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Twenty healthy japanese male wrestlers (18-22 years of age) who participated in the national collegiate ... in, a day after weigh-in (competition day), and 9 days.
Endocrine Journal 2010, 57 (4), 339-345

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Oxidative Stress Associated with Rapid Weight Reduction Decreases Circulating Adiponectin Concentrations Yoshimaro Yanagawa1), 2), Tadashi Morimura1), Katsuhiko Tsunekawa1), Koji Seki1), Takayuki Ogiwara1), Nobuo Kotajima1), Tetsuo Machida1), Shingo Matsumoto3), Takumi Adachi3) and Masami Murakami1) 1)

Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine, Maebashi 371-8511, Japan Ikuei Junior College, Takasaki 370-0011, Japan 3) Graduate School of Health and Sport Science, Nippon Sport Science University, Yokohama 227-0033, Japan 2)

Abstract. The effect of stress associated with acute weight reduction on adipocytokine production is incompletely understood. In the present study, we have investigated the changes in circulating adipocytokine concentrations and urinary concentrations of stress markers in male collegiate wrestlers during acute weight reduction for a competition. Twenty healthy Japanese male wrestlers (18-22 years of age) who participated in the national collegiate wrestling tournament were studied. Body weight, body fat amount, serum testosterone, serum leptin, serum adiponectin, urinary 8-hydroxy-2’deoxyguanosine (8-OHdG) and urinary biopyrrins were analyzed during acute weight reduction for the competition. Body weight, body fat amount and the serum concentrations of testosterone, leptin and adiponectin significantly decreased on the day of weigh-in compared with the levels 12 days before weigh-in. In contrast, urinary concentrations of 8-OHdG and biopyrrins significantly increased on the day of weigh-in compared with the concentrations 12 days before weigh-in. A positive correlation was observed between the serum concentrations of adiponectin and testosterone, and a negative correlation was observed between the concentrations of serum adiponectin and urinary biopyrrins. The present results suggest that rapid weight reduction increases the urinary concentrations of stress markers, which is associated with a decrease in serum concentrations of adiponectin. Key words: Adiponectin, Leptin, 8-OHdG, Biopyrrins

Adipose tissue is not merely a fat storage depot, but it has also been recognized as an endocrine organ capable of producing adipocytokines [1]. Leptin, a well-known adipocytokine produced mainly by white adipose tissue, correlates with fat mass and plays a pivotal role in regulating energy homeostasis and insulin sensitivity [2-5]. Adiponectin, another intensively studied adipocytokine produced exclusively by white adipose tissue [6, 7], inversely correlates with body mass index (BMI) and abdominal fat mass [8]. Adiponectin exerts direct actions on liver, skeletal muscle and cardiovascular system, with prominent roles to improve insulin sensitivity, increase fuel oxiReceived Dec. 16, 2009; Accepted Jan. 15, 2010 as K09E-359 Released online in J-STAGE as advance publication Feb. 24, 2010

Correspondence to: Masami Murakami, Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi 371-8511, Japan E-mail: [email protected]

dation and decrease vascular inflammation [9]. Athletes with weight categories reduce their body weight immediately before a competition and then gain body weight afterward. In this regard, athletes with weight categories, such as amateur wrestlers, presumably suffer from physical and mental stress associated with rapid weight loss. However, the influence of stress associated with rapid weight reduction on physiological functions remains incompletely understood. Recent studies have demonstrated that unstable mental states were observed using the Profile of Mood States test in wrestlers during weight loss for a competition [10]. In addition to the changes in mental states, testosterone concentrations have been reported to decrease as induced by weight loss in young wrestlers during the developmental stage period [11]. It is known that extreme psychological stress is associated with a decrease in serum testosterone concentrations [12], and soldiers participating in a physically and psy-

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chologically stressful training exercise have been reported to show decreased serum testosterone concentrations [13]. Reactive oxygen species (ROS), including superoxide anion radical, hydrogen peroxide and hydroxyl radical are formed continuously in living cells as a consequence of metabolic reactions. Under normal physiological conditions, there is a balance between endogenous oxidants and various antioxidant defenses. The excessive generation of oxidants or a decrease of antioxidants results in an imbalance called oxidative stress. In the presence of oxidative stress, oxidants generated in vivo produce extensive oxidative damage to nucleic acids as well as lipids and proteins. As a result, DNA is constantly damaged and oxidatively modified. 8-hydroxy-2’-deoxyguanosine (8-OHdG) is one of the predominant forms of the oxidative lesions in DNA, which can be measured in human urine. 8-OHdG has been widely used as a biomarker for assessing oxidative DNA damage [14-16]. Bilirubin, a cytotoxic waste product excreted into the bile, has been shown to play a potent antioxidant role in vivo, and is now considered as an important scavenger of ROS [17]. Following oxidation, bilirubin forms hydrophilic metabolites, designated biopyrrins. Biopyrrins have been identified in human urine using anti-bilirubin monoclonal antibodies [18], and are considered to represent markers of oxidative stress [19]. Urinary biopyrrins concentrations are known to increase during surgical stress [20], ischemic heart disease [21], heart failure [22] and psychological stress [23]. Although the relationship between adipocytokines and oxidative stress remains to be elucidated, recent studies have demonstrated that oxidative stress in adipose tissue induces the dysregulated production of adipocytokines including adiponectin [24]. In the present study, we have investigated the changes in circulating adipocytokine concentrations and urinary concentrations of stress markers in male collegiate wrestlers during acute weight reduction for a competition.

Materials and Methods Participants and weight reduction The subjects were 20 healthy male Japanese amateur wrestlers (18-22 years of age) who participated in a national collegiate wrestling tournament in Japan. The subjects represented all the major weight class-

es of wrestling. Twelve days before weigh-in for the competition, they started to reduce food intake. They practiced twice a day regularly including the weight losing period. The morning practice was aerobic training with 4 km running between 0700h and 0800h. The evening practice was training on a wrestling technique and sparring for 2 hours starting at 1630h. Written informed consent to participate in the present study was obtained from all the subjects and the study protocols were approved by the institutional review board. Study protocols and laboratory examinations Body weight, body fat amount, testosterone, stress markers and adipocytokines of the wrestlers were evaluated at a total of five time points: 12 days before weigh-in, 4 days before weigh-in, the day of weighin, a day after weigh-in (competition day), and 9 days after weigh-in. Body fat amount was measured by the bioelectrical impedance method using Physion XP (Physion Inc., Kyoto Japan). Urine and venous blood samples were collected in the morning before the scheduled practice or competition. Urine samples were protected from light and stored at -80 °C until measurement. Serum samples were stored at -30°C until measurement. Urinary creatinine concentrations and serum testosterone concentrations were measured using commercially available kits and automated analyzers at Clinical Laboratory Center of Gunma University Hospital. Measurement of urinary 8-OHdG and biopyrrins concentrations Urinary 8-OHdG concentrations were measured in duplicate using a competitive enzyme linked immunosorbent assay (ELISA) kit (8-OHdG check, Japan Institute for the Control of Aging, Shizuoka, Japan). The intra-assay coefficient of variation (CV) and interassay CV were 1.8% and 4.0%, respectively. The determination range was 0.5 to 200 ng/mL. Data were corrected with the urinary concentrations of creatinine (mg/dL) and expressed as urinary 8-OHdG (ng/mgCr). Urinary biopyrrins concentrations were measured in duplicate using a biopyrrins enzyme immunoassay kit (Shino-test Co., Tokyo, Japan) [18]. The intra-assay CV and inter-assay CV were 3.4% and 8.5%, respectively, and the sensitivity was 0.1 μmol/L. Data were corrected with the urinary concentrations of creatinine (mg/dL) and expressed as urinary biopyrrins (μmol/gCr).

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Table 1. Changes in body weight, BMI, Body fat amount and concentrations of serum testosterone, urinary 8-OHdG, urinary biopyrrins, serum adiponectin and serum leptin of 20 wrestlers at a total of five time points: 12 days before weigh-in, 4 days before weigh-in, the day of weigh-in, a day after weigh-in (the day of competition) and 9 days after weigh-in. -12 days -4 days Weigh-in +1 day +9 days Body weight kg 73.0±11.7 71.8±12.0* 69.3±12.8** 71.9±12.1* 72.8±11.0 25.2±2.8 24.8±2.8* 23.9±3.0** 24.8±2.8* 25.2±2.5 BMI kg/m2 Body fat amount kg 8.6±5.0 8.5±5.0 6.5±4.7** 6.7±4.5** 7.1±4.5** Testosterone ng/mL 6.77±1.53 5.80±1.60* 4.45±1.91** 4.96±1.63** 7.01±1.69 8-OHdG ng/mgCr 5.76±1.45 9.76±3.47** 11.38±2.71** 9.77±2.46** 12.77±3.90** Biopyrrins μmol/gCr 1.23±0.65 1.41±0.71 2.00±1.12** 1.99±1.05* 1.21±0.65 Adiponectin μg/mL 3.14±0.68 2.91±0.86 2.17±0.51** 2.31±0.63** 3.85±1.95 Leptin pg/mL 219.3±309.7 107.8±161.8* 75.6±130.0* 165.2±183.5 251.7±210.9 Data are expressed as mean ± SD, *p