Natural variation in the cytochrome c oxidase subunit 5B OsCOX5B regulates seed vigor by altering energy production in rice

Seed vigor is a crucial trait for the direct seeding of rice.Here we examined the genetic regulation of seed vigor traits in rice,including germination index(GI)and germination potential(GP),using a genome-wide association study approach.One major quantitative trait locus,qGI6/qGP6,was identified simultaneously for both GI and GP.The candidate gene encoding the cytochrome c oxidase subunit 5B(OsCOX5B)was validated for qGI6/qGP6.The disruption of OsCOX5B caused the vigor traits to be significantly lower in Oscox5b mutants than in the japonica Nipponbare wild type(WT).Gene co-expression analysis revealed that OsCOX5B influences seed vigor mainly by modulating the tricarboxylic acid cycle process.The glucose levels were significantly higher while the pyruvic acid and adenosine triphosphate levels were significantly lower in Oscox5b mutants than in WT during seed germination.The elite haplotype of OsCOX5B facilitates seed vigor by increasing its expression during seed germination.Thus,we propose that OsCOX5B is a potential target for the breeding of rice varieties with enhanced seed vigor for direct seeding.

Enhancing metformin-induced tumor metabolism destruction by glucose oxidase for triple-combination therapy

Despite decades of laboratory and clinical trials,breast cancer remains the main cause of cancer-related disease burden in women.Considering the metabolism destruction effect of metformin(Met)and cancer cell starvation induced by glucose oxidase(GOx),after their efficient delivery to tumor sites,GOx and Met may consume a large amount of glucose and produce sufficient hydrogen peroxide in situ.Herein,a pH-responsive epigallocatechin gallate(EGCG)-conjugated low-molecular-weight chitosan(LC-EGCG,LE)nanoparticle(Met–GOx/Fe@LE NPs)was constructed.The coordination between iron ions(Fe3+)and EGCG in this nanoplatform can enhance the efficacy of chemodynamic therapy via the Fenton reaction.Met–GOx/Fe@LE NPs allow GOx to retain its enzymatic activity while simultaneously improving its stability.Moreover,this pH-responsive nanoplatform presents controllable drug release behavior.An in vivo biodistribution study showed that the intracranial accumulation of GOx delivered by this nanoplatform was 3.6-fold higher than that of the free drug.The in vivo anticancer results indicated that this metabolism destruction/starvation/chemodynamic triple-combination therapy could induce increased apoptosis/death of tumor cells and reduce their proliferation.This triple-combination therapy approach is promising for efficient and targeted cancer treatment.

Phylogeny of Apaturinae Butterflies (Lepidoptera: Nymphalidae) Based on Mitochondrial Cytochrome OxidaseⅠ Gene

The phylogenetic relationships of genera in the subfamily Apaturinae were examined using mtDNA sequence data from 1,471 bp of cytochrome oxidase subunit Ⅰ （COI）. The mitochondrial COI gene from a total of 16 species in 11 genera were sequenced to obtain mtDNA data, along with those of 4 species obtained from GenBank, to construct the MP and the NJ trees using Athyma jina, Penthema adelma, Polyura nepenthes, and Charaxes bernardus as outgroups. The transitions at the third codon positions of the COI data set were found saturated, but they were retained for analysis, because they contain the majority of the phylogenetic information. The impacts of equal weight assumptions for all characters in the parsimonious analysis were assessed by potential alternations in clades in response to different transition/transversion weighting schemes. The results indicated four distinct major groups in Apaturinae. Moreover, several well supported and stable clades were found in the Apaturinae. The study also identified undetermined taxon groups whose positions were weakly supported and were subject to changes under different weighting schemes. Within the Apaturinae, the clustering results are approximately identical to the classical morphological classification. The mtDNA data suggest the genus Mimathyma as a monophyletic group. Lelecella limenitoides and Dilipa fenestra have close relationship with very strong support in all phylogenetic trees. It also supports the taxonomic revision of removing several species from Apatura to other genera, namely Mimathyma schrenckii, M. chevana, M. nycteis, Chitoria subcaerulea, C. fasciola, C. pallas, and Helcyra subalba.

The Phylogenetic Relationships among Four Subspecies of the Genus Locusta Based on Sequences of Three Subunit of Cytochrome Oxidase

[Objective] The aim was to explore the phylogenetic relationships among four subspecies of the genus Locusta.[Method] The sequences of three subunits of cytochrome oxidase of Locusta migratoria tibetensis and Locusta migratoria manilensis were amplified and sequenced（COⅠ 1 539 bp,COⅡ 684 bp,CO Ⅲ 792 bp,with the total of 3 015 bp）.The corresponding sequenses of Locusta migratoria migratoria and Locusta migratoria migratorioides were obtained from GenBank and constructed a multiple alignment.Phylogenic trees of four subspecies of L.migratoria were constructed by Neighbor-Joining,Maximum-parsimony and Bayesian,respectively.[Result] The average content of A ＋ T in three subunits of four subspecies was 69.57%;the third site of codon showed the highest A ＋ T content,and the COⅠ had the highest A ＋ T content（87.6%）;The nucleotide substitution mainly occurred at the third site of codon,and the nucleotide replacement rate of CO Ⅱ was the highest.The second site of codon was conservative,so the replacement rate was in the range of 5.9%-15%.The start codon of COⅠ was CCG or ACG.Genetic distances among four subspecies were ranged from 0.001 to 0.076.The relationship between L.m.tibetensis and Locusta migratoria manilensis was the closest,followed by L.m.migratorioides and L.m.migratorioides,while the genetic distance between L.m.tibetensis and L.m.migratorioides was the largest.[Conclusion] The phylogenetic relationships among four subspecies of Locusta migratoria is L.m.tibetensis,L.m.manilensis,L.m.migratoria,L.m.migratorioides.

Cancer and age related colonic crypt deficiencies in cytochrome c oxidase Ⅰ

AIM: To investigate whether defi ciency of expressionof cytochrome c oxidase I (CcOI) in colonic crypts is associated with colon cancer.METHODS: The pattern and level of expression of CcOI in non-neoplastic colonic crypts,and in dysplastic tissues,was assessed using standard immunohis-tochemical methods.Biopsies were obtained from individuals undergoing colonoscopies for screening purposes or for a medically indicated reason.Tissue samples were also obtained from surgical colonic resections.Samples from resections were taken from colonic mucosa 1 and 10 cm from tumors and from the tumors themselves.Samples were evaluated for frequency of crypts with reduced or absent expression of CcOI.In most crypts the loss was apparent throughout the entire crypt,while in a small minority the loss was segmental.The strong immunoreactivity using this monoclonal antibody makes the scoring unambiguous.The percent of crypts with reduced or absent expression of CcOI or (infrequent) segmented loss of expression was then calculated.Data analyses were performed using SPSS statistical package 17.0.RESULTS: The average frequency of CcOI deficient crypts (CcOI-DC) is low in individuals between 20 and 39 years of age,with 0.48% ± 0.40% CcOI-DC for women and 1.80% ± 0.35% for men.CcOI-DC increases after age 40 years,so that between the ages of 40 and 44 years the average frequency of CcOI- DC goes up to 5.89% ± 0.84% in women and 2.15% ± 1.27% in men.By 80-84 years of age,the average frequency of CcOI-DC goes up in women to 15.77% ± 0.97% and in men to 22.6% ± 0.65%.The increases in CcOI-DC from ages 40-44 years compared to 80-84 years in women and men are significantly different with P < 0.01.For women over age 60 years,deficiency of CcOI expression is greater in those women who have had a cancer in their colon.The frequency of CcOI-DC,measured in men,increased in tissues adjacent to colon cancer,being 4.03% ± 0.27% in individuals free of neoplasia in the age range 55-64 yearsand 14.13% ± 0.35% in resected histologically normal tissue of men with cancer in the same age range,P < 0.001.Similar signifi cant differences were noted in older age ranges.The frequency of CcOI-DC crypts in the cecum and sigmoid colon of an individual are signifi cantly correlated,with an R2 = 0.414 for women and R2 = 0.528 for men,P < 0.001.This suggests that the factors determining the level of CcOI deficiency act throughout the colon.Most defective crypts are in clusters of two or more,a likely consequence of crypt fission.In the non-neoplastic margins of cancers,crypts are frequently defi cient for CcOI,and such crypts may appear in large clusters,some containing more than 100 defi cient crypts.CcOI defi ciency is also apparent in colon cancers and sometimes involves a large section of the tumor.Overall,CcOI deficient cells can be visualized in segments of crypts,in whole crypts that increase in frequency with age,in crypts undergoing f ission,in clusters of crypts where the clusters increase in size with age,in increased frequency near tumors,in large clusters in the intimate margins of tumors,and in the tumors themselves.There is no clear dividing line between early stages that can be considered aspects of aging and later stages that can be considered aspects of the progression to cancer.This ambiguity may re ect a rather general situation leading to adult cancer where the early stages of cellular change appear to be relatively innocuous features of the aging process but over decades may evolve into malignancy.CONCLUSION: CcOI defi cient crypts increase in frequency with age,and clusters of defi cient crypts are associated with,and may give rise to,colon cancer.

Identification of the metabolite and cytochrome P450 isoforms involved in rat liver microsomal metabolism of TM208

To identify the metabolite and CYP450 isoforms involved in rat liver microsomal metabolism of TM208. The present study investigated the metabolism of TM208 and the effects of selective CYP450 inhibitors on the metabolism of TM208 in rat liver microsomes. Various specific inhibitors of CYP were used to identify the isoforms of CYP involved in the metabolism of TM208. The inhibitor of CYP2D and that of CYP2B had strong inhibitory effects on TM208 metabolism in a concentration-de- pendant manner, the inhibitor of CYP1A had a modest inhibitory effect, and the inhibitor of CYP3A seemed not to have an obvious inhibitory effect on TM208 metabolism. TM208 might mainly be metabolized by CYP2D and CYP2B in rat liver microsomes.

Phylogeny of Limenitidinae Butterflies (Lepidoptera: Nymphalidae) Inferred from Mitochondrial Cytochrome Oxidase I Gene Sequences

The phylogenetic analyses of the subfamily Limenitidinae are performed based on 1 471 bp of mtDNA cytochrome oxidase subunit I （COI） gene sequence data which were obtained from 21 individuals spanning 9 genera, along with those of 17 species obtained from GenBank, using Apatura iris, Aglais urticae, and Polyura dolon as outgroup species. Although the transitions at the third codon positions of the COI data set were highly saturated, they were still retained for analysis as they contain the majority of the phylogenetic information, and thus, the maximum pasimony （MP） under different weighting schemes and maximum likelihood （ML） trees were reconstructed in this study. The results showed that within this subfamily, the results based on the COI gene sequences are approximately identical to the traditional classification results. However, the clustering of Lexias pardalis and Tanaecia julii within the genus Euthalia as well as the clustering of Phaedyma aspasia within the genus Neptis with weak support are different from that of the current classification scheme made by Chinese scholars. The genus Limenitis is splited into two subclusters in the trees constructed by using MP and ML methods. These results support one of the strongest hypotheses for the tribe relationships within Limenitidinae.

Hippocampal mitochondrial cytochrome C oxidase activity and gene expression in a rat model of chronic cerebral ischemia

The present study established a rat model of chronic cerebral ischemia using bilateral common carotid artery permanent ligation to analyze cytochrome C oxidase activity and mRNA expression in hippocampal mitochondria. Results showed significantly decreased cytochrome C oxidase activity and cytochrome C oxidase II mRNA expression with prolonged ischemia time. Further analysis revealed five mitochondrial cytochrome C oxidase II gene mutations, two newly generated mutations, and four absent mutational sites at 1 month after cerebral ischemia, as well as three mitochondrial cytochrome C oxidase III gene mutations, including two newly generating mutations, and one disappeared mutational site at 1 month after cerebral ischemia. Results demonstrated that decreased cytochrome C oxidase gene expression and mutations, as well as decreased cytochrome C oxidase activity, resulting in energy dysmetabolism, which has been shown to be involved in the DatholoQical Process of ischemic brain iniurv.

Histochemical Study and Microspectrophotometric Analysis of Regional Distribution of Cytochrome Oxidase in Normal Rat Brain.

Histochemical study and determination of cytochrome oxidase (CTO) relative activ-ity with a Leitz MPV-III microspectrophotometer in different regions of normal rat brain werecarried out.9 healthy male Wistar rats were divided randomly into 2 groups;an enzyme activi-ty studied group and a control group with HE staining.It was found that 2 kinds of CTO distri-bution areas exist in the brain of rats;the high activity area including cerebral cortex,corpusstriatum (gray matter),thalamus,cerebellar cortex,etc,and low activity area including corpuscallosum,corpus striatum (white matter),hippocampus,cerebellar white matter,etc.The dif-ference of CTO activity between the 2 areas is statistically significant (P【0.01).Moreover,according to the intensity of positive response to CTO detecting stain,the granular layer of thecerebellum can be classified as cytochrome oxidase richly-contained area(CTORA)orcytochrome oxidase poorly-contained area (CTOPA).The CTO activity of the former issignificantly higher than that of the latter(P【0.01).

Effect of Intestinal Cytochrome P450 3A on Phytochemical Presystemic Metabolism

Phytochemicals, orally administered substances, are found to undergo presystemic metabolism mainly in the intestine. Although early researches confirmed the role of intestinal bacteria in phytochemical presystemic metabolism, along with the development of molecular biology in investigating intestinal metabolism, a breakthrough has been won in research into metabolizing enzymes and transporters in intestine, which demands more attention and further studies. Recently, Cytochrome P450 3A has been found to be the most effective enzyme in mediating both oxidative （Phase Ⅰ ） and conjugative （Phase Ⅱ ） metabolism in the intestine. The present review summarizes the current findings correlated with the effect of intestinal cytochrome P450 3A on phytochemical presystemic metabolism, which provides a good basis for further research on phytochemical pharmacokinetics.

Regio- and Substrate-Specific Oxidative Metabolism of Terpinolene by Cytochrome P450 Monooxygenases in <i>Cupressus lusitanica</i>Cultured Cells

Many of monoterpenes produced in plants contribute to defenses against herbivores, insects and microorganisms. Among those compounds, β-thujaplicin formed in Cupressaceae plants has a unique conjugated seven-membered ring and some useful biological activities, e.g. fungicide, repellent, insecticide and so on. The biosynthesis pathway of β-thujaplicin has not yet been revealed;we have been trying to uncover it using Cupressus lusitanica cultured cells as a model. In our previous study, terpinolene was identified as a potential β-thujaplicin intermediate at the branching point to terpenoids. In this article, terpinolene metabolism in C. lusitanica cultured cells was investigated, and it was shown that the microsomal fraction from cells oxidized terpinolene into the hydroxylated compound, 5-isopropylidene-2-met-hylcyclohex-2-enol (IME). Then, IME was further oxidized by microsomal fraction to the epoxidized compound, 1,6-epoxy-4(8)-p-menthen-2-ol (EMO). These were the only two products detected from the microsomal reactions, respecttively. Moreover, microsomal reactions with monoterpenes other than terpinolene produced nothing detectable. These results show that the enzymes of these reactions had strict substrate specificity and regio-selectivity. Experiments on kinetics and with specific inhibitors confirmed that these reactions were caused by cytochrome P450 monooxygenases, respectively. These results support our hypothesis that terpinolene is a putative intermediate of β-thujaplicin biosynthesis and show that IME and EMO are also putative intermediates.

Retinal ganglion cells of high cytochrome oxidase activity in the rat

Retinal ganglion cells in the rat were studied using the heavy metal intensified oytochrome oxidase and horseradish peroxidase histochemieal methods. The results show that a population of large retinal ganglion cells was consistently observed with the eyto3hrome oxidase staining method in retinas of normal rats or rats which received unilateral thalamotomy at birrth. These oytochrome oxidase rich ganglion cells appeared to have large somata, 3-6 primary dendrites and extensive dendritic arbors, and are comparable to ganglion cells labeled by the wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). However, the morphological details of some of the cells revealed by the cytoahrome oxidase staining method are frequently better than those shown by the HRP histochemieal method. These results suggest that the mit03hondrial enzyme oytoohrome oxidase can be used as a simple but reliable marker for identifying and studying a population of retinal ganglion cells with high metabolie rate in the rat.

Simplifying the Analysis of Enzyme Kinetics of Cytochrome <i>c</i>Oxidase by the Lambert-W Function

Conventional analysis of enzyme-catalyzed reactions uses a set of initial rates of product formation or substrate decay at a variety of substrate concentrations. Alternatively to the conventional methods, attempts have been made to use an integrated Michaelis-Menten equation to assess the values of the Michaelis-Menten KM and turnover kcat constants directly from a single time course of an enzymatic reaction. However, because of weak convergence, previous fits of the integrated Michaelis-Menten equation to a single trace of the reaction have no proven records of success. Here we propose a reliable method with fast convergence based on an explicit solution of the Michaelis-Menten equation in terms of the Lambert-W function with transformed variables. Tests of the method with stopped-flow measurements of the catalytic reaction of cytochrome c oxidase, as well as with simulated data, demonstrate applicability of the approach to de termine KM and kcat constants free of any systematic errors. This study indicates that the approach could be an alternative solution for the characterization of enzymatic reactions, saving time, sample and efforts. The single trace method can greatly assist the real time monitoring of enzymatic activity, in particular when a fast control is mandatory. It may be the only alternative when conventional analysis does not apply, e.g. because of limited amount of sample.

Regulation of cytochrome c oxidase contributes to health and optimal life

The generation of cellular energy in the form of ATP occurs mainly in mitochondria by oxidative phosphorylation.Cytochrome c oxidase(CytOx),the oxygen accepting and rate-limiting step of the respiratory chain,regulates the supply of variable ATP demands in cells by“allosteric ATP-inhibition of CytOx.”This mechanism is based on inhibition of oxygen uptake of CytOx at high ATP/ADP ratios and low ferrocytochrome c concentrations in the mitochondrial matrix via cooperative interaction of the two substrate binding sites in dimeric CytOx.The mechanism keeps mitochondrial membrane potentialΔΨm and reactive oxygen species(ROS)formation at low healthy values.Stress signals increase cytosolic calcium leading to Ca^2+-dependent dephosphorylation of CytOx subunit I at the cytosolic side accompanied by switching off the allosteric ATPinhibition and monomerization of CytOx.This is followed by increase ofΔΨm and formation of ROS.A hypothesis is presented suggesting a dynamic change of binding of NDUFA4,originally identified as a subunit of complex I,between monomeric CytOx(active state with highΔΨm,high ROS and low efficiency)and complex I(resting state with lowΔΨm,low ROS and high efficiency).

Enzymatic Activity of Escherichia Coil Lactate Dehydrogenase and Cytochrome c Oxidase

The catalytic activity of two common bacterial enzymes, lactate dehydrogenase （LDH） and cytochrome c oxidase （COX） from Escherichia coli was examined following bacterial exposure to microwave （MW） radiation under well-defined experimental conditions. The experiments were conducted in a specialized microwave processing apparatus, with an exposure frequency of 18 GHz, and a temperature profile that was restricted to below 40℃ to avoid thermal degradation of the bacteria. The absorbed power was calculated to be 1,500 kW/m3 and the electric field was determined to be 300 Wm. Both values were theoretically confirmed using Computer Simulation Technology （CST） Microwave Studio 3D Electromagnetic Stimulation Software. Results showed that the activity of both enzymes was increased following MW radiation compared to negative controls and thermally treated samples subjected to similar temperature profiles. It is suggested that the increase in COX and LDH enzyme activity could not be explained by conventional heating alone, but was rather a result of micro-thermal effects that incorporated ＇undetectable＇ thermal mechanisms.

Microevolution of Mitochondrial Cytochrome oxidase subunit I in Drosophila melanogaster at ＂Evolution Canyon＂, Israel

We determined the sequence of mitochondrial Cytochrome oxidase subunit I （CO1） in two Drosophila melanogaster strains originating at ＂Evolution Canyon＂, Israel. CO1 nucleotide sequences from two iso-female strains, 2-1 and 6-1, were 1,534 and 1,543 base-pairs, respectively. In each strain, ATAA was used in initiation of translation. Exchange rates for nucleotide and amino acid sequences were larger in the 6-1 strain than in the 2-1 strain when Oregon-R was used as the standard. Non-synonymous exchange rate was larger than synonymous exchange rate among the three strains.

IMMUNOCHEMICAL IDENTIFICATION AND LOCALIZATION OF CYTOCHROME P-450HSjISOZYME, AN ENZYME RELATED TO NITROSAMINE METABOLISM, IN HUMAN GASTRIC MUCOSA AND GASTRIC CARCINOMA

Monoclonal antibody (MAb) to rat liver cyto-chrome P-450j isozyme, an activating enzyme specific to nitrosamine metabolism, was used coupled with immunoblotting, densitometer scanning of SDS-PAGE gels and immunohistochemical technique. The trace P-450HSj isozyme (Mr. 51.5 Kd) was found in human gastric mucosa. It was similar to P-450j in molecular weight, catalytic and immunochemical properties. The concentrations of P-450HSj in mucosa of lesser curvature were higher than those in greater curvature. This might be one of the important reasons that lesser curvature is the commonest area for gastric carcinoma. But there was possibly less P-450HSj in gastric mucosa with cancer. Im-munohistochemically, P-450HSj was discovered in the cytoplasm of some glandular epithelial cells, especially in the glands with hyperplastic and intestinal metaplastic changes adjacent to carcinoma. It was also found in some normal glands and in tumor cells of high-differentiated adenocarcinoma, but not in those of low-differentiated ones. Following subjects are discussed: (1) the method of detecting trace P-450HSj, (2) the rule of distribution of P-450HSj, and (3) the relationship between the isozyme and the occurrence of gastric cancer caused by nitrosa-mines.

Construction of Cox7a2 fluorescent vector and its effect on cytochrome C oxidase activity in mouse Sertoli cell line TM4

Objective To construct Cox7a2 fluorescent vector and study its effect on cytochrome C oxidase ( COX) activity in mouse Sertoli cell line TM4. Methods The coding region of CoxTa2 was amplified from mouse Sertoli cell line TM4 by RT-PCR. PCR product was

Effects of Terpinen-4-ol on Four Metabolic Enzymes and Polyphenol Oxidase (PPO) in Mythimna separta Walker

To study insecticidal mechanism of terpinen-4-ol, a main insecticidal composition in the essential oil of Sabina vulgaris, the 5th instar larvae of Mythimna separta, were investigated with terpinen-4-ol by topical application. The activities of phosphatase, glutathione S-transferase （GSTs）, cytochrome P450 （P450）, and polyphenol oxidase （PPO） of tested insects were determined in all poisoning stages, including exciting stage, convulsing stage, paralysis stage, and recover stage. The result showed that the activities of both acid phosphatase （ACP） and alkaline phosphatase （AKP） in treated insects were induced by terpinen-4-ol, but ACP was inhibited in paralysis stage. The activities of GSTs were inhibited in exciting stage, convulsing stage, and paralysis stage, but gradually recovered in recover stage. O-demethylase activity of cytochrome P450 was inhibited by terpinen-4-ol, and the inhibition rate in all poisoning stages were 26.27, 46.03, 80.24, and 90.22%, respectively. PPO activities were strongly inhibited by terpinen-4-ol both in vitro and in vivo. In conclusion, the activities of P450, GSTs, and PPO could have relation with toxicity of terpinen-4-ol against larvae of the Mythimna separta, but recover stage of the poisoning insects might be related to GSTs induced. As a new insecticide or synergist, terpinen- 4-ol has a potential value in field of insecticide resistance management.

Microdosing cocktail study on the determination and pharmacokinetics of six hepatic cytochrome probes and their metabolites in rat

OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.