2.1. Study design
This was a prospective, observational study including patients participating in the AFFECT EV Metabolite Substudy. AFFECT EV was an investigator-initiated, prospective study conducted at the 1 st Chair and Department of Cardiology, Medical University of Warsaw, Poland . The study protocol, designed in compliance with the Declaration of Helsinki, was approved by the Ethics Committee of the Medical University of Warsaw (approval number KB/112/2016), registered in the Clinical Trials database (NCT02931045), and published previously . All participants provided written informed consent.
2.2. Study participants
Study inclusion and exclusion criteria are listed in Table 1. Patients were eligible for enrolment if they were (i) admitted to the hospital due to the first ST-segment elevation of AMI (STEMI) or non-STEMI (NSTEMI) with an onset of symptoms during the previous 24 hours, and (ii) underwent PCI with stent implantation. STEMI was defined as persistent ST-segment elevation of at least 0.1 mV in at least two contiguous electrocardiography leads, or a new left bundle-branch block . NSTEMI was diagnosed in patients with typical anginal chest pain accompanied by ST-segment changes (ST depression, T-wave changes, transient ST elevation) on electrocardiogram and an elevation of cardiac troponin concentration in the peripheral blood . When the study was initiated, patients with STEMI were pre-treated with clopidogrel before hospital admission. Because antiplatelet therapy with P2Y12 inhibitors affects platelet reactivity, only patients who received clopidogrel prior to PCI were enrolled in the study to obtain a homogenous study group. Since gut metabolites are excreted by the urinary tract, patients with CKD with estimated glomerular filtration rate (eGFR) <45 ml/min/1.73 m2, calculated using the Modification of Diet in Renal Disease (MDRD) formula, were excluded . Because the intestinal metabolism is affected by the state of gastrointestinal tract and its microbiota, patients with acute or chronic gastrointestinal diseases, autoimmune disease, treated with antibiotics within the last 2 months or taking dietary supplements within the last 7 days are excluded from the study .
Healthy volunteers were recruited among the hospital stuff and included people aged 18–99 years, without any medical history of chronic diseases, chronic pharmacotherapy, acute gastrointestinal disease within the last month, antibiotic therapy within the last 2 months and dietary supplements within the last 7 days.
Table 1. Eligibility criteria for the study.
Age ≥ 18 years
First acute myocardial infarction treated with percutaneous coronary intervention with stent implantation
Chronic kidney disease (estimated glomerular filtration rate < 45 mL/min)
Chronic inflammatory disease
Chronic intestinal disease
Acute gastrointestinal disease within the last month
Antibiotic administration in the last 2 months
Dietary supplements in the last 7 days
Pregnancy or breast-feeding
2.3. Trial schedule and blinding
The trial schedule is presented in Figure 1. Blood was collected from patients by an independent operator (CE), who was otherwise not involved in sample analysis. Aggregometry was conducted by an independent operator (AG). During the trial, participants were identified by an individual number, and samples were coded with a sample number. Bacterial metabolite concentration analysis was performed by an independent operator, blinded to clinical data (MU). Statistical analysis was performed by independent operators (PS, KJ) .
All patients received standard treatment after AMI according to the guidelines, including double antiplatelet therapy, β-blocker, angiotensin-converting enzyme inhibitor or angiotensin receptor blocker, aldosterone receptor antagonist, and protein pump inhibitor [18,19].
2.5. Clinical data collection
Data collected at baseline include demographics (age, gender), body mass index, initial diagnosis, and cardiovascular risk factors, including arterial hypertension, diabetes, hyperlipidaemia, and smoking. In addition, routine laboratory parameters were recorded. At discharge, pharmacotherapy was recorded. Data regarding MACE (recurrent AMI, stroke, cardiovascular death, all-cause death) were collected during the phone-call at the median of 3.5-year follow-up call .
2.6. Blood collection and handling
Peripheral venous blood samples were collected from fasting patients at a single time-point (within 24 hours after AMI). Briefly, blood was collected into ethylenediaminetetraacetic acid (EDTA) tubes for metabolites analysis and into hirudin tubes for platelet reactivity analysis. The first 2 mL were disposed of to avoid pre-activation of platelets. Within a maximum of 15 minutes from blood collection, EDTA samples were centrifuged for 15 minutes at 2,500 g. Plasma was frozen at −80°C until analysed .
2.7. Evaluation of gut bacterial metabolite concentration
The concentrations of TMAO and IS were measured using a Waters Acquity Ultra Performance Liquid Chromatograph coupled with a Waters TQ-S Triple-Quadrupole Mass Spectrometer. The mass spectrometer operated in the multiple-reaction monitoring (MRM)-positive electrospray ionization (ESI) mode, as we have previously described .
2.8. Platelet reactivity
Platelet reactivity was assessed by multiple electrode aggregometry using the adenosine diphosphate test (ADP, 6.5 µmol/L) and thrombin receptor-activating peptide-6 (SFLLRN) test (TRAP, 32 µmol/L) was used as a positive control. Unstimulated whole blood was used as a negative control.
The primary end-point of the study were the differences in plasma TMAO and IS concentrations between patients with AMI and healthy volunteers. The secondary end-point was the prognostic value of TMAO and IS for the occurrence of MACE during the median 3.5-year follow-up time. The exploratory end-points was the correlation between TMAO and IS and platelet reactivity .
2.10. Statistical analysis
Statistical analysis was done using IBM SPSS Statistics, version 24.0 (IBM). Qualitative variables were presented as numbers and percentages and compared with the use of Fischer's exact test. A Shapiro–Wilk test was used to assess the normal distribution of continuous variables. Continuous variables were presented as median with interquartile range or as mean and SD and compared using Mann-Whitney U test or an unpaired t-test. The diagnostic ability of gut microbial metabolites to discriminate between patients with and without MACE and the cut-offs were calculated using a receiver operating characteristic (ROC) curve. Logistic regression model incorporating the gut microbial metabolites with significant sensitivity and specificity (area under the ROC curve, AUC) and clinical characteristics were used to determine the best model for MACE prediction. Mortality and other adverse events were reported depictively. A p-value below 0.05 was considered significant .