Abstract
Background
Trimethylamine N‐oxide (TMAO) contributes to cardiovascular disease through its prothrombotic, proatherothrombotic, and proinflammatory effects. We aimed to evaluate whether residual risk of recurrent stroke of TMAO and its precursor choline remain among patients who received dual‐antiplatelet therapy and intensive lipid‐lowering therapy and with a low inflammation level (high‐sensitivity C‐reactive protein <2 mg/L on admission).
Methods and Results
Patients with ischemic stroke or transient ischemic attack were enrolled from the CNSR‐III (Third China National Stroke Registry) in China. Plasma TMAO and choline concentrations at baseline were measured in 9793 participants using liquid chromatography–mass spectrometry. The primary outcome was a new stroke within 1 year. Multivariable‐adjusted hazard ratios were calculated using Cox regression models to investigate the associations of TMAO and choline with stroke recurrence. Among all patients, elevated TMAO and choline levels were associated with an increased risk of recurrent stroke (adjusted hazard ratios, 1.28 [95% CI, 1.12–1.45]; and 1.50 [95% CI, 1.32–1.71], respectively). Moreover, elevated TMAO and choline levels were associated with an increased risk of recurrent stroke among patients who received dual‐antiplatelet therapy (1.65 [95% CI, 1.28–2.13]; and 1.70 [95% CI, 1.32–2.19], respectively), intensive lipid‐lowering therapy (1.49 [95% CI, 1.15–1.94]; and 1.49 [95% CI, 1.15–1.92], respectively), with high‐sensitivity C‐reactive protein <2 mg/L (1.39 [95% CI, 1.14–1.69]; and 1.88 [95% CI, 1.53–2.30], respectively), and concurrently received dual‐antiplatelet therapy, intensive lipid‐lowering therapy and with high‐sensitivity C‐reactive protein <2 mg/L (3.57 [95% CI, 1.73–7.38]; and 2.19 [95% CI, 1.16–4.16], respectively).
Conclusions
TMAO and choline were risk factors for recurrent stroke independent of dual‐antiplatelet therapy, intensive lipid‐lowering therapy at discharge, and low inflammation on admission.
Keywords: (above vs. below the median), choline, ischemic stroke, residual risk, stroke recurrence, trimethylamine N‐oxide
Subject Categories: Ischemic Stroke, Transient Ischemic Attack (TIA)
Nonstandard Abbreviations and Acronyms
- CHANCE
Clopidogrel in High‐Risk Patients With Acute Nondisabling Cerebrovascular Events
- CNSR‐III
The Third China National Stroke Registry
- DAPT
dual‐antiplatelet therapy
- ILT
intensive lipid‐lowering therapy
- LoDoCo
Low Dose Colchicine
- TMAO
trimethylamine‐N‐oxide
- TOAST
the Trial of ORG 10172 in Acute Stroke Treatment
- TST
Treat Stroke to Target
Clinical Perspective.
What Is New?
Trimethylamine N‐oxide and choline were risk factors for recurrent stroke independent of dual‐antiplatelet therapy or intensive lipid‐lowering therapy at discharge and low inflammation on admission.
What Are the Clinical Implications?
Despite administration of evidence‐based secondary stroke prevention, trimethylamine N‐oxide and choline still contributed to residual risk of recurrent stroke, suggesting that intervention strategies to reduce trimethylamine N‐oxide and choline concentrations are needed in the future.
The microbiota‐dependent metabolite trimethylamine‐N‐oxide (TMAO) has been both clinically and mechanistically linked to cardiovascular disease (CVD). 1 , 2 , 3 TMAO synthesis begins with dietary precursors, such as choline, that are abundant in various foods. 4 , 5 Mechanistic studies in both animals and humans have revealed that TMAO contributed to its detrimental effects on CVD, through its prothrombotic, proatherothrombotic, and proinflammatory effects. 4 , 6 , 7 , 8 , 9 , 10 , 11 Elevated plasma levels of TMAO and choline were linked with an increased risk of incident cardiovascular events in patients with ischemic stroke. 5
Recent clinical trials have addressed the above 3 targets of TMAO as the main therapeutic targets—residual thrombotic, 12 residual cholesterol, 13 and residual inflammatory risk 14 —in reducing the residual risk of stroke recurrence. 15 However, in the coming era of dual‐antiplatelet therapy (DAPT), intensive lipid‐lowering therapy (ILT), and anti‐inflammation treatment, it remains unknown whether TMAO and its precursor choline still contribute to the residual risk of recurrent stroke.
The CNSR‐III (Third China National Stroke Registry) recruited patients with acute ischemic stroke and transient ischemic attack (TIA) in China. Herein, using the data from the CNSR‐III, we examined the association of TMAO and choline with the risk of stroke recurrence in the overall cohort and in patients who received DAPT and ILT and with a low inflammation level.
METHODS
Data Availability
The data that support the findings of this study are available from the corresponding author on reasonable request.
Study Design and Population
The CNSR‐III is a nationwide, prospective, multicenter, observational registry for patients presenting to hospitals with acute ischemic cerebrovascular events. Details of the registry database are available in our previous studies. 16 Briefly, consecutive patients were enrolled from 201 hospitals between August 2015 and March 2018 in China. In total, 15 166 patients were enrolled in CNSR‐III, of whom 93.3% had an ischemic stroke (n=14 146) and 6.7% had a transient ischemic attack (n=1020) within 7 days from the onset of symptoms to enrollment. Among the total 201 sites of the CNSR‐III, 171 sites participated in the prespecified gut microbial metabolites substudy.
Baseline Data Collection
The baseline data were collected prospectively using an electronic data capture system by face‐to‐face interviews. The following data were obtained from the registry database: age; sex; systolic blood pressure; body mass index; current smoking; medical history of hypertension, diabetes, coronary heart disease, or stroke; stroke type; stroke subtypes, classified as large‐artery atherosclerosis, and non–large‐artery atherosclerosis according to the TOAST (Trial of ORG 10172 in Acute Stroke Treatment) criteria; National Institutes of Health Stroke Scale score at admission; recombinant tissue plasminogen activator intravenous thrombolytic; laboratory test of low‐density lipoprotein cholesterol, high‐density lipoprotein cholesterol, total cholesterol, triglyceride, and hsCRP (high‐sensitivity C‐reactive protein); estimated glomerular filtration rate; and discharge medication of lipid‐lowering agents, antiplatelet agents, antihypertension agents, and dual‐antiplatelet agents; and metabolites of TMAO and choline. Administration of DAPT was defined as discharge medication of aspirin and clopidogrel treatment. Administration of ILT was defined as persistently receiving statin treatment during hospitalization (atorvastatin 40–80 mg or rosuvastatin 20–40 mg). Low inflammation level was defined as hsCRP <2 mg/L on admission.
Outcome Evaluation
The primary outcome is stroke recurrence (new ischemic stroke and hemorrhagic stroke). Vascular events were confirmed from the treating hospital, and death was confirmed on a death certificate from the attended hospital or the local civil registry.
Plasma Analysis
In this study, blood samples were collected from the 171 study sites that participated in the gut microbial metabolites substudy. Fasting blood samples were collected in serum‐separation tubes and EDTA anticoagulation blood collection tubes within 24 hours of admission. Blood samples were sent to the central laboratory to extract serum, plasma, and white blood cells. Estimated glomerular filtration rate was calculated according to the Chronic Kidney Disease Epidemiology Collaboration creatinine levels. Plasma levels of TMAO and choline were measured using liquid chromatography–mass spectrometry as in our previous studies. 17 The methods used for TMAO and choline measurement are described in Data S1 and Table S1 and S2.
Statistical Analysis
Continuous variables were presented in medians (interquartile ranges) and compared between groups using the nonparametric Kruskal–Wallis test. Categorical variables were presented as percentages and tested by chi‐square test. The primary outcome was the recurrence of stroke within 1 year. The time to all outcome events of each group was presented by using the Kaplan–Meier curves, and the differences between groups were tested by the log‐rank test. The association between TMAO levels and all outcomes during the 1‐year follow‐up period were examined by using the Cox proportional hazard model. Hazard ratios (HRs) with 95% CIs were calculated on univariable (unadjusted) Cox regression models. Adjusted HRs and their 95% CIs were calculated on multivariable Cox regression models. In the second model, we adjusted for age, sex, body mass index, current smoking, heavy drinking, medical history (hypertension, diabetes, stroke, and coronary heart disease), hsCRP, low‐density lipoprotein cholesterol, estimated glomerular filtration rate, National Institutes of Health Stroke Scale score at admission, TOAST subtype, and discharge medications (antiplatelet, antihypertension, hypoglycemic). We added a sensitivity analysis to further investigate the associations of TMAO and choline with recurrent ischemic stroke in the total population, in patients receiving DAPT and ILT and with a low inflammation level (hsCRP <2 mg/L), and in patients concurrently receiving DAPT and ILT and with well‐controlled hsCRP level. Two‐sided P<0.05 was considered statistically significant. The aforementioned statistical analyses were conducted with SAS software version 9.4 (SAS Institute, Inc., Cary, NC). The study protocol was approved by the ethics committees of Beijing Tiantan Hospital and the research board of each participating center according to the principles mentioned in the Declaration of Helsinki. Written informed consents were obtained from all patients or their legally authorized representatives before entering into the study.
RESULTS
Study Participants and Baseline Characteristics
Of the 1516 patients enrolled in the CNSR‐III, after further exclusion of patients with missing data of baseline TMAO, choline and hsCRP levels, discharge medication, and statin treatment during hospitalization, a total of 9793 patients with ischemic stroke or TIA were included in this analysis (Figure S1). Patients included in and those excluded from this analysis were largely comparable (Table S3).
As shown in Table 1, among the 9793 patients, 6673 (68.14%) were men, and the median age was 63.00 years (interquartile range, 54.00–70.00); 9126 (93.19%) patients had an ischemic stroke and 667 (6.81%) patients had a TIA (Table 1). The median TMAO and choline were 1.77 μmol/L (interquartile range, 1.18–2.63 μmol/L) and 13.47 μmol/L (interquartile range, 11.27–16.13 μmol/L), respectively. Among patients who received DAPT in our cohort, after hospital discharge 24.11% of patients adhered to DAPT at 3‐month follow‐up. Among patients who received ILT in our cohort, after hospital discharge 9.26% and 4.23% of patients adhered to ILT at 3‐month and 1‐year follow‐up, respectively. The baseline characteristics of patients grouped by the median levels of TMAO and choline levels are shown in Table S4.
Table 1.
Baseline Characteristics of Study Population
| Characteristics | Total population |
|---|---|
| n=9793 | |
| Demographic characteristics | |
| Age, y, median (IQR) | 63.00 (54.00–70.00) |
| Male, n (%) | 6673 (68.14) |
| BMI (kg/m2), median (IQR) | 24.49 (22.58–26.56) |
| SBP at admission (mm Hg), median (IQR) | 148.50 (135.00–164.00) |
| Current smoker, n (%) | 3079 (31.44) |
| Medical history, n (%) | |
| Hypertension | 6180 (63.11) |
| Diabetes | 2326 (23.75) |
| Coronary heart disease | 1055 (10.77) |
| Stroke | 2227 (22.74) |
| Stroke type, n (%) | |
| TIA | 667 (6.81) |
| Ischemic stroke | 9126 (93.19) |
| NIHSS at admission | |
| NIHSS score 0–3 | 5227 (53.37) |
| NIHSS score ≥4 | 4566 (46.63) |
| TOAST subtype, n (%) | |
| Large‐artery atherosclerosis | 2474 (25.26) |
| Cardioembolic | 618 (6.31) |
| Small‐artery occlusion | 2038 (20.81) |
| Others | 4663 (47.62) |
| TPA, n (%) | |
| Yes | 874 (8.92) |
| No | 8919 (91.08) |
| Laboratory test | |
| Baseline LDL‐C (mmol/L), median (IQR) | 2.32 (1.73–2.99) |
| Baseline HDL‐C (mmol/L), median (IQR) | 0.94 (0.78–1.12) |
| Baseline triglyceride (mmol/L), median (IQR) | 1.37 (1.03–1.87) |
| Baseline TC (mmol/L), median (IQR) | 3.97 (3.31–4.72) |
| Baseline hsCRP (mg/L), median (IQR) | 1.75 (0.81–4.60) |
| eGFR, mL/min per 1.73 m2, median (IQR) | 93.06 (81.89–101.76) |
| Discharge medication, n (%) | |
| Lipid‐lowering agents | 9093 (92.85) |
| Antiplatelet agents | 909 (91.99) |
| Antihypertension agents | 4854 (49.57) |
| Dual‐antiplatelet agents | 3103 (31.69) |
| Metabolites | |
| Choline | 13.47 (11.27–16.13) |
| TMAO | 1.77 (1.18–2.63) |
Continuous data are presented as median (IQR), and categorical variables are presented as percentages. BMI indicates body mass index; eGFR, estimated glomerular filtration rate; hsCRP, high‐sensitivity C‐reactive protein; HDL‐C, high density lipoprotein cholesterol; IQR, interquartile range; LDL‐C, low density lipoprotein cholesterol; NIHSS, National Institutes of Health Stroke Scale; SBP, systolic blood pressure; TC, total cholesterol; TIA, transient ischemic attack; TMAO, trimethylamine N‐oxide; TOAST, Trial of ORG 10172 in Acute Stroke Treatment; and TPA, recombinant tissue plasminogen activator intravenous thrombolytic.
Associations of TMAO and Choline With Stroke Recurrence in the Overall Cohort
As shown in Figure 1A and 1B, the Kaplan–Meier analysis with the log‐rank test showed that participants with high levels of TMAO (TMAO≥median) and choline (choline≥median) had significantly higher cumulative recurrence rates than those with low levels of TMAO (TMAO<median) and choline (choline<median) in the overall cohort (P=0.0002 and P<0.0001, respectively). After multivariable adjustment, high levels of TMAO and choline remained significantly associated with stroke recurrence risk (TMAO adjusted HR, 1.28 [95% CI, 1.21–1.46]; choline adjusted HR, 1.50 [95% CI, 1.31–1.72]; Figure 1C).
Figure 1. The association between elevated plasma TMAO and choline levels with stroke recurrence during the 1‐year follow‐up in the overall cohort.
A, Kaplan–Meier curves for probability of recurrent stroke with elevated TMAO levels during the 1‐year follow up. B, Kaplan–Meier curves for probability of recurrent stroke with elevated choline levels during the 1‐year follow up. C, HRs with 95% CIs for unadjusted model, or following adjustments for traditional risk factors (age; sex; BMI; current smoking; heavy drinking; medical history of hypertension, diabetes, stroke, and CHD; hsCRP; LDL‐C; eGFR; NIHSS score at admission; TOAST subtype; and discharge medications of antiplatelet, antihypertension, and hypoglycemic). BMI indicates body mass index; CHD, coronary heart disease; eGFR, estimated glomerular filtration rate; HR, hazard ratio; hsCRP, high‐sensitivity C‐reactive protein; LDL‐C, low‐density lipoprotein cholesterol; NIHSS, National Institutes of Health Stroke Scale; TMAO, trimethylamine‐N‐oxide; and TOAST, the Trial of ORG 10172 in Acute Stroke Treatment.
Associations of TMAO and Choline With Stroke Recurrence in Patients Received DAPT
Among all the eligible patients, there were 3103 (31.69%) patients who received DAPT (defined as discharge medication of DAPT). As shown in Figure 2A and 2B, the Kaplan–Meier analysis with the log‐rank test showed that participants with high levels of TMAO (TMAO≥median) and choline (choline≥median) had significantly higher cumulative recurrence rates than those with low levels of TMAO (TMAO<median) and choline (choline<median) (P<0.0001 and P<0.0001, respectively). After multivariable adjustment, high levels of TMAO and choline remained significantly associated with stroke recurrence risk (TMAO adjusted HR, 1.65 [95% CI, 1.28–2.13]; choline adjusted HR, 1.70 [95% CI, 1.32–2.19]; Figure 2C).
Figure 2. The association between elevated plasma TMAO and choline levels with stroke recurrence in patients who received dual‐antiplatelet therapy.
A, Kaplan–Meier curves for probability of recurrent stroke with elevated TMAO levels during the 1‐year follow up. B, Kaplan–Meier curves for probability of recurrent stroke with elevated choline levels during the 1‐year follow up. C, HRs with 95% CIs for unadjusted model, or following adjustments for traditional risk factors (age; sex; BMI; current smoking; heavy drinking; medical history of hypertension, diabetes, stroke, and CHD; hsCRP; LDL‐C; eGFR; NIHSS score at admission; TOAST subtype; and discharge medications of antihypertension and hypoglycemic). BMI indicates body mass index; CHD, coronary heart disease; eGFR, estimated glomerular filtration rate; HR, hazard ratio; hsCRP, high‐sensitivity C‐reactive protein; LDL‐C, low‐density lipoprotein cholesterol; NIHSS, National Institutes of Health Stroke Scale; TMAO, trimethylamine‐N‐oxide; and TOAST, the Trial of ORG 10172 in Acute Stroke Treatment.
Associations of TMAO and Choline With Stroke Recurrence in Patients Who Received ILT
Among all the eligible patients, there were 2624 (26.79%) patients who received ILT during hospitalization (40–80 mg for atorvastatin/20–40 mg for rosuvastatin). As shown in Figure 3A and 3B, the Kaplan–Meier analysis with the log‐rank test showed that participants with high levels of TMAO (TMAO≥median) and choline (choline≥median) had a significantly higher cumulative recurrence rate than those with low levels of TMAO (TMAO<median) and choline (choline<median) (P=0.0005 and P=0.0051, respectively). After multivariable adjustment, high levels of TMAO and choline remained significantly associated with stroke recurrence risk (TMAO adjusted HR, 1.49 [95% CI, 1.15–1.94]; choline adjusted HR, 1.49 [95% CI, 1.15–1.92]; Figure 3C).
Figure 3. The association between elevated plasma TMAO, choline levels with stroke recurrence in patients who received intensive lipid‐lowering therapy.
A, Kaplan–Meier curves for probability of recurrent stroke with elevated TMAO levels during the 1‐year follow up. B, Kaplan–Meier curves for probability of recurrent stroke with elevated choline levels during the 1‐year follow up. C, HRs with 95% CIs for unadjusted model, or following adjustments for traditional risk factors (age; sex; BMI; current smoking; heavy drinking; medical history of hypertension, diabetes, stroke, and CHD; hsCRP; eGFR; NIHSS score at admission; TOAST subtype; and discharge medications of antiplatelet, antihypertension, and hypoglycemic). BMI indicates body mass index; CHD, coronary heart disease; eGFR, estimated glomerular filtration rate; HR, hazard ratio; hsCRP, high‐sensitivity C‐reactive protein; LDL‐C, low‐density lipoprotein cholesterol; NIHSS, National Institutes of Health Stroke Scale; TMAO, trimethylamine‐N‐oxide; and TOAST, the Trial of ORG 10172 in Acute Stroke Treatment.
Associations of TMAO and Choline With Stroke Recurrence in Patients With Well‐Controlled hsCRP Level
Among all the eligible patients, there were 5257 (53.68%) with well‐controlled hsCRP level (hsCRP <2 mg/L) on admission. As shown in Figure 4A and 4B, the Kaplan–Meier analysis with the log‐rank test showed that participants with high levels of TMAO (TMAO≥median) and choline (choline≥median) had significantly higher cumulative recurrence rates than those with low levels of TMAO (TMAO<median) and choline (choline<median) (P=0.001 and P<0.0001, respectively). After multivariable adjustment, high levels of TMAO and choline remained significantly associated with stroke recurrence risk (TMAO adjusted HR, 1.39 [95% CI, 1.14–1.69]; choline adjusted HR, 1.88 [95% CI, 1.53–2.30]; Figure 4C).
Figure 4. The association between elevated plasma TMAO, choline levels with stroke recurrence in patients with low inflammation level (hsCRP<2 mg/L).
A, Kaplan–Meier curves for probability of recurrent stroke with elevated TMAO levels during the 1‐year follow up. B, Kaplan–Meier curves for probability of recurrent stroke with elevated choline levels during the 1‐year follow up. C, HRs with 95% CIs for unadjusted model, or following adjustments for traditional risk factors (age; sex; BMI; current smoking; heavy drinking; medical history of hypertension, diabetes, stroke, and CHD; LDL‐C; eGFR; NIHSS score at admission; TOAST subtype; and discharge medications of antiplatelet, antihypertension, and hypoglycemic). BMI indicates body mass index; CHD, coronary heart disease; eGFR, estimated glomerular filtration rate; HR, hazard ratio; hsCRP, high‐sensitivity C‐reactive protein; LDL‐C, low‐density lipoprotein cholesterol; NIHSS, National Institutes of Health Stroke Scale; TMAO, trimethylamine‐N‐oxide; and TOAST, the Trial of ORG 10172 in Acute Stroke Treatment.
Associations of TMAO and Choline With Stroke Recurrence in Patients Who Concurrently Received DAPT and ILT and With Well‐Controlled hsCRP Level
Among all the eligible patients, there were 667 (6.81%) who concurrently received DAPT and ILT and with targeted hsCRP (hsCRP <2 mg/L) levels on admission. As shown in Figure 5A and 5B, the Kaplan–Meier analysis with the log‐rank test showed that participants with high levels of TMAO (TMAO≥median) and choline (choline≥median) had significantly higher cumulative recurrence rates than those with low levels of TMAO (TMAO<median) and choline (choline<median) (P<0.0001 and P=0.0052, respectively). After multivariable adjustment, high levels of TMAO and choline remained significantly associated with stroke recurrence risk (TMAO adjusted HR, 3.57 [95% CI, 1.73–7.38]; choline adjusted HR, 2.19 [95% CI, 1.16–4.16]; Figure 5C).
Figure 5. The association between elevated plasma TMAO, choline levels with stroke recurrence in patients received dual‐antiplatelet therapy and intensive lipid‐lowering therapy and with low inflammation level (hsCRP<2 mg/L) concurrently.
A, Kaplan–Meier curves for probability of recurrent stroke with elevated TMAO levels during the 1‐year follow up. B, Kaplan–Meier curves for probability of recurrent stroke with elevated choline levels during the 1‐year follow up. C, HRs with 95% CIs for unadjusted model, or following adjustments for traditional risk factors (age; sex; BMI; current smoking; heavy drinking; medical history of hypertension, diabetes, stroke, and CHD; eGFR; NIHSS score at admission; TOAST subtype; and discharge medications of antihypertension and hypoglycemic). BMI indicates body mass index; CHD, coronary heart disease; eGFR, estimated glomerular filtration rate; HR, hazard ratio; hsCRP, high‐sensitivity C‐reactive protein; LDL‐C, low‐density lipoprotein cholesterol; NIHSS, National Institutes of Health Stroke Scale; TMAO, trimethylamine‐N‐oxide; and TOAST, the Trial of ORG 10172 in Acute Stroke Treatment.
Kaplan–Meier survival plots stratifying TMAO and choline into low versus high levels (lower than median value versus median value or higher) showed similar trends in total population, in patients who received DAPT and ILT and with a low inflammation level (hsCRP <2 mg/L), and in patients who concurrently received DAPT and ILT and with well‐controlled hsCRP level (Figure S2).
We also investigated the association between elevated plasma TMAO and choline levels with stroke recurrence in patients without DAPT or ILT, or in patients with a high inflammation level (hsCRP ≥2 mg/L), the results of which were consistent with the results in patients who received DAPT or ILT or in patients with a low inflammation level (hsCRP <2 mg/L), or in patients who concurrently received DAPT and ILT and with a well‐controlled hsCRP level (Figure S3).
Sensitivity Analysis
In sensitivity analysis, TMAO and choline still contributed to strong residual risk of recurrent ischemic stroke in total population, in patients who received DAPT and ILT and with a low inflammation level (hsCRP <2 mg/L), and in patients who concurrently received DAPT and ILT and with a well‐controlled hsCRP level (hsCRP <2 mg/L) (Figure S4).
DISCUSSION
In the nationwide, multicenter, prospective registry in China, we observed that TMAO and choline were risk factors for recurrent stroke independent of DAPT or ILT at discharge and low inflammation on admission.
Existing evidence has demonstrated that TMAO and choline are risk factors for CVD, in which the incidence of stroke was included in the major adverse cardiac events. Although some studies have also investigated the association of TMAO and choline with stroke recurrence in the secondary prevention population of ischemic stroke, the results are not consistent. 5 , 18 , 19 Haghikia et al 5 demonstrated that patients with elevated TMAO levels, but not choline, were at higher risk of recurrent stroke over 1‐year follow‐up. However, result of a case–control study from Chinese patients indicated that plasma choline was inversely associated with recurrent stroke in the setting of ischemic stroke. 19 In the present study, we observed that elevated plasma levels of TMAO and choline were associated with an increased risk of recurrent stroke in the cohort of CNSR‐III, which is a nationwide prospective registry for patients presenting to hospitals with acute ischemic cerebrovascular events.
Accumulated evidence from the latest clinical trials of CHANCE (Clopidogrel in High‐Risk Patients With Acute Nondisabling Cerebrovascular Events), 12 TST (Treat Stroke to Target) 13 and LoDoCo (Low Dose Colchicine) 14 have addressed 3 main therapeutic targets—residual thrombotic, residual cholesterol, and residual inflammatory risk—in reducing the residual risk of stroke recurrence. Extensive evidence suggested that TMAO increases CVD risk mainly because of its prothrombotic, 1 proinflammatory, 5 and proatherothrombotic effects. 4 Recently, some studies have reported an association between TMAO and adverse outcomes in patient cohorts that include some individuals on antiplatelet therapy. In the GeneBank cohort, a single‐center cohort of stable patients undergoing elective diagnostic coronary angiography (including both primary and secondary prevention patients), elevated TMAO remained significantly associated with incident major adverse cardiac events major adverse cardiac events in all subjects on antiplatelet therapy. 20 In the multisite Swiss Acute Coronary Syndromes Cohort, a strong association between TMAO and incident major adverse cardiac event risk was observed in a subset on chronic DAPT during follow‐up. 20 Moreover, evidence from the Cleveland Clinic showed that choline supplementation increased both fasting plasma TMAO levels and adenosine diphosphate–dependent platelet aggregation responses at 2 months of oral supplementation of choline. 10 However, few studies have evaluated the association of TMAO and choline with the risk of stroke recurrence in the concurrent presence of DAPT and ILT and with a low inflammation level. Considering that combined DAPT and ILT and anti‐inflammation therapy is likely to be applied to patients with CVD, our study first reported on the association of plasma TMAO and choline with the risk of stroke recurrence in patients with ischemic stroke/TIA in the presence of DAPT and ILT and with hsCRP <2 mg/L. Consistent with the previous studies, we observed a heightened risk of stroke recurrence with elevated TMAO and choline levels in the presence of DAPT. Moreover, heightened risk of stroke recurrence with elevated TMAO and choline levels was also observed in patients in the presence of intensive lipid‐lowering therapy and well controlled hsCRP, respectively. Of note, even in patients who concurrently received DAPT and ILT and with hsCRP <2 mg/L, elevated plasma levels of TMAO and choline still contributed to risk of recurrent stroke.
Dietary choline is the main source of TMAO. The intestinal microbiota anaerobically convert choline to trimethylamine by choline trimethylamine lyase. 1 , 21 , 22 Trimethylamine is further metabolized to TMAO by the flavin‐containing enzyme monooxygenase 3 in the liver. 23 , 24 The underlying mechanisms of the detrimental effect of TMAO mainly including its prothrombotic, 10 proatherothrombotic, 21 and proinflammatory 25 effects. Experimental studies have demonstrated that TMAO is directly linked to platelet responsiveness and in vivo thrombosis potential via cell autonomous effects on circulating platelets. 4 , 10 Moreover, TMAO was involved in modulating lipid homeostasis and could accelerate atherosclerosis in mice. Specifically, TMAO could suppress the reverse cholesterol transport 1 , 21 , 26 and increase the expression in macrophages of scavenger receptors CD36 and SR‐A1, which promote lipid accumulation and foam cell formation. 1 , 27 Additionally, a positive association between the plasma concentration of TMAO and low‐grade inflammation has been observed in a German study. 28 TMAO predicts risk of cardiovascular events in patients with stroke and is related to proinflammatory monocytes. 5 However, our present studies demonstrated that the risk of TMAO and choline remains even in the concurrent presence of DAPT, ILT, and well‐controlled hsCRP levels, which indicated that some other pathological mechanisms of TMAO were involved in the contribution of stroke recurrence. A recent study demonstrated that TMAO could induce vascular endothelial tissue factor in the presence of one or more anti‐platelet drugs, which indicated that endothelial tissue factor expression and extrinsic clotting cascade activation may be involved in the detrimental effect of TMAO. 20 Meanwhile, other mechanisms involved in the pathological effect of TMAO are needed to investigate in the future.
We consider our findings to be of clinical significance. First, considering the contribution of TMAO and choline to the residual risk of recurrent stroke, it is necessary to monitor the plasma level of TMAO and choline and reduce the consumption of choline‐rich foods even in patients with ischemic stroke/TIA who received DAPT and ILT and with a low inflammation level. Second, currently there was no specific inhibitor of TMAO for clinical application. To date, 3,3‐dimethyl‐1‐butanol and halomethylcholines were identified as competitive small‐molecule inhibitors of the gut microbial choline trimethylamine lyase system both in vitro and in vivo, which are capable of lowering plasma TMAO levels, and concurrently ameliorating macrophage cholesterol accumulation, foam cell formation, and atherosclerotic lesion development. 11 , 27 The application of small‐molecule choline trimethylamine lyase inhibitors may further reduce the residual risk of recurrent stroke.
There are some limitations in our current study. First, we measured the plasma level of TMAO and choline only at admission without a sequential dynamic monitor, which could not exclude the possibility that the fluctuation attributable to stress response to the acute ischemic stroke. Second, a causal relationship between TMAO and choline with the risk of stroke recurrence could not be established because of the observational study design. Third, inflammatory markers like hsCRP were measured only at admission without a sequential dynamic monitor, which might be influenced by various acute‐phase reactions. Fourth, this study did not collect stool samples of patients; therefore, we were not able to further analyze the microbiome. Finally, all the participants included in our current cohort are Chinese, which indicates that the conclusion of our study cannot be generalized to other races and ethnicities.
CONCLUSIONS
TMAO and choline were risk factors for recurrent stroke independent of DAPT or ILT at discharge and low inflammation on admission. Future research should focus on efforts to reduce the residual risk caused by TMAO and choline, such as dietary interventions, or targeted inhibitors.
Sources of Funding
This work was supported by grants from the Capital's Funds for Health Improvement and Research (2020‐1‐2041), Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (2019‐I2M‐5‐029), Capital Health Research and Development of Special (2020‐2Z‐20 411), National Natural Science Foundation of China (81970425), National Key R&D Program of China (2020YFA0803700), and Beijing Postdoctoral Research Foundation (2021‐ZZ‐021).
Disclosures
None.
Supporting information
Data S1
Tables S1–S4
Figures S1–S4
Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/JAHA.122.027265
For Sources of Funding and Disclosures, see page 8.
Contributor Information
Lemin Zheng, Email: zhengl@bjmu.edu.cn.
Yongjun Wang, Email: yongjunwang@ncrcnd.org.cn.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data S1
Tables S1–S4
Figures S1–S4
Data Availability Statement
The data that support the findings of this study are available from the corresponding author on reasonable request.