This study is an ancillary analysis of a prospectively acquired database (RICO survey) (16). All consecutive patients admitted to the coronary intensive care unit (ICU) of the Dijon University Hospital (France) from January 2013 to April 2015 for type 1 MI were prospectively included. Patients with prior coronary arterial disease [MI, transluminal angioplasty, unstable angina, or coronary artery bypass graft (CABG) surgery] or chronic kidney disease were excluded. The present study agrees with the ethical guidelines of the Declaration of Helsinki. Informed consent was obtained from the participants before their inclusion in the study, and the Ethics Committee of the University Hospital of Dijon approved the protocol (BIOCARDIS-2016–9205AAO034S02117).

Patient characteristics, including age and gender, were obtained at hospital admission, along with medical history (hypertension, diabetes, smoking, and family history of coronary artery disease, main treatments [antiplatelets, angiotensin 2 receptor blockers (ARB), angiotensin 2 converting enzyme (ACE) inhibitors, statins, and beta-blockers], and clinical data [left ventricular ejection fraction, heart rate (HR), blood pressure, catecholamine administration, and infarction location]. Shock index was defined as the heart rate divided by systolic blood pressure (SBP). Coronary artery disease burden at coronary angiography through multivessel disease and the SYNTAX (17) score were also collected. The Global Registry of Acute Coronary Events (GRACE) risk score (18), a robust prognosis tool following MI, was also calculated.

Blood samples were taken on admission. N-terminal pro-brain natriuretic peptide (Nt-ProBNP, normal value <125 pg/ml), blood lipids [normal range 1.20–2.4 g/L for cholesterol, 0.4–0.6 g/L for HDL cholesterol, and 0.4–1.5 g/L for triglycerides (TG)], glucose (normal range 4.3–6.4 mmol/L), glycated hemoglobin (HbA1c, normal value <6%), and C-reactive protein (CRP) (normal value <4 mg/L) were measured. The troponin Ic peak (normal value <0.1 µg/L) was obtained from three blood samples within 24 h following admission. The estimated glomerular filtration rate (eGFR) was calculated using the chronic kidney disease-epidemiology collaboration formula (CKD-EPI).

Plasma was taken upon ICU admission in an EDTA (ethylenediaminetetraacetic acid) blood collection tube. Blood was centrifugated (2,000×g for 10 min at 4 °C), and plasma was stored at −80 °C. LPS, lipid transfer protein activities [phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP)], and cytokines were retrospectively measured from this plasma collection.

LPS was quantified by measuring one of its components [3-hydroxy myristate (3HM)] using liquid chromatography coupled with tandem mass spectrometry, as previously described (19). Briefly, samples were hydrolyzed for 3 h at 90 °C in the presence of hydrochloric acid to release free fatty acids, which were subsequently extracted with water and ethane/ethyl acetate (3/2 v/v). Dried extracts were dissolved in ethanol and injected onto an SBC18 2.1 mm × 50 mm, 1.8 µm column, connected to an Infinity 1290 HPLC system (Agilent Technologies). Separation of hydroxylated fatty acids was achieved at 45 °C at a flow rate of 0.4 ml/min using ammonium formate (5 mM/0.1% formic acid) as eluent A (55% for 0.5 min) and 95% acetonitrile as eluent B (100% reached in 2.5 min and maintained for 5 min). MS/MS detection was performed in negative mode using a QqQ 6490 triple quadrupole mass spectrometer equipped with a JetStream ESI source to quantify the selected ions as follows: for 3HM, precursor ion 243.2 Da and product ion 59 Da; for 3-hydroxytridecanoic acid (IS), which was used as the internal standard, precursor ion 229.2 Da and product ion 59 Da.

PLTP and CETP activities were measured in undiluted plasma using commercially available fluorescence activity assays (Roar Biomedical), according to the manufacturer's instructions. Incubations were performed at 37 °C for 30 min (for PLTP) or 3 h (for CETP), with fluorescence monitoring (excitation, 465 nm; emission, 535 nm) throughout the incubation period with a Victor2 multilabel counter (PerkinElmer, Waltham, United States). Transfer activities were calculated from the slope of fluorescence increase between 1 and 30 min (for PLTP) or between 1 min and 1 h (for CETP) and expressed as arbitrary fluorescence units (AU).

Cytokines were measured using a Luminex® Human Magnetic assay (R&D Systems, Minneapolis, USA). The assays were performed according to the manufacturer's instructions (including for sample collection and preparation). Plasma was not diluted for this analysis. Standards and samples were analyzed on a Luminex® apparatus (BioPlex 200, Bio-Rad, Munich, Germany) using BioPlex Manager Software (Version 5, Bio-Rad, Hercules, CA, USA). For all cytokines, standard curves ranged from 3.2 to 10,000 pg/ml.

Plasma markers included endotoxin plasma concentration, cytokine plasma concentration, and CRP.

Clinical outcomes included in-hospital major adverse CV events (MACEs), such as CV death, heart failure (defined as Killip class >1), and recurrent myocardial infarction, ventricular and supraventricular arrhythmia, and ICU length of stay. A follow-up at 1 year with the patient, next of kin, or the treating physician was conducted by mail or telephone. Occurrences of MACEs, defined as CV death, recurrent myocardial infarction, hospitalization for heart failure, unscheduled percutaneous coronary intervention (PCI), coronary artery bypass surgery, unstable angina or angina pectoris, were collected.

No sample size calculation was performed for this study. All the patients included in the cohort were analyzed. Dichotomous variables are expressed as n (%), and continuous variables are expressed as means ± standard deviations or medians [interquartile ranges (IQR)]. A Kolmogorov–Smirnov test was performed to assess the normality of continuous variables. Non-normally distributed variables were log-transformed when needed. The Mann–Whitney test or Student's t-test was used to compare continuous data, as appropriate. Pearson correlation analyses (for normally distributed variables) or Spearman correlation analyses (one or two non-Gaussian variables) were performed. The threshold for significance was set at p < 0.05. Multivariate linear regression models were built to estimate LPS levels based on significant variables in univariate analysis, with an inclusion threshold of p < 0.10. Variable selection was stepwise. The normality of residuals and homoscedasticity were checked graphically; the model was also checked for autocorrelations of residuals (Durbin–Watson test) and multicollinearity (variance inflation factor). Missing data were considered at random and were omitted. SPSS version 12.0.1 (IBM Inc., Armonk, NY, USA) was used for all analyses.

Two hundred and forty-five patients were analyzed. Baseline characteristics are presented in Table 1. The mean age was 62 ± 13 years, most patients were men (72%), and the median body mass index (BMI) was 27 (25–29). We identified 92 patients (37.6%) with hypercholesterolemia and 34 (14%) with diabetes. Most patients had ST-segment elevation (Table 2).

The median LPS concentration was 106 (83–131) pmol/L of 3HM. The distribution of LPS concentration in our population is illustrated in Figure 1. Endotoxemia was associated with age, diabetes, and obesity (Table 1, Figure 2), as well as with total cholesterol (r = 0.191, p = 0.003), triglyceridemia (r = 0.201, p = 0.002), and glucose metabolism parameters (blood glucose, HbA1c) (Table 3). LPS was lower in smokers (102.06 pmol/L of 3HM (79.58–121.80) vs. 110.44 pmol/L of 3HM (91.60–132.65), p = 0.22). PLTP activity was positively correlated with LPS concentrations (r = 0.146, p = 0.022). At admission, patients with high endotoxemia had a faster heart rate (r = 0.19, p < 0.01), and a higher shock index (≥0.7) was associated with higher LPS concentrations. In multivariate analysis, triglycerides, total cholesterol, history of diabetes, and age were independently associated with LPS concentration (Table 4).

Interleukin (IL)-6 and CRP were below the detection threshold in most patients (74% and 84%, respectively). We found no association between the measured cytokines [IL-6, IL-8, tumor necrosis factor (TNF)-α] and LPS (p > 0.05). A level of CRP higher than 10 mg/L was not associated with LPS (Table 5).

LPS blood concentration was not associated with initial severity (Table 2) nor with the occurrence of MACE (or any of its components) or all-cause mortality (p = 0.4) during hospital stay, as assessed by GRACE and SYNTAX scores. However, LPS was associated with a longer ICU stay (r = 0.18, p = 0.005) (Table 5).

LPS blood concentration was not associated with the occurrence of 1-year MACE (or any of its components) nor with all-cause mortality (p = 0.9, Table 5).