| Bactrian camel is an unique animal species living in gobi desert and semi-desert grassland. Due to the long time life in harsh and extreme environments, the bactrian camel formed a lot of unique biological characteristics. It would be very helpful to further understand the biological characteristics of bactrian camel if these special metabolic pathways were revealed. CYP enzymes are superfamily enzymes responsible for metabolism of many xenobiotics, endogenous substance and drugs. CYP2D6 is a member of the cytochrome P450 superfamily of enzymes and accounts for only 4% of whole CYP enzyme in animal liver, but plays important role in metabolism of about 20% of drugs. In order to study the characteristics of enzyme kinetics and its sensitivity to the specific inhibitors, the bactrian camel CYP2D6 enzyme was investigated by specific substrate method in vitro.Firstly, liver microsomes of bactrian camels was prepared by modified differential centrifugation method, and the protein concentration, total content of CYP enzyme and content of Cytb5 were mensured by BCA method and CO reduction method, respectively. The results showed that the liver microsomal protein concentration was 5.5650mg/mL±0.5197, total CYP enzyme content was 0.1777nmol/mg ±0.0201 and Cytb5 content was 0.1177 nmol/mg±0.016.Secondly, the HPLC method for testing the specific substrate dextromethorphan hydrobromide of CYP2D6 enzyme and its active metabolite dextrophan was established. Chromatographic condition was 0 min (methanol:ultra pure water=12:88)-10min (methanol:ultra pure water=22:78)-30min (methanol:ultra pure water=40:60), flow rate was 0.5 mL/min, the column temperature was 28℃, the UV detection wavelength was 279 nm, sample volume was 8μL, respectivily. Substrate and liver microsomes was incubated together for five minutes, adding β-NADPH solution and continued to incubate for a certain time. Then the incubation system was detected by HPLC method. The retention time of internal standard-acetaminophen, metabolite-dextrorphan, substrate-dextromethorphan hydrobromide were 6.012 min,8.546 min and 20.404 min, respectively. Under the estabolished chromatographic condition, the incubation system was optimized by testing the concentration of metabilite. The results showed the optimal concentration of liver microsomal protein was 5.5650 mg/mL, the optimal incubation time was 40min and the optimum substrate concentration was 250μg/mL, respectively.Thirdly, the metabolite concentration was quantatively determined by HPLC methods following incubation the different concentrations of substrate in liver microsome, and the parameters of enzyme kinetics were calculated by Origin Pro 8.6 software processing. The results showed that the Vmax value and Km value of CYP2D6 enzyme were 0.1416nmol/min/mg±0.0527 and 12.986μmol/L±0.3572, respectively. Cattle’s Vmax value and Km value of CYP2D6 enzyme were 0.0593 nmol/min/mg±0.0072 and 88.3μmol/L±9.2692 in the same way. The initial judgem, the activity of bactrian camel CYP2D6 enzyme were stronger than CYP2D6 enzyme of cattle.Finally, in order to study whether the quinidine also possess inhibitory effect on CYP2D6 enzyme of bactrian camel, the dynamic concentration of dextrorphan, the main active metabolite of dextromethorphan hydrobromide, was determined by HPLC method following the quinidine, the specific inhibitor of CYP2D6 enzyme, was added to optimized incubation system. Experimental results showed that the dextrorphan concentration was much lower in incubation system with quinidine than that of withaout quenidine. This explained that quinidine inhibited the activity of CYP2D6 enzyme and caused the products decreased significantly. The IC50 value was 2.779 μmol/L±0.0639 and showed a characteristics of time dependently.Therefore, the incubation system of bactrian camel liver microsomal was estabolished and optimized successfully, as well as, the HPLC mehtod with performance of stability, reliability and specificity was estabolished for determining the dextromethorphan hydrobromide, the specific substrate of CYP2D6 enzyme, and its metabolite dextrorphan. The kinetic parameters of bactrian camel CYP2D6 enzyme and the specific inhibitor’s IC50 value of this enzyme were found out firstly in this study. Hence, the experimental results filled the blank in research area of CYP2D6 enzyme activities in vitro, also could provide a valuable information for detection of CYP2D6 enzyme activity in vivo. |
PDF Full Text Request