COST - IMARS Joint Workshop

Cost Action 927 - IMARS

The Maillard Reaction in Food and Medicine

Napoli, 2006 May 24-27

Centro Congressi Ateneo Federico II Via Partenope, 36 - Napoli Wednesday, May 24

13.30: Registration

14.00-18.00: “APPETIZER WORKSHOP”

Three moderated debates about the question: “Are dietary AGEs/ALEs a risk to human health and, if they are, what is the mechanism of action?”

ƒ Motion 1:

Dietary AGEs are a risk to human health Chair: Thomas Henle For the motion: Katerina Sebekova Against the motion: Jenny Ames

ƒ Motion 2:

Dietary ALEs are a risk to human health Chair: Vincent Monnier For the motion: Joseph Kanner Against the motion: John Baynes

Coffee break

ƒ Motion 3:

The mechanism by which dietary AGEs and ALEs pose a risk to human health is via their interaction with RAGE Chair: Paul Thornalley For the motion: Anne-Marie Schmidt Against the motion: Claus Heizmann

Wednesday, May 24

COST927-IMARS WORKSHOP THE MAILLARD REACTION IN FOOD AND MEDICINE

18.15-19.15 Welcome addresses

IMARS President: Vincent Monnier COST action 927 Chair: Vincenzo Fogliano

ƒ Opening lecture: Helmut Erbersdobler

Forty years of Furosine - Forty years of using markers for Maillard Reaction in Food and Nutrition

Thursday, May 25

8.30: Registration

9.00-12.30: Exogenous and endogenous MRPs/AGEs: nutrition and toxicology

Chairs: Karl Heinz Wagner & Veronika Somoza

ƒ 9.00 T. Henle: Antimicrobial and cytotoxic effects of dicarbonyl compounds in honey - risk or benefit?

ƒ 9.25 J. Ames: Effect of dietary heat treatment on the profile of human colonic bacteria in ulcerative colitis: in vitro and in vivo studies

ƒ 9.50 T. Buetler: Mechanistic evaluation of biological effects of dietary AGEs

ƒ 10.15 A. Simm: Influence of the Maillard reaction on ischemia reperfusion injury

10.40-11.00: Coffee Break

ƒ 11.00 K.H. Wagner: Safety assessment of Glucose vs. Fructose based Maillard Reactions Products

ƒ 11.15 L. Bravo: Coffee Melanoidin protects human hepatoma HepG2 against oxidative stress induced by tertbutyl hydroperoxide

ƒ 11.30 F. Tessier: Protective effect of a diet rich in Maillard Reaction products on experimentally induced colitis in mice

ƒ 11.45 P. O´Brien: Dietary AGE and thiamine deficiency increased plasma alpha-oxoaldehyde levels and colonic inflammation

ƒ 12.00 M. Argirova: In vitro effects of model Maillard Reaction product on the mechanical activity of rat gastric smooth muscle

ƒ 12.30 K. Kankova: Relationship between circulating levels of soluble RAGE (sRAGE) and genetic variability in the ager gene in subjects with diabetic nephropathy

12.45-13.45: Lunch

13.45-15.00 Poster session

14.30-18.30: The impact of new food technologies on the MR/AGE formation

Chairs: Eyal Shimoni & Jenny Ames

ƒ 15.00 P. Buera: Water and the kinetics of Maillard reaction: a new perspective

ƒ 15.30 V. Gokmen: Modelling the Formation of Acrylamide in Glucose-Asparagine Model System Using Artificial Neural Network

ƒ 16.00 N. Alt: Influence of new food processing techniques on protein glycation reactions

16.20-16.50: Coffee break

ƒ 16.50 E. Shimoni: Reducing MRP's in liquid foods by ultra-high temperature ohmic heating

ƒ 17.10 B. Cammerer: Shelf life of pea nuts with roasting degree

ƒ 17.30 A. Arnoldi: Technological properties and non-enzymatic browning of lupin-based food products

ƒ 17.50 R. Venkutonis: Effects of heating and natural antioxidants on the content of heterocyclic amines in meat

ƒ 18.10 C. Billaud: Studies of the antioxidant activity of neoformed products from heated cysteine and aldehydes

20.30: Social dinner

Friday, May 26

8.30-12.30: Formation pathways and detection of MRPs/AGEs in food and in vivo

Chairs: Francisco Morales & Susan Thorpe

ƒ 8.30 T. Hofmann: Taste-active glyco-conjugates and their formation upon thermal food processing

ƒ 8.55 P. Thornalley: LC-MS/MS quantitation, hotspot site location and predicted structures of proteins glycated by dicarbonyls - a systems, information-rich approach

ƒ 9.20 J. Baynes: Methionine Sulfoxide, a Sensitive Biomarker of Oxidative Damage to Proteins

ƒ 9.45 R. Stadler: Recent advances in acrylamide analysis, formation and mitigation in foods

10.10-10.35: Coffee Break

ƒ 10.35 S. Thorpe: S-(2-succinyl)cysteine: A novel chemical modification of tissue proteins

ƒ 11.00 I. Blank: Formation of furan and methylfuran under roasting conditions

ƒ 11.15 V. Yaylayan: Vinylogous Amadori rearrangement: consequences in food and in biological systems

ƒ 11.30 I. Birlouez: Simultaneous analysis of reactive lysine, furosine and carboxymethyllysine by GC-MS: Application to a wide range of heat-treated foods

ƒ 11.45 A. Jakas: Synthesis and stability of the early glycation products formed from d-fructose and endogenous opiod peptides

ƒ 12.00 T. Fiedler: Carbohydrate based melanoidins, several alpha-dicarbonyl compounds as direct precursor for the formation of discrete molecular size domains

ƒ 12.15 J. Hajslova: Study of "masked" acrylamide origin

12.30-13.30: Luncheon session The impact of food MRPs on human health

Chairs: Simonetta Salvini & Josephine Forbes

ƒ 12.35 S. Salvini: Problems in the assessment of MRP intake in humans

ƒ 12.50 S. Tuijtelaars: Human exposure of acrylamide in food

ƒ 13.10 N. Pellegrini: Coffee: a protective beverage?

13.30-15.00: Poster session

15.00-18.00: Monitoring and control of the Maillard Reaction in food and in vivo

Chairs: Vincent Monnier & Vincenzo Fogliano

ƒ 15.00 T. Miyata: Advanced glycation end products: Surrogate markers and therapeutic targets for diabetic renal injury

ƒ 15.20 V. Monnier: Ascorbic acid as a Glycating Agent in Vivo

ƒ 15.40 E. Van Schaftingen: Fructosamine-3-kinase and other enzymes involved in protein glycation

ƒ 16.00 V. Fogliano: Amadoriase, a tool for inhibiting protein glycation and food browning

16.20-16.50: Coffee Break

ƒ 16.50 T. Miyazawa: Amadori glycated phosphatidylethanolamine in food and human plasma

ƒ 17.10 R. Nagai: Investigation of pathway for AGE formation in macrophages

ƒ 17.30 Z. Ciesarova: Application of L-Asparaginase for acrylamide mitigation in potato products

ƒ 17.50 S. Sivakami: The roles of dietary factors in preventing glycation mediated damage to DNA in a MG/LYS system

18.15 Report of IMARS activities

18.30-19.30: COST 927 Management Committee meeting (only for MC members) Saturday, May 27

8.30-12.30: AGE related pathologies: Diabetes, Lens and Renal Disease

Chairs: Katerina Sebekova & Naoyuki Taniguchi

ƒ 8.30 N. Taniguchi: Glycation and posttranslational modifications of antioxidative enzymes

ƒ 9.00 A. Stitt: Pathogenic role of substrate-immobilized AGEs in diabetic retinopathy

ƒ 9.25 M. Cooper: Tangled in a web of sugar: AGEs, their actions and reactions in diabetic complications

ƒ 9.50 C. Schalkwijk: AGE/ALEs: non-traditional risk factors for vascular complications

10.15-10.45: Coffee break

ƒ 10.45 G. Stein: Kidney and AGEs

ƒ 11.10 J. De Groot: Advanced Glycation End products in the development of osteoarthritis

ƒ 11.35 M. Pischetsrieder: Effects of Maillard reaction products on NFKB activation and expresion of proinflammatory markers - molecular mechanism

ƒ 12.00 F. Facchiano: Sugar-induced modification of angiogenic growth factors: a structural and functional study

12.20: Meeting Closure

12.40: Lunch and tour to Pompei

Oral programme S1

FORTY YEARS OF FUROSINE – FORTY YEARS OF USING MARKERS FOR MAILLARD REACTION IN FOOD AND NUTRITION

Helmut F. Erbersdobler

Institut für Humanernährung und Lebensmittelkunde, Düsternbrooker Weg 17, D-24105 Kiel

Since about 60 years amino acid analysers were used in order to evaluate protein quality (1). However, soon it was known that this procedure was not reliable in heat damaged foods. It had to be recognized that in usually heated or stored foods especially lysine was less destroyed but nutritionally blocked, a fact which was not considered by the usual determination after acid hydrolysis. One of the first proposals for the determination of available lysine was labelling of the critical ε-amino group of lysine with fluoro- dinitrobenzene (2) which led to a worldwide application of this technique (e.g.3). However, to evaluate the intensity of the heating process it seems to be better to analyse new substances whose formation depends on different heating conditions. Moreover, meanwhile more sensitive indicators are wanted, suited to measuring also a low technical impact on protein quality e.g. in ultra high temperature treated milk. Since its detection forty years ago (4) as a stable indicator of the Amadori products (5,6,7) furosine was used as indicator of thermal damage in food science and medical biochemistry. Its use was enhanced by analytical improvements starting with the proposal of Schleicher et al. (8) and improved by Resmimi et al. (9). Especially after the commercial availability of a pure and stable standard the furosine analyses increased very much and are still in use (e.g.10). Furosine has the disadvantage that it is formed from the Amadori products only at a rate of 30-40%. However, this recovery is reproducible if constant analytical conditions are applied. Moreover, furosine increases not linearly with increasing heat damage since the Amadori products are only intermediates which react to further compounds in the advanced and late Maillard reaction. In more severely heated products furosine values reach a plateau or even decrease. On the other hand, furosine has the big advantage that it is a direct marker for a real existing reaction product of lysine, which is not only of analytical and technical but also of nutritional relevance. Furosine analyses were applied on many food items and especially on dairy products. Some items showed by this way a degree of inactivation up to one third, even to one half. In dairy products also hydroxymethylfurfural (HMF (11) is a common marker resulting also from the Maillard condensation. Data from the literature offer a wide range for this marker which suspect that the HMF determination is insufficiently reproducible between laboratories. However, several studies on UHT treated milks demonstrated the usefulness of the UHT-method as a rapid and simple measure of heat damage. A marker for the advanced and late Maillard reaction, comparable to furosine, is Nε–carboxymethyllysine (CML), first detected 1985 as marker for heated proteins in biological material and 1986 in food proteins (12,13). Although the mechanisms by which CML is formed are not unique like in furosine, CML proved to be a reliable marker of advanced, even late Maillard reaction. In more severely heat treated food items in which furosine levels have already decreased, CML can provide additional information on protein damage. Together with the actual lysine value furosine and CML allow a calculation of total, inactivated and available Lysine. In other papers (14, 15) pyrraline (ε-pyrrole-lysine) was shown to increase progressively to values, indicating that that up to 15% of the lysine residues may have been modified. Pyrraline was found in dairy and bakery products. Also Pentosidine has been found in foods (15) although the range of concentrations was smaller than in furosine or pyrraline. As a derivative of arginine imidazolonylornithine was found in coffee and in bakery products (15). Due to the links between food science and medical biochemistry concerning the Maillard reaction it is likely that more markers will be used in both fields in the future. Important for their preferred use may be 1. analytical access; 2. stability; 3. range of data and 4. biological significance. With respect to these criteria the use of several markers in biology and food science is discussed in detail.

1. Mitchell, H.H., Block, J. (1946) J. Biol. Chem.163, 599; Carpenter, K.J. (1960) Biochem. J. 77, 604 2. Erbersdobler, H., Zucker, H. (1964) Z. Tierphysiol. Tierern. Futtermittelkde. 19, 244 3. Erbersdobler, H., Zucker, H. (1966) Milchwissenschaft, 21, 564 4. Brüggemann, J., Erbersdobler, H. (1968) Z. Lebensmittel- Untersuchung und-Forschung, 137, 137 5. Heyns, K., Heukeshoven, J., Brose, K.-H. (1968) Angew. Chemie 80, 627 6. Finot, P.A., Bricout, J., Viani, R., Mauron, J. (1968) Experientia, 24, 1097 7. Schleicher, E., Wieland, O.H. (1981) J. Clin. Chem. Clin. Biochem. 19, 81 8. Resmimi, P., Pelegrino, L., Batelli,G. (1990) Ital. J. Food Sci. 3, 173 9. Masotti, E., De Noni, I., (2005) Ital. J. Food Sci. 17, 429 10. Keeney, M., Basette, R. (1959) J. Dairy Sci., 6, 945 11. Ahmed, M.U., Thorpe, S.R., Baynes, J.W. (1985) Fed. Proc. 44, 1621 12. Büser, W., Erbersdobler, H. (1966) Milchwissenschaft, 41, 780 13. Chiang, G.H., (1988) J. Agric. Food. Chem. 36, 506 14. Henle, T., Schwarzenbolz, U., Walter, A.W., Klostermeyer, H. (1998) In: O´Brien, J., Nursten, H.E., Crabbe, M.J.C., Ames, J.M. (eds.) (1998) The Maillard Reaction in Food and Medicine, Royal Society of Chemistry, London, pp. 178-183 S2

ANTIMICROBIAL AND CYTOTOXIC EFFECTS OF DICARBONYL COMPOUNDS IN HONEY - RISK OR BENEFIT?

Mavric E., Wittmann S., Lassen A., Müller L., Henle T.

Institute of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany

KEYWORDS: 3-deoxyglucosulose, methylglyoxal, glyoxal, E. coli, S. aureus, HepG2, HT29

Depending on temperature, time, pH or water activity, carbohydrates can undergo various degradation reactions, referred to as “caramelization” or, if amino compunds are involved, “Maillard reactions”. Reaction products are highly reactive 1,2-dicarbonyl compounds such as 3-deoxyglucosulose (3-DG), methylglyoxal (MGO), glyoxal (GO). In foods, 1,2-dicarbonyl compounds play a key role in flavour and colour formation as well as for the modification of proteins. Furthermore, the formation of carbohydrate degradation products is also known for biological systems, where they represent precursors for advanced glycation endproducts (AGEs). Increased levels of glyoxal, methylgloyxal and 3-DG have been reported for diabetic and uremic patients. The possible pathophysiological role of this phenomenon is under debate. Due to their possible cytotoxicity, efforts were made to lower the amount of 1,2-dicarbonyls in fluids used for peritoneal dialysis.

Only very limited information is available concerning the amount of individual 1,2-dicarbonyls in foods. Low amounts of enzymatically formed glyoxal, methylglyoxal and diacetyl were reported for beer and wine. Methylglyoxal and glyoxal are known to form during coffee roasting. Quantitative data for 3- deoxyglucosulose (3-DG) in food are not available.

The purpose of our study was to measure the amount of the 1,2-dicarbonyls together with hydroxymethylfurfural (HMF) in commercially available samples of honey. Furthermore, we investigated whether high amounts of 1,2-dicarbonyl compounds may be related to the well-known antibacterial activity of honey or may induce cytotoxic effects in cell culture experiments.

3-DG, MGO and GO were measured for the first time in 50 commercially available honey samples as the corresponding quinoxalines after derivatization with orthophenylendiamine using RP-HPLC and UV- detection. Compared to also quantified 5-hydroxymethylfural (HMF), which ranged between 0.6 and 44 mg/kg, up to hundredfold higher amounts of 3-deoxyglucosulose, ranging from 80 to 1300 mg/kg, were found. During storage of honey at 35 °C and 45 °C, a linear increase of 3-deoxyglucosulose was observed. Values for glyoxal and methylglyoxal were between 0.2 and 2.7 or 0.4 and 5.4 mg/kg, respectively, and were not affected by storage. For some samples of New Zealand Manuka honeys, however, significant higher amounts of MGO were found, ranging from 48 up to 743 mg/kg. Antibacterial activity of honey and solutions of 1,2-dicarbonyl towards Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were analyzed using an agar well diffusion assay. Minimally concentrations needed for inhibition of bacterial growth (minimally inhibitory concentration, MIC) of MGO were 1.1 mM for both types of bacteria. MIC for GO was 6.9 mM (E. coli) or 4.3mM (S. aureus), respectively. 3-DG showed no inhibition in concentrations up to 60 mM. Whereas most of the honey samples investigated showed no inhibition in dilutions of 80 % (v/v with water) or below, the samples of Manuka honey exhibited antibacterial activity when diluted to 20 to 30 %, which corresponded to MGO concentrations of 1.1 to 1.8 mM. This clearly demonstrates that the pronounced antibacterial activity of New Zealand Manuka honey directly originates from MGO.

We also evaluated the cytotoxic effects of 1,2-dicarbonyls as well as of diluted honey using HepG2 and HT29 cell lines. The cells were exposed to these solutions for 24 h and viability was determined by neutral red assay. Concentrations needed for 50 % inhibition of cell growth (ICG50) were 2.6 mM (HepG2) and 4.7 mM (HT29) for 3-DG, 1.5 mM (HepG2) and 0.9 mM (HT29) for MGO, or 1.4 mM (HepG2) and 1.3 mM (HT29) for GO, respectively. Diluted honey samples also showed cytotoxic effects with ICG50 values ranging from 2.5 to 100 % (v/v with water), but this cytotoxicity was not only dependent on the corresponding content in 1,2-dicarbonyls. These results indicate that honey is a source of bioactive carbohydrate degradation products with physiological implications.

S3

EFFECT OF DIETARY HEAT TREATMENT ON THE PROFILE OF HUMAN COLONIC BACTERIA IN ULCERATIVE COLITIS: IN VITRO AND IN VIVO STUDIES

Jennifer M. Ames (1), Davinia J. S. Hinton (2), Bronwyn V. Prosser (2), M. James C. Crabbe (3), Kieran M. Tuohy (2) and Glenn R. Gibson (2)

(1) School of Biological Sciences, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, U.K, (2) School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6AP, U.K., (3) Luton Institute for Research in the Applied Natural Sciences, Faculty of Creative Arts, Technology and Science, University of Luton, Park Square, Luton LU1 3JU, U.K.

KEYWORDS: protein glycation; Maillard reaction; gut microflora; dietary advanced glycation endproducts

The colon is an authentic organ of health and nutrition. Due to its resident microbiota, it is the most metabolically active organ in the body. The colon is the site of several acute bacterial infections. Various chronic diseases, including inflammatory bowel disease, such as ulcerative colitis, are considered to have a bacterial aetiology. Dietary carbohydrate profoundly affects the gut microflora composition but the effect of dietary protein, including glycated forms, is uncertain. We propose that glycated protein affects the profile of colonic bacteria and that this effect is more pronounced in sufferers of ulcerative colitis, compared to control subjects.

To test the hypothesis, we heated BSA (1 mM) with glucose (0.4 M) at 50 °C for 24 h followed by in vitro mouth, stomach and small intestine digestion. Fermentation of undigested material was performed using a validated, multi-chamber gut model inoculated with mixed faecal bacteria obtained from ulcerative colitis patients or control subjects. Bacteria were enumerated using 16S rRNA sequencing probes.

Compared to healthy subjects, faecal samples from ulcerative colitis patients contained higher numbers of bacteria considered to be detrimental to health, e.g., sulfate-reducing bacteria and clostridia, but lower numbers of bifidobacteria and lactobacilli, which are regarded as conferring health benefits. Following fermentation in the gut model, glycated protein modified the gut flora to a more detrimental profile. In particular, numbers of sulfate-reducing bacteria and bacteroides were higher and this modification was greater for the UC patients compared to the control subjects.

Glycated protein adversely affects the profile of the gut flora in an in vitro gut model. Further in vitro studies using different proteins are called for to confirm the findings. Human intervention studies are required to investigate whether this effect can be replicated in vivo.

S4

MECHANISTIC EVALUATION OF BIOLOGICAL EFFECTS OF DIETARY AGES

Timo Buetler (1), John Newell (1), Thierry Delatour (1), Estelle Leclerc (2), Claus Heizmann (3) and Benoit Schilter (1)

1 Nestlé Research Center, Vers-chez-les-Blanc, Lausanne, Switzerland; 2 Dept. of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, USA; 3 University Children’s Hospital, University of Zurich, Zurich, Switzerland

KEYWORDS: RAGE, model glycation

Advanced glycation endproducts (AGEs) are a diverse class of protein modifications formed by the Maillard reaction and by oxidative stress. They include single modifications of amino acids such as Nε- carboxymethyllysine (CML) or pyrraline or complex protein aggregates formed by intermolecular cross-links such as pentosidine or glucosepan. AGEs have been proposed to act via their receptor RAGE to activate pro-inflammatory cellular responses, thereby contributing to diabetic complications and aging. Because AGEs are also formed during the heating of food, it has recently been suggested that dietary AGEs may contribute to the development of disease and contribute to the aging process. Studies have shown that AGEs are absorbed from the GI tract and can be detected in blood. We set out to investigate whether the molecular complexity of AGEs may influence their interaction with RAGE since dietary AGEs are only absorbed as free or small peptide-bound AGEs. There are indications in the literature (Valencia et al., Diabetologia 47:844, 2004) suggesting that only multiprotein complexed AGEs are able to interact with RAGE. However, these authors showed that this interaction did not result in the activation of cellular responses. We have generated AGEs with single modifications (CML, pyrraline) and cross-linked AGEs containing pentosidine and tested their RAGE binding properties using a BIACORE system with RAGE expressed in yeast. Our results confirm the data reported by Valencia et al. that only cross-linked multiprotein complexed AGEs were able to bind to RAGE at sub-micromolar concentrations (KD 0.1-0.7 µM). CML- or glucose-modified proteins (human serum albumin and β-lactoglobulin) were unable to bind to RAGE. We are currently investigating their effect on RAGE activation using cellular assays.

S5

INFLUENCE OF THE MAILLARD REACTION ON ISCHEMIA REPERFUSION INJURY

Andreas Simm, Babett Bartling, Robert Scheubel, Beatrice Schneider, Rolf-Edgar Silber

Department of Cardio-Thoracic Surgery, Martin-Luther-University Halle-Wittenberg

Acute myocardial infarction is associated with a poor prognosis and a high mortality rate in elderly patients. It is well known that ageing of human myocardium is characterized by functional as well as cellular changes. Those changes within the heart could affect myocardial tolerance to ischemia. Pre-treating a myocardium with brief intervals of ischemia and reperfusion diminishes cardiac damage induced by prolonged ischemia, leading to less myocyte necrosis and better functional recovery. This important phenomenon is called ischemic preconditioning. Investigations on the impact of cardiac aging on preconditioning in right atrial trabeculae of adult (≤55 years) and senescent (≥70 years) patients with coronary artery disease show that ischemic preconditioning has no beneficial effect on the post-ischemic functional recovery of senescent human myocardium. On the other hand, experimental and clinical investigations suggest that blockade of Na+/H+ exchange (NHE) with cariporide provides functional protection during ischemia and reperfusion in mature hearts. Unfortunately, our data show clearly that whereas Cariporide is protective against ischemia-reperfusion injury in adults, it has no benefit on senescent myocardium. It is now well known that age dependent modifications of proteins by carbohydrates (advanced glycation endproducts, AGEs) affect the functionality of organs. To test the involvement of AGEs on ischemic reperfusion injury, atrial trabeculae were treated with glyoxal (a reactive dicarbonyl and intermediate product of AGE formation). Surprisingly, glyoxal treatment protects the myocardium of young as well as old patients from ischemia and reperfusion injury. Biochemical analysis show that glyoxal interfere with the apoptotic machinery by blocking the activity of effector caspases. As glyoxal is generated during the degradation of amino acids as well as the oxidation of glucose, transient modifications of proteins could therefore be an endogenous new pathway to protect cells from short ischemic events. These data also suggest that reversible glycation of proteins, until now only believed to have pathophysiological significance, may be part of a new physiological regulatory network.

S6

SAFETY ASSESSMENT OF GLUCOSE VS. FRUCTOSE BASED MAILLARD REACTIONS PRODUCTS

Karl-Heinz Wagner, Karin Koschutnig, Stefanie Reichhold, Catherine Billaud

Department of Nutritional Sciences, University of Vienna

KEYWORDS: Glucose, Fructose, Glutathione, Cystein, Maillard Reaction Products, AMES Test

Maillard Reaction Products (MRPs) based on fructose/glucose with glutathione or cysteine were shown to inhibit the polyphenoloxidase as antibrowning agents. However, no safety assessment has been done on these MRPs. The aim of the present study was to investigate the antioxidative and antimutagenic effects of glucose or fructose based MRPs in the Ames test.

The MRPs consisted of glucose resp. fructose and cysteine resp. glutathione, which were heated for 4h20min as well as 14h at temperatures of 103°C or 110°C.

During the investigations the plate incorporation assay and the preincubation assay were applied, both with and without metabolic activation (S9). Hydrogen peroxide (H2O2) and tertiär-buthyl hydroperoxide (tBOOH) were used as prooxidatives. The Salmonella typhimurium indicator strains TA 98 and TA 102 were employed, the samples were tested in five different concentrations: 0,05; 0,1; 1,0; 5,5; 11 mg per plate, in order to considerlow physiological conditions but also high concentrations up to the border of solubility.

The his+-revertants glucose-cystein (14h) turned out to be the most reactive one under the influence of H2O2. Without metabolic activation this sample showed antioxidative properties, whereas with the S9-mix it was prooxidative. The other 14h incubated fractions were less reactive. In contrast all 4h20min heated samples were strongly reactive. Regarding all influencing factors fructose-glutathione (4h20min) was the most conspicuous one.

For the inhibition of polyphenoloxidase the employment of glucose-cystein seems to be most expedient considering, however, the formation of mutagenic and prooxidative properties under the influence of H2O2 and S9.

S7

COFFEE MELANOIDIN PROTECTS HUMAN HEPATOMA HEPG2 AGAINST OXIDATIVE STRESS INDUCED BY TERTBUTYL HYDROPEROXIDE

Luis Goya, Laura Bravo, Cristina Delgado-Andrade, José A. Rufián-Henares and Francisco J. Morales

Instituto del Frio (CSIC), Madrid, SPAIN

KEYWORDS: melanoidin, coffee, antioxidant, human hepatoma cells

In the recent years there has been a growing interest in the effect of melanoidins on the human diet and their feasible nutritional, biological and health implications. A whole soluble high molecular weight fraction (named melanoidin) from coffee brew was isolated by ultrafiltration and then digested by simulating a gastric plus pancreatic digestive condition. This digested fraction was partly characterized by capillary zone electrophoresis, gel-filtration and browning. The objective of the present study was to investigate the potential protective effect of the enzymatically treated coffee melanoidin (HMW) on cell viability and redox status of cultured human hepatoma HepG2 cells submitted to oxidative stress induced by 200 µM tert- butylhydroperoxide. Cell concentration of reduced glutathione and malondialdehyde and intracellular generation of reactive oxygen species were used as markers of cellular oxidative status.

Pre-treatment of cultured HepG2 cells with 0.5-10 µg/mL HMW for 2 or 20h completely prevented lactate dehydrogenase leakage from the cells induced by a treatment with tert-butylhydroperoxide for 3h. Besides, pretreatment for 2 or 20h with all doses of HMW (0.5-10 µg/mL) partly prevented the decrease of reduced glutathione and the increase of malondialdehyde evoked by tert-butylhydroperoxide in HepG2 cells. On the contrary, increased reactive oxygen species generation induced by tert-butylhydroperoxide was not prevented when cells were pretreated for 2 or 20h with 0.5-10 µg/mL HMW. The results of the biomarkers analyzed show that treatment for 2 or 20 h of HepG2 cells in culture with concentrations of HMW within the physiological range confers the cells a significant protection against an oxidative insult. In addition, possible mechanisms of action are discussed.

S8

PROTECTIVE EFFECT OF A DIET RICH IN MAILLARD REACTION PRODUCTS ON EXPERIMENTALLY INDUCED COLITIS IN MICE

Fj Tessier, A Craus, A Abdel Nour, S Barry, I Birlouez-Aragon, Pm Anton

Institut Supérieur d'Agriculture de Beauvais, France

Maillard reaction products (MRP) are commonly formed in food during cooking as well as during storage. However little is known about their beneficial or harmful effects on human health. It is also assumed that a large proportion of MRP present in food is not absorbed and therefore simply flows through in the intestinal tract. The aim of this study was to better understand the effect of MRP on the integrity of the gut.

The effects of MRP were investigated in an animal feeding experiment. One hundred and twenty male mice were divided into 3 groups. They received for 3 weeks either regular pellets (groups 1), heated (groups 2) or highly-heated pellets (groups 3) ad libitum. The second week, half of each group was submitted to a dextran sulfate sodium treatment (DSS) to induce experimental colitis. Eight animals of each group were sacrificed on day 14, 18 and 21 of the experiment. Food intake and body weight were monitored throughout the study and the three diets were tested for MRP levels. At sacrifice, blood was drawn to estimate plasmatic levels of AGEs, caecal content was preserved to measure microbiota alteration through multiplex PCR and colonic pieces to study macro- and microscopic changes, MPO activity and other inflammatory mediators by real time PCR

Fluorescence spectroscopy analysis indicated that the level of fluorescent AGEs present in the caecal material was proportional to that ingested. DSS-induced inflammation was significantly reduced in the group of highly-heated food as shown by macro- and microscopic scores, MPO activity and inflammatory measurements whereas moderate heating had no incidence. In non inflamed mice, food heating had no effect. Overall, the results suggest that the protective effect observed with the high-MRP diet might be due to the presence of melanoidins which affects the gut bacterial balance and probably limits the intestinal epithelial oxidative stress.

S9

DIETARY ADVANCED GLYCATION END PRODUCTS AND THIAMIN DEFICIENCY INCREASED PLASMA ALPHA-OXOALDEHYDE LEVELS AND COLONIC INFLAMMATION

Nandita Shangari (1), Flore Depeint (1,2), Rhea Mehta (1), Rudolf Furrer (2), W. Robert Bruce (2), Peter J. O’Brien (1)

(1) Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ont., M5S 2S2,Canada.

KEYWORDS: aberrant crypt foci, advanced glycation end-products, thermolyzed diet, thiamin deficiency

Previously we showed that marginal dietary thiamin deficiency in rats caused aberrant crypt foci (marker of colon carcinogenesis) formation at 160 days without symptoms of clinical beriberi (Cancer Lett. 2003 202(2):125-9). Plasma levels of glyoxal and methylglyoxal (MG) were also increased (FEBS Lett. 2005 579(25):5596-602).

A thermolyzed diet containing advanced glycation end-products (AGEs) and decreased thiamin increases intracellular exposure to alpha-oxoaldehydes and colon carcinogenesis.

4 groups of Fischer 344 rats were given the following diets: Group A was given a AIN93G diet (control diet), Group B AIN93G diet, autoclaved 30 min, 121°C (thermolyzed diet), Group C AIN93G diet with equivalent thiamin as in Group B and Group D AIN93G diet as in Group B + 20 mg/l thiamin in the drinking water. The animals were cycled on 3 weeks of thermolyzed diet and 2 weeks on control diet to restore the vitamin stores that were depleted during thermolysis for 160 days. At a 160 days we measured transketolase activity in red blood cells (thiamin status); glyoxal/MG and TNF-alpha levels in the plasma; glyoxal/MG hydroimidazolone protein adducts and dicarbonyls in the plasma, liver, kidney, brain and adipose tissues; glutathione levels on whole blood, oxidative stress/inflammatory markers in the colon such as macrophages via immunohistochemistry.

Thermolyzed diet resulted in: 1) decreased thiamin status and transketolase activity. 2) increased plasma levels of glyoxal/MG adducts. 3) increased protein dicarbonyls in the liver more than other tissues. 4) lower erythrocyte glutathione levels. 5) an infiltration of macrophages in the colon.

A thermolyzed diet leads to an increased AGEs body burden, decreased thiamin levels thereby decreasing cellular thiamin and increasing genotoxic AGEs (i.e. glyoxal/MG). The increased oxidative stress affected tissues with low levels of antioxidant defenses such as the colon.

S10

IN VITRO EFFECTS OF MODEL MAILLARD REACTION PRODUCT ON THE MECHANICAL ACTIVITY OF RAT GASTRIC SMOOTH MUSCLE

Argirova MD, Stefanova I, Krustev AD.

Department of Chemistry and Biochemistry, Department of Medical Physics and Biophysics, Medical University, Plovdiv, Bulgaria

KEYWORDS: Maillard reaction, melanoidins, gastric smooth muscle txt: Aims: Several investigations suggest that Maillard reaction products (MRPs) bind calcium ions and thus regulate their bioavailibility. On the other hand changes in intracellular calcium level is the major mechanism that influences muscle contractility. This work aims to study the effect of model MRP on the mechanical activity of rat gastric smooth muscle. Methods: Model MRP was obtained from arginine (Arg) and glucose (Glc) under conditions that simulate a low-moisture baking. Total MRP or melanoidin fraction was applied to smooth muscle preparations (SMP) in organ bath. The mechanical activity of SMP was registered isometrically. Membrane potential was measured by the single sucrose-gap method. Results: Total Arg-Glc MRP applied at concentration 1.5 mg/ml caused a reversible contraction of 3.46 ± 1.05 mN (n = 25). The melanoidin fraction (> 3 000 Da) at concentration 1.5 mg/ml caused a contraction of 1.77 ± 0.26 mN (n = 6), suggesting that the melanoidin precursors also can influence the mechanical activity. Study on the concentration effect dependence within the range 0.1 10 mg/ml revealed that at low concentration (up to 4 mg/ml) the melanoidin provoked concentration-dependent contraction, whereas a muscle relaxation was registered at higher melanoidin concentrations. The contraction was preceded by changes in the calcium membrane current and abolished by the calcium channel blockers D-600 and nifedipine. Measurements with Ca2+-selective electrode showed that at concentrations higher than 2.5 mg/ml the melanoidin significantly decreases the concentration of ionized Ca2+. Conclusions: MRP could influence the contractility of gastric smooth muscle through at least two different pathways: 1) At low concentration it causes a depolarization and activation of L-type calcium channels, stimulates the Ca2+-influx, and provoke a contraction. 2) At high concentration the chelating activity of MRP leads to significant depletion of extracellular calcium ions, and most likely underlies the relaxation process observed.

S11

RELATIONSHIP BETWEEN CIRCULATING LEVELS OF SOLUBLE RAGE (sRAGE) AND GENETIC VARIABILITY IN THE AGER GENE IN SUBJECTS WITH DIABETIC NEPHROPATHY

Kankova K, Kalousova M, Hertlova M, Krusova D and Zima T

Dept. of Pathophysiology, Masaryk University, Brno and Institute of Clinical Chemistry and Laboratory Diagnostics, Charles University, Prague, Czech Republic

KEYWORDS: diabetic nephropathy, RAGE, polymorphism, sRAGE, renal failure

Accumulation of Advanced Glycation End products (AGEs) is accelerated in diabetics compared to age- matched non-diabetic subjects and it’s especially pronounced in renal failure. AGEs exert their adverse effects via Receptor of AGE (RAGE). Soluble RAGE is a 50kDa C-terminally truncated form of RAGE functioning as an inhibitor of AGE-mediated signalling events. Decreased levels of supposedly protective sRAGE were ascertained in patients with macrovascular disease and hypertension, however, similar data for diabetic nephropathy are so far lacking. The aim of the study was to analyse the relationship between sRAGE and renal functions as well as genetic variability in AGER gene in subjects with diabetic nephropathy.

Cross-sectional study of a cohort of 255 diabetic patients (36 T1DM, 219 T2DM, approx. 1:1 male-to-female ratio) with variable extent of diabetic nephropathy: 82 with normoalbuminuria, 50 with persistent microalbuminuria, 112 with persistent proteinuria and 10 with ESRD. Plasma sRAGE was assessed by ELISA (Quantikine, RD Systems). Routine biochemical parameters were measured using automatic analysers. sRAGE was significantly higher in subjects with nephropathy than normoalbuminuric patients (P=0.0015, Mann-Whitney), 2154.4±2612 vs. 1622.7±1981.4 pg/ml, respectively. In patients with nephropathy sRAGE positively correlated with its severity (P=0.0028, Spearman). Further, sRAGE positively correlated with age, diabetes duration, S-urea S-creatinine and albumin excretion rate and negatively with glomerular filtration rate (all P<0.05, Spearman). No significant correlation was ascertained with fasting plasma glucose or HbA1c. Further, sRAGE significantly differed among subjects bearing variable number of RAGE2 haplotype copies (i.e. previously identified genetic risk factor of diabetic nephropathy), P<0.05, median test.

Conclusions: Contrary to our anticipations, sRAGE levels are increased in diabetics with nephropathy compared to normoalbuminuric subjects and further rise with decreasing renal function. Whether such increase merely reflects decreased renal clearance or genetically modified up-regulation of sRAGE to protect against AGE-mediated effects remains to be elucidated.

S12

WATER AND THE KINETICS OF MAILLARD REACTION: A NEW PERSPECTIVE

M.P. Buera

Departamento de Industrias. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.

The development of strategies to control the kinetics of Maillard reactions in order to conduct it towards desired results represents a challenge in basic and applied aspects of food science. Temperature and water content of the systems are the variables most widely employed to define and predict the kinetics of the Maillard reaction. However, structural aspects of the matrices where the reaction takes place, state and phase transitions, sorption and solvent properties of water were detected as key aspects to describe the dynamic of this reaction. The analysis of a wide number of published literature (in vegetable and animal tissues, dairy products or model systems) as so as the results from specifically designed experiments, showed that Maillard reaction requires a minimum of water content in order to mobilise the reactants, but it is then immediately inhibited by an excess water over this requirement (which can be associated to the second inflection point of the sorption isotherm). Supplemented phase diagrams demonstrated to be helpful in order to determine the feasibility of phase/state transitions. The reaction occurred below the glass transition temperature (Tg) and both solids-water interactions and structural characteristics of the systems governed the dependence of Maillard reaction rate on relative humidity. The two inflection points of the sorption isotherms were reflected on the dependence of Maillard reaction rate with R.H.. The relative humidity at which the rate of Maillard reaction is maximum can be predicted on the basis of sorption, structural and thermal transition of the matrices where the reaction takes place. Detectable areas for further research involve the study of macroscopic and molecular properties of the materials, such as the incidence of sub Tg relaxations on the kinetics of chemical reactions, or local heterogeneities in water distribution at microscopic length scales.

S13

MODELLING THE FORMATION OF ACRYLAMIDE IN GLUCOSE-ASPARAGINE MODEL SYSTEM USING ARTIFICIAL NEURAL NETWORK

Arda Serpen, Vural Gökmen

Department of Food Engineering, Hacettepe University, Beytepe, 06800 Ankara, Turkey

KEYWORDS: Acrylamide, Maillard reaction, modelling, artificial neural network

After the discovery of acrylamide in thermally processed foods, number of theoretical mechanisms have been proposed for its formation. Most probably, acrylamide in food results largely from the Maillard reaction between amino acids (primarily asparagine) and a reactive carbonyl, proceeding through intermediates that include a Schiff’s base. Several intrinsic and extrinsic factors, such as the initial concentration of reactants and their ratio, temperature and time of processing, have been shown to influence the levels of acrylamide in thermally processed foods. However, the reported data do not allow the development of engineering models for design, evaluation and optimization of food processes due to complex nature of Maillard reaction.

Artificial neural network (ANN) is a data-processing system based on the structure of biological neural system. Predictions with ANN are not like modeling simulation, but outputs are obtained from a learning algorithm based on experimental data. Here, ANN approach was used to predict acrylamide formed in model systems as influenced by various parameters. Concentrations of asparagine (1-10 µmoles), glucose (1-10 µmoles) and glycine (0-10 µmoles), the temperature (150-200 °C) and the time (0-60 min) were the parameters used as the input variables to build the ANN model. The experimental data (320 data points) were used for ANN training using a feed forward back propagation network algorithm. Decision on the use of a given number of hidden layer nodes is complex because it depends on the specific problem being solved. In our study, the architecture with two hidden (9 and 8 nodes) layers and one output layer (1 node) was selected. This network had the best prediction capabilities with a learning rate and momentum of 0.01 and 0.09, respectively. The linear regression coefficient of 1.0 was determined between measured and predicted acrylamide levels for the training data set.

S14

Influence of high hydrostatic pressure (HHP) on the formation of posttranslational protein modifications in arginine and lysine residues

Nadja Alt and Peter Schieberle

Institute for Food Chemistry, Technical University of Munich, Lichtenbergstrasse 4, D-85748 Garching, Germany

High-hydrostatic-pressure (HHP) processing of foods is a new technique mainly used for the inactivation of microorganisms, but also providing products with improved freshness and aroma, as well as alternative functional properties as compared to conventional thermal treatment. The Maillard reaction was previously shown to be influenced by HHP, but up to now, only a few data are available on the formation of posttranslational modified amino acids at HHP. Therefore, the aim of our study was to characterize the lysine and arginine modifications formed and to quantitatively compare their amounts generated either at atmospheric pressure (AP) or at HHP.

In an aqueous model system consisting of Nα-acetylarginine and glucose, first, the formation of arginine modifications at AP vs. HHP were examined. To avoid acidic conditions, a careful analytical procedure for the analysis of arginine modifications was developed using enzymic deacetylation followed by ion exchange chromatography with ninhydrine detection [1]. At HHP, two arginine modifications, namely N5-(5-hydro-5- methyl-4-imidazolon-2-yl)-ornithine and N7-(1-carboxyethyl)-arginine, were identified as major modified amino acids. Quantitative studies showed a significant increase of both arginine compounds with increasing pressure up to 600 MPa.

Approaching more food-like conditions, the influence of HHP on the formation of modified lysine and arginine residues in the model protein β-casein in the presence of reducing sugars at various temperatures was examined. Using LC-ESI-MS analysis, modified peptides could be characterized in tryptic digests of β- casein. Fructoselysine, carboxymethyllysine and pyrraline were detected as modifications formed at lysine residues. The arginine modifications observed were the imidazolinones derived from 2-oxopropanal and 3- desoxyglucosone, as well as N7-(1-carboxyethyl)-arginine. After synthesis of isotopically labeled peptides as internal standards, the formation of lysine and arginine modifications under HHP was quantitatively followed for lysine residue K176 and arginine residue R183. While lysine modifcations were formed at temperatures above 60 °C , but were significantly decreased at HHP, arginine modifications were only generated at temperatures above 90 °C, but were significantly increased with pressure application.

Because 2-oxopropanal was significantly increased with HHP in both model systems, a breakdown of the sugar backbone induced by HHP is undoubtedly the major cause for enhanced levels of amino acids derived from 2-oxopropanal, in particular the imidazolinones and N7-(1-carboxyethyl)-arginine. The data suggest that carboxymethyllysine is formed by a degradation of fructoselysine rather than by the reaction of lysine with glyoxal, because glyoxal was only formed at a very low levels. Also, arginine modifications derived from glyoxal could not be detected in the reaction mixtures.

[1] Alt, N.; Schieberle, P. (2005) Model studies on the influence of high hydrostatic pressure on the formation of glycated arginine modifications at elevated temperatures. J. Agric. Food Chem., 53, 5789- 5797. S15

REDUCING MRP'S IN LIQUID FOODS BY ULTRA-HIGH TEMPERATURE OHMIC HEATING

Eyal Shimoni

Department of Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel

KEYWORDS: Ohmic heating, peritoneal dialysis, glucose degradation.

High quality products are characterized by fresh like look and taste, high nutrients content and long shelf life. Thermal treatment is an essential step in order to kill microorganisms, and to inactivate destructive enzymes in order to extend shelf life. The traditional heating process is based on heat transfer from the outer surface into the product by means of both convection and conduction to achieve absolute assurance that all parts of the product have reached a certain temperature for a defined period of time. This situation may result in over heating of the product, cause loss of flavor and aroma, decrease in nutrients level and browning. Therefore, alternative non-thermal food preservation technologies were developed, including ohmic heating. Ohmic heating involves the flow of current in a continuous electrically conducting food product. The major advantage of electrical heating is the ability to heat materials rapidly and uniformly. Recently, we have demonstrated the efficacy of using ohmic heating to obtain orange juice with high sensorial quality. This technique however, may be of great interest whenever thermal abuse of the product is critical for its quality. Peritoneal dialysis is commonly performed by using pre-prepared dialysis solutions containing, among other things, glucose. As these solutions are prepared in batches of 0.5 to 1 liter, they are thermally treated to achieve commercial sterilization. The length of these thermal treatments at 120oC is 20 to over 40 minutes. As a result a series of glucose degradation products are being formed, which react with the tissue during the dialysis procedure, thus baring a negative effect on the patient and the dialysis process. Our recent work focused on testing the efficacy of ohmic heating as an alternative thermal treatment for continuous sterilization of peritoneal dialysis solutions. For this purpose the process was compared to conventional retort treatment, and the kinetics of glucose degradation products accumulation was measured. The results suggest that this technique can be used to produce solutions with much lower content of glucose degradation products.

S16

SHELF LIFE OF PEA NUTS WITH ROASTING DEGREE

Cämmerer B (1)*, Kroh LW (1), Schneeweiß R (2), Thomann R (2)

1 Institute of Food Chemistry, Technical University Berlin, Berlin, Germany

KEYWORDS: Maillard reaction, deoxyosone, EPR spectroscopy, pea nuts, shelf life

Fat containing, vegetable foods such as pea nuts and sesame seeds possess natural antioxidants e.g. tocopherols which protect fat against deterioration, but thereby were degraded or transformed into inactive compounds. When exposing boosted oxidative stress (e.g. storage at elevated temperatures) antioxidants were consumed rapidly and radical initiated reactions such as fat deterioration reactions take place.

By EPR spectroscopic methods such radical reactions can be detected quantitatively with the help of so called spin traps (lag time measurement) /1/.

During roasting of food via Maillard reaction Maillard reaction products and melanoidins were formed which are known for their antioxidative activity /2/. They can act as radical scavenger and thus are able to interfere with deterioration reactions and to improve shelf life of roasted pea nuts. The degree of Maillard reaction proceeded interrelated to the amount of compounds with antioxidantive properties formed can be estimated by quantitative determination of α-dicarbonyl compounds which are highly reactive intermediates of Maillard reaction /3/.

By correlation of kind and amount of deoxyosones formed with antioxidative stability of pea nuts measured by EPR spectroscopy a prediction of shelf life in dependence of roasting conditions was attempted. The demonstrated tendency is also in accordance with sensory investigations. Results obtained for antioxidant activity in pea nuts measured with conventional methods such as TEAC test or Rancimat test vary only marginal for different roasting degrees and did not allow a correlation with shelf life.

/1/ Uchida M, Suga S,Ono M (1996) J. Am. Soc. Brew. Chem. 54:198-204 /2/ Cämmerer B, Fuchs JI, Kroh LW (2000) Melanoidins in Food and Health vol. 2 159-164 /3/ Hollnagel A, Kroh LW, (1998) Z. Lebenm. Unters. Forsch. A 207: 50-54

S17

TECHNOLOGICAL PROPERTIES AND NON-ENZYMATIC BROWNING OF LUPIN- BASED FOOD PRODUCTS

Alessandra D’Agostina, Donatella Resta, Giovanna Boschin, Anna Arnoldi

Laboratory of Food Chemistry and Mass Spectrometry, Department of Agri-Food Molecular Sciences, University of Milan, Via Celoria 2, I-20133 Milano, Italy.

KEYWORDS: functional food, HMF, furosine, Lupinus albus

Consumers have a growing interest in foods containing components that may be beneficial for health. Two recent papers have reviewed all the data on the possible role of pulses in the prevention of the main risk factors of cardiovascular disease. The genus Lupinus appears to be particularly promising, since recent findings have indicated that a moderate daily intake of white lupin protein leads to the reduction of total and low-density lipoprotein cholesterol levels both in experimental animals and in the clinics. White lupin has some other features well accepted by consumers: there are no genetically modified varieties commercially available and the content of antinutritional components is lower than in soybean. We have therefore started to work on several lupin based food items with the objective to obtain a portfolio of functional food products with satisfactory sensory characteristics.

Since lupin proteins are much richer in lysine than cereals, it may be easily foreseen that lupin-based foods may be greatly affected by the Maillard Reaction (MR) through glycosylation of the lysine side chain. This topic in lupin foods had never been tackled before, as they are very innovative food items. Inside the EU Project Healthy-Profood, several food ingredients and food prototypes were developed. Our task was the investigation of the thermal damage induced by industrial processes, by quantifying two markers of the MR: HMF and furosine. The latter permitted also to calculate the level of non-bioavailable lysine. Our investigation indicated that these items are in line with data reported for standard foods and ingredients. One of the most interesting foods was spaghetti, which was prepared by replacing semolina with different amounts of lupin protein, in order to increase the protein content. Our results indicate that the markers content are quite low and the lysine loss due to MR is only marginal.

S18

EFFECTS OF HEATING AND NATURAL ANTIOXIDANTS ON THE CONTENT OF HETEROCYCLIC AMINES IN MEAT

Vinauskiene R., Venkutonis PR, Damasius J., Skripkauskaite A

Department of Food Technology, Kaunas University of Technology, Lithuania

KEYWORDS: heterocyclic amines, meat, heating, natural antioxidants

The aim of this study was to examine the effects of processing parameters and addition of plant extracts containing strong antioxidants on the formation of heterocyclic amines (HA) in different type of meat during cooking. The parameters for the analysis procedure were selected during the first phase of the study by using the mixtures of reference compounds (DMIP; AαC ; MeAαC; IQ; Trp-P-1; Trp-P-2; PhIP; 7,8- DiMeIQx) and reverse phase analytical HPLC column Synergi PowerRP. Data for calibration curves of reference amines were obtained on Waters ZQ 2000 mas detector operating at 45 V. Further the procedure for the extraction of HAs from meat samples was optimised by using Chromabond XTR and Chromabond PS-H+ cartridges. The effects of heating time and temperature on the content of HAs in poultry and beef was studied and it was found that beef accumulates remarkably higher amounts of HAs during heating as compared with poultry. The amount of HAs increased with the increase of heating time as well. The extract obtained from oregano (Origanum vulgare L.) possessing strong radical scavenging capacity was added to meat at various concentrations and the content of HAs was determined by the elaborated techniques.

S19

STUDY OF ANTIOXIDANT ACTIVITY OF NEOFORMED PRODUCTS FROM HEATED CYSTEINE AND ALDEHYDES

Cheriot Sophie, Billaud Catherine, Nicolas Jacques

Chaire de Biochimie Industrielle et Agro-Alimentaires, Conservatoire National des Arts et Métiers, Paris, France

KEYWORDS: Maillard products; antioxidant; neoformed; cysteine; 5-HMF

In previous works, we found that the soluble part of Maillard reaction products (MRP)made from thiol compounds and glucose contained very powerful inhibitors of enzymatic browning, a phenomenon detrimental to the quality of fresh and minimally processed fruits and vegetables. Because Maillard reaction is a complex whole of reactions between amino and aldehydic or ketonic compounds to produce a wide range of intermediate products, we decided to simplified the model reaction and showed that neoformed products from heated sulfur compounds (cysteine) with some pure aldehydic components (5- hydroxymethylfurfural, furfural and benzaldehyde) exhibited a stronger inhibitory potency toward enzymatic browning of potato, eggplant, apple and mushroom than former MRP. The first part of our research focused on the optimization of the antioxidant activity (AA) of selected mixtures. The effects of the nature and the ratio of the components in the mixture, the pH of cysteine before heating and the heating treatment (combination of duration and temperature) were optimized. In a second part, the stability of the AA for the neoformed products was investigated. AA was kept during storage at 4 °C, 25 °C and 37 °C but lost when exposed to UV radiation. The acidification of the mixtures (e.g heated cysteine / 5-HMF) during storage was favorable to the stability of their antioxidant properties in contrast with alkaline reaction conditions. In order to investigate the structure of the compound(s) involved in the AA, preliminary fractionation and analyses were carried out using HPLC-DAD on the soluble part and CG-MS on dicholoromethane extract of the heated cysteine / aldehyde mixtures.

S20

TASTE-ACTIVE GLYCO-CONJUGATES AND THEIR FORMATION UPON THERMAL FOOD PROCESSING

Hofmann T.

Institute of Food Chemistry, University of Muenster, Germany

KEYWORDS: Maillard reaction, glyco-conjugates, taste, taste enhancer

Reducing carbohydrates and amino acids are well-known to generate intense flavor compounds during thermal food processing. Although a tremendous multiplicity of investigations have been focused towards the volatile aroma compounds formed in course of this so-called Maillard reaction, the molecular information on the non-volatiles inducing gustatory responses is very poor. To fill this lack of information, a screening technique combining instrumental analysis with human psychophysics was developed to sort out the taste-active key compounds from the large number of taste- inactive substances. Application of this so-called taste dilution analysis on thermally processed foods and carbohydrate containing model systems, followed by LC-MS/MS, 1D- and 2D-NMR experiments as well as C-13 labeling studies led to the discovery of various previously unknown, sensory active non-volatiles exhibiting umami-like taste and bitterness, respectively, eliciting chemesthetic effects on the tongue such as, e.g. astringency, or modifying the human sensitivity for the oral chemoreception of basic taste compounds. Most interestingly, these investigations revealed that, besides carbohydrate/amino acid reaction products, sensory active glyco-conjugates are formed by means of Maillard-type reaction chemistry involving organic acids as well as polyphenols. Screening, structure determination, formation pathways, and sensory activity of individual chemosensates will be presented in the lecture. S21

LC-MS/MS QUANTITATION, HOTSPOT SITE LOCATION AND PREDICTED STRUCTURES OF PROTEINS GLYCATED BY DICARBONYLS - A SYSTEMS, INFORMATION-RICH APPROACH

Paul J. Thornalley

Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.

KEYWORDS: LC-MS/MS, proteomics, peptide mapping, albumin, hemoglobin, collagen, methylglyoxal

The application of LC-MS/MS to the quantitation of glycation adducts in physiological systems has shown dicarbonyl-derived advanced glycation endproducts (AGEs) are major glycation adduct residues of proteins and peptides and major glycation free adducts of plasma and urine. They have distinctive links to glycemic control, lipid metabolism, oxidative stress, dicarbonyl stress, renal function and donor age. Proteomics and peptide mapping of dicarbonyl glycation has revealed proteins and arginine residues within them that are hot spots for glycation – leading to concomitant functional impairment. Molecular graphics and bioinformatics approaches predict structural changes and other susceptible sites of dicarbonyl glycation. Overexpression and silencing of glyoxalase 1 provides genetic controls for investigation of cellular effects. Dysfunctional dicarbonyl metabolism is now implicated in vascular disease, ageing and other pathophysiological disorders.

S22

METHIONINE SULFOXIDE, A SENSITIVE BIOMARKER OF OXIDATIVE DAMAGE TO PROTEINS

Brock JWC, Jenkins AJ, Thorpe SR, Baynes JW*

University of South Carolina, Columbia, SC, USA and University of Melbourne, Melbourne, AU

KEYWORDS: methionine sulfoxide, glycoxidation, lipoxidation, apoA-I, HDL

Reactive oxygen species, produced during Maillard reactions of sugars and lipids with protein, irreversibly oxidize ambient proteins. Methionine is particularly susceptible to oxidation, yielding methionine sulfoxide (MetSO). Protein concentrations of MetSO therefore should provide a useful index of exposure of proteins to glycoxidative and lipoxidative stress.

In vitro reactions were conducted by incubation of RNase with ribose or arachidonate under physiological conditions. HDL was obtained from healthy and diabetic subjects by ultracentrifugation. Modified RNase and delipidated HDL were digested with trypsin and analyzed by RP-HPLC-ESI-MS. Chloramine-T (CT) was used as a non-specific oxidant.

MetSO increased in the model protein RNase during aerobic incubation with ribose or arachidonate. The concentration of MetSO was significantly greater than that of AGE/ALEs in glycoxidized and lipoxidized RNase. The same Met residues were oxidized by CT and during glycoxidation and lipoxidation reactions, significantly more MetSO was produced during lipoxidation by arachidonate, compared to glycoxidation by ribose.

Analysis of ApoA-I, the major protein component of HDL, indicated that there was also selective oxidation of Met residues by a non-specific oxidant, such as CT. The MetSO content of ApoA-I from type 1 diabetic patients was, on average 5 times higher than that from non-diabetic controls, but the extent of oxidation of Met was not dependent on the presence of complications.

MetSO is a sensitive indicator of protein modification by glycoxidation and lipoxidation reactions. Lipoxidation is a more likely source of oxidative damage to proteins in the body. The increase in MetSO formation in IDDM patients with and without complications indicates an increase in oxidative stress in diabetes, but independent of the presence of complications. Further studies on methionine oxidation in plasma proteins and correlation of MetSO with progression of complications should improve understanding of the role of oxidative stress in the pathogenesis of diabetic complications.

S23

RECENT ADVANCES IN ACRYLAMIDE ANALYSIS, FORMATION AND MITIGATION IN FOODS

Stadler Richard H.

Nestlé Product Technology Centre, Orbe, Switzerland

KEYWORDS: Acrylamide, analysis, food, coffee, cereals, potatoes, CIAA Toolbox

After the announcement of the occurrence of acrylamide in foods in April 2002, research was conducted to (i) analyse acrylamide in the many different foods of concern (ii) understand its formation, and (iii) investigate possible ways to reduce this undesired Maillard by-product. Today, analytical methods are available to reliably measure acrylamide in all foods of concern, but no single standard method has yet been proposed. Analysts are still “improving” methods, particularly for difficult matrices such as cocoa and coffee. In addition, the conditions of extraction, in especially if these are harsh, may lead to erroneous results due to the formation of AA from precursor molecules. The amino acid asparagine furnishes the backbone of the acrylamide molecule, and the Maillard pathway is today considered the main route common to all foods. However, it has been difficult to identify the salient intermediates, and 3-aminopropionamide (3-APA) has been proposed to play a key role in the formation of acrylamide.

The CIAA (Confederation of the European Food and Drink Industries) established an Acrylamide Technical Expert Group with the main objective of co-ordinating the industry-driven research and sharing the results of the different research initiatives, documented by many publications to date by the group and its members on acrylamide analysis, mechanisms of formation, and mitigation in several peer-reviewed publications. A major milestone in terms of identifying options for interventions that may lead to a reduction of acrylamide in certain foods has been the establishment of the CIAA “Toolbox”. The acrylamide Toolbox is in essence an inventory of interventions, comprised of four major compartments, i.e. natural parameters, product composition (recipes), process technologies and conditions, and final product characteristics (e.g. color). The idea is to share across the different food categories the most promising leads, and possibly benefit from the established knowledge base in neighboring categories.

Recently, the EC together with the Confederation of the European Food and Drink Industries (CIAA), convened a meeting of experts to further develop and update the CIAA acrylamide “Toolbox”. The key achievements pertaining to mitigation in the potato, cereal and coffee sectors will be presented as well as a summary of the future opportunities, gaps and constraints in the different foods. Selected tools may also be of value in reducing acrylamide formation in the domestic cooking environment, and research activities into the contribution of home cooking have recently been launched. However, certain constraints must be acknowledged. In some cases interventions that appear successful in laboratory or modeling experiments do not reflect the same degree of success in a factory environment. Furthermore, in some foods a meaningful reduction in the short to medium term may not be feasible, and certain changes may negatively impact the overall nutritional value of the foods (e.g. by avoiding whole grain) or lead to the formation of other undesired compounds (e.g. 3-monochloropropanediol). Thus, a holistic risk/benefit approach may be warranted instead of scrutiny of a single molecule, particularly for those foods with known health-promoting constituents that may be compromised through certain interventions.

The CIAA Acrylamide Toolbox. ISSN 1782-1584, 23 Sept 2005, http://www.ciaa.be/ciaa Stadler, R.H.; Scholz, G. Nutrition Reviews 2004, 62, 449-467.

S24

S-(2-SUCCINYL)-CYSTEINE: A NOVEL CHEMICAL MODIFICATION OF TISSUE PROTEINS

Blatnik M, Frizzell N, Alt N, Baynes JW, Thorpe SR*

Department of Chemistry and Biochemistry, University of South Carolina, Columbia SC, USA

KEYWORDS: GAPDH, muscle, fumarate, diabetes

Much attention has been focused Maillard reactions between carbonyl compounds and the amino and guanidine groups of lysine and arginine in tissue protein. However, inside cells, the free sulfhydryl group of cysteine is a more reactive nucelophile. We recently detected S-(carboxymethyl)-cysteine as a product of reaction of glyoxal with cysteine residues in protein and report here on the detection of S-(2-succinyl)- cysteine (2SC) in human plasma protein and skin collagen and in rat muscle protein. 2SC, like other AGE/ALES, was increased in muscle protein in diabetic rats.

We hypothesized that fumarate, via a Michael addition reaction, was the physiological source for the formation of 2SC, and that fumarate inhibition of sulfhydryl enzymes might contribute to altered metabolism in diabetes. This hypothesis was tested with the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Fumarate but not succinate inhibited GAPDH in a time and concentration dependent manner, with concomitant formation of 2-SC. MADLI-TOF analysis of tryptic digests of GAPDH showed modification of 3 of the 4 Cys residues of GAPDH. We also observed that GAPDH activity was 30% lower (p = 0.025) in muscle from diabetic compared to control animals (n = 6 for both groups). Based on GC-MS measurements, fumarate was 3.7 fold higher and total 2SC was 4.4 fold higher in the diabetic samples. Studies are in progress to measure the 2SC content of GAPDH from control and diabetic samples.

These results are consistent with fumarate as a physiological source of 2SC in tissue protein and suggest that formation of 2SC at the active site of various sulfhydryl enzymes and transcription factors may contribute to metabolic abnormalities in diabetes.

S25

FORMATION OF FURAN AND METHYLFURAN UNDER ROASTING CONDITIONS

Imre Blank (1,2)*, Julia Märk (1,3), Philippe Pollien (1), Christian Lindinger (1), and Tilmann Märk (3)

1 Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland, 2 Nestle Product Technology Cente (PTC) Orber, 1350 Orbe, Switzerland, 3 Institut für Ionenphysik, Leopold Franzens Universität, 6020 Innsbruck, Austria

KEYWORDS: furan, methylfuran, ascorbic acid, PUFA, Maillard reaction, lipid oxidation, PTR-MS

Furan has recently received attention as a possibly hazardous compound occurring in certain thermally processed foods. Previous model studies have revealed three main precursor systems producing furan upon thermal treatment, i.e. ascorbic acid, Maillard precursors, and polyunsaturated lipids. We employed proton transfer reaction mass spectrometry (PTR-MS) to study the formation of furans under dry roasting conditions. Unambiguous identification and quantification in the headspace was achieved by PTR-MS coupled to GC-MS. Ascorbic acid showed the highest potential to generate furan, followed by glyceryl trilinolenate. Some of the reaction samples generated methylfuran as well, such as Maillard systems containing alanine and threonine as well as lipids based on linolenic acid. Significantly lower yields of furan were obtained from ascorbic acid in an oxygen-free atmosphere (30%) or in the presence of reducing agents (e.g. sulfite, 60%), indicating the important role of oxidaton steps in the furan formation pathway. Already simple binary mixtures of ascorbic acid and amino acids, sugars or lipids reduced furan by 50-95%. These data suggest that more complex reaction systems result in significantly lower furan amounts compared to the individual precursors, most likely due to competing reaction pathways. The formation of furans from the different precursors is discussed. Experiments were performed using labeled precursors to elucidate the formation mechanisms of furan and methylfuran.

S26

VINYLOGOUS AMADORI REARRANGEMENT: CONSEQUENCES IN FOOD AND BIOLOGICAL SYSTEMS.

Yaylayan, VA & Perez, CL

Department of Food Science and Agricultural Chemistry, McGill University

KEYWORDS: Keywords: Vinylogous Amadori rearrangement, pyridoxal, vanillin, salicylaldehyde, HNE

Schiff-base formation of amino acids with aromatic aldehydes have been extensively studied due to their importance as reversible oxygen carriers, as catalysts in the hydrogenation of olefins and for their ability to complex toxic metals. Furthermore, the Schiff bases of primary amines with o- or p-hydoxybenzaldehyde derivatives have been reported to undergo further isomerization to form keto-enamine moiety. This tautomeric shift can be considered as vinylogous Amadori rearrangement, since structurally, the hydroxyaldehyde moieties in o- or p-hydroxybenzaldehydes are vinylogous structures to α-hydroxyaldehyde moiety in the reducing sugars. The corresponding non-aromatic systems such as 4-hydroxyalkenals (i.e.HNE) of lipid oxidation by-products can also be considered as vinylogous moieties to α- hydroxyaldehydes in reducing sugars and are expected to undergo a similar vinylogous Amadori rearrangement in the presence of proteins and contribute to the formation of ALEs. Spectroscopic studies have indicated that food and biologically relevant compounds such as pyridoxal, vanillin, salicylaldehyde, etc. can indeed undergo vinylogous Amadori rearrangement with amino acids. The chemistry of vinylogous Amadori rearrangement will be further explored in this presentation.

S27

SIMULTANEOUS ANALYSIS OF REACTIVE LYSINE, FUROSINE AND CARBOXYMETHYLLYSINE BY GC-MS: APPLICATION TO A WIDE RANGE OF HEAT-TREATED FOODS

Amélie Charissou and Inès Birlouez-Aragon

Institut National Agronomique, Paris-Grignon.

KEYWORDS: heat-treated foods, furosine, CML, reactive lysine, GC-MS

The Maillard reaction has an important impact on the nutritional quality and safety of heat-treated foods justifying the need for pertinent indicators and the development of accurate analytical methods. Lysine is one of the most reactive amino-acid in foods, and its blockage during the reaction provokes a rapid decrease in the nutritional value of the food protein. The early Maillard product, fructosyllysine (FL) is the most abundant derivative accounting for major lysine unavailability. Despite of representing no more than 1%o of Maillard reactive lysine, carboxymethyllysine (CML), formed by oxidative degradation of FL, is an interesting indicator because of possible pro-inflammatory effects after intestinal absorption. This paper proposes a simultaneous quantification of lysine, FL and CML in food proteins by GC-MS after acetylation of the carboxylic groups and acylation of the amino groups followed by single ion monitoring. Reduction of fructosyllysine before hydrolysis is needed for FL levels higher than 100 mg.100g-1. Using CML isotope dilution instead of conventional internal standard allows to decrease the repeatability error, from 5% to 1%. Furosine evaluation by GC-MS is in agreement with the data obtained by HPLC-UV. However, except in the case of simple models, CML levels obtained by GC-MS are much lower than those resulting from ELISA analysis. Some examples are given of the concentration of the three indicators in heat-treated food samples. Furosine and CML are essentially found in samples with high sugar content; no CML was found in low carbohydrate and high fat-containing foods, what is contrary to the data obtained by ELISA. In low treated foods, acid-released lysine was better correlated to furosine, whereas in severely treated foods, it was much better correlated to CML. In conclusion, simultaneous monitoring of reactive lysine, furosine and CML allows a rapid global view of the protein nutritional quality of heat-treated foods.

S28

SYNTHESIS AND STABILITY OF THE EARLY GLYCATION PRODUCTS FORMED FROM D-FRUCTOSE AND ENDOGENOUS OPIOD PEPTIDES

Jakas A*, Horvat S.

Division of Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, Zagreb, Croatia

KEYWORDS: leucine-enkephalin, methionine-enkephalin, D-fructose, Heyns compound, imidazolidinone

Glycation reactions of the endogenous opioid pentapeptides Leu-enkephalin or Met-enkephalin with D- fructose under different conditions (solvent, temperature) were examined. N-(2-Deoxy-D-glucos-2-yl)-L- tyrosylglycylglycyl-L-phenylalanyl-L-leucine/methionine and N-(2-deoxy-D-mannos-2-yl)-L- tyrosylglycylglycyl-L-phenylalanyl-L-leucine/methionine (Heyns compounds), as well as two isomers of the imidazolidinones, N-{[2-(D-arabino-tetritol-1-yl)-4-(4-hydroxybenzyl)-2-hydroxymethyl-5-oxoimidazolidin-1- yl]acetyl}glycyl-L-phenylalanyl-L-leucine/methionine, were obtained as major products. Different reactivity of Leu- and Met-enkephalin in the glycation reaction under identical conditions was observed mainly due to the tendency of methionine residue in Met-enkephalin to be oxidized to sulfoxide. Oxidation depends on the D-fructose concentration: the higher the concentration of D-fructose the faster the oxidation. The influence of N-glycation on the enzymatic and chemical stability of the parent peptide compounds in human serum and phosphate buffer, pH 7.4, was studied. A difference in degradation kinetics was observed not only between the different types of glycated products (Heyns and imidazolidinone compounds), but also between two isomers of the same imidazolidinone.

S29

CARBOHYDRATE BASED MELANOIDINS: SEVERAL α-DICARBONYL COMPOUNDS AS DIRECT PRECURSOR FOR THE FORMATION OF DISCRETE MOLECULAR SIZE DOMAINS

Fiedler Th and Kroh LW.

Institute of Food Technology and Food Chemistry, Department of Food Analysis Technical University of Berlin, Germany

KEYWORDS: Maillard reaction, α-dicarbonyl compounds, melanoidins, 3-deoxyhexosulose, methylglyoxal

Melanoidins are brown colour matters with very heterogeneous structures and different chromophore functional groups. They are end products of the reaction between reducing sugars and amino compounds, so-called Maillard reaction [1]. In literature numerously different structures for melanoidins are described, e.g. melanoidins with furan skeletal structure and little molecular weight and melanoidins with protein crosslink participating pronyl-lysin and a high molecular weight [2]. α-Dicarbonyl compounds can also polymerise in a carbohydrate-based aldol condensation and become brown sugar colours, called melanoidins [3]. During sugar production especially at the evaporation station temperatures up to 130°C act on the sucrose solution. Because of the thermal treatment the formation of reactive intermediates are accelerated and the result is that the sucrose solution contains brown coloured matters, which are enclosed in sugar crystals and affect adversely the quality of white sugar. In high concentrated carbohydrate solutions α-dicarbonyl compounds are as high reactive intermediates against to other structures precursor of melanoidins. With HPLC-DAD and GC/MS 3-deoxyhexosulose, D-glucosone and methylglyoxal could be determined as the most important -dicarbonyls in concentrated carbohydrate solutions [4]. The aim of this research is the characterisation of specific molecular size domains of melanoidins, which are preferable formed in high concentrated sucrose solutions during thermal treatment. The investigations show that α-dicarbonyl compounds are not only important intermediates but also direct precursor for melanoidin formation in high concentrated carbohydrate solutions. Depending on the involvement of several α-dicarbonyl compounds specific molecular size domains were formed, which were determined by size exclusion chromatography (SEC-DAD/RI). Thereby methylglyoxal promotes a high molecular size domain, whereas a lower molecular size domain based on 3-deoxyhexosulose and D- glucosone. Thus it is the first time that molecular size domains of carbohydrate based melanoidins could be directly attached with the involved α-dicarbonyl compounds. With rising pH-value, preferable at high alkaline conditions, the reactivity of all investigated α-dicarbonyls are increasing, whereas always a correlated increasing colour formation could be determined. The presence of an amino compound also leads to an increasing colour formation. A gradation of the degradation and the correlated colour formation of the investigated α-dicarbonyl compounds could be determined. D-glucosone shows the lowest browning activity followed by 3-deoxyhexosulose and methylglyoxal with the highest reactivity. With these new investigations the mechanism of carbohydrate-based melanoidin formation, which describe as an aldol condensation could be approved [3]. Finally it could be concluded, that the melanoidin formation and so the colour formation with high alkaline pH-value is accelerated and under acid conditions the reactivity of -dicarbonyl compounds to colour formation is suppressed.

[1] Ledl F, Schleicher E (1990) Angewandte Chemie 102:597-734 [2] Lindenmeier M, Hofmann T (2004) J Agric Food Chem 52:350-354 [3] Cämmerer B, Jalyschko W, Kroh LW (2002) J Agric Food Chem 50:2083-2087 [4] Fiedler Th, Moritz Th, Kroh LW (2005) Eur Food Res Technol, in press

S30

STUDY OF "MASKED" ACRYLAMIDE ORIGIN

Hajslova J*, Vaclavik L, Novotny L, Dunovska L, Holadova K

Institute of Chemical Technology, Dept. Food Chemistry & Analysis, Prague, CZ

KEYWORDS: masked acrylamide, alkaline extraction, mechanism of release

Recently, Eriksson and Karlsson reported on the increased acrylamide recovery when using alkaline solution for extraction of heat processed foodstuffs. Authors suggested changes in matrix structure to be responsible for releasing “masked” acrylamide. However, since we found elevated acrylamide levels also in alkalinized solution of soluble coffee substitute, the hypothesis on the matrix influence did not seem to represent the only alternative explaining acrylamide increase. To elucidate this phenomenon we conducted a series of model Maillard experiments. The dynamics of free and “total” acrylamide levels in equimolar asparagine - sugar (glucose, fructose and sucrose) mixtures heated at 100, 140, 180°C was measured within 90 min. The largest difference more than one order of magnitude - between the acrylamide content determined in pyrolysate dissolved in water and its concentration measured after alkalinization (pH 12) was observed in glucose - asparagine mixture heated 5 minutes at 140°C. Although absolute concentrations of acrylamide were higher in pyrolysates obtained at 180°C, the amount of ”masked” acrylamide was fairly lower at higher temperature. In all model systems successive slight decrease of both native and 13C1 acrylamide occurred in later phases of heating. In our presentation the results of follow-up experiments aimed at identification of the most probable precursors of “masked” acrylamide will be reported. According to our preliminary observations N-(D-glucos-1-yl)-L-asparaginate and N-(D-fructos-2-yl)-L-asparaginate are potent precursors of alkali extracted acrylamide. However, although release of acrylamide from these N- glycosides was found in alkali and even in neutral solutions, its extent was not as extensive as expected. With regard to this facts we consider their decarboxylated products as the direct intermediates of alkali extracted acrylamide. In any case, deep understanding of "masked" acrylamide issue, mainly its potential release from Maillard precusrors under physiological conditions, is needed to get more realistic data for dietary exposure assessment.

S31

PROBLEMS IN THE ASSESSMENT OF MRP INTAKE IN HUMANS

Simonetta Salvini

Molecular and Nutritional Epidemiology Unit, CSPO - Scientific Institute of Tuscany, Florence, Italy.

KEYWORDS: Maillard Reaction Products, Food Composition Databases, Epidemiological studies

Maillard Reaction Products (MRPs) are a very large and heterogeneous group of compounds, that can be found in food of various origin, and are formed during heat treatment at some stage of the production process, or during home preparation. Assessment of food intake in humans is not an easy task, and various approaches can be considered, depending on the different situations. Several aspects should be considered in designing studies to assess intake: identify the target population; identify the compounds of interest; verify the existence of information on the concentration of the compounds of interest in the food commonly consumed by the target population; identify the dietary assessment method to be used to evaluate intake; and, if needed, validate the instrument to be used. Identification of the compounds of interest is a big challenge. The name MRP include a quite large class of compounds, ranging from substances supposed to have a negative effect on health to compounds that might even have protective effects. The number of compounds that can be classified under this name is very large, and they should in theory all be included in the so called food composition databases (FCDs), instruments that should contain information about the composition of foods is terms of nutrients and non- nutrient compounds. However, these are never exhaustive neither in terms of food included, nor in terms of the compounds considered. FCDs should include all food representative of the diets of the population of interest, but this concept needs to be defined specifically for each situation. Data from such datasets should then be applied to consumption data, collected by the appropriate validated instruments (diaries, recalls, or questionnaires). In other instances laboratory analyses of the compounds of interest might be carried out on samples of duplicate diets, in order to have a direct measure of the concentration of the compounds of interest. The creation of databases including MRP is a challenge that might be considered, but that needs resources and competence in order to be successfully implemented. Similar challenges are faced by researchers devoted to the study of bioactive compounds present in food of vegetable origin, another large and heterogeneous group of molecules.

S32

HUMAN EXPOSURE AND INTERNAL DOSE ASSESSMENTS OF ACRYLAMIDE IN FOOD

Dybing E*, Lalljie SPD**, Tuijtelaars S***

*Norwegian Institute of Public Health, Division of Environmental Medicine, Oslo, Norway, **SEAC - Unilever, Bedford, UK, ***ILSI Europe, Brussels, Belgium

KEYWORDS: Acrylamide, exposure assessment, risk-benefit, mitigation

Since the finding that acrylamide is formed in food during heat processing and preparation of food, much effort has been put into understanding its mechanism of formation, on developing analytical methods and determination of levels in food, and on evaluation of its toxicity and potential human health consequences. A systematic review of key information relevant to exposure assessment was lacking. The European and North American branches of ILSI discussed critical aspects of exposure assessment, parameters influencing the outcome of exposure assessment and summarized the data that was currently available and relevant to the acrylamide exposure assessment to aid the risk characterisation process.

This review provides a framework contributing to the risk assessment of acrylamide in food. It adds to the overall risk assessment framework by focusing especially on exposure assessment and internal dose assessment of acrylamide in food.

The paper reviews the data on acrylamide levels in food including its formation and analytical methods, the determination of human consumption patterns, dietary intake of the general population, estimation of maximum intake levels and identification of groups of potentially high intakes. Furthermore the association of intake levels with biomarkers of exposure and internal dose, considering aspects of bioavailability, is reviewed, and a physiologically-based toxicokinetic (PBTK) model is presented that provides a good description of the kinetics of acrylamide in the rat.

Each of the sections concludes with a summary of remaining gaps and uncertainties. Reduction of acrylamide in food may have an impact on the nutritional quality and safety of food. Appropriate comparison of added risks and /or benefits with the benefit of acrylamide reduction is needed. Consequently, ILSI Europe initiated a project on Risk-benefit Analysis of mitigation measures', with a main aim to understand the impact of mitigation steps on safety, quality, nutritional and organoleptic parameters.

S33

COFFEE: A PROTECTIVE BEVERAGE?

Pellegrini N.

Department of Public Health, University of Parma, Italy.

Cooking and food processing at high temperatures have been showed to generate various kinds of genotoxic substances such as polycyclic aromatic hydrocarbons, N-nitroso compounds and heterocyclic amines. The heterocyclic amines, including acrylamide, are formed in the course of Maillard reactions. Today, there is growing concern about the impact of these substances on human health1. Coffee is one of the most popular and widely consumed beverages through the world and is a rich source of Maillard reaction products (MRPs), developed during the roasting process, and many other ingredients, like polyphenols and caffeine. Even though its high content of MRPs, to date the effects of regular coffee consumption on human health are still conflicting. In fact, some epidemiological studies suggest that consumption of coffee is associated with elevated risk for cardiovascular disease (CVD) such as myocardial infarction2 and acute coronary events3. A J-shaped and U-shaped relation between coffee consumption and the risk of developing acute coronary syndromes4 and CVD incidence3, respectively has been found. On the other hands, several epidemiological studies found that the coffee is associated with reduced plasma γ- glutamyl transpeptidase5, a suggested biomarker for early oxidative stress, reduced risk of symptomatic gallstone disease6, colon cancer7, type II diabetes8 and can reduce the risk of elevated alanine aminotransferase activity in individuals at high risk for liver disease9. The role of caffeine in the modulation of health effect of coffee has been extensively studied for years, even if it is still a controversial issue. Moreover, in the last years, attention has been paid on strong antioxidant activity of MRPs and polyphenols present in coffee. It has been recently demonstrated that the total antioxidant capacity (TAC) of the diet, which describes the ability of the different food antioxidants (independent of their bioavailability) in scavenging preformed free radicals, may be related to efficacy of protection against gastric cancer10 and beneficial effects against inflammatory processes11. Coffee beverages have demonstrated to possess strong antioxidant capacity in vitro in different systems12,13. Due to its high TAC, in several population studies, it has been showed that the main contributor to the TAC of diet is the intake of coffee, which represents the 40-60% of daily mean intake of TAC, overcoming the contribution of well-known antioxidant foods such as fruit and vegetables. In fact, coffee is the major contributor (∼ 64%) to the TAC intake in 61 Norwegians14 as well as in Spanish diet15. Similar results were obtained in our non-diabetic, old-adult population of 285 workers and ex-workers of the food company Barilla where coffee contributed between 37.9 and 54.3% of TAC intake in women and between 26.5 and 36.6% in men, dependently on the TAC assay and the tool of food intake assessment, followed by fruits and vegetables. Until now, little is known about the absorption of antioxidant compounds of coffee, even though it has been demonstrated that coffee affects the lower-gut functions through the action of polyphenols, which increased the antioxidant capacity and the weight of faeces in a group of healthy subjects on a freely-selected diet16, and of MRPs, which are able to modulate the microflora in vitro similarly to the action of lactose and other poorly digestible carbohydrates17. Thus, several questions related to coffee remain open: (i) the high antioxidant effect in vitro of coffee is reflected in a protective effect in vivo and (ii) the antioxidant mechanism could be involved in the protective role emerged in some epidemiological studies.

1Jagerstad M., Skog K., Mut Res 2005, 574, 156–172; 2Tavani A. et al., Eur J Epidemiol 2001, 17, 1131- 1137; 3Happonen P. et al., J Nutr 2004, 134, 2381-2386; 4Panagiotakos D.B. et al., J Nutr 2003, 133, 3228- 3232; 5Tverdal A., Skurtveit S., Ann Epidemiol 2003, 13, 419–423; 6Leitzmann M.F. et al., Gastroenterology 2002, 123, 1823-1830; 7Tavani A., La Vecchia C., Cancer Causes Control 2004, 15, 743–757; 8Salazar- Martinez E. et al., Ann Intern Med 2004, 140, 1-8; 9Homan D.J., Mobarhan S. Nutr Rev 2006, 64, 43-46; 10Serafini M. et al., Gastrenterology 2002, 123, 985-991; 11Brighenti F. et al., Brit J Nutr 2005, 93, 619-625; 12Pellegrini N. et al., J Nutr 2003, 133, 2812-2819; 13Sanchez-Gonzales I. et al., Food Chem 2005, 90, 133- 139; 14Svilaas A. et al., J Nutr 2004, 134, 562-567; 15Saura-Calixto F. et al., Food Chem. 2006, 94, 442-447; 16Garsetti M. et al., Brit J Nutr 2000, 84, 705-710; 17Ames J.M. et al., Brit J Nutr 1999, 82, 489-495. S34

RENOPROTECTION BEYOND BLOOD PRESSURE LOWERING AND INHIBITION OF THE RENIN ANGIOTENSIN SYSTEM

Toshio Miyata

Tokai University School of Medicine, Kanagawa, Japan

KEYWORDS: renoprotection, blood pressure, oxidative stress, metal chelation, AGEs

Angiotensin II receptor blockers (ARB) protect the kidney, heart, and brain, independently of their effect on blood pressure lowering. Unfortunately, the use of ARBs in normotensive patients is curtailed by an attendant hypotension. Tissue protection has been ascribed, at least in part, to the inhibition of oxidative stress and of advanced glycation end product (AGE) formation. In this study, we identify a new oxidative stress and AGE inhibitor, devoid of effect on blood pressure, and assess its in vivo renoprotective effects. Screening of a large chemical library (~1300 compounds) disclosed that edaravone, a drug used to treat cerebral infarction, inhibits in vitro AGE formation. Unfortunately, like most AGE inhibitors, it also traps pyridoxal, limiting its clinical usefulness. A structure-function study of its chemical derivatives traced this entrapment to the alpha-methylene group, whose deletion led to the eventual synthesis of an original compound, 1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-6-methyl-1,3-dihydro-furo[3,4-c]pyridine-7-ol (TM2002). In vitro, TM2002 does not trap pyridoxal. It inhibits AGE formation as well as hydroxyl radicals mediated o-tyrosine formation and transition metals catalyzed oxidation of ascorbic acid (the Fenton reaction). It has no affinity for the human angiotensin II receptor. In rat renal, cardiovascular, and cerebral disease models, TM2002 markedly improves tissue damage without modification of systolic blood pressure. This compound might prove protective in various diseases, in which AGEs and oxidative stress have been implicated.

S35

VITAMIN C MEDIATES LENS CRYSTALLIN AGING BY GLYCATION IN A HUMANIZED TRANSGENIC MOUSE MODEL

V.M. Monnier, X.J.Fan, L. Reneker, S.M.Jarvis.

Depts. of Pathology and Biochemistry, Case Western Reserve University, Dept of Ophthalmology, University of Missouri, Columbia, MO., Research School of Biosciences, University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, United Kingdom

Human lens crystallins become progressively pigmented and crosslinked with age, in part due to glycation reactions by reactive carbonyl compounds. We and others hypothesized this process might be in part mediated by ascorbic acid oxidation products. However, unequivocal demonstration of this concept has not been possible due to the similarity of advanced glycation products (AGEs) from reducing sugars and oxidized vitamin C compounds. Taking advantage from the fact that mice have very low lenticular levels of vitamin C, we created a humanized transgenic mouse lens expressing high levels of the human sodium dependent vitamin C transporter 2 (SVCT2) under control of the mouse alpha-crystallin promoter-chick lens delta crystallin enhancer (denaA). Analysis of the founder line (n=5), F1 and F2 revealed sustained lenticular expression of SVCT2, with a 10-20 fold elevation of ascorbic acid (ASA) (1-3 mM) and dehydroascorbic acid (DHA) (0.1- 0.4 mM) levels compared to wild-type, without impairment of GSH homeostasis in the transgenic lenses. At 3 and 6mos, the ASA-derived glycation products pentosidine and fluorescent K2P crosslink were several fold increased (p<0.0001), while the glucose-derived Amadori product was normal. Longitudinal studies are being carried out to evaulate the long-term impact of accelerated ascorbylation on the crystallin structure and the effect of pharmacological intervention of the process. This study provides unequivocal evidence for the participation of vitamin C degradation products in the chemical aging process of the lens.

S36

FRUCTOSAMINE 3-KINASE AND OTHER ENZYMES INVOLVED IN PROTEIN DEGLYCATION

Van Schaftingen E.

Laboratory of Physiological Chemistry, ICP and Université catholique de Louvain, Brussels, Belgium

KEYWORDS: fructosamine, ribulosamine, kinases, phosphatases

Fructosamine 3-kinase (FN3K) is a recently identified enzyme that phosphorylates both low-molecular- weight and protein-bound fructosamines. Fructosamine 3-phosphates are unstable, breaking down spontaneously to 3-deoxyglucosone, inorganic phosphate and the amino compound that originally reacted with glucose. FN3K is therefore a ‘deglycating’ enzyme. Evidence has been provided that this enzyme indeed removes a significant proportion of the fructosamine residues of hemoglobin in erythrocytes. Recent results obtained with a mouse knock-out model confirm that FN3K acts as a protein deglycating enzyme in tissues.

Unlike FN3K, FN3K-related protein (FN3K-RP) does not act on fructosamines, but it does phosphorylate ketoamines with a D configuration in C3 (ribulosamines, erythrulosamines and, with a lower affinity, psicosamines). The ketoamine 3-phosphates that are formed by FN3K-RP are also unstable and their spontaneous decomposition leads to the regeneration of a free amino group, indicating that FN3K-RP is also a protein repair enzyme. This role has been confirmed in human erythrocytes, which are quite rich in FN3K-RP. Remarkably, the single FN3K-FN3K-RP homologue that is present in fishes, plants and bacteria appears to be also a ribulosamine/erythrulosamine 3-kinase, indicating that the latter corresponds to a more widespread repair mechanism than that catalysed by FN3K.

Ribulosamine and erythrulosamines most likely arise through a reaction of proteins with ribose 5-phosphate and erythrose 4-phosphate, extremely potent glycating agent. The ribulosamine 5-phosphate and erythrulosamine 4-phosphate that are formed in this way must be dephosphorylated by a phosphatase that still needs to be identified. Glucose 6-phosphate is also a potent glycating agent and a phosphatase acting rather specifically on fructosamine 6-phosphates has recently been identified.

In conclusion, protein deglycation appears to involve a whole set of enzymes. A key question for future investigations will be why it is important to free proteins from their sugar adduct rather than replace them with newly synthesized biomolecules.

S37

AMADORIASE, A TOOL FOR INHIBITING PROTEIN GLYCATION AND FOOD BROWNING

Fogliano V., Capuano E., Mennella C., Fedele F., Ferracane R., Lanzuise S., Del Castillo MD#.

Department of Food Science University of Napoli “Federico II”, Napoli, Italy, #Instituto de Fermentaciones Industriales, CSIC, Spain

KEYWORDS: Amadoriase I, Protein glycation, Amadori products, Food browning

Background: Amadori product (AP) is the first stable intermediate of Maillard Reaction in food; the major single modification by the Maillard reaction in vivo and a source of biologically active glycoxidation products leading to protein cross-link in food and biological tissues. Amadoriase I from Aspergillus fumigatus converts AP into deoxyglucosone, hydrogen peroxide and the corresponding primary amine. AP formed on free amino acids are a good substrate for Amadoriase I while it is unable to cleave AP formed on whole proteins. We have previously shown that the enzyme is able to hydrolyze AP on di-tri and tetra peptides and also that its presence during the glycation reaction is able to reduce protein glycation.

Aim: In this work we have investigated the ability of Amadoriase I to inhibit protein glycation on different proteins by monitoring protein glycosylation by MS, and by measuring the concentration of well known markers of Maillard Reaction such as glucosyl-lysine and furosine.

Methods: Recombinant Amadoriase I was produced in E.Coli and purified by histidine-affinity chromatography. Enzyme activity was assessed by deoxyglucosone formation. Glycation inhibition experiments were carried out adding 3 µg mL-1 of enzyme to a solution containing 25 mg mL-1 of different proteins and 1 M of glucose and incubated for different time at 37°C

Results: Recombinant Amadoriase I was produced with a yield of 5 mg L-1 of culture. Its affinity towards dipeptides increased with substrate hydrophobicity. When the Amadoriase I was added during the glycation reaction a significant reduction of the AP formation on peptides was observed. Glycated proteins are poor substrate of the enzyme, however adding Amadoriase during the reaction a significant reduction of the glycated insulin and β-LG was detectable by ESI-MS. The efficacy of the enzyme was higher on the low molecular weight protein (insulin>β-LG>BSA). The results were confirmed by the measure of furosine. After 30 days of incubation of β-LG with glucose the presence of Amadoriase I reduce furosine by 30% while only 10% of reduction was observed using BSA. Preliminary experiments also shown that Amadoriase is able to reduce browning development during storage of dehydrated apple slices and apple puree.

Conclusions: Up to now the possible use of Amadoriase was limited to the measure of the glycation level of human blood proteins. Adding this enzyme to a extensively proteolyzed glycated proteins, the quantification of 3-DG and hydrogen peroxide produced is an indirect indication of the extent of protein glycation. This work suggest a possible use of Amadoriase I to inhibit protein glycation in vivo and in food where a reduction of browning during storage can be obtained.

S38

AMADORI-GLYCATED PHOSPHATIDYLETHANOLAMINE IN FOOD AND HUMAN PLASMA

Teruo Miyazawa

Food & Biodynamic chemistry Lab., Tohoku University

KEYWORDS: Amadori-glycated phosphatidylethanolamine.

Peroxidized phospolipid-mediated cytotoxity involves in diabetic pathophysiology (i.e., high concentration of phosphatidylcholine hydroperoxide, PCOOH, in plasma of type II diabetic patients)1,2). The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (deoxy-D-fructosyl PE, namely Amadori-PE) since Amadori-PE causes membrane lipid peroxidation3). Despite the potential significance of Amadori-PE in pathological signaling4), the accumulation and distribution of Amadori-PE in biological samples as well as in foods has not been fully understood because of lack of a suitable method.

Hence, to determine the presence of Amadori-PE, the carbonyl group of Amadori-PE was ultraviolet (UV)- labeled with 3-methyl-2-benzothiazolinone hydrazone, and the labeled Amadori-PE was analyzed with HPLC-UV (318 nm)5). The HPLC-UV allowed us to show the occurrence of Amadori-PE in many foodstuffs. Since several foods (i.e., infant formula and chocolate) contain a high amount of Amadori-PE, lipid glycation may impair flavor appearance as well as nutritive value of these products. In biological samples, a significant amount of Amadori-PE was detectable in plasma of old rats (50 weeks old). This first suggested that membrane lipid glycation increases with aging. However, we were unable to detect Amadori-PE in human plasma by using HPLC-UV because of insufficient sensitivity.

Consequently, we developed a more sensitive method of Amadori-PE using quadrupole linear ion trap mass spectrometer, Applied Biosystems QTRAP6). When lipid extract from diabetic plasma was directly infused to QTRAP, Amadori-PE molecular species could be screened out by a neutral loss scan mode. Interfacing HPLC with QTRAP enabled the separation and determination of predominant plasma Amadori- PE species in multiple reaction monitoring. Plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Plasma Amadori-PE concentration was proportional to plasma PCOOH concentration (a marker of oxidative stress). These results suggested the simultaneous involvement of both lipid glycation and lipid peroxidation in the diabetes pathogenesis.

1) T. Miyazawa: Free Radic. Biol. Med., 7, 209-217 (1989) 2) M. Kinoshita, S. Oikawa, K. Hayasaka, A. Sekikawa, T. Nagashima, T. Toyota, T. Miyazawa: Clin. Chem., 46, 822-828 (2000) 3) J.H. Oak, K. Nakagawa, T. Miyazawa: FEBS Lett., 481, 26-30 (2000) 4) J.H. Oak, K. Nakagawa, T. Miyazawa: FEBS Lett., 555, 419-423 (2003) 5 J.H. Oak, K. Nakagawa, T. Miyazawa: J. Lipid Res., 43, 523-529 (2002) 6) K. Nakagawa, J.H. Oak, O. Higuchi, T. Tsuzuki, S. Oikawa, H. Otani, M. Mune, H. Cai, T. Miyazawa: J. Lipid Res., 46, 2514-2524 (2005)

S39

INVESTIGATION OF PATHWAY FOR AGE FORMATION IN MACROPHAGES

Ryoji Nagai, Yukio Fujiwara, Yu-ichiro Sakamoto, Naoko Kiyota

Department of Medical Biochemistry, Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University

KEYWORDS: Glycation, Scavenger receptor, Macrophages, Atherosclerosis

We previously demonstrated that hydroxyl radical and peroxynitrite (1) are involved in the formation of Ne- (carboxymethyl)lysine (CML), one of major antigenic AGE structures, from Amadori product. Our subsequent studies have also demonstrated an accumulation of glycolaldehyde-derived AGE structure, named GA-pyridine, in human atherosclerotic lesions (2) and mesangial area (3), suggesting that protein modification by glycolaldehyde may contribute to the pathogenesis of atherosclerosis and diabetic nephropathy. In atherosclerotic lesions of human aortas, AGE is localized in extracellular matrix and intracellular lesion of foamed macrophages. Two interpretations are possible for AGE accumulation inside the macrophages, one is the endocytic uptake of extracellular AGE-proteins by scavenger receptors; the other possibility would be that AGE formation could take place inside the macrophages. In the present study we measured which pathways contribute to AGE accumulation inside macrophages. Although heavy modified AGE-BSA, which was prepared by incubating BSA with 1600 mM glucose for 40 weeks, was recognized by CHO cells overexpressing scavenger receptors such as SR-A, SR-BI, CD36 and LOX-1, mildly modified AGE-BSA, prepared by incubating BSA with 50 mM glucose for 24 weeks, did not show any ligand activity for those receptors. Furthermore, immunochemical study demonstrated that CML and GA- pyridine were accumulated in human monocyte-derived macrophages by maturation for 7 days. Taken together, these results demonstrate that AGE detected inside foamed macrophages in atherosclerotic lesions may be generated inside the cells.

1. Nagai R et al., (2002) Diabetes. 51:2833-2839 2. Nagai R et al., (2002) J. Biol. Chem. 277: 48905-48912 3. Greven WL, Nagai R, et al., (2005) Kid. Int. 68: 595-602

S40

APPLICATION OF L-ASPARAGINASE FOR ACRYLAMIDE MITIGATION IN POTATO PRODUCTS

Ciesarova Z*, Kiss E, Kolek E, Simko P

Food Research Institute, Bratislava, SLOVAKIA

KEYWORDS: acrylamide, L-asparaginase, potato

Acrylamide according to IARC it belongs to 2A group as probably human carcinogenic compound is spontaneously formed during heat treatment in food rich on reducing sugars and amino acids, mainly L- asparagine, as part of Maillard reaction. For mitigation of acrylamide content the application of L- asparaginase was used. This commercial enzyme isolated from E.coli releases NH3-group from non- essential amino acid L-asparagine, which is a precursor of acrylamide formation. At first, L-asparaginase was applied in model system simulating the content of raw potatoes, which consists of starch, water, sugars as glucose, fructose and saccharose, and amino acid L-asparagine. In this case the concentration of L- asparaginase and optimal conditions for its use were studied. Subsequently, L-asparaginase was applied in real systems in two varieties (yellow and rose) of raw potatoes (fresh and stored) and in dried potato powder product. In all these cases the present of L-asparaginase before heat treatment of potato based products caused the significant decrease of acrylamide content in final treated products. For 80 90 % reduction of acrylamide content the doses of 1 2 units of enzyme per 1 g raw potato or 5 10 units of L- asparaginase per 1 g dried potato powder were sufficient. Application of L-asparaginase seems to be an appropriate way for effective mitigation of acrylamide in potato heat treated products.

S41

THE ROLES OF DIETARY FACTORS IN PREVENTING GLYCATION MEDIATED DAMAGE TO DNA IN A MG/LYS SYSTEM.

Suji George & S.Sivakami

Department of Life Sciences, University of Mumbai,Santa Cruz(E), Mumbai,India 400 098.

KEYWORDS: Methyl glyoxal, Lysine, glycation, free radicals, pBR322.

Glycation has been seen as a primary cause of damage to biomacromolecules, and has been implicated in the pathogenesis of diseases like diabetes. In this study the secondary reaction of free radicals generated during glycation was studied using model DNA (pBR322). In order to induce damage, a reaction of Methylglyoxal (MG) with free lysine was used and the abilities of various antiglycation compounds like Aminoguanidine, pyridoxamine and thiamine were studied. The reaction of MG with lysine caused DNA damage, which aggravated on adding Fe+3. Damage was seen to take place at as low concentration as 100 µM of MG and Lysine. Superoxide and hydroxyl radicals were detected in the incubation mixtures. The presence of H2O2 was detected by FOX assay, hydroxyl radical by deoxyribose assay and benzoic acid hydroxylation and superoxide by cytochrome assay. Pyridoxine and pyridoxal-5-phosphate prevented DNA damage. However pyridoxamine was ineffective in preventing the damage at all tested concentrations. This was further supported by its inability to scavenge both superoxide and hydroxyl radicals. Thiamine and thiamine pyrophosphate prevented the damage while tetra hydro folic acid (THF) and folic acid did not. On the contrary THF was itself found to induce DNA damage. Aminoguanidine protected against glycation induced DNA damage, however it itself also caused damage. The AG/metal system was found to produce superoxide and hydroxyl radicals as well as hydrogen peroxide. It appears that AG can form hydrogen peroxide, in a metal catalysed process probably due to prior hydrolysis to semicarbazide and hydrazine. Thus this MG/Lys model system affords a simple method to study damage to macromolecules caused by transient intermediates formed during glycation. The potential of various dietary factors to prevent glycation mediated damage is highlighted.

S42

DIFFERENT REACTIVITY AGAINST GLYCATION AND DIFFERENT IMMUNOREACTIVITY AGAINST MONOCLONAL ANTIBODIES BETWEEN WILD- TYPE AND MUTANT CU,ZN SUPEROXIDE DISMUTASE (SOD) LINKED TO AMYOTROPHIC LATERAL SCLEROSIS.

Taniguchi N, Fujiwara N, Takamiya R, Miyamoto Y and Suzuki K

Department of Biochemistry, Osaka University Graduate School of Medicine, and Department of Biochemistry, 2-2 Yamadaoka Suita, Osaka 565-0871 Japan, Hyogo College of Medicine, 1-1 Mukogawa- cho, Nishinomiya, Hyogo 663-8501, Japan.

KEYWORDS: Glycation, Conformational changes, monoclonal antibodies, FALS mutant Cu,Zn-SOD

Although more than 100 mutations have been identified in the Cu,Zn-SOD in familial amyotrophic lateral sclerosis (FALS), the mechanism responsible for FALS remains unclear. The mutated Cu, Zn-SODs were found to be highly susceptible to glycation compared with the wild-type enzyme as estimated by Western blot analysis using an anti-hexitol lysine antibody. The mutated Cu, Zn-SOD incubated with glucose generated higher levels of hydrogen peroxide than the wild-type enzyme. Mutated Cu, Zn-SODs were also shown to be highly susceptible to fructation, and the fructated mutant also produced higher levels of hydrogen peroxide than the wild type.

We also found that FALS-causing mutant Cu,Zn-SOD proteins, but not wild-type SOD, are barely detected by three monoclonal antibodies (mAbs) in Western blot analyses. The enzyme-linked immunosorbent assay for denatured FALS mutant SODs by dithiothreitol, SDS, or heat treatment also showed a lowered immunoreactivity against the mAbs compared with wild-type SOD. Because all the epitopes of these mAbs are mapped within the Greek key loop (residues 102-115 in human Cu,Zn-SOD), these data suggest that different conformational changes occur in the loop between wild-type and FALS mutant SODs during the unfolding process. The study on the conformational changes in local areas monitoring with mAbs may provide a new insight into the etiology of FALS. These results suggest that high susceptibility to glycation and occurrence of conformational changes in the FALS mutant SODs could be the origin of the oxidative stress associated with neuronal dysfunction in FALS.

S43

AGE-MODIFICATION VASCULAR BASEMENT MEMBRANES AS A BASIS FOR VASODEGENERATION IN DIABETIC RETINOPATHY

Alan Stitt

Centre for Vision Science, School of BioMedical Science, Queen’s University of Belfast, Northern Ireland

KEYWORDS: Maillard reaction, Diabetic retinopathy, vascular basement membrane

Retinopathy remains the most common microvascular complication of diabetes mellitus. As a disease entity it presents major therapeutic problems for the ophthalmologist and despite many decades of intense research it still constitutes a major cause of blindness in the Western world.

This review lecture highlights the aetiology of diabetic retinopathy and proposes a link between disease progression with the formation and accumulation of advanced glycation endproducts (AGEs). As reported in a range of clinical investigations and determined by mechanistic in vitro and in vivo studies, products of Maillard chemistry accumulate in the diabetic retina where they may be effectors of retinal vascular and neural cell dysfunction. Evidence now points towards a pathogenic role for advanced glycation in the initiation and progression of retinopathy and this review will examine the current state of knowledge of AGE- related pathology in the retina at a cellular and molecular level. The lecture will mention how ongoing pharmaceutical strategies to inhibit AGE formation and thereby attenuate their pathogenic influence during chronic hyperglycaemia may play a significant role in the treatment of diabetic retinopathy.

There will be special emphasis placed on AGE-adduct formation on vascular basement membranes (BMs) in vivo and investigative studies on in vitro modeling of cellular interaction with diabetes-modification of BM proteins and how this mediates vasodegeneration in the retinal microvasculature. Unpublished research will be presented on the molecular mechanisms linking diabetic retinopathy-linked pathophysiology in retinal vascular endothelium, pericytes and vasoreparative capacity of circulating endothelial progenitor cells (EPCs). Whilst focused on the retinal microcirculation, the findings have applicability to all microvascular beds and their progressive dysfunction within the diabetic milieu.

S44

TANGLED IN A WEB OF SUGAR: AGES, THEIR ACTIONS AND REACTIONS IN DIABETIC COMPLICATIONS

Josephine Forbes and Mark Cooper

Baker Institute

KEYWORDS: glycation diabetes

Advanced glycation is the irreversible attachment of reducing sugars via the “Maillard reaction” onto the free amino groups of proteins, in particular at lysine and arginine residues. Its physiological roles are thought to include the identification of senescent proteins and hence there is a time dependent accumulation of advanced glycation end products (AGEs) as we grow older. When a protein is labelled with AGEs, it is catabolized by cells into peptides and amino acids and excreted via the kidneys. This process appears to be tightly controlled by AGE clearance receptors such as AGE-R1, AGE-R3 and scavenger receptors such as CD36, SR-AII and SR-BI. Evolution has also provided protection against excess AGE formation by utilisation of glucose as the major metabolic sugar, as it is a slowly reacting reducing sugar. Conditions such as diabetes, however, which have a metabolic overload of a number of reducing sugars, rapidly accelerate AGE formation. In addition, advanced glycation is facilitated by oxidative stress even in the absence of increases in reducing sugar concentrations. Both of these processes contribute to the increased AGE pool seen in diabetes.

Food chemists have seen the value of advanced glycation in foodstuff manufacture by utilising heat in processes such as roasting, brewing, frying and cooking. As part of our western diet, we ingest AGEs of which approximately 70-80% are absorbed, catabolized and excreted over a period of two days. This is a third major source of AGEs and becomes an increasingly important contributor as the clearance of AGEs by the kidneys decreases during diabetes-induced renal impairment.

As AGE levels rise during diabetes, interruption of normal function occurs via three distinct mechanisms. The first involves AGE induced cross-linking of extracellular matrices, which stiffens elastic fibres, disturbs cellular adhesion and prevents renewal of “old” proteins causing structural defects. The second is by intracellular formation of AGEs, which causes generalised cellular dysfunction by interference in enzyme/substrate interactions, signalling and by steric hindrance including conformational changes within proteins and DNA. The third involves the binding of circulating AGEs to specific receptors such as RAGE, the receptor for advanced glycation end products, TOLL-like pattern recognition receptor involved in activation of inflammatory pathways which when chronically activated contributes to excesses in inflammatory molecule production.

Due to the range of dysfunction produced by the accumulation of AGEs in diabetes, there is a growing need for intervention in this process. Indeed, advanced glycation has been implicated as a major therapeutic target in both micro- and macrovascular complications of diabetes and the race is on to produce an inhibitor capable of addressing the many actions of AGEs.

S45

AGE/ALES: NON-TRADITIONAL RISK FACTORS FOR VASCULAR COMPLICATIONS

CG Schalkwijk

Dept of Internal Medicine, University Hospital Maastricht, The Netherlands

The most serious complications in diabetes mellitus are micro- and macrovascular complications such as kidney failure, blindness, neuropathy, stroke, and myocardial infarction. In our view, endothelial dysfunction can be conceptualized as a transducer of risk factors. In various studies we extensively explore how endothelial dysfunction in diabetes relates to the pattern of disease occurrence and which current biochemical mechanisms provide an explanation for the development of endothelial dysfunction. The role of traditional and non-traditional risk factors such as Amadori-albumin and advanced glycation/lipoxidation endproducts (AGE/ALEs) in relation to the initiation of vascular disease is our focus of research. Many aspects of diabetic and also non-diabetic vascular complications are potentially related to the effect of AGEs/ALEs. AGEs/ALEs have been implicated as causal factors in endothelial dysfunction associated with vascular diseases. They accumulate at an accelerated rate in diabetes and in patients with renal failure. A few examples of the role of AGEs/ALEs as non-traditional risk factor for vascular complications will be presented. The association of AGEs/ALEs with vascular complications was studied in a cross-sectional nested case- control study in a large group of type 1 diabetic individuals of the EURODIAB Prospective Complications Study. We found that pulse pressure (a measure of arterial stiffness) was significantly associated with plasma levels of N-(carboxymethyl)lysine (CML) and N-(carboxyethyl)lysine (CEL), as measured with LC- MSMS. To further explore the relationship between AGEs and pulse pressure, we also did studies in spontaneous hypertensive rats (SHR). Data of these experiments will be presented. In these experiments, we found a two-fold higher plasma level of CML in SHR. In both WKY rats and SHR, plasma levels of CML were correlated with pulse pressure. CML staining was five-fold increased in proximal tubuli and as a thin endothelial cap in intrarenal arterioles in SHR. In young SHR, the antihypertensive specific angiotensin II receptor blocker losartan, the mineralocorticoid receptor antagonist spironolactone and the nonselective vasodilator hydralazine significantly lowered blood pressure. As compared to untreated SHR, both losartan and spironolactone, but not hydralazine, decreased CML staining. We conclude from these experiments that the blood pressure increase in young SHR was associated with an increase of CML in renal parenchym and endothelial cells and that CML may contribute to development and complications of hypertension. We also found increased levels of the major AGE CML in human diabetic heart tissue. The six-fold accumulation of CML in the small intramyocardial blood vessels of diabetic hearts as compared to control hearts was much higher in comparison to the differences in staining intensity between renal tissues and lungs in the same subjects. Because diabetes patients have an increased risk of acute myocardial infarction (AMI) , we also examined a putative relationship between CML depositions and AMI. In AMI patients, CML depositions were increased in the small intramyocardial blood vessels and were predominantly colocalized with activated endothelium (E-selectin-positive) in infarction and non-infarction areas. Since in a rat AMI model no CML deposition was found, we speculate that CML more likely play a role in the induction of AMI instead of being a result of AMI. The possible consequence of the high CML deposition in diabetic heart tissue, and of course also in other tissues, is the activation of vascular cells through the AGE receptor RAGE. Thus, CML-RAGE interactions may trigger processes linked to vascular and inflammatory complications that typify disorders in which inflammation is an established component. We hypothesize that CML in diabetic hearts may contribute to the increased risk of heart failure. With these clinical and experimental studies new insights will be given in the significance of the non- traditional risk factor AGEs/ALEs adducts in the burden of cardiovascular disease in patients with diabetes.

S46

KIDNEY AND ADVANCED GLYCATION ENDPRODUCTS (AGES)

Stein G., M. Rüster, Friederike Bötticher, G. Wolf, S. Franke

Dept. Intern Med, Friedrich-Schiller-University of Jena, Germany

Diabetes mellitus and uraemia are both conditions were significantly increased concentration of AGEs are found in tissues and plasma. AGEs and their receptors have now been established as major pathogenic factors promoting disease activity and complications of diabetes mellitus and many other none diabetic renal diseases of inflammatory or sclerosing nature, including chronic allograft nephropathy and amyloidosis. Immunohistochemical staining of kidney sections showed deposition and partially colocalization for Nε-Carboxymethyllysine (CML), Pentosidine (PNT), Imidazolone (IMI), and Pyrraline, with the receptor for AGE (RAGE) and NF-kB in glomeruli, tubulointerstitial space and in arteries. In patients with diabetic nephropathy CML is the major AGE in renal based membrane, and its accumulation involves upregulation of RAGE on podocytes. The elevated serum levels of AGEs or the local de novo syntheses may result in the increased modification of extracellular matrix proteins preferently in nodular lesions composed of types V and VI collagens. The positive immunostaining of tubular cells in almost all the patients regardless of the presence of diabetes may indicate the reabsorbed AGEs by the tubular cells. It appears that activation of Renin-Angiotensin System may contribute to AGE formation through various mechanisms.

The induced specific cellular responses including the release of profibrogenic and proinflammatory cytokines, monocyte migration and activation, gene expression ect. by interacting with RAGE cause to glomerulosclerosis, interstitial fibrosis and tubular atrophy. In this context, the kidney is not only a target of AGEs but also a culprit, because declining renal function entails a rapid increase in plasma concentration of these products. There was no correlation between the immunohistochemical quantification of AGE deposition and the severity of proteinurea or renal insufficiency, but in some diseases the severity of renal of histological proven renal damage has been reported.

Interference with AGE formation has therapeutic potential for preventing the progression of chronic renal diseases. The pathopysiological implications of AGEs, RAGE, and other AGE binding proteins are rapidly expanding, wereas their role in vasculary modelling, chronic inflammatory processes in cellular dysfunction becomes increasingly recognized. However, many important aspects of AGE and their role in renal biology still remain unclear.

S47

ADVANCED GLYCATION ENDPRODUCTS IN THE DEVELOPMENT OF OSTEOARTHRITIS

DeGroot J.

TNO Quality of Life

KEYWORDS: Osteoarthritis, cartilage, Advanced Glycation Endproducts, collagen

Osteoarthritis (OA) is the most common form of arthritis or degenerative joint disease in the world and is a leading cause of chronic disability. As a result of the global demographic changes, OA will become increasingly prevalent among the middle-aged and elderly. Age is the strongest predictor of the disease, but the mechanisms by which ageing contributes to the development of OA remain only partly understood.

Since OA is characterized by the progressive destruction of articular cartilage it was hypothesized that age- related changes in the cartilage extracellular matrix and/or the chondrocytes contribute to the development of OA. Advanced glycation endproducts such as pentosidine, CEL, CML accumulate with age in normal articular cartilage. The accumulation of AGEs affects the biomechanical properties of the tissue: healthy cartilage becomes stiffer and more brittle; hence the accumulation of AGEs increases the susceptibility to damage.

In addition, AGEs affect the remodeling of the cartilage matrix. On the anabolic side, healthy human articular cartilage shows and age-related decrease in proteoglycan synthesis, the correlates to the tissues AGE levels. Collagen synthesis in bovine chondrocytes is suppressed in the presence of AGEs. On the catabolic side, an increase in AGE levels in cartilage results in decreased susceptibility to proteolytic degradation. The overall picture is that the AGE accumulation in cartilage tissue decrease the capacity of healthy cartilage to repair damage and to maintain its integrity, thus increase the susceptibility to OA. Indeed, healthy cartilage of individuals suffering from OA elsewhere in the joint shows elevated AGE levels compared to healthy cartilage of individuals without OA. In canine experimental OA elevation of AGE levels increases the severity of the disease.

It is important that to realize that in studies employing total cartilage explants, one cannot distinguish direct effects of AGEs on chondrocytes from those that are caused by a changed extracellular matrix surrounding the chondrocyte. This becomes clear when isolated chondrocytes are stimulated with AGE-BSA, which activates the cells via the RAGE receptor and induces a catabolic change (increased protease expression) rather than decreased matrix degradation as is mentioned above. Thus in studying the effects of age- related accumulation of AGEs in articular cartilage it is crucial to study the mechanisms involved from a variety of different angles taking into account effects on cells, on the extracellular matrix, and on its interaction.

VERZIJL N, BANK RA, TEKOPPELE JM & DEGROOT J (2003) AGEing and osteoarthritis: a different perspective. Current Opin. Rheum. 15(5):616-622 DEGROOT J, VERZIJL N, WENTING-VAN WIJK MJG, JACOBS KMG, VAN EL B, VAN ROERMUND PM, BANK RA, BIJLSMA JWJ, TEKOPPELE JM & LAFEBER FPJG. (2004) Accumulation of Advanced Glycation Endproducts as a molecular mechanism for aging as a risk factor in Osteoarthritis. Arthritis Rheum. 50(4): 1207-1215. STEENVOORDEN MMC, HUIZINGA TWJ, VERZIJL N, BANK RA, RONDAY HK, LUNING HAF, LAFEBER FPJG, TOES REM & DEGROOT J. (2006) Activation of receptor for advanced glycation end products in osteoarthritis leads to increased stimulation of chondrocytes and synoviocytes. Arthritis & Rheumatism 54(1):253-263.

S48

EFFECTS OF MAILLARD REACTION PRODUCTS ON NF-κB ACTIVATION AND EXPRESSION OF PRO-INFLAMMATORY MARKERS - MOLECULAR MECHANISMS

Muscat S., Muench G.*, Pischetsrieder M.

Institute for Pharmacy and Food Chemistry, University of Erlangen, *Dept. of Biochem. & Mol. Biol., James Cook University, Townsville

KEYWORDS: Maillard reaction, coffee, NF-κB, melanoidins, macrophages, hydrogen peroxide

Maillard reaction products (MRPs) are formed through a non-enzymatic reaction between reducing sugars and proteins or amino acids in heated foods such as bakery products or coffee. "Advanced glycation end products" (AGEs) are formed through similar reactions, although at a lower temperature, in the human body. Increased AGE-levels can be detected in patients with diabetes mellitus (increased glucose level) or renal failure (diminished excretion). AGEs induce an inflammatory response via a RAGE (receptor for advanced glycation end products)-redox sensitive signaling pathway involving the transcription factor Nuclear factor-κB (NF-κB). Little is known whether food-derived MRPs can also trigger such an inflammatory process in the intestine or systemically in the human body after resorption. To investigate the pro-inflammatory properties of food-derived MRPs, macrophages were stimulated with coffee (a food with high MRP content) or a lysine-ribose MRP (produced by incubation at 120°C for 30min). Activation of NF-κB (determined by nuclear translocation) was detected immunochemically. Freshly brewed coffee but not an extract of raw coffee beans led to a 13-fold increase in NF-κB activation. Furthermore, the pro-inflammatory effect of the lysine-ribose MRP was investigated. Stimulation of macrophages with the lysine-ribose MRP led to an 18-fold NF-κB activation compared to the control, and the high molecular weight products, namely melanoidins, were identified as the signal active compounds in this mixture. However, the expression of pro-inflammatory mediators such as cytokines or NO production was not induced by this model MRP. The cellular mechanism of the MRP-induced NF-κB activation was investigated using RAGE-transfected and untransfected human embryonic kidney cells (HEK). No difference in MRP-induced NF-κB activation was found between the RAGE-transfected and untransfected HEK cells. In addition, MRP-generated hydrogen peroxide production up to 450 µM could be measured. Involvement of hydrogen peroxide in MRP-induced NF-κB activation is supported by the fact that it could be inhibited by catalase. In summary, these data suggest that MRPs and coffee activate NF-κB via a cell-independent production of hydrogen peroxide. This pathway does not involve RAGE. MRP-induced NF-κB activation is not sufficient on its own to cause the expression of pro-inflammatory cytokines or the production of NO.

S49

SUGAR-INDUCED MODIFICATION OF ANGIOGENIC GROWTH FACTORS: A STRUCTURAL AND FUNCTIONAL STUDY

Francesco Facchiano (1), Daniela D’Arcangelo (2), Vincenzo Fogliano (3)

1 Istituto Superiore di Sanità, Roma, Italy, 2 Istituto Dermopatico dell’Immacolata, IDI, IRCCS, Roma, Italy, 3University Federico II of Napoli, Napoli, Italy

KEYWORDS: AGE, angiogenesis, diabetes mellitus, FGF, growth factors

New vessel development (angiogenesis) is impaired in diabetes mellitus patients. Therefore aim of the present study was to evaluate the effects of hyperglycemic conditions on structure and function of a number of angiogenic growth factors.

Electrophoretic, mass spectrometry, surface plasmonic resonance analyses and emission fluorescence studies have been carried out to evaluate the structural effects of sugars on Fibroblast Growth Factor-2 (FGF-2), which indicated a marked structural modification of FGF-2 reacting with glucose or fructose, whereas mannitol, used as iso-osmotic control, was ineffective. Glycated-FGF-2 was recognized by anti- AGE antibodies while other angiogenic growth factors (PDGF, PlGF) were not, or markedly less, modified by sugars, suggesting a specific effect. Functional studies in primary cultures of bovine aortic endothelial cells and human umbilical vein cells showed that FGF-2 incubated with glucose was significantly less mitogenic and chemotactic in vitro. Further, FGF-2 lost a significant amount of its angiogenic activity in vivo, as measured in subcutaneous Matrigel assays in mice. The structural modification and the functional effects were time- and sugar-concentration dependent.

These results show that FGF-2, exposed to reducing sugars under conditions resembling diabetic hyperglycemia, is structurally modified and functionally impaired, indicating a possible novel mechanism underlying vascular complications and angiogenesis defects in diabetes mellitus.

Poster session P1

GLYCOGEN STORAGE DISEASE 1A: HYPERLIPIDEMIA AND MICROALBUMINURIA WITHOUT CONSECUTIVE ATHEROSCLEROSIS

N.C. den Hollander (1), D.J. Mulder (1), S.R. Thorpe (2), J.W. Baynes (2), A.J. Smit (1)

1 Dept. of Internal Medicine, University Medical Center Groningen, the Netherlands; 2 Dept. of Biochemistry, University of South Carolina, United States of America

KEYWORDS: Advanced Glycation End products, skin Autofluorescence, Glycogen Storage Disease 1a, Atherosclerosis

Patients with glycogen storage disease type 1a (GSD 1a) do not develop premature atherosclerosis, despite severe hyperlipidemia and microalbuminuria. We hypothesized that this paradox might be explained by a decreased formation of Advanced Glycation Endproducts (AGEs). AGEs are the result of spontaneous reactions between proteins and sugars or lipids. AGEs contribute to aging and chronic diseases like atherosclerosis. Increased AGE formation in diabetes plays a major role in the development of complications. GSD 1a patients might have decreased AGE formation because of their tendency to low blood sugars and their low levels of oxidative stress. The decreased AGE formation could protect them from atherosclerosis.

We measured deposition of specific AGEs (pentosidine, N-(carboxymethyl)lysine (CML) and N- (carboxyethyl)lysine (CEL)) and Collagen Linked Fluorescence (CLF) in skin biopsies of 8 GSD 1a patients and 30 healthy age and sex matched controls. We also measured skin autofluorescence (AF). CLF and skin AF are used to measure AGEs with a characteristic fluorescence pattern. Skin AF was measured with a prototype of the current AGE-Reader, recently developed in Groningen. This is the first technique to measure AGEs non-invasively. Atherosclerosis was determined by measuring the intima media thickness (IMT) of both carotid arteries.

IMT in GSD 1a patients was significantly decreased (p=0.039). The skin samples showed higher CEL levels in the patient group (p=0.008). The amounts of pentosidine and CML were comparable (p=0.40 and p=0.53 respectively). CLF in patients and controls was also comparable (CLF 328/378 p=0.8 and CLF 370/440 p=0.45). Skin AF did not differ between both groups (p=0.26). Skin AF did correlate with CML (r=0.41; p=0.028), CLF 328/378 (r=0.58; p=0.001) and CLF 370/440 (r=0.64; p=0.000). In the control group AF correlated with IMT (r=0.60; p=0.004).

We conclude that the absence of premature atherosclerosis in GSD 1a patients can not be explained by decreased AGE formation as measured in skin. Although the absence of premature atherosclerosis in GSD 1a patients thus stays unexplained, GSD 1a provides an interesting model to discover new facts about the link between hyperlipidemia, microalbuminuria and atherosclerosis, a widely spread and threatening disease in current days. Furthermore, our research introduced the AGE-Reader, a good way to measure AGEs non-invasively and thereby to give AGEs a role in the clinical setting.

P2

ACRYLAMIDE FORMATION FROM DIFFERENT RATIOS OF GLUCOSE AND ASPARAGINE IN GLYCEROL-WATER MIXTURES

Rikke V. Hedegaard, Henrik Frandsen, Kit Granby, Anna Apostolopoulou and Leif H. Skibsted

The Royal Veterinary and agricultural university; Danish Institute for food and Veterinary Research

KEYWORDS: acrylamide, water activity, activation energy

Acrylamide formation from asparagine and glucose in different ratios in glycerol-water mixtures was examined. The formation of acrylamide was found to increase with decreasing water activity and increasing temperature in the aw range (0.33 ≤ aw ≤ 0.71) and temperature range (120ºC ≤ t ≤160ºC) investigated. The initial rate of acrylamide formation was found to be approximately proportional to asparagine concentration for excess of asparagine, but less dependent on excess of glucose. The enthalpy of activation as estimated from the temperature dependence of initial rate increased with decreasing aw despite a higher rate of formation of acrylamide at low aw. For high aw, water dissociation of an intermediate is suspected to be rate determining, while at low aw the increases in activation enthalpy was counteracted more positive entropy of activation and we propose CO2 dissociation to be rate determining at low aw. The results indicate an increase in importance of the entropy contribution of the activation free energy for formation of acrylamide for decreasing aw.

P3

STUDY ON ELECTROCHEMICAL ACTIVITY OF BEER AND MALT

Jarolímová L, Cejpek K and Velíšek J

Institute of Chemical Technology, Technicka 1905, 166 28 Prague 6, Czech Republic

KEYWORDS: Keywords: beer, malt, electrochemical activity

Beer is one of the most popular beverages all over the world. It is an important source of the compounds that possess antioxidative, antimutagenic, anticarcinogenic and anti-inflammatory properties. The antioxidative activity is supposed to come from both naturally occurring components of beer raw materials and process-induced compounds. The components derived from raw material are polyphenols, phenolic acids, thiols, carotenoids and vitamins, originated mostly from barley. The heat-induced antioxidants are believed to include melanoidins and reductones, which are formed by the Maillard reaction during production of malt and brewing. However, the character and antioxidative activity of heat induced active compounds from beer remain mostly uncovered.

The objectives of this study were to evaluate the antiradical activity of seven original kinds of beer and six kinds of malt using HPLC method with electrochemical detection (+0,8V). The most significant active low- molecular compounds of beers and malts were separated and spectrally characterised. The contribution of raw material and technology to both the total electrochemical capacity and particular active compounds was formed. Possible antioxidative activity of these products detected electrochemically was confirmed by the DPPH radical scavenging activity assay. The relationships between colour, electrochemical activity and antiradical activity of the tested malts and beers are evaluated and discussed.

P4 THERAPEUTIC TARGETS FOR PREVENTING CELL DEATH INDUCED BY VARIOUS MECHANISMS OR MAILLARD REACTION INTERMEDIATES USING B VITAMINS AND PHARMACEUTICALS

Rhea Mehta, Nandita Shangari and Peter J.O’Brien

Graduate Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S2S2

Dietary Vitamin Bs are essential for the activity of enzymes required for cellular bioenergetics and macromolecule biosynthesis. Recently, it was discovered that natural intermediates of vitamin B6 (pyridoxamine) or B1 (thiamine pyrophosphate) prevented the development of diabetic nephropathy. Multifunctional cytoprotective effects were proposed, including scavenging Maillard reactive carbonyl intermediates e.g. glyoxal, scavenging reactive oxygen species or scavenging ALE intermediates e.g. acrolein or preventing AGE formation by inhibiting post-Amadori protein modifications.

We have compared the cytoprotective mechanisms of vitamin B1, B5 (pantothenic acid) and B6 intermediates against various types of hepatocyte death using isolated rat hepatocytes: 1) carbonyl stress induced by glyoxal or acrolein 2) oxidative stress induced by hydroperoxides or hydrogen peroxide 3) bioenergetic stress induced by mitochondrial respiratory inhibitors cyanide or rotenone.

Thiamine was best at preventing death induced by glyoxal, cyanide or rotenone suggesting that the Maillard intermediate glyoxal inhibited mitochondrial pyruvate and ketoglutarate dehydrogenases (lipoic acid). However, pantothenic acid was best at preventing acrolein or hydroperoxide induced death suggesting that acrolein contributed to hydroperoxide death by depleting mitochondrial coenzyme A. Pyridoxal was the most effective vitamin B at preventing hydroperoxide induced death and lipid peroxidation or hydrogen peroxide induced death and reactive oxygen species formation or iron induced death and lipid peroxidation suggesting that pyridoxal or a metabolite was an antioxidant, ROS scavenger and iron chelator. Pyridoxamine was best at preventing copper induced death suggesting that pyridoxamine was a copper chelator. Hydralazine was the best therapeutic drug at preventing acrolein or hydroperoxide induced death and lipid peroxidation suggesting that acrolein contributed to hydroperoxide death.

Enzymes requiring vitamin B1 and B6 as coenzymes are vulnerable to cytotoxicity induced by carbonyl stress or Maillard reaction intermediates. Furthermore, cytotoxicity can be prevented by adding vitamin B1 and B6 to the cells.

P5

COPPER-CATALYSED ASCORBATE OXIDATION RESULTS IN GLYOXAL FORMATION AND HEPATOCYTE CYTOTOXICITY

Nandita Shangari (1), Tom S. Chan (2), Peter J. O’Brien (1)

(1) Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ont., M5S 2S2,Canada. (2) Centre de Recherche, CHUM, Hôpital Saint-Luc, 264 Boul. René Levesque Est, Montréal, QC, Canada, H2X 1P1

Previously we showed that 10 µM glyoxal compromises hepatocyte resistance to hydrogen peroxide (H2O2) by increasing GSH oxidation, NADPH oxidation, reactive oxygen species formation, DNA oxidation, protein carbonylation and loss of mitochondrial membrane potential before cytotoxicity ensued (Biochem Pharma In Press).

Ascorbate autoxidation is accelerated in the presence of copper and results in glyoxal formation that compromises hepatocyte resistance to H2O2.

To test this possibility, we used isolated rat hepatocytes and incubated them with ascorbate/copper. Cytotoxicity, glyoxal levels, H2O2, GSH levels and mitochondrial membrane potential were measured. To assess the role of H2O2 on the process of AGE formation we tested the effects of catalase and aminoguanidine on AGE formation on bovine serum albumin (BSA) in the presence of ascorbate/copper.

The ascorbate/copper reaction mixture increased glyoxal and H2O2 formation. Hepatocyte GSH levels were decreased and cytotoxicity ensued after a collapse of the hepatocyte mitochondrial membrane potential. Taurine or glyoxal traps protected hepatocytes against ascorbate/copper-induced cytotoxicity. In cell-free studies with BSA, incubation of BSA with ascorbate resulted in glyoxal-hydroimidazolone (G-H1) formation, which was decreased by both aminoguanidine and catalase.

To our knowledge, this is the first study that illustrates the importance of glyoxal production by transition metal-catalysed ascorbate oxidation. In this study, we have shown that glyoxal formation may both exacerbate oxidative stress derived from the transition metal-catalysed autoxidation of ascorbate as well as increase the rate of G-H1 formation. Understanding this mechanism of toxicity could lead to the development of novel copper chelating drug therapies to treat complications associated with oxidative stress in diabetes (e.g. cataracts).

P6

DIETARY ADVANCED GLYCATION END PRODUCTS AND THIAMIN DEFICIENCY INCREASED PLASMA ALPHA-OXOALDEHYDE LEVELS AND COLONIC INFLAMMATION

Nandita Shangari (1), Flore Depeint (1,2), Rhea Mehta (1), Rudolf Furrer (2), W. Robert Bruce (2), Peter J. O’Brien (1)

(1) Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, Ont., M5S 2S2,Canada.

KEYWORDS: aberrant crypt foci, advanced glycation end-products, thermolyzed diet, thiamin deficiency

Previously we showed that marginal dietary thiamin deficiency in rats caused aberrant crypt foci (marker of colon carcinogenesis) formation at 160 days without symptoms of clinical beriberi (Cancer Lett. 2003 202(2):125-9). Plasma levels of glyoxal and methylglyoxal (MG) were also increased (FEBS Lett. 2005 579(25):5596-602).

A thermolyzed diet containing advanced glycation end-products (AGEs) and decreased thiamin increases intracellular exposure to alpha-oxoaldehydes and colon carcinogenesis.

4 groups of Fischer 344 rats were given the following diets: Group A was given a AIN93G diet (control diet), Group B AIN93G diet, autoclaved 30 min, 121°C (thermolyzed diet), Group C AIN93G diet with equivalent thiamin as in Group B and Group D AIN93G diet as in Group B + 20 mg/l thiamin in the drinking water. The animals were cycled on 3 weeks of thermolyzed diet and 2 weeks on control diet to restore the vitamin stores that were depleted during thermolysis for 160 days. At a 160 days we measured transketolase activity in red blood cells (thiamin status); glyoxal/MG and TNF-alpha levels in the plasma; glyoxal/MG hydroimidazolone protein adducts and dicarbonyls in the plasma, liver, kidney, brain and adipose tissues; glutathione levels on whole blood, oxidative stress/inflammatory markers in the colon such as macrophages via immunohistochemistry.

Thermolyzed diet resulted in: 1) decreased thiamin status and transketolase activity. 2) increased plasma levels of glyoxal/MG adducts. 3) increased protein dicarbonyls in the liver more than other tissues. 4) lower erythrocyte glutathione levels. 5) an infiltration of macrophages in the colon.

A thermolyzed diet leads to an increased AGEs body burden, decreased thiamin levels thereby decreasing cellular thiamin and increasing genotoxic AGEs (i.e. glyoxal/MG). The increased oxidative stress affected tissues with low levels of antioxidant defenses such as the colon.

P7

RENOPROTECTIVE AND VASCULAR EFFECTS OF LR-102, A NOVEL AGE INHIBITOR, IN STZ-DIABETIC RATS

Figarola J, Jandeleit-Dahm K*, Loera S, Rahbar S

City of Hope National Medical Center, Duarte, California, USA and *The Baker Heart Institute, Melbourne, Australia

KEYWORDS: glycation, oxidative stress, diabetes, AGE inhibitors

Increased formation and accumulation of advanced glycation/lipoxidation endproducts (AGEs/ALEs) has been implicated in the pathogenesis of various diabetic complications. Several compounds have been developed as inhibitors of AGE/ALE formation. We investigated whether LR-102 (1,4-benzene-bis[4- methyleneaminophenoxyisobutyric acid]) a new AGE/ALE inhibitor, can prevent development of renal and vascular complications in streptozotocin (STZ)-induced diabetic rats.

Diabetic Sprague-Dawley rats were given LR-102 in their drinking water at 50 mg/L. After 32 weeks, body weight, glycaemic status, urinary albumin and creatinine excretions, and plasma lipids and their lipid peroxides were measured. Kidney histopathology and aortic lesions were examined. AGE/ALE accumulation and RAGE protein expression in kidney and aortic tissues were also determined. The mechanism of action of LR-102 was also examined in vitro.

Administration of LR-102 to diabetic rats significantly inhibited the increase in albuminuria, plasma creatinine, hyperlipidemia and lipid peroxidation in diabetic rats without any effect on hyperglycemia. LR- 102 treatment also reduced diabetes-associated accumulation of AGEs and RAGE protein expression in kidney glomeruli and tubules, AGE-linked crosslinking of tail collagen, and levels of N-(carboxymethyl)lysine (CML) and N-(carboxyethyl)lysine (CEL) in skin collagen. Kidney nitrotyrosine level, an oxidative stress biomarker, was also ameliorated by treatment of the compound in diabetic animals. LR-10R also reduced AGE and RAGE levels in the aorta, and decreased lesions in aorta of diabetic animals. Various in vitro tests showed that the AGE/ALE inhibitory and therapeutic effects of LR-102 could be due to both interaction with reactive carbonyls species, and inhibition of oxidative metabolism, including lipid peroxidation, through metal chelation and free radical scavenging properties.

LR-102 inhibits in vivo AGE/ALE accumulation and RAGE protein expression, as well as protein oxidation and hyperlipidemia in diabetic animals, and could be beneficial in preventing the progression of diabetic nephropathy and atherosclerosis. These findings provide investigators additional therapeutic options for treatment of diabetic nephropathy and possibly other complications of diabetes where AGE/ALE accumulation and oxidative stress are primary contributors.

P8

INCREASES IN PARAOXONASE-1 ACTIVITY AFTER HEMODIALYSIS IN CORRELATES WITH DECREASES IN LOW MOLECULAR WEIGHT AGE.

Gugliucci A, Melhalff K, Kimura S , Kinugasa E, Ogata H , Schulze J and Hermo R

1. Glycation, Oxidation and Disease Laboratory, Department of Basic Sciences, Touro University-California, Vallejo, USA 2. Clinical Pathology Department Showa University School of Medicine, Yokohama, Japan

KEYWORDS: end stage renal disease, atherosclerosis, HDL, glycation

Patients with chronic renal failure show the highest increases in circulating AGEs due to a clearance defect. They also show low levels of paraoxonase activity.

We hypothesized that paraoxonase activity is inhibited in the uremic milieu and that contributes to the lower activity seen in ESRD patients. We reasoned that PON-1 activity should rise after a successful hemodialysis intervention, irrespective of changes in HDL concentration or subclass distribution. Our second hypothesis was that low molecular weight substances in ESRD patients serum but not in control subjects serum inhibit PON-1 activity.

We conducted an intervention study with 22 ESRD patients undergoing hemodialysis in whom paired pre and postdialysis samples were studied along with 30 age-matched control subjects. We measured PON-1 activities, as well as lipid peroxidation, apoA, lipid profiles, HDL-subclasses and AGE products (both > 10 kDa and < 10 kDa) in each of the patients and evaluated the correlations of uremia-associated substances (urea, creatinine) with paraoxonase activity. Ultrafiltrates (cut-off 10 kDa) of both pooled control serum and ESRD serum were incubated with PON-1.

Dialysis produces a 5-40% increment in PON-1 activity (average 20 ± 8%) while no significant changes in either apoA-I or HDL subclasses distribution was observed. This change correlates (r=0.86. p<0.001) with the efficiency of dialysis as determined by either creatinine or urea rate of change. It also correlates (r=0.87. p<0.001) with changes in 350/440 fluorescence and pentosidine fluorescence in the <10 kDa serum fraction. These fractions produce a time and dose dependent inhibition of PON-1 activity (25% ± 3% vs 10 ± 2 % for control, p<0.005).

Our data give both in vitro and in vivo support to an inhibition of PON-1 by uremic toxins that are partially removed by dialysis and suggest that least in part, these toxins are low molecular weight AGE adducts. This research lends support to another mechnism for renal failure-induced atherogenesis and for AGE-related pathogenicity, that would act synergistically with the prevalent HDL, LDL and hemodynamics changes in this disease. Supported by Showa and Touro Universities.

P9

IN VITRO DIGESTIBILITY AND EFFECT ON THROMBIN FORMATION OF SOY PROTEINS GLYCATED WITH FRUCTOSE AND FRUCTANS

M. D. Mesa, J. van de Lagemaat, A. Gil, A. Olano, M. D. Del Castillo

Universidad de Granada; Instituto de Fermentaciones Industriales, CSIC

KEYWORDS: in vitro, gastric digestion, thrombogenicity, Maillard reaction, soy proteins, fructose and fructans

There is a growing interest in soy proteins due to their well-known health benefits. Cardiovascular diseases are one of the main causes of death world-wide. Soy proteins may interact with molecules involved in the coagulation systems preventing advanced artherosclerotic injury. Glycation of proteins modifies both structure and functions of the native proteins. Since there are no papers dealing with the effect of glycation on the in vitro digestibility of soy proteins and the impact of their hydrolysates on thrombin formation facilitated by LDL, these issues were investigated. Soy proteins were glycated with fructose and fructans at 95:C for 1h in 0.5 M phosphate buffer (pH 7.4). Glycation was followed using the OPA assay. Controls consisting of heated and native proteins were also analysed. Samples were digested in vitro, thereby mimicking gastric conditions. The level of digestion was estimated by Kjeldahl analysis, assessing total nitrogen remaining in solution. Digested samples were incubated with coagulation factors (X,V and prothombin), LDL and Ca+2. Subsequently, the in vitro formation of thrombin mediated by LDL was studied. Heat treatment of the native protein resulted in a diminution of around 15% of protein free amino groups, whereas glycation caused a decrease of 90%. Solubility of the protein was unaltered by glycation since soluble total nitrogen values obtained for native (64%) and glycated (60-64%) soy proteins were similar; however, less nitrogen remained in solution when heated proteins were digested (49.7%). Thrombin formation was enhanced by the incubation with hydrolysate of native proteins. Addition of the hydrolysates of heated or glycated soy proteins to coagulation factors caused a lower formation of thrombin than that obtained with native protein. The results indicated that the glycation of soy proteins under the studied conditions did not greatly affect in vitro digestibility and thrombogenicity of LDL.

P10

COMPREHENSIVE ANALYSIS OF GLYCATED HUMAN SERUM ALBUMIN TRYPTIC PEPTIDES BY OFF-LINE LIQUID CHROMATOGRAPHY FOLLOWED BY MALDI ANALYSIS ON A TIME-OF-FLIGHT/CURVED FIELD REFLECTRON MASS SPECTROMETER

A.Lapolla, F.L.Brancia, J.Z.Bereszczak, D.Fedele, L.Baccarin, R.Seraglia, P.Traldi

Dipartimento Scienze Mediche e Chirurgiche, Università di Padova

KEYWORDS: mass spectrometry, non enzymatic glycation

Glycated peptides arising from in vivo digestion of glycated proteins, usually called advanced glycation end product - peptides (AGE peptides) are biologically relevant compounds due to their reactivity towards circulating and tissue proteins. To investigate their structures, in vitro glycation of human serum albumin (HSA) has been performed and followed by enzymatic digestion. Using different MALDI-based approaches, the digestion products obtained have been compared with those arising from enzymatic digestion of the protein. Results obtained using 2,5-dihydroxybenzoic acid (DHB) indicate this as the most effective matrix, leading to an increase in the coverage of the glycated protein. Off-line microbore liquid chromatography prior to MALDI analysis reveals that 63% of the free amino groups amenable to glycation are modified. In addition, the same approach shows the co-presence of underivatized peptides. This indicates that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. The data suggest that the collisionally activated decompositions of singly charged glycated peptides leads to specific fragmentation pathways related to the condensed glucose molecule. These unique product ions can be used as markers to establish the presence of glucose molecules within the peptide ions.

P11

EFFECT OF MAILLARD REACTION DEVELOPMENT ON ANTIOXIDANT PROPERTIES OF DEHYDRATED ONION AND GARLIC DURING STORAGE

Marta Corzo-Martínez, F. Javier Moreno, M. Dolores del Castillo and Mar Villamiel

Instituto de Fermentaciones Industriales (CSIC), C/Juan de la Cierva 3, 28006 Madrid, Spain

KEYWORDS: Maillard reaction, antioxidant activity, garlic, onion

It is well-known that onion (Allium cepa L.) and garlic (Allium sativum L.) are two ingredients with a wide range of applications during the elaboration of foodstuffs. In addition, both vegetables have been traditionally used due to their health benefits being the antioxidant power one of the most relevant. Different compounds such as polyphenols, flavonoids and sulfur derivatives have been described as responsible, in great part, for the antioxidant effect of onion and garlic. However, few information is available on the potential antioxidant property of products derived from the Maillard reaction in this type of food ingredients. Fructosyl-arginine, an early product of the Maillard reaction, has been detected in a concentrated aged garlic extract obtained by storage at room temperature and its potential antioxidant activity has been compared to vitamin C. In this work, the influence of Maillard reaction initial and advanced stages on the overall antioxidant activity (ORyACFL) of dehydrated onion and garlic has been investigated. Dehydrated laboratory prepared samples were stored for 14 days at 30°C without control of aw, 30°C at 0.44 aw and 50°C at 0.44 aw. Neither progress of Maillard reaction nor substantial change in the antioxidant activity were found in dehydrated garlic samples during storage. However, a positive correlation between colour and antioxidant activity of stored onion samples was observed at 50°C. A moderate effect of Amadori compounds, determined as the corresponding 2-furoylmethyl-amino acids, on this activity was also suggested in onion during storage. The results obtained in this work point out the effectiveness of Maillard reaction products as scavengers of peroxyl radicals in dehydrated onion samples.

P12

SIMULTANEOUS ANALYSIS OF CROSSLINKING AND MAILLARD REACTION PRODUCTS IN THERMAL PROCESSED FOOD PROTEINS

L. Bosch, M.L. Sanz, A. Montilla, A. Alegría, R. Farré, M. D. Del Castillo

Instituto de Fermentaciones Indsutriales, CSIC; Universidad de Valencia

KEYWORDS: Carboxy-methyl-lysine, LAL, protein quality

Maillard and cross-linking reactions are key chemical events taking part during food manufacturing reducing protein quality. Various compounds have been proposed as chemical indicators of them such as lysine (Lys), carboxymethyl-lysine (CML) and lysinoalanine (LAL). Lys is an essential amino acid and precursor of CML and LAL. CML (advanced glycation end product) and LAL (crosslinking product) have been described as harmful compounds and commonly determined separately by employing tedious and time consuming procedures. The aim of the present study was to assess the feasibility of a simple gas chromatography method for evaluating protein quality by simultaneous analysis of Lys, CML and LAL. The method was validated employing pure standards substances, model systems and real foods.

Model systems consisting in food proteins (BSA, soy, casein and gluten) and glyoxilic acid were studied to know the influence of source of protein on the rate of the Maillard and crosslinking reactions. Since the nutritional relevance of the milk proteins and their importance as a main food ingredient, the effect of the manufacturing conditions on their deterioration was also studied. Whey permeates were pasteurised (63ºC for 30 min) or sterilized (100ºC for 30 min) and subsequently analysed. Milk-cereal based infant food, boiled eggs, soy drinks, sterilized skimmed milk, condensed milk, powdered milk, evaporated milk, bread, dry powdered crepes, cocoa-milkshake were also studied.

The rate of both reactions strongly depended on the nature of the involved protein. Thermally conditions employed for milk processing scarcely contributed to the evolution of the Maillard reaction up to advanced stages and protein crosslinking. The method allowed the simultaneously analysis of Lys, CML and LAL in food samples. Levels of CML of 2.15-23.8 mg/100 g were measured in stored milk cereal infant foods, soy drinks, boiled eggs and dry powdered crepes. LAL was formed during boiling of eggs.

P13

ASSESSMENT OF THE MAILLARD REACTION OF FRUCTOSE AND LYSINE BY ANALYSIS OF 2-FUROYLMETHYL-AMINO ACIDs.

M. D. del Castillo, J.M. Silván, M. Amigo, F.J. Moreno, A. Olano

Instituto de Fermentaciones Industriales, CSIC

KEYWORDS: Maillard reaction, Heyns compounds, 2-furoylmethyl-amino acids.

Maillard reaction is the process by which reducing sugars react non-enzymatically with protein residues of lysine and arginine. This reaction is relevant both in food science and biology. Since aldose reducing sugar such as glucose and lactose are major carbonyl compounds in biology and food, there has been considerable interest in studying their glycation products. Particular attention has been dedicated to the initial glycation product from the reaction of aldose and lysine, also known as Amadori products. Less information related to the condensation products of ketose such as fructose with lysine leading to glucoselysine also called Heyns product is available. The aim of the present research was to evaluate the usefulness of the analysis of the type-Heyns products of lysine with fructose and fructans as 2-furoylmethyl amino acids, which are widely employed for the indirect measurement of Amadori compounds.

Model systems of e-amino-lysine and ketose (fructose and fructans (DP 2-8) in methanol were heated under reflux until pale yellow colour was developed. Formation of Heyns compounds were detected by electrospray ionisation-mass spectrometry (ESI-MS). Samples were thermal digested with 8M HCL to obtain the corresponding 2-furoylmethyl amino acid (2-FM-AA) and analysed by ion-pair RP-HPLC. The identity of Heyns compounds was also confirmed by RP-HPLC-MS analysis of their 2-FM-AA.

Analysis by ESI-MS showed the presence of the ions (M + H)+ 309.2 and 471.3 due to the Heyns compounds mono-fructosyl-Lys and di-fructosyl-Lys, respectively. Glycation products of lysine with fructose and fructans were observed by RP-HPLC in the studied samples. Also Heyns products of soy proteins with these sugars have been measured following this analysis. In agreement, it can be concluded that ion-pair RP-HPLC is adequate to trace Heyns compounds. Further investigations should be conducted to compare the yield of formation of 2-furoylmethyl amino acids derivatives from Heyns and Amadori products.

P14

PENTOSIDINE AND ENDOTHELIN 1 IN PATIENTS UNDERGOING CHRONIC HAEMODIALYSIS

Fiammetta Monacelli, Patrizio Odetti, Daniela Storace, Cristina Robaudo, Simona Rossi, Giacomo Deferrari, Tommaso Barreca.

Department of Internal Medicine and Medical Specialties (DIMI) University of Genova

KEYWORDS: Pentosidine, Endothelin 1 ,uremia

Advanced glycation end products (AGEs) accumulate in blood and tissues of patients with Chronic Renal Failure (CRF) undergoing chronic hemodialysis and play an important role in the pathogenesis of uraemic complications. Endothelin 1 (ET1), a 21-aminoacids peptide with vasoconstrictor and mitogen properties, is an important factor involved in the endothelial dysfunction occurring in uremia. Both AGEs and ET1 circulating levels have been reported to be increased in chronic renal failure.

To evaluate a possible correlation between AGEs circulating levels and ET1 secretion in CRF patients receiving chronic hemodyalitic treatment .

The plasma concentration of free and bound pentosidine (HPLC method) and endothelin-1 (RIA method) were measured before the hemodialysis session in 40 CRF non diabetic patients (22 males and 18 females; 54 ± 3 years) undergoing chronic hemodialysis since at least 1 year. Forty age sex matched normal subjects constituted the control group.

The bound and free levels of pentosidine were significantly higher in hemodialysed patients in comparison to the normal subjects (Bound pentosidine (0.2-2.0 pmol/mg protein and free pentosidine 0-5 pmol/m in uraemic patients and 1.2+/- 0.2 in normal subjects). Endothelin-1 was too significantly (P<0.01) increased in CRF patients (10.56±044 pmol/ml in CRF patients and 2.68±0.32 pmol/ml in normal subjects). A significant positive correlation was shown between total and free pentosidine and endothelin-1 plasma values.

The strict correlation between pentosidine and endothelin-1 provides a further link of the detrimental role of AGEs on vascular endothelium, contributing to hypertension onset and to other cardiovascular complications occurring in CRF patients.

P15

HANDLING OF AGES BY THE KIDNEY - EFFECT OF EXPERIMENTAL NEPHRECTOMY AND URETERAL LIGATION IN THE RAT

Ahmed N* (1), Sebekova K (2), Sebekova K Jr (2) Heidland A (3), Thornalley PJ (1)

(1) Department of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom; (2) Institute of Preventive and Clinical Medicine, Medical University, Bratislava, Slovakia (Slovak Republic) and (3) Department of Internal Medicine, University of Wuerzburg, Wuerzburg, Germany.

KEYWORDS: AGE residues, AGE free adducts, renal clearance, renal failure, oxidation, nitration

The aim of this study was to investigate the effect of experimental kidney removal (bilateral nephrectomy, BNX) and blocking of urine collection (ureteral ligation (BUL) on the plasma levels of advanced glycation end products (AGEs) in rats. We measured AGE residues of plasma protein and AGE free adducts in plasma ultrafiltrate of BNX and BUL rats and compared them with the sham-operated controls (CTRL). AGEs were determined quantitatively by liquid chromatography with tandem mass spectrometric detection in plasma 72 h after induction of acute renal failure; n = 4. Serum creatinine (micromolar) was: CTRL 40 ± 3, BNX 1030 ± 52 and BUL 980 ± 55. AGE free adducts were increased markedly in BNX and moderately or not significantly in BUL. AGE free adduct concentrations (nM): N-carboxymethyl-lysine (CML) - CTRL 466 ± 107, BNX 4808 ± 705 (+10-fold, P<0.01), and BUL 1886 ± 200 (+4-fold, P<0.01); N-carboxyethyl- lysine (CEL) - CTRL 58 ± 8, BNX 900 ± 216 (+16-fold, P<0.01), and BUL426 ± 48 (+9 fold, P<0.01); glyoxal-derived hydroimidazolone (G-H1) - CTRL 197 ± 65, BNX 1012 ± 250 (+5-fold, P<0.01), and BUL 707 ± 64 (+4-fold, P<0.01); methylglyoxal-derived hydroimidazolone (MG-H1) - CTRL 72 ± 32, BNX 462 ± 72 (+6-fold, P<0.01), and BUL 68 ± 25; and 3-deoxyglucosone-derived hydroimidazolone (3DG-H) - CTRL 88 ± 32, BNX 714 ± 44 (8-fold, P<0.01), and BUL 176 ± 65. These and other glycation, oxidation and nitration free adduct analytes increased progressively during the 3-day post-operation period. The marked increase in AGE free adducts of BNX rats confirms that renal elimination is a major fate of these uremic toxins. The smaller increases of AGE free adduct concentrations in BUL rats, with respect to BNX rats, suggests the ligated kidneys in BUL retain the ability to remove AGE free adducts from the circulation.

P16

NON-ENZYMATIC GLYCOSYLATION OF BOVINE BETA-LACTOGLOBULIN WITH GALACTO-OLIGOSACCHARIDES

M. L. Sanz (1), A. Olano (2), R. A. Rastall (3) and F. J. Moreno (2)

(1) Instituto de Química Orgánica General (CSIC). (2) Instituto de Fermentaciones Industriales (CSIC). C/ Juan de la Cierva, (3) 28006 Madrid (Spain). 3 Department of Food Biosciences The University of Reading RG6 6AP Reading (UK)

KEYWORDS: beta-lactoglobulin; galacto-oligosaccharides; glycation; Maillard reaction

Galacto-oligosaccharides (GOS) are well-known prebiotic ingredients which can be included in the manufacture of new functional dairy products. Due to their reducing nature, they are able to participate in the Maillard reaction with amino acids, peptides and/or proteins during the food processing. The aim of this work was to study the non-enzymatic glycosylation of bovine β-lactoglobulin (β-lg) with GOS at weight ratios of 1 : 1 and 1 : 3 under controlled conditions (aw = 0.44, 40ºC for 23 days). A mixture of carbohydrates containing GOS (degree of polimerization 3-7), lactose, glucose and galactose was the original source employed. Prior to storage, mono- and disaccharides were removed by adsorption onto activated charcoal. RP-HPLC-UV profile of β-lg was modified as consequence of its glycation from the second day of incubation. Glycosylation extent of β-lg was evaluated by formation of 2-ε-furoylmethyl-lysine (furosine) following acid hydrolysis. After 16 days of incubation, 2.4 and 2.7 mg of furosine/100 mg of β-lg were found for 1 : 1 and 1 : 3 weight ratios, respectively. Moreover, a decrease in tri- and tetrasaccharides was observed by anion-exchange chromatography with pulsed-amperometric detection upon glycation. These results were confirmed by MALDI-TOF-MS. Further studies are planned to determine the prebiotic activity of these conjugates.

This work was financed by the project AGL2004-07227-CO2-02 supported by the Ministerio Español de Educación y Ciencia.

P17

CONSUMER ATTITUDES TOWARDS HIGH PRESSURE PROSESSING OF FOOD

Lampila P*, Lähteenmäki L.

Consumer studies, VTT - Technical Research Centre of Finland, Finland

KEYWORDS: consumer attitudes, high pressure processing

Consumer acceptance is one of the key issues in the future success when new processes are introduced to the food processing. Although consumers are not typically focused on processing method itself but rather to the benefits and risks associated with it. Consumer attitudes towards low temperature high pressure processing were investigated through a consumer survey in Spain, the Netherlands, Belgium and Finland (n = 936).

Generally attitudes towards high pressure processing were neutral, even thought the term was unfamiliar for most consumers. High pressure processing was not regarded as unnatural which gives a good starting point for its future adoption. The relative importance of choice criteria was studied with conjoint analysis. Processing method was considered less important than price or environmental impact. However, there was a group of consumers who put more emphasis on the processing method. Not having to raise the price and possible environmental benefits seem to be the most crucial factors for promoting the acceptance for high- pressure technology as a new processing method.

P18

THE IDENTIFICATION OF SOME FURAN DERIVATIVES IN GLUCOSE-LYSINE MODEL SYSTEM BY GAS CHROMOGRAPHY/ MASS SPECTOMETRY

Maria Cioroi

University Dunarea de Jos of GALATI, Romania

KEYWORDS: Maillard reaction, furan derivatives, LIKENS-NICKERSON apparatus, CG-SM, glucose, lysine.

Furan derivatives are important in the aroma of the food products subject to thermic processing. They are formed as a result of the interaction of the sugars with the amino acids in the Maillard reaction. One of the principal effects of the Maillard reaction is the change in sensory characteristics of a food, by the production of colour and flavour compounds.

This study presents the results of the separation and identification of some furan derivatives generated in the glucose-lysine model system.

The system was thermal treated at 100 °C in Likens-Nickerson apparatus. The volatile compounds, extracted with ethyl ether - pentane, were separated by means of Gas-Chromatography method; then they were identified by means of GC-MS method.

The system revealed a decreasing of pH during the heat treatment at the same time with the apparition of the volatile compounds. In these circumstances, there are prevalently formed the furan and furan derivatives, all having caramel flavor.

P19

5-HYDROXYMETHILFURFURAL - A POSSIBLE INDICATOR IN THE STANDARDIZATION OF THE ANALYSIS METHOD TO CONTROL THE PREZERVATION ABILITIES OF THE THERMALLY PROCESSED FOOD

Maria Cioroi

University Dunarea de Jos, Galati, Romania

KEYWORDS: Maillard Reaction,5-hydroxymethilfurfural (HMF),GC-SM, HPLC, spectrophotometry UV-VIS

The Maillard reaction (MR) involves the reaction of carbonyl groups of monosaccharide or polysaccharides and amino groups of amino acids or proteins. There is increasing recognition of the role of the MR in food science and technology. The MR plays an important role in the development of color, aroma, texture, and nutritional value of food products. This work is mainly focused on the preparation of model MR product (MRP) of amino acid/monosaccharide and to present the results from the gas chromatographic analysis and the mass spectrometry in order to identify the 5-HMF in the glucose-lysine model system. The time dynamic quantitative estimation of HMF is made by the HPLC method. Studies were carried out on the glucose- lysine model of 1M concentration and 1:1 mole ratio treated in different time intervals at 100 °C temperature.

Taking into account that 5-HMF has an absorption peak 280 nm spectrophotometric measurements were made relative to the DO variation in the preset heat treatment time intervals. Correlating the results from experiments a significant increase in the amount of 5-HMF is noticed which confirms the direct contribution of the monosaccharide in the primary phase of Maillard reaction only in the presence of the proton agent, the α-amino acid, respectively.

It is thus shown that with the glucose-lysine model the amount of 5-HMF is much higher than in the glucose solution alone at pH=7. This makes it obvious the importance of the chemical interactions between glucose and amino acid.

P20

SUPPRESSION OF ACRYLAMIDE FORMATION BY SOME CATIONS IN GLUCOSE- ASPARAGINE MODEL SYSTEM

Vural Gökmen (1), Hamide Z. Şenyuva (2)

(1) Department of Food Engineering, Hacettepe University, Beytepe, 06800 Ankara, Turkey , (2) Ankara Test and Analysis Laboratory, Scientific and Technical Research Council of Turkey, Ankara 06330, Turkey

KEYWORDS: Acrylamide, HMF, cation, Maillard reaction

High concentrations of acrylamide found in common fried and baked foods attained considerable public concern since it has been classified as a probable human carcinogen. These findings gave rise to legal authorities to initiate concepts for minimization of acrylamide in commercial and homemade foods. This study aimed to find a viable approach for this concept and to show the effect of cations on the formation of acrylamide in model system. The effects of mono- (Na+ and K+), di- (Ca2+, Mg2+ and Zn2+) and trivalent cations (Fe3+) on the formation of acrylamide were studied in glucose-asparagine model system. The reaction mixture was composed of 10 µmoles of asparagine and glucose. The amounts of cations in the reaction mixture were adjusted 0, 1, 5, 10 and 20 µmoles. The reactants were transferred into tightly closed pyrolysis tubes (25 ml) with limited amount of water (100 µl) to promote the physical interaction of fructose and asparagine. The reactions were performed at 150°C for 20 min. Effectiveness of cations on the elimination of acrylamide formation in glucose-asparagine system was in the order of Fe3+ > Mg2+ > Zn2+ and Ca2+ > K+ > Na+. At amounts equivalent to those of acrylamide precursors, added cations, except of Na+ were found to prevent acrylamide formation greater than 97%. It was observed that increased amounts of cations decreased the amount of N-glycosyl asparagine which is the key intermediate leading to acrylamide. However, increasing the amounts of cations gave rise in the amounts of hydroxymethylfurfural and furfural formed in the model system.

P21

EFFECT OF BARLEY COFFEE ON THE ADHESIVE PROPERTIES OF ORAL STREPTOCOCCI

Papetti A*, Tarsi R, Daglia M, Grisoli P, Dacarro C, Pruzzo C, Gazzani G

Dept. Pharmaceutical Chemistry, University of Pavia (Italy)- Dept. of Biology, University of Genoa (Italy)- Dept. Experimental and Applied Pharmacology, University of Pavia (Italy)

KEYWORDS: barley coffee, streptococci, adherence, functional food, anti-adhesive properties

Rapid advances in scientific knowledge supporting the vital role of diet foods in health and disease prevention have been made recently. Caries is an infectious disease for which Streptococcus mutans is considered the principal etiological agents in humans. S. mutans adhesion to tooth surfaces is a crucial step in dental caries initiation and development. A promising approach for caries prevention is based on the inhibition of such process. Some beverages and foods are known to protect against S. mutans colonization of dental surfaces; we have recently shown that roasted coffee interferes with S. mutans adsorption to hydroxyapatite (HA). In the present works the antibacterial and antiadhesive properties of barley coffee (BC), a beverage obtained by roasted barley grains and frequently used as coffee substitute, was studied and components responsible for the found activities were identified and isolated. Three experimental approaches were followed to evaluate the effects of BC sub-lethal concentrations (sub-MICs) on S. mutans adherence to saliva-coated HA beads: BC and bacteria were added simultaneously to HA beads; streptococci were pre-treated with BC before addition of bacteria; streptococci grown in the presence of BC were added to HA. All treatments caused a statistically significant, dose-dependent inhibition of S. mutans sucrose-dependent and sucrose-independent adherence to HA;. BC components were fractioned by dialysis; the MM <1000 Da fraction, containing polyphenols, zinc and fluoride ions, and the MM >100KDa fraction, consisting of a brown potent antioxidant melanoidin, showed anti-adhesive properties. The high MM melanoidin was not found in the natural barley, indicating that it originates in barley grains during the roasting process. These results suggest that BC consumption might influence colonization of tooth surfaces by cariogenic bacteria.

P22

IN VITRO ANTIOXIDANT ACTIVITY OF DIETS RICH IN MAILLARD REACTION PRODUCTS

Isabel Seiquer, Beatriz Ruiz Roca, and María Pilar Navarro

Unidad de Nutrición, Estación Experimental del Zaidín (CSIC), 18008 Granada, Spain

KEYWORDS: antioxidant activity, DPPH assay, TBARS, diets

During food processing many antioxidant compounds are lost to some extent, while others (Maillard reaction products, MRPs) are generated. Antioxidant properties of MRPs have been studied in foods or in model systems, but rarely in a whole diet, where a big variety of antioxidants and prooxidants may be present. The aim of this work was to investigate the in vitro antioxidant activity of a diet rich in MRPs, determining the capacity to scanvenge free radicals and to inhibit lipid peroxidation.

1)Two 7-days menus were elaborated, with a similar energy and nutrient composition but different food processing whenever possible: white diet (WD) and brown diet (BD), low and rich in MRPs, respectively. Initial content of some natural antioxidants in the diets (vitamin C, E and A) was calculated using a computer program based on the Spanish food composition tables. The higher values of HMF content and percentage of relative fluorescence intensity in the BD confirmed the advanced development of the MR. 2) Antioxidant activity was tested in lyophilized samples of the diets by the free radical 1,1-diphenyl-2-picryl- hydrazyl (DPPH) method and measuring the thiobarbituric acid reactive substances (TBARS) using a lipid emulsion system.

The initial content of vitamin E was slightly higher in the WD, but the other antioxidant vitamins did not differ between diets. Both diets showed similar values of scavenging effect. In contrast, the ability to reduce oxidation in a linoleic acid emulsion was increased in the more processed diet.

The likely loss of some natural antioxidants due to thermal process in the BD seems to be counter-balanced by the formation of intermediate and advanced MRPs with antioxidant activity. The formed compounds could contribute to the radical scavenging and oxidation reduction properties of the whole diet. P23

PROTEOLYSIS OF MILK PROTEINS GLYCATED WITH LACTOSE IN MODEL SYSTEMS

Trine Kastrup Dalsgaard, Jacob Holm Nielsen and Lotte Bach Larsen

Danish Institute of Agricultural Sciences, Department of Food Science, Research Centre Foulum, P.O. Box 50, DK-8830 Tjele, Denmark.

KEYWORDS: milk proteins, glycation, lactose, proteolysis, chymosin, plasmin, cathepsin D

Four different milk proteins (β-casein, α-casein, κ-casein, β-lactoglobulin) and the model peptide (VHFFKNIVTARTP) were glycated with lactose by incubation at 65°C at pH 6.8 for four days. Lactoferrin was incubated at 55°C due to lower thermal stability for this protein. The glycation was monitored by measurement of the level of free primary amino groups by a fluorescent probe. After four days of glycation no further changes in the level of free primary amino groups were observed, and the proteins were enzymatically hydrolysed by the three milk relevant proteases plasmin, cathepsin D and chymosin. The enzymatic hydrolysis was performed at 30°C and pH 6.8 for plasmin and at pH 5.0 for the two aspartic proteases: cathepsin D and chymosin. The hydrolysis by plasmin was highly negatively affected by glycation in all substrates. This effect could be explained by modification of lysine residues, being the preferred cleaving site for plasmin, but also the residue glycated with lactose. Cleavage of β-casein, α- casein, κ-casein, β-lactoglobulin by cathepsin D and chymosin was not affected by glycation, whereas decreased hydrolysis was observed for lactoferrrin and the model peptide after glycation with lactose for hydrolysis with these enzymes.

P24

IN VIVO FLUORESCENCE OF ADVANCED GLYCATION ENDPRODUCTS IN HUMAN SKIN INCREASES WITH AGE AND IN SMOKERS, IN CONTRAST TO AROMATIC AMINO ACIDS.

H Corstjens*(1), A Neven (1), G Eyckmans (1), B Gruner, L Declercq (1) and D Maes (2)

(1) Estee Lauder Companies, Oevel, Belgium (2) Estee Lauder Companies, Melville, NY

The accumulation of advanced glycation endproducts (AGEs) has been implicated in the ageing and photoaging of proteins and lipids. From in vitro experiments it is very well known that some glycated modifications show fluorescent properties. This feature has been extensively used to detect and quantify AGEs in various experimental settings. In the current experiments we investigated whether in vivo skin fluorescence, used as a marker for AGE accumulation in human skin, was affected by chronological age and smoking behaviour. We gathered in vivo fluorescence intensity data on the ventral forearm of 94 panellists, 78 being non-smokers and 16 being smokers. Fluorescence intensity was attributed to the aromatic amino acid tryptophan (λex/λem 295/345nm), AGEs (λex/λem 370/440nm) and elastin or collagen cross-links (λex/λem 440/520nm). Skin colour (CIE Lab colour space and individual typology angle) was measured on the same test site. There was a strong inverse correlation between the fluorescence intensities and skin colour, probably due to absorbance and/or scattering of the fluorescence excitation or emission light by pigmented skin. Multiple linear regression techniques were therefore used to simultaneously evaluate in vivo fluorescence as a function of skin colour, panellist age and smoking behaviour. Taking into account the differences in individual skin colour, the fluorescence intensity attributed to AGEs and elastin-collagen cross-links increased as a function of panellist age. Comparison of non- smokers to smokers revealed elevated fluorescence intensities attributed to glycation in the latter group. Fluorescence linked to tryptophan was neither affected by chronological age nor by smoking. These data support that AGEs, measured as in vivo fluorescence intensity, accumulate in human skin as a function of chronological age. The in vivo skin fluorescence was also associated with smoking behaviour, suggesting increased AGE accumulation in smokers.

P25

STUDIES ON THE ANTRADICAL ACTIVITY AND IRON CHELATING POWER OF GLUCOSE LYSINE MAILLARD REACTION PRODUCTS

Isabel Seiquer, Beatriz Ruiz Roca, and María Pilar Navarro

Unidad de Nutrición, Estación Experimental del Zaidín (CSIC), 18008 Granada, Spain

KEYWORDS: DPPH assay, antioxidant activity, iron chelating power

The antioxidant activity attributed to some Maillard reaction products (MRPs) may be due to different mechanisms, as their ability to scanvenge free radicals or their affectivity as metal chelating agents. The intensity of the heating process greatly affects antioxidant properties of the MRPs, but data on this sense are controversial. The aim of this work was to evaluate the effect of the time of heating on antioxidant activity of MRPs from a glucose-lysine model system. Furthermore, the ability of the compounds to chelate iron was evaluated.

Equimolar mixtures of glucose-lysine were heated at 100ºC for different times (15 to 90 min). The samples were characterized by development of the brown color at 420 nm, pH values and free lysine by HPLC. 2) The scavenging ability of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical by the glucose-lysine MRPs was determined at different concentrations. Results were compared with those of commercial antioxidants commonly used in food industry (BHT and α-tocopherol). 3) Iron-chelating power was measured by the method of Yoshimura et al., slightly modified.

The absorbance of the diluted samples increased with the time of heating. On the contrary, pH values and free lysine decreased. DPPH scavenging activity was higher for the mixture glucose-lysine less heated (15 min), and decreased progressively in the more heated and coloured samples (30, 60 and 90 min). Moreover, a progressive decrease in the scavenging effect with the samples concentration was observed. However, the antiradical activity of commercial antioxidants increased with concentration. All the heated mixtures showed strong iron chelating ability, but, in this case, the more heating time, the greater metal chelating power.

The reaction conditions strongly affect the properties of the formed MRPs. In our experimental conditions, development of browning influence in different manner the mechanisms involved in the antioxidant activity of the final products

P26

ISOLATION, PURIFICATION AND CHARACTERIZATION OF HISTIDINO- THREOSIDINE, A NOVEL MAILLARD REACTION CROSSLINK FROM THREOSE, LYSINE AND HISTIDINE

Zhenyu Dai, Wei Chen, and Vincent M. Monnier.

Departments of Biochemistry and Pathology , Case Western Reserve University

Ascorbic acid mediated glycation has been implicated in the aging of lens crystallins. Searching to identify novel crosslinks, we have isolated a novel acid-labile yellow chromophore from the incubation of lysine, histidine and threose and identified its chemical structure by LC-MS, LC-MS/MS, 1H, DEPT and two dimensional NMR. This new cross-link exhibits a UV absorbance maximum at 305nm and a molecular mass of 451 Da. One- and two dimensional NMR suggests the structure to be 2-amino-5-(3-((4- (2-amino-2-carboxyethyl)-1H-imidazol-1-yl)methyl)-4-(1,2-dihydroxyethyl)-2-formyl-1H-pyrrol-1 yl)pentatonic acid, a cross-link between lysine and histidine with addition of two threose molecules. We assigned the compound the trivial name histidino-threosidine. Systemic incubation revealed that histidino-threosidine can be formed from fructose, glyceraldehyde, methylglyoxal, glycolaldehyde, ascorbic acid, and dehydroascorbic acid, but at a much higher yield with degradation products of ascorbic acid---threose, erythrose, and erythrulose. Histidino-threosidine has both novel and as well as features reminiscent of previously described Maillard reaction compounds. It has a surprising similar structure with a simple adduct- - formyl threosyl pyrrole observed by Nagaraj and Monnier. The involvement of histidine in the crosslink is new what concerns Maillard reaction products, but has been previously reported with 4-hydroxynonenal. Other studies point to the possible beneficial effects and utilization of histidine as an anti-cataract agent.

P27

HIGH MOLECULAR WEIGHT COFFEE MELANOIDINS: CHARACTERIZATION OF DIFFERENT POPULATIONS

Bekedam E.K., Schols H.A., Smit G., van Boekel M.A.J.S.

Laboratory of Food Chemistry and Product Design and Quality Management Group, Department of Agrotechnology and Food Science, Wageningen University, Wageningen, The Netherlands

KEYWORDS: coffee, melanoidins, ethanol precipitation, anion exchange chromatography, CE

The structure of coffee melanoidins is extremely complex. Although several researchers have studied the formation and structure of these compounds, the structure of melanoidins remains largely unknown. Unfortunately, still no method is available to quantify these melanoidins unambiguously. In our research we focussed on the removal of non-melanoidin material by chromatographic procedures in order to study the presence of different melanoidin populations as well as the characterization of the compounds bound to melanoidins.

An attempt was made to quantify the melanoidin content present in various coffee fractions by determination of the absorption at 405 nm per gram of coffee fraction. A brew prepared from roasted coffee beans was fractionated by diafiltration (NMWC 3 kDa) and ethanol precipitation. It was found that the high molecular weight (HMw) fraction of the brew was rich in melanoidins and contains 26% of the melanoidins present in the coffee brew; the HMw fraction represents 15 w/w% of the brew dry matter. Also, it was found that 60% of the HMw melanoidins was soluble at high ethanol concentrations (>64% EtOH) suggesting that the melanoidins possess a less hydrophilic character. The content and composition of other coffee components in these fractions, like sugars, proteins, and phenols were also monitored. Especially proteins and phenols were present in relatively high extends in the melanoidin rich fractions. These findings suggest that especially phenols and proteins might be (covalently) bound to melanoidins.

Another important finding was that almost all (>95%) of the high molecular weight melanoidins were negatively charged, and was retained on an anion exchange chromatography (AEC) column.

Both AEC and capillary electrophoresis using laser induced fluorescence detection point out that the HMw brew consists of various melanoidin populations.

The interaction between the different components on molecular level will be discussed.

P28

LC/MS-MS AND GC/HRTOF-MS METHODS FOR ANALYSIS OF ACRYLAMIDE IN FOODSTUFFS

L. Vaclavmk, T. Cajka, L. Dunovska and J. Hajslova nstitute of Chemical Technology, Department of Food Chemistry and Analysis, Technicka 3 166 28 Prague 6, Czech Republic

KEYWORDS: acrylamide, GC-MS, LC/MS-MS

Acrylamide represents food contaminant presence of which in heat proccesed foodstuffs has been proven only recently. This compound is classified as human carcinogen by the International Agency for Research on Cancer (IARC) and is therefore of health concern for consumers.

Several analytical methods have been implemented for a determination of acrylamide in various foodstuffs. Most of procedures employ LC/MS-MS systems with triplequadrupole analyzer for obtaining low detection limits. SPE cartridges are often used to remove interfering impurities from crude extract prior to determinative step. In most of methods using GC/MS bromination of acrylamide is carried out in the first step to improve its detection parameters (higher, hence more selective m/z values of target analyte are obtained in this way, however sample handling is rather laborious).

In our study, two simple alternative methods employing either LC/MS-MS or GC/HRTOF-MS were developed. Both of them enable direct determination of target analyte. Fundamental simplification of sample preparation step was achieved by introduction of QuEChERS strategy that uses dispersive solid-phase extraction. Performance characteristics of both analytical approaches for various matrices are compared. GCT high-resolution time-of-flight mass spectrometer (HRTOF-MS, Micromass, UK) and Quattro Premier MS/MS (Micromass, UK) respectively, were used for experiments.

P29

HMF UPTAKE AND TRANSPORT FROM ENRICHED CULTURE MEDIUM AND BREAKFAST CEREALS IN CACO-2 CELLS

Cristina Delgado-Andrade (1), Isabel Seiquer (2), José A. Rufián-Henares (1), María Pilar Navarro (2) and Francisco J. Morales (1)

1 Instituto del Frío (CSIC), 28040 Madrid, Spain; 2 Unidad de Nutrición, Estación Experimental del Zaidín (CSIC), 18008 Granada, Spain

KEYWORDS: hydroxymethylfurfural, uptake, transport, Caco-2

Hydroxymethylfurfural (HMF), a common intermediate in the Maillard reaction also formed by sugar degradation, has been detected in many foods. Its toxicological relevance is still uncertain, since in vitro studies on genotoxicity/mutagenicity have given controversial results. A previous step to analyse the impact of HMF consumption on health may be the study of its uptake/transport in the Caco-2 cell line, as a model to predict the in vivo availability. The aim of this work was to test the HMF uptake/transport from HMF- added culture medium and from in vitro digested breakfast cereals.

1)The culture medium was added with three different levels of HMF until final concentrations of 130, 65 and 13 mg/L HMF (DMEM-1, -2 and -3, respectively). 2) HMF was analysed in three different breakfast cereals (A, B and C), before and after the in vitro digestion (in the supernatants obtained). 3) Caco-2 cells were seeded into bicameral chambers (Transwell). On day 21 post-seeding, test solutions (DMEM-1, -2, -3 and supernatants of A, B and C digested samples) were added to the apical chambers; a transport solution, HMF free, placed in the basolateral chamber and cell cultures adequately incubated for 2h. After incubation HMF transported across the cell monolayer and that internalised into the cells during the experiment was measured by HPLC.

After the in vitro digestion, only a 13.2, 56.4 and 46.5% of the HMF present in A, B and C samples was soluble. The uptake and the transport of HMF by the cells were demonstrated in the enriched culture medium and in the digested breakfast cereals. In the last ones, the initial concentration of HMF was not directly related to the soluble fraction after digestion neither to the %HMF transported, showing, as expected, an effect of the sample composition in the availability of HMF.

P30

MAGNESIUM-DEPENDENT PHOSPHATASE-1 IS A PROTEIN-FRUCTOSAMINE-6- PHOSPHATASE POTENTIALLY INVOLVED IN DEGLYCATION

Fortpied J, Maliekal P, Vertommen D and Van Schaftingen E

Laboratory of Physiological Chemistry, ICP and Université Catholique de Louvain, Brussels, Belgium

KEYWORDS: glycation, glucose 6-phosphate, protein phosphatase, deglycation

Fructosamine-3-kinase (FN3K) is a protein-repair enzyme responsible for the removal of fructosamines, which are the products of a spontaneous reaction of glucose with amines. We show that glucose 6- phosphate (G6P) is a more powerful glycating agent than glucose and fructosamine 6-phosphates (FN6P) presumably arise spontaneously in tissues. As FN3K does not act on FN6P, we investigated whether tissues contain a FN6P phosphatase. We found such an enzyme in several rat tissues. The FN6P phosphatase was purified from skeletal muscle and identified as Magnesium-Dependent Phosphatase-1 (MDP-1), an enzyme of the haloacid dehalogenase family with a putative protein-tyrosine phosphatase function. Human recombinant MDP-1 acted on protein-FN6P with a catalytic efficiency > 10-fold higher than those observed with its next best substrates, arabinose 5-phosphate and free fructoselysine 6-phosphate, and > 100-fold higher than with protein-phosphotyrosine. It had no detectable activity on fructosamine 3- phosphates. MDP-1 dephosphorylated up to 75% of the FN6P residues that are present on lysozyme after incubation of this protein with G6P, and dephosphorylation by MDP-1 leads to a substrate for FN3K. We conclude that MDP-1 may act physiologically in conjunction with FN3K to free proteins from the glycation products derived from G6P.

P31

ACCUMULATION OF GLYCATED PROTEINS IN FRUCTOSAMINE-3-KINASE DEFICIENT MICE

Maria Veiga-da-Cunha (1), Patrick Jacquemin (2), Ghislain Delpierre (1), Frédéric Lemaigre (2) and Emile Van Schaftingen (1)

Laboratories of (1) Physiological Chemistry and (2) Hormone and Metabolic Research Unit, Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium

KEYWORDS: Fructosamine 3-kinase, glycation, fructoselysine, protein repair

Fructosamines are formed during the non-enzymatic reaction of glucose with amines, including those present on proteins. We inactivated in mice fructosamine-3-kinase (FN3K), a recently identified intracellular enzyme that phosphorylates free and protein-bound fructose-?-lysines. FN3K-/- mice looked healthy and had normal blood glucose and serum fructosamine levels. However, their level of hemoglobin-bound fructosamines was about 2.5-fold higher than that of control or FN3K+/- mice. Other intracellular proteins were also significantly more glycated in FN3K-/- mice in erythrocytes (1.8 to 2.2-fold) and in brain, kidney, liver and skeletal muscle (1.2 to 1.8-fold), indicating that, in vivo, FN3K removes fructosamines from intracellular proteins. The urinary excretion of free fructose-?-lysine was 10-20 fold higher in fed mice compared to 36 h-starved mice and did not differ between fed FN3K+/+ and FN3K-/- mice, indicating that food is the main source of urinary fructose-?-lysine in these mice and that FN3K does not participate in the metabolism of food-derived fructose-?-lysine. However, in starved animals, the urinary excretion of fructose- ?-lysine was 2.5-fold higher in FN3K-/- mice than in FN3K+/+ or FN3K+/- mice. Furthermore, a marked increase (5 to 13-fold) was observed in the concentration of free fructose-?-lysine in tissues of fed FN3K-/- mice compared to control mice, indicating that FN3K participates in the metabolism of endogenously- produced fructose-?-lysine. Taken together, these data indicate that FN3K serves both as a protein repair enzyme and in the metabolism of endogenously produced free fructose-?-lysine.

P32

MILK-CEREAL BASED INFANT FOODS: FORMATION OF FUROSINE DURING STORAGE

Bosch L, Alegría A, Farré R*.

Nutrition and Food Chemistry, Faculty of Pharmacy, University of Valencia, Burjassot, Valencia, Spain.

KEYWORDS: Furosine; infant foods; Maillard reaction; storage

Milk-cereal based infant foods have a long shelf-life and can be consumed for up to one year after their manufacture. Their composition and the shelf-life favour the development of the Maillard reaction (MR) that reduces quality of proteins. The aim of this work was to study the formation of furosine during storage of infant foods to evaluate the development of the earliest stages of MR. Studies were made on three (A,B,C) different milk-cereal based infant foods containing 88% milk and 8.8% cereals stored at 25º, 30º and 37ºC during 9 months. All three had the same composition, except for 0.9% of honey in B, and 1.1% of fruit in C. Furosine was determined by high-performance liquid chromatography with UV detection at 280nm. Furosine values obtained were in the range 10.5-11.9 mg/100g sample for infant foods just after the manufacture process and in the range 14.5-21.1 mg/100g sample at the last month of storage. ANOVA test was applied to furosine contents in order to detect differences between the infant foods studied, the storage temperature and the storage period. A gradual increase in furosine contents was observed during the storage period and a statistically significant (p≤0.05) difference in furosine content was observed according to the storage temperature. Furosine values were higher and statistically different for infant foods containing honey than in samples without them. There was an interaction between storage time and temperature or type of sample: the formation of furosine during the storage period was faster at higher temperature and depended on the studied infant food (A, B, C). To study the possible influence of storage time on furosine contents a simple regression analysis was carried out. A lineal model that can explain 61% of the variability of furosine was obtained.

P33

SMAD1 PLAYS A PIVOTAL ROLE FOR THE DEVELOPMENT OF DIABETIC NEPHROPATHY

Takeshi Matsubara, Hideharu Abe, Noriyuki Iehara, Akira Mima, Hidenori Arai, Toru Kita, and Toshio Doi

Department of Nephrology, Geriatric Medicine, Cardiovascular Medicine Kyoto University, and Department of clinical biology and Medicine, Tokushima University

KEYWORDS: Smad1, Type IV collagen, Diabetic nephrology

Mesangial matrix expansion is the hallmark in diabetic nephropathy. However, the precise mechanism for the development of mesangial matrix expansion has remained unknown. The key component involved in mesangial matrix expansion is ±1/±2 type IV collagen (Col4). We have reported Col4 is transcriptionally regulated by Smad1 under advanced glycation end products (AGEs). Using chromatin immunoprecipitation and reporter assay, we observed that Smad1 directly regulated transcription for Col4 through the binding to the promoter of Col4. Smad1 was significantly induced and activated in AGE-treated mesangial cells, and suppression of Smad1 resulted in a decrease of AGE-induced Col4 over-production.

To confirm a crucial role of Smad1 for the development of diabetic nephropathy in vivo, we then constructed Streptozotocin-induced (STZ) diabetic rats at the age of 32 weeks, and examined the glomerular expression of Smad1 by Western blotting and immunohistochemistry. The glomerular expression of Smad1 was significantly increased in STZ rats. We also found that increased Smad1 was localized mostly in the glomerular mesangial area. Next, we examined the relationship between glomerular expression of Smad1 and the severity of mesangial matrix expansion. The glomerular expression of Smad1 was closely correlated with the glomerular accumulation of AGEs, glomerular expression of Col4, and the severity of mesangial matrix expansion in diabetic rats. We also demonstrated that glomerular expression of Smad1 was closely correlated with the glomerular expression of smooth muscle alpha actin (±-SMA), which is a key molecule in the phenotypic modulation of mesangial cells. In cultured mesangial cells, over-expression of Smad1 up-regulated the transcriptional activity of ±-SMA in addition to Col4.

In conclusion, these findings indicate that Smad1 is a novel direct regulator of Col4 under AGE stimulation, and contributes to the phenotypic modulation of mesangial cells. Thus, Smad1 plays a pivotal role for the development of diabetic nephropathy.

P34

FUNCTIONAL ANALYSES OF NEUTROPHIL-LIKE DIFFERENTIATED CELL LINES ON A HYPERGLYCEMIA MODEL

Oya-Ito T*, Naitou H, Masuda S, Ohashi N, Kinae N

Graduate School of Nutritional and Environmental Sciences, University of Shizuoka

KEYWORDS: inflammatory diseases, high glucose condition, neutrophil-like differentiated human myeloid leukemia cell lines, reactive oxygen species (ROS) production, myeloperoxidase MPO activity

Hyperglycemia is a major factor associated with diabetic complications. Diabetes may also complicated by increased susceptibility to recurrently bacterial infection. Neutrophils under the hyperglycemia are thought to involve in the susceptibility, but its functional mechanisms are still unknown. We report here the functional changes in neutrophil-like differentiated human myeloid leukemia cell lines (HL-60, THP-1, and NB-4) under a high glucose condition as a hyperglycemia model. HL-60 and NB-4 cells were differentiated with ATRA, and THP-1 cells were differentiated with ATRA and GM-CSF. The cells underwent neutrophil- like differentiation as judged by morphological changes, nitroblue tetrazolium assay and FACS analysis with anti-CD11b, -CD14 and -CD15 antibodies. Then, these 3 cells were treated with 35 mM of glucose for 24 hrs, and the reactive oxygen species (ROS) production, myeloperoxidase (MPO) activity, amount of intracellular GSH, morphology, and apoptosis were examined. In differentiated HL-60 cells, the treatment with high glucose induced ROS production after PMA stimulation, enhanced MPO activity, and slightly reduced the loss of intracellular GSH by PMA stimulation. Intracellular ROS of differentiated THP-1 cells cultured under the high glucose condition were abnormally increased by PMA stimulation. Furthermore, the high glucose exposure of differentiated NB-4 cells enlarged vacuoles and delayed apoptosis. Since ROS generation was induced only after PMA stimulation in the high glucose-treated cells, the high glucose condition appears to cause the excessive response of neutrophil-like cells to the stimulation rather than direct induction of ROS production. These extraordinary sensitivities may play an important role in the development of inflammatory diseases.

P35

CHANGES IN REDUCING POWER DURING PRODUCTION OF PRUNES

Cejpek K, Jarolímová L, Velíšek J.

Department of Food Chemistry and Analysis, Institute of Chemical Technology, Prague, Czech Republic

KEYWORDS: prunes, Maillard reaction, reducing activity, antioxidants

Prunes, i.e. dried plums, are generally believed to be an excellent source of antioxidants in human diet. During drying of plums, substantial changes in the composition of antioxidants may occur as a result of various transformation reactions (hydrolysis, oxidation, the Maillard reaction, etc.).

In this paper, reducing activity of fresh plums, and commercially available and traditionally prepared prunes was evaluated using HPLC method with electrochemical detection (+0.8 V). Compared on a dry matter basis, the changes are not significant if mild drying (60°C, up to 12 h) is employed during the production of commercially prepared prunes with sorbate for soft and moist fruit. However, somewhat chewer dried plums prepared according to the traditional recipes (60-90°C, up to 18 h) revealed substantial increase of reducing activity, mostly due to much faster Maillard reaction. One major relatively polar, low-molecular, process- induced product derived from hexoses was revealed in traditionally prepared prunes. Its presumptive antioxidant activity was confirmed by DPPH radical assay and beta-carotene bleaching method. The effect of variable parameters of drying, sample treatment, and plum quality on the stability and formation of active compounds was evaluated.

P36

ANGIOTENSIN II-DEPENDENT ACTIVATION OF SRC AND SMAD1 IS CRUCIAL FOR THE DEVELOPMENT OF DIABETIC NEPHROPATHY

Akira Mima

Department of Nephrology, Kyoto University, Kyoto, Japan

KEYWORDS: AGEs type IV collagen Smad1 angiotensin II

We have reported that Smad1 was significantly induced along with type IV collagen (Col4) in advanced glycation end products (AGEs) treated mesangial cells and that it played a key role for the development of diabetic glomerulosclerosis in vivo. Angiotensin II (AII) plays a pivotal role in the development of diabetic nephropathy, and AII receptor blocker (ARB) is used to inhibit the progression of diabetic nephropathy. However, little is known about the molecular mechanism by which ARB prevents glomerular sclerosis. Therefore, the aim of this study is to examine whether AII activates Smad1 or what is the signaling mechanism involved in AII-mediated Smad1 activation in mesangial cells. We examined whether AII can modulate Smad1-mediated signaling involved in glomerulosclerosis in diabetic nephropathy in vivo and in vitro.

We used STZ rats. The rats were divided into four groups: control, control treated with olmesartan, STZ (DM+vehicle), STZ treated with olmesartan (DM+ARB). Mesangial PAM and Col4 positive areas as well as the glomerular expression of α-SMA were then measured after 24 weeks.

In DM+vehicle, the mesangial sclerotic area and the glomerular expression of Col4, α-SMA, Smad1 and Src were increased compared with control rats. ARB treatment significantly inhibited mesangial sclerotic areas and these expressions.

To determine whether AII can activate Smad1 and increase the synthesis of Col4, cultured mesangial cells were incubated with AII. AII stimulated the phosphorylation of Smad1 and Src, and the synthesis of Col4. ARB inhibited the phosphorylation of Smad1 and Src and the expression of Col4 after AII stimulation. Moreover, PP2 can inhibit AII-induced phosphorylation of Src and Smad1.

These findings indicate that that AII can modulate Smad1-mediated signaling in diabetic glomerulosclerosis via a Src-dependent pathway, and that ARB can prevent the glomerular structural changes in the early phase of diabetic nephropathy through the inhibition of Src and Smad1.

P37

ACROLEIN, A LIPOPEROXIDATION PRODUCT, CAUSES APOPTOSIS IN RETINAL PERICYTES

Zhang X, Lai Y, McCance D, Uchida K, McDonald D, Gardiner TA, Stitt AW, Curtis TM*

Centre of Vision Sciences, Queen’s University of Belfast, UK

KEYWORDS: diabetes, retinopathy, acrolein, apoptosis

Lipoperoxidation products may be associated with the pathogenesis of diabetic microvascular complications, including retinopathy. Acrolein is a major lipid peroxidation product that is known to be elevated in diabetic patients. Retinal pericyte apoptosis is a pathophysiological hallmark of diabetic retinopathy. In the present study we demonstrate that acrolein induces retinal pericyte apoptosis in vitro and we have begun to unravel the molecular mechanisms through which this occurs.

Cultured bovine retinal pericytes were incubated with acrolein (10, 25 & 50 µM) for 24 h. Pericyte apoptosis was visualised and measured by confocal scanning laser microscopy and FACs analysis respectively, using annexin V or JC-1. Intracellular oxidants were detected using DCFH-DA. Caspase activity was quantified using luminometric assay kits and NF-kB (p65 subunit) activity was determined with a transcription factor assay kit. Acrolein adduct generation in the presence of antioxidants was measured by competitive ELISA using the monoclonal antibody 5F6.

Acrolein caused a dose-dependent increase in cytotoxicity, with >50% apoptosis at the highest concentration compared to non-exposed cells (p<0.01). Acrolein-induced cell death was associated with enhanced generation of reactive oxygen species with levels 1.8 and 2.8 fold above basal values for the two highest acrolein concentrations (p<0.01). Interestingly, the activity of caspase 3/7, 8 and 9 declined as a function of the acrolein level, with values falling below 40% of those measured in control cells at a concentration of 50 µM (p<0.01). The activity of the anti-apoptotic transcription factor, NF-kB also decreased with increasing concentrations of acrolein (p<0.01). The antioxidants ascorbic acid, superoxide dismutase and catalase partially reversed the generation of oxidants by acrolein (p<0.01), but failed to elevate NF-kB activity. None of these agents directly scavenged acrolein.

These results suggest that acrolein-induced apoptosis in retinal pericytes appears to be caspase- independent and may, at least in part, involve direct attenuation of NF-kB activity by acrolein. Acrolein generation in vivo may contribute to the pathogenesis of diabetic retinopathy.

P38

ELEVATED HAEMOGLOBIN LEVELS OF N(EPSILON)-CARBOXYMETHYLLYSINE AND ACROLEIN ADDUCTS ARE ASSOCIATED WITH PROLIFERATIVE DIABETIC RETINOPATHY.

Zhang X, Lai Y, McCance D, Uchida K, McDonald D, Gardiner TA, Stitt AW, Curtis TM*

Centre of Vision Sciences, Queen’s University of Belfast, UK

KEYWORDS: CML, acrolein, serum, hemoglobin, diabetes, retinopathy

To investigate whether serum and hemoglobin levels of the advanced glycation/lipoxidation end product (AGE/ALE), N(epsilon)-carboxymethyllysine (CML), and the ALE, acrolein, are associated with the severity of retinopathy in diabetic patients.

Serum and hemoglobin levels of CML and acrolein were measured by means of a novel competitive ELISA in Type 1 and Type 2 diabetic patients (n=176; age 60.15+/-1.27 years) with no retinopathy, background retinopathy or proliferative retinopathy, and in a non-diabetic control group (n=51; age 52.49+/-2.52 years). Patients with macroalbuminuria were excluded from the study.

Serum levels of CML and acrolein were significantly increased in diabetic patients compared to healthy subjects (mean respective values were 138+/-3 and 121+/-7 ug/ml, p<0.01 for CML and 22+/-0.5 and 19+/- 0.9 ug/ml, p<0.05 for acrolein). However, serum levels of these adducts failed to associate with the severity of retinopathy (p>0.05; one-way ANOVA). Hemoglobin levels of CML and acrolein were also elevated in diabetic patients (mean respective values for diabetic and control subjects were 13.3+/-0.4 and 10.72+/-0.4 AU/mg, p<0.01 for CML and 0.36+/-0.02 and 0.23+/-0.04 AU/mg, p<0.01 for acrolein). While no differences in hemoglobin CML and acrolein levels were apparent between control individuals and diabetic patients without retinopathy or with background retinopathy, these adducts were increased in patients with proliferative retinopathy (CML: 14.2+/-1.5 AU/mg p<0.05 vs control subjects; acrolein: 0.42+/-0.03 AU/mg, p<0.01 vs controls).

Our data suggest hemoglobin levels of CML and acrolein may provide a useful risk marker for proliferative diabetic retinopathy and that elevated intracellular ALE/AGE formation may be involved in the pathogenesis of the advanced stages of the disease.

P39

SAFETY ASSESSMENT OF GLUCOSE VS. FRUCTOSE BASED MAILLARD REACTIONS PRODUCTS

Karl-Heinz Wagner, Karin Koschutnig, Stefanie Reichhold, Catherine Billaud

Department of Nutritional Sciences, University of Vienna

KEYWORDS: Glucose, Fructose, Glutathione, Cystein, Maillard Reaction Products, AMES Test

Maillard Reaction Products (MRPs) based on fructose/glucose with glutathione or cysteine were shown to inhibit the polyphenoloxidase as antibrowning agents. However, no safety assessment has been done on these MRPs. The aim of the present study was to investigate the antioxidative and antimutagenic effects of glucose or fructose based MRPs in the Ames test.

The MRPs consisted of glucose resp. fructose and cysteine resp. glutathione, which were heated for 4h20min as well as 14h at temperatures of 103°C or 110°C.

During the investigations the plate incorporation assay and the preincubation assay were applied, both with and without metabolic activation (S9). Hydrogen peroxide (H2O2) and tertiär-buthyl hydroperoxide (tBOOH) were used as prooxidatives. The Salmonella typhimurium indicator strains TA 98 and TA 102 were employed, the samples were tested in five different concentrations: 0,05; 0,1; 1,0; 5,5; 11 mg per plate, in order to considerlow physiological conditions but also high concentrations up to the border of solubility.

The his+-revertants glucose-cystein (14h) turned out to be the most reactive one under the influence of H2O2. Without metabolic activation this sample showed antioxidative properties, whereas with the S9-mix it was prooxidative. The other 14h incubated fractions were less reactive. In contrast all 4h20min heated samples were strongly reactive. Regarding all influencing factors fructose-glutathione (4h20min) was the most conspicuous one.

For the inhibition of polyphenoloxidase the employment of glucose-cystein seems to be most expedient considering, however, the formation of mutagenic and prooxidative properties under the influence of H2O2 and S9.

P40

LUPIN PROTEIN ISOLATES WITH VALUABLE TECHNOLOGICAL PROPERTIES AND MINIMUM THERMAL DAMAGE.

Jürgen Bez (1), Michael Schott (1), Udo Knauf (1), Donatella Resta (2), Alessandra D’Agostina (2), Giovanna Boschin (2), Anna Arnoldi (2)

(1) Fraunhofer Institute for Process Engineering and Packaging (IVV), Department of Process Engineering, Giggenhauserstr. 35, 85354 Freising, Germany

KEYWORDS: Keywords: lupin protein isolate, available lysine, functional food, food ingredients

Seeds of Lupinus albus (white lupin) have been used as food by the Mediteranean populations for over 3,000 years. The high protein content (about 35-40 %) indicates that this legume has the potential to become a useful source of protein concentrates and isolates, to be exploited for example in replacement for milk, egg, or meat proteins. The favourable features of lupin protein isolates are related to some key techno-functional properties, i.e. emulsifying and foaming properties and the optimization of cost in comparison to most animal proteins. In addition, recent literature has pointed out some possible health benefits: a moderate daily intake of lupin protein led to a reduction of total and low density lipoprotein cholesterol, when administered to an established animal model of hyperlipidemia, a vegetable beverage based on lupin confirmed similar effects in a clinical study.

The Fraunhofer IVV research team has developed an innovative process for the separation of native protein products from lupin seeds. With this new process, efficient protein enrichment and purification were accomplished in pilot plant scale. This technology delivers two types of high value protein isolates with improved technological, sensory and nutritional characteristics: lupin protein isolate type E (LPI-E), with a useful emulsifying capacity, and lupin protein isolate type F (LPI-F), with a high capability of foam formation and stabilization. A relevant objective of the process optimisation was the reduction of the thermal damage in order to compete with other commercial food ingredients. This was done by measuring two classical molecular markers of non-enzymatic browning: Nε-furoylmethyl-L-lysine (furosine) and 5-hydroxymethyl-2- furancarboxaldehyde (HMF).

The spray-drying stage was particularly critical for inducing some thermal damage, but a careful selection of the conditions permitted to obtain ingredients where the bioavailability of lysine was only marginally impaired. The reproducibility of the protein extraction process was tested on two different lupin varieties.

P41

GENERATION OF THE BREAD FLAVOR COMPONENT 6-ACETYL-1,2,3,4- TETRAHYDROPYRIDINE IN PROLINE/1,3-DIHYDROXYACETONE MODEL SYSTEMS

An Adams and Norbert De Kimpe

Department of Organic Chemistry, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium

KEYWORDS: Maillard model reactions, 6-acetyl-1,2,3,4-tetrahydropyridine, 2,3-dihydro-1H-pyrrolizines, proline Maillard model reactions

The production of 6-acetyl-1,2,3,4-tetrahydropyridine, an important Maillard flavour compound, in the model reaction of proline and 1,3-dihydroxyacetone was investigated in detail, with the intention of using different reaction conditions to optimize the production of the characteristic bread flavour compound. Various 2,3- dihydro-1H-pyrrolizines were identified as side products in the reaction, the most important ones being 5- acetyl-6-methyl-2,3-dihydro-1H-pyrrolizine and 5-acetyl-6-hydroxymethyl-2,3-dihydro-1H-pyrrolizine. Reaction parameters such as the temperature, the reaction time, and the amount and type of reducing agent and sugar degradation fragment were studied in detail. Control of the reaction conditions and of the various additives in the reaction enabled an optimization of bread flavour formation with minimal formation of undesired side products. A maximum yield of 6-acetyl-1,2,3,4-tetrahydropyridine of 2.7 mol % from proline and 1,3-dihydroxyacetone in the presence of two equivalents of sodium bisulfite was noted at 130 °C. Since the presence of one equivalent of sodium bisulfite increased the production of 6-acetyl-1,2,3,4- tetrahydropyridine about 100-fold, its role as reducing agent and stabilizer of the bread flavour compound was established. The fundamental importance of the intermediate occurrence of 2-oxopropanal and 1- hydroxy-2-propanone in the formation of 6-acetyl-1,2,3,4-tetrahydropyridine and of 2,3-dihydro-1H- pyrrolizine side products was underlined.

P42

VALIDATION OF A PREDICTION MODEL FOR DETERMING OF THE TYPE OF HEAT- TREATED OIL AND ITS CONTENT IN POLAR AND POLYMERIC COMPOUNDS USING FRONT-FACE FLUORESCENCE

J Rizkallah, P Vigneron and I Birlouez-Aragon

Institut National Agronomique, PARIS-GRIGNON.

KEYWORDS: Fluorescence, polar compounds, polymers, chemocetrics, frying, vegetable oils

Vegetable oils (VO) suffer deep changes during heating and frying, some of which are deleterious for health. Thermoxidative degradation of triglycerides give rise to polymers (PM) and polar compounds (PC) respectively, both corresponding to a complex mixture of modified triglycerides. The content of PO is regulated so that vegetable oils with more than 25% PC are not considered as edible. For a rapid screening of fried oils edibility, SMEs and regulation instances use analytical kits allowing simple and rapid assessment of PO, but not always with accurate results. On the other hand conventional analysis of PM and PC are time consuming and need big solvent volumes. Front face fluorescence spectroscopy, a rapid and low cost technique was tested as a possible new tool to screen the PO and PM content of various VO. Peanut, rapeseed, sunflower and palm olein oils were analysed after 5 to 30 cycles of heating or frying at 190°C. Four main fluorophores mixtures were identified which were influenced by the type of oil and the heat treatment. The samples optical properties also differed according to their chemical composition and degradation level. So, in addition to the concatenated fluorescence spectra, synchronous spectra were used for further analysis to take these optical interferences into account. Multivariate analysis allowed to extract the specific information related to the VO type, the number of cycles applied and the PO and PM content. A comparison of the accuracy and robustness of the predictive models obtained using various pre- treatment tools allowed selecting the best method to either discriminate the different VO or to predict the PO and PM content with a mean error lower than 5% (RMSEP). The model was then applied to real samples provided by restaurants and the performance of the new method was compared to that of other rapid kits.

P43

PRELIMINARY STUDIES ON PHENOTHIAZINES-MEDIATED REVERSAL OF MULTIDRUG RESISTANCE IN MOUSE LYMPHOMA AND COLO 320 CELLS

Pajak B. (1), Molnar J. (2), Engi H. (2), Orzechowski A. (1)

(1) Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw Agricultural University, ul. Nowoursynowska 159, 02–776 Warsaw, Poland (2) Institute of Medical Microbiology and Immunobiology, Faculty of Medicine, University of Szeged, Dom ter 10, H-6720 Szeged, Hungary

KEYWORDS: P-glycoprotein, multidrug resistance, phenothiazines, flow cytometry

The reversal of multidrug resistance (MDR) in some neoplastic cells is the major failure of the present medicine. To assess the possible application in anticancer therapy the ability of phenothiazine derivatives to inhibit transport activity of P-glycoprotein in the resistant mouse lymphoma and MDR/COLO 320 cells was studied. A rhodamine 123 efflux from the above-mentioned neoplastic cells in the presence of tested compounds was examined by flow cytometry. Two of the phenothiazine derivatives namely perphenazine and prochlorperazine dimaleate proved to be effective inhibitors of rhodamine efflux. Other tested phenothiazine derivatives (promethazine hydrochloride, oxomemazine, methotrimeprazine maleate, trifluoropromazine hydrochloride, trimeprazine) also modulated intracellular drug accumulation in either resistant cell line, however, they exerted additional cytotoxic effect. Differences observed upon treatment with tested compounds on intracellular drug accumulation could be the outcome of differences in phenothiazine’s chemical structure which is crucial for the drug-cell membrane interactions. The results of this study provide information about a new group of compounds that give promise to reverse MDR in tumor cells.

P44

LACTOSE UREIDE,LACTOSE DERIVATIVE OF UREA, IN THERMALLY PROCESSED MILK AND MILK PRODUCTS

Kyozo Suyama and Atsusi Sasaki

Department of Nutrition, Sendai University, Miyagi, Japan

KEYWORDS: lactose ureide, lactose, urea, milk

Lactose derivative of urea, lactose ureide (LU), was newly found to be form in thermally processed milk and milk products in this experiment. First, the non-reducing carbohydrate was separated by the method developed in our laboratory from the water soluble fraction of powdered skimmed milk and was identified as LU using ESI-MS (m/z 385.1[M + H+]), 1H, 13C, 1H1H-COSY and HSQC nmrs. For HPLC analysis, synthesis of LU was carried out, and LU thus obtained was benzoylated by benzoic anhydride to afford hepta-O-benzoyl lactose ureide (mp 137-139C, m/z 1113 [M + H+], λmax 229nm; ε 8.1037 x 107).

Crude non-reducing carbohydrates fraction of thermally processed milks and milk products were directly benzoylated and then benzoilated LU was analyzed by HPLC on ODS column. The content of LU in powdered skimmed milks (5.0 ± 1.1 mg/l) were almost three times higher than that of UHT milk (1.5 ± 0.5 mg/l), and trace amount in law milk. When law milk was heated at 110C for different times, UL content increased in time-course. It was thought from the results that LU was formed by the Maillard reaction between lactose and urea in milk and milk products. It was known that human intestine is not able to digest LU. The health effect of LU is not known in the present time.

P45

FORMATION OF 5-HYDROXYMETHYLFURFURAL AND 5-HYDROXYMETHYL- FURAN-2-CARBOXYLIC ACID IN COFFEE AND IN MODEL SYSTEMS

Murkovic M, Bornik M

Graz University of Technology, Institute for Food Chemistry and Technology

KEYWORDS: HMF, coffee, 5-hydroxymethylfurfuric acid, model system

5-Hydroxymethylfurfural (HMF) has become a substance of interest since recent results show a possible carcinogenic potential in consequence of a metabolic activation by sulfotransferases. HMF is formed either by acid catalysed degradation of reducing sugars or via the Maillard reaction. This work shows that HMF is formed during roasting of coffee. Several types of roasted coffee were analysed which contained from 300 to 900 mg/kg of HMF. Additionally to HMF 5-hydroxymethyl-furan-2-carboxylic acid (HMF-A) is formed. Depending on the roasting conditions the concentration of the acid is ca. 200 to 400 mg/kg.

The formation of HMF and HMF-A is investigated during roasting of coffee and in heated mixtures of carbohydrates with amino acids.

P46

THE ADVANCED GLYCATED END PRODUCT (AGE) Nε-CARBOXYMETHYLLYSINE (CML) AS A POTENTIAL PREDICTOR OF CARDIOVASCULAR EVENTS AND RENAL OUTCOMES IN PATIENTS WITH TYPE 2 DIABETIC KIDNEY DISEASE

Martin Busch, Sybille Franke, Gunter Wolf, Antje Brandstädt*, Undine Ott, Jens Gerth, Lawrence G. Hunsicker" and Guenter Stein for the Collaborative Study Group

Department of Internal Medicine III and *Institute of Medical Statistics, University of Jena, Germany, "Department of Internal Medicine, University of Iowa College of Medicine, Iowa

KEYWORDS: chronic renal failure, cardiovascular risk, advanced glycation end-products, CML, type 2 diabetes, IDNT study

Advanced glycation end-products (AGEs) are implicated in the pathogenesis of vascular damage, especially in diabetes and renal insufficiency. The oxidatively formed AGE Nε-carboxymethyllysine (CML) is commonly thought to be a marker of oxidative stress. This study was designed to determine whether elevated serum levels of CML are related to a higher risk for cardiovascular or renal events in type 2 diabetics with nephropathy. Methods: 450 patients of the Irbesartan in Diabetic Nephropathy Trial (IDNT) cohort (mean age 58 ± 8.2 years, 137 female, 313 male) with a mean GFR of 48.2 ml/min (MDRD formula) were followed for 2.6 years. Serum levels of CML were measured with an ELISA assay. The relationship between CML, traditional risk factors and CV and renal events was tested in Cox proportional hazards models. Results: The mean serum level of CML was 599.9 ± 276.0 ng/mL, that of HbA1C 7.5 ± 1,6 %. 143 CVE occurred during follow-up; a total of 74 patients died, 44 of cardiovascular causes. Final multivariate analysis showed age (RR 1.87, CI 1.13-3.11, P = 0.016 for the highest quartile comp. to the lowest), a history of prior cardiovascular events (RR 1.96, CI 1.35-2.85, P < 0.0005) and the urine albumin-creatinine ratio (RR 1.29, CI 1.11-1.50 per doubling, P = 0.001) to be independent risk factors for a first cardiovascular event, but not CML concentrations. CML concentration was also not significantly correlated with renal outcomes. Conclusion: Serum level of CML could not be identified as an independent risk factor for cardiovascular or renal outcomes in the examined population. This suggests that traditional risk factors, mainly albuminuria, play a more important role for these endpoints than CML. However, a potential role of CML in modifying risk factors cannot be ruled out.

P47

EFFECT OF GLYCATION ON FIBROBLAST GROWTH FACTOR-2 INDUCED ANGIOGENESIS: RELEVANCE TO DIABETIC WOUND HEALING

Y.Duraisamy, M.Slevin, C.A.Smith, J.Gaffney and N.Ahmed

School of Biology, Chemistry and Health Science, Manchester Metropolitan University, Manchester M1 5GD, United Kingdom

KEYWORDS: glycation, fibroblast growth factor-2, angiogenesis, wound healing

Impaired wound healing in diabetic patients is characterised by reduced angiogenesis. Fibroblast growth factor-2 (FGF-2) is stored intracellularly in a variety of cell types including endothelial cells. Upon cellular injury, FGF-2 is released and promotes angiogenesis. This study investigated the kinetics of FGF-2 glycation and compared its ability with that of native FGF-2 to activate mitogenesis, capillary tube formation and signal transduction pathways in cultured endothelial cells. FGF-2 was glycated in vitro by exposure to 0.25 M glucose-6-phosphate for 24-72 hours and the extent of glycation determined by matrix assisted laser desorption ionisation mass spectrometry (MALDI). After 24, 48 and 72 hours of incubation with glucose-6- phosphate, there was evidence of glycation with a mass increases corresponding to addition of 2.7, 4 and 8.7 glucose-6-phosphate residues respectively. When glycated FGF-2 (100 pg/ml-5 ng/ml) was added to cultured endothelial cells, it reduced their proliferation by 25 to 40% compared to native FGF-2. Similarly, glycated FGF-2 (100pg/ml-1 ng/ml) reduced capillary tube formation by 60 to 90% when compared to native FGF-2. Glycated FGF-2 caused a reduction in signal transduction molecules associated with mitogenesis, in particular mitogen activated protein kinase (MAPK) and early response kinase (ERK-1, ERK-2). Total binding of glycated FGF-2 to the cell surface was significantly reduced in endothelial cells analysed by FACS compared to native FGF-2. Using 125I-labelled differentially washed samples, it was possible to demonstrate a significant reduction in glycated FGF-2 binding to the high affinity tyrosine kinase receptor compared to native FGF-2. In conclusion, glycated FGF-2 has a reduced ability to bind to tyrosine kinase recptors and activate signal transduction pathways responsible for both mitogenesis and capillary tube formation. Glycation of FGF-2 in vivo may explain the mechanism underlying impaired wound healing in diabetes.

P48

INVESTIGATIONS ON THE GENERATION OF α-DICARBONYLS FROM REDUCING SUGARS IN PRESENCE OF BAKING AGENTS AND THEIR IMPACT ON ACRYLAMIDE FORMATION.

T. M. Amrein, L. Andres, F. Escher, R. Amadò

Institute of Food Science and Nutrition, ETH Zurich, CH-8092 Zurich, Switzerland

KEYWORDS: acrylamide, dicarbonyls, baking agent, ammonium hydrogencarbonate

Acrylamide has neurotoxic and carcinogenic properties and is formed during heating of various food products. Free asparagine and reducing sugars are considered to be the main precursors for acrylamide formation in food. The baking agent ammonium hydrogencarbonate enhances the acrylamide formation in bakery products which contain substantial amounts of reducing sugars. Two hypotheses on the promoting effect of ammonium hydrogencarbonate are shown and discussed. The first hypothesis focuses on amidation of acrylic acid by ammonia originating from this baking agent. Experiments with 15N-labelled baking agent showed that this formation pathway is not relevant in gingerbread as an example for a bakery product containing large amounts of reducing sugars and ammonium hydrogencarbonate. The second hypothesis considers the potential formation of reactive α-dicarbonyl compounds from reducing sugars in the presence of baking agents. Preliminary results on the impact of time, temperature, pH, and type of baking agent on the formation of glyoxal, methylglyoxal, and 2,3-butanedione are shown. In addition, preliminary results regarding the formation of acrylamide from asparagine under mild conditions in the presence of these α-dicarbonyls are presented.

P49

HYPERGLYCEMIA LEADS TO ACCUMULATION OF INTRACELLULAR ADVANCED GLYCATION END PRODUCTS (AGE) AND IMPAIRMENT OF PROTEASOME ACTIVITY

Markus Queisser (1), Erwin D. Schleicher (2), Klaus T. Preissner (1)

(1) Department of Biochemistry, School of Medicine, University of Giessen, Giessen, (2) Departments of Medicine IV and Neurology, University of Tübingen, Tübingen

KEYWORDS: intracellular AGE, Proteasome

Hyperglycemia leads to accumulation of toxic AGE products but also found in diverse pathological settings, such as diabetes, inflammation, arteriosclerosis and during aging. Little is known about the intracellular activities of AGE and their contribution to chronic vascular diseases. Since the ubiquitin-proteasome system functions in cellular quality control by degrading misfolded or damaged proteins that form potentially toxic aggregates, the relation between hyperglycemia, AGE accumulation and proteasome activity was studied.

In A549 epithelial lung and Hek293 endothelial cells formation of intracellular AGE was found to be, dependent to time, and related to glucose concentration. While hyperglycemia provoked reducation of proteasomal trypsin- and chymotrypsin-like activity, peptidyl-glutamyl activity was not affected. A clear reduction of the 19S regulatory proteasome complex was found, while no change of the catalytic 20S subunit was occurred.

While nonglycated BSA become degraded by the 20S proteasome, glycated BSA was proteolysis resistant in vitro.

Under hyperglycemia intracellular AGE formation leads to AGE products accumulation, reduction of the proteasomal trypsin- and chymotrypsin-like activity and down regulation of the 19S complex. Incubation with the 20S proteasome shows that AGEs are proteolysis resistant by the 20S proteasome in vitro.

All together, these results provide evidence for alteration in the ubiquitin-proteasome system after inflammatory response under hyperglycemia, suggesting the impaired activity of the proteasome what leads consequent to intracellular AGE accumulation.

P50

SKIN AUTOFLUORESCENCE, A NON-INVASIVE MARKER FOR ADVANCED GLYCATION END PRODUCTS IS INCREASED IN PATIENTS WITH AGE RELATED MACULAR DEGENERATION.

J. Mulder (1), M. Bieze (1), R. Graaff (2), A.J. Smit (1), J.M.M Hooymans (3)

(1) Department of Medicine, (2) Department of Biomedical-Engineering, (3) Department of Ophthalmology

KEYWORDS: Advanced Glycation End products, skin Autofluorescence, Age related Macular Degeneration

Skin Autofluorescence (AF) is a non-invasive marker for glycaemic and oxidative stress derived Advanced Glycation Endproducts (AGEs) in tissue. AGEs have been implicated to play a role in the pathophysiology of Age related Macular Degeneration (AMD) in the eye. It has been demonstrated that systemic inflammation is associated with AMD, however it is unclear whether AMD is also accompanied by enhanced systemic AGE formation.

The aim of this study was to investigate whether skin AF, as a systemic measure for AGEs, is increased in patients with AMD compared with age matched healthy control subjects.

Skin AF was assessed in 75 patients with AMD scheduled for photodynamic therapy (mean age 78 ± 7 years; male: 40%; smoking: 53%; diabetes: 9%; hypertension: 32%; prior cardiovascular disease: 20%) and in 64 healthy controls (mean age 63 ± 17 years; male: 58%; smoking: 5%; hypertension: 19%) of which 31 were matched with AMD patients on age and gender (mean age 77 ± 5 years; p=0.72 compared with AMD). Patients with impaired renal function, heart failure, active inflammatory disease, or a dark skin type were excluded. Skin AF was defined as the average light intensity in the range of 420 600 nm divided by average light intensity in the range of 300-420 nm (emission/excitation) and was measured on the lower arm, using a prototype of the current AGE-Reader.

Skin AF was increased in AMD compared with matched controls (2.51 ± 0.59 * 10-2 AU vs. 2.23 ± 0.68 * 10-2 AU; p < 0.035) In a stepwise linear regression model including all patients and controls AMD was, in addition to age, independently associated with skin AF, even after controlling for age, diabetes, smoking, prior cardiovascular disease, hypertension, and gender (β 0.21; 95% confidence interval, 0.03-0.39; P=0.024).

Skin AF is increased in patients with AMD, independent of cardiovascular risk factors. These data may contribute to further understanding of the role of systemic inflammation, oxidative stress, and AGE formation as mediators of AMD.

P51

THE INFLUENCE OF EXOGENOUSLY DERIVED ADVANCED GLYCATION END PRODUCTS ON THE 15 MONTHS PROGRESSION OF SKIN AUTOFLUORESCENCE IN SUBJECTS AT HIGH RISK FOR CARDIOVASCULAR DISEASE

D.J. Mulder (1), G.M. Mulder (1), B.M. Jochemse (1), R. Graaff (2), A.J. Smit (1)

(1) Department of Medicine, (2) Department of Biomedical-Engineering, University Medical Center Groningen, The Netherlands.

KEYWORDS: Advanced Glycation End products, skin Autofluorescence, Dietary Glycotoxins, Smoking

Skin Autofluorescence (AF) is a validated non-invasive marker for Advanced Glycation End products (AGEs) in tissue. In addition to in vivo formation of AGEs, associated with diabetes and renal disease, exogenously derived glycotoxins (i.e. tobacco smoke and diet) may also contribute to tissue AGE accumulation and promote atherosclerosis. The aim of this study was to investigate which factors contribute to skin AF progression in subjects at high risk for cardiovascular disease.

Skin AF was assessed at baseline and after 15 months in 167 subjects (age 63.1±6.2 years; male 54%) with at least 3 cardiovascular risk factors, but without a history of cardiovascular disease. Skin AF was defined as the average light intensity in the range of 420 600 nm divided by average light intensity in the range of 300-420 nm (emission/excitation) and was measured on the lower arm, using a prototype of the current AGE-Reader. Seventy-four subjects recorded their dietary habits in a 3 day food diary, from which the average intake of total calories and fat, carbohydrate, and protein percentage of total calories were estimated. Additionally, the average intake of dietary AGEs was estimated from these diaries, based on an AGE assessment questionnaire, kindly provided by Dr. Uribarri (ref: Goldberg, J Am Diet Assoc. 2004).

Skin AF did not change significantly after in 15 months (2.21 ± 0.43 vs 2.15 ± 0.40; p=NS) in the total group. After correction for baseline skin AF levels, smoking (r2=0.19; p=0.008), age (r2=0.18; p=0.012), diabetes (β, 0.16; p=0.019), and leukocyte count (r2=0.16; p=0.024), but not kidney function, blood pressure, or obesity were independent predictors of skin AF progression (r2=0.34; p<0.001). In the subgroup of subjects that recorded their dietary pattern, total calorie intake, percentages of fat, protein, or carbohydrates, or dietary AGEs did not predict skin AF progression significantly.

Smoking behaviour, old age, diabetes, and inflammation are independent predictors of skin AF progression over 15 months. Dietary habits, including high intake of glycotoxins, do not influence skin AF progression significantly. These data may provide further insight in the role of in vivo formed AGEs and exogenously derived glycatoxins in the accumulation of AGEs in tissue.

P52

THE INFLUENCE OF VARIETY AND STORAGE ON THE FORMATION OF ACRYLAMIDE DURING BAKING OF DRIED POTATO POWDER AND OIL.

Wicklund T. (1), Dimitrijevic S. (1), Molteberg E.L . (2), Segtnan V.E. (3), Knutsen S.H. (3)

(1) Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Ås, Norway (2) Bioforsk Øst, Apelsvoll, 2849 Kapp, Norway (3) Matforsk, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway

Acrylamide is formed during heating of food material such as frying, deep frying or baking. Most important is the combined temperature and time input. Small changes in frying temperatures and time might introduce relatively large changes in the acrylamide content in the final products. Secondly it is well established that the levels of reducing sugars (Glucose and Fructose) are the most important chemical precursors influencing the end content of acrylamide in potato based products. Aspargine is also crucial for the formation and is therefore classified as a precursor. In potato the variation in asparagine is of less importance since the concentration of reducing sugars are normally considered to be the limiting factor. During cold storage of potato, which is applied to improve shelf life and depress sprouting, especially the contents of reducing sugars are accumulating. Therefore stored potato used during winter/spring tend to give rise to more acrylamide when heat-treated. In addition growth locality as well as techniques such as haulm killing might influence the content of precursors. In this present poster communication the attention is on the actual content of precursors in the two varieties ‘Peik’ and ‘Saturna’ and the corresponding acrylamide formation. A model system was set up in order to study the influence of the precursor constituents of the potato material only. Freeze-dried potato powders (2,5 g) were mixed with palm oil (1 g) in a small aluminium dish. All samples were baked simultaneously for a constant time (10 min) and temperature (165 °C) in a baking oven. The content of sugars and asparagine was recorded before heating and acrylamide was determined on the finished samples. Simultaneously colour were measured in L*a*b* units with a colorimeter. The analysis shoved that the relationship between acrylamide formation and the initial sugar content were different for the two cultivars studied, especially after different storage times. In addition it was shown that for samples prior to any storage there were linear relationships between the asparagine content and acrylamide formation. For the unstored samples the acrylamide content varied linearly with a* except from samples that originally were high and low in their asparagine content. The results suggest that the potential for acrylamide formation can not be predicted from the content of reducing sugars alone. Also other factors that might change during storage may have importance. Results are evaluated by multivariate statistics.

P53

AMINOAZAARENES, AZAARENES, POLYNUCLEAR AROMATIC HYDROCARBONS AND OKSYCHOLESTEROLS IN HEAT TREATED MEAT.

Janoszka B.*, Blaszczyk U.**, Warzecha L.**

*Department of Chemistry;** Department of Biochemistry in Zabrze, Medical University of Silesia in Katowice, Poland

KEYWORDS: heterocyclic amines, acridines, benzo(a)pyrene, 7-ketocholesterol

Introduction/aims: Thermally treated meat may contain different promuta- and cancerogenic compounds. Heterocyclic amines are products of Maillard reaction of sugars/aminoacids/creatin(in)e, azaarenes and polynuclear aromatic hydrocarbons (PAH) may be formed as the result of thermal decomposition of aminoacids, proteins and fat however, oxycholesterols are the oxidation products of cholesterol. Meat samples (pork and beef, without any additives or spices and filled with onion) prepared by grilling or frying were investigated. Methods: Tandem solid phase extraction (Extelut NT 20/PRS cation exchanger/C-18 phase) was used to isolate aminoazaaerenes, azaarenes and PAH fractions. Liquid extraction and SPE (silicagel/florisil) were used to oxycholesterols fraction separation. The analysis of selected compounds belonging to the four investigated groups has been performed by HPLC with DAD and fluorescence detection, GC/MS and planar chromatography with densitomeric detection. Results: The analyzed meat samples contained aminoazaarenes (IQ, MeIQ, MeIQx, 4,8-DiMeIQx, PhIP) azaarenes (benzoacridines and dibenzoacridines) and PAHs (5 compounds, among other benzo(a)pyrene) in the range from 0 to 28 ng/g. The concentration of two oxycholesterols (7- hydroxycholesterol and 7-ketocholesterol) was higher up to 440 ng/g. Meat sample prepared with use of onion contained lower amounts of the analyzed mutagens. Conclusion: Vegetable additives may reduce the concentration of procancerogenic compounds formed in meat as the result of heat treatment.

AUTHOR INDEX Abdel Nour A. S8 Ciesarova Z. S40 Abe H. P33 Cioroi M. P18, P19 Adams A. P41 Cooper M. S44 Ahmed N. P15, P47 Corstjens H. P24 Alegría A. P12, P32 Corzo-Martínez M. P11 Alt N. S14, S24 Crabbe M. S3 Amadò R. P48 Craus A. S8 Ames J. S3 Curtis T. P37, P38 Amigo M. P13 D’Agostina A. S17, P40 Amrein T. P48 D’Arcangelo D. S49 Andres L. P48 Dacarro C. P21 Anton P. S8 Daglia M. P21 Apostolopoulou A. P2 Dai Z. P26 Arai H. P33 Dalsgaard T. P23 Argirova M. S10 Damasius J. S18 Arnoldi A. S17, P40 De Kimpe N. P41 Baccarin L. P10 Declercq L. P24 Barreca T. P14 Deferrari G. P14 Bartling B. S5 DeGroot J. S47 Baynes J. S22, S24, P1 Del Castillo M. S37, P9, P11, Bekedam E. P27 P12, P13 Bereszczak J. P10 Delatour T. S4 Bez J. P40 Delgado-Andrade C. S7, P29 Bieze M. P50 Delpierre G. P31 Billaud C. S6, S19, Den Hollander N. P1 P39 Depeint F. S9, P6 Birlouez-Aragon I. S8, S27, P42 Doi T. P33 Blaszczyk U. P53 Dunovska L. S30, P28 Blank I. S25 Duraisamy Y. P47 Blatnik M. S24 Dybing E. S32 Bornik M. P45 Engi H. P43 Bosch L. P12, P32 Erbersdobler H. S1 Boschin G. S17, P40 Escher F. P48 Bötticher M. S46 Eyckmans G. P24 Brancia F. P10 Facchiano F. S49 Brandstädt A. P46 Fan X. S35 Bravo L. S7 Farré R. P12, P32 Brock J. S22 Fedele D. P10 Bruce W. S9, P6 Fedele F. S37 Buera P. S12 Ferracane R. S37 Buetler T. S4 Fiedler T. S29 Busch M. P46 Figarola J. P7 Cajka T. P28 Fogliano V. S37, S49 Cämmerer B. S16 Forbes J. S44 Capuano E. S37 Fortpied J. P30 Cejpek K. P3, P35 Frandsen H. P2 Chan T. P5 Franke S. S46, P46 Charissou A. S27 Frizzell N. S24 Chen W. P26 Fujiwara N. S42 Cheriot S. S19 Fujiwara Y. S39 Furrer R. S9, P6 Krustev A. S10 Gaffney J. P47 Lähteenmäki L. P17 Gardiner T. P37, P38 Lai Y. P37, P38 Gazzani G. P21 Lalljie S. S32 George S. S41 Lampila P. P17 Gerth J. P46 Lanzuise S. S37 Gibson G. S3 Lapolla A. P10 Gil A. P9 Larsen L. P23 Gökmen V. S13, P20 Lassen A S2 Goya L. S7 Leclerc E. S4 Graaff R. P50, P51 Lemaigre F. P31 Granby K. P2 Lindinger C. S25 Grisoli P. P21 Loera S. P7 Gruner B. P24 Maes D. P24 Gugliucci A. P8 Maliekal P. P30 Hajslova J. S30, P28 Märk J. S25 Hedegaard R. P2 Märk T. S25 Heidland A. P15 Masuda S. P34 Heizmann C. S4 Matsubara T. P33 Henle T. S2 Mavric E. S2 Hermo R. P8 McCance D. P37, P38 Hertlova M. S11 McDonald D. P37, P38 Hinton D. S3 Mehta R. S9, P4, P6 Hofmann T. S20 Melhalff K. P8 Holadova K. S30 Mennella C. S37 Hooymans J. P50 Mesa M. P9 Horvat S. S28 Mima A. P33, P36 Hunsicker L. P46 Miyamoto Y. S42 Iehara N. P33 Miyata T. S34 Janoszka B P53 Miyazawa T. S38 Jacquemin P. P31 Molnar J. P43 Jakas A. S28 Molteberg E. P52 Jandeleit-Dahm K. P7 Monacelli F. P14 Jarolímová L. P3, P35 Monnier V. S35, P26 Jarvis S. S35 Montilla A. P12 Jenkins A. S22 Morales F. S7, P29 Jochemse B. P51 Moreno F. P11, P13, P16 Kalousova M. S11 Muench G. S48 Kankova K. S11 Mulder D. P1, P51 Kimura S. P8 Mulder G. P51 Kinae N. P34 Mulder J. P50 Kinugasa E. P8 Müller L. S2 Kiss E. S40 Murkovic M. P45 Kita T. P33 Muscat S. S48 Kiyota N. S39 Nagai R. S39 Knauf U. P40 Naitou H. P34 Knutsen S. P52 Navarro M. P22, P25, P29 Kolek E. S40 Neven A. P24 Koschutnig K. S6, P39 Newell J. S4 Kroh L. S16, S29 Nicolas J. S19 Krusova D. S11 Nielsen J. P23 Novotny L. S30 Shangari N. S9, P4, P5, P6 O’Brien P. S9, P4, P5, P6 Shimoni E. S15 Odetti P. P14 Silber R. S5 Ogata H. P8 Silván J. P13 Ohashi N. P34 Simko P. S40 Olano A. P9, P13, P16 Simm A. S5 Orzechowski A. P43 Sivakami S. S41 Ott U. P46 Skibsted L. P2 Oya-Ito T. P34 Skripkauskaite A. S18 Pajak B. P43 Slevin M. P47 Papetti A. P21 Smit A. P1, P50, P51 Pellegrini N. S33 Smit G. P27 Perez C. S26 Smith C. P47 Pischetsrieder M. S48 Stadler R. S23 Pollien P. S25 Stefanova I. S10 Preissner K. P49 Stein G. S46, P46 Prosser B S3 Stitt A. S43, P37, P38 Pruzzo C. P21 Storace D. P14 Queisser M. P49 Suyama K. P44 Rahbar S. P7 Suzuki K. S42 Rastall R. P16 Takamiya R. S42 Reichhold S. S6, P39 Taniguchi N. S42 Reneker L. S35 Tarsi R. P21 Resta D. S17, P40 TessierF. S8 Rizkallah J. P42 Thomann R. S16 Robaudo C. P14 Thornalley P. S21, P15 Rossi S. P14 Thorpe S. S22, S24, P1 Rufián-Henares J. S7, P29 Traldi P. P10 Ruiz Roca B. P22, P25 Tuijtelaars S. S32 Rüster M. S46 Tuohy K. S3 Sakamoto Y. S39 Uchida K. P37, P38 Salvini S. S31 Vaclavik L. S30 Sanz M. P12, P16 Vaclavmk L. P28 Sasaki A. P44 Van Boekel M. P27 Schalkwijk C. S45 Van de Lagemaat J. P9 Scheubel R. S5 Van Schaftingen E. S36, P30, P31 Schilter B. S4 Veiga-da-Cunha M. P31 Schieberle P. S14 Velíšek J. P3, P35 Schleicher E. P49 Venkutonis P. S18 Schneeweiß R. S16 Vertommen D. P30 Schneider B. S5 Vigneron P. P42 Schols H. P27 Villamiel M. P11 Schott M. P40 Vinauskiene R. S18 Schulze J. P8 Wagner K. S6, P39 Sebekova K Jr. P15 Warzecha L. P53 Sebekova K. P15 Wicklund T. P52 Segtnan V. P52 Wittmann S. S2 Seiquer I. P22, P25, P29 Wolf G. S46, P46 Şenyuva H. P20 Yaylayan V. S26 Seraglia R. P10 Zhang X. P37, P38 Serpen A. S13 Zima T. S11