PURPOSE: Usefulness of mouse liver S9 fraction was evaluated for the measurement of the metabolites in the in vitro metabolism study of 18F-labeled radiotracers. MATERIALS AND METHODS: Mouse liver S9 fraction was isolated at an early step in the course of microsome preparation. The in vitro metabolism studies were carried out by incubating a mixture containing the radiotracer, S9 fraction and NADPH at 37 degrees C, and an aliquot of the mixture was analyzed at the indicated time points by radio-TLC. Metabolic defluorination was further confirmed by the incubation with calcium phosphate, a bone mimic. RESULTS: The radiotracer [18F]1 underwent metabolic defluorination within 15 min, which was consistent with the results of the in vivo method and the in vitro method using microsome. Radiotracer [18F]2 was metabolized to three metabolites including 4-[18F]fluorobenzoic acid within 60 min. It is likely that the one of these metabolites at the origin of radio-TLC was identical with the one that obtained from the in vivo and in vitro (microsome) method. Compared with the in vitro method using microsome, the method using S9 fraction gave a similar pattern of the metabolites but with a different ratio, which can be explained by the presence of cytosol in the S9 fraction. CONCLUSION: These results suggest that the findings of the in vitro metabolism studies using S9 fraction can reflect the in vivo metabolism of novel radiotracers in the liver. Moreover, this method can be used as a tool to determine metabolic defluorination along with calcium phosphate absorption method.
Absorption ; Animals ; Calcium ; Cytosol ; Liver* ; Metabolism* ; Mice* ; Microsomes ; NADP

Objective To study the metabolic transformation pathways of 4F-MDMB-BUTINACA in vivo by establishing zebrafish models. Methods Six adult zebrafish were randomly divided into blank control group and experimental group, with three fish in each group. After the zebrafish in the experimental group were exposed to 1 μg/mL 4F-MDMB-BUTINACA for 24 h, they were transferred to clean water and cleaned three times, then pretreated for instrumental analysis. The zebrafish in blank control group were not exposed to 4F-MDMB-BUTINACA. Mass spectrometry and structural analysis of 4F-MDMB-BUTINACA and its metabolites were conducted by liquid chromatography-high resolution mass spectrometry and Mass Frontier software. Results A total of twenty-six metabolites of 4F-MDMB-BUTINACA were identified in zebrafish, including eighteen phase Ⅰ metabolites and eight phase Ⅱ metabolites. The main metabolic pathways of phase Ⅰ metabolites of 4F-MDMB-BUTINACA in zebrafish were ester hydrolysis, N-dealkylation, oxidative defluorination and hydroxylation, while the main metabolic pathway of phase Ⅱ metabolites was glucuronidation. Conclusion Metabolite Md24 （ester hydrolysis） and Md25 （ester hydrolysis combined with dehydrogenation） would be recommended to be potentially good biomarkers for abuse of 4F-MDMB-BUTINACA.
Animals ; Cannabinoids ; Chromatography, Liquid ; Illicit Drugs ; Microsomes, Liver/chemistry* ; Zebrafish

No abstract available.
Catechols ; Cytochrome P-450 Enzyme System ; Cytochromes ; Fatty Alcohols ; Humans ; Liver ; Microsomes, Liver

It is known that dehydroepiandrosterone (DHEA) shows a dual effect, prooxidant or antioxidant, depending on the dosage or physiological status of animals. The purpose of this study was to determine the effects of DHEA administration at low dose on lipid peroxidation, protein carbonylation and fatty acid composition in liver. Sprague Dawley male rats were fed either corn oil diet containing 15% corn oil or fish oil diet containing 2% corn oil + 13% sardine oil, with or without 0.2% DHEA for 9 weeks. Atherogenic index and hepatic triglyceride and cholesterol levels were significantly reduced by DHEA administration in rats fed with fish oil diet. Hepatic lipid peroxide product (TBARS) and protein carbonyl levels were significantly higher in rats fed with fish oil diet than in rats fed with corn oil diet. However, DHEA administration significantly reduced the hepatic thiobarbituric acid-reactive substance (TBARS) and conjugated diene levels in rats fed with fish oil diet. Contents of C16 : 0, C16 : 1, C20 : 5 and C22 : 6 in hepatic microsome were higher in rats fed with fish oil diet than in rats fed with corn oil diet, and contents of C18 : 2 and C20 : 4 were lower than in rats fed with corn oil diet. DHEA administration significantly increased C16 : 0 and C18 : 3 contents and reduced C18 : 2 content in rats fed with corn oil diet, while it increased C16 : 0 and C18 : 1 and reduced C20 : 5 and C22 : 6 in rats fed with fish oil diet. On overall, DHEA administration increased saturated fatty acid (SFA) and reduced polyunsaturated fatty acid (PUFA) in hepatic microsome, thereby PUFA/SFA ratio was significantly (p < 0.0001) reduced without the change of n-3/n-6 ratio. Taken together, low dose of DHEA administration lowered PUFA/SFA ratio in hepatic microsomal membranes and also showed antioxidative effect especially in fish oil-induced highly oxidative stress condition through blocking increases of C20 : 5 and C22 : 6 contents.
Animals ; Cholesterol ; Corn Oil ; Dehydroepiandrosterone* ; Diet ; Humans ; Lipid Peroxidation ; Liver* ; Male ; Membranes ; Microsomes ; Microsomes, Liver* ; Oxidative Stress ; Protein Carbonylation ; Rats* ; Triglycerides

We have investigated the effect of cholestasis on the hepatic thiosulfate sulfurtransferase (rhodanese) and UDP-glucuronosyltransferase (UDP-GT) activities in rats. Rhodanese activities in the liver cytosol, mitochondria and microsomal fractions as well as in the rat serum, and UDP-GT activity in the microsome have been investigated for a period of 42 days after common bile duct (CBD) ligation. The cytosolic rhodanese activity showed a significant decrease between the first through the 42nd day, and the mitochondrial activity showed a significant decrease between the 7th through the 42nd day after CBD ligation compared to the activities from the sham operated control, respectively. In the case of microsomal preparation, both rhodanese and UDP-GT also showed significant decrease in their activities after the ligation for the former enzyme between the 14th and the 42nd days, and for the latter enzyme between the third and 42nd days, respectively. On the other hand, the serum rhodanese activity increased markedly soon after the ligation, exhibiting the peak activity after 1 day of CBD ligation with about 4.6-fold increment. The activity subsequently decreased gradually reaching to the control level at the 42nd day post-ligation. Enzyme kinetic parameters of hepatic rhodanese and UDP-GT were analyzed using sodium thiosulfate and p-nitrophenol as substrates, respectively, with the preparations from the 28th day post-ligation. The results indicated that although the K-m values of these enzymes were about the same as the sham-operated control, the V-max values of the both enzymes decreased significantly. These results, therefore, suggest that the biosynthesis of rhodanese and UDP-GT have been reduced in response to cholestasis, and that the elevation of rhodanese activity in the serum is most likely due to leakage from the liver subsequent to CBD ligation.
Animals ; Cholestasis ; Common Bile Duct* ; Cytosol ; Hand ; Ligation* ; Liver* ; Microsomes ; Mitochondria ; Rats* ; Sodium ; Thiosulfate Sulfurtransferase*

The reasonable prediction of metabolic clearance rate from the humanized in vitro system is valuable in drug discovery, which is commonly used in the identification and optimization of compounds that mostly like to process appropriate pharmacokinetic characteristics in humans. A detailed development of the general theory and, models underlying the prediction of in vivo hepatic drug metabolism from in vitro data were presented. Furthermore, the accuracy when extrapolating from in vitro data considering the in vitro-in vivo correlation, method-logical issues and potential solutions were discussed as well. This review can give us a better insight into exploring methods whereby human clearance can be accurately predicted from in vitro data in the process of new drug development.
Animals ; Humans ; Metabolic Clearance Rate ; Microsomes, Liver ; metabolism ; Models, Biological ; Pharmaceutical Preparations ; metabolism ; Pharmacokinetics

OBJECTIVETo study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes.

METHODSThe metabolites of CARV were identified by a hydrolysis reaction with beta-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosylisothiocyanate.

RESULTSTwo CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of K(m) and V(max) for (S)-CARV and (R)-CARV enantiomers were (118+/-44) micromol/L, (2 500+/-833) pmol/(min.mg protein) and (24+/-7) micromol/L, (953+/-399) pmol/(min.mg protein), respectively.

CONCLUSIONThese results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.

Carbazoles ; metabolism ; China ; Glucuronic Acid ; metabolism ; Glucuronides ; metabolism ; Microsomes, Liver ; metabolism ; Propanolamines ; metabolism ; Stereoisomerism

OBJECTIVETo obtain the information on the glucuronidation of Ginkgo flavonoid and the interaction profile of Ginkgo flavones with other drugs in vitro.

METHODSGinkgo flavonoids (quercetin, isorhamnetin and keampferol) and other drugs were co-incubated with rat hepatic microsome at 25 degree; the residual concentrations of flavonoids were determined by HPLC. The enzymatic parameters of quercetin, isorhamnetin and keampferol metabolism were assessed. The interactions between flavonoids and these drugs on glucuronidation were observed.

RESULTThe K(m) values were ( 24+/-0.05), (148+/-0.09) and (110+/-0.03) micromol/L and the V(max) values were (60+/-0.21), (48+/-0.02) and (34+/-0.02) micromol x g(-1) x min(-1) for quercetin, isorhamnetin and kaempferol, respectively. The IC(50) of nifedipine propafenone ipriflavone and diphenytriazol on flavonoids metabolism were 54 - 70, 69 - 122, 85 - 98 and 210 - 362 micromol, respectively. The inhibition constants (Ki) of diphenytriazol propafenone and ipriflavone on quercetin, isorhamnetin and keampferol metabolism were (57.6, 50.5, 33.1) (33.6, 59.5, 45.2) and(13.7,24.0,15.7) microg/ml respectively. The ratio [I]/[Ki] of the plasma concentration and inhibition constant for propafenone was 0.002 - 0.003.

CONCLUSIONThe metabolic level of quercetin is the strongest among three Ginkgo flavonoids. Nifedipine propafenone and ipriflavone inhibit the metabolism of quercetin, isorhamnetin and keampferol at different levels. Because of the interaction between Ginkgo flavonoids with nifedipine, caution must be taken when two drugs are used together clinically.

Animals ; Drug Interactions ; Flavonoids ; metabolism ; Ginkgo biloba ; Glucuronides ; metabolism ; Microsomes, Liver ; metabolism ; Rats

The mass spectrometry-based metabolomics method was used to systematically investigate the formation of celastrol metabolites,and the effect of celastrol on endogenous metabolites. The mice plasma,urine and feces samples were collected after oral administration of celastrol. Ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry( UPLC-QTOF-MS) was applied to analyze the exogenous metabolites of celastrol and its altered endogenous metabolites. Mass defect filtering was adopted to screen for the exogenous metabolites of celastrol. Multivariate statistical analysis was used to identify the endogenous metabolites affected by celastrol. Celastrol and its eight metabolites were detected in urine and feces of mice,and 5 metabolites of them were reported for the first time. The hydroxylated metabolites were observed in the metabolism of both human liver microsomes and mouse liver microsomes. Further recombinant enzyme experiments revealed CYP3 A4 was the major metabolic enzyme involved in the formation of hydroxylated metabolites. Urinary metabolomics revealed that celastrol can affect the excretion of intestinal bacteria-related endogenous metabolites,including hippuric acid,phenylacetylglycine,5-hydroxyindoleacetic acid,urocanic acid,cinnamoylglycine,phenylproplonylglycine and xanthurenic acid. These results are helpful to elucidate the metabolism and disposition of celastrol in vivo,and its mechanism of action.
Animals ; Chromatography, High Pressure Liquid ; Humans ; Mass Spectrometry ; Metabolomics ; Mice ; Microsomes, Liver ; metabolism ; Triterpenes ; metabolism ; pharmacokinetics

Improving analytical throughput is the focus of many quantitative workflows being developed for early drug discovery. For drug candidate screening, it is common practice to use ultra-high performance liquid chromatography (U-HPLC) coupled with triple quadrupole mass spectrometry. This approach certainly results in short analytical run time; however, in assessing the true throughput, all aspects of the workflow needs to be considered, including instrument optimization and the necessity to re-run samples when information is missed. Here we describe a high-throughput metabolic stability assay with a simplified instrument set-up which significantly improves the overall assay efficiency. In addition, as the data is acquired in a non-biased manner, high information content of both the parent compound and metabolites is gathered at the same time to facilitate the decision of which compounds to proceed through the drug discovery pipeline.
Chromatography, High Pressure Liquid ; methods ; Data Mining ; Humans ; Mass Spectrometry ; methods ; Microsomes, Liver ; metabolism