Analysis On The Protein Adduct Of The Food Carcinogen Heterocyclic Aromatic Amines As Biomarker

Heterocyclic aromatic amines(HAAs)are a class of low-molecular organic compounds produced by high-temperature processing of protein-rich substances,which have carcinogenic effect.With the improvement of the quality of life and the aggravation of air pollution,HAAs are everywhere in daily environment.Most of the Food-borne HAAs are predominant,and HAAs also exist in natural environments such as smoke and atmospheric particles.But HAAs are only at part per billion(nanograms per gram)in meat products.The separation and detection of trace substances in complex systems have became current research hotspot.Based on the carcinogenic effects of HAAs,the assessment of intake of HAAs in human will also be a hot topic in future research.This study explored the levels of HAAs in common processed meat products,the HAA-protein adduct that may be present in human and their quantitative methods.It was mainly divided into the following three parts:(1)Heterocyclic aromatic amines(HAAs)in meat products were analyzed by optimizing liquid-liquid extraction(LLE)and solid-phase extraction(SPE)combined with high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).The limit of detection(LOD)and limit of quantitation(LOQ)of the six HAAs in meat products were as low as 0.0006 ng/g and 0.0021 ng/g,respectively.The effects of common cooking methods(oven roasting and deep-frying)on the formation of HAAs in the most common meat products(chicken,pork,beef,grass carp)were studied.The results showed that the formation of HAAs in meats and fish by the same cooked method increaseed with the increased processing temperature;deep-frying treatment of meats and fish produced more HAAs than oven roasting treatment when similar processing temperatures employed.2-amino-1-methyl-6 phenylimidazo[4,5-b]pyridine(PhIP)and 2-amino-3,8-dimethylimidazo[4,5-f]quinolone(Me IQx)were the most abundant HAAs formed in roasted and deep-fried samples,respectively.When meats and fish were roasted at the same temperature(275?),roasted chicken contained the highest amount of total HAAs,followed by roasted pork,grass carp and beef.The highest concentration of PhIP was detected in roasted chicken cooked at275?,up to 341.15±7.87 ng/g.Whereas,deep-fried meats and fish treated at the same temperature exhibited slight differences in total HAAs at relative low levels.The highest concentration of Me IQx(7.44±0.51 ng/g)formed in deep-fried products was found in fried beef cooked at 200?.In summary,PhIP was more widely distributed and more toxic in the roasting and frying.Therefore,PhIP was selected as the object of further study.(2)PhIP was selected as the research object,and the N-hydroxylated metabolite of PhIP,2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine(HONH-PhIP),was synthesized based on its metabolic pathway in human.The metabolites of PhIP were prepared by chemical synthesis technology and high-performance liquid phase(HPLC)real-time monitoring mean,and solid phase extraction(SPE)technology was used to purify and enrich the metabolites of PhIP.The commerical human serum albumin(HSA)may be oxidized during storage,so it was reduced with the reducing agent?-mercaptoethanol.The adduct formed between HONH-PhIP and reduced HSA was purified with a gel filtration column.The adduct was hydrolyzed by trypsin and chymotrypsin,and its structure was characterized by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).According to the detected modified peptide chain LQQC*PFEDHVK(C-[S=O]-PhIP),the reaction site of PhIP-HSA sulfenamide adduct was on the sulfhydryl group of its Cysteine 34(Cys³⁴).This adduct may be one of the main adducts formed by PhIP and HSA in human.(3)PhIP-HSA sulfinamide adducts can be used as biomarkers to assess the relationship between PhIP intake and cancer risk.The quantitative method of PhIP-HSA sulfinamide adduct was established based on its instability under acidic conditions.PhIP-HSA sulfinamide adducts were quantified by the pretreatment method of acid hydrolysis experiment and the high sensitivity detection method of HPLC-MS/MS.By setting the acid hydrolysis time 1h and 3h,explored the optimal hydrolysis time of the acid hydrolysis experiment.The results showed that the acid hydrolysis time of 1h was sufficient to finish almost complete hydrolysis.The adduct was hydrolyzed under weakly acidic conditions(0.16N,37?,1h),and the hydrolysate PhIP was detected using HPLC-MS/MS to quantify the PhIP-HSA sulfinamide adduct.The recovery rate of this quantitative method were between 57%and 92%,the limit of detection(LOD)were 18fg adduct/mg albumin,and the limit of quantification(LOQ)were 60fg adduct/mg albumin,down to part per trillion(picograms per gram).There was a linear relationship between the amount of theoretically added adduct and the amount of PhIP recovered by acid hydrolysis(N=1.0117n+3.2563,r2=0.9987).At the same time,it also lays the foundation for the detection of PhIP in cancer patient samples,and further evaluates the relationship between PhIP intake and cancer risk.