2Armed Forces Institute of Rehabilitation Medicine/Cardiac Rehab, Rawalpindi, Pakistan
Method: It was a diagnostic validation case-control study carried out at the Clinical Pathology Laboratories of the Army Medical College, Rawalpindi, Pakistan, from May, 2013 to Jan, 2014. Two hundred and fifty subjects aged < 45 years scheduled to undergo coronary angiography were consecutively screened. Out of these ninetyeight patients with > 70% stenosis, in at least one coronary vessel on angiography, were labeled to have PCAD. An equal number of angio-negative subjects were taken as controls from the study group. Serum IL- 10, IL- 18 and TNF alpha were measured using Enzyme Linked Immuno-sorbent Assay (ELISA), hS CRP on Immulite 1000 and serum cholesterol, Triglycerides and High Density Lipoprotein (HDL) by colorimetric methods. Statistical analysis was done using SPSS-17 and MedCalc software.
Results: Total 196 subjects consisting of 98 PCAD patients and 98 angio-negative controls participated in the study. Mean ± SD age of PCAD patients was 40.7 ± 4.23yrs (89 males and 9 females) while in controls it was 35.1 ± 7.55yrs (93 males and 5 females). Serum hS CRP had highest area under curve AUC (95% confidence interval) of 0.936 (0.89- 0.97) while AUC (95% CI) of IL-18, TNF alpha and IL-10 were 0.853 (0.79-0.90), 0.731 (0.659-0.796) and 0.574 (0.497- 0.649) respectively. Sensitivity-specificity of hS CRP and IL-18 at cut off value of 3.18 pg/ml and 200pg/ml were 86%-91% and 77%-81% respectively while that of IL-18/IL-10 ratio and IL-10 at cut off values > 138.9 and > 0.3pg/ml were 68-64% and 20-100% respectively. Significant correlation was observed between hS CRP (p < 0.01), IL- 18 (p < 0.01) and TNF alpha (p < 0.05).
Conclusion: hS CRP and IL-18 have the best diagnostic potential among the cytokines for detection of PCAD with high sensitivity and specificity. IL-18/IL-10 ratio had moderate significance in the diagnosis of PCAD although it correlated well with the disease burden. IL-10 had 100% specificity highlighting its role in the diagnosis of the disease but its low sensitivity is a limiting factor. Thus, hS CRP and IL-18 are promising potential biochemical parameters aiding in the clinical diagnosis of Premature Coronary Artery Disease.
Keywords: Diagnosis; Heart disease; hS CRP; Interleukin- 18; Interleukin- 10; TNF alpha
IL-18 induces the expression of pro-inflammatory cytokines and Chemokines such as Interleukin-6 (IL-6), IL-8 and Monocyte Chemo attractant Protein-1 (MCP-1) [3]. TNF alpha is also a proinflammatory cytokine which is up regulated in ischemia inducing activation of xanthine oxidase and production of O2*−, leading to dysfunctional coronary endothelium [4]. Samnegard et al. [5] demonstrated that the systemic concentrations of IL-18 and TNF alpha were higher in post myocardial patients as compared to the controls. Moreover, individuals with elevated levels of TNF alpha were at increased risk for acute myocardial infarction and CAD [6]. High sensitive C-reactive protein (hS CRP) is an established proinflammatory biomarker for the detection of individuals at a risk of coronary artery disease [7]. Several studies have also shown that increased levels of fibrinogen, CRP and IL-6 are associated not only with the risk of coronary artery disease but also with its clinical course, progression and severity [8]. Interleukin-10 (IL- 10) on the other hand is an anti-inflammatory cytokine which is associated with a humoral immune response that acts by limiting the local inflammatory response which provides stability to the atherosclerotic lesion [9]. We sought to assess the role of cytokine mediators IL-18, TNF alpha, hS CRP, IL-10, IL-18/IL-10 ratio and TNF alpha/IL-10 ratio in the diagnosis of PCAD patients.
Enzyme Linked Immuno-sorbent Assay (ELISA) technique was used for measuring the concentrations of serum IL-10, IL- 18, and TNF alpha using human IL-10, IL-18, and TNF alpha, (Bender med Systems, Austria) commercial kits with monoclonal antibodies. The calculated overall intra assay Coefficient of Variation (CV) for IL-18, TNF alpha and IL-10 was 6.5%, 6.0% and 3.2% respectively while the limit of detection was 9pg/ml, 2.3pg/ ml and 0.1pg/ml respectively. The inter assay CV was 3.4%, 3.8% and 3.0% for IL-18, TNF alpha and IL-10 respectively. Analysis of hS CRP was done by a chemiluminescent immunoassay on Immulite 1000 using kit provided by Siemens (UK). The interassay coefficient of variation (CV) was 3.5%. Serum cholesterol was measured by cholesterol oxidase method (CHOD-POD) and serum triglyceride was measured by GPO-POD Colorimetric method. All the analytes were run on Selectra E (Vital Scientific, Netherland) using kits provided by Pioneer Diagnostics (USA). CV of the method was < 1%.
Coronary angiography was performed by trained cardiologists by Jut kin technique using a quantitative coronary angiographic system. Degree of atherosclerosis was calculated using the Gensini score [10]. All coronary angiograms were evaluated by the cardiologists who are unaware of the biochemical analysis results to avoid bias.
ROC analysis of biomarkers in PCAD patients and controls revealed that Area under curve (AUC) and 95% (CI) of hS CRP 0.936 (0.89-0.97) was the highest closely followed by IL-18: 0.853 (0.79-0.90). Area under curve (AUC) and 95% (CI) of TNF alpha, IL-18/IL-10 ratio and IL-10 were 0.731 (0.659-0.796), 0.667 (0.592-0.737) and 0.574 (0.497-0.649) respectively [Figure 1]. The diagnostic odds ratio of hS CRP was highest at a cutoff level of 3.18 mg/L. The sensitivity and specificity of hS CRP at this cutoff for the diagnosis of PCAD was 86% and 91% respectively.
Parameters |
Cases (n = 98) |
Controls (n = 98) |
p- value |
Age (years) |
40 ± 4.23 |
35 ± 7.55 |
0.76 |
Gender(M/F) |
89 / 9 |
93 / 5 |
0.63 |
Weight (Kg) |
74.1 ± 11.7** |
67.7 ± 10.1 |
0.0009 |
Height (m) |
1.68 ± 0.06 |
1.69 ± 0.08 |
0.85 |
BMI (Kg/ m²) |
26.32 ± 3.7** |
23.6 ± 3.5 |
< 0.002 |
Positive Diabetes n (%) |
36 (37)** |
3(4) |
< 0.01 |
Positive Premature CAD Family History n(%) |
43( 44)** |
2 (3) |
< 0.01
|
Positive DM family history n (%) |
36(37)** |
11(15) |
< 0.01 |
Smoker’s n(%) |
60 (61)* |
38 (39) |
< 0.05 |
Total Cholesterol (mmol/l) |
4.9 ± 3.77 |
4.5 ± 1.38 |
0.56 |
Similarly, we observed that the sensitivity and specificity of IL-18 was also very high for the diagnosis of PCAD. Positive correlation has been shown between IL-18 and carotid intimamedia thickness and coronary plaque area [19]. This is probably because IL-18 over expression induces atheroma formation while the endogenous inhibitor of IL-18 slows plaque development [20]. Li et al. [21] demonstrated that IL-18 had AUC of 0.86 for detecting and predicting thin cap fibro-atheromas as confirmed on intravascular optical coherence tomography. On the other hand Martins et al. [22] found only marginal significance of IL-18 for prediction of CAD with or without adverse events.
TNF alpha and IL-18/IL-10 ratio showed moderate diagnostic performance for the diagnosis of PCAD. According to Branen et al. [23] TNF alpha is actively involved in the progression of atherosclerosis and its inhibition reduces atherosclerosis in mice. TNF alpha is being considered to be a sensitive and specific biomarker in the early diagnosis of metabolic syndrome which increases the risk to develop PCAD [24]. TNF alpha levels have shown significant association with the atherosclerotic burden consistently [25]. Chalikias et al. [26] demonstrated that an imbalance between the pro-and anti-inflammatory forces leads to plaque disruption and recurrent cardiovascular accidents making IL-18/IL-10 an independent predictor of adverse events
Biomarkers |
SN (%) |
95%CI |
SP (%) |
95%CI |
LR+ |
LR- |
DOR |
hS- CRP (mg/ L) > 3.14 |
85.14 |
75-92.3 |
90.82 |
83.3-95.7 |
9.27 |
0.16 |
58 |
> 3.18* |
86.49 |
76.5-93.3 |
90.82 |
83.3-95.7 |
9.42 |
0.15 |
62 |
> 3.2 |
86.49 |
76.5-93.3 |
89.80 |
82.0-95.0 |
8.48 |
0.15 |
57 |
IL-18 (pg/ ml) ≥ 195 |
70.27 |
58.5-80.3 |
82.65 |
73.7-89.6 |
4.05 |
0.36 |
11 |
> 200* |
77.03 |
65.8-86.0 |
80.61 |
71.4-87.9 |
3.97 |
0.28 |
14 |
> 210 |
78.38 |
67.3-87.1 |
78.57 |
69.1-86.2 |
3.66 |
0.28 |
13 |
TNF alpha(pg/ ml ) > 4.5 |
56.76 |
44.7-68.2 |
83.67 |
74.8-90.4 |
3.48 |
0.52 |
6.7 |
> 4.6* |
58.11 |
46.1-69.5 |
83.67 |
74.8-90.4 |
3.56 |
0.50 |
7.1 |
> 4.8 |
58.11 |
46.1-69.5 |
80.61 |
71.4-87.9 |
3.00 |
0.52 |
5.8 |
IL-18/IL-10 ratio >135.7 |
66.22 |
54.3-76.8 |
64.29 |
54.0-73.7 |
1.85 |
0.53 |
3.5 |
> 138.9* |
67.57 |
55.7-78.0 |
64.29 |
54.0-73.7 |
1.89 |
0.50 |
3.8 |
> 140 |
67.57 |
55.7-78.0 |
63.27 |
52.9-72.8 |
1.84 |
0.51 |
3.6 |
IL-10 (pg/ml) > 0.25 |
13.51 |
6.7-23.5 |
100.0 |
96.3-100 |
5.5 |
0.86 |
6.4 |
> 0.3* |
20.27 |
11.8-31.2 |
100.0 |
96.3-100 |
5.35 |
0.80 |
6.7 |
> 0.4 |
21.62 |
12.9-32.7 |
95.92 |
89.9-98.9 |
5.3 |
0.82 |
6.5 |
Significant positive correlation was observed between hS CRP and IL-18 highlighting the role and probable synergism of proinflammatory cytokine mediators in the diagnostic evaluation and pathogenesis of PCAD. Yamaoka-Tojo et al. [26] suggests that CRP induces the production of IL-18 further enhancing the pro-inflammatory component of the cytokine cascade. Moderate correlation was observed between hS CRP and TNF alpha also. This is in agreement with certain studies which suggest close correlation between hS CRP and TNF alpha [27]. One of the major strengths of our study is that we have independently established the reference interval of novel cytokine mediators in our study and have included angio-negative individuals as healthy controls thus allowing for a better comparison between patients and controls. It is also the first kind of its study to the best of our knowledge which has studied the diagnostic accuracy of proand anti-inflammatory cytokines in Premature Coronary artery disease patients who are young (< 45 years) and whose number is increasing alarmingly in Pakistan and South Asia. Moreover, we have seen that while IL-18 and hS CRP have high diagnostic performance for diagnosis of CAD and may be used in combination with clinical assessment for better therapeutic management. The major limitation of our study is its small sample size and the fact that we have included stable PCAD patients. We therefore recommend that future studies be carried out in multiple centers on larger patient populations and should also include acute coronary syndrome cases of PCAD for a better assessment of the diagnostic efficacy of cytokines in PCAD patients.
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Parameters |
B |
SE |
Exp(B) (95%CI) |
Sig. |
IL- 18 (pg/ ml) |
0.020 |
0.003 |
1.020 (1.004- 1.019)** |
0.005 |
IL- 10 (pg/ ml) |
0.310 |
0.182 |
1.062 ( 0.842- 2.037)* |
0.04 |
TNF- alpha (pg/ ml) |
0.609 |
0.174 |
1.723 (1.274- 2.523)** |
0.001 |
hS- CRP (mg/ dl) |
0.450 |
0.124 |
1.561(1.253-2 .145)** |
0.000 |
Parameters |
B |
SE |
Exp(B) (95%CI) |
Sig. |
IL- 18 (pg/ ml) |
0.020 |
0.003 |
1.020(1.004 - 1.019)** |
0.005 |
IL- 10 (pg/ ml) |
0.310 |
0.182 |
1.062( 0.842 - 2.037)* |
0.04 |
TNF- alpha (pg/ ml) |
0.609 |
0.174 |
1.723(1.274 - 2.523)** |
0.001 |
hS- CRP (mg/ dl) |
0.450 |
0.124 |
1.561(1.253 - 2.145)** |
0.000 |