Development and validation of a sensitive liquid chromatography-tandem mass spectrometry method for the determination of paeoniflorin in rat brain and its application to pharmacokinetic study.

A sensitive and specific method was developed and validated for the determination of paeoniflorin in rat brain with liquid chromatography-tandem mass spectrometry. Sample pretreatment involved protein precipitation following solid-phase extraction. Paeoniflorin and geniposide (internal standard) were separated isocratically on a Waters Symmetry C18 column (150 mm x 2.1 mm i.d., 5 microm), using a mobile phase of methanol/water with 0.1% formic acid (50:50, v/v) at a flow-rate of 200-300 microL/min in 4min. A Finngan LTQ tandem mass spectrometer equipped with electrospray ionization source was operated in the positive ion mode. Selective reaction monitoring was performed to quantify paeoniflorin and the internal standard at m/z transitions of 503-->381 and 411-->231, respectively. A good linearity was found in the range of 2-500 ng/mL (R(2)=0.9939). The intra- and inter-batch assay precisions (coefficient of variation, CV) at 5, 50 and 400 ng/mL (n=5) ranged from 6.3% to 9.7% and 1.2% to 7.2%, respectively, and the accuracies were from 95.9% to 101.6% and 99.4% to 102.9%, respectively. The mean recoveries of paeoniflorin were 81.2%, 80.9% and 82.3% at 5, 50 and 400 ng/mL (n=5), respectively, and the mean recovery of the internal standard was 76.7% with a concentration of 50 ng/mL (n=5). Stability studies showed that paeoniflorin was stable in different conditions. Finally, the method was successfully applied to the pharmacokinetic study of paeoniflorin in rat brain following a single subcutaneous administration (10 mg/kg) to rats.

Heme-reversible impairment of CYP2B1/2 induction in heme-depleted rat hepatocytes in primary culture: translational control by a hepatic alpha-subunit of the eukaryotic initiation factor kinase?

The role of heme in the phenobarbital-mediated induction of CYP2B1/2 was reexamined in rat hepatocytes in monolayer culture, acutely depleted of heme by treatment with either 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) or N-methylprotoporphyrins (NMPP). The findings revealed that such acute hepatic heme depletion markedly impaired CYP2B1/2 protein induction, an effect that was reversible by heme resupplementation. However, TaqMan analyses of hepatic mRNA isolated from these heme-depleted cells revealed that this impairment was not due to faulty transcriptional activation of either CYP2B1 or CYP2B2 gene expression as previously proposed, thereby confirming literature reports that heme is not a transcriptional regulator of the CYP2B1/2 gene. In contrast, the rate of de novo CYP2B1/2 protein synthesis was found to be dramatically inhibited in both DDEP- and NMPP-treated hepatocytes. Concurrently, a marked (>80%) suppression of de novo hepatocellular protein synthesis was also observed, along with a significantly enhanced phosphorylation of the alpha-subunit of the eukaryotic initiation factor eIF2 (eIF2alpha), as monitored by the phosphorylated eIF2alpha/total eIF2alpha ratio in these heme-depleted cells. Indeed, the parallel reversal of all these three effects by heme supplementation suggests that this impaired CYP2B1 induction most likely stems from blocked translational initiation resulting from the activation of a heme-sensitive eIF2alpha kinase. Such global suppression of hepatic protein synthesis may disrupt a myriad of vital cellular functions, thereby contributing to the clinical symptoms of acute hepatic heme-deficient states such as the hepatic porphyrias.

Phenotype distribution and gender-related differences of CYP2E1 activity in a Chinese population.

1. The aim was to investigate the phenotype distribution characteristic and gender-related differences of CYP2E1 activity in a healthy Chinese population. 2. Two hundred and three healthy Chinese subjects (105 men, 98 women) were enrolled in this study. 3. CYP2E1 activity was determined by plasma 6-hydroxychlorzoxazone-to-chlorzoxazone concentration ratio (CHZ-MR) 4h after chlorzoxazone dosing. The concentrations of 6-hydroxychlorzoxazone and chlorzoxazone in plasma were detected by reversed-phase HPLC. 4. The results showed an almost 9-fold variation of CYP2E1 activity at a range of from 0.23 to 1.99. The coefficient of variation CY % was demonstrated with skewness and kurtosis of the ratios in the studied population were 44%, 0.96 and 1.10, respectively. 5. CYP2E1 activity was normally distributed in logarithmic form of 6-OH-CHZ/CHZ as evaluated by Kolmogorov-Smirnov test of normality (p = 0.20). Probit plots of the CYP2E1 activity index of men shifted to the right as compared with that of women. The mean CHZ-MR in men was significantly higher than that in women (0.76 +/- 0.30 versus 0.60 +/- 0.28, p < 0.001), and this difference still existed when normalized by weight (0.73 +/- 0.28 versus 0.66 +/- 0.32, p = 0.016). Body weight correlated positively with CYP2E1 activity in the total group(r < 0.212, p < 0.01).

Inducibility of CYP1A2 by omeprazole in vivo related to the genetic polymorphism of CYP1A2.

AIMS: To evaluate the effect of the CYP1A2*1C and CYP1A2*1F polymorphisms on the inducibility of CYP1A2 by omeprazole in healthy subjects. METHODS: Mutations of CYP2C19 and CYP1A2 were identified by PCR-RFLP. Omeprazole, 120 mg day-1, was given to 12 extensive metabolizers (EM) with respect to CYP2C19 (six CYP1A2*1F/CYP1A2*1F and six CYP1A2*1C/CYP1A2*1F of CYP1A2) for 7 days. CYP1A2 activity was determined on three occasions, namely on day 1, day 9 and day 16 using the caffeine plasma index (the ratio of the concentrations of paraxanthine to caffeine), 6 h after oral administration of 200 mg caffeine. RESULTS: There was a significant difference (P = 0.002) between the caffeine ratios for CYP1A2*1F/CYP1A2*1F and CYP1A2*1C/CYP1A2*1F genotypes on day 9, but not on day 1 or day 16 (P > 0.05). The changes in the ratios from day 9 to day 1 (48% +/- 20%vs 19% +/- 20%) and from day 9 to day 16 (50% +/- 31%vs 15% +/- 22%) were significantly different (P < 0.05) between the CYP1A2*1F/CYP1A2*1F and CYP1A2*1C/CYP1A2*1F genotypes. CONCLUSION: The CYP1A2*1C and CYP1A2*1F genetic polymorphisms influenced the induction of CYP1A2 activity in vivo by omeprazole.

Phenotypic polymorphism of CYP2A6 activity in a Chinese population.

AIMS: To investigate the distribution characteristics of CYP2A6 activity in a Chinese population and to examine the sex-related differences in CYP2A6 activities. METHODS: One hundred and twenty healthy volunteers, 63 men and 57 women, were included in the study. Cytochrome P450 (CYP) 2A6 activity was measured using the ratio of urinary 7-hydroxycoumarin (7-OHC) excreted in 8 h after a coumarin dose. The concentrations of 7-OHC in urine were determined using high performance liquid chromatography. RESULTS: A 300-fold interindividual variation of CYP2A6 activity was shown in the studied Chinese population. The coefficient of variation of CYP2A6 activity was 27.2%. A Kolmogorov-Smirnov test indicated a non-normal distribution of CYP2A6 activity ( P<0.001). Probit plots of CYP2A6 activity revealed a bimodal distribution with breakpoint of activity index near 0.47. The percentage of poor metabolizers (PMs) was 13.3% (95% confidence interval 7.3%-19.4%) in this population. Residual analysis also supported bimodality ( P<0.01). The CYP2A6 activities of females were obviously higher than those of males when the activity index was less than 0.74, although no statistically significant difference in the activity index of CYP2A6 between males and females was found. However, there was no sex-related difference in the incidence of PMs ( P>0.5). CONCLUSIONS: There are pronounced interindividual variations and phenotypic polymorphism of CYP2A6 activities in the Chinese population.