Acrylamide Is Formed in the Maillard Reaction

brief communications

*Institute for Zoo and Wildlife Research, Research Unit, 33612 Cestas cedex, France 185 °C, no acrylamide was detected (detec- Department of Evolutionary Genetics, #Hudson River Foundation for Science and tion limit, 0.5 mg mol11). Glutamine and 10252 Berlin, Germany Environmental Research, New York, aspartic acid gave only trace quantities of e-mail: [email protected] New York 10011, USA acrylamide (0.5–1 mg mol11).

†Institute of Zoology, University of Rostock, 1. Ludwig, A., May, B., Debus, L. & Jenneckens, I. Genetics 156, When a dry mixture of asparagine and 18051 Rostock, Germany 1933–1947 (2000). glucose was reacted at 185 °C (that is, ‡Department of Environmental Medicine, 2. King, T. L., Lubinski, B. A. & Spidle, A. P. Conserv. Genet. 2, without buffer solution), only 25 mg mol11 103–119 (2001). New York University School of Medicine, Tuxedo, 3. Brown, J. T., Beckenbach, A. T. & Smith, M. J. Mol. Biol. Evol. acrylamide was formed. Although the dry New York 10987, USA 10, 326–341 (1993). reaction is a realistic system with which to §German Archaeological Institute, 4. Magnin, E. Le Naturaliste Canadien 91, 5–20 (1964). 5. Artyukhin, E. & Vecsei, P. J. Appl. Ichthyol. 15, 35–37 (1999). simulate the later stages of baking and toast- Eurasian Division, 14195 Berlin, Germany 6. Borodin, N. Trans. Am. Fish. Soc. 55, 184–190 (1925). ing of food, it is less efficient because the ||Institute for Animal Breeding and Genetics, 7. Quantz, H. Mitt. Dt. Seefischerei-Vereins 19, 176–204 (1903). reactants are incompletely mixed in the Supplementary information accompanies this communication on University of Göttingen, 37075 Göttingen, Germany Nature’s website. absence of a solvent. Trace quantities of acryl- ¶Cemagref, Inland Living Aquatic Resources Competing financial interests: declared none. amide were produced under these conditions from glutamine and aspartic acid, but not from any of the other amino acids apart from methionine, which yielded 5 mg mol11. Food chemistry these intermediates (Fig. 1), in which the To test for the involvement of Strecker amino acid is decarboxylated and deami- degradation in the the production of acryl- Acrylamide is formed in nated to form an aldehyde. amide, we used 2,3-butanedione instead of We investigated whether this reaction glucose in these reactions (butanedione is the Maillard reaction could provide a possible route to acrylamide. one of several dicarbonyl compounds eports of the presence of acrylamide The amino acid asparagine should be a formed in the Maillard reaction). Acryl- in a range of fried and oven-cooked particularly suitable reactant as it already has amide was produced when asparagine was Rfoods1,2 have caused worldwide con- an amide group attached to a chain of two allowed to react with butanedione both in a cern because this compound has been carbon atoms. We therefore performed a dry system (40 mg mol11) and in buffer classified as probably carcinogenic in series of Maillard reactions between glucose (63 mg mol11). Heating asparagine on its humans3. Here we show how acrylamide and asparagine, as well as with other amino own at 185 °C did not produce acrylamide, can be generated from food components acids that do not have the correct carbon confirming the requirement for the dicar- during heat treatment as a result of the backbone for acrylamide (Fig. 1). bonyl reactant and Strecker degradation. Maillard reaction between amino acids Significant quantities of acrylamide Again, there was no significant prod- and reducing sugars. We find that (221 mg per mol of amino acid) were uction of acrylamide in either system asparagine, a major amino acid in pota- found when an equimolar mixture of from butanedione and the other amino toes and cereals, is a crucial participant asparagine and glucose was reacted at acids, with the exception of methionine in the production of acrylamide by this 185 °C in phosphate buffer in a sealed glass (6 mg mol11 in the dry system). The Streck- pathway. tube. The temperature dependence of acryl- er aldehyde formed from methionine is Products of the Maillard reaction are amide formation from asparagine indicates methional, but acrolein can also be formed, responsible for much of the flavour and that this is favoured above 100 °C and that together with ammonia: subsequent oxida- colour generated during baking and roast- very high temperatures are not necessary tion of acrolein to acrylic acid followed by ing. An important associated reaction is the (Fig. 2). In similar reactions with glucose amidation could then generate acrylamide Strecker degradation of amino acids by and glycine, cysteine or methionine at (Fig. 1). However, this reaction might be limited by its requirement for ammonia, R 500 O R Z N 1 which reacts readily with carbonyls and ZCHNH C 1 C C 2 – H2O H other Maillard intermediates.

+ ) C C C C R R –1 400 O OH O 2 O O O 2 The almost exclusive formation of acryl- Amino acid Dicarbonyl amide from asparagine could explain the compound H – CO2 300 occurrence of acrylamide in cooked plant- H N 2 R1 Z based foods, such as cereals and potato, CH Amino C N R 200 4 C 1 which are rich in this particular amino acid . C ketone H In potato used for the manufacture of potato O R2 C H O R2 Acrylamide (mg mol 100 + H O crisps, the dominant free amino acid is 2 11 H R CO CO R + asparagine (940 mg kg , representing 40% 1 2 5 Z C Strecker 0 of the total amino-acid content ); in wheat aldehyde NH3 + CH3 SH + O 100 120 140 160 180 200 11 CHO flour it is present at 167 mg kg , corre- CH2 CH Temperature (°C) Acrolein sponding to 14% of the total free amino Figure 2 Temperature-dependent formation of acrylamide (mg acids (our unpublished results), and a high- H2N CH2 CH COOH 11 C CH CHO per mol of amino acid) from asparagine (0.1 mmol) and glucose protein rye variety contains 173 mg kg 2 ? NH3 6 O (0.1 mmol) in 0.5 M phosphate buffer (100 m l, pH 5.5) heated in (18% of the total free amino acids) . CH CH COO– NH + ? 2 4 a sealed glass tube for 20 min. Error bars represent standard Our findings indicate that Maillard H N 2 deviations (n43). Acrylamide produced in the reaction was reactions involving asparagine can produce C CH CH2 O extracted with ethyl acetate and analysed by gas chromatography acrylamide and might explain the increased Acrylamide with mass spectrometry after derivatization to 2,3-dibromo- concentrations of acrylamide in certain Figure 1 Proposed pathways for the formation of acrylamide after propanamide7, using 2-methylacrylamide as the internal standard. plant-derived foods after cooking. Strecker degradation of the amino acids asparagine and methion- Selected ion monitoring was used to detect the analytes, with Donald S. Mottram*, Bronislaw L. ine in the presence of dicarbonyl products from the Maillard m/z 150 and 152 for acrylamide and m/z 120 and 122 for methyl- Wedzicha†, Andrew T. Dodson* reaction. In asparagine, the side chain Z is –CH2CONH2; in acrylamide. The presence of acrylamide in selected samples was *School of Food Biosciences, The University of methionine, it is –CH2CH2SCH3. confirmed in full mass spectra. Reading, Whiteknights, Reading RG6 6AP, UK

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†Procter Department of Food Science, University of acids (Asn, Gln, Met, Cys) with an equimolar ) Leeds, Leeds LS2 9JT, UK amount of D-fructose, D-galactose, lactose or 1,000

sucrose all led to a significant release of acryl- ed ino acid ino 1. Rosen, J. & Hellenas, K.-E. Analyst 127, 880–882 (2002). rm 100

2. Tareke, E., Rydberg, P., Karlsson, P., Eriksson, S. & Törnqvist, M. amide, with comparable yields from each fo

l am l o J. Agric. Food Chem. 50, 4998–5006 (2002). sugar. No acrylamide was detected when any id 3. IARC IARC Monographs on the Evaluation of Carcinogenic Risks 10

of these carbohydrates was heated alone. m er

to Humans 60, 389 (1994). l p l

To test whether early Maillard products crylam o 4. Belitz, H.-D. & Grosch, W. Food Chemistry (Springer, A 1

New York, 1999). such as N-glycosides could be acrylamide m µ 5. Martin, F. L. & Ames, J. M. J. Agric. Food Chem. 49, ( precursors in thermal decomposition reac- 0.1 3885–3892 (2001). tions, we measured the yields of acrylamide 510203060 6. Dembinski, E. & Bany, S. J. Plant Physiol. 138, 494–496 (1991). Time (min) at 180 °C 7. Castle, L. J. Agric. Food Chem. 41, 1261–1263 (1993). after pyrolysis (ti420 min, 180 °C) of Competing financial interests: declared none. 0.2 mmol of four different N-glycosides b OH (Fig. 1b). Yields were significant (in m mol O HO per mol N-glycoside: compound 1, 1 HO 2 NH NH2 1,3055323; 2, 1,4195278; 3,1452.7; and 4, OH Food chemistry 8.151.5) and comparable to those released CO2K O from the amino-acid and reducing-sugar 1 Acrylamide from Maillard precursors under the same O conditions. Furthermore, compound 1 was HO reaction products HO 2 NH NH confirmed as an intermediate in the OH 2 he discovery of the adventitious for- asparagine/glucose reaction by high-reso- 1 CO K O HO 2 mation of the potential cancer-causing lution mass-spectrometric analysis of a 2 agent acrylamide in a variety of foods methanol extract of the pyrolysate. T OH during cooking1,2 has raised much concern3,4, On the basis of structural considera- O O but the chemical mechanism(s) governing tions, asparagine or the N-glycosides 1 and HO 1 HO 2 NH its production are unclear. Here we show 2 could be direct precursors of acrylamide OH NH that acrylamide can be released by the under pyrolytic conditions. Condensation 2 13 CO2K thermal treatment of certain amino acids of asparagine with C6-labelled glucose 3 (asparagine, for example), particularly in confirmed that the amino acid is the carbon OH combination with reducing sugars, and of source of acrylamide. Upon pyrolysis, for- O HO early Maillard reaction products (N-glyco- mation of the corresponding N-glycoside 1 HO 2 NH S sides)5. Our findings indicate that the probably facilitates the decarboxylation OH Me Maillard-driven generation of flavour and step and heterolytic cleavage of the nitro- CO2K colour in thermally processed foods can — gen–carbon bond to liberate acrylamide 4 under particular conditions — be linked to (CH25CHCONH2). Although decarboxyla- Figure 1 Production of acrylamide from N-glycosides. a, Loga- the formation of acrylamide. tion is favoured at higher temperatures, the rithmic-scale plot of the formation of acrylamide over time in We heated 20 amino acids individually N-glycosidic bond seems to facilitate the pyrolysates of glucose with glutamine (triangles), asparagine at 180 °C for 30 min and found that acryl- deamination step. (squares) or methionine (circles). Each data point represents the amide is formed under these conditions Further evidence to support this pathway average of n43 independent determinations; the coefficient of from methionine and from asparagine to acrylamide production is provided by the variation was less than 25%. For acrylamide analysis (by liquid (3.651.4 and 0.5650.05 µmol acrylamide 98.6% incorporation of nitrogen-15 label chromatography coupled to electrospray ionization tandem mass 15 13 per mol amino acid, respectively; all results into acrylamide after the pyrolysis of N- spectrometry), pyrolysates were supplemented with C3-acryl- are averages of n46 independent determi- amide-labelled asparagine with glucose; there amide (50 ng), then suspended in hot water (more than 90 7C), nations unless stated otherwise). was no incorporation into acrylamide when sonicated and filtered before being applied to a solid-phase When pyrolysed at 180 °C with an 15N-a-amino-labelled asparagine was used in extraction cartridge (OASIS HLB, 0.2 g). Acrylamide eluted with equimolar amount of glucose, asparagine in the same reaction. Results from similar iso- 20% methanol was separated on a Shodex RSpak DE-613 particular generates significant amounts of tope-labelling experiments (not shown) to polymer column with isocratic solvent flow. Detection by mass acrylamide, reaching an average of determine the route of acrylamide formation spectrometry was in the multiple-reaction monitoring mode with 11 368 m mol mol after an incubation time (ti) from different N-glycosides produced by glu- the characteristic fragmentation transitions for acrylamide (m/z of 30 min. If asparagine monohydrate was cose pyrolysis with glutamine or methionine 72➝55, 72➝27, 72➝54) and confirmed by ion ratios (55/54 used in the incubation or water was added to are less clear-cut, which suggests that other and 55/27). Further details are available from the authors. the reaction (0.05 ml) before thermolysis, the pathways (such as that for homolytic cleav- b, Chemical structures of the potassium salts of N-(D-glucos-1-yl)- release of acrylamide was enhanced nearly age) might also lead to acrylamide. L-asparagine (1), N-(D-fructos-2-yl)-L-asparagine (2), N-(D-glucos- 11 threefold (9605210 m mol mol ), or over The N-glycosidic bond is labile in the 1-yl)-L-glutamine (3) and N-(D-glucos-1-yl)-L-methionine (4). 1,700 times the amount formed from presence of water6 or under acidic and neu- asparagine alone under the same conditions. tral pH conditions7, hydrolysing rapidly to potential progenitors of acrylamide. Reaction of methionine and glutamine the reducing sugar and amino acid. At higher Richard H. Stadler, Imre Blank, Natalia with equimolar amounts of glucose at pH, however, N-glycosides can be isolated as Varga, Fabien Robert, Jörg Hau, Philippe A. 180 °C also increased the formation of bimolecular complexes in the presence of Guy, Marie-Claude Robert, Sonja Riediker acrylamide, which occurred rapidly in each polyvalent alkaline or transition-metal ions8. Nestlé Research Center, Nestec, Vers-chez-les-Blanc, case (ti45 min; Fig. 1a). Cysteine was found In food-processing systems that incorporate 1000 Lausanne 26, Switzerland to liberate acrylamide after condensation conditions of high temperature and water e-mail: [email protected] with glucose (2.050.8 m mol mol11 at loss, N-glycoside formation could be 1. Swedish National Food Agency website http://www.slv.se ti430 min and 180 °C). favoured; when this condensation occurs 2. Rosén, J. & Hellenäs, K.-E. The Analyst 127, 880–882 (2002). Investigating the role of different carbo- between reducing sugars and certain amino 3. WHO FAO/WHO Consultation on the Health Implications of hydrates in the formation of acrylamide, we acids, a direct pathway is opened up to Acrylamide in Food (Geneva, 25–27 June 2002)

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