Journal of Neuroimmunology 354 (2021) 577526

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Journal of Neuroimmunology
journal homepage: www.elsevier.com/locate/jneuroim

Gut microbial metabolite TMAO portends prognosis in acute

ischemic stroke
Jianli Zhang a, 1, *, Liankun Wang b, 1, Jinle Cai a, Aidi Lei a, Caiwen Liu a, Ruidian Lin a, Li Jia c, **,
Yingzi Fu d, ***
a
Department of Neurology, Xiamen Fifth Hospital, Xiamen, China
b
Department of Neuroelectrophysiology, Heilongjiang Province Hospital, Harbin, China
c
Center of Health Laboratory Technology, Public Health Department of Harbin Medical University, Harbin, China
d
Department of Neurology, Heilongjiang Province Hospital, Harbin, China

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Over the recent years, the role of trimethylamine N-oxide (TMAO) as a gut-derived metabolite
Trimethylamine N-oxide mediating cardiovascular disease pathogenesis has been under particularly intense scrutiny. The aim was to
Gut microbial metabolism explore whether TMAO levels were associated with clinical severity or functional outcome in Chinese patients
Ischemic stroke
with ischemic stroke.
Prognosis
Mortality
Methods: This is a single-center, prospective cohort study from Xiamen, China. We examined the relationship
between fasting TMAO and 2 of its nutrient precursors, choline and betaine, vs. 3-month functional outcome and
mortality in 351 first-ever patients with acute ischemic stroke.
Results: The median value of the plasma level of TMAO was 6.1 μM (IQR, 3.7–9.9 μM), which was higher than in
those control cases (4.0; 2.4–5.9 μM). Patients with a poor outcome and nonsurvivors had significantly increased
TMAO levels on admission (P < 0.001). Following adjustments for traditional risk factors, increased TMAO
concentrations remained predictive of both poor outcome and mortality risks in stroke patients [e.g., quartiles 4
vs 1, odd ratio 5.65 (95% CI, 2.87–13.45), P < 0.001; and 5.84 (95% CI, 3.05–16.12), P < 0.001, respectively]. In
multivariate analysis, TMAO was an independent predictor of functional outcome and mortality and improved
the prognostic accuracy of the NIHSS to predict functional outcome (combined areas under the curve, 0.82; 95%
CI 0.77–0.89, P = 0.003) and mortality (combined areas under the curve, 0.85; 95% CI: 0.80–0.90, P = 0.002).
Conclusions: Fasting plasma concentrations of gut microbial TMAO are higher in patients with ischemic stroke
and portend higher poor functional outcome events and mortality.

1. Introduction microbiota to cardiometabolic diseases with mechanistic links to gut

China has 2.5 million new stroke cases and 1.6 million stroke mor­
tality each year, and 7.5 million stroke survivors, which has exceeded
heart disease to become the leading cause of death and adult disability
(Tu et al., 2017). A previous study suggested that in 2016, the global
lifetime risk of stroke from the age of 25 years onward was approxi­
mately 25%, and China had the highest estimated risk (39.3%; 95%
uncertainty interval, 37.5 to 41.1) (The GBD, 2016).
Recent studies indicate a pathophysiological contribution of gut
microbial choline metabolism (Tang et al., 2017). Trimethylamine-N-
oxide (TMAO) is a circulating organic compound produced by dietary
L-carnitine and choline metabolism, which was recently found to induce
atherosclerosis in rodents (Stubbs et al., 2016) directly. Intestinal bac­
teria metabolize both L-carnitine and choline to trimethylamine, a
metabolite that is absorbed from the intestine and subsequently oxidized
via hepatic flavin monooxygenase enzymes to form TMAO (LangDH
et al., 1998). Wang et al. (Wang et al., 2011) reported that plasma
concentrations of metabolites of dietary phosphatidylcholine, including

* Correspondence to: J. Zhang, No.101, Min’an Road, Xiang’an District, Xiamen City, Fujian Province, China.
** Correspondence to: L. Jia, No.157, Baojian Road, Nangang District, Harbin City, Heilongjiang Province, China.
*** Correspondence to: Y. Fu, Guo Ge Li street 405, Nangang District, Harbin, Heilongjiang Province, China.
E-mail addresses: zhangjianli1223@163.com (J. Zhang), jiali1013@163.com (L. Jia), yingzibest@126.com (Y. Fu).
1
Jianli Zhang and Liankun Wang contributed equally to this work as co-first authors.

https://doi.org/10.1016/j.jneuroim.2021.577526
Received 15 October 2020; Received in revised form 17 February 2021; Accepted 17 February 2021
Available online 20 February 2021
0165-5728/© 2021 Elsevier B.V. All rights reserved.
J. Zhang et al.
choline, betaine, and TMAO, were associated with atherosclerotic car­
diovascular risks in patients. In animal models, they also confirmed that
those biomarkers mechanistically linked to atherosclerosis development
(Wang et al., 2011).
Elevated TMAO levels have been shown to predict future risk of
major adverse cardiac events (MACE), an increased prevalence of car­
diovascular diseases (CVD), and have shown a relationship with the
number of diseased coronary vessels (Wang et al., 2011; Tang et al.,
2013; Koeth et al., 2014). A meta-analysis found that higher circulating
TMAO may independently predict the risk of subsequent cardiovascular
events and mortality (Qi et al., 2018). Interestingly, a higher plasma
TMAO level was suggested to be associated with worse long-term sur­
vival in patients with chronic kidney disease (Tang et al., 2015) and with
heart failure (Tang et al., 2014; Troseid et al., 2015).
Furthermore, one study demonstrated a graded relation between
TMAO levels and the risk of subsequent cardiovascular events in patients
with recent prior ischemic stroke (Haghikia et al., 2018). Another study
proposed that higher TMAO levels were associated with increased risk of
the first stroke in hypertensive patients (Nie et al., 2018). Also, TMAO
was an independent predictor of ischemic stroke, potentially refining
stroke stratification in patients with atrial fibrillation (Liang et al.,
2019). Shafi et al. (Shafi et al., 2017) reported that TMAO had a linear
increase in risk with cardiovascular mortality in white hemodialysis
patients, but not in black patients, suggesting the role of TMAO might
differ by race and ethnic groups. However, most prior studies came from
Western countries. Research on China populations, where dietary habits
are not exactly the same as those of Western countries, is limited. Thus,
in this study, the aim was to explore whether TMAO levels were asso­
ciated with clinical severity or functional outcome in Chinese patients
with ischemic stroke.
2. Patients and methods
2.1. Patients
This was a prospective cohort study at one hospital in Fujian (Xiamen
Fifth Hospital), China. During March 2017–June 2018, consecutive
patients with first-ever ischemic stroke (symptoms onset was less than
24 h) were included. Ischemic stroke was diagnosed and identified ac­
cording to World Health Organization recommendations. Exclusion
criteria were: (1) malignant tumor; (2) metabolic syndrome; (3) renal
and liver insufficiency; (4) any surgical procedure within the previous
month; (5) inability to consent (e.g., dementia); (6) Other neurological
diseases (such as intracerebral hemorrhage, cerebral hemorrhage, Par­
kinson’s disease, and Alzheimer’s disease) and (7) presence of cardio­
genic shock, sepsis, pneumonia; acute coronary syndromes (ACS).
One hundred and fifty age and gender-matched healthy volunteers
from our hospital medical center were assigned to as the healthy control
group. All control subjects were also clinically examined by a neurolo­
gist (not an author) to exclude any sub-clinical stroke features. The
median age of those control cases was 66 (IQR, 55–74) years, and 50%
were men.
2.2. Clinical variables and laboratory testing
At baseline, demographic data (age and sex), body mass index (BMI),
and history of conventional vascular risk factors (smoking, hyperten­
sion, type 2 diabetes mellitus, hypercholesterolemia, atrial fibrillation,
coronary heart disease, previous myocardial infarction, peripheral
vascular disease, and transient ischemic attack) were collected. Pre-
stroke therapy, including oral anticoagulants, antiplatelet agents, anti­
hypertensive treatment, and statins, as well as acute treatment (IV
thrombolysis and/or mechanical thrombectomy), was also recorded.
National Institutes of Health Stroke Scale (NIHSS) was used to
evaluate clinical severity at admission by a neurologist (Tu et al., 2014).
Ischemic stroke was classified according to Trial of Org 10,172 in Acute
2
Journal of Neuroimmunology 354 (2021) 577526
Stroke Treatment (TOAST) criteria: (1) large-artery arteriosclerosis; (2)
cardioembolism; (3) small-artery occlusion; (4) the other causative
factor, and (5) undetermined causative factor (Adams Jr et al., 1993).
Functional impairment was assessed at 3-month using the modified
Rankin scale (mRS), and a good functional outcome was defined as mRS
of 0–2 points, while a poor outcome was defined as 3–6 points (Bonita
and Beaglehole, 1988). The secondary end-point was all-cause mortality
within 3-month. The diagnosis of stroke was based on the results of a
strict neurological examination by magnetic resonance imaging (MRI).
The infarct volume was calculated using the formula 0.5 × a × b × c
(where a is longest dimension in axis x, b is longest perpendicular
dimension to axis x (y), and c is total length in z dimension) (Sims et al.,
2009).
2.3. Blood collection and quantification
The plasma of all patients was collected under fasting at 8:00 on the
first morning of admission for TMAO determination (within 0–6 [n =
78], 6–12 [n = 95], 12–24 [n = 88], and 24–48 [n = 90] hours from
symptom onset). Plasma blood samples were collected using EDTA tubes
and immediately processed and frozen at − 80 ◦ C until analysis. TMAO,
choline, and betaine levels in plasma were determined using stable
isotope dilution high-performance liquid chromatography with online
electrospray ionization tandem mass spectrometry (LC/MS/MS) on an
AB SCIEX 5500 triple quadrupole mass spectrometer using d9-(tri­
methyl)-labelled internal standards as described previously (Stubbs
et al., 2016). TMAO and d9-TMAO were monitored in the positive
multiple reaction monitoring mass spectrometry mode using charac­
teristic precursor–production transitions, including m/z 76/58 and m/z
85/66, respectively. To calculate the TMAO concentration, various
TMAO standards were added to control plasma to prepare the calibra­
tion curves. The assay described shows good inter- and intraday repro­
ducibility (all CVs < 6.0%), accuracy (>98.5% across low, mid, and high
values). Other biomarkers were also tested by standard assays, such as
serum levels of C-reactive protein (CRP), glucose, and interleukin-6 (IL-
6). An estimated glomerular filtration rate (eGFR; in mL/min per 1.73
m2) was calculated using the Modification of Diet in Renal Disease study
equation (Levey et al., 1999).
2.4. Statistical analyses
The results of the categorical variable (percentage) and continuous
variable (median value [interquartile ranges, IQRs]) were described.
Chi-square and Mann-Whitney U tests were applied for comparing
groups. Bivariate correlation was analyzed using Spearman’s rank
correlation.
The relationship between TMAO level and clinical severity (dichot­
omized as NIHSS<6 and NIHSS≥6) (Daubail et al., 2013) or functional
outcomes (mortality) was assessed by logistic regression. The results
were described as odds ratios (OR) with 95% confidence intervals (CI).
Adjustments were made for individual traditional risk factors including
age, sex, BMI, traditional risk factors, NIHSS and Infarct volume at
admission, stroke subtype, pre-stroke treatment, acute stroke treatment,
eGFR, plasma levels of glucose, CRP, IL-6, Choline, Betaine, and TMAO.
We also divided plasma TMAO levels into quartiles.
The accuracy of the TMAO level for predicting the outcome of stroke
was evaluated with Receiver operating characteristic (ROC) curves, and
results were calculated with the Area under the curve (AUC). The clin­
ical value of adding TMAO to the existing risk factors for predicting the
outcome of stroke was calculated with the integrated discrimination
improvement (IDI) index and net reclassification improvement (NRI)
index (Pencina et al., 2011). Furthermore, to study the ability of TMAO
for mortality prediction, we calculated Kaplan–Meier survival curves
and stratified patients by TMAO cut-off, which had been defined by the
ROC curve. All statistics were analyzed with SPSS software. The ROC R
package (version 1.0–2) and GraphPad Prism 5.0 were also applied for
J. Zhang et al.
plotting. P < 0.05 was defined as statistical significance.
2.5. Ethics
The study’s design was reviewed and approves by the investigational
review board of the Xiamen Fifth Hospital, according to the principles of
the Declaration of Helsinki. Informed consent was obtained from pa­
tients or their relatives (patients unable to communicate) prior to their
inclusion in this study.
3. Results
3.1. Patients
From 503 screened patients with suspected first-ever ischemic
stroke, acute ischemic stroke was diagnosed in 388 patients, and 351
completed 3-month follow-up and were finally included (Fig. 1).
The median value of the plasma level of TMAO was 6.1 μM (IQR,
3.7–9.9 μM), which was higher than in those control cases (4.0; 2.4–5.9
μM), Fig. 2A. The included patients’ median age was 66 (IQR, 55–74)
years, and 50.4% were men. On admission, the median NIHSS score and
infarct volumes were 5 (IQR, 3–12) points and 15.5(6.2–24.1) ml,
respectively. A total 49 patients (14.0%) received IV thrombolysis and/
or mechanical thrombectomy. In these patients, the median TMAO level
was lower than that observed in other patients (4.4 μM [IQ, 2.6–7.3] vs.
6.3 μM [4.0–10.1], P = 0.003). In addition, 39 out of those 49 patients
who received IV thrombolysis and/or mechanical thrombectomy were
successfully reperfused after thrombectomy. In these patients, the me­
dian TMAO level was no different from that of non-reperfused patients
(4.4 μM [IQ, 2.5–7.5] vs. 4.4 μM [2.8–6.9], P = 0.92). A poor functional
outcome was found in 117 patients (33.3%) with a median mRS score of
Fig. 1. Study profile/flow sheet of the study.
3
Journal of Neuroimmunology 354 (2021) 577526
4 (IQR, 3–6). Forty-seven patients died, and the mortality rate was
13.4% (95%CI:9.8%–17.0%). The general information of the stroke
patients was presented (Table 1).
3.2. Main results
3.2.1. TMAO and severity of stroke
As a continuous variable, a modest correlation was obtained between
NIHSS score and plasma TMAO level (r = 0.413; P < 0.001). At
admission, there were 184 minor strokes (52.4%, NIHSS<6). The me­
dian TMAO levels in those patients were lower compared to those of
patients with moderate-to-high stroke (4.9 μM [IQR, 2.9–6.6] vs. 8.6 μM
[5.2–11.6], Z = 7.521; P < 0.001). In univariate and multivariable
models, when TMAO was increased by each 1 μM, the unadjusted and
adjusted (variables including age, sex, BMI, traditional risk factors,
infarct volume at admission, stroke subtype, pre-stroke treatment, eGFR,
plasma levels of glucose, CRP, IL-6, Choline, and Betaine) risk of
moderate-to-high stroke were increased by 21% (OR = 1.21 [95%CI:
1.13–1.30], P < 0.001) and 10% (1.10 [1.03–1.21], P = 0.005),
respectively. Also, TMAO levels paralleled lesion size. There was a
positive correlation between infarct volume and plasma TMAO level (r
= 0.244; P < 0.001). Furthermore, correlation analysis showed that
plasma TMAO level positively corrected with age, IL-6, Choline, and
Betaine, while negative with eGFR (P < 0.05, all). Patients of the male
sex, older age, suffered from diabetes, and received glucose-lowing
drugs were more likely to had higher levels of TMAO(p < 0.05).
3.2.2. Copeptin and functional outcome after 3 months
TMAO plasma levels in patients with a poor outcome were signifi­
cantly higher than those in patients with a good outcome (5.0 [IQR,
3.0–7.0] vs. 9.4 [IQR, 6.2–12.2] μM; P < 0.001; Fig. 2B). In univariate
J. Zhang et al.
logistic regression analysis, with an unadjusted OR of 1.35 (95% CI,
1.25–1.46), TMAO is strongly associated with poor functional outcomes.
After adjusting for all other significant outcome predictors, the OR
adjusted for all other risk factors was 1.21 (95% CI, 1.07–1.35), sug­
gesting that TMAO was still an independent factor for predicting func­
tional outcome, Table 2. As shown in Table 2, age, stroke severity, large-
vessel occlusive, IL-6, and acute treatment remained significant outcome
predictors, unlike all others assessed. Interestingly, Choline and Betaine
were outcome predictors in univariate analysis. However, this trend
disappeared in the multivariable models.
The poor outcome was distributed across TMAO quartiles, from
11.2% (first quartile, Q1) to 62.1% (fourth quartile, Q4). In the analysis
of multivariable models, the second, third, and fourth quartiles (Q2, Q3,
and Q4) of TMAO levels were compared against Q1 (Fig. 3). The results
showed that TMAO levels in Q3 and Q4 were correlated with functional
outcome, and the poor outcome risk was increased by 202% (OR 3.02,
95% CI 1.34–6.12) and 465% (5.65, 2.87–13.45), respectively (Fig. 3).
The independence of the correlation between TMAO and functional
outcome was tested by the likelihood ratio test (P = 0.001). In addition,
plasma choline and betaine levels in Q4 (vs. Q1) were correlated with
functional outcome (Sup table I).
The ROC curve was applied for choosing the cut-off value of TMAO in
predicting functional outcome. An optimal amount of 5.8 μM produced a
sensitivity of 57.5% and a specificity of 74.9%, with AUC of 0.78 (95%
CI 0.72–0.83, Fig. 4A), which showed a significantly greater discrimi­
natory ability as compared with age, CRP, and IL-6, and was within the
range of the NIHSS score (Table 3). In a combined model, the AUC of the
NIHSS score could be increased with TMAO (0.82; 95% CI 0.77–0.89, P
= 0.003). Furthermore, TMAO improved the established risk factors
(AUC of the combined model, 0.83; 95% CI, 0.78–0.90; p < 0.001). This
improvement was stable in an internal 5-fold cross-validation that
resulted in an average AUC (standard error) of 0.805 (0.032) for the
established risk factors and 0.834(0.026) for the combined model, cor­
responding to a difference of 0.029 (0.008). The NRI statistic results
indicated that the correct reclassification of functional outcomes could
be improved after adding TMAO to the existing risk factors (P = 0.02).
Furthermore, this result also is confirmed with the IDI statistic (P =
0.01) (Table 4).
4
Journal of Neuroimmunology 354 (2021) 577526
Fig. 2. Plasma levels of TMAO in different
groups. (A) Plasma levels of TMAO in pa­
tients with ischemic stroke and controls. (B)
Plasma levels of TMAO in patients with a
good outcome and poor outcome. A good
functional outcome was defined as an mRS
score of 0 to 2 points, while poor outcome
was defined as 3–6 points. (C) TMAO levels
in survivors and nonsurvivors of stroke. All
data are medians and interquartile ranges
(IQR). P values refer to Mann-Whitney U
tests for differences between groups. TMAO,
Trimethylamine N-oxide.
3.2.3. TMAO and death within 3 months
Copeptin plasma levels in 47 patients who died were nearly two
times as compared with patients who survived (10.6 [IQR, 8.5–13.2] vs.
5.8 [IQR, 3.4–8.5] μM; p < 0.001; see Fig. 2C). In univariate and
multivariate logistic regression analysis models, TMAO was correlated
with all-cause mortality, and the mortality risk was increased by 36%
(OR 1.36, 95% CI: 1.23–1.49) 24% (1.24, 1.06–1.38), respectively. As
shown in Table 2, age, stroke severity, IL-6, and acute treatment
remained significant mortality predictors, unlike all others assessed.
Again, Choline and Betaine were mortality predictors in univariate
analysis. However, this trend disappeared in the multivariable models.
All-cause mortality was distributed across TMAO quartiles, between
3.4% (Q1) to 32.2% (Q4). In multivariable models, Q2, Q3, and Q4 of
TMAO plasma levels were compared against Q1 (Fig. 5). The results
suggested that TMAO levels in Q4 were correlated with all-cause mor­
tality, and the risk was increased by 484% (OR 5.84, 95% CI
3.05–16.12). The results showed that TMAO levels in Q2 and Q3 were
not correlated with mortality, and the OR were 2.29(0.83–6.03) and
0.89(0.43–3.87), respectively (Fig. 5). The independence of the TMAO
correlation with mortality was also confirmed by the likelihood ratio test
(P = 0.005). In addition, plasma choline and betaine levels in Q4 (vs.
Q1) were correlated with mortality (Sup table I).
The ROC curve was also applied for exploring the cut-off value of
TMAO in predicting mortality. An optimal amount of 7.8 μM produced a
sensitivity of 80.9% and a specificity of 72.0%, with AUC of 0.80 (95%
CI 0.74–0.87; Fig. 4B), which showed a significantly greater discrimi­
natory ability as compared with age, CRP, and IL-6, and was within the
range of the NIHSS score (Table 3). In a combined model, the AUC of the
NIHSS score could be increased with TMAO (0.85; 95% CI: 0.80–0.90, P
= 0.002). Furthermore, TMAO improved the established risk factors
(AUC of the combined model, 0.86; 95% CI, 0.80–0.93; P < 0.001). This
improvement was stable in an internal 5-fold cross-validation that
resulted in an average AUC (standard error) of 0.837 (0.025) for the
established risk factors and 0.861(0.020) for the combined model, cor­
responding to a difference of 0.024 (0.005). The NRI statistic results
indicated that functional outcomes’ correct reclassification could be
improved after adding TMAO to the existing risk factors (P = 0.003).
However, this result could not be confirmed with the IDI statistic (P =
0.11) (Table 4).
J. Zhang et al. Journal of Neuroimmunology 354 (2021) 577526

Table 1 Table 2
Characteristics of stroke patients and control cases. Predictors of poor functional outcome and all-cause mortality.
Ischemic Control cases Predictor variable OR (95%CI) P
stroke a
Multivariate Analysis for Functional Outcome
N 351 150 TMAO (increase per unit) 1.21(1.07–1.35) <0.001
Age(years), median (IQR) 66(55–74) 66(55–74) IL-6 (increase per unit) 1.30(1.18–1.52) 0.005
Male, n (%) 177(50.4) 75(50.0) Age (increase per unit) 1.05(1.02–1.08) 0.003
BMI, kg/m2, median (IQR) 25.6 24.8 Stroke severity, NIHSS (>5) 4.45(2.85–8.16) <0.001
(23.7–27.4) (22.5–26.2) Large-vessel occlusive VS. other 8.15 0.004
Prior vascular risk factors, n (%) (2.15–20.44)
Hypertension 224(63.8) 62(41.3) IV thrombolysis and/or mechanical 0.45(0.23–0.70) <0.001
Diabetes 77(21.9) 25(16.7) thrombectomy
Hypercholesterolemia 85(24.2) 33(22.0) Multivariate analysis for mortalitya
Atrial fibrillation 71(21.1) 2(1.3) TMAO (increase per unit) 1.24(1.06–1.38) <0.001
Prior TIA 42(12.0) 3(2.0) IL-6 (increase per unit) 1.28(1.10–1.64) 0.015
Smoking 61(17.4) 25(16.7) Age (increase per unit) 1.06(1.03–1.08) 0.001
PVD 15(4.3) 1 (0.7) Stroke severity, NIHSS (>5) 5.05(2.75–8.77) <0.001
Pre-stroke treatment, n (%) IV thrombolysis and/or mechanical 0.40(0.19–0.64) <0.001
Antihypertensive 218(62.1) 38(25.3) thrombectomy
Anticoagulant 38(10.8) 5(3.3)
Antiplatelet agents 65(18.5) 6(4.0) OR, odds ratio; NIHSS, National Institutes of Health Stroke Scale; IL-6, inter­
Statins 72(20.5) 6(4.0) leukin-6; CRP, C–reactive protein; TMAO, Trimethylamine N-oxide; eGFR,
Acute treatment, n (%) estimated glomerular filtration rate.
a
IV thrombolysis 43(12.2) – Multivariable model included all of the following variables: age, sex, BMI,
Mechanical thrombectomy 10(2.8) – traditional risk factors, NIHSS and Infarct volume at admission, stroke subtype,
IV thrombolysis and/or mechanical 49(14.0) – pre-stroke treatment, acute stroke treatment, eGFR, plasma levels of glucose,
thrombectomy CRP, IL-6, Choline, Betaine and TMAO. OR expressed as OR (95% confidence
Stroke causative factors, n (%)
interval).
Small-vessel occlusive 75(21.4) –
Large-vessel occlusive 73(20.8) –
Cardioembolic 135(38.5) – 5. Discussions
Other 45(12.8) –
Unknown 23(6.6)
TMAO is an oxidized metabolite mediated by gut microbial meta­
–
Laboratory findings, median (IQR)
Glucose level, mmol/l 5.95 5.68 bolism of choline-containing lipids and carnitine-based molecules
(5.05–7.29) (4.95–6.38) (Suzuki et al., 2017). The gut microbial TMAO pathway has been shown
C–reactive protein, mg/l 6.5(3.8–11.6) 4.4(2.6–7.3) to possess numerous clinical and mechanistic links with the promotion
IL-6, pg/ml 7.8(5.5–9.4) 4.9(3.5–7.4)
of atherosclerosis (Wang et al., 2011; Miao et al., 2015) and with mor­
eGFR, ml⋅min− 1⋅(1.73⋅m2)− 1
83(60–97) 72(55–88)
TMAO, μM 6.1(3.7–9.9) 4.0(2.4–5.9) tality and adverse cardiovascular outcomes for cardiorenal disorders (e.
Choline, μM 10.1(7.2–12.6) 8.2(5.9–10.3) g., coronary artery disease (Wang et al., 2011; Senthong et al., 2016),
Betaine, μM 43.5 35.2 heart failure (Troseid et al., 2015) and chronic kidney disease (Tang
(20.7–54.1) (15.4–47.3) et al., 2015; Rhee et al., 2013)) and cerebrovascular diseases (Yin et al.,
NIHSS at admission, median (IQR) 5(3− 12)
2015).
–
Infarct volume, ml. median (IQR) 15.5(6.2–24.1) –
This study was the first population-based study on the correlation
NIHSS, National Institutes of Health Stroke Scale; TIA, transient ischemic attack; between circulating TAMO levels and functional outcome(mortality)
IL-6, interleukin-6; IQR, interquartile ranges; TMAO, Trimethylamine N-oxide; risk in Chinese patients with ischemic stroke. This study’s findings were
PVD, peripheral vascular disease; eGFR, estimated glomerular filtration rate.
as follows: First, plasma levels of TMAO were dose-dependently asso­
ciated with 3-month poor functional outcome and all-cause mortality
The time to death was analyzed by Kaplan–Meier survival curves risk among patients with ischemic stroke. Furthermore, TMAO
based on TMAO optimal cut-off value, which had been defined by the improved the prognostic accuracy of the NIHSS to predict functional
ROC curve(7.8 μM). The Kaplan–Meier estimates of all-cause mortality outcome and mortality. Second, we confirmed previous reports that
stratified by baseline TMAO (elevated vs. normal) was shown in Fig. 6. suggested patients with ischemic stroke had higher TMAO than healthy
Patients with elevated TMAO had shorter survival than patients with controls. Third, increased fasting TMAO concentrations were observed
normal TMAO (P < 0.001, log-rank test). The OR between those two to be associated with stroke severity according to the NIHSS score and
groups (elevated vs. normal) was 3.48 (95% CI 1.58–6.46). lesion size. Fourth, we observe that the prognostic value of increased
choline and betaine are only observed among those with increased
4. Sub-group analysis TMAO.
A previous study finished by Haghikia et al. (Haghikia et al., 2018)
we also conducted analyses separately among cases who experienced reported a graded relation between TMAO levels and the risk of subse­
with eGFR <90 mL/min/1.73 m2 and ≥ 90 mL/min/1.73 m2. In the quent cardiovascular events in patients with recent prior ischemic
multivariate regression analysis, the data suggested that for each 1 μM stroke, suggesting that TMAO-related increase of proinflammatory
increase of plasma concentration of TMAO, the association with poor monocytes may add to an elevated cardiovascular risk of patients with
outcome was stronger among cases who experienced with eGFR increased TMAO levels. Consistent with our findings, Suzuki et al.
<90 mL/min/1.73 m2 (OR = 1.25, 95%CI: 1.10–1.38; P < 0.001) versus (Suzuki et al., 2017) reported that TMAO levels showed an association
≥90 mL/min/1.73 m2 (OR = 1.16, 95%CI: 1.03–1.33; P = 0.005). with poor prognosis at two years and superiority over current bio­
Interestingly, the predictive value of TMAO to predict all-cause mor­ markers for patients hospitalized due to acute myocardial infarction.
tality in cases who experienced with eGFR <90 mL/min/1.73 m2 (OR = Elevated levels were associated with poor prognosis at one year, and a
1.30, 95%CI: 1.13–1.43; p < 0.001) was stronger than in ≥90 mL/min/ combination of TMAO and NT-pro-BNP provided additional prognostic
1.73 m2 (1.18 [1.03–1.40]; P = 0.008). information in acute heart failure (Suzuki et al., 2016). Similarly, Tang
et al. (Tang et al., 2014) reported that high TMAO levels were observed
in patients with HF, and elevated TMAO levels portended higher long-

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J. Zhang et al. Journal of Neuroimmunology 354 (2021) 577526

Fig. 3. Multivariate analyses for poor functional outcome according to TMAO quartiles in stroke patients (first quartile as the reference). The numbers in the first
column represent stroke patients, and the second column represents patients with poor outcome. §Multivariable model included all of the following variables: age,
sex, BMI, traditional risk factors, NIHSS and Infarct volume at admission, stroke subtype, pre-stroke treatment, acute stroke treatment, eGFR, plasma levels of
glucose, CRP, IL-6, Choline, and Betaine. OR was expressed as OR (95% confidence interval). NIHSS, National Institutes of Health Stroke Scale; CRP, C-reactive
protein; BMI, body mass index; IL-6, interleukin-6; TMAO, Trimethylamine N-oxide.

Fig. 4. ROC curves were utilized to evaluate the accuracy of plasma TMAO levels to predict functional outcome and mortality. (A) ROC curves were used to assess
the accuracy of plasma TMAO levels to predict poor functional outcomes. (B) ROC curves were utilized to assess the accuracy of plasma TMAO levels to predict
mortality. A poor outcome was defined as an mRS score of 3 to 6 points. TMAO, Trimethylamine N-oxide; ROC, receiver operating characteristic.

Table 3 Table 4
Prediction of poor functional outcomes and morality according to ROC. Plasma levels of TMAO at admission prediction of functional outcome and all-
Parameter AUC 95% CI p
cause mortality with AUROC.
End points AUROC
Prediction of functional outcome
TMAO 0.78 0.72 0.83 – TMAO Risk Risk Incremental NRI(P) IDI(P)
NIHSS 0.75 0.70 0.79 0.072 factorsa factors area (P)b
Age 0.70 0.64 0.75 0.003 with
IL-6 0.65 0.59 0.70 <0.001 TMAOa
CRP 0.63 0.56 0.69 <0.001
Combined score (TMAO/NIHSS) 0.82 0.77 0.89 0.011 Functional 0.78 0.81 0.83 0.03(0.01) 0.18 0.03
Prediction of mortality (0.02) (0.01)
TMAO 0.80 0.74 0.87 – Morality 0.80 0.84 0.86 0.02(0.02) 0.28 0.02
NIHSS 0.82 0.77 0.89 0.15 (0.003) (0.11)
Age 0.73 0.67 0.78 0.005 NIHSS, National Institutes of Health Stroke Scale; CRP, C-reactive protein; BMI,
IL-6 0.70 0.65 0.76 <0.001
body mass index; IL-6, interleukin-6; TMAO, Trimethylamine N-oxide.
CRP 0.69 0.63 0.74 <0.001 a
Established risk factors including: age, sex, BMI, traditional risk factors,
Combined score (TMAO/NIHSS) 0.85 0.80 0.90 <0.001
NIHSS and Infarct volume at admission, stroke subtype, pre-stroke treatment,
AUC, area under the curve; CI, confidence interval; CRP, C-reactive protein; acute stroke treatment, eGFR, plasma levels of glucose, CRP, IL-6, Choline, and
NIHSS, National Institutes of Health Stroke Scale; IL-6, interleukin-6; TMAO, Betaine.
Trimethylamine N-oxide. b
Comparison of AUROCs: established risk factors without TMAO levels vs.
established risk factors with TMAO levels.

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J. Zhang et al. Journal of Neuroimmunology 354 (2021) 577526

Fig. 5. Multivariate analyses for mortality according to TMAO quartiles in stroke patients (first quartile as the reference). The numbers in the first column represent
stroke patients, and the second column represents non-survivors. §Multivariable model included all of the following variables: age, sex, BMI, traditional risk factors,
NIHSS and Infarct volume at admission, stroke subtype, pre-stroke treatment, acute stroke treatment, eGFR, plasma levels of glucose, CRP, IL-6, Choline, and Betaine.
OR was expressed as OR (95% confidence interval). NIHSS, National Institutes of Health Stroke Scale; CRP, C-reactive protein; BMI, body mass index; IL-6, inter­
leukin-6; TMAO, Trimethylamine N-oxide.

Fig. 6. Kaplan-Meier analysis of all-cause mortality according to the TMAO cut-off value (defined by the ROC curve). The patients with elevated TMAO had a higher
risk of mortality (log-rank test P < 0.001). TMAO, Trimethylamine N-oxide; ROC, receiver operating characteristic.

term mortality risk independent of traditional risk factors and car­
diorenal indexes. Increased TMAO concentrations correlate with coro­
nary atherosclerosis burden and may associate with long-term mortality
in patients with chronic kidney disease (CKD) undergoing coronary
angiography (Stubbs et al., 2016). Another study also suggested that
plasma TMAO levels were elevated in patients with CKD and portend
lower long-term survival (Tang et al., 2015).
Similarly, fasting plasma concentrations of TMAO were higher in
diabetic patients and portend higher major adverse cardiac events
(MACE) and mortality risks independent of other factors (Tang et al.,
2017). Although TMAO levels were found to be increased in hypergly­
cemic subjects and patients with impaired kidney function in other
studies, they were not associated with all-cause mortality, cardiovas­
cular death, or hospitalizations (Kaysen et al., 2015). Another study
showed that TMAO was associated with all-cause mortality in subjects
with any degree of renal function impairment, but not in subjects with
normal renal function (Gruppen et al., 2017).
Furthermore, one study showed that high choline and betaine levels
are only associated with a higher risk of future MACE with a concomi­
tant increase in TMAO (Wang et al., 2014), confirmed by our results.
Also, Gruppen et al. (Gruppen et al., 2017) found that TMAO was
associated with all-cause mortality, particularly in subjects with eGFR
<90 mL/min/1.73 m2. We also found that the prognostic value of TMAO
was more pronounced in subjects with eGFR <90 mL/min/1.73 m2. one

7
J. Zhang et al.
study found that higher levels of TMAO were not independently asso­
ciated with cardiovascular death after adjustment for age and eGFR
(Skagen et al., 2016). However, we found that TMAO was still associated
with mortality after adjustment for age and eGFR.
In this study, we found that ischemic stroke had higher TMAO than
healthy controls. A previous study found that higher TMAO levels were
associated with increased risk of the first stroke in hypertensive patients
(Nie et al., 2018). However, it should be noted that while the majority of
studies in different cohorts have observed a positive association between
systemic TMAO levels and incident cardiovascular risks (Shafi et al.,
2017; Senthong et al., 2016), others have not (Kaysen et al., 2015;
Mueller et al., 2015). One study showed that stroke and transient
ischemic attack patients showed significant dysbiosis of the gut micro­
biota, and their blood TMAO levels were decreased (Yin et al., 2015).
Similarly, a European study of 339 patients undergoing coronary angi­
ography found no association between plasma TMAO and prevalent
coronary artery disease or incident events over an 8-year follow-up
period. (Mueller et al., 2015). In a North American study of 817
healthy mixed-race volunteers aged 33 to 45, plasma TMAO levels were
not associated with incident coronary artery calcification over a 10-year
follow-up period (Meyer et al., 2016). In this study, the median levels of
TMAO in stroke and controls were 6.1 μM and 4.0 μM. A previous study
in the Chinese population reported that the average and median TMAO
levels of stroke/TIA patients were only 2.68uM and 1.91 uM, respec­
tively, comparable to quartile 1 in a US cohort (Koeth et al., 2013). The
range of TMAO levels in different study groups was vast (Supplementary
materials). These apparently conflicting data are likely to have resulted
from various issues, including differences in study methodology, diet,
ethnicity, environment, and prevalent patterns of gut microbiome
composition in study subjects.
Alterations to the normal gut microbiota or gut dysbiosis caused by
acute brain lesions can affect neuroinflammatory and immune responses
in the brain and worsen neurological function (Singh et al., 2016).
Commensal gut microbiota exerts protection against ischemic damage,
and their depletion or dysbiosis increases poststroke mortality in mice
and influences stroke outcome (Winek et al., 2016). In ischemic stroke,
gut dysbiosis via stress-mediated intestinal paralysis can alter T-cell
homeostasis, promote migration of T cells from the intestine to the
ischemic brain, and increase proinflammatory responses resulting in
poor stroke outcome (Benakis et al., 2016). In ischemic stroke, gut
dysbiosis, and increased bacterial counts of Lactobacillus ruminis sub­
group in the fecal gut microbiota was associated with elevated systemic
inflammation and altered the balance in favor of proinflammatory
mechanism (Arpaia et al., 2013).
Altered TMAO levels have been associated with impaired cardiac
function, heart attack, and heart failure (Zhu et al., 2016). The role of
elevated TAMO in stroke prognosis might, through the following
mechanisms: (1) One study suggested that aging increases circulating
TMAO levels, which may impair eNOS-derived NO bioavailability by
increasing vascular inflammation and oxidative stress, contributing to
aging-associated endothelial dysfunction in aged rats (Li et al., 2017). In
part, endothelial dysfunction resulting from excessive production of
reactive oxygen species and inflammation is an essential mechanism of
cerebrovascular damage (Moskowitz et al., 2010). Recent studies
demonstrated that TMAO promotes vascular inflammation and oxida­
tive stress, inhibits eNOS expression and activity, and reduces NO pro­
duction, which is associated with endothelial dysfunction and
atherosclerosis (Wang et al., 2011; Seldin et al., 2016; Chen et al., 2016).
(2) TMAO mediated vascular inflammation by activating the nucleotide-
binding oligomerization domain-like receptor family pyrin
domain–containing 3 (NLRP3) inflammasome via the sirtuin-3 (SIRT3)–
superoxide dismutase (SOD)–mitochondrial reactive oxygen species
(mtROS) signaling pathway (Chen et al., 2017). (3) One study showed
that gut microbes, through the generation of trimethylamine N-oxide
(TMAO), directly contribute to platelet hyperreactivity and enhanced
thrombosis potential (Zhu et al., 2016), and direct exposure of platelets
8
Journal of Neuroimmunology 354 (2021) 577526
to TMAO enhanced sub-maximal stimulus-dependent platelet activation
from multiple agonists through augmented Ca (2+) release from intra­
cellular stores (Zhu et al., 2016). TMAO promotes an imbalance in
cholesterol uptake and effluxes from macrophages, leading to an
abundance of pro-atherogenic foam cells migrating to the arterial wall
(Wang et al., 2011; Koeth et al., 2013).
Some study limitations need to be considered. (1) only a single time-
point blood draw after an overnight fast was available, and we did not
have dietary history to confirm or refute the impact of diet on TMAO. (2)
the relatively small number of mortalities end-points (N = 47) and lack
of data on cause of death, preventing us from obtaining a more nuanced
understanding of the relationship between TMAO and cardiovascular
mortality. (3) the potential for confounding by unmeasured risk factors,
such as dietary patterns and proteinuria. Specifically, we did not have
any information regarding gastrointestinal symptoms and pathology or
prior antibiotic use or knowledge of previous supplements other than
those taken on the day of enrollment. Thus, the effects of antibiotic use
in modulating gut microbiome–mediated TMAO synthesis will need to
be incorporated in future studies in this field. (4) in this study, only
ischemic strokes were included. Interestingly, one study demonstrated
that TMAO has a stronger association with hemorrhagic stroke than
ischemic stroke (Nie et al., 2018). (5) The variants of the FMO3 gene,
which could provide opportunities to assess the impact of reduced
TMAO production on CV disease, were not tested in this study. Inter­
estingly, in a separate cohort, no association was identified between 471
FMO gene polymorphisms and plasma TMAO levels in 3865 subjects
referred for cardiac evaluation (Hartiala et al., 2014). (6) TMAO plasma
level was measured at only a single time point, and we did not test
TMAO in other time-point and cerebral spinal fluid samples. One study
suggested that TMAO can be assessed in human CSF (Del Rio et al.,
2017). (7) It is not known whether decreasing systemic TMAO using
antibiotics or diet improve the stroke outcome in humans. Observational
studies challenge the proposed relationships between dietary choline or
betaine intakes with CVD outcomes (Meyer and Shea, 2017). Unfortu­
nately, as a cross-sectional study, the obtained results could not prove
any causal relationship.
6. Clinical perspective
In this study, the data suggests that the elevated serum level of gut
microbial TMAO is a potential useful biomarker for predicting poor
prognosis, and therefore it is useful for therapeutic decision making in
patients with ischemic stroke. Whether TMAO reduction treatment
could improve stroke prognosis need further conform.
7. Conclusions
Fasting plasma concentrations of gut microbial TMAO are higher in
patients with ischemic stroke and portend higher poor functional
outcome events and mortality risks independent of traditional risk fac­
tors. The present studies suggest a potential clinical utility for clearance
of gut microbiome generated uremic toxins as a possible adjunct therapy
for stroke. Future large-scale studies specifically designed to examine
the predictive value of TMAO for cardiovascular and mortality outcomes
in stroke populations will be necessary to determine the generalizability
of our initial observations to these separate groups.
Conflict of interest disclosures
None.
Funding/support
This work was supported by the Xiamen Health Care Guidance
Program (No. 3502Z20209227).
J. Zhang et al. Journal of Neuroimmunology 354 (2021) 577526

Acknowledgment containing monooxygenase 3 as a potential player in diabetes-associated

atherosclerosis. Nat. Commun. 6, 6498.
Moskowitz, M.A., Lo, E.H., Iadecola, C., 2010. The science of stroke: mechanisms in
All authors have contributed significantly and agreed with the con­ search of treatments. Neuron. 67, 181–198.
tent of the manuscript. We are grateful to all the people who participated Mueller, D.M., Allenspach, M., Othman, A., et al., 2015. Plasma levels of trimethylamine-
in our study. We also thank Andrea Baird, MD, from Edanz Editing China N-oxide are confounded by impaired kidney function and poor metabolic control[J].
Atherosclerosis 243 (2), 638–644.
(www.liwenbianji.cn/ac), for editing the English text of a draft of this Nie, J., Xie, L., Zhao, B., et al., 2018. Serum trimethylamine N-oxide concentration is
manuscript. positively associated with first stroke in hypertensive patients[J]. Stroke 49 (9),
2021–2028.
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