Identifying changes in serum metabolites before the occurrence of acute myocardial infarction (AMI) is an important approach for finding novel biomarkers of AMI. In this prospective cohort study, serum samples obtained from patients at risk of AMI (n = 112) and non-risk controls (n = 89) were tested using high-resolution metabolomics (HRM). Partial least-squares discriminant analysis (PLS-DA), along with univariate analysis using a false discovery rate (FDR) of q = 0.05 were performed to discriminate metabolic profiles and to determine significantly different metabolites between healthy control and AMI risk groups. PLS-DA significantly separated the AMI risk sera from control sera. The metabolites associated with amino acid biosynthesis, 2-oxocarboxylic acid, tryptophan, and amino sugar and nucleotide sugar metabolism pathways were mainly elevated in patients at risk of AMI. Further validation and quantification by MS/MS showed that tryptophan, carnitine, L-homocysteine sulfinic acid (L-HCSA), and cysteic acid (CA) were upregulated, while L-cysteine and L-cysteine sulfinic acid (L-CSA) were downregulated, specifically among AMI risk sera. Additionally, these discriminant metabolic profiles were not related to hypertension, smoking or alcoholism. In conclusion, detecting upregulated L-HCSA and CA along with carnitine among patients at risk for AMI could serve as promising non-invasive biomarkers for early AMI detection.

The samples were randomized and analyzed using C18 Synchronis aQ, 1.9 μm, 1002.1 mm (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The column and autosampler temperature were maintained at 45 °C and 10 °C, respectively. Solvent A consisting of 0.1% formic acid in water and solvent B consisting of 0.1% formic acid in acetonitrile were used as mobile phases. The injection volume and flow rate were 5 µL and 0.4 mL/min, respectively. The HPLC gradient was programmed as follows: 95% water for 1 min, a linear decrease to 55% water over 8 min, a descending gradient to 10% water over 3 min, a 90 sec-min hold, and return to 95% water over 0.1 min. The electrospray ionization detector was operated with a curtain gas of 35 psi at 250 °C, supplied at 14 mL/min, and sheath gas temperature of 250 °C, supplied at a flow rate of 11 mL/min. An equal volume of blank sample consisting of 100% ACN was randomly inserted among the real sample queue to be processed as a needle wash and to equilibrate the column, as well as avoid contamination among real samples.All samples were run in triplicate and data for each ionization technique were acquired in positive ion mode.The standards and serum samples were first scanned in the mass range (m/z) 50–1,000. Collision energy of 0, 5, 10, 15, and 20 V was then used to produce the highly abundant fragment ions of the putative metabolites during product-ion analysis in positive mode. Concentrations of identified metabolites in serum samples were quantified by making a calibration curve of each standard compound with at least eight appropriate concentration levels. The limit of detection (LOD) and limit of quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise (S/N) ratio of 3 and 10, respectively. Sera samples were run in triplicate and the data from triplicated run were averaged to calculate the final concentrations

Quantified (micromolar) concentrations of L-tryptophan, carnitine, L-homocysteine sulfinic acid, cysteic acid, L-cysteine, and L-cysteine sulfinic acid were analysed in serum samples from patients at risk of acute myocardial infarction (represented as MI) and control subjects (represented as control or C). HSA represents L-homocysteine sulfinic acid, CA represents cysteic acid, and CSA represents L-cysteine sulfinic acid.