Exploring potential serum levels of Homocysteine, interleukin‐1 beta, and apolipoprotein B 48 as new biomarkers for patients with ischemic stroke

Abstract Background Stroke is the second leading cause of death worldwide with heterogeneous characteristics. The subtypes of stroke are due to different pathophysiological regulations and causes. This study aimed to investigate the correlation of serum levels of apolipoprotein B 48, interleukin‐1β and Homocysteine with BMI in patients with ischemic stroke (IS). Methods Over one hundred controls (120) and an equal number of IS patients, including 31 women and 89 men, were recruited to participate in the case‐control study conducted at Imam Reza Hospital (Tabriz, Iran) from February 2019 to March 2020. We measured serum levels of apolipoprotein B 48, interleukin‐1β, and Homocysteine. Receiver operating characteristic analysis (ROC) was performed to evaluate the diagnostic value of these indices in patients and control groups. Results The mean serum levels of apolipoprotein B 48, interleukin‐1β, and Homocysteine, were significantly increased in the experimental group compared to the control group with a p‐value of 0.001. The ROC curve analysis showed that the area under the curve for apo B48, IL −1β, hs‐CRP, and Homocysteine serum levels were 0.94, 0.98, 0.99, and 1, respectively. Conclusions The results of our current study show that the determination of serum levels of apolipoprotein B 48, interleukin‐1β, and Homocysteine can potentially be used to monitor and diagnose IS patients. However, there was no statistically significant correlation between serum levels of apolipoprotein B 48, interleukin 1β and Homocysteine and BMI in the patient group. However, there was a statistically significant inverse correlation between serum levels of high‐sensitivity C‐reactive protein (hs‐CRP) and BMI in the patient group.


| INTRODUC TI ON
Stroke is the second leading cause of death in industrialized countries. It is estimated that nearly 15 million people worldwide fall victim to a stroke, of whom 5 million die and the remainder suffer from permanent disability. 1,2 Stroke is a multifaceted disease with different subtypes that have various physiopathologic etiologies and mechanisms. A moderate decline in the total number of deaths from strokes has been observed in industrialized countries.
Nevertheless, stroke remains the leading cause of death and mortality worldwide. 3 IS is a condition in which the blood supply to the brain fails due to the formation of blood clots in the vessels and lack of nutrient and oxygen supply, resulting in brain tissue impairment. 3 However, appropriate preventive/therapeutic strategies include lifestyle modification, dietary guidelines, and pharmaceutical interventions. The prevalence of stroke remains high, especially in developed countries with a significant financial burden. 4 It should be noted that predicting the outcome of IS is of paramount importance to clinicians, researchers, and patients. Previous clinical prognostic approaches have failed to accurately predict the outcome of IS in patients. In this context, blood markers associated with pathophysiological aspects of IS, including inflammation, glial and neuronal damage, cardiovascular disease, and hemostasis, could be considered to improve the utility of clinical prognostic approaches. 5 Diabetes mellitus and obesity are potential risk factors for cardiovascular disease.
High blood triglyceride levels, also known as hypertriglyceridemia (HTG), are a typical dyslipidemia. HTG favors the development and promotion of atherosclerosis, which eventually leads to CVD. Therefore, HTG is a potential predisposing factor for IS. 6 In this context, numerous studies have attempted to uncover the correlation between IS and HTG. Numerous studies have shown that triglyceride levels after eating (without fasting) may be a more reliable biomarker for IS and atherosclerosis disease than triglyceride levels during fasting. 7 A variety of factors contribute to the increase in triglyceride levels after a meal. However, normalization/standardization has not been considered in the measurement of triglyceride concentration after a meal. An alternative screening for atherosclerotic disease and IS may be apolipoprotein B 48 (apo B48). Enterocytes also called absorptive cells that line the small intestine, produce apo B48, an element necessary for forming chylomicrons. In each chylomicron particle, there is one Apo B48. 6 In addition, some studies have shown that fasting apo-B 48 level can be used as a surrogate marker for measuring lipidemia in the non-fasting state. Therefore, the fasting apo-B 48 level could be a potential biological marker for atherosclerotic diseases such as CVD and IS. [6][7][8] Homocysteine is considered a causative factor in atherogenesis and thrombus formation by causing endothelial impairment, vascular smooth muscle proliferation, and coagulopathy.
Abnormally elevated blood levels of Homocysteine, also known as hyperhomocysteinemia (HH), have been associated with CVD and cerebrovascular disorders. 9 It has been reported that HH is an independent predisposing factor for IS in adolescent Asians. 10 One of the essential proinflammatory cytokines is interleukin-1 (IL −1), which plays an important role during the inflammatory process. IL −1α and IL −1β belong to the IL −1 superfamily. 11 Numerous studies have shown that IL −1β is a neurotoxic element in IS models and accelerates patients with acute IS. 12 Some studies have found that IL −1 is a critical chemical compound involved in hypoxia, ischemia, stress responses, and infection. IL −1β leads to inflammatory responses in the brain by stimulating cells to produce neurotoxic elements, resulting in necrosis of brain tissue. 11,12 High-sensitivity C-reactive protein (hs-CRP) is a sensitive marker of arterial wall inflammation and tissue damage. 13,14 CRP is a glycoprotein produced by the liver that plays a critical role in developing atherosclerotic disease in the heart and cerebral circulation. In addition, elevated hsCRP levels have been associated with acute stroke as a sign of infection and inflammation. 15,16 Although body mass index (BMI) is a known risk factor for vascular disease, the impact of BMI on stroke has been debated. Several studies have shown that obesity is associated with better functional outcomes after stroke. It is unknown whether obesity plays a role in stroke patients. 17 The present study examined the correlation between serum concentrations of apo B48, IL −1β, and Homocysteine with BMI in IS patients.

| Study population
This study is a retrospective case-control study. We selected 155 prototypes. We excluded samples from patients with the previous concurrent IS, neurologic disease (epilepsy), infarcts, hypothyroidism, circulatory disorders, pregnancy, cerebral sinus thrombosis, postnatal condition, ischemic CVD, brain tumors, renal disease, use of oral contraceptives, use of drugs affecting serum concentrations of folic acid, B12, and Homocysteine, deficiency of vitamins including folic acid and B12, and history of migraine. The control group was selected from patients whose presence of IS could be excluded by a neurologist based on MRI and CT results. The inclusion criteria were primary diagnosis of acute IS, completion of questionnaires, and consent to participate in the study. The control group was selected from patients in whom IS was excluded by a neurologist based on MRI and CT results.
The control group, consisting of 120 subjects (31 women and 89 men) without stroke and neurological diseases and degeneration of the nervous system, and the experimental group, consisting of 120 subjects (31 women and 89 men) affected by IS, were studied in a case-control study at Imam Reza Hospital, Tabriz, Iran, during 2019 (February)-2020 (March). Investigators registered patients at baseline IS; magnetic resonance imaging (MRI) and computed tomography (CT) were used for radiological diagnosis IS. Neurologists confirmed the disease. Infarct size was assessed with a threshold of 2.0 cm (an infarct larger than 2.0 cm was considered IS). 18 Ninetyfive patients had IS right cerebral hemisphere disease, and 25 patients suffered from left cerebral hemisphere disease IS.

| Ethics statement
The study described here was approved by the Ethics Committee of Tabriz University of Medical Sciences (IR. TBZMED. REC.1398.483), and patients also provided written informed consent.

| Blood sample collection
The sampling place was the laboratory of Imam Reza Hospital in Tabriz, and sampling was performed from 2019 (February) to 2020 (March). After fasting for 12 hours, 10 ml of venous blood was collected by venipuncture (by an expert in laboratory science) and centrifuged at 1200 g after a clotting time of 1-2 h at room temperature. The serum obtained from the blood samples was immediately poured into EP tubes and stored at 70°C for analysis.

| BMI measurements
BMI was determined by measuring the height and weight of patients, and a BMI greater than/equal to 30 kg/m 2 was considered obesity. 21

| Statistical analysis
Graph Pad Prism 8 was used to analyze the data. The Kolmogorov-Smirnov test was also used to assess the normality of the (quantitative) variables. Mean ± SD and percentage frequency were used to indicate quantitative and qualitative variables, respectively. In addition, logistic regression and Mann-Whitney U examination were used for data analysis. ROC curves served the purpose of identifying the most appropriate thresholds (stopping values) to distinguish IS patients from healthy individuals. Spearman test was performed to evaluate the correlation of serum concentrations of Apo B48, IL −1β and Homocysteine with BMI in both groups (patients or healthy subjects). In the study groups, a Pvalue of less than 0.05 was considered to indicate a significant difference.

| Demographic data of the subject
The study included 120 samples from each group (120 from patients and 120 from healthy subjects). As shown in Table 1, there were no statistically significant differences between the groups with respect to age, smoking, diabetes, and sex. However, there were significant differences between groups for BMI and hypertension, with p-values <0.001.

| Assessing the concentrations of biochemical factors
As Table 2 shows, there were remarkable differences in serum concentrations of HDL-C, chol, folic acid, vitamin B12, apo B48, and IL −1β in IS patients and healthy subjects. However, there were no raemarkable differences in the serum concentrations of TG and LDL in both groups.

| Assessing the serum concentrations of Homocysteine
As Figure 1 shows, serum Homocysteine level was significantly in-

| Assessing the serum concentrations of apo B48
As Figure 1 shows, serum concentrations of apo B48 were significantly increased in IS patients (4.7 ± 0.25 μg/ml) compared to those in the healthy subjects group (3.9 ± 0.49 μg/ml) with a P-value of 0.0001.

| Assessing the serum concentrations of hs-CRP
As shown in Figure 1, serum concentrations of hs-CRP were significantly elevated (2.7 ± 0.21 mg/L) in the IS patients compared to the healthy group (1.7 ± 0.6 mg/L) with a p-value of 0.0001.

| Assessing the serum concentrations of IL-1β
As shown in Figure 1, serum concentrations of IL −1β were significantly elevated (21.8 ± 1.5 Pg/ml) in the IS patients compared to the healthy group with a p-value of 0.0001. with IS as well as healthy individuals ( Figure 2). Table 4 shows the cut-off value of IL −1β, hs-CRP, apo B48, and Homocysteine according to the Youden index results.

| Spearman's correlation
The results of the Spearman correlation test showed that there were only small and statistically non-significant correlations between serum levels of apo-B48, IL −1β and Homocysteine and BMI in IS patients.
However, there was a small but statistically significant inverse correlation between BMI and serum levels of hs-CRP, according to Table 5.

| DISCUSS ION
The current study examined the correlation between serum con- However, a review of the United Kingdom population found that Homocysteine is generally correlated with low vascular complications. 9,27 Serum Homocysteine levels have been reported to increase with age. 28 However, no association between Homocysteine and age was found in the present study. The patients who participated in this study were predominantly over 50 years of age. Thus, from a statistical point of view, aging did not affect the results of this study.
Elevated hsCRP levels (>3.0 mg/L) were found in 32% of the study group. In addition, elevated hs-CRP levels independently predicted recurrent stroke during a one-year follow-up period. In a randomized trial, there was no association between hsCRP levels and antiplatelet drugs. 29 Our results showed that serum hs-CRP levels increased significantly in IS patients compared with the healthy group. Consequently, this biomarker can be used to monitor the therapeutic process.
In the current study, we found that fasting levels of Apo B 48 were elevated in IS patients. Other studies have also found cytokine In addition, numerous studies have described plausible mechanisms. apo B48 contains a binding residue to proteoglycans of the arterial wall. 31 Consequently, apo B48 has been observed in plaques obtained from carotid and femoral endarterectomy specimens formed during atherosclerosis. 32 In vitro studies have also shown that chylomicron remnants are generally taken up by murine peritoneal macrophages and human monocyte-derived macrophages via different mechanisms. 33 Suggesting that fasting apo B48 may be a novel biomarker for IS and a new therapeutic intervention target. In Western countries with a very high BMI, the reduced risk of developing IS was not observed. The increased risk of IS or hemorrhagic stroke in the male population with BMI above the standard range (22 to 23 kg/ m 2 ) was rejected after adjustment for blood pressure, glucose, and cholesterol, suggesting that BMI may have a significant impact on the subtypes of stroke in the population. 34 Other studies have also shown a correlation between different subtypes of stroke and BMI. 35,36 However, numerous studies on the obesity paradox have shown that obesity and being overweight can be risk factors for vascular complications. This study showed that the serum concentration of IL −1β was strikingly increased in IS patients compared with healthy individuals. Reportedly, the expression level of IL −1β is high in cerebral infarction patients, characterized by various neurological deficits compared to mild cerebral infarction patients. 30 Other studies have also found that after brain injury, cytokine levels are increased due to increased cytokine production by neurons, glia, and inflammatory cells. 37

ACK N OWLED G M ENTS
The authors thank the Neurosciences Research Center of Tabriz University of Medical Sciences for their kind cooperation and abidance.

CO N FLI C T S O F I NTE R E S T
There are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
AN and MN contributed to the study design; DL contributed to the analysis and interpretation of the data; VK, BS, and AN contributed to revising the manuscript content; and AN approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data obtained in the study can be accessed upon request.