Pro-atherosclerotic actions

There has been a focus on roles of TMAO in atherosclerosis and vascular dysfunction. At substantially elevated concentrations, TMAO is pro-inflammatory, impairs vascular function and structure, and may up-regulate scavenger receptors (SR) while inhibiting reverse cholesterol transport (RCT)^{(Reference Seldin, Meng and Qi6,Reference Chen, Zhu and Ran7,Reference Sun, Jiao and Ma19,Reference Koeth, Wang and Levison20)} (Fig. 3). High TMAO concentrations can also induce platelet hyper-reactivity, enhancing thrombotic potential via amplified intracellular Ca²⁺ release^{(Reference Zhu, Gregory and Org21)}. A 10-fold elevation in serum TMAO with choline supplementation in mice (1 % w/w, about 12× normal) also augments platelet reactivity to ADP, though failing to influence other indices including surface phosphatidylserine content in ADP-stimulated platelets, levels of Von Willebrand factor, α granule release, or baseline pro-thrombotic microvesicle release^{(Reference Roberts, Gu and Buffa22)}. These mixed actions could contribute to vascular disease when TMAO exceeds 10–20 µm, for example in heart failure (HF) and chronic kidney disease (CKD); however, involvement in the initial pathogenesis of atherosclerosis and CHD is questionable^{(Reference Wang, Roberts and Buffa23)}.

Fig. 3. Putative pro-inflammatory and -atherosclerotic actions of trimethylamine-N-oxide (TMAO). Pronounced elevations in TMAO to >10–20 µm, for example in advanced heart failure or chronic kidney disease, may be sufficient to modify multiple determinants of inflammation and atherosclerosis, as detailed here. Whether levels of circulating TMAO in obesity, diabetes or CHD are sufficient to significantly influence these processes is presently unclear. CD36, cluster of differentiation 36; eNOS, endothelial nitric oxide synthase; ICAM-1, intracellular adhesion molecule-1; MCP-1; monocyte chemoattractant protein 1; NF-κB; nuclear factor κ-light-chain-enhancer of activated B cells; ox-LDL, oxidised-LDL; ROS, reactive oxygen species; SR, scavenger receptor; SRA, scavenger receptor A, VCAM-1, vascular adhesion protein-1.

Studies in genetic models predisposed to disease indicate that major elevations in TMAO with high-level carnitine supplementation (1–1·3 % in drinking water) promote atherogenesis. Carnitine supplementation exaggerates atherosclerosis in ApoE^–/– mice, an effect countered by broad-spectrum antibiotics^{(Reference Koeth, Wang and Levison20)} and the choline analogue inhibitor of TMA lyase, 3,3-dimethyl-1-butanol (DMB)^{(Reference Wang, Roberts and Buffa23)}. Pro-atherosclerotic effects of intermittent hypoxia/hypercapnia (relevant to sleep apnoea) in ApoE^–/– and LDL receptor knockout mice are also partially DMB sensitive^{(Reference Xue, Zhou and Poulsen24)}. These observations support some role for TMAO in high-risk settings where both propensity to atherosclerosis and TMAO concentrations are artificially augmented; however, their broader relevance is less clear. A word of caution is also warranted regarding the applicability of the ApoE^–/– model to human atherosclerosis, given lack of the cholesteryl ester transfer protein important in human RCT. Contrasting these studies, analysis of outcomes in cholesteryl ester transfer protein-transfected ApoE^–/– mice supports a protective rather than detrimental role for TMAO^{(Reference Collins, Drazul-Schrader and Sulpizio25)}. Other work reveals no relationship between TMAO, plasma cholesterol and atherogenesis in mice fed normal diets^{(Reference Veeravalli, Karu and Scott26)}, or in ApoE^–/– mice fed high-fat diets^{(Reference Geng, Yang and Wang27)}. Differences in TMAO within inbred strains of mice are also estimated to explain less than 10 % of the variance in atherosclerosis^{(Reference Bennett, de Aguiar Vallim and Wang28)}.

Vascular dysfunction and senescence

Recent studies also suggest that very high concentrations of TMAO may impair eNOS- or EDHF-dependent vascular control, and facilitate vessel remodelling and senescence^{(Reference Li, Chen and Gua8,Reference Li, Gua and Wu39-Reference Matsumoto, Kojima and Takayanagi41)} . Elevated TMAO can increase formation of ROS that degrade NO, and may also inhibit eNOS expression, compounding vascular dysfunction (Fig. 3)^{(Reference Sun, Jiao and Ma19)}. Sun et al. ^{(Reference Sun, Jiao and Ma19)} found that 100–300 µm TMAO suppresses endothelial eNOS expression and NO release, an effect involving ROS-dependent activation of the NLRP3 inflammasome^{(Reference Sun, Jiao and Ma19)}. Additionally, Matsumoto et al. ^{(Reference Matsumoto, Kojima and Takayanagi41)} report a modest inhibitory effect of 300 µm TMAO on EDHF-mediated relaxation in the femoral (but not superior mesenteric) artery. Suggestive of some vascular influences, chronic TMAO infusion in rats to achieve a 58 µm plasma concentration did not modify blood pressure, yet prolonged the hypertensive effect of angiotensin II^{(Reference Ufnal, Jazwiec and Dadlez42)}. Whether this reflects a direct vascular influence is unclear. Supporting TMAO-dependent vascular dysfunction in disease, inhibition of TMA production with DMB reportedly preserves aortic relaxation and eNOS phosphorylation, and eliminates vascular inflammation and oxidative stress in a rat model of CKD associated with a doubling of plasma TMAO^{(Reference Li, Gua and Wu39)}. This study, remarkable in implicating a modest elevation in TMA/TMAO as the sole driver of inflammation, oxidative stress and eNOS and vascular dysregulation in CKD, requires broader confirmation and reconciliation both with evidence of other mechanistic involvement (including nuclear factor erythroid 2-related factor 2 (Nrf2) suppression, inducible NO synthase (iNOS) induction, altered parathyroid hormone and Ca²⁺ handling) and with the excessive TMAO concentrations required to induce such effects in vitro. There is other support for TMAO-dependent vascular damage in CKD, with vessel calcification in CKD patients correlating with TMAO concentrations above 100 µm, and evidence that 100–600 µm TMAO can promote vascular calcification and expression of bone-related and inflammatory molecules in vitro and in vivo ^{(Reference Zhang, Li and Yang37)}.

Ageing is an independent risk factor for CVD and involves vascular remodelling and progressive reductions in endothelial function and NO bioavailability, coupled with increased cyclo-oxygenase (COX)-derived vasoconstrictors. Circulating TMAO increases with age in humans and rodents^{(Reference Li, Chen and Gua8,Reference Ke, Li and Zhao40,Reference Li, Ke and Zhan43,Reference Brunt, Gioscia-Ryan and Richey44)} , and recent work indicates that inhibition of TMAO formation reduces^{(Reference Li, Chen and Gua8)} whereas TMAO supplementation mimics or accelerates^{(Reference Ke, Li and Zhao40)} ageing-dependent vascular dysfunction and inflammation in rodents. Pro-senescence effects were associated with exaggerated oxidative stress, suppression of sirtuin-1 and activation of p53/p21/Rb signalling^{(Reference Ke, Li and Zhao40)}. Antibiotic treatment in ageing mice also improves vascular reactivity, in association with a fall in circulating TMAO; however, the role of TMAO was not directly tested^{(Reference Brunt, Gioscia-Ryan and Richey44)}. These studies, together with evidence for TMAO-dependent brain ageing^{(Reference Li, Ke and Zhan43)}, suggest elevated TMAO could promote biological ageing, predisposing to strongly age-dependent diseases such as CHD and CKD. Indeed, Li et al. ^{(Reference Li, Chen and Gua8)} report that a moderate elevation in TMAO from about 6 to 14 µm is solely responsible for age-dependent vascular dysfunction, inflammation and eNOS depletion, with the aged vascular phenotype in senescent rats reversed by DMB treatment. This surprising observation also demands broader confirmation, and reconciliation with evidence for diverse mechanistic elements of vascular ageing. More fundamentally, TMAO concentrations required to modify viability and senescence markers in human endothelial cells^{(Reference Ke, Li and Zhao40)} are so extreme (100 mm) as to have no biological or pathological relevance in humans or animal models.

Myocardial dysfunction

Although there are relatively few studies of the myocardial effects of TMAO, emerging evidence suggests cardiac sensitivity to concentrations that are approached or surpassed in HF and CKD. These pronounced elevations in TMAO may thus promote cardiac dysfunction, as suggested by select effects of DMB in diseased but not healthy animals^{(Reference Chen, Zheng and Feng10,Reference Li, Sun and Zhang45)} . In addition to evidence of TMAO-dependent exaggeration of cardiac fibrosis and dysfunction in disease models^{(Reference Organ, Otsuka and Bhushan46)}, TMAO may disrupt myocyte structure, contractile function and energy metabolism, and promote oxidative stress^{(Reference Savi, Bocchi and Bresciani47,Reference Makrecka-Kuka, Volska and Antone48)} . Inhibitory effects on contraction, relaxation and Ca²⁺ dynamics are evident with 20 µm TMAO^{(Reference Savi, Bocchi and Bresciani47)}, although the latter effects appeared concentration-independent in the limited range tested. However, other work indicates that 0·3–3 µm TMAO disrupts T-tubule organisation and Ca²⁺ handling in isolated murine cardiomyocytes^{(Reference Jin, Ji and Zuo49)}, suggesting a curious scenario in which concentrations more than 10-fold lower than circulating levels in healthy mice^{(Reference Chen, Zheng and Feng10)} are significantly damaging. Distinct from these studies, recent work supports modest positive inotropic effects of TMAO when applied at extremely high (0·3–3 mm) levels^{(Reference Oakley, Vallejo and Wang50)}. Further work is needed to clarify the cardiac functional influences of relevant concentrations of TMAO.

Potential impacts of TMAO on cardiac energy metabolism are supported by TMAO-dependent inhibition of mitochondrial pyruvate and fatty acid oxidation, also apparent in vitro with 20 µm TMAO^{(Reference Makrecka-Kuka, Volska and Antone48)}. Another recent study suggests a threshold of about 100 µm TMAO for inhibition of oxidoreductase capacity in cultured cardiomyoblasts, although TMA appeared substantially more potent, reducing metabolic capacity at 1–10 µm ^{(Reference Jaworska, Hering and Mosieniak51)}. It has also been reported that exogenously applied TMAO can destabilise atrial electrophysiology to promote fibrillation (potentially via inflammation)^{(Reference Yu, Meng and Huang52)}; however, concentrations mediating this effect are difficult to ascertain. There is also some indirect evidence that myocardial protection with physical activity may be inhibited by TMAO levels exceeding 20 µm ^{(Reference Zhang, Meng and Yu13)}; however, involvement of TMAO was not tested and is inconsistent with a lack of effect of TMAO supplementation.

Apparent sensitivities to about 20 µm TMAO suggest that cardiac dysfunction could arise as a direct result of elevations observed in HF and CKD, or following acute myocardial infarction (AMI). Renal hypo-perfusion and neuroendocrine changes characteristic of HF probably contribute to the associated rise in TMAO^{(Reference Stubbs, House and Ocque15,Reference Missailidis, Hällqvist and Qureshi53)} . Ageing, obesity, insulin resistance and diabetes^{(Reference Shan, Sun and Huang54)} – associated with and promoting HF – may additionally contribute to increased TMAO, for example via altered flavin-containing mono-oxygenase 3 (FMO3) expression^{(Reference Brunt, Gioscia-Ryan and Richey44,Reference Miao, Ling and Manthena55,Reference Schugar, Shih and Warrier56)} . These TMAO changes could constitute an important cardio-renal linkage, with TMAO functioning as a uraemic toxin that promotes cardiac and renal dysfunction and remodelling^{(Reference Ronco, Haapio and House57)} (Fig. 2). Elevated TMAO does appear to worsen pressure overload-induced HF in mice^{(Reference Organ, Otsuka and Bhushan46)}, and promotes renal inflammation and fibrosis^{(Reference Li, Chen and Gua8,Reference Sun, Yin and Liu11)} . Causal involvement in cardiac dysfunction and remodelling is supported by the observation that TMAO is elevated to about 20 µm in a rodent model of obesity, with DMB preventing diastolic dysfunction, inflammation and cardiac fibrosis independently of metabolic disruption^{(Reference Chen, Zheng and Feng10)}. The observation that myocardial impacts of dietary obesity appear entirely dependent on a 2-fold rise in TMAO (to about 20 µm) has important implications, though awaits confirmation. Li et al. ^{(Reference Li, Sun and Zhang45)} present evidence that post-infarct HF in rats may also involve an associated increase in circulating TMAO (to about 25 µm), with cardiac outcomes improved upon DMB treatment^{(Reference Li, Sun and Zhang45)}. Nonetheless, involvement of circulating TMAO in the pathogenesis of HF awaits more extensive analysis, as does its potential role as a uraemic toxin in the cardio-renal syndrome. There is contrary evidence, for example, that chronic elevations in TMAO may actually protect against myocardial dysfunction and fibrosis^{(Reference Huc, Drapala and Gawrys58)}.

Implied cardiovascular pathogenicity of trimethylamine N-oxide

The nature of reported TMAO–CVD associations raises important questions regarding causality: the TMAO concentrations linked to major increases in CVD risk or adverse outcomes are well within ranges reported in healthy cohorts (Table 1), and orders of magnitude lower than those mediating pathological effects in experimental models^{(Reference Seldin, Meng and Qi6,Reference Chen, Zhu and Ran7,Reference Sun, Jiao and Ma19,Reference Koeth, Wang and Levison20)} . For example, it seems implausible that the TMAO–CVD association reported by Wang et al. ^{(Reference Wang, Klipfell and Bennett2)} reflects causal involvement, as it implies that a rise in TMAO from <1 to 5 µm increases CVD risk 4-fold, despite these levels falling within the range observed in healthy subjects, and no evidence of pathological responses to ≤5 µm TMAO. Similarly, the data of Tang et al. ^{(Reference Tang, Wang and Levison14)} implies that a modest 40 % rise in TMAO from 3·5 µm to 5 µm significantly promotes CVD; the association in Mente et al. ^{(Reference Mente, Chalcraft and Ak17)} suggests that a rise from <1 to 5 µm increases risk of CVD about 9-fold; and Mafune et al. ^{(Reference Mafune, Iwamoto and Tsutsumi18)} link small elevations in TMAO from <3 to 4, 5 and 6 µm to increases in diseased coronaries from 0 to 1, 2 and 3 in surgical patients with CVD^{(Reference Mafune, Iwamoto and Tsutsumi18)}. While association between TMAO and thrombosis risk suggests markedly increased thrombotic events with a rise in TMAO from 2 to 6 µm, evidence supports a 10–100 µm threshold for functional effects of TMAO in platelets^{(Reference Zhu, Gregory and Org21)}. Meta-analysis suggests a somewhat lower sensitivity of all-cause mortality, which increases about 8 % per 10 µm increment in plasma TMAO^{(Reference Schiattarella, Sannino and Toscano72)}, and highlights an unexplained disparity between TMAO levels and outcomes: mortality exhibited an unexpected 10-fold greater dependence on TMAO in the low 1–10 µm v. higher concentration ranges. In short, were these varied associations reflective of causal involvement of TMAO as an early driver of disease, normal variance observed in TMAO concentrations across healthy populations^{(Reference Li, Ke and Zhan43,Reference Wang, Levison and Hazen59,Reference Cho, Taesuwan and Malysheva62,Reference Meyer, Benton and Bennett65)} would be predicted to profoundly increase CVD risk and mortality. However, there is little evidence for pathological effects of ≤10 µm TMAO on inflammation, atherosclerosis or thrombosis, and no pathobiological basis for markedly higher dependence of mortality on <10 µm v. >10 µm TMAO. These findings contrast evidence of a higher TMAO threshold for cardiometabolic risk approaching 10 µm ^{(Reference Barrea, Annunziata and Muscogiuri78)}. Fundamentally complicating matters, the basis of the variance in TMAO in healthy populations and of elevated concentrations with CVD risks, co-morbidities and disease itself remain unclear, constraining capacity to address the question of TMAO causal involvement.

Dietary substrates

Excessive intake of substrates for TMA generation may contribute to elevations in circulating TMAO, influenced by gut microbiota profiles^{(Reference Cho, Taesuwan and Malysheva62)}. That said, evidence that major TMAO substrates protect against cardiometabolic disease^{(Reference Millard, Musani and Dibaba82)} and that red meat intake is a weak CVD risk^{(Reference Haring, Gronroos and Nettleton83-Reference Wang, Lin and Ouyang85)} appears difficult to reconcile with an important mechanistic role for TMAO in CVD. Generation of TMAO increases with intake of choline and carnitine substrates for TMA generation^{(Reference Rohrmann, Linseisen and Allenspach86)} (Fig. 1). Increased formation occurs when sufficient choline/carnitine reaches regions of bacterial TMA production – the caecum appears to have the highest TMA generation capacity in mouse models^{(Reference Koeth, Wang and Levison20,Reference Koeth, Levison and Culley87)} , though a recent report (albeit for a single human subject) suggests that TMA may be generated and absorbed specifically in the small intestine^{(Reference Stremmel, Schmidt and Schuhmann88)}. Such localisation, if confirmed, could increase the diet sensitivity of TMA/TMAO generation in humans v. rodents. Following gut formation and absorption, the amine is oxidised by hepatic FMO3 to TMAO (Fig. 1)^{(Reference Koeth, Wang and Levison20,Reference Lang, Yeung and Peter89,Reference Dolphin, Janmohamed and Smith90)} . Additional forms of FMO in humans (FMO1, FMO2, FMO4, FMO5) do not appear to participate significantly in TMA oxidation^{(Reference Fennema, Phillips and Shephard91)}, whereas recent work indicates that FMO1 is responsible for about 10 % of TMA conversion in mice^{(Reference Veeravalli, Karu and Scott26)}.

Choline

Foods rich in lecithin, such as eggs, milk, liver, red meat, poultry and seafoods, are major dietary sources of choline^{(Reference Wang, Klipfell and Bennett2)}, which can be derived from fat-soluble phosphatidylcholine (the dominant source) and sphingomyelin, and water-soluble phosphocholine, glycerophosphocholine and free choline^{(Reference Wiedeman, Barr and Green92)}. The abundant choline moiety within bile presents another potential TMA substrate^{(Reference Roberts, Gu and Buffa22)}. Choline, which contains a trimethylammonium moiety, is directly converted to TMA by gut bacteria, or may be initially metabolised to γ-butyrobetaine before subsequent conversion^{(Reference Koeth, Levison and Culley87)}. There appears to be a threshold intake beyond which choline reaches the large intestine in sufficient quantities for bacterial metabolism^{(Reference West, Shih and Wang93,Reference Ufnal, Zadlo and Ostaszewski94)} . Choline is absorbed from the small intestine via transport proteins that are 50 % saturated at 200–300 µm in mice^{(Reference Sheard and Zeisel95,Reference Sanford and Smyth96)} . With transporter saturation, choline can reach distal sites of bacterial conversion to TMA and dimethylamine^{(Reference Zeisel, DaCosta and Fox97)}, though preliminary evidence of TMA generation within the small intestine^{(Reference Stremmel, Schmidt and Schuhmann88)} suggests that transit to the large intestine may be unnecessary. While details of choline transport and metabolism to TMA and TMAO in humans remain to be clarified, consumption of two hard-boiled eggs (about 250 mg of choline each) together with a 250 mg radiolabelled phosphatidylcholine supplement transiently doubles plasma TMAO^{(Reference Tang, Wang and Levison14)}, similar to other studies of egg consumption^{(Reference Cho, Taesuwan and Malysheva62,Reference Miller, Corbin and da Costa98)} (Table 2). Consistent with transience of diet-induced TMAO changes, baseline plasma TMAO in healthy subjects is not altered with 8 weeks’ consumption of six eggs per week^{(Reference West, Shih and Wang93)}. In high-fat-fed mice, supplementation with 1·2 % (w/w) of the alternative choline source sphingomyelin also increases serum TMAO, although concentrations are relatively low and insufficient to influence atherosclerosis^{(Reference Chung, Wang and Bursill99)}. Summarising studies of choline feeding: high intakes 10- to 15-fold above normal can elevate TMAO in animals^{(Reference Seldin, Meng and Qi6,Reference Li, Gua and Wu39,Reference Tang, Wang and Kennedy76)} , as can dietary supplementation in humans^{(Reference Tang, Wang and Levison14,Reference Zhu, Wang and Tang100)} (Table 2); however, dietary elevations appear transient, and continuous high-level intakes may be necessary to chronically elevate TMAO to pathological levels. This may involve longer-term effects of dietary substrates on TMA-generating bacteria. Further analysis of choline’s role in influencing TMAO levels and CVD is warranted, since studies indicate dietary choline may mitigate against CVD^{(Reference Millard, Musani and Dibaba82)} or exert no effect^{(Reference Meyer and Shea101,Reference Nagata, Wada and Tamura102)} , contrary to proposed pathogenicity of TMAO.

Table 2. Effects of diet or supplementary interventions on plasma trimethylamine N-oxide (TMAO) concentrations*

NA, not available.

* Summary of human studies examining dietary influences or effects of dietary interventions on plasma TMAO concentrations. Levels of TMAO are shown as concentration or as cumulative AUC over defined periods post-interventions, and are variably expressed as: means and standard deviations, or as means or medians and 95 % CI.

Hepatic flavin-containing mono-oxygenase 3

Circulating TMAO concentrations are dependent on TMA oxidation by hepatic FMO3, and correlate with FMO3 expression in mice and human subjects^{(Reference Wang, Klipfell and Bennett2)}.The N-oxygenation of TMA via FMO3 proceeds with a K _m of 28 μm and a k _cat (substrate to endproduct conversions per unit time) of >30/min, based on analysis of heterologously expressed FMO in vitro ^{(Reference Lang, Yeung and Peter89)}. Thus, rapid conversion to TMAO will occur in a substrate-dependent manner in healthy individuals (TMA about 20–25 µm)^{(Reference Jaworska, Hering and Mosieniak51)}. There is evidence that hepatic FMO3 expression or activity is sensitive to sex steroids^{(Reference Bennett, de Aguiar Vallim and Wang28,Reference Falls, Ryu and Cao138,Reference Esposito, Varriale and D’Angelo139)} , dietary factors^{(Reference Cashman, Xiong and Lin140)} and energy intake^{(Reference Astafev, Patel and Kondratov141,Reference Guo, Shen and Li142)} , NO signalling^{(Reference Tanino, Bando and Komada143)} and the transcriptional regulator CCAAT/enhancer-binding protein β^{(Reference Klick, Shadley and Hines144)}. Hepatic FMO3 expression also increases postnatally in humans, with an apparent plateau after early adulthood^{(Reference Koukouritaki, Simpson and Yeung145,Reference Xu, Bhatt and Yeung146)} . Conversely, in female mice hepatic FMO3 transcript may decline following this phase of maturational up-regulation^{(Reference Fu, Csanaky and Klaassen147)}. While there is some debate regarding hepatic FMO expression in male rodents, an ageing-dependent increase in FMO3 protein is reported in male mice^{(Reference Brunt, Gioscia-Ryan and Richey44,Reference Astafev, Patel and Kondratov141,Reference Guo, Shen and Li142)} . Sex is a particularly strong determinant of FMO3 expression patterns, a linkage not strictly consistent with involvement of TMAO in CVD risk: hepatic FMO3 is substantially higher in female v. male mice and human subjects^{(Reference Bennett, de Aguiar Vallim and Wang28)}, paralleled by dimorphism in TMAO concentrations in mice^{(Reference Li, Chen and Gua8,Reference Bennett, de Aguiar Vallim and Wang28,Reference Ke, Li and Zhao40)} and to a lesser extent humans^{(Reference Shimizu, Cashman and Yamazaki148)}, yet females enjoy protection against CVD over much of the lifespan. Pregnancy further up-regulates FMO3^{(Reference Hukkanen, Dempsey and Jacob149)}, and trimethylaminuria (a disorder in which TMA conversion to TMAO is impaired, leading to odour in sweat, urine and breath from rising TMA) is also exacerbated in women during menstruation^{(Reference Hukkanen, Dempsey and Jacob149)}. Circadian patterns of FMO3 expression may also differ markedly between sexes, a dimorphism reduced by cardioprotective energy restriction which up-regulates FMO3 in males, ‘feminising’ the transcriptional profile^{(Reference Astafev, Patel and Kondratov141,Reference Guo, Shen and Li142)} . However, there remains some debate regarding hepatic expression of FMO3 in male mice: there is evidence that FMO3 mRNA is below detection limits^{(Reference Fu, Csanaky and Klaassen147)} or detectable at 0·1–10 % of the levels in females^{(Reference Astafev, Patel and Kondratov141,Reference Guo, Shen and Li142)} ; and that FMO3 protein is not detectable in males^{(Reference Chen, Yi and Zhang9,Reference Bennett, de Aguiar Vallim and Wang28)} , or lowly expressed and augmented with adenoviral delivery, energy restriction or ageing^{(Reference Brunt, Gioscia-Ryan and Richey44,Reference Astafev, Patel and Kondratov141,Reference Guo, Shen and Li142)} . These observations raise questions regarding the utility of male murine models in assessing the role of FMO3 and TMAO in human disease.

Expression of FMO3 is modified by chronic disease and CVD risk factors, including renal dysfunction and disease^{(Reference Johnson, Prokopienko and West150)}, inflammation^{(Reference Zhang, Chaluvadi and Reddy151)}, insulin resistance and diabetes^{(Reference Wang, Klipfell and Bennett2,Reference Tang, Wang and Levison14,Reference Shan, Sun and Huang54,Reference Miao, Ling and Manthena55)} , ageing^{(Reference Brunt, Gioscia-Ryan and Richey44,Reference Astafev, Patel and Kondratov141,Reference Guo, Shen and Li142)} , pollutants and hepatic toxins^{(Reference Petriello, Hoffman and Sunkara152)}. Elevations in TMAO itself may up-regulate FMO3 expression, with evidence of up to 5-fold induction in male mice supplemented with TMAO^{(Reference Ke, Li and Zhao40)}. Though untested (and questions remain regarding hepatic FMO3 in male mice), this suggests that a potentially detrimental positive feedback could emerge in disease settings associated with significantly elevated TMAO. Kidney dysfunction and disease not only reduce TMAO excretion but increase FMO3 expression in mice^{(Reference Johnson, Prokopienko and West150)}, further promoting elevations in TMAO. Expression of FMO3 is also increased with insulin resistance in both mice and humans^{(Reference Wang, Klipfell and Bennett2,Reference Tang, Wang and Levison14,Reference Shan, Sun and Huang54,Reference Miao, Ling and Manthena55)} , providing a basis for TMAO elevations in diabetes and a link between TMAO and CVD^{(Reference Wang, Klipfell and Bennett2,Reference Tang, Wang and Levison14,Reference Shan, Sun and Huang54,Reference Miao, Ling and Manthena55)} . Plasma TMAO in CHD patients undergoing coronary intervention can be independently linked to diabetes, together with age and BMI^{(Reference Dambrova, Latkovskis and Kuka64)}. Whether FMO3-dependent elevations in TMAO might contribute to diabetes remains to be established, though FMO3 knockdown reduces hyperglycaemia in a murine model of hepatic insulin resistance^{(Reference Miao, Ling and Manthena55)}, and circulating TMAO is predictive of insulin resistance with obesogenic feeding in macaques^{(Reference Li, Chen and Liu81)}. On the other hand, Liao et al. ^{(Reference Liao, McManus and Hughes153)} report beneficial effects of FMO3 on glucose homeostasis independent of the insulin-signalling pathway. Furthermore: hepatic TMAO concentrations decline and renal concentrations are unaltered in the db/db mouse model of diabetes^{(Reference Xu, Zhang and Cai154)}; a low-glycaemic index diet increases TMAO^{(Reference Barton, Navarro and Buas155)} despite known benefit in obesity, diabetes and on CVD risk^{(Reference Augustin, Franceschi and Jenkins156)}; and metformin increases TMAO levels while reducing blood glucose in type 2 diabetic patients^{(Reference Huo, Cai and Lu157)}. Disruption of glycaemic control and emergence of the metabolic syndrome in mice is also independent of TMAO levels and insensitive to inhibition of TMA generation^{(Reference Roberts, Gu and Buffa22)}. Interestingly, anti-diabetic and cardioprotective resveratrol up-regulates FMO3 yet reduces TMAO levels via microbiota remodelling^{(Reference Chen, Yi and Zhang9,Reference Bennett, de Aguiar Vallim and Wang28)} , an effect associated with reduced atherosclerosis in ApoE^–/– mice^{(Reference Chen, Yi and Zhang9)}. Conversely, recent work indicates that resveratrol does not influence plasma TMAO in mice on a choline-supplemented (1 % w/w) diet^{(Reference Roberts, Gu and Buffa22)}.

Importantly, links between FMO3, TMAO and disease development are clouded by TMAO-independent effects of the enzyme. Knockdown of FMO3 improves cholesterol balance while augmenting hepatic stress and inflammation in cholesterol-fed mice independently of TMAO levels and TMA generation^{(Reference Warrier, Shih and Burrows158)}. Metabolic effects of FMO3 manipulation in LDL receptor knockout mice are TMA/TMAO independent^{(Reference Bennett, de Aguiar Vallim and Wang28,Reference Shih, Wang and Lee159)} . Furthermore, while FMO3-dependent elevations in TMAO are suggested to promote tissue ageing^{(Reference Li, Chen and Gua8,Reference Ke, Li and Zhao40,Reference Li, Ke and Zhan43)} , and FMO3 expression may rise with age^{(Reference Brunt, Gioscia-Ryan and Richey44)}, recent evidence indicates that increased FMO3 expression is protective, exerting anti-ageing effects that mimic those of energy restriction^{(Reference Guo, Shen and Li142)}. Thus, not only does FMO3 exert beneficial effects, but linkages between FMO3 and disease do not necessarily reflect a role for TMAO.

Cellular transport and renal excretion

Renal function is a key determinant of circulating TMAO, contributing to elevations in inter-related CKD^{(Reference Stubbs, House and Ocque15,Reference Mafune, Iwamoto and Tsutsumi18,Reference Missailidis, Hällqvist and Qureshi53,Reference Xu, Xia and Lu68)} , CVD^{(Reference Wang, Klipfell and Bennett2,Reference Mafune, Iwamoto and Tsutsumi18,Reference Tang, Wang and Shrestha60)} , diabetes^{(Reference Dambrova, Latkovskis and Kuka64)} and ageing^{(Reference Li, Chen and Gua8,Reference Ke, Li and Zhao40,Reference Missailidis, Hällqvist and Qureshi53)} . The cellular influx of TMAO may occur predominantly via organic cation transporter 2, which facilitates renal tubular TMAO uptake^{(Reference Teft, Morse and Leake80)}. Efflux occurs via ATP-binding cassette family transporters, with genetic variation in the ABCG2 transporter potentially contributing to altered TMAO concentrations in dyslipidaemic subjects^{(Reference Teft, Morse and Leake80)}. The ABCG2 gene has oestrogen and hypoxia response elements, is down-regulated by parathyroid hormone and up-regulated by aryl hydrocarbon receptors and DNA methylation, and polymorphisms influence renal excretion^{(Reference Kusuhara and Sugiyama160,Reference Horsey, Cox and Sarwat161)} .

In healthy volunteers with normal kidney function, fractional renal clearance of TMAO exhibits a broad dynamic range^{(Reference Wang, Bergeron and Levison79)}, while patients with renal dysfunction exhibit significantly elevated plasma TMAO as a consequence of functional deficit^{(Reference Stubbs, House and Ocque15,Reference Missailidis, Hällqvist and Qureshi53,Reference Al-Waiz, Mitchell and Idle162,Reference Bell, Lee and Lee163)} . Elevations in TMAO in CKD and HF involve changes in glomerular filtration rate and renal function^{(Reference Missailidis, Hällqvist and Qureshi53)}, with renal medullary damage with hypoperfusion additionally increasing TMAO^{(Reference Doucet, Dutheil and Petit164)}. Recent findings suggest that chronic ingestion of red meat may reduce excretion of TMAO^{(Reference Wang, Bergeron and Levison79)}. Though speculative, detrimental feedback may also emerge at the level of kidney function: as for uraemic toxins in the cardio-renal syndrome, increases in circulating TMAO due to renal hypoperfusion/injury with atherosclerosis, CHD and HF may surpass a pathological threshold to exacerbate vascular^{(Reference Li, Chen and Gua8,Reference Sun, Yin and Liu11)} and myocardial dysfunction^{(Reference Savi, Bocchi and Bresciani47,Reference Makrecka-Kuka, Volska and Antone48)} , renal inflammation, fibrosis and dysfunction^{(Reference Li, Chen and Gua8,Reference Sun, Yin and Liu11,Reference Tang, Wang and Kennedy76)} (Fig. 2). Though this remains to be directly tested, elevations in TMAO predict mortality in CKD^{(Reference Missailidis, Hällqvist and Qureshi53,Reference Tang, Wang and Kennedy76)} , and mimicking TMAO concentrations in advanced CKD and HF (80–100 µm) with either choline (1·0 % total) or TMAO (0·12 %) supplementation promotes renal fibrosis and injury in mice^{(Reference Tang, Wang and Kennedy76)}. On the other hand, others report no relationship between TMAO and mortality or cardiovascular outcomes in end-stage renal disease^{(Reference Kaysen, Johansen and Chertow165)}.

The gut microbiota and human trimethylamine N-oxide concentrations

Up to about 75 % of the variance in plasma TMAO in healthy humans appears unexplained, with meat and fish consumption estimated to account for <15 % and renal function <5 % of variance^{(Reference Krüger, Merz and Rist61)}. Age is an important factor, with substantial age-dependent elevations in TMAO in animal models and humans^{(Reference Li, Chen and Gua8,Reference Ke, Li and Zhao40,Reference Li, Ke and Zhan43)} . Curiously, diet has been estimated to be a surprisingly weak determinant of the variance in TMAO in healthy humans^{(Reference Krüger, Merz and Rist61,Reference Teft, Morse and Leake80)} , and analysis of the heritability of high TMAO concentrations indicates that host genetics also plays a minor role^{(Reference Xu, Wang and Li166)}, consistent with failure of gene association analysis to identify TMAO-linked loci^{(Reference Hartiala, Bennett and Tang167)}. This poor understanding of the control of TMAO levels reflects in part our incomplete (though evolving) understanding of the roles and control of gut bacterial populations in determining TMAO concentrations. Since circulating TMAO is critically dependent on gut bacteria, elevations may reflect microbiota remodelling with diet and disease. The microbiota of vegans has minimal TMAO-generation capacity^{(Reference Koeth, Wang and Levison20)}, and Cho et al. ^{(Reference Cho, Taesuwan and Malysheva62)} present evidence that distinct bacterial profiles govern TMAO generation in healthy male omnivores, identifying ‘high’ v. ‘low’ producers (42 and 58 % of volunteers, respectively). A substantial proportion of the population may thus, owing to specific microbiota profiles, be specifically susceptible to diet-dependent elevations in TMAO. Apparently paradoxical benefits of TMAO substrate intake (choline, carnitine, seafood) on cardiovascular health may also reflect dominant influences of microbiota makeup and remodelling.

The fundamental importance of the microbiota is evidenced by absence of urinary TMA in germ-free mice, and its reduction with antibiotic treatment in conventional mice^{(Reference al-Waiz, Mikov and Mitchell168)}. Effects of antibiotics on carnitine-dependent elevations in TMAO confirm microbiota dependence in humans: in female omnivores, increases in plasma TMAO with carnitine intake are abolished by antibiotics and restored after 3 weeks’ recovery for microbiota re-population^{(Reference Koeth, Wang and Levison20)}. Subsequent investigations confirm the essential role of the microbiota in humans^{(Reference Wang, Klipfell and Bennett2,Reference Tang, Wang and Levison14)} , and differences in TMAO generation in vegans v. non-vegans further emphasise the microbiota’s importance: carnitine challenge increases plasma and urinary TMAO in omnivores but not vegans, a dimorphism eliminated by antibiotic treatment^{(Reference Koeth, Wang and Levison20)}. This suggests that intake of animal protein favours carnitine/choline-metabolising bacteria^{(Reference Koeth, Wang and Levison20,Reference Ferguson169)} , while a non-meat diet low in these substrates and relatively enriched with betaine (a less favourable TMA substrate^{(Reference Wang, Tang and Buffa170,Reference Ross, Zangger and Guiraud171)} ) promotes non-TMA-generating species. Recent identification of distinct bacterial profiles in high and low TMAO producers (high and low responses to dietary substrate)^{(Reference Cho, Taesuwan and Malysheva62)}, and effects of red v. white meat on bacterial carnitine v. choline metabolism^{(Reference Wang, Bergeron and Levison79)}, further confirm the importance of microbiota changes in driving TMAO elevations in humans.

Fennema et al. ^{(Reference Fennema, Phillips and Shephard91)} provide a comprehensive review and summary detailing bacterial species involved in the formation of TMA. Bacteria including Prevotella, Deferribacteres and Teneriticutes species can metabolise choline and carnitine to TMA, while Bacteroides appears less effective than Prevotella ^{(Reference Koeth, Wang and Levison20,Reference Chistiakov, Bobryshev and Kozarov69)} . A recent cross-sectional analysis of 1653 multi-ethnic participants linked circulating TMAO to the abundance of thirteen genera (six Firmicutes, three Bacteroidetes, three Protobacteria, one Fusobacteria), including Prevotella, Mitsuokella, Fusobacterium, Desulfovibrio, Bilophila and Ruminococcaceae and Lachnospiraceae family members^{(Reference Fu, Hullar and Randolph67)}. Studies indicate that omnivores possess more Firmicutes than Bacteroidetes species and a less diverse microbiota compared with vegans/vegetarians, who also have relatively lower Clostridium species^{(Reference Cho, Taesuwan and Malysheva62,Reference Matijasic, Obermajer and Lipoglavsek172,Reference Kabeerdoss, Devi and Mary173)} . In general, meat intake appears to promote Bacteroides, Alistipes, Ruminococcus, Clostridia and Bilophila, while decreasing Bifidobacterium species^{(Reference Tomova, Bukovsky and Rembert174)}. Other work links a vegan diet to decreased Clostridium v. enrichment of Anaerostipes caccae and Lachnobacterium species in subjects with the metabolic syndrome^{(Reference Smits, Kootte and Levin175)}. Although a majority of TMA production occurs within the gut, it is also worth noting that formation can also occur in the mouth via Streptococcus sanguis metabolism of choline^{(Reference Chao and Zeisel176)}. Recent studies identify three biochemical paths for bacterial TMA production, involving the genes cutC ^{(Reference Craciun and Balskus177)}, cntA ^{(Reference Zhu, Jameson and Crosatti178)} and YeaW ^{(Reference Koeth, Levison and Culley87)}. Genomic assessment of TMA production potential in human bacteria, based on these pathways, identifies Firmicutes, Proteobacteria and Actinobacteria species, whereas these paths appear absent in Bacteroidetes ^{(Reference Martinez-del Campo, Bodea and Hamer179,Reference Falony, Vieira-Silva and Raes180)} . Interestingly, in terms of observed increases in circulating TMAO with ageing, Brunt et al. ^{(Reference Brunt, Gioscia-Ryan and Richey44)} report select ageing-dependent changes in mouse gut phyla (increased Proteobacteria, Verrucomicrobia, and candidate division TM7) and genera (increased Bacteroides, Akkermansia and pro-inflammatory, TMA-generating Desulfovibrio; and reductions in several Firmicutes).

Interactions between diet, microbiota makeup and TMAO generation remain to be better detailed in humans, with findings somewhat contradictory. For example, some studies indicate that vegetarian (but not vegan) diets can increase relative proportions of Prevotella, known to produce TMA^{(Reference Wu, Chen and Hoffmann181,Reference Ruengsomwong, Korenori and Sakamoto182)} . One study found that plasma TMAO did not differ significantly between vegans and lacto-ovo-vegetarians^{(Reference Obeid, Awwad and Keller183)}, though the period the groups adhered to these diets was not provided. Recent evidence indicates that red v. white meat may selectively increase bacterial metabolism of carnitine (not choline) to TMA, while reducing renal TMAO excretion^{(Reference Wang, Bergeron and Levison79)}. Further studies are needed to clarify roles of microbiota constituents in governing TMA and thus TMAO production, and the influences of animal- and plant-derived nutrients. It is important that broader biochemical influences of TMA-generating bacteria are also detailed to better understand how the gut influences cardiovascular health – additional co-modified factors may contribute to or influence associations between TMAO and CVD. This is critical in addressing the key question of whether elevated TMAO is a biomarker of microbiota changes secondary to different CVD risk factors and co-morbidities, or is a causal factor contributing to CVD development.

Manipulating trimethylamine N-oxide in CVD

As discussed above, modulation of TMAO accumulation might be beneficial in limiting disease progression and impacts in those with existing CVD, or at particularly high risk (including high TMAO producers^{(Reference Cho, Taesuwan and Malysheva62)}). Reductions in TMAO in CVD might be achievable via modulation of both diet and the gut microbiota, though unknowns and challenges arise with each approach. Given the importance of the gut microbiota in determining TMAO production^{(Reference Wang, Klipfell and Bennett2,Reference Tang, Wang and Levison14,Reference Koeth, Wang and Levison20,Reference Cho, Taesuwan and Malysheva62)} , its manipulation is an obvious candidate for lowering TMAO in those with CVD. Unfortunately, this is not presently feasible, as what constitutes a healthy microbiota and the roles of microbiota composition in governing TMAO concentrations are not fully understood, and our ability to selectively remodel the gut microbiota (suppressing/promoting individual species or their functionality) is not currently feasible. Antibiotics suppress TMAO production and TMAO-dependent atherosclerosis^{(Reference Koeth, Wang and Levison20,Reference Koeth, Levison and Culley87)} ; however, this ‘shotgun’ approach is not viable given broad impacts on microbiota and host health, immunity and antibiotic resistance^{(Reference Brown and Hazen186)}. Ongoing investigations into bacterial control of TMAO generation may reveal strategies for manipulating the biota genetically, pharmacologically or via other means. For example, a recent study shows that halogen-substituted choline analogues alter the caecal microbiota and suppress elevations in TMA, TMAO and thrombosis associated with choline supplementation^{(Reference Roberts, Gu and Buffa22)}. Many drugs can modify the gut microbiota^{(Reference Imhann, Bonder and Vich Vila187,Reference Rogers and Aronoff188)} , and a recent study indicates a quarter of 1000 drugs examined possess antibiotic-like side effects^{(Reference Maier, Pruteanu and Kuhn189)}. While this broadly supports the potential for pharmacological manipulation of the microbiota, the challenge of specificity (and untoward side effects) remains. This hurdle might be overcome with our evolving understanding of bacterial TMAO production (for example, identification of biochemical paths, and gene determinants such as cutC ^{(Reference Craciun and Balskus177)}, cntA ^{(Reference Zhu, Jameson and Crosatti178)} and YeaW ^{(Reference Koeth, Levison and Culley87)}), revealing molecular targets for microbiota-directed genetic or pharmacological therapy.

Probiotics offer a low-cost and -risk approach to microbiota manipulation; however, findings are equivocal, particularly in a CVD context. Supplementation with Lactobacillus casei Shirota for >3 months (19·5 × 10⁹ colony-forming units/d) failed to influence TMAO production in patients with the metabolic syndrome^{(Reference Tripolt, Leber and Triebl190)}, and 3-month probiotic supplementation in haemodialysis patients (9 × 10¹³ colony-forming units/d, including Streptococcus thermophilus, Lactobacillus acidophilus and Bifidobacteria longum) failed to influence plasma TMAO^{(Reference Borges, Stenvinkel and Bergman191)}. Meta-analyses support some cardiovascular benefits of probiotics, though outcomes are modest^{(Reference Khalesi, Sun and Buys192-Reference Khalesi, Bellissimo and Vandelanotte194)}. Unexpectedly, a small double-blind randomised controlled trial of faecal microbiota transplantation from vegans into metabolic syndrome patients successfully modified intestinal microbiota composition yet failed to alter TMAO production^{(Reference Smits, Kootte and Levin175)}. It was unclear what period of time donors adhered to vegan diets, thus whether a vegan microbiota phenotype was established, although shifts between meat and vegetarian diets induce rapid microbiota changes^{(Reference David, Maurice and Carmody195)}. While FMO3 inhibition might also appear to be a target for manipulating TMAO in patients with CVD, sufficiently effective reductions in FMO3 lead to TMA accumulation and trimethylaminuria^{(Reference Mitchell and Smith196)}. More fundamentally, FMO3 mediates diverse effects on metabolism, tissue stress and ageing independently of TMA/TMAO^{(Reference Guo, Shen and Li142,Reference Warrier, Shih and Burrows158)} .