J Cardiol 2006 Oct; 48（4）: 183 – 191

Redox Imbalance in Patients With
Coronary Artery Disease Showing
Progression of Atherosclerotic
Lesions
Rie

TSURU, MD

Yukihiro

HOJO, MD

Miyuki

GAMA, MDOsamu

MIZUNO, MD

Takaaki KATSUKI, MD
Kazuyuki

SHIMADA, MD, FJCC

Abstract ─────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────────
Objectives. To clarify the relationship between changes in redox balance and the development of newcoronary lesions in patients with coronary artery disease（CAD）.
Methods. We studied 82 CAD patients（70 males and 12 females, mean age 61.8 ± 9.2 years）who
underwent repeated coronary angiography within 1 year after percutaneous coronary intervention. Levelsof serum lipid peroxide, erythrocyte glutathione peroxidase activity, and the redox state of erythrocyte（ratio of reduced to oxidized glutathione, the GSH/GSSG ratio）were measured at the time of follow-up
coronary angiography. According to the development of significant stenotic lesions, we divided thepatients into two groups : 57 patients without the development of new stenotic lesions（group A）and 25
patients showing new significant stenotic lesions within 1 year
（group B）.
Results. The serum lipid peroxide level in group B was significantly higher than those of group A（2.61 ± 0.32 vs 1.74 ± 0.16 nmol/ml, p ＜ 0.01）. Erythrocyte glutathione peroxidase activity did not differ
significantly between two groups. The erythrocyte GSH/GSSG ratio in group B was significantly lower
than that of group A（83 ± 9.6 vs 126 ± 7.3, p ＜ 0.01）. The sensitivity and specificity of GSH/GSSG ratio
to detect CAD patients with the development of significant coronary stenosis were 80.0% and 61.4%,
respectively.
Conclusions. CAD patients who showed development of new coronary lesions within 1 year haveincreased oxidative stress and imbalanced erythrocyte redox state. The GSH/GSSG ratio, an indicator for
redox balance, could be a useful marker to identify high-risk CAD patients.
──────────────────────────────────────────────────────────────────────────────────────────────────────────────────────J Cardiol 2006 Oct ; 48
（4）
: 183−191

Key Words
Stress oxidative
Atherosclerosis
Antioxidants enzyme
Interventional cardiology percutaneous coronary intervention
Coronary artery disease

INTRODUCTION
A growing body of evidence indicates that reactive oxygen species（ROS）play an important role inthe development of cardiovascular diseases such as
atherosclerosis,1）heart failure,2−4）and hyperten-sion.5,6）Oxidative stress is defined as an imbalance
between production and degradation of ROS, suchas the superoxide radical, hydrogen peroxide, thehydroxyradical, peroxinitrite and lipid peroxide（LPO）. LPO, one of the free radicals generated inhuman tissues, may be important in the develop-──────────────────────────────────────────────
自治医科大学 循環器内科 : 〒 329−0498 栃木県下野市薬師寺 3311−1
Department of Cardiology, School of Medicine, Jichi Medical University, Tochigi
Address for correspondence : HOJO Y, MD, Department of Cardiology, School of Medicine, Jichi Medical University, Yakushiji
3311−1, Shimotsuke, Tochigi 329−0498 ; E-mail : yhojo@jichi.ac.jp
Manuscript received February 8, 2006 ; revised June 16, 2006 ; accepted July 14, 2006

183

184

Tsuru, Hojo, Gama et alment of liver disease, atherosclerosis, degeneration
and aging. 7−10）To eliminate ROS, a number ofantioxidant enzymes such as superoxide dismutase,catalase and various reductases have been developed in higher organisms. Among antioxidant
enzymes, glutathione peroxidase（GPX）has a critical role in quenching LPO.
In mammalian tissues, GPX and glutathione constitute a major antioxidant defense system. The
reduced form of glutathione（GSH）
is a major cellular antioxidant that provides a proton（H ＋）
for GPX
to reduce LPO and hydrogen peroxide（ H 2O 2）.
During this process, GSH is converted to oxidized
glutathione（ GSSG）, and GSSG is reduced into
GSH by glutathione reductase（GR）receiving H ＋
from β-nicotinamide adenine dinucleotide phosphate（NADPH）and is recycled in the glutathione
system（Fig. 1）. Excess oxidative stress causes
accumulation of GSSG, leading to a decrease in the
GSH/GSSG ratio.
In the clinical situation, we often observe development of new coronary lesions in patients withcoronary artery disease（CAD）. Development ofcoronary stenosis may induce a recurrence of
myocardial ischemia/infarction, critical ventriculararrhythmia, acute pump failure, and sudden cardiac
death. Thus, the disease progression may be a critical problem in patients with CAD even after treatment with drug-eluting stents. In addition, we
sometimes experience patients with rapid development of stenotic lesions in coronary arteries within
1 year after diagnosis.11）To date, there are few clinical markers to identify these high-risk CAD
patients.
In this study, we investigated the relationshipbetween markers of redox system and the development of new lesions in CAD patients who had
already undergone percutaneous coronary intervention（PCI）. We found increased oxidative stress andimbalance of oxidant/antioxidant system in patientswith CAD who showed new stenotic lesions within
1 year after PCI.
SUBJECTS AND METHODS
Subjects
This study complied with the Declaration of
Helsinki. The Ethics Committee of Jichi Medical
University approved the protocol of this study. All
patients enrolled in this study gave informed consent. Patients examined by coronary angiography in
our university hospital between October 2002 andFig. 1 Glutathione system
Glutathione, a tripeptide（γ-glutamylcysteinylglycine）,
is a major cellular antioxidant. Reduced glutathione（GSH）provides protons（H ＋）for glutathione peroxidase（GPX）that reduces hydrogen peroxide（H2O2）andlipid peroxide（LOOH）to H2O and LOH, respectively.
During this process, GSH becomes oxidized to glutathione（GSSG）. Then GSSG is reduced by glutathione
reductase and nicotinamide adenine dinucleotide phosphate（NADPH）. When cells are exposed to oxidative
stress, the ratio of GSH/GSSG decreases as a consequence of GSSG accumulation.

April 2005 were included in this study. We selectedpatients who had already undergone PCI of thecoronary arteries within 1 year. We identified 82
patients who had undergone second coronary
angiography for routine follow-up or examination
for the recurrence of symptoms during the study
period.
Blood collection
Cardiac catheterization was performed according
to the standard diagnostic procedure. Under 1%
lidocaine hydrochloride local anesthesia, thefemoral or radial artery was cannulated with a 5 Frsheath. Heparin（3,000 U）was administered intravenously before coronary angiography. Nonioniccontrast media（ioxaglate）was used for all patients.
The coronary stenosis was accurately evaluated by
selective coronary angiography. Stenotic lesions in
the coronary arteries treated by PCI（PCI-related
stenosis）were excluded from the study. Patientswith chronic renal failure, liver dysfunction, malignancy, and hematological disorders were also
excluded from the study.
Arterial blood samples were obtained through a 5
Fr-catheter from the radial or femoral artery. Afterthe initial 3 ml of blood was discarded, blood samples were gently obtained with syringes containing
EDTA. Blood was centrifuged and the plasma was
J Cardiol 2006 Oct; 48（4）: 183 – 191

Redox Imbalance in Coronary Artery Diseasestored at − 80 °C until the assay. Erythrocyte GPX
activity was measured as previously described.12）
Briefly, red blood cells were washed with ice-cold
physiological saline three times and 5μl of thesample was lysed into 250μl of distilled water.
Samples were then frozen until the assay. Our preliminary study revealed that freezing did notchange GPX activity（data not shown）.
Erythrocyte lysate, GSH, GR, and NADPH weremixed together in a 96-well plate. Enzyme reaction
was initiated by adding t-butyl hydroperoxide andabsorbance at 340 nm was monitored for 3 min at
30 °C. Change in absorbance at 340 nm from 1 to 3
min was measured and GPX activity was calculated. Serum LPO levels were measured by the colorimetric method as described previously using
methylcarbamoyl 1-3,7-dimethylamino-10 H-phenothiazine.13）The redox state of erythrocyte（theGSH/GSSG ratio）was measured by 5,5-dithio-bis（2-nitrobenzoic acid）-enzyme recycling assay（GSH/GSSG-412 assay kit, Oxis Research）according to the manufacturer s instructions. The % coefficient of variance values of intra- and inter-assay
kit were 3.11 and 3.18, respectively. Quantitative
coronary angiography（ QCA）was performed by
QCA-CMS software（MEDIS）.
Statistical analysis
Data are expressed as mean ± SEM unless otherwise indicated. Comparisons between the twogroups were analyzed by unpaired t-test. Differenceof distribution was analyzed by χ square test.
Changes in stenosis rate of coronary arteries
between two groups were analyzed by two-way
ANOVA with repeated measurement. Multivariate
stepwise regression analysis was performed by
Stat-View version 5.0（SAS Institute）. Receiveroperating characteristic（ROC）curve analysis was
performed by MedCalc（MedCalc Software, version
8.0.2.0）. Values of p ＜ 0.05 were considered significant.
RESULTS
Profiles of study subjects
The 82 CAD patients included 70 males and 12
females aged from 36 to 79［mean 61.8 ± 9.2（SD）］years. Fifty six patients had hypertension
（68%）
, 29
patients had diabetes（35%）, 49 patients had hyperlipidemia（60%）, 47 patients were current smokers（57%）, and 18 patients had a family history ofCAD（22%）. The baseline coronary artery diseases
J Cardiol 2006 Oct; 48（4）: 183 – 191

185

were acute coronary syndromes in 16 patients andstable angina pectoris in 66, including 41 patientswith one vessel, 31 with two vessel and 10 withthree vessel disease. Two patients had received
coronary bypass surgery. Forty two（51%）patients
had old myocardial infarction. Left ventricular ejection fraction ranged from 23 to 84%（mean 61.5 ±
1.6%）.
Comparison between patients with or without
progression of coronary stenosis
According to the development of coronary stenosis, we divided the patients into two groups : 57
patients without development of new lesions（groupA）
and 25 patients showing new significant stenotic
lesions［ over 75% by the American Heart
Association（ AHA）classification］within 1 year
（group B）. Table 1 shows the clinical characteristics of the two groups. Mean observation period
was not significantly different between two groups.
Age, distribution of sex, incidence of major coronary risk factors, severity of the CAD, left ventricular ejection fraction, and incidence of previous
myocardial infarction were not significantly different between the two groups. Body mass index ingroup B was significantly higher than that in group
A. As shown in Table 2, systolic and diastolicblood pressure, total cholesterol, high-density
lipoprotein cholesterol, low-density lipoproteincholesterol, fasting blood glucose, and hemoglobin
A1c levels, and duration of diabetes mellitus were
not significantly different between the two groups.
High sensitive C-reactive protein level in group B
was significantly higher than that in group A. The
number of patients taking angiotensin receptor
blocker（ ARB）/angiotensin converting enzyme（ACE）inhibitor in group A was significantly higher than that in group B.
Table 3 summarizes the changes in stenosis of
PCI-treated lesions and new lesions analyzed by
QCA. Sixty two lesions were treated in group A
and 25 in group B. There was no significant difference in changes in stenosis of PCI-treated lesions
between the two groups（F ＝ 1.26, p ＝ 0.26）.
As shown in Fig 2, the mean serum LPO level ingroup B was significantly higher than that in groupA（2.61 ± 0.32 vs 1.74 ± 0.16 nmol/ml, p ＜ 0.01）.
The mean erythrocyte GPX activity did not differ
significantly between groups A and B（2.06 ± 0.08
vs 2.00 ± 0.08 U/g protein, p ＝ 0.66）. The meanerythrocyte GSH/GSSG ratio in group B was sig-

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Tsuru, Hojo, Gama et al

Clinical characteristics of the study subjects（1）Table 1

Group A
（n＝57）

Group B
（n＝25）

Observation period（months）

7.1±2.9

6.1±2.6Age（yr）

61.7±8.5

62.0±10.8

Sex（male/female）

52/5

18/7

Body mass index（kg/m2）24.0±0.4

25.4±0.7＊

Hypertension

40
（70%）

16（64%）

Diabetes mellitus

19
（33%）

10（40%）

Hyperlipidemia

31
（54%）

18（72%）

Current smokers

35
（61%）

12（48%）

Family history of coronary artery disease

13
（23%）

5（20%）

1/2/3 vessel disease

34/16/7

7/15/3

Left ventricular ejection fraction（%）

62.1±1.960.3±3.1

Previous myocardial infarction

29（51%）

13（52%）

Continuous values are mean±SEM. ＊p＜0.05 vs group A.
Group A : Patients with coronary artery disease without progression of coronary atherosclerosis.
Group B : Patients with coronary artery disease with progression of coronary atherosclerosis.

Table 2Clinical characteristics of the study subjects（2）Group A
（n＝57）

Group B
（n＝25）

Systolic

135±3.1

130±1.9

Diastolic78±1.971±3.3

191±4.9202±12.0

Blood pressure（mmHg）

Total cholesterol（mg/dl）
Triglyceride（mg/dl）

134±10.5160±24.7

High-density lipoprotein cholesterol（mg/dl）

47.6±1.7

46.6±2.0

Low-density lipoprotein cholesterol（mg/dl）

120±4.3

131±9.5

Fasting blood glucose（mg/dl）

129±6.5131±10.6

Hemoglobin A1c（%）

6.0±0.16

6.1±0.24

History of diabetes mellitus（yr）

2.0±0.52

3.1±1.2

1,439±420

4,940±2,199＊

Beta blockers

25（44%）

10（40%）

ARB/ACE inhibitors†
42（74%）

11（44%）

Calcium blockers

23（40%）

14（56%）

Statins30（52%）

16（64%）High sensitive C-reactive protein（ng/ml）
Medication

Continuous values are mean±SEM. ＊p＜0.05 vs group A. †p＜0.05 vs group B.
ARB＝angiotensin receptor blocker ; ACE＝angiotensin converting enzyme.

nificantly lower than that in group A（83 ± 9.6 vs
126 ± 7.3, p ＜ 0.01）.
Relationship between the GSH/GSSG ratio andcoronary risk factors
To examine the factors that affect the erythrocyte
GSH/GSSG ratio, we performed multivariate step-

wise regression analysis to examine the factors that
affect the GSH/GSSG ratio. We set the GSH/GSSG
ratio as a responsible parameter and age, sex, severity of CAD（the number of stenotic major coronary
arteries）, left ventricular ejection fraction, bloodpressure, total cholesterol, fasting blood sugar,
hemoglobin A1c, smoking, body mass index and the
J Cardiol 2006 Oct; 48（4）: 183 – 191

Redox Imbalance in Coronary Artery Disease

drugs received（β blocker, ARB/ACE inhibitors,
calcium blockers and statins）as explanatory variables. In this model, administration of ARB/ACE
inhibitors was a significant independent predictorof the GSH/GSSG ratio（F ＝ 7.64, p ＝ 0.007, r2 ＝0.083,β＝＋ 0.31）.

A

ROC curve analysis
We found increased LPO levels and decreased
GSH/GSSG ratio in CAD patients with progressive
coronary lesions within 1 year after PCI. To analyze whether these markers can be used to detect
these high-risk CAD patients, we performed ROC
curve analysis. As shown in Table 4, the
GSH/GSSG ratio predicted progression of coronary
atherosclerosis in CAD patients within 1 year witha sensitivity of 80.0% and specificity of 61.4%.
Serum LPO level also predicted the progression of
new coronary lesions, but with lower sensitivity（72.0%）and higher specificity（ 64.9%）than the
GSH/GSSG ratio. The erythrocyte GPX levels predicted progression of disease with a high sensitivity
Table 3187

B

C

Changes in stenosis of coronary artery lesions
analyzed by quantitative coronary angiography
Group A
（n＝57）

Group B
（n＝25）

62

25

Number of PCI-treated lesions
PCI-treated lesions
Before PCI（%）

91.0±1.594.2±1.9

Follow-up CAG（%）

26.0±1.9

16.1±5.8

Fig. 2 Comparison of redox parameters between two
groups of patients with coronary artery disease＊p ＜ 0.01 vs group A. † p ＜ 0.05 vs group A.
A : Serum level of lipid peroxide in group B was significantly higher than that in group A .
B : Erythrocyte glutathione peroxidase activity did not
differ significantly between groups A and B.
C : Ratio of reduced to oxidized glutathione（GSH/GSSG ratio）in group B was significantly lower
than that in group A.
Abbreviations as in Fig. 1.

New lesions
Before PCI（%）

37.5±5.0

Follow-up CAG（%）

85.6±2.6

Continuous values are mean±SEM.
PCI＝percutaneous coronary intervention ; CAG＝coronary
angiography.

Table 4

Results of receiver-operating characteristic curve analysis

Parameters

Cut off value

Sensitivity

Specificity

Area under curve

GPX（U/g protein）

1.73

88.0%

29.8%

0.522

LPO
（nmol/ml）

1.60

72.0%

64.9%

0.696

GSH/GSSG

107.4

80.0%

61.4%

0.728

LPO＝lipid peroxide. Other abbreviations as in Fig. 1.

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Tsuru, Hojo, Gama et al

GSH/GSSG
LPO
GPX

Fig. 3 Receiver-operating characteristic curve analysisof redox parameters
Receiver-operating curve analysis of redox parameters
for the detection of new significant stenotic lesions.
Horizontal axis indicates values of 100 − specificity（%）. Vertical axis shows values of sensitivity（%）.
Abbreviations as in Fig. 1.

（88.0%）but a low specificity（29.8%）after 1 yearof PCI. The highest area under the curve wasobserved in the ROC curve when the GSH/GSSG
ratio was used for the parameter. Fig. 3 shows the
ROC curves of GSH/GSSG, LPO and GPX.
DISCUSSION
The present study investigated changes in redox
states in patients with CAD. We hypothesized that
CAD patients with progression of coronary atherosclerosis（ progressors ）have imbalanced redox
state. Serum total cholesterol level was similar
between the two groups, but progressors showedelevated serum LPO levels and their erythrocytes
were more oxidized. These results suggest that disorder of redox balance is important in the progression of coronary atherosclerosis. The GSH/GSSG
ratio may be a simple and useful marker to detect
high-risk CAD patients such as progressors. The
results of multivariate regression analysis showedthat receiving ARB/ACE inhibitors increases theerythrocyte GSH/GSSG ratio, suggesting that inhibition of the renin-angiotensin system makes thecellular redox state less oxidative. The present
study showed that the administration of ARB/ACE
inhibitors was more common in non-progressors
than in progressors. Angiotensin Ⅱ activates
NADPH oxidase, a ROS-producing enzyme, in
vascular smooth muscle cells. 14）Thus, a more

active renin-angiotensin system generates excess
ROS that makes the cellular redox state oxidative.
Although there is still no evidence that inhibition of
the renin-angiotensin system prevents the progression of atherosclerosis, population-based studies
might disclose the benefit of ARB/ACE inhibitors
in the near future.
Our results suggest that regulation of the cellular
redox state is critical to prevent rapid progressionof atherosclerosis. Inhibition of the reninangiotensin system might be one method to makethe cellular redox state less oxidative by decreasing
ROS generation. In vitro experiments showed that
N-acetylcysteine is a potent substance to reduce
intracellular glutathione. However, there are still noestablished drugs to regulate the cellular redox
state. Our preliminary data indicate that anti-oxidant vitamins such as vitamin C and vitamin E did
not change the cellular redox state（ data notshown）. In fact, there is no evidence that antioxidant vitamins reduce cardiovascular events. 15）
Exercise increases antioxidant enzyme activities,16）
so physical training is one method to modulate thecellular redox state.
In clinical situations, we often experiencepatients with rapid progression of coronary atherosclerosis, sometimes occurring within months. The
present study tried to detect these high-risk CAD
patients because progression of coronary atherosclerosis may induce critical ischemic events.
Serum LPO levels are increased in patients with
CAD.17,18）The present study clarified that progressors are exposed to excess oxidative stress and consequently their cellular redox state is more oxidized
than that of non-progressors. Experimental studies
have also shown that intracellular signal transduction is enhanced when cells are oxidized.19）In fact,
progressors showed significantly higher high sensitive C-reactive protein levels. Thus the inflammatory responses induced by atherogenic stimuli are
more pronounced if cells or tissues are more oxidized. Therefore, vascular tissues in progressors arethought to be susceptible to inflammatory stimuli
and this inflammatory response may induce the progression of stenosis.20）In addition, the redox statesof macrophages are critical role in their activation,
which initiates the formation of atheromatous
plaques in blood vessels.21−23）
A number of studies have reported decreased
GPX activity in erythrocytes or platelets in CAD
patients.24−28）Recently, decreased erythrocyte GPX
J Cardiol 2006 Oct; 48（4）: 183 – 191

Redox Imbalance in Coronary Artery Diseaseactivity was independently associated with anincreased risk of cardiovascular diseases.29）In thepresent study, we did not find a significant difference in erythrocyte GPX activity between progressors and non-progressors. We have alreadyobserved decreased erythrocyte GPX activity of
CAD patients with disease progression more than 1
year after the first PCI（unpublished observation）.
We speculate that progression of coronary atherosclerosis is more closely related to oxidative stressthan changes in the antioxidant system within 1
year after PCI. In contrast, the decreased antioxidant system provides a greater contribution to disease progression after the first year of PCI. Thus
disease mechanisms might be different within andafter 1 year. The increased oxidative stress might
accelerate accumulation of macrophages to atheromatous plaques, transformation of vascular smoothmuscle cells and rapid proliferation of plaque composing cells, leading to the progression of atherosclerosis. In contrast, cellular antioxidant enzyme
might have a central role in the prevention ofchronic progression of atherosclerosis caused byaging. The balance of redox states appears to havecritical roles in both situations. Previously, we
reported that GSH/GSSG ratio is related to left ven要

tricular function after acute myocardial
infarction.30）It is possible that cellular redox stateis critical in the prognosis of cardiovascular diseases. Interestingly, changes in stenosis in PCItreated lesions were not dependent on the cellular
redox state. The clinical application of the drug
eluting stent might be responsible for these results.
This study investigated the relationship between
disease progression and redox markers in a retrospective design. We should investigate the relationship between redox parameters and the prognosis of
CAD patients in a prospective study with a larger
population. We still do not know the relationshipbetween redox imbalance and atheromatous coronary plaque rupture, fatal arrhythmic events, anddeterioration of heart failure in CAD patients.
Further studies are needed to clarify these points.
The results of the present study suggest that therapies reducing serum lipid levels and controlling
redox balance may improve the prognosis of highrisk CAD patients.
Acknowledgement
We thank Ms. Takako Takagi for her excellent technical assistance. We also thank David Lee and Michel B. Tsang for fruitful
discussion.

約病変進行を示す冠動脈疾患患者における酸化還元状態の不均衡

目

都留

利恵

北條

行弘

蒲

幸

水 野

修

勝木

孝明

島田

和幸

的 : 冠動脈疾患患者において新規冠動脈病変の進行と酸化還元状態の不均衡との関連を明ら

かにする．
方

法 : 経皮的冠動脈形成術を施行後，1 年以内に再冠動脈造影を受けた冠動脈疾患患者 82 例

（男性 70 例，女性 12 例，平均年齢 61.8 ± 9.2 歳）を対象とした．血清過酸化脂質と，再冠動脈造影の際，赤血球グルタチオンペルオキシダーゼ活性および赤血球酸化還元状態の指標である還元型/
酸化型グルタチオン比（GSH/GSSG 比）を測定した．新規病変進行の有無により対象を 2 群に分けた（A 群 : 病変進行のみられなかった患者 57 例，B 群 : 新規有意狭窄病変が出現した患者 25 例）．
結

果 : B 群の血清過酸化脂質値は A 群よりも有意な高値を示した（2.61 ± 0.32 vs 1.74 ±0.16 nmol/ml，p ＜ 0.01）．赤血球グルタチオンペルオキシダーゼ活性では 2 群間で有意な差はな
かった．B 群の赤血球 GSH/GSSG 比は A 群と比べ有意な低値を示した（83 ± 9.6 vs 126 ± 7.3，p ＜0.01）．新規冠動脈病変出現検出における GSH/GSSG 比の感度および特異度はおのおの 80.0% と61.4% であった．
結論 : 新規病変が出現する冠動脈疾患患者では赤血球酸化還元状態の不均衡がみられた．酸化

還元状態の指標である GSH/GSSG 比はこのようなハイリスク冠動脈疾患患者の検出に有用である

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189

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