Search results for "hydroxylation"
showing 10 items of 102 documents
Improved sample preparation for the testosterone hydroxylation assay using disposable extraction columns
1992
The preparation of samples for injection into a high-performance liquid chromatograph from assay mixtures for the determination of cytochrome P-450-dependent testosterone hydroxylation has been substantially facilitated. By replacing the multiple cumbersome extraction steps of the conventional method with a single column extraction the time for sample preparation was reduced from hours to minutes. The new procedure also yields better recoveries for most of the testosterone metabolites than the original protocol. The use of extraction columns for sample preparation allows the simultaneous treatment of a large number of samples or even the automation of the whole assay procedure. The modified…
Impact of the post-treatment conditions of parent silica on the silanization of n-octadecyl bonded silica packings in reversed-phase high-performance…
2001
Native mesoporous silica beads were subjected to a sequence of post-treatment procedure including hydrochloric acid treatment, calcination and subsequent rehydroxylation. The post-treated silica beads were converted into RP-18 silica by silanization with monochloro- and dimethoxy-n-octadecylsilanes, respectively. The influence of post-treatments and silanization conditions on the physico-chemical characteristics and on the chromatographic behaviour of the RP-silicas was studied. Also the changes of the pore structural parameters and the silanol group densities during the post-treatment and silanization were assessed.
High Expression of Human CYP2C in Immortalized Human Liver Epithelial Cells
2010
Cell lines stably expressing high levels of single isozymes of human CYP2C genes (CYP2C8, CYP2C9, CYP2C18 and CYP2C19) have been successfully generated by transfecting liver epithelial human cells (THLE) with an appropriate expression vector. To this aim, cDNAs encoding for each CYP2C gene were inserted by blunt-ended cloning into the unique insertion site of the singular expression vector pCMVneo. The recombinant pCMV2C8, pCMV2C9, pCMV2C18 and pCMV2C19 vectors were liposome-mediated transfected into THLE cells. The resulting transgenic cells, designated as T5-2C8, T5-2C9, T5-2C18 and T5-2C19, were cloned and the expression of the ectopic gene, mRNA and protein, was investigated by RT-PCR a…
Thermally Induced Structural Modification of Silica Nanoparticles Investigated by Raman and Infrared Absorption Spectroscopies
2010
We report an experimental investigation by Raman and infrared (IR) absorption spectroscopies on the structural modifications induced by isochronal thermal treatments on amorphous SiO2 nanoparticles (fumed silica). In particular, three different commercial types of this material, characterized by particle mean diameters of 7, 14, and 40 nm, were subjected to thermal treatments from 100 up to 1000 °C. We found that some properties of fumed silica, such as the SiOSi mean bond angle, ring size distribution, and surface adsorbed water content, are drastically different from those of common bulk silica materials and intimately related to the particles' dimension. The SiOSi mean bond angle, probed…
Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms
1992
Primary steps in the metabolism of caffeine and theophylline are cleavage of methyl groups and/or hydroxylation at position 8, mediated by cytochromes P450. V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2 and P450IIBI and human P450IA2 and rat liver epithelial cells expressing murine P450IA2 were used to overcome problems arising in the proper allocation of metabolic pathways to specific isoforms by conventional techniques. These cell lines were exposed to caffeine and/or theophylline, and concentrations of metabolites formed in the medium were determined by HPLC. Caffeine was metabolized by human, rat and murine P45…
Applications of stable V79-derived cell lines expressing rat cytochromes P4501A1, 1A2, and 2B1.
1992
1. Chinese hamster V79-derived cell lines, stably expressing cytochromes P4501A1, 1A2, and 2B1 activities, were constructed by genetic engineering in continuation of our work to establish a battery of V79 derived cell lines designed to study the metabolism of xenobiotics. 2. Cell lines XEM1 and XEM2, expressing cytochrome P4501A1, were capable of the O-dealkylation of 7-ethoxycoumarin and the hydroxylation of benzo[a]pyrene. 3. Cell lines XEMd.MZ and XEMd.NH, expressing P4501A2, were shown to hydroxylate 17 beta-estradiol and 2-aminofluorene. 4. Cell line SD1, expressing cytochrome P4502B1, was able to hydroxylate testosterone stereo- and regio-specifically at the 16 alpha and 16 beta posit…
Deoxysarpagine Hydroxylase — A Novel Enzyme Closing a Short Side Pathway of Alkaloid Biosynthesis in Rauvolfia
2002
Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase…
Hepatic metabolism of diclofenac: role of human CYP in the minor oxidative pathways.
1999
The aim of this study was to re-examine the human hepatic metabolism of diclofenac, with special focus on the generation of minor hydroxylated metabolites implicated in the idiosyncratic hepatotoxicity of the drug. Different experimental approaches were used: human hepatocytes, human microsomes, and engineered cells expressing single human CYP (cytochromes P450). Human hepatocytes formed 3'-hydroxy-, 4'-hydroxy-, 5-hydroxy- 4',5-dihydroxy-, and N,5-dihydroxydiclofenac, as well as several lactams. Formation of 4'- and 5-hydroxydiclofenac by human liver microsomes followed a Michaelis-Menten kinetics (Km 9 +/- 1 microM; Vmax 432 +/- 15 pmol/min/mg and Km 43 +/- 5 microM; and Vmax 15.4 +/- 0.6…
DEVELOPMENTAL ASPECTS OF DRUG METABOLISM
1977
Publisher Summary This chapter discusses the developmental aspects of drug metabolism and those of other biological phenomena that are twofold. Development is described by examining various species of different levels of biological evolution or by studying the ontogenetic evolvement of the features in question in one animal species or groups of related species. A review on diene-organochlorine insecticides epitomizes the fact that insects, birds, and fish possess the enzymatic mechanisms for epoxidation of these insecticides to only a slightly less degree than do mammals. Thus, the ability to oxidize foreign compounds does not seem to be restricted to animals of higher organization, and con…
Crystal structure of human gamma-butyrobetaine hydroxylase.
2010
Gamma-butyrobetaine hydroxylase (GBBH) is a 2-ketoglutarate-dependent dioxygenase that catalyzes the biosynthesis of l-carnitine by hydroxylation of gamma-butyrobetaine (GBB). l-carnitine is required for the transport of long-chain fatty acids into mitochondria for generating metabolic energy. The only known synthetic inhibitor of GBBH is mildronate (3-(2,2,2-trimethylhydrazinium) propionate dihydrate), which is a non-hydroxylatable analog of GBB. To aid in the discovery of novel GBBH inhibitors by rational drug design, we have solved the three-dimensional structure of recombinant human GBBH at 2.0A resolution. The GBBH monomer consists of a catalytic double-stranded beta-helix (DBSH) domai…