ISSN: 1301-2193 E-ISSN: 1308-9846
  • Turkish Journal of
    Endocrinology and Metabolism

Metabolic syndrome (MetS) is characterized by a group of metabolic and anthropometric abnormalities including excess weight, hyperglycemia, hypertension, low concentration of high-density lipoprotein cholesterol (HDL-C) and hypertriglyceridemia (1-3). Oxidative stress is known to play a major role in the pathogenesis of MetS (1,2,4). Oxidative stress means an imbalance between production of reactive oxygen species and antioxidant defense system that buffers the oxidative damage (5) and is considered to be one of the main causes of molecular damage to cellular and tissue structures (6). Serum total antioxidant status (TAS) combines the concentrations of individual antioxidants such as vitamin C and E, B-carotene, and thiol groups as well as their synergism. TAS is sensitive to the changes in plasma antioxidant levels and degrees of oxidative stress (7). Ischemia-modified albumin (IMA) measured by the albumin cobalt binding test was used originally as a marker of myocardial ischemia (8). Currently, however, IMA is regarded as a marker of oxidative stress related to ischemia-reperfusion in any organ, because it was found to be high in various clinical entities associated with oxidative stress such as ischemic stroke (9). The aim of this study was to evaluate IMA level, TAS and total oxidant status (TOS), as well as endocrine and metabolic parameters in patients with MetS and healthy controls.
 Materials and Methods
We studied 52 MetS patients (11 male, 41 female; mean age: 56.74±12.75 years) and 36 healthy subjects (36 female; mean age: 26.47±6.52 years). The patients had been referred to the Endocrinology and Metabolism Disease outpatient clinic at Diskapi Education and Research Hospital during the period between July 2009 and December 2009. The protocol was approved by the local ethics committee. All patients gave written informed consent. MetS was defined according to the revised National Cholesterol Education Program-Adult Treatment Panel III criteria (10). The individual components were as follows: waist circumference  88 cm for women or  102 cm for men; glucose level  100 mg/dL; blood pressure  130/80 mmHg or on medication for hypertension; HDL-C level <40 mg/dL (men) or <50 mg/dL (women) and triglyceride level   150 mg/dL. Smokers were excluded from the study. The MetS patients were divided into two groups according to presence of diabetes mellitus (DM). The sample size was calculated with 80% statistical power and 95% confidence interval according to the study by Chawla et al. (11). There were 24 patients in the diabetic group and 28 patients in the non-diabetic group. Oxidative stress markers and metabolic factors were compared between the groups. Diabetes mellitus definition criteria were as follows: a fasting plasma glucose level of 126 mg/dL or greater on at least two occasions; plasma glucose of 200 mg/dL or greater in 2-h 75-g oral glucose tolerance test (OGTT), or the need for insulin or an oral hypoglycemic drug to control glucose levels. The control group (n=36) consisted of healthy subjects who were admitted to Endocrinology and Metabolism Department for check-up without any systemic disorder. Their health status was determined by medical history, physical examination and complete blood chemistry. Weight and height were measured in light clothing without shoes. Body mass index (BMI) was calculated dividing the weight by square of height (kg/m2). Waist circumference was measured at the narrowest level between the costal margin and the iliac crest.
Biochemical Evaluation
Blood samples were drawn from each patient after a 12-h overnight fast for the determination of hormones, lipid profile, high-sensitivity C-reactive protein (hs-CRP), glucose, TAS, TOS and IMA levels. Plasma glucose was measured by glucose oxidase/peroxidase method (Gordion Diagnostic, Ankara, Turkey). Levels of total- cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) were determined by enzymatic colorimetric assays using spectrophotometry (BioSystems S.A., Barcelona (Spain)). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald formula.  Serum hs-CRP was determined using high-sensitivity CRP immunonephelometry (BN, Dade-Behring; Marburg, Germany).
Ischemia- Modified Albumin (IMA)
After obtaining blood samples in plain tubes containing separation gel, the samples were allowed to clot for 30 minutes and centrifuged before separating the serum.  The samples were then immediately frozen and stored at –80 °C for IMA assay. Reduced albumin cobalt binding capacity (IMA level) was analyzed using the rapid and colorimetric method of Bar-Or et al. (8).  Two hundred μL of patient serum was placed into glass tubes, and 50 μL of 0.1% cobalt chloride (Sigma, CoCl2.6H2O) in H2O was added. After gentle shaking, the solution was left for 10 minutes in order to ensure sufficient cobalt-albumin binding. Fifty microliters of dithiothreitol (DTT) (Sigma, 1.5 mg/mL H2O) was added as a colorizing agent and the reaction was quenched 2 min later by adding 1.0 mL of 0.9% NaCl. A colorimetric control was prepared. For the colorimetric control samples, 50 μL of distilled water was substituted for 50 μL of 1.5 mg/mL DTT. Specimen absorbencies were analyzed at 470 nm by a spectrophotometer (Beckman DU530, California). The color of the DTT-containing specimens was compared with that of the colorimetric control tubes. The results were reported as absorbance units (ABSUs). 
Measurement of Total Antioxidative Capacity of Plasma
The TAS of the plasma was determined using a novel automated measurement method developed by Erel (12).
Measurement of TOS
TOS of serum was determined using a novel automated measurement method as previously described (13).
Statistical Analysis
Collected data were analyzed using SPSS version 15. Continuous data were shown as mean±SD.  Normally distributed variables were compared by using the Students t-test, whereas non- normally distributed data were compared by Mann-Whitney U test. ANCOVA was used to compare adjusted means. Pearsons correlation coefficient was applied to determine associations. A  p-value lower than 0.05 was accepted as statistically significant.
Results
We examined the clinical and endocrinological parameters of both diabetic/non-diabetic patients with MetS and healthy control subjects (Table 1).  We studied 52 MetS patients (mean age: 56.74±12.75 years) and 36 healthy participants (mean age: 26.47±6.52 years). As expected, the levels of fasting blood glucose (FBG), total cholesterol, TG, LDL-C, hs-CRP, BMI, and waist circumference were significantly higher in the MetS group than in the control group (Table 2). There was no statistically significant difference between the groups in terms of IMA level, TAS and TOS (p=0.208, p=0.305, and p=0.079, respectively, Table 2). No statistically significant correlation was found between the parameters (p>0.05) in correlation analysis.
Diabetic MetS Patients 
No statistically significant difference was determined for serum IMA levels in diabetic MetS patients and control group (p=0.769). Serum TOS values were significantly lower in patients with diabetic MetS than in control subjects (p=0.003). Plasma TAS values were similar in diabetic MetS patients and control group (p= 0.99). Although median TAS and TOS values were lower in diabetic MetS patients, no significant difference was observed between diabetic and non diabetic MetS groups in terms of IMA level, TAS and TOS values (Table 3).
Age-adjusted IMA, TAS, TOS and CRP Levels
a - MetS and Control group
Age-adjusted mean IMA levels were similar between the groups (0.26±0.01 in MetS patients, 0.28±0.01 in control group (p=0.31)).  Age-adjusted TOS values were lower in the MetS group (1.29±0.17 in MetS group, 2.04±0.21 in control group (p=0.028)). Age-adjusted TAS values were similar between the groups (2.1±0.18 in MetS group, 2.1±0.25 in control group (p=0.87)).
b- Diabetic and Non-diabetic MetS Group
Age-adjusted mean IMA levels were higher in diabetic than non-diabetic MetS patients (0.27±0.011 versus 0.24±0.015, p=0.048). Age-adjusted mean TOS values were similar between diabetic and non-diabetic MetS patients (1.19±0.187 in diabetic MetS group, 1.5±0.246 in non-diabetic MetS group (p=0.243)). Age-adjusted mean TAS values were similar between diabetic and non-diabetic MetS patients (1.92±0.199 in diabetic MetS group, 2.4±0.277 in non-diabetic MetS group (p=0.086)). Median hs-CRP level was higher in diabetic patients. Age- adjusted hs-CRP values remained significantly different between diabetic and non-diabetic MetS patients (p=0.025).
Discussion
In this study, we have tried to show the changes in oxidative stress markers in MetS patients and healthy participants. To date, there are conflicting data about the association between IMA, inflammation, ischemia and metabolic parameters in risky patients (14-24). A study from Brazil has found that MetS group showed higher levels of IMA and the authors concluded that IMA levels could be associated with cardiometabolic risks and represent a possible indication of microvascular dysfunction in MetS patients (20). In our study, the mean IMA level was similar between metS and control groups. This was thought to be due to the younger age of control subjects. Kaefer et al. had shown higher levels of IMA and hs-CRP in type 2 diabetes patients and concluded that hyperglycemia and inflammation reduce the capacity of albumin to bind cobalt, resulting in higher IMA levels (22). Consistent with these results, it was found that the mean IMA and hs-CRP levels were higher in diabetic patients than non-diabetic patients in our study. However, in another study, IMA was estimated in 60 newly diagnosed type 2 DM patients and 30 healthy controls along with HbA1c and other investigations (to rule out vascular complications). There was no significant change in IMA levels in type 2 diabetic patients as compared to controls. The authors concluded that IMA levels are not affected in type 2 DM before the onset of vascular complications (23). In our study, the mean IMA level was higher in diabetic MetS patients and it was also unexpectedly higher in the control subjects than in non-diabetic MetS patients. This was thought to be due to high metabolic rate in young healthy participants. Thus, IMA was thought to be discriminative only for MetS patients with and without DM. Data suggest that CRP may promote endothelial dysfunction and complement activation and may be directly involved in the atherogenic process (25,26). Elevated CRP level, reflecting increased proinflammatory activity, is associated with increase in plaque vulnerability and propensity to thrombosis, thus, is thought to be a risk factor for future cardiovascular events (27). In our study, the mean hs-CRP level was significantly higher in MetS group than in control participants and was also higher in diabetic MetS patients than in non-diabetic MetS patients. Present data strengthen the idea that there is an increased risk of cardiovascular events in MetS group, and patients with DM have the highest risk. In our study, an oxidative stress marker IMA was not found to be significantly different between patients with MetS and healthy controls. Other markers, TAS and TOS, were similar between the groups. Presence of DM in MetS patients was associated with higher age-adjusted IMA levels. Absence of difference between diabetic patients and controls may be caused by higher IMA levels due to higher metabolic rate in younger subjects, and this study suggests that IMA cut-offs should be determined according to patients age. There are few studies about the association between MetS and TAS/TOS values. One of these includes psoriatic MetS groups.  In that study, patients with plaque-type psoriasis with (n=25) or without (n=27) MetS has been matched for age and sex to an equally sized control group (n=25). As a result, serum TAS and TOS values have not been statistically significant in any of the three groups (28). In our study, there was no significant difference between the groups in terms of TAS and TOS values. In recent study, TAS was significantly lower in both MetS and obese groups compared to controls. It was concluded that oxidative stress is altered in non-diabetic MetS and non-MetS obese patients, but paraoxonase and arylesterase activities seem not to be affected and this result may be due to the absence of diabetes, the most severe form of altered carbohydrate metabolism (29). In the present study, the mean TAS was highest in the non-diabetic MetS group. This data strengthen the idea that TAS activity seems to be affected by the carbohydrate metabolism, so TAS is reduced in diabetic patients. The mean TAS value was also found to be lower in the control group in our study. It may be due to the younger age of control participants. This finding suggests that TAS increases with age, but presence of diabetes inhibits this increase.  Further studies are needed to show the association between oxidative stress markers, DM, cardiovascular diseases and MetS. The correlation between duration of MetS, severity of metabolic disorders in MetS, oxidative stress and cardiovascular risk are needed to be shown in further studies. 
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