Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 38
Filter
1.
China Journal of Chinese Materia Medica ; (24): 591-598, 2021.
Article in Chinese | WPRIM | ID: wpr-878883

ABSTRACT

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.


Subject(s)
Animals , Deer/metabolism , Gelatin , Glycosylation , Hydroxylation , Lysine/metabolism , Protein Processing, Post-Translational , Tandem Mass Spectrometry
2.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 939-945, 2016.
Article in English | WPRIM | ID: wpr-812537

ABSTRACT

The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3-6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3-6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC values being 35.59 ± 5.37 μmol·L (Caco-2), 24.04 ± 4.70 μmol·L (MCF-7), and 22.24 ± 5.19 μmol·L (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.


Subject(s)
Humans , Antineoplastic Agents , Chemistry , Metabolism , Biotransformation , Cell Line, Tumor , Diterpenes , Chemistry , Metabolism , Gibberella , Metabolism , Hydroxylation , Molecular Structure , Mortierella , Metabolism , Stereoisomerism
3.
Braz. j. microbiol ; 46(1): 261-264, 05/2015. tab, graf
Article in English | LILACS | ID: lil-748265

ABSTRACT

The monoterpenoid 1,8-cineole is obtained from the leaves of Eucalyptus globulus and it has important biological activities. It is a cheap natural substrate because it is a by-product of the Eucalyptus cultivation for wood and pulp production. In this study, it was evaluated the potential of three filamentous fungi in the biotransformation of 1,8-cineole. The study was divided in two steps: first, reactions were carried out with 1,8-cineole at 1 g/L for 24 h; afterwards, reactions were carried out with substrate at 5 g/L for 5 days. The substrate was hydroxylated into 2-exo-hydroxy-1,8-cineole and 3-exo-hydroxy-1,8-cineole by fungi Mucor ramannianus and Aspergillus niger with high stereoselectivity. Trichoderma harzianum was also tested but no transformation was detected. M. ramannianus led to higher than 99% of conversion within 24 h with a starting high substrate concentration (1 g/L). When substrate was added at 5 g/L, only M. ramannianus was able to catalyze the reaction, but the conversion level was 21.7% after 5 days. Both products have defined stereochemistry and could be used as chiral synthons. Furthermore, biological activity has been described for 3-exo-hydroxy-1,8-cineol. To the best of our knowledge, this is the first report on the use of M. ramannianus in this reaction.


Subject(s)
Aspergillus niger/metabolism , Cyclohexanols/metabolism , Eucalyptus/chemistry , Monoterpenes/metabolism , Mucorales/metabolism , Hydroxylation , Time Factors , Trichoderma/metabolism
4.
Chinese Journal of Biotechnology ; (12): 939-954, 2015.
Article in Chinese | WPRIM | ID: wpr-240574

ABSTRACT

Erythromycin A is a clinically important macrolide antibiotic with broad-spectrum activity. Its biosynthesis involves the formation of the 14-membered skeleton catalyzed by polyketide synthases, and the modification steps such as hydroxylation, glycosylation and methylation. Based on the understanding of the biosynthetic mechanism, it is reliable to genetically manipulate the erythromycin A-producing strain for production improvement and structure modification. In this paper, we reviewed the progress regarding erythromycin A in high-producing strain construction and chemical structure derivation, to provide insights for further development.


Subject(s)
Anti-Bacterial Agents , Chemistry , Erythromycin , Chemistry , Glycosylation , Hydroxylation , Methylation , Multigene Family , Polyketide Synthases , Metabolism
5.
Chinese Journal of Biotechnology ; (12): 1481-1485, 2014.
Article in Chinese | WPRIM | ID: wpr-345576

ABSTRACT

In order to discover the steroid biotransformation ability of filamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1 per thousand, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids


Subject(s)
Androstenedione , Metabolism , Aspergillus niger , Metabolism , Biotransformation , Fermentation , Hydroxylation , Industrial Microbiology
6.
Acta Pharmaceutica Sinica ; (12): 151-157, 2014.
Article in Chinese | WPRIM | ID: wpr-297999

ABSTRACT

Prolyl-4-hydroxylase domain (PHDs) family is one of the most important regulatory factors in hypoxic stress. PHD2 plays a critical role in cells and tissues adaptation to the low oxygen environment. Its hydroxylation activity regulates the stability and transcriptional activity of the hypoxia-inducible factor 1 (HIF-1), which is the key factor in response to hypoxic stress. Subsequently, PHD2 acts as an important factor in oxygen homeostasis. Studies have shown that PHD2, through its regulation on HIF-1, plays an important role in the post-ischemic neovascularization. Furthermore, under hypoxic condition, PHD2 also regulates other pathways that positively regulate angiogenesis factors HIF-1 independently. Moreover, recently, several evidences have also shown that PHD2 also affects tumor growth and metastasis in a tumor microenvironment. Based on these facts, PHD2 have been considered as a potential therapeutic target both in treating ischemic diseases and tumors. Here, we review the molecular regulation mechanism of PHD2 and its physiological and pathological functions. We focus on the role of PHD2 in both therapeutic angiogenesis for ischemic disease and tumor angiogenesis, and the current progress in utilizing PHD2 as a therapeutic target.


Subject(s)
Animals , Humans , Hydroxylation , Hypoxia-Inducible Factor 1 , Metabolism , Hypoxia-Inducible Factor-Proline Dioxygenases , Physiology , Neoplasms , Metabolism , Pathology , Therapeutics , Neovascularization, Pathologic , Metabolism , Pathology , Tumor Microenvironment , Vascular Diseases , Pathology , Therapeutics
7.
Chinese Journal of Biotechnology ; (12): 147-156, 2014.
Article in Chinese | WPRIM | ID: wpr-279533

ABSTRACT

Combined with method of ketoconazole resistance screening, a 7alpha,15alpha-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7alpha,15alpha-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4 x 7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4 x 7H2O 0.015, MgSO4 x 7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7alpha,15alpha-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.


Subject(s)
Biotransformation , Colletotrichum , Metabolism , Dehydroepiandrosterone , Chemistry , Fermentation , Hydroxylation , Industrial Microbiology , Mutation
8.
Biomedical and Environmental Sciences ; (12): 138-147, 2013.
Article in English | WPRIM | ID: wpr-320358

ABSTRACT

<p><b>OBJECTIVE</b>To compare the nature of the metabolites formed from the phase I metabolism (hydroxylation and oxidation) and phase II metabolism (glutathionyl conjugation) of PCBs that have different chlorine substitution patterns. To discuss the structure-activity relationships and metabolic mechanisms of PCBs.</p><p><b>METHODS</b>4-Cl-biphenyl (PCB3), 4,4'-Cl-biphenyl (PCB15), 3,4,3',4'-Cl-biphenyl (PCB77) were used for in vitro metabolic study. LC/MS and UV-Vis studies were performed for metabolites identification.</p><p><b>RESULTS</b>The cytochrome P-450 catalyzed hydroxylation rate decreased as the number of chlorine substitutions increased. In this reaction, PCB3 was fully metabolized, approximately half of the PCB15 was metabolized and PCB77 was not metabolized at all. The oxidation rate of PCB15-HQ was higher than that of PCB3-HQ under various oxidation conditions. The LC/MS and UV-Vis data suggest that in the conjugation reaction of PCB15-Q and GSH, the Michael addition reaction occurs preferentially over the displacement reaction.</p><p><b>CONCLUSION</b>The metabolic profiles of polychlorinated biphenyls (PCBs) are dramatically affected by chlorine substitution patterns. It is suggested that the metabolic profiles of PCBs are related to their chlorine substitution patterns, which may have implications for the toxicity of PCB exposure.</p>


Subject(s)
Hydroxylation , Molecular Structure , Oxidation-Reduction , Polychlorinated Biphenyls , Chemistry
9.
Chinese Journal of Biotechnology ; (12): 1590-1598, 2013.
Article in Chinese | WPRIM | ID: wpr-242434

ABSTRACT

Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated by a hydroxylase. To obtain the critical hydroxylase, a novel hydroxylase gene was isolated from Amycolatopsis sp. CGMCC1149 by Degenerate PCR and Self-Formed Adaptor PCR and expressed in Escherichia coli. BLAST sequence analysis revealed that the gene belonged to cytochrome P450 gene superfamily and could encode a 403-amino-acid protein with a molecular weight of 44.8 kDa. The secondary structure prediction result showed that this protein contained many typical functional regions of P450, such as oxygen binding site, ion-pair region and heme binding region. Meanwhile, a catalytic function verification system was constructed by NADH, ferredoxin and ferredoxin reductase which could catalyze lovastatin hydroxylation into the target product. These would be helpful for further studies in large-scale production of wuxistatin.


Subject(s)
Actinomycetales , Genetics , Amino Acid Sequence , Butyrates , Metabolism , Cloning, Molecular , Cytochrome P-450 Enzyme System , Genetics , Metabolism , Hydroxylation , Industrial Microbiology , Lovastatin , Metabolism , Molecular Sequence Data
10.
Biomolecules & Therapeutics ; : 487-492, 2013.
Article in English | WPRIM | ID: wpr-202589

ABSTRACT

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (k cat/K m) for lauric acid hydroxylation mainly due to an increase in K m. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in k cat and an increase in K m. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.


Subject(s)
Animals , Cats , Humans , Arachidonic Acid , Blood Pressure , Clinical Coding , Cytochrome P-450 Enzyme System , Escherichia coli , Fatty Acids, Unsaturated , Hydroxylation , Liver , Luciferases , Luminescence , Mass Screening , Mutagenesis
11.
Acta Pharmaceutica Sinica ; (12): 1374-1379, 2011.
Article in Chinese | WPRIM | ID: wpr-232980

ABSTRACT

Sinisan is a widely used traditional Chinese medicine (TCM) in treating various diseases; however, the in vivo metabolic profile of its multiple components remains unknown. In this paper, ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was applied to identify the metabolites of Sinisan extract in rat plasma, urine, feces and bile after intragastric administration. Using MS(E) and mass defect filter techniques, 41 metabolites of 10 parent compounds (naringin, naringenin, hesperidin, neohesperidin, liquiritin, liquiritigenin, glycyrrhizic acid, glycyrrhetinic acid, saikosaponin a and saikosaponin d) were detected and tentatively identified. It was shown by our results that these compounds was metabolized to the forms of hydroxylation, glucuronidation, sulfation, glucuronidation with sulfation and glucuronidation with hydroxylation in vivo.


Subject(s)
Animals , Male , Rats , Administration, Oral , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Metabolism , Pharmacokinetics , Flavanones , Metabolism , Pharmacokinetics , Glucosides , Metabolism , Pharmacokinetics , Glycyrrhizic Acid , Metabolism , Pharmacokinetics , Hesperidin , Metabolism , Pharmacokinetics , Hydroxylation , Plants, Medicinal , Chemistry , Random Allocation , Rats, Sprague-Dawley , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
12.
Korean Journal of Pediatrics ; : 51-54, 2011.
Article in English | WPRIM | ID: wpr-36580

ABSTRACT

Vitamin D is present in two forms, ergocalciferol (vitamin D2) produced by plants and cholecalciferol (vitamin D3) produced by animal tissues or by the action of ultraviolet light on 7-dehydrocholesterol in human skin. Both forms of vitamin D are biologically inactive pro-hormones that must undergo sequential hydroxylations in the liver and the kidney before they can bind to and activate the vitamin D receptor. The hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D], plays an essential role in calcium and phosphate metabolism, bone growth, and cellular differentiation. Renal synthesis of 1,25(OH)2D from its endogenous precursor, 25-hydroxyvitamin D (25OHD), is the rate-limiting and is catalyzed by the 1alpha-hydroxylase. Vitamin D dependent rickets type I (VDDR-I), also referred to as vitamin D 1alpha-hydroxylase deficiency or pseudovitamin D deficiency rickets, is an autosomal recessive disorder characterized clinically by hypotonia, muscle weakness, growth failure, hypocalcemic seizures in early infancy, and radiographic findings of rickets. Characteristic laboratory features are hypocalcemia, increased serum concentrations of parathyroid hormone (PTH), and low or undetectable serum concentrations of 1,25(OH)2D despite normal or increased concentrations of 25OHD. Recent advances have showed in the cloning of the human 1alpha-hydroxylase and revealed mutations in its gene that cause VDDR-I. This review presents the biology of vitamin D, and 1alpha-hydroxylase mutations with clinical findings.


Subject(s)
Animals , Humans , Biology , Bone Development , Calcitriol , Calcium , Cholecalciferol , Clone Cells , Cloning, Organism , Dehydrocholesterols , Ergocalciferols , Hydroxylation , Hypocalcemia , Kidney , Liver , Muscle Hypotonia , Parathyroid Hormone , Receptors, Calcitriol , Rickets , Seizures , Skin , Ultraviolet Rays , Vitamin D , Vitamins
13.
Acta Pharmaceutica Sinica ; (12): 573-580, 2011.
Article in English | WPRIM | ID: wpr-348916

ABSTRACT

Abstract: The activities of four CYP450 enzymes (CYP3A, 1A2, 2El and 2C) and the mRNA expression levels of CYP1A2, 2El, 2Cll and 3A1 in rat liver were determined after Wistar rats were orally administered with brucine (BR) at three dosage levels (3, 15 and 60 mg.kg-1 per day) and the high dose of BR combined with glycyrrhetinic acid (GA, 25 mg.kg-1 per day) or liquiritin (LQ, 20 mg.kg-1 per day) for 7 consecutive days. Compared with the control, brucine caused 24.5% and 34.6% decrease of CYP3A-associated testosterone 6beta-hydroxylation (6betaTesto-OH) and CYP2C-associated tolbutamide hydroxylation (Tol-OH), respectively, and 146.1% increase of CYP2El-associated para-nitrophenol hydroxylation (PNP-OH) at the high dose level. On the other hand, (BR+GA) caused 51.4% and 33.5% decrease, respectively, of CYP2El-associated PNP-OH and CYP1A2-associated ethoxyresorufin-O-de-ethylation (EROD) as compared with the high dose of BR group. Meanwhile, (BR+LQ) caused 41.1% decrease of CYP2El-associated PNP-OH and 37.7% increase of CYP2C-associated Tol-OH. The results indicated that the co-administration of BR with GA or LQ had effect on mRNA expression and activities of the CYP450 enzymes mentioned above to some extent, and the in vivo antagonism of LQ on BR-induced CYPs adverse effects and the in vivo inhibitory action of GA on CYP2E1 and 1A2 might play an important role in the detoxification of Radix Glycyrrhizae against Strychnos nux-vomica L.


Subject(s)
Animals , Male , Rats , Aryl Hydrocarbon Hydroxylases , Genetics , Metabolism , Cytochrome P-450 CYP1A1 , Metabolism , Cytochrome P-450 CYP1A2 , Genetics , Metabolism , Cytochrome P-450 CYP2E1 , Genetics , Metabolism , Cytochrome P-450 CYP3A , Genetics , Metabolism , Cytochrome P-450 Enzyme System , Genetics , Metabolism , Cytochrome P450 Family 2 , Flavanones , Pharmacology , Gene Expression Regulation, Enzymologic , Glucosides , Pharmacology , Glycyrrhetinic Acid , Pharmacology , Hydroxylation , Liver , Metabolism , Nitrophenols , Metabolism , Plants, Medicinal , Chemistry , RNA, Messenger , Metabolism , Rats, Wistar , Steroid 16-alpha-Hydroxylase , Genetics , Metabolism , Steroid Hydroxylases , Metabolism , Strychnine , Pharmacology , Strychnos nux-vomica , Chemistry , Tolbutamide , Metabolism
14.
Biol. Res ; 41(1): 81-92, 2008. ilus, tab, graf
Article in English | LILACS | ID: lil-490635

ABSTRACT

CYP2E1 enzyme is related to nonalcoholic steatohepatitis (NASH) due to its ability for reactive oxygen species production, which can be influenced by polymorphisms in the gene. The aim of this study was to investigate hepatic levels, activity, and polymorphisms of the CYP2E1 gene to correlate it with clinical and histological features in 48 female obese NASH patients. Subjects were divided into three groups: (i) normal; (ii) steatosis; and (iii) steatohepatitis. CYP2E1 protein level was assayed in microsomes from liver biopsies, and in vivo chlorzoxazone hydroxylation was determined by HPLC. Genomic DNA was isolated for genotype analysis through PCR. The results showed that liver CYP2E1 content was significantly higher in the steatohepatitis (45 percent; p=0.024) and steatosis (22 percent; p=0.032) group compared with normal group. Chlorzoxazone hydroxylase activity showed significant enhancement in the steatohepatitis group (15 percent, p=0.027) compared with the normal group. c2 rare allele of RsallPstl polymorphisms but no C allele of Dral polymorphism was positively associated with CHZ hydroxylation, which in turn is correlated with liver CYP2E1 content (r=0.59; p=0.026). In conclusion, c2 allele is positively associated with liver injury in NASH. This allele may determine a higher transcriptional activity of the gene, with consequent enhancement in pro-oxidant activity of CYP2E1 thus affording liver toxicity.


Subject(s)
Adult , Female , Humans , /metabolism , Fatty Liver/enzymology , Hepatitis/enzymology , Liver/enzymology , Obesity/enzymology , Case-Control Studies , Chromatography, High Pressure Liquid , Chlorzoxazone/metabolism , /genetics , Fatty Liver/pathology , Gene Frequency , Genotype , Hepatitis/pathology , Hydroxylation/genetics , Liver/pathology , Obesity/pathology , Polymorphism, Genetic
15.
Journal of Southern Medical University ; (12): 717-719, 2007.
Article in English | WPRIM | ID: wpr-337357

ABSTRACT

The aromatic-ring-hydroxylating dioxygenase is a key enzyme that initiates the biodegradation of polycyclic aromatic hydrocarbons in bacteria. In the present study, a novel dioxygenase sequence was cloned from Terrabacter sp. FLO using a genome walking method. The dioxygenase was cloned into pET17 and actively expressed in E.coli BL21 (DE3) in co-expression with electron transfer chain proteins. The recombinant dioxygenase was found to transform phenanthrene, fluorene, pyrene and fluoranthene into the cis-dihydrodiol metabolites.


Subject(s)
Actinomycetales , Genetics , Bacterial Proteins , Genetics , Metabolism , Biodegradation, Environmental , Cloning, Molecular , Dioxygenases , Genetics , Metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Metabolism , Fluorenes , Metabolism , Hydroxylation , Molecular Sequence Data , Phenanthrenes , Metabolism , Polycyclic Aromatic Hydrocarbons , Metabolism , Pyrenes , Metabolism , Recombinant Proteins , Metabolism
16.
Article in English | IMSEAR | ID: sea-17866

ABSTRACT

BACKGROUND AND OBJECTIVES: Phenytoin, a widely used anti-epileptic drug, is metabolized mainly by CYP2C9 (90%) and partly by CYP2C19 (10%) to its major metabolite 5-(para-hydroxyphenyl)-5- phenylhydantoin (p-HPPH). The CYP2C9 and CYP2C19 genes encoding these enzymes are polymorphically expressed and most of the variants result in decreased metabolism of the respective substrates. The present study was undertaken to investigate the influence of the CYP2C9*2 and *3 as well as CYP2C19*2 and *3 variant genotypes on phenytoin hydroxylation in healthy subjects from south India. METHODS: A total of 27 healthy, unrelated, subjects were administered a single oral dose of 300 mg phenytoin. Four hours later, 5 ml of blood was collected and genotyped for CYP2C9*1, *2, *3, CYP2C19*1, *2 and *3 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Phenytoin and the major metabolite p-HPPH were estimated by reverse phase HPLC. The metabolic ratio was calculated as concentration of phenytoin/p-HPPH. RESULTS: A significant correlation was observed between the CYP2C9 genotype and metabolic ratio of phenytoin/p-HPPH (r = 0.472, 95% CI 0.100 to 0.728; P = 0.01). While no association was found with CYP2C19 alone, a significant correlation was observed between the combined CYP2C9 and CYP2C19 genotypes and phenytoin metabolic ratio (r = 0.507, 95% CI 0.146 to 0.749; P< 0.01). INTERPRETATION AND CONCLUSION: CYP2C9*2 and *3 mutant alleles caused decreased hydroxylation of phenytoin in vivo, whereas the mutant alleles of CYP2C19 played only a minor role in the metabolism of phenytoin in subjects of our study. The results of present preliminary study needs to be confirmed with a larger sample.


Subject(s)
Adult , Anticonvulsants/metabolism , Aryl Hydrocarbon Hydroxylases/genetics , Female , Genotype , Humans , Hydroxylation , India , Male , Mixed Function Oxygenases/genetics , Phenytoin/metabolism , Polymorphism, Single Nucleotide
17.
Chinese Journal of Biotechnology ; (12): 662-666, 2006.
Article in Chinese | WPRIM | ID: wpr-286231

ABSTRACT

The 11-hydroxylation of 16alpha,17alpha-epoxy-4-pregenene-3,20-dione as a useful intermediate for the preparation of hormones can be achieved by the mycelium of Absidia coerulea at higher conversion rate than using other strains. In this paper 16alpha,17alpha-epoxy-4-pregenene-3,20-dione mixed with a little water, beta-cyclodextrin, Tween-80 was introduced into the fermentation broth after ultrasonication to increase pseudo-water-solubility of the hydrophobic substrate. This pseudo-crystallo feed could avoid the toxicity of organic solvents and was more available for the microbial transformation. The multi layer feed-forward neural network was used to setup a model which indicated the relationship between medium and feed components and the conversion rate. Particle swarm optimization (PSO), which was a stochastic global optimization algorithm and of which the convergence speed was high, was applied to obtain the optimal concentration of the medium and feed components. At optimum conditions with the pseudo-crystallo feed, the conversion rate of 16alpha,17alpha-epoxy-4-pregenene-3,20-dione at an initial concentration of 10 g/L was 87.5% in shaking flasks. The conversion rate of the substrate was up to 86.6% at higher concentration of 20 g/L feed in a 3.7 L fermentor.


Subject(s)
Absidia , Metabolism , Fermentation , Hydroxylation , Pregnenediones , Metabolism
18.
Acta Pharmaceutica Sinica ; (12): 1004-1009, 2006.
Article in Chinese | WPRIM | ID: wpr-294898

ABSTRACT

<p><b>AIM</b>To identify the hydroxylate metabolites and its sulfate conjugates of daidzein in rat urine.</p><p><b>METHODS</b>Urine samples from 0 - 24 h were collected after single ig dose of 500 mg x kg(-1) daidzein to each of six rats. The urine samples were purified by SPE column (SPE C18) and analyzed with liquid chromatographic-tandem electrospray ionization ion trap mass spectrometry (LC-ESI/MS(n)) for potential metabolites.</p><p><b>RESULTS</b>Several new hydroxylate metabolites and its sulfate conjugates were found and identified in rat urine.</p><p><b>CONCLUSION</b>LC-ESI/MS(n) is proved to be a simple, rapid, sensitive and specific technique for identification of the hydroxylate metabolites and its sulfate conjugates of daidzein in rat urine.</p>


Subject(s)
Animals , Male , Rats , Chromatography, Liquid , Methods , Hydroxylation , Isoflavones , Chemistry , Metabolism , Urine , Molecular Structure , Phytoestrogens , Chemistry , Metabolism , Urine , Rats, Sprague-Dawley , Reproducibility of Results , Seeds , Chemistry , Soybeans , Chemistry , Spectrometry, Mass, Electrospray Ionization , Methods , Sulfates , Metabolism , Tandem Mass Spectrometry , Methods
19.
Acta Pharmaceutica Sinica ; (12): 912-915, 2005.
Article in Chinese | WPRIM | ID: wpr-253542

ABSTRACT

<p><b>AIM</b>To identify the drug-metabolizing enzymes involved in the hydroxylation of the new anti-inflammatory and anodyne imrecoxib.</p><p><b>METHODS</b>Imrecoxib was incubated with heterologous expression human cytochrome P450 (rCYPs) in vitro, and metabolites and remained parent drug were detected with liquid chromatography-multistage mass spectrometry. The contribution of 4 CYPs in the hydroxylation metabolism of imrecoxib was evaluated by total normalized rate (TNR) method.</p><p><b>RESULTS</b>Imrecoxib is metabolized by CYP2C9, CYP2D6 and CYP3A4, with the rate of 62.5%, 21.1% and 16.4%, respectively.</p><p><b>CONCLUSION</b>CYP2C9 is the major enzyme involved in imrecoxib hydroxylation metabolism.</p>


Subject(s)
Aryl Hydrocarbon Hydroxylases , Metabolism , Cyclooxygenase 2 Inhibitors , Metabolism , Cytochrome P-450 CYP2C9 , Cytochrome P-450 CYP2D6 , Metabolism , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System , Metabolism , Hydroxylation , Pyrroles , Metabolism , Spectrometry, Mass, Electrospray Ionization , Sulfides , Metabolism
20.
Acta Pharmaceutica Sinica ; (12): 892-896, 2004.
Article in Chinese | WPRIM | ID: wpr-241416

ABSTRACT

<p><b>AIM</b>To identify the cytochrome P450 (CYP) isoform (s) involved in daidzein mono-hydroxylated metabolites using human liver microsomes.</p><p><b>METHODS</b>Kinetic analysis of the rates of formation of mono-hydroxylated metabolites of daidzein, including 7,8,4'-trihydroxyisoflavone (7,8,4'-THI), 7,3',4'-trihydroxyisoflavone (7,3',4'-THI) and 6,7,4'-trihydroxyisoflavone (6,7,4'-THI), was performed using human liver microsomes (HLM) and recombinant enzymes at substrate concentrations ranging from 0.5 to 400 micromol x L(-1). Nine selective inhibitors or substrate probes specific for different CYP isoforms were applied for screening the isoform(s) responsible for mono-hydroxylated metabolism of daidzein.</p><p><b>RESULTS</b>Michaelis-Menten kinetic parameters were best fitted to one-component enzyme kinetic model. The mean Km (micromol x L(-1) ) and V(max) (micromol x g(-1) x min(-1)) values were 27 +/- 10 and 4. 8 +/- 2.1, 54 +/- 22 and 2.3 +/- 1.0, 51 +/- 29 and 2.2 +/- 0.8, for the formation rates of 7,8,4'-THI, 7,3',4'-THI, and 6,7,4'-THI, respectively. Furafylline, the CYP1A2 specific inhibitor, estrogen, and monoclonal antibody raised against human CYP1A2 (MAB-1A2) apparently inhibited the formation of mono-hydroxylated metabolites, The IC50 of Fur for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI was 1.0, 0.9 and 0. 8 mol x L(-1), respectively. The IC50 of estrogen for the formation of 7,3',4'-THI, 6,7,4'-THI and 7,8,4'-THI were 51, 60 and 64 mol x L(-1) respectively. The IC50 of MAB-1A2 for the formation of the mono-hydroxylated products was 1 mol x L(-1), but neither other selective inhibitor nor substrate probes, including coumarin (CYP2D6), sulphaphenzole ( CYP2C9/10), omeprazole ( CYP2C19), quinidine (CYP2D6), diethyldithiocarbamate (CYP2E1), troleandomycin (CYP3A4) and keteconazole (CYP3A4), did so with human liver microsomes.</p><p><b>CONCLUSION</b>The in vitro studies indicated that daidzein mono-hydroxylated products were principally metabolized by CYP1A2 in human.</p>


Subject(s)
Humans , Cytochrome P-450 CYP1A2 , Metabolism , Cytochrome P-450 CYP1A2 Inhibitors , Estrogens , Pharmacology , Hydroxylation , Isoflavones , Metabolism , Microsomes, Liver , Metabolism , Theophylline , Pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL