Current Status on Acrylamide Research in Food - Analytics, Formation, Toxicity -

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Charleston, August 29 September 01, 2004

1 Current Status on Acrylamide Research in Food - Analytics, Formation, Toxicity - AA = f(c, T, a W, t, p, p) 8 th International Symposium on the Maillard Reaction, Charleston, August 29 September 01, 2004 Imre Blank estlé Research Centre, Vers-chez-les-Blanc, Switzerland

2 ow it started Swedish ational Food Agency (Tareke et al., JAFC, April 2002): igh concentrations of acrylamide in highly heated, carbohydrate-rich foods, particularly in fried and baked foods like French fries, potato crisps and crisp bread. o acrylamide was found in raw and boiled foods. Molecule C 3 5, MW = Melting point 84.5 C Boling point 125 C (25 mm g) Solubility (water) g/l (30 C) Stability Polymerization above m.p. Reactivity Reacts with acids, bases, oxidizing agents Use Exposure Polyacrylamide production (clarifying water, gels for laboratories, soil stabilization, etc.) Dermal adsorption (solution), inhalation (dry monomer, aerosol), ingestion (food) 2

3 Scope Progress in Safety Assessment Toxicity Dietary intake Progress in Analytics: Analytical methods ccurrence in food Progress in Formation and Mitigation Mechanisms and precursors Food chemistry and physics Reduction of acrylamide Conclusions and utlook 3

4 S 1 Reactivity and metabolism 2 CP2E1 2 Epoxidation to glycidamide (plasma half life of 2 h, oral administration in rat). Reaction with glutathione (Michael-type nucleophilic addition), excreted via the urine. AA and glycidamide form covalent adducts with DA and proteins (e.g. haemoglobin biomarkers). nly glycidamide-da adducts identified in vivo. 4 (Friedman, J. Agric. Food Chem., 2003)

5 S 2 Toxic effects 2 CP2E1 2 eurotoxicity Acrylamide is a human neurotoxin, described for occupational exposure. Mechanism: Disruption of axonal transport and interference with synaptic transmission. AEL = 0.2 mg/kg bw/day, LAEL = 1.0 mg/kg bw/day (rat subchronic drinking water study). (agmar et al., 2001) 5

6 S 3 Toxic effects 2 CP2E1 2 Mutagenicity AA induces gene mutations and chromosomal aberrations in germ cells, somatic cells, cultured cells in vitro and induces cell transformation in mouse cell lines. (Dearfield et al., Mut. Res., 1995) nly glycidamide is mutagenic in bacterial test systems. Dietary intake too low to cause damage (epidemiological study needed). Cancerogenicity AA produces cancer in various organs (mammary gland, testis, thyroid) at high concentrations (1-2 mg/kg bw/day) in long-term exp. animal studies. o link between dietary acrylamide and some cancers in humans. (Mucci et al., Brit. J. Cancer, 2003; Int. J. Cancer, 2004; Pelucchi et al., Int. J. Cancer, 2003) Teratogenicity AA is not teratogenic. 6

7 S 4 Exposure Low level human exposure to AA via food from food packaging and polyacrylamide treated drinking water. igher exposures documented in occupational settings in the chemical industry. Low dietary exposure μg/kg bw/day (FA/W) 0.43 μg/kg bw/day (FDA/TP) 0.5 μg/kg bw/day (Svensson et al.) o ADI fixed Average exposure 35 μg/day owever, exposure to young individuals (13-18 years) by a factor of 3 higher. 7 (Svensson et al., Food Chem. Toxicol., 2003)

8 S 5 Safety assessment: Current status Classification Group 2A carcinogen = probably carcinogenic to humans (International Agency for Research on Cancer) Category 2 carcinogen and category 2 mutagen (EU) o link between dietary acrylamide and some cancers. (Mucci et al., Brit. J. Cancer, 2003; Pelucchi et al., Int. J. Cancer, 2003) eurotoxic with LAEL = 1.0 mg/kg bw/day o neurotoxic effects to be expected from dietary AA (Dybing & Sanner, Toxicol. Sci., 2003) W o recommendation for change in dietary habits Food should not be cooked excessively Balanced and varied diet recommended 8

9 A 1 Quantification by GC-MS and LC-MS Review: Wenzl et al., Food Add. Contam. 20, (2003) Approach: Use of internal standards labeled with stable isotopes (e.g. 13 C 3 -AA, 2 3 -AA) Isotope Dilution Assay GC-MS(CI) Methods: GC-MS, usually after derivatization, e.g. bromination (Castle et al., 1991) e.g. silylation (Lagalante & Felter, 2004) LC-MS, usually w/o derivatisation (Becalski et al., 2003; Riediker & Stadler, 2003; Jezussek & Schieberle, 2003) 9

10 A 2 Typical amounts of acrylamide in food Food AA (μg/kg) Median Food AA (μg/kg) Potato crisps French fries Fried potato Biscuits, wafers < Crisp bread < Breakfast cereals < Popcorn Bread < Coffee powder Boiled potato <30 Boiled rice <30 Meat balls <30 Potato crisps French fries Fried potato 1270 Biscuits, wafers Crisp bread Breakfast cereals Gingerbread Bread Coffee powder Snacks Beer, malt Fish products (Svensson et al., J. Agric. Food Chem., 2003) (Friedman, J. Agric. Food Chem., 2003)

11 A 3 Extraction and clean-up are critical steps Improved RC metod - Flow diagramme - Key Steps Protein precipitation 2 g (60 C, 10 ml 2 ) Ultra Turrax EtAc extract Carrez I & II (1 ml each) rganic layer Swirling Aqueous layer (6mL) (acl, 1.8g, EtAc, 13 ml) C 2 Cl 2 (5 ml) Stirring, Centrifugation Centrifugation Salting-out + 2 ml 2 Stirring, Evaporation, 2 2 x Extraction 2 Multimode (SPE, 0.5 g ) Elute 2 Conc. 40 C Eluate, Aliquote 180 ul + 90 ul Me SPE clean-up LC-ESI-MS/MS (Delatour et al., J. Agric. Food Chem., 2004) 11

12 A 4 Improved RC method (LC-MS/MS) (Delatour et al., 2004) 12

13 A 5 Cocoa powder (130 µg/kg) Transition m/z taken for quantitation LoD : 4.6 µg/kg LoQ : 7.9 µg/kg Repeatability (CV %) 15.8 (12.7 µg/kg) 6.1 (304 µg/kg) 5.4 (2504 µg/kg) Recovery (%): 43 (12.7 µg/kg) 54 (304 µg/kg) 51 (2504 µg/kg) 13

14 A 6 Analytics: Current status Analytical capacities available today suffice Majority of methods based on LC-MS/MS Proficiency tests have highlighted problems with certain matrices (e.g. cocoa, coffee) and methods (e.g. GC/MS without derivatization) Urgent need for fully validated reference method(s) as well as rapid screening methods. 14

15 F 1 Formation: Possible pathways to acrylamide (Gertz & Klostermann, Eur. J. Lipid Sci. Technol., 2002) (asuhara et al., J. Agric. Food Chem., 2003) (Stadler et al., Chem. Res. Toxicol., 2003) only ~5% of the yield compared to Asn Procter & Gamble (Sanders et al., Sept. 2002) Univ. Reading / Leeds (Mottram et al., ct. 2002) estlé Research (Stadler et al., ct. 2002) Food control laboratory (Weisshaar et al., ov. 2002) Cantonal laboratory (Biedermann et al., Dec. 2002) 15

16 F 2 Strecker aldehyde versus -glycoside (Mottram, 2002) 2 (Stadler, 2002) 2 C Acrylamide (mmol/mol) t= 5 min Glc/Asn Temperature ( C) 2 2 Acry la m id e ( mmol/mol) T= 100 C Time (min) (Stadler et al., J. Agric. Food Chem., 2004) 16

17 F 3 Decarboxylated Amadori comp. X X (aylayan, 2003) 2 (Zyzak, 2003) 2 ΔT ΔT X X + Vinylogous compound (Styrene) X + X + X m/z 251 Strecker aldehyde Strecker alcohol 2 2 (Blank et al., ACS Syposium, 2004) 17

18 F 4 X C 2 + X C X C Amino acid Carbonyl -Glycosyl conjugate Amadori compound AA (mmol/mol) - 2 I 2 C 2 Glc 0.6 R R' C R Asn R R C Asn ~ X + Schiff base betaine -C 2 - X Schiff base II X + Azomethine ylide X X xazolidin-5-one -C 2 2 X Aminoketone + (Blank et al., ACS Meeting, 2004) Decarboxylated Amadori compound Vinylogous compound 18

19 F 5 3-Aminopropionamide (Zyzak, 2003) 2 X + X APA + Glc (mmol/mol) APA 2 19 (Granvogl et al., J. Agric. Food Chem., 2004)

20 F 6 Formation: Current status Route/Intermediate Reference Glycoconjugates of Asparagine (Asn) -Glycosides of Asn AA Stadler et al., 2002 Schiff base of Asn AA Zyzak et al., 2003 Decarboxylation of the Schiff base + aylayan et al β-elimination of the Amadori product Blank et al., Aminopropionamide from Asn Zyzak et al., APA AA Granvogl et al., 2004 Acrylic acid and ammonia Acrylic acid + 3 AA (~5% of yield from Asn) Stadler et al., 2003 Acrolein + 3 AA asuhara et al., Propenal Acrylic acid Vattem & Shetty, 2003 Polyacrylamide is not a precursor (175 C) Ahn et al., 2003 ther amino acids aylayan et al.,

21 M 1 Mitigation: Effect of the type of sugar Acrylamide amount in the sample (measured by PLC-DAD) 2.0 ΑΑ (mmol/mol Asn) Fru/ Asn Gal/ Asn Pyrolysis (180 C, 5 min) Glc/ Asn ypothesis: Molecular mobility plays a key role in AA formation by solid-state Maillard reactions. eat flow [W/g] Fructose is more efficient in generating acrylamide under low moisture conditions! Chemical reactivity of sugar does not explain this phenomenon DSC of different sugar/asn 2 mixtures (open reaction system, 5 C/min) pure Asparagine monohydrate Crystallisation water release Fructose Glucose Temperature [ C] Acrylamide formation depends on melting point of the sugars. Galactose (Robert et al., J. Agric. Food Chem., in press) 21

22 M 3 Effect of moisture and p in model systems Acrylamide (mmol/mol Asn) Acrylamide (mmol/mol Asn) A B Water content (μl) p 3 p 4 p 6 p 8 p 9 Fructose is more reactive than glucose in generating acrylamide from asparagine. Moisture does influence acrylamide formation. Fructose is more reactive than glucose. ptimum for acrylamide formation at p 8. Low pk a value of Asn (8.9), i.e. α- 2 less protonated (Blank et al., unpublished, 2004) 22

23 M 4 Effect of p in potatoes omogenized potatoes T= 180 C, t= 25 min omogenized potatoes t= 25 min (p ~5.8) p 6.2 p 6.2 French fries 190 C, 6.5 min (Rydberg et al., J. Agric. Food Chem., 2003) p 5.2 p 4.9 (Jung et al., J. Food Sci., 2003) 23

24 M 5 AA (mmol/mol Asn) Effect of reaction time and temperature Glc/Asn 5 min min Temperature ( C) Reaction time and temperature are covariant parameters. igh amounts of acrylamide formed at C, depending on reaction time. (Robert et al., in press) Fried potatoes SVR= 0.3 mm -1 SVR= 0.8 mm -1 Acrylamide amounts in potatoes depend on surface-to-volume ratio (SVR) and processing time and temperature. 24 (Taubert et al., 2004)

25 M 6 Mitigation: Current status Parameter Relative effect Interpretation More asparagine +++ Direct precursor (e.g. potato) More red. sugars +++ Initiating Maillard reaction Add amino acids (Gly) -- Consumption of red. sugars igher T ++/- Pyrolysis / decomposition Longer t ++/- Pyrolysis / decomposition p +/- p 8 optimum, low p: less AA Antioxidants? o clear effect Radical initiators? o clear effect Water binding -- Reduced pyrolysis Asparaginase --- Removal of asparagine (patent) Fermentation -- Lowering the p (patent) (Rydberg et al., J. Agric. Food Chem., 2003) 25

26 Conclusion and outlook Importance to understand the unexpected presence of acrylamide in food Major pathways have been elucidated Investigate ways to reduce overall intake via food Further research on safety assessment of acrylamide in food needed (epidemiological studies) Insufficient data to justify changes in dietary advice eed to work in partnership and co-ordinate the research 26

27 Acknowledgments rganization committee J. Baynes et al. Analytics Safety assessment T. Goldmann, S. Riediker, G. Scholz R. Stadler,. Varga Chemistry Physics T. Davidek, S. Devaud, P. Pollien, M.-I. Alonso, I. Bauwens, G. Vuataz F. Robert, F. Saucy Thanks for your attention 27

Chemical and Physical Aspects of Acrylamide Formation in Low Moisture Systems

Chemical and Physical Aspects of Acrylamide Formation in Low Moisture Systems 227 th ACS Meeting on Acrylamide, Anaheim, March 30 th, 2004 I. Blank, T. Davidek, T. Goldmann, P. Pollien, F. obert,. Stadler,