Elmer Original Article J Clin Med Res • 2011;3(5):247-251 ress The Association Between Reactive Oxygen Metabolites and Metabolic Syndrome in Asymptomatic Japanese Men

Background The association between the oxidative status and metabolic syndrome (MetS) should be studied in various populations with various oxidative stress-related markers. The aim of this cross-sectional study was to investigate the association between oxidative status, as assessed by the reactive oxygen metabolites (d-ROMs) test, and MetS in asymptomatic Japanese men, in relation to age. Methods The serum d-ROMs levels were measured in cardiovascular disease-free, non-smoking, non-medicated males (n = 140), who were divided into groups as follows: Group 1, < 60 years (n = 75, mean age 46 ± 9 [SD] years), and Group 2, ≥ 60 years (n = 65, mean 68 ± 6 years). The MetS was determined by the NCEP-ATP recommendations with minor modifications for a Japanese population. Results There was no significant difference in the d-ROMs levels between the subjects with and without MetS in Group 2 (≥ 60 years), but the subjects with MetS (n = 38, 324 ± 59 U. Curr.) exhibited significantly higher d-ROMs levels than those without MetS (n = 37, 290 ± 49 U. Curr., P < 0.01) in Group 1 (< 60 years). These differences did not change even after adjustments for basic confounders. Conclusions These results suggest that oxidative status, as assessed by the d-ROMs, can be enhanced among asymptomatic younger, but not older, Japanese males with MetS. Further studies are required to establish the observed associations. Keywords Oxidative stress; Reactive oxygen species; D-ROMs; Obesity; Metabolic syndrome


Introduction
When cardiometabolic risk factors such as obesity, hyperglycemia, dyslipidemia and hypertension are clustered, cardiovascular disease (CVD) risks are remarkably increased [1]. While metabolic syndrome (MetS) represents a clustering disorder of such risk factors, a deeper understanding of the underlying mechanisms of the contribution of MetS to CVD is needed to control CVD events [2]. One of the major pathophysiologies of MetS is an oxidative condition caused by the overproduction and inactivation of reactive oxygen species (ROS) [3]. Although numerous studies have examined oxidative status in MetS subjects using oxidative stress-related markers, these results are not always consistent [4][5][6][7][8][9][10][11][12]. This can partly be due to differences in not only the studied populations but also in the markers used; namely, we note the weaker associations in healthier relative to diseased populations [4,7,11] and a different signifi cance of markers used on MetS traits is reported even in the same population [10]. Recently, the reactive oxygen metabolites (d-ROMs, Diacron, Italy) test was developed to quantify oxidative status by measuring the hydroperoxidation of organic compounds (occurring in lipids, proteins, nucleic acids, etc.) [13,14], and is also used in medical settings [15][16][17]. However, the literature on determining oxidative status using this test in MetS subjects is rare.
Another debatable point is a difference in the clinical relevance of MetS on CVD between genders and age; namely, there may be a higher CVD risk in men with MetS than women [18] and minimal MetS-CVD linkage in relatively older populations [12,19,20]. Therefore, the aim of this study was to investigate the association between the levels of d-ROMs and MetS properties in an asymptomatic Japanese Manuscript accepted for publication August 16, 2011 a male population, in considering different age groups.

Methods
A total of 140 non-smoking, non-medicated male participants were recruited during routine check-ups in health education classes and outpatient clinics. This population was divided into two groups by age: Group 1, < 60 years of age and Group 2, ≥ 60 years of age (Table 1). Eligible subjects had no histories of cardiovascular, thyroid, hematological, renal, hepatic, or collagenous diseases. The presence of MetS was diagnosed with at least 3 out of 5 of the following criteria according to the National Cholesterol Education Program Adult Treatment Panel (NCEP-ATP) III recommendations [2]: 1) obesity identifi ed by body mass index (BMI) ≥ 25.0 kg/m 2 as a surrogate for a Japanese population [21]; 2) increased blood pressure (BP) identifi ed by systolic BP (SBP) ≥ 130 mmHg and/or diastolic BP (DBP) ≥ 85 mmHg; 3) hypertriglyceridemia identifi ed by serum triglyceride (TG) ≥ 1.69 mmol/L; 4) low high-density lipoprotein cholesterol (HDL-C) identifi ed by serum HDL-C < 1.04 mmol/L, and 5) hyperglycemic status identifi ed by a fasting plasma glucose (FPG) ≥ 6.1 mmol/L. The study was approved by the institutional ethics committee, and all subjects gave their informed consent.
In addition to BMI, the SBP and DBP levels were determined in the seated subject's right-arm with a mercury sphygmomanometer. Blood was sampled after an overnight fast with no alcohol consumption or exercise during the preceding day. The serum TG and FPG levels were measured using enzymatic methods, and the serum low-density lipoprotein (LDL-C) and HDL-C levels were measured using homogeneous methods (Sekisui Co. Ltd., Tokyo, Japan).
The d-ROMs values were obtained using a kinetic spectrophotometric assay (F.R.E.E system; Diacron, Italy) with the intra-and inter-assay coeffi cients of variation of 2.1% and 3.1%, respectively [13,14]. Briefl y, serum samples were mixed with a buffered solution, and a chromogenic substrate was added into the mixture. The mixture was centrifuged and then incubated in the thermostatic block of the system. The absorbance was then recorded at 505 nm. Measurements are expressed as U. Carr., and 1 U. Carr. corresponds to 0.08 mg/dL H 2 O 2 (the reference value is suggested to be approximately 250 -300 U. Carr.).
Data are expressed as the means ± SD or medians plus interquartile range. Differences between two groups were analyzed using the unpaired t-test. The infl uence of MetS (as a fi xed variable) on d-ROMs (as a dependent variable) was examined using a general linear model analysis with adjustments for basic confounders (i.e., age or LDL-C that is not included in the diagnostic criteria of MetS but can contribute to the development of CVD and oxidative status). The values of TG were log-transformed for the analyses because of the skewed distribution. A P-value of < 0.05 was considered to be statistically signifi cant.

Results
The clinical characteristics between age-stratifi ed groups are shown in Table 1. Group 2 (≥ 60 years) showed signifi cantly higher age and SBP levels and a lower BMI level than Group 1 (< 60 years). Although Group 2 had high FPG and d-ROMs levels relative to Group 1, these differences did not reach statistically signifi cant levels.
The prevalence of MetS was 51% and 57% in Groups 1 and 2, respectively, as shown in Table 2. There were no sig- nifi cant differences in the d-ROMs levels between each criterion of MetS in either group (Table 2). However, while there was no signifi cant difference in the d-ROMs levels between the subjects with and without MetS in Group 2 (P = 0.26), the subjects with MetS exhibited a signifi cantly higher d-ROMs level than those without MetS in Group 1 (P < 0.01). The difference in the d-ROMs levels by MetS remained statistically signifi cant, even after adjusting for age (F = 7.46, P < 0.01) and for age and LDL-C (F = 11.11, P < 0.01).

Discussion
The present study revealed a signifi cantly higher d-ROMs level in Japanese male subjects with MetS than those without MetS in Group 1 (< 60 years), but not Group 2 (≥ 60 years). It would therefore be valuable to add new fi ndings to our current knowledge regarding the association between MetS and oxidative status, by using the d-ROMs test that differed from the test described in previous studies [4][5][6][7][8][9][10][11][12].
The fi rst notable fi nding of this study is confi rmation that, although no signifi cant differences in the d-ROMs levels were observed between each criterion of MetS, MetS clearly expressed the hyperoxidative status of the d-ROMs. This is in agreement with the clustering concept of MetS (a disorder clustering of each criterion) to remarkably increase the CVD risks [1,2]. Fat and systemic ROS, which stem from obese adipocytes and unfavorable lifestyles, are associated with the augmentation of NADPH-oxidase and suppression of antioxidative enzymes, and these pathways can directly lead to the initiation/development of MetS via cell damage and indirectly via dysregulated adipocytokines [3,22]. The progression/exacerbation of the MetS critera further promotes a vicious circle with the addition of oxidative stress induced at atherosclerotic sites [3]. The biological mechanisms can account for the present fi nding of a signifi cant increase of the d-ROMs levels in MetS subjects. In addition, although the previous studies suggested a signifi cant increase of the d-ROMs in hypertensive subjects [15] and obese subjects [17], weak differences in the d-ROMs between solo criterion groups observed in the present study might partially be explained by the differences in studied populations (the present study population was of a relatively good state and/or mild state, even though the subjects showed the diseased levels, as seen in Table 1).
The second notable fi nding of this study is confi rmation that a signifi cant increase of the d-ROMs levels in MetS was clearly observed in the younger Group 1, rather than the older Group 2. Previous studies indicated weak magnitudes of MetS on CVD in elderly populations [19,20], while another study reported a signifi cant positive relationship between MetS and oxidative status in an elderly population [12]. Although the precise reasons for these fi ndings remain to be unclear, our present study seems to support the former studies [19,20]. Because aging per se is an oxidative condition (in fact, Group 2 tended to show somewhat high d-ROMs levels relative to Group 1, as seen in Table 1) [23], it may be diffi cult or confusing to distinguish clearly the oxidative status between pathologies in older subjects. This seems to be an important view to control MetS-related CVD events, if the potential magnitudes of MetS on CVD can differ by age. The possibility that the infl uence of MetS on CVD can be modulated by age merits further investigations. The present study had a few limitations. The cross-sectional design did not allow for determining the cause-andeffect relationship. The study was conducted in male participants only and in relatively small sample sizes. Future studies with larger populations including females and prospective/interventional designs are needed.
In summary, the present study showed signifi cantly higher d-ROMs levels in asymptomatic Japanese male subjects with MetS than those without MetS in Group 1 (< 60 years), but not Group 2 (≥ 60 years). The oxidative status can be enhanced under the clustering concept of MetS in younger men in particular, which may imply an age-modulated effect of MetS on CVD. Further studies are necessary to establish the observed associations.

Confl ict of Interest
None