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ESR techniques for the detection of nitric oxide in vivo as an index of endothelial function

Bruno Fink1, Sergey Dikalov2, Nelli Fink1

Noxygen Science Transfer & Diagnostics GmbH, 79215 Elzach, Germany

Emory University School of Medicine, Department of Cardiology, Atlanta, GA 30322, USA Correspondence: Bruno Fink, e-mail: info@noxygen.de

Abstract:

Plasma nitrite/nitrate levels reflect oxidation of formed nitric oxide (NO_) but are not indicative of endothelial nitric oxide synthase (eNOS) function due to interference by dietary nitrates and reactive oxygen species (ROS). Nitrosyl hemoglobin (NOHb), a metabolic product of nitric oxide, may better correlate with bioavailable NO_but it may depend on the activity of different nitric oxide synthase (NOS) isoforms and may be affected by dietary nitrite/nitrate. We examined the correlation between vascular endothelial NO_release and circulating blood levels of NOHb. We measured NOHb in blood using electron spin resonance (ESR) spectrometry and also quantified vascular production of NO_using colloid Fe(DETC) and ESR in mouse and human venous blood before and after treatment with theb -blocker carvedilol. Exclusively the inhibition with L-NAME and not the treatment with the selective neuronal nitric oxide synthase (nNOS) inhibitor, N-AANG or with the selective inducible nitric oxide synthase (iNOS) inhibitor, 1400W, halved NOHb formation, which reflects the complete inhibition of NO_ release by aortic endothelium. The relationship between NOHb and NO_production by the endothelium (0.23 µM NOHb to 3.73 µM/hour of NO_per mg of aorta dry weight) was found to be identical for both C57Blk/6 mice and for mice with vascular smooth muscle-targeted expression of p22phox associated with strong increase in eNOS activity. Furthermore, the treatment of patients with cardiovascular diseases with carvedilol for 3 weeks increases up to 2 times the circulating NOHb concentration. These results demonstrate the important role of eNOS in the formation of circulating NOHb and suggest that NOHb can be used as a noninvasive marker of endothelial NO_productionin vivo.

Keywords:

nitrosyl haemoglobin, endothelial dysfunction, oxidative stress, nitric oxide

Abbreviations: 1400W – N-(3-aminomethyl)benzylacetamid- ine, AAD – amino acid diet, BAECs – bovine aorta endothelial cells, BD-5755 – low nitrate/nitrite diet, C57Blk/6 – wild-type mice, CP-H –1-hydroxy-3-carboxy-pyrrolidine, DETC – dieth- yldithiocarbamate, eNOS – endothelial nitric oxide synthase, ESR – electron spin resonance, GTN – nitroglycerin, LD-5001 – regular labor diet, L-NAME –NM-nitro-L-arginine methyl es- ter, MAHMA-NONOate – (Z)-1-(N-Methyl-N-[6-(N-methyl- ammoniohexyl)amino])-diazen-1-ium-1,2-diolate, MGD – N-(dithio- carbamoyl)-N-methyl-D-glucamine, NaSO"– sodium dithionite, N-AANG – (4S)-N-(4-amino-5[aminoethyl]aminopentyl)-N’- nitroguanidine, nNOS – neuronal nitric oxide synthase, NO nitric oxide, NOHb – nitrosyl hemoglobin, O2 – superoxide, ONOO– – peroxynitrite, ROS – reactive oxygen species, VSMCs – vascular smooth muscle cells

Introduction

A growing interest in nitric oxide (NO) called for re- liable and sensitive techniques for its quantification bothin vitro and in vivo. Developing the in vivo tech- nique was the most challenging demand. Mostin vivo studies investigating endothelial function relied on ni- trite/nitrate measurements in blood plasma [27]. Ni- trite and nitrate are metabolic products of NOand are used to quantify NO by its biotransformation of nitrates [14, 28, 35]. Unfortunately, nitrite/nitrate plasma levels are strongly affected by the dietary con- sumption of nitrite/nitrate, which is difficult to mini-

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mize even in laboratory conditions. Moreover, the ni- trite/nitrate level does not reflect bioactive amount of NOsince the inactivation of NOby superoxide and other oxidants leads to the formation of nitrite/nitrate [34, 36]. A method forin vivo NOdetection based on the formation of nitrosyl hemoglobin (NOHb) in the reaction of deoxyhemoglobin (Hb) with NOhas been described [1, 12]. Therefore, to overcome the limita- tions of nitrite/nitrate assay, we used electron spin resonance (ESR) to analyze NOHb in frozen blood [16, 24].

The process of NOtransfer into erythrocytes is of critical biological importance because it controls plasma NObioavailability and diffusional distance of endothelial-derived NO. It has been suggested that NO under physiological conditions is consumed rather than conserved by reaction with oxyhemoglo- bin [17]. Indeed, rate constants imply that most of the NOwill react with oxyhemoglobin,producing nitrate and methemoglobin, while only a minor fraction will form NOHb detectable by ESR.

NO is a well-known ligand of deoxygenated he- moglobin [18]. Paramagnetic properties of NOHb have been previously studied with the use of ESR and recent in vitro and in vivo ESR studies demonstrated

the presence of NOHbin vivo during inflammation, drug metabolism, and treatment with statins [23, 24, 31, 37]. The lifetime of NOHb ranges from 12 min to 20 h with an average lifetime of 4 h, therefore, it can be accumulated in the blood in substantial concentra- tions [5]. While an electrochemical assay for meas- urement of NOHb was recently developed [32], ESR remains the most direct and unambiguous method for NOHb measurement. A growing body of evidence supports the idea that NOHb can be used as a marker of bioavailable NO in vivo for identification of sources releasing NO (Scheme 1). ESR analysis of 5- coordinate NOHb permits the highly specific detec- tion of low levels of NOHb during hypertension and other diseases [5, 9, 10].

NO production in vivo can also be analyzed ex vivo in various tissues. Previously, NOproduction in rabbit and mouse aorta has been measured by colloid iron diethyldithiocarbamate, Fe(DETC)2 and ESR spectroscopy [20–22]. This chapter details the ESR techniques for measuring blood NOHb and NOpro- duction in tissues with colloid Fe(DETC)2. It shows the association of blood NOHb with endothelial NO production.

Materials and Methods

Animals

C57Blk/6 (wild-type) and eNOS KO mice were ob- tained from Jackson Laboratories (Bar Harbor, ME).

Studies were performed on 12- to 18-week-old male mice. Mice overexpressing the NADPH oxidase p22phox subunit in vascular smooth muscle cells (VSMC) (Tgp22smcmice) were created by cloning the p22phoxcDNA downstream of these cells [26]. Inves- tigation of in vivo effects of selective eNOS, iNOS, and nNOS inhibitors on NOHb formation and NO production in the aorta of C57Blk/6, p22phox overex- pressed and eNOS KO mice were performed using subcutaneous injection of 1400W (N-(3-amino- methyl)benzylacetamidine), or N-AANG ((4S)-N-(4- amino-5[aminoethyl]aminopentyl)-N’-nitroguanidine) (1 mg/kg in 0.9% NaCl) twice a day. On the day of the study, mice were injected with 100 U heparin/25 g BW ip 5 min before euthanasia with CO2. During preparation, the vessels were maintained in chilled

NOHb concentration as an index of endothelial function

Bruno Fink et al.

Sources of NO in vivo

Endogenous eNOS iNOS nNOS mNOS

NO

L -NIO, L -NAME

1400 W

N-AAANG

Exogenous Nitrate

Nitrite

oxidation

Nitrosothiols

S-nitrosati on

?

LD 5001 (33/5 ppm)

BD 5755(12/3ppm) AAD 44181(10/2 ppm)

HbFe2NO

nitrosylation k = 107M–1s–1

?

Scheme 1. Sources of NOin vivo: The picture summarizes the knowl- edge about the sources of NOin vivo with intended purposes of un- derstanding the mechanisms responsible for release of NO and ef- fects on circulating NOHb concentrations. In the picture, we used the following abbreviations: eNOS – endothelial nitric oxide synthase;

iNOS – inducible nitric oxide synthase; nNOS – neuronal nitric oxide synthase; mNOS – mitochondrial nitric oxide synthase; L-NIO – (L-N#-(1-imunoethyl)ornitine); 1400 W – (N-(3-aminomethyl) benzyla- cetamidine); N-AAANG – ((4S)-N-(4-Amino-5[aminoethyl]aminopen- tyl)-N’-nitroguanidine); LD-5001 – regular labor diet; BD-5755 – low-nitrate/nitrite diet; AAD – amino acid diet

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Chemicals

FeSO4, DETC, and calcium ionophor (A-23187) were obtained from Sigma-Aldrich (St. Louis, MO). L-NAME, L-NIO, 1400W, and N-AANG were purchased from EMD Biosciences (San Diego, CA). Spin probe CPH was purchased from Noxygen Science Transfer &

Diagnostics GmbH (Elzach, Germany). The modified Krebs-HEPES buffer (KHB) for vessel studies was composed of 99.01 mmol/l NaCl, 4.69 mmol/l KCl, 2.50 mmol/l CaCl2, 1.20 mmol/l MgSO4, 25 mmol/l NaHCO3, 1.03 mmol/l K2HPO4, 20 mmol/l Na-HEPES, and 5.6 mmol/l D-glucose, pH 7.35. All other chemi- cals were obtained from Sigma-Aldrich (St. Louis, MO, USA) and were of the ultra pure grade.

Preparation of colloid Fe(DETC)2

The preparation of Fe2+(DETC)2 stock solution was performed according to Kleschyov et al. [20]. The formed 0.8 mM Fe(DETC)2 colloid solution was yellow-brown in color and was used immediately af- ter preparation.

Detection of NO formation in cultured cells

Bovine aortic endothelial cells (BAECs, BioWhit- taker, Inc. MD) were cultured in Medium 199 con- taining 10% fetal calf serum supplemented with L- glutamine (2 mM), 1% solution of vitamins, strepto- mycin (20 g/ml) and penicillin (20 units/ml). On the day prior to the study, the fetal calf serum concentra- tion was reduced to 5%. Confluent BAECs from pas- sages 4–5 were used for experiments. On the day of the study, the cells were rinsed with 3 ml of chilled Krebs/HEPES-buffer, preloaded within 15 min with Fe(DETC)2and then exposed to laminar shear stress as described by Davies et al. [6] at 37°C for 30 min.

The ESR spectrometer settings were as follows: mi- crowave power, 40 mW; modulation amplitude, 5 G;

center field, 2.03 g; sweep width, 90 G; conversion time, 160 milliseconds; time constant, 40 millisec- onds; number of scans, 32; sweep time, 10.24 sec- onds. The amount of detected NO was determined from the calibration curve for integral intensity of the ESR signal of NO-Fe2+(MGD)2 prepared at various

Incubation of aorta with colloid Fe(DETC)

2

The preparation of aorta section was performed ac- cording to Fink B et al. [7]. Samples were stored for up to six months at –80°C before ESR analysis. The ESR spectrometer settings were as follows: microwave power, 40 mW; modulation amplitude, 5 G; center field, 2.03 g; sweep width, 90 G; conversion time, 160 milliseconds; time constant, 40 milliseconds; number of scans, 32; sweep time, 10.24 seconds.

Preparation of blood for measurements of NOHb

Animals

Heparinized blood was collected by a 26 G needle from the right ventricle of the CO2-euthanized mice.

The venous blood was frozen in liquid nitrogen and kept at –80°C before measurements. The three-line hyperfine spectrum of the 5-coordinate complex of NO with hemoglobin was recorded with an X-band EMX series ESR spectrometer (Bruker Biospin GmbH, Karlsruhe, Germany) using a high sensitivity SHQ microwave cavity in finger Dewar filled with liquid nitrogen. ESR spectrometer settings were as follows: microwave power, 10 mW; modulation am- plitude, 5 G; center field, 2.01 g; sweep width, 240 G;

conversion time, 655 milliseconds; time constant, 5.24 seconds; number of scans, 2; sweep time, 336 seconds.

Humans

The human venous blood was collected from vena cu- bitalis using 2 ml Sarsted/Li-Heparin syringes under minimal arm occlusion. After the 5 min centrifugation of blood at room temperature at 1600 × g, the blood was frozen in liquid nitrogen and kept at –80°C before measurements. The concentration of NOHb was re- corded with an X-band EPR spectrometer Alexis (Bruker Biospin GmbH, Karlsruhe, Germany) using a high sensitivity SHQ microwave cavity and finger Dewar filled with liquid nitrogen. ESR spectrometer settings were as follows: microwave power, 40 mW;

modulation amplitude, 10 G; center field, 2.01 g;

sweep width, 240 G; conversion time, 320 millisec-

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onds; time constant, 80 milliseconds; number of scans, 48; sweep time, 20.48 seconds. The amount of de- tected NOwas determined from the calibration curve for intensity of the ESR signal of erythrocytes treated with known concentrations of nitrite (1–25 µM) and Na2S2O4(20 mM) [24].

Study population

In the study, we include the outpatients with docu- mented peripheral vascular disease in age of 57 ± 7 years and essential hypertension (147 ± 9 mmHg sys- tolic and 97 ± 6 mmHg diastolic blood pressure). Be- fore beginning of treatment all patients were not treated with the antihypertensive medication. All study participants had a medical examination and an ECG; both of which were abnormal. They were not allowed to have supplementary vitamin intake. Diet and life style had to be unchanged 2 weeks before and during the study. All study participants gave written informed consent. In order to investigate the magni- tude of the potential effects of carvedilol on produc- tion of ROS and circulating NOHb concentration, we compared the values before and after treatment for 4 weeks (25 mg carvedilol twice a day).

Analysis of GTN bioconversion in blood

The venous blood (1 ml) was mixed with 10 mg of Na2S2O4and 10 µg of nitroglycerin (GTN). After in- cubation for 30 min at 37°C with permanent gentle shaking (10 rpm), the sample was frozen in liquid ni- trogen and kept at –80°C before measurements of formed NOHb.

Quantification of reactive oxygen species (ROS)

Cyclic hydroxylamine 1-hydroxy-3-carboxy-pyrro- lidine (CP-H) was used for the quantification of reac- tive oxygen species (ROS; e.g. O2or ONOO). Dur- ing the reaction of CP-H with ROS, stable (lifetime more than 4 h), reducing agent-resistant nitroxide radicals are formed [8]. The amount of trapped ROS radicals was obtained by quantifying the concentra- tion of the corresponding nitroxide radical 3-carboxy- proxyl (CP). Quantification of ROS released from blood samples (non-stimulated ROS formation) was performed after incubation at 37°C for 30 min using 500 µM CP-H and the low-field component of elec- tron spin resonance (ESR) spectra in 50 µl quartz cap-

illaries (Noxygen Science Transfer & Diagnostics GmbH, Elzach, Germany). In order to inhibit transi- tion metal-catalyzed oxidation of spin traps, CP-H was used in the presence of deferoxamine. The ESR spectrometer settings were as follows: microwave power, 20 mW; modulation amplitude, 2 G; center field, 1.99 g; sweep width, 50 G; conversion time, 80 milliseconds; time constant, 40 milliseconds; number of scans, 10; sweep time, 5.12 seconds.

Results

Relation of NOHb concentration to production of NOin aorta

Various papers described the release of NOfrom en- dothelial cells using the value obtained in unstimu- lated, resting cells. We report the release of NOunder physiological conditions induced by laminar shear stress in comparison with stimulation by 10 µM cal- cium ionophor and under resting conditions (Fig. 1).

The cultured bovine aorta endothelial cells (BAECs) respond to laminar shear stress with the near the same NO release (129 ± 9 µM/mg protein) as the cells treated with calcium ionophor (Ca-channel opener, 121 ± 10 µM/mg protein in 30 min). The release of NOunder resting conditions in BAECs was as usual in mice aorta section up to 3.5 times less (34 µM/mg protein) in comparison to shear stress. Therefore, we investigated production of NO induced by calcium

NOHb concentration as an index of endothelial function

Bruno Fink et al.

basal laminar shear A-23187 0

50 100 150

* *

NO-release(µM/mg-protein)

Fig. 1. Response of cultured bovine aorta endothelial cells to laminar shear stress and calcium ionophore stimulation: Detection of NO_- production was performed after exposure of BAECs to laminar shear stress and stimulation with 10 µM of calcium ionophor using ESR and Fe(DETC)2 colloid. Data are mean (n = 4). * p < 0.05vs. basal

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ionophore in aorta endothelium of C57Blk/6 mice in comparison to p22phoxoverexpressed mice which ex- pressed up to 3 times more eNOS and in aortas from eNOS KO mice. To identify the enzyme responsible for production of NO, we treated the mice with spe- cific NOS inhibitors L-NAME, 1400W, or N-AAANG (Fig. 2) and measured NOHb concentration (Fig. 3) in the blood and NO production in the aorta endothe- lium. Of note, IC50 for targeted enzyme can be a thousand-fold lower than IC50 for nontargeted enzyme. For example, 1400W inhibits iNOS with Kd= 7nM, which is 5000-fold lower than the value

pressed mice. The in vivo treatment with selective iNOS and nNOS inhibitors 1400W and N-AANG re- spectively, did not affect the production of NO in aorta endothelium. As expected,we did not detect any production of NO in eNOS KO mice (Fig. 2).

Finally, we determined the relationship between NOHb and endothelial NOproduction as a ratio of L-NAME inhibitable NOHb (Fig. 3) to NO produc- tion in aorta (Fig. 2). This ratio (0.23 µM NOHb to 3.73 µM/h of NO per mg of aorta dry weight) was found to be identical for both C57Blk/6 mice and for mice with a vascular smooth muscle-targeted expres- sion of p22phox [19].

Of note, the NOHb level in eNOS KO mice was significantly higher in comparison to the C57 Blk/6 mice (Fig. 3). However, the NOHb in eNOS KO mice was inhibited by the iNOS inhibitor 1400W which did not affect NOHb in the C57 Blk/6 mice (Fig. 3).

Association of blood NOHb with production of ROS and GTN-bioconversion in human blood

Follow the knowledge of relation between circulating NOHb concentration and production of NOin endo- thelium, we tested the effects of theb-blocker carve- dilol treatment on changes in circulating NOHb and on bioconversion of nitroglycerin in blood. The treat- ment with the b-blocker carvedilol (25 mg twice a day) increased the circulating NOHb by up to 2

C57Blk/6 p22phox eNOS-KO

0 4 8

* *

AortaNO-production (µmole/mgdryweight)

Fig. 2. Effects of selective eNOS, iNOS, nNOS inhibitors on produc- tion of NO in aortic segments of C57Blk/6, p22phox overexpressed, and eNOS KO mice. The production of NO was measured after stimu- lation with 10 µM of calcium ionophore afterin vivo pretreatment with L-NAME (100 mg/l in drinking water), after a two-day pretreatment with the selective iNOS inhibitor 1400W (hatched bars), and after two-day pretreatment with the selective nNOS inhibitor N-AAANG (vertical striped bars)

C57Blk/6 p22phox eNOS-KO

0.0 0.4 0.8

control + L-NAME + 1400W + N-AAANG

*

*

*

*

NO-Hbconcentration(µM)

Fig. 3. Effects of selective eNOS, iNOS, nNOS inhibitors on formation of circulating of NOHb concentration in blood of C57Blk/6, p22phox overexpressed, and eNOS KO mice. The NOHb concentrations were measured afterin vivo pretreatment with L-NAME (100mg/l in drink- ing water, fasciated bars), after a two-day pretreatment with the se- lective iNOS inhibitor 1400W (hatched bars), and after two-day pre- treatment with the selective nNOS inhibitor N-AAANG (vertical striped bars)

Before After

0 10 20 30

0 10 20 GTN bioconversion 30 NOHb-concentration

33G

GTN-bioconversion(µM) NOHb-concentration(nM)

Fig. 4. Effects of carvedilol treatment on concentration of circulating NOHb and on bioconversion of nitroglycerin in patients with cardio- vascular diseases. Detection of circulating NOHb (hatched bars) and analysis of bioconversion of nitroglycerin (white bars) was per- formed before and after 4 weeks of treatment with carvedilol (25 mg twice a day) using ESR. Insert represents the typical tracing of NOHb measurements in blood of patients after treatment with carvedilol

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times from 8.9 ± 1.9 nM to 21.7 ± 2.5 nM (Fig. 4) and was associated with the drop in the systolic (–5.7 ± 2.1 mmHg) and diastolic (–4.3 ± 2.6 mmHg) blood pressure. Similarly to the changes in circulating NOHb concentration, we observed the increase in the metabolic capacity of organic nitrate nitroglycerin (GTN-bioconversion) in blood of patients with car- diovascular diseases. The GTN-bioconversion after 4 weeks of treatment with carvedilol increased from 9.6® ± 1.4 µM to 20.5 ± 1.1 µM (Fig. 4). Therefore, we observed the significant decrease from 9.6 ± 1.0 µM to 6.1 ± 0.6 µM in production of ROS in blood of pa- tients with cardiovascular diseases after treatment with the carvedilol (Fig. 5) as well as the increase in the reduced SH-group concentration in plasma (data not shown).

Discussion

In this work,we demonstrated the use of circulating NOHb concentrations as a noninvasive parameter of endothelial function/nitric oxide synthase activity in animal model related to endothelial function in hu- mans. It has been reported that nitrite can be reduced to nitric oxide by deoxyhemoglobin and cause vaso- dilatation in the human circulation under hypoxic condition [4]. It is possible that a part of the residual amount of NOHb can be derived from either endoge-

nous or exogenous of nitrite/nitrate in case of low- nitrite diet which contains 12 mg/kg nitrate and 3 mg/kg nitrite (Scheme 1). Alternatively, NOS inhi- bition may increase in vivo reduction of exogenous and endogenous nitrite to NO, which was recently demonstrated in hypoxic tissues [2]. Various intracel- lular compartments, such as endoplasmic reticulum, and mitochondria are also involved in bioconversion of nitrates [24]. It is clear that NOHb is strongly asso- ciated with vascular NO production. Stimulation of the vascular tissue with calcium ionophore (Fig. 1) reflects the physiological NOproduction in the vas- culature, showing characteristic triplet ESR signal (g = 2.035; AN= 12.6 G) [7].

With the use of highly selective NOS inhibitors, it is possible to differentiate the contribution of other enzymes for example iNOS as the source of NOHb (Fig. 2), which is likely a compensatory effect of the lack of eNOS in transgenic mice (Fig. 3). Our results suggest that NOHb can be used not only as an index of in vivo NO production by eNOS under normal conditions but also as an evidence of increased activ- ity of iNOS under inflammation induced for example by treatment with LPS [25]. Under normal physio- logical conditions iNOS and nNOS do not contribute to NOHb formation. Although a low-nitrate diet is es- sential for analysis of NOHb, the fact that NOHb re- flects bioavailable levels of vascular NO makes NOHb useful as a noninvasive marker of endothelial function.

Our results support an important role of eNOS in the formation of NOHb in of human subjects (Fig. 4).

Unlike other NO assays, the formation of NOHb is proportional to the bioavailable amount of NO [31].

This allowed us to study the effect of hypertension on NOHb content in the red blood cells, showing the sig- nificant decrease in NOHb in cardiovascular diseased patients. The low production rate of NOoccurs in en- dothelium of patients with cardiovascular diseases mostly due to the increase in ROS (O2, ONOO) pro- duction in the endothelium which results in endothe- lial dysfunction and all following consequences (hy- pertension, atherosclerosis, infarct, stroke etc.) [3, 13, 15, 38].

The hypertension can be treated with carvedilol, a non-selectiveb-blocker with antioxidant properties, which also prevented nitrate tolerance induced by the increased production of ROS. Our findings (Fig. 4 and 5) clearly demonstrate that blood pressure reduc- tion within 4 weeks improves the endothelial function

NOHb concentration as an index of endothelial function

Bruno Fink et al.

before after

0 4 8 12

ROSformation(µM)

Fig. 5. Effect of carvedilol treatment on formation of ROS in cardio- vascular diseased patients. ROS formation in blood of CAD patients was analyzed using spin probe CPH (500 µM) before (white bar) and after (hatched bar) 4 weeks of treatment with theb-blocker carvedilol (25 mg twice a day).

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of rennin and might result in a decrease in angiotensin II levels. This, in turn, may inhibit NADH/NADPH oxidase activity and superoxide production. Finally, the reduction of oxidative stress at the same time re- sults in improvement of metabolic capacity of blood to convert GTN which was also shown in other publi- cations [11, 29].

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Received:

December 15, 2006, in revised form: December 27, 2006

NOHb concentration as an index of endothelial function

Bruno Fink et al.

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