Hyperhomocysteinemia in association with male gender, advanced age, uric acid and metabolic syndrome: A retrospective cross-sectional study

Background Elevated homocysteine level has been proposed as a risk factor for cardiovascular disease. The aim of this study is to evaluate the connection between hyperhomocysteinemia with other factors for early detection of possible cardiovascular disease. Methods The data in this retrospectively designed cross-sectional study was retrieved from the health examination database in a medical center located in southern Taiwan in 2016. The correlation of hyperhomocysteinemia with sex, age, body mass index, waist circumference, lipid profile, blood pressure, uric acid, high sensitivity C-reactive protein and lipoprotein were investigated. Both simple and multiple stepwise logistic regression models were used for the assessment of their connection. Results A total of 878 subjects with mean age of 55.4 ± 10.8 years were included in the current study and 73 (9.1%) participants had elevated homocysteine levels. Simple logistic regression analysis showed elevated Hcy was significantly associated with sex (OR 0.11, 95% CI 0.04-0.31, P < 0.001), age (OR 1.03, 95% CI 1.00-1.05, P < 0.035), waist circumference (OR 1.04, 95% CI 1.02-1.06, P = 0.001), HDL-C (OR 0.97, CI 0.95-0.99, P = 0.011), triglyceride (OR 1.00, CI 1.00-1.01, P = 0.002), SBP (OR 1.02, CI 1.01-1.03, P = 0.004), DBP (OR 1.04, CI 1.02-1.06, P = 0.001), sugar level (OR 1.01, CI 1.00-1.02, P = 0.021), uric acid (OR 1.39, CI 1.20-1.60, P < 0.001) and the presence of metabolic syndrome (OR 1.81, CI 1.12-2.93, P = 0.016). Multiple stepwise logistic regression analysis was applied and the result showed that male gender (OR 0.15, 95% CI 0.05-0.43,

Background Cardiovascular disease is one of the most common leading causes of mortality and morbidity globally. In Taiwan, cardiac disease was the second major cause of death following by malignant neoplasm in 2016. Well-established risk factors for cardiovascular disease include hypertension, dyslipidemia, diabetes mellitus and obesity. Homocysteine (Hcy), an amino acid produced during the metabolism of methionine, is considered as an independent factor for cardiovascular disease [1].
Previous studies concluded that Hcy exerts adverse effects on endothelium and smooth muscle cells [2], resulting in endothelial dysfunction, vascular smooth muscle cells proliferation, oxidative stress, increased collagen synthesis and arterial stiffness [2]. Besides Hcy, inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP) are proposed as novel predictors for cardiovascular disease based on the concept that inflammation serves as a major role in the progression of atherothrombosis. The predictive role of hs-CRP for cardiovascular disease has been established by previous research [3]. Furthermore, oxidative stress is regarded as a contributor to cardiac and vascular abnormalities [4]. Xanthine oxidase, an enzyme participating in the uric acid synthesis, is suggested to be one of the sources of reactive oxygen species. The upregulation of xanthine oxidase and consequent elevation of uric acid is considered as a potential biomarker for cardiovascular disease [5].
We conducted a cross-sectional study to investigate the association between hyperhomocysteinemia and other cardiovascular risk factors mentioned above. The aim of this article is to provide evidence for early identification of asymptomatic populations at high risk of cardiovascular disease with Hcy and other serum biomarkers.

Subjects
This retrospective, cross-sectional study was conducted by applying data from a health examination database in a medical center located in southern Taiwan from Information from the subjects including sex, age, height (cm), weight (kg), waist circumference (cm), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were obtained by trained nurses. Body mass index (BMI) was calculated by using the formula as weight in kilograms divided by the height in meters squared.

Definition of hyperhomocysteinemia and metabolic syndrome
The normal concentration of Hcy in this study was defined as ≤ 15 µmol/L and hyperhomocysteinemia was considered > 15 µmol/L [6]. The definition of metabolic syndrome (MetS) was adopted from the National Cholesterol Education Program-Third Adult Treatment Panel (NCEP-ATP III) [7]. Subjects were diagnosed with MetS when they met any three or more of the following five components: 1) abdominal obesity (waist circumference > 90 cm in men or > 80 cm in women, the recommended criteria for Asian Americans), 2) high fasting blood glucose level (≥100 mg/dL), 3) high triglyceride level (≥150 mg/dL), 4) high SBP (≥ 130 mmHg) or DBP (≥ 85 mmHg), or 5) low HDL-C level (≤ 40 mg/dL in men or ≤ 50 mg/dL in women).

Data analysis
The data were expressed as mean ± standard deviation for continuous variables and percentages for categorical variables. The 2 -test was used for comparing categorical characteristics of subjects and the Student`s t-test was used for the comparison of continuous variables of subjects. Univariate and multivariate logistic regression were applied for analyzing the odds ratio (OR) of significant factors associated with hyperhomocysteinemia. A P value < 0.05 was considered as statistically significant. The SPSS software version 22.0 (IBM Corp., Armonk, NY, USA) was used for all analyses.

Table 1. Baseline characteristics
Subjects were categorized into two groups by Hcy level (Table 2). Of the total 878, hyperhomocysteinemia was observed in 73 subjects. In participants with elevated Hcy level, the proportion of males was significantly higher than in a normal sample of Hcy level participants (94.5% versus 34.0%, P < 0.001). Besides, participants with high plasma Hcy had significantly higher waist circumference, triglyceride, SBP, DBP, sugar AC and significantly lower HDL-C as compared with those with normal plasma Hcy. The percentage of those with metabolic syndrome in the high Hcy group was also significantly higher than in the normal Hcy group (46.0% versus 32.5%, P = 0.015). Participants with elevated Hcy were more likely to be older and have higher uric acid level than control group subjects. BMI, total cholesterol, LDL-C, hs-CRP, lipoprotein showed no significant difference between the two groups.  of 4660 Chinese in an urban community, with data that showed hyper-homocysteine state was independently related to elevated triglyceride [10]. The mechanism behind the relation between homocysteinemia and triglyceride has been proposed by several animal research studies. Experimental studies have suggested that Hcyrelated endoplasmic reticulum stress increases the expression of sterol regulatory element-binding proteins (SREBPs), which further promote cholesterol and triglyceride synthesis [11]. The result was reproduced in an animal experiment by 8 using methionine diet-fed mice, revealing elevated plasma triglyceride in increased plasma Hcy mice [12]. However, the relation between Hcy and triglyceride is not so consistent. Yadav et al. conducted a study involving 60 ischemic heart disease patients and failed to find a significant relationship between Hcy level and lipid profiles [13]. Another study enrolling 155 type 2 diabetes mellitus subjects also reported no significant association regarding hyperhomocysteinemia and high plasma triglyceride level [14].
Despite the inconsistent findings on the effect of Hcy, we provide a strong relation between elevated Hcy and triglyceride level in the current research. Some crosssectional studies discussing the association between Hcy and lipid profiles other than triglyceride showed a negative relationship with HDL-C [9, 10, 15-19] and apolipoprotein [15][16][17]20] but positive relationship with LDL-C [18]. The mechanism of Hcy related to decreased HDL-C and apolipoprotein has been demonstrated in animal models. Hcy down-regulated peroxisome proliferator-activated receptor (PPAR ) and apolipoprotein AI protein level [21]. The result caused the suppression of lecithin-cholesterol acyltransferase substrate, which led to HDL maturation inhibition [22]. Significant relationship was noted between hyperhomocysteinemia and metabolic syndrome in our simple logistic regression analysis; however, the studies discussing the connection between Hcy and metabolic syndrome showed conflicting results. Seven hundred and twenty-two patients undergoing medical checkup in Korea revealed lack of association [23]. On the contrary, Esteghamati et al. found that homocysteine was correlated with metabolic syndrome based on a prospective study composed of 5893 subjects in Iran [24]. In our multiple stepwise logistic regression analysis, only some of the components of metabolic syndrome such as triglycerides and systolic blood pressure were independently significantly 9 associated with elevated plasma homocysteine level. The differences between our analysis and prior studies were likely because of the adjusting variables we used, as our study also incorporated other cardiovascular disease risk factors including uric acid and CRP.
The connection between hyperhomocysteinemia and uric acid is explained by the methionine-homocysteine cycle [25]. In this cycle, methionine is converted to Sadenosyl-homocysteine (SAH), which becomes Hcy and adenosine, with adenosine then being degraded to uric acid. Therefore, an increase in Hcy leads to an increase in uric acid level; this result is consistent with our analysis. Similar findings were also observed in other research studies [26,27]. On the other hand, sex difference was also found to be an independent factor for elevated Hcy level in the current study, the mechanism of which might be related to the sex difference in Hcy metabolism. Normally, Hcy is metabolized by 2 different pathways: remethylation and transsulfuration [26]. A study involving 11 healthy young men and premenopausal women without cardiac risk factors conducted by Fukagawa et al.
reported a significant higher rate of remethylation and a tendency toward higher rates of transmethylation in women than in men [28]. Therefore, it is reasonable to anticipate that Hcy level is higher in men than women.
An Indian study composed of 1243 healthy subjects confirmed the sex difference by showing a significantly higher level of Hcy over women [29]. In addition, creatine is synthesized from guanidinoacetate during the formation of SAH in the methionine-Hcy cycle. Creatine and subsequent creatinine synthesis is related to muscle mass, which provides an explanation for higher Hcy levels in men than women [30].
Hormonal difference has also been proposed as a reason contributing to the different Hcy levels in males and females. A clinical study with transsexuals showed that male-to-female subjects who were treated with ethinyl estradiol and antiandrogen had significantly decreasing levels of Hcy. On the contrary, female-tomale transsexuals who underwent testosterone treatment had increased plasma Hcy [31]. The age-related elevation of Hcy could be explained by the physiologicallyassociated renal function decline. A previous study concluded that glomerular filtration rates served as an important role in Hcy and creatinine clearance [32].
Possible mechanisms regarding the relationship between Hcy and blood pressure is that Hcy induces endothelial injury and oxidative stress, which leads to impaired nitric oxide release [33], and the consequences of these effects contribute to the raise of blood pressure.
There are several limitations in the current study. Firstly, the study is retrospectively designed, so further studies are needed to determine any causation.
Secondly, the subjects were recruited from a single medical center, which might not represent the general population in Taiwan. Thirdly, participants who received medications for hypertension or hyperlipidemia were not excluded, so medication use might affect the result of our analysis.

Conclusions
Elevated serum homocysteine is associated with sex, age, uric acid and metabolic syndrome components. Among these factors, male gender, advanced age, triglyceride, systolic blood pressure and uric acid were profoundly associated with hyperhomocysteinemia. Further prospective cohort studies are needed for validating the influence on cardiovascular event prediction with homocysteine and its associates.

Ethics approval and consent to participate
The study was approved by the Chang Gung Medical Foundation Institutional Review Board. In this retrospective study, informed consent was unnecessary because the information of all participants was anonymized and de-identified.

Consent for publication
Not applicable

Competing interests
The authors declare that they have no competing interests.

Funding
No financial support was received for this project.