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PubMed2004

Analysis of polyaniline oligomers by laser desorption ionization and solventless MALDI.

While direct laser desorption ionization of soluble polyaniline dried onto metal sample plates results in mass spectra that are similar to previously shown electrospray ionization data of similar samples, laser desorption of unsolubilized solid polyaniline results in major fragmentation of the phenyl rings. Solventless MALDI, a recently developed technique for insoluble or slightly soluble species, involves the use of only solid analyte and matrix during sample preparation. Solventless MALDI of solid polyaniline results in mass spectra that are similar to the direct laser desorption ionization spectra of the soluble oligomers with some larger molecular weight oligomers also being detected. Based on the matrix used, different series of polyaniline with dissimilar end groups are detected. The matrix also affects the percentages of benzenoid and quinoid units in the oligomers. Thus, solventless MALDI appears to be a promising new technique for the mass spectrometric analysis of low solubility, but industrially important, polyanilines.

Aniline Compounds↗
PubMed2004

Development of medium pressure laser ionization, MPLI. Description of the MPLI ion source.

A novel pulsed valve/ion source combination capable of time-resolved sampling from atmospheric pressure has been developed for use with laser ionization time of flight mass spectrometry. The source allows ionization extremely close to the nozzle of the pulsed valve, enabling ultra-sensitive detection of a number of compounds, e.g., NO, at mixing ratios <1 pptV. Furthermore, at analyte mixing ratios in the ppbV range, the temporal resolution of the system is in the sub-second regime, allowing time-resolved monitoring of highly dynamic and complex mixtures, e.g., human breath or reacting chemical mixtures in atmospheric smog chamber experiments. Rotational temperatures of approximately 50 K have been observed for analytes seeded in the supersonic jet expansion at a distance of 1 mm downstream of the nozzle orifice. The refinement of the original ion source has drastically reduced the impact of reflected laser light and the resultant electron impact signals previously observed. The general applicability of this technique is demonstrated here by coupling the source to commercially available as well as home-built time-of-flight mass spectrometers. Finally, we discuss the MPLI technique in view of the very recently introduced atmospheric pressure laser ionization (APLI) as well as the traditional jet-REMPI approach.

Atmospheric Pressure↗
PubMed2005

Ion-ion and ion-molecule reactions at the surface of proteins produced by nanospray. Information on the number of acidic residues and control of the number of ionized acidic and basic residues.

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 microM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1-10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an n(MAX) which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A- form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution.

Acetates↗
PubMed2004

Simultaneous determination of clozapine, olanzapine, risperidone and quetiapine in plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry.

UNLABELLED: Clozapine (CLZ), olanzapine (OLZ), risperidone (RIP) and quetiapine (QTP) have been widely used in the treatment of schizophrenia. However, no study (or little study) has been conducted to determine the four drugs simultaneously by the use of high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI). OBJECTIVE: To develop a sensitive method for simultaneous determination of CLZ, OLZ, RIP and QTP in human plasma by HPLC-MS/ESI. METHODS: The analytes were extracted twice by ether after samples had been alkalinized. The HPLC separation of the analytes was performed on a MACHEREY-NAGEL C(18) (2.0 mm x 125 mm, 3 microm, Germany) column, using water (formic acid: 2.70 mmol/l, ammonium acetate: 10 mmol/l)-acetonitrile (53:47) as mobile phase, with a flow-rate of 0.16 ml/min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. RESULTS: The calibration curves were linear in the ranges of 20-1000 ng/ml for CLZ and QTP, 1-50 ng/ml for OLZ and RIP, respectively. The average extraction recoveries for all the four analysts were at least above 80%. The methodology recoveries were higher than 91% for the analysts. The intra- and inter-day R.S.D. were less than 15%. CONCLUSION: The method is accurate, sensitive and simple for routine therapeutic drug monitoring (TDM) and for the study of the pharmacokinetics of the four drugs.

Antipsychotic Agents↗
PubMed2005

Simultaneous determination of fluoxetine, citalopram, paroxetine, venlafaxine in plasma by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI).

UNLABELLED: Fluoxetine, citalopram, paroxetine and venlafaxine have been widely used in the treatment of depression. However, no study has been conducted to determine the four drugs simultaneously by high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI). OBJECTIVE: To establish a new, rapid and sensitive HPLC-MS/ESI method for simultaneous determination and screening in human plasma of the four most commonly prescribed nontricyclic antidepressants: fluoxetine, citalopram, paroxetine and venlafaxine. METHODS: The analytes in plasma were extracted by solid-phase-extraction column after samples had been alkalinized. The HPLC separation of the analytes was performed on a MACHEREY-NAGEL C(18) (250 mmx4.6 mm, 5 microm, Germany) column, using water (formic acid 0.6 per thousand, ammonium acetate: 30 mmol/l)-acetonitrile (35:65, v/v) as mobile phase, with a flow-rate of 0.85 ml/min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. RESULTS: The calibration curves were linear in the 5.0-1000.0 ng/ml range for all compounds, all of them with coefficients of determination above 0.9900. The average extraction recoveries for all the four analytes were above 73.2%. The methodology recoveries were higher than 95.0%. The limits of detection (LODs) were 0.5, 0.3, 0.3 and 0.1 ng/ml for fluoxetine, citalopram, paroxetine and venlafaxine, respectively. The intra- and inter-day variation coefficients were less than 15.0%. CONCLUSION: The method is accurate, sensitive and simple for routine therapeutic drug monitoring (TDM) as well as toxicologic screening, and for the study of the pharmacokinetics and metabolism of the four drugs.

Antidepressive Agents, Tricyclic↗
PubMed2006

High performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine and its three metabolites in human plasma.

A high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-MS/ESI) method for simultaneous determination of venlafaxine (VEN) and its three metabolites O-desmethylvenlafaxine (ODV), N-desmethylvenlafaxine (NDV) and N,O-didesmethylvenlafaxine (DDV) in human plasma has been developed and validated. Estazolam was used as the internal standard. The compounds and internal standard were extracted from plasma by a liquid-liquid extraction. The HPLC separation of the analytes was performed on a Thermo BDS HYPERSIL C18 (250 mm x 4.6 mm, 5 microm, USA) column, using a gradient elution program with solvents constituted of water (ammonium acetate: 30 mmol/l, formic acid 2.6 mmol/l and trifluoroacetic acid 0.13 mmol/l) and acetonitrile (60:40, V/V) at a flow-rate of 1.0 ml/min. All of the analytes were eluted within 6 min. The compounds were ionized in the electrospray ionization (ESI) ion source of the mass spectrometer and were detected in the selected ion recording (SIR) mode. Calibration curves in spiked whole blood were linear from 4.0-700 ng/ml, 2.0-900 ng/ml, 3.0-800 ng/ml and 2.0-700 ng/ml for VEN, ODV, NDV and DDV, respectively, all of them with coefficients of determination above 0.9991. The average extraction recoveries for all the four analytes were above 77%. The methodology recoveries were higher than 91%. The limits of detection were 0.4, 0.2, 0.3, and 0.2 ng/ml for VEN, ODV, NDV and DDV, respectively. The intra- and inter-day variation coefficients were less than 11%. The method is accurate, sensitive and reliable for the pharmacokinetic study of venlafaxine as well as therapeutic drug monitoring (TDM).

Antidepressive Agents, Second-Generation↗
PubMed2006

Ionizing radiation to prevent arterial intimal hyperplasia at the edges of the stent: induces necrosis and fibrosis.

BACKGROUND: Although ionizing radiation has been proposed for the prevention of intimal hyperplasia in coronary and peripheral arteries in multicenter clinical trials, information is lacking on how irradiation affects arterial histology after stenting and especially how it affects the edges of the stent. We investigated intimal hyperplasia recasting with histological changes in arterial wall at the edges of the stent after arterial stenting followed by adequate external radiation for the prevention of intimal hyperplasia in pigs. MATERIALS AND METHODS: The aorta was experimentally stented in 30 pigs who were then assigned to two groups: irradiation with 20 Gy and a control group with no irradiation. The aorta was resected for morphometric and histological studies 6 weeks after procedure. RESULTS: Intimal thickness was reduced and the intima/media ratio was significantly lower in irradiated groups than in control pigs. In the irradiated group histological examination at the edges of the stent showed thin neointimal proliferation with an intact endothelium. In all sections analyzed in the 20-Gy irradiated group the vascular media at 45 days contained necrotic areas and fibrosis with calcifications. CONCLUSIONS: After arterial injury, adequate ionizing radiation effectively reduces neointimal thickening. Irradiation-induced histological changes include previously undetected recasting with necrosis and fibrosis at the arterial edges of the stent. The parietal recasting we observed in animal arteries irradiated at high doses is unclear and a cause of concern especially after clinical spontaneous dissection was recently reported. The use of ionizing radiation for the prevention of arterial restenosis awaits confirmation with a long-term follow-up including specific experimental histological analyses.

Animals↗
PubMed2005

Analysis of ionizing radiation-induced foci of DNA damage repair proteins.

Repair of DNA double-strand breaks by homologous recombination requires an extensive set of proteins. Among these proteins are Rad51 and Mre11, which are known to re-localize to sites of DNA damage into nuclear foci. Ionizing radiation-induced foci can be visualized by immuno-staining. Published data show a large variation in the number of foci-positive cells and number of foci per nucleus for specific DNA repair proteins. The experiments described here demonstrate that the time after induction of DNA damage influenced not only the number of foci-positive cells, but also the size of the individual foci. The dose of ionizing radiation influenced both the number of foci-positive cells and the number of foci per nucleus. Furthermore, ionizing radiation-induced foci formation depended on the cell cycle stage of the cells and the protein of interest that was investigated. Rad51 and Mre11 foci seemed to be mutually exclusive, though a small subset of cells did show co-localization of these proteins, which suggests a possible cooperation between the proteins at a specific moment during DNA repair.

Animals↗
PubMed2005

Effect of ionizing radiation on the pteridine metabolic pathway and evaluation of its cytotoxicity in exposed hospital staff.

Investigations carried out to estimate the effect of long-term occupational exposure to low levels of external ionizing radiation indicated that exposed hospital staff showed an increase in chromosome aberrations. The purpose of this study was to evaluate whether genomic instability or an alteration in pteridine synthesis could be used as a marker of the potential hazard of ionizing radiation in hospital workers. Twenty gamma-radiation- and 33 X-ray-exposed technicians working in radiotherapy and radio-diagnostic units were included in this study, along with 22 healthy matched individuals. Plasma concentrations of nitrite plus nitrate (NO(x)) were measured to estimate reactive nitrogen species. Urinary neopterin, biopterin and creatinine concentrations were measured by high-performance liquid chromatography to determine metabolic activity along the pteridine pathway. Sister chromatid exchange was used as a measure of mutagenicity. Apoptosis was evaluated morphologically and also with a DNA-fragmentation test. The plasma NO(x) levels of both gamma-radiation- and X-ray-exposed technicians were significantly higher than those of the healthy controls (p<0.05). While the urinary biopterin concentrations were significantly higher in radiation-exposed groups compared with the healthy subjects (p<0.05), urinary neopterin concentrations remained unchanged. The apoptosis rates of gamma-radiation- and X-ray-exposed workers were significantly elevated in comparison with those in the control group (both p<0.05). Also, the increase in sister chromatid exchange frequency was significant in each of the radiation-exposed groups (exposed groups versus controls; p<0.05). These results indicate that long-term exposure to low-dose ionizing radiation, even below the permitted levels, could result in increased oxidative stress, which may lead to DNA damage and mutagenicity.

Adult↗
PubMed2004

Matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF) mass spectrometry in lipid and phospholipid research.

The interest in the analysis of lipids and phospholipids is continuously increasing due to the importance of these molecules in biochemistry (e.g. in the context of biomembranes and lipid second messengers) as well as in industry. Unfortunately, commonly used methods of lipid analysis are often time-consuming and tedious because they include previous separation and/or derivatization steps. With the development of "soft-ionization techniques" like electrospray ionization (ESI) or matrix-assisted laser desorption and ionization time-of-flight (MALDI-TOF), mass spectrometry became also applicable to lipid analysis. The aim of this review is to summarize so far available experiences in MALDI-TOF mass spectrometric analysis of lipids. It will be shown that MALDI-TOF MS can be applied to all known lipid classes and the characteristics of individual lipids will be discussed. Additionally, some selected applications in medicine and biology, e.g. mixture analysis, cell and tissue analysis and the determination of enzyme activities will be described. Advantages and disadvantages of MALDI-TOF MS in comparison to other established lipid analysis methods will be also discussed.

Brain Chemistry↗
PubMed1997

Matrix-assisted laser desorption ionization mass spectrometry for the analysis of monosulfated oligosaccharides.

Sulfated oligosaccharides are an important class of compounds in the field of glycobiology. Mass spectrometric analysis of these molecules is challenging due to their readiness to dissociate in sample preparation and their tendency to fragment during ionization. Moreover, their presence in small quantity in biological systems poses additional problems. We report the development of a mass spectrometric method based on matrix-assisted laser desorption ionization (MALDI) in a time-lag focusing time-of-flight mass spectrometer for the analysis of monosulfated oligosaccharides. It is found that coumarin 120 is an excellent matrix for the analysis of monosulfated disaccharides, whereas the use of a mixture of coumarin 120 and 6-aza-2-thiothymine is very effective for the ionization of sulfated trisaccharides and tetrasaccharides including those containing N-acetylneuraminic acid. Molecular ions for a series of synthetic sulfo/sialo beta Gal(1-->3)GlcNAc and beta Gal(1-->4)GlcNAc structures can thus be observed with subpicomole detection sensitivity using a uniform microcrystal matrix/sample preparation procedure. It is demonstrated that, with this matrix formulation, the presence of a high amount of sodium chloride or sodium phosphate buffer, which is often the case for the HPLC fractionated samples, does not deteriorate the MALDI performance. The analysis of mixtures containing different types of oligosaccharides is also examined. It is found that different classes of oligosaccharides require different matrix preparation methods.

Azo Compounds↗
PubMed2001

Analysis of triacylglycerol positional isomers in food products as brominated derivatives by high-performance liquid chromatography coupled with a flame ionization detection.

Reversed-phase HPLC resolution and HPLC-flame ionization detection quantitation of model triacylglycerol positional isomer pairs (important in the study of food formulation lipids) after facile conversion to brominated derivatives is reported. The positional isomers in the triacylglycerol pairs were at least 98% resolved from each other during reversed-phase HPLC. Triacylglycerol quantitation obtained by HPLC-flame ionization detector was checked against standard positional isomer pairs known by mass. The flame ionization detection area percent gave absolute error range of 0.3-1.6% per triacylglycerol.

Bromine↗
PubMed2001

Ionizing radiation affects 26s proteasome function and associated molecular responses, even at low doses.

BACKGROUND AND PURPOSE: Ionizing radiation is known to activate certain signal transduction pathways, the regulation of which could involve post-transcriptional as well as transcriptional mechanisms. One of the most important post-transcriptional pathways in eukaryotic cells is the ATP- and ubiquitin-dependent degradation of proteins by the 26s proteasome. This process controls initiation of many cellular stress responses, as well as inflammatory responses under control of the transcription factor NF-kappaB. The literature on the relationship between radiation and inflammation seems somewhat paradoxical. At high doses, radiation is generally pro-inflammatory. On the other hand, low dose radiation has a long history of use in the treatment of inflammatory disease. This suggests the involvement of multiple mechanisms that may operate differentially at different dose levels. MATERIALS AND METHODS: In this paper, the ability of different doses of ionizing radiation to directly affect 26s proteasome activity was tested in ECV 304 cells. Proteasome activity, IkappaBalpha protein levels, and NF-kappaB activation were monitored. RESULTS: Inhibition of chymotrypsin-like 20s and 26s proteasome activity was observed immediately after low- and high-dose irradiation either of cells or purified proteasomes. The inhibitory effect was independent of the availability of the known endogenous proteasome inhibitor heat shock protein 90 (hsp90). Levels of IkappaBalpha, a physiological 26s proteasome substrate, were increased only at low doses (0.25 Gy) and unaltered at higher doses whereas only the highest doses (8 and 20 Gy) activated NF-kappaB. CONCLUSIONS: We conclude that the proteasome is a direct target of ionizing radiation and suggest that inhibition of proteasome function provides a molecular framework within which low dose anti-inflammatory effects of radiation, and radiation-induced molecular responses in general, should be considered.

Cells, Cultured↗
PubMed2002

Enhancement of nestin protein-immunoreactivity induced by ionizing radiation in the forebrain ependymal regions of rats.

Expression of nestin was immunohistochemically examined in the forebrains of rats receiving ionizing radiation. Nestin-immunoreactive cells were predominately distributed in ependymal regions. Nestin-immunoreactivity in ependymal regions of irradiated rats increased significantly from 1 to 4 weeks after ionizing radiation compared with that of controls. Double immunofluorescence confirmed that about 94% of nestin-positive cells exhibited glial fibrillary acidic protein-immunoreactivity and a minor population of them showed Ki-67-immunoreactivity in these regions. The results have provided evidence for up-regulation of nestin expression induced by ionizing radiation in ependymal cells, suggesting that these reactive ependymal cells may be involved in remodeling and repairing processes of brain irradiation injury.

Animals↗
PubMed1987

A nine-year evaluation of emergency department personnel exposure to ionizing radiation.

Emergency department personnel experience potential occupational hazards from exposure to ionizing radiation (x-rays). To assess this risk, ionizing radiation exposure was analyzed during a nine-year period for 128 ED personnel. The group consisted of 21 physicians, 92 nurses, and 15 ancillary personnel. Exposure was measured for both penetrating and nonpenetrating radiation using standard film dosimeter badges. Film badge use compliance was 66.7% for physicians, 86.2% for nurses, and 86.7% for ancillary personnel. Penetrating radiation exposure averaged 0.12 mrem/month for physicians, 0.70 mrem/month for nurses, and 0 mrem/month for ancillary personnel, all less than the average natural background exposure. We concluded that if standard radiation precautions are taken, the occupational risk from ionizing radiation exposure to personnel in the ED is minimal, and that routine monitoring of radiation exposure of ED personnel is unnecessary.

Allied Health Personnel↗
PubMed2003

Modulation of intercellular communication mediated at the cell surface and on extracellular, plasma membrane-derived vesicles by ionizing radiation.

The plasma membrane is a dynamic organelle whose function includes receptor-mediated signal transduction into the cell. Conversely, the plasma membrane is the origin of inter-cellular signaling. In addition to expressing and releasing growth factors in a soluble form(through exocytosis) and via proteolysis of cell surface components, membrane ligands may signal nearby cells through juxtacrine stimulation or by the exfoliation or shedding of plasma membrane-derived vesicles. Ionizing radiation (IR) has a profound effect on plasma membrane structure and function. IR-induced ultrastructural alterations are mediated via lipid interaction with water radiolysis products (e.g., hydroxyl radicals, hydrogen radicals, and hydrated electrons). Ionizing radicals act directly on lipid molecules to promote lipid hydro-peroxides and lipid hydroperoxide breakdown products (e.g., alpha, beta unsaturated aldehydes) that contribute to altered plasma membrane lipid composition. A change in lipid composition increases membrane lipid microviscosity and results in membrane fenestrations that enhance permeability to small molecules and ions. Reactive ionizing species also stimulate sphingomyelinase activity, leading to sphingomyelin hydrolysis and ceramide generation that further contributes to altered membrane lipid composition and cellular apoptosis. In addition, exposure to IR results in impaired rate of and cumulative shedding of plasma membrane-associated growth factors. Mechanisms of exfoliation are reviewed for normal cells and the impact of radiation on modulating signal transduction mediated by exfoliation is summarized.

Apoptosis↗
PubMed1999

Expression of synaptic proteins in the developing rat cerebellum following ionizing radiation.

Various proteins regulating neurotransmission release and synaptic vesicle exocytosis have been implicated in axonal elongation and synaptic maturation. In the present study, immunohistochemistry to the presynaptic membrane proteins syntaxin-I and synaptosomal-associated protein of 25 kDa (SNAP-25) synaptic vesicle-associated proteins synaptophysin and synapsin-I and the neuronal maturation and axonal growth-related protein GAP-43, has been carried out in the normal developing cerebellum and following a single dose of ionizing radiation (2 Gy gamma-rays) at postnatal day 1. Our aim has been to learn about the morphological and possible functional modalities that occur during the progression of neuronal connectivity in normal and abnormal development. Expression of all these proteins is associated with the arrival of afferents in the subcortical white matter and with the maturation of the internal granule cell layer and molecular layer during normal development. In addition, SNAP-25 and GAP-43 are strongly expressed in granule cells of the external granule cell layer, thus suggesting that these proteins are involved in cell elongation of granule cells. Apoptosis appears at 3 h and peaks at 6 h following ionizing radiation. Radiation-induced apoptosis in the external granule cell layer produces a transient decrease in the expression of SNAP-25 and GAP-43 in the external granule cell layer. The external granule cell layer recovers at 48 h and external granule cells of proliferating cells also express SNAP-25 and GAP-43, thus indicating that proliferating cells in this layer are equipped with proteins involved in cell elongation. Furthermore, expression of synaptophysin, synapsin-I, syntaxin-I and SNAP-25 is the same in the cerebellum of irradiated and normal rats from this time to adulthood (3 months). These results point to the likelihood that recovery of the cerebellar cortex occurs following a single exposure of ionizing radiation during postnatal development.

Aging↗
PubMed2001

Involvement of the pathway phosphatidylinositol-3-kinase/AKT-1 in the establishment of the survival response to ionizing radiation.

Ionizing radiation is one of the agents inducing activation of DNA repair, cell cycle arrest, apoptosis and cell death. Here we report evidence for an enhanced activity of DNA polymerase beta, one of the repair enzymes, concomitant to the activation of the pathway phosphatidylinositol-3-kinase/AKT-1 (PI-3-kinase/AKT-1), which delivers a survival signal in Friend erythroleukemia cells exposed to 15 Gy. Significantly, the preincubation of the cellls with PI-3-kinase inhibitors wortmannin and LY 294002, disactivating this pathway, sensitizes the cells to ionizing radiation by further reducing the rate of proliferation without substantial variations of the number of dead cells. Thus, we suggest a role for these enzymes in maintaining survival programs upon exposure to ionizing radiation and in giving to these cells a chance to recover from this stress.

Androstadienes↗