Study On The Metabolism Of Sodium Dehydroacetate In Rats Based On CYP450 Subenzymes

Sodium dehydroacetate（Na-DHA）is a commonly used preservative in the food,feed,and cosmetics industries.Studies have shown that Na-DHA can cause coagulation problems in rats and chickens,and there are noticeable gender differences.In rats,Na-DHA is primarily excreted in its original form,and female rats have higher blood concentrations than male rats,but the reasons for these differences are not yet fully understood.To investigate the CYP450 enzymes involved in Na-DHA metabolism and its main metabolites,we conducted an in vitro rat liver microsomal incubation model and in vivo experiments in rats,using specific inhibitors.We also detected the expression levels of CYP450 enzymes in the livers of male and female rats through RT-PCR and analyzed the main metabolites in rats by Q-TOF-LC/S.This study provides preliminary insights into the gender differences in the blood concentration-coagulation function relationship of Na-DHA and has important implications for its clinical use.Select furafyl line,ketoconazole,quinidine,tetcyclidine,chlormethiazole as specific inhibitors of CYP1A2,CYP3A2,CYP2D1,CYP2C11 and CYP2E1,respectively;nifedipine,aminopyrine,dextromethorphan,omeprazole and clozapine as probe substrates of these 5 enzymes.In vitro rat liver microsome incubation model,the incubation conditions of 0.1 mg·L（-1） liver microsome concentration,5 mg·L（-1） Na-DHA concentration and 3 h incubation time were determined.25 μM CYP450 inhibitors could reduce the metabolism of corresponding probe substrates by about 40%.With or without specific inhibitors,different concentrations of Na-DHA or probe substrates were incubated with female or male rat liver microsomes respectively.The concentrations of Na-DHA or probe substrates were detected by HPLC.The CYP450 enzyme types metabolizing Na-DHA were analyzed.After inhibition of CYP1A2,CYP3A2 and CYP2D1 by furafylline,ketoconazole and quinidine,the metabolism of Na-DHA by female or male rat liver microsomes decreased significantly（P<0.05）in a concentration-dependent manner.After inhibition of CYP2C11 and CYP2E1,there was no significant effect on the metabolism of Na-DHA（P>0.05）.It indicates that CYP1A2,CYP3A2 and CYP2D1 are mainly involved in the metabolism of Na-DHA by in vitro liver microsome incubation system.The metabolism of Na-DHA by female rat liver microsomes is less than that of male rat liver microsomes,showing significant gender differences.In vivo rat experiments,female or male rats were intragastric gavaged the above 5 specific inhibitors of CYP450 enzymes（furafylline 5 mg·kg（-1）,ketoconazole 25 mg·kg（-1）,tetcyclidine 10 mg·kg（-1）,quinidine 10 mg·kg（-1）,chlormethiazole 25 mg·kg（-1））and CMC-Na for 3 days,and then intragastric gavage with 10 mg·kg（-1） of the above 5 CYP450 enzyme probe substrates or 200 mg·kg（-1） Na-DHA.① Blood was collected at different time points after NaDHA administration,and the serum drug concentrations of probe substrates or Na-DHA were detected by HPLC.Pharmacokinetic curves were drawn and pharmacokinetic parameters were calculated to analyze the CYP450 subenzyme types metabolizing Na-DHA in rats.The results showed that after specific inhibition of CYP450 subenzymes,the serum drug concentrations of probe substrates corresponding to each enzyme in the blank control group increased significantly（P<0.05）,indicating that the selected inhibitor concentrations could effectively inhibit the corresponding enzyme activity.After inhibition of CYP1A2,CYP3A2 and CYP2D1,the Na-DHA concentrations in the serum of female or male rats were significantly higher than those in the blank control group（P<0.05）;the Na-DHA concentrations in the serum of female rats were significantly higher than those of male rats（P<0.05）,and pharmacokinetic parameters of Na-DHA in female rats such as t1/2,Cmax,AUC（0-t）and AUC（0-∞）were significantly increased（P<0.05）;Vd or CL decreased significantly（P<0.05）.However,after inhibition of CYP2C11 and CYP2E1,there were no significant changes in Na-DHA concentrations and pharmacokinetic parameters in the serum of female or male rats（P>0.05）.②RT-PCR was used to detect the mRNA expression of 5 CYP450 enzymes in normal rat liver.The expression of Cyp1a2,Cyp3a2,Cyp2d1 and Cyp2c11 in female rat liver was significantly lower than that in male rat liver（P<0.05）,and the expression of Cyp2e1 showed no significant difference between female and male rats.③In HPLC analysis,two main metabolites M1 and M2 of Na-DHA were found in rat serum.After Na-DHA administration,the blood concentration of Na-DHA decreased gradually with time,while the concentrations of metabolites M1 and M2 increased gradually.Q-TOFLC/MS was used to analyze the metabolites of Na-DHA in rat serum.It was speculated that the molecular formula of M1 was C8H8O5 and the structural formula was 3-acetyl-6-methyl2,3-dihydropyran-2,4-dione;the molecular formula of M2 was C₆H₅O₃N and the structural formula was 3-imino-6-methyl-2,3-dihydropyran-2,4-dione.In summary,CYP1A2,CYP3A2 and CYP2D1 are the major enzymes metabolizing NaDHA in rats in vivo or in vitro liver microsomes.There are obvious gender differences in the metabolism of Na-DHA in rats.The metabolism of Na-DHA in female rats is less than that in male rats.M1 and M2 are the major metabolites of Na-DHA in rats in vivo.The results are important for understanding the metabolism,anticoagulant effects and gender differences of Na-DHA,and can also provide a theoretical basis for the rational clinical application of Na-DHA.