Biomedical subjects

Irving W Wainer

Publications and source records attributed to Irving W Wainer.

At least 19 recordsLinked to original sources

PubMed2006

Simultaneous determination of buprenorphine, norbuprenorphine and the enantiomers of methadone and its metabolite (EDDP) in human plasma by liquid chromatography/mass spectrometry.

A previously reported enantioselective LC-MS assay for the determination of (R)- and (S)-methadone [Met] and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine [EDDP] (the primary metabolite of Met) has been adapted for use in the simultaneous determination of the plasma concentrations of Met, EDDP, buprenorphine (Bu) and norbuprenorphine (norBu). All of the target compounds were separated within 15 min using an alpha1-acid glycoprotein chiral stationary phase, a mobile phase composed of acetonitrile: ammonium acetate buffer [10 mM, pH 7.0] in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min at 25 degrees C. Deuterium labeled compounds were used as internal standards [d4-Bu, d3-norBu, (R,S)-d3-Met and (R,S)-d3-EDDP] and linear relationships between peak height ratios and drug concentrations were obtained for Bu and norBu in the range 0.2-12 ng/ml with correlation coefficients greater than 0.999. The relative standard deviations (%R.S.D.) for the intra- and inter-day precision of the method were <4.5% and for accuracy was <4.0%. The method was validated and used to analyze plasma samples obtained from opioid dependent methadone-maintained adults enrolled in a research study.

Buprenorphine↗

PubMed2006

Conformational mobility of immobilized proteins.

Cellular membrane fragments have been immobilized on the surface of a silica-based liquid chromatographic support and on the surface of glass capillaries to create immobilized receptor and drug transporter columns. These columns have included phases containing one subtype of the nicotinic receptor (alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4) and the P-glycoprotein transporter. A key question in the application of these columns to drug discovery and development is the ability of the immobilized receptor or transporter to undergo ligand and/or co-factor induced conformational changes. Using frontal affinity chromatographic techniques and non-linear chromatographic techniques it has been demonstrated that the immobilized nicotinic receptors undergo agonist-induced conformational shifts from the resting to desensitized states with corresponding changes in binding affinities and enantioselectivities. Ligand-induced allosteric interactions and ATP-driven conformational changes have also been demonstrated with the immobilized Pgp stationary phase. The results demonstrate that the immobilized proteins retained their ability to undergo conformational mobility and that this is an attractive alternative to allow for the full characterization of multiple protein conformations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

On-line drug metabolism in capillary electrophoresis. 1. Glucuronidation using rat liver microsomes.

A rat liver microsome pseudostationary phase has been used for the on-line capillary electrophoresis monitoring of glucuronidation. Uridine diphosphate glucuronosyltransferase (EC 2.4.1.17) containing microsomes was isolated from rat liver and directly injected onto neutrally coated capillary containing polymeric replaceable gels followed by injection of the substrate mixture. On-line glucuronidation was observed within 15 min without any sample preparation. The factors affecting the separation of glucuronides and parent compounds were investigated by varying the applied electric fields and the size (length and internal diameter) of capillary. The Michaelis-Menten parameters (Km and Vmax) for the glucuronidation of 4-methyl-7-hydroxy coumarin and 4-nitrophenol were determined using the CE method and by off-line microsomal incubation. No significant differences were observed for Km and Vmax values for 4-methyl-7-hydroxycoumarin and 4-nitrophenol between on-line and off-line glucuronidation of these two compounds. This method was also used to determine the inhibition constant (IC50 value) for the competitive inhibition of morphine glucuronidation by codeine, IC50 (on-line) = 170 vs 580 microM (off-line). The results demonstrate that this method can be used to screen for the glucuronidation of test compounds and should reduce the time required for this screening process.

Animals↗

PubMed2006

Uniformly-sized, molecularly imprinted polymers for nicotine by precipitation polymerization.

Uniformly-sized, molecularly imprinted polymers (MIPs) for (S)-nicotine have been prepared by a precipitation polymerization method using methacrylic acid (MAA) or 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer and divinylbenzene (DVB) as a cross-linker in a mixture of toluene and acetonitrile. The (S)-nicotine-imprinted MAA-co-DVB polymers were monodispersed microspheres of about 4 microm in diameter, while the TFMAA-co-DVB ones were gel-like. Molecular recognition abilities of the former MIPs were evaluated for nicotine and its structurally related compounds in liquid chromatography using a mixture of sodium phosphate buffer and acetonitrile as the eluent. Enantioseparation of nicotine was attained using the (S)-nicotine-imprinted MAA-co-DVB polymers. Furthermore, they could selectively trap nicotine in cigarette smoke extracts.

Chemical Precipitation↗

PubMed2006

Non-competitive inhibitory activities of morphinan and morphine derivatives at the alpha 3 beta 4 Neuronal nicotinic acetylcholine receptor determined using nonlinear chromatography and chemometric techniques.

PURPOSE: A series of morphine and morphinan derivatives were chromatographed on a column containing immobilized cellular membranes from a cell line expressing the alpha 3 beta 4 neuronal nicotinic acetylcholine receptor (alpha 3 beta 4 nAChR). METHODS: The results were analyzed using chemometric and molecular modeling techniques in order to predict the noncompetitive inhibitory (NCI) activity of these compounds, the molecular basis for the predicted activity and the binding sites of the inhibitors. RESULTS: The data demonstrated that seven of seven morphinans were NCIs and bound in the central lumen of the nAChR while only 2 of 13 morphine derivatives had NCI activity and these compounds most likely bound at the quinacrine binding site on the nAChR. The predicted activities were confirmed using functional inhibition studies. CONCLUSIONS: The results indicate that this approach can be used to rapidly assess pharmacological activity and to guide new drug design.

Binding Sites↗

PubMed2006

Intensive therapy with growth factor support for patients with Ewing tumor metastatic at diagnosis: Pediatric Oncology Group/Children's Cancer Group Phase II Study 9457--a report from the Children's Oncology Group.

PURPOSE: Prognosis is poor for Ewing sarcoma patients with metastasis at diagnosis. We intensified a five-drug therapy (ifosfamide, etoposide alternated with vincristine, doxorubicin, and cyclophosphamide) using filgrastim but not stem-cell support. We studied topotecan alone and combined with cyclophosphamide in therapeutic windows before the five-drug therapy. A randomly assigned proportion of patients received amifostine as a cytoprotective agent. PATIENTS AND METHODS: Eligible patients were < or = 30 years old and had histologically proven Ewing sarcoma or primitive neuroectodermal tumor (PNET) and metastasis at diagnosis. Chemotherapeutic cycles began every 21 days, after recovery from toxicities. RESULTS: One hundred ten of the 117 patients enrolled were eligible. Thirty-six patients received initial topotecan. Three had partial responses (PRs), and 17 had progressive disease (PD). Thirty-seven patients were administered topotecan and cyclophosphamide; 21 of these patients achieved PR, and one patient had PD. In a randomly assigned group of 69 patients, amifostine did not provide myeloprotection, which was measured by absolute neutrophil count, platelet count, or cycle intervals. The best responses to the overall therapy included 45 complete responses, 41 PRs, stable disease in 14 patients, and PD in five patients. For all patients, the 2-year event-free survival (EFS) rate was 24% (+/- 4%), and the overall survival rate was 46% (+/- 5%). For the 39 patients with isolated pulmonary metastases, the 2-year EFS rate was 31% (+/- 7%) compared with 20% (+/- 5%) for patients with more widespread disease. CONCLUSION: Topotecan had limited activity in patients with Ewing sarcoma or PNET metastatic at diagnosis. The topotecan-cyclophosphamide combination was active. Amifostine was not myeloprotective. Overall results showed no improvement compared with previous studies.

Adolescent↗

PubMed2006

Enantioselective separation and online affinity chromatographic characterization of R,R- and S,S-fenoterol.

BACKGROUND: rac-Fenoterol is a beta2-adrenoceptor agonist (beta2-AR) used in the treatment of asthma. It has two chiral centers and is marketed as a racemic mixture of R,R'- and S,S'-fenoterol (R-F and S-F). Here we report the separation of the R-F and S-F enantiomers and the evaluation of their binding to and activation of the beta2-AR. METHODS: R-F and S-F were separated from the enantiomeric mixture by chiral chromatography and absolute configuration determined by circular dichroism. Beta2-AR binding was evaluated using frontal affinity chromatography with a stationary phase containing immobilized membranes from HEK-293 cells that express human beta2-AR and standard membrane binding studies using the same membranes. The effect of R-F and S-F on cardiomyocyte contractility was also investigated using freshly isolated adult rat cardiomyocytes. RESULTS: Chiral chromatography of rac-fenoterol yielded separated peaks with an enantioselectivity factor of 1.21. The less retained peak was assigned the absolute configuration of S-F and the more retained peak R-F. Frontal chromatography using membrane-bound beta2-AR as the stationary phase and rac-3H-fenoterol as a marker ligand showed that addition of increasing concentrations of R-F to the mobile phase produced concentration-dependent decreases in rac-3H-fenoterol retention, while similar addition of S-F produced no change in rac-3H-fenoterol retention. The calculated dissociation constant of R-F was 472 nM and the number of available binding sites 176 pmol/column, which was consistent with the results from the membrane binding study 460 +/- 55 nM (R-F) and 109,000 +/- 10,400 nM (S-F). In the cardiomyocytes, R-F increased maximum contractile response from (265 +/- 11.6)% to (306 +/- 11.8)% of resting cell length (P < 0.05) and reduced EC50 from -7.0 +/- 0.270 to -7.1 +/- 0.2 log[M] (P < 0.05), while S-F had no significant effect. DISCUSSION: Previous studies have shown that rac-fenoterol acts as an apparent beta2-AR/G(s) selective agonist and fully restores diminished beta2-AR contractile response in cardiomyocytes from failing hearts of spontaneously hypertensive rats (SHR). Here we report the separation of the enantiomers of rac-fenoterol and that R-F is the active component of rac-fenoterol. Further evaluation of R-F will determine if it has enhanced selectivity and specificity for beta2-AR/G(s) activation and if it can be used in the treatment of congestive heart failure.

Animals↗

PubMed2005

Development and characterization of an immobilized enzyme reactor (IMER) based on human glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies.

Immobilized enzyme reactors (IMERs) for on-line enzymatic studies are useful tool to select specific inhibitors and may be used for direct determination of drug-receptor binding interactions and for the rapid on-line screening to identify specific inhibitors. This technique has been shown to increase the stability of enzymes. The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi and it has become a key target in the drug discovery program for Chagas' disease. Crystallographic studies have indicated that there are significant inter-species differences in GAPDH activity and sensitivity. For example the active sites of GAPDH in T. cruzi and humans differ by a substitution of ASP(210) (T. cruzi) by Leu(194) in human. Based on this information we initiated the study to develop optimal conditions for the covalent immobilization of the human GAPDH enzyme on a modified capillary support (400 mm x 0.10 mm). The chromatographic separation of NAD from NADH was achieved using a RP-Spherex-diol-OH (10 cm x 0.46 cm, 10 microm, 100 A) column. By using multidimensional HPLC chromatography system it was possible to investigate the activity and kinetic parameters of the GAPDH-IMER. The values obtained for D-GA3P and NAD were K(m)=3.5+/-0.2 mM and 0.75+/-0.04 mM, respectively, and were compared with values obtained with the free enzyme. The activity of the immobilized GAPDH has been preserved for over 120 days.

Chromatography↗

PubMed2005

The covalent immobilization of microsomal uridine diphospho-glucuronosyltransferase (UDPGT): initial synthesis and characterization of an UDPGT immobilized enzyme reactor for the on-line study of glucuronidation.

The microsomal fraction of rat liver containing uridine diphospho-glucuronosyltransferase (UDPGT; EC 2.4.1.17) has been covalently immobilized on a high performance chromatographic support. In this study Nucleosil Si-500 silica was converted into diol-bonded silica and subsequently converted into an aldehyde form through oxidation with sodium periodate. The microsomal fraction was immobilized via Schiff base formation followed by reduction with sodium cyanoborohydride. The resulting immobilized enzyme reactor (IMER) was placed in a multi-dimensional chromatographic system which utilized a mixed mode (C18 and anion exchange) column to trap the parent compound and glucuronide and a C18 column to separate the substrate and product. The IMER system was used for the online glucuronidation of 4-methylumbelliferone (4Me7OHC) and acetaminophen (APAP). The Michaelis-Menten kinetic parameters (Km and Vmax) associated with the formation of 4Me7OHC and APAP glucuronides demonstrated that the immobilization had not significantly affected the enzymatic activity of the UDPGT relative to the non-immobilized enzyme. The IMER retained enzymatic activity for more than 6 weeks. The results of this study demonstrate an easy and convenient way to identify compounds which may be glucuronidated and to synthesize and characterize the resulting products.

Animals↗

PubMed2005

Direct chromatographic determination of dissociation rate constants of ligand-receptor complexes: assessment of the interaction of noncompetitive inhibitors with an immobilized nicotinic acetylcholine receptor-based liquid chromatography stationary phase.

A liquid chromatographic stationary phase containing immobilized membranes from a cell line expressing the alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) has been used to assess dissociation rate constants (kd) of 12 noncompetitive inhibitor-nAChR complexes. The pharmacological effects of the noncompetitive inhibitors, expressed as percent recovery of activity at 7 min and 4 h postexposure to the inhibitor, were also determined. The results demonstrate that the kd values correlated with the pharmacological effect and that this approach can be used to identify molecular structures associated with differences in kd values. The method can be adapted for use with membrane-bound receptors, ion channels, and transporters and represents a direct and facile technique for the assessment of dissociation rate constants (kd) of ligand-receptor complexes.

Binding, Competitive↗

PubMed2005

Determination of total and free concentrations of the enantiomers of methadone and its metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine) in human plasma by enantioselective liquid chromatography with mass spectrometric detection.

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been validated for the determination of total and free plasma concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP, the primary metabolite of Met), using their respective deuterium-labeled compounds as internal standards [(R,S)-d3-Met and (R,S)-d3-EDDP]. For total drug determinations, 1 ml human plasma was extracted, using a cation-exchange solid-phase extraction cartridge; the eluate was evaporated, reconstituted in the mobile phase, and injected into the LC-MS system. The free fractions of Met and EDDP were determined, using 500 microl of plasma, which were placed in an ultrafiltration device and centrifuged at 2000 x g until 250 microl of filtrate was collected. The filtrate was extracted as described above and analyzed. Enantioselective separations were achieved using an alpha1-acid glycoprotein chiral stationary phase, a mobile phase composed of acetonitrile-ammonium acetate buffer [10 mM, pH 7.0] (18:82, v/v), a flow rate of 0.9 ml/min at 25 degrees C. Under these conditions, enantioselective separations were observed for Met (alpha = 1.30) and EDDP (alpha = 1.17) within 15 min. Met, EDDP, [2H3]-Met and [2H3]-EDDP were detected using selected ion monitoring at m/z 310.30, 278.20, 313.30, and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for Met in the range 1.0-300.0 ng/ml, and for EDDP from 0.1 to 25.0 ng/ml with correlation coefficients greater than 0.999, where the lower limit of quantification (LLOQ) was 1 ng/ml for Met and 0.1 ng/ml for EDDP. The relative standard deviation (R.S.D.) expressed as R.S.D. for the intra- and inter-day precision of the method were < 5.3% and the R.S.D. for accuracy was < 5.0%. The method was used to analyze plasma samples obtained from patients enrolled in a Met-maintenance program.

Calibration↗

PubMed2005

Qualitative assessment of IC50 values of inhibitors of the neuronal nicotinic acetylcholine receptor using a single chromatographic experiment and multivariate cluster analysis.

It has been widely demonstrated that affinity chromatography can be used to derive binding affinities, and that these affinities can be correlated to data obtained using standard techniques such as membrane binding, ultrafiltration and equilibrium dialysis. The purpose of this study is to evaluate the use of immobilized nicotinic acetylcholine receptor stationary phase in chromatographic experiments to assess the functional activity of series of noncompetitive inhibitors (NCIs) as reflected in their IC50 values. Chromatographically determined retention values and computer generated molecular descriptors were obtained for 29 compounds and the data were analyzed by cluster analysis. The approach qualitatively ranked the test compounds as efficient NCIs (low IC50 values) or poor NCIs (high IC50 values). The data obtained with the 29 compounds used in this study demonstrate that the experimental approach had been able to place 25 of these compounds in the correct IC(50) clusters. To our knowledge, this is the first relationship established between chromatographic retention and IC50 for membrane-bound receptors. These results suggest that the chromatographic approach may be useful in development of lead drug candidates including the determination of off-target binding.

Chromatography, Affinity↗

PubMed2005

The synthesis and initial characterization of an immobilized DNA unwinding element binding (DUE-B) protein chromatographic stationary phase.

The DNA unwinding element binding protein (DUE-B) plays a key role in DNA replication. The DUE-B protein has been immobilized on a liquid chromatography support and the resulting immobilized protein column was used for the on-line screening of a series of steroids. The DUE-B protein was expressed with an added C-terminal sequence of six adjacent histidine residues, a His6-tag and immobilized on a chiral ligand exchange support, the CLC-L column, using Ni2+ as the coordinating metal ion. The chromatographic retentions of 12 steroids were determined on the DUE-B/CLC-L column. The magnitudes of the steroid-immobilized DUE-B interactions, reflected by the observed retention times, correlated to the effect of the steroids in the cell-free replication system, i.e. the longer the retention, the greater the increase in DNA replication. The coefficient of determination for the %DNA activities linear relation to retention time was 0.9694. The data suggest that the DUE-B/CLC-L phase can be used for on-line pharmacological studies. The results also indicated that His-tagged proteins can be directly immobilized on the CLC-L stationary phase and the resulting columns used as rapid screens for the isolation and identification of small molecule or protein ligands from complex biological or chemical mixtures.

Chromatography, Liquid↗

PubMed2005

Conformational mobility of immobilized alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nicotinic acetylcholine receptors.

Four affinity chromatography stationary phases have been developed based upon immobilized nicotinic acetylcholine receptor (nAChR) subtypes, the alpha3beta2, alpha3beta4, alpha4beta2, and alpha4beta4 nAChRs. The stationary phases were created using membranes from cell lines expressing the subtypes and an immobilized artificial membrane stationary phase. The immobilized nAChRs were characterized using frontal chromatography with the agonist epibatidine as the marker. The observed binding affinities for the agonists epibatidine, nicotine, and cytisine were consistent with reported values, indicating that the nAChRs retained their ability to bind agonists. The noncompetitive inhibitors (NCIs) of the nAChR (R)- and (S)-mecamylamine, phencylcidine, dextromethoprphan, and levomethorphan were also chromatographed on the columns using nonlinear chromatography techniques. The studies were carried out before and after exposure of the columns to epibatidine. The NCI retention times increased after exposure to epibtatidine as did the enantioselective separation of mecamylamine and methorphan. The results indicate that the immobilized nAChRs retained their ability to undergo agonist-induced conformational change from the resting to the desensitized states. The columns provide a unique ability to study the interactions of NCIs with both of these conformational states.

Alkaloids↗

PubMed2004

On-line screening of conformationally constrained nicotines and anabasines for agonist activity at the alpha3beta4- and alpha4beta2-nicotinic acetylcholine receptors using immobilized receptor-based liquid chromatographic stationary phases.

Liquid chromatography columns containing stationary phases based upon immobilized nicotinic acetylcholine receptors (nAChRs) were used to screen a series of conformationally constrained nicotine and anabasine derivatives for agonist activity. The alpha3beta4 nAChR and alpha4beta2 nAChR subtypes were used to prepare the chromatographic columns and [(3)H] epibatidine dihydrochloride ([(3)H] EB) was used as the marker ligand. Single displacement experiments were conducted with the test ligands and with nicotine and carbachol. Nicotine was used as an internal control for compounds with agonist activity and carbachol was used as an internal control for compounds with very weak agonistic activity (K(d) > 4700 nM for alpha3beta4). The displacement of [(3)H] EB by each of the test compounds and internal controls was calculated and expressed as Deltaml. Functional studies were then conducted using a stably transfected cell line that expresses the alpha3beta4 nAChR and EC(50) values were determined for the test compounds and the internal controls. A comparison of the Deltaml and EC(50) values indicated that 9/11 compounds had been correctly identified as agonists or non-agonists of the alpha3beta4 nAChR. A similar comparison could not be made for the alpha4beta2 nAChR, since the intact cell line was not available for testing. The results of the study suggest that the immobilized nAChR columns can be used for the rapid on-line screening of compounds for their relative affinities for the immobilized receptor and as an initial determination of qualitative functional activities.

Anabasine↗

PubMed2004

G-protein-coupled receptor chromatographic stationary phases. 2. Ligand-induced conformational mobility in an immobilized beta2-adrenergic receptor.

Membranes from a HEK-293 cell line expressing the beta(2)-adrenergic receptor (beta(2)-AR) have been immobilized on an artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in on-line chromatographic system. Frontal displacement affinity chromatography was used to determine the dissociation constants (K(d)) of CGP 12177A (552.6 nM) and (S)-propranolol (84.3 nM). Zonal displacement chromatography using CGP 12177A as the marker and racemic mixtures of the antagonists nadolol and propranolol demonstrated that the immobilized beta(2)-AR retained its ability to specifically bind these compounds. Similar experiments with (R)- and (S)-propranolol demonstrated that the immobilized receptor retained its enantioselectivity as (S)-propranolol displaced the CGP 12177 marker to a great extent that the (R)-enantiomer. The addition of the agonist butoxamine to the mobile phase increased the retention of the CGP-12177A as did the addition of the agonist fenoterol. These results indicate that the immobilized beta(2)-AR retained its ability to undergo ligand-induced conformational changes. The data from this study suggest that the immobilized beta(2)-AR can be used to screen for ligand binding interactions in both the resting and active states of the receptor.

Adrenergic beta-Antagonists↗

PubMed2004

Interaction of noncompetitive inhibitors with an immobilized alpha3beta4 nicotinic acetylcholine receptor investigated by affinity chromatography, quantitative-structure activity relationship analysis, and molecular docking.

A large number of drug substances act as noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor (nAChR) by blocking the ion flux through the channel. An affinity chromatography technique has been developed for investigating the interactions between NCIs and the alpha3beta4 subtype of neuronal nAChR. The data obtained from the chromatographic study were used to construct QSAR models of the NCI-nAChR binding with both electronic and steric parameters observed as important descriptors. A molecular model of the transmembrane domain of the alpha3beta4 subtype of nAChR was constructed and used to simulate the docking of a series of NCIs. A key aspect of the model was the discovery of the cleft produced by the incorporation of the bulky phenylalanine moiety into the nonpolar section of the lumen by the beta4 subunit. Quantitatively, the results of docking simulations modeled the experimental affinity data better than QSAR results. The computational approach, combined with the modeling of NCI-nAChR interaction by affinity chromatography, can be used to predict possible toxicities and adverse interactions.

Amino Acid Sequence↗

PubMed2004

Immobilized enzyme reactors based upon the flavoenzymes monoamine oxidase A and B.

Monoamine oxidase (MAO) catalyzes the oxidative deamination of amines. The enzyme exists in two forms, MAO-A and MAO-B, which differ in substrate specificity and sensitivity to various inhibitors. Membrane fractions containing either expressed MAO-A or MAO-B have been non-covalently immobilized in the hydrophobic interface of an immobilized artificial membrane (IAM) liquid chromatographic stationary phase. The MAO-containing stationary phases were packed into glass columns to create on-line immobilized enzyme reactors (IMERs) that retained the enzymatic activity of the MAO. The resulting MAO-IMERs were coupled through a switching valve to analytical high performance liquid chromatographic columns. The multi-dimensional chromatographic system was used to characterize the MAO-A (MAO-A-IMER) and MAO-B (MAO-B-IMER) forms of the enzyme including the enzyme kinetic constants associated with enzyme/substrate and enzyme/inhibitor interactions as well as the determination of IC(50) values. The results of the study demonstrate that the MAO-A-IMER and the MAO-B-IMER can be used for the on-line screening of substances for MAO-A and MAO-B substrate/inhibitor properties.

Bioreactors↗