Research goals for folate and related B vitamin in Europe.

In the past decade, the understanding of folate bioavailability, metabolism and related health issues has increased, but several problems remain, including the difficulty of delivering the available knowledge to the populations at risk. Owing to the low compliance of taking folic acid supplements, for example, among women of child-bearing age who could lower the risk of having a baby with a neural tube defect, food-based strategies aimed at increasing the intake of folate and other B-group vitamins should be a priority for future research. These should include the development of a combined strategy of supplemental folate (possibly with vitamin B(12)), biofortification using engineered plant-derived foods and micro-organisms and food fortification for increasing folate intakes in the general population. Currently, the most effective population-based strategy to reduce NTDs remains folic acid fortification. However, the possible adverse effect of high intakes of folic acid on neurologic functioning among elderly persons with vitamin B(12) deficiency needs urgent investigation. The results of ongoing randomized controlled studies aimed at reducing the prevalence of hyperhomocysteinemia and related morbidity must be available before food-based total population approaches for treatment of hyperhomocysteinemia can be recommended. Further research is required on quantitative assessment of folate intake and bioavailability, along with a more thorough understanding of physiological, biochemical and genetic processes involved in folate absorption and metabolism.

Folates and dairy products: a critical update.

In recent years, folates have come into focus due to their protective role against child birth defects, for example, neural tube defects. In addition, folates may have a protective role to play against coronary heart disease and certain forms of cancer. During the last few years most countries have established increased recommended intakes of folates, for example, between 300-400 microg per day for adults. This review of folates in milk and dairy products compares some recent data based on high pressure liquid chromatography (HPLC) analyses and radioprotein-binding assays, with previous data based on microbiological assays. All three methods show similar ranges for folates in cow's milk, 5-10 microg per 100 g, the variation being due to seasonal variations. Data on folates in fermented milk (buttermilk and yogurt) are also similar for these methods. Different starter cultures, however, might explain some of the variations in folate content and folate forms. Most cheese varieties contain between 10 microg and 40 microg folate per kg, with slightly higher values for whey cheese. Ripened soft cheeses may contain up to 100 microg folate per 100 g. Most previous and recent studies using HPLC indicate that 5-methyl-tetrahydrofolate (5-methyl-THF) is the major folate form in milk, but more studies are needed concerning folate forms in other, especially fermented dairy products. Relatively new data on actual concentrations in different dairy products show folate-binding proteins (FBP) to occur in unprocessed milk, but also in pasteurised milk, spray-dried skim milk powder and whey. In contrast, UHT milk, fermented milk and most cheeses only contain low levels or trace amounts.

Dietary heterocyclic amines and cancer of the colon, rectum, bladder, and kidney: a population-based study.

BACKGROUND: Heterocyclic amines formed in cooked meat and fish are carcinogenic in animal models and form DNA adducts in human beings. We undertook a study to assess whether these substances are related to the risks of cancer in the large bowel and urinary tract. METHODS: In a population-based case-control study, cases were identified from the Swedish cancer registry. Controls were randomly selected from the population register. Information on intake of various foods and nutrients was assessed by questionnaire, with photographs of foods cooked at various temperatures. We measured the content of heterocyclic amines in foods cooked under these conditions. FINDINGS: Information was retrieved from 553 controls, 352 cases of colon cancer, 249 cases of rectal cancer, 273 cases of bladder cancer, and 138 cases of kidney cancer. The response rate was 80% for controls and 70% for cases. The estimated daily median intake of heterocyclic amines was 77 ng for controls, and 66 ng, 63 ng, 96 ng, and 84 ng for cases with cancer of the colon, rectum, bladder, and kidney, respectively. The relative risk for the intake of heterocyclic amines (highest vs lowest quintile) was 0.6 (95% CI 0.4-1.0) for colon cancer, 0.7 (0.4-1.1) for rectal cancer, 1.2 (0.7-2.1) for bladder cancer, and 1.0 (0.5-1.9) for kidney cancer. Seven cases, but no controls, had an estimated daily intake of heterocyclic amines above 1900 ng. INTERPRETATION: Intake of heterocyclic amines, within the usual dietary range in this study population, is unlikely to increase the incidence of cancer in the colon, rectum, bladder, or kidney. For daily intakes above 1900 ng, our data are consistent with human carcinogenicity, but the precision was extremely low.

Heterocyclic amines in process flavours, process flavour ingredients, bouillon concentrates and a pan residue.

Seven process flavours, five process flavour ingredients, four bouillon concentrates and a pan residue were analysed for mutagenic/carcinogenic heterocyclic amines. To improve chromatographic efficiency for samples with complex matrixes (process flavours, pan residues, etc.), a new additional purification method was designed. The following polar heterocyclic amines were detected: 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in one sample (3.4 ng/g), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) in two samples (0.7-2.0 ng/g), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in four samples (1.0-13.8 ng/g), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) in three samples (1.3-2.9 ng/g), 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline (7,8-DiMeIQx) in one sample (0.3 ng/g), and traces of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in two samples. 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) was not identified in any of the samples. The following non-polar heterocyclic amines were detected: 2-amino-9H-pyrido[2,3-b]indole (AalphaC) in one sample (0.4 ng/g), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) in one sample (20.3 ng/g), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in two samples (1.4-1.7 ng/g), and traces of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in two samples. Of the co-mutagenic heterocyclic amines, 1-methyl-9H-pyrido[3,4-b]indole (harman) was identified in 15 of 17 samples (3.3-755 ng/g), and 9H-pyrido[3,4-b]indole (norharman) in 16 of 17 samples (1.2-176 ng/g). The polar heterocyclic amines were detected only in the samples of animal and mixed animal plus vegetable origin, while the non-polar heterocyclic amines were identified in samples of animal, mixed animal plus vegetable and pure vegetable origin.

Inhibitory effect of threo-9,10-dichlorostearic acid on the mutagenic activity of MeIQx, 2-AF and B[a]P in the Ames/Salmonella test.

The mutagenic activity of threo-9,10-dichlorostearic acid, one of the chlorinated fatty acids identified in fish lipids, was examined in the Ames/Salmonella test. No mutagenic activity was found on any of the Salmonella typhimurium strains TA 98, TA 100 and TA 102, either with or without S9 activation. On the other hand, dichlorostearic acid showed an inhibitory effect on the mutagenic activity of the indirectly-acting mutagens 2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-aminofluorene (2-AF) and benzo[a]pyrene (B[a]P) using strain TA 98 in the presence of S9. However, no inhibition was observed when mixing MeIQx and S9 before the addition of dichlorostearic acid. Furthermore, dichlorostearic acid did not show any inhibitory effect on the mutagenic activity of the directly-acting mutagen 4-nitroquinoline-N-oxide (4NQO) using the tester strains TA 98 and TA 100. We, therefore, suggest that dichlorostearic acid interacts with the enzymes of the S9 mix, thereby dose-dependently inhibiting the transformation of MeIQx, 2-AF and B[a]P into their active forms.

Carcinogenic heterocyclic amines in model systems and cooked foods: a review on formation, occurrence and intake.

Frying or grilling of meat and fish products may generate low ppb levels of mutagenic/carcinogenic heterocyclic amines (HAs). Many heterocyclic amines are formed via the Maillard reaction from creatine, free amino acids and monosaccharides; compounds naturally occurring in protein-rich foods of animal origin. The formation and yield of HAs are dependent on physical parameters, such as cooking temperature and time, cooking technique and equipment, heat and mass transport, and on chemical parameters, especially the precursors to HAs. This paper reviews the current knowledge on the formation of HAs in cooked foods and model systems, and summarizes data on the content of HAs in various cooked foods, and estimates of the dietary intake of HAs. It should be noted that the presence of carcinogens of other types in food (e.g. nitrosamines, aromatic amines, cholesterol oxide products) and that their generation during frying and grilling are outside the scope of this review.

Analysis of nonpolar heterocyclic amines in cooked foods and meat extracts using gas chromatography-mass spectrometry.

Heat processing of muscle foods gives rise to the formation of mutagenic and carcinogenic heterocyclic amines, often at ng/g levels. A gas chromatographic-mass spectrometric (GC-MS) technique was introduced for the analysis of nonpolar heterocyclic amines in common cooked meats, pan residues, and meat extracts after solid-phase extraction. The mutagenic heterocyclic amines 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-9H-pyrido[2,3-b]indole (A alpha C) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C) were identified in several samples in amounts up to 8 ng/g. Also the comutagenic substances 1-methyl-9H-pyrido [3,4-b]indole (harman) and 9H-pyrido[3,4-b]indole (norharman) were detected in the samples in amounts up to almost 200 ng/g. The GC-MS method can be applied without derivatisation of the sample. The technique offers high chromatographic efficiency, yielding detection limits for pure references in the range 0.1-2 ng per injection.

Mobility of alpha-tocopherol and BHT in LDPE in contact with fatty food simulants.

The migration/sorption behaviour of butylated hydroxytoluene (BHT) and alpha-tocopherol was studied in packaging material in contact with fatty food simulants. Two low-density polyethylene (LDPE) films, containing either BHT or alpha-tocopherol as antioxidants, were stored in contact with sunflower oil or 95% (v/v) ethanol. The antioxidant content was monitored in the films throughout a period of 7 weeks. The migration of alpha-tocopherol into the food simulants was slower than that of BHT. Since alpha-tocopherol was transferred from the film to the simulant to a lesser extent, it is considered to be a more stable antioxidant than BHT when used in an LDPE film in contact with 95% ethanol or sunflower oil.

Assessment of the human exposure to heterocyclic amines.

Heterocyclic amines are possible human carcinogens and fried meat is an important source of exposure in the Western diet. To study the effect of heterocyclic amines in humans, accurate assessment of individual food consumption is essential. Parameters influencing the intake include the amount and type of meat ingested, frequency of consumption, cooking method, cooking temperature and the duration of cooking. The aim of the present study was to develop a practical method for assessing individual intakes of specific heterocyclic amines in a large sample of people. This has been done by combining information on food consumption and laboratory findings of heterocyclic amines in food products. Diet was assessed using a semi-quantitative food frequency questionnaire including photos of fried meat and, in all, 22 dishes were cooked and chemically analyzed. The method was employed in an elderly population in Stockholm to estimate the daily mean intake of the five heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The total daily intake ranged from none to 1816 ng, with a mean intake of 160 ng, which is well below estimates reported previously. Highest amounts ingested were of PhIP (mean 72, range 0-865 ng/day) and MeIQx (mean 72, range 0-1388 ng/day), followed by DiMeIQx (mean 16, range 0-171 ng/day), while MeIQ and IQ were ingested only in very small amounts (mean <1 ng/day).

Trigonelline, a naturally occurring constituent of green coffee beans behind the mutagenic activity of roasted coffee?

Trigonelline and amino acids are natural components in green coffee beans. Model systems mimicking coffee roasting were used to produce heated samples of trigonelline, amino acids and glucose. Trigonelline and amino acids were heated separately or in combinations for 20 min at 250 degrees C. The results of bacteria mutation assays (Salmonella typhimurium strains TA 98, YG 1024 and YG 1029) showed that trigonelline, alone or in combination with most of the single amino acids and mixtures of amino acids, yielded potent mutagenic activity. Of the singly heated compounds, the highest mutagenic activity was found for trigonelline. The mutagenic activity detected with metabolic activation of the heated trigonelline samples indicated that the mutagenic compounds might be amines; however, higher mutagenic activity was found for trigonelline and its combinations without metabolic activation, which suggests that other types of mutagens (direct-acting) were predominant. High-performance liquid chromatographic analysis of some of the heated samples did not reveal the presence of any known mutagenic heterocyclic amine.

Polar and non-polar heterocyclic amines in cooked fish and meat products and their corresponding pan residues.

Fourteen cooked dishes with their corresponding pan residues were analysed for polar and non-polar heterocyclic amines using HPLC. The choice of foods, including beef, pork, poultry, game, fish, egg and sausages, was based on an investigation of an elderly population in Stockholm participating in an analytical epidemiological case-control study on cancer risks after intake of heterocyclic amines. The food items were prepared using normal household cooking practices, and to reflect the wide range of surface browning of the cooked dishes that would be encountered in this population, four cooking temperatures were used in the range 150-225 degrees C. For all food samples, the total amount of heterocyclic amines formed at 150 degrees C was less than 1 ng/g cooked product, and at 175 degrees C less than 2 ng/g. The highest concentrations of heterocyclic amines were detected in fillet of pork, reindeer meat and chicken breast fried at 200 and 225 degrees C and their corresponding pan residues. The total sum of 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine was about 1 microgram per 100 g portion (including pan residues) for reindeer meat and chicken breast, and between 1.9 and 6.3 micrograms per 100-g portion for fillet of pork. PhIP was the most abundant heterocyclic amine, identified in 73 of 84 samples, and the highest concentration of PhIP, 32.0 ng/g, was found in the pan residue from fillet of pork cooked at 225 degrees C. The non-polar heterocyclic amines 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole were detected in the range of 0.5-7.4 ng/g in most foods cooked at 225 degrees C, and also in meat sauce prepared at 200 and 175 degrees C. The other heterocyclic amines tested for: 2-amino-3-methylimidazo-[4,5-f]quinoline, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-6-methyl-pyrido-[1,2-a:3',2'-d]-imidazole and 2-aminodipyrido-[1,2-a:3',2'-d]imidazole, were present only at very low or non-detectable levels. The low recoveries of the amino-alpha-carbolines 2-amino-9H-pyrido[2,3-b]indole and 2-amino-3-methyl-9H-pyrido[2,3-b]indole made it impossible to quantify them. However, the co-mutagenic substances 1-methyl-9H-pyrido-[3,4-b]indole and 9H-pyrido[3,4-b]indole were detected at levels of about 1-30 ng/g in most of the dishes cooked at 200 and 225 degrees C.

Folate and folate-binding protein content in dairy products.

Recent findings suggest a protective role for folates in the reduction of neural tube defects and possibly also coronary heart disease and cancer. Consequently, an increase in the daily intake of folates is warranted, which emphasizes the need for quantitative as well as qualitative measurements of dietary folates. Milk plays an important part in the food chain in many Western countries today. Several studies suggest that folate-binding proteins might have an impact on folate absorption and therefore their concentrations are also important. The mean concentration of the predominant form of folate, 5-methyltetrahydrofolate (5-CH3THF), was determined using HPLC in thirteen selected dairy products; skim milk powder, two pasteurized milks, UHT milk, two fermented milks, three whey products and four different cheeses. All results were corrected for recovery by spiking the samples with 5-CH3THF. Effects of storage of dairy products on 5-CH3THF concentrations were also investigated; generally small and insignificant fluctuations were found, except for hard cheese, in which 5-CH3THF decreased significantly. There was a significant seasonal variation in the folate concentration of pasteurized milk which peaked in the summer months. The concentrations of folate-binding protein in skim milk powder and pasteurized milk analysed using an enzyme-linked immunosorbent assay were similar. UHT milk and fermented milk, both of which are processed at temperatures > 90 degrees C, contained significantly lower concentrations of folate-binding protein.

Effect of milk processing on the concentration of folate-binding protein (FBP), folate-binding capacity and retention of 5-methyltetrahydrofolate.

The main objective of this study was to investigate the effects of pasteurisation, UHT processing and fermentation on the concentration of folate-binding proteins (FBP) and their folate binding capacity in comparison with the retention of the most predominant folate from, 5-CH3THF. The amount of folate-binding protein (FBP) was analysed using enzyme-linked immunosorbent assay (ELISA). Unprocessed milk and pasteurised milk were found to contain similar amounts, 211 and 168 nmol/l, of FBP, respectively. UHT-processed milk and Yoghurt naturelle, both processed at temperatures above 90 degrees C, contained only 5.2 and 0.2 nmol/l FBP, respectively. As an indication of the protein-binding capacity free and protein-bound folates were analysed after charcoal treatment using the radio-protein binding assay method (RPBA). These results indicated that all folates in unprocessed milk and pasteurised milk were protein-bound, while folates in UHT-processed milk and Yoghurt naturelle occurred freely which is supported by our findings on FBP. High-performance liquid chromatography analysis indicated that unprocessed milk, pasteurised milk, UHT-processed milk and Yoghurt naturelle contained 44.8 +/- 2.1 (n = 10), 41.1 +/- 0.9 (n = 10), 36.1 +/- 1.8 (n = 10) and 35.6 +/- 9.1 micrograms/l (n = 10) 5-methyltetrahydrofolates (5-CH3THF), respectively, after deconjugation. Corresponding values for total milk folates analysed using radio-protein binding assay were 80.4 +/- 0.9 (n = 10), 64.2 +/- 2.7 (n = 10), 48.2 +/- 1.8 (n = 10) and 54.0 +/- 8.2 micrograms/l (n = 10), respectively. Hence, both methods indicated significant (P < 0.05) losses of 5-CH3THF as a result of pasteurisation, UHT processing and fermentation, compared with unprocessed milk. In spite of apparent discrepancies in folate concentrations obtained using the two different methods, these results support the equimolar ratio of FBP and folates in unprocessed and pasteurised milk when data on 5-CH3THF, obtained using HPLC were corrected for differences in recovery. Thus, heat processing of milk not only reduced the amount of 5-CH3 THF significantly, but also changed the concentration of FBP and the folate-binding capacity of FBP, which may have implications on the bioavailability of milk folates.

Influence of frying fat on the formation of heterocyclic amines in fried beefburgers and pan residues.

The influence of six frying fats (butter, margarine, margarine fat phase, liquid margarine, rapeseed oil and sunflower seed oil) on the formation of mutagenic/carcinogenic heterocyclic amines (HAs) during the frying of beefburgers was investigated. Frying was performed at 165 and 200 degrees C (i.e. under conditions that represented normal household cooking practices). The fried beefburgers and their corresponding pan residues were purified using solid-phase extraction and analysed for HAs using HPLC with photodiode array UV and fluorescence detection. The HAs 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 9H-pyrido[3,4-b]indole (norharman) and 1-methyl-9H-pyridol[3,4-b]indole (harman) were recovered. The amount increased with the temperature, and the content of HAs in the pan residue was much higher than in the corresponding beefburger. The amounts of MeIQx ranged from 0.2 to 1.6 ng/g in the beefburgers and from 0.8 to 4.3 ng/g in the pan residues. DiMeIQx ranged from undetectable to 0.4 ng/g in the beefburgers and from 0.4 to 1.3 ng/g in the residues. PhIP ranged from 0.08 to 1.5 ng/g in the meat and from 0.4 to 13.3 ng/g in the residues. The total amount of HAs in meat and pan residue combined was significantly lower after frying in sunflower seed oil or margarine than after frying with the other fats. The observed differences in MeIQx and DiMeIQx formation could be explained in terms of oxidation status (peroxide and anisidine value) and antioxidant content (vitamin A, vitamin E and tocopherols/tocotrienols) using partial least squares analysis.

Influence of amino acids on the formation of mutagenic/carcinogenic heterocyclic amines in a model system.

Mixtures of creatinine, glucose and various single amino acids were heated at 180 degrees C for 10 min in an aqueous model system. The heated mixtures all showed mutagenic activity, ranging from 80 to 2400 TA98 revertant colonies/mumol creatinine with metabolic activation. Testing of HPLC fractions for mutagenic activity showed each mixture to contain several mutagenic components, some of which corresponded to known heterocyclic amines and others to unknown compounds. The presence of 2-amino-3-methyl-imidazo[4,5-f]quinoxaline, 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline and 2-amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline in most of the samples was established using HPLC with photodiode array detection and liquid chromatography/mass spectrometry with electrospray interface and single ion monitoring. In addition, 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole and the co-mutagenic compounds 9H-pyrido[3,4-b]indole and 1-methyl-9H-pyrido[3,4-b]indole were detected in some samples.

Effect of cooking temperature on the formation of heterocyclic amines in fried meat products and pan residues.

Frequent consumers of meat have an increased risk of colorectal cancer and possibly also of breast, stomach, pancreas and urinary bladder cancer. Bacon, 'Falusausage', ground beef, meatballs, pork belly, pork chops and sliced beef account for more than one-third of the intake of fried meat of the population of Stockholm of age 50-75. These dishes were fried at four temperatures (150, 175, 200 and 225 degrees C) representing normal household cooking practices in Stockholm. Heterocyclic amines in these dishes were analysed using solid-phase extraction and HPLC. The heterocyclic amines 2-amino-3-methylimidazo[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 (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were recovered. The formation of IQ was favoured by moderate cooking temperatures; IQ was detected in one meat sample cooked at 150 degrees C and in some pan residues. The yield of MeIQx, DiMeIQx and PhIP increased with the temperature. For several of the meat dishes, the content of heterocyclic amines in the pan residue was as large or larger than for corresponding piece of meat. The highest levels of MeIQx were 23.7 ng/g in the meat and 23.3 ng/g in the pan residue. Corresponding data for DiMeIQx were 2.7 and 4.1 ng/g and for PhIP 12.7 and 82.4 ng/g. The study leaves little doubt that mutagenic heterocyclic amines are ingested by the population of Stockholm, and added to previous epidemiological studies from the same area, the combined data are consistent with human carcinogenicity of heterocyclic amines. However, analytical epidemiological studies are needed before any statement on causality can be made.

Characterization of mutagenic activity in instant hot beverage powders.

Extracts of several grain-based coffee-substitute blends and instant coffees were mutagenic in the Ames/Salmonella test using TA98, YG1024, and YG1029 with metabolic activation. The beverage powders induced 150 to 500 TA98 and 1,150 to 4,050 YG1024 revertant colonies/g, respectively. Increased sensitivity was achieved using strain YG1024. No mutagenic activity was found in instant hot cocoa products. The mutagenic activity in the beverage powders was shown to be stable to heat and the products varied in resistance to acid nitrite treatment. Differential bacterial strain specificity, and a requirement for metabolic activation suggest that aromatic amines are present. Characterization of the mutagenic activity, using HPLC and the Ames test of the collected fractions, showed the coffee-substitute blends and instant coffees contain several mutagenic compounds. Known heterocyclic amines are not responsible for the major part of the mutagenic activity. The main mutagenic activity in grain-based coffee-substitute blends and instant coffees is due to several unidentified compounds, which are most likely aromatic amines.

Factors affecting the formation and yield of heterocyclic amines.

The main food mutagens found in cooked meat products are heterocyclic amines. The formation of these heterocyclic amines has been shown to require three classes of precursors: (i) creatine/creatinine, (ii) free amino acids or dipeptides, and (iii) sugar. The mutagen forming reactions were investigated in model systems and several heterocyclic amines (IQx, MeIQx, DiMeIQx, TriMeIQx and PhIP) were identified and quantified. A number of mutagenic fractions produced in the model system are not yet identified, among them one having a mass number of 217. A participation of the Maillard reaction in the formation of the heterocyclic amines was proposed ten years ago. Since then, the support for this route has increased, especially in a recent study in which carbon atoms from 14C-labelled glucose were shown to be incorporated into MeIQx and DiMeIQx. Other studies have shown sugar to either enhance or inhibit the yield of mutagenic activity depending on its molar ratio relative to the other reactants. Thus, the Maillard reaction can be utilized both to enhance and inhibit the formation of the heterocyclic amines. Olive oil and corn oil were found to almost double the yield of MeIQx in the model system after heating for 30 minutes at 180 degrees C. When adding deep-fat frying oil of different oxidation status in the model system, the yield of MeIQx was not affected by oxidation status or presence of vitamin E, but the lipids enhanced the formation of MeIQx by 30% after heating at 180 degrees C for 30 minutes. Artificial production of free radical reactions by adding FeSO4, hydrogen peroxide and oxygen to the model system doubled the yield of MeIQx.

Occurrence of mutagenic/carcinogenic heterocyclic amines in meat and fish products, including pan residues, prepared under domestic conditions.

Some typical Swedish meat and fish products, e.g. bacon, beefburgers, meatballs, Baltic herring, salmon, smoked fish, black pudding and sausages, and their corresponding pan residues, were analysed by HPLC for their content of mutagenic/carcinogenic heterocyclic amines (HAs). The products were cooked using recommended domestic cooking conditions concerning temperature, time and frying equipment. The amount of HAs was low in most products, though the amount was higher in the pan residues, especially in the pan residue from the frying of Falun sausage, which contained 18.5 ng HAs/g cooked product. Mostly MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline) and 4,8-DiMeIQx (2-amino-3,4,8-trimethylimidazo[4,5-f]-quinoxaline) were found, being 0.03-2.8 ng MeIQx/g and n.d.-3.4 ng 4,8-DiMeIQx/g cooked product in the food products and 0.05-7.3 ng MeIQx/g and n.d.-2.8 ng 4,8-DiMeIQx/g cooked product in the pan residues. High levels of IQ (2-amino-3-methylimidazo[4,5-f]quinoline), 10.5 ng/g, were only found in well-done bacon and a correlation was seen between fat content and IQ formation. Low levels of MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) and PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) were found in the foods.

Influence of oxidized deep-frying fat and iron on the formation of food mutagens in a model system.

The effects of oxidized fats, iron and tocopherol content on the yield and species of mutagenic heterocyclic amines were studied using a model system. A mixture of glycine (0.9 mmol), creatinine (0.9 mmol) and glucose (0.45 mmol) was heated for 10 and 30 min at 180 degrees C, with the addition of iron and fats. The mutagens formed were identified and quantified using HPLC. (2-Amino-3-methylimidazo[4,5-f]-quinoxaline) (IQx), 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 formed in the model mixtures. The addition of iron (FeSO4) or oxidized fats to the model system did not affect the species of food mutagens formed, but the iron addition more than doubled the amount of MeIQx. The oxidation status of the fat added to the model system had little effect on the formation of MeIQx. The fat content was shown to affect the mutagen formation significantly, especially after heating for 30 min. No difference in yield of MeIQx was observed in the presence of tocopherol and tocotrienol at naturally occurring concentrations.