Simultaneous quantitative analysis of sphingoid base 1-phosphates in biological samples by o-phthalaldehyde precolumn derivatization after dephosphorylation with alkaline phosphatase

This paper describes a simultaneous analytical method for the measurement of sphingoid base 1-phosphates and sphingoid bases from a variety of biological samples. This method consists of two steps of sample pretreatment: the enzymatic dephosphorylation of sphingoid base 1-phosphates by alkaline phosphatase (APase) and the subsequent analysis of o-phthalaldehyde (OPA) derivatives of the liberated sphingoid bases by HPLC. By introducing C17-sphingosine 1-phosphate and C17-sphingosine as internal standards, not only phytosphingosine 1-phosphate, sphingosine 1-phosphate, and sphinganine 1-phosphate but also phytosphingosine, sphingosine, and sphinganine present in a sample could be quantified in 12 min on a C18 reversed-phase column with a simple mobile phase of acetonitrile:deionized distilled water (90:10, v/v). With this HPLC method, we could reproducibly analyze the levels of sphingoid base 1-phosphates over a broad range of concentrations from 0.5 to 100.0 pmol from various biological samples including serum, cultured cells, and rat tissue homogenates. The conversion of sphingoid base 1-phosphates into sphingoid bases increased the stability of the OPA adducts. Thus, this indirect measurement of sphingoid base 1-phosphates increased the sensitivity and reproducibility of the method. This HPLC method was also used to measure the changes in the levels of sphingoid base 1-phosphates in cultured cells after treatment with 1,25-(OH)2D3, a sphingosine kinase activator, or with fumonisin B1, a sphinganine N-acyltransferase inhibitor.     

Chemical properties and stereoisomerism of heterogeneous long chain bases in lysosphingolipids by positive ion fast atom bombardment mass spectrometry and carbon-13 NMR spectroscopy

Positive ion fast atom bombardment (FAB) mass spectrometry of galactopsychosine and glucopsychosine was capable of showing not only the pseudo molecular ion peaks, but also various fragment ion peaks such as protonated sphingosine and its fragment ions. The percent distribution of sphingosine and dihydrosphingosine in each lysosphingolipid was determined by GLC of the trimethyl-silylated derivatives of long chain bases after methanolysis and was comparable to the relative intensities of ion peaks derived from the sphingosine and dihydrosphingosine groups. The FAB mass spectra showed that during the fast atom bombardment the sphingosine more preferentially gave rise to one and/or two fragment ions by loss of one and/or two molecules of water than the dihydrosphingosine did. The stereoisomerism of sphingosylphosphorylcholine containing mainly L-threo-sphingosine could be reconfirmed by carbon-13 NMR spectroscopy. Furthermore, although the carbon-13 NMR signals of sphingosine C-1, C-2, C-3, C-4, and C-5 showed significant chemical shift differences between D-erythro and L-threo-sphingosines of lysosphingolipids, it was concluded that the signal position of sphingosine C-3 was the most important for the determination of D-erythro and L-threo configuration in the long chain base moieties of lysosphingolipids.     

Synthesis of (R)- and (S)-3,3,3-trifluoro-2-phenylpropanoyl isocyanates and their use as reactive analogues of Mosher's acid

The title compounds have been prepared from the respective 3,3,3-trifluoro-2-methoxy-2-phenylpropanoic acids (MTPA) by a three-step synthesis with MTPA chloride and MTPA amide as reaction intermediates. The requested compounds were obtained in high chemical yields without any change in optical purity during the preparation. To ascertain the usefulness of this auxiliary agent in the chiral analysis, isomeric 3,3,3-trifluoro-2-phenylpropanoyl isocyanates were subjected in NMR tubes to noncatalyzed reactions with 16 different commercially available chiral alcohols. The steric arrangement of all diastereomers prepared correlated well with their NMR spectral nonequivalence data (Deltadelta), thus demonstrating the regular sign distribution of Deltadelta of particular groups according to the devised molecular model. The usefulness of the novel derivatization is discussed.     

Development of an electrochemical assay for 2,6-dinitrotoluene, based on a screen-printed carbon electrode, and its potential application in bioanalysis, occupational and public health

Screen-printed carbon electrodes (SPCEs) have been successfully exploited as disposable sensors for the measurement of 2,6-dinitrotoluene (2,6-DNT) using a stripping voltammetric method. Initial investigations were undertaken using cyclic voltammetry (CV) to characterise the redox behaviour at the SPCEs. Further studies were then performed to deduce the optimum applied potential and accumulation time for the stripping voltammetric procedure. In addition, a study was carried out to ascertain whether small volumes of samples could be reliably used for analysis. From these studies it was shown that a 100 microl aliquot of sample could be analysed and the calibration plot was linear from 161 ng ml(-1) to 137 microg ml(-1) (R(2)=0.9991), the former concentration being the detection limit. The effects of the major components of human saliva at concentrations normally present were investigated. Of the individual components tested, only Cl(-) and albumen were found to interfere. The presence of the latter could be easily overcome by the addition of (NH(4))(2)SO(4). An interference study was also carried out on some inorganic and organic species that may be present in water samples. The sensors were evaluated by carrying out 2,6-DNT determinations on spiked and unspiked human saliva, dust wipe and potable water samples. Mean recoveries of 47.5, 73.4 and 102.4% were obtained; coefficients of variation of 7.88, 6.63 and 6.42% were calculated for a concentration of 9.1 microg ml(-1) in water, 10.6 microg ml(-1) saliva samples, and 141.1 ng cm(-2) for dust wipe samples, respectively. The performance characteristics show that the method holds promise and reliable data may be obtained for 2,6-DNT in bioanalysis and public health.     

Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting.

The use of fluorescence-activated flow cytometric cell sorting to obtain highly enriched populations of viable target bacteria was investigated. Preliminary studies employed mixtures of Staphylococcus aureus and Escherichia coli. Cells of S. aureus, when mixed in different proportions with E. coli, could be selectively recovered at a purity in excess of 90%. This was possible even when S. aureus composed only approximately 0.4% of the total cells. Cell sorting was also tested for the ability to recover E. coli from natural lake water populations and sewage. The environmental samples were challenged with fluorescently labelled antibodies specific for E. coli prior to cell sorting. Final sample purities of greater than 70% were routinely achieved, as determined by CFU. Populations of E. coli released into environmental samples were recovered at greater than 90% purity. The use of flow cytometry and cell sorting to detect and recover viable target bacteria present at levels of less than 1% within an indigenous microflora was also demonstrated.     

Detection of Rubisco and mycotoxins as potential contaminants of a plantibody against the hepatitis B surface antigen purified from tobacco

Antibodies have been one of the proteins widely expressed in tobacco plants for pharmaceutical purposes, which demand contaminant free preparations. Rubisco constitutes 40-60% of tobacco leaf soluble proteins; therefore it is the major potential protein contaminant of plantibodies, while mycotoxins are toxic compounds that could be introduced during the biomass production and post-harvest stages with important consequences to human health. The objective of this paper was to investigate whether Rubisco and mycotoxins are present in Plantibody HB-01 preparations used in the immunopurification of the hepatitis B surface antigen. Rubisco was purified from Nicotiana tabacum yielding 154 microg of protein per gram of leaves and purity over 95%. Among mouse monoclonal antibodies generated against this enzyme, the CBSS.Rub-2 was selected for its immunodetection. It recognizes a conserved sequential epitope of Rubisco large subunit with an affinity constant of 0.13 x 10(8)M(-1). Rubisco quantification limit was 1 microg spreading to the measurement of this contaminant less than 4% of plantibodies samples. Additionally, according to a Reverse Phase-HPLC used to measure the level of adventitiously introduced contaminants, it can be concluded that aflatoxins B1, B2, G1 and G2 were undetected in the purified Plantibody HB-01 samples.     

Development and validation of a gas chromatography-mass spectrometry method for the determination of isoimperatorin in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

Isoimperatorin is one of the major furanocoumarins isolated from the dried root of Angelica dahuricae Benth.et Hook. The aim of the present study is to develop a procedure based on gas chromatography-mass spectrometry (GC-MS) to describe the analysis of isoimperatorin in rat plasma and tissue. The method was set up and adapted for the analysis of small biological samples taken from rats. Biological samples were extracted by liquid-liquid extraction. Extracted compounds were acetic ether/light petroleum (1:2). They were separated by GC on a DB-5MS analytical column and determined by a quadrupole mass spectrometer detector operated under selected ion monitoring mode. Excellent linearity was found between 0.027-5.32 microg/mL (r >0.99) for plasma samples and 0.108-21.28 microg/g (r >0.99) for the tissue samples. The limit of detection (LOD) was 1.0 ng/mL or 1.0 ng/g (three times signal/noise ratio). Within- and between-day precisions expressed as the relative standard deviation (RSD) for the method were 2.81-5.22% and 4.72-6.52%, respectively. The method recoveries for all samples were >80%. The main pharmacokinetic parameters obtained were T(max)=(1.06+/-0.12)h, C(max)=(0.72+/-0.14) microg/mL, AUC=(2.11+/-0.29)h microg/mL and K(a)=(1.76+/-0.13)/h. The concentrations of isoimperatorin in rat liver, heart, cerebellum and cerebrum were higher than those in other organs. The results presented here clearly indicate that this proposed method could be applicable to investigate the pharmacokinetic and tissue distribution of isoimperatorin in rats after administration.     

Monoclonal antibody AA4, which inhibits binding of IgE to high affinity receptors on rat basophilic leukemia cells, binds to novel alpha-galactosyl derivatives of ganglioside GD1b

Mouse monoclonal antibody AA4 inhibits the binding of IgE to high affinity IgE receptors on the rat basophilic leukemia cell line RBL-2H3. As shown by immunostaining of thin layer chromatograms, antibody AA4 binds avidly to two disialogangliosides (antigen I and antigen II) that occur in this cell line. The two antigens were purified by anion exchange chromatography followed by short-bed continuous thin-layer chromatography. About 230 micrograms of antigen I and 60 micrograms of antigen II were obtained from 20 g (wet weight) of leukemia cells. The structures of both purified antigens were determined to be alpha-galactosyl derivatives of the ganglioside GD1b by fast atom bombardment-mass spectrometry, by chemical ionization-mass spectrometry of permethylated samples, by gas chromatography-mass spectrometry of partially methylated alditol acetates, and by treatment with exoglycosidases and mild acid hydrolysis. The structure of antigen I is: (formula; see text) Antigen II has an additional alpha-galactosyl residue as follows: (formula; see text) The ceramide of antigen I contains approximately equal amounts of C24:0, C22:0, C20:0, C18:0, and C16:0 N-acyl fatty acids. The ceramide base is predominantly sphingosine along with a small amount of dihydrosphingosine. In contrast, the ceramide of antigen II contains mainly C24:0 N-acyl fatty acid with much lower amounts of C22:0, C20:0, and C18:0 fatty acids. Moreover, the ceramide base is approximately 55% sphingosine and 45% dihydrosphingosine. No unsaturated N-acyl fatty acids were detected in either antigen.     

Antifungal activity of thyme (Thymus vulgaris L.) essential oil and thymol against moulds from damp dwellings

AIMS: To characterize antifungal activities of essential oil of thyme (Thymus vulgaris L.) and pure thymol, as comparative substance, on different mould species isolated from damp dwellings. METHODS AND RESULTS: Fifty samples of wall scrapes were collected from damp dwellings in Zagreb, the capital of Croatia. The members of the following mould genera were recovered from the samples: Aspergillus (44%), Penicillium (18%) Alternaria, Ulocladium, Absidia and Mucor (8%) Cladosporium, Trichoderma and Rhizopus (6%), and Chaetomium (2%). Two strains of Stachybotrys chartarum were isolated from damp dwellings in Slovakia. Antifungal activities of the thyme essential oil, which contains p-cymene (36.5%), thymol (33.0%) and 1,8-cineole (11.3%) as main components, and pure thymol were determined by the dilution method and exposure to vaporous phase of the oil. Minimum inhibitory concentrations (MIC) of both thymol and essential oil were bellow 20 microg ml(-1), except for Mucor spp. (50.20 microg ml(-1)). Thymol exhibited approximately three-times stronger inhibition than essential oil of thyme. The vaporous phase of the thyme essential oil (82 microg l(-1)) in glass chambers strongly suppressed the sporulation of moulds during 60 days of exposure. CONCLUSION: The thyme essential oil possesses a wide range spectrum of fungicidal activity. The vaporous phase of the oil exhibited long-lasting suppressive activity on moulds from damp dwellings. SIGNIFICANCE AND IMPACT OF THE STUDY: Essential oil of thyme and thymol could be used for disinfection of mouldy walls in the dwellings in low concentration.     

Synthesis of All Four Possible Stereoisomers of 1-Phenyl-2,3-butanediol and Both Enantiomers of 3-Hydroxy-4-phenyl-2-butanone to Determine the Absolute Configuration of the Natural Constituents

Enantioselective syntheses of all the possible stereoisomers of 1-phenyl-2,3-butanediol (erythro isomer 1 and threo isomer 2) and of 3-hydroxy-4-phenyl-2-butanone 3, the odor components of wisteria flowers, was accomplished via Sharpless asymmetric epoxydation. The absolute configurations of 1–3 were determined by an HPLC analysis of the corresponding MTPA esters of synthetic samples.     

Simultaneous determination of various aromatic amines and metabolites of aromatic nitro compounds in urine for low level exposure using gas chromatography-mass spectrometry

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (<0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (<0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.     

Comparative quantification of sphingolipids and analogs in biological samples by high-performance liquid chromatography after chloroform extraction

Sphingosine 1-phosphate (S1P) is an extra- and intracellular messenger that specifically activates five G-protein-coupled cell surface receptors designated S1P_1-5. The S1P₁ receptor is particularly important for the maintenance of immune surveillance by regulating egress of lymphocytes from thymus and secondary lymphoid organs. S1P is generated through phosphorylation of sphingosine which is catalyzed by sphingosine kinase types 1 and 2. The immunosuppressant and sphingosine analog Fingolimod (2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol, FTY720) can also be phosphorylated and induces lymphopenia by downregulating cell surface expression of the S1P₁ receptor on lymphocytes. To analyze the role of S1P in lymphocyte circulation and distribution we established a high-performance-liquid-chromatography-based method for parallel detection and quantification of Fingolimod, sphingosine, and dihydrosphingosine together with their phosphorylated derivatives Fingolimod-phosphate, S1P, and dihydrosphingosine 1-phosphate. Phosphorylated and nonphosphorylated lipids were efficiently isolated from biological samples such as cells, tissues, serum, plasma, and media by simple chloroform extraction. Fluorescence labeling with 9-fluorenylmethyl chloroformiate ensured high selectivity and enhanced sensitivity for sphingolipid detection. The described method provides an accurate approach to investigate phosphorylation, dephosphorylation, hydrolyzation, and dehydrolyzation of sphingolipids and analogs. In addition it works independently from enzymatic conversions, measuring actual concentrations rather than enzymatic activities.     

Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA

Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 microg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 microg pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 degrees C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (approximately 100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process.     

ISOLATION AND CHARACTERIZATION OF GLUCOCEREBROSIDES FROM ADULT RAT BRAIN1

Abstract— By chromatography on borate-coated silicic acid, glucocerebrosides, galactocerebrosides, sulfatides and sphingomyelins from brain tissue could be efficiently separated. Adult rat brain was found to contain 54.1 ± 1.5 nmol of glucocerebrosides per gram fresh weight. Ninety percent of the glucocere-broside fatty acids were palmitate, stearate and oleate; fatty acids with chain lengths above C₂₀ were virtually absent. No hydroxy fatty acids were found. The long chain bases of adult rat brain glucocerebrosides consisted of 74.6% C₁₈-sphingosine, 24.4% C₁₈-sphinganine and 1.1% C₂₀-sphingosine. These results are compared to those obtained from glucocerebrosides from immature rat brains (Abe & Norton , 1974) and discussed in respect to changes occurring during brain development.     

Isolation and structural characterization of glycosphingolipids of in vitro propagated human umbilical vein endothelial cells

To investigate in detail the expression of glycosphingolipids (GSLs) on endothelial cells, 4.85 x 10(9) human umbilical vein endothelial cells (HUVECs) were cultivated in a 2 l bioreactor using microcarriers as a support for anchorage dependent growing cells. Neutral GSLs and gangliosides were isolated and their structures were determined by TLC immunostaining, fast atom bombardment-mass spectrometry (FAB-MS) of the native GSLs, and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates. GbOse4Cer, GbOse3Cer, and LacCer, all carrying mainly C24- and C16-fatty acid beside C18-sphingosine, were detected as the major neutral GSLs (36%, 23%, and 15% of the total orcinol stain, respectively); GlcCer, nLcOse4Cer, and nLcOse6Cer were expressed to substantial minor amounts (9%, 12%, and 5% of the total orcinol stain, respectively). TLC immunostaining revealed the presence of lipid bound Lewisx antigen, whereas the isomeric Lewisa structure was detectable only in very low quantities. GM3(Neu5Ac) with C18-sphingosine was the major ganglioside constituting about 90% of the whole ganglioside fraction. The fatty acid composition was determined by GC-MS of fatty acid methyl esters, indicating the predominance of C24- and C16-substituted GM3(Neu5Ac), followed by C18- and C22-substituted species. Terminally alpha2-3 sialylated neolacto-series ganglioside IV3Neu5Ac-nLcOse4Cer was the second most abundant ganglioside in HUVECs (8% of the total resorcinol stain), and IV6Neu5Ac-nLcOse4Cer and VI3Neu5Ac-nLcOse6Cer (together less than 2% of total resorcinol stain) were found in minor quantities. Lipid bound sialyl Lewisx antigen with poly-N-acetyllactosaminyl chains, and traces of gangliotetraose-type gangliosides GM1 and GD1a were identified by TLC immunostaining. The expression of dominant neutral GSLs LacCer, GbOse3Cer, and GbOse4Cer, and of ganglioside GM3(Neu5Ac) was assayed by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The complete structural characterization of GSLs from HUVECs contributes to our understanding about their functional role, not only of the carbohydrate but also of the lipid moiety, as receptors for bacterial toxins, as cell surface antigens of cellular interaction and as receptors for blood components and macromolecules of the extracellular matrix.     

Determination of total aluminum, chromium, copper, iron, manganese, and nickel and their fractions leached to the infusions of black tea, green tea, Hibiscus sabdariffa, and Ilex paraguariensis (mate) by ETA-AAS

Total aluminum, chromium, copper, iron, manganese, and nickel were determined in black tea, green tea, Hibiscus sabdariffa, and Ilex paraguariensis (mate) by electrothermal atomic absorption spectrometry after nitric/perchloric acid digestion. In each case, one ground sample of commercially available leafy material was prepared and three 0.5-g subsamples were run in parallel. The infusions were also analyzed and the percentage of each element leached into the liquor was evaluated. The obtained results indicated that hibiscus and mate contained lower levels of aluminum (272±19 μg/g and 369±22 μg/g, respectively) as referred to black tea (759±31 μg/g) or green tea (919±29 μg/g) and suggested that mate drinking could be a good dietary source of essential micronutrient manganese (total content 2223±110 μg/g, 48.1% leached to the infusion). It was also found that the infusion of hibiscus could supply greater amounts of iron (111±5 μg/g total, 40.5% leached) and copper (5.9±0.3 μg/g total, 93.4% leached) as compared to other infusions. Moreover, it was found that the percentage of element leached to the infusion was strongly related to the tannins content in the beverage (correlation coefficients >0.82 with the exception for nickel); for lower tannins level, better leaching was observed.     

Structural investigations and biological activity of inositol sphingophospholipids from Phytophthora capsici

Inositol sphingophospholipids that protect pepper (Capsicum annuum c.v. Yolo Wonder) against pathogen have been isolated by chromatographic methods from the mycelium of Phytophthora capsici. The structure of the major compound was determined by chemical methods and mass spectrometry. Phosphodiester bond cleavage of the phospholipid by mild alkaline hydrolysis liberated a ceramide which contained a C16-sphingosine. This long-chain base was identified by gas chromatography and mass spectrometry of its trimethylsilyl derivative. One of the amide-linked fatty acids was found to be 4-hydroxy-2 docosenoic acid. Fast-atom-bombardment mass spectrometry and fast-atom-bombardment collison-induced tandem mass spectrometry were used to characterize the ceramide as N(4-hydroxy-2-docosenoyl)C16-sphingosine. These sphingolipids have a protective effect on cotyledons of young peppers against necrotic lesions induced by the pathogen P. capsici.     

Isolation of circulating human monocytes with high purity for proteomic analysis

We describe a simple method for isolation of human blood monocytes with the high purity (95-98%) required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to paramagnetic beads. The elution conditions of the positive selection step were modified to avoid contamination with albumin. This method is compatible with flow cytometry which was used to assess the purity of the cell population. From 28 mL of blood, 10(7) monocytes with > 96% purity are routinely obtained. From the isolated monocytes 200-250 microg of protein could be recovered. The whole method can be performed in three hours. Similar results were obtained using a negative selection step but with lower purity (92%), increased cost and longer time. After solubilization of monocytes, the proteins were analyzed by two-dimensional gel electrophoresis (2-DE) in the 3-10, 4-7, 6-9 and 6-11 pH range. DNA was the main contaminant that interfered with the 2-DE and it was removed by treatment with DNAse. Image analysis of gels allowed the reproducible detection and quantification of 1500 spots in the 4-7 pH range and more than 2000 spots in total by combining (overlapping) 2-D gels in the 4-7, 6-9 and 6-11 pH range. This method is useful for clinical studies of monocytes from a large number of patients due to its rapidity and reproducibility, which permits comparative analysis of normal versus pathological samples and which allows follow up of the expressed proteins of monocytes from each patient.     

New gas chromatographic-mass spectrometric method for the determination of urinary pyrethroid metabolites in environmental medicine

We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean-up, NaOH was added to the organic phase and the carboxylic acids were re-extracted into the aqueous phase. After acidification and extraction into n-hexane again, the metabolites were then derivatised to volatile esters using N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples.