Acrylamide from Maillard Reaction Products

brief communications e-mail: [email protected] found that pyrolysing any of these amino a 10,000

†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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http://www.who.int/fsf/ 4. European Commission Scientific Committee on Food (SCF) Opinion of the Scientific Committee on Food on New Findings Alice Regarding the Presence of Acrylamide in Food Zugspitze (SCF/CS/CNTM/CONT/4 Final, 3 July 2002) (2,950 m) http://europa.eu.int/comm/food/fs/scf/index_en.html 5. Ledl, F. & Schleicher, E. Angew. Chem. Int. Ed. Engl. 29, Bob 565–594 (1990). Westliche 6. Paulsen, H. & Pflughaupt, H. in The Carbohydrates — S karwendespitze Chemistry and Biochemistry (eds Pigman, W. & Hortin, D.) (2,244 m) Vol. 1B, 881–927 (Academic, New York, 1980). 7. Von Euler, H. & Brunius, E. Chem. Ber. 59, 1581–1585 (1926). 8. Chen, J., Pill, T. & Beck, W. Z. Naturforsch. B 44, D(45°,0) 459–464 (1989). Alice Competing financial interests: declared none. 23.4 km M L' TF BSR PBS A A D(45°,1) Quantum cryptography D(0°,1)

A step towards global L PBS 4 LDs Fast pulse D(0°,0) Computer key distribution Computer generator Mobile phone Bob arge random bit-strings known as link ‘keys’ are used to encode and decode Lsensitive data, and the secure distribu- Figure 1 Overview of the experiment against a relief map of the trial site. In the Alice module, four separate lasers (LDs) encode the four tion of these keys is essential to secure com- polarizations based on a random bit-string fed from the Alice computer. They are combined in a spatial filter (A,A) using a conical mirror 1 munications across the globe . Absolutely (M) and a lens (L). The beam expands to 50 mm and is collimated in an output lens (L8). In the Bob module, a telescope (T) collects the secure key exchange2 between two sites has light, which is filtered (F) and then spilt in a polarization-insensitive beam-splitter (BS), passing on to polarizing beam-splitters (PBS) and now been demonstrated over fibre3 and four photon-counting detectors (D). One polarizing beam-splitter is preceded by a 45° polarization rotator (R). A click in one of the photon- 4–6 free-space optical links. Here we describe counting detectors D(u, B) sets the bit value B and the measurement basis u. the secure exchange of keys over a free- space path of 23.4 kilometres between two verify the security of the channel. Low error ‘trusted courier’ carrying a long random mountains. This marks a step towards rates due to background light detection and bit-string, the key, from one location to the accomplishing key exchange with a near- polarization settings are securely eliminated other. Our experiment paves the way for Earth orbiting satellite and hence a global by using classical error-correcting codes sent the development of a secure global key- key-distribution system. over the mobile-telephone link. distribution network based on optical links The security of our key-exchange system In the long-range experiment, Alice was to low-Earth-orbit satellites. We note that a is guaranteed by encoding single photons located at a small experimental facility on 10-kilometre key-exchange experiment has using two sets of orthogonal polarizations. the summit of Zugspitze in southern recently been announced7. Our transmitter module (Alice; Fig. 1) Germany, and Bob was on the neighbour- C. Kurtsiefer*, P. Zarda*, M. Halder*, incorporates a miniature source of polariza- ing mountain of Karwendelspitze, 23.4 km H. Weinfurter*, P. M. Gorman†, tion-coded faint pulses (approximating away. At this distance, the transmitted P. R. Tapster †, J. G. Rarity† single photons; C.K., P.Z., M.H. and H.W., beam was 1–2 m in diameter and was *Ludwig-Maximilian University, 80799 Munich, unpublished results), where 0° or 45° polar- only weakly broadened by air-turbulence Germany ization encode binary zero, and 90° or 135° effects at this altitude. Lumped optical loss- †Photonics Department, QinetiQ, Malvern , code binary one. These light pulses are es of about 18–20 decibels were measured Worcestershire WR14 3PS, UK expanded and collimated in a simple and, using faint pulses containing 0.1 e-mail: [email protected] telescope to a beam of about 50 mm and photons per bit, the detected bit rate at 1. Singh, S. The Code Book (Anchor, New York, 1999). then accurately aligned on the receiver Bob was 1.5–2 kilobits per second (receiver 2. Bennett, C. H. et al. J. Cryptol. 5, 3–28 (1992). (Bob; Fig. 1), a 25-cm-diameter commercial efficiency of 15%). 3. Gisin, N., Ribordy, G., Tittel, W. & Zbinden, H. Rev. Mod. Phys. telescope. Light is collected and focused Operating at night with filters of 10-nm 74, 145–196 (2002). 4. Buttler, W. T. et al. Phys. Rev. Lett. 84, 5652–5655 (2000). onto a compact four-detector photon- bandwidth reduced the background 5. Rarity, J. G., Gorman, P. M. & Tapster, P. R. Electron. Lett. counting module (Fig. 1). A detection in counts, and errors appeared in less than 37, 512–514 (2001). any one detector then has an associated bit 5% of key bits. After sifting and error 6. Rarity, J. G., Gorman, P. M. & Tapster, P. R. J. Mod. Opt. value, measurement basis (0° or 45°) and correction, net key exchange rates were 48, 1887–1901 (2001). 7. Hughes, R. J., Nordholt, J. E., Derkacs, D. & Peterson, C. G. detection time. The bit values then form a hundreds of bits per second. In a series of New J. Phys. 4, 43.1–43.14 (2002). raw key string. Valid bits are measured in experiments, several hundreds of kilobits Competing financial interests: declared none. the same basis as that in which they were of identical key string were generated at encoded. Alice and Bob. Alice and Bob use a standard communi- In associated experiments in poorer visi- cations channel, such as a mobile telephone, bility, we showed that key exchange could to ascertain which bits arrived (many are be carried out when transmission losses erratum lost) and which measurement basis was used, were up to 27 decibels, but improvements Cognitive change and the APOE ;4 allele then they both discard the invalid bits — in receiver efficiency and background I. J. Deary, M. C. Whiteman, A. Pattie, J. M. Starr, which leaves them with nearly identical counts should take us beyond 33 decibels. C. Hayward, A. F. Wright, A. Carothers, L. J. Whalley random bit-strings, the sifted key. Eaves- With this performance, key exchange to Nature 418, 932 (2002) dropping measurements on the single near-Earth orbit (500–1,000 km range) In the second sentence of the seventh paragraph of this photons disturb the encoding and introduce should become possible. communication, the MMSE scores are incorrectly speci- errors of up to 25%, so Alice and Bob test for Until now, the principal method of fied as less than or equal to 28; these should read as errors in a short section of sifted key to high-security key exchange has been the greater than or equal to 28.

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