Capillary zone electrophoresis for determination of carbohydrate-deficient transferrin in human serum

Carbohydrate-deficient transferrin (CDT) is the most specific marker for diagnosis of chronic excessive alcohol consumption and includes the serum transferrin (Tf) isoforms with two or less sialic acid residues (di-, mono-, and asialo-Tf). To monitor serum CDT, we developed a capillary zone electrophoresis (CZE) method based on the dynamic capillary coating with diethylenetriamine (DETA). The separation was performed in a bare fused-silica capillary (50 microm ID, 57 cm in length), applying a voltage of 25 kV and a temperature of 40 degrees C. Using a 100 mmol/L borate buffer, pH 8.4 with 3 mmol/L DETA, the Tf isoforms (asialo- to pentasialo-Tf) were resolved within 16 min. Enzymatic cleavage of sialic acid residues with neuraminidase and immunosubtraction were used to identify CDT isoforms. The relative amount of CDT expressed as area % of disialo-Tf isoform related to the area of tetrasialo-Tf in 50 healthy donors (24 males and 26 females; aged 25-50 years) was 3.15 +/- 0.76% (mean +/- SD). The comparison between CDT values obtained by this CZE procedure and the "Axis-Shield %CDT" kit gave r = 0.644, p < 0.001 (n = 290). This easy to use and inexpensive CZE procedure could be an ideal tool to investigate CDT proteins for clinical or forensic purposes.     

Capillary zone electrophoresis with a dynamic double coating for analysis of carbohydrate-deficient transferrin in human serum: impact of resolution between disialo- and trisialotransferrin on reference limits

Capillary electrophoresis with a dynamic double coating formed by charged polymeric reagents represents a very effective tool for the separation of iron-saturated transferrin (Tf) isoforms and thus the determination of carbohydrate-deficient transferrin (CDT) in human serum. The resolution between di- and trisialo-Tf is dependent on the applied voltage and capillary temperature. With a 50 microm inside diameter (ID) capillary of about 60 cm total length mounted into the P/ACE MDQ, 28 kV and 40 degrees C, the resolution of the two Tf isoforms is shown to be between 1.0 and 1.4, whereas with reduced voltage and/or temperature, increased resolution at the expense of elongated run times is observed. Best data with complete resolution (Rs > or = 1.4) are obtained at 20 kV and 30 degrees C. For the determination of CDT in serum, incomplete separation of di- and trisialo-Tf is demonstrated to have an impact on the reference limits. Analysis of the sera of 54 healthy individuals with no or moderate alcohol consumption and using valley-to-valley peak integration, the upper (lower) reference limits for CDT in relation to total Tf at the two power levels are 1.33 (0.52) and 1.57 (0.81)%, respectively, representing intervals that are significantly different (P < 0.001). Furthermore, the reference intervals are shown to be strongly dependent on the peak integration approach used. Valley-to-valley peak integration should only be employed for conditions with complete resolution between disialo- and trisialo-Tf.     

Evaluation and optimization of capillary zone electrophoresis with different dynamic capillary coatings for the determination of carbohydrate-deficient transferrin in human serum

Serum transferrin (Tf) comprises several isoforms with up to two complex oligosaccharide chains containing zero to eight sialic acid residues and neutral sugars. The major glycoform, known as tetrasialo-Tf, contains four sialic acid residues and accounts for about 80% of whole Tf in human serum. Carbohydrate-deficient transferrin (CDT) encompasses isoforms that are deficient in carbohydrate chains and consequently in sialic acid residues (including asialo-, monosialo- and disialo-Tf) and is a well known marker for chronic alcohol abuse. Recently capillary zone electrophoresis (CZE) has been reported as a tool extremely effective for the simultaneous, individual, quantitative determination of CDT isoforms. Three CZE methods that feature different dynamic capillary coatings were evaluated and optimized for CDT determination in human serum of alcohol abusers and control subjects. CZE separation was performed in alkaline borate buffers after serum sample saturation with iron, electropherograms were detected at 200 nm, data were evaluated as % area of disialo-Tf in relation to tetrasialo-Tf and peak identification was accomplished via relative migration times to tetrasialo-Tf, immunosubtraction and enzymatic sequential cleavage of sialic acid residues. Dynamic capillary coatings with diaminobutane, spermine and a double coating produced by commercially available proprietary agents were investigated and found to be suitable for determination of CDT in human serum. For all three approaches, best results were obtained in 50 microm I.D. fused-silica capillaries of 50 cm effective length and a capillary cartridge temperature of 20-25 degrees C. Using 3 mM 1,4-diaminobutane or 0.02 mM spermine in a borate-based running buffer of pH 8.3 provided data of remarkable similarity with resolution of di-, tri-, tetra- and pentasialo-Tf within 15-18 min. With the double coating, asialo-Tf and Tf isoforms with two to six sialic acid residues were baseline separated. Compared to the two amine-based procedures, the run times were found to be somewhat shorter, the detector signals higher, the applied power level significantly lower and the reproducibility better.     

Capillary zone electrophoresis determination of carbohydrate-deficient transferrin using the new CEofix reagents under high-resolution conditions

Capillary zone electrophoresis (CZE) with a dynamic double coating based on the new CEofix reagents is shown to provide high-resolution separations of serum transferrin (Tf) isoforms, a prerequisite for the monitoring of unusual and complex Tf patterns, including those seen with genetic variants and disorders of glycosylation. A 50 microm I.D. fused-silica capillary of 60 cm total length, an applied voltage of 20 kV and a capillary temperature of 30 degrees C results in 15 min CZE runs of high assay precision and thus provides a robust approach for the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-Tf and disialo-Tf in relation to total Tf) in human serum. Except for selected samples of patients with severe liver diseases and sera with high levels of paraproteins, interference-free Tf patterns are detected. Compared with the use of the previous CEofix reagents for CDT under the same instrumental conditions, the resolution between disialo-Tf and trisialo-Tf is significantly higher (1.7 versus 1.4). The CDT levels of reference and patient sera are comparable, suggesting that the new assay can be applied for screening and confirmation analyses. The high-resolution CZE assay represents an attractive alternative to HPLC and can be regarded as a candidate of a reference method for CDT.     

Determination of carbohydrate-deficient transferrin in human serum by two capillary zone electrophoresis methods and a direct immunoassay: comparison of patient data

Data obtained with two CZE assays for determining carbohydrate-deficient transferrin (CDT) in human serum under routine conditions, the CAPILLARYS CDT and the high-resolution CEofix (HR-CEofix) CDT methods, are in agreement with patient sera that do not exhibit interferences, high trisialo-transferrin (Tf) levels or genetic variants. HR-CEofix CDT levels are somewhat higher compared to those obtained with the CAPILLARYS method and this bias corresponds to the difference of the upper reference values of the two assays. The lower resolution between disialo-Tf and trisialo-Tf observed in the CAPILLARYS system (mean: 1.24) compared to HR-CEofix (mean: 1.74) is believed to be the key for this difference. For critical sera with high trisialo-Tf levels, genetic variants, or certain interferences in the beta-region, the HR-CEofix approach is demonstrated to perform better than CAPILLARYS. However, the determination of CDT with the HR-CEofix method can also be hampered with interferences. Results with disialo-Tf values larger than 3% in the absence of asialo-Tf should be evaluated with immunosubtraction of Tf and possibly also confirmed with another CZE method or by HPLC. Furthermore, data gathered with the N Latex CDT direct immunonephelometric assay suggest that this assay can be used for screening purposes. To reduce the number of false negative results, CDT data above 2.0% should be confirmed using a separation method.     

Monitoring human serum transferrin by capillary zone electrophoresis with end-column amperometric detection

Capillary zone electrophoresis was employed for the determination of human serum transferrin using end-column amperometric detection with a carbon fiber microelectrode at a constant potential of 1.9 V vs. saturated calomel electrode (SCE). The optimum conditions of separation and detection are 7.5 x 10(-4) mol/L Tris-3.44 x 10(-4) mol/L HCl for the buffer solution, 20 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, respectively. The limit of detection is 6.7 x 10(-8) mol/L or 440 amol (S/N = 2). The relative standard deviations are 0.67% for the migration time and 1.5% for the electrophoretic peak current. The method was applied to the determination of transferrin in human serum. The recovery is between 93-104%.     

Capillary zone electrophoresis of proteins applying ionic liquids for dynamic coating and as background electrolyte component

The use of ionic liquids in capillary electrophoresis, either as coating material or as components of the background electrolyte needs systematic standardization to set up optimal conditions. Excellent separation of the proteins was achieved using 1-ethyl-3-methylimidazolium tetrafluoroborate ([emim][BF₄]) or 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF₄]) ionic liquids using the properly made ionic-liquid–water binary mixtures for the experiments. The binary mixture has a distinctly stable and well perceptible low pH, which depends on the concentration of the ionic liquid, and on the preparation time of the mixture. Optimal conditions for the electrophoretic separation were obtained upon a multivariate analysis of the experimental parameters (applied voltage, migration time, concentration, and type of the ionic liquid). The standardized condition provides a low electroendosmotic flow toward the anode, which, however, did not hinder the proteins to migrate toward the cathode. The migration of cytochrome c, lysozyme, myoglobin, trypsin, and apo-transferrin at a pH around 2, far below the isoelectric points of the proteins, showed RSD values of the migration times less than 7.5% and less than 6.5% when using [emim][BF₄] or [bmim][BF₄], respectively, either in run-to-run or day-to-day experiments. The determination of the extent of the EOF is not possible with the commonly used EOF markers, due to interaction with the ionic-liquid constituents. The interaction of the ionic liquids with the proteins influences the migration order in zone electrophoresis. This method has been applied successfully for the analyses of real biological samples such as proteins from egg whites and human tears.     

Role of chemical structure in stereoselective recognition of beta-blockers and H1-antihistamines by human serum transferrin in capillary zone electrophoresis

Studies on chiral resolution of beta-blocker and H1-antihistamine drugs by CZE using human serum transferrin are described. The drugs with different structures passed a pseudostationary protein zone in a coated capillary applying the partial filling method for the chiral separation. In this study we screened 15 compounds; most of them showed longer migration time, indicating an interaction with transferrin. Stereoselective interaction was observed only for five beta-blockers (celiprolol, talinolol, mepindolol, bopindolol, and oxprenolol) and for one H1-antihistamine (brompheniramine). The most important finding was that very small differences in the chemical structure of the drug resulted in significant changes in the stereoselective recognition. Resolution of mepindolol enantiomers was observed showing the essential role of one methyl group compared to pindolol, which is not resolved by transferrin. Bopindolol, also a derivative of pindolol having bigger difference in the structure, showed more experienced separation. The very slight difference between alprenolol and oxprenolol was also revealed with these methods, since only oxprenolol enantiomers, having an extra oxygen in the structure, are resolved. Determining the migration order of the eutomers and distomers (chlorpheniramine, brompheniramine) we can deduct conclusions about the role of serum proteins in the delivery of drugs within the body.     

Capillary electrophoresis frontal analysis for the study of flavonoid interactions with human serum albumin

Capillary electrophoresis based on the principles of frontal analysis (CE-FA) was used to characterize the binding of flavonoids to human serum albumin (HSA) at near-physiological conditions: 67 mM phosphate buffer (pH 7.4), temperature 36.5 °C. The studied flavonoids (flavone, rutin, quercitrin) displayed moderate affinities toward the human serum albumin with binding constants in the range 10³−10⁴ M⁻¹. The binding of the flavonoids to the protein noticeably depended on their lipophilicity and decreased in the case of glycosylation. The corresponding thermodynamic parameters characterized the acting forces between the HSA and flavonoids as mainly hydrophobic forces and electrostatic interactions. Based on the results of the displacement experiments, the binding of the flavonoids took place at site I of the HSA molecule. The results demonstrated by CE-FA were similar to those obtained by fluorescence spectroscopy. The developed method proved to be a reliable alternative to conventional methods, providing a lot of useful parameters for characterization of ligand–protein interactions.     

Determination of pKa values by capillary zone electrophoresis with a dynamic coating procedure

CZE allows to measure the acidic dissociation constant (pKa) of many drug substances. However, determining the EOF intensity may be time-consuming, especially at a low pH. In order to overcome this drawback, a dynamic coating procedure of the capillary was carried out to increase microEOF, and thus to reduce the analysis time. In addition, this coating procedure enhanced migration time stability. The effective mobilities of 15 compounds were measured at different pH, producing pK'a values dependent on BGE ionic strength. The latter values were corrected with the activity coefficient to obtain a "true" pKa value. The 15 investigated compounds were (i) five acids: namely, salicylic acid, benzoic acid, ketoprofen, phenobarbital, and phenol, (ii) four bases: lidocaine, propafenone, propranolol, and quinine, (iii), five ampholytes: sulfanilamide, sulfabenzamide, sulfadimethoxine, sulfadoxine, and sulfisoxazole, and (iv) one zwitterion: cetirizine. The range of determined pKa values was between 1.2 and 11.2, and close to the pKa values available from the literature.     

Transferrin immunoextraction for determination of carbohydrate‐deficient transferrin in human serum by capillary zone electrophoresis

CZE‐based assays for carbohydrate‐deficient transferrin (CDT) in which serum is mixed with an Fe(III) ion‐containing solution prior to analysis are effective approaches for the determination of CDT in patient samples. Sera of patients with progressed diseases, however, are prone to interferences comigrating with transferrin (Tf) that prevent the proper determination of CDT by CZE in these samples. The need of a simple and economic approach to immunoextract Tf from human serum prompted us to investigate the use of a laboratory‐made anti‐Tf spin column containing polyclonal rabbit anti‐human Tf antibodies linked to Sepharose 4 Fast Flow beads. This article reports extraction column manufacturing and column characterization with sera having normal and elevated CDT levels. The developed procedure was applied to a number of relevant hepatology and dialysis patient samples and could thereby be shown to represent an effective method for extraction and concentration of all Tf isoforms. Furthermore, lipemic sera were delipidated using a mixture of diisopropyl ether and butanol prior to immunoextraction. CDT could unambiguously be determined in all pretreated samples.     

Mapping of stereoselective recognition sites on human serum transferrin by capillary electrophoresis and molecular modelling

Stereoselective recognition of chiral compounds can be used for mapping of surface interaction sites on proteins. Iron-free human serum transferrin is a suitable chiral selector in capillary electrophoresis used in native form in solution. Separation of optical isomers of tryptophan-methylester, tryptophan-ethylester and tryptophan-butylester and various drugs were studied in capillary zone electrophoresis applying a distinct transferrin zone prior to sample injection. Changes in the electrophoretic patterns (i.e., in the migration properties) of the molecules reflected the possible interactions with the protein. The tryptophan derivatives and eight drugs possessed stereoselective interactions, seven drugs showed interactions without appreciable chiral separation, and the others did not present any direct complexation with the protein molecules. Molecular modelling was performed to characterize the binding areas at the iron binding site of iron-free transferrin. The docking of tryptophan derivatives on transferrin showed that the R-enantiomers possess a stronger complexation with transferrin, whereas the S-enantiomers are bound by weaker interactions, which is in excellent agreement with the capillary electrophoresis results, where the R-enantiomers were always retarded stronger by transferrin. A ranking of drugs by the lipo score parameter of the docking shows an accordance with the stereoselective interactions by the protein.     

Improved capillary electrophoresis method for the determination of carbohydrate-deficient transferrin in patient sera

Capillary zone electrophoresis (CZE) with a dynamic double coating formed by charged polymeric reagents represents an effective tool for the separation of iron-saturated transferrin (Tf) isoforms and thus the determination of carbohydrate-deficient transferrin (CDT, sum of asialo-, monosialo- and disialo-Tf in relation to total Tf) in human serum. Using the CEofix-CDT reagents, a 50 microm inner diameter (ID) capillary of 60 cm total length and the P/ACE MDQ under optimized instrumental conditions (20 kV and 30 degrees C) is demonstrated to provide outstanding assay precision for the determination of CDT in human serum. For CDT levels of 1.0% and 4.5%, precision relative standard deviation (RSD) values (n = 8) were determined to be < 3.0% and < 1.5%, respectively. During the first year of operation under routine conditions, more than 600 patient samples were analyzed in a total of 62 sets of runs. Except for selected samples of patients with severe liver diseases, interference-free Tf patterns were detected. Asialo-Tf was not detected in control sera and in patient sera with a CDT level < 1.70%, but became detectable in 89.6% of sera with > 2.3% disialo-Tf. Monosialo-Tf was only detected in two sera containing > 13.3% CDT. The optimized CZE assay was applied to confirm positive CDT results produced by an immunoassay during long-term monitoring of a patient which led to the determination of the elimination kinetics of asialo-Tf, disialo-Tf, and CDT after an episode of high alcohol consumption (estimated apparent half lifes of 4.86, 7.24, and 6.74 days, respectively). The optimized CZE assay with an upper reference limit for CDT of 1.70% represents an attractive alternative to high-performance liquid chromatography (HPLC). It features simpler sample preparation, faster analysis time, and higher isoform resolution compared to the most recent HPLC approach and can thus be regarded as a new candidate of a reference method for CDT.     

Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase

Cellobiohydrolase (CBH) is an important enzyme for the conversion of lignocellulosic biomass to ethanol. This work separated the glycoforms of CBH possessing different numbers of neutral mannoses using capillary zone electrophoresis (CZE) in a 50 mM, pH 7.5 phosphate buffer. The method analysed CBH in an intact form using a polyacrylamide coated fused silica capillary without requiring additives or labelling of the enzyme. The migration time of the major peak was found to be 21.6±0.1 min (n=3) and the approach is suitable for testing of batch-to-batch consistency of CBH. Ease-of-use, automation and speed are the other benefits due to which the use of CZE for analysing glycoforms of CBH was concluded to be ideal.     

毛细管区带电泳法快速测定人血血清蛋白

人血血清蛋白电泳分析是临床上诊断多种疾病的常用生化指标 ,也是临床实验室检查的常规项目。目前采用的方法有醋酸纤维薄膜电泳和琼脂糖电泳 ,尤以前者为主。由于醋酸纤维薄膜电泳法操作繁琐 ,每一步骤均需手工完成 ,影响因素较多 ,初学者往往不易掌握 ,且测定的重复性较差。高效毛细管电泳是近几年来迅速发展起来的分离分析技术 [1,2 ]。用该技术分析人血清蛋白国内外虽有报道 ,但由于人血清中蛋白质组分甚为复杂 ,采用不同的分离方法和条件可出现不同数量的组分峰 ( 6、7个甚至 1 0个以上组分 ) ,与目前临床上常用的醋酸纤维薄膜电泳图谱…     

Capillary electrophoresis enzyme immunoassay for alpha-fetoprotein and thyroxine in human serum with electrochemical detection

A novel CE-based enzyme immunoassay (CE-EIA) method was developed in o-aminophenol (OAP)-H(2)O(2)-horseradish peroxidase (HRP) system and applied to benign liver disease and hyperthyroidism research in the clinical practical field. In the presented method, after the enzyme immunoreaction, the HRP-labeled antibody or HRP-labeled antigen catalyzed the enzyme substrate OAP and H(2)O(2). The product of the enzymatic catalysis reaction 2-aminophenoxazine-3-one (AP) was determined using electrochemical detection on a Pt electrode at the outlet of the reaction capillary. Factors influencing the performance, including running buffer concentration, separation, and detection voltage, were investigated to the optimum conditions. Noncompetitive and competitive models were utilized to detect alpha-fetoprotein (AFP) and thyroxine (T(4)) in human sera, respectively. The linear ranges and the detection limits (S/N = 3) were from 1.5 to 66.6 ng/mL and 0.48 ng/mL for AFP, and from 1.7 to 260.0 ng/mL and 1.0 ng/mL for T(4). The results of this method were linear proportional to those of spectrophotometric ELISA method, giving a good prospect for a new clinical diagnostic instrument.     

Capillary zone electrophoresis of albumin-depleted human serum using a linear polyacrylamide-coated capillary: separation of serum alpha-and beta-globulins into individual components

The separation of human serum globulins into individual components was investigated by capillary zone electrophoresis (CZE) using a linear polyacrylamide-coated capillary at pH 7.4. Prior to CZE analysis of globulin components present in serum, it was found that it was necessary to remove albumin. Preparation of albumin-depleted human serum with a HiTrap Blue column allowed the detection of alpha- and beta-globulin components as a series of peaks. Almost all the peaks, both narrow and broad, observed in CZE analysis could be assigned to six globulin components (alpha1-acid-glycoprotein, alpha1 -antitrypsin, haptoglobin, alpha2-macroglobulin, Gc-globulin, and transferrin) by using the technique of antibody-based indirect detection. The CZE results, obtained from serum preparations from three healthy adults and six patients, showed that the CZE system might be capable of detecting qualitative differences among individuals with regard to individual globulin components.     

Analysis of genetic variants of transferrin in human serum after desialylation by capillary zone electrophoresis and capillary isoelectric focusing

Capillary electrophoresis analysis of transferrin in human serum is used to assess genetic variants after desialylation with neuraminidase and iron saturation to reduce the complexity of the transferrin pattern and thus facilitate the recognition of transferrin polymorphisms. Asialo‐transferrin forms are analyzed by capillary zone electrophoresis using assay conditions as for the monitoring of carbohydrate‐deficient transferrin or by capillary isoelectric focusing in a pH 5–8 gradient which requires immunoextraction of transferrin prior to analysis. With the carrier ampholytes used, peaks for iron saturated and iron depleted transferrin are monitored which indicates complexation of iron ions by carrier ampholytes. For BC, CD, and BD genetic variants, the expected peaks for B, C, and D forms of transferrin were detected with both methods. Monitoring of CC patterns revealed three cases, namely those producing double peaks in both methods, a double peak in capillary isoelectric focusing only and a double peak in capillary zone electrophoresis only. For all samples analyzed, data obtained by capillary isoelectric focusing could be confirmed with gel isoelectric focusing. The two capillary electrophoresis methods are shown to represent effective tools to assess unusual transferrin patterns, including genetic variants with dissimilar abundances of the two forms.