Introduction

Hyperuricemia is commonly considered as an underlying cause of gout, which is characterized by the precipitation of extracellular uric acid in joints [1]. However, earlier studies have explored the role of elevated plasma serum uric acid (SUA) in the onset of cardiovascular diseases (CVD) and other conditions like hypertension [2], diabetes [3] and renal disorders [4]. Uric acid activates various cellular mechanisms, which lead to hypertension and diabetes. Complications of hypertension and diabetes are major contributors in CVD related deaths. According to the estimates of World Health Organization (WHO), 31% of the global deaths are caused by CVD.

Of all the CVD deaths, ~ 90% are caused owing to coronary artery disease, which is characterized by the decreased blood supply to the heart tissues resulting from the narrowing or blockage of the coronary arteries (vessels that supply blood to the myocardium). Vascular homeostasis is of prime importance in maintaining cardiovascular health [5]. Hypertension and diabetes cause damage to the blood vessels which results in vascular injury. High SUA also causes vascular injury by promoting oxidative stress, activating renin angiotensin system (RAAS) [6], reducing mitochondrial DNA and depleting intracellular ATP concentration [7].

SUA can induce oxidative stress by activating nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidases (major source of reactive oxygen species in vascular cells) [8] and inactivating endothelial nitric oxide synthase [9]. The reduced bioavailability of nitric oxide results in impaired vasodilation, increased vasoconstriction and endothelial dysfunction [10]. Furthermore, SUA also activates pro-inflammatory factors and RAAS which alter vascular biology, thus promote plaque formation which decrease the blood supply to the heart tissues [11].

Various studies have shown a positive correlation of hyperuricemia with the onset of CVD; however, age, gender, ethnicity, life-style, dietary habits and other concomitant diseases like hypertension, diabetes and dyslipidemia affect the contribution of uric acid in CVD risk assessment [11]. The influence of these confounding factors makes the association of hyperuricemia and CVD controversial [12, 13]. Thus, aim of the current study was to elucidate the role of SUA in raising the risk of CVD independent of other key confounding factors like age, gender, hypertension, diabetes, dietary and life-style habits.

Materials and methods

For this retrospective observational study, clinical and biochemical data of 502 human subjects of both genders (52% males and 48% females) with normouricemia (n = 266) and hyperuricemia (n = 236) were analyzed. SUA and CVD related data for these subjects were taken from our previously collected cardio-metabolic disorders patients cohort [14,15,16] who were enrolled from March to November, 2017 during their outpatient visits or hospitalization in coronary care units of Allied hospital, Faisalabad and Faisalabad Institute of Cardiology (FIC), Faisalabad, Pakistan. All enrolled subjects were adults (age > 18 years) suffering from cardiovascular diseases (CVD) and/or hypertension and/or diabetes. Pregnant and lactating women, subjects having chronic infections or cancer were not included.

Cardiac disease was diagnosed by an expert cardiologist, on the basis of physical examination and electrocardiogram (ECG). Blood pressure was measured in comfortably seated position after 5 min of rest. According to American College of Cardiology/American Heart Association guidelines, hypertension was defined as either blood pressure is more than 130/80 mmHg or patient is already on antihypertensive drugs [17]. Subjects with type 2 diabetes mellitus recruited for this study were already confirmed to have diabetes according to the American Diabetes Association criteria [18]. Hyperuricemia was defined as SUA level is > 7.0 mg/dL in males and > 6.0 mg/dL in females [19, 20] and data was analyzed according to this criterion as reported in current study. However, additionally, cut-off values (> 5.1 mg/dL for females and > 5.6 mg/dL for males) for SUA according to URic acid Right for heArt Health (URRAH) study were also used for analysis (This data is provided in supplementary Table S1 and Table S2).

Informed oral and/or written consent was taken from all subjects. The institutional (National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan) ethics review committee approved this study (copy of approval letter is available as supplementary file, S1). All procedures or protocols followed for human research, were in accordance with the guidelines of Declaration of Helsinki.

A questionnaire related to biodemographic details (age, gender, and address), disease history, medication, dietary habits (weekly intake of vegetables, pulses, meat and daily intake of sugar, salt, tea and soft drinks) and life-style (level of physical activity per day) was recorded for all patients. CVDs were divided into three clinical subtypes by expert cardiologist based on clinical phenotypes as; acute coronary syndrome (ACE), myocardial infarction (MI) and heart failure (HF). Blood sample (3–5 ml) was taken and serum was separated from the clotted blood by centrifugation at 3500 rpm for 10 min. All the samples were stored at -20oC until further analysis.

Biochemical analysis

Uric acid and other clinically important biochemical parameters including random blood glucose, total cholesterol, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and triglycerides were measured on a semi-automated clinical chemistry analyzer (Micro-lab 300) using commercial kits (Merck Inc.) by following the protocols provided by the vendor.

Statistical analysis

All continuous variables are expressed as mean ± standard deviation and categorical variables are presented as number (percentage). The normality of all variables was checked by Shapiro Wilk test or Kolmogorov-Smirnov test. Parametric tests like independent t test or ANOVA was used for normally distributed variables; however, for non-normal variables, non-parametric tests including Mann-Whitney U test or Kruskal-Wallis test were performed by SPSS version 20. Chi-square test was also used to check the association between categorical variables. Uric acid was further divided into quartiles and Pearson/Spearman correlation was applied to evaluate the strength and direction of association between the serum uric acid and CVD. Regression analysis were also done to analyze the risk of disease associated with serum uric acid.

Results

Analysis of serum uric acid levels among cardiovascular disease subtypes

Descriptive statistics showed that SUA concentration was gradually increasing from non-cardiac subjects (6.0 ± 2.4 mg/dL) to ACS (6.6 ± 2.6 mg/dL), MI (7.3 ± 2.7 mg/dL) and HF (9.3 ± 2.5 mg/dL) patients (Fig. 1). The results of Kruskal-Wallis test revealed that SUA levels are significantly different among CVD phenotypes (χ2 = 42, p < 0.001). Analysis revealed risk of HF increased from 2 to 14% (Table 1) with an elevation in SUA concentration. Similar results were obtained when analyses were done according to URRAH cut-off values for SUA (Table S1, supplementary data).

Fig. 1
figure 1

Serum uric acid concentration increases with severity of cardiovascular disease subtypes. CVD; cardiovascular diseases, ACS; acute coronary syndrome, MI; myocardial infarction, HF; Heart failure

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Association of SUA with CVD phenotypes

Chi-square test also showed a significant association of SUA with CVD phenotypes (p < 0.001) (Table 1). Further analysis of direction and strength of this association by Spearman correlation revealed a significant positive association of SUA with CVD (rho = 0.149, p = 0.001) which indicates that increased serum concentration of SUA also increases the risk of CVD.

Table 1 Analysis of clinical and biochemical data among normouricemia and hyperuricemia
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SUA associated risk assessment of cardiovascular diseases

SUA levels were further divided into quartiles (< 5.0 mg/dL, ≥ 5.0 and < 7.0 mg/dL, ≥ 7.0 and < 9.0 mg/dL, ≥ 9.0 mg/dL) and characteristics of study population were assessed in these groups (Table 2). This table represents very interesting results for HDL-C, LDL-C and cardiovascular phenotypes. Concentration of HDL-C increased linearly with an increase in SUA concentration, however, this trend was opposite for LDL-C. Similarly, frequency of ACS was decreasing with an increase in SUA levels, while prevalence of heart failure was increasing with an increase in SUA levels from Q1 to Q4. This table shows statistically strong link between HDL-C, LDL-C, cardiac phenotypes and SUA. Prevalence of diabetes and hypertension was also significantly different between SUA quartiles.

Table 2 Distribution of clinical and biochemical parameters between SUA quartiles
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Furthermore, multinomial regression analyses were also carried out to find the SUA associated risk of CVD. The results showed that SUA levels above physiological range (> 7.0 mg/dL), which increased the risk of CVD by 1.7 times [OR: 1.78 (CI: 1.28–2.48) p = 0.001] and this risk raised up to 2.37 folds in group with SUA above 9.0 mg/dL [OR: 2.37 (CI: 1.72–3.27) p < 0.001]. Moreover, this association remained significant in [3rd quartile: OR: 1.58 (CI: 1.09–2.28) p = 0.016 and 4th quartile: OR: 2.12 (CI: 1.48–3.03) p < 0.001] after adjusting for confounding factors including age, gender, total cholesterol, HDL-C, LDL-C, triglycerides, hypertension and diabetes. Furthermore, SUA associated CVD risk remained significant [3rd quartile: OR: 1.73 (CI: 1.01–2.98) p = 0.048 and 4th quartile: OR: 2.85 (CI:1.67–4.85) p < 0.001] after including the life style and dietary habits in adjusting parameters (Table 3). Almost similar results were obtained by multinomial regression analysis of SUA groups defined according to URRAH cut off values for CVD (Table S2, supplementary data). Hence, it could be inferred that SUA is associated with CVD and it can act as an independent risk factor for CVD.

Table 3 Multivariate regression analyses of serum uric acid quartiles and cardiovascular diseases
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Discussion

Current study revealed that elevated serum uric acid (SUA) concentration is positively associated with risk of cardiovascular diseases independent of other confounding factors like age, gender, life style and dietary habits. Severity of cardiovascular disease increases with an increase in SUA, even this association was not influenced by diabetes and hypertension which are well-known risk factors for CVD.

In previous years, several large-scale studies including NHANES I epidemiologic follow-up study, URRAH study, Brisighella Heart Study and AMORIS study have been conducted to check the association between SUA and cardiovascular diseases. Studies from URRAH (Uric Acid Right for Heart Health) database demonstrated that serum uric acid increases the risk of fatal cardiac events [21] and identified cut-off SUA value for reduced CVD event is < 5.26 mg/ dL in women and 5.49 mg/dL in men [22]. Similarly, a sub-study of Apolipoprotein Mortality Risk Study (AMORIS) with follow-up of 11.8 years including 417,734 human subjects concluded that risk of cardiac events (acute myocardial infarction, ischemic and hemorrhagic stroke) increases with an increase in SUA concentration [23]. Evaluation of 1,557 subjects in Brisighella Heart Study also concluded that SUA can act as a predictor for electrocardiographically diagnosed myocardial infarction, left ventricular hypertrophy and tachyarrhythmias [24].

Apart from these studies, a meta-analysis of 21 cohort studies demonstrated that SUA is associated with CVD events in both high risk and healthy subjects, however; the association was stronger in high risk subjects only [25]. Likewise, a meta-analysis of 402,997 subjects demonstrate that SUA can marginally increase the risk of coronary heart disease [26] which weakens the independent association of SUA with CVD. Similarly, a brief review by Wannamethee [27] and experimental studies on British population [28] and Framingham heart study participants [29] reported that the association of SUA with risk of coronary heart disease events is influenced by CVD risk factors. A prospective cohort study conducted on participants of NHANES stated that SUA can increase the risk of CVD caused mortality in diabetes [30]. However, another cross-sectional study of NHANES diabetic participants with Mendelian randomization analysis failed to establish a causal relationship between SUA and CVD [31]. Another study conducted on obese subjects also supported previous studies [32]. An increase in severity of coronary artery disease with a rise in SUA concentration was also observed in studies on Chinese, Turkish, Korean and Pakistani population [21, 33,34,35].

High prevalence (53%) of hyperuricemia among CVD patients is also demonstrated in the current study. These findings are comparable to another Pakistani study [36] which explain that SUA is associated with risk factors of Heart failure (HF) and measurement of SUA concentration can help to identify high-risk HF patients. In the present study, an increase in HF risk was also observed in hyperuricemia group. Apart from the studies on adult subjects, a study from Brazil showed the association of SUA with the risk factors of CVD in children of age 6–17 years [37].

Few reports from China and Korea also demonstrated an association between hyperuricemia and arterial fibrillation [38,39,40]. Nonetheless, a study by Wheeler et al. including more than 9000 incident cases and ~ 155,000 controls from eight countries concluded that SUA is unlikely to predict the coronary heart disease risk in general population [41]. Furthermore, a cohort study including 11,009 participants of National Health and Nutrition Examination Survey III also stated that SUA is not a predictor for CVD and coronary heart disease mortality. These studies make the association between SUA and CVD controversial.

Moreover, all the conflicting studies were conducted on distinct populations with varying life styles and dietary habits [21, 42,43,44,45]. These differences in the study conditions can greatly influence the independent association of SUA with CVD. These studies strengthen the role of dietary habits and life style in influencing the association between SUA and CVD. As per literature review, no said study has evaluated the association of SUA with CVD independent of differential influence of life style and dietary habits. The current study has shown that SUA increases the risk of CVD by ~ 2.85 fold after adjusting for classical confounding factors along with additional factors including life-style and dietary habits, which is the main strength of present study.

At the molecular level, this controversy could be due to the anti-oxidant pro-oxidant paradigm of SUA [13]. The conditions responsible for the anti-oxidant – oxidant shift are not clearly known. It is suggested that in hydrophilic environment, SUA acts as an anti-oxidant while in hydrophobic environment it shifts its function to pro-oxidant molecule [46]. Studies have shown that SUA increases the expression of inflammatory markers [47], activates renin angiotensin aldosterone system (RAAS) [48] and decreases bioavailability of nitric oxide (a vasodilator) [49]. Nitric oxide and RAAS play an important role in vascular homeostasis and cardiac function [49].

Raised levels of SUA can disturb this balance by inducing nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) activation [50] and decreasing bioavailability of nitric oxide by directly reacting with it [49] or converting it to peroxynitrite [51]. These effects of hyperuricemia result in increased production of reactive oxygen species (ROS), which is major cause of endothelial dysfunction and CVD [52]. At low levels, ROS play a role in redox signaling by modulating proteins and DNA, while increased levels of ROS can result in cellular damage and enhance ROS generation [53]. Increased ROS generation can further disturb the vascular homeostasis and leads to impaired cardiac function [51]. Thus, elevated levels of SUA can act as pro-oxidant and increase the risk of CVD incidence and severity.

Although, current study demonstrates a strong association between SUA and CVD, however, this study does not provide information about the onset of CVD in subjects with higher SUA levels. Further longitudinal studies on large sample size should be conducted in future to assess the risk of CVD onset owing to hyperuricemia.

Conclusion

This study revealed a strong association between serum uric acid levels and risk of cardiovascular diseases. Risk of heart failure increases gradually with an increase in serum uric acid levels. Hence, subjects – apparently asymptomatic for cardio-metabolic conditions but having higher serum uric acid levels should be examined for cardiovascular health, so that such diseases could be prevented, managed or treated in early stages.