Cardiovascular diseases (CVDs) remain a leading cause of death worldwide and their prevalence is increasing in the general population [1, 2]. The non-ST-segment elevation myocardial infarction (NSTEMI) is a frequent type of CVDs undergoing coronary intervention and reported with two-fold higher long-term adverse outcomes than patients with ST-segment elevation myocardial infarction (STEMI) [3, 4]. Nevertheless, sufficient consideration attributed to the reduction of the traditional risk factors including hyperlipidemia, smoking, hypertension, diabetes mellitus, and treatment with novel pharmacotherapies only decreased 30% of adverse outcomes related with CVDs [5, 6, 7]. Hence, identification of novel pathogenic risk factor related to CVDs has important public health significance for disease prevention and early stratification [8].

Recently, trimethylamine N-oxide (TMAO) metabolite gained widespread attention and purposed to play a potential role in adverse outcomes and pathogenesis for CVDs [9, 10, 11, 12, 13]. A significant association of the TMAO with the atherosclerotic burden in coronary vessels has been reported in STEMI and stable coronary artery disease (CAD) [14, 15]. However, the relationship between high coronary atherosclerotic burden and TMAO is yet to be explored in NSTEMI patients. In present study, we examined the relationship of the plasma TMAO levels with metrics that reflect coronary atherosclerotic burden, including the number of diseased coronary vessels and clinical risk scores including the SYNTAX and GENSINI scores in newly diagnosed NSTEMI patients.

In this prospective cohort, the mean age (60.1 $\pm$ 12 versus 56 $\pm$ 7.7, P = 0.083) and male (83.6% versus 77.1%, P = 0.290) were noted in NSTEMI and Healthy groups respectively.

The baseline characteristics of the NSTEMI group stratified by MVD, SYNTAX and GENSINI scores are shown in Table 1. The fifty-two patients (71.2%) had MVD and twenty-one patients (28.7%) had SVD. The median SYNTAX score 23 (IQR: 11.5-29) and median GENSINI score 38 (IQR: 20-64) were noted. Comparable baseline characteristics were seen between MVD versus SVD, and intermediate-high risk (score $\ge$ 23) versus low risk (score 23) SYNTAX and GENSINI scores (P $>$ 0.05).

The median plasma TMAO level (0.59 $\mu$M; interquartile range [IQR]: 0.43-0.78) was significantly higher in the NSTEMI group than the individuals in Healthy group (0.42 $\mu$M; IQR: 0.33-0.64; P = 0.006; Fig. 1). Within the NSTEMI group, significantly higher plasma TMAO levels in the MVD versus SVD (P = 0.002), and intermediate-high risk (score $\ge$ 23) versus low risk (score 23) of SYNTAX (P = 0.003) and GENSINI (P = 0.005) were observed (Fig. 2 and Table 1). There was a strong positive correlation between plasma TMAO levels and SYNTAX score (Spearman’s correlation: r = 0.28, P = 0.016), GENSINI score (Spearman’ correlation: r = 0.26, P = 0.025), and the number of coronary vessels affected (Spearman’s correlation: r = 0.25, P = 0.034).

Fig. 1.

TMAO levels in NSTEMI and Healthy groups. TMAO, trimethylamine N-oxide.

Fig. 2.

Comparison of TMAO levels in Low versus Intermediate-High Atherosclerotic Burden. TMAO levels in low risk versus intermediate-high risk SYNTAX (A) and GENSINI (B) scores, and number of vessel disease (C). MVD, multivessel disease; SVD, single vessel disease; TMAO, trimethylamine N-oxide.

Multivariate logistic regression adjusting analysis found that plasma TMAO level was independently associated with MVD (odds ratio [OR]: 5.94, P = 0.005), and intermediate-high risk (score $\ge$ 23) SYNTAX (OR: 3.61, P = 0.013) and GENSINI scores (OR: 4.60, P = 0.008) following adjustment of traditional risk factors (including age, sex, hypertension, diabetes mellitus, smoking, LDL, TG, eGFR, hs-CRP, and BMI) as displayed in Table 2. Additionally, the AUC for TMAO predicted high atherosclerotic burden as MVD (AUC: 0.73, P = 0.002), and intermediate-high risk (score $\ge$ 23) SYNTAX (AUC: 0.70, P = 0.003) and GENSINI scores (AUC: 0.70, P = 0.005) as illustrated in Fig. 3.

Fig. 3.

Relationship of TMAO with High Atherosclerotic Burden. The ROC curves of TMAO for predicting a high atherosclerotic burden as intermediate-high risk SYNTAX (A) and GENSINI (B) scores, and MVD (C). AUC, area under the receiver-operating characteristic curve; Cl, confidence interval; MVD, multivessel disease.

The main finding of this study was a significant association between plasma TMAO levels and high coronary atherosclerotic burden as stratified by number of disease vessels and clinical risk scores including SYNTAX and GENSINI scores in NSTEMI patients. Moreover, TMAO independently predicted the MVD, and intermediate-high risk (score $\ge$ 23) SYNTAX and GENSINI scores, even following traditional risk factors adjustment. In all, TMAO predicts high coronary atherosclerotic burden as quantified above.

Atherosclerosis is a significant clinical problem leading to ischemia in different parts of the vasculature [19, 20, 21]. In this occurs in the coronary vessels, then CAD with calcification [22], partial or total vessel occlusion [23, 24, 25], leading to gradual reduction or absent coronary blood flow [21, 26]. This can manifest as silent asymptomatic disease, angina pectoris [27, 28], NSTEMI or STEMI [29, 30, 31], with adverse consequences such as arrhythmias, heart failure, and death [32]. Therefore, there is an increasing interest to develop predictive risk models for accurate risk stratification, which may include clinical signs or symptoms, imaging results and biomarkers [33]. The TMAO is a gut metabolite, generated from dietary nutrients such as choline, L-carnitine, betaine, and phosphatidylcholine [9, 34, 35]. There is growing appreciation that TMAO has mechanistic links to proatherogenic effect via increased macrophage foam cell formation, activation of the inflammatory and platelet hyperactivity pathways, impaired cholesterol and bile acid transport [9, 10, 11, 12, 34, 35, 36, 37]. These all impaired factors are associated with high-risk CVDs including stroke, atherosclerosis, atrial fibrillation, heart failure, and chronic kidney disease [9, 10, 11, 12, 20, 34, 35, 36, 37, 38]. Based on experimental animal studies, TMAO accelerates atherosclerotic progression by inhibiting reverse cholesterol transport, enhancing platelet activity and thrombosis, and activating macrophages, while targeting production of TMAO inhibit pathogenesis of atherosclerotic [9, 10, 34, 35, 36, 37]. Recent clinical data demonstrated a close relationship of TMAO with markedly high short and long-term adverse outcomes related to CVDs even following adjustment for confounding risk variables [11, 12, 13, 38]. More recently, Yoriko et al. further appraised that long-term increases in TMAO were linked to a higher risk for CVDs and repeated evaluation of TMAO over 10 years increased the early identification of individuals with a greater CVD-related risk [39].

This study is the first, to the best of the authors’ knowledge, to evaluate the relationship between TMAO and coronary atherosclerotic burden in NSTEMI patients. Our study further contributes to earlier findings those suggested a significant link between plasma TMAO and atherosclerotic burden in STEMI and stable CAD patients [14, 15]. Sheng et al. showed a significant association between plasma TMAO levels and high atherosclerotic burden as ascertained by the SYNTAX score and MVD in STEMI [14]. Senthong et al. noted that strong predictability of TMAO for high coronary atherosclerotic burden as evaluated via SYNTAX score stable CAD [15]. Lately, Guo et al. also observed a positive linked of TMAO level with of a high atherosclerotic burden particularly severe coronary artery lesion ($\ge$ 90% stenosis) compared to mild coronary artery lesion in CAD patients [40].

The strength of our study that TMAO was evaluated in multiple parameters for quantification and stratification of coronary atherosclerotic burden including clinical risk scores such as SYNTAX and GENSINI, and MVD as compared to prior studies evaluated TMAO in only SYNTAX score and/or MVD [14, 15]. A high SYNTAX and GENSINI scores, and MVD are associated with poor prognosis and considered as clinical markers of coronary atherosclerotic burden [3, 17, 41]. Notably, the median SYNTAX score 23 (IQR: 11.5-29) in NSTEMI patients recruited in the present cohort were seemingly higher than STEMI patients described by Sheng et al. 18 (IQR: 11-23.5) and stable CAD patients by Senthong et al. 11 (IQR: 4-18.5), which might be partially attributed to higher proportion of patients (56.1%) in intermediate-high risk SYNTAX score category in the present cohort compared to Sheng et al. (27.8%) and Senthong et al. (18.1%) [14, 15]. Moreover, our study included NSTEMI patients with significant coronary lesion ($\ge$ 70% stenosis) as compared to prior studies $\ge$ 50% [14, 15] and that might further strengthened our observation for association of TMAO with significant coronary lesion ($\ge$ 70% stenosis). In fact, Guo et al. reported strong correlation of TMAO for server coronary artery stenosis compared to mild coronary artery stenosis [40]. Furthermore, our study observed a lower concentration of TMAO in patients with NSTEMI than previously reported studies in other types of CAD [14, 15, 40]. Indeed, Wang et al. reported a wide range of TMAO concentrations (lower limit 0.05 $\mu$M to upper limit 200 $\mu$M) with $\ge$ 95% accuracy determination by isotope dilution high-performance liquid chromatography among 4000 subjects including both healthy individuals and cardiovascular patients [18]. These differences may be caused by many factors associated with different populations particularly demographic characteristics including age, sex, and ethnicity, different types of CAD and criteria for severity, Eastern and Western dietary habits, and composition of gut microbiota [42, 43, 44, 45, 46]. Furthermore, Liu et al. reported significant alteration in gut microbiota profiles in different CAD subtypes of patients and atherosclerotic severity, however they did not observe these changes in TMAO but purposed accordingly to their findings [42]. Therefore, more investigations that cover all these points are required for further explanation.

Recently, there is accumulating evidence to suggest TMAO is a clinically relevant target or marker for risk stratification and short or long-term prognostic outcome related in CVDs [11, 12, 14, 15]. Moreover, inhibition of TMAO has been identified as a novel therapeutic approach for the prevention of atherosclerosis progression compared with existing traditional treatment [10, 11, 12, 37, 39, 43, 44, 45, 46]. In light of these promising findings, implication of TMAO may establish early diagnosis, risk stratification, optimal treatment, and preventive strategies for CVDs [10, 11, 12, 37, 39, 43, 44, 45, 46].

In conclusion, our study demonstrated that plasma TMAO levels were significantly associated with a high coronary atherosclerotic burden as quantified by the number of diseased vessels, and clinical risk scores including SYNTAX and GENSINI scores in patients with NSTEMI. We encouraged and warranted future large, randomized trial to evaluate our observed results in NSTEMI patients for the clinical endpoints.

CAD, coronary artery disease; CAG, coronary angiography; CVDs, cardiovascular diseases; ECG, electrogram; MVD, multiple vessels disease; NSTMEI, non-ST-segment myocardial infarction; STEMI, ST-segment myocardial infarction; SVD, single vessel disease; SYNTAX, SYNergy between percutaneous coronary intervention with TAXus and cardiac surgery; TMAO, trimethylamine N-oxide.

Lili W, Junlei C, and Bin Waleed K were involved to research design, protocol, submission to ethical committee, and trial registration. Bin Waleed K, Pengnong C, Tu H, Ding L, Yangkong L, Houqing Z, Qiying C, and Aimei S contributed to literature review, patients selection accordingly to inclusion and exclusion criteria, informed consent, and data collection and interpretation of data. Yunlong X, Shulin W, Tse G, Xintao L, and Afrasyab A underwent statistical analysis and manuscript writing. All authors are involved in final draft of the manuscript, read and approved the final manuscript.

The study was conducted in accordance with the Declaration of Helsinki and the Ethics Committee of Fuwai Hospital Chinese Academy of Medical Sciences Shenzhen (Ethics approval number: YN201901) and registered clinical study (ChiCTR1900022366) as SZ-NSTEMI. The study can be identified http://www.chictr.org.cn/showprojen.aspx?proj=37821. Written informed consent was obtained from all participants.

We thanked all participated individuals in this study.

This study was funded by the Science Technology and Innovation Commission of Shenzhen Municipality (ZDSYS20190902093409851), Guangdong Innovation Platform of Translational Research for Cerebrovascular Diseases, and National Natural Science Foundation of China (81971634).

The authors declare no conflicts of interest.