Screening of aromatase inhibitors in traditional Chinese medicines by electrophoretically mediated microanalysis in a partially filled capillary

An electrophoretically mediated microanalysis method with partial filling technique was developed for screening aromatase inhibitors in traditional Chinese medicine. The in‐capillary enzymatic reaction was performed in 20 mM sodium phosphate buffer (pH 7.4), and sodium phosphate buffer (20 mM, pH 8.0) was used as a background electrolyte. A long plug of coenzyme reduced β‐nicotinamide adenine dinucleotide 2′‐phosphate hydrate dissolved in the reaction buffer was hydrodynamically injected into a fused silica capillary followed by the injection of reaction buffer, enzyme, and substrate solution. The reaction was initiated with a voltage of 5 kV applied to the capillary for 40 s. The voltage was turned off for 20 min to increase the product amount and again turned on at a constant voltage of 20 kV to separate all the components. Direct detection was performed at 260 nm. The enzyme activity was directly assayed by measuring the peak area of the produced β‐nicotinamide adenine dinucleotide phosphate and the decreased peak area indicated the aromatase inhibition. Using the Lineweaver–Burk equation, the Michaelis–Menten constant was calculated to be 50 ± 4.5 nM. The method was applied to the screening of aromatase inhibitors from 15 natural products. Seven compounds were found to have potent AR inhibitory activity.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

A rapid and sensitive CE method with field-enhanced sample injection and in-capillary derivatization for selenomethionine metabolism catalyzed by flavin-containing monooxygenases

A rapid and sensitive electrophoretically mediated microanalysis method with field-enhanced sample injection (FESI) for in-capillary derivatization was developed to determine selenomethionine (SeMet) and selenomethionine selenoxide (SeOMet). Phthalic anhydride (PA) was selected as the derivatization reagent due to the fast reaction at room temperature and the stability of derivatives. The in-capillary derivatization was accomplished by electrophoretically mixing PA and sample plugs. PA reagent was introduced hydrodynamically into the capillary, whereas the sample solution was injected electrokinetically, thus allowing a selective preconcentration of the analytes by FESI. For FESI, the optimum sample solvent was 2 mM borate solution. The borate buffer was suitable for both in-capillary derivatization and separation of the derivatives. The combination of electrophoretically mediated microanalysis with FESI for in-capillary derivatization was successfully achieved with about 800-fold concentration sensitivity enhancement compared to direct CE-UV detection in the same setup. The present method is miniaturized and fully automated, which ensures the on-line derivatization, stacking, separation and detection in 10 min. Finally, the developed method was successfully applied to measure enzyme activities by analyzing the reaction mixtures of SeMet with human flavin-containing monooxygenases (FMO). The results showed that both FMO1 and FMO3, but not FMO5 could catalyze the Se-oxygenation of SeMet.     

Study of substrate inhibition by electrophoretically mediated microanalysis in partially filled capillary

Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. In the case of haloalkane dehalogenase, a model enzyme selected for this study, the enzymatic reaction was performed in 20 mM glycine buffer (pH 8.6) whereas 20 mM beta-alanine-hydrochloric acid buffer (pH 3.5) was used as a background electrolyte in combination with direct detection at 200 nm. The whole study was performed on poorly soluble brominated substrate--1,2-dibromoethane. As a result it was first necessary to find the compromise between the concentrations of the enzyme and the substrate preserving both the adequate sensitivity of the assay and at the same time the attainable substrate solubility. By means of the developed EMMA methodology we were able to determine the Michaelis constant (K(M)) as well as the substrate inhibition constant (K(SI)). The value of K(M) and K(SI) obtained were 7.7+/-2.5 mM and 1.1+/-0.4 mM, respectively. Observation of the substrate inhibition of haloalkane dehalogenase by 1,2-dibromoethane is in accordance with previous literature data.     

Electrophoretically mediated microanalysis with partial filling technique and indirect or direct detection as a tool for inhibition studies of enzymatic reaction

The inhibition of the model enzyme, haloalkane dehalogenase from Sphingomonas paucimobilis, was investigated by a combination of electrophoretically mediated microanalysis with a partial filling technique, followed by indirect or direct detection. In this setup, part of the capillary is filled with a buffer suitable for the enzymatic reaction (20 mM glycine buffer, pH 8.6) whereas the rest of the capillary is filled with the background electrolyte optimal for separation of substrates and products. Two different background electrolytes and corresponding detection approaches were used to show the versatility of the developed method. The inhibition effect of 1,2-dichloroethane on the dehalogenation of brominated substrate 1-bromobutane was studied by means of 10 mM chromate - 0.1 mM cetyltrimethylammonium bromide (pH 9.2) in combination with indirect detection or 20 mM beta-alanine - hydrochloric acid (pH 3.5) in combination with direct detection. The method was used to estimate the inhibition constant K(I) (0.44 mM by indirect detection and 0.63 mM by of direct detection) and to determine the inhibition type. Compared to spectrophotometric and other discontinuous assays, the method is rapid, can be automated, and requires only small amount of reagents that is especially important in the case of enzymes and inhibitors.     

Electrophoretically mediated microanalysis of a nicotinamide adenine dinucleotide-dependent enzyme and its facile multiplexing using an active pixel sensor UV detector

An electrophoretically mediated microanalysis (EMMA) method has been developed for yeast alcohol dehydrogenase and quantification of reactant and product cofactors, NAD and NADH. The enzyme substrate ethanol (1% (v/v)) was added to the buffer (50 mM borate, pH 8.8). Results are presented for parallel capillary electrophoresis with a novel miniature UV area detector, with an active pixel sensor imaging an array of two or six parallel capillaries connected via a manifold to a single output capillary in a commercial CE instrument, allowing conversions with five different yeast alcohol dehydrogenase concentrations to be quantified in a single experiment.     

Development of a CD‐MEKC method for investigating the metabolism of tamoxifen by flavin‐containing monooxygenases and the inhibitory effects of methimazole,nicotine and DMXAA

A selective and low‐cost CD‐MEKC method under acidic conditions was developed for investigating the N‐oxygenation of tamoxifen (TAM) by flavin‐containing monooxygenases (FMOs). The inhibitory effects of methimazole (MMI), nicotine and 5,6‐dimethylxanthenone‐4‐acetic acid (DMXAA) on the given FMO reaction were also evaluated; 100 mM phosphate buffer (pH 8.6) was used for performing the enzymatic reaction and the separation of TAM and its metabolite tamoxifen N‐oxide (TNO) was obtained with a BGE consisting of 100 mM phosphoric acid solution adjusted to pH 2.5 with triethanolamine containing 50 mM sodium taurodeoxycholate, 20 mM carboxymethyl β‐CD and 20% ACN. The proposed method was applied for the kinetics study of FMO1 using TAM as a substrate probe. A Michaelis–Menten constant (K_m) of 164.1 μM was estimated from the corrected peak area of the product, TNO. The calculated value of the maximum reaction velocity (V_max) was 3.61 μmol/min/μmol FMO1; 50% inhibitory concentration and inhibition constant (K_i) of MMI, the most common alternate substrate FMO inhibitor, were evaluated and the inhibitory effects of two other important FMO substrates, nicotine and DMXAA, a novel anti‐tumour agent, were investigated.     

Interaction of cyclophilin and cyclosporins monitored by affinity capillary electrophoresis.

The affinity capillary electrophoretic separation of the complex of the enzyme cyclophilin (Cyp) with the immunosuppressive drug cyclosporin A (CsA) from uncomplexed Cyp and CsA in phosphate buffer (pH 8) under non-denaturing conditions by equilibrium-mixture analysis is reported. Using a new approach combining mobility-shift analysis and electrophoretically mediated microanalysis the binding constant of rhCyp18 to CsA and derivatives was estimated.     

Study of enzymatic reaction by electrophoretically mediated microanalysis in a partially filled capillary with indirect or direct detection

Electrophoretically mediated microanalysis (EMMA), in combination with a partial filling technique and indirect or direct detection, is described for the study of enzymes reacting with the high mobility inorganic or organic anions as substrates or products. Part of the capillary is filled with a buffer optimized for the enzymatic reaction, the rest of the capillary with the background electrolyte being optimal for the separation of substrates and products. With haloalkane dehalogenase, chosen as a model enzyme, the enzymatic reaction was performed in a 20 mM glycine buffer (pH 8.6). Because of the wide substrate specificity of this enzyme, utilizing chlorinated as well as brominated substrates and producing either nonabsorbing chloride or absorbing bromide ions, two different background electrolytes and detection approaches were adopted. A 10 mM chromate-0.1 mM cetyltrimethylammonium bromide background electrolyte (pH 9.2) was used in combination with indirect detection and 20 mM beta-alanine-hydrochloric acid (pH 3.5) in combination with direct detection. The Michaelis constant (K(m)) of haloalkane dehalogenase for 1-bromobutane was determined. The K(m) values 0.59 mM estimated by means of indirect detection method and 0.17 mM by means of direct detection method were comparable with the value 0.13 mM estimated previously by gas chromatography.     

Kinetic study of gamma-glutamyltransferase activity by electrophoretically mediated microanalysis combined with micellar electrokinetic capillary chromatography

The use of capillary electrophoresis for the determination of gamma-glutamyltransferase (GGT) activity with gamma-glutamyl-p-nitroanilide (Glu-p-NA) as a substrate was investigated. The reaction velocity was quantified spectrophotometrically by the corrected peak area of the product p-nitroaniline (pNA) at 380 nm. Micelles composed of sodium deoxycholic acid were used in the background electrolyte in order to obtain a baseline separation between the substrate and the product. The presence of the micelles did not influence the enzymatic reaction. The electrophoretic system was used, not only for the separation and quantitation of the different reaction compounds but also for the in-capillary mixing of the enzyme and substrate plugs. This methodology is known as electrophoretically mediated microanalysis (EMMA). With the developed in-capillary activity assay an average Michaelis constant (K(M)) for GGT was calculated to be 2.09 mM (RSD = 7.3%, n = 3), a value consistent with previously reported values.     

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

CE was used for the first time to study the two‐substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine‐5′‐triphosphate were in‐capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine‐5′‐triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero‐potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine‐5′‐diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose‐1,6‐bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in‐capillary mixed using an electrophoretically mediated microanalysis approach.     

Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes

An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of l-methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-l-methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc-l-methionine sulfoxide and the product Fmoc-l-methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-l-methionine-(S)-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc-l-methionine-(R)-sulfoxide by human methionine sulfoxide reductase B. The Michaelis–Menten constant, K _m, and the maximum velocity, v _max, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-(R)-sulfoxide is a substrate of human methionine sulfoxide reductase B.

Study of atypical kinetic behaviour of cytochrome P450 2C9 isoform with diclofenac at low substrate concentrations by sweeping‐MEKC combination

In view of the fact that several studies have shown that diclofenac hydroxylation by cytochrome P450 2C9 deviated from Michaelis–Menten kinetics at low substrate concentrations, sweeping combined with MEKC was applied for the kinetic study of this pharmacologically important reaction. A 50 μm fused silica capillary (56 cm effective length) was used to carry out all separations. 70 mM SDS in 20 mM phosphate 20 mM tetraborate buffer, pH 8.6, was used as the BGE. Injection was accomplished by the application of 50 mbar (5 kPa) pressure to the sample vial for 52 s. Separation was performed at 22 kV (positive polarity), with a capillary temperature of 25°C and detection at 200 nm. The higher sensitivity of the sweeping‐MEKC combination compared with the simple MEKC method enabled this reaction to be fitted to a Hill kinetic model and confirmed the findings of other authors. A Michaelis constant of 2.91±0.10 μM, maximum reaction velocity of 9.16±0.16 nmol/min/nmol and Hill coefficient of 1.66±0.08 were determined. This value of Hill coefficient confirms the presence of a positive cooperativity at low diclofenac concentrations and supports the hypothesis of two substrates binding at or near the active site.     

Determination of nitrate and nitrite in rat brain perfusates by capillary electrophoresis

A fast and simple method for the direct, simultaneous detection of nitrite (NO(2) (-)) and nitrate (NO(3) (-)) in rat striatum has been developed using a capillary electrophoresis separation of low-flow push-pull perfusion samples. The method was optimized primarily for nitrite because nitrite is more important physiologically and is found at lower levels than nitrate. We obtained a complete separation of NO(2) (-) and NO(3) (-) in rat striatum within 1.5 min. Optimal CE separations were achieved with 20 mM phosphate, 2 mM cetyltrimethylammonium chloride (CTAC) buffer at pH 3.5. The samples were injected electrokinetically for 2 s into a 40 cm x 75 microm ID fused-silica capillary. The separation voltage was 10 kV (negative polarity), and the injection voltage was 16 kV (negative polarity). UV detection was performed at 214 nm. The limits of detection obtained at a signal-to-noise ratio (S/N) of 3 for nitrite and nitrate were 0.96 and 2.86 microM. This is one of the fastest separations of nitrite and nitrate of a biological sample ever reported. Interference produced by the high physiological level of chloride is successfully minimized by use of CTAC in the run buffer.     

Experimental design optimization of the capillary electrophoresis separation of leucine enkephalin and its immune complex

To optimize the capillary electrophoretic separation conditions for leucine enkephalin (LE) and the immune complex of the LE and anti-LE reaction, an analysis using a three-level, three-factorial Box-Behnken design was performed. Three separation parameters, buffer pH (X(1)), buffer concentration (X(2)), and applied voltage (X(3)), were chosen to observe the effect on separation responses. The responses were theoretical plate number, migration time of the LE peak, and resolution between the peaks. The optimum conditions and process validation were determined using statistical regression analysis and surface plot diagrams. The capillary electrophoresis optimum separation conditions were established to be 75 mM phosphate buffer at pH 7.00 with an applied separation voltage of 15 kV. By using the analysis technique, the prediction of responses was satisfactory and process verification yielded values within the +/-5% range of the predicted efficiency.     

Determination of synthesized alpha-vitamin E by micellar electrokinetic chromatography

We developed a micellar electrokinetic chromatography method (MEKC) for the direct determination of the content of synthesized alpha-vitamin E. It was found that under the optimum separation conditions 7 mM borate + 14 mM phosphate + 15 mM sodium dodecyl sulfate (SDS) + 10 mM sodium cholate (NaCh) + 8% acetonitrile (pH 9.2) with UV detection wavelength at 214 nm, 16 kV constant voltage, and 26 degrees C constant temperature, alpha-vitamin E and its isomers can be baseline separated and alpha-vitamin E was quantitatively analyzed. In addition, the sample recovery, the limit of detection and the repeatability of the method were investigated. The influence of various parameters on the separation such as SDS concentration, NaCh concentration, buffer pH and acetonitrile percentage were also discussed.     

Kinetic study of cytochrome P450 3A4 activity on warfarin by capillary electrophoresis with fluorescence detection

The use of capillary electrophoresis (CE) for the determination of cytochrome P450 3A4 (CYP3A4) activity with R-warfarin as a substrate was investigated. CYP3A4 activity was determined by the quantitation of the product, 10-hydroxywarfarin, based on separation by CE. The separation conditions were as follows: capillary, 80.5 cm (75 microm i.d., 60 cm effective length); 50 mM sodium phosphate buffer (pH 6.5); 23 kV (90 microA) applied voltage; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 37 degrees C. With the developed CYP3A4 activity assay and the Lineweaver-Burk equation, the Michaelis-Menten parameters Km and Vmax for formation of 10-hydroxywarfarin from R-warfarin in the presence of CYP3A4 were calculated to be 166 +/- 12 microM and 713 +/- 14 pmol/min/nmol (or 91.4 pmol/min/mg) CYP3A4, respectively.     

High-performance capillary electrophoresis of proteins from the fluid lining of the lungs of rats exposed to perfluoroisobutylene.

Measurements of the biochemical constituents in the fluid lining of the lung can be used for diagnosing and assessing lung disorders. To facilitate such measurements, a high-performance capillary electrophoresis (HPCE) method has been developed by which the proteins in lung fluid can be analyzed. The lung fluid was obtained by a bronchoalveolar lavage procedure using 48 ml of physiological saline to wash out the lung fluid of rats. The proteins were precipitated from the fluid with 10 volumes of acetone and concentrated by dissolution in 2 ml of water containing 0.2% of trifluoroacetic acid. Aliquots of these samples (5 microliters) were then injected into a Bio-Rad HPE-100 capillary electrophoresis instrument fitted with a 50 cm x 50 microns I.D. coated capillary filled with 0.1 M phosphate buffer (pH 2.5). With phosphate buffer in the outlet electrode chamber (cathode) and water in the inlet electrode chamber (anode), the proteins were loaded into the capillary electrophoretically for 10 s at 10 kV constant voltage. The inlet electrode chamber was then filled with phosphate buffer and HPCE was performed at 8 kV constant voltage. Six major protein fractions were resolved in 35 min, and were detected by UV absorption at 200 nm. The procedure was used to compare the lung fluid proteins of normal untreated rats with those of rats exposed by inhalation to perfluoroisobutylene (PFIB) at a concentration of 100 mg/m3. It was found that PFIB induced pulmonary edema involving a translocation of blood compartment proteins into the lung's alveolar compartment. Comparison of the HPCE fractions with similar fractions obtained by high-performance liquid chromatography confirmed albumin, transferrin and IgG as three major proteins translocated into the alveolar space after PFIB exposure.     

The use of a highly sulfated cyclodextrin for the simultaneous chiral separation of amphetamine-type stimulants by capillary electrophoresis

We investigated the simultaneous chiral separation of nine amphetamine type stimulants (dl-norephedrine, dl-norpseudoephedrine, dl-ephedrine, dl-pseudoephedrine, dl-amphetamine, dl-methamphetamine, dl-methylenedioxyamphetamine (MDA), dl-methylenedioxymethamphetamine (MDMA), and dl-methylenedioxyethylamphetamine (MDEA)) by capillary electrophoresis using highly sulfated gamma-cyclodextrin (SU(XIII)-gamma-CD) as a chiral selector. Three different approaches using SU(XIII)-gamma-CD with 50 mM phosphate background electrolyte were designed; (I) high CD concentration (10 mM SU(XIII)-gamma-CD) at neutral pH (pH 7.0) in the normal polarity mode, (II) low CD concentration (1.0 mM) at low pH (pH 2.6) in the normal polarity mode and (III) high CD concentration at low pH (pH 2.6) in the reversed-polarity mode. In mode (II), the effects of adding three neutral CDs (beta-CD, dimethyl-beta-CD and hydroxypropyl-beta-CD) were also investigated. The best separation was obtained after optimizing mode (III) as follows: run buffer of 10 mM SU(XIII)-gamma-CD with 50 mM phosphate background electrolyte at pH 2.6, applied voltage of -12 kV and capillary temperature of 15 degrees C.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.