Pharmacokinetics And Metabonomics Of Three Drugs In Largemouth Bass

Largemouth Bass(Micropterus salmoides),native to North America,was introduced to China in 1980s and now has been widely bred in China.Largemouth bass have high nutritional value and economic value for its white and delicate meat,delicious taste,fast growth and fishing convenience.The production of Largemouth bass was 352 thousand tons in China in 2014.The domestication of wild species and many years'artificial culture have result the resistance reduction and caused the prevail of the fish disease.In order to control and cure the illness caused by microorganism,antibiotics were widely used for treatment,however the treatment of the disease by using antibiotics in largemouth bass was rarely reported at home and abroad.Therefore,it is necessary to carry out research on antibiotics in vivo metabolism of largemouth bass.The pharmacokinetics,biological transformation and metabonomics of florfenicol,doxycycline and enrofloxacin were studied in largemouth bass and the main results were as follows:(1)Based on biochemical reaction and 16s rRNA gene sequencing,the dominant bacteria of illness of largemouth bass was identified as Plesiomonas shigelloides(PS).According to VITEK 2,64 of the biochemical reactions were analysis and the results showed that,10 strains isolated strains were consistent with the biochemical characteristics except 2 strains.By the artificial infection test and toxicity test,the half lethal dose(LD50)was calculated to 3.47 × 106 CFU/mL.Plesiomonas shigelloides was sensitive to cefoperazone,aztreonam,florfenicol,tetracycline,norfloxacin,florfenicol and doxycycline in drug sensitivity test.(2)The pharmacokinetics of enrofloxacin,florfenicol and doxycycline were studied by using high performance liquid chromatography of largemouth bass.For the 5d 30 mg/kg b.w oral dose administration of florfenicol,doxycycline and enrofloxacin,the plasma,muscle and liver of largemouth bass were sampled after final dose in different times and the concentrations of florfenicol,doxycycline and enrofloxacin were determined in tissues of largemouth bass.The data processed by 3P97 pharmacokinetic software and the results shows as follows:At 25 ± 2 ?,three kinds of drugs in tissues of largemouth bass could be described as One compartment model.After continuous 5d oral dose of 30 mg/kg b.w of florfenicol,the peak times of florfenicol in plasma,muscle and liver of largemouth bass were 4h,4h and 4h,respectively.The absorption half-lives were:1.591 h,2.109 h,and 1.599 h,respectively.The elimination half-lives were:15.087h,55.862h and 27.680 h,respectively.The areas under curve(AUC)of the concentration time curve were 124.09 g/mL·h,219.83 g/g·h and 156.37 g/g·h,respectively.The peak times of doxycycline in plasma,muscle and liver of largemouth bass were 2 h,8 h and 4 h,respectively.The absorption half-lives were 1.975 h,3.590 h,and 2.019 h,respectively.The elimination half-lives were 22.549 h,26.452 h and 43.890 h,respectively.The areas under curve(AUC)of the concentration time curve were 195.14 g/mL·h,269.48 g/g·h and 336.71 g/g·h,respectively.The peak times of enrofloxacin in plasma,muscle and liver of largemouth bass were 4 h,4 h and 4 h,respectively.The absorption half-lives were 0.998 h,2.176 h,and 1.892 h,respectively.The elimination half-lives were 8.494 h,23.840 h and 37.53 h,respectively.The areas under curve(AUC)of the concentration time curve were 132.23 g/mL h,273.50 g/g h and 247.12 g/g·h,respectively.The withdrawal periods of the three drugs were as follows:7 days,15 days and 15 days.(3)The vivo biotransformations of enrofloxacin and florfenicol and doxycycline in largemouth bass were studied by using the technology of ultra performance liquid chromatography tandem time-of-flight mass spectrometry(UPLC-TOF/MS).After single intraperitoneal injections 150 mg/kg b.w dose of florfenicol,enrofloxacin or doxycycline,the liver of largemouth bass was sampled after 24 h compared to blank samples.The possible metabolites were determined by MS(Q1)and be confirmed by MS/MS(Q2).The results showed that the metabolite of florfenicol was amine metabolites.Metabolite of enrofloxacin were ciprofloxacin and taurine conjugates and there was no detection of doxycycline metabolite.(4)The pharmaco-metabonomics of plasma in largemouth bass after a dose of 30 mg/kg b.w of enrofloxacin was studied by using UPLC-TOF/MS.The plasma of enrofloxacin was sampled at 0 h,24 h,48 h,96 h and 168 h after intraperitoneal injections compared to blank samples.The pharmaco-metabonomics data were determined by UPLC-TOF/MS with positive and negative ion scan and processed by Angilent MPP software.The data were processed by peak alignment,quality control,data extraction by MPP software,cluster analysis and principal component analysis.The result shows as follows:Administration of 0 h after the sample and the control of small gr-oups,could be merged into a group of treatment.Compared with the control group,the sample of 24 h had the biggest change.The difference between 48 h,96 h and 168 h after administration was small,which could be incorporated into a group of sample treatment.Therefore,samples co uld be divided into 3 groups,ORIGINAL,MAX CHANGE and RETURN,respectively.According to the screening of volcano map,209 changed su-bstances between ORIGINAL and MAX CHANGE,213 kinds of chan-ged materials between MAX CHANGE and RETURN and 83 changed materials between ORIGINAL and RETURN were found,respectively.Through further screening,there were 14 kinds of compounds which got concentration changed in all of ORIGINAL,MAX CHANGE and RET-URN.After Database matching,3 kinds of compounds,including Cyclic dAMP,11-(3-acetoxy-1-propynyl)-1,25-dihydroxy-9,11-didehydrov-itamin D3 and Pantetheine could be potential biomarkers in plasma of largemouth bass to indicate the intake of enrofloxacin,which need further tests to determine.