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PubMed2003

The effect of lipophilicity of spin-labeled compounds on their distribution in solid lipid nanoparticle dispersions studied by electron paramagnetic resonance.

Solid lipid nanoparticles (SLN) constitute an attractive drug carrier system. The aim of this study was to investigate the influence of lipophilicity and structure of different model molecules on their distribution in SLN dispersions. SLN composed of glyceryl tripalmitate as lipid and soybean lecithin and poloxamer 188 as stabilizers were prepared by a melt-emulsification process. PC(10,3), MeFASL(10,3), C(14)-Tempo, and Tempol were incorporated into SLN as spin-labeled compounds. The partition of SP between triglyceride and water was determined experimentally by electron paramagnetic resonance (EPR) and compared with calculated partition coefficients. The distribution of molecules in SLN dispersions was determined from the parameters of EPR spectra, from the reduction kinetics of the spin-labeled compounds with sodium ascorbate, and by computer simulation of EPR spectral line shapes. The experimentally obtained partition coefficients increase in the order Tempol < MeFASL(10,3) < C(14)-Tempo, showing the same trend as the partition coefficients calculated according to Rekker. In SLN dispersions, it was estimated that the ratio of SP between solid lipid core, phospholipid layers (deeper in SLN layer or in liposomes and closer to the surface of SLN), and water is for Tempol 0:0:100, for C(14)-Tempo 46:54(20:34):0, for MeFASL(10,3) 34:65(38:27):1, and for PC(10,3) 10:89(26:3:60):1.

Electron Spin Resonance Spectroscopy↗
PubMed1993

Calcium- and lead-activated morphological changes in human erythrocytes: a spin label study of the cytoplasm.

Stimulation of the human red blood cell (RBC; erythrocyte) by Ca2+ or Pb2+ ions induced potassium efflux (the so-called Gárdos effect). Time-dependent changes in the volume and shape of the RBCs were monitored on the basis of mean cell volume and light scattering. Changes in the RBC cytoplasm were followed using electron paramagnetic resonance (EPR), after labeling of the cytoplasmic glutathione with 2,2,6,6-tetramethyl-4-maleimidopiperidine-N-oxyl. The RBC could still be activated in vitro with Ca2+ (plus ionophore A23187) or Pb2+ ions. Pb2+ retained its stimulatory-inhibitory activity. No linear relationship between the reciprocal of cell volume and the mobility (correlation time) of the probe was found over the morphological range covered upon Ca2+ activation. The response of the probe is considered to be mainly governed by the microviscosity due to hemoglobin and any influence by cytoskeleton products could not be discerned. Pretreatment of the RBC with N-ethylmaleimide (1 mM for 30 min) at room temperature prevented spin labeling. However, the RBC still responded to the ion activation, whereas this effect was eliminated after pretreatment at 37 degrees C. EPR of spin-labeled cells, before and after reaching the sphero-echinocytic state, resulted in similar final spectra. Spectra were also observed from RBCs fixed in various states with glutaraldehyde.

Calcimycin↗
PubMed1996

In vivo spin-label murine pharmacodynamics using low-frequency electron paramagnetic resonance imaging.

A novel, very-low-frequency electron paramagnetic resonance (EPR) technique is used to image the distribution of several nitroxides with distinct pharmacologic compartment affinities in the abdomens of living mice. Image acquisition is sufficiently rapid to allow a time sequence of the distribution for each compound. The spectra and concentrations of these nitroxides are imaged with the use of spectral-spatial imaging to distinguish a single spatial dimension. Liver and bladder of the mouse anatomy are distinguished by this technique. After an intraperitoneal injection of the spin-label probes, a shift in the distribution of the compounds from the upper abdomen (primarily liver) to the lower abdomen (primarily bladder) is observed. The time dependence of the shift in regional distribution depends on the structural properties of the side chain attached to the spin label. These results indicate that this application of in vivo electron paramagnetic resonance imaging will provide a new method of magnetic resonance imaging for determination of pharmacodynamics in the body of an intact animal.

Animals↗
PubMed1981

Interaction of some charged amphiphilic drugs with phosphatidylcholine vesicles. A spin label study of surface potential effects.

Adsorptions of amphiphilic drugs (propranolol, alprenolol, metoprolol and tetracaine) to phosphalidylcholine vesicles in media of different pH and NaCl concentrations have been studied. A positively charged spin label amphiphile, N,N-di-methyl-N-nonyl-N-tempoylammoniumbromide, was used to follow the variation in the surface potential by ESR. Competition experiments between the probe molecule and the drugs were carried out. A spin-labeled analogue of propranolol was also employed. We have analysed the results in terms of the theory for the diffuse double layer (Gouy-Chapman) and treated various equilibrium models. A weak, specific adsorption of chloride ions was introduced. For the charged forms of the drugs simulations of the experiments by numerical solution of the system of equations in a satisfactory way furnished intrinsic binding constants, independent of surface potential effects. The common electrostatic surface potential is mainly ruling the competition, and not the number of surface vacancies.

Journal Article↗
PubMed1985

Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids.

We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.

Crotalid Venoms↗
PubMed2006

Dual vessel arterial spin labeling scheme for regional perfusion imaging.

Regional perfusion imaging (RPI) based on pulsed arterial spin labeling and angulated inversion slabs has been recently proposed. The technique allows mapping of individual brain perfusion territories of the major feeding arteries and could become a valuable clinical tool for evaluation of patients with cerebrovascular diseases. Here we propose a new labeling scheme for RPI where lateral and posterior circulations are labeled simultaneously. Two scans instead of three are sufficient to obtain the same perfusion territories as in the original approach, allowing for a 33% reduction in the total RPI protocol time. Moreover, the position of the inversion slabs with respect to vascular anatomy facilitates the planning and allows potentially better labeling efficiency. The new approach was tested on seven healthy volunteers and compared to the original labeling scheme. The results showed that the same perfusion territories and regional CBF values can be obtained.

Algorithms↗
PubMed1991

Cholesterol modulation of lipid-protein interactions in liver microsomal membrane: a spin label study.

ESR spectra of spin probes were used to monitor lipid-protein interactions in native and cholesterol-enriched microsomal membranes. In both systems composite spectra were obtained, one characteristic of bulk bilayer organization and another due to a motionally restricted population, which was ascribed to lipids in a protein microenvironment. Computer spectral subtractions revealed that cholesterol modulates the order/mobility of both populations in opposite ways, i.e., while the lipid bilayer region gives rise to more anisotropic spectra upon cholesterol enrichment, the spectra of the motionally restricted population become indicative of increased mobility and/or decreased order. These events were evidenced by measurement of both effective order parameters and correlation times. The percentages of the motionally restricted component were invariant in native and cholesterol-enriched microsomes. Variable temperature studies also indicated a lack of variation of the percentages of both spectral components, suggesting that the motionally restricted one was not due to protein aggregation. The results correlate well with the effect of cholesterol enrichment on membrane-bound enzyme kinetics and on the behavior of fluorescent probes [Castuma & Brenner (1986) Biochemistry 25, 4733-4738]. Several hypothesis are put forward to explain the molecular mechanism of the cholesterol-induced spectral changes.

Animals↗
PubMed1981

Organization of membrane lipids and proteins in human En(a-) erythrocytes that lack the major sialoglycoprotein, glycophorin A. A spin-label study.

Membrane fluidity was studied by electron-spin-resonance techniques in human En(a-) erythrocytes that lack the major membrane sialoglycoprotein, glycophorin A. By using stearic acid spin labels with a doxyl group in the C-12 or C-15 positions, we demonstrated that the hydrophobic core in these cells was more fluid than in normal cells. Surface-located regions in isolated En(a-) membranes, when probed with stearic acid labelled in the C-5 position, appeared more stable than in normal membranes. In isolated En(a-) membranes, protein motion was decreased when probed with a nitroxide derivative of maleimide. After incubation with anti-(glycophorin A) antibodies protein motion and membrane fluidity were increased in normal membranes. This effect was observed also after spectrin depletion, which by itself increased protein motion but decreased membrane fluidity in the hydrophobic core of the membrane. The results show that membrane proteins influence the fluidity of membrane lipids.

Blood Group Antigens↗
PubMed1987

Spinach leaf ribulose-5-phosphate kinase: examination of sulfhydryls by chemical modification and spin-labeling.

Methodology has been developed for complete or selective modification of the cysteinyl sulfhydryls of ribulose-5-phosphate (Ru5P) kinase. Using native enzyme, iodoacetate modifies four sulfhydryls with varying levels of completeness. The most reactive sulfhydryl in the native enzyme can be selectively titrated with iodoacetate; complete loss of activity occurs. Composition and N-terminal analyses of the peptide bearing this essential sulfhydryl indicate that the alkylated residue (Cys-16) is identical to the site modified by other modification reagents (M. A. Porter and F. C. Hartman (1986) Biochemistry 25, 7314-7318). In the presence of ATP, a nonessential sulfhydryl of the native enzyme is carboxymethylated. The peptide bearing this modified cysteine has been isolated and its composition and N-terminal sequence determined. Enzyme that is carboxymethylated in the presence of ATP retains activity and can be oxidatively inactivated in a reversible fashion. This suggests that the cysteine targeted by iodoacetate in the presence of ATP is not a residue that participates in regulation of enzyme activity. Using a spin-labeled analog of iodoacetate, both essential and nonessential cysteines have been selectively modified. ESR measurements suggest that the environment of these cysteines is not highly constrained. Modest effects on spin-label mobility are observed upon occupancy of Ru5P or ATP sites on the modified enzyme. These effects are dependent on the presence of divalent cations, suggesting that a binary enzyme-cation complex must form prior to productive enzyme-substrate interactions.

Amino Acids↗
PubMedDate not supplied

The efficiency of adiabatic inversion for perfusion imaging by arterial spin labeling.

Velocity-driven adiabatic inversion is an attractive method for labeling arterial blood spins for quantitative perfusion imaging. To quantify perfusion and to optimize experimental parameters, an accurate estimate of labeling efficiency is required. We present theoretical and numerical methods to calculate the labeling efficiency over a wide range of experimental and physiologic parameters. The results are compared to experimental measurements in vivo. Inversion efficiency was found to be higher than previously assumed and relatively insensitive to flow velocity and the amplitude of the RF irradiation used for inversion. Assuming laminar flow, labeling efficiencies of greater than 90% are easily obtainable over a broad range of flow velocities. For applications where RF power deposition is a limiting factor such as at high field strengths, labeling efficiency can be maintained by reducing the labeling gradient. These results further illustrate the capability of adiabatic inversion to effectively label flowing blood.

Adult↗
PubMed1985

Kinetics of phospholipid transfer between liposomes (neutral or negatively charged) and high-density lipoproteins: a spin-label study of early events.

The kinetics of spin-labeled phosphatidylcholine transfer between vesicles and HDL particles exhibited a two-phase process, as seen by ESR spectroscopy. The results were analyzed by considering several possible steps in the overall transfer, whose aspects were also studied: (i) micellar complex formation after HDL apolipoprotein-vesicle mixture, (ii) the rate of PC transfer from the micellar complex to HDL, (iii) the rate of the reverse reaction between overloaded HDL particles and other particles such as HDLs, LDLs, and lipid vesicles. The results agree most convincingly with a mechanism in which the diffusion of phospholipids into the HDL-endogenous lipids is the limiting step, occurring as a two-step process. In addition, we observed a negative charge effect on the lipid transfer rates and yields.

Electron Spin Resonance Spectroscopy↗
PubMed1987

Spin label study of erythrocyte deformability. Ca2+-induced loss of deformability and the effects of stomatocytogenic reagents on the deformability loss in human erythrocytes in shear flow.

The Ca2+-induced loss of deformability in human erythrocytes and the recovery of the lost deformability by stomatocytogenic reagents were investigated by means of a new flow electron paramagnetic resonance (EPR) spin label method, which provides information on deformation and orientation characteristics of spin labeled erythrocytes in shear flow. The Ca2+-induced loss of deformability is attributed mainly to the increase in intracellular viscosity resulting from efflux of intracellular potassium ions and water (Gardos effect). Partial recovery of the lost deformability is demonstrated in the presence of stomatocytogenic reagents, such as chlorpromazine, trifluoperazine, W-7, and calmidazolium (R24571). The recovery can not be explained solely by suppression of the Gardos effect due to the reagents. Incorporation of an optimal amount of the reagents into the membrane appears to compensate for the membrane modification due to Ca2+ ions to restore a part of the lost deformability.

Calcimycin↗
PubMed1976

Symmetry and asymmetry of the pyruvate dehydrogenase complexes from Azotobacter vinelandii and Escherichia coli as reflected by fluorescence and spin-label studies.

Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotobacter vinelandii and Escherichia coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non-equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A. vinelandii. Reduction of the complex with Mg2+, thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label [N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide] introduced in such a way proves the existence of at least two different micro-environments around the lipoyl moieties in complex isolated from A. vinelandii. Electron paramagnetic resonance spectra of the specifically spin-labelled complexes from E. coli and A. vinelandii, when dissolved in tricine [N-tris(hydroxymethyl)-methylglycine] buffer, show interactions of at least two electron spins with each other, which indicate that the lipoyl moieties are rather close together. Fluorescent label [N-(1-anilinonaphthyl-4)maleimide] is specifically attached to the lipoyl moiety of the high-Mr transacetylase of the freshly isolated complex from A. vinelandii. From the large differences in the apparent lifetimes tau p and tau m, as detected by phase fluorimetry, it is shown that this fluorscent label is distributed in different micro-environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-Mr transacetylase, indicate different conformations of the complex from A. vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high-Mr transacetylase of A. vinelandii are both non-symmetrically distributed within the complex.

Acetyltransferases↗
PubMed2003

Investigating magnetically aligned phospholipid bilayers with various lanthanide ions for X-band spin-label EPR studies.

This paper reports the EPR spectroscopic characterization of a model membrane system that magnetically aligns with a variety of different lanthanide ions in the applied magnetic field (<1 T) of an X-band EPR spectrometer. The ability to align phospholipid bilayer systems is valuable because the anisotropic spectra provide a more detailed and complete description of the structural and motional properties of the membrane-associated spin label when compared to randomly dispersed EPR spectra. The nitroxide spin probe 3beta-doxyl-5alpha-cholestane (cholestane or CLS) was inserted into the bilayer discs to demonstrate the effects of macroscopic bilayer alignment through the measurement of orientational dependent hyperfine splittings. The effects of different lanthanide ions with varying degrees of magnetic susceptibility anisotropy and relaxation properties were examined. For X-band EPR studies, the minimal amounts of the Tm(3+), Yb(3+), and Dy(3+) lanthanide ions needed to align the phospholipid bilayers were determined. Power saturation EPR experiments indicate that for the sample compositions described here, the spin-lattice relaxation rate of the CLS spin label was increased by varying amounts in the presence of different lanthanide (Gd(3+), Dy(3+), Er(3+), Yb(3+), and Tm(3+)) ions, and in the presence of molecular oxygen. The addition of Gd(3+) caused a significant increase in the spin-lattice relaxation rate of CLS when compared to the other lanthanide ions tested.

Electron Spin Resonance Spectroscopy↗
PubMed1987

Orientation dependence and motional properties of spin labels in cardiac and skeletal muscle fibres.

Orientation dependence and rotational motion of maleimide spin labels attached to the fast reacting thiol sites of myosin were studied in glycerinated cardiac and skeletal muscle fibres in rigor and in relaxing medium. The probe order in skeletal muscle was shown to be about one order of magnitude higher than that in cardiac muscle. In skeletal muscle in rigor the orientational order is static on the time scale of the saturation transfer electron paramagnetic resonance measurement (ST EPR, rotational correlation time of the label is greater than 1 ms), but in cardiac muscle fibres, a disorder was observed which was at least partly dynamical, the rotational correlation time being about 100 microseconds. In relaxing solution the degree of order of probe molecules in both types of muscle was strongly reduced at and above the resting length. The disorder was at least partly dynamical on the ST EPR time scale, the apparent rotational correlation times being 200 microseconds for skeletal muscle and 60 microseconds for cardiac muscle, respectively. According to the results of ST EPR the rotational behavior of cross-bridges was identical in cardiac and skeletal muscle in relaxing medium.

Adenosine Triphosphatases↗
PubMedDate not supplied

Spin label EPR-based characterization of biosystem complexity.

Following the widely spread EPR spin-label applications for biosystem characterization, a novel approach is proposed for EPR-based characterization of biosystem complexity. Hereto a computational method based on a hybrid evolutionary optimization (HEO) is introduced. The enormous volume of information obtained from multiple HEO runs is reduced with a novel so-called GHOST condensation method for automatic detection of the degree of system complexity through the construction of two-dimensional solution distributions. The GHOST method shows the ability of automatic quantitative characterization of groups of solutions, e.g. the determination of average spectral parameters and group contributions. The application of the GHOST condensation algorithm is demonstrated on four synthetic examples of different complexity and applied to two physiologically relevant examples--the determination of domains in biomembranes (lateral heterogeneity) and the study of the low-resolution structure of membrane proteins.

Algorithms↗
PubMed2006

Model-free arterial spin labeling quantification approach for perfusion MRI.

In this work a model-free arterial spin labeling (ASL) quantification approach for measuring cerebral blood flow (CBF) and arterial blood volume (aBV) is proposed. The method is based on the acquisition of a train of multiple images following the labeling scheme. Perfusion is obtained using deconvolution in a manner similar to that of dynamic susceptibility contrast (DSC) MRI. Local arterial input functions (AIFs) can be estimated by subtracting two perfusion-weighted images acquired with and without crusher gradients, respectively. Furthermore, by knowing the duration of the bolus of tagged arterial blood, one can estimate the aBV on a voxel-by-voxel basis. The maximum of the residue function obtained from the deconvolution of the tissue curve by the AIF is a measure of CBF after scaling by the locally estimated aBV. This method provides averaged gray matter (GM) perfusion values of 38 +/- 2 ml/min/100 g and aBV of 0.93% +/- 0.06%. The average CBF value is 10% smaller than that obtained on the same data set using the standard general kinetic model (42 +/- 2 ml/min/100 g). Monte Carlo simulations were performed to compare this new methodology with parametric fitting by the conventional model.

Algorithms↗
PubMed1980

Effects of aliphatic alcohols and aldehydes on fluidity of spin-labeled synaptosomal plasma membranes.

Synaptosomal plasma membranes from Swiss-Webster mice were spin-labeled with 5-doxylstearic acid. By electron paramagnetic resonance techniques an order parameter was measured. A decrease in the order parameter reflected an increase in membrane fluidity. In confirmation of our previous work, ethanol decreased membrane order. Pentanol also showed a concentration-related decrease in the order parameter in the range of 23 to 87 mM. At equimolar concentrations, pentanol was about 9 times more effective than ethanol. Acetaldehyde had no effect on membrane fluidity at concentrations that might be found in vivo during ethanol oxidation (23 or 227 microM) but a very high concentration, 2.3 mM, produced a small decrease in the order parameter comparable to the effect of an intoxicating concentration of ethanol. Valeraldehyde also had a slight fluidizing effect and appeared very roughly three times more potent than acetaldehyde. This study shows that the fluidizing effects of ethanol are not shared by its aldehyde metabolite at relevant concentrations. The fluidizing effects of aldehydes are slight but they increase with concentration and with chain length.

Alcohols↗