The symbiotic relationship between humans and microbes is important for maintaining good health. And according to mounting evidence, dysfunctional relationships could increase susceptibility to disease (1). Here, I will use the example of trimethylamine [N-oxide] (TMA[O]), a molecule mediated through metabolism of dietary components by gut microbes, to illustrate the complexity of the microbiome.
TMAO can be measured in biofluids and, in 2011, was found to be elevated in the plasma of patients diagnosed with coronary artery disease (2). Later, it was demonstrated to be elevated in patients at higher risk of major adverse cardiac events (for example, stroke, myocardial infarction) within three years (3). Most systemically circulating TMAO is formed by metabolism of dietary components, such as L-carnitine and free choline, by the gut microbiota (4). These molecules are readily available in red meat and dairy, and TMAO has been identified as a possible mediator in the link between red meat and cardiovascular disease. But the relationship is complex. Paradoxically, TMAO is present in relatively high quantities in fish, yet populations with seafood-rich diets are considered at lower risk of heart disease than other western populations (5). We, and others, are attempting to unravel the relationship between diet, TMAO and heart disease. TMAO is a non-volatile small molecule (molecular weight 75.11), and liquid chromatography-mass spectrometry (LC-MS) methods have been developed to measure circulating concentrations in plasma and serum, and excreted concentrations in urine. Though previous methods have predominantly employed multiple reaction monitoring on triple-quadrupole MS systems, our lab has developed a protocol employing the quadrupole-traveling wave-time of flight setup on a Waters Synapt G-2S instrument (6). The inclusion of a dilution step, using an isotopically labeled internal standard (D9-TMAO), allows a highly specific and selective analysis of samples with accurate quantification. Additionally, the inherent ability for selected/multiple reaction monitoring measurements using LC-MS allows for simultaneous analysis of other molecules related to gut microbial metabolism, without loss of sensitivity or selectivity. For example, analyses may include additional molecules, such as L-carnitine, choline, betaine and ɣ-butyrobetaine, allowing an improved understanding of the dynamics and kinetics of these molecular/metabolic relationships. Using these methods, we have shown that elevated levels of TMAO are associated with poor prognosis in acute hospitalizations of heart failure (7) and myocardial infarction (8). These experiments support previous data from gene knockout mice models, which showed that high levels of TMAO induced atherosclerosis (9) and worsened conditions associated with heart failure (for example, left ventricular ejection fraction) (10). Interestingly, we (and others) have also reported a strong correlation between circulating TMAO levels and markers of renal dysfunction. It is crucial that we ascertain whether elevated TMAO levels cause increased cardiovascular risk, or whether elevated TMAO is a side effect of renal dysfunction (11). In the latter case, increases in TMAO may be a surrogate biomarker for severity of cardiovascular/renal disease, rather than a direct cause. I’m confident that ongoing studies into the metabolic pathways involved will give us the evidence we need to establish the nature of these relationships. Whether TMAO acts as a direct toxin on human cardiac/renal tissue or exists merely as a surrogate biomarker, this small molecule offers valuable prognostic information for a range of cardiovascular conditions, and we hope eventually to see it in clinical use.
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References
- J Aron-Wisnewsky, K Clémont, “The gut microbiome, diet, and links to cardiometabolic and chronic disorders”, Nat Rev Nephrol, 12, 169–181 (2016). Z Wang, et al., “Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease”, Nature, 472, 57–63 (2011). WH Tang, et al., “Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk”, N Engl J Med, 368, 1575–1584 (2013). BJ Bennett et al., “Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation”, Cell Metab,17, 49–60 (2013). R Estruch et al., “Primary prevention of cardiovascular disease with a Mediterranean diet”, New Engl J Med, 368, 1279–1290 (2013). LM Heaney et al., “High mass accuracy assay for trimethylamine N-oxide using stable-isotope dilution with liquid chromatography coupled to orthogonal acceleration time of flight mass spectrometry with multiple reaction monitoring”, Anal Bioanal Chem, 408, 797–804 (2016). T Suzuki et al., “Trimethylamine N-oxide and prognosis in acute heart failure”, Heart 102, 841–848 (2016). T Suzuki et al., “Trimethylamine N-oxide and risk stratification after acute myocardial infarction”, Clin Chem, 63, 420–428 (2017). RA Koeth et al., “Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis”, Nat Med, 19, 576–585 (2013). CL Organ et al., “Choline diet and its gut microbe-derived metabolite, trimethylamine N-oxide, exacerbate pressure overload-induced heart failure”, Circ Heart Fail, 9, e002314 (2016). MA Bain et al., “Accumulation of trimethylamine and trimethylamine-N-oxide in end-stage renal disease patients undergoing haemodialysis”, Nephrol Dial Transplant, 21, 1300–1304 (2006).
