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published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Glycation is a series of chemical interactions occurring in food and biological systems between reducing sugars and proteins leading to the formation of Advanced Glycation End products (AGEs). Ingestion of dietary AGEs and/or their formation in-vivo are mainly associated with cardiovascular and other age-related diseases and complications of long term diabetes. Potential strategies to prevent AGE formation can help to reduce risk factors associated with thermal processing of many foods. The overall objective of this research was focused on the identification of potential AGE inhibitors and investigation of their activity in glucose and ribose-based model systems containing lysozyme. The carboxylic acid functional group was chosen as a potential candidate based on their ability to interact with Schiff bases in addition to their ability to form amide bonds and carboxylate salts with the lysine side chains of proteins. The model systems were incubated with and without selected carboxylic acids (maleic, acetic, oxalic and citraconic) at 50°C for 12, 24 and 48h at pH 6.5. The effect of carboxylic acids on the glycation of lysozyme was studied by electrospray ionization mass spectrometry (ESI-MS). The experimental results showed that none of the carboxylic acids were able to form amide linkages with lysozyme under the experimental conditions and only maleic acid was able to form carboxylate salts, however, oxalic acid was the only acid able to interact with the Schiff base and form 1,3-oxazolidine-4,5-dione intermediate and thus hinder its rearrangement into Amadori product and consequently inhibit glycation. As a result the percentage of free or unreacted lysozyme was the highest in oxalic acid model systems (9.4% in the case of glucose, 7.1% in the case of ribose system) and was even higher than the control systems (6.0% in the case of glucose, 1.2% in the case of ribose system) of both glucose and ribose. In addition, all carboxylic acids were able to modify the relative distribution of different glycoforms generated during the incubation period however oxalic acid was the most efficient in shifting the distribution of glycoforms to lower molecular weight clusters which can additionally contribute to its anti-glycation activity.

Standards of Amadori rearrangement products (ARP) were synthesized for the purpose of developing analytical techniques and performing mechanistic studies related to their thermal decomposition. Several synthetic strategies were explored. An HPLC analytical system with a diode array detector was coupled to a fluorometer and an electrochemical detector, in order to detect simultaneously and on-line, a wide variety of degradation products of ARPs and to follow their kinetics. The potential of such a system to analyze complex Maillard mixtures was demonstrated. The kinetics of the reaction of glucose with morpholine (a Strecker inactive analogue of proline was used in order to simplify the kinetics) to produce Amadori morpholine was studied under experimental conditions that minimize side reactions and maximize Amadori product formation. At specific time intervals, the samples were analyzed for the presence of reactants and Amadori product by the multidetector HPLC system. Color and fluorescence were also measured. The data obtained were used to calculate the rate constants for the formation and degradation of Amadori product. A mechanistic model that statistically fitted the kinetic data was proposed. To further understand the details of the decomposition mechanism of Amadori proline, different mass spectrometric experiment were performed. High resolution, linked-field scan and neutral loss experiments have indicated that 1-((2$ sp prime$-carboxyl)pyrrolidinyl)-1-deoxy- scD-fructose (Proline Amadori product) followed two main pathways of fragmentation under electron impact conditions; one initiated by the ring oxygen and the other by the amino acid nitrogen, producing two well stabilized fragment ions; oxonium and imminium ions. In addition, ortho-elimination reactions initiated by O or N-centered radical sites were shown to produce the most intense peaks and diagnostically important ions for the identification of Amadori products. However this approach can only pro

The reactions of the early and intermediate stages of protein glycation were studied using bovine serum albumin and hen egg white lysozyme as model proteins, and D-glucose and D-fructose as model reducing sugars. The reactions were studied under low moisture and limiting sugar conditions in the presence and absence of oxygen in a closed system. Several analytical methods, including the fluorescamine assay, total Maillard fluorescence and solubility measurement, size exclusion chromatography, and electrospray ionization mass spectrometry were used to follow the glycation reaction. The effect of glycation on the structure of proteins was also studied by electrospray ionization mass spectrometric peptide mapping. The results showed that under low moisture conditions, the initial rate of glucation was 5 to 6 times higher than the initial rate of fructation, both in the presence and in the absence of oxygen. The presence of oxygen in the reaction system induced selectivity of the glycation of the protein amino groups during the initial stages of the reaction, due to competition between glycation and glycoxidation. The reaction, however, occurred at a higher rate in the presence of oxygen than in its absence, especially during the latter stages of the reaction. The more reactive glycoxidation products, formed during the initial stages of the reaction also participated in the glycation reaction and increased the involvement of arginine residues in the glycation reaction. The glycation reaction also resulted in a heterogeneous mixture of protein glycoforms with varying degrees and extent of glycation. The heterogeneity of the glycated species was further complicated by the involvement of glycoxidation products in the glycation reaction, resulting in a distribution of different classes of glycoconjugates. Limited glycation improved protein solubility and increased solvent accessibility into the interior of the protein, thereby, increasing protein digestibility. Electrospr

Thesis (Ph.D.) -- University of Rhode Island, 2008. / Typescript. Includes bibliographical references (leaves 107-114).

Master of Science / Food Science Institute / J. Scott Smith / Heterocyclic amines (HCAs) are potentially carcinogenic and highly mutagenic byproducts of the Maillard browning reaction that form specifically in high temperature cooked meat products. Consumption of HCAs has been associated with various cancers including prostate, breast, colon, and pancreatic cancers and efforts have been made to understand formation and inhibition of these compounds. Chemical model systems are a preferred method to study the in vitro formation and inhibition of HCAs as the complex matrix effects found in meat are eliminated. Two black pepper extracts were evaluated for their efficacy on PhIP formation in model systems, but no significant results were observed. Secondly, four Maillard reaction variables were evaluated for their effect on formation of five HCAs (IQ, IQx, MeIQ, MeIQx, and 4,8-DiMeIQx) in chemical model systems with an effort to define an ideal model system. Precursor molar concentration (0.2/0.2, 0.4/0.4, 0.6/0.6, and 0.8/0.8 mmol), water percentage (0, 5, 10, and 15%), sugar type (fructose, galactose, glucose, and lactose), and sugar molar amount (quarter, half, equi, and double molar) were the four Maillard variables examined in the study. Additionally, four antioxidants (butylated hydroxyanisole (BHA), epigallocatechin gallate (EGCG), rosmarinic acid, and naringenin) were evaluated for their effect on HCA formation in chemical model systems. All four Maillard variables had a significant effect (p < 0.05) on the formation of HCAs in the model system, with an interaction effect occurring between water percentage and precursor concentration. The four antioxidants had no effect on the formation of HCAs in the model system. A model system containing 0.6/0.6/1.2 mmol of threonine, creatinine, and glucose, with 15% water was determined to be the best representative chemical model system for the formation of HCAs commonly formed in meats.

Orientadores: Maria Cecilia de Figueiredo Toledo / Tese (doutorado) Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T21:23:15Z (GMT). No. of bitstreams: 1 Arisseto_AdrianaPavesi_D.pdf: 1259436 bytes, checksum: 3d905ca51bf22b95fe5a0c5cb696018c (MD5) Previous issue date: 2007 / Resumo: Uma importante descoberta de pesquisadores suecos em abril de 2002 mostrou que acrilamida, uma substância provavelmente carcinogênica a seres humanos, pode ser formada em determinados alimentos que são submetidos a tratamento térmico em altas temperaturas. No presente estudo, 111 amostras de diferentes categorias de alimentos foram coletadas em supermercados, lojas de ¿fast-foods¿ e restaurantes da cidade de Campinas-SP, entre os meses de setembro de 2004 e abril de 2006, e analisadas para verificar a presença de acrilamida. As amostras foram selecionadas em função dos resultados divulgados por outros países e incluíram, além de produtos à base de batata, trigo e café, alimentos tipicamente brasileiros à base de mandioca e milho, processados em altas temperaturas. Os níveis de acrilamida foram determinados por cromatografia líquida de alta eficiência e espectrometria de massas em série (LC-MS/MS), conforme método previamente desenvolvido. Como a aplicação deste método em matrizes de cacau não apresentou resultados satisfatórios, o mesmo foi modificado através da inclusão de uma etapa de precipitação de proteínas e de alterações no procedimento de limpeza, o que resultou em melhor desempenho nesta matriz. As concentrações de acrilamida determinadas nas amostras analisadas confirmam que produtos à base de batata, tais como batatas fritas e batatas chips, biscoitos e café são os alimentos que apresentam os maiores níveis de acrilamida. Em etapa posterior, a ingestão potencial diária deste contaminante foi estimada combinando-se os dados analíticos de ocorrência de acrilamida obtidos no presente estudo com dados de consumo dos alimentos analisados, disponíveis para a população em geral e para uma população de adolescentes da cidade de Piracicaba-SP. Os valores médios de ingestão estimados para ambas as populações (0,14 e 0,12 µg/kg de peso corpóreo/dia, respectivamente) são inferiores aos valores relatados para populações de países da América do Norte e Europa (0,3 a 2 µg/kg de peso corpóreo/dia), o que pode ser parcialmente atribuído ao fato de que as estimativas de ingestão nestes países levaram em conta a contribuição de um maior número de alimentos. Dessa forma, é importante que mais amostras e grupos de alimentos sejam investigados para que, futuramente, possa ser calculada a contribuição da dieta total como fonte de acrilamida para a população brasileira e avaliados os possíveis riscos à saúde relacionados à exposição a este contaminante / Abstract: An important discovery of Swedish researchers in April 2002 showed that acrylamide, a probable carcinogen to humans, can be formed in certain foods which are submitted to thermal treatment at high temperatures. In the present study, 111 samples of different food categories were collected at supermarkets, fast-food restaurants and restaurants, in Campinas-SP, between September 2004 and April 2006, and analysed to verify the presence of acrylamide. The samples were selected according to results reported in other countries and included, beyond potato- and wheat-based products and coffee, typical Brazilian foods made from cassava and maize, and processed at high temperatures. The levels of acrylamide were determined by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS), according to a method previously developed. As the application of this method in cocoa matrices did not presented satisfactory results, there was a need to modify it by the inclusion of a protein precipitation step and changes in the clean-up procedure, which improved its performance in this matrix. The concentrations of acrylamide determined in analysed samples confirm that potato-based products, such as French fries and potato chips, biscuits and coffee are the foods containing the highest levels of acrylamide. In a next step, the potential daily intake of this contaminant was estimated by combining analytical data on the occurrence of acrylamide obtained in the present study with data on food consumption for the analysed foods, available for the general population and for a population of adolescents from the city of Piracicaba-SP. The mean intakes estimated for both populations (0.14 e 0.12 µg/kg body weight/day, respectively) are below the values reported for populations from North American and European countries (0.3 to 2 µg/kg body weight/day), which may be partially attributed to the fact that intake estimates conducted in these countries took into account the contribution of a greater number of foods. In this way, it is important that more samples and food groups be investigated so that, in the future, it may be possible to calculate the contribution of the total diet as source of acrylamide for the Brazilian population, and to assess the possible risks to health with regard to the exposure to this contaminant / Doutorado / Doutor em Ciência de Alimentos

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