Association between Serum Zinc and hs-CRP Concentrations in Different Metabolic Syndrome Phenotypes

Background: Metabolic syndrome (MetS) is associated with an increased risk of cardiovascular disease and all-cause mortality. Zinc (Zn) is an essential trace element for the synthesis, storage, and release of insulin. The relationship between serum Zn and MetS is controversial. In this study we have evaluated whether serum Zn levels are associated with hsCRP level according to MetS phenotypes. Method: A total of 9493 individuals (3768 men and 5635 women) were recruited as part of the Mashhad Stroke and Heart Association Disorder (MASHAD) study. We measured the concentration of serum Zn by ame atomic absorption (Varian AA240FS) and we divided the subjects into quartile of serum Zn. We used SPSS version 18 statistical analyses for all. GraphPad Prism 6 for gures was used. Results: In this study there were signicant differences between quartiles of Zn according to sex. The results showed that the level of serum hs-CRP were higher in subjects with metabolic syndrome who also had a serum Zn >95 µg/dl. In subjects with serum Zn <70 µg/dl, an increase in serum hs-CRP was associated with an increased risk of MetS by 2.2%, 1.022(CI: 1.01-1.034). Moreover, among subjects in the rst, second and third quartiles of serum Zn, some phenotypes of metabolic syndrome (WHB, WHT, WTG, WTB, WHBG, WHTG, and WTBG) were associated with a risk of increasing hs-CRP level (p< 0.05). Conclusion: Serum hs-CRP concentrations were related to MetS phenotypes. This relationship was modied by serum Zn level. According to the number of MetS components serum Zn levels decreased as the number of MetS component increased, and low serum zinc levels (Q1) showed an overall greater prevalence of MetS.


Introduction
Metabolic syndrome (MetS), a cluster of metabolic risk factors including glucose intolerance, atherogenic dyslipidemia, elevated blood pressure and abdominal obesity, is associated with an increased risk of cardiovascular disease (CVD) and all-cause mortality (1)(2)(3). The prevalence of MetS has been increasing rapidly worldwide (4) and this has become a major medical concern. Zinc (Zn) is an essential trace element, involved in the synthesis, storage, and release of insulin (5,6). This mineral is also a fundamental component required for the synthesis of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase (7). Therefore, Zn de ciency may induce abnormal insulin metabolism and oxidative stress, which are important factors for the pathophysiology of diabetes and metabolic syndrome (MetS) (8-10).
The relationship between serum Zn and MetS is controversial. Higher levels of dietary Zn have been reported to be protective against the development of MetS in speci c populations (11,12). An inverse relationship between serum Zn concentration and MetS was also reported in women (13). In contrast, a longitudinal study reported that higher serum Zn concentration are a predictive factor for MetS (14).
The potential role of oxidative stress and chronic in ammation in MetS has also been reported, and increased oxidative stress or the presence of chronic in ammation may affect the development of MetS (15)(16)(17). Despite the critical roles of insulin resistance and/or oxidative stress and chronic in ammation in MetS pathogenesis (16,17) and the functions of Zn related to insulin resistance (7,8), oxidative stress (9), or chronic in ammation (10,13), studies on the link between MetS and body zinc status are scarce and the results are controversial. Furthermore, to our knowledge the association between MetS phenotypes and serum Zn levels is con icting. Therefore, we evaluated whether or not serum Zinc levels are associated with hsCRP level according to MetS phenotypes.

Study population
In this study 9403 individuals (3768 men and 5635 women) were investigated as part of the Mashhad Stroke and Heart Association Disorder (MASHHAD) study. They were initially selected using a random clustering method during the period of 2007-2008, as previously described (18). The full agreement of all participants was taken using the protocols approved by the Mashhad University of Medical Sciences (MUMS) Ethics Committee. The criteria for entering and leaving the study of MASHAD and the general characteristics of sample population such as marital and occupational status, educational level, drug use, biochemical measurements and anthropometrics was reported previously (18). Several biochemical characteristics of participants were measured at baseline. Then serum samples were aliquoted into 0.5 mL tube and stored at -80 o C. The subjects were then divided into groups with or without MetS, de ned by the IDF criteria (19). In this study we divided participants into groups, metabolic syndrome (3398 (28.4%)) and healthy (6005 (51.4%)) subjects, then we measured serum Zn in all subjects. We were also able to de ne eleven metabolic syndrome phenotypes based on these criteria: WHG (waist circumference, HDL, glucose), WHB (waist circumference, HDL, blood pressure), WHT (waist circumference, HDL, TG), WBG (waist circumference, blood pressure, glucose), WTG (waist circumference, TG, glucose), WTB (waist circumference, TG, blood pressure), WHBG (waist circumference, HDL, blood pressure, glucose), WHTG (waist circumference, HDL, TG, glucose), WHTB (waist circumference, HDL, TG, blood pressure), WTBG (waist circumference, TG, blood pressure, glucose) and WHTBG (waist circumference, HDL, TG, blood pressure, glucose) according to IDF criteria.

Demographic, anthropometric and metabolic data
For all the participants, systolic blood pressure (mm Hg), diastolic blood pressure( mm Hg), serum high-density lipoprotein cholesterol( mg/dl), triglycerides ( mg/dl), blood glucose(mg/dl), waist circumference(cm), highsensitivity C-reactive protein (mg/l) and zinc intake(mg/day) were measured. Waist circumference was measured with a tape measure to the nearest millimeter. Systolic and diastolic blood pressure (SBP and DBP) was measured twice using the same standard sphygmomanometer. Biochemical parameters such as high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), high-sensitivity C-reactive protein (hsCRP) and serum glucose were determined by the methods describe previously(18)

Measurement of serum zinc
Sera were diluted with nitric acid 1.5% at the ratio of 1:10. The concentration of serum Zn was measured with Flame atomic absorption (Varian AA240FS). Zn standard curves were made using Zn standard (Merck and Pharmacy Company). The accuracy of the methods for Zn was 93 ± 4.8%, which was evaluated through measuring the con rmed reference material (Merck KGaA 64271 Darmstadt, Germany) comprising known values (1000 ± 2 mg / L) Zn. Also for Zn the intra-assay and inter-assay coe cient of variation (CV) were 1.5 ± 0.2% and 2.6 ± 0.4%.
Participants were divided into different groups based on serum zinc. Serum zinc was de ned as quartiles and based on this we investigated various factors in different zinc groups.

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All statistical analyses were undertaken using SPSS version 18(SPSS Inc. Chicago, IL, USA). The normality of the data was assessed using the Kolmogorov-Smirnov test. Descriptive statistics including mean, frequency, and standard deviation (SD) were de ned for all variables and expressed as mean ± standard deviation (SD) for variables with normally distribution or median ± IQR for not normally distributed variables. One-Way ANOVA or Kruskal-wallis H was determined. Bonferonni corrections were made for multiple comparisons. Chi-square or Fisher exact tests were used to evaluate the categorical parameters. Logistic regression analysis with sex included as model covariates was used to evaluate the relation between increasing hs-CRP level in MetS according to zinc quartiles. All the analyses were two-sided and P values less than or equal to 0.05 were considered signi cant.
GraphPad Prism 6 for gures was used.

General characteristics of the subjects
Of the total group (9493), 3768 were male and 5635 were female, and there were signi cant differences between the quartiles of zinc according to sex (p < 0.05) ( Table 1). µg/dl)), 47.82 ± 7.81 (Q3 (78-95 µg/dl)) and 47.23 ± 6.91(Q4 (> 95 µg/dl)), respectively. The results indicated that in subjects who had serum zinc between 78 and 95. DBP and HDL-C was higher and there was a signi cant difference between these groups (p < 0.05). However, in subjects with serum zinc > 95 µg/dl waist circumference was lower and hs-CRP was signi cantly higher than other groups (p < 0.05) ( Table 1). Figure 1, shows serum hs-CRP level for the individuals with and without metabolic syndrome according to serum Zn quartiles. The second and third quartiles were merged because they had similar values and were within normal range. The serum hs-CRP was higher in subjects with MetS in forth quartile (zinc > 95 µg/dl) (p < 0.05) (Fig. 1).

Serum hs-CRP level according to Zinc Quartiles in Metabolic Syndrome
Serum hs-CRP levels in WHB, WBG, WTB, WHTB, WTBG and WHTBG phenotypes of metabolic syndrome was signi cantly higher versus other phenotypes (p < 0.05) ( Table 2) in the rst quartile; in second and third quartile WTB, WHTG, WHTB and WHTBG had the higher level of hs-CRP (p < 0.05); and in forth quartile WHT had the higher level of hs-CRP (p < 0.05) ( Table 2).

Risk of increasing hs-CRP level according to Zinc Quartiles in Metabolic Syndrome
In subjects with a serum zinc < 70 µg/dl, an increase in serum hs-CRP was associated with an elevated risk of MetS (an increase of one unit (1 mg/l) increased the chance of MetS by 2.2% time (OR: 1.022 (CI: 1.01-1.034)) (p < 0.05), subjects with zinc between 70-95 µg/dl, an increase of serum hs-CRP by one unit (1mg/l) can increase the chance of getting metabolic syndrome by 1.1% time (OR: 1.011 (CI: 1.006-1.019) (p < 0.05) in serum hs-CRP level in metabolic syndrome (Fig. 2).
Moreover, among subjects in the rst quartile of serum Zn, some phenotypes of metabolic syndrome (WHB, WHT, WTG, WTB, WHBG, WHTG, and WTBG) were associated with a risk of increasing hs-CRP level (p < 0.05) ( Table 3). In the second and third quartiles of zinc WHB, WHT, WBG, WTB, WHTG, WTBG, and WHTBG and in the fourth quartile WHG had risk of rising hs-CRP level in metabolic syndrome (p < 0.05) ( Table 3).  (20). In agreement with our study, Jin-A Seo showed that with increasing metabolic syndrome components, serum Zn levels fell (21). Also, Freitas et al showed that serum zinc and zinc uptake in subjects with MetS are lower than healthy subjects and they had adequate zinc and moreover, an increase urinary zinc excretion (22).
Mean serum zinc in a population may re ect the status of dietary zinc intakes, and could be used as an indicator of zinc de ciency of the population (23).
The relationship between serum Zn and other metabolic parameters is controversial. Several studies have evaluated the association of MetS with serum Zn concentrations. In a few studies, men and women with MetS had a higher and lower level of serum zinc than those without, respectively (1, 3, 23). In contrast, other studies reported no relationship between serum zinc concentration and MetS (1, 2). Other factors not included in the clinical de nition of MetS, such as chronic in ammation or oxidative stress, may lead to the development of MetS (15,16). In ammatory cytokines released tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), stimulate C-reactive protein production in the liver, during the MetS (17).
Zn a cofactor for antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase, decrease reactive oxygen species(ROS) generation and induces metallothionein, which decreases the burden of ROS (1), suggesting that a decrease in body zinc status may contribute to the development of MetS. In addition, chronic in ammation or oxidative stress may contribute to the decreased serum zinc levels.
The relationship between serum Zn and hs-CRP with MetS was investigated in previous studies, but the strength of this current study was the sample size, and the assessment of serum Zn and hs-CRP in different MetS phenotypes.
Although, one of the limitation of this study is the cross-sectional, design so we need more cohort study to con rm decreasing zinc level can elevated hs-CRP concentration according to metabolic syndrome components.
Declarations Figure 1 serum hs-CRP level according to serum zinc quartiles in individuals with Metabolic Syndrome; (Negative: No Metabolic Syndrome, Positive: Metabolic Syndrome); Mann-Whitney test has been done; * p< 0.01