Doppler Ultrasound Study on the Effects of Different Blood Uric Acid Levels on the Carotid Artery Intima-media Thickness in Patients with Gout

Objective : Doppler ultrasonography is used to study the effects of different serum uric acid (SUA) levels in primary gout (PG) on the carotid artery intima - media thickness (CIMT) and its risk factors and to provide clinical evidence for early prevention and treatment of systematic gout damage. Methods: We obtained information of 237 patients with PG (intermittent period) and estimated the association between gout and common carotid artery intima-media thickness (CCAIMT), carotid plaque formation, and blood flow resistance index (RI)) using binary logistic regression. Results: The CCAIMT of patients with PG was significantly correlated with different SUA levels, age, course of the disease, blood routine laboratory parameters etc ( P < 0.05). The CCAIMT of patients with PG combined with the tophi, secondary hypertension, or diabetes group was significantly different from that in the uncombined group ( P < 0.05). The age, disease course, body mass index (BMI), blood routine indexes, liver function, renal function, and lipid metabolism-related indexes of patients with gout combined with the carotid plaque group and without the carotid plaque group were significantly different ( P < 0.05). Age (OR, 95% CI = 1.136, 1.073-1.202), disease course (OR, 95% CI = 1.236, 1.084-1.410), and low-density lipoprotein cholesterol (LDL-C) (OR, 95% CI= 6.982, 2.460-19.816) were risk factors for CCAIMT of more than 0.0858cm, whereas SUA (OR, 95% CI = 0.994, 0.988-0.999) and Lymphocyte percentage (LY%) (OR, 95% CI = 0.842, 0.723-0.980) were protective factors. Age was a risk factor for carotid plaque (OR, 95% CI = 1.137, 1.071-1.207), and SUA was a protective factor (OR, 95% CI = 0.992, 0.986-0.998). Moreover, secondary hypertension (OR, 95% CI = 3.496, 1.442-8.479), PLT (OR, 95% CI = 1.005, 1.001-1.010), and SUA (OR, 95% CI = 1.003, 1.000-1.005) were risk factors for common carotid artery RI of ≥ 0.7 in patients with gout. Conclusion: Age, disease course, blood routine indexes, renal function, and lipid metabolism of patients with gout with different SUA levels were closely related to carotid intima-media diseases. Age, disease course, and LDL-C were the risk factors for carotid artery intima-media lesions in patients with PG, whereas SUA and LY% might be protective factors. Inflammatory indexes: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR).


Introduction
Gout is a metabolic and inflammatory/immune disease caused by impaired purine metabolism and/or decreased uric acid excretion. The continuous increase in blood uric acid causes urate (monosodium urate, MSU) crystals to precipitate and deposit in the joints or other tissues [1,2]. The global prevalence of gout ranges from < 1% to 6.8%, and the incidence is 0.58-2.89 per 1,000 people, with males having a higher prevalence than females. In addition, there are ethnic differences in the disease, and the prevalence increases with age [3]. MSU crystals can not only act on joints but also on multiple target organs, such as blood vessels, the heart, and the kidneys, and are closely related to atherosclerosis, hypertension, metabolic syndrome, and chronic kidney disease [2,4,5,9]. Presently, joint fluid or tophi tissue puncture to detect MSU crystals is the gold standard for diagnosing gouty arthritis, but this is an invasive test. The gout classification standards formulated in 2015 by the American College of Rheumatology (ACR) and the European League Against Rheumatism (EULAR) and the EULAR updated gout diagnosis recommendations in 2018 emphasize the importance of ultrasound in the diagnosis of gout [1,2]. Ultrasound detection of peripheral arteries has been widely used in clinical practice and has become an important auxiliary method for detecting atherosclerosis [6]. As a superficial artery, the carotid artery can reflect the degree of systemic atherosclerosis. Studies have shown that carotid artery intima-media thickness (CIMT) is one of the indicators that reflect the degree of carotid arteriosclerosis with high sensitivity and specificity. Two changes occur in atherosclerosis: the early change is intima-media thickening, and the late change is atherosclerotic plaque formation [15]. The common carotid artery resistive index (CCARI) is affected in the early stage of the pathological process of cardiovascular disease, which reflects disease progression. Therefore, changes in the CCARI are a key indicator of pathological changes in important vascular regions [7,8].
Studies have shown that serum uric acid (SUA) is significantly related to carotid artery diseases [9][10][11][12], but some studies have shown that SUA is not significantly related to carotid artery diseases [4,5,13,14]. The aforementioned studies were mostly based on pure blood uric acid levels or studies on non-gout patients. Thus, the effects of different blood uric acid levels on CIMT in patients with primary gout remain unclear. This study was conducted to investigate the effects of different blood uric acid levels on CIMT in patients with primary gout using Doppler ultrasound technology and to provide clinical evidence for the diagnosis and treatment of gout system damage.
Participants and methods

Participants
In this study, 237 patients with primary gout (intermittent period) were enrolled. The inclusion criteria were as follows: patients with primary gout that met the 2015 ACR/EULAR gout diagnostic criteria [2]; patients aged 16-90 years; patients who did not receive uric acid-lowering and other related treatments within 3 months. The exclusion criteria were as follows: patients with secondary gout caused by myeloproliferative disease, tumor radiotherapy, chemotherapy, and kidney disease; patients with a history of cervical radiotherapy, carotid artery intervention or surgery, and carotid artery trauma; patients with rheumatoid arthritis, reactive arthritis, psoriatic arthritis, spondyloarthritis, or other autoimmune diseases; patients with severe heart and kidney diseases, blood infections, vasculitis, and other infectious diseases; patients with serious diseases of other systems affecting the investigation and patients who refuse to sign informed consent. This study was approved by the Medical Ethics Committee of the First Affiliated Hospital of Chengdu Medical College, and all research subjects provided informed consent.
recorded. Height and weight were measured, and BMI was calculated. All subjects signed the informed consent.

Blood routine indexes and biochemical indexes
Fasting venous blood and clean midstream urine were collected from the patients in the morning (at least 8 hours of overnight dietary restriction). The blood routine indexes (Automatic blood analyzer: Japan Sysmex XN-9000) and biochemical indexes (Automatic biochemical analyzer: Japan Hitachi Limited 7600, Erythrocyte sedimentation rate analyzer: USA Vital Diagnostics Monitor-100, Automatic urine analyzer: Japan Sysmex UF-4000) were detected. All Inflammatory indexes: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR).

Cervical ultrasound examination
The PHILIPS EPIQ7c and Mindray resona8 ultrasonic imaging instrument was used for ultrasound inspection, with a linear array probe frequency of 9-12 MHz using the cervical blood vessel inspection mode. The patient underwent bilateral carotid artery ultrasound examination in the supine position under a calm breathing state. The gray-scale imaging mode was used to scan the carotid artery first by transverse section and then longitudinal section. Scanning was started from the bifurcation of the innominate artery on the right side and the beginning of the common carotid artery on the left side. The carotid artery (proximal, middle, and distal segment), internal carotid artery, and external carotid artery were continuously scanned. While avoiding plaque and clearly showing the front and rear walls, the CIMT (thickest part) was measured. If no obvious thick spot is observed by the naked eye, the intima-media thickness of the common carotid artery (CCAIMT) of 1-1.5 cm below the bifurcation of the common carotid artery was measured ( Fig.   1-A), and the average of three measurements was calculated. Color Doppler flow imaging was used to observe the blood flow filling state of the aforementioned arteries. Pulse Doppler ultrasound was used to measure the peak systolic velocity, end diastolic velocity, and resistive index (RI) of the common and internal carotid arteries ( Fig. 1-C). The sampling volume was fixed at 2 mm, placed in the brightest part of the center of the blood vessel. The best sound beam blood flow angle (< 60°) was set, the blood flow spectrum of three cardiac cycles was measured, and the average value was calculated. The aforementioned ultrasound detection was performed on the left and right carotid arteries, and the average values of bilateral CCAIMT, common carotid artery (RI) CCARI, and internal carotid artery resistive index (ICARI) were calculated. Plaque was defined as a thickness of ≥ 0.15cm or more than 50% of the surrounding intima-media thickness (IMT) [15] ( Fig. 1-B). In this study, the average CCAIMT values of greater than 75% were regarded as abnormal, and the average CCARI and average ICARI values of ≥ 0.70 were regarded as abnormal.

Statistical analysis
Statistical Package for the Social Sciences version 22.0 was used for all statistical analyses. In the baseline characteristics, categorical variables were expressed as counts and percentages, and continuous variables were expressed as mean ± standard deviation or quartiles. The Mann-Whitney rank sum test was used to evaluate the differences between two independent samples, spearman correlation analysis was used to test the correlation between different parameters, and binary logistic regression analysis was used to evaluate the risk factors of carotid artery diseases. P-values of less than 0.05 were used to indicate that the difference is statistically significant.

Correlation analysis between carotid ultrasound parameters and general clinical features and laboratory indicators
In the gout patients, CCAIMT was positively correlated with age, disease duration of gout, NE, UREA, CysC, β2-MG, CRP, ESR (P < 0.05), and negatively correlated with RBC, HGB, PLT, LY %, ALT, TBIL, DBIL, SUA, TP, ALB (P < 0.05). (Table 4) The CCAIMT of the patients with gout in the secondary hypertension group was greater than that of the patients with gout in the non-secondary hypertension group; the CCAIMT of the patients with gout in the secondary diabetes group was greater than that of the patients with gout in the non-secondary diabetes group; and the CCAIMT of the patients in the combined tophi group was greater than that of the patients in the non-combined tophi group. The aforementioned differences were statistically significant (P < 0.05) (Fig. 2).   were greater than those in the no plaque group (P < 0.05). In addition, the BMI, RBC, HGB, PLT, LY %, ALT, AST, SUA, UUA, TP and ALB of the plaque group were lower than those in the no plaque group (P < 0.05) (Fig. 3). A: Age and disease duration of the plaque group were higher than those of the non-plaque group, and BMI was lower than that of the non-plaque group (P < 0.001, < 0.001, 0.007, respectively). B: RBC, HGB, PLT, LY% in the plaque group were all lower than those in the non-plaque group, and NE was greater than that in the non-plaque group (P < 0.001, < 0.001, 0.009, 0.004, 0.036, respectively). C: ALT and AST in the plaque group were lower than those in the non-plaque group (P < 0.001, 0.033, respectively). D: UREA, CREA, CysC and β2-MG in the plaque group were all higher than those in the non-plaque group, and SUA and UUA were lower than those in the non-plaque group (P < 0.001, 0.029, < 0.001, < 0.001, 0.015, 0.029, respectively). E: TP and ALB in the plaque group were lower than those in the non-plaque group (all P < 0.001). F: FPG and ESR in the plaque group were higher than those in the non-plaque group (P = 0.049, < 0.001, respectively).
A plaque in a neck vessel was considered positive. The indicators related to plaque, combined with hypertension, diabetes, the presence or absence of tophi, BMI, LDL-C, and HDL-C, were included in the regression model, and a multivariate binary logistic regression analysis was performed. The model was tested using the Hosmer-Lemeshow and showed a good fit (P = 0.972).
An ICARI of ≥ 0.700 was defined as positive. The indicators that were correlated with the ICARI in the Spearman correlation analysis, combined with the presence or absence of hypertension, diabetes, tophi, age, course of the disease, and BMI, were incorporated into the regression model, and a multivariate binary logistic regression analysis was performed. However, no variables had a statistically significant impact on the ICARI (P > 0.05).

Discussion
This study found that the carotid ultrasound indexes of patients with primary gout and the CCAIMT of gout patients with different SUA levels were closely related to their age, course of the disease, blood routine indexes, renal function, lipid metabolism, and other indexes.

CCAIMT and Plaque
1.1 This study found that in patients with primary gout, SUA, and LY% reduced the risk of carotid artery diseases. However, some studies have shown [4,13,14,19] that the CIMT is not associated with SUA. Panagiota Drivelegka et al. [5] have not shown a significant correlation between the SUA quartile and CIMT or carotid plaque, regardless of gender. In addition, Mariano Andrés [16] et al. have found that the course of gout and the presence of tophi were not significantly related to the increase in the CIMT and plaque formation. However, José M. A. Wijnands, Arrigo F.G.
Furthermore, a study based on patients with metabolic syndrome by Shun-Sheng Wu et al. [12] has shown that SUA is positively correlated with the CIMT. Moreover, Rada Gancheva et al. [18] have shown that in patients with asymptomatic hyperuricemia and gout, age and gouty arthritis increased the risk of CIMT abnormality (CIMT of more than 0.90 mm), and the presence of tophi was the most effective risk factor. However, asymptomatic hyperuricemia did not affect the CIMT.
The results of this study showed that SUA reduced the risk of having a CCAIMT of more than 0.0858 cm and carotid plaque formation in patients with gout, which was different from the results of previous studies. The study by Maxwell SRJ et al. [20] has shown that increasing the uric acid levels might be beneficial to patients with cardiovascular disease because of the antioxidant property of uric acid. Philip K. Tan et al. [21] have found that primates have increased their affinity for uric acid during the evolution process and, thus, inferred that maintaining a high level of SUA might provide an advantage to our primate ancestors. In addition, a multicenter study in Japan by Atsushi Tanaka et al. [22] has shown that compared with non-drug treatment, treatment with febuxostat, a uric acid-lowering agent, of patients with asymptomatic hyperuricemia for 24 months did not delay the progression of carotid atherosclerosis. In this study, SUA in patients with primary gout showed a negative effect on the CCAIMT, which might be related to its antioxidant effect. This put forward new thinking on the standard of uric acid-lowering treatment for patients with gout, especially for elderly patients, whether the standard of uric acid-lowering treatment should be appropriately relaxed to improve the protective effect of uric acid on blood vessels.
However, the sample size needs to be further expanded in the future to confirm this conclusion.
In addition, the results of this study showed that LY% was a protective factor for CCAIMT, and LY% could reduce the risk of CCAIMT thickening. Studies have shown that macrophages and LY % play a key role in the development of atherosclerosis and can provide signals for the potential pro-inflammatory background of atherosclerosis [23]. The results of this study were inconsistent with the aforementioned pathological process. However, in the study by Mucahid Yılmaz et al. [24], the neutrophil/lymphocyte ratio (NLR) was an index reflecting high neutrophil levels (indicating inflammation) and lymphopenia induced by physiological stress. In addition, the NLR level of the coronary artery disease group was higher than that of the normal coronary artery group, indicating that low LY% levels were an index that was not conducive to the coronary artery.
In this study, the increase in LY% in patients with gout helped reduce the probability of CCAIMT lesions, which was consistent with the study by Mucahid Yılmaz. However, few studies have focused on the relationship between LY% and arteriosclerosis in patients with primary gout. This study may provide a new direction for future research, hoping to provide a more comprehensive reference index for preventing and treating clinical gout vascular complications.
Age and LDL-C increasing the risk of atherosclerosis has been confirmed by many studies [23,25], and the results of this study also confirmed this. In addition, the results of this study showed that the course of gout increased the risk of CCAIMT thickening, which was inconsistent with the results of the study by Mariano Andrés [16]. The reason might be that the course of gout in this study was positively correlated with age (age and disease course were Spearman-related: R = 0.312; P < 0.001).

Different SUA levels and CCAIMT
The study by Shinji Kishimoto et al. [26] has proven that Hct, HGB, and RBC were related to the brachial artery IMT in male patients, and the increase in Hct, HGB, and RBC content might reduce the risk of cardiovascular diseases. The correlation analysis between different SUA levels and the CCAIMT in this study showed that the RBC count in the patients with gout in the Q1-Q4 groups were negatively correlated with the CCAIMT, and the HGB level of the patients with gout in the Q2-Q4 groups were negatively correlated with the CCAIMT. We could infer that within the normal range, relatively high RBC and HGB levels were beneficial to the CCAIMT in patients with gout with different SUA levels.
In this study, the CysC of the patients with gout in the Q1-Q4 groups were positively correlated with the CCAIMT, and the β-2MG of the patients with gout in the Q2-Q4 groups were positively correlated with the CCAIMT. Regarding CysC, a study by Chiu-Shong Liu et al. [27] has shown that CysC and CIMT have a strong linear positive correlation in Taiwanese residents. to protect patients with primary gout from cervical vascular damage is clinically possible.
A study by Shigeru Hayashi et al. [35] has shown that the CIMT is positively correlated with age, creatinine, BUN, and C-reactive protein and negatively correlated with TP and ALB in patients with atherosclerosis, which was consistent with the results of this study. It is worth mentioning that creatinine is not associated with the overall CCAIMT in this study, whereas it is positively correlated with the CCAIMT in the Q3 and Q4 groups (SUA levels of more than 510 mmol/L). It might be inferred from this that patients with gout with high SUA levels should be alert to creatinine levels to better prevent vascular complications.

CCARI
A series of studies involving patients with asymptomatic hyperuricemia and gout by Rada N.
Gancheva [17,18] have shown that hypertension significantly increased the risk of CCARI abnormalities, the presence of tophi significantly increased the possibility of having a CCARI of ≥ 0.7 in patients with gout, and gout with tophi and hyperuricemia had no significant effect on the risk of CCARI abnormalities. The results of this study showed that both SUA and hypertension were risk factors for having a CCARI of ≥ 0.7, which is consistent with the results of previous studies. This study suggests that treating gout early is essential.
Several studies have shown that [36][37][38][39][40][41], in addition to hemostasis, platelets are also involved in the process of atherosclerosis. Constantinos Bakogiannis et al. [40] have found that platelets could release various chemokines and recruit and activate immune cells during vascular injuries, such as monocytes, neutrophils, and T lymphocytes, thereby playing a role in vascular inflammation and atherosclerosis. Studies by Norbert Gerdes, Tessa J. Barrett et al. [38,41] have also shown that platelets promote atherosclerosis. This study found that PLT was a risk factor for having a CCARI of ≥ 0.7 in patients with gout, which was consistent with the role of PLT in previous studies. The results of this study suggest that we can indirectly predict the impact of platelets on the CCARI by detecting their changes during the diagnosis and treatment of gout.

ICARI
The results of study by the José Alexandre Addeo Cipolli [19] et al. have shown that SUA is not significantly correlated with the CIMT in men and women. After adjusting for potential confounding factors, regression analysis has shown that the ICARI was independently associated with SUA and hyperuricemia in women. The results of the regression analysis in this study did not find risk factors for ICARI in patients with gout, but the proportion of women in this study was small, which might be the reason for the inconsistency with the results of this study and previous studies.

Conclusion
In summary, this study found that the CCAIMT in patients with primary gout with different SUA levels was closely related to blood routine indexes, renal function, lipid metabolism, and other indicators. Especially for patients with gout with high SUA levels, the CCAIMT was positively correlated with several renal function-related indicators, among which creatinine might be an observed index of carotid artery disease in patients with gout with high SUA level. In patients with primary gout, age, course of the disease, and LDL-C might be risk factors for carotid intima-media diseases, suggesting that we should pay attention to early diagnosis and treatment of gout to prevent carotid intima-media-related lesions. Meanwhile, SUA and LY% are possible protective factors for carotid arterial intima-media lesions in patients with primary gout. The study can be expanded in the future to provide a new reference for the standard of uric acid treatment for vascular protection in patients with primary gout. The limitations of this study were that the sample size was small and the proportion of women was low. In the future, the sample size could be expanded and the gender ratio could be further studied to confirm the results of this study.