Journal of chromatography. B, Biomedical sciences and applications最新文献

S-Nitrosoalbumin (SNALB) and S-[15N]nitrosoalbumin (S[15N]ALB) were prepared by various methods, purified and isolated by a novel selective extraction procedure using HiTrapBlue Sepharose affinity columns and characterized by various techniques including SDS-PAGE electrophoresis, UV-Vis spectroscopy and gas chromatography-mass spectrometry (GC-MS). S-Nitrosylation of albumin in freshly obtained human plasma by unlabeled and 15N-labeled butylnitrite at neutral pH revealed the purest preparations. For GC-MS analysis, SNALB and S[15N]ALB were treated with HgCl2 to obtain nitrite and [15N]nitrite, respectively, which were then analysed as their pentafluorobenzyl derivatives. S[15N]ALB preparations were standardized by GC-MS using nitrite as internal standard. S[15N]ALB was prepared and isolated at concentrations of 188+/-43 microM (mean +/- SD, n = 8) at a final yield of about 45%, an isotopic purity of 98%, and SDS-PAGE electrophoretic purity of 90%. 15N-Labeled SNALB was used to study its metabolism in human blood. The half-life of S[15N]ALB (25 microM) in human heparinized blood in vitro was determined by GC-MS as 5.5 h. The GC-MS method described here could be useful for the quantitative determination of SNALB in human plasma using S[15N]ALB as an internal standard.

A gas chromatographic-mass spectrometric method for the quantitative determination of S-nitrosoalbumin (SNALB) in human plasma is described. The method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S15NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, Hg2+ -catalysed conversion of the S-nitroso groups to nitrite and [15N]nitrite, respectively, followed by their derivatization to the pentafluorobenzyl derivatives and quantification by GC-MS. Mean recovery of SNALB and S15NALB from plasma was 45%. Mean precision and accuracy within the range 0-10 microM was 95% and 99%, respectively. The limit of quantitation was determined as 100 nM at a precision of 93.8% and an accuracy of 94.8%. Considerable improvement of method sensitivity is possible by eliminating nitrite present in the elution buffer. The limit of detection was 0.2 nM corresponding to 67 amol of S15NALB. In 0.4-ml aliquots of plasma samples from healthy humans, endogenous SNALB was determined at concentrations of 181+/-150 nM (mean +/- SD, n = 23). External addition of SNALB to these plasma samples at 2 microM and 5 microM serving as quality control samples resulted in quantitative recovery of SNALB. Our results show that SNALB occurs in human plasma at concentrations at least one-order of magnitude smaller than those reported in the literature from measurements using chemiluminescence.

Two-dimensional electrophoresis has rapidly become the method of choice for resolving complex mixtures of proteins. Since the technique was pioneered in 1975, 2-D gel methods have undergone a series of enhancements to optimize resolution and reproducibility. Recent improvements in the sensitivity of mass spectrometry have allowed the direct identification of polypeptides from 2-D gels by a procedure termed "mass profiling". In combination, these two techniques have made possible the characterization of the complete collection of gene products, or proteome, of an organism. Proteomes are increasingly being documented as interactive informational databases available on the World Wide Web (WWW). This availability of organismic global protein patterns will no doubt be an invaluable resource aiding the discovery of diagnostic and therapeutic disease markers.

While annotated two-dimensional (2D) gel electrophoresis databases contain thousands of proteins, they do not represent the entire genome. High-molecular-mass proteins in particular are conspicuously absent from such databases. Filamin is prototypical of this class of proteins since it is a dimer with relative molecular mass (Mr) of 520000 containing at least 240 potential phosphorylation sites. Filamin is not readily separated by current 2D procedures, and is difficult to study with respect to cycles of phosphorylation-dephosphorylation. Novel technologies are needed to identify biochemical pathways impinging upon such targets. The success of immunofluorescence microscopy as a research tool can be attributed in part to the fact that proteins redistribute in response to a variety of physiological stimuli. Comparable quantitative methods are required in proteome analysis. Three components are necessary for development of an approach that is capable of screening for protein redistribution events: (1) subcellular fractionation, (2) protein labeling and (3) data acquisition. An integrated approach is presented that utilizes differential detergent fractionation combined with reversible, luminescent protein stains and analytical imaging for high-throughput analysis of signal transduction events leading to protein subcellular redistribution. The procedure has been successfully implemented to rapidly define key second messenger pathways leading to endothelial cell junctional permeability and to guide in the design of a new family of peptide-based anti-inflammatory drugs.

Magnetic separation is an emerging technology using magnetism, sometimes in combination with conventional separation or identification methods, to purify cells, cell organelles and biologically active compounds (nucleic acids, proteins, xenobiotics) directly from crude samples. Several magnetic separation procedures have been developed to isolate target cells specifically. The purpose of this short review is to summarize various methodologies, strategies and materials which can be employed for the selection and separation of target cells with the help of magnetic field and thus to help the novices in this field to be able to orient themselves in vast amount of literature available. Immunomagnetic separations employing specific antibodies to label the target cells represent the most often used approach and are discussed in detail.

Chloroformates with simplest alkyls, i.e. methyl, ethyl or isobutyl, already known as favourable reagents for treating amino groups in gas chromatography for years, were revealed randomly as exceptionally rapid esterification agents. Unlike the rather poor results achieved with chloroformate-mediated ester formation in organic chemistry, the pyridine-catalyzed esterification of carboxylic acids appeared to proceed at the analytical microscale smoothly. Along with the catalyzer, an alcohol should also be present in the medium, accompanied by acetonitrile or water, according to the character of the compounds treated. Reaction conditions were optimized for various classes of carboxylic acids and a uniquely rapid derivatization of amino acids in aqueous ethanol was shown to be possible. Most of the analytes, e.g. acidic metabolites in physiological fluids, could be treated directly in the aqueous matrix. A simultaneous analysis of, e.g., amino and fatty acids or amines and their acidic catabolytes was proven to be possible. Along with the low-molecular-mass reagents, still some others, i.e. the hexyl, menthyl or pentafluorobenzyl ones, found their application fields. Results of optimized reaction conditions and a wide range of applications of chloroformate-mediated derivatization in various disciplines have been summarized in this review.

In this paper, the HPLC method for the measurement of nitrite and nitrate in serum of humans newly reported by E1 Menyawi et al. is discussed, especially in regard to the extremely low nitrate levels measured in serum of healthy humans. From the discussion, it is concluded that: (1) Biogenic amines at physiological concentrations do not significantly interfere with the batch Griess assay. (2) The HPLC method of E1 Menyawi et al. does not reveal accurate levels for serum nitrate. (3) In serum and plasma of healthy humans, nitrate ranges within 15-70 microM. (4) Exogenous NG-nitro-L-arginine analogs can interfere with the measurement of nitrate in human plasma and urine by the batch Griess assay, chemiluminescence and GC-MS; interferences can be effectively eliminated by solid-phase extraction on cation-exchangers.

This contribution presents a framework for the rational design of affinity sorbents based on cellulose materials as a support. A three-level evaluation procedure, utilizing the knowledge of physical, chemical and engineering theories, is discussed, which integrates the design of support, affinity sorbent and chromatographic contactor. The principal support properties, such as morphological, diffusional, hydrodynamic, mechanical or ligand-binding properties, are presented and literature data on them are surveyed.

An assay method for pregnenolone and its 3-stearate in rat brains has been developed using LC-atmospheric pressure chemical ionization (APCI) isotope dilution MS. The extraction of the rat brain homogenate containing deuterated internal standards (ISs) with organic solvent followed by silica gel mini-column chromatography gave two fractions containing pregnenolone and its 3-stearate together with the respective IS. Each fraction was derivatized into 3-acetate-20-methyloxime and 20-methyloxime, respectively, followed by silica gel mini-column chromatography to remove the excess reagents, and then each derivatized fraction was applied onto reversed-phase LC-APCI-MS. The method was applied to the determination of these steroids in the gray matter and olfactory bulbs of rat brains, which were divided into control and acute stressed specimens. Although pregnenolone in both regions of the rat brains increased more than five times after stress, its 3-stearate decreased after stress.