ACRYLAMIDE IN FOOD chemical structure

bài báo này viết về cấu trúc của arylamide và điều kiện hình thành arylamide. cách làm giảm sự hình thành đó.

Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 723 ISSN: 1579-4377 ACRYLAMIDE IN FOOD (Chemical Structure of Acrylamide) Semih Ötles 1 and Serkan Ötles 2 1 Ege University, Food Engineering Department, Izmir, Turkey 2 Izmir Institute of Technology, Chemical Engineering Department, Izmir, Turkey KEYWORDS Acrylamide, food ABSTRACT Acrylamide is a versatile organic compound that finds its way into many products in our everyday life. Acrylamide is a monomer of polyacrylamide. The monomer with form of acrylamide is toxic to the nervous system, a carcinogen in laboratory animals and a suspected carcinogen in humans. The multiple unit or polymeric form is not known to be toxic. The monomeric acrylamide is primarily used in research laboratories for gel preparation. The acrylamide gel is used for electrophoresis, a technique for protein separation. It is also used to produce grout, dyes, contact lenses and in the construction of dams, tunnels and sewers. Acrylamide polymers are used as additives for water treatment, flocculants, paper making aids, thickeners, soil conditioning agents, textiles (permenant – press fabrics), production of organic chemicals and ore and crude oil processing. Although polyacrylamide is not toxic, a small amount of the acrylamide monomer may leach from the polymer. Acrylamide and its analogues have been widely used since the last century for various chemical and environmental applications and can be formed by heating of biological material derived from plant tissues. This compound, identified previously as a potential industrial hazard, has now been found in many cooked foods. Reports of the presence of acrylamide in a range of fried and oven-cooked foods have caused world-wide concern because of its probable carcinogenicity in humans. INTRODUCTION Acrylamide and its analogues have been widely used since the last century for various chemical and environmental applications. Some of the common uses of acrylamide are in the paper, dyes, cosmetics and toiletry industry. It is produced commercially as an intermediate in the production and synthesis of polyacrylamides. Acrylamides have also been used as flocculants for clarifying drinking water and for waste water treatment. They are also a component of tobacco smoke, which gave the earliest indication that it can be formed by heating of biological material. Extensive studies have been done on acrylamide on its mutagenicity and carcinogenicity in bacterial, animal and human systems. Acrylamide has been shown to be non – mutagenic in Salmonella – microsome test systems. Acrylamide is known to produce neuropathy in both human and experimental animals. Ötles et al. EJEAFChe, 3 (5), 2004. [723-730] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 724 There are some analysis methods to be able to see the levels of acrylamide in food stuff. They are sampling, extraction, gas chromatography/mass spectrometry, liquid chromatography/tandem mass spectrometry and identification of acrylamide. The health impairment caused by acrylamide hinges on its carcinogenic and genotoxic impact. Acrylamide causes cancer in animals. While there are no scientific reasons to doubt this risk in humans, in principle, it cannot be reliably estimated, at present, how high the risk of contracting cancer is in humans after the intake of acrylamide-containing foods (Anon., 2004a-d). In this report, it is aimed to investigate the analysis methods of acrylamide, acrylamide’s health damaging properties and its exposure and also to investigate the what acrylamide consumers can do. WHAT IS ACRYLAMIDE? Acrylamide is a versatile organic compound that finds its way into many products in our everyday life. Acrylamide exists in two forms as a monomer and polyacrylamide as a polymer. The single unit form of acrylamide is toxic to the nervous system, a carcinogen in laboratory animals, and a suspected carcinogen in humans. The multiple unit or polymeric form is not known to be toxic (Giese, 2002; Konings et al., 2003; Richmont and Borrow, 2003; Tyl and Crump, 2002; Vattem and Shetty, 2003). The Chemical Structure of Acrylamide Acrylamide is a chemical intermediate (monomer) used in the synthesis of polyacrylamides. This monomer occurs in a white flowing crystalline form it is odourless, flake – like crystals, it is soluble in water, ethanol, methanol, dimethyl ether and acetone, it is not soluble in heptane and benzene. Figure 1, shows the chemical structure of acrylamide. Figure 1. Structure of Acrylamid It readily polymerises on reaching melting point or exposure to UV light. Solid acrylamide is stable at room temperature, but may polymerise violently when melted or exposed to oxidating agents. Table 1, shows the chemical properties of acrylamide. Ötles et al. EJEAFChe, 3 (5), 2004. [723-730] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 725 Table 1. Chemical Properties of Acrylamide Synonyms: 2-Propenamide; ethylene carboxamide; acrylic amide; vinyl amide CAS No: 79-06-1 Molecular weight: 71.09 Chemical Formula: CH 2 CHCONH 2 Boiling Point: 125 ° C Melting Point: 87.5 ° C (183 ° F) Application Areas of Acrylamide Acrylamide is used in the manufacture of plastics, including some food packaging, and in the production of synthetic rubber and some copolymers also it is used in water purification. When added to water, it coagulates and traps suspended solids that can then be easily removed during the treatment of drinking water. Acrylamide does not bind to soil but is degraded by micro-organisms within a few days in soil and water. The acrylamide that does not coagulate remains in the water as a contaminant that, under U.S. Environmental Protection Agency regulations, must be present at less than half a part per billion (0.5 ppb). The monomeric acrylamide is primarily used in research laboratories for gel preparation (Giese, 2002; Simonne and Archer, 2002). The acrylamide gel is used for electrophoresis, a technique that uses flat gels of polyacrylamide to separate and isolate DNA and other bio – molecules. It is also used to produce grout, dyes, contact lenses, and in the construction of dams, tunnels, and sewer. Acrylamide polymers are used as flocculants, paper making aids, thickeners, soil conditioning agents, textiles (permanent-press fabrics), production of organic chemicals, and ore and crude oil processing. Because of the large application areas of acrylamide, the annual production of acrylamide in the EU is 80,000 to 100,000 tonnes (Simonne and Archer, 2002). METHODS OF ANALYSIS By current standards of analytical science, the recent findings of acrylamide in foodstuffs are reliable. None of the methods used to measure acrylamide in foodstuffs has yet been fully validated by inter- laboratory collaborative trials. However, most methods fulfil the requirements of single – laboratory (“in- house”) validation and accreditation. (AOAC, 2002; Simonne and Archer, 2002; Tareke et al., 2000) Sampling Levels of acrylamide can vary considerably in foods, seemingly due to the processing or cooking conditions used and the temperature achieved. Consequently, there can be considerable variability from product to product and even concentration hot-spots within an individual food item. The whole food item or package should be homogenised before sub – sampling and a representative portion taken for analysis. For the foodstuffs investigated to date, there have been no problems reported of significant losses during storage and homogenisation of the sample prior to analysis (Tareke et al., 2000). According to the Swedish Food Administration, fried potato products and bread contributed most to the exposure of acrylamide. For all foods selected, information on the most used brands sold and on market shares is obtained from commodity boards and from a commercial market research agency. On the basis of market shares, the most current brands are selected. For each brand, three different production codes are sampled. In case of private labels, some different sales locations with the highest market shares are sampled. If acrylamide concentrations in foods are supposed to be subject to regional variations, like bread and chips from fish and chips stands, samples are collected from the most important sales-channels in three regions. All samples are homogenised before analysis. Secondly, to investigate acrylamide levels in other food sources, single samples are selected from foods, which are exposed to heat during industrial Ötles et al. EJEAFChe, 3 (5), 2004. [723-730] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 726 processing. If acrylamide appeared to be present in these samples, more brands and production codes are analysed (Tareke et al., 2000). Table 2. Acrylamide Levels (µg/kg) in Various Foodstuffs (Svensson et al., 2003) Food/Product Group Nº of Samples Mean Median Min-max Potato crisps 11 1360 980 330–2300 French fries 7 540 410 300–1100 Fried potato/baked products 8 310 300 34–688 Cookies/biscuits/wafers 11 300 230 <30–640 Crisp bread/thin unleavened bread 21 300 135 <30–1900 Bread 21 50 40 <30–160 Breakfast cereals 14 220 100 <30–1400 Tortilla crisps 3 150 150 120–180 Popcorn 3 500 390 365–715 Coffee (medium roast; mixed brands; as ready for consumption) 2 25 25 25 Wholemeal .our 1–2 <30 Rye flour <30 Oat flakes/rolled <30 Oat-bran <30 Oatmeal porridge <30 Gruel; instant <30 Gruel; oat based instant <30 Gruel for adults; instant <230 Potato (boiled) <30 Spaghetti (mixed products) <30 Rice (mixed products) <30 Rice (boiled + microwave) <30 Pancake (mixed products) <30 Waffles (mixed products) 42 Fish fingers (mixed products) 30 Chickenbits (restaurant) 39 Deep fried fish (restaurant) 39 Meat balls (mixed products) 64 Bacon (raw) <30 Eggs; fried <30 Cauliflower gratin (restaurant) <30 Vegetarian schnitzel <30 Taco shells (+ microwave) <30 Danish pastry <30 Pistachio garland bun <30 Soft biscuit <30 Soft ginger biscuit <30 Sponge cake <30 American muffins <100 Some scientists made an experiment in University of Stockholm in 2002. More than 130 samples were collected from supermarkets in Uppsala in the spring of 2002. The analytical survey comprised crisp bread, bread, flour, pasta, rice, fish, chicken, bacon, meatballs, sausages, vegetarian schnitzels, eggs, biscuits, cookies, Danish pastries, buns, muffins, breakfast cereals, porridge, gruel and coffee and ready prepared meals such as pizza, pancakes, waffles and products made of potatoes (French fries, potato crisps and fried potatoes) or corn. Table 2 lists the results of the analytical survey (Svensson et al., 2003). Ötles et al. EJEAFChe, 3 (5), 2004. [723-730] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 727 Extraction Free acrylamide is extracted from the sample using cold or hot water. It is demonstrated, by adding known amounts of acrylamide standard to the sample before extraction, that these extraction procedures give complete recovery. Many sample extracts can be analyzed directly, however some sample types benefit from further cleanup and concentration of the extract. It is desirable to add an internal standard to the food sample at the outset, as an internal standard compensates for any recovery losses in these steps and helps to ensure that results are reliable (Chakrabarti and Ungeheuer, 2002; Tareke et al., 2000). In an experiment, samples were homogenised and analysed fresh or stored at 20° C until analysis. To homogenised samples (4 g), water (40 ml) and an internal standard (deuterium labelled acrylamide) were added. Samples were extracted by means of a homogeniser, centrifuged and filtered on solid phase extraction columns. The filtrates were collected and passed through a centrifuge spin filter until a sufficient volume had been obtained for analysis with LC–MS–MS (Chakrabarti and Ungeheuer, 2002; Tareke et al., 2000). Analysis Gas chromatography/mass spectrometry (GC-MS) Although acrylamide can be analysed as such, without derivatization, when using GC-MS, the molecule is normally brominated to form a derivative that has improved GC properties. The acrylamide derivative is identified by its retention time and by the ratio of characteristic MS ions. Once the identity of acrylamide has been established in a particular type of food, it may be possible to use gas chromatography with electron capture detection (ECD) or other selective detection techniques to routinely monitor levels, although with this analytical technique the identification rests on the retention time alone. The lowest level that can be measured when using GC-MS is in the range of 5 to 10 µg/kg (Chakrabarti and Ungeheuer, 2002) Liquid chromatography/tandem mass spectrometry (LC-MS-MS) The LC–MS–MS system consisted of a triple quadrupole. Because there are concerns about possible artefact formation during the bromination procedure, LC-MS-MS methods are developed for the direct analysis of acrylamide without the need to derivatize. Identification of the substance is by its retention time and by the relative ion intensities. The limit of measurement using LC-MS/MS is about 20 to 50 µg/kg. (Chakrabarti and Ungeheuer, 2002) In the experiment they prepared extracts as described before, were analysed according to the method developed by Rose´ n and Hellena¨ s, using liquid chromatography tandem mass spectrometry with a graphitised carbon column, water as mobile phase, and a triple quadrupole. The overall method was validated for a concentration range of 30–10 000 mg kg_1 (accuracy 91–102%, relative standard deviation 3–21%) (Chakrabarti and Ungeheuer, 2002; Tareke et al., 2000) Identification of Acrylamide When the same food sample is extracted and analysed by both methods describe, there is generally excellent agreement between the results and the putative acrylamide fulfils the identification criteria in both techniques. This provides adds confidence in the qualitative and quantitative results to date. By modern standards of analytical evidence, the identification of acrylamide in foodstuffs is highly reliable (Chakrabarti and Ungeheuer, 2002) Ötles et al. EJEAFChe, 3 (5), 2004. [723-730] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 728 HEALTH – DAMAGING PROPERTIES OF ACRYLAMIDE The health impairment caused by acrylamide hinges on its carcinogenic and genotoxic impact. Acrylamide causes cancer in animals. While there are no scientific reasons to doubt this risk in humans, in principle, it cannot be reliably estimated, at present, how high the risk of contracting cancer is in humans after the intake of acrylamide-containing foods. In principle, the so-called Alara Principle, i.e. as low as reasonably achievable, applies to genotoxic and carcinogenic substances. The Scientific Committee on Food of the European Commission also raised this claim in its opinion on acrylamide in foods dated 3 July 2002. Due to insufficient data, the fixing of limits is currently neither toxicologically justifiable nor technically realisable (Chakrabarti and Ungeheuer, 2002; Mottram et al., 2002; Raloff, 2002). In 2002, The Uppsala Ethical Committee had an animal experiment to be able to see the health – damaging properties of acrylamide. Male CBA-CA mice, aged 7–8 weeks and weighing approximately 20–25 g, were bought from B & K Universal, Sollentuna, Sweden. In the first experiment the mice were kept in an animal house at EBC, Uppsala University. In the second experiment they were kept at the locality of the National Food Administration, Sweden. The mice were allowed free access to solid food and tap water, and were provided with 12 h of light and 12 h darkness. Temperature and humidity were correctly adjusted to be optimal for the animals (Zetterberg, 2003). The investigation is divided in two separate experiments. In the first experiment, the studied dose interval was 0–100 mg/kg b.w. In the second experiment, the studied dose interval was 0–30 mg/kg b.w. In the first experiment, 49 male CBA mice were involved in this experiment. All animals were intraperitoneally injected once. The injection volume was 10 ∝l/g of mouse. Three mice constituted the positive control group and were given a dose of 1 mg/kg b.w. of colchicine dissolved in PBS. The other 46 mice were divided into 22 groups with different doses of AA. AA was dissolved in PBS. Figure 2. The Frequency of Micronucleated of Male CBA Mice Given 22 Different Doses (single injections) of Acrylamide (Zetterberg, 2003) In the second experiment, 35 male CBA mice were treated with different doses of AA. The procedure was identical to that in Experiment 1, but the doses were 0, 1, 3, 6, 12, 24, and 30 mg/kg b.w., with five animals in each of the groups. The injection volumes were the same as in Experiment 1. Ötles et al. EJEAFChe, 3 (5), 2004. [723-730] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 729 After injection, blood samples were collected under light anaesthesia with fluothane from the orbital plexus of each animal at 42 h after the injections (the same procedure in both experiments). Directly after the collection of blood, the animals were killed by cervical dislocation. The results from the analysis in peripheral blood (fMPCE) are shown in figure 2. In Experiment 1, each value represents the mean of two animals, except for the control group: 0 and 75 mg/kg b.w. group (three animals each). From each AA-treated animal, in general 280,000 PCE were analysed. Due to the production of cell doublets, some of the parallel samples were excluded from the analysis, resulting in less than four parallel samples from some of the mice. In Experiment 2, each mean value represents five animals treated with the same AA dose. From each animal, in general 350,000 PCE were analysed. In both experiments, the frequency of PCE in peripheral blood was found to be almost the same for all AA dose groups which indicated that no depression of the cell proliferation occurred at these doses. No sign of sickness or decreased activity of the mice was noticed during the experiments. DISCUSSION Acrylamide concentrations for different food items or food groups were determined by various authors. In some cases the foodstuffs analysed are well characterized, but in other cases the results were assigned to a product group and no further details regarding the specific food item were provided. Therefore additional studies on the acrylamide levels in specific foods on the country base are necessary. REFERENCES 1. Anonymous.2004a. http://www.cfsan.fda.gov/~dms/acryposn/sld001.html 2. Anonymous.2004b. www.cfsan.fda.gov/~dms/acrylami.html 3. Anonymous.2004c. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=52015 4. Anonymous.2004d. http://who.int/fsf/Acrylamid_Summaryreport.pdf 5. AOAC International, Inside Laboratory Management 2002; 5 : 8 – 9 6. Chakrabarti T, Ungeheuer, P. Health Implications of Acrylamide in Food, Food Safety. Report of a Joint FAO/WHO Consultation. FAO, Geneva 2002 : 1-34 7. Giese, J. Acrylamide in Foods. Food Technology 2002; 56(10), 71-72 8. Konings, E, Baars, A, van Klaveren, D, Spanjer, M, rensen, P, Hiemstra, M, van Kooij, J, 9. Peters, P. Acrylamide Exposure from Foods of the Dutch Population and an Assessment of the Consequent Risks. Food and Chemical Toxicology 2003; 41 : 1569-1579 10. Mottram, DS, Wedzicha, BL, Dodson, A. Acrylamide is formed in the Maillard reaction. Nature 2002; 419 : 448-449 11. Raloff, J. Launches Acrylamide Investigations. Science News 2002; 162 : 15 12. Richmond, P, Borrow, R. Acrylamide in Food. The Lancet 2003; 361(2) : 361-362 13. Simonne, H, Archer, L. Acrylamide in Foods: A review and Update. University of Florida Extension 2002; 10: 1-3 Ötles et al. EJEAFChe, 3 (5), 2004. [723-730] Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377 730 14. Svensson, K., Abramsson, L., Becker, W., Glynn, A., Hellenas, K., Lind, Y., Rosen, J. Dietary intake of acrylamide in Sweden. Food and Chemical Toxicology, 2003; 41: 1581-1586 15. Tareke, E, Rydberg, P, Karlsson, P, Ericksson, S, Törnquist, M. Acrylamide: A cooking carcinogen? Chem Res Toxicol 2000; 13 : 517-522 16. Tyl, R, Crump, K. Acrylamide in Food. Food Standards Agency 2003; 5 : 215-222 17. Vattem, A, Shetty, K. Acrylamide in Food: a Model for Mechanism of Formation and its Reduction. Innovative Food Science and Emerging Technologies 2003; 4: 331-338 18. Zetterberg, L.A. The dose-response relationship at very low doses of acrylamide is linear in the flow cytometer-based mouse micronucleus assay. Mutation Research 2003; 535: 215-222 . high the risk of contracting cancer is in humans after the intake of acrylamide- containing foods. In principle, the so-called Alara Principle, i.e. as low. The Chemical Structure of Acrylamide Acrylamide is a chemical intermediate (monomer) used in the synthesis of polyacrylamides. This monomer occurs in a