New sample preparation method for the capillary electrophoretic determination of adenylate energy charge in human erythrocytes (original) (raw)
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Analytical Chemistry, 2002
Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of human red blood cells (RBCs) using the split-flow technique for interfacing CE to MS. By using a long (∼125cm) and narrow (∼15-µm-i.d.) capillary, the four major proteins of the RBC, which are hemoglobin (Hb, r-and-chains, 900 amol/chain), carbonic anhydrase I (CAI, ∼7 amol/cell), and carbonic anhydrase II (CAII, ∼0.8 amol/cell), were separated from each other and detected at low-attomole levels in one run and minimal sample preparation. Under these conditions, the detection limits for CAI and CAII in lysed RBCs were ∼20 and ∼44 amol, respectively. The ∼20-amol detection limit of CAI was confirmed by the CE/ESI-MS analysis of three intact RBCs that had been drawn into the capillary under a microscope. A shorter capillary (∼55 cm long) provided faster analysis time but did not separate CAII from the-chain of hemoglobin, causing the CAII signal to be masked by the background chemical noise generated by the ∼1000× molar excess of the-chain. Under this condition, the CAII detection limit increased to ∼500 amol. From three methods of sample introduction (injection of lysed blood, injection of intact cells under microscope, and injection of intact cells suspended in saline solution), injection of lysed blood provided the optimum sensitivity. It was found that a background electrolyte (BGE) containing 0.1% acetic acid in water worked best for the analysis of intact cells, while a BGE containing 0.1% acetic acid in water + acetonitrile (50/50 by volume) worked best for the analysis of lysed blood.
Capillary electrophoresis and its application in the clinical laboratory
Clinica Chimica Acta, 2003
Over the past 10 years, capillary electrophoresis (CE) is an analytical tool that has shown great promise in replacing many conventional clinical laboratory methods, especially electrophoresis and high performance liquid chromatography (HPLC). The main attraction of CE was that it was fast, used small amounts of sample and reagents, and was extremely versatile, being able to separate large and small analytes, both neutral and charged. Because of this versatility, numerous methods for clinically relevant analytes have been developed. However, with the exception of the molecular diagnostic and forensic laboratories CE has not had a major impact. A possible reason is that CE is still perceived as requiring above-average technical expertise, precluding its use in a laboratory workforce that is less technically adept. With the introduction of multicapillary instruments that are more automated, less technique-dependent, in addition to the availability of commercial and cost effective test kit methods, CE may yet be accepted as a instrument routinely used in the clinical laboratories. Thus, this review will focus on the areas where CE shows the most potential to have the greatest impact on the clinical laboratory. These include analysis of proteins found in serum, urine, CSF and body fluids, immunosubstraction electrophoresis, hemoglobin variants, lipoproteins, carbohydrate-deficient transferrin (CDT), forensic and therapeutic drug screening, and molecular diagnostics. D
Analytical Biochemistry, 2004
Intracellular redox and energetic status play a crucial role in cardiovascular diseases and metabolic disorders. The physiological status of reducing agents, such as NADPH and NADH, and of high-energy molecules, such as ATP, is required for antioxidant system activity. For these reasons, an accurate measurement of adenine and pyridine nucleotides is fundamental. In this study we examined the preanalytical phase of reduced pyridine (RPN) and adenine and oxidized pyridine (AOPN) nucleotide assay in human whole blood. DiVerent experimental conditions were applied to RPN alkaline and AOPN acid extracts to Wnd the best analytical performance. Our results show that a good RPN and AOPN linearity (r from 0.994 to 0.999), recovery (near to 100%), and precision (coeYcient of variation 05%) were obtained when supernatant from acid and ultraWltrate from alkaline extracts were neutralized, frozen, and thawed just before HPLC injection. Since NADH decays rapidly at ¡80°C, RPN levels must be assayed within 72 h while AOPN can be stored for 1 month at the same temperature. An accurate and quantitative method for nucleotide determination can be obtained by applying the preanalytical conditions proposed in this study.
Clinica Chimica Acta, 1985
An anion-exchange high performance liquid chromatography (HPLC) method is described for the quantitation of intracellular purine and pyrimidine nucleotides. With an ammonium phosphate salt and pH gradient, complete separation is achieved of all major nucleotides and several interfering substances, such as dehydroascorbic acid and NAD. For optimal resolution of the monophosphates, strict control of the equilibration pH is essential. To prevent interference by a degradation product of NADPH with the determination of GDP, the pH of the high-ionic strength buffer has to be in the range of 4.9-5.0. The use of radially compressed, prepacked cartridges filled with Partisil-10 SAX appeared to be a fast and cheap alternative for expensive stainless-steel columns. The use of ammonium phosphate buffers, in combination with precolumns filled with pellicular silica and SAX resin, and interim EDTA washes prevents baseline shift. This allows analysis at 0.01 Absorbance Units Full Scale during the entire column lifetime (about 180 analyses), which is sufficiently sensitive for the quantitation of low levels of nucleotides, especially when the amount of sample is limited. The usefulness of the presented chromatographic system is demonstrated by the quantitation of the nucleotides in extracts of lymphocytes and neutrophils from the blood of healthy human donors. With this method nucleotide concentrations were measured, with a within-assay variation of 5-10% and an inter-donor variation of 10%.
An Overview of Capillary Electrophoresis (CE) in Clinical Analysis
2019
The development and general applications of capillary electrophoresis (CE) in the field of clinical chemistry are discussed. It is shown how the early development of electrophoresis was closely linked to clinical testing. The rise of gel electrophoresis in clinical chemistry is described, as well as the eventual developments that lead to the creation and the use of modern CE. The general principles of CE are reviewed and the potential advantages of this method in clinical testing are examined. Finally, an overview is presented of several areas in which CE has been developed and is currently being explored for use with clinical samples.
Capillary electrophoresis and the clinical laboratory
ELECTROPHORESIS, 2006
Over the past 15 years, CE as an analytical tool has shown great promise in replacing many conventional clinical laboratory methods, such as electrophoresis and HPLC. CE's appeal was that it was fast, used very small amounts of sample and reagents, was extremely versatile, and was able to separate large and small analytes, whether neutral or charged. Because of this versatility, numerous methods have been developed for analytes that are of clinical interest. Other than molecular diagnostic and forensic laboratories CE has not been able to make a major impact in the United States. In contrast, in Europe and Japan an increasing number of clinical laboratories are using CE. Now that automated multicapillary instruments are commercially available along with cost-effective test kits, CE may yet be accepted as an instrument that will be routinely used in the clinical laboratories. This review will focus on areas where CE has the potential to have the greatest impact on the clinical laboratory. These include analyses of proteins found in serum and urine, hemoglobin (A1c and variants), carbohydrate-deficient transferrin, forensic and therapeutic drug screening, and molecular diagnostics.
Journal of Chromatography B, 2003
Cell extraction and further sample preparation for nucleotide pool analysis using capillary electrophoresis was faster and simpler using volatile extraction solvents (e.g. organic solvents and de-ionized water) compared to the commonly applied acids dissolved in water (e.g. perchloric acid and trichloracetic acid). Temperature had to be controlled during the whole sample preparation process to prevent degradation, and extracts had to be cleaned from proteins and other large molecules prior to capillary electrophoretic analysis to improve reproducibility. Capillary electrophoresis using borate and cyclodextrins in the background electrolyte was used for determining 11 cellular nucleotides simultaneously. In order to optimize the assay, 0-100% acetonitrile, 0-100% ethanol, and 0-100% methanol in de-ionized water were applied to extract nucleotides from mouse lymphoma cells, and nucleotide yields, recovery, and reproducibility were compared. The assay met the commonly accepted validation limits for biological fluids, if 20-80% acetonitrile in water and 40-60% ethanol in water were used as extraction solvents.
Nucleotide and Nucleotide Sugar Analysis in Cell Extracts by Capillary Electrophoresis
CHIMIA International Journal for Chemistry, 2016
In biotechnological processes the intracellular level of nucleotides and nucleotide sugars have a direct impact on the post-translational modification (glycosylation) of the therapeutic protein products and on the exopolysaccharide pattern of the cells. Thus, they are precursors and also key components in the production of glycoproteins and glycolipids. All four nucleotides (at different phosphorylation stages) and their natural sugar derivatives coexist in biological samples. Their relative ratios depend on the actual conditions under which the cells are grown. Therefore, their simultaneous determination at different time points and different cell culture conditions in biotechnological samples is of interest in order to develop the optimal cell culture process. In our study capillary electrophoresis (CE) combined with UV detection @ 260 nm was selected for the separation and quantification of the complex nucleotide mixture of the structurally very similar nucleotides and nucleotide sugars in cell extracts. The high separation efficiency of CE as well as its insensitivity to the complex cell matrix makes this method superior to commonly used HPLC methods. In our study eleven nucleotides and six nucleotide sugars were analyzed. A robust and reproducible analysis system was developed. As background electrolyte borate (40 mM, pH 9.5) was used containing 1% PEG (MW 35'000 Da) which enhanced resolution. In order to obtain high reproducibility in terms of migration time, mandatory for the unambiguous identification of the single compounds in the complex cell extract mixtures, dynamic coating was also employed. The method was tested for CHO cell extracts where three sugar nucleotides and seven nucleotides were identified and quantified using GDP-Glc as internal standard.