Review Article Imaging of Nitric Oxide by Magnetic Resonance … · 2019. 7. 31. · Review Article...

Review ArticleIn Vivo Imaging of Nitric Oxide byMagnetic Resonance Imaging Techniques

Rakesh Sharma1 Jeong-Won Seo2 and Soonjo Kwon3

1 Center for Nanomagnetics and Biotechnology Tallahassee FL 32310 USA2Department of Ophthalmology Hallym University Dongtan Sacred Heart Hospital Hwaseong 445-907 Republic of Korea3 Department of Biological Engineering Inha University 100 Inharo Nam-gu Incheon 402-751 Republic of Korea

Correspondence should be addressed to Soonjo Kwon soonjokwoninhaackr

Received 23 June 2014 Accepted 28 June 2014 Published 17 July 2014

Academic Editor Ki-Joon Jeon

Copyright copy 2014 Rakesh Sharma et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Nitric oxide (NO) biosensors are novel tools for real-time bioimaging of tissue oxygen changes and physiological monitoringof tissue vasculature Nitric oxide behavior further enhances its role in mapping signal transduction at the molecular levelSpectrometric electron paramagnetic resonance (EPR) and fluorometric imaging are well known techniques with the potential forin vivo bioimaging of NO In tissues NO is a specific target of nitrosyl compounds for chemical reaction which provides a uniqueopportunity for application of newly identified NO biosensors However the accuracy and sensitivity of NO biosensors still need tobe improved Another potential magnetic resonance technique based on short term NO effects on proton relaxation enhancementis magnetic resonance imaging (MRI) and someNO biosensors may be used as potent imaging contrast agents for measurement oftumor size byMRI combined with fluorescent imagingThe present review provides supporting information regarding the possibleuse of nitrosyl compounds as NO biosensors in MRI and fluorescent bioimaging showing their measurement limitations andquantitative accuracy These new approaches open a perspective regarding bioimaging of NO and the in vivo elucidation of NOeffects by magnetic resonance techniques

1 Introduction

Nitric oxide (NO) as a metabolic nitrogen compound inbound gas form plays an important role in physiological reg-ulation of the cardiovasculature in our body [1 2] Sinceendothelium-derived relaxing factor (EDRF) was first identi-fied in 1980 biological and chemical evidence has suggestedthat EDRF is nitric oxide (NO) a potent vasodilator [3]NO is released through the intermittent catalytic actionof constitutive NO synthase (cNOS) [4] In addition largetransient production ofNOat sites of inflammation is derivedfrom inducible NO synthase (iNOS) and related to hostdefense against infection [5] In vivo imaging of NO as abiosensor is an emerging monitoring technique that employsEPR fluoroscopy and MRI [6] The success of this methoddepends on visualizing free radical distribution of in vivospin-trapped NO NO imaging techniques primarily utilizemagnetic resonance (MR) electron paramagnetic resonance(EPR) spectrometry and fluorometry NO is a diatomic

free radical that contains one unpaired electron derivedfrom L-arginine via the catalytic action of NOS The in situvisualization of NO using bioimaging techniques providesinformation pertaining to the production and diffusionprocesses of NO [7 8] Real-time bioimaging techniquesusing EPR fluorescent indicators chemiluminescence real-time MRI and functional MRI (fMRI) have recently beenintroduced [9ndash11]

Physiological Basis of Bioimaging of NO NO is synthesizedby neuronal NOS endothelial NOS which is commonlyreferred to as cNOS and other types of iNOS specific tomacrophages andmicroglia through stimulation by cytokinesand endotoxins at sites of inflammation NOS isoforms aregenerally classified as either cNOS (Ca2+) or iNOS (Ca2+independent) [12] NO is a highly unstable molecule thatis rapidly oxidized into nitrite (NO2minus) and nitrate (NO3minus)in the presence of oxygen especially in the liquid phaseSynthesizedNO combines with oxygenwithin themembrane

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2014 Article ID 523646 13 pageshttpdxdoiorg1011552014523646

2 Journal of Nanomaterials

NH NH NH

O

L-Arginine L-Citrulline

NADPH NADPH

Heme

Heme

FAD

FADFMN FMN

CAM

CAM

Cell membrane

NO synthase (NOS)

BH4

BH4

H2N

H3N+ COOminus

+NO∙

N120596-OH-L-arginine

NndashOHNH2+

H2N H2N

H3N+ COOminus H3N+ COOminus

and O2

Figure 1 Proposed mechanism of NO production from epithelial cells NO∙ is released by several isoforms of the enzyme NOS whichcatalyzes the 5-electron oxidation of the guanidino nitrogen moiety of a nonaromatic amino acid (L-arginine) to citrulline via N120596-hydroxy-L-arginine The enzyme utilizes O

2and NADPH as cosubstrates and thiol tetrahydrobiopterin (BH4) FAD and FMN as cofactors NOS is

unique among eukaryotic enzymes in being a dimeric calmodulin-dependent or calmodulin-containing isozyme

and blood [13] As shown in Figure 1 NO is released from L-arginine via catalytic action of themembrane-bound enzymeNO synthase (NOS) NO is a physiologically importantmediator in metabolically active organs and tissues as wellas in neurotransmitters in central and peripheral neurons invivo [14]

11 Bioimaging of NO in Cardiovascular System

111 Electrochemical Measurement of NO NO biosensorswere initially developed for high resolution electrochemicalmeasurement methods of NO by several groups [15ndash17]These NO biosensors enable evaluation of dynamic changesin NO concentration in solutions and tissues in responseto agonists NO-generating reagents and physical stimuli[18ndash20] However their electrochemical applications areprimarily limited to short term recording of NO sensitivemyocardial changes for monitoring the effect of cardiacpotassium channel blockers [21] Use of this technique is alsolimited due to its poor sensitivity

112 NO Biosensing by Electron Paramagnetic Resonance(EPR) Spectrometry EPR spectroscopy is a specific techniquefor measurement of in vivoNO free radicals by spin-trappingcompounds (spin-traps) [22] A number of derived pyrrolineoxide and dithiocarbamate compounds have been shown tobe potential EPR spin-trap biosensors (Table 1)

113 NO Biosensing by Fluorometry Recently reported flu-orescent indicators allow real-time bioimaging of NO with

high spatial and temporal resolution Diaminorhodaminesand diaminofluorescein compounds undergo specific reac-tions with NO in cardiovascular tissues and may serve aspotential biosensors in fluoroscopy [23 24]

114 Spin-Trapping Technique in NO Biosensing The pre-sence of NO radicals at greater than the EPR detection limit(01ndash001120583M) can be detected by nitrone traps 55-dimethyl-1-pyrroline-119873-oxide (DMPO) 5-diethoxyphosphoryl-5-methyl-1-pyrroline-119873-oxide (DEPMPO) 120572-phenyl-119873-tert-butylnitrone (PBN) and 120572-(4-pyridyl-1-oxide)-119873-tert-buty-lnitrone (POBN) as well as by nitroso traps 2-methyl-2-nitrosopropane (MNP) and 35-dibromo-4-nitrosobenze-nesulfonic acid (DBNBS) [25ndash28]

115 In Vivo EPR Detection by Biosensing Free Radicals EPRspectrometers operating at S-band (16ndash4GHz) and L-band (04ndash16GHz) microwave frequency and at radiofrequency (02ndash04GHz) are utilized for in vivo measure-ments of the whole body of small animals The electronicconfiguration of NO with 11 valence electrons is(K2K2)(2s120590b)2(2s120590lowast)2(2p120587b)4(2p120590b)2(2p120587lowast)1 NO is a freeradical with one unpaired electron in the antibonding120587 orbital therefore EPR is considered to be the mostappropriate tool for its detection The electronic groundstate of NO is expressed by the term symbol

2

prod1232

[8 22 29] EPR signals from large biological samples cannotbe detected with a conventional X-band spectrometerdue to its poor sensitivity and detection limit However

Journal of Nanomaterials 3

Table 1 Various complexes as nitric oxide biosensors and NO bioimaging contrast applications and limitations

Complexes bound with NO NO bioimaging contrast applications and limitations(N-Methyl-D-glucamine)2-Fe(II)-NO complex EPR low contrast and MRI high contrast5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide NMR imaging with good possibility55-Dimethyl-1-pyrroline N-oxide (DMPO) EPR spin-trap with possibility of EPR imaging5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide EPR spin-trap with possibility of EPR imaging120572-Phenyl-N-tert-butylnitrone (PBN) EPR spin-trap with possibility of EPR imaging120572-(4-Pyridyl-1-oxide)-N-tert-butylnitrone EPR spin-trap with possibility of EPR imagingnitroso traps 2-methyl-2-nitrosopropane (MNP) EPR spin-trap with possibility of EPR imaging35-Dibromo-4-nitrosobenzenesulfonic acid EPR spin-trap with possibility of EPR imaging3-Carbamoyl-2255-tetramethylpyrrolidine-1-yloxyl EPR spin-trap with possibility of EPR imagingDithiocarbamate derivatives (Fe-DTCs) EPR spin-trap with possibility of EPR imagingPyrrolidine dithiocarbamate (PDTC) EPR spin-trap with possibility of EPR and MRIN-Methyl-D-glucamine dithiocarbamate (MGD) EPR spin-trap with possibility of EPR imagingN-(Dithiocarboxy)sarcosine (DTCS) EPR spin-trap with possibility of EPR imagingN-Methyl-L-serine dithiocarbamate (MSD) EPR spin-trap with possibility of EPR imagingL-Proline dithiocarbamate (ProDTC) EPR bioimagingDisulfiram (disulfide of DETC) EPR bioimagingDiglutathionyl dinitrosyl iron complex [DNIC-(GS)2] EPR bioimagingFe(III)(DTCS)3 and NO-Fe(II)(MGD)2 MRI EPR and chemiluminescence bioimagingFe-DETC trap EPR imaging of cultured alveolar cell[14N]ISDN or [15N]ISDN EPR-CT bioimagingDinitrosyl dithiolate iron complex EPR-CT bioimagingNO-Fe(DTC)2 EPR-CT bioimaging(MGD)2-Fe(II)-NO complex MRI NMR and EPR bioimagingDiaminonaphthalene DAN Fluorescent biosensorDichlorofluorescein DCFH Fluorescent biosensorIron(II)-quinoline pendant cyclam Heme fluorescent reporter biosensorCo complex [Co(NO)2(

RDATI)] Fluorescent biosensorCheletropic traps FNOCTs ESR and fluorophobic bioimagingDiaminofluoresceins DAFs FluorometryDiaminorhodamines DARs Fluorometry

EPR spectrometers operating at lower frequency arenow applied to in vivo measurements of EPR signalsfrom the whole body of small animals In this method anitro-compound spin-trap generates a frequency sensitiveelectron resonance signal due to the hydroxyl ion changein spin-trap energy [30] By application of a suitable analogto digital simulation (Monte Carlo simulation) all digitizedsimulations generate a colorful spot image at differentlocations that results in a whole body in vivo animal image[31] In vivo EPR imaging experiments using 4-hydroxy-2266-tetramethylpiperidine-1-yloxyl(4-hydroxy-TEMPO)and 3-carbamoyl-2255-tetramethylpyrrolidine-1-yloxyl(carbamoyl-PROXYL) have been extensively developedas a promising approach to oximetry for noninvasivemeasurement of tissue oxygen statusThis technique also hasthe possibility of physiological oxygen imaging applicationto NO-based fMRI in the near future For in vivo detectionof hydroxyl radical using a DEPMPO spin-trap in miceiron complexes with dithiocarbamate derivatives (Fe-DTCs)were used as spin-traps for NO adduct [NO-Fe(DTC)

2]

[32ndash34]

116 Trapping Target Complexes of Nitric Oxide Thecomplexform of NO (eg nitrosothiol) has a relatively longer half-lifethan free NO NO is rapidly oxidized into nitrite or nitrate inthe presence of oxygen especially in the liquid or tissue phaseand must be trapped by chemical NO biosensors to monitorits physiological concentration In previous studies differentlevels of NO concentrations were measured in differentorgans or tissues as tissue specific NO targets (Table 2)Figure 2 shows the different levels of effector moleculesor free radical induced cyclic guanosine monophosphate(cGMP) which are proportional to NO concentration Wepreviously reported guanylate and adenylate cyclase activityand intracellular levels of cGMP proportional to NO con-centration in alveolar epithelial cells and hepatocytes [3536] NO concentration is proportional to the intracellularspecific tissue responses that reflect the measurable physicalproperties of tissue metabolic state detectable using routinespin-trap imaging modalities of CT EPR MRI and opticaltechniques NO concentration in cardiovascular tissue is inthe range of nanomoles and picomoles These new biosensorapproaches have opened a new realm of nanomolar and


Table 2 In tissues concentrations of NO are shown in differentorgans or tissues up to the level of nanomolL and picomolL

Specific organstissues Mean concentrations ReferencesBrain 250 pmolL [112 113]Postischemic muscle 227 nmolL [109]Breast cancer tissue 36ndash70mmolL [103]Serum 225 nmolL [116]Liver 600 nmolL [104]Endothelium 120ndash68 nmolL [106]Alveoli 1500 pbb [117]Erythrocytes 438 to 1460mmolL [118]Articular cartilage 20ndash140 nmolmg protein [105]Subchondral bone 045ndash29 120583molmg protein [105]Trabecular bone 05ndash075 120583molmg protein [105]

NO biosensors

Hormone Effector molecules(high levels of NO

derived from iNOS)

GTP

GTP

GDP

Second messenger

Receptor

Response(detected by EPR MRI and optical

Guanylatecyclase

G120572

G120573 G120574

cGMP

imaging techniques)

Figure 2 Nitric oxide-induced cascade with a role of guanylatecyclase to generate physiological response detectable by NO boundbiosensor compounds (modified from the reference) [115] GTPguanosine triphosphateGDP guanosine diphosphate cGMP cyclicguanosinemonophosphate G

120572 G protein alpha G

120573 G protein beta

and G120574 G protein gamma

picomolar scale molecular imaging that has yet to be fullydeveloped

12 NO Specific Spin-Trapping and Biosensing Active Groupsin Bioimaging Different types of NO biosensors have activechemical groups that capture nitric oxide at various levels ofsensitivity in the body The ability to capture NO depends onthe active chemical structure of nitrone or cheletropic groupsand the oxidation state of inorganic iron elements present inthe biosensing molecule The oxidation state of nitric oxideis crucial to its capturing property and its sensitivity as abioimaging target Accordingly various trapping chemicalsare routinely used

121 NO-Trapping Reagents The major classes of compo-unds are nitrone (DMPO) and nitroso (MNP DBNBS)

spin-traps NO cheletropic traps (NOCTs) o-quinodi-methane 2-phenyl-4455-tetramethylimidazoline-1-yloxyl-3-oxide (PTIO) ferrous iron complexes such as Hbs andFe-DTC complexes Fe(III) hemoproteins and porphyrincomplexes [37ndash40] Consider

Fe (III) (Heme) + 2NO 997888rarr NO minus Fe (II) (Heme) +NO+

(1)

Cytochromes 1198881015840 are hemoproteins common in denitrifyingand photosynthetic bacteria that have high affinity for NOand can be used as biosensors for NO Recently NO-selectivephotometric or electrochemical biosensors have been devel-oped using cytochrome 1198881015840 immobilized on an optical fiber orelectrode or encapsulated in sol-gel glass [41]

122 Dithiocarbamates Pyrrolidine dithiocarbamate(PDTC) N-methyl-D-glucamine dithiocarbamate (MGD)N- (dithiocarboxy)sarcosine (DTCS) N-methyl-L-serinedithiocarbamate (MSD) L-proline dithiocarbamate(ProDTC) disulfiram (disulfide of DETC) NN-diethyldi-thiocarbamate (DETC) and diglutathionyl dinitrosyl ironcomplex [DNIC-(GS)

2] are major nitric oxide biosensor

com pounds Pyrrolidine dithiocarbamate (PDTC) inhibitsoxidative activation of nuclear transcription factor 120581B (NF-120581B) to develop immunity stress responses inflammationglial and neuronal function and the inhibition of apoptosisTherefore inhibition of NF-120581B activation by PDTC andDETC causes various biological phenomena includinginhibition of iNOS expression inhibition of apoptosisin thymocytes leukemic cells and L929 fibroblasts andinduction of heme oxygenase-1 gene expression [42ndash49]

123 Iron-Dithiocarbamate Complexes as NO-TrappingReagents NO-Fe(II)(DTCS)

2(gt100mM) and NO-Fe(II)

(MGD)2(lt1mM) showed specific ability for NO-trapping

of NO-Fe(II)(MGD)2and NO-Fe(II)(DTCS)

2complexes

[50ndash52]

124 Reactions of NO NO+ NOminus and NO2minus with Fe-

DTC Complexes NO reacts with disulfiram derivativeFe(II)(DTC)

2complex to form NO-Fe(II)(DTC)

2as the pri-

mary product and Fe(III)(DTC)3as a secondary product as

shown below Consider

NO + Fe (II) (DTC)2997888rarr NO minus Fe (II) (DCT)

2 (2)

Fe (III) (DETC)3+ NO 997888rarr NO minus Fe (II) (DETC)

2

+ DETClowast(3)

Basically there are three mechanisms responsible for thereductive nitrosylation of Fe(III)(DETC)

3complexes to form

Fe(III)(DETC)2[53] Consider

2DETClowast 997888rarr bis (DETC) (4)


The reductive nitrosylation of Fe(III)(MGD)3in the presence

of thiols generates NO-Fe(III)(MGD)2 which is further

oxidized into NO-Fe(II)(MGD)2products Consider

2NO + Fe (III) (MGD)2

H2O(lowastOH)997888997888997888997888997888997888997888997888rarr NO minus Fe (II) (MGD)

2

+NO2

minus

(5)

NO + Fe (III) (MGD)2

Reducing Equivalent997888997888997888997888997888997888997888997888997888997888997888997888997888997888rarr NO

minus Fe (II) (MGD)2

(6)

The reducing equivalent refers to endogenous reducingagents such as ascorbate hydroquinone and thiol MRIEPR optical and chemiluminescence LC-electrospray massspectroscopy showed that bioimaging of NO could beaccomplished using Fe(III)(DTCS)

3and NO-Fe(II)(MGD)

2

[54] In another mechanism unreacted Fe(III)(DTCS)3

donates an electron to the NO-Fe(III) complex to form NO-Fe(II)(DTCS)

2and Fe(IV)(DTCS)

3in the presence of ascor-

bate and glutathione suggesting that Fe-DTC complexes areefficient as NO traps in vivo and should be suitable for in vivoreal-time measurements of NO [55] Consider

[Fe (III) (DTCS)3]3minus

+NO 997888rarr [NO minus Fe (III) (DTCS)3]3minus

(7)

[NO minus Fe (III) (DTCS)3]3minus

+ [Fe (III) (DTCS)3]3minus

997888rarr [NO minus Fe (II) (DTCS)2]2minus

+ [Fe (IV) (DTCS)3]2minus

+ DTCS2minus

(8)

[Fe (IV) (DTCS)3]2minus

+ DTCS2minus 997888rarr [Fe (III) (DTCS)3]3minus

+1

2[bis (DTCS)]2minus

(9)

The overall reaction is as follows


+ NO 997888rarr [NO minus Fe (II) (DTCS)2]2minus

+1

2[bis (DTCS)]2minus

(10)

TheoxidizedNOminus nitroxyl ion donormoleculewith Fe-MGDcomplex and Fe(II)(MGD)

2complex is EPR which can be

used to visibly distinguish NO and reduced to molecularNO2

minus under low pH conditions such as tissue ischemia [56]

13 In Vitro and Ex Vivo EPR Detection of NO Using Fe-DTC Traps Current studies of nitric oxide bioimaging aremainly focused on detection ofNO in cultured cells and usingEPR and fluoroscopy methods to generate images of smallanimals The following sections provide an account of bothdetection and bioimaging of NO in cultured cells tissues andorgans

131 In Vitro Detection of NO from Cultured Cells UsingChemiluminescence Method NO is highly unstable in thepresence of oxygen and is rapidly converted into NO

2

minus andNO3

minus in the liquid phase To detect NO both NO2

minus andNO3

minus were converted into NO using a reducing agent (vana-dium (III) chloride) In cultured cells NO was measuredbased on the chemiluminescence (Model 280 NOA SieversInc Boulder CO) To achieve high conversion efficiency thereduction was performed at 90∘C [4 5]

132 Detection of NO in Resected Tissues and Organs Inthe last decade Fe-DETC traps have also been applied tomeasure NO concentrations in the liver kidney intestinespleen heart and lung as well as in regenerating rat livermouse stomach during adaptive relaxation and rat jejunumand ileum under ischemia reperfusion [57ndash59]The Fe-MGDtrap detectedNO formation fromnitrovasodilators includingglyceryl trinitrate isosorbide dinitrate (ISDN) and SNP [6061]

14 In Vivo EPR Detection and Imaging of NO in Living SmallAnimals In vivo EPR bioimaging is used for visualizationof iron bound nitrone and dithiocarbamates NMR imag-ing provides higher resolution than EPR imaging enablingobservation of spatial distribution of nitrone free radicalsdue to NMR sensitivity to iron paramagnetic behavior in thebody

141 Instrumentation and Imaging Techniques for In Vivo EPRMeasurements The three-dimensional EPR image (ie EPR-CT) was constructed based on Lauterburrsquos method [62] Inthis method a pair of magnetic field gradient coils for the 119909-119910- and 119911-axes are attached to the surface of the main magnetto obtain one set of EPR-CT images The microwave circuitwas constructed with a signal source a VSWR bridge aphase shifter a preamplifier and a double-balancedmixer forhomodyne detection The projection spectra were obtainedby changing the angles of the field gradient sequentially undera fixed gradient intensity in one plane The direction of thefield gradient was rotated in 20∘ steps and projections werecollected The obtained data for nine spectra of each two-dimensional projection constituted a three-dimensional setof images Arbitrary slice planes (ie CT images) can be cutfrom the three-dimensional data Thus data on 81 projectionspectra were needed under the selected field gradients toobtain one set of EPR-CT images [63 64]

142 In Vivo EPR Detection of Endogenous NO In vivo real-time detection of NO in the mouse tail was reported using aFe-MGD trap and a Fe-DETC trap with an L-band (114GHz)EPR spectrometer In vivo NO detection at the head regionmodels of sepsis and bacterial meningitis by the Fe-DETCtrap for NO-Fe(DETC)

2suggested that the NO-Fe(DETC)

2

signal is dependent on iNOS induced by IFN-120574 [65ndash71]

143 In Vivo EPR Imaging of Endogenous NO In vivo EPRimaging was used by applying the Fe-DTC traps for three-dimensional EPR imaging of NO in ischemia-hypoxia EPR


images from the frozen resected brain were obtained byemploying a Fe-DETC trap and an EPR imaging system witha microwave frequency of 12 GHz [72] An in vivo EPRimaging system with 700MHz microwave unit was designedfor bioimaging with a two-gap and loop-gap resonator usingNO-Fe(DTC)

2complexes as a spin probe or an imaging

agent [73] Our continued interest in in vivo EPR and MRIimaging of endogenously produced NO in the abdominalregion in a mouse encouraged us to use a NO-Fe-DTCS trapto image free radicals Another approach focused on using[14N]ISDN or [15N]ISDN to image the liver and kidney Amore complex multimodal imaging set of EPR-CT images inthe 119911-119909 plane showed the upper abdomen of a mouse due to15N substitution in the liver [74]

15 EPR Detection and Imaging of EndogenousNO-Relevant Complexes

151 Nitrosylheme Complexes Produced from Infused NitriteAn approach employing a combination of nitrite compoundand a Fe-MGD trap to detect NO in ischemia using L-band(13 GHz) EPR imaging of a heart subjected to cardiopul-monary arrest was reported [8]

152 Dinitrosyl Dithiolate Iron Complex Administered asa Spin Probe Dinitrosyl dithiolate iron complex (DNIC)consists of paramagnetic molecules that exhibit a charac-teristic EPR spectrum in both the solution state at roomtemperature and the frozen state in tissues at low temperaturePhysiologically DNIC and nitrosothiol (RSNO) compoundsstabilize and transport NO in biological systems In vivoreal-time detection and three-dimensional EPR-CT imagingof DNIC-(GS)

2in the abdomen in a 700MHz EPR system

showed NO in blood and NO delivery to the abdomen andliver [75]

153 NO-Fe(DTC)2Complexes as Spin Probes and Imaging

Reagents Paramagnetic NO-Fe(DTC)2complexes serve as

spin probes or imaging reagents for in vivo EPR imagingUsing these compounds a 700MHz EPR-CT system gen-erated a two-dimensional image of blood circulation in thecoronal section of the rat head (spatial resolution = 60mm)in which the high-intensity area (ventral side) was clearlydistinguished from the low-intensity area (dorsal side) [73]EPR-CT imaging in the mouse abdomen was accomplishedusing NO-Fe(DTCS)

2 NO-Fe(MGD)

2 and NO-Fe(DETC)

2

complexes as spin probes with the 700MHz EPR systemEPR-CT images showed the utility of NO-Fe(DTCS)

2and

NO-Fe(DTC)2complexes (spatial resolution 36mm) [73]

16 Approaches to NO Evaluation by Magnetic ResonanceImaging (MRI) Techniques Recently EPR-NMR techniquesemploying a proton-electron-double-resonance-imaging(PEDRI) hybrid technique showed enhancement of protonNMR signal intensity in the presence of radicals throughthe Overhauser effect or relaxation of neighboring protonssuch as the nitrosyl iron complex This method may beuseful as a functional MRI contrast agent specific for NO in

living organisms [76ndash79] L-Arginine increased the cerebralblood volume in hypertensive rats while ISDN increasedboth tumor blood flows on the NO images via magneticresonance techniques [11] We propose that another use ofNO exposure to hemoglobin may be capturing fMRI BOLDsignal hyperintensities on T1- T2- and T2lowast-weighted imagesdue to the addition of aqueous NO nitrite or dithionite andnitrite to the hemoglobin in the blood that is metHb andNO-Hb However additional studies are needed to confirmthis

Multimodal InVivoNOSpin-TrappingMRI-EPRExperimentsIn Vivo MRI imaging of Fe(II)-chelate spin-trapped nitricoxide by N-methyl-D-glucamine dithiocarbamate- (MGD-)NO mapping revealed radical distribution to localize nitricoxide in liver [80 81] Synthase (iNOS) is the main source ofNO At the optimal concentration of (MGD)

2-Fe(II) [MGD

100mM Fe 20mM] MR images on a GE 2-T CSI and IBMPC20 MiniSpect measured millimolar relaxivity of (MGD)2-Fe(II)-NO at parameters of TR 500msec TE 10msec NEX2 4mm slice thickness 1mm slice gap field of view 12 times3 times 12 cm and matrix 256 times 256 A 20MHz Jeol JES-FG2XGEPR spectrometer (microwave frequency 94GHz incidentmicrowave power 20mW 100 kHz modulation amplitude2G sweep width 100G scan time 2min) was used for EPRimaging

Several assumptions were made regarding multimodalin vivo NO spin-trapping MRI-EPR experiments (1) spin-trapped NO is stable in vivo (2) its contrast enhancementproperties in MRI have been assessed and (3) simultaneousvisualization and mapping of free radicals are possible byMRI The NO complex (MGD)

2-Fe(II)-NO is stable in

tissues and organs for MRI imaging and subsequent L-bandEPR measurements The liver is the most sensitive to NOcomplex upon X-band EPR [80 81] The (MGD)

2-Fe(II)-

NO complex shows remarkably strong proton relaxationenhancement because of its paramagnetic properties Thestrong magnetic moment of the unpaired electron promotesboth spin lattice and spin-spin relaxation of the surround-ing water protons resulting in a decrease in their spin-lattice (T1) and spin-spin (T2) relaxation times These effectscan be exploited to enhance signal intensity in T1 or T2weighted MR images in vivo in areas in which NO is trapped[78 79]

The NO complex acts as an effective intrinsic contrastagent enhancing its contrast in the images of several organsMRI analyses have shown that the NO complex can be apotentially usefulNO specificcontrast agentMapping the siteof NO generation is possible by L-band EPR combined withMRI spin-trapping for the direct detection of NO radicals invivo Here we propose a multimodal MRI-EPR-fluorometryapproach to map NO radicals within tissues and organs atmuch higher spatial resolution The spin-trapped adduct(MGD)

2-Fe(II)-NO a NMR contrast agent has the potential

to providemuch higher spatial resolution than with EPR NOis known to bind to iron compounds to form generally stablecomplexes such as (MGD)

2-Fe(II)-NO In vivo hemoglobin

is normally a natural NO spin-trap Specifically NO tends tobind with hemoglobin or to oxidize the hemoglobin after


which it was converted to nitrosyl-hemoglobin or methe-moglobin both of which are paramagnetic speciesWhen thebrain is stimulated to generate NO it is quite possible that(paramagnetic) nitrosyl-hemoglobin andmethemoglobin areformed Signal intensity enhancement in functional MRI(fMRI) is believed to result from changes in blood flowHowever for blood flow independent effects in MRI theparamagnetic relaxation from spin-trapped NO might pro-vide a new fMRI contribution using 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) This multimodalmethodology not only is suitable for mapping NO but alsomight be valid for other important free radicals in vivo whencombined with appropriate spin-trapping reagent techniques[11 82 83]

17 Fluorometric Imaging of NO by Fluorescent Probes for NO

171 Diaminonaphthalene (DAN) NO is readily oxidizedinto NO

2

minus and NO3

minus as final products in the presenceof O2 The fluorometric assay for the quantification of

NO2

minusNO3

minus up to 10 nM excited at 375 nm and emittedat 415 nm is based on the reaction of NO

2

minus with 23-diaminonaphthalene (DAN) to form the fluorescent product1-(H)-naphthotriazole (NAT) This method can serve as atool for defining the role of NOS in both normal andpathophysiological processes However the method cannotbe adapted for NO bioimaging because it causes seriousdamage to living cells [84]

172 Dichlorofluorescein (DCFH) 27-Dichlorofluorescein(DCFH) is oxidized by NO to dichlorofluorescein Thiscompound is a nonfluorescent species that may be used inmonitoring of intracellular NO formed in neuronal cells butis not suitable for bioimaging [85] DCFH has been shownto readily react with all reactive oxygen species Hence theoverall fluorescence level of DCFH would be specific to alllevels of reactive oxygen species and not exclusive to anyindividual of them [86]

173 Iron Complexes The iron(II)-quinoline pendant cyc-lam a fluorescent probe for NO is not convenient forNO detection in biological systems This probe mimics theactivation site of guanylate cyclase if used as a fluorescentbiosensor of NO The iron(II) complex further showed poorfluorescence emission at 460 nmwhichwas quenched byNOfrom NO releasing agents 2266-Tetramethylpiperidine-N-oxyl (TEMPO) labeled with acridine and Fe(II)(DTCS)

2

complex can be used to monitor direct production of NO inbiological systems but has not yet been applied in bioimaging[87 88]

174 Heme Domain with Fluorescent Reporter Dye Cyto-chrome 1198881015840 labeled with a fluorescent reporter dye containingfluorescent microspheres can serve as a ratiometric sensorof intracellular macrophage NO levels in phagocytosis NO-selective sensors were reported as a heme domain of guany-late cyclase (sGC) labeled with a fluorescent reporter dyeThe fluorescence intensity indicated the sGC heme domainrsquos

characteristic binding ofNOThe formation ofNO fromNOSin endothelial cells has a detection limit of 8120583MNO [89 90]

175 Cobalt Complex [Co(NO)2(RDATI)] Aminotropon-

iminates (H RATIs) with a dansyl fluorophore serve as a fluo-rescent NO biosensor and paramagnetic Co2+ complexesquench the fluorescence The [Co(NO)

2(RDATI)] increases

fluorescence intensity which is ideal for fluorescent NOsensing but not for bioimaging [91]

176 Fluorescent NO Cheletropic Trap (FNOCTs) FNOCTsreact with NO in a formal cheletropic reaction NO wasdetected by this method in alveolar macrophages [92]

18 Fluorescein Biosensors as NO Bioimaging Probes

181 Diaminofluoresceins (DAFs) Diaminofluoresceins(DAFs) are used as novel probes for NO DAFs change totriazole forms (DAF-Ts) with changes in the absorbancemaxima of NO fluorescence intensity due to the conversionof DAF-2 to DAF-2 T by NO in the presence of O

2 Major

compounds include DAF-4M1 4M2 5M1 and 5M2fluorinated fluorescein derivatives These compounds arederived amino acids (DAN) with aromatic groups Howeveruse of these compounds in NO bioimaging is in its infancybecause DAN leaks easily through cell membranes afterloading Nevertheless the use of esterified DAN has shownpromise in studies of NO bioimaging [93 94] DAF hasemerged as a reliable fluorophore for real-time NO detectionin live cells However DAF is highly cytotoxic Thus DAF-based measurements are only accurate if measured withinfirst 15ndash20 minutes Beyond that it significantly affects theviability of the cells In some of the research studies toovercome the cytotoxicity of DAF DAF was coincubatedwith serumThis approach significantly improved the qualityof cells following DAF incubation However most of theDAFs had reacted with serum to form fluorescent productwhich imposed limitations related to reproducibility of themeasurement

182 Diaminorhodamines (DARs) Fluorescent rhodamine Bfluorophore imaging with DAR-1 AM DAR-1 EE DAR-MDAR-M AM and DAR-4M has shown little success [95]

183 Emission Mechanism DAF shows the photoinducedelectron transfer (PET) process of fluorescence quenchingor reduced fluorescence of the fluorophore The mechanismwas reported using 9-[2-(3-carboxy) naphthyl]-6-hydroxy-3H-xanthen-3-one (NX) and 9-[2-(3-carboxy) anthryl]-6-hydroxy-3H-xanthen-3-one (AX) NX is highly fluorescentwhereas AX is almost nonfluorescent [96]

19 Biological Applications of DAFs and DARs

191 Cardiovascular Tissue Current studies are focusingon fast real-time nitric oxide biosensing by electrochemical


methods as recently reported by electron transfer acrossmul-tiassembly of hemoglobin-montmorillonite with polymer asbiosensors with high reproducibility [97] Low nitric oxidelevels are considered potent markers of sickling and majorfactors responsible for the inability of red blood cells to relaxarteries and oxygen deprivation Nitric oxide is now on themarket as a nutrient supplement Nitric oxide levels wereelevated following in vivo correction of cardiac ischemiaand NO capture was detected by a nanobiosensor (Nafionm-phenylenediamine and resorcinol) based amperometrictechnique [98] The nitric oxide content in arteries wasdetermined by measuring superoxide anion from superoxidedismutase enzyme at levels of up to 10 nM nitric oxide byusing an enzyme biosensor based amperometric method

Nitric oxide is an organically produced signaling mole-cule that regulates blood pressure clots that cause stroke andheart attack and atherosclerosis This molecule penetratesacross membranes with biological signals and messagesinfluencing every organ including the lungs liver stomachgenitals and kidneys New technological developments suchas nanotechnology have led to great advances in nitric oxidebiosensing through use of fiber optic chemical sensingcarbon nanotubes and metalloporphyrin biosensors [99ndash102]

192 Breast Cancer Tissue Recently nitric oxide was evalu-ated as an angiogenesis marker in breast cancer patients withthe potential for generation of a biomarker of prognosis [103]

193 Liver The major application of liver bioimaging wasestablished by nitric oxide and asymmetric dimethylargininein human alcoholic cirrhosis [104] The mouse abdomenwas imaged in three dimensions by localization of NO-richregions in the liver [74]

194 Bone and Cartilage The use of NO biosensor in boneand cartilage application is still in its infancy and limited Arecent report indicated the possibility of usingNO biosensorsas a method of detection of NO in bone and for cartilagecharacterization [105]

195 Endothelial Cells DAF-FM is a useful tool for visualiz-ing the temporal and spatial distribution of intracellular NOEndogenous ATP plays a central role in HTS-induced NOSin BAEC Endothelial cNOS a Ca2+calmodulin-dependentenzyme is critical to vascular homeostasis and generates adetectable basal level of NO production at low extracellularCa2+ Actin microfilaments in PAEC regulate L-argininetransport which can affect NO production by PAEC DAR-4M should be useful for bioimaging of samples that havestrong autofluorescence [106 107]

196 Smooth Muscle Cells DAF-2 DA and DAF-FM Tenhance fluorescence intensity [108] This sensitive methodenables their use for detection of spontaneous and substanceP (active coronary artery protein) induced NO release fromisolated porcine coronary arteries This NO release was

entirely dependent on the NOS activity in vascular endothe-lial cells Furthermore fluorescence images of culturedsmooth muscle cells in the rat urinary bladder were capturedafter loading with DAF-FMDA [109] In cells pretreated withcytokines the fluorescence intensity increasedwith time afterDAF-FM loading

197 Brain DAF-2 DA was used for direct detection of NOin theCA1 region of the hippocampus by imaging techniquesDAF-FM DA was also applied to imaging of NO generatedin rat hippocampal slices [110 111] Recently the use of NObioimaging for assessment of cortical impact injury wasevaluated and physiological concentrations of target NOweremonitored [112 113]

198 Ion Channels Voltage-gated Na+ channels and themechanisms by which they enable signaling across cardiactissue are not well understood However NO is an endoge-nous regulator of persistent Na+ current NMDA-receptor-(NMDAR-) associated ion channel has been reported to bemodulated by exogenous and endogenous NO EndogenousS-nitrosylation may regulate ion channel activity [114]

New Emerging Information Regarding Nitric Oxide Nitricoxide plays a unique role in the body and its rapid real-timebiosensing and measurement may reveal a great deal of newinformation in time In the body nitric oxide is known to

(i) fight bacteria viruses and parasites(ii) suppress proliferation of some types of cancer cells(iii) prevent serious complications in diabetic patients

particularly in association with impaired blood flow(iv) play a major role in memory(v) act as a neurotransmitter(vi) increase sexual functioning(vii) act as a powerful antioxidant deactivating free radi-

cals that contribute to cancer diabetes heart diseaseand stroke

NO plays important roles in inflammatory processesFor example increased expression of iNOS mRNA causesincreasedNOproduction at sites of inflammationDrosophilautilizes components of the NOcGMP signaling pathwayand chemical sensors are specific to endothelial nitric oxideand nitric oxide synthase enzyme systems A new class ofbiosensors that are multifunctional and multimodal has theability to perform as nitric oxide detectors and to monitortissue response to nitric oxide synthase biochemical mech-anisms Recently DAF-2 DA has been reported as a usefulbiosensor of hypoxia Adenovirus-mediated gene transfer ofeNOS in adrenal zona glomerulosa (ZG) cells results in theexpression of active endothelial NOS enzyme decreasingaldosterone synthesis Moreover 120574-irradiation at doses of 2ndash50Gy stimulates the expression of iNOS which is accom-panied by an increase in the fluorescence of DAF-2 NOproduction by mitochondrial NOS plays a role in respirationas well as apoptosis in PC 12 and COS-1 cells DAF-2 can


be used to image real-time intracellular NO production inretina specific synapsesKalanchoe daigremontiana andTaxusbrevifolia showed NO-induced apoptosis upon application ofDAF-2 DA while L-NMMA suppressed NO production andapoptosis [114]

2 Conclusion

This review highlights the biosensing of NO by multimodalin vivo EPRMRIfluorometry based on the potential useof NO biosensors Fluorescent biosensors such as DAFsand DARs visualize the production of intracellular NO andenable observation of the temporal and spatial distributionof intracellular NO as a nitric oxide map Additionally thecurrently available data indicate that more attention shouldbe given to in vivo real-time imaging of NO which couldbe developed based on a combination of EPR and NMRtechniques as NO sensitive fMRI Amperometric and elec-trochemical methods using nanotechnology and advancedelectronics appear to be a breakthrough in nitric oxide real-time measurement Currently DAFs and DARs are goodcandidates for bioimaging of NO in terms of specificity sen-sitivity and handling Therefore the NO detection methoddepends on reactive oxygen species such as NO

2

minus NO3

minusROS superoxide hydrogen peroxide and ONOOminus to yieldany fluorescent product Ratiometric probes are other optionsfor intensity measurements Overall further studies on thedevelopment of novel ratiometric NO bioimaging probes arewarranted

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

Funding was provided by Inha University Research Grant(INHA-47289-01) Republic of Korea

References

[1] A Suhardja ldquoMechanisms of disease roles of nitric oxideand endothelin-1 in delayed cerebral vasospasm produced byaneurysmal subarachnoid hemorrhagerdquo Nature Clinical Prac-tice Cardiovascular Medicine vol 1 no 2 pp 110ndash116 2004

[2] K M Naseem ldquoThe role of nitric oxide in cardiovasculardiseasesrdquoMolecular Aspects of Medicine vol 26 no 1-2 pp 33ndash65 2005

[3] R F Furchgott ldquoIntroduction to EDRF researchrdquo Journal ofCardiovascular Pharmacology vol 22 supplement 7 pp S1ndashS21993

[4] S Kwon R L Newcomb and S C George ldquoMechanisms ofsynergistic cytokine-induced nitric oxide production in humanalveolar epithelial cellsrdquo Nitric Oxide vol 5 no 6 pp 534ndash5462001

[5] S Kwon and S C George ldquoSynergistic cytokine-induced nitricoxide production in human alveolar epithelial cellsrdquo NitricOxidemdashBiology and Chemistry vol 3 no 4 pp 348ndash357 1999

[6] F Hyodo R Murugesan K Matsumoto et al ldquoMonitoringredox-sensitive paramagnetic contrast agent by EPRI OMRIandMRIrdquo Journal ofMagnetic Resonance vol 190 no 1 pp 105ndash112 2008

[7] H Fujii and L J Berliner ldquoNitric oxide prospects and perspec-tives of in vivo detection by L-band EPR spectroscopyrdquo Physicsin Medicine and Biology vol 43 no 7 pp 1949ndash1956 1998

[8] P Kuppusamy R A Shankar V M Roubaud and J L ZweierldquoWhole body detection and imaging of nitric oxide generationinmice following cardiopulmonary arrest detection of intrinsicnitrosoheme complexesrdquo Magnetic Resonance in Medicine vol45 no 4 pp 700ndash707 2001

[9] X Zhang ldquoReal time and in vivo monitoring of nitric oxideby electrochemical sensorsmdashfrom dream to realityrdquo Frontiers inBioscience vol 9 pp 3434ndash3446 2004

[10] K Liu G Ning and X Zheng ldquoIn vivo detection of nitricoxide in rat hippocampusrdquo in Proceedings of the 27th AnnualInternational Conference of the Engineering in Medicine andBiology Society (IEEE-EMBS rsquo05) pp 1039ndash1042 ShanghaiChina September 2005

[11] F Di Salle P Barone H Hacker F Smaltino and M DrsquoIschialdquoNitric oxide-haemoglobin interaction a new biochemicalhypothesis for signal changes in fMRIrdquo NeuroReport vol 8 no2 pp 461ndash464 1997

[12] X Liu M J S Miller M S Joshi H Sadowska-Krowicka D AClark and J R Lancaster Jr ldquoDiffusion-limited reaction of freenitric oxide with erythrocytesrdquo Journal of Biological Chemistryvol 273 no 30 pp 18709ndash18713 1998

[13] J R Lancaster Jr and L J Ignarro Nitric Oxide Biology andPathobiology Academic Press San Diego Calif USA 2000

[14] J L Dinerman C J Lowenstein and S H Snyder ldquoMolecularmechanisms of nitric oxide regulation potential relevance tocardiovascular diseaserdquo Circulation Research vol 73 no 2 pp217ndash222 1993

[15] T Malinski and Z Taha ldquoNitric oxide release from a single cellmeasured in situ by a porphyrinic-based microsensorrdquo Naturevol 358 no 6388 pp 676ndash678 1992

[16] K Shibuki ldquoAn electrochemical microprobe for detecting nitricoxide release in brain tissuerdquo Neuroscience Research vol 9 no1 pp 69ndash76 1990

[17] Z Taha F Kiechle and T Malinski ldquoOxidation of nitric oxideby oxygen in biological systems monitored by porphyrinicsensorrdquo Biochemical and Biophysical Research Communicationsvol 188 no 2 pp 734ndash739 1992

[18] S Mochizuki Y Chiba Y Ogasawara et al ldquoDirect in situevaluation of nitroglycerin-derived nitric oxide production inthe canine and rat vascular walls at high temporal and spatialresolutionsrdquo Cardiovascular Engineering vol 1 no 2 pp 85ndash912001

[19] S Mochizuki M Goto Y Chiba Y Ogasawara and F KajiyaldquoFlow dependence and time constant of the change in nitricoxide concentration measured in the vascular mediardquo Medicaland Biological Engineering and Computing vol 37 no 4 pp497ndash503 1999

[20] P Vallance S Patton K Bhagat et al ldquoDirect measurement ofnitric oxide in human beingsrdquo The Lancet vol 346 no 8968pp 153ndash154 1995

[21] D J Pinsky S Patton S Mesaros et al ldquoMechanical transduc-tion of nitric oxide synthesis in the beating heartrdquo CirculationResearch vol 81 no 3 pp 372ndash379 1997

[22] L J Berliner and H Fujii ldquoIn vivo spin trapping of nitric oxiderdquoAntioxidants amp Redox Signaling vol 6 no 3 pp 649ndash656 2004


[23] N Suzuki H Kojima Y Urano K Kikuchi Y Hirata and TNagano ldquoOrthogonality of calcium concentration and ability of4 5-diaminofluorescein to detect NOrdquoThe Journal of BiologicalChemistry vol 277 pp 47ndash49 2002

[24] X Ye S S Rubakhin and J V Sweedler ldquoSimultaneous nitricoxide and dehydroascorbic acid imaging by combining diami-nofluoresceins and diaminorhodaminesrdquo Journal of Neuro-science Methods vol 168 no 2 pp 373ndash382 2008

[25] A L Kleschyov P Wenzel and T Munzel ldquoElectron paramag-netic resonance (EPR) spin trapping of biological nitric oxiderdquoJournal of Chromatography B Analytical Technologies in theBiomedical and Life Sciences vol 851 no 1-2 pp 12ndash20 2007

[26] J Weaver S Porasuphatana P Tsai T Budzichowski and GMRosen ldquoSpin trapping nitric oxide from neuronal nitric oxidesynthase a look at several iron-dithiocarbamate complexesrdquoFree Radical Research vol 39 no 10 pp 1027ndash1033 2005

[27] P G Winyard I A Knight F L Shaw et al ldquoChapter 8 dete-rmination of s-nitrosothiols in biological and clinical samplesusing electron paramagnetic resonance spectrometry with spintrappingrdquoMethods in Enzymology vol 441 pp 151ndash160 2008

[28] M Ziaja J Pyka A MacHowska A Maslanka and P MPlonka ldquoNitric oxide spin-trapping and NADPH-diaphoraseactivity inmature rat brain after injuryrdquo Journal ofNeurotraumavol 24 no 12 pp 1845ndash1854 2007

[29] P Kuppusamy P Wang A Samouilov and J L Zweier ldquoSpatialmapping of nitric oxide generation in the ischemic heart usingelectron paramagnetic resonance imagingrdquoMagnetic Resonancein Medicine vol 36 no 2 pp 212ndash218 1996

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[33] E Linares S Giorgio and O Augusto ldquoInhibition of in vivoleishmanicidal mechanisms by tempol nitric oxide down-regulation and oxidant scavengingrdquo Free Radical Biology andMedicine vol 44 no 8 pp 1668ndash1676 2008

[34] K J Liu M Miyake T Panz and H Swartz ldquoEvaluation ofDEPMPO as a spin trapping agent in biological systemsrdquo FreeRadical Biology amp Medicine vol 26 no 5-6 pp 714ndash721 1999

[35] S Kwon and R Sharma ldquoRole of effectors on hypoxia dueto nitric oxide production in human alveolar epithelial cellsand oxygen depletion in human hepatocytes Part II PossibleMechanism of Cancerrdquo Cancer Research Journal vol 4 pp 85ndash101 2009

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[37] Y R Chen C L Chen X Liu H Li J L Zweier and R PMason ldquoInvolvement of protein radical protein aggregationand effects on NO metabolism in the hypochlorite-mediatedoxidation of mitochondrial cytochrome crdquo Free Radical Biologyand Medicine vol 37 no 10 pp 1591ndash1603 2004

[38] Y Chen C ChenW Chen et al ldquoFormation of protein tyrosineortho-semiquinone radical and nitrotyrosine from cytochromec-derived tyrosyl radicalrdquo The Journal of Biological Chemistryvol 279 no 17 pp 18054ndash18062 2004

[39] A FVaninAHuisman E SG Stroes F C deRuijter-HeijstekT J Rabelink and E E van Faassen ldquoAntioxidant capacityofmononitrosyl-iron-dithiocarbamate complexes implicationsfor NO trappingrdquo Free Radical Biology andMedicine vol 30 no8 pp 813ndash824 2001

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[43] W Eberhardt D Kunz and J Pfeilschifter ldquoPyrrolidine dithio-carbamate differentially affects interleukin 1120573- and cAMP-induced nitric oxide synthase expression in rat renal mesangialcellsrdquo Biochemical and Biophysical Research Communicationsvol 200 no 1 pp 163ndash170 1994

[44] F J Bedoya M Flodstrom and D L Eizirik ldquoPyrrolidinedithiocarbamate prevents IL-1-induced nitric oxide synthasemRNA but not superoxide dismutase mRNA in insulin pro-ducing cellsrdquo Biochemical and Biophysical Research Communi-cations vol 210 no 3 pp 816ndash822 1995

[45] J T Wolfe D Ross and G M Cohen ldquoA role for metals andfree radicals in the induction of apoptosis in thymocytesrdquo TheFEBS Letters vol 352 no 1 pp 58ndash62 1994

[46] R Bessho K Matsubara M Kubota et al ldquoPyrrolidine dithio-carbamate a potent inhibitor of nuclear factor 120581B (NF-120581B) acti-vation prevents apoptosis in human promyelocytic leukemiaHL-60 cells and thymocytesrdquo Biochemical Pharmacology vol48 no 10 pp 1883ndash1889 1994

[47] C Victor Jongeneel ldquoBcl-2 protects from oxidative damage andapoptotic cell death without interfering with activation of NF-120581B by TNFrdquo FEBS Letters vol 351 no 1 pp 45ndash48 1994

[48] Y Hattori K Akimoto Y Murakami and K Kasai ldquoPyrroli-dine dithiocarbamate inhibits cytokine-induced VCAM-1 geneexpression in rat cardiac myocytesrdquo Molecular and CellularBiochemistry vol 177 no 1-2 pp 177ndash181 1997

[49] B M Altura and A Gebrewold ldquoPyrrolidine dithiocarbamateattenuates alcohol-induced leukocyte-endothelial cell interac-tion and cerebral vascular damage in rats possible role of activa-tion of transcription factor NF-120581B in alcohol brain pathologyrdquoAlcohol vol 16 no 1 pp 25ndash28 1998

[50] SV PaschenkoVVKhramtsovM P SkatchkovV F Plyusninand E Bassenge ldquoEPR and laser flash photolysis studies ofthe reaction of nitric oxide with water soluble NO trap Fe(II)-proline-dithiocarbamate complexrdquo Biochemical and BiophysicalResearch Communications vol 225 no 2 pp 577ndash584 1996

[51] H Nakagawa N Ikota T Ozawa T Masumizu and MKohno ldquoSpin trapping for nitric oxide produced in LPS-treatedmouse using various new dithiocarbamate iron complexeshaving substituted proline and serine moietyrdquo Biochemistry and


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[52] H Fujii T Yoshimura and H Kamada ldquoESR studies of A1119906and

A2119906

oxoiron(IV) porphyrin 120587-cation radical complexes Spincoupling between ferryl iron and A

1119906A2119906

orbitalsrdquo InorganicChemistry vol 35 no 8 pp 2373ndash2377 1996

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[56] J L Zweier P Wang A Samouilov and P KuppusamyldquoEnzyme-independent formation of nitric oxide in biologicaltissuesrdquo Nature Medicine vol 1 no 8 pp 804ndash809 1995

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[59] A M Komarov J H Kramer I T Mak and W B WeglickildquoEPR detection of endogenous nitric oxide in postischemicheart using lipid and aqueous-soluble dithiocarbamate-ironcomplexesrdquo Molecular and Cellular Biochemistry vol 175 no1-2 pp 91ndash97 1997

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[69] Y S Kim and M G Tauber ldquoNeurotoxicity of glia activatedby gram-positive bacterial products depends on nitric oxideproductionrdquo Infection and Immunity vol 64 no 8 pp 3148ndash3153 1996

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[73] T Yoshimura ldquoIn vivo EPR detection and imaging of endoge-nously produced and exogenously supplied nitric oxiderdquo Ana-lytical Sciences vol 13 pp 451ndash454 1997

[74] S Fujii Y Suzuki T Yoshimura and H Kamada ldquoIn vivothree-dimensional EPR imaging of nitric oxide productionfrom isosorbide dinitrate in micerdquo The American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 274 no5 pp G857ndashG862 1998

[75] T Ueno Y Suzuki S Fujii A F Vanin and T Yoshimura ldquoInvivo distribution and behavior of paramagnetic dinitrosyl dithi-olato iron complex in the abdomen of mouserdquo Free RadicalResearch vol 31 no 6 pp 525ndash534 1999

[76] M A Foster I Seimenis and D J Lurie ldquoThe application ofPEDRI to the study of free radicals in vivordquo Physics in Medicineand Biology vol 43 no 7 pp 1893ndash1897 1998

[77] A Mulsch D J Lurie I Seimenis B Fichtlscherer and MA Foster ldquoDetection of nitrosyl-iron complexes by proton-electron-double-resonance imagingrdquo Free Radical Biology andMedicine vol 27 no 5-6 pp 636ndash646 1999

[78] S Rast A Borel L Helm E Belorizky P H Fries andA E Merbach ldquoEPR spectroscopy of MRI-related Gd(III)complexes simultaneous analysis of multiple frequency andtemperature spectra including static and transient crystal fieldeffectsrdquo Journal of the American Chemical Society vol 123 no11 pp 2637ndash2644 2001

[79] A E Merbach and E Toth The Chemistry of Contrast Agentsin Medical Magnetic Resonance Imaging John Wiley amp SonsChichester UK 2001

[80] T Yoshimura H Yokoyama S Fujii F Takayama K Oikawaand H Kamada ldquoIn vivo EPR detection and imaging ofendogenous nitric oxide in lipopolysaccharide-treated micerdquoNature Biotechnology vol 14 no 8 pp 992ndash994 1996

[81] A F Vanin P I Mordvintcev S Hauschildt and A MulschldquoThe relationship between L-arginine-dependent nitric oxidesynthesis nitrite release and dinitrosyl-iron complex formationby activated macrophagesrdquo Biochimica et Biophysica Acta vol1177 no 1 pp 37ndash42 1993


[82] Y Kotake ldquoContinuous and quantitative monitoring of rate ofcellular nitric oxide generationrdquo Methods in Enzymology vol268 pp 222ndash229 1996

[83] A F Vanin ldquoIron diethyldithiocarbamate as spin trap for nitricoxide detectionrdquoMethods in Enzymology vol 301 pp 269ndash2791999

[84] T P Misko R J Schilling D Salvemini W M Moore and MG Currie ldquoA fluorometric assay for the measurement of nitritein biological samplesrdquo Analytical Biochemistry vol 214 no 1pp 11ndash16 1993

[85] P G Gunasekar B G Kanthasamy J L Borowitz and GE Isom ldquoMonitoring intracellular nitric oxide formation bydichlorofluorescin in neuronal cellsrdquo Journal of NeuroscienceMethods vol 61 no 1-2 pp 15ndash21 1995

[86] O Myhre J M Andersen H Aarnes and F Fonnum ldquoEvalua-tion of the probes 2101584071015840-dichlorofluorescin diacetate luminoland lucigenin as indicators of reactive species formationrdquoBiochemical Pharmacology vol 65 no 10 pp 1575ndash1582 2003

[87] N Soh Y Katayama and M Maeda ldquoA fluorescent probe formonitoring nitric oxide production using a novel detectionconceptrdquo Analyst vol 126 no 5 pp 564ndash566 2001

[88] X Ye S S Rubakhin and J V Sweedler ldquoDetection of nitricoxide in single cellsrdquo Analyst vol 133 no 4 pp 423ndash433 2008

[89] S L R Barker H A Clark S F Swallen R Kopelman AW Tsang and J A Swanson ldquoRatiometric and fluorescence-lifetime-based biosensors incorporating cytochrome c1015840 and thedetection of extra- and intracellular macrophage nitric oxiderdquoAnalytical Chemistry vol 71 no 9 pp 1767ndash1772 1999

[90] S L Barker Y ZhaoMAMarletta andRKopelman ldquoCellularapplications of a sensitive and selective fiber-optic nitric oxidebiosensor based on a dye-labeled heme domain of solubleguanylate cyclaserdquoAnalytical Chemistry vol 71 no 11 pp 2071ndash2075 1999

[91] K J Franz N Singh and S J Lippard ldquoMetal-based NOsensing by selective ligand dissociation this workwas supportedby a grant from the national science foundation and a fel-lowship to NS from the undergraduate research opportunityprogram (MIT) We thank Prof Roger Tsien for valuable dis-cussions at the inception of this projectrdquoAngewandte ChemiemdashInternational Edition vol 39 pp 2120ndash2122 2000

[92] P Meineke U Rauen H de Groot H-G Korth and RSustmann ldquoNitric oxide detection and visualization in biolog-ical systems Applications of the FNOCT methodrdquo BiologicalChemistry vol 381 no 7 pp 575ndash582 2000

[93] L J Ignarro J M Fukuto J M Griscavage N E Rogers andR E Byrns ldquoOxidation of nitric oxide in aqueous solution tonitrite but not nitrate comparison with enzymatically formednitric oxide from L-argininerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 90 no17 pp 8103ndash8107 1993

[94] H Kojima N Nakatsubo K Kikuchi et al ldquoDetection andimaging of nitric oxide with novel fluorescent indicatorsdiaminofluoresceinsrdquo Analytical Chemistry vol 70 no 13 pp2446ndash2453 1998

[95] H Kojima M Hirotani N Nakatsubo et al ldquoBioimaging ofnitric oxide with fluorescent indicators based on the rhodaminechromophorerdquo Analytical Chemistry vol 73 no 9 pp 1967ndash1973 2001

[96] R Y Tsien A Waggoner and J B Pawley Handbook ofBiological Confocal Microscopy Plenum Press New York NYUSA 1995

[97] J Pang C Fan X Liu T Chen andG Li ldquoA nitric oxide biosen-sor based on the multi-assembly of hemoglobinmontmo-rillonitepolyvinyl alcohol at a pyrolytic graphite electroderdquoBiosensors and Bioelectronics vol 19 no 5 pp 441ndash445 2003

[98] J K Park P H Tran J K T Chao R Ghodadra R Rangarajanand N V Thakor ldquoIn vivo nitric oxide sensor using non-conducting polymer-modified carbon fiberrdquo Biosensors andBioelectronics vol 13 no 11 pp 1187ndash1195 1998

[99] K Balasubramanian and M Burghard ldquoBiosensors based oncarbon nanotubesrdquo Analytical and Bioanalytical Chemistry vol385 no 3 pp 452ndash468 2006

[100] T Imanishi A Kuroi H Ikejima et al ldquoEffects of pioglitazoneon nitric oxide bioavailability measured using a catheter-typenitric oxide sensor in angiotensin II-infusion rabbitrdquoHyperten-sion Research vol 31 no 1 pp 117ndash125 2008

[101] R Wadsworth E Stankevicius and U Simonsen ldquoPhysiolog-ically relevant measurements of nitric oxide in cardiovascularresearch using electrochemical microsensorsrdquo Journal of Vas-cular Research vol 43 no 1 pp 70ndash85 2006

[102] D H P Hedges D J Richardson and D A Russell ldquoElec-trochemical control of protein monolayers at indium tin oxidesurfaces for the reagentless optical biosensing of nitric oxiderdquoLangmuir vol 20 no 5 pp 1901ndash1908 2004

[103] D Konukoglu M S Turhan V Celik and H Turna ldquoRelationof serum vascular endothelial growth factor as an angiogenesisbiomarker with nitric oxide amp urokinase-type plasminogenactivator in breast cancer patientsrdquo Indian Journal of MedicalResearch vol 125 no 6 pp 747ndash751 2007

[104] P Lluch B Torondel P Medina et al ldquoPlasma concentrationsof nitric oxide and asymmetric dimethylarginine in humanalcoholic cirrhosisrdquo Journal of Hepatology vol 41 no 1 pp 55ndash59 2004

[105] M R van der Harst S Bull P A J Brama A Barneveld PR van Weeren and C H A van de Lest ldquoNitrite and nitro-tyrosine concentrations in articular cartilage subchondralbone and trabecular bone of normal juvenile normal adultand osteoarthritic adult equine metacarpophalangeal jointsrdquoJournal of Rheumatology vol 33 no 8 pp 1662ndash1667 2006

[106] P Kleinbongard A Dejam T Lauer et al ldquoPlasma nitriteconcentrations reflect the degree of endothelial dysfunction inhumansrdquo Free Radical Biology and Medicine vol 40 no 2 pp295ndash302 2006

[107] H Kojima Y Urano K Kikuchi T Higuchi Y Hirata andT Nagano ldquoFluorescent indicators for imaging nitric oxideproductionrdquo Angewandte Chemie International Edition vol 38pp 3209ndash3212 1999

[108] Y Itoh F H Ma H Hoshi et al ldquoDetermination andbioimaging method for nitric oxide in biological specimens bydiaminofluorescein fluorometryrdquo Analytical Biochemistry vol287 no 2 pp 203ndash209 2000

[109] F Stoffels F Lohofener M Beisenhirtz F Lisdat and RButtemeyer ldquoConcentration decrease of nitric oxide in thepostischemicmuscle is not only caused by the generation ofO

2rdquo

Microsurgery vol 27 no 6 pp 565ndash568 2007[110] H Kojima N Nakatsubo K Kikuchi et al ldquoDirect evidence

of NO production in rat hippocampus and cortex using a newfluorescent indicator DAF-2 DArdquo NeuroReport vol 9 no 15pp 3345ndash3348 1998

[111] O Yermolaieva N Brot H Weissbach S H Heinemann andT Hoshi ldquoReactive oxygen species and nitric oxide mediateplasticity of neuronal calcium signalingrdquo Proceedings of the


National Academy of Sciences of the United States of Americavol 97 no 1 pp 448ndash453 2000

[112] C N Hall and D Attwell ldquoAssessing the physiological concen-tration and targets of nitric oxide in brain tissuerdquo Journal ofPhysiology vol 586 no 15 pp 3597ndash3615 2008

[113] L Cherian and C S Robertson ldquoL-arginine and free radicalscavengers increase cerebral blood flow and brain tissue nitricoxide concentrations after controlled cortical impact injury inratsrdquo Journal of Neurotrauma vol 20 no 1 pp 77ndash85 2003

[114] Y Choi L Tenneti D A Le et al ldquoMolecular basis of NMDAreceptor-coupled ion channel modulation by S-nitrosylationrdquoNature Neuroscience vol 3 no 1 pp 15ndash21 2000

[115] Y Wang-Rosenke H Neumayer and H Peters ldquoNO signalingthrough cGMP in renal tissue fibrosis and beyond key pathwayand novel therapeutic targetrdquo Current Medicinal Chemistry vol15 no 14 pp 1396ndash1406 2008

[116] L Corzo R Zas S Rodrıguez L Fernandez-Novoa and RCacabelos ldquoDecreased levels of serum nitric oxide in differentforms of dementiardquo Neuroscience Letters vol 420 no 3 pp263ndash267 2007

[117] H Shin P Condorelli and S C George ldquoExamining axialdiffusion of nitric oxide in the lungs using heliox and breathholdrdquo Journal of Applied Physiology vol 100 no 2 pp 623ndash6302006

[118] F A Recchia T R Vogel and T H Hintze ldquoNO metabolitesaccumulate in erythrocytes in proportion to carbon dioxide andbicarbonate concentrationrdquo American Journal of PhysiologymdashHeart and Circulatory Physiology vol 279 no 2 pp H852ndashH856 2000

Submit your manuscripts athttpwwwhindawicom

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

CorrosionInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Polymer ScienceInternational Journal of



CeramicsJournal of


CompositesJournal of

NanoparticlesJournal of



International Journal of

Biomaterials


NanoscienceJournal of

TextilesHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Journal of

NanotechnologyHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

CrystallographyJournal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


CoatingsJournal of

Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



MetallurgyJournal of


BioMed Research International

MaterialsJournal of


Nano

materials


Journal ofNanomaterials


NH NH NH

O

L-Arginine L-Citrulline

NADPH NADPH

Heme

Heme

FAD

FADFMN FMN

CAM

CAM

Cell membrane

NO synthase (NOS)

BH4

BH4

H2N

H3N+ COOminus

+NO∙

N120596-OH-L-arginine

NndashOHNH2+

H2N H2N

H3N+ COOminus H3N+ COOminus

and O2

Figure 1 Proposed mechanism of NO production from epithelial cells NO∙ is released by several isoforms of the enzyme NOS whichcatalyzes the 5-electron oxidation of the guanidino nitrogen moiety of a nonaromatic amino acid (L-arginine) to citrulline via N120596-hydroxy-L-arginine The enzyme utilizes O

2and NADPH as cosubstrates and thiol tetrahydrobiopterin (BH4) FAD and FMN as cofactors NOS is

unique among eukaryotic enzymes in being a dimeric calmodulin-dependent or calmodulin-containing isozyme

and blood [13] As shown in Figure 1 NO is released from L-arginine via catalytic action of themembrane-bound enzymeNO synthase (NOS) NO is a physiologically importantmediator in metabolically active organs and tissues as wellas in neurotransmitters in central and peripheral neurons invivo [14]

11 Bioimaging of NO in Cardiovascular System

111 Electrochemical Measurement of NO NO biosensorswere initially developed for high resolution electrochemicalmeasurement methods of NO by several groups [15ndash17]These NO biosensors enable evaluation of dynamic changesin NO concentration in solutions and tissues in responseto agonists NO-generating reagents and physical stimuli[18ndash20] However their electrochemical applications areprimarily limited to short term recording of NO sensitivemyocardial changes for monitoring the effect of cardiacpotassium channel blockers [21] Use of this technique is alsolimited due to its poor sensitivity

112 NO Biosensing by Electron Paramagnetic Resonance(EPR) Spectrometry EPR spectroscopy is a specific techniquefor measurement of in vivoNO free radicals by spin-trappingcompounds (spin-traps) [22] A number of derived pyrrolineoxide and dithiocarbamate compounds have been shown tobe potential EPR spin-trap biosensors (Table 1)

113 NO Biosensing by Fluorometry Recently reported flu-orescent indicators allow real-time bioimaging of NO with

high spatial and temporal resolution Diaminorhodaminesand diaminofluorescein compounds undergo specific reac-tions with NO in cardiovascular tissues and may serve aspotential biosensors in fluoroscopy [23 24]

114 Spin-Trapping Technique in NO Biosensing The pre-sence of NO radicals at greater than the EPR detection limit(01ndash001120583M) can be detected by nitrone traps 55-dimethyl-1-pyrroline-119873-oxide (DMPO) 5-diethoxyphosphoryl-5-methyl-1-pyrroline-119873-oxide (DEPMPO) 120572-phenyl-119873-tert-butylnitrone (PBN) and 120572-(4-pyridyl-1-oxide)-119873-tert-buty-lnitrone (POBN) as well as by nitroso traps 2-methyl-2-nitrosopropane (MNP) and 35-dibromo-4-nitrosobenze-nesulfonic acid (DBNBS) [25ndash28]

115 In Vivo EPR Detection by Biosensing Free Radicals EPRspectrometers operating at S-band (16ndash4GHz) and L-band (04ndash16GHz) microwave frequency and at radiofrequency (02ndash04GHz) are utilized for in vivo measure-ments of the whole body of small animals The electronicconfiguration of NO with 11 valence electrons is(K2K2)(2s120590b)2(2s120590lowast)2(2p120587b)4(2p120590b)2(2p120587lowast)1 NO is a freeradical with one unpaired electron in the antibonding120587 orbital therefore EPR is considered to be the mostappropriate tool for its detection The electronic groundstate of NO is expressed by the term symbol

2

prod1232

[8 22 29] EPR signals from large biological samples cannotbe detected with a conventional X-band spectrometerdue to its poor sensitivity and detection limit However






2]

[32ndash34]





NO biosensors


derived from iNOS)

GTP

GTP

GDP

Second messenger

Receptor


Guanylatecyclase

G120572

G120573 G120574

cGMP

imaging techniques)










(1)









2complexes

[50ndash52]




2as the pri-




2 (2)


2

+ DETClowast(3)


3complexes to form









2

+NO2

minus

(5)




(6)


3and NO-Fe(II)(MGD)

2



2and Fe(IV)(DTCS)





(7)





+ DTCS2minus

(8)



+1

2[bis (DTCS)]2minus

(9)




+1

2[bis (DTCS)]2minus

(10)







2

minus andNO3


minus andNO3







2















2 NO-Fe(MGD)

2 and NO-Fe(DETC)

2


2and





2-Fe(II) [MGD





2-Fe(II)-











2

minus and NO3


NO2

minusNO3


2




2






2(RDATI)] increases





2 Major
























2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








2]

[32ndash34]





NO biosensors


derived from iNOS)

GTP

GTP

GDP

Second messenger

Receptor


Guanylatecyclase

G120572

G120573 G120574

cGMP

imaging techniques)










(1)









2complexes

[50ndash52]




2as the pri-




2 (2)


2

+ DETClowast(3)


3complexes to form









2

+NO2

minus

(5)




(6)


3and NO-Fe(II)(MGD)

2



2and Fe(IV)(DTCS)





(7)





+ DTCS2minus

(8)



+1

2[bis (DTCS)]2minus

(9)




+1

2[bis (DTCS)]2minus

(10)







2

minus andNO3


minus andNO3







2















2 NO-Fe(MGD)

2 and NO-Fe(DETC)

2


2and





2-Fe(II) [MGD





2-Fe(II)-











2

minus and NO3


NO2

minusNO3


2




2






2(RDATI)] increases





2 Major
























2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






NO biosensors


derived from iNOS)

GTP

GTP

GDP

Second messenger

Receptor


Guanylatecyclase

G120572

G120573 G120574

cGMP

imaging techniques)










(1)









2complexes

[50ndash52]




2as the pri-




2 (2)


2

+ DETClowast(3)


3complexes to form









2

+NO2

minus

(5)




(6)


3and NO-Fe(II)(MGD)

2



2and Fe(IV)(DTCS)





(7)





+ DTCS2minus

(8)



+1

2[bis (DTCS)]2minus

(9)




+1

2[bis (DTCS)]2minus

(10)







2

minus andNO3


minus andNO3







2















2 NO-Fe(MGD)

2 and NO-Fe(DETC)

2


2and





2-Fe(II) [MGD





2-Fe(II)-











2

minus and NO3


NO2

minusNO3


2




2






2(RDATI)] increases





2 Major
























2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials









2

+NO2

minus

(5)




(6)


3and NO-Fe(II)(MGD)

2



2and Fe(IV)(DTCS)





(7)





+ DTCS2minus

(8)



+1

2[bis (DTCS)]2minus

(9)




+1

2[bis (DTCS)]2minus

(10)







2

minus andNO3


minus andNO3







2















2 NO-Fe(MGD)

2 and NO-Fe(DETC)

2


2and





2-Fe(II) [MGD





2-Fe(II)-











2

minus and NO3


NO2

minusNO3


2




2






2(RDATI)] increases





2 Major
























2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials















2 NO-Fe(MGD)

2 and NO-Fe(DETC)

2


2and





2-Fe(II) [MGD





2-Fe(II)-











2

minus and NO3


NO2

minusNO3


2




2






2(RDATI)] increases





2 Major
























2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials







2

minus and NO3


NO2

minusNO3


2




2






2(RDATI)] increases





2 Major
























2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





















2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





2 Conclusion


2

minus NO3




Acknowledgment


References
























































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




































A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials






A2119906


1119906A2119906




























































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials
































2rdquo




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



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Review Article Imaging of Nitric Oxide by Magnetic Resonance … · 2019. 7. 31. · Review Article...

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