OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After ad...OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.展开更多
BACKGROUND The coronavirus disease 2019(COVID-19),a pandemic contributing to more than 105 million cases and more than 2.3 million deaths worldwide,was described to be frequently accompanied by extrapulmonary manifest...BACKGROUND The coronavirus disease 2019(COVID-19),a pandemic contributing to more than 105 million cases and more than 2.3 million deaths worldwide,was described to be frequently accompanied by extrapulmonary manifestations,including liver dysfunction.Liver dysfunction and elevated liver enzymes were observed in about 53%of COVID-19 patients.AIM To gain insight into transcriptional abnormalities in liver tissue of severe COVID-19 patients that may result in liver dysfunction.METHODS The transcriptome of liver autopsy samples from severe COVID-19 patients against those of non-COVID donors was analyzed.Differentially expressed genes were identified from normalized RNA-seq data and analyzed for the enrichment of functional clusters and pathways.The differentially expressed genes were then compared against the genetic signatures of liver diseases including cirrhosis,fibrosis,non-alcoholic fatty liver disease(NAFLD),and hepatitis A/B/C.Gene expression of some differentially expressed genes was assessed in the blood samples of severe COVID-19 patients with liver dysfunction using qRT-PCR.RESULTS Analysis of the differential transcriptome of the liver tissue of severe COVID-19 patients revealed a significant upregulation of transcripts implicated in tissue remodeling including G-coupled protein receptors family genes,DNAJB1,IGF2,EGFR,and HDGF.Concordantly,the differential transcriptome of severe COVID-19 liver tissues substantially overlapped with the disease signature of liver diseases characterized with pathological tissue remodeling(liver cirrhosis,Fibrosis,NAFLD,and hepatitis A/B/C).Moreover,we observed a significant suppression of transcripts implicated in metabolic pathways as well as mitochondrial function,including cytochrome P450 family members,ACAD11,CIDEB,GNMT,and GPAM.Consequently,drug and xenobiotics metabolism pathways are significantly suppressed suggesting a decrease in liver detoxification capacity.In correspondence with the RNA-seq data analysis,we observed a significant upregulation of DNAJB1 and HSP90AB1 as well as significant downregulation of CYP39A1 in the blood plasma of severe COVID-19 patients with liver dysfunction.CONCLUSION Severe COVID-19 patients appear to experience significant transcriptional shift that may ensue tissue remodeling,mitochondrial dysfunction and lower hepatic detoxification resulting in the clinically observed liver dysfunction.展开更多
A dual wavelength differential first derivative spectrophotometric method has been developed to standardize the concentration of a saturated aqueous solution of carbon monoxide (CO) as the standard and to identify and...A dual wavelength differential first derivative spectrophotometric method has been developed to standardize the concentration of a saturated aqueous solution of carbon monoxide (CO) as the standard and to identify and to determine CO formed during the microsomal metabolism of xenobiotics in vitro. The method can significantly eliminate the background interference in the assay media and increase the quantitative accuracy and the sensitivity. There is a good linear relationship between CO concentration in the range of 2～10 μmol·L 1 CO and the distance D between the first derivative peak at 415 nm amd valley at 426 nm with r=0.9999(n=5),the regression equation being C (mmol·L 1 )=17.6D 0.4, the detection limit lower than 0.1 μmol·L 1 CO. The average recoveries of CO from the assay system and the sample were 102.1%, RSD=2.9% (n=7) and 79.7%, RSD=6.8% (n=12),respectively. The RSD of within day was 4.4%(n=18),and the RSD of day to day was 6.1%(n=16). By this method, four trihaloanilines and one trihalobenzene were tested, the results showed that only 2,4,5 trifluoroaniline could be converted to CO by the incubation with rat hepatic microsomes, NADPH and oxygen, the ability of phenobarbital or dexamethasone to induce rat hepatic microsomes to catalyze CO formation was 3 or 8 times higher than that of the control.展开更多
基金The project supported by National Natural Science Foundation of China(81473579,81273654,81102879)the National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2013ZX09103002-022)
文摘OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.
基金The University of Sharjah,No.CoV19-0308,No.CoV19-0307 and No:1901090254Sharjah Research Academy,No:MED001Al-Jalila Foundation Seed Grant,No.AJF202019.
文摘BACKGROUND The coronavirus disease 2019(COVID-19),a pandemic contributing to more than 105 million cases and more than 2.3 million deaths worldwide,was described to be frequently accompanied by extrapulmonary manifestations,including liver dysfunction.Liver dysfunction and elevated liver enzymes were observed in about 53%of COVID-19 patients.AIM To gain insight into transcriptional abnormalities in liver tissue of severe COVID-19 patients that may result in liver dysfunction.METHODS The transcriptome of liver autopsy samples from severe COVID-19 patients against those of non-COVID donors was analyzed.Differentially expressed genes were identified from normalized RNA-seq data and analyzed for the enrichment of functional clusters and pathways.The differentially expressed genes were then compared against the genetic signatures of liver diseases including cirrhosis,fibrosis,non-alcoholic fatty liver disease(NAFLD),and hepatitis A/B/C.Gene expression of some differentially expressed genes was assessed in the blood samples of severe COVID-19 patients with liver dysfunction using qRT-PCR.RESULTS Analysis of the differential transcriptome of the liver tissue of severe COVID-19 patients revealed a significant upregulation of transcripts implicated in tissue remodeling including G-coupled protein receptors family genes,DNAJB1,IGF2,EGFR,and HDGF.Concordantly,the differential transcriptome of severe COVID-19 liver tissues substantially overlapped with the disease signature of liver diseases characterized with pathological tissue remodeling(liver cirrhosis,Fibrosis,NAFLD,and hepatitis A/B/C).Moreover,we observed a significant suppression of transcripts implicated in metabolic pathways as well as mitochondrial function,including cytochrome P450 family members,ACAD11,CIDEB,GNMT,and GPAM.Consequently,drug and xenobiotics metabolism pathways are significantly suppressed suggesting a decrease in liver detoxification capacity.In correspondence with the RNA-seq data analysis,we observed a significant upregulation of DNAJB1 and HSP90AB1 as well as significant downregulation of CYP39A1 in the blood plasma of severe COVID-19 patients with liver dysfunction.CONCLUSION Severe COVID-19 patients appear to experience significant transcriptional shift that may ensue tissue remodeling,mitochondrial dysfunction and lower hepatic detoxification resulting in the clinically observed liver dysfunction.
文摘A dual wavelength differential first derivative spectrophotometric method has been developed to standardize the concentration of a saturated aqueous solution of carbon monoxide (CO) as the standard and to identify and to determine CO formed during the microsomal metabolism of xenobiotics in vitro. The method can significantly eliminate the background interference in the assay media and increase the quantitative accuracy and the sensitivity. There is a good linear relationship between CO concentration in the range of 2～10 μmol·L 1 CO and the distance D between the first derivative peak at 415 nm amd valley at 426 nm with r=0.9999(n=5),the regression equation being C (mmol·L 1 )=17.6D 0.4, the detection limit lower than 0.1 μmol·L 1 CO. The average recoveries of CO from the assay system and the sample were 102.1%, RSD=2.9% (n=7) and 79.7%, RSD=6.8% (n=12),respectively. The RSD of within day was 4.4%(n=18),and the RSD of day to day was 6.1%(n=16). By this method, four trihaloanilines and one trihalobenzene were tested, the results showed that only 2,4,5 trifluoroaniline could be converted to CO by the incubation with rat hepatic microsomes, NADPH and oxygen, the ability of phenobarbital or dexamethasone to induce rat hepatic microsomes to catalyze CO formation was 3 or 8 times higher than that of the control.
