Metabolic Study Of Antiviral Drug Ribavirin In Laying Hens

The illegal veterinary drug residue levels and illicitly adding some drugs have become prominent problems that affect the animal-derived food safety,it’s a serious threat to human health and safety,so it’s crucial to effectively monitor and supervise the use of illicit drugs and residue levels.At present,the medicine antetype was often tested in residues monitoring,the drugs often do not exist in parent drug form but were metabolized after taken in the animal body,so it is necessary to make clear drug residue target and target tissue in animal body,which will provide a scientific basis for effective monitoring.Ribavirin is a kind of antiviral drug for human purposes.In practical breeding,the phenomenon that prohibited ribavirin is often used is still serious,although ribavirin is forbidden to use as a veterinary in the 560^th proclamation by the ministry of agriculture.The metabolites of Ribavirin in the poultry is not yet clear,and the method standard has not been established.Laying hens was used as the object of study in this paper,to provide theoretical basis and method for the monitoring and supervision of the prohibited using of ribavirin,we studied the in vitro metabolism of ribavirin in blood cells and in vivo metabolism of ribavirin in laying hens,then concluded that the possible metabolites of ribavirin in laying hens.The main research contents and results are as follows:1.The establishment and verification of metabolic model of blood cells in chicken.The blood cells of laying hens was separated firstly in this part.The growth condition of blood cells were compared that in different medium（L-15,M199 and RPMI 1640）and different concentration（0,5%,10%and 15%）fetal bovine serum.The cell vitality was tested by MTT.And the metabolic model of blood cells was verified through the ribavirin dosing experiment.The results showed that:the blood cells cultured in vitro better by using L-15 medium supplemented with 10%fetal bovine serum;when the blood cells were cultured3 hours was the best time of dosing;the reported metabolites of ribavirin quickly occurred after the cells were dosed 0.5 h,and then present certain regularity.It indicates that the metabolic model of blood cells is effective.2.In vitro metabolic study on ribavirin in chicken by the metabolic model of blood cells.In this part,the LightSight software was used to analyse the possible metabolites of ribavirin in the organism.On the basis of established metabolic model of chicken blood cells,ribavirin was dosed after the cell cultured 3hours,and the culture media was analyzed by Q-TOF at the incubating time（0.5 h,1 h,2 h,3 h）.We found five metabolites of ribavirin,including TCONH₂,RTCOOH and RDP that have been reported in the mammalian body,and also found the glucuronidation metabolite（MRBV-3 is RBV-3,5-DGlcA）and sulfation metabolite（MRBV-4 is RBV-5-SO₃H）of ribavirin,it was proved that not only the deglycosylation or amide hydrolysis and phosphorylation metabolic pathways of ribavirin exist in blood cells of laying hens,but also the metabolic pathway that is not reported in mammals as glucuronidation and sulfation of ribavirin occurred in blood cells of laying hens.3.In vivo metabolic study of ribavirin in plasma of laying hens.In this section the ribavirin was dosed to the laying hens,then the plasma was gathered at 1-4 hours after ribavirin was taken by the laying hens,the blank samples and dosed samples were processed at the same time,then analyzed by Q-TOF LC/MS.We found 5 kinds of metabolites of ribavirin.Three kinds of the metabolites have been reported in mammals,which are TCONH₂,RTCOOH and TCOOH,it was well proved that the metabolic pathways of deglycosylation or amide hydrolysis of ribavirin exist in laying hens.Similar to the results of in vitro metabolic experiment of blood cells we also found the sulfation metabolites RBV-5-SO₃H（MRBV-N5 is MRBV-4）,it indicates that the sulfation metabolic pathway of ribavirin really exists in the laying hens.In addition,we also found the acetylation metabolite of ribavirin RBV-CH₃CO（MRBV-N4）,that is different from the results of in vitro metabolic study in blood cells.