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  • 1
    ISSN: 0935-6304
    Keywords: Capillary electrophoresis ; Run-to-run reproducibility ; Capillary thermostatting ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Run-to-run sample separation reproducibility has been compared on two commercial high performance capillary electrophoresis units which differ in the mode by which the capillary temperature is thermostatted. Three standard analytes, differing dramatically in molecular character and size, were used for the analysis: benzoic acid, a 14 amino acid peptide from human chorionic gonadotropin, and ribonuclease A represent, respectively, small stable organic molecules, small peptides with little or no secondary structure, and proteins with secondary structure. These standards were evaluated with regard to reproducibility of migration time, peak area, and peak height. The analyses, performed in buffers of optimum pH for the separations, demonstrated that the liquid and forced air convection thermostatted systems both performed extremely well. The reproducibility, as judged by the percent coefficient of variance (% CV) of replicate analyses, was generally found to be less than 1 % (migration time); the reproducibility decreased in the order migration time 〉 peak height 〉 peak area. Whereas the absolute % CV values for MTrel (migration relative to a standard) observed with the liquid thermostatted system were 2- to 4-fold lower than those observed with the forced air convection thermostatted system, there was little statistically significant difference between the two. As expected, the data indicated a reduction in reproducibility as the complexity of the analyte increased, perhaps as the result of an increased potential for wall interactions. Comparing separations in which low (≍1 watt/meter [W/m] of capillary) and high (〉5 W/m) Joule heat was generated by altering the sodium chloride content of the buffer revealed few statistically significant differences in the reproducibility obtained from the two systems. With these particular standard analytes and their respective buffer systems, there appears to be little difference between forced air convection and liquid thermostatting of the capillary.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0173-0835
    Keywords: Nitrite ; Nitrate ; Reduction ; Capillary electrophoresis ; Chemiluminescence detection ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Production of nitrates and nitrites is a common step in many methodologies used to measure nitric oxide (NO) and NO-derived products in biological fluids. We report conditions that allow the rapid separation and quantification of nitrite from nitrate ions in biological fluids by capillary ion electrophoresis (CIE). CIE can be used to directly quantify nitrites and nitrates near the millimolar range. To detect lower levels, we have used CIE to monitor the reduction of nitrites and nitrates to NO for chemiluminescence detection. For reduction reactions, we directly compared the ability of three commonly used agents  -  potassium iodide (KI), mercuric chloride (HgCl2) and vanadium chloride (VCl3)  -  to reduce nitrite and nitrate ions to NO. Nitrites/nitrates can be efficiently reduced to NO at 37°C using vanadium chloride (100%) or HgCl2 (80%). However, these CE-derived conditions cannot simply be extrapolated to chemiluminescence measurements. Vanadium (III) yields high background in the photomultiplier that diminishes the sensitivity of chemiluminescence measurement to that outside of physiological ranges. We find that reactions carried out at 37°C in 2 M HCl using HgCl2 is efficient using both techniques.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 0173-0835
    Keywords: Capillary electrophoresis ; Serum proteins ; Immunosubtraction ; Immunofixation electrophoresis ; Monoclonal protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The capabilities of capillary electrophoresis (CE) for serum protein electrophoresis and immunotyping have been demonstrated. CE-based systems specifically designed for serum protein electrophoresis and immunotyping via immunosubtraction (IS) are now available and are being evaluated for efficiency, specificity and sensitivity by several groups. The use of CE for serum protein electrophoresis and immunotyping (IS) in the clinical laboratory compares well with agarose gel electrophoresis (AGE) and immunofixation (IF) for the detection and characterization of monoclonal proteins. In addition to routine use, this technology is useful for a subset of serum samples that are difficult to interpret with conventional technology. In this study, sera abnormalities difficult to detect/interpret by AGE-IF are subdivided into four categories: (i) patients with polyclonal increases in immunoglobulin, (ii) point of application artifacts, (iii) abnormalities in the beta region, and (iv) patients with free light chains. CE is superior to AGE for evaluating samples characterized by the above abnormalities. Sera containing monoclonal proteins within a polyclonal increase are easier to detect by CE as well as being easier to type by IS than by IF. Point-of-application artifacts, periodically observed with AGE, do not exist on CE since the point of detection is remote from the point of application. Enhanced resolution in the beta region allows for increased detection of monoclonal proteins migrating in this region. Some free light chains are undetected by CE as a result of no apparent abnormalities on the CE serum protein profile and, thus, still require IF for detection. CE detects more serum electrophoretic abnormalities than AGE in this clinically important group of patients with Bence Jones proteinemia.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0173-0835
    Keywords: Capillary electrophoresis ; Carbohydrate-deficient transferrin ; Transferrin ; Sialic acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The presence of specific transferrin (Tf) glycoforms in human serum has been shown to correlate with certain clinical syndromes. Hence, the ability to separate and quantitatively measure the various forms of human transferrin has become increasingly important. As a means of evaluating the potential for developing a rapid capillary electrophoresis-based assay for the analysis of carbohydrate-deficient transferrins (CDTs), the capillary zone electrophoretic (CZE) analysis of Tfs from several species was evaluated using uncoated capillaries and a separation augmented with cationic additives. With bovine Tf, five peaks (representing different sialylated forms) were partially resolved in borate and baseline-resolved when 1,4-diaminobutane was added to the buffer. These same conditions were found to be inadequate for the resolution of the sialo-forms from other species. Some success was achieved using α,γ-bis-quaternary ammonium alkanes instead of the 1,4-diaminobutane and optimizing the pH for each of the species Tfs. Human Tf was found to be resolved in an uncoated capillary equilibrated with a borate buffer containing millimolar concentrations of decamethonium bromide as a buffer additive. Under these conditions, resolution of the various sialoforms from the iron-saturated Tf was possible and the glycoforms were found to migrate differently than their iron-depleted counterparts. Despite the resolution achievable under these conditions, the lengthy analysis time is incompatible with the requirements for a clinical CZE-based assay.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 0173-0835
    Keywords: Capillary electrophoresis ; Polymer solutions ; Polymerase chain reaction ; T-cell receptor (TCRγ) ; Gene rearrangements ; Laser-induced fluorescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The rapid increase in the number of DNA-based clinical diagnostic procedures, particularly polymerase chain reaction (PCR)-based assays, has generated interest in analytical techniques that are less time-consuming and labor-intensive than traditional procedures such as polyacrylamide gel electrophoresis (PAGE). Capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection, which allows for rapid and sensitive detection of DNA fragments in an automated format, is well-suited for DNA-based clinical assays. In this study, we demonstrate the potential of CE-LIF for the detection of PCR products from T-cell receptor gamma (TCRγ) gene rearrangements present in monoclonal populations of lymphocytes. The presence of monoclonal populations of T-cells is associated (but not always synonymous) with lymphocytic malignancies. Analysis of 31 patient samples, as well as sensitivity controls, demonstrated that CE-LIF detection of monoclonal lymphocytic populations is comparable to that of PAGE-SYBR Green I staining, and that CE-LIF detection can be accomplished in less than 20 min. These preliminary results illustrate the potential feasibility of a CE-based diagnostic assay for the detection of T-cell gene rearrangements.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 0173-0835
    Keywords: Clinical analysis ; Protein ; Capillary electrophoresis ; Serum ; Hemoglobin ; Immunoglobulin ; Isozymes ; Lipoprotein ; Glycoprotein ; Transferrin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Most electrophoretic analyses of proteins in the clinical laboratory are currently carried out by electrophoresis in acrylamide or agarose gels. This labor-intensive method is now being challenged by capillary electrophoresis (CE) because of its potential for automated, rapid, high efficiency separations. The automatability of CE itself, combined with its amenability to interfacing with other robotized functions, positions this technology perfectly for the analysis of proteins in physiological matrices, such as serum, urine, and cerebrospinal fluid. The focus of this overview is to familiarize the clinical scientist with the research demonstrating the applicability of the technology to the clinical protein laboratory.
    Additional Material: 6 Ill.
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  • 7
    ISSN: 0173-0835
    Keywords: Capillary electrophoresis ; Bacteriorhodopsin ; Hydrophobic peptide mapping ; Solid-phase extraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The structural investigation of G protein-coupled receptors has been hindered by the lack of techniques to effectively resolve the hydrophobic peptides obtained by chemical or proteolytic cleavage, as well as the minute amounts of protein typically isolated. We have developed a capillary electrophoresis method for efficient separation of hydrophobic peptides using a cyanogen bromide digest of bacteriorhodopsin as a model for these clinically important membrane proteins. This procedure includes (i) solubilization of the protein digest in acetic acid; and (ii) electrophoresis using an acetic acid-based buffer system augmented by acetonitrile and hexane sulfonic acid, in a Polybrenecoated fused silica capillary. The potential for detection sensitivity to be increased at least 100-fold by use of on-line solid-phase extraction on C18-silica is shown. This approach is potentially useful for peptide fingerprinting of sparse and extremely hydrophobic membrane receptors.
    Additional Material: 7 Ill.
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  • 8
    ISSN: 0173-0835
    Keywords: Iothalamate ; Capillary electrophoresis ; Glomerular filtration rate ; Renal function ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The conditions for quantitative measurement of nonisotopic iothalamate meglumine (Conray) in urine and plasma by capillary zone electrophoresis (CE) have been developed. The impetus for developing this methodology was to replace the traditional [125I]iothalamate glomerular filtration rate (GFR) marker assay, a routine tool in the measurement of kidney function. This new approach for measuring kidney function is attractive since it avoids the cost of administration of radioisotopic compounds to patients, as well as the cost associated with purchase and disposal of isotopic compounds and contaminated samples. The concentration of iothalamate in urine and plasma determined by CE can be used directly to calculate GFR. The GFR in patients injected with [125I]iothalamate and nonisotopic iothalamate simultaneously showed an excellent correlation (0.998) with between-day coefficient of variation of 2.30% and a recovery of 102% and 98%, respectively, when added to urine and plasma. Interference from drugs and other urinary compounds is eliminated with this method. Collectively, this study has shown that CE is a cost-effective alternative to the current methodology for measuring GFR.
    Additional Material: 8 Ill.
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