The Siriraj Institutional Review Board (SIRB) of the Faculty of Medicine Siriraj Hospital, Mahidol University approved this study (COA no. Si 354/2016), the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki, and all included patients gave informed written consent to participate. This prospective cohort study enrolled 305 consecutive patients in whom CAD was suspected, and they all underwent coronary angiogram during January 2017 to June 2019. We excluded patients who might have other non-cardiac causes of elevated inflammatory biomarkers, such as rheumatoid arthritis, allergic asthma, cancer, gingival fibroblasts, neuropathy of Alzheimer’s disease, post-transplantation, inflammatory bowel disease, and active infections. We also excluded patients who refused to participate in the study, which involved consenting to blood testing at baseline and at the 6-month follow-up. Baseline clinical characteristics, presenting symptoms, previous history of cardiovascular intervention, basic laboratory test results, current medication used, and coronary angiogram results were obtained.
Measurement of high-sensitivity troponin T (hs-TnT) and high-sensitivity C-reactive protein (hs-CRP)
Blood samples for determination of hs-TnT and hs-CRP levels were obtained at baseline before coronary angiography in lithium heparin vacutainer tubes, and then transported to our center’s Central Laboratory (certified by ISO 15189 accreditation). Plasma hs-TnT levels were measured by an immunoassay using electrochemiluminescence technology (Elecsys Troponin T hs STAT; Roche Diagnostics, Rotkreuz, Switzerland) on a cobas 8000 (e 602) analyzer during January 2017 to June 2019 (Elecsys Troponin T hs STAT e602: Limit of blank: 3 pg/mL, Limit of detection: 5 pg/mL, and Limit of quantification: 13 pg/mL), and on a cobas 8000 (e 801) analyzer during December 2017 to June 2019 (Elecsys Troponin T hs STAT e801: Limit of blank: 2.5 pg/mL, Limit of detection: 3 pg/mL, and Limit of quantification: 13 pg/mL). Hs-CRP concentrations were measured by latex-enhanced immunoturbidimetric assays (Roche Diagnostics, Rotkreuz, Switzerland) on a cobas 8000 (c 502) analyzer. The assay has a lower limit of detection of 0.15 mg/L, and an interassay coefficient of variation < 10% at concentrations of 0.3 mg/L. Method validation of the assays was performed according to CLSI EP 15-A3, CLSI EP 09-A3, and CLSI EP 06-A3 guidelines.
Liquid chromatography–mass spectrometry (LC–MS) analysis of L-tryptophan (L-Trp) and L-kynurenine (L-Kyn)
The kynurenine (Kyn) to tryptophan (Trp) ratio (KTR) was used as a surrogate indicator of IDO activity. Blood samples for determination of Kyn and Trp levels were obtained at baseline before coronary angiogram and at the 6-month follow-up. Indoleamine 2,3 dioxygenase (IDO) activity as reflected by Kyn/Trp ratio was calculated. Kyn and TRP were detected according to the method described by Yong and Lau [12], with minor modifications using high-performance liquid chromatography (HPLC) (Thermoseparation products with UV/VIS Detectors, Fremont, CA, USA). Serum Kyn and Trp levels were measured by reversed-phase HPLC. After overnight fasting, 2 cc of venous was blood drawn from each participant. Collected samples were centrifuged at 2,000 revolutions per minute (rpm) for 10 min within 30 min of collection, and the sample was stored at -80 degrees Celsius until analysis. Frozen serum was thawed at room temperature (RT). The thawed sample was deproteinized by adding an equal volume of 5% (v/v) perchloric acid. The acidified serum was vortexed, allowed to sit at RT for 10 min to precipitate the protein, and was then centrifuged for 10 min at 9000 rpm. Twenty µL of supernatant was injected into the HPLC column for analysis. Tryptophan, kynurenine, and kynurenine acid were determined by reversed-phase HPLC, as previously described [12].
Coronary angiogram was performed according to standard protocol. Two cardiologists who were unaware of the biomarker results reviewed and interpreted the results of coronary angiogram. Significant CAD was defined as visibly obvious luminal coronary obstruction > 70% in at least 1 major epicardial vessel. The percent and degree of coronary artery stenosis was recorded. Location of significant CAD also obtained according to American Heart Association classification. Percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) was performed if needed according to standard protocol. The clinical outcome of myocardial infarction, recurrent angina, hospitalization from angina, revascularization, and death at 1 year was prespecified.
Statistical analysis
Sample size was calculated based on the study from Wirleitner [13]. The mean of IDO activity level in healthy control and patient with significant CAD was 28.1 \(\pm\) 5.15 and 36.3 \(\pm\) 13.0 µmol/mmol. We assumed the significant CAD patients has mean IDO activity level 20% greater than healthy control (28.1 × 1.2 = 33.72 µmol/mmol). P-values less than 0.05 were considered to indicate significant differences and the power of test was 90%. The proportion in healthy control’s group to significant CAD patient’s group was 1: 3. A total of 304 samples were obtained by 76 of healthy controls and 228 of significant CAD patients. Categorical data were presented as frequency and percentage. Continuous variables were reported as mean ± standard deviation or median (minimum, maximum) depending on the distribution of data. Categorical data were compared using chi-square test or Fisher’s exact test, and continuous data were compared using Student’s t-test (normality) or Mann–Whitney U test (non-normality). Patient survival was estimated using Kaplan–Meier survival analysis, and log-rank test was used to compare survival between groups. The best cut-off value for the statistical significance of variables was determined by receiver operating characteristic (ROC) curve analysis, and validity was assessed by the area under the curve (AUC). A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using PASW Statistics v.22.0 (SPSS, Inc., Chicago, IL, USA).