Tryptophan/kynurenine and Neopterin Levels as Promising Inflammatory Biomarkers for Diagnosis of Asymptomatic Carotid Artery Stenosis

doi:10.21203/rs.3.rs-4554784/v1

Asymptomatic carotid artery stenosis is usually detected by physicians in patients, coincidentally, during an ultrasound examination of the neck. Therefore, measurable biomarkers in blood are needed to define the presence and severity of atherosclerotic plaque in patients to identify and manage it. We hypothesized that biomarkers that indicate pathways related to the pathogenesis of atherosclerosis could be used to identify the presence and severity of plaque in patients. For this purpose, we determined the levels of participants' inflammatory and oxidative stress biomarkers. On the other hand, kynurenine/tryptophan and neopterin levels were measured as relatively new biomarkers of inflammation in this study. Our study included 57 patients diagnosed with asymptomatic carotid artery stenosis and 28 healthy volunteers. Blood kynurenine and tryptophan levels were measured with LCMS/MS. The ELISA assay was used to measure blood catalase, total superoxide dismutase, glutathione peroxidase, malondialdehyde, and neopterin levels. According to our results, while the kynurenine and neopterin levels were higher, the tryptophan levels were lower in patients. Furthermore, the kynurenine to tryptophan ratio, which reflects IDO-1 activity, was higher in patients. On the other hand, catalase, total superoxide dismutase, and malondialdehyde levels were higher, while the glutathione peroxidase activity was lower in patients. Increasing the kynurenine/tryptophan ratio and neopterin level in patients with asymptomatic carotid artery stenosis have been associated with an inflammatory state. The oxidative stress and inflammatory response biomarkers may be an effective diagnostic and prognostic tool for asymptomatic carotid artery stenosis.

Biological sciences/Biochemistry

Health sciences/Biomarkers

Health sciences/Neurology

Asymptomatic carotid artery stenosis

inflammatory biomarkers

oxidative stress biomarkers

tryptophan/kynurenine pathway

neopterin

Asymptomatic carotid artery stenosis is frequently characterized by atherosclerotic constriction of the carotid artery without a history of ischemic stroke, transient ischemic attack, or other neurologic symptoms related to the carotid arteries [1, 2, 3]. Carotid artery stenosis is responsible for around 10–20% of all ischemic strokes leading cause of death and disability worldwide [4, 5]. This occurs through two primary mechanisms: hemodynamic impairment in cases of considerable stenosis and thromboembolism from an atherosclerotic plaque, regardless of the extent of stenosis. The degree of stenosis is a relevant risk factor for ischemic stroke [4]. Narrowing between 50% and 69% is classified as moderate stenosis, while narrowing exceeding 70% is typically classified as severe [6]. Certainly, carotid artery stenosis primarily focuses on avoiding strokes; it is important to identify and treat the degree of carotid artery stenosis to avert complications [7]. The current gold-standard diagnostic tool for carotid artery stenosis is Doppler ultrasound. However, a robust and clinically validated biomarker in the blood that may accurately diagnose patients with carotid artery stenosis and predict unfavorable outcomes in these individuals has not yet been identified [8]. Atherosclerosis, a chronic and local immune system response, constitutes the underlying pathology of carotid artery stenosis [9, 10]. The most promising biomarkers are the ones that closely correlate with the pathophysiological process of the disease. In this context, focusing on the pathways involved in the initiation and progression of atherosclerosis may enable the identification of new diagnostic and prognostic biomarkers.

Studies have demonstrated that heightened levels of inflammation indicate an initiation and prognosis in atherosclerosis [11–13]. Numerous biomarkers associated with inflammation, such as C-reactive protein and interleukin-6, have been identified as novel targets for monitoring atherosclerosis and cardiovascular risk [11, 14]. The kynurenines and neopterin are one of these research topics, mainly due to their role in inflammatory processes. The kynurenines, metabolites of the kynurenine pathway, which is the main pathway of metabolism of essential amino acid tryptophan, have been shown to have immune-modulatory properties and to be associated with inflammatory diseases [15–18]. The pro-inflammatory cytokine IFN-γ, associated with immune responses at all stages of atherosclerosis, regulates the kynurenine pathway in macrophage and dendric cells. Some kynurenines are specified to have atherogenic effects by modulating the immune system [15, 19–21]. But, while an increasing number of studies indicate alterations in the kynurenine pathway in cardiovascular diseases [21–27] the precise role of endogenous kynurenines in their initiation and progression remains unclear. Also, neopterin, which is a pteridine derived from guanosine triphosphate, is produced by macrophages and dendritic cells by IFN-γ stimuli [28–30]. Studies have shown that plasma neopterin levels increased in patients with coronary artery disease [31–34], unstable angina [35], and stable angina pectoris [32, 36, 37]. Extensive expression of neopterin within atherosclerotic lesions in the arteries was evidenced [37, 38].

As known, atherosclerosis begins with the accumulation of lipoproteins, especially LDL, in the arterial wall, with subsequent LDL oxidation into oxidized LDLs [19, 39, 40]. Besides the importance of this process, the imbalance between oxidants and antioxidants is also important for the progression of atherogenesis [41]. The overproduction of reactive oxygen species results in oxidative stress, a significant contributing factor to the development and progression of atherosclerosis. Reactive oxygen species are crucial in preserving vascular health due to their potent signaling abilities. Nevertheless, reactive oxygen species also triggers pro-atherogenic mechanisms such as inflammation, endothelial dysfunction, and disrupted lipid metabolism. Additionally, the failure or overwhelming of endogenous antioxidant systems is a common feature in atherosclerosis that promotes oxidative stress [42].

The release of inflammatory chemokines and cytokines that lead to the overactivation of the inflammatory response and the overgeneration of reactive oxygen species that lead to a state of oxidative stress are highlighted as major risk factors for the development and progression of atherosclerosis. Therefore, we focused on alterations in inflammatory and oxidative stress biomarker levels in patients diagnosed with asymptomatic carotid artery stenosis. Additionally, kynurenine/tryptophan and neopterin levels were measured as relatively new inflammatory biomarkers to determine their diagnostic and prognostic value in carotid artery stenosis.

2.1 Study Groups

This study included 57 patients diagnosed with asymptomatic carotid artery stenosis and 28 healthy volunteers admitted to the Neurology Outpatient Clinic of Eskişehir Osmangazi University Hospital from December 2021 to November 2023. Using a standardized questionnaire, we obtained information about demographic variables, previous disease history, and smoking through face-to-face clinic interviews. Patients who have been diagnosed with an inherited metabolic disease, who have undergone major surgery or trauma within the previous 3 months, who have chronic inflammatory disease, congestive heart failure, liver failure, hematological disorders, kidney failure, or any history of malignancy were excluded from the study. Smoking was defined as smoking more than 3 cigarettes per day for more than six months. Hypertension and diabetes were defined as the subject having been previously diagnosed with the disease in the hospital or taking the medication regularly. Obesity status was determined using body mass index (BMI). BMI was calculated as weight (kg) divided by height squared (m²). Individuals with a BMI over 30 kg/m² were classified as obese. Demographic data of the participants are presented in Table 1.

Carotid artery stenosis was diagnosed through Doppler ultrasonography performed by vascular neurologists within dedicated stroke units at the hospital. Doppler ultrasonography is the noninvasive diagnostic technique most frequently employed to identify carotid artery stenosis. Neurologists with at least 5 years of experience in vascular and interventional neurology meticulously determined patients' treatment protocols, considering factors such as patients' comorbidities, stenosis levels, characteristics of stenotic plaque, and medication history. As a result of the evaluation, the appropriate treatment that maximizes each patient's life expectancy was determined. All asymptomatic patients were divided into three groups based on their treatment. The first group was defined as healthy volunteers without carotid artery stenosis (Group 1). The second group was defined as patients with stenosis medical follow-up by drug treatment (Group 2). The third group was defined as patients with stenosis who underwent stenting (Group 3). The fourth group was defined as patients with stenosis who underwent carotid endarterectomy (Group 4).

2.2 Ethical Approval

The research protocol received ethical approval from the Eskişehir Osmangazi University Non-Interventional Clinical Research Ethics Committee (No. 2021/12, No. 08). The Helsinki Declaration conducted the study. Participants provided informed consent before their involvement in the study.

2.3 Biochemical Parameters

Blood samples were collected from patients before medical treatment and carotid artery interventional management.

Blood samples for total cholesterol, triglyceride, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and C-reactive protein (CRP) were taken in the morning and analyzed using standardized procedures at the hospital's central biochemistry laboratory. Tryptophan and kynurenine levels were measured using LC-MS/MS (Shimadzu Corporation, Kyoto, Japan). Indoleamine 2,3-dioxygenase-1 (IDO-1) enzyme activity was calculated using serum kynurenine and tryptophan levels and presented as µmol kynurenine to mmol kynurenine.

Neopterin, catalase, total superoxide dismutase, glutathione peroxidase, and malondialdehyde levels were determined using commercially available human enzyme-linked immunosorbent assay (ELISA) kits. The neopterin ELISA kit was purchased from Bioassay Technology Laboratory (Shanghai Korain Biotech Co., Ltd, Shanghai, China). In contrast, the others were purchased from Elabscience Biotechnology Inc. (Elabscience Biotechnology Co., Ltd, Texas, USA). All ELISAs were performed according to the manufacturer’s instructions with commercially available kits.

2.4 Tryptophan and Kynurenine Analysis

Trichloroacetic acid, formic acid, acetonitrile, and potassium phosphate dibasic trihydrate (Merck KGaA, Darmstadt, Germany) were purchased from Sigma-Aldrich. L-tryptophan and L-kynurenine (Cayman Chemical Company, Michigan, USA) were also used. All reagents utilized in the experiment were of analytical grade or higher purity, and water of HPLC grade was used to prepare all aqueous solutions.

LCMS/MS analysis was performed using a LCMS-8040 system. Tryptophan calibration standards were prepared in the 10 - 100 μmol/L range by sequential dilution of solutions. Kynurenine calibration standards were prepared in the 0,4 - 8 μmol/L range by sequential dilution of solutions. A total 400 µL plasma sample was diluted with 400 µL phosphate buffer. The samples were deproteinized by adding 100 µL 2 M trichloroacetic acid solution. After vortexing, the samples were centrifuged at 13000 rpm. The supernatants were transferred into vials and injected into the LC-MS/MS system. A mixture of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid (65/35, v/v) was used as the mobile phase. The total injection volume was 0.3 µL, and the flow rate was 0.3 mL/min. Chromatographic separation was performed using an isocratic gradient with Nucleosil HPLC Column (Teknokroma Brisa LC2 C18 Column 3 μm 15 X 0,46 cm., Teknokroma Analytica S.A., Barcelona, Spain) maintained at 40 °C. Multiple-reaction monitoring (MRM) method in positive mode for LCMS analysis was performed, and the parameters were given in Table 2.

2.5 Statistical analysis

IBM SPSS Statistics v. 23 (IBM Corporation- International Business Machines Corporation of Armonk, New York, USA) was utilized for statistical analysis. The Shapiro-Wilk test was employed to assess the normal distribution of the variables. Descriptive analyses were presented using mean and standard deviation data and were compared using Pearson's Chi-Square, One-Way Analysis of Variance (ANOVA), and Fisher-Freeman-Halton. The ANOVA test was used to compare more than two groups in independent groups. When the ANOVA test was statistically significant, the post hoc Tukey and LSD test was performed. The Pearson correlation test evaluated the relationships between parameters and biochemical results. All tests were defined as statistically significant at a P-value less than 0.05.

3.1 Lipid profiles of participants included in the study

The levels of triglycerides, HDL, and LDL did not differ significantly between the groups. Compared to the healthy volunteers, a statistically significant reduction in total cholesterol levels was identified in patients. The potential cause for the reduced cholesterol levels observed in patients could be the pharmaceutical interventions employed by the patients to manage their dyslipidemia. In fact, during in-person interviews, it was determined that patients diagnosed with carotid artery stenosis were using at least one antihyperlipidemic drug (Table 3).

3.2 Inflammatory biomarkers of participants included in the study

When CRP levels were compared to healthy volunteers, increased CRP levels were observed in patients, but this difference was not considered statistically significant. Neopterin levels were increased in patients compared with healthy volunteers; moreover, a statistically significant increase was observed in carotid endarterectomy patients. Additionally, the neopterin level was statistically significantly higher in the carotid endarterectomy patients than in other patients. Tryptophan levels were statistically significantly decreased in patients compared to the healthy volunteers. On the other hand, kynurenine levels were increased in patients compared to the healthy volunteers; however, a statistically significant increase was observed in the carotid endarterectomy patients. Additionally, the kynurenine level was statistically significantly higher in the carotid endarterectomy patients than in other patients. When groups were compared regarding the kynurenine/tryptophan ratio that reflects IDO-1 activity, increases were observed in the patients compared to the healthy volunteers; however, statistically significant increases were observed in the carotid stenting and endarterectomy patients. Additionally, the kynurenine/tryptophan ratio was statistically significantly higher in the carotid endarterectomy patients than in other patients. (Table 4).

3.3 Oxidative stress biomarkers of participants included in the study

While increases in catalase activities were noted in the patients compared to the healthy volunteers, these increases were statistically significant in the patients. Additionally, catalase activity was statistically significantly higher in the carotid endarterectomy patients than in other patients. Increases in total superoxide dismutase activity were noted in the patients compared to the healthy volunteers, and these increases were statistically significant in the medical follow-up and carotid endarterectomy patients. Decreases in glutathione peroxidase activity were noted in the patients compared to the healthy volunteers, but these decreases were statistically significant in the medical follow-up and carotid stenting patients. While malondialdehyde levels were increased in the patients compared to the healthy volunteers, these increases were statistically significant in the carotid endarterectomy patients (Table 5).

Atherosclerosis is a chronic inflammatory condition that primarily affects the walls of large and medium arteries, including the carotid arteries [43]. Many factors contribute to the pathogenesis of atherosclerosis, the most important of which is inflammation [44]. Inflammation plays a significant role in all stages of atherosclerosis [45, 46]. Therefore, several biomarkers linked to vascular inflammation have been discovered as new targets for monitoring atherosclerosis. The CRP is a well-researched and widely accepted biomarker that indicates inflammation [14, 47]. The CRP is proven value as a predictive marker for cardiovascular events, independently and in combination with other parameters [48]. Many studies have examined the relationship between CRP levels and carotid artery stenosis. These studies found that increased CRP levels were associated with carotid artery stenosis [49-52]. In our study, although plasma CRP levels increased in patients, this increase was not considered statistically significant. At this point, attention should be paid to this biological indicator's poor specificity [14, 47]. In conclusion, while CRP has been extensively studied as a marker of inflammation and cardiovascular risk, its specificity for atherosclerosis has been called into question. To enhance risk assessment and management in atherosclerotic disease, there is a growing consensus in the scientific community for the development and validation of new biomarkers that can provide more targeted and accurate information on the presence and progression of atherosclerosis. Hence, this research is based on pathways that are involved in inflammatory processes.

At this point, it should be emphasized that immune responses in atherosclerosis are regulated by cytokines that affect all stages of the disease [19, 40]. Cytokines can be produced by almost all cell types in this context, particularly macrophages. Several cytokines, like TNF-α and IFN-γ, play crucial roles in this network by stimulating the expression of other cytokines, including IL-6 and IL-8 [53]. It has been reported that the kynurenine pathway, which is the main pathway responsible for the metabolism of the essential amino acid tryptophan, regulated by cytokines, plays a crucial role in the regulation of vascular inflammation [15, 19, 20, 54]. A relationship between IDO-1 activity, the main enzyme that converts tryptophan to kynurenine, and systemic chronic low-grade immune-mediated inflammation has been demonstrated in the development of atherosclerosis [18]. In response to inflammatory signals, the proinflammatory cytokine IFN-γ activates IDO-1 in macrophages and other cells [16-18, 55]. Under inflammatory conditions, activation of IDO-1 leads to increased metabolism of tryptophan to kynurenine [18, 56]. For biomonitoring of the metabolism of tryptophan to kynurenine, the ratio of blood kynurenine concentration to blood tryptophan concentration is used, and this ratio reflects IDO-1 activity [21, 55, 57, 58]. In studies, metabolites of the tryptophan-kynurenine pathway and especially the increased kynurenine/tryptophan ratio, so IDO-1 activity have been associated with the presence of atherosclerotic plaque [18, 56, 59]. Our study determined that IDO-1 activity, reflected by the plasma kynurenine/tryptophan ratio, was increased in patients with asymptomatic carotid artery stenosis compared to healthy volunteers. Additionally, more IDO-1 activity was observed in carotid endarterectomy and stenting patients compared to others. Therefore, IDO-1 activity can be considered a biomarker that can be used to determine both the presence and degree of atherosclerotic plaque. Studies have determined that increased carotid artery intima/media thickness is accompanied by an increased kynurenine/tryptophan ratio [60, 61]. Also, studies have observed that IDO-1 activity is increased in patients with cardiovascular diseases compared to healthy volunteers [62-66]. Prospective study results are interpreted as kynurenine pathway metabolites may be useful in predicting the onset and progression of atherosclerotic pathologies. The results point out that tryptophan levels tend to decrease, and kynurenine levels increase in disease states [21, 59, 67-70].

Neopterin is a metabolite of guanosine triphosphate (GTP). Neopterin is formed in activated macrophages after the induction of GTP cyclohydrolase I by IFN-γ [65, 71]. Thus, neopterin levels significantly increase in any condition involving immune response activation and inflammation [71]. Studies conducted in recent years have drawn attention to the relationship between the increase in plasma neopterin levels and carotid artery stenosis [72-75]. Our study measured neopterin levels higher in patients with asymptomatic carotid artery stenosis than healthy volunteers. In addition, more neopterin levels were noted in carotid endarterectomy patients than in other patients. Therefore, neopterin level may also indicate both the presence and degree of stenosis.

Oxidative stress is a widely recognized factor in the development of atherosclerosis, which co-occurs with the activation of pro-inflammatory signaling pathways [76]. Oxidative modification of LDL accumulated in the artery wall as a critical process of atherosclerosis initiates atherosclerosis development, promoting the vascular inflammatory response [42, 77, 78]. Regarding vascular inflammatory response, migrating immune cells to the arterial wall produces further reactive oxygen species. Therefore, vascular inflammation and oxidative stress cannot separate each other in the development and progression of atherosclerotic plaque [76]. As mentioned above, currently, there is no agreement on the definition of an optimal biomarker that can predict the risk of stroke in patients with carotid artery stenosis. Therefore, targeting biomarkers of oxidative stress as a causal factor of atherosclerosis appears to be a plausible approach. Oxidative stress can be monitored using different biomarkers, such as malondialdehyde, one of the lipid oxidation products. Also, previous research has established a correlation between atherosclerosis and an elevation in malondialdehyde level, a byproduct of lipid peroxidation [77, 79, 80]. Our study also drew attention to increased malondialdehyde levels in patients with asymptomatic carotid artery stenosis compared to healthy volunteers. On the other hand, antioxidants are crucial in preventing oxidant formations, intercepting oxidants after they have formed, and repairing damage caused by oxidants [81]. Superoxide dismutase, glutathione peroxidases, and catalase, as some of the key enzymes of the antioxidant system, regulate the formation and degradation of reactive oxygen species in vascular cells [78]. Our results demonstrated lower glutathione peroxidase activity but higher superoxide dismutase and catalase activities in patients with asymptomatic carotid artery stenosis compared to healthy volunteers. Compared to healthy individuals, the altered activities of these enzymes in patients with carotid stenosis suggest a dysregulation in the antioxidant defense system, potentially contributing to the pathogenesis of asymptomatic carotid artery stenosis. At this point, it may be specified that homeostatic up-regulation of the antioxidant enzyme system in response to increased free radicals may occur to prevent vascular damage [82, 83]. In summary, the dysregulation of antioxidant enzyme activities, specifically lower glutathione peroxidase and higher malondialdehyde, superoxide dismutase, and catalase activities, in patients with asymptomatic carotid artery stenosis compared to healthy volunteers highlights the potential role of oxidative stress in the development and progression of carotid artery disease. Further exploration of these enzyme systems and their implications in carotid stenosis could offer valuable insights for developing innovative diagnostic and therapeutic approaches for this condition. On the other hand, it was also observed that oxidative stress was induced more in carotid endarterectomy patients than in other patients. Therefore, biomarkers of oxidative stress may also indicate the degree of stenosis. In addition, it is obvious that the balance between reactive oxygen species and antioxidant enzymes is disrupted in patients with asymptomatic carotid artery stenosis.

Our study has identified kynurenine, tryptophan, and neopterin as significant biomarkers for asymptomatic carotid artery stenosis, 10-20% of all ischemic strokes leading cause of death and disability worldwide. These results may provide new insight into developing a more effective diagnosis and treatment strategy to heal and/or prevent the progression of carotid artery stenosis. Additionally, the data mentioned above suggest that oxidative stress was elevated in patients, as evidenced by the notable alterations in the levels of oxidative stress markers. Certainly, our results need to be supported by extensive clinical trials. Finally, it may be said that investigating the therapeutic capacity of immunomodulation in carotid artery stenosis may be considered an important approach.

Credit authorship contribution statement

SI, OA, and GG conceived and supervised the project. OA and MY examined the carotid arteries through Doppler ultrasonography in patients and healthy volunteers. SL and ABK performed LC-MS analyses. ABK performed ELISA assays. ABK statically analyzed the results. SI and ABK wrote the original draft. All authors reviewed and edited the paper.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data used in this study were initially collected in Excel format and subsequently converted to SPSS format for statistical analysis. If you wish to access the research data, please send an email to [email protected] and the data will be provided upon request.

Acknowledgments

This research was supported by the Scientific Research Projects of Anadolu University (Project number 2111S214). We thank Prof. A. Ozcan Ozdemir for his methodology assistance and comments that greatly improved the work.

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Table 1

Demographic data of the participants
	Group 1 (n = 28)	Group 2 (n = 27)	Group 3 (n = 18)	Group 4 (n = 12)	p-value
Gender, n (%); Male Female	10 (35.7) 18 (64.3)	15 (55.6) 12 (44.4)	12 (66.7) 6 (33.3)	9 (75) 3 (25)	0.069*
Age (average ± SD)	60 ± 0.23	66 ± 0.15	65 ± 0.18	74 ± 0.22	< 0.05**
Smoking, n (%); Yes No	5 (17.9) 23 (82.1)	10 (37.0) 17 (63.0)	6 (33.3) 12 (66.7)	2 (16.7) 10 (83.3)	0.320*
Comorbid Diseases, n (%); Diabetes mellitus Obesity Hypertension	4 (14.3) 9 (32.1) 15 (53.6)	6 (22.2) 8 (29.6) 18 (66.7)	7 (38.9) 2 (11.1) 7 (38.9)	6 (50.0) 0 (0.0) 7 (58.3)	0.068* 0.067*** 0.337*
Values are presented as mean ± standard deviation. Differences between all patient subgroups at a p < 0.05 level. When the ANOVA test was statistically significant, the post hoc LSD test was performed. Group I, healthy volunteers; Group 2, patients for drug treatment; Group 3, patients for carotid stenting; Group 4, patients for carotid endarterectomy. Pearson's chi-square, * One-Way ANOVA, *** Fisher-Freeman-Halton test.

Table 2

MRM data for tryptophan and kynurenine
	Precursor ion	Daughter ion	Dwell time ms	Q1 pre-Bias (V)	CE	Q3 pre-Bias (V)
L-Tryptophan	205.10	188.05	100.0	-15.0	-12.0	-19.0
	205.10	146.05	100.0	-15.0	-22.0	-27.0
	205.10	117.90	100.0	-10.0	-27.0	-25.0
L-Kynurenine	209.10	192.15	100.0	-10.0	-8.0	-12.0
	209.10	146.05	100.0	-23.0	-22.0	-27.0
	209.10	94.05	100.0	-14.0	-17.0	-18.0

Table 3

Lipid profiles of participants included in the study
	Group I	Group II	Group III	Group IV
Parameters	Mean ±SD	Mean ±SD	Mean ±SD	Mean ±SD
Total cholesterol (mg dL⁻)	222.35 ± 39.51	183.48 ± 42.98 (*)	173.11 ± 50.54 (*)	153.22 ± 18.40 (*)
Triglyceride (mg dL⁻)	168.24 ± 76	126.62 ± 41.59	141.11 ± 58.16	142.36 ± 70.33
LDL (mg dL⁻)	146.53 ± 36.41	119.05 ± 35.86	114.58 ± 44.17	124.58 ± 45.38
HDL (mg dL⁻)	44.00 ± 6.98	46.43 ± 9.03	41.33 ± 12.26	43.80 ± 11.27
Values are presented as mean ± standard deviation. One-way analysis of variance (ANOVA), a parametric test, was used to examine differences between all participants at the p < 0.05 level. When the ANOVA test was statistically significant, a post hoc Tukey test was applied. Group I, healthy volunteers; Group 2, patients for drug treatment; Group 3, patients for carotid stenting; Group 4, patients for carotid endarterectomy. Abbreviations: LDL, Low-density lipoprotein; HDL, High-density lipoprotein.
* Different from Group 1. (p < 0.05)

Table 4

Inflammatory biomarkers of participants included in the study
	Group I	Group II	Group III	Group IV
Parameters	Mean ±SD	Mean ±SD	Mean ±SD	Mean ±SD
CRP (mg L⁻)	1.98 ± 1.18	2.37 ± 0.96	2.24 ± 1.38	3.13 ± 2.28
NEOP (nmol L⁻)	3.18 ± 0.99	3.23 ± 0.68	2.85 ± 0.97	4.28 ± 1.41 (*, +, !)
KYN (µmol L⁻)	2.01 ± 0.22	2.04 ± 0.22	2.08 ± 0.44	2.51 ± 0.59 (*, +, !)
TRP (µmol L⁻)	45.38 ± 7.82	37.17 ± 6.44 (*)	36.40 ± 6.61 (*)	30.07 ± 6.82 (*)
IDO activity	45.66 ± 4.68	47.36 ± 5.48	52.37 ± 5.27 (*)	66.73 ± 7.76 (*, +, !)
Values are presented as mean ± standard deviation. One-way analysis of variance (ANOVA), a parametric test, was used to examine differences between all participants at the p < 0.05 level. When the ANOVA test was statistically significant, a post hoc Tukey test was applied. Group I, healthy volunteers; Group 2, patients for drug treatment; Group 3, patients for carotid stenting; Group 4, patients for carotid endarterectomy. Abbreviations: CRP, C-reactive protein; NEOP, neopterin; KYN, kynurenine; TRP, tryptophan; IDO, plasma kynurenine level/tryptophan value.
* Different from Group 1. (p < 0.05)
+ Different from Group 2. (p < 0.05)
! Different from Group 3. (p < 0.05)

Table 5

Oxidative stress biomarkers of participants included in the study
	Group I	Group II	Group III	Group IV
Parameters	Mean ±SD	Mean ±SD	Mean ±SD	Mean ±SD
CAT (U mL⁻)	36.66 ± 12.37	193.46 ± 74.88 (*)	177.28 ± 50.71 (*)	284.13 ± 74.7 (*, +, !)
T-SOD (U mL⁻)	52.03 ± 10.45	61.49 ± 9.46 (*)	60.47 ± 13.40	63.78 ± 9.16 (*)
GSH-Px (U mL⁻)	1008.78 ± 188.38	630.56 ± 230.18 (*)	584.94 ± 185.73 (*)	731.14 ± 202.83
MDA (ng mL⁻)	80.75 ± 23.41	82.40 ± 29.98	85.69 ± 39.30	90.66 ± 27.22 (*)
Values are presented as mean ± standard deviation. One-way analysis of variance (ANOVA), a parametric test, was used to examine differences between all participants at the p < 0.05 level. When the ANOVA test was statistically significant, a post hoc Tukey test was applied. Group I, healthy volunteers; Group 2, patients for drug treatment; Group 3, patients for carotid stenting; Group 4, patients for carotid endarterectomy. Abbreviations: CAT, catalase; T-SOD, total superoxide dismutase; MDA, malondialdehyde; GSH-Px, glutathione peroxidase.
* Different from Group 1. (p < 0.05)
+ Different from Group 2. (p < 0.05)
! Different from Group 3. (p < 0.05)

No competing interests reported.

Tryptophan/kynurenine and Neopterin Levels as Promising Inflammatory Biomarkers for Diagnosis of Asymptomatic Carotid Artery Stenosis

Status:

Version 1

Abstract

1. INTRODUCTION

2. MATERIALS AND METHODS

3. RESULTS

4. DISCUSSION

5. CONCLUSION

Declarations

References

Tables

Additional Declarations

Status:

Version 1