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Gut Microbial Metabolite Trimethylamine N-Oxide Is Related to Thrombus Formation in Atrial Fibrillation Patients
ARTICLE IN PRESS CLINICAL INVESTIGATION
Gut Microbial Metabolite Trimethylamine N-Oxide Is Related to Thrombus Formation in Atrial Fibrillation Patients Dingxu Gong, MD, PhD1, Lin Zhang, MD, PhD2, Ying Zhang, MD3, Fang Wang, MD, PhD4, Zhenwen Zhao, PhD5 and Xianliang Zhou, MD, PhD2 1 Department of Cardiac Surgery; 2 Department of Cardiology; 3 Peking Union Medical College, Department of Cardiology; 4 Department of Clinical Laboratory, Fuwai Hospital Chinese Academy of Medical Science and the National Center for Cardiovascular Disease of China, Beijing, China; 5 Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of Sciences, University of Chinese Academy of Sciences, Beijing, China
ABSTRACT Background: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia. Patients with AF are prone to forming cardiac thrombi. Elevated serum trimethylamine N-oxide (TMAO) levels are associated with increased thrombosis risk. No previous studies have examined the relationship between serum TMAO levels and thrombus formation in AF patients. Materials and Methods: A total of 117 consecutive rheumatic heart disease patients with AF were enrolled. The patients were divided into 2 groups: patients with thrombi (n = 25) and patients without thrombi (n = 92). Platelet function tests were performed by light transmittance aggregometry. Serum TMAO, betaine and choline levels were quantified by liquid chromatography combined with tandem mass spectrometry. Results were compared between the 2 groups. The correlation between serum TMAO levels and thrombi formation was examined. Results: No remarkable differences in demographic characteristics were found between the 2 groups. Serum TMAO levels were significantly higher in the thrombus group (4.55 UM [3.19-4.83] vs. 3.53 UM [2.96-4.25], P = 0.01). Enhanced platelet hyperreactivity was more likely in the thrombus group. Receiver operating characteristic analysis showed the diagnostic potential of serum TMAO levels to identify thrombus formation, with an area under the curve of 0.661 (P = 0.01, 95% confidence interval: 0.52-0.80). Binary regression analyses showed that serum TMAO had potent predictive power for thrombus formation (P < 0.01, 95% CI of 1.21-3.08). Conclusions: Elevated serum TMAO levels were predictive of thrombus formation in AF patients. Our results highlight the usefulness of serum TMAO levels in identifying individuals with increased susceptibility to thrombus formation, allowing development of precise thrombus prevention strategies. Key Indexing Terms: Trimethylamine N-oxide; Atrial fibrillation; Platelet function; Thrombus formation. [Am J Med Sci 2019;&(&):1–7.]
INTRODUCTION
trial fibrillation (AF) is the most prevalent cardiac arrhythmia, and it increases the risk of morbidity and mortality because of a substantially increased risk of stroke and systemic thromboembolism.1 In patients with AF, the incidence of embolism is 7 times higher when compared with individuals with sinus rhythm.2 In rheumatic heart disease patients, AF and thrombus formation are common. The reason some AF patients develop thrombi while others do not is unknown. The intestinal microbiota in our body is composed of trillions of nonpathogenic organisms. It plays a great role
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in the digestion and absorption of many nutrients.3 The intestinal microbial communities form a “plastic” endocrine organ that interfaces with the host and contributes to the pathogenesis of cardiovascular diseases.4 Foods such as meat, egg yolks and high-fat dairy products are rich in the lipid phosphatidylcholine, and they are believed to be the major dietary sources for choline. Catabolism of choline and other trimethylamine (TMA)containing species (for example, betaine) by intestinal microbes forms TMA.5 TMA is absorbed and metabolized by hepatic flavin monooxygenase enzymes to form TMAO.6 An association between elevated blood TMAO levels and increased thrombosis risk has been recently
Copyright © 2019 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved. www.amjmedsci.com www.ssciweb.org
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ARTICLE IN PRESS Gong et al
identified.7 Platelet activation and aggregation are key steps in thrombus formation.8 The gut microbial metabolite TMAO can enhance platelet hyperreactivity,7 and enhanced platelet reactivity is associated with both thrombus formation and the extent of end-organ injury.9 Gut microbiota can also regulate thrombus formation via Toll-like receptor-2.10 Although many environmental factors converge to promote thrombus formation, we studied the relationship between microbe metabolite TMAO and thrombus formation in AF patients.
METHODS Participants and Study Design The present study was a prospective observational cohort study. It was approved by a local institutional review board. Between April 2015 and August 2016, 117 consecutive valvular heart disease patients with AF were enrolled after exclusion. The exclusion criteria were as follows: informed consent not obtained; antiplatelet drug usage; had the end-stage renal disease; New York Heart Association cardiac function greater than or equal to class III and lung or liver function impairment. The enrolled patients were then divided into 2 groups: group I, AF patients with thrombus formation (n = 25), and group II, AF patients without thrombus formation (n = 92). Baseline characteristics of the 2 groups are summarized in Table 1. Platelet function and serum TMAO, betaine and choline levels were measured and compared between the 2 groups. Blood Specimen Collection Blood samples were drawn from each participant at 6 AM before breakfast. All the blood samples were obtained from the peripheral vein. For the analysis of platelet function, citrated whole blood (3 mL) was sent to the lab within 60 minutes. For the quantification of TMAO, choline and betaine, specimens were stored in sealed vials at 80°C after centrifugation. Platelet Function Test Light transmittance aggregometry is considered the gold standard for determining platelet function.11 Platelet aggregation levels induced by arachidonic acid (AA, 0.5 mmol/L), adenosine diphosphate (ADP, 10 mmol/L), collagen (COL, 0.2 mg/mL) and adrenaline (ADR, 0.4 mg/ mL) were measured with light transmittance aggregometry (automatic platelet aggregation analyzer LBY-NJ4A, Techlink Biomedical, Beijing, China) in platelet-rich plasma in this study. The citrated whole blood was centrifuged at 120 £ g for 5 minutes, and platelet-rich plasma was obtained. The blood-citrate mixture was then subjected to further centrifugation at 850 £ g for 5 minutes to obtain platelet-poor plasma. Platelets were stimulated with AA, ADP, COL and ADR, respectively. Platelet aggregation levels were expressed as the 2
maximal percent change in light transmittance from the baseline, using platelet-poor plasma as a reference.
Quantification of TMAO, Choline and Betaine Ultra−high-performance liquid chromatography combined with tandem mass spectrometry was used for quantification of TMAO, choline and betaine. All of the biological samples were extracted based on the methanol method, as we previously reported.12 In brief, 25 mL of human plasma was added into 1,000 mL MeOH containing 100 pmol of 1-stearoyl-2-hydroxy-sn-glycero-3phosphocholine (12:0 LPC, internal standard). After vortex and centrifugation at 13,225 £ g for 10 minutes at room temperature, the supernatants were collected for direct analysis of TMAO, choline and betaine. Supernatants (5 mL) were analyzed by injection onto a silica column (2.1 mm £ 100 mm, 5 mm). A discontinuous gradient was generated to resolve the analytes by mixing solvent A (10 mmoL/L ammonium formate, Ph = 3.0) with solvent B (acetonitrile) at different ratios. Chromatographic conditions and mass spectrometry methods were based on the ultra−high-performance liquid chromatography combined with tandem mass spectrometry method built by Ocque.13 The intraday variation coefficient was 2.13-2.94%. The interday variation coefficient was 6.80-10.13%. The recovery rate was 91.44103.63%. When the serum was stored at 4°C, the concentration of supernatants changed within 10% within 24 hours.
Clinical Data Collection All clinical data were collected with the medical record system of Fuwai Hospital by trained study personnel. Baseline demographic characteristics, variables that can affect platelet function and thrombus formation were collected.
Statistical Analysis Statistical analyses were performed to compare baseline characteristics, platelet function, plasma TMAO, choline and betaine levels among different patient groups. Continuous variables are summarized by means and standard deviations (or medians and interquartile range if the distribution was skewed). Numbers and percentages were used to summarize categorical data. The chi-squared test was used to compare the difference for categorical variables, and the Student's t test or Wilcoxon rank-sum test were used for continuous variables. Receiver operating characteristic (ROC) analyses and binary regression analyses were performed to determine the correlation between serum TMAO and thrombus formation. All reported P values are 2-sided, and a P value less than 0.05 was considered statistically significant. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME & NUMBER & & 2019
ARTICLE IN PRESS Gut Microbial Metabolite and Thrombus Formation
TABLE 1. Baseline characteristics. Characteristics Men, n (%) Age, years, median (IQR) BMI Hb (g/L) Platelet count (£109/L) CAD, n (%) Diabetes, n (%) Hypertension, n (%) Heart failure, n (%) Hyperlipidemia, n (%) Previous stroke, n (%) Serum creatinine (mmol/L) LDL (mmol/L) HDL (mmol/L) Triglyceride (mmol/L)
Group I (AF with thrombus, n = 25)
Group II (AF without thrombus, n = 92)
P Value
9 (36.00%) 57 (50-64) 23.28 § 2.28 139.84 § 17.48 180.12 § 61.50 2 (8.00%) 1 (4.00%) 3 (12.00%) 0 (0.00%) 5 (20.00%) 2 (8.00%) 79.79 § 18.61 2.69 § 0.39 1.81 § 0.47 1.20 § 0.40
39 (42.40%) 56 (50-62) 23.80 § 3.21 140.16 § 15.54 193.00 § 58.12 3 (3.30%) 2 (2.20%) 11 (12.00%) 6 (6.50%) 7 (7.60%) 5 (5.40%) 76.97 § 16.21 2.67 § 0.31 1.71 § 0.49 1.28 § 0.34
0.57 0.70 0.49 0.93 0.33 0.29 0.52 1.00 0.34 0.13 0.64 0.46 0.82 0.37 0.30
Abbreviations: AF, atrial fibrillation; BMI, body mass index; CAD, coronary artery disease; Hb, hemoglobin; HDL, high-density lipoproteins; LDL, low-density lipoproteins.
RESULTS Baseline Characteristics We excluded 11 patients according to the exclusion criteria. A total of 117 patients were enrolled for final analysis. Table 1 shows the baseline demographic and clinical characteristics. We found no remarkable differences in the demographic characteristics between the 2 groups (Table 1). Platelet counts were comparable between these 2 groups (180.12 § 61.50 £ 109/L in Group I vs. 193.00 § 58.12 £ 109/L in Group II, P = 0.33).
Patients With Thrombus Formation Showed Higher TMAO, Choline and Betaine Levels Circulating TMAO is the most common gut microbiota metabolite. Choline and betaine are precursors of TMAO. Previous studies have shown elevated gut microbiota metabolite TMAO levels to be associated with elevated platelet reactivity and thrombosis risk.4,7 In our study, the gut microbiota metabolites levels between the AF patients with thrombus formation and without thrombus formation were compared, and we found that AF patients with thrombus (group I) formation showed higher microbial metabolite levels. Circulating TMAO, choline and betaine levels were obviously higher in patients with thrombus formation (TMAO, group I: 4.55 [3.19-4.83] mM, group II: 3.53 [2.96-4.25] mM, P = 0.01;
betaine, group I: 72.58 [62.88-86.72] mM, group II: 63.51 [50.92-72.68] mM, P = 0.02 and choline, group I: 16.96 [11.24-21.82] mM, group II: 13.65 [9.22-17.50] mM, P = 0.01; Table 2). Patients With Higher TMAO Levels Had Enhanced Platelet Hyperreactivity To study the relationship between the gut microbiota metabolite TMAO levels and platelet reactivities, platelet functions were determined by light transmittance aggregometry and compared between the 2 groups. Patients in group I with higher microbial metabolites levels had enhanced platelet hyperreactivity. The maximal percent changes of platelet aggregation were higher in group I patients (COL, group I: 70.97 [47.66-81.16], group II: 41.74 [30.54-54.14], P < 0.01; ADR, group I: 64.04 [47.32-80.14], group II: 39.41 [25.81-50.76], P < 0.01; ADP, group I: 55.31 [43.49-64.01], group II: 36.42 [27.37-44.97], P < 0.01 and AA, group I: 68.61 [57.3480.32], group II: 37.19 [25.45-47.95], P < 0.01; Table 3). Regression analyses identified that serum TMAO was related to enhanced platelet hyperreactivity when stimulated with ADP (P < 0.05, 95% CI of 1.21-3.08). Spearman correlation plot analysis found that TMAO serum levels are positively related to platelet aggregation percentages (Figure 1). The correlation coefficient between serum TMAO levels and platelet aggregation percentages
TABLE 2. Serum TMAO, betaine and choline levels. Variables TMAO, mM, median (IQR) Betaine, mM, median (IQR) Choline, mM, median (IQR)
Group I (AF with thrombus)
Group II (AF without thrombus)
P Value
4.55 (3.19-4.83) 72.58 (62.88-86.72) 16.96 (11.24-21.82)
3.53 (2.96-4.25) 63.51 (50.92-72.68) 13.65 (9.22-17.50)
0.01 0.02 0.01
Abbreviations: AF, atrial fibrillation; IQR, interquartile range; TMAO, trimethylamine-N-oxide.
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TABLE 3. Platelet function (platelet aggregation test). Agonists Collagen (0.2 mg/mL), (%), median (IQR) Adrenaline (0.4 mg/mL), (%), median (IQR) ADP (10 mmol/L), (%), median (IQR) Arachidonic acid (0.5 mmol/L), (%), median (IQR)
Group I (AF with thrombus)
Group II (AF without thrombus)
70.97 (47.66-81.16) 64.04 (47.32-80.14) 55.31 (43.49-64.01) 68.61 (57.34-80.32)
41.74 (30.54-54.14) 39.41 (25.81-50.76) 36.42 (27.37-44.97) 37.19 (25.45-47.95)
P Value <0.01 <0.01 <0.01 <0.01
Abbreviations: AF, atrial fibrillation; ADP, adenosine diphosphate; IQR, interquartile range.
of each agonists are as follows: COL, 0.28 (P < 0.01); ADR, 0.31 (P < 0.01); ADP, 0.53 (P < 0.01) and AA, 0.32 (P < 0.01).
Higher TMAO Level Was Related to Thrombus Formation ROC analysis was used to determine the ability of TMAO to identify patients at risk of thrombus formation. ROC analysis showed the diagnostic potential of
serum TMAO levels to identify thrombus formation, with an area under the curve of 0.66 (P = 0.01, 95% CI: 0.52-0.80) and relatively high sensitivity and specificity (Figure 2). After adjusting for other factors associated with thrombus formation (including platelet count, hyperlipidemia, diabetes, hypertension and heart failure), binary regression analyses identified serum TMAO as a significant predictor of thrombus formation in AF patients (P < 0.01, 95% CI of 1.213.08).
FIGURE 1. Spearman correlation plot analysis of trimethylamine-N-oxide (TMAO) serum levels with platelet aggregation percentages when stimulated with collagen (A), adrenaline (B), adenosine diphosphate (ADP) (C), arachidonic acid (AA) (D).
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ARTICLE IN PRESS Gut Microbial Metabolite and Thrombus Formation
FIGURE 2. Receiver operating characteristic curve analysis of the diagnostic ability of serum trimethylamine-N-oxide (TMAO) level for thrombus formation.
TMAO Increases ADP-Induced Platelet Aggregation in Healthy Donors To show the direct causality between TMAO and platelet hyperreactivity, plasma samples from 20 healthy donors were collected. ADP-induced platelet aggregation was measured after pretreated by TMAO (100 mM, the same concentration as used by Zhu et al).7 A significantly higher platelet aggregation percentage was observed when compared with itself before the addition of TMAO (50.89 § 13.95 vs. 29.83 § 7.24, P < 0.01).
DISCUSSION
The present study demonstrated, for the first time, the relationship between gut microbe metabolite, TMAO and thrombus formation in cardiac valvular disease patients with AF. We enrolled 117 consecutive AF patients and divided them into 2 groups: a thrombus group and a nonthrombus group. We then compared gut microbe metabolites levels (including TMAO, betaine and choline) and platelet hyperreactivity between the 2 groups. We found that AF patients with thrombus formation had higher circulating TMAO, betaine and choline levels (Table 2) as well as enhanced platelet hyperreactivity (Table 3). Higher TMAO levels were related to thrombus formation in AF patients.
The mechanisms of thrombus formation in AF patients are complex and involve many risk factors. Congestive heart failure, hypertension, advanced age, diabetes mellitus, prior thromboembolism, vascular disease and female sex are all related to thrombus formation in AF patients.14 The reasons why some AF patients develop thrombi while others do not when taking the same treatments is not known. Recently, the idea that microbes within our gut participate in meta-organismal signaling pathways has raised significant interest. Higher circulating TMAO levels are related to enhanced platelet hyperreactivity.7 In accordance with this, we found that patients with higher TMAO levels have elevated platelet aggregation levels (Table 3). Elevated TMAO levels can also increase thrombosis risk.7 After adjusting serum TMAO levels for other factors that are associated with thrombus formation, binary regression analyses also identified serum TMAO as a significant predictor of thrombus formation in AF patients (P < 0.01, 95% CI of 1.21-3.08). The Spearman correlation of TMAO serum levels with the percentage of platelet aggregation was weak for COL, ADR and AA stimulation and was moderate for ADP stimulation (Figure 1); this result may be because of the relatively small sample size. In a previous study, Perez-Gomez et al found that combined antiplatelet medication plus moderate-
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intensity anticoagulation therapy significantly decreased vascular events compared with anticoagulation alone and was proved to be safe in AF patients.15 Azzam and Zagloul revealed that in valvular heart disease patients with AF, platelet microparticle levels were elevated.16 In the present study, we found that elevated serum TMAO was associated with enhanced platelet hyperreactivity and thrombosis risk in valvular AF patients. Therefore, combined antiplatelet plus anticoagulation therapy may be considered in AF patients with elevated gut microbial metabolites levels. Genetic variation only accounts for the development of cardiovascular diseases in no more than 20% of cases.17,18 It is obvious that environmental factors play a predominant role in these pathologic processes. Ingested foods are the most common environmental factors humans encounter daily. Catabolism of choline and betaine by intestinal microbes forms TMA, which is then absorbed and rapidly metabolized by the hepatic flavin monooxygenase enzymes to form TMAO.19 Foods such as egg yolk, milk, liver, red meat, poultry, shellfish and fish are rich in the lipid phosphatidylcholine, and they are believed to be the major dietary sources for choline, betaine and TMAO production.5 High intake of dietary lipid phosphatidylcholine was reportedly associated with elevated circulating choline, betaine and TMAO levels.19 Elevated circulating TMAO levels were related to thrombus formation in this study. Therefore, to reduce thrombosis risk, valvular AF patients with higher circulating TMAO levels may be informed to modify their dietary habits to consume less TMAO precursors. Gut microbes participate in modulating TMAO responses to dietary precursors.20 However, TMAO responses are highly variable. Recently, Cho et al found that a greater ratio of Firmicutes to Bacteroidetes exhibiting a greater response to dietary TMAO precursor intake.21 This finding indicates that TMAO production is an individualized process depending upon patients’ gut microbiomes. As such, pharmaceutical strategies that increase gut microbiota diversity and restore the symbiotic relationship between gut microbes and patients should be adopted in valvular AF patients, who have higher circulating gut microbial metabolites levels. In conclusion, we found that valvular AF patients with thrombus formation had higher circulating microbe metabolite levels and enhanced platelet hyperreactivity. These findings are significant, as circulating microbe metabolites may be used to identify individuals with increased thrombus susceptibility. Our results have implications for evaluating the risks associated with thrombosis and for developing precise thrombus prevention strategies. Several limitations should be noted when extrapolating results from this study. First, the sample size of this cohort was relatively small. Only patients with AF were enrolled. A multicenter study to enroll more participants is required in the future. Second, because of the limitations of clinical sample collection, a thorough mechanistic study was not performed. 6
AUTHOR CONTRIBUTIONS D.G. and L.Z. conceived of the presented idea. X.Z. developed the theory. Y.Z., F.W. and Z.Z. verified the analytical methods and performed the computations. All authors discussed the results and contributed to the final manuscript.
ACKNOWLEDGMENTS The authors would like to thank Xueli Yang from the Division of Biostatistics at the National Center for Cardiovascular Diseases of China for his assistance in statistical analysis. In addition, the authors thank all doctors and nurses at the Adult Cardiac Surgery Center at Fuwai Hospital for their participation.
REFERENCES 1. January CT, Wann LS, Alpert JS, et al. American College of Cardiology/ American Heart Association Task Force on Practice G. 2014 AHA/ACC/ HRS guideline for the management of patients with atrial fibrillation: a report of the American College of Cardiology/American Heart Association Task Force on practice guidelines and the heart rhythm society. J Am Coll Cardiol. 2014;64. e1-76. 2. Dewar HA, Weightman D. A study of embolism in mitral valve disease and atrial fibrillation. Br Heart J. 1983;49:133–140. 3. Backhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–1920. 4. Tang WH, Wang Z, Levison BS, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013;368: 1575–1584. 5. Zeisel SH, Mar MH, Howe JC, et al. Concentrations of choline-containing compounds and betaine in common foods. J Nutr. 2003;133:1302– 1307. 6. Lang DH, Yeung CK, Peter RM, et al. Isoform specificity of trimethylamine N-oxygenation by human flavin-containing monooxygenase (FMO) and P450 enzymes: selective catalysis by FMO3. Biochem Pharmacol. 1998;56:1005–1012. 7. Zhu W, Gregory JC, Org E, et al. Gut microbial metabolite tmao enhances platelet hyperreactivity and thrombosis risk. Cell. 2016;165:111–124. 8. Tantry US, Bonello L, Aradi D, et al. Consensus and update on the definition of on-treatment platelet reactivity to adenosine diphosphate associated with ischemia and bleeding. J Am Coll Cardiol. 2013;62:2261–2273. 9. Frossard M, Fuchs I, Leitner JM, et al. Platelet function predicts myocardial damage in patients with acute myocardial infarction. Circulation. 2004;110:1392–1397. €ckel S, Kiouptsi K, Lillich M, et al. Gut microbiota regulate hepatic 10. Ja von Willebrand factor synthesis and arterial thrombus formation via Toll-like receptor-2. Blood. 2017;130(4):542–553. 11. Linnemann B, Schwonberg J, Mani H, et al. Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary. J Thromb Haemost. 2008;6:677–683. 12. Zhao Z, Xu Y. An extremely simple method for extraction of lysophospholipids and phospholipids from blood samples. J Lipid Res. 2010;51:652–659. 13. Ocque AJ, Stubbs JR, Nolin TD. Development and validation of a simple UHPLC-MS/MS method for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine. J Pharm Biomed Anal. 2015;109:128–135. 14. Gage BF, Waterman AD, Shannon W, et al. Validation of clinical classification schemes for predicting stroke: results from the national registry of atrial fibrillation. JAMA. 2001;285:2864–2870. 15. Perez-Gomez F, Alegria E, Berjon J, et al. Comparative effects of antiplatelet, anticoagulant, or combined therapy in patients with valvular and nonvalvular atrial fibrillation: a randomized multicenter study. J Am Coll Cardiol. 2004;44:1557–1566. 16. Azzam H, Zagloul M. Elevated platelet microparticle levels in valvular atrial fibrillation. Hematology. 2009;14:357–360.
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17. Ardissino D, Berzuini C, Merlini PA, et al. Influence of 9p21.3 genetic variants on clinical and angiographic outcomes in early-onset myocardial infarction. J Am Coll Cardiol. 2011;58:426–434. 18. Ripatti S, Tikkanen E, Orho-Melander M, et al. A multilocus genetic risk score for coronary heart disease: case-control and prospective cohort analyses. Lancet. 2010;376:1393–1400. 19. Zhang AQ, Mitchell SC, Smith RL. Dietary precursors of trimethylamine in man: a pilot study. Food Chem Toxicol. 1999;37:515–520. 20. Koeth RA, Wang Z, Levison BS, et al. Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013;19:576–585. 21. Cho CE, Taesuwan S, Malysheva OV, et al. Trimethylamine-N-oxide (TMAO) response to animal source foods varies among healthy young men and is influenced by their gut microbiota composition: a randomized controlled trial. Mol Nutr Food Res. 2017;61(1).
Submitted February 11, 2019; accepted September 13, 2019. The study was supported by the National Natural Science Foundation of China (NSFC) (81500238 and 81700282), Peking Union Medical College (PUMC) Youth Fund, and Fundamental Research Funds for the Central Universities (3332016017, 3332016014 and 3332018195). The authors have no conflicts of interest to disclose. The first three authors (DG, LZ and YZ) contributed equally to this work. Correspondence: Dingxu Gong, MD, PhD, Department of Cardiac Surgery, Fuwai Hospital Chinese Academy of Medical Science and the National Center for Cardiovascular Disease of China, Beijing 10027, China (E-mail: [email protected]).
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Gut Microbial Metabolite Trimethylamine N-Oxide Is Related to Thrombus Formation in Atrial Fibrillation Patients Dingxu Gong, MD, PhD1, Lin Zhang, MD, PhD2, Ying Zhang, MD3, Fang Wang, MD, PhD4, Zhenwen Zhao, PhD5 and Xianliang Zhou, MD, PhD2 1 Department of Cardiac Surgery; 2 Department of Cardiology; 3 Peking Union Medical College, Department of Cardiology; 4 Department of Clinical Laboratory, Fuwai Hospital Chinese Academy of Medical Science and the National Center for Cardiovascular Disease of China, Beijing, China; 5 Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of Sciences, University of Chinese Academy of Sciences, Beijing, China
ABSTRACT Background: Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia. Patients with AF are prone to forming cardiac thrombi. Elevated serum trimethylamine N-oxide (TMAO) levels are associated with increased thrombosis risk. No previous studies have examined the relationship between serum TMAO levels and thrombus formation in AF patients. Materials and Methods: A total of 117 consecutive rheumatic heart disease patients with AF were enrolled. The patients were divided into 2 groups: patients with thrombi (n = 25) and patients without thrombi (n = 92). Platelet function tests were performed by light transmittance aggregometry. Serum TMAO, betaine and choline levels were quantified by liquid chromatography combined with tandem mass spectrometry. Results were compared between the 2 groups. The correlation between serum TMAO levels and thrombi formation was examined. Results: No remarkable differences in demographic characteristics were found between the 2 groups. Serum TMAO levels were significantly higher in the thrombus group (4.55 UM [3.19-4.83] vs. 3.53 UM [2.96-4.25], P = 0.01). Enhanced platelet hyperreactivity was more likely in the thrombus group. Receiver operating characteristic analysis showed the diagnostic potential of serum TMAO levels to identify thrombus formation, with an area under the curve of 0.661 (P = 0.01, 95% confidence interval: 0.52-0.80). Binary regression analyses showed that serum TMAO had potent predictive power for thrombus formation (P < 0.01, 95% CI of 1.21-3.08). Conclusions: Elevated serum TMAO levels were predictive of thrombus formation in AF patients. Our results highlight the usefulness of serum TMAO levels in identifying individuals with increased susceptibility to thrombus formation, allowing development of precise thrombus prevention strategies. Key Indexing Terms: Trimethylamine N-oxide; Atrial fibrillation; Platelet function; Thrombus formation. [Am J Med Sci 2019;&(&):1–7.]
INTRODUCTION
trial fibrillation (AF) is the most prevalent cardiac arrhythmia, and it increases the risk of morbidity and mortality because of a substantially increased risk of stroke and systemic thromboembolism.1 In patients with AF, the incidence of embolism is 7 times higher when compared with individuals with sinus rhythm.2 In rheumatic heart disease patients, AF and thrombus formation are common. The reason some AF patients develop thrombi while others do not is unknown. The intestinal microbiota in our body is composed of trillions of nonpathogenic organisms. It plays a great role
A
in the digestion and absorption of many nutrients.3 The intestinal microbial communities form a “plastic” endocrine organ that interfaces with the host and contributes to the pathogenesis of cardiovascular diseases.4 Foods such as meat, egg yolks and high-fat dairy products are rich in the lipid phosphatidylcholine, and they are believed to be the major dietary sources for choline. Catabolism of choline and other trimethylamine (TMA)containing species (for example, betaine) by intestinal microbes forms TMA.5 TMA is absorbed and metabolized by hepatic flavin monooxygenase enzymes to form TMAO.6 An association between elevated blood TMAO levels and increased thrombosis risk has been recently
Copyright © 2019 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved. www.amjmedsci.com www.ssciweb.org
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identified.7 Platelet activation and aggregation are key steps in thrombus formation.8 The gut microbial metabolite TMAO can enhance platelet hyperreactivity,7 and enhanced platelet reactivity is associated with both thrombus formation and the extent of end-organ injury.9 Gut microbiota can also regulate thrombus formation via Toll-like receptor-2.10 Although many environmental factors converge to promote thrombus formation, we studied the relationship between microbe metabolite TMAO and thrombus formation in AF patients.
METHODS Participants and Study Design The present study was a prospective observational cohort study. It was approved by a local institutional review board. Between April 2015 and August 2016, 117 consecutive valvular heart disease patients with AF were enrolled after exclusion. The exclusion criteria were as follows: informed consent not obtained; antiplatelet drug usage; had the end-stage renal disease; New York Heart Association cardiac function greater than or equal to class III and lung or liver function impairment. The enrolled patients were then divided into 2 groups: group I, AF patients with thrombus formation (n = 25), and group II, AF patients without thrombus formation (n = 92). Baseline characteristics of the 2 groups are summarized in Table 1. Platelet function and serum TMAO, betaine and choline levels were measured and compared between the 2 groups. Blood Specimen Collection Blood samples were drawn from each participant at 6 AM before breakfast. All the blood samples were obtained from the peripheral vein. For the analysis of platelet function, citrated whole blood (3 mL) was sent to the lab within 60 minutes. For the quantification of TMAO, choline and betaine, specimens were stored in sealed vials at 80°C after centrifugation. Platelet Function Test Light transmittance aggregometry is considered the gold standard for determining platelet function.11 Platelet aggregation levels induced by arachidonic acid (AA, 0.5 mmol/L), adenosine diphosphate (ADP, 10 mmol/L), collagen (COL, 0.2 mg/mL) and adrenaline (ADR, 0.4 mg/ mL) were measured with light transmittance aggregometry (automatic platelet aggregation analyzer LBY-NJ4A, Techlink Biomedical, Beijing, China) in platelet-rich plasma in this study. The citrated whole blood was centrifuged at 120 £ g for 5 minutes, and platelet-rich plasma was obtained. The blood-citrate mixture was then subjected to further centrifugation at 850 £ g for 5 minutes to obtain platelet-poor plasma. Platelets were stimulated with AA, ADP, COL and ADR, respectively. Platelet aggregation levels were expressed as the 2
maximal percent change in light transmittance from the baseline, using platelet-poor plasma as a reference.
Quantification of TMAO, Choline and Betaine Ultra−high-performance liquid chromatography combined with tandem mass spectrometry was used for quantification of TMAO, choline and betaine. All of the biological samples were extracted based on the methanol method, as we previously reported.12 In brief, 25 mL of human plasma was added into 1,000 mL MeOH containing 100 pmol of 1-stearoyl-2-hydroxy-sn-glycero-3phosphocholine (12:0 LPC, internal standard). After vortex and centrifugation at 13,225 £ g for 10 minutes at room temperature, the supernatants were collected for direct analysis of TMAO, choline and betaine. Supernatants (5 mL) were analyzed by injection onto a silica column (2.1 mm £ 100 mm, 5 mm). A discontinuous gradient was generated to resolve the analytes by mixing solvent A (10 mmoL/L ammonium formate, Ph = 3.0) with solvent B (acetonitrile) at different ratios. Chromatographic conditions and mass spectrometry methods were based on the ultra−high-performance liquid chromatography combined with tandem mass spectrometry method built by Ocque.13 The intraday variation coefficient was 2.13-2.94%. The interday variation coefficient was 6.80-10.13%. The recovery rate was 91.44103.63%. When the serum was stored at 4°C, the concentration of supernatants changed within 10% within 24 hours.
Clinical Data Collection All clinical data were collected with the medical record system of Fuwai Hospital by trained study personnel. Baseline demographic characteristics, variables that can affect platelet function and thrombus formation were collected.
Statistical Analysis Statistical analyses were performed to compare baseline characteristics, platelet function, plasma TMAO, choline and betaine levels among different patient groups. Continuous variables are summarized by means and standard deviations (or medians and interquartile range if the distribution was skewed). Numbers and percentages were used to summarize categorical data. The chi-squared test was used to compare the difference for categorical variables, and the Student's t test or Wilcoxon rank-sum test were used for continuous variables. Receiver operating characteristic (ROC) analyses and binary regression analyses were performed to determine the correlation between serum TMAO and thrombus formation. All reported P values are 2-sided, and a P value less than 0.05 was considered statistically significant. THE AMERICAN JOURNAL OF THE MEDICAL SCIENCES VOLUME & NUMBER & & 2019
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TABLE 1. Baseline characteristics. Characteristics Men, n (%) Age, years, median (IQR) BMI Hb (g/L) Platelet count (£109/L) CAD, n (%) Diabetes, n (%) Hypertension, n (%) Heart failure, n (%) Hyperlipidemia, n (%) Previous stroke, n (%) Serum creatinine (mmol/L) LDL (mmol/L) HDL (mmol/L) Triglyceride (mmol/L)
Group I (AF with thrombus, n = 25)
Group II (AF without thrombus, n = 92)
P Value
9 (36.00%) 57 (50-64) 23.28 § 2.28 139.84 § 17.48 180.12 § 61.50 2 (8.00%) 1 (4.00%) 3 (12.00%) 0 (0.00%) 5 (20.00%) 2 (8.00%) 79.79 § 18.61 2.69 § 0.39 1.81 § 0.47 1.20 § 0.40
39 (42.40%) 56 (50-62) 23.80 § 3.21 140.16 § 15.54 193.00 § 58.12 3 (3.30%) 2 (2.20%) 11 (12.00%) 6 (6.50%) 7 (7.60%) 5 (5.40%) 76.97 § 16.21 2.67 § 0.31 1.71 § 0.49 1.28 § 0.34
0.57 0.70 0.49 0.93 0.33 0.29 0.52 1.00 0.34 0.13 0.64 0.46 0.82 0.37 0.30
Abbreviations: AF, atrial fibrillation; BMI, body mass index; CAD, coronary artery disease; Hb, hemoglobin; HDL, high-density lipoproteins; LDL, low-density lipoproteins.
RESULTS Baseline Characteristics We excluded 11 patients according to the exclusion criteria. A total of 117 patients were enrolled for final analysis. Table 1 shows the baseline demographic and clinical characteristics. We found no remarkable differences in the demographic characteristics between the 2 groups (Table 1). Platelet counts were comparable between these 2 groups (180.12 § 61.50 £ 109/L in Group I vs. 193.00 § 58.12 £ 109/L in Group II, P = 0.33).
Patients With Thrombus Formation Showed Higher TMAO, Choline and Betaine Levels Circulating TMAO is the most common gut microbiota metabolite. Choline and betaine are precursors of TMAO. Previous studies have shown elevated gut microbiota metabolite TMAO levels to be associated with elevated platelet reactivity and thrombosis risk.4,7 In our study, the gut microbiota metabolites levels between the AF patients with thrombus formation and without thrombus formation were compared, and we found that AF patients with thrombus (group I) formation showed higher microbial metabolite levels. Circulating TMAO, choline and betaine levels were obviously higher in patients with thrombus formation (TMAO, group I: 4.55 [3.19-4.83] mM, group II: 3.53 [2.96-4.25] mM, P = 0.01;
betaine, group I: 72.58 [62.88-86.72] mM, group II: 63.51 [50.92-72.68] mM, P = 0.02 and choline, group I: 16.96 [11.24-21.82] mM, group II: 13.65 [9.22-17.50] mM, P = 0.01; Table 2). Patients With Higher TMAO Levels Had Enhanced Platelet Hyperreactivity To study the relationship between the gut microbiota metabolite TMAO levels and platelet reactivities, platelet functions were determined by light transmittance aggregometry and compared between the 2 groups. Patients in group I with higher microbial metabolites levels had enhanced platelet hyperreactivity. The maximal percent changes of platelet aggregation were higher in group I patients (COL, group I: 70.97 [47.66-81.16], group II: 41.74 [30.54-54.14], P < 0.01; ADR, group I: 64.04 [47.32-80.14], group II: 39.41 [25.81-50.76], P < 0.01; ADP, group I: 55.31 [43.49-64.01], group II: 36.42 [27.37-44.97], P < 0.01 and AA, group I: 68.61 [57.3480.32], group II: 37.19 [25.45-47.95], P < 0.01; Table 3). Regression analyses identified that serum TMAO was related to enhanced platelet hyperreactivity when stimulated with ADP (P < 0.05, 95% CI of 1.21-3.08). Spearman correlation plot analysis found that TMAO serum levels are positively related to platelet aggregation percentages (Figure 1). The correlation coefficient between serum TMAO levels and platelet aggregation percentages
TABLE 2. Serum TMAO, betaine and choline levels. Variables TMAO, mM, median (IQR) Betaine, mM, median (IQR) Choline, mM, median (IQR)
Group I (AF with thrombus)
Group II (AF without thrombus)
P Value
4.55 (3.19-4.83) 72.58 (62.88-86.72) 16.96 (11.24-21.82)
3.53 (2.96-4.25) 63.51 (50.92-72.68) 13.65 (9.22-17.50)
0.01 0.02 0.01
Abbreviations: AF, atrial fibrillation; IQR, interquartile range; TMAO, trimethylamine-N-oxide.
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TABLE 3. Platelet function (platelet aggregation test). Agonists Collagen (0.2 mg/mL), (%), median (IQR) Adrenaline (0.4 mg/mL), (%), median (IQR) ADP (10 mmol/L), (%), median (IQR) Arachidonic acid (0.5 mmol/L), (%), median (IQR)
Group I (AF with thrombus)
Group II (AF without thrombus)
70.97 (47.66-81.16) 64.04 (47.32-80.14) 55.31 (43.49-64.01) 68.61 (57.34-80.32)
41.74 (30.54-54.14) 39.41 (25.81-50.76) 36.42 (27.37-44.97) 37.19 (25.45-47.95)
P Value <0.01 <0.01 <0.01 <0.01
Abbreviations: AF, atrial fibrillation; ADP, adenosine diphosphate; IQR, interquartile range.
of each agonists are as follows: COL, 0.28 (P < 0.01); ADR, 0.31 (P < 0.01); ADP, 0.53 (P < 0.01) and AA, 0.32 (P < 0.01).
Higher TMAO Level Was Related to Thrombus Formation ROC analysis was used to determine the ability of TMAO to identify patients at risk of thrombus formation. ROC analysis showed the diagnostic potential of
serum TMAO levels to identify thrombus formation, with an area under the curve of 0.66 (P = 0.01, 95% CI: 0.52-0.80) and relatively high sensitivity and specificity (Figure 2). After adjusting for other factors associated with thrombus formation (including platelet count, hyperlipidemia, diabetes, hypertension and heart failure), binary regression analyses identified serum TMAO as a significant predictor of thrombus formation in AF patients (P < 0.01, 95% CI of 1.213.08).
FIGURE 1. Spearman correlation plot analysis of trimethylamine-N-oxide (TMAO) serum levels with platelet aggregation percentages when stimulated with collagen (A), adrenaline (B), adenosine diphosphate (ADP) (C), arachidonic acid (AA) (D).
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FIGURE 2. Receiver operating characteristic curve analysis of the diagnostic ability of serum trimethylamine-N-oxide (TMAO) level for thrombus formation.
TMAO Increases ADP-Induced Platelet Aggregation in Healthy Donors To show the direct causality between TMAO and platelet hyperreactivity, plasma samples from 20 healthy donors were collected. ADP-induced platelet aggregation was measured after pretreated by TMAO (100 mM, the same concentration as used by Zhu et al).7 A significantly higher platelet aggregation percentage was observed when compared with itself before the addition of TMAO (50.89 § 13.95 vs. 29.83 § 7.24, P < 0.01).
DISCUSSION
The present study demonstrated, for the first time, the relationship between gut microbe metabolite, TMAO and thrombus formation in cardiac valvular disease patients with AF. We enrolled 117 consecutive AF patients and divided them into 2 groups: a thrombus group and a nonthrombus group. We then compared gut microbe metabolites levels (including TMAO, betaine and choline) and platelet hyperreactivity between the 2 groups. We found that AF patients with thrombus formation had higher circulating TMAO, betaine and choline levels (Table 2) as well as enhanced platelet hyperreactivity (Table 3). Higher TMAO levels were related to thrombus formation in AF patients.
The mechanisms of thrombus formation in AF patients are complex and involve many risk factors. Congestive heart failure, hypertension, advanced age, diabetes mellitus, prior thromboembolism, vascular disease and female sex are all related to thrombus formation in AF patients.14 The reasons why some AF patients develop thrombi while others do not when taking the same treatments is not known. Recently, the idea that microbes within our gut participate in meta-organismal signaling pathways has raised significant interest. Higher circulating TMAO levels are related to enhanced platelet hyperreactivity.7 In accordance with this, we found that patients with higher TMAO levels have elevated platelet aggregation levels (Table 3). Elevated TMAO levels can also increase thrombosis risk.7 After adjusting serum TMAO levels for other factors that are associated with thrombus formation, binary regression analyses also identified serum TMAO as a significant predictor of thrombus formation in AF patients (P < 0.01, 95% CI of 1.21-3.08). The Spearman correlation of TMAO serum levels with the percentage of platelet aggregation was weak for COL, ADR and AA stimulation and was moderate for ADP stimulation (Figure 1); this result may be because of the relatively small sample size. In a previous study, Perez-Gomez et al found that combined antiplatelet medication plus moderate-
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intensity anticoagulation therapy significantly decreased vascular events compared with anticoagulation alone and was proved to be safe in AF patients.15 Azzam and Zagloul revealed that in valvular heart disease patients with AF, platelet microparticle levels were elevated.16 In the present study, we found that elevated serum TMAO was associated with enhanced platelet hyperreactivity and thrombosis risk in valvular AF patients. Therefore, combined antiplatelet plus anticoagulation therapy may be considered in AF patients with elevated gut microbial metabolites levels. Genetic variation only accounts for the development of cardiovascular diseases in no more than 20% of cases.17,18 It is obvious that environmental factors play a predominant role in these pathologic processes. Ingested foods are the most common environmental factors humans encounter daily. Catabolism of choline and betaine by intestinal microbes forms TMA, which is then absorbed and rapidly metabolized by the hepatic flavin monooxygenase enzymes to form TMAO.19 Foods such as egg yolk, milk, liver, red meat, poultry, shellfish and fish are rich in the lipid phosphatidylcholine, and they are believed to be the major dietary sources for choline, betaine and TMAO production.5 High intake of dietary lipid phosphatidylcholine was reportedly associated with elevated circulating choline, betaine and TMAO levels.19 Elevated circulating TMAO levels were related to thrombus formation in this study. Therefore, to reduce thrombosis risk, valvular AF patients with higher circulating TMAO levels may be informed to modify their dietary habits to consume less TMAO precursors. Gut microbes participate in modulating TMAO responses to dietary precursors.20 However, TMAO responses are highly variable. Recently, Cho et al found that a greater ratio of Firmicutes to Bacteroidetes exhibiting a greater response to dietary TMAO precursor intake.21 This finding indicates that TMAO production is an individualized process depending upon patients’ gut microbiomes. As such, pharmaceutical strategies that increase gut microbiota diversity and restore the symbiotic relationship between gut microbes and patients should be adopted in valvular AF patients, who have higher circulating gut microbial metabolites levels. In conclusion, we found that valvular AF patients with thrombus formation had higher circulating microbe metabolite levels and enhanced platelet hyperreactivity. These findings are significant, as circulating microbe metabolites may be used to identify individuals with increased thrombus susceptibility. Our results have implications for evaluating the risks associated with thrombosis and for developing precise thrombus prevention strategies. Several limitations should be noted when extrapolating results from this study. First, the sample size of this cohort was relatively small. Only patients with AF were enrolled. A multicenter study to enroll more participants is required in the future. Second, because of the limitations of clinical sample collection, a thorough mechanistic study was not performed. 6
AUTHOR CONTRIBUTIONS D.G. and L.Z. conceived of the presented idea. X.Z. developed the theory. Y.Z., F.W. and Z.Z. verified the analytical methods and performed the computations. All authors discussed the results and contributed to the final manuscript.
ACKNOWLEDGMENTS The authors would like to thank Xueli Yang from the Division of Biostatistics at the National Center for Cardiovascular Diseases of China for his assistance in statistical analysis. In addition, the authors thank all doctors and nurses at the Adult Cardiac Surgery Center at Fuwai Hospital for their participation.
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17. Ardissino D, Berzuini C, Merlini PA, et al. Influence of 9p21.3 genetic variants on clinical and angiographic outcomes in early-onset myocardial infarction. J Am Coll Cardiol. 2011;58:426–434. 18. Ripatti S, Tikkanen E, Orho-Melander M, et al. A multilocus genetic risk score for coronary heart disease: case-control and prospective cohort analyses. Lancet. 2010;376:1393–1400. 19. Zhang AQ, Mitchell SC, Smith RL. Dietary precursors of trimethylamine in man: a pilot study. Food Chem Toxicol. 1999;37:515–520. 20. Koeth RA, Wang Z, Levison BS, et al. Intestinal microbiota metabolism of l-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013;19:576–585. 21. Cho CE, Taesuwan S, Malysheva OV, et al. Trimethylamine-N-oxide (TMAO) response to animal source foods varies among healthy young men and is influenced by their gut microbiota composition: a randomized controlled trial. Mol Nutr Food Res. 2017;61(1).
Submitted February 11, 2019; accepted September 13, 2019. The study was supported by the National Natural Science Foundation of China (NSFC) (81500238 and 81700282), Peking Union Medical College (PUMC) Youth Fund, and Fundamental Research Funds for the Central Universities (3332016017, 3332016014 and 3332018195). The authors have no conflicts of interest to disclose. The first three authors (DG, LZ and YZ) contributed equally to this work. Correspondence: Dingxu Gong, MD, PhD, Department of Cardiac Surgery, Fuwai Hospital Chinese Academy of Medical Science and the National Center for Cardiovascular Disease of China, Beijing 10027, China (E-mail: [email protected]).
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