Formation And Inhibition Of N~ï¿¡-(Carboxymethyl)lysine During Glycation Reaction In Food Model Systems

Maillard reaction not only changes the structure and function of food components, butalso generated a lot of harmful substances in food processing. Among those complex Maillardreaction products, Nε－(Carboxymethyl)lysine(CML)as the representative of advancedglycation end products (AGEs) has affinity relationship to diabetes and other diseases, CMLas a potentially harmful sector has attracted more and more attention. Correctly, theinternational food science and technology research focused on the physiology in medicine,analysis and toxicity of CML, however, the mechanism of formation of CML in foodprocessing was still unclear. The aim of this study was to evaluate the performance of formingCML during glycation reaction in food model systems, and then discuss the strategies oncontrolling, inhibiting and eliminating CML in food processingFirstly, a rapid and accurate HPLC-MS detection method was used to detect CML infood model system. The HPLC-MS detection method had good reproducibility and highaccuracy, the limit of quantification (LOQ) of CML detection was achieved50ng/mL. Thedetection method was used for the determination of CML formed in food model system in thepaper.Then, the performance of forming CML during glycation reaction in food model systemswas also evaluated. The study showed that reducing sugars in the Maillard reaction was moreconducive to form CML.The order of reactivity for the formation of CML was lactose>glucose> sucrose. Microwave heating treatment in food model systems produced more CMLthan traditional heating treatment.Secondly, we discussed the formation of CML intermediate glyoxal and fructoselysine,and made a relationship between the two intermediates. Fructoselysine had the capacity toproduce glyoxal, the result showed fructoselysine not only was the intermediate of CML, butalso was the precursors of CML intermediate glyoxal. Because CML intermediate glyoxal wasformed at all stages of glycation in the Maillard reaction, so CML may be called as a productof the glycation process in theory.In this study, carbohydrate modul labeling technique (CAMOLA)was used to explainthe formation of the CML intermediate and the complex chemical reactions pathway offorming CML. The traditional view was the carboxymethyl of CML was only formed from C－1and C－2of glucose residues, however, we found the source of carboxymethyl of CML had two ways:(1)C－1and C－2of glucose residues in fructoselysine were involved in theformation of carboxymethyl of CML;(2)C－1and C－2of glucose residues, C－3and C－4of glucose residues, C－5and C－6of glucose residues were involved in the formationof glyoxal during glucose oxidative cleavage, and then glyoxal and lysine formed CML, sothe carboxymethyl of CML was from C－1and C－2of glucose residues, C－3and C－4ofglucose residues, C－5and C－6of glucose residues. The result expanded the theory offormation of CML in food processing.During the glucose-amino acid model system, the formation of CML fromfructoselysine was great higher than from glyoxal.Combined with the concentration of13Clabeled CML and CML intermediate formation, fructoselysine was the main intermediate, andglyoxal was the minor intermediate of CML,the formation of fructoselysine was the mainpathway to form CML, the paper overturned the traditional view that carbonyl compoundsglyoxal is the main intermediate of CML.The traditional view indexed that the pathway offorming CML was mainly through the intermediate products glyoxal, we found forming CMLthrough the intermediate product of fructose lysine was the main path.Furthermore, a multi－stage kinetics model was also discussed for formation of CML.Glucose and lysine, intermediates fructoselysine and glyoxal, the reaction end product CMLand melanoidins,the parameters of the rate constants and activation energies were estimatedin the kinetics model. The reaction rate constant of fructoselysine was great higher thanglyoxal, and the activation energy Ea of fructoselysine was lower than glyoxal, the resultshowed the formation of fructoselysine was easier than glyoxal, and fructoselysine wasfavorer to form CML.We found fructoselysine was a major intermediate of CML.Lastly, we also examined the inhibition effect of CML by some inhibitors in glucose－lysine model systems, the inhibitors were three vitamins (vitamin B1, vitamin C and vitaminE), two flavonoids (rutin and quercetin) and reducing agent sodium borohydride sodium.Ascorbic acid and tocopherol did not affect inhibition of CML. There was a competition ofcarbonyl reducing sugar competition between the vitamin B1and amino acids or amino lysineresidues of protein, inhibiting the generation of CML. Sodium borohydride reduction has astrong ability to reduce carbonyl compounds, not only to reduce dicarbonyl compoundsglyoxal, but also to reduce the main intermediate fructoselysine to terminate the oxidation ofCML forming. Rutin and quercetin inhibited the formation of CML via vicinyl dihydroxylgroups, which has a strong antioxidant capacity. We also discussed the pathway of inhibiting CML by rutin in a multi－stage kinetics model. Rutin inhibited the formation of CML mainlyoccurred in the second phase of the glycosylation reaction, the formation of the intermediateproduct and transformed into the stage of CML.