CLINICAL INVESTIGATION
Association of Nitrotyrosine Levels
With Cardiovascular Disease
and Modulation by Statin Therapy
Mehdi H. Shishehbor, DO
Ronnier J. Aviles, MD
Marie-Luise Brennan, PhD
Xiaoming Fu, MS
Marlene Goormastic, MPH
Gregory L. Pearce, MS
Noyan Gokce, MD
John F. Keaney, Jr, MD
Marc S. Penn, MD, PhD
Dennis L. Sprecher, MD
Joseph A. Vita, MD
Stanley L. Hazen, MD, PhD
N
ITRIC OXIDE IS A VASODILA-
tor and inhibitor of platelet
aggregation, leukocyte ad-
hesion, and smooth muscle
cell proliferation.1-3 However, under
pathological conditions, nitric oxide
may be converted into potent nitrat-
ing oxidants that promote oxidative
damage, cell injury, and conversion of
low-density lipoprotein (LDL) into an
atherogenic form.1-5 One pathway for
generating nitric oxide–derived oxi-
dants involves interaction with super-
oxide anion, leading to formation of
peroxynitrite. Peroxynitrite is a po-
tent oxidant that promotes nitration of
protein tyrosine residues producing a
distinctive “molecular fingerprint” for
nitric oxide–derived oxidants, nitroty-
rosine.6,7 An alternative mechanism for
generating nitric oxide–derived oxi-
dants involves myeloperoxidase,7-11 a
leukocyte-derived enzyme enriched in
atherosclerotic lesions that serves as an
©2003 American Medical Association. All rights reserved.
Context Formation of nitric oxide–derived oxidants may serve as a mechanism linking
inflammation to development of atherosclerosis. Nitrotyrosine, a specific marker for pro-
tein modification by nitric oxide–derived oxidants, is enriched in human atherosclerotic
lesions and low-density lipoprotein (LDL) recovered from human atheroma.
Objectives To determine whether systemic levels of nitrotyrosine are associated with
the prevalence of coronary artery disease (CAD) and are modulated by hydroxymeth-
ylglutaryl coenzyme-A reductase inhibitor (statin) therapy.
Design, Setting, and Patients A case-control and interventional study at 2 ur-
ban tertiary-care referral centers; recruitment for each was from June 1, 2001, until
January 1, 2002. For the case-control study, 100 case-patients with established CAD
and 108 patients with no clinically evident CAD were recruited consecutively. In the
interventional study, participants aged 21 years or older with hypercholesterolemia
(LDL cholesterol ⱖ130 mg/dL [ⱖ3.5 mmol/L]) underwent nutrition and exercise coun-
seling. Those whose levels did not decrease with 6 to 8 weeks were enrolled in the
study (n=35). For 12 weeks, they received 10 mg/d of oral atorvastatin therapy.
Main Outcome Measures In the case-control study, the association between systemic
levels of protein-bound nitrotyrosine, CAD risk, and presence of CAD. In the interven-
tional study, the change in nitrotyrosine, lipoprotein, and C-reactive protein (CRP) levels.
Results Nitrotyrosine levels were significantly higher among patients with CAD (me-
dian 9.1 µmol/mol [interquartile range, 4.8-13.8 µmol/mol] tyrosine vs 5.2 µmol/mol
[interquartile range, 2.2-8.4 µmol/mol]; P⬍.001). Patients in the upper quartile of ni-
trotyrosine (29%; P⬍.001) had a higher odds of CAD compared with those in the low-
est quartile (unadjusted odds ratio, 6.1; 95% confidence interval, 2.6-14.0; P⬍.001). In
multivariate models adjusting for Framingham Global Risk Score and CRP, upper quar-
tiles of nitrotyrosine remained associated with CAD (odds ratio, 4.4; 95% confidence
interval, 1.8-10.6; P⬍.001). Statin therapy reduced nitrotyrosine levels significantly (25%;
P⬍.02) with a magnitude similar to reductions in total cholesterol levels (25%; P⬍.001)
and LDL particle number (29%; P⬍.001) yet were independent of alterations in lipo-
proteins and inflammatory markers like CRP.
Conclusions The findings from this preliminary study indicate that nitrotyrosine lev-
els are associated with the presence of CAD and appear to be modulated by statin
therapy. These results suggest a potential role for nitric oxide–derived oxidants as in-
flammatory mediators in CAD and may have implications for atherosclerosis risk as-
sessment and monitoring of anti-inflammatory actions of statins.
JAMA. 2003;289:1675-1680 www.jama.com
Author Affiliations: Departments of Cell Biology (Drs and Vita), Boston University School of Medicine, Bos-
Shishehbor, Aviles, Brennan, Penn, Sprecher, and Ha- ton, Mass.
zen and Ms Fu), Cardiovascular Medicine (Drs Aviles, Financial Disclosure: Dr Hazen is named as co-
Penn, Sprecher, and Hazen and Ms Goormastic and inventor on pending patents filed by the Cleveland
Mr Pearce) and the Center for Cardiovascular Diag- Clinic Foundation that relate to the use of biomark-
nostics and Prevention (Drs Shishehbor, Aviles, Bren- ers for inflammatory and cardiovascular diseases.
nan, Penn, Sprecher, and Hazen and Ms Goormastic), Corresponding Author and Reprints: Stanley L. Ha-
the Cleveland ClinicFoundation, Cleveland, Ohio; and zen, MD, PhD, Cleveland Clinic Foundation, Cleve-
Evans Department of Medicine (Drs Gokce, Keaney, land, OH 44195 (e-mail: hazens@ccf.org).
(Reprinted) JAMA, April 2, 2003—Vol 289, No. 13 1675
NITROTYROSINE LEVELS AND ATHEROSCLEROSIS
independent predictor of cardiovascu-
lar risk.10 Although pathways that form
nitrotyrosine have been mechanisti-
cally linked to atherosclerosis devel-
opment, and nitrotyrosine levels are en-
riched in human atheroma,12,13 a role
for plasma or serum levels of nitroty-
rosine as a predictor of human coro-
nary artery disease (CAD) has not yet
been examined.
The pleiotropic effects of hydroxy-
methylglutaryl coenzyme-A reductase
inhibitors (statins) are an area of in-
tense interest.14,15 The pathways lead-
ing to generation of nitric oxide–
derived oxidants and nitrotyrosine
formation all require the formation of
superoxide or its dismutation prod-
uct, hydrogen peroxide.5-7,11 The ma-
jor enzymatic sources for superoxide
anion formation within vascular tis-
sues involve leukocyte and vascular cell
NAD(P)H oxidase complexes.5 Statins
have been shown to inhibit superox-
ide anion formation within vascular
cells by inhibiting isoprenylation of key
NAD(P)H oxidase components,15,16 sug-
gesting that statins may cause signifi-
cant reductions in nitrotyrosine forma-
tion. Alternatively, statin-induced
inhibition in isoprenylation of the small
G-protein Rho leads to increased lev-
els of endothelial cell nitric oxide syn-
thase and enhanced nitric oxide pro-
duction.15,17 The net effect of statin
therapy on formation of nitric oxide–
derived oxidants in vivo is unclear. This
study was performed to determine
whether CAD is associated with in-
creased systemic levels of nitrotyro-
sine and whether statin therapy would
reduce these levels.
METHODS
Case-Control Study
We enrolled 208 individuals from 2
venues in Boston, Mass, from June 2001
until January 2002. Consecutive pa-
tients presenting to the cardiology sec-
tion of the Boston Medical Center were
enrolled. Simultaneously (and to in-
crease recruitment of controls), con-
secutive area residents responding to
advertisements in a community news-
paper were recruited. The 100 case pa-
1676 JAMA, April 2, 2003—Vol 289, No. 13 (Reprinted)
tients had a history of CAD, defined as
documented myocardial infarction,
coronary artery bypass graft surgery,
percutaneous coronary intervention, or
a stenosis of 50% or greater in 1 or more
major coronary vessels on angiogra-
phy. Some of the patients with CAD also
had peripheral arterial disease (PAD),
defined as an ankle-brachial index less
than 0.9, intermittent claudication,
and/or documented stenoses of periph-
eral or carotid arteries by angiogra-
phy, magnetic resonance imaging, or ul-
trasound. Controls had no clinical
history or symptoms suggestive of CAD
or PAD. All participants gave written
informed consent, and the institu-
tional review board of Boston Medical
Center approved the study protocol.
Prospective Intervention Study
We enrolled 35 consecutive patients
from the Preventive Cardiology Clinic
at the Cleveland Clinic Foundation from
June 2001 until January 2002. Patients
who were at least 21 years old, had no
clinical evidence of CAD, PAD, or dia-
betes mellitus, were naive to statin
therapy, and had low-density lipopro-
tein cholesterol (LDL-C) levels that were
130 mg/dL or higher (ⱖ3.3 mmol/L) re-
ceived counseling on nutritional and ex-
ercise interventions. If after 6 to 8 weeks
LDL-C remained at 130 mg/dL or higher
(ⱖ3.3 mmol/L), patients were eligible
for enrollment in the study. Fasting
morning plasma samples were col-
lected prior to initiation of therapy (base-
line) and following 12 weeks of ator-
vastatin therapy (10 mg/d). All patients
enrolled completed the study. Exclu-
sion criteria included liver disease, re-
nal insufficiency, or changes in medi-
cal therapy during the treatment period.
All patients gave written informed con-
sent, and the institutional review board
at the Cleveland Clinic Foundation ap-
proved the study protocol.
Laboratory Analysis
General. Blood samples were col-
lected into serum separator tubes (case-
control study) or EDTA tubes (inter-
ventional study) from patients who had
fasted overnight. Samples were centri-
©2003 American Medical Association. All rights reserved.
fuged at 3500 rpm for 10 minutes,
plasma/serum were recovered, and ali-
quots were stored at −80°C until analy-
sis. Personnel blinded to clinical data
performed all laboratory measure-
ments. Lipoprotein/lipid profiles and
high-sensitivity C-reactive protein
(CRP) measurements were performed
as previously described.18,19
Nitrotyrosine. Protein-bound nitro-
tyrosine levels were determined by stable
isotope dilution liquid chromatography–
electrospray ionization tandem mass
spectrometry-based methods using an
ion trap mass spectrometer (LCQ Deca,
ThermoFinigann, San Jose, Calif ),
as previously described.11 Synthetic 3-
nitro-[ 13 C 6 ]tyrosine (2 pmol) and
[13C915N1]tyrosine (2 nmol) were added
to protein pellets both as internal stan-
dards and to simultaneously monitor ni-
trotyrosine, tyrosine, and potential ar-
tifactual formation of nitrotyrosine
during analyses.11 Nitrotyrosine con-
tent in samples is expressed as the mo-
lar ratio between nitrotyrosine and the
precursor amino acid tyrosine. Protein-
bound nitrotyrosine levels in plasma re-
producibly were greater than that ob-
served in serum, suggesting clotting
factors represent preferential targets for
nitration. Control studies demon-
strated protein-bound nitrotyrosine lev-
els within plasma of healthy subjects are
stable over time, with coefficients of vari-
ance less than 15% for multiple inde-
pendent repeated measures on differ-
ent days or for comparisons made during
more than 3 months apart.
Statistical Analysis
Case-Control Study. Nitrotyrosine lev-
els were not normally distributed (Sha-
piro-Wilk test). Consequently, quartile-
based methods were used for analyses,
and summary measures were pre-
sented as median and interquartile
range. Comparisons between cases and
controls were made with ␹2 tests for cat-
egorical measures and Wilcoxon rank-
sum tests for continuous measures.
Trends were assessed with Cochran-
Armitage tests.
Logistic regression models (SAS Sys-
tem, SAS Institute, Cary, NC) were used
 NITROTYROSINE LEVELS AND ATHEROSCLEROSIS

to calculate odds ratios (ORs) associ- Nitrotyrosine Levels and CAD. Ni- ing triglycerides (r=0.14, P =.03), and
ated with the second, third, and highest trotyrosine levels were higher in pa- CRP levels (r =0.15, P =.02); however,
quartile of nitrotyrosine compared with tients with CAD compared with con- these associations were small in mag-
the lowest quartile for the indicated out- trols (median values, 9.1 µmol/mol nitude and accounted for less than 5%
comes. Single adjustments were made for tyrosine vs 5.2 µmol/mol tyrosine, re- of the observed variance in nitrotyro-
individual traditional CAD risk factors spectively; P⬍.001; Table 1). Rates of sine. There was no significant correla-
(age, sex, diabetes, hypertension, smok- CAD increased with higher nitrotyro- tion between nitrotyrosine and LDL-C,
ing [ever or current]), family history, sine quartiles (26% vs 58%, lowest vs HDL-C, or total cholesterol. Partici-
total cholesterol, LDL-C, high-density li- highest quartiles; P⬍.001 for trend). Pa- pants with diabetes had higher nitro-
poprotein cholesterol [HDL-C], triglyc- tients in the highest quartile of nitro- tyrosine levels than those who did not
erides, CRP), and a modified Framing- tyrosine levels had increased risk of have diabetes (median values, 9.6 µmol/
ham Global Risk Score,10 alone and with CAD compared with patients in the mol tyrosine vs 5.7 µmol/mol tyro-
CRP. Hosmer-Lemeshow goodness-of- lowest quartile (TABLE 2; the unad- sine, respectively; P⬍.001).
fit tests were used to evaluate appropri- justed nitrotyrosine fourth quartile OR, Adjusted Models for Nitrotyrosine
ate model fit. Associations among con- 6.1; 95% CI, 2.6-14.0; P⬍.001). CAD and CAD. Nitrotyrosine levels re-
tinuous variables were assessed with use rates were also higher with increasing mained significantly associated with CAD
of Spearman rank-correlation coeffi- CRP quartiles (25% vs 50%, lowest vs following individual adjustments for age;
cient. Associations among categorical highest quartiles; P⬍.001 for trend). sex; history of diabetes; current smok-
variables were assessed using Wil- The proportion of patients with CAD ing; history of hypertension; and levels
coxon rank-sum tests. was higher among those in the upper of HDL-C, LDL-C, triglyceride, and CRP
The study was planned to have at quartile of both nitrotyrosine and CRP with minimal changes observed in ad-
least 100 patients per group, based on levels compared with patients in the justed ORs and CIs (data not shown). Af-
a logistic regression power calculation lower quartiles (76% vs 7%; P⬍.001). ter adjustment for the Framingham
demonstrating that 198 patients would Nitrotyrosine Levels and CAD Risk Global Risk Score, nitrotyrosine re-
provide 80% power (␣ = .05) to detect Factors. Nitrotyrosine levels corre- mained a robust predictor of presence of
an OR of 2.0 for elevated nitrotyrosine lated with age (r = 0.14, P = .03), fast- CAD (Table 2, Model 1; adjusted nitro-
(upper quartile).
Interventional Study. Wilcoxon Table 1. Baseline Characteristics by Coronary Artery Disease Status
rank-sum test was used to analyze the Patients With
differences between measurements at Control Patients CAD P
baseline and 12 weeks. Spearman rank- Characteristic (n = 108) (n = 100) Value
correlation coefficients were used to Age, median (IQR), y 44 (35-53) 59 (53-67) ⬍.001
Women, No. (%) 76 (70) 46 (46) ⬍.001
assess associations between both base-
Body mass index, median (IQR) 28 (24-33) 27 (24-33) .80
line and atorvastatin-induced changes in
Risk factors, No. (%)
nitrotyrosine levels, lipoprotein profile Diabetes mellitus 3 (3) 34(34) ⬍.001
measures, and CRP levels. Approxi- Hypertension 36 (33) 63 (63) ⬍.001
mate 95% confidence intervals (CIs) were Family history of CAD 18 (17) 48 (48) ⬍.001
found using Fisher r-to-z transform. Mul- Current smoker 53 (49) 73 (73) ⬍.001
tiple regression analyses were per- Cholesterol, median (IQR), mg/dL
formed to determine factors associated Total 199 (180-225) 196 (168-212) .03
with changes in nitrotyrosine levels. High-density lipoprotein 59 (46-67) 53 (39-67) .58
Low-density lipoprotein 119 (99-147) 99 (49-128) ⬍.001
RESULTS Triglycerides, median (IQR), mg/dL 106 (68-148) 148 (125-198) ⬍.001
Case-Control Study C-reactive protein, median (IQR), mg/dL 0.19 (0.08-0.46) 0.49 (0.32-1.50) ⬍.001
Patient Demographics. Baseline char- Nitrotyrosine, median (IQR), µmol/mol tyrosine 5.2 (2.2-8.4) 9.1 (4.8-13.8) ⬍.001
acteristics of study participants are Therapy, No. (%)
Antiplatelet 5 (5) 84 (84) ⬍.001
shown in TABLE 1. As expected, pa-
Angiotensin-converting enzyme inhibitors 10 (9) 40 (40) ⬍.001
tients with CAD were older, more likely
Angiotensin II receptor blockers 2 (2) 3 (3) .59
to be men, and more likely to have hy- ␤-Blockers 12 (11) 72 (72) ⬍.001
pertension, diabetes mellitus, or fam- Calcium channel blockers 6 (6) 18 (18) .005
ily history of CAD. Patients with CAD Nitrates 3 (3) 15 (15) ⬍.01
also had increased triglyceride levels Statins 3 (3) 39 (39) ⬍.001
and CRP levels, and they were more Abbreviations: CAD, coronary artery disease; IQR, interquartile range.
likely to use lipid-lowering drugs and SI conversion factors: To convert high-density lipoprotein, low-density lipoprotein, and total cholesterol from mg/dL to
mmol/L, multiply by 0.0259; triglycerides, multiply by 0.0113.
other cardiovascular medications.
©2003 American Medical Association. All rights reserved. (Reprinted) JAMA, April 2, 2003—Vol 289, No. 13 1677
NITROTYROSINE LEVELS AND ATHEROSCLEROSIS

tyrosine 4th quartile OR, 5.4; 95% CI, sine to multivariable prediction models with clinical evidence of atheroscle-
2.0-14.3; P⬍.001). Addition of CRP to that included established markers of car- rotic burden (ie, determine if the cor-
the model had little effect on the OR for diovascular risk (eg, Model 2, Table 2) relation of nitrotyrosine levels with ath-
nitrotyrosine as a predictor of CAD sta- significantly added to prediction for pres- erosclerosis is even stronger in patients
tus (Table 2, Model 2; adjusted nitroty- ence of CAD (␹2 =10.42; P⬍.001). with more extensive atherosclerosis
rosine fourth quartile OR, 4.4; 95% CI, Nitrotyrosine Levels and Athero- [CAD plus PAD]). Patients with CAD
1.8-10.6; P⬍.001). Likelihood ratio tests sclerosis Burden. We also examined plus PAD demonstrated increases in
confirmed that introducing nitrotyro- whether nitrotyrosine levels correlate prevalence of atherosclerosis with in-
creasing nitrotyrosine quartiles (3%
Table 2. Odds Ratios of Cardiovascular Disease Risk According to Quartiles of Nitrotyrosine vs 46%, lowest vs highest quartiles;
Quartiles P⬍.001 for trend). Within this athero-
P sclerosis-laden group, nitrotyrosine
Characteristics 1 2 3 4 Value† served as the strongest independent pre-
Nitrotyrosine, µmol /mol ⬍3.6 3.6 − 6.31 6.32 − 10.04 ⱖ10.05 dictor associated with atherosclerosis
tyrosine
risk following multivariable adjust-
CAD, Cases (n = 100), Controls (n = 108)
ments with Framingham Global Risk
No. of cases 17 19 26 38
Score and CRP (adjusted nitrotyro-
No. of controls 38 29 27 14
sine fourth quartile OR, 25.4; 95% CI,
Odds ratio, (95% CI)
Unadjusted 1.0 1.4 (0.6-3.3) 2.2 (1.0-4.7) 6.1 (2.6-14.0) ⬍.001 2.8-274; P⬍.001 [Table 2]; adjusted
Model 1‡ 1.0 2.3 (0.8-6.3) 2.1 (0.8-5.3) 5.4 (2.0-14.3) ⬍.001 Framingham Global Risk Score OR,
Model 2§ 1.0 2.3 (0.3-2.0) 1.1 (0.4-3.0) 4.4 (1.8-10.6) ⬍.001 1.25; 95% CI, 1.15-1.36; P⬍.001; ad-
CAD Without PAD, Cases (n = 64), Controls (n = 108) justed CRP fourth quartile OR, 5.0; 95%
No. of cases 16 10 12 26 CI, 2.1-16.6; P⬍.001).
No. of controls 38 29 27 14
Odds ratio, (95% CI)
Interventional Study
Unadjusted 1.0 0.8 (0.3-2.0) 1.1 (0.5-2.6) 4.4 (1.8-10.6) ⬍.001 In the case-control study of the entire
Model 1‡ 1.0 0.8 (0.3-2.5) 1.0 (0.4-2.8) 4.4 (1.6-11.7) ⬍.001 cohort (n = 208), nitrotyrosine levels
Model 2§ 1.0 1.0 (0.3-3.2) 1.1 (0.4-3.0) 4.0 (1.4-11.4) ⬍.001 demonstrated a tendency toward being
CAD Plus PAD, Cases (n = 36), Controls (n = 108) lower in patients taking statins (P=.06),
No. of cases 1 9 14 12 suggesting that statin therapy may re-
No. of controls 38 29 27 14 duce nitrotyrosin levels. This was di-
Odds ratio (95% CI) rectly assessed in an interventional
Unadjusted 1.0 12.2 (1.5-102) 19.0 (2.4-153) 32.6 (3.9-274) ⬍.001
study comprised of 35 patients with a
Model 1‡ 1.0 17.1 (1.9-156) 17.6 (2.0-151) 26.3 (2.9-238) ⬍.001
mean (SD) of 54 (10) years, 49% of
Model 2§ 1.0 17.2 (1.9-158) 17.3 (2.0-149) 25.4 (2.8-274) ⬍.001
whom were men. TABLE 3 shows the
Abbreviations: CAD, coronary artery disease; PAD, peripheral artery disease.
*Unadjusted odds ratios (ORs) and confidence intervals (CIs) were calculated using logistic regression models com- lipid and lipoprotein levels, CRP, and
paring risk of each of the upper 3 quartiles for a given parameter to the lowest quartile. nitrotyrosine levels at baseline and af-
†P for trend across quartiles.
‡Model 1 consisted of Framingham Global Risk Score and nitrotyrosine quartiles. ter taking orally 10 mg/d of atorvas-
§Model 2 consisted of Framingham Global Risk Score, C-reactive protein, and nitrotyrosine quartiles.
tatin for 12 weeks. Atorvastatin treat-
ment reduced total cholesterol levels by
Table 3. Lipid Levels, High-Sensitivity C-Reactive Protein, and Nitrotyrosine at Baseline and 25%, LDL-C by 39%, and apolipopro-
After 12 Weeks of Treatment With Atorvastatin*
tein B-100 by 29%. Statin-induced re-
Mean (SD) ductions in plasma nitrotyrosine lev-
Baseline 12 Weeks P els (25%; P = .02) were similar in
Variables (n = 35) (n = 35) Value magnitude to decreases in total choles-
Nitrotyrosine (µmol/mol tyrosine) 15 (7) 11 (5) .02 terol and LDL particle number (ie, apo-
C-reactive protein, mg/dL 0.26 (0.32) 0.23 (0.33) .10 lipoprotein B-100). A nonsignificant
Cholesterol, mg/dL trend toward statin-induced reduc-
Total 253 (27) 190 (28) ⬍.001
High-density lipoprotein 56 (12) 58 (12) .21
tions in CRP levels was also observed
Low-density lipoprotein 169 (22) 103 (29) ⬍.001
(11% reduction; P =.10).
Triglycerides, mg/dL 146 (90) 132 (81) .22
No significant correlations were
Apolipoprotein B-100, mg/dL 135 (17) 96 (21) ⬍.001
noted between baseline levels of nitro-
Conversion factors: To convert high-density lipoprotein, low-density lipoprotein, and total cholesterol from mg/dL to tyrosine, lipid parameters, and CRP.
mmol/L, multiply by 0.0259; triglycerides, multiply by 0.0113. Furthermore, no significant correla-
*P values represent comparisons between mean baseline and 12-week levels for the indicated variables.
tions were noted between statin-
1678 JAMA, April 2, 2003—Vol 289, No. 13 (Reprinted) ©2003 American Medical Association. All rights reserved.

induced changes in nitrotyrosine vs
changes in lipoprotein and inflamma-
tory markers including total choles-
terol level (95% CI; ␳, −0.23 to 0.43),
LDL-C (95% CI; ␳, −0.2 to 0.45),
HDL-C (95% CI; ␳, −0.18 to 0.47), or
CRP (95% CI, ␳ −0.22 to 0.44). In mul-
tivariable regression analysis, there was
no significant association between
change in nitrotyrosine levels and
changes in levels of total cholesterol,
LDL-C, HDL-C, and CRP (F-ra-
tio=0.71; P=.60).
COMMENT
The results of these preliminary stud-
ies suggest that nitrotyrosine, a marker
specific for protein modification by ni-
tric oxide–derived oxidants, may serve
as an inflammatory marker for CAD.
Systemic levels of protein-bound ni-
trotyrosine were associated with pres-
ence of CAD even following multivari-
able adjustments for traditional CAD
risk factors and CRP. Importantly, statin
therapy promoted significant reduc-
tions in nitrotyrosine levels that were
similar in magnitude to reductions in
total cholesterol and LDL particle num-
ber. Moreover, reductions in nitroty-
rosine promoted by statin therapy were
independent of reductions in lipid pa-
rameters and CRP. Taken together,
these results suggest that nitrotyro-
sine measurements may prove useful
both in assessing CAD status and for
monitoring the anti-inflammatory ef-
fects of statins.
Numerous lines of evidence support
potential links between formation of ni-
tric oxide–derived oxidants and devel-
opment of CAD. Current evidence sug-
gests that an imbalance between
superoxide and nitric oxide formation
within diseased artery walls leads to a
functional deficiency of nitric oxide, and
consequent generation of nitric oxide-
derived oxidants such as peroxynitrite
and myeloperoxidase-generated reac-
tive nitrogen species.1-5,9-11 Enhanced for-
mation of superoxide in diseased ar-
tery walls may occur through vascular,
endothelial (eg, nox), and leukocyte-
derived NAD(P)H oxidase com-
plexes,20,21 as well as nitric oxide syn-
©2003 American Medical Association. All rights reserved.
NITROTYROSINE LEVELS AND ATHEROSCLEROSIS
thase that is “uncoupled.”22 Superoxide
thus formed may interact with nitric ox-
ide, resulting in formation of nitrating
oxidants. Similarly, myeloperoxidase,
a leukocyte-derived heme protein
enriched in human atheroma,23,24 cata-
lytically consumes nitric oxide as a
physiological substrate.9,25 Organ cham-
ber studies and studies with myeloper-
oxidase knockout mice suggest that
peroxidase enrichment at sites of
inflammation, such as within the sub-
endothelial space of a diseased vessel
wall, inhibits nitric oxide-dependent va-
sodilation responses through mecha-
nisms that lead to nitrotyrosine forma-
tion.26,27 Human monocytes have been
shown to use both peroxynitrite and
myeloperoxidase-dependent path-
ways for generating nitric oxide–
derived oxidants, as monitored by
nitrotyrosine formation.7 One conse-
quence of these reactions appears to be
the oxidative conversion of LDL into a
high uptake form for macrophages,
leading to cholesterol accumulation
and foam cell formation.4 Alternative
mechanisms have linked nitric oxide–
derived oxidants to activation of
matrix metalloproteases and develop-
ment of unstable plaques28 and devel-
opment of prothrombic states.29,30 The
potential contributions of nitric oxide–
derived oxidants to CAD develop-
ment are thus numerous and varied.
In this study, therapy with low-
dose atorvastatin significantly re-
duced nitrotyrosine levels. Statins pro-
mote systemic effects that extend
beyond simply lowering cholesterol lev-
els.1,14,15 Statin-induced inhibition in su-
peroxide formation has been shown in
cultured vascular smooth muscle cells.16
We therefore hypothesized that signifi-
cant reductions in nitrotyrosine would
be noted since pathways leading to for-
mation of nitric oxide–derived oxi-
dants invariably require superoxide
generation. The mechanism for de-
creased superoxide formation appears
to involve inhibition of isoprenylation
of the protein Rac, a key NAD(P)H oxi-
dase component that normally re-
quires isoprenylation for appropriate
translocation to the plasma mem-
(Reprinted) JAMA, April 2, 2003—Vol 289, No. 13 1679
brane surface during cell stimula-
tion.15,16 Thus, in contrast to the mod-
est alterations in CRP typically noted
relative to those observed for lipopro-
tein and cholesterol levels,31-33 these re-
sults demonstrated that nitrotyrosine
reductions were comparable in magni-
tude to those noted for total choles-
terol or LDL particle number with ad-
ministration of low-dose statin (Table
3). The growing appreciation of the
pleiotropic actions of statins has un-
derscored the requirement for new mea-
sures that quantify the anti-inflamma-
tory properties of this widely used class
of drugs. Our study suggests that sys-
temic nitrotyrosine levels may serve as
an independent measure of the anti-
inflammatory actions of statins.
A corollary to these findings is that
low-dose atorvastatin therapy pro-
motes potent systemic antioxidant ef-
fects by suppressing formation of ni-
tric oxide–derived oxidants. Further
studies of the systemic antioxidant ac-
tions promoted by statin therapy are
warranted. Recent randomized trials
with antioxidant vitamins, particu-
larly ␣-tocopherol, have failed to dem-
onstrate benefit against cardiovascu-
lar disease,34,35 and it is notable that
␣-tocopherol is relatively ineffective at
blocking the effects of nitric oxide–
derived oxidants.36-38 It is tempting to
speculate that interventional studies
with statins, which have repeatedly
demonstrated clinical benefits, have in
fact also been “antioxidant” trials that
used therapeutic agents able to sup-
press generation of more clinically rel-
evant nitric oxide–derived oxidants.
Elevated nitrotyrosine levels in pa-
tients with diabetes were recently re-
ported,39 a finding also observed in our
cohort. Postprandial elevations in ni-
trotyrosine levels following consump-
tion of a high fat or high glucose meal
that were attenuated following simva-
statin therapy were also recently re-
ported.40 Although nitrotyrosine en-
richment in human atherosclerotic
lesions is well known from both im-
munohistochemical and mass spec-
trometry-based studies,12,13 our study is
the first, to our knowledge, that di-
NITROTYROSINE LEVELS AND ATHEROSCLEROSIS
rectly correlates systemic levels of ni-
trotyrosine with presence of CAD and
response to statin therapy. The cross-
sectional (rather than longitudinal) de-
sign of the case-control study and the
pilot nature of the intervention study
are important limitations of our study.
However, the results point toward
promising potential clinical utility in
use of nitrotyrosine levels as an ad-
junct for CAD risk stratification and
monitoring of anti-inflammatory ac-
tions of statin therapy. Further evalu-
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