) by high-performance liquid chromatography (HPLC) with coulometric electrode array detection. The compounds are separated on reversed phase columns (LiChroCart Superspher 60 RP-select B, 250 mm x 2 mm, 4 microm and LiChrospher 60 RP-select B, 250 mm x 4 mm, 5 microm) using mobile phases consisting of acetonitrile/buffer/distilled water and detected at eight working electrodes at potentials between +190 and +680 mV against modified palladium electrodes. In the context of an EU-interlaboratory exercise, the method was applied to analyse a standardised test solution and--after isolation of the analytes by several clean-up steps--for the analysis of standardised beef extract and grilled meat. Further, the method could be applied for the analysis of HAs in suspensions of bacteria and rat urine without any sample preparation step beyond sample dilution. The data obtained show that HPLC with coulometric electrode array detection gives accurate results.     

Effects of municipal waste compost and sewage sludge on proton binding behavior of humic acids from Portuguese sandy and clay loam soils

The effects of amendment with municipal solid waste compost (MSWC) and sewage sludge (SS) on acid-base properties of soil humic acids (HAs) were investigated. For this purpose, HAs were isolated from MSWC and SS and two different Portuguese soils, one sandy and the other clay loam, either unamended or amended with MSWC or SS at a rate of 60 t ha(-1), and analysed by potentiometric titrations at various ionic strengths (0.01, 0.05, 0.1 and 0.3M) over the pH range from 3.5 to 10.5. All titration data were fitted with the NICA-Donnan model and the variations of model parameters between the various HA samples were discussed. The HAs from MSWC and SS had lower acidic functional group contents and higher proton binding affinities than the control soil HAs. Amending soils with MSWC and SS determined a decrease of acidic functional group contents and an increase on proton binding affinities of soil HAs. These effects were more evident in SS-amended soil HAs than in MSWC-amended soil HAs, and in clay loam soil HA than in sandy soil HA.     

Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells

Aim of the present study was the investigation of the genotoxicity of amino-alpha-carboline (AalphaC) in human derived cells and of its organ-specific effects in laboratory rodents. This heterocyclic amine (HA) is contained in fried meat and fish in higher concentrations than most other cooked food mutagens. In the present experiments, AalphaC caused dose-dependent induction of micronuclei in the human derived hepatoma cell line HepG2 at concentrations > or =50 microM. In contrast, no significant effects were seen in Hep3B, another human hepatoma cell line, which may be explained by the concurrent lower activity of sulfotransferase (SULT), an enzyme playing a key role in the activation of AalphaC. A positive result was also obtained in the single cell gel electrophoresis (SCGE) assay in peripheral human lymphocytes, but the effect was only significant at the highest concentration (1000 microM). In Fischer F344 rats and ICR mice, the liver was the main target organ for the formation of DNA adducts (at > or =50 mg/kg bw), and in lungs and colon substantially lower levels were detected. Identical organ specificity as in the DNA adduct measurements was seen in SCGE assays with rats, whereas in mice the most pronounced induction of DNA migration was observed in the colon. Comparison of our results with data from earlier experiments indicate that the genotoxic potency of AalphaC is equal to that of other HAs, which are contained in human foods in much smaller amounts. Therefore, our findings can be taken as an indication that the human health risk caused by exposure to AalphaC is higher than that of other HAs that are formed during the cooking of meat and fish.     

Blue cotton, Blue Rayon and Blue Chitin in the analysis of heterocyclic aromatic amines--a review

Heterocyclic amines (HCAs) are a group of compounds formed when protein-rich foods, such as meat or fish, are prepared under normal cooking conditions, such as frying, grilling, or broiling. To evaluate and estimate the risks associated with HCAs contained in the diet, it is important to determine the levels in cooked foods, and the levels of HCAs and metabolites in the body. HCAs are normally found at low amounts in a complex matrix, which necessitates a good purification method and a sensitive detection system. The objective of this review was to briefly present the current knowledge on the use of Blue Cotton, Blue Rayon and Blue Chitin in the analysis of HCAs.     

Chromatographic and related techniques for the determination of aromatic heterocyclic amines in foods

Some 20 years ago, Japanese scientists discovered a new group of highly toxic compounds, classified as heterocyclic aromatic amines, from broiled and grilled meat and fish products. Numerous studies have shown that most HAs are mutagenic and carcinogenic, and the safety of HA-containing foods has become a concern for the public. To date, more than 20 different mutagenic and/or carcinogenic heterocyclic amines have been identified in foods. This paper reviews the analysis of foods for HAs with 145 references. We survey some of the numerous methods available for the chromatographic analysis of heterocyclic amines and highlight the recent advances. We discuss chromatographic and related techniques, including capillary electrophoresis, and their coupling to mass spectrometry for the determination of these contaminants in foods. In addition, the review summarises data on the content of HAs in various cooked foods.     

Influenza virus hemagglutinin-induced cell-planar bilayer fusion: quantitative dissection of fusion pore kinetics into stages

Cells expressing the influenza virus hemagglutinin (HA) fuse to planar bilayer membranes under acidic conditions. After an electrically quiescent perfusion stage (Q), a fusion pore forms that enlarges in three subsequent stages. A repetitively flickering pore stage (R) develops into a securely open stage (S) that exhibits conductances ranging from a few to tens of nS. The pore then expands to a terminal stage (T) with a large conductance on the order of one microSiemens. We have studied how virus strain, HA receptors in the target bilayer membrane, and cytoskeleton affect the time a fusion pore remains in each stage. These intervals are referred to as waiting times. In the quiescent stage, waiting times were very sensitive to the virus strain and presence of gangliosides (HA receptors) in the bilayer. When bilayers contained gangliosides, the waiting times in the Q stage for cells infected with the PR/8/34 strain of virus were exponentially distributed, whereas waiting times for cells infected with the Japan/305/57 strain were not so distributed. Without gangliosides, the waiting time distribution for PR/8/34 infected cells was complex. The waiting times in the R and S stages of pore growth were exponentially distributed under all tested conditions. Within the R stage, we analyzed the kinetics of the flickering pore by fitting the open and closed time distributions with a sum of two exponentials. Neither the open and closed time distributions nor the flickering pore conductance distributions were appreciably affected by virus strain or gangliosides. Colchicine and cytochalasin B increased the flicker rates, without affecting the waiting time in the R stage. We conclude that variations in amino acid sequences of the HAs and the presence of gangliosides as receptors within the target membrane critically affect the kinetics of fusion pore formation, but do not affect subsequent stages.     

Structural Alteration of Humic Acids by Pseudomonas spp. from Deep Terrestrial Subsurface Diatomite Formations in Northernmost Japan

Bacterial utilization of humic acids (HAs) was examined under aerobic conditions using Pseudomonas spp. from diatomite from a depth of 250 m below ground level in the Horonobe Underground Research Laboratory. HA decolorization and bacterial aggregation were observed during cultivation when an auxiliary carbon source was added. High-performance size-exclusion chromatography showed that high-molecular-weight HAs were produced. Fourier transform infrared spectroscopy indicated that carboxyl groups and polysaccharide-related substances in HAs were eliminated, while aliphatic structural units and amide groups were added to HAs. These results suggested that Pseudomonas spp. utilize and alter the molecular structure of HAs under aerobic conditions caused by the construction of underground facilities.