Biomonitoring of dietary heterocyclic amines and metabolites in urine by liquid phase microextraction: 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a possible biomarker of exposure to dietary PhIP.

Heterocyclic amines (HCAs) are mutagenic/carcinogenic compounds formed at ng/g levels during frying meat or fish. The effect of the normal intake of dietary HCAs in humans and their involvement in the etiology of cancer are currently unknown. In this work, a new extraction method, liquid phase microextraction (LPME) with hollow fibers, and LC-MS/MS have been used for the first time to determine HCAs and metabolites in nonspiked human urine following a single meal of chicken cooked at 180 °C for 6 min. The total intake of HCAs was estimated to be 6 µg, of which 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) accounted for about 1 µg. The concentrations of PhIP in nonhydrolyzed urine samples ranged from 11.7 to 59.4 pg/g. The total amount of PhIP in urine ranged between 9.3 and 21.1 ng, which corresponds to 0.91-2.1% of the ingested PhIP. In addition, the urine levels of 4'-OH-PhIP (2-amino-1-methyl-6-(4'-hydroxy)phenylimidazo[4,5-b]pyridine) and 5-OH-PhIP (2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine) also showed a narrow variation between the samples. The analysis of urine samples after acid hydrolysis did not give additional information but showed a notable increase in norharman in some cases. The obtained results suggest PhIP in urine as a possible biomarker of exposure to HCAs and the LPME and LC-MS/MS method as an appropriate strategy to biomonitor HCAs in urine.

Application of the BRAFO tiered approach for benefit-risk assessment to case studies on heat processing contaminants.

The aim of the European Funded Project BRAFO (benefit-risk analysis of foods) project was to develop a framework that allows quantitative comparison of human health risks and benefits of foods based on a common scale of measurement. This publication describes the application of the BRAFO methodology to three different case studies: the formation of acrylamide in potato and cereal based products, the formation of benzo(a)pyrene through smoking and grilling of meat and fish and the heat-treatment of milk. Reference, alternative scenario and target population represented the basic structure to test the tiers of the framework. Various intervention methods intended to reduce acrylamide in potato and cereal products were evaluated against the historical production methods. In conclusion the benefits of the acrylamide-reducing measures were considered prevailing. For benzo(a)pyrene, three illustrated alternative scenarios were evaluated against the most common smoking practice. The alternative scenarios were assessed as delivering benefits, introducing only minimal potential risks. Similar considerations were made for heat treatment of milk where the comparison of the microbiological effects of heat treatment, physico-chemical changes of milk constituents with positive and negative health effects was assessed. In general, based on data available, benefits of the heat treatment were outweighing any risks.

Hollow fibre-supported liquid membrane extraction and LC-MS/MS detection for the analysis of heterocyclic amines in urine samples.

Heterocyclic amines (HCAs) are potent mutagens/carcinogens to which humans are frequently exposed through the consumption of cooked meat and fish food. The effect of normal intake of HCAs and their role in the aetiology of human cancer is unknown. To some extent, limitations of the existing analytical methods in monitoring the low levels of HCAs in biological samples have hindered obtaining conclusive results. In this study, a method for the analysis of HCAs in human urine has been studied to detect HCAs and metabolites at levels resulting from consumption of food cooked at ordinary conditions. The analytical method consisted of extraction and clean-up by the novel technique liquid-phase microextraction combined with LC-MS/MS. The effect of pH during the extraction and hydrolysis step was examined. High sensitivity was achieved when the extraction was performed in raw urine adjusted to pH 5.5, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine being detected from 2 pg/g urine, levels comparable with a normal exposure. Good reproducibility and repeatability was obtained for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, below 9% using isotopic dilution. The performance of the method on 9H-pyrido[3,4-b]indole, 2-amino-1-methyl-6-(4'-hydroxyphenyl)imidazo[4,5-b]pyridine and 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo[4,5-b]pyridine was also studied.

Determination of heterocyclic aromatic amines in human urine by using hollow-fibre supported liquid membrane extraction and liquid chromatography-ultraviolet detection system.

A hollow-fibre supported liquid membrane (HF-SLM) extraction method has been developed for determination of 11 heterocyclic aromatic amines (HCAs) in human urine samples by using high performance liquid chromatography (HPLC) equipped with an ultraviolet (UV) absorbance detector. These compounds were extracted from an alkaline urine sample (donor phase) into the organic solvent residing in the pores of a polypropylene hollow fibre and then back extracted into an acidic solution (acceptor phase) inside the lumen of the hollow fibre. After extraction, HCAs were analyzed by injecting the analyte enriched acceptor phase into the HPLC. The analyte enrichment factors ranged between 241 and 339 obtained in a 90 min extraction time, and method detection limits (MDL) ranged between 0.1 and 0.5 microg L(-1) with relative standard deviation (RSD) values between 3.4% and 11%. The extraction technique employed in this work is easy to use and rapid as it involves only a few minutes manipulation of each sample. It is the most economical sample preparation/preconcentration technique to our knowledge as compared to other microextraction techniques.

Do both heterocyclic amines and omega-6 polyunsaturated fatty acids contribute to the incidence of breast cancer in postmenopausal women of the Malmö diet and cancer cohort?

Heterocyclic amines (HAs), formed when meat and fish are cooked at high temperatures, have been linked to mammary gland cancer in rats, and some epidemiological studies indicate increased breast cancer risk by consumption of well-done meat. The epidemiological evidence linking HAs per se to breast cancer is however sparse, especially from prospective studies. Moreover, high-fat diets rich in omega-6 polyunsaturated fatty acids (PUFAs) have produced higher frequencies of HA-induced mammary gland tumors in rats compared to those fed low-fat diets. The aim was to evaluate prospectively if intake of HAs is associated with breast cancer incidence, and if the association is independent of omega-6 PUFA intakes. Among women 50 years or older at baseline from the population-based prospective Malmö Diet and Cancer cohort (n = 11,699), 430 women were diagnosed with incident invasive breast cancer during a mean follow-up of 10.4 years. Information on dietary habits was collected by a modified diet history method. Cox proportional hazards regression estimated hazard ratios (HRs) and 95% confidence intervals (CIs) of breast cancer associated with energy-adjusted intakes of HAs and omega-6 PUFA. Intakes of HAs were not associated with breast cancer incidence (HR, 0.94; 95% CI, 0.69-1.28, for highest compared to lowest quintile). In individuals with low HA intakes, a significant increased risk was observed among those with high intakes of omega-6 PUFAs. In conclusion, intakes of HAs are not associated with breast cancer incidence in this Swedish cohort, but dietary patterns very high in omega-6 PUFA may promote breast cancer development.

A simplified hollow-fibre supported liquid membrane extraction method for quantification of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in urine and plasma samples.

A simple and easy-to-use extraction procedure has been optimised, validated, and applied for extraction of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in urine and spiked plasma samples. PhIP is a carcinogenic and mutagenic heterocyclic aromatic amine that is formed during cooking of meat and fish. The novelty of the extraction procedure lies in using a short piece of narrow capillary-like microporous hollow-fibre (HF) membrane as extraction device. The HF membrane was filled with a few microlitres of acidic solution and the membrane pores were impregnated with an organic extraction solvent. Therefore, the technique was called hollow-fibre supported liquid membrane (HF-SLM) extraction. The HF extraction device was then supported by a syringe needle and directly immersed in urine (1.4 mL) or plasma (0.3 mL) previously made alkaline by adding 0.5 mol L(-1) NaOH solution to give a final volume of 1.6 mL. The operation of the HF-SLM extraction at the optimal conditions resulted in a PhIP extraction efficiency of 74% from both spiked urine and plasma, corresponding to enrichment factors of 126 and 27, respectively. For 90 min extraction time, limits of detection and quantification were, respectively, 8 and 25 pg mL(-1) for urine and 6 and 11 pg mL(-1) for plasma. Within-day repeatability (n = 6) and between-day reproducibility (n = 3) were, respectively, 5% and 13% for urine and 6% and 7% for plasma. Analysis of urine samples collected for 12 h after a volunteer had eaten 250 g well-done chicken showed the PhIP concentration was 124 +/- 21 pg mL(-1), calculated assuming an extraction efficiency of 74%.

On-line capillary based quantitative analysis of a heterocyclic amine in human urine.

A high through-put miniaturised separation-quantification method for the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in urine was developed. The limit of detection was of 0.65 fmol (0.14 pg) injected or 65 pM. Heterocyclic aromatic amines are mutagenic and carcinogenic compounds formed at low levels in protein-rich food during cooking. Due to the low concentrations and the high complexity of the matrix in which they exist (food, blood, and urine), and the often small sample volumes (capillary blood; urine, blood and milk from small animals), very sensitive and selective analytical methods are required for their detection. Miniaturization was accomplished by micro solid-phase extraction in capillaries with blue chitin as solid-phase, coupled on-line (in-capillary) to capillary electrophoresis with nanospray tandem mass spectrometric detection. This new technique provided a total analysis time of only 15 min, including extraction and separation, together with low sample and solvent consumption. Blue chitin showed high tolerance towards the urine matrix. Urine collected 12h after consumption of fried chicken contained 1.8 nmol L(-1) (0.40 pg microL(-1)) of PhIP.

Genotoxicity of heat-processed foods.

Gene-environment interactions include exposure to genotoxic compounds from our diet and it is no doubt, that humans are regularly exposed to e.g. food toxicants, not least from cooked foods. This paper reviews briefly four classes of cooked food toxicants, e.g. acrylamide, heterocyclic amines, nitrosamines and polyaromatic hydrocarbons. Many of these compounds have been recognised for decades also as environmental pollutants. In addition cigarette smokers and some occupational workers are exposed to them. Their occurrence, formation, metabolic activation, genotoxicity and human cancer risk are briefly presented along with figures on estimated exposure. Several lines of evidence indicate that cooking conditions and dietary habits can contribute to human cancer risk through the ingestion of genotoxic compounds from heat-processed foods. Such compounds cause different types of DNA damage: nucleotide alterations and gross chromosomal aberrations. Most genotoxic compounds begin their action at the DNA level by forming carcinogen-DNA adducts, which result from the covalent binding of a carcinogen or part of a carcinogen to a nucleotide. The genotoxic and carcinogenic potential of these cooked food toxicants have been evaluated regularly by the International Agency for Research on Cancer (IARC), which has come to the conclusion that several of these food-borne toxicants present in cooked foods are possibly (2A) or probably (2B) carcinogenic to humans, based on both high-dose, long-term animal studies and in vitro and in vivo genotoxicity tests. Yet, there is insufficient scientific evidence that these genotoxic compounds really cause human cancer, and no limits have been set for their presence in cooked foods. However, the competent authorities in most Western countries recommend minimising their occurrence, therefore this aspect is also included in this review.

Heterocyclic amines in some Swedish cooked foods industrially prepared or from fast food outlets and restaurants.

Accurate assessment of human intake of mutagenic/carcinogenic heterocyclic amines (HAs) is necessary for epidemiological studies and future risk assessment. Using questionnaires, the frequency of consumption of specific dishes can be obtained at an individual level and linked to analyzed concentrations of different compounds in corresponding dishes. Some typical Swedish cooked meat dishes, hamburgers and kebab, industrially prepared or from fast food outlets and restaurants, were analyzed regarding their content of 11 different HAs. The amount of each of these compounds was below 0.1 ng/g cooked weight in most of the industrially prepared products. The total amount of HAs was highest in the kebab samples. The intake of HAs from 200 g of the dishes was estimated to range from not detectable levels to 0.6 microg. The results of the present study indicate that the content of HAs in a specific dish may vary with origin, and that the concentrations of HAs in commercial fried meat products are generally low, although some of these food items may contain elevated amounts.

In-capillary micro solid-phase extraction and capillary electrophoresis separation of heterocyclic aromatic amines with nanospray mass spectrometric detection.

A miniaturised technique to analyse and detect heterocyclic aromatic amines (HAs) using micro solid-phase extraction ( microSPE) coupled on-line (in-capillary) to capillary electrophoresis (CE) separation with nanospray (nESI) mass spectrometry (MS) detection has been developed. HAs are mutagenic and carcinogenic compounds formed at low levels in protein-rich food during cooking. Due to the low concentrations of HAs and the high complexity of the matrix in which they exist, sensitive and selective analytical methods are required for quantification. microSPE was performed on a packed bed of C(18 )particles inside the CE capillary, which minimised the dead volume. The on-line coupling of SPE, CE and nESI-MS reduced the time for extraction and identification to less than half an hour, which will allow for screening of several samples per day. The new technique provides short analysis time, low sample and solvent consumption, and HAs in standard solutions were easily detected at 12-17 fmol injections, and in spiked urine samples at 750-810 fmol injections.

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.

Blue chitin columns for the extraction of heterocyclic amines from cooked meat.

Mutagenic/carcinogenic heterocyclic amines (HAs) are formed at low levels (ng/g) during heat processing of protein-rich food such as meat and fish. The complex matrix requires effective extraction and purification methods. Blue Chitin columns were used for the extraction of HAs from fried chicken fillets and the samples were analysed with LC-MS. Several HAs were identified at levels ranging from 0.04 to 0.10 ng/g. The use of Blue Chitin columns provides a simple and fast method for the extraction of HAs from meat samples.

Natural variations of precursors in pig meat affect the yield of heterocyclic amines--effects of RN genotype, feeding regime, and sex.

Pig meat shows natural variations in the concentrations of precursors of heterocyclic amines (HCAs), which may affect formation of HCAs in cooked pig meat. To study this, 26 pigs with an inherent genetic variation (carriers and noncarriers of the RN(-) allele) were subjected to different feeding regimes (conventional feed compared with feed composed according to organic standards). In addition, the effect of sex (castrated males or females) was considered when assessing chemical and technological meat quality parameters. Concentrations of precursors of HCAs, i.e., creatine, residual glycogen, dipeptides, and free amino acids, were analyzed in the raw meat, and the levels of some HCAs (4,8-DiMeIQx, MeIQx, PhIP, harman, and norharman) were then determined in fried meat patties prepared from these pigs. The RN genotype most affected technological meat quality parameters and the level of precursors of HCAs, especially the level of residual glycogen, where carriers of the RN(-) allele showed levels four times as high as those of noncarriers (75.3 +/- 2.6 compared with 17.2 +/- 2.4 micromol/g meat, least-squares means +/- SE). The increased level of residual glycogen resulted in about 50% lower amounts of total mutagenic HCAs in cooked meat compared with cooked meat from normal pigs. Fried meat from carriers of the RN(-) allele obtained darker crust color than meat from noncarriers. Feeding regime and sex did not significantly affect the chemical composition of the meat or the formation of HCAs.