Characteristics of patients
Overall, 1726 consecutive patients with suspected functionally relevant coronary artery disease (fCAD) were included in this analysis (Supplemental Fig. 1), 478 patients (28%) were adjudicated to have fCAD. A total of 421 (24%) patients underwent coronary angiography with 284 patients within the 3 months after enrolment. Anamnesis revealed 764 (44%) to have known history of CAD. During follow-up, 88 patients experienced an incident MI, 223 patients died overall, 115 of which died due to cardiovascular reasons (CV death). Characteristics of patients stratified by fCAD are shown in Table 1. Stratification of patients by experience of either an AMI, all-cause or CV death, is shown in Supplemental Table 1. Patients with fCAD tended to be older and a higher proportion were male. A significant proportion of patients had diabetes, hypertension, and a history of cardiovascular disease. To portrait renal state of the patients, baseline eGFR and cystatin-C were added to the baseline tables. As expected, eGFR was significantly lower in patients with fCAD and cystatin-C significantly higher in patients with fCAD.
Circulating TMAO, betaine, choline and carnitine concentrations
Median TMAO concentrations were significantly higher in patients with fCAD than in those without fCAD (5.33 IQR [3.55, 8.80] µM vs. 4.66 IQR [3.08, 7.24] µM, p < 0.001, Fig. 1A). Stratification by a history CAD revealed that this phenomenon was exclusively present in patients without known CAD (5.36 IQR [3.71, 8.57] µM vs 4.38 IQR [2.94, 6.83] µM, p < 0.001, Fig. 1B). Similar findings emerged for the three precursors betaine, choline and carnitine (Fig. 1C–H).
In patients with normal eGFR (n = 689), TMAO and the three precursors were significantly higher in patients adjudicated to have fCAD (Supplemental Fig. 2A–D). The baseline characteristics of this subgroup are presented in Supplemental Table 2.
TMAO concentrations were significantly, albeit weakly, correlated with hs-cTnT (Spearman’s rho 0.32, p < 0.001), NT-proBNP (Spearman’s rho 0.22, p < 0.001) and age (Spearman’s rho 0.25, p < 0.001). These correlations were weaker for betaine, choline and carnitine (Supplemental Fig. 3).
Diagnostic performance for the detection of fCAD
In the overall cohort, diagnostic accuracy for detection of fCAD was quantified and showed modest but statistically significant value for TMAO with a ROC AUC of (0.56, 95% CI 0.53–0.59, p < 0.001) and its precursors (betaine 0.55, 95% CI 0.52–0.58, p = 0.002; choline 0.54, 95% CI 0.51–0.57, p = 0.007; carnitine: 0.55, 95% CI 0.51–0.58; p = 0.001; Supplemental Fig. 4). Similar findings emerged in the subgroup of patients without known CAD (TMAO 0.59, 95% CI [0.54–0.63], p < 0.001; betaine 0.54, 95%-CI [0.50–0.58], p = 0.039; choline: 0.56, 95% CI [0.51–0.60], p = 0.006; carnitine 0.57, 95% CI [0.52–0.61], p = 0.002; Supplemental Fig. 4B). TMAO and its precursors did not significantly increase the AUC provided by the quantitative clinical assessment by the treating physician before (AUC 0.61, 95% CI [0.58–0.64]) and after (AUC 0.65, 95% CI [0.62–0.68]) stress testing (p > 0.05 for all comparisons of VAS + TMAO (or precursors) versus VAS alone, Supplemental Fig. 4C + 4D). TMAO (OR 1.19, 95% CI [1.08–1.31], p < 0.001) and its precursors were significant predictors of fCAD in the univariable model, with TMAO remaining a significant predictor even after adjusting for age, sex and CAD history (OR 1.12, 95% CI [1.01–1.24], p = 0.036, Supplemental Table 3). When adjusting for renal function (i.e., taking Cystatin-C into the model) or when adjusting for further for pre-defined patient characteristics, cardiovascular risk factors and medical history (model 2), neither TMAO nor its precursors remained significant predictors. In patients with normal eGFR, TMAO and the three precursors were only significant predictors in the univariable model but were no longer significant after adjusting further (Supplemental Table 3).
Prognostic performance for incident major adverse cardiac events
The median follow-up time was 1827.5 days (IQR [756, 1908]). The cumulative event incidence for 5-years death was 12.9% (223 events), for 5-years CV death 6.7% (115 events) and for 5-years MI 5.1% (88 events), and the composite 5-years MACE (MI and cardiovascular death) was 11.8% (203 events).
In Kaplan–Meier analyses, TMAO concentrations above the median had substantially higher all-cause, CV mortality and mace events versus patients below the median (P < 0.001, each; Supplemental Fig. 5 and 6). Similar results emerged for choline and carnitine.
Kaplan–Meier analyses revealed that stratification according to hs-TnT and TMAO concentrations below/above the URL provided incremental prognostic value with the highest all-cause and CV mortality observed in patients with both hs-cTnT and TMAO above the URL (Fig. 2A).
Subgroup analysis (n = 919) of marker-combination with low and normal eGFR levels, Kaplan–Meier survival analyses revealed significantly highest cumulative all-cause death events in patients with low eGFR combined with a TMAO above the median (Fig. 2B).
Time-dependent ROC curve analysis showed a consistent and moderate-to-good discriminative performance of TMAO and choline for 5-years all-cause death (e.g. TMAO at 2-years AUC: 0.67, 95% CI [0.61–0.73]; Choline at 2-years AUC: 0.64, 95%-CI [0.57–0.70]) and 5-years CV death (TMAO at 2-year, AUC: 0.73, 95% CI [0.65–0.81]; Choline at 2-year AUC: 0.67, 95% CI [0.59, 0.76]), and modest accuracy for the other markers and endpoints (Fig. 3). Furthermore, Cox-regression analyses on continuous log-transformed markers are shown in Table 2. TMAO, Carnitine and Choline remained independent predictors in the full model without adjustment of renal function (model 2). When adjusting further for renal function (model 2 + cystatin-c) both TMAO and carnitine remained independent predictors for 5-years CV death. In the cox-regression analysis performed on median stratified marker (Supplemental Table 4), only TMAO remained a significant predictor of all-cause death and CV death, even when adjusted fully and taking renal function into account (model 2 + cystatin-c).