tempo and tempol

We have developed novel photopolymer gels to function as separators in blood collection tubes. By incorporating antioxidants such as α-tocopherol and nitroxides (TEMPO and TEMPOL), the new formulation can be sterilized with electron beam or gamma rays at a dose level of 17 kGy, without inducing premature curing of the photopolymers. For the blood separator gels that contain α-tocopherol, our results show that α-tocopherol plays a decisive role in impeding C-centered free radical propagation reactions through an H

Topics: alpha-Tocopherol; Animals; Antioxidants; Cyclic N-Oxides; Gamma Rays; Gels; Materials Testing; Polymers; Spin Labels; Sterilization; Ultraviolet Rays; X-Rays

Known since the ancient times, cotton continues to be one of the essential materials for the human civilization. Cotton fibers are almost pure cellulose and contain both crystalline and amorphous nanodomains with different physicochemical properties. While understanding of interactions between the individual cellulose chains within the crystalline phase is important from a perspective of mechanical properties, studies of the amorphous phase lead to characterization of the essential transport parameters, such as solvent diffusion, dyeing, drug release, and toxin absorption, as well as more complex processes of enzymatic degradation. Here, we describe the use of spin probe electron paramagnetic resonance methods to study local polarity and heterogeneous viscosity of two types of unprocessed cotton fibers, G. hirsutum and G. barbadense, harvested in the State of North Carolina, USA. These fibers were loaded with two small molecule nitroxide probes that differ in polarity-Tempo and its more hydrophilic derivative Tempol-using a series of polar and non-polar solvents. The electron paramagnetic resonance spectra of the nitroxide-loaded cotton fibers were analyzed both semi-empirically and by least-squares simulations using a rigorous stochastic theory of electron paramagnetic resonance spectra developed by Freed and coworkers. A software package and least-squares fitting protocols were developed to carry out automatic simulations of multi-component electron paramagnetic resonance spectra in both first-derivative and the absorption forms at multiple resonance frequencies such as X-band (9.5 GHz) and W-band (94.3 GHz). The results are compared with the preceding electron paramagnetic resonance spin probe studies of a commercial bleached cotton sheeting carried out by Batchelor and coworkers. One of the results of this study is a demonstration of a co-existence of cellulose nanodomains with different physicochemical properties such as polarity and microviscosity that are affected by solvents and temperature. Spin labeling studies also revealed a macroscopic heterogeneity in the domain distribution along the cotton fibers and a critical role the cuticular layer is playing as a barrier for spin probe penetration. Finally but not lastly, the simultaneous multi-component least-squares simulation method of electron paramagnetic resonance spectra acquired at different resonant frequencies and the display forms (e.g., absorption and first-derivative displays) and the str

Topics: Cellulose; Cotton Fiber; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Least-Squares Analysis; Solvents; Spin Labels; Temperature; Viscosity

The reactivity of small molecules in emulsions is believed to depend on their partitioning between phases, yet this is hard to verify experimentally in situ. In the present work, we use electron paramagnetic resonance (EPR) spectroscopy to simultaneously measure the distribution and reactivity of a homologous series of lipophilized spin probes in an emulsion.. 4-Hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPOL) was derivatized with saturated fatty acids to create a series of spin probes with increasing lipophilicity (C4-, C8-, C12-, and C16-TEMPO). The probes were added to a 10 wt.% tetradecane-in water emulsions (d32∼190 nm) stabilized with sodium caseinate (1 wt.% in the aqueous phase, pH 7). The distribution of the probes between phases was measured by electron paramagnetic resonance (EPR) spectroscopy.. TEMPOL partitioned into the aqueous phase, C4-TEMPO distributed between the lipid and aqueous phases (69% and 31% respectively) while the more lipophilic probes dissolved exclusively within the lipid droplets. Interestingly, the more lipophilic probes initially precipitated upon their addition to the emulsion, and only slowly redistributed to the droplets over hours or days, the rate of which was dependent on their carbon chain length. The reactivity of the probes with aqueous an aqueous phase reductant (ascorbate) generally depended on the proportion in the aqueous phase (i.e., TEMPOL>C4-TEMPO>C8-TEMPO∼C12-TEMPO∼C16-TEMPO).

Topics: Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Emulsions; Spin Labels

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.

Topics: Animals; Cryopreservation; Cryoprotective Agents; Cyclic N-Oxides; Male; Oxidative Stress; Semen Analysis; Semen Preservation; Sheep; Spermatozoa; Spin Labels

The main objective was to study the effects, on sperm function, of the addition of two superoxide dismutase (SOD) analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. Twelve alpaca semen samples were collected using an artificial vagina and then diluted at a 1:3 ratio in an extender based on skim milk, egg yolk, and fructose. Each semen sample was divided into three equal parts to form the following groups: control, Tempo (1 mM), and Tempol (1 mM). Groups were cooled to 5 °C in 90 minutes (-1 °C in 3 minutes); when samples reached approximately 10 °C, SOD analogues were added to the respective groups. At 5 °C, ethylene glycol (final concentration, 0.1 M) was added to each group. After 30 minutes at 5 °C, samples were loaded in 0.25 mL plastic straws, placed in liquid nitrogen vapor for 15 minutes, and then plunged. Percentages of sperm motility, functional sperm membrane integrity, and viable sperm with intact acrosomes were evaluated before and after freeze-thaw using visual analysis, the hypoosmotic swelling test, and the double-stain trypan blue/giemsa technique, respectively. The Terminal deoxymucleotidyl transferase dUTP Nick End Labeling assay was performed for evaluation of sperm DNA fragmentation of frozen-thawed sperm. Sperm motility was higher (P < 0.05) in the Tempol and Tempo groups than in the control group (mean, 22.1%, 19.7%, and 11.2%, respectively), with similar results for functional sperm membrane integrity. Additionally, DNA fragmentation was lower (P < 0.05) in the Tempol group (16.7%) than in the control group (38.8%). Viable sperm with intact acrosomes were not affected by the use of SOD analogues. There was a negative correlation (r = -0.58) between DNA fragmentation of alpaca sperm and sperm motility after freeze-thawing, but DNA damage was neither related to functional membrane integrity nor viable sperm with intact acrosomes. We concluded that DNA fragmentation and loss of motility during cryopreservation of alpaca sperm could be partially prevented by supplementation of the semen extender with 1 mM Tempo or Tempol.

Topics: Acrosome; Animals; Antioxidants; Camelids, New World; Cryopreservation; Cyclic N-Oxides; DNA Fragmentation; Male; Semen; Semen Preservation; Sperm Motility; Spin Labels; Superoxide Dismutase

Electron spin resonance spectroscopy and mass spectrometry are two analytical methods that are very rarely used in combination. In this paper, we will show that the methods complement one another in the example of the distribution of stable nitroxide radicals in human skin, including the spatial resolution of these distribution processes. There are many ESR investigations dealing with this subject, but unfortunately, they are all limited to the detection of paramagnetic species. The combination with MS allows the successful examination of the distribution profile of the main biotransformation product of the nitroxide radicals, the respective "ESR-silent" hydroxylamines. In order to maintain the biological state of the sample material as far as possible, atmospheric pressure matrix-assisted laser desorption/ionization with ion trap detection has been used for the mass spectrometric investigations. The results validate the former findings of the strong reduction of stable free radicals by biological material; moreover, the diamagnetic species formed during these processes have been identified.

Topics: Antioxidants; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Female; Humans; Mass Spectrometry; Nitrogen Oxides; Skin; Spin Labels

The molecular dynamics of spin-labeled compounds included into the solid phase of cyclodextrins (CDs) has been studied using conventional (X-band) ESR at 9 GHz and high-field high-frequency (HFHF) ESR at 240 and 170 GHz. The patterns of axial rotation at these higher frequencies are clear just by inspection of the spectrum, unlike the case for 9 GHz spectra. That is HFHF ESR is sensitive to molecular motion about the diffusion axis collinear with the X, Y or Z-direction of the magnetic g- and A-tensors of the nitroxide moiety (referred to, respectively, as X, Y or Z-rotation). For doxyl stearic acids (Z-rotation) and TEMPOyl caprylate (X-rotation) included in beta- and gamma-CDs we were able to determine the rate of molecular motion and the corresponding potential barriers. We emphasize that determining the rate of Z-rotation by ESR is feasible only using HFHF ESR. For the X-rotation case we suggest that the motion of the nitroxide moiety consists of fast small-angle librations about the magnetic X-axis superimposed by rotational diffusion about the same axis. The potential barrier of 1.7 Kcal mol(-1) for this rotational diffusion is unusually low. A fascinating feature of TEMPO derivatives included in beta-CD is the detectable molecular motion at temperatures below 77 K. For the other CD-spin probe systems, we used multifrequency analysis to assign the conformations of spin-labeled molecules. A dramatic spectral change for 16-sasl in beta- and gamma-CDs at approximately 260 K corresponds to a tilting of the position of the nitroxide moiety on the rotating molecule relative to the long diffusion axis, while for TEMPO derivatives in gamma-cyclodextrin below 200 K, we observe a rapid transition from fast to very slow rotational motion. More complex features are best studied by means of multifrequency ESR experiments. The visual clarity and the simplicity of analysis of the ESR spectra shown in this work should provide a benchmark for future studies of molecular motion by HFHF ESR.

Topics: beta-Cyclodextrins; Caprylates; Cyclic N-Oxides; Cyclodextrins; Electron Spin Resonance Spectroscopy; Fatty Acids; gamma-Cyclodextrins; Models, Molecular; Palmitic Acids; Spin Labels; Stearic Acids; Temperature

The possibilities of electron spin resonance (ESR) and electron spin resonance imaging (ESRI) for investigating the properties of the spin probes TEMPO and TEMPOL in two-phase systems have been examined in the systems water/n-octanol, Miglyol/Miglyol, and Precirol/Miglyol. Phases and regions of the phase boundary could be mapped successfully by means of the isotropic hyperfine coupling constants, and, moreover, the quantification of rotational and lateral diffusion of the spin probes was possible. For the quantitative treatment of the micropolarity, a simplified empirical model was established on the basis of the Nernst distribution and the experimentally determined isotropic hyperfine coupling constants. The model does not only describe the summarized micropolarities of coexisting phases, but also the region of the phase boundary, where solvent molecules of different polarities and tendencies to form hydrogen bonds compete to interact with the NO group of the spin probe.

Topics: Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Hydrogen Bonding; Models, Chemical; Phase Transition; Solvents; Spin Labels

The spin probes 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and 2,2,6,6-tetramethyl-4-trimethylammoniumpiperidine-1-oxylIodide (CAT-1) are examined in a number of ionic liquids based on substituted imidazolium cations and tetrafluoroborate and hexafluorophosphate anions, respectively. The reorientation correlation times tau(R) of the spin probes in these systems have been determined by complete spectra simulation and, for rapid reortientation, by analysis of the intensities of the hyperfine lines of the electron spin resonance (ESR) spectra. A comparison of the results with those from the model system glycerol/water and selected organic solvents is made. Additions of diamagnetic and paramagnetic ions allow the conclusion that salt effects and spin exchange are present, and that both are superimposed by motional effects. Specific interactions in the ionic liquids, as well as between the spin-probe molecules and the constituents of the ionic liquids are reflected in the spectra of the spin probes, depending on their molecular structure.

Topics: Anions; Cations; Chemistry, Physical; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Ions; Magnetics; Models, Chemical; Molecular Conformation; Oxides; Spin Labels; Temperature; Time Factors

Intensity of EPR spectra of stable organic free radicals, nitroxides, is decreasing with time if the radicals are dissolved in beer. The process is determined by a chemical reaction of nitroxide reduction by components naturally present in beer. Kinetics can be described as a simple irreversible first order with respect to both nitroxide and one reducing agent. Effective concentration of the reducing agent and the corresponding reaction rate constant has been determined. It is demonstrated that the nitroxide reduction is sensitive to the presence of solvent-accessible SH groups of proteins present in beer. It is proposed that quantitative analysis of reduction kinetics of small water-soluble nitroxide radicals such as TEMPO and TEMPOL can be used to assess the reducing power of beer. The effect of accelerated aging of beer achieved at elevated temperatures on nitroxide reduction kinetics is demonstrated.

Topics: Beer; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Kinetics; Oxidation-Reduction; Spin Labels; Sulfhydryl Compounds; Triacetoneamine-N-Oxyl

Stable nitroxide radicals are potent antioxidants and are among the most effective non-thiol radioprotectants, although they react with hydroxyl radicals more slowly than typical phenolic antioxidants or thiols. Surprisingly, the reduced forms of cyclic nitroxides, cyclic hydroxylamines, are better reductants yet have no radioprotective activity. To clarify the reason for this difference, we studied the kinetics and mechanisms of the reactions of nitroxides and their hydroxylamines with (*)OH radicals and with OH-adducts by using pulse radiolysis, fluorimetric determination of phenolic radiation products, and electron paramagnetic resonance spectrometric determination of nitroxide concentrations following radiolysis. Competition kinetics with phenylalanine as a reference compound in pulse radiolysis experiments yielded rate constants of (4.5 +/- 0.4) x 10(9) M(-1) s(-1) for the reaction of (*)OH radical with 2,2,6,6-tetramethylpiperidine-N-oxyl (TPO), 4-hydroxy-TPO (4-OH-TPO), and 4-oxo-TPO (4-O-TPO), (3.0 +/- 0.3) x 10(9) M(-1) s(-1) for deuterated 4-O-TPO, and (1.0 +/- 0.1) x 10(9) M(-1) s(-1) for the hydroxylamine 4-OH-TPO-H. The kinetic isotope effect suggests the occurrence of both (*)OH addition to the aminoxyl moiety of 4-O-TPO and H-atom abstraction from the 2- or 6-methyl groups or from the 3- and 5-methylene positions. This conclusion was further supported by final product analysis, which demonstrated that (*)OH partially oxidizes 4-O-TPO to the corresponding oxoammonium cation. The rate constants for the reactions of the nitroxides with the OH-adducts of phenylalanine and terephthalate have been determined to be near 4 x 10(6) M(-1) s(-1), whereas the hydroxylamine reacted at least 50 times slower, if at all. These findings indicate that the reactivity toward (*)OH does not explain the differences between the radioprotective activities of nitroxides and hydroxylamines. Instead, the radioprotective activity of nitroxides, but not of hydroxylamines, can be partially attributed to their ability to detoxify OH-derived secondary radicals.

Topics: Antioxidants; Azides; Cyclic N-Oxides; Hydroxyl Radical; Hydroxylamines; Kinetics; Nitrogen Oxides; Spin Labels

Bull sperm are exposed to aerobic conditions during processing before freezing, and they have little endogenous antioxidant to protect them against reactive oxygen species that may be present. Seventeen laboratory studies and two field trials were conducted with 174 semen collections from bulls in an artificial breeding cooperative. More than 250 combinations and concentrations of reduced glutathione (GSH), superoxide dismutase (SOD), ascorbic acid, hypotaurine (HPT), 2,2,6,6-tetramethylpeperidine-1-oxyl (Tempo) and 4-hydroxy-2, 2, 6, 6-tetramethylpeperidine (Tempol) were tested by adding these compounds to fresh semen, and to a whole milk (WM) glycerol extender. Semen packaged in straws in the WM extender was frozen with liquid nitrogen. The motility of frozen-thawed sperm during storage at 25 or 5 degrees C after freezing was compared with semen stored without freezing. Antioxidants generally were not beneficial, except the percentage of motile sperm was improved by 6-11% units (P<0.05) when sperm were stored unfrozen or after freezing when 0.5mM of GSH with or without SOD was added. In two field trials, non-return rates were 71.9, 69.5 and 70.9% (P>0.05) with WM containing 0.0, 0.5 and 1.0mM of GSH, respectively, and 74.0 and 73.9% with WM and WM plus 0.5mM of GSH and 100U/ml of SOD (P>0.05). WM contains an abundant supply of casein which is an antioxidant, and additional antioxidants were ineffective in improving motility of sperm immediately after freezing and thawing or in affecting fertility. However, sperm responses were different in egg yolk-Tris extender. Sperm in this egg yolk extender tolerated substantial concentrations of Tempo and Tempol compared with toxic effects in WM (P<0.05). Therefore, optimal combinations of antioxidants tested here may have more useful applications in organizations using an egg yolk-based semen extender.

Topics: Animals; Antioxidants; Ascorbic Acid; Cattle; Cryopreservation; Cyclic N-Oxides; Egg Yolk; Fertility; Glutathione; Glycerol; Male; Milk; Oxidation-Reduction; Reactive Oxygen Species; Semen Preservation; Sperm Motility; Spermatozoa; Spin Labels; Superoxide Dismutase; Taurine

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.

Topics: Amidines; Animals; Cattle; Copper; Cyclic N-Oxides; DNA; DNA Damage; DNA, Bacterial; DNA, Recombinant; Free Radical Scavengers; Free Radicals; Heterocyclic Compounds, 2-Ring; Hydrogen Peroxide; Hydroxylamines; Iron; Oxidants; Oxidative Stress; Piperidines; Quinolones; Spin Labels; Triazoles

We previously reported on the ability of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a ultraviolet A (UVA)-absorbing sunscreen, to generate free radicals upon illumination, and as a consequence, to inflict strand breaks in plasmid DNA in vitro. This study has now been extended to determine the effects of Parsol 1789 and DBM on proteins, under UVA illumination, with the sole purpose of gaining more knowledge on the photobiological effects of sunscreen chemicals. Parsol 1789 (100 microM) caused a 2-fold increase in protein carbonyl formation (an index of oxidative damage) in bovine serum albumin (BSA) when exposed to illumination, and this damage was both concentration- and time-dependent. The degree of protein damage was markedly reduced by the presence of free radical scavengers, namely piperidinic and indolinonic nitroxide radicals, in accordance with our previous study. Vitamin E had no effect under the conditions used. The results obtained corroborate the fact that Parsol 1789 generates free radicals upon illumination and that these are, most probably, responsible for the protein damage observed under the conditions used in our system. However, at present, we cannot extrapolate from these results the relevance to human use of sunscreens; therefore, further studies should be necessary to determine the efficacy at the molecular and cellular level of this UVA-absorber in order to ascertain protection against photocarcinogenic risk.

Topics: Animals; Antioxidants; Benzoates; Chalcones; Cyclic N-Oxides; Free Radicals; Kinetics; Mice; Nitrogen Oxides; Oxidation-Reduction; Propiophenones; Serum Albumin, Bovine; Spin Labels; Sunscreening Agents; Ultraviolet Rays; Vitamin E

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.

Topics: Antioxidants; Benzoates; Chalcones; Cyclic N-Oxides; DNA; DNA Damage; DNA, Bacterial; Electron Spin Resonance Spectroscopy; Humans; Indoles; Kinetics; Nitrogen Oxides; Spin Labels; Sunscreening Agents; Ultraviolet Rays

Metabolism of different nitroxides with piperidine structure used as spin labels in electron spin resonance (ESR) studies in vitro and in vivo was investigated in human keratinocytes of the cell line HaCaT by GC and GC-MS technique combined with S-band ESR. Besides the well known reduction of the nitroxyl radicals to the ESR silent hydroxylamines as primary products our results indicate the formation of the corresponding secondary amines. These reductions are inhibited by the thiol blocking agent N-ethylmaleimide and by the strong inhibitors of the thioredoxin reductase (TR) 2-chloro-2,4-nitrobenzene and 2,6-dichloroindophenol. The competitive inhibitor TR inhibitor azelaic acid and the cytochrome P-450 inhibitor metyrapone lack any effects. The rates of reduction to the hydroxylamines and secondary amines were dependent on the lipid solubility of the nitroxides. Therefore, it can be assumed that the nitroxides must enter the cells for their bioreduction. The mostly discussed intracellular nitroxide reducing substances ascorbic acid and glutathione were unable to form the secondary amines. In conclusion, our results suggest that the secondary amine represents one of the major metabolites of nitroxides besides the hydroxylamine inside keratinocytes formed via the flavoenzyme thioredoxin reductase most probably. Further metabolic conversions were detected with 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl and the benzoate of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl as substrates.

Topics: Cell Line; Cell Survival; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Gas Chromatography-Mass Spectrometry; Humans; Keratinocytes; Molecular Structure; Nitrogen Oxides; Piperidines; Spin Labels; Structure-Activity Relationship

Measurements of 7Li nuclear spin-lattice relaxation times are made at applied magnetic field strengths from 0.25 mT to 7.05 T, in order to determine directly the form of the frequency-dependent spectral densities that modulate relaxation. This magnetic resonance dispersion (MRD) technique provides detailed information regarding molecular dynamics down to the picosecond time scale. 7Li MRD measurements made on aqueous lithium ion in the presence of small concentrations of nitroxide free radicals give direct evidence supporting the formation of a coordination complex. The dipole-dipole electron-nuclear coupling is modulated by both translational and rotational diffusive motions, and both of these contributions are resolved. However, scalar coupling arising from the presence of paramagnetic electron spin density at the nucleus dominates the nuclear relaxation. Changes in the pH and free radical moiety are compared with dynamical variables, geometric constraints, and formation constants obtained from a model of nuclear relaxation. The calculated bimolecular formation constants are on the order of 2 x 10(-3) M-1, and the relative accuracy of this parameter is tested.

Topics: Algorithms; Cyclic N-Oxides; Diffusion; Electron Spin Resonance Spectroscopy; Free Radicals; Hydrogen-Ion Concentration; Isotopes; Lithium; Magnetics; Models, Chemical; Molecular Conformation; Rotation; Spectrum Analysis; Spin Labels; Water

Tempol and tempo are stable free radical nitroxides that possess antioxidant properties. In this study, we examined the effects of these compounds on components of the mitogen-activated protein kinase signal transduction cascade. Tempo treatment (15 min) of MDA-MB 231 human breast cancer cells resulted in significant levels of tyrosine phosphorylation of several as yet unidentified proteins compared with equimolar concentration of tempol (10 mM). Both compounds caused tyrosine phosphorylation and activation of Raf-1 protein kinase (30 min, 2-3-fold). Interestingly, however, only tempol caused increased extracellular signal-regulated kinase 1 activity (2 h, approximately 3-fold). On the other hand, tempo, but not tempol, potently activated stress-activated protein kinase (2 h, >3-fold). Consistent with these data, tempol was found to be noncytotoxic, whereas tempo induced apoptotic cell death (2 h, >50%). Tempo treatment also resulted in significant elevation of ceramide levels at 30 min (54% over control) and 1 h (71% over control) posttreatment, preceding stress-activated protein kinase activation and apoptosis. These data suggest that in the absence of an environmental oxidative stress, tempol and tempo elicit distinct cellular signaling pathways. The recognition of the molecular mechanisms of nitroxide action may have important implications for biological effectiveness of these compounds.

Topics: Antioxidants; Apoptosis; Breast Neoplasms; Calcium-Calmodulin-Dependent Protein Kinases; Ceramides; Cyclic N-Oxides; Enzyme Activation; Humans; JNK Mitogen-Activated Protein Kinases; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Phosphorylation; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-raf; Signal Transduction; Spin Labels; Tumor Cells, Cultured

The EPR spectra of nitroxide spin labels have been simulated as a function of microwave field, H1, taking into account both magnetic field modulation and molecular rotation. It is found that the saturation of the second integral, S, of the first harmonic in-phase absorption spectrum is approximated by that predicted for slow-passage conditions, that is, S approximately H1/1 + PH21, in all cases. This result is independent of the degree of inhomogeneous broadening. In general, the fitting parameter, P, depends not only on the T1 and T2 relaxation times, but also on the rate of molecular reorientation and on the modulation frequency. Calibrations for determining the relaxation times are established from the simulations. For a given modulation frequency and molecular reorientation rate, the parameter obtained by fitting the saturation curves is given by 1/P = a + 1/gamma2eT1 . Teff2, where Teff2 is the effective T2. For molecular reorientation frequencies in the range 2 x 10(7)-2 x 10(8) s-1, Teff2 is dominated by the molecular dynamics and is only weakly dependent on the intrinsic T02, allowing a direct estimation of T1. For reorientation frequencies outside this range, the (T1T2) product may be determined from the calibrations. The method is applied to determining relaxation times for spin labels undergoing different rates of rotational reorientation in a variety of environments, including those of biological relevance, and is verified experimentally by the relaxation rate enhancements induced by paramagnetic ions.

Topics: Absorption; Algorithms; Calibration; Computer Simulation; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Forecasting; Glycerol; Lipid Bilayers; Lipids; Magnetics; Membrane Fluidity; Microwaves; Models, Chemical; Molecular Structure; Rotation; Spin Labels; Time Factors; Triacetoneamine-N-Oxyl

Free radicals are associated with post-ischaemic intestinal injury and contribute to major clinical problems primarily in premature infants. Various antioxidative means and modes of intervention, previously tested, have demonstrated only limited efficacy.. To study the protective activity of the stable nitroxide radical 4-OH, 2,2,6,6-tetramethylpiperidine-1-oxyl (TPL) and its respective hydroxylamine (TPL-H) against ischaemia/reperfusion (I/R) injury.. An isolated loop of ileum was created in laboratory male Sabra rats and constantly perfused with warmed normal saline. Intestinal injury was elicited through clamping of the superior mesenteric rat artery followed by reperfusion. Either TPL or TPL-H was given intravenously immediately before ischaemia or reperfusion and continuously afterwards. The rate of mucosal to lumen clearance of para-aminohippurate (PAH) was used to evaluate intestinal mucosal injury. Serum and perfusate levels of both TPL and TPL-H were measured using electron paramagnetic resonance spectrometry.. The increase in intestinal permeability induced by I/R was significantly inhibited by both TPL and TPL-H. The nitroxide was effective also when given immediately before reperfusion.. Through a continuous exchange, TPL and TPL-H act as self-replenishing antioxidants and thus protect from intestinal injury. This demonstrates the potential of the family of nitroxide antioxidants against oxidative stress in general and I/R injury in particular.

Topics: Animals; Antioxidants; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Intestinal Absorption; Intestinal Mucosa; Intestine, Small; Male; p-Aminohippuric Acid; Rats; Rats, Inbred Strains; Reperfusion Injury; Spin Labels

The present study focused on protective activity of two six-membered-ring nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-Tempo (Tempol), against radiation damage to acyl chain residues of egg phosphatidylcholine (EPC) of small unilamellar vesicles (SUV). SUV were gamma-irradiated (10-12 kGy) under air at ambient temperature in the absence and presence of nitroxides. Acyl chain composition of the phospholipids before and after irradiation was determined by gas chromatography. Both Tempo and Tempol effectively and similarly protected the acyl chains of EPC SUV, including the highly sensitive polyunsaturated acyl chains, C20:4, C22:5, and C22:6. The conclusions of the study are: (a) The higher the degree of unsaturation in the acyl chain, the greater is the degradation caused by irradiation. (b) The fully saturated fatty acids palmitic acid (C16) and stearic acid (C18) showed no significant change in their levels. (c) Both Tempo and Tempol provided similar protection to acyl chain residues. (d) Nitroxides' lipid-bilayer/aqueous distribution is not validly represented by their n-octanol/saline partition coefficient. (e) The lipid-bilayer/aqueous partition coefficient of Tempo and Tempol cannot be correlated with their protective effect. (f) The nitroxides appear to protect via a catalytic mode. Unlike common antioxidants, such as alpha-tocopherol, which are consumed under irradiation and are, therefore, less effective against high radiation dose, nitroxide radicals are restored and terminate radical chain reactions in a catalytic manner. Furthermore, nitroxides neither yield secondary radicals upon their reaction with radicals nor act as prooxidants. Not only are nitroxides self-replenished, but also their reduction products are effective antioxidants. Therefore, the use of nitroxides offers a powerful strategy to protect liposomes, membranes, and other lipid-based assemblies from radiation damage.

Topics: Antioxidants; Arachidonic Acid; Cell Membrane; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Fatty Acids, Unsaturated; Free Radicals; Gamma Rays; Lipid Bilayers; Lipid Peroxidation; Liposomes; Molecular Structure; Nitrogen Oxides; Phosphatidylcholines; Reactive Oxygen Species; Spin Labels

The present study aims to determine the effect of bilayer composition on oxidative damage and the protection against it in lipid multicomponent membranes. Irradiation damage in 200-nm liposomes and the protection provided by the nitroxide radicals, 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo) and 4-hydroxy-2,2,6,6-tetramethylpiperidine--1-oxyl (Tempol) were assessed by monitoring several chemical and physical parameters. Liposomes were prepared in four different lipid compositions (mole ratios), DPPC:DPPG 10:1; DPPC:DPPG:cholesterol 10:1:4; EPC:EPG 10:1; and EPC:EPG:cholesterol 10:1:4, and gamma-irradiated with a dose of 32 kGy. Lipid degradation was determined by HPLC and GC analyses, whereas size and differential scanning calorimetry measurements were used to monitor physical changes in the liposomal dispersions. The results indicate that: (1) addition of 5 mM Tempo or Tempol, or freezing of the sample inhibited radiation-induced lipid degradation; (2) Tempo and Tempol caused neither physical nor chemical changes in the liposomal dispersions; and (3) both nitroxides prevented or reduced some of the radiation-induced changes in thermotropic characteristics of the liposomes, preventing a shift in the temperature of the maximum of the main phase transition.

Topics: Acylation; Cryopreservation; Cyclic N-Oxides; Free Radicals; Gamma Rays; Hydrogen-Ion Concentration; Lipid Peroxidation; Liposomes; Phospholipids; Radiation-Protective Agents; Spin Labels

Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike the others, the apolar TEMPO radical does not enter the cavity but instead binds to ferritin, presumably at a hydrophobic region of the protein. The kinetic data indicate that diffusion is not purely passive, the driving force coming not only from the concentration gradient between the inside and outside of the protein but also from charge interactions between the diffusant and the protein. A model for diffusion is derived that describes the observed kinetics. First-order half-lives for diffusion into the protein of 21-26 min are observed, suggesting that reductant molecules with diameters considerably larger than approximately 9 A would probably enter the protein cavity too slowly to mobilize iron efficiently by direct interaction with the mineral core.

Topics: Animals; Binding Sites; Biophysical Phenomena; Biophysics; Cyclic N-Oxides; Diffusion; Electrochemistry; Electron Spin Resonance Spectroscopy; Ferritins; Free Radicals; Horses; Kinetics; Nitrogen Oxides; Spin Labels; Spleen

Nitroxides are antioxidant compounds that have been shown to provide radioprotection in vivo and in vitro. Radioprotection in vivo is limited by toxicity, which appears to be neurologic in nature. To further evaluate the toxicity of these compounds, three representative nitroxides, Tempol, Tempamine, and Tempo, were examined in slices of guinea pig hippocampus. Each nitroxide increased the population spike and caused potentiation of excitatory postsynaptic potential--spike coupling. Repetitive activity and epileptiform activity were observed at the highest concentrations of Tempo and Tempamine. Tempol was the least toxic compound in this system, followed by Tempamine and Tempo. Additional studies are necessary to further define the effects of nitroxides on the central nervous system and to develop strategies to mitigate these effects.

Topics: Animals; Antioxidants; Cyclic N-Oxides; Electrophysiology; Epilepsy; Evoked Potentials; Guinea Pigs; Hippocampus; In Vitro Techniques; Male; Microelectrodes; Nitrogen Oxides; Spin Labels

By stopped-flow kinetic analysis, we have directly evaluated the superoxide dismutase (SOD) activity of a number of organic nitroxides and iron- and manganese-based complexes that have been attributed with having SOD activity based upon competition experiments with cytochrome c. In 60 mM HEPES buffer, pH 8.1, or 50 mM potassium phosphate buffer, pH 7.8, Mn(II) and manganese complexes of desferal had no detectable SOD activity by stopped-flow analysis (catalytic rate constant (kcat) < 10(5.5) M-1 s-1), whereas Mn(II) and manganese complexes of desferal inhibited the reduction of cytochrome c by superoxide generated by the xanthine/xanthine oxidase system. Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (FeTPEN) was eight times more active than Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine(Fe-TPAA) in the cytochrome c assay, but only FeTPAA catalyzed the first-order decay of superoxide (kcat = 2.15 x 10(6) M-1 s-1) by stopped-flow. Fe(III)-tetrakis(4-N-methylpyridyl)porphine (FeTMPP) was active at low micromolar concentrations in both the cytochrome c and stopped-flow assays. At high micromolar concentrations, the organic nitroxides 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPOL) were inhibitory in the cytochrome c assay, but showed no detectable SOD activity by stopped-flow. None of the tested compounds inhibited xanthine oxidase activity as shown by the measurement of urate production. Under the conditions of the cytochrome c assay, FeTPEN, TEMPO, and TEMPOL oxidized reduced cytochrome c which rationalizes the false positives for these compounds in this assay. The inhibitory activities of Mn(II) and the manganese desferal complexes in the cytochrome c assay appear to be due to a stoichiometric, not catalytic, reaction with superoxide as catalytic amounts of these agents do not induce a first-order decay of superoxide as shown by stopped-flow.

Topics: Cyclic N-Oxides; Cytochrome c Group; Ferric Compounds; Ferrous Compounds; In Vitro Techniques; Kinetics; Manganese; Oxidation-Reduction; Spin Labels; Superoxide Dismutase; Superoxides

It is well known that oxygen enhances the relaxation of free radical EPR probes through spin lattice and Heisenberg spin-spin interactions with consequent effect on the line height and width. The two relaxation processes have opposing effects on the signal heights and depend on the concentration of oxygen, the incident microwave power, and the presence of other paramagnetic species. During EPR studies of chemical, biochemical, and cellular processes involving free radicals, molecular oxygen has significant magnetic influence on the EPR signal intensity of the free radical species under investigation in addition to affecting the rates of production of the primary species and the stability of the spin adduct nitroxides. These effects are often overlooked and can cause artifacts and lead to erroneous interpretation. In the present study, the effects of oxygen and ferricyanide on the EPR signal height of stable and persistent spin adduct nitroxides at commonly employed microwave powers were examined. The results show that under commonly adopted EPR spectrometer instrumental conditions, artifactual changes in the EPR signal of spin adducts occur and the best way to avoid them is by keeping the oxygen level constant using a gas-permeable cell.

Topics: Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Ferricyanides; Free Radicals; Microwaves; Oxygen; Spin Labels; Triacetoneamine-N-Oxyl

The mechanism of luminescence quenching by spin labels was investigated in aqueous solution by steady-state and time-resolved luminescence techniques. Water-soluble nitroxide radicals strongly quenched the luminescence emitted by Tb3+ chelates and by fluorescein, either free or conjugated to proteins. The following features of the quenching reaction were established: (I) the rate constant for quenching of triplet-state Tb3+ by nitroxides was about 4 orders of magnitude smaller (ca. 10(5) M-1 s-1) than those of the singlet-state probes; (II) the quenchers reduced the excited-state lifetime of both probes; (III) the rate constants for quenching of both probes were found to be apparently independent of the temperature (between 6 and 42 degrees C) and viscosity (up to 60 mPa.s) of the solutions; (IV) both singlet and triplet quenching rates were sensitive to solvent polarity; (V) there is a small but significant spectral overlap between the absorption band of weekly absorbing nitroxide radicals and the emission spectra of luminophores, the extent of which, however, does not correlate with the extent of quenching; (VI) the quenching rate declines sharply with an increasing luminophore to nitroxide distance. The distance dependence of the quenching rate showed a satisfactory fit to an exponential function. These findings indicate that the quenching reaction is dominated by an electron exchange between the excited singlet- or triplet-state luminophore and the nitroxide radical rather than controlled by diffusional properties of the reactants.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cyclic N-Oxides; Edetic Acid; Kinetics; Luminescent Measurements; Solutions; Spectrometry, Fluorescence; Spin Labels; Terbium; Water

Inhibition of lipid peroxidation by nitroxide radicals and their corresponding hydroxylamines was investigated. The nitroxides were either oxazolidines or piperidines, differing in substitution of the backbone of the molecule (a five or six-membered ring structure, respectively). Concentration requirements for 50% inhibition of microsomal lipid peroxidation varied from 340 to 6 microM for the nitroxides, and from 120 to 3 microM for the hydroxylamines, correlating with lipophilicity and chemical structure. Intramembrane concentrations required for 50% inhibition was independent of lipophilicity when peroxidation was initiated with ADP-Fe2+ but increased with lipophilicity when peroxidation was initiated with t-butylhydroperoxide. During studies of the kinetics of the inhibition, two modes were seen: a delay or a decreased rate of the process. The former mode was seen with the more lipophilic inhibitors. The mechanism of inhibition was similar for all nitroxides and consisted of the following three major components: blocking of primary initiation, prevention of secondary (peroxide-dependent) initiation, and scavenging of various lipoid radicals in the membrane, the major mode of action of the hydroxylamines. Inhibitory efficiency was interpreted in terms of steric hindrance, diffusibility, regeneration of inhibitor, and ability to interact with hydrophilic sites in a hydrophobic environment.

Topics: Adenosine Diphosphate; Animals; Cyclic N-Oxides; Free Radicals; Kinetics; Lipid Peroxidation; Microsomes, Liver; Molecular Structure; Nitrogen Oxides; Oxazoles; Rats; Rats, Inbred Strains; Spin Labels; Structure-Activity Relationship

The effect of elevated temperatures on the permeability of non-electrolyte spin labels, hydrophilic TEMPOL and more hydrophobic TEMPO across the porcine lymphocyte membrane was investigated. In the range of 41-44 degrees C, temperature-induced changes in the permeation constant were lower for TEMPO than TEMPOL. The data obtained may suggest that the permeability of spin labels across the membrane is sensitive to changes of temperature especially above 43 degrees C.

Topics: Animals; Cell Membrane Permeability; Cells, Cultured; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Hot Temperature; Lymphocytes; Spin Labels; Swine

Erythrocytes from normal mice and mice infected with the malarial parasite Plasmodium berghei reduce the water-soluble spin probes 2,2,6,6-tetramethylpiperidine-4-hydroxy-N-oxyl (TEMPOL), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) and 2,2,6,6-tetramethylpiperidine-4-keto-N-oxyl (TEMPONE) at similar rates under both air and N2 atmospheres. The ESR signal of the lipid-soluble spin probe 5-doxyl-stearate is stable on incorporation into erythrocytes from normal mice. In contrast, parasitized red cells reduce this nitroxide probe, at a rate which increases with the level of parasitemia. Inhibitors of electron transport such as KCN and NaN3, increase the rate of reduction. We propose that nitroxide reduction occurs via the electron transport chain in the parasite. The antimalarial drug primaquine causes reduction of both water-soluble and lipid-soluble spin probes. This action of primaquine is independent of its ability to release H2O2 from oxyhemoglobin, and is ascribed to the ability of primaquine to accelerate flux through the hexose monophosphate shunt. The increased production of NADPH results in increased rates of reduction of the nitroxide radicals. Methylene blue, which also increases flux through the shunt, is even more effective than primaquine at reducing the nitroxides. Chloroquine has no such effect. Parasitized mice treated with chloroquine six hours prior to ESR measurements show less nitroxide reducing capacity than do untreated mice. Chloroquine is known to decrease flux through the hexose monophosphate shunt. The metabolic influences of the two antimalarial drugs are, thus, quite different.

Topics: Air; Animals; Chloroquine; Cyclic N-Oxides; Electron Transport; Erythrocytes; Free Radicals; Hydrogen Peroxide; Male; Mice; Nitrogen; Oxidation-Reduction; Pentose Phosphate Pathway; Plasmodium berghei; Primaquine; Solubility; Spin Labels; Triacetoneamine-N-Oxyl

The paper presents some results on the effect of PCMB and NEM on the transport of non-electrolyte spin labels: TEMPO and TEMPOL across non-irradiated and irradiated porcine erythrocyte. Irradiated erythrocytes exhibited increased inhibitory effect of thiol reactive compounds in the TEMPO and TEMPOL transport compared to non-irradiated erythrocytes.

Topics: Animals; Cell Membrane Permeability; Chloromercuribenzoates; Cyclic N-Oxides; Erythrocyte Membrane; Ethylmaleimide; Gamma Rays; p-Chloromercuribenzoic Acid; Spin Labels; Swine

The addition of cupric ions caused decreased permeability to hydrophilic molecules and increased permeability to hydrophobic molecules. These results suggest that TEMPOL penetrates the erythrocyte in a different way than TEMPO. Penetration of TEMPOL is controlled by-SH groups, while TEMPO probably diffuses through the lipid bilayer. Cupric ions increase the permeability of erythrocyte membranes to both non-electrolytes in vivo.

Topics: Animals; Carps; Cell Membrane Permeability; Copper; Copper Sulfate; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Erythrocyte Membrane; Spin Labels

Carp erythrocytes were treated with p-chloromercuribenzoate or N-ethylmaleimide. It was observed that these thiol-group inhibitors decreased the transport of spin-labelled hydrophilic compound, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and increased the transport rate of more hydrophobic 2,2,6,6-tetramethylpiperidine-1-oxyl.

Topics: Animals; Biological Transport; Carps; Cell Membrane Permeability; Cyclic N-Oxides; Erythrocyte Membrane; Kinetics; Piperidines; Spin Labels; Structure-Activity Relationship; Sulfhydryl Reagents

Four different thiol reagents: p-chloromercuribenzoic acid (pCMB), mercuric chloride (HgCl2), N-ethylmaleimide (NEM), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were employed as agents modifying the transport of a hydrophilic and hydrophobic non-electrolyte spin labels: 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) into bovine erythrocytes. Gamma-irradiation of erythrocytes amplified the effects of pCMB, HgCl2 and NEM of inhibition of TEMPOL transport and attenuated them in the case of TEMPO transport. These results suggest that the transport of TEMPOL across the erythrocyte membrane is controlled by both superficially and more deeply located membrane -SH groups while only superficial -SH groups control the transport of TEMPO. The lower extent of inhibition of TEMPO transport indicates a higher contribution of diffusion through the lipid phase to the transport of TEMPO across the erythrocyte membrane as compared with TEMPOL.

Topics: Animals; Cattle; Cell Membrane Permeability; Chloromercuribenzoates; Cobalt Radioisotopes; Cyclic N-Oxides; Dithionitrobenzoic Acid; Erythrocyte Membrane; Erythrocytes; Ethylmaleimide; Gamma Rays; Mercuric Chloride; Mercury; p-Chloromercuribenzoic Acid; Spin Labels; Sulfhydryl Reagents

Topics: Biological Transport; Cell Membrane Permeability; Cyclic N-Oxides; Down Syndrome; Erythrocyte Membrane; Erythrocytes; Humans; Kinetics; Spin Labels

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.

Topics: Animals; Cattle; Cell Membrane Permeability; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Erythrocyte Aging; Erythrocyte Membrane; Erythrocytes; Membrane Lipids; Membrane Proteins; Spin Labels

The effect of ionizing radiation on the non-electrolyte, anion and cation permeability of the erythrocyte membrane was studied by measurement of the reduction rate of appropriate nitroxyl derivatives. Irradiation of bovine erythrocytes in the dose-range of 2-50 krad resulted in a regular dose-dependent increase in the reduction rates of a cation (TEMPO-choline) and a hydrophobic non-electrolyte (TEMPO), and non-regular changes in the reduction rate of a hydrophilic non-electrolyte (TEMPOL). The permeation constant for TEMPO-choline also showed a non-regular response to radiation, similar to the response pattern of other red blood cell parameters. These results also demonstrate that the effects of radiation on the transport of various solutes can be used as a means of distinguishing between different channels of membrane transport.

Topics: Animals; Biological Transport; Cattle; Choline; Cyclic N-Oxides; Dose-Response Relationship, Radiation; Erythrocyte Membrane; Erythrocytes; Extracellular Space; Gamma Rays; Kinetics; Mathematics; Piperidines; Spin Labels