Comparison of inflammatory biomarkers between peripheral artery disease patients and healthy individuals

Ahmet Deniz Kaya; Esma Nur Metin Kaya; İhsan Alur

doi:10.5606/e-cvsi.2024.1694

Abstract

Objectives

This study aimed to compare inflammatory markers such as fibrinogen, C-reactive protein, and white blood cell count between patients with peripheral artery disease (PAD) and healthy individuals and investigate whether there is a relationship between low-grade inflammation and PAD.

Patients and methods

This case-control study was conducted with 162 individuals (107 males, 55 females; mean age: 52.5±13.7 years; range, 24 to 87) between January 2023 to January 2024. Eighty-seven of these participants were diagnosed with PAD by lower extremity color Doppler ultrasonography and computed tomography angiography, and the remaining 75 were healthy individuals. Biochemical results of patients and control groups were examined.

Results

Comparing the groups, statistical significance (p<0.05) was found according to sex, age, hypertension, diabetes mellitus, smoking, blood glucose levels, blood creatinine levels, estimated glomerular filtration rate, high-density lipoprotein, triglyceride, fibrinogen, white blood cell count, and C-reactive protein levels. In the group of PAD patients, male sex, hypertension, diabetes mellitus, and smoking were more prevalent, along with higher levels of glucose, creatinine, triglyceride, fibrinogen, white blood cell count, and C-reactive protein.

Conclusion

Inflammation biomarkers, such as fibrinogen and C-reactive protein, were found to be significantly higher in the PAD group, indicating that the low-grade inflammation hypothesis may play a role in PAD. Large-scale, prospective, randomized controlled studies on this subject are needed.

Keywords:

C-reactive protein, fibrinogen, low-grade inflammation, peripheral artery disease, white blood cells

Introduction

Peripheral artery disease (PAD) occurs due to atherosclerosis, which causes stenosis or constriction in the main arteries feeding the lower extremity.

Peripheral artery disease includes arterial stenosis or occlusion caused by atheromatous plaques, thrombosis, arterial inflammation, arterial dilation/aneurysm, or external pressure.[1] Development of PAD is closely associated with classical cardiovascular risk factors such as diabetes mellitus (DM), dyslipidemia, smoking, hypertension (HT), and advanced age.[1] Endothelial dysfunction is one of the earliest anomalies in the development of atherosclerosis.[2,3] Inflammation is an immune system reaction that helps defends the host by repairing damaged tissues and eliminating toxic agents. If this reaction becomes chronic and continues at a low grade, it can cause increased toxic activity of immune cells and tissue damage.[4] Low-grade inflammation is defined by increased levels of C-reactive protein (CRP) in the blood. C-reactive protein is one of the main biomarkers of inflammation in the body. The higher the level of CRP measured in the blood, the more inflammation there is in the body. Low-grade inflammation can be defined by CRP levels of 3 to 10 mg/L in the blood.[5,6] Low-grade inflammation causes changes in the homeostasis of the organism and eases the onset of chronic diseases such as DM, atherosclerosis, heart failure, PAD, obesity, metabolic syndrome, polycystic ovary syndrome, depression, periodontitis, osteoarthritis, and cancer.[5,6] Inflammation, besides atherosclerosis, can have a major role in the progression of PAD. The increase in inflammatory biomarkers can predict low-grade inflammation. These patients can be diagnosed and treated before disease reaches severe grades. Furthermore, preventing and treating low-grade inflammation in this group of patients can stop or slow down the progression of the disease. Yener et al.[7] found that neutrophil-to-lymphocyte ratio and platelet-to-lymphocyte ratio were high in patients with type D lesions according to the TASC (Trans-Atlantic Inter-Society Consensus Document on Management of Peripheral Arterial Disease) classification in PAD.

They suggested that inflammation may play a role in the development of atherosclerosis in atherosclerotic PAD. The present study aimed to compare the levels of inflammatory biomarkers such as CRP, fibrinogen, and white blood cell (WBC) count in patients with PAD and healthy individuals.

Methods

This case-control study was conducted with 162 individuals (107 males, 55 females; mean age: 52.5±13.7 years; range, 24 to 87) at the Ağrı Training and Research Hospital between January 2023 to January 2024. Eighty-seven of the participants were diagnosed with PAD, and the remaining 75 were healthy individuals. Biochemical results of both groups were examined. Having symptoms resembling PAD and existence of known cardiovascular risk factors were the inclusion criteria for the patient group. The exclusion criteria were age under 18 years, morbid obesity, cancer, and chronic disease such as heart failure, kidney failure, liver failure, and rheumatologic disease. These patients were not included in the study since the inflammation present in chronic diseases increases inflammatory markers. Age, sex, DM, HT, dyslipidemia, smoking, fasting blood glucose level, creatinine, estimated glomerular filtration rate (eGFR), total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, fibrinogen, and CRP levels and WBC, monocyte, lymphocyte, and platelet counts were recorded. Diabetes mellitus, HT, and dyslipidemia were diagnosed according to relevant guidelines.[8-10] A written informed consent was obtained from each patient. The study protocol was approved by the Ağrı İbrahim Çeçen University, Ethics Committee (date: 29.03.2024, no: E-98270).

The study was conducted in accordance with the principles of the Declaration of Helsinki. Peripheral artery disease was diagnosed by lower extremity color Doppler ultrasonography or computed tomography angiography.

Statistical analysis

A power analysis was calculated according to creatinine levels using G*Power version 3.1.9.7 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). A power of 87% was found according to n1=75 (0.82±0.34), n2=87 (1.16±0.83), alpha=0.05, and effect size (d)=0.49.

Data were analyzed using IBM SPSS version 27.0 (IBM Corp., Armonk, NY, USA) and MedCalc version 15.8 (MedCalc Software, Ostend, Belgium).

While evaluating the data, in addition to descriptive statistical methods (frequency, percentage, mean, standard deviation, median, minimum-maximum, and interquartile range), the chi-square test was used to compare qualitative data. The suitability of the data for normal distribution was evaluated with the Kolmogorov-Smirnov test, skewness-kurtosis, and graphical methods (histograms, Q-Q plots, stem-and-leaf plots, and boxplots). In this study, for the evaluation of normally distributed quantitative data, the independent samples t-test was used. For the evaluation of data that did not show normal distribution, the Mann-Whitney U test was used.

Receiver operating characteristic curves were used to determine the distinctiveness of the variables, and binary logistic regression was used to determine risk ratios. The level of statistical significance was set at p<0.05.

Results

In comparisons made according to groups; it was found that there was a statistically significant difference (p<0.05) between the groups in terms of sex, age, HT, DM, smoking, fasting blood glucose, creatinine, eGFR, HDL, triglyceride, fibrinogen, and CRP levels and WBC count (Table 1).

In the PAD group, it was found that the male sex, HT, DM, and smoking rates were higher, along with older age and higher levels of fasting blood glucose, creatinine, triglyceride, fibrinogen, WBCs, and CRP (Table 2). There was no statistically significant difference between the groups in terms of other variables (p>0.05). Variables that were considered to be more clinically significant and did not have a high correlation between the variables that showed differences in pairwise comparisons between groups (sex, age, HT, DM, smoking, glucose, creatinine, eGFR, HDL, triglyceride, fibrinogen, WBCs, monocytes, lymphocytes, platelets, and CRP) were included in the model. The backward stepwise method was used in the analysis, and the model was terminated at the ninth step. In this model, approximately 87% of the dependent variable (PAD group) could be explained (Nagelkerke R2=0.866). According to this model, there was a statistically significant relationship between PAD status and sex, age, glucose, creatinine, fibrinogen, monocytes, platelets, and CRP (p<0.05; Table 3).

Peripheral Artery Disease (PAD) is approximately 21.52 times more prevalent in men. It is 1.07 times more common in individuals with higher age, 1.03 times more common in those with elevated glucose levels, and 0.34 times more frequent in those with increased creatinine levels. PAD is also 1.02 times more likely in individuals with higher fibrinogen levels, 1764.11 times more prevalent in those with elevated monocyte counts, 1.01 times more common in those with higher platelet counts, and 1.28 times more frequent in individuals with elevated CRP levels (Table 3). A prediction table was created according to the model. Eighty-two of 87 patients (94.3%) with PAD were predicted correctly, and 71 of 75 patients (94.7%) without PAD were predicted correctly. The overall accuracy rate was found to be 94.4% (Table 4). Variables that were considered to be more clinically significant and did not have a high correlation between the variables that showed differences in pairwise comparisons between groups (fibrinogen, WBC count, and CRP) were included in the model. The backward stepwise method was used in the analysis, and the model was finalized at the second step. Approximately 67% of the dependent variable (PAD group) could be explained in this model (Nagelkerke R2=0.665). According to this model, there was a statistically significant relationship between PAD status and fibrinogen and CRP values (p<0.05; Figures Figure 1-Figure 5). Those with high fibrinogen values were approximately 1.02 times more likely to have PAD than those without, and those with high CRP values were approximately 1.18 times more likely to have PAD than those without (Table 5). A prediction table was created according to the created model. Eighty of 87 patients (92.0%) with PAD were predicted correctly, while 69 of 75 patients (92.0%) without PAD were predicted correctly. The overall accuracy rate was found to be 92.0% (Table 6).

As a result of the evaluations conducted using ROC analysis on the variables found to be different in pairwise comparisons. The cutoff points of different variables were as follows: fasting blood glucose, >110 mg/dL (area under the curve [AUC]=0.741, 95% confidence interval [CI]: 0.667-0.807, p<0.001); creatinine, >0.94 (AUC=0.731, 95% CI: 0.656-0.798, p<0.001); eGFR, ≤83 (AUC=0.803, 95% CI: 0.733-0.861, p<0.001); HDL, ≤43.8 (AUC=0.620, 95% CI: 0.540-0.695, p=0.007); triglycerides, >154.4 (AUC=0.661, 95% CI: 0.583-0.734, p<0.001); fibrinogen, >373 (AUC=0.923, 95% CI: 0.871- 0.959, p<0.001); monocytes, >0.51 (AUC=0.740, 95% CI: 0.665-0.805, p<0.001); and CRP, >4.76 (AUC=0.781, 95% CI: 0.709-0.842, p<0.001) (Table 7).

Discussion

Our findings demonstrated a statistically significant difference in inflammation biomarkers, such as fibrinogen, CRP, and WBC count, in the PAD group compared to the control group. Proposed mechanisms by which low-grade inflammation may affect atherogenesis include increased foam cell formation via monocyte accumulation and LDL cholesterol deposition in the arterial wall.[11,12] Low-grade inflammation has been associated with an increased risk of cardiovascular events and cardiovascular mortality in various populations.[13] In a study conducted in acute myocardial infarction patients with and without DM, high-sensitivity CRP (hsCRP) was shown to predict in-hospital outcomes and two-year mortality.[14] It has also been shown that reducing low-grade inflammation in patients with coronary artery disease with or without type 2 DM reduces the residual risk of cardiovascular events.[13] In our study, the rate of DM in the PAH group was 47.1%.

This finding is statistically significant (p<0.001) and is compatible with the literature (Table 2). One study reported a bidirectional relationship between DM and periodontitis, with hyperglycemic individuals having a higher prevalence of periodontitis compared to normoglycemic individuals. It has been suggested that low-grade inflammation and periodontitis increase the levels of proinflammatory mediators in the serum, which may lead to insulin resistance and diabetes.[15] It has been reported that DM and insulin resistance also trigger low-grade inflammation, and systemic inflammation is a risk factor for cardiovascular diseases, including PAD.[5,6]

One study demonstrated that HT is associated with lower-grade systemic inflammation indices in hypertrophic cardiomyopathy patients and that the adverse effect of HT in hypertrophic cardiomyopathy patients is a result of systemic effects rather than changes in cardiac function since left ventricular systolic and diastolic dysfunction measurements did not differ between hypertensive and normotensive patients.[16] In a review, it was stated that low-grade inflammation plays an important role in the relationship between the immune system and angiotensin II-induced HT.[17] It has been emphasized that monocyte/macrophage cells play an important role in vascular inflammation and interaction with the arterial wall throughout the progression of HT.[17] In our study, hypertension is statistically absent in 36% of patients with PAD (p<0.001; Table 2). One of the risk factors for PAH is dyslipidemia.[1] The risk of early atherosclerotic cardiovascular disease dramatically increases in patients with lipid disorders.[8] Recent studies have shown a definite link between systemic low-grade inflammation and obesity and dyslipidemia, with both disorders being associated with endothelial dysfunction/activation, a proinflammatory and prothrombotic state of the endothelium leading to the infiltration of leukocytes into the arterial wall.[18-20] In our study, the average triglyceride value in the PAD group was 161.60 mg/dL (98.70-243.40 mg/dL), with statistical significance (p<0.001; Table 2).

Smoking, one of the other risk factors of PAD, is one of the most important preventable factors causing this disease.[1] Cigarette smoke extracts activate the NLRP3 inflammasome via reactive oxygen species, resulting in the downstream release of interleukin (IL)-1 beta, IL-18, and other inflammatory factors, leading to functional changes such as autophagy, pyrolysis, and apoptosis in endothelial cells.[19-21] According to our findings, the rate of smokers in the PAD group was 23% and this results was compatible with the literature (Table 2). In their study investigating the effect of physical activity on inflammation in PAD patients, Christofaro et al.[22] found that high hsCRP values were associated with inflammation and that hsCRP values significantly decreased in patients who engaged in regular physical activity. Jacobs et al.,[23] in their study on patients with metabolic syndrome, investigated CRP, IL-6, sVCAM-1 (soluble vascular cell adhesion molecule-1), sICAM-1 (soluble intercellular adhesion molecule-1), and serum amyloid A levels. They found a partial association between metabolic syndrome and the prevalence of coronary artery disease and PAD severity, suggesting that this relationship was mediated by low-grade inflammation. In our study, the mean fibrinogen 462.58±88.09 mg/dL and mean CRP 7.21±9.7 (3.74-9.43) mg/L values were high and were statistically significant (p<0.001). We observed other noteworthy findings in this study. For example, the mean monocyte and lymphocyte values were higher in the PAD group (0.55±0.21 and 2.82±1.57, respectively), with statistical significance (p<0.001).

Barhoumi et al.[17] demonstrated that monocyte activation plays a role in vascular inflammation and low-grade inflammation.

This study had several limitations, including a small sample size and a retrospective design.

Additionally, it would have been more valuable if other inflammatory biomarkers, such as tumor necrosis factor-alpha and IL-6, had been analyzed in this study.

In conclusion, inflammatory biomarkers such as fibrinogen, CRP, and WBC count were found to be statistically significant in the PAD group, indicating that the low-grade inflammation hypothesis may play a role in PAD. If the role of low-grade inflammation in PAD is proven in future large-scale, prospective, randomized controlled trials, anti-inflammatory treatment modalities may be incorporated into PAD treatment.

Data Sharing Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Author Contributions: Study design, overwiew: A.D.K.; Writing and references: E.N.M.K.; Statistics: İ.A.

Conflict of Interest: The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding: The authors received no financial support for the research and/or authorship of this article.

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