Feasibility Study Of The Protein Adducts Of Heterocyclic Aromatic Amines For Related Cancer Risk Assessment

Heterocyclic aromatic amines（HAAs）are polycyclic aromatic compounds that produce carcinogenicity and mutagenicity during thermal processing of fish,red meat,poultry meat and other high-protein foods.The evaluation of the relationship between Haas intake and related cancer risk is the focus of epidemiological research.Due to the different dietary structure and cooking methods of people,the daily intake of heterocyclic aromatic amines cannot be effectively quantified.Therefore,it is necessary to select and determine appropriate biomarkers to evaluate the intake of heterocyclic aromatic amines in order to explore the relationship between the intake level of Haas and the related cancer risk.After HAAs enters the human body,the metabolites produced by a series of reactions can combine with human serum albumin（HSA）to form HAA-HSA adducts.2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine（PhIP）is the most abundant HAA when frying and roasting red meat.In this study,PhIP-HSA sulfinamide adduct were selected as biomarkers,and an ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）method was established for qualitative and quantitative study of PhIP-HSA sulfinamide adduct in human plasma,so as to further study the association between dietary intake of PhIP and related cancer risk.The research is divided into the following three parts:（1）To establish and optimize the quantitative analysis method of PhIP-HSA sulfinamide adducts in human plasma.PhIP-HSA sulfinamide adduct is easy to hydrolyze and separate the corresponding PhIP under weak acidic conditions.Through the detection of PhIP,the PhIP-HSA sulfinamide adducts in human blood can be quantified to evaluate the intake of heterocyclic aromatic amines.The preparation of PhIP-HSA sulfinamide adducts is produced by the reaction of PhIP N-oxidation metabolites2-nitro-1-methyl-6-phenylimidazo[4,5-b]pyridine（NO2 PhIP）and2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine（honh PhIP）with HSA in vitro.PhIP-HSA sulfinamide adducts are quantified by detecting the PhIP containing peptide chain produced by enzymatic hydrolysis and PhIP produced by acid hydrolysis.The comparison between the two methods shows that the sensitivity of the method for detecting acid hydrolysate PhIP is much higher than that for detecting peptide chains containing PhIP.Therefore,the method of UPLC-MS/MS for detecting PhIP,the acid hydrolysate of PhIP-HSA sulfinamide adducts,is used to evaluate the content of PhIP-HSA sulfinamide adducts in human blood.By selecting different affinity columns and optimizing the extraction conditions,the extraction rate of HSA in human blood was improved;At the same time,the acid hydrolysis efficiency was effectively improved by optimizing the acid hydrolysis conditions.The concentration of hydrochloric acid hydrolysis was 16.0,and the optimized result was M;Reaction time:1 h.The efficiency of acid hydrolysis is as high as 96%.The recoveries of the internal standard[²H₃]-PhIP in all samples analyzed on the same day and on the next day were between 70～77%,indicating that the detection method has good repeatability.The content of different concentrations of PhIP-HSA sulfinamide adducts showed a linear positive correlation with the signal intensity of PhIP,y=1.0117x+3.2563,R²=0.9987。The difference between the actual detection amount and the theoretical addition amount of PhIP-HSA sulfinamide adducts is less than 13%.The detection limit is as low as 0.005 fmol/Mg HSA and the quantitative limit is as low as 0.015 fmol/Mg HSA.Therefore,this method has high sensitivity and can detect the very low content of PhIP HSA sulfonamide adduct in human body.（2）Establishment and quantitative study of detection method of free PhIP in human blood.After the PhIP in the food enters the human body,it is combined with HSA in the plasma through a series of metabolism to form PhIP-HSA sulfinamide adducts.Some PhIP may not participate in metabolism,exist freely in the blood and then be discharged from the body.The detection of PhIP-HSA sulfinamide adducts in blood can only reflect the PhIP involved in metabolism.Therefore,the analysis and evaluation of PhIP not involved in metabolism in blood is helpful to comprehensively evaluate the intake of PhIP.The detection of free PhIP in blood is to remove the protein in plasma,then enrich and purify it with SPE,and conduct quantitative analysis with UPLC-MS/MS.The results show that the sedimentation effect is the best when the content of ethanol is 90%.The standard curve of the method is y=2E+06x-5406.1,R2=0.9999,the recovery is 73.09～88.42%,the detection limit is as low as 0.003 fmol/m L plasma and the quantitative limit is as low as 0.009 fmol/m L plasma.Human plasma samples from patients with rectal cancer（15）and colon cancer（3）were analyzed.PhIP was not detected in all human plasma samples.This shows that the content of free PhIP in human plasma is lower than the detection limit of the method.（3）Quantitative study of PhIP-HSA sulfinamide adducts in human blood.Plasma samples from normal people（15）,patients with rectal cancer（15）,patients with colon cancer（12）and mixed（10）were collected.The mixed samples were the same as a single sample,which was composed of plasma samples from 4cancer patients.The optimized UPLC-MS/MS detection method was used to evaluate the content of PhIP-HSA sulfinamide adducts in human plasma samples.The results showed that PhIP-HSA sulfinamide adducts were not detected in the blood of healthy people.The samples that detected PhIP-HSA sulfinamide adductss included rectal cancer plasma samples（4）,colon cancer plasma samples（2）,rectal cancer plasma mixed samples（2）and colon cancer plasma mixed samples（3）.The content of PhIP-HSA sulfinamide adducts in these plasma samples ranged from 0.12 to 0.23fmol/Mg HSA.PhIP-HSA sulfinamide adducts were detected in the blood of 16%（6/37）of cancer patients,indicating that it is feasible to use PhIP-HSA sulfinamide adducts as biomarkers to study the intake of heterocyclic aromatic amines and related cancer analysis.