| introductionKetamine is a N-methyl-D-aspartate receptor antagonist used in clinical practice since 1970 for its anesthetic, sedative, and analgesic properties. It is frequently used for induction of anesthesia in short term surgical operations, in maintaining anesthesia with other anesthetic agents, and as a postoperative pain relief agent in intensive care units. In recent years, there is increasing evidence of ketamine abuse, many cases of illegal ketamine consumption and dependence was reported. Ketamine is N-demethylated by cytochrome P450 (P450) enzymes in the liver intonorketamine. The identification of the enzymes responsible for ketamine metabolism is of great importance in clinical practice. Yanagihara et al investigated the metabolism of ketamine in human liver microsomes at clinically relevant concentrations. Pooled human liver microsomes were used. N-demethylation of ketamine was correlated with orphenadrine , a specific inhibitor to CYP2B6, inhibited the N-demethylation of ketamine in human liver microsomes to the degree of 60-70%. They demonstrate that CYP2B6 is the principal enzyme responsible for ketamine N-demethylation in human liver microsomes at therapeutic concentrations of the drug. In a recent study (Youssef et al, 2002), the human liver microsomal enzyme CYP2C9, CYP2B6 and CYP3A4 were identified as the main P450 isoform responsible for the N-demethylation of ketamine in pooled human liver microsomes obtained from 20 donors, at a ketamine concentration of 0.005 mM. In light of these studies, and in the view of the growing interest of ketamine both as a therapeutic agent and as a drug of abuse, the knowledge of the identity and the contribution of P450 enzymes to N-demethylation of ketamine in humans, at clinically relevant concentrations, is highly desired.In the family of cytochrome P450, there were three main subfamilies (CYP1, CYP2, CYP3) which played an important role in metabolism of most of xenobiotics. The three subfamilies composed of 70% of P450 totally in the liver. CYP2C9, CYP2B6 and CYP3A4 had higher activities of catalysis. CYP2C9 was responsible for many xenobiotics metabolisms, including drugs, precarcinogen, preintoxicant and mutagenic agents. And CYP2C9 catalysed 12 percent of clinically common used medicine. Genetic polymorphism, inhibition and conduction of enzyme and physiological factors could change activities of P450, so that pharmacokinetics and potency of drugs would be changed and interactions of drugs occured.Gene of CYP2C9 located in chromosome 10q24.2 with totally 55kb, which included nine exons and eight introns and incoded 490 amino acids. There were twelve gene polymorphisms in the open encode area (CYP2C9* 1 to CYP2C9* 12). The base A was instead of the base C, which resulted in Leu359 instead of Ile359. The research about modulation of CYP2C9 expression at gene level was needed.In the present study, we used the specific substrate and inhibitor of CYP2C9 to demonstrate the catalysis action of CYP2C9 on ketamine, By CYP2C9 genotyping, we hoped to reveal the impact of CYP2C9 gene polymorphism on the content of P450, activity of enzyme CYP2C9 and metabolism of ketamine. It was helpful to elucidate mechanism of ketamine pharmacokinetics and predict individual difference.Materials1. Chemicals and reagentsKetamine standard and bupivacaine were products of Shanghai Bolly Biology Technology Co, LTD. Chemical specific substrate, tolbutamide, and inhibitor sulfaphenazole to CYP2C9 and NADPH were purchased from Sigma - Aldrich (St. Quentin, France). The PCR primers, PCR reagents were purchased from shanghai Sangon Biological Engineering Technology & Services Co. NsiI restriction enzyme was a product of Huamei Biology Technology Co .The marker was purchased from Takara (Japan).2. InstrumentationThe HPLC system (HP1160 chromatography, G1311 pump,G1314A UVspectrophotometer, U.S.A); Hypersil ODS2 analytical column(Dalian,China); UV 300spectrophotometer (Pye-unicam/spectronic, England) ;Power Pac 1000 electrophoresis-apparatus (Bio-Rad, U.S.A) ;Gene Amp PCR System 9700 PCR amplification device(Perkin-Elmer, U.S.A) ;BIS 303PC gel image analyzing system (DNR, Israel).Methods1. Catalysis of CYP2C9 in the metabolism of ketamine in human livermicrosomes.Human liver microsomes from twenty cases were obtained from the hepatic tissues of patients who undertoke hepatic partial excision under general anesthesia in the General Hospital of Shenyang Command of PLA, the First Affiliated Hospital and Shengjing Affiliated Hospital of China Medical University. The hepatic tissues were frozened in liquid nitrogen rapidly within 10 min and then stored at refrigerator(—70℃).Liver microsomes were prepared by the differential centrifugation and then stored at refrigerator(—70℃).Protein content of microsomes were measured with modified Lowry's method.Enzyme Incubation Conditions: The typical incubation mixture consisted of 0.4 mg/ml human liver microsomes, 100 mM potassium phosphate buffer(pH 7.4), and substrate in a final volume of 0.5 ml incubated in polypropylene tubes, at 37℃. After preincubation for 3 min, the reactions were initiated by the addition of 1 mM NADPH and incubated in 0, 5, 10, 20, 40, 60min. At the end of incubation time, 1 ml of cold 0.1 M NaOH was added to stop the enzymatic activity.The change of ketamine concentration in an incubation mixture with human liver microsomes was determined by high performance liquid chromatography (HPLC), to calculate the rate constants of metabolism of ketamine. The correlation of these rate constants with rates of metabolism of CYP2C9 selective substrate tolbutamide, and the effect of CYP2C9 specific inhibitor sulfaphenazole on ketamine metabolism were examined. The mobile phase: acetonitrile: KH2PO4: triethylamine (40:60:0.02). The flow rate was 1ml/min. Fluorescence detection wavelength was 211nm.2. Impact of CYP2C9 polymorphism on the content of P450 andactivities of CYP2C9 in human liver microsomes.Two hundred and three cases were chosen, who were picked up for hepatic partial excision under general anesthesia in the General Department of the General Hospital of Shenyang Command of PLA, the First Affiliated Hospital and Shengjing Affiliated Hospital of China Medical University from March, 2006 to March, 2007.Two-milliliter venous blood was taken after anesthesia for DNA estration and genotyping. Genotyping was performed by multiplex polymerase chain reaction (PCR) and restriction enzyme digestion with NsiI. Two different groups were created according to genotype, wild-type and mutation-type. The content of P450 and activities of CYP2C9 were compared for the two groups.3. Impact of CYP2C9 polymorphism on the metabolism of ketaminein human liver microsomes.Resources of two hundred and three cases and the work of genotyping were the same as those mentioned above in the second part. Two different groups were created according to genotype, wild-type and mutant-type. Compare the metabolic rates of ketamine in the two groups. Ketamine metabolism test in the hepatic microsomes was performed as that of mentioned above in the first part.4. Pharmacokinetic analysis and statistical treatment.Data was expressed as mean standard deviation. Statistical analyses ware performed with the SPSS11.5 software. Regression analysis was done for the relationship of metabolic rates of ketamine and tobutamide. Pairs-Student tests were used for the comparisons of the differences between the two groups, group of wild-type and group mutant-type. P<0.05 was considered as the significant difference.Results1. Analysis of ketamine concentration in human liver microsome atthe different time points.HPLC result showed that sample concentration had good relation with peak area. On the condition of this study ketamine was totally separated with internal standard bupivacaine and suffered no endogenous material intervention. Retention time of ketamine and internal standard bupivacaine was 3.4 and 5.8min respectively. Degree of accuracy, precision and extraction recovery rate fitted the demand of biological sample detection.2. Measurement of the metabolic rate of ketamine in human livermicrosome.The metabolic rate of ketamine in the twenty cases of microsomes was 8.2nmol·min-1·mg-1 protein ( 7.3-10.8 ) on average. The most rapid rate was 11.2nmol·min-1·mg-1 protein and was about double to that of the slowest rate, which was 5.4nmol·min-1·mg-1 protein. The average rate of ketamine metabolism showed obvious correlation to that of tobutamide (activity probe of CYP2C9) (r=0.886, P<0.01). After addition of sulfaphenazole (the specific inhibitor of CYP2C9), the average metabolic rate of ketamine(5.4nmol·min-1·mg-1protein) was slower than that without sulfaphenazole (P<0.01). The inhibition degree was 33.6%.3. CYP2C9 genotypingAll PCR products were specific 170bp fragments. PCR products of CYP2C9 mutant type (CYP2C9*3) had one NSil catalytic site, and was separated into 140bp and 30bp segments by enzymolysis. Therefore wild type had one segment 170bp, the mutant heterozygote had three segments 170bp, 140bp and 30bp, and the mutant homozygote had two segments 140bp and 30bp. In the two hundred and three cases, one hundred and ninety-six cases belonged to wild type; seven cases belonged to mutation heterozygote and mutation homozygote was not detected.4. Impact of CYP2C9 polymorphism on the content of P450 in humanliver microsomes.The range of content of P450 was 0.4-1.0nmol/mg it fitted the demand of content of P450. For the group of wild type , the content of P450 was 0.76nmol/mg and for the other group, mutation group, was 0.81nmol/mg. (P>0.05)5. Impact of CYP2C9 polymorphism on the activity of CYP2C9 inhuman liver microsomes.For the two groups (wild type group and mutation group), the metabolic rate of tobutamide was 1.7nmol·min-1·mg-1 protein and 0.6nmol·min-1·mg-1 protein respectively. (P<0.01)6. Impact of CYP2C9 polymorphism on the metabolism of ketaminein human liver microsomes.The metabolic rate of ketamine was 7.9nmol·min-1·mg-1 protein for the wild type group and 5.3 nmol·min-1·mg-1 protein for the mutation group. (P<0.05)DiscussionThe results of this study suggested that CYP2C9 participated ketamine metabolism. The specific inhibitor of CYP2C9 sulfaphenazole decreased the metabolic rate of ketamine.In the past, many kinds of methods were reported to detect the concentration of ketamine in blood plasm and urine, but it was the first time to detect the concentration of ketamine in human liver microsome in our country. We used the HPLC method to detect the concentration of ketamine in human liver microsome in this study. The ketamine and internal standard bupivacaine peak shape was good .The ketamine was totally separated with internal standard bupivacaine and suffered no endogenous material intervention. Degree of accuracy, precision and extraction recovery rate fitted the demand of biological sample detection.Plenty of investigations showed CYP2C9 had several mutant alleles. The most important was CYP2C9*1, CYP2C9*2 and CYP2C9*3. Single nucleotide substitutions were known to result in a different amino acid at one of four sites in cytochrome P4502C9 (CYP2C9) namely: residue 144: Arg/Cys; residue 358: Tyr/Cys; residue 359: Ile/Leu and residue 417: Gly/Asp. It was likely that functional changes occurring as a result of the Ile359Leu transition were responsible for the activity of CYP2C9. If it was Leu instead of Ile, the structure of enzyme CYP2C9 changed and capability of catalysis decreased.Researches about pharmacogenetics tried to increase safety and availability of medicine, decrease the occurrence of side effect It was helpful to individualization of treatment and preventive treatment. Unfortunately, it was lack of investigations about economics of pharmacogenetics and outcome of patients, which was very necessary to be sure that the patients profited from pharmacogenetics and medical cost would be decreased, Therefore, investigations about cost comparison of genotyping should be regarded as the most effective process to decrease medical cost. It was more significant that the detection of pharmacogenetics must be effective and convenient.Conclusion1. CYP2C9 was responsible for metabolism of ketamine. The specific inhibitor of CYP2C9 sulfaphenazole decreased the metabolic rate of ketamine.2. CYP2C9 polymorphism had no effect on the content of P450 in human lover microsome.CYP2C9 polymorphism decreased activity of CYP2C9.3. CYP2C9 polymorphism had an effect on metabolism of ketamine in human lover microsome. CYP2C9 polymorphism was an important genetic factor to produce individual differences of ketamine pharmaco-kenetics.
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