[The new technologies for the analytical examination of the embryonic metabolome and its prospects]. / Nové technologie a perspektivy analýzymetabolomu embrya.

OBJECTIVE: To review current technologies for the analytical examination of the embryonic metabolome and its perspectives. DESIGN: Review article. SETTING: Department of Gynecology and Obstetrics, Faculty of Medicine, Masaryk University, and University Hospital, Brno, Department of Biochemistry, Faculty of Science and CEITEC, Masaryk University, Brno. METHODS AND RESULTS: Nowadays, very sensitive analytical technologies are available. They enable exact measurement of various molecules - products of embryo metabolism during first days of cultivation. The capillary electrophoresis is one of promising method. Recent studies analysed metabolic differences between embryos that result in a pregnancy and those that do not. Amino acid levels, glucose or pyruvate in the embryo culture media were analysed most frequently. However, results of these studies are ambiguous. CONCLUSIONS: The capillary electrophoresis with contactless conductivity detection may provide a useful data of the embryonic metabolome. A comprehensive analysis of the used culture medium may represent a valuable adjunct to morphological criteria for enhanced rates of implantation and delivery.

Application of capillary electrochromatography using macroporous polyacrylamide columns for the analysis of lignans from seeds of Schisandra chinensis.

Capillary electrochromatography (CEC) using polymer-based monolithic stationary phase has been developed as a promising method for the determination of lignans of Schisandra chinensis. The columns were prepared by in situ copolymerisation of acrylamide, N,N'-methylenebisacrylamide, vinylsulfonic acid and lauryl acrylate in presence of poly(ethylene glycol) as a porogenic agent. The columns [33 cm (24.5 cm effective length) x 75 microm I.D.] were successfully used to analyse and quantify the major lignans in extract of the seeds of Schisandra chinensis. Good separations were achieved in less than 35 min. The calibration graphs were linear in the range 0.025-1.0 mg/ml of given lignan with correlation coefficients between 0.9951 and 0.9996. The inter-day reproducibility of the peak area were below 3.9% and the inter-day reproducibility of the migration time were below 4.2%. The results of quantitative CEC analyses were compared with those obtained by reversed-phase HPLC, the levels of schizandrin, gomisin A, gomisin N and wuweizisu C determined by CEC were in a good agreement with those determined by HPLC.

Analysis of thiocyanate in biological fluids by capillary zone electrophoresis.

A new sensitive and simple method has been developed for the determination of thiocyanate in human serum, urine and saliva. The determinations were performed in a fused-silica capillary [64.5 cm (56 cm effective length) x 75 microm] using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity), temperature of capillary 25 degrees C and direct detection at 200 nm. Serum samples were 10-times diluted with deionised water and deproteinised with acetonitrile in the ratio 1:2. Urine and saliva samples need only 20-fold dilution with deionised water. The proposed method was successfully applied to the determination of thiocyanate in various human serum, saliva and urine samples.

Application of capillary zone electrophoresis to study the properties of rhodanese from Acidithiobacillus ferrooxidans.

A new capillary zone electrophoretic method was applied to the assay of enzymic activity of rhodanese from Acidithiobacillus ferroxidans. The enzyme activity determined by capillary zone electrophoresis was compared with that determined by discontinuous spectrophotometry, the values obtained being in good agreement. The method was also used to evaluate Michaelis constants of cyanide and thiocyanate as substrates; a new approach was developed to solve the problem with variable ionic strength of the samples. The pH and temperature optima for the enzyme were also determined.

Specific thiol determination by micellar electrokinetic chromatography and on-column detection reaction with 2,2'-dipyridyldisulfide.

A new method for specific determination of glutathione using micellar electrokinetic chromatography and on-column reaction with 2,2'-dipyridyldisulfide is described. 2,2'-Dipyridyldisulfide and a sample of glutathione are injected consecutively into the capillary as two discrete plugs separated with a short plug of background electrolyte. Due to the differences in the mobilities of the 2,2'-dipyridyldisulfide and glutathione, on-column mixing and reaction occur. Glutathione is in this reaction quantitatively transformed into a mixed disulfide concomitantly with formation of an equimolar amount of the 2-thiopyridone which is further separated by micellar electrokinetic chromatography and determined spectrophotometrically at 343 nm. The concentration of glutathione is thus estimated indirectly from the result of 2-thiopyridone determination.

Determination of haloalkane dehalogenase activity by capillary zone electrophoresis.

A new sensitive method has been developed for the determination of haloalkane dehalogenase activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of product - bromide or chloride ions - was monitored by sequential capillary zone electrophoresis runs. The determinations were performed in a 75 microm fused-silica capillary using 5 mM chromate, 0.5 mM tetradecyltrimethylammonium bromide (pH 8.4) as a background electrolyte, separation voltage 15 kV (negative polarity) and indirect detection at sample wavelength 315 nm, reference wavelength 375 nm for brominated and chlorinated substrates, respectively 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, separation voltage 18 kV (negative polarity) and direct detection at 200 nm for brominated substrates. The temperature of capillary was in both cases 25 degrees C. The method is rapid, can be automated, and requires only small amount of enzyme preparation and substrate.

Determination of pyrroloquinoline quinone by capillary zone electrophoresis.

A new method for the determination of pyrroloquinoline quinone by capillary zone electrophoresis has been developed. Separation conditions have been optimised with the respect to different parameters including pH and ionic strength of the background electrolyte, separation voltage and temperature of the capillary. A buffer consisting of 50 mM beta-alanine-HCl pH 3.0 was found to be the most suitable electrolyte for this separation. An applied voltage of 25 kV (negative polarity) and a temperature of 25 degrees C gave the best analysis of pyrroloquinoline quinone. The linear detection range for concentration versus peak area for the assay is from 5 to 500 microM (correlation coefficient 0.9998) with a detection limit of 0.1-0.2 microM. The inter-day reproducibility of the peak area was 2.5% and the inter-day reproducibility of the migration time was below 0.18%.

Determination of rhodanese enzyme activity by capillary zone electrophoresis.

A new sensitive method has been developed for the determination of rhodanese activity. The enzymatic reactions were carried out directly in thermostatted autosampler vials and the formation of SCN- was monitored by sequential capillary zone electrophoretic runs. The determinations were performed in a 75-micron fused-silica capillary using 0.1 M beta-alanine-HCl (pH 3.50) as a background electrolyte, a separation voltage of 18 kV (negative polarity), a capillary temperature of 25 degrees C and direct detection at 200 nm. Short-end injection or long-end injection procedures were used for sample application. The method is rapid, able to be automated and requires only small amounts of sample and substrates, which is especially important in the case of highly toxic cyanide. The developed capillary electrophoretic method also has great potential for thiocyanate determinations in other applications.

Use of thiopropyl sepharose for the synthesis of an adsorbent for the affinity chromatography of glutathione S-transferase.

Thiopropyl Sepharose 6B in the 2-thiopyridyl-activated form was used for the reversible immobilisation of reduced glutathione (GSH). The resulting affinity matrix was successfully tested as a sorbent for the partial purification of glutathione S-transferase (GST) from pig kidney. The specific elution of the enzyme was performed with 10 mM GSH in Tris-HCl buffer (pH 7.8), non-specific elution with 20 mM dithiotreitol (DTT) in the same buffer.

Detection of quinoproteins after electrophoresis in the presence of urea or SDS.

A method for the direct detection of quinoproteins in gels after electrophoresis in the presence of urea or SDS has been developed. The conditions of sample preparation and detection were optimized to achieve maximum sensitivity. The method is suitable for the detection of quinoproteins in complex protein mixtures as well as for the characterization of certain enzyme preparations.

Purification and properties of malate dehydrogenase from Paracoccus denitrificans.

Affinity chromatography on immobilized Cibacron Blue (Matrex Gel Blue A) gel permeation chromatography on UltroPac TSK G 3000 SWG column and ion-exchange chromatography on "Mono Q" column were used to purify the malate dehydrogenase (MDH) from P. denitrificans to electrophoretic homogeneity. The last two purification steps were performed in FPLC system. The enzyme having a specific activity of about 2300 nkat/mg protein was obtained with an approximate 70% yield. MDH is a dimer with a molecular mass of 80,000 +/- 10,000 and an isoelectric point of 4.85 +/- 0.05. Absorption, fluorescence and CD-spectra were also measured and basic kinetic parameters were obtained for the homogeneous enzyme. The present paper also suggests the possibility of using the prepared enzyme for the determination of aspartate transferase (AST) in blood serum.

Improved chromatographic purification of pea seedlings diamine oxidase.

An improved and simplified purification procedure has been developed for the isolation of diamine oxidase from pea seedlings (DAO EC 1.4.3.6). It involves ammonium sulphate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography and size-exclusion chromatography. The homogeneity of the final enzyme preparations and molecular weight were determined by size-exclusion chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS PAGE). The isoelectric point of 7.35 +/- 0.05 was determined by chromatofocusing and by polyacrylamide gel isoelectric focusing.

Pea (Pisum sativum) diamine oxidase contains pyrroloquinoline quinone as a cofactor.

Diamine oxidase was prepared from pea (Pisum sativum) seedlings by a new purification procedure involving two h.p.l.c. steps. We studied the optical and electrochemical properties of the homogeneous enzyme and also analysed the hydrolysed protein by several methods. The data presented here suggest that the carbonyl cofactor of diamine oxidase is firmly bound pyrroloquinoline quinone.