The synergistic stimulating effect of combined intake of carbohydrate and protein on plasma insulin concentration has been reported previously. However, it remains unclear whether the amount of protein ingested after exercise affects the concentrations of plasma insulin and amino acids. This study of trained men compared the effects of post-exercise co-ingestion of carbohydrate plus different amounts of whey protein hydrolysates (WPHs) with carbohydrate alone on (1) blood biochemical parameters of carbohydrate metabolism during the post-exercise phase, and (2) endurance performance. Eight trained men exercised continuously for 70 min. Immediately after exercise and 30, 60, 90, and 120 min later, the participants received supplements containing: (1) 17.5 g carbohydrate, (2) 3.0 g WPHs and 17.5 g carbohydrate (L-WPH), or (3) 8.0 g WPHs and 17.5 g carbohydrate (H-WPH). After a 2-h recovery period, the participants performed an endurance performance test. The concentrations of blood glucose were lower and plasma insulin significantly higher in the H-WPH trial compared with the carbohydrate trial. The concentrations of plasma amino acids were increased in a dose-dependent manner following ingestion of different amounts of WPHs with carbohydrate. Endurance performance was not significantly different between the three trials. Co-ingestion of carbohydrate and H-WPH was more effective than ingestion of carbohydrate alone for stimulating insulin secretion and increasing the availability of plasma amino acids. These results suggest that plasma concentrations of amino acids during the recovery period are determined by the amount of dietary protein ingested, and that it is necessary to increase the concentration of plasma amino acids above a certain level to stimulate insulin secretion.

The present study elucidated the effects of habitual rowing exercise on arterial stiffness and plasma levels of the vasoconstrictor endothelin-1 and the vasodilator nitric oxide (NO) in older men. Eleven rowers (68.0 +/- 1.6 years) and 11 sedentary control older men (64.9 +/- 1.1 years) were studied. Peak oxygen uptake (36.0 +/- 1.7 vs. 27.7 +/- 1.9 ml . kg(-1) . min(-1)), leg press power (1346 +/- 99 vs. 1077 +/- 68 W), and HDL-cholesterol (75 +/- 5 vs. 58 +/- 3 mg . ml(-1)) were higher and triglyceride (78 +/- 9 vs. 120 +/- 14 mg . ml(-1)) was lower in rowers than in control participants (all P < 0.05). Arterial stiffness indices (carotid beta-stiffness and cardio-ankle vascular index) and plasma endothelin-1 and NOx (nitrite+nitrate) levels did not differ between the two groups. These results suggest that habitual rowing exercise in older men is associated with high muscle power and aerobic capacity, and favourable blood lipid profile without affecting arterial stiffness or plasma levels of endotheline-1 and NO.

The synergistic stimulating effect of combined intake of carbohydrate and protein on plasma insulin concentration has been reported previously. However, it remains unclear whether the amount of protein ingested after exercise affects the concentrations of plasma insulin and amino acids. This study of trained men compared the effects of post-exercise co-ingestion of carbohydrate plus different amounts of whey protein hydrolysates (WPHs) with carbohydrate alone on (1) blood biochemical parameters of carbohydrate metabolism during the post-exercise phase, and (2) endurance performance. Eight trained men exercised continuously for 70 min. Immediately after exercise and 30, 60, 90, and 120 min later, the participants received supplements containing: (1) 17.5 g carbohydrate, (2) 3.0 g WPHs and 17.5 g carbohydrate (L-WPH), or (3) 8.0 g WPHs and 17.5 g carbohydrate (H-WPH). After a 2-h recovery period, the participants performed an endurance performance test. The concentrations of blood glucose were lower and plasma insulin significantly higher in the H-WPH trial compared with the carbohydrate trial. The concentrations of plasma amino acids were increased in a dose-dependent manner following ingestion of different amounts of WPHs with carbohydrate. Endurance performance was not significantly different between the three trials. Co-ingestion of carbohydrate and H-WPH was more effective than ingestion of carbohydrate alone for stimulating insulin secretion and increasing the availability of plasma amino acids. These results suggest that plasma concentrations of amino acids during the recovery period are determined by the amount of dietary protein ingested, and that it is necessary to increase the concentration of plasma amino acids above a certain level to stimulate insulin secretion.

Objective: Depletion of glycogen stores is associated with fatigue during both sprint and endurance exercises and therefore it is considered important to maintain adequate tissue stores of glycogen during exercise. The aims of the present study in rats were therefore to investigate the effects of preexercise supplementation with carbohydrate and whey protein hydrolysates (WPH) on glycogen content, and phosphorylated signaling molecules of key enzymes that regulate glucose uptake and glycogen synthesis during exercise.
Methods: Male SD rats were used in the study (n = 7/group). Prior to exercise, one group of rats was sacrificed, whereas the other groups were given either water, glucose, or glucose plus WPH solutions. After ingestion of the test solutions, glycogen-depleting exercise was carried out for 60 min. The rats were then sacrificed and the triceps muscles excised quickly.
Results: Compared to water or glucose only, preexercise ingestion of glucose plus WPH caused a significant attenuation of muscle glycogen depletion during the postexercise period. Coingestion of glucose and WPH also significantly lowered phosphorylated glycogen synthase levels compared to ingestion of water only. In the glucose plus WPH group, the levels of phosphorylated Akt were increased significantly compared to the group ingesting water only, while the levels of phosphorylated PKC were significantly higher than in the groups ingesting only water or glucose.
Conclusion: Taken together, these results indicate that, compared to ingestion of glucose or water only, preexercise ingestion of carbohydrate plus WPH activates skeletal muscle proteins of key enzymes that regulate glucose uptake and glycogen synthesis during exercise, thereby attenuating exercise-induced glycogen depletion. (C) 2011 Elsevier Inc. All rights reserved.

Energy requirements can be estimated from resting energy expenditure (REE). However, little is known about factors influencing REE in Japanese female athletes. This study was performed to evaluate the relationship between REE and body composition in Japanese female athletes with a wide range of body sizes. Ninety-three athletes (age 20.3 +/- 1.2y, height 162.8 +/- 6.4 cm, body weight (BW) 57.0 +/- 9.2 kg, fat-free mass (FFM) 45.4 +/- 6.2 kg) were classified into three groups according to BW: small-size (S) (n=34), medium-size (M) (n=34), and large-size (L) (n=25). Systemic and regional body compositions (skeletal muscle (SM), fat mass (FM), bone mass (BM), and residual mass (RM)) were estimated by dual energy X-ray absorptiometry (DXA). Measured resting energy expenditure (REEm) was evaluated by indirect calorimetry. Marked differences were found in REEm (S: 1,111 +/- 150, M: 1,242 +/- 133, L: 1,478 +/- 138 kcal/d), and systemic and regional body compositions among the three groups. REEm was strongly correlated with PPM, and absolute values of RM and SM increased significantly according to body size. There was good agreement between REEm and estimated REE (REEe) from the specific metabolic rates of four major organ tissue level compartments. These data indicate that REE for female athletes can be attributed to changes in organ tissue mass, and not changes in organ tissue metabolic rate. That is, change in REE can be explained mainly by the change in PPM, and REE can be assessed by PPM in female athletes regardless of body size.

The number of lean young women has been increasing. Fear of being fat may induce unnecessary attempts to reduce body weight, which can cause several types of illness. Many investigations have demonstrated dysfunction of the hypothalamus and metabolic differences in patients with anorexia nervosa. However, it is unclear whether there are any differences in physical characteristics between women with lower body weight and no illness compared to those of normal body weight. In this study, we investigated the differences in body composition, biochemical parameters, and resting energy expenditure (REE) between young women with low and normal body mass index (BMI). Twenty lean women (BMI < 18.5 kg/m(2)) and 20 normal women (18.5 <= BMI < 25 kg/m(2)) were recruited for this study. Body composition, biochemical parameters, and REE (REEm: measurement of REE) were measured, and the REE (REEe: estimation of REE) was estimated by using a prediction model. Marked differences were found in body composition. All of the values of blood analysis were in the normal ranges in both groups REEm (kcal/d and kcal/kg BW/d) was significantly lower in lean than in normal women, but there were no significant differences in the REEm to fat free mass (PPM) ratio between the two groups. In addition, there was good agreement between REEm and REEe obtained from the specific metabolic rates of four tissue organs. These data indicate that the lean women without any illness have normal values of biochemical parameters and energy metabolism compared to women with normal BMI.

Habitual moderate-to-vigorous-intensity physical activity attenuates arterial stiffening. However, it is unclear whether light physical activity also attenuates arterial stiffening. It is also unclear whether light physical activity has the same effects in fit and unfit individuals. This cross-sectional study was performed to determine the relationships between amount of light physical activity determined with a triaxial accelerometer and arterial stiffness. A total of 538 healthy men and women participated in this study. Subjects in each age category were divided into either high-light or low-light physical activity groups based on daily time spent in light physical activity. Arterial stiffness was measured by carotid-femoral pulse wave velocity. Two-way ANOVA indicated a significant interaction between age and time spent in light physical activity in determining carotid-femoral pulse wave velocity (P<0.05). In the older group, carotid femoral pulse wave velocity was higher in the low-light physical activity level group than in the high-light physical activity level group (945 +/- 19 versus 882 +/- 16 cm/s; P<0.01). The difference remained significant after normalizing carotid-femoral pulse wave velocity for amounts of moderate and vigorous physical activity. The carotid-femoral pulse wave velocity (r=-0.47; P<0.01) was correlated with daily time spent in light physical activity in older unfit subjects. No relationship was observed in older fit subjects. These results suggested that longer time spent in light physical activity is associated with attenuation of arterial stiffening, especially in unfit older people. (Hypertension. 2010;56:540-546.)

Exercise training induces various adaptations in skeletal muscles. However, the mechanisms remain unclear. In this study, we conducted 2D-DIGE proteomic analysis, which has not yet been used for elucidating adaptations of skeletal muscle after high-intensity exercise training (HIT). For 5 days, rats performed HIT, which consisted of 14 20-s swimming exercise bouts carrying a weight (14% of the body weight), and 10-s pause between bouts. The 2D-DIGE analysis was conducted on epitrochlearis muscles excised 18 h after the final training exercise. Proteomic profiling revealed that out of 800 detected and matched spots, 13 proteins exhibited changed expression by HIT compared with sedentary rats. All proteins were identified by MALDI-TOF/MS. Furthermore, using western immunoblot analyses, significantly changed expressions of NDUFS1 and parvalbumin (PV) were validated in relation to HIT. In conclusion, the proteomic 2D-DIGE analysis following HIT-identified expressions of NDUFS1 and PV, previously unknown to have functions related to exercise-training adaptations.

The effect of protein fractionation on the bioavailability of amino acids and peptides and insulin response and whether the protein source influences these effects in humans are poorly understood. This study compared the effects of different sources and degrees of hydrolysis of dietary protein, independent of carbohydrate, on plasma amino acid and dipeptide levels and insulin responses in humans. Ten subjects were enrolled in the study, with five subjects participating in trials on either soy or whey protein and their hydrolysates. Protein hydrolysates were absorbed more rapidly as plasma amino acids compared to nonhydrolyzed protein. Whey protein also caused more rapid increases in indispensable amino acid and branched-chain amino acid concentrations than soy protein. In addition, protein hydrolysates caused significant increases in Val-Leu and lle-Leu concentrations compared to nonhydrolyzed protein. Whey protein hydrolysates also induced significantly greater stimulation of insulin release than the other proteins. Taken together, these results demonstrate whey protein hydrolysates cause significantly greater increases in the plasma concentrations of amino acids, dipeptides, and insulin.

Recent studies showed that a combination of carbohydrate and protein was more effective than carbohydrate alone for replenishing muscle glycogen after exercise. However, it remains to be unclear whether the source or degree of hydrolysis of dietary protein influences post-exercise glycogen accumulation. The aim of this study was to compare the effect of dietary protein type on glycogen levels in the post-exercise phase, and to investigate the effects of post-exercise carbohydrate and protein supplementation on phosphorylated enzymes of Akt/PKB and atypical PKCs. Male Sprague-Dawley rats, trained for 3 days, swam with a 2% load of body weight for 4 h to deplete skeletal muscle glycogen. Immediately after the glycogen-depleting exercise, one group was killed, whereas the other groups were given either glucose or glucose plus protein (whey protein, whey protein hydrolysates (WPH), casein hydrolysates or branched-chain amino acid (BCAA) solutions. After 2 h, the rats were killed, and the triceps muscles quickly excised. WPH caused significant increases in skeletal muscle glycogen level (5.01 +/- A 0.24 mg/g), compared with whey protein (4.23 +/- A 0.24 mg/g), BCAA (3.92 +/- A 0.18 mg/g) or casein hydrolysates (2.73 +/- A 0.22 mg/g). Post-exercise ingestion of glucose plus WPH significantly increased both phosphorylated Akt/PKB (131%) and phosphorylated PKC zeta (154%) levels compared with glucose only. There was a significant positive correlation between skeletal muscle glycogen content and phosphorylated Akt/PKB (r = 0.674, P < 0.001) and PKC zeta (r = 0.481, P = 0.017). Post-exercise supplementation with carbohydrate and WPH increases skeletal muscle glycogen recovery by activating key enzymes such as Akt/PKB and atypical PKCs.

This study investigated regulation of autophagy in slow-twitch soleus and fast-twitch plantaris muscles in fasting-related atrophy. Male Fischer-344 rats were subjected to fasting for 1, 2, or 3 days. Greater weight loss was observed in plantaris muscle than in soleus muscle in response to fasting. Western blot analysis demonstrated that LC3-II, a marker protein for macroautophagy, was expressed at a notably higher level in plantaris than in soleus muscle, and that the expression level was fasting duration-dependent. To identify factors related to LC3-II enhancement, autophagy-related signals were examined in both types of muscle. Phosphorylated mTOR was reduced in plantaris but not in soleus muscle. FOXO3a and ER stress signals were unchanged in both muscle types during fasting. These findings suggest that preferential atrophy of fast-twitch muscle is associated with induction of autophagy during fasting and that differences in autophagy regulation are attributable to differential signal regulation in soleus and plantaris muscle. (C) 2010 Elsevier Inc. All rights reserved.

We previously reported that 14 bouts of exhaustive high-intensity intermittent training [20 s periods of swimming while carrying a weight (14% of body weight), separated by pauses of 10 s] is the highest stimuli in terms of exercise training-induced glucose transporter 4 (GLUT-4) expression in rat epitrochlearis (EPI) muscles. In the present study, we found that the GLUT-4 protein content in the skeletal muscle of male Sprague-Dawley rats (age 5 weeks old; body weight 90-110 g) that underwent intermittent exercise training of 3 and 14 bouts of 20 s swimming for 5 days was increased over age-matched sedentary control rats by 75 and 71%, respectively, 18 h after the last bout of exercise. These results suggest that GLUT-4 content in rat EPI muscle increases dramatically after very short (60 s) and nonexhaustive high-intensity intermittent exercise training.

Arterial stiffening, hypertension and left ventricular (LV) remodelling are associated with increased risk of cardiovascular disease. Cardiorespiratory fitness is associated with cardiovascular function and reduced risk of cardiovascular disease. This cross-sectional study was carried out to determine the relationships between cardiorespiratory fitness, arterial stiffness, blood pressure (BP) and LV remodelling in women. On the basis of peak oxygen uptake, a total of 159 premenopausal (young) and postmenopausal (older) women were categorized into either low (unfit) or high (fit) cardiorespiratory fitness groups. The arterial stiffness and LV remodelling were measured by brachial-ankle pulse wave velocity (baPWV) and carotid augmentation index (AI) and LV relative wall thickness (RWT). Two-way analysis of variance indicated a significant interaction between age and cardiorespiratory fitness in baPWV, carotid AI, BP and RWT. In the older group, arterial stiffness (baPWV; 1401 +/- 231 vs 1250 +/- 125cms(-1), P<0.01, AI; 32.9 +/- 9.9 vs 24.8 +/- 10.1%, P<0.01), systolic blood pressure (SBP) (130 +/- 22 vs 117 +/- 15mmHg, P<0.01) and RWT (0.47 +/- 0.08 vs 0.42 +/- 0.04, P<0.05) in fit women were lower than in unfit women. In older women, RWT was significantly related to baPWV (r = 0.46, P<0.01), carotid AI (r = 0.29, P<0.05), SBP (r = 0.57, P<0.01). (V) over dot(2peak) (r = -0.32, P<0.05). In young women, they were not significant correlations, except for a weak correlation between RWT and SBP (r = 0.21, P<0.05). These results suggest that higher cardiorespiratory fitness is associated with lower arterial stiffness, BP and RWT in older women. Journal of Human Hypertension (2010) 24, 197-206; doi:10.1038/jhh.2009.57; published online 16 July 2009

CAO Z., N. MIYATAKE. M. HIGUCHI, M. MIYACHI, K. ISHIKAWA-TAKATA, and I. TABATA. Predicting (V)over dotO(2max) with an Objectively Measured Physical Activity in Japanese women. Med. Sci. Sports Exerc., Vol. 42. No. 1 pp. 179-186, 2010. Purpose: To investigate the use of the accelerometer-determined physical activity (PA) intensity variables as the objective PA variables for estimating (V)over dotO(2max) in Japanese adult women. Methods: The subjects Of this Study were 148 Japanese women aged 20 to 69 yr. Maximal oxygen Uptake was measured with a maximal incremental test oil a bicycle ergometer. Daily step Counts (SC) and file amount Spent ill moderate to vigorous PA (MVPA) and Vigorous PA (VPA) were measured using accelerometer-based activity monitors for 7 consecutive days. Using data of age. SC, MVPA, or VPA, and either body mass index (BMI) or waist circumference (WC), the nonexercise (V)over dotO(2max) prediction models Were derived as BMI model(MVPA), WC models(MVPA), BMI models(VPA), and WC models(VPA), and cross-validated by using two separate cross-validation procedures. Results: SC, MVPA, and VPA were significantly related to (V)over dotO(2max) (r = 0.43, r = 0.52, and r = 0.58, respectively), The Multiple correlation coefficients for the BMI and WC models(MVPA) were 0.83and 0.85, respectively and for the BMI and WC models(MVPA), they were 0.85 and 0.86. respectively. The SEE Was 3.3 and 3.1 mL.kg(-1).min(-1) for the BMI and WC models(MVPA), respectively, and it Was 3.1 and 3.0 mL.kg(-1).min(-1) for the BMI and WC models(VPA), respectively, All regression models demonstrated a high level of cross-validity Supported by the minor shrinkage of the coefficient of determination and the increment of SEE in file predicted residual sum of squares procedure, and by small constant errors for the Subgroups of age, SC, and (V)over dotO(2max) between 25 and 35 mL.kg(-1).min(-1). Conclusions: This study demonstrated that multiple regression models using data of MVPA or VPA were useful in predicting for Japanese adult women.

This study examined the effects of maintaining euhydration by ingesting fluids with or without carbohydrate on subjective responses of untrained men during prolonged exercise in a hot environment. Six healthy untrained subjects completed 90 min of cycling exercises at 55% maximal oxygen consumption (V(O2max)) in a hot environment (temperature: 28 degrees C. humidity: 50%) under three different experimental conditions. During the first trial. subjects did not ingest fluids during exercise (dehydration (DH) trial). In the second and third trials, subjects received mineral water (MW) and hypotonic fluid containing carbohydrate (HF), respectively, in amounts equaling their weight loss in the DH trial. At the end of exercise, the overall rating of perceived exertion (RPE-O) was lower in the MW and HF trials than in the DH trial (14.3+/-1.0 and 13.7+/-0.6 vs 17.7+/-1.0. p<0.05. respectively). RPE-cardiovascular and RPE-legs were lower at the end of exercise in the HF trial compared with the DH trial. V(O2), heart rate (HR), and rectal temperature increased during exercise in the three trials. At the end of exercise, the drift in V(O2) was lower in the MW and HF trials than in the DH trial (304+/-41 and 339+/-40 vs 458+/-33 mL, p<0.05. respectively). HR at the end of exercise in the HF trial was lower than in the DH. trial (158+/-5 vs 173+/-7 bpm. p<0.05). These results suggest that maintaining euhydration during prolonged exercise in untrained men could attenuate RPE-O and that hypotonic electrolyte-carbohydrate solution could attenuate RPE-legs during exercise.

It has recently been reported that a 4-wk high-fat diet gradually increases skeletal muscle peroxisome proliferator activated receptor (PPAR) gamma coactivator-1 alpha (PGC-1 alpha) protein content, which has been suggested to regulate GLUT-4 gene transcription. However, it has not been reported that a high-fat diet enhances GLUT-4 mRNA expression and protein content in skeletal muscle, suggesting that an increase in PGC-1 alpha protein content is not sufficient to induce muscle GLUT-4 biogenesis in a high-fat fed animal. Therefore, we first evaluated the relationship between PGC-1 alpha and GLUT-4 expression in skeletal muscle of rats fed a high-fat diet for 4 wk. The PGC-1 alpha protein content in rat epitrochlearis muscle significantly increased by twofold after the 4-wk high-fat diet feeding. However, the high-fat diet had no effect on GLUT-4 protein content and induced a 30% decrease in GLUT-4 mRNA expression in rat skeletal muscle (p<0.05). To clarify the mechanism by which a high-fat diet downregulates GLUT-4 rnRNA expression, we next examined the effect of PPAR delta activation. which is known to occur in response to a high-fat diet. on GLUT-4 rnRNA expression in L6 myotubes. Incubation with 500 nm GW501516 (PPAR delta activator) for 24 h significantly decreased GLUT-4 rnRNA in L6 myotubes. Taken together, these findings suggest that a high-fat diet downregulates GLUT-4 mRNA, possibly through the activation of PPAR delta, despite an increase in PGC-1 alpha protein content in rat skeletal muscle, and that a posttranscriptional regulatory mechanism maintains GLUT-4 protein content in skeletal muscle of rats fed a high-fat diet.

Reduced response to reactive hyperemia (RH) in the extremities reflects impaired endothelium-dependent vasodilation of the microvasculature. The aims of the present study were to determine whether resistance training and a combination of aerobic and resistance training increase the endothelial vasodilation of the forearm assessed by RH. A total of 39 young men were assigned to either high-intensity resistance training (HIR; six types of exercises, 80% 1RM x 10 repetitions x 3 sets, n = 14) or moderate-intensity resistance training (MIR; six types of exercises, 50% 1RM x 16 repetitions x 3 sets, n = 14) or a combination of high-intensity resistance training and moderate-intensity endurance training (COMBO; HIR and 60% maximal heart rate x 30 min, n = 11) groups. We measured forearm blood flow response to RH before and after 4 months of exercise intervention. All training groups increased maximal strength in all muscle groups tested (all P < 0.05). After 4 months of training, the forearm blood flow during RH increased significantly in the MIR and COMBO groups, from 57 +/- 4 to 66 +/- 7 ml/min per 100 ml tissue and from 59 +/- 6 to 74 +/- 8 ml/min per 100 ml tissue, respectively (both P < 0.05). There was no change in the response to RH in the HIR groups. In conclusion, the findings in this study demonstrate that combined resistance and aerobic training may affect the vasoreactivity response to RH in the forearm, but not resistance training alone.

Sarcopenia is characterized by increased regenerating myofibres and decreased myofibre size. Sarcopenia progression might be partially regulated by ageing-related signals associated with necrotic fibre disruption and nuclear apoptosis. This study sought to identify ageing-related signals in aged atrophying skeletal muscle by comparison with unloaded muscle atrophy in adults. Adult (6-month) and old (32-month) rats were used. Some adult rats were subjected to 2 weeks of hindlimb unloading (6-month-HU). Histological analysis found that regenerating fibres increased by about 30-fold only in 32-month aged soleus muscle compared with 6-month rats. The number of apoptotic DNA fragmented nuclei was increased by 3.9-fold in 6-month-HU and 2.8-fold in 32-month rats. Cleaved caspase-3 was observed at high levels on basal membranes and in nuclei in 32-month rats. By Western blot analysis additional ageing-related signals could be identified since (1) phosphorylated Bcl-2 content was increased in both cytosolic and mitochondrial fractions; (2) ER stress signal proteins caspase-12, CHOP/GADD153, and GRP78 were increased; and (3) stress-inducible chaperone HSP70 was decreased in soleus muscle from 32-month but not changed in 6-month-HU rats. We conclude that activation of ageing-related signals may mediate necrotic myofibre disruption and nuclear apoptosis induction that contribute to progression of sarcopenia. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Objective: To evaluate the possibility that measurement of the magnitude and distribution of fundamental somatic heat-producing units using dual-energy X-ray absorptiometry (DXA) can be used to estimate resting energy expenditure (REE) in both young and elderly women with different aerobic fitness levels.
Subjects and methods: Peak oxygen uptake (VO(2) peak) and REE(m) were directly measured in 116 young (age: 22.3 +/- 2.1 years) and 72 elderly (63.3 +/- 6.4 years) women. The subjects were divided into four groups according to categories of age and VO(2) peak; young: high fitness (YH, n = 58); low fitness (YL, n = 58); elderly: high fitness (EH, n = 37) and low fitness (EL, n = 35). Using DXA, systemic and regional body compositions were measured, and REE(e) was estimated from the sum of tissue organ weights multiplied by corresponding metabolic rate.
Results: Although there were remarkable differences in systemic and regional body compositions, no significant differences were observed between REE(m) and REE(e) in the four groups. REE(e) significantly correlated with REE(m) in elderly as well as young women; the slopes and intercepts of the two regression lines were statistically not different between the elderly and young groups (elderly: y = 0.60x+472, r = 0.667; young: y = 0.78x+250, r = 0.798; P < 0.001, respectively). A Bland-Altman analysis did not indicate bias in calculation of REE for all the subjects.
Conclusion: These results suggest that REE can be estimated from tissue organ components in women regardless of age and aerobic fitness.

Objective: To investigate the relationships between regional skeletal muscle mass (SM mass) and bone mineral indices and to examine whether bone mineral content (BMC) normalized to SM mass shows a similar decrease with age in young through old age.
Subjects/Methods: One hundred and thirty-eight young and postmenopausal women aged 20-76 years participated in this study and were divided into three groups: 61 young women, 49 middle-aged postmenopausal women and 28 older postmenopausal women. Muscle thickness (MTH) was determined by ultrasound, and regional SM mass (arm, trunk and leg) was estimated based on nine sites of MTH. Whole-body and regional lean soft tissue mass (LSTM), bone mineral density (BMD) and BMC (whole body, arms, legs and lumbar spine) were measured using dual-energy X-ray absorptiometry.
Results: Ultrasound spectroscopy indicated that SM mass is significantly correlated with site-matched regional bone mineral indices and these relationships correspond to LSTM. The BMC and BMD in older women were significantly lower than those in middle-aged women. When BMC was normalized to site-matched regional SM mass, BMC normalized to SM mass in arm and trunk region were significantly different with age; however, whole-body and leg BMC normalized to SM mass showed no significant difference between middle-aged and older postmenopausal women.
Conclusions: The age-related differences in BMC were found to be independent of the ageing of SM mass in the arm and trunk region. However, differences in BMC measures of the leg and whole body were found to correspond to age-related decline of SM mass in postmenopausal women.

In earlier studies we showed that dietary whey protein increased skeletal muscle and liver glycogen content in exercise-trained rats. However, little is known about whether ingredients of whey protein stimulate skeletal muscle glycogen accumulation. The aim of this Study was to identify bioactive peptides in whey protein hydrolysates (WPH) which stimulated glucose uptake and glycogen synthesis rate in skeletal muscles. Branched-chain amino acid (BCAA)-containing dipeptides in WPH were identified using LC/MS/MS. L6 myotubes and isolated epitrochlearis muscles were used for the glucose uptake assays. The myotubes and muscles were incubated with or without 1 mM dipeptides, LY294002 a phosphoinositide 3-kinase (PI3-kinase) inhibitor. or GF102903X an atypical protein kinase C (aPKC) inhibitor, followed by measurement of 2-deoxyglucosc uptake. Isolated muscles were incubated for 3 h with or without 1 mM Ile-Leu to determine glycogen synthesis rate. The BCAA-containing dipeptides, Ile-Val, Leu-Val, Val-Leu, Ile-Ile, Leu-Ile, Ile-Leu, and Ile-Leu were detected in the WPH. by LC/MS/MS. These dipeptides caused significant stimulation in glucose uptake rate in the L6 myotubes. Ile-Leu. the main component in WPH, also stimulated glucose uptake in isolated skeletal muscles. Stimulation of glucose uptake by Ile-Leu was completely inhibited by treatment with either LY294002, or GF109203X in both L6 cells and isolated muscles. Ile-Leu increased glycogen contents in isolated muscles. These results suggest that BCAA-containing bioactive dipeptides in WPH stimulate glucose uptake in skeletal Muscles via the P13-kinase and aPKC pathways, resulting in increased skeletal muscle glycogen contents.

The purpose of the study was to develop a new non-exercise VO(2max) prediction model using a physical activity (PA) variable determined by pedometer-determined step counts (SC, steps day(-1)) in Japanese women aged 20-69 years old. Eighty-seven and 102 subjects were used to develop the prediction model, and to validate the new model, respectively. VO(2max) was measured using a maximal incremental test on a bicycle ergometer. SC was significantly related to VO(2max) (partial correlation coefficient r = 0.40, P < 0.001) after adjusting for BMI (kg m(-2)) and age (years). When the new prediction equation developed by multiple regression to estimate VO(2max) from age, BMI, and SC (R = 0.71, SEE = 5.3 ml kg(-1) min(-1), P < 0.001) was applied to the Validation group, predicted VO(2max) correlated well with measured VO(2max) (r = 0.81, P < 0.001), suggesting that SC is a useful PA variable for non-exercise prediction of VO(2max) in Japanese women.

The aim of this cross-sectional study was to compare body composition and risk factors of lifestyle-related diseases between young and older male rowers and sedentary controls. Healthy males aged 19-73 years participated in the study, and were divided into four groups: 26 young rowers, 24 senior rowers, 23 young sedentary controls, and 22 senior sedentary controls. Total and regional lean soft tissue, fat mass, and bone mineral density were measured using dual-energy X-ray absorptiometry. The HDL-cholesterol of senior rowers (67.4 +/- 13.4 mg . dl(-1)) was significantly (P < 0.05) higher than that of senior sedentary controls (59.2 +/- 11.9 mg . dl(-1)), while HDL-cholesterol was similar in senior rowers and young rowers (66.1 +/- 10.8 mg . dl(-1)). Arm, leg, and trunk lean soft tissue mass were significantly higher in senior rowers (5.6 +/- 0.6 kg, 18.2 +/- 1.8 kg, and 27.3 +/- 3.2 kg respectively) than in senior sedentary controls (5.1 +/- 0.4 kg, 16.3 +/- 1.4 kg, and 24.6 +/- 1.7 kg respectively; P < 0.05). Bone mineral density was also significantly higher in senior rowers than in senior sedentary controls (ribs, lumbar spine, and pelvic segments; P < 0.05). We conclude that age-related increases in the risk of lifestyle-related diseases, such as osteoporosis and sarcopenia, are attenuated in male rowers. These results suggest that regular rowing exercise may have a positive influence in the prevention of lifestyle-related diseases in older Japanese people.

CC-chemokine receptor 2 (CCR2) and its ligand, monocyte chemotactic protein-1 (MCP-1, also known as CCL2), are crucial for the recruitment of monocytes/macrophages to sites of inflammation. We conducted a series of experiments to investigate the relationship between stress, monocyte CCR2 expression and migration activity. First, we collected peripheral blood mononuclear cells (PBMC) from untrained subjects (n = 8) and measured CCR2 expression on CD14(+) monocytes cultured with cortisol, epinephrine and norepinephrine. Second, we collected PBMC from the subjects before and after they cycled for 60 min at 70% peak O-2 uptake (V-O2peak), and measured alterations in CCR2 expression on monocytes following exercise. Third, we cultured PBMC with serum obtained before and after exercise and the glucocorticoid antagonist RU-486 to determine the effect of cortisol on CCR2 expression in vitro. Last, we measured the ability of PBMC treated with serum or cortisol to migrate through membrane filters in response to CCL2. Cortisol (but not epinephrine or norepinephrine) increased CCR2 expression on monocytes in a dose- and time-dependent manner. Exercise did not influence CCR2 expression on PBMC, whereas incubation of PBMC with post-exercise serum significantly increased CCR2 expression. Both cortisol and post-exercise serum increased the migration of PBMC toward CCL2. The increase in CCR2 expression on PBMC following stimulation with cortisol and serum was blocked by the glucocorticoid receptor antagonist RU-486. In conclusion, cortisol released during exercise increased monocyte CCR2 expression and migration activity in vitro. These alterations may influence inflammation and regeneration of damaged tissue after acute stress. (C) 2008 Elsevier Inc. All rights reserved.

The present study was performed to test the hypothesis that the blood pressure (BP) response to resistance exercise in middle-aged men with stiffening arteries is greater than that in young men with compliant arteries. The BP responses to acute dynamic resistance exercise (leg press) at individual relative (low, moderate and high) and absolute intensities were investigated in both young and middle-aged men. A total of 21 sedentary healthy normotensive men, 21-25 years of age (young) and 41-59 years of age (middle-aged), were included in the study. At rest, the arterial compliance (simultaneous ultrasound and applanation tonometry) and muscle strength (leg press) were lower, and indices of arterial stiffness and BP were higher in the middle-aged men than in the young men (p < 0.05). There were no significant differences in height, body mass, or heart rate between the two groups. During exercise, the systolic BP of the middle-aged men at 80% one-repetition maximum (1RM) was significantly lower than that of the young men for the last half of the exercise period (p < 0.05). The amounts of change in systolic and diastolic BP from baseline to the end of resistance exercise were lower in the middle-aged men than in the young men at individual relative intensities (p < 0.05) and at individual absolute intensity. In contrast to our hypothesis, these findings indicated that the BP response during dynamic resistance exercise using large muscle groups may be attenuated in middle-aged men relative to young men.

Resistance training is a popular mode of exercise, but may result in stiffening of the central arteries. Changes in carotid artery diameter were determined using the cold pressor test (CPT), which results in production of nitric oxide via sympathetic activation and is one of the novel methods available for assessing endothelial function in the carotid artery. To investigate the effect of resistance training on endothelial function, we designed a cross-sectional study of carotid arterial vasoreactivity to CPT in men participating in regular resistance training with increased carotid arterial stiffness compared with age-matched control subjects. Twelve resistance-trained middle-aged men (age 38.7 +/- 1.7 years) and 17 age-matched control subjects (age 36.8 +/- 1.2 years) were studied. The direction and magnitude of changes in carotid artery diameter were measured by B-mode ultrasonography during sympathetic stress induced by submersion of the foot in ice slush for 90 s. Carotid arterial beta-stiffness index, and systolic and mean arterial blood pressure were higher (7.7 +/- 0.7 versus 6.0 +/- 0.4 arbitrary units, 116 +/- 2 versus 131 +/- 4 mmHg and 86 +/- 2 versus 95 +/- 2 mmHg, respectively, all P < 0.05) in the resistance training group compared with control subjects. There were, however, no significant differences in the amount or percentage change in carotid artery diameter in CPT between the two groups (resistance training group, 0.33 +/- 0.07 mm and 5.2 +/- 1.1%; control group, 0.37 +/- 0.06 mm and 5.8 +/- 0.9%, respectively). These findings suggest that while carotid arterial stiffening and higher blood pressure are observed in regular resistance-trained men, these are not associated with abnormalities in carotid arterial vasoreactivity to sympathetic stimulus, which implies intact endothelial function.

It has been demonstrated in a previous study that resting energy expenditure (REE) is associated with adiponectin levels in the blood. However, body composition was not taken into consideration in that study. The purpose of the present study was to again investigate the relationship between blood adipocytokines and REE, adjusted by body composition, in both young and elderly women. REE and blood adipocytokines were measured in 115 young (age: 22.3 +/- 2.1 y, BMI: 21.3 +/- 1.9 kg/ml) and 71 elderly (63.4 +/- 6.5 y, 22.9 +/- 2.3 kg/m(2)) women. Dual energy X-ray absorptiometry was used to measure percent body fat. Fat mass and fat free mass (FFM) were calculated. REE (kcal/d and kcal/kg BW/d) was lower in elderly women than in young women, but no significant difference was observed in REE, expressed as kcal/kg FFM/d, between the two groups. Although elderly women had a higher percent body fat and higher serum leptin concentrations than young women, plasma adiponectin concentrations did not differ between young and elderly women. In elderly women, REE (kcal/d) was significantly and inversely correlated with plasma adiponectin concentration (r = -0.386, p<0.001), but REE expressed per kilogram of BW or FFM was not significantly correlated. Furthermore, no significant correlation was observed between REE (kcal/d) and concentrations of plasma adiponectin or serum leptin, after adjusting for potential confounders such as body composition and hormones, in either age group. These results suggest that adipocytokines do not influence REE in adult women.

Ventilatory threshold (VT) is an important predictor of cardiorespiratory fitness, such as peak oxygen uptake (<(V)over dot >(O2peak)) and is a valuable index of aerobic exercise intensity. However, little is known about the role of skeletal muscle ( SM) mass in the ageassociated decline of VT. Therefore, the present study was performed to investigate the effects of age on cardiopulmonary fitness normalised for regional SM mass in 1,463 Japanese men and women, and to determine the relevance of VT normalised to SM mass based on age and gender. Total, trunk and thigh SM mass were measured using an ultrasound method, while <(V)over dot >(O2pea)k and VT were determined during treadmill walking. <(V)over dot >(O2peak) was estimated using the predicted maximum heart rate ( HR) and the HR-<(V)over dot >(O2) relationship for sub- maximal treadmill walking. There were significant negative correlations between VT normalised for body mass and age in men and women ( P < 0.001). Age- associated declines were also observed in VT normalised for body mass in both men and women; however, VT normalised for SM mass was not significantly different with age. Significant correlations were also observed between thigh SM mass and VT in both men and women. These results suggest that thigh SM mass is closely associated with <(V)over dot >(O2peak) and/ or VT in both men and women, and the decrease in VT with age is predominantly due to an age- related decline of SM mass. Moreover, this study provides normative cardiorespiratory fitness data regarding VT normalised SM mass in healthy men and women aged 20 - 80 years.

The basal metabolic rate (BMR) of Japanese females in their twenties (F2006) was compared with that of females of the same age measured in the 1950s (F1950; Nagamine and Suzuki, 1964). The subjects measured during 2004-2006 were 83 females (F2006) with no exercise habits. BMR was measured by using indirect calorimetry, and the body composition was assessed by dual-energy X-ray absorptiometry (DXA).<br>While the height and weight of F2006 were respectively significantly higher and heavier than those of F1950, BMR of F2006 (1, 110±112kcal/day) was not significantly different from that of F1950 (1, 132kcal/day). In addition, there was no difference in lean body mass (LBM) between the two groups. On the other hand, BMR per body weight of F2006 (21.5±2.1kcal/kg/day) was significantly lower than that of F1950 (23.1kcal/kg/day). BMR per body weight of F2006 was correlated with LBM per body weight (%LBM, r=0.51, p<0.001), although BMR per LBM of F2006 was not different from that of F1950.<br>These data suggest that BMR per body weight of the current young females of Japan is lower than that of the young females of the same age measured in the 1950s, during which data for establishing the BMR reference value in "Dietary Reference Intakes for Japanese, 2005" was obtained. Furthermore, the difference in BMR per body weight between the two groups can be explained by the difference in %LBM.

Although it is clear that rowers have a large muscle mass, their distribution of muscle mass and which of the main motions in rowing mediates muscle hypertrophy in each body part are unclear. We examine the relationships between partial motion power in rowing and muscle cross-sectional area of the thigh, lower back, and upper arms. Sixty young rowers (39 males and 21 females) participated in the study. joint positions and forces were measured by video cameras and rowing ergometer software, respectively. One-dimensional motion analysis was performed to calculate the power of leg drive, trunk swing, and arm pull motions. Muscle cross-sectional areas were measured using magnetic resonance imaging. Multiple regression analyses were carried out to determine the association of different muscle cross-sectional areas with partial motion power. The anterior thigh best explained the power demonstrated by leg drive (r(2) = 0.508), the posterior thigh and lower back combined best explained the power demonstrated by the trunk swing(r(2) = 0.493), and the elbow extensors best explained the power demonstrated by the arm pull (r(2) = 0.195). Other correlations, such as arm muscles with leg drive power (r(2) = 0.424) and anterior thigh with trunk swing power (r(2) = 0.335), were also significant. All muscle cross-sectional areas were associated with rowing performance either through the production of power or by transmitting work. The results imply that rowing motion requires a well-balanced distribution of muscle mass throughout the body.

The combined intervention of isoflavone intake and walking exercise over 1 year in postmenopausal Japanese women exhibited a trend for a greater effect on prevention of bone loss at the total hip and Ward's triangle regions.
Introduction: The additive effects of isoflavones and exercise on bone and lipid metabolism have been shown in estrogen-deficient animals. In this study, we determined the effects of isoflavone intake, walking exercise, and their interaction on bone, fat mass, and lipid metabolism over I year in postmenopausal Japanese women.
Materials and Methods: A total of 136 postmenopausal women at &lt; 5 years after the onset of menopause were randomly assigned to four groups: (1) placebo, (2) walking (45 minutes/day, 3 days/week) with placebo, (3) isoflavone intake (75 mg of isoflavone conjugates/day), and (4) combination of isoflavone plus walking. BMD, fat mass, serum lipid, and serum and urinary isoflavone concentrations were assessed.
Results: A significant main effect of isoflavone on the reduction in trunk fat mass was obtained at 12 months. Significant main effects of walking on the reduction in fat mass in the whole body and the trunk were observed at 3, 6, and 12 months and that in the legs and arms at 6 and 12 months. Serum high-density lipoprotein (HDL)-cholesterol concentration significantly increased by 12 months after the walking and the combined intervention. After 12 months, a significant main effect of isoflavone on BMD was observed only at Ward's triangle. Walking prevented bone loss at the total hip and the Ward's triangle to significant degrees. The effect of the combined intervention on BMD at total hip and Ward's triangle regions was greater than that of either alone. No significant interaction was observed between isoflavone and walking in any measurements recorded during the study.
Conclusions: Our study suggest that combined intervention of 75 mg/day of isoflavone intake and walking exercise 3 times/week for I year showed a trend for a greater effect on BMD at total hip and Ward's triangle regions than either alone. Intervention with isoflavone in postmenopausal Japanese women showed a modest effect on BMD compared with those in Westerners. Further studies over longer treatment duration that include assessment of BMD at various regions are necessary to ascertain the clinical significance of the combined intervention of isoflavone plus walking in postmenopausal women.

Cooperative effects of isoflavones and exercise on bone and lipid metabolism have been exhibited in estrogen-deficient animals; however, results from clinical trials have not been published. In this study, we determined the effects of isoflavone intake and walking and their interaction on bone and lipid metabolism in postmenopausal women over 24 weeks. The bioavailability and metabolism of isoflavones (daidzein in particular) were also examined to clarify the mechanism of their bone-protective effects in humans. One hundred twenty-eight Subjects were randomly assigned to 4 groups: placebo; placebo combined with walking (3 times per week); isoflavone intake (75 mg of isoflavones conjugates per day); and isoflavone combined with walking. The Subjects were classified by equol status (producers or nonproducers) as identified using production of equol from daidzein in fecal Culture. Bone mineral density (BMD), body composition, and serum concentrations of isoflavones were assessed. Scrum high-density lipoprotein cholesterol concentration significantly increased (6.1%, P = .03), and fat mass in the whole body significantly decreased (-4.3%, P = .0003) from the baseline in the combined intervention group. There were no significant differences in BMD between baseline and postintervention in any of the treatment groups. However, the percent changes in BMD in equol producers were -0.53% and +0.13% in the sub-whole body and total hip, respectively. This was significantly different compared with -1.35 and -1.77 for the sub-whole body and total hip, respectively, in nonproducers in the isoflavone group (P = .049 and .040, respectively). The mean scrum equol concentration was significantly higher in equol producers than in nonproducers in the isoflavone groups, but not in the placebo group. The combination of isoflavones and exercise exhibited favorable effects on serum lipid and body composition of postmenopausal women. The findings of this study suggest that the preventive effects of isoflavones on bone loss depend on the individual's intestinal flora for equol production. (c) 2006 Elsevier Inc. All rights reserved.

The basal metabolic rate (BMR) in 46 fit older people (age: 66&plusmn;3yrs., height: 163.3&plusmn;9.5cm, body weight (BW): 59.0&plusmn;10.4kg; mean&plusmn;SD) was evaluated in relation to the body composition (body fat mass (FM): 15.3&plusmn; 4.0kg, lean body mass (LBM): 43.7&plusmn;9.5kg). BMR for all the subjects was 1, 328&plusmn;218kcal/day, 22.6&plusmn;1.9kcal/kgBW/day, and 30.9&plusmn;3.6kcal/kgLBM/day. BMR (kcal/day) was significantly correlated with BW (r=0.87, p<0.001) and LBM (r=0.85, p<0.001). The height, BW, BMI and LBM were significantly higher in the males than in the females (p<0.01, respectively). No difference was apparent in FM between the two sexes. BMR (kcal/day) was higher in the males than in the females (p<0.01), but the males had lower BMR in terms of kcal/kgBW/day and kcal/kgLBM/day than the females (p<0.05 and p<0.01, respectively). No significant difference was apparent in the adjusted BMR (kcal/(kgLBM&plusmn;24.5)/day) between the males and females. A multiple-regression analysis showed the most powerful predictor to be LBM and the second most powerful to be FM of BMR in fit older people.

We tested the hypothesis that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, folic acid deficiency and riboflavin deficiency, independently or interactively, are important determinants of genomic stability, cell death, cell proliferation and homocysteine (Hcy) concentration in 9-d human lymphocyte cultures. Lymphocytes of seven wild-type (CC) and seven mutant (TT) homozygotes were cultured under the four possible combinations of deficiency and sufficiency of riboflavin (0 and 500 nmol/L) and folic acid (20 and 100 nmol/L) at a constant L-methionine concentration of 50 mumol/L. Viable cell growth was 25% greater in TT than in CC cells (P &lt; 0.05) and 32% greater at 100 nmol/L folic acid than at 20 nmol/L folic acid (P = 0.002). The comprehensive cytokinesis-block micronucleus assay was used to measure micronuclei (MNi; a marker for chromosome breakage and loss), nucleoplasmic bridges (NPB; a marker of chromosome rearrangement) and nuclear buds (NBUD, a marker of gene amplification). The MNi levels were 21% higher in TT cells than in CC cells (P &lt; 0.05) and 42% lower in the high folic acid medium than in the low folic acid medium (P &lt; 0.0001). The NBUD levels were 27% lower in TT cells than in CC cells (P &lt; 0.05) and 45% lower in the high folic acid medium than in the low folic acid medium (P &lt; 0.0001). High riboflavin concentration (500 nmol/L) increased NBUD levels by 25% (compared with 0 nmol/L riboflavin) in folate-deficient conditions (20 nmol/L folic acid medium; P &lt; 0.05), and there was an interaction between folic acid and riboflavin that affected NBUD levels (P = 0.042). This preliminary investigation suggests that MTHFR C677T polymorphism and riboflavin affect genome instability; however, the effect is relatively small compared with that of folic acid.

We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019–2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14–16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-β hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.<br>

We tested the hypothesis that methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism, folic acid deficiency and riboflavin deficiency, independently or interactively, are important determinants of genomic stability, cell death, cell proliferation and homocysteine (Hcy) concentration in 9-d human lymphocyte cultures. Lymphocytes of seven wild-type (CC) and seven mutant (TT) homozygotes were cultured under the four possible combinations of deficiency and sufficiency of riboflavin (0 and 500 nmol/L) and folic acid (20 and 100 nmol/L) at a constant L-methionine concentration of 50 mumol/L. Viable cell growth was 25% greater in TT than in CC cells (P &lt; 0.05) and 32% greater at 100 nmol/L folic acid than at 20 nmol/L folic acid (P = 0.002). The comprehensive cytokinesis-block micronucleus assay was used to measure micronuclei (MNi; a marker for chromosome breakage and loss), nucleoplasmic bridges (NPB; a marker of chromosome rearrangement) and nuclear buds (NBUD, a marker of gene amplification). The MNi levels were 21% higher in TT cells than in CC cells (P &lt; 0.05) and 42% lower in the high folic acid medium than in the low folic acid medium (P &lt; 0.0001). The NBUD levels were 27% lower in TT cells than in CC cells (P &lt; 0.05) and 45% lower in the high folic acid medium than in the low folic acid medium (P &lt; 0.0001). High riboflavin concentration (500 nmol/L) increased NBUD levels by 25% (compared with 0 nmol/L riboflavin) in folate-deficient conditions (20 nmol/L folic acid medium; P &lt; 0.05), and there was an interaction between folic acid and riboflavin that affected NBUD levels (P = 0.042). This preliminary investigation suggests that MTHFR C677T polymorphism and riboflavin affect genome instability; however, the effect is relatively small compared with that of folic acid.

We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019–2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14–16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-β hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.<br>

We evaluated the impact of bilateral leg extension power and fat-free mass on 2000 m rowing ergometer performance in 332 young oarsmen (age 21+/-2 years, height 1.76+/-0.05 m, body mass 62+/-6 kg; mean+/- s ). The 2000 m rowing performance time was correlated with height (1.62-1.93 m; R-2 = 0.23, P &lt;0.001), body mass (53-95 kg; R-2 = 0.53, P &lt;0.001), fat-free mass (47-82 kg; R-2 = 0.58, P &lt;0.001) and bilateral leg extension power (1202-3302 W; R-2 = 0.38, P &lt;0.001). Multiple regression analysis selected fat-free mass and bilateral leg extension power as regressor variables. Fat-free mass explained 58% of the variability in rowing performance and the inclusion of bilateral leg extension power improved the power of prediction by 5%. The results suggest that rowing involves almost every muscle in the body and that bilateral leg extension power is very important during this activity.

We evaluated the impact of bilateral leg extension power and fat-free mass on 2000 m rowing ergometer performance in 332 young oarsmen (age 21+/-2 years, height 1.76+/-0.05 m, body mass 62+/-6 kg; mean+/- s ). The 2000 m rowing performance time was correlated with height (1.62-1.93 m; R-2 = 0.23, P &lt;0.001), body mass (53-95 kg; R-2 = 0.53, P &lt;0.001), fat-free mass (47-82 kg; R-2 = 0.58, P &lt;0.001) and bilateral leg extension power (1202-3302 W; R-2 = 0.38, P &lt;0.001). Multiple regression analysis selected fat-free mass and bilateral leg extension power as regressor variables. Fat-free mass explained 58% of the variability in rowing performance and the inclusion of bilateral leg extension power improved the power of prediction by 5%. The results suggest that rowing involves almost every muscle in the body and that bilateral leg extension power is very important during this activity.

The purpose of this study was to test the hypothesis that regular exercise training in elderly people affects the type 1/type 2 balance. Nine elderly women who train by walking (mean+/-S.E.M., age 63+/-1 years, VO2 peak 32.2+/-1.0 ml kg(-1) min(-1)), 12 age-matched untrained women (63+/-1 years, 27.8+/-0.9 ml kg(-1) min(-1)), and nine young untrained women (26+/-1 years, 37.8+/-1.3 ml kg(-1) min(-1)) participated in the study. We measured interferon gamma (IFN-gamma), IL-2, and IL-4 in CD4+ and CD8+ T cells, to compare type I and type 2 T cells. The number of CD4+ cells expressing intracellular IFN-gamma (CD4+/IFN-gamma cell) significantly higher in the elderly trained than in the young (P &lt; 0.01). The number of CD8+/IL-2 cells was significantly higher in the elderly trained than in the elderly untrained (P &lt; 0.05). The number of CD8+/IL-4 cells was significantly higher in the young than in the elderly untrained (P &lt; 0.01). No significant differences were apparent in the IFN-gamma/IL-4 ratio within CD4+ and CD8+ cells from the three groups. These results might indicate that the effect of age on the balance of type 1/type 2 T cells was stronger than the effect of physical training. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

The purpose of this study was to test the hypothesis that regular exercise training in elderly people affects the type 1/type 2 balance. Nine elderly women who train by walking (mean+/-S.E.M., age 63+/-1 years, VO2 peak 32.2+/-1.0 ml kg(-1) min(-1)), 12 age-matched untrained women (63+/-1 years, 27.8+/-0.9 ml kg(-1) min(-1)), and nine young untrained women (26+/-1 years, 37.8+/-1.3 ml kg(-1) min(-1)) participated in the study. We measured interferon gamma (IFN-gamma), IL-2, and IL-4 in CD4+ and CD8+ T cells, to compare type I and type 2 T cells. The number of CD4+ cells expressing intracellular IFN-gamma (CD4+/IFN-gamma cell) significantly higher in the elderly trained than in the young (P &lt; 0.01). The number of CD8+/IL-2 cells was significantly higher in the elderly trained than in the elderly untrained (P &lt; 0.05). The number of CD8+/IL-4 cells was significantly higher in the young than in the elderly untrained (P &lt; 0.01). No significant differences were apparent in the IFN-gamma/IL-4 ratio within CD4+ and CD8+ cells from the three groups. These results might indicate that the effect of age on the balance of type 1/type 2 T cells was stronger than the effect of physical training. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

For older people exercise intensity is often determined based on heart rate (HR) or the percentage of maximal HR (%HRmax). This study evaluated oxygen uptake ((V) over circleO(2)) and HR during ergometry rowing (combined arm and leg; sitting exercise) and treadmill running (leg; upright exercise) for 15 older people [age, (mean +/- SD) 62 +/- 3 years]. The HR was lower during ergometry rowing than during treadmill running at a blood lactate concentration of 4 mmol 1(-1) (151 +/- 4 beat min(-1) versus 160 +/- 5 beat min(-1), P &lt; 0(.)05) and at a maximal effort (171 +/- 7 beat min(-1) versus 177 +/- 7 beat min(-1), P &lt; 0(.)05). This was the case although the (V) over circleO(2) was higher during ergometry rowing than during treamill running both at a blood lactate concentration of 4 mmol 1(-1) (3(.)0 +/- 0(.)4 l min(-1) versus 2(.)7 +/- 0(.)4 l min(-1), P &lt; 0(.)05) and at a maximal effort (3(.)4 +/- 0(.)4 l min versus 3(.)1 +/- 0(.)3 l min P &lt; 0(.)05). %HRmax and %HR reserve were lower during ergometry rowing than during treadmill running. The results suggest that, in prescription of rowing for older people, the relation between HR and (V) over circleO(2) for rowing and the attenuated HR response to rowing should be taken into consideration.

There is evidence that estrogen plays an important role in skeletal tissue in males as well as females. We have reported that phytoestrogens, such as genistein, selectively act on bone and exhibit cooperative effects on bone mass when combined with exercise in ovariectomized mice. In this study, we examined whether both interventions exhibit cooperative effects on bone loss in androgen-deficient mice similar to those in estrogen-deficient mice. Male mice aged 7 wk were either sham operated or orchidectomized (ORX) and divided into six groups: 1) sham; 2) ORX; 3) ORX and treated with genistein (0.4 mg/day) subcutaneously; 4) ORX, exercised on a treadmill daily for 30 min/day at 12 m/min; 5) ORX, given genistein, and exercised (ORX+ExG); and 6) ORX and treated with 17beta-estradiol (E-2). Four weeks after the intervention, seminal vesicle weight strikingly decreased in ORX mice, and it was not affected by administration of genistein or E-2. Bone mineral density of whole femur was significantly reduced by ORX, and bone loss was prevented by the combined intervention. Histomorphometric analysis showed that bone volume and trabecular thickness in the distal femoral cancellous bone were significantly lower in the ORX group than in the Sham group, and they were completely restored in the ORX+ExG group, as in the ORX with E-2 group. These results indicate that the combined intervention of moderate exercise and a low dose of genistein administration shows an additive effect in preventing bone loss in ORX mice similar to that in ovariectomized mice.

For older people exercise intensity is often determined based on heart rate (HR) or the percentage of maximal HR (%HRmax). This study evaluated oxygen uptake ((V) over circleO(2)) and HR during ergometry rowing (combined arm and leg; sitting exercise) and treadmill running (leg; upright exercise) for 15 older people [age, (mean +/- SD) 62 +/- 3 years]. The HR was lower during ergometry rowing than during treadmill running at a blood lactate concentration of 4 mmol 1(-1) (151 +/- 4 beat min(-1) versus 160 +/- 5 beat min(-1), P &lt; 0(.)05) and at a maximal effort (171 +/- 7 beat min(-1) versus 177 +/- 7 beat min(-1), P &lt; 0(.)05). This was the case although the (V) over circleO(2) was higher during ergometry rowing than during treamill running both at a blood lactate concentration of 4 mmol 1(-1) (3(.)0 +/- 0(.)4 l min(-1) versus 2(.)7 +/- 0(.)4 l min(-1), P &lt; 0(.)05) and at a maximal effort (3(.)4 +/- 0(.)4 l min versus 3(.)1 +/- 0(.)3 l min P &lt; 0(.)05). %HRmax and %HR reserve were lower during ergometry rowing than during treadmill running. The results suggest that, in prescription of rowing for older people, the relation between HR and (V) over circleO(2) for rowing and the attenuated HR response to rowing should be taken into consideration.

There is evidence that estrogen plays an important role in skeletal tissue in males as well as females. We have reported that phytoestrogens, such as genistein, selectively act on bone and exhibit cooperative effects on bone mass when combined with exercise in ovariectomized mice. In this study, we examined whether both interventions exhibit cooperative effects on bone loss in androgen-deficient mice similar to those in estrogen-deficient mice. Male mice aged 7 wk were either sham operated or orchidectomized (ORX) and divided into six groups: 1) sham; 2) ORX; 3) ORX and treated with genistein (0.4 mg/day) subcutaneously; 4) ORX, exercised on a treadmill daily for 30 min/day at 12 m/min; 5) ORX, given genistein, and exercised (ORX+ExG); and 6) ORX and treated with 17beta-estradiol (E-2). Four weeks after the intervention, seminal vesicle weight strikingly decreased in ORX mice, and it was not affected by administration of genistein or E-2. Bone mineral density of whole femur was significantly reduced by ORX, and bone loss was prevented by the combined intervention. Histomorphometric analysis showed that bone volume and trabecular thickness in the distal femoral cancellous bone were significantly lower in the ORX group than in the Sham group, and they were completely restored in the ORX+ExG group, as in the ORX with E-2 group. These results indicate that the combined intervention of moderate exercise and a low dose of genistein administration shows an additive effect in preventing bone loss in ORX mice similar to that in ovariectomized mice.

We have reported that strenuous physical exercise causes a decrease in protein-bound sulfhydryl groups (p-SHs), such as albumin cysteine residues, in human plasma (Inayama et al., 1996, Life Sci., 59, 573-578). We further investigated the fate of plasma protein thiols after moderate exercise. Six untrained healthy female volunteers ran for 30-min at the individual ventilatory threshold. We observed an increase in protein cysteine mixed disulfides (p-S-Cys) after running, as evidenced by reducing plasma proteins with dithiothreitol to detect the increase of cysteine, along with the concomitant decrease in p-SHs in plasma. However, plasma protein-bound glutathione (GSH) and S-nitroso-protein were undetectable before and after exercise. Test tube experiments suggest that p-S-Cys are probably formed by the hydrolysis of protein GSH mixed disulfides by gamma-glutamyltranspeptidase and peptidase, and/or by the oxidative addition of p-SHs to cystine.

We have reported that strenuous physical exercise causes a decrease in protein-bound sulfhydryl groups (p-SHs), such as albumin cysteine residues, in human plasma (Inayama et al., 1996, Life Sci., 59, 573-578). We further investigated the fate of plasma protein thiols after moderate exercise. Six untrained healthy female volunteers ran for 30-min at the individual ventilatory threshold. We observed an increase in protein cysteine mixed disulfides (p-S-Cys) after running, as evidenced by reducing plasma proteins with dithiothreitol to detect the increase of cysteine, along with the concomitant decrease in p-SHs in plasma. However, plasma protein-bound glutathione (GSH) and S-nitroso-protein were undetectable before and after exercise. Test tube experiments suggest that p-S-Cys are probably formed by the hydrolysis of protein GSH mixed disulfides by gamma-glutamyltranspeptidase and peptidase, and/or by the oxidative addition of p-SHs to cystine.

We evaluated effects of age and rowing on concentrations of lipids and lipoprotein cholesterols in the blood. Maximal oxygen uptake ((V)over dot O-2max), and concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured in 17 oarsmen [mean (SD)] [age 64 (4) years, body mass 69 (6) kg] and in sedentary men [age 65 (3) years, body mass 70 (7) kg] who were matched on the basis of body size. Also the variables were obtained from young oarsmen [age 22 (2) years, body mass 70 (4) kg] and young sedentary men [age 22 (3) years, body mass 69 (7) kg]. The percentage body fat of the older oarsmen was lower than that of the older sedentary men [18 (4)% compared to 23 (4)%, P&lt;0.05], but it was similar to that of the young sedentary men [17 (4)%]. Although older oarsmen possessed a lower (V)over dot O-2max than the young oarsmen [3.0 (0.4) 1.min(-1) compared to 4.1 (0.3) 1.min(-1), P&lt;0.01], they showed a (V)over dot O-2max, similar to that of the young sedentary men [3.1 (0.5) 1.min(-1)] but a higher value than obtained from the older sedentary men [2.2 (0.3) 1.min(-1), P&lt;0.05]. Although the indices of risk factors for coronary artery disease in the older oarsmen were higher than those in the young oarsmen [LDL-C/HDL-C 1.7 (0.2) compared to 1.3 (0.4), TC/HDL-C 3.1 (0.2) compared to 2.6(0.4), P&lt;0.05], they were lower than those in both the older [2.1 (0.3), 3.6 (0.3), P&lt;0.05] and the Young sedentary men [2.1 (0.4), 3.5 (0.4), P&lt;0.05]. The results suggest that rowing is an appropriate type of exercise for the promotion of health.

We evaluated effects of age and rowing on concentrations of lipids and lipoprotein cholesterols in the blood. Maximal oxygen uptake ((V)over dot O-2max), and concentrations of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured in 17 oarsmen [mean (SD)] [age 64 (4) years, body mass 69 (6) kg] and in sedentary men [age 65 (3) years, body mass 70 (7) kg] who were matched on the basis of body size. Also the variables were obtained from young oarsmen [age 22 (2) years, body mass 70 (4) kg] and young sedentary men [age 22 (3) years, body mass 69 (7) kg]. The percentage body fat of the older oarsmen was lower than that of the older sedentary men [18 (4)% compared to 23 (4)%, P&lt;0.05], but it was similar to that of the young sedentary men [17 (4)%]. Although older oarsmen possessed a lower (V)over dot O-2max than the young oarsmen [3.0 (0.4) 1.min(-1) compared to 4.1 (0.3) 1.min(-1), P&lt;0.01], they showed a (V)over dot O-2max, similar to that of the young sedentary men [3.1 (0.5) 1.min(-1)] but a higher value than obtained from the older sedentary men [2.2 (0.3) 1.min(-1), P&lt;0.05]. Although the indices of risk factors for coronary artery disease in the older oarsmen were higher than those in the young oarsmen [LDL-C/HDL-C 1.7 (0.2) compared to 1.3 (0.4), TC/HDL-C 3.1 (0.2) compared to 2.6(0.4), P&lt;0.05], they were lower than those in both the older [2.1 (0.3), 3.6 (0.3), P&lt;0.05] and the Young sedentary men [2.1 (0.4), 3.5 (0.4), P&lt;0.05]. The results suggest that rowing is an appropriate type of exercise for the promotion of health.

This study evaluated whether the heart rate (HR) response to exercise depends on body position and on the active muscle mass. The HR response to ergometer rowing (sitting and using both arms and legs) was compared to treadmill running (upright exercise involving mainly the legs) using a progressive exercise intensity protocol in 55 healthy men [mean (SD) height 176 (5) cm, body mass 71 (6) kg, age 21 (3) years]. During rowing HR was lower than during running at a blood lactate concentration of 2 mmol.l(-1) [145 (13) compared to 150 (11) beat-min(-1), P &lt; 0.05], 4 mmol.l(-1) [170 (10) compared to 177 (13) beat-min(-1), P &lt; 0.05], and 6 mmol.l(-1) [182 (10) compared to 188 (10) beat-min(-1), P &lt; 0.05]. Also during maximal intensity rowing, HR was lower than during maximal Intensity running [194 (9) compared to 198 (11) beat-min(-1), P &lt; 0.05]. These results were accompanied by a higher maximal oxygen uptake during rowing than during running [rowing compared to running, 4.50 (0.5) and 4.35 (0.4) l.min(-1), respectively, P &lt; 0.01]. Thus, the oxygen pulse, as an index of the stroke volume of the heart, was higher during rowing than during running at any given intensity. The results suggest that compared to running, the seated position and/or the involvement of more muscles during rowing facilitate venous return and elicit a smaller HR response for the same relative exercise intensity.

This study evaluated whether the heart rate (HR) response to exercise depends on body position and on the active muscle mass. The HR response to ergometer rowing (sitting and using both arms and legs) was compared to treadmill running (upright exercise involving mainly the legs) using a progressive exercise intensity protocol in 55 healthy men [mean (SD) height 176 (5) cm, body mass 71 (6) kg, age 21 (3) years]. During rowing HR was lower than during running at a blood lactate concentration of 2 mmol.l(-1) [145 (13) compared to 150 (11) beat-min(-1), P &lt; 0.05], 4 mmol.l(-1) [170 (10) compared to 177 (13) beat-min(-1), P &lt; 0.05], and 6 mmol.l(-1) [182 (10) compared to 188 (10) beat-min(-1), P &lt; 0.05]. Also during maximal intensity rowing, HR was lower than during maximal Intensity running [194 (9) compared to 198 (11) beat-min(-1), P &lt; 0.05]. These results were accompanied by a higher maximal oxygen uptake during rowing than during running [rowing compared to running, 4.50 (0.5) and 4.35 (0.4) l.min(-1), respectively, P &lt; 0.01]. Thus, the oxygen pulse, as an index of the stroke volume of the heart, was higher during rowing than during running at any given intensity. The results suggest that compared to running, the seated position and/or the involvement of more muscles during rowing facilitate venous return and elicit a smaller HR response for the same relative exercise intensity.

Exercise is known to induce the oxidation of blood low-molecular-weight (LMW) thiols such as reduced glutathione (GSH). We previously reported that full-marathon running induced a decrease in human plasma levels of protein-bound sulfhydryl groups (p-SHs). Moderate exercise, a 30-min running at the intensity of the individual ventilatory threshold, performed by untrained healthy females caused a significant decrease in erythrocyte levels of p-SHs (mostly hemoglobin cysteine residues) and LMW thiols, but their levels returned to each baseline by 2 h. No significant change in plasma LMW thiols was observed. However, plasma levels of p-SHs significantly decreased after running and remained unchanged after 24 h. These results suggest that moderate exercise causes the oxidation of blood thiols, especially protein-bound thiols. (C) 2002 Elsevier Science Inc. All rights reserved.

Exercise is known to induce the oxidation of blood low-molecular-weight (LMW) thiols such as reduced glutathione (GSH). We previously reported that full-marathon running induced a decrease in human plasma levels of protein-bound sulfhydryl groups (p-SHs). Moderate exercise, a 30-min running at the intensity of the individual ventilatory threshold, performed by untrained healthy females caused a significant decrease in erythrocyte levels of p-SHs (mostly hemoglobin cysteine residues) and LMW thiols, but their levels returned to each baseline by 2 h. No significant change in plasma LMW thiols was observed. However, plasma levels of p-SHs significantly decreased after running and remained unchanged after 24 h. These results suggest that moderate exercise causes the oxidation of blood thiols, especially protein-bound thiols. (C) 2002 Elsevier Science Inc. All rights reserved.

We evaluated the effects of rowing on the morphology and function of the leg extensor muscle in old people. The area and the power of the leg extensor muscle were measured in 15 oarsmen - age [mean (SD)] 65 (3) years
height 171 (4) cm, body mass 68 (6) kg - and in 15 sedentary men - age 66 (4) years, height 170 (4) cm, body mass 67 (7) kg - who were matched on the basis of their body size. The leg extensor muscle area of the oarsmen was larger than that of the sedentary men [77.8 (5.4) vs 68.4 (5.1) cm2, P &lt
0.05]. Also the bilateral leg extension power of the oarsmen was larger than that of the sedentary men [1,624 (217) vs 1,296 (232) W, P &lt
0.05]. Thus, the leg extension power per the leg extensor muscle area was not significantly different between two groups [20.9 (2.0) vs 19.9 (2.1) W·cm-2) and leg extension power was correlated to the leg extensor muscle area (59-89 cm 2, r = 0.74, P &lt
0.001). Also the 2,000-m rowing ergometer time of the oarsmen [495 (14) s
range 479-520 s] was related to leg extensor muscle area (68-89 cm2, r = 0.63, P &lt
0.01). The results suggest that rowing prevents age-related muscle wasting and weakness. © Springer-Verlag 2002.

We evaluated the effects of rowing on the morphology and function of the leg extensor muscle in old people. The area and the power of the leg extensor muscle were measured in 15 oarsmen - age [mean (SD)] 65 (3) years
height 171 (4) cm, body mass 68 (6) kg - and in 15 sedentary men - age 66 (4) years, height 170 (4) cm, body mass 67 (7) kg - who were matched on the basis of their body size. The leg extensor muscle area of the oarsmen was larger than that of the sedentary men [77.8 (5.4) vs 68.4 (5.1) cm2, P &lt
0.05]. Also the bilateral leg extension power of the oarsmen was larger than that of the sedentary men [1,624 (217) vs 1,296 (232) W, P &lt
0.05]. Thus, the leg extension power per the leg extensor muscle area was not significantly different between two groups [20.9 (2.0) vs 19.9 (2.1) W·cm-2) and leg extension power was correlated to the leg extensor muscle area (59-89 cm 2, r = 0.74, P &lt
0.001). Also the 2,000-m rowing ergometer time of the oarsmen [495 (14) s
range 479-520 s] was related to leg extensor muscle area (68-89 cm2, r = 0.63, P &lt
0.01). The results suggest that rowing prevents age-related muscle wasting and weakness. © Springer-Verlag 2002.

We developed an analytical method for measuring tea catechins in plasma by solid-phase extraction (SPE), followed by HPLC with a coulometric electrochemical detector. The plasma was mixed with an equal volume of acetonitrile to precipitate protein, and catechins in the resulting supernatant were extracted by SPE, using a C 18 cartridge. To correct the extraction efficiency, ethyl gallate was simultaneously added with acetonitrile as an internal standard. Plasma samples were treated in microtubes, and evaporation and SPE were performed by the use of a vacuum centrifuge and vacuum manifold for SPE. The use of these instruments allowed the handling of a large number of samples simultaneously. In this method, (-)-epicatechin (EC), (-)-epicatechin-3-O-gallate (ECg), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-O-gallate (EGCg), and ethyl gallate could be detected as a single peak with high sensitivity. For an analysis of the conjugated form of catechins, plasma samples were treated with glucuronidase and sulfatase. Type H-2 beta-glucuronidase effectively digested the conjugated forms, and the enzyme also converted EGCg and ECg to their nongallated form. When the concentrations of catechins in plasma were analyzed in subjects who took a single dose of catechin liquid, the concentration of free EGCg in plasma reached a maximum of 300 nM at 1 h after intake; those of the other free form of catechins increased only slightly after the intake. The concentration of total catechins (free+conjugated forms) in plasma increased up to 2 h after the intake.

We developed an analytical method for measuring tea catechins in plasma by solid-phase extraction (SPE), followed by HPLC with a coulometric electrochemical detector. The plasma was mixed with an equal volume of acetonitrile to precipitate protein, and catechins in the resulting supernatant were extracted by SPE, using a C 18 cartridge. To correct the extraction efficiency, ethyl gallate was simultaneously added with acetonitrile as an internal standard. Plasma samples were treated in microtubes, and evaporation and SPE were performed by the use of a vacuum centrifuge and vacuum manifold for SPE. The use of these instruments allowed the handling of a large number of samples simultaneously. In this method, (-)-epicatechin (EC), (-)-epicatechin-3-O-gallate (ECg), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-O-gallate (EGCg), and ethyl gallate could be detected as a single peak with high sensitivity. For an analysis of the conjugated form of catechins, plasma samples were treated with glucuronidase and sulfatase. Type H-2 beta-glucuronidase effectively digested the conjugated forms, and the enzyme also converted EGCg and ECg to their nongallated form. When the concentrations of catechins in plasma were analyzed in subjects who took a single dose of catechin liquid, the concentration of free EGCg in plasma reached a maximum of 300 nM at 1 h after intake; those of the other free form of catechins increased only slightly after the intake. The concentration of total catechins (free+conjugated forms) in plasma increased up to 2 h after the intake.

We reported that genistein, a soybean isoflavone, prevents bone loss caused by estrogen deficiency, without undesirable effects on the uterus. In this study, we examined cooperative effects of genistein administration and running exercise on bone mass in ovariectomized (OVX) mice. Female mice aged 7 weeks were either sham-operated or OVX and divided into six groups: (1) sham; (2) OVX; (3) OVX, treated with genistein at a submaximal dose (0.4 mg/day) subcutaneously (G); (4) OVX, exercised on a treadmill daily for 30 minutes/day at 12 m/minute on a 10 degrees uphill slope (Ex); (5) OVX, given genistein and exercised (ExG); and (6) OVX, treated with 17 beta -estradiol (0.03 mug/day) in the same manner as genistein (E-2). Four weeks after intervention, bone mass was estimated by dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Bone mineral density (BMD) of the whole femur measured by DXA was higher in both the G and the Ex groups than in the OVX group. Furthermore, BMD in the ExG group was significantly higher than that in the groups receiving either intervention alone. Bone area in distal region of the femur was significantly higher in Ex and ExG groups as compared with those in the OVX and G groups. pQCT analysis showed that the cross-sectional areas (CSAs) and periosteum perimeter at midshaft of the femur did not differ in the sham and OVX groups but were significantly higher in Ex and ExG groups. Histomorphometric analysis showed that bone formation rate/bone surface (BFR/BS) was significantly higher in both Ex and ExG groups as compared with that in non-exercised groups. The bone volume (BV/TV) in the distal femoral cancellous bone was lower in the OVX than that in the sham group, and it was restored completely in the ExG group, as in the E-2 group. Thickness of the trabecular bone (Tb.Th) was higher in Ex and ExG groups than that in the OVX and G groups. These results indicate that the combined intervention of moderate exercise and the submaximal dose of genistein administration show a cooperative effect in preventing bone loss in OVX mice.

We reported that genistein, a soybean isoflavone, prevents bone loss caused by estrogen deficiency, without undesirable effects on the uterus. In this study, we examined cooperative effects of genistein administration and running exercise on bone mass in ovariectomized (OVX) mice. Female mice aged 7 weeks were either sham-operated or OVX and divided into six groups: (1) sham; (2) OVX; (3) OVX, treated with genistein at a submaximal dose (0.4 mg/day) subcutaneously (G); (4) OVX, exercised on a treadmill daily for 30 minutes/day at 12 m/minute on a 10 degrees uphill slope (Ex); (5) OVX, given genistein and exercised (ExG); and (6) OVX, treated with 17 beta -estradiol (0.03 mug/day) in the same manner as genistein (E-2). Four weeks after intervention, bone mass was estimated by dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Bone mineral density (BMD) of the whole femur measured by DXA was higher in both the G and the Ex groups than in the OVX group. Furthermore, BMD in the ExG group was significantly higher than that in the groups receiving either intervention alone. Bone area in distal region of the femur was significantly higher in Ex and ExG groups as compared with those in the OVX and G groups. pQCT analysis showed that the cross-sectional areas (CSAs) and periosteum perimeter at midshaft of the femur did not differ in the sham and OVX groups but were significantly higher in Ex and ExG groups. Histomorphometric analysis showed that bone formation rate/bone surface (BFR/BS) was significantly higher in both Ex and ExG groups as compared with that in non-exercised groups. The bone volume (BV/TV) in the distal femoral cancellous bone was lower in the OVX than that in the sham group, and it was restored completely in the ExG group, as in the E-2 group. Thickness of the trabecular bone (Tb.Th) was higher in Ex and ExG groups than that in the OVX and G groups. These results indicate that the combined intervention of moderate exercise and the submaximal dose of genistein administration show a cooperative effect in preventing bone loss in OVX mice.

This study was performed to assess the effects of short-term, extremely high-intensity intermittent exercise training on the GLUT-4 content of rat skeletal muscle. Three- to four-week-old male Sprague-Dawley rats with an initial body weight ranging from 45 to 55 g were used for this study. These rats were randomly assigned to an 8-day period of high-intensity intermittent exercise training (HIT), relatively high-intensity intermittent prolonged exercise training (RHT), or low-intensity prolonged exercise training (LIT). Age-matched sedentary rats were used as a control. In the HIT group, the rats repeated fourteen 20-s swimming bouts with a weight equivalent to 14, 15, and 16% of body weight for the first 2, the next 4, and the last 2 days, respectively. Between exercise bouts, a 10-s pause was allowed. RHT consisted of five 17-min swimming bouts with a 3-min rest between bouts. During the first bout, the rat swam without weight, whereas during the following four bouts, the rat was attached to a weight equivalent to 4 and 5% of its body weight for the first 5 days and the following 3 days, respectively. Rats in the LIT group swam 6 h/day for 8 days in two 3-h bouts separated by 45 min of rest. In the first experiment, the HIT, LIT, and control rats were compared. GLUT-4 content in the epitrochlearis muscle in the HIT and LIT groups after training was significantly higher than that in the control rats by 83 and 91%, respectively. Furthermore, glucose transport activity, stimulated maximally by both insulin (2 mU/ ml) (HIT: 48%, LIT: 75%) and contractions (25 10-s tetani) (HIT: 55%, LIT: 69%), was higher in the training groups than in the control rats. However, no significant differences in GLUT-4 content or in maximal glucose transport activity in response to both insulin and contractions were observed between the two training groups. The second experiment demonstrated that GLUT-4 content after HIT did not differ from that after RHT (66% higher in trained rats than in control). In conclusion, the present investigation demonstrated that 8 days of HIT lasting only 280 s elevated both GLUT-4 content and maximal glucose transport activity in rat skeletal muscle to a level similar to that attained after LIT, which has been considered a tool to increase GLUT-4 content maximally.

This study was performed to assess the effects of short-term, extremely high-intensity intermittent exercise training on the GLUT-4 content of rat skeletal muscle. Three- to four-week-old male Sprague-Dawley rats with an initial body weight ranging from 45 to 55 g were used for this study. These rats were randomly assigned to an 8-day period of high-intensity intermittent exercise training (HIT), relatively high-intensity intermittent prolonged exercise training (RHT), or low-intensity prolonged exercise training (LIT). Age-matched sedentary rats were used as a control. In the HIT group, the rats repeated fourteen 20-s swimming bouts with a weight equivalent to 14, 15, and 16% of body weight for the first 2, the next 4, and the last 2 days, respectively. Between exercise bouts, a 10-s pause was allowed. RHT consisted of five 17-min swimming bouts with a 3-min rest between bouts. During the first bout, the rat swam without weight, whereas during the following four bouts, the rat was attached to a weight equivalent to 4 and 5% of its body weight for the first 5 days and the following 3 days, respectively. Rats in the LIT group swam 6 h/day for 8 days in two 3-h bouts separated by 45 min of rest. In the first experiment, the HIT, LIT, and control rats were compared. GLUT-4 content in the epitrochlearis muscle in the HIT and LIT groups after training was significantly higher than that in the control rats by 83 and 91%, respectively. Furthermore, glucose transport activity, stimulated maximally by both insulin (2 mU/ ml) (HIT: 48%, LIT: 75%) and contractions (25 10-s tetani) (HIT: 55%, LIT: 69%), was higher in the training groups than in the control rats. However, no significant differences in GLUT-4 content or in maximal glucose transport activity in response to both insulin and contractions were observed between the two training groups. The second experiment demonstrated that GLUT-4 content after HIT did not differ from that after RHT (66% higher in trained rats than in control). In conclusion, the present investigation demonstrated that 8 days of HIT lasting only 280 s elevated both GLUT-4 content and maximal glucose transport activity in rat skeletal muscle to a level similar to that attained after LIT, which has been considered a tool to increase GLUT-4 content maximally.

To determine the influence of rowing exercise on serum lipid and lipoprotein profiles, forty-one collegiate male rowers were compared with thirty-one sedcntary men. Compared with sedentary men, male rowers were taller (P<0.01),^-heavier (P<0.001), had higher body mass index (BMI)(P<0.05), and a larger proportion of lean body mass (LBM)(P<0.001). However, the rowers had a similar percentage of body fat as that of sedentary men (11±4 vs. 13±5 %, mean±SD). Male rowers had higher maximal oxygen uptake (VO_<2max> 61±4 ml・kg^<-1>・min^<-1>) and leg extension power (33.7±5W・kg^<-1>) than sedentary men (48±6 ml・kg^<-1>・min^<-1>, 25±6 W・kg^<-1>, P<0.001, respectively). The blood variables were within the normal range, except for an elevated creatine phosphokinase (CPK) in male rowers. Male rowers had lower total cholesterol (TC 3.95±0.62 vs. 4.77±0.64 mmol・L^<-1>, P<0.001), low-density lipoprotein cholesterol (LDLC 2.01±0.51 vs. 2.85±0.59 mmol・L^<-1>, P<0.001) and the ratio of LDLC to high-density lipoprotein cholesterol (HDLC) (1.41±0.48 vs. 2.00±0.56 unit, P<0.001) than sedentary men. There was similar HDLC levels between the rowers and sedentary men (1.49±0.31 vs. 1.47±0.22 mmol・L^<-1>). Thus, the findings of the current cross-sectional study suggest the possibility that regularly performed rowing exercise, which increases both VO_<2max> and LBM, may induce a favorable reduction of serum LDLC in men.

To determine the influence of rowing exercise on serum lipid and lipoprotein profiles, forty-one collegiate male rowers were compared with thirty-one sedcntary men. Compared with sedentary men, male rowers were taller (P<0.01),^-heavier (P<0.001), had higher body mass index (BMI)(P<0.05), and a larger proportion of lean body mass (LBM)(P<0.001). However, the rowers had a similar percentage of body fat as that of sedentary men (11±4 vs. 13±5 %, mean±SD). Male rowers had higher maximal oxygen uptake (VO_<2max> 61±4 ml・kg^<-1>・min^<-1>) and leg extension power (33.7±5W・kg^<-1>) than sedentary men (48±6 ml・kg^<-1>・min^<-1>, 25±6 W・kg^<-1>, P<0.001, respectively). The blood variables were within the normal range, except for an elevated creatine phosphokinase (CPK) in male rowers. Male rowers had lower total cholesterol (TC 3.95±0.62 vs. 4.77±0.64 mmol・L^<-1>, P<0.001), low-density lipoprotein cholesterol (LDLC 2.01±0.51 vs. 2.85±0.59 mmol・L^<-1>, P<0.001) and the ratio of LDLC to high-density lipoprotein cholesterol (HDLC) (1.41±0.48 vs. 2.00±0.56 unit, P<0.001) than sedentary men. There was similar HDLC levels between the rowers and sedentary men (1.49±0.31 vs. 1.47±0.22 mmol・L^<-1>). Thus, the findings of the current cross-sectional study suggest the possibility that regularly performed rowing exercise, which increases both VO_<2max> and LBM, may induce a favorable reduction of serum LDLC in men.

We investigated the influence of a single exhaustive bout of downhill running on oxidative damage to DNA and changes of antioxidant vitamin concentrations in rats. Plasma vitamin E levels were unchanged up to 48 hr postexercise. However, plasma ascorbic acid (AA) levels increased after the exercise, then decreased thereafter. This increase corresponded to a marked decrease in AA concentration in the adrenal glands. The activity of hepatic l-gulono-gamma -lactone oxidase, which catalyzes AA synthesis, was unaltered after the exercise. The weight of the adrenal glands was significantly increased 24 hr postexercise. These results indicate that the change in the plasma AA concentration after vigorous exercise was due mainly to the release of AA from the adrenal glands. The plasma creatine phosphokinase (CPK) activity and white blood cell (WBC) count increased 3 to 6 hr postexercise. Over this same period, a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine in DNA, increased in the WBC, but not in the foreleg muscle. Lipid peroxide and vitamin E levels were also unchanged in the foreleg muscle. There was a positive correlation between CPK activity in the plasma and DNA damage in the WBC, suggesting that the DNA damage in the WBC was closely related with muscle damage due to exercise. (J. Nutr. Biochem. 11:401-407, 2000) (C) Elsevier Science Inc. 2000. All rights reserved.

We investigated the influence of a single exhaustive bout of downhill running on oxidative damage to DNA and changes of antioxidant vitamin concentrations in rats. Plasma vitamin E levels were unchanged up to 48 hr postexercise. However, plasma ascorbic acid (AA) levels increased after the exercise, then decreased thereafter. This increase corresponded to a marked decrease in AA concentration in the adrenal glands. The activity of hepatic l-gulono-gamma -lactone oxidase, which catalyzes AA synthesis, was unaltered after the exercise. The weight of the adrenal glands was significantly increased 24 hr postexercise. These results indicate that the change in the plasma AA concentration after vigorous exercise was due mainly to the release of AA from the adrenal glands. The plasma creatine phosphokinase (CPK) activity and white blood cell (WBC) count increased 3 to 6 hr postexercise. Over this same period, a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine in DNA, increased in the WBC, but not in the foreleg muscle. Lipid peroxide and vitamin E levels were also unchanged in the foreleg muscle. There was a positive correlation between CPK activity in the plasma and DNA damage in the WBC, suggesting that the DNA damage in the WBC was closely related with muscle damage due to exercise. (J. Nutr. Biochem. 11:401-407, 2000) (C) Elsevier Science Inc. 2000. All rights reserved.

Exercise induced chromosomal damage was evaluated in trained and untrained subjects, who performed treadmill running at 85% of maximal oxygen uptake for 30 min. The subjects had their peripheral blood taken before, immediately after and 30 min after the running test for the analysis of lymphocyte chromosomal damage that was evaluated by micronucleus assay. The blood samples were also subjected to X-ray irraddiation in vitro to examine the modification of exercise induced chromosomal damage by a secondarily induced oxidative stress. Spontaneous chromosomal damage in lymphocytes did not significantly increase at least until 30 min after the running both in the trained and untrained subjects. However, the X-ray-induced chromosomal damage was significantly enhanced at 30 min after the running in the untrained group, but not in the trained group. The ratio of X-ray-induced/spontaneous chromosomal damage also tended to increase after the running only in the untrained group. These preliminary results suggest that intensive exercise induced Very slight chromosomal damage only in the untrained group. which could be intensified by the secondarily induced oxidative stress.

Exercise induced chromosomal damage was evaluated in trained and untrained subjects, who performed treadmill running at 85% of maximal oxygen uptake for 30 min. The subjects had their peripheral blood taken before, immediately after and 30 min after the running test for the analysis of lymphocyte chromosomal damage that was evaluated by micronucleus assay. The blood samples were also subjected to X-ray irraddiation in vitro to examine the modification of exercise induced chromosomal damage by a secondarily induced oxidative stress. Spontaneous chromosomal damage in lymphocytes did not significantly increase at least until 30 min after the running both in the trained and untrained subjects. However, the X-ray-induced chromosomal damage was significantly enhanced at 30 min after the running in the untrained group, but not in the trained group. The ratio of X-ray-induced/spontaneous chromosomal damage also tended to increase after the running only in the untrained group. These preliminary results suggest that intensive exercise induced Very slight chromosomal damage only in the untrained group. which could be intensified by the secondarily induced oxidative stress.

Recently (K. Kawanaka, I. Tabata, and M. Higuchi. J. Appl. Physiol. 83: 429-433, 1997), we demonstrated that glucose transport activity after repeated 10-s-long in vitro tetani in rat epitrochlearis (Epi) muscle was negatively correlated with the postcontraction muscle glycogen concentration. Therefore, we examined whether high-intensity intermittent swimming, which depletes muscle glycogen to a lower level than that observed after ten 10-s-long in vitro tetani, elicits higher glucose transport than that observed after ten 10-s-long in vitro tetani, which has been regarded as the exercise-induced maximal stimulus for glucose transport. In male rats, 2-deoxy-D-glucose transport rate in Epi muscle after eight bouts of high-intensity intermittent swimming with a weight equal to 18% of body mass (exercise duration: 20 s, rest duration between exercise bouts: 40 s) was higher than that observed after the ten 10-s-long tetani (2.25 +/- 0.08 vs. 1.02 +/- 0.16 mu mol.ml intracellular water(-1).20 min(-1)). Muscle glycogen concentration in Epi after eight bouts of high-intensity intermittent swimming was significantly lower than that observed after ten 10-s-long in vitro tetani (7.6 +/- 0.5 vs. 14.8 +/- 1.4 mu mol glucose/g muscle). These observations show that the high-intensity intermittent swimming increases glucose transport in rat Epi to a much higher level than that induced by ten 10-s-long in vitro tetani, which has been regarded as the exercise-related maximal stimulus for glucose transport. Furthermore, this finding suggests that the lower muscle glycogen level after high-intensity intermittent swimming than after in vitro tetani may play a role, because there was a significant negative correlation between glucose transport and muscle glycogen concentration in Epi after high-intensity swimming and in vitro tetani.

Recently (K. Kawanaka, I. Tabata, and M. Higuchi. J. Appl. Physiol. 83: 429-433, 1997), we demonstrated that glucose transport activity after repeated 10-s-long in vitro tetani in rat epitrochlearis (Epi) muscle was negatively correlated with the postcontraction muscle glycogen concentration. Therefore, we examined whether high-intensity intermittent swimming, which depletes muscle glycogen to a lower level than that observed after ten 10-s-long in vitro tetani, elicits higher glucose transport than that observed after ten 10-s-long in vitro tetani, which has been regarded as the exercise-induced maximal stimulus for glucose transport. In male rats, 2-deoxy-D-glucose transport rate in Epi muscle after eight bouts of high-intensity intermittent swimming with a weight equal to 18% of body mass (exercise duration: 20 s, rest duration between exercise bouts: 40 s) was higher than that observed after the ten 10-s-long tetani (2.25 +/- 0.08 vs. 1.02 +/- 0.16 mu mol.ml intracellular water(-1).20 min(-1)). Muscle glycogen concentration in Epi after eight bouts of high-intensity intermittent swimming was significantly lower than that observed after ten 10-s-long in vitro tetani (7.6 +/- 0.5 vs. 14.8 +/- 1.4 mu mol glucose/g muscle). These observations show that the high-intensity intermittent swimming increases glucose transport in rat Epi to a much higher level than that induced by ten 10-s-long in vitro tetani, which has been regarded as the exercise-related maximal stimulus for glucose transport. Furthermore, this finding suggests that the lower muscle glycogen level after high-intensity intermittent swimming than after in vitro tetani may play a role, because there was a significant negative correlation between glucose transport and muscle glycogen concentration in Epi after high-intensity swimming and in vitro tetani.

After running training, which increased GLUT-4 protein content in rat skeletal muscle by &lt;40% compared with control rats, the training effect on insulin-stimulated maximal glucose transport (insulin responsiveness) in skeletal muscle was short lived (24 h). A recent study reported that GLUT-4 protein content in rat epitrochlearis muscle increased dramatically (similar to 2-fold) after swimming training (J.-M. Ren, C. F. Semenkovich, E. A. Gulve, J. Gao, and J. O. Holloszy. J. Biol. Chem. 269, 14396-14401, 1994). Because GLUT-I protein content is known to be closely related to skeletal muscle insulin responsiveness, we thought it possible that the training effect on insulin responsiveness may remain for &gt;24 h after swimming training if GLUT-4 protein content decreases gradually from the relatively high level and still remains higher than control level for &gt;24 h after swimming training. Therefore, we examined this possibility. Male Sprague-Dawley rats swam 2 h a day for 5 days with a weight equal to 2% of body mass. Approximately 18, 42, and 90 h after cessation of training, GLUT-I protein concentration and 2-[1,2-H-3]deoxy-D-glucose transport in the presence of a maximally stimulating concentration of insulin (2 mU/ml) were examined by using incubated epitrochlearis muscle preparation. Swimming training increased GLUT-4 protein concentration and insulin responsiveness by 87 and 85%, respectively, relative to age-matched controls when examined 18 h after training. Forty-two hours after training, GLUT-4 protein concentration and insulin responsiveness were still higher by 52 and 51%, respectively, in muscle from trained rats compared with control. GLUT-4 protein concentration and insulin responsiveness in trained muscle returned to sedentary control level within 90 h after training. We conclude that 1) the change in insulin responsiveness during detraining is directly related to muscle GLUT-4 protein content, and 2) consequently, the greater the increase in GLUT-4 protein content that is induced by training, the longer an effect on insulin responsiveness persists after the training.

After running training, which increased GLUT-4 protein content in rat skeletal muscle by &lt;40% compared with control rats, the training effect on insulin-stimulated maximal glucose transport (insulin responsiveness) in skeletal muscle was short lived (24 h). A recent study reported that GLUT-4 protein content in rat epitrochlearis muscle increased dramatically (similar to 2-fold) after swimming training (J.-M. Ren, C. F. Semenkovich, E. A. Gulve, J. Gao, and J. O. Holloszy. J. Biol. Chem. 269, 14396-14401, 1994). Because GLUT-I protein content is known to be closely related to skeletal muscle insulin responsiveness, we thought it possible that the training effect on insulin responsiveness may remain for &gt;24 h after swimming training if GLUT-4 protein content decreases gradually from the relatively high level and still remains higher than control level for &gt;24 h after swimming training. Therefore, we examined this possibility. Male Sprague-Dawley rats swam 2 h a day for 5 days with a weight equal to 2% of body mass. Approximately 18, 42, and 90 h after cessation of training, GLUT-I protein concentration and 2-[1,2-H-3]deoxy-D-glucose transport in the presence of a maximally stimulating concentration of insulin (2 mU/ml) were examined by using incubated epitrochlearis muscle preparation. Swimming training increased GLUT-4 protein concentration and insulin responsiveness by 87 and 85%, respectively, relative to age-matched controls when examined 18 h after training. Forty-two hours after training, GLUT-4 protein concentration and insulin responsiveness were still higher by 52 and 51%, respectively, in muscle from trained rats compared with control. GLUT-4 protein concentration and insulin responsiveness in trained muscle returned to sedentary control level within 90 h after training. We conclude that 1) the change in insulin responsiveness during detraining is directly related to muscle GLUT-4 protein content, and 2) consequently, the greater the increase in GLUT-4 protein content that is induced by training, the longer an effect on insulin responsiveness persists after the training.

We improved the analytical method for the detection of ascorbic acid in plasma by high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD) to be more selective and rapid than the protocol previously used. Main improvements are as follows. Applied potential of ECD to obtain the maximal response for ascorbic acid was +450 mV versus Ag/AgCl, but it was reduced to +350 mV. In that condition, uric acid did not respond to ECD, and only ascorbic acid was detected. EDTA contained in sample extraction/stabilizing solution gave the peak after the ascorbic acid. The addition of EDTA (0.2 mM) to the mobile phase eliminated the EDTA peak. These two improvements gave the chromatogram in which the peak that appeared from the plasma sample was only ascorbic acid, and shorten the sample run time. Ascorbic acid in plasma was unstable even though the plasma was treated with methanol/EDTA: it decreased from 1.5h at 4 degrees C. However, the treated sample, which was placed at -14 degrees C until the analysis was performed, gave the reliable ascorbic acid value at least up to 6h. The data obtained from the HPLC-ECD method was consistent with those from the hydrazine method.

We improved the analytical method for the detection of ascorbic acid in plasma by high-performance liquid chromatography (HPLC) with an electrochemical detector (ECD) to be more selective and rapid than the protocol previously used. Main improvements are as follows. Applied potential of ECD to obtain the maximal response for ascorbic acid was +450 mV versus Ag/AgCl, but it was reduced to +350 mV. In that condition, uric acid did not respond to ECD, and only ascorbic acid was detected. EDTA contained in sample extraction/stabilizing solution gave the peak after the ascorbic acid. The addition of EDTA (0.2 mM) to the mobile phase eliminated the EDTA peak. These two improvements gave the chromatogram in which the peak that appeared from the plasma sample was only ascorbic acid, and shorten the sample run time. Ascorbic acid in plasma was unstable even though the plasma was treated with methanol/EDTA: it decreased from 1.5h at 4 degrees C. However, the treated sample, which was placed at -14 degrees C until the analysis was performed, gave the reliable ascorbic acid value at least up to 6h. The data obtained from the HPLC-ECD method was consistent with those from the hydrazine method.