Association between glutathione peroxidase‐3 activity and carotid atherosclerosis in patients with type 2 diabetes mellitus

Abstract Background and Aims Deficiency of glutathione peroxidase 3 (GPx3) has been recognized as an independent risk factor for cardiovascular events. However, little is known regarding the role of GPx3 in carotid atherosclerosis, which is ubiquitously observed in type 2 diabetes mellitus (T2DM). This study aimed to investigate the relationship between GPx3 activity and carotid atherosclerosis among patients with T2DM. Methods From January 2018 to December 2018, 245 consecutive patients with T2DM were enrolled in this observational study. Assessment of serum GPx3 activity was performed after admission. We also used carotid ultrasound to measure the mean carotid intima–media thickness (CIMT) and to assess the presence of carotid plaque. Results Of the 245 patients, the median serum GPx3 activity was 22.5 U/ml (interquartile range, 12.4–35.9 U/ml). Carotid plaque was observed in 113 (46.1%) patients, and mean CIMT was 0.8 ± 0.1 mm. Univariate analysis showed that age, smoking, previous coronary heart disease, carotid plaque, and level of mean CIMT and hypersensitive C‐reactive protein were significantly associated with decreasing tertile of GPx3. Furthermore, after adjusting for all potential confounders by multivariable logistic regression analysis, PGx3 activity was significantly and independently associated with the mean CIMT (β = −.406, p = .002) and carotid plaque (first tertile of GPx3, odds ratio, 1.870, 95% confidence intervals, 1.124–3.669, p = .024). Conclusions This study demonstrated that serum GPx3 activity was inversely associated with mean CIMT and carotid plaque, suggesting that lower GPx3 activity may be an independent predictor for carotid atherosclerosis in T2DM.

Inflammation and oxidative stress are milestones of the atherosclerosis and are supposed to take place in the initiation, progression, and rupture of lipid-rich lesions (Viña, Borras, Abdelaziz, Garcia-Valles, & Gomez-Cabrera, 2013;Yang et al., 2017). Several serological biomarkers of oxidative stress, such as matrix metalloproteinases, lipid hydroperoxides, and NADPH oxidase, have been reported to be associated with atherosclerosis (Tibaut et al., 2019).
GPx3 deficiency has been found to be associated with increases of peroxide-related oxidants, suppression of insulin signaling (Chung et al., 2009), and subsequent chronic diseases such as cardiovascular events (Pastori et al., 2016) and chronic kidney disease (Pang et al., 2018). However, the relationship between GPx3 activity and carotid atherosclerosis has not been well established. Therefore, we performed this hospital-based observational study in a cohort of patients with T2DM to investigate the relationship between GPx3 activity with CIMT and carotid plaque.

| Baseline data collection
Data collection was performed using a standardized questionnaire by trained physician after admission. Variables were recorded as follows: demographic characteristics (age and gender), vascular risk factors (hypertension, hyperlipidemia and smoking), previous medication (antihypertensive, antidiabetic, antiplatelet and statin medication), and clinical data (history of cardiovascular disease, body mass index, systolic blood pressure, and diastolic blood pressure).
Body mass index was calculated as the weight in kilograms divided by the square of height in meters. Hypertension was defined as systolic blood pressure level ≥140 mmHg and/or diastolic blood pressure level ≥90 mmHg on three occasions, a self-reported physician diagnosis of hypertension, or receiving antihypertensive medication.

| Biochemical measurements
Venous blood samples were drawn into standardized tubes containing an anticoagulant within 24 hr after admission. For assessing the GPx3, the specimens were immediately separated by centrifugation at 1,500 r/min for 10 min and the isolated serum frozen at −80°C for subsequent analysis. The measurement of GPx3 activity was conducted by colorimetric assay kit (Abcam) following the manufacturers' instructions. All measurements were performed by duplicate, and inter-and intra-assay coefficients of variation were 6.0% and 4.1%, respectively. The lower functional detection limit of the assay is 0.5 mU/ml. We also performed laboratory tests including fasting blood glucose, hemoglobin A1c (HbA1c), high-sensitivity C-reactive protein (hs-CRP), homocysteine (HCY), and serum lipid status.

| Carotid ultrasonography
Carotid ultrasonography on each subject was performed by a highresolution and linear-array transducers (Philips iU22; Philips), with a linear probe at frequency of 4.0-9.0 Hz. The IMT of the common carotid artery, carotid bulb, and internal carotid artery were measured in both arteries. For the common carotid artery, both near and far walls were examined 10 mm proximal to the bulb. Only far walls were examined in bulb and internal carotid artery. Mean CIMT was calculated by averaging the left and right IMT. Plaque was defined as a focal structure that encroaches into the arterial lumen of ≥0.5 mm or 50% of the surrounding IMT value (Ng, Lim, Kang, Kadir, & Tai, 2016;Touboul et al., 2007). All ultrasonographic examination was performed by a single certified sonographer who was blinded to clinical data.

| Statistical analysis
The statistical analysis was performed using SPSS version 22.0 (SPSS Inc.). We performed the Kolmogorov-Smirnov test to evaluate the normality of continuous variables. Continuous variables were presented as mean ± standard deviation or median (interquartile range), and categorical data were expressed as n (%).
Differences in baseline characteristics among the GPx3 tertile were tested using the analysis of variance or Kruskal-Wallis test for continuous variables, and Pearson's chi-square test for categorical variables. We performed logistic regression models to detect the risk factors for increasing value of mean CIMT and carotid plaque.

| RE SULTS
A total of 245 patients with T2DM were included for the final analy- The median GPx3 value was 22.5 U/ml, with tertile levels as follows: first tertile <18.5 U/ml, second tertile 18.5-32.6 U/ml, and third tertile >32.6 U/ml. The baseline characteristics were compared between the groups set according to serum GPx3 tertile (Table 1).
Furthermore, after adjusting for all potential confounders (age, smoking, previous ischemic stroke, previous coronary heart disease, and body mass index, low-density lipoprotein, hs-CRP, HbA1c and HCY level), lower GPx3 activity was identified as an independent predictor of increased mean CIMT (β = −.370, p = .002).  of T2DM patients. Furthermore, these associations remained significant even after adjusting for potential confounders, indicating that decreased GPx3 activity may play a crucial role in carotid atherosclerosis.

| D ISCUSS I ON
It is well-accepted that atherosclerosis is a pathophysiological process involving of chronic inflammation and oxidative stress, which may cause the vascular endothelium and lead to the cardiovascular diseases. GPx functions as the major antioxidant molecule in the peripheral blood. It may also exert an important anti-atherogenic effect. To date, several studies have analyzed the GPx activity in human subjects and its prognostic value in atherosclerotic diseases (Buijsse et al., 2017;Lapenna et al., 1998;Pastori et al., 2016). In carotid atherosclerotic plaques, GPx1 activity is found decreased or absent, which may further induce the development of more severe lesions in humans (Lapenna et al., 1998). Moreover, decreased serum GPx3 activity is associated with increased cardiovascular mortality in individuals with low HDL (Buijsse et al., 2017), suggesting that a low antioxidant status predisposing to poor vascular outcomes.
Recently, the serum GPx3 activity has been measured in participants with atrial fibrillation and the results showed that decreasing GPx3 activity may be a predictor of fatal and nonfatal cardiovascular complications (Pastori et al., 2016). However, little is known about the role of GPx3 in carotid atherosclerosis. Our present study indicates that the reduced GPx3 activity was involved in the deterioration of mean CIMT and the presence of carotid plaque, in support of the linking between GPx3 activity and the pathogenesis of carotid atherosclerosis in T2DM subjects. According to previous studies, it has been suggested that GPx3 is increased in subjects with insulin resistance, overweight or obese, and metabolic syndrome (Baez-Duarte et al., 2012. However, we did not find any cross-sectional association of GPx3 activity with fasting blood glucose (Spearman's correlation analysis, r = .018, p = .778) and body mass index (Spearman's correlation analysis, r = −.089, p = .281). These discrepancies might be explained at least in part that the expression of GPx3 is induced by different disease stage (Chung et al., 2009).
Although the underlying mechanism of the regulation of GPx3 activity and its physiological and pathophysiological roles in carotid atherosclerosis remains limited, several speculations may explain this association. Experimental evidences showed that GPx-3 can catabolize hydrogen peroxide under circumstances of normal metabolism or oxidative insult, therefore preserving the vascular endothelium-derived vasorelaxation and anti-atherosclerosis (Takebe et al., 2002). Nonetheless, the prolonged oxidative stress keeps consuming antioxidants, leading to oxidants augment and endothelial dysfunction (Redón et al., 2003). Moreover, in GPx3deficient mice models, vascular endothelial damage is also observed (Wolin, 2011). Thus, GPx3 may be a determinant mediator in diseases which may impair endothelial function and result in atherosclerosis. Furthermore, GPx3 plays a role in inflammatory responses.
Decreased GPx3 activity has been observed in patients with systemic inflammatory response syndrome and possessed a predictive value for this syndrome (Manzanares et al., 2009). As reported, pro-inflammatory cytokines can regulate GPx3 production (Agnani et al., 2011;Montague et al., 1998). We therefore assume that low In conclusion, our study suggested that decreasing serum GPx3 activity is associated with the severity of carotid atherosclerosis in T2DM patients. Further large-sample prospective studies are needed to confirm the causal relationship and evaluate the underlying mechanism between serum GPx3 and carotid atherosclerosis.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest.

AUTH O R CO NTR I B UTI O N S
PL designed the study, conducted statistical analysis, and wrote the draft of the manuscript. WS and GZ conducted statistical analysis and acquired data. CQ and YL acquired data. YX and XY conceived and designed the study, conducted statistical analysis, and supervised this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.