This prospective case-control study involved 115 consecutively enrolled and consented young CAD patients who presented with acute STEMI. Inclusion criteria: acute STEMI patients (aged < 45 years); for the disease diagnosis, we employed the European Society of Cardiology guidelines and its 4^th MI definition16. The study was conducted over six months, from April 2022 to September 2022. The study sample size was calculated with a power of 0.95 and a hypothetical effect size of 0.33, using G Power software. Patients were admitted to the Department of Cardiology, Assiut University Hospitals, Assiut, Egypt. As a control group, 55 age-matched and apparently healthy volunteers were recruited. We excluded: STEMI patients over 45 years, those with chronic diseases (diabetes, hepatic or renal failure), COVID-19 patients, and addicts (for drugs and/or alcohol). The patient group is part of our previous cohort study that investigated the implication of PCSK-9 in premature CAD pathogenesis17.

The study was carried out in compliance with the Helsinki Declaration principles after being approved by the Institutional Review Board, Faculty of Medicine, Assiut University (Approval#: IRB17200689). Volunteering participants signed the informed consent form after being briefed on the nature of the study and their roles and rights.

After recording the clinical data, the socio-demographic and anthropometric characteristics of the participants, the presence of traditional risk factors (family history, smoking, hypertension, dyslipidemia, and body mass index [BMI; kg/m²]), and the relevant routine laboratory findings were retrieved. On admission, resting 12-lead surface ECG recording, echocardiography, and percutaneous coronary intervention (primary PCI) were conducted. All coronary lesions with >50% diameter stenosis in a vessel >1.5 mm in diameter were assessed by interventional cardiologists for each patient to determine the SYNTAX score (SS), based on the SS calculator18. According to SS, the disease severity was stratified into three levels: SS ≤ 12, >12–≤21.5, and >21.519. Healthy volunteers were subjected to history taking, physical examination, and assessment of their BMI.

After admission, 5 mL of peripheral venous blood was collected from each participant in a plain tube, left to clot, centrifuged at 3000 rpm for 10 minutes, and the separated serum was stored at -20 °C. We used commercially available specific quantitative ELISA kits to assess serum protein biomarkers: 1) Cardiac troponin T (cTnT) and creatine phosphokinase-MB (CK-MB) (Cat.# ab223860 and ab193696; Abcam, UK); and, 2) CRP (with 0.44 pg/mL sensitivity), TNF-α, and IL-10 (Cat.# EL0036Hu, SL1761Hu, and SL0967Hu, Sunlong Biotech Co., Hangzhou, China), as instructed by the manufacturer. We used quantitative colorimetric assay kits (Spectrum, Cairo, Egypt) to assess lipid profile and relevant organ functions [aspartate aminotransferase (AST; REF: 260-002), alanine aminotransferase (ALT; REF: 264-002), alkaline phosphatase (ALP; REF: 216-001), creatine phosphokinase (CPK; REF: 238-002), creatinine (REF: 235-001), urea/BUN (REF: 319-001), total cholesterol (REF: 230006), high-density lipoprotein cholesterol (HDL-C; REF: 266003), triglycerides (REF: 314005), and bilirubin (total and direct; REF 225-001), as instructed]. We calculated LDL-C to be = Total Cholesterol–Triglycerides/5–HDL-C. Total anti-oxidative capacity (TAC; U/mL) measured by Fe³+-2,3,6-tri (2-pyridyl)-1,3,5-triazine reagent, and lipid peroxidation (LPO; μmol/L) measured as malondialdehyde (MDA), after the reaction with thiobarbituric acid, were colorimetrically assayed (Cat# AK0455-100T-96S and AK0694-50T-58S, Sunlong Biotech Co., Hangzhou, China). Serum nitric oxide (NO; nmol/mL) was estimated as nitrite using Griess reagent against the sodium nitrite standard20. Serum reduced glutathione (GSH; μg/mL) concentration was estimated by the DTNB reagent21, 22. Estimation of serum L-ascorbic acid (μg/mL) was done using Folin phenol reagent vs. L-ascorbic acid standard23.

The nominal data were expressed as frequency (percentage), and Chi²-test was used to compare them between groups. The continuous data were expressed as mean and standard error (SE), and the Student’s t-test or one-way analysis of variance (ANOVA), with Bonferroni post hoc test, was used to compare their means among groups. Receiver Operating Characteristic (ROC) curve analysis was used to determine the area under the curve (AUC) for each of CRP, TNF-α, and IL-10 to determine their sensitivity and specificity to discriminate patients from healthy controls at several cutoff points. A probability (P value) of <0.05, at a confidence level of 95%, was considered statistically significant. Data entry, analysis, and presentation were done using the Statistical Package for the Social Sciences (SPSS) version 20 (IBM, Armonk, New York, USA).

Table 1

	Patients (n=115)	Control (n=55)	P-value
Age, year	38 ± 0.4	38 ± 0.3	0.63
BMI, kg/m2	26 ± 0.4	24 ± 0.2	> 0.001
Sex	Male: 108, (94.7%) Female: 6, (5.3%)	Male: 50, (90.9%) Female: 5, (9.1%)	0.66
Smoking	Smokers: 92, (80%)	Smokers: 6, (10.9%)	> 0.001
Hypertention	Hypertensive: 20, (17.3%)	Hypertensive ( 0 )	0.14
Family history of PCAD	Yes: 19, (16.5%)	Yes: 2, (3.6%)	0.19
Family history of hyperlipidimia	Yes: 15, (13%)	Yes: 4, (7.2%)	0.73
History of previous PCI	Yes: 11, (9.5%)	Yes ( 0 )	0.20
Main CBC parameters
WBCs, 109/µL	10.2 ± 0.3	5.5 ± 0.1	> 0.001
Platelets, 109/L	276.7± 7.8	380.2 ± 4.0	> 0.001
Hb, g/L	144 ± 16	142 ± 18	0.32
Liver biomarkers
Total bilirubin, µmol/L	6.5 ± 0.1	6.3 ± 0.2	0.56
Direct bilirubin, µmol/L	2.2 ± 0.07	2.1 ± 0.1	0.63
AST, µKat/L	0.31 ± 0.01	0.31 ± 0.01	0.87
ALT, µKat/L	0.29 ± 0.01	0.28 ± 0.01	0.63
ALP, µKat/L	1.05 ± 0.02	1.06 ± 0.04	0.84
Kidney biomarkers
Creatinie, µmol/L	68.3 ± 1.4	55.4 ± 1.3	0.41
BUN, mmol/L	6.0 ± 0.4	3.4 ± 0.1	> 0.001
Lipid profile
LDL-C, mmol/L	3.2 ± 0.08	3.1 ± 0.1	0.44
Total cholesterol, mmol/L	4.8 ± 0.1	4.5 ± 0.1	0.14
HDL-C, mmol/L	1.1 ± 0.02	1.5 ± 0.03	> 0.001
Triglycerides, mmol/L	3.5 ± 0.3	3.0 ± 0.2	=0.04
Cardiac biomarkers
c-Tnt, µg/L	82.5 ± 16.4	0.02 ± 0.01	> 0.001
CK-MB, µg/L	1118.5 ± 83.3	2.1 ± 0.08	> 0.001
CPK, µKat/L	21.9 ± 1.7	0.9 ± 0.03	> 0.001

Continuous data was expressed in the form of mean ± SE (compared with Student’s t test), while nominal data was expressed in form of frequency (percentage) (compared with Chi2 test). P value was significant if ≤ 0.05.

The age comparison of our participants was not significantly different (38 ± 0.3 years for healthy controls versus 38 ± 0.4 years for patients). BMI was significantly higher among patients compared to controls (26 ± 0.4 versus 24 ± 0.2 kg/m²; P > 0.001). While the majority of our patients were smokers, smokers constituted a minority among controls (80% versus 10.9%; P > 0.001). Hypertensive patients constituted 16.5%, and for family history, 13% had hyperlipidemia and 16.5% had premature CAD in their families. Percutaneous coronary intervention (PCI) was performed on 9.5% of our patients (Table 1).

The patient group demonstrated non-significantly higher serum levels of total cholesterol (4.8 ± 0.1 mmol/L) and LDL-C (3.2 ± 0.08 mmol/L) compared to healthy controls (4.5 ± 0.1 mmol/L and 3.1 ± 0.1 mmol/L, respectively). Healthy controls had significantly greater HDL-C levels (1.5 ± 0.03 mmol/L versus 1.1 ± 0.02 mmol/L; P > 0.001) and lower triglyceride levels (3.0 ± 0.2 mmol/L versus 3.5 ± 0.3 mmol/L; P = 0.04) compared to the premature STEMI patients. Significantly high levels of CPK, CK-BM, and cTnT were observed in patients compared to healthy controls (P > 0.001) (Table 1).

Table 2

	Patients (n = 115)	Control (n = 55)	P-value
CRP mg/L	38.7 ± 1.9	0.8 ± 0.04	> 0.001
TNF-α pg/mL	68.4 ± 4.7	10.8 ± 0.7	> 0.001
IL-10 pg/mL	11.9 ± 0.8	8.7 ± 0.4	=0.001
TNF-α/IL-10	6.8 ± 0.5	1.7 ± 0.2	> 0.001

Continuous data was expressed in the form of mean ± SE (compared with Student’s t test). P-value was significant if ≤ 0.05

Figure 1

Serum CRP levels were significantly higher in premature STEMI patients than healthy controls (38.7 ± 1.9 versus 0.8 ± 0.04 mg/L; P > 0.001). Serum levels of TNF-α (68.4 ± 4.7 pg/mL versus 10.8 ± 0.7 pg/mL; P > 0.001) and IL-10 (11.9 ± 0.8 pg/mL versus 8.7 ± 0.4 pg/mL; P = 0.001) were significantly higher in patients compared to healthy controls. The TNF-α/IL-10 ratio was significantly higher in premature STEMI patients than controls (6.8 ± 0.5 versus 1.7 ± 0.2; P > 0.001) (Table 2). CRP levels positively correlated with TNF-α levels (r = 0.580 and P > 0.001) (Figure 1).

Table 3

Parameters	Patients (n = 115)	Control (n = 55)	P-value
T-AOC, U/mL	25.5 ± 0.9	14.9 ± 0.4	> 0.001
NO, nmole/mL	10.5 ± 0.3	8.1 ± 0.3	> 0.001
GSH, µg/mL	3.8 ± 0.1	5.2 ± 0.2	> 0.001
L-ascorbic acid, µg/mL	7.8 ± 0.3	7.3 ± 0.3	0.172
LPO, µmole/L	15.8 ± 0.4	4.1 ± 0.2	> 0.001

Continuous data was expressed in the form of mean ± SE (compared with Student’s t test). P value was significant if ≤ 0.05.

Table 4

Parameter	SS ≤ 12	12 < SS ≤ 21.5	SS > 21.5	p-value
CRP (mg/L)	30.6 ± 2.5	46.3 ± 2.9	53.7 ± 9	> 0.001
TNF-α (pg/mL)	49.3 ± 4.3	87.3 ± 8.6	88.6 ± 17.4	> 0.001
IL-10 (pg/mL)	11.9 ± 1.5	11.7 ± 0.5	14.9 ± 4.4	0.80
TNF-α/IL-10	5.9 ± 0.7	7.6 ± 0.7	6.5 ± 0.9	0.23
T-AOC (U/mL)	26.0 ± 1.2	25.8 ± 1.5	26.2 ± 6.3	0.99
NO (nmole/mL)	9.8 ± 0.5	11.0 ± 0.5	13.5 ± 1.1	=0.04
GSH (µg/mL)	3.8 ± 0.2	4.0 ± 0.2	4.3 ± 0.7	0.70
L-ascorbic acid (µg/mL)	7.3 ± 0.5	8.8 ± 0.6	6.2 ± 1.7	0.09
LPO (µmole/L)	15.7 ± 0.5	16.3 ± 0.7	16.5 ± 0.7	0.08

Continuous data was expressed in the form of mean ± SE (compared with ANOVA test). P-value was significant if ≤ 0.05.

Figure 2

Serum T-AOC levels were significantly higher in premature STEMI patients than healthy controls (25.5 ± 0.9 versus 14.9 ± 0.4 U/mL; P > 0.001). The higher serum levels of NO (10.5 ± 0.3 versus 8.1 ± 0.3 nmol/mL; P > 0.001) and the lower GSH levels (3.8 ± 0.1 µg/mL versus 5.2 ± 0.2 µg/mL; P > 0.001) were significantly different when comparing patients and controls. Serum vitamin C levels were non-significantly higher in patients than controls (7.8 ± 0.3 versus 7.3 ± 0.3 µg/mL; P = 0.17). Serum LPO levels were markedly higher in patients than in the healthy group (15.8 ± 0.4 versus 4.1 ± 0.2 µmol/L, P > 0.001) (Table 3).

There were significant progressive increases in serum levels of CRP (P > 0.001), TNF-α (P > 0.001), and NO (P = 0.04) with the progress in disease severity assessed by SS (Table 4).

ROC was utilized to determine the cutoff levels, sensitivity, specificity, and area under the curve (AUC) to investigate the ability of CRP, TNF-α, IL-10, and the TNF-α/IL-10 ratio to differentiate premature ST-segment elevation myocardial infarction (STEMI) patients from healthy controls. A serum CRP cut-off point >1.45 pg/mL had a sensitivity of 97% and a specificity of 99.5% for the prediction of premature STEMI, with an AUC of 0.980. A serum TNF-α cutoff point >22.31 pg/mL displayed a sensitivity of 83% and a specificity of 99.5% in predicting premature STEMI, with an AUC of 0.970. Serum IL-10 levels >11.15 pg/mL displayed a sensitivity of 50% and a specificity of 85% for the prediction of premature STEMI, with an AUC of 0.670. A serum TNF-α/IL-10 ratio cutoff point >3.55 displayed a sensitivity of 77% and a specificity of 93% for the prediction of premature STEMI, with an AUC of 0.910 (Figure 2, Table 5).