Intact blood-brain barrier during spontaneous attacks of migraine without aura: a 3T DCE-MRI study.

BACKGROUND AND PURPOSE: The integrity of the blood-brain barrier (BBB) has been questioned in migraine, but BBB permeability has never been investigated during spontaneous migraine attacks. In the present study, BBB permeability during spontaneous attacks of migraine without aura was investigated compared to an interictal state. METHODS: Seventy-four patients suffering from migraine without aura were recruited to participate in this cross-sectional dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) study. The patients were instructed to report at the hospital for DCE-MRI scan during and outside of a spontaneous migraine attack. The primary end-point was a difference in the BBB permeability (ml/100 g/min) between the attack and the headache-free days. The permeability was assessed in five different regions of interest (ROIs) located in the anterior, middle and posterior cerebral area, brain stem, posterior pons and whole brain. The paired samples t test was used to compare Ki (permeability) values between the attack and headache-free days. RESULTS: Nineteen patients completed the study. Median time from onset of migraine attack to scan was 6.5 h (range 4.0-15.5 h). No change in the mean BBB permeability (ml/100 g/min) was found between the attack and the headache-free days in any of the measured ROIs. No relationship between the pain side or intensity and BBB permeability was found in 15 patients with unilateral pain during the examined attack. CONCLUSIONS: It was demonstrated that the BBB permeability during spontaneous migraine attacks without aura was unchanged.

Fluorescence-detected X-ray magnetic circular dichroism of well-defined Mn(II) and Ni(II) doped in MgO crystals: credential evaluation for measurements on biological samples.

L(2,3)-edge X-ray magnetic circular dichroism (XMCD) spectra have been measured for the well-defined dilute Ni(II) and Mn(II) ions doped into a MgO crystal, with sub-Kelvin dilution refrigerator cooling and 2 T magnetic field magnetization. A 30-element Ge array X-ray detector has been used to measure the XMCD for these dilute ions, whose concentrations are 1400 ppm for Ni(II) and 10,000 ppm for Mn(II). Large XMCD effects have been observed for both Ni(II) and Mn(II), and multiplet simulation described the observed spectra. The fluorescence-detected L-edge absorption spectrum and XMCD of Ni(II) in MgO are comparable with both theoretical calculations and the total electron yield measured ions in similar chemical environments, at least qualitatively validating the use of the sensitive fluorescence detection technique for studying XMCD for dilute 3d metal ions, such as various metalloproteins. Sum rule analyses on the XMCD spectra are also performed. In addition, these XMCD measurements have also been used to obtain the sample's magnetization curve and the beamline's X-ray helicity curve. This study also illustrated that bend magnet beamlines are still useful in examining XMCD on dilute and paramagnetic metal sites.

Synchrotron X-ray analyses demonstrate phosphate-bound gadolinium in skin in nephrogenic systemic fibrosis.

BACKGROUND: Nephrogenic systemic fibrosis (NSF) is an incurable, debilitating disease found exclusively in patients with decreased kidney function and comprises a fibrosing disorder of the skin and systemic tissues. The disease is associated with exposure to gadolinium (Gd)-based contrast agents (GBCA) used in magnetic resonance imaging (MRI). Tissue samples from many patients with NSF contain micron-sized insoluble Gd-containing deposits. However, the precise composition and chemical nature of these particles is unclear. OBJECTIVES: To clarify the precise chemical structure of the Gd-containing deposits in NSF tissues. METHODS: Autopsy skin tissues from a patient with NSF were examined in situ using synchrotron X-ray fluorescence (SXRF) microscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy and in correlation with light microscopy and the results of scanning electron microscopy /energy dispersive spectroscopy analyses. RESULTS: The insoluble Gd deposits were shown to contain Gd no longer coordinated by GBCA chelator molecules but rather in a sodium calcium phosphate material. SXRF microscopy shows a clear correlation between Gd, Ca and P. EXAFS spectroscopy shows a very different spectrum from the GBCAs, with Gd­P distances at 3·11 A and 3·11 A as well as Gd­Gd distances at an average of 4·05 A, consistent with a GdPO4 structure. CONCLUSIONS: This is the first direct evidence for the chemical release of Gd from GBCA in human tissue. This supports the physical­chemical, clinical and epidemiological data indicating a link between stability and dose of GBCA to the development of NSF.

The manganese site of the photosynthetic water-splitting enzyme.

As the originator of the oxygen in our atmosphere, the photosynthetic water-splitting enzyme of chloroplasts is vital for aerobic life on the earth. It has a manganese cluster at its active site, but it is poorly understood at the molecular level. Polarized synchrotron radiation was used to examine the x-ray absorption of manganese in oriented chloroplasts. The manganese site, in the "resting" (S1) state, is an asymmetric cluster, which probably contains four manganese atoms, with interatomic separations of 2.7 and 3.3 angstroms; the vector formed by the 3.3-angstrom manganese pair is oriented perpendicular to the membrane plane. Comparisons with model compounds suggest that the cluster contains bridging oxide or hydroxide ligands connecting the manganese atoms, perhaps with carboxylate bridges connecting the 3.3-angstrom manganese pair.

Identification and characterization of zinc binding sites in protein kinase C.

Metal ion coordination in the regulatory domain of protein kinase C (PKC) is suggested by the conservation of six cysteines and two histidines in two homologous regions found therein. By monitoring x-ray fluorescence from a purified sample of rat PKC beta I overexpressed in insect cells, direct evidence has been obtained that PKC beta I tightly binds four zinc ions (Zn2+) per molecule. Extended x-ray absorption fine structure (EXAFS) data are best fit by an average Zn2+ coordination of one nitrogen and three sulfur atoms. Of the plausible Zn2+ coordination models, only those featuring nonbridged Zn2+ sites accommodate the EXAFS data and all of the conserved potential ligands.

X-ray absorption spectroscopy of oriented cytochrome oxidase.

The polarized X-ray absorption spectra of the copper, iron and zinc sites of mitochondrial cytochrome oxidase in oriented membrane multilayers have been examined. The copper X-ray absorption edge spectra indicate the presence of a tetragonal copper, which we assign as CuB, oriented with the long axis approximately orthogonal to the membrane normal. We have also detected the presence of a relatively long (2.6 A) Cu-S or Cu-Cl interaction, which we assign to a copper-thioether (probably Met210) coordination at the CuA site, with the bond oriented along the membrane normal. The coordination of the zinc, the iron and the CuB heme a3 binuclear site are discussed.

Spectroscopic properties of the hydroxylase of methane monooxygenase.

The hydroxylase component of methane monooxygenase (EC 1.14.13.25), which catalyzes the oxidation of methane to methanol, has been studied by visible, electron spin resonance and X-ray spectroscopies. The enzyme appears to possess a mu-oxo- or mu-hydroxo-bridged binuclear iron site, with no sulfur ligands to the cluster. Each Fe has 4-6 oxygen (or nitrogen) ligands, at an average distance of 1.92 +/- 0.03 A. The Fe-Fe distance is 3.05 +/- 0.05 A. Essentially all of the irons are in the Fe3+ state as the enzyme is prepared, but reduction with N-methylphenazonium methosulfate generates ESR-detectable states that appear to emanate from mixed-valence binuclear sites. One of these, with gav near 1.85, displays typical Curie law microwave saturation behavior, but the other, gav near 1.73, has a very potent method of spin-relaxation. Together they account for approximately 0.6 spins per molecule.

Nitrogen chemical structure in DNA and related molecules by X-ray absorption spectroscopy.

The electronic environment of nitrogen in nucleic acid bases, nucleotides, polynucleotides and DNA has been studied, for the first time using X-Ray Absorption Near-Edge Spectroscopy (XANES). Generally, the spectra of these complex molecules consist of low energy bands corresponding to 1s-->pi* transitions and high energy bands corresponding to 1s-->sigma* transition, as illustrated using several nitrogen model compounds. The 1s-->pi* transitions show particular sensitivity to the chemical environment of the nitrogen. Oxygen substitution on ring carbon atoms generally results in a significant blue shift of the lowest 1s-->pi* bands while halogen substitution results in a small blue shift. These observations illustrate the significance of the disturbance of the aromatic ring system produced by exocyclic carbonyl groups. Direct substitution on the nitrogen frequently results in significant spectral perturbations. Differences between the spectra of the polynucleotides and the sums of spectra of the individual nucleotides point to the effects of hydrogen-bonding in complementary double-helix structures. The XANES spectrum of a DNA sample with a known ratio of the polynucleotides is equivalent to the weighted sum of the spectra of individual polynucleotides, indicating that the difference in base stacking interactions produces negligible spectral effects. The variability of nitrogen K-edge spectra in these samples and in protein may be useful for chemically specific imaging using X-ray microscopes.

Effect of cyanide binding on the copper sites of cytochrome c oxidase: an X-ray absorption spectroscopic study.

Cu x-ray absorption spectroscopy (XAS) has been used to investigate the effect of cyanide treatment on the structures of the copper sites in beef heart cytochrome c oxidase. The Cu K-edge spectrum changes significantly upon cyanide binding to resting state enzyme, as does the Cu extended x-ray absorption fine structure (EXAFS) spectrum. The Cu EXAFS Fourier transfer (FT) exhibits an enhanced peak for the cyanide-treated enzyme in the region containing the Cu...Fe peak in the resting state FT (at R' approximately equal to 2.6-2.7 A). This peak in the cyanide-treated sample is hypothesized to arise from "outer shell" scattering from a linear Cu-cyanide moiety, suggesting cyanide binding to CuB only (CuB 2+-CN-) or cyanide bridging between the Fe of heme a3 and CuB (Fe3+-(CN-)-CuB 2+).

Molybdenum EXAFS of the Desulfovibrio gigas Mo(2Fe-2S) protein--structural similarity to "desulfo" xanthine dehydrogenase.

The molybdenum EXAFS of the Mo(2Fe-2S) protein from Desulfovibrio gigas has been examined using fluorescence detection and synchrotron radiation. In the oxidized form the molybdenum environment is found to contain two terminal oxo groups and two long (2.47 A) Mo-S bonds. Evidence was also found for an oxygen or nitrogen donor ligand at 1.90 A. Addition of dithionite to the oxidized enzyme results in loss of a terminal oxo group, perhaps due to protonation. In addition, a 0.1 A contraction in the Mo-S bond lengths is observed. The behavior of both oxidized and dithionite-treated forms is similar to that observed previously with "desulfo" xanthine oxidase.

X-ray absorption studies of the copper-beta domain of rat liver metallothionein.

Rat liver metallothionein contains two domains, each of which enfolds a separate metal-thiolate cluster. The binding stoichiometry of these clusters depends on the particular metal ion bound. In the aminoterminal beta domain the cluster can accommodate either three Cd(II) ions or six Cu(I) ions. The Cd ions are known to be coordinated in a tetrahedral geometry. In order to better understand the binding of Cu ions in this domain, the Cu-beta domain fragment of metallothionein was prepared and investigated by x-ray absorption spectroscopy. Quantitative analysis of the EXAFS data indicates copper-sulfur distances of 2.25 +/- 0.03 A. The EXAFS amplitudes and distance results are most consistent with trigonal coordination. A trigonal biprism is proposed for the Cu6Cys9 complex in which Cu occupies each vertex and cysteinyl sulfur bridges at each of the nine edges.

Formate dehydrogenase molybdenum and tungsten sites--observation by EXAFS of structural differences.

Preliminary EXAFS data has been collected on the molybdenum (K-edge) in C. pasteurianum formate dehydrogenase and the tungsten (LIII-edge) in C. thermoaceticum formate dehydrogenase. In the presence of dithionite, the tungsten enzyme was devoid of W = O bonds, and exhibited average W-(O, N) and W-S bond lengths of 2.13 +/- 0.03 A and 2.39 +/- 0.03 A, respectively. In sharp contrast, the C. pasteurianum molybdenum site has three Mo = O bonds with an average bond length of 1.74 +/- 0.03 A. It is also the first molybdenum enzyme found lacking Mo-S bonds, and does not appear to be redox active in the presence of formate or dithionite. Model compounds WO2(8-hydroxyquinoline)2 = WO2(ox)2, and WO2(8 mercaptoquinoline)2 = WO2(tox)2, were also examined. Respective predicted bond lengths for WO2(ox)2 and WO2(tox)2 were W = O of 1.71, 1.73 A; W-N of 2.31, 2.29 A; W-O or W-S of 1.92 or 2.40 A, with estimated uncertainties of +/- 0.03 A.

Refinement of the nickel site structure in Desulfovibrio gigas hydrogenase using range-extended EXAFS spectroscopy.

We have reexamined the Ni EXAFS of oxidized, inactive (as-isolated) and H(2) reduced Desulfovibrio gigas hydrogenase. Better spatial resolution was achieved by analyzing the data over a 50% wider k-range than was previously available. A lower k(min) was obtained using the FEFF code for phase shifts and amplitudes. A higher k(max) was obtained by removing an interfering Cu signal from the raw spectra using multiple energy fluorescence detection. The larger k-range allowed us to better resolve the Ni-S bond lengths and to define more accurately the Ni-O and Ni-Fe bond lengths. We find that as-isolated, hydrogenase has two Ni-S bonds at approximately 2.2 A, but also 1-2 Ni-S bonds in the 2.35+/-0.05 A range. A Ni-O interaction is evident at 1.91 A. The as-isolated Ni-Fe distance cannot be unambiguously determined. Upon H(2) reduction, two short Ni-S bonds persist at approximately 2.2 A, but the remaining Ni-S bonds lengthen to 2.47+/-0.05 A. Good simulations are obtained with a Ni-Fe distance at 2.52 A, in agreement with crystal structures of the reduced enzyme. Although not evident in the crystal structures, an improvement in the fit is obtained by inclusion of one Ni-O interaction at 2.03 A. Implications of these distances for the spin-state of H(2) reduced H(2)ase are discussed.

Abnormal blood-brain barrier permeability in normal appearing white matter in multiple sclerosis investigated by MRI.

OBJECTIVES: To investigate whether blood-brain barrier (BBB) permeability is disrupted in normal appearing white matter in MS patients, when compared to healthy controls and whether it is correlated with MS clinical characteristics. METHODS: Dynamic contrast-enhanced MRI was used to measure BBB permeability in 27 patients with MS and compared to 24 matched healthy controls. RESULTS: Permeability measured as K(trans) was significantly higher in periventricular normal appearing white matter (NAWM) and thalamic gray matter in MS patients when compared to healthy controls, with periventricular NAWM showing the most pronounced difference. Recent relapse coincided with significantly higher permeability in periventricular NAWM, thalamic gray matter, and MS lesions. Immunomodulatory treatment and recent relapse were significant predictors of permeability in MS lesions and periventricular NAWM. Our results suggest that after an MS relapse permeability gradually decreases, possibly an effect of immunomodulatory treatment. CONCLUSIONS: Our results emphasize the importance of BBB pathology in MS, which we find to be most prominent in the periventricular NAWM, an area prone to development of MS lesions. Both the facts that recent relapse appears to cause widespread BBB disruption and that immunomodulatory treatment seems to attenuate this effect indicate that BBB permeability is intricately linked to the presence of MS relapse activity. This may reveal further insights into the pathophysiology of MS.

Structural aspects of the copper sites in cytochrome c oxidase. An X-ray absorption spectroscopic investigation of the resting-state enzyme.

Copper K-edge X-ray absorption spectroscopy (XAS) has been used to investigate the structural details of the coordination environment of the copper sites in eight resting-state samples of beef heart cytochrome c oxidase prepared by different methods. The unusual position and structure of the resting-state copper edge spectrum can be adequately explained by the presence of sulfur-containing ligands, with a significant amount of S----Cu(II) charge transfer (i.e., a covalent site). Quantitative curve-fitting analysis of the copper extended X-ray absorption fine structure (EXAFS) data indicates similar average first coordination spheres for all resting-state samples, regardless of preparation method. The average coordination sphere (per 2 coppers) mainly consists of 6 +/- 1 nitrogens or oxygens at an average Cu-(N,O) distance of 1.99 +/- 0.03 A and 2 +/- 1 sulfurs at an average Cu-S distance of 2.28 +/- 0.02 A. Quantitative curve-fitting analysis of the outer shell of the copper EXAFS indicates the presence of a Cu...Fe interaction at a distance of 3.00 +/- 0.03 A. Proposed structures of the two copper sites based on these and other spectroscopic results are presented, and differences between our results and those of other published copper XAS studies [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] are discussed.

X-ray Raman spectroscopy of carbon in asphaltene: light element characterization with bulk sensitivity.

X-ray Raman spectra of the carbon K-edge have been recorded using 6.461 keV radiation for a petroleum asphaltene. By comparison with coronene, graphite, and paraffin standards, the asphaltene spectrum is seen to be composed of contributions from saturated and aromatic carbon species. The information contained in the carbon K-edge was extracted with bulk (approximately 1 mm) sensitivity, because the Raman method used hard X-rays. This helps alleviate concerns about surface artifacts that frequently occur with soft X-ray spectroscopy of light elements. X-ray Raman spectroscopy shows great potential for characterization of light elements in fuels, catalysts, and other complex materials under chemically relevant conditions.

High-resolution X-ray fluorescence and excitation spectroscopy of metalloproteins.

A spectrograph has been developed with sufficient efficiency to make high-resolution fluorescence experiments on metalloproteins possible. The resolution of this spectrometer can reach 0.45 eV at 7.1 keV emission energy. The focus images of this multiple curved-crystal array spectrometer are presented. The chemical sensitivity of Kbeta emission spectra can be used to identify chemical states, and the spin-polarized near-edge structure provides a new measure of the spin density. The high-resolution fluorescence metalloprotein studies should become routine with third-generation synchrotron facilities, and the strength of both site and spin selectivity should complement the structural information from other spectroscopies.

X-ray absorption spectroscopy study of the LixFePO4 cathode during cycling using a novel electrochemical in situ reaction cell.

The extraction and insertion of lithium in LiFePO4 has been investigated in practical Li-ion intercalation electrodes for Li-ion batteries using Fe K-edge X-ray absorption spectroscopy (XAS). A versatile electrochemical in situ reaction cell was utilized, specifically designed for long-term X-ray experiments on battery electrodes during the lithium-extraction/insertion process in electrode materials for Li-ion batteries. The electrode contained about 7.7 mg of LiFePO4 on a 20 microm-thick Al foil. In order to determine the charge compensation mechanism and structural perturbations occurring in the system during cycling, in situ X-ray absorption fine-structure spectroscopy (XAFS) measurements were conducted on the cell at a moderate rate using typical Li-ion battery operating voltages (3.0-4.1 V versus Li/Li+). XAS studies of the LiFePO4 electrode measured at the initial state (LiFePO4) showed iron to be in the Fe(II) state corresponding to the initial state (0.0 mAh) of the battery, whereas in the delithiated state (FePO4) iron was found to be in the Fe(III) state corresponding to the final charged state (3 mAh) of the battery. The X-ray absorption near-edge structure (XANES) region of the XAS spectra revealed a high-spin configuration for the two states [Fe(II), d6 and Fe(III), d5]. The XAFS data analysis confirmed that the olivine structure of the LiFePO4 and FePO4 is retained by the electrodes, which is in agreement with the X-ray diffraction observations on these compounds. The XAFS data that were collected continuously during cycling revealed details about the response of the cathode to Li insertion and extraction. These measurements on the LiFePO4 cathode show that the material retains good structural short-range order leading to superior cycling.

Spinach carbonic anhydrase: investigation of the zinc-binding ligands by site-directed mutagenesis, elemental analysis, and EXAFS.

The enzyme carbonic anhydrase has been well characterized in mammalian systems, but the structural properties of the plant isozymes remain elusive. To investigate the nature of the zinc-binding site in spinach carbonic anhydrase, we targeted potential zinc ligands for mutagenesis and examined the resulting enzymes for catalytic activity and stoichiometric zinc binding. In addition, we examined the wild-type protein using extended X-ray absorption fine structure analysis. Our results suggest that spinach carbonic anhydrase utilizes a Cys-His-Cys-H2O ligand scheme to bind the zinc ion at the active site.