Oxidative Stress Markers and the Risk of Incident Stroke and Ischemic Heart Disease: A Case-Cohort Study

Article information

J Stroke. 2026;28(2):273-282

Publication date (electronic) : 2026 May 8

doi : https://doi.org/10.5853/jos.2025.05162

Shuai Guo¹, Hitomi Kimura², Kazumasa Yamagishi^,1,3, Tomomi Kihara^1,4, Isao Muraki¹, Yoshihiro Kokubo⁵, Isao Saito⁶, Hiroshi Yatsuya⁷, Hiroyasu Iso², Taiki Yamaji^,8, Manami Inoue⁹, Shoichiro Tsugane^9,10, Norie Sawada⁹, Motoki Iwasaki^8,9, the JPHC Study Group

¹Department of Public Health Medicine, Institute of Medicine, and Health Services Research and Development Center, University of Tsukuba, Tsukuba, Japan

²Institute for Global Health Policy Research, Bureau of Global Health Cooperation, Japan Institute for Health Security, Tokyo, Japan

³Department of Public Health, Juntendo University Graduate School of Medicine, Tokyo, Japan

⁴Division of Public Health, Department of Planning and Coordination, Bureau of Health Security and Management, Japan Institute for Health Security, Tokyo, Japan

⁵Department of Preventive Cardiology, National Cerebral and Cardiovascular Center, Suita, Japan

⁶Department of Public Health and Epidemiology, Faculty of Medicine, Oita University, Yufu, Japan

⁷Department of Medicine, Graduate School of Medicine, Nagoya University, Nagoya, Japan

⁸Division of Epidemiology, National Cancer Center Institute for Cancer Control, Tokyo, Japan

⁹Division of Cohort Research, National Cancer Center Institute for Cancer Control, Tokyo, Japan

¹⁰Graduate School of Public Health, International University of Health and Welfare, Tokyo, Japan

Correspondence: Kazumasa Yamagishi Department of Public Health Medicine, Institute of Medicine, and Health Services Research and Development Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan Tel: +81-298532695 E-mail: yamagishi.kazumas.ge@u.tsukuba.ac.jp

Co-correspondence: Taiki Yamaji Division of Epidemiology, National Cancer Center Institute for Cancer Control, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan Tel: +81-0335475201 E-mail: tyamaji@ncc.go.jp

Received 2025 October 14; Revised 2026 February 6; Accepted 2026 February 24.

Abstract

Introduction

Oxidative stress, a process that occurs when the production of reactive oxygen species (ROSs) exceeds the antioxidant capacity of the body [1], plays a pivotal role in the development of inflammation, hypertension, and atherosclerosis [2,3]. These conditions are key mechanisms underlying the pathogenesis of cardiovascular disease (CVD). Over recent years, the role of oxidative stress in the pathophysiology of CVD has been well established, providing a theoretical foundation for identifying biomarkers of oxidative stress as potential tools for the early detection of CVD [4].

Numerous markers have been developed to evaluate oxidative stress including markers of lipid peroxidation (isoprostane and malondialdehyde) [5,6], and markers of DNA and RNA oxidation (8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine) [7]. Although these markers have been used in some clinical studies [5-7], the specific measurement of such biomarkers in large population studies is currently challenging due to the technical complexity and cost of the procedures involved [8,9]. Recently, two easy and low-cost assays for oxidative stress biomarkers have been developed: the derivatives of reactive oxygen metabolites (d-ROMs) test and the biological antioxidant potential (BAP) test [10-12]. The d-ROMs test measures the total amount of hydroperoxides, metabolic by-products formed when ROS oxidize essential biomolecules such as lipids, proteins, amino acids, and nucleic acids. This test has been widely utilized in clinical studies to investigate the association between oxidative stress and the prognoses of patients with CVD [13,14], diabetes [15], and colorectal cancer [16]. The BAP test evaluates the capacity of a plasma sample to reduce ferric ions, thus providing a simple, rapid, and cost-effective measurement of antioxidant capacity [17]. The efficiency and practicality of the d-ROMs and BAP tests render then popular tools for clinical research [18-20]. Several studies have investigated associations between d-ROMs, BAP and the risk factors for CVD, including visceral fat and the thickness of the intimamedia in the carotid artery [18,21]. Only one study, utilizing a nested case-control design, reported a positive association between d-ROMs levels and the risk of myocardial infarction (MI) and stroke [22]. These authors found that the levels of total thiol, a marker of redox control status, were inversely associated with the risk of total stroke in healthy European populations [22]. However, the authors did not investigate the association between d-ROMs, thiol levels and different subtypes of stroke. Furthermore, researchers have yet to investigate for an association between BAP levels and the risk of CVD.

In the present study, we investigated the association between plasma markers of oxidative stress and the risk of incident stroke, including different subtypes of stroke (ischemic stroke, subarachnoid hemorrhage, and intraparenchymal hemorrhage), and ischemic heart disease (IHD). This study was conducted in a healthy Japanese population using a case cohort design and employed two oxidative stress biomarkers: d-ROMs and BAP.

Methods

Study population and design

The Japan Public Health Center-based prospective study (JPHC study) is a large-scale prospective study conducted across 11 public health center areas in Japan and consists of two cohorts initiated in 1990 (cohort I) and 1993 (cohort II). At baseline, 140,420 residents aged 40–69 years participated in the survey. Questionnaires on lifestyle and dietary habits were distributed to the participants. In addition, blood samples were acquired and health check-up information was collated. During the follow-up period, the participants were monitored for relocation, vital status, the occurrence of cancer, and CVD (stroke and IHD). Specific details of the JPHC study have been published elsewhere [23].

Following a 5-year interval, a follow-up survey was conducted in 1995 for cohort I and 1998–1999 for cohort II, during which questionnaires, blood samples, and health check-up information were collected. A case-cohort study design was used in this follow-up survey to investigate the associations between d-ROMs, BAP, and incident CVD (Figure 1). Participants were restricted to a base cohort of 29,423 participants (10,672 males and 18,751 females) in nine out of 11 public health center areas, and two metropolitan public health center areas were excluded because information relating to incident CVD was not collected in these areas. In this follow-up survey, participants completed questionnaires and provided samples of venous blood. In the source cohort, 1,805 incident CVD cases (stroke, n=1,497; IHD, n=331) occurred during the follow-up period. After excluding participants with a self-reported history of stroke (n=60), MI (n=45), or angina (n=50); those with occurrences of stroke or IHD before the follow-up survey (n=126); and those without the necessary d-ROMs or BAP data (n=3), 1,521 cases were retained.

Figure 1.

Flow diagram for the exclusion criteria. JPHC, Japan Public Health Center; PHC, Public Health Center; CVD, cardiovascular disease; IHD, ischemic heart disease; MI, myocardial infarction; d-ROMs, derivatives of reactive oxygen metabolites; BAP, biological antioxidant potential.

We randomly selected 4,946 participants of the source cohort (17% of the source cohort population) as a sub-cohort. After excluding participants with a self-reported history of stroke (n=30), MI (n=25), angina (n=72), stroke, or IHD before the follow-up survey (n=54), as well as those without the necessary d-ROMs or BAP data (n=4), 4,761 participants remained in the sub-cohort. The final case cohort study included 4,761 sub-cohort participants and 1,521 participants with incident stroke or IHD (244 participants were included in the sub-cohort).

Results

Compared with the sub-cohort participants, those who developed stroke or IHD tended to be older, male, current smokers, and to consume ≥150 g of ethanol per week (Table 1). Additionally, these participants were more likely to have a history of hypertension. Participants who developed stroke were less likely to walk and consume fruit, had a history of diabetes mellitus, and had higher d-ROMs values. Those who developed IHD were less likely to consume vegetables.

Table 1.

Baseline characteristics of cases of stroke and ischemic heart disease, and the sub-cohort in the JPHC study

Tables 2 and 3 present sex-specific baseline characteristics of the sub-cohort participants according to d-ROMs and BAP quartiles, respectively. Males and females in the highest d-ROMs quartile tended to be older and to consume ≥150 g of ethanol per week. These participants were also less likely to consume coffee on a daily basis. When considering males, those in the highest quartile had a lower BMI, were more likely to be current smokers, and had a history of hypertension. When considering females, those in the highest quartile had a higher BMI, were more likely to receive treatment for hypercholesterolemia, and were less likely to drink green tea on a daily basis. In contrast, males and females in the highest BAP quartile tended to be younger, consumed more fruit, and were less likely to be current smokers or daily coffee drinkers. They also had a higher BMI. Males in the highest BAP quartile were more likely to receive treatment for hypercholesterolemia and women were more likely to engage in exercise at least once a week.

Table 2.

Sex-specific characteristics of sub-cohort participants according to d-ROMs quartiles in the JPHC study, 1995–1999

Table 3.

Sex-specific characteristics of sub-cohort participants according to BAP quartiles in the JPHC study, 1995–1999

With a median follow-up of 14.1 years, 1,271 strokes occurred (1,061 outside and 210 within the sub-cohort), including 781 ischemic strokes (647 outside and 134 within the sub-cohort), 146 subarachnoid hemorrhages (128 outside and 18 within the sub-cohort), and 340 intraparenchymal hemorrhages (282 outside and 58 within the sub-cohort). In addition, 265 IHD cases were identified (229 outside and 36 within the sub-cohort).

As shown in Table 4, d-ROMs were positively and linearly associated with the risk of total stroke, ischemic stroke, IHD, and the composite outcome of ischemic stroke and IHD but not with subarachnoid hemorrhage or intraparenchymal hemorrhage (Model 2). Positive associations were consistently observed across subtypes of ischemic stroke (Supplementary Table 1). Further adjustment for potential mediators, including a history of hypertension, diabetes, and treatment for hypercholesterolemia, in Model 3 did not materially alter the associations.

Table 4.

Hazard ratios and 95% confidence intervals for stroke, stroke subtypes, and ischemic heart disease, according to d-ROMs quartiles (Q) in the JPHC study

BAP levels were not associated with the risk of CVD outcomes (Table 5 and Supplementary Table 2).

Table 5.

Hazard ratios and 95% confidence intervals for stroke, stroke subtypes, and ischemic heart disease, according to BAP quartiles (Q) in the JPHC study

In this study, d-ROMs and BAP were used to evaluate oxidative and antioxidant capacities, respectively. Because oxidative balance is maintained in the body, each measure may be influenced by the other, and simultaneous adjustment for both parameters could obscure true associations. Therefore, d-ROMs and BAP were not included in the same primary model. Notably, including both parameters together did not alter our results (data not shown). In addition, given that the d-ROMs/BAP ratio may reflect the oxidative balance, we conducted additional analysis using the d-ROMs/BAP ratio; these data are shown in Supplementary Table 3. The associations between d-ROMs/BAP ratio and the risk of disease were almost identical to those between d-ROMs and risk; stronger associations were not expected.

Discussion

In this large-scale, population-based cohort study, we identified positive and linear associations between d-ROMs and the risks of total stroke, ischemic stroke, and IHD, but not hemorrhagic stroke. No significant association was found between BAP and the risk of any outcome. This is the first study to comprehensively evaluate the association between oxidative stress biomarkers and CVD risk in the general population.

Our findings are consistent with those of a previous nested case-control study conducted in a European population aged 45–85 years (476 MI cases and 2,380 controls; 454 stroke cases and 2,270 controls) [22]. The authors of this previous study also reported positive associations between d-ROMs levels and the risks of both outcomes, with multivariable HRs per 100 U.CARR increase in d-ROMs of 1.21 (95% CI: 1.05–1.40) for MI and 1.17 (95% CI: 1.01–1.35) for stroke [22].

Our present findings align with established mechanistic evidence that oxidative stress, as quantified by d-ROMs, drives atherosclerosis [28]. Notably, we detected a positive association between d-ROMs levels and both ischemic stroke and IHD, but not with subarachnoid or intraparenchymal hemorrhages. Oxidative stress is known to impair the functionality of vascular endothelial cells, platelets, and erythrocytes, thereby initiating a cascade of events that promote the development of atherosclerosis and thrombus formation [29,30]. In contrast to the occlusive nature of ischemic stroke and IHD, intraparenchymal hemorrhage typically arises from the acute rupture of structurally compromised small arteries [31], while subarachnoid hemorrhage primarily originates from the rupture of intracranial aneurysms [32]. Our present findings suggest that d-ROMs, as a biomarker of oxidative stress, may serve as a specific marker for identifying the risk of occlusive vascular events rather than of hemorrhagic stroke.

No associations were detected between BAP levels and the risk of CVD outcomes. The reason for this lack of association remains uncertain, although we anticipated an inverse association owing to potential antioxidant action. The effective action of most antioxidants depends on interactions between enzymatic and non-enzymatic systems. As the BAP assay primarily reflects non-enzymatic and low-molecular-weight antioxidants, it may not adequately capture antioxidant pathways that are relevant to atherosclerosis [33], potentially explaining the null association observed between BAP levels and the risk of CVD outcomes.

This study has several limitations that should be considered when interpreting our findings. First, although a previous study demonstrated that blood samples for d-ROMs and BAP analysis remained stable when stored at -80°C for up to 60 months [34], the plasma samples used in the present study were stored at -80°C for approximately 20 years. Prolonged storage may have led to autooxidation, potentially affecting the assessment of oxidative and antioxidant capacities. Specifically, autooxidation could have resulted in the underestimation of plasma antioxidant levels. However, a previous cross-sectional study of Japanese men and women with a mean age of 47±11 years reported mean±SD values of 321±63 U.CARR for d-ROMs and 2,829±247 μmol/L for BAP; in the present study, we achieved similar values (333±60 for U.CARR and 2,848±266 μmol/L for BAP). These findings suggest that the influence of long-term storage on auto-oxidation was smaller than expected. Second, we measured d-ROMs and BAP levels at a single time point; this may have led to exposure misclassification during follow-up. Because this potential misclassification is assumed to be non-differential, it would generally exert bias on the effect estimates towards null. Third, because the d-ROMs test reflects global oxidative status, this test cannot distinguish the specific contributions of lipid, protein, or nucleic acid oxidation to overall plasma oxidative stress, thus limiting mechanistic interpretation. Nevertheless, its simplicity and high levels of reproducibility make this test a valuable tool for the early identification of individuals with an elevated risk of CVD. Fourth, because this study was conducted solely in the Japanese population, the generalizability of the findings to other ethnic groups may be limited.

Despite these limitations, the present study had notable strengths. Long-term follow-up allowed the observation of more outcome events, thus enhancing the robustness of the analysis. Furthermore, the distinction between stroke subtypes enabled a more detailed exploration of the influence of oxidative stress on ischemic and hemorrhagic stroke.

Conclusions

Our analyses identified a positive association between d-ROMs levels and the risk of ischemic stroke and IHD, but not hemorrhagic stroke. Biomarkers related to oxidative status may be useful for the early risk identification of ischemic CVD.

Supplementary materials

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