Changes of thioredoxin, oxidative stress markers, inflammation and muscle/renal damage following intensive endurance exercise.

Thioredoxin (TRX) is a 12 kDa protein that is induced by oxidative stress, scavenges reactive oxygen species (ROS) and modulates chemotaxis. Furthermore it is thought to play a protective role in renal ischemia/reperfusion injury. Complement 5a (C5a) is a chemotactic factor of neutrophils and is produced after ischemia/reperfusion injury in the kidney. Both TRX and C5a increase after endurance exercise. Therefore, it may be possible that TRX has an association with C5a in renal disorders and/or renal protection caused by endurance exercise. Accordingly, the aim of this study was to investigate relationships among the changes of urine levels of TRX, C5a and acute kidney injury (AKI) caused by ischemia/reperfusion, inflammatory responses, and oxidative stress following intensive endurance exercise. Also, we applied a newly-developed measurement system of neutrophil migratory activity and ROS-production by use of ex vivo hydrogel methodology with an extracellular matrix to investigate the mechanisms of muscle damage. Fourteen male triathletes participated in a duathlon race consisting of 5 km of running, 40 km of cycling and 5 km of running were recruited to the study. Venous blood and urine samples were collected before, immediately following, 1.5 h and 3 h after the race. Plasma, serum and urine were analyzed using enzyme-linked immunosorbent assays, a free radical analytical system, and the ex vivo neutrophil functional measurement system. These data were analyzed by assigning participants to damaged and minor-damage groups by the presence and absence of renal tubular epithelial cells in the urinary sediments. We found strong associations among urinary TRX, C5a, interleukin (IL)-2, IL-4, IL-8, IL-10, interferon (IFN)-γ and monocyte chemotactic protein (MCP)-1. From the data it might be inferred that urinary TRX, MCP-1 and ß-N-acetyl-D-glucosaminidase (NAG) were associated with renal tubular injury. Furthermore, TRX may be influenced by levels of IL-10, regulate chemotactic activity of C5a and IL-8, and control inflammatory progress by C5a and IL-8. In the longer duration group (minor-damage group), circulating neutrophil count, plasma concentration of myeloperoxidase (MPO) and serum concentration of myoglobin were markedly increased. In the higher intensity group (damaged group), neutrophil activation and degranulation of MPO might be inhibited, because not only was ROS production observed to be higher, but also antioxidant capacity and antiinflammatory cytokines were increased. Critically, the newlydeveloped ex vivo methodology corroborated the neutrophil activation levels in the two groups of participants.

Urinary excretion of cytokines versus their plasma levels after endurance exercise.

It has been consistently shown that circulating levels of interleukin (IL)-6, IL-8, IL-1 receptor antagonist (IL-1ra) and IL-10 increase remarkably following endurance exercise longer than 2 h such as marathon and triathlon races. However, no studies have compared changes in the plasma and urinary levels of these cytokines after endurance exercise, including the recovery period. In the present study, we investigated kinetic changes in the urinary excretion of cytokines following endurance exercise up to 3 h after exercise to evaluate the magnitude of change in comparison to the plasma levels and to explore the possible biological significance and the mechanisms of cytokine dynamics following exercise. Fourteen male athletes participated in a duathlon race consisting of 5 km of running, 40 km of cycling, and 5 km of running. Venous blood and urine samples were collected before, immediately after, 1.5 h and 3 h after the race. Plasma and urine were analyzed using enzyme-linked immunosorbent assays (ELISA). Plasma concentrations of lL-1beta, IL-1ra, IL-6, IL-8, IL-10 and monocyte chemotactic protein (MCP)-1 increased significantly after the race, whereas tumour necrosis factor (TNF)-alpha, IL-2, IL-4, IL-12 and interferon (IFN)-gamma did not change significantly. Urinary concentrations of lL-1beta, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFN-gamma and MCP-1 increased significantly after the race. When the urine concentrations were adjusted by creatinine concentration, urine volume and sampling time, the increases of lL-2, IL-4, IL-8, IL-10, IFN-gamma and MCP-1 were evident and these were notably present in urine of the stressed athletes suffering from renal tubular epithelial damage. The present study provides new evidence that the kinetics and magnitude of changes in urinary cytokine concentrations differ from plasma cytokine concentrations following endurance exercise, especially, in the recovery period several hours after exercise, and that the damaged kidney might be responsible at least in part for the kinetics of some cytokines. Urinary cytokines may be sensitive biomarkers of the impact of exhaustive exercise workload on renal damage and inflammation in the recovery period after endurance exercise.

IL-17, neutrophil activation and muscle damage following endurance exercise.

The T-cell subset Th17 is induced partly by interleukin (IL)-6 and activated by IL-23, and produces a proinflammatory cytokine IL-17. Since IL-6 increases dramatically following long-lasting endurance exercise, this response may also stimulate the induction of IL-17 and IL-23 after exercise. The aim of this study was to clarify the dynamics of IL-17 in association with endurance exercise-induced muscle damage and inflammatory responses. Fourteen male triathletes participated in a duathlon race consisting of 5 km of running, 40 km of cycling and 5 km of running. Venous blood and urine samples were collected before, immediately after 1.5 h and 3 h after the race. Plasma and urine were analyzed using enzyme-linked immunosorbent assays (ELISA). Haematological and biochemical variables such as neutrophil activation marker (myeloperoxidase: MPO), muscle damage marker (myoglobin: Mb) and soluble receptor activator of nuclear factor (NF)-KB ligand (sRANKL) were also determined to estimate the biological and pathological significance. Plasma concentrations oflL-6 (+26.0x), MPO (+3.2x) and Mb (+4.9x) increased significantly immediately after the race and IL-17 and IL-23 tended to increase. Furthermore, plasma concentrations of IL-12p40 and sRANKL increased significantly after the race. The measured parameters related to Thl 7 cytokines in the urinary output were closely correlated with each other and muscle damage marker. These findings suggest that IL-17 induced by IL-6 and activated by IL-23 or other IL-17 producing-cells and IL-23 might promote neutrophil activation and muscle damage following prolonged endurance exercise.

Antihypertensive effect of salt-free soy sauce, a new fermented seasoning, in spontaneously hypertensive rats.

UNLABELLED: In this study, we focused on the antihypertensive effect of newly fermented salt-free soy sauce (SFS) in spontaneously hypertensive rats (SHRs). A 13-wk oral administration of SFS was performed daily in 8- to 21-wk-old SHRs with a dose of 200 mg/kg of body weight/d. The systolic (SBP) and diastolic (DBP) blood pressures were measured for each week during the experimental period. As a result, a significant (P < 0.01) BP lowering effect was observed from the 5th d to the end of the experiment in the SFS group compared with the control group (DeltaSBP(21-wk): 27 mmHg, DeltaDBP(21-wk): 20 mmHg). In contrast to such substantial effect by SFS intake, angiotensin I-converting enzyme activities in blood and local organs as well as pharmaceutical parameters such as serum Na(+) or K(+) level did not show any significant difference between in both SFS and control groups. Vasoconstriction experiment using thoracic aorta rings from 21-wk SHRs provided an interesting result that the rings from the SFS group evoked a >2-fold higher increase in the angiotensin II-stimulated constrictive response compared with the rings from the control group (P = 0.012), which suggested that the SFS-intake would be effective in possessing a higher vessel tone. PRACTICAL APPLICATION: In this study, we investigated the antihypertensive effect of newly fermented SFS in SHRs. As a result of 13-wk SFS-administration, we found a significant antihypertensive effect of the SFS. This finding strongly demonstrates that the developed SFS would be greatly beneficial for health and useful for health-related industries.

Effects of alpha-glucosylhesperidin on the peripheral body temperature and autonomic nervous system.

We studied the effects of alpha-glucosylhesperidin (G-Hsp) on the peripheral body temperature and autonomic nervous system in humans. We first conducted a survey of 97 female university students about excessive sensitivity to the cold; 74% of them replied that they were susceptible or somewhat susceptible to the cold. We subsequently conducted a three-step experiment. In the first experiment, G-Hsp (500 mg) was proven to prevent a decrease in the peripheral body temperature under an ambient temperature of 24 degrees C. In the second experiment, a warm beverage containing G-Hsp promoted blood circulation and kept the finger temperature higher for a longer time. We finally used a heart-rate variability analysis to study whether G-Hsp changed the autonomic nervous activity. The high-frequency (HF) component tended to be higher, while the ratio of the low-frequency (LF)/HF components tended to be lower after the G-Hsp administration. These results suggest that the mechanism for temperature control by G-Hsp might involve an effect on the autonomic nervous system.

Effect of 4G-alpha-glucopyranosyl hesperidin on brown fat adipose tissue- and cutaneous-sympathetic nerve activity and peripheral body temperature.

Changes in the activity of the autonomic nervous system are good indicators of alterations in physiological phenomena such as the body temperature, blood glucose, blood pressure. Hesperidin, a flavanone known as vitamin P, has been shown to reduce the levels of serum lipids, cholesterol, and blood pressure. However, hesperidin is not water-soluble and is not well absorbed from the intestine. G-hesperidin (4G-alpha-glucopyranosyl hesperidin) is more water-soluble and more rapidly absorbed than hesperidin. In order to clarify the functions of G-hesperidin, we examined the effects of oral administration of G-hesperidin on interscapular brown adipose tissue-sympathetic nerve activity (BAT-SNA) and cutaneous sympathetic nerve activity (CASNA) in rats weighing about 300 g. In this study, we found that oral administration of 60 mg of G-hesperidin increased the BAT-SNA but decreased the CASNA in urethane-anesthetized rats. Since an elevation in BAT-SNA increases heat production (i.e. body temperature (BT)) and a decrease in CASNA increases cutaneous perfusion, we examined whether oral administration of G-hesperidin had an effect on the peripheral BT in rats. Consequently, we observed that the subcutaneous BT at the caudal end of the back after oral administration of 60 mg of G-hesperidin was significantly higher than the subcutaneous BT after oral administration of water in conscious rats. These findings suggest that G-hesperidin enhances the BAT-SNA and suppresses the CASNA resulting in an increase in the peripheral BT, probably by an increase in the thermogenesis in the BAT and an elevation in the cutaneous blood flow.

Identification of ACE-inhibitory peptides in salt-free soy sauce that are transportable across caco-2 cell monolayers.

In present study, we aimed to identify angiotensin I-converting enzyme (ACE)-inhibitory peptides from a salt-free soy sauce (SFS), a newly developed antihypertensive seasoning obtained by Aspergillus oryzae fermentation of soybean in the absence of salt, which can be transported through caco-2 cell monolayers. Through an Ussing transport investigation of SFS across caco-2 cell monolayers, three di-peptides, Ala-Phe, Phe-Ile and Ile-Phe, were successfully identified from the SFS as transportable inhibitory peptides. Ala-Phe and Ile-Phe, but not Phe-Ile, exhibited ACE-inhibitory activity with IC(50) values of 165.3 microM and 65.8 microM, respectively. Kinetic studies revealed that Ile-Phe (Km: 3.1 mM, P(app): 2.4 x 10(-6) cm/s) exhibited greater affinity toward the transport compared with Ala-Phe (K(m): 48.1 mM, P(app): 1.4 x 10(-6) cm/s) and Phe-Ile (K(m): 12.7 mM, P(app): 1.4 x 10(-6) cm/s).

Simultaneous determination of Angiotensin I-converting enzyme inhibitory peptides in tryptic casein hydrolysate by high-performance liquid chromatography combined with a replicate heart-cut column-switching technique.

A replicate heart-cut column-switching HPLC method combined with two switching valves was newly developed for the simultaneous determination of three antihypertensive peptides (Ala-Phe, Tyr-Pro, and Trp-Tyr) in tryptic casein hydrolysate in one run-in assay. After a first separation on an octadecyl silane (ODS) column, heart-cuts of each peptide were individually separated on a subsequent analytical ODS column: 26% acetonitrile for Ala-Phe and Tyr-Pro (32% for Trp-Tyr) in 0.1% trifluoroacetic acid containing 10 mM sodium 1-octanesulfonate at 0.8 mL/min. Ala-Phe, Tyr-Pro, and Trp-Tyr in casein hydrolysate were determined within 70 min to be 0.377 +/- 0.037 mg/g, 2.50 +/- 0.26 mg/g, and 0.096 +/- 0.008 mg/g, respectively.