Journal of the American College of Cardiology
© 2006 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 47, No. 3, 2006
ISSN 0735-1097/06/$32.00
doi:10.1016/j.jacc.2005.06.089
Plasma Nitroso Compounds Are
Decreased in Patients With Endothelial Dysfunction
Christian Heiss, MD,* Thomas Lauer, MD,† André Dejam, MD,*
Petra Kleinbongard, PHD,* Sandra Hamada, MS,* Tienush Rassaf, MD,*
Simone Matern, BS,* Martin Feelisch, PHD,‡ Malte Kelm, MD*
Aachen and Duesseldorf, Germany; and Boston, Massachusetts
We investigated whether plasma nitros(yl)ated species (RXNOs) that mediate systemic nitric
oxide (NO) bioactivity are depleted in individuals with cardiovascular risk factors and
endothelial dysfunction.
BACKGROUND Endothelium-derived NO acts not only as a regional messenger but exerts significant systemic
effects via formation of circulating RXNOs delivering NO to sites of impaired production.
METHODS
Endothelial function was assessed in 68 patients with one to four major cardiovascular risk
factors (RF) and 39 healthy control subjects (C) by measurement of flow-mediated dilation
(FMD) of the brachial artery using high-resolution ultrasound. In parallel, plasma RXNOs
were determined by reductive gas phase chemiluminescence.
RESULTS
Increasing numbers of risk factors were accompanied by a progressive decrease in FMD: 6.5 ⫾
0.4% (C); 4.7 ⫾ 0.5% (one RF); 2.8 ⫾ 0.4% (two RF); 2.2 ⫾ 0.4% (three RF); and 1.0 ⫾ 0.3%
(four RF). Progressively impaired vascular function was associated with a concomitant decrease
in plasma RXNOs (p ⬍ 0.01): 39 ⫾ 2 nmol/l (C); 30 ⫾ 2 nmol/l (one RF); 24 ⫾ 3 nmol/l
(two RF); 22 ⫾ 3 nmol/l (three RF); and 15 ⫾ 2 nmol/l (four RF), with univariate correlation
between FMD and RXNO (r ⫽ 0.41, p ⬍ 0.001). In a multivariate regression model, RXNO
was an independent predictor of endothelial function.
CONCLUSIONS Endothelial dysfunction in patients with cardiovascular risk factors is associated with
decreased levels of circulating RXNOs. Plasma RXNOs may be diagnostically useful markers
of NO bioavailability and a surrogate index of endothelial function. Whether the observed
decrease in concentration reflects impaired NO formation, accelerated decomposition, and/or
consumption of RXNOs and whether these processes play a causal role in the pathophysiology of arteriosclerosis remain to be investigated. (J Am Coll Cardiol 2006;47:573–9)
© 2006 by the American College of Cardiology Foundation
OBJECTIVES
Arteriosclerosis is the major cause for chronic vascular
diseases such as coronary artery disease, cerebrovascular
disease, and peripheral arterial occlusive disease. The key
event in the pathogenesis of arteriosclerosis is believed to be
dysfunction of the endothelium and disruption of endothelial homeostasis, predisposing blood vessels to vasoconstriction, inflammation, leukocyte adhesion, thrombosis, and
proliferation of vascular smooth muscle cells.
See page 580
Endothelium-derived nitric oxide (NO) is an essential
short-lived signaling molecule important for vascular homeostasis. Decreased NO bioactivity leads to accelerated
atherogenesis. Until recently, it was believed that the bioactivity of NO is limited to close temporal and spatial
From the *Department of Cardiology, Pneumology, and Vascular Medicine,
RWTH Aachen, Aachen, Germany; †Department of Cardiology, Pneumology, and
Angiology, Heinrich-Heine University, Duesseldorf, Germany; and the ‡Whitaker
Cardiovascular Institute, Boston University School of Medicine, Boston,
Massachusetts. Dr. Kelm received a grant from the Deutsche Forschungsgemeinschaft (SFB 612); Dr. Lauer received a grant from Klinische Forschungskommission of the Heinrich-Heine University Duesseldorf; and Dr. Feelisch received a
grant from the National Institutes of Health (HL69029). Drs. Heiss and Lauer
contributed equally to this work. Dr. Heiss is currently affiliated with the Division
of Cardiology, UCSF, San Francisco, California.
Manuscript received May 5, 2005; revised manuscript received June 3, 2005,
accepted June 27, 2005.
proximity of the endothelium and that NO is a mere
autocrine/paracrine effector (i.e., that it can only travel short
distances in the bloodstream (1) before it becomes inactivated). Recent studies challenge this concept by suggesting that
free NO is in equilibrium with a pool of various NOcontaining compounds in blood that have a bioactivity that
resembles that of authentic NO. Inhaled and intravenously
applied exogenous NO is transported and delivered along the
vascular tree to dilate distal conduit arteries and the microvasculature (2,3). While we have recently reported that this
“circulating NO pool” can be modulated by dietary intervention (4), the chemical identity of those species is currently
unclear. Initially, the circulating pool was believed to be mainly
comprised of nitrosothiols, in particular S-nitrosoalbumin
(⬎80%), and to a lesser extent S-nitrosohemoglobin,
S-nitrosoglutathione, and S-nitrosocysteine (5). Consistent
with this view, intravenous application of authentic
S-nitrosothiols leads to vasodilation (2), inhibition of
platelet activation (6), abolition of carotid embolization
(7), and protection from ischemia/reperfusion injury (8).
More recently, however, N-nitroso proteins and ironnitrosyl complexes (9,10), as well as nitrite (11,12) and
potentially nitrated lipids (13), have also been implicated
to serve as plasma NO stores.
The measurement of flow-mediated dilation (FMD) of
the brachial artery (BA) as a noninvasive endothelial func-
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Heiss et al.
Plasma Nitroso Species in Endothelial Dysfunction
JACC Vol. 47, No. 3, 2006
February 7, 2006:573–9
that a lower concentration of this bioactive NO pool in
human plasma correlates with the degree of endothelial
dysfunction as measured by FMD.
Abbreviations and Acronyms
BA
⫽ brachial artery
eNOS ⫽ endothelial nitric oxide synthase
FMD ⫽ flow-mediated dilation
GTN ⫽ glycerol trinitrate
NO
⫽ nitric oxide
RXNO ⫽ sum of circulating nitric oxide species
(synonym: circulating NO pool)
METHODS
tion test in humans was introduced in 1992 by Celermajer
et al. (14), and this approach has now been used by
numerous groups throughout the world to monitor endothelial function. This ultrasonographic method quantifies
the dilation of conduit arteries in response to physiologically relevant increases in laminar shear stress induced by
ischemic dilation of the downstream microvasculature.
Increases in shear stress (i.e., the tangential force exerted
by the flow of blood over the surface of the endothelium)
lead to a rapid activation of endothelial NO synthase
(eNOS) with consecutive increases in NO formation.
Accordingly, FMD is largely abolished after NOS inhibition, and, therefore, provides a valuable “read-out” of
local vascular NO availability.
Using this approach, it has been shown that the conditions associated with the major cardiovascular risk factors
have an additive negative impact on endothelial function
that is observable well before clinically apparent vascular
complications. A depletion of the “circulating NO pool”
under these conditions may contribute to the progression of
arteriosclerosis and trigger or aggravate cardiovascular emergencies. Whether the concentration of circulating NO species
is indeed decreased in individuals with endothelial dysfunction, however, is currently unknown. We, therefore, sought
to investigate the hypothesis that the circulating sum of NO
species (RXNOs) are progressively depleted in individuals with
increasing numbers of major cardiovascular risk factors and
Study subjects. We studied 68 patients with one to four
major cardiovascular risk factors and 39 healthy subjects
without clinical evidence for other cardiovascular risk factors
recruited from the outpatient clinic of the department of
internal medicine. Patients with reduced left ventricular
function or signs of heart failure, valvular disease, and
arrhythmogenic disorders were excluded. Major cardiovascular risk factors were hypertension, hypercholesterolemia,
smoking, or diabetes mellitus. Hypertension was defined by a
systolic blood pressure ⬎140 mm Hg, diastolic blood pressure
⬎90 mm Hg (15), or current antihypertensive medication.
According to American Diabetes Association guidelines (16),
diabetes mellitus was defined by glucose levels in plasma ⬎126
mg/dl (fasting), ⬎200 mg/dl (2 h after oral glucose tolerance),
⬎200 mg/dl (any time in combination with diabetic symptoms), or current antidiabetic medication.
Hypercholesterolemia was defined by total cholesterol
levels ⬎240 mg/dl, low-density lipoprotein cholesterol levels
⬎160 mg/dl, or cholesterol-lowering medication (17). Coronary artery disease was diagnosed by coronary angiography.
The clinical characteristics are summarized in Table 1. Patients
were asked to refrain from smoking and stay fasted from 8:00
PM of the night before and until completion of investigations. Current medication (Table 2) was discontinued for
⬎18 h before and during the investigations (i.e., “night” and
“morning” medication was withheld). Informed consent was
obtained from all study subjects before enrollment. The
study protocol was approved by the local ethics committee
of the Heinrich-Heine University of Duesseldorf.
NO species, nitrite, and nitrate in plasma. Before vascular measurements were performed, blood was drawn from
Table 1. Clinical Characteristics of the Study Population
No. of Cardiovascular
Risk Factors
Age, yrs
Gender, M/F
BMI, kg/m2
Hypertension
Hypercholesterolemia
Diabetes mellitus
Smoker
Coronary artery disease
Mean arterial pressure, mm Hg
Total cholesterol, mg/dl
LDL cholesterol, mg/dl
HDL cholesterol, mg/dl
Triglycerides, mg/dl
Glucose, mg/dl
Pack-yrs
0 (Control)
1
2
3
4
n ⴝ 39
n ⴝ 31
n ⴝ 10
n ⴝ 14
n ⴝ 13
41.7 ⫾ 2.9
19/20
23.1 ⫾ 0.4
n⫽0
n⫽0
n⫽0
n⫽0
n⫽1
93 ⫾ 1
200 ⫾ 4
137 ⫾ 4
68 ⫾ 2
90 ⫾ 6
90 ⫾ 1
0†
45.1 ⫾ 2.7
16/15
24.0 ⫾ 0.5
n⫽4
n⫽9
n⫽1
n ⫽ 17
n⫽2
90 ⫾ 2
209 ⫾ 8
147 ⫾ 7
68 ⫾ 4
110 ⫾ 91
90 ⫾ 2
8.9 ⫾ 2.0*
53.5 ⫾ 4.6
7/3
27.3 ⫾ 0.9*†
n⫽4
n⫽7
n⫽1
n⫽6
n⫽4
96 ⫾ 3
214 ⫾ 16
150 ⫾ 13
57 ⫾ 6
146 ⫾ 18
97 ⫾ 7
13.2 ⫾ 4.3*
60.6 ⫾ 2.0*†
11/3
27.0 ⫾ 0.7*†
n ⫽ 13
n ⫽ 13
n⫽4
n ⫽ 12
n ⫽ 13
101 ⫾ 2†
245 ⫾ 44
144 ⫾ 10
48 ⫾ 5*†
432 ⫾ 261*†
111 ⫾ 9*†
29.8 ⫾ 4.1*†
68.4 ⫾ 2.4*†
12/1
28.6 ⫾ 1.1*†
n ⫽ 13
n ⫽ 13
n ⫽ 11
n ⫽ 13
n ⫽ 13
100 ⫾ 3†
195 ⫾ 2
135 ⫾ 12
56 ⫾ 2
207 ⫾ 59
136 ⫾ 15*†
38.9 ⫾ 4.6*†
Data expressed as mean ⫾ SE. p values from analysis of variance (ANOVA). *p ⬍ 0.05 vs. control; †p ⬍ 0.05 vs. one risk factor.
BMI ⫽ body mass index; HDL ⫽ high-density lipoprotein; LDL ⫽ low-density lipoprotein.
p Value
(ANOVA)
⬍0.001
⬍0.001
0.002
0.289
0.587
0.002
0.021
⬍0.001
⬍0.001
Heiss et al.
Plasma Nitroso Species in Endothelial Dysfunction
JACC Vol. 47, No. 3, 2006
February 7, 2006:573–9
575
Table 2. Current Medication of the Study Population
No. of Cardiovascular
Risk Factors
ACE inhibitors
ATII receptor antagonists
Beta-blockers
Diuretics
Calcium-channel antagonists
Nitrates
Acetylsalicylic acid
Statins
Oral antidiabetics
Insulin
0 (Control)
1
2
3
4
n ⴝ 39
n ⴝ 31
n ⴝ 10
n ⴝ 14
n ⴝ 13
0
0
0
0
0
0
1 (5)
0
0
0
2 (7)
0
0
2 (7)
4 (13)
1 (3)
2 (7)
2 (7)
0
1 (3)
4 (40)
2 (20)
4 (40)
2 (20)
1 (10)
2 (20)
3 (30)
3 (30)
1 (10)
0
10 (71)
1 (7)
10 (71)
2 (14)
2 (14)
4 (29)
12 (86)
8 (57)
1 (7)
1 (7)
10 (77)
1 (8)
12 (92)
5 (38)
3 (23)
4 (31)
13 (100)
9 (67)
3 (23)
1 (8)
Data expressed as n (% of group).
ACE ⫽ angiotensin-converting enzyme; ATII ⫽ angiotensin II.
the antecubital vein. All measurements were performed after
overnight fasting of more than 12 h between 8:00 and 9:00 AM.
Plasma levels of nitros(yl)ated NO species (RXNO: the sum
of S-nitrosothiols, N-nitrosamines, iron-nitrosyl species)
and nitrite were determined using a triiodide/ozone-based
chemiluminescence assay, essentially as described (18,19). A
high reproducibility of RXNO measurements has been
shown previously for standards (19). In addition, we have
performed repeated measurements of plasma RXNOs on
two separate days at the same time of the day in fasted
healthy volunteers (n ⫽ 20) and found an intrasubject
variation (i.e., averaged deviation between measurements of
1.8 ⫾ 9%). Nitrate was quantified after enzymatic reduction
to nitrite by nitrate reductase using flow-injection analysis
(20). All analyses were performed by independent investigators blinded to the clinical history of study subjects.
Endothelium-dependent and endothelium-independent
vasodilation. Endothelium-dependent dilation of the BA
was measured noninvasively by high-resolution ultrasound
(SONOS 5500, Agilent Technologies, Palo Alto, California, with a 15-MHz linear-array transducer) using standard
techniques (21). Briefly, baseline data for diameter and
blood-flow velocity of the BA were quantified after 10 min
of supine rest in an air-conditioned room (21°C) 1 to 2 cm
above the elbow. Then a blood pressure cuff was placed
around the forearm distal to the cubital fossae and inflated
to 200 mm Hg for a period of 5 min. Diameter and blood
flow velocity were measured immediately after deflation of
the cuff (hyperemic blood flow) as well as 60, 75, 90, and
120 s later. Maximal BA diameter observed during this time
period was used to calculate FMD. Endothelium-independent
dilation of BA was quantified 4 min after sublingual administration of 400 ␮g glycerol trinitrate (GTN) (Nitrolingual
mite, Pohl, Germany). All ultrasound scans were performed
by the same operator using the same equipment. An
automated analysis system was used to measure diameters
(Brachial Analyzer, Medical Imaging Applications, Iowa
City, Iowa) yielding low variabilities of our methodology
described elsewhere (21). Internal quality control was performed by an independent investigator blinded to the
protocol (96% approved); FMD and endothelium-
independent dilation were expressed as a percentage change
from baseline. To estimate the relative proportion of FMD
compared to the maximally achievable diameter after GTN,
the FMD/GTN ratio was calculated and expressed as
percentage for each individual. Blood flow was calculated by
multiplication of cross-sectional area and mean velocity at
each time point.
Statistical analysis. Results are expressed as means ⫾ SE.
Comparisons between groups were analyzed by analysis of
variance (ANOVA) and, if significant, consecutive post-hoc
test (Bonferroni). P values from Bonferroni post-hoc tests
were corrected for 10 pairwise comparisons. Linear relationships between continuous variables were calculated using
Pearson’s r. A multivariate regression analysis was performed, to estimate the predictive value of plasma RXNO
concentrations to explain the variability of vascular function
as a surrogate for cardiovascular risk. Established parameters
known to affect FMD were also included in the model.
Standardized coefficients were calculated as a measure for
the relative predictive value. Statistical significance was
assumed if a null hypothesis could be rejected at p ⫽ 0.05.
All analyses were performed with SPSS 11.0.1 (SPSS Inc.,
Chicago, Illinois).
RESULTS
Effect of cardiovascular risk factors on vascular parameters. Endothelial function measured by FMD was significantly impaired in individuals with increasing numbers of
cardiovascular risk factors (one risk factor: 4.7 ⫾ 0.4%; two
risk factors: 2.8 ⫾ 0.4%; three risk factors: 2.2 ⫾ 0.4%; four
risk factors: 1.0 ⫾ 0.3%) compared to control subjects (6.5 ⫾
0.4%; p ⬍ 0.005 Bonferroni; Fig. 1A), irrespective of the
nature of risk for cardiovascular disease. Flow-mediated
dilation in individuals with one risk factor was significantly
greater than in those with three and four risk factors (p ⫽
0.003, Bonferroni). Endothelium-independent dilation in
response to GTN was similarly impaired: 14.5 ⫾ 1.0% (zero
risk factors); 12.3 ⫾ 1.0% (one risk factor); 10.6 ⫾ 1.3%
(two risk factors); 9.1 ⫾ 1.9% (three risk factors); 6.8 ⫾
1.0% (four risk factors) (ANOVA: p ⬍ 0.05). However, the
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Heiss et al.
Plasma Nitroso Species in Endothelial Dysfunction
JACC Vol. 47, No. 3, 2006
February 7, 2006:573–9
Figure 1. Endothelial function and plasma nitric oxide species are decreased in individuals with increasing numbers of cardiovascular risk factors.
(A) Flow-mediated dilation (FMD) and (B) percentage of FMD compared to glycerol trinitrate (GTN)-induced dilation of brachial artery (FMD/GTN
ratio) are significantly lower in individuals with cardiovascular risk factors. (C) Nitros(yl)ated species (RXNO) in plasma, representing the circulating pool
of bioactive nitric oxide, are also significantly attenuated with increasing numbers of risk factors. Columns ⫽ mean values; error bars ⫽ SE. P values are
Bonferroni-corrected for 10 pairwise comparisons.
significantly lowered FMD/GTN ratio indicates preferential
endothelial dysfunction (Fig. 1B). The baseline diameter was
significantly increased in the groups with three (5.1 ⫾ 0.2 mm)
and four risk factors (5.1 ⫾ 0.2 mm) compared to subjects
with zero to two risk factors (control: 4.0 ⫾ 0.1 mm; one
risk factor: 4.3 ⫾ 0.1 mm; two risk factors: 4.7 ⫾ 0.3 mm;
p ⬍ 0.001 Bonferroni). No significant differences were seen
in terms of blood flow at baseline, during hyperemia, or
Doppler flow reserve (data not shown).
Effect of cardiovascular risk factors on NO-related metabolites in plasma. In individuals without risk factors,
RXNO plasma concentration amounted to 39 ⫾ 2 nmol/l.
Increasing numbers of risk factors were associated with a
significant reduction in plasma RXNO levels: 30 ⫾ 2 nmol/l
(one risk factor); 24 ⫾ 3 nmol/l (two risk factors); 22 ⫾ 3
nmol/l (three risk factors); and 16 ⫾ 2 nmol/l (four risk
factors) (p ⬍ 0.001 Bonferroni; Fig. 1C). Also, RXNO was
significantly different in the groups with one and four risk
factors (p ⫽ 0.003 Bonferroni). Plasma nitrite concentrations followed a similar trend without reaching statistical
significance: 142 ⫾ 13 (control); 125 ⫾ 13 (one risk factor);
131 ⫾ 23 (two risk factors); 118 ⫾ 17 (three risk factors);
and 106 ⫾ 19 (four risk factors) nmol/l (ANOVA: p ⫽
0.821). Nitrate concentrations were not significantly
different between groups: 21 ⫾ 3 ␮mol/l (control); 23 ⫾
3 ␮mol/l (one risk factor); 26 ⫾ 5 ␮mol/l (two risk
factors); 32 ⫾ 7 ␮mol/l (three risk factors); and 23 ⫾ 4
␮mol/l (four risk factors) (ANOVA: p ⫽ 0.800).
Endothelial function correlates with circulating NO
pool. There was a highly significant correlation between
FMD and plasma RXNO concentration (r ⫽ 0.41; p ⬍
0.001; Fig. 2) suggesting an association between endothelial
dysfunction and circulating NO pool.
RXNOs as an independent predictor of endothelial dysfunction. In order to establish RXNO concentration as an
independent predictor of endothelial function, we performed a multivariate linear regression analysis including
baseline characteristics known to affect vascular function
(age, gender, body mass index, total cholesterol, plasma
glucose, mean arterial blood pressure, pack years, diameter of
BA, nitrite) and current medication with angiotensinconverting enzyme inhibitors and statins. As shown in Table
3, the independent predictor variables for FMD were
gender, age, RXNO concentration, and BA diameter accounting for 53% of the total variability of FMD (adjusted
R2 ⫽ 0.526; p ⬍ 0.001). The magnitudes of standardized
coefficients for the independent predictors were in the same
range as gender and age suggesting a comparable predictive
value.
DISCUSSION
The key findings of the present study are that: 1) the plasma
pool of RXNOs is depleted in patients with cardiovascular
Figure 2. Vascular endothelial function correlates with circulating nitric
oxide (NO) pool. Endothelial function, measured as NO-dependent
flow-mediated dilation of the brachial artery, correlates with the concentration of circulating nitroso/nitrosyl species (RXNO) in plasma (r ⫽ 0.41,
p ⬍ 0.001). Circles ⫽ individual values; filling ⫽ number of major
cardiovascular risk factors (RF).
Heiss et al.
Plasma Nitroso Species in Endothelial Dysfunction
JACC Vol. 47, No. 3, 2006
February 7, 2006:573–9
Table 3. Multivariate Linear Regression Analysis
FMD
Standardized
Coefficient
Gender
Age
BMI
Mean arterial blood pressure
Pack-yrs
Total cholesterol
Plasma glucose
RXNO
Nitrite
Brachial artery diameter
ACE inhibitors
Statins
Adjusted R2
Significance (ANOVA)
0.224
⫺0.189
⫺0.039
0.091
⫺0.010
⫺0.048
⫺0.161
0.163
0.093
⫺0.377
⫺0.189
⫺0.066
0.526
p Value
0.029
0.045
0.706
0.267
0.920
0.552
0.052
0.044
0.212
0.001
0.077
0.527
⬍0.001
ACE ⫽ angiotensin-converting enzyme; ANOVA ⫽ analysis of variance; BMI ⫽
body mass index; FMD ⫽ flow-mediated dilation; RXNO ⫽ sum of circulating nitric
oxide species.
risk factors; and that 2) RXNO is an independent predictor
of endothelial function in a multivariate regression model.
The earliest studies of endothelial control of vasomotion
used quantitative coronary angiography to examine the vasomotor response of the epicardial coronary artery during infusion of acetylcholine (22) or increased blood flow (23). In
healthy individuals, the endothelium responds to these
stimuli by releasing vasodilator factors, in particular NO.
Patients with angiographically documented coronary artery
disease display impaired FMD and a vasoconstrictor response to acetylcholine, which likely reflects loss of NO and
unopposed muscarinic receptor activation at the level of the
vascular smooth muscle. Notably, studies suggest that endothelial function detected noninvasively in the BA correlates with function in conduit coronary arteries (24).
As in the coronary circulation, endothelial function in the
brachial circulation is impaired in the setting of traditional
and novel risk factors and responds to interventions known
to reduce cardiovascular disease risk (25). Traditional cardiovascular risk factors as diverse as smoking, aging (26),
hypercholesterolemia, hypertension, hyperglycemia, and a
family history of premature atherosclerotic disease are all
associated with attenuation/loss of endothelium-dependent
vasodilation in both adults and children. More recently
recognized risk factors such as obesity, elevated C-reactive
protein, and chronic systemic infection are also associated
with endothelial dysfunction. Corroborating several studies
by Celermajer et al. (14) and other groups, we observed an
inverse association between the cumulative number of cardiovascular risk factors and FMD of the BA.
Although case-control studies indicate an association
between endothelial dysfunction and acute coronary syndromes (27), more convincing evidence for a pathogenic
role of the former is provided by studies demonstrating that
endothelial function identifies patients at increased risk for
future events. Numerous published studies involving ⬎2,000
577
patients with atherosclerosis have proven the prognostic value
of endothelial vasomotor dysfunction (25). These studies
strongly and consistently demonstrate that endothelial dysfunction identifies patients who have increased risk for
cardiovascular events in the short and long run. Primary and
secondary preventive therapies (e.g., exercise therapy,
angiotensin-converting enzyme inhibitors, and statins) are
believed to mediate their positive prognostic effect in large
part by increasing endothelial function. In our study, we did
not observe significant contributions of body mass index or
current medication with angiotensin-converting enzyme
inhibitors or statins to the variability of endothelial function
in our multivariate regression model. This may either be due
to the small n-number or the significantly greater predictive
value of other included parameters. Thus, endothelial dysfunction appears to be a systemic process that can be
identified in different vascular beds, including those remote
from the coronary and cerebral circulation where the majority of actual events occur.
The present study is the first to report that RXNO levels
in plasma are lower in individuals with one or more
cardiovascular risk factors compared to controls. The importance of RXNOs results from the very similar biological
actions exhibited by both NO and RXNOs in vivo as well as
in vitro (28). Cannon et al. (3) have provided the first
evidence for the intravascular delivery of bioactive NO
species in humans. Inhaled NO restored blood flow and
vascular resistance of the forearm during regional inhibition
of NO synthesis. Intravenous infusion of NO in healthy
volunteers increased plasma RXNOs and induced systemic
hemodynamic effects at the level of both conduit and resistance vessels, as reflected by dilator responses in the BA and
forearm microvasculature. Infusion of S-nitrosoglutathione
exerted similar effects, and the observed changes in RXNO
concentrations correlated with the vasodilator effects exerted
by NO and S-nitrosoglutathione (2). Furthermore, RXNOs
inhibit platelet aggregation (29) and abolish embolization
from carotid plaques (30).
The RXNO levels reported in the literature range from
the low nanomolar up to the micromolar range. This may be
due to differences in the methodology applied to measure
S-nitrosothiols and N-nitrosamines, sample preparation and
handling, choice of standards, and detection methods
(5,9,10,19,31,32). In view of the fact that the chemical identity
of most of these species is currently unknown, we intentionally
opted to measure the sum of all nitroso and nitrosyl species
detectable under these conditions attempting a groupspecific analysis as opposed to quantification of select species
of unclear physiological role (19). Regardless of the absolute
levels of RXNOs, the significant difference between the
patient groups in relation to the impairment of endothelial
function remains (31). Nevertheless, future studies are
needed to identify relevant individual bioactive compounds
comprising RXNOs.
The decreased pool of circulating NO with increasing
cardiovascular risk load may be secondary to endothelial
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Heiss et al.
Plasma Nitroso Species in Endothelial Dysfunction
dysfunction reflected by the impaired FMD. This is underscored by the univariate correlation between FMD and the
circulating NO pool and the multivariate analysis with
RXNO being a significant predictor for FMD independent
of age, gender, body mass index, blood pressure, cholesterol,
glucose, baseline diameter of the BA, pack years, current
statin and angiotensin-converting enzyme inhibitor therapy
with comparable magnitude as gender and age. Corroborating our previous findings, the baseline diameter was also a
significant predictor (33). Also, cigarette smoke contains
large amounts of nitrogen oxides (34); the subgroup of
smokers without other cardiovascular risk factors showed
decreased RXNOs and FMD. Potentially, the reduced
circulating NO pool may accelerate atherogenesis and predispose individuals to clinical arteriosclerotic syndromes
characterized by impaired regional NO production, such as
peripheral and coronary artery disease. Whether decreased
levels of RXNO are a consequence of reduced endogenous
NO synthesis and/or bioavailability or that of an accelerated
breakdown and/or NO consumption is currently unclear. In
addition to a diminished NO production, alterations in
NOS expression, co-factor, and/or substrate availability may
also contribute directly to oxidative stress by producing
superoxide as a consequence of uncoupling of NOS. Thus,
although the sources of oxidative stress may differ and
several different enzymatic and biochemical mechanisms can
disrupt normal NO signaling, a central problem appears to
be a shift in NO/nitroso-redox balance away from “physiologic” heme nitrosylation and S-nitrosation to one of
enhanced nitrosation/nitration of protein targets under
conditions of oxidative stress. Whether plasma RXNO
concentration represents a valid surrogate marker for cardiovascular risk burden or oxidative stress and qualifies as a
prognostic factor cannot be concluded from the present
study and remains to be investigated.
In newer publications, nitrite, one of the major oxidative
metabolites of NO, was implicated to be both an indicator
for NOS activity (35) and a circulating NO donor that may
selectively donate NO to hypoxic vascular beds (11). Thus,
apart from RXNOs, nitrite within the blood and tissues
(11,36,37) may evolve as an important contributor to the
circulating pool of bioactive NO. Although in the present
study no significant differences in mean plasma nitrite levels
were observed between individual groups, there was clearly
an inverse relationship between nitrite levels and the number of cardiovascular risk factors. The fact that RXNOs, but
not nitrite, predicts endothelial dysfunction in the multivariate regression model in this relatively small sample size may
be explained by several differences in biochemical features of
both compounds. The short half-life of plasma nitrite in
blood, which is a consequence of its rapid transition (38)
into red blood cells and surrounding tissues (19), contributes
to its usefulness to index acute changes in eNOS activity
(35). Although the absolute levels are conserved throughout
many mammalian species (36), the intraindividual variability still poses a problem because diet, NO donor drugs, renal
JACC Vol. 47, No. 3, 2006
February 7, 2006:573–9
function, gut bacteria (39), and other NOS-independent
confounders may influence baseline levels and thus require
relatively large n-values to detect clear differences between
patient populations. Because of its longer half-life and
apparent robustness against influence by NOS-independent
dietary confounders such as nitrate (40), RXNOs may be a
more suitable diagnostic marker for long-term changes in
eNOS activity. Nevertheless, new analytical methods capable of differentiating between individual RXNO components and an identification of the physiological roles of these
species are necessary. Despite these obstacles, a composite
view and analysis of the constituents of the circulating NO
pool is a promising first step toward the development of new
biochemical approaches to assess endothelial dysfunction.
Conclusions. From the data presented herein, we conclude
that endothelial dysfunction is associated with a depletion of
circulating nitroso/nitrosyl species in plasma, which is likely
to contribute to the increased risk for major cardiovascular
events in individuals with endothelial dysfunction. Measuring circulating nitroso/nitrosyl species may help in identifying individuals at risk and serve as a therapeutic surrogate
marker in the future. Further studies are needed to establish
whether the plasmatic RXNO pool represents a valuable
parameter that allows optimal dose titration of therapeutic
agents aimed at targeting endothelial dysfunction.
Acknowledgment
The authors wish to thank Gabi Schoder for her technical
assistance.
Reprint requests and correspondence: Dr. Malte Kelm, Department of Cardiology, Pneumology, and Vascular Medicine, RWTH
Aachen, Pauwelsstraße 30, 52074 Aachen, Germany.
E-mail: [email protected].
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