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.

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

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

Table 1 Chemical Properties of Acrylamide

Synonyms: 2-Propenamide; ethylene carboxamide; acrylic amide; vinyl amide

Molecular weight: 71.09

Chemical Formula: CH2CHCONH2

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 – inter-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

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)

Crisp bread/thin unleavened bread 21 300 135 <30–1900

Coffee (medium roast; mixed brands; as ready for consumption) 2 25 25 25

Cauliflower gratin (restaurant) <30

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)

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)

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

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

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