Simultaneous Determination Of Dietary Acrylamide Mercapturic Acid Adducts And Chemoprotection In Vivo

Many in vitro research accomplishments regarding formation pathways and mitigation mechanism have been achieved since acrylamide contaminant was found in heat processing foods. At present, few researches have been carried out to focus on the metabolism and chemopretection of acrylamide in vivo. Studies on the metabolism and kinetics of acrylamide to reveal the toxicity of acrylamide become a challengable and hotspot research project. This study systematically investigated the effect of catechins on the metabolism and chemoprotection of acrylamide in vivo based on the levels of urine mercapturic acid adducts as acrylamide biomarkers.Acrylamide mercapturic acid adducts and corresponding isotope-labelled internal standards were successfully synthesized. Acrylamide or glycidamide was capable of reacting with N-acety-L-cyteineto get N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and a pair of isomers [N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine (GAMA) and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-L-cysteine iso-GAMA], repectively. AAMA was then oxidized to N-acetyl-S-(2-carbamoylethyl)-L-cysteine-sulfoxide (AAMA-sul) in the presence of hydrogen peroxide (H2O2). The synthesis of corresponding isotope-labelled internal standards consisted of two steps. In the first step, acrylamide or glycidamide reacted with cysteine to generate acrylamide-cysteine conjugates and a pair of isomers (glycidamide-cysteine and iso-glycidamide-cysteine conjugates), repectively. In the second step, all the conjugates were acetylated by D[6]-acetic anhydride to generate D[3]-AAMA, D[3]-GAMA and D[3]-iso-GAMA. D[3]-AAMA was then transformed to D[3]-AAMA-sulfoxide by oxidation of H2O2. All the synthesized target compouds were separated and purified by HPLC (Capcell C18,20×250mm i.d.,5μm). The accurate m/z and the structure of the purified adducts were confirmed and identified through ultra high performance liquid chromatography quadrupole time of flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS). Simultaneously, the possible fragmentation pattern of acrylamide mercapturic acid adducts and corresponding isotope-labelled internal standards were investigated, which was used for the following method development for the determination of acrylamide mercapturic acid adducts.An analytical method of isotope dilution ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was established to simultaneously quantify the acrylamide mercapturic acid adducts in urine of rats and humans. The instrument was operated using electron spray ionization in negative mode (ESI-). The human urine was mixed with formic acid/formate ammonia buffer system to precipitate impurity and then passed through solid phase extraction (SPE) cartridges for the enrichment and purification. The rat urine was directly diluted without further SPE clean-up procedures. The chromatographic analysis of four acrylamide mercapturic acid adducts in processed urine samples was performed on a Waters ACQUTITY UPLC(?) HSS T3column (2.1x150mm i.d.,1.8μm), using0.1%fomic acid/acetonitrile as the flow phase. The developed method was extensively validated by precision, repeatability and recovery tests. The recovery rates were92.25%-117.28%and92.23%-114.00%for the analytes in human urine and rat urine, respectively, both with the relative standard deviation (RSD) range of3.19%-12.58%. The UHPLC-MS/MS method was further validated via the analysis the urine of ten non-smoker volunteers within the age range of18-24.The results showed that the concentrations of AAMA, GAMA, iso-GAMA and AAMA-sul were20.1-80.1ng/mL,3.1-16.3ng/mL,3.5-12.9ng/mL and13.8-34ng/mL, respectively. The method was subsequently applied to the study on the metabolism of acrylamide in rats.Sprague Dawley (SD) rats were selected as experimental animals for the investigation of the metabolism and toxicokinetics of acrylamide in female and male rats orally administered with low, medium and high dose levels of acrylamide. Considering AAMA, GAMA and iso-GAMA as biomarkers, the first-order elimination kinetic model curve of acrylamide was investigated and the elimination constant and half-life period were calculated. The half-life period of three acrylamide mercapturic acid adducts ranged within12hours for both female and male rats with different gavaged doses. For both female and male rats, the molar ratio of cumulative excretion of acrylamide mercapturic acid adducts in urine to the gavaged dose level of acrylamide and the molar ratio of GAMA to AAMA in high gavaged dose (50mg/kg bw) was lower than corresponding molar ratos in low gavaged dose (1mg/kg bw), of which60%of the gavaged acrylamide was excreted as the form of mercapturic acid adducts. The molar ratio of GAMA to AAMA in the urine of male rats was higher than the ratio in the urine of female rats.SD female and male rats orally administered with the medium dose level (10mg/kg bw) of acrylamide were selected as the control groups in the chemoprevention study. Three representative cathchins, i.e. tea polyphenols, esterified catechin (EGCG) and non-esterified catechin (EC), were selected to investigate the metabolism and chemoprotection of acrylamide. The results indicated that three catechins could promote the acrylamide excretion through the form of mercapturic acid adducts. Compared to control groups, the molar ratio of cumulative excretion of acrylamide mercapturic acid adducts in urine to the gavaged dose of acrylamide in the tea polyphenols, EGCG and EC intervention groups increased by24.6%,14.7%and22.0%, respectively. Our study revealed that EC mainly contributes to the excretion of acrylamide via its reduction pathway while tea polyphenols mainly contributes to the excretion of acrylamide excretion via its oxidation pathway. This finding indicated that the3-hydroxyl functional group of flavanols may play an important role in the chemoprotection of acrylamide in vivo.