Genome-Wide Identification and Characterization of the Shaker-Type K+ Channel Genes in Prunus persica (L.) Batsch.

Shaker-type K+ channels are critical for plant K+ acquisition and translocation that play key roles during plant growth and development. However, molecular mechanisms towards K+ channels are extremely rare in fruit trees, especially in peach. In this study, we identified 7 putative shaker-type K+ channel genes from peach, which were unevenly distributed on 5 chromosomes. The peach shaker K+ channel proteins were classified into 5 subfamilies, I-V, and were tightly clustered with pear homologs in the phylogenetic tree. Various cis-acting regulatory elements were detected in the promoter region of the shaker-type K+ channel genes, including phytohormone-responsive, abiotic stress-responsive, and development regulatory elements. The peach shaker K+ channel genes were expressed differentially in distinct tissues, and PpSPIK was specifically expressed in the full-bloom flowers; PpKAT1 and PpGORK were predominantly expressed in the leaves, while PpAKT1, PpKC1, and PpSKOR were majorly expressed in the roots. The peach shaker K+ channel genes were differentially regulated by abiotic stresses in that K+ deficiency, and ABA treatment mainly increased the shaker K+ channel gene expression throughout the whole seedling, whereas NaCl and PEG treatment reduced the shaker K+ channel gene expression, especially in the roots. Moreover, electrophysiological analysis demonstrated that PpSKOR is a typical voltage-dependent outwardly rectifying K+ channel in peach. This study lays a molecular basis for further functional studies of the shaker-type K+ channel genes in peach and provides a theoretical foundation for K+ nutrition and balance research in fruit trees.

Genome-Wide Identification and Evolutionary Analysis of Argonaute Genes in Hexaploid Bread Wheat.

Argonaute (AGO) proteins play a pivotal role in plant growth and development as the core components of RNA-induced silencing complex (RISC). However, no systematic characterization of AGO genes in wheat has been reported to date. In this study, a total number of 69 TaAGO genes in the hexaploid bread wheat (Triticum aestivum cv. Chinese Spring) genome, divided into 10 subfamilies, were identified. Compared to all wheat genes, TaAGOs showed a significantly lower evolutionary rate, which is consistent with their high conservation in eukaryotes. However, the homoeolog retention was remarkably higher than the average, implying the nonredundant biological importance of TaAGO genes in bread wheat. Further homoeologous gene expression bias analyses revealed that TaAGOs may have undergone neofunctionalization after polyploidization and duplication through the divergent expression of homoeologous gene copies, to provide new opportunities for the generation of adaptive traits. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) analyses indicated that TaAGO gene expression was involved in response to heat, drought, and salt stresses. Our results would provide a theoretical basis for future studies on the biological functions of TaAGO genes in wheat and other gramineous species.

The Chloroplastic Small Heat Shock Protein Gene KvHSP26 Is Induced by Various Abiotic Stresses in Kosteletzkya virginica.

Small heat shock proteins (sHSPs) are a group of chaperone proteins existed in all organisms. The functions of sHSPs in heat and abiotic stress responses in many glycophyte plants have been studied. However, their possible roles in halophyte plants are still largely known. In this work, a putative sHSP gene KvHSP26 was cloned from K. virginica. Bioinformatics analyses revealed that KvHSP26 encoded a chloroplastic protein with the typical features of sHSPs. Amino acid sequence alignment and phylogenetic analysis demonstrated that KvHSP26 shared 30%-77% homology with other sHSPs from Arabidopsis, cotton, durian, salvia, and soybean. Quantitative real-time PCR (qPCR) assays exhibited that KvHSP26 was constitutively expressed in different tissues such as leaves, stems, and roots, with a relatively higher expression in leaves. Furthermore, expression of KvHSP26 was strongly induced by salt, heat, osmotic stress, and ABA in K. virginica. All these results suggest that KvHSP26 encodes a new sHSP, which is involved in multiple abiotic stress responses in K. virginica, and it has a great potential to be used as a candidate gene for the breeding of plants with improved tolerances to various abiotic stresses.

Effect of CYP4F2, VKORC1, and CYP2C9 in Influencing Coumarin Dose: A Single-Patient Data Meta-Analysis in More Than 15,000 Individuals.

The cytochrome P450 (CYP)4F2 gene is known to influence mean coumarin dose. The aim of the present study was to undertake a meta-analysis at the individual patients level to capture the possible effect of ethnicity, gene-gene interaction, or other drugs on the association and to verify if inclusion of CYP4F2*3 variant into dosing algorithms improves the prediction of mean coumarin dose. We asked the authors of our previous meta-analysis (30 articles) and of 38 new articles retrieved by a systematic review to send us individual patients' data. The final collection consists of 15,754 patients split into a derivation and validation cohort. The CYP4F2*3 polymorphism was consistently associated with an increase in mean coumarin dose (+9% (95% confidence interval (CI) 7-10%), with a higher effect in women, in patients taking acenocoumarol, and in white patients. The inclusion of the CYP4F2*3 in dosing algorithms slightly improved the prediction of stable coumarin dose. New pharmacogenetic equations potentially useful for clinical practice were derived.

Two new sesquiterpenes from the aerial parts of Schizonepeta tenuifolia.

Two new sesquiterpenes named petafolias A-B were isolated from the aerial parts of Schizonepeta tenuifolia. Their structures were elucidated by various spectroscopic techniques (UV, IR, MS, CD, 1D, and 2D NMR).

Profiling of microRNAs under wound treatment in Aquilaria sinensis to identify possible microRNAs involved in agarwood formation.

Agarwood, a kind of highly valued non-timber product across Asia, is formed only when its resource trees--the endangered genus Aquilaria are wounded or infected by some microbes. To promote the efficiency of agarwood production and protect the wild resource of Aquilaria species, we urgently need to reveal the regulation mechanism of agarwood formation. MicroRNAs (miRNAs) are a group of gene expression regulators with overwhelming effects on a large spectrum of biological processes. However, their roles in agarwood formation remain unknown. This work aimed at identifying possible miRNAs involved in the wound induced agarwood formation. In this study, the high-throughput sequencing was adopted to identify miRNAs and monitor their expression under wound treatment in the stems of A. sinensis. The miR171, miR390, miR394, miR2111, and miR3954 families remained at the reduced level two days after the treatment. 131 homologous miRNAs in the 0.5 h library showed over three-fold variation of read number compared with the control library, of which 12 exhibiting strong expression alterations were further confirmed by real-time quantitative PCR. Target prediction and annotation of the miRNAs demonstrated that the binding, metabolic process, catalytic activity, and cellular process are the most common functions of the predicted targets of these newly identified miRNAs in A.sinensis. The cleaveage sites of three newly predicted targets were verified by 5'RACE.

[Induction of hairy roots and plantlet regeneration of Bupleurum chinense DC].

In this study, the induction of hairy roots of Bupleurum chinense DC. was explored and established after experiments at different conditions: A. rhizogenes A4 was used to infect the leaves bases of B. chinense tube seedlings. The explants were co-cultured on Phytagel-solidified media for 3 days and then, were turned into solid media, similar with the co-culture media except that bacteriostat was added. After 10 days, rootlets began to appear and after 4 to 5 weeks, rootlets can be converted into liquid shaking culture stage. Plants regeneration from hairy root was useful for the research of new germplasm production and the variety improvement breeding. In the present study, the regenerated plants were obtained. One approach was to continuously culture under light conditions the seedlings which parting off spontaneously from the hairy roots during liquid shaking culture. The other approach was to culture the callus-like tissues produced by hairy roots with the optimized regeneration media for the induction of regenerated plants. The results of present study provide a technique to induce hairy roots and plantlet regeneration of B. chinense and this technique is helpful for the researches on metabolism, especially on the Agrobacterium-mediated genetic transformation of B. chinense.

Two new triterpenoid saponins from the root of Platycodon grandiflorum.

Two new triterpenoid saponins, named platycodon A (3-O-ß-D-glucopyranosyl-16-O-ß-D-glucopyranosyl-2ß,3ß,16ß,21ß-tetrahydroxyolean-12-en-28-oic acid) and platycodon B (3-O-ß-D-glucopyranosyl-16-O-ß-D-xylopyranosyl-2ß,3ß,16ß,21ß-tetrahydroxyolean-12-en-28-oic acid) were isolated from the roots of Platycodon grandiflorum. The structures of these compounds were elucidated by means of various spectroscopic analyses. Compounds 1 and 2 were tested in HepG-2, A549 and DU145 human cancer cell lines and showed remarkable cytotoxic activities against HepG-2 and A549 cancer cell lines with IC(50) values ranging from 4.9 to 9.4 µM, but they displayed weak cytotoxic activities against DU145 cancer cell line with IC(50) values greater than 10 µM.

[Studies on high efficient plant regeneration system from anther callus of Bupleurum chinense].

The callus of Bupleurum chinense with anthers at the stage of uninucleate was induced. After several subcultures, anther calli of B. chinense were cultured at 20 MS culture mediums with different plant hormones to differentiate into plantlets. Differentiation of callus was detected after 21 and 49 days to select the most effective medium. There were 19 culture mediums in which anther callus could differentiate into plantlets with differentiation rate range from 3% to 60% , and most less than 20%. MS + KT 0.5 mg x L(-1) + sucrose 30 g c L(-1) + phytagel 5 g x L(-1) was the best differentiation medium with the differentiation rate of 60%, followed by MS + ZT 1.0 mg x L(-1) + sucrose 30 g x L(-1) + phytagel 5 g x L(-1) with the differentiation rate of 58%. Then plantlets were transferred to rooting medium to obtain whole plant. All plantlets could root in the rooting medium of MS + sucrose 30 g x L(-1) + phytagel 5 g x L(-1) and 1/2 MS + NAA 0.5 mg x L(-1) + sucrose 30 g x L(-1) + phytagel of 5 g L(-1) with the rooting rate of 100%. As a result, the high efficient and stable plant regeneration system was established from anther callus of B. chinense.

The potential influence of 5-aminosalicylic acid on the induction of myelotoxicity during thiopurine therapy in inflammatory bowel disease patients.

OBJECTIVE: To investigate the potential influence of 5-aminosalicylic acid (5-ASA) on the induction of myelotoxicity during thiopurine therapy in inflammatory bowel disease patients. METHODS: (a) The retrospective study included inflammatory bowel disease patients treated with azathioprine (AZA)/6-mercaptopurine (6-MP). Thiopurine methyltransferase (TPMT) activity and 6-thioguanine nucleotide (6-TGN) levels were detected at stable medication points. (b) The prospective study was performed in active disease patients: 4 weeks of AZA 50 mg/day followed by concomitant 5-ASA 3 g/day for another 4 weeks. 6-TGN was analyzed at weeks 4 and 8. RESULTS: (a) Of the 139 retrospective study patients, 45 were on AZA/6-MP+5-ASA and 94 on AZA/6-MP alone. The myelotoxicity rates were 47 and 16%, respectively. Multivariates regression analysis indicated that the administration of concomitant 5-ASA was the only risk factor associated with myelotoxicity (odds ratio=3.45, 95% confidence interval 1.31-9.04, P=0.01). (b) Thiopurine methyltransferase activity was not significantly different between patients on AZA/6-MP+5-ASA and patients on AZA/6-MP alone (P=0.78). (c) 6-TGN levels were significantly higher in samples on AZA/6-MP+5-ASA than those on AZA/6-MP (P=0.003) alone. (d) Sixteen patients completed the prospective study. After 4 weeks on AZA 50 mg/day, 6-TGN levels of 13 patients were less than 230 pmol/8×10 RBC. After another 4 weeks' cotreatment with mesalazine 3 g/day, 12 patients had 6-TGN levels at least 230 pmol/8×10 RBC, five patients had 6-TGN levels at least 420 pmol/8×10 RBC, and two of these five patients developed myelotoxicity. CONCLUSION: The risk of thiopurine-induced myelotoxicity markedly increases in patients treated with combined 5-ASA and 2 mg/kg/day AZA therapy, which may be correlated to the increase in 6-TGN. 50 mg daily AZA when concomitant 5-ASA might help maintain an effective 6-TGN level without increasing the risk of myelotoxicity.

[Cloning of UGT gene of Bupleurum chinense and construction of over expressing and RNAi transgenic vectors].

OBJECTIVE: To clone the full-length cDNA of a uridine diphosphate glycosyltransferase (UGT) gene which may be involved in saikosaponin biosynthesis of Bupleurum chinense, and construct the transgenic vectors for over expression and RNAi of the cloned UGT. These works will provide foundation for further its function analysis by transgene study. METHOD: RAGE and LD-PCR were used to clone the full-length cDNA of the UGT, on the basis of its partial cDNA sequence obtained from our previous 454-sequenced dataset. The ORF and partial sequences of the UGT were PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted into pCAMBIA-SUPER 1 300 and pHANNIBAL. The recombinant pHANNIBAL were digested with Not I and then were inserted into a binary vector, pART27. Finally, the transgenic vectors for over expression and RNAi of the cloned UGT were constructed. RESULT: The full-length cDNA of a UGT were cloned from B. chinense. The recombinant vectors for over expression and RNAi of the UGT were obtained. CONCLUSION: Our works on full-length cDNA cloning and transgenic vectors construction provide a substantial foundation for follow-up biofunction analysis of the UGT through transgenic research.

Hypoxanthine guanine phosphoribosyltransferase activity is related to 6-thioguanine nucleotide concentrations and thiopurine-induced leukopenia in the treatment of inflammatory bowel disease.

BACKGROUND: Thiopurine drugs are widely used in the treatment of inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and adverse events occurrence. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the effects of TPMT and hypoxanthine guanine phosphoribosyltransferase (HPRT) activities on 6-thioguanine nucleotides (6-TGNs) concentrations and thiopurine-induced leukopenia in patients with IBD. METHODS: Clinical data and blood samples were collected from 120 IBD patients who were receiving azathioprine (AZA)/6-mercaptopurine (6-MP) therapy. Erythrocyte TPMT, HPRT activities and 6-TGNs concentrations were determined. HPRT activity and its correlation with TPMT activity, 6-TGNs level, and leukopenia were evaluated. RESULTS: The HPRT activity of all patients ranged from 1.63-3.33 (2.31 ± 0.36) µmol/min per g Hb. HPRT activity was significantly higher in patients with leukopenia (27, 22.5%) than without (P < 0.001). A positive correlation between HPRT activity and 6-TGNs concentration was found in patients with leukopenia (r = 0.526, P = 0.005). Patients with HPRT activity > 2.70 µmol/min per g Hb could have an increased risk of developing leukopenia (odds ratio = 7.47, P < 0.001). No correlation was observed between TPMT activity and HPRT activity, 6-TGNs concentration, or leukopenia. CONCLUSIONS: High levels of HPRT activity could be a predictor of leukopenia and unsafe 6-TGN concentrations in patients undergoing AZA/6-MP therapy. This could partly explain the therapeutic response or toxicity that could not be adequately explained by the polymorphisms of TPMT.

Simultaneous determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry - application to Chinese systemic lupus erythematosus patients.

BACKGROUND: A pharmacogenomics study of cyclophosphamide in systemic lupus erythematosus patients is being conducted in our laboratory in which the plasma concentrations of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide should be assayed rapidly and sensitively. METHODS: A rapid, stable and sensitive liquid chromato-graphy/electrospray ionization tandem mass spectrometry method was developed to simultaneously determine cyclophosphamide and 4-hydroxycyclophosphamide in human plasma with ifosfomide as an internal standard. After a protein precipitation with cold acetonitrile and stabilization of 4-hydroxycyclophosphamide by ansyldrazine and extraction with ethyl acetate, separation was performed on a C18 3.5 µm 2.1 × 50 mm column with mobile phase of acetonitrile and water (50:50, v/v) with 0.1% formic acid at 200 µL/min. The chromatographic run time was 3 min. RESULTS: The linear calibration curves ranged from 5 to 5000 ng/mL for cyclophosphamide and 5-500 ng/mL for 4-hydroxycyclophosphamide. The recoveries of the liquid extraction were 54.5%-58.5% for cyclophosphamide and 103.5%-105.5% for 4-hydroxycyclophosphamide. The lower limit of quantification was 5 ng/mL for both analytes. The intra- and inter-day precision was <15% for quality control samples at 4000, 500, 50 ng/mL for cyclophosphamide and 4-hydroxycyclophosphamide at 400, 100, 20 ng/mL. The method was applied in this pharmacogenomics study in Chinese systemic lupus erythematosus patients treated with low-dose cyclophosphamide. CONCLUSIONS: The method was efficient with shorter running time and lower limit of quantification compared to previous reports and has been successfully applied in this pharmacogenomics study.

Effect of Wuzhi tablet (Schisandra sphenanthera extract) on the pharmacokinetics of paclitaxel in rats.

Wuzhi tablet (WZ, registration no. in China: Z20025766) is a preparation of an ethanol herb extract of Wuweizi (Schisandra sphenanthera) containing 7.5 mg Schisantherin A per tablet. It was reported recently that WZ could significantly increase the blood concentrations of tacrolimus, which might be due to the inhibitory effect of WZ and its ingredients on P-gp and/or CYP450 activity. Paclitaxel is a substrate of the efflux transporter P-gp, and is mainly metabolized by CYP450 enzymes in the liver. Therefore, the purpose of this study was to investigate whether and how WZ affects the pharmacokinetics of paclitaxel in rats. After pretreatment with WZ, there were significant increases in the AUC(0-24h) of oral paclitaxel (from 280.8 ± 97.3 to 543.5 ± 115.2 h ng/mL; p < 0.05) and C(max) (from 44.6 ± 16.4 to 86.8 ± 16.1 ng/mL; p < 0.05). The pharmacokinetic data for i.v. paclitaxel with WZ showed a relatively small (when compared against oral paclitaxel) but still significant increase in AUC(0-24h) (from 163.6 ± 22.1 to 212.7 ± 17.7 h ng/mL; p < 0.05) and a decrease in clearance (from 3.2 ± 0.6 to 2.2 ± 0.3 L/h/kg; p < 0.05). Thus, the presence of WZ improved the systemic exposure of paclitaxel in rats. The herb-drug interaction between WZ and paclitaxel should be taken into consideration in clinical use.

Paracetamol metabolism and related genetic differences.

Paracetamol (acetaminophen) is a worldwide used analgesic and antipyretic drug. It is metabolised via several metabolic pathways, including glucuronidation, sulfation, oxidation, hydroxylation, and deacetylation: Hepatic and other organ damage may occur, especially in overdose, because of the accumulation of a toxic metabolite. Intersubject and ethnic differences have been reported in paracetamol metabolism activation, suggesting possible differences in susceptibility to toxicity and in pain alleviation, linked to different pharmacogenetic profiles. This article aims at reviewing, in the literature, the links between paracetamol metabolism and enzyme genotypes in the context of toxic side effects and efficacy of paracetamol in therapeutics.

CYP4F2 rs2108622: a minor significant genetic factor of warfarin dose in Han Chinese patients with mechanical heart valve replacement.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT * Genetic polymorphisms of VKORC1 and CYP2C9 are known to influence warfarin dosage. * Recent studies among Caucasians showed that polymorphisms of CYP4F2 also play a role in warfarin pharmacogenetics. * The contribution of CYP4F2 variants to the variability inwarfarin dose requirement in Chinese subjects remains to be investigated. WHAT THIS STUDY ADDS * This research was to study the effect of CYP4F2 variants on warfarin requirements in the Han Chinese population. * This study developed a multiple regression model including CYP2C9, VKORC1 3673G>A, CYP4F2 genotypes and age, weight, combination use of amiodarone which could explain 56.1% of the individual variability in warfarin dose CYP4F2 could explain 4% of the variance in warfarin dose. * We found that one novel genotypic polymorphism 5417G>T for Asp36Tyr, which was identified as an important marker of warfarin resistance, was absent in the Han Chinese population in our study. AIMS The objective of this study was to assess the effect of the CYP4F2 on the daily stable warfarin dose requirement in Han Chinese patients with mechanical heart valve replacement (MHVR). METHODS From March 2007 to November 2008, 222 Han Chinese MHVR patients were recruited in our study. VKORC1 3673G>A, 5417G>T, CYP2C9*3 and CYP4F2 rs2108622 were genotyped by using the polymerase chain reaction restriction fragment length polymorphism method (PCR-RFLP). Polymorphisms of VKORC1 9041G>A were detected by direct sequencing. Multiple linear regression analysis was used to investigate the contribution of CYP4F2. RESULTS The CYP4F2 rs2108622 CT/TT group took a significantly higher stable warfarin dose (3.2 mg day(-1)) than the CC group (2.9 mg day(-1), 95% CI 0.2, 1.0, P= 0.033). The multiple linear regression model included VKORC1 3673G>A, CYP2C9, CYP4F2 genotypes and clinical characteristics. The model could explain 56.1% of the variance in stable warfarin dose in Han Chinese patients with MHVR. CYP4F2 contributed about 4% to the variance in the warfarin dose. There was no variation in the SNPs of VKORC1 5417G>T. CONCLUSION CYP4F2 is a minor significant factor of individual variability in the stable warfarin dose in Han Chinese patients with MHVR. The effect of CYP2C9 and VKORC1 genotypes on variability in the stable warfarin dose had also been confirmed.

Enhancement of oral bioavailability of paclitaxel after oral administration of Schisandrol B in rats.

Paclitaxel is a substrate of the efflux transporters such as P-glycoprotein, and is mainly metabolized by the liver. Schisandrol B (Sch B), one of the active components in Schisandra, has been reported to be able to inhibit the activity of P-gp and CYP3A. It might be possible that Sch B would alter the pharmacokinetic behavior of paclitaxel. Therefore, the purpose of this study was to investigate the effect of Sch B on the pharmacokinetics of paclitaxel administered orally and intravenously in rats. Paclitaxel were administered to rats orally (30 mg/kg) or intravenously (0.5 mg/kg) with or without the concomitant administration of Sch B (10 or 25 mg/kg). Oral pharmacokinetic parameters of paclitaxel were significantly altered when pretreated with Sch B. There were significant increases in AUC(0-24h) (from 297.7+/-110.3 to 838.9+/-302.1 h*ng/ml; p<0.05) and C(max) (from 51.7+/-20.1 to 136.4+/-35.5 ng/ml; p<0.05) in the presence of Sch B (25 mg/kg). The pharmacokinetic parameters for i.v. paclitaxel were not significantly affected by Sch B in contrast to that of oral administration. Since the presence of Sch B enhanced the systemic exposure of paclitaxel, their pharmacokinetic interaction should be taken into consideration. As the oral bioavailability of paclitaxel was increased about 3-fold in the presence of Sch B, the concomitant use of Sch B may provide a benefit in the oral delivery of paclitaxel.

Use of transgenic mice in UDP-glucuronosyltransferase (UGT) studies.

Transgenic mouse models are useful to understand the function and regulation of drug-metabolizing enzymes in vivo. This article is intended to describe the general strategies and to discuss specific examples on how to use transgenic, gene knockout, and humanized mice to study the function as well as genetic and pharmacological regulation of UDP-glucuronosyltransferases (UGTs). The physiological and pharmacological implications of transcription factor-mediated UGT regulation will also be discussed. The UGT-regulating transcription factors to be discussed in this article include nuclear hormone receptors (NRs), aryl hydrocarbon receptor (AhR), and nuclear factor erythroid 2-related factor 2 (Nrf2).

Single nucleotide polymorphisms of the pregnane x receptor gene in Han Chinese and a comparison with other ethnic populations.

The pregnane X receptor (PXR/NR1I2) gene is a master regulator for a number of cytochrome P450s (CYPs) and drug transporters. This study aimed to detect the single nucleotide polymorphisms (SNPs) of the PXR gene in Han Chinese (n = 186) and to compare the frequencies of polymorphisms of the PXR gene with those in Caucasian and African Americans reported in the literature. The SNPs of the PXR gene were analyzed using the polymerase chain reaction (PCR) and direct sequencing analysis. The mutant frequencies of A11156C and T11193C in Han Chinese were 55% (95% confidence interval (CI): 0.49-0.61) and 59% (95% CI: 0.52-0.64), respectively, higher than those of Caucasian Americans (16 and 16%, respectively) and African Americans (33 and 30%, respectively). However, the reported SNPs in exons 2 and 4 (PXR*2,*3,*4,*6,*9,*10,and *11) were not detected in Han Chinese. These results indicate that there are marked differences in the mutant frequencies of A11156C and T11193C of PXR between Han Chinese and other ethnic groups. The mutant frequency in the coding region (exons 2 and 4) of PXR was very low in Han Chinese. Further studies are needed to determine the impact of common SNPs of PXR in Han Chinese and other ethnic populations on the phenotypic activity of cytochrome P450s and drug transporters transactivated by PXR.

Determination of levonorgestrel in human plasma by liquid chromatography-tandem mass spectrometry method: application to a bioequivalence study of two formulations in healthy volunteers.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to determine levonorgestrel in human plasma was developed and fully validated. After hexane-ethyl acetate (70:30, v/v) induced extraction from the plasma samples, levonorgestrel was subjected to LC/MS/MS analysis using electro-spray ionization. The MS system was operated in the selected reaction monitoring mode. Chromatographic separation was performed on a Hypersil BDS C18 column (i.d. 2.1x50 mm, particle size 3 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 0.25-90 ng/mL for levonorgestrel. The lower limit of quantification of the method was 0.25 ng/mL for levonorgestrel. The intra- and inter-batch precision was 3.7-10.2 and 5.1-12.9%, respectively, for all quality control samples at concentrations of 0.5, 6.0 and 45.0 ng/mL. These results indicate that the method was efficient with a simple preparation procedure and a very short running time (2.0 min) for levonorgestrel compared with those methods reported in the literature and had high selectivity, acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method was successfully used for a bioequivalence study of two tablet formulations of levonorgestrel in healthy volunteers.