Serum retinol-binding protein 4 and the risk of ischemic stroke in Egyptian patients with hypothyroidism

Ischemic stroke (IS) is one of the major causes of disability and


Introduction
Emerging evidence demonstrated that stroke, a leading course of death or disability, shares many risk factors with coronary artery disease (CAD), such as age, smoking, hypertension, diabetes mellitus, inactivity, overweight or obesity, and dyslipidemia [1,2].Compelling evidence explored that ischemic stroke (IS) is the most preventable disease.Although CAD and stroke share risk factors, published data on the association between hypothyroidism and stroke are insufficient and conflicting [3].
A growing body of evidence has corroborated that stroke is an important cause of adult mortality and morbidity; however, its pathogenesis is still unknown.Substantial pieces of evidence implicate proinflammatory cytokines as a critical mediator in the pathophysiology of IS [4].
Retinol-binding protein 4 (RBP4) is an ∼21 kDa secreted protein that transports retinol (vitamin A) in circulation [5].RBP4 has been well known as an important adipokine that contributes to insulin resistance in both rodent and human [6,7].Recent clinical studies have also linked higher circulating RBP4 to cardiovascular diseases, including hypertension [8,9], heart failure [10,11], and atherosclerosis [12].RBP4 binds to transthyretin (TTR) forming a protein complex that prevents glomerular filtration and reduces the renal clearance of RBP4 [13].Lowering TTR could decrease circulating levels of RBP4 by promoting its This is an open access journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as appropriate credit is given and the new creations are licensed under the identical terms.
renal clearance [14].However, it remains unclear whether increased RBP4 is a cause or a result of hypothyroidism.Thus, we carried out the present study to evaluate serum RBP4 in patients with hypothyroidism and to assess the association of serum RBP4 with susceptibility of noncardioembolic stroke.To the best of our knowledge, this is the first study that explored the correlation of serum RBP4 with susceptibility of IS in patients with hypothyroidism.

Patients and methods
This case-control study was conducted at Internal Medicine, Neurology and Clinical Pathology Departments, Faculty of Medicine, Zagazig University.After approval from the Medical Ethical Committee of the Faculty of Medicine, Zagazig University, all participants signed an informed written consent.Ninety patients with hypothyroidism [patients with elevated thyroid-stimulating hormone (TSH) and low thyroid hormones levels diagnosed as hypothyroid patients] were included in this study, who were divided into nonstroke group (n=60) and stroke group (n=30), in addition to 50 healthy controls; all participants were matched regarding age, sex, and ethnic origin.
The patients were chosen with the following inclusion and exclusion criteria.The inclusion criteria were focal or global neurological deficit lasting greater than 24 h on initial neurological evaluation.Computed tomography (CT) scan of the brain showed evidence of cerebral ischemia.However, the exclusion criteria were nonischemic etiology such as hemorrhagic stroke (patients with intracerebral hemorrhage, subarachnoid hemorrhage).Patients with a history of respiratory disease, cancer, severe hepatic and renal diseases, acute illness, hormonal therapy, any active inflammatory diseases, alcoholism, carotid artery surgery, and chronic heart failure were excluded from the study.
All patients in the study were subjected to the following: thorough history taking and full clinical assessment including a general and neurological examination.Stroke severity within 72 h of the onset of symptoms was assessed using the National Institute of Health Stroke Scale [15].CT scan of the brain was performed for each patient to exclude intracranial hemorrhage and to diagnose cerebral infarction including its site and size.If the CT scan result was negative, it was repeated after 72 h.The size of the lesion was calculated according to the formula 0.5×A×B×C [where A and B are the largest perpendicular diameters measured on CT and C is the slice thickness (10 mm).

Blood sampling and laboratory testing
Blood samples were drawn from all participants after an overnight fast.Sera were separated after 1 h longstanding and stored at −80°C.We determined the fasting plasma glucose (FPG) using the glucose oxidase method (Roche Cobas 8000-e507; Roche Diagnostics, Basel, Switzerland) (Spinreact, Girona, Spain).Total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured by routine enzymatic methods (Roche Cobas 8000-e507).Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula [16].2.2 Assay of thyroid function.

Measurement of serum retinol-binding protein 4
Serum RBP4 was measured with commercially available ELISA kits according to the manufacturer's instructions (Abnova, Abnova Corporation, Taipei City, Taiwan) [12].

Statistical analysis
We examined the general characteristics of the study population using mean (SD) for continuous variables and numbers (%) for categorical variables.Pearson's correlation coefficient was used to assess the association between serum RBP4 and clinical as well as biochemical tests.Receiver operating characteristic (ROC) analysis was performed to assess the potential accuracy of serum RBP4, the area under the curve (AUC), and the cutoff values for differentiating of hypothyroidism and stroke among the studied population.A linear regression analysis was done to detect the main predictors of serum RBP4 values in hypothyroidism group.We considered P to be significant at less than 0.05 with a 95% confidence interval (CI).All analyses were conducted using SPSS version 21 (Chicago, Illinois, USA).

Results
Clinical, anthropometric, and laboratory characteristics of all studied participants Among the studied participants, 90 patients with hypothyroidism (11 males and 69 females; mean age, 48.7±12.3years) were enrolled.The control group consisted of 50 healthy participants (28 males and 22 females; mean age, 49.5±14.14years).Hypothyroidism cases had significantly higher values of systolic and diastolic blood pressures compared with the control group.Moreover, the values of BMI, FPG, TC, triglyceride (TG), LDL, and TSH were increased compared with controls.On the contrary, patients with hypothyroidism had significantly lower values of HDL, FT4, and FT3 (P<0.001*)(Table 1).
Clinical, anthropometric, and laboratory characteristics of hypothyroidism groups, as shown in Table 2.
Stroke group had significantly higher values of systolic blood pressure, diastolic blood pressure, BMI, FPG, TG, TSH, and TG than non-IS group (P<0.001*).On the contrary, there were significantly lower values of FT3, FT4, and HDL in stroke group compared with nonstroke group (P<0.001*).

Pearson's correlation between serum retinol-binding protein 4 (μg/ml) with other parameters
There was a positive correlation between serum RBP4 and BMI, TC, TG, FPG, as well as TSH (Fig. 2).However, there were significantly negative correlations between serum RBP4 level and HDL and FT3 as well as FT4 (P<0.001*)(Table 3).

Linear regression analyses in patients with hypothyroidism
Linear regression analysis test was done to assess the main independent parameters associated with serum RBP4.Our results showed that diastolic and systolic blood pressures, TG, FT3, as well as TSH were independently correlated with serum RBP4 (Table 4).
The accuracy of serum retinol-binding protein 4 (μg/ml) for discriminating hypothyroidism group from control group using receiver operating characteristic curve analysis We further investigated the potential diagnostic value of serum RBP4 by ROC curves, as presented in Fig. 3.In the studied groups, when we discriminate hypothyroidism from controls, the cutoff values were 12.25 and the AUC was 0.909 (95% CI: 0.861-0.957).Additionally, the sensitivities and the specificities were 96.7 and 74.5%, respectively.
The accuracy of serum retinol-binding protein 4 level for discriminating ischemic stroke group from the nonstroke group by receiver operating characteristic curve analysis Regarding the diagnostic value of serum RBP4 level for discriminating IS group from the nonstroke group by ROC curves as presented in Fig. 4; among patients with hypothyroidism when we tried to discriminate IS group from nonstroke group, the cutoff value was 11.4, and the AUC was 0.822 (95% CI: 0.737-0.906).Additionally, the sensitivities and the specificities were 94.3 and 96%, respectively.

Discussion
Mounting evidence indicates that stroke is the third leading cause of death.The mortality rate after an ischemic incident is very high 30% and survivors almost always face disabilities that require costly long-term care [17].
RBP4 is a protein synthesized mainly by hepatocytes and adipocytes [18].RBP4 could promote the abnormal proliferation and migration of vascular smooth muscle cells, which is important for the formation of coronary atherosclerosis [19].Moreover, high level of RBP4 facilitated macrophage-derived foam cell formation through activating cholesterol uptake, and thus accelerated atherosclerosis progression [20].
Even though omics studies observed increased RBP4 level in patient with CAD [21,22], some other studies demonstrated no significant relationship between serum RBP4 and cardiovascular risk [23,24].Despite the high mortality and morbidity associated with IS, currently established therapies are limited.Thus, the current study aimed to evaluate serum RBP4 in patients with hypothyroidism and to assess the association of serum RBP4 with susceptibility of IS.
The results of the current study revealed that hypothyroid patient had significantly higher values of cardiometabolic risks compared with controls.Even more importantly, serum RBP4 levels were significantly higher in the hypothyroid group compared with controls.
Concordance with our finding, Choi et al. [25] observed that RBP4 level was elevated in hypothyroidism independently of obesity in elderly participants with normal glucose tolerance.
Similar to our findings, Kokkinos et al. [26] detected that serum RBP4 levels were significantly higher in clinical and subclinical hypothyroidism.Moreover, Güdücü et al. [27] detected higher levels of RBP4 in association with free testosterone and TSH in postmenopausal women.
RBP4 binds to TTR forming a protein complex that prevents glomerular filtration and reduces the renal clearance of RBP4.Thus, lowering TTR could decrease circulating levels of RBP4 by promoting its renal clearance [14].
The results presented herein are innovative as this study performs a robust evaluation of serum RBP4  as a diagnostic biomarker of IS.Noteworthy, our results confirmed that serum RBP4 levels were significantly higher in IS compared with the non-IS group.Interestingly, they were positively correlated with other vascular and metabolic risks and TSH.
The results of Chaker et al. [28] found no association between subclinical hypothyroidism and overall risk of stroke events or fatal stroke.In stratified analyses, younger participants, particularly those under the age of 50 years, had increased stroke risk, although the number of events was small.
Interestingly, Chaker et al. [28] found that patients with a TSH level of 7.0-9.9mIU/l also had an increased risk of fatal stroke compared with their euthyroid counterparts.
We in this study attempted to pierce out the independent correlation with serum RBP4.Our results showed that there was a positive correlation between serum RBP4 and BMI, TC, TG, FPG, as well as TSH.However, there was a significantly negative correlation between serum RBP4 level with HDL and FT3 as well as FT4.Linear regression analysis test was done to assess the main independent parameters associated with serum RBP4.Our results showed that diastolic and systolic blood pressures, as well as postprandial glucose and HbA1c, were independently correlated with serum RBP4.
In Chaker et al. [23], RBP4 levels were positively correlated with TC, LDL-C, and BMI, which are all risk factors for CAD.Interestingly, other studies reported that increased circulating RBP4 is not only correlated with established cardiovascular risk factors such as obesity and dyslipidemia but also correlated with the prevalence of atherosclerotic diseases and CAD [13].The exact mechanism by which increased circulating RBP4 promotes the development of CAD remains unclear.In endothelial cells, RBP4 could induce inflammation and mitochondrial dysfunction, both of which play key roles in atherogenesis [24].

Conclusion
The current study provides evidence of higher levels of serum RBP4 in hypothyroidism, especially in patients with IS, associated with metabolic and glucose abnormalities, and, thus, it could be a promising predictive biomarker of IS in hypothyroidism.

Conflicts of interest
There are no conflicts of interest.

Table 2
Clinical and laboratory characteristics of case groups