Plasma succinate and the risks of cardiovascular events and recurrent stroke after ischemic stroke: A nested case-control study

To the Editor: Stroke is among the leading public health problems in China, with high rates of recurrent stroke, cardiovascular events and mortality, resulting in a considerable medical and economic burden.[1] The identification of new molecular markers and therapeutic targets is necessary to better refine the risk prediction of cardiovascular events and develop new treatment strategies for ischemic stroke. Succinate is a tricarboxylic acid (TCA) cycle intermediate and has received considerable attention for its involvement in various signaling pathways associated with cardiovascular diseases.[2–4] Recent studies have reported that succinate is a novel mediator that promotes atherosclerotic lesion progression, fibroblast activation, and apoptosis resistance. Experimental studies have suggested that the accumulation of succinate is a universal metabolic signature of ischemia in a range of tissues,[5] and the attenuation of succinate accumulation during cerebral ischemia by pharmacological inhibition could effectively ameliorate brain injury, decrease the volume of infarcted brain tissue, and prevent a decline in neurological and sensorimotor functions in murine models of cerebral ischemia.[6,7] However, population-based evidence of the prognostic significance of circulating succinate levels in patients with ischemic stroke is rare. We performed a nested case-control study within the China Antihypertensive Trial in Acute Ischemic Stroke (CATIS) to investigate the relationships of plasma succinate with cardiovascular events and recurrent stroke in patients with ischemic stroke.[8] CATIS is a multicenter randomized controlled trial which aimed to investigate the efficacy of immediate blood pressure (BP) reduction after acute ischemic stroke (NCT01840072). A total of 4071 patients with acute ischemic stroke in 26 hospitals across China from August 2009 to May 2013 were recruited. This study was approved by the ethical committees of Soochow University (No. 2007IRB2) and all the participating hospitals. All participants or their immediate family members provided written consent. The study participants were followed up at 24 months after stroke by trained neurologists and nurses, who were unaware of the baseline characteristics and treatment assignment. The primary endpoint was cardiovascular events after ischemic stroke, including vascular death, non-fatal stroke, non-fatal myocardial infarction, hospitalization and treatment for angina, congestive heart failure, and peripheral arterial disease. A total of 371 (9.1%) cardiovascular events were identified. We further excluded 68 cases without succinate data from the 371 cases. For each included case, we selected a control from the eligible participants who were free of recurrent cardiovascular events at 24 months. Cases and controls were matched by age (±1 year), sex, and treatment group. A total of 303 cardiovascular events (including 247 recurrent strokes) and 303 matched controls were finally included [Supplementary Figure 1, https://links.lww.com/CM9/C450]. Blood samples were collected within 24 h of patients’ hospital admission after at least 8 h of fasting. Serum and plasma samples were separated and immediately frozen at −80°C until analysis. Plasma succinate levels were measured via ultra-performance liquid chromatography coupled with a tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA). The measurement of plasma succinate was performed with stringent quality control by experienced technicians blinded to the baseline characteristics and clinical outcomes. Baseline data on demographic characteristics, clinical features, medical history were collected at admission using a standard questionnaire. Stroke severity was evaluated using the NIH Stroke Scale (NIHSS) by trained neurologists at hospital admission. Ischemic stroke was classified as large artery atherosclerosis (embolus/thrombosis), cardiac embolism (embolic), or small artery occlusion lacunae (lacunar) according to symptoms and imaging data.[9] We divided the participants into three groups according to the tertiles of plasma succinate levels, with cut-off points based on the distribution among controls. There were 171 participants (28.2%) in tertile 1 (T1), 215 participants (35.5%) in tertile 2 (T2), and 220 participants (36.3%) in tertile 3 (T3). Conditional logistic regression models were fit to evaluate the associations of plasma succinate with cardiovascular events and recurrent stroke. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) for the highest tertile compared with the lowest tertile and for 1-SD increments of log-transformed succinate. We assessed the predictive performance of plasma succinate by adding it to the basic model constructed with conventional risk factors for unfavorable outcomes after stroke. All P-values were two-tailed, and a significance level of 0.050 was used. Statistical analysis was conducted using SAS statistical software (version 9.4; SAS Institute, Cary, NC). Most baseline characteristics of the cases and controls were balanced [Supplementary Table 1, https://links.lww.com/CM9/C450]. Continuous variables are expressed as the means with standard deviation (SDs) or as medians with interquartile ranges (Q1–Q3), categorical variables are expressed as n (%). There were 396 men (65.3%) and 210 women (34.7%) with a mean age of 63.6 years (SD, 10.6 years) and a median succinate concentration of 6.5 μmol/L (IQR, 5.3–8.4 μmol/L; Supplementary Table 1, https://links.lww.com/CM9/C450). Plasma succinate was positively associated with cardiovascular events and recurrent stroke after ischemic stroke [Table 1]. Compared with those in the lowest tertile of succinate, the multivariable-adjusted ORs for the highest tertile cardiovascular events and recurrent stroke were 1.64 (95% CI: 1.05–2.57) and 1.66 (95% CI: 1.01–2.73), respectively. Each 1-SD increase in log-transformed succinate was associated with a 34% (95% CI: 11–62%) higher risk for subsequent cardiovascular events and a 44% (95% CI: 15–80%) for recurrent stroke. Restricted cubic spline analyses suggested linear associations of succinate levels with cardiovascular events and recurrent stroke (P for linearity <0.050 for both; Supplementary Figure 2, https://links.lww.com/CM9/C450). Table 1 - Associations of plasma succinate levels and risks of cardiovascular events and recurrent stroke after ischemic stroke. Items Tertiles of succinate P trend Per 1-SD increase in log-succinate T1 (n = 171) T2 (n = 215) T3 (n = 220) Cardiovascular events Median (μmol/L) 4.7 (4.2–5.2) 6.2 (5.8–6.8) 9.3 (7.9–11.8) Event (%) 70 (40.9) 115 (53.5) 118 (53.6) 0.017 303 (50.0) Model 1 1.00 1.65 (1.09–2.49) 1.62 (1.09–2.41) 0.021 1.36 (1.14–1.61) Model 2 1.00 1.65 (1.03–2.65) 1.64 (1.05–2.57) 0.040 1.34 (1.11–1.62) Recurrent stroke Median (μmol/L) 4.7 (4.2–5.2) 6.2 (5.8–6.8) 9.3 (7.9–11.8) Event (%) 60 (35.1) 88 (40.9) 99 (45.0) 0.049 247 (40.8) Model 1 1.00 1.31 (0.83–2.06) 1.52 (0.98–2.36) 0.063 1.26 (1.05–1.50) Model 2 1.00 1.45 (0.87–2.41) 1.66 (1.01–2.73) 0.050 1.44 (1.15–1.80) Data are expressed as n (%), or medians (Q1–Q3). Conditional logistic regression models were used to calculate the ORs (95% CIs). Model 1 was unadjusted. Model 2 was adjusted for current smoking, alcohol consumption, BMI, systolic and diastolic BP, HsCRP, history of hypertension and diabetes mellitus, use of antihypertensive and lipid-lowering medications, family history of stroke, ischemic stroke subtype, time from onset to hospital, and admission NIHSS scores. BMI: Body mass index; BP: Blood pressure; CI: Confidence interval; HsCRP: High-sensitive C-reactive protein; NIHSS: National Institutes of Health Stroke Scale; ORs: Odds ratios; SD: Standard Deviation. In this nested case–control study, we found that elevated baseline succinate levels were associated with an increased risk of both cardiovascular events and recurrent stroke, independent of stroke severity, medical history, and ischemic stroke subtype. In addition, plasma succinate offered substantial risk discrimination and reclassification improvements for cardiovascular events and recurrent stroke [Supplementary Table 3 and Supplementary Figure 3, https://links.lww.com/CM9/C450]. These findings suggest that succinate might serve as a prognostic predictor for ischemic stroke. Experimental evidence has shown that accumulated succinate during ischemia is localized to ischemic lesions in murine models of heart attack and stroke. Targeting succinate metabolism could produce multiple neuroprotective effects on brain injury and is considered as a novel therapeutic approach for ischemia reperfusion injury in ischemic stroke. This study provides prospective, population-based evidence supporting the prognostic significance of plasma succinate in the setting of acute ischemic stroke. Several biological mechanisms have been proposed, including inflammatory responses, oxidative stress, and energy metabolism. Hypoxia, ischemia, and subsequent reperfusion injury leading to endothelial dysfunction and vascular occlusion may also be the underlying mechanisms.[2,3] Our findings can help to refine future risk prediction of cardiovascular events and enhance the understanding of the pathogenesis of ischemic stroke; therefore, these findings have important clinical implications. It is of great clinical interest to determine whether targeting succinate homeostasis with specific inhibitors would improve cardiovascular outcomes after ischemic stroke. Several limitations should be considered when our findings are interpreted. First, the CATIS excluded patients with a BP of 220/120 mmHg or who were treated with intravenous thrombolytic therapy, so selection bias might be a concern. Second, owing to the observational study design, we could not establish a causal relationship, although multiple key confounders were controlled. Third, all participants were exclusively Chinese ischemic stroke patients, and the findings should be extrapolated to other populations with caution. In conclusion, elevated plasma succinate was associated with an increased risk of cardiovascular events and recurrent stroke and provided incremental prognostic value for risk discrimination and reclassification after ischemic stroke. Acknowledgments The authors thank the study participants and their relatives and the clinical staff at all participating hospitals for their support and contribution to this project. Funding This study was supported by the National Natural Science Foundation of China (Nos. 82273706 and 82220108001), the Suzhou Science and Technology Project (No. SYW2024067), Interdisciplinary Basic Frontier Innovation Program of Suzhou Medical College of Soochow University (No. YXY2302013), and the Project of MOE Key Laboratory of Geriatric Diseases and Immunology (No. JYN202406). Conflicts of interest None.