Gut Microbiota Metabolites and Risk of Major Adverse Cardiovascular Disease Events and Death: A Systematic Review and Meta‐Analysis of Prospective Studies

In the paper

“The burden of CVD is high among patients with chronic kidney disease (CKD) or end‐stage renal disease,47 and the circulating TMAO is predominantly excreted by the kidneys.42”

What the sources say

• Partly supported

  The review states that TMAO is excreted in urine and accumulates in plasma when renal function is impaired (review series section; page unavailable). This supports renal excretion, but the passage does not establish that the kidneys excrete circulating TMAO predominantly. (LLM, conf=0.85)

  cites [42] Tang WH, Hazen SL. The contributory role of gut microbiota in cardiovascular disease. J Clin Invest. 2014; 124: 4204– 4211. 25271725 PMC4215189 sourceExperimental — accuracy not yet measured

In the paper

“Nonetheless, median values of TMAO concentrations in healthy individuals were reported as ranges of 3 to 6 μmol/L,8, 17, 18, 19 and the study by Shafi et al22 consisted of hemodialysis patients with the substantially high mean vale of TMAO (101.9 μmol/L among total participants).”

What the sources say

• Supported

  The Results (Baseline Characteristics) report a median TMAO concentration of 3.5 μmol/L in the healthy control cohort, within the cited 3–6 μmol/L range. The paper gives 2.3–5.7 μmol/L as that cohort’s interquartile range, not as a range of medians. (LLM, conf=0.91)

  cites [8] Tang WH, Wang Z, Fan Y, Levison B, Hazen JE, Donahue LM, Wu Y, Hazen SL. Prognostic value of elevated levels of intestinal microbe‐generated metabolite trimethylamine‐N‐oxide in patients with heart failure: refining the gut hypothesis. J Am Coll Cardiol. 2014; 64: 1908– 1914. 25444145 PMC4254529 source
• Supported

  Table 1 reports a median TMAO concentration of 5.8 for the 62 healthy controls, within the cited range of 3–6. Section 3.2 identifies this group as healthy controls; the table’s full observed range is separate from the median. (LLM, conf=0.88)

  cites [17] Skagen K, Troseid M, Ueland T, Holm S, Abbas A, Gregersen I, Kummen M, Bjerkeli V, Reier‐Nilsen F, Russell D, Svardal A, Karlsen TH, Aukrust P, Berge RK, Hov JE, Halvorsen B, Skjelland M. The carnitine‐butyrobetaine‐trimethylamine‐N‐oxide pathway and its association with cardiovascular mortality in patients with carotid atherosclerosis. Atherosclerosis. 2016; 247: 64– 69. 26868510 source
• Partly supported

  The Results section, ‘Renal function was a major determinant of TMAO-levels’ (page unavailable), says TMAO in 80 healthy controls was presented as a median in Fig. 1, but the text does not give that median. Table 3 reports 5.5 μmol/L after transplantation, and the paper says transplantation reduced TMAO to control levels; that is consistent with the claimed range, but it does not establish a healthy-control median of 3–6 μmol/L. (LLM, conf=0.76)

  cites [18] Missailidis C, Hallqvist J, Qureshi AR, Barany P, Heimburger O, Lindholm B, Stenvinkel P, Bergman P. Serum trimethylamine‐N‐oxide is strongly related to renal function and predicts outcome in chronic kidney disease. PLoS One. 2016; 11: e0141738. 26751065 PMC4709190 sourceExperimental — accuracy not yet measured
• Not checked yetcites [19] Stubbs JR, House JA, Ocque AJ, Zhang S, Johnson C, Kimber C, Schmidt K, Gupta A, Wetmore JB, Nolin TD, Spertus JA, Yu AS. Serum trimethylamine‐N‐oxide is elevated in CKD and correlates with coronary atherosclerosis burden. J Am Soc Nephrol. 2016; 27: 305– 313. 26229137 PMC4696571 source

In the paper

“Of the 22 studies, 2 only investigated combinations of TMAO and choline, betaine,9 or l‐carnitine4; thus we did not include the 2 studies4, 9 in the analysis for TMAO, although these studies were eligible for the analysis of TMAO precursors and risk of MACE.”

What the sources say

• Not checked yetcites [9] Wang Z, Tang WH, Buffa JA, Fu X, Britt EB, Koeth RA, Levison BS, Fan Y, Wu Y, Hazen SL. Prognostic value of choline and betaine depends on intestinal microbiota‐generated metabolite trimethylamine‐N‐oxide. Eur Heart J. 2014; 35: 904– 910. 24497336 PMC3977137 source
• Not checked yetcites [4] Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, Smith JD, Didonato JA, Chen J, Li H, Wu GD, Lewis JD, Warrier M, Brown JM, Krauss RM, Tang WHW, Bushman FD, Lusis AJ, Hazen SL. Intestinal microbiota metabolism of L‐carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013; 19: 576– 585. 23563705 PMC3650111 source

In the paper

“Of the 19 data points, the study in blacks by Shafi et al22 affected the heterogeneity; removal of the data in blacks22 resulted in a pooled RR of 1.70 (95% CI, 1.52, 1.91; P<0.001; P heterogeneity=0.9; I2=0%; fixed‐effect model; Figure S1A).”

What the sources say

• Not checked yetcites [22] Shafi T, Powe NR, Meyer TW, Hwang S, Hai X, Melamed ML, Banerjee T, Coresh J, Hostetter TH. Trimethylamine N‐oxide and cardiovascular events in hemodialysis patients. J Am Soc Nephrol. 2017; 28: 321– 331. 27436853 PMC5198291 source

In the paper

“The mean or median value of TMAO ranged from 1.74 to 103.8 μmol/L across the 22 prospective studies listed in Table 1.”

In the paper

“Of the total 22 publications, multivariate‐adjusted RRs were available for 21 (95%) publications; of the 19 data points used for the main analysis, 79% (n=15) were multivariate‐adjusted data.”

In the paper

“All of the studies included clinical cohorts, including patients at higher cardiovascular risk, that is, enriched for baseline heart disease,3, 8, 12, 13, 14, 15, 16, 17, 21 kidney disease,10, 11, 18, 19, 20, 22, 23 peripheral artery disease,25 or diabetes mellitus.7, 26”

What the sources say

• Not checked yetcites [3] Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, Wu Y, Hazen SL. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013; 368: 1575– 1584. 23614584 PMC3701945 source
• Not checked yetcites [8] Tang WH, Wang Z, Fan Y, Levison B, Hazen JE, Donahue LM, Wu Y, Hazen SL. Prognostic value of elevated levels of intestinal microbe‐generated metabolite trimethylamine‐N‐oxide in patients with heart failure: refining the gut hypothesis. J Am Coll Cardiol. 2014; 64: 1908– 1914. 25444145 PMC4254529 source
• Not checked yetcites [12] Tang WH, Wang Z, Shrestha K, Borowski AG, Wu Y, Troughton RW, Klein AL, Hazen SL. Intestinal microbiota‐dependent phosphatidylcholine metabolites, diastolic dysfunction, and adverse clinical outcomes in chronic systolic heart failure. J Card Fail. 2015; 21: 91– 96. 25459686 PMC4312712 source

In the paper

“In results of a sensitivity analysis omitting 1 study at a time and calculating the pooled RRs for the remainder of 18 studies (the data in blacks were not included), the pooled RRs ranged from 1.65 (95% CI, 1.47, 1.86) to 1.75 (95% CI, 1.55, 1.98), and no other study was identified as an influential outlier across the 18 studies (P heterogeneity ranged 0.8–0.9; I2 was consistently 0%).”

In the paper

“Subsequently, the remaining 19 articles published from 2013 to 2017 assessed the RRs of TMAO (either as a categorical or continuous variable) and risk of MACE.”

In the paper

“We identified 24 potentially eligible articles, including 2 cross‐sectional studies and 22 prospective studies (Figure 1).”

In the paper

“Associations appeared to be stronger among studies with participants who had generally lower BMI, or lower prevalence of smoking, although such differences did not attain statistical significance.”

In the paper

“Second, the definition of elevated TMAO levels was difference across individual studies.”

In the paper

“We observed the highly consistent positive associations between TMAO levels and the outcomes across studies, even among studies with low (<5.0 μmol/L) concentrations of TMAO at baseline, particularly after excluding the data among blacks.”

In the paper

“We also performed a stratified analysis using a cut‐off value of 8.0 μmol/L. We observed similarly increased risks of MACE regardless of studies with <8.0 (range, 3.0–7.9) or ≥8.0 (range, 20.41–98.4) μmol/L mean/median TMAO levels. Again, there was no heterogeneity between low or high TMAO concentrations at baseline.”

In the paper

“Fifth, most of the study participants were from Europe and the United States and included mainly (approximately of 65–90% of total study participants) whites,10, 19, 20, 23 so that our results might not be applicable to other race/ethnic groups such as Asians and Africans.”

What the sources say

• Not checked yetcites [10] Kaysen GA, Johansen KL, Chertow GM, Dalrymple LS, Kornak J, Grimes B, Dwyer T, Chassy AW, Fiehn O. Associations of trimethylamine N‐oxide with nutritional and inflammatory biomarkers and cardiovascular outcomes in patients new to dialysis. J Ren Nutr. 2015; 25: 351– 356. 25802017 PMC4469547 source
• Not checked yetcites [19] Stubbs JR, House JA, Ocque AJ, Zhang S, Johnson C, Kimber C, Schmidt K, Gupta A, Wetmore JB, Nolin TD, Spertus JA, Yu AS. Serum trimethylamine‐N‐oxide is elevated in CKD and correlates with coronary atherosclerosis burden. J Am Soc Nephrol. 2016; 27: 305– 313. 26229137 PMC4696571 source
• Not checked yetcites [20] Kim RB, Morse BL, Djurdjev O, Tang M, Muirhead N, Barrett B, Holmes DT, Madore F, Clase CM, Rigatto C, Levin A, Can PI. Advanced chronic kidney disease populations have elevated trimethylamine N‐oxide levels associated with increased cardiovascular events. Kidney Int. 2016; 89: 1144– 1152. 27083288 source
• Not checked yetcites [23] Robinson‐Cohen C, Newitt R, Shen DD, Rettie AE, Kestenbaum BR, Himmelfarb J, Yeung CK. Association of FMO3 variants and trimethylamine N‐oxide concentration, disease progression, and mortality in CKD patients. PLoS One. 2016; 11: e0161074. 27513517 PMC4981377 source

In the paper

“In addition, there were 5 studies with extremely high mean/median TMAO values (>20 μmol/L).”

In the paper

“The associations did not significantly differ according to the past histories of CVD, prevalence of diabetes mellitus, kidney dysfunction, follow‐up duration, or TMAO levels at baseline.”

In the paper

“Again, after removal of the data in blacks, there was no heterogeneity across the studies (pooled RR, 1.72; 95% CI, 1.50, 1.97; P heterogeneity=0.7; I2=0%; Figure S1B).”

In the paper

“The protocol of the present study was registered with the PROSPERO database: CRD42016052185.”

In the paper

“There is no standard definition for MACE, and the definition varies by study, whereas most studies include MI and death to define MACE.30”

What the sources say

• Not checked yetcites [30] Kip KE, Hollabaugh K, Marroquin OC, Williams DO. The problem with composite end points in cardiovascular studies: the story of major adverse cardiac events and percutaneous coronary intervention. J Am Coll Cardiol. 2008; 51: 701– 707. 18279733 source

In the paper

“Two reviewers (Y.H. and W.M.) independently extracted data on study design and population characteristics and compared results to ensure consistency. Inconsistencies in data were resolved by a third reviewer (L.Q.).”

In the paper

“We assessed publication bias by using Begg's and Egger's tests and visual inspection of the funnel plot if 10 or more studies were available.35”

What the sources say

• Not checked yetcites [35] Sterne JA, Gavaghan D, Egger M. Publication and related bias in meta‐analysis: power of statistical tests and prevalence in the literature. J Clin Epidemiol. 2000; 53: 1119– 1129. 11106885 source

In the paper

“Heterogeneity was assessed by the Cochran Q test and I2 statistic; low, moderate, and high I2 values were considered to be 25%, 50%, and 75%, respectively.32, 33”

What the sources say

• Not checked yetcites [32] Higgins JP. Commentary: heterogeneity in meta‐analysis should be expected and appropriately quantified. Int J Epidemiol. 2008; 37: 1158– 1160. 18832388 source
• Not checked yetcites [33] Higgins JP, Thompson SG, Deeks JJ, Altman DG. Measuring inconsistency in meta‐analyses. BMJ. 2003; 327: 557– 560. 12958120 PMC192859 source

In the paper

“There is no standard definition for MACE, and the definition varies by study, whereas most studies include MI and death to define MACE.30”

What the sources say

• Not checked yetcites [30] Kip KE, Hollabaugh K, Marroquin OC, Williams DO. The problem with composite end points in cardiovascular studies: the story of major adverse cardiac events and percutaneous coronary intervention. J Am Coll Cardiol. 2008; 51: 701– 707. 18279733 source

In the paper

“We used a fixed‐effect model, using the method of Mantel and Haenszel when there was no significant heterogeneity, and a random effect model when heterogeneity was significant.34”

What the sources say

• Not checked yetcites [34] Lau J, Ioannidis JP, Schmid CH. Quantitative synthesis in systematic reviews. Ann Intern Med. 1997; 127: 820– 826. 9382404 source

In the paper

“Quality assessment was performed using to the Newcastle–Ottawa quality assessment scale.31”

What the sources say

• Not checked yetcites [31] Wells GA, Shea B, O'Connell D, Peterson J, Welch V, Losos M, Tugwell P. The Newcastle‐Ottawa Scale (NOS) for assessing the quality of nonrandomised studies in meta‐analyses. 2014 Available at: http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp. Accessed May 7, 2016.

In the paper

“TMAO is a small organic compound, mainly derived from choline (found in foods such as red meat, fish, poultry, and eggs), which is metabolized to produce trimethylamine (TMA) by microbiota1, 4 and then to TMAO by the hepatic enzyme, flavin monooxygenase 3.1, 5”

What the sources say

• Not checked yetcites [1] Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, Feldstein AE, Britt EB, Fu X, Chung YM, Wu Y, Schauer P, Smith JD, Allayee H, Tang WH, DiDonato JA, Lusis AJ, Hazen SL. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011; 472: 57– 63. 21475195 PMC3086762 source
• Not checked yetcites [4] Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, Smith JD, Didonato JA, Chen J, Li H, Wu GD, Lewis JD, Warrier M, Brown JM, Krauss RM, Tang WHW, Bushman FD, Lusis AJ, Hazen SL. Intestinal microbiota metabolism of L‐carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013; 19: 576– 585. 23563705 PMC3650111 source
• Not checked yetcites [5] Bennett BJ, de Aguiar Vallim TQ, Wang Z, Shih DM, Meng Y, Gregory J, Allayee H, Lee R, Graham M, Crooke R, Edwards PA, Hazen SL, Lusis AJ. Trimethylamine‐N‐oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab. 2013; 17: 49– 60. 23312283 PMC3771112 source

In the paper

“There is no standard definition for MACE, and the definition varies by study, whereas most studies include MI and death to define MACE.30”

What the sources say

• Not checked yetcites [30] Kip KE, Hollabaugh K, Marroquin OC, Williams DO. The problem with composite end points in cardiovascular studies: the story of major adverse cardiac events and percutaneous coronary intervention. J Am Coll Cardiol. 2008; 51: 701– 707. 18279733 source

In the paper

“The reference lists of identified articles were also scanned to identify any other relevant studies.”

In the paper

“Recent studies by Wang and Tang et al reported that circulating concentrations of trimethylamine N‐oxide (TMAO), a metabolite derived from the gut microbiota, were predictive of prevalent cardiovascular diseases (CVDs)1 and future cardiovascular events3 in clinical cohorts.”

What the sources say

• Not checked yetcites [1] Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, Feldstein AE, Britt EB, Fu X, Chung YM, Wu Y, Schauer P, Smith JD, Allayee H, Tang WH, DiDonato JA, Lusis AJ, Hazen SL. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature. 2011; 472: 57– 63. 21475195 PMC3086762 source
• Not checked yetcites [3] Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, Wu Y, Hazen SL. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med. 2013; 368: 1575– 1584. 23614584 PMC3701945 source

In the paper

“We followed the standard criteria for conducting meta‐analyses of observational studies and reporting the results.29”

What the sources say

• Not checked yetcites [29] Stroup DF, Berlin JA, Morton SC, Olkin I, Williamson GD, Rennie D, Moher D, Becker BJ, Sipe TA, Thacker SB. Meta‐analysis of observational studies in epidemiology: a proposal for reporting. Meta‐analysis Of Observational Studies in Epidemiology (MOOSE) group. JAMA. 2000; 283: 2008– 2012. 10789670 source

In the paper

“We searched MEDLINE (PubMed) and Embase up to December 6, 2016 for eligible studies with a search strategy that combined text word and Medical Subject Headings identifying reports relating blood concentrations of TMAO and the MACE.”

In the paper

“No language restrictions were applied.”

In the paper

“We also estimated dose‐response associations based on data for categories of TMAO levels on median dose, number of cases and participants, and effect estimates with corresponding SEs using the generalized least‐squares for trend estimation method of Greenland and Longnecker.36”

What the sources say

• Not checked yetcites [36] Greenland S, Longnecker MP. Methods for trend estimation from summarized dose‐response data, with applications to meta‐analysis. Am J Epidemiol. 1992; 135: 1301– 1309. 1626547 source

In the paper

“It has also been reported that morality risk may differ between black and white dialysis patients.46”

What the sources say

• Not checked yetcites [46] Kucirka LM, Grams ME, Lessler J, Hall EC, James N, Massie AB, Montgomery RA, Segev DL. Association of race and age with survival among patients undergoing dialysis. JAMA. 2011; 306: 620– 626. 21828325 PMC3938098

In the paper

“Fourth, the gut microbiota are affected by environmental factors37, 38 such as dietary intakes,39, 40, 41 which may, in turn, influence blood levels of TMAO and its precursors.4”

What the sources say

• Not checked yetcites [37] Scalbert A, Brennan L, Manach C, Andres‐Lacueva C, Dragsted LO, Draper J, Rappaport SM, van der Hooft JJ, Wishart DS. The food metabolome: a window over dietary exposure. Am J Clin Nutr. 2014; 99: 1286– 1308. 24760973 source
• Not checked yetcites [38] Wong JM. Gut microbiota and cardiometabolic outcomes: influence of dietary patterns and their associated components. Am J Clin Nutr. 2014; 100(suppl 1): 369S– 377S. 24898225 source
• Not checked yetcites [39] Claesson MJ, Jeffery IB, Conde S, Power SE, O'Connor EM, Cusack S, Harris HM, Coakley M, Lakshminarayanan B, O'Sullivan O, Fitzgerald GF, Deane J, O'Connor M, Harnedy N, O'Connor K, O'Mahony D, van Sinderen D, Wallace M, Brennan L, Stanton C, Marchesi JR, Fitzgerald AP, Shanahan F, Hill C, Ross RP, O'Toole PW. Gut microbiota composition correlates with diet and health in the elderly. Nature. 2012; 488: 178– 184. 22797518 source
• Not checked yetcites [40] Kong LC, Holmes BA, Cotillard A, Habi‐Rachedi F, Brazeilles R, Gougis S, Gausseres N, Cani PD, Fellahi S, Bastard JP, Kennedy SP, Dore J, Ehrlich SD, Zucker JD, Rizkalla SW, Clement K. Dietary patterns differently associate with inflammation and gut microbiota in overweight and obese subjects. PLoS One. 2014; 9: e109434. 25330000 PMC4203727 source
• Not checked yetcites [41] Cotillard A, Kennedy SP, Kong LC, Prifti E, Pons N, Le Chatelier E, Almeida M, Quinquis B, Levenez F, Galleron N, Gougis S, Rizkalla S, Batto JM, Renault P; Consortium ANRM, Dore J, Zucker JD, Clement K, Ehrlich SD. Dietary intervention impact on gut microbial gene richness. Nature. 2013; 500: 585– 588. 23985875 source
• Not checked yetcites [4] Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, Smith JD, Didonato JA, Chen J, Li H, Wu GD, Lewis JD, Warrier M, Brown JM, Krauss RM, Tang WHW, Bushman FD, Lusis AJ, Hazen SL. Intestinal microbiota metabolism of L‐carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013; 19: 576– 585. 23563705 PMC3650111 source

In the paper

“Third, similar to other observational studies, we could not exclude the possibility of residual confounding attributed to unmeasured factors.”

In the paper

“Emerging evidence has shown associations of gut microbiota with obesity.48, 49, 50, 51”

What the sources say

• Not checked yetcites [48] Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, Sogin ML, Jones WJ, Roe BA, Affourtit JP, Egholm M, Henrissat B, Heath AC, Knight R, Gordon JI. A core gut microbiome in obese and lean twins. Nature. 2009; 457: 480– 484. 19043404 PMC2677729 source
• Not checked yetcites [49] Ley RE, Turnbaugh PJ, Klein S, Gordon JI. Microbial ecology: human gut microbes associated with obesity. Nature. 2006; 444: 1022– 1023. 17183309 source
• Not checked yetcites [50] Zhernakova A, Kurilshikov A, Bonder MJ, Tigchelaar EF, Schirmer M, Vatanen T, Mujagic Z, Vila AV, Falony G, Vieira‐Silva S, Wang J, Imhann F, Brandsma E, Jankipersadsing SA, Joossens M, Cenit MC, Deelen P, Swertz MA, Weersma RK, Feskens EJ, Netea MG, Gevers D, Jonkers D, Franke L, Aulchenko YS, Huttenhower C, Raes J, Hofker MH, Xavier RJ, Wijmenga C, Fu J. Population‐based metagenomics analysis reveals markers for gut microbiome composition and diversity. Science. 2016; 352: 565– 569. 27126040 PMC5240844 source
• Not checked yetcites [51] Sonnenburg JL, Backhed F. Diet‐microbiota interactions as moderators of human metabolism. Nature. 2016; 535: 56– 64. 27383980 PMC5991619 source

In the paper

“However, detailed assessment of dietary intakes was not available in all the included studies; this limited our abilities to investigate the potential roles of dietary factors in the associations of gut microbiota metabolites and the outcomes.”

In the paper

“We carefully assessed the influence of several potential confounders and traditional risk factors for CVDs.”

In the paper

“A study4 showed that plasma l‐carnitine concentrations predicted MACE independent of traditional cardiovascular risk factors, but the significant association disappeared after adjustment for plasma TMAO concentration.”

What the sources say

• Not checked yetcites [4] Koeth RA, Wang Z, Levison BS, Buffa JA, Org E, Sheehy BT, Britt EB, Fu X, Wu Y, Li L, Smith JD, Didonato JA, Chen J, Li H, Wu GD, Lewis JD, Warrier M, Brown JM, Krauss RM, Tang WHW, Bushman FD, Lusis AJ, Hazen SL. Intestinal microbiota metabolism of L‐carnitine, a nutrient in red meat, promotes atherosclerosis. Nat Med. 2013; 19: 576– 585. 23563705 PMC3650111 source

In the paper

“In our analyses, the associations of these correlated gut microbiota metabolites could not be mutually adjusted; therefore, we could not determine whether the TMAO or its precursors were independently associated with the outcomes.”